Clinical Metabolomics

Applications in
Genetic Diseases

Anas M. Abdel Rahman

Editor

123
Clinical Metabolomics Applications
in Genetic Diseases
Anas M. Abdel Rahman
Editor

Clinical Metabolomics
Applications in Genetic
Diseases
Editor
Anas M. Abdel Rahman
Metabolomics Section, Department of Clinical Genomics, Center for Genome Medicine
King Faisal Specialist Hospital & Research Center (KFSHRC)
Riyadh, Saudi Arabia

ISBN 978-981-99-5161-1    ISBN 978-981-99-5162-8 (eBook)

[Link]

© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore
Pte Ltd. 2023
This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether
the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of
illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and
transmission or information storage and retrieval, electronic adaptation, computer software, or by similar
or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication
does not imply, even in the absence of a specific statement, that such names are exempt from the relevant
protective laws and regulations and therefore free for general use.
The publisher, the authors, and the editors are safe to assume that the advice and information in this book
are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the
editors give a warranty, expressed or implied, with respect to the material contained herein or for any
errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.

This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd.
The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721,
Singapore
Contents


The Advanced Technology and Clinical Application in Metabolomics������    1
Anas M. Abdel Rahman

Mass Spectrometry-Based Metabolomics for the Clinical Laboratory������   17
Joshua A. Dubland
 etabolomics: A Pipeline for Biomarker Discovery in Genetic Diseases��   43
M
Lina A. Dahabiyeh and Refat M. Nimer

Bioinformatic Tools for Clinical Metabolomics��������������������������������������������   71
David S. Wishart

Untargeted Metabolomics in Newborn Screening����������������������������������������   97
Joshua Manor and Sarah H. Elsea
Untargeted Metabolomics, Targeted Care:
The Clinical Utilities of Bedside Metabolomics�������������������������������������������� 117
Joshua Manor and Sarah H. Elsea

Metabolomics in the Study of Human Mitochondrial Diseases������������������ 147
Rajaa Sebaa, Mary-Ellen Harper, Ruqaiah Al-Tassan,
Mohammed Al-Owain, and Anas M. Abdel Rahman
Metabolomics of Rare Endocrine, Genetic Disease:
A Focus on the Pituitary Gland���������������������������������������������������������������������� 173
Afshan Masood, Abeer Malkawi, Anas M. Abdel Rahman,
and Mohamed Siaj
Metabolomics and Genetics of Rare Endocrine Disease:
Adrenal, Parathyroid Glands, and Cystic Fibrosis�������������������������������������� 189
Afshan Masood, Abeer Malkawi, Mohamed Siaj,
and Anas M. Abdel Rahman

Metabolomic Role in Personalized Medicine: An Update���������������������������� 207
Minnie Jacob and Anas M. Abdel Rahman

v
vi Contents


Lipidomic Profiling in Clinical Practice Using LC-MS�������������������������������� 225
Núria Amigó Grau and Pablo Ortiz Betes
Bringing Human Serum Lipidomics to the Forefront of
Clinical Practice: Two Clinical Diagnosis Success Stories �������������������������� 239
Núria Amigó Grau and Pablo Ortiz Betes
LC-MS-Based Population Metabolomics:
A Mini-Review of Recent Studies and Challenges
from Sample Collection to Data Processing�������������������������������������������������� 269
Myriam Mireault and Lekha Sleno
Metabolomics and Transcriptomic Approach to
Understand the Pathophysiology of Interstitial Lung Disease�������������������� 301
Sanjukta Dasgupta, Anindita Bhattacharya, Priyanka Choudhury,
Nilanjana Ghosh, Tanisha Das, Sushmita Roychowdhury,
Riddhiman Dhar, and Koel Chaudhury
Transferring Metabolomics to Portable Diagnostic Devices:
Trending in Biosensors������������������������������������������������������������������������������������ 327
Shimaa Eissa
The Advanced Technology and Clinical
Application in Metabolomics

Anas M. Abdel Rahman

Abstract Metabolomics identifies and quantifies small molecules (metabolites)

using high-throughput techniques. The biological system metabolome integrates
metabolomics data in conjugation with metabolic pathways, including other omics
datasets, to produce a network of endogenous metabolites (metabotype) associated
with the phenotypes. Nuclear magnetic resonance (NMR) spectroscopy and mass
spectrometry are the main analytical techniques in combination with some separa-
tion techniques such as capillary electrophoresis, ultra-high-pressure liquid chro-
matography, and gas chromatography. The drastic improvement in the detection
sensitivity and accuracy of the analytical techniques has widened the covered
metabolomics. The comprehensive coverage of metabolomics becomes more inte-
grated with other omics datasets to understand the system-level phenotypic changes
and provide insight into the mechanisms that underlie various physiological condi-
tions and diseases. This chapter highlights analytical methods for clinical metabo-
lomics research and personalized medicine. Several innovative clinical metabolomics
projects have reached up to patient services are discussed in this chapter.

Keywords Metabolomics · Chromatography · Lipidomics · Biomarker discovery ·

Mass spectrometry (MS) · Nuclear magnetic resonance (NMR)

Abbreviations

4MOP 4-Methyl-2-oxopentanoic acid

BHBA β-hydroxybutyric acid
BSTFA N,O-bis(trimethylsilyl)trifluoroacetamide
CE Capillary electrophoresis

A. M. Abdel Rahman (*)

Metabolomics Section, Department of Clinical Genomics, Center for Genome Medicine,
King Faisal Specialist Hospital & Research Center (KFSHRC), Riyadh, Saudi Arabia
e-mail: aabdelrahman46@[Link]

© The Author(s), under exclusive license to Springer Nature Singapore Pte 1

Ltd. 2023
A. M. Abdel Rahman (ed.), Clinical Metabolomics Applications in Genetic
Diseases, [Link]
2 A. M. Abdel Rahman

CI Chemical ionization
EHMN Edinburgh human metabolic network
ESI Electrospray ionization
FFPE Formalin-fixed paraffin-embedded
GSEA Gene set enrichment analysis
HIES Hyper-IgE syndromes
HILIC Hydrophilic interaction liquid chromatography
HRM High-resolution metabolomics
ICR Ion cyclotron resonance
LC-MS Liquid chromatography-mass spectrometry
LGPC Linoleoylglycerophosphocholine
LIT Linear quadrupole ion trap
NAFLD Nonalcoholic fatty liver disease
NMR Nuclear magnetic resonance
QIT Quadrupole ion trap
ROC Receiver operating characteristic
SMPDB Small Molecule Pathway Database
TOF Time of flight

1 Introduction

The history of metabolomics started from the essential biochemical genetics’ tech-
niques used routinely for patient diagnostics. The first metabolomics study was con-
ducted in 1984 by Jermey Nicholson using nuclear magnetic resonance (NMR) and
in 1995 by liquid chromatography-mass spectrometry (LC-MS) by Gary Siuzdak at
Scripps Research Institute [1, 2]. Metabolomics and lipidomics are the techniques
for studying the end product of the genetic makeup of living cells. Both techniques
cover small molecules with a size below 1500 Da. This class of molecules covers
endogenous and exogenous molecules, including drugs, food additives, microbiome
secretome, and environmental exposome, with more than 217,920 compounds
based on the human Metabolome Database (HMDB 5.0) [3].
In the last couple of decades, along with the advancement of MS and NMR,
metabolomics and lipidomics have drastically populated different fields, including
clinical research and drug development. Multiple disease models have been used to
study the unique metabolomics profiles such as primary cell lines, mouse, and
human biological materials (e.g., serum, plasma, tissue) [4, 5]. The use of metabo-
lomics in medicine extends chronic diseases such as chronic kidney disease (CKD)
[6], diabetes [7, 8], and rare syndromes with single gene deficiencies such as
DOCK8 deficiency causing hyper-IgE syndromes (HIES) [9, 10].
In clinical research, metabolomics has shown to be a great choice for studying
the disease mechanism, diagnostic and prognostic biomarkers, and therapeutic tar-
gets. More than 95% of the available clinical tests in the medical laboratory, 85% of
the known drugs, and 50% of the genetic diseases are based on small molecules,
The Advanced Technology and Clinical Application in Metabolomics 3

making metabolomics a great translational tool to improve the patient’s quality of

life. The technology of studying metabolomics has drastically improved in the last
couple of decades alongside the cheminformatics and bioinformatic tools. For
instance, mass spectrometry has been developed to increase the analyzers’ mass-­
resolving power and quantitative sensitivities by advancing the hardware, such as
using ion funnels in quadrupole time-of-flight (QTOF) analyzers [11]. However, the
pre-analytical, analytical, and post-analytical limitations associated with the instinct
of metabolites still need to be considered in any metabolomics research to repro-
duce the findings.
This chapter introduces metabolomics and lipidomics as major analytical tech-
nologies and their applications in clinical research and personalized medicine.

2 Mass Spectrometry

Mass spectrometry separates molecules based on their mass-to-charge ratio (m/z) in the
gas phase under low pressure. At a given energy, molecules with the lowest m/z pass the
analyzer the fastest. The MS performance can be evaluated based on resolution, preci-
sion, accuracy, and sensitivity. The MS resolution, the ability of the analyzer to separate
ions and calculate as m/∆m, is based mainly on the complexity of the mixture and the
length of the analysis path. The mass precision is based on the isotope abundance mea-
surement reproducibility, represented as the coefficient of variation (CV%) for multiple
measurements of the same sample. The MS accuracy is more challenging than precision
evaluation, where the analysis has to be throughout interlaboratory standards. The accu-
racy of MS is known as the proximity of the experimental measurement to the true and
exact mass (measurement error). The minimum sample size to obtain the optimal mass
accuracy and precision is known as the sensitivity of MS.
The separation techniques enhanced the analytical performance of the MS and
the accuracy of molecular annotation by introducing analytes in groups based on
their physicochemical properties and reducing the matrix effect. Liquid chromatog-
raphy (LC), gas chromatography (GC), and capillary electrophoresis (CE) are the
main separation techniques hyphenated to MS used in metabolomics and lipido-
mics. Each technique has performance capabilities based on target molecules and
the separation approach, such as in LC reversed-phase (RP) chromatography [12–
15]. LC and CE analyze polar nonvolatile molecules without pretreatment or
derivatization. RP LC is a dominating technique for metabolomics profiling.
However, for the highly polar compounds, ion-pairing reagents are used for the
stationary phase hydrophobicity and enhance their column retention for better anal-
ysis. In high-resolution and untargeted metabolomics analysis, ion-pairing reagents
are used in negative mode ionization. Once switched to positive, ion-pairing reagent
will drastically develop ion suppression and ion source contamination. Hydrophilic
interaction liquid chromatography (HILIC) was found as an alternative to ion-­
pairing chromatography, where the polar stationary phase combines organic and
aqueous mobile phases [16–18].
4 A. M. Abdel Rahman

Capillary electrophoresis has been used widely to analyze polar and charged
metabolites, where the molecular separation is based on electrophoretic mobility
(charge-to-size ratio). Although both CE and HILIC are suitable for polar and
charged molecules, HILIC is more sensitive due to the column capacity, while
CE’s peak is more efficient. The potential use of CE-MS in metabolomics has
been reviewed [13]. The limitations of using CE are attributed to the lack of stan-
dardization of the small specimen load (poor sensitivity) and migration time
variability.
The metabolomics throughput and depth of coverage have drastically increased
after adding another dimension of post-ionization gas-phase separation using ion
mobility (IM) to MS analyzers. The IM separation is based on molecular size, where
the confidence of metabolites annotation is increased by adding the collision cross-­
section (CCS) as a molecular descriptive [19, 20]. In addition, IM plays a crucial
role in lipidomics by enhancing the lipids’ complex separation, improving isomer
resolution, and increasing confidence in molecular identification and characteriza-
tion [21].
Combining multiple chromatographic and IM techniques with high-resolution
mass spectrometry and a data extraction algorithm is known as high-resolution
metabolomics (HRM) [22]. The physiochemical properties and abundance hetero-
geneity of the cellular metabolome make the analytical tools used to study their
expression quite challenging. Any research group aims to expand the metabolomics
coverage to reach the low abundant molecules by combining multiple chromato-
graphic approaches in different detection modes. The author’s laboratory experi-
ence uses an alternating strategy: two chromatographic systems (RP and HILIC)
and two ionization polarities (positive and negative) to cover the maximum number
of metabolites. Several classes of molecules can be overlapped but in different sen-
sitivities (Fig. 1).
Electrospray ionization (ESI) is the most common technique in both LC-MS and
CE-MS use in metabolomics research. Moreover, the ESI ionization is considered
soft to prevent uncontrolled in-source fragmentation, which is ideal for biomolecu-
lar analysis. The retention variations to using unbuffered mobile phase and ion sup-
pression are the main restraints in using ESI in omics applications, mainly
metabolomics (more details are covered in chapter “Metabolomics: A Pipeline for
Biomarker Discovery in Genetic Diseases”).
GC-MS is one of the most efficient and reproducible metabolomics platforms to
match the standard of commercial and “in-house” established libraries and data-
bases. As an analytical tool, GC-MS is considered robust, excellent separation capa-
ble, selective, sensitive, and reproducible to cover a large group of metabolites.
Electron ionization (EI) and chemical ionization (CI) are GC-MS’s main ionization
techniques, producing fragments and molecular ion spectra for the target molecule.
However, EI is the most informative ionization technique compatible with metabo-
lomics’ most available libraries and databases. Low molecular weight (50–600 Da)
and volatile compounds are the most likely compounds that can be analyzed using
GC-MS. The polar, thermolabile, and nonvolatile compounds can be analyzed using
GC-MS after using some derivatization reagents to collect an informative analytical
The Advanced Technology and Clinical Application in Metabolomics 5

Fig. 1 The
chromatographic option to
increase the detection
coverage of metabolites
using LC-ESI-HRMS

signal [12]. The volatome or volatilome, a comprehensive and untargeted study of

the expression of volatile compounds, was used interchangeably using GC-MS. For
instance, the nonvolatile compounds can be analyzed on GC-MS after derivatization
based on the functional groups (e.g., carboxylic, alcohol, amines, and thiol).
Alkylation, acylation, and silylation are the common derivatization groups, whereas
trimethylsilylation (TMS) is the most comprehensive to cover many functional
groups. N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) is the most popular
metabolomics and produces minimal “artifacts” compared to other silylation
reagents [12].
Linear quadrupole ion trap (LIT), three-dimensional quadrupole ion trap (QIT),
orbitrap, time of flight (TOF), and ion cyclotron resonance (ICR), all of these use
the static or dynamic magnetic/electric field that are the main mass analyzers used
in targeted and untargeted metabolomics and lipidomics studies. The proper selec-
tion of the mass analyzer depends on the resolution, mass range, scan rate, and
detection limit (his part has been covered extensively in chapter “Metabolomics: A
Pipeline for Biomarker Discovery in Genetic Diseases”). The choice of analytical
technique depends on the research objectives, experimental design, and the biologi-
cal sample queued for investigation. For unsupervised and discovery projects, it is
highly recommended to use two or more independent or hyphenated techniques in
multiple ionization polarities and separation modes to achieve a wide-ranging pro-
file of metabolites.
6 A. M. Abdel Rahman

3 Nuclear Magnetic Resonance

Nuclear magnetic resonance (NMR) is a spectroscopic analytical technique used to

identify and quantify organic molecules based on hydrogen, carbon, nitrogen, flour,
and phosphorus nuclei. The nuclei of the elements carry a charge that creates a
magnetic dipole when the proton and neutron spins are not paired. The resulting
magnetic dipole generates a spin axis along with the nucleus’ spin axis, where the
magnitude of this dipole is a fundamental nuclear property (nuclear magnetic
moment, μ). The charge distribution is a function of the internal structure of the
nuclei. The transitions between nuclear spins should be observable once a magnetic
field is applied perpendicular to a nuclear magnetic energy level. Hydrogen NMR
(1H-NMR) is the most commonly targeted nucleus in analyzing samples of biologi-
cal origin due to its natural abundance. NMR also targets other atoms, such as
carbon and phosphorus, for additional information on a specific class of metabolites.
Initially, NMR has played a crucial role in metabolomics for molecular character-
ization and structural elucidation, followed by expression profiling and dynamic
determination. The sensitivity and element-selective detection of the NMR to the
nuclear spin made it one of the major tools in metabolomics studies. Coupling NMR
with mass spectrometry enhances metabolomics profiling and identifications. NMR
has proven its role in medical diagnosis and clinical research that targets the protein
[23], lipid [24], and metabolites biomarkers in addition to the whole microbiological
species (reviewed in chapter “Bringing Human Serum Lipidomics to the Forefront
of Clinical Practice: Two Clinical Diagnosis Success Stories”) [24]. Despite the ana-
lytical sensitivity, NMR is considered one of the highly robust techniques to quan-
tify the naturally abundant metabolites in biological matrices for diagnostic purposes.
The NMR analytical workflow starts by extracting metabolites from the biologi-
cal matrices and dissolving the dry extracts with NMR technique-compatible sol-
vents. Post-acquisition, the NMR spectral data generated for each metabolite are
univariate and multivariate analyzed. Then, the significant features are annotated
using public libraries such as the Human Metabolome Database (HMDB) [3]. This
part can be further explored in chapter “Metabolomics in the Study of Human
Mitochondrial Diseases” by David Wishart.
In conclusion, NMR complements the MS by analyzing the isobaric, hard-to-be
ionized, or structurally unknown compounds [25]. The metabolic transformation’s
dynamic and mechanism can be depicted using stable isotope labels-NMR (reviewed
extensively by Markley et al. 2017) [26].

4 Metabolomics Data Analysis

The association of metabolic dysregulation with the clinical phenotype and

the feasibility of being a diagnostic biomarker are explored further in multiple
packages by developing a model. The validation approach considers
The Advanced Technology and Clinical Application in Metabolomics 7

predictive accuracy, sensitivity, and specificity. For instance, the receiver

operating characteristic (ROC)’s area under the curve (AUC) represents the
probability of the classifier ranking a randomly chosen positive sample higher
than a randomly chosen negative one. ROC curve avoids systematic bias and
is the most used performance assessment method, where the ROC’s perfect
classifier (AUC = 1) while a random classifier will obtain AUC close to 0.5.
An AUC > 0.7 is often considered the minimal performance for a biomarker
test to be clinically useful [27]. The classification models are followed with a
validation process to estimate the model’s performance to a new set of sam-
ples, particularly when a small set of samples are used in the discovery cohort.
Permutation analysis and cross-validation testing are the two main approaches
for validation [28].
The connection between the discovered metabolomics panel in a discovery
cohort with the disease etiology is usually achieved through the pathway and net-
work analyses. Several computational platforms such as the Kyoto Encyclopedia of
Genes and Genomes (KEGG) [29–31], Small Molecule Pathway Database
(SMPDB) [32], Edinburgh Human Metabolic Network (EHMN) [33], Wiki
Pathways [34], and MetaCyc [35] provide extensive information of a large number
of metabolic pathways. The metabolic pathways-based methods are known as
metabolite set enrichment analysis (MSEA). They are based on the gene set enrich-
ment analysis (GSEA) approach, which was designed for pathway analysis of gene-­
expression data [36, 37].

5 Advancement of Metabolomics Application

in Clinical Research

Decades of advancement in clinical chemistry are mainly based on biochemical

analyses, where separation science and molecular spectroscopy techniques play the
primary role. Metabolomics is considered an advanced version of the biochemical
genetics and clinical biochemistry platforms, where newborn screening, and urinary
organic acid profile using the GC-MS platform, is the first clinical application of
metabolomics. The advancement of the MS techniques and the data analysis pipe-
lines enhanced the discovery rate of metabolic biomarkers and translational poten-
tial for clinical usage. Several chapters in this book will cover some clinical
applications of metabolomics, such as endocrinology (reviewed in detail in chapters
“Metabolomics and Genetics of Rare Endocrine Disease: Adrenal, Parathyroid
Glands, and Cystic Fibrosis” and “Metabolomics Role in Personalized Medicine:
An Update”), mitochondrial diseases (reviewed in detail in chapter “Lipidomic
Profiling in Clinical Practice Using LC-MS”), the inborn error of metabolism
(reviewed in detail in chapters “Bioinformatics Tools for Clinical Metabolomics”
and “Untargeted Metabolomics in Newborn Screening”), etc. Some applications are
highlighted in this section.
8 A. M. Abdel Rahman

Clinical metabolomics research is based mainly on study design, replication,

clinical data collection, specimen collection, storage, metabolism quenching, meta-
bolic extraction, and instrumental acquisition (reviewed in detail in chapters
“Metabolomics: A Pipeline for Biomarker Discovery in Genetic Diseases” and
“Metabolomics of Rare Endocrine, Genetic Disease: A Focus on the Pituitary
Gland”). These factors have shown in multiple studies a drastic role in generating a
reproducible and sensitive metabolic biomarker for disease diagnosis and treatment
management. In 2019, a quality assurance (QA) and quality control (QC) consor-
tium (mQACC) was established to engage the community in addressing the quality
challenges in untargeted metabolomics [38].
In this decade, metabolomics has been extensively applied in oncology, where
many studies have focused on potential cancer biomarkers discovery for diagnostic,
prognostic, therapy, and prevention.
Depending upon the genetic alterations, patient-tailored therapy by cancer’s
genomic and epigenomic characteristics has become possible. Many studies that
have been conducted on cancer metabolome, estrogen receptor, and Her2/neu status
in breast cancer are some known examples in oncology [39–42].
Metabolomics has also unfolded different branches of medicine, including peri-
natology, prenatal care, and maternal-fetal medicine. Prenatal medicine pregnancy
is a critical state with various aspects to consider, including the mother’s and child’s
well-being. Perinatal pathologies include chromosomal aberration, preterm delivery
(PTD), congenital heart defects, spina bifida, chorioamnionitis, and low birth
weight. Many studies have been conducted for biomarker discovery in aneuploidy
screening, preeclampsia, fetal growth restriction, preterm labor, and delivery
[43–45].

5.1 Oncology

The Warburg effect was the first to report the metabolic arm of cancer by converting
glucose to lactate at a higher rate than normal cells [46]. This effect moves the main
source of cellular energy from the mitochondria to the cytoplasm, which shifts the
cell from an inert entity producing ATP to rapidly dividing cells. The metabolic
nature of cancer progression produces many amino acids, lipids, and nucleotides, to
produce the cellular biomass for the highly proliferating cells. Metabolomics has a
great role in cancer personalized medicine which has been extensively reviewed
[47]. As the frozen tissue is quite limited, an alternative formalin-fixed paraffin-­
embedded (FFPE) option is still viable for cancer metabolic biomarkers, mainly for
tumor classification [48]. This protocol combines sensitive targeted metabolomics
that covers cancer’s most important pathways, such as glycolysis, TCA, and pentose
phosphate pathways. For instance, more than 30 endogenous metabolites in breast
cancer have been reported, including cholate levels (resulting from increased phos-
phocholine), low glycerophosphocholine, and low glucose, as potential biomarkers.
These biomarkers improve the sensitivity and sensibility up to 83–100% in
The Advanced Technology and Clinical Application in Metabolomics 9

detecting malignancy more than tumor size, lymph node hormonal status, and his-
tology [49].
Elevated levels of glutamine, glycine, cysteine, and threonine were reported in
ovarian cancer studies, and tryptophan, histidine, and phenylalanine were degraded
significantly (reviewed by Turkoglu et al.) [50]. The profile based on the next-­
generation metabolomics in lung cancer was reviewed extensively as biomarkers for
diagnosis, pathogenesis, classifications, and precision medicine. Most LC metabo-
lomics markers are involved in the upregulation and reprogramming of the TCA and
glycolysis pathways and the upregulation of phospholipid metabolic pathways and
fatty syntheses (reviewed by Turkoglu et al.) [51]. Serum lactic acid, progesterone,
homocysteine, 3-hydroxybutyrate, linoleic acid, stearic acid, myristic acid, threo-
nine, and valine levels were reported to be significantly dysregulated in endometrial
cancer (EC) [52, 53]. A group of metabolites has shown great concordance with
colorectal cancer (CC) recurrence, prognosis, and survival [54]. Most of these
metabolites play roles in perturbing cellular respiration and carbohydrate metabo-
lism (i.e., TCA cycle and anaerobic respiration), lipid metabolism (i.e., fatty acid
oxidation), amino acid metabolism (i.e., histidine, methionine, and tryptophan), and
nucleotide metabolites (i.e., uracil, p-cresol, etc.) [54]. Cancer as a metabolic dis-
ease has been reviewed recently by Wishart [55].
The cancer metabolic biomarkers (oncometabolomics) open the venue for utiliz-
ing mass spectrometry in translational medicine. Removing tumor tissue accurately
with minimally invasive, especially in brain tumors, is now closely using iKnife
technology. These surgical cutters connected to desorption electrospray ionization
(DESI) mass spectrometry were developed by Graham Cooks groups at Purdue
University and Nathalie Agar at Harvard Medical School. This ambient ionization
technique mediated a medical cutter and mass spectrometry collecting analytical
signals for oncometabolite to accurately diagnose tissue removed during brain sur-
gery [56]. This approach reduces postsurgical complications, speeds up surgery, and
minimizes the subjectivity of conventional pathology. Zoltán Takáts at Imperial
College London capitalized on this technology and started to build an oncometabolite-­
based library from multiple laboratories using rapid evaporative ionization mass
spectrometry (REIMS). In BC, 63 phospholipids and 6 triglyceride species were
responsible for 24 spectral differences between normal and tumor tissues, increas-
ing the diagnostic sensitivity and specificity to 93.4% and 94.9%, respectively,
using REIMS [57].

5.2 Prenatal Medicine

Pregnancy is a very natural process and complicated from a research standpoint to

consider the mother and the baby’s well-being. A wide range of physiological changes
is required throughout the gestation in maternal, which requires circulating some
metabolites such as triglyceride, cholesterol, and lipids to satisfy in utero fetal devel-
opment from energy and catabolic needs. Multiple complications are known to be
10 A. M. Abdel Rahman

associated with pregnancy, e.g., bleeding, ectopic pregnancy, miscarriage or fetal loss,
placental complication, and preeclampsia or eclampsia. These complications are
associated with overlapped risk factors such as the mother’s age, body mass index,
and medical history. Preeclampsia is the leading pregnancy complication that affects
about 5–8% of pregnant women worldwide. Gestational high blood pressure is the
main preeclampsia characterization, and it is combined with protein in the urine,
occasionally fluid retention, and in some severe cases, seizures, coma, and death.
Oxidative stress is the common metabolic pathway in most prenatal disorders,
except preeclampsia and congenital anatomic defects. Acylcarnitine and amino acid
metabolism are altered in both preeclampsia and CAD, while taurine metabolic
dysregulation is unique in patients with preeclampsia. Preterm labor and deliveries
alter bile acid and inflammation metabolism. Some energy metabolism (e.g., pen-
tose phosphate pathways, ketone body production) is dysregulated in other preg-
nancy complications, including single gene disorders.
Throughout the pregnancy, noninvasive (e.g., history analysis, ultrasound, mater-
nal serum analysis) and invasive (e.g., fetal samples of the placenta) assessments
might be needed. Developing noninvasive tests with lower false-positive rates is
fundamental in prenatal medicine, where metabolomics is potentially useful.
Metabolomics has great potential for evaluating several biomarkers in a single,
rapid, relatively low-cost, controlled experiment. This technology identifies the tar-
geted pathway altered through multiple intermediates unsupervised, which can iden-
tify the fetal or pregnancy disorders using simultaneously different compartments
(e.g., maternal, placental, and fetal). However, overinterpretation and high false dis-
covery rates, with unestablished cutoff values for the target analytes, are the biggest
limitation of metabolomics in prenatal medicine. Multiple metabolomics studies
have been conducted on prenatal cases covering normal pregnancy in different tri-
mesters [43–45]. Aneuploidy screening, preeclampsia [58], fetal, preterm labor and
delivery [45], congenital anatomic defects (CAD), and single gene disorders [43].
The cause of preeclampsia is not known, and mothers with a history should be
under continuous monitoring for any potential risk. Several metabolic biomarkers
were reported in the literature to predict the early onset of preeclampsia from con-
trol, such as taurine and asparagine [58]. Metabolomics Diagnostics (Metabolomic
Diagnostics), an Ireland-based, deep-tech medical diagnostics company, specializes
in developing novel biomarker-based diagnostic solutions. This company estab-
lished a preeclampsia screening program after screening more than 1000 pregnant
women to validate a panel of biomarkers for preeclampsia prediction. Their panel of
metabolites (PrePsia) is now executed as a prototype screening test to identify
women at increased risk of developing preeclampsia in early pregnancy.

6 Frontiers in Metabolomics

Metabolomics platforms have been drastically advanced in the past decade, with
great translational potential in medicine. The biological fluids and materials are
used between the study groups in the standard clinical metabolomics pipeline.
The Advanced Technology and Clinical Application in Metabolomics 11

Single-cell metabolomics has shown promising results that explain the cellular het-
erogeneity based on the phenotypical differences to help understand the cause of
cellular biochemical activity with implications for health and disease [59]. Single-­
cell metabolomics experiments with low sample volume are based on a shotgun-like
approach. This motivates the developments of the metabolomics pipelines such as
the nano-DESI, ambient ionization technique, droplet-based microextraction, single
probe with duel bore capillary, etc. Single-cell metabolomics has great potential for
studying the cellular biochemical changes in a specific genetic disease that is hard
to notice in biological fluids, such as mitochondrial disorders (reviewed in detail in
chapter “Lipidomic Profiling in Clinical Practice Using LC-MS”).
Molecular imprinting polymer (MIP)-based electrochemical sensor is greatly
applied in targeted metabolomics toward developing point-of-care tests (POCT)
[60]. Having this line of research and technique advancement encourages the appli-
cation scientist to continue in biomarker discovery at multiple levels, including
monitoring health statuses such as metabolic disorders and those with critical meta-
bolic risk. This part of the research is covered extensively in chapter “Transferring
Metabolomics to Portable Diagnostic Devices: Trending in Biosensors”.
Together, the advancement of the biosensor of targeted metabolomics and the
metabolomics biomarker discovery opens the venue toward real-time health moni-
toring, mainly for metabotyping individuals for well-being, nutritional, and person-
alized medicine. Urine metabolomics in multiple studies has shown a real-time
behavior regardless of the analytical platform, from sample collection to analysis
limitations [61]. By enabling patients to access continuous measurement and wear-
able devices to diagnose and control their illnesses precisely, metabolomics will
have a great opportunity in this field by multiplexing the monitoring to avoid the
confounding effects in each case.
Metabolomics is gaining significant interest in medicine and clinical diagnosis,
where multiple novel assays have been licensed to provide metabolomics-based diag-
nostic services. Metabolon and Baylor College of Medicine have worked in the last
decades in developing Global MAPSTM, a semiquantitative metabolomics profiling,
to determine the disruption related to specific biochemical abnormalities. More
details regarding their experience in detecting some metabolic diseases are detailed in
chapters “Bioinformatics Tools for Clinical Metabolomics” and “Untargeted
Metabolomics in Newborn Screening”. Quantose®IR and Quantose®IGT are metab-
olomics-based clinical assays for insulin resistance (IR) and impaired glucose toler-
ance (IGT) identification in patients with multiple conditions, such as type 2 diabetes.
In addition to insulin, Quantose®IR is based on a panel of biomarkers comprised of a
small organic acid (α-hydroxybutyric acid (AHB)) and two lipids (oleic acid and
linoleoylglycerophosphocholine (LGPC)) [62]. The algorithm scoring was devel-
oped based on a nondiabetic cohort from 13 European countries, where the score
cutoff is 63 between insulin sensitivity and resistance. This test is performed using
LC-MSMS for treatment monitoring, such as pioglitazone [62]. Quantose®IGT iden-
tifies the prediabetes risk based on the IGT. The Quantose®IGT scores were devel-
oped based on α-hydroxybutyric acid (AHB), 4-methyl-2-­oxopentanoic acid (4MOP),
oleic acid, linoleoylglycerophosphocholine (LGPC), β-hydroxybutyric acid (BHBA),
serine, and pantothenic acid (vitamin B5) levels in the sample [63].
12 A. M. Abdel Rahman

Nonalcoholic fatty liver disease (NAFLD) is a highly prevalent progressive

chronic disease in which the liver displays histological features like those induced
by excessive alcohol intake but in the absence of alcohol consumption. NAFLD is
very common in people with diabetes and the obese. Its early identification is impor-
tant due to the burden of the disease and the fact that NAFLD often presents with
only mild or no symptoms. OWLiver® Care and OWLiver® are noninvasive assays
for fatty liver screening and NASH diagnosis. These two tests use highly sensitive
laboratory processes to determine the form of disease present in the patient’s liver.
These tests determine the risk of developing NASH, reflected in the patient’s life-
style and medical management. This part is covered extensively in chapter “Bringing
Human Serum Lipidomics to the Forefront of Clinical Practice: Two Clinical
Diagnosis Success Stories”.

References

1. Holmes E, Antti H. Chemometric contributions to the evolution of metabonomics: mathemati-

cal solutions to characterising and interpreting complex biological NMR spectra. Analyst.
2002;127(12):1549–57.
2. Cravatt BF, Prospero-Garcia O, Siuzdak G, Gilula NB, Henriksen SJ, Boger DL, et al.
Chemical characterization of a family of brain lipids that induce sleep. Science (New York,
NY). 1995;268(5216):1506–9.
3. Wishart DS, Guo A, Oler E, Wang F, Anjum A, Peters H, et al. HMDB 5.0: the human metabo-
lome database for 2022. Nucleic Acids Res. 2022;50(D1):D622–31.
4. Kennedy AD, Wittmann BM, Evans AM, Miller LAD, Toal DR, Lonergan S, et al.
Metabolomics in the clinic: a review of the shared and unique features of untargeted metabo-
lomics for clinical research and clinical testing. J Mass Spectrom. 2018;53(11):1143–54.
5. Abdel Rahman AM, Pawling J, Ryczko M, Caudy AA, Dennis JW. Targeted metabolomics
in cultured cells and tissues by mass spectrometry: method development and validation. Anal
Chim Acta. 2014;845:53–61.
6. Jacob M, Nimer RM, Alabdaljabar MS, Sabi EM, Al-Ansari MM, Housien M, et al.
Metabolomics profiling of nephrotic syndrome towards biomarker discovery. Int J Mol Sci.
2022;23(20):12614.
7. Aleidi SM, Dahabiyeh LA, Gu X, Al Dubayee M, Alshahrani A, Benabdelkamel H, et al.
Obesity connected metabolic changes in type 2 diabetic patients treated with metformin. Front
Pharmacol. 2020;11:616157.
8. Hocher B, Adamski J. Metabolomics for clinical use and research in chronic kidney disease.
Nat Rev Nephrol. 2017;13(5):269–84.
9. Jacob M, Gu X, Luo X, Al-Mousa H, Arnaout R, Al-Saud B, et al. Metabolomics distinguishes
DOCK8 deficiency from atopic dermatitis: towards a biomarker discovery. Metabolites.
2019;9(11):274.
10. Jans JJM, Broeks MH, Verhoeven-Duif NM. Metabolomics in diagnostics of inborn metabolic
disorders. Curr Opin Syst Biol. 2022;29:100409.
11. Heinemann J. Machine learning in untargeted metabolomics experiments. Methods Mol Biol.
2019;1859:287–99.
12. Beale DJ, Pinu FR, Kouremenos KA, Poojary MM, Narayana VK, Boughton BA, et al. Review
of recent developments in GC-MS approaches to metabolomics-based research. Metabolomics.
2018;14(11):152.
The Advanced Technology and Clinical Application in Metabolomics 13

13. Ramautar R. Capillary electrophoresis-mass spectrometry for clinical metabolomics. Adv Clin
Chem. 2016;74:1–34.
14. Zhou J, Yin Y. Strategies for large-scale targeted metabolomics quantification by liquid
chromatography-­mass spectrometry. Analyst. 2016;141(23):6362–73.
15. Gordillo R. Supercritical fluid chromatography hyphenated to mass spectrometry for metabo-
lomics applications. J Sep Sci. 2021;44(1):448–63.
16. Spagou K, Tsoukali H, Raikos N, Gika H, Wilson ID, Theodoridis G. Hydrophilic interac-
tion chromatography coupled to MS for metabonomic/metabolomic studies. J Sep Sci.
2010;33(6–7):716–27.
17. Virgiliou C, Gika HG, Theodoridis GA. HILIC-MS/MS multi-targeted method for metabolo-
mics applications. Methods Mol Biol. 2018;1738:65–81.
18. Tang DQ, Zou L, Yin XX, Ong CN. HILIC-MS for metabolomics: an attractive and comple-
mentary approach to RPLC-MS. Mass Spectrom Rev. 2016;35(5):574–600.
19. Zhang X, Quinn K, Cruickshank-Quinn C, Reisdorph R, Reisdorph N. The application of ion
mobility mass spectrometry to metabolomics. Curr Opin Chem Biol. 2018;42:60–6.
20. Mairinger T, Causon TJ, Hann S. The potential of ion mobility-mass spectrometry for non-­
targeted metabolomics. Curr Opin Chem Biol. 2018;42:9–15.
21. Zandkarimi F, Brown LM. Application of ion mobility mass spectrometry in lipidomics. Adv
Exp Med Biol. 2019;1140:317–26.
22. Uppal K. Models of metabolomic networks. In: Wolkenhauer O, editor. Systems medicine.
Oxford: Academic Press; 2021. p. 134–42.
23. Hu Y, Cheng K, He L, Zhang X, Jiang B, Jiang L, et al. NMR-based methods for protein analy-
sis. Anal Chem. 2021;93(4):1866–79.
24. Marchand J, Martineau E, Guitton Y, Le Bizec B, Dervilly-Pinel G, Giraudeau P. A mul-
tidimensional (1)H NMR lipidomics workflow to address chemical food safety issues.
Metabolomics. 2018;14(5):60.
25. Wishart DS. NMR metabolomics: a look ahead. J Magn Reson. 1997;2019(306):155–61.
26. Markley JL, Brüschweiler R, Edison AS, Eghbalnia HR, Powers R, Raftery D, et al. The future
of NMR-based metabolomics. Curr Opin Biotechnol. 2017;43:34–40.
27. Xia J, Broadhurst DI, Wilson M, Wishart DS. Translational biomarker discovery in clinical
metabolomics: an introductory tutorial. Metabolomics. 2013;9(2):280–99.
28. Wikoff WR, Anfora AT, Liu J, Schultz PG, Lesley SA, Peters EC, et al. Metabolomics analysis
reveals large effects of gut microflora on mammalian blood metabolites. Proc Natl Acad Sci U
S A. 2009;106(10):3698–703.
29. Kanehisa M, Goto S. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res.
2000;28(1):27–30.
30. Kanehisa M, Furumichi M, Tanabe M, Sato Y, Morishima K. KEGG: new perspectives on
genomes, pathways, diseases and drugs. Nucleic Acids Res. 2017;45(D1):D353–61.
31. Kanehisa M, Furumichi M, Sato Y, Ishiguro-Watanabe M, Tanabe M. KEGG: integrating
viruses and cellular organisms. Nucleic Acids Res. 2021;49(D1):D545–51.
32. Jewison T, Su Y, Disfany FM, Liang Y, Knox C, Maciejewski A, et al. SMPDB 2.0: big
improvements to the small molecule pathway database. Nucleic Acids Res. 2014;42(Database
issue):D478–84.
33. Hao T, Ma HW, Zhao XM, Goryanin I. Compartmentalization of the Edinburgh Human
Metabolic Network. BMC Bioinform. 2010;11:393.
34. Kelder T, van Iersel MP, Hanspers K, Kutmon M, Conklin BR, Evelo CT, et al. WikiPathways:
building research communities on biological pathways. Nucleic Acids Res. 2012;40(Database
issue):D1301–7.
35. Caspi R, Foerster H, Fulcher CA, Kaipa P, Krummenacker M, Latendresse M, et al. The
MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of Pathway/
Genome Databases. Nucleic Acids Res. 2008;36(Database issue):D623–31.
36. Tilford CA, Siemers NO. Gene set enrichment analysis. Methods Mol Biol. 2009;563:99–121.
14 A. M. Abdel Rahman

37. Deng L, Ma L, Cheng KK, Xu X, Raftery D, Dong J. Sparse PLS-based method for overlap-
ping metabolite set enrichment analysis. J Proteome Res. 2021;20(6):3204–13.
38. Beger RD, Dunn WB, Bandukwala A, Bethan B, Broadhurst D, Clish CB, et al. Towards
quality assurance and quality control in untargeted metabolomics studies. Metabolomics.
2019;15(1):4.
39. Zhao X, Modur V, Carayannopoulos LN, Laterza OF. Biomarkers in pharmaceutical research.
Clin Chem. 2015;61(11):1343–53.
40. Ni Y, Xie G, Jia W. Metabonomics of human colorectal cancer: new approaches for early diag-
nosis and biomarker discovery. J Proteome Res. 2014;13(9):3857–70.
41. Liesenfeld DB, Habermann N, Owen RW, Scalbert A, Ulrich CM. Review of mass
spectrometry-­based metabolomics in cancer research. Cancer Epidemiol Biomarkers Prev.
2013;22(12):2182–201.
42. Armitage EG, Barbas C. Metabolomics in cancer biomarker discovery: current trends and
future perspectives. J Pharm Biomed Anal. 2014;87:1–11.
43. Monni G, Atzori L, Corda V, Dessolis F, Iuculano A, Hurt KJ, et al. Metabolomics in prenatal
medicine: a review. Front Med. 2021;8:645118.
44. McKeating DR, Fisher JJ, Perkins AV. Elemental metabolomics and pregnancy outcomes.
Nutrients. 2019;11(1):73.
45. Carter RA, Pan K, Harville EW, McRitchie S, Sumner S. Metabolomics to reveal biomark-
ers and pathways of preterm birth: a systematic review and epidemiologic perspective.
Metabolomics. 2019;15(9):124.
46. Warburg O. On respiratory impairment in cancer cells. Science. 1956;124(3215):269–70.
47. Cacciatore S, Loda M. Innovation in metabolomics to improve personalized healthcare. Ann N
Y Acad Sci. 2015;1346(1):57–62.
48. Kelly AD, Breitkopf SB, Yuan M, Goldsmith J, Spentzos D, Asara JM. Metabolomic profiling
from formalin-fixed, paraffin-embedded tumor tissue using targeted LC/MS/MS: application
in sarcoma. PLoS One. 2011;6(10):e25357.
49. Bathen TF, Geurts B, Sitter B, Fjøsne HE, Lundgren S, Buydens LM, et al. Feasibility of MR
metabolomics for immediate analysis of resection margins during breast cancer surgery. PLoS
One. 2013;8(4):e61578.
50. Turkoglu O, Zeb A, Graham S, Szyperski T, Szender JB, Odunsi K, et al. Metabolomics
of biomarker discovery in ovarian cancer: a systematic review of the current literature.
Metabolomics. 2016;12(4):60.
51. Yu L, Li K, Zhang X. Next-generation metabolomics in lung cancer diagnosis, treatment and
precision medicine: mini review. Oncotarget. 2017;8(70):115774–86.
52. Troisi J, Sarno L, Landolfi A, Scala G, Martinelli P, Venturella R, et al. Metabolomic signature
of endometrial cancer. J Proteome Res. 2018;17(2):804–12.
53. Njoku K, Sutton CJ, Whetton AD, Crosbie EJ. Metabolomic biomarkers for detection, progno-
sis and identifying recurrence in endometrial cancer. Metabolites. 2020;10(8):314.
54. Zhang F, Zhang Y, Zhao W, Deng K, Wang Z, Yang C, et al. Metabolomics for biomarker dis-
covery in the diagnosis, prognosis, survival and recurrence of colorectal cancer: a systematic
review. Oncotarget. 2017;8(21):35460–72.
55. Wishart D. Metabolomics and the multi-omics view of cancer. Metabolites. 2022;12(2):154.
56. Eberlin LS, Norton I, Orringer D, Dunn IF, Liu X, Ide JL, et al. Ambient mass spectrometry
for the intraoperative molecular diagnosis of human brain tumors. Proc Natl Acad Sci U S
A. 2013;110(5):1611–6.
57. St John ER, Balog J, McKenzie JS, Rossi M, Covington A, Muirhead L, et al. Rapid evaporative
ionisation mass spectrometry of electrosurgical vapours for the identification of breast pathol-
ogy: towards an intelligent knife for breast cancer surgery. Breast Cancer Res. 2017;19(1):59.
58. Kuc S, Koster MP, Pennings JL, Hankemeier T, Berger R, Harms AC, et al. Metabolomics pro-
filing for identification of novel potential markers in early prediction of preeclampsia. PLoS
One. 2014;9(5):e98540.
The Advanced Technology and Clinical Application in Metabolomics 15

59. Duncan KD, Fyrestam J, Lanekoff I. Advances in mass spectrometry based single-cell metabo-
lomics. Analyst. 2019;144(3):782–93.
60. Ozcelikay G, Kaya SI, Ozkan E, Cetinkaya A, Nemutlu E, Kır S, et al. Sensor-based MIP
technologies for targeted metabolomics analysis. TrAC Trends Anal Chem. 2022;146:116487.
61. Miller IJ, Peters SR, Overmyer KA, Paulson BR, Westphall MS, Coon JJ. Real-time health
monitoring through urine metabolomics. NPJ Digit Med. 2019;2:109.
62. Cobb J, Gall W, Adam KP, Nakhle P, Button E, Hathorn J, et al. A novel fasting blood test for
insulin resistance and prediabetes. J Diabetes Sci Technol. 2013;7(1):100–10.
63. Gall WE, Beebe K, Lawton KA, Adam KP, Mitchell MW, Nakhle PJ, et al. alpha-­
hydroxybutyrate is an early biomarker of insulin resistance and glucose intolerance in a non-
diabetic population. PLoS One. 2010;5(5):e10883.
Mass Spectrometry-Based Metabolomics
for the Clinical Laboratory

Joshua A. Dubland

Abstract In the clinical laboratory, analysis of small molecules using mass spectrom-
etry (MS) primarily encompasses the targeted and quantitative determination of
known biomarkers for disease diagnosis and monitoring, general health status evalu-
ation, toxicology, and therapeutic drug monitoring. Although there are exceptions,
MS-based assays in the clinical laboratory typically involve the utilization of analyte-
specific calibration curves for quantitation, and stable isotope-labeled internal stan-
dards to correct for any sample preparation and instrument-related variability. A
clinical MS-based assay usually consists of a relatively small panel of biomarkers in a
certain diagnostic context that are compatible with the same sample preparation pro-
tocol. Alternatively, the term metabolomics generally refers to the comprehensive and
systematic large-scale profiling of small molecules within a biological system.
Targeted and quantitative MS-based assays containing relatively large panels of small
molecules (metabolites) are routinely utilized for newborn screening (NBS), bio-
chemical genetics testing, and toxicology. Broad nontargeted metabolomics investiga-
tions have found some utility in the aforementioned testing areas, but are not currently
commonplace in the clinical laboratory. This chapter discusses current state-of-the-art
MS instrumentation, describes several applications, and provides implementation
considerations for MS-based metabolomics in the clinical laboratory.

Keywords Mass spectrometry · Metabolomics · Clinical laboratory ·

Instrumentation · Applications · Validation

J. A. Dubland (*)
Department of Pathology and Laboratory Medicine, BC Children’s Hospital Research
Institute, University of British Columbia, Vancouver, BC, Canada
e-mail: [Link]@[Link]

© The Author(s), under exclusive license to Springer Nature Singapore Pte 17

Ltd. 2023
A. M. Abdel Rahman (ed.), Clinical Metabolomics Applications in Genetic
Diseases, [Link]
18 J. A. Dubland

Abbreviations

AC Alternating current
APCI Atmospheric chemical ionization
APPI Atmospheric pressure photoionization
CCS Collisional cross section
CI Chemical ionization
CID Collision-induced dissociation
Da Dalton
DC Direct current
EI Electron ionization
ESI Electrospray ionization
eV Electron volt
FTICR Fourier-transform ion cyclotron resonance
FWHM Full width at half maximum
GC Gas chromatography
HILIC Hydrophilic interaction liquid chromatography
HPLC High-performance liquid chromatography
HRMS High-resolution mass spectrometry
ICP Inductively coupled plasma
IEMs Inborn errors of metabolism
IMS Ion mobility spectrometry
kV Kilovolt
LC Liquid chromatography
m/z Mass-to-charge ratio
MALDI Matrix-assisted laser desorption/ionization
MRM Multiple-reaction monitoring
MS Mass spectrometry
MS/MS Tandem mass spectrometry
QTOF Quadrupole time-of-flight
RF Radiofrequency
TOF Time-of-flight
UPLC Ultra-high-performance liquid chromatography

1 Introduction

Mass spectrometry (MS) is a powerful tool for investigating biological processes

and has been extensively utilized for metabolomics, which is the comprehensive
and systematic large-scale analysis of small molecules within biological systems.
MS-based metabolomics can provide insights into the biochemical status and flux in
both the healthy and diseased states. Metabolomics investigations can be either tar-
geted or nontargeted (or a mixture of these approaches).
Targeted metabolomics has been used in both research and clinical diagnostic
laboratories, and focuses on identifying and accurately quantifying small metabolite
Mass Spectrometry-Based Metabolomics for the Clinical Laboratory 19

panels (typically <10 analytes). For targeted metabolomics, analytes have been pre-
selected and the analytical approach has gone through some form of validation to
ensure the accuracy of the results. Targeted and quantitative analysis of relatively
large small molecule panels (>10 analytes) using MS has found applications in new-
born screening (NBS), biochemical genetics testing, and toxicology.
Alternatively, nontargeted (global) metabolomics aims to detect as many specific
metabolites or metabolomic features as possible within a given sample. The term
“metabolomic feature” refers to a certain mass-to-charge ratio (m/z) at a unique chro-
matographic retention time. Nontargeted metabolomics can involve the following
or combinations of the following: (1) m/z pattern recognition and relative signal com-
parisons between samples, (2) quantitative or semiquantitative analyses using calibra-
tion curves and/or surrogate calibrants, and (3) spectral comparisons with MS
libraries to try and identify some or all of the metabolomic features. Nontargeted
metabolomics analyses can lead to the discovery of new metabolites and potential bio-
markers. Confirmatory analyses are still needed, including chromatographic retention
time and m/z comparisons with synthesized or commercially available compounds,
and/or nuclear magnetic resonance (NMR) spectroscopy to elucidate the chemical
structure of isolated metabolite chromatographic fractions. Large-­scale nontargeted
(global) MS-based metabolomics investigations have been utilized primarily for
research purposes and have not been widely utilized in the clinical diagnostic laboratory.
This chapter starts with a description of the basic concepts of a mass spectrom-
eter and instrumental interfacing with several chromatographic techniques. Next,
some current examples of MS-based metabolomics in the clinical diagnostic labora-
tory are described. A brief discussion on guidance documentation and validation
considerations for small molecule MS assays in the regulated clinical setting is
then provided.

2 Components of a Mass Spectrometer

On a simplistic level, a mass spectrometer consists of three components: an ion

source, a mass analyzer, and a detector. The ion source first generates charged mol-
ecules (ions) in the gas phase from the sample being analyzed. The mass analyzer
component separates and isolates the ions based on their m/z. The abundance of
isolated ions at specific m/z's are then recorded by a detector. A mass spectrometer
requires low pressure (high vacuum) to direct ions through the instrument and
remove contaminants [1]. Specialized software is used to control the mass spec-
trometer and analyze data.

2.1 Sample Ionization

A variety of ionization techniques are available for introducing charged molecules

into a mass spectrometer. Currently, the most utilized methods of ionization include
electron ionization (EI), chemical ionization (CI), electrospray ionization (ESI),
20 J. A. Dubland

atmospheric chemical ionization (APCI), and matrix-assisted laser desorption/ion-

ization (MALDI). A mixture of ionization approaches is needed to improve metabo-
lite coverage for global metabolomics analysis of biological specimens (extensively
reviewed in chapter “The Advanced Technology and Clinical Application in
Metabolomics”) [2].
EI is frequently utilized for ionization and fragmentation of thermally stable
volatile small molecules. It is a process where a beam of electrons (typically having
a kinetic energy of 70 eV) is emitted from a heated filament and collides with gas-­
phase neutral molecules to generate charged ions and fragments. This process must
occur in a vacuum in order to avoid oxidation and beam scattering. The electron
beam abstracts one electron from a neutral molecule, generating a radical cation,
which then breaks into smaller, either charged or neutral, fragments. Neutral frag-
ments are eliminated or removed via a vacuum on the EI chamber walls. Positively
charged ions are drawn out of the source using an electric field, and the beam of ions
is focused [3]. The generated EI fragment spectra (ions are typically separated and
isolated by a quadrupole mass analyzer prior to detection) are useful for quantita-
tion, structural characterization, and library matching. EI is a very common ioniza-
tion and fragmentation method used for coupling gas chromatography (GC) with a
mass spectrometer (GC-MS).
CI is a soft ionization technique (lower energy than EI) where a proton is added
or removed from the neutral molecule using a reagent gas such as methane or
ammonia. An electron beam produces a number of ionized and reactive reagent gas
species, which transfer a proton to the neutral molecule of interest and generate a
product ion. Generally, there is minimal fragmentation, and a protonated molecular
ion is abundantly present in the mass spectra. Typically, CI is operated in positive
ion mode (addition of a proton), as most neutral molecules can form positive ions
through reactive intermediates. CI can also be conducted in negative ion mode if the
molecules being ionized can stabilize a negative charge from electron capture, such
as carboxylic acid groups or highly electronegative halogens [4, 5]. A CI source can
be used in GC-MS, but is less routinely utilized than EI.
ESI is also a soft ionization technique and operates at atmospheric pressure. It is
the standard ionization source utilized when liquid chromatography (LC) is coupled
to a mass spectrometer (LC-MS). The ESI source exists in many variations, but con-
sists of the same general components. The liquid sample first passes through a capil-
lary inside a sample probe. An electrical voltage of less than 5 kV is applied to the
capillary, leading to the generation of charged droplets as the liquid exits the capillary
[1]. A heated nebulization gas (usually nitrogen) moving parallel to the sample capil-
lary is used to help facilitate liquid droplet formation, directionality, and evaporation.
Charged liquid droplets generate smaller liquid droplets as they move through the
atmospheric region, which then completely evaporate (desolvate), resulting in the for-
mation of gas-phase ions. Singly or multiply charged ions can be generated. After
moving through the atmospheric region, the ions enter the mass spectrometer via a
small orifice in a sampling cone that has a voltage applied to it. The capillary that
gener­ates charged droplets can be directly pointed toward the sampling cone orifice or
positioned orthogonally to it. One or more cones (skimmers) in a series are sometimes
Mass Spectrometry-Based Metabolomics for the Clinical Laboratory 21

utilized. A neutral gas running perpendicular to the direction of ions moving through
the cone orifice is used to prevent contamination of the MS. ESI works well with polar
compounds that can hold a charge or exist in a charged state [4, 6].
In the APCI source, no voltage is applied to the capillary needle. Instead, the
heated nebulization gas alone, flowing parallel to the capillary, generates solvent
droplets as the solvent exits the tip of the capillary. The nebulization gas helps with
the desolvation of the liquid droplets and is typically heated to higher temperatures
than in ESI. The solvent in the vapor becomes charged from being in close proximity
to a corona discharge needle that has a current (microamps typically) applied to it.
The charge on the solvent (e.g., water, methanol, acetonitrile) is transferred to mole-
cules in the vapor to produce ions. Like ESI, APCI operates at atmospheric pressure.
APCI can provide more efficient ionization than ESI for nonpolar molecules. Both
ESI and APCI are considered soft ionization techniques and generate minimal analyte
fragmentation compared to EI. In-source fragmentation can occur for some mole-
cules with ESI and APCI as a result of the high heat (between 300 and 650 °C) and/
or the electric charge applied. LC is most often coupled to MS using ESI or APCI
sources [1, 4, 7]. The solvents (mobile phases) carrying analytes from the LC into the
ESI or APCI source are frequently made basic or acidic using modifiers such as for-
mic acid, ammonium hydroxide, or a pH buffer such as ammonium formate to
enhance the addition or removal of a proton (charge). Solvent modification can
increase the MS signal intensity as more ions may be generated in the source and
then enter the MS.
A variation of APCI much less commonly utilized is atmospheric pressure pho-
toionization (APPI). In APPI, a photon-emitting lamp generates ions instead of a
corona discharge needle. An ionizable dopant such as toluene or acetone is com-
monly infused in parallel to the nebulization gas for APPI. The dopant increases
charge exchange or proton transfer to the molecule(s) being analyzed [8, 9]. Like
APCI, APPI is generally used for the analysis of nonpolar analytes. In some cases,
using APPI can lead to increased analyte ionization and higher signal intensities
than APCI.
Matrix-assisted laser desorption/ionization (MALDI) is an ionization technique
where a pulsed laser generates ions from a sample on which a UV-absorbing matrix
has been applied. The matrix often acts as a proton donor to generate charged ana-
lytes. MALDI is a soft ionization technique that produces mostly singly charged
ions [5]. MALDI is often used to generate ions for imaging MS of proteins and
small molecules in tissue samples [10, 11]. Each MALDI laser shot at a certain
location on a tissue section provides a mass spectrum comparable to a pixel in digi-
tal imaging. The combined analysis of many pixels provides spatial distribution
information about ions across a tissue. An alternative surface ionization technique
to MALDI is desorption electrospray ionization (DESI). With DESI, a charged sol-
vent is directed toward the surface of a sample instead of a laser, and the charged
solvent desorbs ions directly from the sample [5]. It is, therefore, a combination of
ESI and surface desorption approaches. Interestingly, DESI has potential applica-
tions for real-time surgical tissue analysis as it is performed at atmospheric pressure
and has simplified sample preparation relative to MALDI [12].
22 J. A. Dubland

Direct analysis in real time (DART) is a rapid analysis approach that also pro-
duces ions directly from the surface of a sample without the need for the addition of
any matrix or time-consuming sample preparation. With DART, an electrical current
is applied to a heated inert gas to generate excited neutral species, which are directed
at the sample of interest. The electronically excited inert gas (e.g., helium) interacts
with water vapor and other gases at atmospheric pressure to generate reagent ions,
which then cause chemical ionization of the analytes on the sample’s surface. Gas-
phase ions generated from the surface of the specimen then enter the MS for analy-
sis [13]. DART can be performed in an open environment and is well suited for trace
analysis screening purposes (e.g., drugs of abuse, plant material, explosives) [14]
and has also been utilized for metabolite profiling [15].

2.2 Mass Analyzers

A variety of mass analyzers are commercially available, the most common of which
are the quadrupole, time-of-flight (TOF), ion trap, orbitrap, and Fourier-transform
ion cyclotron resonance (FTICR) mass analyzers. Mass analyzers vary in their m/z
range, mass accuracy and resolution, and acquisition speed. At a very basic level,
mass analyzers can be classified as either beam or trapping-type instruments. In
beam instruments, ions make a single pass through the mass analyzer and are
recorded using a detector. In trapping instruments, ions are confined using combina-
tions of electric and magnetic fields, which are manipulated in order to measure
selected ions. Beam instruments generally operate on the timescale of microseconds
to milliseconds, whereas trapping instruments can operate from milliseconds to
minutes (or even longer). Quadrupole and TOF mass analyzers are beam instru-
ments, whereas ion trap, orbitrap, and FTICR instruments are trapping instruments.
Although the development of the first mass spectrometers date back over 100 years
[16–18], the basic utilization of electric and magnetic fields for separating and iso-
lating ions remain the same. It is common for several types of mass analyzers to be
combined within the same instrument and incorporate collision cell(s). These tech-
nical arrangements allow for the analysis of intact charged metabolites (precursors)
and charged fragments of metabolites (products), which is critical for targeted anal-
yses as well as nontargeted investigations.

2.2.1 Quadrupole

The quadrupole mass analyzer consists of four symmetrically arranged cylindrical

metal rods with an ion flight path in the center that is parallel to the rods’ direction
(the Z-axis). Both alternating current (AC) and direct current (DC) are applied to
pairs of rods directly opposing each other in the configuration. One set of opposing
rods has a positive AC potential applied (X-Z plane), while at the same time, the
Mass Spectrometry-Based Metabolomics for the Clinical Laboratory 23

other set of opposing rods has a negative potential applied (Y-Z plane). This means
that the AC or radio frequency (RF) waveform applied to one set of rods is 180° out
of phase with the waveform applied to the other set of rods. At the same time, the
DC potential of one set of opposing electrodes is positive (X-Z plane), while the
other is negative (Y-Z plane). The applied voltages affect the flight path of ions
between the rods as they traverse the device. When the AC potential is positive, the
ions are accelerated onto the central Z-axis. In contrast, when the AC potential is
negative, the ions are defocused and accelerated toward the rods. Depending on the
voltage applied, ions either travel along the length of the quadrupole and pass
through or are eliminated by colliding with the rods [19, 20].
In the X-Z plane, heavier ions will be mostly influenced by the positive DC
potential and not affected by the high-frequency AC potential. Lower mass ions, on
the other hand, will be significantly affected in the X-Z plane by the AC potential
and, if they are light enough, may collide with the rods and be eliminated. In the Y-Z
plane, heavier ions will be defocused from the central Z-axis. They may be elimi-
nated on the Y-Z rods as they will mainly feel the destabilizing effect of the negative
DC potential and not the high-frequency AC potential. However, lighter ions will be
focused onto the central Z-axis in the Y-Z plane by the high-frequency AC potential
and pass through the quadrupole.
In essence, the opposing rods in the X-Z plane act as a high-pass mass filter as
only higher m/z ions will be transmitted and lower m/z ions will strike the X-rods
and then be removed as neutral species by the turbo pumps. Conversely, the oppos-
ing rods in the Y-Z plane act as a low-pass mass filter transmitting only lower m/z
ions and eliminating higher m/z ions [19]. Mathieu’s equation describes the stability
or instability of certain m/z ions in the X- and Y-coordinates in relation to AC and
DC voltages on electrodes of opposite potential [21, 22]. The combination of the
high-pass and low-pass mass filters creates a narrow band pass window (Δ m/z) of
ions transmitted through the quadrupole. Changing the amplitude of the RF voltage
can select (tune) for different masses to be transmitted through the quadrupole. A
complete mass spectrum can be obtained by simultaneously varying the amplitude
of the AC and DC voltages applied to the quadrupole rods but keeping the DC/RF
ratio fixed. The mass resolution can be varied by changing the DC/RF ratio [19].
Generally, parameters are set such that the band pass window (Δ m/z) is constant
across the entire m/z range, which provides sufficient mass resolution to separate
isotopes of small molecules that are singly charged. Mass resolution is calculated as
[(m/z)/(Δ m/z)]. The quadrupole mass analyzer can act as either a mass filter (i.e.,
isolate single Δ m/z’s as set by the user) or as a scanning instrument (i.e., scan Δ m/z
across a range as set by the user) [4].
The benefits of quadrupole mass analyzers include high sensitivity, a large
dynamic range, fast positive and negative mode polarity switching, small size,
robustness, and low cost. Limiting factors are a mass range typically less than
2000 m/z, low mass accuracy (greater than 100 ppm), and a mass resolution of 0.5
to 1 Dalton (Da) full width at half the maximum (FWHM) [1, 4]. One Da is equiva-
lent to 1/12 of the mass of carbon 12 in its lowest energy state. Some quadrupole
analyzers can reach resolutions of ≤0.1 Da at FWHM with specially designed
24 J. A. Dubland

quadrupole rods [23]. A quadrupole mass analyzer is commonly combined in series

with another quadrupole mass analyzer, but is also frequently combined with ion
trap (termed a QTRAP) or TOF (termed a QTOF) mass analyzers.

2.2.2 Triple Quadrupole

Triple quadrupole or tandem mass spectrometers (MS/MS) are a very common

instrument found in the clinical laboratory for quantitative analysis as a result of their
high specificity and sensitivity. The development of the triple quadrupole mass spec-
trometer was initially reported in 1978 by Yost and Enke [24]. A triple quadrupole
mass spectrometer consists of the following: a quadrupole mass filter (Q1), a low
energy collision cell usually consisting of an RF-only quadrupole (Q2), another
quadrupole mass filter (Q3), and then an electron multiplier to detect transmitted ions.
The tandem mass spectrometer has several modes of operation: multiple-­reaction
monitoring (MRM), product ion scan, precursor ion scan, and neutral loss scan. The
most utilized mode of operation for quantitative analysis is MRM. In MRM mode,
a single mass is selected in Q1, fragmented in the Q2 collision cell, and then a prod-
uct ion is selected in Q3 and detected. The instrument can acquire one MRM at a
time for a set amount of time, which is termed the dwell time. MRM dwell times are
on the order of milliseconds (typically between 10 and 300 ms). A product ion scan
selects a certain m/z in Q1, fragments the ion in Q2, and obtains a product ion spec-
trum by scanning an m/z range in Q3. A precursor ion scan is the reverse of a prod-
uct ion scan. A fragment ion m/z is selected in Q3, and Q1 is scanned for all precursor
m/z’s that give that Q3 fragment ion. This mode is useful for analyzing compounds
with a known common fragment ion. In a neutral loss scan, both Q1 and Q3 are
scanned, looking for a common neutral mass difference (loss) between precursor
and product ions [4, 25].
The Q2 collision cell is usually a low-energy collision-induced dissociation
(CID) device that contains an inert gas such as argon or nitrogen. Ions transmitted
from Q1 are given kinetic energy as they enter the CID device, collide with the inert
gas, and are fragmented. Collision energies are typically between 1 and 100 eV. The
Q2 quadrupole is operated in an RF-only mode (i.e., all ions are transmitted) by
removal of the DC potential on the quadrupole rods [19, 26]. Although CID is the
most common fragmentation method utilized in a triple quadrupole MS, other
methods such as electron capture dissociation or surface-induced dissociation frag-
mentation methods are possible [27].

2.2.3 TOF

A TOF is a beam type mass analyzer where m/z is determined from the time it takes
for an ion with a certain kinetic energy to travel through a long tube under a vacuum.
An electrostatic field first accelerates packets of ions entering the TOF. The ions all
obtain the same kinetic energy and then are separated over a drift path and
Mass Spectrometry-Based Metabolomics for the Clinical Laboratory 25

registered by a detector. Lower mass ions arrive at the detector first. The ion flight
path length limits a TOF device’s resolution. To increase the flight path and not the
overall size of the instrument flight tube, the flight path can be reflected multiple
times within the tube [28, 29]. The TOF mass analyzer is a pulsed instrument by
nature but is frequently paired with a continuous incoming ion beam from ioniza-
tion sources such as ESI, APCI, or EI. In order to extract packets of pulsed ions from
the continuous incoming ion beam, the TOF drift tube is placed orthogonally to it.
Ion packets are then accelerated and injected into the TOF low-pressure drift tube in
pulses [30]. A TOF mass analyzer is also often paired with MALDI, which is already
a pulsed incoming ion beam.
The TOF instrument is a high-resolution mass spectrometer (HRMS) that can
measure the exact mass of an ion, typically within a mass error of five parts per mil-
lion (ppm). This high-resolution mass measurement allows the TOF mass analyzer
to be useful in identifying unknown compounds by matching the measured exact
mass to a library of compounds of known molecular weight. Often, a quadrupole
mass analyzer followed by a collision cell is placed between the ionization source
and the TOF mass analyzer in order that high resolution fragment ion spectra can
also be collected. This instrumental arrangement is called a QTOF [31]. A TOF
mass analyzer has a very fast acquisition rate of microseconds, making it compati-
ble with being placed after a quadrupole mass analyzer that has an acquisition rate
of milliseconds. The quadrupole allows for initial low-resolution selection of spe-
cific ions or a range of ions, followed by fragmentation of those ions in the collision
cell and then high-resolution measurement of those fragments by TOF. Metabolomics
studies are often performed using a QTOF. The combination of measuring both the
exact mass of intact precursor ions and the exact mass of the fragmentation (product
ions) spectra greatly increases the spectral library matching performance. In addi-
tion, LC or GC is usually performed prior to molecules entering the MS in order to
separate compounds and thereby improve spectral quality. The LC or GC retention
time data also can be informative for identifying unknown molecules in combina-
tion with the exact mass spectra [4, 20].
Benefits of a TOF mass analyzer include high-resolution mass measurement
capabilities, high spectral acquisition rates, very large mass measurement ranges,
relative simplicity, durability, and having relatively reasonable cost. Disadvantages
include a lower analytical sensitivity range than quadrupole mass analyzers, the
requirement for a highly controlled instrument temperature environment, and lower
resolution capabilities than other HRMS systems such as orbitrap or FTICR
instruments.

2.2.4 Ion Traps

An ion trap is a mass analyzer that traps and stores ions using electric or magnetic
fields. Several configurations of ion traps exist and are discussed briefly in the fol-
lowing section. These include the 3D-type quadrupole ion trap, 2D-type linearity
ion trap, orbitrap, and ion cyclotron resonance mass analyzer.
26 J. A. Dubland

The 3D-type quadrupole ion trap (also called a Paul ion trap) functions similarly
to a quadrupole mass analyzer as oscillating RF fields and DC voltages are utilized.
Quadrupole ion traps differ in their configuration though, as they consist of a
hyperbolic ring electrode situated between two symmetrical hyperbolic entrance
and exit end cap electrodes. Ions enter and exit the device through holes in the end
caps. Unlike a quadrupole, which is a mass filter in 2D space, the ion trap accumu-
lates ions confined in a circular 3D space between the electrodes. Storage of
selected ion species or a certain mass range can be set in the ion trap. The incoming
ion beam is first trapped, and then, ions are subsequently scanned out based on
their m/z by manipulating the electric fields [22, 25]. Ion traps have high sensitivity
but are limited in the overall number of ions that can accumulate due to space
charging effects, which restricts the dynamic signal range. Since ions are accumu-
lated in the ion trap spectral skewing from chromatographic peak elution does not
affect the mass analysis. Notably, a 3D-type quadrupole ion trap is able to perform
multiple-stage fragmentation (MSn) experiments. All ions except the ion to be frag-
mented are first ejected from the trap. Then, the ion of interest is fragmented by
collisional activation, and the products are subsequently detected. The fragmenta-
tion and analysis process can then be repeated at higher orders. MSn experiments
are useful for structural investigations of molecules, but higher fragmentation
orders lead to a loss of signal intensity [20, 22, 32].
A 2D-type linear ion trap consists of a quadrupole with the addition of electro-
static plates on both ends of the device that generate a stopping potential to trap
ions. The linear ion trap is unique because it can function as a stand-alone quadru-
pole mass analyzer or an ion trap. Relative to 3D-type quadrupole (Paul type) ion
traps, the linear ion trap has a higher capacity for storing ions [33, 34]. Linear ion
traps have been paired in series following a standard quadruple mass analyzer (a
hybrid instrumental arrangement called a QTRAP). In this configuration, the hybrid
instrument can operate as a regular quadrupole mass analyzer (i.e., generate stan-
dard MRM data) or utilize the trapping features of the linear ion trap such as
enhanced MS scan, enhanced product ion scan, enhanced resolution scan, enhanced
multiply charged scan, and generation of MS3 ion fragmentation data [35–38].
The orbitrap is a high-resolution mass analyzer where ions are trapped in orbit
around a central spindle-like electrode in electrostatic fields. The general princi-
ples of orbitraps in use today are based on the ion trap device called the Kingdon
trap that was reported in 1923 [39]. Commercial orbitrap instruments were devel-
oped by Makarov et al. in the early 2000s [40, 41]. An orbitrap mass analyzer is
made up of a central spindle-like electrode surrounded by outer cup-shaped elec-
trodes. An ion packet from an orthogonally positioned curved linear ion trap
(called a C-trap) is injected into the orbitrap through a small slit in the outer cup-
shaped electrodes. An incoming ion beam initially fills the C-trap and then the ion
packet is pulsed orthogonally out of the C-trap into the orbitrap mass analyzer. The
ions that enter the orbitrap are bent around the central axial electrode using a radial
electric field and with the correct choice of parameters ions continue to orbit the
central electrode. At the same time, an axial electric field induces harmonic axial
ion oscillations. An image current signal (meaning a current induced by ions
Mass Spectrometry-Based Metabolomics for the Clinical Laboratory 27

passing by a conductor) from the frequency of the harmonic axial ion oscillations
is then recorded by outer electrodes on the orbitrap device acting as receiver plates.
The frequency of the oscillations is mathematically related to the m/z of the ion.
After Fourier transformation of the image current signal, a mass spectrum is
obtained [40–42]. The resolution of the orbitrap mass analyzer decreases as the
scan speed increases and also as the m/z increases [43]. In some incidences, resolu-
tion may need to be reduced in order to increase the orbitrap scan speed. In com-
parison, a TOF mass analyzer obtains relatively lower mass resolution than an
orbitrap, but the resolution is independent of detection time and m/z.
Orbitrap mass analyzers have also been coupled with other devices, such as
quadrupoles, linear ion traps, and collision cells in order to increase selectivity and
perform MSn fragmentation for structural analysis [41, 44]. The coupling of differ-
ent devices is facilitated via the C-trap, which can also serve as a T-device. Instrument
configurations can vary, but generally, the ion beam first passes through a quadru-
pole mass filter or a linear ion trap (having fragmentation capabilities) and then
enters the C-trap. From the C-trap, ions can be sent orthogonally to the orbitrap
mass analyzer or pass straight through the C-trap to a high collision-induced disso-
ciation (HCID) cell or linear ion trap where fragmentation is induced. The ions are
then returned to the C-trap, where they are orthogonally injected into the orbitrap to
generate high-resolution mass spectra [41]. In this way, both high-resolution precur-
sor and product ion spectra can be obtained with various experiments. Other molec-
ular fragmentation methods have been utilized with orbitrap mass analyzers, such as
electron transfer dissociation (ETD), which utilizes reagent anions to interact with
peptide cations. ETD has helped facilitate in-depth analysis of peptides and post-
translational protein modifications [41, 45].
The FTICR mass analyzer currently offers the highest resolution and mass accu-
racy of commercially available mass spectrometers and works on the basis that ions
within a magnetic field undergo cyclical motion (cyclotron motion). FTICR mass
analyzers consist of a Penning trap, where a strong uniform magnetic field is applied
to induce cyclotron motion of ions in the plane perpendicular to the magnetic field
lines (the radial plane). A ring and endcap electrodes are used to generate a
weak quadrupolar electric field, which traps ions in the axial plane. Within the
Penning trap, ions undergo three independent motions: cyclotron, magnetron, and
axial. Cyclotron motion is the large circular motion of ions in the plane perpendicu-
lar to the magnetic field lines (the radial plane). Magnetron motion is an additional
slow circular drifting motion of ions in the radial plane (a drifting of the cyclotron
motion centre). Axial motion is the harmonic oscillation of ions along the magnetic
field lines. Excitation electrodes generate a sweeping RF potential that excites ions
to larger cyclotron orbits in the radial plane so that they pass in close proximity to a
pair of detection electrodes. Similar to an orbitrap mass analyzer, an image current
is recorded by the pair of detection electrodes and then Fourier transformation is
performed to generate mass spectra. The m/z of an ion is inversely proportional to
the ion cyclotron frequency. Resolution can be improved by increasing the magnetic
field strength or by increasing the scan time. FTICR instruments can also perform
MSn experiments [46–51].
28 J. A. Dubland

3 Chromatographic Separations Interfacing with MS

Physical separation of small molecules for MS analysis is important for metabolo-

mic investigations in order to reduce matrix effects, separate interferences, and
resolve both isomeric and isobaric compounds. Standard chromatographic tech-
niques, as mentioned earlier, include liquid chromatography (LC) and gas chroma-
tography (GC). Capillary electrophoresis (CE) has also been utilized. A somewhat
newer separation tool for MS-based metabolomics is ion mobility, where ions are
separated in the gas phase by interactions with a neutral gas in the presence of an
electric field. A brief discussion of these separation approaches is provided here.

3.1 Liquid Chromatography

LC is an important laboratory technique that separates a mixture into individual

components based on the interaction between a liquid and a solid stationary phase.
Generally, LC refers to a separation that is performed in a column packed with a
stationary phase. Thin layer chromatography (TLC), though, is also a type of LC
where the stationary phase is coated on a sheet of inert material (such as aluminum
or glass), and the liquid is drawn up the plate based on capillary action. LC columns
use pressurized liquid to speed up chromatographic separations. Two general types
of instruments are currently available, the high-performance liquid chromatography
(HPLC) system and the ultra-high-performance liquid chromatography (UPLC)
system. HPLC operates in a system pressure range generally less than 6000 psi,
whereas UPLC operates in a pressure range of less than 18,000 psi. Due to the
higher system pressures for UPLC, the use of stainless steel tubing is required.
Sample introduction for HPLC and UPLC is done using an autosampler. A sam-
pling needle is first used to take up a small amount (typically 1–20 μL) of sample
from a prepared liquid specimen in a vial or plate within the autosampler. Pressurized
mobile phases (MPs) then transfer the sample via a fix-loop or flow-through injector
onto an LC column. Analytes are separated on the column based on their interaction
with the MPs and the column’s stationary phase. Once analytes have eluted from the
column they are transferred via tubing to the MS source for ionization (typically
ESI or APCI, as described earlier in this chapter).
HPLC and UPLC columns are small, typically on the order of 30–150 mm in
length and 1–4.6 mm in diameter. The adsorbent particle sizes for HPLC are typi-
cally between 3 and 5 μm in diameter. UPLC columns utilize particle diameters
below 2 μm, which improves chromatographic efficiency (peak dispersion).
Chromatographic efficiency is inversely proportional to the particle size. Smaller
particle sizes increase system pressure, as the particle size is also inversely propor-
tional to the column back pressure. The use of UPLC columns, which have high
efficiency, can increase chromatographic resolution (sharper and narrower peaks)
and simultaneously reduce sample analysis time. Higher flow rates can be utilized
Mass Spectrometry-Based Metabolomics for the Clinical Laboratory 29

since the system can tolerate higher back pressures. UPLC paired with MS is a pre-
ferred approach for metabolomics investigations [52].
Generally, synthetic silica polymers are used as the adsorbent packing material
in LC columns. Pore sizes of the silica polymer particles are typically in the
100–300 Å range. The smaller the pore size, the larger the surface area available on
the particle to interact with analytes. Smaller pore sizes are generally utilized for
small molecules, whereas larger pore sizes are preferable for larger molecules such
as proteins. The silica polymers packed into an LC column are usually covalently
modified using alkyl chains (e.g., C8 or C18 chain lengths) or other functional
groups (e.g., phenyl, cyano, amide, amine, fluorophenyl) to improve selectivity. In
reversed-phase chromatography, a nonpolar stationary phase (e.g., C18 modified
silica) is utilized. The mobile phase composition is ramped from high aqueous
(polar) to high organic (nonpolar) solvent content. The organic solvent competes
with nonpolar hydrophobic analytes for interaction with the nonpolar adsorbent sta-
tionary phase. Analytes generally elute in order of decreasing polarity as the mobile
phase composition is increased from high aqueous to high organic solvent content.
In normal-phase chromatography, the stationary phase is polar (e.g., bare silica or
amide-modified silica), and the mobile phase composition is nonpolar. The mobile
phase can be ramped to higher polarity content in order to elute polar analytes.
Nonpolar compounds generally elute first in normal-phase chromatography.
Normal-phase chromatography is not frequently utilized with HPLC or UPLC. If
the stationary phase is bare silica, organic solvents nonmiscible with water are gen-
erally utilized as water can become highly adsorbed to the silica and cause chro-
matographic variability. Polar-modified silica (e.g., amide modification) is a
normal-phase chromatography approach where water can be tolerated in the mobile
phase. Another normal-phase type approach that has also been developed is called
hydrophilic interaction liquid chromatography (HILIC). In HILIC separations,
polar hydrophilic compounds are retained more than nonpolar hydrophobic com-
pounds by the stationary at high nonpolar solvent content. The stationary phase is
polar and hydrophilic. As the water (polar) content in the mobile phase increases,
polar hydrophilic compounds elute off the column. The mobile phases used for
HILIC generally require buffers such as ammonium formate or ammonium acetate
to improve chromatographic peak shape. Additionally, acid or base modifiers such
as formic acid or ammonium hydroxide are often added to mobile phases to enhance
either positive or negative mode ionization in the MS source [53].

3.2 Gas Chromatography

GC is a gas-phase separation technique where a gaseous sample mixture is separated

into components using a narrow hollow metal tube filled with a porous silica-­based
stationary phase. A neutral gas such as helium or hydrogen is used to move the vola-
tile analytes through the column as a temperate ramp or constant temperature is
applied to the column using a temperature-controlled oven. GC oven temperatures
30 J. A. Dubland

can reach up to 350 °C, and columns must be able to withstand these high tempera-
tures. MS is frequently used for detecting eluting analytes (typically using an EI
source at 70 eV, as discussed earlier) when a mass spectrum is required for analyte
confirmation or library searching. Other methods include flame ionization detection
and thermal conductivity detection. GC columns are usually very long, being on the
order of 10 m or more in length [54]. The first reports of combining GC with MS
date back to the late 1950s [55]. GC-MS is a robust and well-utilized method of
analyzing specimens for metabolic investigations and allows for the identification of
unknown analytes using well-developed 70 eV EI libraries, but can only analyze
volatile compounds. To enhance the volatilization of molecules derivatization is fre-
quently required. A common method is silylation to generate trimethylsilyl (TMS)
derivatives from alcohols, carboxylic acids, and amines. Ketone groups form TMS
derivatives following initial treatment with hydrazine. Sample preparation of bio-
logical specimens for GC-MS analysis typically involves liquid-liquid extraction
protocols that are generally longer than sample preparation techniques required for
LC-MS analysis of the same specimens. Urine organic acid profiling by GC-MS is a
standard targeted and nontargeted metabolomics assay utilized in biochemical
genetics laboratories to investigate inborn errors of metabolism (IEMs) [56].

3.3 Capillary Electrophoresis

CE can be interfaced with MS and is an emerging method of separating analytes

prior to MS analysis. It is a technique based on the movement of ions in a high elec-
tric field. Analytes move from one end of the capillary to the other based on their
size and charge. CE is particularly useful for the separation of polar and charged
molecules. ESI is a common mode of interfacing CE with MS [57].

3.4 Ion Mobility Spectrometry

Ion mobility spectrometry (IMS) is an analysis technique where molecules are sepa-
rated based on their mobility through an inert buffer gas such as nitrogen or helium
in the presence of an electric field. IMS generates an analytical output called a col-
lisional cross section (CCS) value that provides additional complementary data to
m/z determined by MS. The CCS value is a measure, in square Ångströms (Å2), of
the interaction of a molecule with the buffer gas. CCS values depend on the mole-
cules’ specific size, shape, and charge and also vary based on the buffer gas utilized
in the device. Several different types of IMS devices are commercially available.
These include drift tube ion mobility spectrometry (DTIMS), traveling wave ion
mobility spectrometry (TWIMS), structures for lossless ion manipulations (SLIM),
field asymmetric waveform ion mobility spectrometry (FAIMS), and trapped ion
mobility spectrometry (TIMS) [58–61].
Mass Spectrometry-Based Metabolomics for the Clinical Laboratory 31

IMS devices operate on the millisecond timescale and are, therefore, well suited
to being incorporated into an MS instrument (termed IMS-MS), which are often
additionally paired with either LC or GC front-end separations. IMS is a useful
analytical technique for increasing the separation of isomers and interferences in
addition to LC and GC, or it can also operate as a stand-alone separation method.
An IMS-MS instrument paired with initial chromatographic separation can provide
retention time, m/z, and CCS data that is useful for metabolomics investigations [59,
62]. Ion mobility spectrometry has not been utilized in the clinical laboratory to any
great extent thus far, but in the future it may offer ways to increase selectivity,
shorten analysis times, and potentially reduce reliance on traditional LC or GC sep-
arations [63].

4 Current Clinical Metabolomic Applications

MS has been utilized for diagnostic testing in several areas of clinical pathology
including clinical biochemistry, microbiology, anatomical pathology, toxicology,
and newborn screening and biochemical genetics testing (Fig. 1) [64–67]. Generally,
clinical quantitative diagnostic assays that utilize MS consist of targeted small pan-
els of analytes where sample preparation and instrument parameters can be fairly
easily optimized. Applications of MS for clinical biochemistry include quantitative
LC-MS/MS analysis for steroids, vitamins, therapeutic drug monitoring of small
molecules and antibodies, and protein biomarkers for various disorders [64, 66].
These types of quantitative clinical assays typically consist of a single analyte or a
small panel of analytes. Larger profiling panel assays have also been developed to
improve patient diagnosis. For example, a 26 analyte panel urine steroid profiling
assay by LC-HRMS has been reported [68]. Trace metal analysis is also done by
inductively coupled plasma MS (ICP-MS) [69]. MS is utilized in microbiology for
the qualitative identification of microorganisms by MALDI-QTOF MS via spectral
matching of ionizable proteins and peptides present in a bacterial culture to a vali-
dated MS library [70]. In anatomical pathology, the use of MALDI-QTOF MS for
spatial imaging analysis of small molecules, proteins, and peptides in cancer and
other tissue biopsies is an emerging area [71]. MS has also been used during surger-
ies to guide the removal of tumor tissues [72].
In toxicology, LC-MS/MS, GC-MS, and LC-HRMS are routinely used for drug
screening and investigation of toxic chemical exposures. ICP-MS is also utilized to
investigate exposure to toxic levels of metals [73]. Detecting known and unknown
drug substances in the body involves a combination of targeted and nontargeted
approaches. LC-HRMS and GC-MS assays utilize analytical standards and spectral
library matching to identify known and unknown drugs and toxic substances.
LC-MS/MS is utilized for targeted quantitative analysis. Additionally, the use of
large panel targeted and nontargeted MS assays to detect drug metabolites and
determine the effect of drugs on biochemical pathway metabolite formation may
find applications in clinical toxicology to improve workflows [74–77]. Global
32 J. A. Dubland

Fig. 1 Mass spectrometry applications to diagnostic testing by clinical pathology division. (i)
Divisions of clinical pathology that MS has been applied to. (ii) Applications of MS in each divi-
sion. (iii) MS platforms typically utilized in each division. Abbreviations: FIA-MS/MS flow injec-
tion analysis-tandem mass spectrometry, GC-MS gas chromatography-mass spectrometry, ICP-MS
inductively coupled plasma-mass spectrometry, LC-HRMS liquid chromatography-high-resolution
mass spectrometry, LC-MS/MS liquid chromatography-tandem mass spectrometry, MALDI-QTOF
MS, matrix-assisted laser desorption/ionization-quadrupole time-of-flight mass spectrometry.

analysis looking at exogenous xenobiotic metabolites formed in the body, in addi-

tion to the effect of drug exposure on metabolites normally found in the body, is part
of multidisciplinary metabolomics. Metabolomics being the large-scale systematic
analysis of small molecules (metabolites) in a biological system.
Newborn screening and biochemical genetics is the clinical pathology division
that currently heavily utilizes large-scale MS-based metabolomics investigations of
biochemical pathway metabolites (both targeted large panels and nontargeted anal-
yses) for diagnosing and monitoring IEMs. Targeted large panel assays primarily
analyze for amino acids, acylcarnitines, organic acids, and purines and pyrimidines.
Organic acid analysis also typically involves spectral library searching for nontar-
geted identification of unknowns. Other tests utilizing MS include very long chain
fatty acids for analysis of peroxisomal disorders [78, 79], steroids for analysis of
sterol biosynthesis and steroidogenesis disorders [80–83], glycans and intact trans-
ferrin for analysis of congenital disorders of glycosylation [84], oligosaccharide and
Mass Spectrometry-Based Metabolomics for the Clinical Laboratory 33

glycosaminoglycan analysis [85, 86], and lysosomal enzyme activity testing for
lysosomal storage disorders [87].
Amino acids are the building blocks of life and are utilized to synthesize peptides
and proteins in the body, such as enzymes, hormones, and neurotransmitters. They
are also key energy sources and metabolic intermediates in biological pathways,
and can be recycled when required. There are 21 amino acids that are utilized to
generate proteins, and 9 of these are called “essential” amino acids as the body can-
not synthesize them. Essential amino acids must be obtained in the diet [88].
Mutations in genes involved in amino acid catabolism can cause metabolic pathway
blockages in the body, leading to toxic elevations in certain amino acids and metab-
olites [88, 89]. Deficiency in the synthesis pathways of amino acids caused by gene
mutations can also occur [90]. As such, the measurement of amino acids in blood,
plasma, urine, and cerebral spinal fluid is of essential importance for both diagnos-
ing IEMs and subsequent patient monitoring [91]. Amino acid analysis for NBS is
discussed later on. As part of the diagnostic workup, gene sequencing for patients
suspected of an IEM is frequently performed in addition to the biochemical
assessment.
Traditionally, deproteinized amino acids have been analyzed by ion exchange
chromatography (IEC) with post-column ninhydrin derivatization for colorimetric
detection [92, 93]. IEC has been considered the gold standard for amino acid analy-
sis for over half a century. This approach involves lengthy sample analysis time
(more than 1 h of analysis time per sample), lacks specificity and analytical resolu-
tion, and has minimal calibration. Many clinical laboratories have, therefore, moved
away from IEC in favor of LC-MS/MS amino acid analysis approaches. LC-MS/
MS approaches include pre-column or post-column amino acid derivatization, and
underivatized analyses using ion pairing reagents in the LC mobile phases [94–97].
Assays must be able to cover large analytical ranges as amino acid concentrations
span three orders of magnitude. LC-MS/MS provides a much faster sample analysis
time relative to IEC, which can facilitate urgent patient care needs and improve
laboratory workflows.
Acylcarnitine analysis is done to help diagnose mitochondrial β-oxidation and
organic acid metabolism disorders [98, 99]. Typically, this is performed by flow
injection analysis paired with MS/MS (FIA-MS/MS) for urine and serum speci-
mens. Either derivatized or nonderivatized approaches can be used. Acylcarnitine
analysis for NBS is discussed later on. Acylcarnitine isomers have also been sepa-
rated using LC-MS/MS to increase specificity [100].
Urine organic acid analysis is traditionally done by GC-MS and covers a broad
range of IEMs arising from enzyme or transporter deficiencies [56]. Disorders can
affect many biochemical pathways, including the metabolism of organic acids,
amino acids, carbohydrates, fatty acids, sterols, and purines and pyrimidines. It pro-
vides targeted analysis of known biomarkers and also nontargeted screening capa-
bilities through spectral library searching (fragmentation energy of 70 keV is
standard for GC-MS library generation and searching). Elevated levels of excreted
metabolites and observed metabolite patterns are used to both identify and monitor
disorders. Normalization of values to urine creatinine levels is required for
34 J. A. Dubland

diagnostic evaluation. Analysis of urinary organic acids by GC-MS generally

requires lengthy liquid-liquid extraction, derivatization to enhance organic acid
volatilization, and long chromatography run times to separate analytes and interfer-
ences (typically greater than 30 min per sample). Recently, an LC-QTOF MS
method was reported, allowing for simplified sample preparation without
the need for sample derivatization [101].
Purines and pyrimidines are important molecules that help form DNA and RNA,
act as metabolic regulators and intermediates, and provide an energy store [102].
Metabolic disorders of purine and pyrimidine metabolism are usually screened in a
panel assay by LC-MS/MS [103, 104], identifying disorders by abnormal urinary
excretion levels of these molecules. This analysis typically does not require sample
derivatization as LC, and not GC, is utilized. Similar to urine organic acids, normal-
ization to creatinine levels is required for urinary purine and pyrimidine analysis.
NBS programs aim to identify infants in the first few days after birth that are at
high risk of having an IEM that may not be easily identifiable otherwise (i.e., having
a lack of initial signs and symptoms of the disorder without a blood test). Early
detection of certain disorders, such as phenylketonuria, allows for prompt treatment
intervention in order to prevent death and other health complications. Dried blood
spot (DBS) cards are typically utilized for NBS as they are relatively easy to collect
via a heal prick from a newborn and provide sufficient analyte stability for the card
to be transported at room temperature to a testing location. DBS cards are punched
using an automated hole punch to provide a small sample for testing. Acylcarnitines
and amino acids from DBS cards are analyzed simultaneously for NBS by FIA-MS/
MS quantitation [105]. Derivatization with butanol hydrochloride is frequently uti-
lized for NBS FIA-MS/MS. The initial (first-tier) FIA-MS/MS analysis lacks suf-
ficient diagnostic specificity for some disorders and a follow-up (second-tier) test is
needed to increase the overall positive predictive value without reducing screening
sensitivity. Second-tier tests provide more specific biomarkers and/or separate inter-
ferences from the analysis. Typically, second-tier tests involve LC separations fol-
lowed by MS/MS detection. The current necessity for lack of chromatographic
separation for first-tier NBS prior to introduction into the MS is the sheer daily
volume of specimens that must be analyzed [106, 107]. FIA-MS/MS analysis is less
than 2 minutes a sample, whereas second-tier analyses can range anywhere from 2
to 15 minutes a sample. From the first-tier testing a smaller cohort of specimens is
flagged for second-tier analysis, which allows for a more manageable screening
workflow. Of note, a recent publication has outlined a strategy to combine first-tier
and second-tier NBS using microfluidic capillary electrophoresis paired with
HRMS analysis [108].
It is possible to develop large-scale targeted LC-HRMS or low-resolution
LC-MS/MS clinical metabolomics panels for evaluations of IEMs (reviewed com-
prehensively in chapters “Bioinformatics Tools for Clinical Metabolomics” and
“Untargeted Metabolomics in Newborn Screening”) [56, 97, 101, 103, 109, 110].
Nontargeted metabolomics has limited clinical application to date outside urine
organic acids analysis, which is typically performed by GC-MS. The advantage of
large panel analyses is the efficient utilization of instrument time and a broader
snapshot of the biological phenotype. A disadvantage of using very large metabolite
Mass Spectrometry-Based Metabolomics for the Clinical Laboratory 35

panels is that quality control issues arising from both the sample preparation and
the instrumental analysis can occur more frequently. Analyte recovery can vary
greatly when using a universal sample preparation approach. Obtaining optimal
chromatographic peak shape may not be possible as columns are designed to work
optimally with different classes of molecules. Matrix effects leading to analyte ion
suppression or enhancement need to be considered. Using a large number of inter-
nal standards to correct matrix effects can be problematic from a technical perspec-
tive. In some situations, benefits and disadvantages need to be taken into
consideration regarding either condensing or expanding existing small molecule
test panels. Ultimately the approach must be beneficial to patient care and clinical
laboratory workflows.

5 Guidance Documents and Validation Hurdles

Several guidance documents exist for validating small molecule MS assays [111,
112]. These documents describe analytical criteria that should be demonstrated in a
method validation to prove that the assay is acceptable for use in the clinical labora-
tory. Assay parameters to be included in a method validation are sensitivity, speci-
ficity, linearity, analytical range, the limit of quantitation, the limit of detection,
recovery, matrix effects, accuracy, precision, and sample stability. Evaluation of
normal ranges, clinical diagnostic decision-making points, and abnormal ranges
must be performed. Frequently, a method comparison of the new assay vs. another
assay in use at a different laboratory is also conducted using real specimens [113,
114]. Targeted MS-based assays almost always involve the use of stable-labeled
internal standards. Post-validation assay performance is monitored using a system
suitability sample that is run prior to starting a batch of samples, in addition to QC
samples analyzed during the run. Involvement in available external proficiency pro-
grams is also often done for assay performance monitoring.
Guidance strategies are currently available and work well, for single analyte or
small panel assays in the clinical setting. However, for larger panel targeted metabo-
lomic assays (such as some mentioned in the prior section), sample and batch
acceptance criteria are not as straightforward. A fit-for-purpose approach, in some
cases, is required. The clinical importance of certain biomarkers in the context of
assay performance must be taken into consideration.
Nontargeted global metabolomics investigations are not generally utilized out-
side urine organic acids analysis in the routine clinical laboratory [56]. A major goal
of many nontargeted metabolomics investigations is to identify differentially pres-
ent biomarkers or biomarker signatures that may be informative of a disease or
health state. Accurate quantitation in nontargeted metabolomics investigations is
not generally a main goal [115]. The incorporation of HRMS nontargeted metabo-
lomics investigations with clinically validated targeted small molecule panels may
provide further health insights. Any potential biomarkers identified in nontargeted
analyses should be validated in a fit-for-purpose manner and not by themselves
drive clinical decisions. Harmonization of nontargeted metabolomics approaches
36 J. A. Dubland

and the use of proficiency testing specimens [116, 117] will help improve results
and data consistency from different laboratories [118–120]. A guidance document
has also been published regarding combining and reporting ion mobility data from
different instruments and buffer gases utilized [121].

6 Summary and Future Outlook

MS is an important analysis technique for metabolomics investigations. Targeted

and nontargeted metabolomics analyses provide valuable insights into phenotype
changes. These biochemical investigations provide complementary data to genomic
analyses in relation to disease evaluation and overall health status. Typically, screen-
ing and diagnostic workflows in the clinical laboratory involve biochemical analy-
ses first, followed by genome investigations when a genetic disorder is suspected
from the biochemical profile. The order of investigations may change in the future
as the cost of genomic testing continues to decrease. At some point in the future
genomic analyses may be performed first, screening for known gene mutations and
variants of unknown significance (VUS) associated with various metabolic disor-
ders. Biochemical testing would then follow the genetic workup to evaluate for the
presence of known disease biomarkers and provide further comprehensive (targeted
and/or nontargeted) metabolic profiling as needed. Utilizing internal standards,
quality controls, and external proficiency monitoring for biochemical MS assays is
important to ensure the quality of the clinical laboratory data.
Further guidance documents are needed regarding approaches to acceptance cri-
teria for large panel targeted MS-based metabolomics analyses, as current docu-
ments are aimed at small panels of analytes. Nontargeted metabolomics investigations
have yet to be widely implemented in the routine clinical laboratory. Still, they may
provide an investigative means in scenarios where existing targeted diagnostic test-
ing has not provided clear answers. Harmonization of methodologies, validation cri-
teria, and proficiency testing specimens for nontargeted MS-based
metabolomics investigations is needed in the future. Incorporating nontargeted
analyses into the clinical setting may facilitate the discovery of new biomarkers that
can be validated and included in targeted analyte panels.

References

1. Kaklamanos G, Aprea E, Theodoridis G. Mass spectrometry: principles and instrumentation.

In: Caballero B, Finglas PM, Toldrá F, editors. Encyclopedia of food and health. Oxford:
Academic Press; 2016. p. 661–8.
2. Nordström A, Want E, Northen T, Lehtiö J, Siuzdak G. Multiple ionization mass spectrom-
etry strategy used to reveal the complexity of metabolomics. Anal Chem. 2008;80(2):421–9.
3. Gross JH. Electron ionization. In: Gross JH, editor. Mass spectrometry: a textbook. Berlin:
Springer; 2004. p. 193–222.
Mass Spectrometry-Based Metabolomics for the Clinical Laboratory 37

4. Rockwood AL, Kushnir MM, Clarke NJ. Chapter 2 - Mass spectrometry. In: Rifai N,
Horvath AR, Wittwer CT, editors. Principles and applications of clinical mass spectrometry.
Amsterdam: Elsevier; 2018. p. 33–65.
5. Bhardwaj C, Hanley L. Ion sources for mass spectrometric identification and imaging of
molecular species. Nat Prod Rep. 2014;31(6):756–67.
6. Konermann L, Ahadi E, Rodriguez AD, Vahidi S. Unraveling the mechanism of electrospray
ionization. Anal Chem. 2013;85(1):2–9.
7. Covey TR, Thomson BA, Schneider BB. Atmospheric pressure ion sources. Mass Spectrom
Rev. 2009;28(6):870–97.
8. Robb DB, Covey TR, Bruins AP. Atmospheric pressure photoionization: an ionization
method for liquid chromatography−mass spectrometry. Anal Chem. 2000;72(15):3653–9.
9. Raffaelli A, Saba A. Atmospheric pressure photoionization mass spectrometry. Mass
Spectrom Rev. 2003;22(5):318–31.
10. Seeley EH, Caprioli RM. MALDI imaging mass spectrometry of human tissue: method chal-
lenges and clinical perspectives. Trends Biotechnol. 2011;29(3):136–43.
11. Reyzer ML, Caprioli RM. MALDI-MS-based imaging of small molecules and proteins in
tissues. Curr Opin Chem Biol. 2007;11(1):29–35.
12. Calligaris D, Norton I, Feldman DR, Ide JL, Dunn IF, Eberlin LS, et al. Mass spectrometry
imaging as a tool for surgical decision-making. J Mass Spectrom. 2013;48(11):1178–87.
13. Gross JH. Direct analysis in real time—a critical review on DART-MS. Anal Bioanal Chem.
2014;406(1):63–80.
14. Sisco E, Forbes TP. Forensic applications of DART-MS: a review of recent literature. Forensic
Chem. 2021;22:100294.
15. Chernetsova ES, Morlock GE. Ambient desorption ionization mass spectrometry (DART,
DESI) and its bioanalytical applications. Bioanal Rev. 2011;3(1):1–9.
16. Münzenberg G. Development of mass spectrometers from Thomson and Aston to present. Int
J Mass Spectrom. 2013;349–350:9–18.
17. Nier KA, Yergey AL, Jane GP. A general chronicle of mass spectrometry and guide to the
scope of the volume. In: Gross ML, Caprioli RM, editors. The encyclopedia of mass spec-
trometry. Boston: Elsevier; 2016. p. 7–12.
18. Nier KA. Dempster’s descendants—the core of the development of mass spectrometry. J
Mass Spectrom. 2020;55(8):e4353.
19. Miller PE, Denton MB. The quadrupole mass filter: basic operating concepts. J Chem Educ.
1986;63(7):617.
20. Li C, Chu S, Tan S, Yin X, Jiang Y, Dai X, et al. Towards higher sensitivity of mass spectrom-
etry: a perspective from the mass analyzers. Front Chem. 2021;9:813359.
21. Stafford GC, Kelley PE, Syka JEP, Reynolds WE, Todd JFJ. Recent improvements in and
analytical applications of advanced ion trap technology. Int J Mass Spectrom Ion Process.
1984;60(1):85–98.
22. Paul W. Electromagnetic traps for charged and neutral particles (Nobel Lecture). Angew
Chem Int Ed Engl. 1990;29(7):739–48.
23. Yang L, Amad M, Winnik WM, Schoen AE, Schweingruber H, Mylchreest I, et al.
Investigation of an enhanced resolution triple quadrupole mass spectrometer for high-­
throughput liquid chromatography/tandem mass spectrometry assays. Rapid Commun Mass
Spectrom. 2002;16(21):2060–6.
24. Yost RA, Enke CG. Selected ion fragmentation with a tandem quadrupole mass spectrometer.
J Am Chem Soc. 1978;100(7):2274–5.
25. Thomas SN. Chapter 10 - Mass spectrometry. In: Clarke W, Marzinke MA, editors.
Contemporary practice in clinical chemistry. 4th ed. New York: Academic Press; 2019.
p. 171–85.
26. Yost RA, Enke CG, McGilvery DC, Smith D, Morrison JD. High efficiency collision-induced
dissociation in an RF-only quadrupole. Int J Mass Spectrom. 1979;30(2):127–36.
38 J. A. Dubland

27. Sleno L, Volmer DA. Ion activation methods for tandem mass spectrometry. J Mass Spectrom.
2004;39(10):1091–112.
28. Plaß WR, Dickel T, Scheidenberger C. Multiple-reflection time-of-flight mass spectrometry.
Int J Mass Spectrom. 2013;349–350:134–44.
29. Mamyrin BA. Time-of-flight mass spectrometry (concepts, achievements, and prospects). Int
J Mass Spectrom. 2001;206(3):251–66.
30. Coles J, Guilhaus M. Orthogonal acceleration — a new direction for time-of-flight mass
spectrometry: fast, sensitive mass analysis for continuous ion sources. TrAC Trends Anal
Chem. 1993;12(5):203–13.
31. Allen DR, McWhinney BC. Quadrupole time-of-flight mass spectrometry: a paradigm shift
in toxicology screening applications. Clin Biochem Rev. 2019;40(3):135–46.
32. Johnson JV, Yost RA, Kelley PE, Bradford DC. Tandem-in-space and tandem-in-time mass
spectrometry: triple quadrupoles and quadrupole ion traps. Anal Chem. 1990;62(20):2162–72.
33. Clarke W. Chapter 1 - Mass spectrometry in the clinical laboratory: determining the need and
avoiding pitfalls. In: Nair H, Clarke W, editors. Mass spectrometry for the clinical laboratory.
San Diego: Academic Press; 2017. p. 1–15.
34. Douglas DJ, Frank AJ, Mao D. Linear ion traps in mass spectrometry. Mass Spectrom Rev.
2005;24(1):1–29.
35. Cabruja M, Priotti J, Domizi P, Papsdorf K, Kroetz DL, Brunet A, et al. In-depth triacylg-
lycerol profiling using MS3 Q-trap mass spectrometry. Anal Chim Acta. 2021;1184:339023.
36. Collings BA. Fragmentation of ions in a low pressure linear ion trap. J Am Soc Mass
Spectrom. 2007;18(8):1459–66.
37. Lv S, Wang H, Yan Y, Ge M, Guan J. Quantification and confirmation of four aflatoxins using
a LC–MS/MS QTRAP system in multiple reaction monitoring, enhanced product ion scan,
and MS3 modes. Eur J Mass Spectrom. 2020;26(1):63–77.
38. Hopfgartner G, Varesio E, Tschäppät V, Grivet C, Bourgogne E, Leuthold LA. Triple quad-
rupole linear ion trap mass spectrometer for the analysis of small molecules and macromol-
ecules. J Mass Spectrom. 2004;39(8):845–55.
39. Kingdon KH. A method for the neutralization of electron space charge by positive ionization
at very low gas pressures. Phys Rev. 1923;21(4):408–18.
40. Makarov A. Electrostatic axially harmonic orbital trapping: a high-performance technique of
mass analysis. Anal Chem. 2000;72(6):1156–62.
41. Hecht ES, Scigelova M, Eliuk S, Makarov A. Fundamentals and advances of orbitrap mass
spectrometry. In: Encyclopedia of analytical chemistry. New York: Wiley. p. 1–40.
42. Zubarev RA, Makarov A. Orbitrap mass spectrometry. Anal Chem. 2013;85(11):5288–96.
43. Crutchfield CA, Clarke W. Chapter 12 - High resolution accurate mass (HRAM) mass spec-
trometry. In: Nair H, Clarke W, editors. Mass spectrometry for the clinical laboratory. San
Diego: Academic Press; 2017. p. 247–59.
44. Makarov A, Denisov E, Kholomeev A, Balschun W, Lange O, Strupat K, et al.
Performance evaluation of a hybrid linear ion trap/orbitrap mass spectrometer. Anal Chem.
2006;78(7):2113–20.
45. Kim MS, Pandey A. Electron transfer dissociation mass spectrometry in proteomics.
Proteomics. 2012;12(4–5):530–42.
46. Guan S, Marshall AG. Ion traps for Fourier transform ion cyclotron resonance mass spec-
trometry: principles and design of geometric and electric configurations. Int J Mass Spectrom
Ion Process. 1995;146–147:261–96.
47. Nikolaev EN, Kostyukevich YI, Vladimirov GN. Fourier transform ion cyclotron resonance
(FT ICR) mass spectrometry: theory and simulations. Mass Spectrom Rev. 2016;35(2):219–58.
48. Blaum K, Eliseev S, Sturm S. Perspectives on testing fundamental physics with highly
charged ions in penning traps. Quantum Sci Technol. 2021;6(1):014002.
49. Ostrander CM, Arkin CR, Laude D. Central ring electrode for trapping and excitation/
detection in Fourier transform ion cyclotron resonance mass spectrometry. J Am Soc Mass
Spectrom. 2001;12(1):30–7.
Mass Spectrometry-Based Metabolomics for the Clinical Laboratory 39

50. Marshall AG, Hendrickson CL. Fourier transform ion cyclotron resonance detection: prin-
ciples and experimental configurations. Int J Mass Spectrom. 2002;215(1):59–75.
51. Marshall AG, Hendrickson CL, Jackson GS. Fourier transform ion cyclotron resonance mass
spectrometry: a primer. Mass Spectrom Rev. 1998;17(1):1–35.
52. Perez de Souza L, Alseekh S, Scossa F, Fernie AR. Ultra-high-performance liquid chro-
matography high-resolution mass spectrometry variants for metabolomics research. Nat
Methods. 2021;18(7):733–46.
53. Žuvela P, Skoczylas M, Jay Liu J, Baṃczek T, Kaliszan R, Wong MW, et al. Column charac-
terization and selection systems in reversed-phase high-performance liquid chromatography.
Chem Rev. 2019;119(6):3674–729.
54. Bartle KD, Myers P. History of gas chromatography. TrAC Trends Anal Chem.
2002;21(9):547–57.
55. Gohlke RS. Time-of-flight mass spectrometry and gas-liquid partition chromatography. Anal
Chem. 1959;31(4):535–41.
56. Gallagher RC, Pollard L, Scott AI, Huguenin S, Goodman S, Sun Q. Laboratory analysis
of organic acids, 2018 update: a technical standard of the American College of Medical
Genetics and Genomics (ACMG). Genet Med. 2018;20(7):683–91.
57. Ramautar R. Capillary electrophoresis-mass spectrometry for clinical metabolomics. Adv
Clin Chem. 2016;74:1–34.
58. Chouinard CD, Wei MS, Beekman CR, Kemperman RHJ, Yost RA. Ion mobility in clinical
analysis: current progress and future perspectives. Clin Chem. 2016;62(1):124–33.
59. Delvaux A, Rathahao-Paris E, Alves S. Different ion mobility-mass spectrometry coupling
techniques to promote metabolomics. Mass Spectrom Rev. 2022;41(5):695.
60. Dodds JN, Baker ES. Ion mobility spectrometry: fundamental concepts, instrumentation,
applications, and the road ahead. J Am Soc Mass Spectrom. 2019;30(11):2185–95.
61. Ibrahim YM, Hamid AM, Deng L, Garimella SVB, Webb IK, Baker ES, et al. New fron-
tiers for mass spectrometry based upon structures for lossless ion manipulations. Analyst.
2017;142(7):1010–21.
62. Paglia G, Astarita G. Metabolomics and lipidomics using traveling-wave ion mobility mass
spectrometry. Nat Protoc. 2017;12(4):797–813.
63. Dubland JA. Lipid analysis by ion mobility spectrometry combined with mass spectrometry:
a brief update with a perspective on applications in the clinical laboratory. J Mass Spectrom
Adv Clin Lab. 2022;23:7–13.
64. Fung AWS, Sugumar V, Ren AH, Kulasingam V. Emerging role of clinical mass spectrometry
in pathology. J Clin Pathol. 2020;73(2):61.
65. Strathmann FG, Hoofnagle AN. Current and future applications of mass spectrometry to the
clinical laboratory. Am J Clin Pathol. 2011;136(4):609–16.
66. Adaway JE, Keevil BG, Owen LJ. Liquid chromatography tandem mass spectrometry in the
clinical laboratory. Ann Clin Biochem. 2015;52(1):18–38.
67. Jannetto PJ, Fitzgerald RL. Effective use of mass spectrometry in the clinical laboratory. Clin
Chem. 2016;62(1):92–8.
68. Hines JM, Bancos I, Bancos C, Singh RD, Avula AV, Young WF, et al. High-resolution,
accurate-mass (HRAM) mass spectrometry urine steroid profiling in the diagnosis of adrenal
disorders. Clin Chem. 2017;63(12):1824–35.
69. Nuttall KL, Gordon WH, Ash KO. Inductively coupled plasma mass spectrometry for trace
element analysis in the clinical laboratory. Ann Clin Lab Sci. 1995;25(3):264–71.
70. Croxatto A, Prod’hom G, Greub G. Applications of MALDI-TOF mass spectrometry in clini-
cal diagnostic microbiology. FEMS Microbiol Rev. 2012;36(2):380–407.
71. Arentz G, Mittal P, Zhang C, Ho YY, Briggs M, Winderbaum L, et al. Chapter 2 - Applications
of mass spectrometry imaging to cancer. In: Drake RR, McDonnell LA, editors. Advances in
cancer research, vol. 134. New York: Academic Press; 2017. p. 27–66.
40 J. A. Dubland

72. Basu SS, Stopka SA, Abdelmoula WM, Randall EC, Gimenez-Cassina Lopez B, Regan MS,
et al. Interim clinical trial analysis of intraoperative mass spectrometry for breast cancer sur-
gery. NPJ Breast Cancer. 2021;7(1):116.
73. Mbughuni MM, Jannetto PJ, Langman LJ. Mass spectrometry applications for toxicology.
EJIFCC. 2016;27(4):272–87.
74. Szeremeta M, Pietrowska K, Niemcunowicz-Janica A, Kretowski A, Ciborowski
M. Applications of metabolomics in forensic toxicology and forensic medicine. Int J Mol
Sci. 2021;22(6):3010.
75. Plumb RS, Stumpf CL, Granger JH, Castro-Perez J, Haselden JN, Dear GJ. Use of liquid
chromatography/time-of-flight mass spectrometry and multivariate statistical analysis shows
promise for the detection of drug metabolites in biological fluids. Rapid Commun Mass
Spectrom. 2003;17(23):2632–8.
76. Steuer AE, Brockbals L, Kraemer T. Untargeted metabolomics approaches to improve case-
work in clinical and forensic toxicology—“where are we standing and where are we head-
ing?”. WIREs Forensic Sci. 2022;4(4):e1449.
77. Keen B, Cawley A, Reedy B, Fu S. Metabolomics in clinical and forensic toxicology, sports
anti-doping and veterinary residues. Drug Test Anal. 2022;14(5):794–807.
78. Al-Dirbashi OY, Santa T, Rashed MS, Al-Hassnan Z, Shimozawa N, Chedrawi A, et al. Rapid
UPLC-MS/MS method for routine analysis of plasma pristanic, phytanic, and very long chain
fatty acid markers of peroxisomal disorders. J Lipid Res. 2008;49(8):1855–62.
79. Valianpour F, Selhorst JJ, van Lint LE, van Gennip AH, Wanders RJ, Kemp S. Analysis
of very long-chain fatty acids using electrospray ionization mass spectrometry. Mol Genet
Metab. 2003;79(3):189–96.
80. Schwarz E, Liu A, Randall H, Haslip C, Keune F, Murray M, et al. Use of steroid profiling by
UPLC-MS/MS as a second tier test in newborn screening for congenital adrenal hyperplasia:
the Utah experience. Pediatr Res. 2009;66(2):230–5.
81. Janzen N, Sander S, Terhardt M, Steuerwald U, Peter M, Das AM, et al. Rapid steroid hor-
mone quantification for congenital adrenal hyperplasia (CAH) in dried blood spots using
UPLC liquid chromatography-tandem mass spectrometry. Steroids. 2011;76(13):1437–42.
82. Herman GE, Kratz L. Disorders of sterol synthesis: beyond Smith-Lemli-Opitz syndrome.
Am J Med Genet C Semin Med Genet. 2012;160c(4):301–21.
83. Storbeck K-H, Schiffer L, Baranowski ES, Chortis V, Prete A, Barnard L, et al. Steroid
metabolome analysis in disorders of adrenal steroid biosynthesis and metabolism. Endocr
Rev. 2019;40(6):1605–25.
84. Abu Bakar N, Lefeber DJ, van Scherpenzeel M. Clinical glycomics for the diagnosis of con-
genital disorders of glycosylation. J Inherit Metab Dis. 2018;41(3):499–513.
85. Forni G, Malvagia S, Funghini S, Scolamiero E, Mura M, Della Bona M, et al. LC-MS/MS
method for simultaneous quantification of heparan sulfate and dermatan sulfate in urine by
butanolysis derivatization. Clin Chim Acta. 2019;488:98–103.
86. Huang R, Cathey S, Pollard L, Wood T. UPLC-MS/MS analysis of urinary free oligosac-
charides for lysosomal storage diseases: diagnosis and potential treatment monitoring. Clin
Chem. 2018;64(12):1772–9.
87. Gelb MH, Lukacs Z, Ranieri E, Schielen P. Newborn screening for lysosomal storage dis-
orders: methodologies for measurement of enzymatic activities in dried blood spots. Int J
Neonatal Screen. 2019;5(1):1.
88. Aliu E, Kanungo S, Arnold GL. Amino acid disorders. Ann Transl Med. 2018;6(24):471.
89. Ferreira CR, Rahman S, Keller M, Zschocke J, Group IA. An international classification of
inherited metabolic disorders (ICIMD). J Inherit Metab Dis. 2021;44(1):164–77.
90. de Koning TJ. Amino acid synthesis deficiencies. J Inherit Metab Dis. 2017;40(4):609–20.
91. Phipps WS, Jones PM, Patel K. Chapter 2 - Amino and organic acid analysis: essential tools
in the diagnosis of inborn errors of metabolism. In: Makowski GS, editor. Advances in clini-
cal chemistry, vol. 92. Amsterdam: Elsevier; 2019. p. 59–103.
Mass Spectrometry-Based Metabolomics for the Clinical Laboratory 41

92. Berridge BJ Jr, Chao WR, Peters JH. Analysis of plasma and urinary amino acids by ion-­
exchange column chromatography. Am J Clin Nutr. 1971;24(8):934–9.
93. Spackman DH, Stein WH, Moore S. Automatic recording apparatus for use in chromatogra-
phy of amino acids. Anal Chem. 1958;30(7):1190–206.
94. Carling RS, McDonald BA, Austin D, Burden D, Correia J, Leung J, et al. Challenging the
status quo: a comparison of ion exchange chromatography with liquid chromatography-mass
spectrometry and liquid chromatography-tandem mass spectrometry methods for the mea-
surement of amino acids in human plasma. Ann Clin Biochem. 2020;57(4):277–90.
95. Waterval WAH, Scheijen JLJM, Ortmans-Ploemen MMJC, Habets-van der Poel CD, Bierau
J. Quantitative UPLC-MS/MS analysis of underivatised amino acids in body fluids is a reli-
able tool for the diagnosis and follow-up of patients with inborn errors of metabolism. Clin
Chim Acta. 2009;407(1):36–42.
96. Piraud M, Vianey-Saban C, Petritis K, Elfakir C, Steghens JP, Bouchu D. Ion-pairing reversed-­
phase liquid chromatography/electrospray ionization mass spectrometric analysis of 76
underivatized amino acids of biological interest: a new tool for the diagnosis of inherited dis-
orders of amino acid metabolism. Rapid Commun Mass Spectrom. 2005;19(12):1587–602.
97. Le A, Ng A, Kwan T, Cusmano-Ozog K, Cowan TM. A rapid, sensitive method for quantita-
tive analysis of underivatized amino acids by liquid chromatography-tandem mass spectrom-
etry (LC-MS/MS). J Chromatogr B Analyt Technol Biomed Life Sci. 2014;944:166–74.
98. Miller MJ, Cusmano-Ozog K, Oglesbee D, Young S, Committee ALQA. Laboratory analysis
of acylcarnitines, 2020 update: a technical standard of the American College of Medical
Genetics and Genomics (ACMG). Genet Med. 2021;23(2):249–58.
99. Millington DS, Stevens RD. Acylcarnitines: analysis in plasma and whole blood using tan-
dem mass spectrometry. Methods Mol Biol. 2011;708:55–72.
100. Peng M, Fang X, Huang Y, Cai Y, Liang C, Lin R, et al. Separation and identification of
underivatized plasma acylcarnitine isomers using liquid chromatography–tandem mass spec-
trometry for the differential diagnosis of organic acidemias and fatty acid oxidation defects.
J Chromatogr A. 2013;1319:97–106.
101. Körver-Keularts IMLW, Wang P, Waterval HWAH, Kluijtmans LAJ, Wevers RA, Langhans
C-D, et al. Fast and accurate quantitative organic acid analysis with LC-QTOF/MS facilitates
screening of patients for inborn errors of metabolism. J Inherit Metab Dis. 2018;41(3):415–24.
102. Nyhan WL. Disorders of purine and pyrimidine metabolism. Mol Genet Metab.
2005;86(1):25–33.
103. Monostori P, Klinke G, Hauke J, Richter S, Bierau J, Garbade SF, et al. Extended diagnosis
of purine and pyrimidine disorders from urine: LC MS/MS assay development and clinical
validation. PLoS One. 2019;14(2):e0212458.
104. Hartmann S, JrG O, Schmidt C, Langhans C-D, Garbade SF, Burgard P, et al. Comprehensive
detection of disorders of purine and pyrimidine metabolism by HPLC with electrospray ion-
ization tandem mass spectrometry. Clin Chem. 2006;52(6):1127–37.
105. Chace DH, Kalas TA, Naylor EW. Use of tandem mass spectrometry for Multianalyte screen-
ing of dried blood specimens from newborns. Clin Chem. 2003;49(11):1797–817.
106. Chace DH, Hannon WH. Impact of second-tier testing on the effectiveness of newborn
screening. Clin Chem. 2010;56(11):1653–5.
107. Gramer G, Hoffmann GF. Second-tier strategies in newborn screening – potential and limita-
tions. Med Genet. 2022;34(1):21–8.
108. Austin Pickens C, Isenberg SL, Cuthbert C, Petritis K. Combining first and second-tier new-
born screening in a single assay using high-throughput Chip-based capillary electrophoresis
coupled to high-resolution mass spectrometry. Clin Chem. 2021;67(12):1709–20.
109. Jacob M, Malkawi A, Albast N, Al Bougha S, Lopata A, Dasouki M, et al. A targeted metab-
olomics approach for clinical diagnosis of inborn errors of metabolism. Anal Chim Acta.
2018;1025:141–53.
42 J. A. Dubland

110. Miller MJ, Kennedy AD, Eckhart AD, Burrage LC, Wulff JE, Miller LA, et al. Untargeted
metabolomic analysis for the clinical screening of inborn errors of metabolism. J Inherit
Metab Dis. 2015;38(6):1029–39.
111. U.S. Department of Health and Human Services FaDA, Center for Drug Evaluation and
Research (CDER), Center for Veterinary Medicine (CVM), editor. Bioanalytical method
validation: guidance for industry; 2018.
112. CLSI. Liquid chromatography-mass spectrometry methods; approved guideline. CLSI docu-
ment C62-A. Wayne, PA: Clinical and Laboratory Standards Institute; 2014.
113. Wu AHB, French D. Implementation of liquid chromatography/mass spectrometry into the
clinical laboratory. Clin Chim Acta. 2013;420:4–10.
114. Rappold BA. Review of the use of liquid chromatography-tandem mass spectrometry in clini-
cal laboratories: part I-development. Ann Lab Med. 2022;42(2):121–40.
115. Naz S, Vallejo M, García A, Barbas C. Method validation strategies involved in non-targeted
metabolomics. J Chromatogr A. 2014;1353:99–105.
116. Simón-Manso Y, Lowenthal MS, Kilpatrick LE, Sampson ML, Telu KH, Rudnick PA, et al.
Metabolite profiling of a NIST standard reference material for human plasma (SRM 1950):
GC-MS, LC-MS, NMR, and clinical laboratory analyses, libraries, and web-based resources.
Anal Chem. 2013;85(24):11725–31.
117. Bowden JA, Heckert A, Ulmer CZ, Jones CM, Koelmel JP, Abdullah L, et al. Harmonizing
lipidomics: NIST interlaboratory comparison exercise for lipidomics using SRM 1950–
metabolites in frozen human plasma. J Lipid Res. 2017;58(12):2275–88.
118. Beger RD. Interest is high in improving quality control for clinical metabolomics: setting
the path forward for community harmonization of quality control standards. Metabolomics.
2018;15(1):1.
119. Liu KH, Nellis M, Uppal K, Ma C, Tran V, Liang Y, et al. Reference standardization for quan-
tification and harmonization of large-scale metabolomics. Anal Chem. 2020;92(13):8836–44.
120. Ulmer CZ, Maus A, Hines J, Singh R. Challenges in translating clinical metabolomics data
sets from the bench to the bedside. Clin Chem. 2021;67(12):1581–3.
121. Gabelica V, Shvartsburg AA, Afonso C, Barran P, Benesch JLP, Bleiholder C, et al.
Recommendations for reporting ion mobility mass spectrometry measurements. Mass
Spectrom Rev. 2019;38(3):291–320.
Metabolomics: A Pipeline for Biomarker
Discovery in Genetic Diseases

Lina A. Dahabiyeh and Refat M. Nimer

Abstract Genetic disorders (GD) affect hundreds of millions of lives worldwide,

leading to specific molecular perturbations in the metabolic profile within a certain
biological matrix. Metabolomic studies use advanced technologies (nuclear mag-
netic resonance (NMR) and mass spectrometry (MS)) with bioinformatics to iden-
tify and quantify a set of small molecules (such as carbohydrates, nucleic acids,
amino acids, and lipids) present in a biological system. As metabolites represent the
downstream product of gene and protein activity, they most likely reflect the pheno-
type of an organism at a specific time. Metabolomic studies provide novel insights
into the underlying disease pathophysiological mechanisms, evaluate the progress
of the disease, and identify unique biomarkers for the prediction of disease and
therapeutic outcomes. This chapter highlights the applications of metabolomic tech-
niques for biomarker discovery in GD. It discusses the workflow followed, the
methods used for sample analysis and data interpretation, and the major challenges
and limitations in applying the metabolomic approach for biomarker discovery in
GD. Moreover, the chapter provides an overview of the biomarkers identified in
four GD: cystic fibrosis, Down syndrome, sickle cell anemia, and glycogen storage
disorders, highlighting the promising role of metabolomics in clinical applications.

Keywords Biomarker · Genetic diseases · Cystic fibrosis · Metabolomics · Mass

spectrometry · NMR · Down syndrome

L. A. Dahabiyeh (*)
Department of Pharmaceutical Sciences, School of Pharmacy, The University of Jordan,
Amman, Jordan
e-mail: [Link]@[Link]
R. M. Nimer (*)
Department of Medical Laboratory Sciences, Jordan University of Science and Technology,
Irbid, Jordan
e-mail: rmnimer@[Link]

© The Author(s), under exclusive license to Springer Nature Singapore Pte 43

Ltd. 2023
A. M. Abdel Rahman (ed.), Clinical Metabolomics Applications in Genetic
Diseases, [Link]
44 L. A. Dahabiyeh and R. M. Nimer

Abbreviations

BALF Bronchoalveolar lavage fluid

CF Cystic fibrosis
CFTR Cystic fibrosis transmembrane conductance regulator
CID Collision-induced dissociation
DBS Dried blood spot
DDA Data-dependent acquisition
DIA Data-independent acquisition
EBC Exhaled breath condensate
GC Gas chromatography
GD Genetic disorders
GSDs Glycogen storage disorders
LOD Limit of detection
LOQ Limit of quantification
MS Mass spectrometry
NMR Nuclear magnetic resonance
OPLS-DA Orthogonal partial least squares-discriminant analysis
PCA Principal component analysis
PLS-DA Partial least squares-discriminant analysis
SCD Sickle cell disease
VIP Variable importance in projection

1 Introduction

Hundreds of millions of lives are affected by an estimated 10,000 unique genetically

determined diseases [1]. Genetic disorders (GD) refer to any disease caused by
mutations in one or more genes. Although GDs are individually rare, they account
for approximately 80% of rare disorders [2]. Additionally, genetic material is con-
sidered a risk factor for several frequent complex multifactorial disorders, including
cancer, asthma, heart disease, and diabetes [2, 3]. As with any other illness, GD will
result in specific alterations in the profile of the biological molecules within a cer-
tain biological matrix, such as biofluids, cells, and tissues. Measurements of these
biomolecules can be used to identify disease biomarkers and aid in understanding
the underlying molecular mechanisms of the disease.
The last few decades have witnessed increasing interest in omics studies: genom-
ics, transcriptomics, proteomics, and metabolomics (Fig. 1). They represent
advanced and promising measurement approaches for disease biomarker discovery.
Metabolomics is the global identification and quantification of a set of small mole-
cules, less than 1500 Da (such as carbohydrates, nucleic acids, amino acids, and
lipids) present in a biological system [4]. As metabolites represent the downstream
product of gene and protein activity, they most likely reflect the phenotype of an
organism at a specific time [5]. Metabolomic studies provide novel insights into the
Metabolomics: A Pipeline for Biomarker Discovery in Genetic Diseases 45

Fig. 1 The four omics sciences genomics, transcriptomics, proteomics, and metabolomics in sys-
tems biology approach. Metabolomics represents the downstream output of the genome, and
together with environmental factors and the microbiome, it can reflect the phenotype of an
organism

underlying disease pathophysiological mechanisms, evaluate the progress of the

disease, and identify unique biomarkers for the prediction of disease and therapeutic
outcomes [6–10].
Global metabolomic studies use advanced technologies with bioinformatics.
Two main analytical technologies are employed: nuclear magnetic resonance
(NMR) and mass spectrometry (MS) [8, 11]. Advances in MS coupled with chroma-
tography (LC-MS) have improved the efficiency and reliability of metabolite profil-
ing. Over the years, LC-MS-based metabolomics has witnessed tremendous
improvements in sensitivity, mass resolution, metabolome coverage, and data pro-
cessing, enabling reliable identification of diverse metabolites even within complex
biological samples [12]. The application of metabolomics in biomedical research
has identified biochemical pathways and has discovered diverse sets of potential
biomarkers that have enhanced the understanding of the molecular mechanisms in
several diseases and aided in their diagnosis, including diabetes [13, 14], cancer [15,
16], and GD [17]. A biomarker is a biomolecule (i.e., gene, protein, or metabolite-­
based substance) that indicates an abnormal condition within a subject. Biomarkers
can be used in clinical and medical settings to determine specific disorders (known
as diagnostic biomarkers), monitor disease progression (prognostic biomarkers),
indicate probable response to therapy (predictive biomarkers), and indicate the risk
46 L. A. Dahabiyeh and R. M. Nimer

of developing a disease (predisposition biomarkers) [18]. Metabolomics allows the

identification of tens of potential biomarkers that need to undergo analytical (with
other analytical platforms such as polymerase chain reactions (PCR) or immune
affinity-based assays) and clinical validations before being approved to be used in
the clinic. Metabolome profiling of biofluids can be combined with whole-genome
profiling of single nucleotide polymorphisms to identify many gene-metabolite
associations simultaneously in metabolomic genome-wide association studies [19].
This chapter highlights the applications of metabolomic techniques for bio-
marker discovery in GD. The chapter discusses the workflow followed and the
methods used for sample analysis and data interpretation in the metabolomic
approach. Moreover, the chapter provides an overview of the most interesting recent
biomarker discovered in the most frequent GD, such as cystic fibrosis, Down syn-
drome, and sickle cell anemia, highlighting the promising role of metabolomics in
clinical applications. The current challenges and limitations in biomarker discovery
in GD using the metabolomic approach will also be covered.

2 Sample Preparation in Metabolomics

Careful consideration of different aspects of metabolomic studies, such as study

design, is vital to ensure high-quality data and reproducible biomarker generation.
Following standardized protocols during sample collection and processing and
using validated analytical methods to analyze samples will result in robust and
reproducible analyses [20].
Experimental design in metabolomic studies may differ depending on the study’s
objective, available resources, and the type of biological sample. However, the gen-
eral experimental workflow is the same. The typical workflow in metabolomics
includes sample preparation and metabolite extraction, metabolite separation using
chromatography (liquid (LC) or gas chromatography (GC)), metabolite analysis
using MS or NMR spectroscopy, metabolite identification, and data processing
using uni- and multivariate analyses for the discovery of potential biomarkers
(Fig. 2).

2.1 Sample Collection

In metabolomic experiments, at least two samples (two conditions) are typically

compared, one being the control (or reference) group. Biospecimens used for bio-
marker discovery are preferably collected from large case-control studies or cohorts
with defined inclusion and exclusion criteria and complete information on the clini-
cal and demographic characteristics, as much as possible.
Poorly defined groups, non-matched confounding factors, or heterogeneous
samples are potential pitfalls in the study design and may negatively affect the study
Metabolomics: A Pipeline for Biomarker Discovery in Genetic Diseases 47

Fig. 2 Workflow followed in untargeted metabolomic analysis

outcomes. Poor study design during sample collection is one of the reasons that
might explain the poor progression of discovered biomarkers toward clinical appli-
cability [18].

2.2 Sample Type

Biomarker research aims to discover a biomarker using a noninvasive (e.g., urine,

tears, saliva) or minimally invasive (e.g., blood and serum or plasma) sample and, if
possible, try to avoid invasive samples (e.g., tissue). Blood is the most commonly
used biofluid in metabolomic studies since it is easy to collect (compared to tissues).
Its composition is stable and reflects the state of the body at the time of collection
[21]. Blood is considered the most commonly used sample type in many GD,
including cystic fibrosis (CF) and sickle cell disease [22–24]. Serum, plasma, sweat,
dried blood spots, and epithelial cell cultures from cohorts of patients with CF [24–
28] were used to identify novel metabolic abnormalities associated with CF and
discover a panel of potential biomarkers for the disease. Metabolomic studies
involving Down syndrome, the most common human chromosomal aberration,
mainly used amniotic fluid from fetuses with Down syndrome [29, 30]. Cultured
fibroblasts and liver tissues were used in metabolomic studies conducted on glyco-
gen storage disease (GSD) and glucose-6-phosphatase deficiency mouse models,
respectively [31, 32]. Inherited genetic mutations play a major role in about 5–10%
48 L. A. Dahabiyeh and R. M. Nimer

of all cancers. Many metabolomic studies on cancer patients mainly used tissue
specimens to monitor disease progression, identify different disease stages, and dis-
cover biomarker candidates in breast and colon cancers [33–35].

2.3 Metabolite Extraction

The matrix of the biological sample has a certain level of interferences (such as salts
and proteins) that can affect the operation of the analytical instrument. Therefore,
sample preparation is crucial in metabolomic analyses and might be a major source
of variability [36]. To ensure informative and accurate metabolite profile outcomes
in global metabolomics, the sample preparation method should be (a) unselective to
maximize metabolite coverage, (b) sensitive and compatible with the analytical
approach followed, (c) simple and fast to avoid metabolite degradation and mini-
mize variability, (d) reproducible, and (e) include a metabolism quenching step to
stop any biochemical reactions after the time of sampling [37].
In the metabolomic study of serum or plasma, proteins will be precipitated first
with organic solvent (such as methanol, acetonitrile, and acetone or a combination),
followed by metabolite extraction. Among the most commonly used extraction
methods are (a) liquid-liquid separation (with specified ratios of solvents), where
metabolites of interest are separated into an immiscible solvent, (b) using a column
or solid-phase extraction (SPE) approach to trap the metabolites, and (c) selective
solubilization [38, 39]. Analysis involving GC-MS will typically include a step of
derivatization before metabolic profiling. Among the solvents used in extraction,
methanol was the best in metabolite coverage and method reproducibility [39]. On
the other hand, sample preparation for NMR analysis is much simpler than MS-based
approaches and requires a deuterated solvent and a chemical shift reference.
Metabolite extraction during metabolite profiling using the LC-MS approach in CF,
Down syndrome, and sickle cell disease mainly employed a solvent extraction
method [23, 29, 40, 41]. It offers a simple and rapid extraction approach with excel-
lent metabolome coverage. However, the latter highly depends on the solvents used
[37]. To ensure system stability and aid in metabolite identification, stable isotope-­
labeled quality control internal standards are added during sample preparation, as in
the metabolite profiling of sickle cell disease patients [22].

3 Technologies Used in Metabolomics

Current technologies used in metabolomic research involve MS and NMR

spectroscopy.
Metabolomics: A Pipeline for Biomarker Discovery in Genetic Diseases 49

3.1 Mass Spectrometry (MS)

Mass spectrometry (MS) is an indispensable analytical tool and a vital technology

in metabolomic studies. It is widely applied due to its high sensitivity (typically at
the pg level) and fast data acquisition speed. During the last decades, MS has moved
into the front line of metabolomic research. Several well-established methods have
identified potential biomarkers in GD from blood plasma-derived samples, cells,
and tissues [29, 32, 34, 42, 43]. Advances in the MS analysis were achieved due to
enhancements in the sensitivity, ionization, mass accuracy, and the high-throughput
capabilities of different mass analyzers and the hyphenation of MS to different sep-
aration techniques, such as LC and GC, to allow for the analysis of complex biologi-
cal samples. Common MS analyzers include quadrupole (Q; acts as a mass filter),
high-resolution accurate mass analyzers, time-of-flight (TOF), and Orbitraps. To
provide quality assurance for MS analysis and data acquisition, quality control (QC)
samples, prepared from pooling aliquots of all samples to be analyzed, are random-
ized for analysis among the study samples [44]. Another approach used to compen-
sate for the effect of ion suppression and increase the reliability of the generated
data is to spike the samples with stable isotope-labeled internal standard structural
analogs as internal standards, which is mainly applied in the targeted metabolomics
[45, 46].

3.2 Nuclear Magnetic Resonance (NMR) Spectroscopy

NMR spectroscopy, including 1H NMR, is used to profile metabolites for biomarker

discovery and develop metabolic fingerprints for disease diagnosis and monitoring
response to treatment due to its fundamental quantitative nature. NMR-based
metabolomics has been applied to profile plasma and urine samples from subjects
with Down syndrome and normal controls [47, 48] and plasma samples from sickle
cell disease patients with normal albuminuria and patients with moderately or
severely increased albuminuria [49]. The same approach was used to identify
plasma metabolic phenotypes of children with autism spectrum disorder, idiopathic
developmental delay, and Down syndrome compared to typically developed con-
trols [50]. Unlike MS, NMR allows structural verification/identification of known
and unknown metabolites and quantitation from the same measurement, requiring
minimal sample preparation. However, NMR methods have lower sensitivity,
dynamic range, and metabolite coverage when compared to LC-MS-based meth-
ods [12].
50 L. A. Dahabiyeh and R. M. Nimer

4 Metabolite Identification and Statistical Analysis

Advanced software combined with rich databases highly impacted advancement in

metabolomic research. Typically, acquired data will be cleaned up to remove artifact
peaks or peaks with poor repeatabilities, such as peaks detected in less than 50% QC
samples or peaks with high variability (e.g., CV > 30% in QC). Normally, metabo-
lite identification is performed by matching accurate masses of the detected peaks
with metabolite in specialized databases and libraries, the retention times (RT) of
authentic standards, and/or the MS/MS fragmentation database [41]. The confi-
dence in metabolite identification in GD follows the general recommendations
based on the Chemical Analysis Working Group Metabolomics Standards Initiative
recommendation, where the confidence is assigned as level 1–4 (L1–4) [51, 52].
Different metabolomic studies in GD used multivariate analysis and univariate
analysis to identify potential biomarkers of the disease [28–30, 47, 49]. For multi-
variate analysis, imported datasets are Pareto scaled, and principal component anal-
ysis (PCA), partial least squares-discriminant analysis (PLS-DA), and orthogonal
partial least squares-discriminant analysis (OPLS-DA) are used for modeling the
differences between the study groups. The robustness of the generated models is
monitored by the fitness of the model (R2X) for PCA (R2Y) for PLS-DA and
OPLS-DA and predictive ability (Q2) values. The model yielding large R2X and
R2Y (close to 1) and Q2 values of ~0.5 indicates a robust model [53]. Mass ions are
responsible for the class separation between diseased and control groups specified
using variable importance in projection (VIP) of the generated PLS-DA or OPLS-DA
model. Metabolites with VIP scores above 1.0 are considered important for the
model and responsible for differentiating between samples [54]. An unpaired two-­
tailed Student’s t-test is used for univariate analysis to identify significantly altered
metabolites between the two compared groups. False discovery rate (FDR)-corrected
p-value of less than 0.05 is considered significant.

5 Approaches Followed in Metabolomics

for Biomarker Discovery

The key to discovering new biomarkers depends on the quantification, relative or

absolute, of the changes in the levels of the metabolites in the study versus control
samples. Two main approaches can be applied in metabolomic analyses: untargeted
metabolomics (sometimes referred to as “shotgun metabolomics”) and targeted
[55, 56].
Metabolomics: A Pipeline for Biomarker Discovery in Genetic Diseases 51

5.1 Untargeted Approach

Global untargeted metabolomics aims to capture all metabolites in a specific bio-

logical sample. In MS-based metabolomics, data can be acquired either in data-­
dependent acquisition (DDA) or data-independent acquisition (DIA). DDA is the
classic LC-MS/MS method for scanning metabolites in metabolomic profiling
experiments and generating further MS information for identification. In this
approach, MS measures ions in duty cycles. A quick MS1 (the first mass analyzer)
survey scan for all the detectable ions will be performed in each duty cycle. Second,
the top ions with the highest intensities will be selected for MS2 (the second mass
analyzer) analyses. Third, a series of MS2 scans will be done for fragment ions of
each preselected target ion [57]. DIA has gained increased interest in recent years
due to the advancement in computer algorithms and speed. This data acquisition
does not rely on the MS1 survey scans to detect top precursor ions for fragmentation
and subsequent product ion scan. Although DIA can increase MS/MS information,
it cannot generate high-quality precursor-specific MS2 spectra [58]. For untargeted
global metabolomics, high-resolution accurate-mass MS instruments, such as TOF
and Orbitrap coupled to quadrupole (Q) mass filter, are preferred [44]. Knowledge
of accurate masses facilitates ion identification across different samples using online
or commercially available databases.
Most of the metabolomic studies applied in GD used an untargeted approach for
biomarker discovery. LC coupled with a QTOF mass spectrometer with DDA or
DIA was used to profile metabolites of the amniotic fluid of fetuses with Down
syndrome [30], the amniotic fluid of women carrying a fetus with Down syndrome
[29], and blood from 292 participants with Down syndrome [40]. On the other hand,
a hybrid Q-Orbitrap mass spectrometer was used to identify plasma metabolites
implicated in sickle cell disease clinical heterogeneity [22]. Metabolomic discovery
untargeted screening was also used to identify metabolites associated with neutro-
philic inflammation in bronchoalveolar lavage fluid (BALF) supernatant from pre-
school children with CF [59] and to identify perturbed metabolites in human sickle
erythrocytes compared with human non-sickle erythrocytes [60, 61].
Although identifying specific biomarker candidates is the ultimate goal of the
untargeted metabolomic approach, this approach will need better reproducibility
and the detection of a high number of false positives in the presence of deficiencies
in the experimental design. The latter will have a negative impact on the reproduc-
ibility of the method. Therefore, it is essential to have quality control over the ana-
lytical method by (a) randomization of the sequence of the samples, (b) analysis of
blank and pooled QC samples, (c) monitoring mass accuracy of internal standards
during the run, and (d) checking retention time and peak intensity for spiked inter-
nal standards [62, 63].
52 L. A. Dahabiyeh and R. M. Nimer

5.2 Targeted Approach

The targeted analysis will focus on a specific set of defined metabolites. Hence, it
offers an improved limit of detection (LOD) and limit of quantification (LOQ) com-
pared to global metabolomic methods. The approach can provide absolute quantifi-
cation (when analyzed with standards) of tens-hundreds of metabolites
simultaneously in a highly specific and sensitive manner. Triple quadruple MS
instruments are the preferred analyzers in targeted quantification as they offer high
sensitivity, particularly when running multiple reaction monitoring (MRM) modes
[44, 64]. MRM requires the selection of precursor-to-fragment transitions; MS1
(Q1) will selectively filter ions of a particular mass over charge m/z (within a certain
resolution) corresponding to the intact targeted metabolite. The precursor ions are
then subjected to fragmentation using collision-induced dissociation (CID) in a col-
lision cell (q2). Finally, the fragment ions of the target analyte with high ion inten-
sity and specificity are filtered through MS2 (Q3), generating a signal of the MRM
transition [65].
Due to its high specificity and sensitivity, MS-based targeted metabolomics can
be used to quantify low-abundance metabolites. It can also be applied for verifying
and validating candidate biomarkers, especially in easily accessible blood samples
and body fluids. It is an essential step for translating disease biomarkers into clinical
practice. Targeted MS metabolomics was employed to quantify extra- and intracel-
lular metabolites in cultured fibroblasts from healthy controls and patients with the
GSD Ia, GSD Ib, and GSD III [32], relevant metabolites in BALF and blood sam-
ples (spiked with isotopically labeled internal standard) from children with CF [27,
59, 66, 67], and plasma metabolites of normal individuals and patients with sickle
cell disease [23].

6 Biomarker Discovery and Validation

A biomarker is a characteristic that can be objectively evaluated and measured as an

indicator of a normal biological process, a pathogenic process, or a pharmacologic
response to treatment [68]. Biomarkers are essential to early disease detection and
provide useful predictive and monitoring information for more precise diagnoses
and successful patient treatment and management. With the pivotal roles of bio-
markers in clinical applications and the recent development of new technologies,
biomarker discovery has been a topic of intensive research [69].
Metabolomics is the closest biomolecule to the phenotype. Therefore, metabo-
lome analysis may be useful for identifying diagnostic and predictive biomarkers.
With the growing availability of metabolome analysis, researchers’ attention has
shifted toward analyzing biofluids and tissues to find alterations in metabolites that
can potentially be novel biomarkers of diseases. The general premise was relatively
straightforward: identify as many metabolites as possible in a particular kind of
Metabolomics: A Pipeline for Biomarker Discovery in Genetic Diseases 53

biofluid obtained from diseased patients and compare them to those found in healthy
individuals matched for age, sex, and other factors. Once potential candidates are
identified; the key step is validation, which produces disease biomarkers for even-
tual clinical applications.
Metabolomic biomarker discovery has led to identifying numerous potential
candidates for disease diagnosis, prognosis, and prediction of response to therapy.
The field of metabolomics has significantly developed over the last 10–15 years.
However, very few potential biomarkers discovered have been validated clinically
and are regularly utilized in clinical practice [70]. One challenge that has delayed
the translation of the most promising biomarker candidates into a clinical setting is
validation [71, 72]. Due to the large physicochemical varieties of the metabolome,
validating metabolite characteristics is still regarded as a bottleneck [4]. Potential
biomarkers should be further validated with large-scale studies [69]. However, one
of the obstacles in biomarker validation is the limited access to large and indepen-
dent cohort samples [72].
At the discovery level, metabolomic technologies permit only a few (20–100)
samples to be analyzed in each class sampled from two independent populations.
Then, the study is repeated at the internal validation stage to validate the previous
findings from the discovery study. This step is needed to see if the proposed bio-
marker can distinguish disease states in a cohort similar to the discovery cohort. In
the external validation stage, a larger sample size in the hundreds to thousands is
analyzed; however, only a small number (e.g., one to ten) of metabolites are mea-
sured [73, 74]. One crucial issue often overlooked in biomarker development and
validation is proper patient recruitment, simply because more samples need to be
analyzed in the biomarker validation stage than in the biomarker discovery stage.
During external validation, the biomarker must be evaluated in a population typical
of the population for whom the test is designed; thus, ethnicity and place of origin
should be considered. In addition, a suitable control group for the biomarker pur-
pose must also be established [70]. Notably, the test and control groups must have
precisely the same collection conditions with similar demographics. Confounding
variables, such as sex and age, should always be matched; other confounders, such
as experimental conditions, diet, and drug interactions, should also be controlled
during the discovery and validation of a metabolomic biomarker [75, 76].
After the discovery phase, targeted studies can be carried out to validate the
results. However, in patients with nonspecific phenotypes, untargeted metabolomics
shows promise as a validation tool for variants of unknown significance in candidate
genes for genetic disorders [77]. Moreover, biomarker validation studies can be
performed using affinity multiplexing assays [78]. In a second independent study,
NMR spectroscopy was used to validate metabolites from exhaled breath conden-
sate of patients with unstable CF, stable CF, and healthy subjects [79]. Purine
metabolism and protein catabolism pathways have been found as biomarkers for
neutrophilic airway inflammation in CF patients.
Furthermore, these multiple metabolic pathways were validated by an indepen-
dent cohort [80]. Calprotectin and urinary glucose tetrasaccharide for hepatic GSDs
and Pompe disease are examples of biomarkers approved for clinical use in
54 L. A. Dahabiyeh and R. M. Nimer

GD. Nevertheless, most of the reported biomarkers for GSD were performed with
few patients and needed clinical validation to transfer to routine use [81].

7 Application of Metabolomics in Disease Biomarker

Discovery in Genetic Disorders

One of the greatest and ongoing challenges in genetics is the ability to predict the
phenotypes of individuals from their genotypes. In GD, the genetic variants may
alter an individual’s metabolome; this link between genetic variants with changes in
the metabolome may help predict novel phenotypes [82]. Metabolomics differs
from other “omics” sciences in linking gene and environmental interactions. As a
result, scientists may investigate gene-environment interactions by studying metab-
olites and metabolism [83, 84].
Our focus in this chapter will be on cystic fibrosis, Down syndrome, sickle cell
anemia, and glycogen storage disorders.

7.1 Cystic Fibrosis

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the

gene that codes for the cystic fibrosis transmembrane conductance regulator (CFTR)
[85]. More than 2000 mutations have been identified and reported in the CFTR1
(CF Mutation Database) [86]. CF is a disease that affects various organ systems
ranging from lung illness to liver disease and poor response to current treatments
[87]. Although CF-causing mutations in the CFTR gene are known, there is signifi-
cant clinical variability in phenotype level, and the etiology of many symptoms is
unknown. Therefore, metabolic investigations were carried out to provide a deep
understanding of how CFTR mutations cause disease consequences and aid in find-
ing novel biomarkers [17]. A summary of selected studies and their main findings is
presented in Table 1.
In a study, Quinn et al. [88] compared metabolite levels in the sputum of partici-
pants known to have CF with exacerbation during treatment and posttreatment and
in stable groups [88]. They utilized LC-MS/MS to evaluate the impact of exacerba-
tions on the sputum metabolome. Platelet-activating factor (PAF) and a related
monacylglycerophosphocholine lipid were identified as possible biomarkers of cys-
tic fibrosis pulmonary exacerbations (CFPE) [88]. Targeted metabolomic profiling
on 39 dried blood spot (DBS) samples identified sorbitol as an important indicator
for the mucoviscidosis seen in patients with CF. Esther et al. [59] conducted a study
with 20 BALF samples analyzed by LC-MS/MS and GS-MS and then validated 34
extra BALF samples with targeted MS [59]. They verified that metabolites associ-
ated with adenyl purine metabolism, dipeptides, cellular energy, and lipids
Metabolomics: A Pipeline for Biomarker Discovery in Genetic Diseases 55

Table 1 A summary of the literature on applying metabolomics for biomarker discovery in CF

Sample
Sample Method size Main findings References
Sputum LC-MS/MS 11 Platelet-activating factor (PAF) and a Quinn
related et al. [88]
monacylglycerophosphocholine lipid
are elevated in CF patients with
pulmonary exacerbations
DBS GC/MS and 69 26 metabolites were significantly Al-Qahtani
LC-MSMS differentially expressed and et al. [27]
characterized by amino acid,
glycolysis, mitochondrial,
peroxisomal metabolism, and sorbitol
pathways
CF patients have a significantly lower
level of osmolyte (sorbitol) than
healthy individuals, indicating that
their sorbitol pathway is disturbed,
which may explain the mucoviscidosis
found in those with CF
BALF GC/MS and 20/ 34 for Early structural lung disease was Esther
LC-MSMS validation predicted by findings that involved et al. [59]
the catabolism of proteins, oxidative
stress, and the metabolism of
adenosine products. The enzymes
associated with adenosine metabolism
were also elevated in those with early
disease samples. Metabolites and
pathways altered with neutrophilic
inflammation and destructive lung
disease
Serum A chemical 69 Metabolites changed between CF Masood
isotope labeling mutational classes II–VI and III– et al. [66]
liquid IV. The highly sensitive biomarkers
chromatography-­ for CF, 3,4-dihydroxymandelate-3-O-
mass sulfate and 5-aminopentanoic acid,
spectrometry were found in all three analyses
Serum GC/MS and 62 Several pathways were found to be Joseloff
LC-MSMS different in CF, indicating decreased et al. [26]
activity in the oxidation of fatty acids
CF has lower ketone bodies, lower
medium chain carnitines, higher
dicarboxylic acids, and lower
2-hydroxybutyrate from amino acid
metabolism
(continued)
56 L. A. Dahabiyeh and R. M. Nimer

Table 1 (continued)
Sample
Sample Method size Main findings References
Primary Untargeted Three The levels of purine nucleotides were Wetmore
human metabolomic/ separate significantly reduced in CF cells. et al. [25]
airway UHLC/MS/MS cohorts Decreased glucose metabolism in CF
epithelial cells may exacerbate oxidative stress
cell and limit epithelial cell response to
cultures environmental stress
DBS Nontargeted 152 N-glycated amino acids, oxidized DiBattista
capillary glutathione disulfide, and et al. [89]
electrophoresis-­ nicotinamide were differentially
mass expressed in normal birth weight CF
spectrometry neonates without meconium ileus
(CE-MS) compared to the control group
Sputum Untargeted LC/ 34 Pseudomonas aeruginosa Moyne
HRMS “metabotypes,” antibiotic resistance et al. [90]
and virulence phenotypes, and
clinical exacerbations were
significantly associated
Sweat Nontargeted 68 Several metabolites associated with Macedo
capillary asymptomatic CF infants were et al. [28]
electrophoresis-­ identified in sweat, including
mass asparagine and glutamine. Both
spectrometry pilocarpic acid, a synthetic sweat
(CE-MS) stimulant, and mono(2-ethylhexyl)
phthalic acid, a natural sweat
stimulant, were secreted in
significantly lower concentrations in
CF infants than in unaffected CF
screen positive controls
Exhaled UPLC-MS 35 4-hydroxycyclohexylcarboxylic acid Zang et al.
breath and pyroglutamic acid were used to [92]
condensate distinguish acute pulmonary
exacerbation (APE) samples from
stable CF samples. Lactic acid and
pyroglutamic acid accurately
distinguished pre-APE samples from
stable CF samples and matched the
APE signature when projected onto
the APE vs. stable CF model.
Post-APE samples had a metabolomic
signature more similar to stable CF
samples
Serum UPLC-MS 30 Tryptophan-kynurenine, nitric oxide, Muhlebach
bile acids, and microbial-derived et al. [41]
amino acid metabolites were altered
in serum to distinguish the pre- from
post-exacerbation state
Metabolomics: A Pipeline for Biomarker Discovery in Genetic Diseases 57

Table 1 (continued)
Sample
Sample Method size Main findings References
BALF NMR 11 Alteration in amino acids and lactate Wolak
may help distinguish the high- from et al. [91]
the low-inflammation groups
Plasma UPLC-MS 52 Fatty acid, amino acid, and Alvarez
carbohydrate metabolism differed et al. [24]
between baseline vitamin D and
placebo-treated CF patients
The amino acid pathways in CF
patients treated with vitamin D3
versus placebo were altered. Several
tricarboxylic acid cycle intermediates
increased, while amino acid-related
metabolites decreased in the placebo
group but not in the vitamin D3 group
Exhaled GC-TOF-MS 105 Volatile organic compounds (VOCs) Robroeks
breath such as C16 polyunsaturated et al. [93]
hydrocarbon in the exhaled breath of
CF patients can distinguish between
CF and non-CF patients and between
CF patients with and without
Pseudomonas

pathways are altered with neutrophilic inflammation and destructive lung disease
[59]. Metabolites related to amino acids, di-, and tri-peptides, glutathione, gluta-
mine, glutamate, and arginine metabolism pathways have also been described as
potential biomarkers among the CF mutational classes (II–VI) and between the
class III and IV through analyses of serum samples by chemical isotope-labeled
MS-based metabolomic approach [66]. Joseloff et al. [26] applied LC-MS/MS and
GC-MS in two groups of children with CF and non-CF lung disease. They reported
an alteration in cellular energy metabolism in CF, potentially reflecting mitochon-
drial dysfunction, which may be useful in differentiating CF from non-CF lung
diseases [26]. Metabolomic profiling showed differences in CF and non-CF primary
lung epithelial cells [25]. The levels of glucose, sorbitol, glycerophosphocholine,
and various glycolytic intermediates, including glucose 6-phosphate, fructose
6-phosphate, and lactate, were significantly reduced in CF cells compared with the
non-CF cells [25].
Many studies have revealed that different biomarkers, based on metabolite pro-
files, could be detected in a range of biological samples, including blood [89], spu-
tum [88, 90], BALF [80, 91], exhaled breath condensate (EBC) [92, 93], cell culture
[25], sweat [28], plasma [24], and serum [26, 41, 66]. Biomarkers in the polyamine
(e.g., putrescine, spermidine) metabolism highlighted significant associations
between Pseudomonas aeruginosa “metabotypes,” expression of antibiotic resis-
tance and virulence phenotypes, and frequency of clinical exacerbations in CF
patients [90]. The level of ophthalmic acid was found to be downregulated, along
58 L. A. Dahabiyeh and R. M. Nimer

with amino acids (serine, threonine, proline, and glycine) present in neonatal DBS
[89]. Mono(2-ethylhexyl) phthalic acid in sweat samples from positive CF infants
was differentially downregulated compared to non-CF [28]. Zang et al. [92] reported
a distinctive profile for CF in exhaled breath condensate, including two metabolic
discriminant characteristics, 4-hydroxycyclohexylcarboxylic acid and pyroglutamic
acid, between APE and stable CF samples. Moreover, lactic acid and pyroglutamic
acid were found to differentiate stable CF from pre-APE [92].

7.2 Down Syndrome

Down syndrome (DS) is the most common chromosomal-related GD. The presence
of an extra-human copy of chromosome 21 characterizes DS. DS patients are char-
acterized by the facial appearance and various complications, including intellectual
disabilities, mental and growth retardation, vision problems, hearing loss, infec-
tions, hypothyroidism, blood disorders, and cardiovascular abnormalities [94, 95].
Even though the anatomical and physiological abnormalities in DS are well known,
and the genetic etiology of DS has been identified, understanding the exact cellular
mechanisms linking genotype to phenotype is the major challenge. With the pres-
ence of well-established procedures for testing DS, such as ultrasonographic meth-
ods and amniocentesis, which looks for chromosome disorders, the diagnosis of DS
based adequately on reliable biomarkers is underdeveloped [30, 96].
DS genotype affects the functional phenotype leading to changes in metabolo-
mic profiles. Therefore, metabolomics seems to be a promising tool for identifying
disease-related biomarkers. To date, few metabolomic studies have been conducted
to study and discover novel biomarkers in DS disease. The urinary metabolome of
122 maternal urines has been analyzed using LC-MS and revealed that dihydroura-
cil was significantly elevated in the urine of women with a DS-affected pregnancy.
In contrast, progesterone was decreased in a DS-affected pregnancy compared with
normal pregnancies [97].
Parfieniuk et al. [29] reported significant differences in the level of methylhisti-
dine, hexanoylcarnitine, diacetylspermine, and p-cresol sulfate when comparing
amniotic fluid of 13 women with fetal DS with 13 healthy fetuses [29]. Diaz et al.
[98] conducted a study on urine levels of 2-ketoglutarate, 1-methylhistidine,
3-hydroxybutyrate, 4-OH-hippurate, and dimethylamine and were able to distin-
guish the pregnant women carrying a baby with a chromosomal abnormality from
the control group using NMR method [98]. Furthermore, alterations of cortisol lev-
els, free amino acids (arginine, histidine, and glutamate), and pregnenolone sulfate
in amniotic fluid from fetuses with DS were observed and validated in a study con-
ducted by Haung et al. [30] using UPLC-MS. NMR-based metabolomic profiles of
plasma samples from 129 people with DS and 46 healthy controls showed a dra-
matic difference in the 7 metabolites that may be used to distinguish between the DS
and healthy groups [30]. However, the metabolomic patterns examined cannot be
linked to the degree of intellectual disability (ID) [48]. Caracausi et al. [47]
Metabolomics: A Pipeline for Biomarker Discovery in Genetic Diseases 59

analyzed the plasma and urine metabolome from 67 DS patients and 29 healthy
controls using NMR and reported distinguished metabolic patterns in DS attribut-
able mainly to mitochondrial dysmetabolism [47]. Moreover, a study using untar-
geted metabolomic analysis of amniotic fluid samples from women having normal
and DS fetuses identified alterations in several steroid hormones and their deriva-
tives, glutathione catabolites coupled, gamma-glutamyl amino acids, phospholipid
catabolites, sugars, and dicarboxylic acids [99].

7.3 Sickle Cell Disease

Sickle cell disease (SCD) is a set of red blood cell hereditary disorders caused by
structural hemoglobin (Hb) abnormalities known as sickle hemoglobin (HbS). A
single nucleotide change in the gene-producing β-globin is the cause of SCD. The
sixth position of the β-globin in hemoglobin S (HbS) is replaced by valine instead
of glutamic acid. SCD is caused by a homozygous HbS condition (HbSS) or is the
result of having inherited HbS with additional hemoglobin mutations such as beta0
thalassemia (HbS-beta0 that), which refers to the absence of production of beta-­
globin, HbC (HbSC), or beta+ thalassemia mutations (HbS-beta+thal) where the
beta gene makes low levels of globin [100]. Sickle cell anemia (SCA) is a condition
where a mutation in the β-globin gene on chromosome 11 may occur. This is an
autosomal recessive condition. The sickle cell trait is characterized by the appear-
ance of long polymers of deoxygenated HbS (deoxyHbS), resulting in sickle-shaped
erythrocytes and the eventual vascular hemolysis [100]. SCD symptoms include
anemia, acute chest syndrome, stroke, transient ischemia events, severe vaso-­
occlusive discomfort, severe pain, and splenic sequestration. In addition, SCD may
cause problems in the central nervous system, lungs, kidneys, and gastrointestinal
system [100, 101].
Currently, metabolomics is aiding scientists in precisely measuring functional
phenotypes that arise from changes in genomic, transcriptomic, and proteomic lev-
els. Therefore, this approach can be applied to identify new potential candidates’
diagnostic, prognostic, and therapeutic biomarkers for SCD. In this context, several
studies concerning the application of metabolomics in SCD have been conducted.
Dembélé et al. [102] presented the results of metabolomic profiling of patients
experiencing vaso-occlusive crises compared to their sickle cell disease baselines.
To determine the differences between these two disease states, a standardized tar-
geted metabolomic method was used for samples from 40 individuals, including
plasma and erythrocyte fractions. They found that metabolic signatures in the
plasma were especially notable for their differences in nitric oxide metabolism,
which hints at connections with pain. In addition, during the crisis, red blood cells
had extensive alterations in phospholipids, indicating significant membrane remod-
eling [102].
Several metabolomic studies were conducted on the transgenic mouse model of
SCD. A list of 251 metabolites associated with 8 pathways, including glycolysis,
60 L. A. Dahabiyeh and R. M. Nimer

the pentose phosphate pathway (PPP), amino acids, nucleotides, xenobiotics, lipids,
fatty acids, and carbohydrates, were significantly changed in SCD mouse blood
[23]. In addition, the elevation of several metabolites was reported in SCD blood
mice, for instance, nucleosides (including adenosine), lipids (such as sphingosine-­1-­
phosphate [S1P], lysophospholipids, and free acyl fatty acids), and glycolytic inter-
mediates (such as 2,3-BPG) [23, 103, 104].
Analysis of the red blood cell metabolome from 28 adult patients with the HbSS
(hemoglobin SS) genotype in a steady state and comparing it to 24 healthy adults
(HbAA) showed that 31 metabolites in key metabolic pathways (e.g., glycolysis,
pentose phosphate, glutathione, ascorbate, polyamines, carnitine, creatine, and
other amino acids) were significantly altered [60].

7.4 Glycogen Storage Disorders

Glycogen storage disorders (GSDs) are a group of metabolic abnormalities in gly-

cogen metabolism resulting from deficiencies in glycogen production or degrada-
tion enzymes (Inborn Metabolic Diseases: Diagnosis and Treatment). GSDs are
rare GD that usually primarily influence the liver, muscles, or the two.
Until now, studies conducted to find clinical biomarkers for GSDs have been
limited. Among the 19 types of GSDs classified based on enzyme deficiency and
affected tissue, glycogen storage disease type Ia (GSDIa, von Gierke disease) is
considered the most common type that occurs in about 1 out of every 100,000 live
births [105]. Tamara et al. [106] noted alterations in energy production pathways,
such as the tricarboxylic acid cycle, creatine metabolism, urea cycle, amino acid,
purine/pyrimidine metabolism, and enzyme cofactors, such as biotin. This study
was conducted on 14 plasma samples from adult GSDI patients compared to 31
healthy controls utilizing LC-MS/MS [106]. Similar findings were reported by
Farah et al. [31] using cell culture and a mouse model of GSDIa. The previous study
reported oxidative phosphorylation dysfunction, abnormalities in TCA cycle metab-
olites, reduced mitochondrial membrane potential, and dysfunctional mitochondrial
ultrastructure [31].
To differentiate between metabolite profiles from GSDI, GSD III subtypes, and
healthy controls, cultured fibroblasts from the three groups were analyzed by tar-
geted LCMS/MS [25]. Malfunctions in energy production pathways (glycolysis,
Krebs cycle, succinate) and decreased creatinine and antioxidant defense of the
cysteine and glutathione systems in GSDIa and GSD IIII have been detected [25].
For GSDII (Pompe disease), urine glucose tetrasaccharide has been authorized for
clinical usage [107]. However, most of the discovered biomarkers for GSDs lack
clinical validation.
A summary of selected studies and their main findings for DS, SCD, and GSDs
are presented in Table 2.
Metabolomics: A Pipeline for Biomarker Discovery in Genetic Diseases 61

Table 2 A summary of the literature on applying metabolomics for biomarker discovery in Down
syndrome (DS), sickle cell disease (SCD,) and glycogen storage disorders (GSDs)
Sample
Sample Method size Main findings References
1. Down syndrome (DS)
Urine ZIC-HILIC MS 122 Dihydrouracil is elevated in the urine of Trivedi
women with a DS-affected pregnancy, et al. [97]
while progesterone is decreased
Amniotic LC-MSMS 26 There were significant differences in the Parfieniuk
fluid levels of four metabolites: et al. [29]
methylhistidine, hexanoylcarnitine,
diacetylspermine, and p-cresol sulfate,
which may be linked to the improper
nervous system and muscle development
Urine NMR 300 Urine levels of 2-ketoglutarate, Diaz et al.
spectroscopy 1-methylhistidine, 3-hydroxybutyrate, [98]
4-OH-hippurate, and dimethylamine
have been shown to differentiate
pregnant women carrying a baby with a
chromosomal abnormality from the
control group
Amniotic LC-QTOF-MS 50 Significant differences between DS Huang
fluid fetuses and controls in porphyrin, bile et al. [30]
acids, amino acids, and hormone
metabolites such as
taurochenodeoxycholate, l-arginine, and
taurocholate. They also found
differences in l-histidine and
glycocholic acid metabolites
Plasma NMR 175 Alterations in the level of 7 metabolites Antonaros
spectroscopy may be used to distinguish between the et al. [48]
DS and healthy groups. No association
between the differences in metabolites
and the degree of ID
Plasma NMR 96 Several significantly altered metabolites Caracausi
and urine spectroscopy of Down syndrome correlate with et al. [47]
alteration of mitochondrial metabolism
Amniotic UPLC-MS 42 There were many different metabolites Liu et al.
fluid found in the amniotic fluid of DS [99]
pregnancies compared to normal
pregnancies, including lower levels of
several steroid hormones and their
derivatives, higher levels of glutathione
catabolites, and lower levels of
gamma-glutamyl amino acids
(continued)
62 L. A. Dahabiyeh and R. M. Nimer

Table 2 (continued)
Sample
Sample Method size Main findings References
2. Sickle cell disease (SCD)
Red blood Targeted 40 The involvement of metabolites not Dembélé
cells and quantitative previously identified in sickle cell et al. [102]
plasma using LC-MS/ disease, such as hexoses, acyl-carnitines,
MS and -aminoadipate all help explain how
sickle cell disease affects energetic
metabolism
Red blood LC/GC-MS 44 human Erythrocyte sphingosine kinase 1 Zhang
cells and and 6 mice SPHK1-mediated elevation of et al. [23]
plasma sphingosine-1-phosphate (S1P)
contributes to sickling and disease
progression
Whole LC/GC-MS 37 human Increased erythrocyte cytosolic Wu et al.
blood and and 12 phospholipase A2 (cPLA2) is a key [103]
plasma mice factor in the imbalanced lands cycle
seen in SCD erythrocytes, as well as the
increased erythrocyte LysoPC and
circulating arachidonic acid seen in SCD
mice
Whole UHPLC-MS 52 The concentrations of reduced Darghouth
blood glutathione (GSH) and oxidized et al. [60]
glutathione (GSSG) were reduced in
HbS cells, while their precursors
(glutamine, glutamate, and glycine)
were increased. N-acetylglutathione
(NAG) was found to be decreased in
HbS cells, as were two ascorbate
metabolism metabolites, diketogulonic
acid and threonolactone
3. Glycogen storage disorders (GSDs)
Plasma LC-MS/MS 45 Changes in metabolites in various Tamara
metabolic pathways, such as the et al. [106]
tricarboxylic acid cycle, creatine
metabolism, urea cycle, amino acid, and
purine/pyrimidine metabolism, as well
as enzyme cofactors such as biotin these
metabolic changes were seen in both
GSD subtypes (Ia and Ib)
Skin Targeted Skin Energy production pathways (glycolysis, Hannibal
fibroblast LCMS/MS fibroblasts Krebs cycle, succinate) were et al. [32]
cell lines dysfunctional in GSDIa and GSD III
Urine UPLC-MS/MS 79 Urinary glucose tetrasaccharide (Glc4) Chien et al.
determination may be useful in the [107]
follow-up of a positive newborn Pompe
disease screening result. A high Glc4
indicates an infantile phenotype
Metabolomics: A Pipeline for Biomarker Discovery in Genetic Diseases 63

8 Current Trends and Future Perspective

The field of metabolomics has recently seen a rapid expansion in clinical research,
with goals ranging from elucidating disease pathogenesis to discovering clinical
biomarkers. As a result, a new technology utilizing NMR and MS to assess metabo-
lite profiles in clinical samples has been introduced to detect the disease’s biomarker
molecules and metabolic effects or its treatment [108].
It is difficult to determine GD’s pathogenic mechanisms because of their com-
plexity and effects on multiorgan systems. Furthermore, it appears that GD does not
have a single biomarker that can distinguish between patients and accurately reflect
the pathology of the disease. In this context, metabolomic analysis of clinical sam-
ples may be an excellent tool for distinguishing biomarkers from therapeutic tar-
gets [109].
Recently, the field of metabolomics has made significant contributions to study-
ing GD. This is due to the ability of metabolomics, in contrast to other “omics”
sciences, to link gene and environmental interactions. It represents the genome’s
downstream output and the upstream input of the surrounding environment [110].
As a result of its ability to detect subtle changes in large datasets through compre-
hensive metabolic measurements, metabolomics is a practical application in GD for
clinical biomarker discovery [111].
Technological advancements in MS have made it possible to detect tens of thou-
sands of signals in complex biological systems, allowing the detection of a wide
range of metabolites in a single test and opening up new opportunities in the preci-
sion and personalized medicine [112]. Computing tools, databases, and big data
analytics are evolving quickly, allowing a complete annotation and identification of
these signals. Thus, it will be possible to discover new molecules, classes of com-
pounds, or metabolism pathway configurations that have not previously been con-
sidered in studies of GD. Public databases for metabolomics are being established,
and they will apply to a wide range of GD and therapeutic areas shortly.
Current biomarker discovery strategies on GD frequently rely on identifying
alterations in metabolites and the association of these altered metabolites with a
specific disease. When it comes to GD, the exact mechanism of these metabolites
and the functional role of metabolite biomarkers are frequently unknown and under-
studied. An integrated biomarker discovery platform must be supplemented with
genomic, transcriptomic, and proteomic data to be effective.
Despite significant progress in metabolomic research over the past decade, most
identified biomarkers still need to replace existing clinical tests. A potential bio-
marker must be confirmed and validated using hundreds of specimens before being
used in a clinical setting. It must also be reproducible, specific, and sensitive to be
approved. Robust experimental designs, data acquisition, data mining, and bio-
marker validation must be performed to successfully translate the results of a
metabolomic experiment to clinical use in GD. In addition, future methodology
development in sample preparation and analysis is still necessary to achieve com-
prehensive metabolome studies.
64 L. A. Dahabiyeh and R. M. Nimer

9 Conclusions

Genetic diseases have widely varied in pathophysiological consequences and sever-

ity of the disease. Currently, the available treatments and markers for properly man-
aging GD complications are limited. Additionally, the diagnosis and follow-up of
GD still depend on examining chromosomes or DNA and routine laboratory tests,
which are invasive or nonspecific. Hence, there is a high demand to identify and
validate novel biomarkers that may have diagnostic, prognostic, and therapeutic
values, improving the management and treatment of GD.
Metabolomic research provides insight into the human metabolome under spe-
cific disease states. Recently, metabolomics has shown promise as a readout of
genes and the environment at a particular time. Developing a combination of dis-
ease biomarkers by metabolomic study is a promising approach, particularly
because many of these diseases are heterogeneous and multifactorial. However,
identified potential biomarkers should be validated (analytical and clinical valida-
tion) and confirmed using an independent sample set to ensure that they are specific
to the disease state and are detected due to variability within the biological sample
of patients. The ability of metabolomic studies to produce a detailed disease charac-
terization allows personalized disease management and treatment, which are the
basis of precision medicine.

References

1. Boycott KM, Hartley T, Biesecker LG, Gibbs RA, Innes AM, Riess O, et al. A diagnosis for
all rare genetic diseases: the horizon and the next frontiers. Cell. 2019;177(1):32–7.
2. Jackson M, Marks L, May GHW, Wilson JB. The genetic basis of disease. Essays Biochem.
2018;62(5):643–723.
3. Ropers HH. New perspectives for the elucidation of genetic disorders. Am J Hum Genet.
2007;81(2):199–207.
4. Zhou B, Xiao JF, Tuli L, Ressom HW. LC-MS-based metabolomics. Mol BioSyst.
2012;8(2):470–81.
5. Rinschen MM, Ivanisevic J, Giera M, Siuzdak G. Identification of bioactive metabolites
using activity metabolomics. Nat Rev Mol Cell Biol. 2019;20(6):353–67.
6. Tolstikov V, Moser AJ, Sarangarajan R, Narain NR, Kiebish MA. Current status of metab-
olomic biomarker discovery: impact of study design and demographic characteristics.
Metabolites. 2020;10(6):224.
7. Zhang AH, Sun H, Wang XJ. Saliva metabolomics opens the door to biomarker discovery,
disease diagnosis, and treatment. Appl Biochem Biotechnol. 2012;168(6):1718–27.
8. Dahabiyeh LA, Malkawi AK, Wang XH, Colak D, Mujamammi AH, Sabi EM, et al.
Dexamethasone-induced perturbations in tissue metabolomics revealed by chemical isotope
labeling LC-MS analysis. Metabolites. 2020;10(2):42.
9. Dahabiyeh LA, Mahmoud NN, Al-Natour MA, Safo L, Kim DH, Khalil EA, et al.
Phospholipid-gold nanorods induce energy crisis in MCF-7 cells: cytotoxicity evaluation
using LC-MS-based metabolomics approach. Biomolecules. 2021;11(3):364.
Metabolomics: A Pipeline for Biomarker Discovery in Genetic Diseases 65

10. Dahabiyeh LA, Mujammami M, Arafat T, Benabdelkamel H, Alfadda AA, Rahman AMA. A
metabolic pattern in healthy subjects given a single dose of metformin: a metabolomics
approach. Front Pharmacol. 2021;12:705932.
11. Aranibar N, Borys M, Mackin NA, Ly V, Abu-Absi N, Abu-Absi S, et al. NMR-based metab-
olomics of mammalian cell and tissue cultures. J Biomol NMR. 2011;49(3–4):195–206.
12. Ren JL, Zhang AH, Kong L, Wang XJ. Advances in mass spectrometry-based metabolomics
for investigation of metabolites. RSC Adv. 2018;8(40):22335–50.
13. Pallares-Mendez R, Aguilar-Salinas CA, Cruz-Bautista I, del Bosque-Plata L. Metabolomics
in diabetes, a review. Ann Med. 2016;48(1–2):89–102.
14. Razquin C, Toledo E, Clish CB, Ruiz-Canela M, Dennis C, Corella D, et al. Plasma lipi-
domic profiling and risk of type 2 diabetes in the PREDIMED trial. Diabetes Care.
2018;41(12):2617–24.
15. Chen ZH, Li ZH, Li HR, Jiang Y. Metabolomics: a promising diagnostic and therapeutic
implement for breast cancer. Onco Targets Ther. 2019;12:6797–811.
16. Chen XL, Zhu Y, Jijiwa M, Nasu M, Ai JM, Dai SM, et al. Identification of plasma lipid
species as promising diagnostic markers for prostate cancer. BMC Med Inform Decis Mak.
2020;20:223.
17. Liessi N, Pedemonte N, Armirotti A, Braccia C. Proteomics and metabolomics for cystic
fibrosis research. Int J Mol Sci. 2020;21(15):5439.
18. Quezada H, Guzman-Ortiz AL, Diaz-Sanchez H, Valle-Rios R, Aguirre-Hernandez J. Omics-­
based biomarkers: current status and potential use in the clinic. Bol Med Hosp Infant Mex.
2017;74(3):219–26.
19. Robinette SL, Holmes E, Nicholson JK, Dumas ME. Genetic determinants of metabolism in
health and disease: from biochemical genetics to genome-wide associations. Genome Med.
2012;4:30.
20. Goossens N, Nakagawa S, Sun XC, Hoshida Y. Cancer biomarker discovery and validation.
Transl Cancer Res. 2015;4(3):256–69.
21. Wishart DS. Metabolomics for investigating physiological and pathophysiological processes.
Physiol Rev. 2019;99(4):1819–75.
22. Ilboudo Y, Garrett ME, Bartolucci P, Brugnara C, Clish CB, Hirschhorn JN, et al. Potential
causal role of L-glutamine in sickle cell disease painful crises: a Mendelian randomization
analysis. Blood Cells Mol Dis. 2021;86:102504.
23. Zhang YJ, Berka V, Song AR, Sun KQ, Wang W, Zhang WR, et al. Elevated sphingosine-­1-­
phosphate promotes sickling and sickle cell disease progression. J Clin Investig.
2014;124(6):2750–61.
24. Alvarez JA, Chong EY, Walker DI, Chandler JD, Michalski ES, Grossmann RE, et al. Plasma
metabolomics in adults with cystic fibrosis during a pulmonary exacerbation: a pilot random-
ized study of high-dose vitamin D-3 administration. Metabolism. 2017;70:31–41.
25. Wetmore DR, Joseloff E, Pilewski J, Lee DP, Lawton KA, Mitchell MW, et al. Metabolomic
profiling reveals biochemical pathways and biomarkers associated with pathogenesis in cys-
tic fibrosis cells. J Biol Chem. 2010;285(40):30516–22.
26. Joseloff E, Sha W, Bell SC, Wetmore DR, Lawton KA, Milburn MV, et al. Serum metabo-
lomics indicate altered cellular energy metabolism in children with cystic fibrosis. Pediatr
Pulmonol. 2014;49(5):463–72.
27. Al-Qahtani W, Jabar MA, Masood A, Jacob M, Nizami I, Dasouki M, et al. Dried
blood spot-based metabolomic profiling in adults with cystic fibrosis. J Proteome Res.
2020;19(6):2346–57.
28. Macedo AN, Mathiaparanam S, Brick L, Keenan K, Gonska T, Pedder L, et al. The sweat
metabolome of screen-positive cystic fibrosis infants: revealing mechanisms beyond impaired
chloride transport. ACS Cent Sci. 2017;3(8):904–13.
29. Parfieniuk E, Pietrowska K, Samczuk P, Kretowski A, Ciborowski M, Zbucka-Kretowska
M. Amniotic fluid metabolic fingerprinting indicated metabolites which may play a
66 L. A. Dahabiyeh and R. M. Nimer

role in the pathogenesis of foetal Down syndrome - a preliminary report. Ginekol Pol.
2021;92(3):188–94.
30. Huang J, Mo JH, Zhao GL, Lin QY, Wei GH, Deng WN, et al. Application of the amniotic
fluid metabolome to the study of fetal malformations, using Down syndrome as a specific
model. Mol Med Rep. 2017;16(5):7405–15.
31. Farah BL, Sinha RA, Wu YJ, Singh BK, Lim A, Hirayama M, et al. Hepatic mitochondrial dys-
function is a feature of Glycogen Storage Disease Type Ia (GSDIa). Sci Rep. 2017;7:44408.
32. Hannibal L, Theimer J, Wingert V, Klotz K, Bierschenk I, Nitschke R, et al. Metabolic pro-
filing in human fibroblasts enables subtype clustering in glycogen storage disease. Front
Endocrinol. 2020;11:579981.
33. Tang XH, Lin CC, Spasojevic I, Iversen ES, Chi JT, Marks JR. A joint analysis of metabolo-
mics and genetics of breast cancer. Breast Cancer Res. 2014;16(4):415.
34. Long ZP, Zhou JD, Xie K, Wu Z, Yin HH, Daria V, et al. Metabolomic markers of colorectal
tumor with different clinicopathological features. Front Oncol. 2020;10:981.
35. Yusof HM, Ab-Rahim S, Suddin LS, Saman MSA, Mazlan M. Metabolomics profiling on dif-
ferent stages of colorectal cancer: a systematic review. Malays J Med Sci. 2018;25(5):16–34.
36. Alvarez-Sanchez B, Priego-Capote F, de Castro MDL. Metabolomics analysis II. Preparation
of biological samples prior to detection. Trends Analyt Chem. 2010;29(2):120–7.
37. Vuckovic D. Current trends and challenges in sample preparation for global metabolomics
using liquid chromatography-mass spectrometry. Anal Bioanal Chem. 2012;403(6):1523–48.
38. Pinu FR, Villas-Boas SG. Extracellular microbial metabolomics: the state of the art.
Metabolites. 2017;7(3):43.
39. Brauer R, Leichtle AB, Fiedler GM, Thiery J, Ceglarek U. Preanalytical standardization of
amino acid and acylcarnitine metabolite profiling in human blood using tandem mass spec-
trometry. Metabolomics. 2011;7(3):344–52.
40. Mapstone M, Gross T, Macciardi F, Cheema A, Petersen M, Head E, et al. Metabolic cor-
relates of prevalent mild cognitive impairment and Alzheimer’s disease in adults with
Down syndrome. Alzheimers Dement (Amst). 2020;12(1):e12028. [Link]
dad2.12028.
41. Muhlebach MS, Sha W, MacIntosh B, Kelley TJ, Muenzer J. Metabonomics reveals altered
metabolites related to inflammation and energy utilization at recovery of cystic fibrosis lung
exacerbation. Metabol Open. 2019;3:100010. [Link]
42. Dahabiyeh LA. The discovery of protein biomarkers in pre-eclampsia: the promising role of
mass spectrometry. Biomarkers. 2018;23(7):609–21.
43. Rahman AMA, Pawling J, Ryczko M, Caudy AA, Dennis JW. Targeted metabolomics in
cultured cells and tissues by mass spectrometry: method development and validation. Anal
Chim Acta. 2014;845:53–61.
44. Steuer AE, Brockbals L, Kraemer T. Metabolomic strategies in biomarker research-new
approach for indirect identification of drug consumption and sample manipulation in clinical
and forensic toxicology? Front Chem. 2019;7:319.
45. Wei R, Li GD, Seymour AB. High-throughput and multiplexed LC/MS/MRM method for
targeted metabolomics. Anal Chem. 2010;82(13):5527–33.
46. Jacob M, Malkawi A, Albast N, Al Bougha S, Lopata A, Dasouki M, et al. A targeted metab-
olomics approach for clinical diagnosis of inborn errors of metabolism. Anal Chim Acta.
2018;1025:141–53.
47. Caracausi M, Ghini V, Locatelli C, Mericio M, Piovesan A, Antonaros F, et al. Plasma and
urinary metabolomic profiles of Down syndrome correlate with alteration of mitochondrial
metabolism. Sci Rep. 2018;8:2977.
48. Antonaros F, Ghini V, Pulina F, Ramacieri G, Cicchini E, Mannini E, et al. Plasma metabo-
lome and cognitive skills in Down syndrome. Sci Rep. 2020;10(1):10491.
49. Elsherif L, Pathmasiri W, McRitchie S, Archer DR, Ataga KI. Plasma metabolomics analy-
sis in sickle cell disease patients with albuminuria - an exploratory study. Br J Haematol.
2019;185(3):620–3.
Metabolomics: A Pipeline for Biomarker Discovery in Genetic Diseases 67

50. Orozco JS, Hertz-Picciotto I, Abbeduto L, Slupsky CM. Metabolomics analysis of children
with autism, idiopathic-developmental delays, and Down syndrome. Transl Psychiatry.
2019;9:243.
51. Sumner LW, Amberg A, Barrett D, Beale MH, Beger R, Daykin CA, et al. Proposed mini-
mum reporting standards for chemical analysis. Metabolomics. 2007;3(3):211–21.
52. Sumner LW, Lei ZT, Nikolau BJ, Saito K, Roessner U, Trengove R. Proposed quantitative
and alphanumeric metabolite identification metrics. Metabolomics. 2014;10(6):1047–9.
53. Eriksson L, Andersson PL, Johansson E, Tysklind M. Megavariate analysis of environ-
mental QSAR data. Part I - a basic framework founded on principal component analysis
(PCA), partial least squares (PLS), and statistical molecular design (SMD). Mol Divers.
2006;10(2):169–86.
54. Yin PY, Wan DF, Zhao CX, Chen J, Zhao XJ, Wang WZ, et al. A metabonomic study of
hepatitis B-induced liver cirrhosis and hepatocellular carcinoma by using RP-LC and HILIC
coupled with mass spectrometry. Mol BioSyst. 2009;5(8):868–76.
55. Kind T, Tsugawa H, Cajka T, Ma Y, Lai ZJ, Mehta SS, et al. Identification of small molecules
using accurate mass MS/MS search. Mass Spectrom Rev. 2018;37(4):513–32.
56. Ghanbari R, Sumner S. Using metabolomics to investigate biomarkers of drug addiction.
Trends Mol Med. 2018;24(2):197–205.
57. Michalski A, Cox J, Mann M. More than 100,000 detectable peptide species elute in single
shotgun proteomics runs but the majority is inaccessible to data-dependent LC-MS/MS. J
Proteome Res. 2011;10(4):1785–93.
58. Boxler MI, Schneider TD, Kraemer T, Steuer AE. Analytical considerations for (un)-tar-
geted metabolomic studies with special focus on forensic applications. Drug Test Anal.
2019;11(5):678–96.
59. Esther CR, Turkovic L, Rosenow T, Muhlebach MS, Boucher RC, Ranganathan S, et al.
Metabolomic biomarkers predictive of early structural lung disease in cystic fibrosis. Eur
Respir J. 2016;48(6):1612–21.
60. Darghouth D, Koehl B, Madalinski G, Heilier JF, Bovee P, Xu Y, et al. Pathophysiology of
sickle cell disease is mirrored by the red blood cell metabolome. Blood. 2011;117(6):E57–66.
61. Adebiyi MG, Manalo JM, Xia Y. Metabolomic and molecular insights into sickle cell disease
and innovative therapies. Blood Adv. 2019;3(8):1347–55.
62. Valdes A, Lucio-Cazana FJ, Castro-Puyana M, Garcia-Pastor C, Fiehn O, Marina
ML. Comprehensive metabolomic study of the response of HK-2 cells to hyperglycemic
hypoxic diabetic-like milieu. Sci Rep. 2021;11(1):5058.
63. Barupal DK, Zhang Y, Shen T, Fan SL, Roberts BS, Fitzgerald P, et al. A comprehensive
plasma metabolomics dataset for a cohort of mouse knockouts within the International
Mouse Phenotyping Consortium. Metabolites. 2019;9(5):101.
64. Boja ES, Fehniger TE, Baker MS, Marko-Varga G, Rodriguez H. Analytical validation con-
siderations of multiplex mass-spectrometry-based proteomic platforms for measuring protein
biomarkers. J Proteome Res. 2014;13(12):5325–32.
65. Yao XD, Bajrami B, Shi Y. Ultrathroughput multiple reaction monitoring mass spectrometry.
Anal Chem. 2010;82(3):794–7.
66. Masood A, Jacob M, Gu X, Abdel Jabar M, Benabdelkamel H, Nizami I, et al. Distinctive
metabolic profiles between cystic fibrosis mutational subclasses and lung function.
Metabolomics. 2021;17(1):4.
67. Zardini Buzatto A, Abdel Jabar M, Nizami I, Dasouki M, Li L, Abdel Rahman AM. Lipidome
alterations induced by cystic fibrosis, CFTR mutation, and lung function. J Proteome Res.
2020;20(1):549–64.
68. Fidock M, DeSilva B. Bioanalysis of biomarkers for drug development. Bioanalysis.
2012;4(20):2425–6.
69. Zhang A, Sun H, Yan G, Wang P, Wang X. Metabolomics for biomarker discovery: moving to
the clinic. Biomed Res Int. 2015;2015:354671.
68 L. A. Dahabiyeh and R. M. Nimer

70. Marchand CR, Farshidfar F, Rattner J, Bathe OF. A framework for development of useful
metabolomic biomarkers and their effective knowledge translation. Metabolites. 2018;8(4):59.
71. López-López A, López-Gonzálvez A, Barker-Tejeda TC, Barbas C. A review of validated
biomarkers obtained through metabolomics. Expert Rev Mol Diagn. 2018;18(6):557–75.
72. Nagana Gowda A, G, Raftery D. Biomarker discovery and translation in metabolomics. Curr
Metabolomics. 2013;1(3):227–40.
73. Dunn WB, Broadhurst DI, Atherton HJ, Goodacre R, Griffin JL. Systems level studies of
mammalian metabolomes: the roles of mass spectrometry and nuclear magnetic resonance
spectroscopy. Chem Soc Rev. 2011;40(1):387–426.
74. Usher-Smith J, Harshfield A, Saunders C, Sharp S, Emery J, Walter F, et al. External valida-
tion of risk prediction models for incident colorectal cancer using UK Biobank. Br J Cancer.
2018;118(5):750–9.
75. Putri SP, Yamamoto S, Tsugawa H, Fukusaki E. Current metabolomics: technological
advances. J Biosci Bioeng. 2013;116(1):9–16.
76. Emwas A-HM, Salek RM, Griffin JL, Merzaban J. NMR-based metabolomics in human
disease diagnosis: applications, limitations, and recommendations. Metabolomics.
2013;9(5):1048–72.
77. Almontashiri NAM, Zha L, Young K, Law T, Kellogg MD, Bodamer OA, et al. Clinical
validation of targeted and untargeted metabolomics testing for genetic disorders: a 3 year
comparative study. Sci Rep. 2020;10(1):9382.
78. Ali SE, Farag MA, Holvoet P, Hanafi RS, Gad MZ. A comparative metabolomics approach
reveals early biomarkers for metabolic response to acute myocardial infarction. Sci Rep.
2016;6(1):36359.
79. Montuschi P, Paris D, Melck D, Lucidi V, Ciabattoni G, Raia V, et al. NMR spectroscopy
metabolomic profiling of exhaled breath condensate in patients with stable and unstable cys-
tic fibrosis. Thorax. 2012;67(3):222–8.
80. Esther CR Jr, Coakley RD, Henderson AG, Zhou Y-H, Wright FA, Boucher RC. Metabolomic
evaluation of neutrophilic airway inflammation in cystic fibrosis. Chest. 2015;148(2):507–15.
81. Molares-Vila A, Corbalán-Rivas A, Carnero-Gregorio M, González-Cespón JL,
Rodríguez-Cerdeira C. Biomarkers in glycogen storage diseases: an update. Int J Mol Sci.
2021;22(9):4381.
82. Swain-Lenz D, Nikolskiy I, Cheng J, Sudarsanam P, Nayler D, Staller MV, et al. Causal
genetic variation underlying metabolome differences. Genetics. 2017;206(4):2199–206.
83. Balashova EE, Maslov DL, Lokhov PG. A metabolomics approach to pharmacotherapy per-
sonalization. J Pers Med. 2018;8(3):28.
84. Jacob M, Lopata AL, Dasouki M, Abdel Rahman AM. Metabolomics toward personalized
medicine. Mass Spectrom Rev. 2019;38(3):221–38.
85. Tsui L-C, Dorfman R. The cystic fibrosis gene: a molecular genetic perspective. Cold Spring
Harb Perspect Med. 2013;3(2):a009472.
86. Cystic Fibrosis Mutation Database [updated April 25, 2011; cited 2021, available from:
[Link]
87. Ciuca IM, Marian P, Monica M. Biomarkers in cystic fibrosis lung disease–a review. Rom J
Anaesth Intensive Care. 2020;27(2):34.
88. Quinn RA, Lim YW, Mak TD, Whiteson K, Furlan M, Conrad D, et al. Metabolomics
of pulmonary exacerbations reveals the personalized nature of cystic fibrosis disease.
PeerJ. 2016;4:e2174.
89. DiBattista A, McIntosh N, Lamoureux M, Al-Dirbashi OY, Chakraborty P, Britz-McKibbin
P. Metabolic signatures of cystic fibrosis identified in dried blood spots for newborn screen-
ing without carrier identification. J Proteome Res. 2019;18(3):841–54.
90. Moyne O, Castelli F, Bicout DJ, Boccard J, Camara B, Cournoyer B, et al. Metabotypes of
Pseudomonas aeruginosa correlate with antibiotic resistance, virulence and clinical outcome
in cystic fibrosis chronic infections. Metabolites. 2021;11(2):63.
Metabolomics: A Pipeline for Biomarker Discovery in Genetic Diseases 69

91. Wolak JE, Esther CR Jr, O’Connell TM. Metabolomic analysis of bronchoalveolar lavage
fluid from cystic fibrosis patients. Biomarkers. 2009;14(1):55–60.
92. Zang X, Monge ME, McCarty NA, Stecenko AA, Fernández FM. Feasibility of early detec-
tion of cystic fibrosis acute pulmonary exacerbations by exhaled breath condensate metabo-
lomics: a pilot study. J Proteome Res. 2017;16(2):550–8.
93. Robroeks CM, van Berkel JJ, Dallinga JW, Jöbsis Q, Zimmermann LJ, Hendriks HJ, et al.
Metabolomics of volatile organic compounds in cystic fibrosis patients and controls. Pediatr
Res. 2010;68(1):75–80.
94. Gardiner K, Herault Y, Lott IT, Antonarakis SE, Reeves RH, Dierssen M. Down syndrome:
from understanding the neurobiology to therapy. J Neurosci. 2010;30(45):14943–5.
95. Bull MJ. Health supervision for children with down syndrome. Am Acad Pediatr.
2011;128:393.
96. Kolialexi A, Tsangaris GT, Papantoniou N, Anagnostopoulos AK, Vougas K, Bagiokos V,
et al. Application of proteomics for the identification of differentially expressed protein mark-
ers for Down syndrome in maternal plasma. Prenat Diagn. 2008;28(8):691–8.
97. Trivedi DK, Iles RK. Shotgun metabolomic profiles in maternal urine identify potential
mass spectral markers of abnormal fetal biochemistry–dihydrouracil and progesterone in the
metabolism of Down syndrome. Biomed Chromatogr. 2015;29(8):1173–83.
98. Diaz SO, Barros AS, Goodfellow BJ, Duarte IF, Galhano E, Pita C, et al. Second trimester
maternal urine for the diagnosis of trisomy 21 and prediction of poor pregnancy outcomes. J
Proteome Res. 2013;12(6):2946–57.
99. Liu X, Quan S, Fu Y, Wang W, Zhang W, Wang X, et al. Study on amniotic fluid metabolism
in the second trimester of trisomy 21. J Clin Lab Anal. 2020;34(3):e23089.
100. Bakshi N, Morris CR. The role of the arginine metabolome in pain: implications for sickle
cell disease. J Pain Res. 2016;9:167.
101. Kassim AA, Galadanci NA, Pruthi S, DeBaun MR. How I treat and manage strokes in sickle
cell disease. Blood. 2015;125(22):3401–10.
102. Dembélé KC, Veyrat-Durebex C, Guindo A, Chupin S, Tessier L, Goïta Y, et al. Sickle cell
disease: metabolomic profiles of vaso-occlusive crisis in plasma and erythrocytes. J Clin
Med. 2020;9(4):1092.
103. Wu H, Bogdanov M, Zhang Y, Sun K, Zhao S, Song A, et al. Hypoxia-mediated impaired
erythrocyte lands’ cycle is pathogenic for sickle cell disease. Sci Rep. 2016;6(1):29637.
104. Zhang Y, Dai Y, Wen J, Zhang W, Grenz A, Sun H, et al. Detrimental effects of adenosine
signaling in sickle cell disease. Nat Med. 2011;17(1):79–86.
105. Özen H. Glycogen storage diseases: new perspectives. World J Gastroenterol.
2007;13(18):2541.
106. Tamara M, Martin P, Matthias G, Barbara P, Matthias B, Hochuli M, editors. Untargeted
plasma metabolomics identifies broad metabolic perturbations in glycogen storage disease
type I. 22nd European Congress of Endocrinology, 2020: BioScientifica.
107. Chien Y-H, Goldstein JL, Hwu W-L, Smith PB, Lee N-C, Chiang S-C, et al. Baseline urinary
glucose tetrasaccharide concentrations in patients with infantile-and late-onset Pompe dis-
ease identified by newborn screening. JIMD Rep. 2014;19:67–73.
108. Kang J, Zhu L, Lu J, Zhang X. Application of metabolomics in autoimmune diseases: insight
into biomarkers and pathology. J Neuroimmunol. 2015;279:25–32.
109. Jafari A, Babajani A, Rezaei-Tavirani M. Multiple sclerosis biomarker discoveries by pro-
teomics and metabolomics approaches. Biomark Insights. 2021;16:11772719211013352.
110. Sasaki K, Sagawa H, Suzuki M, Yamamoto H, Tomita M, Soga T, et al. Metabolomics plat-
form with capillary electrophoresis coupled with high-resolution mass spectrometry for
plasma analysis. Anal Chem. 2018;91(2):1295–301.
111. Zhang X-w, Li Q-h, Dou J-j. Mass spectrometry-based metabolomics in health and medical
science: a systematic review. RSC Adv. 2020;10(6):3092–104.
112. Dettmer K, Aronov PA, Hammock BD. Mass spectrometry-based metabolomics. Mass
Spectrom Rev. 2007;26(1):51–78.
Bioinformatic Tools for Clinical
Metabolomics

David S. Wishart

Abstract Clinical metabolomics may be used for the discovery of novel disease
biomarkers, the diagnosis of known diseases, and the understanding or rationaliza-
tion of disease mechanisms. This chapter introduces readers to some of the bioinfor-
matic tools that can be used to facilitate clinical metabolomics and provide key
insights into disease processes. In particular, readers will be introduced to several
important bioinformatic tools and resources in clinical metabolomics that are widely
used for metabolite identification, for biomarker discovery, for disease diagnosis,
and for understanding disease mechanisms. These will include discussions on the
Metabolomics Standards Initiative (MSI), software tools for metabolite identifica-
tion and quantification (such as Bayesil and XCMS), data resources for metabolite
annotation such as the Human Metabolome Database (HMDB) and MarkerDB (a
biomarker database), data analysis and biomarker discovery tools such as
MetaboAnalyst, and resources for interpreting or characterizing disease mecha-
nisms, such as the Small Molecule Pathway Database (SMPDB). Using these and
other well-known bioinformatic resources, clinicians are now able to make more
precise diagnoses, integrate multiple types of omics data, and provide the frame-
work for making metabolomics and integral part of precision medicine.

Keywords Metabolomics · Software · Review · Bioinformatics · Clinical

applications

D. S. Wishart (*)
Department of Computing Science, University of Alberta, Edmonton, AB, Canada
Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada
e-mail: dwishart@[Link]

© The Author(s), under exclusive license to Springer Nature Singapore Pte 71

Ltd. 2023
A. M. Abdel Rahman (ed.), Clinical Metabolomics Applications in Genetic
Diseases, [Link]
72 D. S. Wishart

1 Introduction

Metabolomics is a branch of “omics” science that is focused on the comprehensive

characterization of the small molecule metabolites in the metabolome. Clinical
metabolomics involves the application of metabolomic techniques toward discov-
ery, diagnosis, and monitoring of human diseases. Clinical metabolomics can also
be used to explore and understand the molecular basis to disease and disease mecha-
nisms. As highlighted throughout this book, metabolomics is being successfully
used in many areas of medicine and medical genetics, including the diagnosis and
monitoring of inborn errors of metabolism (IEMs), the detection of various endo-
crine disorders, the characterization of neurodegenerative diseases, and the diagno-
sis of cancers and cardiovascular diseases. The success of metabolomics in these
many diverse areas of medicine and medical genetics lies in the fact that metabolites
represent the downstream products of upstream events occurring within the genome,
the transcriptome, and the proteome. Indeed, metabolites are sometimes called the
“canaries of the genome.” Just as canaries were used by miners in the 1800s to serve
as sensitive indicators of toxic gases in coal mines, metabolites can serve as remark-
ably sensitive indicators of problems in the genome. Indeed, a single base change in
a gene can lead to a 10,000-fold change in the concentrations of certain metabolites
[1]. This exceptional sensitivity is the basis to newborn screening, in which metabo-
lite tests have been used to detect IEMs (such as phenylketonuria) for many decades
[2]. Metabolite concentrations are not only very sensitive to what goes on in the
genome, but they are also very sensitive to what goes on in the environment. Indeed,
metabolite concentrations are heavily influenced by nutrition, physical activity,
exposure to environmental chemicals, the time of day, or the even the outside tem-
perature [3, 4].
Because metabolites are the end products of complex interactions happening
inside the cell (the genome and the transcriptome) and events happening outside the
cell (the environment), metabolomics is ideal for assessing the interactions between
genes and the environment (i.e., measuring the phenotype). Therefore, metabolo-
mics offers clinicians and medical researchers an ideal route to measure and moni-
tor both human phenotypes and human diseases in “real time.” This gives
metabolomics an important advantage over genomics. While the genome can tell
you what might happen, the metabolome actually tells you what is happening.
As highlighted throughout this book, there are two distinct “flavors” of metabo-
lomics: (1) targeted metabolomics and (2) untargeted metabolomics. Targeted
metabolomics is focused on the identification (and often absolute quantification) of
a specific, predefined collection or category of metabolites in a tissue, biofluid, or
biological matrix. Because of its ability to achieve absolute quantification, targeted
metabolomics is widely used in clinical medicine, biomarker testing/discovery, and
disease monitoring. On the other hand, untargeted metabolomics involves the broad,
unbiased identification of the maximum number metabolites or metabolic features
in a tissue, biofluid, or biological matrix. Untargeted metabolomics is not reliably
quantitative, and so it is more widely used in early-stage biomarker discovery or
Bioinformatic Tools for Clinical Metabolomics 73

hypothesis generation applications. Both targeted and untargeted metabolomics can

be, and are currently, used in clinical metabolomics. Both can be used to discover
biomarkers or biomarker profiles, and both methods can be performed with standard
metabolomic platforms such as liquid chromatography mass spectrometry (LC-­
MS), gas chromatography mass spectrometry (GC-MS), and nuclear magnetic reso-
nance (NMR) systems. All three of these analytical platforms are capable of
separating, detecting, and characterizing hundreds, even thousands of chemicals in
complex chemical mixtures. In almost all cases, when NMR, GC-MS, or LC-MS
instruments are used to analyze clinical materials, they produce spectra or chro-
matograms consisting of many hundreds to thousands of peaks.
The quantity of data generated by these platforms necessitates the use of comput-
ers and a wide variety of bioinformatic software tools. The main bioinformatic chal-
lenges in metabolomics, and clinical metabolomics in particular, are (1) determining
which peaks in these spectra match to which chemical compounds (metabolite iden-
tification); (2) detecting which compounds are significantly altered in concentration
or abundance (determining metabolite significance); (3) determining which metab-
olites or combination of metabolites can serve as robust disease biomarkers (bio-
marker discovery); and (4) understanding the biological and genetic context for the
observed metabolic changes (finding disease mechanisms or causes).
This chapter is intended to provide an overview to some of the bioinformatic
tools that can be used to facilitate clinical metabolomics and provide key insights
into disease processes. In particular, readers will be introduced to a number of soft-
ware tools, data resources, and data standards for facilitating compound identifica-
tion (for both targeted and untargeted metabolomics), for detecting which
compounds are significantly altered in abundance, for identifying and assessing
metabolite biomarkers or biomarker panels, and for understanding biological and
genetic context of the observed metabolite changes. These will include discussions
on the Metabolomics Standards Initiative (MSI), software tools for metabolite iden-
tification and quantification (such as AMIX, Bayesil, AMDIS, and XCMS), data
resources for metabolite annotation such as the Human Metabolome Database
(HMDB) and MarkerDB (a biomarker database), data analysis and biomarker dis-
covery tools such as MetaboAnalyst, and resources for interpreting or characteriz-
ing disease mechanisms, such as the Small Molecule Pathway Database (SMPDB).

2 Bioinformatic Tools for Metabolite Identification

Depending on the method used (targeted vs. untargeted) and the type of compounds
being measured (lipids vs. non-lipids), different levels of metabolite identification
can be achieved. According to the Metabolomics Standards Initiative (MSI) [5],
there are actually four levels of metabolite identification: (1) positively identified
compounds, (2) putatively identified compounds, (3) compounds putatively identi-
fied to be part of a compound class, and (4) unknown compounds. Positively identi-
fied compounds correspond to those chemicals that have a name, a known structure,
74 D. S. Wishart

a CAS (Chemical Abstracts Services) number, or an InChI (International Chemical

Identifier) string. To fall into this category, a compound must be identified using a
purified, authentic standard collected under identical or near-identical data collec-
tion conditions. For targeted metabolomic studies, most metabolites are identified at
a Level 1 standard. On the other hand, for most untargeted studies, achieving Level
1 identification is rare. Putatively identified compounds (Level 2) correspond to
those where the compound is identified based on a spectral match (i.e., MS/MS) or
an alternative parameter (i.e., retention time and exact mass) match to a reference
database value. In these cases, an authentic standard is not available, meaning that
there is some ambiguity about the compound’s true identity. Certainly, if the com-
pound is known to exist in a human biofluid as indicated by numerous literature
reports or data resources such as the HMDB, these Level 2 compound identifica-
tions are much stronger and may be considered “near positive.”
The third level of compound identification is typical of many lipids, where the
exact structure of the compound cannot be determined, but it is known to be a spe-
cific class of lipid (a phospholipid or triglyceride) or a lipid where the total mass of
the acyl chains is known, but the type of position of the individual acyl chains is not
known (i.e. the phosphatidyl choline PC(38:3)). Level 3 identification is common
for untargeted MS-based lipidomic studies. The fourth level of compound identifi-
cation is the “unknown” category. Again, this is usually only seen with untargeted
metabolomic studies. In many cases, a compound is labeled as an “unknown” sim-
ply because the investigator has not been very thorough in their analyses or because
their software/database being used for compound identification is inadequate,
incomplete, or too small.

2.1 Bioinformatic Tools for Metabolite Identification Via NMR

NMR-based metabolomics has been used in clinical metabolomics for a number of

years. These include applications in diagnosing IEMs [6], detecting novel genetic
disorders [7], and for lipoprotein profiling [8, 9]. There are three methods for per-
forming metabolite identification by NMR. One is to manually spike the suspected
compound into the sample, collect the NMR spectrum of the spiked biofluid, and
confirm that the spiked compound changes the observed NMR spectrum in the
expected manner. This method is obviously slow and less than ideal for analyzing
large numbers of samples or identifying large numbers of metabolites. The second
is to use manual chemical shift “lookup” tables to match observed NMR chemical
shifts with known chemical shifts. Many NMR labs compile their own chemical
shift reference tables to perform manual assignments. However, there are now sev-
eral online NMR spectral databases that contain both experimentally measured and
(accurately) predicted NMR spectra for thousands of human metabolites. These
include the Human Metabolome Database or HMDB [10], the Natural Products
Magnetic Resonance Database or NP-MRD [11], and the BioMagResBank or
BMRB [12]. The HMDB is a particularly important metabolomic resource for
Bioinformatic Tools for Clinical Metabolomics 75

clinical metabolomics. It currently contains the largest collection (>250,000) of

known human metabolites along with detailed information on their structures,
descriptions, names and synonyms, biological pathways, biofluid concentrations,
and disease associations along with their corresponding MS and NMR spectra (to
aid in compound identification). In particular, the HMDB has experimentally mea-
sured and predicted 1H and 13C NMR spectra for more than 220,000 human metabo-
lites at NMR spectrometer frequencies ranging from 300 MHz to 1000 MHz. While
somewhat smaller and less clinically relevant, the NP-MRD has experimentally
measured and computationally predicted 1H and 13C NMR spectra for nearly 90,000
metabolites and natural products at NMR spectrometer frequencies ranging from
300 MHz to 1000 MHz. The BMRB has nearly 1000 commonly occurring metabo-
lites with experimentally measured 1H and 13C NMR spectra at 400 MHz and
600 MHz. All of these databases support web-based, automated metabolite identifi-
cation and NMR spectral searches and/or peak matching.
The third approach to perform metabolite identification via NMR spectroscopy
is to use spectral deconvolution. Spectral deconvolution is a computational approach
that involves taking a complex spectrum consisting of a mixture with many chemi-
cals and simplifying it into individual spectra of its “pure” chemical components
(Fig. 1).
This process requires a specially constructed spectral database as well as care-
fully developed spectral fitting software. The spectral database used in NMR spec-
tral deconvolution typically consists of reference 1D 1H or 13C NMR spectra of the
pure compound(s) that are known or expected to be in the biological sample of

Fig. 1 An image illustrating spectral deconvolution. On the left side is a simplified depiction of
how an NMR mixture spectrum could be decomposed into three separate “pure” spectra from three
separate compounds (A, B, and C). Summing the three spectra together produces the spectrum of
the mixture at the top. On the right side of this image is a spectral deconvolution performed on a
real NMR spectrum with the list of identified compounds and their concentrations
76 D. S. Wishart

interest. These reference NMR spectra must be collected under exactly the same
conditions (same temperature, same solvent, same salt, same pH) under which the
biological sample was analyzed.
The fact that most metabolites have distinct, almost invariant chemical shift “fin-
gerprints” made up of several compound-specific peaks is one reason why spectral
deconvolution works so well for NMR. Furthermore, given that most metabolites
have many NMR peaks helps to alleviate the problem of spectral redundancy. To put
it in another way, it is highly unlikely that any two distinct compounds will have the
same number of peaks, chemical shifts, peak intensities, spin couplings, or line
shapes in their NMR spectra. Another reason why spectral deconvolution works so
well with NMR is because NMR peak intensities provide precise information about
compound concentrations. In other words, accurate spectral matches not only lead
to exact compound IDs, but they also lead to exact or near-exact concentration
determinations.
There are several commercial programs that support NMR spectral deconvolu-
tion for metabolite identification, including AMIX (Bruker) and NMR Suite
(Chenomx) (for small molecule metabolites). These software packages have large
NMR spectral reference libraries consisting of hundreds of metabolites. Newer ver-
sions of these packages such as Bruker B.I. QUANT and Bruker FoodScreener as
well as NMR Suite (Version 7 and above) now support semiautomatic deconvolu-
tion for higher-throughput analysis. In addition to small molecule metabolite analy-
sis for clinical applications, several other companies, including LipoScience,
Bruker, and Nightingale, have begun to offer lipoprotein analyses of serum or
plasma samples through an automated or semiautomated spectral deconvolution.
These programs or services generate quantitative clinically useful data on high-,
medium-, and low-density lipoprotein particles (HDL, MDL, and LDL) from human
plasma samples.
In addition to these commercial programs or commercial services for clinically
based NMR metabolomics, several freely available academic programs have been
developed to perform semiautomatic compound identification or quantification via
NMR. These include Batman [13], AQuA [14], ASICS 2.0 [15], and rDolphin [16].
Unfortunately, these programs do not support automated data processing, which
means a separate software package such as NMRPipe [17] or NMRFx [18] must be
used to manually process the data prior to analysis. This requirement for manual
processing significantly slows the analysis (hours per spectrum) and adds an ele-
ment of human error to the analysis pipeline.
Recently, two new NMR spectral deconvolution programs called Bayesil [19]
and MagMet [20] have been introduced. These programs support fully automated
NMR metabolite identification and quantification. Both Bayesil and MagMet can
perform fully automated data processing and spectral deconvolution of one-­
dimensional (1D) 1H NMR spectra to identify and quantify upward of 50 to 60
compounds in 3–4 min. Just as with the Chenomx NMR Suite, Bayesil and MagMet
work with most NMR instrument models and field strengths but are limited to ana-
lyzing specific biofluids such as serum, plasma, or fecal water. Both Bayesil (http://
Bioinformatic Tools for Clinical Metabolomics 77

[Link]/) and MagMet ([Link] are freely accessible through web

servers.

2.2 Bioinformatic Tools for Metabolite Identification

Via GC-MS

GC-MS has been used in clinical chemistry for more than 50 years [21]. Indeed,
many clinical labs continue to routinely use GC-MS as their go-to platform for
metabolite profiling. GC-MS can be used to identify and quantify amino acids,
organic acids, hormones, and other important clinical biomarkers. As a result,
GC-MS can be used to diagnose and monitor many IEMs or other genetic disorders.
While most clinical chemists are reluctant to call GC-MS analysis a form metabo-
lomics, the simple fact is that clinical GC-MS is one of the most robust and most
widely used metabolomic platforms in clinical chemistry.
Just as with NMR-based metabolomics, compound identification via GC-MS is
best done through a form of spectral deconvolution. A typical GC-MS spectrum or
total ion chromatogram (TIC) from a metabolite mixture will consist of dozens of
sharp peaks (corresponding to ion counts) covering an elution time of about
30–45 min. Each peak often consists of one or more EI (electron ionization) mass
spectra arising from one or more compounds. A variety of commercial GC-MS data
analysis tools such as AMDIS, which stands for Automated Mass Spectral
Deconvolution and Identification System [22], MassHunter (Agilent), ChromaTOF
(Leco), and AnalyzerPro (SpectralWorks) can be used to identify and quantify
metabolites. Once the EI-MS spectra are extracted, metabolite identification is per-
formed in a similar manner to what is done for NMR. Namely, the extracted EI-MS
spectra from the biofluid are compared, one at a time, to EI-MS spectral reference
libraries containing the EI-MS spectra of thousands of pure, derivatized, and authen-
ticated compounds. This process is done semiautomatically with users making
metabolite identification calls based on the information and spectral image overlays
that the computer programs provide.
There are three key factors that ultimately determine the quality of a compound
identification by GC-MS: (1) the quality of the extracted query spectrum, (2) the
quality of the spectral matching algorithm, and (3) the quality and comprehensive-
ness of the reference spectral database. Unlike NMR, where “false-positive” peaks
are extremely rare, GC-MS is frequently plagued with an abundance of false-­
positive peaks. In some cases, up to 50% of features seen in GC-MS spectra are
fragments, adducts, or derivatives of either the column matrix, the derivatization
reagents, or of the metabolites themselves. Different software packages tend to han-
dle these spectral artifacts differently. A study by Lu et al. [23] compared three
commonly used GC-MS deconvolution packages (AMDIS, ChromaTOF, and
AnalyzerPro) using a defined mixture of 35 compounds with widely varying con-
centrations. It was found that both the AMDIS and ChromaTOF packages produced
78 D. S. Wishart

unusually high numbers of false positives or false/impure spectra, while the

AnalyzerPro package generally performed best.
Ultimately, the main factor driving the success in compound identification by
GC-MS is the size and quality of the EI-MS spectral reference database. The most
common and widely used EI-MS resource is the NIST (National Institute of
Standards and Technology) EI-MS spectral database. The latest release contains
EI-MS spectra for more than 300,000 compounds or derivatized compounds along
with retention index (RI) values for another 140,000 compounds. However, most of
the NIST compounds are not metabolites nor are they derived from biological mate-
rials. The paucity of real metabolites in the NIST library can lead to a number of
false-positive identifications, especially if authentic standards are not used to verify
the identity of a given compound. On the other hand, the HMDB [10] provides a
much larger collection of human metabolites (>250,000) along with a much larger
library of corresponding GC-MS data, including almost 2.3 million predicted and
experimental EI-MS spectra and nearly seven million retention indices. In particu-
lar, the latest version of the HMDB currently contains 6,696,000 accurately pre-
dicted RI values for 26,880 parent compounds (and 2.1 million TMS and TBDMS
derivatives of those parent compounds) and 2,282,000 predicted and experimental
EI-MS spectra. These spectra and RI data can be readily searched, separately or
together. However, it is important to note that the quality of most predicted EI-MS
spectra is not yet sufficiently high to achieve 100% identification accuracy.
Therefore, identifications made using predicted EI-MS data or predicted RI data
should always be viewed as putative identifications (Level 2).

2.3 Bioinformatic Tools for Metabolite Identification

Via LC-MS

Over the past three decades, LC-MS has become the most popular analytical plat-
form for clinical chemistry and clinical metabolomics [2, 24]. Indeed, most new-
born screening activities in the developed world are done using triple quadrupole
(QQQ) tandem mass (MS/MS) spectrometers [2]. Relative to NMR or GC-MS,
LC-MS methods offer much greater sensitivity, more comprehensive compound
detection, and generally higher throughput. These advantages largely explain its
growing popularity. Most LC-MS methods adopted in clinical chemistry laborato-
ries employ targeted approaches that use authentic, isotopically labelled standards
to simultaneously identify and quantify a small (15–25) number of high-priority
metabolites. Most of these targeted LC-MS methods use defined tables of specific
metabolite retention times (for their given liquid chromatography system),
metabolite-­specific multiple reaction monitoring (MRM) or single reaction moni-
toring (SRM) peak lists, and multipoint calibration curves for metabolite identifica-
tion and quantification. A number of different software packages from various
LC-MS vendors are available to facilitate targeted LC-MS analysis. These include
Bioinformatic Tools for Clinical Metabolomics 79

Analyst (from Sciex), MassHunter (from Agilent), Progenesis QI (from Waters),

and TraceFinder/LCQUAN (from Thermo Fisher). In addition to these vendor-­
specific packages, Biocrates Life Sciences provides a vendor-independent software
package, called MetIDQ with its targeted metabolomic kits to perform semiauto-
mated MRM-based metabolite identification and quantification. Modern, targeted
LC-MS-based metabolomic software and methods typically allow the identification
and quantification of up to 700 metabolites in a given sample in less than 30 min.
Untargeted LC-MS-based metabolomics is normally reserved for biomarker dis-
covery as opposed to biomarker testing. A typical LC-MS spectrum from an untar-
geted metabolomic study will consist of many sharp peaks (corresponding to ion
counts) covering an elution time of about 10–35 min. Each peak may consist of one
or more ESI (electrospray ionization) m/z values arising from one or more com-
pounds. As a result, untargeted LC-MS metabolomic studies can easily generate a
huge number of spectral features or putative compounds (>10,000). This is many
times more than what is seen by NMR or GC-MS. Many of these LC-MS features
turn out to be noise peaks, column contaminants, insource fragments, adducts, and
isotopic variants. As a result, untargeted LC-MS data typically requires a consider-
able amount of post-processing and peak consolidation to reduce the number of
peaks to a reliable, countable number (preferably <2000 putative compounds).
Untargeted LC-MS data is often further complicated by the fact that liquid chro-
matographic data is substantially more variable from run to run than NMR or
GC-MS data. As a result, metabolomic data acquired via untargeted LC-MS tech-
niques typically requires additional de-noising, spectral alignment, and spectral
averaging to ensure that the correct peaks are being picked and compared. This kind
of spectral processing requires sophisticated software that either comes bundled
with the LC-MS instrument or which is designed, written, and distributed by highly
specialized MS laboratories. Examples of some of the instrument-specific tools
include Mass Frontier (Thermo Fisher), MassHunter (Agilent), XCMS-Plus (Sciex),
Profile Analysis (Bruker), and Progenesis (Waters). There are also a number of
platform-­independent freeware systems including XCMS [25], MS-DIAL [26], and
MzMine2 [27]. All of these software packages support chromatographic and MS
spectral alignment, peak finding, multivariate statistics (for data reduction), parent
ion mass matching, molecular formula calculation, and MS/MS spectral matching.
Metabolite identification via accurate parent ion mass (or more correctly the
mass-to-charge, m/z) measurement requires the use of very high-resolution MS
instruments such as quadrupole time-of-flight (QTOFs), Orbitraps, or Fourier-­
Transform Ion Cyclotron Resonance (FT-ICR) spectrometers. If a parent ion mass
is measured to 4–5 decimal places, which corresponds to a mass accuracy of
<5 ppm, it is usually possible to determine the ion’s molecular formula and its puta-
tive identity (Level 3 identification) through a chemical formula calculator. Several
commercial MS chemical formula calculators exist such as SigmaFit (Bruker),
Formula Predictor (Shimadzu), and MassHunter (Agilent) as well as a number of
freeware packages including 7-Golden-Rules [28] and SIRIUS [29]. By including
restrictions on the types of elements typically found in metabolites as well as
requirements on hydrogen/carbon ratios and isotopic abundances, it is often
80 D. S. Wishart

possible to reduce the number of feasible chemical formulas even further [30].
Unfortunately, even with these improvements, parent ion-based metabolite identifi-
cation is still very risky as there are often many masses or molecular formulae that
can still match dozens of metabolites in existing compound databases.
The preferred route of metabolite identification for most untargeted LC-MS
metabolomic studies is to use both parent ion (or formula) matching and MS/MS
spectral matching. MS/MS spectra, with their characteristic fragmentation patterns,
provide very useful structural information about molecules. Successful LC-MS/MS
spectral matching is critically dependent on having instrument-specific or condition-­
specific MS/MS product ion fragment libraries. Many of these libraries are bundled
with the instrument-specific software packages mentioned earlier. On the other
hand, commercial MS/MS databases, such as the NIST20 database and METLIN
[31], as well as public MS/MS databases such as MassBank of North America
(MoNA), [32], and HMDB [10] are normally used by the freeware packages
(XCMS, MS-DIAL, and MzMine) to perform MS/MS spectral matching. Table 1
provides list of MS/MS databases with experimentally acquired (and predicted)
MS/MS spectral data and their relative size.
The challenge with using experimentally acquired MS/MS spectra from these
MS databases is that each compound is often represented by dozens of different
MS/MS spectra collected on different MS instruments under different ionization
conditions or at different collision energies. So, while the number of experimentally
collected MS spectra is large, the actual number of unique (parent) compounds
represented by this diverse collection is quite small. Indeed, it is thought that the
current experimental MS/MS spectral collection represents <20% of known or
expected human metabolites. Given the striking shortage of experimentally col-
lected MS/MS spectra, a number of investigators have started to use computational
tools to predict MS/MS spectra for individual compounds where no experimental
MS/MS spectra exist [33, 34]. Many of these in silico predicted MS/MS spectral
libraries are now available through the HMDB [10] and the CFM-ID database [34].
These data resources support direct MS/MS spectral searches (to find specific com-
pounds) as well as neutral loss searches (to find related compounds). Other

Table 1 A list of MS/MS spectral databases with the reported numbers of compounds and MS/
MS spectra
Number of
Database name compounds Number of MS/MS spectra
Metlin 870,000 2,510,000 (experimental)
CFM-ID 216,890 1,771,460 (predicted and
experimental)
MassBank of North America 223,614 658,790 (experimental and
(MoNA) predicted)
NIST20 MS/MS 31,808 1,300,000 (experimental)
MassBankEU 15,055 89,769 (experimental)
GNPS 11,947 584,567 (experimental)
mzCloud 11,495 3,243,574 (experimental)
Bioinformatic Tools for Clinical Metabolomics 81

computational MS/MS spectral interpretation tools, including CSI:FingerID [35]

and SIRIUS4 [36], allow users to input an experimental MS/MS spectrum and will
generate a structure match or a structure class match without the need to match
against any predicted MS/MS spectra. These programs use a technique called chem-
ical or spectral fingerprint analysis rather than spectral prediction [34].
Even with the best MS/MS spectral databases (experimentally acquired or com-
putationally predicted) and the best chemical/spectral fingerprint analysis tools, it is
still quite difficult to confidently identify (MSI Level 2) more than 400–500 metab-
olites via untargeted LC-MS-based metabolomics. While the instrumental times for
most untargeted metabolomic LC-MS assays are relatively quick (15–20 min per
sample), the data analysis times are often quite slow. Indeed, they often run for sev-
eral hours per sample as most workflows require considerable manual inspection
and manual intervention.

3 Bioinformatic Tools for Detecting Metabolite Differences

Regardless of whether targeted or untargeted approaches are used, one of the central
goals of any clinical metabolomic study is to determine which peaks or which
metabolites are significantly different for those individuals with a disease or condi-
tion relative to healthy controls. This comparison between healthy concentration
values versus diseased concentration values is how most known disease biomarkers
are measured and how new disease biomarkers are discovered. In clinical metabo-
lomics, there are three routes for determining which metabolites are significantly
different or significantly differentiating. These include (1) reference-based metabo-
lite differentiation, (2) multivariate metabolite differentiation, and (3) multivariate
peak differentiation.
Reference-based metabolite differentiation involves comparing quantitatively
measured metabolite concentrations in a given biofluid for a diseased individual
(measured via targeted metabolomic methods) with healthy, age-specific and sex-­
specific reference metabolite concentrations for the same biofluid, as reported in a
database or a reference textbook. Reference-based metabolite differentiation is
ideal for diagnosing individuals or for identifying biomarkers in individuals afflicted
with rare diseases, such as IEMs, or other genetic disorders. This approach is the
most widely used method for detecting significant metabolite differences in clinical
metabolomics.
The second approach, called multivariate metabolite differentiation, detects
metabolite differences by conducting case-versus-control studies using targeted
metabolomic methods. This involves quantitatively measuring specific “named”
metabolites from biofluid samples collected from multiple individuals with the dis-
ease of interest and biofluid samples from multiple healthy individuals (age and sex
matched) without the disease. This approach, which requires the use of multivariate
statistical techniques, is ideal for discovering multiple metabolite biomarkers for a
given disease and for creating robust multi-marker profiles or multi-marker models.
82 D. S. Wishart

The third approach to detecting metabolite differences is specific to untargeted

metabolomics. Like the second approach, it uses a case-versus-control experimental
design and multivariate statistics, but the goal is to use multivariate statistics in
combination with relative peak intensity differences (as opposed to absolute con-
centrations of fully identified metabolites) to identify which peaks or features in the
untargeted dataset are differentially abundant. The goal is to reduce the initial list of
thousands of unidentified features or peaks to a more manageable list of a few dozen
features that exhibit strong differences between cases and controls. From this
smaller list of yet-to-be-identified features, it is possible to use various techniques
(spectral matching, spike-in experiments, etc.) to identify the actual metabolites
showing the most significant concentration changes. Multivariate peak differentia-
tion is well suited for novel biomarker discovery and early-stage or putative bio-
marker identification. However, these putative markers must ultimately be validated
using a targeted metabolomic technique that quantitatively measures the presump-
tive metabolites.

3.1 Bioinformatic Tools for Reference-Based

Metabolite Differentiation

Reference-based metabolite differentiation requires a large and reliable set of

human reference metabolite concentrations for different ages, sexes and biofluids.
Currently the most complete set of healthy metabolite reference values for different
biofluids for different ages and sexes is the Human Metabolome Database or HMDB
[10]. The HMDB is widely regarded as the most complete open-access database on
human metabolites and their disease associations. Currently the HMDB contains
reference concentration values for 3073 metabolites in serum/plasma, 1757 metabo-
lites in urine, 447 metabolites in cerebrospinal fluid, 883 metabolites in saliva and
1805 metabolites in feces. These values include the literature sources, age group or
age range of the measured cohort and sex (if available). In many cases, multiple
values are provided as different analytical methods can lead to slight differences in
the reported concentration values. Abnormal concentrations are also reported in the
HMDB, along with the associated conditions and the corresponding literature
sources. Currently, the HMDB contains abnormal metabolite concentration data for
more than 660 IEMs and other genetic disorders. Users can easily query the HMDB
via its web interface with a specific metabolite name, metabolite structure or metab-
olite InChI identifier to obtain the corresponding biofluid concentrations and explore
what else is known about the queried metabolite. Likewise, users may also query or
browse the HMDB by disease or condition names and the results will provide the
lists of altered metabolites associated with that condition.
Another useful source of reference metabolite concentrations and reference-­
based metabolite differentiation is MarkerDB [37]. MarkerDB is the world’s most
comprehensive open-access biomarker database. It contains more than 26,600
Bioinformatic Tools for Clinical Metabolomics 83

genetic, protein, and metabolite biomarkers for 670 human disorders or conditions.
MarkerDB not only provides metabolite concentration data for healthy individuals
(with information on age- and sex-specific values), but it also provides data on the
unhealthy metabolite concentrations (in different biofluids), descriptions of the
associated disorders, detailed descriptions of the metabolites, and even biomarker
performance indications, such as biomarker sensitivity and specificity. In this
regard, the disease and biomarker data in MarkerDB is probably more complete
than the data in HMDB.
Both HMDB and MarkerDB are primarily designed for querying or browsing a
small number (<3) of metabolites and assessing their concentration differences rela-
tive to healthy normal values. If multiple (10 or more) metabolites need to be que-
ried to determine if the observed metabolite concentrations are significantly different
from normal, it is possible to use an online metabolomic web server called
MetaboAnalyst [38]. In particular, the Single Sample Profiling (SSP) option within
MetaboAnalyst’s enrichment analysis (EA) module allows users to enter long lists
of metabolite names and concentrations which are then compared against those val-
ues reported in the HMDB for a wide variety of biofluids. Metabolites that are
higher (H) or lower (L) than the normal reference values are flagged in the resulting
output. Hyperlinks to the HMDB compound database entries are also provided. The
SSP option with MetaboAnalyst provides users a fast and convenient route to per-
form reference-based metabolite differentiation from targeted metabolomic data. It
also allows important metabolite features (i.e., those marked with H or L) to gener-
ate a biomarker profile for a given disease.

3.2 Bioinformatic Tools for Multivariate Metabolite/

Peak Differentiation

When conducting biomarker discovery or biomarker validation studies in clinical

metabolomics, it is standard practice to perform well-powered case-versus-control
studies. Case-control studies often involve hundreds of subjects, thousands of bio-
fluid samples, and the collection of very large metabolomic datasets. As highlighted
earlier, any single targeted metabolomic assay can easily generate hundreds of
named metabolites and metabolite concentrations. Likewise, an untargeted metabo-
lomic assay can easily generate thousands of un-named “features” or peaks along
with their corresponding relative peak intensities. Because the number of variables
in these types of clinical studies is so large, special statistical methods must be used
to help manage the data, differentiate up- or downregulated metabolites, and reduce
the problems of overlap, false positives, and significance. In particular, the tech-
niques that must be used are called multivariate statistics. In multivariate (short for
multiple variable) statistics, the variables are called “dimensions.” One of the pri-
mary objectives of multivariate statistics is to reduce the number of variables or
dimensions so that the problem can be tackled more simply using traditional
84 D. S. Wishart

univariate statistics, such as Student’s t-tests or ANOVA techniques. Multivariate

statistics uses a class of mathematical techniques called dimensional reduction
methods to make multivariate data look more like univariate (single variable) data.
Dimensional reduction allows one to identify the key components in a large multi-
variate dataset that contain the maximum amount of information or maximize the
differences among groups. As a result, dimensional reduction reduces a long list of
metabolites to a shorter list of the most significant metabolites. This is the essence
of multivariate feature/metabolite differentiation. The most common form of dimen-
sional reduction is called principal component analysis or PCA.

3.3 Principal Component Analysis

Principal component analysis (PCA) is an unsupervised clustering technique.

Clustering is the process of grouping a set of objects in such a way that objects in
the same group are more similar to each other than to those in other groups.
Clustering helps distinguish groups, such as cases and controls, from one another
based on their metabolic parameters. In a more formal “mathematical” sense, PCA
determines an optimal linear transformation for a collection of data points such that
the properties of that set of data points are most clearly displayed along a small
number of coordinate (or principal) axes. Simply put, PCA allows metabolomic
researchers to easily plot, visualize, and cluster multiple lists of metabolites and
their concentrations based on linear combinations of their shared features. PCA is
most commonly used in clinical metabolomics to determine whether one or more
samples are different from another. It also allows one to identify which variables or
metabolites contribute most to this difference and whether those metabolites con-
tribute in the same way (i.e., are correlated) or independently (i.e., uncorrelated)
from each other. PCA is particularly appealing because it allows one to visually
detect sample clusters or groupings. In particular, the results of a PCA are usually
discussed in terms of scores and loadings. The scores represent the original data in
the new coordinate system, and the loadings are the weights applied to the original
data during the projection process. Plotting out the data using two sets of scores
(one for the X axis and one for the Y axis) will produce a “scores” plot. The “weight-
ings” of the individual components correspond to a PCA “loadings” plot. With
untargeted metabolomic data, the loadings plot can be used to narrow down the list
of features or peaks to just a few important ones that need to be identified. This
makes PCA ideal for reducing the number of features in untargeted metabolomic
data from 1000s to just a few dozen or less. It can also help reduce the list of metab-
olites in targeted metabolomic studies from 100 s to just a dozen or fewer.
Furthermore, PCA can be used to identify the most important or most informative
metabolites required to generate a biomarker profile for a given disease.
PCA can be easily conducted using a variety of free or nearly free software pro-
grams such as MatLab or the R project ([Link] using R’s prcomp
or princomp commands. PCA can also be performed using freely available,
Bioinformatic Tools for Clinical Metabolomics 85

downloadable software packages such as XCMS [25], MS-DIAL [26], MAVEN

[39], and GALAXY-M [40], which are frequently used for processing LC-MS data.
Freely available web servers are also available that support PCA and other common
multivariate statistical techniques. The most widely used web server for multivariate
statistical analysis in metabolomics is MetaboAnalyst [38]. MetaboAnalyst, which
is freely available, provides an easy-to-use graphical interface that allow users to
simply point and click to perform complex multivariate statistical operations or to
generate colorful, interactive graphs or tables. Nearly one-third of all published
metabolomic papers use MetaboAnalyst in the metabolomic data analysis pipeline.

3.4 Partial Least Squares Discriminant Analysis

PCA is not the only multivariate statistical approach that can be used to identify
important metabolites or reduce the number of spectral features. Another type of
multivariate statistical method that can be used for this purpose is known as super-
vised classification. Supervised classifiers are programs or algorithms that require
that information about the class identities must be provided in advance of running
the analysis. In other words, prior knowledge about which samples belong to the
“cases” and which samples belong to the “controls” is used to label each of the
samples. Examples of supervised classifiers include SIMCA (soft independent
modeling by class analogy), PLS-DA (partial least squares discriminant analysis),
and OPLS-DA (orthogonal projections to latent structures discriminant analysis).
All of these techniques can be used to help convert extensive NMR, LC-MS/MS,
and GC-MS metabolite lists (for targeted metabolomics) or their corresponding
spectral features (for untargeted metabolomics) into much shorter lists of highly
significant metabolites and/or features.
PLS-DA or partial least squares discriminant analysis is often used when PCA
techniques do not generate sufficiently distinct clusters or sufficiently distinct
metabolite sets. In particular, PLS-DA can be used to enhance the separation
between data points in a PCA “scores” plot by essentially rotating the PCA compo-
nents such that a maximum separation among classes is obtained. This enhanced
separation allows one to better understand which variables are most responsible for
separating the observed (or apparent) classes. Care must be taken in using PLS-DA
methods because these classification techniques can be overtrained. That is, PLS-DA
can create convincing clusters or classes that have no statistical meaning (i.e., they
over-fit the data). The best way of avoiding these problems is to use permutation
(random relabeling) approaches to ensure that the data clusters derived by PLS-DA
are real and robust. A number of freely available metabolomic software packages
and web servers, such as MetaboAnalyst, are able to perform these permutation
tests. Another way of quantitatively assessing a PLS-DA model is to report R2 and/
or Q2 values. Both R2 and Q2 are typically reported by metabolomic web servers and
software packages such as MetaboAnalyst. R2 is the correlation index and refers to
the goodness of fit or the explained variation, while Q2 refers to the predicted
86 D. S. Wishart

variation or quality of prediction. A poorly fit model will have an R2 of 0.2 or 0.3,
while a nicely fit model will have an R2 of 0.7 or 0.8. In practice, Q2 typically tracks
very closely to R2. However, if the PLS-DA model becomes over-fit, Q2 reaches a
maximum value and then begins to fall. Generally, a Q2 > 0.5 if considered good,
while a Q2 of 0.9 is outstanding.
If a robust PLS-DA model can be generated, the set of important metabolites
(generated via targeted metabolomics) or features (generated via untargeted metab-
olomics) arising from the variable importance plot (VIP) can be more easily inter-
preted than those determined via a PCA loading plot. PLS-DA is generally among
the most powerful and useful methods for reducing the number of features in untar-
geted metabolomic data from 1000s to just a few dozen or less. PLS-DA is also very
effective in reducing the list of important or differential metabolites in targeted
metabolomic studies from 100 s to just a dozen or fewer. Furthermore, PLS-DA can
be used to identify the most important or most informative metabolites required to
generate a biomarker profile for a given disease. The utility of PLS-DA in bio-
marker development and discovery is discussed in the next section.

4 Bioinformatic Tools for Biomarker Discovery

One of the principal goals of clinical metabolomics is to discover and/or measure

metabolite biomarkers of human disease. Biomarkers are typically defined as objec-
tively measurable biological characteristics that can be used to diagnose, monitor,
or predict the risk of disease [41]. For example, blood glucose is a standard chemi-
cal biomarker for monitoring diabetes, while serum creatinine is a chemical marker
for kidney function. Many traditional clinical chemistry biomarkers consist of just a
single measured entity. However, metabolomics has allowed clinicians to measure
multiple chemicals at once. This means it is now possible to measure multiple bio-
markers or develop multi-biomarker panels to predict or diagnose diseases with
greatly improved sensitivity and specificity. Indeed, it has long been common prac-
tice among physicians to combine multiple physiological biomarkers
(age + BMI + triglyceride level + cholesterol level = cardiac disease risk) to improve
biomarker sensitivity and specificity. Now, with metabolomics, it is possible to cre-
ate diagnostic or predictive models from multiple metabolites, which can be used to
classify individuals into specific groups (i.e., healthy vs. diseased) with much
improved sensitivity and specificity.
Sensitivity and specificity have very formal definitions in biomarker studies and
the biomarker literature. In standard case vs. control studies, sensitivity (Sn) is
mathematically defined as Sn = TP/(TP + FN), and specificity (Sp) is mathemati-
cally defined as Sp = TN/(TN + FP), where TP is the number of true positives, TN
is the number of true negatives, FN is the number of false negatives, and FP is the
number of false positives. Sensitivity (also known as the true positive rate) can be
considered as the probability of a positive test result given that a subject has an
actual positive outcome. Specificity (also known as the true negative rate) can be
Bioinformatic Tools for Clinical Metabolomics 87

considered as the probability of a negative test result given that a subject has an
actual negative outcome. For instance, if a biomarker or biomarker panel has a sen-
sitivity of 0.95 and a specificity of 0.60, this indicates that if a patient has a test score
that is above the decision boundary there is a 95% chance that the patient is cor-
rectly diagnosed with the disease/condition; but if the test score is below the deci-
sion boundary, then there is only a 60% chance that the patient is correctly classified
as being healthy. A promising biomarker must have both high sensitivity (i.e., to
give a positive test result when the disease is actually present) and high specificity
(i.e., to give a negative test result when the disease is absent).
One of the best ways to observe how a decision boundary affects sensitivity and
specificity is through a receiver operator characteristic (ROC) curve. A ROC curve
shows how the sensitivity and specificity change as the classification decision
boundary is varied across the range of available biomarker scores. Because an ROC
curve depicts the performance of a biomarker test over the complete range of pos-
sible decision boundaries, it allows the optimal specificity and associated sensitivity
to be determined by visual inspection. When one evaluates a biomarker using a
ROC curve, there is no need to be worried about the “data normality” of either the
predicted positive or negative score distributions nor whether the two distributions
have equal numbers of subjects or equal variance. As a result, ROC curve analysis
is widely considered to be the most objective and statistically valid method for bio-
marker performance evaluation [42].
ROC curves are often summarized into a single metric known as the “Area Under
the Curve” (AUC or AUROC). The AUROC indicates a biomarker model’s ability
to discriminate between cases (positive examples) and non-cases (negative exam-
ples.). If all positive cases are ranked before negative ones (i.e., a perfect classifier),
the AUC is 1.0. An AUC of 0.5 is equivalent to randomly classifying subjects as
either sick or healthy (i.e., the classifier is of no practical utility). A rough guide for
assessing the utility of a biomarker based on its AUROC is as follows: 0.9–1.0 = excel-
lent; 0.8–0.9 = good; 0.7–0.8 = fair; 0.6–0.7 = poor; and 0.5–0.6 = fail (see Fig. 2).
Currently, the most useful tool for biomarker discovery, biomarker selection, and
for performing sensitivity/specificity analysis (via ROC curve analysis) with metab-
olomic data is MetaboAnalyst [38]. In particular, the MetaboAnalyst biomarker
module supports three common ROC-based analysis modes: (1) classical univariate
ROC curve analysis, (2) multivariate ROC curve exploration, and (3) manual bio-
marker model creation and evaluation. The most popular and useful option is the
multivariate ROC curve exploration which supports automated multi-biomarker
selection and optimization using Monte Carlo cross validation (MCCV). This
allows the biomarker panel’s AUROC to be maximized while minimizing the num-
ber of biomarkers being used. MetaboAnalyst will typically generate several bio-
marker models with different numbers of metabolites and different AUROCs to
allow users some choice over what biomarker panel matches their biomarker
requirements or performance expectations.
Four different biomarker modeling options are currently offered with
MetaboAnalyst’s Biomarker module: (1) partial least squares discriminant analysis
(PLS-DA), (2) support vector machine (SVM), (3) random forests, and (4) logistic
88 D. S. Wishart

Fig. 2 A depiction of several different ROC curves for different biomarker tests with the area
under the ROC curves indicated. On the bottom left is an example of a perfect biomarker with a
perfect ROC curve having an AUROC of 1.0. On the top left is an example of an excellent bio-
marker profile with an AUROC of 0.9. On the top right is an example of a moderately good bio-
marker profile with an AUROC of 0.7. On the bottom left is an example of a random biomarker
with no predictive or diagnostics capability

regression. The most useful of these four options is the logistic regression model as
it provides an equation, or set of equations, incorporating metabolite concentrations
that can be universally used for calculating cutoff thresholds or decision boundaries.
MetaboAnalyst also generates a number of useful graphs, ROC curves, confidence
intervals, and charts to help users assess the selected biomarkers and biomarker
models. The simplicity with which biomarker models can be developed (mostly via
point-and-click operations) and rich graphical support in MetaboAnalyst within its
Biomarker module makes it the ideal tool for biomarker discovery in clinical
metabolomics.
It is important to note that the metabolomic data being uploaded into
MetaboAnalyst’s Biomarker module should be absolutely quantitative. As with
most analytical methods supported by MetaboAnalyst, the metabolite data uploaded
Bioinformatic Tools for Clinical Metabolomics 89

into the biomarker module must be properly normalized, scaled, and transformed so
that metabolite values are comparable and therefore more robustly analyzable.

5 Bioinformatic Tools for Biomedical Interpretation

and Data Integration

Biomarker identification is usually limited to disease diagnosis or disease prediction

[37]. Biomarkers are not necessarily intended to help uncover the underlying cause
of the disease or explain specific disease mechanisms. Of course, metabolic bio-
markers may be strongly associated with disease mechanisms, but without some
kind of biological interpretation or some kind of biomedical context, association
does not imply causation, nor does it lead to mechanistic insights. To properly deter-
mine disease causes or disease mechanisms from clinical metabolomic data, it is
often necessary to turn to metabolic pathways or to integrate both genomic and
metabolomic data together. Metabolite interpretation via pathway analysis often
involves determining whether the identified metabolites belong to a single pathway
or a smaller set of related pathways. In many cases, this requires searching or read-
ing carefully through various online metabolic pathway databases.
Metabolic pathway databases provide a centralized collection of schematic path-
ways that depict the current state of the knowledge regarding metabolic (catabolic,
anabolic, or signaling) processes that occur within a cell, tissue, or organism.
Pathway databases combine large collections of carefully curated metabolite data,
with large amounts of carefully collected protein and/or genetic data through a
series of illustrated enzyme-mediated reactions, receptor-mediated signaling pro-
cesses, or protein-aided transport activities. These represent the key molecular and
cellular activities that underlie all physiological processes. Because pathway data-
bases combine multi-omic (metabolomic, proteomic, genomic) data together along
with general information about physiological or biological consequences, these
databases can play a key role in the biological analysis or biomedical interpretation
of metabolomic data.
Some of the most popular small molecule pathway databases include KEGG
[43], the Reactome database [44], the “Cyc” databases [45], WikiPathways [46], the
Small Molecule Pathway Database or SMPDB [47], and PathBank [48]. A number
of commercial pathway databases also exist such as BioCarta, TransPath (from
BioBase Inc.), and Ingenuity Pathway Analysis (from Ingenuity Systems Inc.). The
most useful pathway database for clinical metabolomics is SMPDB as it offers that
largest number and most diverse pathways specific to human biology and human
diseases. In particular, SMPDB contains 150 signaling pathways, 20,250 disease
pathways (covering many IEMs and genetic disorders), 468 drug pathways, and
27,800 metabolic (catabolic/anabolic) pathways.
Most pathway databases support interactive image mapping with hyperlinked
information content that allows users to view chemical information (if a compound
90 D. S. Wishart

is clicked) or brief summaries of genes and/or proteins (if a protein or enzyme is

clicked). Almost all pathway databases support some kind of limited text search,
and a few, such as Reactome, SMPDB, and the “Cyc” databases, support the map-
ping of gene, protein, and/or metabolite expression data onto pathway diagrams.
Only a few pathway databases, such as SMPDB, provide their pathway data in com-
mon, machine-readable data exchange formats such as BioPAX [49], SBML
[Systems Biology Markup Language] [50], or SBGN-ML [Systems Biology
Graphical Notation Markup Language] [51].
Nearly all of the major pathway databases used in metabolomics today (KEGG,
the Reactome database, the “Cyc” databases, WikiPathways, and SMPDB) permit
users to upload metabolite data and generate highlighted pathway plots indicating
the location of key metabolites in a given pathway. Unfortunately, most metabolite/
metabolism databases (such as KEGG, the Cyc databases, WikiPathways, Reactome)
only contain anabolic or catabolic pathways associated with endogenous metabo-
lites. Almost no information is provided on metabolite signaling pathways, disease
pathways, metabolic diseases (such as phenylketonuria), or drug action pathways
(how aspirin works). As a result, many metabolomic pathway analyses are limited
to interpreting complex metabolite data in only the simplest of terms. An important
exception to this is SMPDB. SMPDB resource contains hundreds of human-specific
pathways including dozens of signaling pathways as well as hundreds of disease
and drug pathways. Currently, SMPDB is the only open-access database that covers
such a broad diversity of human disease or disease mechanism pathways – espe-
cially for small molecules. This makes SMPDB one of the most popular tools for
interpreting and integrating clinical metabolomic data.
While pathway visualization can provide some important qualitative insight into
the biological roles for metabolites detected in a clinical metabolomic study, it is
also important to remember that more quantitative tools for pathway analysis also
exist. In particular, pathway enrichment and pathway topological analysis are two
quantitative methods that can be quite helpful. MetaboAnalyst offers several
advanced pathway enrichment analysis procedures along with pathway topological
analysis to help identify the most relevant metabolic pathways involved in a given
clinical metabolomic study. The pathway analysis module in MetaboAnalyst uses
simple point and click operations to support three types of analyses: (1) pathway
enrichment analysis, (2) pathway topological analysis, and (3) pathway impact
analysis. Pathway enrichment analysis can be done using either overrepresentation
analysis or via metabolite set enrichment analysis using Fishers’ exact test, the
hypergeometric test, and GlobalAncova [52]. Pathway topological analysis is based
on the centrality measures of a metabolite in a given metabolic network. Centrality
is a quantitative measure of the position of a metabolite relative to the other metabo-
lites in a pathway. Centrality can be used to estimate a metabolite’s relative impor-
tance or role in a pathway or network diagram. MetaboAnalyst uses relative
“betweenness” centrality and “out-degree” centrality to calculate the relative impor-
tance of a metabolite. Centrality means that metabolites located on the periphery of
a pathway or those that are involved in side reactions have little consequence and
are not particularly “central.” On the other hand, metabolites that are in pathway
Bioinformatic Tools for Clinical Metabolomics 91

bottlenecks or those that serve as hubs or precursors for many reactions are more
“central.” By calculating the topological importance of different metabolites in a
given pathway, as well as the enrichment of certain metabolites in a pathway, it is
possible to calculate a pathway impact score.
By plotting the pathway impact score against the number of significant metabo-
lites appearing that pathway, it is possible to generate a plot that illustrates the most
important pathways detected from a set of significantly altered metabolites in a
given metabolomic experiment (Fig. 3).
In this example, the X-axis displays the pathway impact score, while the Y-axis
displays the level of enrichment. The size of the colored circles represents the num-
ber of metabolites in the illustrated pathway, and the color of the circle indicates its
overall significance (with red being most significant and pale yellow being least
significant). By clicking on the colored circles, it is possible to see more details
about the pathway name, the pathway components, and their topological relation-
ships. Within MetaboAnalyst, each detected metabolite is also “clickable” so that a
box-and-whisker plot can be generated that illustrates the metabolite concentrations
and range between the “case” and “control” samples.
In addition to pathway analysis, there are also a number of other approaches that
can be used to interpret, visualize, or explore clinical metabolomic data. One par-
ticularly useful approach involves using a technique called metabolite set

Fig. 3 An example of a pathway impact diagram from MetaboAnalyst. For the graph on the left
side of the image, the X-axis displays the pathway impact score, while the Y-axis displays the level
of enrichment. The size of the colored circles represents the number of metabolites in the illus-
trated pathway, and the color of the circle indicates its overall significance (with red being most
significant and pale yellow being least significant). By clicking on the colored circles, it is possible
to see more details about the pathway name, the pathway components, and their topological rela-
tionships. This expanded view is shown on the right side of the image with the pathway diagram
being taken from KEGG and the individual metabolites being identified with KEGG identifiers
92 D. S. Wishart

enrichment or MSEA [53]. MSEA is a form of functional enrichment analysis simi-

lar to gene set enrichment analysis (GSEA). For metabolite set enrichment to be
effective, one usually needs a comprehensive database of metabolic pathways, a
database of healthy/diseased metabolite concentrations, or a database with associa-
tions between metabolites and SNPs or metabolites and gene expression levels.
Ideally, a good MSEA system should have all of these databases and support all of
these functional analyses. In this regard, the MSEA module in MetaboAnalyst actu-
ally has all of these databases and functional tools, making it particularly useful for
clinical interpretation. Another approach to interpreting clinical metabolomic data
is to combine it with gene expression or protein expression data [54]. There are a
number of bioinformatic tools that support this kind of integration. One example is
MetScape [55]. MetScape is a plugin for the widely used open-source network anal-
ysis and visualization tool called Cytoscape. MetScape supports the interactive,
network-­based exploration and visualization of both metabolite and gene expres-
sion data by integrating both the KEGG and EHMN (Edinburgh human metabolic
network) databases. MetScape allows users to identify enriched pathways from
gene/metabolite expression profiling data, build and analyze gene/metabolite net-
works, and interactively visualize changes in gene/metabolite data. Another inte-
grated “omics” approach that offers similar capabilities is called Integrated
Metabolomic and Expression Analysis or INMEX [54]. This web-based tool is now
available through MetaboAnalyst. Like MetScape, INMEX makes use of the KEGG
pathway database as well as a number of pathways from SMPDB.
How these software tools and resources are used and how the data is eventually
interpreted depends somewhat on the knowledge of the user. Naïve analyses per-
formed by a naïve individual will lead to naïve interpretation. Taking the time to
read the literature and to discover what else is known (genetically or metabolically)
about a given disease or condition will allow for a much more efficient use of the
software and a much more intelligent interpretation of the data. In this regard, it is
always important to remember that bioinformatics should always be used as an aid
to support and extend one’s own biochemical and biological knowledge.

6 Summary

This chapter has provided a high-level overview of the bioinformatic resources

needed to analyze clinical metabolomic data. As highlighted at the beginning of this
chapter, the main bioinformatic challenges in clinical metabolomics are (1) metabo-
lite identification, (2) determining metabolite significance, (3) biomarker discovery,
and (4) finding disease mechanisms or causes. To address these challenges, we
introduced and discussed a number of software tools, data resources, and data stan-
dards for facilitating compound identification, for detecting which compounds are
significantly altered in abundance, for identifying and assessing metabolite bio-
markers or biomarker panels, and for understanding biological and genetic context
of the observed metabolite changes. In particular, we discussed the Metabolomics
Bioinformatic Tools for Clinical Metabolomics 93

Standards Initiative (MSI) for compound identification and introduced software

tools for metabolite identification and quantification for NMR, GC-MS, and LC-MS/
MS (such as AMIX, Bayesil, AMDIS, and XCMS). We also discussed data resources
for metabolite annotation such as the Human Metabolome Database (HMDB) and
MarkerDB, as well as data analysis and biomarker discovery tools such as
MetaboAnalyst. Finally, we closed the chapter with a discussion on different bioin-
formatic resources for interpreting or characterizing disease mechanisms, such as
the Small Molecule Pathway Database (SMPDB).
The field of clinical metabolomics has grown considerably over the past 10 years,
and detailed descriptions of all the bioinformatic tools and resources that have been
developed for clinical metabolomics could easily fill several books. This chapter is
only intended to serve as an introduction so that individuals who are interested in
pursuing clinical metabolomics and using bioinformatic tools for clinical metabolo-
mics can better appreciate what is available, what is possible, and what still needs to
be done.

References

1. Wishart DS, Tzur D, Knox C, Eisner R, Guo AC, Young N, Cheng D, Jewell K, Arndt D,
et al. HMDB: the human metabolome database. Nucleic Acids Res. 2007;35(Database
issue):D521–6.
2. Levy PA. An overview of newborn screening. J Dev Behav Pediatr. 2010;31:622–31.
3. Bassini A, Cameron LC. Sportomics: building a new concept in metabolic studies and exercise
science. Biochem Biophys Res Commun. 2014;445:708–16.
4. Brown SA. Circadian metabolism: from mechanisms to metabolomics and medicine. Trends
Endocrinol Metab. 2016;27:415–26.
5. Sumner LW, Amberg A, Barrett D, Beale MH, Beger R, Daykin CA, Fan TWM, Fiehn O, et al.
Proposed minimum reporting standards for chemical analysis. Metabolomics. 2007;3:211–21.
6. Moolenaar SH, Engelke UF, Wevers RA. Proton nuclear magnetic resonance spectroscopy
of body fluids in the field of inborn errors of metabolism. Ann Clin Biochem. 2003;40(Pt
1):16–24.
7. Verrips A, Hoefsloot LH, Steenbergen GC, Theelen JP, Wevers RA, Gabreëls FJ, van Engelen
BG, van den Heuvel LP. Clinical and molecular genetic characteristics of patients with cere-
brotendinous xanthomatosis. Brain. 2000;123(Pt 5):908–19.
8. Otvos JD, Jeyarajah EJ, Bennett DW, Krauss RM. Development of a proton nuclear magnetic
resonance spectroscopic method for determining plasma lipoprotein concentrations and sub-
species distributions from a single, rapid measurement. Clin Chem. 1992;38(9):1632–8.
9. Soininen P, Kangas AJ, Würtz P, Suna T, Ala-Korpela M. Quantitative serum nuclear magnetic
resonance metabolomics in cardiovascular epidemiology and genetics. Circ Cardiovasc Genet.
2015;8:192–206.
10. Wishart DS, Guo A, Oler E, Wang F, Anjum A, Peters H, Dizon R, Sayeeda Z, Tian S,
Lee BL, et al. HMDB 5.0: the human metabolome database for 2022. Nucleic Acids Res.
2022a;50(D1):D622–31.
11. Wishart DS, Sayeeda Z, Budinski Z, Guo A, Lee BL, Berjanskii M, Rout M, Peters H, Dizon
R, Mah R, et al. NP-MRD: the natural products magnetic resonance database. Nucleic Acids
Res. 2022b;50(D1):D665–77.
94 D. S. Wishart

12. Markley JL, Ulrich EL, Berman HM, Henrick K, Nakamura H, Akutsu H. BioMagResBank
(BMRB) as a partner in the worldwide protein data Bank (wwPDB): new policies affecting
biomolecular NMR depositions. J Biomol NMR. 2008;40:153–5.
13. Hao J, Liebeke M, Astle W, De Lorio M, Bundy JG, Ebbels TMD. Bayesian deconvolution
and quantification of metabolites in complex 1D NMR spectra using BATMAN. Nat Protoc.
2014;9:1416–27.
14. Röhnisch HE, Eriksson J, Mullner E, Agback P, Sandstrom C, Moazzami AA. AQuA: an auto-
mated quantification algorithm for high-throughput NMR-based metabolomics and its applica-
tion in human plasma. Anal Chem. 2018;90:2095–102.
15. Lefort G, Liaubet L, Marty-Gasset N, Canlet C, Vialaneix N, Servien R. Joint automatic
metabolite identification and quantification of a set of (1)H NMR spectra. Anal Chem.
2021;93:2861–70.
16. Cañueto D, Gómez J, Salek RM, Correig X, Cañellas N. rDolphin: a GUI R package for
proficient automatic profiling of 1D (1)H-NMR spectra of study datasets. Metabolomics.
2018;14:24.
17. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A. NMRPipe: a multidimensional
spectral processing system based on UNIX pipes. J Biomol NMR. 1995;6:277.
18. Norris M, Fetler B, Marchant J, Johnson BA. NMRFx processor: a cross-platform NMR data
processing program. J Biomol NMR. 2016;65:205–16.
19. Ravanbakhsh S, Liu P, Bjorndahl TC, Mandal R, Grant JR, Wilson M, Eisner R, Sinelnikov I,
Hu X, Luchinat C, Greiner R, Wishart DS. Accurate, fully-automated NMR spectral profiling
for metabolomics. PloS One. 2015;10:e0124219.
20. Foroutan A, Fitzsimmons C, Mandal R, Berjanskii MV, Wishart DS. Serum metabolite
biomarkers for predicting residual feed intake (RFI) of Young Angus bulls. Metabolites.
2020;10:491.
21. Jellum E, Stokke O, Eldjarn L. Combined use of gas chromatography, mass spectrometry, and
computer in diagnosis and studies of metabolic disorders. Clin Chem. 1972;18:800–9.
22. Stein SE. An integrated method for spectrum extraction and compound identification from gas
chromatography/mass spectrometry data. J Am Soc Mass Spectrom. 1999;10:770–81.
23. Lu H, Liang Y, Dunn WB, Shen H, Kell DB. Comparative evaluation of software for decon-
volution of metabolomics data based on GC-TOF-MS. Trends Anal Chem. 2008;27:215–27.
24. Ismail IT, Showalter MR, Fiehn O. Inborn errors of metabolism in the era of untargeted metab-
olomics and lipidomics. Metabolites. 2019;9:242.
25. Smith CA, Want EJ, O'Maille G, Abagyan R, Siuzdak G. XCMS: processing mass spectrom-
etry data for metabolite profiling using nonlinear peak alignment, matching, and identification.
Anal Chem. 2006;78:779–87.
26. Tsugawa H, Cajka T, Kind T, Ma Y, Higgins B, Ikeda K, Kanazawa M, VanderGheynst J, Fiehn
O, Arita M. MS-DIAL: data-independent MS/MS deconvolution for comprehensive metabo-
lome analysis. Nat Methods. 2015;12:523–6.
27. Pluskal T, Castillo S, Villar-Briones A, Oresic M. MZmine 2: modular framework for pro-
cessing, visualizing, and analyzing mass spectrometry-based molecular profile data. BMC
Bioinformatics. 2010;11:395.
28. Kind T, Fiehn O. Seven Golden rules for heuristic filtering of molecular formulas obtained by
accurate mass spectrometry. BMC Bioinformatics. 2007;8:105.
29. Dührkop K, Scheubert K, Böcker S. Molecular Formula Identification with
SIRIUS. Metabolites. 2013;3:506–16.
30. Kind T, Fiehn O. Advances in structure elucidation of small molecules using mass spectrom-
etry. Bioanal Rev. 2010;2:23–60.
31. Tautenhahn R, Cho K, Uritboonthai W, Zhu Z, Patti GJ, Siuzdak G. An accelerated workflow
for untargeted metabolomics using the METLIN database. Nat Biotechnol. 2012;30:826–8.
32. Kind T, Tsugawa H, Cajka T, Ma Y, Lai Z, Mehta SS, Wohlgemuth G, Barupal DK, Showalter
MR, Arita M, Fiehn O. Identification of small molecules using accurate mass MS/MS search.
Mass Spectrom Rev. 2017;37(4):513–32.
Bioinformatic Tools for Clinical Metabolomics 95

33. Allen F, Greiner R, Wishart DS. Competitive fragmentation modeling of ESI-MS/MS spectra
for putative metabolite identification. Metabolomics. 2015;11:98–110.
34. Wang F, Liigand J, Tian S, Arndt D, Greiner R, Wishart DS. CFM-ID 4.0: more accurate
ESI-MS/MS spectral prediction and compound identification. Anal Chem. 2021;93:11692–700.
35. Dührkop K, Shen H, Meusel M, Rousu J, Böcker S. Searching molecular structure databases
with tandem mass spectra using CSI:FingerID. Proc Natl Acad Sci U S A. 2015;112:12580–5.
36. Dührkop K, Fleischauer M, Ludwig M, Aksenov AA, Melnik AV, Meusel M, Dorrestein PC,
Rousu J, Böcker S. SIRIUS 4: a rapid tool for turning tandem mass spectra into metabolite
structure information. Nat Methods. 2019;16:299–302.
37. Wishart DS, Bartok B, Oler E, Liang KYH, Budinski Z, Berjanskii M, Guo A, Cao X,
Wilson M. MarkerDB: an online database of molecular biomarkers. Nucleic Acids Res.
2021;49(D1):D1259–67.
38. Chong J, Soufan O, Li C, Caraus I, Li S, Bourque G, Wishart DS, Xia J. MetaboAnalyst
4.0: towards more transparent and integrative metabolomics analysis. Nucleic Acids Res.
2018;46(W1):W486–94.
39. Melamud E, Vastag L, Rabinowitz JD. Metabolomic analysis and visualization engine for
LC-MS data. Anal Chem. 2010;82:9818–26.
40. Davidson RL, Weber RJ, Liu H, Sharma-Oates A, Viant MR. Galaxy-M: a Galaxy workflow
for processing and analyzing direct infusion and liquid chromatography mass spectrometry-­
based metabolomics data. Gigascience. 2016;5:10.
41. Biomarkers Definitions Working Group. Biomarkers and surrogate endpoints: preferred defini-
tions and conceptual framework. Clin Pharmacol Ther. 2001;69(3):89–95.
42. Soreide K. Receiver-operating characteristic curve analysis in diagnostic, prognostic and pre-
dictive biomarker research. J Clin Pathol. 2009;62:1–5.
43. Kanehisa M, Goto S, Sato Y, Kawashima M, Furumichi M, Tanabe M. Data, information,
knowledge and principle: back to metabolism in KEGG. Nucleic Acids Res. 2014;42(Database
issue):D199–205.
44. Croft D, O'Kelly G, Wu G, Haw R, Gillespie M, Matthews L, Caudy M, Garapati P, Gopinath
G, Jassal B, et al. Reactome: a database of reactions, pathways and biological processes.
Nucleic Acids Res. 2011;39:D691–7.
45. Karp PD, Riley M, Saier M, Paulsen IT, Paley SM, Pellegrini-Toole A. The EcoCyc and
MetaCyc databases. Nucleic Acids Res. 2000;28:56–9.
46. Kelder T, van Iersel MP, Hanspers K, Kutmon M, Conklin BR, Evelo CT, Pico
AR. WikiPathways: building research communities on biological pathways. Nucleic Acids
Res. 2012;40(Database issue):D1301–7.
47. Jewison T, Su Y, Disfany FM, Liang Y, Knox C, Maciejewski A, Poelzer J, Huynh J, Zhou
Y, Arndt D, et al. SMPDB 2.0: big improvements to the small molecule pathway database.
Nucleic Acids Res. 2014;42(Database issue):D478–84.
48. Wishart DS, Li C, Marcu A, Badran H, Pon A, Budinski Z, Patron J, Lipton D, Cao X, Oler E,
et al. PathBank: a comprehensive pathway database for model organisms. Nucleic Acids Res.
2020;48(D1):D470–8.
49. Strömbäck L, Lambrix P. Representations of molecular pathways: an evaluation of SBML, PSI
MI and BioPAX. Bioinformatics. 2005;21:4401–7.
50. Gillespie CS, Wilkinson DJ, Proctor CJ, Shanley DP, Boys RJ, Kirkwood TB. Tools for the
SBML community. Bioinformatics. 2006;22:628–9.
51. van Iersel MP, Villéger AC, Czauderna T, Boyd SE, Bergmann FT, Luna A, Demir E, Sorokin
A, Dogrusoz U, Matsuoka Y, et al. Software support for SBGN maps: SBGN-ML and
LibSBGN. Bioinformatics. 2012;28:2016–21.
52. Xia J, Wishart DS. MetPA: a web-based metabolomics tool for pathway analysis and visualiza-
tion. Bioinformatics. 2010a;26:2342–4.
53. Xia J, Wishart DS. MSEA: a web-based tool to identify biologically meaningful patterns in
quantitative metabolomic data. Nucleic Acids Res. 2010b;38(Web Server issue):W71–7.
96 D. S. Wishart

54. Xia J, Fjell CD, Mayer ML, Pena OM, Wishart DS, Hancock RE. INMEX--a web-based tool
for integrative meta-analysis of expression data. Nucleic Acids Res. 2013;41(Web Server
issue):W63–70.
55. Karnovsky A, Weymouth T, Hull T, Tarcea VG, Scardoni G, Laudanna C, Sartor MA,
Stringer KA, Jagadish HV, Burant C, Athey B, Omenn GS. Metscape 2 bioinformatics tool
for the analysis and visualization of metabolomics and gene expression data. Bioinformatics.
2012;28:373–80.
Untargeted Metabolomics in Newborn
Screening

Joshua Manor and Sarah H. Elsea

Abstract Since its inception six decades ago, newborn screening has been lauded
as a highly successful and cost-effective public health program by identifying disor-
ders at the presymptomatic stage, enabling early disease-modifying intervention
that otherwise invariably leads to death or permanent damage if treated at the symp-
tomatic stage. The advent of multiplex high-throughput assays involving chroma-
tography coupled with mass spectroscopy enabled the analysis of multiple disorders
in a single run, vastly increasing the repertoire of screened disorders while keeping
the cost nearly the same. Industrialized countries provide unified screening for more
than 50 conditions, compared to about a dozen, a mere decade ago. Inevitably, we
now screen, in essence, more than we know how to treat. Nonetheless, as a constant
flow of new therapies breaks ground, providing accurate diagnostic data is vital for
patient outcomes. Breaking the diagnostic barrier can mean new research, new
drugs, and ultimately increased survival. In this chapter, we overview the concept of
untargeted metabolomics as applied to newborn screening, how it fares compared to
the well-standardized tests of the targeted screening, and its ability to screen for
more disorders that are currently “unscreenable.”

Keywords Untargeted metabolomics · Inborn error of metabolism (IEM) · Mass

spectrometry (MS) · Dried blood spot (DBS) · Newborn screening (NBS) ·
Biomarker discovery

J. Manor
Metabolic Disease Unit, Haim Sheba Medical Center, Edmond and Lily Safra Children’s
Hospital, Sheba Medical Center, Tel Hashomer, Israel
S. H. Elsea (*)
Department of Molecular and Human Genetics, Baylor College of Medicine,
Houston, TX, USA
e-mail: [Link]@[Link]

© The Author(s), under exclusive license to Springer Nature Singapore Pte 97

Ltd. 2023
A. M. Abdel Rahman (ed.), Clinical Metabolomics Applications in Genetic
Diseases, [Link]
98 J. Manor and S. H. Elsea

1 Population-Wide Untargeted Screening

Variant reclassification, as demonstrated in Chap. 3, is not only important for

accurate genetic counseling but also to guide management in treatable hereditary
disorders, which is best exemplified in IEMs. IEMs constitute a large group of
disorders of inherited defects in metabolic pathways and, if left untreated, can
lead to significant morbidity and mortality [1]. In a global effort to reduce the
IEM morbidity burden and to improve patients’ outcomes, a more than half-­
century-­long effort of newborn screening bore fruition in many countries. What
started in the 1960s as a single disorder screening assay has evolved into an
extended 50+ IEM screening machinery for each newborn [2]. This expansion
was made possible primarily with the advent of MS-based targeted metabolomics
(TM), allowing multiplexed screening in a single sample and facilitating the
overall screening process. This is in contrast to the ad hoc methodology—each
for a single disorder per test—e.g., electrophoresis isoelectric focusing for hemo-
globinopathies and enzyme-linked immunosorbent assay (ELISA) for thyroxine
(T4) quantification. The multiplexed assays have also altered the traditional view
of NBS. From screening for disorders that (a) are occurring at significant fre-
quency, (b) can be screened inexpensively, (c) include effective treatment that can
avert significant morbidity, and (d) cannot be screened based on other signs and
symptoms presented at birth [2], screening is concentrating now on only the sec-
ond principal, and many disorders (some are exceedingly rare) are also caught in
the wider net, despite having inadequate treatment options. One can conceive an
“expanded” NBS not simply as a longer list of disorders that are screened at birth
but actually as a screening procedure for disorders that are now screened for pre-
symptomatically, merely because technology allows it. With the rapid growth of
precision medicine, improved drug delivery, improved blood-brain barrier pene-
tration, and the constant deluge of clinical trials, the need for expanded screening
may prove beneficial quicker than previously imagined. To that end, a NBS
expansion can mean moving forward from TM to untargeted metabolomics (UM).
So, our next question is how does a qualitative UM compared with quantitative
TM when it comes to disease screening?
UM, by probing for not only a relevant subset of predefined metabolites (e.g.,
tyrosine levels in tyrosinemia patients), can identify up to 1000s [3] to 10,000s [4,
5] unique metabolites in each sample. Coupled with high-throughput separation
techniques like high-resolution liquid chromatography (HPLC) or gas chromatogra-
phy (GC), successful identification of disease-defining metabolites is made by dem-
onstrating a statistically significant alteration (elevations or reductions), exceeding
a −2 ≤ log2 ≥ 2 ratio, meaning a four-fold change from the normal range.
Untargeted Metabolomics in Newborn Screening 99

2 Screening for IEM Using UM

Miller et al. employed both GC and LC coupled to single-quadruple MS on 120

patients diagnosed with 21 different IEMs and positively identified 20 IEMs in 112
patients by showing perturbation of 2–20 metabolites in relevant pathways for each
IEM [6]. Coene et al. applied a HPLC in tandem to quadrupole time-of-flight MS as
a single platform on a cohort of 46 IEMs, identifying the key metabolites in 42 [4].
Bonte et al. utilized pentafluorophenylpropyl phase-based HPLC with Orbitrap MS
in a cohort of 33 IEMs, identifying successfully 31 [5]. Haijes et al. applied direct
infusion MS without chromatography-based separation, circumventing the need to
create an experimental library for analytes’ retention times [7]. Screens were per-
formed on both dried blood spots (DBS) and plasma. Two additional cohorts were
constructed for urine samples [8, 9] with biomarkers from 41 IEMs successfully
detected using liquid chromatography coupled to either quadruple time-of-flight
(QTOF) or high-resolution MS. Correct identification was reported for 90–95% of
the IEMs. Results are detailed together in Table 1.
Importantly, UM is performed well on the “can’t miss” diagnoses. Propionic
acidemia (MIM 606054), an autosomal recessive inborn error of metabolism of
propionic acid (PA), is due to a defective propionyl-CoA carboxylase. It results in
the toxic accumulation of PA derived from the catabolism of methionine, valine,
isoleucine, threonine, and odd-chain fatty acids. NBS reveals elevated propionylcar-
nitine (also referred to as C3), and confirmatory testing will show elevations of
2-methylcitrate and 3-OH-propionate. When utilizing UM, researchers found eleva-
tions in both primary and secondary metabolites while noticing a decrease in
2-methylmalonyl carnitine, an analyte associated with the product of propionyl-­
CoA carboxylase. Similar picture was obtained for the other two main organic aci-
demias, methylmalonic aciduria (MIM 251000) and isovaleric aciduria (MIM
243500). For the aminoacidopathy, maple syrup urine disease (MSUD), in which
the keto acids of deaminated leucine, isoleucine, and valine cannot be further catab-
olized resulting in a severe encephalopathic disorder of hyperleucinosis, plasma
was assessed from seven individuals. In these patients, isoleucine, the disease-­
pathognomonic biomarker alloisoleucine, and the corresponding analytes of the
keto acids were found to be elevated, while downstream products were decreased:
3-hydroxyisobutyrate (valine catabolite), isovalerylcarnitine (isoleucine catabolite),
and hydroxyisovalerylcarnitine (leucine catabolite), demonstrating a diagnostic bio-
chemical profile for this disorder (a more complete profile that cannot be obtained
by TM alone). For another can’t miss diagnosis, medium-chain acyl-CoA dehydro-
genase deficiency (MCADD, MIM 201450), an autosomal recessive disorder of
β-oxidation of medium-chain fatty acids, not all known analytes were discovered,
but a near pathognomonic picture with elevations of hexanoylcarnitine (C6) and
octanoylcarnitine (C8) and the dicarboxylic acid suberic and sebacic acid was
obtained, pointing out the diagnosis rather easily. The same was true for phenylke-
tonuria, tyrosinemia type 1 (MIM 276700), and histidinemia (MIM 235800), while
the finding of elevated tetradecenoylcarnitine was detected in four cases of very
Table 1 Successful identification of inborn errors of metabolism (IEM) based on untargeted metabolomics (UM) [4–9, 11, 12, 26–28]
Common diagnostic markers (in bold—when analytes
100

Group Disease MIM used for NBS-DBS) P U DBS RUSP

Disorders of branched- Propionic acidemia 606054 Propionylcarnitine (P), propionylglycine (U), + + CR
chain amino acid 3-hydroxypropionic acid (U), methylcitric acid (P, U)
metabolism Methylmalonyl-CoA mutase 251000 Propionylcarnitine (P), methylmalonic acid (P, U), + + CR
deficiency 3-hydroxypropionic acid (U), methylcitric acid (P, U)
Isovaleric acidemia 243500 Isovalerylcarnitine (P), isovaleric acid (P), + + + CR
isovalerylglycine (U), 3-hydroxyisovaleric acid (U)
3-methylcrotonyl-CoA 210200 Hydroxyisovalerylcarnitine (P), + + + CR
carboxylase deficiency type 1 and 3-methylcrotonylglycine (U)
or 2 210210
2-methyl-3-hydroxybutyryl- 300256 2-methyl-3-hydroxybutyrylcarnitine (P), tiglylglycine + SE
CoA dehydrogenase (P, U)
deficiencyA
2-methylbutyryl-CoA 610006 2-methylbutyrylcarnitine (P, U) + + SE
dehydrogenase deficiency
3-hydroxy-3-methylglutaryl- 246450 Hydroxyisovalerylcarnitine (P), methylglutarylcarnitine + + CR
CoA lyase deficiency (P), 3-hydroxy-3-­methylglutaric acid (U),
3-hydroxyisovaleric acid (P, U), 3-methylglutaconic acid
(P, U)
Methylmalonyl-CoA epimerase 251120 Methylmalonic acid (U) + NO
deficiencyB
Malonyl-CoA decarboxylase 248360 Malonylcarnitine (P), malonic acid (U) + + SE
deficiency
Combined methylmalonic and 614265 Propionylcarnitine (P), malonylcarnitine (P), + NO
malonic aciduria (ACSF3 methylmalonic acid (P, U), malonic acid (U)
deficiency)C
Methylmalonate semialdehyde 614105 3-hydroxyisobutyric acid (U), 3-aminoisobutyric acid (U) + NO
dehydrogenase deficiencyD
Maple syrup urine disease 248600 Leucine (P), isoleucine (P), valine (P), alloisoleucine (P), + +E + CR
J. Manor and S. H. Elsea

2-oxoisocaproic acid (U), 2-oxo-3-methylvaleric acid

(U), 2-oxoisovaleric acid (U)
 Common diagnostic markers (in bold—when analytes
Group Disease MIM used for NBS-DBS) P U DBS RUSP
Disorders of fatty acid Short-chain acyl-CoA 201470 Butyrylcarnitine (P), methylsuccinic acid (P, U), + NO
oxidation and transport dehydrogenase deficiencyF ethylmalonic acid (P, U)
Medium-chain acyl-CoA 201450 Octanoylcarnitine (P), hexanoylcarnitine (P), + + + CR
dehydrogenase deficiency decenoylcarnitine (P), decanoylcarnitine (P),
hexanoylglycine (U), suberylglycine (U),
3-phenylpropionylglycine (U)
Very-long-chain acyl-CoA 201475 Tetradecenoylcarnitine (P), C6-C10 dicarboxylic acids + + CR
dehydrogenase deficiency (U) [e.g., sebacic acid, adipic acid]
Long-chain 3-hydroxyacyl- 609016 Hydroxyhexadecanoylcarnitine (P), 3-hydroxy + + CR
CoA dehydrogenase deficiency octadecenoylcarnosine (P), C6-C14 dicarboxylic acids
(U)
Carnitine palmitoyltransferase 255120 Carnitine (P), reduction in other acylcarnitines, C6-C10 0 +G + SE
I deficiency dicarboxylic acids (U) [e.g., sebacic, suberic, and adipic
Untargeted Metabolomics in Newborn Screening

acids]
Carnitine palmitoyltransferase 600649 Hexadecanoylcarnitine (P), stearoylcarnitine (P), + + SE
II deficiencyH 608836 oleoylcarnitine (P), Linoleoylcarnitine (P)
255110
(continued)
101
Table 1 (continued)
102

Group Disease MIM Common diagnostic markers (in bold—when analytes P U DBS RUSP
used for NBS-DBS)
Common diagnostic markers (in bold—when analytes
Group Disease MIM used for NBS-DBS) P U DBS RUSP
Disorders of ammonia Carbamoyl phosphate 237300 Ammonia (P), glutamine (P), arginine (P) +I NO
detoxification synthetase I deficiency
Ornithine transcarbamylase 300461 Ammonia (P), glutamine (P), arginine (P), orotic acid (U) + 0 NO
deficiency
Citrullinemia type 1 215700 Citrulline (P, U), ammonia (P), glutamine (P), orotic acid + + CR
(U)
Argininosuccinic acid lyase 207900 Citrulline (P), argininosuccinate (P, U), ammonia (P), + CR
deficiency glutamine (P), orotic acid (P, U)
Arginase deficiency 207800 Arginine (P), ammonia (P) glutamine (P) + SE
Hyperammonemia- 238970 Ornithine (P), homocitrulline (U) + +/− NO
hyperornithinemia-
homocitrullinuria syndrome
Disorders of Phenylketonuria, 261600 Phenylalanine (P), phenylpyruvic acid (U) + + + CR
phenylalanine hyperphenylalaninemia
metabolism
Disorders of tyrosine Tyrosinemia type 1 276700 Tyrosine (P), succinylacetone (P, U) + + J CR
metabolism Alkaptonuria 203500 Homogentisate (U) 0 + NO
Hawkinsinuria 140350 4-hydroxycyclohexylacetate (U), hawkinsin (U) + NO
Disorders of sulfur Homocystinuria 236200 Methionine (P), homocysteine (P, U) + + CR
amino acid and sulfide Methionine adenosyltransferase 250850 Methionine (P), S-adenosylmethionine (P), methionine + + NO
metabolism I/III deficiency sulfoxide (U)
Ethylmalonic encephalopathyF 602473 C4-butyrylcarnitine (P), 2-methylbutyrylcarnitine (P), + NO
ethylmalonic acid (P, U), methylsuccinic acid (P, U)
J. Manor and S. H. Elsea
 Common diagnostic markers (in bold—when analytes
Group Disease MIM used for NBS-DBS) P U DBS RUSP
Disorders of lysine Glutaric acidemia type 1 231670 Glutarylcarnitine (P), glutaric acid (U, P), + + +/−K CR
metabolism 3-hydroxyglutaric acid (U)
Pyridoxine-dependent epilepsy 266100 l-α-aminoadipic semialdehyde (P,U), pipecolic acid (P) + NO
Hyperlysinemia type 1 or 2L 268700 Lysine (P, U), saccharopine (P, U) + NO
238700
Disorders of proline and Hyperprolinemia type 2 239510 Proline (P, U), hydroxyproline (P, U), pyrroline-5- + NO
ornithine metabolism carboxylate, (P, U)
Ornithine aminotransferase 613349 Ornithine (P, U), lysine (U), arginine (U) + + NO
deficiency
Disorders of serine and Phosphoglycerate 601815 Low CSF serine, low fasting plasma serine + 0M NO
glycine metabolism dehydrogenase deficiency
Glycine encephalopathyN 605899 Glycine (P) + + NO
Disorder of histidine Histidinemia 235800 Histidine (P, U) + NO
Untargeted Metabolomics in Newborn Screening

metabolism
Disorders of purine Adenylosuccinate lyase 103050 Succinyladenosine (P, U), + + NO
metabolism deficiency succinylaminoimidazolecarboxamide riboside (SAICA
riboside) (P, U)
Hypoxanthine guanine 300322 Uric acid (P, U), hypoxanthine (P, U) + NO
phosphoribosyltransferase and
deficiency 300323
Disorders of pyrimidine Uridine monophosphate 258900 Orotic acid (P, U) + + NO
metabolism synthase deficiency
β-Ureidopropionase 613161 N-carbamoyl-β-alanine (P, U), N-carbamoyl-β- + NO
deficiencyO aminoisobutyric acid (P)
Disorders of carnitine Primary carnitine deficiency 212140 Low free carnitine (P, U) + + CR
metabolism Trimethyllysine hydroxylase 300872 Low free carnitine (P) + NO
epsilon deficiency
(continued)
103
Table 1 (continued)
104

Group Disease MIM Common diagnostic markers (in bold—when analytes P U DBS RUSP
used for NBS-DBS)
Common diagnostic markers (in bold—when analytes
Group Disease MIM used for NBS-DBS) P U DBS RUSP
Disorders of ketone β-Ketothiolase T2 deficiencyA 203750 2-methyl-3-hydroxybutyrylcarnitine (P, U), + + CR
body metabolism tiglylglycine (P, U), 2-methylacetoacetic acid (U)
3-hydroxy-3-methylglutaryl- 605911 4-hydroxy-6-methyl-2-pyrone (U), 3-OH-dicarboxylic + NO
CoA synthase 2 deficiency acids (U)
Disorders of biotin Holocarboxylase synthetase 253270 Hydroxyisovalerylcarnitine (P), + CR
metabolism deficiency 3-methylcrotonylglycine (U), 3-hydroxypropionic acid
(U), lactate (P)
Disorders of cobalamin Cobalamin-related disorderP See commentP + CR
metabolism
Disorders of folate Glutamate 229100 Formiminoglutamate (P, U), hydantoin-5-propionate (U) + NO
metabolism formiminotransferase
deficiency
Methylenetetrahydrofolate 236250 Homocysteine (P) 0 + NO
reductase deficiencyQ
Disorders of riboflavin Multiple acyl-CoA 231680 Glutarylcarnitine (P), isovalerylcarnitine (P), C6-C18 + + SE
metabolism dehydrogenase deficiency esters (P), ethylmalonic acid (U), isovaleric acid (U),
glutaric acid (U), isovalerylglycine (U), hexanoylglycine
(U)
Disorders of Molybdenum cofactor 252150, S-sulfocysteine (P, U), taurine (P, U), xanthine (P, U), low +/− NO
molybdenum deficiencyR 252160, uric acid (P)
metabolism 615501
Xanthinuria type 2R 603529 Hypoxanthine (P, U), xanthine (P, U) + NO
J. Manor and S. H. Elsea
 Common diagnostic markers (in bold—when analytes
Group Disease MIM used for NBS-DBS) P U DBS RUSP
Disorders of Aromatic l-amino acid 608643 High 3-O-methyldopa (CSF, P) and S + NO
neurotransmitters decarboxylase deficiency 5-hydroxytryptophan (CSF); low homovanillic acid (CSF,
P), 5-hydroxyindoleacetic acid (CSF, P), 3-methoxy-4-
hydroxyphenyl glycol (CSF), and dopamine-3-O-sulfate
(P)
Succinic semialdehyde 271980 4-hydroxybutyric acid (U), γ-aminobutyric acid (P) + NO
dehydrogenase deficiency
Disorders of creatine Guanidinoacetate 612736 Guanidinoacetate (P, U), low creatine (P, U) +T + NO
metabolism methyltransferase deficiency
Disorders of amino acid Lysinuric protein intolerance 222700 Lysine (U), ornithine (U), arginine (P, U), citrulline (P), +U + +/− NO
transport orotic acid (U), glutamine (P), ammonia (P)
Disorders of the pentose Transaldolase deficiency 606003 Sedoheptulose (U), sedoheptulose-7-P (U), erythronic +V + NO
phosphate pathway and acid (U), erythritol (U), ribitol (U), arabitol (U)
glutathione synthesis Transketolase deficiency 617044 Arabitol (P,U), erythritol (P, U), ribitol (P, U) + + NO
Untargeted Metabolomics in Newborn Screening

Glutathione synthetase 266130 5-oxoproline (U) + NO

deficiency
Disorders of cholesterol Mevalonic aciduria 610377 Mevalonic acid (U) 0 + NO
and bile acid synthesis Cerebrotendinous 213700 Cholestanol (P) + NO
xanthomatosis
α-Methylacyl-CoA racemase 604489 (25R)-3a,7a,12a-trihydroxy-5b-cholestan-26-oic acid + NO
deficiency (THCA) (P), (25R)-3a,7a-dihydroxy-5b-cholestan-26-oic
acid (DHCA) (P), pristanic acid (S)
Disorders of galactose Galactosemia 230400 Galactose-1-phosphate (P), galactose (P), galactitol (P, +W CR
and fructose metabolism U)
Glycerate kinase deficiency 610516 d-glycerate (P, U) + NO
(continued)
105
Table 1 (continued)
106

Common diagnostic markers (in bold—when analytes

Group Disease MIM used for NBS-DBS) P U DBS RUSP
Aminoacylase deficiency Aminoacylase I deficiency 609924 N-acetylalanine (U), N-acetylglutamic acid (U), + + N
N-acetylglycine (U)
Canavan disease 271900 N-acetylaspartic acid (P, U) + + N
(aspartoacylase deficiency)
Peroxisomal disorders Refsum disease 266500 Phytanic acid (P), pristanic acid (P), pipecolic acid (P, U) +X N
Primary hyperoxaluria type 1 259900 Oxalic acid (P, U), glycolic acid (P, U) +Y N
Oligosaccharidosis β-Mannosidosis 248510 − + N
Disorders of the Krebs Fumarate hydratase deficiency 606812 2-ketoglutarate (U), lactate (P), succinic acid (U), fumaric + N
cycle acid (U)
GTP-specific succinyl-CoA 245400 Propionylcarnitine (P), methylmalonic acid (P, U) +/−Z + N
ligase α subunit deficiency
Disorders of Mitochondrial 603041 Thymidine (P, U), deoxyuridine (P, U) + N
mitochondrial nucleotide neurogastrointestinal
pool maintenance encephalopathy (MNGIE)
Congenital disorders of N-acetylneuraminic acid 610442 N-acetyl-d-mannosamine (P, U) + N
glycosylation phosphate synthase deficiency
MIM accession number of the (online) Mendelian inheritance in man, P plasma (sample), U urine, S serum, DBS dried blood spot, RUSP Secretary of the
Department of Health and Human Services (HHS) Recommended Uniform Screening Panel, + successful identification, 0 failed to identify, +/− identification
of only some diagnostic metabolites
For RUSP, CR denotes a core recommendation, SE, secondary recommendation, and NO, does not appear in the list
A—2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency and β-ketothiolase deficiency cannot be differentiated based on elevated analytes: tiglylgly-
cine, tiglylcarnitine, and 2-methyl-3-hydroxybutyric acid
B—Altered analytes for methylmalonyl-CoA epimerase deficiency are propionylcarnitine and 3-OH-propionic acid, which are also elevated in other disorders
branched-chain amino acid metabolism, e.g., the more prevalent propionic academia
C—Combined methylmalonic and malonic aciduria was identified by the elevation of methylmalonic acid without elevation of malonic acid, making this
metabolic profile nonspecific
D—Elevation of 3-hydroxy-isobutyric acid is suggestive of methylmalonate semialdehyde dehydrogenase deficiency as it does not accumulate in 3-hydroxy-
J. Manor and S. H. Elsea

isobutyryl-CoA hydrolase deficiency (HIBCH, MIM 611283)

Table 1 (continued)
E—In urine, MSUD was identified by elevations of 2-hydroxy-3-methlypentanoic acid, 2-hydroxy-3-methylbutyric acid, and 2-oxoisocaproic acid, which are
different analytes than the “traditional” MSUD analytes of alloisoleucine and leucine (and in addition, valine and isoleucine)
F—Currently, the benign short-chain acyl-CoA dehydrogenase deficiency and the severe ethylmalonic encephalopathy are identified similarly with elevations
in ethylmalonic acid, isobutyrylglycine, and methylsuccinic acid
G—Identification of CPT1 deficiency was achieved based on elevations of medium-chain dicarboxylic acids which are the detectable metabolite in urine in
this condition; however, these findings are nonspecific for this condition and can also indicate medium-chain acyl-CoA dehydrogenase deficiency. In addition,
pyrrole-2-carboxylic acid was significantly elevated in the urine, a finding not known to be associated with CPT1 but rather with hyperprolinemia type 2
[29, 30]
H—Carnitine palmitoyltransferase II deficiency cannot be distinguished based on metabolic profiling from carnitine acylcarnitine translocase deficiency
(MIM 212138)
I—Altered analytes for carbamoyl phosphate synthetase I deficiency include elevation of glutamine and glutamate; both are nonspecific indicators for compen-
sated hyperammonemic status
J—Tyrosinemia of uncertain type was correctly identified by DBS with elevation of 4-hydroxyphenyllactic acid, 4-hydroxyphenyllacetic acid, 4-hydroxyphe-
nylpyruvic acid, and tyrosine but without succinylacetone, fitting for a diagnosis of tyrosinemia type 2 or type 3
K—DBS detected elevations in glutarylcarnitine, similar to traditional targeted screening, but failed to detect the pathognomonic 3-hydroxyglutaric acid
Untargeted Metabolomics in Newborn Screening

(which was detected readily in plasma and urine)

L—Elevations in lysine, N-acetyl-lysine, and l-pipecolic acid cannot distinguish between the two forms of hyperlysinemia
M—Failure of DBS to detect reduction in serine and glycine is attributed to supplementation. However, one should remember that low levels are more difficult
to detect on DBS as the lower limit of normal can approach 0
N—Only 50% of cases of glycine encephalopathy (nonketotic hyperglycinemia) were detected by the elevation of glycine, due to only mild elevations in
the samples
O—Identification of β-ureidopropionase deficiency was made based on elevations of dihydrouracil and dihydrothymine. This profile can also fit dihydropyrim-
idinase deficiency (MIM 222784)
P—Several disorders of intracellular cobalamin metabolism can fit the screening results of elevation of 2-methylmalonylcarnitine, propionylcarnitine, and
2-methylcitrate, a metabolic profile of methylmalonic acidemia (MMA). No homocysteine was detected in plasma, which is not detected in many assays as it
requires an additional unique processing. According to HHS RUSP, screening for cobalamin defects should be performed by screening for MMA. Disorders of
intracellular cobalamin metabolism resulting in isolated MMA elevations (without elevations in homocysteine) are cobalamin A (MMAA, MIM 251100),
cobalamin B (MMAB, MIM 251110), and several cases of cobalamin D (MIM 277410)
Q—Identification of methylenetetrahydrofolate reductase (MTHFR) deficiency was achieved by elevation of homocysteine derivatives (homocysteine thiolac-
tone and homocysteine). This is not specific for MTHFR deficiency and can be found also in other re-methylation defects, such as cobalamin G (MIM 250940),
cobalamin E (MIM 236270), and some cobalamin D (MIM 277410) defects. See also comment P above for homocysteine detection
107
Table 1 (continued)
108

R—Consistently found altered in MoCD in the cohorts above is elevation of xanthine. Only one plasma sample presented, in addition, statistically significant
reduction in uric acid and elevation of S-sulfocysteine [7]. Other samples did not show statistically significant reduction in uric acid including samples from
DBS. Elevation of xanthine and reduction of uric acid can also be seen in xanthinuria type 1 (MIM 278300) and type 2 (MIM 603592)
S—Successful identification of this disorder was previously reported [31] by identification of elevated 3-methoxytyrosine (3-O-methyldopa) in plasma, a
methylation product of l-DOPA by catechol-O-methyltransferase (COMT). 3-methoxytyrosine is elevated when l-DOPA is not further metabolized to dopa-
mine due to defective aromatic l-amino acid decarboxylase (AADC)
T—Guanidinoacetate failed detection in two cohorts but was identified in a third
U—Lysinuric protein intolerance was identified successfully in two studies while two others failed. LPI diagnostic rates increase when combined with a
urine sample
V—In one case, only sedoheptulose was detected in the sample. This finding is not specific for transaldolase deficiency and can be seen in isolated sedoheptu-
lose kinase deficiency (MIM 617213), a benign condition but also due to a common copy number variant found in patients with cystinosis (MIM 219800)
W—In plasma, only galactitol was identified in a single case of galactosemia. Galactitol will be elevated also in galactokinase deficiency (MIM 230200) and
galactose epimerase deficiency (MIM 230350). In urine, elevations in galactose were also seen, giving a similar nonspecific picture
X—Identification of Refsum disease (phytanoyl-CoA hydroxylase deficiency) was made based on elevation of phytanic acid. Elevation of pipecolic acid was
not seen. Elevation in phytanic acid can also be seen in peroxisomal biogenesis defects (Zellweger spectrum disorder) and rhizomelic chondrodysplasia punc-
tata (MIM 215100)
Y—Identification of primary hyperoxaluria type 1 was made based on elevation of glycolic acid. These elevations can also be seen in fumarate hydratase
deficiency (in which fumaric acid will be elevated) or d-2-hydroxyglutaric acid (in which 2-OH-glutaric acid will be elevated)
Z—Elevated MMA was detected in plasma, indicating a disorder of elevated MMA. In urine, the additional detection of elevated lactate creates a more specific
diagnosis of this disorder of mitochondrial DNA depletion
J. Manor and S. H. Elsea
Untargeted Metabolomics in Newborn Screening 109

Table 2 Examples of diagnostic metabolites detected by untargeted metabolomics leading to non-­

benign IEMs that are not routinely screened by NBS
Metabolite(s) Condition(s) MIM Specimen
N-acetylalanine, Aminoacylase I deficiency 609924 Plasma,
N-acetylglutamate, urine
N-acetylmethionine,
N-acetylleucine
N-acetylaspartate Canavan disease 271900 Plasma
Orotic acid Uridine monophosphate synthase 613891 Plasma,
deficiency (primary), urea cycle (primary) urine
defects (secondary)
Mevalonate Mevalonic aciduria 610377 Urine
Homocysteine thiolactone/ Methylenetetrahydrofolate reductase 236250 Plasma,
homocysteinea deficiency, cobalamin-related 236270 DBS
disorders 250940
277380
277400
277410
309541
Xanthine, S-sulfocysteine Molybdenum cofactor deficiency 252150 Plasma
types A, B and C, isolated sulfite 252160
oxidase deficiency 615501
272300
Succinyladenosine Adenylosuccinate lyase deficiency 103050 Plasma
3-methoxytyrosine Aromatic l-amino acid 608643 Plasma,
(3-O-methyldopa) decarboxylase deficiency urine
Guanidinoacetate↑, Guanidinoacetate methyltransferase 612736 Plasma,
+/− ↓creatine deficiency DBS
Proline and ornithine Ornithine aminotransferase 258870 Plasma,
deficiencyb DBS
↓Lysine, ↓ornithine, Lysinuric protein intolerance 222700 Plasma
↓arginine
Glycine Nonketotic hyperglycinemia 605899 Plasma,
DBS
Sedoheptulose Transaldolase deficiency, rare cases 606003 Plasma
of cystinosis
↓Serine, ↓glycine Phosphoglycerate dehydrogenase 256520 Plasma
deficiencyc 601815
Cholestane-3,7,12,25,25-­ Cerebrotendinous xanthomatosis 213700 Plasma
pentol
Di- and tri-hydroxy-5b-­ α-Methylacyl-CoA racemase 614307 Plasma
cholestan-26-oic acidsd deficiency
N-acetyl-d-mannosamine N-acetylneuraminic acid phosphate 610442 Plasma
synthase deficiency
Phytanic acide Peroxisomal biogenesis defects, 266500 Plasma
Refsum disease (Refsum)
Fumaric acid Fumarate hydratase deficiency 606812 Urine
110 J. Manor and S. H. Elsea

Table 2 (continued)
Metabolite(s) Condition(s) MIM Specimen
4-Hydroxy-6-methyl-2-­­ 3-hydroxy-3-methylglutaryl-CoA 605911 Urine
pyroned synthase 2 deficiency
2-Pyrrolidinone, succinamic GABA transaminase deficiency 613163 Plasma
acid
UM untargeted metabolomics, IEM inborn error of metabolism, NBS newborn screening, ↓ reduc-
tion in metabolite; otherwise, alteration is elevation
a
With normal levels of methionine. Reported only by some laboratories as the detection of homo-
cysteine requires special preparatory steps
b
Elevations of proline and ornithine may not be seen in ornithine aminotransferase deficiency dur-
ing the neonatal period due to substrate stoichiometry
c
Similar metabolomics picture can be seen in phosphoserine aminotransferase 1 deficiency
(PSAT1D, MIM 616038), which can present similarly to phosphoglycerate dehydrogenase
deficiency
d
Not routinely reported by all laboratories
e
Alterations may also include the elevation of pipecolic acid

long-chain acyl-CoA dehydrogenase deficiency (VLCADD, MIM 201475) in one

study [4], although undetected in two cases from another study [6]. Homocysteine
and homocysteine thiolactone are both accumulating in cystathionine β-synthase
deficiency (homocystinuria, MIM 236200) and 5,10-methylenetetrahydrofolate
reductase deficiency (MTHFR, MIM 607093) [1, 10]. These two disorders were
successfully diagnosed in DBS by demonstration of significant elevation in one of
the two analytes in conjunction with elevation of methionine (a “can’t miss” diag-
nosis) in the former but not the latter. Homocysteine was only elevated in DBS, as
in plasma metabolomics homocysteine is not detected using high-throughput meth-
odology for sample prep, which does not allow for the pretreatment required for
proper analysis.
Patients with β-ketothiolase deficiency (T2 deficiency, MIM 203750), a disorder
of isoleucine breakdown and ketone utilization deficiency, were found to have ele-
vation of tiglylglycine and 2-methyl-3-hydroxybutyric acid with no elevation of
2-methylacetoacetic acid, making this metabolic profile indistinguishable from
2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency (HSD17B10 defi-
ciency, MIM 300438). Contrarily, for the single patient with 2-methyl-3-­
hydroxybutyric deficiency in the cohorts, tiglylglycine was elevated, but
2-methyl-3-hydroxybutyric was not. This suggests that tiglylglycine is a sensitive
marker for both of these two distal isoleucine breakdown disorders; however, spe-
cific biomarkers for either disorder are lacking.
An important group of disorders that are only partially screened for in NBS are
urea cycle defects. Most notably is the severe ornithine transcarbamylase deficiency,
an X-linked urea cycle defect (OTCD, MIM 300461) for which early detection is
crucial for improved outcomes, yet this condition is difficult to detect due to limita-
tion on measuring low quantities (of citrulline in the case of OTCD) and extraction
of orotic acids in high-throughput methods. In one case of OTCD, significant eleva-
tions of uridine were found, which is a secondary disease biomarker. The elevation
Untargeted Metabolomics in Newborn Screening 111

in uridine stems from shunting of carbamoyl phosphate from the urea cycle into the
pyrimidine biosynthesis pathway via the orotic acid intermediate; however, orotic
acid was not elevated in the two patients with OTCD. For citrin deficiency (citrul-
linemia type 2, MIM 603471 and 605814), citrulline was elevated along with amino
acid markers of liver injury (tyrosine and its degradation products
4-­hydroxyphenyllactic acid 4-hydroxyphenylpruvic acid, lysine, arginine, and
5′-S-methyl-5′-thioadenosine), a common finding in neonatal presentation of this
disorder. In a patient with hyperornithinemia-hyperammonemia-homocitrullinuria
syndrome (HHH, MIM 238970), a secondary UCD caused by mitochondrial orni-
thine transporter defect, only general markers for urea cycle dysfunction were iden-
tified (elevations in uracil and orotic acid), yet this finding should merit a focused
investigation for UCDs. Homocitrulline and ornithine were both elevated in plasma
UM of a 30-year-old patient with HHH, [11], while it failed to detect elevations in
citrulline in a patient with citrin deficiency in another study [12]. For OTCD, DBS
samples showed orotic acid elevation in one of two cases; however, no significant
elevations in uridine or uracil were found, in contrast to plasma, where both were
elevated. Nonetheless, these results are somewhat encouraging giving hope for
expanded DBS screening for the most common urea cycle defect [2], with severe
presentation in males and no current screening protocols.
A few important examples demonstrate the increased diagnostic repertoire of
UM (see Table 2). Lysinuric protein intolerance (LPI, 222700) is a disorder caused
by defective cationic amino acid (CAA) transport at the basolateral membrane of
epithelial cells in the kidney and intestine leading to increased renal excretion of
CAA leading to the depletion of mainly lysine, arginine, and ornithine, the latter
leading to secondary urea cycle defect (UCD) which can result in hyperammone-
mia. UM in two patients identified excess glutamine, a marker for compensated
hyperammonemia and reduction of plasma CAA (although mild in one study). In
urine, low glutarylcarnitine and N6-trimethyllysine were recorded, with mildly ele-
vated N6-acetyllysine [9]; the latter two are products of lysine catabolism. Urine
levels of CAA did not differ significantly from the control.
Successful identifications of four severe nucleotide degradation IEMs have been
reported, two pyrimidine degradation disorders, β-ureidopropionase deficiency
(MIM 606673), in plasma, and dihydropyrimidine dehydrogenase deficiency (MIM
274270), in urine, and two purine metabolism syndromes: X-linked hypoxanthine-­
guanine phosphoribosyltransferase deficiency (causing either Lesch-Nyhan syn-
drome (MIM 300322) or HPRT-related hyperuricemia (MIM 300323)) in two
patients (one each in plasma and in urine) and adenylosuccinate lyase deficiency
(MIM 608222) in the other two (also one each in plasma and in urine). These results
are promising, as currently purine and pyrimidine metabolism defects can cause a
severe neurological phenotype which may be amenable for treatment but currently
are not routinely screened among presymptomatic newborns. Moreover, elevation
of xanthine and xanthosine (purine catabolites) and low uric acid (the degradation
product of xanthine) were found in five patients, three with molybdenum cofactor
deficiency (MoCD, MIM 252150) and two with xanthinuria type 2 (MIM 603592).
The former disorder is a severe, rapidly progressive encephalopathy due to
112 J. Manor and S. H. Elsea

deficiency in the molybdenum and biopterin cofactors, the latter a benign disorder
of accumulation of xanthine due to defect in xanthine oxidase and aldehyde oxidase
(AO, affecting mainly N1-methylnicotinamide degradation). Of note, elevations in
the specific MoCD marker S-sulfocysteine was seen in only one sample, while thio-
sulfate and taurine were not detected. On DBS, increased levels of α-amino adipic
semialdehyde (AASA) were also seen, which are also observed in sulfite oxidase
deficiency (MIM 272300) and AASA deficiency (antiquitin deficiency, also called
pyridoxine-dependent epilepsy, MIM 266100) [13], both are disorders bearing
importance of early screening. For the MoCD samples without elevated
S-sulfocysteine, both patients were under treatment; however, a profile containing
elevations in xanthine and xanthosine and reduction of uric acid is expected to
include sulfa-containing analytes, as well. Identification of these metabolites can
facilitate the diagnosis of MoCD, which for its most common genetic cause a thera-
peutic option is available, and avoidance of permanent damage can be achieved if
therapy is initiated within days after birth [14]. 3-phosphoglycerate dehydrogenase
(3-PGDH) deficiency (Neu-Laxova syndrome, MIM 606879), a severe neurodevel-
opmental due to serine biosynthesis defect, is characterized by low plasma levels of
serine and glycine, and while this profile was correctly captured in the plasma, it
was not observed in DBS, presumably due to treatment.
Cerebrotendinous xanthomatosis (CTX, MIM 213700), an autosomal recessive
bile acid synthesis disorder resulting in a neurodegeneration and premature athero-
sclerosis due to diffuse xanthomata, was successfully identified in five patients due
to elevations in cholestane-3,7,12,24,25-pentol, a specific marker for CTX [15].
These results would allow early treatment with chenodeoxycholic acid early in the
course of the disease and can prevent neurodegeneration in a disorder otherwise
diagnosed well into the development of systemic symptoms. Elevations of phytanic
acids were seen in one patient with adult Refsum disease (MIM 266500), a disease
of defective α-oxidation of phytanoyl esters into pristanic esters, resulting in vision
decline, hearing loss, polyneuropathy, ichthyosis, and cardiac conduction defects.
Early recognition can initiate low phytanic acid diet can improve significantly the
overall prognosis of this disorder that is usually diagnosed after the appearance of
symptoms [1].
Unlike organic acidemias, amino acidopathies, and urea cycle disorders, the
screening for organelle-based diseases, such as storage diseases, lags behind with
currently only two types of mucopolysaccharidoses being screened [16]. UM offers
hope as a highly sensitive screening tool for additional metabolites that can indicate
abnormal intra-organelle catabolism, which are pathognomonic for certain disor-
ders. For lysosomes, β-mannosidosis, a neurodegenerative lysosomal storage disor-
der (LSD) due to a defect in removal of β-d-mannose from glycoproteins (MIM
248510), was successfully identified by elevation of the pathognomonic mannosyl-­
β1,4-N-acetylglucosamine (GlcNAc-Man). In the mitochondria, mitochondrial
neurogastrointestinal encephalopathy (MNGIE, MIM 603041) is a multisystem dis-
order of mitochondrial DNA depletion causing severe gastrointestinal (GI) dys-
motility with peripheral neuropathy, hearing and vision impairment, and early death
in the fourth decade [17]. On average, diagnosis is delayed by 12 years [18] due to
Untargeted Metabolomics in Newborn Screening 113

the initial nonspecific GI symptoms due to deficiency of thymidine phosphorylase.

Here, UM screening assists in early diagnosis, with elevations of thymidine and
deoxyuridine, and, thus, can reduce morbidity with the emergence of new thera-
pies [17].
In the cohorts of urine samples [8, 9], two patients with aromatic l-amino acid
decarboxylase deficiency (AADCD, MIM 608643), a monoamine neurotransmitter
synthesis defect, had significant elevations of vanillactic acid, which is expected to
increase with the accumulation of l-DOPA, a substrate of AADC. In addition,
4-hydroxybutyric acid was elevated in two patients with succinic semialdehyde
dehydrogenase deficiency (SSADHD, MIM 271980), a γ-aminobutyric acid
(GABA) catabolism defect. Both conditions are severe and are not typically
screened, with emerging gene therapy options for the former [19, 20]. Additionally,
adenosine deaminase deficiency, a purine degradation disorder that is the fourth
most common etiology for severe combined immunodeficiency (SCID, MIM
102700) [21], was successfully screened by the identification of 2-deoxyadenosine
and 2-deoxyinosine [9].
Few notable limitations of UM are worth mentioning. For GAMT deficiency,
which converts guanidinoacetate (GAA) and S-adenosylmethionine (SAM) into
creatine and S-adenosylhomocysteine (SAH), only one study out of the three suc-
cessfully identified GAA accumulation and reduction of creatine in plasma.
However, more recently, an increased sensitivity to GAA is reported by laborato-
ries, which may lead to improved identification of GAMT deficiency. In plasma,
homogentisic acid was not detected in the case of alkaptonuria, a tyrosine break-
down disorder causing a degenerative joint and cartilage disorder (MIM 203500),
and similarly, mevalonic acid was not detected for mevalonic aciduria, a heteroge-
neous multisystemic disorder of cholesterol synthesis (MIM 610377 and 260920).
In urine samples, however, both homogentisic acid and mevalonic acid were
detected readily [8]. For LPI, not all cohorts detected low levels of CAA, leaving the
nonspecific hyperglutaminemia in plasma the only consistent finding in these cases.
In urine, alteration of lysine metabolites was noted (low N6-trimethyllysine and high
N6-acetyllysine), as mentioned above.
Importantly, despite all methods grouped under “UM,” different preparation
techniques yielded different metabolic profiles and detection rates of the IEMs.
Examples are LPI, VLCADD, GAMT deficiency, and alkaptonuria, in which the
primary metabolites CAA, C14:1, GAA, and homogentisic acid were found only in
some studies but not in others. Additionally, while UM detected up to 10,000 ana-
lytes per sample [4], only about 300–400 analytes were matched to known library
of reference and analyzed for perturbations to create metabolic profiling [4, 5].
Large population screening, such as newborn screening, must maintain consistency
in the selection of derivatives to be detected; the same small group of representative
molecules must be detected in each and every sample. To be effectively used for
large population screening, steps for streamlining sample collection and preparation
must be undertaken, as well efforts to reduce unreliable peak integration in the anal-
ysis process, supporting semiautomated result interpretation [22]. One approach is
114 J. Manor and S. H. Elsea

the implementation of automated interpretation pipeline [23] or utilization of

machine learning to more clearly define biochemical phenotypes [24, 25].

Glossary

Dried blood spot (DBS) A method of whole blood sample collection, in which a
small amount of fresh blood is blotted onto an absorbent filter paper, followed by
drying. This method provides a convenient storage and shipment platform and
is widely used for newborn screening. Typically, a small punch from the DBS
paper is eluted with phosphate-buffered saline, availing the sample for testing.
Multiplex assay An assay measuring simultaneously multiple analytes in a single
testing. These tests are becoming more popular in the metabolic sciences where
several similar analytes are tested for alterations from the normal range, e.g.,
urine polyols for evaluation of the pentose phosphate pathway, urine glycosami-
noglycans for the diagnosis of mucopolysaccharidoses, or carbohydrate moieties
for congenital disorders of glycosylation.

References

1. Saudubray JM, Baumgartner MR, Walter JH. Inborn metabolic diseases: diagnosis and treat-
ment. Berlin, Heidelberg: Springer; 2016.
2. Lee B, Scaglia F. Inborn errors of metabolism : from neonatal screening to metabolic path-
ways. New York, NY: Oxford University Press; 2015.
3. Wright Muelas M, Roberts I, Mughal F, O'Hagan S, Day PJ, Kell DB. An untargeted metabo-
lomics strategy to measure differences in metabolite uptake and excretion by mammalian cell
lines. Metabolomics. 2020;16(10):107.
4. Coene KLM, Kluijtmans LAJ, van der Heeft E, Engelke UFH, de Boer S, Hoegen B, et al.
Next-generation metabolic screening: targeted and untargeted metabolomics for the diagnosis
of inborn errors of metabolism in individual patients. J Inherit Metab Dis. 2018;41(3):337–53.
5. Bonte R, Bongaerts M, Demirdas S, Langendonk JG, Huidekoper HH, Williams M, et al.
Untargeted metabolomics-based screening method for inborn errors of metabolism using
semi-automatic sample preparation with an UHPLC-Orbitrap-MS Platform. Metabolites.
2019;9(12):289.
6. Miller MJ, Kennedy AD, Eckhart AD, Burrage LC, Wulff JE, Miller LA, et al. Untargeted
metabolomic analysis for the clinical screening of inborn errors of metabolism. J Inherit Metab
Dis. 2015;38(6):1029–39.
7. Haijes HA, Willemsen M, Van der Ham M, Gerrits J, Pras-Raves ML, Prinsen H, et al. Direct
infusion based metabolomics identifies metabolic disease in patients’ dried blood spots and
plasma. Metabolites. 2019;9(1):12.
8. Körver-Keularts I, Wang P, Waterval H, Kluijtmans LAJ, Wevers RA, Langhans CD, et al. Fast
and accurate quantitative organic acid analysis with LC-QTOF/MS facilitates screening of
patients for inborn errors of metabolism. J Inherit Metab Dis. 2018;41(3):415–24.
9. Kennedy AD, Miller MJ, Beebe K, Wulff JE, Evans AM, Miller LA, et al. Metabolomic pro-
filing of human urine as a screen for multiple inborn errors of metabolism. Genet Test Mol
Biomarkers. 2016;20(9):485–95.
Untargeted Metabolomics in Newborn Screening 115

10. Jakubowski H, Boers GH, Strauss KA. Mutations in cystathionine beta-synthase or methy-
lenetetrahydrofolate reductase gene increase N-homocysteinylated protein levels in humans.
FASEB J. 2008;22(12):4071–6.
11. Liu N, Xiao J, Gijavanekar C, Pappan KL, Glinton KE, Shayota BJ, et al. Comparison of
untargeted metabolomic profiling vs traditional metabolic screening to identify inborn errors
of metabolism. JAMA Netw Open. 2021;4(7):e2114155.
12. Almontashiri NAM, Zha L, Young K, Law T, Kellogg MD, Bodamer OA, et al. Clinical valida-
tion of targeted and untargeted metabolomics testing for genetic disorders: a 3 year compara-
tive study. Sci Rep. 2020;10(1):9382.
13. Mills PB, Footitt EJ, Ceyhan S, Waters PJ, Jakobs C, Clayton PT, et al. Urinary AASA excre-
tion is elevated in patients with molybdenum cofactor deficiency and isolated sulphite oxidase
deficiency. J Inherit Metab Dis. 2012;35(6):1031–6.
14. Schwahn BC, Van Spronsen FJ, Belaidi AA, Bowhay S, Christodoulou J, Derks TG, et al.
Efficacy and safety of cyclic pyranopterin monophosphate substitution in severe molybdenum
cofactor deficiency type a: a prospective cohort study. Lancet. 2015;386(10007):1955–63.
15. Shefer S, Dayal B, Tint GS, Salen G, Mosbach EH. Identification of pentahydroxy bile alco-
hols in cerebrotendinous xanthomatosis: characterization of 5beta-cholestane-3alpha, 7alpha,
12alpha, 24xi, 25-pentol and 5beta-cholestane-3alpha, 7alpha, 12alpha, 23xi, 25-pentol. J
Lipid Res. 1975;16(4):280–6.
16. Jalal K, Carter RL, Barczykowski A, Tomatsu S, Langan TJ. A roadmap for potential improve-
ment of newborn screening for inherited metabolic diseases following recent developments
and successful applications of bivariate Normal limits for pre-symptomatic detection of MPS
I, Pompe disease, and Krabbe disease. Int J Neonatal Screen. 2022;8(4):61.
17. Bax BE. Mitochondrial neurogastrointestinal encephalomyopathy: approaches to diagnosis
and treatment. J Transl Genet Genom. 2020;4:1–16.
18. Danjou M, Guardia D, Geoffroy PA, Seguy D, Cottencin O. Mitochondrial neuro-gastro-­
intestinal encephalopathy (MNGIE): when and how to suspect it in front of an atypical
anorexia nervosa? Encéphale. 2016;42(6):574–9.
19. Tai CH, Lee NC, Chien YH, Byrne BJ, Muramatsu SI, Tseng SH, et al. Long-term efficacy and
safety of eladocageneexuparvovec in patients with AADC deficiency. Mol Ther. 2021;30:509.
20. Merola A, Kobayashi N, Romagnolo A, Wright BA, Artusi CA, Imbalzano G, et al. Gene
therapy in movement disorders: a systematic review of ongoing and completed clinical trials.
Front Neurol. 2021;12:648532.
21. Routes J, Verbsky J. Newborn screening for severe combined immunodeficiency. Curr Allergy
Asthma Rep. 2018;18(6):34.
22. Chetnik K, Petrick L, Pandey G. MetaClean: a machine learning-based classifier for reduced
false positive peak detection in untargeted LC-MS metabolomics data. Metabolomics.
2020;16(11):117.
23. Hoegen B, Zammit A, Gerritsen A, Engelke UFH, Castelein S, van de Vorst M, et al.
Metabolomics-based screening of inborn errors of metabolism: enhancing clinical application
with a robust computational pipeline. Metabolites. 2021;11(9):568.
24. Heinemann J. Machine learning in untargeted metabolomics experiments. Methods Mol Biol.
2019;1859:287–99.
25. García-Pérez P, Zhang L, Miras-Moreno B, Lozano-Milo E, Landin M, Lucini L, et al. The
combination of untargeted metabolomics and machine learning predicts the biosynthesis of
phenolic compounds in Bryophyllum medicinal plants (genus Kalanchoe). Plants (Basel).
2021;10(11):2430.
26. Odom JD, Sutton VR. Metabolomics in clinical practice: improving diagnosis and informing
management. Clin Chem. 2021;67(12):1606–17.
27. Alaimo JT, Glinton KE, Liu N, Xiao J, Yang Y, Reid Sutton V, et al. Integrated analysis of
metabolomic profiling and exome data supplements sequence variant interpretation, classifica-
tion, and diagnosis. Genet Med. 2020;22(9):1560–6.
116 J. Manor and S. H. Elsea

28. Shayota BJ, Donti TR, Xiao J, Gijavanekar C, Kennedy AD, Hubert L, et al. Untargeted metab-
olomics as an unbiased approach to the diagnosis of inborn errors of metabolism of the non-­
oxidative branch of the pentose phosphate pathway. Mol Genet Metab. 2020;131(1–2):147–54.
29. Heacock AM, Adams E. Formation and excretion of pyrrole-2-carboxylate in man. J Clin
Invest. 1974;54(4):810–8.
30. Wajner M, Wannmacher CM, Purkiss P. High urinary excretion of N-(pyrrole-2-carboxyl) gly-
cine in type II hyperprolinemia. Clin Genet. 1990;37(6):485–9.
31. Atwal PS, Donti TR, Cardon AL, Bacino CA, Sun Q, Emrick L, et al. Aromatic L-amino acid
decarboxylase deficiency diagnosed by clinical metabolomic profiling of plasma. Mol Genet
Metab. 2015;115(2–3):91–4.
Untargeted Metabolomics, Targeted Care:
The Clinical Utilities of Bedside
Metabolomics

Joshua Manor and Sarah H. Elsea

Abstract The second decade of the twenty-first century saw a quiet revolution in
the field of inborn errors of metabolism. Decades of extensive research into meta-
bolic pathways of physiologically active cells and tissues, along with an improved
resolution of high-throughput screening capabilities, brought forth the clinical
metabolome. Clinicians can now take a metabolic snapshot while assessing their
patients and receive invaluable information on pathological processes, rule in or
rule out a proposed diagnosis, highlight early signs of decompensation, assess
response to treatment, explore new disease biomarkers, and even suggest novel
treatment options. In this chapter, we review the major strengths of clinical metabo-
lomics as a diagnostic aid and its capabilities in promoting novel biomarker discov-
ery. We also provide an outlook for how next-gen interpretation modalities (such as
machine learning) are expected to revolutionize this field further to benefit patients
worldwide.

Keywords Clinical metabolomics · Inborn error of metabolism (IEM) · Mass

spectrometry (MS) · Dried blood spot (DBS) · Newborn screening (NBS) ·
Biomarker discovery

J. Manor
Metabolic Unit, Haim Sheba Medical Center, Edmond and Lily Safra Children’s Hospital,
Tel Hashomer, Israel
S. H. Elsea (*)
Department of Molecular and Human Genetics, Baylor College of Medicine,
Houston, TX, USA
e-mail: [Link]@[Link]

© The Author(s), under exclusive license to Springer Nature Singapore Pte 117
Ltd. 2023
A. M. Abdel Rahman (ed.), Clinical Metabolomics Applications in Genetic
Diseases, [Link]
118 J. Manor and S. H. Elsea

1 Introduction

In recent years, clinical practice has seen a dramatic increase in the utilization of
targeted and untargeted metabolomics (TM and UM, respectively) [1, 2].
Metabolomics has been termed “the stethoscope of the twenty-first century” with
broad applications in many fields of medicine: In oncology, it is utilized for bio-
markers; in neurology, for severity stratification of neurodegenerative disorders; in
endocrinology for diabetes modulators; in rheumatology and cardiology where cer-
tain metabolites may improve prognostication of chronic diseases (e.g., osteoarthri-
tis and atherosclerosis); and in gastroenterology, where metabolomics may assist in
differentiating between Crohn’s disease and ulcerative colitis [3]. UM has been used
in multifactorial disorders to assist in risk assessment and early diagnosis, such as
the observation that increased plasma levels in three out of five aromatic and
branched-chain amino acids (isoleucine, leucine, valine, tyrosine, and phenylala-
nine) confers a fivefold increased likelihood for developing type 2 diabetes [4] or
that increased plasma levels of glycocholate, taurocholate, and glycochenodeoxy-
cholate are associated with nonalcoholic fatty liver disease (NAFLD). In contrast,
decreased plasma levels of free carnitine, butyrylcarnitine, methyl-butyryl carnitine,
and cysteine-glutathione are seen in nonalcoholic steatohepatitis (NASH) [5].
Similarly, breast cancer showed a pattern of increased total choline-containing sub-
stances and decreased glycerophosphocholine in the plasma [6], correlating malig-
nancy to a glycerophosphocholine-to-phosphocholine ratio switch [7].
Naturally, UM can highlight alterations in complex “metabolic disorders” and
refine our understanding of metabolic flux in health and disease. The untargeted
global assessment can further provide a high-resolution cellular homeostasis map
and multivariant perturbation metrics that can assist in prognostication, the need for
intervention, and the effect of a therapeutic modality. From a clinical perspective, a
rare disease is a primary focus, and areas of influence for untargeted metabolomics
include improving diagnostic rates, allowing affordable high-throughput disease
screening, and identifying novel disease biomarkers that can be translated to the
clinic. UM can contribute significantly toward a facilitated diagnosis and direct tar-
geted treatment, thus enabling a reduction of the traditionally high morbidity and
mortality associated with the under-recognition and undertreatment of metabolic
disorders.

2 Metabolomics Joins the Diagnostic Front Seat

Clinicians justifiably consider exome sequencing (ES) to be the panacea of all diag-
nostic dilemmas, particularly when routine laboratory studies and tissue biopsies
fail to establish a diagnosis. Indeed, ES has ushered in a new diagnostic era. Thanks
to the reduced cost of sequencing, massive utilization of ES is now widely available,
turning it into first-tier clinical testing for diagnostic evaluation of developmental
Untargeted Metabolomics, Targeted Care: The Clinical Utilities of Bedside Metabolomics 119

delay and other congenital anomalies [8–10]. ES is perhaps most heavily relied
upon in the neonatal intensive care unit (ICU), in which a third of admitted patients
are due to genetic causes [11]. ES is also heavily relied upon for primary mitochon-
drial disorders (PMD) [12], for which there is a notorious lack of biomarkers with
adequate specificity [13]. This is further exemplified by the staggering finding of
normal muscle respiratory chain enzymes in 10–20% of cases with mitochondrial
myopathy undergoing invasive diagnostic procedures [14].
Nonetheless, as intriguing as it may appear, a diagnosis relying solely on ES
without additional laboratory results lowers the test’s pretest probability and
increases the likelihood of false-positive results [15]. The American College of
Medical Genetics and Genomics (ACMG) recommends that effort should be made
to avoid using the pathogenicity of a variant as the sole evidence of a Mendelian
disease but rather should be used in conjunction with other clinical information
[16]. Perhaps, the best demonstration of the chasm between the diagnostic promise
of ES and reality is variants of uncertain significance (VUS) [17], perceived as a
“challenge” in ~1/3 of the articles reporting ES results [18]. With inconclusive
molecular data, generating a snapshot of metabolites during illness creates a func-
tional bioassay for candidate metabolic pathways. It provides an independent tool
for ruling in or ruling out suspected diagnoses. Following the ACMG variant inter-
pretation algorithm [16], metabolomic data fall into the functional data category by
providing “well-established functional studies to show deleterious [or non-­
deleterious] effect,” which is considered strong supportive information for the
pathogenic or benign nature of a variant. Metabolomic data, therefore, can push the
needle from the neutral zone (a VUS) to the actionable zone [19, 20]. It is, neverthe-
less, of utmost importance to report only significant variations in metabolites to
avoid reporting fluctuations stemming merely from dietary changes, environmental
factors, drug exposure, and normal daily changes in metabolism. However, the defi-
nition of significance is still laboratory dependent [21]. It is also of equal impor-
tance to provide detailed clinical information and the nutritional and therapeutic
status of the patient to the performing laboratory to optimize the data analysis.
Given that a VUS is a common finding in ES, estimated to occur in 30–80% of clini-
cally indicated ES tests [22, 23], and comprises ~30% of variants found by targeted
sequencing for suspected inborn errors of metabolism (IEMs) [24], a variant valida-
tion and classification tool alongside molecular diagnostics is critical. UM serves as
a valuable biochemical, functional validation instrument that can be integrated into
clinical care.

2.1 Monoamine Synthesis

The utilization of UM for VUS reclassification was demonstrated by Atwal et al. in

a diagnostic odyssey of an 11-month-old boy presenting with intellectual disability
and hypotonia with episodes of generalized stiffening [25]. Initially diagnosed with
cerebral palsy, exome sequencing showed two missense VUS in DCC, which
120 J. Manor and S. H. Elsea

encodes aromatic-l-amino acid decarboxylase (AADC), a key enzyme in mono-

amine synthesis. AADC deficiency (AADCD) results in a severe neurometabolic
disorder due to the combined deficiency of serotonin, dopamine, norepinephrine,
and epinephrine. The onset of the disease is typically in the first months of life.
However, the phenotypic spectrum of disease is broad and includes hypotonia, ocu-
logyric crises, ptosis, dystonia, hypokinesia, developmental delays, and autonomic
dysfunction [26]. Diagnosis is typically made by abnormal monoamine metabolites
in CSF, followed by molecular confirmation or enzyme analysis in plasma. Elevation
of 3-O-methyldopa (3-OMD), also termed 3-methoxytyrosine (3-MT), a catabolite
of dihydroxyphenylalanine (l-DOPA), indicating a blockage in the conversion of
l-DOPA into dopamine. For the patient described in Atwal et al., UM showed a
plasma level of 3-MT 69 times higher than the control population.
Further confirmation of the UM findings was achieved by demonstrating abso-
lute levels of 3-MT in CSF > three-fold the upper limit of normal, along with unde-
tectable levels of the dopamine and serotonin derivatives, homovanillic acid (HVA),
and 5-hydroxy indoleacetic acid (5-HIAA), respectively. Neurotransmitter analysis
in CSF has been considered the gold standard for diagnosing AADC deficiency. Yet,
this work presents that UM can attain similar results in plasma without requiring an
invasive procedure. At times of atypical presentation and a wide differential diagno-
sis, UM can cast a wide net in a single test and may render specific diagnostic tests
and procedures, each for a single suspected disease, inessential. The presentation
can be atypical for the late-onset subgroup of patients with AADCD, including
milder symptoms of hypotonia, dystonia, and fatigue [27]. As symptoms of AADC
uniformly present in the first years of life [28, 29], a differential diagnosis may
include “dopa-responsive dystonia,” with a trial of l-DOPA initiated as part of the
diagnostic workup [30]. In light of this treatment approach [31], an additional work
examined the ability of UM to differentiate an AADCD metabolic profile from other
l-DOPA-treated conditions, as 3-MT will be elevated in both cases [32]. Two
patients with AADCD before initiation of l-DOPA showed similar elevations of
3-MT as five patients with non-AADCD pathology (Z-scores of +5.88 and +7.65,
respectively). However, reduced levels of dopamine 3-O-sulfate (D3OS) and vanil-
lylmandelic acid (VMA) downstream of the AADC enzyme were seen in AADCD
patients. In contrast, non-AADCD patients had elevated levels of D3OS and
3-methoxytyramine sulfate (3-MTS), showing a surplus of dopamine following
treatment with L-DOPA. As these results were obtained from plasma and not from
CSF, these data again demonstrate the robustness of the metabolic profile of AADCD
obtained noninvasively.

2.2 Ornithine Metabolism

Diagnosis by variant reclassification was also made in a 7-year-old male with global
developmental delay (GDD), ADHD, epilepsy, and ectodermal abnormalities [33].
The boy was found to have a novel, presumed splice-site VUS in ODC1. This gene
Untargeted Metabolomics, Targeted Care: The Clinical Utilities of Bedside Metabolomics 121

encodes ornithine decarboxylase, converting ornithine into putrescine, the first step
of the spermidine pathway. UM showed excessive N-acetylputrescine, a metabolite
of putrescine, confirming a gain-of-function variant in this gene, consistent with a
diagnosis of Bachmann-Bupp syndrome (MIM 619075), an autosomal dominant
disorder of neurodevelopment characterized by GDD, macrocephaly, white matter
and callosal abnormalities, spasticity, seizures, and ectodermal defects (alopecia,
cutaneous vascular malformations) [34, 35].

2.3 NAD(P)HX Repair System

VUS reclassification can also direct treatment for metabolic disorders amenable to
the intervention. The NAD(P)HX repair system is a highly conserved two-enzyme
system that restores damaged NAD(P)H [36]. In an acidic and hyperthermic envi-
ronment, NADH and NADPH can both undergo hydration into NAD(P)HX nonen-
zymatically [37]. Without the repair system, the inactive NAD(P)HX accumulates
and depletes the NAD+ pool under cellular stress. Biallelic pathogenic variants in
either of the genes coding for the two enzymes, NAXD and NAXE, cause a rapidly
progressive neurometabolic disorder triggered by inflammatory stress (mostly
febrile illness), bearing high mortality in the first decade of life (MIM 618321 and
617186, respectively) [38–40]. A case of a fever-triggered encephalomyopathy cri-
sis in a 16-year-old adolescent demonstrated a small deletion encompassing the first
two exons of NAXD, in trans to a missense VUS in the exon 1-intron 1 splice donor,
suspected to alter both splicing and the mitochondrial localization of NAXD, which
contains the mitochondrial targeting sequence in its first exon [41]. Plasma UM dur-
ing metabolic crisis demonstrated NAD+ depletion and led the medical team to initi-
ate niacin therapy. Under therapy, clinical status improved, and baseline
metabolomics demonstrated repletion of NAD+. Not only did UM biochemically
support the pathogenicity of the variant, but it also provided monitoring data for the
targeted treatment. These results were further validated in a case of NAXE defi-
ciency, for which evidence of depletion of NAD+ derivatives was presented during a
crisis. Niacin treatment appeared to prevent metabolic decompensation during a
subsequent febrile illness while on niacin supplementation (Fig. 1).

2.4 Riboflavin Metabolism

Diagnosis and treatment guidelines were also provided in a case of a 2-year-old

male diagnosed with hypoplastic macrocytic anemia (hemoglobin of 5.5 g/dL and
mean corpuscular volume of 107.1 fL, range 10.5–14.0 g/dL and 76–90 fL, respec-
tively) who later developed ataxia and nystagmus in the context of respiratory syn-
cytial virus (RSV) infection [42]. Brain magnetic resonance imaging (MRI) showed
enhancement of cranial nerves III and V, diffuse intramedullary T2 hyperintensity of
122 J. Manor and S. H. Elsea

Fig. 1 NAD(P)HX repair system deficiency and plasma untargeted metabolomics. Top: NAD+
utilization (red) and repletion by biosynthesis (green) pathways are shown. When the repair system
is defective (left, black), correction of spontaneously converted NADH to NADHX back to NADH
(blue) is impaired, lowering available NAD+ and, thus, lowering utilization products while increas-
ing upstream biosynthesis markers (quinolinate). Bottom: Plasma metabolomic profiles of defi-
ciency in the NAD(P)HX repair system when patients are under inflammatory stress. Two patient
profiles are shown, one for each repair system enzyme (NAXD, NAXE), with the effect of NAD+
depletion on selected analytes (red bars) and after supplementation with niacin (green bars). Taken
together, in both NAXD and NAXE deficiencies, these results point to NAD+ depletion during
inflammatory stress that is amenable to correction with substrate repletion. Bottom: Left panel—
At the time of acute inflammatory stress, the red bars show the metabolomic profile from a patient
with NAD(P)HX dehydratase deficiency (NAXD, EC [Link]), demonstrating the absence of
1-methylnicotinamide, marked deficiency in N1-methyl-2-pyridine-5-carboxamide, and increased
quinolinate (red bars). At 11 months’ post-crisis, the patient underwent another UM profiling
(green bars), showing a reversal of these alterations while under niacin treatment. Bottom: Right
panel—Shown are the same key molecules in plasma from a patient with NAD(P)HX epimerase
deficiency (NAXE, EC [Link]) demonstrating similar trends during an acute inflammatory crisis
(red bars). Repeat UM profiling at 9 months’ post-crisis while on niacin supplementation showed
a reversal of these alterations (green bars)

the entire cord, and cauda equina nerve root thickening and enhancement. ES
showed a pathogenic variant and a novel VUS predicted to cause an in-frame single
amino acid deletion (p.Phe153del) in SLC52A2, an intestinal basolateral and a
blood-brain barrier riboflavin transporter (MIM 607882). UM showed elevated C6
(hexanoylcarnitine), C8 (octanoylcarnitine), C10 (decanoylcarnitine), and C10:1
(decenoylcarnitine), supporting the pathogenicity of the in-frame deletion variant,
in addition to elevations in 2-hydroxyglutarate, methyl succinate, and ethylmalo-
nate, common secondary alterations in short- and medium-chain fatty acid oxida-
tion defects. These perturbations can also be detected on TM, yet the value of UM
was nicely demonstrated by showing additional perturbations in metabolic path-
ways that are related to riboflavin deficiency. Riboflavin is the precursor of flavin
adenine dinucleotide (FAD), a necessary cofactor for kynurenine-3-­monooxygenase,
the de novo NAD+ biosynthesis pathway member; in the patient, the proximal kyn-
urenine was increased, and the distal picolinate was decreased. Reduction of
Untargeted Metabolomics, Targeted Care: The Clinical Utilities of Bedside Metabolomics 123

5,10-methylene tetrahydrofolate into 5-methyltetrahydrofolate is also dependent on

FAD, leading to inhibition of the one-carbon cycle and recycling of sulfur-­containing
amino acid, ultimately resulting in elevations in sarcosine, dimethylglycine, methio-
nine, and glycine. These values normalized upon initiation of high-dose riboflavin
(70 mg/kg/day), with a resolution of the anemia and nystagmus and improvement in
ataxia. While ataxia and nystagmus are common findings, the highly suggestive
upper body proximal muscle weakness with prominent neck flexion and the early
symptom of dysphagia were not seen; however, macrocytic anemia is a rare finding
[43], making a diagnosis based on clinical suspicion very difficult. Indeed, SLC52A2
deficiency, also called Brown-Vialetto-Van Laere syndrome 2 (MIM 614707), is a
difficult diagnosis to make, with an average time to diagnosis of more than 2 years,
a significant delay for a disorder manifesting in the first decade of life [43]. For
example, normocytic anemia was misdiagnosed in a toddler as pure red cell aplasia
overlooking an insidious weakness and areflexia and delaying the diagnosis of
SLC52A2 deficiency for 3 years [44].

2.5 Histidine Metabolism

Not only for VUS can interpretation by UM assist in diagnostic dilemmas.

Deficiency of urocanase (also called urocanate hydratase), an enzyme participating
in the histidine deamination breakdown pathway, was previously considered to be
associated with intellectual disability [45, 46]. Intellectual disability (ID) is a rela-
tively common clinical finding, with a 0.8–3.7% prevalence in the pediatric popula-
tion [47, 48]. Common disorders can manifest independently in rare diseases. A
connection can be falsely established due to (a) small cohorts (an inherent problem
of rare disorders), (b) consanguinity increasing independently the prevalence of
IEM [49, 50] and ID [51, 52], and (c) publication bias, resulting from a top-down
sequencing of patients presenting with a multitude of symptoms. In a recent work,
plasma and urine UM showed a significant increase in both cis- and trans-urocanate
and imidazole propionate in two asymptomatic patients with biallelic pathogenic
variants in UROC1 and normal intellect and with no other significant metabolic
alterations [53] (Fig. 2). UM helped expand our understanding of the benign nature
of urocanase deficiency (MIM 276880) and the need to pursue a genetic diagnosis
for ID in patients with urocanase deficiency, per standard guidelines [10].

2.6 The Diagnostic Rate Among Inborn Error of Metabolism

A direct comparison of TM (plasma amino acids [PAA], acylcarnitine profile [ACP],

and urine organic acids [UOA]) to UM showed that the latter has an overall ~sixfold
higher diagnostic rate for IEM (7.1% vs. 1.3%) [19]. That result may not be unex-
pected, yet the power of UM was demonstrated by the variety of diagnoses it allows
124 J. Manor and S. H. Elsea

Fig. 2 Metabolomic map of the histidine catabolism pathway in a patient with urocanate hydra-
tase (EC [Link], urocanase) deficiency in (a) plasma and (b) urine. The affected arm of the
pathway, highlighted in green arrows in both plasma and urine, shows the accumulation of analytes
immediately upstream to the enzymatic block, trans-urocanate and cis-urocanate. These accumula-
tions then result in the subsequent accumulation of imidazole propionate (in equilibrium with
trans-urocanate) and, to a lesser degree, 1-methylhistidine. In urine, the deficiency of the down-
stream analytes, hydantoin-5-propionate and glutamate, are also shown (blue circles). The color,
diameter, and shading of each circle are proportional to the Z-score. Red circles indicate analytes
in excess (Z-score > +2); blue circles represent deficient analytes (Z-score < −2); pink circles
indicate analyte excess with a Z-score > +1.5 < +2. Black circles indicate analytes within
−1.5 ≤ Z-score ≤ +1.5. Gray circles indicate analytes that are not measured in this assay. (Adapted
from Glinton et al., 2018. Urocanate hydratase (urocanase) is indicated by the black “no entry” sign)

over TM. Diagnoses included disorders of synthesis of neurotransmitters, choles-

terol, and peroxisomal biogenesis. γ-aminobutyric acid-(GABA)-transaminase defi-
ciency (MIM 613163). This early infantile epileptic encephalopathy with a
movement disorder and hypersomnolence due to GABA catabolism defect, was
diagnosed in a 1-year-old patient with hypotonia and movement disorder and com-
pound heterozygous VUS in ABAT, which encodes GABA-transaminase. UM
showed 2-pyrrolidinone, succinamic acid, and succinimide elevations that were not
seen on UOA. These three metabolites are reliably detected in both CSF and plasma,
in contrast to CSF GABA alone, the gold standard for diagnosis but prone to false-
negative results if not handled appropriately [27]. Specifically, 2-­pyrrolidinone is a
butyrolactam spontaneously converted from GABA when the latter is not broken
down to succinic semialdehyde (SSA) by GABA-transaminase. 2-pyrrolidinone is
converted to succinimide and succinamic acid [54], making these three metabolites
alternative biomarkers. The same metabolic profile was seen in a 6-year-old with
global developmental delay (GDD) and movement disorder who was also homozy-
gous for a VUS and two other patients presenting with encephalopathy, seizures,
cortical blindness, motor developmental delay, hypotonia, strabismus, ataxia, and
intellectual disability [19, 54].
Untargeted Metabolomics, Targeted Care: The Clinical Utilities of Bedside Metabolomics 125

A diagnosis of Smith-Lemli-Opitz (MIM 270400), a disorder of cholesterol bio-

synthesis, was made in a typically presenting 10-year-old male with microcephaly,
hypotonia, developmental delays, and a congenital cardiac defect. In that patient,
7-dehydrocholesterol was elevated, while cholesterol was decreased. Initially, a
diagnosis could be ascertained by comparing this profile to the near identical profile
of a molecularly confirmed 3-year-old boy [19] and later confirmed molecularly. In
a different study, such a diagnosis was excluded in a 28-year-old patient with devel-
opmental delays by demonstrating normal 8-dehydrocholesterol and
7-­dehydrocholesterol, downgrading a VUS to likely benign [55].
The latter study presented 16 cases of either homozygous or compound hetero-
zygous VUS in which metabolomics assisted in variant reclassification after ES was
nondiagnostic. Noteworthy in this study are (1) a case of a patient presenting with
early-onset recurrent nephrolithiasis, urosepsis, and transfusion-dependent anemia,
showing an excess of orotic acid in the urine (a metabolite not easily detected in
UOA); thus, the two missense VUS were reclassified to pathogenic and likely
pathogenic, and treatment with uridine monophosphate was initiated; and (2) a case
of psychomotor retardation and retinitis pigmentosa with increased urinary
5-­oxoproline excretion, and despite negative ES, the finding that prompted targeted
sequencing of the GSS gene, demonstrating homozygosity for a deep intronic vari-
ant for this highly heterogeneous condition, 5-oxoprolinase deficiency (MIM
260005).
UM performed on plasma from a 17-year-old with agenesis of the corpus callo-
sum, autism spectrum disorder, lactic acidosis, hyperammonemia, and electrolyte
abnormalities revealed reduced pantothenate, carnitine, and carnitine derivatives
due to SLC5A6 deficiency, confirmed by biallelic VUS identified by subsequent
exome sequencing [19]. SLC5A6 is a cellular cotransporter of pantothenate, biotin,
and α-lipoic acid in the intestine and blood-brain barrier [56]. SLC5A6 deficiency
leads to early-onset neurodegenerative disorder and also includes failure to thrive,
acquired microcephaly, movement disorder, immunodeficiency, gastrointestinal
dysfunction, and osteopenia. Early treatment with high-dose pantothenate, biotin,
and α-lipoic acid seems to improve outcomes [57, 58]. UM also confirmed the diag-
nosis of medium-chain acyl-CoA dehydrogenase deficiency, argininemia, and
X-linked glycerol kinase deficiency (GKD, MIM 307030), in which molecular
diagnoses were inconclusive or missing, among other conditions [19].
To directly analyze the contribution of UM to the interpretation of variants iden-
tified in ES, Alaimo et al. examined clinical samples with both ES and UM testing
that were sent for diagnostic purposes in a cohort of 170 patients [20]. This cohort
was similarly primarily pediatric, with>90% presenting with neurological symp-
toms. The researchers identified 145 variants in 74 patients in their cohort in 73
genes associated with an IEM. Based on the metabolomic data, the 12.3% diagnos-
tic rate in this cohort facilitated the reclassification of 27 variants (19%). Of the
reclassified variants, 24 VUS were reclassified as either likely pathogenic (n = 15)
or likely benign (n = 9), while an additional three variants were upgraded from
likely pathogenic to pathogenic. Classifications were done according to the ACMG
guidelines [16]. For 17 additional pathogenic variants, the study presented
126 J. Manor and S. H. Elsea

confirmatory metabolic perturbations. One case upgraded a homozygous VUS to a

likely pathogenic variant, making the diagnosis of guanidinoacetate methyltransfer-
ase (GAMT) deficiency likely in the patient and allowing the clinician to focus on
the treatment for this disorder of cerebral creatine deficiency. Moreover, for autoso-
mal recessive conditions in which sequencing revealed only heterozygous variants,
UM ruled out the suspected condition in ~60% of cases by showing a normal metab-
olite profile for the metabolic pathway in question, thus excluding the possibility of
a biochemically symptomatic carrier possessing a second disease-causing allele not
detected due to the constraints of ES.

2.7 Automation of Data Interpretation

Similarly to ES diagnostic capabilities, no discussion of UM clinical diagnostic

capabilities will be complete without discussing the interpretation of the (untar-
geted) data. The ability to correctly diagnose a condition based on UM heavily
depends on the art of data interpretation. Rather than the convoluted manual analy-
sis of metabolomic data (with or without genomic information), automated bioin-
formatic tools provide means for pattern recognition and thus hold a great promise
of an improved matching between datasets and a particular disease or pathway. One
approach applies the clique problem in a metabolomic graph. A clique is defined as
an interconnected group of metabolites (nodes), and small highly connected cliques
are extracted based on computational analysis bounding cliques’ p values [59].
Across 539 plasma samples, this connect-the-dots (CTD) approach reproduced
accurate diagnosis of 16 different IEMs [60]. The top-down bioinformatic approach
seeks to identify causative genes by providing a likelihood scoring system based on
heuristic algorithms predicting the effects they expect to exert on the -omic dataset.
Several metrics achieved prioritization of candidate genes based on UM. In cross-­
omics, a metabolomic study performed using dried blood spots (DBS) [61], candi-
date genes were prioritized based on their distance from the perturbed metabolite
(where each reaction accounts for 1 step), with a narrowing process of including
metabolites that participate in only limited amount of reactions (“uniqueness”) and
limiting metabolites to only those with a significant Z-score (“significance”) to gen-
erate gene-specific metabolite sets. Successful prioritization was achieved by con-
sidering metabolites up to four reactions away from the primary reaction, uniqueness
of up to 15 reactions, and significance of >+3 or <−3. In metPropagate [62], each
protein is assigned a rank based on its associated metabolite enrichment, and
dynamic ranking is propagated with the protein’s functional linkage network. This
approach successfully prioritized causative genes (within the top 20th centile) in
20% of IEMs in the study group and 82% (9/11) in the test group of neurometabolic
disorders. This algorithm outperformed Exomiser [63], a causative variant
Untargeted Metabolomics, Targeted Care: The Clinical Utilities of Bedside Metabolomics 127

prioritization tool based on human phenotype ontology data and standard pathoge-
nicity prediction (variant prevalence, conservation, and information from model
organisms and inheritance patterns). In another approach, Reafect [64] assigned a
score based on cumulative pathway perturbations, including metabolites with sub-­
significant Z-scores, correctly predicting the causative gene in the top fifth centile in
80% of the 76 patients harboring 36 different IEMs. When combined with the del-
eteriousness predictive score, Combined Annotation Dependent Depletion (CADD)
[65], specificity further increased. Another promising approach is the implementa-
tion of a siamese neural network, weighing in the tandem computational metabolic
network (for predicting metabolite flux) and machine learning (ML, for matching
metabolic networks to diseases) trained by the ML algorithm [66]. The model used
a single simulated profile for each disease with real data points from only 2% of the
diseases to outperform a generic algorithm that prioritizes causative genes based on
distance from real data profiles (using the L1 Manhattan metrics). Limitations to
ML include the quality of the training set and the generalizability of metabolic pro-
files to novel diseases. Nonetheless, implementing predictive ML-based algorithms
promises to reduce the number of patient-derived samples required for disease dis-
covery (smaller cohorts), a vital prerequisite in the world of rare diseases such as
IEMs [59].

3 Metabolomic Fingerprinting: Identifying Diseases’

Biometrics and Finding New Disease Biomarkers

Biomarkers are crucial for the effective screening, designation, and diagnostic con-
firmation or exclusion of diseases by UM. Biomarkers are also critical in monitor-
ing an affected patient’s metabolic status and treatment efficacy. Clinical biomarkers
for IEMs are abundant [67], although most are derived from TM. Many biomarkers
are assessed by routine biochemical tests, such as ammonia, lactate, uric acid, and
cholesterol, or from TM, such as UOA (e.g., trimethylamine in fish odor syndrome)
or PAA (high phenylalanine in PKU). To increase specificity, either identification of
disease-specific biomarkers can assist in the diagnosis (or ruling out) of a disease,
such as allo-isoleucine in MSUD or argininosuccinic acid in argininosuccinate
lyase (ASL) deficiency, or the identification of several non-pathognomonic metabo-
lites, such as low levels of lysine, ornithine, and arginine in plasma amino acids
suggestive of LPI, or the elevation of propionylcarnitine on ACP, methylmalonic
acid in UOA, low methionine on PAA, and homocysteine in the blood, indicative of
an intracellular cobalamin utilization defect. UM can instigate both strategies. By
its untargeted nature, UM has the potential to uncover biomarkers that were not
known to be associated with an IEM or not visible by targeted testing.
128 J. Manor and S. H. Elsea

3.1 Peroxisome Biogenesis

Peroxisome biogenesis disorders in the Zellweger spectrum (PBD-ZSD) are a het-

erogeneous group of genetic disorders caused by mutations in genes responsible for
normal peroxisome assembly and functions [68]. The majority of cases are due to
biallelic pathogenic variants in PEX1 (61%), followed by PEX6 (15%) and PEX12
(8%), with additional contribution from at least another 10 PEX genes [69], an
important group of proteins essential for the assembly of peroxisomes and the rec-
ognition and transport of cytoplasmic proteins that contain peroxisomal targeting
sequence [70]. The severe end of PBD-ZSD includes neuronal migration defects
with leukodystrophy, neonatal onset seizures, hypotonia, failure to thrive, liver dys-
function, bony stippling respiratory insufficiency, cataracts, sensorineural hearing
loss, and renal cortical microcysts; the mild end of the spectrum includes develop-
mental delays and intellectual disability that can be mild and slowly progressive
retinopathy and sensorineural hearing loss [68, 69, 71]. Severe cases can be screened
by elevation of the very-long-chain fatty acid (VLCFA), C26:0-­
lysophosphatidylcholine (DBS, plasma), phytanic and pristanic acids (plasma);
reduction in plasmalogens (plasma, erythrocyte membranes); increase in pipecolic
acid (plasma, urine); and increase in the bile acids, dihydroxycholestanoate, and
trihydroxycholestanoate (plasma, urine) [69]. Intermediate and mild cases may
show only subtle alterations, and in combination with subtle clinical symptoms,
screening for PBD-ZSD may not be performed. In a cohort of 19 mild to intermedi-
ate PBD-ZSD pediatric patients, Wangler et al. showed a distinct PBD-ZSD plasma
metabolome (Fig. 3), with elevated levels of long-chain dicarboxylic acids, pipe-
colic acid, and the bile acid derivative 7α-hydroxy-3-oxo-4-cholestenoic acid and
with reductions in phosphatidylcholines, phosphatidylethanolamines, and plasmal-
ogens [71]. Pipecolic acid and the lysophospholipid 1-lignoceroyl-GPC (24:0) were
most strikingly elevated with a Z-score of +3.7. Less anticipated changes included
dicarboxylic acids of 16–22 carbons. UM also proposed novel biomarkers, with a
reduction in nine sphingomyelin species. Reduction of several sphingomyelins in
the clinical UM database, excluding PBD-ZSD, was observed in only 2% (interest-
ingly, one of which was diagnosed with bifunctional protein deficiency, which can
mimic PBD-ZSD). Moreover, the plasma elevations in pipecolic acid and reduc-
tions in the sphingomyelins attenuated with age, an expected finding correlating
milder phenotypes among older surviving patients.

3.2 Urea Cycle

The urea cycle is the principal mechanism for the clearance of waste nitrogen
resulting from protein turnover, the sole source of endogenous production of argi-
nine, ornithine, and citrulline, and a principal component of the nitric oxide (NO)
production pathway [72]. The urea cycle is also connected with TCA anaplerosis
Untargeted Metabolomics, Targeted Care: The Clinical Utilities of Bedside Metabolomics 129

0 1 2 3 4 5 6 7 8 9
ZSD < 10 y of age

Lysoplasmalogen 7.81E-7
Plasmalogen 1.17E-9
Sphingolipid Metabolism 3.34E-9
Phosphatidylcholine (PC) 2.27E-7
Fatty Acid, Dicarboxylate 0.14
Methionine, Cysteine, SAM and Taurine Metabolism 0.27
Lysophospholipid 0.23
Androgenic Steroids 0.49
Glycine, Serine and Threonine Metabolism 0.49
Purine Metabolism, Adenine containing 0.62
Diacylglycerol 0.69
Endocannabinoid 0.69
Phospholipid Metabolism 0.69
TCA Cycle 0.75
Lysine Metabolism 0.75
Urea cycle; Arginine and Proline Metabolism 0.74
Leucine, Isoleucine and Valine Metaboilsm 0.76
Primary Bile Acid Metabolism 0.83
Phosphatidylethanolamine (PE) 0.83
Secondary Bile Avid Metabolism 0.89
Histidine Metabolism 0.92
Gamma-glutamyl Amino Acid 0.92
Tyrosine Metabolism 0.92
Dipeptide 0.94
Fatty Acid Metabolism(Acyl Carnitine) 0.95

0 1 2 3 4 5 6 7 8 9

Dipeptide 0.04

Lysoplasmalogen 0.17

Fatty Acid Metabolism(Acyl Carnitine) 0.05

ZSD > 10 y of age

Histidine Metabolism 0.27

Tyrosine Metabolism 0.29

Urea cycle; Arginine and Proline Metabolism 0.30

Methionine, Cysteine, SAM and Taurine Metabolism 0.32

Food Component/Plant 0.45

Leucine, Isoleucine and Valine Metabolism 0.48

Fig. 3 Plasma metabolomic footprint of peroxisome biogenesis disorder-Zellweger spectrum dis-

order (PBD-ZSD). Top: Left panel. Early diagnosis (age <10 years) of PBD-ZSD shows a more
significant metabolic phenotype, with enrichment of lysoplasmalogens, plasmalogens, sphingo-
lipid metabolism, and phosphatidylcholines, reaching statistical significance (colored bars) for
patients when compared to control population. Top: Right panel. The relative deficiencies of most
of these lipids in PBD-ZSD in children under the age of 10 years are represented by the blue circles
in the metabolomic tree of lipid analytes (lipidomics). Bottom: Left panel. Later diagnosis of
PBD-ZSD (age >10 years) exhibits a more subtle metabolic phenotype. Bottom: Right panel. The
metabolomic fingerprint of PBD-ZSD in individuals >10 years of age showed mild perturbations
in which reductions of the plasmalogens and lysoplasmalogens did not reach statistical signifi-
cance. The lipidomic tree shows considerably fewer perturbations when compared to plasma
obtained from a younger patient. Trees were rendered using Cytoscape ([Link]
org). (Figure adapted from Wangler et al. 2018)

via the alternative synthesis of fumarate. UCDs include eight different IEMs
resulting from defects in any one of the six enzymes or two transporters involved
in the hepatic removal of ammonia as waste nitrogen by its conversion to urea and
excretion by the kidneys [73]. Mortality and morbidity primarily contribute to
neurological damage resulting from hyperammonemia (HA) and the elevation of
other neurotoxic intermediates of metabolism [74]. A typical presentation of a
UCD includes neonatal hyperammonemic crisis; however, nontypical presenta-
tions of later-onset HA, acute liver dysfunction, intellectual disability, or insidious
pyramidal signs of the lower extremities with minimal HA crises (in arginase
deficiency) have been described [27]. It has been suggested that arginase defi-
ciency, with its unique presentation compared to other UCDs, exerts a neurotoxic
130 J. Manor and S. H. Elsea

effect by the guanylation of glycine, with the excess arginine serving as a guani-
dine donor rather than acute HA crises [75]. In a cohort of 13 patients with arginin-
emia, Burrage et al. demonstrated an increase in additional guanidine compounds,
namely, N-acetylarginine, homoarginine, argininate, and 2-oxoarginine, to a
greater degree than guanidinoacetate (GA) [76]. Guanidine compound (GC) tox-
icity is believed to be derived from the observed in vitro neurotoxicity: diminished
response to the inhibitory GABA and glycine neurotransmitters [77], promotion
of non-apoptotic cell death and axonal hypersprouting [78], and inhibition of Na+/
K+-ATPase activity and glutamate uptake, and decrease in antioxidant defense in
the rat brain [79]. Other toxic effects of GC include ethanol-induced liver injury,
stimulated osteoclastogenesis, generation of reactive oxygen species (ROS), and
modulation of cerebral cortex potentials [79]. In GAMT deficiency, GA is the
main GC accumulating and phenotypically exerting a greater degree of intellec-
tual disability, refractory epilepsy, and dystonia. At the same time, pyramidal
signs are less dominant as compared to arginase deficiency. While these results
may indicate an important role of GA in the developing brain, another important
modifier between the two disorders is the creatine level, which is normal in argi-
nase deficiency and low in GAMT deficiency [80]. Peripheral administration of
polyethylene glycol amalgamated to (PEGylated) arginase, now in advanced
stages of development, will help elucidate further pathomechanistic insights by
the peripheral reduction of arginine excess without restoring urea cycle function
in the liver.
The same study by Burrage and colleagues also examined a UM profile of OTC
deficiency, a severe X-linked disorder bearing high morbidity and mortality among
males and affected females. In this UCD, morbidity is attributed to HA crises, and
biomarkers are scarce, making this disorder “unscreenable.” In this study, 83% of
patients (10/12) with a history of hyperammonemia (excluding females with no
such history) showed a significant elevation (Z-score >+2) of either orotate or uri-
dine. Other biomarkers of increased pyrimidine metabolism (due to the shunting
of the cabamoylphosphate from the dysfunctional urea cycle), β-ureidopropionate,
and uracil were not significantly elevated. For screening purposes, the sensitivity
of these markers is ~60% (10/17), and the specificity is also low, given shared
pathway perturbation with other UCD and pyrimidine metabolism defects; how-
ever, these results point to an important consideration for biochemical testing,
indicating such abnormalities in cases of uncertain diagnosis given the sensitive
nature of this disorder. The authors are aware of a case of a 1.5-year-old female
presenting with fulminant liver failure and nonspecific liver biopsy for which only
uracil was elevated in urine on traditional metabolic screening. Based on that
result, a presumptive diagnosis of OTC deficiency was made, and targeted treat-
ment was promptly provided; molecular testing later confirmed the suspected
diagnosis. Both citrullinemia and ASL deficiency did not show unique metabolic
fingerprints beyond the accumulation of citrulline and argininosuccinic acid,
respectively [76 ].
Untargeted Metabolomics, Targeted Care: The Clinical Utilities of Bedside Metabolomics 131

3.3 Pyruvate Kinase

Pyruvate kinase deficiency (PKD, MIM 266200) is the most common form of inher-
ited anemia due to glycolytic defects. It results in a spectrum of hemolytic anemia
that can result in infantile-onset transfusion-dependent anemia or a milder form of
compensated anemia [81]. A diagnostic gap exists for PKD due to the unsatisfactory
performance of activity assays, a genetic composition complicating molecular diag-
nosis (variants in regulatory elements or effector genes such as KLF1), and the
confounding effect of frequent blood transfusion on these methods [82]. In a cohort
of 16 patients with PKD (against 32 controls), van Dooijeweert et al. showed three
groups of metabolites in DBS that differed between the two cohorts: glycolytic
products phosphoenolpyruvate and 2- and 3-phosphoglycerate, as one might expect,
along with polyamines, such as spermine, spermidine, N1-acetylspermidine, and
putrescine, which are associated with red blood cell (RBC) membrane integrity, and
acylcarnitines such as methylmalonylcarnitine and propionylcarnitine which are
involved in turnover and repair of the RBC membrane [83]. Principal core analysis
showed a separation of metabolic profiles between the two groups. Interestingly, the
mildly affected patients with no history of transfusion dependence or splenectomy
more closely resembled the control group, followed by transfusion dependence
(severe phenotype), probably due to the frequent retrieval of donor RBC, and the
splenectomized patients (moderate phenotype) were furthest away from control.
Based on these findings, a machine learning algorithm trained on a subset of the
cohort could predict the disease in 94% of cases.

3.4 Glucose Transporter 1

UM can also reveal a metabolic fingerprinting of disease-modifying treatments,

aiding the monitoring of both therapeutic efficacy and disease progression. The
brain glucose transporter GLUT1 facilitates the diffusion of glucose to brain tissue
to compensate for insufficient passive diffusion, given the elevated requirement for
glucogenic energy production by that tissue. Heterozygous pathogenic variants in
SLC2A1, which encodes GLUT1, result in a brain energy failure syndrome
(GLUT1DS) caused by impaired glucose transport and result in a spectrum of phe-
notypes including epileptic encephalopathy, intellectual disability, acquired micro-
cephaly, ataxia, action limb dystonia, chorea, and tremor in its severe form and
paroxysmal ataxia in its mild end. Diagnosis is made by demonstrating a low CSF
concentration of glucose in the presence of plasma normoglycemia and/or identifi-
cation of a pathogenic variant in SLC2A1 [84]. Aside from probable energy deple-
tion, it was suggested that depletion of glycolysis intermediates might play a role
in the disorder’s pathogenesis; however, it is unknown to what extent, as in vitro
132 J. Manor and S. H. Elsea

models include near-complete abrogation of the transporter [85]. The sole thera-
peutic option available is a classic ketogenic diet (CKD). By altering the brain
energy fuel into mainly ketone bodies, this therapy dramatically affects seizures
and can improve cognitive outcomes. However, diet discontinuation was reported
in up to 10% of patients due to side effects [86], and low compliance led experts to
recommend alternative low carbohydrate diets (such as the modified Atkins diet)
for adolescents and adults [87] and even exploring amylopectin-based diet of the
low glycemic index [88]. Monitoring CKD is based on serum β-hydroxybutyrate
levels, as measuring urine ketones—a convenient and qualitative measure of aceto-
acetate—has a limited role in diet monitoring [87]. A more comprehensive picture
created by UM performed on six treated patients with GLUT1DS [89] showed
elevations in β-hydroxybutyrate, β-hydroxybutyrylcarnitine, β-methyladipate, and
N-acetylglycine. Other elevated derivatives were α-ketobutyrate, β-hydroxylaurate,
10-nonadecenoate, margarate, 15-methylpalmitate, and α-aminoheptanoate.
Furthermore, pathway analysis using Kruskal-Wallis analysis (comparing pathway
metabolite perturbations vs. non-pathway metabolite perturbations) showed
involvement of long-chain fatty acids, phospholipids, acylcarnitines, and, to a
lesser degree, sphingolipids, mono-hydroxy fatty acids, and polyunsaturated fatty
acids. In accordance with fatty acid utilization, free carnitine levels were low, while
carnitine-bound metabolites were elevated. CSF UM of three patients prior to CKD
onset revealed low levels of glycerol 3-phosphate, an intermediary metabolite in
lipid metabolism, and an increased level of isocitrate, which can indicate a TCA
dysfunction [89]. These results supply a CKD metabolic profile with a more com-
plete ketosis map, which can assist in diet fine-tuning, e.g., increasing the fat-to-­
carbohydrate ratio to increase overall ketosis or examining the effect of decreasing
the ratio in reduced diet tolerability. The results can also guide the need for supple-
mental carnitine due to increased secondary excretion in ketosis, which, although
it is considered a benign supplementary agent, can also exacerbate GI-related
symptoms in patients under this GI-unfriendly diet and can also independently
elevate plasma trimethylamine N-oxide (TMAO) levels, an atherosclerotic agent
[90, 91].

3.5 Serine Metabolism

Serine biosynthesis defects occur due to deficiency in either of the three enzymes
converting 3-phosphoglycerate into serine, phosphoglycerate dehydrogenase
(PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase
(PSP), and result in a severe neurometabolic disorder including severe intellectual
disability, ataxia, nystagmus, epilepsy, hypertonia/spasticity, microcephaly, and
poor growth. Deficiencies in enzymes of the serine de novo biosynthesis pathway
result in low plasma and CSF levels of serine. Low serine impedes the synthesis of
sphingolipids (from serine and palmitoyl-CoA), phosphatidylserine, d-serine (an
agonist to the ionotropic glutamate receptor N-methyl-d-aspartate (NMDA)), and
Untargeted Metabolomics, Targeted Care: The Clinical Utilities of Bedside Metabolomics 133

5,10-methylenetetrahydrofolate (by converting serine to glycine) [92]. This multi-­

pathway effect could explain neurodevelopmental abnormalities associated with
this disorder; however, a more direct association has not been clinically demon-
strated. In four children with two serine biosynthesis defects, PGDH deficiency
(three children) and PSAT deficiency (one child), Glinton et al. demonstrated an
abnormal phospholipid profile at the time of diagnosis and, upon treatment, normal-
ization thereof. Before initiation of treatment, serine and glycine were both
decreased in all patients, and multiple phospholipids were reduced, including spe-
cies of the mono- and di-unsaturated 18-carbon phosphatidylcholine and phospha-
tidylethanolamine in three or four of the patients [93]. Multiple sphingolipid species
were reduced in two patients, most notably sphingomyelin. Except for phospholip-
ids, no other compounds were found to be altered (either reduced or elevated) con-
sistently in all patients evaluated [93]. The majority of these abnormalities
normalized under supplementation. These results emphasize the need for early
treatment. De novo synthesis of phosphatidylcholine (PC) and phosphatidylethanol-
amine (PE) from choline, ATP, cytidine triphosphate (CTP), and diacylglycerol by
choline kinase, phosphocholine cytidylyltransferase, and choline transferase (also
referred to as the Kennedy pathway) may, in fact, be inadequate at the time of rapid
growth or neuronal differentiation in utero and require supplementation of PE and
PC from phosphatidylserine pool (by mitochondrial phosphatidylserine decarbox-
ylase). Another possibility is serine palmitoyltransferase (SPT) substrate promiscu-
ity, leading to the condensation of alanine and palmitoyl-CoA into
1-deoxy-sphinganine (instead of 1-dehydro-sphinganine when serine is sufficient).
Indeed, decreased sphingomyelin and increased 1-deoxy-sphingomyelin have been
reported in targeted metabolomics applied for primary serine biosynthesis defects
and secondary serine deficiency due to mitochondrial disorders [94]. Interestingly,
a DBS sample from one patient at 38 hours of life showed markedly reduced serine,
which could have served as abnormal NBS, as discussed above [93].

3.6 Pentose Phosphate Pathway and Polyol Metabolism

The pentose phosphate pathway is an important cytosolic pathway that converts

glucose into ribulose-5-phosphate and produces reduced nicotinamide adenine
dinucleotide phosphate (NADPH) for restoration of the antioxidant glutathione. Its
oxidation product, ribulose-5-phosphate, can serve as a nucleotide building block or
be further converted into phosphorylated mono-sugars that can serve as glycolytic
intermediates [95]. The nonoxidative portion includes four enzymes, ribulose
5-phosphate isomerase and reductase, transaldolase (TALDO), and transketolase
(TKT). Common features of TALDO deficiency (MIM 606003) are hepatospleno-
megaly, anemia, thrombocytopenia, renal tubulopathy, heart abnormalities, and
cholestatic liver dysfunction that can develop into cirrhosis [96]. For TKT defi-
ciency (MIM 617044), manifestations include short stature, developmental delays,
and congenital heart defects [27]. These manifestations are nonspecific; further,
134 J. Manor and S. H. Elsea

considering the potential reversibility of these two disorders (despite no currently

available treatment), high-yield screening can facilitate diagnosis and improve out-
comes. While TALDO deficiency was indicated by UM screening by identification
of sedoheptulose, a single polyol that can point out an abnormality within the non-
oxidative portion of PPP, Shayota et al. successfully demonstrated high-specificity
multiple polyol alterations in plasma and urine metabolomics for the diagnosis of
these two enzyme deficiencies (see Table 1) [97]. Two novel biomarkers were shown
for both disorders (erythronate and ribonate). Secondary alterations in purine and
pyrimidine metabolites, as seen in this work, are most probably due to defect in
ribulose-5-phosphate processing.

Table 1 TM versus UM findings in two disorders of the nonoxidative portion of the pentose
phosphate pathway [97]
Metabolic Targeted metabolomics Untargeted metabolomics
pathway Plasma Urine Plasma Urine
TALDO Polyol Arabitol Arabitol Arabitol/xylitol Arabitol/xylitola
a

deficiency Erythritol Sedoheptulose Ribitol Ribitol

Ribitol Erythritol Erythronate
Xylitol Sedoheptulose Ribonate
Sedoheptulose Erythronate
Ribonate
Tryptophan – – Kynurenate Quinolinate
Xanthurenate Xanthurenate
Purine – – Xanthosine
Pyrimidine N-­carbamoylaspartate
TCA – – α-Ketoglutarate Succinate
Fumarate
Malate
TKT Polyol Arabitol None Arabitol/xylitol Arabitol/xylitol
deficiency Erythritol Ribitol Ribitol
Ribitol Erythritol Erythritol
Xylitol Ribose Erythronate
Sedoheptulose Erythronate Ribonate
Ribonate
Tryptophan – – Kynurenine Kynurenine
Xanthurenate 3-hydroxykynurenine
Quinolinate
Indolelactate
Purine – – Inosine Xanthosine
Guanosine
Pyrimidine N-­carbamoylaspartate
Underline—a newly discovered disease biomarker
a
Arabitol could not be easily distinguished from xylitol or arabonate from xylonate by this UM
platform, given the similar structure and molecular weight
Untargeted Metabolomics, Targeted Care: The Clinical Utilities of Bedside Metabolomics 135

3.7 Metabolomics of Muscular Diseases

The muscular disease has a broad differential diagnosis and multiple pathological
mechanisms. An important etiological group is the mitochondrial myopathies, with
can have early- or late-onset and acute or subacute course and are progressive in
nature [14]. Buzkova et al. compared the metabolomic profiles of mitochondrial
myopathies and ataxias (lumping the sporadic inflammatory disorder inclusion
body myositis, which also affects mitochondrial functioning) in comparison to non-­
mitochondrial neuromuscular diseases [98]. This study utilized TM of 94 metabo-
lites but is included herein, given its broad application and interest in disease
fingerprinting. The first group demonstrated alterations in the transsulfuration path-
way, including elevated cystathionine (1.9–4.1-fold increase) and a less consistent
reduction in taurine. In contrast, the second group was characterized by normal
levels of cystathionine, depletion of nicotinamide (−1.7-fold change), and increased
creatine (2.1-fold change). Alterations were also found in carbohydrate metabolism
leading the authors to propose a quad-biomarker set of elevations in sorbitol, ala-
nine, myoinositol, and cystathionine, producing an area under the curve similar to
fibroblast growth factor-21 (FGF-21), lactate, and pyruvate, to distinguish the mito-
chondrial origin of myopathy (with an overall 76% sensitivity, 95% specificity). A
particular mitochondrial myopathy, mitochondrial encephalomyopathy, lactic aci-
dosis, and stroke-like episodes (MELAS, MIM 540000) showed elevations in car-
bohydrate derivatives (sorbitol, glucuronate, myoinositol, and sucrose), decreased
arginine, and an increase in transsulfuration intermediates (cystathionine,
γ-glutamyl-cysteine S-adenosylmethionine, and glutamate) with a decrease in ade-
nosine, guanidinoacetate, and betaine. In another UM study of MELAS patients,
Sharma et al. demonstrated novel amino acid, acylcarnitine, and fatty acid biomark-
ers [99]: N-lactoyl attached to the branched-chain amino acids leucine, isoleucine,
or valine or to the aromatic amino acids phenylalanine and tyrosine; β-hydroxy
acylcarnitines of even-length C10:0 to C16:0 (C10:0, C12:0, C14:0, C16:0); and
β-hydroxy fatty acids of even-length C8:0 to C14:0. These biomarkers were associ-
ated with the degree of diseased severity (per Karnofsky performance score).
Interestingly, β-OH-C16:0 carnitine showed a severity correlation similar to the
well-accepted growth/differentiation factor-15 (GDF-15) [100], while β-hydroxy
acylcarnitines and β-hydroxy fatty acids correlated with ventricular lactate levels,
and the N-lactoyl-amino acids correlated with urine heteroplasmy. Table 2 summa-
rizes selected metabolic pathways in which more than 10% of metabolites are sig-
nificantly altered in the individual disorders (two-sample t-test for significance).
Table 2 Plasma metabolic pathways in which ≥10% of metabolites are altered compared to control in selected mitochondrial myopathies and ataxias,
136

compared to non-­mitochondrial myopathies [98]

Group Family Disorder OMIM Gene Altered metabolic pathways
Mitochondrial mtDNA IOSCA 271245 TWNK Primary bile acids; nitrogen; alanine, aspartate, and glutamine; cysteine and
depletion methionine; histidine; butanoate; d-glutamine and d-glutamate; cyanoamino
acids; glutathione
MIRAS 607459 POLG Cysteine and methionine; alanine aspartate and glutamine; taurine and
hypotaurine; histidine; β-alanine; nitrogen; aminoacyl-tRNA; cyanoamino
acids; pantothenate and CoA
Single mtDNA PEO 530000 Sporadic Plasma: Alanine, aspartate, and glutamine; cysteine and methionine;
deletion 258450 mtDNA nitrogen; glycine serine and threonine; d-glutamine and d-glutamate;
157640 deletion arginine and proline; aminoacyl-tRNA
609286 TWNK
POLG Muscle tissue (invasive): Cyanoamino acids; branched-chain amino acids;
nitrogen; pantothenate and CoA; alanine aspartate and glutamine; cysteine
and methionine; aminoacyl-tRNA; histidine
Mitochondrial MELASa 540000 m.3243A>G d-arginine and d-ornithine; taurine and hypotaurine; cysteine and
tRNA defects methionine; alanine, aspartate, and glutamine; arginine and proline;
aminoacyl-­tRNA; glutathione
Immune Inclusion 147421 Sporadic Nitrogen; glycine serine and threonine; cysteine and methionine;
dysregulation body pyrimidine; arginine and proline; glutathione; primary bile acid; histidine
myositis
Non-­ Neuromuscular diseases AD or AR Arginine and proline; folate (one-carbon pool); glutathione; glycine, serine,
mitochondrial DMPK and threonine; histidine; butanoate; β-alanine
ZNF9
CAPN3
PABPN1
SMN1
TIA1
Or unknown
Pathways are presented in decreasing order of alteration
J. Manor and S. H. Elsea

mtDNA mitochondrial DNA, IOSCA infantile-onset spinocerebellar ataxia, MIRAS mitochondrial recessive ataxia syndrome, MELAS mitochondrial encepha-
lomyopathy, lactic acidosis, and stroke-like episodes
a
Sharma et al.
Untargeted Metabolomics, Targeted Care: The Clinical Utilities of Bedside Metabolomics 137

4 Future Perspectives

We shall end our discussion of bedside UM with a futuristic perspective of UM in

the service of autism spectrum disorder (ASD). The autism spectrum is a range of
neurodevelopmental conditions exhibiting persistent deficits in reciprocal social
interaction and restricted, repetitive patterns of behavior, interests, or activities.
Historically, ASD gained an independent psychiatric status from schizophrenia only
in the second half of the twentieth century (the word autism was initially coined to
describe severe schizophrenia) [101] and became a spectrum only at the end of that
century. We now appreciate a 60–80% heritability of ASD [102, 103], with an esti-
mated genetic or genomic etiology in 30–40% of cases [104]. Within the group of
ASD, due to a known genetic variant, an estimated 3–5% are due to an IEM [105].
Examples include [106] amino acidopathies (PKU, homocystinuria,
S-adenosylhomocysteine hydrolase deficiency, MSUD, UCD), organic acidurias
(PA, L-2-hydroxyglutaric aciduria), cholesterol biosynthesis defects (Smith-Lemli-
Opitz syndrome), disorders of neurotransmitter synthesis or degradation (SSA defi-
ciency; SSADH deficiency), disorders of purine metabolism (ADSLD, Lesch-Nyhan
syndrome), cerebral creatine deficiency syndromes (GAMT deficiency, creatine
transporter defect), disorders of folate transport and metabolism (cerebral folate
deficiency, MTHFR deficiency), lysosomal storage disorders (mucopolysaccharido-
sis type III, neuronal ceroid lipofuscinoses (NCL), Niemann-Pick disease type C),
CTX, MELAS, Wilson disease, and several types of neurodegeneration with brain
iron accumulation, among others. Moreover, multiple lines of evidence have sug-
gested biochemical alterations in children with ASD compared to peers. Alterations
can derive from the large intestine microbiome, showing differential fecal content
for isopropanol, p-cresol, and short-chain fatty acids. Mitochondrial dysfunction
can result in complex I, IV, or V deficiencies in children with ASD (up to 7%).
Alterations were also demonstrated in some cases of glutathione reduction in the
CNS [107]. While attempts to find unifying markers of autism demonstrated inter-
esting candidates, they failed to show consistency across multiple UM platforms
and ASD cohorts. Glutaric acid, arginine, histidine (and its catabolites), taurine,
β-alanine, and succinic acid were most consistently elevated, along with a reduction
of creatine and creatinine [107]. In a cohort of 52 pregnant women whose children
developed ASD, compared to 62 control pregnant women in an NMR-based UM,
differences were found in glycosphingolipid metabolism, N-glycan and pyrimidine
metabolism, bile acid pathways, and C21-steroid hormone biosynthesis but with
only a mild perturbation in each metabolite [108]. The latter two candidates are of
interest due to the involvement of cholesterol metabolism, as ASD is highly preva-
lent among patients with Smith-Lemli-Opitz syndrome, a disorder of cholesterol
synthesis. Abnormal bile acid synthesis can cause perturbations in taurine, and
pyrimidine synthesis defects can alter β-alanine. However, a lesson learned from
these works is that ASD may represent shared neurodevelopmental outcomes of
quite different neurometabolic processes, complicating the search for biomarkers,
as the case separation from control may be shadowed by intragroup variability
138 J. Manor and S. H. Elsea

[109]. Instead, a machine learning approach to recognize a pattern of altered seem-

ingly unrelated metabolites in a study group could allow the discovery of heteroge-
neous biomarkers, indicating a high-risk status for ASD, Alzheimer’s disease,
Parkinson’s disease, or other conditions, and, once defined, may also be then tar-
geted for treatment and/or monitoring. Such a learning process was performed on
UM data from a cohort of 500 children with ASD (against 200 controls) [110]. The
group was halved into study and discovery groups. The study group yielded 34
groups sharing metabolic phenotypes with a specificity of ≥95%. Those groups
were then clustered into six metabolic groups, each with an abnormal ratio of either
α-ketoglutarate, 4-hydroxyproline, glycine, lactate/pyruvate, ornithine, or succinic
acid compared to other metabolites. These clusters were then validated in the dis-
covery group. When screening for the clusters together, this assay had 53% sensitiv-
ity and 91% specificity for indicating “high risk” for ASD. These outcomes
demonstrate the potential for a higher level of complexity in data interpretation.
Identification of such abnormalities by the physician ordering an “ASD risk stratifi-
cation” test may be a difficult task. Still, computer-assisted data analysis could flag
such abnormal results and promote a revolution in the field of developmental
neuroscience.
While UM offers an opportunity for discovery both inside and outside clinical
settings, its use in the clinical lab supports broad screening for inborn errors of
metabolism well beyond the newborn screen, supports the development of meta-
bolic profiles for a disease that may be monitored during treatment, and, when inte-
grated with genomics, provides a precision medicine approach to the diagnosis of
rare disease.

Glossary

Dried blood spot (DBS) A method of whole blood sample collection in which a
small amount of fresh blood is blotted onto an absorbent filter paper, followed by
drying. This method provides a convenient storage and shipment platform and
is widely used for newborn screening. Typically, a small punch from the DBS
paper is eluted with phosphate-buffered saline, availing the sample for testing.
Elevation/reduction (of a metabolite) In the context of this chapter, a metabolite
is considered reduced (insufficient) or elevated (in excess) when UM reveals a
Z-score ≥+2 or ≤−2. The Z-score is the number of standard deviations that a data
point differs from the population means, representing the relative level of a given
metabolite. Raw values for individual metabolites are log2-transformed, and the
relative Z-score is calculated compared to a lab-specific reference population
[91, 111–113].
Inborn error of metabolism (IEM) A heterogeneous group of mostly inherited
disorders involving a failure of the certain metabolic pathway(s) to break down
or store biomolecules (typically carbohydrates, lipids, or amino acids) in the cell.
Untargeted Metabolomics, Targeted Care: The Clinical Utilities of Bedside Metabolomics 139

Although any given inborn error of metabolism is rare, taken as a group, inborn
errors of metabolism occur in 1 in 2000 births [114].
Molecular confirmation A suspected diagnosis, as suggested by biochemical test-
ing such as UM, is said to be molecularly confirmed when genomic sequenc-
ing reveals pathogenic variants, either monoallelic for autosomal dominant or
X-linked disorders or biallelic for recessive disorders in the gene associated with
the metabolic abnormality. Sequencing can be targeted for the specific gene(s) or
untargeted as exome or genome sequencing. In the latter case, further confirma-
tion by Sanger sequencing may be performed to validate the variants identified.
Reference population A lab-specific reference population created by performing
UM on samples received in the clinical laboratory, with careful inclusion and
exclusion of clinical samples to ensure pathways and analytes are covered for
comprehensive clinical assessment. Raw data for each metabolite are median
scaled, log2 transformed, extreme outliers removed, and Z-scores generated based
on the mean and standard deviation in this reference population [91, 111–113].
Traditional screening methods Screening tools for certain common abnormalities
indicative of diseases. While there is no definition per se for traditional screen-
ing methods, they usually include organic acids measured in urine (urine organic
acids, UOA); measurements of standard amino acids in plasma (PAA); and car-
nitine conjugates of fatty acids (acylcarnitine profile, ACP). ACP and PAA are
examples of TM where predefined biochemicals are quantitatively measured
compared to known standards. UOA is a semiquantitative, untargeted analysis
in which analytes are qualitatively compared against a few laboratory-specific
internal standards. UOA and PAA are typically performed by liquid chromatog-
raphy (LC) and/or gas chromatography (GC) coupled with mass spectrometry
(MS). In contrast, ACP is performed by tandem MS/MS, where indicators are
aimed at detecting carnitine daughter ions.

References

1. Zhang A, Sun H, Yan G, Wang P, Wang X. Metabolomics for biomarker discovery: moving to
the clinic. Biomed Res Int. 2015;2015:354671.
2. Clish CB. Metabolomics: an emerging but powerful tool for precision medicine. Cold Spring
Harb Mol Case Stud. 2015;1(1):a000588.
3. Ashrafian H, Sounderajah V, Glen R, Ebbels T, Blaise BJ, Kalra D, et al. Metabolomics: the
stethoscope for the twenty-first century. Med Princ Pract. 2021;30(4):301–10.
4. Wang TJ, Larson MG, Vasan RS, Cheng S, Rhee EP, McCabe E, et al. Metabolite profiles and
the risk of developing diabetes. Nat Med. 2011;17(4):448–53.
5. Kalhan SC, Guo L, Edmison J, Dasarathy S, McCullough AJ, Hanson RW, et al. Plasma
metabolomic profile in nonalcoholic fatty liver disease. Metabolism. 2011;60(3):404–13.
6. Mimmi MC, Finato N, Pizzolato G, Beltrami CA, Fogolari F, Corazza A, et al. Absolute quan-
tification of choline-related biomarkers in breast cancer biopsies by liquid chromatography
electrospray ionization mass spectrometry. Anal Cell Pathol (Amst). 2013;36(3–4):71–83.
7. Glunde K, Jacobs MA, Bhujwalla ZM. Choline metabolism in cancer: implications for diag-
nosis and therapy. Expert Rev Mol Diagn. 2006;6(6):821–9.
140 J. Manor and S. H. Elsea

8. Lionel AC, Costain G, Monfared N, Walker S, Reuter MS, Hosseini SM, et al. Improved
diagnostic yield compared with targeted gene sequencing panels suggests a role for whole-­
genome sequencing as a first-tier genetic test. Genet Med. 2018;20(4):435–43.
9. Srivastava S, Love-Nichols JA, Dies KA, Ledbetter DH, Martin CL, Chung WK, et al.
Meta-analysis and multidisciplinary consensus statement: exome sequencing is a first-tier
clinical diagnostic test for individuals with neurodevelopmental disorders. Genet Med.
2019;21(11):2413–21.
10. Manickam K, McClain MR, Demmer LA, Biswas S, Kearney HM, Malinowski J, et al.
Exome and genome sequencing for pediatric patients with congenital anomalies or intel-
lectual disability: an evidence-based clinical guideline of the American College of Medical
Genetics and Genomics (ACMG). Genet Med. 2021;23:2029.
11. Baxter SK, King MC. A time to sequence. JAMA Pediatr. 2017;171(12):e173435.
12. Theunissen TEJ, Nguyen M, Kamps R, Hendrickx AT, Sallevelt S, Gottschalk RWH, et al.
Whole exome sequencing is the preferred strategy to identify the genetic defect in patients
with a probable or possible mitochondrial cause. Front Genet. 2018;9:400.
13. Lehtonen JM, Auranen M, Darin N, Sofou K, Bindoff L, Hikmat O, et al. Diagnostic value of
serum biomarkers FGF21 and GDF15 compared to muscle sample in mitochondrial disease.
J Inherit Metab Dis. 2021;44(2):469–80.
14. Mancuso M, Klopstock T. Diagnosis and management of mitochondrial disorders. Berlin:
Springer International; 2019.
15. Friedman JM, Jones KL, Carey JC. Exome sequencing and clinical diagnosis, vol. 324.
JAMA; 2020. p. 627.
16. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guide-
lines for the interpretation of sequence variants: a joint consensus recommendation of the
American College of Medical Genetics and Genomics and the Association for Molecular
Pathology. Genet Med. 2015;17(5):405–24.
17. Cooper GM. Parlez-vous VUS? Genome Res. 2015;25(10):1423–6.
18. Bertier G, Hétu M, Joly Y. Unsolved challenges of clinical whole-exome sequencing: a sys-
tematic literature review of end-users’ views. BMC Med Genet. 2016;9(1):52.
19. Liu N, Xiao J, Gijavanekar C, Pappan KL, Glinton KE, Shayota BJ, et al. Comparison of
untargeted metabolomic profiling vs traditional metabolic screening to identify inborn errors
of metabolism. JAMA Netw Open. 2021;4(7):e2114155.
20. Alaimo JT, Glinton KE, Liu N, Xiao J, Yang Y, Reid Sutton V, et al. Integrated analysis of
metabolomic profiling and exome data supplements sequence variant interpretation, classifi-
cation, and diagnosis. Genet Med. 2020;22(9):1560–6.
21. Amendola LM, Jarvik GP, Leo MC, McLaughlin HM, Akkari Y, Amaral MD, et al. Performance
of ACMG-AMP variant-interpretation guidelines among nine Laboratories in the Clinical
Sequencing Exploratory Research Consortium. Am J Hum Genet. 2016;98(6):1067–76.
22. Trujillano D, Bertoli-Avella AM, Kumar Kandaswamy K, Weiss ME, Köster J, Marais A,
et al. Clinical exome sequencing: results from 2819 samples reflecting 1000 families. Eur J
Hum Genet. 2017;25(2):176–82.
23. Shashi V, McConkie-Rosell A, Schoch K, Kasturi V, Rehder C, Jiang YH, et al. Practical
considerations in the clinical application of whole-exome sequencing. Clin Genet.
2016;89(2):173–81.
24. Park KJ, Park S, Lee E, Park JH, Park JH, Park HD, et al. A population-based genomic
study of inherited metabolic diseases detected through newborn screening. Ann Lab Med.
2016;36(6):561–72.
25. Atwal PS, Donti TR, Cardon AL, Bacino CA, Sun Q, Emrick L, et al. Aromatic L-amino acid
decarboxylase deficiency diagnosed by clinical metabolomic profiling of plasma. Mol Genet
Metab. 2015;115(2–3):91–4.
26. Wassenberg T, Molero-Luis M, Jeltsch K, Hoffmann GF, Assmann B, Blau N, et al. Consensus
guideline for the diagnosis and treatment of aromatic l-amino acid decarboxylase (AADC)
deficiency. Orphanet J Rare Dis. 2017;12(1):12.
Untargeted Metabolomics, Targeted Care: The Clinical Utilities of Bedside Metabolomics 141

27. Saudubray JM, Baumgartner MR, Walter JH. Inborn metabolic diseases: diagnosis and treat-
ment. Berlin, Heidelberg: Springer; 2016.
28. Pearson TS, Gilbert L, Opladen T, Garcia-Cazorla A, Mastrangelo M, Leuzzi V, et al. AADC
deficiency from infancy to adulthood: symptoms and developmental outcome in an interna-
tional cohort of 63 patients. J Inherit Metab Dis. 2020;43(5):1121–30.
29. Fusco C, Leuzzi V, Striano P, Battini R, Burlina A, Spagnoli C. Aromatic L-amino acid
decarboxylase (AADC) deficiency: results from an Italian modified Delphi consensus. Ital
J Pediatr. 2021;47(1):13.
30. Luc QN, Querubin J. Clinical management of dystonia in childhood. Paediatr Drugs.
2017;19(5):447–61.
31. Kim R, Jeon B, Lee WW. A systematic review of treatment outcome in patients with Dopa-­
responsive dystonia (DRD) and DRD-plus. Mov Disord Clin Pract. 2016;3(5):435–42.
32. Pappan KL, Kennedy AD, Magoulas PL, Hanchard NA, Sun Q, Elsea SH. Clinical metabolo-
mics to segregate aromatic amino acid decarboxylase deficiency from drug-induced metabo-
lite elevations. Pediatr Neurol. 2017;75:66–72.
33. Almontashiri NAM, Zha L, Young K, Law T, Kellogg MD, Bodamer OA, et al. Clinical
validation of targeted and untargeted metabolomics testing for genetic disorders: a 3 year
comparative study. Sci Rep. 2020;10(1):9382.
34. Rodan LH, Anyane-Yeboa K, Chong K, Klein Wassink-Ruiter JS, Wilson A, Smith L, et al.
Gain-of-function variants in the ODC1 gene cause a syndromic neurodevelopmental disorder
associated with macrocephaly, alopecia, dysmorphic features, and neuroimaging abnormali-
ties. Am J Med Genet A. 2018;176(12):2554–60.
35. Bupp CP, Schultz CR, Uhl KL, Rajasekaran S, Bachmann AS. Novel de novo pathogenic
variant in the ODC1 gene in a girl with developmental delay, alopecia, and dysmorphic fea-
tures. Am J Med Genet A. 2018;176(12):2548–53.
36. Marbaix AY, Noël G, Detroux AM, Vertommen D, Van Schaftingen E, Linster CL. Extremely
conserved ATP- or ADP-dependent enzymatic system for nicotinamide nucleotide repair. J
Biol Chem. 2011;286(48):41246–52.
37. Marbaix AY, Tyteca D, Niehaus TD, Hanson AD, Linster CL, Van Schaftingen E. Occurrence
and subcellular distribution of the NADPHX repair system in mammals. Biochem
J. 2014;460(1):49–58.
38. Van Bergen NJ, Guo Y, Rankin J, Paczia N, Becker-Kettern J, Kremer LS, et al. NAD(P)HX
dehydratase (NAXD) deficiency: a novel neurodegenerative disorder exacerbated by febrile
illnesses. Brain. 2019;142(1):50–8.
39. Kremer LS, Danhauser K, Herebian D, Petkovic Ramadža D, Piekutowska-Abramczuk D,
Seibt A, et al. NAXE mutations disrupt the cellular NAD(P)HX repair system and cause a
lethal Neurometabolic disorder of early childhood. Am J Hum Genet. 2016;99(4):894–902.
40. Van Bergen NJ, Walvekar AS, Patraskaki M, Sikora T, Linster CL, Christodoulou J. Clinical
and biochemical distinctions for a metabolite repair disorder caused by NAXD or NAXE
deficiency. J Inherit Metab Dis. 2022;45(6):1028–38.
41. Manor J, Calame DG, Gijavanekar C, Tran A, Fatih JM, Lalani SR, et al. Niacin therapy
improves outcome and normalizes metabolic abnormalities in an NAXD-deficient patient.
Brain. 2022;145(5):e36–40.
42. Pillai NR, Amin H, Gijavanekar C, Liu N, Issaq N, Broniowska KA, et al. Hematologic
presentation and the role of untargeted metabolomics analysis in monitoring treatment for
riboflavin transporter deficiency. Am J Med Genet A. 2020;182(11):2781–7.
43. Amir F, Atzinger C, Massey K, Greinwald J, Hunter LL, Ulm E, et al. The clinical journey
of patients with riboflavin transporter deficiency type 2. J Child Neurol. 2020;35(4):283–90.
44. Liu Z, Peng Q, Li J, Rao C, Lu X. BVVLS2 overlooked for 3 years in a pediatric patient
caused by novel compound heterozygous mutations in SLC52A2 gene. Clin Chim Acta.
2021;523:402–6.
45. Kalafatic Z, Lipovac K, Jezerinac Z, Juretic D, Dumic M, Zurga B, et al. A liver urocanase
deficiency. Metabolism. 1980;29(11):1013–9.
142 J. Manor and S. H. Elsea

46. Espinós C, Pineda M, Martínez-Rubio D, Lupo V, Ormazabal A, Vilaseca MA, et al.

Mutations in the urocanase gene UROC1 are associated with urocanic aciduria. J Med Genet.
2009;46(6):407–11.
47. Camp BW, Broman SH, Nichols PL, Leff M. Maternal and neonatal risk factors for mental
retardation: defining the ‘at-risk’ child. Early Hum Dev. 1998;50(2):159–73.
48. Boyle CA, Yeargin-Allsopp M, Doernberg NS, Holmgreen P, Murphy CC, Schendel
DE. Prevalence of selected developmental disabilities in children 3-10 years of age: the
metropolitan Atlanta developmental disabilities surveillance program, 1991. MMWR CDC
Surveill Summ. 1996;45(2):1–14.
49. Keyfi F, Nasseri M, Nayerabadi S, Alaei A, Mokhtariye A, Varasteh A. Frequency of inborn
errors of metabolism in a northeastern Iranian sample with high consanguinity rates. Hum
Hered. 2018;83(2):71–8.
50. Afzal RM, Lund AM, Skovby F. The impact of consanguinity on the frequency of inborn
errors of metabolism. Mol Genet Metab Rep. 2018;15:6–10.
51. Saad HA, Elbedour S, Hallaq E, Merrick J, Tenenbaum A. Consanguineous marriage and
intellectual and developmental disabilities among Arab Bedouins children of the Negev
region in southern Israel: a pilot study. Front Public Health. 2014;2:3.
52. Jamra R. Genetics of autosomal recessive intellectual disability. Med Genet. 2018;30(3):323–7.
53. Glinton KE, Levy HL, Kennedy AD, Pappan KL, Elsea SH. Untargeted metabolomics iden-
tifies unique though benign biochemical changes in patients with pathogenic variants in
UROC1. Mol Genet Metab Rep. 2019;18:14–8.
54. Kennedy AD, Pappan KL, Donti T, Delgado MR, Shinawi M, Pearson TS, et al.
2-Pyrrolidinone and Succinimide as clinical screening biomarkers for GABA-transaminase
deficiency: anti-­ seizure medications impact accurate diagnosis. Front Neurosci.
2019;13:394.
55. Ferreira EA, Veenvliet ARJ, Engelke UFH, Kluijtmans LAJ, Huigen M, Hoegen B, et al.
Diagnosing, discarding, or de-VUSsing: a practical guide to (un)targeted metabolomics as
variant-transcending functional tests. Genet Med. 2023;25(1):125–34.
56. Subramanian VS, Constantinescu AR, Benke PJ, Said HM. Mutations in SLC5A6 associ-
ated with brain, immune, bone, and intestinal dysfunction in a young child. Hum Genet.
2017;136(2):253–61.
57. Byrne AB, Arts P, Polyak SW, Feng J, Schreiber AW, Kassahn KS, et al. Identification
and targeted management of a neurodegenerative disorder caused by biallelic mutations in
SLC5A6. NPJ Genom Med. 2019;4:28.
58. Holling T, Nampoothiri S, Tarhan B, Schneeberger PE, Vinayan KP, Yesodharan D, et al.
Novel biallelic variants expand the SLC5A6-related phenotypic spectrum. Eur J Hum Genet.
2022;1-11:439.
59. Thistlethwaite LR, Petrosyan V, Li X, Miller MJ, Elsea SH, Milosavljevic A. CTD: an
information-­theoretic algorithm to interpret sets of metabolomic and transcriptomic pertur-
bations in the context of graphical models. PLoS Comput Biol. 2021;17(1):e1008550.
60. Thistlethwaite LR, Li X, Burrage LC, Riehle K, Hacia JG, Braverman N, et al. Clinical
diagnosis of metabolic disorders using untargeted metabolomic profiling and disease-specific
networks learned from profiling data. Sci Rep. 2022;12(1):6556.
61. Kerkhofs M, Haijes HA, Willemsen AM, van Gassen KLI, van der Ham M, Gerrits J, et al.
Cross-omics: integrating genomics with metabolomics in clinical diagnostics. Metabolites.
2020;10(5):206.
62. Graham Linck EJ, Richmond PA, Tarailo-Graovac M, Engelke U, Kluijtmans LAJ, Coene
KLM, et al. metPropagate: network-guided propagation of metabolomic information for pri-
oritization of metabolic disease genes. NPJ Genom Med. 2020;5:25.
63. Smedley D, Jacobsen JO, Jäger M, Köhler S, Holtgrewe M, Schubach M, et al. Next-generation
diagnostics and disease-gene discovery with the exomiser. Nat Protoc. 2015;10(12):2004–15.
64. Bongaerts M, Bonte R, Demirdas S, Huidekoper HH, Langendonk J, Wilke M, et al.
Integration of metabolomics with genomics: metabolic gene prioritization using metabolo-
mics data and genomic variant (CADD) scores. Mol Genet Metab. 2022;136(3):199–218.
Untargeted Metabolomics, Targeted Care: The Clinical Utilities of Bedside Metabolomics 143

65. Rentzsch P, Schubach M, Shendure J, Kircher M. CADD-splice-improving genome-wide vari-

ant effect prediction using deep learning-derived splice scores. Genome Med. 2021;13(1):31.
66. Messa GM, Napolitano F, Elsea SH, di Bernardo D, Gao X. A Siamese neural network
model for the prioritization of metabolic disorders by integrating real and simulated data.
Bioinformatics. 2020;36(Suppl_2):i787–i94.
67. Garg U, Smith LD. Biomarkers in inborn errors of metabolism: clinical aspects and labora-
tory determination. Amsterdam: Elsevier Science; 2017.
68. Braverman NE, Raymond GV, Rizzo WB, Moser AB, Wilkinson ME, Stone EM, et al.
Peroxisome biogenesis disorders in the Zellweger spectrum: an overview of current diagno-
sis, clinical manifestations, and treatment guidelines. Mol Genet Metab. 2016;117(3):313–21.
69. Steinberg SJ, Raymond GV, Braverman NE, Moser AB. Zellweger spectrum disorder. In:
Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH, Gripp KW, et al., editors.
GeneReviews(®). Seattle, WA: University of Washington; 1993.
70. Kim PK, Hettema EH. Multiple pathways for protein transport to peroxisomes. J Mol Biol.
2015;427(6 Pt A):1176–90.
71. Wangler MF, Hubert L, Donti TR, Ventura MJ, Miller MJ, Braverman N, et al. A metabo-
lomic map of Zellweger spectrum disorders reveals novel disease biomarkers. Genet Med.
2018;20(10):1274–83.
72. Ah Mew N, Simpson KL, Gropman AL, Lanpher BC, Chapman KA, Summar ML. Urea
cycle disorders overview. In: Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH,
Gripp KW, et al., editors. GeneReviews(®). Seattle, WA: University of Washington; 1993.
73. Stone WL, Basit H, Jaishankar GB. Urea cycle disorders. In: StatPearls. Treasure Island, FL:
StatPearls; 2022.
74. Sen K, Whitehead M, Castillo Pinto C, Caldovic L, Gropman A. Fifteen years of urea cycle
disorders brain research: looking back, looking forward. Anal Biochem. 2022;636:114343.
75. Amayreh W, Meyer U, Das AM. Treatment of arginase deficiency revisited: guanidinoacetate
as a therapeutic target and biomarker for therapeutic monitoring. Dev Med Child Neurol.
2014;56(10):1021–4.
76. Burrage LC, Thistlethwaite L, Stroup BM, Sun Q, Miller MJ, Nagamani SCS, et al.
Untargeted metabolomic profiling reveals multiple pathway perturbations and new clinical
biomarkers in urea cycle disorders. Genet Med. 2019;21(9):1977–86.
77. De Deyn PP, Marescau B, Macdonald RL. Guanidino compounds that are increased in hyper-
argininemia inhibit GABA and glycine responses on mouse neurons in cell culture. Epilepsy
Res. 1991;8(2):134–41.
78. Hanna-El-Daher L, Béard E, Henry H, Tenenbaum L, Braissant O. Mild guanidinoacetate
increase under partial guanidinoacetate methyltransferase deficiency strongly affects brain
cell development. Neurobiol Dis. 2015;79:14–27.
79. Ostojic SM. Safety of dietary Guanidinoacetic acid: a villain of a good guy? Nutrients.
2021;14(1):75.
80. Ingoglia F, Chong JL, Pasquali M, Longo N. Creatine metabolism in patients with urea cycle
disorders. Mol Genet Metab Rep. 2021;29:100791.
81. Grace RF, Bianchi P, van Beers EJ, Eber SW, Glader B, Yaish HM, et al. Clinical spectrum
of pyruvate kinase deficiency: data from the pyruvate kinase deficiency natural history study.
Blood. 2018;131(20):2183–92.
82. Bianchi P, Fermo E, Glader B, Kanno H, Agarwal A, Barcellini W, et al. Addressing the
diagnostic gaps in pyruvate kinase deficiency: consensus recommendations on the diagnosis
of pyruvate kinase deficiency. Am J Hematol. 2019;94(1):149–61.
83. Van Dooijeweert B, Broeks MH, Verhoeven-Duif NM, Van Beers EJ, Nieuwenhuis EES,
Van Solinge WW, et al. Untargeted metabolic profiling in dried blood spots identifies disease
fingerprint for pyruvate kinase deficiency. Haematologica. 2021;106(10):2720–5.
84. Wang D, Pascual JM, De Vivo D. Glucose transporter type 1 deficiency syndrome. In:
Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH, Gripp KW, et al., editors.
GeneReviews(®). Seattle, WA: University of Washington; 1993.
144 J. Manor and S. H. Elsea

85. Tang M, Monani UR. Glut1 deficiency syndrome: new and emerging insights into a proto-
typical brain energy failure disorder. Neurosci Insights. 2021;16:26331055211011507.
86. Wibisono C, Rowe N, Beavis E, Kepreotes H, Mackie FE, Lawson JA, et al. Ten-year single-­
center experience of the ketogenic diet: factors influencing efficacy, tolerability, and compli-
ance. J Pediatr. 2015;166(4):1030–6.e1.
87. Klepper J, Akman C, Armeno M, Auvin S, Cervenka M, Cross HJ, et al. Glut1 deficiency syn-
drome (Glut1DS): state of the art in 2020 and recommendations of the international Glut1DS
study group. Epilepsia Open. 2020;5(3):354–65.
88. Almuqbil M, Go C, Nagy LL, Pai N, Mamak E, Mercimek-Mahmutoglu S. New paradigm
for the treatment of glucose transporter 1 deficiency syndrome: low glycemic index diet and
modified high amylopectin cornstarch. Pediatr Neurol. 2015;53(3):243–6.
89. Cappuccio G, Pinelli M, Alagia M, Donti T, Day-Salvatore DL, Veggiotti P, et al. Biochemical
phenotyping unravels novel metabolic abnormalities and potential biomarkers associated
with treatment of GLUT1 deficiency with ketogenic diet. PloS One. 2017;12(9):e0184022.
90. Miller MJ, Bostwick BL, Kennedy AD, Donti TR, Sun Q, Sutton VR, Elsea SH. Chronic
Oral L-Carnitine Supplementation Drives Marked Plasma TMAO Elevations in Patients
with Organic Acidemias Despite Dietary Meat Restrictions. JIMD Rep. 2016;30:39–44.
[Link] Epub 2016 Mar 3. PMID: 26936850; PMCID:
PMC5110437.
91. Kennedy AD, Miller MJ, Beebe K, Wulff JE, Evans AM, Miller LA, et al. Metabolomic pro-
filing of human urine as a screen for multiple inborn errors of metabolism. Genet Test Mol
Biomarkers. 2016;20(9):485–95.
92. El-Hattab AW. Serine biosynthesis and transport defects. Mol Genet Metab.
2016;118(3):153–9.
93. Glinton KE, Benke PJ, Lines MA, Geraghty MT, Chakraborty P, Al-Dirbashi OY, et al.
Disturbed phospholipid metabolism in serine biosynthesis defects revealed by metabolomic
profiling. Mol Genet Metab. 2018;123(3):309–16.
94. Ferreira CR, Goorden SMI, Soldatos A, Byers HM, Ghauharali-van der Vlugt JMM, Beers-­
Stet FS, et al. Deoxysphingolipid precursors indicate abnormal sphingolipid metabolism in
individuals with primary and secondary disturbances of serine availability. Mol Genet Metab.
2018;124(3):204–9.
95. Litwack G. Chapter 7 - Glycogen and Glycogenolysis. In: Litwack G, editor. Human bio-
chemistry. Boston: Academic Press; 2018. p. 161–81.
96. Banne E, Meiner V, Shaag A, Katz-Brull R, Gamliel A, Korman S, et al. Transaldolase defi-
ciency: a new case expands the phenotypic Spectrum. JIMD Rep. 2016;26:31–6.
97. Shayota BJ, Donti TR, Xiao J, Gijavanekar C, Kennedy AD, Hubert L, et al. Untargeted
metabolomics as an unbiased approach to the diagnosis of inborn errors of metabo-
lism of the non-oxidative branch of the pentose phosphate pathway. Mol Genet Metab.
2020;131(1–2):147–54.
98. Buzkova J, Nikkanen J, Ahola S, Hakonen AH, Sevastianova K, Hovinen T, et al. Metabolomes
of mitochondrial diseases and inclusion body myositis patients: treatment targets and bio-
markers. EMBO Mol Med. 2018;10(12):e9091.
99. Sharma R, Reinstadler B, Engelstad K, Skinner OS, Stackowitz E, Haller RG, et al.
Circulating markers of NADH-reductive stress correlate with mitochondrial disease severity.
J Clin Invest. 2021;131(2):e136055.
100. Yatsuga S, Fujita Y, Ishii A, Fukumoto Y, Arahata H, Kakuma T, et al. Growth differentiation
factor 15 as a useful biomarker for mitochondrial disorders. Ann Neurol. 2015;78(5):814–23.
101. Evans B. How autism became autism: the radical transformation of a central concept of child
development in Britain. Hist Human Sci. 2013;26(3):3–31.
102. Sandin S, Lichtenstein P, Kuja-Halkola R, Hultman C, Larsson H, Reichenberg A. The heri-
tability of autism Spectrum disorder. JAMA. 2017;318(12):1182–4.
103. Colvert E, Tick B, McEwen F, Stewart C, Curran SR, Woodhouse E, et al. Heritability
of autism Spectrum disorder in a UK population-based twin sample. JAMA Psychiatry.
2015;72(5):415–23.
Untargeted Metabolomics, Targeted Care: The Clinical Utilities of Bedside Metabolomics 145

104. Schaefer GB, Mendelsohn NJ. Clinical genetics evaluation in identifying the etiology of
autism spectrum disorders: 2013 guideline revisions. Genet Med. 2013;15(5):399–407.
105. Ghaziuddin M, Al-Owain M. Autism spectrum disorders and inborn errors of metabolism: an
update. Pediatr Neurol. 2013;49(4):232–6.
106. Žigman T, Petković Ramadža D, Šimić G, Barić I. Inborn errors of metabolism associated with
autism Spectrum disorders: approaches to intervention. Front Neurosci. 2021;15:673600.
107. Glinton KE, Elsea SH. Untargeted metabolomics for autism spectrum disorders: current sta-
tus and future directions. Front Psych. 2019;10:647.
108. Ritz B, Yan Q, Uppal K, Liew Z, Cui X, Ling C, et al. Untargeted metabolomics screen of
mid-pregnancy maternal serum and autism in offspring. Autism Res. 2020;13(8):1258–69.
109. Courraud J, Ernst M, Svane Laursen S, Hougaard DM, Cohen AS. Studying autism using untar-
geted metabolomics in newborn screening samples. J Mol Neurosci. 2021;71(7):1378–93.
110. Smith AM, Natowicz MR, Braas D, Ludwig MA, Ney DM, Donley ELR, et al. A metabo-
lomics approach to screening for autism risk in the Children’s autism metabolome project.
Autism Res. 2020;13(8):1270–85.
111. Ford L, Kennedy AD, Goodman KD, Pappan KL, Evans AM, Miller LAD, et al. Precision
of a clinical metabolomics profiling platform for use in the identification of inborn errors of
metabolism. J Appl Lab Med. 2020;5(2):342–56.
112. Kennedy AD, Pappan KL, Donti TR, Evans AM, Wulff JE, Miller LAD, et al. Elucidation of
the complex metabolic profile of cerebrospinal fluid using an untargeted biochemical profil-
ing assay. Mol Genet Metab. 2017;121(2):83–90.
113. Kennedy AD, Wittmann BM, Evans AM, Miller LAD, Toal DR, Lonergan S, et al.
Metabolomics in the clinic: a review of the shared and unique features of untargeted metabo-
lomics for clinical research and clinical testing. J Mass Spectrom. 2018;53(11):1143–54.
114. Waters D, Adeloye D, Woolham D, Wastnedge E, Patel S, Rudan I. Global birth prevalence
and mortality from inborn errors of metabolism: a systematic analysis of the evidence. J Glob
Health. 2018;8(2):021102.
Metabolomics in the Study of Human
Mitochondrial Diseases

Rajaa Sebaa, Mary-Ellen Harper, Ruqaiah Al-Tassan, Mohammed Al-Owain,

and Anas M. Abdel Rahman

Abstract Mitochondria are dynamic cellular organelles playing many biological

roles that are fundamentally required for cellular functions. The primary role of
mitochondria is ATP production through oxidative phosphorylation (OXPHOS).
Mitochondria are found in nearly all cell types, and their number within cells varies
in a tissue−/organ-dependent manner. Tissues/organs characterized by high-energy
demands contain abundant mitochondria, and these tissues/organs are most fre-
quently affected when their mitochondria are dysfunctional. The resulting patholo-
gies can be generally referred to as mitochondrial diseases (MDs). MDs can be
caused by nuclear or mitochondrial DNA mutations in genes encoding mitochon-
drial proteins, including OXPHOS proteins. Also, MDs can be developed through
nongenetic mechanisms such as those involving environmental factors, mitotoxicity
drugs, oxidative stress, and aging. MDs can appear over the entire life span. Patients
with particular MDs present a wide range of heterogeneous phenotypes with differ-

R. Sebaa
Department of Medical Laboratories, College of Applied Medical Sciences, Shaqra
University, Al-Dawadmi, Saudi Arabia
M.-E. Harper
Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine,
University of Ottawa, Ottawa, ON, Canada
Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, ON, Canada
R. Al-Tassan
Department of Internal Medicine, King Abdullah Hospital, Bisha, Saudi Arabia
Department of Medical Genomics, Centre for Genomic Medicine, King Faisal Specialist
Hospital and Research Center, Riyadh, Saudi Arabia
M. Al-Owain
Department of Medical Genomics, Centre for Genomic Medicine, King Faisal Specialist
Hospital and Research Center, Riyadh, Saudi Arabia
A. M. Abdel Rahman (*)
Metabolomics Section, Department of Clinical Genomics, Center for Genome Medicine,
King Faisal Specialist Hospital & Research Center (KFSHRC), Riyadh, Saudi Arabia
e-mail: AabdelRahman46@[Link]

© The Author(s), under exclusive license to Springer Nature Singapore Pte 147
Ltd. 2023
A. M. Abdel Rahman (ed.), Clinical Metabolomics Applications in Genetic
Diseases, [Link]
148 R. Sebaa et al.

ent levels of disease severity. The wide variety of leading causes and heterogenous
phenotypes of MDs make diagnosing MDs notoriously challenging. Despite these
challenges, multiple diagnostic examinations and tools, including family history,
phenotypic examinations, neurological imaging, biochemical tests, and genetic
analyses, have collectively enhanced the diagnosis of MDs. As a result of the diag-
nostic limitations and drawbacks, there have been demands for developing new
diagnostic approaches capable of detecting metabolic perturbations of MDs used as
metabolic biosignatures. For that reason, the metabolomic approach, the study of
small metabolites ≤1500 daltons, has recently garnered attention. While metabolo-
mics offers significant advances, it is recommended that data sets be integrated with
other diagnostic approaches. This chapter reviews the application of metabolomic
analyses in studying human MDs.

Keywords Mitochondria · Mitochondrial diseases · Mass spectrometry · Nuclear

Magnetic Resonance (NMR) Spectroscopy · Untargeted metabolomics · Targeted
metabolomics · Metabolic biosignatures

Abbreviations
ANT Adenine nucleotide translocase
ATP Adenosine triphosphate
BAT Brown adipose tissue
CAT Catalase
CE-MS Capillary electrophoresis-coupled mass spectrometry
CIL Chemical isotope labeling
CPEO Chronic progressive external ophthalmoplegia
DRP1 Dynamin-related protein-1
ETF Electron transfer flavoprotein
FIS1 Fission protein-1
GC-MS Gas chromatography-coupled mass spectrometry
GPxs Glutathione peroxidases
GSH Glutathione
IMM Inner mitochondrial membrane
IMS Intermembrane space
KSS Kearns-Sayre syndrome
LC-MS Liquid chromatography-coupled mass spectrometry
LHON Leber hereditary optic neuropathy
MDs Mitochondrial diseases
MELAS Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like
episodes
MERRF Myoclonic epilepsy with ragged-red fibers
MFF Mitochondrial fission factor
MFN1 Mitofusins-1
MFN2 Mitofusins-2
MiD49 Mitochondrial dynamic proteins of 49 kDa
Metabolomics in the Study of Human Mitochondrial Diseases 149

MiD51 Mitochondrial dynamic proteins of 51 kDa

MNGIE Mitochondrial neurogastrointestinal encephalopathy
MS Mass spectrometry
mtDNA Mitochondrial DNA
NAC N-acetylcysteine
NARP Neuropathy, ataxia, and retinitis pigmentosa
nDNA Nuclear DNA
NMR Nuclear magnetic resonance
OMM Outer mitochondrial membrane
OPA1 Optic atrophy-1
OXPHOS Oxidative phosphorylation
PMF Proton motive force
PMS Pearson marrow pancreas syndrome
ROS Reactive oxygen species
rRNA Ribosomal RNA
SOD Superoxide dismutase
SPG7 Hereditary spastic paraplegia 7
TCA Tricarboxylic acid cycle
TFAM Transcription factor A of mitochondria
TFB2M Mitochondrial transcription factor B2
tRNA Transfer RNA
Trx Thioredoxin
UCP1 Uncoupling protein-1
VUS Variants of uncertain significance
WES Whole exome sequencing
WGS Whole genome sequencing

1 Introduction

Mitochondria are organelles most commonly referred to as the powerhouses of cells

due to their fundamental function of ATP production to fuel the energy-demanding
process in cells. Mitochondria transduce energy substrates into adenosine triphos-
phate (ATP) in key metabolic tissues [1]. Mitochondria are found in nearly all cell
types, although their numbers vary in a tissue−/organ-dependent manner. Tissues
characterized by high energy demands have many mitochondria, but those with less
energy demand have fewer mitochondria to match their cellular energy require-
ments [2]. Despite the variations in mitochondrial content across different tissues,
certain factors can increase or decrease the mitochondrial number in adaptive pro-
cesses when cells undergo physiological changes such as exercise, dietary energy
surfeit or deficit, tissue growth or atrophy, and aging [3, 4]. The role of mitochon-
dria in energy transduction is widely understood. However, mitochondria are also
involved in other vital biological functions, including reactive oxygen species
(ROS) production, redox signaling, Ca+2 hemostasis, and cellular apoptosis [5, 6].
150 R. Sebaa et al.

Moreover, mitochondrial functions are influenced by certain properties such as

mitochondrial morphology, ultrastructure, and dynamics [7, 8].
Depending on cell type and physiological and energetic status, mitochondria
present diverse lengths from 0.5 to 10 μm and shapes, but they share the main struc-
tural components [9]. Mitochondria have five main compartments, including the
outer mitochondrial membrane (OMM), inner mitochondrial membrane (IMM),
intermembrane space (IMS), cristae, and matrix. The OMM and IMM are mainly
composed of phospholipids and proteins to support the function of these mem-
branes, with the IMM having a far greater membrane protein density than the
OMM. The specific compositions of phospholipids and proteins in the OMM and
IMM determine the degree of the integrity and permeability of these membranes.
The OMM is relatively permeable, transporting low molecular metabolites, solutes,
and ions from the cytoplasm into the IMS. The IMM is highly selectively imperme-
able to most solutes and metabolites as this structural feature of the IMM is impor-
tant to allow OXPHOS to occur.
Furthermore, the IMM is associated with a wide range of transporters and protein
shuttles to support the many mitochondrial metabolic and bioenergetic pathways [10,
11]. Aqueous regions defined by the membranes are the IMS and matrix. While IMS
is present between OMM and IMM, the matrix is defined and enveloped by the
IMM. Both the IMS and matrix are important for metabolic events and pathways. The
matrix contains many enzymes involved in metabolic pathways such as the tricarbox-
ylic acid cycle, fatty acid β-oxidation, ketogenesis, amino acid metabolism, urea
cycle, hormone synthesis, etc. Also, the matrix houses the circular mitochondrial
DNA (mtDNA), which exclusively encodes mitochondrial components and its genetic
machinery elements such as ribosomes and RNA (more details mentioned below).
The fifth mitochondrial compartment is the cristae, defined as the folds of IMM into
the matrix to increase the surface area of IMM for enhancing mitochondrial metabolic
activity and ATP production and to allow important protein-­protein interactions [12].
The mitochondrial structure is illustrated in (Fig. 1).
Concerning mitochondrial morphology, mitochondria exist in many morpholo-
gies, such as short oval or spherical tubules, long elongated tubules, or reticular
networks. Mitochondrial morphology is distinctively different across cell/tissue
types. Moreover, multiple morphologies of mitochondria can be seen with one cell

Fig. 1 Mitochondrial
structure consists of outer
mitochondrial membrane
(OMM), inner
mitochondrial membrane
(IMM), intermembrane
space (IMS), matrix,
cristae, and circular
mitochondrial genome
(mtDNA)
Metabolomics in the Study of Human Mitochondrial Diseases 151

type [13–15]. Under various physiological and pathological conditions, mitochon-

dria continuously remodel their morphology to allow the cells to adapt [16, 17].
Mitochondria are highly dynamic and continuously undergo events of fission and
fusion. Mitochondrial dynamics maintain mitochondrial health, morphology, size,
and numbers [18, 19]. The morphological changes are induced when cells are trig-
gered by certain stressors or undergo energetic changes and are required during
cellular adaptation [9, 19]. Mitochondrial dynamics is under the control of GTPase
proteins that mediate the events of fission and fusion. Mitochondrial fission is the
fragmentation of one mitochondrion into two or more mitochondria. It requires the
recruitment of a cytosolic protein called dynamin-related protein-1 (DRP1) through
the action of dynamic proteins, including mitochondrial fission protein-1 (FIS1),
mitochondrial fission factor (MFF), and mitochondrial dynamic proteins of 49 and
51 kDa (MiD49 and MiD51). Mitochondrial fission is important for quality control
as the fragmented, damaged mitochondria are removed. This is referred to as
mitophagy, the selective removal of damaged mitochondria by autophagy [20–22].
In contrast to mitochondrial fission, mitochondrial fusion promotes the joining of
two separate mitochondria by merging OMM, IMM, and matrix to make one elon-
gated organelle. Mitochondrial fusion needs the action of several GTPase proteins,
including mitofusin (MFN1) and mitofusin-2 (MFN2), to promote OMM fusion
and optic atrophy-1 (OPA1) protein for IMM fusion [23, 24]. Fission and fusion
should be balanced to maintain a healthy mitochondrial reticulum in cells; other-
wise, mitochondrial dysfunction occurs and contributes to the development of mito-
chondrial diseases (MDs).
From the genetic aspect, mitochondria have the exceptional feature of being the
only cellular organelle possessing DNA beyond that found in the nucleus. mtDNA
is maternally inherited since paternal sperm mitochondria are targeted and destroyed
after egg fertilization during embryogenesis. mtDNA is circular, and there are
100–10,000 copies per cell. mtDNA contains 37 genes encoding only 13 mitochon-
drial protein subunits involved in OXPHOS, 2 ribosomal RNA (rRNA), and 22
transfer RNA (tRNA) for intra-mitochondrial protein synthesis [25, 26]. In contrast,
approximately 1500 mitochondrial proteins are encoded by nuclear DNA (nDNA),
translated into the cytoplasm, and imported to the mitochondria [27]. As mitochon-
dria have their transcriptional machinery, mtDNA is colocalized with transcriptional
factors, which are nuclear-encoded, such as transcription factor A of mitochondria
(TFAM) and mitochondrial transcription factor B2 (TFB2M), to initiate and regu-
late mtDNA transcription and eventually synthesize OXPHOS subunits [28].
mtDNA is particularly susceptible to mutations compared to nDNA due to the lim-
ited repair mechanisms and the high exposure to ROS emission in mitochondria.
Mutations in mitochondrial protein-encoding genes lead to dysfunctional mitochon-
dria and various MDs (mentioned below).
152 R. Sebaa et al.

2 Mitochondrial Metabolic Pathways

Mitochondria are hubs of cellular metabolism. They are involved in many crucial
metabolic pathways, including the tricarboxylic acid cycle (TCA), fatty acid
β-oxidation, acyl-carnitine metabolism, urea cycle, amino acid degradation, keto-
genesis/ketolysis, steroidogenesis, and OXPHOS as shown in (Fig. 2). These path-
ways produce small molecular weight intermediate molecules called metabolites,
which are also important for cellular proliferation, signaling, survival, and function.
Defects in mitochondrial metabolism are associated with alterations in the level of
metabolites, which can directly or indirectly affect the physiology of tissues/organs
in the body [12, 29].
The OXPHOS system in the IMM consists of NADH dehydrogenase (complex
I), succinate dehydrogenase (complex II), cytochrome c reductase (complex III),
cytochrome c oxidase (complex IV), and ATP synthase (complex V) [30]. In detail,
in the presence of oxygen, mitochondria convert chemical energy stored in energy
substrates, such as pyruvate, acyl-CoA, ketone bodies, etc., into ATP through cou-
pled OXPHOS systems mediated via complex V. These energetic metabolites
undergo oxidative reactions producing reducing agents such as nicotinamide ade-
nine dinucleotide hydrogen (NADH + H) and flavin adenine dinucleotide dihydro-
gen (FADH2). These reduced coenzymes then provide electrons either to certain
OXPHOS complexes, including complex I and complex II or other IMM-bounded

Fig. 2 Simplified illustration of various metabolic pathways taking place in mitochondria

Metabolomics in the Study of Human Mitochondrial Diseases 153

proteins in conjunction with OXPHOS, such as electron transfer flavoprotein (ETF).

When electrons are accepted by complex I, complex II, or ETF, they are subse-
quently passed to coenzyme Q, a mobile fat-soluble electron carrier found in the
IMM. Then, coenzyme Q transfers the electrons to complex III, which are then
transferred to complex IV through cytochrome C. Ultimately, the electrons reduce
oxygen to produce water. While electron flow happens along complexes I–IV, pro-
tons are pumped from the matrix to IMS through complex I, complex III, and com-
plex IV, contributing to the generation of an electrochemical gradient called proton
motive force (PMF) located across the IMM. Subsequently, free energy stored in
PMF drives protons back to the mitochondria matrix through complex V, resulting
in the production of ATP, as shown in (Fig. 3) [30–33].
The coupling of electron flow (and oxygen consumption) from ATP production
is far from perfect. A certain amount of uncoupling occurs through proton leaks in
all cell types [34]. However, one highly metabolic tissue called brown adipose tissue
(BAT) that uncoupled OXPHOS has the physiological function of heat production
(thermogenesis) instead of ATP production. BAT has low levels of ATP synthase but
high amounts of uncoupling protein 1 (UCP1). UCP1 belongs to a gene family of
UCPs, and in BAT, it mediates proton leak for thermoregulatory functions. When
UCP1 is activated, oxidative reactions upstream of UCP1 support a high influx of
reducing equivalents (i.e., electrons) into the electron transfer system in complexes
I–IV. This thereby results in proton pumping from the matrix into IMS, causing the
formation of PMF, which is rapidly dissipated through UCP1-mediated proton leak

Fig. 3 Mitochondrial-coupled OXPHOS pathways

154 R. Sebaa et al.

activity. Thermogenesis results from the upstream oxidative reactions [35]. This is
depicted in (Fig. 4). Interestingly, there are other uncoupling proteins named UCP2
and UCP3, which protect against oxidative damage and facilitate fatty acid oxida-
tion in the tissues/cells [36]. UCP2 is found in most tissue types, while UCP3 is
predominantly found in the skeletal muscle and BAT, but their levels of expression
are approximately two orders of magnitude lower than the expression of UCP1 in
BAT [37, 38]. Adenine nucleotide translocase (ANT), which normally exchanges
cytosolic ADP for mitochondrial ATP, has been shown to cause proton leak across
the IMM and thereby possibly protect against oxidative damage [38–42].
During coupled or uncoupled OXPHOS pathways, ROS, which are byproducts
of aerobic metabolism, can be formed. ROS include superoxide anion (O2−), hydro-
gen peroxide (H2O2), and hydroxyl radicals (OH·). The last are reactive molecules
and free radicals derived from molecular oxygen produced in mitochondria.
Although they are needed at certain physiological levels to support cellular signal-
ing and transduction, increased and uncontrolled levels of ROS are detrimental,
causing pathological effects on cells/tissues such as proteins, lipids, and DNA [43–
45]. Therefore, cells/tissues have antioxidant systems that neutralize excessive
amounts of ROS. The antioxidant systems can be enzymatic and nonenzymatic
based. The enzymatic antioxidant system contains several enzymes, including
superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPxs), and

Fig. 4 Mitochondrial uncoupled OXPHOS pathways

Metabolomics in the Study of Human Mitochondrial Diseases 155

thioredoxin (Trx). Nonenzymatic-based antioxidant systems include reduced gluta-

thione (GSH), GSH precursors and reducers, such as N-acetylcysteine (NAC), and
vitamins such as vitamins C, A, and E and their derivatives. Accordingly, the anti-
oxidant systems are fundamental as a protective cellular mechanism against oxida-
tive damage. Thus, impairments in the antioxidant mechanisms can lead to oxidative
stress due to the imbalance between ROS and antioxidant systems. Oxidative stress
can lead to pathological conditions, including MDs [46–49].

3 Mitochondrial Diseases

As described above, MDs can be developed because of defects in mitochondrial

properties, including mitochondrial genome, metabolism, structure, and dynamics.
MDs are heterogeneous and complex diseases sharing the common feature of mito-
chondrial dysfunction. MDs are mainly caused directly by inherited mutations in
genes encoding mitochondrial OXPHOS proteins, regardless of whether genes in
nDNA or mtDNA encode the proteins. MDs can also develop because of inherited
alterations in non-OXPHOS mitochondrial proteins, which are essentially required
for mitochondrial function. In addition, nongenetic factors such as environmental
stressors, myotoxicity drugs, oxidative stress, and aging can progressively cause
dysfunctional mitochondria leading to eventually MDs during a lifetime [50].
Examples of MDs developed by either genetic or nongenetic causes are mito-
chondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS),
chronic progressive external ophthalmoplegia (CPEO), neuropathy, ataxia, and reti-
nitis pigmentosa (NARP), myoclonic epilepsy with ragged-red fibers (MERRF),
Leber hereditary optic neuropathy (LHON), Kern-Sayre syndrome (KSS), Pearson
marrow pancreas syndrome (PMS), mitochondrial neurogastrointestinal encepha-
lopathy (MNGIE), Leigh syndrome, Alpers-Huttenlocher Syndrome, Barth
Syndrome, Parkinson’s disease, Alzheimer’s disease, autism, Huntington’s disease,
amyotrophic lateral sclerosis, Wilson’s disease, Charcot-Marie-Tooth type 2 K,
hereditary spastic paraplegia 7 (SPG7), Friedreich’s ataxia, schizophrenia, sepsis,
cardiovascular diseases, cancers, diabetes, and metabolic syndromes [51–55].
Expectedly, new MDs are about to develop and continue to be discovered. The prev-
alence of MDs is not accurately recorded. It has been challenging to precisely deter-
mine the prevalence of MDs due to the following reasons: First, the identification of
MDs accounted only for diagnostic patients who have undergone molecular testing
for the monogenic MDs, which does not account for other unidentified and/or sus-
pected individuals of MDs, especially for those who had mitochondrial defect with-
out developing obvious symptoms of MD during their lifetimes. Second, one
particular MD can show heterogenous phenotypes and symptoms in different
patients, which makes the symptoms of that particular MD not firm. Third, MDs can
develop from different factors, including genetic and nongenetic factors, and for
nongenetic factors, there is a possibility that new factors might arise with time.
156 R. Sebaa et al.

Thus, the current prevalence of MDs is probably underestimated. A better esti-

mate of the true prevalence of MDs will lead to increased efforts to diagnose and
effectively treat the diseases accurately. To do so, there have been increasing global
efforts from the research and clinical community to develop clinical approaches for
diagnosing patients with MDs, contributing to the correction of underestimated MD
prevalence.
Currently, studies have reported that MDs observed in children account for
approximately 5 individuals per 100,000 population [56]. Another study reported
that the prevalence of MDs in adults is estimated to be one individual per 5000 [57].
MDs are currently more common than previously thought, requiring an early diag-
nosis to enhance the health outcomes of patients at an early stage and provide them
with proper therapeutic interventions.

4 Diagnostic Tools for MDs

Various clinical approaches are utilized for diagnosing MDs, ranging from simple
to sophisticated methods. Suspected MD patients undergo a series of examinations
and tests. Examinations are extensive and include investigations of a patient’s fam-
ily medical history, clinical phenotypes, biochemical parameters, molecular genetic
tests, tissue biopsies, and neurological abnormalities. All these examinations have
usually been considered in diagnosing MDs because they give a comprehensive
picture of pathological status at distinct levels. Consensus-based recommendations
written by the Mitochondrial Medicine Society mention the optimal diagnostic
tools that can be used for MDs involving biochemical tests, genetic analyses, tissue
biopsy examinations, and neuroimaging as they explained the purposes of each
approach to verify the diagnosis of MDs [58].
Following the initial investigation of the medical family history and clinical phe-
notypes of the suspected MD patients, biochemical tests are usually performed on
biological fluids such as blood, urine, and CFS samples to measure the levels of
certain metabolites commonly disrupted in MDs, including pyruvate, lactate, acyl-­
carnitines, ketone bodies, amino acids, and organic acids [58]. Lactate levels and the
ratio of lactate/pyruvate are commonly increased when mitochondria are defective
as pyruvate utilization by the mitochondria decreases. Moreover, ketone bodies are
often perturbed in MD patients. In addition, there are alterations in the level of
amino acids and organic acids in MD patients acting as an indication of mitochon-
dria dysfunction [59]. All the biochemical analyses mentioned above are not spe-
cific and exclusive to MDs because these alterations are a common feature in most
MDs and also could be seen in non-MDs.
Thus, supportive and specific analyses are needed to perform in addition to the
biochemical tests to improve the diagnostic investigation of MDs, involving neuro-
logical imaging of the central nervous system and collecting tissue biopsies from
MD patients for enzymatic analyses of OXPHOS and other mitochondrial proteins.
These methods show their great ability to inform about alterations in mitochondria
Metabolomics in the Study of Human Mitochondrial Diseases 157

in MDs cases; however, these methods have certain pitfalls as neurological imaging
detect abnormalities in the brain and nerve system developed only in particular, but
not all, MD patients [58]. In addition, enzymatic assays provide valuable functional
analyses of mitochondria, although they require an invasive tissue biopsy procedure.
In addition, genetic testing has been extensively included in the diagnostic
criteria of MDs since most MDs are inherited diseases. Genetic testing discovers
the genetic mutations leading to MDs. The implication of genetic testing has been
done for MDs through testing for common pathogenic variants in nDNA or
mtDNA genes associated with MDs by sequencing the whole mtDNA or nDNA
via whole genome sequencing (WGS) and/or whole exome sequencing (WES).
MDs that were identified by multiple genetic testing approaches include those
MDs developed by various pathogenic variants in genes coding for the following
proteins, NADH dehydrogenase, pyruvate dehydrogenase complex component X
(PDHX), ethylmalonic encephalopathy 1 protein (ETHE1), mitochondrial inner
membrane protein (MPV17), mitochondrial carnitine/acylcarnitine carrier protein
(SLC25A20), mitochondrial fission factor (MFF), F-Box and leucine-rich repeat
protein 4 (FBXL4), elaC ribonuclease Z 2 (ELAC2), protein PET100 (PET100),
iron-sulfur cluster assembly 2 (ISCA2), mitochondrial-processing peptidase sub-
unit alpha (PMPCA), metal cation symporter ZIP8 (SLC39A8), mitochondrial
coenzyme A transporter (SLC25A42), ATP-dependent zinc metalloprotease
(YME1L1), mitochondrial intermediate peptidase (MIPEP), mitochondrial cal-
cium uptake protein 2 (MICU2), cytochrome c oxidase subunit 5A (COX5A),
ubiquinone biosynthesis methyltransferase COQ5 (COQ5), and nundid hydrolase
2 (NUDT2) [60–74].
Regardless of the substantial implications of genetic approaches in identifying
MDs, the genetic approaches have certain diagnostic downsides. Specifically, these
genetic tests require reading out an unlimited number of genes, and the roles of
many genes are still not fully understood. Finding mutations in metabolism-related
genes that are functionally unknown, called variants of uncertain significance
(VUS), could be misleading in the context of MDs because it is not fully understood
if these unknown mutations affect health. Furthermore, mutations identified by
genetic tests might be secondary findings of other diseases not discovered or devel-
oped yet and unrelated to MDs of interest, which is considered a false-positive dis-
covery. In addition, genetic testing cannot be used for those MDs that are developed
by nongenetic factors over a patient’s lifetime [75, 76].
Consequently, based on the previous issues associated with the genetic
approaches, there is still a definitive need to perform alternative functional analyses
of suspected VUS found in metabolism-related genes to ensure whether these
genetic mutations truly cause MDs. Also, these alternative functional analyses could
help examine the pathogenic effects of nongenetic factors leading to MDs. Since
mitochondria work as a hub of metabolism and as metabolite producers, metabolo-
mics, which detect small metabolites ≤1500 Daltons through high-throughput mass
spectrometry, can predict mitochondrial status in health and disease. Metabolomics
has recently gained great attention because of its high potential to be used as a diag-
nostic tool for all types of MDs, either genetic or nongenetic MDs. The strengths of
158 R. Sebaa et al.

using metabolomics as a diagnostic tool are as follows: First, metabolomics mea-

sures intermediates or products of ongoing metabolic pathways, and their measure-
ments directly reflect the snapshot readouts of physiological conditions, which
make metabolomics more real functional analyses of metabolism compared to other
omic approaches. Thus, if there are any metabolic perturbations, as seen in MDs,
they could be detected by the metabolomic approach. Second, metabolomics shows
high sensitivity with extraordinary capabilities for identifying distinct metabolic
biosignatures/profiles of MDs, reflecting the disrupted metabolic pathways in MD
conditions. Third, metabolomic data analysis is easier than other omic data because
of the limited number of metabolites that currently can be accurately measured
compared to the huge numbers of genes or proteins that can be accurately measured.
Fourth, compared to other approaches, metabolomics is not expensive, requires
relatively little time, and provides comprehensive biological measurements. All
these reasons encourage researchers and scientists to focus on applying metabolo-
mics to MDs.

5 Metabolomics of MDs

Several metabolomic studies have been conducted to ultimately identify diagnostic

biomarkers/biosignatures of MDs using different biological samples with various
mass spectrometry (MS) machines. Herein, we mentioned examples of these metab-
olomic studies conducted for MDs (Table 1). For instance, a study performed gas
chromatography-­coupled mass spectrometry (GC-MS)-based metabolomic analy-
ses on plasma samples and skeletal muscle fibers collected from patients diagnosed
with mitochondrial myopathy/progressive external ophthalmoplegia disease show-
ing elevated levels of certain metabolites such as cystathionine, glutamic acid, ser-
ine, and arachidyl carnitine compared to the samples collected from the controls
[77]. Also, GC-MS-based metabolomic analyses of urine samples collected from
children diagnosed with deficiencies in OXPHOS proteins revealed increased levels
of organic acids, including fumaric acid, glutaric acid, lactic acid, malic acid, and
others, compared to the control children [78]. Another study used a metabolomic
approach as a pre-screening tool to identify metabolic biosignatures of MD patients
who need to undergo tissue biopsies. In this way, MD patients who do not need a
tissue biopsy do not need to undergo the invasive procedure [79]. Their study would
help to select the proper MD patient who needs to undergo tissue biopsies. Smuts
et al. identified biosignatures in urine samples collected from patients with deficient
OXPHOS to be used as indicators before the tissue biopsies. They performed untar-
geted nuclear magnetic resonance (NMR). They targeted GC-MS on OXPHOS-­
deficient patients’ urine samples. They found six organic acids (lactic, succinic,
2-hydroxybutyric, 3-hydroxybutyric, 3-hydroxyisovaleric, and 3-hydroxy-3-­
methylglutaric acids), six amino acids (alanine, glycine, glutamic acid, serine, tyro-
sine, and α-aminoadipic acid), and creatine as biosignatures of these patients that
need to undergo tissue biopsies for the validation of MDs [79].
Metabolomics in the Study of Human Mitochondrial Diseases 159

Table 1 Examples of metabolomic studies done to screen metabolomic biosignatures of MDs by

using various biological samples and metabolomic approaches
Biological
MD name specimen Major findings Technique Ref.
Mitochondrial Plasma Elevated levels of certain GC-MS Nikkanen
myopathy/ samples metabolites such as et al. [77]
progressive and cystathionine, glutamic acid,
external skeletal serine, and
ophthalmoplegia muscle arachidoyl-carnitine
disease fibers
Deficiencies in Urine Increased levels of organic GC-MS Reinecke
OXPHOS samples acids, including fumaric acid, et al. [78]
proteins glutaric acid, lactic acid, malic
acid, and others
Deficient Urine Altered six organic acids Untargeted Smuts et al.
OXPHOS in samples (lactic, succinic, NMR and [79]
muscles 2-hydroxybutyric, targeted GC-MS
3-hydroxyisobutyric,
3-hydroxyisovaleric and
3-hydroxy-3-methylglutaric
acids), six amino acids
(alanine, glycine, glutamic
acid, serine, tyrosine, and
α-aminoadipic acid), and
creatine
French-Canadian Urine and Altered 45 metabolite markers, Targeted Thompson
Leigh syndrome plasma including alanine, asparagine, GC-MS and Legault
samples ketones, acylcarnitines, LC-MS et al. [80]
succinate, kynurenine, lactate,
and pyruvates
MELAS Plasma Elevated levels of pyruvate, Targeted and Sharma
samples lactate, malate, alanine, untargeted MS et al. [81]
α-hydroxybutyrate, N-lactoyl-
amino acids, β-hydroxy
acylcarnitines, and β-hydroxy
fatty acids
MELAS and Urine Lower levels of 4-cresyl NMR Hall et al.
MIDD with renal samples sulfate, S-methyl-cysteine-­ spectroscopy [82]
dysfunction sulfoxide,
N-methylnicotinamide, and
hippuric acid
LHON Fibroblasts Decreases in amino acids, Targeted LC-MS Chao de la
spermidine, putrescine, Barca et al.
isovaleryl-carnitine, propionyl- [83]
carnitine, and five
sphingomyelin species but
increases in ten
phosphatidylcholine species
(continued)
160 R. Sebaa et al.

Table 1 (continued)
Biological
MD name specimen Major findings Technique Ref.
Barth syndrome Plasma Perturbations in creatinine, NMR Sandlers
samples fatty acids, methionine, and spectroscopy et al. [84]
proline
Alterations in acylcarnitines, Targeted LC-MS
bigeneric amines, PC/lysoPC,
and amino acids
KSS Urine Increased levels of pyruvate, Targeted Semeraro
samples fumarate, and GC-MS et al. [85]
3-hydroxybutyrate
PMS Urine Increased levels of lactate,
samples 3-hydroxybutyrate,
3-hydroxyisobutyrate,
fumarate, pyruvate,
2-hydroxybutyrate, 2-methyl-­
2,3-dihydroxybutyrate,
3-methylglutarate, 2-ethyl-3-­
hydroxypropionate,
3-methylglutaconate, malate,
and tiglylglycine
MELAS Urine Increases in caproic/caprylic Targeted Esterhuizen
samples acid, 2-hydroxyglutaric acid, LC-MS/MS and et al. [86]
butyric/valeric/2-­ untargeted
hydroxybutyric/3-methyl-2-­ GC–MS and
oxovaleric acid, 4-pentenoic NMR
acid, acetylcarnitine, spectroscopy
propionylcarnitine, taurine,
acetic acid but decreased
metabolites pyruvic acid,
glycerol, carbamate,
2,5-furandicarboxylic acid,
fumaric acid, pseudouridine,
glycolic acid, and arabinose
MIDD Urine Increases in myoinositol,
samples 2-hydroxyglutaric acid,
4-pentenoic acid, glucuronic
acid, 2-hydroxyisovaleric acid,
glucose, 2-ethylhydracrylic
acid, and 3-hydroxyisobutyric
acid. Metabolites’ decrease
includes glycolic acid,
sarcosine, 1,2-ethandiol,
3-methylphenol,
2,5-furandicarboxylic acid,
homocysteine, arabinose, and
pseudouridine
Myopathy Urine Increases in 2-hydroxyglutaric
samples acid, 4-pentenoic acid,
3-methylphenol,
2-ethylhydracrylic acid, and
creatine but decreased glycolic
acid
Metabolomics in the Study of Human Mitochondrial Diseases 161

Table 1 (continued)
Biological
MD name specimen Major findings Technique Ref.
Parkinson’s Plasma and Altered pattern of metabolites Untargeted Stoessel
disease CSF involved in the metabolism of LC-MS and MS/ et al. [87]
samples glycerophospholipid, MS
sphingolipid, acylcarnitine,
and amino acids
Parkinson’s Urine Altered metabolites related to Untargeted Luan et al.
disease samples tryptophan and tyrosine LC-MS [88]
metabolism
Alzheimer’s CSF Altered patterns of choline, Untargeted Ibáñez et al.
disease samples dimethylarginine, arginine, capillary [89]
valine, proline, serine, electrophoresis
mass
histidine, creatine, carnitine,
and suberylglycine spectrometry
(CE-MS)
Medium-chain Dried Altered levels of certain amino Targeted LC-MS Scolamiero
acyl-coenzyme A blood spots acids and acylcarnitines et al. [90]
dehydrogenase
deficiency
disease
Long-chain Dried Decreases in lysine, valine, Targeted Jacob et al.
acyl-coenzyme A blood spots glycerol, and niacinamide but LC-MS/MS [91]
dehydrogenase and serum increases in glutamine succinic
deficiency samples acid and guanosine
disease
Type 2 diabetes Urine Decreases in Targeted LC-MS Salihovic
(T2D) samples t3-hydroxyundecanoyl-­ et al. [92]
carnitine
Insulin resistance Serum Insulin resistance altered Chemical isotope Gu et al.
samples amino acids such as amino labeling (CIL) [93]
acids (Asn, Gln, and his), liquid
methionine (met) sulfoxide, chromatography-­
2-methyl-3-hydroxy-5-­­ mass
formylpyridine-4-­carboxylate, spectrometry
serotonin, L-2-amino-3- (LC-MS)
oxobutanoic acid, and
4,6-dihydroxyquinoline
Type 2 diabetes Distinct amino acids, amino
acid metabolites, and
dipeptides for T2D
162 R. Sebaa et al.

A cohort of French-Canadian Leigh syndrome patients with mutations in the

LRPPRC gene causing defects in one of the OXPHOS proteins was involved in a
metabolomic study. Urine and plasma samples were collected from the Leigh syn-
drome patients and were analyzed using targeted GC-MS and liquid chromatography-­
coupled mass spectrometry (LC-MS). As a result, they found 45 outstanding
metabolic biomarkers, including alanine, asparagine, ketones, acylcarnitines, suc-
cinate, kynurenine, lactate, and pyruvates, altered compared to controls (Thompson
[80]). Additionally, another research group recruited mitochondrial encephalomy-
opathy lactic acidosis and stroke-like episode (MELAS) syndrome and maternally
inherited diabetes and deafness (MIDD) patients with renal dysfunction for identi-
fication of urinary metabolic markers via NMR spectroscopy-based metabolomics
and found that these patients distinguished from controls by having lower levels of
4-cresyl sulfate, S-methyl-cysteine-sulfoxide, N-methylnicotinamide, and hippuric
acid [82]. Recently, Sharma et al. performed metabolomic analyses on plasma sam-
ples collected from MELAS and controls by using targeted and untargeted MS,
revealing that elevated levels of pyruvate, lactate, malate, alanine, α-hydroxybutyrate,
N-lactoyl-amino acids, β-hydroxy acylcarnitines, and β-hydroxy fatty acids [81].
Another example of metabolomic study is those conducted for Barth syndrome.
They identified dysregulated metabolic markers and pathways underlying Barth
syndrome using human plasma samples showing perturbations in creatinine, fatty
acids, methionine, and proline detected by NMR. At the same time, LC-MS revealed
metabolic alterations found in acylcarnitines, bigeneric amines, PC/lysoPC, and
amino acids in patients compared to controls [84].
Furthermore, a targeted LC-MS-based metabolomic approach was applied to
fibroblasts taken from LHON patients to uncover metabolites affected in this dis-
ease used as biomarkers, including decreases in amino acids, spermidine, putres-
cine, isovaleryl-carnitine, propionyl-carnitine, and five sphingomyelin species but
increases in ten phosphatidylcholine species [83]. Also, metabolomic studies of
PMS and KSS were performed recently using a targeted GC-MS approach on urine
samples collected from PMS and KSS patients. Semeraro et al.’s study revealed that
abnormal alterations in urinary organic acids were detected in both PMS and
KSS. Still, the alterations are more pronounced in PMS patients than in KSS. They
found that urine samples from KSS had increased levels of pyruvate, fumarate, and
3-hydroxybutyrate, while urinary metabolites detected in PMS patients indicated
elevated levels in lactate, 3-hydroxybutyrate, 3-hydroxyisobutyrate, fumarate, pyru-
vate, 2-hydroxybutyrate, 2-methyl-2,3-dihydroxybutyrate, 3-methylglutarate,
2-ethyl-3-hydroxypropionate, 3-methylglutaconate, malate, and tiglylglycine [85].
Very recently, Esterhuizen et al. attempted to clinically distinguish between three
types of MDs, including MELAS, MIDD, and myopathy, by identifying disease-­
specific metabolic profiles. They used combined multi-metabolomic approaches of
targeted LC-MS/MS, untargeted GC-MS, and NMR spectroscopy to comprehen-
sively investigate metabolites in urine samples collected from MELAS, MIDD, and
myopathy patients compared to individual disease-matched controls. Although they
found some metabolic similarities in identified metabolites shared in the three dis-
eases, their metabolomic data shows that each of the three diseases has certain
Metabolomics in the Study of Human Mitochondrial Diseases 163

unique metabolites compared to their controls. Compared to controls, MELAS

patients showed increased caproic/caprylic acid, 2-hydroxyglutaric acid, butyric/
valeric/2-hydroxybutyric/3-methyl-2-oxovaleric acid, 4-pentenoic acid, acetylcar-
nitine, propionylcarnitine, taurine, and acetic acid but decreased metabolites pyru-
vic acid, glycerol, carbamate, 2,5-furandicarboxylic acid, fumaric acid,
pseudouridine, glycolic acid, and arabinose. Furthermore, MIDD patients showed
increased myoinositol, 2-hydroxyglutaric acid, 4-pentenoic acid, glucuronic acid,
2-hydroxyisovaleric acid, glucose, 2-ethylhydracrylic acid, 3-hydroxyisobutyric
acid although decreased metabolites were shown including glycolic acid, sarcosine,
1,2-ethandiol, 3-methylphenol, 2,5-furandicarboxylic acid, homocysteine, arabi-
nose, pseudouridine. Lastly, they showed that myopathy patients revealed increased
2-hydroxyglutaric acid, 4-pentenoic acid, 3-methylphenol, 2-ethylhydracrylic acid,
and creatine but decreased glycolic acid [86].
In addition to the previous studies, Stoessel et al. focused on the metabolic
changes in Parkinson’s disease, in which they performed untargeted LC/MS and
MS/MS analysis to profile metabolic changes in plasma and CSF samples collected
from Parkinson’s disease patients. They uncovered a perturbed pattern of metabo-
lites involved in the metabolism of glycerophospholipid, sphingolipid, acylcarni-
tine, and amino acids as Parkinson’s disease biosignatures [87]. In concordance
with the previous Parkinson’s disease study, another research group studied
Parkinson’s disease and discovered alterations in metabolites related to tryptophan
and tyrosine metabolism and other pathways using untargeted LC-MS approaches
applied to urine samples from Parkinson’s disease patients [88]. Moreover, Ibáñez
et al. used untargeted metabolomic profiling based on capillary electrophoresis-­
coupled mass spectrometry (CE-MS) to identify Alzheimer’s disease biosignatures
in CSF samples collected from Alzheimer’s disease patients for the prediction of the
disease and its progression at distinct stages. They found choline, dimethylarginine,
arginine, valine, proline, serine, histidine, creatine, carnitine, and suberylglycine as
potential Alzheimer’s disease progression markers [89]. Also, Scolamiero et al. ana-
lyzed dried blood spots (DBS) from patients diagnosed with medium chain acyl-­
coenzyme A dehydrogenase deficiency disease via targeted LC-MS to identify
metabolic profiling associated with the disease condition. Their results showed that
the level of certain acylcarnitine (C2, C6, C8, and C10) was perturbed distinctively
in these affected patients compared to the controls, potentially used as diagnostic
markers [90]. In addition, another research group performed a metabolomic study
on patients diagnosed with long-chain acyl-coenzyme A dehydrogenase deficiency
disease by performing targeted LC-MS analyses on dried blood spots and serum
samples collected from the patients showing that there were alterations in metabo-
lites in patients’ samples compared to the controls. Examples of altered metabolites
are decreases in lysine, valine, glycerol, and niacinamide but increases in glutamine
succinic acid and guanosine [91].
Furthermore, diabetes was extensively studied in different biological samples
with different MS approaches; thus, example studies are mentioned. A recent study
conducted nontargeted LC-MS-based metabolomics to profile urine metabolites in
patients diagnosed with type 2 diabetes (T2D), resulting in the discovery of a
164 R. Sebaa et al.

promising biomarker t3-hydroxyundecanoyl-carnitine, which was decreased with

T2D [92]. In addition, another study done by another research group revealed
metabolite biosignatures related to insulin resistance and T2D. This study collected
serum samples from normal, obese T2D participants for metabolic profiling using
chemical isotope labeling (CIL) and LC-MS. Their results showed that certain bio-
markers associated with insulin resistance, such as amino acids (asparagine, gluta-
mine, histidine), methionine sulfoxide,
2-methyl-3-hydroxy-5-formylpyridine-4-­ carboxylate, serotonin, L-2-amino-3-
oxobutanoic acid, and 4,6-dihydroxyquinoline and other biomarkers for T2D,
including amino acids, amino acid metabolites, and dipeptides [93].
Last but not least, there is a growing body of recent studies demonstrating the
application of metabolomic technologies in sepsis [94–96], cardiovascular diseases
[97, 98], and cancers [99–102].

6 Conclusion

The application of metabolomics can be promising for the diagnosis and prognosis
of MDs in humans. However, it has been reported that metabolomic analyses have
certain challenges and limitations that need to be considered. For instance, metabo-
lomic analyses can be conducted by multiple mass spectrometry approaches, includ-
ing NMR, GC-MS, and LC-MS. All of them are used for various purposes due to
their different functionalities, metabolite coverage, and experimental design. Each
approach has advantages and disadvantages. For example, NMR is suitable for sig-
nificant volume samples, is nondestructive to samples, does not require sample
preparations, and gives reproducible data. However, NMR is not as sensitive as
other techniques, such as GC-MS and LC-MS, which can detect a large range of
metabolites with very tiny amounts of sample. GC-MS and LC-MS function based
on the polarity of metabolites, as GC-MS is mainly used more for nonpolar metabo-
lites. Thus LC-MS is the preferable approach for polar metabolites.
Nevertheless, MS approaches have certain drawbacks, such as lower reproduc-
ibility, higher-cost expensive techniques, and time-consuming sample preparation
steps. Thus, no single approach can comprehensively detect all metabolites with
various chemical and physical properties in biological samples within one analyti-
cal run. Other limitations are related to metabolomic data preprocessing and spec-
tral library databases. Examples of issues during metabolomic data processing
missing data can be seen, and that is due to several reasons, such as the concentra-
tion of metabolites being either lower or higher than the detection limit of the
machine. Another limitation of metabolomics is the availability of metabolite spec-
tral libraries for data analyses and interpretation. NMR and LC-MS approaches
have limited libraries compared to GC-MS libraries.
Many of the challenges associated with metabolomics can be overcome by com-
bining various approaches to detect and cover a comprehensive range of metabo-
lites. Furthermore, metabolomics shows the powerful capability and potential in
Metabolomics in the Study of Human Mitochondrial Diseases 165

diagnosing MDs as it represents real perturbations associated with MDs.

Nonetheless, it is best to integrate metabolomic diagnostic data with other diagnos-
tic approaches (i.e., patient and family histories, physical examinations, clinical bio-
chemical and histological tests, and enzymatic assays). Finally, research in this area
and the corresponding field development will improve with the involvement of
larger patient cohorts. Increased global collaborations between many clinical com-
munities will strengthen and validate the finding of metabolic biosignatures of
human MDs and improve diagnostic platforms.

References

1. Elston T, Wang H, Oster G. Energy transduction in ATP synthase. Nature.

1998;391(6666):510–3. [Link]
2. Veltri KL, Espiritu M, Singh G. Distinct genomic copy number in mitochondria of dif-
ferent mammalian organs. J Cell Physiol. 1990;143(1):160–4. [Link]
jcp.1041430122.
3. Spiegelman BM. Transcriptional control of mitochondrial energy metabolism through the
PGC1 coactivators. In: Mitochondrial biology: new perspectives. Chichester: John Wiley &
Sons; 2007. p. 60–9. [Link]
4. Tzameli I. The evolving role of mitochondria in metabolism. Trends Endocrinol Metab.
2012;23(9):417–9. [Link]
5. Friedman JR, Nunnari J. Mitochondrial form and function. Nature. 2014;505(7483):335–43.
[Link]
6. Herst PM, Rowe MR, Carson GM, Berridge MV. Functional mitochondria in health and dis-
ease. Front Endocrinol. 2017;8:296. [Link]
7. McBride HM, Neuspiel M, Wasiak S. Mitochondria: more than just a powerhouse. Curr Biol.
2006;16(14):R551–60. [Link]
8. Shaw JM, Nunnari J. Mitochondrial dynamics and division in budding yeast. Trends Cell
Biol. 2002;12(4):178–84. [Link]
9. Trushina E. A shape shifting organelle: unusual mitochondrial phenotype determined with
three-dimensional electron microscopy reconstruction. Neural Regen Res. 2016;11(6):900–1.
[Link]
10. Gohil VM, Greenberg ML. Mitochondrial membrane biogenesis: phospholipids and proteins
go hand in hand. J Cell Biol. 2009;184(4):469–72. [Link]
11. Kühlbrandt W. Structure and function of mitochondrial membrane protein complexes. BMC
Biol. 2015;13(1):89. [Link]
12. Bowsher CG, Tobin AK. Compartmentation of metabolism within mitochondria and plastids.
J Exp Bot. 2001;52(356):513–27. [Link]
13. Glancy B, Hartnell LM, Malide D, Yu Z-X, Combs CA, Connelly PS, Subramaniam S,
Balaban RS. Mitochondrial reticulum for cellular energy distribution in muscle. Nature.
2015;523(7562):617–20. [Link]
14. Glancy B, Kim Y, Katti P, Willingham TB. The functional impact of mitochondrial struc-
ture across subcellular scales. Front Physiol. 2020;11:541040. [Link]
fphys.2020.541040.
15. Yoon Y. Sharpening the scissors. Cell Biochem Biophys. 2004;41(2):193–205. [Link]
org/10.1385/[Link].
16. Ding W-X, Li M, Biazik JM, Morgan DG, Guo F, Ni H-M, Goheen M, Eskelinen E-L, Yin
X-M. Electron microscopic analysis of a spherical mitochondrial structure*. J Biol Chem.
2012;287(50):42373–8. [Link]
166 R. Sebaa et al.

17. Navaratnarajah T, Anand R, Reichert AS, Distelmaier F. The relevance of mitochondrial

morphology for human disease. Int J Biochem Cell Biol. 2021;134:105951. [Link]
org/10.1016/[Link].2021.105951.
18. Busch KB, Kowald A, Spelbrink JN. Quality matters: how does mitochondrial network
dynamics and quality control impact on mtDNA integrity? Philos Trans R Soc Lond B Biol
Sci. 2014;369(1646):20130442. [Link]
19. Mishra P, Chan DC. Metabolic regulation of mitochondrial dynamics. J Cell Biol.
2016;212(4):379–87. [Link]
20. Ashrafi G, Schwarz TL. The pathways of mitophagy for quality control and clearance of
mitochondria. Cell Death Differ. 2013;20(1):31–42. [Link]
21. Losón OC, Song Z, Chen H, Chan DC. Fis1, Mff, MiD49, and MiD51 mediate Drp1 recruit-
ment in mitochondrial fission. Mol Biol Cell. 2013;24(5):659–67. [Link]
mbc.E12-­10-­0721.
22. Otsuga D, Keegan BR, Brisch E, Thatcher JW, Hermann GJ, Bleazard W, Shaw JM. The
dynamin-related GTPase, Dnm1p, controls mitochondrial morphology in yeast. J Cell Biol.
1998;143(2):333–49.
23. Chen H, Detmer SA, Ewald AJ, Griffin EE, Fraser SE, Chan DC. Mitofusins Mfn1 and Mfn2
coordinately regulate mitochondrial fusion and are essential for embryonic development. J
Cell Biol. 2003;160(2):189–200. [Link]
24. Song Z, Ghochani M, McCaffery JM, Frey TG, Chan DC. Mitofusins and OPA1 mediate
sequential steps in mitochondrial membrane fusion. Mol Biol Cell. 2009;20(15):3525–32.
[Link]
25. Gustafsson CM, Falkenberg M, Larsson N-G. Maintenance and expression of mam-
malian mitochondrial DNA. Annu Rev Biochem. 2016;85:133. [Link]
annurev-­biochem-­060815-­014402.
26. Wai T, Ao A, Zhang X, Cyr D, Dufort D, Shoubridge EA. The role of mitochondrial DNA copy
number in mammalian Fertility1. Biol Reprod. 2010;83(1):52–62. [Link]
biolreprod.109.080887.
27. Rath S, Sharma R, Gupta R, Ast T, Chan C, Durham TJ, Goodman RP, Grabarek Z, Haas
ME, Hung WHW, Joshi PR, Jourdain AA, Kim SH, Kotrys AV, Lam SS, McCoy JG, Meisel
JD, Miranda M, Panda A, Mootha VK. MitoCarta3.0: an updated mitochondrial pro-
teome now with sub-organelle localization and pathway annotations. Nucleic Acids Res.
2021;49(D1):D1541–7. [Link]
28. D’Souza AR, Minczuk M. Mitochondrial transcription and translation: overview. Essays
Biochem. 2018;62(3):309–20. [Link]
29. Demine S, Reddy N, Renard P, Raes M, Arnould T. Unraveling biochemical pathways affected
by mitochondrial dysfunctions using metabolomic approaches. Meta. 2014;4(3):831–78.
[Link]
30. Wallace DC, Fan W, Procaccio V. Mitochondrial energetics and therapeutics. Annu Rev
Pathol. 2010;5:297–348. [Link]
31. Dimroth P, Kaim G, Matthey U. Crucial role of the membrane potential for ATP synthesis by
F(1)F(o) ATP synthases. J Exp Biol. 2000;203(1):51–9. [Link]
32. Mitchell P, Moyle J. Chemiosmotic hypothesis of oxidative phosphorylation. Nature.
1967;213(5072):137. [Link]
33. Watmough NJ, Frerman FE. The electron transfer flavoprotein: ubiquinone oxidore-
ductases. Biochim Biophys Acta. 2010;1797(12):1910–6. [Link]
bbabio.2010.10.007.
34. Divakaruni AS, Brand MD. The regulation and physiology of mitochondrial proton leak.
Physiology (Bethesda). 2011;26(3):192–205. [Link]
35. Cannon B, Nedergaard J. Brown adipose tissue: function and physiological significance.
Physiol Rev. 2004;84(1):277–359. [Link]
Metabolomics in the Study of Human Mitochondrial Diseases 167

36. Mailloux RJ, Harper M-E. Uncoupling proteins and the control of mitochondrial reactive oxy-
gen species production. Free Radic Biol Med. 2011;51(6):1106–15. [Link]
freeradbiomed.2011.06.022.
37. Brand MD, Esteves TC. Physiological functions of the mitochondrial uncoupling proteins
UCP2 and UCP3. Cell Metab. 2005;2(2):85–93. [Link]
38. Nicholls DG. Mitochondrial proton leaks and uncoupling proteins. Biochim Biophys Acta.
2021;1862(7):148428. [Link]
39. Bertholet AM, Chouchani ET, Kazak L, Angelin A, Fedorenko A, Long JZ, Vidoni S, Garrity
R, Cho J, Terada N, Wallace DC, Spiegelman BM, Kirichok Y. H+ transport is an integral
function of the mitochondrial ADP/ATP carrier. Nature. 2019;571(7766):515–20. [Link]
org/10.1038/s41586-­019-­1400-­3.
40. Bevilacqua L, Seifert EL, Estey C, Gerrits MF, Harper M-E. Absence of uncoupling protein-3
leads to greater activation of an adenine nucleotide translocase-mediated proton conduc-
tance in skeletal muscle mitochondria from calorie restricted mice. Biochim Biophys Acta.
2010;1797(8):1389–97. [Link]
41. Echtay KS, Esteves TC, Pakay JL, Jekabsons MB, Lambert AJ, Portero-Otín M, Pamplona R,
Vidal-Puig AJ, Wang S, Roebuck SJ, Brand MD. A signalling role for 4-hydroxy-2-­nonenal
in regulation of mitochondrial uncoupling. EMBO J. 2003;22(16):4103–10. [Link]
org/10.1093/emboj/cdg412.
42. Klumpe I, Savvatis K, Westermann D, Tschöpe C, Rauch U, Landmesser U, Schultheiss
H-P, Dörner A. Transgenic overexpression of adenine nucleotide translocase 1 protects
ischemic hearts against oxidative stress. J Mol Med (Berl). 2016;94(6):645–53. [Link]
org/10.1007/s00109-­016-­1413-­4.
43. Ayala A, Muñoz MF, Argüelles S. Lipid peroxidation: production, metabolism, and signaling
mechanisms of malondialdehyde and 4-Hydroxy-2-Nonenal. Oxidative Med Cell Longev.
2014;2014:e360438. [Link]
44. Cadet J, Davies KJA, Medeiros MH, Di Mascio P, Wagner JR. Formation and repair of oxi-
datively generated damage in cellular DNA. Free Radic Biol Med. 2017;107:13–34. https://
[Link]/10.1016/[Link].2016.12.049.
45. Davies MJ. Singlet oxygen-mediated damage to proteins and its consequences.
Biochem Biophys Res Commun. 2003;305(3):761–70. [Link]
S0006-­291X(03)00817-­9.
46. Christofidou-Solomidou M, Muzykantov VR. Antioxidant strategies in respiratory medicine.
Treat Respir Med. 2006;5(1):47–78. [Link]
47. Holzerová E, Prokisch H. Mitochondria: much ado about nothing? How dangerous is
reactive oxygen species production? Int J Biochem Cell Biol. 2015;63:16–20. [Link]
org/10.1016/[Link].2015.01.021.
48. Murphy MP. How mitochondria produce reactive oxygen species. Biochem J. 2009;417(Pt
1):1–13. [Link]
49. Shan Y, Schoenfeld RA, Hayashi G, Napoli E, Akiyama T, Iodi Carstens M, Carstens EE,
Pook MA, Cortopassi GA. Frataxin deficiency leads to defects in expression of antioxidants
and Nrf2 expression in dorsal root ganglia of the Friedreich’s ataxia YG8R mouse model.
Antioxid Redox Signal. 2013;19(13):1481–93. [Link]
50. Sulaiman SA, Mohd Rani Z, Mohd Radin FZ, Abdul Murad NA. Advancement in the diag-
nosis of mitochondrial diseases. J Transl Genet Genom. 2020;4(3):159–87. [Link]
org/10.20517/jtgg.2020.27.
51. Chinnery PF. Primary mitochondrial disorders overview. In: Adam MP, Mirzaa GM, Pagon
RA, Wallace SE, Bean LJ, Gripp KW, Amemiya A, editors. GeneReviews®. Seattle:
University of Washington; 1993; [Link]
52. Khajuria K, Khajuria V, Sawhney V. SECONDARY MITOCHONDRIAL
DYSFUNCTION. Int J Pharm Pharm Sci. 2021;14:14–9. [Link]
ijpps.2021v13i3.40335.
168 R. Sebaa et al.

53. Niyazov DM, Kahler SG, Frye RE. Primary mitochondrial disease and secondary mitochon-
drial dysfunction: importance of distinction for diagnosis and treatment. Mol Syndromol.
2016;7(3):122–37. [Link]
54. Rötig A, Cormier V, Blanche S, Bonnefont JP, Ledeist F, Romero N, Schmitz J, Rustin P,
Fischer A, Saudubray JM. Pearson’s marrow-pancreas syndrome. A multisystem mito-
chondrial disorder in infancy. J Clin Invest. 1990;86(5):1601–8. [Link]
JCI114881.
55. Schlieben LD, Prokisch H. The dimensions of primary mitochondrial disorders. Front Cell
Dev Biol. 2020;8:1441. [Link]
56. Skladal D, Halliday J, Thorburn DR. Minimum birth prevalence of mitochondrial respira-
tory chain disorders in children. Brain. 2003;126(8):1905–12. [Link]
brain/awg170.
57. Gorman GS, Schaefer AM, Ng Y, Gomez N, Blakely EL, Alston CL, Feeney C, Horvath R,
Yu-Wai-Man P, Chinnery PF, Taylor RW, Turnbull DM, McFarland R. Prevalence of nuclear
and mitochondrial DNA mutations related to adult mitochondrial disease. Ann Neurol.
2015;77(5):753–9. [Link]
58. Parikh S, Goldstein A, Koenig MK, Scaglia F, Enns GM, Saneto R, Anselm I, Cohen BH,
Falk MJ, Greene C, Gropman AL, Haas R, Hirano M, Morgan P, Sims K, Tarnopolsky M, Van
Hove JLK, Wolfe L, DiMauro S. Diagnosis and management of mitochondrial disease: a con-
sensus statement from the mitochondrial medicine society. Genet Med. 2015;17(9):689–701.
[Link]
59. Rodenburg RJT. Biochemical diagnosis of mitochondrial disorders. J Inherit Metab Dis.
2011;34(2):283–92. [Link]
60. Al Aqeel AI, Rashid MS, Pn Ruiter J, Ijlst L, Ja Wanders R. A novel molecular defect of the
carnitine acylcarnitine translocase gene in a Saudi patient. Clin Genet. 2003;64(2):163–5.
[Link]
61. Al-Hassnan ZN, Al-Dosary M, Alfadhel M, Faqeih EA, Alsagob M, Kenana R, Almass R,
Al-Harazi OS, Al-Hindi H, Malibari OI, Almutari FB, Tulbah S, Alhadeq F, Al-Sheddi T,
Alamro R, AlAsmari A, Almuntashri M, Alshaalan H, Al-Mohanna FA, Kaya N. ISCA2
mutation causes the infantile neurodegenerative mitochondrial disorder. J Med Genet.
2015;52(3):186–94. [Link]
62. Baertling F, Al-Murshedi F, Sánchez-Caballero L, Al-Senaidi K, Joshi NP, Venselaar H, van
den Brand MA, Nijtmans LG, Rodenburg RJ. Mutation in mitochondrial complex IV subunit
COX5A causes pulmonary arterial hypertension, lactic acidemia, and failure to thrive. Hum
Mutat. 2017;38(6):692–703. [Link]
63. Boycott KM, Beaulieu CL, Kernohan KD, Gebril OH, Mhanni A, Chudley AE, Redl D, Qin
W, Hampson S, Küry S, Tetreault M, Puffenberger EG, Scott JN, Bezieau S, Reis A, Uebe
S, Schumacher J, Hegele RA, McLeod DR, Abou Jamra R. Autosomal-recessive intellectual
disability with cerebellar atrophy syndrome caused by mutation of the manganese and zinc
transporter gene SLC39A8. Am J Hum Genet. 2015;6(97):886–93. [Link]
ajhg.2015.11.002.
64. Eldomery MK, Akdemir ZC, Vögtle F-N, Charng W-L, Mulica P, Rosenfeld JA, Gambin T,
Gu S, Burrage LC, Al Shamsi A, Penney S, Jhangiani SN, Zimmerman HH, Muzny DM,
Wang X, Tang J, Medikonda R, Ramachandran PV, Wong L-J, Sutton VR. MIPEP recessive
variants cause a syndrome of left ventricular non-compaction, hypotonia, and infantile death.
Genome Med. 2016;8(1):106. [Link]
65. Haack TB, Kopajtich R, Freisinger P, Wieland T, Rorbach J, Nicholls TJ, Baruffini E, Walther
A, Danhauser K, Zimmermann FA, Husain RA, Schum J, Mundy H, Ferrero I, Strom TM,
Meitinger T, Taylor RW, Minczuk M, Mayr JA, Prokisch H. ELAC2 mutations cause a mito-
chondrial RNA processing defect associated with hypertrophic cardiomyopathy. Am J Hum
Genet. 2013;93(2):211–23. [Link]
66. Hartmann B, Wai T, Hu H, MacVicar T, Musante L, Fischer-Zirnsak B, Stenzel W, Gräf R,
van den Heuvel L, Ropers H-H, Wienker TF, Hübner C, Langer T, Kaindl AM. Homozygous
Metabolomics in the Study of Human Mitochondrial Diseases 169

YME1L1 mutation causes mitochondriopathy with optic atrophy and mitochondrial network
fragmentation. elife. n.d.;5:e16078. [Link]
67. Heberle LC, Tawari AAA, Ramadan DG, Ibrahim JK. Ethylmalonic encephalopathy—report
of two cases. Brain Dev. 2006;28(5):329–31. [Link]
68. Jobling RK, Assoum M, Gakh O, Blaser S, Raiman JA, Mignot C, Roze E, Dürr A, Brice
A, Lévy N, Prasad C, Paton T, Paterson AD, Roslin NM, Marshall CR, Desvignes J-P,
Roëckel-Trevisiol N, Scherer SW, Rouleau GA, Yoon G. PMPCA mutations cause abnormal
mitochondrial protein processing in patients with non-progressive cerebellar ataxia. Brain.
2015;138(6):1505–17. [Link]
69. Malicdan MCV, Vilboux T, Ben-Zeev B, Guo J, Eliyahu A, Pode-Shakked B, Dori A, Kakani
S, Chandrasekharappa SC, Ferreira C, Shelestovich N, Marek-Yagel D, Pri-Chen H, Blatt
I, Niederhuber JE, He L, Toro C, Taylor RW, Deeken J, Anikster Y. A novel inborn error of
the coenzyme Q10 biosynthesis pathway: cerebellar ataxia and static encephalomyopathy
due to COQ5 C-methyltransferase deficiency. Hum Mutat. 2018;39(1):69–79. [Link]
org/10.1002/humu.23345.
70. Ramadan DG, Head RA, Al-Tawari A, Habeeb Y, Zaki M, Al-Ruqum F, Besley GTN, Wraith
JE, Brown RM, Brown GK. Lactic acidosis and developmental delay due to deficiency of
E3 binding protein (protein X) of the pyruvate dehydrogenase complex. J Inherit Metab Dis.
2004;27(4):477–85. [Link]
71. Shamseldin HE, Alasmari A, Salih MA, Samman MM, Mian SA, Alshidi T, Ibrahim N,
Hashem M, Faqeih E, Al-Mohanna F, Alkuraya FS. A null mutation in MICU2 causes abnor-
mal mitochondrial calcium homeostasis and a severe neurodevelopmental disorder. Brain.
2017;140(11):2806–13. [Link]
72. Shamseldin HE, Smith LL, Kentab A, Alkhalidi H, Summers B, Alsedairy H, Xiong Y,
Gupta VA, Alkuraya FS. Mutation of the mitochondrial carrier SLC25A42 causes a novel
form of mitochondrial myopathy in humans. Hum Genet. 2016;135(1):21–30. [Link]
org/10.1007/s00439-­015-­1608-­8.
73. Spinazzola A, Santer R, Akman OH, Tsiakas K, Schaefer H, Ding X, Karadimas CL, Shanske
S, Ganesh J, Di Mauro S, Zeviani M. Hepatocerebral form of mitochondrial DNA deple-
tion syndrome: novel MPV17 mutations. Arch Neurol. 2008;65(8):1108–13. [Link]
org/10.1001/archneur.65.8.1108.
74. Yavuz H, Bertoli-Avella AM, Alfadhel M, Al-Sannaa N, Kandaswamy KK, Al-Tuwaijri W,
Rolfs A, Brandau O, Bauer P. A founder nonsense variant in NUDT2 causes a recessive neu-
rodevelopmental disorder in Saudi Arab children. Clin Genet. 2018;94(3–4):393–5. https://
[Link]/10.1111/cge.13386.
75. Ferguson E. The strengths and weaknesses of whole-genome sequencing; 2020. p. 3.
76. Petersen B-S, Fredrich B, Hoeppner MP, Ellinghaus D, Franke A. Opportunities and chal-
lenges of whole-genome and -exome sequencing. BMC Genet. 2017;18(1):14. [Link]
org/10.1186/s12863-­017-­0479-­5.
77. Nikkanen J, Forsström S, Euro L, Paetau I, Kohnz RA, Wang L, Chilov D, Viinamäki J,
Roivainen A, Marjamäki P, Liljenbäck H, Ahola S, Buzkova J, Terzioglu M, Khan NA,
Pirnes-Karhu S, Paetau A, Lönnqvist T, Sajantila A, Suomalainen A. Mitochondrial DNA
replication defects disturb cellular dNTP pools and remodel one-carbon metabolism. Cell
Metab. 2016;23(4):635–48. [Link]
78. Reinecke CJ, Koekemoer G, van der Westhuizen FH, Louw R, Lindeque JZ, Mienie LJ,
Smuts I. Metabolomics of urinary organic acids in respiratory chain deficiencies in children.
Metabolomics. 2012;8(2):264–83. [Link]
79. Smuts I, van der Westhuizen FH, Louw R, Mienie LJ, Engelke UFH, Wevers RA, Mason
S, Koekemoer G, Reinecke CJ. Disclosure of a putative biosignature for respiratory chain
disorders through a metabolomics approach. Metabolomics. 2013;9(2):379–91. [Link]
org/10.1007/s11306-­012-­0455-­z.
80. Legault JT, Strittmatter L, Tardif J, Sharma R, Tremblay-Vaillancourt V, Aubut C, Boucher G,
Clish CB, Cyr D, Daneault C, Waters PJ. A metabolic signature of mitochondrial dysfunction
170 R. Sebaa et al.

revealed through a monogenic form of Leigh syndrome. Cell Rep. 2015;13(5):981–9. https://
[Link]/10.1016/[Link].2015.09.054.
81. Sharma R, Reinstadler B, Engelstad K, Skinner OS, Stackowitz E, Haller RG, Clish CB,
Pierce K, Walker MA, Fryer R, Oglesbee D, Mao X, Shungu DC, Khatri A, Hirano M, De
Vivo DC, Mootha VK. Circulating markers of NADH-reductive stress correlate with mito-
chondrial disease severity. J Clin Invest. 2021;131(2):136055. [Link]
JCI136055.
82. Hall AM, Vilasi A, Garcia-Perez I, Lapsley M, Alston CL, Pitceathly RDS, McFarland R,
Schaefer AM, Turnbull DM, Beaumont NJ, Hsuan JJ, Cutillas PR, Lindon JC, Holmes
E, Unwin RJ, Taylor RW, Gorman GS, Rahman S, Hanna MG. The urinary proteome
and metabonome differ from normal in adults with mitochondrial disease. Kidney Int.
2015;87(3):610–22. [Link]
83. Chao de la Barca JM, Simard G, Amati-Bonneau P, Safiedeen Z, Prunier-Mirebeau D, Chupin
S, Gadras C, Tessier L, Gueguen N, Chevrollier A, Desquiret-Dumas V, Ferré M, Bris C,
Kouassi Nzoughet J, Bocca C, Leruez S, Verny C, Miléa D, Bonneau D, Reynier P. The
metabolomic signature of Leber’s hereditary optic neuropathy reveals endoplasmic reticulum
stress. Brain. 2016;139(11):2864–76. [Link]
84. Sandlers Y, Mercier K, Pathmasiri W, Carlson J, McRitchie S, Sumner S, Vernon
HJ. Metabolomics reveals new mechanisms for pathogenesis in Barth syndrome and intro-
duces novel roles for Cardiolipin in cellular function. PLoS One. 2016;11(3):e0151802.
[Link]
85. Semeraro M, Boenzi S, Carrozzo R, Diodato D, Martinelli D, Olivieri G, Antonetti G,
Sacchetti E, Catesini G, Rizzo C, Dionisi-Vici C. The urinary organic acids profile in sin-
gle large-scale mitochondrial DNA deletion disorders. Clin Chim Acta. 2018;481:156–60.
[Link]
86. Esterhuizen K, Lindeque JZ, Mason S, van der Westhuizen FH, Rodenburg RJ, de Laat P,
Smeitink JAM, Janssen MCH, Louw R. One mutation, three phenotypes: novel m ­ etabolic
insights on MELAS, MIDD and myopathy caused by the m.3243A > G mutation.
Metabolomics. 2021;17(1):10. [Link]
87. Stoessel D, Schulte C, Teixeira dos Santos MC, Scheller D, Rebollo-Mesa I, Deuschle C,
Walther D, Schauer N, Berg D, Nogueira da Costa A, Maetzler W. Promising metabolite
profiles in the plasma and CSF of early clinical Parkinson’s disease. Front Aging Neurosci.
2018;10:51. [Link]
88. Luan H, Liu L-F, Meng N, Tang Z, Chua K-K, Chen L-L, Song J-X, Mok VCT, Xie L-X, Li
M, Cai Z. LC–MS-based urinary metabolite signatures in idiopathic Parkinson’s disease. J
Proteome Res. 2015;14(1):467–78. [Link]
89. Ibáñez C, Simó C, Martín-Álvarez PJ, Kivipelto M, Winblad B, Cedazo-Mínguez A,
Cifuentes A. Toward a predictive model of Alzheimer’s disease progression using capil-
lary electrophoresis–mass spectrometry metabolomics. Anal Chem. 2012;84(20):8532–40.
[Link]
90. Scolamiero E, Cozzolino C, Albano L, Ansalone A, Caterino M, Corbo G, di Girolamo
MG, Di Stefano C, Durante A, Franzese G, Franzese I, Gallo G, Giliberti P, Ingenito L,
Ippolito G, Malamisura B, Mazzeo P, Norma A, Ombrone D, Ruoppolo M. Targeted metabo-
lomics in the expanded newborn screening for inborn errors of metabolism. Mol BioSyst.
2015;11(6):1525–35. [Link]
91. Jacob M, Malkawi A, Albast N, Al Bougha S, Lopata A, Dasouki M, Abdel Rahman AM. A
targeted metabolomics approach for clinical diagnosis of inborn errors of metabolism. Anal
Chim Acta. 2018;1025:141–53. [Link]
92. Salihovic S, Broeckling CD, Ganna A, Prenni JE, Sundström J, Berne C, Lind L, Ingelsson
E, Fall T, Ärnlöv J, Nowak C. Non-targeted urine metabolomics and associations with
prevalent and incident type 2 diabetes. Sci Rep. 2020;10(1):16474. [Link]
s41598-­020-­72456-­y.
Metabolomics in the Study of Human Mitochondrial Diseases 171

93. Gu X, Al Dubayee M, Alshahrani A, Masood A, Benabdelkamel H, Zahra M, Li L, Abdel

Rahman AM, Aljada A. Distinctive metabolomics patterns associated with insulin resis-
tance and type 2 diabetes mellitus. Front Mol Biosci. 2020;7:411. [Link]
fmolb.2020.609806.
94. Bu Y, Wang H, Ma X, Han C, Jia X, Zhang J, Liu Y, Peng Y, Yang M, Yu K, Wang
C. Untargeted Metabolomic profiling of the correlation between prognosis differences and
PD-1 expression in sepsis: a preliminary study. Front Immunol. 2021;12:740. [Link]
org/10.3389/fimmu.2021.594270.
95. Lin S-H, Fan J, Zhu J, Zhao Y-S, Wang C-J, Zhang M, Xu F. Exploring plasma metabolomic
changes in sepsis: a clinical matching study based on gas chromatography–mass spectrom-
etry. Ann Transl Med. 2020;8(23):1568. [Link]
96. Wang J, Sun Y, Teng S, Li K. Prediction of sepsis mortality using metabolite biomarkers in
the blood: a meta-analysis of death-related pathways and prospective validation. BMC Med.
2020;18(1):83. [Link]
97. Cavus E, Karakas M, Ojeda FM, Kontto J, Veronesi G, Ferrario MM, Linneberg A, Jørgensen
T, Meisinger C, Thorand B, Iacoviello L, Börnigen D, Woodward M, Schnabel R, Costanzo
S, Tunstall-Pedoe H, Koenig W, Kuulasmaa K, Salomaa V, BiomarCaRE consortium.
Association of circulating metabolites with risk of coronary heart disease in a European
population: results from the biomarkers for cardiovascular risk assessment in Europe
(BiomarCaRE) consortium. JAMA Cardiol. 2019;4(12):1270–9. [Link]
jamacardio.2019.4130.
98. Griffin JL, Atherton H, Shockcor J, Atzori L. Metabolomics as a tool for cardiac research. Nat
Rev Cardiol. 2011;8(11):630–43. [Link]
99. Debik J, Euceda LR, Lundgren S, Gythfeldt HVDL, Garred Ø, Borgen E, Engebraaten O,
Bathen TF, Giskeødegård GF. Assessing treatment response and prognosis by serum and tis-
sue metabolomics in breast cancer patients. J Proteome Res. 2019;18(10):3649–60. https://
[Link]/10.1021/[Link].9b00316.
100. Feng LR, Barb JJ, Regan J, Saligan LN. Plasma metabolomic profile associated with fatigue
in cancer patients. Cancer Med. 2021;10(5):1623–33. [Link]
101. Guida F, Tan VY, Corbin LJ, Smith-Byrne K, Alcala K, Langenberg C, Stewart ID,
Butterworth AS, Surendran P, Achaintre D, Adamski J, Amiano P, Bergmann MM, Bull CJ,
Dahm CC, Gicquiau A, Giles GG, Gunter MJ, Haller T, Johansson M. The blood metabolome
of incident kidney cancer: a case–control study nested within the MetKid consortium. PLoS
Med. 2021;18(9):e1003786. [Link]
102. Wojakowska A, Chekan M, Widlak P, Pietrowska M. Application of metabolomics in thyroid
cancer research. Int J Endocrinol. 2015;2015:e258763. [Link]
Metabolomics of Rare Endocrine, Genetic
Disease: A Focus on the Pituitary Gland

Afshan Masood, Abeer Malkawi, Anas M. Abdel Rahman, and Mohamed Siaj

Abstract Endocrine diseases and disorders are influenced by individual genetic

and environmental factors that directly influence metabolite levels revealing unique
metabolomic disease profiles. In the last few years, metabolomic-derived biomark-
ers and quantitative traits have helped identify the underlying mechanisms for many
rare endocrine diseases and demonstrated a high potential for use in precision medi-
cine. Using the metabolomic platform has also provided disease-specific biomark-
ers for diagnosis and monitoring treatment beyond the use of conventional
biochemical immunoassays. Although the development of metabolomic profiling in
pituitary disorders is at an early stage, recent advances have shown it to be a promis-
ing approach for identifying specific disease biomarkers in cases of growth hor-
mone disorders (acromegaly, short stature) and Cushing’s syndrome. Implementing
high-performance metabolomic analysis techniques in pituitary disease will be a
helpful clinical tool for significantly improving diagnosis and, potentially, the thera-
peutic approach by identifying highly specific disease biomarkers and novel molec-
ular pathogenic mechanisms.

Keywords Rare endocrine disease · Rare genetic disease · Metabolomics ·

Biomarkers · Pituitary adenomas · Growth hormone · Acromegaly · Short stature ·
Pituitary dwarfism · Cushing’s syndrome

A. Masood · A. Malkawi · M. Siaj (*)

Department of Chemistry and Biochemistry, Université du Québec à Montréal (UQAM),
Montreal, QC, Canada
e-mail: [Link]@[Link]
A. M. Abdel Rahman (*)
Metabolomics Section, Department of Clinical Genomics, Center for Genome Medicine,
King Faisal Specialist Hospital & Research Center (KFSHRC), Riyadh, Saudi Arabia
e-mail: AabdelRahman46@[Link]

© The Author(s), under exclusive license to Springer Nature Singapore Pte 173
Ltd. 2023
A. M. Abdel Rahman (ed.), Clinical Metabolomics Applications in Genetic
Diseases, [Link]
174 A. Masood et al.

Abbreviations

CS Cushing’s syndrome
DMA Dimethylamine
GH Growth hormone
GPR101 G protein-coupled receptor 101
GWAS Genome-wide association
IR Insulin resistance
LC-MS Liquid chromatography-mass spectrometry
NMR Nuclear magnetic resonance
PA Pituitary adenomas
rhGH Recombinant human growth hormone
UPLC Q-TOF Ultra-performance liquid chromatography quadrupole time
of flight

1 Introduction

Endocrinology is a branch of medicine that deals with studying the endocrine sys-
tem. The endocrine system comprises a system of specialized ductless glands
responsible for synthesizing, storing, and releasing their secretions, that is, hor-
mones, directly into circulation. Classically, hormone action occurs at different
peripheral target tissues having receptors for the specific hormones. In addition to
the classical endocrine signaling, hormones also act on neighboring cells (paracrine
effect), on the cell secreting the hormone itself (autocrine effect), or locally within
the cell without actually getting released from it (intracranial effect).
Hormonal action can be broadly described as important in controlling and coordi-
nating whole-body metabolism and maintaining body homeostasis. Hormones are
responsible for maintaining energy balance, regulating metabolic pathways, repro-
duction, growth and development, and response to injury, stress, and environmental
factors. The hormonal synthesis, production, and secretion are tightly regulated pro-
cesses, broadly, through complex regulatory feedback control loops or axes between
different endocrine glands. The feedback mechanisms control the circulating concen-
trations of the hormones within a tight physiological range necessary for optimal hor-
mone action at the cellular level. The most well known among these are those under
hypothalamic-pituitary control, such as the hypothalamo-pituitary-thyroid axis and
hypothalamo-pituitary-adrenal axis that control and regulate thyroid and adrenal hor-
mone action via the regulatory and organ-stimulating hormones. Endocrine dysfunc-
tion arises as a result of either subnormal (e.g., hypothyroid) or excess production
(e.g., hyperthyroid) of hormones or due to peripheral resistance to hormone action
(e.g., insulin resistance (IR)), which leads to different disease states.
Clinical evaluation of endocrine dysfunction is generally based on measuring
single effector hormones using immunoassays in the clinical laboratory. Although
these methods are well established and widely used in different hospital laborato-
ries, single hormone measurements, as commonly perceived, do not completely
Metabolomics of Rare Endocrine, Genetic Disease: A Focus on the Pituitary Gland 175

represent disease or hormonal defects [1]. The complexity in endocrine disease

diagnosis arises as virtually all complex processes are regulated at any point with
one hormone whose functions are closely intertwined. Therefore, measurement of
single hormone levels may not be sufficient in identifying disease. The immunoas-
says are further limited in their detection capabilities due to their narrow specificity,
sensitivity, and a high coefficient of variation between the different immunoassays
for the same hormone [2, 3]. With the advent of deeper genome sequencing, an
increasing number of endocrine diseases are grouped as a single disease with a het-
erogeneous presentation but are now identified as distinct disorders based on their
genetic makeup. In the present chapter, we will look at the metabolomics of diseases
of the pituitary gland.

2 Endocrine Diseases and Genetics

Although the paradigm of studying hormonal action and related diseases focused on
the concept of one anatomical gland and its associated hormone, this view is chang-
ing with many interrelated complex interactions being identified between the differ-
ent hormones. Hormones do not act in isolation; a single hormone affects multiple
organs and functions, and several hormones control each function. Identification of
various genetic polymorphisms conveying an increased risk of developing endo-
crine disease has been identified through genome-wide association (GWAS) stud-
ies. These studies have greatly improved knowledge and genetic testing, becoming
a significantly important component for the thorough clinical diagnostic workflow
in endocrine diagnostics and the routinely performed biochemical laboratory analy-
sis [4, 5].
Recent studies using GWAS have identified many genetic mutations (such as
single nucleotide polymorphisms, allelic loss or gene amplification, and epigenetic
changes, usually by promoter methylation chromosomal defects) and their variants
that are known to lead to endocrine disorders and pathogenic or benign neoplastic
endocrine disease conditions [5–9]. This has been facilitated through clinical genetic
studies in patients carrying mutations with well-characterized presentations and
manifestations. These genetic mutations have been identified in genes involved in
regulating the cell cycle, growth factors, signaling pathways, and hormone recep-
tors. The genetic mutations may either result in gain of function or loss of function
of the specific genes and their downstream protein products. These defects in pro-
teins translate into biochemical enzymatic defects resulting in alterations in the hor-
monal levels due to disorders of hormone synthesis or receptor uptake, to name a
few, resulting in endocrine disease [9–13].
Additionally, advances in cellular biology and transcriptomic studies have shown
the significant role that hormones play in regulating gene expression. Mutations in
the germline result in familial diseases, while somatic mutations may present with
expression only within specific tissues. Evaluation of rare endocrine genes besides
the characterization of the hormonal defects requires a comprehensive genetic
workup [4, 5].
176 A. Masood et al.

Genetic diagnostic testing in rare endocrine conditions has become important for
achieving an early molecular diagnosis and identifying presymptomatic individuals.
Although significant, genetic evaluation is not without disadvantages as it is highly
specialized and not easily available routinely. Recent advances in OMIC technolo-
gies, specifically in metabolomics, have shown that changes in the metabolite pat-
tern reflect the associated disease conditions [14]. The disease is often seen to
exceed the influence of clinical factors. The variations in many metabolites have
been heritable and linked to distinct loci identified by GWAS [15]. Genetic variants,
particularly of the enzyme coding genes, are associated with changes in the proteins
regulating the homeostasis of key lipids, carbohydrates, or amino acid metabolisms
and metabolites.
Over the last decade, the understanding of physiological and pathological pro-
cesses underpinning endocrine and endocrine-related disease has significantly
expanded, aided by advances in mass spectrometry (MS) approaches and novel
molecular biological and computational tools. The metabolic compound dysregula-
tion is associated with changes in physiology which are consequences of a unique
expression of the genes. A small variation in protein expression can significantly
affect the metabolic pathway activity with alterations in concentrations of metabo-
lites related to the relevant proteins. Thus, compared to both the proteome and tran-
scriptome, metabolomic profile is far more sensitive. The development of the
high-throughput omic platforms has helped identify the specific metabolites altered
with endocrine diseases and more so in the cases of rare genetic, endocrine diseases.
Determining metabolomic features as causes of endocrine disorders provides a bet-
ter holistic opportunity to understand the related pathophysiology and develop
approaches for personalized therapy [16].

3 Metabolomics of Endocrine Disease

Endocrine diseases deeply impact the human metabolome as hormonal defects

affect the functions of enzymes, causing alterations in the normal metabolic activity
of tissues or glands, leading to disease. Metabolomics has contributed significantly
to endocrinology by providing means to understand better the inner workings of
individual cells. It also helped to understand the interactions between different
organ systems and providing insight into physiological and pathological processes.
The main application of metabolomics in endocrine disease has been clinical
research dealing with disease metabolomic profiling and biomarker discovery for
diagnostic purposes. Plasma profiling of the disease and control states has allowed
the characterization of a comprehensive set of metabolites in biological samples.
Fluctuations in concentration of these small molecules, in response to different
stimuli, serve as markers for monitoring of disease. Two principal analytical plat-
forms have been most used to analyze metabolites in body fluids, primarily deriva-
tives of blood (serum, plasma), tissues, and urine. These include nuclear magnetic
resonance (NMR) spectroscopy and liquid/gas chromatography coupled with mass
Metabolomics of Rare Endocrine, Genetic Disease: A Focus on the Pituitary Gland 177

spectrometry (LC-MS). The analysis of lipid metabolites was enhanced by using

ultra-performance liquid chromatography quadrupole time-of-flight (UPLC Q-TOF)
MS and GC-MS. TMS major complementary approaches have been applied. One
involves the targeted analysis of metabolites, which measures the concentrations of
known disease-associated metabolites. The second is an untargeted hypothesis-
driven approach that provides a global profile of a large set of metabolites associ-
ated with the disease semiquantitatively.
Alterations in metabolite levels or a specific group of metabolites reflect the
intricate relationships between genes and their products, as well as the activity of
related enzymes within metabolic pathways either physiologically or in a disease
state. In combination with genetics, untargeted metabolomics has provided addi-
tional information to determine the association between genetic variants and dis-
ease. Combining the clinical (phenotype) with the different molecular datasets
(OMIC technologies) in a bioinformatic-based clinical decision-making system
also has the advantage of advancing personalized medicine. These advances are in
understanding the pathophysiology, diagnosis, stratification, management, and
assessment of treatment efficacy in endocrinology. Metabolomics adds to this
milieu by including metabolic information, furthering the accuracy of diagnosis in
individuals and discovering novel metabolites as reported in the human metabolome
database with the disease. For each metabolic gene, prediction models can then be
developed for predicting changes in concentrations of metabolites in different bio-
logical fluids (serum, plasma, urine, saliva, etc.) or tissue specimens with various
genetic mutations (Table 1).

3.1 Metabolomics of Hypothalamus and Pituitary

Gland Dysfunction

The hypothalamus along with pituitary gland together constitutes a functional unit
responsible for secreting regulatory hormones that exert control over the functions
of nearly all the endocrine glands. The pituitary is the master regulatory gland of the
body, acting through several hypothalamic-pituitary-target organ regulatory axes.
Any defects arising within these regulatory systems, synthesis, storage, or release of
the hormones impact the human body’s overall physiological functions, leading to
disorders and diseases.
Primary dysfunction of the pituitary gland is uncommon. The majority of the
cases of hypo-, hyper-, or panhypopituitarism arise secondary to the presence of
either neoplastic or benign tumors such as adenomas. Pituitary adenomas (PA) are
benign neoplasms that are highly heterogeneous with varying subtypes and accounts
for nearly 15% of primary tumors arising in the brain. They are either secretory
based on the increase or decrease in the hormones secreted by the affected cell types
or nonsecretory. Inherited genetic mutations, although rare, have been identified by
GWAS and are known to lead to the development of benign PA, with several genetic
Table 1 The different endocrine glands with the associated disease, gene locus, and major metabolites altered with the disease
Endocrine Metabolomic
178

gland Hormones Disease Genes platform Major significant metabolites References

Pituitary GH defects GH acromegaly AIP, GNAS, GPR101, GC-MS Serine, dihydrocoumarin, glyceric [24–26]
USP8, USP48, and (untargeted), acid, L-dithiothreitol,
BRAF. MEN1, LC-MS (targeted), 5-aminovaleric acid, N-acetyl-L-
PRKAR1A, GPR101, and 1H-NMR glutamic acid, gluconic acid,
SDHx, CDKN1B, and (untargeted) mono-olein, and dimethylamine (↑)
MAX BCAA, lysine, D-erythronolactone,
taurine, carbamoyl-­aspartic acid,
mucic acid, and lactate (↓)
Short stature PIT-1, HESX13, NMR (untargeted) Citrate, lactate, mannitol, alanine, [32]
SOX3, FGFR3, and and DMA (↑). Creatine, creatinine,
PAPP-A2 TMAO, and acetoacetate (↓)
GC-MS (untargeted) Threonic acid, cysteine, cystine, and [31]
palmitoleic acid (↓). Glutamic acid,
aspartic acid, hypoxanthine-­like,
uridine, and glyceric acid (↑)
NMR (untargeted) Citrate, phenylalanine, creatinine, [33]
and tyrosine (↑)
Glucose, serine, betaine, inositol,
lysine, glycerol, and glutamine (↓)
ACTH increase Cushing’s disease USP8 LC-MS/MS 11-Deoxycortisol, cortisol, [38, 40]
(targeted) cortisone, corticosterone,
11-deoxycorticosterone,
androstenedione, 18-oxocortisol,
DHEA, DHEA-SO4, and
aldosterone (↓)
Adrenal gland Cortisol Cushing’s PRKACA and LC-MS/MS 2-Hydroxybutyric acid, aminoadipic [39]
syndrome ARMC5 (targeted) acid, L-aspartic acid,
3-hydroxyphenyl acetic acid,
hypoxanthine, 4-pyridoxic acid,
quinolinic acid, sucrose, xanthine,
A. Masood et al.

glucose 6-phosphate, deoxycholic

acid, and 3-methyladipate (direction
of change unspecified)
Metabolomics of Rare Endocrine, Genetic Disease: A Focus on the Pituitary Gland 179

mutations identified as single gene mutations. These have been identified for single
genes encoding for guanine nucleotide-binding protein G(s) subunit alpha (GNAS)
and G protein-coupled receptor 101 (GPR101), aryl hydrocarbon receptor-­
interacting protein (AIP), and deubiquitinase genes: USP8, USP48, and BRAF. PA
can also occur in familial syndromes, including MEN1, SDHx (mutated in the PA,
pheochromocytomas, and paragangliomas), von Hippel-Lindau, DICER1,
PRKAR1A (mutated in Carney complex), succinate dehydrogenase-related familial
PA, GPR101 (involved in X-linked acrogigantism), neurofibromatosis type 1 (NF1),
and Lynch syndromes [17]. The phenotype of the secretory PA depends on the
affected cell types and the hormones secreted; growth hormone (GH)-secreting
somatotroph adenomas result in acromegaly, corticotroph cell adenomas-secreting
corticotropin hormone result in Cushing’s disease, lactotroph cell adenomas-­
secreting prolactin result in hyperprolactinemia, and thyrotropin-secreting adeno-
mas result in hyperthyroidism. The nonsecreting adenomas, on the other hand,
manifest as incidental pituitary sellar masses and lead to hypogonadism. The clini-
cal diagnosis of pituitary disease, irrespective of the cell type affected, is challeng-
ing. This is because of the wide range of presenting symptoms, nonspecific changes
in single-hormone measurements, the need for adopting stimulation or suppression
testing, and the requirement for imaging studies. Metabolomic approaches using
GC/LC-MS have profiled pituitary-related diseases and identified biomarkers dif-
ferentiating between patients with PA and healthy controls. Plasma secretory plasma
metabolomic profiling revealed significant alterations in the metabolism of amino
acid, specifically in alanine, glutamate (metabolites of the glutamate oxidationcy-
cle), and aspartate (metabolite of the urea cycle involved in gluconeogenesis), and
increase in homocysteine levels. Increased homocysteine levels in patients with PA
are considered to be the cause for increased cytotoxicity and oxidative stress [18].
In addition to the findings in plasma, pituitary tissue metabolomic profiling was
also done on tissue sections removed postoperatively from patients with gonadotro-
pin- and PRL-secreting PA. The metabolomic profile showed differential regulation
of phosphoethanolamine, glutamate, glutamine, N-acetyl aspartate, aspartate, and
myoinositol, which are known metabolites involved in the regulation of the central
nervous system. Levels of these metabolites distinctly differentiated between the
two adenomas with PRL-secreting PA showing a decrease in levels of phosphoetha-
nolamine, N-acetyl aspartate, and myoinositol while aspartate, glutamate, and glu-
tamine levels were increased. Women with PRL-secreting PA showed higher
estrogen metabolites and 17-ketosteroids in the urine. The increase in these metabo-
lites was most likely due to reduced enzymatic actions of 3-beta-­hydroxysteroid
dehydrogenase and 5α-reductase enzymes among these patients. These patients also
showed characteristic increase in the ratios of levels between 5-beta and 5-alpha-
hydrogensteroids and delta 5 and delta 4 steroid ratios which served as novel mark-
ers for disease detection [19, 20].
180 A. Masood et al.

3.2 Growth Hormone Secretion Defects

The primary cause of GH defects is pituitary adenomas that secrete aberrant

GH. They can appear as single (isolated) tumors or as component of other systemic
conditions as multiple endocrine neoplasia type 1 or type 4, Carney complex,
McCune-Albright syndrome, or in association with pheochromocytoma and para-
ganglioma. GH acts via the somatotropic axis consisting of GH, GH receptors,
insulin-­like growth factors (IGF) 1 and 2, their associated carrier proteins, recep-
tors, and releasing factors, which regulate growth and body composition. GH secre-
tion defects can manifest clinically as an increase in its circulating levels leading to
acromegaly or a decrease that leads to short stature.

3.2.1 Growth Hormone Excess: Acromegaly

An excess of GH or its chronic hypersecretion results in acromegaly or gigantism, a

rare endocrine disease that is debilitating and leads to multiple comorbidities with
reduction in life expectancy. The uncontrolled production of GH stimulates the liver
to increasingly synthesize and secrete insulin-like growth factor-1 (IGF-1) which in
turn influences metabolic changes in various organ systems and stimulates somatic
(internal organs, tissues, bones, and muscles) overgrowth and development of
comorbidities, including cardiovascular and malignant diseases. In skeletal mus-
cles, GH exerts an anabolic impact that increases amino acid intake and protein
synthesis and decreases protein oxidation. This action shifts the normal metabolism
from mainly utilizing glucose and protein substrates to oxidation of lipid oxidation,
altering the linear body growth and organ growth during childhood. The resulting
clinical phenotype of these patients presents with growth acceleration, enlarged feet
and hands, increased appetite, and broadening and coarsening of the facial features.
The primary cause of pituitary gigantism in nearly 50% of individuals is an
underlying genetic variant or mutation [21, 22]. In 95% of cases, acromegaly occurs
sporadically because of adenomas of somatotroph cells that are characterized by
excessive secretion of GH and IGF-1. Fifty percent of these tumors are attributable
to familial germline mutations in aryl hydrocarbon receptor-interacting protein
(AIP) and probable G-protein-coupled receptor 101 (GPR101) genes. On the other
hand, cases with pituitary hyperplasia are a rarity and are usually encountered as
part of syndromes such as the McCune-Albright syndrome, Carney complex dis-
ease, or X-linked acrogigantism. Besides the mutations seen in AIP, germline muta-
tions have also been documented in other genes such as GPR101, PRKAR1A,
CDKN1B, GNAS, MAX, SDHx, and MEN1. Somatic mutations have been shown
to be related to mutations in GNAS gene and account for 40% of tumors [23].
Presently the diagnosis of active acromegaly is made by determining elevations
in GH levels, after a standard oral glucose tolerance testing, and in levels of IGF-1
when compared to age and gender matched controls. Confirmation of the diagnosis
is through radiological MRI evaluation that validates the findings of an adenoma.
Metabolomics of Rare Endocrine, Genetic Disease: A Focus on the Pituitary Gland 181

Biochemical evaluations of GH and IGF-1 levels are sometimes inconsistent, and an

increase in GH levels is not paralleled with that of IGF-1. The heterogeneous clini-
cal presentation of the disease lacks early warning signs, and the inconsistencies in
the measurements of GH and IGF-1 levels lead to delay or misdiagnosis. Molecular
genetic testing using chromosomal microarray analysis or single gene duplication
studies has helped identify cases with germline or somatic mutations [22].
In addition to genetic testing, metabolomic analysis has aided the diagnosis of
acromegaly by creating a metabolomic fingerprint of the disease and identifying
characteristic metabolites that differentiate patients with active disease from con-
trols. Elevations were noted in gluconeogenic substrates (glycerol, lactate, propio-
nate, and glucogenic amino acids such as valine and isoleucine); serine,
5-aminovaleric acid, dihydrocoumarin, mono-olein, N-acetyl-L-glutamic acid, gly-
ceric acid, L-dithiothreitol, and gluconic acid were observed in the metabolomic
profile in active acromegaly compared with normal controls. In contrast, serum lev-
els of D-erythronolactone, taurine, carbamoyl-aspartic acid, and mucic acid were
decreased. Among the different metabolites, glyceric acid and taurine had the high-
est sensitivities to discriminate between patients with acromegaly from normal con-
trols [24, 25].
Patients with active disease having increased GH but normal IGF-1 levels showed
significantly lower levels of essential branched-chain amino acids (BCAAs) (valine,
isoleucine), lysine, and lactate, whereas levels of the metabolite dimethylamine
were higher. The inverse correlation between valine and isoleucine was seen with
higher GH levels and not with IGF-1 indicating that lower BCAA levels, which
represent the main metabolic fingerprint of acromegaly, were influenced by GH
rather than IGF-1 as the primary mediator [26]. Metabolomic analysis in patients
with acromegaly also highlighted significant alterations in major metabolic path-
ways in these groups of patients. The pathways affected as a result of active disease
included glycerolipid, glyoxylate, taurine, hypotaurine, dicarboxylate, and pyrimi-
dine pentose phosphate pathway. Dysregulation of these pathways was proposed to
be the underlying reason that supported hyperplasia of tissues observed in different
organs, due to excess GH secretion [19]. An increase in gluconeogenic metabolites
along with decreased BCAA levels detected was suggested to be a result of an
accelerated consumption of BCAAs. Assessing levels of BCAAs, besides identify-
ing the active disease, were also beneficial in monitoring the response to therapy.
Metabolomic profiling in patients with acromegaly, with associated cardiovascu-
lar dysfunction, identified distinct changes in amino acids and in plasma lipids.
Specific perturbations were noted in the glycerophospholipid, sphingolipid, and
metabolites involved in the linoleic acid metabolic pathways. Levels of phosphati-
dyl ethanolamine (PE) (22:6/16:0) positively correlated with changes in left ven-
tricular mass, while lysophosphatidyl choline (LysoPC) (16:0) was positively
correlated with alterations in fractional shortening and left ventricle ejection frac-
tion [27]. Patients with active acromegaly also present with increased insulin resis-
tance (IR) accompanied with lowered hepatocellular lipids and cholesterol. This is
in stark contrast to other IR-associated metabolic diseases which are commonly
seen associated with fatty liver disease. The lower hepatic cholesterol levels reflect
182 A. Masood et al.

the decreased liver fat content, a lower unsaturated-to-saturated lipid ratio, and
decreased levels of different carnitine species, namely, plasma butyryl carnitine
hexanoyl carnitine [25]. Among the various FFA species, levels of cholesteryl esters
(CEs, 18:3) were lower. In contrast, within phosphocholine (PC) lipids, increased
levels of LPC (18:0), as well as decreased PC (36:5), ether PC (38:6), PC (40:7),
and PC (42:5), were noted, and in the sphingomyelin class, SM (36:0) was signifi-
cantly lower. The hepatic lipid content decreased from increased hepatic ATP syn-
thesis due to excess GH.

3.2.2 Growth Hormone Deficiency

Insufficient production of GH leads to GH deficiency or pituitary dwarfism, a rare

endocrine disease condition. The causes of GHD can be inherited, congenital, or
acquired. The usual manifestation of the disease is during childhood, with children
clinically presenting with abnormally short stature but normal body proportions.
Lately, the importance of GH has also been observed in adult patients, especially
concerning its effect on lipid profile, body composition, bone mass, and cognition
[28]. Adult growth hormone deficiency is a well-defined clinical condition that is
characterized by abnormal body composition, poor quality of life, dyslipidemia,
and increased risk of cardiovascular disease and death [29]. Genetic analysis by
whole genome sequencing has identified multiple genes that affect the normal
development of the pituitary, hypothalamic signaling or influence the actions of GH
and IGF-1 in target tissues by altering binding to its receptors. Isolated GH defi-
ciency can arise due to mutations or defects in the growth hormone (GH1) gene
itself or the GH-releasing hormone receptor which lead to either classical GH defi-
ciency or produce physiologically inactive GH. More rarely, growth hormone defi-
ciency can result from mutations in HESX13, SOX3, FGFR3 gene, and
pregnancy-associated plasma protein (PAPP)-A2 [30].
The diagnosis of GHD is based on laboratory evaluation, through radiological
imaging examinations and the insulin hypoglycemia test, which is the gold standard
test in adults. The biochemical evaluation is done through measuring levels of GH,
IGF-1, and IGFBP-3 (insulin-like growth factor binding protein 3) as indirect mark-
ers of growth hormone action. Although these markers are used routinely, they lack
sensitivity, and among them, IGF-1 is a least sensitive marker and a poor diagnostic
indicator of GHD.
Untargeted metabolomic profiling using GC-MS identified numerous metabo-
lites and potential biomarkers for diagnosing GHD and was also used for monitor-
ing recombinant human growth hormone (rhGH) replacement therapy in these
patients. Levels of 13 metabolites including threonic acid, cystine, cysteine, palmi-
toleic acid, glutamic acid, glyceric acid, aspartic acid, uridine, and hypoxanthine-­
like were reported to be significantly dysregulated in adult patients with GHD. On
the other hand, successful treatment with rhGH showed a reversal in glutamic acid,
glyceric acid, hexadecanoic acid, and palmitoleic acid to normal control levels [31].
The effectiveness of growth hormone treatment was similarly assessed in a patient
Metabolomics of Rare Endocrine, Genetic Disease: A Focus on the Pituitary Gland 183

with a rare pituitary-specific positive transcription factor (PIT-1) genetic defect that
caused short stature. PIT-1 is responsible for the normal development of the anterior
pituitary gland. Urine metabolomic profile from this patient using NMR-based
metabonomics showed that higher levels of creatine, creatinine, lactate, and
trimethylamine-­N-oxide and lower levels of urinary citrate, dimethylamine (DMA),
and alanine observed before GH treatment were reversed after treatment and
returned to normal values [32]. It was also possible to determine the metabolic dif-
ferences between children with short stature (SS) and healthy controls, using NMR-­
based blood metabolomic analysis, to identify metabolites which can serve as
potential biomarkers for the diagnosis of SS. Serum levels of creatinine, citrate,
phenylalanine, and tyrosine were increased, and levels of inositol, lysine, glycerol,
glucose, betaine, serine, and glutamine were lower in comparison to normal con-
trols. These dysregulated metabolites were associated with disturbances in meta-
bolic pathways related to carbohydrate and amino acid metabolism [33].
Metabolomic profiling was also used to monitor therapy and assess the metabolic
effects of IGF-1 treatment in PAPPA2-deficient patients. The metabolomic analysis
highlighted profound changes in lipid and protein metabolism after replacement. An
increase in BCAA, hydroxyproline, glutamic acid, glutamine, and asparagine was
noted along with a decrease in levels of glycerol and FFA, palmitate, oleate, arachi-
donate, linoleate, palmitoleate, and stearate [34]. Metabolomics in GHD extends
beyond identifying metabolites as biomarkers for disease diagnosis as it also helped
to assess the risk of patients for developing cardiovascular disease. The identified
metabolites have been linked in adults with untreated GHD to a number of CV risk
factors, including morphological changes in the heart, metabolic alterations, and
visceral obesity [26]. More recently, epigenetics and metabolomics have been
increasingly developed to detect distinctive fingerprints, which could predict an
increased risk of CVD in patients with GHD.
Because of its anabolic effects on protein metabolism and muscle growth, ath-
letes have used GH to improve their performance. Another aspect of metabolomics
has been its extension toward analyzing biological fluids, urine, and plasma samples
for evidence of doping. In the treated groups, application of direct discriminant
analysis distinguished the treatments and was also used to classify them accord-
ingly [35].

3.3 Hypercortisolism (Cushing’s Syndrome and Cushing’s

Disease)

Hypercortisolism is a term that encompasses an increase in cortisol production and

is a feature of both Cushing’s syndrome (CS) and Cushing’s disease. It can be clas-
sified into three types: ACTH-dependent (due to pathology of the pituitary gland),
ectopic ACTH secretion, and ACTH-independent (due to adrenal pathology). The
most common pituitary pathology is adenoma of the pituitary gland affecting the
184 A. Masood et al.

corticotrophic cells resulting in glucocorticoid (cortisol) excess production from the

adrenal glands. The uncontrolled ACTH secretion from a pituitary adrenocortico-
trophic adenoma is a rare pathology and is termed as endogenous Cushing’s disease.
Clinically cortisol excess manifests by a well-characterized group of signs and
symptoms that include centripetal obesity, hypertension, muscle weakness, moon
face, fatigue, diabetes mellitus, cognitive difficulties skin changes, osteoporotic
fractures, headaches, and changes in mood. In the female patients, menstrual irregu-
larities and hirsutism (female) are also additionally seen [36]. Diagnosis of
Cushing’s syndrome (CS) (and Cushing’s disease) is a tedious multistep process
requiring verification of hypercortisolism as the first step, followed by identifying
the cause of hyperfunctioning of adrenocortical axis. This is further hampered by
the poor diagnostic utility of the glucocorticoid metabolic pathway indices such as
plasma cortisol levels, urinary-free cortisol, and other clinically measurable bio-
chemical parameters, such as 11-deoxycortisol. Assessing the cause of hypercorti-
solism is challenging as each factor in the differential diagnosis of the disease needs
to be ruled out.
Metabolomic evaluation of patients suspected of CS, with clinical features of
hypercortisolism but indeterminate pituitary imaging, identified many affected met-
abolic pathways. These mainly involved metabolic pathways related to fatty acids,
amino sugars, carbohydrates (glycolysis/gluconeogenesis), purines, glycated nucle-
otides, amino acids (glutamate, alanine, aspartate, and lysine), vitamin B metabo-
lism, aminoacyl-tRNA biosynthesis, and starch and sucrose metabolism [37].
Patients with ACTH-secreting pituitary adenomas showed distinct changes in
plasma metabolite levels identified using LC-MS/MS compared to the control
group. These included 2-hydroxybutyric acid, 3-hydroxyphenyl acetic acid, hypo-
xanthine, L-aspartic acid, aminoadipic acid, 4-pyridoxic acid, quinolinic acid,
deoxycholic acid, xanthine, 3-methyladipate, and sucrose and glucose 6-phosphate.
Besides the plasma and urine metabolomics, tissue metabolomic analysis from
ACTH-secreting pituitary adenoma showed increased short-chain fatty acids (hexa-
noic, capric, heptanoic, octanoic, and nonanoic fatty acids) [38]. At the same time,
glucose-6-phosphate was decreased compared to the control group [19]. Short- and
medium-chain acylcarnitines, branched-chain and aromatic amino acids, and poly-
amine levels were also lower in patients with hypercortisolism. Independently, the
severity of hypercortisolism is linked to changes in intermediate metabolism, which
are consistent with skewed protein synthesis and catabolism and incomplete
β-oxidation, indicating the presence of metabolic inflexibility exists [19, 39].
Differentiating between the different subtypes of hypercortisolism is also clini-
cally challenging. Using metabolomic approaches through serum and urine steroid
profiling has helped identify sensitive markers in patients with CS. Steroid metabo-
lite profiling has shown promise for accurately subtyping patients with CS. The
profiling revealed that patients with ACTH-dependent CS (caused by ACTH-­
secreting pituitary adenomas or ectopic malignancies) have elevated androgens and
related metabolites, whereas patients with CS having glucocorticoid hypersecretion
from the adrenals which is autonomous or independent from ACTH have lower
levels [38, 40]. Targeted metabolomic analysis using a combination panel of ten
Metabolomics of Rare Endocrine, Genetic Disease: A Focus on the Pituitary Gland 185

plasma steroids was shown to provide a sensitive panel of markers for diagnosis of
CS with a high discriminatory capacity between the three subtypes of CS. The vari-
ous steroids in the panel (11-deoxycortisol, cortisol, cortisone, corticosterone,
11-deoxycorticosterone, androstenedione, 18-oxocortisol, DHEA, DHEA-SO4,
and aldosterone) demonstrated significantly distinct profile patterns among patients
with ACTH-independent and ACTH-dependent forms of CS. From the metabolites
mentioned earlier, increases in cortisol levels, its precursor 11-deoxycortisol and
derivative 21-deoxycortisol, cortisone, corticosterone, 11-deoxycorticosterone, and
decrease in plasma aldosterone and 18-oxocortisol differentiated between patients
with and without CS. Higher levels of 11-deoxycorticosterone and 11-deoxycortisol
were higher in patients with pituitary and adrenal CS, while patients with ectopic
CS disease showed lower plasma levels of 18-oxocortisol and aldosterone. The
plasma levels of adrenal androgens, androstenedione, DHEA, and DHEAS were
higher in pituitary and ectopic CS compared to adrenal CS. On the other hand, ecto-
pic CS had highest increase in glucocorticoids.
The urine metabolomic profile was also evaluated in patients with CS and showed
a higher urinary excretion of DHEA and DHEAS metabolites in pituitary disease
compared to adrenal CS [19, 37]. The ratio of cortisol to metabolites of cortisone
was higher, indicating the suppression of activity of the cortisol-inactivating enzyme
HSD11B2. The unrestricted action of cortisol on the mineralocorticoid receptor
explains the clinical symptoms of hypertension and hypokalemia commonly seen in
these patients.
Cushing’s syndrome leads to various metabolic dysfunctions, including cardio-
vascular disease due to a proatherogenic shift in the circulating lipids. Metabolomic
analysis identified increased urinary ceramides, glycerophospholipids, and sphin-
golipid metabolites, independently associated with higher urinary-free cortisol. In
the study by Vega-Beyhart, patients with CS showed significant alterations in 93
metabolites showing an increase in sulfur containing amino acids, ceramides, triac-
ylglycerols, cholesteryl esters, and glycerophospholipids. On the other hand, a
decrease was seen in essential and nonessential AA, polyunsaturated fatty acids,
conjugated bile acids, and second messenger glycerolipid concentrations. Twenty-­
four-­hour urinary-free cortisol was associated with alterations in the concentration
of many lipids and amino acid metabolites. The identified metabolites comprising
of ten amino acids and ten lipid metabolites demonstrated an AUC-ROC of 95% and
served as a metabolic signature for the classification of CS. The identified amino
acids, acylcarnitines, ceramides, and glycerophospholipid markers were suggested
as potential biomarkers of cardiovascular risk in patients with CS [41].

4 Conclusion

Metabolomic profiling of endocrine diseases with its high-power discrimination is

a powerful tool for the clinical investigation and diagnosis of rare pituitary endo-
crine diseases. The realm of metabolomics, in managing pituitary diseases, will in
186 A. Masood et al.

the future extend beyond identifying metabolites as biomarkers for disease diagno-
sis and towards personalized medicine. This approach will aim to deliver targeted
individualized therapy that optimizes the effectiveness of treatments and avoid
unwanted adverse effect reactions or procedures. Establishing disease-specific
metabolite panels on a chip could further revolutionize the diagnostic industry.
Achieving this milestone would require further studies with larger populations,
meta-analysis, and a uniform standardization of analytical and statistical steps to
yield reproducible results.

References

1. Wallaschofski H. What will metabolomics studies mean to endocrinology? J Endocrinol.

2012;215(1):1–2.
2. Barth JH. An evidence-base for laboratory endocrinology? Clin Biochem Rev.
2008;29(3):97–101.
3. Hillebrand JJ, Wickenhagen WV, Heijboer AC. Improving science by overcoming laboratory
pitfalls with hormone measurements. J Clin Endocrinol Metab. 2021;106(4):e1504–12.
4. De Sousa SM, et al. Genetic testing in endocrinology. Clin Biochem Rev. 2018;39(1):17–28.
5. Eggermann T, et al. Genetic testing in inherited endocrine disorders: joint position paper of
the European reference network on rare endocrine conditions (Endo-ERN). Orphanet J Rare
Dis. 2020;15(1):144.
6. Goodarzi MO. Genetics of common endocrine disease: the present and the future. J Clin
Endocrinol Metab. 2016;101(3):787–94.
7. Hirschhorn JN, et al. A comprehensive review of genetic association studies. Genet Med.
2002;4(2):45–61.
8. Song ZJ, et al. The genome-wide mutational landscape of pituitary adenomas. Cell Res.
2016;26(11):1255–9.
9. Fang Q, et al. Genetics of combined pituitary hormone deficiency: roadmap into the genome
era. Endocr Rev. 2016;37(6):636–75.
10. Hwangbo Y, Park YJ. Genome-wide association studies of autoimmune thyroid diseases, thy-
roid function, and thyroid cancer. Endocrinol Metab (Seoul). 2018;33(2):175–84.
11. Rahmioglu N, et al. Variability of genome-wide DNA methylation and mRNA expression pro-
files in reproductive and endocrine disease related tissues. Epigenetics. 2017;12(10):897–908.
12. Persani L, et al. GENETICS IN ENDOCRINOLOGY: genetic diagnosis of endocrine dis-
eases by NGS: novel scenarios and unpredictable results and risks. Eur J Endocrinol.
2018;179(3):R111–23.
13. Eriksson D, et al. GWAS for autoimmune Addison’s disease identifies multiple risk loci and
highlights AIRE in disease susceptibility. Nat Commun. 2021;12(1):959.
14. Jacob M, et al. Metabolomics toward personalized medicine. Mass Spectrom Rev.
2019;38(3):221–38.
15. Rhee EP, et al. A genome-wide association study of the human metabolome in a community-­
based cohort. Cell Metab. 2013;18(1):130–43.
16. Macel M, Van Dam NM, Keurentjes JJ. Metabolomics: the chemistry between ecology and
genetics. Mol Ecol Resour. 2010;10(4):583–93.
17. Gadelha MR, Kasuki L, Korbonits M. The genetic background of acromegaly. Pituitary.
2017;20(1):10–21.
18. Bicikova M, et al. Aminothiols in human brain tumors. Clin Chem Lab Med. 2006;44(8):978–82.
19. Pinzariu O, Georgescu B, Georgescu CE. Metabolomics-a promising approach to pituitary
adenomas. Front Endocrinol (Lausanne). 2018;9:814.
Metabolomics of Rare Endocrine, Genetic Disease: A Focus on the Pituitary Gland 187

20. Lee SH, Nam SY, Chung BC. Altered profile of endogenous steroids in the urine of patients
with prolactinoma. Clin Biochem. 1998;31(7):529–35.
21. Rostomyan L, et al. Clinical and genetic characterization of pituitary gigantism: an interna-
tional collaborative study in 208 patients. Endocr Relat Cancer. 2015;22(5):745–57.
22. Iacovazzo D, et al. Germline or somatic GPR101 duplication leads to X-linked acrogigantism:
a clinico-pathological and genetic study. Acta Neuropathol Commun. 2016;4(1):56.
23. Boguslawska A, Korbonits M. Genetics of acromegaly and gigantism. J Clin Med.
2021;10(7):1377.
24. Yu H, et al. Metabolic profiling of acromegaly using a GC-MS-based nontargeted metabolo-
mic approach. Endocrine. 2020;67(2):433–41.
25. Fellinger P, et al. Increased ATP synthesis might counteract hepatic lipid accumulation in acro-
megaly. JCI Insight. 2020;5(5):e134638.
26. Biagetti B, et al. Metabolic fingerprint of acromegaly and its potential usefulness in clinical
practice. J Clin Med. 2019;8(10):1549.
27. Wang M, et al. High-performance liquid chromatography-mass spectrometry-based lipid
metabolite profiling of acromegaly. J Clin Endocrinol Metab. 2020;105(3):e1075.
28. De San-Martin BS, et al. Metabolomics as a potential tool for the diagnosis of growth hormone
deficiency (GHD): a review. Arch Endocrinol Metab. 2021;64(6):654–63.
29. Molitch ME, et al. Evaluation and treatment of adult growth hormone deficiency: an Endocrine
Society clinical practice guideline. J Clin Endocrinol Metab. 2011;96(6):1587–609.
30. Argente J, et al. Genetics of growth disorders-which patients require genetic testing? Front
Endocrinol (Lausanne). 2019;10:602.
31. Hoybye C, et al. Metabolomics: a tool for the diagnosis of GH deficiency and for monitoring
GH replacement? Endocr Connect. 2014;3(4):200–6.
32. Abd Rahman S, et al. Urine metabonomic profiling of a female adolescent with PIT-1 muta-
tion before and during growth hormone therapy: insights into the metabolic effects of growth
hormone. Growth Hormon IGF Res. 2013;23(1–2):29–36.
33. Xu R, et al. Metabolomic analysis reveals metabolic characteristics of children with short stat-
ure caused by growth hormone deficiency. Clin Sci (Lond). 2019;133(6):777–88.
34. Mastrangelo A, et al. Metabolomics changes in patients with PAPP-A2 deficiency in response
to rhIGF1 treatment. Growth Hormon IGF Res. 2018;42-43:28–31.
35. Narduzzi L, et al. Applying metabolomics to detect growth hormone administration in athletes:
proof of concept. Drug Test Anal. 2020;12(7):887–99.
36. Nieman LK, et al. The diagnosis of Cushing’s syndrome: an Endocrine Society clinical prac-
tice guideline. J Clin Endocrinol Metab. 2008;93(5):1526–40.
37. Oklu R, et al. Identification of small compound biomarkers of pituitary adenoma: a bilateral
inferior petrosal sinus sampling study. J Neurointerv Surg. 2014;6(7):541–6.
38. Arlt W, et al. Steroid metabolome analysis reveals prevalent glucocorticoid excess in primary
aldosteronism. JCI Insight. 2017;2(8):e93136.
39. Di Dalmazi G, et al. Cortisol-related metabolic alterations assessed by mass spectrometry
assay in patients with Cushing’s syndrome. Eur J Endocrinol. 2017;177(2):227–37.
40. Eisenhofer G, et al. Plasma steroid metabolome profiling for diagnosis and subtyping patients
with Cushing syndrome. Clin Chem. 2018;64(3):586–96.
41. Vega-Beyhart A, et al. Endogenous cortisol excess confers a unique lipid signature and meta-
bolic network. J Mol Med (Berl). 2021;99(8):1085–99.
Metabolomics and Genetics of Rare
Endocrine Disease: Adrenal, Parathyroid
Glands, and Cystic Fibrosis

Afshan Masood, Abeer Malkawi, Mohamed Siaj, and Anas M. Abdel Rahman

Abstract Recent advances in metabolomic technologies and methodologies have

identified significant metabolites related to rare endocrine disease conditions of the
adrenal gland (hyperaldosteronism, primary adrenal insufficiency), parathyroid
(hypoparathyroidism), and cystic fibrosis. Metabolomic profiling combined with
genomics is increasingly being employed for improving understanding, clinical
diagnosis, and management of these clinically challenging conditions. Advances in
gas and liquid chromatography combined with tandem mass spectrometry (GC/LC–
MS/MS) techniques have improved the profiling of steroid metabolites. Significant
alterations in levels of these metabolites demonstrate the potential to serve as spe-
cific markers of disease, help in their stratification, and contribute toward moving to
personalized medicine.

Keywords Rare endocrine disease · Genetic disease · Metabolomics · Primary

aldosteronism · Primary adrenal insufficiency hyperaldosteronism · Parathyroidism
Pheochromocytoma · Paragangliomas · Cystic fibrosis

A. Masood · A. Malkawi · M. Siaj (*)

Department of Chemistry and Biochemistry, Université du Québec à Montréal (UQAM),
Montreal, QC, Canada
e-mail: [Link]@[Link]
A. M. Abdel Rahman (*)
Metabolomics Section, Department of Clinical Genomics, Center for Genome Medicine,
King Faisal Specialist Hospital & Research Center (KFSHRC), Riyadh, Saudi Arabia
e-mail: AabdelRahman46@[Link]

© The Author(s), under exclusive license to Springer Nature Singapore Pte 189
Ltd. 2023
A. M. Abdel Rahman (ed.), Clinical Metabolomics Applications in Genetic
Diseases, [Link]
190 A. Masood et al.

Abbreviations

ATP1A1 ATPase Na+/K + -transporting subunit alpha 1

ATP2B3 ATPase plasma membrane Ca2+ transporting 3
CACNA1H Calcium voltage-gated channel subunit alpha 1 H
CACNA1H Calcium voltage-gated channel subunit alpha 1 H
CASR G protein-coupled calcium-sensing receptor
CCND1/PRAD1 Cyclin D1
CDKN1C Cyclin-dependent kinase inhibitor 1C
CFTR CF transmembrane conductance regulator
CHD7 Chromodomain helicase DNA binding protein 7
CLCN2 Chloride voltage-gated channel 2
CSDE1 Cold shock domain-containing E1
CTNNB1 Catenin beta 1
CYP11B2 Cytochrome P450 family 11 subfamily B member 2
DAX-1 (NR0B1) SF-1 Nuclear receptor subfamily 0 group B member 1
DLST Dihydrolipoamide S-succinyltransferase
FH Fumarate hydratase
GATA3 GATA binding protein 3
GCM2 Glial cells missing transcription factor 2
GNA11 G protein subunit alpha 11
H3F3A H3.3 histone A
HIF2A Hypoxia-inducible factor 1 subunit alpha
HRAS HRas proto-oncogene, GTPase
IDH Isocitrate dehydrogenase (NADP(+)) 1
IRP1 Iron regulatory protein
KCNJ5 Potassium inwardly rectifying channel subfamily J
member 5
MAML3 Mastermind-like transcriptional coactivator 3
MDH2 Malate dehydrogenase 2
NF1 Neurofibromin 1
NR5A1 Nuclear receptor subfamily 5 group A member 1
P450scc/CYP11A1 Cytochrome P450 family 11 subfamily A member 1
PHD1 Prolyl hydroxylase 1
POLE1 DNA polymerase epsilon, catalytic subunit
PTH Parathyroid hormone
RET Ret proto-oncogene
SAMD9 Sterile alpha motif domain containing 9
SDHx Succinate dehydrogenase complex iron-sulfur subunit B
SEMA3E Semaphorin 3E
SGPL1 Sphingosine-1-phosphate lyase 1
SLC25A11d Solute carrier family 25 member 13
SOX3 SRY-Box transcription factor 3
TMEM127 Transmembrane protein 127
VHL/EPAS Von Hippel–Lindau tumor suppressor
Metabolomics and Genetics of Rare Endocrine Disease: Adrenal, Parathyroid Glands… 191

1 Introduction

Over the course of the past 10 years, research based on metabolomics has grown
significantly and emerged as a promising instrument for clinical diagnostics as well
as for improving our comprehension of the physiological and pathological pro-
cesses that are the foundation of study for endocrine-related and rare diseases. In
this chapter, we looked at how metabolomics helped diagnose, treat, and follow up
rare endocrine disease of the pituitary gland. Beyond the pituitary, metabolomics
has also been applied in disease stratification and management and in identifying
biomarkers with applications in disease prediction, diagnosis, prognosis, and
therapy.

2 Metabolomics of Adrenal Dysfunction

The adrenal glands play an important role in regulation of body homeostasis.

Anatomically, they are made up of the cortex and medulla that secrete hormones
involved in maintaining electrolyte and mineral balance, control metabolic path-
ways, provide response to stress (cortisol production in the adrenal cortex and cat-
echolamines in adrenal medulla), and are crucial for sexual differentiation (through
producing steroid hormones in the adrenal cortex). Diseases of the adrenal glands
result in the resistive synthesis of glucocorticoids, sex hormones, and catechol-
amines (epinephrine and norepinephrine). Excessive circulating glucocorticoid
(cortisol) levels, independent of ACTH, primarily arise from the adrenal gland dis-
ease (CS), while increased secretion of corticotropin (ACTH)-dependent cortisol is
primarily due to disease of the pituitary and in some cases due to other glands. CS
accounts for 15% of the cases while a majority is 70% due to CD and other causes
including ectopic production is 15% [1]. The associated genetic defects and metab-
olite changes related to endogenous and exogenous hypercortisolism were covered
in the previous chapter. Adrenal gland dysfunction also results in disorders of aldo-
sterone synthesis (hyperaldosteronism or Conn’s syndrome and adrenal insuffi-
ciency or Addison’s disease), steroidogenesis, and the synthesis of sex hormones.
Rare forms of these conditions arise due to germline mutations resulting in benign
adrenal tumors or adrenocortical adenomas having an overall incidence in the gen-
eral population of 1–2 cases per million [2]. Clinical evaluation of these disorders
of adrenal steroidogenesis and disease requires measurement of specific hormonal
levels, radiological imaging, and histopathology of the biopsy specimens. Advances
in GC–MS and LC–MS/MS techniques have greatly improved the diagnosis of
these diseases by metabolomic identification and quantification of the steroid
metabolome that includes steroid hormones along with metabolic derivatives in
bodily fluids (e.g., serum, urine) for clinical (diagnostics and treatment monitoring)
as well as research purposes.
192 A. Masood et al.

2.1 Hyperaldosteronism (Conn’s Syndrome)

Primary aldosteronism (PA) is characterized by inappropriate and excessive secretion

of the adrenal steroid hormone and aldosterone (hyperaldosteronism), the main min-
eralocorticoid hormone responsible for salt and water reabsorption, as well as
increased potassium and proton secretion from the kidneys. Among the primary
causes of PA is aldosterone-producing adenomas that present clinically as secondary
endocrine hypertension. The nonneoplastic rare causes of PA (5%) are due to familial
hyperaldosteronism (FH I–IV) and bilateral adrenal hyperplasia (BAH) [3, 4]. The
advent of next-generation sequencing (NGS) technology and its wider application
determined a largely genetic basis for the rare (5%) causes of PA through the identifi-
cation of overlapping set of genes carrying numerous disease-­causing germline muta-
tions. These genome-wide association studies (GWAS) identified germline variants in
CACNA1H (encoding a subunit of T-type voltage-gated calcium channel, CaV3.2),
KCNJ5 (potassium inwardly rectifying channel subfamily J member 5), CYP11B2
(encoding aldosterone synthase), CACNA1D (calcium channel voltage-dependent
L-type alpha-1D subunit), and CLCN2 (encoding voltage-­gated chloride channel
ClC-2) [5–8]. Metabolomic approaches and metabolome profiling using gas chroma-
tography–mass spectrometry (GC–MS) and ultra-­HPLC–tandem mass spectrometry
(UHPLC–MS/MS) have comprehensively profiled the steroid metabolite profiling in
sera and urine of patients and have found applications in the areas of personalized
medicine for diagnostic purpose and prediction of prognosis. Coupling metabolomics
together with genomics using GWAS provides a platform for employing integrated
OMICS toward understanding and identifying the clinical phenotype. These genetic
variations with their resulting metabolic alterations have created the metabolic pheno-
types termed “genetically determined metabotypes” that is now being considered as a
diagnostic feature [9]. Clinically, the characteristic presentation of PA is an increase in
blood pressure, that is, hypertension along with disturbances in the electrolyte levels.
When compared to patients with primary hypertension, these patients are at a higher
risk of developing cardiovascular and kidney disease [10, 11], making an early diag-
nosis vital. Aside from these complications, there is also the need to differentiate
between the different PA subtypes and unilateral or bilateral disease as clinical man-
agement of both conditions differs; the former is managed surgically while the latter
is managed medically.
A metabolomic approach using liquid chromatography with tandem mass spec-
trometry (LC–MS/MS) successfully quantified adrenal steroids. The multi-steroid
signatures associated with steroid biosynthesis and metabolism disorders showed a
high level of diagnostic accuracy to differentiate between PA and adrenal hyperpla-
sia that have similar presentations. Levels of cortisol derivatives (18-­hydroxycortisol
and 18-oxocortisol) were elevated in patients with PAs, carrying KCNJ5 mutations,
in comparison to those with adrenal hyperplasia. Urinary 18-hydroxycortisol
showed a high accuracy in distinguishing between the two conditions [12]. On the
other hand, patients with adrenal hyperplasia showed elevated levels of plasma
dehydroepiandrosterone (DHEA), DHEA-S, cortisol, and corticosterone [13].
Metabolomics and Genetics of Rare Endocrine Disease: Adrenal, Parathyroid Glands… 193

Targeted metabolomic analysis by LC-MS/MS using a 32-metabolite steroid panel

that included 11-deoxycorticosterone, aldosterone, cortisol, 11-deoxycortisol,
21-deoxycortisol, corticosterone, progesterone, 17-hydroxyprogesterone,
18-­oxocortisol, 18-hydroxycortisol, cortisone, pregnenolone, androstenedione,
DHEA, and DHEA-S was also used. Levels of 18-oxocortisol and 18-­hydroxycortisol
were found to have distinctively higher levels in cases of FH I and III compared to
controls and also showed a high level of correlation with histological features of
adenoma. In addition, mutations in CTNNB1, coding for β-catenin, have been iden-
tified in 2–5% of cases with aldosterone-producing benign adenoma. A somatic
mutation in CLCN2, coding for the chloride channel ClC-2 (chloride channel pro-
tein 2) mutated in familial hyperaldosteronism type II (FH-II) and early-onset PA,
has recently been identified. Previous studies have shown that CYP11B2 can con-
vert 11-deoxycortisol efficiently to 18-hydroxycortisol and 18-oxocortisol, while
CYP11B1 can synthesize only 18-hydroxycortisol [5, 14, 15] (Table 1).
Hyperaldosteronism is suggested to cause inflammation and metabolic dys-
regulation and contribute to development of cardiovascular disease. Metabolomic
profiling in patients with Conn’s disease revealed significant alterations in levels
of triglyceride concentrations, large VLDL particles with urate concentrations,
and derivatives of the linoleic acid metabolism pathway [16]. Steroid profiling has
also revealed high production of the “hybrid” cortisol metabolites 18-hydroxycor-
tisol and 18-oxocortisol in patients with rare, familial forms of PA associated with
specific genetic errors (CYP11B1/CYP11B2 hybrid gene, KCNJ5 mutations) [8,
17, 18]. The levels of these hybrid metabolites also served as markers to differen-
tiate between PA and BAH. Specifically, the 18-oxocortisol/cortisol ratio in adre-
nal vein samples and urinary 18-hydroxycortisol levels showed sufficient
diagnostic accuracy to distinguish APAs from BAHs of patients [19, 20]. In addi-
tion to the clear elevation of plasma 18-oxocortisol in PAs, increased levels of
plasma cortisol, corticosterone, DHEA, and DHEA-S were documented in patients
with BAH [13]. Differences between the metabolite levels among the varying
subtypes were also demonstrated using in situ metabolomics and demonstrated
that levels of 18-­oxocortisol and 18-hydroxycortisol negatively correlate with the
CYP11B1. The steroid profiles were also correlated with their respective geno-
types. The PAs carrying KCNJ5 mutations presented significantly higher levels of
18-oxocortisol in both adrenal vein and peripheral plasma samples than all other
PAs, while PAs harboring ATPase mutations displayed the highest peripheral con-
centrations of aldosterone, cortisol, 11-deoxycorticosterone, and corticosterone.
At the same time, patients with CACNA1D-mutated PAs had lower aldosterone
and corticosterone concentrations than all other groups [21]. Distinct molecular
signatures between KCNJ5- and CACNA1D-mutated PAs involving metabolites
of steroidogenesis as well as purine metabolism KCNJ5 carriers displayed signifi-
cantly higher levels of 18-­hydroxycortisol and 18-oxocortisol when compared to
CACNA1D carriers. Activation of purine metabolism was observed in KCNJ5
mutant APAs, with a significant increase in adenosine monophosphate and diphos-
phate [14].
Table 1 The different endocrine glands with the associated disease, the gene locus, and the major metabolites altered with the disease
194

Metabolomic
Endocrine Genes and chromosomal platform and
gland Hormones Disease locus approach Major significant metabolites References
Adrenal Aldosterone Primary KCNJ5, CACNA1D, LC–MS/MS 18-Hydroxycortisol and [13, 17]
gland hyperaldosteronism ATP1A1, ATP2B3, (targeted) and steroid 18-oxocortisol, tetrahydro-18-­
CTNNB1, CYP11B2, profiling using oxocortisol, aldosterone, cortisol,
CLCN2, and CACNA1H GC-MS and LC–MS/ 11-deoxycorticosterone, and
MS (untargeted) corticosterone (↑)
Long-chain acylcarnitines C18:1,
C18:2, ornithine, and spermidine
ACTH Adrenal insufficiency DAX-1 (NR0B1) SF-1, GC-MS, LC-MS/MS, Total cortisol metabolites, 11β-­ [17, 24, 27]
decrease NR5A1, CDKN1C, SAMD9, 1HNMR, and steroid HSD1 11β-HSD2, and 5α- and
POLE1, P450scc/ profiling (targeted) 5β-reductase (↓)
CYP11A1, and SGPL1
Pheochromocytoma/ NF1, RET, TMEM127, ESI–LC–MS/MS Succinate, citrate, isocitrate, [29, 38]
paraganglioma HRAS, SDHx, FH, IDH, (targeted), LC–MS cis-aconitate (↑), fumarate, and
H3F3A, HIF2A, MDH2, 2-hydroxyglutarate (↓)
PHD1, IRP1, SLC25A11d, 2-Oxoglutarate/malate carrier and
DLST, VHL/EPAS, and glutamic-oxaloacetic transaminase 2
CSDE1 MAML3 (↓)
Parathyroid PTH Hypoparathyroidism GCM2, SOX3, CHD7, UHPLC–MS Adenosine, inosine, hypoxanthine, [45]
SEMA3E, GATA3, PTH, (untargeted) guanosine, and xanthine (↓) N-acetyl
CASR, and GNA11 aspartate
A. Masood et al.
Pancreas Cystic fibrosis CFTR GC/LC–MS Sorbitol and cholesteryl esters [21, 49, 50,
(untargeted) Purine nucleotides, adenosine, 55–57]
UHLC–MS/MS and inosine, hypoxanthine, and
GC–MS (untargeted) guanosine (↑)
Oxidized glutathione,
S-lactoylglutathione,
S-nitrosoglutathione, and
ophthalmate (↓)
ATP1A1 ATPase Na+/K + -transporting subunit alpha 1, ATP2B3 ATPase plasma membrane Ca2+ transporting 3, CACNA1D calcium voltage-gated channel
subunit alpha 1 D, CACNA1H calcium voltage-gated channel subunit alpha 1 H, CACNA1H calcium voltage-gated channel subunit alpha 1 H, CASR G protein-­
coupled calcium-sensing receptor, CDKN1C cyclin-dependent kinase inhibitor 1C, CFTR CF transmembrane conductance regulator, CHD7 chromodomain
helicase DNA binding protein 7, CLCN2 chloride voltage-gated channel 2, CSDE1 cold shock domain containing E1, CTNNB1 catenin beta 1, CYP11B2
cytochrome P450 family 11 subfamily B member 2, DAX-1 (NR0B1) SF-1 nuclear receptor subfamily 0 group B member 1, DLST dihydrolipoamide
S-succinyltransferase, FH fumarate hydratase, GATA3 GATA binding protein 3, GCM2 glial cells missing transcription factor 2, GNA11 G protein subunit
alpha 11, H3F3A H3.3 histone A, HIF2A hypoxia-inducible factor 1 subunit alpha, HRAS HRas proto-oncogene, GTPase, IDH isocitrate dehydrogenase
(NADP(+)), 1IRP1 iron regulatory protein, KCNJ5 potassium inwardly rectifying channel subfamily J member 5, MAML3 mastermind-like transcriptional
coactivator 3, MDH2 malate dehydrogenase 2, NF1 neurofibromin 1, NR5A1 nuclear receptor subfamily 5 group A member 1, P450scc/CYP11A1 cytochrome
P450 family 11 subfamily A member 1, PHD1 prolyl hydroxylase, POLE1 DNA polymerase epsilon, catalytic subunit, PTH parathyroid hormone, PTPN22
protein tyrosine phosphatase non-receptor type 22, RET Ret proto-oncogene, SAMD9 sterile alpha motif domain containing 9, SDHx succinate dehydrogenase
complex iron sulfur subunit B, SEMA3E semaphorin 3E, SGPL1 sphingosine-1-phosphate lyase 1, SLC25A11d solute carrier family 25 member 13, SOX3
SRY-Box transcription factor 3, TMEM127 transmembrane protein 127, UPLC–MS/MS ultra-PLC–tandem mass spectrometry, VHL/EPAS Von Hippel-Lindau
tumor suppressor
Metabolomics and Genetics of Rare Endocrine Disease: Adrenal, Parathyroid Glands…
195
196 A. Masood et al.

2.2 Primary Adrenal Insufficiency

Primary adrenal insufficiency (PAI), a deficiency of glucocorticoid and mineralo-

corticoid production, is a relatively rare life-threatening condition due to autoim-
mune disorders and enzymatic defects. The patients clinically present with skin and
mucous membrane hyperpigmentation, craving for salt, failure to thrive, depression,
and fatigue. PAI is caused due to pathology within the adrenal glands which results
in stimulation of the hypothalamo–pituitary axis and the renin–angiotensin–aldoste-
rone system regulatory feedback loop. PAI is typically diagnosed by measuring
levels of ACTH and proopiomelanocortin peptides, which are elevated in addition
to inappropriately low cortisol secretion. A delayed diagnosis of PAI is linked with
an adverse quality of life and raises the patient’s risk of an adrenal crisis that might
be fatal. Recent studies have implicated several genetic mutations in the pathophysi-
ology of the disease. These include defects in the nuclear receptors DAX-1 (NR0B1),
steroidogenic factor-1 (SF-1/NR5A1), CDKN1C and SAMD9 or loss of POLE1,
P450scc/CYP11A1 insufficiency, and sphingosine-1-phosphate lyase-1 (SGPL1)
defects [22]. Treatment of PAI conventionally is modeled around corticosteroid
replacement therapy that is conventionally administered three times a day [23].
Metabolomic studies in PAI are limited in the literature. In a study, metabolite
profiling was carried out in the sera or urine for disease identification and stratifica-
tion and for evaluating optimal replacement therapy. The natural circadian rhythm
of cortisol cycle cannot be entirely replicated by current glucocorticoid replacement
regimens, which leads to either over- or under-replacement. The urinary cortisol
metabolome was assessed to determine optimal cortisol replacement in patients
with PAI. The metabolic profile of patients using two hydrocortisone replacement
therapies were compared, namely, the once-a-day dual-release hydrocortisone
(DHC) and three-times-a-day hydrocortisone (TID-HC) therapy. In the 24-h urine
samples, total cortisol metabolites decreased after DHC therapy compared to
TID-HC and were more in line with the usual control levels. 11-β-Hydroxysteroid
dehydrogenase (11β-HSD) type 1 activity dropped with DHC compared to TID-HC
therapy, whereas 11-β HSD2 activity fell with TID-HC but returned to normal with
DR-HC. Moreover, 5α- and 5β-reduced metabolites were decreased with DR-HC
compared to TID-HC. Patients undergoing traditional TID-HC replacement treat-
ment with enhanced 11β-HSD1 activity exhibits significant alterations in the urine
cortisol metabolome, which may explain the adverse metabolic profile in patients
with PAI. Its shift toward normalcy with DHC therapy might serve as an indicator
for more favorable metabolic outcomes [17, 24]. Aside from providing means for
optimizing therapeutic dosage, the metabolomic approach was also used to measure
the effects of glucocorticoid therapy and identify biomarkers related to its action.
Serum metabolic profiling was also undertaken in PAI patients, using GC–MS and
LC–MS, during glucocorticoid therapy and after its withdrawal to assess response
to therapy. The differentially expressed metabolites identified were amino acids
(tyrosine, tryptophan, asparagine), malic acid, lactic acid, and uracil. The metabo-
lism of tryptophan, which modulates mood and energy homeostasis, is regulated by
Metabolomics and Genetics of Rare Endocrine Disease: Adrenal, Parathyroid Glands… 197

glucocorticoids through the kynurenine pathway. Metabolomic analysis was able to

identify that administering high doses of glucocorticoid, especially after a treatment
of 10-week treatment, resulted in decreased tryptophan levels by influencing the
kynurenine pathway [25, 26]. Hence, metabolomics assisted in assessing the thera-
peutic effects, allowing for the individualization of approaches and optimization of
glucocorticoid therapy.

2.3 Metabolomics of Pheochromocytoma

Pheochromocytomas (PCC) and paragangliomas (PPGLs) are a group of rare het-

erogeneous neuroendocrine tumors that arise from either the adrenal medullary
chromaffin cells or from outside the adrenal gland in the neural crest cells (sympa-
thetic and parasympathetic paraganglia). Most PPGLs are benign tumors, with an
incidence of approximately of one per million population per year [27]. The charac-
teristic clinical phenotype of these patients is associated with features of excess
circulating catecholamine levels due to increased synthesis or release. The present-
ing signs and symptoms classically range from a triad consisting of sweating, head-
aches, and palpitations to nonspecific symptoms such as weight loss, nausea,
tiredness, or flushing [27, 28]. The diverseness and nonspecificity of the clinical
manifestations, heterogeneity of these tumors regarding the age of presentation, and
differences in their location make an early clinical diagnosis of PCC difficult [29].
PCC and PPGLs have the highest degree of heritability, where PPGLs carrying a
germline mutation account for 30–40%. More than 20 susceptibility genes with
varying mutations have been identified as predisposing factors to this condition,
placing it among the rare genetic endocrine conditions. The metabolic phenotypes
in PCC are based on the affected specific gene/protein [30] that determines the
secretory profile, molecular features, metabolic changes, clinical outcomes, and
potential for malignancy [31].
PCC and PPGLs have been classified based on their inheritance, multiple endo-
crine neoplasia type 2 (MEN2), familial Von Hippel–Lindau (VHL) syndrome and
less commonly neurofibromatosis type 1 (NF1)) or sporadic [30], or by molecular
pathway subtypes, kinase signaling subtype (RET, transmembrane protein 127
(TMEM127), mutations in the NF1, and HRAS genes), pseudohypoxia (Von
Hippel–Lindau (VHL/EPAS)-related and tricarboxylic acid cycle (TCA)-related
mutations, and Wnt-altered subtype (CSDE1 somatic mutations and mastermind-­
like transcriptional coactivator 3 (MAML3) fusion genes)). The TCA-related PPGL
subtype consists of tumors having mutations in the succinate dehydrogenase sub-
units A–D (SDHx), fumarate hydratase (FH), and isocitrate dehydrogenase (IDH).
In addition, other genetic mutations in the H3F3A, malate dehydrogenase 2
(MDH2), PHD1, IRP1, SLC25A11, and DLST were identified with a lower fre-
quency and have not yet been included in the Cancer Genome Atlas. PCC and
PPGLs are also classified into clusters based on their secretory profile as adrenergic
and noradrenergic clusters. The noradrenergic pseudohypoxic phenotype (secreting
198 A. Masood et al.

norepinephrine and normetanephrine (NMN)) constitutes cluster 1. It includes

tumors with SDHx mutations, along with VHL, FH, (MDH2), hypoxia-induced fac-
tor (HIF2α), and IDH mutations and the newly identified SLC25A11 [32]. Tumors
with the adrenergic phenotype (secreting epinephrine and metanephrine (MN)),
which are associated with abnormal kinase signaling pathways and include muta-
tions in the genes rearranged during transfection (RET), NF1, TMEM127, kinesin
family member 1B (KIF1B), and MYC-associated factor X (MAX), make up clus-
ter 2. Cluster 3 is associated with the Wnt signaling pathway; it includes somatic
mutations of cold shock domain-containing E1 (CSDE1) and mastermind-like tran-
scriptional coactivator 3 (MAML3) fusion genes [28, 32].
The gold standard for diagnosis relies on biochemical measurements of urinary
or plasma products of the catecholamine degradation, noradrenaline (MN), adrena-
line (NMN), and dopamine (methoxytyramine (MTY)) [33]. Measurements of
plasma-free MN have been proven in several independent investigations to have
diagnostic sensitivity surpassing 96% and specificity between 85 and 100%. An
alternate method with a comparable degree of diagnostic sensitivity is provided by
urinary-fractionated MN [28]. The measurement of plasma and urine MN by LC–
MS/MS is presently widely accepted in the USA and many other laboratories as the
gold standard approach [28, 34]. In addition to laboratory measurement, all patients
with documented PCC and PPGLs should have genetic determination of PPGL phe-
notype as part of the diagnostic panel. It is very common to find mixed phenotypes
of both adrenergic and dopaminergic secreting tumors in comparison to either
adrenergic or dopaminergic ones. Each of these phenotypes has been linked to
mutations in different genes. The genetic mutations in TMEM127 gene have been
associated with only the adrenergic tumors while mutations in the KIF1B, MAX,
RET, and NF1 genes have been associated with tumors with adrenergic mixed phe-
notype. On the other hand, it is known that extra-adrenal PPGLs having noradrener-
gic and dopaminergic phenotypes have mutations in PHD1/PHD2, HIF2A, SDHx,
SDHAF2, FH, and IDH genes. The majority of PPGL tumors with HIF2A and
VHL-mutated are typically noradrenergic while predominantly dopaminergic
secreting tumors are known to be commonly associated with SDHx mutations [33].
Although confirmatory, genetic testing can be complex and, in many cases, unavail-
able at all centers. This potentially leads to delayed or inconclusive diagnosis [30,
35, 36]. Due to the probable increased risk of metastatic illness in these patients, it
becomes crucial for an effective clinical management to distinguish early on
between tumors with underlying germline mutations and those that are sporadic
[37]. In these instances, quantifying metabolites can help verify functionality and
identify underlying mechanisms and factors for germline or somatic mutations in
patients with unresolved genetic testing results.
Metabolomic studies have helped to bridge this gap by identifying metabolites
that have not only helped in detailing the metabolic pathways affected by the dis-
ease but also to differentiate between the phenotypes, stratify the disease, and pro-
pose metabolites that are amenable to diagnostic applications. The tumor
metabolomic profile distinguishes these different subtypes of tumors to classify
patients with PGLs as sporadic or hereditary. Untargeted metabolomic approaches
Metabolomics and Genetics of Rare Endocrine Disease: Adrenal, Parathyroid Glands… 199

aided in profiling the disease pathology and associating the changes with the differ-
ent variants of genetic mutations. On the other hand, targeted metabolomic
approaches using the identified metabolites have also been studied to determine
their impact on metabolism and utilize them as diagnostic markers in clinical labo-
ratories [29] for monitoring therapeutic response potential conversion metastases
[38]. Surgical resection of the PPGL with normalization of catecholamine levels
was associated with significant changes in the metabolites. Following surgery levels
of glycerophospholipids (phosphatidylcholine diacyl (PC aa) 42:0, phosphatidyl-
choline acyl-alkyl (PCae) 42:5, PCae (44:5), and PCae (44:6) and hexoses were
lower, while levels of amino acids (biogenic amines), namely, histidine and creati-
nine, were demonstrated to be higher [29]. The metabolomic profile in each of the
different genetic variants of these tumors was also deciphered. Around 15–25% of
all PCC/PPGLs were linked to defects in the Krebs cycle enzymes, SDH, FH, MDH,
and IDH, with SDH faults, being the most frequent. The 2-­oxoglutarate/malate car-
rier, glutamic-oxaloacetic transaminase 2, and others have more recently been
linked to hereditary PPGL as regulators of mitochondrial metabolites [36]. The
PCC/PGLs associated with mutations in the pseudo hypoxic cluster (cluster 1) were
associated with the hypoxia-inducible factor (HIF) signaling pathway and involved
mutations in genes encoding the HIF2A, succinate dehydrogenase subunits or their
assembly factors (SDHx [SDHA, SDHB, SDHC, SDHD]), succinate dehydroge-
nase complex assembly factor 2 (SDHAF2), Von Hippel–Lindau tumor suppressor
(VHL), and egl-9 prolyl hydroxylases 1 and 2 (EGLN1/EGLN2). The pathogenic
mutations in these genes lead to an accumulation of their related metabolites, that is,
succinate, fumarate, or 2-hydroxyglutarate, which in turn were responsible for
tumor development.
Distinct differences were noted in metabolites and pathways related to oxygen
sensing, hypermethylation, DNA repair, and overexpression of certain transporters
and receptors; notably Krebs cycle enzymes have been through the genetic investi-
gations in PCC/PGL tumors [39–41]. Metabolomic profiling identified differential
regulation of metabolites between the various genetic causes of PCC/
PGL. Metabolomic analysis of PCC/PGL arising from mutations in SDHx revealed
a decrease in activity of SDH (mitochondrial electron transport chain complex II)
enzyme and other TCA cycle metabolites, including fumarate glutamate and aspar-
tate with elevated succinate levels [37]. The ratio of two metabolites, succinate to
fumarate, was determined as a novel metabolic marker to detect paraganglioma with
underlying SDHB/D mutations [38, 42]. Moreover, in tumors linked to SDHx muta-
tions, glutamate levels and ATP/ADP/AMP values were shown to be lower with
modest but significant changes in levels of histidine, threonine, and lysoPC (C28:0)
[32, 42]. Significant correlations were also noted between plasma MN and total
urine catecholamine levels with the sum of detected hexoses (reflecting glucose)
which were found [33], along with the increase in levels of glutamine [40].
Significant alterations were noted in isocitrate, cis-aconitate, and citrate levels in
patients with mutations in FH. In contrast, IDHx mutations were characterized by
higher citrate, isocitrate, and cis-aconitate levels [37]. These differences could serve
as potential biomarkers for early diagnosis of disease.
200 A. Masood et al.

3 Metabolomics of Parathyroid Dysfunction

The parathyroid glands are four small pea-sized glands behind the thyroid gland
secreting parathyroid hormone (PTH). The primary endocrine glands maintain cal-
cium and phosphorus homeostasis with other hormones, including vitamin D and
fibroblast growth factor (FGF23). PTH regulation occurs mainly between three
organs, the intestine, kidney, and bone. A complex interplay occurs between PTH,
active vitamin D (1,25(OH)2D), and calcium sensor receptors (CaSRs) that main-
tain serum calcium concentration within a narrow physiological range to maintain
mineral homeostasis. Dysfunction of the parathyroid glands occurs as a primary
disease of the gland or secondary to other diseases such as chronic kidney disease.
Parathyroid dysfunction results in inappropriate parathyroid hormone (PTH) pro-
duction, resulting in abnormal calcium homeostasis. Phenotypically, it can manifest
as either an increase, hyperparathyroidism, or a decrease, hypoparathyroidism, in
circulating PTH levels. Primary parathyroid dysfunction is relatively rare compared
to secondary causes and can be seen as an isolated condition or component of a
complex endocrine syndrome. Hypocalcemia and hyperphosphatemia are the char-
acteristic hallmarks of primary hypoparathyroidism, which is caused by inadequate
quantities of circulating parathyroid hormone.

3.1 Hypoparathyroidism

Incidental hypoparathyroidism is a rare disorder with an estimated prevalence

of 0.25 per 1000 individuals. Hypoparathyroidism is clinically characterized by
decreased parathyroid hormone levels resulting in hypocalcemia that directly
impacts calcium and phosphorus homeostasis and the bone. It can occur as an
isolated condition or as part of a complex endocrine syndrome. The most com-
mon cause of hypoparathyroidism is transient postsurgical hypoparathyroidism
resulting from a functional impairment or surgical resection of parathyroid
glands after acute manipulation during neck surgery. Other causes include
impairment of PTH action, pseudo-hypoparathyroidism, and genetic causes.
Numerous somatic or germline mutations have been identified, leading to dys-
genesis of the parathyroid gland or an inability of the parathyroid glands to
secrete PTH. These include mutation in the PTH gene, GCM2, SOX3, CASR,
GNA11, TBX1, CHD7, GATA3, and TBCE [43, 44]. Rare genetic defects
involving the transient receptor potential ion channel (TRMP6) and tight-junc-
tion gene claudins 16 and 19 have been identified, resulting in the abnormal
homeostasis of magnesium leading to hypoparathyroidism (23). The cause of
low magnesium levels is attributed to nutritional deficiencies or chronic dis-
eases, including T2DM, hypertension, and renal conditions, which either
decrease secretion of PTH or increase resistance to the actions of PTH in the
bone and kidneys. Metabolomic studies evaluating changes in the metabolites
Metabolomics and Genetics of Rare Endocrine Disease: Adrenal, Parathyroid Glands… 201

within hypoparathyroidism are limited. A single study by Paprocka et al. in

children with hypoparathyroidism identified alterations in N-acetyl aspartate by
1H magnetic resonance spectroscopy [45]. Further metabolomic studies are
needed to identify the different metabolites altered with hypoparathyroidism.

4 Exocrine Pancreatic Dysfunction: Metabolomics

of Cystic Fibrosis

Metabolomics has also been important in studying the pathology of cystic fibrosis,
which leads to endocrine-related complications of the pancreas. Cystic fibrosis is a
lethal autosomal recessive disorder arising from mutations in the cystic fibrosis
transmembrane conductance regulator (CFTR) gene expressed in the apical mem-
branes of various epithelial cells. It is a cAMP-regulated channel that conducts ATP
and regulates several apical membrane-associated channels, including the sodium,
chloride, and potassium channel along with regulating release of bicarbonate. The
disease represents an example of a monogenic defect with over 2000 mutations that
leads to characteristic multisystemic disease. Besides the characteristic pulmonary
manifestations, patients with CF show endocrine defects in the pancreas and the
reproductive system. The most common cause of the pathology is blockage of endo-
crine ducts due to the thickened secretions. CF mutations can be grouped as those
causing severe or mild disease and are further categorized as one of six classes;
classes I–III represent severe disease, while mild mutations are classes IV–VI.
Recently, metabolomics has been utilized as an invaluable tool to study the
changes in the metabolic profiles in CF, understand the pathophysiology, and eluci-
date the different metabolic pathways altered with CF [46, 47]. The gold standard
for initial newborn screening is a measurement of immunoreactive trypsinogen
(IRT) in dry blood spots (DBSs), followed by targeted CFTR mutation analysis and
confirmation with abnormally elevated sweat chloride. Our laboratory identified
significant differences in 26 metabolites involved in peroxisomal, amino acids, sor-
bitol, glycolysis, and mitochondrial metabolic pathways. A distinct and interesting
finding was the decrease in the osmolyte sorbitol in adult patients with CF patients
compared to healthy controls. In order to maintain correct cellular activities and cell
survival, organic osmolytes are crucial for regulating cell volume and fluid balance.
The perturbation in the sorbitol pathway was identified as a causative factor for the
mucoviscidosis [48]. A reduction in the sorbitol levels, and glycerol phosphorylcho-
line, another osmolyte, was noted in an untargeted metabolomic analysis of primary
human airway epithelial cell culture in CF patients. Additionally, significant altera-
tions were noted in the purine nucleotides, adenosine, inosine, hypoxanthine, and
guanosine, which may regulate cellular responses via purinergic signaling.
Reductions were also seen in metabolites related to glutamate, including oxidized
glutathione levels, in S-lactoylglutathione, S-nitrosoglutathione, and ophthal-
mate [49].
202 A. Masood et al.

Metabolomic profiling identified differences in patients with different grades and

severity of the disease and between the functional classes of CF. Distinct metabo-
lites were identified that related to clinical phenotype and lung function. We identi-
fied specific metabolites between the different CF functional classes using chemical
isotope-labeled LC–MS-based metabolomics. The metabolomic profile was
assessed between CF and controls, between the different mutation classes of CF,
and specifically among classes III and IV. Significant alterations were seen in gluta-
thione, glutamine, glutamate, and arginine metabolism, amino acids, and di- and
tripeptides. The significant metabolites include gamma-glutamylglutamic acid,
1-aminopropan-2-ol, cystathionine, ophthalmate, and serotonin. An above-average
FEV1% level of lung function was associated with decreased glutamic acid and
increased guanosine levels. Metabolomic profiling, between the three analyses,
demonstrated alterations in several amino acids and dipeptides governing glutathi-
one metabolism and identified two metabolites in common between the analyses.
These metabolites, namely, 3,4-dihydroxymandelate-3-O-sulfate and
5-­aminopentanoic acid, could serve as biomarkers for CF [50].
Moreover, serum metabolomics was employed to evaluate CF bacterial lung ill-
ness in the preform post-exacerbation stage and identify which systemically mea-
surably connected pathways were impacted throughout recovery. Bile acids, amino
acid metabolites generated from microorganisms, increases in the lipid classes of
glycerophospholipid, glycerolipids, cholesterol, phospholipids, and the class of
sphingolipids were among the compounds and pathways affected. The resolution of
the exacerbation was characterized by alterations of the tryptophan–kynurenine
pathway, decreased polyamines, a reduction in lipid markers such as fatty acids (n6/
n3), and increased in nitric oxide pathway metabolites [51]. On the other hand,
metabolites altered with acute pulmonary exacerbation in CF patients demonstrated
lower essential amino acids, L-arginine, and oxoproline levels than healthy controls.
This decrease was mainly attributed to the skeletal muscle wasting, poor protein
intake, increased amino acid utilization, and decreased intestinal absorption of pro-
teins leading to an overall protein-deficient state [52, 53].
In addition to the derangements in the amino acids, patients with CF also showed
abnormal lipid metabolism for most lipid subclasses, with significant plasma eleva-
tions in odd-chain and polyunsaturated fatty acyl lipids and a decrease in the plasma
levels of several species of lysophosphatidylcholine (18:0, 18:2, 20:3, and 20:5) and
phosphatidylcholine (36:5, O-38:0, 38:4, 38:5, 38:6, and P-40:1). Plasma phospho-
lipid signatures were found to discriminate between mild and severe forms of CF. In
contrast, levels of phosphatidic acids and diacylglycerols were particularly affected
by different genotypic mutation classes. A biomarker panel of five oxidized lipids
successfully differentiated patients with reduced lung function. Four species of PC
(36:3, 36:5, 38:5, and 38:6) were consistently downregulated in severe vs. mild
patients, while sphingolipid SM(d18:0) was significantly increased in all patients
[54]. The lung function of CF patients is often assessed through forced vital expira-
tory capacity (FEV1) measurements using FEV1% or FEV1/FVC ratio. Lipid
Metabolomics and Genetics of Rare Endocrine Disease: Adrenal, Parathyroid Glands… 203

fractions of the PUFA (C20:3n-9, C20:5n-3, C22:5n-3, and C22:6n-3) positively

correlated with FEV1, along with PC (32:2) and PC (36:4), and oleoyl ethanolamide
was negatively correlated with FEV1 progression. Lower PC(32:2), PC(38:5), and
C18:3n-3, triacylglycerols higher cholesterol, and cholesterol esters were noted in
chronically infected patients [55–57].
Metabolomic analysis was also carried out in other body fluids, including spu-
tum, saliva, sweat, urine, bronchoalveolar lavage fluid, and exhaled breath analysis.
These studies were generally aimed at identifying the differences in metabolite pat-
terns to unravel the underlying pathophysiological mechanisms of CF and evaluate
the effectiveness of treatment modalities. A recent study identified the changes in
the lung microbial composition through untargeted metabolomic analysis of the
sputum and exhaled breath. Patients with homozygous Phe508del genotype usually
receive treatment with combination therapy lumacaftor and ivacaftor. Lumacaftor
targets CFTR class II mutations specifically, while Ivacaftor improves the gating
(class III) or conduction (class IV) defect in the mutant channels.
CFTR modulators improve CFTR function significantly by partly restoring the
function of the chloride channel and improving transport of epithelial fluid in the
airways. Besides improving lung function, treatment with CFTR modulators alters
the pulmonary microbiome by reducing the abundance of the bacteria, for example,
Pseudomonas aeruginosa. Metabolomic analysis by GC-TOF/MS showed changes
in concentrations of the metabolite phenyl pyruvate in the sputum. On the other
hand, the breath metabolome showed alterations in volatile organic compounds
such as 4-ethylbenzanoic acid 2-pentyl ester, suggesting a strong link between oxi-
dative stress and inflammation [58]. Untargeted metabolomic profiling of sweat
between carriers and cases showed significant alterations in purine derivatives,
organic acids, dipeptides, amino acids, and amino acid derivatives, in affected
patients, and alterations in levels of asparagine and glutamine, in asymptomatic
patients [59]. Patients with CF also present with lung disease characterized by bron-
chial inflammation due to chronic bacterial infection. The resulting inflammatory
response is predominantly dominated by neutrophils. Metabolomic studies were
used to identify and quantify the metabolites in the bronchoalveolar lavage fluid
samples from these patients. A targeted metabolomic approach identified and quan-
tified metabolites related to proteins, metabolism of purines, polyamines, and nico-
tinamide which correlated strongly with the clinical markers and neutrophil counts
[60]. In addition to these body fluids, the urine metabolomic profile was also stud-
ied. The urinary metabolome in CF although heterogeneous showed metabolic
alteration that were distinct when compared to non-CF groups. A targeted metabo-
lomic study in the urine revealed an altered methyl status and oxidative stress in
children with CF using NMR. Additionally, a subgroup of these children with pan-
creatic insufficiency showed a considerable rise of phthalate chemicals in their urine
NMR spectra in comparison to children with CF who did not have pancreatic insuf-
ficiency [47, 61, 62].
204 A. Masood et al.

5 Conclusion and Future Perspectives

Metabolomics has slowly made inroads into many aspects of patient care and has
shown its relevance in understanding disease pathophysiology, diagnosis, and thera-
peutic monitoring. It provides a bridge between knowledge accumulated from basic
science to clinical research as it considers the individual’s metabolic characteristics.
Combining the clinical (phenotype) with the metabolomic and genomics data will
aid the clinical decision-making process by providing more sensitive and specific
analyte panels for diagnostic testing. The potential of this omic approach is to fur-
ther advance in bringing an era of personalized medicine in endocrinology.

References

1. Nieman LK, Ilias I. Evaluation and treatment of Cushing’s syndrome. Am J Med.

2005;118(12):1340–6.
2. Mourtzi N, et al. Unravelling the genetic basis of primary aldosteronism. Nutrients.
2021;13(3):875.
3. Young WF Jr. Diagnosis and treatment of primary aldosteronism: practical clinical perspec-
tives. J Intern Med. 2019;285(2):126–48.
4. Monticone S, et al. GENETICS IN ENDOCRINOLOGY: the expanding genetic horizon of
primary aldosteronism. Eur J Endocrinol. 2018;178(3):R101–11.
5. Azizan EA, et al. Somatic mutations in ATP1A1 and CACNA1D underlie a common subtype
of adrenal hypertension. Nat Genet. 2013;45(9):1055–60.
6. Seidel E, Schewe J, Scholl UI. Genetic causes of primary aldosteronism. Exp Mol Med.
2019;51(11):1–12.
7. Funder JW, et al. The management of primary aldosteronism: case detection, diagnosis,
and treatment: an endocrine society clinical practice guideline. J Clin Endocrinol Metab.
2016;101(5):1889–916.
8. Storbeck KH, et al. Steroid metabolome analysis in disorders of adrenal steroid biosynthesis
and metabolism. Endocr Rev. 2019;40(6):1605–25.
9. Tokarz J, et al. Endocrinology meets metabolomics: achievements, pitfalls, and challenges.
Trends Endocrinol Metab. 2017;28(10):705–21.
10. Hundemer GL, et al. Cardiometabolic outcomes and mortality in medically treated primary
aldosteronism: a retrospective cohort study. Lancet Diabetes Endocrinol. 2018;6(1):51–9.
11. Hundemer GL, et al. Renal outcomes in medically and surgically treated primary aldosteron-
ism. Hypertension. 2018;72(3):658–66.
12. Spyroglou A, et al. Transcriptomics, epigenetics, and metabolomics of primary aldosteronism.
Cancers (Basel). 2021;13(21):5582.
13. Eisenhofer G, et al. Mass spectrometry-based adrenal and peripheral venous steroid profiling
for subtyping primary aldosteronism. Clin Chem. 2016;62(3):514–24.
14. Murakami M, et al. In situ metabolomics of aldosterone-producing adenomas. JCI Insight.
2019;4(17):e130356.
15. De Sousa K, et al. Genetic, cellular, and molecular heterogeneity in adrenals with aldosterone-­
producing adenoma. Hypertension. 2020;75(4):1034–44.
16. van der Heijden C, et al. Vasculometabolic and inflammatory effects of aldosterone in obesity.
J Clin Endocrinol Metab. 2020;105(8):2719.
17. Papathomas TG, Nose V. New and emerging biomarkers in endocrine pathology. Adv Anat
Pathol. 2019;26(3):198–209.
Metabolomics and Genetics of Rare Endocrine Disease: Adrenal, Parathyroid Glands… 205

18. Ulick S, Chu MD. Hypersecretion of a new corticosteroid, 18-hydroxycortisol in two types of
adrenocortical hypertension. Clin Exp Hypertens A. 1982;4(9–10):1771–7.
19. Nakamura Y, et al. 18-oxocortisol measurement in adrenal vein sampling as a biomarker for
subclassifying primary aldosteronism. J Clin Endocrinol Metab. 2011;96(8):E1272–8.
20. Mulatero P, et al. 18-hydroxycorticosterone, 18-hydroxycortisol, and 18-oxocortisol in the diag-
nosis of primary aldosteronism and its subtypes. J Clin Endocrinol Metab. 2012;97(3):881–9.
21. Williams TA, et al. Genotype-specific steroid profiles associated with aldosterone-producing
adenomas. Hypertension. 2016;67(1):139–45.
22. Fluck CE. MECHANISMS IN ENDOCRINOLOGY: update on pathogenesis of primary adre-
nal insufficiency: beyond steroid enzyme deficiency and autoimmune adrenal destruction. Eur
J Endocrinol. 2017;177(3):R99–R111.
23. Nowotny H, et al. Therapy options for adrenal insufficiency and recommendations for the
management of adrenal crisis. Endocrine. 2021;71(3):586–94.
24. Espiard S, et al. Improved urinary cortisol metabolome in addison disease: a prospective trial
of dual-release hydrocortisone. J Clin Endocrinol Metab. 2021;106(3):814–25.
25. Chantzichristos D, et al. Identification of human glucocorticoid response markers using inte-
grated multi-omic analysis from a randomized crossover trial. elife. 2021;10:10.
26. Sorgdrager FJH, et al. Hydrocortisone affects fatigue and physical functioning through
metabolism of tryptophan: a randomized controlled trial. J Clin Endocrinol Metab.
2018;103(9):3411–9.
27. Hescot S, et al. Prognosis of malignant pheochromocytoma and paraganglioma (MAPP-­
Prono study): a European network for the study of adrenal tumors retrospective study. J Clin
Endocrinol Metab. 2019;104(6):2367–74.
28. Darr R, et al. Pheochromocytoma—update on disease management. Ther Adv Endocrinol
Metab. 2012;3(1):11–26.
29. Erlic Z, et al. Metabolic impact of pheochromocytoma/paraganglioma: targeted metabolomics
in patients before and after tumor removal. Eur J Endocrinol. 2019;181(6):647–57.
30. Neumann HPH, Young WF Jr, Eng C. Pheochromocytoma and paraganglioma. N Engl J Med.
2019;381(6):552–65.
31. Fishbein L, et al. Comprehensive molecular characterization of pheochromocytoma and para-
ganglioma. Cancer Cell. 2017;31(2):181–93.
32. Eijkelenkamp K, et al. Clinical implications of the oncometabolite succinate in SDHx-­
mutation carriers. Clin Genet. 2020;97(1):39–53.
33. Mercado-Asis LB, et al. Pheochromocytoma: a genetic and diagnostic update. Endocr Pract.
2018;24(1):78–90.
34. Lenders JW, et al. Pheochromocytoma and paraganglioma: an endocrine society clinical prac-
tice guideline. J Clin Endocrinol Metab. 2014;99(6):1915–42.
35. Richter S, et al. Krebs cycle metabolite profiling for identification and stratification of pheo-
chromocytomas/paragangliomas due to succinate dehydrogenase deficiency. J Clin Endocrinol
Metab. 2014;99(10):3903–11.
36. Dwight T, et al. Metabolomics in the diagnosis of pheochromocytoma and paraganglioma.
Horm Metab Res. 2019;51(7):443–50.
37. Richter S, et al. Metabolome-guided genomics to identify pathogenic variants in isocitrate
dehydrogenase, fumarate hydratase, and succinate dehydrogenase genes in pheochromocy-
toma and paraganglioma. Genet Med. 2019;21(3):705–17.
38. Lendvai N, et al. Succinate-to-fumarate ratio as a new metabolic marker to detect the presence
of SDHB/D-related paraganglioma: initial experimental and ex vivo findings. Endocrinology.
2014;155(1):27–32.
39. Chae YC, et al. Landscape of the mitochondrial Hsp90 metabolome in tumours. Nat Commun.
2013;4:2139.
40. Jochmanova I, Pacak K. Pheochromocytoma: the first metabolic endocrine cancer. Clin Cancer
Res. 2016;22(20):5001–11.
206 A. Masood et al.

41. de Cubas AA, et al. DNA methylation profiling in pheochromocytoma and paraganglioma
reveals diagnostic and prognostic markers. Clin Cancer Res. 2015;21(13):3020–30.
42. Imperiale A, et al. A new specific succinate-glutamate metabolomic hallmark in SDHx-related
paragangliomas. PLoS One. 2013;8(11):e80539.
43. Li D, et al. Heterozygous mutations in TBX1 as a cause of isolated hypoparathyroidism. J Clin
Endocrinol Metab. 2018;103(11):4023–32.
44. Gordon RJ, Levine MA. Genetic disorders of parathyroid development and function.
Endocrinol Metab Clin N Am. 2018;47(4):809–23.
45. Paprocka J, et al. Neurological picture and 1H MRS in 4 children with hypoparathyroidism.
Przegl Lek. 2005;62(7):680–4.
46. Liessi N, et al. Proteomics and metabolomics for cystic fibrosis research. Int J Mol Sci.
2020;21(15):5439.
47. Muhlebach MS, Sha W. Lessons learned from metabolomics in cystic fibrosis. Mol Cell
Pediatr. 2015;2(1):9.
48. Al-Qahtani W, et al. Dried blood spot-based metabolomic profiling in adults with cystic fibro-
sis. J Proteome Res. 2020;19(6):2346–57.
49. Wetmore DR, et al. Metabolomic profiling reveals biochemical pathways and biomarkers asso-
ciated with pathogenesis in cystic fibrosis cells. J Biol Chem. 2010;285(40):30516–22.
50. Masood A, et al. Distinctive metabolic profiles between cystic fibrosis mutational subclasses
and lung function. Metabolomics. 2021;17(1):4.
51. Muhlebach MS, et al. Metabonomics reveals altered metabolites related to inflamma-
tion and energy utilization at recovery of cystic fibrosis lung exacerbation. Metabol Open.
2019;3:100010.
52. Alvarez JA, et al. Plasma metabolomics in adults with cystic fibrosis during a pulmonary
exacerbation: a pilot randomized study of high-dose vitamin D3 administration. Metabolism.
2017;70:31–41.
53. Grasemann H, et al. Decreased systemic bioavailability of L-arginine in patients with cystic
fibrosis. Respir Res. 2006;7:87.
54. Zardini Buzatto A, et al. Lipidome alterations induced by cystic fibrosis, CFTR mutation, and
lung function. J Proteome Res. 2021;20(1):549–64.
55. Guerrera IC, et al. A novel lipidomic strategy reveals plasma phospholipid signatures associ-
ated with respiratory disease severity in cystic fibrosis patients. PLoS One. 2009;4(11):e7735.
56. Ollero M, et al. Plasma lipidomics reveals potential prognostic signatures within a cohort of
cystic fibrosis patients. J Lipid Res. 2011;52(5):1011–22.
57. Grothe J, et al. Plasma phosphatidylcholine alterations in cystic fibrosis patients: impaired
metabolism and correlation with lung function and inflammation. Cell Physiol Biochem.
2015;35(4):1437–53.
58. Neerincx AH, et al. Lumacaftor/ivacaftor changes the lung microbiome and metabolome in
cystic fibrosis patients. ERJ Open Res. 2021;7(2):00731.
59. Macedo AN, et al. The sweat metabolome of screen-positive cystic fibrosis infants: revealing
mechanisms beyond impaired chloride transport. ACS Cent Sci. 2017;3(8):904–13.
60. Esther CR Jr, et al. Metabolomic evaluation of neutrophilic airway inflammation in cystic
fibrosis. Chest. 2015;148(2):507–15.
61. Innis SM, et al. Choline-related supplements improve abnormal plasma methionine-­
homocysteine metabolites and glutathione status in children with cystic fibrosis. Am J Clin
Nutr. 2007;85(3):702–8.
62. Keller BO, Davidson AG, Innis SM. Phthalate metabolites in urine of CF patients are associated
with use of enteric-coated pancreatic enzymes. Environ Toxicol Pharmacol. 2009;27(3):424–7.
Metabolomic Role in Personalized
Medicine: An Update

Minnie Jacob and Anas M. Abdel Rahman

Abstract Metabolomics is a rapidly evolving omic technology in personalized

medicine and has been extensively valued because it involves prescribing the right
medicine to the right patient. The breathtaking boost in metabolomic technology
has paved the huge potential for its application in personalized medicine. Correlating
the metabolic phenotype of individuals into subgroups that respond differently is
also becoming a reality through metabolomics. The perception of the metabotype
has emerged and played a crucial role in developing a personalized healthcare sys-
tem. Metabotypes are groups of individuals defined based on their similarities in
metabolic profiles. Metabolomics has been utilized in the therapeutic outcomes of
drugs, thereby mapping the metabolic profiles of the patients with their responses.
In contrast, the efficacy and toxicity of drugs can be predicted in the pharmaco-
metabolomic method to provide the theoretical basis for individualized medical
treatment. This chapter overviews clinical metabotyping, disease biomarker discov-
ery, and pharmacometabolomics toward personalized medicine, improving drug
efficacy. These three approaches enhance the understanding of the disease’s patho-
physiological mechanisms and the metabolic side effects of drugs on human bodies.

Keywords Personalized medicine · Metabolomics · Biomarker discovery ·

Pharmacometabolomics · Metabotyping

M. Jacob · A. M. Abdel Rahman (*)

Metabolomics Section, Department of Clinical Genomics, Center for Genome Medicine,
King Faisal Specialist Hospital and Research Center (KFSHRC), Riyadh, Saudi Arabia
e-mail: aabdelrahman46@[Link]

© The Author(s), under exclusive license to Springer Nature Singapore Pte 207
Ltd. 2023
A. M. Abdel Rahman (ed.), Clinical Metabolomics Applications in Genetic
Diseases, [Link]
208 M. Jacob and A. M. Abdel Rahman

Abbreviations

3-OHKY 3-hydroxykyenurinine
CAR Chimeric antigen receptor
CIL-LC/MS Chemical isotope labeling liquid mass spectrometry
DBS Dried blood spots
DILI Idiosyncratic drug-induced liver injury
DIPP Diabetes prediction and prevention
DOCK8 Dedicator of cytokinesis 8
EPA Environment Protection Agency
FDA Food and Drug Administration
IEM Inborn errors of metabolism
LC-MSMS Liquid chromatography-tandem mass spectrometry
MSI-CE-MS Multisegment injection-capillary electrophoresis-mass
spectrometry
NGS Next-generation sequencing
NMR Nuclear magnetic resonance
NUDT15 Nudix hydrolase 15
PC Pancreatic cancer
REIMS Rapid evaporative ionization mass spectrometry
SRM Selected reaction monitoring
SSRIs Selective serotonin reuptake inhibitors
ToF Time of flight
TPMT Thiopurine methyltransferase

1 Introduction

The swift growth in metabolomics leads to a recharged enthusiasm for cellular

metabolism and the role of small molecules in many biological processes.
Metabolomics’ advanced analysis combined with sophisticated computational tech-
niques is used for molecular characterization and relative expression [1].
Metabolome, a collection of small molecules (<15,000 Da), is the most sensitive
biomolecule as regards to physiological, biological, and environmental changes [2].
Integrating metabolomics’ relative expression with the pathway analyses helps
understand disease pathophysiology and mechanisms. Metabolite expression is
associated with the genetic blueprint, sequence variation, RNA transcription, and
protein translational processes interacting with environmental exposure and reflect-
ing genotype, especially for multifactorial diseases. Among genomics and pro-
teomics, metabolomics perhaps is closely linked to the phenotype. Hence, it provides
Metabolomic Role in Personalized Medicine: An Update 209

valuable information on healthy and pathophysiological conditions and the response

to an external stimulus, such as treatment and environmental exposures [2]. The
metabolomics’ relative expression helps to know the pathophysiology of the disease
at the molecular level, identify the biomarkers of disease prediction and diagnosis,
assess disease progression, interpret the influence of the environment and lifestyle,
and lastly assess the disease toxicity and related adverse reactions [3–5].
The research community accepted using more than one omic technique to read a
disease phenotype’s complexity functionally. Healthcare systems worldwide are
shifting from the traditional “one-size-fits-all” approach to proactive medical mod-
els. Such models are becoming predictive, preventive, personalized, and participa-
tory, known as “Proactive P4 Medicine” [6]. However, integrating multiple omic
datasets is promising in P4 personalized medicine, including developing and tailor-
ing effective therapies for individual patients [7, 8].
Considering multiple layers of biochemical reactions between genotypes and
phenotypes, different degrees of sensitivity, stability, and physiological and envi-
ronmental influences are unique for each omic molecule. Accordingly, metabolites
are the most functional biomolecules to use as biomarkers, given that the biological
connection between the phenotype and genotype has been proven. More than 95%
of clinical assays in medical laboratories are based on measuring small molecules.
Integrating multi-omics is an approach that allows small molecules through
upstream biology to eventually be used for clinical services.
Multiple international projects have invested in their population and genomic
profiling after developing next-generation sequencing (NGS), for example, the
Genome Canada, Genome England, and Saudi Human Genome Project. However,
the widespread use of this advanced technology in routine clinical diagnoses is still
encumbered by multiple obstacles, including cost and data analysis. Combining
genomic data with a more functional layer of data, such as epigenomics, transcrip-
tomics, proteomics, and metabolomics, will overcome most of the limitations asso-
ciated with the exclusive use of genomic profiling.
The terms “personalized medicine” and “precision medicine” are used inter-
changeably, and in addition to a comprehensive clinical profile (phenotype), they
refer to the long-term collection of multiple layers of data in various fields, includ-
ing genomics (genotype), transcriptomics, proteomics, metabolomics, micro-
biomics, and exosomes. This approach can help healthcare providers customize
care management, decisions, and recommendations based on a highly accurate esti-
mation of individual patients’ risks and potential outcomes. Personalized or strati-
fied medicine enables clinicians to efficiently prescribe the right medicine to the
right patient at the right time, thereby improving healthcare quality and reducing
unnecessary diagnostic testing and therapies. Precision medicine can be defined as
“treatments targeted to the individual patients based on genetic biomarker, pheno-
type or physiological characteristics of a given patient from other patients with
similar clinical presentations” [9, 10].
210 M. Jacob and A. M. Abdel Rahman

Irrespective of the diagnoses’ complexities, significant advancements are still

required for proper diagnosis, prognosis, therapeutic monitoring, and clinical man-
agement. The sensitivity and specificity of the available biomarkers for the particu-
lar disease determine the diagnostic efficiency. Typically, liquid
chromatography-tandem mass spectrometry (LC-MS/MS) has been used for many
decades for several inborn errors of metabolism (IEM) diagnosis, where population-­
based screening programs usually rely on high-quality biomarkers [11]. However,
there are no biomarkers with perfect diagnostic performance, so a combination of
multiple biomarkers (including ratios) is used for disease screening and diagnostic
purposes. For example, in the newborn screening for phenylketonuria (PKU), both
phenylalanine (Phe) levels and the ratio of phenylalanine-to-tyrosine (Phe/Tyr) are
quite remarkable, and using them together reduces the false favorable/negative
detection rates drastically [12]. Screening for medium-chain acyl Co-A dehydroge-
nase (MCAD) deficiency relies on the combined profile of elevated acylcarnitines
(C6, C8, C10, C10:1), although they have different weights. Regardless of the natu-
ral expression in different matrices, metabolites vary quantitatively based on the
tumors’ types and locations, which determines the suitable metabolic biomarker
particularly. Accordingly, personalized medicine can provide a complete and inte-
grated picture of the most related metabolites to the phenotype.
This chapter discusses the updates on metabolomics’ role in personalized medi-
cine’s main applications: disease metabotyping, biomarker discovery, and
pharmacometabolomics.

2 Disease Metabolic Profiling “Metabotyping”

The grouping of individuals based on their metabolic and phenotype characteristics

into coherent subgroups is referred to as metabolic phenotypes or metabotypes. The
distinctive metabolic profile of the physiological changes during disease progres-
sion and medical intervention enables an understanding of the connections of
metabotypes to individual factors. Modifying the metabotypes and responding indi-
vidually under a given intervention is the ultimate goal of developing the personal-
ized diagnostic profile [13, 14]. These physiological-based assays display distinctive
metabolic profiles associated with the disease’s main cause, clinical manifestation,
treatment and management, and health outcomes.
Metabolomic Role in Personalized Medicine: An Update 211

Metabolomics is a rapidly emerging tool that helps analyze these metabotypes

using the newly developed analytical strategies that pave the way to annotate several
metabolites. For example, in type I diabetes and many diseases, physiological sys-
tems play a pivotal role in the progression of a disease. The pathogenesis and etiol-
ogy require an integrative system biology approach, which requires intensive data
acquisition, refinement, and validation of mathematical models that include the con-
tributing factors [15].
The BABYDIAB study in Germany represents metabolic profiles related to age
at the onset of islet autoimmunity. Islet autoantibodies appear before developing
type I diabetes in children. Still, those developing before 2 years of age are not the
same as those developing later in childhood [16]. Metabolic profiling of these two
subgroups showed significant differences in the profiles relative to age and islet
autoantibody status. A twofold lower concentration of methionine was observed in
children who developed autoantibodies by 2 years, compared with those who devel-
oped autoantibodies in their late childhood or those found to be autoantibody nega-
tive. The critical role of methionine is highlighted in the pathways related to the
development of islet autoantibodies in early infancy [17]. This research was in
agreement with the Diabetes Prediction and Prevention (DIPP) study [18], where
most of the children progressed to islet autoimmunity before 2 years of age. In these
two studies, it would be interesting to know if the early detected metabotypes that
precede autoimmunity are specific to children who later progress to type I diabetes
or were mainly found in children who progress to one or more islet autoantibodies.
Another example was a study on overweight obesity, where obesity could be pre-
dicted in children by studying the urolithin metabotypes, which led to early bio-
markers related to obesity [19].
Metabotype profiling of patients diagnosed with cystic fibrosis (CF) using dried
blood spot (DBS) samples reported intermediate byproducts associated with
patients’ genotypes and phenotypes [20, 21]. The identified metabolic profile
included 26 significantly differentially expressed metabolites involving the amino
acids, glycolysis, mitochondrial and peroxisomal metabolism, and sorbitol path-
ways. Specifically, the osmolyte (sorbitol) was remarkably downregulated in CF
patients compared to healthy controls indicating perturbation in the sorbitol path-
way, which may be responsible for the mucoviscidosis seen in patients with CF
[22]. These findings may be supported by the clinical utility of inhaled mannitol and
hypertonic saline in patients with CF. A few examples of metabotypes in various
genetic disorders are described in Table 1.
Table 1 Metabotypes in genetic disorders
212

Genetic disorders Class Disease/gene Metabotypes Perturbed key metabolites Reference

Single gene Autosomal Huntington’s disease/HTT Huntington’s disease patients and controls Arginine, citrulline, glycine, d-serine, cholesterol esters, [23]
disease dominant diacylglycerides, triacylglycerides, phosphatidylcholines,
phosphatidylethanolamines, sphingomyelin, and NAD+
Acute intermittent Patients with asymptomatic acute intermittent Glycolysis and energy-conversion pathways, notably [24]
porphyria/HMBS porphyria (aAIP) and familial or sporadic porphyria acetate, citrate, and pyruvate
cutanea tarda
Tuberous sclerosis complex 2/ Before, during, and after 6 months of treatment 5′-Methylthioadenosine and arginine and [25]
TCS2 with sirolimus and hydroxychloroquine adenosylmethionine decarboxylase 1
Autosomal Hyper-IgE syndrome_DOCK8 DOCK8 and AD 3-Anthranilic acid, aspartic acid, hypotaurine, guanosine, [26]
recessive deficiency/DOCK8 leucyl-phenylalanine, and glycyl-phenylalanine
Cystic fibrosis/CFTR Cystic fibrosis and healthy controls Sorbitol, glutamic acid, pipecolic acid, fructose-1,6- [21, 22]
bisphosphate, hypoxanthine, and lactate
Sickle cell disease/HBB Sickle cell patients and controls 2,3-Bisphosphoglycerate, glutathione, carnitines, choline, [27]
and increased expression of metabolic precursors
involved in polyamine synthesis
Phenylketonuria/PAH Phenylketonuria patients and healthy controls LDL2 Apo-B, LDL2 particle number, LDL2 [28]
phospholipids, LDL2 cholesterol, VLDL5-free
cholesterol, VLDL5 cholesterol, VLDL5 phospholipids,
and VLDL4-free cholesterol, tyrosine, glutamine,
creatinine, and citric and glutamic acids
Mucopolysaccharidosis-type VI MPS VI patients and healthy control Dermatan sulfate, amino acids, carnitine, arginine- [29]
(MPS VI)/ARSB proline, histidine, and glutathione metabolism
Hereditary Hereditary hemochromatosis patients and 20 Serum phosphatidylcholine (PC) and [30]
hemochromatosis/HFE healthy subjects phosphatidylethanolamine
Mitochondrial Myoclonic epilepsy with ragged MERRF patients and controls Sodium, phosphate, and magnesemia levels, N-methyl [31]
disease red fibers (MERRF) nicotinamide, and hippurate
Mitochondrial encephalopathy, MELAS patients and controls Glutathione GSH, glutathione disulfide, and cysteine [32]
lactic acidosis, and stroke-like
episodes (MELAS)
M. Jacob and A. M. Abdel Rahman
Chromosomal Down syndrome Down syndrome patients and healthy controls Pyruvate, citrate, succinate, lactate, leucine, [33]
abnormalities phenylalanine, and formate
Turner syndrome Girls with turner syndrome and ge-matched girls BCAAs such as methionine, phenylalanine, lysine, [34]
with obesity tryptophan, histidine, tyrosine, alanine and ornithine,
arginine, tyrosine, glutamic acid, citrulline, and alanine
Multifactorial Type 1 diabetes Age and islet autoantibody status Triglycerides and polyunsaturated fatty acids containing [17]
diseases phospholipids
Obesity Children and adolescents Urolithin metabotypes (UM-A, UM-B, and UM-0), the [19]
top-ten contributing SNPs being rs1801253-ADRB1,
rs4343-ACE, rs8061518-­FTO, rs1130864-CRP,
rs659366-UCP2, rs6131-SELP, rs12535708-LEP,
rs1501299-­ADIPOQ, rs708272-CETP, and
rs2241766-ADIPOQ
Celiac disease Celiac disease patients and healthy controls Triacylglycerols and phosphatidylcholines [35]
Rheumatoid arthritis Rheumatoid arthritis and healthy controls Phospholipids, diol and its derivatives, arsonoacetate, [36]
oleanolic acid acetate, docosahexaenoic acid methyl
ester, linolenic acid, and eicosatrienoic acid derivatives
Cancer Pre-cachectic patients and weight-stable cancer α-Fetoprotein, carcinoembryonic antigen, carbohydrate [37]
patients antigen 15–3, carbohydrate antigen 242, carbohydrate
antigen 50, cytokeratin 19 fragments, procalcitonin, and
Metabolomic Role in Personalized Medicine: An Update

interleukin-6. Altered levels of direct bilirubin, total

bilirubin, total cholesterol, total bile acid, alkaline
phosphatase, γ-glutamyltransferase alanine
aminotransferase, aspartate aminotransferase,
α-hydroxybutyric dehydrogenase, and lactate
dehydrogenase. Biochemical biomarkers include direct
bilirubin, total bilirubin, total bile acid, alkaline
phosphatase, γ-glutamyltransferase, aspartate
aminotransferase, α-hydroxybutyric dehydrogenase, and
lactate dehydrogenase
213
214 M. Jacob and A. M. Abdel Rahman

3 Personalized Medicine and Biomarker Discovery

The eventual goal of personalized medicine is to enable clinicians to prescribe the

right medicine with maximum efficacy and minimal toxicity by predicting disease
onset among populations, thereby improving healthcare quality and reducing unnec-
essary diagnostic testing and therapies. The well-tested medicine may lead to the
right treatment at the right time but not necessarily the right one for that individual
[9]. President Barack Obama introduced a precision medicine initiative in January
2015 called “All of US” ([Link]/precisionmedicine) that enrolled
over one million Americans, and they were expected to share the data generated
over 10 years from sequencing, electronic medical records, personal reported infor-
mation, and digital health technologies.
Biomarker discovery uses a combination of technologies to capture the data,
which is then translated to select biomarkers that most reliably detect the disease.
Firstly, a biospecimen has to be analyzed using a robust method ensuring consistent
performance. The emerging biomarkers need further evaluation and validation
against a larger sample size to ensure the highest data quality, depending on the
clinical model. The validated biomarkers should be sensitive and reliable enough to
distinguish patients from healthy subjects.
A metabolomic biomarker-based device called an intelligent knife (Iknife) was
introduced recently, which is based on rapid evaporative ionization mass spectrom-
etry (REIMS) technology. Iknife can discriminate cancer from normal tissue in dif-
ferent tumor sites, including the brain, breast, ovaries, and colon [38].
Sensor technology is evolving at a fast rate. It can be well translated into clinical
biomarker discovery, wherein targeted biomarkers for a condition can be monitored
or detected by wearable sensors by the end-user and then uploaded the data for the
researcher to build a model for further prediction and response to the therapeutic
intervention. An example is the mPower study for Parkinson’s disease [36], wherein
the aspects were investigated through surveys and frequent sensor-based recordings
from participants with and without Parkinson’s disease. Similar metabolomic data
can be streamlined to look at a panel of biomarkers detectable by sensor chips capa-
ble of reporting and interpreting the real-time data. Yet another metabolomic
approach published by Blasco et al. (2017) characterized plasma levels in phenylke-
tonuria on a multiplatform, where the commonly dysregulated metabolites were
glutamine, arginine, succinate, and alpha aminobutyric acid, in addition to the
pathophysiological mechanisms like protein synthesis, energetic metabolism, and
oxidative stress, thereby confirming specific metabolic signature related to tyrosine
and phenylalanine concentrations (Table 2) [43].
Table 2 Application of metabolomics in different disease conditions
Metabolomic platform
Disease/condition Targeted Untargeted Biomarkers/major findings Matrix Platform Reference
Propionic and √ Propionyl-carnitine and gamma-butyrobetaine Plasma ToF-MS [39]
methylmalonic acidemia
Diabetes mellitus √ √ 1,5-Anhydroglucitol Saliva LC-MSMS [40]
Gestational diabetes Glycine, serine, and threonine metabolism, steroid Serum LC-MSMS [41]
mellitus hormone biosynthesis, tyrosine metabolism, QToF-MS
glycerophospholipid metabolism, and fatty acid
metabolism
Diabetes mellitus 3-Indoxy sulfate, glycerophospholipids, free fatty Blood LC-MS and [42]
acids, and bile acids NMR
Phenylketonuria √ Tyrosine and phenylalanine Urine NMR/GC-MS/ [43]
amino acid
analyzer
Cystic fibrosis Sorbitol DBS LC-MSMS [22]
Metabolomic Role in Personalized Medicine: An Update

√
√ 3,4-Dihydroxymandelate-3-O-sulfate and Serum CIL-LC/MS [20, 21]
5-aminopentanoic acid. Ophthalmic acid, MSI-­CE-­MS
nicotinamide, deoxycarnitine, and a few amino acids
√ Lactic acid and 4-hydroxy cyclohexyl carboxylic acid Exhaled UPLC-MS/MS [44]
breath
condensate
HIES √ Hypotaurine, 3-hydroxy anthranilic acid, and Serum CIL-LC/MS [26]
glycyl-phenylalanine
CIL-LC/MS chemical isotope labeling liquid mass spectrometry, NMR nuclear magnetic resonance, ToF time of flight, DOCK8 dedicator of cytokinesis 8.
MSI-CE-MS multisegment injection-­capillary electrophoresis-mass spectrometry
215
216 M. Jacob and A. M. Abdel Rahman

Recently, a clinical validation study was reported on 12 disease groups belonging

to the study of IEM groups which included disorders in the metabolism of amino
acids, fatty acids, ketones, purines, pyrimidines, carbohydrates, porphyrias, neu-
rotransmitters, vitamins, cofactors, and creatine. Remarkably, even mild metabolite
patterns are seen in mild multiple acyl-CoA dehydrogenase deficiencies (GA-II),
and maple syrup urinary disease (MSUD) could be differentiated easily in this
study [45].
Jacob et al. (2019) identified seven positively identified metabolites, distinguish-
ing DOCK8 deficiency from atopic dermatitis (AD) patients. Aspartic acid and
3-hydroxy anthranilic acid (3HAA, a tryptophan degradation pathway intermediate)
were upregulated in DOCK8 deficiency, whereas hypotaurine, leucyl-­phenylalanine,
glycyl-phenylalanine, and guanosine were downregulated. Hypotaurine, 3-hydroxy
anthranilic acid, and glycyl-phenylalanine were identified as potential biomarkers
specific to DOCK8 deficiency [26] (Table 2).
Currently, personalized medicine faces a few challenges in obtaining approval
for routine use from regulatory agencies and healthcare stakeholders before
using them routinely in clinics. Moreover, tailored or personalized therapies can
be expensive, such as autologous chimeric antigen receptor (CAR) T cell trans-
plant therapies for certain types of cancer [46] and mutation-specific medicines
to treat CF patients [47, 48]. Before the biomarkers can be utilized as novel
therapy, there is a need for rigorous, standardized protocols and pipelines for
biomarker discovery, analysis, validation, and reporting. The pipeline shows
that a metabolomic study can be performed either in a targeted or untargeted
manner, depending on the metabolomic platform. Untargeted analysis covers
and attempts to identify all detected peaks above the noise threshold using scan
mode and utilizes both annotated and unannotated peak information for statisti-
cal analysis. The targeted analysis seeks only known metabolites detected by
selected reaction monitoring (SRM). The identified metabolite biomarkers asso-
ciated with disease development and progression may pave the way for discov-
ering predictive, diagnostic, and prognostic biomarkers and monitoring
therapeutic outcomes after validation and prototype assay development. These
results demonstrate that robust metabolomics has the potential as a noninvasive
strategy and is a promising screening tool to evaluate the potential of these
metabolites in the early diagnosis of patients and provide new insight into
pathophysiological mechanisms (Fig. 1).
Metabolomic Role in Personalized Medicine: An Update 217

Fig. 1 Biomarker development pipeline workflow

4 Pharmacometabolomics

Pharmacometabolomics was introduced by Clayton et al. in 2006 [49] as an evolv-

ing research field aiming to achieve a tailored therapeutic regime and focuses on
predicting or evaluating responses to drug treatments based on their metabolic fin-
gerprints. Pharmacometabolomics can be used interchangeably with pharmacome-
tabolomics, which predicts drug effects based on a mathematical model of predose
metabolite profiles [49] and monitoring drug metabolic pathway alteration. It is
based on metabolic phenotypes or metabotypes, which are the ultimate result of
genetic, physiological, chemical, and environmental influences [50]. In
218 M. Jacob and A. M. Abdel Rahman

pharmacometabolomics, gut microbiome and environment exposures reveal infor-

mation about the metabotypes and treatment outcomes, thereby creating metabolic
signatures to find potential biomarkers that might inform treatment outcomes. It
also provides tools for mapping drug effects and revealing pathways contributing to
drug response phenotypes.

4.1 Prediction of Treatment Outcomes

New pathways can be identified for therapeutic discovery by comparing the

metabolomes of patients with different clinical parameters like slow vs. fast pro-
gressors. For example, in the case of depression, comparing metabolic signatures
of fast-­acting drugs like ketamine vs. slow-acting drugs like SSRIs (selective
serotonin reuptake inhibitors) can define more effective therapies [51].
Pharmacometabolomic studies have identified biomarkers associated with drug
metabolism responders, nonresponders, or patients with adverse drug responses.
Models can be constructed with baseline samples or treatment samples, as sample
collection during or after drug exposure is possible. Such treatment samples may
be useful for finding mechanisms associated with drugs that do not cause a posi-
tive or negative effect for several months after the initial dosing period. For
instance, increased cardiotoxicity risk is seen for many years after treating cancer
patients with chemotherapy drugs [52]. Similarly, prolonged latency is observed
in a few patients who suffer from idiosyncratic drug-induced liver injury
(DILI) [53].
Sixty-eight thousand chemicals out of a total of 81,000 parent chemicals were
registered under the Toxic Substances Control Act as per the report from the
Environment Protection Agency (EPA). Most of these entities are registered without
including the biotic and abiotic transformation products, estimated to be around a
million exposure in a lifetime [54]. Although an exogenous biomarker can be mea-
sured using specific and sensitive methods, a comprehensive approach covering
hundreds to thousands of molecules is quite challenging without using high-­
throughput techniques such as metabolomics. The endogenous metabolites, nutri-
ents and lipids, phytochemicals, pharmaceuticals, and environmental exposures
(xenobiotics) can be covered using exposome-based metabolomics, the only tool to
explore the environment and genetic integration to understand the disease risk and
development. The xenobiotics and their transformed products are contributed to the
host’s phenotype through micro- and macroscale interactions with endogenous pro-
cesses. On the other hand, genetic polymorphisms reflect the xenobiotic clearance
and bioactivity across the study population, which is worth to be evaluated and
integrating.
Metabolomic Role in Personalized Medicine: An Update 219

4.2 Integrating Drug Metabolism Pathway Alteration

Pharmaceuticals are exogenous molecules that drastically perturb endogenous

metabolism, instantly reflecting the phenotype. Pharmacometabolomics plays an
important role in our lives because the response to drugs in humans and animals is
different in all individuals depending on their predose phenotypes, which are influ-
enced by their genomes, environment, and microbiome. Pharmacometabolomic
experiments mainly establish a correlation between variations in an individual’s
predose biofluid metabolite profile and their post-drug dose responses, using differ-
ent matrices like blood, urine, or plasma.
In patients, a drug interaction is expected to return the metabolic profile to a
healthy state in case of a positive therapeutic response. However, ineffective treat-
ment in patients might shift the metabolomic profile to a toxic response, such as the
accumulation of thiopurine drugs. These toxic metabolites result from variant detec-
tion on thiopurine methyltransferase (TPMT) and nudix hydrolase 15 (NUDT15)
genes. Thus, metabolomics significantly can be involved in all the drug discovery
phases, from identifying the therapeutic target to therapeutic monitoring. The drug
exposome, including the factors of drug administration route, frequency, formula-
tion, and ADME (absorption, distribution, metabolism, and excretion), is essential
for data mining in any environmental exposome studies [55].
Numerous patients experience little or no efficacy, or sometimes even toxicity,
due to their prescribed drugs. As per the study of Lazarou et al., over two million
people in American hospitals face serious adverse drug reactions requiring hospital-
ization or leading to permanent disability [56]. Gebregiworgis et al.’s studies
focused on pancreatic cancer (PC) cells that respond or develop resistance to gem-
citabine treatment. This study compared the wild type and resistant type of PC cell
lines before and after treatment with gemcitabine, which revealed unique metabolic
changes differentiating the response or the acquired resistance to the drug. The
resistant type was combined with stable-isotope labeling experiments using
13
C6-glucose, primarily derived for nucleotide synthesis to compensate for gem-
citabine activity. In the wild type, glucose is directed toward glycolysis after treat-
ment [57].
The first pharmacometabolomic study on patients demonstrated that the predose
serum levels of lactate, alanine, and percentage body fat could predict weight gain
in a group of 21 breast cancer patients undergoing 5-fluorouracil, cyclophospha-
mide, or epirubicin-based chemotherapy [58]. Weight gain is the risk factor for
reoccurrence. Predictive tests could be very useful in these patients. The levels of
3-hydroxykyenurinine (3-OHKY) could predict the severity of clinical symptoms
during the early stages in schizophrenic patients [59]. Low predose levels of
3-OHKY were predictive of greater responsibility in the fourth week with one or
more antipsychotic drugs.
220 M. Jacob and A. M. Abdel Rahman

In a study by Clayton et al. (2009), a group of volunteers was administered acet-

aminophen and showed increased levels of urinary p-cresol sulphonate acetamino-
phen (a metabolite of acetaminophen) [60]. Before taking the drug, individuals with
high urine p-cresol sulphonate showed a low concentration of sulfonated acetamin-
ophen in the subsequent urine sample, suggesting that p-cresol sulphonate acts as a
competitive inhibitor of acetaminophen. This confirms that each individual is
unique, with certain metabolic pathways involved in the catabolism of the drug,
hence focusing on the importance of decoding individual metabotypes before the
first dose.
Pharmacometabolomics can play a pivotal role in improving personalized medi-
cine or stratified healthcare for clinicians to help choose the optimal treatment for
the subsets of patients. Interestingly, the Critical Path Opportunities Report pub-
lished by the FDA (Food and Drug Administration) considered pharmacometabolo-
mics a fundamental part of the early phases of drug development [61].
Rapidly improving analytical technologies have taken metabolisms to a great
level, but the clinical use of pharmacometabolomics is quite slow. To better under-
stand or predict individual patient responses to the drug, the combination of phar-
macogenomics and pharmacometabolomics provides genetic, drug metabolite,
systemic metabolite, and environmental information [51, 62, 63].
Pharmacometabolomics has been used to discover provisional and/or safety bio-
markers, which can help in patient selection during clinical trials. In this decade, the
need of the hour is to understand how environmental factors influence drug
responses. Pharmacometabolomics is important in providing information on xeno-
biotic, endogenous, and gut-microbe metabolites present in a patient before, during,
and after drug exposure.

5 Conclusion

In summary, identifying the metabotypes in a particular state of health will likely

benefit the patient’s health. Metabotyping helps identify metabolically similar sub-
populations or patient subgroups responding differently to nutritional or drug inter-
ventions, which can help, for example, tailor a dietary recommendation for a
particular subgroup of the obese population. Since the metabolome is complex, it
requires complementary analytical methodologies to explain the underlying bio-
logical processes, as global lifestyle and genetic components play a decisive role.
Metabolomics is expected to revolutionize the pattern of biochemical information
used to assess health and disease states, as it promises a better prognosis for the
disease. Metabolomics-based personalized medicine helps detect altered metabolic
pathways correlated with a given health condition and its progression over time.
Pharmacometabolomics has the potential to accelerate drug development by
identifying clinical development processes at an early stage, and it is the integrated
outcome of genomics, proteomics, and environmental influences on the organism
which help in providing vital information on drug responses not easily expressed by
Metabolomic Role in Personalized Medicine: An Update 221

other omics. Early planning and identification of potential metabolic signatures,

ethics consideration, education, and sample processing are vital inclusions in phar-
macometabolomic principles in clinical development, which can shorten clinical
development timelines and lower the overall developmental costs. The integrated
analysis of data obtained from different platforms (metabolomics, genomics, and
proteomics) can help characterize the treatment effects and benefit the personalized
medicine field, thereby improving treatment selection for the patients. Further
in vivo and in vitro studies are required to understand better the biological mecha-
nism underlying metabolic changes that ultimately lead to the discovery of sensitive
and specific biomarkers. Specimen stability and pre-analytical issues are crucial in
accomplishing a successful biomarker discovery.
In the near future, significant improvements in imaging technologies and predic-
tion algorithms will provide immense knowledge and differentiate between healthy
and disease conditions. Medical databases must be developed and populated with
large-scale metabotyping data for various diseases. Pharmacometabolomics plays a
crucial role in shortening the clinical development timelines, bringing down the
overall cost, and benefiting the healthcare system through overall development and
translational effectiveness. The interplay of pharmacogenomics, metabolomics, and
pharmacometabolomics is quite fascinating, as the identification of the genetic
components of the metabolome seems to be the key to the discovery of many bio-
markers. “Pharmacometabolomics-informed pharmacogenomics” is expected to
give new insight and contribute toward finding the right metabotypes leading to
personalized medicine.

References

1. German JB, Hammock BD, Watkins SM. Metabolomics: building on a century of biochemis-
try to guide human health. Metabolomics. 2005;1(1):3–9.
2. Wishart DS, Tzur D, Knox C, Eisner R, Guo AC, Young N, et al. HMDB: the human metabo-
lome database. Nucleic Acids Res. 2007;35(Database issue):D521–6.
3. Kohler I, Verhoeven A, Derks RJ, Giera M. Analytical pitfalls and challenges in clinical metab-
olomics. Bioanalysis. 2016;8(14):1509–32.
4. Jacob M, Lopata AL, Dasouki M, Abdel Rahman AM. Metabolomics toward personalized
medicine. Mass Spectrom Rev. 2017;38:221.
5. Nicholson JK, Holmes E, Kinross JM, Darzi AW, Takats Z, Lindon JC. Metabolic phenotyping
in clinical and surgical environments. Nature. 2012;491(7424):384–92.
6. Hood L, Flores M. A personal view on systems medicine and the emergence of proac-
tive P4 medicine: predictive, preventive, personalized and participatory. New Biotechnol.
2012;29(6):613–24.
7. Jacob M, Lopata AL, Dasouki M, Abdel Rahman AM. Metabolomics toward personalized
medicine. Mass Spectrom Rev. 2019;38(3):221–38.
8. Wishart DS. Metabolomics: the principles and potential applications to transplantation. Am J
Transpl. 2005;5(12):2814–20.
9. Jameson JL, Longo DL. Precision medicine--personalized, problematic, and promising. N
Engl J Med. 2015;372(23):2229–34.
222 M. Jacob and A. M. Abdel Rahman

10. Abdel Rahman AM, Lopata AL, Randell EW, Helleur RJ. Absolute quantification method and
validation of airborne snow crab allergen tropomyosin using tandem mass spectrometry. Anal
Chim Acta. 2010;681(1–2):49–55.
11. Villoria JG, Pajares S, López RM, Marin JL, Ribes A. Neonatal screening for inherited meta-
bolic diseases in 2016. Semin Pediatr Neurol. 2016;23(4):257–72.
12. Bhattacharya K, Wotton T, Wiley V. The evolution of blood-spot newborn screening. Transl
Pediatr. 2014;3(2):63–70.
13. Nicholson JK, Lindon JC, Holmes E. ‘Metabonomics’: understanding the metabolic responses
of living systems to pathophysiological stimuli via multivariate statistical analysis of biologi-
cal NMR spectroscopic data. Xenobiotica. 1999;29(11):1181–9.
14. Holmes E, Loo RL, Stamler J, Bictash M, Yap IK, Chan Q, et al. Human metabolic phenotype
diversity and its association with diet and blood pressure. Nature. 2008;453(7193):396–400.
15. Marinković T, Sysi-Aho M, Orešič M. Integrated model of metabolism and autoimmune
response in β-cell death and progression to type 1 diabetes. PLoS One. 2012;7(12):e51909.
16. Ziegler AG, Nepom GT. Prediction and pathogenesis in type 1 diabetes. Immunity.
2010;32(4):468–78.
17. Pflueger M, Seppänen-Laakso T, Suortti T, Hyötyläinen T, Achenbach P, Bonifacio E, et al.
Age- and islet autoimmunity-associated differences in amino acid and lipid metabolites in
children at risk for type 1 diabetes. Diabetes. 2011;60(11):2740–7.
18. Oresic M, Simell S, Sysi-Aho M, Näntö-Salonen K, Seppänen-Laakso T, Parikka V, et al.
Dysregulation of lipid and amino acid metabolism precedes islet autoimmunity in children
who later progress to type 1 diabetes. J Exp Med. 2008;205(13):2975–84.
19. Cortés-Martín A, Colmenarejo G, Selma MV, Espín JC. Genetic polymorphisms, mediter-
ranean diet and microbiota-associated urolithin metabotypes can predict obesity in childhood-­
adolescence. Sci Rep. 2020;10(1):7850.
20. Masood A, Jacob M, Gu X, Abdel Jabar M, Benabdelkamel H, Nizami I, et al. Distinctive meta-
bolic profiles between Cystic Fibrosis mutational subclasses and lung function. Metabolomics.
2021;17(1):4.
21. DiBattista A, McIntosh N, Lamoureux M, Al-Dirbashi OY, Chakraborty P, Britz-McKibbin
P. Metabolic signatures of cystic fibrosis identified in dried blood spots for newborn screening
without carrier identification. J Proteome Res. 2019;18(3):841–54.
22. Al-Qahtani W, Abdel Jabar M, Masood A, Jacob M, Nizami I, Dasouki M, et al. Dried
blood spot-based metabolomic profiling in adults with cystic fibrosis. J Proteome Res.
2020;19(6):2346–57.
23. McGarry A, Gaughan J, Hackmyer C, Lovett J, Khadeer M, Shaikh H, et al. Cross-sectional
analysis of plasma and CSF metabolomic markers in Huntington’s disease for participants of
varying functional disability: a pilot study. Sci Rep. 2020;10(1):20490.
24. Luck M, Schmitt C, Talbi N, Gouya L, Caradeuc C, Puy H, et al. Correction to: urinary meta-
bolic profiling of asymptomatic acute intermittent porphyria using a rule-mining-based algo-
rithm. Metabolomics. 2018;14(3):21.
25. Tang Y, El-Chemaly S, Taveira-Dasilva A, Goldberg HJ, Bagwe S, Rosas IO, et al. Alterations
in polyamine metabolism in patients with lymphangioleiomyomatosis and tuberous sclerosis
complex 2-deficient cells. Chest. 2019;156(6):1137–48.
26. Jacob M, Gu X, Luo X, Al-Mousa H, Arnaout R, Al-Saud B, et al. Metabolomics distin-
guishes DOCK8 deficiency from atopic dermatitis: towards a biomarker discovery. Meta.
2019;9(11):274.
27. Adebiyi MG, Manalo JM, Xia Y. Metabolomic and molecular insights into sickle cell disease
and innovative therapies. Blood Adv. 2019;3(8):1347–55.
28. Cannet C, Pilotto A, Rocha JC, Schäfer H, Spraul M, Berg D, et al. Lower plasma cholesterol,
LDL-cholesterol and LDL-lipoprotein subclasses in adult phenylketonuria (PKU) patients
compared to healthy controls: results of NMR metabolomics investigation. Orphanet J Rare
Dis. 2020;15(1):61.
29. Tebani A, Abily-Donval L, Schmitz-Afonso I, Piraud M, Ausseil J, Zerimech F, et al. Analysis
of mucopolysaccharidosis type VI through integrative functional metabolomics. Int J Mol Sci.
2019;20(2):446.
Metabolomic Role in Personalized Medicine: An Update 223

30. Seeßle J, Gan-Schreier H, Kirchner M, Stremmel W, Chamulitrat W, Merle U. Plasma

Lipidome, PNPLA3 polymorphism and hepatic steatosis in hereditary hemochromatosis.
BMC Gastroenterol. 2020;20(1):230.
31. Hall AM, Vilasi A, Garcia-Perez I, Lapsley M, Alston CL, Pitceathly RD, et al. The urinary
proteome and metabonome differ from normal in adults with mitochondrial disease. Kidney
Int. 2015;87(3):610–22.
32. Homma K, Toda E, Osada H, Nagai N, Era T, Tsubota K, et al. Taurine rescues mitochondria-­
related metabolic impairments in the patient-derived induced pluripotent stem cells
and epithelial-­ mesenchymal transition in the retinal pigment epithelium. Redox Biol.
2021;41:101921.
33. Antonaros F, Ghini V, Pulina F, Ramacieri G, Cicchini E, Mannini E, et al. Plasma metabolome
and cognitive skills in Down syndrome. Sci Rep. 2020;10(1):10491.
34. Bugajska J, Berska J, Wójcik M, Starzyk JB, Sztefko K. Metabolic fingerprint of turner syn-
drome. J Clin Med. 2020;9(3):664.
35. Sen P, Carlsson C, Virtanen SM, Simell S, Hyöty H, Ilonen J, et al. Persistent alterations in
plasma lipid profiles before introduction of gluten in the diet associated with progression to
celiac disease. Clin Transl Gastroenterol. 2019;10(5):1–10.
36. Carlson AK, Rawle RA, Wallace CW, Adams E, Greenwood MC, Bothner B, et al. Global
metabolomic profiling of human synovial fluid for rheumatoid arthritis biomarkers. Clin Exp
Rheumatol. 2019;37(3):393–9.
37. Yang QJ, Zhao JR, Hao J, Li B, Huo Y, Han YL, et al. Serum and urine metabolomics study
reveals a distinct diagnostic model for cancer cachexia. J Cachexia Sarcopenia Muscle.
2018;9(1):71–85.
38. Tzafetas M, Mitra A, Paraskevaidi M, Bodai Z, Kalliala I, Bowden S, et al. The intelligent
knife (iKnife) and its intraoperative diagnostic advantage for the treatment of cervical disease.
Proc Natl Acad Sci U S A. 2020;117(13):7338–46.
39. Wikoff WR, Gangoiti JA, Barshop BA, Siuzdak G. Metabolomics identifies perturbations in
human disorders of propionate metabolism. Clin Chem. 2007;53(12):2169–76.
40. Halama A, Kulinski M, Kader SA, Satheesh NJ, Abou-Samra AB, Suhre K, et al. Measurement
of 1,5-anhydroglucitol in blood and saliva: from non-targeted metabolomics to biochemical
assay. J Transl Med. 2016;14(1):140.
41. Liu T, Li J, Xu F, Wang M, Ding S, Xu H, et al. Comprehensive analysis of serum metabolites in
gestational diabetes mellitus by UPLC/Q-TOF-MS. Anal Bioanal Chem. 2016;408(4):1125–35.
42. Suhre K, Meisinger C, Döring A, Altmaier E, Belcredi P, Gieger C, et al. Metabolic footprint
of diabetes: a multiplatform metabolomics study in an epidemiological setting. PLoS One.
2010;5(11):e13953.
43. Blasco H, Veyrat-Durebex C, Bertrand M, Patin F, Labarthe F, Henique H, et al. A multiplat-
form metabolomics approach to characterize plasma levels of phenylalanine and tyrosine in
phenylketonuria. JIMD reports. 2017;32:69–79.
44. Zang X, Monge ME, Gaul DA, McCarty NA, Stecenko A, Fernández FM. Early detection of
cystic fibrosis acute pulmonary exacerbations by exhaled breath condensate metabolomics. J
Proteome Res. 2020;19(1):144–52.
45. Steinbusch LKM, Wang P, Waterval H, Stassen F, Coene KLM, Engelke UFH, et al. Targeted
urine metabolomics with a graphical reporting tool for rapid diagnosis of inborn errors of
metabolism. J Inherit Metab Dis. 2021;44(5):1113–23.
46. Fry TJ, Shah NN, Orentas RJ, Stetler-Stevenson M, Yuan CM, Ramakrishna S, et al. CD22-­
targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted
CAR immunotherapy. Nat Med. 2018;24(1):20–8.
47. Davis PB, Yasothan U, Kirkpatrick P. Ivacaftor. Nat Rev Drug Discov. 2012;11(5):349–50.
48. Gulland A. Cystic fibrosis drug is not cost effective, says NICE. BMJ. 2016;353:i3409.
49. Clayton TA, Lindon JC, Cloarec O, Antti H, Charuel C, Hanton G, et al. Pharmaco-­
metabonomic phenotyping and personalized drug treatment. Nature. 2006;440(7087):1073–7.
50. Holmes E, Wilson ID, Nicholson JK. Metabolic phenotyping in health and disease. Cell.
2008;134(5):714–7.
224 M. Jacob and A. M. Abdel Rahman

51. Kaddurah-Daouk R, Weinshilboum R. Metabolomic signatures for drug response phenotypes:

pharmacometabolomics enables precision medicine. Clin Pharmacol Ther. 2015;98(1):71–5.
52. Jain D, Ahmad T, Cairo M, Aronow W. Cardiotoxicity of cancer chemotherapy: identification,
prevention and treatment. Ann Transl Med. 2017;5(17):348.
53. Mosedale M, Watkins PB. Drug-induced liver injury: advances in mechanistic understanding
that will inform risk management. Clin Pharmacol Ther. 2017;101(4):469–80.
54. Niedzwiecki MM, Walker DI, Vermeulen R, Chadeau-Hyam M, Jones DP, Miller GW. The
exposome: molecules to populations. Annu Rev Pharmacol Toxicol. 2019;59:107–27.
55. Lipinski CA. Drug-like properties and the causes of poor solubility and poor permeability. J
Pharmacol Toxicol Methods. 2000;44(1):235–49.
56. Lazarou J, Pomeranz BH, Corey PN. Incidence of adverse drug reactions in hospitalized
patients: a meta-analysis of prospective studies. JAMA. 1998;279(15):1200–5.
57. Gebregiworgis T, Bhinderwala F, Purohit V, Chaika NV, Singh PK, Powers R. Insights
into gemcitabine resistance and the potential for therapeutic monitoring. Metabolomics.
2018;14(12):156.
58. Keun HC, Sidhu J, Pchejetski D, Lewis JS, Marconell H, Patterson M, et al. Serum molecu-
lar signatures of weight change during early breast cancer chemotherapy. Clin Cancer Res.
2009;15(21):6716–23.
59. Condray R, Dougherty GG Jr, Keshavan MS, Reddy RD, Haas GL, Montrose DM, et al.
3-Hydroxykynurenine and clinical symptoms in first-episode neuroleptic-naive patients with
schizophrenia. Int J Neuropsychopharmacol. 2011;14(6):756–67.
60. Clayton TA, Baker D, Lindon JC, Everett JR, Nicholson JK. Pharmacometabonomic identifi-
cation of a significant host-microbiome metabolic interaction affecting human drug metabo-
lism. Proc Natl Acad Sci U S A. 2009;106(34):14728–33.
61. Woodcock J, Woosley R. The FDA critical path initiative and its influence on new drug devel-
opment. Annu Rev Med. 2008;59:1–12.
62. Abo R, Hebbring S, Ji Y, Zhu H, Zeng ZB, Batzler A, et al. Merging pharmacometabolomics
with pharmacogenomics using ‘1000 Genomes’ single-nucleotide polymorphism imputation:
selective serotonin reuptake inhibitor response pharmacogenomics. Pharmacogenet Genomics.
2012;22(4):247–53.
63. Oh J, Yi S, Gu N, Shin D, Yu KS, Yoon SH, et al. Utility of integrated analysis of pharma-
cogenomics and pharmacometabolomics in early phase clinical trial: a case study of a new
molecular entity. Genomics Informa. 2018;16(3):52–8.
Lipidomic Profiling in Clinical Practice
Using LC-MS

Núria Amigó Grau and Pablo Ortiz Betes

Abstract The advances in lipidomic profiling techniques in the last two decades
have significantly improved our understanding of the biological processes involved
in health and disease. Currently, many lipidomic profiling applications are moving
from research laboratories to clinical application, as lipidomics has the potential to
be implemented into the clinical routine, complementing traditional clinical factors
to improve the diagnosis, to stratify risk post-diagnosis, and to support treatment
monitoring of both pharmaceutical and lifestyle interventions.
In this chapter, we describe how clinical environment is opening the door to the
implementation of lipidomic-based applications for predicting and monitoring a
wide range of metabolic diseases. We present a use case based on LC-MS lipido-
mics already operating in hospital clinical workflows for Non-Alcoholic Fatty Liver
Disease assessment, exemplifying the potential of precision medicine approaches
for personalized diagnostics.

Keywords Lipidomics · LC-MS lipidomics · NMR metabolomics · Advanced

lipoprotein profiling · Nonalcoholic fatty liver disease · Cardiovascular risk
assessment · Cardiovascular diseases · Precision medicine

N. Amigó Grau (*)

Biosfer Teslab, Av. Universitat 1, Reus, Spain
Department of Basic Medical Sciences, Universitat Rovira i Virgili (URV), Institut
d’Investigació Sanitària Pere Virgili (IISPV), Av. Universitat 1, Reus, Spain
Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas
Asociadas (CIBERDEM), Instituto de Salud Carlos III (ISCIII), Madrid, Spain
e-mail: namigo@[Link]
P. Ortiz Betes
Biosfer Teslab, Av. Universitat 1, Reus, Spain

© The Author(s), under exclusive license to Springer Nature Singapore Pte 225
Ltd. 2023
A. M. Abdel Rahman (ed.), Clinical Metabolomics Applications in Genetic
Diseases, [Link]
226 N. Amigó Grau and P. Ortiz Betes

Abbreviations

ApoB Apoprotein B
AUC Area under the curve
AUROC Area under the receiver operating characteristic curve
BMI Body mass index
CIBERDEM Centro de Investigación Biomédicas en Red de Diabe
tes y Enfermedades Metabólicas
CLIA Clinical Laboratory Improvement Amendments
CMD Cardiometabolic diseases
CVD Cardiovascular disease
DAG Diacylglycerol
DG Diglycerides
EAS European Atherosclerosis Society
ESC European Society of Cardiology
FFAs Free fatty acids
HCC Hepatocarcinoma
HDL High-density lipoprotein
IDL Intermediate-density lipoproteins
IISPV Institut d’Investigació Sanitària Pere Virgili
IMT Intima–media thickness
IVD In vitro diagnostics
LC-MS Liquid chromatography-mass spectrometry
LDL Low-density lipoprotein
LITMUS Liver Investigation Testing Marker Utility in Steatohepatitis
LMWM Low-molecular-weight metabolites
NAFLD Nonalcoholic fatty liver disease
NASH Nonalcoholic steatohepatitis
NMR Nuclear magnetic resonance
NPV Negative predictive value
OWL One-way liver
PC Phosphatidylcholine
PPV Positive predictive value
PUFAs Polyunsaturated fatty acids
RCT Reverse cholesterol transport
ROC Receiver operating characteristics
VLDL Very-low-density lipoprotein
TAG Triacylglycerol
TG Triglycerides
US FDA US Food and Drug Administration
WHO World Health Organization
Lipidomic Profiling in Clinical Practice Using LC-MS 227

1 Introduction

Metabolomics is an emerging approach in the systems biology field especially in

clinical trials and is considered the closest biology system to the phenotypes.
Metabolites are the products of complex molecular pathways (including genom-
ics, transcriptomics, and proteomics). Therefore, metabolomic analysis is a promis-
ing strategy for identifying disease-associated biomarkers: metabolites, small
molecules, and end products produced as a result of interactions between genes and
environmental factors.
There are two possible approaches for discovering new biomarkers. The first is a
classic approach, whereby a biomarker is sought that explains the pathophysiology
of metabolic diseases, such as glucose quantification to determine whether a person
has diabetes or the quantification of LDL-C for assessing someone’s risk of cardio-
vascular diseases (CVD). The second approach involves searching for metabolic
patterns that are characteristic of the physiology of a metabolic disease [1]. In this
second approach, high-performance techniques such as liquid chromatography-­
mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) are used to
carry out metabolomic studies without prior knowledge of the metabolites’ involve-
ment or role in the disease mechanism or physiology [2], since the metabolites are
the final product of the interactions between gene and protein expression and envi-
ronmental exposure and consequently of the biochemical activity that characterize
part of the patient’s metabolism.
The metabolomic approach compares the metabolomic profiles of fluids or tis-
sues of a patient with those of a healthy subject to identify which metabolites are
expressed differently [3].
Metabolomics generally includes all kinds of molecules constituting a biological
matrix, and lipidomics is the study of specific lipids. In particular, lipidomics
involves the study of the structure, function, and metabolism of lipids in living
organisms [4]. Lipidomics is a broad field that encompasses analysis of the com-
plete set of lipids present in a biological sample and the pathways and enzymes
involved in their synthesis, degradation, and modification.
Various analytical techniques are used in lipidomics, such as mass spectrometry,
NMR spectroscopy, and chromatography, to identify and quantify the different lipid
species present in a sample. These techniques allow researchers to obtain a detailed
picture of the lipid content of a sample and to study the changes in lipid levels in
response to different conditions or treatments [5].
Lipidomics has a wide range of applications in various fields, including medi-
cine, nutrition, and environmental science. It is used to study the role of lipids in
health and disease, and the mechanisms through which lipids affect cellular pro-
cesses and signaling pathways, and to identify potential therapeutic targets for treat-
ing lipid-related disorders [6].
228 N. Amigó Grau and P. Ortiz Betes

Compared to other omic techniques, such as proteome or genome analysis, the

lipidome is much more complex because of the enormous structural diversity com-
prising both linear and coupled macromolecules [5]. This diversity increases the
number of lipid molecules and the complexity of their carriers, lipoproteins. For this
reason, analyzing the lipidome and lipoproteins is very challenging from a technical
standpoint. In recent years, LC-MS and NMR have proved to be important analyti-
cal tools for metabolomic studies in biological fluids. Unlike other techniques,
LC-MS and NMR can quantify many molecular entities simultaneously and effec-
tively [7, 8]. These techniques are widely used in both routine clinical laboratories
and large epidemiological studies.
This chapter discusses lipidomic platforms for clinical applications using
LC-MS. It explores their benefits, challenges, difficulties, and future opportunities.
Application of these technologies in the clinical environment has opened the door to
their future implementation for predicting and monitoring numerous diseases. The
technologies analyzed in the present chapter are already being applied in clinics,
exemplifying the potential of precision medicine approaches for personalized
diagnostics.

2 Lipids and Lipoproteins: A Biochemical Approach

2.1 General Concepts

Lipids are a diverse group of organic molecules that are important for many biologi-
cal functions and essential to life. They include fats, oils, waxes, and other water-­
insoluble compounds [9].
Lipids are synthesized by living cells through a process called lipid biosynthesis.
This process involves condensing fatty acids with glycerol or other alcohols to form
triglycerides, the primary component of fats. Alternatively, lipids can also be syn-
thesized by modifying existing lipids, such as by adding a phosphate group to form
a phospholipid or adding a carbohydrate group to form a glycolipid.
The structure of lipids is characterized by their hydrophobic nature, which arises
from the presence of long, nonpolar hydrocarbon chains. This nonpolarity allows
lipids to interact with other molecules and form aggregates, such as micelles or lipid
bilayers, which can serve as structural components of cell membranes [10].
In general, the structure of lipids in plasma is that of a submicroscopic oil droplet
containing an outer layer of phospholipids, unesterified cholesterol, and proteins,
with a core of neutral lipids, predominately cholesterol esters and triglycerides,
named lipoproteins [11]. The lipoprotein classes differ in their lipid composition
(being either cholesterol [C]-rich or triglyceride [TG]-rich), their protein composi-
tion (apoprotein), and their density (which is proportional to their protein/lipid
ratio). Based on protein composition, there are two types of lipoproteins, those that
Lipidomic Profiling in Clinical Practice Using LC-MS 229

contain ApoB and those that contain ApoA. Based on lipid composition, there are
also two types of lipoproteins, those that are C-rich and those that are TG-rich.
Lipids have many functions in living organisms, from energy reserve to insula-
tion, as well as being a structural component of every cell and tissue. They also play
important roles in cellular signaling and regulation. This structural diversity is mir-
rored by the enormous variation in their physiological function. The abundance of
individual lipid molecular species in plasma may indicate the variety of specific
human diseases.
Around 4500 metabolites have been detected/identified in the human serum
metabolome to date: half are phospholipids and over a thousand are glycerolipids
(triglycerides TG, diglycerides DG, and monoacylglycerols) [12]. In other words,
lipids make up approximately three-quarters of the known human serum metabolome.
To date, thousands of distinct molecular species have been quantified, covering
the six main mammalian lipid categories: fatty acyls, glycerolipids, glycerophos-
pholipids, sphingolipids, sterol lipids, and prenol lipids. The number of metabolites
keeps increasing, as each of these types of lipid can exist in multiple forms [13]. In
addition, lipids can undergo chemical modifications, such as the addition of a phos-
phate group or a carbohydrate group, which can result in the creation of new lipid
species. This structural diversity is particularly relevant within the sphingolipid and
glycerophospholipid categories, principally determined by variations in fatty acid
content and head groups.

2.2 Lipids Mirror Present and Future Metabolic Health: Two

Sides of the Same Problem

Abnormal plasma lipids and lipoproteins are important risk factors for metabolic
and cardiovascular diseases. Metabolic diseases are increasing exponentially world-
wide, and their complications, in cardiovascular and liver diseases, are the leading
cause of mortality worldwide [14]. They have a common characteristic: their etiol-
ogy is linked to excess fat and associated inflammation. Nonalcoholic fatty liver
disease (NAFLD), associated with obesity and excess fat in the liver, is increasingly
common around the world, especially in Western countries, and affects approxi-
mately a quarter of the population in countries such as the USA [15]. Symmetrically,
arterial health is compromised by excess fat, which accelerates the progression of
arteriosclerosis, the underlying cause of myocardial infraction or stroke, the leading
cause of death in all developed countries [14]. Both liver disease and atherosclerosis
can develop and progress more rapidly when accompanied by several well-known
risk factors and comorbidities, such as obesity and dyslipidemia.
Lipids circulate in the blood in the form of lipoprotein particles, including chy-
lomicrons, very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL),
and high-density lipoproteins (HDL). Some parameters, such as cholesterol and
230 N. Amigó Grau and P. Ortiz Betes

triglyceride levels, are assessed routinely, and physicians regularly prescribe lipid-­
lowering drugs to patients with dyslipidemia [16].
The duration of exposure to high lipid levels is also a crucial risk factor in car-
diometabolic health, especially for metabolic disorders that start early in childhood.
Evidence indicates that atherosclerosis begins in childhood with the accumulation
of lipids in the intima of arteries to form fatty streaks [17]. Nearly all children have
at least some degree of aortic fatty streaks by 3 years of age [18]. These fatty streaks
increase after 8 years of age, with atherosclerotic plaques being found in the coro-
nary arteries during adolescence [19]. Clusters of risk factors in childhood predict
the presence of risk factors in adults [20].
Carotid ultrasonography screening for subclinical arteriosclerosis has been vali-
dated in observational, longitudinal, and randomized clinical studies. Those results
were significantly correlated with intravascular coronary ultrasonography, coronary
angiography, and pathologic findings of arterial lesions in healthy and CVD patients,
and longitudinal studies have demonstrated that increased lipid alterations and
intima–media thickness in young adults are associated with cardiovascular risk fac-
tors in childhood [21, 22].
The contribution of LDL-associated cholesterol to the development of CVD has
been well described. While LDL particle accumulation increases the atherosclerotic
process, HDL helps remove excess cholesterol by reverse cholesterol transport.
Low levels of HDL are associated with high cardiovascular risk [23].
Conversely, in circulating chylomicrons and VLDL, triglycerides undergo hydro-
lysis to generate a pool of free fatty acids (FFAs), which are used as an energy
source in tissues. Excess FFAs are stored in adipocytes, favoring the expansion and
dysfunction of adipose tissue, increasing insulin resistance and diabetes. This pro-
cess is associated with abnormally high plasma levels of saturated FFAs, allowing
increasing their uptake into hepatocytes to exceed metabolic requirements, which
leads to hepatic steatosis and inflammation [24].

3 Clinical Relevance of the Lipidome

Lipids participate in many biological processes, so it is not surprising that defects

in lipoprotein homeostasis are the direct cause of many diseases and therefore be
considered markers of the disease. Detailed knowledge of the composition and
concentration of plasma lipidome is expected to expand our diagnostic capabili-
ties and improve the pharmacological evaluation and efficacy of prescribed ther-
apy. A deep analysis of the lipidome might reflex altered synthesis of specific lipid
species or identify abnormal underlying pathological lipoprotein patterns.
Knowledge about the role of lipids and lipoproteins in disease mechanisms is
constantly growing as more information on lipids and lipoprotein physiology
becomes clear.
Lipidomic Profiling in Clinical Practice Using LC-MS 231

3.1 Lipids and NAFLD: Introduction Through LC-MS

Nonalcoholic fatty liver disease (NAFLD) includes a wide spectrum of disorders

ranging from benign lipid accumulation in the liver (steatosis) to a more compli-
cated clinical stage when fat induces hepatic inflammation and hepatocyte necrosis
producing a new stage called nonalcoholic steatohepatitis (NASH) also named as
steatohepatitis and, eventually, when fibrosis progression is added to fat and inflam-
mation (“NASH at risk”) [25]. The final stage of disease progression is cirrhosis
and/or hepatocarcinoma (HCC) [26]. Interestingly, another potential evolution from
NASH directly to HCC without any significant fibrosis contribution has also been
described [27].
The exact cause of NAFLD is not known. Many factors contribute to this condi-
tion, such as excessive food intake, obesity, type 2 diabetes, and dyslipidemia, but
not all patients develop NAFLD/NASH, and not all patients with NAFLD/nonalco-
holic steatohepatitis (NASH) suffer from these conditions [28].
The pathophysiology of NAFLD is quite complex, and the progression from
hepatic steatosis to the different stages of this condition is not completely under-
stood. However, lipid metabolic changes, including the production of lipotoxic spe-
cies in the liver, could be responsible for disease progression in NAFLD [27].
LC-MS allows the tracking of more than 400 different lipid species in the liver
and serum. Changes in many of these metabolites were followed in several trans-
verse cohort studies [29–35], giving robust data about the lipidomic signature of the
different clinical stages of NAFLD, from liver steatosis to steatohepatitis and
advanced fibrosis. In the past 20 years, research studies in serum lipidomics have
successfully identified biomarkers to differentiate the stages of NAFLD [29].
The first attempt to diagnose fatty liver through lipidomics in humans was
reported by Puri et al. in 2007. They analyzed the hepatic lipid profiles of subjects
with normal liver histology, steatosis, and NASH [30]. The study showed that there
was no difference in the FFAs between the three groups. The triacylglycerol (TAG)
and diacylglycerol (DAG) levels were increased, and the phosphatidylcholine (PC)
content was decreased in NAFLD, which suggests that PC hydrolysis may contrib-
ute to DAG and TAG accumulation in fatty liver and thereby increased lipogenesis
in NAFLD.
These findings were confirmed by Kotronen and García-Cañaveras through
semi-quantification of the full range of lipids using LC-MS [31, 32]. Total lysophos-
pholipids, DAG, and TAG were elevated in NAFLD. The stearic-to-oleic acid ratio
was decreased in NAFLD, indicative of increased TAG biosynthesis, and NAFLD is
also characterized by an increase in DAG and a reduction in polyunsaturated fatty
acids (PUFAs).
An additional pilot study (42 biopsy samples) and a pivotal validation study (467
biopsy samples) were published by Barr et al. in 2010 and 2012, respectively,
whereby LC-MS was used to identify lipidomic signatures associated with NAFLD
232 N. Amigó Grau and P. Ortiz Betes

progression [33]. A variety of lipid biomarkers were identified that correlated with
NAFLD progression. In the second study, which included 467 biopsy samples with
normal liver (n = 90) or diagnosed with NAFLD (steatosis, n = 246; NASH, n = 131),
approximately 540 circulating metabolites were analyzed, including amino acids,
FFA, DAG, TG, PC, PE, PI, ceramides, SM, cholesteryl esters, and bile acids.
Analysis of the lipidomic data allowed the definition of a robust BMI-dependent
lipidomic signature that reliably and accurately differentiated liver steatosis from
NASH. The area under the curve (AUC) was 0.84 for lean/pre-obese, 0.85 for obese,
and 0.87 for morbidly obese patients. More recently, using this same cohort of
patients, a set of 25 BMI-dependent lipid profiles was established that could differ-
entiate between steatosis and NASH with AUC values of 0.99, 0.90, and 0.91 for
lean/pre-obese, obese, and morbidly obese patients, respectively [34].
Distinguishing between simple steatosis and NASH is relevant for differentiating
between a generally benign condition and one with increased morbidity and mortal-
ity [35]. Therefore, there is an unmet need for noninvasive biomarkers that are
robust, reliable, and cost-effective for patients with NAFLD.
Mayo et al. reanalyzed the lipidomic data from Barr’s pivotal trial and a new
cohort of 192 biopsy samples from NAFLD patients, culminating in the validation
of a BMI-dependent algorithm with 20 TGs. The area under the receiver operating
characteristic curve (AUROC) versus biopsy for the discrimination between NASH
and NAFLD was 0.95 with sensitivity, specificity, positive predictive value, and
negative predictive value of 0.83, 0.94, 0.89, and 0.90, respectively [34].
Bril et al. evaluated the lipidomic signature in 220 patients with type 2 diabetes
mellitus to differentiate between steatosis and NASH. They found an AUROC of
0.79 (95% CI 0.68–0.90) in patients with adequate glycemic control. However, this
differentiation was quite poor in patients with high insulin resistance or poor glyce-
mic control [36].
Validation of the lipidomic signatures in such important cohorts indicates that
lipidomic markers play a significant role in elucidating the phenotypic stages of
NAFLD. Therefore, LC-MS is not just for basic research: it is a very efficient tool
for providing clinically relevant information for patient management. The contribu-
tion of lipidomics to understand NAFLD complexity was well evaluated in a review
by Masoodi et al. [29].

4 Applications of LC-MS-Based Lipidomics

in Clinical Practice

4.1 The OWLiver® Test

The one-way liver (OWLiver®) panel is an in vitro diagnostic test based on the
serum lipidomic signature of patients with NAFLD. This panel can stratify this
condition into four clinically relevant stages: normal liver, steatosis, NASH, and
Lipidomic Profiling in Clinical Practice Using LC-MS 233

“NASH at risk” (NASH + F2 or more). The panel was developed by OWL metabo-
lomics in Spain as a CE-certified IVD for over 8 years.
The company collects serum samples and biopsies from different cohorts of
NAFLD patients in Spain, the Czech Republic, Chile, Mexico, Israel, and the USA
(Florida and New York). Together with the biopsy readings and clinical and analyti-
cal information, they have built up an impressive database consisting of more than
1200 NAFLD patients.
Furthermore, the panel was selected for the two major international consortia:
Noninvasive Biomarkers of Metabolic Liver Diseases (NIMBLE) and Liver
Investigation: Testing Marker Utility in Steatohepatitis (LITMUS) (US and EU con-
sortia created for the validation of noninvasive diagnosis of NAFLD spectrum) [37].
Currently, the OWLiver panel is available to most Spanish hospitals, and its use
is fully reimbursed for the Health Service of the Basque Country. As the panel
obtained self-certificate CE marked, it is available for most EU countries. CLIA test
is expected to be launched in the USA in late 2023.
The panel applies three different algorithms to the lipidomic signatures obtained
after the injection of a small volume (10 μl) into an LC-MS. All three algorithms are
BMI-dependent, while two of them are ALT- and AST-dependent. The final output
of the panel is the classification of the patient into one of the four mentioned diag-
nostic categories.
The first algorithm, based on BMI, ALT, AST, and 12 complex lipids, identifies
patients with “NASH at risk.” Accuracy in comparison with biopsy results in an
AUC close to 0.8, which compares very well with the other noninvasive alternatives
in development. The selection of this group of patients is clinically very relevant
since they would benefit greatly from early treatment.
The second algorithm, based on BMI, ALT, AST, and 16 complex lipids, identi-
fies patients with NASH, which correlates with an AUC versus biopsy of close to
0.8. This classification is also clinically important for patients’ prognosis since the
morbidity–mortality for this category is higher than for patients with normal liver
and simple steatosis. Only NIS 4 has shown comparable accuracy in diagnosing
NASH [38]. To date, no other noninvasive procedures with the capability to recog-
nize the inflammatory component of this condition have been reported.
Based on BMI and 11 triglycerides, the third algorithm differentiates NAFLD
from normal liver with an AUC close to 0.9 versus biopsy [34]. The accuracy is very
close to that observed with Fibroscan. Although this category is not very clinically
relevant since a simple echography can provide the same information, it could be
useful for epidemiological or population studies because it only requires a blood test.
The first results from NIMBLE were published in 2022 after blinded evaluation
of more than 1000 samples from NAFLD patients and exhibited the same accuracy
as observed by Mayo et al. [34].
The OWLiver panel, combined with the Fibrosis-4 score (FIB4), is widely in
hepatology units used to select “NASH at-risk” patients. This combination reduces
false negatives from FIB 4, identifying most of the patients suffering from NASH
and fibrosis stage 2 or above [34].
234 N. Amigó Grau and P. Ortiz Betes

Endocrinology units are also very interested in identifying “NASH at-risk” and
NASH patients because the technology allows them to carefully monitor overweight
type 2 diabetes patients during their potential long-term progression.
Finally, knowing whether these metabolite changes are reversed when patients
improve or even return to normality is vital. Some longitudinal studies are currently
being carried out by two major consortia, NIMBLE and LITMUS, to confirm if the
original lipidomic signature is recovered when patients improve.
In conclusion, the accuracy shown by the different lipidomic signatures in the
OWLiver panel was very competitive compared with biopsy, and the panel is com-
plementary to the other noninvasive tools developed in parallel for the same pur-
pose, such as NIS 4 (based on microRNA), Fibroscan, and MRI [38].

4.2 Other Diseases

LC-MS lipidomics has enormous potential for improving the diagnosis and treat-
ment/understanding of complex disease, as was already shown with NAFLD. Several
other conditions have been explored using LC-MS lipidomics in the last decade
with promising results. Selected studies are presented in Table 1 to show the hetero-
geneity of the conditions and the potential of the diagnostic tools under development.

Table 1 Potential clinical applications of LC-MS

Biomarkers for disease diagnosis
Idiopathic Seijo et al. [39] showed that a subset of five metabolites differentiates patients
noncirrhotic with idiopathic noncirrhotic portal hypertension from patients with liver
portal cirrhosis and healthy volunteers (AUROC = 0.8871 [0.838–0.924]). Using
hypertension high and low cut-off values, the model can diagnose or exclude idiopathic
non-cirrhosis portal hypertension, respectively
Multiple Villoslada et al. [40] identified metabolomic signatures for classifying patients
sclerosis versus controls with high accuracy, as well as for classifying patients with a
medium to high disability (EDSS 3.0). Among them, sphingomyelin and
lysophosphatidylethanolamine were the metabolites that showed a more robust
pattern in the time series analysis for discriminating between patients and
controls
Alzheimer’s Olarazan et al. [41] developed a panel with seven metabolites that could
disease differentiate Alzheimer’s disease patients from those with amnestic mild
cognitive impairment. The final panel consisted of seven metabolites: Three
amino acids (glutamic acid, alanine, and aspartic acid), one non-esterified fatty
acid (22:6n-3, DHA), one bile acid (deoxycholic acid), one
phosphatidylethanolamine [PE (36:4)], and one sphingomyelin [SM (39:1)]
Colorectal Cubiella et al. [42] evaluated the fecal levels of 105 metabolites and found 18
cancer that were significantly altered in patients with advanced neoplasia compared to
controls. The combinations of seven metabolites, ChoE (18:1), ChoE (18:2),
ChoE (20:4), PE (16:0/18:1), SM (d18:1/23:0), SM (42:3), and TG (54:1),
discriminated advanced neoplasia patients from healthy controls. These seven
metabolites were employed to construct a predictive model that provides an
AUC value of 0.821 for cancer diagnosis
Lipidomic Profiling in Clinical Practice Using LC-MS 235

5 Summary and Future Outlook

The advances in lipidomic profiling techniques in the last decades have significantly
improved our understanding of the biological processes involved in health and dis-
ease. Currently, many lipidomic profiling applications are moving from research
laboratories to clinical application. However, lipidomics has the potential to widely
assist in the routine diagnosis of disease, to stratify risk post-diagnosis, and to moni-
tor the efficacy of both pharmaceutical and lifestyle interventions.
The global objective is to implement these precise and personalized technologies
in an impactful way, to scale and make them accessible and effective to help health-
care providers around the world to be time and cost-effective. After technical devel-
opment and validation, clinical lipidomic platforms will have to be approved by the
relevant regulatory body in the country of use, as has already been done for the first
platforms in the EU and USA.
Clinical and Implementation Risks Lipidomic applications are based on highly
innovative technologies for bio-screening purposes. Although metabolomics has
repeatedly been demonstrated to be cost-effective for CVD management, the price
of the technology surpasses that of the gold standard, which is a significant barrier
to implementation in stressed healthcare systems.
Moreover, lipidomic applications are primarily focused on cardiometabolic dis-
eases (CMD). Lifestyle interventions can address obesity and concomitant meta-
bolic alterations. Innovative solutions may be addressed after adherence to
therapeutic improvement and lifestyle interventions, and complementary technolo-
gies can appear before the present solutions are broadly applied. However, personal-
ized systems could still be applied to create patient awareness about personalized
health status, favoring the adoption of a healthy lifestyle routine.
Regulatory Difficulties Although regulatory bodies currently include novel tech-
nologies, large amounts of time and resources are required for approval by a notifi-
cation body. Particularly for US FDA certification, if there is no precedent, novel
technologies may experience serious delays before clinical introduction, even when
promoted by previous IVD-CE marketing equivalent technology, threatening the
economic sustainability of the whole technological development. Based on solu-
tions presented in this chapter, lipidomics has already crossed the regulatory barri-
ers under the risk classification ISO 13485:2016, requiring technical file preparation,
CE declaration, and registry with the European Competent Authority. For future
lipidomic applications, the previous regulatory strategy might be useful. The techni-
cal documentation must provide evidence of conformance with the essential require-
ments of 98/79/EC and the imminent regulation (EU) 2017/746 on in vitro medical
devices.
Despite the difficulties, the use of lipidomic technologies as diagnostic, prognos-
tic, and evaluation tools is expected to expand enormously as a result of the develop-
ments in modern medicine.
236 N. Amigó Grau and P. Ortiz Betes

Lipidomic-based applications are expected to increase awareness regarding the

need for diagnostic and prognostic technologies for other diseases. Lipidomics will
help with the clinical assessment of different pathologies, spread the importance of
metabolomic-based approaches, and ensure uptake of the already developed appli-
cations by clinicians, hospital decision-makers, and regulators.
The introduction of advanced molecular profiling is aligned with the global strat-
egy to incorporate personalized screening tools to define better monitoring and
therapeutic strategies that will improve healthcare systems and empower clinicians
with more detailed information, allowing an early therapeutic response and thus
reducing the development of life-threatening symptoms.

References

1. Monteiro MS, Carvalho M, Bastos ML, Guedes de Pinho P. Metabolomics analysis for bio-
marker discovery: advances and challenges. Curr Med Chem. 2013;20:257–71. [Link]
org/10.2174/092986713804806621.
2. Rhee EP, Gerszten RE. Metabolomics and cardiovascular biomarker discovery. Clin Chem.
2012;58:139–47. [Link]
3. Zhang A, Sun H, Yan G, Wang P, Wang X. Metabolomics for biomarker discovery: moving to
the clinic. Biomed Res Int. 2015;2015:354671. [Link]
4. Dennis EA. Lipidomics joins the omics evolution. Proc Natl Acad Sci U S A. 2009;106:2089–90.
[Link]
5. Quehenberger O, et al. Lipidomics reveals a remarkable diversity of lipids in human plasma. J
Lipid Res. 2010;51:3299–305. [Link]
6. Meikle TG, Huynh K, Giles C, Meikle PJ. Clinical lipidomics: realizing the potential of lipid
profiling. J Lipid Res. 2021;62:100127. [Link]
7. Burla B, et al. MS-based lipidomics of human blood plasma: a community-initiated position
paper to develop accepted guidelines. J Lipid Res. 2018;59:2001–17. [Link]
jlr.S087163.
8. Li J, Vosegaard T, Guo Z. Applications of nuclear magnetic resonance in lipid analyses: an
emerging powerful tool for lipidomics studies. Prog Lipid Res. 2017;68:37–56. [Link]
org/10.1016/[Link].2017.09.003.
9. Havel RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrif-
ugally separated lipoproteins in human serum. J Clin Invest. 1955;34:1345–53. [Link]
org/10.1172/jci103182.
10. Magos L. C. Lentner (ed.). Geigy Scientific Tables, 8th edition. Vol. 1. Units of Measurement.
Body Fluids. Composition of the Body. Nutrition. 1981, 298 pp. Vol. 2. Introduction to
Statistics. Statistical Tables. Mathematical Formulae. 1982, 241 pp. Vol. 3. Physical Chemistry.
Composition of the Blood. Haematology. Human Somatometric Data. 1984, 359 pp. Vol. 4.
Biochemistry. Metabolism of Xenobiotics. Inborn Error of Metabolism. Pharmacogenetics and
Ecogenetics. 1986, 330 pp. Ciba-Geigy, Basel, £12.50 each volume. Distributed in U.K. by
Farrand Press. J Appl Toxicol. 1987;7:413. [Link]
11. Fahy E, et al. A comprehensive classification system for lipids. J Lipid Res. 2005;46:839–61.
[Link]
12. Psychogios N, et al. The human serum metabolome. PloS one. 2011;6:e16957. [Link]
org/10.1371/[Link].0016957.
13. Quehenberger O, Dennis EA. The human plasma lipidome. N Engl J Med. 2011;365:1812–23.
[Link]
Lipidomic Profiling in Clinical Practice Using LC-MS 237

14. Roth GA, et al. Global burden of cardiovascular diseases and risk factors, 1990-2019: update
from the GBD 2019 study. J Am Coll Cardiol. 2020;76:2982–3021. [Link]
jacc.2020.11.010.
15. Riazi K, et al. The prevalence and incidence of NAFLD worldwide: a systematic review
and meta-analysis. Lancet Gastroenterol Hepatol. 2022;7:851–61. [Link]
s2468-­1253(22)00165-­0.
16. Langlois MR, et al. Quantifying atherogenic lipoproteins for lipid-lowering strate-
gies: consensus-­ based recommendations from EAS and EFLM. Clin Chem Lab Med.
2020;58:496–517. [Link]
17. McGill HC Jr. The pathogenesis of atherosclerosis. Clin Chem. 1988;34:B33–9.
18. Napoli C, Pignalosa O, de Nigris F, Sica V. Childhood infection and endothelial dysfunction:
a potential link in atherosclerosis? Circulation. 2005;111:1568–70. [Link]
Cir.0000161816.52136.66.
19. Stary HC, et al. A definition of the intima of human arteries and of its atherosclerosis-prone
regions. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis,
American Heart Association. Circulation. 1992;85:391–405. [Link]
cir.85.1.391.
20. Bao W, Srinivasan SR, Wattigney WA, Berenson GS. Persistence of multiple cardiovascular
risk clustering related to syndrome X from childhood to young adulthood. The Bogalusa Heart
Study. Arch Intern Med. 1994;154:1842–7.
21. Peters SA, et al. Extensive or restricted ultrasound protocols to measure carotid intima-media
thickness: analysis of completeness rates and impact on observed rates of change over time. J
Am Soc Echocardiogr. 2012;25:91–100. [Link]
22. Li S, et al. Childhood cardiovascular risk factors and carotid vascular changes in adulthood: the
Bogalusa Heart Study. JAMA. 2003;290:2271–6. [Link]
23. Ouimet M, Barrett TJ, Fisher EA. HDL and reverse cholesterol transport. Circ Res.
2019;124:1505–18. [Link]
24. Sandesara PB, Virani SS, Fazio S, Shapiro MD. The forgotten lipids: triglycerides, remnant
cholesterol, and atherosclerotic cardiovascular disease risk. Endocr Rev. 2019;40:537–57.
[Link]
25. Choi SS, Diehl AM. Hepatic triglyceride synthesis and nonalcoholic fatty liver disease. Curr
Opin Lipidol. 2008;19:295–300. [Link]
26. Page JM, Harrison SA. NASH and HCC. Clin Liver Dis. 2009;13:631–47. [Link]
org/10.1016/[Link].2009.07.007.
27. Day CP, James OF. Steatohepatitis: a tale of two “hits”? Gastroenterology. 1998;114:842–5.
[Link]
28. Bellentani S, Scaglioni F, Marino M, Bedogni G. Epidemiology of non-alcoholic fatty liver
disease. Dig Dis. 2010;28:155–61. [Link]
29. Masoodi M, et al. Metabolomics and lipidomics in NAFLD: biomarkers and non-invasive
diagnostic tests. Nat Rev Gastroenterol Hepatol. 2021;18:835–56. [Link]
s41575-­021-­00502-­9.
30. Puri P, et al. A lipidomic analysis of nonalcoholic fatty liver disease. Hepatology
(BaltimoreMd.). 2007;46:1081–90. [Link]
31. Kotronen A, et al. Hepatic stearoyl-CoA desaturase (SCD)-1 activity and diacylglycerol but
not ceramide concentrations are increased in the nonalcoholic human fatty liver. Diabetes.
2009;58:203–8. [Link]
32. García-Cañaveras JC, Donato MT, Castell JV, Lahoz A. A comprehensive untargeted metabo-
nomic analysis of human steatotic liver tissue by RP and HILIC chromatography coupled to
mass spectrometry reveals important metabolic alterations. J Proteome Res. 2011;10:4825–34.
[Link]
33. Barr J, et al. Obesity-dependent metabolic signatures associated with nonalcoholic fatty liver
disease progression. J Proteome Res. 2012;11:2521–32. [Link]
238 N. Amigó Grau and P. Ortiz Betes

34. Mayo R, et al. Metabolomic-based noninvasive serum test to diagnose nonalcoholic steato-
hepatitis: results from discovery and validation cohorts. Hepatol Commun. 2018;2:807–20.
[Link]
35. Mishra A, Younossi ZM. Epidemiology and natural history of non-alcoholic fatty liver disease.
J Clin Exp Hepatol. 2012;2:135–44. [Link]
36. Bril F, et al. Use of a metabolomic approach to non-invasively diagnose non-alcoholic fatty
liver disease in patients with type 2 diabetes mellitus. Diabetes Obes Metab. 2018;20:1702–9.
[Link]
37. Rasmussen DGK, et al. NAFLD and NASH biomarker qualification in the LITMUS con-
sortium - lessons learned. J Hepatol. 2022;78:852. [Link]
38. Sanyal AJ, Shankar S, Yates KP, Daly E, Dehn CA, Bolognese JA, Neuschwander-Tetri BA,
Kowdley KV, Vuppalanchi RK, Guy CA, Tonascia JA, Samir AE, Sirlin CB, Sherlock SP,
Fowler KJ, Heymann H, Kamphaus TN, Loomba R, Calle RA, for the NIMBLE Project Team.
AASLD meeting. 2021.
39. Seijo S, Lozano JJ, Alonso C, Miquel R, Berzigotti A, Reverter E, García-Pagán
JC. Metabolomics as a diagnostic tool for idiopathic non-cirrhotic portal hypertension. Liver
Int. 2016;36(7):1051–8.
40. Villoslada P, et al. Metabolomic signatures associated with disease severity in multiple
sclerosis. Neurol Neuroimmunol Neuroinflamm. 2017;4:e321. [Link]
nxi.0000000000000321.
41. Olazarán J, et al. A blood-based, 7-metabolite signature for the early diagnosis of Alzheimer’s
disease. J Alzheimers Dis. 2015;45:1157–73. [Link]
42. Cubiella J, et al. Targeted UPLC-MS metabolic analysis of human faeces reveals novel low-­
invasive candidate markers for colorectal cancer. Cancers (Basel). 2018;10:300.
Bringing Human Serum Lipidomics
to the Forefront of Clinical Practice: Two
Clinical Diagnosis Success Stories

Núria Amigó Grau and Pablo Ortiz Betes

Abstract The present chapter describes two clinical applications based on LC-MS
and NMR lipidomics that have already been introduced into clinical workflows to
better stratify metabolic health, including staging nonalcoholic fatty liver disease
according to a specific lipid signature for the disease progression and improving the
cardiovascular disease risk based on advanced lipoprotein profiling.
The chapter includes a list of potential applications based on the same technolo-
gies and details the envisaged risks and limitations.
The implications of developing advanced high-throughput technologies for clini-
cal applications go much further, such as accelerating the deployment of lipidomic-­
based assessments in the healthcare system, favoring true disruption through precise
and personalized medicine based on global bio-screening approaches.

Keywords Nuclear magnetic resonance (NMR) · Metabolomics · Clinical

diagnosis · Cardiovascular diseases (CVD) · Lipidomics · Personalized medicine

Abbreviations

ALT Alanine transaminase

ApoA/B Apolipoprotein A/B
AST Aspartate transaminase

N. Amigó Grau (*)

Biosfer Teslab, Reus, Spain
Department of Basic Medical Sciences, Institut d’Investigació Sanitària Pere Virgili (IISPV),
Av. Universitat, Reus, Spain
Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas
Asociadas (CIBERDEM), Instituto de Salud Carlos III (ISCIII), Madrid, Spain
e-mail: namigo@[Link]
P. Ortiz Betes
Biosfer Teslab, Reus, Spain

© The Author(s), under exclusive license to Springer Nature Singapore Pte 239
Ltd. 2023
A. M. Abdel Rahman (ed.), Clinical Metabolomics Applications in Genetic
Diseases, [Link]
240 N. Amigó Grau and P. Ortiz Betes

AUC/ AUROC Area under the ROC curve

BMI Body mass index
CIBERDEM Centro de Investigación Biomédicas en Red de Diabetes y
Enfermedades Metabólicas
CLIA Clinical Laboratory Improvement Amendments
CMD Cardiometabolic disease
CMR Cardiometabolic risk
EAS European Atherosclerosis Society
ESC European Society of Cardiology
FFA Free fatty acids
HCC Hepatocellular carcinoma
HDL High-density lipoproteins
IDL Intermediate-density lipoproteins
IISPV Institut d’Investigació Sanitària Pere Virgili
IMT Intima-media thickness
IRAS Insulin resistance atherosclerosis
IVD In vitro diagnostic
LC Liquid chromatography-mass
LDL Low-density lipoproteins
LITMUS Liver Investigation: Testing Marker Utility in Steatohepatitis
LMWM Low-molecular-weight metabolites
MAFLD Metabolic-associated fatty liver disease
MESA Multi-Ethnic Study of Atherosclerosis
ML Machine learning
MUFAs Monounsaturated fatty acids
NASH Nonalcoholic steatohepatitis
NIMBLE Noninvasive biomarkers of metabolic liver disease
NPV Negative predictive value
PPV Positive predictive value
RUO Research use only
VLDL Very-low-density lipoproteins
WHO World Health Organization

1 Introduction

Metabolomics is an omic approach and is closest to phenotypes because metabolites

are the end products of complex and transverse molecular pathways (including
genomics, transcriptomics, and proteomics). Therefore, metabolomic analysis is a
promising strategy for identifying disease-associated biomarkers: metabolites,
small molecules, and end products of the interaction between genes and environ-
mental factors [1].
In the search for new biomarkers, there are two main possible approaches that
can be taken. The first is a classic approach based on searching for a biomarker that
Bringing Human Serum Lipidomics to the Forefront of Clinical Practice: Two Clinical… 241

explains the pathophysiology of metabolic diseases, such as glucose quantification

to determine whether a person has diabetes or the quantification of LDL-C for
assessing the risk of cardiovascular diseases (CVDs). The second approach involves
searching for metabolic patterns that are characteristic of the physiology of a meta-
bolic disease [2]. In this second approach, high-performance analytical techniques,
especially liquid chromatography-mass spectrometry (LC-MS) or nuclear magnetic
resonance (NMR), are used to analyze the metabolome without prior knowledge of
the metabolites’ involvement or role in the disease mechanism or physiology [3].
Since the metabolites are the final products of the interactions between the expres-
sion of genes and proteins and environmental exposure, and consequently of the
biochemical activity that gives rise to the phenotype of the measured sample, they
thus characterize part of the patient’s metabolism. The metabolomic approach com-
pares the metabolites in fluids or tissues of a patient with those of a healthy subject
to see which metabolites are expressed differently [4].
Metabolomics generally includes a wide range of molecules within a biological
matrix. The term “lipidomics” should be adopted when the studied metabolites are
specifically lipids. In particular, lipidomics is the study of the structure, function,
and metabolism of lipids in living organisms [5]. It is a broad field that encompasses
the measurement of all of the lipids present in a biological sample as well as the
pathways and enzymes involved in their synthesis, degradation, and modification.
Various analytical techniques are applied in lipidomics to identify and quantify
the different lipid species present in a sample, especially mass spectrometry, NMR
spectroscopy, and gas or liquid chromatography. These techniques allow research-
ers to get a detailed picture of the lipids that are present in a sample and to study
changes in lipid levels in response to different conditions or treatments [6].
Applications of lipidomics have been reported in a range of different areas,
including medicine, nutrition, and environmental science. It is used to study the
roles of lipids in health and disease, understand the mechanisms through which
lipids affect cellular processes and signaling pathways, and identify potential thera-
peutic targets for treating lipid-related disorders [7].
Lipidome analysis is much more complex than other omic techniques, such as
proteome or genome analysis, because of the enormous structural diversity of lipids,
which can differ in both their linear and coupled macromolecular compositions [6].
This diversity increases the number of lipid molecules and the complexity of their
carriers, lipoproteins. Thus, lipidome and lipoprotein analysis is technically very
challenging. LC-MS and NMR are indispensable analytical tools for metabolomic
studies in biological fluids. LC-MS and NMR have the advantage of being able to
quantify and identify a large number of compounds simultaneously, reproducibly,
and effectively [8, 9]. These techniques are often used in large epidemiological
studies and are starting to be applied routinely in clinical laboratories.
This chapter is focused on LC-MS- and 1H-NMR-based lipidomic platforms for
clinical applications, exploring their benefits, challenges, difficulties, and future
opportunities. The application of these technologies in the clinical environment has
opened the doors to the future use of such advanced technologies for predicting and
monitoring several diseases. The technologies analyzed in the present chapter are
242 N. Amigó Grau and P. Ortiz Betes

already applied in clinics, exemplifying the potential of precision medicine

approaches for personalized diagnostics.

2 Lipids and Lipoproteins: A Biochemical Approach

2.1 General Concepts

Lipids are a diverse group of organic molecules that are essential to life and many
biological functions. They include fats, oils, waxes, and other water-insoluble com-
pounds [10].
Lipids are synthesized by living cells through a process called lipid biosynthesis.
This process involves condensing fatty acids with glycerol or other alcohols to form
triglycerides, the primary component of fats. Lipids can also be synthesized by
modifying existing lipids, such as by adding a phosphate group to form a phospho-
lipid or adding a carbohydrate group to form a glycolipid [11].
The structure of lipids is characterized by their hydrophobic nature, which arises
from the presence of long, nonpolar hydrocarbon chains. This nonpolarity allows
lipids to interact with each other’s molecules and form aggregates, such as micelles
or lipid bilayers, which can serve as structural components of cell membranes [12].
Lipids are generally found in plasma as lipoproteins, which are macromolecular
complexes with a hydrophobic core of neutral lipids, mainly cholesterol esters and
triglycerides, surrounded by a hydrophilic layer of phospholipids, unesterified cho-
lesterol, and proteins [13]. Lipoproteins can be classified based on their density,
protein composition (apoprotein), or lipid composition (rich in cholesterol or tri-
glycerides). Considering the protein composition, there are two types of lipopro-
teins, ApoA and ApoB.
Lipids have many functions in living organisms: from energy reserve to insula-
tion, as well as being a structural component of every cell and tissue. They are also
important for cellular signaling and regulation [14]. The diversity of lipid structures
is reflected by a wide range of physiological functions they perform. The levels of
particular lipids in plasma can be used to diagnose diseases.
So far, approximately 4500 metabolites have been identified in human serum.
Around half of these metabolites are phospholipids while over a thousand are glyc-
erolipids (triglycerides [TG], diglycerides [DG], and monoacylglycerols) [15].
Thus, lipids make up approximately 75% of the known human serum metabolome.
Analysts have already quantified thousands of distinct species of lipids, includ-
ing fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, and
prenol lipids, representing the six main categories of lipids found in mammals.
Moreover, the number is continually increasing as each of these types of lipids can
exist in multiple forms [16]. In addition, lipids can undergo chemical modifications,
such as the addition of a phosphate group or a carbohydrate group, which can result
in the creation of new lipid species. Sphingolipids and glycerophospholipids are
Bringing Human Serum Lipidomics to the Forefront of Clinical Practice: Two Clinical… 243

particularly diverse in structure, due to variations in fatty acid content and

head groups.

2.2 Lipids Mirror Present and Future Metabolic Health: Two

Sides of the Same Problem

Abnormal levels of lipids and lipoproteins in the blood are major risk factors for
metabolic and cardiovascular diseases. Metabolic diseases are increasing exponen-
tially worldwide, and their complications, at cardiovascular and liver levels, are the
leading cause of mortality worldwide [17]. They have a common characteristic:
their etiology is linked to excess fat and associated inflammation. Nonalcoholic
fatty liver disease (NAFLD) is associated with obesity and excess fat in the liver,
and its prevalence is increasing around the world, especially in Western countries,
currently affecting approximately a quarter of people in countries such as the USA
[18]. Symmetrically, arterial health is compromised by an excess of fat, which
accelerates the progression of arteriosclerosis, the underlying disease of heart dis-
ease and stroke and the leading cause of death in all developed countries [17]. Both
liver disease and atherosclerosis can develop and progress more rapidly when
accompanied by several well-known risk factors and comorbidities, such as obesity
and dyslipidemia.
Lipids circulate in the blood in the form of lipoprotein particles, ranging from
chylomicrons, as the largest lipoprotein, to very-low-density lipoproteins (VLDL),
low-density lipoproteins (LDL), and high-density lipoproteins (HDL), which are
the smallest. Health services routinely check cholesterol and triglyceride, and doc-
tors frequently prescribe lipid-lowering drugs to treat patients with dyslipid-
emia [19].
The duration of exposure to high lipid levels is also a crucial risk factor in car-
diometabolic health, dramatically increasing the risk of major cardiovascular events,
especially for metabolic disorders that start early in childhood. Atherosclerosis typi-
cally begins in childhood [20]. Fatty streaks, buildups of lipids in the intima of
arteries, are present in nearly all children by 3 years of age [21]. After 8 years of age,
these fatty streaks increase, and atherosclerotic plaques form the coronary arteries
during adolescence [22]. Children with clusters of risk factors for cardiovascular
diseases are likely to have those same risk factors as adults [23].
Carotid ultrasonography screening for subclinical arteriosclerosis has been vali-
dated in observational, longitudinal, and randomized clinical assays. Those results
were significantly correlated with intravascular coronary ultrasonography, coronary
angiography, and pathologic findings of arterial lesions in healthy and CVD patients,
and longitudinal studies have demonstrated that increased lipid alterations and
intima–media thickness (IMT) in young adults are linked to cardiovascular risk fac-
tors in childhood [24, 25].
244 N. Amigó Grau and P. Ortiz Betes

The contribution of LDL-associated cholesterol to the development of CVD has

been well described. While LDL particle accumulation increases the atherosclerotic
process, HDL helps eliminate excess cholesterol by reverse cholesterol transport.
Low levels of HDL are associated with high cardiovascular risk [26].
Conversely, in circulating chylomicrons and VLDL, triglycerides undergo hydro-
lysis, producing a pool of free fatty acids (FFAs), which are used by tissues as an
energy source. Adipocytes store excess FFAs, favoring the expansion and dysfunc-
tion of adipose tissue, increasing insulin resistance and diabetes. This process is
associated with excessive levels of saturated FFAs in plasma, increasing their uptake
in hepatocytes to exceed metabolic requirements, which leads to hepatic steatosis
and inflammation [27].

3 Clinical Relevance of the Lipidome

Lipids play important roles in many biological processes, so it is not surprising that
many diseases can be caused by defects in lipoprotein homeostasis, but it does mean
that lipids can be used as markers of the disease. Expanding our knowledge of the
composition and concentration of lipid metabolites in the plasma lipidome will lead
to improved diagnostic capabilities, as well as enhancing pharmacological evalua-
tion and the efficacy of prescribed treatments [28]. A deep analysis of the lipidome
might reflex altered synthesis of specific lipid species or identify abnormal underly-
ing pathological lipoprotein patterns. Our understanding of the roles played by lip-
ids and lipoproteins in disease mechanisms is constantly growing as more
information on lipids and lipoprotein physiology becomes available, further high-
lighting the physiological importance of the lipidome composition and transport,
which is strictly regulated and interrelated to cellular response.

3.1 Lipids and NAFLD: Introduction to LC-MS

Nonalcoholic fatty liver disease (NAFLD) covers a wide range of disorders, from
benign lipid accumulation in the liver (steatosis) to a more complicated clinical
stage when fat induces hepatic inflammation and hepatocyte necrosis producing a
new stage called nonalcoholic steatohepatitis (NASH) also named as steatohepati-
tis, when fibrosis progression is added to fat and inflammation. The final stage of
disease progression is cirrhosis and/or hepatocarcinoma (HCC) [29]. Interestingly
another potential evolution from NASH directly to HCC without any significant
fibrosis contribution has also been described [30].
The exact cause of NAFLD is not known. Many factors contribute to this condi-
tion, such as excessive food intake, obesity, type 2 diabetes, and dyslipidemia, but
not all patients develop NAFLD/NASH, and not all patients with NAFLD/NASH
suffer from one of these conditions [30, 31].
Bringing Human Serum Lipidomics to the Forefront of Clinical Practice: Two Clinical… 245

The pathophysiology of NAFLD is quite complex, and the progression from

hepatic steatosis to the different stages of this condition is not completely under-
stood. However, lipid metabolic changes, including the production of lipotoxic spe-
cies in the liver, could be responsible for disease progression in NAFLD [30].
LC-MS allows the tracking of more than 400 different lipid species in the liver
and serum. Changes in many of these metabolites were followed in several trans-
verse cohorts giving robust data about the lipidomic signature of the different clini-
cal stages of NAFLD, from liver steatosis to steatohepatitis and advanced fibrosis.
Lipids are highly likely to be involved in both the origin and the progression of
the disease, so serum lipidomics has, in the last 20 years, been one of the most suc-
cessful research lines in identifying markers to differentiate different stages of
NAFLD [32].
The first attempt to diagnose NAFLD in humans using lipidomics was reported
by Puri et al. in 2007 [33]. The researchers analyzed the lipid levels in the livers of
people with normal liver tissue, fatty liver disease, and nonalcoholic steatohepatitis
(NASH) and found no differences in the FFA contents in the three groups. However,
in NAFLD both the TG and DG levels were higher, while the PC level was decreased,
which suggests that PC hydrolysis may contribute to DG and TG accumulation in
the fatty liver. The SFA-to-MUFA ratio generally decreased across multiple classes
of lipids, providing evidence for increased lipogenesis in NAFLD.
These findings were confirmed in two small series by Kotronen and García-­
Cañaveras through semiquantitative analysis of the full range of lipids by LC-MS
[34, 35]. Total lysophospholipids, DG, and TG were found to be elevated in NAFLD,
while the stearic-to-oleic acid ratio was lower, indicating increased TG biosynthe-
sis. Increased DG and reduced PUFA are also characteristic of NAFLD.
An additional pilot study (42 biopsied patients) and a pivotal validation one (467
biopsied patients) were published by Barr et al. in 2010 and 2012, respectively, that
clearly identified lipidomic signatures associated with NAFLD progression using
LC-MS [36, 37].
Especially relevant is Barr’s 2012 study that included 467 biopsied individuals,
comprising 90 with normal liver and 377 diagnosed with NAFLD (steatosis,
n = 246; NASH, n = 131). In this study, they analyzed 540 circulating metabolites,
including amino acids, FA, DG, TG, PC, PE, PI, ceramides, SM, cholesteryl esters,
and bile acids. After analyzing the lipidomic data, the authors established a robust
“body mass index-dependent lipidomic signature” for reliably and accurately dis-
tinguishing liver steatosis from NASH. The areas under the curve (AUC) for lean/
pre-obese, obese, and morbidly obese patients were 0.84, 0.85, and 0.87, respec-
tively. Subsequently, a set of 25 BMI-dependent lipids was established using this
same cohort of patients, allowing differentiation between steatosis and NASH with
AUC of 0.99, 0.90, and 0.91 for lean/pre-obese, obese, and morbidly obese patients,
respectively [38].
It is important to distinguish between simple steatosis and NASH because simple
steatosis is a generally benign condition, while NASH is a more serious condition
that can lead to increased morbidity and mortality [39].
246 N. Amigó Grau and P. Ortiz Betes

Mayo et al. reanalyzed the lipidomic data from the cohort from Barr’s pivotal
trial [36] and a new cohort of 192 biopsied NAFLD patients, which allowed them to
establish and validate a BMI-dependent algorithm with 20 TGs. The algorithm was
able to distinguish between NASH and NAFLD with a high degree of accuracy,
with an AUROC of 0.95 versus biopsy. The sensitivity, specificity, positive predic-
tive value, and negative predictive value of the test were 0.83, 0.94, 0.89, and 0.90,
respectively [38].
Using the lipidomic signature in 220 patients with type 2 diabetes mellitus, Bril
et al. found an AUROC of 0.79 (95% CI 0.68–0.90) in patients with adequate gly-
cemic control for discrimination between steatosis and NASH. However, the dis-
crimination ability was poor in patients with high insulin resistance or poor glycemic
control [40].
Validation of the lipidomic signatures in such important cohorts indicates that
lipidomic markers will have a major impact in elucidating the phenotypic stages of
NAFLD. Therefore, LC-MS is not just for use in basic research: it has also become
a very efficient tool for providing clinically relevant information for patient man-
agement. The review by Masoodi et al. gives an in-depth discussion of the contribu-
tion of LC-MS to lipidomics and the complexity of NAFLD [32].

3.2 Lipoproteins and CVD: Introduction to NMR

According to data from the World Health Organization (WHO), CVDs are the lead-
ing cause of mortality and morbidity in developed countries. They are a major bur-
den on society, leading to a significant reduction in the quality of life and health, as
well as healthcare costs [17]. These diseases can be present for many years before
becoming clinically apparent, making clinical management difficult. Thus, early
CVD risk identification is important to delay and prevent its onset.
Traditionally, CVDs have been diagnosed based on the analysis of risk factors
such as smoking, high (total and LDL) cholesterol, high blood pressure, obesity,
sedentary lifestyles, or type 2 diabetes. However, it has not been possible to accu-
rately identify all individuals at risk for cerebrovascular accidents or complications
using such risk factors. Unfortunately, unexpected acute ischemic events still occur
at high rates, both in patients known to have arteriosclerosis and subjects thought to
be healthy [41].
LDL cholesterol (LDL-C) is the most important lipid factor for assessing an
individual’s cardiovascular risk. However, many individuals with CVD have
normal LDL-C levels [42]. LDL-C concentrations are often normal or only
slightly elevated in people with metabolic disorders such as diabetes, obesity, or
metabolic syndrome. At the same time, the level of LDL particles (LDL-P) is
raised owing to the presence of smaller particles, lower cholesterol levels, and
higher atherogenicity, so they are able to access the arterial wall easily. Among
the LDL particles, the smallest and densest can easily infiltrate the arterial wall
and stick to the extracellular matrix proteoglycans [43]. Small LDL-P
Bringing Human Serum Lipidomics to the Forefront of Clinical Practice: Two Clinical… 247

significantly increase the risk of CVD but are not considered by the traditional
risk estimation methods.
Beyond LDL-P, lipoproteins rich in triglycerides, including VLDL, intermediate-­
density lipoproteins (IDL), and cholesterol remnants, favor the development of ath-
erosclerosis as a complementary source of cholesterol and promote inflammation.
In particular, lipoproteins below 70 nm in diameter can cross the vascular endothe-
lial barrier and interact with macrophages exacerbating inflammatory mechanisms
[44]. Remnant cholesterol transported by triglyceride-rich particles shows a CVD
predictive value even higher than that of LDL cholesterol (LDL-C) [45].
The basic and generally used lipid panel for CV risk assessment does not offer
an approach to the real protagonists of atherogenicity, the lipoprotein particles [46].
The proatherogenic particle concentration can be indirectly characterized by deter-
mining the plasma apolipoprotein B (ApoB) levels [47]. ApoB-100 is the major
apolipoprotein of atherogenic lipoproteins. Each lipoprotein particle has only one
ApoB molecule, from chylomicrons and VLDL to LDL. For the same amount of
LDL-C, a higher concentration of ApoB in plasma ApoB indicates the presence of
more atherogenic particles and smaller lipoproteins [48]. The latest guidelines on
dyslipidemia and cardiovascular prevention from the European Society of
Cardiology (ESC) and the European Atherosclerosis Society (EAS) recognize that
the LDL-C level is a surrogate for the level of atherogenic particles and that lipid-­
lowering drugs achieve their beneficial effects by reducing the number of athero-
genic particles [49]. These guidelines propose the determination of the ApoB level
as a surrogate marker for CVD closer to the number of LDL particles and set out
certain concentrations of ApoB as secondary therapeutic goals. Despite this, the
concentration of ApoB does not distinguish between triglyceride-rich particles and
LDL particles.
For this reason, advanced lipoprotein testing is being developed into a new diag-
nostic system, helping health specialists improve the control of cardiovascular risk,
providing an accurate picture of the complete lipid profile, and establishing person-
alized therapy for patients [50].
Advanced lipoprotein profiling using NMR allows the determination in one
NMR run of the basic lipid profile, including total cholesterol, LDL, HDL, non-­
HDL, and triglycerides, as well as a more advanced lipoprotein profile that includes
the lipid composition, particle size, and concentration of the main lipoprotein
classes (VLDL, LDL, and HDL). The complete characterization of the blood lipo-
protein profile contributes to personalized preventive and therapeutic decisions in
estimating and addressing the CVD risk [51]. This comprehensive characterization
of the lipoprotein profile facilitates the detection of individuals with an increased
CVD risk [52].
The NMR technique for lipoprotein profiling was set in 1992 and has repeatedly
proved helpful measuring a wide spectrum of cardiovascular risk factors in a high-­
throughput operational mode [53]. NMR allows us to obtain advanced metabolic
profiling that includes the detailed lipoprotein profile and a complementary set of
information related to the cardiometabolic status (glycoprotein profile and the con-
centrations of low-molecular-weight metabolites [LMWM], among others).
248 N. Amigó Grau and P. Ortiz Betes

In particular, NMR biomarker profiling has repeatedly demonstrated cost-­

effectiveness and high performance and has been used for both preclinical screening
and in vitro diagnostics (IVD)-by-NMR discovery and validation [54, 55]. Except
for some applications, NMR is broadly considered a research use only (RUO)
device. However, the development of the technology over the last decade has facili-
tated the implementation of NMR metabolomics as an IVD system in the clinical
workflow. The specific application of NMR lipoprotein profiling for cardiovascular
risk assessment has recently been introduced in advanced lipid units in some coun-
tries, favoring true disruption to a preventive, predictive, and precise medicine
approach to help with diagnosis and the development of effective, safe medications
and doses that are tailored to patients’ individual lipidomic profiles [42].

4 Measurement Techniques for Characterization of Lipid

Species and Lipoproteins

4.1 Main Techniques Used to Measure Lipid Species

for Cardiometabolic Health Assessment

Various MS-based methods can be used to analyze plasma lipids: LC-MS, direct
flow injection, and direct-infusion/shotgun MS (DIMS) are the most common
approaches. LC-MS/MS is typically applied for the targeted analysis of very low
abundancy (nanomoles per liter range) lipid mediators (e.g., eicosanoids, special-
ized pro-resolving mediators, oxysterols). Meanwhile, DIMS as well as LC-MS and
LC-MS/MS can be used for analyzing lipid classes with higher abundancy, in the
high micromoles per liter to millimoles per liter range (e.g., glycerolipids, glycero-
phospholipids, cholesterol esters, and ceramides).
As mentioned, in plasma, lipids are often found bound to soluble carrier proteins
(e.g., albumin) or associated with multiprotein assemblies (lipoproteins). Therefore,
a single lipid recovery protocol is unlikely to be effective for all analytical
approaches. This is because different lipid families require different extraction and
analysis protocols. For example, some lipids are more stable than others and can be
extracted using milder methods. Additionally, different lipids require different ana-
lytical techniques. Furthermore, some analytical approaches require that the lipids
be kept in their native state, which means that they cannot be disrupted by the
extraction or analysis process. This is important because the structure of the lipids
can provide important information about their function.
Therefore, truly comprehensive lipidome analysis requires multiple analytical
platforms (each suited for a subset of lipid classes) to be applied in parallel, particu-
lar sample preparation techniques and non-destructive approaches for lipoprotein
structure profiling [56]. Frequently, plasma lipidome analysis only includes lipid
classes that can be determined in one run using the only mass spectrometer that the
researchers have available and supplemented by structural lipoprotein analysis
using NMR spectroscopy [57].
Bringing Human Serum Lipidomics to the Forefront of Clinical Practice: Two Clinical… 249

It is important, however, to achieve cost-effectiveness through high-throughput

approaches. MS is applied for more detailed characterization of the lipid family. MS
characterization is based on mass difference, while NMR characterization of lipids
is based on the spectroscopically different response of the lipids to an external mag-
netic field according to the size of the lipoprotein particle carrying them.
In this section, we will report two novel clinical applications based on MS and
NMR methods for quantifying circulating lipids and lipoproteins as robust and reli-
able tools for biomedical diagnosis of liver and cardiometabolic diseases.
The two analytical techniques included in the present chapter for performing high-
throughput lipid analysis in cardiometabolic health assessment are liquid
chromatography-­mass spectrometry (LC-MS) and NMR spectroscopy [58]. Both
techniques have advantages and disadvantages [59]. MS can resolve more compounds
than NMR, has a higher sensitivity, and requires a smaller sample volume [60].
However, MS requires standards and quality control samples for absolute quantifica-
tion and reproducibility. NMR is highly reproducible and intrinsically quantitative
and can be scaled for use in different laboratories [61, 62]. NMR can immediately
provide qualitative and quantitative information, including lipoproteins.
Historically, it has been challenging to perform wide-scale lipid profiling. This is
because lipid metabolites have a variety of physical properties, which require the
use of different purification systems and complex technical procedures. However,
the evolution of lipidomics has led to the development of new analytical platforms,
particularly in mass spectrometry. These new platforms have streamlined the proce-
dures involved in lipid profiling, allowing for the analysis of many more lipid mol-
ecules in greater detail.

4.2 The Complementarity of Serum/Plasma LC/MS and NMR

for Lipoprotein Analysis

Mass spectrometry coupled with chromatography (LC-MS) is the most common

technique used for analyzing the lipidome due to its sensitivity and selectivity.
When LC is combined with a high-resolution accurate mass instrument (e.g.,
Orbitrap or time-of-flight instruments), large numbers of analytes can be analyzed
at the same time. Typically, reversed-phase chromatography is used to separate the
analytes before reaching the mass spectrometer, which determines their structure
and concentration. Various chromatographic columns are available for separating
lipids depending on their chemical structure, such as their lipid class (e.g., phospha-
tidylcholine or phosphatidylethanolamine head groups) and fatty acyl composition
(e.g., chain length and degree of unsaturation) [58, 63–66].
On the other side, NMR is an excellent technique for profiling biofluids under physi-
ological temperature conditions and is especially adept at characterizing complex solu-
tions. Minimal sample preparation is required for profiling serum/plasma samples by
NMR. Large numbers of plasma/serum samples can be collected from patients at a high
frequency in a standard clinical routine, allowing the detailed characterization of
250 N. Amigó Grau and P. Ortiz Betes

Table 1 Strengths and LC-MS NMR

weaknesses of LC-MS and High throughput + +++
NMR for lipidomic analysis
Sensitivity +++ +
Equipment cost + ++
Implementation level + ++
Versatility ++ +
Robustness + +
Scalability + ++
Absolute concentration +a +++
Labor intensity + +++
Specific application field NAFLD CVD
a
Only when the standard is available

dynamic metabolic events. One of the advantages of NMR metabolomics is that it can
be used to generate quantitative profiles of solute-state fluids with minimal sample prep-
aration. This allows for a naturalistic, largely unbiased view of their composition that
closely represents the in vivo state. In metabolic research, NMR spectroscopy allows
comprehensive metabolic profiling associated with different metabolic conditions and
inflammation: from small molecules (known as the aqueous metabolome) to large mac-
romolecular complexes (advanced lipoprotein testing based on NMR technology and
NMR glycoprotein profiling) from intact plasma or serum, as well as the characteriza-
tion of different lipid species from lipid plasma or serum extracts.
The clinically relevant information that can be found in 1H NMR spectra of
blood plasma goes far beyond standard lipid panels, including a set of parameters
associated with lipoprotein profiling for cardiometabolic health (size, composition,
and particle concentration) and other parameters, including glycoproteins, LMWM,
and some lipid species. Since the vascular wall releases molecules into the blood-
stream that reflect the patient pathological processes, the concentrations of these
molecules participating in pathological processes could be potential biomarkers for
the future appearance of diseases with which to establish predictive mathematical
models that can be used in clinics.
As mentioned in the previous section, measuring lipids and lipoproteins using
LC-MS and NMR can be complementary, presenting different strengths and weak-
nesses (Table 1).
The sensitivity of LC-MS exceeds that of NMR by an order or magnitude and
hence the higher number of visible metabolites and potential versatility. However,
lipoprotein profiling should be performed using NMR. Moreover, NMR exhibits
low experimental variability between laboratories. The main source of variability in
NMR is intra-operator and intra-day. NMR can measure a quantitative lipoprotein
profile over time for individual longitudinal studies.

4.3 LC-MS Lipidomics and 1H NMR Lipoprotein Profiling

Technical Aspects (Table 2)
Bringing Human Serum Lipidomics to the Forefront of Clinical Practice: Two Clinical… 251

Table 2 LC-MS Lipidomics and 1H NMR Lipoprotein Profiling Specifications

LC-MS NMR
Sample Serum and plasma samples derived Different biochemical protocols are applied to
handling from blood should be frozen at the serum and plasma fractions derived from blood
LC-MS lab and immediately samples after collection
aliquoted following a platform-­ The coagulation factors (i.e., fibrinogen) and
specific protocol depending on the blood cells are removed from serum by
lipid class(es) that will be analyzed
centrifugation
Plasma is typically obtained from blood
samples that have had an anticoagulant agent
added (i.e., heparin or EDTA), which produce
high-intensity peaks (EDTA) or overlapping
signals (heparin) in the NMR spectra, so serum
can be preferable to plasma for some NMR
applications
Storage Samples should be stored at −20 °C Storage must be considered when analyzing
for less than 10 days or −70 °C or lipoproteins and other plasma/serum
−80 °C if stored for longer periods metabolites by NMR
[67] Samples can be stored at 2–4 °C for up to
7 days or at −20 °C for up to 1–2 months
However, as some enzymes, for example,
plasma esterase, are still active at −20 °C,
samples should be stored at −70 °C or −80 °C
for longer periods. Good lipoprotein stability
in frozen samples stored for more than
10 years has been reported in some cases [68]

5 LC-MS Lipidomics and 1H NMR Lipoprotein Analysis

in Clinical Practice

5.1 Clinical Application of LC-MS: The OWLiver® Test

The OWLiver® panel is an in vitro diagnostic test based on the serum lipidomic
signature of patients with NAFLD. This panel can stratify this condition into four
clinically relevant stages: normal liver, steatosis, NASH, and “NASH at risk”
(NASH + F2 or more). The panel was developed by OWL metabolomics in Spain
and has been applied as a CE-certified IVD for over 8 years.
The company collects serum samples and biopsies from different cohorts of
NAFLD patients in Spain, the Czech Republic, Chile, Mexico, Israel, and the USA
(Florida and New York). Combining the biopsy readings with the clinical and ana-
lytical data, they have established an impressive database including data for more
than 1200 NAFLD patients.
Furthermore, the panel was selected for the two major international consortia:
Non-invasive Biomarker of Metabolic Liver Diseases (NIMBLE) and Liver
Investigation Testing Marker Utility in Steatohepatitis (LITMUS) [69] (US and EU
consortia created for the validation of noninvasive diagnostic of NAFLD stages).
Currently, the OWLiver panel is available to most Spanish hospitals and its use
is fully reimbursed for the Health Service of the Basque Country. A CLIA lab test is
252 N. Amigó Grau and P. Ortiz Betes

also available for most EU countries interested in the panel, and the CLIA test is
expected to launch in the USA in 2023.
The panel applies three different algorithms to the lipidomic signatures obtained
after the injection into an LC-MS of a very low amount of serum (~10 μl). All three
algorithms are BMI-dependent, and two of them also need the patient’s alanine
amino transferase (ALT) and aspartate amino transferase (AST). The final output of
the panel is the classification of the patient into one of the four mentioned diagnostic
categories.
The first algorithm, based on BMI, ALT, AST, and 12 complex lipids, identified
patients suffering from “NASH at risk.” Accuracy in comparison with biopsy results
in an AUC close to 0.8, which compares very well with the other noninvasive alter-
natives in development. The diagnosis of this group of patients is clinically very
relevant since they are the ones that would benefit the most from earlier treatment.
The second algorithm, based on BMI, ALT, AST, and 16 complex lipids, identi-
fies patients with NASH, with an AUC versus biopsy of close to 0.8. This classifica-
tion is also clinically important for the patients’ prognosis since the
morbidity–mortality of this category is higher than for patients with normal liver
and simple steatosis. Only NIS 4 has shown comparable accuracy in diagnosing
NASH [70]. To date, no other noninvasive procedure has been able to recognize the
inflammatory component of this condition.
Based on BMI and 11 triglycerides, the third algorithm differentiates NAFLD
from normal liver patients with an AUC close to 0.9 versus biopsy [38]. The accu-
racy is very close to that observed with Fibroscan. Although this category is not very
clinically relevant since a simple echography can provide the same information, it
could be useful for epidemiological or population studies because it only requires a
blood test.
The first results from NIMBLE were published in 2022 after evaluation in blind
more than 1000 samples from NAFLD patients and exhibited the same accuracy as
observed by Mayo et al. [38, 70].
The main use of the OWLiver panel in hepatology units is in combination with
FIB4 to select “NASH at-risk” patients. This combination reduces false negatives
from FIB 4, identifying most of the patients suffering from NASH and fibrosis stage
2 or more [38, 71].
Endocrinology units are also very interested in identifying “NASH at-risk” and
NASH patients because the technology allows them to carefully monitor overweight
type 2 diabetes patients during their potential long-term progression.
Finally, knowing if these metabolite changes are reversed when patients improve
or even return to normality is vital. Some longitudinal studies are currently being
carried out by two major consortia, NIMBLE and LITMUS, to confirm if the origi-
nal lipidomic signature is recovered once the patient recovers.
In conclusion, the accuracy shown by the different lipidomic signatures in the
OWLiver panel was very competitive compared with biopsy, and the panel is com-
plementary to the other noninvasive tools developed in parallel with the same pur-
pose, such as NIS 4 (based on microRNA, Fibroscan, and MRI) [70].
Bringing Human Serum Lipidomics to the Forefront of Clinical Practice: Two Clinical… 253

5.2 Clinical Application of 1H NMR: The Liposcale Test

Although lipoprotein particle size and number of particles are not routinely mea-
sured in clinical practice, they can be efficiently and simultaneously assessed using
NMR spectroscopy. Lipoprotein profiling by using NMR was set two decades
before and has proved helpful concerning a wide spectrum of metabolic risk fac-
tors [72].
Lipoprotein determination by NMR has been established for some years, in both
basic research and using commercially available tests; most approaches (one-­
dimensional approaches) are limited to analysis of lipids and lipoproteins using
empirical models based on correlations between the raw NMR data and laboratory
biochemical measurements.
The analysis of lipoproteins by 1D 1H NMR spectroscopy is based on particle
size. The lipid methyl groups that are transported within the lipoproteins resonate at
slightly different frequencies depending on the lipoprotein that transports them,
with smaller particles resonating at lower frequencies. Therefore, it is possible to
quantify lipoproteins by decomposing the NMR signal of the methyl group of the
lipids in individual signals. This method, commercialized by Liposcience (recently
acquired by LabCorp), provides the concentrations of particles of the major lipopro-
tein classes and subclasses from indirectly estimated size, as it is based on a library
of one-dimensional NMR spectra of previously isolated lipoprotein classes and an
algorithm, which adjusts the NMR signal depending on samples being analyzed.
Therefore, an advanced lipoprotein profile based on NMR allows for the deter-
mination of both the basic lipid profile, including total cholesterol, LDL, HDL,
non-HDL, and triglycerides, as well as a more advanced lipoprotein profile, which
includes lipid composition, particle size, and concentration of the main lipoprotein
classes (VLDL, LDL, and HDL) and the concentrations of nine subclasses of lipid
particles. The complete characterization of the blood lipoprotein profile contributes
to personalized preventive and therapeutic decisions in estimating and addressing
the CVD risk. This comprehensive characterization of the lipoprotein profile facili-
tates the identification of individuals with an increased CVD risk.
The application of an advanced lipoprotein profile to identify the situations
described in the following section is particularly relevant.

5.2.1 Individuals with Discordant Levels of LDL-C and LDL-P

LDL particles are highly heterogeneous in size and lipid content. This heterogeneity
has led to the definition of two phenotypes or patterns associated with a greater or
lesser risk of CVD, depending on the relationship between the levels of LDL-C
and LDL-P:
LDL-C > LDL-P or those individuals with particularly large LDL particles with
a higher cholesterol content and lower CVD risk.
254 N. Amigó Grau and P. Ortiz Betes

Fig. 1 Incidence of
cardiovascular events
according to the
stratification of the MESA
study population
concerning LDL-C/LDL-P
levels. (Adapted from
Otvos et al. [73])

LDL-C < LDL-P or those individuals with especially small LDL particles with a
lower cholesterol content and increased risk.
A relevant report by Otvos and colleagues published in the Journal of Clinical
Lipidology, including approximately 6000 individuals from the prospective Multi-­
Ethnic Study for Atherosclerosis (MESA), showed that the number of cardiovascu-
lar and cerebrovascular accidents accumulated over the years is significantly
associated with the level of LDL-P but not with the level of LDL-C if these two
magnitudes show discrepancies [73]. As shown in Fig. 1, the incidence of CVD in
individuals with especially large LDL particles (and therefore levels of
LDL-C > LDL-P) was observed to be significantly lower than in the group of indi-
viduals with small particles.
For individuals with a low LDL-P concentration, the CVD risk is overestimated
if the traditional LDL-C factor is used. In contrast, for individuals with high LDL-P
concentrations but normal LDL-C levels, the CVD risk is underestimated [73].

5.2.2 Lipoprotein Profiles Associated with the Future Development

of Type 2 Diabetes and Insulin Resistance

Type 2 diabetes mellitus has been classified as the epidemic of the twenty-first cen-
tury both for its growing magnitude and its impact on CVD. Determining the factors
associated with the onset of diabetes before it starts (prediabetic signs) is a chal-
lenge representing a breakthrough in CVD prevention.
In this sense, Dr. Samia Mora [74] and Dr. Rafael Carmena [75] argue that
advanced lipoprotein characterization helps to identify individuals at higher risk of
developing cardiometabolic diseases by allowing the identification of a characteris-
tic lipoprotein profile years before the onset of clinical manifestations.
This concept has been reinforced in other relevant works, such as the report pub-
lished in Circulation of the IRAS (Insulin Resistance Atherosclerosis Study) [76].
Bringing Human Serum Lipidomics to the Forefront of Clinical Practice: Two Clinical… 255

The study showed that NMR can be used to identify lipoprotein profiles that are
associated with an increased risk of developing cardiometabolic diseases, such as
type 2 diabetes and insulin resistance. These profiles show high concentrations of
large VDL particles and small HDL particles. This abnormal distribution between
lipoprotein subclasses is reflected in an increased VLDL size and a decreased size
of HDL. Early detection of these particular patterns can help prevent the future
development of chronic hyperglycemia.
In any case, the measurement of lipoprotein profiles is of great interest to those
patients who are exhibiting [77]:
• Family history of atherosclerotic cardiovascular risk
• Elevated triglycerides
• Low levels of HDL-C
• Metabolic syndrome
• Diabetes mellitus
• Secondary prevention: recurrent events due to atherosclerotic cardiovascular risk
despite intervention in lifestyle changes and/or administration of lipid-­
lowering therapy
A recent study by Puig-Jove et al. [78] in the journal Revista Española de
Cardiología provides a clinical overview of the use of 1H NMR serum lipidomics
to directly assess the number, composition, and size of the different lipoprotein
particles in other areas of cardiovascular research, such as premature cardiovascular
disease (CVD) or heart failure.
Several methodological approaches have been commercially developed to quan-
tify blood lipoproteins from NMR spectra. The first commercially available was
based on a deconvolution method (LipoProfile, LabCorp Inc., USA) and includes
decomposition of the NMR signal into individual signals obtained from a library of
NMR spectra for isolated lipoproteins. Another alternative based on linear regres-
sion modeling for lipid prediction was developed by Mika Ala-Korpela and is cur-
rently commercialized by the Nightingale Health Ltd., Finland [79, 80]. These
approaches do not allow direct quantification of the particle size and provide an
indirect measurement of the concentration of particles and estimation of the size.
As an alternative to the abovementioned one-dimensional 1H NMR methods,
two-dimensional 1H NMR appears as a second-generation approach for lipoprotein
characterization. Diffusion Ordered Nuclear Magnetic Resonance Spectroscopy
(DOSY-NMR) is a type of NMR experiment that can be used to measure the hydro-
dynamic characteristics of molecules. This includes the diffusion coefficient, which
is a measure of how fast the molecules move through a solution. The diffusion coef-
ficient of each subclass of lipoprotein can be measured using DOSY-NMR. The
sizes of the different lipoprotein subclasses can then be calculated directly from the
diffusion coefficients using the Stokes-Einstein equation. However, it is important
to directly measure the size of the lipoprotein particles since this is used to calculate
the number of lipoprotein particles.
Therefore, the two-dimensional 1H NMR approach allows direct calculation of
the sizes and gives more precise particle concentrations than can be obtained using
256 N. Amigó Grau and P. Ortiz Betes

one-dimensional NMR methods by showing better correlations between [1] the

LDL-P and ApoB content of isolated LDL fractions, [2] the VLDL-P and ApoB
content of isolated VLDL fractions, and [3] the HDL-P and ApoA-I content of HDL
fractions [81].
This methodology was developed recently (Liposcale®, CE marked) and is cur-
rently commercialized in Spain ([Link]). The technology is also avail-
able as a CLIA lab test for most EU countries. The launch in the USA is also
expected for 2023. This advanced lipoprotein profiling method determines the num-
ber of lipoprotein particles from each of the three main classes of lipoproteins
(VLDL, LDL, and HDL) and their three subclasses (large, medium, and small). The
size of each lipoprotein class is also determined, as well as the cholesterol and tri-
glyceride content in each fraction, including remnant cholesterol.

5.2.3 Other Diseases and Applications

LC-MS-based and NMR lipidomics has enormous potential to contribute to improv-

ing the treatment, understanding and diagnosis of complex disease, as was already
shown for NAFLD. Several other conditions have been explored in the last decade
with promising results. A short selection of studies is discussed now to show the
heterogeneity of the conditions and the potential of the diagnostic tools under devel-
opment (Table 3).
The earlier examples show the diversity and dimension of the lipidomic and
metabolomic fields for clinical applications. Still, they need to follow a long path-
way to clinical use. As shown in the section discussing the OWLiver panel, these
candidates should be validated in big cohorts from a wide range of patients, and
“revalidation” in independent cohorts will be required for international
recognition.
The field of potential applications for NMR metabolomics is certainly broad:
beyond advanced lipoprotein profiling, 1H NMR technology is capable of simulta-
neously detecting the presence of a set of blood metabolites (and/or combinations
thereof), such as glycoproteins, LMWM (amino acids, sugars), and lipid species
that are associated with chronic inflammatory processes and the risk of development
of other metabolic diseases and that can therefore be used as early biomarkers in
different diseases complementing lipoprotein profiling.
Protein Glycation A change in the protein glycosylation pattern has been identi-
fied as a major event that occurs during the transition from healthy to diseased tis-
sue, with significant changes being observed during chronic inflammatory processes,
such as obesity, metabolic syndrome, polycystic ovaries syndrome, or rheumatoid
arthritis [72, 84–86].
Recently, glyc-A, an NMR-derived biomarker, has been shown to be an indepen-
dent risk factor for CVD inflammatory diseases, as well as being linked with the
residual risk of CVD and death in patients treated with low LDL cholesterol levels
[87–90].
Bringing Human Serum Lipidomics to the Forefront of Clinical Practice: Two Clinical… 257

Table 3 Potential clinical applications of LC-MS

Biomarkers for disease diagnosis
Idiopathic Seijo et al. showed that a subset of five metabolites can differentiate patients
noncirrhotic with idiopathic noncirrhotic portal hypertension from patients with liver
portal cirrhosis and healthy volunteers (AUROC = 0.8871 [0.838–0.924]). The model
hypertension can diagnose or exclude idiopathic non-cirrhosis portal hypertension using
high or low cut-off values, respectively
Multiple Villoslada et al. [82] identified metabolomic signatures for distinguishing
sclerosis patients from controls with high accuracy and for classifying patients with a
medium to high disability (EDSS 3.0). From their time series analysis,
sphingomyelin and lysophosphatidylethanolamine were reported to be the
most robust metabolites for discriminating between patients and controls
Alzheimer’s Olarazan et al. developed a panel with seven metabolites that could
disease differentiate Alzheimer’s disease patients from those with amnestic mild
cognitive impairment. Seven metabolites were used in the final panel: Three
amino acids (glutamic acid, alanine, and aspartic acid), one non-esterified fatty
acid (22:6n-3, DHA), one bile acid (deoxycholic acid), one
phosphatidylethanolamine [PE (36:4)], and one sphingomyelin [SM (39:1)]
Colorectal Cubiella et al. [83] measured the levels of 105 metabolites in feces, and found
cancer that 18 were significantly different in patients with advanced neoplasia
compared to controls. Seven metabolites, namely, ChoE (18:1), ChoE (18:2),
ChoE (20:4), PE (16:0/18:1), SM (d18:1/23:0), SM (42:3), and TG (54:1),
could be used to discriminate advanced neoplasia patients from healthy
controls. A predictive model establishing using these metabolites exhibited an
AUC value of 0.821 for cancer diagnosis
Alcoholic In a study with 90 patients with alcoholic hepatitis and alcoholic cirrhosis,
hepatitis Michelena et al. were able to build an algorithm using only 4 metabolites to
diagnose alcoholic hepatitis with an AUC value of 0.932. With another four
metabolites, it was possible to select alcoholic hepatitis patients with poor
prognosis

LMWM and Specific Lipid Families Dysregulated lipid metabolism is an impor-

tant and well-known risk factor in cardiovascular diseases. Understanding the whole
lipidome signature in vascular pathophysiology is a current challenge in CVD
research [91]. On the other hand, aqueous metabolites, including branched-chain
and aromatic amino acids, FFAs, and some low-molecular-weight metabolite prod-
ucts of energetic and nitrogenate metabolism, like glycerol, are predictors of insulin
resistance and the development of hyperglycemia and type 2 diabetes. It was
reported that insulin resistance is linked to higher plasma glutamate levels but lower
plasma glutamine levels and glutamine/glutamate ratios. The study also found that
an excess of glutamine in blood relative to glutamate was associated with a reduced
risk of incidence for T2DM [92].
The clinical development of high-throughput NMR for biomedical screening has
recently experienced a significant expansion. Now, it is possible to obtain advanced
metabolic profiling that includes the detailed lipoprotein profile and a complemen-
tary set of information related to the cardiometabolic status and inflammation,
including a systemic glycoprotein profile and the concentrations of LMWM simul-
taneously. The whole analysis is compatible with the clinical requirements
258 N. Amigó Grau and P. Ortiz Betes

regarding time, reproducibility, and scalability. It is expected to open a new horizon

in evaluating metabolism and position lipidomics toward precision medicine.
In that sense, the present and future of NMR clinical applications—complement-
ing the CE-IVD Liposcale® test for lipoprotein profiling—seems to be a winning
strategy shortly approaching affordable refined bioscreening tools to the patients
(Table 4).

Table 4 Potential clinical applications of NMR

Biomarkers for disease events and risk prediction
CVD Phenylalanine and MUFAs are predictive for a high risk of CVD events.
Omega-6 fatty acids and docosahexaenoic acid levels are inversely
associated with the risk of a CVD event [93]
The 5-year risk of Glycoprotein acetylation, albumin, and VLDL particle size [94]
death
DM2 Eight amino acids with glycemia, branched-chained and aromatic amino
acids, alanine, and glutamine were predictive of diabetes risk. Ketones
acetoacetate and β-hydroxybutyrate, lipids, and lipoprotein subclass
measures are associated with glycemia and type 2 diabetes risk [95–97]
Inborn errors NMR-based screening of newborn urine is currently the optimum method
for detecting inherited errors of metabolism [98, 99]
Prognosis for HIV A baseline NMR serum metabolomic signature is associated with
patients immunological CD4(+) T-cell recovery in HIV-infected patients [100]
Chronic kidney Lipoprotein alterations [101, 102]
disease
Clinical oncology Branched-chain amino acids, lipoprotein alterations, and glycoprotein
and cancer research profile alteration [103–105]
Parkinson’s Glycoprotein alterations and HDL composition [106, 107]
cognitive
impairment
Drug interventions
Statin therapy Lower levels of small VLDL particles and remnant cholesterol, in addition
to the LDL-lowering effects [108]
Hormone therapy Changes in many fatty acids and amino acids [109]
PPAR-α/PPAR-γ Lower proatherogenic profile and remnant cholesterol, in addition to the
agonist for TG-lowering effects and inflammatory GlycA markers profile [110]
treatment of
NAFLD
Metabolic risk factor characterization
Adiposity Causal effects on numerous metabolic measures: Branched-chain and
aromatic amino acids, omega-6 fatty acids, and glycoprotein acetylation,
as well as multiple sizes and lipid classes of lipoprotein [111]
Insulin resistance Lipoprotein subclass profiling [112]
Birth weight Metabolic signature as the metabolite association pattern with higher
adiposity or future cardiometabolic risk [113–115]
Menopause/aging Glutamine, tyrosine, and isoleucine, in addition to the atherogenic
lipoprotein pattern [116]
Alcohol Biomarkers for alcohol intake beyond routine lipids, including adverse
consumption associations with omega-6 fatty acids, MUFAs, glutamine, and citrate
[117]
Vitamin D Large VLDL and small LDL subclasses and related measures, for
example, serum triglycerides [118]
Bringing Human Serum Lipidomics to the Forefront of Clinical Practice: Two Clinical… 259

6 Concluding Remarks and Future Perspective

The advances in lipidomic profiling techniques in the last two decades have opened
up a new era of research into the role of lipids in health and disease, significantly
improving our understanding of the underlying biological processes involved. More
recently, the related technologies in specific fields, such as NMR lipoprotein profil-
ing and LC-MS liver health evaluation, reached the clinical environment after high-­
throughput sample preparation, and screening became feasible. These technologies
have been shown to have clinical utility in several applications and applied to the
analysis of large cohort clinical trials and are cost-effective in the diagnosis of sub-
clinical CVD and metabolic-related conditions, such as metabolic-associated fatty
liver disease (MAFLD) evaluation and stratification.
Currently, many lipidomic profiling applications are crossing the barrier from
research to clinical application. The technology is now being applied in some
advanced clinical settings, and it has the potential to be widely used in the future
to diagnose diseases, stratify risk, and monitor the efficacy of all kinds of treat-
ments. Although the technology and techniques are still under development, lipi-
domic profiling has the potential to revolutionize the way we diagnose and treat
diseases. For example, it could be used to identify biomarkers for diseases that are
currently difficult to diagnose, such as cancer and Alzheimer’s disease. It could
also be used to stratify risk for diseases, so that we can better target treatments to
those who are most likely to benefit. Additionally, it could be used to monitor the
efficacy of treatments, so that clinicians can check that they are working and that
patients are not experiencing any adverse side effects. The widespread use of this
technology has the potential to improve the health of millions of people around
the world.
The global objective is to implement NMR and LC-MS lipidomics as precise and
personalized technologies in an impactful way, to scale and make them accessible
and effective to help improve the time-efficiency and cost-effectiveness of health-
care providers around the world. Once clinical lipidomic platforms have been devel-
oped and validated, they will need to be approved by the relevant regulatory body in
the country where they will be used, as has already been done for the first platforms
in the EU and USA. This is an important procedure, and it helps to ensure that the
platforms are safe and effective.
Specific applications based on MS and NMR are already accessible around the
world. However, the cost of the equipment is high, and the potential of the technol-
ogy is greater than the currently developed applications. The next step is to integrate
lipidomic and lipoprotein profiling into clinical practice. For this to be achieved, the
standardization of sample preparation is essential to ensure the accuracy and repro-
ducibility of results. Moreover, the streamlining of analytical procedures will make
lipidomic profiling more accessible to a wider range of laboratories, and the estab-
lishment of metabolite databases will allow for the rapid identification of lipids and
the comparison of results between different laboratories.
260 N. Amigó Grau and P. Ortiz Betes

6.1 Envisaged Risks and Limitations of Clinical Lipidomics

Despite the technological advances, the implementation of new technologies into

the clinical routine is not high-throughput. We have identified the principal risks for
introducing lipidomics into clinical workflows into either clinical and implementa-
tion risks related to the adoption of current LC-MS and NMR approaches and tech-
nical and regulatory risks.
Clinical and Implementation Risks Lipidomic applications are based on highly
innovative technologies for bioscreening purposes. Although lipidomics has repeat-
edly been demonstrated to be cost-effective for CVD management, the price of the
technology surpasses that of the gold standard, which is a significant barrier in
stressed healthcare systems.
Moreover, lipidomic applications are primarily focused on cardiometabolic dis-
eases (CMD). However, lifestyle interventions and lifestyle changes can easily
address obesity and concomitant metabolic alterations; the more expensive applica-
tions are unlikely to be easily implemented. Innovative solutions may be addressed
after adherence to therapeutic improvement and lifestyle interventions, and comple-
mentary technologies can appear before the present solutions are broadly applied.
However, personalized systems could still be applied to create patient awareness
about personalized health status, favoring the adoption of a healthy lifestyle.
Technological Risks Molecular signatures based on LC-MS-NMR are not suffi-
cient to explain the variability associated with cardiometabolic diseases and fail to
identify individuals at higher risk of cardiometabolic risk (CMR). Alternatively,
developments may produce faster diagnostic tests based on other technologies.
However, based on previous evidence-based literature, lipidomic approaches have
demonstrated discriminatory capacity among several diseases. They can be used for
advanced lipoprotein testing (relevant for CVD events), which has already been
recommended in the clinical guidelines for risk management in specific patients in
countries such as the USA, Canada, and Spain. Moreover, these technologies could
still be applied to future metabolomic profiling in pharmacological interventions,
clinical trials, or other clinical cost-effective applications (CVD, inborn errors).

Regulatory Difficulties Although regulatory bodies currently encourage novel

technologies, large amounts of time and resources are required for approval by a
regulatory body. Particularly for USFDA certification, if there is no precedent, novel
technologies may experience serious delays before clinical introduction, even when
promoted by previous IVD-CE marketing equivalent technology, threatening the
economic sustainability of the whole technological development. Based on solu-
tions presented in this chapter, lipidomics has already crossed the regulatory barri-
ers under the risk classification ISO 13485:2016, requiring technical file preparation,
CE declaration, and registry with the European Competent Authority. For future
lipidomic applications, the established regulatory strategy might be useful. The
technical documentation must provide evidence of conformance with the essential
Bringing Human Serum Lipidomics to the Forefront of Clinical Practice: Two Clinical… 261

requirements of 98/79/EC and the imminent regulation (EU) 2017/746 on in vitro

diagnostic medical devices.
Despite the difficulties, the use of lipidomic technologies as diagnostic, prognos-
tic, and evaluation tools is expected to expand enormously as a result of the develop-
ment of modern medicine.
Lipidomic-based applications are expected to increase awareness regarding the
need for diagnostic and prognostic technologies for other diseases. Lipidomics will
help clinical assessment of different pathologies, spread the importance of
metabolomic-­based approaches, and ensure uptake of the already developed appli-
cations by clinicians, hospital decision-makers, and regulators.
The introduction of advanced molecular profiling is aligned with the global strat-
egy to incorporate personalized screening tools to define better monitoring and
treatment strategies that will improve healthcare systems and empower clinicians
with more detailed information, allowing an early therapeutic response and thus
reducing the development of life-threatening symptoms.
The presented lipidomic-based technologies for liver disease and lipoprotein
characterization exemplify the possibility of deploying advanced molecular screen-
ing tools in the clinical workflow. Beyond these two examples, using “omic”
approaches to unravel the complexity of diseases will accelerate the understanding
of diseases, help identify altered mechanisms that have not been described previ-
ously, discover prognostic biomarkers, monitor health status, and select better treat-
ments and therapies facilitating the healthcare transformation toward personalized
medicine.

References

1. Idle JR, Gonzalez FJ. Metabolomics. Cell Metab. 2007;6(5):348–51.

2. Monteiro MS, Carvalho M, Bastos ML, Guedes de Pinho P. Metabolomics analysis for bio-
marker discovery: advances and challenges. Curr Med Chem. 2013;20(2):257–71.
3. Rhee EP, Gerszten RE. Metabolomics and cardiovascular biomarker discovery. Clin Chem.
2012;58(1):139–47.
4. Zhang A, Sun H, Yan G, Wang P, Wang X. Metabolomics for biomarker discovery: moving to
the clinic. Biomed Res Int. 2015;2015:354671.
5. Dennis EA. Lipidomics joins the omics evolution. Proc Natl Acad Sci U S
A. 2009;106(7):2089–90.
6. Quehenberger O, Armando AM, Brown AH, Milne SB, Myers DS, Merrill AH, et al.
Lipidomics reveals a remarkable diversity of lipids in human plasma. J Lipid Res.
2010;51(11):3299–305.
7. Meikle TG, Huynh K, Giles C, Meikle PJ. Clinical lipidomics: realizing the potential of lipid
profiling. J Lipid Res. 2021;62:100127.
8. Burla B, Arita M, Arita M, Bendt AK, Cazenave-Gassiot A, Dennis EA, et al. MS-based
lipidomics of human blood plasma: a community-initiated position paper to develop accepted
guidelines. J Lipid Res. 2018;59(10):2001–17.
9. Li J, Vosegaard T, Guo Z. Applications of nuclear magnetic resonance in lipid analyses: an
emerging powerful tool for lipidomics studies. Prog Lipid Res. 2017;68:37–56.
262 N. Amigó Grau and P. Ortiz Betes

10. Havel RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifu-
gally separated lipoproteins in human serum. J Clin Invest. 1955;34(9):1345–53.
11. Yeagle PL. Lipid regulation of cell membrane structure and function. FASEB
J. 1989;3(7):1833–42.
12. Magos L. C. Lentner (ed.). Geigy Scientific Tables, 8th edition. Vol. 1. Units of Measurement.
Body Fluids. Composition of the Body. Nutrition. 1981, 298 pp. Vol. 2. Introduction
to Statistics. Statistical Tables. Mathematical Formulae. 1982, 241 pp. Vol. 3. Physical
Chemistry. Composition of the Blood. Haematology. Human Somatometric Data. 1984,
359 pp. Vol. 4. Biochemistry. Metabolism of Xenobiotics. Inborn Error of Metabolism.
Pharmacogenetics and Ecogenetics. 1986, 330 pp. Ciba-Geigy, Basel, £12.50 each volume.
Distributed in U.K. by Farrand Press. J Appl Toxicol. 1987;7(6):413.
13. Fahy E, Subramaniam S, Brown HA, Glass CK, Merrill AH Jr, Murphy RC, et al. A compre-
hensive classification system for lipids. J Lipid Res. 2005;46(5):839–61.
14. Sunshine H, Iruela-Arispe ML. Membrane lipids and cell signaling. Curr Opin Lipidol.
2017;28(5):408–13.
15. Psychogios N, Hau DD, Peng J, Guo AC, Mandal R, Bouatra S, et al. The human serum
metabolome. PloS One. 2011;6(2):e16957.
16. Quehenberger O, Dennis EA. The human plasma lipidome. N Engl J Med.
2011;365(19):1812–23.
17. Roth GA, Mensah GA, Johnson CO, Addolorato G, Ammirati E, Baddour LM, et al. Global
burden of cardiovascular diseases and risk factors, 1990-2019: update from the GBD 2019
study. J Am Coll Cardiol. 2020;76(25):2982–3021.
18. Riazi K, Azhari H, Charette JH, Underwood FE, King JA, Afshar EE, et al. The preva-
lence and incidence of NAFLD worldwide: a systematic review and meta-analysis. Lancet
Gastroenterol Hepatol. 2022;7(9):851–61.
19. Langlois MR, Nordestgaard BG, Langsted A, Chapman MJ, Aakre KM, Baum H, et al.
Quantifying atherogenic lipoproteins for lipid-lowering strategies: consensus-based recom-
mendations from EAS and EFLM. Clin Chem Lab Med. 2020;58(4):496–517.
20. McGill HC Jr. The pathogenesis of atherosclerosis. Clin Chem. 1988;34(8b):B33–9.
21. Napoli C, Pignalosa O, de Nigris F, Sica V. Childhood infection and endothelial dysfunction:
a potential link in atherosclerosis? Circulation. 2005;111(13):1568–70.
22. Stary HC, Blankenhorn DH, Chandler AB, Glagov S, Insull W Jr, Richardson M, et al. A
definition of the intima of human arteries and of its atherosclerosis-prone regions. A report
from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart
Association. Circulation. 1992;85(1):391–405.
23. Bao W, Srinivasan SR, Wattigney WA, Berenson GS. Persistence of multiple cardiovascular
risk clustering related to syndrome X from childhood to young adulthood. The Bogalusa
Heart Study. Arch Intern Med. 1994;154(16):1842–7.
24. Peters SA, den Ruijter HM, Palmer MK, Grobbee DE, Crouse JR 3rd, O’Leary DH, et al.
Extensive or restricted ultrasound protocols to measure carotid intima-media thickness:
analysis of completeness rates and impact on observed rates of change over time. J Am Soc
Echocardiogr. 2012;25(1):91–100.
25. Li S, Chen W, Srinivasan SR, Bond MG, Tang R, Urbina EM, et al. Childhood cardiovas-
cular risk factors and carotid vascular changes in adulthood: the Bogalusa Heart Study.
JAMA. 2003;290(17):2271–6.
26. Ouimet M, Barrett TJ, Fisher EA. HDL and reverse cholesterol transport. Circ Res.
2019;124(10):1505–18.
27. Sandesara PB, Virani SS, Fazio S, Shapiro MD. The forgotten lipids: triglycerides, remnant
cholesterol, and atherosclerotic cardiovascular disease risk. Endocr Rev. 2019;40(2):537–57.
28. Choi SS, Diehl AM. Hepatic triglyceride synthesis and nonalcoholic fatty liver disease. Curr
Opin Lipidol. 2008;19(3):295–300.
29. Page JM, Harrison SA. NASH and HCC. Clin Liver Dis. 2009;13(4):631–47.
30. Day CP, James OF. Steatohepatitis: a tale of two “hits”? Gastroenterology. 1998;114(4):842–5.
Bringing Human Serum Lipidomics to the Forefront of Clinical Practice: Two Clinical… 263

31. Bellentani S, Scaglioni F, Marino M, Bedogni G. Epidemiology of non-alcoholic fatty liver

disease. Dig Dis. 2010;28(1):155–61.
32. Masoodi M, Gastaldelli A, Hyötyläinen T, Arretxe E, Alonso C, Gaggini M, et al.
Metabolomics and lipidomics in NAFLD: biomarkers and non-invasive diagnostic tests. Nat
Rev Gastroenterol Hepatol. 2021;18(12):835–56.
33. Puri P, Baillie RA, Wiest MM, Mirshahi F, Choudhury J, Cheung O, et al. A lipidomic analy-
sis of nonalcoholic fatty liver disease. Hepatology. 2007;46(4):1081–90.
34. García-Cañaveras JC, Donato MT, Castell JV, Lahoz A. A comprehensive untargeted
metabonomic analysis of human steatotic liver tissue by RP and HILIC chromatography
coupled to mass spectrometry reveals important metabolic alterations. J Proteome Res.
2011;10(10):4825–34.
35. Kotronen A, Seppänen-Laakso T, Westerbacka J, Kiviluoto T, Arola J, Ruskeepää AL, et al.
Hepatic stearoyl-CoA desaturase (SCD)-1 activity and diacylglycerol but not ceramide con-
centrations are increased in the nonalcoholic human fatty liver. Diabetes. 2009;58(1):203–8.
36. Barr J, Caballería J, Martínez-Arranz I, Domínguez-Díez A, Alonso C, Muntané J, et al.
Obesity-dependent metabolic signatures associated with nonalcoholic fatty liver disease pro-
gression. J Proteome Res. 2012;11(4):2521–32.
37. Barr J, Vázquez-Chantada M, Alonso C, Pérez-Cormenzana M, Mayo R, Galán A, Caballería
J, Martín-Duce A, Tran A, Wagner C, Luka Z, Lu SC, Castro A, Le Marchand-Brustel
Y, Martínez-Chantar ML, Veyrie N, Clément K, Tordjman J, Gual P, Mato JM. Liquid
chromatography-­mass spectrometry-based parallel metabolic profiling of human and mouse
model serum reveals putative biomarkers associated with the progression of nonalcoholic
fatty liver disease. J Proteome Res. 2010;9(9):4501–12.
38. Mayo R, Crespo J, Martínez-Arranz I, Banales JM, Arias M, Mincholé I, et al. Metabolomic-­
based noninvasive serum test to diagnose nonalcoholic steatohepatitis: results from discovery
and validation cohorts. Hepatol Commun. 2018;2(7):807–20.
39. Mishra A, Younossi ZM. Epidemiology and natural history of non-alcoholic fatty liver dis-
ease. J Clin Exp Hepatol. 2012;2(2):135–44.
40. Bril F, Millán L, Kalavalapalli S, McPhaul MJ, Caulfield MP, Martinez-Arranz I, et al. Use
of a metabolomic approach to non-invasively diagnose non-alcoholic fatty liver disease in
patients with type 2 diabetes mellitus. Diabetes Obes Metab. 2018;20(7):1702–9.
41. Sachdeva A, Cannon CP, Deedwania PC, Labresh KA, Smith SC Jr, Dai D, et al. Lipid levels
in patients hospitalized with coronary artery disease: an analysis of 136,905 hospitalizations
in get with the guidelines. Am Heart J. 2009;157(1):111–7.e2.
42. Langlois MR, Chapman MJ, Cobbaert C, Mora S, Remaley AT, Ros E, et al. Quantifying
atherogenic lipoproteins: current and future challenges in the era of personalized medicine
and very low concentrations of LDL cholesterol. A consensus statement from EAS and
EFLM. Clin Chem. 2018;64(7):1006–33.
43. Tehrani DM, Zhao Y, Blaha MJ, Mora S, Mackey RH, Michos ED, et al. Discordance of
low-density lipoprotein and high-density lipoprotein cholesterol particle versus cholesterol
concentration for the prediction of cardiovascular disease in patients with metabolic syn-
drome and diabetes mellitus (from the multi-ethnic study of atherosclerosis [MESA]). Am J
Cardiol. 2016;117(12):1921–7.
44. Borén J, Williams KJ. The central role of arterial retention of cholesterol-rich apolipoprotein-­
B-­containing lipoproteins in the pathogenesis of atherosclerosis: a triumph of simplicity. Curr
Opin Lipidol. 2016;27(5):473–83.
45. Castañer O, Pintó X, Subirana I, Amor AJ, Ros E, Hernáez Á, et al. Remnant cholesterol,
not LDL cholesterol, is associated with incident cardiovascular disease. J Am Coll Cardiol.
2020;76(23):2712–24.
46. Ference BA, Graham I, Tokgozoglu L, Catapano AL. Impact of lipids on cardiovascular
health: JACC health promotion series. J Am Coll Cardiol. 2018;72(10):1141–56.
47. Cole TG, Contois JH, Csako G, McConnell JP, Remaley AT, Devaraj S, et al. Association of
apolipoprotein B and nuclear magnetic resonance spectroscopy-derived LDL particle num-
264 N. Amigó Grau and P. Ortiz Betes

ber with outcomes in 25 clinical studies: assessment by the AACC Lipoprotein and Vascular
Diseases Division Working Group on Best Practices. Clin Chem. 2013;59(5):752–70.
48. Pichler G, Amigo N, Tellez-Plaza M, Pardo-Cea MA, Dominguez-Lucas A, Marrachelli VG,
et al. LDL particle size and composition and incident cardiovascular disease in a South-­
European population: the Hortega-Liposcale Follow-up Study. Int J Cardiol. 2018;264:172–8.
49. Mach F, Baigent C, Catapano AL, Koskinas KC, Casula M, Badimon L, et al. 2019 ESC/EAS
guidelines for the management of dyslipidaemias: lipid modification to reduce cardiovascular
risk. Eur Heart J. 2020;41(1):111–88.
50. Masana L, Ibarretxe D. Magnetic resonance-assessed lipoprotein profile. The time has come
for its clinical use. Rev Esp Cardiol (Engl ed). 2022;75(1):5–8.
51. Aday AW, Lawler PR, Cook NR, Ridker PM, Mora S, Pradhan AD. Lipoprotein par-
ticle profiles, standard lipids, and peripheral artery disease incidence. Circulation.
2018;138(21):2330–41.
52. Mora S, Buring JE, Ridker PM. Discordance of low-density lipoprotein (LDL) choles-
terol with alternative LDL-related measures and future coronary events. Circulation.
2014;129(5):553–61.
53. Otvos JD, et al. Development of a proton nuclear magnetic resonance spectroscopic method
for determining plasma lipoprotein concentrations and subspecies distributions from a single,
rapid measurement. Clin Chem. 1992;38:1632.
54. Folse HJ, Goswami D, Rengarajan B, Budoff M, Kahn R. Clinical-and cost-effectiveness of
LDL particle-guided statin therapy: a simulation study. Atherosclerosis. 2014;236(1):154–61.
55. Allaire J, Vors C, Couture P, Lamarche B. LDL particle number and size and cardiovascular
risk: anything new under the sun? Curr Opin Lipidol. 2017;28(3):261–6.
56. Li M, Yang L, Bai Y, Liu H. Analytical methods in lipidomics and their applications. Anal
Chem. 2014;86(1):161–75.
57. Wishart DS. Quantitative metabolomics using NMR. TrAC Trends Anal Chem.
2008;27(3):228–37.
58. Cajka T, Fiehn O. Comprehensive analysis of lipids in biological systems by liquid
chromatography-­mass spectrometry. Trends Analyt Chem. 2014;61:192–206.
59. Jeremy K, Nicholson JCL. Systems biology: metabonomics. Nature. 2008;455(7216):1054–6.
60. DeFilippis AP, Trainor PJ, Hill BG, Amraotkar AR, Rai SN, Hirsch GA, Rouchka EC,
Bhatnagar A. Identification of a plasma metabolomic signature of thrombotic myocardial
infarction that is distinct from non-thrombotic myocardial infarction and stable coronary
artery disease. PLoS One. 2017;12(4):e0175591.
61. Gallo V, Intini N, Mastrorilli P, Latronico M, Scapicchio P, Triggiani M, et al. Performance
assessment in fingerprinting and multi component quantitative NMR analyses. Anal Chem.
2015;87:6709–17.
62. Eckhart AD, Beebe K, Milburn M. Metabolomics as a key integrator for “omic” advancement
of personalized medicine and future therapies. Clin Trans Sci. 2012;5:285–8.
63. Cajka T, Fiehn O. Increasing lipidomic coverage by selecting optimal mobile-phase modi-
fiers in LC–MS of blood plasma. Metabolomics. 2016;12(34).
64. Isaac G, McDonald S, Astarita G. Lipid separation using UPLC with charged surface hybrid
technology. Milford, MA: Waters Corp.
65. Ulmer CZ, Patterson RE, Koelmel JP, Garrett TJ, Yost RAA. Robust lipidomics workflow for
mammalian cells, plasma, and tissue using liquid-chromatography high- resolution tandem
mass spectrometry. Methods Mol Biol. 2017;1609:91–106.
66. Haider A, Wei Y-C, Lim K, Barbosa AD, Liu C-H, Weber U, Mlodzik M, Oras K, Collier
S, Hussain MM, et al. PCYT1A regulates phosphatidylcholine homeostasis from the inner
nuclear membrane in response to membrane stored curvature elastic stress. Dev Cell.
2018;45:481–95.
67. Pawula M, Hawthorne G, Smith GT, Hill HM. Best practice in biological sample collection,
processing, and storage for LC-MS in bioanalysis of drugs. In: Handbook of LC-MS bio-
analysis: best practices, experimental protocols, and regulations; 2013. p. 139–64.
Bringing Human Serum Lipidomics to the Forefront of Clinical Practice: Two Clinical… 265

68. Loo RL, Lodge S, Kimhofer T, Bong SH, Begum S, Whiley L, Holmes E. Quantitative in-
vitro diagnostic NMR spectroscopy for lipoprotein and metabolite measurements in plasma
and serum: recommendations for analytical artifact minimization with special reference to
COVID-19/SARS-CoV-2 samples. J Proteome Res. 2020;19(11):4428–41.
69. Rasmussen DGK, Anstee QM, Torstenson R, Golding B, Patterson SD, Brass C, Karsdal
MA. NAFLD and NASH Biomarker Qualification in the LITMUS consortium–Lessons
learned. J Hepatol. 2022. 2023;78(4):852–65. [Link]
70. Sanyal AJ, Yates KP, Daly E, Dehn CA, Neuschwander-Tetri B, Kowdley KV, Vuppalanchi
RK, Tonascia JA, Samir AE, Sirlin CB, Fowler KJ, Heymann H, Kamphaus TN, Loomba R,
Calle RA; the NIMBLE Project Team. Primary results of the nimble stage 1-NASH CRN
study of circulating biomarkers for nonalcoholic steatohepatitis and its activity and fibrosis
stage. AASLD meeting. 2021.
71. Noureddin MM, et al. Si llega a tiempo la cita ponemos esa, si no referenciamos el abstract
poblicado en J of Hepatology por los mismos autores. Hepatology. 2023.
72. Connelly MA, Gruppen EG, Wolak-Dinsmore J, et al. GlycA, a marker of acute phase gly-
coproteins, and the risk of incident type 2 diabetes mellitus: PREVEND study. Clin Chim
Acta. 2016;452:10–7.
73. Otvos JD, Mora S, Shalaurova I, Greenland P, Mackey RH, Goff DC Jr. Clinical implica-
tions of discordance between LDL cholesterol and LDL particle number. J Clin Lipidol.
2011;5(2):105–13.
74. Mora S, Otvos JD, Rifai N, Rosenson RS, Buring JE, Ridker PM. Lipoprotein particle pro-
files by nuclear magnetic resonance compared with standard lipids and apolipoproteins in
predicting incident cardiovascular disease in women. Circulation. 2009;119:931–9.
75. Carmena R, Duriez P, Fruchart JC. Atherogenic lipoprotein particles in atherosclerosis.
Circulation. 2004;109(23 Suppl 1):III2–7.
76. Festa A, Williams K, Hanley AJ, et al. Nuclear magnetic resonance lipoprotein abnormali-
ties in prediabetic subjects in the Insulin Resistance Atherosclerosis Study. Circulation.
2005;111(25):3465–72.
77. Pintó X, Masana L, Civeira F, et al. Consensus document of an expert group from the Spanish
Society of Arteriosclerosis (SEA) on the clinical use of nuclear magnetic resonance to assess
lipoprotein metabolism (Liposcale®). Clin Investig Arterioscler. 2020;32(5):219–29.
78. Puig-Jové C, Castelblanco E, Falguera M, et al. Advanced lipoprotein profile in individuals
with normal and impaired glucose metabolismPerfil lipoproteico avanzado en individuos con
metabolismo glucémico normal y alterado. Rev Esp Cardiol (Engl Ed). 2022;75(1):22–30.
79. Aru V, Lam C, Khakimov B, Hoefsloot HCJ, Zwanenburg G, Lind MV, Schäfer H, van
Duynhoven J, Jacobs DM, Smilde AK, Engelsen SB. Quantification of lipoprotein profiles
by nuclear magnetic resonance spectroscopy and multivariate data analysis. Trends Analyt
Chem. 2017;94:210–9.
80. Ala-Korpela M, Korhonen A, Keisala J, et al. 1H NMR-based absolute quantitation of human
lipoproteins and their lipid contents directly from plasma. J Lipid Res. 1994;35(12):2292–304.
81. Mallol R, Amigó N, Rodríguez MA, et al. Liposcale: a novel advanced lipoprotein test based
on 2D diffusion-ordered 1H NMR spectroscopy. J Lipid Res. 2015;56(3):737–46.
82. Villoslada P, Alonso C, Agirrezabal I, Kotelnikova E, Zubizarreta I, Pulido-Valdeolivas I,
et al. Metabolomic signatures associated with disease severity in multiple sclerosis. Neurol
Neuroimmunol Neuroinflamm. 2017;4(2):e321.
83. Cubiella J, Clos-Garcia M, Alonso C, Martinez-Arranz I, Perez-Cormenzana M, Barrenetxea
Z, et al. Targeted UPLC-MS metabolic analysis of human faeces reveals novel low-invasive
candidate markers for colorectal cancer. Cancers (Basel). 2018;10(9):300.
84. Carmona-Maurici J, Amigó N, Cuello E, et al. Bariatric surgery decreases oxidative stress and
protein glycosylation in patients with morbid obesity. Eur J Clin Investig. 2020;50(11):e13320.
85. Moncayo S, Insenser M, Martínez-García MÁ, et al. Acute-phase glycoprotein profile
responses to different oral macronutrient challenges: influence of sex, functional hyperan-
drogenism and obesity. Clin Nutr. 2021;40(3):1241–6.
266 N. Amigó Grau and P. Ortiz Betes

86. Fuertes-Martín R, Taverner D, Vallvé JC, et al. Characterization of 1H NMR plasma gly-
coproteins as a new strategy to identify inflammatory patterns in rheumatoid arthritis. J
Proteome Res. 2018;17(11):3730–9.
87. Fuertes-Martín R, Correig X, Vallvé JC, Amigó N. Human serum/plasma glycoprotein analy-
sis by 1H-NMR, an emerging method of inflammatory assessment. J Clin Med. 2020;9(2):354.
88. McGarrah RW, Kelly JP, Craig DM, et al. A novel protein glycan-derived inflammation bio-
marker independently predicts cardiovascular disease and modifies the association of HDL
subclasses with mortality. Clin Chem. 2017;63(1):288.
89. Holmes MV, Millwood IY, Kartsonaki C, et al. Lipids, lipoproteins, and metabolites and risk
of myocardial infarction and stroke. J Am Coll Cardiol. 2018;71(6):620–32.
90. Otvos JD, Guyton JR, Connelly MA, et al. Relations of GlycA and lipoprotein particle sub-
species with cardiovascular events and mortality: a post hoc analysis of the AIM-HIGH trial.
J Clin Lipidol. 2018;12(2):348–55.
91. Kohno S, Keenan AL, Ntambi JM, Miyazaki M. Lipidomic insight into cardiovascular dis-
eases. Biochem Biophys Res Commun. 2018;504(3):590–5.
92. Cheng S, Rhee EP, Larson MG, Lewis GD, McCabe EL, Shen D, et al. Metabolite pro-
filing identifies pathways associated with metabolic risk in humans. Circulation.
2012;125(18):2222–31.
93. Würtz P, Havulinna AS, Soininen P, et al. Metabolite profiling and cardiovascular event risk:
a prospective study of 3 population-based cohorts. Circulation. 2015;131(9):774–85.
94. Würtz P, Kangas AJ, Soininen P, Lawlor DA, Davey Smith G, Ala-Korpela M. Quantitative
serum nuclear magnetic resonance metabolomics in large-scale epidemiology: a primer on-­
omic technologies. Am J Epidemiol. 2017;186(9):1084–96.
95. Wang TJ, Larson MG, Vasan RS, et al. Metabolite profiles and the risk of developing diabe-
tes. Nat Med. 2011;17(4):448–53.
96. Ahola-Olli AV, Mustelin L, Kalimeri M, et al. Circulating metabolites and the risk of type 2
diabetes: a prospective study of 11,896 young adults from four Finnish cohorts. Diabetologia.
2019;62(12):2298–309.
97. Hameed A, Mojsak P, Buczynska A, Suleria HAR, Kretowski A, Ciborowski M. Altered
metabolome of lipids and amino acids species: a source of early signature biomarkers of
T2DM. J Clin Med. 2020;9(7):2257.
98. Mussap M, Zaffanello M, Fanos V. Metabolomics: a challenge for detecting and monitoring
inborn errors of metabolism. Ann Transl Med. 2018;6(17):338.
99. Embade N, Cannet C, Diercks T, et al. NMR-based newborn urine screening for optimized
detection of inherited errors of metabolism. Sci Rep. 2019;9:13607.
100. Malo AI, Rull A, Girona J, et al. Glycoprotein profile assessed by 1H-NMR as a global
inflammation marker in patients with HIV infection. A prospective study. J Clin Med.
2020;9(5):1344.
101. Zhang Y, Zhang S, Wang G. Metabolomic biomarkers in diabetic kidney diseases—a system-
atic review. J Diabetes Complicat. 2015;29(8):1345–51.
102. Bermudez-Lopez M, Perpiñan H, Amigo N, et al. Advanced lipoprotein parameters could
better explain atheromatosis in non-diabetic chronic kidney disease patients. Clin Kidney
J. 2021;14(12):2591–9.
103. Elebo N, Omoshoro-Jones J, Fru PN, et al. Serum metabolomic and lipoprotein profiling of
pancreatic ductal adenocarcinoma patients of African ancestry. Meta. 2021;11(10):663.
104. Mayers JR, Wu C, Clish CB, et al. Elevation of circulating branched-chain amino acids is an
early event in human pancreatic adenocarcinoma development. Nat Med. 2014;20(10):1193–8.
105. Schmidt DR, Patel R, Kirsch DG, Lewis CA, Vander Heiden MG, Locasale JW. Metabolomics
in cancer research and emerging applications in clinical oncology. Cancer J Clin.
2021;71(4):333–58.
106. Laguna A, Xicoy H, Tolosa E, et al. Serum metabolic biomarkers for synucleinopathy conver-
sion in isolated REM sleep behavior disorder. npj Parkinsons Dis. 2021;7:40.
Bringing Human Serum Lipidomics to the Forefront of Clinical Practice: Two Clinical… 267

107. Rademacher TD, Meuth SG, Wiendl H, Johnen A, Landmeyer NC. Molecular biomarkers
and cognitive impairment in multiple sclerosis: state of the field, limitations, and future
direction–a systematic review and meta-analysis. Neurosci Biobehav Rev. 2023;146:105035.
108. Langlois MR, Chapman MJ, Cobbaert C, et al. Quantifying atherogenic lipoproteins: current
and future challenges in the era of personalized medicine and very low concentrations of LDL
cholesterol. A consensus statement from EAS and EFLM. Clin Chem. 2018;64(7):1006–33.
109. Karppinen JE, Törmäkangas T, Kujala UM, et al. Menopause modulates the circulating metab-
olome: evidence from a prospective cohort study. Eur J Prev Cardiol. 2022;29(10):1448–59.
110. Gawrieh S, Noureddin M, Loo N, et al. Saroglitazar, a PPAR-α/γ agonist, for treat-
ment of NAFLD: a randomized controlled double-blind phase 2 trial. Hepatology.
2021;74(4):1809–924.
111. O’Keeffe LM, Bell JA, O’Neill KN, et al. Sex-specific associations of adiposity with cardio-
metabolic traits in the UK: a multi–life stage cohort study with repeat metabolomics. PLoS
Med. 2022;19(1):e1003636.
112. Iida M, Harada S, Takebayashi T. Application of metabolomics to epidemiological studies of
atherosclerosis and cardiovascular disease. J Atheroscler Thromb. 2019;26(9):747–57.
113. Miranda J, Simões RV, Paules C, et al. Metabolic profiling and targeted lipidomics in small
for gestational age and foetal growth restriction. Sci Rep. 2018;9:13614.
114. Youssef L, Simões RV, Miranda J, et al. Paired maternal and fetal metabolomics reveal a differ-
ential fingerprint in preeclampsia versus fetal growth restriction. Sci Rep. 2021;11(1):14422.
115. Algaba-Chueca F, Maymó-Masip E, Ballesteros M, et al. Cord blood advanced lipoprotein
testing reveals an interaction between gestational diabetes and birth-weight and suggests a
new early biomarker of infant obesity. Biomedicine. 2022;10(5):1033.
116. Panyard DJ, Yu B, Snyder MP. The metabolomics of human aging: advances, challenges, and
opportunities. Sci Adv. 2022;8(42):eadd6155.
117. Silva RA, Pereira TCS, Souza AR, Ribeiro PR. 1H NMR-based metabolite profiling for bio-
marker identification. Clin Chim Acta. 2020;502:269–79.
118. Bashir NA, Bashir AAM, Bashir HA. Effect of vitamin D deficiency on lipid profile. Am J
Lab Med. 2019;4(1):11–8.
LC-MS-Based Population Metabolomics:
A Mini-Review of Recent Studies
and Challenges from Sample Collection
to Data Processing

Myriam Mireault and Lekha Sleno

Abstract Metabolomics aims to identify and quantify metabolites in biological

samples to understand better biological changes resulting from lifestyle, environ-
ment, or disease. This is challenging due to the structural diversity of the metabo-
lites and the complexity of samples of interest, such as blood and urine, useful in
population studies to study biological changes in large cohorts. The limited number
of commercially available standards and incomplete metabolite spectral databases
impedes the identification of many metabolites. Furthermore, the need for more
standardization in sample preparation, analysis, and interpretation of data is an
important issue that can influence results in large cohort studies. Variations or errors
occurring during the pre-analytical stage can highly affect levels of metabolites. In
this mini-review, we outline the challenges associated with population metabolomic
studies and show an overview of current practices in the field with some case studies.

Keywords Population studies · Metabolomics · Sample collection · Analytical

considerations · Data processing · Mini-review · Liquid chromatography · Mass
spectrometry

Abbreviations

APCI Atmospheric pressure chemical ionization

ASQ Age and stage questionnaire
BMI Body mass index
CI Chemical ionization
COMETS COnsortium of METabolomic Studies
CV Coefficient of variation

M. Mireault · L. Sleno (*)

Chemistry Department/CERMO-FC, Université du Québec à Montréal (UQAM),
Montreal, QC, Canada
e-mail: [Link]@[Link]

© The Author(s), under exclusive license to Springer Nature Singapore Pte 269
Ltd. 2023
A. M. Abdel Rahman (ed.), Clinical Metabolomics Applications in Genetic
Diseases, [Link]
270 M. Mireault and L. Sleno

DNA Deoxyribonucleic acid

EDIH Empirical dietary index of hyperinsulinemia
EDTA Ethylenediaminetetraacetic acid
EI Electron ionization (electron impact)
ESI Electrospray ionization
FDR False discovery rate
GC-MS Gas chromatography-mass spectrometry
HDL High-density lipoprotein
HMDB Human metabolome database
IS Internal standard
LC-MS Liquid chromatography-mass spectrometry
LOD Limit of detection
LTR Long-term reference
MALDI Matrix-assisted laser desorption/ionization
MAR Missing at random
MCAR Missing completely at random
MNAR Missing not at random
MS Mass spectrometry
NIST National Institute of Standards and Technology
NMR Nuclear magnetic resonance
OPLS-DA Orthogonal partial least-squares discriminant analysis
PBS Phosphate-buffered saline
PCA Principal component analysis
QA Quality assurance
QC Quality control
QqQ Triple quadrupole
QqTOF Quadrupole time of flight
QRILC Quantile regression imputation of left-censored data
RNA Ribonucleic acid
ROC Receiver operating characteristic curve
SRM Standard reference material
VLDL Very-low-density lipoprotein
WGCNA Weighted gene co-expression network analysis

1 Introduction

Metabolomics is one of the most recent branches of “omic” science, studying the
molecules involved in the structure, function, or dynamics of a cell, tissue, or organ-
ism [1, 2]. It encompasses the qualitative and quantitative analysis of metabolites
present in a biological sample to interrogate metabolic pathways. It is possible with
metabolomics to investigate potential therapeutic targets and identify biomarkers
for a disease’s early detection and progression [3, 4]. Small molecule metabolites
(less than 1500 Da) are intermediates or end products of metabolism and comprise
LC-MS-Based Population Metabolomics: A Mini-Review of Recent Studies… 271

a large variety of chemical structures [2, 5]. They are also considered to correlate
more with the phenotype of a tissue or organism than proteins and genes, thereby
allowing fast detection of biological changes [5].
The metabolome’s structural diversity increases the analyses’ complexity com-
pared to the proteome, genome, and transcriptome (Fig. 1). Proteins are combina-
tions of 20 amino acids, while DNA and RNA are each composed of 4 nucleotides.
Metabolites are formed from different endogenous or exogenous compounds and do
not have predefined structures [6]. Isomeric metabolites have different structures
but identical exact mass, increasing the difficulty of metabolomic analyses [5].
The choice of a biological system can influence the metabolome. Urine and
blood are commonly used in metabolomics because of their wide range of metabo-
lites and their accessibility, as well as having the ability to represent an overall pic-
ture of metabolism from different organs and tissues [7]. Measuring polar metabolites
in urine can inform on the major elimination pathways from food, drugs, and envi-
ronmental contaminants [8]. Blood is composed of polar and non-polar molecules,
excreted or secreted by tissues [9]. Thus, these two biofluids offer a global view of
a person’s exposure and health status [7].
In metabolomics, two approaches can be employed; targeted, and untargeted
analysis. The targeted approach quantifies a limited number of known metabolites
in a sample. However, the small number of known metabolites and commercially
available pure reference standards limits this approach in epidemiological studies.
The untargeted analysis provides a more comprehensive view by identifying known
and unknown metabolites. It requires a nonselective and reproducible preparation to

Fig. 1 The complexity of the different omic sciences, showing structural diversity involved in
metabolomic studies
272 M. Mireault and L. Sleno

avoid any change in the metabolic profile [10]. Due to a lack of standardization of
protocols, several pre-analytical, analytical, and post-analytical approaches may
influence the results of such studies. Common practices and challenges related to
epidemiological studies using urine and blood will be discussed in this chapter.

2 Pre-Analytical Factors

The pre-analytical steps (Figs. 2 and 3) greatly impact the results, corresponding to
60–80% of laboratory errors [11, 12]. Due to the presence of enzymes, metabolism
continues even after biological samples are collected unless properly quenched,
resulting in varying levels of some compounds [7]. Sample degradation is also an
important factor to consider when analyzing metabolites with limited stability.
Therefore, sample collection, preparation, and storage must be considered crucial
steps in metabolomic studies [13].

Fig. 2 Pre-analytical steps involved in metabolomic analyses of blood samples

Fig. 3 Pre-analytical steps to consider for urine sample

LC-MS-Based Population Metabolomics: A Mini-Review of Recent Studies… 273

2.1 Blood Samples

Whole blood, plasma, or serum is usually prepared for metabolomic studies. When
plasma samples are collected, anticoagulants, such as ethylenediaminetetraacetic
acid (EDTA), sodium citrate, and heparin, are added to inhibit the coagulation cas-
cade [7, 14]. EDTA and sodium citrate are chelators of calcium which acts as a
cofactor in the cascade, while heparin activates antithrombin [14]. The choice of
one anticoagulant over another does not appear to significantly affect the levels of
metabolites [13]. Nevertheless, citrate and EDTA can be detected by mass spec-
trometry and interfere with the analysis of metabolites of interest. Citrate can also
alter the sample pH and reduce the yield of metabolite extraction. Unlike the other
two anticoagulants, heparin is not detectable with MS, but it is often added as lith-
ium or sodium salts that can form adducts during electrospray ionization [7].
Special precautions must be taken when collecting blood samples to avoid hemo-
lysis. Erythrocytes can release intracellular components, including hemoglobin, and
alter the metabolic profile of plasma and serum [7]. Other pre-analytical factors
influencing metabolite levels include sample collection time, delays, and ambient
temperature [7, 13].
Samples are often stored for a long period of time in an epidemiological study.
However, storage for over 5 years at −80 °C can significantly change metabolite
concentrations. It is preferable to store samples in liquid nitrogen as aliquots to
avoid repeated freeze-thaw cycles that can alter the metabolic profile [7, 13].

2.2 Urine Samples

Urine differs from other biofluids in that it is easily collected in large quantities [7,
8]. However, collection at different times of the day and with or without fasting may
interfere with the urinary metabolic profile [7, 13]. Metabolites from the diet are
also more likely to be detected in urine than in other biofluids [15]. Therefore, it is
preferable to standardize the time of collection and obtain samples after fasting [13].
Bacteria can also alter urinary metabolite levels in samples, which grow rapidly
even after samples are collected [7]. Centrifugation and filtration can remove bacte-
ria and cellular debris. However, high-speed pre-centrifugation can break the cells
and cause the release of cellular components, which can alter the metabolic profile.
Mild centrifugation at 1000–3000 g is preferred to remove contaminants without
damaging cells. Filtration can sometimes also lead to a loss of metabolites [16].
Storing samples at 4 °C or lower immediately after sample collection inhibits bacte-
rial growth [7]. In the long term, it is preferable to store samples in liquid nitrogen
or at −80 °C and in smaller aliquots to limit freeze/thaw cycles and reduce the
potential for metabolite degradation [7, 13].
The concentration of metabolites in urine can also vary significantly between
samples depending on hydration status and other variables [17]. Creatinine concen-
trations or osmolarity can be used to normalize urine samples. However, creatinine
274 M. Mireault and L. Sleno

levels can vary depending on different factors, including age, gender, ethnicity, diet,
muscle mass, exercise level, time of day, and disease states. Osmolarity would pro-
vide better separation of biological groups compared with creatinine but is less
available in practice. Specific gravity, generally used as an alternative to osmolarity,
corresponds to the ratio between the density of urine and water measured at constant
temperature by refractometry [17]. Pre-acquisition normalization has been shown to
provide better results than post-acquisition normalization, with a combination of
both being even better [13], for instance, normalizing urine dilution steps during
sample preparation based on creatinine concentration followed by a second normal-
ization of raw data using either endogenous signals in the urine or average ion inten-
sity for the chromatographic run.

3 Quality Assurance and Quality Control

Like many aspects of metabolomics, quality standards are not well defined. Poor
data quality management leads to biased results, wasted resources, and can damage
the credibility of this field of study [18]. Quality assurance (QA) establishes the
prerequisites (equipment maintenance, personnel training, etc.) to ensure the quality
and reproducibility of results, while quality control (QC) is important to make sure
that no bias occurs during sample preparation and analysis [18–20]. The QC proce-
dure includes using several control samples, including blanks, pooled QC or intra-­
study QC samples, long-term reference (LTR) or intra-laboratory QC samples, and
standard reference material (SRM)/interlaboratory QC samples. Internal standards,
technical replicates, and random sample analysis are also often used [18]. However,
each laboratory uses different methods, which makes it difficult to harmonize results
[18, 20]. Long et al. developed a list of recommendations for good laboratory prac-
tices (QA and QC) to ensure quality results [19]. They are classified into five steps
(pre-pre-analytical, pre-analytical, analytical, post-analytical, and post-post-­
analytical), and each is accompanied by commonly made errors.
During collection, the most common errors are due to the misidentification of
samples or improper collection, resulting in sample hemolysis [12]. Centrifugation
conditions (time and speed), handling (sorting, pipetting/aliquoting), storage, and
inappropriate transport of samples are also errors often observed in the medical
environment and can have an impact on the metabolic profile [11, 12, 19]. Although
less frequent, analytical errors can also occur [12]. They are caused by equipment
malfunction, interference from an endogenous or exogenous compound, or a failure
not detected by quality control. Therefore, it is important to perform regular main-
tenance on the equipment and use various QC samples to ensure the quality of the
data generated [19]. Following data acquisition, there may also be erroneous valida-
tion, incorrect data entry, or misinterpretation of results [19]. Thus, errors can occur
at different stages of the analysis process. However, many of them are human errors,
and it is important to prioritize staff training to avoid them.
LC-MS-Based Population Metabolomics: A Mini-Review of Recent Studies… 275

4 Identification of Metabolites

4.1 Confidence Levels

Confidence levels ensure the quality of metabolite identification. They classify from
0 to 4, with 0 being the highest confidence level. This level requires determining the
three-dimensioanl structure and the complete stereochemistry of the metabolites of
interest. Level 1 uses a reference standard to identify metabolites from two orthogo-
nal techniques, such as MS/MS spectrum and retention time. Level 2 allows a pos-
sible identification of metabolites by comparing information from two orthogonal
techniques with the literature or a database. Level 3 determines a possible class of
metabolites based on at least one technique. Finally, level 4 does not allow the iden-
tification, but the compound remains present in the sample after extraction and can
be quantified [19, 21].
To ensure the identification of metabolites present in samples, it is always prefer-
able to use reference standards (level 1) [5, 21]. However, obtaining a complete set
of metabolite standards can be an arduous and expensive task. Initiatives for creat-
ing metabolite standard mixes are an interesting concept to reduce the cost of indi-
vidual standards. A comparison of MS/MS spectra and retention times to those
present in databases allows, at best, a level 2 confidence [22].

4.2 Databases for Metabolite Identification

Several databases, such as Metlin, HMDB, MassBank, and NIST, are available to
support metabolite identification. These can contain endogenous metabolites, exog-
enous compounds, and transformation products from food, microbiome, drugs,
plants, and pollutants [23]. Nevertheless, metabolite identification remains com-
plex, and it is estimated that less than 25% of MS/MS spectra can be identified due
to a low number of metabolites listed in these databases and the limited number of
commercially available pure reference standards [5, 23].
Some isomeric metabolites have a distinct structure but identical molecular for-
mulae and, therefore, the same exact mass. The presence of these isomers can lead
to false discovery rates (FDRs) due to the similarity between many MS/MS spectra
present in databases [5]. A low FDR allows reliable annotation of a small number of
metabolites, while a high FDR allows annotation of a larger number of metabolites
whose quality may be poor [24]. Thus, FDRs are generally higher in large databases
because they have many MS/MS spectra for the same compound. Each device pro-
vides a different spectrum, and the diversity of the methods used leads to many
redundancies [5]. It is generally accepted that the best scenario would be to compare
spectra obtained under the exact conditions as the metabolomic data was acquired.
Different platforms can show variable fragmentation, especially if the collision
276 M. Mireault and L. Sleno

energies, for instance, are not the same. Another important concept would be the
difference between instruments having varying mass resolutions. A low-resolution
system, such as a triple quadrupole, allows only unit resolution to be achieved;
therefore, molecular formulae of fragment ions detected cannot be confirmed.
MassBank and NIST contain MS/MS spectra from different instruments (QqTOF,
Orbitrap, QqQ, ion-trap) and different ionization techniques (ESI, EI, CI, APCI, and
MALDI) [23]. HMDB also contains spectra from different devices but uses only
electrospray ionization (ESI) [25]. Metlin is a database whose spectra are acquired
on the electrospray-quadrupole-time-of-flight (QqTOF) platform from the Agilent
Technologies. The spectra have been generated in positive and negative modes at
three distinct collision energies (10, 20, and 40 V) [23, 26].

4.3 Data Processing Tools

Data processing is an essential and usually very time-consuming step in untargeted

metabolomics. For this purpose, many tools are available, both commercially or
freely online, using script platforms (such as R and python) [24, 27]. XCMS,
MZmine, and MS-DIAL are commonly used in metabolomics. They allow filtering,
feature detection, alignment, annotation, and identification of metabolites [28–30].
XCMS and MZmine also include statistical tests, while only XCMS allows the
analysis of metabolic pathways and data integration with proteomics and genomics
[22, 28, 29]. Other metabolomic analysis tools are available and have been dis-
cussed in various reviews [21, 22, 24]. However, all these softwares use different
algorithms, resulting in a lack of reproducibility and consistency. Studies have
shown differences in the number of features detected and in statistical analysis
between tools, resulting in the identification of false-positive biomarkers [31–34].
Hohrenk et al. [33] tested four different softwares (MZmine2, enviMass, Compound
Discoverer, and XCMS online) and found that only 10% of identified features
were common.

5 Analytical Challenges

5.1 Variation of Metabolites

Metabolite variability is an important issue in epidemiological studies. It can arise

from inter- and intra-subject variability or lack of technical reproducibility [4].
Metabolites can vary according to different factors, including age, gender, BMI,
pregnancy, diet, ethnicity, smoking, environment, and physical activity [15, 35, 36].
Therefore, obtaining a large cohort that could correct all these criteria is difficult.
Several authors report having an insufficient number of participants in the limita-
tions of their study, despite a very large number of participants [37–42].
LC-MS-Based Population Metabolomics: A Mini-Review of Recent Studies… 277

5.2 Missing Values

Missing values are a key factor to consider when analyzing the results. They result
from metabolite signals in some samples and are absent in others. This may be due
to biological variation between two groups of samples (control vs. disease) or exter-
nal factors, such as pre-analytical conditions or chemical instability [4]. Missing
values from technical factors are divided into three classes, missing not at random
(MNAR) values, missing at random (MAR) values, and missing completely at ran-
dom (MCAR) values, also called abundance-dependent missing values [43, 44].
MCAR values result from random errors acquired during data acquisition, such as
reduced ionization efficiency or ion suppression [43]. MAR values are more general
and are difficult to distinguish from MCAR, as they are often combined [44]. They
may result from poor peak detection or deconvolution of two co-eluting peaks [45].
Finally, the MNAR values correspond to metabolites with concentrations below the
limit of detection [43].
In epidemiological studies, missing values are often replaced by half or a fraction
of the lowest detected value [4]. However, the different types of missing values may
influence the choice of approach to adopt. Random forest is considered the best
approach for MCAR/MAR, while quantile regression imputation of left-censored
data (QRILC) is preferred for MNAR [45]. When the missing value is from a bio-
logical factor, a change may distort the results; therefore, it would be better, in this
case, to keep these values as null values [4]. Therefore, the choice of method consid-
ers the authors’ opinion on the nature of the missing values, which can be verified
by comparing the different results obtained using these methods [46].
Following the missing data processing, the metabolites usually undergo a log
transformation to normalize the results [4]. This method allows us to obtain a
Gaussian-like distribution in the metabolite variables and to compare the statistical
analyses [46]. However, this approach cannot be applied to all metabolites, and a
comparison of the distribution of values must be performed before and after the
transformation. When there is no change, it is better to keep the initial values [46].

6 Statistical Analysis

Several statistical analyses are used in epidemiological studies. Nevertheless, some

tests seem to be used more than others. According to a survey of laboratories partici-
pating in the COnsortium of METabolomics Studies (COMETS), univariate regres-
sion is mostly used in metabolomic analysis [4]. Specifically, it is the linear
regression that associates a metabolite with an outcome. It is combined with multi-
ple correction tests, such as Benjamini-Hochberg, to determine the false discovery
rate or Bonferroni correction to control the false positive or type 1 error [4, 46].
However, the Bonferroni correction is a conservative approach and can increase the
risk of false negatives or type 2 error [47]. Multivariable analysis of metabolites
278 M. Mireault and L. Sleno

with covariates and principal component analysis (PCA) are also widely used [4].
Unlike linear regression, PCA does not identify a specific biomarker. However, it
allows the correlation between a set of metabolites and an outcome [46].
The choice of statistical tests will depend on the objective of the study. Many
researchers use the area under the Receiver Operating Characteristic Curve (ROC)
to determine the quality of a biomarker, while partial correlation is the most com-
mon method for analyzing intercorrelations between different metabolites.
Laboratories that have performed network analysis [4] used the weighted gene co-­
expression network analysis (WGCNA) and MetaboAnalyst, for a freely available
and user-friendly data analysis online platform. Recently, a recent version of
MetaboAnalyst (5.0) has been released online and provides better support for statis-
tical analysis (univariate and multivariate) and functional analysis (enrichment,
pathway, and functional meta-analysis) [48, 49].

7 Selected Case Studies

Since Oliver et al. [50] introduced the metabolome concept in 1998, metabolomic
studies related to disease and new biomarkers have continued to increase [5].
Cancers (breast, colon, lung) [39, 51–55], autism spectrum disorder [56], hyperin-
sulinemia [38], eating disorders [41], chronic kidney disease [57], depression [58],
osteoporosis [59], metabolic syndrome [60], ovarian reserve dysfunction [61], and
obesity [62] are few examples of diseases that are the focus of population-based
metabolomic studies. They perform metabolomic profiling to predict disease inci-
dence or progression. Kelly et al. [56] showed that plasma metabolite levels could
predict autism in children aged 8 years with good sensitivity and specificity.
Tryptophan and tyrosine metabolic pathways would be associated with better ages
and stages questionnaire (ASQ) communication scores. Tabung et al. [38] evaluated
the correlation between plasma metabolites and the empirical dietary index of
hyperinsulinemia (EDIH). They showed increased levels of diacylglycerol, triacyl-
glycerols, C10:2 carnitine, and C18:2 sphingomyelins and decreased phospholipid
levels of trigonelline, and eicosapentaenoate would correlate with elevated EDIH
[38]. Another study performed metabolic profiling of plasma in 7-year-old children.
It showed that high levels of VLDL, triglycerides, apolipoprotein-B/apolipoprotein-
­A, and monounsaturated fatty acids ratio could decrease the probability of develop-
ing anorexia nervosa at the age of 18, while high levels of HDL, docosahexaenoic
acid, polyunsaturated fatty acid ratio, and fatty acid unsaturation could increase this
risk [41].
Other population-based studies focus more on factors leading to metabolite vari-
ability in different biological matrices to characterize them better. Wang et al. per-
formed metabolic profiling in plasma [63] and urine [64] to identify diet-related
biomarkers. They associated 238 plasma metabolites with 74 food groups and 513
urinary metabolites with 79 food groups. Darst et al. [65] instead studied the effect
LC-MS-Based Population Metabolomics: A Mini-Review of Recent Studies… 279

of age and sex on the metabolic profile of plasma. They found that 623 metabolites
were associated with age, of which 29 steroids decreased with age while levels of
most fatty acids and sphingolipids increased. Furthermore, 695 metabolites were
associated with sex, 55% of which decreased in women (mainly steroids and
amino acids).
Nevertheless, for each study, information regarding the pre-analytical procedures
(collection, preparation, or storage sample) and methods to ensure quality control or
data processing is often lacking. This increases the difficulty of comparing two
studies with each other. Thus, the experimental approach used in some metabolomic-­
related population studies is summarized in Tables 1, 2 and 3.

7.1 Pre-Analytical Procedures

For sample collection, it was common for patients to be fasting (2 h overnight).

Plasma samples were collected in tubes containing EDTA. Only the study by Larkin
et al. [53] used lithium heparin, whereas three studies did not specify the anticoagu-
lant used. Several studies did not describe the steps involved in the preparation of
plasma and serum samples. In general, centrifugation parameters, time delays, or
any other information that could influence the levels of metabolites when obtaining
either of these two matrices were not specified.
Conversely, some studies shared not only all of this information but also the aver-
age total time required for sample preparation [40, 52, 65, 66], an important factor
for the variability of blood metabolites. The studies did not use pre-centrifugation
and filtration for urine samples to remove bacteria. Some of them kept the samples
at 4 °C and at a lower temperature, or in some cases, it is simply not defined. Two
of five studies normalized metabolite concentrations to osmolality. Only one per-
formed a creatinine normalization, which was not indicated in the other two studies.
For all biofluids, samples were generally aliquoted before being stored. In the long
term, samples were stored at −80 °C to avoid changes in metabolite levels. Some
studies opted for storage in liquid nitrogen or at −70 °C.

7.2 Data Acquisition

Liquid chromatography-mass spectrometry (LC-MS) is the most used analytical

method. Nuclear magnetic resonance (NMR) is also widely used, while only three
studies used gas chromatography-mass spectrometry (GC-MS). To ensure the qual-
ity of the results, the most common method was to use a pool of samples injected
several times. The coefficient of variation (CV) was then determined from this pool.
Duplicates or triplicates were also used to assess inter- or intra-batch variability.
Lau et al. [66] used several techniques to ensure the quality of their results. Internal
Table 1 Cancer-related metabolomic population studies
280

Data Data
Subject Sample Cohort pre-acquisition Data acquisition post-acquisition Statistical analyses Outcomes of study Reference
Breast Serum 174 women – Fasting blood NMR – MetaboAnalyst – Log-­transformation – Alanine, leucine, tyrosine, [51]
cancer (aged 35–64) sample – Missing values – Wilcoxon rank-sum valine, lactic acid, pyruvic
– Aliquoted half of the LOQ tests acid, triglycerides, lipid
– Stored in liquid – > 20% under – Frequencies main fraction, and 11
N2 LOQ excluded – Pearson’s chi- VLDL lipid subfractions
squared tests were inversely associated
– Conditional logistic with high MBD BC cases
regression models – Acetic acid was directly
– Benjamini-­Hochberg associated with high
– PCA MBD cases
– OPLS-DA – Phenylalanine, tyrosine,
and tryptophan pathway
emerged MBD BC cases
M. Mireault and L. Sleno
 Data Data
Subject Sample Cohort pre-acquisition Data acquisition post-acquisition Statistical analyses Outcomes of study Reference
Breast Serum 62 Asian Collection UHPLC-QTOF – Agilent Mass – Log-­transformation – All altered amino acids [54]
cancer females – Incubated at r.t. MS Profiler software – Autoscaling were upregulated, while
for 30 min – Leucine – In-house library, SIMCA-P all cardiolipin (CL)
– Centrifuged at encephalin used METLIN and – PCA species are downregulated
3000 r/min, as lock mass for HMDB – OPLS-DA in the TNBC samples
5 min internal mass MetScape MetaboAnalyst 4.0 – Glycerophospholipid
– Stored at calibration – Correlation-­ – Univariate statistical metabolism, aminoacyl-­
−80 °C, – Pooled QC based metabolic analysis tRNA biosynthesis, and
Metabolite sample injected networking MetScape valine, leucine, and
extraction every eight analysis – Pearson’s correlation isoleucine biosynthesis
– Protein samples – MetaboAnalyst coefficient were significantly altered
precipitation by 4.0 for – Debiased squared – dUMP,
MeOH metabolic partial correlation L-octanoylcarnitine,
– Centrifuged at pathway SPSS L-proline, lysoPC (22:1),
12000 rpm, analysis – ROC curve PS (22:0/0:0), and uric
15 min at 4 °C (KEGG) acid correlated with a
– Samples kept at five-year survival rate in
4 °C throughout TNBC patients
analysis

(continued)
LC-MS-Based Population Metabolomics: A Mini-Review of Recent Studies…
281
Table 1 (continued)
282

Data Data
Subject Sample Cohort pre-acquisition Data acquisition post-acquisition Statistical analyses Outcomes of study Reference
Cancer Plasma 293 – Fasting blood NMR – Assignments by R package – OPLS-DA models [53]
participants (2 h) – Pooled samples reference to – OPLS-DA separated unwell patients
(age ≥ 40) – Lithium-­ literature values – Two-sided with solid tumor
heparin tubes – HMDB Kolmogorov-­ diagnoses from those with
– Left to stand at Smirnov test noncancer diagnoses with
room – ROC curves an AUC of 0.91,
temperature – Pearson’s correlation sensitivity of 94%, and a
– Centrifugation specificity of 82%
at 2200 × g, – OPLS-DA models also
10 min separated patients with a
– Stored at −80 ° metastatic cancer from
C those with nonmetastatic
– Defrosted on cancer with an AUC of
ice 0.91, sensitivity of 94%,
– Mixed with and specificity of 88%
NMR buffer
– Centrifugation
at 16,000 × g,
3 min
M. Mireault and L. Sleno
 Data Data
Subject Sample Cohort pre-acquisition Data acquisition post-acquisition Statistical analyses Outcomes of study Reference
Colorectal Urine 52 – Aliquoted – Creatinine – MetaboAnalyst – Log-­transformation – Levels of acetone, [39]
cancer participants – Stored at normalization software 4.0 – Autoscaled arginine, 2.3.-butanediol,
(cachexia) −80 °C – IS GC-MS – Benjamini-Hochberg and 2.3.-
GC-MS – MZMine 2.0 – One-way ANOVA dihydroxybutyrate
– Pooled QC – HMDB and – Pearson’s chi- decreased in cachectic
samples (every PubChem squared test compared to non-
batch) – Authentic – Pearson’s partial cachectic patients
– Kovats retention reference correlation – Glycerol phosphate
mixtures (every standard coefficients shuttle metabolism and
batch) NMR – OPLS-DA glycine, ketone body
– LOESS function – Bruker TopSpin metabolism, and serine
– %RSD software with metabolism were the top
– Analyzed paired zero filling 3 pathways
samples in same – DataChord
analytical spectrum miner
batches software
NMR – Chenomx
– QC samples library/HMDB
were run at the – Authentic
beginning and reference
end of each standard
analytical batch
– Citrate was
measured in
duplets
LC-MS-Based Population Metabolomics: A Mini-Review of Recent Studies…

(continued)
283
Table 1 (continued)
284

Data Data
Subject Sample Cohort pre-acquisition Data acquisition post-acquisition Statistical analyses Outcomes of study Reference
Lung Urine 564 – Sterilized cup LC-MS and NMR LC-MS R package, SAS, and – 5-Methyl-2-furoic acid [55]
cancer never- containing – Pooled QC – Progenesis SIMCA-P+ software was significantly
smoking ascorbic acid samples (every (nonlinear – Log- transformation associated with lower
females (aged (antioxidant) five patient dynamics) – PCA lung cancer risk
52–66) – Kept at 0 °C to samples) – XCMS software – OPLS-DA – 25% of the association
−4 °C – CV >30%, – Wilcoxon rank sum between soy consumption
– Processed features were test and higher lung cancer
within 6 h excluded – Fisher exact test risk was significantly
– Storage at – In-house – Separate mediated via 5- methyl-2-
−70 °C databases, unconditional furoic acid
HMDB and logistic regression – 1-carbon metabolism,
METLIN models nucleotide metabolism,
NMR – Multivariable linear oxidative stress, and
– MATLAB regression model inflammation were
R2012b – Mediation analysis associated with lung
– Normalized by – Spearman correlation cancer risk
probabilistic coefficients
quotient – Benjamini-
Missing data Hochberg
– Metabolites
were excluded
from all
analyses
Mummichog
analysis
– Pathway
enrichment
M. Mireault and L. Sleno
Table 2 Population metabolomic studies related to other diseases
Subject Sample Cohort Data pre-acquisition Data acquisition Data post-acquisition Statistical analysis Outcomes of study Reference
Autism spectrum Plasma 806 children – Separated LC-MS – In-house library R package – Tryptophan and tyrosine [56]
disorder (ages 1 and – Stored at – 80 °C – Two batches sent 6 Missing value – Log-­transformation metabolism were associated
3 years) months apart – 5 0% or more – PCA with better communication
– Normalized to metabolites were – PLS-DA score
sample mass excluded – Bonferroni – Plasma metabolite level
correction provides a high sensitivity
– ROC (88.9%) and specificity
(84,5%) for predicting
autism
Stool 806 children – Stored at −80 °C LC-MS – In-house library
(age 3 years) – Precipitation with MeOH – One batch Missing value
– Five aliquots – Normalized to – S/N < 10 or with
sample mass missing levels >10%
were excluded
Hyperinsulinemia Plasma 1919 women – Fasting blood sample LC-MS – Authentic reference – Multivariable-­ – Mainly phospholipids as [38]
(aged 50–79) (12 h) – Pooled plasma standards or reference adjusted linear well as trigonelline and
– EDTA tube reference samples samples regression models eicosapentaenoate
– Stored at −70 °C – Running every 20 – Coefficients of decreased with increasing
– Shipment on dry ice samples variation (CVs) EDIH scores
Missing value – Mainly diacylglycerol and
– Signal-to-noise triacylglycerols as well as
ratio < 10 C10:2 carnitine and C18:2
– Half the lowest sphingomyelins increased
observed value with EDIH scores
(continued)
LC-MS-Based Population Metabolomics: A Mini-Review of Recent Studies…
285
Table 2 (continued)
286

Subject Sample Cohort Data pre-acquisition Data acquisition Data post-acquisition Statistical analysis Outcomes of study Reference
Eating disorders Plasma 2929 children – Non-fasting NMR R package – Elevated VLDL, [41]
(7 years old) – EDTA tube – Sample standard – Logistic regression triglycerides, Apo-B/A, and
deviation (SD) monounsaturated fatty acids
ratio at age 7 were
associated with lower odds
of anorexia nervosa at
age 18
– Elevated HDL,
docosahexaenoic acid, and
fatty acid unsaturation at
age 7 were associated with
higher risk for anorexia
nervosa at 18 years
Chronic kidney Serum or 454 Trained personal under GC-MS – Agilent Fiehn GC/MS R package and SPSS – D-malic acid, [57]
disease (CKD) plasma participants strict quality control – Three technical metabolomics RTL – Cox regression acetohydroxamic acid, and
(mean age Collection replicates library models butanoic acid were
68 ± 12) – Fasting blood (overnight) – Blank: Deionized – NIST library 11 – Multivariable independently associated
– Prepared water – Agilent MassHunter models with death
– Stored in liquid N2 – QC (serum pool) workstation – Unadjusted and – Docosahexaenoic acid was
– Serum samples thawed on samples everyday quantitative analysis adjusted fine and inversely associated to
ice Agilent’s MassHunter gray models mortality
Extraction plasma software – ROC curves – Lactose and 2-O-glycerol-
metabolites (diabetes) – IS d27-myristic acid – Partial correlations α-D-­­galactopyranoside
– Solvent mixture using the RTL system MetaboAnalyst were associated to end
– Spiked with IS MetaboAnalyst Hypergeometric test stage renal disease (ESRD)
– Centrifuged at – Pathway analysis adjusted risk
15.800 × g, 15 min at 4 ̊C – KEGG homo sapiens – Tyrosine showing an
library inverse relationship with
Missing value ESRD
>50% below LOD were
excluded
M. Mireault and L. Sleno
Subject Sample Cohort Data pre-acquisition Data acquisition Data post-acquisition Statistical analysis Outcomes of study Reference
Depression Plasma 10,145 – Fasting blood (overnight) NMR Missing values: R package – Higher levels of Apo B, [58]
control – EDTA tubes – Sample handling to Metabolite values in – Log transformation VLDL, triglycerides,
5283 Centrifugation and stored data processing is subjects with outlying – Random effects diglycerides,
depressed at −80 °C highly standardized concentrations (6 5 SD) meta-analyses monounsaturated fatty
persons or and fully automated were additionally set as – I2 with 95% acids, fatty acid chain
Centrifugation at 4 °C and – Unaware of missing confidence intervals length, glycoprotein
stored at −20 °C depression cases vs. – Wald tests acetyls, tyrosine, and
control status – FDR method isoleucine were associated
– Bayesian modeling with increased odds of
depression
– Lower levels of HDL,
acetate, and Apo A1 were
associated with increased
odds of depression
Osteoporosis and Serum 320 – Fasting blood LC-MS – Biocrates proprietary SAS – In males, glutarylcarnitine, [59]
bone mineral participants – EDTA MetIQ TM software – Log-­transformation hydroxy sphingomyelin
density (BMD) – Centrifugation – Semi- quantitation – PCA C16:1, sphingomyelin
– Frozen at −80 °C was applied to the – PLS-DA C18:0, lysine, and serine
– AbsoluteIDQ p180 kit lipid quantitation – Wilcoxon test were associated with
based on isotopic lipid – χ2 test, osteoporosis
IS – Random forest – In postmenopausal females,
Missing value classifier method acetylcarnitine,
– >20% below LOD – ROC curves phosphatidylcholine diacyl,
were excluded phosphatidylcholine
acyl-alkyl, and
hydroxyproline were
associated with
osteoporosis
– ROC curve for BTMs and
LC-MS-Based Population Metabolomics: A Mini-Review of Recent Studies…

serum metabolites
increased significantly
compared to BTMs only
(continued)
287
Table 2 (continued)
288

Subject Sample Cohort Data pre-acquisition Data acquisition Data post-acquisition Statistical analysis Outcomes of study Reference
Metabolic Plasma 115 – Fasting plasma LC-MS/MS LC-MS SIMCA software – In the univariate analyses, [60]
syndrome participants – EDTA Reference interface – Profinder™ software – PCA the metabolic syndrome
Metabolite extraction – Purine m/z 121.05 package – OPLS (score) was associated with
– H2O: MeOH (1:9) and m/z 119.03632 – In-house libraries – 95% confidence multiple individual
– Incubated for 2 min – HP-0921 m/z – Metabolite and lipid interval using the metabolites, including
– Stored on ice for 2 h 922.0098 and m/z signals were jackknife method valeryl carnitine, pyruvic
– Centrifuged at 18620 966.000725 normalized to the – CV-ANOVA acid, lactic acid, alanine,
RCF, 10 min at 4 °C – Positive and total peak area – Permutation and lipids (diglyceride)
Lipid extraction negative modes, GC-MS analyses
– CHCl3: MeOH (2:1) respectively – MATLAB R package
– Stored at room GC-MS/MS – In-house library – Univariate linear
temperature for 60 min – Normalization to IS regression
– Centrifuged at 18620 – Log-transformation
RCF, 3 min at 4 °C
Functional Serum 398 women – Non-fasted blood NMR – Quantification is R package – Anti-Müllerian hormone [61]
ovarian reserve (ages 18–45) – Centrifuged achieved through – Multivariable linear (AMH) showed positive
– Frozen at −80 °C three molecular regression associations with HDL,
– Assays within 1 year of windows from each – Scaled to standard omega-6, and
storage sample deviation (SD) units polyunsaturated fatty acids
– No freeze/thaw cycles – PERCH NMR – Bonferroni and the amino acids
software isoleucine, leucine, and
– Serum extract tyrosine and negatively with
metabolites are scaled acetate
via the total – Antral follicle count (AFC)
cholesterol was positively associated
with alanine, glutamine,
and glycine
M. Mireault and L. Sleno
Subject Sample Cohort Data pre-acquisition Data acquisition Data post-acquisition Statistical analysis Outcomes of study Reference
Obesity Plasma 1020 children CHOP study LC-MS – Quantification of SM R package – Only SM 32:2 was [62]
(ages 5.5, 8, – Fasted blood CHOP study and 32:2 was achieved by – Linear models significantly associated
and 10) – Centrifugation GINIplus/LISA study comparison to regressing with BMI z-score in all
– Frozen at −70 °C – 6 QC samples per commercially – Bivariate linear four populations
UBCS batch available standard SM models – Alanine showed the
– Fasted blood (>10 h) – Intra-batch (d18:1/18:0) – Multiple linear strongest positive
– Aliquoted CV < 0.2, the batch – Metabolite were models association with HOMA,
– Frozen at −80 °C was included excluded if >1.5 x SD – Partial R2 while acylcarnitines and
GINIplus/LISA study – QC > 50% of the from second highest – Cochran’s Q31 nonesterified fatty acids
– Aliquoted batches, metabolite value – Bonferroni were negatively associated
– Frozen at −80 °C was included with HOMA
For all, sent on dry ice and UBCS – SM d18:2/14:0 is a
re-stored at −80 °C – Six QC samples powerful marker for
Metabolite extraction measured twice molecular changes in
– Protein precipitation with – QC > 35% CV, childhood obesity
MeOH, containing IS metabolites were
– Centrifuged at 4000 rpm, excluded
10 min at room
temperature

(continued)
LC-MS-Based Population Metabolomics: A Mini-Review of Recent Studies…
289
Table 3 Population metabolomic studies related to age, sex, BMI, pregnancy, and lifestyle habits
290

Subject Sample Cohort Data pre-acquisition Data acquisition Data post-acquisition Statistical analysis Outcomes of study Reference
Age and sex Plasma 1212 participants Plasma metabolomics LC-MS/MS – Metabolon’s hardware Metabolomics – 623 were associated with [65]
(mean age 61) – Fasting blood in EDTA – Pooled matrix sample and software – Median-­scaled age; 29 steroid lipids
tubes – Blank: Extracted water – In-house library – Log-­transformation significantly decreased
– Centrifuged at samples – Identified by future – Pearson r while higher levels of
3000 rpm, 15 min at RT – Cocktail of QC acquisition of a SAS most fatty acid,
– Aliquoted and – Instrument performance matching purified – Linear mixed effects sphingolipids and amino
frozen −80 °C, 30 min monitoring standard or by classical regression models acids increased with age
– Shipped on dry ice – Aided chromatographic structural analysis – – 695 were associated with
– Stored at −80 °C alignment – Metabolites with Benjamini-­ sex; most steroid lipids
Metabolite extraction – Median relative standard interquartile range of Hochberg and amino acids were in
– Recovery standards deviation (RSD) zero excluded from Genomics lower levels while most
– Protein precipitation Study samples were analyses – PCA fatty acids were higher in
with MeOH randomized Missing metabolite – Pearson r women
– Centrifugation QC samples spaced evenly values GCTA – The heritabilities of
– Stored under N2 among the injections – Imputed to the lowest h2 of each metabolite metabolites ranged
Genomics level of detection for dramatically
– DNA was extracted each metabolite (0.2–99.2%)
using PUREGENE®
DNA isolation kit
M. Mireault and L. Sleno
Subject Sample Cohort Data pre-acquisition Data acquisition Data post-acquisition Statistical analysis Outcomes of study Reference
Sex-specific Urine 369 participants – Stored at −80 °C LC-MS/MS – In-house library SAS software and R – Ten metabolites [67]
associations of (aged 16–18) – Protein precipitation by – Pools of sample were –N  ormalization with software associated with both BMI
BMI and body MeOH used as quality controls block correction – Log-transformation and BF
fat – Normalization by urine – ICA – Eleven metabolites
osmolality – Linear regression associated with only BF
Missing value model and nine with only BMI
– >20% below LOD – enjamini-Hochberg – None of these
were excluded associations was in
– Random forest method females
built into “mice” – Strong sexual
package dimorphism in the
relationship between
body composition and
the urine metabolome
(continued)
LC-MS-Based Population Metabolomics: A Mini-Review of Recent Studies…
291
Table 3 (continued)
292

Subject Sample Cohort Data pre-acquisition Data acquisition Data post-acquisition Statistical analysis Outcomes of study Reference
Age, sex, BMI Serum 1192 children – Collection in silica LC-MS/MS LC-MS/MS (serum) R package Sex: [66]
and dietary (aged 6–11) plastic tubes – AbsoluteIDQ p180 – MetIDQTM software – Log-transformation – Urinary isoleucine was at
habits – Inverted gently for 6–7 kit with IS Missing value: – Metabolome-wide lower while 5-oxoproline
times – Blank PBS samples – CV > 30% association study and tyrosine were higher
– Spun at 2500 g, 15 min (three replicates) > 30% below LOD (MWAS) in males
at 4 °C – Randomized samples – Multiple linear – Neurotransmitter
– Median processing time – NIST SRM 1950 plasma regression models serotonin in serum was
(collection to freezing): reference material (4 – Bonferroni higher in males while
1.8 h replicates) correction serine, lysine, ornithine,
– Median time between – QC material (two – Partial R2 approach acylcarnitines, and three
last meal and replicates, SeraLab, – PCA sphingolipids were
collection: 3.3 h S-123-M-27485) – Pearson’s higher in females
–QCs from manufacturer correlation Age:
in three concentrations coefficients – Creatinine
CVs: NIST SRM 1950 – Cytoscape software BMI z-score:
Urine – Collection: Evening and NMR – MATLAB and the MetScape – Urinary
morning – Randomised – Probabilistic quotient plugin application 4-deoxyerythronic acid,
– Kept in fridge overnight – Pooled urine samples normalisation urinary valine, serum
– Transported in from 20 individuals – HMDB carnitine, acylcarnitines,
temperature-controlled CVs: Pooled QC – ChenomxNMRsuite glutamate, BCAA valine,
environment 7.1 profiler lysoPC, and
– Aliquoted and frozen Missing value: sphingolipids
within 3 h – CV > 30% Dietary metabolite:
– Centrifuged at > 30% below LOD – Urinary creatine and
13,000 g, 10 min at serum PC with meat
4 °C – Serum PC with fish
– Urinary hippurate with
vegetables and urinary
proline betaine
– Hippurate with fruit
Population-specific
variance was better
captured in the serum than
in the urine profile
M. Mireault and L. Sleno
Subject Sample Cohort Data pre-acquisition Data acquisition Data post-acquisition Statistical analysis Outcomes of study Reference
BMI Serum 304 – Fasting blood (≥10 h) LC-MS/MS – In-house library – Log-transformation – 50 BMI-correlated [68]
postmenopausal – Processed – Pooled QC standard – Normalization to the SAS metabolites
women (aged – Stored at −86 °C within – CVs calculated for 38 run day – Partial Pearson –B  MI-related metabolites
50–74) 12 h samples using single Missing value correlations were more strongly
– Several recovery pooled QC sample – >90% below LOD Bonferroni correction correlated with fat mass
standards were excluded than lean mass
– Protein precipitation by – Minimum observed – Eight metabolites that
MeOH value of that specific were correlated with fat
metabolite mass or lean mass but
not BMI
Diet Urine 648 participants – 24 h urine collections LC-MS/MS – In-house library – Log-transformation – 708 diet-metabolite [64]
(mean age – Refrigerate or with – Triplicates of 44 – Normalization by – Autoscaled associations were
52.2 ± 9.4) cooler packs participant samples osmolality – Pearson’s partial identified
– Aliquoted – Interclass correlation Missing value: correlation – 513 unique metabolites
– Frozen and shipped on coefficients (ICCs) – ICC < 0.5 were – Bonferroni correlated with 79 food
dry ice excluded – ROC curve using R groups/items
– Storage in liquid N2 – >90% below LOD package
– Precipitate proteins were excluded
with MeOH –Imputed with the
minimum

Diet Plasma 671 participants – Fasting blood (8 h) LC-MS/MS – In-house library – Log-transformation – A total of 677 [63]
(mean age – EDTA – Duplicates of 60 – Each metabolite was – Autoscaled diet-metabolite
52.3 ± 9.5) – Refrigerated participant samples divided by its daily – Pearson’s partial associations
– Centrifugation – Interclass correlation median correlation – 238 plasma metabolites
– Aliquoted coefficients (ICCs) – Excluded – Missing – Bonferroni were associated with 74
– Shipped on dry ice values were assigned – ROC curve using R food groups/items
– Storage in liquid N2 the minimum detection package
– Protein precipitation by value
LC-MS-Based Population Metabolomics: A Mini-Review of Recent Studies…

MeOH

(continued)
293
Table 3 (continued)
294

Subject Sample Cohort Data pre-acquisition Data acquisition Data post-acquisition Statistical analysis Outcomes of study Reference
Pregnancy Serum 8774 women – Fasting serum NMR – PERCH NMR R package – White European women [40]
– Processed within 2.5 h software – Multivariable linear (WEs) had higher levels
– Centrifuged at – Repeated analyses regression of most lipoprotein,
3500 rpm, 10 min at including only cholesterol, glycerides,
room temperature complete case data to phospholipids,
– Stored at −80 °C test whether any monosaturated fatty
missing data were acids, pyruvate, glycerol,
altering the results and creatinine
– South Asian women had
higher levels of glucose,
linoleic acid, omega-6
and polyunsaturated fatty
acids, and most amino
acids
– Higher BMI and having
GD had higher levels of
lipoprotein, triglycerides,
mostly with stronger
associations in WEs
M. Mireault and L. Sleno
Subject Sample Cohort Data pre-acquisition Data acquisition Data post-acquisition Statistical analysis Outcomes of study Reference
Breast cancer Plasma 1319 participants – EDTA tubes NMR Missing values: R package and Stata Fruit and vegetable [52]
preventive (aged 30–74) – Stored at 4 °C – Half minimum value of – Log-transformation consumption
lifestyle – Centrifuged at that metabolic measure – Multivariate – Negative associations
behaviors 1300 g,10 min at 4 °C in total population imputations by between glycoprotein
with brake on – CV: Duplicate samples chained equation acetyls, an inflammation-
– Stored at −80 °C (5%) (MICE) related metabolite with
(19–24 h after blood – Linear regression lower BMI
collection) – Pearson pairwise – Positive association
– Free of hemolysis and correlations between polyunsaturated
lipidemia MetScape fatty acids
– Conditional BMI
correlation networks – Positive associations
between HDL with lower
BMI
Alcohol consumption
– Positive association of
ApoA1
BMI and fruit and
vegetable consumption
were generally consistent
but were opposite with
alcohol consumption
LC-MS-Based Population Metabolomics: A Mini-Review of Recent Studies…
295
296 M. Mireault and L. Sleno

standards (IS), blanks with PBS, different control samples (NIST SRM 1950, com-
mercially available serum QCs and QCs provided by the manufacturer), and ran-
domized batches were used for serum samples.

7.3 Post-Acquisition Data Processing

Missing values are due to the absence of metabolites in some samples. When a
metabolite is not detected in several samples (20–90%), it is usually excluded from
the analysis. In some cases, the missing value is replaced by a fraction of the lowest
detected value or the minimum value. However, it is not always specified whether
this is the limit of detection (LOD) or the value detected in a sample set. The study
by McClain et al. [68] used the random forest method built into the “mice” package
to handle missing data. Several studies use an in-house library to identify metabo-
lites, which allows for reducing the FDR and obtaining MS/MS spectra specific to
the method used. Online databases are also used. The most common was HMDB,
followed by METLIN and NIST. Studies have combined in-house libraries and
online databases to increase the number of identified metabolites. Some studies
confirm with commercially available standards to achieve a level 1 confidence. Prior
to statistical analyses, most studies perform a log transformation to obtain a Gaussian
distribution. Subsequently, the most used statistical tests were the principal compo-
nent analysis (PCA), the area under the receiver operating characteristic (ROC)
curve, and the multivariate linear regression model. Other analyses, such as orthog-
onal partial least-squares discriminant analysis (OPLS-DA), Pearson correlation
coefficient, and Pearson partial correlation coefficient, were also present in several
studies. Many studies have performed multiple correction tests, with a slight prefer-
ence for the Bonferroni correction. Thus, despite the diversity of methods used,
some approaches seem to be more common than others.

8 Conclusion

Recently, population-based metabolomic studies have increased significantly due to

their potential to predict, diagnose, and monitor disease progression [5, 69].
However, due to the lack of standardization of protocols, these studies use different
approaches, which may lead to limited reproducibility and consistency between
results. Therefore, it is essential to establish standards for a specific workflow for
untargeted metabolic analysis. Furthermore, to get a complete view of the biological
mechanisms involved, it is preferable to integrate metabolomics with other “omic”
sciences, such as proteomics, transcriptomics, and exposomics.
LC-MS-Based Population Metabolomics: A Mini-Review of Recent Studies… 297

References

1. Vailati-Riboni M, Palombo V, Loor JJ. What are omics sciences? In: Periparturient diseases of
dairy cows; 2017. p. 1–7.
2. Aderemi AV, et al. Metabolomics: a scoping review of its role as a tool for disease biomarker
discovery in selected non-communicable diseases. Meta. 2021;11(7):418.
3. Gika HG, et al. Current practice of liquid chromatography-mass spectrometry in metabolomics
and metabonomics. J Pharm Biomed Anal. 2014;87:12–25.
4. Playdon MC, et al. Metabolomics analytics workflow for epidemiological research: perspec-
tives from the consortium of metabolomics studies (COMETS). Meta. 2019;9(7):145.
5. Cui L, Lu H, Lee YH. Challenges and emergent solutions for LC-MS/MS-based untargeted
metabolomics in diseases. Mass Spectrom Rev. 2018;37(6):772–92.
6. Villas-Boas SG, et al. Mass spectrometry in metabolome analysis. Mass Spectrom Rev.
2005;24(5):613–46.
7. Kirwan JA, et al. Preanalytical processing and biobanking procedures of biological samples
for metabolomics research: a white paper, community perspective (for “precision medicine
and pharmacometabolomics task group”-the metabolomics society initiative). Clin Chem.
2018;64(8):1158–82.
8. Bouatra S, et al. The human urine metabolome. PLoS One. 2013;8(9):e73076.
9. Psychogios N, et al. The human serum metabolome. PLoS One. 2011;6(2):e16957.
10. Chen Y, et al. Methods used to increase the comprehensive coverage of urinary and plasma
metabolomes by MS. Bioanalysis. 2016;8(9):981–97.
11. Lippi G, et al. Preanalytical variability: the dark side of the moon in laboratory testing. Clin
Chem Lab Med. 2006;44(4):358–65.
12. Szecsi PB, Odum L. Error tracking in a clinical biochemistry laboratory. Clin Chem Lab Med.
2009;47(10):1253–7.
13. Stevens VL, et al. Pre-analytical factors that affect metabolite stability in human urine, plasma,
and serum: a review. Metabolites. 2019;9(8):156.
14. Denery JR, Nunes AA, Dickerson TJ. Characterization of differences between blood sample
matrices in untargeted metabolomics. Anal Chem. 2011;83(3):1040–7.
15. Wu J, Gao Y. Physiological conditions can be reflected in human urine proteome and metabo-
lome. Expert Rev Proteomics. 2015;12(6):623–36.
16. Bernini P, et al. Standard operating procedures for pre-analytical handling of blood and urine
for metabolomic studies and biobanks. J Biomol NMR. 2011;49(3–4):231–43.
17. Wu Y, Li L. Sample normalization methods in quantitative metabolomics. J Chromatogr
A. 2016;1430:80–95.
18. Evans AM, et al. Dissemination and analysis of the quality assurance (QA) and quality con-
trol (QC) practices of LC-MS based untargeted metabolomics practitioners. Metabolomics.
2020;16(10):113.
19. Long NP, et al. Toward a standardized strategy of clinical metabolomics for the advancement
of precision medicine. Meta. 2020;10(2):51.
20. Beger RD, et al. Towards quality assurance and quality control in untargeted metabolomics
studies. Metabolomics. 2019;15(1):4.
21. Blazenovic I, et al. Software tools and approaches for compound identification of LC-MS/MS
data in metabolomics. Meta. 2018;8(2):31.
22. Spicer R, et al. Navigating freely-available software tools for metabolomics analysis.
Metabolomics. 2017;13(9):106.
23. Vinaixa M, et al. Mass spectral databases for LC/MS- and GC/MS-based metabolomics: state
of the field and future prospects. TrAC Trends Anal Chem. 2016;78:23–35.
24. Misra BB. New software tools, databases, and resources in metabolomics: updates from 2020.
Metabolomics. 2021;17(5):49.
25. Wishart DS, et al. HMDB 4.0: the human metabolome database for 2018. Nucleic Acids Res.
2018;46(D1):D608–17.
298 M. Mireault and L. Sleno

26. Kind T, et al. Identification of small molecules using accurate mass MS/MS search. Mass
Spectrom Rev. 2018;37(4):513–32.
27. Cajka T, Fiehn O. Toward merging untargeted and targeted methods in mass spectrometry-­
based metabolomics and lipidomics. Anal Chem. 2016;88(1):524–45.
28. Tautenhahn R, et al. XCMS online: a web-based platform to process untargeted metabolomic
data. Anal Chem. 2012;84(11):5035–9.
29. Pluskal T, et al. MZmine 2: modular framework for processing, visualizing, and analyzing
mass spectrometry-based molecular profile data. BMC Bioinform. 2010;11(1):1–11.
30. Tsugawa H, et al. MS-DIAL: data-independent MS/MS deconvolution for comprehensive
metabolome analysis. Nat Methods. 2015;12(6):523–6.
31. Gurdeniz G, et al. The effect of LC-MS data preprocessing methods on the selection of plasma
biomarkers in fed vs. fasted rats. Metabolites. 2012;2(1):77–99.
32. Chen Y, et al. Assessment of data pre-processing methods for LC-MS/MS-based metabolo-
mics of uterine cervix cancer. Analyst. 2013;138(9):2669–77.
33. Hohrenk LL, et al. Comparison of software tools for liquid chromatography-high-resolution
mass spectrometry data processing in nontarget screening of environmental samples. Anal
Chem. 2020;92(2):1898–907.
34. Coble JB, Fraga CG. Comparative evaluation of preprocessing freeware on chromatography/
mass spectrometry data for signature discovery. J Chromatogr A. 2014;1358:155–64.
35. Thevenot EA, et al. Analysis of the human adult urinary metabolome variations with age, body
mass index, and gender by implementing a comprehensive workflow for univariate and OPLS
statistical analyses. J Proteome Res. 2015;14(8):3322–35.
36. Lawton KA, et al. Analysis of the adult human plasma metabolome. Pharmacogenomics.
2008;9(4):383–97.
37. Perng W, et al. Urate and nonanoate mark the relationship between sugar-sweetened bever-
age intake and blood pressure in adolescent girls: a metabolomics analysis in the ELEMENT
cohort. Meta. 2019;9(5):100.
38. Tabung FK, et al. Identifying metabolomic profiles of Insulinemic dietary patterns. Meta.
2019;9(6):120.
39. Ose J, et al. Multiplatform urinary metabolomics profiling to discriminate cachectic from
non-cachectic colorectal cancer patients: pilot results from the ColoCare study. Meta.
2019;9(9):178.
40. Taylor K, et al. Differences in pregnancy metabolic profiles and their determinants between
white European and South Asian women: findings from the born in Bradford cohort. Meta.
2019;9(9):190.
41. Santos Ferreira DL, et al. Associations between blood metabolic profile at 7 years old and
eating disorders in adolescence: findings from the Avon longitudinal study of parents and chil-
dren. Meta. 2019;9(9):191.
42. Ellul S, et al. Metabolomics: population epidemiology and concordance in Australian children
aged 11-12 years and their parents. BMJ Open. 2019;9(Suppl 3):106–17.
43. Karpievitch YV, Dabney AR, Smith RD. Normalization and missing value imputation for
label-free LC-MS analysis. BMC Bioinform. 2012;13(16):1–9.
44. Lazar C, et al. Accounting for the multiple natures of missing values in label-free quantitative
proteomics data sets to compare imputation strategies. J Proteome Res. 2016;15(4):1116–25.
45. Wei R, et al. Missing value imputation approach for mass spectrometry-based metabolomics
data. Sci Rep. 2018;8(1):663.
46. Antonelli J, et al. Statistical workflow for feature selection in human metabolomics data. Meta.
2019;9(7):143.
47. Andrade C. Multiple testing and protection against a type 1 (false positive) error using the
Bonferroni and Hochberg corrections. Indian J Psychol Med. 2019;41(1):99–100.
48. Pang Z, et al. MetaboAnalyst 5.0: narrowing the gap between raw spectra and functional
insights. Nucleic Acids Res. 2021;49(W1):W388–96.
LC-MS-Based Population Metabolomics: A Mini-Review of Recent Studies… 299

49. Xia J, et al. MetaboAnalyst: a web server for metabolomic data analysis and interpretation.
Nucleic Acids Res. 2009;37(Web Server issue):W652–60.
50. Oliver SG, et al. Systematic functional analysis of the yeast genome. Trends Biotechnol.
1998;16(9):373–8.
51. Bendinelli B, et al. Prediagnostic circulating metabolites in female breast cancer cases with
low and high mammographic breast density. Sci Rep. 2021;11(1):13025.
52. Qi J, et al. Metabolomics and cancer preventive behaviors in the BC generations project. Sci
Rep. 2021;11(1):12094.
53. Larkin JR, et al. Metabolomic biomarkers in blood samples identify cancers in a mixed popu-
lation of patients with nonspecific symptoms. Clin Cancer Res. 2022;28:1651.
54. Li L, et al. Metabolomics-based discovery of molecular signatures for triple negative breast
cancer in Asian female population. Sci Rep. 2020;10(1):370.
55. Seow WJ, et al. Association of untargeted urinary metabolomics and lung cancer risk among
never-smoking women in China. JAMA Netw Open. 2019;2(9):e1911970.
56. Kelly RS, et al. Metabolomics and communication skills development in children; evidence
from the ages and stages questionnaire. Meta. 2019;9(3):42.
57. Titan SM, et al. Metabolomics biomarkers and the risk of overall mortality and ESRD in CKD:
results from the Progredir cohort. PLoS One. 2019;14(3):e0213764.
58. Bot M, et al. Metabolomics profile in depression: a pooled analysis of 230 metabolic markers
in 5283 cases with depression and 10,145 controls. Biol Psychiatry. 2020;87(5):409–18.
59. Wang J, et al. Discovery of potential biomarkers for osteoporosis using LC-MS/MS metabolo-
mic methods. Osteoporos Int. 2019;30(7):1491–9.
60. Surowiec I, et al. Metabolomic and lipidomic assessment of the metabolic syndrome in Dutch
middle-aged individuals reveals novel biological signatures separating health and disease.
Metabolomics. 2019;15(2):23.
61. Al Rashid K, et al. Association of the functional ovarian reserve with serum metabolomic pro-
filing by nuclear magnetic resonance spectroscopy: a cross-sectional study of ~ 400 women.
BMC Med. 2020;18(1):247.
62. Hellmuth C, et al. An individual participant data meta-analysis on metabolomics profiles for
obesity and insulin resistance in European children. Sci Rep. 2019;9(1):5053.
63. Wang Y, et al. Identification and reproducibility of plasma metabolomic biomarkers of habitual
food intake in a US diet validation study. Meta. 2020;10(10):382.
64. Wang Y, et al. Identification and reproducibility of urinary Metabolomic biomarkers of habit-
ual food intake in a cross-sectional analysis of the cancer prevention Study-3 diet assessment
sub-study. Meta. 2021;11(4):1.
65. Darst BF, et al. Longitudinal plasma metabolomics of aging and sex. Aging (Albany NY).
2019;11(4):1262.
66. Lau CE, et al. Determinants of the urinary and serum metabolome in children from six
European populations. BMC Med. 2018;16(1):202.
67. Brachem C, et al, Oluwagbemigun K. Associations of BMI and body fat with urine metabo-
lome in adolescents are sex-specific: a cross-sectional study. Metabolites. 2020;10(8).
68. McClain KM, et al. Body composition and metabolomics in the Alberta physical activity and
breast cancer prevention trial. J Nutr. 2022;152(2):419–28.
69. Zanetti KA. The future of metabolomic profiling in population-based research: opportunities
and challenges. J Anal Bioanal Tech. 2014;5(4):2.
Metabolomics and Transcriptomic
Approach to Understand
the Pathophysiology of Interstitial Lung
Disease

Sanjukta Dasgupta, Anindita Bhattacharya, Priyanka Choudhury,

Nilanjana Ghosh, Tanisha Das, Sushmita Roychowdhury, Riddhiman Dhar,
and Koel Chaudhury

Abstract Interstitial lung diseases (ILD) are a heterogeneous group of parenchy-

mal pulmonary disorders that result from varying degrees of inflammation or fibro-
sis in the lung interstitium, that is, the septum between alveoli and the blood
capillaries. The clinical presentation of ILD is complex and the diagnosis is often
challenging. Therefore, the need to establish disease-specific molecular fingerprints
to better understand the underlying pathogenesis is well realized. “Omics” is a pow-
erful tool that collectively depicts and quantifies biomolecules, including key
genomic, transcriptomic, proteomic, and metabolomic signatures, and discloses
their dynamic interactions within an organism. Metabolomics is a branch of omics
that identifies numerous small molecules from body fluids or tissues and holds
immense potential for early diagnosis, therapeutic monitoring, and understanding
of disease pathophysiology. Another evolving popular omic field is transcriptomics,
which identifies key genetic regulations and posttranscriptional modifications trig-
gering diseases. The findings of 17 original articles on metabolomics and 63 on
transcriptomics of ILD reported are discussed. Though each omic dataset provides
valuable information, integrating these platforms offers an overall snapshot of the
interplay between the candidate molecules and genes, thereby paving the path for
highlighting the genotype-to-phenotype relationship and assisting in making more
effective treatment decisions for complex diseases.

S. Dasgupta · A. Bhattacharya · P. Choudhury · N. Ghosh · T. Das · K. Chaudhury (*)

School of Medical Science and Technology, Indian Institute of Technology Kharagpur,
Kharagpur, India
e-mail: koel@[Link]
S. Roychowdhury
Fortis Hospital, Kolkata, India
R. Dhar
Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, India

© The Author(s), under exclusive license to Springer Nature Singapore Pte 301
Ltd. 2023
A. M. Abdel Rahman (ed.), Clinical Metabolomics Applications in Genetic
Diseases, [Link]
302 S. Dasgupta et al.

Keywords Interstitial lung diseases · Metabolomics · Transcriptomics ·

Systems biology

1 Introduction

Interstitial lung disease (ILD) is an umbrella term that encompasses about 300
parenchymal pulmonary disorders, resulting from varying degrees of inflammation
or fibrosis in the lung interstitium, that is, the septum between alveoli and the blood
capillaries. A schematic diagram of a healthy vs. ILD lung is shown in Fig. 1.
Numerous studies across the globe have reported the incidence, prevalence, and
relative frequency of ILD. The annual incidence of ILD varies between 1 and 31.5
per 100,000 [1]. The incidence and prevalence vary among populations, likely due
to differences in study design, data collection, and incorrect recognition of the dis-
ease subtypes [2]. ILD is classified based on clinical, radiological, and histopatho-
logical features. The latest classification focuses on recognizing the underlying
etiology since this often impacts both prognostication and management decisions.
ILD mainly consists of disorders of known causes [collagen vascular disease,
hypersensitivity pneumonitis (HP)] as well as disorders of unknown/idiopathic
causes [idiopathic interstitial pneumonia (IIP), sarcoidosis] [3]. ILD registries com-
prising patients from Western countries suggest that idiopathic pulmonary fibrosis
(IPF) and sarcoidosis are the most common phenotypes. However, the ILD registry
of India indicates HP to be the most common, which accounts for nearly 50% of all
ILD cases [4].

Fig. 1 Healthy lung vs. interstitial lung disease (created using [Link])
Metabolomics and Transcriptomic Approach to Understand the Pathophysiology… 303

The emerging field of metabolomics, in which many small molecules from body
fluids or tissues can be identified, holds immense potential for early diagnosis, ther-
apeutic monitoring, and understanding of disease pathophysiology. Over the past
two decades, nuclear magnetic resonance (NMR) spectroscopy and gas chromatog-
raphy (GC)/liquid chromatography (LC) coupled with mass spectrometry (MS)
combined with chemometric analysis have emerged as principal analytical tech-
niques for use in metabolomics. Several biofluids including cerebrospinal fluid
(CSF), bronchoalveolar lavage fluid (BALF), bile, seminal fluid, amniotic fluid,
synovial fluid, gut aspirate, serum/plasma, saliva, exhaled breath condensate (EBC),
and urine contain hundreds to thousands of detectable metabolites which have been
extensively studied so far [5]. More recently, metabolic profiling of intact tissue and
extracts of lipid and aqueous metabolites are gaining increasing importance for
detection of biomarkers.
Another branch of popular omic science is transcriptomics, which provides
detailed information about gene regulation in normal and diseased conditions. Two
key contemporary techniques commonly used for transcriptomic analysis are
hybridization-based microarray techniques, which quantify a set of predetermined
sequences, and next-generation sequencing (NGS), which uses high-throughput
sequencing to capture all sequences [6]. In the last decade, these two transcriptomic
approaches have been utilized most widely to understand the underlying disease
pathogenesis at both molecular and genetic levels and also for molecular diagnosis
and clinical therapy. Human biofluids including amniotic fluid, aqueous humor,
ascites, bile, BALF, breast milk, CSF, colostrum, gastric fluid, pancreatic cyst fluid,
plasma, saliva, seminal fluid, serum, sputum, stool, synovial fluid, sweat, tears,
urine, and tissues are widely used for transcriptomic studies to identify biomarkers
of several diseases [7–9].

2 Types of ILD

ILD, as mentioned earlier, refers to a group of lung diseases ranging from occa-
sional self-limited inflammatory processes to severe debilitating fibrosis of the lung
parenchyma. There are varied causes of ILD, which generally result from a range of
environmental, occupational, recreational, or drug-related exposures or could arise
from the various systemic autoimmune or connective tissue diseases (CTD) [10].
Classification of different types of ILD is shown in Fig. 2. A few of the common
ILD subtypes are described in the present section.
304 S. Dasgupta et al.

IPF

iNSIP

Desqualmative IP

RB-ILD
IIPs
COP

Acute interstitial pneumonia

Idiopathic pleuroparenchymal fibroelastosis

Idiopathic lymphocytic interstitial pneumonia

ILD RA-ILD
Autoimmune ILD
SSC-ILD
HP

Sarcoidosis
Drug induced ILD

Occupational and environmental exposure-related

Other ILD other ILD

Fig. 2 Classification of different types of interstitial lung disease (Cottin et al. 2018) [3]. ILD
interstitial lung disease, IIP idiopathic interstitial pneumonia, IPF idiopathic pulmonary fibrosis,
iNSIP idiopathic nonspecific interstitial pneumonia, RB-ILD respiratory bronchiolitis-associated
ILD, COP cryptogenic organizing pneumonia, RA-ILD rheumatoid arthritis-associated ILD, SSC-­
ILD systemic sclerosis-associated ILD, HP hypersensitivity pneumonitis

2.1 Idiopathic Interstitial Pneumonia (IIP)

The cause of IIP, comprising of diffuse parenchymal lung diseases, remains

unknown. IIP is characterized by varying degrees of inflammation and fibrosis in
the lung interstitium. These characteristics split IIP into eight clinicopathologic
entities, that is, IPF, nonspecific interstitial pneumonia (NSIP), cryptogenic organiz-
ing pneumonia (COP), acute interstitial pneumonia, respiratory bronchiolitis-­
associated interstitial lung disease, desquamative interstitial pneumonia, lymphoid
interstitial pneumonia, and idiopathic pleuroparenchymal fibroelastosis [11].
Among all IIPs, IPF is the most common phenotype characterized by fibroblastic
foci and the presence of inflammation and honeycombing in the lung parenchyma.
Metabolomics and Transcriptomic Approach to Understand the Pathophysiology… 305

2.2 Autoimmune ILD

Autoimmune ILD is caused specifically by autoimmune disorders, which involve

the body’s immune system attacking the lungs. This ILD group gradually develops
and emerges over a long period of time. The symptoms of this ILD include difficulty
in breathing, dry cough, and shortness of breath. Connective tissue disease-related
ILD (CTD-ILD), rheumatoid arthritis-associated ILD (RA-ILD), and systemic
sclerosis-­associated ILD (SSC-ILD) are the common types of autoimmune ILD
[12, 13].

2.3 Hypersensitivity Pneumonitis (HP)

HP, also referred to as extrinsic alveolar alveolitis, is a complex subtype of ILD aris-
ing from repeated exposure to certain antigens, most commonly avian, microbial
(especially molds), or chemical. HP is the third most prevalent ILD after IPF and
CTD-ILD. The inhaled antigen triggers type III and type IV hypersensitivity reac-
tions, which causes the damage of alveolar epithelial cells. An impaired repair
mechanism may result in fibroblast activation, deposition of collagen by the destruc-
tion of extracellular matrix, and parenchymal architecture [14]. The major forms of
HP are acute, subacute, and chronic. Acute and subacute HP is mainly characterized
by influenza-like symptoms, such as cough, dyspnea, and fever, developing after
2–9 h of antigen exposure. The chronic form of HP arises from repetitive, low-level
exposure to the causative agent. Still, the identity of the causative antigen may
remain unknown in more than half the cases. Chronic HP patients slowly develop
fibrosis in the lung interstitium and are associated with a significantly high mortality
rate [15].

2.4 Sarcoidosis

Sarcoidosis is a systemic, inflammatory disease resulting from an unknown origin.

Chronic immune response to an idiopathic antigen may lead to sarcoidosis in genet-
ically susceptible subjects. Almost 90% of sarcoidosis patients have pulmonary
involvement. Dry cough, chest tightness, chronic dyspnea on exertion, shortness of
breath, wheezing, hypoxemia, and decline in pulmonary function are the common
signs and symptoms of sarcoidosis. Near about 20% of sarcoidosis patients develop
pulmonary fibrosis, that is, stage IV sarcoidosis which is associated with high mor-
tality [16].
306 S. Dasgupta et al.

2.5 Occupational and Environmental Exposure-Related

Other ILDs

Long-term exposure to occupational or environmental antigens could cause certain

types of ILD via pulmonary and systemic inflammation and oxidative stress. Many
different types of mineral dust, such as silica, asbestos, beryllium, coal mine dust,
metal, and organic dust, including mold spores, can also affect the lung airways,
either by a direct wound or through reactive oxygen molecules. Common conditions
include asbestosis, which is associated with asbestos fibers, and silicosis, which is
caused by free crystalline silicon dioxide or silica particles [17, 18].

3 Metabolomics: An Emerging Tool in Clinical Research

Metabolomics, one of the newest omics science, is an evolving field in clinical

research. Metabolomics is the scientific study of metabolic fingerprints that all cel-
lular processes leave behind in a biological sample [19]. It provides a snapshot of
the metabolic state of an individual at a given point in time. On the other hand,
“metabonomics,” a term first coined by Jeremy Nicholson, refers to “the quantita-
tive measurement of the dynamic multiparametric metabolic response of living sys-
tems to pathophysiological stimuli or genetic modification” [20, 21]. The terms
“metabolomics” and “metabonomics” are often used interchangeably. Among the
different omic approaches, metabolomics is considered to modulate best and depict
the molecular phenotype of health and disease [22]. Thus, it is increasingly becom-
ing a useful and powerful tool for the investigation of complex diseases with unclear
etiology, enabling the discovery of novel biomarkers, which, in turn, aid in the pre-
vention and early diagnosis of diseases. Metabolomics can also monitor the effect
of pharmacotherapy, allowing clinicians to choose the best treatment option for
patients suffering from potentially devastating disorders. The two analytical tech-
niques popularly used have their own advantages and disadvantages. While mass
spectrometry can analyze a wider range of metabolites and is more sensitive, it
results in the destruction of the analyzed sample. NMR spectroscopy, on the other
hand, is highly reproducible and does not destroy the sample; however, sensitivity
is limited [23, 24]. Over the years, application of metabolomics in diseases is rap-
idly growing, and recent studies exploring the metabolic profiles of various human
samples, including but not limited to plasma, serum, urine, BALF, exhaled breath,
saliva, and tissues, bring this technology closer to the patients’ bedside, thereby
enhancing its clinical utility. A schematic representation of the metabolomic work-
flow is shown in Fig. 3.
Metabolomics and Transcriptomic Approach to Understand the Pathophysiology… 307

Fig. 3 Schematic representation of the metabolomic workflow (created using [Link])

3.1 Metabolomics in ILD

Several attempts have been made to understand the metabolic status of ILD patients
and identify prospective biomarkers in lung tissues and various body fluids using a
nontargeted and targeted metabolomic approach. Studies utilizing the metabolomic
approaches to investigate ILD are summarized in Table 1.
308 S. Dasgupta et al.

Table 1 A summary of studies exploring different types of ILD in humans using metabolomic
approach
IPF
Biological
sample Technique Main findings References
Tissue NMR Lactic acid levels significantly elevated in IPF lung [25]
(untargeted) tissue, suggested to be a key driver of myofibroblast
differentiation, as well as onset and progression of
fibrotic disorders
Tissue MS Alterations in glycolytic, adenosine triphosphate [26]
(untargeted) degradation, glutathione biosynthesis, and ornithine
aminotransferase pathways indicated in lung tissues
of IPF patients
Tissue MS (targeted) Free fatty acid dysregulation in IPF lungs; stearic [27]
acid suggested exhibiting antifibrotic effect in IPF
Tissue MS Dysregulation in sphingolipid metabolic pathway, [28]
(untargeted) arginine pathway, glycolysis, TCA cycle, and
mitochondrial β-oxidation; dysregulated haem, bile
acid, and glutamate/aspartate metabolism suggested
to play a crucial role in IPF pathogenesis
Exhaled MS Distinct metabolic profile with 58 discriminatory [29]
breath (untargeted) metabolites identified in EBC of IPF patients
Exhaled MS (targeted) Significantly increased expression levels of proline, [30]
breath 4-hydroxyproline, alanine, valine, leucine/isoleucine,
and allysine were detected in exhaled breath of IPF
patients
Plasma MS (targeted) 62 altered lipids, including 24 types of [31]
glycerophospholipids, 30 types of glycerolipids, 3
types of sterol lipids, 4 types of sphingolipids, and 1
type of fatty acid identified in the plasma of IPF
patients
Plasma MS Lysophosphatidylcholine (lysoPC) and several fatty [32]
(untargeted) acids, including palmitoleic acid, oleic acid, and
linoleic acid, significantly upregulated, whereas
dihydrotestosterone significantly downregulated in
IPF patients
Plasma MS (targeted) Discrimination between stable and progressive IPF [33]
patients based on differences in plasma levels of
triglycerides and phosphatidylcholine; this difference
further confirmed in lung tissue of IPF
Serum MS LysoPC was found to be significantly dysregulated in [34]
(untargeted) IPF patients, indicating its potential as a biomarker
for diagnosis and monitoring of IPF
HP
Serum, NMR Three metabolites, including lactate, pyruvate, and [35]
EBC, and (untargeted) proline, significantly altered in all three biofluids
BALF
Metabolomics and Transcriptomic Approach to Understand the Pathophysiology… 309

Table 1 (continued)
IPF
Biological
sample Technique Main findings References
Sarcoidosis
Serum NMR Three major pathways, including fatty acid [36]
(untargeted) metabolism, glycolysis/TCA cycle, and
homocysteine/methylamine, altered in sarcoidosis
Plasma NMR Distinct metabolomic and metallomic profiles were [37]
(untargeted) observed in veterans with sarcoidosis as compared to
and MS civilians, with levels of magnesium, calcium,
(targeted) aluminium, titanium, and iron increased in
sarcoidosis
RA-ILD
Serum MS Four serum metabolites (mannosamine, alliin, [38]
(untargeted) kynurenine, and 2-hydroxybutyric acid) exhibit
better performance in distinguishing types of RA
patients with acute-onset diffuse ILD (AoDILD) as
compared to existing AoDILD markers, KL-6 and
SP-D
Serum MS Significantly altered expression of decanoic acid, [39]
(untargeted) glycerol, and morpholine was observed on
comparing RA-ILD (usual interstitial pneumonia-­
associated RA and NSIP-associated RA) and RA
patients without any chronic lung disease
Silicosis
Plasma MS L-arginine and kynurenine associated with severity [40]
(untargeted of silicosis with a predictive role in disease
and targeted) monitoring
Lymphangioleiomyomatosis
Cell line MS Targeting E2-dependent cellular metabolic pathways [41]
(untargeted) may have favorable therapeutic effects on
lymphangioleiomyomatosis patients

4 Transcriptomics: A Promising Omic Approach

Transcriptome analysis utilizes high-throughput methods to study the complete set

of RNA transcripts produced by the genome under specific circumstances. It covers
all types of transcripts, including mRNAs, miRNAs, and different types of long
noncoding RNAs (lncRNAs). Transcriptome analysis gives us an overview of all
genes’ expression levels and enables us to understand the physiology of the cell.
More precisely, it also discloses key regulations of biological processes triggering
diseases. While microarrays are generally less complex and easier to use than NGS,
the latter is associated with greater flexibility, high throughput, and high discovery
potential. A schematic representation of the transcriptomic workflow is shown
in Fig. 4.
310 S. Dasgupta et al.

Fig. 4 Schematic representation of the transcriptomic workflow (created using [Link])

4.1 Transcriptomics in ILD

Various studies have been performed to understand the transcriptomic signatures of

ILD patients and identify prospective biomarkers in lung tissues and various bioflu-
ids using NGS and microarray techniques. Despite increasing interest and effort
invested by clinicians and scientists during the last decade, the etiology of ILD
remains elusive and controversial.
As mentioned earlier, IPF is characterized by remodeling or scarring of the air-
way epithelium. The activated extracellular matrix (ECM)-produced myofibroblasts
play a key role in the process of fibrotic tissue remodeling. Advances in transcrip-
tomic techniques have allowed high-throughput analysis and discovery of gene
deregulation in IPF. Several studies using lung tissues have reported that IPF is
associated with variances in the expression levels of genes such as CCL8 [42],
CXCL14 [43], CXCL4 and CXCL12 [44], NOTCH2 [45], TGF-β1 and RhoA kinase
[46], REVERBα [47], IL-1β [48], FLIL33 and POU2AF1 [49], FOXL1 [50],
COL6A3, and POSTN [51]. Microarray analysis of peripheral blood by Abe et al.
(2020) has shown dysregulated PDGF B, VEGF B, and FGF 2. The authors con-
firmed their findings using ELISA, western blot, immunofluorescence, and 3H thy-
mine uptake assays. Xia and co-workers (2021) recently utilized weighted gene
co-expression network analysis (WGCNA) of BALF samples and could associate
four genes, TLR2, CCR2, HTRA1, and SFN, with disease prognosis.
Pathway enrichment analysis based on dysregulated genes highlights the associ-
ated biological pathways, molecular functions, and cellular components. This
Metabolomics and Transcriptomic Approach to Understand the Pathophysiology… 311

method identifies all biological pathways enriched in a gene list more than would be
expected by chance. The KEGG pathway tool maps the pathways associated with
dysregulated genes in a specific disease. Pathway enrichment analysis of IPF
patients revealed that the differentially expressed genes were majorly associated
with myofibroblast differentiation and massive ECM deposition. The transcriptomic
signatures of fibroblasts suggest that characterization of lung proteins, specifically
lung fibrotic ECM, helps determine its composition and define targetable molecules
for advanced stages of fibrosis. Boesch and his team (2020) isolated fibrosis-­specific
mesenchymal stem cell-like cells from lung tissue of IPF subjects and observed that
the differentially expressed genes were enriched with hypoxia, fibrosis, and bacte-
rial colonization factors which are the typical hallmarks of pulmonary fibrosis. They
found that the cells isolated from IPF patients express genes associated with activat-
ing canonical TGF-β, HIPPO/YAP, PI3K/AKT, p53, and WNT signaling cascades,
which are activated in an integrated network. Another interesting study by Hsu and
co-authors (2011) suggested that IPF lungs enriched in fibrosis-related genes,
insulin-­like growth factor signaling, and caveolin-mediated endocytosis. This
microarray analysis also highlighted the common molecular signatures between
lung tissue and fibroblasts of these patients.
Like IPF, HP is associated with matrix remodeling and formation of fibrosis.
There exist only two studies where transcriptomics has been used to explore genetic
alterations in HP. Sarcoidosis, as mentioned earlier, is an immune-mediated multi-
system disease characterized by the formation of non-caseating granuloma. Multiple
pro-inflammatory signaling pathways, including IFN-γ/STAT-1, IL-6/STAT-3, and
NF-κB, have been implicated in mediating macrophage activation and granuloma
formation in sarcoidosis. Utilizing RT-PCR, Christophi et al. (2014) have demon-
strated that IL-6, COX-2, MCP-1, IFN-γ, T-bet, IRF-1, Nox2, IL-33, and eotaxin-1
hold potential for differential diagnosis between sarcoidosis, suture, and fungal
granulomas. In another recent study, Lepzien and co-workers (2021) have shown
that allogeneic T cell proliferation increased after coculture with monocytes and
dendritic cells of sarcoidosis patients. The authors also found that mainly T-bet and
RORγt-expressing T cells produce IFN-γ. Monocytes from sarcoidosis patients can
activate and polarize T cells towards Th1 and Th17.1 cells. In a comparative study
between sarcoidosis and IPF, cluster analysis of BALF cells showed elevated mRNA
expression of genes associated with ribosome biogenesis in sarcoidosis patients.
Clusters formed by genes with altered mRNA expression in patients with IPF could
be implicated in cell migration and adhesion processes, metalloproteinase expres-
sion, and negative regulation of cell proliferation. Various studies highlighting the
transcriptome fingerprints and associated pathways in different ILD subtypes are
summarized in Table 2.
312 S. Dasgupta et al.

Table 2 A summary of studies exploring different types of ILD in humans using transcriptomic
approach
IPF
Biofluid Technique Findings Reference
Plasma, Microarray CCL8 is a key molecule for differential diagnosis [42]
BALF, of IPF and can also predict survival
And tissues
Tissue NGS Differentially expressed genes in IPF are [43]
associated with fibrosis, hypoxia, bacterial
colonization, and pulmonary fibrosis metabolism
Tissue Microarray TGF-β1, RhoA kinase, and the TSC2/RHEB axis [44]
form major signaling clusters associated with
collagen gene expression in IPF
Tissue NGS Specific connective tissue-related genes including [45]
alpha-smooth muscle actin, fibrillin, fibronectin,
tenascin C, osteopontin, chains of highly
abundant structural collagens and other collagens,
multiple matrix metalloproteinases, and Wilms
tumor protein are elevated in IPF
Tissue Microarray TGF-β1 increases the risk of developing IPF in [46]
smokers
Tissue NGS Notch signaling regulates the maintenance of an [47]
expanded pool of secretory primed basal cells in
the distal lung of IPF patients
Tissue Microarray IPF lungs are enriched with fibrosis-related gene, [48]
insulin-like growth factor signaling, and
caveolin-mediated endocytosis
Tissue Microarray Lower expression of cell migration-inducing and [49]
hyaluronan-binding protein in pirfenidone-treated
IPF patients
Tissue Microarray IPF lungs are enriched with cell adhesion, [50]
molecule binding, chemical homeostasis,
surfactant homeostasis, and receptor binding
genes
Tissue NGS Elevated expression of numerous immune, [45]
inflammation, and extracellular matrix-related
mRNAs observed in IPF
Tissue NGS Alternative splicing COL6A3 and POSTN may [51]
be involved in the pathogenesis of IPF
Tissue Microarray Twist1 as a regulator of noncanonical NF-κB [52]
signaling through CXCL12 may have a
profibrotic effect in IPF
Tissue Microarray CXCL14 and CXCL4 may be involved in the [53]
activation of fibroblasts within IPF lungs and are
involved in disease pathogenesis
Tissue Microarray A significant upregulation of EGFR, both at [54]
protein and mRNA level, was observed in IPF,
fibrotic NSIP, and COP compared with controls
Tissue NGS MMP7 is differentially expressed in IPF patients [55]
Metabolomics and Transcriptomic Approach to Understand the Pathophysiology… 313

Table 2 (continued)
IPF
Biofluid Technique Findings Reference
Tissue NGS Hypoxia and TGF-β1 synergistically increase [56]
myofibroblast marker expression in IPF
Tissue NGS Discrete types of macrophages expressing (1) [57]
monocyte markers and (2) higher levels of
FABP4, INHBA, SPP1, and MERTK present in
IPF lungs
Tissue NGS FOXL1 can control a wide array of genes that [58]
potentiate fibroblast function, including TAZ/YAP
signature genes and PDGF receptor-α in IPF
Tissue NGS POU2AF1 regulates fibrosis in IPF [59]
Tissue NGS FLIL33 overexpression and stimulation with [60]
TGF-β differentially regulates the fibroblast
transcriptome in IPF
Tissue Microarray Genes associated with cell adhesion, molecule [50]
binding, chemical homeostasis, surfactant
homeostasis, and receptor binding are
dysregulated in lungs of IPF patients
Tissue Microarray LncRNAs are crucial regulators of proliferation [61]
and inflammation in human lung fibroblasts,
suggesting their possible involvement in the
lower inflammatory response in IPF
Tissue NGS Increased CD44 is a characteristic of IPF [62]
mesenchymal progenitor cells
Tissue NGS Following TGF-β1 stimulation, collagen secretion [63]
is elevated in IPF patients
Tissue and NGS The expression of GDF15 is increased in IPF and [64]
plasma is associated with the progression of the disease
Tissue NGS Altered basaloid cells that express basal [65]
epithelial, mesenchymal, senescence, and
developmental markers are located at the
myofibroblast foci edge. Ectopically expanded
cell populations are observed in vascular
endothelial cells
Tissue Microarray CXCL12, collagen 3A1, MMP2, and MMP14 are [66]
upregulated in fibrotic ILD, including IPF, NSIP,
organizing pneumonia, and alveolar fibroelastosis
as compared with controls
Tissue NGS IPF fibroblast transcriptional signatures indicate [67]
enrichment of WNT, TGF-β, and ECM genes and
downregulation of miR-29b-3p, miR-138-5p, and
miR-146b-5p
Tissue Microarray Pathways associated with vascular proliferation, [68]
WNT signaling, and apoptosis are dysregulated
in IPF arterioles
(continued)
314 S. Dasgupta et al.

Table 2 (continued)
IPF
Biofluid Technique Findings Reference
Tissue NGS Alveolar type 1 (AT1), AT2, and conducting [69]
airway selective markers are frequently
co-expressed by IPF cells, and aberrant activation
of canonical signaling via TGF-β, HIPPO/YAP,
p53, WNT, and AKT/PI3K is predicted via
pathway analysis
Tissue Microarray Expression of cilium genes appears to identify [70]
two unique molecular phenotypes
Of IPF/UIP, which may affect therapeutic
responsiveness
BALF Microarray IPF is associated with cell migration, cell [71]
adhesion, metalloproteinase expression, and
negative regulation of cell proliferation
BALF Microarray TLR2, CCR2, HTRA1, and SFN are involved in [72]
the prognosis of IPF
Peripheral Microarray PDGF B, VEGF B, and FGF 2 genes are [73]
blood associated with IPF
Peripheral Microarray YBX3, UTRN, hsa_circ_0001924, and FENDR [74]
blood could be potential diagnostic biomarkers of IPF
Peripheral Microarray Increased circulating FUT3 level is associated [75]
blood with reduced risk of IPF
PBMC, NGS Type I IFN pathway is the key regulator for [76]
monocytes, driving chronic inflammation and fibrosis in IPF
and serum
Nasal biopsy NGS Pathways related to immune response and [77]
inflammatory signaling are elevated in IPF
patients
Tissue Fluorescence-­ IGF-1 signaling, ERK/MAPK signaling, protein [78]
based RNA ubiquitination, PI13/AKT signaling, cardiac
quantitation b-adrenergic signaling, actin-cytoskeleton
assay signaling, integrin signaling, and NRF2-mediated
oxidative stress response pathways are associated
with IPF
HP
Tissue NGS HP is associated with specific genes, including [79]
CXCL9, an IFN-γ-inducible chemokine, and
ligand for CXCR3
Tissue NGS Antigen presentation and extracellular matrix-­ [80]
associated transcriptomic signatures are present
in mild HP cases, whereas B cells are
predominant in fibrotic HP
Sarcoidosis
Tissue Microarray Multiple pro-inflammatory signaling pathways [81]
mediate macrophage activation and granuloma
formation in sarcoidosis
(continued)
Metabolomics and Transcriptomic Approach to Understand the Pathophysiology… 315

Table 2 (continued)
IPF
Biofluid Technique Findings Reference
Tissue NGS STAB1, HBEGF, and NOTCH4 genes are [82]
associated with sarcoidosis pathogenesis
BALF cells Microarray Increased mRNA gene expression associated with [71]
ribosome biogenesis and proteasome apparatus
observed in sarcoidosis patients
BAL Microarray Cathepsin S is significantly upregulated in [83]
sarcoidosis
BAL NGS In four sarcoidosis endotypes (hilar [84]
lymphadenopathy, extraocular involvement,
chronic stage, and multiorgan involvement
condition), elevated acute T-cell response, PI3K
pathways, increased immune response pathways,
and increased IL-1 and IL-18 immune and
inflammatory responses are observed
Blood and NGS Monocytes of sarcoidosis patients can activate [85]
BAL and polarize T cells toward Th1 and Th17.1
Blood and NGS Monocytes/monocyte-derived cells increased in [86]
BAL blood and BAL of sarcoidosis compared to
healthy controls
Blood Microarray Interferon-inducible neutrophil-driven blood [87]
transcriptional signature observed in sarcoidosis
PBMC and Microarray Alterations in TLR2 signaling pathway and [88]
BAL cells downstream of NF-κB apoptosis and proliferation
evidenced in sarcoidosis
PBMC NGS Dysfunctional p53, cell death, and TNFR2 [89]
signaling associated with sarcoidosis
PBMC, Microarray Molecular pathways, regulated by IL-13, which [90]
in vitro helps in activated M2 macrophage polarization, is
granuloma, associated with the pathogenesis of sarcoidosis
and tissue
SSC-ILD
Tissue NGS Mesenchymal cell population including [91]
SPINT2hi, MFAP5hi, few WIF1hi fibroblasts,
and a new large myofibroblast population may be
actively involved in the regulation of disease
pathogenesis
Tissue NGS Cellular stress pathways are upregulated in [92]
SSC-ILD, a population of KRT5-/KRT17+
aberrant basaloid cells representing markers of
epithelial-mesenchymal transition and cellular
senescence identified in the disease for the first
time
Tissue Microarray Increased expression of TGF-β response [93]
signature is the key regulator of fibrosis
formation in fibrotic SSC-ILD
(continued)
316 S. Dasgupta et al.

Table 2 (continued)
IPF
Biofluid Technique Findings Reference
Tissue Microarray Targeting IL-6 trans-signaling, IGFBP2, IGFL2, [94]
and the coagulation cascade represent potential
therapeutic strategies against the disease
Skin biopsy Microarray SELP, MMP 3, and CCL2 which are involved in [95]
the adhesion and extravasation of inflammatory
cells are associated with SSC-ILD
Serum Microarray Hepatic fibrosis, granulocyte and agranulocyte [96]
adhesion, and diapedesis are associated with
SSC-ILD
Silicosis
Tissue NGS Several critical genes, including MUC5AC and [97]
FGF10, serve as potential drug targets in silicosis
Cell line NGS Transcription factors, EGR2 and BHLHE40, are [98]
upregulated while TBX2, NR1H3, NR2F1,
PPAR-γ, and EPAS1 are downregulated, which
may play a crucial regulatory role in disease
pathogenesis
Dermatomyositis-associated ILD
Blood NGS PLAUR may play an important role in disease [99]
pathogenesis by regulating the neutrophil-­
associated immune response

5 Integration of Metabolomic
and Transcriptomic Fingerprints

As mentioned earlier, clinical metabolomics is primarily used to identify low

molecular weight compounds differentially expressed in a particular disease. In
contrast, transcriptomics identifies the complete set of dysregulated RNAs associ-
ated with a disease. Integration of metabolomic and transcriptomic signatures has
emerged as a popular application-driven method for investigating underlying dis-
ease mechanisms, monitoring disease progression, and identifying potential bio-
markers [100–102]. The omic tools highlight alterations in genotype and phenotype
and provide complementary information about genetic alterations, protein synthe-
sis, metabolism, and cellular function. Pathways and network connections further
reflect the association between key metabolites and candidate transcripts.
Biological pathway networks reveal hidden patterns in unstructured data by con-
verting them into logically structured and visually evident representations, with
nodes representing genes and metabolites and edges suggesting relationships
between nodes and clusters with similar chemical activities. VANTED [103], VisAnt
Metabolomics and Transcriptomic Approach to Understand the Pathophysiology… 317

[104], Impala [105], and Metscape2 [106] are some of the network-based visualiza-
tion tools that interface with public databases. In addition, Arena3D allows users to
envision three-dimensional biological networks [107]. Interactive editing is fre-
quently performed for small biological networks. However, for major networks,
automated layout web tools, that is, Cytoscape [108], NAViGaTOR [109], and
Cerebral [110], are more convenient. Alternatively, pathway visualization tools
highlight the biochemical activities and different interactive pathways in experi-
mental datasets. Pathguide offers an overview of nearly 190 web-usable network
databases and biological pathways [110]. Arakawa and his team have developed a
pathway visualization tool for KEGG-based pathways. Users can capture system-
atic features of biological activity by visualizing pathways at the level of different
omic data representations [111]. Paintomics, another software program, analyzes
the expression of genes and concentration of metabolite data and displays it on
KEGG pathway maps [112]. ProMeTra can display dynamic data and accept anno-
tated images in SVG format [113]. In plants, KaPPa-View and MapMan show the
number of metabolites and transcripts for preset route blocks [114, 115]. Other tools
like MAYDAY enable viewing expression data in a genomic context with any meta-
data [116], and PaVESy creates personalized pathways using proteins and metabo-
lites provided by the user [117]. A schematic representation of integrated
metabolomic and transcriptomic workflow is shown in Fig. 5.
In a recent study, our group used NMR coupled with chemometric analysis to
identify the unique metabolic fingerprints in BALF of HP subjects. A total of six
metabolites were found to be significantly altered in HP compared to non-HP con-
trols [35]. Next, we considered NGS data of lung tissues from HP patients and
controls, reported in the NCBI-GEO public database by Furusawa et al., and

Fig. 5 Schematic representation of integrated metabolomic and transcriptomi data (created using
[Link], STITCH database, and Graph pad prism version 7)
318 S. Dasgupta et al.

performed bioinformatic analysis. A total of 555 genes were dysregulated (373

upregulated and 182 downregulated) in HP cases. An interaction network between
the six candidate metabolites and most significantly altered genes (five upregulated
and five downregulated) was established utilizing the Search Tool for Interactions of
Chemicals (STITCH) database. The metabolite-gene interaction by STITCH dem-
onstrated 19 nodes connected via 16 edges. The clustering coefficient of the net-
work was found to be 0.768 (protein-protein interaction enrichment p-value:
0.0838). Overall pathway overrepresentation analysis was performed by integrating
the candidate metabolites and transcripts utilizing IMPaLA version 12. Glycolysis
and phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) signaling path-
ways emerged to be most significantly associated with the pathogenesis of HP. These
findings are encouraging, since association of these pathways in chronic HP is well
established. Since glycolysis is the key energy driving force for myofibroblast dif-
ferentiation and formation of fibrosis, perturbation of glycolysis seems likely [118].
The involvement of PI3K-AKT pathway is also evidenced in bleomycin-induced
pulmonary fibrosis. It is hypothesized that PI3K-AKT plays a central role in fibrosis
development [119, 120]. A novel insight into the pathogenesis of HP is envisioned
by integrating the findings of the two omic platforms.

6 Challenges and Future Scope

Most of the omic-driven studies conducted on ILD so far have included a small
number of patients, which is quite understandable considering that ILD is a severe
condition with a short average life expectancy. Power and sample size estimation,
however difficult, would be useful because the low sample size is connected with
statistical errors and risks of overfitting and misleading calculations. Since omic
output is highly dynamic, clinical variables such as physiological status, age, gen-
der, and treatment may influence the findings. Hence, baseline characteristics of
recruited ILD subjects need to be closely matched. Lack of a rigorous subject selec-
tion approach could also result in discovering markers that are not exclusive to ILD
subtypes. It is observed that only a few groups have included healthy controls in
their omic-driven research on ILD. Also, nonuniformity in including smokers and
nonsmokers is frequently observed while comparing disease populations with
healthy controls. This makes unbiased comparisons and conclusions impossible. A
few groups were also unable to validate ILD candidate markers, which is crucial for
biomarker identification. In fact, one of the main reasons why most of the omic-­
based disease markers identified so far have not made it to clinical practice is due to
a lack of adequate validation trials. Another observation that warrants attention
while using omics is that different research groups identify different biomarkers in
the same biofluid for a particular disease. This is not surprising given the fact that
factors such as sampling methods, sample collection, handling and preparation,
instrumentation, and data mining protocols tend to vary from one setup to another.
To generate robust and reproducible data, the practices and procedures should be
Metabolomics and Transcriptomic Approach to Understand the Pathophysiology… 319

standardized and rigorously followed across all clinics and research laboratories.
Metabolic flux analysis is crucial to obtain insight into dysregulated cellular metab-
olism caused by disease perturbations. It is expected that stratifying ILD patients
based on disease severity and subtypes will significantly improve metabolome and
transcriptome coverage. Assessment of sensitivity, specificity, and clinical relevance
of the differentially expressed molecules is also recommended. For a reliable and
unbiased diagnosis of this severe pulmonary disease, large-scale, well-designed,
multicentric clinical studies and recruitment of suitable controls are recommended.
The ultimate focus of metabolomic and transcriptomic data integration is identi-
fying key metabolic and genetic factors that contribute significantly to disease etiol-
ogy. Integrated omics is more than a collection of tools; it is a comprehensive
paradigm for interpreting multi-omic datasets in a way that can provide new insights
into basic biology, as well as health and disease. Machine learning approaches for
multi-omic data analyses is an emerging trend for exploring molecular pathways in
detail and drawing a holistic representation of a given phenotype using all biologi-
cal and clinical information of an individual. One of the major advantages is incor-
porating biological domain knowledge into the machine learning models as
inductive biases to reduce data overfitting. Additionally, as omic tools evolve, they
need to be user-friendly, interoperable, and effective for computationally intensive
analyses. Machine learning methods offer novel techniques to integrate such omic
datasets. With the emerging precision medicine initiative, where disease prevention
and management take into account the variability in genes, environment, and life-
style of each individual in contrast to the conventional one-size-fits-all approach,
integration of clinical data with the patients’ metabolome and genetic makeup will
provide an in-depth understanding of disease pathophysiology and facilitate design-
ing of targeted therapies for individuals, thereby revolutionizing precision medicine-­
based decision-making in the clinic.

Acknowledgments Figures were created with [Link].

References

1. Rivera-Ortega P, Molina-Molina M. Interstitial lung diseases in developing countries. Ann

Glob Health. 2019;85(1):4.
2. ERS. Interstitial lung diseases. In: ERS European lung White-book. Chapter 22. ERS; 2015.
[Link] Accessed 15 Sep 2016.
3. Cottin V, Hirani NA, Hotchkin DL, Nambiar AM, Ogura T, Otaola M, Skowasch D, Park
JS, Poonyagariyagorn HK, Wuyts W, Wells AU. Presentation, diagnosis and clinical
course of the spectrum of progressive-fibrosing interstitial lung diseases. Eur Respir Rev.
2018;27(150):180076.
4. Singh S, Collins BF, Sharma BB, Joshi JM, Talwar D, Katiyar S, Singh N, Ho L, Samaria JK,
Bhattacharya P, Gupta R. Interstitial lung disease in India. Results of a prospective registry.
Am J Respir Crit Care Med. 2017;195(6):801–13.
5. Gowda GN, Zhang S, Gu H, Asiago V, Shanaiah N, Raftery D. Metabolomics-based methods
for early disease diagnostics. Expert Rev Mol Diagn. 2008;8(5):617–33.
320 S. Dasgupta et al.

6. Hulstaert E, Morlion A, Cobos FA, Verniers K, Nuytens J, Eynde EV, Yigit N, Anckaert J,
Geerts A, Hindryckx P, Jacques P. Charting extracellular transcriptomes in the human bio-
fluid RNA atlas. Cell Rep. 2020;33(13):108552.
7. El-Mogy M, Lam B, Haj-Ahmad TA, McGowan S, Yu D, Nosal L, Rghei N, Roberts P, Haj-­
Ahmad Y. Diversity and signature of small RNA in different bodily fluids using next genera-
tion sequencing. BMC Genomics. 2018;19(1):1–24.
8. Ferrero G, Cordero F, Tarallo S, Arigoni M, Riccardo F, Gallo G, Ronco G, Allasia M,
Kulkarni N, Matullo G, Vineis P. Small non-coding RNA profiling in human biofluids and
surrogate tissues from healthy individuals: description of the diverse and most represented
species. Oncotarget. 2018;9(3):3097.
9. Godoy PM, Bhakta NR, Barczak AJ, Cakmak H, Fisher S, MacKenzie TC, Patel T, Price
RW, Smith JF, Woodruff PG, Erle DJ. Large differences in small RNA composition between
human biofluids. Cell Rep. 2018;25(5):1346–58.
10. Guler SA, Corte TJ. Interstitial lung disease in 2020: a history of progress. Clin Chest Med.
2021;42(2):229–39.
11. Oliveira DS, Araújo JDA, Paiva AFL, Ikari ES, Chate RC, Nomura CH. Idiopathic interstitial
pneumonias: review of the latest American Thoracic Society/European Respiratory Society
classification. Radiol Bras. 2018;51(5):321–7.
12. Antoniou KM, Margaritopoulos G, Economidou F, Siafakas NM. Pivotal clinical dilem-
mas in collagen vascular diseases associated with interstitial lung involvement. Eur Respir
J. 2009;33(4):882–96.
13. Mathai SC, Danoff SK. Management of interstitial lung disease associated with connective
tissue disease. BMJ. 2016;352:h6819.
14. Takemura T, Akashi T, Ohtani Y, Inase N, Yoshizawa Y. Pathology of hypersensitivity pneu-
monitis. Curr Opin Pulm Med. 2008;14(5):440–54.
15. Sforza GGR, Marinou A. Hypersensitivity pneumonitis: a complex lung disease. Clin Mol
Allergy. 2017;15(1):1–8.
16. Nardi A, Brillet PY, Letoumelin P, Girard F, Brauner M, Uzunhan Y, Naccache JM, Valeyre
D, Nunes H. Stage IV sarcoidosis: comparison of survival with the general population and
causes of death. Eur Respir J. 2011;38(6):1368–73.
17. Gulati M, Redlich CA. Asbestosis and environmental causes of usual interstitial pneumonia.
Curr Opin Pulm Med. 2015;21(2):193.
18. Leung CC, Yu ITS, Chen W. Silicosis. Lancet. 2012;379(9830):2008–18.
19. Oliver SG, Winson MK, Kell DB, Baganz F. Systematic functional analysis of the yeast
genome. Trends Biotechnol. 1998;16(9):373–8.
20. Nicholson JK, Lindon JC, Holmes E. ‘Metabonomics’: understanding the metabolic
responses of living systems to pathophysiological stimuli via multivariate statistical analysis
of biological NMR spectroscopic data. Xenobiotica. 1999;29(11):1181–9.
21. Faber JH, Malmodin D, Toft H, Maher AD, Crockford D, Holmes E, Nicholson JK,
Dumas ME, Baunsgaard D. Metabonomics in diabetes research. J Diabetes Sci Technol.
2007;1(4):549–57.
22. Guijas C, Montenegro-Burke JR, Warth B, Spilker ME, Siuzdak G. Metabolomics activ-
ity screening for identifying metabolites that modulate phenotype. Nat Biotechnol.
2018;36(4):316–20.
23. Li S, Todor A, Luo R. Blood transcriptomics and metabolomics for personalized medicine.
Comput Struct Biotechnol J. 2016;14:1–7.
24. Schreier C, Kremer W, Huber F, Neumann S, Pagel P, Lienemann K, Pestel S. Reproducibility
of NMR analysis of urine samples: impact of sample preparation, storage conditions, and ani-
mal health status. Biomed Res Int. 2013;2013:878374.
25. Kottmann RM, Kulkarni AA, Smolnycki KA, Lyda E, Dahanayake T, Salibi R, Honnons S,
Jones C, Isern NG, Hu JZ, Nathan SD. Lactic acid is elevated in idiopathic pulmonary fibro-
sis and induces myofibroblast differentiation via pH-dependent activation of transforming
growth factor-β. Am J Respir Crit Care Med. 2012;186(8):740–51.
Metabolomics and Transcriptomic Approach to Understand the Pathophysiology… 321

26. Kang YP, Lee SB, Lee JM, Kim HM, Hong JY, Lee WJ, Choi CW, Shin HK, Kim DJ, Koh
ES, Park CS. Metabolic profiling regarding pathogenesis of idiopathic pulmonary fibrosis. J
Proteome Res. 2016;15(5):1717–24.
27. Kim HS, Yoo HJ, Lee KM, Song HE, Kim SJ, Lee JO, Hwang JJ, Song JWW. Stearic
acid attenuates profibrotic signalling in idiopathic pulmonary fibrosis. Respirology.
2021;26(3):255–63.
28. Zhao YD, Yin L, Archer S, Lu C, Zhao G, Yao Y, Wu L, Hsin M, Waddell TK, Keshavjee S,
Granton J. Metabolic heterogeneity of idiopathic pulmonary fibrosis: a metabolomic study.
BMJ Open Respir Res. 2017;4(1):e000183.
29. Rindlisbacher B, Strebel C, Guler S, Kollár A, Geiser T, Fiedler GM, Leichtle AB, Bovet C,
Funke-Chambour M. Exhaled breath condensate as a potential biomarker tool for idiopathic
pulmonary fibrosis—a pilot study. J Breath Res. 2017;12(1):016003.
30. Gaugg MT, Engler A, Bregy L, Nussbaumer-Ochsner Y, Eiffert L, Bruderer T, Zenobi R,
Sinues P, Kohler M. Molecular breath analysis supports altered amino acid metabolism in
idiopathic pulmonary fibrosis. Respirology. 2019;24(5):437–44.
31. Yan F, Wen Z, Wang R, Luo W, Du Y, Wang W, Chen X. Identification of the lipid bio-
markers from plasma in idiopathic pulmonary fibrosis by lipidomics. BMC Pulm Med.
2017;17(1):1–12.
32. Nambiar S, Tan DBA, Clynick B, Bong SH, Rawlinson C, Gummer J, Corte TJ, Glaspole I,
Moodley YPP, Trengove R. Untargeted metabolomics of human plasma reveal lipid markers
unique to chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. Proteom
Clin Appl. 2021;15(2–3):e2000039.
33. Nambiar S, Clynick B, How BS, King A, Walters EH, Goh NS, Corte TJ, Trengove R, Tan D,
Moodley Y. There is detectable variation in the lipidomic profile between stable and progres-
sive patients with idiopathic pulmonary fibrosis (IPF). Respir Res. 2021;22(1):1–8.
34. Rindlisbacher B, Schmid C, Geiser T, Bovet C, Funke-Chambour M. Serum metabolic profil-
ing identified a distinct metabolic signature in patients with idiopathic pulmonary fibrosis–a
potential biomarker role for LysoPC. Respir Res. 2018;19(1):1–12.
35. Dasgupta S, Ghosh N, Choudhury P, Joshi M, Chowdhury SR, Bhattacharyya P, Chaudhury
K. NMR metabolomic and microarray-based transcriptomic data integration identifies unique
molecular signatures of hypersensitivity pneumonitis. Mol Omics. 2022;18(2):101–11.
36. Geamanu A, Gupta SV, Bauerfeld C, Samavati L. Metabolomics connects aberrant bioen-
ergetic, transmethylation, and gut microbiota in sarcoidosis. Metabolomics. 2016;12(2):35.
37. Banoei MM, Iupe I, Bazaz RD, Campos M, Vogel HJ, Winston BW, Mirsaeidi M. Metabolomic
and metallomic profile differences between veterans and civilians with pulmonary sarcoid-
osis. Sci Rep. 2019;9(1):1–12.
38. Furukawa H, Oka S, Shimada K, Hashimoto A, Komiya A, Matsui T, Fukui N, Tohma
S. Serum metabolomic profiles of rheumatoid arthritis patients with acute-onset diffuse inter-
stitial lung disease. Biomark Insights. 2019;14:1177271919870472.
39. Furukawa H, Oka S, Shimada K, Okamoto A, Hashimoto A, Komiya A, Saisho K, Yoshikawa
N, Katayama M, Matsui T, Fukui N. Serum metabolomic profiling in rheumatoid arthritis
patients with interstitial lung disease: a case–control study. Front Med. 2020;7:599794.
40. Xue C, Wu N, Fan Y, Ma J, Ye Q. Distinct metabolic features in the plasma of patients
with silicosis and dust-exposed workers in China: a case–control study. BMC Pulm Med.
2021;21(1):1–10.
41. Sun Y, Gu X, Zhang E, Park MA, Pereira AM, Wang S, Morrison T, Li C, Blenis J, Gerbaudo
VH, Henske EP. Estradiol promotes pentose phosphate pathway addiction and cell survival
via reactivation of Akt in mTORC1 hyperactive cells. Cell Death Dis. 2014;5(5):e1231.
42. Lee JU, Cheong HS, Shim EY, Bae DJ, Chang HS, Uh ST, Kim YH, Park JS, Lee B, Shin
HD, Park CS. Gene profile of fibroblasts identify relation of CCL8 with idiopathic pulmonary
fibrosis. Respir Res. 2017;18(1):1–12.
322 S. Dasgupta et al.

43. Boesch M, Baty F, Brutsche MH, Tamm M, Roux J, Knudsen L, Gazdhar A, Geiser T, Khan
P, Hostettler KE. Transcriptomic profiling reveals disease-specific characteristics of epithelial
cells in idiopathic pulmonary fibrosis. Respir Res. 2020;21(1):1–9.
44. Guillotin D, Taylor AR, Platé M, Mercer PF, Edwards LM, Haggart R, Miele G, McAnulty
RJ, Maher TM, Hynds RE, Jamal-Hanjani M. Transcriptome analysis of IPF fibroblastic foci
identifies key pathways involved in fibrogenesis. Thorax. 2021;76(1):73–82.
45. Luzina IG, Salcedo MV, Rojas-Peña ML, Wyman AE, Galvin JR, Sachdeva A, Clerman A,
Kim J, Franks TJ, Britt EJ, Hasday JD. Transcriptomic evidence of immune activation in
macroscopically normal-appearing and scarred lung tissues in idiopathic pulmonary fibrosis.
Cell Immunol. 2018;325:1–13.
46. Checa M, Hagood JS, Velazquez-Cruz R, Ruiz V, Garcia-De-Alba C, Rangel-Escareño C,
Urrea F, Becerril C, Montaño M, García-Trejo S, Lira JC. Cigarette smoke enhances the expres-
sion of profibrotic molecules in alveolar epithelial cells. PLoS One. 2016;11(3):e0150383.
47. Carraro G, Mulay A, Yao C, Mizuno T, Konda B, Petrov M, Lafkas D, Arron JR, Hogaboam
CM, Chen P, Jiang D. Single-cell reconstruction of human basal cell diversity in normal and
idiopathic pulmonary fibrosis lungs. Am J Respir Crit Care Med. 2020;202(11):1540–50.
48. Hsu E, Shi H, Jordan RM, Lyons-Weiler J, Pilewski JM, Feghali-Bostwick CA. Lung tissues
in patients with systemic sclerosis have gene expression patterns unique to pulmonary fibro-
sis and pulmonary hypertension. Arthritis Rheum. 2011;63(3):783–94.
49. Kwapiszewska G, Gungl A, Wilhelm J, Marsh LM, Puthenparampil HT, Sinn K, Didiasova
M, Klepetko W, Kosanovic D, Schermuly RT, Wujak L. Transcriptome profiling reveals
the complexity of pirfenidone effects in idiopathic pulmonary fibrosis. Eur Respir
J. 2018;52(5):1800564.
50. Lee JU, Son JH, Shim EY, Cheong HS, Shin SW, Shin HD, Baek AR, Ryu S, Park CS, Chang
HS, Park JS. Global DNA methylation pattern of fibroblasts in idiopathic pulmonary fibrosis.
DNA Cell Biol. 2019;38(9):905–14.
51. Nance T, Smith KS, Anaya V, Richardson R, Ho L, Pala M, Mostafavi S, Battle A, Feghali-­
Bostwick C, Rosen G, Montgomery SB. Transcriptome analysis reveals differential splicing
events in IPF lung tissue. PLoS One. 2014;9(3):e92111.
52. Tan J, Tedrow JR, Nouraie M, Dutta JA, Miller DT, Li X, Yu S, Chu Y, Juan-Guardela B,
Kaminski N, Ramani K. Loss of Twist1 in the mesenchymal compartment promotes increased
fibrosis in experimental lung injury by enhanced expression of CXCL12. J Immunol.
2017;198(6):2269–85.
53. Rodriguez LR, Emblom-Callahan M, Chhina M, Bui S, Aljeburry B, Tran LH, Novak R,
Lemma M, Nathan SD, Grant GM. Global gene expression analysis in an in vitro fibroblast
model of idiopathic pulmonary fibrosis reveals potential role for CXCL14/CXCR4. Sci Rep.
2018;8(1):1–12.
54. Tzouvelekis A, Ntolios P, Karameris A, Vilaras G, Boglou P, Koulelidis A, Archontogeorgis
K, Kaltsas K, Zacharis G, Sarikloglou E, Steiropoulos P. Increased expression of epidermal
growth factor receptor (EGF-R) in patients with different forms of lung fibrosis. Biomed Res
Int. 2013;2013:654354.
55. Vukmirovic M, Herazo-Maya JD, Blackmon J, Skodric-Trifunovic V, Jovanovic D, Pavlovic
S, Stojsic J, Zeljkovic V, Yan X, Homer R, Stefanovic B. Identification and validation of dif-
ferentially expressed transcripts by RNA-sequencing of formalin-fixed, paraffin-­embedded
(FFPE) lung tissue from patients with idiopathic pulmonary fibrosis. BMC Pulm Med.
2017;17(1):1–12.
56. Senavirathna LK, Huang C, Pushparaj S, Xu D, Liu L. Hypoxia and transforming growth fac-
tor β1 regulation of long non-coding RNA transcriptomes in human pulmonary fibroblasts.
Physiol Rep. 2020;8(1):e14343.
57. Morse C, Tabib T, Sembrat J, Buschur KL, Bittar HT, Valenzi E, Jiang Y, Kass DJ, Gibson K,
Chen W, Mora A. Proliferating SPP1/MERTK-expressing macrophages in idiopathic pulmo-
nary fibrosis. Eur Respir J. 2019;54(2):1802441.
Metabolomics and Transcriptomic Approach to Understand the Pathophysiology… 323

58. Miyashita N, Horie M, Suzuki HI, Saito M, Mikami Y, Okuda K, Boucher RC, Suzukawa
M, Hebisawa A, Saito A, Nagase T. FOXL1 regulates lung fibroblast function via multiple
mechanisms. Am J Respir Cell Mol Biol. 2020;63(6):831–42.
59. McDonough JE, Ahangari F, Li Q, Jain S, Verleden SE, Herazo-Maya J, Vukmirovic M,
DeIuliis G, Tzouvelekis A, Tanabe N, Chu F. Transcriptional regulatory model of fibrosis
progression in the human lung. JCI Insight. 2019;4(22):e131597.
60. Luzina IG, Fishelevich R, Hampton BS, Courneya JP, Parisella FR, Lugkey KN, Baleno
FX, Choi D, Kopach P, Lockatell V, Todd NW. Full-length IL-33 regulates Smad3 phos-
phorylation and gene transcription in a distinctive AP2-dependent manner. Cell Immunol.
2020;357:104203.
61. Hadjicharalambous MR, Roux BT, Csomor E, Feghali-Bostwick CA, Murray LA, Clarke
DL, Lindsay MA. Long intergenic non-coding RNAs regulate human lung fibroblast func-
tion: implications for idiopathic pulmonary fibrosis. Sci Rep. 2019;9(1):1–15.
62. Beisang DJ, Smith K, Yang L, Benyumov A, Gilbertsen A, Herrera J, Lock E, Racila E,
Forster C, Sandri BJ, Henke CA. Single-cell RNA sequencing reveals that lung mesenchymal
progenitor cells in IPF exhibit pathological features early in their differentiation trajectory.
Sci Rep. 2020;10(1):1–12.
63. Roach KM, Sutcliffe A, Matthews L, Elliott G, Newby C, Amrani Y, Bradding P. A model of
human lung fibrogenesis for the assessment of antifibrotic strategies in idiopathic pulmonary
fibrosis. Sci Rep. 2018;8(1):1–15.
64. Zhang Y, Jiang M, Nouraie M, Roth MG, Tabib T, Winters S, Chen X, Sembrat J, Chu Y,
Cardenes N, Tuder RMM. GDF15 is an epithelial-derived biomarker of idiopathic pulmonary
fibrosis. Am J Phys Lung Cell Mol Phys. 2019;317(4):L510–21.
65. Adams TS, Schupp JC, Poli S, Ayaub EA, Neumark N, Ahangari F, Chu SG, Raby BA,
DeIuliis G, Januszyk M, Duan Q. Single-cell RNA-seq reveals ectopic and aberrant lung-­
resident cell populations in idiopathic pulmonary fibrosis. Sci Adv. 2020;6(28):eaba1983.
66. Jonigk D, Stark H, Braubach P, Neubert L, Shin HO, Izykowski N, Welte T, Janciauskiene
S, Warnecke G, Haverich A, Kuehnel M. Morphological and molecular motifs of fibrosing
pulmonary injury patterns. J Pathol Clin Res. 2019;5(4):256–71.
67. Mullenbrock S, Liu F, Szak S, Hronowski X, Gao B, Juhasz P, Sun C, Liu M, McLaughlin
H, Xiao Q, Feghali-Bostwick C. Systems analysis of transcriptomic and proteomic profiles
identifies novel regulation of fibrotic programs by miRNAs in pulmonary fibrosis fibroblasts.
Genes. 2018;9(12):588.
68. Patel NM, Kawut SM, Jelic S, Arcasoy SM, Lederer DJ, Borczuk AC. Pulmonary arteriole
gene expression signature in idiopathic pulmonary fibrosis. Eur Respir J. 2013;41(6):1324–30.
69. Xu Y, Mizuno T, Sridharan A, Du Y, Guo M, Tang J, Wikenheiser-Brokamp KA, Perl AKT,
Funari VA, Gokey JJ, Stripp BR. Single-cell RNA sequencing identifies diverse roles of epi-
thelial cells in idiopathic pulmonary fibrosis. JCI Insight. 2016;1(20):e90558.
70. Yang IV, Coldren CD, Leach SM, Seibold MA, Murphy E, Lin J, Rosen R, Neidermyer AJ,
McKean DF, Groshong SD, Cool C. Expression of cilium-associated genes defines novel
molecular subtypes of idiopathic pulmonary fibrosis. Thorax. 2013;68(12):1114–21.
71. Paplińska-Goryca M, Goryca K, Misiukiewicz-Stępień P, Nejman-Gryz P, Proboszcz M,
Górska K, Maskey-Warzęchowska M, Krenke R. mRNA expression profile of bronchoalveo-
lar lavage fluid cells from patients with idiopathic pulmonary fibrosis and sarcoidosis. Eur J
Clin Investig. 2019;49(9):e13153.
72. Xia Y, Lei C, Yang D, Luo H. Construction and validation of a bronchoalveolar lavage cell-­
associated gene signature for prognosis prediction in idiopathic pulmonary fibrosis. Int
Immunopharmacol. 2021;92:107369.
73. Abe S, Sato S, Aono Y, Azuma M, Kishi M, Koyama K, Takahashi N, Kagawa K, Kawano
H, Nishioka Y. Functional analysis of human fibrocytes derived from monocytes reveals their
profibrotic phenotype through paracrine effects. J Med Investig. 2020;67(1.2):102–12.
324 S. Dasgupta et al.

74. Di Mauro S, Scamporrino A, Fruciano M, Filippello A, Fagone E, Gili E, Scionti F, Purrazzo

G, Di Pino A, Scicali R, Di Martino MT. Circulating coding and long non-coding RNAs as
potential biomarkers of idiopathic pulmonary fibrosis. Int J Mol Sci. 2020;21(22):8812.
75. Nakanishi T, Cerani A, Forgetta V, Zhou S, Allen RJ, Leavy OC, Koido M, Assayag
D, Jenkins RG, Wain LV, Yang IV. Genetically increased circulating FUT3 level leads to
reduced risk of idiopathic pulmonary fibrosis: a Mendelian randomisation study. Eur Respir
J. 2021;59:2003979.
76. Fraser E, Denney L, Antanaviciute A, Blirando K, Vuppusetty C, Zheng Y, Repapi E,
Iotchkova V, Taylor S, Ashley N, St Noble V. Multi-modal characterization of monocytes in
idiopathic pulmonary fibrosis reveals a primed type I interferon immune phenotype. Front
Immunol. 2021;12:226.
77. Sala MA, Balderas-Martínez YI, Buendía-Roldan I, Abdala-Valencia H, Nam K, Jain M,
Bhorade S, Bharat A, Reyfman PA, Ridge KM, Pardo A. Inflammatory pathways are upreg-
ulated in the nasal epithelium in patients with idiopathic pulmonary fibrosis. Respir Res.
2018;19(1):1–10.
78. Boon K, Bailey NW, Yang J, Steel MP, Groshong S, Kervitsky D, Brown KK, Schwarz MI,
Schwartz DA. Molecular phenotypes distinguish patients with relatively stable from progres-
sive idiopathic pulmonary fibrosis (IPF). PLoS One. 2009;4(4):e5134.
79. Furusawa H, Cardwell JH, Okamoto T, Walts AD, Konigsberg IR, Kurche JS, Bang TJ,
Schwarz MI, Brown KK, Kropski JA, Rojas M. Chronic hypersensitivity pneumonitis, an
interstitial lung disease with distinct molecular signatures. Am J Respir Crit Care Med.
2020;202(10):1430–44.
80. De Sadeleer LJ, McDonough JE, Schupp JC, Yan X, Vanstapel A, Van Herck A, Everaerts S,
Geudens V, Sacreas A, Goos T, Aelbrecht C. Lung microenvironments and disease progres-
sion in fibrotic hypersensitivity pneumonitis. Am J Respir Crit Care Med. 2021;205(1):60–74.
81. Christophi GP, Caza T, Curtiss C, Gumber D, Massa PT, Landas SK. Gene expression pro-
files in granuloma tissue reveal novel diagnostic markers in sarcoidosis. Exp Mol Pathol.
2014;96(3):393–9.
82. Casanova NG, Gonzalez-Garay ML, Sun B, Bime C, Sun X, Knox KS, Crouser ED,
Sammani N, Gonzales T, Natt B, Chaudhary S. Differential transcriptomics in sarcoidosis
lung and lymph node granulomas with comparisons to pathogen-specific granulomas. Respir
Res. 2020;21(1):1–12.
83. Tanaka H, Yamaguchi E, Asai N, Yokoi T, Nishimura M, Nakao H, Yoneda M, Ohtsuka
Y, Konno S, Yamada N. Cathepsin S, a new serum biomarker of sarcoidosis discovered
by transcriptome analysis of alveolar macrophages. Sarcoidosis Vasc Diffuse Lung Dis.
2019;36(2):141.
84. Vukmirovic M, Yan X, Gibson KF, Gulati M, Schupp JC, DeIuliis G, Adams TS, Hu B,
Mihaljinec A, Woolard TN, Lynn H. Transcriptomics of bronchoalveolar lavage cells identi-
fies new molecular endotypes of sarcoidosis. Eur Respir J. 2021;58(6):2002950.
85. Lepzien R, Nie M, Czarnewski P, Liu S, Yu M, Ravindran A, Kullberg S, Eklund A,
Grunewald J, Smed-Sörensen A. Pulmonary and blood dendritic cells from sarcoidosis
patients more potently induce IFNγ-producing Th1 cells compared with monocytes. J Leukoc
Biol. 2021;2021:1–10.
86. Lepzien R, Liu S, Czarnewski P, Nie M, Österberg B, Baharom F, Pourazar J, Rankin G,
Eklund A, Bottai M, Kullberg S. Monocytes in sarcoidosis are potent TNF producers and
predict disease outcome. Eur Respir J. 2021;58(1):2003468.
87. Bloom CI, Graham CM, Berry MP, Rozakeas F, Redford PS, Wang Y, Xu Z, Wilkinson KA,
Wilkinson RJ, Kendrick Y, Devouassoux G. Transcriptional blood signatures distinguish
pulmonary tuberculosis, pulmonary sarcoidosis, pneumonias and lung cancers. PLoS One.
2013;8(8):e70630.
88. Kachamakova-Trojanowska N, Jazwa-Kusior A, Szade K, Kasper L, Soja J, Andrychiewicz
A, Jakiela B, Plutecka H, Sanak M, Jozkowicz A, Sladek K. Molecular profiling of regulatory
T cells in pulmonary sarcoidosis. J Autoimmun. 2018;94:56–69.
Metabolomics and Transcriptomic Approach to Understand the Pathophysiology… 325

89. Garman L, Pelikan RC, Rasmussen A, Lareau CA, Savoy KA, Deshmukh US, Bagavant H,
Levin AM, Daouk S, Drake WP, Montgomery CG. Single cell transcriptomics implicate novel
monocyte and T cell immune dysregulation in sarcoidosis. Front Immunol. 2020;11:567342.
90. Locke LW, Crouser ED, White P, Julian MW, Caceres EG, Papp AC, Le VT, Sadee W,
Schlesinger LS. IL-13–regulated macrophage polarization during granuloma formation in an
in vitro human sarcoidosis model. Am J Respir Cell Mol Biol. 2019;60(1):84–95.
91. Jung SM, Park KS, Kim KJ. Integrative analysis of lung molecular signatures reveals
key drivers of systemic sclerosis-associated interstitial lung disease. Ann Rheum Dis.
2021;81(1):108–16.
92. Valenzi E, Bulik M, Tabib T, Morse C, Sembrat J, Bittar HT, Rojas M, Lafyatis R. Single-cell
analysis reveals fibroblast heterogeneity and myofibroblasts in systemic sclerosis-associated
interstitial lung disease. Ann Rheum Dis. 2019;78(10):1379–87.
93. Valenzi E, Tabib T, Papazoglou A, Sembrat J, Trejo Bittar HE, Rojas M, Lafyatis R. Disparate
interferon signaling and shared aberrant basaloid cells in single-cell profiling of idiopathic
pulmonary fibrosis and systemic sclerosis-associated interstitial lung disease. Front Immunol.
2021;12:960.
94. Lindahl GE, Stock CJ, Shi-Wen X, Leoni P, Sestini P, Howat SL, Bou-Gharios G, Nicholson
AG, Denton CP, Grutters JC, Maher TM. Microarray profiling reveals suppressed interferon
stimulated gene program in fibroblasts from scleroderma-associated interstitial lung disease.
Respir Res. 2013;14(1):1–14.
95. Renaud L, da Silveira WA, Takamura N, Hardiman G, Feghali-Bostwick C. Prominence
of IL6, IGF, TLR, and bioenergetics pathway perturbation in lung tissues of scleroderma
patients with pulmonary fibrosis. Front Immunol. 2020;11:383.
96. Assassi S, Wu M, Tan FK, Chang J, Graham TA, Furst DE, Khanna D, Charles J, Ferguson
EC, Feghali-Bostwick C, Mayes MD. Skin gene expression correlates of severity of intersti-
tial lung disease in systemic sclerosis. Arthritis Rheum. 2013;65(11):2917–27.
97. Hutter C, Kauer M, Simonitsch-Klupp I, Jug G, Schwentner R, Leitner J, Bock P, Steinberger
P, Bauer W, Carlesso N, Minkov M. Notch is active in Langerhans cell histiocytosis and con-
fers pathognomonic features on dendritic cells. Blood. 2012;120(26):5199–208.
98. Pang J, Luo Y, Wei D, Cao Z, Qi X, Song M, Liu Y, Li Z, Zhang J, Li B, Chen J. Comparative
transcriptome analyses reveal a transcriptional landscape of human silicosis lungs and pro-
vide potential strategies for silicosis treatment. Front Genet. 2021;12:652901.
99. Yao W, Yang P, Qi Y, Jin L, Zhao A, Ding M, Wang D, Li Y, Hao C. Transcriptome analysis
reveals a protective role of liver X receptor alpha against silica particle-induced experimental
silicosis. Sci Total Environ. 2020;747:141531.
100. Chen J, Zhang R, Xie M, Luan C, Li X. Transcriptome sequencing identifies PLAUR as an
important player in patients with dermatomycitis-associated interstitial lung disease. Front
Genet. 2021;12:784215.
101. Johnson CH, Ivanisevic J, Siuzdak G. Metabolomics: beyond biomarkers and towards mech-
anisms. Nat Rev Mol Cell Biol. 2016;17(7):451–9.
102. Chambers DC, Carew AM, Lukowski SW, Powell JE. Transcriptomics and single-cell RNA-­
sequencing. Respirology. 2019;24(1):29–36.
103. Junker BH, Klukas C, Schreiber F. VANTED: a system for advanced data analysis and visu-
alization in the context of biological networks. BMC Bioinform. 2006;7(1):1–13.
104. Kamburov A, Cavill R, Ebbels TM, Herwig R, Keun HC. Integrated pathway-level analysis of
transcriptomics and metabolomics data with IMPaLA. Bioinformatics. 2011;27(20):2917–8.
105. Hu Z, Hung JH, Wang Y, Chang YC, Huang CL, Huyck M, DeLisi C. VisANT 3.5: multi-­
scale network visualization, analysis and inference based on the gene ontology. Nucleic
Acids Res. 2009;37(Web Server issue):W115–21.
106. Karnovsky A, Weymouth T, Hull T, Tarcea VG, Scardoni G, Laudanna C, Sartor MA, Stringer
KA, Jagadish HV, Burant C, Athey B. Metscape 2 bioinformatics tool for the analysis and
visualization of metabolomics and gene expression data. Bioinformatics. 2012;28(3):373–80.
326 S. Dasgupta et al.

107. Pavlopoulos GA, O’Donoghue SI, Satagopam VP, Soldatos TG, Pafilis E, Schneider
R. Arena3D: visualization of biological networks in 3D. BMC Syst Biol. 2008;2(1):1–7.
108. Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B,
Ideker T. Cytoscape: a software environment for integrated models of biomolecular interac-
tion networks. Genome Res. 2003;13(11):2498–504.
109. McGuffin MJ, Jurisica I. Interaction techniques for selecting and manipulating subgraphs in
network visualizations. IEEE Trans Vis Comput Graph. 2009;15(6):937–44.
110. Barsky A, Munzner T, Gardy J, Kincaid R. Cerebral: visualizing multiple experimental condi-
tions on a graph with biological context. IEEE Trans Vis Comput Graph. 2008;14(6):1253–60.
111. Arakawa K, Kono N, Yamada Y, Mori H, Tomita M. KEGG-based pathway visualization tool
for complex omics data. In Silico Biol. 2005;5(4):419–23.
112. García-Alcalde F, García-López F, Dopazo J, Conesa A. Paintomics: a web based tool
for the joint visualization of transcriptomics and metabolomics data. Bioinformatics.
2011;27(1):137–9.
113. Neuweger H, Persicke M, Albaum SP, Bekel T, Dondrup M, Hüser AT, Winnebald J,
Schneider J, Kalinowski J, Goesmann A. Visualizing post genomics datasets on custom-
ized pathway maps by ProMeTra–aeration-dependent gene expression and metabolism of
Corynebacterium glutamicum as an example. BMC Syst Biol. 2009;3(1):1–14.
114. Tokimatsu T, Sakurai N, Suzuki H, Ohta H, Nishitani K, Koyama T, Umezawa T, Misawa
N, Saito K, Shibata D. KaPPA-view. A web-based analysis tool for integration of transcript
and metabolite data on plant metabolic pathway maps. Plant Physiol. 2005;138(3):1289–300.
115. Thimm O, Bläsing O, Gibon Y, Nagel A, Meyer S, Krüger P, Selbig J, Müller LA, Rhee SY,
Stitt M. MAPMAN: a user-driven tool to display genomics data sets onto diagrams of meta-
bolic pathways and other biological processes. Plant J. 2004;37(6):914–39.
116. Symonsy S, Zipplies C, Battke F, Nieselt K. Integrative systems biology visualization with
MAYDAY. J Integr Bioinform. 2010;7(3):1–14.
117. Lüdemann A, Weicht D, Selbig J, Kopka J. PaVESy: pathway visualization and editing sys-
tem. Bioinformatics. 2004;20(16):2841–4.
118. Xie N, Tan Z, Banerjee S, Cui H, Ge J, Liu RM, Bernard K, Thannickal VJ, Liu G. Glycolytic
reprogramming in myofibroblast differentiation and lung fibrosis. Am J Respir Crit Care
Med. 2015;192(12):1462–74.
119. Hsu HS, Liu CC, Lin JH, Hsu TW, Hsu JW, Su K, Hung SC. Involvement of ER stress, PI3K/
AKT activation, and lung fibroblast proliferation in bleomycin-induced pulmonary fibrosis.
Sci Rep. 2017;7(1):1–11.
120. Zhao H, Li C, Li L, Liu J, Gao Y, Mu K, Chen D, Lu A, Ren Y, Li Z. Baicalin alleviates
bleomycin induced pulmonary fibrosis and fibroblast proliferation in rats via the PI3K/AKT
signaling pathway. Mol Med Rep. 2020;21(6):2321–34.
Transferring Metabolomics to Portable
Diagnostic Devices: Trending in Biosensors

Shimaa Eissa

Abstract Metabolic biomarkers are very popular in clinical laboratories, where

more than 95% of the clinical assays are based on small molecules. Biosensors can
offer a faster, simpler, and cheaper alternative to conventional analytical assays as
they can make the metabolic biomarkers more accessible in a high-throughput fash-
ion for point-of-care testing. As research continues in different aspects of the bio-
sensor design, more sensitive and selective biosensors are being developed for
various metabolic biomarkers for diagnostic applications. In this chapter, we pro-
vide a brief overview of the current biosensor designs and formats for applications
in metabolomics. Various biorecognition receptors and transducers used in the
development of biosensors for metabolic biomarkers are discussed. Major advances
in the biosensors for metabolites such as the use of aptamers as new recognition
receptors as well as the utilization of nanomaterials as transducers are highlighted.
The developments of multiplexed array biosensors for the simultaneous detection of
multiple biomarkers and wearable biosensors are discussed as emerging diagnostic
tools in metabolomics. The challenges and future perspectives in the use of biosen-
sors in metabolomics are discussed.

Keywords Biosensing platforms · Metabolic biomarkers · Point-of-care

biosensors · Metabolomics · Electrochemical biosensors · Optical biosensors ·
Multiplexed array biosensors · Detection

S. Eissa (*)
Department of Chemistry, Khalifa University of Science and Technology,
Abu Dhabi, United Arab Emirates
Advanced Materials Chemistry Center (AMCC), Khalifa University of Science and
Technology, Abu Dhabi, United Arab Emirates
e-mail: [Link]@[Link]

© The Author(s), under exclusive license to Springer Nature Singapore Pte 327
Ltd. 2023
A. M. Abdel Rahman (ed.), Clinical Metabolomics Applications in Genetic
Diseases, [Link]
328 S. Eissa

Abbreviations

Aptasensors Aptamer-based biosensors

HQ Hydroquinone
MS Mass spectrometry
NMR High-resolution nuclear magnetic resonance spectroscopy
POCT Point-of-care testing
SELEX Systematic evolution of ligand by exponential enrichment
TMB/H2O2 Tetramethylbenzidine/hydrogen peroxide

1 Introduction

The development of fast, accurate, and reliable diagnostic methods is crucial to

improving the clinical course of any disease. Metabolomics offers new opportuni-
ties to discover biomarkers associated with complex diseases [1]. Detecting these
low molecular weight metabolic biomarkers in biological samples can provide use-
ful information about the pathology and the progress of the disease [2]. Moreover,
it can provide insight into the response of the patients to specific treatment and helps
to understand the mechanism of the disease. Therefore, metabolomics has become
a powerful tool in clinical research for discovering biomarkers in disease diagnosis
[1]. With the recent advancements in the bionanotechnology field, various promis-
ing metabolomic technologies are being developed to identify new therapeutic tar-
gets and improve the disease prognosis and diagnosis [3]. Advances in bioinformatics,
mass spectrometry (MS), high-resolution nuclear magnetic resonance spectroscopy
(NMR), and ultra-performance liquid chromatography methods have led to a drastic
improvement in the reliability and efficiency of metabolic profiling [4]. Moreover,
the coupling of mass spectrometry with chromatography has enabled more compre-
hensive coverage of metabolic biomarkers [4].
Biosensors are analytical devices that gained significant interest over the last
decades as it offers a lower cost, simpler, faster, and potentially more versatile alter-
native to traditional analytical techniques. Biosensing devices are of high impor-
tance in metabolomic applications due to their potential to provide simple and
noninvasive measurements of various biomarkers in biofluids such as saliva, blood,
sweat, tears, and urine. There are various types of biosensors which can be used for
different applications. A typical biosensor contains three main units: a biorecogni-
tion receptor such as antibody, enzyme, or aptamer which is responsible for the
specific binding with the target analyte; a transducer such as optical, electrochemi-
cal, or mass-based detection techniques which can transform the binding event
between the recognition receptor and the analyte into a measurable signal; and a
signal detector which reports and displays the biosensor signal. The potential utility
of biosensors in metabolomics is evident from the rapidly increasing reported bio-
sensing platforms for various metabolic biomarkers for point-of-care testing. In the
next section, the different recognition receptors and transducers used in the biosen-
sors for metabolites are discussed. Moreover, the integration of microfluidics with
Transferring Metabolomics to Portable Diagnostic Devices: Trending in Biosensors 329

array multiplexed biosensors as well as flexible materials for wearable biosensors as

promising improvements in the biosensors for metabolites is highlighted.

2 Biorecognition Receptors Used in the Biosensors

for Metabolic Biomarkers

The selection of the proper bioreceptor is a major element in the biosensor design.
The choice of the biorecognition receptor used to fabricate the biosensor depends
mainly on the type of target analyte under investigation. Antibodies are mainly used
for the detection of proteins, whereas ssDNA can be used for the detection of
genomic sequences by hybridization. However, some antibodies have been also pro-
duced against some metabolites. Enzymes are widely used as bioreceptors in bio-
sensors for some small molecules where the enzyme is used to catalyze specific
biochemical reaction. However, since low molecular weight metabolites can have
various structural and chemical properties, the choice of specific recognition recep-
tors for each individual metabolite can be challenging. Various suitable bioreceptors
are used for different types of metabolites. Specific enzymes for some metabolites
like glucose, lactate, and uric acid have been used for the development of biosen-
sors. Antibodies for other metabolites have been also developed and used for the
fabrication of immunosensors such as hormones. Aptamers have appeared in the
recent years as potential alternative to antibodies for the biosensor’s development,
particularly for small molecules. Therefore, aptamers hold considerable promise in
the metabolomic field because of their low cost, easier in vitro synthesis, and high
stability. In the next subsections, we will discuss various bioreceptors, which have
been used for the development of biosensing platforms for the detection of different
metabolites.

2.1 Enzyme-Based Biosensors

Enzymes are selective bioreceptors that are mainly used to develop catalytic biosen-
sors for certain target analytes. The enzyme catalyzes a reaction leading to the for-
mation or disappearance of an electroactive product which can be detected using an
electrochemical technique such as amperometry [5, 6]. Enzyme-based biosensors
are easy to construct and can provide sensitive and rapid analysis. However, there
are no available specific enzymes for many target molecules. Moreover, the enzyme
stability is limited as it gradually loses activity over time, and thus, the shelf lifetime
of the enzyme-based biosensors is short.
Monitoring blood glucose, one of the most common metabolic biomarkers, was
the main driving force for the research work to develop enzymatic biosensors for
medical applications [5, 7, 8]. Blood glucose has been established as a biomarker
for diagnosis of diabetes. The development of biosensors to detect glucose level has
started since almost 60 years [8]. Significant effort has been made to improve the
330 S. Eissa

glucose biosensor technology in terms of sensitivity, reliability, stability, and porta-

bility. The historical development of glucose biosensors has been described in detail
in several reviews [5, 7–9]. In summary, glucose biosensors are divided into three
generations. The first-generation enzyme biosensors were oxygen-based, whereas
the second-generation are mediator-based. However, the third-generation glucose
biosensors are the directly coupled enzyme electrodes. The continuous emergences
of new nanostructures and nanocomposites have led to the developments of various
glucose biosensors with improved electron transfer efficiency and electrocatalytic
activity [10].
Similarly, other enzyme-based biosensors for the detection of different metabo-
lites have been later developed such as lactate [11, 12], xanthine [13, 14], caffeine
[13], glycolic acid [15], bile acids [16, 17], L-arginine [18], choline [19], and bio-
sensors. Lactate is produced when glucose is broken down and is commonly used as
avital biomarker in medical monitoring [20]. The concentration of lactate in blood
often rises during exercises such as running; however, it can be also altered due to
hemorrhage, trauma, and ischemia. Lactate is also used as a biomarker for condi-
tions such as bacterial meningitis or acidosis [21]. Various enzymes have been used
as biorecognition receptors in lactate biosensors such as lactate monooxidase, lac-
tate oxidase, lactate dehydrogenase, and cytochrome b [6]. Mediators such as
NAD+/NADH and ferricyanide are sometimes used in these enzymatic electro-
chemical biosensors leading to the production of a current after applying certain
potential that is measured amperometrically [6]. The uric acid is a metabolite used
as indicator of gout which is produced by the breaking down of purine nucleotides
with xanthine oxidase enzyme. The detection of xanthine is also of increasing medi-
cal interest. Thus, several amperometric biosensors using xanthine oxidase have
been developed for the detection of xanthine [14].
The detection of choline is important in clinical practice, especially in the early
diagnosis of some brain disorders such as Parkinson’s and Alzheimer’s diseases
[22]. Many enzyme-based amperometric biosensors for choline have been devel-
oped. In these biosensors, choline oxidase was used, and the detection was based on
amperometric monitoring of hydrogen peroxide produced when choline oxidase
catalyzes the reaction. Hydrogen peroxide reacts either directly with the redox
mediator [23–26], or its reaction is catalyzed by a second enzyme such as horserad-
ish peroxidase to enhance the current signal [27, 28]. Different nanomaterials have
been integrated in these enzyme-based biosensors for choline such as carbon nano-
tubes [19, 26, 29], gold nanoparticles, and graphene [27].

2.2 Antibody-Based Biosensors

The production of antibodies against low molecular weight compounds is usually

challenging. Because of their small size, most metabolites are not usually immuno-
genic and need to be conjugated with larger carrier protein before using in immuniz-
ing the animals. Moreover, most of the antibodies for small molecules suffer from
low specificity issues. However, some successful examples of antibodies produced
Transferring Metabolomics to Portable Diagnostic Devices: Trending in Biosensors 331

for metabolites have been reported. Antibodies against various hormones such as
progesterone, testosterone, cortisol, 11-deoxycortisol, and diethylstilbestrol have
been produced. Table 1 shows the reported biosensors for metabolic biomarker
which used antibody as recognition receptor. Serafín et al. [30] have reported the
development of a biosensor based on an anti-progesterone antibody for the detec-
tion of progesterone in saliva (Fig. 1). A competitive amperometric biosensor was
fabricated on a low-cost disposable electrode which allowed a fast (45 min) and
sensitive detection of progesterone with a LOD of 5 pg/mL. Good selectivity against
other hormones such as testosterone, corticosterone, cortisol, and
17-β-ethynylestradiol was shown with comparable results to commercial
ELISA. Other studies have shown the use of anti-progesterone antibody for the
development of electrochemical immunosensors utilizing a thionine/graphene oxide
composite achieving LOD of 6.3 pg/mL [31] and gold nanoparticles yielding LODs
of 430 pg/mL [32] and 80 pg/mL [33].
Several biosensors have utilized antibody against cortisol for the development of
biosensors to measure the concentration of cortisol in buffer [34–37], artificial saliva
[38], real human saliva [39, 40], and interstitial fluid [39, 41]. Different approaches were
used in these immunosensors to minimize the matrix effect especially when cortisol was
detected in human saliva such as dilution [39] or using fluid control system [42].
Kämäräinen et al. [40] have utilized anti-cortisol antibody to develop a competi-
tive electrochemical disposable immunosensor for the detection of cortisol in human
saliva using cortisol-alkaline phosphatase conjugate and screen-printed electrodes
showing good sensitivity, reproducibility, and repeatability. Moreover, the results of
the cortisol immunosensor were comparable with ultra-high pressure liquid
chromatography-­tandem mass spectrometry.
Many studies have shown the integration of antibody against estradiol in several
immunosensors for the detection of estradiol [43, 44]. The reported immunosensors
have mainly utilized a competitive assay where a protein-estradiol conjugate was
employed to compete with the free estradiol molecules on the sample for the anti-
body immobilized on the sensor surface. Enzymes such as alkaline phosphatase [43,
44] and horse radish peroxidase [45, 46] or bovine serum albumin [47] were conju-
gates with estradiol; in some of these studies, a competitive immunosensor was
developed. A non-labeled competitive immunosensor for estradiol has been also
reported using hydroquinone as redox marker and differential pulse voltammetry
for the detection [48]. Ojeda et al. [46] have described the integration of an antibody
in a competitive electrochemical immunosensor for estradiol hormone. The immu-
nosensor was fabricated on a screen-printed electrode modified with streptavidin on
which a biotinylated anti-estradiol was immobilized. A competitive assay was per-
formed using horse radish peroxidase-labeled estradiol, and the detection was
achieved amperometrically employing hydroquinone as redox mediator. The immu-
nosensor exhibited good sensitivity with a LOD of 0.77 pg/mL as well as good
selectivity against other hormones. Moreover, this estradiol immunosensor showed
good applicability in spiked serum and urine samples.
Antibody for testosterone was produced and utilized in the fabrication of some
immunosensors. Eguílaz et al. [49] have reported the development of an amperom-
etry immunosensor based on disposable screen-printed carbon electrodes and
332 S. Eissa

Table 1 Immunosensors for different metabolites

Target Limit of
metabolite Material Transducer detection Reference
Progesterone Magnetic microbeads Amperometry 5 pg/mL [30]
Progesterone Thionine-graphene Amperometry 6.3 pg/mL [31]
Oxide composites
Progesterone Colloidal Amperometry 430 pg/mL [32]
gold-graphite-Teflon
Progesterone Gold nanoparticles Amperometry 80 pg/mL [33]
Cortisol Dithiobis (succinimidyl Electrochemical 1 pM [34]
propionate)-modified gold impedance spectroscopy
microarray
Cortisol Polyaniline-protected gold Cyclic voltammetry 1 pM [37]
nanoparticles
Cortisol Low temperature co-fired Cyclic voltammetry 10 pM [36]
ceramic (LTCC)-based
microfluidic system
Cortisol Single-walled, carbon Chemiresistor 1 pg/mL [38]
nanotube
Cortisol Graphite Square wave 1.7 ng/mL [40]
voltammetry
Estradiol Carbon Amperometry 50 pg/mL [44]
Estradiol Gold nanoparticles Amperometry 6 pg/mL [45]
Estradiol Carbon Amperometry 0.77 pg/mL [46]
Estradiol Gold nanoparticle Square wave 18 pg/mL and [47]
thiolated protein voltammetry and 26 pg/mL
G-scaffold electrochemical
impedance spectroscopy
Testosterone Magnetic beads Amperometry 1.7 pg/mL [49]
Testosterone Carbon Amperometry 26 pg/mL and [50]
1.8 pg/mL in
buffer and
urine
Vitamin D3 Cellulose acetate fibers Amperometry 10 ng/mL [54]
Vitamin D3 Magnetite nanoparticles Differential pulse 0.12 ng/mL [55]
incorporated into voltammetry
electrospun
polyacrylonitrile
nanofibers
Vitamin D3 Gold-platinum bimetallic Differential pulse 4.9 pg/mL [56]
nanoparticle-coated voltammetry
3 -(aminopropyl)
triethoxysilane
Vitamin D3 Gold nanoparticles Surface plasmon 1 μg/mL [57]
Resonance
Vitamin D3 Nanostructured cerium Differential pulse 4.63 ng/mL [58]
(IV) oxide (nCeO2) voltammetry
Transferring Metabolomics to Portable Diagnostic Devices: Trending in Biosensors 333

Fig. 1 (a) Scheme of the design of progesterone amperometric immunosensor. (Reprinted with
permission from Ref. [30]). (b) Schematic illustration of the selection process of sepiapterin
aptamer and the working principle of the competitive biosensor using square wave voltammetry
detection. (Reprinted with permission from Ref. [87])

protein A-functionalized magnetic beads. The antibody was immobilized onto the
magnetic beads, and competitive assay was performed using HRP-testosterone con-
jugate. The amperometric detection was achieved after the addition of H2O2 using
hydroquinone (HQ) as redox mediator showing a LOD of 1.7 pg/mL. The immuno-
sensor showed good selectivity against other steroid hormones and was successfully
applied for the detection of testosterone in spiked human serum samples. A recom-
binant Fab fragment was also used for the fabrication on another electrochemical
immunosensor using screen printed electrode and was applied for the detection of
testosterone in bovine urine [50]. A competitive assay was utilized using
334 S. Eissa

HRP-labeled testosterone and tetramethylbenzidine/hydrogen peroxide (TMB/

H2O2) substrate for the signal development. The detection was realized using chro-
noamperometry at +100 mV.
Vitamin D3 is one of the metabolic biomarkers that has significant physiological
functions. Vitamin D3 deficiency is considered a global health issue because of its
serious health impact and correlation with various diseases [51] such as cardiovas-
cular diseases, bone disorder, diabetes, infections, tuberculosis, osteoarthritis,
hypertension, cancer, and even COVID-19 [52, 53]. Thus, antibodies for vitamin D3
have been produced and employed for the fabrication of various immunosensors to
monitor vitamin D3 level [54–58]. Chauhan et al. [54] have described the fabrication
of a low-cost and eco-friendly immunosensor for the detection of 25-hydroxy vita-
min-­D3 using disposable conducting paper substrate decorated with electrospun cel-
lulose acetate fibers. The detection was realized using a chronoamperometric
technique showing a LOD of 10.0 ng/mL. Moreover, the immunosensor was applied
for the detection of vitamin D3 in serum samples exhibiting good agreement with
the results obtained from ELISA. The same research group has also reported that
the development of another electrochemical immunosensor for vitamin D3 detec-
tion using magnetite nanoparticles incorporated electrospun polyacrylonitrile nano-
fibers. The immunosensor has shown a LOD of 0.12 ng/mL [55]. Kaur et al. [56]
have also developed an electrochemical immunosensor for the detection of vitamin
D3 using gold-platinum bimetallic nanoparticle-coated 3-(aminopropyl)triethoxysi-
lane on fluorine tin oxide glass electrode. The vitamin D3 antibody was attached to
the electrode covalently via glutaraldehyde as a cross linker. The immunosensor
showed good sensitivity with a LOD of 0.49 pg/mL. Carlucci et al. [57] have
reported the detection of vitamin D3 using both electrochemical and surface plas-
mon resonance immunosensors showing LODs of 10 ng/mL and 45 ng/mL, respec-
tively. Recently, Chauhan et al. [58] have described the fabrication of carbon
cloth-based immunosensor for detection of 25-hydroxy vitamin D3 using a label-­
free format. The immunosensor was prepared by depositing nanostructured cerium
(IV) oxide on carbon cloth followed by the immobilization of anti-vitamin D3 anti-
bodies. The immunosensor exhibited good sensitivity with a LOD of 4.63 ng/mL
and a fast response time of 15 min. The immunosensor was successfully applied in
real serum samples and demonstrated good agreement with the conventional
ELISA [58].

2.3 Aptamer-Based Biosensors

Aptamers are single-stranded DNA or RNA which were firstly reported in 1990 [59]
and widely considered as promising alternative to the gold standard antibodies in
biosensing applications [60–62]. Many aptamers have been identified and tested
against various analytes in the last two decades. However, only few aptamers have
reached the commercialization stage for therapeutic and diagnostic applications.
Aptamers have been widely identified and successfully applied for several
Transferring Metabolomics to Portable Diagnostic Devices: Trending in Biosensors 335

diagnostic applications showing great promise for point-of-care testing (POCT)

[63–67]. Aptamers offers several advantages over antibodies such as their high sta-
bility, ease of in vitro production, and simplicity of synthesis at very low cost. These
remarkable advantages, along with the ease of their immobilization and regenera-
tion as well as their small size, have made them excellent candidates for the devel-
opment of aptamer-based biosensors (aptasensors). Since metabolites are small
molecule health markers, their detection by conventional antibody-based methods
can be challenging. Thus, the identification of aptamers against metabolites for
health-related and diagnostic applications has recently received considerable atten-
tion. Aptamers are usually identified through a process called systematic evolution
of ligand by exponential enrichment (SELEX) [68]. The selection is realized by
exposing nucleic acid library which consists of random sequences with the target
analyte. Then, a partitioning step is performed to separate the bound from the
unbound sequences followed by amplification of the bound sequences using a poly-
merase chain reaction. The amplified DNA is then purified and used to start a new
SELEX cycle. The selection cycles are repeated from 10 to 20 times until the DNA
pool is enriched with the highest affinity binders to the target.
Several aptamers have been selected against hormones such as estradiol [69, 70],
progesterone [71, 72], cortisol [73, 74], thyroxine [75], 11-deoxycortisol [76], tes-
tosterone [77], and vasopressin [78, 79]. These aptamers have been exploited for the
development of several biosensors for point-of-care diagnostic applications. The
increase of progesterone levels can lead to several health issues. Therefore, the
detection of progesterone in clinical samples is very important to protect the public
health. Jiménez et al. [71] have described the selection, identification, and charac-
terization of high binding affinity DNA aptamers against progesterone using in vitro
selection. Electrochemical impedance spectroscopy and fluorometric assays were
utilized to determine the dissociation constants. The highest affinity aptamer has
shown dissociation constant of 17 nM without any significant cross-reactivity to
similar analogues such as 11-norethisterone and 17β-estradiol. The aptamer was
then immobilized on gold electrode, and a complementary short sequence was
hybridized to the aptamer at different sites to optimize the signal gain of the aptas-
ensor. The detection relied on the conformational change of the aptamer upon bind-
ing with the analyte as confirmed by circular dichroism spectroscopy. The aptasensor
has shown excellent sensitivity with a LOD of 0.90 ng/mL. This aptamer has been
optimized in another study [72] and used to fabricate a fluorescence-based biosen-
sor for the detection of progesterone. The aptamer was truncated in this study to
eliminate the nonbinding region which had negative impact on the binding affinity
of the aptamer to progesterone. Fluorescence mapping was performed to enhance
the affinity and the specificity of the aptamer with 16-fold increase in the dissocia-
tion constant compared to the original aptamer. The truncated aptamer was then
used in a displacement fluorescence assay where it was hybridized at different sites
to fluorescein- and quencher-labelled complementary DNA sequences to form
duplex structures. The detection of progesterone was realized via displacement of
the complementary sequence which causes enhancement of the fluoresce signal.
336 S. Eissa

Despite of the numerous advantages of aptamers as bioreceptors, the selection of

aptamers against various metabolites is still limited mainly due to the tediousness
and high cost of the traditional SELEX process. Eissa et al. [76] have reported a new
method for the selection of aptamers against 11-deoxycortisol hormone. The selec-
tion was based on an electrochemical method which enabled a cost-effective, effi-
cient, and rapid enrichment process. In this method, the hormone molecules were
immobilized on a gold electrode which was then used as the solid support for the
SELEX method. The enrichment of the DNA during the selection process was mon-
itored using square wave voltammetry in a label-free format unlike the conventional
SELEX protocol for small molecules which often requires the use of fluorescently
labeled DNA. High-affinity aptamers against 11-deoxycortisol hormone were suc-
cessfully selected after eight cycles showing dissociation constants at the subnano-
molar level. The selected aptamer was utilized to fabricate an electrochemical
biosensor for the detection of 11-deoxycortisol showing very high sensitivity. This
aptasensor has shown good selectivity and successful supplication in spiked serum
samples.
Akki et al. [70] have reported the selection of DNA aptamers against the
endocrine-­disrupting compounds: 17β-estradiol and 17α-ethynylestradiol using
SELEX. The selected aptamers have demonstrated good affinity with dissociation
constants of 0.6 and 0.5 μM for 17β-estradiol and 17α-ethynylestradiol, respec-
tively. The authors found that the selected aptamer against 17β-estradiol has shown
good specificity against 17α-ethynylestradiol. Similarly, one of the selected aptam-
ers against 17α-ethynylestradiol showed good specificity against 17β-estradiol and
the similar analogue, estrone. It is very important to study the selectivity when
selecting aptamers against small molecules and to evaluate to which extent the
aptamer binds to other structurally similar compounds.
The stress hormone, cortisol, is one of the most important metabolites synthe-
sized by the stimulation of adrenal cortex upon stress. Cortisol has immunosuppres-
sive and anti-inflammatory effects as well as a strong impact on blood pressure,
heart rate, and reproductive and digestive activities. A selection method for an
aptamer against the stress biomarker cortisol based on tunable stringency magnetic
bead was described by Martin et al. [74]. After 15 rounds of selection, the enriched
pool showed a single sequence with high copy number. A next-generation sequenc-
ing analysis indicated a correlation between the number of aptamer copies and
enhanced affinity to the target under certain conditions. Two methods were used for
the estimation. The highest-affinity aptamer has shown dissociation constant of 6.9
and 16.1 μM by equilibrium and microscale thermophoresis methods, respectively.
A gold nanoparticle assay incorporating the selected aptamer was performed show-
ing good discrimination between cortisol and other structurally related biomarkers:
epinephrine, norepinephrine, and cholic acid.
In another study, an in silico approach of molecular docking was used to investi-
gate the interactions between aptamers and cortisol [73]. The tertiary conforma-
tional structures of ten aptamers were studied against cortisol and other related
hormones. It was found that the hydrophobic interactions of cortisol with the
Transferring Metabolomics to Portable Diagnostic Devices: Trending in Biosensors 337

aptamer have the major impact on the binding compared to the hydrogen bonding
according to the docking results.
RNA has the capability to bind to a wide range of small molecules which can be
exploited in many applications in molecular biology. Riboswitches are examples of
RNA domains which can serve as bioreceptors that bind to specific metabolite.
Despite that most of the aptamers selected against metabolic biomarkers are DNA
based, some RNA aptamers have been also reported. LEVESQUE et al. [75] have
reported the identification of thyroxine-specific aptamers using SELEX showing
good affinity and selectivity for thyroxine over its inactive derivative, thyronine.
Transcripts with site-specific modified nucleotides, mutational studies, circular
dichroism, and binding shift assays were used to investigate the binding of the
aptamer to thyroxine. This study suggested that the iodine moiety in the thyroxine
molecule is the main binding site to the aptamer.
The selection of an aptamer against the main sex hormone, testosterone, has been
reported [77]. Testosterone is responsible for the regulation of various physiological
processes in males such as growth of skeletal muscles and bones and other male sex
characteristics. Thus, the accurate detection of testosterone levels in biological flu-
ids is highly important. The selection of testosterone aptamer has been conducted
using classical SELEX via immobilizing the target on magnetic beads and perform-
ing counterselections against other steroids with similar chemical structures [77].
Ten aptamer sequences were identified using next-generation sequencing showing
dissociation constants in the nanomolar range. The conformational change of the
aptamers upon binding with testosterone was studied using circular dichroism.
An aptamer has been selected against the nine-amino acid peptide hormone,
vasopressin [78–80]. This hormone is considered a biomarker in patients with hem-
orrhagic shocks as it plays an important role in enhancing peripheral vascular resis-
tance which leads to an increase in arterial blood pressure. The applicability of
aptamers in biological fluids especially blood is often limited by their instability due
to the presence of nucleases. However, the selection of single-stranded DNA stable
and nuclease-resistant aptamer against vasopressin has been reported [78–80]. The
aptamer has been also used to develop biosensors for the detection of vasopressin
using different designs and transducers [78, 81]. Williams et al. [79] have reported
the selection of an aptamer for the enantiomer of vasopressin to identify a mirror-­
image DNA aptamer (enantiomer) that binds with vasopressin with high stability to
nucleases. The enantiomer of the aptamer was synthesized and showed high stabil-
ity and good bioactivity as vasopressin antagonist in cell culture.
Graphene oxide-based SELEX has been used to select aptamer against
25-hydroxy vitamin D3 [82]. This immobilization-free method has led to the identi-
fication of high-affinity and specificity aptamers. The affinity of the aptamers was
investigated using both isothermal titration and gold nanoparticle-based colorimet-
ric assay. From the selected aptamer pool, 9 sequences showed good affinity out of
16 aptamer candidates. The aptamer which showed the highest binding affinity to
25-hydroxy vitamin D3 with a dissociation constant of 11 nM has been utilized to
develop gold nanoparticle-based colorimetric biosensor showing a LOD of
1 μM. The conformation change of the aptamer upon binding with the target was
338 S. Eissa

also confirmed using circular dichroism analysis as well as by utilizing a displace-

ment assay with both magnetic beads and streptavidin-coated 96-well plates.
Many metabolic biomarkers are used for the diagnosis of genetic diseases.
Among them, sepiapterin level has been used as indicator of a rare inborn genetic
error of neurotransmitter metabolism called sepiapterin reductase deficiency [83–
85]. This genetic disease is characterized by cognitive and motor abnormalities. The
early diagnosis of such neurotransmitter diseases is crucial to enhance the treatment
and avoid the progression of the disease [86]. The detection of sepiapterin is chal-
lenging because there is no available specific antibody for sepiapterin in market
until now. We have reported recently the selection and identification of DNA aptam-
ers which binds specifically to sepiapterin using SELEX [87] (Fig. 1). Few aptamers
have been identified exhibiting high affinity and specificity with dissociation con-
stants in the nanomolar range. The aptamer with the highest affinity to sepiapterin
was used to fabricate a competitive electrochemical biosensor. The detection was
achieved via competition of the free sepiapterin in the sample with immobilized
analyte on gold electrode for binding to the free aptamer. Square wave voltammetry
technique was used for the detection showing high sensitivity and selectivity against
closely related molecules.
Metabolomics has also led to the discovery of biomarkers associated with asthma
pathogenesis in serum. Among these biomarkers, it was reported that a decreased
level of arginine can be used as indicator of asthma [88]. Yuan et al. [89] have
reported the development of an aptamer-based biosensor for the chiral recognition
of arginine enantiomers. The biosensor was based on fluorescence detection using
gold nanoparticles on which fluorophore-labeled aptamers were immobilized

Fig. 2 A schematic of fluorescence-based biosensor for the detection of D, L- arginine. The fluo-
rescence of the labeled aptamers was quenched when the aptamer adsorbs on gold nanoparticles
due to the fluorescence resonance energy transfer. The binding of the aptamer with D, L- arginine
leads to desorption of the aptamers from the gold surface and thus enhancement in the fluorescence
intensity. (Reprinted with permission from [89])
Transferring Metabolomics to Portable Diagnostic Devices: Trending in Biosensors 339

(Fig. 2). The detection was achieved by following the increase in the fluorescence
intensity when the aptamers bind to the targets leading to the release of the fluores-
cence label from the gold nanoparticles.

3 Transducers for Detection of Metabolic Biomarkers

Many different transducers are used for the development of various biosensors. The
most popular transducers used in most of the biosensor’s designs are the optical,
electrochemical, thermal, and mass-based transducers. Each of these detection tech-
niques has its own advantages and disadvantages and can be better suited for the
detection of different target analytes.
Several biosensors have been reported for the detection of metabolic biomarkers
for the point-of-care diagnosis of diseases [16, 35, 41, 67, 76, 90]. Considerable
research effort has been particularly devoted toward the fabrication of electrochemi-
cal and optical biosensors for the detection of different types of metabolites. The
integration of various nanomaterials, such as carbon nanotubes, graphene, carbon
nanofibers, magnetic nanoparticles, and gold nanoparticles into these biosensors,
has led to significant improvements in their analytical performance because of their
large surface area, high electrical conductivity, and chemical stability [91]. In the
next subsections, we will focus mainly on the electrochemical and optical detection
techniques used in the biosensor designs for different metabolites.

3.1 Electrochemical Detection

Electrochemical detection is the main detection approach used in most of the bio-
sensors as transducer via monitoring the electrochemical signal generated when the
analyte binds to the recognition receptor. These electrochemical signals can be a
measurable charge accumulation or potential (potentiometry), current (amperome-
try/voltammetry), conductivity (conductometry), or resistance and capacitance
(Electrochemical impedance spectroscopy). Electrochemical detection methods in
biosensors offer several advantages such as their high sensitivity, low cost, ease of
use, and capability of miniaturization. These advantages have led to significant
applications of electrochemical biosensors in point-of-care testing as they are easier
to implement in integrated biosensors and operate than optical techniques. Moreover,
the advances in the screen-printing technology have led to a wide range of applica-
tions of screen-printed electrodes in point-of-care diagnostic biosensors.
Electrochemical detection techniques used in the biosensors for metabolites are
mostly voltammetry/amperometry and electrochemical impedance spectroscopy.
Thus, these techniques will be discussed in the next sections.
340 S. Eissa

3.1.1 Amperometry/Voltammetry

Voltammetric and amperometric techniques are used to measure the current result-
ing from an electrochemical oxidation or reduction processes when a potential is
applied to a working electrode versus a reference electrode. In the voltammetric
techniques, the potential is scanned over a set potential range resulting in a current
response in the form of a peak, whereas in the amperometry, a constant potential is
maintained, and the generated current is monitored directly with time. The most
used voltammetric methods in biosensors are the cyclic voltammetry, differential
pulse voltammetry, linear sweep voltammetry, and square wave voltammetry.
Amperometric methods are primarily used for biocatalytic/enzymatic biosensors,
whereas voltammetric-based detection is often used on affinity biosensors (immu-
nosensors and aptasensors).
Several metabolites have been detected using amperometric- [15–19, 92] or
voltammetric-based biosensors [76, 87] utilizing various electrodes and nanomate-
rials. Since most of the amperometric−/voltammetric-based detection methods are
mainly used in enzyme-based biosensors, they were described in more detail in
Sect. 2.1.

3.1.2 Electrochemical Impedance Spectroscopy

Electrochemical impedance spectroscopy is an electrochemical technique which

used to measure the capacitive and resistive properties of an electrode upon pertur-
bation of a system with a small amplitude sinusoidal ac excitation signal typically
of 2–10 mV. The current response is then determined with changing frequency over
a wide range, and the result is displayed in the form of an impedance spectrum.
Impedance-based detection is mainly used in affinity biosensor showing good
advantages in terms of sensitivity and nondestructive nature. Several impedance-­
based biosensors for different metabolites have been reported [71, 93, 94]. For
instance, impedance-based biosensor for the detection of parathyroid hormone for
the diagnosis of thyroid cancer, hypoparathyroidism, and hyperparathyroidism has
been described [93]. The biosensor was fabricated by immobilizing antibody for
parathyroid on poly amidoamine dendrimer which was attached to gold electrode.
The detection was achieved via monitoring the change in the charge transfer resis-
tance of the electrode upon binding of the hormone with the immunosensor. The
linear range of the biosensor was from 10 to 60 fg/mL. Immunosensor was used to
detect parathyroid levels in artificial serum samples.
Transferring Metabolomics to Portable Diagnostic Devices: Trending in Biosensors 341

3.2 Optical Detection

The detection of the binding event between the recognition receptor and the target
in the biosensors through monitoring the change in the optical signals is very popu-
lar. The optical detection methods are usually based on the use of an optically active
label conjugated to the target molecule. These labels can be a simple fluorophore or
quencher molecules for fluorescence detection or nanoparticle tags for colorimetric
or surface plasmon resonance-based biosensors.
A fluorescence-based aptasensor for arginine has been reported [89]. The detec-
tion is based on the quenching of the carboxyfluorescein-labeled aptamer when it is
adsorbed on the negatively charged gold nanoparticles because of the fluorescence
resonance energy transfer effect. However, when the D- or L-arginine exist in the
sample, the aptamer binds to the target causing a change in the conformation of the
aptamer. This prevents the fluorescence label from being adsorbed on the gold
nanoparticles leading to a recovery in the fluorescence intensity. In this study, it was
shown that the increase in the fluorescence signal was stronger when L-arginine was
used compared to D-arginine. An aptamer-based surface-enhanced Raman spectros-
copy biosensor for the detection of vasopressin was reported [81]. Densely packed
metal nanotube arrays prepared using an anodized alumina nanoporous membrane
were used to create the active substrate. The integration of the membranes with a
polydimethylsiloxane microfluidic device has demonstrated good sensitivity with a
LOD of 5.2 μU/mL. Colorimetric-based detection of cortisol is simple and low cost
and does not require the use of sophisticated equipment as the results can be seen by
the naked eye. Four different chromogens (sulfuric acid, Porter-Silber reagent,
Prussian blue, and blue tetrazolium) have been used for the detection of cortisol in
artificial saliva and human sweat. This method has shown comparable sensitivity to
the electrochemical biosensors. The simplicity of the colorimetric detection makes
them suitable for point-of-care testing of different metabolites [95]. Colorimetric-­
based aptasensor was reported for the detection of progesterone in human serum
and urine [96]. This method was based on the alteration of the aggregating proper-
ties of the gold nanoparticles upon addition of progesterone, aptamer, and the cat-
ionic surfactant, hexadecyltrimethylammonium bromide. When the aptamer binds
to progesterone, the surfactant causes aggregation of the gold nanoparticles leading
to a change of the color of the solution from red to blue. Progesterone has been also
detected colorimetrically using lateral flow assay [97]. A specific aptamer for pro-
gesterone was immobilized on gold nanoparticles, and a biotin-labeled complemen-
tary DNA sequence was then hybridized with the aptamer. The test line was coated
with streptavidin which allows the capture of the biotinylated gold nanoparticles –
aptamer duplex. However, upon binding of the aptamer with the target progester-
one, a displacement of the biotinylated complementary DNA has occurred which
prevented the capture of the gold particles on the test line. This aptamer-based lat-
eral flow assay method has led to sensitive detection of progesterone in the nanomo-
lar range as well as good selectivity against other hormones.
342 S. Eissa

4 Advances in the Development of Biosensors

for Metabolic Biomarkers

Recent advances in nanotechnology have accelerated the improvements in the

development of biosensors for metabolic biomarkers in point-of-care diagnosis.
Multiplexed biosensors offered high-throughput simultaneous screening of various
targets which is highly important in metabolomics. Particularly, the ease of fabrica-
tion of multiple individually addressable arrays of working electrodes on small
chips has led to the development of many multiplexed electrochemical biosensors
[98]. Microfluidics enables the use of very small sample volumes for detection, and
thus, it is perfectly suited for the diagnostic point-of-care biosensors where a small
blood sample is often used [99–101]. Zhao et al. [102] have reported the develop-
ment of a microfluidic paper-based multiplexed electrochemical biosensor array for
the simultaneous detection of three metabolic biomarkers, glucose, lactate, and uric
acid. Channels were fabricated on chromatographic paper via solid wax printing,
and the silver connections and carbon electrodes were printed on the surface of the
paper using screen printing. An array of eight biosensors were developed, and the
signals were detected using a portable handheld custom-made potentiostat.
Simultaneous measurements for multiple analytes were successfully recorded, and
the analytical performance of the device was comparable with the commercial
platforms.
We have reported the development of a multiplexed electrochemical immuno-
sensor for the simultaneous detection of the metabolites: morphine, benzoylecgo-
nine, and tetrahydrocannabinol in urine [103]. Gold nanoparticle-modified
screen-printed carbon array electrodes were utilized on which specific antibodies
for the three metabolites were immobilized. A competitive assay was used for the
detection by using bovine serum albumin-conjugated analytes. The multiplexed
biosensor showed fast response and high sensitivity and selectivity.
Wearable biosensors are gaining significant interest because of their potential to
provide noninvasive and real-time measurements of biomarkers of different metab-
olites in biofluids, such as saliva, sweat, and tears [104]. Wearable biosensors com-
bine the microfluidic sampling, multiplexed biosensing, and transport systems with
flexible materials to form miniaturized and easily operating detection tool (Fig. 3).
Recent wearable biosensors for healthcare monitoring have been recently reviewed
by Kim et al. [104].
Recently, a wearable lactate electrochemical biosensor for sweat analysis has
been reported [11]. An outer plasticized polymeric layer containing the tetradodecy-
lammonium tetrakis (4-chlorophenyl) borate salt has been used to prevent the lac-
tate oxidase enzyme from being in direct contact with the sample which significantly
reduced the effect of temperature and pH. The authors reported that their biosensor
showed higher sensitivity than other reported biosensors with good selectivity and
reproducibility. The analytical performance of this wearable biosensor was compa-
rable with ion chromatography method. The biosensor was also applied for the
detection of lactate in sweat on three different body locations (forehead, back, and
Transferring Metabolomics to Portable Diagnostic Devices: Trending in Biosensors 343

b c

Fig. 3 (a) flexible screen-printed electrode array. (b) The enzymatic biosensing mechanism of
glucose detection and implantation of wearable biosensor. (c) Schematic of the integrated wearable
biosensor for the continuous monitoring of glucose on a rabbit. (Reprinted with permission
from [105])

thigh) showing good performance indicating that it holds great promise toward
other healthcare applications.

5 Conclusions and Future Perspectives

The development of biosensors for metabolic biomarkers offers a faster, simpler,

and lower cost alternative to the conventional analytical assays. Several biosensors
for various metabolites have been developed for point-of-care diagnosis. In this
chapter, we reviewed the different biosensor designs used in metabolomics.
Extensive research has been devoted for the development of enzyme-based biocata-
lytic sensors for some metabolites used in medical diagnosis such as glucose, lac-
tate, and xanthene. Various types of nanoparticles have been integrated in these
biosensors to enhance their analytical performance which resulted in high sensitiv-
ity and rapid analysis. However, there are no specific enzymes for many metabo-
lites. Moreover, the enzyme stability is limited as it gradually loses activity over
time which represents a major challenge in the enzymatic biosensors. Therefore,
recent research has focused on the development of other biorecognition receptors
344 S. Eissa

such as antibodies and aptamer to fabricate affinity biosensors for various metabo-
lites. The continuous identification of new aptamers that binds specifically to vari-
ous types of metabolites is highly needed and can open the door for the development
of sensitive biosensing platforms for point-of-care diagnosis. Optical and electro-
chemical detections have been used in most of the reported biosensors for metabolic
biomarkers. Particularly, electrochemical biosensors offer great promise because of
their low cost, high sensitivity, ease of integration into portable biosensors, and
capability of multiplexing which make them ideal for high-throughput screening in
metabolomics. Advances in the development of wearable biosensors have been also
highlighted in this chapter as they provide continuous and noninvasive measure-
ments for different metabolites in body fluids. Yet, large cohort studies of these
wearable biosensors are required for validation to reach clinical acceptance.

References

1. Zhang A, Sun H, Yan G, Wang P, Wang X. Metabolomics for biomarker discovery: moving to
the clinic. Biomed Res Int. 2015;2015:354671. [Link]
2. Song Q, Zhang A-h, Yan G-l, Liu L, Wang X-J. Technological advances in current metabo-
lomics and its application in tradition Chinese medicine. RSC Adv. 2017;7(84):53516–24.
[Link]
3. Griffiths WJ, Koal T, Wang Y, Kohl M, Enot DP, Deigner HP. Targeted metabolomics for bio-
marker discovery. Angew Chem Int Ed Engl. 2010;49(32):5426–45. [Link]
anie.200905579.
4. Clarke CJ, Haselden JN. Metabolic profiling as a tool for understanding mechanisms of tox-
icity. Toxicol Pathol. 2008;36(1):140–7. [Link]
5. Wang J. Glucose biosensors: 40 years of advances and challenges. Electroanalysis.
2001;13(12):983–8. [Link]
AID-­ELAN983>[Link];2-­#.
6. Ronkainen NJ, Halsall HB, Heineman WR. Electrochemical biosensors. Chem Soc Rev.
2010;39(5):1747–63. [Link]
7. Yoo EH, Lee SY. Glucose biosensors: an overview of use in clinical practice. Sensors (Basel).
2010;10(5):4558–76. [Link]
8. Juska VB, Pemble ME. A critical review of electrochemical glucose sensing: evolution of
biosensor platforms based on advanced nanosystems. Sensors (Basel). 2020;20(21):6013.
[Link]
9. Oliver NS, Toumazou C, Cass AEG, Johnston DG. Glucose sensors: a review of current and
emerging technology. Diabet Med. 2009;26(3):197–210. [Link]
1.2008.02642.x.
10. Taguchi M, Ptitsyn A, McLamore ES, Claussen JC. Nanomaterial-mediated biosen-
sors for monitoring glucose. J Diabetes Sci Technol. 2014;8(2):403–11. [Link]
org/10.1177/1932296814522799.
11. Xuan X, Pérez-Ràfols C, Chen C, Cuartero M, Crespo GA. Lactate biosensing for reli-
able on-body sweat analysis. ACS Sens. 2021;6(7):2763–71. [Link]
acssensors.1c01009.
12. Hiraka K, Tsugawa W, Asano R, Yokus MA, Ikebukuro K, Daniele MA, et al. Rational
design of direct electron transfer type l-lactate dehydrogenase for the development of
multiplexed biosensor. Biosens Bioelectron. 2021;176:112933. [Link]
bios.2020.112933.
Transferring Metabolomics to Portable Diagnostic Devices: Trending in Biosensors 345

13. Raj MA, John SA. Simultaneous determination of uric acid, xanthine, hypoxanthine and
caffeine in human blood serum and urine samples using electrochemically reduced gra-
phene oxide modified electrode. Anal Chim Acta. 2013;771:14–20. [Link]
aca.2013.02.017.
14. Pundir CS, Devi R. Biosensing methods for xanthine determination: a review. Enzym Microb
Technol. 2014;57:55–62. [Link]
15. Tsiafoulis CG, Prodromidis MI, Karayannis MI. Development of amperometric biosensors
for the determination of glycolic acid in real samples. Anal Chem. 2002;74(1):132–9. https://
[Link]/10.1021/ac0106896.
16. Tian G, Ding M, Xu B, He Y, Lyu W, Jin M, et al. A novel electrochemical biosensor for
ultrasensitive detection of serum total bile acids based on enzymatic reaction combined with
the double oxidation circular amplification strategy. Biosens Bioelectron. 2018;118:31–5.
[Link]
17. Dong E, Zheng J, Liu H. Bile acid detection by biosensors-a review. Sheng Wu Gong Cheng
Xue Bao. 2020;36(12):2779–90. [Link]
18. Verma N, Singh AK, Singh M. L-arginine biosensors: a comprehensive review. Biochem
Biophys Rep. 2017;12:228–39. [Link]
19. Magar HS, Ghica ME, Abbas MN, Brett CMA. A novel sensitive amperometric cho-
line biosensor based on multiwalled carbon nanotubes and gold nanoparticles. Talanta.
2017;167:462–9. [Link]
20. Weis BK, Balshaw D, Barr JR, Brown D, Ellisman M, Lioy P, et al. Personalized exposure
assessment: promising approaches for human environmental health research. Environ Health
Perspect. 2005;113(7):840–8. [Link]
21. Boldt J. Clinical review: hemodynamic monitoring in the intensive care unit. Crit Care.
2002;6(1):52–9. [Link]
22. Sánchez-Paniagua López M, Hervás Pérez JP, López-Cabarcos E, López-Ruiz
B. Amperometric biosensors based on choline oxidase entrapped in polyacrylamide micro-
gels. Electroanalysis. 2007;19(2–3):370–8. [Link]
23. Zhang H, Yin Y, Wu P, Cai C. Indirect electrocatalytic determination of choline by monitoring
hydrogen peroxide at the choline oxidase-prussian blue modified iron phosphate nanostruc-
tures. Biosens Bioelectron. 2012;31(1):244–50. [Link]
24. Ricci F, Amine A, Palleschi G, Moscone D. Prussian blue based screen printed biosensors
with improved characteristics of long-term lifetime and pH stability. Biosens Bioelectron.
2003;18(2):165–74. [Link]
25. Shi H, Yang Y, Huang J, Zhao Z, Xu X, Anzai J, et al. Amperometric choline biosensors pre-
pared by layer-by-layer deposition of choline oxidase on the Prussian blue-modified platinum
electrode. Talanta. 2006;70(4):852–8. [Link]
26. Keihan AH, Sajjadi S, Sheibani N, Moosavi-Movahedi AA. A highly sensitive choline
biosensor based on bamboo-like multiwall carbon nanotubes/ionic liquid/Prussian blue
nanocomposite. Sensors Actuators B Chem. 2014;204:694–703. [Link]
snb.2014.08.039.
27. Deng K, Zhou J, Li X. Noncovalent nanohybrid of ferrocene with chemically reduced
graphene oxide and its application to dual biosensor for hydrogen peroxide and choline.
Electrochim Acta. 2013;95:18–23. [Link]
28. Razola SS, Pochet S, Grosfils K, Kauffmann JM. Amperometric determination of choline
released from rat submandibular gland acinar cells using a choline oxidase biosensor. Biosens
Bioelectron. 2003;18(2–3):185–91. [Link]
29. Song Z, Huang J-D, Wu B-Y, Shi H-B, Anzai J-I, Chen Q. Amperometric aqueous sol–
gel biosensor for low-potential stable choline detection at multi-wall carbon nanotube
modified platinum electrode. Sensors Actuators B Chem. 2006;115(2):626–33. [Link]
org/10.1016/[Link].2005.10.030.
30. Serafín V, Martínez-García G, Aznar-Poveda J, Lopez-Pastor JA, Garcia-Sanchez AJ, Garcia-­
Haro J, et al. Determination of progesterone in saliva using an electrochemical ­immunosensor
346 S. Eissa

and a COTS-based portable potentiostat. Anal Chim Acta. 2019;1049:65–73. [Link]

org/10.1016/[Link].2018.10.019.
31. Dong X-X, Yuan L-P, Liu Y-X, Wu M-F, Liu B, Sun Y-M, et al. Development of a progester-
one immunosensor based on thionine-graphene oxide composites platforms: improvement
by biotin-streptavidin-amplified system. Talanta. 2017;170:502–8. [Link]
talanta.2017.04.054.
32. Carralero V, González-Cortés A, Yáñez-Sedeño P, Pingarrón JM. Nanostructured progesterone
immunosensor using a tyrosinase–colloidal gold–graphite–teflon biosensor as amperometric
transducer. Anal Chim Acta. 2007;596(1):86–91. [Link]
33. Monerris MJ, Arévalo FJ, Fernández H, Zon MA, Molina PG. Integrated electrochemi-
cal immunosensor with gold nanoparticles for the determination of progesterone. Sensors
Actuators B Chem. 2012;166-167:586–92. [Link]
34. Arya SK, Chornokur G, Venugopal M, Bhansali S. Dithiobis(succinimidyl propionate)
modified gold microarray electrode based electrochemical immunosensor for ultrasensitive
detection of cortisol. Biosens Bioelectron. 2010;25(10):2296–301. [Link]
bios.2010.03.016.
35. Kumar A, Aravamudhan S, Gordic M, Bhansali S, Mohapatra SS. Ultrasensitive detection of
cortisol with enzyme fragment complementation technology using functionalized nanowire.
Biosens Bioelectron. 2007;22(9):2138–44. [Link]
36. Vasudev A, Kaushik A, Tomizawa Y, Norena N, Bhansali S. An LTCC-based microfluidic
system for label-free, electrochemical detection of cortisol. Sensors Actuators B Chem.
2013;182:139–46. [Link]
37. Arya SK, Dey A, Bhansali S. Polyaniline protected gold nanoparticles based mediator and
label free electrochemical cortisol biosensor. Biosens Bioelectron. 2011;28(1):166–73.
[Link]
38. Tlili C, Myung NV, Shetty V, Mulchandani A. Label-free, chemiresistor immunosensor for
stress biomarker cortisol in saliva. Biosens Bioelectron. 2011;26(11):4382–6. [Link]
org/10.1016/[Link].2011.04.045.
39. Arya SK, Chornokur G, Venugopal M, Bhansali S. Antibody modified gold micro array elec-
trode based electrochemical immunosensor for ultrasensitive detection of cortisol in saliva
and ISF. Procedia Eng. 2010;5:804–7. [Link]
40. Kämäräinen S, Mäki M, Tolonen T, Palleschi G, Virtanen V, Micheli L, et al. Disposable
electrochemical immunosensor for cortisol determination in human saliva. Talanta.
2018;188:50–7. [Link]
41. Venugopal M, Arya SK, Chornokur G, Bhansali S. A realtime and continuous assessment
of cortisol in ISF using electrochemical impedance spectroscopy. Sensors Actuators A Phys.
2011;172(1):154–60. [Link]
42. Yamaguchi M, Matsuda Y, Sasaki S, Sasaki M, Kadoma Y, Imai Y, et al. Immunosensor with
fluid control mechanism for salivary cortisol analysis. Biosens Bioelectron. 2013;41:186–91.
[Link]
43. Volpe G, Fares G, Delli Quadri F, Draisci R, Ferretti G, Marchiafava C, et al. A disposable
immunosensor for detection of 17beta-estradiol in non-extracted bovine serum. Anal Chim
Acta. 2006;572(1):11–6. [Link]
44. Pemberton RM, Mottram TT, Hart JP. Development of a screen-printed carbon electro-
chemical immunosensor for picomolar concentrations of estradiol in human serum extracts. J
Biochem Biophys Methods. 2005;63(3):201–12. [Link]
45. Liu X, Wong DK. Picogram-detection of estradiol at an electrochemical immunosensor with
a gold nanoparticle|protein G-(LC-SPDP)-scaffold. Talanta. 2009;77(4):1437–43. [Link]
org/10.1016/[Link].2008.09.027.
46. Ojeda I, López-Montero J, Moreno-Guzmán M, Janegitz BC, González-Cortés A, Yáñez-­
Sedeño P, et al. Electrochemical immunosensor for rapid and sensitive determination of estra-
diol. Anal Chim Acta. 2012;743:117–24. [Link]
Transferring Metabolomics to Portable Diagnostic Devices: Trending in Biosensors 347

47. Liu X, Duckworth PA, Wong DK. Square wave voltammetry versus electrochemical imped-
ance spectroscopy as a rapid detection technique at electrochemical immunosensors. Biosens
Bioelectron. 2010;25(6):1467–73. [Link]
48. Kuramitz H, Matsuda M, Thomas JH, Sugawara K, Tanaka S. Electrochemical immunoas-
say at a 17β-estradiol self-assembled monolayer electrode using a redox marker. Analyst.
2003;128(2):182–6. [Link]
49. Eguílaz M, Moreno-Guzmán M, Campuzano S, González-Cortés A, Yáñez-Sedeño P,
Pingarrón JM. An electrochemical immunosensor for testosterone using functionalized mag-
netic beads and screen-printed carbon electrodes. Biosens Bioelectron. 2010;26(2):517–22.
[Link]
50. Conneely G, Aherne M, Lu H, Guilbault GG. Development of an immunosensor for the
detection of testosterone in bovine urine. Anal Chim Acta. 2007;583(1):153–60. [Link]
org/10.1016/[Link].2006.09.062.
51. Wang H, Chen W, Li D, Yin X, Zhang X, Olsen N, et al. Vitamin D and chronic diseases.
Aging Dis. 2017;8(3):346–53. [Link]
52. Demir M, Demir F, Aygun H. Vitamin D deficiency is associated with COVID-19 positiv-
ity and severity of the disease. J Med Virol. 2021;93(5):2992–9. [Link]
jmv.26832.
53. Weir EK, Thenappan T, Bhargava M, Chen Y. Does vitamin D deficiency increase the
severity of COVID-19? Clin Med (Lond). 2020;20(4):e107–e8. [Link]
clinmed.2020-­0301.
54. Chauhan D, Solanki PR. Hydrophilic and insoluble electrospun cellulose acetate fiber-­
based biosensing platform for 25-Hydroxy vitamin-D3 detection. ACS Appl Polym Mater.
2019;1(7):1613–23. [Link]
55. Chauhan D, Gupta PK, Solanki PR. Electrochemical immunosensor based on magne-
tite nanoparticles incorporated electrospun polyacrylonitrile nanofibers for vitamin-D(3)
detection. Mater Sci Eng C Mater Biol Appl. 2018;93:145–56. [Link]
msec.2018.07.036.
56. Kaur A, Kapoor S, Bharti A, Rana S, Chaudhary GR, Prabhakar N. Gold-platinum bime-
tallic nanoparticles coated 3-(aminopropyl)triethoxysilane (APTES) based electrochemical
immunosensor for vitamin D estimation. J Electroanal Chem. 2020;873:114400. [Link]
org/10.1016/[Link].2020.114400.
57. Carlucci L, Favero G, Tortolini C, Di Fusco M, Romagnoli E, Minisola S, et al. Several
approaches for vitamin D determination by surface plasmon resonance and electrochemi-
cal affinity biosensors. Biosens Bioelectron. 2013;40(1):350–5. [Link]
bios.2012.07.077.
58. Chauhan D, Yadav AK, Solanki PR. Carbon cloth-based immunosensor for detection
of 25-hydroxy vitamin D3. Microchim Acta. 2021;188(4):145. [Link]
s00604-­021-­04751-­y.
59. Ellington AD, Szostak JW. In vitro selection of RNA molecules that bind specific ligands.
Nature. 1990;346(6287):818–22. [Link]
60. Song S, Wang L, Li J, Fan C, Zhao J. Aptamer-based biosensors. TrAC Trends Anal Chem.
2008;27(2):108–17. [Link]
61. Marrazza G. Aptamer sensors. Biosensors (Basel). 2017;7(1):5. [Link]
bios7010005.
62. Ruscito A, DeRosa MC. Small-molecule binding aptamers: selection strategies, characteriza-
tion, and applications. Front Chem. 2016;4:14. [Link]
63. Zhou W, Jimmy Huang P-J, Ding J, Liu J. Aptamer-based biosensors for biomedical diagnos-
tics. Analyst. 2014;139(11):2627–40. [Link]
64. Eissa S, Siddiqua A, Chinnappan R, Zourob M. Electrochemical selection of a DNA aptamer,
and an impedimetric method for determination of the dedicator of cytokinesis 8 by self-­
assembly of a thiolated aptamer on a gold electrode. Microchim Acta. 2019;186(12):828.
[Link]
348 S. Eissa

65. Eissa S, Zourob M, et al. Sci Rep. 2017;7(1):1016. [Link]

s41598-­017-­01226-­0.
66. Almusharraf AY, Eissa S, Zourob M. Truncated aptamers for total and glycated hemoglobin,
and their integration into a graphene oxide-based fluorometric method for high-­throughput
screening for diabetes. Microchim Acta. 2018;185(5):256. [Link]
s00604-­018-­2789-­3.
67. Prante M, Segal E, Scheper T, Bahnemann J, Walter J. Aptasensors for point-of-care detec-
tion of small molecules. Biosensors. 2020;10(9):108. [Link]
68. Musheev MU, Krylov SN. Selection of aptamers by systematic evolution of ligands by
exponential enrichment: addressing the polymerase chain reaction issue. Anal Chim Acta.
2006;564(1):91–6. [Link]
69. Rather JA, Khudaish EA, Kannan P. Graphene-amplified femtosensitive aptasensing of
estradiol, an endocrine disruptor. Analyst. 2018;143(8):1835–45. [Link]
C7AN02092A.
70. Akki SU, Werth CJ, Silverman SK. Selective aptamers for detection of estradiol and ethy-
nylestradiol in natural waters. Environ Sci Technol. 2015;49(16):9905–13. [Link]
org/10.1021/[Link].5b02401.
71. Contreras Jiménez G, Eissa S, Ng A, Alhadrami H, Zourob M, Siaj M. Aptamer-based label-­
free impedimetric biosensor for detection of progesterone. Anal Chem. 2015;87(2):1075–82.
[Link]
72. Alhadrami HA, Chinnappan R, Eissa S, Rahamn AA, Zourob M. High affinity truncated
DNA aptamers for the development of fluorescence based progesterone biosensors. Anal
Biochem. 2017;525:78–84. [Link]
73. Zulkeflee Sabri M, Abdul Hamid AA, Sayed Hitam SM, Abdul Rahim MZ. In-silico study
of single-strand DNA aptamers against cortisol as a potential biomarker receptor in thera-
peutics and diagnostics. Mater Today Proc. 2020;31:A90–A7. [Link]
matpr.2020.12.1080.
74. Martin JA, Chávez JL, Chushak Y, Chapleau RR, Hagen J, Kelley-Loughnane N. Tunable
stringency aptamer selection and gold nanoparticle assay for detection of cortisol. Anal
Bioanal Chem. 2014;406(19):4637–47. [Link]
75. Lévesque D, Beaudoin J-D, Roy S, Perreault J-P. In vitro selection and characterization of
RNA aptamers binding thyroxine hormone. Biochem J. 2007;403(1):129–38. [Link]
org/10.1042/BJ20061216.
76. Eissa S, Siddiqua A, Chinnappan R, Zourob M. Electrochemical SELEX technique for the
selection of DNA aptamers against the small molecule 11-Deoxycortisol. ACS Appl Bio
Mater. 2019;2(6):2624–32. [Link]
77. Skouridou V, Jauset-Rubio M, Ballester P, Bashammakh AS, El-Shahawi MS, Alyoubi
AO, et al. Selection and characterization of DNA aptamers against the steroid testosterone.
Microchim Acta. 2017;184(6):1631–9. [Link]
78. He P, Oncescu V, Lee S, Choi I, Erickson D. Label-free electrochemical monitoring of vaso-
pressin in aptamer-based microfluidic biosensors. Anal Chim Acta. 2013;759:74–80. https://
[Link]/10.1016/[Link].2012.10.038.
79. Williams KP, Liu XH, Schumacher TN, Lin HY, Ausiello DA, Kim PS, et al. Bioactive
and nuclease-resistant L-DNA ligand of vasopressin. Proc Natl Acad Sci U S
A. 1997;94(21):11285–90. [Link]
80. Purschke WG, Eulberg D, Buchner K, Vonhoff S, Klussmann S. An L-RNA-based aquaretic
agent that inhibits vasopressin in vivo. Proc Natl Acad Sci U S A. 2006;103(13):5173. https://
[Link]/10.1073/pnas.0509663103.
81. Huh YS, Erickson D. Aptamer based surface enhanced raman scattering detection of vaso-
pressin using multilayer nanotube arrays. Biosens Bioelectron. 2010;25(5):1240–3. https://
[Link]/10.1016/[Link].2009.09.040.
Transferring Metabolomics to Portable Diagnostic Devices: Trending in Biosensors 349

82. Lee BH, Nguyen VT, Gu MB. Highly sensitive detection of 25-HydroxyvitaminD3 by using
a target-induced displacement of aptamer. Biosens Bioelectron. 2017;88:174–80. [Link]
org/10.1016/[Link].2016.08.011.
83. Echenne B, Roubertie A, Assmann B, Lutz T, Penzien JM, Thöny B, et al. Sepiapterin reduc-
tase deficiency: clinical presentation and evaluation of long-term therapy. Pediatr Neurol.
2006;35(5):308–13. [Link]
84. Blau N, Bonafé L, Thöny B. Tetrahydrobiopterin deficiencies without Hyperphenylalaninemia:
diagnosis and genetics of DOPA-responsive dystonia and sepiapterin reductase deficiency.
Mol Genet Metab. 2001;74(1):172–85. [Link]
85. Blau N. Disorder of tetrahydrobiopterin and related biogenic amines. The metabolic and
molecular bases of inherited disease. 2000.
86. Assmann B, Surtees R, Hoffmann GF. Approach to the diagnosis of neurotransmitter dis-
eases exemplified by the differential diagnosis of childhood-onset dystonia. Ann Neurol.
2003;54(S6):S18–24. [Link]
87. Eissa S, Alkhaldi S, Chinnappan R, Siddiqua A, Abduljabbar M, Abdel Rahman AM, et al.
Selection, characterization, and electrochemical biosensing application of DNA aptamers for
sepiapterin. Talanta. 2020;216:120951. [Link]
88. Jung J, Kim SH, Lee HS, Choi GS, Jung YS, Ryu DH, et al. Serum metabolomics
reveals pathways and biomarkers associated with asthma pathogenesis. Clin Exp Allergy.
2013;43(4):425–33. [Link]
89. Yuan H, Huang Y, Yang J, Guo Y, Zeng X, Zhou S, et al. An aptamer-based fluorescence
bio-sensor for chiral recognition of arginine enantiomers. Spectrochim Acta A Mol Biomol
Spectrosc. 2018;200:330–8. [Link]
90. Imani S, Bandodkar AJ, Mohan AMV, Kumar R, Yu S, Wang J, et al. A wearable chemical–
electrophysiological hybrid biosensing system for real-time health and fitness monitoring.
Nat Commun. 2016;7(1):11650. [Link]
91. Malhotra BD, Ali MA. Nanomaterials in biosensors: fundamentals and applications. Nanomat
Biosens. 2018;1:1–74. [Link]
92. He C, Xie M, Hong F, Chai X, Mi H, Zhou X, et al. A highly sensitive glucose biosen-
sor based on gold nanoparticles/bovine serum albumin/Fe3O4 biocomposite nanoparticles.
Electrochim Acta. 2016;222:1709–15. [Link]
93. Özcan HM, Sezgintürk MK. Detection of parathyroid hormone using an electrochemical
impedance biosensor based on PAMAM dendrimers. Biotechnol Prog. 2015;31(3):815–22.
[Link]
94. Saxena R, Srivastava S. A sensitive and one-step quantification of thyroid stimulating hor-
mone using nanobiosensor. Mater Today Proc. 2019;18:1351–7. [Link]
matpr.2019.06.600.
95. Tu E, Pearlmutter P, Tiangco M, Derose G, Begdache L, Koh A. Comparison of colorimetric
analyses to determine cortisol in human sweat. ACS Omega. 2020;5(14):8211–8. [Link]
org/10.1021/acsomega.0c00498.
96. Du G, Wang L, Zhang D, Ni X, Zhou X, Xu H, et al. Colorimetric aptasensor for progester-
one detection based on surfactant-induced aggregation of gold nanoparticles. Anal Biochem.
2016;514:2–7. [Link]
97. Alnajrani MN, Alsager OA. Lateral flow aptasensor for progesterone: competitive tar-
get recognition and displacement of short complementary sequences. Anal Biochem.
2019;587:113461. [Link]
98. Eissa S, Zourob M. Ultrasensitive peptide-based multiplexed electrochemical biosensor for
the simultaneous detection of listeria monocytogenes and Staphylococcus aureus. Microchim
Acta. 2020;187(9):486. [Link]
99. Jayamohan H, Sant HJ, Gale BK. Applications of microfluidics for molecular diagnostics.
Methods Mol Biol. 2013;949:305–34. [Link]
350 S. Eissa

100. Nikoleli G-P, Siontorou CG, Nikolelis DP, Bratakou S, Karapetis S, Tzamtzis N. Chapter
13—biosensors based on microfluidic devices lab-on-a-chip and microfluidic technology. In:
Nikolelis DP, Nikoleli G-P, editors. Nanotechnology and biosensors. Amsterdam: Elsevier;
2018. p. 375–94.
101. Zhao C, Liu X. A paper-based microfluidic device for multiplexed electrochemical detec-
tion of biomarkers. 2013 International Conference on Manipulation, Manufacturing and
Measurement on the Nanoscale. 2013. p. 162–5.
102. Zhao C, Thuo MM, Liu X. A microfluidic paper-based electrochemical biosensor array for
multiplexed detection of metabolic biomarkers. Sci Technol Adv Mater. 2013;14(5):054402.
[Link]
103. Eissa S, Almthen RA, Zourob M. Disposable electrochemical immunosensor array for the
multiplexed detection of the drug metabolites morphine, tetrahydrocannabinol and benzoy-
lecgonine. Microchim Acta. 2019;186(8):523. [Link]
104. Kim J, Campbell AS, de Ávila BE-F, Wang J. Wearable biosensors for healthcare monitoring.
Nat Biotechnol. 2019;37(4):389–406. [Link]
105. Jin X, Li G, Xu T, Su L, Yan D, Zhang X. Fully integrated flexible biosensor for wear-
able continuous glucose monitoring. Biosens Bioelectron. 2022;196:113760. [Link]
org/10.1016/[Link].2021.113760.