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New Perspectives in Monitoring Lung Inflammation
Analysis of Exhaled Breath Condensate 1st Edition Paolo
Montuschi (Editor) Digital Instant Download
Author(s): Paolo Montuschi (Editor)
ISBN(s): 9781134345427, 0415324653
Edition: 1
File Details: PDF, 4.51 MB
Year: 2013
Language: english
NEW PERSPECTIVES IN
MONITORING
LUNG INFLAMMATION
Analysis of Exhaled
Breath Condensate
TF1734_C00.fm Page ii Tuesday, September 14, 2004 1:01 PM
NEW PERSPECTIVES IN
MONITORING
LUNG INFLAMMATION
Analysis of Exhaled
Breath Condensate

EDITED BY

Paolo Montuschi, M.D.

Catholic University of the Sacred Heart
Rome, Italy

CRC PR E S S
Boca Raton London New York Washington, D.C.
CRC Press
Taylor & Francis Group
6000 Broken Sound Parkway NW, Suite 300
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© 2013 by Taylor & Francis Group, LLC

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TF1734_C00.fm Page v Tuesday, September 14, 2004 1:01 PM

Dedication

To my father, Ferdinando, and my mother, Laura,

with everlasting gratitude.
TF1734_C00.fm Page vi Tuesday, September 14, 2004 1:01 PM
TF1734_C00.fm Page vii Tuesday, September 14, 2004 1:01 PM

Preface
There is increasing interest in exhaled breath condensate, a noninvasive method to
study airway inflammation. Exhaled breath consists of a gaseous phase containing
volatile substances (e.g., nitric oxide and carbon monoxide) and a liquid phase,
termed exhaled breath condensate, that contains aerosol particles in which nonvol-
atile substances (e.g., inflammatory mediators) have been identified. Analysis of
exhaled breath condensate is potentially useful for monitoring airway inflammation
and pharmacological therapy. Identification of selective profiles of inflammatory
markers in exhaled breath condensate in different lung diseases might be relevant
to differential diagnosis. Given its noninvasiveness, this method might be suitable
for longitudinal studies in patients with lung disease, including children.
This book provides an introduction to the analysis of exhaled breath condensate.
To provide an overview of lung inflammation, basic and clinical pharmacology of
leukotrienes, prostanoids, cytokines, chemokines, and tachykinins in the respiratory
system is presented.
Current knowledge on the physicochemical properties of exhaled breath con-
densate and its formation in the airways is presented, and the methods for collection
of exhaled breath condensate are described. Particular emphasis is put on the meth-
odological issues because they are essential for future development of this technique.
The number of molecules identified in exhaled breath condensate is rapidly increasing.
Information on biomarkers and/or classes of biomarkers of airway inflammation that
have been measured in this biological fluid are presented in single chapters. One
chapter takes into consideration the potential applications of analysis of exhaled
breath condensate in children. At present, the quantification of lung inflammation
is mainly based on invasive or semi-invasive methods or on measurement of inflam-
matory biomarkers in biological fluids that are likely to reflect systemic rather than
lung inflammation. Assessment of lung inflammation is relevant for diagnosis and
management of inflammatory lung diseases because the inflammatory process often
precedes the onset of symptoms and decrease in lung function. The availability of
sensitive noninvasive biomarkers of lung inflammation might indicate the need for
beginning anti-inflammatory therapy at an earlier stage and have a significant impact
on the management of lung diseases including asthma and chronic obstructive
pulmonary disease. Measurement of inflammatory biomarkers in exhaled breath
condensate might provide more sensitive end points for clinical trials in respiratory
medicine — a uniquely valuable approach to establish the effectiveness of dose
regimens of current drugs and a rational basis for assessing new pharmacological
therapies. The potential implications of analysis of exhaled breath condensate for
diagnosis and therapy of respiratory diseases are thoroughly discussed in this book,
and indications for future research in this area are provided.
TF1734_C00.fm Page viii Tuesday, September 14, 2004 1:01 PM

Standardization and validation of analysis of exhaled breath condensate currently

are the main priority in this research area. At present, the lack of a standardized
technique makes it difficult to compare the results from different laboratories. Robust
analytical methodology usually precedes the application of a new technique. In the
case of exhaled breath condensate, the initial enthusiasm, the search for new mole-
cules in this biological fluid, and the availability of immunoassays for several
inflammatory mediators led researchers to overlook a rigorous analytical approach,
leaving open the question of the specificity and reliability of measurements. Because
several methodological issues still need to be addressed, whether and when the
analysis of exhaled breath condensate will be applicable to the clinical setting is
difficult to predict. However, considering the importance of inflammation in the
pathophysiology of lung disease, the relative lack of noninvasive methods for moni-
toring airway inflammation and therapy, and the relevance of its potential applications,
further research on exhaled breath condensate analysis is warranted. Identification
of breath “fingerprints” might open a new era in respiratory medicine. These are the
promises. Future research will attest whether they are a reality.
TF1734_C00.fm Page ix Tuesday, September 14, 2004 1:01 PM

Editor
Paolo Montuschi was born in Rome in 1964 and is Associate Professor of Phar-
macology in the Faculty of Medicine of the Catholic University of the Sacred Heart
in Rome, Italy. He is also Senior Registrar in the Clinical Pharmacology Unit of the
University Hospital Agostino Gemelli in Rome. He graduated in Medicine in 1989.
He was Fellow in Pharmacology from 1990 to 1994 when he became Assistant
Professor of Pharmacology. In 1996, he was Visiting Scientist at the Centers for
Disease Control (CDC), in Morgantown, West Virginia. From 1997 to 2000, he was
Visiting Clinical Research Fellow at Imperial College, School of Medicine, at the
National Heart and Lung Institute, Department of Thoracic Medicine, in London,
United Kingdom.
He was awarded two NATO-Italian Research Council Fellowships in 1996 and
1998, and two Italian Research Council Fellowships in 1997 and 1999. He served
as a Temporary Adviser of the World Health Organization/International Programme
on Chemical Safety, in Geneva, Switzerland.
His scientific activity is focused on basic and clinical pharmacology of the
respiratory system.
He is author or coauthor of more than 130 publications, 40 of them as full papers
in peer-reviewed journals. He serves as a reviewer of the American Journal of
Respiratory and Critical Care Medicine, Thorax, and European Respiratory Journal.
TF1734_C00.fm Page x Tuesday, September 14, 2004 1:01 PM
TF1734_C00.fm Page xi Tuesday, September 14, 2004 1:01 PM

Contributors
Peter J. Barnes Richard M. Effros
National Heart and Lung Institute Department of Medicine
Imperial College School of Medicine Harbor-UCLA Medical Center
London, England Torrance, California

Christopher A. Bates Jon L. Freels

National Jewish Medical and Research Respiratory Science Center
Center The University of Arizona Medical
Denver, Colorado Center
Tucson, Arizona
Julie Biller and
Department of Medicine Carl T. Hayden VA Medical Center
Medical College of Wisconsin Phoenix, Arizona
Milwaukee, Wisconsin Benjamin Gaston
Pediatric Respiratory Medicine
P.N. Richard Dekhuijzen University of Virginia School of
Department of Pulmonary Diseases Medicine
University Medical Centre Charlottesville, Virginia
Nijmegen, The Netherlands
Umur Hatipoǧlu
Katelijne O. De Swert Department of Medicine
Department of Respiratory Diseases University of Illinois at Chicago
Ghent University Hospital Chicago, Illinois
Ghent, Belgium
John F. Hunt
Louise E. Donnelly Department of Pediatric Respiratory
National Heart and Lung Institute Medicine
Imperial College School of Medicine University of Virginia School of
London, England Medicine
Charlottesville, Virginia
Marshall Dunning Quirijn Jöbsis
Department of Medicine Department of Pediatrics
Medical College of Wisconsin University Hospital Maastricht
Milwaukee, Wisconsin Maastricht, The Netherlands

Ryszard Dworski Guy F. Joos

Center for Lung Research Department of Respiratory Diseases
Vanderbilt University Medical Center Ghent University Hospital
Nashville, Tennessee Ghent, Belgium
TF1734_C00.fm Page xii Tuesday, September 14, 2004 1:01 PM

Sergei A. Kharitonov Philippe P.R. Rosias

Department of Thoracic Medicine Department of Pediatrics
National Heart and Lung Institute University Hospital Maastricht
Imperial College of Medicine Maastricht, The Netherlands
London, England
Israel Rubinstein
Departments of Medicine and
Paolo Montuschi
Biopharmaceutical Sciences
Department of Pharmacology
Colleges of Medicine and Pharmacy
Faculty of Medicine
University of Illinois
Catholic University of the Sacred Heart
and VA Chicago Health Care Sytem
Rome, Italy
Chicago, Illinois

Romain A. Pauwels Reza Shaker

Department of Respiratory Diseases Department of Medicine
Ghent University Hospital Medical College of Wisconsin
Ghent, Belgium Milwaukee, Wisconsin

R. Stokes Peebles, Jr. James R. Sheller

Center for Lung Research Center for Lung Research
Vanderbilt University Medical Center Vanderbilt University Medical Center
Nashville, Tennessee Nashville, Tennessee

Philip E. Silkoff
Richard A. Robbins 224 Spruce Tree Road
Respiratory Science Center Radnor, Pennsylvania
The University of Arizona Medical
Center Suzanne L. Traves
Tucson, Arizona Department of Thoracic Medicine
and National Heart and Lung Institute
Carl T. Hayden VA Medical Center Imperial College
Phoenix, Arizona London, England

Wendy J.C. van Beurden

Department of Pulmonary Diseases
Catharina Hospital Eindhoven
Eindhoven, The Netherlands
TF1734_C00.fm Page xiii Tuesday, September 14, 2004 1:01 PM

Contents
Chapter 1 Exhaled Breath Condensate: A New Approach to Monitoring
Lung Inflammation...............................................................................1
Peter J. Barnes

Chapter 2 Analysis of Exhaled Breath Condensate: Methodological Issues ....11

Paolo Montuschi

Chapter 3 Exhaled Breath Condensate: Formation and Physicochemical

Properties............................................................................................31
Richard M. Effros, Julie Biller, Marshall Dunning, and Reza Shaker

Chapter 4 Isoprostanes, Prostanoids, and Leukotrienes in Exhaled Breath

Condensate .........................................................................................53
Paolo Montuschi

Chapter 5 Hydrogen Peroxide in Exhaled Breath Condensate..........................67

Wendy J.C. van Beurden and P.N. Richard Dekhuijzen

Chapter 6 Measurement of Exhaled Breath Condensate pH: Implications

for Pathophysiology and Monitoring of Inflammatory Airway
Diseases ..............................................................................................73
Benjamin Gaston and John F. Hunt

Chapter 7 Nitric Oxide-Derived Markers in Exhaled Breath Condensate ........85

Sergei A. Kharitonov

Chapter 8 Analysis of Exhaled Breath Condensate in Children......................105

Quirijn Jöbsis and Philippe P.R. Rosias

Chapter 9 Exhaled Breath Condensate: Comparisons with Other Methods

for Assessing Lung Inflammation....................................................113
Christopher A. Bates and Philip E. Silkoff
TF1734_C00.fm Page xiv Tuesday, September 14, 2004 1:01 PM

Chapter 10 Analysis of Exhaled Breath Condensate: Potential Implications

for Diagnosis and Therapy of Lung Diseases .................................123
Umur Hatipoǧlu and Israel Rubinstein

Chapter 11 Future of Exhaled Breath Condensate.............................................139

Jon L. Freels and Richard A. Robbins

Chapter 12 The Role of Leukotrienes and Prostanoids in Airway

Inflammation ....................................................................................149
Ryszard Dworski, R. Stokes Peebles, Jr., and James R. Sheller

Chapter 13 Neurogenic Inflammation in the Airways: Role of Tachykinins ....167

Guy F. Joos, Katelijne O. De Swert, and Romain A. Pauwels

Chapter 14 Cytokines and Chemokines in Airway Inflammation .....................183

Suzanne L. Traves and Louise E. Donnelly

Index ......................................................................................................................211
TF1734_book.fm Page 1 Wednesday, August 25, 2004 4:57 PM

1 Exhaled Breath
Condensate: A New
Approach to Monitoring
Lung Inflammation
Peter J. Barnes

CONTENTS

I. Introduction ......................................................................................................1
A. Noninvasive Markers ..............................................................................2
B. Volatile Gases .........................................................................................2
II. Exhaled Breath Condensate .............................................................................2
A. Origin......................................................................................................3
B. Factors Affecting Measurements............................................................4
C. Markers of Oxidative Stress...................................................................4
D. Markers of Nitrative Stress ....................................................................5
E. Inflammatory Mediators .........................................................................5
III. Future Developments .......................................................................................6
A. Use in Clinical Practice..........................................................................6
B. Guidelines ...............................................................................................6
C. Increased Sensitivity of Assays..............................................................6
D. On-Line Measurements ..........................................................................6
E. Proteomics ..............................................................................................7
IV. Conclusions ......................................................................................................7
Further Reading .........................................................................................................7

I. INTRODUCTION
Inflammation plays a critical role in the pathophysiology of many pulmonary dis-
eases, but traditionally it has been difficult to measure this in airway and peripheral
lung disease because invasive methods, such as bronchial biopsy, lung biopsy, or
bronchoalveolar lavage, have been necessary. This has been a particular problem in
patients with severe disease and with children. The invasiveness of these measure-

0-415-32465-3/05/$0.00+$1.50
© 2005 by CRC Press LLC 1
TF1734_book.fm Page 2 Wednesday, August 25, 2004 4:57 PM

2 New Perspectives in Monitoring Lung Inflammation

ments also means that repeated measurements are not possible, so it is difficult to
follow disease progression or the response to therapy.

A. NONINVASIVE MARKERS
Traditionally, asthma control has been monitored by symptoms and lung function
measurements, particularly peak expiratory flow. Airway inflammation underlies
asthma symptoms but, as discussed above, is difficult to measure directly because
this involves invasive procedures. More recently, the less invasive procedure of
sputum induction has been introduced.1 Although this is more acceptable to patients,
some patients find it unacceptable and it is not possible to obtain adequate samples
from other patients. It also is particularly difficult to apply in young children. The
procedure of sputum induction with hypertonic saline itself induces airway inflam-
mation, so it cannot be repeated frequently.2,3
This has led to a search for less invasive ways to measure airway and lung
inflammation to aid diagnosis, to assess response to anti-inflammatory treatments,
to predict loss of disease control, and to assess the response to novel treatments.
There is increasing evidence that measurement of biomarkers in the breath may
reflect pulmonary disease. Direct sampling from the lung has major advantages
compared with sampling from the blood or urine, when dilution and metabolism of
inflammatory markers arising in the lungs make interpretation very difficult. Many
biomarkers in exhaled breath have now been explored.4 This is a rapidly advancing
field with the potential for enormous clinical impact. It provides new opportunities
to explore the underlying inflammatory process in asthma, chronic obstructive pul-
monary disease (COPD), interstitial lung disease, as well as providing a potential
means of monitoring systemic disease.

B. VOLATILE GASES
Most progress has been made with volatile gases that are detected in the gaseous
phase, including nitric oxide (NO), carbon monoxide (CO), and hydrocarbons, such
as ethane. Exhaled NO has been particularly intensively investigated and appears to
be a useful marker of asthmatic inflammation, so that it is useful for monitoring
response to corticosteroid therapy and ensuring adequate control of the underlying
inflammatory disease.5 Measurements of exhaled NO are still a research tool but are
increasingly being used in the clinic. As less expensive analyzers are developed, it
is likely that this measurement will become much more widely used; with advances
in technology, it may even be possible for patients to measure NO at home.

II. EXHALED BREATH CONDENSATE

Exhaled breath condensate (EBC) contains many biomarkers of inflammation and
oxidative stress6,7 (Table 1.1). The principle of sampling the airways by EBC is that
mediators from airways are released from the airway lining fluid, carried up by
exhaled breath, and subsequently collected by condensation of the exhalate by
TF1734_book.fm Page 3 Wednesday, August 25, 2004 4:57 PM

Exhaled Breath Condensate 3

TABLE 1.1
Inflammatory Markers in Exhaled
Breath Condensate
Markers of Oxidative and Nitrative Stress
Hydrogen peroxide
8-Isoprostane
Thiobarbituric acid
Nitrite/nitrate
3-Nitrotyrosine
S-Nitrosothiols

Inflammatory Mediators
Leukotriene B4
Cysteinyl-leukotrienes
Prostaglandins
Histamine
Adenosine
Interleukin-4
Interleukin-6
Interleukin-8
Interferon-γ
Hydrogen ions (pH)

breathing into a cooled tube. EBC collection appears to be a simple and non-invasive
method to sample the lower respiratory tract, which can be performed in young children
and severely ill patients and can be repeated several times with no adverse effects.
There are now numerous publications on EBC, but published studies have been char-
acterized by a variation in mediator levels and methods used for collection of EBC,
much of which are likely to be related to the nature of EBC formation. This has led
to the setting up of a task force by the European Respiratory Society and the American
Thoracic Society to make recommendations about how measurements of EBC should
be standardized and to facilitate research into the methodological issues surrounding
EBC collection. This has been very important in facilitating research and clinical
development in exhaled NO.8
The first studies identifying surface-active properties, including pulmonary sur-
factant, of exhaled condensate were published in Russian in the 1980s,9,10 and since
then an increasing number of inflammatory mediators, oxidants, and ions have been
identified in EBCs.

A. ORIGIN
Potentially, EBC is derived from the mouth (oral cavity and oropharynx), respiratory
tract, and alveoli, but the origin of individual biomarkers in EBC has not yet been
determined. It is assumed that airway surface liquid becomes aerosolized during
TF1734_book.fm Page 4 Wednesday, August 25, 2004 4:57 PM

4 New Perspectives in Monitoring Lung Inflammation

turbulent airflow, so that the content of the condensate reflects the composition of
airway surface liquid, although large molecules may not aerosolize as well as small
soluble molecules. It is possible that some lipid mediators may be volatile at body
temperature and dissolve in the condensate during collection. Much more research
is needed to establish the origin of biomarkers in EBC and to compare its compo-
sition with airway lining fluid.

B. FACTORS AFFECTING MEASUREMENTS

Several methods of condensate collection have been described. The most common
approach is to ask the subject to breathe tidally via a mouthpiece through a non-
rebreathing valve in which inspiratory and expiratory air is separated. During expi-
ration, the exhaled air flows through a condenser, which is cooled to 0°C by melting
ice or to –20°C by a refrigerated circuit, and breath condensate is then collected
into a cooled collection vessel. A low temperature may be important for preserving
labile markers such as lipid mediators during the collection period, which usually
takes between 10 and 15 min to obtain 1 to 3 ml of condensate. Exhaled condensate
may be stored at –70°C and is subsequently analyzed by gas chromatography and/or
extraction spectrophotometry, or by immunoassays (enzyme-linked immunosorbent
assay [ELISA]).
Salivary contamination may influence the levels of several markers detectable
in EBC. Thus, high concentrations of some eicosanoids (thromboxane B2, leukot-
riene [LTB4], prostaglandin [PGF2α]), but low levels of prostaglandin E2 (PGE2) and
prostacyclin have been found in saliva, so it is important to minimize and monitor
salivary contamination. Subjects should rinse their mouth before collection and keep
the mouth dry by periodically swallowing their saliva. Salivary contamination, mea-
sured by amylase concentration of condensate, should be monitored routinely. In
most of the studies reported, amylase has been measured in condensate and no
salivary contamination has been detected.11,12 The quantity of exhaled condensate is
dependent on the ventilation volume per unit time (minute volume), but this does
not affect the concentration of mediators.13,14
The major component of EBC is condensed water vapor and this may lead to
variable dilution of EBC constituents. This is likely to be less of a problem with volatile
mediators, but a greater problem with soluble mediators, such as hydrogen peroxide
and proteins that are present in airway lining fluid. This is one of the factors contrib-
uting to the variability in measurements and a dilution marker is needed to correct
for this variable dilution. Cation concentrations, urea, and conductivity (after removal
of ammonium ions) have been suggested as a way of correcting for this variable water
vapor dilution,15 but more studies are needed to test the validity of this approach.

C. MARKERS OF OXIDATIVE STRESS

Exhaled hydrogen peroxide (H2O2) levels are increased in adults and children with
asthma, particularly those with severe disease,16–19 and are correlated with airway
hyperresponsiveness.20 The concentrations of H2O2 in exhaled condensate are
TF1734_book.fm Page 5 Wednesday, August 25, 2004 4:57 PM

Exhaled Breath Condensate 5

reduced in asthmatic patients after treatment with inhaled corticosteroids.21 The

concentration of H2O2 is also increased in patients with COPD22 and bronchiectasis.11
However, this marker is relatively unstable and there is a large diurnal variability.23
8-Isoprostane is a more stable marker of oxidative stress that is formed nonen-
zymatically by oxidation of arachidonic acid. Levels of 8-isoprostane are increased
in exhaled condensate of asthmatic patients and are correlated with disease severity.24
8-Isoprostane is also increased in patients with COPD and cystic fibrosis.13,25 8-Iso-
prostane may be well suited to assessment of oxidative stress in asthma and could
be used to assess the effects of antioxidant therapies.

D. MARKERS OF NITRATIVE STRESS

Elevated levels of nitrite and nitrate are detectable in exhaled breath of patients with
asthma and COPD and in normal smokers.26–28 Nitrosothiols, formed from an inter-
action of NO with low-molecular-weight thiols such as glutathione or with thiol
groups on cysteine residues of proteins, are also elevated in asthma.28 S-Nitrosothiols
have bronchodilator effects, and deficiency of S-nitrosothiol concentrations in the
airways have been linked causally to asthmatic respiratory failure in children.29 The
interaction of NO with superoxide anions formed from oxidative stress results in
the formation of peroxynitrite, which is unstable but interacts with tyrosine residues
in proteins to form stable 3-nitrotryosine. 3-Nitrityrosine levels are increased in
exhaled breath of patients with asthma and cystic fibrosis.30,31 It is likely that
increased 3-nitrotyrosine levels in the breath might occur during exacerbations of
asthma when oxidative stress increases.

E. INFLAMMATORY MEDIATORS
Several mediators of inflammation have been detected in the breath of patients with
inflammatory airway diseases. There is an increase in the levels of LTB4 and cys-
teinyl-leukotrienes (cys-LTs) in asthmatic patients.31–34 LTB4 concentrations in EBC
appear to be relatively reproducible and are measurable even in normal subjects,
making changes with disease easier to interpret.35 In COPD patients there is a greater
increase of exhaled LTB4 than seen in asthma, but no increase in cys-LTs (as in
asthma) and an increase in exhaled PGE2.36 There is an additional increase in exhaled
LTB4 concentrations during exacerbations.37 Several other mediators, including ATP
and adenosine, also might be detectable in the breath.6 Even cytokines might be
detectable in EBC, and in children with asthma there is an increase in the concen-
trations of interleukin (IL)-4 and a decrease in interferon-γ compared with normal
children38 and an increase in IL-6 concentrations in smokers.39 There is also an
increase in the peptide endothelin-1 and vitronectin in patients with interstitial lung
disease.40 However, the variability of cytokine measurements is high, presumably
because these proteins are carried in the droplets. Many cytokines, including IL-8,
IL-13, and tumor necrosis factor α, are not detectable in EBC, although they are
readily detectable in sputum. Acidification of EBC (low pH) also might indicate acute
inflammation in patients with asthma41 and COPD.42
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6 New Perspectives in Monitoring Lung Inflammation

III. FUTURE DEVELOPMENTS

A. USE IN CLINICAL PRACTICE
Exhaled breath condensate is a simple, entirely noninvasive procedure that can be
used in patients with severe disease and in children older than 4 years. The apparatus
is relatively simple to use and operate and is inexpensive, making it possible to
conduct studies in a general practice setting that might be particularly appropriate
for common lung diseases, such as asthma and COPD. In a recent study, we measured
EBC of COPD patients during and following an exacerbation, demonstrating an
increase in exhaled 8-isoprostane and LTB4, presumably reflecting the increase in
oxidative stress and neutrophilic inflammation.37 Interestingly, we demonstrated a
very slow recovery to baseline values, indicating that the increase in inflammation
associated with an acute exacerbation is very prolonged in COPD. Measurements
can be used repeatedly so that the kinetics of drugs can be investigated. The mea-
surement of specific mediators and biomarkers means that it might be used to explore
the effects of specific inhibitors, as well as anti-inflammatory effects. For example,
measurement of LTB4 or cys-LTs can be used to study the efficacy of a 5-lipoxygenase
inhibitor, or exhaled 8-isoprostane can be used to study the effect of an antioxidant.

B. GUIDELINES
It is clear that EBC contains many potential biomarkers. It is important to optimize
their measurement and study the clinical value of monitoring biomarkers in the
breath in a variety of lung diseases and to establish the reproducibility of these
measurements. This is a complex task — each biomarker needs to be considered
differently because of differing solubility, stability, volatility, and amount.

C. INCREASED SENSITIVITY OF ASSAYS

One of the current limitations of EBC measurements is the low concentration of
many biomarkers so that their measurement is limited by the sensitivity of assays.
It is likely that more sensitive assays will be developed as more potent antibodies are
developed and new molecular detection techniques are introduced. Metabonomics is
a recent technique that might be particularly applicable to EBC analysis. Metabo-
nomics involves the detection of hundreds of thousands of metabolites in a biological
fluid, usually using high-resolution nuclear magnetic resonance (NMR) spectrometry
or liquid chromatography/mass spectrometry. Powerful pattern recognition computer
programs recognize patterns of metabolites that are sensitive to disease, effects of
treatment, and disease severity. Metabonomics of EBC (a breathogram) might there-
fore prove to be useful in screening lung diseases, following disease progression,
predicting responses to treatment, and monitoring response to therapy.43,44

D. ON-LINE MEASUREMENTS
A relative disadvantage of EBC measurements is that they require a subsequent
analysis; it is likely that there will be important advances in on-line detection of
TF1734_book.fm Page 7 Wednesday, August 25, 2004 4:57 PM

Exhaled Breath Condensate 7

particular biomarkers using sensitive biosensors. For example, it is possible to detect

H2O2 on-line (in real time) using a silver electrode or by coating a platinum electrode
or polymer with horseradish peroxidase.45,46 Similar enzyme detector systems also
might be developed for real-time monitoring of various lipid mediators, including
8-isoprostane, prostaglandins, and leukotrienes. It is relatively easy to monitor the
pH of EBC; this is readily amenable to real-time detection. Several molecular
biosensors are in development and have the potential to detect very low concentra-
tions of various relevant biomarkers.47 Ultimately, it might be desirable to collect
EBC to monitor patients in clinical practice using disposable detector sticks.

E. PROTEOMICS
Proteomics, which applies high-resolution gel electrophoresis or mass spectrometry
to detect multiple proteins in biological samples, might also be a useful approach
to analyze the proteins in EBC. This might reveal disease-specific patterns and lead
to the identification of novel proteins for detection of disease and identification of
new therapeutic targets.48 However, several technical problems have to be overcome
before this becomes a useful approach.

IV. CONCLUSIONS
EBC is an exciting new approach to monitoring inflammatory lung diseases. The
technique might have great potential in the future. Far more information is needed
about the technical aspects of measurement, and we need to understand better the
factors that affect the measurements so that reliable and reproducible measurements
can be made. Because the technique is relatively inexpensive, it might be useful in
large clinical studies and in clinical practice. This could require the development of
on-line measurements. In the future, it might be possible to detect multiple bio-
markers in EBC to aid diagnosis, to predict the most effective therapies, and to
monitor the response to treatment in a variety of pulmonary diseases.

FURTHER READING
1. Parameswaran, K. et al., Clinical judgement of airway inflammation versus sputum
cell counts in patients with asthma, Eur. Respir. J., 15, 486, 2000.
2. Magnussen, H. and Holz, O., Monitoring airway inflammation in asthma by induced
sputum, Eur. Respir. J., 13, 5, 1999.
3. Nightingale, J.A., Rogers, D.F., and Barnes, P.J., Effect of repeated sputum induction
on cell counts in normal volunteers, Thorax, 53, 87, 1998.
4. Kharitonov, S.A. and Barnes, P.J., Exhaled markers of pulmonary disease, Am. J.
Respir. Crit. Care Med., 163, 1693, 2001.
5. Kharitonov, S.A. and Barnes, P.J., Clinical aspects of exhaled nitric oxide, Eur.
Respir. J., 16, 781, 2000.
6. Montuschi, P. and Barnes, P.J., Analysis of exhaled breath condensate for monitoring
airway inflammation, Trends Pharmacol. Sci., 23, 232, 2002.
7. Mutlu, G.M. et al., Collection and analysis of exhaled breath condensate in humans,
Am. J. Respir. Crit. Care Med., 164, 731, 2001.
TF1734_book.fm Page 8 Wednesday, August 25, 2004 4:57 PM

8 New Perspectives in Monitoring Lung Inflammation

8. Kharitonov, S.A., Alving, K., and Barnes, P.J., Exhaled and nasal nitric oxide mea-
surement: recommendations, Eur. Respir. J., 10, 1683, 1997.
9. Kurik, M.V. et al., Physical properties of a condensate of exhaled air in chronic
bronchitis patients, Vrach. Delo, 37, 1987.
10. Sidorenko, G.I., Zborovskii, E.I., and Levina, D.I., Surface-active properties of the
exhaled air condensate (a new method of studying lung function), Ter. Arkh., 52, 65,
1980.
11. Loukides, S. et al., Elevated levels of expired breath hydrogen peroxide in bron-
chiectasis, Am. J. Respir. Crit. Care Med., 158, 991, 1998.
12. Scheideler, L. et al., Detection of nonvolatile macromolecules in breath, Am. Rev.
Respir. Dis., 148, 774, 1993.
13. Montuschi, P. et al., Exhaled 8-isoprostane as a new non-invasive biomarker of
oxidative stress in cystic fibrosis, Thorax, 55, 205, 2000.
14. Reinhold, P. et al., Breath condensate — a medium obtained by a noninvasive method
for the detection of inflammation mediators of the lung, Berl Munch. Tierarztl.
Wochenschr., 112, 254, 1999.
15. Effros, R.M. et al., A simple method for estimating respiratory solute dilution in
exhaled breath condensates, Am. J. Respir. Crit. Care Med., 2003 (in press).
16. Antczak, A. et al., Increased hydrogen peroxide and thiobarbituric acid-reactive
products in expired breath condensate of asthmatic patients, Eur. Respir. J., 10, 1235,
1997.
17. Dohlman, A.W., Black, H.R., and Royall, J.A., Expired breath hydrogen peroxide is
a marker of acute airway inflammation in pediatric patients with asthma, Am. Rev.
Respir. Dis., 148, 955, 1993.
18. Horváth, I. et al., Combined use of exhaled hydrogen peroxide and nitric oxide in
monitoring asthma, Am. J. Respir. Crit. Care Med., 158, 1042, 1998.
19. Jöbsis, Q. et al., Hydrogen peroxide in exhaled air is increased in stable asthmatic
children, Eur. Respir. J., 10, 519, 1997.
20. Emelyanov, A. et al., Elevated concentrations of exhaled hydrogen peroxide in asth-
matic patients, Chest, 120, 1136, 2001.
21. Antczak, A. et al., Inhaled glucocorticosteroids decrease hydrogen peroxide level in
expired air condensate in asthmatic patients, Respir. Med., 94, 416, 2001.
22. Dekhuijzen, P.N.R. et al., Increased exhalation of hydrogen peroxide in patients with
stable and unstable chronic obstructive pulmonary disease, Am. J. Respir. Crit. Care
Med., 154, 813, 1996.
23. van Beurden, W.J.C. et al., Variability of exhaled hydrogen peroxide in stable COPD
patients and matched healthy controls, Respiration, 69, 211, 2002.
24. Montuschi, P. et al., Increased 8-isoprostane, a marker of oxidative stress, in exhaled
condensate of asthma patients, Am. J. Respir. Crit. Care Med., 160, 216, 1999.
25. Montuschi, P. et al., Exhaled 8-isoprostane as an in vivo biomarker of lung oxidative
stress in patients with COPD and healthy smokers, Am. J. Respir. Crit. Care Med.,
162, 1175, 2000.
26. Balint, B. et al., Increased nitric oxide metabolites in exhaled breath condensate after
exposure to tobacco smoke, Thorax, 56, 456, 2001.
27. Ganas, K. et al., Total nitrites/nitrate in expired breath condensate of patients with
asthma, Respir. Med., 95, 649, 2001.
28. Corradi, M. et al., Increased nitrosothiols in exhaled breath condensate in inflamma-
tory airway diseases, Am. J. Respir. Crit. Care Med., 163, 854, 2001.
29. Gaston, B. et al. Bronchodilator S-nitrosothiol deficiency in asthmatic respiratory
failure, Lancet, 351, 1317, 1998.
TF1734_book.fm Page 9 Wednesday, August 25, 2004 4:57 PM

Exhaled Breath Condensate 9

30. Balint, B. et al., Increased nitrotyrosine in exhaled breath condensate in cystic fibrosis,
Eur. Respir. J., 17, 1201, 2001.
31. Hanazawa, T., Kharitonov, S.A., and Barnes, P.J., Increased nitrotyrosine in exhaled
breath condensate of patients with asthma, Am. J. Respir. Crit. Care Med., 162, 1273,
2000.
32. Antczak, A. et al., Increased exhaled cysteinyl-leukotrienes and 8-isoprostane in
aspirin-induced asthma, Am. J. Respir. Crit. Care Med., 166, 301, 2002.
33. Csoma, Z. et al., Increased leukotrienes in exhaled breath condensate in childhood
asthma, Am. J. Respir. Crit. Care Med., 166, 1345, 2002.
34. Montuschi, P. and Barnes, P.J., Exhaled leukotrienes and prostaglandins in asthma,
J. Allergy Clin. Immunol., 109, 615, 2002.
35. Montuschi, P. et al., Validation of leukotriene B4 measurements in exhaled breath
condensate, Inflamm. Res., 52, 69, 2003.
36. Montuschi, P. et al., Exhaled leukotrienes and prostaglandins in COPD. Thorax, 58,
585, 2003.
37. Biernacki, W.A., Kharitonov, S.A., and Barnes, P.J., Increased leukotriene B4 and
8-isoprostane in exhaled breath condensate of patients with exacerbations of COPD,
Thorax, 58, 294, 2003.
38. Shahid, S.K. et al., Increased interleukin-4 and decreased interferon- in exhaled breath
condensate of children with asthma, Am. J. Respir. Crit. Care Med., 165, 1290, 2002.
39. Carpagnano, G.E. et al., Increased inflammatory markers in the exhaled breath con-
densate of cigarette smokers, Chest, 124, 1386, 2003.
40. Carpagnano, G.E. et al., Increased vitronectin and endothelin-1 in the breath conden-
sate of patients with fibrosing lung disease, Respiration, 70, 154, 2003.
41. Hunt, J.F. et al., Endogenous airway acidification. Implications for asthma patho-
physiology, Am. J. Respir. Crit. Care Med., 161, 694, 2000.
42. Kostikas, K. et al., pH in expired breath condensate of patients with inflammatory
airway diseases, Am. J. Respir. Crit. Care Med., 165, 1364, 2002.
43. Brindle, J.T. et al., Rapid and noninvasive diagnosis of the presence and severity of
coronary heart disease using 1H-NMR-based metabonomics, Nat. Med, 8, 1439, 2002.
44. Nicholson, J.K. et al., Metabonomics: a platform for studying drug toxicity and gene
function, Nat. Rev. Drug Discov., 1, 153, 2002.
45. Razola, S.S. et al., Hydrogen peroxide sensitive amperometric biosensor based on
horseradish peroxidase entrapped in a polypyrrole electrode, Biosens. Bioelectron.,
17, 921, 2002.
46. Thanachasai, S. et al., Novel hydrogen peroxide sensors based on peroxidase-carrying
poly[pyrrole-co-[4-(3-pyrrolyl)butanesulfonate]] copolymer films, Anal. Sci., 18, 773,
2002.
47. Nakamura, H. and Karube, I., Current research activity in biosensors, Anal. Bioanal.
Chem. 377, 446, 2003.
48. Zhu, H., Bilgin, M., and Snyder, M., Proteomics, Annu. Rev. Biochem., 72, 783, 2003.
TF1734_book.fm Page 10 Wednesday, August 25, 2004 4:57 PM
TF1734_book.fm Page 11 Wednesday, August 25, 2004 4:57 PM

2 Analysis of Exhaled
Breath Condensate:
Methodological Issues
Paolo Montuschi

CONTENTS

I. Introduction ....................................................................................................11
II. Experimental Setup ........................................................................................12
III. What Is Measured ..........................................................................................16
IV. Methodological Issues....................................................................................19
A. Flow Dependence and Influence of Ventilation Patterns.....................19
B. Origin(s) of Exhaled Markers ..............................................................20
C. Organ Specificity of Exhaled Breath Condensate ...............................22
D. Dilution Reference Indicators ..............................................................22
E. Nasal Contamination ............................................................................22
F. Saliva Contamination ...........................................................................23
G. Influence of Different Factors on Exhaled Markers ............................24
H. Diurnal and Day-to-Day Variability of Exhaled Markers ...................24
I. Sample Handling ..................................................................................25
J. Validation of Analytical Methods ........................................................25
V. Advantages and Limitations ..........................................................................26
VI. Conclusions and Future Directions ...............................................................26
Acknowledgment .....................................................................................................27
Further Reading .......................................................................................................27

I. INTRODUCTION
There has been growing interest in the identification of biomarkers for inflammatory
airway diseases. Ideally, these biomarkers should (1) identify those patients who are
more susceptible to the disease; (2) reflect the degree of pulmonary inflammation
and the disease severity; (3) be reproducible in stable clinical conditions; (4) be
noninvasive (i.e., easy to obtain for repeated measurements in the longitudinal
follow-up of the patients); (5) be elevated during exacerbations; (6) be useful for
monitoring pharmacological therapy; and (7) be of prognostic value.1 Inflammation
has an important pathophysiological role in lung diseases such as asthma and chronic

0-415-32465-3/05/$0.00+$1.50
© 2005 by CRC Press LLC 11
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12 New Perspectives in Monitoring Lung Inflammation

obstructive pulmonary disease (COPD).2–5 The assessment of lung inflammation is

relevant to the management of respiratory diseases because it may indicate that
pharmacological intervention is required in an early stage in the disease process.
Moreover, monitoring airway inflammation might be useful in the follow-up of
patients with lung disease, and for guiding drug treatment. At present, quantification
of inflammation in the lungs is based on invasive methods such as the analysis of
bronchoalveolar lavage (BAL) fluid and bronchial biopsies,6 semi-invasive methods
such as sputum induction,7 and the measurement of inflammatory markers in plasma
and urine, which are likely to reflect systemic rather than lung inflammation. Exhaled
breath consists of a gaseous phase that contains volatile substances (e.g., nitric oxide,
carbon monoxide, and hydrocarbons) and a liquid phase, termed exhaled breath
condensate (EBC), that contains aerosol particles in which nonvolatile substances
have been identified.8 Since its discovery in the exhaled air of humans in the early
1990s, measurement of exhaled nitric oxide (NO) has become a widely used method
for monitoring airway inflammation in patients with asthma who are not being treated
with corticosteroids.9
Recently, attention has focused on analyzing EBC as a noninvasive method for
studying the composition of the fluid that lines the respiratory tract.10,11 Using urea
(a freely diffusible molecule) as a marker, it has been shown that a measurable
fraction (1 in 24 parts) of the EBC in healthy subjects is derived from aerosolized
airway lining fluid.12 EBC analysis of inflammatory markers is a noninvasive method
that has the potential to be useful for monitoring lung inflammation in patients with
respiratory diseases, including children.10,11 Given that it is completely noninvasive,
EBC also is suitable for longitudinal studies and for monitoring the efficacy of
pharmacological therapy. Moreover, different biomarkers might reflect different
aspects of lung inflammation or oxidative stress, which is an important component
of inflammation. Identification of selective profiles of biomarkers in different inflam-
matory airway diseases might be relevant to differential diagnosis in respiratory
medicine. However, several methodological issues, such as standardization of sample
collection and validation of analytical methods, need to be addressed before this
approach can be applicable to the clinical setting. This chapter describes the exper-
imental setup for EBC sample collection, discusses methodological issues, presents
advantages and limitations of EBC analysis, and proposes future directions of this
promising method.

II. EXPERIMENTAL SETUP

The collection of EBC samples is easy to perform. In most studies, the equipment
is homemade and generally consists of a mouthpiece with a one-way valve connected
to a collecting system that is placed in either ice or liquid nitrogen to cool the breath.8
The collecting system is a condensing chamber with a double wall of glass, the inner
wall of which is cooled by ice (Figure 2.1)13; alternatively, jacketed cooling pipes
or tubes in buckets of ice have been used.8 Generally, subjects wear a noseclip and
are asked to breathe tidally for 15 min through a mouthpiece connected to the
condenser. Exhaled air enters and leaves the chamber through one-way valves at the
inlet and outlet while the chamber is kept closed. If the condenser consists of two
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Analysis of Exhaled Breath Condensate: Methodological Issues 13

FIGURE 2.1 Schematic representation of EBC collecting systems. (A) Homemade EBC
collecting system, which consists of a glass condensing chamber that contains a double wall
of glass for which the inner side of the glass is cooled by ice. EBC is collected between the
two glass surfaces and drops to the bottom of the outer glass container in a liquid form.
(A) Schematic representation of a commercially available condenser (EcoScreen). Frozen
EBC is collected in the collecting vial, as indicated by the arrow. (Modified from Montuschi,
P., Nature Reviews Drug Discovery, Vol. 1, Indirect Monitoring of Lung Inflammation, Mac-
millan Magazines, 2002, pp. 238–242. With permission.)

glass containers, EBC is collected between the two glass surfaces and drops at the
bottom of the outer glass container in a liquid form.13 Usually, 1.0 to 2.5 ml of EBC
is collected depending on respiratory parameters (minute ventilation, respiratory
rate, tidal volume), condenser material and temperature, and turbulence of airflow.
Commercially manufactured condensing chambers are also available.14,15 The
EcoScreen® (Jaeger Toennies, Hoechberg, Germany) is an electric refrigerated sys-
tem consisting of a mouthpiece with a one-way valve and a collecting system
connected to a power supply by an extendable arm (Figure 2.1). Subjects sitting
upright on a chair breathe through the mouthpiece that is connected to a valve block
in which inspiratory and expiratory air are separated. The valve block, in turn, is
connected to the collecting system (lamellar condenser and sample collection vial)
that is inserted into a cooling cuff maintained at a cold temperature by a refrigerator.
According to the information provided by the manufacturers, the temperature inside
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14 New Perspectives in Monitoring Lung Inflammation

FIGURE 2.2 The RTube is a portable device that consists of a disposable polypropylene
condensation chamber with an exhalation valve that also serves as a syringe-style plunger to
pull fluid off the condenser walls. Cooling is achieved by placing an aluminum cooling sleeve
over the disposable polypropylene tube.

the cooling cuff is maintained at –10˚C. During expiration, the air flows through the
lamellar condenser, condenses on the inner wall of the lamellar condenser, and drops
into the vial. Currently, there is no evidence of advantages of this condenser over
homemade devices, except for the possible immediate freezing of the samples, which
can be important for chemically unstable compounds, such as leukotriene (LT) E4.
However, because the temperature inside the cooling cuff is probably higher than
–10˚C, is not maintained at –10˚C throughout the test, and/or other possible technical
problems, samples usually are collected in a liquid or in a mixed liquid/frozen form.
Inconsistencies in collection of samples (liquid, frozen, or liquid/frozen) may affect
the concentrations of labile compounds in EBC and explain part of the variability
in their concentrations reported by different studies. In any case, even if the sample
is collected frozen, when measuring more than one marker, the sample must be
thawed at the time of collection to make sufficient aliquots. The high cost of this
commercial condenser also should be taken into account when considering a large-
scale application of this technique.
The RTube (Respiratory Research, Inc., Charlottesville, VA), another commer-
cially available condenser, is a portable device that consists of a disposable polypropy-
lene condensation chamber with an exhalation valve that also serves as a syringe-style
plunger to pull fluid off the condenser walls (Figure 2.2).16 Cooling is achieved by
placing an aluminum cooling sleeve over the disposable polypropylene tube (Figure
2.2). The temperature of the cooling sleeve can be chosen by the investigator. The
device inherently prevents salivary contamination, and no detectable concentrations
of amylase in EBC have been reported.16 An attachable microbial filter (0.3 µm) is
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Analysis of Exhaled Breath Condensate: Methodological Issues 15

provided to prevent transmission of infectious particles. The RTube is particularly

suitable for pH measurement in EBC samples, which requires deaeration for removal
of carbon dioxide.16 A separate device, the pHTube (Respiratory Research, Inc.),
can be used for this purpose.16 The RTube has three advantages: (1) it is portable,
allowing for collection of EBC samples at home, which is particularly suitable for
longitudinal studies or when collection of several samples a day is required; (2) the
EBC sample contained in the polypropylene tube can be stored in the freezer at
home; and (3) a sample of EBC for pH measurement can be collected in as little as
1 min.16 However, EBC samples need to be brought to the laboratory for biochemical
assays, and storage conditions in freezers at home should be as similar as possible
to those in the laboratory, particularly when chemically unstable mediators are to
be measured.
Careful sterilization of the EBC equipment is essential for avoiding cross-
contamination. However, collection of EBC from patients with severe infections
(e.g., methicillin-resistant Staphylococcus aureus and Burkholderia cepacia) should
be avoided. Although standardized procedures for sterilization of condensers are not
available, it is reasonable to leave the EBC collecting system in an antibacterial
solution (e.g., 1% aqueous solution of sodium hypochlorite) for at least 1 h. This
procedure limits the number of samples that can be collected, unless several devices
are available. This is particularly relevant when very expensive parts of the condenser
need to be sterilized, such as the valve block in the EcoScreen condenser. A possible
approach to overcome this problem is the use of completely disposable material
such as with the RTube or some homemade condensers. After sterilization, the EBC
collecting system needs to be washed thoroughly with water to remove completely
the antiseptic solution and dried accurately to avoid dilution of EBC. Some inves-
tigators use Teflon (E.I. du Pont de Nemours & Company, Inc., Wilmington, DE)
tubes or Teflon-coated lamellar condensers as in the EcoScreen to avoid adhesion
of biomolecules to the EBC collecting system walls and, therefore, an artifactual
decrease in their concentrations in the EBC samples. A condenser with borosilicate
glass coating may be superior to silicone, aluminium, polypropylene, and Teflon for
detection of albumin in EBC.17 Moreover, the physicochemical effects of the steril-
ization procedures on the EBC collecting system materials are not currently known
and studies to clarify this important issue are required. It needs to be clarified whether
the sterilizing solutions interact and damage the coating material of the collecting
devices. Technical improvements in the design of new condensers would allow
measurement of flow rate, total exhaled volume, tidal volume, respiratory rate, and
minute ventilation. A device that can be applied to the condenser for measuring
respiratory parameters during the collection of EBC has recently been manufactured
by Jaeger Toennies. Alternatively, the EcoScreen can be connected to a pneumo-
tachograph and a computer for online recording of respiratory parameters. Condensers
should also be designed to collect simultaneously several frozen aliquots that will
allow different markers to be measured without thawing the whole sample. The
possibility of using selective sensors to make on-line measurements of hydrogen
peroxide18 and possibly other specific inflammatory mediators in the breath is cur-
rently under investigation, but, because of the expected high cost, it is unlikely that
this method will be used routinely.
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16 New Perspectives in Monitoring Lung Inflammation

III. WHAT IS MEASURED

Several biomolecules, including markers of inflammation, have been measured in
the EBC of healthy subjects, and of patients with different inflammatory lung
diseases (Table 2.1 and Figure 2.3). In most studies, exhaled markers were measured
by immunoassays that still need to be validated with more specific analytical methods,
such as gas chromatography/mass spectrometry (GC/MS), liquid chromatography/
mass spectrometry (LC/MS), or high-performance liquid chromatography (HPLC).
These methods also will provide a more accurate analytical determination of the
concentrations of the different biomarkers in EBC. The presence of 8-isoprostane
and prostaglandin E2 (PGE2) in EBC has been confirmed by GC/MS.19 Malondial-
dehyde and other aldehydes in EBC have been measured by LC/MS,20 whereas
adenosine21 and reduced glutathione22 in EBC can be detected by HPLC. pH values
in EBC from patients with acute asthma are more than 2 log orders lower than
normal and normalize with corticosteroid therapy.23 Measurement of EBC pH is
highly reproducible and could prove to be useful clinically for diagnosis and for
monitoring therapy.24 pH values in EBC from patients with other lung diseases have
been reported,23,25 but the biological significance of these findings is currently
unknown.
A wide interindividual variability in the total protein content in EBC (from
undetectable to 1.4 mg) has been reported.26 The reasons for these findings are not
known, and efforts should be made to establish the protein concentrations in EBC
under standard conditions. In one study, protein concentrations in EBC in 20 healthy
subjects averaged 2.3 mg/dl. Amylase concentrations were undetectable in the EBC
of five subjects and very low (mean concentration of 4.67 units/ml, or less than
0.01% of that in saliva) in the EBC of the others, indicating little salivary contam-
ination of EBC.27 Proteins in nasal (collected through a “free touch” technique by
negative pressure) and oral EBC were separated by two-dimensional electrophoresis,
but they were not identified.28
Using immunoassays, cytokines such as inteleukin-1β, tumor necrosis factor-α,
interleukin-8, interleukin-4, and interferon-γ26,29,30 have been measured in EBC in
healthy subjects and in patients with different inflammatory airway diseases. How-
ever, details on the specificity of the immunoassays that were used in these studies
are not available and the measurements still need to be validated by more specific
analytical methods. Moreover, concentrations of cytokines in EBC reported in these
studies were close to the detection limit of the assay, casting doubts on the reliability
of these findings. However, because of its biological importance, the issue of the
presence of cytokines in EBC deserves further investigation. One study reported
the presence of DNA in EBC in healthy subjects and patients with lung cancer, and
proposed the possibility of amplifying DNA by the polymerase chain reaction
(PCR).31 Recently, Vogelberg et al.32 failed to detect Pseudomonas aeruginosa and
B. cepacia by PCR in the EBC of patients with cystic fibrosis. Whether gene
expression analysis in EBC will have diagnostic relevance is unknown.
Comparisons between absolute concentrations of exhaled markers that have been
reported by different studies is difficult at present because of (1) differences in the
EBC collection procedures and sample handling; (2) differences in the analytical
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Analysis of Exhaled Breath Condensate: Methodological Issues 17

TABLE 2.1
Markers of Inflammation in EBC
Marker Analytical Method
Hydrogen ions pH meter or pH microelectrode23,24,39
Electrolytes (sodium, potassium, calcium, Ion-selective electrodes,27 ion chromatography33
magnesium, chloride)

Isoprostanes
8-Isoprostane (8-iso-PGF2α) GC/MS,19 EIA,13,15,55–57 RIA35,51–53

Prostanoids
PGE2 GC/MS,19 EIA,36,60 RIA35,51–53
PGF2α EIA36
PGD2 EIA36
Thromboxane B2 EIA,36 RIA21,52,61

Leukotrienes
LTB4 EIA14,36,55–59,62,63
LTE4 EIA36,63
Cysteinyl-LTs (LTC4/LTD4/LTE4) EIA14,55,56,62

NO-Derived Products
S-Nitrosothiols Spectrophotometry,50 fluorometric assay64
3-Nitrotyrosine EIA14,65
Nitrite Spectrophotometry,29,47,50,66 fluorometric assay64,65
Nitrate Fluorometric assay64,65

Others
Hydrogen peroxide Spectrophotometry,49,67,68 fluorometric assay,34,48,69
chemiluminescence70
Adenosine HPLC21
Glutathione HPLC,22 enzymatic recycling assay71
Aldehydes (e.g., malondialdehyde) GC/MS20,22
TBARS 49

Cytokines
IL-1β EIA26
TNF-α RIA26
IL-4 ELISA30
IL-8 ELISA29
Interferon-γ EIA30
DNA PCR31

Abbreviation: EIA, enzyme immunoassay; ELISA, enzyme-linked immunosorbent assay;

TBARS, thiobarbituric acid reactive substances.

Source: Montuschi, P., Nature Reviews Drug Discovery, Vol. 1, Indirect Monitoring of Lung
Inflammation, Macmillan Magazines, 2002, pp. 238–242. With permission.
TF1734_book.fm Page 18 Wednesday, August 25, 2004 4:57 PM

18 New Perspectives in Monitoring Lung Inflammation

L-Arginine
Citrulline
Thiols
NOS RS-NO

GSH
NADPH NO–3
NO
oxidase CI-
O2 Na+
O–2
NO–2
SOD K+
Mg++
NO2 ONOO–
H2O2 Ca++
Tyr
MPO
H+
NO2 NO–2 3-NT
Polyunsaturated fatty acids

ROS 8-IP
ROS adenosine
Arachidonic
Aldehydes acid
TBARS

5-LO COX
TX
synthase
5-HETE 5-HPETE PGG2/PGH2 TXA2
LTA Hydrolysis
synthase Isomerases
LTA4 LTC4 TXB2
LTA4 synthase
hydrolase
PGE2 PGF2α PGD2
LTC4
LTB4 γ-GTP

LTD4
Dipeptidase

LTE4

FIGURE 2.3 Biomarkers of airway inflammation and/or oxidative stress that have been measured in EBC in healthy
subjects and in patients with airway inflammatory diseases. NO is derived from the amino acid L-arginine by the enzyme
nitric oxide synthase NOS. Two isoforms of NOS, constitutive and inducible NOS, have been identified in different cell
types within the respiratory tract, including airway and alveolar epithelial cells, macrophages, neutrophils, eosinophils,
mast cells, and vascular endothelial and smooth muscle cells. NO in exhaled air is derived mainly from the airways.
When free radical production is increased, NO can combine with superoxide anion (O2–) to form highly reactive
peroxynitrite (ONOO–). This, in turn, can result in nitrosation of either tyrosine or tyrosine residues in proteins to form
3-nitrotyrosine (3-NT). NO can also combine with thiols, such as glutathione or thiol residues in albumin, to produce
RS-NO or be metabolized to nitrite (NO2–) and nitrate (NO3–), which are end-products of NO metabolism. O2– can be
converted to hydrogen peroxide (H2O2) by superoxide dismutase (SOD). 8-Isoprostane (8-IP) is formed by reactive
oxygen species (ROS)-catalyzed peroxidation of arachidonic acid. LTs such as LTB4 and cysteinyl-leukotrienes (LTC4,
LTD4, and LTE4) are products of 5-lipoxygenase (5-LO) activity. Prostaglandins (PGE2, PGD2, and PGF2α) and throm-
boxane A2 (TXA2) are formed by cyclooxygenase (COX). Two COX isoforms (not shown) are known. The pathway
leading to PGI2 synthesis is not shown. PGD2, PGF2α, and TXA2, the stable hydrolysis product of TXB2, are only
detectable in some healthy subjects and/or patients with asthma and COPD. EBC from patients with acute asthma has
a pH over 2 log orders lower than normal. Electrolytes, adenosine, glutathione (GSH), aldehydes, and cytokines (not
shown) have been detected in EBC. Abbreviations: -GTP, -glutamyl-transpeptidase; 5-HETE, 5-hydroeicosatetraenoic
acid; 5-HPETE, 5-hydroperoxyeicosatetraenoic acid; MPO, myeloperoxidase; NADPH oxidase, reduced nicotinamide
adenine dinucleotide phosphate oxidase; TBARS, thiobarbituric acid reactive substances. (Modified from Montuschi,
P. and Barnes, P.J., Analysis of exhaled breath condensate for monitoring airway inflammation, in Trends in Pharmaco-
logical Sciences, Vol. 23, Elsevier, New York, 2002, pp. 232–237. With permission.)
TF1734_book.fm Page 19 Wednesday, August 25, 2004 4:57 PM

Analysis of Exhaled Breath Condensate: Methodological Issues 19

techniques used (enzyme immunoassays, radioimmunoassays, GC/MS, HPLC);

(3) incomplete characterization of factors that affect EBC analysis; (4) differences in
clinical characteristics of study groups (diagnostic criteria, disease severity, treat-
ment); and (5) interindividual biological variability. Based on electrolyte concentra-
tions, changes in respiratory solutes can also result from variations in the dilution of
respiratory droplets, as has been shown in healthy subjects.27 For this reason, the use
of reference indicators such as urea, electrolytes, or conductivity has been proposed.33

IV. METHODOLOGICAL ISSUES

To allow comparisons of data from different research centers and to assess the clinical
usefulness of EBC analysis, standardization of sample collection and validation of
the analytical techniques for measuring each inflammatory biomarker are required.
Several methodological issues need to be addressed, including flow dependence,
time dependence, and influence of ventilation patterns; origin(s) of exhaled markers;
organ specificity of EBC; nasal contamination; saliva and sputum contamination;
identification of a dilution marker to ascertain to what extent EBC reflects the
composition of airway lining fluid and to adjust for possible variations in the dilution
of respiratory droplets; influence of temperature, humidity, and collecting-system
materials; reproducibility studies (intrasubject, diurnal variability); comparisons of
collection devices; storage issues; the need for sample pretreatment; and the develop-
ment of sensitive, specific, and reproducible analytical methods to be used routinely.

A. FLOW DEPENDENCE AND INFLUENCE OF VENTILATION PATTERNS

One study has shown that hydrogen peroxide concentrations in EBC depend on
expiratory flow rate in healthy adults (n = 15).34 Mean ± standard error concentrations
of hydrogen peroxide in EBC at flow rates of 140, 69, and 48 ml/sec were 0.12 ±
0.02, 0.19 ± 0.02, and 0.32 ± 0.03 µM, respectively, indicating that exhaled hydrogen
peroxide levels are flow dependent.34 In six healthy adults, the concentrations of
MDA in EBC collected at flow rates of 200, 150, 100, and 50 ml/sec were similar,20
indicating that exhaled MDA levels are not flow dependent. In four healthy children,
no differences were observed among MDA and glutathione concentrations in EBC
samples collected at different flow rates.22 To study the influence of ventilation patterns
on 8-isoprostane, a marker of lipid peroxidation, and PGE2 concentrations in EBC,
we asked 15 healthy adults to breathe at three different expiratory minute ventilations
(10, 20, and 30 l/min) for 10, 10, and 5 min, respectively.35 Mean 8-isoprostane and
PGE2 concentrations in EBC were similar, indicating that the concentrations of these
eicosanoids in EBC are not influenced by expiratory minute ventilation.35 Likewise,
leukotriene B4 (LTB4) (41.2 ± 3.7 vs. 43.9 ± 4.1 pg/ml) and LTE4 (12.8 ± 1.5 vs.
13.9 ± 2.3 pg/ml) concentrations in EBC were similar in five healthy subjects who
breathed at 14 and 28 breaths/min for 15 min, maintaining the same tidal volume.36
Other researchers have shown that nitrite and total protein concentrations in EBC
are unaffected by respiratory pattern37 and that ethanol levels in EBC samples
collected after tidal breathing and deep inspiration and expiration are similar.38 Taken
together, these data indicate that different markers in EBC behave differently regarding
TF1734_book.fm Page 20 Wednesday, August 25, 2004 4:57 PM

20 New Perspectives in Monitoring Lung Inflammation

flow dependence and ventilation patterns and, therefore, each marker should be
considered and studied individually.
For several markers, there are no data on the influence of airflow and/or venti-
lation patterns on their levels in EBC and additional studies are warranted. Moreover,
the influence of airflow and ventilation patterns on the concentrations of EBC
markers has been studied in healthy subjects and these data cannot be directly
extrapolated to patients with inflammatory lung diseases. Studies on mechanically
ventilated patients might clarify the influence of minute ventilation on the concen-
trations of inflammatory markers in EBC. In one study, the correlation between
minute ventilation and 8-isoprostane concentrations in EBC was not significant
(p < .07).19 Assessing the flow dependence of different biomarkers is important for
the standardization and reproducibility of measurements. On the basis of the flow
dependence, EBC sampling will be performed either at a constant flow rate for a
fixed time, or at a constant total exhaled volume for a variable time.
The duration of the EBC collection does not influence the concentrations of
8-isoprostane,19 whereas conflicting results have been reported for MDA. In one
study, MDA concentrations in EBC samples collected in 10 and 20 min were similar
(16.4 ± 2.4 vs. 17.1 ± 1.7 nmol/L),20 whereas in another study the MDA levels
decreased significantly with prolonged sampling time (5 min, 0.32 pmol/sec vs.
10 min, 0.18 pmol/sec, p = .020, vs. 15 min, 0.11 pmol/sec, p = .0039).39 Hydrogen
peroxide concentrations in EBC were also reduced as sampling time was prolonged
(2 min, 1.2 pmol/sec vs. 4 min, 1.0 pmol/sec vs. 10 min, 0.7 pmol/sec vs. 15 min,
0.6 pmol/sec).40 EBC concentrations of unstable compounds (e.g., cysteinyl-leuko-
trienes [cys-LTs] or hydrogen peroxide) are most likely to be affected by sampling
time. Additional studies are required to establish the effect of duration of collection
on different biomolecules and to establish the ideal sampling time for each of them.
The volume of EBC collected depends on total exhaled volume, ventilation rate,
and breath test duration. In healthy subjects, the volumes of EBC collected in a
15-min test were higher at 28 breaths/min compared with those at 14 breaths/min
(2.4 ± 0.3 ml vs. 1.4 ± 0.2 ml, p < .01).35 In 10 healthy subjects, there was a correlation
between expiratory minute ventilation and EBC volume (r = 0.7, p < .001), although
the condenser efficiency decreased at the higher expiratory minute ventilation val-
ues.37 The mean volume of EBC during 10 and 20 min of tidal breathing was 1.22 ±
0.1 and 2.1 ± 0.1 ml, respectively.20 The mode of inhalation (oral vs. nasal) does
not influence the concentrations of adenosine in EBC in young healthy subjects
(8.3 ± 4.3 vs. 7.9 ± 3.9 nM, respectively), whereas the volume of EBC collected is
increased when subjects inhale through their noses and exhale through their mouths
without wearing a noseclip (2321 ± 736 vs. 1746 ± 400 µl, respectively).21 This is
probably due to an increase in minute ventilation when the sample collection is
performed without a noseclip.21

B. ORIGIN(S) OF EXHALED MARKERS

There are three aspects related to the origin of markers in EBC: (1) their cellular
source(s); (2) the compartment of the respiratory system in which they are primarily
TF1734_book.fm Page 21 Wednesday, August 25, 2004 4:57 PM

Analysis of Exhaled Breath Condensate: Methodological Issues 21

produced (airways vs. alveolar region); and (3) the extent to which EBC reflects the
composition of airway lining fluid.
EBC analysis is not suitable for studying the inflammatory cells in the respiratory
system, and identifying the cellular source(s) of exhaled markers requires invasive
techniques such as bronchial biopsies. Indirect information on the cellular source(s)
of exhaled biomolecules can be obtained by correlating their concentrations in EBC
with inflammatory cell counts in sputum. However, a correlation between an exhaled
marker and a cell type does not necessarily indicate that the mediator is released by
that cell type. In contrast, a lack of correlation could be due to increased cell activation
and consequent enhanced release of mediator without increased cell counts. Flow
dependence might provide information on the origin of inflammatory mediators in
the respiratory system (airways as opposed to alveolar).34 If the concentrations of a
biomarker in EBC are dependent on expiratory flow rate, this could indicate that the
biomarker is mainly produced within the airways, given that flow rate affects the
time available for its accumulation in the respiratory tract, and therefore its concen-
trations in the EBC.34 In contrast, it is likely that flow rate has a minor role at the
level of the alveolar region, so the alveolar contribution to the exhaled markers
should not depend on it.34 Based on these assumptions, hydrogen peroxide in EBC
should primarily be derived from the airways,34 given that it is flow dependent. In
contrast, 8-isoprostane,35 PGE2,35,36 aldehydes,20 and glutathione22 should be pro-
duced mainly within the alveolar region because their concentrations in the EBC
are not influenced by expiratory flow. However, these studies were performed in
healthy subjects and there is no adequate information for patients with inflammatory
lung diseases. In these patients, the increased numbers and/or activation of selective
inflammatory cell types in different compartments of the respiratory system (airways
vs. alveolar region) might have a different impact on the concentrations of a specific
biomarker in EBC compared with healthy subjects. Moreover, the effects of flow
rate on the levels of exhaled markers are probably more complex, given that turbulent
flow and high current velocity in the airways can facilitate formation of aerosols
containing nonvolatile inflammatory mediators (e.g., lipids, proteins) and, therefore,
increase their concentrations in the EBC.16
Studies of potential flow dependence of breath condensate solutes are more
complicated than those pertaining to exhaled nitric oxide because slow or fast flows
cannot be maintained easily for the time usually required for EBC collection.16 The
origin of nonvolatile inflammatory mediators, which are probably transported as
aerosols (e.g., protein markers) in EBC, can also be studied by identifying cell-
specific markers such as surfactant proteins. One study using ethanol as a model
compound indicates that both bronchial and alveolar compartments contribute to the
formation of EBC, but the ratio between the two compartments depends on venti-
lation patterns, with an apparent shift toward the alveolar region during forced
expiration.38 Studies on the mechanisms of formation of EBC are necessary to
establish to what extent EBC reflects the composition of the airway lining fluid and
could be used to investigate lung pathophysiology. The physicochemical properties
and mechanism(s) of formation of EBC and its relations with airway lining fluid
are discussed in Chapter 3.
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22 New Perspectives in Monitoring Lung Inflammation

C. ORGAN SPECIFICITY OF EXHALED BREATH CONDENSATE

Whether the concentrations of inflammatory mediators in EBC reflect lung inflam-
mation or overall systemic productions of these compounds is still unknown. It is
possible that inflammatory mediators produced at sites remote from the lung could
circulate in plasma and enter the pulmonary epithelial lining fluid, particularly under
conditions of increased permeability.19 In this regard, studies aimed at measuring
inflammatory mediators in EBC in patients with pathophysiological conditions in
which a role for systemic inflammation has been implicated (e.g., systemic lupus
erythematosus) might help clarify this question. Comparisons between concentrations
of inflammatory markers in EBC and in biological fluids reflecting systemic produc-
tion of inflammatory mediators such as plasma and/or urine might also be useful.

D. DILUTION REFERENCE INDICATORS

Most EBC collected in cooled condensers is derived from water vapor. However,
the presence of nonvolatile compounds in EBC suggests that droplets of the respi-
ratory fluid also have been collected.27 Calculation of respiratory fluid solute con-
centrations from EBC requires estimation of the dilution of respiratory droplets by
water vapor.27 Based on condensate EBC electrolyte concentrations, it has been
estimated that respiratory fluid represents between 0.01 and 2% of EBC volumes.27
Although increases in the levels of inflammatory mediators that have been reported
in different lung diseases could reflect increases in the concentrations of these
markers in the respiratory fluid, they also could reflect increased droplet formation.33
Part of the variation in nonvolatile compound concentrations in EBC also could be
related to differences in the dilution of respiratory droplets by water vapor.27 How-
ever, the selective increase of inflammatory mediators in EBC and the lack of
correlation between EBC concentrations of structurally related compounds are not
consistent with this evidence. Reference indicators that remain relatively unchanged
in the respiratory fluid and, ideally, similar in concentration to those in plasma, are
required to estimate the dilution of respiratory droplets in EBC.33 Recently, it has
been shown that measurements of conductivity can be used to estimate airway
electrolyte concentrations and the dilution of respiratory droplets by water vapor
after most of the ammonia has been removed by lyophilization.33 This technique
provides a reliable method of estimating the total concentrations of ions in EBC and
the dilution of respiratory droplets by the water vapor.

E. NASAL CONTAMINATION
Inflammatory mediators such as LTs and PGs are formed in the nose and paranasal
sinuses under both physiological and pathological conditions (e.g., rhinitis)41,42 and
can enter the oral expiratory air through the posterior nasopharynx. It is important
to rule out nasal contamination of EBC samples, which can be achieved by measuring
biomarker concentrations in three different experimental settings: inhaling and exhal-
ing without a noseclip, inhaling and exhaling with a noseclip to increase the chance
of nasal contamination, and exhaling against a resistance to ensure soft palate closure
TF1734_book.fm Page 23 Wednesday, August 25, 2004 4:57 PM

Analysis of Exhaled Breath Condensate: Methodological Issues 23

and minimize nasal contamination. As the concentration of nitric oxide in the

nasopharynx might be high relative to that recovered in the lower airways, nasal
contamination must be avoided when measuring exhaled nitric oxide. This is gen-
erally achieved by exhaling against a resistance to ensure closure of the soft palate.
In a similar manner, concentrations of nitric oxide metabolites such as nitrite and
nitrate in EBC could be affected by nasal contamination. However, wearing a
noseclip during EBC collection to increase the chance of nasal contamination does
not affect the concentrations of nitrite and nitrate in this biological fluid.43 However,
collection of EBC when subjects have breathed against a resistance are needed to
exclude definitively a possible nasal contribution to nitrite and nitrate concentrations
in EBC. In healthy subjects, adenosine, ammonia, and thromboxane B2 concentra-
tions in EBC are not influenced by the mode of inhalation (oral vs. nasal).21 Similar
results were reported in patients with allergic rhinitis.21 It is not currently known if
the mode of inhalation during the collection of EBC influences the concentrations
of other biomolecules.

F. SALIVA CONTAMINATION
Eicosanoids and other biomolecules are present at high concentrations in saliva.44,45
A simple method to exclude saliva contamination of the EBC consists of measuring
amylase concentrations in the samples, with the absence of amylase indicating an
absence of saliva.46 Amylase concentrations were generally undetectable in all or
most of the samples tested.21,27,46–48 In those samples in which amylase concentrations
were detectable, they averaged less than 0.01% of those in saliva.27 In contrast,
sodium ion concentrations in EBC averaged 32% of those in saliva.27 Moreover,
adenosine and thromboxane B2 concentrations in EBC samples obtained through
tracheostomy in mechanically ventilated patients or through the mouth in healthy
subjects were similar.21 Taken together, these data indicate that saliva contamination
of the EBC, if it occurs, is not a major issue, at least with the solutes discussed
above. Relatively few of the droplets captured in the condensate are formed in the
mouth; most are presumably formed in the respiratory tract.26 However, at least three
aspects need to be considered: (1) most of the ammonia in the EBC is formed in
the mouth, probably as ammonium gas, as indicated by undetectable levels of
ammonia in EBC obtained from patients with tracheostomies27 (these findings indi-
cate that potential saliva contamination of the EBC samples should be considered
separately for each marker); (2) the chance of saliva contamination of EBC samples
might differ from one condenser to the other (e.g., presence of saliva filters); and
(3) for most biomarkers, the concentrations in saliva are much higher than those in
EBC, so that even a small saliva contamination might influence their concentrations
in EBC. This can be particularly relevant to measurements of LTs, PGs, and iso-
prostanes in EBC, given that their concentrations are in the range of picograms per
milliliter. Measurement of viscosity also can be used to exclude saliva contamination.
At present, measurement of amylase concentrations in each EBC sample to exclude
gross saliva contamination seems to be a reasonable approach, but more sensitive
methods are required to address this point.
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24 New Perspectives in Monitoring Lung Inflammation

G. INFLUENCE OF DIFFERENT FACTORS ON EXHALED MARKERS

There are no data on the influence of temperature and humidity on EBC measure-
ments. Depending on the physical and chemical properties of the different inflam-
matory mediators, the collecting system materials might affect their concentrations
in EBC. After instillation of three concentrations of 8-isoprostane (50, 250, and
500 pg/ml) in a collecting system consisting of Teflon-lined tubing (Tygon; Norton
Performance Plastics, Akron, OH), there was a fractional loss of yield ranging from
24 to 30%, likely due to lipid adsorption to the tubing.19 However, the linear recovery
of 8-isoprostane indicates lack of ex vivo lipid peroxidation within the collection
system.19 After nebulization of the same three concentrations of 8-isoprostane, the
loss in yield was greater, presumably due both to lipid adsorption to the nebulizer
apparatus and incomplete condensation of the airborne droplets.19 For analysis of
eicosanoids, glass or polypropylene collecting systems should be used to avoid
adsorption to the containers. A collecting system coated with borosilicate glass seems
to be the most suitable for collecting albumin.17 Smoking affects 8-isoprostane,15
hydrogen peroxide,49 and S-nitrosothiols50 concentrations in EBC. The influence of
age, sex, respiratory maneuvers (e.g., spirometry), airway caliber, circadian rhythm,
infections, and medications on EBC markers is largely unknown.

H. DIURNAL AND DAY-TO-DAY VARIABILITY OF EXHALED MARKERS

Reproducibility studies are needed to establish intrasubject variability (diurnal and
day to day) of biomarker measurements. Hydrogen peroxide shows diurnal variations
in healthy subjects.49 We recently assessed the day-to-day reproducibility of 8-iso-
prostane and PGE2 measurements in EBC by collecting three EBC samples on days
1, 3, and 7 from healthy volunteers.35 8-Isoprostane and PGE2 in EBC were measured
by radioimmunoassays (RIAs) that were developed in our laboratory and were
previously validated by reversed-phase high-performance liquid chromatography
(RP-HPLC), providing evidence for their specificity.51 Measurements of 8-isopros-
tane (intraclass correlation coefficient = .95) and PGE2 (intraclass correlation coef-
ficient = .90) in EBC in healthy subjects were highly reproducible.35 We also assessed
day-to-day RIA measurements of 8-isoprostane, PGE2, and thromboxane B2 in EBC
in 15 children with asthma.52,53 Repeatability of RIAs was expressed as limits of
agreement according to analyses described by Bland and Altman.54 Day-to-day
radioimmunoassay measurements of 8-isoprostane (limits of agreement, 5 and
–5 pg/ml) and PGE2 (limits of agreement, 5.1 and –5.1 pg/ml)52 in EBC were highly
reproducible as well as RIA measurements of thromboxane B2 (limits of agreement,
–3.7 and 4.7 pg/ml),52 which was detectable only in some asthmatic children. In
children with asthma, measurements of LTE4 and LTB4 in EBC with commercially
available enzyme immunoassays (Cayman Chemicals, Ann Arbor, MI) were less
reproducible than RIA for prostanoids as indicated by their limits of agreement
(LTE4, 12.4 and –15.6 pg/ml; LTB4, 9.8 and –8.4 pg/ml; C. Mondino et al., unpub-
lished data). The mean coefficient of variation for MDA measured in two samples
in 8 subjects was 8.2%.20
TF1734_book.fm Page 25 Wednesday, August 25, 2004 4:57 PM

Analysis of Exhaled Breath Condensate: Methodological Issues 25

I. SAMPLE HANDLING
There are no studies on the stability of biomolecules in EBC. Information on the
stability of the different inflammatory markers in EBC and standardization of sample
storage conditions are important for study planning. In particular, the effect of
freezing and thawing, temperature, and storage time on the stability of the different
compounds should be established.

J. VALIDATION OF ANALYTICAL METHODS

The presence of 8-isoprostane in EBC has been confirmed by stable isotope dilution
in conjunction with GC/MS in selected ion mode.19 The presence of PGE2 in EBC
has also been confirmed by GC/MS but analytical details are not available.19 We
have recently provided evidence for the specificity of 8-isoprostane and PGE2
measurements in EBC by RIAs developed in our laboratory.51 In this study, RIAs
for 8-isoprostane and PGE2 were qualitatively validated by RP-HPLC.51 Because of
their biological importance in asthma and other inflammatory airway diseases, there
is increasing interest in measuring LTs in EBC. A wide variability in EBC concen-
trations have been reported for both LTB4 and cys-LTs even in healthy subjects.36,55–57
In view of the fact that LTs, particularly cys-LTs, are chemically unstable com-
pounds, part of this variability could be due to collection and/or storage of EBC
samples. Efforts should be made to optimize the recovery for LTs in EBC (suitable
collecting device material, snap freezing of EBC samples, and storage of EBC
samples at –80˚C for a short time before measurement). However, part of the
variability in LT concentrations in EBC could be related to analytical methods,
including specificity, sensitivity, and reproducibility of techniques. In all the studies
on LTs in EBC published so far, LTs were measured by commercially available
enzyme immunoassays for which robust data on their specificity and reproducibility
are not available. We have recently provided evidence for the specificity of a com-
mercially available enzyme immunoassay for LTB4 by demonstrating identical chro-
matographic behavior of LTB4-like immunoreactivity in the EBC with the respective
standard.58 Some studies reported concentrations of LTB4 and cys-LTs in EBC close
to the detection limit of the immunoassays,56,59 casting doubts on the reliability of
these findings and on their interpretation. Enzyme immunoassays for LTs generally
work well in buffer, but their behavior in EBC is not known and should be studied
carefully. Most of the EBC markers usually have been measured by immunological
techniques. Validation by more specific analytical methods (e.g., HPLC, GC/MS, or
LC/MS) is critical for providing definitive evidence for the presence of inflammatory
biomarkers in EBC and for a more quantitative assessment of their concentrations.
Quantitative comparisons between immunoassays and independent assay methods
(GC/MS, HPLC) are a prerequisite for large-scale use of immunoassays to measure
biomolecules in EBC. Considering that reference analytical techniques such as
GC/MS are very expensive, time-consuming, and not suitable for routine use, vali-
dation of immunoassays for inflammatory biomarkers may contribute to the devel-
opment of EBC analysis in respiratory medicine.
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26 New Perspectives in Monitoring Lung Inflammation

V. ADVANTAGES AND LIMITATIONS

Measurements of inflammatory mediators in EBC provide insights into the patho-
physiology of inflammatory lung diseases. Because it is completely noninvasive,
EBC analysis is well accepted by patients, applicable to children, useful for outpa-
tient or even domiciliary monitoring of lung inflammation as portable and disposable
collecting systems will become widely available, suitable for longitudinal studies
and patient follow-up, and potentially useful for monitoring drug therapy. Charac-
terization of selective profiles of exhaled markers (e.g., eicosanoids) might be rele-
vant to the differential diagnosis of inflammatory airway diseases. However, EBC
analysis requires standardization and validation of the analytical techniques; the
origin (airways vs. alveolar region) and cellular sources of exhaled markers are not
known; in contrast to sputum induction and BAL fluid, analysis of lung inflammatory
cells is not possible; and definite evidence that EBC analysis reflects lung rather
than systemic inflammation is required.

VI. CONCLUSIONS AND FUTURE DIRECTIONS

Studies that aim to clarify the methodological issues that are discussed in this chapter
are the main priority in this research area. Future research should also include (1) the
identification of reference values for the different inflammatory markers in healthy
adults and children; (2) large longitudinal studies to ascertain if sequential measure-
ments in the individual patient reflect the degree of lung inflammation and/or disease
severity; (3) studies of the relationships between EBC markers and symptoms, lung
function, and other indices, and/or methods for quantifying airway inflammation
(e.g., sputum and BAL analysis, exhaled nitric oxide); (4) the identification of levels
and profiles of exhaled markers in different lung diseases; (5) studies on the relative
concentrations of biomarkers in other fluids that reflect lung inflammation (sputum
supernatants, BAL fluid); (6) controlled studies to establish the usefulness of EBC
analysis for guiding pharmacological treatment in inflammatory airway diseases,
given that the available studies on the effects of drugs on EBC markers are mainly
cross-sectional; (7) studies to determine the usefulness of EBC analysis for predict-
ing treatment response and assessment of new therapies; (8) the identification of
other inflammatory mediators; (9) the possible identification of markers of lung
cancer or infectious lung diseases (e.g., proteins, nucleic acids); (10) studies to
determine the feasibility of gene expression analysis in EBC; (11) studies on drug
disposition in EBC (e.g., antibiotics); and (12) studies on formation of EBC, its
origin in the respiratory system, and its relationships with airway lining fluid.
Whether and when EBC analysis will be applicable to the clinical setting is
difficult to predict. However, considering the importance of inflammation in the
pathophysiology of lung diseases such as asthma and COPD, the relative lack of
noninvasive methods for monitoring airway inflammation and therapy, and the rel-
evance of its potential applications, additional research on EBC analysis is warranted.
Identification of breath “fingerprints” may open a new era in respiratory medicine.
These are the promises. Future research will say if they are reality.
TF1734_book.fm Page 27 Wednesday, August 25, 2004 4:57 PM

Analysis of Exhaled Breath Condensate: Methodological Issues 27

ACKNOWLEDGMENT
Supported by Catholic University of the Sacred Heart, Fondi di Ateneo 2002–2003.

FURTHER READING
1. Wielders, P.L.M.L. and Dekhuijzen, P.N.R., Disease monitoring in chronic obstructive
pulmonary disease: is there a role for biomarkers? Eur. Respir. J., 10, 2443, 1997.
2. Barnes, P.J., Chung, K.F., and Page, C.P., Inflammatory mediators of asthma: an
update, Pharmacol. Rev., 50, 515, 1998.
3. Suki, B., Lutchen, K.R., Ingenito, E.P., On the progressive nature of emphysema,
Am. J. Respir. Crit. Care Med., 168, 516, 2003.
4. Barnes, P.J., Chronic obstructive pulmonary disease, N. Engl. J. Med., 343, 269, 2000.
5. Rutgers, S.R. et al., Markers of active airway inflammation and remodelling in chronic
obstructive pulmonary disease, Clin. Exp. Allergy, 31, 193, 2001.
6. Adelroth, E., How to measure airway inflammation: bronchoalveolar lavage and
airway biopsies, Can. Respir. J., 5, 18A, 1998.
7. Berlyne, G.S. et al., A comparison of exhaled nitric oxide and induced sputum as
markers of airway inflammation, J. Allergy Clin. Immunol., 106, 638, 2000.
8. Mutlu, G.M. et al., Collection and analysis of exhaled breath condensate in humans.
Am. J. Respir. Crit. Care Med., 164, 731, 2001.
9. Kharitonov, S.A. and Barnes, P.J., Exhaled markers of pulmonary disease, Am. J.
Respir. Crit. Care Med., 163, 1693, 2001.
10. Montuschi, P., Indirect monitoring of lung inflammation, Nat. Rev. Drug Discov., 1,
238, 2002.
11. Montuschi, P. and Barnes, P.J., Analysis of exhaled breath condensate for monitoring
airway inflammation, Trends Pharmacol. Sci., 23, 232, 2002.
12. Dwyer, T.M., Expired breath condensate (EBC) and the ultimate disposition of airway
surface liquid (ASL), Am. J. Respir. Crit. Care Med., 163, A406, 2001.
13. Montuschi, P. et al., Increased 8-isoprostane, a biomarker of oxidative stress, in
exhaled condensate of asthma patients, Am. J. Respir. Crit. Care Med., 160, 216, 1999.
14. Hanazawa, T., Kharitonov, S.A., and Barnes, P.J., Increased nitrotyrosine in exhaled
breath condensate of patients with asthma, Am. J. Respir. Crit. Care Med., 162, 1273,
2000.
15. Montuschi, P. et al., Exhaled 8-isoprostane as an in vivo biomarker of lung oxidative
stress in patients with COPD and healthy smokers, Am. J. Respir. Crit. Care Med.,
162, 1175, 2001.
16. Hunt, J., Exhaled breath condensate: an evolving tool for non-invasive evaluation of
lung disease, J. Allergy Clin. Immunol., 110, 28, 2002.
17. Rosias, P.P.R. et al., Inner coating of condenser systems influences detection of
albumin in exhaled breath condensate, Eur. Respir. J., 22, Suppl. 45, 280s, 2003.
18. Gajdocsi, R. et al., The reproducibility and accuracy of on-line hydrogen peroxide
bioassay and the decreasing effect of storage on H2O2 concentration, Eur. Respir. J.,
22, Suppl. 45, 279s, 2003.
19. Carpenter, C.T., Price, P.V., and Christman, B.W., Exhaled breath condensate isopros-
tanes are elevated in patients with acute lung injury or ARDS, Chest, 114, 1653, 1998.
20. Corradi, M. et al., Aldehydes in exhaled breath condensate of patients with chronic
obstructive pulmonary disease, Am. J. Respir. Crit. Care Med., 167, 1380, 2003.
TF1734_book.fm Page 28 Wednesday, August 25, 2004 4:57 PM

28 New Perspectives in Monitoring Lung Inflammation

21. Vass, G. et al., Comparison of nasal and oral inhalation during exhaled breath con-
densate collection, Am. J. Respir. Crit. Care Med., 167, 850, 2003.
22. Corradi, M. et al., Aldehydes and glutathione in exhaled breath condensate of children
with asthma exacerbation, Am. J. Respir. Crit. Care Med., 167, 395, 2003.
23. Hunt, J.F. et al., Endogenous airway acidification. Implications for asthma patho-
physiology, Am. J. Respir. Crit. Care Med., 161, 694, 2000.
24. Vaughan, J. et al., Exhaled breath condensate pH is a robust and reproducible assay
of airway acidity, Eur. Respir. J., 22, 889, 2003.
25. Kostikas, K. et al., pH in expired breath condensate of patients with inflammatory
airway diseases, Am. J. Respir. Crit. Care Med., 165, 1364, 2002.
26. Scheideler, L. et al., Detection of nonvolatile macromolecules in breath: a possible
diagnostic tool?, Am. Rev. Respir. Dis., 148, 778, 1993.
27. Effros, R.M. et al., Dilution of respiratory solutes in exhaled condensates, Am. J.
Respir. Crit. Care Med., 165, 663, 2002.
28. Griese, M., Noss, J., and von Bredow, C., Protein pattern of exhaled breath condensate
and saliva, Proteomics, 2, 690, 2002.
29. Cunningham, S. et al., Measurement of inflammatory markers in the breath conden-
sate of children with cystic fibrosis, Eur. Respir. J., 15, 955, 2000.
30. Shahid, S.K. et al., Increased interleukin-4 and decreased interferon-gamma in
exhaled breath condensate of children with asthma, Am. J. Respir. Crit. Care Med.,
165, 1290, 2002.
31. Gessner, C. et al., Amplification of DNA from breath condensate of volunteers and
patients with non-small cell lung cancer (NSCLC), Am. J. Respir. Crit. Care Med.,
163, A482, 2001.
32. Vogelberg, C. et al., Orally and nasally exhaled nitric oxide and nitrite in asthmatic
and cystic fibrosis patients, Eur. Respir. J., Suppl. 45, 131s, 2003.
33. Effros, R.M. et al., A simple method for estimating respiratory solute dilution in
exhaled breath condensates, Am. J. Respir. Crit. Care Med., 2003 (in press).
34. Schleiss, M.B. et al., The concentration of hydrogen peroxide in exhaled air depends
on expiratory flow rate, Eur. Respir. J., 16, 1115, 2000.
35. Montuschi, P. et al., Methodological aspects of exhaled prostanoid measurements,
Eur. Respir. J., Suppl. 45, 18s, 2003.
36. Montuschi, P. and Barnes, P.J., Exhaled leukotrienes and prostaglandins in asthma,
J. Allergy Clin. Immunol., 109, 615, 2002.
37. McCafferty, J.B. et al., Effect of varying respiratory pattern on exhaled breath con-
densate collection, Eur. Respir. J., Suppl. 45, 37s, 2003.
38. Rickmann, J. et al., Breath condensate reflects different compartments of respiratory
tract depending on ventilation pattern, Am. J. Respir. Crit. Care Med., 163, A407,
2001.
39. Larstad, M., Torén, K., and Olin, A.-C., Influence of sampling time on malondialde-
hyde levels and pH in exhaled breath condensate, Eur. Respir. J., Suppl. 45, 38s, 2003.
40. Svensson, S., Olin, A.-C., and Torén, K., Sampling time is important for the collection
of hydrogen peroxide in exhaled breath condensate (EBC), Eur. Respir. J., Suppl.
45, 78s, 2003.
41. Howarth, P.H., Leukotrienes in rhinitis, Am. J. Respir. Crit. Care Med., 161, S133,
2000.
42. Wang, D.Y., Smitz, J., and Clement, P., Prostaglandin D2 measurement in nasal
secretions is not a reliable marker for mast cell activation in atopic patients, Clin.
Exp. Allergy, 25, 1228, 1995.
TF1734_book.fm Page 29 Wednesday, August 25, 2004 4:57 PM

Analysis of Exhaled Breath Condensate: Methodological Issues 29

43. Chladék, J. et al., Short-term variation and influence of a nose clip on nitrite and
nitrate levels in the exhaled breath condensate, Eur. Respir. J., Suppl. 45, 267s, 2003.
44. Zakrzewski, J.T. et al., Lipid mediators in cystic fibrosis and chronic obstructive
pulmonary disease, Am. Rev. Respir. Dis., 136, 779, 1987.
45. McKinney, E.T. et al., Plasma, urinary, and salivary 8-epi-prostaglandin F2α levels in
normotensive and preeclamptic pregnancies, Am. J. Obstet. Gynecol., 183, 874, 2000.
46. Montuschi, P. et al., Exhaled 8-isoprostane as a new non-invasive biomarker of
oxidative stress in cystic fibrosis, Thorax, 55, 205, 2000.
47. Ho, L.P., Innes, J.A., and Greening, A.P., Nitrite levels in breath condensate of patients
with cystic fibrosis is elevated in contrast to exhaled nitric oxide, Thorax, 53, 680,1998.
48. Ho, L.P. et al., Expired hydrogen peroxide in breath condensate of cystic fibrosis
patients, Eur. Respir. J., 13, 103, 1999.
49. Nowak, D. et al., Exhalation of H2O2 and thiobarbituric acid reactive substances
(TBARs) by healthy subjects, Free Radic. Biol. Med., 15, 178, 2001.
50. Corradi, M. et al., Increased nitrosothiols in exhaled breath condensate in inflamma-
tory airway diseases, Am. J. Respir. Crit. Care Med., 163, 854, 2001.
51. Montuschi, P. et al., Validation of 8-isoprostane and prostaglandin E2 measurements
in exhaled breath condensate, Inflamm. Res., 52, 502, 2003.
52. Montuschi, P.et al., Profile of prostanoids in exhaled breath condensate in childhood
asthma, Eur. Respir. J., Suppl. 45, 400s, 2003.
53. Baraldi, E. et al., Increased exhaled 8-isoprostane in childhood asthma, Chest, 124,
25, 2003.
54. Bland, M.J., and Altman, D., Statistical methods for assessing agreement between
two methods of clinical measurement, Lancet, 1, 307, 1986.
55. Antczak, A. et al., Increased exhaled cysteinyl-leukotrienes and 8-isoprostane in
aspirin-induced asthma, Am. J. Respir. Crit. Care Med., 166, 301, 2002.
56. Baraldi, E. et al., Cysteinyl leukotrienes and 8-isoprostane in exhaled breath conden-
sate of children with asthma exacerbations, Thorax, 58, 505, 2003.
57. Biernacki, W.A., Kharitonov, S.A., and Barnes, P.J., Increased leukotriene B4 and
8-isoprostane in exhaled breath condensate of patients with exacerbations of COPD,
Thorax, 58, 294, 2003.
58. Montuschi, P. et al., Validation of leukotriene B4 measurements in exhaled breath
condensate, Inflamm. Res., 52, 69, 2003.
59. Carpagnano, G.E. et al., Increased inflammatory markers in the exhaled breath con-
densate of cigarette smokers, Chest, 124, 1386, 2003.
60. Kostikas, K. et al., Prostaglandin E2 in expired breath condensate of patients with
asthma, Eur. Respir. J., 22, 743, 2003.
61. Huszar, E. et al., Thromboxane in exhaled breath condensate in healthy volunteers
and in patients with asthma, Eur. Respir. J., Suppl. 45, 293s, 2003.
62. Csoma, Z. et al., Increased leukotrienes in exhaled breath condensate in childhood
asthma, Am. J. Respir. Crit. Care Med., 166, 1345, 2002.
63. Mondino, C. et al., Leukotrienes in exhaled breath condensate in childhood asthma,
Am. J. Respir. Crit. Care Med., 167, A445, 2003.
64. Balint, B. et al., Increased nitric oxide metabolites in exhaled breath condensate after
exposure to tobacco smoke, Thorax, 56, 456, 2001.
65. Balint, B. et al., Increased nitrotyrosine in exhaled breath condensate in cystic fibrosis,
Eur. Respir. J., 17, 1201, 2001.
66. Gessner, C. et al., Exhaled breath condensate nitrite and its relation to tidal volume
in acute lung injury, Chest, 124, 1046, 2003.
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30 New Perspectives in Monitoring Lung Inflammation

67. Dekhuijzen, P.N.R. et al., Increased exhalation of hydrogen peroxide in patients with
stable and unstable chronic obstructive pulmonary disease, Am. J. Respir. Crit. Care
Med., 154, 813, 1996.
68. Horváth, I. et al., Combined use of exhaled hydrogen peroxide and nitric oxide in
monitoring asthma, Am. J. Respir. Crit. Care Med., 158, 1042, 1998.
69. Jöbsis, Q. et al., Hydrogen peroxide in exhaled air of healthy children: reference
values, Eur. Respir. J., 12, 483, 1998.
70. Zappacosta, B. et al. A fast chemiluminescent method for H2O2 measurement in
exhaled breath condensate, Clin. Chim. Acta, 310, 187, 2001.
71. Diegel, H. et al., Glutathione in breath condensate samples of healthy individuals and
patients with obstructive lung disease, Am. J. Respir. Crit. Care Med., 163, A408,
2001.
Exploring the Variety of Random
Documents with Different Content
themselves, that the greater part of the conquered nation had fallen
away from Christianity.
Many causes had conspired to produce this lamentable condition
of affairs. The geographical isolation of the Peninsula, which has
always had a tendency to preserve unaltered the mental and
physical characteristics of its people, has also had no unimportant
influence upon the national faith. That country, even at the time of
the Saracen invasion, was Christian only in name. It had never
wholly discarded its Pagan forms or traditions. It was the last
kingdom of Europe to nominally accept the new religion. Its creed
had long been heterodox, and that creed it had abandoned, without
remonstrance or regret, at the command of its sovereign. The
despotic power of the hierarchy had never been able to abolish the
ceremonies of Pagan antiquity which were incorporated with the
ritual of the Church. The population, the offspring of a score of
nations, each of which worshipped different divinities and was
familiar with the fraudulent pretensions of many sacerdotal claimants
to inspiration, was inclined to discredit and deride them all. To such
a society religious professions and formalities were naturally matters
of indifference. A nation which could spontaneously abandon the
heresy of Arius would hardly hesitate to embrace the monotheistic
doctrines of Mohammed. By the Moslems, so far as their tributaries
were concerned, no open inducements were offered for apostasy.
The practice of Islam discouraged the active proselytism advocated
by other sects. The conversion of a Christian tributary, unless he had
violated the law, must be voluntary, and the obligation, once
assumed, could never be renounced.
The favor enjoyed by the renegade was, however, a far more
powerful incentive than any that the promises of the ministers of
religion could evoke. The apostate was at once received into full
social communion with his former masters. He was eligible to the
highest political and military honors. In theory, at least, no stigma
could attach to his former condition or antecedents. The equality of
all men who professed belief in its dogmas was, as is well known,
the cardinal principle of the law of the Prophet.
 To the slave, these considerations appealed with peculiar force.
Tens of thousands of this oppressed and degraded caste had been
transferred, at a single stroke by the fortunes of war, from the hands
of one master to those of another. A host of captives had been taken
in battle. In the minds of but few of these unfortunates the
obligations of religion were deeply founded. While emancipation did
not invariably follow the profession of the faith of Islam, it usually
did; and the condition of the slave was always greatly improved by
this concession to the prejudices of him who regulated his conduct
and controlled his destiny. In view of these facts, there is little
wonder that multitudes of slaves embraced the Mussulman
doctrines.
The religious freedom of the Christians under Moslem rule was
mainly dependent on the prejudices of their own clergy, the
character of the dominant faction, and the temper of the sovereign.
The provisions of the treaties which guaranteed their privileges were
at first strictly observed. The general influx of fanatical foreigners, in
time, however, created a strong public sentiment against the
proscribed tributaries. They were sometimes deprived of their
houses of worship. Arbitrary contributions were frequently exacted
from them. On one occasion, the Christians of Cordova were
compelled to pay into the treasury the sum of a hundred thousand
pieces of gold, nearly a million and a quarter dollars. The revenues
of the Church were so impaired by these grievous impositions, that
ecclesiastics were often forced to engage in commercial pursuits to
provide for the pressing necessities of their order. Some carried the
manufactures of Cordova to Germany. Others journeyed as peddlers
through France. The trading priest of Moorish Spain was well known
in the markets of Genoa and Constantinople. Persons in clerical garb
were no longer safe in public places. In the time of the Almoravides,
when a Christian passed through the streets, the crowd shrank from
contact with him as from one stricken with the plague. Religious
processions were pelted by mobs of hooting children, and those who
took part in them were fortunate if they escaped without serious
personal injury. The ringing of the church-bells provoked the loud
threats and curses of intemperate zealots. The breaking up of a
congregation during Holy Week was often the signal for a riot. The
vengeance of Allah upon the idolater was invoked by the scoffing
bystanders when the corpse of a Christian was consigned to the
grave.
The clergy, against whom these insults were principally aimed,
were naturally exasperated by the indignity suffered by their creed
and their profession. Their ignorance, in spite of the example and
the benefits of Moslem civilization ever before their eyes, was not
less dense than that of their brethren of Catholic Europe. With every
opportunity to familiarize themselves with the tenets of Islam, and
thoroughly conversant with Arabic, they steadfastly declined to
honor the alleged revelations of the Prophet with their attention or
perusal. Their opinions on this subject they obtained from the
writings of fanatical monks, fully as ignorant as, and even more
bigoted than, themselves. The sage conclusion which they arrived at
from these researches was that the doctrines of the most
uncompromising of monotheists and image-breakers were Pagan
and idolatrous.
Apprehensive of violence if they ventured to show themselves in
public, they remained almost constantly in the seclusion of their
dwellings. Even the sacred calls of duty remained unanswered.
Often, for weeks, mass was not celebrated. The pulpit and the
confessional were deserted. The dying passed away unshriven.
Maddened by rage and terror, they were scarcely accessible even to
their sympathizing parishioners, who themselves incurred the risk of
ill-treatment from the populace in their visits to the episcopal palace
and the parsonage. Brooding over their wrongs, encouraged by the
promises and exhortations of the Fathers of the Church, wresting the
texts of Scripture to their purpose, fasting many consecutive days,
praying for hours at a time, exhausted by penance, their enthusiasm
became wrought up to the highest pitch. From such a condition the
progress to martyrdom is easy.
 The persecution of the Christians of Spain was inflicted, for the
most part, under the reigns of Abd-al-Rahman II. and Mohammed.
The annoyances to which they were subjected were by no means so
serious as they subsequently became, when the influence of the
Africans preponderated. The word persecution, implying as it does
the tyrannical abuse of superior power, is not applicable to the
circumstances under which the Mozarabes were sent to the scaffold.
They were rather criminals than martyrs. They voluntarily offered
themselves for the sacrifice. They denounced the religion of Islam as
false and idolatrous. They reviled the name of the Prophet. They
rushed into the mosques. When the voice of the muezzin resounded
from the minaret, they crossed themselves, and cried out, “Save us,
O Lord, from the call of the Evil One, both now and in eternity!” In
their eagerness to court destruction, they pushed their way into the
tribunals, and, in the presence of the judge, gave utterance to their
blasphemies. Even the majesty of the throne was not respected by
these frantic enthusiasts. St. Pelayus called the Khalif a dog to his
face. St. Isaac, not content with heaping abuse on Mohammed,
grossly insulted the Grand Kadi of Cordova. Such offences were
capital under the law, and admitted of neither extenuation nor
pardon.
At first, the magistrates, moved by astonishment and compassion,
refused to condemn persons whose actions seemed attributable only
to intoxication or insanity. But the deluded wretches would accept no
indulgence. Thrown into prison, they continued their revilings. Their
spurious zeal, mistaken constancy, and self-inflicted tortures
produced many imitators. Their cells became places of pilgrimage.
From them each day went forth new candidates for pious
consideration, fresh victims for the executioner. Some were hanged,
others beheaded. Not a few were burned at the stake and their
ashes cast into the river. The bitter feelings engendered by religious
controversy were not confined to Mohammedans. The ties of blood
seemed for a time forgotten or ignored. The hiding-places of the
accused were revealed by their own kindred. Brothers and sisters
denounced each other for the sake of the property they might
inherit. But the punishment only aggravated the evil. The number of
martyrs constantly multiplied. A great many of these came from the
laity. Youths of tender age excited the wonder and admiration of the
devout by the boldness of their utterances and the unflinching
courage with which they met their fate. Delicate women walked
barefoot for leagues, nominally to share the glory of dying for the
Faith, in reality to solicit the infliction of the extreme penalty of
violated law.
The contagion of example spread fast through the Christian
community of Cordova. No distinction was now so honorable as to
stand in the foremost rank of the blasphemers of the Prophet. In this
pious and meritorious performance, the secular clergy were,
however, not conspicuous. Their lives were entirely too precious to
be endangered so long as members of their flocks were eager to
demonstrate their willingness to die for a perverted religious
principle, involving an unprovoked breach of the contract from which
they derived security of worship, life, and property. In secret, they
promoted the increasing madness by prayer and vehement
exhortation. The impulse to the spirit of spontaneous martyrdom
was not a little stimulated by the honors paid to the victims.
Independent of both Roman and Asturian influence, the Andalusian
hierarchy conferred without delay the distinction of canonization
upon each aspirant for celestial glory. Their remains were conveyed
to the churches, where they at once began to disclose their
supernatural powers by response to prayer, by the cure of disease,
by the working of portentous and astonishing miracles.
The Moslem authorities were appalled by the strange conduct of
their tributaries, insensible alike to the inducement of clemency or
the dread of punishment. In the hope of abating the evil by
summary measures, Abd-al-Rahman II. authorized, by public edict,
any one to kill on the instant a Christian who was guilty of
blasphemy. This decree, while not fully accomplishing its object,
lessened the number of applicants for martyrdom and produced a
great increase of apostates and fugitives.
 But the mania which impelled the most fanatical to self-sacrifice
was far from infecting the entire Christian population of the capital.
There were many who looked with disapproval upon a course which
must eventually result in the oppression of their sect, in the increase
of its burdens, in the curtailment of its privileges. They foresaw that
the acts of a few irresponsible individuals would ere long be
regarded by the Moslem government as the authorized policy of the
Church. Many Christians held office under the administration. It was
only a question of time, if these disturbances continued, when they
would be dismissed from their employments. The khalifate was then
at the height of its power. If an uprising provoked by the clergy
should occur, as seemed not improbable, the entire tributary sect
might be exterminated; and, indeed, this measure had already been
vehemently urged by the intolerant African marabouts. In any event,
there would be arbitrary taxation, confiscation, violence, exile. In
their extremity, the more sober-minded of the Christians petitioned
the Khalif to summon a council, whose decision might be
authoritative and final in determining the duty of the people in the
present emergency.
All the prelates in the jurisdiction of the khalifate were accordingly
convoked. Abd-al-Rahman appointed as his representative an official
named Gomez, prominent in the administration, nominally attached
to the Christian communion, but of suspicious morals and of more
than suspicious orthodoxy. He was a man of fine education,
conspicuous talents, polished manners, insufferable pride, and
enormous wealth. The head of the faction which had, in vain,
endeavored to check the increasing disposition to martyrdom which
menaced the destruction of his sect, he had incurred the
unmeasured hatred of the clergy. Realizing fully the fatal
consequences of the insane acts of his co-religionists if unrestrained,
his interest concurred with his inclination to repress the dangerous
manifestations of their intemperate zeal before it became too late.
With great ability and eloquence he presented his views to the
council. The assembled prelates, awed by the government and
possessing little sympathy for those who were destroying the credit
of their order, were not disinclined to condemn these fanatical
suicides. But here a serious difficulty arose. The martyrs had been
canonized. Their relics had already demonstrated their sanctity by
the production of miracles. Their bodies were enshrined in the
shadow of the altar; their deeds and their sufferings were now a
part of the history of the Church. It was therefore manifestly
impolitic, as well as sacrilegious, to attempt to deprive them of the
rank in the celestial hierarchy which had been conferred by the
infallible wisdom of God. A middle course was possible. The council,
silent upon past martyrdoms, prohibited them in the future. Like all
temporizing measures intended to correct deeply rooted abuses, this
evasion of the issue left matters worse than before. The extremists,
headed by St. Eulogius, declared that the real sentiment of the
council manifestly ran counter to the one it expressed, as it did not
pronounce deserving of censure the acts of those who had suffered
for the Faith. The priests continued to arouse the zeal of their
misguided parishioners; enthusiasts continued to outrage the
sanctity of the mosques and the dignity of the tribunals, and the
executions went relentlessly on. Recafred, Archbishop of Cordova,
exasperated by the contempt with which the decree of the council
had been received, heartily co-operated with the Moslems in the
punishment of the offenders, now under the ban of both the
government and the Church. Many recalcitrant priests were seized
and thrown into prison. Others eluded with the greatest difficulty the
search of the authorities. Among the latter was St. Eulogius, with
whom, as well as with many of his holy brethren, the merits of
martyrdom seemed most glorious when obtained by the sufferings of
others. These vigorous measures filled the souls of the elect with
terror. A few escaped to the Asturias. A considerable number,
including some who had been loudest in their praise of the saints
and apparently most eager to emulate their example, apostatized.
The so-called persecution, begun under Abd-al-Rahman II. and
continued under Mohammed, lasted eight years. The works of
contemporaneous ecclesiastical writers conclusively establish the fact
that it was provoked by the violence of the Christians themselves. It
is apparent from the same authorities that its effects and importance
were grossly exaggerated. The Memorial of the Saints, by Eulogius,
the last and most eminent of the alleged victims of Moslem tyranny,
contains the names of comparatively few martyrs. But forty-four are
mentioned by the erudite historian Florez, whose diligent industry
has collated the voluminous records bearing upon the hagiology of
that time, as having been executed at Cordova. Several of these
were women, between whom and their male companions in
suffering and glory, the pious chronicler naïvely declares,
“mysterious affinities” existed.
With the decline of the empire, the prevalence of anarchy, and the
ascendency of the Berbers, the condition of the Spanish Christians
became more and more distressing. The suspension of the laws
afforded every facility for their oppression. Their churches were torn
down. Their property was confiscated. The descendants of the
partisans of Ibn-Hafsun maintained a correspondence with the
Castilian enemy. Alfonso of Aragon traversed the Peninsula from the
Ebro to the sea, at the invitation of the Mozarabes of Granada. Ten
thousand of the latter attended him in his retreat. The vengeance
exacted of their treacherous vassals by the Moors of that kingdom
was terrible. The expedition was productive of not less unhappy
results at Cordova. Nearly every church was destroyed, the
Christians were tortured, despoiled of their possessions, and
deported in a body to Africa.
At the beginning of the twelfth century, the misfortunes of the
maltreated sectaries had reached their culmination. The Almohades,
when not dominated by the marabouts, were inclined to be tolerant.
The Arab chronicles which treat of the Moorish principalities do not
mention the subject of persecution, and no Christian records of that
time have been preserved. The Mozarabes of the kingdom of
Granada enjoyed the largest liberty. In Sicily, during the entire period
of Moslem supremacy, martyrdoms were exceedingly rare.
Considering the widely extended apostasy which followed the
Arab conquest, it is remarkable, if viewed only from a worldly stand-
point, that the entire Christian population of the Peninsula did not
become Mohammedan. There is no doubt that those who remained
consistently steadfast in the faith were in a decided minority. No
inconsiderable number of proselytes was recruited from the patrician
class. Among the great body of serfs and slaves, there were few who
were not willing to renounce their religion for the certain enjoyment
of liberty and the flattering prospect of future ease or distinction.
The mass of the tributaries of the province of Seville had early
abandoned the Christian communion, and during the reign of Abd-al-
Rahman II. a magnificent mosque was built for their especial
accommodation. The majority of the prisoners taken in war
embraced without hesitation the doctrines of Islam. Leaving out of
consideration the influence of that Divine Power which must have
preserved its servants under the severest trials, circumstances of a
political or social character may have arisen to prevent the wholesale
apostasy of a nation.
And such was indeed the case. The treatment to which the
renegades were subjected is a single instance of many, most
important in determining the causes of the decline of proselytism. In
this class, the freedmen largely preponderated in numbers.
Notwithstanding the nominal equality of the renegade granted by his
former masters in the beginning, this equality was now never
conceded. The stigma of servitude which attached to the majority
became the unjust reproach of the caste. While many were sincere
in their belief, others took small pains to disguise the interested
motives which had prompted their conversion. The knowledge of this
fact impelled the Moslems to treat all converts with the greatest
indignity. They were publicly insulted. Opprobrious epithets were
heaped upon them. Even those whose ancestors had ranked with
the most distinguished of the Gothic aristocracy were not exempt
from the sneers of the Mussulman rabble. Possession of vast wealth,
reputation for genius, taste, or learning, afforded no immunity from
outrage by the vilest of mankind. It was rare that a renegade, no
matter how conspicuous his abilities, obtained a responsible office in
the government. Even the Christian stood a far better chance of
official promotion by the followers of the Prophet than the recent
proselyte to Islam. It was not in the nature of a numerous and
powerful caste, smarting under unmerited humiliation and conscious
of its strength, to calmly submit to such injustice. Nor was it long
before this destructive policy, which, like many of the evils that
afflicted the Mussulman domination, had its origin in Arab pride,
produced momentous political results. It encouraged treasonable
correspondence with the Christians of the North. It raised up spies in
every community. It provoked the bloody revolt of the southern
suburb of Cordova during the reign of Al-Hakem I. It recruited the
armies of Ibn-Hafsun, who for thirty years defied the power of the
khalifate. The renegades, who outnumbered all other classes
combined, lacked only organization and leadership to have driven
their haughty oppressors into the sea. When the power of the Arab
faction was destroyed, their condition was improved, but the ardor
of proselytism had vanished. Such experiences tended rather to
confirm than to weaken the faith of the hesitating.
Other causes contributed to the prevalent apathy. The semi-
theocratical character of the Moslem constitution implied to all
believers the active exertion of supernatural power. The head of the
government was at the same time the Successor and the
Representative of the Prophet. A system which claims divine
superiority should by all means be free from turmoil, from vices,
from schism; its infallibility should be demonstrated by the pre-
eminent wisdom of its decrees; its banners should never be lowered.
Yet Islam was rent by faction and controversy. Rival princes, on
every side, asserted their conflicting pretensions. In the confusion of
warring sects, it was always impossible to distinguish the heretic
from the orthodox. The Mussulman armies had often retired in
disgrace from before the half-savage and ill-equipped Asturian
mountaineers. Tried by the standards of mediæval ignorance,
standards founded upon unity of purpose and invincibility in war,
Islamism was no better than the creeds it had supplanted.
Again, the results of Moslem civilization, whose benefits were
apparent to the least discerning, were not derived from the efforts of
the devout. The theologians, without exception, were
obstructionists. They decried learning. They denounced philosophy.
To them the elegant pursuits of literature were an abomination. As a
rule, they had nothing in common with the scholars of Cordova,
renowned for their wit, their politeness, their culture. Their persons
were neglected, their manners uncouth, their language coarse,
ungrammatical, and insolent. In their opinion a madman was
inspired, and a scientific instrument a device of Satan.
Not so, however, with the eminent instructors who directed the
public mind of the nation, who imparted knowledge to eager pilgrims
from foreign lands. It was to their lectures that the young Christians
delighted to repair. There was no subject on which they were not
competent to discourse; no topic which they did not elucidate with
their learning and adorn with their eloquence. They were, almost to
a man, what would be called in our day agnostics. Some were
acknowledged atheists. Others inclined to the Pantheism of India.
None mentioned without a contemptuous smile the celestial origin of
the Koran or the claims of the Prophet to divine inspiration.
The University of Cordova was the seat of the literary faction
whose influence was long paramount in the empire. Although its
exercises were sometimes held in the Great Mosque, it had no
sympathy with religion or its ministers. Its infidel teachings had for
generations been the reproach of the pious faquis and the
abhorrence of the Catholic clergy. Its doors were open to the
studious of every race; its honors were bestowed upon the
meritorious scholar, without regard to his belief or his ancestry. In its
great library, the Mussulman, the Christian, the Buddhist, and the
Jew pursued their researches in generous rivalry or friendly co-
operation.
Under such unfavorable circumstances, it is not surprising that the
conversion of Christians to Islamism was permanently arrested.
Outrages upon proselytes, frequent insurrections, confusion of
doctrines, vulgarity of theologians, infidelity of those best qualified to
determine the value of established opinions, and the unrestricted
enjoyment of educational facilities were serious impediments, rather
than incentives, to a change of religious belief.
The fierce hostility that has always been manifested by the
Apostolic Church against every kind of profane learning—the
outgrowth of the tremendous power successfully exerted for many
centuries to degrade the mind, to pervert the understanding, to
dwarf the noble faculty of reason—had no terrors for the more
enlightened part of the Christian population of the khalifate. There,
in the presence of the unrivalled achievements of Moslem genius,
the stern intolerance of Patristicism could not stand before the liberal
policy of Islam and the daily application of the lofty sentiment of its
Prophet, “Whoso pursues the road of knowledge, God will direct him
to the road of Paradise. Verily, the superiority of a learned man over
a mere worshipper is like that of the full moon over all the stars!”
The exhibition of universal charity, of broad philanthropy, of
educational advantages impartially bestowed, as contrasted with the
narrow maxims of their own communion; the overwhelming
superiority of Mussulman civilization; the powerful influence of daily
intercourse and example; the prodigious augmentation of
commercial prosperity and worldly grandeur; the alluring prospect of
carnal pleasures, while they might not conduce to proselytism,
nevertheless undermined the faith and constancy of the Christian
youth.
The teachings of the philosophers of Cordova were not propitious
to the maintenance of either established dogma or ecclesiastical
superiority; and the clergy saw, with undisguised dismay, the
growing prevalence of lukewarmness and skepticism. The
predominance of the Spanish Arabs in every branch of literary
culture, their eminent success in arms, their intelligence, their valor,
their courtesy, the seductive power of their splendor and their
opulence had far more effect upon the minds of the rising
generation of Christians than the delusive promises and impotent
anathemas proclaimed every week from a thousand pulpits. And,
indeed, the contrast presented by the two rival religions was most
striking to the unprejudiced seeker after truth. On the one hand was
the church, with its resounding vaults and its gloomy and sepulchral
crypt; the monastery, with its privations; the reliquaries, with their
offensive hoards of withered flesh and mouldering bones; the
inconsistencies of a system which inculcated charity and commanded
persecution; the inexorable tyranny of the priesthood; the systematic
discouragement of learning; the confessional with its enforced
revelation of secrets; the mass with its monotonous services and its
ritual in an unknown tongue; the penance with its sufferings and
humiliation. On the other hand rose the mosque, light, airy,
beautiful; its graceful minaret pointing towards the heavens; its
court shaded by palm- and orange-trees, redolent with the mingled
fragrance of a thousand exotics, musical with the plashing of crystal
waters; its walls covered with a maze of intricate and brilliant
stuccoes; its ceiling emblazoned with the golden texts of the Koran;
its sanctuary sparkling with mosaics, whose exquisite tracery rivalled
the fabled creations of the genii; the sermon, intelligible to the most
humble and untutored listener; the prayer, remarkable for
earnestness, simplicity, reverence. On this side were exhibited the
factitious virtues and revolting license inseparable from the unnatural
condition of celibacy; the sacrifice of every diversion that renders
health attainable or existence attractive; the morose austerity of
monastic solitude; the ill-concealed excesses by which human nature
attempts to indemnify itself for the restraints imposed by organized
hypocrisy; the solicited martyrdom of the half-crazed zealot; the
savage pursuit of infidels and schismatics; the sanctified example of
ecclesiastical ignorance, moral abasement, and physical impurity. On
the other were the delights of the harem; the physical and mental
vigor derived from constant exercise of the muscular system and the
intellectual faculties; the benefits arising from the practice of
frequent ablution; the palatial appointments of the public bath; the
innumerable conveniences invented or adopted by a society ever
alert to grasp every new idea, to profit by every past experience; the
advantages of a method of education unparalleled in excellence and
unapproached by even the wisest teachers of antiquity; the vast
libraries, filled with the stores of ancient learning; the lectures of the
lyceum; the curious experiments of scientific observers; the
entertaining scenes of social festivity; the animated disputations of
learned assemblies.
The jurisprudence of the orthodox believer was basely subservient
to the claims of superstition. His cause was determined by the
uncertain results of judicial combat, by the oaths of prejudiced
compurgators, by the frivolous ordeals of water and fire. The sectary
of Mohammed was tried by the kadi, a magistrate governed by
established principles of law, and bound by religious as well as by
temporal considerations to an impartial administration of justice.
When a Christian became ill, attempts were made to exorcise the
evil spirit to which his sufferings were attributed by binding him to
the altar, by the invocation of saints, by the application of relics and
consecrated amulets. The Moslem was conveyed to the hospital
provided and maintained by royal beneficence; the cause of his
complaint was ascertained; and during his stay he received
gratuitously the assiduous attentions of the nurse and the intelligent
care of the surgeon.
While the priest-ridden peasantry of the Pyrenees and the Rhone
denounced the Saracen as a foe of God and a scourge to humanity,
the Christian who lived in security under his government, enjoyed his
favor, shared his hospitality, profited by his instruction, knew but too
well the calumny of these assertions, and that their maligned object
exhibited upon occasion all the noble attributes of a faithful friend
and a brave and chivalrous enemy. The dissensions of the Arabs, and
their ungenerous treatment of those who voluntarily embraced their
faith, were largely instrumental in preventing the amalgamation of
races, even then far on the way towards accomplishment. Had not
these causes intervened, only a few centuries would probably have
elapsed before the subject nation, already closely united with the
predominant caste by the bonds of marriage, consanguinity, and
interest, by intimate mercantile associations, by the powerful
influence of habits, education, and language, might have become
thoroughly Mohammedanized. As it was, a greater affinity always
existed between the Christian vassals of the Spanish khalifs and their
lords than between the members of the several factions of the Arabs
themselves, whose inextinguishable hatred, the fruit of countless
generations of hostility, eventually compassed the destruction of
their empire.
 CHAPTER XXVI
THE MORISCOES

1492–1609
State of the Kingdom after the Conquest—Superiority of the Moors—
Policy of the Crown—Introduction of the Holy Office—
Administration of Talavera—His Popularity—He is superseded by
Ximenes—The Two Great Spanish Cardinals—Their Opposite
Characters—Influence on Their Age—Violence of Ximenes—He
burns the Arabic Manuscripts—Insurrection of the Moriscoes—
Rout in the Sierra Bermeja—Bigotry of Isabella—The Moors under
Charles V.—Persecution by the Clergy and the Inquisition under
Philip II.—War in the Alpujarras—Ibn-Ommeyah—Operations of
Don John of Austria—Removal of the Moors of Granada—Death of
Ibn-Ommeyah—Ibn-Abu becomes King—Siege of Galera—
Atrocities of the Campaign—Fate of Ibn-Abu—Condition of the
Moriscoes in Spain—They are Exiled by Philip III.—Their
Sufferings—Effect of their Banishment upon the Prosperity of the
Kingdom.
The close of the Reconquest left the Spanish monarchy in a
condition of physical and financial collapse. The maintenance of a
great army for ten years, with the resultant casualties of battle,
exposure, and disease, had sensibly diminished the population. The
treasury had long been depleted. The Queen had pawned her jewels
to the bankers of Valencia and Barcelona. Wealthy subjects had been
induced to advance funds to the government by methods equivalent
to confiscation, and which held out but slender hopes of ultimate
reimbursement. National credit was practically destroyed. The
absence of the more industrious citizens in military service, the
incorrigible idleness of those who remained, had impaired the
pursuit of agriculture, upon which the resources of the kingdom
depended. Had it not been for the taxes and extraordinary
contributions levied upon the Jewish and Moslem tributaries, the war
could not have been prosecuted to a successful conclusion. These
two sects, which occupied an anomalous position in the body politic,
numbered over two million. Although so inferior in numbers, they
engrossed the trade and controlled the personal property of the
Peninsula. The Jew, who practised with enormous profit the
congenial but unpopular profession of usury, converted his gains into
money and jewels. The Mudejar, who, after the Conquest, gave
place to the Morisco, mindful of the Koranic precept which inculcates
industry as a virtue and stigmatizes idleness as a crime, was the
most laborious and successful of agriculturists, the most skilful of
artisans. Representatives of these two classes directed the
operations of the largest mercantile houses in the principal cities,
and the commerce of the entire country was practically in their
hands. Their prosperity was regarded with an evil eye by their
Castilian masters, and the Moslem was especially the object of this
animadversion. For generations the former had pursued the glorious
but brutalizing calling of arms. With them, every occupation that
implied or necessitated the performance of manual labor was
considered undignified and degrading. Centuries of unremitting
warfare had impressed upon the whole nation a military character,
with its inevitable concomitants of pride, tyranny, and insolence; and
these sentiments were intensified a hundred-fold by racial hatred
and sectarian prejudice. From the earliest times the Moors had been
regarded as interlopers, scarcely entitled to the ordinarily
indisputable rights of conquest. The acquisition of their domain by
Spanish prowess was always considered as the recovery of former
inalienable possession, not as new territory wrested from an
adversary by dint of superior strength and valor. The establishment
of the Catholic faith was, in the opinion of adroit casuists, an
additional argument in favor of their title, for it was held that the
consecration of altars to Christianity conferred rights which could
never be abrogated through occupation by infidels. With the
inconsistency of ignorance, the Castilians asserted their title both by
inheritance and prescription. They forgot that Spain had ever been
the rich prize for which almost every warlike nation of the ancient
world had contended. The Visigoths overran and ravaged it in the
fifth century, and their occupancy, derived solely from conquest,
lasted three hundred years. Then came the Saracens, whose
domination, obtained in precisely the same manner, required about
the same length of time for the conquest, but endured for more than
twice as long. It was evident, therefore, to every mind not obscured
by prejudice, that the title of the Moslems, even from the Spanish
point of view, was better than that of their conquerors. In more than
one respect, indeed, had the followers of Mohammed claims upon
the country of their adoption as well as upon the gratitude and
admiration of mankind. Their industry and enterprise had developed
beyond all precedent the wonderful resources of the Peninsula. Its
prosperity had never been so great, its people so happy, its
sovereigns so renowned, as at the meridian of the Moslem power. In
intellectual attainments, and the skilful adaptation of scientific
principles to the practical affairs of life, the subjects of the khalifate
far surpassed all their contemporaries. The civilization—if it is worthy
of the name—which the Saracens overthrew was infinitely inferior to
the one that they created. The Visigoths had scarcely emerged from
barbarism. Their monarchs attempted to emulate, in their
magnificence and luxury, the brilliant court of the Eastern Empire,
and to supply, by the splendor and richness of the materials, the
glaring deficiencies in skill and workmanship which characterized the
productions of their artisans. They never discarded the savage
customs engendered and perpetuated by ages of violence and
injustice. Sedentary and industrial occupations were repugnant to
the genius of a people whose national traditions from time
immemorial had breathed a spirit of truculence and war. And yet,
even in their chosen field, they at once demonstrated their inferiority
to an enemy who had hardly completed his apprenticeship in arms.
After the Conquest, the insignificant number of Christians saved
by the inaccessible fastnesses of the Asturias from Mohammedan
subjection had little left but their swords and their independence.
Their previous habits had unfitted them for labor. The ungenerous
nature of the soil and the severity of the climate offered few
inducements for tillage. They had, therefore, no resource but war by
which to maintain their existence and repair their broken fortunes.
Their children were reared in ignorance and under conditions
favorable to the development of the highest degree of ferocity and
fanaticism. They were taught to regard their enemies as monsters,
unworthy of the name and attributes of humanity, and having
nothing in common with the remainder of mankind but an erect form
and the capacity of speech. In the course of time, greater familiarity
with their adversaries insensibly produced a change of feeling, and
many of these absurd and unjust prejudices were modified or
entirely discarded. Numerous Mohammedan customs were adopted,
especially by the nobility of Castile, whose inherent profligacy
especially inclined them to the forbidden and unorthodox license of
the seraglio. Moslem kings were not infrequently appointed arbiters
of disputes between Christian princes of the blood. In arms, in
manners, in costume, in amusements, the despised infidel furnished
models to the proud and boorish descendants of Pelayus and his
mountaineers. Even the language was contaminated. Thousands of
terms familiar to the reader of the Koran were incorporated
unchanged into its comprehensive vocabulary, and the noble and
sonorous Castilian idiom remains to-day almost one-third Arabic. The
system of warfare, the evolutions of cavalry, the adoption of lighter
armor, all exhibited the effect of the pervading Moorish influence.
Architects from Granada were employed by Castilian monarchs in the
construction of palaces, and even by orthodox prelates in the
ornamentation of cathedrals. It was the custom of many sovereigns
in those turbulent times to intrust their safety to a body-guard of
Saracen mercenaries, who could neither be intimidated nor
corrupted. The honors paid to deceased Castilian royalty by the
Moslems were not inferior to those with which the obsequies of the
greatest emirs were celebrated. The court of Granada went into
mourning for Ferdinand III., and a guard of Moorish nobles escorted
his remains to the tomb. Henry IV. gave audience to ambassadors
seated upon a divan and supported by cushions, in the traditional
Saracen fashion. The tilt of reeds and the bull-fight, the exercises of
the grand arena, which, requiring the greatest address and agility,
were so popular with the Spanish chivalry, superseded the ruder and
more dangerous exhibitions of the tournament. In innumerable
examples, in every phase of the public and domestic life of the
Christians, the influence of Mohammedan association was
manifested. It is a curious fact, as already stated, that, in spite of
this, the deep-seated prejudices of the two races, so far from being
eradicated, were scarcely even perceptibly modified.
Notwithstanding intermarriages, the formal and elaborate display of
public courtesy, the frequency of appeals to royal arbitration, the
adoption of official ceremonials by one people, the voluntary
solicitation of protection by the other, all appearances of amity were
fallacious, and a feeling of irreconcilable hostility constantly prevailed
between the two races. Both reduced their prisoners to slavery, a
condition which generally implied the most inhuman treatment. The
captives taken by the Castilians were branded upon the forehead, a
mark of degradation which could never be erased; the slaves of the
Moslems were confined in damp and unwholesome dungeons, and
compelled to labor daily in the construction of mosques and
fortifications. It was no unusual occurrence, when a place had
provoked the animosity of either by an obstinate resistance, for the
entire population, irrespective of age or sex, to be ruthlessly put to
the sword. In the heat of conflict, quarter was seldom expected.
Despite the omnipresent and irrefutable evidences of superior
knowledge, refinement, and culture, the arrogant and conceited
Castilians always stigmatized their adversaries as barbarians. With
them, implicit belief in and attachment to the Roman Catholic faith
was the infallible touchstone of civilization. Whatever they did not
understand they attributed to magic. The mysterious accents of the
Arabic language, and the intricate manner in which its characters
were combined in the inscriptions which adorned the public edifices,
aroused in the minds of the ignorant suspicions of sorcery, with its
accompaniments of talismans, amulets, charms, and incantations.
The magnificent architectural works of Arab genius were attributed
to infernal agency, as beyond the efforts of unaided human power;
an opinion still entertained by the Spanish peasantry, who not only
firmly believe that the Moslem palaces were constructed by evil
spirits, but also ascribe the origin of the gigantic, and apparently
eternal, monuments of classic antiquity to the hands of the devil
himself.
Besides the inveterate prejudices arising from antagonistic faiths
and protracted warfare, other circumstances intervened to preclude
the fusion of the two races after the Conquest. The Spaniard, with
characteristic pride, asserted the superiority and predominance of
his race and origin, and the slightest suspicion of Moorish blood
constituted a blemish which no political or military distinction was
ever able to eradicate. The industry of the Mudejares, their frugality,
their clannishness, the seclusion of their women, aroused
unfavorable comment among a people whose prejudices associated
these practices with the name of an hereditary and implacable
enemy. It had long been a subject of universal complaint that the
larger proportion of the wealth of the kingdom was possessed by
these unpopular tributaries. The idle Castilian, whose ancestors had
for twenty-three generations subsisted by rapine, could not regard
with indifference the plodding industry that conferred upon a
subjugated and misbelieving race those substantial benefits which
he had always been taught to regard as the birthright of a Christian.
It was also publicly stated, to the prejudice of the tributary Moors,
that even when they renounced their faith they still adhered to their
former laborious habits; that none of them ever entered convents or
monasteries; and that their contributions to the Church were not of
the value to be expected from the zeal and generosity of sincere
proselytes. Their conversion did not bring with it that indulgence and
those privileges to which their ghostly instructors assured them they
would be entitled; it did not even confer immunity from insult. Until
the reign of Henry II. the Mudejares were exempt from the
inconvenience of wearing a distinctive mark indicative of their social
condition, which, long before imposed upon the Jews, was justly
considered a badge of ignominy. After that time, however, they were
required to wear upon their caps and turbans a blue crescent “of the
size of an orange,” which constantly brought upon them the affronts
of children, and not infrequently the taunts and violence of a
fanatical populace. In spite of the serious restrictions imposed upon
the Mudejares, and the enormous contributions levied upon their
industry, they continued to prosper, and at the time of the surrender
of Granada they were the most valuable subjects of the Spanish
Crown. Policy, based upon a sense of weakness, had long repressed
the avarice and envy of the Castilian sovereigns in their relations
with a class whose skill and labor were the principal sources of the
opulence of the realm. The time had now come when all restraint
could be cast aside without danger, and royal aggression, not only
sanctioned but suggested and encouraged by ecclesiastical authority,
could violate every obligation, human and divine, that had been
entered into with a conquered people, whose principal crime was
their prosperity, and whose independence had been voluntarily
relinquished under solemn treaties which had absolutely guaranteed
their personal safety and the unmolested exercise of their civil and
religious rights and privileges. A most pernicious maxim, but one
entirely consonant with the prevailing sentiments of the age, had
been recently adopted, and declared by the highest ecclesiastical
authority susceptible of unlimited application. This was that, the
original conquest of the Peninsula by the Moors partaking of the
nature of an usurpation, or rather of a theft obtained by violence, all
treaties or engagements entered into with the descendants of the
invaders were valid only so long as the Christians chose to observe
them, as having been dictated by necessity and contracted with
persons outside the pale of the law. The peculiar casuistry, which
deduced from Biblical precedent and the exterminating wars of the
Jews analogies whose application wrought such havoc among the
conquered nations of Spain and the New World, found no difficulty in
the acceptation of the broader, and consequently even more
atrocious, principle that no faith whatever was to be kept with
infidels. Ecclesiastical ingenuity has never invented more potent
weapons for the attainment of absolutism than these two maxims,
which, rigorously applied, demonstrated their temporary and
apparent efficacy by the utter extermination of millions of nominal
enemies of the Spanish monarchy.
 By the union of Castile and Aragon and the Conquest of Granada
national unity had been secured; it now remained to place the
religious establishment of the kingdom upon the same advantageous
footing. The Inquisition, an engine of tremendous power, whose
operations were attended by the most gratifying results, had, for
more than two centuries, been employed in subduing recalcitrant
heretics, procuring conversions, and replenishing the exhausted
coffers of Church and State. First introduced into Aragon from
France, its efforts were mainly directed against the Jews, whose
wealth had brought upon them a convenient suspicion of heresy.
The main objects of the Inquisition were in reality secular and
political. That hideous institution aimed at the establishment of
unquestioned sovereignty by the instruments of persecution.
Religious dogmas, while nominally of vital importance in its
procedure, were but pretexts by which the clergy, and indirectly the
orthodox monarch, profited in the acquirement and consolidation of
irresponsible authority. The stifling of human thought, the
suppression of every branch of knowledge, the prohibition of the
exercise of private judgment, the infinite multiplication of offences
against religion, the minute gradation of penances, many of them of
barbarous and incredible severity, were all means to the
accomplishment of one base and ignoble end. The theological aspect
of the Inquisition has engrossed the attention of historians to the
exclusion of its genuine but concealed objects. That the punishment
of heresy was not the real mission of its tribunal is proved by the
fact that its sentences were frequently suspended, commuted, or
abrogated by the sovereign, conditional on the payment of money.
The rich were the especial objects of its hostility; the denunciation of
a wealthy person was equivalent to conviction; and if a Hebrew or a
Moslem, he could hardly escape the extreme penalty. The mystery of
its organization, its unexpected arrests, its secret procedure, its
frightful dungeons, the fiendish cruelty of the tortures it inflicted,
and the atrocities of its public exhibitions—which partook of the
nature of religious festivals, and, with shocking inconsistency, were
supposed to be devoted to popular recreation—struck terror into
every community and every family.
 The successful prosecution of heresy by the Inquisition, as well as
the financial advantages it promised, and the increase of
ecclesiastical and royal power which followed its establishment,
appealed forcibly to the bigoted and arbitrary mind of the Spanish
Queen. Not so, however, with Ferdinand, whose experience with that
dread tribunal had caused him to regard its operations with disfavor,
and who had rendered his orthodoxy liable to suspicion by intrusting
to Jewish bankers the administration of the finances of the Crown of
Aragon. His remonstrances were, however, unheeded by his
obstinate and despotic consort. The Kingdom of Castile had always
enjoyed an unquestioned preponderance of authority and prestige in
the affairs of the Peninsula. The compact which consolidated the two
great realms into one empire expressly conferred upon Isabella the
exclusive control of all matters relating to ecclesiastical jurisdiction.
The right of presentation to benefices—long asserted by Castilian
princes as a royal prerogative, and whose exercise, denounced by
the Papacy as an usurpation, had repeatedly brought upon them the
censures of the Holy See—invested the Queen with a power of vast
and indefinable extent over the members of the Roman Catholic
hierarchy, who owed their offices to her generosity, and whose
revenues were largely dispensed in accordance with her advice. Her
policy and her apparent interest induced her, therefore, to consent to
the introduction of the Holy Office; and its tribunal was established
at Cordova, under the direction of Tomas de Torquemada, first
Inquisitor-General of the kingdom, a name of awful prominence in
the history of Spanish persecution.
The capitulation of Granada had been concluded with every
indication of sincerity, and with the most solemn assurances with
which it is possible to invest the provisions and confirm the faith of
treaties. The unsuspecting Moslems did not long remain in ignorance
of the duplicity of their conquerors. Excesses were publicly
committed by licentious cavaliers, who, instead of undergoing the
penalty of death adjudged for such offences, escaped with a gentle
reprimand, and were even conspicuously distinguished by the favor
of their royal mistress. The seclusion of domestic life, so jealously
guarded by Mohammedan custom, was unceremoniously invaded
upon the most frivolous pretexts by the rude and insolent soldiery.
The mosques, whose possession had been especially guaranteed by
the articles of the treaty, were one after another seized and
consecrated to the Christian worship. For these flagrant breaches of
trust, the stupid and remorseless bigotry of Isabella was largely
responsible. The city had hardly passed into the hands of the
conquerors, before the advisability of forcible conversion began to be
seriously discussed, and the Queen listened with pleasure to
suggestions of indiscriminate and compulsory baptism. The efforts of
priestly avarice and intolerance, secure in the royal support, began
to encroach more and more upon the acknowledged rights of these
unfortunate victims of persecution, until a revolution broke out,
which threatened the integrity of the newly acquired dominions, and
required the entire resources of the kingdom to suppress it. The
government of Granada had been left in the hands of three men,
whose excellent qualifications, previous experience, and inborn
sense of justice rendered them eminently qualified for the difficult
task to which they had been assigned. The famous Count of Tendilla
was appointed Captain-General of the province. The interests of the
Church were committed to Hernando de Talavera, Archbishop of
Granada, a prelate in whose mind fanaticism never attained
predominance over the noble impulses which assert the dignity of
human nature; and whose liberality, rare in his age and profession,
never refused indulgence and compassion to those of different blood
or hostile faith. To these two representatives of royal and
ecclesiastical authority was added as an adviser, and an interpreter
of the treaty of capitulation, which he himself had drafted, Hernando
de Zafra, secretary of the Catholic sovereigns, a man of talent,
intelligence, and spotless integrity, who enjoyed the confidence of
his superiors, and who, while conspicuously devout, was far less
tinctured with the prejudices of the time than his theological
education and previous associations would seem to imply.
Under the administration of these three dignitaries, the territory
of Granada once more assumed an appearance of prosperity. Their
probity won the confidence of the Moors, which had been shaken by
the arbitrary and indefensible proceedings following the surrender.
The capital, fallen into neglect and decay during years of
insurrection and war, was repaired; new streets were opened,
sanitary regulations were enforced, the markets were again crowded
with traders; the Vega, long the scene of desolation, began to
blossom once more under the patient hands of the industrious
laborer. While a high sense of honor and an unusual diplomatic tact
obtained for the Count of Tendilla the respect of his dependents, it
was upon the disposition of the Archbishop that the security of the
government and the pacification of the Moslems principally
depended. The first great difficulty was, in reality, not with the latter,
but with the Christian colonists, who had received, in recompense
for real or fictitious services, establishments in the city, and whose
licentious conduct provoked the animosity of the vanquished, and
rendered the streets unsafe at night for wayfarers of every
description.
The conduct of the Archbishop was beyond all praise. He
endeavored by every conceivable means to improve the condition of
his diocese, to revive decaying industry, and to promote the friendly
relations of the two races whose previous traditions made complete
fusion impossible. He dispensed at all times the most unbounded
and discerning charity. He caused public works to be inaugurated, by
which the needy poor were provided with employment. His apostolic
zeal never stooped to the violence of persecution; his appeals were
made to reason alone; and his subordinates, for the effectual
performance of their duties, were compelled to learn the Arabic
language, in which he himself, although far advanced in years,
became sufficiently proficient to employ it successfully for the noble
purposes of religious instruction. From the printing-presses,
established by his munificence, issued sumptuous volumes printed in
Castilian and Arabic, whose perusal might not only arouse the
interest of old believer and recent proselyte, but could not fail to
alike confirm the faith and facilitate the intercourse of both Christian
and Moslem. Under his direction schools were founded; rituals and
works embodying the doctrines and discipline of the Church
translated; and regular conferences organized, wherein, at stated
intervals, the comparative merits of the Christian and Mohammedan
creeds were publicly discussed by learned theologians of both
religions.
This excellent prelate, whose virtues are the more conspicuous
and admirable when contrasted with the generally dissolute
character of the ecclesiastics of the Spanish court, voluntarily
renounced the larger portion of the emoluments of his office,
reserving only what was sufficient for his immediate necessities, and
dispensing with the pomp which the dignitaries of the hierarchy were
accustomed to assume in the exercise of their calling. Two hundred
and fifty persons shared daily the hospitality of his table; his bounty
was enjoyed alike by officials of the highest rank, by Moors of every
degree, by pilgrims and travellers soliciting alms. In his visits to the
sick and the unfortunate he permanently impaired his health.
Recognizing the importance of a consistent example, he instituted
extensive reforms among the clergy. Their luxury was repressed,
their intemperate zeal restrained, the systematic observance of their
duties compelled, and those vices which had long been the scandal
of the pious were either entirely checked, or, driven from public
view, were forced into seclusion for their indulgence. In every
possible manner he attempted to relieve the oppressive burdens
imposed upon his parishioners by the fiscal regulations. His notaries
were forbidden to collect the fees, which formed an important part
of the revenues of the archiepiscopal see. He interposed his
authority to prevent illegal and oppressive exactions by the tax-
collectors. In his sermons, and by the exertion of his authority, he
discouraged the practice of professional mendicity, the scourge and
the disgrace of both Catholic and Mussulman countries.
With the secular and the ecclesiastical power vested in the hands
of such men as the Count of Tendilla and Hernando de Talavera, the
greatest results could not fail of accomplishment. The manners of
the Spaniards were insensibly reformed. Such was the public
tranquillity, that a mere handful of soldiers sufficed for the garrison
of the Alhambra and the guard of the captain-general. The pious and
unselfish example of the Archbishop soon bore fruit. Great numbers
of Moors voluntarily signified their desire to become Christians. In
one day three thousand were baptized, not one of whom ever
afterwards recanted. These conversions were not obtained through
suggestions of temporal advantage or the influence of fear; nor were
the proselytes admitted to communion without previous instruction
in the doctrines they were expected to profess or the duties they
would be required to perform. The affection and respect of the
Moslems for their instructor and friend were unbounded. They called
him the “Holy Faqui of the Christians.” The churches were found
unable to accommodate the increasing numbers of converts, and
altars and pulpits were erected in the three principal squares of the
city; the nightly brawls excited by the turbulent soldiers of fortune,
domiciled by the Conquest in the Moorish capital, became more and
more infrequent; a sense of security began to prevail in the
community; the relations of noble and vassal were modified, to the
decided advantage of the latter; ancient prejudices, confirmed by
the enmity of centuries, were softened; and the political union of the
two peoples, which could only be effected by a just and conciliatory
policy, and upon which, in fact, depended the future prosperity of
the Peninsula, seemed at length to offer a flattering prospect of
realization.
Under these favorable auspices, for the space of several years,
order, tranquillity, and contentment reigned in Granada. The
courteous and equitable, but firm, administration of the governor;
the blameless life, the humble piety, the sympathetic interest of the
Archbishop had awakened the love and compelled the obedience of
the tributary Moslems, who compared with wonder and gratification
the operation of a system of kindness and justice with the arbitrary
and violent measures of the despotism to which they had heretofore
always been accustomed. During that period many important and
tragic events transpired. Al-Zagal, oppressed with years and
calamities and broken in spirit, had gone into voluntary exile.
Boabdil, by means of an ignoble and treacherous device, whose
adoption was alike unworthy of a monarch and a Christian, had been
deprived of the principality for which he had bartered his crown and
forced to retire into Africa. Every important provision of the
capitulation had been repeatedly violated, and only the tact of those
who controlled the government of Granada had prevented the most
serious consequences. The Jews, under circumstances of
unspeakable cruelty, had been expelled from the kingdom. In the
hierarchy changes had taken place which boded no good to the
heretic and the suspected apostate. Cardinal Mendoza, Primate of
Spain, had died, and Francisco Ximenes de Cisneros, a Franciscan
friar and the confessor of the Queen, had been promoted to that
exalted dignity, whose power and emoluments rivalled those of the
crown. The life, the associations, the studies of this man had
developed a mind whose feelings were in perfect accordance with
the narrow and intolerant spirit of the age. Without indulgence for
the inherent weakness of human nature, without patience to await
the effect of the deliberate and rational methods of discussion which
promote religious conviction, absolutely devoid of generosity, of
tenderness, of sympathy, he regarded unquestioning obedience to
the Church as the most imperative of all obligations and mortification
of the flesh as the most meritorious of virtues. He had recently
secured the appointment of Diego de Deza, one of his creatures, to
the place of Inquisitor-General, which gave him absolute control of
the operations of the Holy Office.
The characters of the two great churchmen who in succession
dictated the policy of the crown, though widely different in many
respects, in general faithfully represent the prevalent ideas and
aspirations of every class of society in the kingdom. The aim of both
was religious unity, which during the long crusade against the infidel
had usurped the place and depreciated the worth of patriotism. Both
governed the sovereign, and with the sovereign the monarchy. Both
filled the highest ecclesiastical office in the Peninsula, an office
second in dignity and power only to the Papacy. Both were zealous
patrons of the Inquisition. One recommended the expulsion of the
Jews. The other inaugurated the persecution of the Moriscoes. Both
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