VISION

Why don’t we make it simple and comprehensive?! All high

yield concepts are included in an ordered, simple manner with
illustrated videos.

Ahmed Shebl
USMLE STEP 1

Biochemistry

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 USMLE ENDPOINT SERIES BY DR AHMED SHEBL Biochemistry

INTRO:

 Comprehensive explanation for ALL HIGH YIELD topics and Question banks in USMLE step
1.
 ONE BOOK for
1. First aid book
2. USMLE World QUESTION bank
3. Pathoma
4. High yield principles in Kaplan qbank
5. Boards & beyond.
6. Important notes from Kaplan videos
7. Becker videos & qbank
8. Illustrated SIMPLE images & slides.
 Topics arranged in systemwise manner according to the first aid book.
 Available flashcards for every single information will be provided on the group.
 Evaluation questions for every online session.
 You can have simple, illustrated explanation for every single information through ONLINE
COURSE subscription.

AUTHOR

 Dr. Ahmed Shebl,

1. Cardiothoracic surgery resident,
2. Scored 248 on USMLE step one.
3. 2 year-experience in teaching USMLE STEP 1.

CONTACT US

 for more information, explanation videos and online course subscription at this link:
 FB group: https://www.facebook.com/groups/173082529766502/
 FB account: https://www.facebook.com/ahmed.shebl1
 E-mail: [email protected]
 Mobile: 00201060690750

DEDICATION

To my Mom and Dad, no words can explain what u did for me.

With special gratitude to Eman, the best wife I can imagine. You have been a gift from the beginning.

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MOLECULAR BIOLOGY
OVERVIEW:
 Replication is the process by which DNA is duplicated. This process is undertaken
prior to cell division to ensure that all daughter cells contain the same genetic
information as the parent cell.
 The information in DNA is transmitted to RNA via a process called transcription.
 RNA synthesizes protein through the process of translation, which largely controls
the appearance and function (i.e., the phenotype) of cells and organisms.

NUCLEIC ACID STRUCTURE

 DNA and RNA are the two major types of nucleic acids.
 They are composed of nucleotides which are molecules with three distinct parts:
 A five-carbon sugar, either ribose or deoxyribose. The carbons of the pentose
sugar are numbered 1' through 5', clockwise, beginning with the carbon
attached to the nitrogenous base.
 A nitrogenous base.
 One or more phosphate groups (attached to the 5' pentose carbon).

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 How to recognize the sugar on the diagram? It has no double bond.

 The 2' position differentiates between ribose & deoxyribose. If it has OH group, it is ribose.
If not  deoxyribose.
 The 3' position  attaches to the next nucleotide.
 The 5' position  attached to phosphate.
 The phosphate group links the 2 adjacent nucleotides by 3′-5′phosphodiester bond.

 The nitrogenous base: There are two types of nitrogenous bases found in
nucleic acids: purines and pyrimidines.
1. The purines:
 Include adenine [A], guanine [G], xanthine, and
hypoxanthine.
PURines (A,G)  PURe As Gold.
 Composed of two rings.
 Deamination of adenine (contains amino group) makes
guanine.
 Uric acid is a metabolic breakdown product of purines.

2. The pyrimidines:
 Include cytosine [C], thymine [T], and uracil [U].
PYrimidines (C,U,T)  CUTthe PY(pie).
 [3 pyramids  3 bases].
 Composed of only one ring.
 Thymine has a methyl.
 Uracil
o A precursor for both thymine and cytosine.
o Amination of uracil makes cytosine.
o Methylation of uracil makes thymine.
 The methyl group is given by folate. So, in
folate deficiency  ↓thymine  ↓ DNA 
blasts fail to mature  megaloblastic anemia.
 5-FU inhibits methylation.
 Uracil found in RNA; thymine in DNA.

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 NucleoSide = base + (deoxy)ribose (Sugar).

 Nucleosides are named according to the attached nitrogenous base.
 These are: Adenosine, Thymidine, Guanosine, Uridine, and Cytidine.
 NucleoTide = base + (deoxy)ribose + phosphaTe; linked by
3′-5′phosphodiester bond.
 Named according to the number of attached phosphates. For example, the
nucleotides formed from the nucleoside adenosine include:
1 phosphate = adenosine monophosphate (AMP).
2 phosphates = adenosine diphosphate (ADP).
3 phosphates = adenosine triphosphate (ATP).

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BASE PAIRING

 Guanine and cytosine (G-C pair)  three

hydrogen bonds.
 Adenine and thymine (A-T pair) in DNA
 two hydrogen bonds.
 Adenine and uracil (A-U pair) in RNA 
two hydrogen bonds.
 G-C bond (3 H bonds) stronger than A-T
bond (2 H bonds).
 ↑ G-C content  ↑ melting temperature
of DNA. ―C-G bonds are like Crazy
Glue.‖
 Complementary base pairing character:
 In a double stranded DNA molecule, the total number of (A) nucleotides
is equal to the total number of (T) nucleotides.
 Similarly, the total number of (G) nucleotides is equal to the total number
of (C) nucleotides.

 RNA is usually single-stranded (an important exception

is the class of dsRNA viruses discussed in Microbiology).
 RNA base pairing is typically intramolecular,
that is, one part of a strand pairs with another
part of the same strand.
 This allows individual RNA molecules to form
unique three-dimensional structures such as the
hairpin loop shown in Figure (G-C rich
region).
 Hairpin loop will stop the transcription in
prokaryotes.
 Remember the RNA base pairs are G-C and A-U.

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Q1. A molecular biologist in lab is analyzing the DNA, founded that 10% G, 30% C, 40% A, 20% T.
What is the most likely structure?
A. Bacterial chromosome.
B. Viral genome.
C. Plasmid.
D. Mitochondrial chromosome.
E. Nuclear genome.

Answer: the correct answer is B. As the percentage of G is not equal to C and also A% ≠ T% so, it is not a
nuclear genome  not a dsDNA. Instead it is a single stranded nucleic acid (ssDNA as it has thymine not
uracil) which is a feature of some viral genomes. Bacterial chromosome, plasmid and mitochondria 
dsDNA.

Q2. In HCV genome, if there is 30% G in the sample:

A. 30% C
B. 25 % A
C. 35 % T
D. 35 % U
E. can’t answer.
Answer: the correct answer is E. we can’t predict it as HCV is NOT double stranded rather it is ssRNA.
HBV instead is a dsDNA virus.

CHROMATIN STRUCTURE:
 DNA exists in the condensed, chromatin form in order to fit into the nucleus.
 Negatively charged DNA loops twice around positively charged histone octamer to
form nucleosome.
 The nucleosome consists of DNA wrapped around a complex of proteins called
histones. ―Beads on a string.‖
 Histones are rich in the amino acids lysine and arginine  giving the histones its
+ve charge.
 H1 binds to the nucleosome and to ―linker DNA,‖ thereby stabilizing the
chromatin fiber.
 H1 helps to organize the DNA into a polynucleosome or nucleofilament.
 It makes it more condensed (30 nm)  resistant to nucleases.
 In mitosis, DNA condenses to form chromosomes.
 DNA and histone synthesis occur during S phase.

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CHROMATIN
 DNA + packaging proteins.
 Chromatin is found in two distinct forms: Heterochromatin & euchromatin.
 10 nm chromatin  (H2A, H2B, H3, H4)2
 30 nm chromatin  H1 + (H2A, H2B, H3, H4)2

HETEROCHROMATIN
 Condensed (HeteroChromatin = Highly Condensed.)
 Appears darker on EM (labeled H in A).
 Transcriptionally inactive, sterically inaccessible, less likely to be digested by
endonucleases.
 ↑ Methylation, ↓ acetylation.
 Examples:
 Barr bodies (inactive X chromosomes).
 Centromeres.
 Nuclear membrane.

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EUCHROMATIN
 Less condensed. Appears lighter on EM (labeled E in A).
 Transcriptionally active, sterically accessible.
Eu = true, ―truly transcribed.‖ Euchromatin is Expressed.

DNA METHYLATION
 Template strand cytosine and adenine are methylated in DNA replication, which
allows mismatch repair enzymes to distinguish between old and new strands in
prokaryotes.
 DNA methylation at CpG islands represses transcription.
 Example: genomic imprinting.
 CpG Methylation Makes DNA Mute.

HISTONE METHYLATION
 Usually reversibly represses DNA transcription, but can activate it in some cases
depending on methylation location.
 Histone Methylation Mostly Makes DNA Mute.

HISTONE ACETYLATION
 Histone acetylation  ↑ negative charge on histones  ↓ attachment to the (–ve)
DNA  ↓ condensation  more active  allowing gene expression. (the same is
phosphorylation).
 Relaxes DNA coiling, allowing for transcription.
 Histone Acetylation makes DNA Active.

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Q. Endonucleases activation leads to fragmentation of DNA in apoptosis. Which of the

following gets broken first?
A. Barr body.
B. Nucleosomes.
C. Polynucleosomes.
D. Centromeres.

Answer: the correct answer is B. Nucleosomes are less condensed (10 nm fiber) as they have no
H1 making them more susceptible for endonucleases. Polynucleosome is thick 30 nm fiber more
resistant to digestion. Barr body is the inactivated X-chromosome in the females which is very
condensed  not expressed and less likely to be digested. Centromeres are heterochromatin in the
center of the chromosomes (highly condensed). Nuclear membrane is the dark area around the
nucleus because it is heterochromatin  highly condensed.

Mitochondrial DNA (mtDNA):

 The most common non-nuclear DNA found in eukaryotic cells.
 It resembles prokaryotic DNA  small circular chromosomes.
 Maternally derived. As a result, diseases arising from mutations in mtDNA are
transmitted from the mother to all of her offspring.
 Mitochondria can be identified on electron microscopy by their characteristic
double membrane and wavy cristae.
 Mutations involving mtDNA or nuclear DNA that codes for mitochondrial proteins
can cause a variety of mitochondrial disorders, including Leigh syndrome and
MELAS.

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DE NOVO PYRIMIDINE AND PURINE SYNTHESIS

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 What are the amino acids needed for pyrimidine & purine synthesis?
 For pyrimidine synthesis  Aspartate & glutamine.
 For purine synthesis  Glycine & Aspartate & glutamine.
 What is the precursor for all pyrimidines?
 Orotic acid.
 What is the 1st nucleotide synthesized in pyrimidine pathway?
 UMP.
 What is the rate limiting step in purine synthesis?
 PRPP amidotransferase.

Various immunosuppressive, antineoplastic, and antibiotic drugs function by

interfering with nucleotide synthesis:
 Disrupt pyrimidine synthesis:
 Leflunomide:
 Inhibits dihydroorotate dehydrogenase.
 Methotrexate (MTX), trimethoprim (TMP), and pyrimethamine:
 Inhibit dihydrofolate reductase (↓ deoxythymidine monophosphate
[dTMP]) in humans, bacteria, and protozoa, respectively.
 5-fluorouracil (5-FU):
 Forms 5-F-dUMP, which inhibits thymidylate synthase (↓ dTMP).
 Disrupt purine synthesis:
 6-mercaptopurine (6-MP)and its prodrug azathioprine:
 Inhibit de novo purine synthesis by ↓PRPP amidotransferase.
 Mycophenolate and ribavirin:
 Inhibit inosine monophosphate dehydrogenase.
 Disrupts purine and pyrimidine synthesis:
 Hydroxyurea:
 Inhibits ribonucleotide reductase.
 At lower doses, it can be used for sickle cell anemia patients by
↑HbF which prevents sickling.

When sulfamethoxazole is added to trimethoprim, the effect is a synergistic inhibition of

tetrahydrofolate synthesis through two different steps:
- Sulfamethoxazole inhibits PABA  folic acid.
- Trimethoprim inhibits DHF  THF.

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Hereditary orotic aciduria:

 Cause:
 Autosomal recessive disorder of de novo pyrimidine synthesis (↓UMP
synthase).
 C/P:
 Physical and mental retardation (eg. low height/weight, delayed developmental
milestones).
 Megaloblastic anemia (eg. elevated mean corpuscular volume, low reticulocyte
count) due to ↓ hematopoiesis from ↓pyrimidine synthesis.
 Elevated urinary orotic acid levels  crystallizes (orange color crystals in the
diaper of the baby), and causes urinary obstruction
 Treatment:
 Uridine supplementation can bypass this enzymatic defect and improve
symptoms as uridine is converted to UMP via nucleoside kinases.
 Differential diagnosis of orotic aciduria:

Hereditary orotic aciduria Ornithine transcarbamylase deficiency

Autosomal recessive X-linked recessive

Defect in pyrimidine synthesis (↓UMP Defect in urea cycle

synthase)
Megaloblastic anemia No megaloblastic anemia

No hyperammonemia hyperammonemia

Folate deficiency:
 Folate deficiency  ↓ thymidylate synthase  ↓dTMP which limits DNA synthesis and
promotes megaloblastosis and erythroid precursor cell apoptosis.
 Because thymidine supplementation can moderately increase dTMP levels, it can reduce
erythroid precursor cell apoptosis.

Gout occurs with increased frequency in patients with activating mutations involving PRPP
synthetase due to increased production and degradation of purines.

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PURINE SALVAGE DEFICIENCIES

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ADENOSINE DEAMINASE DEFICIENCY:

 Autosomal recessive.
 ADA is required for degradation of adenosine and deoxyadenosine.
 In ADA deficiency, ↑ dATP  toxicity in lymphocytes.
 One of the major causes of autosomal recessive SCID.

LESCH-NYHAN SYNDROME:
 X-linked recessive.
 Pathogenesis:
 Defective purine salvage due to absent HGPRT, which converts
hypoxanthine to IMP and guanine to GMP  ↓AMP & IMP & GMP 
no negative feedback inhibition on PRPP amidotransferase  ↑ Activity
of PRPP amidotransferase to supply a sufficient quantity of purine
nucleotides  excess uric acid production.
 ↓HGPRT  ↓GTP; a cofactor for THB  ↓THB; a cofactor for aromatic
amino acids hydroxylases  ↓dopamine in brain  spastic cerebral
palsy & self-mutilation.
HGPRT:
 Findings:
 Intellectual disability, self-mutilation, Hyperuricemia, Gout, Pissed
off (aggression, self-
aggression, dystonia.
mutilation), Retardation
 Hyperuricemia (orange ―sand‖ [sodium
(intellectual disability),
urate crystals] in diaper), gout. DysTonia.
 Treatment:
 Allopurinol.
 Febuxostat (2nd line).
 Rasburicase.

Rasburicase:
Recombinant version of urate oxidase, an enzyme present in many mammals, but not in humans.

It catalyzes the conversion of uric acid to allantoin which is 5-10 times more soluble than uric
acid.

Rasburicase is effective in preventing and treating hyperuricemia and the resulting renal
manifestations of tumor lysis syndrome.

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Predisposing factors for Hyperuricemia:

 Weak acids compete with uric acid for excretion  hyperuricemia
 Lactic acidosis (chronic alcoholism).
 Drugs: loop & thiazide diuretics and aspirin.
 Phosphate trapping: Conditions with lack of phosphorus 
hypothanxine can’t be converted to IMP as in
 Von-Gierk disease.
 Galactosemia.
 Hereditary fructose intolerance.
 Glucose 6-phosphatase deficiency.

GENETIC CODE FEATURES:

UNAMBIGUOUS:
Each codon specifies only 1 amino acid.

DEGENERATE/REDUNDANT:
Most amino acids are coded by multiple codons.
There are 61 codons that code for only 20 amino acids.
Wobble:
 Codons that differ in 3rd, ―wobble‖ position may code for the same
tRNA/amino acid.
 Specific base pairing is usually only required in the first 2 nucleotide positions
of mRNA codon.
 For instance, the codons GGU, GGC, GGA and GGG all correspond to the
amino acid glycine.
 Each tRNA molecule is specific for a given amino acid. Many tRNA
anticodons can bind to a few different codons coding for the same amino acid.
Exceptions:
 Methionine and tryptophan encoded by only 1 codon (AUG and UGG,
respectively).

COMMALESS, NONOVERLAPPING:
Read from a fixed starting point as a continuous sequence of bases.
Exceptions: some viruses.

UNIVERSAL
Genetic code is conserved throughout evolution.
Exception in humans: mitochondria.

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DNA REPLICATION
 Eukaryotic DNA replication is more complex than the prokaryotic process but uses
many enzymes analogous to those listed below.

Origin of replication (Ori)

 Particular consensus sequence of base pairs in genome where DNA
replication begins. AT-rich sequences (such as TATA box regions) are found
in promoters and origins of replication.
 May be single (prokaryotes) or multiple (eukaryotes).

Replication fork
 Y-shaped region along DNA template where leading and lagging strands are
synthesized.
Helicase
 Unwinds DNA template at replication fork by breaking the hydrogen bonds
between the nitrogenous bases. Helicase Halves DNA.

Single-stranded binding proteins

 Prevent strands from reannealing.

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DNA topoisomerases
 Create a single- or double-stranded break in the helix to add or remove
supercoils.
 Topoisomerase I  cut one strand of the double helix supercoiled DNA.
 Topoisomerase II  cut 2 strands of the double helix supercoiled DNA.

 Irinotecan/topotecan inhibits eukaryotic topoisomerase I.

 Etoposide/teniposide inhibits eukaryotic topoisomerase II.
 Fluoroquinolones inhibit prokaryotic topoisomerase II (DNA gyrase) and
topoisomerase IV.

Primase (DNA-dependent RNA polymerase)

 Makes an RNA primer on which DNA polymerase III can initiate
replication.
 The primer: Short piece of RNA with an open 3' hydroxyl, which the DNA
polymerase can elongate.
 RNA primers are required for replication but not transcription.
 RNA primers are removed later on by DNA polymerase I.

DNA polymerase III

 Prokaryotes only. Elongates leading strand by adding deoxynucleotides to the
3′end.
 DNA polymerase uses the 3 ' hydroxyl group of the RNA primer as a starting
point for synthesis.
 Elongates lagging strand until it reaches primer of preceding fragment.
 Has 5′  3′ synthesis and proofreads with 3′  5′exonuclease.
 3′  5′ exonuclease activity ―proofreads‖ each added nucleotide.
 Drugs blocking DNA replication often have a modified 3′OH, thereby
preventing addition of the next nucleotide (―chain termination‖).

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The 5'3' exonuclease activity

removes RNA primers as well
as damaged DNA segments. (Only
DNA polymerase I).

The 3'5' exonuclease activity

performs a proofreading function. (All
three prokaryotic DNA polymerases).

DNA polymerase I
 Prokaryotic only.
 Degrades RNA primer; replaces it with DNA.
 Same functions as DNA polymerase III, also excises RNA primer with 5′ 
3′ exonuclease.
DNA ligase
 Catalyzes the formation of a phosphodiester bond within a strand of double-
stranded DNA.
 Joins Okazaki fragments. Ligase Links DNA.
Telomerase
 Eukaryotes only.
 A reverse transcriptase (RNAdependent DNA polymerase) that adds DNA
(TTAGGG) to 3′ ends of chromosomes to avoid loss of genetic material with
every duplication.
―Telomerase TAGs for Greatness and Glory‖.
 Often dysregulated in cancer cells, allowing unlimited replication.

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 In both prokaryotes and eukaryotes, DNA replication is:

 Semiconservative: meaning that each daughter molecule of DNA consists of
one strand of parental DNA and one strand of newly synthesized daughter
DNA.

 Involves both continuous and discontinuous (Okazaki fragment) synthesis.

 Occurs in the 5′  3′direction.

DNA POLYMERASES

Prokaryotic (Bacterial) Cells Eukaryotic Cells

DNA polymerase I  degrades the RNA DNA polymerase α  Priming and initial
primers (has a 5′ exonuclease activity) and synthesis.
fills in the resulting gap.
DNA polymerase II  participates in DNA DNA polymerase β  DNA repair.
repair.
DNA polymerase III  does the majority DNA polymerase γ  mitochondrial DNA
of synthesis and proofreading. replication.
DNA polymerase δ  Lagging strand
synthesis.
DNA polymerase ε  Leading strand
synthesis and also DNA repair.

 Nucleases break phosphodiester bonds (not phosphodiesterases!!)

 Exonucleases remove nucleotides by breaking the phosphodiester bond of the
first (5'  3') or last (3'  5') nucleotide in a strand of DNA.
 Endonucleases break phosphodiester bonds in the middle of a DNA strand.

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TELOMERES (EUKARYOTES)

 Long stretches of repetitive sequences (TTAGGG) at the end of chromosomes.

 Telomeres are progressively shortened with each cell division.
 When the length is exhausted, the cells often become quiescent or undergo apoptosis
(programmed cell death).
 Thus, the length of telomeres is one factor that determines the life span of a cell.
 The human genome includes a gene that encodes the enzyme telomerase which is a
human reverse transcriptase (RNA dependent DNA polymerase) which has a "built-
in" RNA template.
 If this gene is expressed in a cell, telomerase will be able to complete the
replication of the telomeres so that the chromosomes in the cell will not
shorten, thus conferring on the cell a sort of immortality.
 This is advantageous in several circumstances:
 During embryonic and fetal life, when very high rates of cell division are
required to form a healthy newborn from a single fertilized ovum.
 Throughout life in stem cells that may also have a high rate of cell division,
such as the pluripotent stem cells that replace red and white blood cells.
 In many types of cancer cells, the gene for telomerase has been re-activated
inappropriately.

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Syndromes of premature aging such as Bloom syndrome are associated with shortened telomeres.

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TRANSCRIPTION
 Process by which an RNA copy is made from a DNA
template.
 Conceptually, the process is similar to making multiple
copies of a page (the RNAs) from a book (the DNA
gene), using a copying machine (an RNA polymerase).
 Actinomycin D or α-amanitin block transcription by
inhibiting RNA polymerase II from elongation of the
transcription.

Amatoxins are found in a variety of poisonous mushrooms (eg. Amanita phalloides,

known as death cap) and are potent inhibitors of RNA polymerase II (halting mRNA
synthesis).
Symptoms typically start 6-24 hours after ingestion and include abdominal pain, vomiting, and
severe, cholera-like diarrhea that may contain blood and mucus.
Severe poisoning can lead to acute hepatic and renal failure.
Urine testing for α-amanitin can confirm suspected amatoxin poisoning.

 In prokaryotes, single RNA polymerase that forms all types of RNA.

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 TYPES OF RNA

1. Heterogeneous nuclear RNA (hnRNA) and messenger RNA (mRNA):

a. Initial transcript is called heterogeneous nuclear RNA (hnRNA).
b. Transcribed by RNA polymerase II.
c. hnRNA is then modified and becomes mRNA.
d. The following processes occur in the nucleus:
i. Capping of 5′ end (addition of 7-
methylguanosine cap)
ii. Polyadenylation of 3′ end (≈ 200 A’s).
iii. Splicing out of introns.
e. Capped, tailed, and spliced transcript is called mRNA.
f. mRNA is transported out of the nucleus into the cytosol, where it is translated.
g. mRNA quality control occurs at cytoplasmic processing bodies (P-bodies),
which contain exonucleases, decapping enzymes, and microRNAs; mRNAs
may be stored in P-bodies for future translation.
h. Poly-A polymerase does not require a template.
AAUAAA = polyadenylation signal.

Transport
• Capping outside the Translation
Transcription or
DNA
by RNA pol II
hnRNA • Polyadenylation mRNA nucleus to processing
• Splicing
the by P-bodies
cytoplasm

2. Ribosomal RNA (rRNA):

a. Important structures in protein synthesis (translation).
b. Transcribed in the nucleolus by RNA polymerase I except the 5S rRNA,
whose gene is transcribed by RNA polymerase III in the nucleoplasm.

3. Small nuclear RNA (snRNA):

a. This RNA is produced by RNA polymerase II and combines with protein
components to form snRNP (small nuclear ribonucleoprotein).
b. These snRNPs, also known as spliceosomes, catalyze the removal of introns
from the primary transcript.
4. Transfer RNA (tRNA):
a. tRNA is responsible for decoding RNA during translation to form proteins.
b. Transfer RNA is produced by RNA polymerase III.

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 GENES AND NON-CODING REGIONS OF DNA

DNA

Coding Non coding

regions regions

Responsible for regulation

Genes which are of gene expression (when,
expressed where, how much gene
are expressed)

 GENE STRUCTURE

 A gene consists of:

 Transcription unit
 Promoter
 Introns & Exons
 Enhancers and silencers
 Terminator

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THE TRANSCRIPTION UNIT:

 Begins with the first nucleotide introduced into the RNA, by convention
referred to as the +1 base.
 If a nucleotide lies before (5' or upstream) the +1 base, it is given a negative
number and it is not transcribed into the new RNA. Similarly, if the
nucleotide has a positive number, you should recognize that it is within the
transcription unit and will be found in the RNA transcript.
 DNA coding strand is identical to the mRNA (except T for U).
 DNA template is complementary and antiparallel to the mRNA.

PROMOTER
 Most of our DNA is non-coding. The RNA polymerase needs a marker for
these small coding regions. This marker is the promoter; the site where RNA
polymerase II and multiple other transcription factors (general transcription
factors) bind to DNA.
 On/Off switch regulator of gene expression  Gene transcription begins
when RNA polymerase II attaches promoter site in a process facilitated by
numerous general transcription factors (In eukaryotes, RNA polymerase II
alone is unable to recognize the TATA box).
 Promoter mutations  ↓gene expression by altering the ability of
RNA polymerase II and transcription factors to bind.
 Though promoters are not directly translated into protein,
 The promoter is approximately 100 bp long with two important sequences:
 TATA box:  25 bp before (5' or upstream) the transcription start
site.
 CAAT box:  75 bp before (5' or upstream) the transcription start
site.

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INTRONS VS EXONS:
 Exons (Expressed sequences) contain the actual genetic information coding for
protein.
 Introns (Intervening sequences) are intervening Introns are intervening
noncoding segments of DNA. sequences and stay in the
 Different exons are frequently combined by alternative nucleus, whereas exons exit
and are expressed.
splicing to produce a larger number of unique proteins.
 Abnormal splicing variants are implicated in
Between the 1st exon and +1
oncogenesis and many genetic disorders (eg, β-
thalassemia). bp there is an untranslated
region is called 5` UTR.

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ENHANCERS AND SILENCERS (MODULATORS)

 Enhancers
1) DNA sequences that bind specific proteins Promoter is ON/OFF switch
called transcription factors (specific). whereas enhancers and
2) Location (anywhere): silencers are modulators of
 Enhancers can be located upstream
gene transcription.
or downstream from the gene being
transcribed, and may be near the
gene or thousands of base pairs away (Enhancers have been
identified both within introns of the gene being transcribed as well
as on separate chromosomes).
3) When an enhancer binds with a transcription factor, the presence of the
complex increases the possibility that RNA polymerase will bind to the
promoter and begin transcription of that gene.

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 Silencer
1) Has the opposite effect on transcription.
2) When a silencer sequence binds with a transcription factor, the probability
of RNA polymerase binding with a promoter decreases and thus the rate of
transcription of that gene decreases.

TERMINATOR
 Transcription stops at a terminator sequence.
 Transcription termination in prokaryotes:

Rho-independent (intrinsic) Rho-dependent

Rho protein is not used. Rho protein is used.

Terminator sequence causes the RNA to When Rho protein is formed, it

form a hairpin stem-and-loop structure causes RNA polymerase to
that makes RNA pol to stop transcription. dissociate from DNA.

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Q. What is correct about promoter? The answer is B. promoter isn’t transcribed,

A. It will be transcribed but not translated.
translated or expressed but rather replicated as a
B. It is replicated.
C. It is expressed. portion of the DNA. Promoter does not exist more
D. It is not transcribed but translated. than -100 bp upstream.
E. It is at -450 bP.
Q. A patient with phenylketonuria is found to have deficiency in phenylalanine hydroxylase
enzyme. A lysine residue is replaced by a glutamate, changing a positive charge to a negative one
in a critical position of the protein sequence. The mutation causing this patient’s disease most
likely occurred in which part of the DNA?
A. Enhancer.
B. Exon.
C. Intron.
D. Poly-A tail.
E. Promoter
The answer is B. Exon is the only part of the DNA that CODE for proteins. This is a missense
mutation.

RNA PROCESSING (EUKARYOTES)

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 5' CAPPING:  CO-TRANSCRIPTIONAL

 This cap has two functions:
1) The 5' cap protects the RNA from nucleases
Bacteria have no 5` cap,
(5` exonucleases).
2) Allow the binding of ribosome assembly instead it has 5` UTR which acts
when translation of the mRNA begins in the as a binding site for ribosomes.
cytoplasm.
This 5` UTR sequence in
bacteria is called Shine
Dalgarno sequence.

 3' POLYADENYLATION:  POST-TRANSCRIPTIONAL

 Functions:
1) Protect the RNA from degradation by 3' The poly-A tail is not transcribed
exonucleases.
from DNA, but rather added as a
2) Transport of the mRNA to the cytoplasm.
3) Extends the half-life of the RNA. posttranscriptional modification
downstream of a consensus
sequence (usually "AAUAAA")

 INTRON REMOVAL AND SPLICING OF PRE-MRNA:

1. The introns (non-coding) need to be removed from the hnRNA so that the protein
coding exons are placed together in the right sequence for proper protein coding.
2. Splicing is performed by spliceosomes, which consist of snRNPs plus proteins.
3. Synthesis of snRNP also occurs in the nucleus, catalyzed by RNA polymerase II.

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4. The spliceosome recognizes the starting and ending sequences of an intron, which
are always GT and AG, respectively.
5. The introns form a loop ―Lariat-shaped (looped) intermediate‖.
6. Lariat is released to precisely remove intron and join 2 exons.
 Antibodies to spliceosomal snRNPs (antiSmith antibodies) are highly
specifc for SLE.
 Anti-U1 RNP antibodies are highly associated with mixed connective
tissue disease (MCTD).

 Example of alternative splicing:

 Apo B gene gives Apo B100 lipoprotein in the liver while gives Apo B48 in the
gut.

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 MICRORNAS
 MicroRNAs (miRNA) are small, conserved, noncoding RNA molecules that
posttranscriptionally regulate gene expression by targeting the 3′ untranslated
region of specific mRNAs for degradation or translational repression.
 Abnormal expression of miRNAs contributes to certain malignancies (eg, by
silencing an mRNA from a tumor suppressor gene).

5` UTR in
mRNA

Initiation of
translation

Eukaryotes Prokaryotes

Shine-
Kozak
Dalgarno
sequence
sequence

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The Kozak consensus sequence:

Occurs on eukaryotic mRNA and is defined by the following sequence: (gcc)gccRccAUGG, where R is
either adenine or guanine. When the methionine codon (AUG) is positioned near the beginning of a
mRNA molecule and is surrounded by the Kozak sequence, it serves as the initiator for translation (i.e
mRNA binding to ribosomes). Among other factors, a purine (G or A) positioned three bases
upstream from the AUG appears to be a key factor in this initiation process.

A mutation in which guanine (G) is replaced by cytosine (C) in this particular position of the (β-globin
gene has been associated with thalassemia intermedia.

Hypochromic, microcytic anemia is the classic laboratory finding in patients with thalassemia. Red
blood cell morphology is quite variable depending on the type of thalassemia and can include marked
anisopoikilocytosis, target cell formation, tear drop cells, and/or Heinz bodies.

 TRANSCRIPTION FACTORS:

Transcription
factors

DNA-binding proteins that

modify expression of genes

General TFs Specific TFs

Bind enhancers in response

Bind promoter --> allows
to specific signals such as
RNA polymerase to bind and
hormones,
initiate transcription -->
growth factors, or
basal transcription
developmental cues.

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Some important specific transcription factors:

PAX3 gene
 Function:
 Active in neural crest cells directing differentiation of neural crest cells to
form specialized tissues.
 Playing an important role in early myogenesis.
 Mutation:  Waardenburg Syndrome:
 Type 1:
 Pigmentary abnormalities (white forelock, heterochromia iridis, patchy
hypopigmentation of skin).
 Sensorineural hearing loss
 Dystopia canthorum
 No limb abnormalities
 Type 2: Similar to Type 1, but also upper limb abnormalities.

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Sonic Hedgehog Gene (SHH)

 Function:
 Organization of the brain.
 Growth of digits on limbs.
 Mutation:
 Holoprosencephaly: Failure of forebrain of the
embryo to separate into two hemsipheres.
 Defects in the face.

Homeobox genes

 Code for DNA-binding transcription factors that play an important role in

morphogenesis.
 Morphogenesis is the proper formation and placement of tissues, organs and
structural elements of the body.
 Mutations of these genes caused limbs and appendages to develop in the incorrect
locations.

START AND STOP CODONS

mRNA start codons

 AUG (or rarely GUG). AUG inAUGurates protein synthesis.
 In Eukaryotes,
 Codes for methionine, which may be removed before translation is
completed.
 In Prokaryotes,
 Codes for N-formylmethionine (fMet).
 fMet stimulates neutrophil chemotaxis.

mRNA stop codons

 The stop codons do not code for amino acids.
 Instead, when the ribosome encounters a stop codon,
releasing factors bind to the ribosome and stimulate
release of the formed polypeptide chain and dissolution
of the ribosome-mRNA complex.

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Expression of genes per mRNA:

 Eukaryotes:
 Monocistronic  expression of one gene per mRNA (a single coding
region)  mRNA codes for one protein.
 Prokaryotes:
 Polycistronic  several express portions of mRNA for different proteins
(one mRNA codes for different proteins.) These express portions in the
prokaryotic mRNA are called ―Operons‖.

LAC OPERON (E. COLI)

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 The lac operon consists of:

 Regulatory gene (lac I).
 Produces a repressor protein, when bound to the operator region,
prevents binding of RNA polymerase to the promoter region, thus
decreasing transcription of the lac Z, lac Y, and lac A genes.
 Lactose-containing media causes a conformational change in the
repressor protein, preventing its attachment to the operator region and
↑transcription of the lac operon structural genes.
 Glucose-containing media results in reduced expression of the lac
operon.
 ↑Glucose  ↓cAMP  ↓ binding of catabolite activator
protein (CAP) to a site slightly upstream from the promoter
region  ↓ transcription of the lac operon structural genes.
 Promoter region (lac p):
 Binding site for RNA polymerase during In summary, the lac operon
the initiation of transcription. is regulated by 2 distinct
 Operator region (lac o).
mechanisms:
 A binding site for the repressor proteins
1. Negatively by binding of
 prevents binding of RNA polymerase
the repressor protein to the
to the promoter region, thus decreasing
operator locus
transcription.
2. Positively by cAMP-CAP
 Three structural genes (lac Z, lac Y, and lac
binding upstream from the
A).
promoter region
 The lac Z gene codes for (β-
galactosidase, which is responsible for
the hydrolysis of lactose to glucose and galactose)

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 The lac Y gene codes for permease which allows lactose to enter the
bacterium.
 Classic example of a genetic response to an environmental change.
 Glucose is the preferred metabolic substrate in E coli, but when glucose is absent and
lactose is available, the lac operon is activated to switch to lactose metabolism.
 Mechanism of shift:
 Low glucose  ↑ adenylate cyclase activity  ↑ generation of cAMP from
ATP  activation of catabolite activator protein (CAP)  ↑ transcription.
 High lactose  unbinds repressor protein from repressor/operator site  ↑
transcription.

t-RNA

STRUCTURE

 75–90 nucleotides, 2º structure, cloverleaf form,

anticodon end is opposite 3′ aminoacyl end.
 All tRNAs, both eukaryotic and prokaryotic, have
CCA at 3′ end along with a high percentage of
chemically modified bases.
 The amino acid is covalently bound to the 3′ end of
the tRNA. CCA Can Carry Amino acids.
 T-arm:
 Contains the TΨC (ribothymidine,
pseudouridine, cytidine) sequence
necessary for tRNA-ribosome binding.
 T-arm Tethers tRNA molecule to
ribosome.
 D-arm:
 Contains dihydrouridine residues necessary for tRNA recognition by the
correct aminoacyl tRNA synthetase.
 D-arm Detects the tRNA by aminoacyl-tRNA synthetase.

 Acceptor stem:
 The 5′-CCA-3′ sequence  used as a recognition sequence by
enzymes.
 The 3' end is the amino acid binding site.

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CHARGING

 During protein synthesis, tRNA acts as an adaptor molecule between the codons
found on mRNA and the amino acids being incorporated into the polypeptide chain.
 The sequence of amino acids in a polypeptide chain is dictated by binding of the
tRNA anticodon to its complementary codon on the mRNA molecule being translated.
 Amino acid activation and attachment to the 3' end of tRNA is catalyzed by
aminoacyl-tRNA synthetases (AA-tRNA synthetases).
 Each amino acid/tRNA pair has a specific AA-tRNA synthetase that links them
together. These enzymes are responsible for ensuring that each amino acid binds to
the tRNA with the proper anticodon.
 Aminoacyl-tRNA synthetase activation and binding sites are highly specific for their
correct amino acids and tRNA molecules. Additionally, some AA-tRNA synthetases
can ''proofread" their specific tRNA molecules and hydrolyze the amino acid bond
when their tRNAs are incorrectly charged.
 Erroneous amino acid/tRNA coupling by the AA-tRNA synthetase causes the wrong
amino acid to be incorporated into the growing polypeptide chain.
 The error rate for AA-tRNA synthetases is thus very low at less than 1 error per 10 4
charges.

 Aminoacyl-tRNA synthetase (1 per amino acid; ―matchmaker‖; uses ATP) scrutinizes

amino acid before and after it binds to tRNA.
 If incorrect, bond is hydrolyzed.
 The amino acid-tRNA bond has energy for formation of peptide bond.
 A mischarged tRNA reads usual codon but inserts wrong amino acid.
 Aminoacyl-tRNA synthetase and binding of charged tRNA to the codon are responsible
for accuracy of amino acid selection.

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RIBOSOMES:

 The only rRNA not transcribed in the nucleolus by RNA polymerase I is the 5S
rRNA, which is produced by RNA polymerase III in the nucleoplasm.
 The prokaryotic small subunit 16s is complementary and antiparallel to the Shine
DalGarno sequence of mRNA (5`UTR).

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PROTEIN SYNTHESIS (TRANSLATION)

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Initiation
 Eukaryotic initiation factors (eIFs) identify either
the 5′ cap or an internal ribosome entry site
(IRES).
 IRES can be located at many places in an mRNA
(most often 5′ UTR).
 The eIFs then help assemble the 40S ribosomal
subunit with the initiator tRNA and are released
when the mRNA and the ribosomal
60S subunit assemble with the complex.
 Requires GTP (initiated by GTP hydrolysis)

Elongation
 Aminoacyl-tRNA binds to A site (except for
initiator methionine). requires an elongation factor
and GTP
 rRNA (―ribozyme‖) catalyzes peptide bond
formation, transfers growing polypeptide to amino
acid in A site.
 Ribosome advances 3 nucleotides toward 3′ end of mRNA, moving peptidyl tRNA
to P site (translocation).
Termination
 Stop codon is recognized by release factor, and completed polypeptide is released
from ribosome.
 Requires GTP.

Q. How many high energy bonds are needed for each amino acid added during protein
synthesis? 4 GTP bonds; so in hypoxic conditions  ↓energy  ↓GTP  ↓protein
synthesis. (The ribosomes detaches themselves from the endoplasmic reticulum.)

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Antibiotics that inhibit protein synthesis:

Bacteria that inhibit protein synthesis:

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PROTEIN FOLDING:

SECONDARY STRUCTURE: (H BONDS)

1) In patients with Alzheimer disease, beta-amyloid protein loses its alpha-
helical configuration and forms beta-sheets, which are less soluble and
therefore prone to aggregating. Aggregations of beta-sheets are the primary
component of the extracellular senile (neurotic) plaques found in
Alzheimer patients.
2) The conversion of alpha-helices to beta-sheets involves the breaking and
reforming of hydrogen bonds.

TERTIARY STRUCTURE
 Three-dimensional form that a polypeptide assumes after the
secondary structure is in place (e.g. globulins and fibrillary
proteins). This is determined by a number of different forces
or interactions:
 Covalent bonds between side chains (e.g.,
disulfide bonds between two cysteine amino
acids.)
 Ionic bonds between oppositely charged
amino acids.
 Hydrogen bonds between polar side chains and/or backbone
amino and carboxyl groups.
 Hydrophobic interactions between aliphatic amino acids.

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 Disulfide bonds are very strong covalent bonds between two cysteine residues
within the same polypeptide chain that enhance a protein’s ability to withstand
denaturation.

QUATERNARY STRUCTURE
1) As tertiary structure proteins but with
multiple subunits.
2) Example: Hemoglobin A with 2 alpha
subunits + 2 beta subunits.

What helps that folding?

Chaperon proteins

CHAPERONE PROTEIN

 Intracellular protein involved in facilitating and/or maintaining protein folding.

 For example, in yeast, heat shock proteins (eg, HSP60) are expressed at high
temperatures to prevent protein denaturing/misfolding.

 Misfolded proteins are destroyed by ubiquitination and proteolysis in structures

named proteasomes (see next).

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CO- AND POSTTRANSLATIONAL MODIFICATIONS

N-terminal hydrophobic signal sequence (Co-transcriptional)

 Proteins that are translated in the cytoplasm, are needed to be targeted to the RER
to be secreted outside the cell or to the cell membrane.
 N-terminal hydrophobic signal sequence target the protein to the RER to undergo
modifications before secretion.
 Signal recognition particle (SRP) binds to the signal sequence and mediates the
attachment of the translation complex to the outer surface of the RER where
translation continues.

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 N-glycosylation of proteins:
1) Occurs in both RER (modified in Golgi).
2) On the amino acid Asparagine.
3) Need Dolichol-P as a cofactor, which comes from cholesterol synthesis.
 O-glycosylation of proteins:
1) Occurs in Golgi only.
2) On the amino acid Serine & Threonine.

 Phosphorylation of mannose:
1) Occurs in the Golgi.
2) By phosphotransferase enzyme.
3) Phosphorylated mannose residues will be targeted to lysosomes to make
lysosomal enzymes.

Phosphotransferase deficiency (I-cell disease/mucolipidosis type II):

 Without this enzyme, the lysosomal enzymes will not be targeted to be stored in the
lysosomes. Instead it will be in the cytoplasm & extracellular  autodigestion of the
cell  often fatal in childhood.
 It is a lysosomal storage disease. Absence of key lysosomal enzymes in lysosomes 
phagocytosis is not completed  Inclusion bodies within cells (secondary lysosomes
filled with indigestible material)
 High levels of lysosomal enzymes in blood or serum.
 Results in coarse facial features, clouded corneas, restricted joint movement, umbilical
hernia and macroglossia.

Trimming (posttranscriptional)
 Removal of N- or C-terminal propeptides from zymogen to generate mature protein
(eg, trypsinogen to trypsin).

Covalent alterations (posttranscriptional)

 Phosphorylation, glycosylation, hydroxylation, methylation, acetylation, and
ubiquitination.

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COLLAGEN
 Most abundant protein in the human body.
 Extensively modified by posttranslational modification.
 Organizes and strengthens extracellular matrix.
Be (So Totally) Cool, Read Books.

Type I Most common (90%)—Bone (made Type I: bone.

by osteoblasts), Skin, Tendon, dentin, ↓ Production in osteogenesis
fascia, cornea, late wound repair. imperfecta type I.

Type II Cartilage (including hyaline), vitreous Type II: cartwolage.

body, nucleus pulposus.

Type III Reticulin—skin, blood vessels, Type III: deficient in the

uterus, fetal tissue, granulation tissue. uncommon, vascular type of
Ehlers-Danlos syndrome (ThreE
D).
Type IV Basement membrane, basal lamina, Defective in Alport syndrome;
lens. Type IV: under the floor targeted by autoantibodies in
(basement membrane). Goodpasture syndrome.

 COLLAGEN SYNTHESIS AND STRUCTURE

1) Synthesis:
a. Translation of collagen α chains (preprocollagen)—usually Gly-X-Y
(X and Y are proline or lysine).
b. Glycine content best reflects collagen synthesis (collagen is 1⁄3
glycine).
c. Glycine is the most abundant amino acid in collagen.
2) Hydroxylation:
a. Hydroxylation of specific proline and lysine Paget disease  ↑bone
residues. turnover  ↑destruction
b. Requires vitamin C; deficiency  scurvy.
of collagen 
3) Glycosylation:
↑hydroxyproline in urine.
a. Glycosylation of pro-α-chain hydroxylysine
residues and formation of procollagen via
hydrogen and disulfide bonds (triple helix of 3 collagen α chains).
b. Problems forming triple helix  osteogenesis imperfecta.
4) Exocytosis:
a. Exocytosis of procollagen into extracellular space.
5) Proteolytic processing:
a. Cleavage of disulfide-rich terminal regions of procollagen by
extracellular peptidases  insoluble tropocollagen.
b. Problems with cleavage  Ehlers-Danlos syndrome.

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6) Cross-linking:
a. Reinforcement of many staggered tropocollagen molecules by
covalent lysine-hydroxylysine cross-linkage (by copper containing
lysyl oxidase) to make collagen fibrils.
b. Problems with cross-linking  Ehlers-Danlos syndrome, Menkes
disease.

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 OSTEOGENESIS IMPERFECTA
 Genetic bone disorder (brittle bone disease) caused by a variety of gene defects (most
commonly COL1A1 and COL1A2).
 Most common form is autosomal dominant with ↓ production of otherwise normal
type I collagen.
 Manifestations can include:
1) Multiple fractures with minimal trauma A
B; may occur during the birth process.
May be confused with child abuse.
2) Blue sclera C due to the translucent
connective tissue over choroidal veins.
3) Some forms have tooth abnormalities,
including opalescent teeth that wear easily
due to lack of dentin (dentinogenesis imperfecta).
4) Hearing loss (abnormal ossicles).
 Treat with bisphosphonates to ↓ fracture risk.

 EHLERS-DANLOS SYNDROME
 Caused by a deficiency in procollagen peptidase, the
enzyme that cleaves terminal propeptides from procollagen in
the extracellular space. Impaired propeptides removal results
in the formation of soluble collagen that does not properly
crosslink causing hyperextensible skin, tendency to bleed
(easy bruising), and hypermobile joints A.
 May be associated with joint dislocation, berry and aortic
aneurysms, organ rupture.
 Multiple types:
1) Hypermobility type (joint instability): most common type.
2) Classical type (joint and skin symptoms): caused by a mutation in type V
collagen (eg, COL5A1, COL5A2).
3) Vascular type (vascular and organ rupture): deficient type III collagen.
 Inheritance and severity vary. Can be autosomal dominant or recessive.

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 MENKES DISEASE
 X-linked recessive connective tissue disease caused by impaired copper absorption
and transport due to defective Menkes protein (ATP7A).
 Leads to ↓ activity of lysyl oxidase (copper is a necessary
cofactor).
 Results in:
1) Brittle, ―kinky‖ hair.
2) Growth retardation.
3) Hypotonia.

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ELASTIN
 Stretchy protein within skin, lungs, large arteries, elastic ligaments, vocal cords,
ligamenta flava (connect vertebrae  relaxed and stretched conformations).
 Rich in nonhydroxylated proline, glycine, and lysine residues, vs the hydroxylated
residues of collagen.
 Tropoelastin is secreted into the extracellular space where it interacts with
microfibrils called fibrillin; a glycoprotein that forms a sheath around elastin.
 Cross-linking takes place extracellularly and gives elastin its elastic properties.
 Broken down by elastase, which is normally inhibited by α1-antitrypsin.
 α1-Antitrypsin deficiency results in excess elastase activity, which can cause
emphysema.

 Changes with aging:

 ↓ Dermal collagen and elastin.
 ↓ Synthesis of collagen fibrils; crosslinking remains normal.

Elastin differs from collagen in a number of ways:

1. Proline and lysine residues are less hydroxylated in elastin.
2. Whereas triple helix formation is the basis of the collagen molecule, elastin
does not form triple helices.
3. Triple helix formation in collagen is initiated by hydroxylation, glycosylation
and interchain disulfide bridges at the C-terminus of procollagen molecule.
These modifications do not occur in the formation of elastin molecules.

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 MARFAN SYNDROME
 Autosomal dominant connective tissue disorder affecting skeleton, heart, and eyes.
 FBN1 gene mutation on chromosome 15 results in defective fibrillin, a glycoprotein
that forms a sheath around elastin.
 Fibrillin in the extracellular space acts as a scaffold for deposition of elastin extruded
from connective tissue cells.
 Findings:
 Skeleton: tall with long extremities; pectus carinatum (more specific) or
pectus excavatum; hypermobile joints; long, tapering fingers and toes
(arachnodactyly).
 Heart: cystic medial necrosis of aorta; aortic incompetence and dissecting
aortic aneurysms; floppy mitral valve.
 Eyes: Subluxation of lenses, typically upward and temporally (Look up at
a ceiling fan.)

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MUTATIONS IN DNA

Types of
mutations

point
frameshift
(inframe)

Splice site
Silent Missense Nonsense
mutation

 Point mutations: Base substitutions, where one

base is substituted with another base.
 Transition—purine to purine (eg, A to G)
or pyrimidine to pyrimidine (eg, C to T).
 Transversion—purine to pyrimidine (eg,
A to T) or pyrimidine to purine (eg, C to G).
 Severity of damage: silent << missense < nonsense < frameshift.

SILENT MUTATIONS
 Nucleotide substitution but codes for same (synonymous) amino acid; often
base change in 3rd position of codon (tRNA wobble).
 For example, a single base substitution in UCA to UCU will not result in any
change in protein structure because both code for the same amino acid, serine.

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MISSENSE MUTATIONS
 Nucleotide substitution resulting in changed amino acid.
 Two types:
 Conservative Mutation: Converts the encoded amino acid to another
with similar properties (substitution of aliphatic AA with another
aliphatic AA).
 Nonconservative Mutation: Converts the encoded amino acid to
another with different properties.

 Examples:
 Sickle cell disease (substitution of glutamic acid ―-ve charged‖ with
valine ―no charge‖)  nonconservative.
 HbC disease (substitution of glutamic acid with lysine)
 Typically asymptomatic and often have mild hemolytic anemia
and splenomegaly.

NONSENSE MUTATIONS
 Nucleotide substitution resulting in early stop codon (UAG, UAA, UGA).
 Usually results in early termination of translation  small nonfunctional
truncated (↓size) proteins.
Stop the nonsense!

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SPLICE SITE MUTATION

 The spliceosome recognizes specific sequences (GU and AG) at the
beginning and end of each intron. Thus, mutation of these sites in the
corresponding DNA could block intron splicing and prevent the formation of
the proper mRNA.
 This could significantly reduce gene expression.
 Mutations in splice sites affect the accuracy of intron removal from hnRNA
during posttranscriptional processing.
 If a splice site is lost through mutation, spliceosomes may:
 Delete nucleotides from the adjacent exon.
 Leave nucleotides of the intron in the processed mRNA.
 Use the next normal upstream or downstream splice site, deleting an
exon from the processed mRNA.
Example of splice site
 Mutation at a splice site  retained intron in the
mRNA  Larger, nonfunctional protein but mutation: mutation in the 5`
often retain the immunoreactivity of the normal end of exon 2 results in its
protein (binding to antibodies). entire deletion  splice
 EXAMPLES:
acceptor mutation.
 β-thalassemia, Gaucher disease, and Tay-
Sachs.

FRAMESHIFT MUTATIONS
 Deletion or insertion of a number of nucleotides not divisible by 3, resulting
in misreading of all nucleotides downstream.
 Alter the reading frame of the genetic code, resulting in the formation of
non-functional proteins; may be shorter or longer, and its function may be
disrupted or altered.
 Examples:
 Duchenne muscular dystrophy,
 Tay-Sachs disease.
 Frameshift mutations involving the alpha globin genes can cause alpha
thalassemia, which results in the production of beta tetramers
(hemoglobin H).

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 If the insertion is a multiple of three nucleotides (3, 6, 9, etc.), this simply adds
amino acids to the protein  trinucleotide repeat insertions ―In-frame
mutation‖
 Fragile X syndrome repeats CGG.
 Huntington repeats CAG.
 Some forms of adult onset muscular dystrophy contain tri- or
tetranucleotide repeat insertions of CTG and CCTG.

 Somatic mutations are not transmitted to offspring BUT if it happened during

gametogenesis, it will be transmitted to offspring.

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DNA REPAIR

 SINGLE STRAND
 Nucleotide excision repair: (pyrimidine dimers)
 Specific endonucleases release the oligonucleotides containing damaged
bases.
 DNA polymerase and ligase fill and reseal the gap, respectively.
 Occurs in G1 phase of cell cycle.
 Example:
 Absence of UV-specific endonuclease; this prevents repair of
pyrimidine dimers that are formed as a result of ultraviolet light
exposure.

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BASE EXCISION REPAIR:

 Several types of insults can alter the DNA
bases. For example, dietary nitrites can
deaminate C, A and G. There are also
spontaneous changes, such as deamination
of C to U.
 If these abnormal bases are not removed
and replaced with the correct base, DNA
mutations and carcinogenesis may result.
 Base-specific Glycosylase removes altered
base and creates AP site
(apurinic/apyrimidinic).
 One or more nucleotides are removed by
AP-Endonuclease, which cleaves the
5′end.
 Lyase cleaves the 3′end.
 DNA Polymerase-β fills the gap and DNA
Ligase seals it.
 Occurs throughout cell cycle.
 Important in repair of spontaneous/toxic
deamination.
―GEL PLease‖

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MISMATCH REPAIR:
 Newly synthesized strand is recognized,
mismatched nucleotides are removed, and
the gap is filled and resealed.
 Occurs predominantly in G2 phase of cell
cycle.
 Defective in Lynch syndrome (hereditary
non polyposis colorectal cancer
[HNPCC]).

Lynch syndrome (hereditary non polyposis colon

cancer):
 Autosomal dominant disease caused by
defective DNA mismatch repair.
 DNA replication occurs with a high degree of
fidelity because mismatched nucleotides are
repaired through the proofreading activity of
DNA polymerases delta and epsilon.
 The mismatch repair system involves several
genes, including hMSH2 and hMLH1, which
code for components of the human MutS and
MutL homologs, which recognize the
mismatched base pairs and cut them with their
additional endonuclease activity.
 Mutations in these 2 genes account for around
90% of cases of Lynch syndrome

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Steps of mismatch
repair
Mismatch repair begins with MutS
homolog detecting a mismatch on the
newly created daughter strand, which is
distinguished from the parent strand by
occasional nicks in the phosphodiester
bonds.

MutL homolog is then recruited, and the

resulting complex slides along the DNA
molecule until 1 of the daughter strand
nicks is encountered. At this point,
exonuclease 1 is loaded onto and
activated by the repair complex.

The daughter strand is then degraded

backward past the initial mismatch point,
leaving a variable gap of single-stranded
DNA that is stabilized by ssDNA-binding
protein. The complex then dissociates
while DNA polymerase delta loads at
the 3' end of the discontinuity and begins
synthesizing a new daughter strand
segment. Finally, DNA ligase I seals the
remaining nick to complete the repair
process.

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 DOUBLE STRAND

NONHOMOLOGOUS END JOINING:

 Brings together 2 ends of DNA fragments to repair double-stranded
breaks.
 No requirement for homology.
 Some DNA may be lost.
 Defective in ataxia telangiectasia, breast/ovarian cancers with
BRCA1mutation, and Fanconi anemia.
 UW: Therapeutic ionizing radiation (eg. gamma rays, x-rays), commonly used
to treat or palliate several types of cancer, can cause cell death through 2 major
mechanisms:
 DNA double-strand breakage  breakage of both strands is generally
required, as single strand breaks are readily repaired by polymerases.
 Free radical formation  Reactive oxygen species are formed by
ionization of water: oxygen free radicals are then able to cause cellular and
DNA damage.

Exposure to ionizing radiation causes double-stranded DNA breaks that are repaired
by end-joining repair mechanisms.

BRCA1 and BRCA2 in particular are involved in repair of double-stranded DNA

breaks.

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HOMOLOGOUS RECOMBINATION
 Requires two homologous DNA duplexes.
 A strand from the damaged dsDNA is repaired using a complementary
strand from the intact homologous dsDNA as a template.
 Restores duplexes accurately without loss of nucleotides.
 Defective in breast/ovarian cancers with BRCA1 mutation.

DNA REPAIR DEFECTS

 FANCONI ANEMIA
 dsDNA breaks can’t be repaired  sensitivity to damage caused by DNA
crosslinking agents.
 They also exhibit congenital skeletal malformations and progressive aplastic
anemia.
 They are susceptible to developing acute myeloid leukemia (AML).
 ATAXIA TELANGIECTASIA
 dsDNA breaks caused by a defect in the ATM gene, which codes for a protein
critical to the cell response to a several forms of stress, including double-strand
breaks in DNA.
 Sensitivity to damage caused by x-rays.
 Clinical picture:
 They have numerous oculocutaneous telangiectasias (on eyes and skin).
 Progressive cerebellar ataxia.
 Immunodeficient.
 Susceptible to lymphoid malignancies.

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GENETICS
DNA FRAGMENTATION:

 PALLINDROMES:
 DNA sequence cleaved by restriction
endonuclease.
 Every restriction endonuclease search for a
selective DNA sequence to cut.
 The palindrome sequence is read same
backwards & forwards (usually 4 or 6 bp).

 Why we cut DNA?

1) The spliced part of DNA is used as a cloned
gene that is introduced into the bacteria
vector (Recombinant DNA) and then
express it in bacteria for commercial use
(e.g. insulin, HBV vaccine)
2) Gene therapy.

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GENE THERAPY

 Introduction of a normal copy of the gene that is defective into the tissues that give
rise to the pathology of the genetic disease.
 Requirements & major problems:
1) Gene transfer requires a delivery vector (retrovirus, adenovirus, and
liposome).
2) Targeting the right tissue.
3) The normal gene is not inherited by offspring ―ethics‖.
4) Integrating the gene correctly.
5) Regulating the expression of genes.

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Ex vivo gene replacement therapy:

 Cells are modified outside the body, then transplanted
back to the body.
 Examples:
 SCID  IL-gamma chain & ADA.
 Hyperammonemia  Ornithine
transcarbamaylase.

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BLOTTING PROCEDURES
Blotting techniques have been developed to identify DNA, RNA, and proteins from
complex mixtures of those substances.

Steps of blotting:

‫ هتعمل اٌه ؟‬B-globin chain ‫ حضرتك عاٌز تشوف الجٌن الخاص بال‬
DNA ‫ عشان تدوب الهٌستون وٌتبقالك ال‬detergent ‫ هتاخد اي خلٌة من الجسم وتحط علٌها‬
5`3` ‫ بتاع الجٌن وٌكون من‬SEQUENCE ‫ هتفتح الالب بتاعك وتجٌب موقع جوجل وتدور على ال‬
complementary probe to ‫ وتقولهم "سالم علٌكم من فضلك انا عاٌز‬probes ‫ تتصل بشركة تصنٌع ال‬
the gene
‫ هٌقوللك‬sequence ‫ هتقوللهم اٌوة وتملٌه ال‬sequence ‫ خدمة العمال هترد علٌك وتقوللك انت عارف ال‬
 ‫بعد كده معاك فلوس كتٌرة تقوله اٌوة‬
 ‫ ومعاه شوٌة جل وورقة بٌضا هللا ٌخلٌك‬restriction endonucleases ‫ وتقوللهم كمان انا محتاج شوٌة‬
ً‫ هٌمسك فٌها وٌنور ف‬probe ‫ هٌقطع الجٌن اللً انت عاٌز تدور علٌه باطوال مختلفة وكل حتة ال‬RI ‫ ال‬
‫الجل وتشوف الحتة اللً مسك فٌها‬
‫ على الجل وتوصله بالكهربا‬probe ‫ مع الكروموسومات ومعاهم ال‬RI ‫ هتحط‬
‫ الجزأ اللً انت بتدور علٌه هٌتقطع حتت وكل حتة ماسك فٌها‬.‫ الجل فٌه قطبٌن موجب تحت وسالب فوق‬
ً‫ لكن الل‬DNA is –ve ‫ الحتت دي لما تحطها على الجل هتروح ناحٌة القطب الموجب الن ال‬. probe
‫هٌروح اسرع ناحٌة القطب الموجب هو اللً حجمه صغٌر بمعنى ان الحجم الصغٌر هتالقٌه تحت ناحٌة‬
.‫الموجب والحجم الكبٌر فوق ناحٌة السالب النه تقٌل فً الحركة‬
 Fragmentation: the large DNA is fragmented by specific endonucleases into
several pieces of multiple sizes.
 These –ve charged DNA fragments are then put on the top of the gel electrophoresis
to be allowed to migrate to the bottom with different levels according to the size of
each fragment which is determined by the number of bp of each fragment.
 The small fragments move fast to the bottom & large fragments move slowly and
will be stuck at the top.
 Transfer them to a membrane (filter).
 Get a radioactive probe which is complementary and antiparallel to what you are
interested in.
 Banding pattern will appear if the probe find what you are searching for.

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Types of probes:
 Single gene probes:
 Bind to the 2 alleles of the gene making two bands and can’t differentiate
them.
 Not able to study recessive diseases or carrier states of dominant diseases.
 ASO ―allele specific oligonucleotides‖ probes:
 8-10 nucleotides long.
 One to recognize the normal allele, one to recognize the diseased allele
 can differentiate between the 2 alleles  detect point mutations.
 Broadly specific probes:
 Bind in the repeats of the spacer DNA. These repeats are different
between individuals; so they can detect the individuals DNA.

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Types of blotting:

Southern blot  DNA sample is enzymatically cleaved into smaller pieces, which are
separated on a gel by electrophoresis, and then transferred to a filter.
 Filter is exposed to radiolabeled DNA probe that recognizes and anneals
to its complementary strand.
 Resulting double-stranded, labeled piece of DNA is visualized when
filter is exposed to film.
Northern blot  Similar to Southern blot, except that an RNA sample is electrophoresed.
Useful for studying mRNA levels, which are reflective of gene
expression.
Western blot  Sample protein is separated via gel electrophoresis and transferred to a
membrane.
 Labeled antibody is used to bind to relevant protein (eg, confirmatory
test for HIV after ⊕ ELISA).
Southwestern  Identifies DNA-binding proteins (eg, transcription factors) using
blot labeled oligonucleotide probes.

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 Examples of proteins that are able to bind DNA include:

 Transcription factors, steroids, thyroid proteins, vitamin D receptors, retinoic
acid receptors, DNA transcription and replication proteins, and others.
 N-myc is a transcription factor that is, by definition, capable of binding DNA.
Because of its DNA-binding ability, N-myc can be detected by DNA probes.
 The MYC proteins are mammalian transcription factors.
 C-myc is the protein that is overexpressed in Burkitt lymphoma.
 C-Jun and c-Fos are nuclear transcription factors that directly bind DNA via a
leucine zipper motif. The genes that code for c-Jun and c-Fos are proto-
oncogenes, genes that can become oncogenes following a mutation or with
constitutive expression.

 SOUTHERN BLOTS AND RESTRICTION FRAGMENT LENGTH

POLYMORPHISMS

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 In SCA, there is a point mutation on the MstII

palindrome on beta globin gene on
chromosome 11  the endonucleases cut at
larger fragments.

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 NORTHERN BLOT:
 Analyze RNA and measure gene expression.
 Notice in figure 6-l.lG that the gene is heavily expressed in brain and testes, and to
a lesser extent in lung and heart (example of FMR1 gene of fragile X syndrome.)
 The different lengths of the transcripts observed could be due to alternative RNA
splicing.

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Note that heavily expression of the

FMR1 gene “familial mental
retardation” in the brain and testes
explain the presentation of mental
retardation and macroorchidism of
this disease. The different size of the
gene expression in the heart is due
to alternative spicing.

 Every nucleated cell in our body have insulin gene which can be studied with
southern blot; but the expression of this gene only in pancreas is studied by northern
blot.

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MICROARRAYS
 Thousands of nucleic acid sequences are arranged in
grids on glass or silicon.
 DNA or RNA probes are hybridized to the chip, and
a scanner detects the relative amounts of
complementary binding.
 Used to profile gene expression levels of thousands
of genes simultaneously to study certain diseases
and treatments.
 Used mainly for profiling of tumors.
 Able to detect single nucleotide polymorphisms
(SNPs) and copy number variations (CNVs) for a
variety of applications including genotyping, clinical
genetic testing, forensic analysis, cancer mutations,
and genetic linkage analysis.
Q. A physician would like to determine the global
patterns of gene expression in two different types of
tumor cells in order to develop the most appropriate form
of chemotherapy for each patient. Which of the
following techniques would be most appropriate for this
purpose?
A. Southern blot.
B. Northern blot.
C. Western blot.
D. ELISA.
E. Microarray.
Correct answer = E. Microarray analysis allows the
determination of mRNA production (thus, gene
expression) from thousands of genes at once. A Northern
blot only measures mRNA production from one gene at a
time. Western blots and ELISA measure protein
production (also gene expression), but only from one
gene at a time. Southern blots are used to analyze DNA,
not gene expression.

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POLYMERASE CHAIN REACTION

 The polymerase chain reaction (PCR) is a technique that can amplify & make many
copies of a specific segment of DNA through a process similar to replication.
 Useful as a diagnostic tool (eg, neonatal HIV, herpes encephalitis).

 PCR requires four components:

 A DNA template.
 Two specific primers those are antiparallel and complementary to the DNA
sequence flanking the interval of interest. These two primers bind opposite
strands and face each other across this interval.
 Nucleotide triphosphates.
 Heat-stable polymerase from thermophilic bacteria.
 PCR is a three-step process:
 The DNA is denatured (strands separated) by heating to 95°C.
 The primers are allowed to anneal to the DNA by lowering the temperature to
55°C to 65°C.
 The temperature is raised to 72°C, the temperature at which the bacterial DNA
polymerase is most active, to allow polymerization of DNA starting at each
primer.
 This three-step process is then repeated for a number of cycles (usually 20 to
40). The amount of DNA produced grows exponentially, resulting in many
copies of the region of interest.

 UW: What must be known in order to perform PCR?

 Nucleotides sequence of the regions flanking the target DNA (i.e the area of
which the primers will bind).
 PCR doesn’t require that the sequence of targeted DNA to be known.

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 Uses of PCR include:

 Identification of a sequence in a patient sample: e.g., diagnosis of infectious
diseases by identification using primers that bind to and amplify the nucleic acid
sequences of viruses and bacteria.
 Direct mutation testing.
 Quantification of sequences: e.g., RT-PCR in HIV viral load analysis.
 Genotyping of short tandem repeat polymorphisms.
 Recombinant DNA applications.

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 PCR FOR HIV:

 The initial step for diagnosis of HIV is ELIZA (antibody detection) and
confirmed by Western blot.
 We use PCR for diagnosis in special conditions:
1) Too early, at risk.
2) Neonatal HIV testing  as antibody tests are inconclusive due to
maternally-derived transplacental IgG.
3) Immunosuppressed individuals who will have weak antibody against
HIV.

 REVERSE TRANSCRIPTASE PCR

 Used to monitor a known HIV patient  used to measure the viral load: the
concentration of virions circulating in the blood.

HIV

Prognosis
Diagnosis
(viral load)

PCR RT-PCR

 GENETIC FINGERPRINTING (PATERNITY TESTING)

 When repetitive sequences (Short Tandem Repeat Polymorphisms, or STRPs
or micro satellites) occur outside of coding regions, variable expansions of these
sequences do not affect the function of any genes.
 STRPs become highly polymorphic in populations and can be used in paternity
testing and forensic medicine to develop a genetic fingerprint.
 Example: in chromosome 7 there is a sequence repeat of GATA. This sequence
is repeated 6 times in father and 10 times in mother. The child will have a
chromosome 7 with two chromatids; the first from the mother (6 repeats) & the
second from the father (10 repeats)

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 Consider figure 6- 1.2C: In case 1, the tested male could be the father of the
child, whereas in case 2, the tested male could not be the father of the child
because neither of his bands is shared with the child.

Review questions:
1. Two sets of parents were friends in a small town and had babies on the same day.
The wristbands of the two similar-looking infants (A and B) were inadvertently
mixed at the pediatric care unit. In order to accurately identify the parents of the
respective infants, PCR analysis was performed on samples of blood taken from the
two infants and both sets of parents (Father 1 and Mother 1 versus Father 2 and
Mother 2). Shown below is the analysis of the PCR products by gel electrophoresis.

What is the best conclusion from the analysis?

(A) A is the child of Parents 1.
(B) A is the child of Parents 2.
(C) B is the child of Parents 1.
(D) Father 1 (F1) could be the father of both infants.
(E) Father 2 (F2) could be the father of both infants.

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2. Paternal relationship between a man and infant can be best determined by the
technique commonly referred to as DNA fingerprinting. Which of the following
sequences is most conveniently analyzed in a DNA fingerprint?
(A) Histocompatibility loci
(B) Centromeres
(C) Microsatellite tandem repeats (STRs)
(D) Restriction enzyme sites
(E) Single-copy sequences
3. Sickle cell anemia is caused by a missense mutation in codon 6 of the β-globin gene.

A man with sickle cell disease and his phenotypically normal wife request genetic
testing because they are concerned about the risk for their unborn child. DNA
samples from the man and the woman and from fetal cells obtained by amniocentesis
are analyzed using the PCR to amplify exon 1 of
the β-globin gene. Which 12-base nucleotide sequence was most likely used as a
specific probe complementary to the coding strand of the sickle cell allele?
(A) CCTCACCTCAGG
(B) CCTGTGGAGAAG
(C) GGACACCTCTTC
(D) CTTCTCCACAGG
(E) CTTCTCCTCAGG

Answers

1. Answer: A. Among the conclusions offered, only A is consistent with the results on the
blot. Infant A’s pattern shows a PCR product (lower on the blot) matching F1 and another
PCR product (higher on the blot) matching M1. Neither of infant A’s PCR products match F2
(choices B and E). The upper PCR product in infant B’s pattern does not match with either
F1 or M1 (choices C and D). Although unlikely given the situation, another possibility is
consistent with the blot. Infant A could be the child of M2 and F1, although this is not offered
as an option.
2. Answer: C. STR sequences are amplified using a PCR and analyzed by gel
electrophoresis. Although RFLP analysis could potentially be used for this
purpose, it is not the method of choice.
3. Answer: D. The complementary probe will be antiparallel to the coding
strand of the mutant allele, with all sequences written 5′ → 3′.

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 SEQUENCING DNA FOR MUTATION TESTING

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CRISPR/CAS9 “FA 2019”

 A genome editing tool derived from bacteria.

 Consists of:
1) Guide RNA (gRNA), which is complementary to a target DNA sequence.
 Can be designed to target any DNA sequence.
2) Endonuclease (Cas9), which makes a single or double-strand break at the target site.
 Function:
1) Break imperfectly repaired by nonhomologous end joining (NHEJ)  accidental
frameshift mutations (" knock-out").
2) A donor DNA sequence can be added to fill in the gap using homology-directed
repair (HOR) (" knock-in").
 Not used clinically. Potential applications include removing virulence factors from
pathogens, replacing disease-causing alleles of genes with healthy variants, and specifically
targeting tumor cells.

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FLOW CYTOMETRY
 Laboratory technique to assess size,
granularity, and protein expression
(immunophenotype) of individual cells in a
sample.
 Cells are tagged with antibodies specific to
surface or intracellular proteins.
 Antibodies are then tagged with a unique
fluorescent dye.
 Sample is analyzed one cell at a time by
focusing a laser on the cell and measuring
light scatter and intensity of fluorescence.
 Data are plotted either as histogram (one
measure) or scatter plot (any two measures,
as shown).
 In illustration:
 Cells in left lower quadrant ⊝ for
both CD8 and CD3.
 Cells in right lower quadrant ⊕ for
CD8 and ⊝ for CD3.
 Right lower quadrant is empty because all CD8-expressing cells also express
CD3.
 Cells in left upper quadrant ⊕ for CD3 and ⊝ for CD8.
 Cells in right upper quadrant ⊕ for CD8 and CD3 (red + blue  purple).
 Commonly used in workup of:
 Hematologic abnormalities (eg, paroxysmal nocturnal hemoglobinuria, fetal
RBCs in mother’s blood).
 Immunodeficiencies (eg, CD4 cell count in HIV).

ENZYME-LINKED IMMUNOSORBENT ASSAY (ELIZA)

 Immunologic test used to detect the presence of either a specific antigen (eg, HBsAg)
or antibody (eg, anti-HBs) in a patient’s blood sample.
 Detection involves the use of an antibody linked to an enzyme.
 Added substrate reacts with enzyme, producing a detectable signal.
 Can have high sensitivity and specificity, but is less specific than Western blot.
 Direct ELISA tests for the antigen directly, while indirect ELISA tests for the
antibody (thus indirectly testing for the antigen).

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KARYOTYPING
 Visualizing chromosomes for abnormalities in number and size.
 A process in which metaphase chromosomes are stained, ordered, and numbered
according to morphology, size, arm-length ratio, and banding pattern (arrows in A
point to extensive abnormalities in a cancer cell).
 Can be performed on a sample of blood, bone marrow, amniotic fluid, or placental
tissue.
 Used to diagnose chromosomal imbalances (eg, autosomal trisomies, sex
chromosome disorders).

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FLUORESCENCE IN SITU HYBRIDIZATION (FISH)

 Fluorescent DNA or RNA probe binds to specific gene site of interest on
chromosomes (arrows in A point to abnormalities in a cancer cell, whose karyotype is
seen above; each fluorescent color represents a chromosome specific probe).
 Used for specific localization of genes and direct visualization of chromosomal
anomalies at the molecular level.
 Microdeletion—no fluorescence on a chromosome compared to fluorescence
at the same locus on the second copy of that chromosome.
 Translocation—fluorescence signal that corresponds to one chromosome is
found in a different chromosome.
 Duplication—extra site of fluorescence on one chromosome relative to its
homologous chromosome.

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CLONING METHODS
 Cloning is the production of a recombinant DNA molecule that is self-perpetuating.
 Steps:
1. Isolate eukaryotic mRNA (post-RNA processing) of interest.
2. Expose mRNA to reverse transcriptase to produce cDNA (lacks introns).
3. Insert cDNA fragments into bacterial plasmids containing antibiotic resistance
genes.
4. Transform recombinant plasmid into bacteria.
5. Surviving bacteria on antibiotic medium produce cloned DNA (copies of
cDNA).
6. This process allows bacteria to be used to produce large amounts of a protein
of interest.

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GENE EXPRESSION MODIFICATIONS

 TRANSGENIC MICE
 If a cloned gene is introduced into a fertilized ovum or embryonic stem cell, a
laboratory animal can be produced that can be a model for human disease.
 The introduced gene is called a transgene.
 If the process is used to intentionally delete a gene, the result is referred to as a
knockout animal.
 We use the germ cells or embryonic stem cells of the animal for gene insertion or
deletion.
 Transgenic strategies in mice involve:
1) Random insertion of gene into mouse genome.
2) Targeted insertion or deletion of gene through homologous recombination
with mouse gene.

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 CRE-LOX SYSTEM
 Used to carry out deletions, insertions, translocations and inversions at specific
sites in the DNA of cells.
 It allows the DNA modification to be targeted to a specific cell type or be
triggered by a specific external stimulus.
 Can inducibly manipulate genes at specific developmental points (eg, to study a
gene whose deletion causes embryonic death).

 RNA INTERFERENCE
 dsRNA is synthesized that is complementary to the mRNA sequence of interest.
 When transfected into human cells, dsRNA separates and promotes degradation
of target mRNA, “knocking down” gene expression.

RNA silencing RNA interference is an important

(RNA mechanism by which short (20-30
interference) base pair) non-coding RNA
sequences (eg microRNA and small
interfering RNA) induce
posttranscriptional gene silencing by
small
microRNA base-pairing with complementary
interfering RNA
(miRNA) sequences within
(siRNA)
target mRNA molecules.

siRNA:
 Synthetic siRNA sequences can be introduced into cells to silence specific
pathogenic genes (e.g. c-Myc oncogene) and are being explored as possible
therapeutic agents.

miRNA:
 The human genome encodes over 1000 miRNA genes, each one capable of
repressing hundreds of target genes. Altered expression of even a few miRNA
genes can lead to cellular dysregulation and has been implicated in the
development of many diseases; including hematologic and solid malignancies.
 After being transcribed, miRNA undergoes processing in the nucleus to form a
double-stranded precursor that is then exported into the cytoplasm.
 There, the precursor is cleaved into a short RNA helix by a ribonuclease protein
called dicer.
 The individual strands are then separated and incorporated into RNA-induced
silencing complex (RISC). This multiprotein complex uses its associated miRNA
as a template to bind to complementary sequences found on target mRNAs.
 An exact match generally results in mRNA degradation, but a partial match
also causes translational repression by preventing ribosome and transcription
factor binding.

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GENETICS
MODES OF INHERITANCE

 AUTOSOMAL DOMINANT
 Often due to defects in structural genes.
 The disease is in every generation.
 Both males and females are affected.
 Often pleiotropic (multiple apparently unrelated effects)
and variably expressive (different between individuals).
 Family history crucial to diagnosis.
 Recurrence risk: (probability that offspring will express a genetic disease)
 With one affected (heterozygous) parent, on average, ½ of children
affected.

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 AUTOSOMAL RECESSIVE
 Often due to enzyme deficiencies.
 Usually seen in only 1 generation (the disease
skips generations).
 Commonly more severe than dominant disorders;
patients often present in childhood.
 ↑ Risk in consanguineous families.
 With 2 carrier (heterozygous) parents, on average:
 ¼ of children will be affected
(homozygous).
 ½ of children will be carriers.
 ¼ of children will be neither affected nor
carriers.

Sickle cell anemia is an

autosomal recessive
hemoglobinopathy. In order for
a child to have sickle cell
disease, both parents must be
earners. Hemoglobin
electrophoresis can be used to
determine the carrier status of
a prospective parent who has
no history of sickle cell
anemia.

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 X-LINKED RECESSIVE
 The gene of interest is located on the X chromosome.
 No male-to-male transmission.
 Affected males will always produce unaffected sons and carrier daughters.
 Carrier females have a 50% chance of producing an affected son or carrier
daughter.
 Skips generations.
 Commonly more severe in males.
 Females usually must be homozygous to be affected.
 Females with Turner syndrome (45,XO) are more likely to have an X-linked
recessive disorder.

 X-LINKED DOMINANT
 Transmitted through both parents.
 Mothers transmit to 50% of daughters and sons.
 Fathers transmit to all daughters but no sons (no male-to-male transmission).
 Hypophosphatemic rickets:
 Formerly known as vitamin D–resistant rickets.
 Inherited disorder resulting in ↑ phosphate wasting at proximal tubule.
 Results in rickets-like presentation.
 Other examples: fragile X syndrome, Alport syndrome.

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 MITOCHONDRIAL INHERITANCE
 Transmitted only through the mother  if the mother is affected, all the
offspring will be affected.
 No male-to-male transmission because sperm mitochondria do not pass into the
ovum during fertilization  if the father is affected, no children will be affected.
 Variable expression in a population or even within a family due to heteroplasmy.
 Mitochondrial diseases: (affect highly aerobic tissues like muscles & neurons)
 MELAS syndrome:
 mitochondrial encephalopathy, lactic acidosis, and stroke-like
episodes
 Often present with myopathy, lactic acidosis, and CNS disease.
 2° to failure in oxidative phosphorylation anaerobic
↑lactic acid.
 Leber hereditary optic neuropathy:
 This disorder results in degeneration of the retinal ganglion
cells and optic nerve resulting in progressive, bilateral loss of
vision in young adults.
 Myoclonic epilepsy with ragged red muscle fibers (MERRF).
 Most common mitochondrial disease.
 Myoclonic seizures and myopathy associated with exercise.
 Muscle biopsy often shows ―ragged red fibers‖ (due to
accumulation of diseased mitochondria).

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FA 2018: Rett syndrome

 Sporadic disorder seen almost exclusively in girls (affected males die in utero or
shortly after birth).
 Most cases are caused by de novo mutation of MECP2 on X chromosome.
 Symptoms of Rett syndrome usually appear between ages 1–4 and are characterized
by regression (Retturn) in motor, verbal, and cognitive abilities; ataxia; seizures;
growth failure; and stereotyped handwringing.

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 POLYGENIC INHERITANCE:

 TRINUCLEOTIDE REPEAT EXPANSION DISEASES

 Huntington disease, myotonic dystrophy, fragile X syndrome, and Friedreich
ataxia. Try (trinucleotide) hunting for my fragile cagefree eggs (X).
 May show genetic anticipation (disease severity ↑ and age of onset ↓ in
successive generations).

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EXAMPLES OF IMPORTANT INHERITED DISEASES:

AUTOSOMAL DOMINANT D ISEASES

NEUROFIBROMATOSIS TYPE 1:
 Autosomal dominant disease caused by a mutation in the NF1 gene (tumor suppressor
gene), located on chromosome 17.
 It has 100% penetrance (All patients who inherit the gene develop the disease), but the
presentation is highly variable.
 Clinical picture:
1) Skin:
a) Cafe-au-lait spots (arrows) are hyperpigmented lesions with either
smooth or irregular borders.
b) Axillary or inguinal freckles is another cutaneous feature of NF-1.
2) Neurofibromas:
a) Short, sessile, or pedunculated lesions that vary in size.
b) They are commonly multiple and distributed throughout the body.
3) Eye:
a) Optic nerve gliomas may occur and cause visual loss.
b) Lisch nodules are pigmented asymptomatic hamartomas of the iris.
4) Bony abnormalities:
a) These include sphenoid dysplasia, congenital pseudoarthrosis, and scoliosis.
5) Other associated tumors:
a) Meningiomas, astrocytomas, gliomas, and pheochromocytomas.

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NEUROFIBROMATOSIS TYPE 2 (THE MORE "CENTRAL” FORM)

 Mutation on NF2 gene on chromosome 22; type 2 = 22.
 Characterized by
1) Bilateral acoustic neuromas.
2) Meningiomas.
3) Schwannomas of the dorsal roots in the spinal cord.
4) Juvenile cataracts.

MULTIPLE ENDOCRINE NEOPLASIAS:

 Mucosal neuromas:
1) Unencapsulated,
thickened proliferations
of neural tissue.
2) Flesh-colored nodules
on the lips and tongue.
 Early recognition of MEN
2B  prophylactic
thyroidectomy.
 MEN 1 is associated with
MEN1 gene, MEN 2A and
2B are associated with RET
gene.

VON HIPPEL-LINDAU DISEASE

 Autosomal dominant disorder caused by deletion of the VHL gene on chromosome 3p.
 Characterized by:
1) Cerebellar  hemangioblastomas  ↑erythropeotin  polythycemia.
2) Renal & suprarenal:
a) Bilateral clear cell renal carcinoma  40% of these patients, and is a
major cause of death.
b) Pheochromocytomas.
3) Liver cysts.
4) Retinal hemangioma.

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HEREDITARY HEMORRHAGIC TELANGIECTASIA (OSLER-WEBER-RENDU

SYNDROME)

 Inherited disorder of blood vessels.

 Findings:
1) Branching skin lesions (telangiectasias).
2) Recurrent epistaxis, GI bleeding, hematuria.
3) Skin discolorations.
4) Arteriovenous malformations (AVMs).

TUBEROUS SCLEROSIS
 An autosomal dominant neurocutaneous disorder with multiorgan system involvement.
 Characterized by:
1) CNS:  Neurocutaneous
a) Seizures, and mental retardation. diseases:
b) Calcified periventricular hamartomas (tubers) 1. NF
2) Cutaneous: 2. Tuberous sclerosis
a) Angiofibromas (adenoma sebaceum). 3. VHL
b) Shagreen patches (Rough, raised patches as 4. Sturge Weber
orange peel, in the lumbosacral region). syndrome
c) Asch leef patches (hypopigmented).

3) Multiorgan include:
a) Renal  bilateral angiomyolipoma or cysts.
b) Cardiac  rhabdomyoma.
c) Variety of other hamartomas.

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HUNTINGTON DISEASE:
 Genetics:
1. Autosomal dominant caused by an increase in the number of CAG
trinucleotide repeats in the gene that codes for the huntingtin protein on
chromosome 4 ―Hunting 4 food.‖
2. Abnormal huntingtin causes increased histone deacetylation; this
prevents the transcription of certain genes that code for neurotrophic
factors, contributing to neuronal cell death. As a result, one of the
treatment options under investigation includes histone deacetylase
inhibitors that help upregulate survival genes.
 Findings:
1. Triad of movement disorder (chorea), behavioral abnormalities
(aggressiveness, apathy or depression), and dementia.
2. Most patients develop symptoms in their 40s or 50s. An earlier age of
onset is associated with a larger number of trinucleotide repeats.
3. Caudate atrophy, ↑ dopamine, ↓ GABA, ↓ ACh in the brain.

LI-FRAUMENI SYNDROME
 Abnormalities in TP53  multiple malignancies at an early age.
 Also known as SBLA cancer syndrome (sarcoma, breast, leukemia, adrenal
gland).

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AUTOSOMAL RECESSIVE DISEASES

CYSTIC FIBROSIS:

Genetics  Autosomal recessive; defect in CFTR gene on chromosome 7;

commonly a deletion of 3 base pairs of Phe508.
 Most common lethal genetic disease in Caucasian population.
Pathophysiology  CFTR encodes an ATP-gated Cl- channel that secretes Cl- in lungs
and GI tract, and reabsorbs Cl- in sweat glands.
 Most common mutation  misfolded protein  protein retained in
RER and not transported to cell membrane, causing ↓ Cl- (and H2O)
secretion; ↑ intracellular Cl- results in compensatory ↑ Na+
reabsorption via epithelial Na+ channels  ↑ H2O reabsorption 
abnormally thick mucus secreted into lungs and GIT.
 ↑ Na+ reabsorption also causes more negative transepithelial
potential difference.

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Labs  ↑ Cl- concentration (> 60 mEq/L) in sweat is diagnostic.

 If the sweat chloride test is equivocal, measurement of nasal
transepithelial potential difference and genetic testing for CFTR
mutations should be performed to confirm the diagnosis.
 Can present with contraction alkalosis and hypokalemia (ECF effects
analogous to a patient taking a loop diuretic) because of ECF H2O/Na+
losses and concomitant renal K+/H+ wasting.
 ↑ Immunoreactive trypsinogen (newborn screening).

Symptoms &  Pulmonary:

complications  Recurrent pulmonary infections (eg, S aureus [early infancy],
P aeruginosa [adolescence]).
 Obstructive lung disease & chronic bronchitis  bronchiectasis
 reticulonodular pattern on CXR, opacification of sinuses.
 Nasal polyps, clubbing of nails.
 GIT:
 Meconium ileus in newborns (MCCO meconium ileus).
 Pancreatic insufficiency, malabsorption with steatorrhea, fat-
soluble vitamin deficiencies (A, D, E, K),
 Biliary cirrhosis, liver disease.
 Infertility in men:
 Although spermatogenesis is usually normal, almost all males
with CF are unable to secrete semen ―azoospermia‖ due to
congenital bilateral absence of the vas deferens) and
 Subfertility in women:
 Amenorrhea, abnormally thick cervical mucus.
 CFTR mutations are likely responsible for abnormal
development of Wolffian structures  vasal agenesis and
defective sperm transport.
 UW: Patients with CF produce eccrine sweat that contains high
concentrations of sodium and chloride compared to normal individuals
 easy to collapse with exercise.

Treatment  Multifactorial: chest physiotherapy, albuterol, aerosolized dornase

alfa (DNAse), and hypertonic saline facilitate mucus clearance.
 N-acetylcysteine to loosen mucus plugs (cleaves disulfde bonds within
mucus glycoproteins)
 Azithromycin used as anti-inflammatory agent.
 Ibuprofen slows disease progression.
 Pancreatic enzymes for insufficiency.
 FA 2018: In patients with Phe508 deletion: combination of lumacaftor
(corrects misfolded proteins and improves their transport to cell
surface) and ivacaftor (opens Cl– channels  improved chloride
transport).

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X-LINKED DISEASES:

FRAGILE X SYNDROME
Genetics  XLD disease  males who inherit the full mutation will manifest the
features of FXS, whereas females with one abnormal X chromosome
will have a milder and variable presentation.
 Trinucleotide repeat (CGG) in FMR1 gene  hypermethylation  ↓
expression.
 When the cells of affected individuals are cultured in folate-deficient
medium, the area of increased repeats does not stain and appears
"broken." hence the name "fragile X."
NB: Trinucleotide repeat disorder (CGG)n. Chin (protruding), Giant Gonad.
Epidemiology  Most common cause of inherited intellectual disability and autism.
 2nd most common cause of genetically associated mental deficiency
(after Down syndrome).
Findings  Post-pubertal macroorchidism (enlarged testes).
 Long face with a large jaw, large everted ears.
 Neuropsychiatric findings
1. Developmental delay (earliest manifestations)
2. Intellectual disability
3. Attention deficit-hyperactivity disorder.
4. Anxiety
 Mitral valve prolapse.
 Patients often have hyperlaxity of the joints in the hand.
Diagnosis  Southern blot analysis is used to measure the degree of methylation and
determine the number of CGG repeats.
 Cytogenetic studies (chromosomal analysis) typically show a small
gap near the tip of the long arm of the X chromosome.

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MUSCULAR DYSTROPHIES
 DUCHENNE MUSCULAR DYSTROPHY:

Genetics  X-linked recessive disorder typically due to frameshift or nonsense

mutations  truncated or absent dystrophin protein  progressive
myofiber damage. (Duchenne = deleted dystrophin.)
 Dystrophin gene (DMD) is the largest protein-coding human gene 
↑ chance of spontaneous mutation.
 Dystrophin helps anchor muscle fibers, primarily in skeletal and
cardiac muscle.
 It connects the intracellular cytoskeleton (actin) to the transmembrane
proteins α- and β-dystroglycan, which are connected to the
extracellular matrix (ECM).
 Loss of dystrophin results in myonecrosis.
Findings  Onset before 5 years of age.
 Weakness begins in pelvic girdle muscles and progresses superiorly 
Gower sign & Waddling gait.
 Pseudohypertrophy of calf muscles due to fibrofatty replacement of muscle A.
 Dilated cardiomyopathy is common cause of death.

Diagnosis  ↑ CK and aldolase are seen.

 Genetic testing confirms diagnosis.

 BECKER MUSCULAR DYSTROPHY:

Genetics  X-linked recessive disorder typically due to nonframeshift deletions in

dystrophin gene (partially functional instead of truncated).
 Deletions can cause both Duchenne and Becker muscular dystrophies.
 2⁄3 of cases have large deletions spanning one or more exons.
Findings  Less severe than Duchenne.
 Onset in adolescence or early adulthood.

 MYOTONIC TYPE 1
1) Autosomal dominant.
2) CTG trinucleotide repeat expansion in the myotonic dystrophy protein kinase
gene DMPK  abnormal expression of myotonin protein kinase  myotonia,
muscle wasting, cataracts, testicular atrophy, frontal balding, and arrhythmia.
Cataracts, Toupee (early balding in men), Gonadal atrophy.

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GOWER SIGN
 Patient uses upper extremities to help stand up.
 Classically seen in Duchenne muscular dystrophy, but also seen in other muscular
dystrophies and inflammatory myopathies (eg, polymyositis).

GENETIC TERMS

TERM DEFINITION EXAMPLE

Locus Location of a gene on a chromosome.
Allele Different forms of a gene. If an individual
has the same allele on both homologous
chromosomes, they are said to be
homozygous for that allele. If the individual
has different alleles, they are said to be
heterozygous.
Genotype A description of the alleles carried at a
particular locus.
Phenotype Physically observable features.
(Appearance or function). The final
manifestations of the genotype.
Codominance Both alleles contribute to the phenotype of Blood groups A, B, AB;
the heterozygote. α1-antitrypsin deficiency.

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Variable Every diseased person in the pedigree 2 patients with

expressivity (genotypically diseased) has the disease but neurofibromatosis type 1
to different degrees. (NF1) may have varying
At least they have SOME pathology. disease severity (variable
number of tumors & size of
the tumors).
For example, a patient with
Marfan syndrome may have
only tall stature while
another patient with the
same genetic defect will
have tall stature aortic root
dilation , and lens
dislocation.
Incomplete Not all individuals with a mutant genotype BRCA1 gene mutations do
penetrance show the mutant phenotype. not always result in breast or
Some people with the mutant genotype have ovarian cancer.
the disease & the others have NO Mostly in autosomal
pathology. dominant diseases as
retinoblastoma,
hemochromatosis.
Recurrence risk is affected
if the penetrance is
incomplete.

Pleiotropy One gene is mutated and multiple organs Untreated phenylketonuria

are affected. (PKU) manifests with light
Most syndromic genetic illnesses exhibit skin, intellectual disability,
pleiotropy. and musty body odor.
Cystic fibrosis also exhibit
multiple organ affection.

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Anticipation Increased severity or earlier onset of Trinucleotide repeat

disease in succeeding generations. diseases (eg, Huntington
disease).

Loss of If a patient inherits or develops a mutation Retinoblastoma and the

heterozygosity in a tumor suppressor gene, the ―two-hit hypothesis,‖ Lynch
complementary allele must be deleted/ syndrome (HNPCC), Li-
mutated before cancer develops. This is not Fraumeni syndrome.
true of oncogenes (only one hit is enough.)
Dominant Exerts a dominant effect. A heterozygote Mutation of a transcription
negative produces a nonfunctional altered protein factor in its allosteric site.
mutation that also prevents the normal gene product Nonfunctioning mutant can
from functioning. still bind DNA, preventing
wild-type transcription factor
from binding.

Linkage Tendency for certain alleles at 2 linked loci

disequilibrium to occur together more or less often than
expected by chance. Measured in a
population, not in a family, and often varies
in different populations.

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Mosaicism Process by which an organism can have McCune-Albright

two or more populations of cells within syndrome—due to mutation
the body with slightly different affecting G-protein signaling.
genotypes. Presents with unilateral café-
‫مش كل خاليا الجسم الواحد شبه بعضها‬ au-lait spots with ragged
Somatic mosaicism—mutation arises edges, polyostotic fibrous
from mitotic errors after fertilization and dysplasia, and at least one
propagates through multiple tissues or endocrinopathy (eg,
organs. precocious puberty).
Gonadal mosaicism—mutation only in Lethal if mutation occurs
egg or sperm cells. If parents and before fertilization (affecting
relatives do not have the disease, all cells), but survivable in
suspect gonadal (or germline) patients with mosaicism.
mosaicism. Another example if 2 identical
twins, one has Down
syndrome and the other one is
normal.
Locus Mutations at different loci can produce a Albinism.
heterogeneity similar phenotype. (Different locations
of the gene).
Allelic Different mutations in the same locus 1- Hemophilia may be caused
heterogeneity produce the same phenotype. by several different mutations
May be different in severity but the same that vary in the alteration of
disease. the factor VIII gene product.
It can’t happen in the same family as Missense mutations will cause
the same mutation or allele are less severe disease than
transmitted. nonsense mutations, which
cause the production of
truncated, nonfunctional
molecules.
2- β-thalassemia.
Heteroplasmy Having different organellar genomes (eg, Mitochondrial myopathy
mutated and wild-type of mitochondrial
DNA) within a single cell.
The higher proportion of defective
mitochondrial genomes within the cells,
the higher the severity of the disease.
Resulting in variable expression in
mitochondrially inherited disease.
Uniparental Offspring receives 2 copies of a Uniparental is euploid (correct
disomy chromosome from 1 parent and no copies number of chromosomes), not
from the other parent. aneuploid. Most occurrences
HeterodIsomy (heterozygous) indicates a of uniparental disomy (UPD)
meiosis I error. IsodIsomy (homozygous)  normal phenotype.
indicates a meiosis II error or postzygotic Consider UPD in an individual
chromosomal duplication of one of a pair manifesting a recessive
of chromosomes, and loss of the other of disorder when only one parent
the original pair. is a carrier

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IMPRINTING
 At some loci, only one allele is active; the other is
inactive (imprinted/inactivated by methylation). It is a
normal process.
 With one allele inactivated, deletion of the active
allele  disease.
 Phenomenon in which an offspring's genes are
expressed in a parent-specific manner.
 Both Prader-Willi and Angelman syndromes are due
to mutation or deletion of genes on chromosome 15.

Prader-Willi syndrome AngelMan syndrome

Maternal imprinting: gene Paternal imprinting: gene
from mom is normally silent from dad is normally silent
and Paternal gene is deleted/ and Maternal gene is
mutated. deleted/mutated.
Results in hyperphagia, Results in inappropriate
obesity, intellectual laughter (―happy puppet‖),
disability, hypogonadism, seizures, ataxia, and severe
and hypotonia. intellectual disability.
25% of cases due to 5% of cases due to paternal
maternal uniparental uniparental disomy
disomy (two maternally (two paternally imprinted
imprinted genes are received; genes are received; no
no paternal gene received). maternal gene received).

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Lyonization:
 A process that converts the inactive X chromosome into condensed
heterochromatin, which can be identified on microscopy as a compact body at the
periphery of the nucleus (Barr body).
 Disease carrier females are variably affected depending on the pattern of
inactivation of the X chromosome carrying the mutant vs normal gene.
 Example: in Lesch-Nyhan (XLR), carrier females inherited the mutant gene from
the mother. 50% of the cells will have ↓HGPRT enzyme as the normal gene on the
X-chromosome is inactivated & 50% of the cells will have normal HGPRT.

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HARDY-WEINBERG POPULATION GENETICS

 The Hardy-Weinberg principle is based on the binomial theorem: p2 + 2pq + q2 = 1.
1) For normal allele (A), the frequency in the population is p.
2) For the mutant allele (a), the frequency in the population is q.
3) Because there are assumed to be only 2 alleles, p + q = 1.
4) The frequency of the homozygote A = p2, the heterozygote Aa = 2pq, and the
frequency of the mutant homozygote a = q2.

 Application in the cases of modes of inheritance.

1) Autosomal recessive inheritance:
a. For a disorder with an incidence of 1/10000 persons, q2 = 1/10000 and q
= 1/100.
b. p = 1 – 1/100 or 99/100, and the frequency of AA is p2 = 98/100.
c. The frequency of the heterozygote is 2pq ~ 1/50.
d. Carrier frequency: Heterozygous individuals (disease carriers) have
only 1 mutant allele (genotype is either pq or qp) so in general, carrier
frequency = 2pq. For rare autosomal recessive disorders, p = 1 therefore,
the probability of being a carrier approximates to 2x frequency of the
mutant allele or 2q.
2) Autosomal dominant inheritance:
a. The incidence of the disorder in the population = 2pq.
b. The frequency of the mutant allele = half the incidence.
c. For simplicity, the homozygous affected individuals are ignored because
such diseases are typically lethal.
3) X-linked inheritance:
a. The frequency of an X-linked recessive disease in males = q and in
females = q2.
b. The carrier frequency in females is 2pq.

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 Hardy-Weinberg law assumptions include:

 No mutation occurring at the locus.
 Natural selection is not occurring.
 Completely random mating.
 No net migration.

CHROMOSOMAL ABNORMALITIES

Chromosomal
abnormalities

Numerical Structural

Euploid Cells (multiple of

Aneuploidy 23 chromosomes) Translocations

Deletions/
autosomal sex chromosomes tripleudy (69)
duplications

Monosomy
(incopmpitible with Trisomy Monosomy (Turner) tetrapleudy inversions
life)

Incompitible with life (16)

MCCO of 1st trimester trisomy (Kleinfilter) ring chromosomes
trisomy

Compitable with life

isochromosomes
(13, 18, 21)

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 STRUCTURAL ALTERATIONS OF THE CHROMOSOMES:

 Types:
1. Balanced alterations (90%) result in no loss or gain of the genetic material.
2. Unbalanced alterations (10%) result in loss or gain of the genetic material.
 If these structural alterations that occurs in:
a. Germline  transmitted to offspring.
b. Somatic cell  cancer.

1- RECIPROCAL TRANSLOCATION:
 Occurs when genetic material is exchanged between nonhomologous
chromosomes; for example, chromosomes 2 and 8.

 If it occurs in the germline of an individual:

 No problem as there are still 2 copies of the genes.
 This individual is ―reciprocal translocation carrier‖
 Carriers are typically identified during:
 Evaluation for recurrent pregnancy loss or infertility.
 After the diagnosis a fetus or a child with an unbalanced
translocation.
 If it occurs in somatic cells:  give rise to cancers.

 CML ―chronic myeloid leukemia‖:

 t(9;22)  This translocation fuses the BCR gene on
chromosome 22 to the ABL gene on chromosome 9, resulting
in formation of the oncogenic BCRABL fusion gene on the
shortened chromosome 22  continuous activation of
tyrosine kinase.
 Imatinib is used as tyrosine kinase inhibitor treating CML.
 Karyotype analysis shows elongation of chromosome 9.

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 Burkitt lymphoma:
 C-myc proto-oncogene is a specific transcription factor, regulates
expression of 15% of all genes. Degraded by ubiquitin-proteasomes
(t1/2 = 30 min.)
 t(8;14)  Ig heavy chain gene near c-myc  inhibits
ubiquitination.

 Acute promyelocytic leukemia (APML):

 t(15;17)  translocation between the retinoic acid receptor alpha
(RARα) gene on chromosome 17 and the promyelocytic leukemia
(PML) gene on chromosome 15.
 Fusion of these 2 genes produces a chimeric gene product
(PML/RARα) which codes for an abnormal retinoic acid receptor.
 This abnormal fusion gene inhibits promyelocyte differentiation
and triggers the development of APML.
 Management is with all-trans retinoic acid.

 Follicular non-Hodgkin lymphoma:

 t(14;18)  moves the Bcl-2 (B-cell lymphoma-2) protooncogene
from chromosome 18 to chromosome 14, near the site of the
immunoglobulin heavy chain enhancer element.
 Bcl-2 is considered a protooncogene because it has anti-apoptotic
effects.
 When this protooncogene is positioned near the immunoglobulin
enhancer element the resultant Bcl-2 overexpression allows for
cell immortality.

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2- ROBERTSONIAN TRANSLOCATION
 Involves the loss of the short arm (P arms) of the acrocentric chromosomes
(chromosomes with centromeres near their ends  13, 14, 15, 21, and 22.) then
the q arms are fused at the centromere.

 No essential genes are lost by losing the short arms of these chromosomes.
 Still have 2 copies of genes  no problem in this individual but could be in the
offspring.
 Karyotyping: 45 XY, -14, -21, +t(14q; 21q) or 45 XX, -14, -21, +t(14q; 21q)

 Balanced translocations
normally do not cause any
abnormal phenotype.
 Unbalanced
translocations can result in
miscarriage, stillbirth, and
chromosomal imbalance
(eg, Down syndrome, Patau
syndrome).

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3- DELETIONS:
 Type of deletions:
 Chromosomal  Turner 45, XO.
 Single gene deletions  Prader-Willi, Angelman syndromes
(chromosome 15).
 Microdeletions  Cri-du-chat, Williams, DiGeorge syndromes.
 Codon deletions  Cystic fibrosis.
 Nucleotide deletions  Duchenne (Frameshift).

CRI-DU-CHAT SYNDROME:
 Congenital microdeletion of short arm of chromosome 5 (46,XX or
XY, -5p).
 Findings:
 High-pitched crying/ meowing. (Cri du chat = cry of the cat.)
 Microcephaly, moderate to severe intellectual disability.
 Epicanthal folds.
 Cardiac abnormalities (VSD).

WILLIAMS SYNDROME:
 Congenital microdeletion of long arm of chromosome 7 (deleted
region includes elastin gene).
 Findings:
 Distinctive ―elfin‖ facies. Think Will Ferrell in Elf.
 Intellectual disability, well-developed verbal skills.
 Extreme friendliness with strangers.
 Hypercalcemia (↑ sensitivity to vitamin D).
 Cardiovascular problems (eg, supravalvular aortic stenosis,
renal artery stenosis).

22Q11 DELETION SYNDROMES:

 Microdeletion at chromosome 22q11.

 Due to aberrant development of 3rd and 4th branchial pouches.
 Findings:
 Variable presentations including Cleft palate, Abnormal facies,
Thymic aplasia  T-cell deficiency, Cardiac defects (T4), and
Hypocalcemia 2° to parathyroid aplasia. CATCH-22.
Patients typically present with hypocalcemic tetany and recurrent viral and
fungal infections due to T-cell deficiency.

 DiGeorge syndrome—thymic, parathyroid, and cardiac defects.

 Velocardiofacial syndrome—palate, facial, and cardiac defects.

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4- ISOCHROMOSOME
 When a chromosome divides along the axis perpendicular to its normal axis of
division  two copies of one arm but no copy of the other.
 Karyotyping: 46,X,i(Xq)
 All isochromosomes are lethal except the isochromosome-X  Turner syndrome
(22% of turner syndrome are due to isochromosomes)

 NON-DISJUNCTION:
 Failure of chromosome pairs to separate properly during cell division.
 This could be due to a failure of homologous chromosomes to separate in
meiosis I or a failure of sister chromatids to separate during meiosis II or
mitosis.
 This is a major cause of aneuploidy.

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o Non-disjunction detection by RFLP analysis:

 Restriction fragment length polymorphism (RFLP) analysis shows that both

parents demonstrate 2 bands. Each parental band represents a homologous
chromosome 21.
 The child A has 3 bands indicating that he has 3 different versions of
chromosome 21 that he obtained from his parents. He received the lower band
from the father and both of the upper bands from the mother. The fact that he
received 2 different bands from the mother indicates that he inherited both of
her homologous chromosomes. Thus, the problem occurred in the mother
during meiosis I. In fact, the vast majority of Down syndrome cases arise due
to nondisjunction during maternal meiosis I.
 The child B analysis reveal only two bands: a single band from the father and
a darker, thicker band from the mother. The darker, thicker band signifies
the inheritance of both sister chromatids, which will produce equal-size
restriction fragments (but twice the normal amount.)

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AUTOSOMAL TRISOMIES

 DOWN SYNDROME (TRISOMY 21)

 Incidence:
 1:700. Most common viable chromosomal disorder.
 Most common cause of genetic intellectual disability.
 Findings:

System Findings
affected
CNS  Mental retardation (IQ range is 20-70).
 Early Alzheimer disease.
Head & neck  Upslanting palpebral fissures, epicanthal folds, flat nasal bridge.
 speckled iris (Brushfield spots)
 open mouth with protruding tongue
Musculoskeletal  Hypotonia, atlanto-axial instability,
 short hands with single transverse palmar crease (Simian crease)
 Gap between 1st 2 toes.
CVS  Endocardial cushion defects (atrioventricular septal defects).
 VSD, ASP, PDA , ToF
GIT  Duodenal atresia or stenosis, annular pancreas.
 Tracheoesophageal fistula.
 Hirschsprung's disease.
 Omphalocele, imperforate anus.
Immunologic  Impaired cellular immunity, high incidence of infections and
autoimmune disorders and malignancies.
Hematologic  AML-M7 (500x increased risk) and ALL (10-20x increased risk) in
childhood.
Endocrine  Hypothyroidism, hyperthyroidism, type I diabetes mellitus.
 Infertility in males.
 Why Down syndrome is associated with early-
onset Alzheimer disease?
 The Amyloid precursor protein gene is
located on chromosome 21.
 The extra copy of APP present in Down
syndrome is thought to accelerate amyloid
accumulation and lead to early-onset AD.

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Inheritance in down syndrome

Inheritance Pathogenesis Recurrence Number of
risk chromosomes
Meiotic  Extra copy of chromosome 21 Based on 47
nondisjunction present in every cell. maternal age. chromosomes.
(95%)
Unbalanced  All or part of additional High if balanced 46
(Robertsonian) chromosome 21 attached to translocation is chromosomes.
translocation another chromosome. present in one
(4%)  Most typically between parent.
chromosomes 14 and 21.

Mosaicism  Some (not all) cells have an Based on

(1%) extra copy of chromosome 21. maternal age.
 Nondisjunction event in early
embryonic life
(postfertilization mitotic
error).
 Affected individuals have 2
cell lines; 1 with a normal
genotype, and 1 with trisomy
21.
 The proportion of affected
cells determines the severity of
DS features.
 Diagnosis of Dawn syndrome:
 First-trimester commonly shows:
 Ultrasound:
 ↑ Nuchal translucency (excessive skin at the posterior neck)
 Hypoplastic nasal bone & flat facial features.
 Maternal serum:
 ↓ Serum pregnancy-associated plasma protein A (PAPP-A).
 ↑ Free β-hCG.
 Second-trimester quad screen at 15-18 weeks gestation shows:
 ↓ α-fetoprotein, ↓ estriol  due to suboptimal fetal tissue function.
 ↑ β-hCG, ↑ inhibin A  compensatory placental hyperfunction.
 The diagnosis is confirmed by:
 Karyotyping fetal cells in the amniotic fluid (amniocentesis).

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 EDWARDS SYNDROME (TRISOMY 18)

Epidemiology  Death usually occurs by age 1. Incidence 1:8000.

 2nd most common autosomal trisomy resulting in live birth (most
common is Down syndrome).
Findings  PRINCE Edward—Prominent occiput, Rocker-bottom feet,
Intellectual disability, Nondisjunction, Clenched fists (with
overlapping fingers), low-set Ears, micrognathia (small jaw),
congenital heart disease.
Diagnosis  First trimester:
 ↓ PAPP-A and free β-hCG.
 Second-trimester quad screen at 15-18 weeks gestation shows:
 ↓ α-fetoprotein, ↓ β-hCG.
 ↓ Estriol, ↓ or normal inhibin A.

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 PATAU SYNDROME (TRISOMY 13)

 Death usually occurs by age 1. Incidence 1:15,000.
 Findings:
 Severe intellectual disability, microcephaly, holoProsencephaly,
microphthalmia.
 Cleft liP/Palate,
 Polydactyly, rockerbottom feet.
 Cutis aPlasia.
 Polycystic kidney disease.
 Congenital heart disease.
 Diagnosis:
 First-trimester pregnancy screen shows ↓ free β-hCG, ↓ PAPP-A.

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KLINEFELTER SYNDROME

Genetics  Caused by a meiotic nondisjunction event during parental gametogenesis

that results in a 47,XXY karyotype.
 Variants include 46.XY/47.XXY mosaicism and 48.XXXY.
 In general, patients with higher numbers of X chromosomes are more likely
to have more severe manifestations.
Findings  The disorder is usually not diagnosed until puberty when the characteristic
physical signs begin to develop.
 The major features are as follows:
1. Primary testicular failure:
a. Due to hyalinization and fibrosis of the seminiferous tubules.
b. This results in small, firm testes and azoospermia (infertility)
Leydig cell dysfunction also occurs and leads to testosterone
deficiency.
c. Gonadotropin (FSH, LH) levels are increased secondary to
gonadal failure.
2. Testosterone deficiency results in
a. Eunuchoid body habitus.
b. tall stature
c. Gynecomastia.
d. Facial and body hair is sparse or absent
e. Muscle mass is decreased.
3. Mild intellectual disability is seen in some patients, although the
majority have normal intelligence.
4. Psychosocial abnormalities (eg. lack of insight, poor judgment) are
also common.

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TURNER SYNDROME

Genetics  Most commonly caused by paternal meiotic nondisjunction during

gametogenesis.
 The loss of the parental X chromosome in the sperm results in a missing X-
chromosome in most or all cells.
 Turner has 3 forms:
1. Complete loss of an X chromosome (45.XO): most of patients.
2. Mosaicism:
a. Missing the X chromosome in some of cells.
b. This is known as mosaic Turner syndrome (45, X / 46, XX).
c. Due to non-disjunction in mitosis after fertilization.
3. Abnormal X-chromosome:
a. Some patients have both X chromosomes, but one is
abnormally shaped, missing some genetic material, or has
structural abnormalities (eg, X fragments, isochromosomes).
 The loss of the X chromosome results in a missing SHOX gene, which is
responsible for long bone growth. Therefore, patients with Turner syndrome
typically have short stature.
Findings  Short stature, shield chest (broad, with widely spaced nipples), and webbed
neck (broad neck with low hairline),
 Streak ovaries (degeneration of the ovarian follicles with replacement by
fibrotic tissue)  ↓Estrogen will lead to:
1. Primary amenorrhea.
2. ↓Breast development.
 Cystic hygromas and lymphedema occur due to abnormalities of lymphatic
outflow in Turner syndrome. The swelling decreases with age.
 Coarctation of the aorta, bicuspid aortic valve.

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GENETIC DISORDERS BY CHROMOSOME

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RECOMBINATION AND LINKAGE:

 Linkage: alleles of 2 loci that likely to be inherited together.

 Recombination: is gene exchange that occurs through the crossing over of two
double-stranded DNA molecules.
 Crossing over between alleles makes the chance of 2 different alleles to be
inherited together less.
 Recombination frequency:
 The closer to 0%, the closer the alleles in a haplotype to each other.

Review question:

A family with an autosomal dominant disorder is typed for a 2

allele marker, which is closely linked to the disease locus. Based on
the individuals in Generation III, what is the recombination rate
between the disease locus and the marker locus?
(A) 0
(B) 0.25
(C) 0.50
(D) 0.75
(E) 1.0
(F) The marker is uninformative
Answer: A. In this pedigree, the disease allele is consistently transmitted with the 1 allele.
There is no case in this small number of individuals where recombination between these two
loci has occurred. Therefore, in Generation III, there is no recombination seen in any of the
four individuals. Receiving the one allele always goes together with receiving the disease
gene. Linked markers can be ―uninformative‖ (choice E) in some pedigrees if, for example,
the same alleles are expressed in all family members. In such a case, it would be impossible
to determine any recombination frequency.

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PROTO-ONCOGENES & TUMOR SUPPRESSOR GENES

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CELLULAR BIOCHEMISTRY
CELL CYCLE

 The cell cycle is the process that a cell goes through to divide into two daughter
cells.
 The cell cycle consists of five phases (S, G2, M, G1, and G0):
1) G1 phase:
 Synthesis of proteins, organelles.
 Expression of genes related to DNA synthesis.
 Length varies depending on conditions.
 Mitogens:
 Extracellular signaling molecules, usually proteins.
 Stimulate cell division.
 Function via cyclin dependent kinases (Cdks).
 Growth factor:
 Stimulates growth in size.
 Some molecules both mitogens and GFs.
 Terms sometimes used interchangeably.
2) S (synthesis) phase:
 The period of DNA replication during which the cell is making a
second copy of its DNA.
 Chromosomes  two sister chromatids.
3) G2 phase:
 Check for replication errors & prepare for mitosis.

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4) M (mitosis) phase:
 Shortest phase of cell cycle.
 The period during which the cell is actually dividing.
 This phase is traditionally divided into five stages based on the
microscopic appearance of the nuclear membrane and the
chromosomes:
 Prophase:
 Chromosomes condense.
 The nuclear membrane dissolves.
 Spindle fibers appear.
 Prometaphase:
 Spindle fibers attaches to condensed chromosomes.
 Metaphase:
 The condensed chromosomes line up in the center of
the cell.
 Anaphase:
 The chromosomes migrate to either side of the cell,
pulled by microtubules.
 Telophase:
 The chromosomes decondense.
 Separate nuclear membrane reforms around each set of
chromosomes.
 Cytokinesis: cytoplasm splits in two.

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5) G0 phase:
 Quiescent phase outside of the cell cycle in which no cell division
takes place (non-dividing state).
 Permanent cells are in this phase.
 May occur in absence of mitogen stimulation of cells.

 This process is regulated by cyclins, cyclin-dependent kinases (CDKs), and tumor

suppressors.

REGULATION OF CELL CYCLE

 Cells regulate progression through ―checkpoints‖
1) Also called ―restriction points‖.
 G1-S (prior to S phase entry).
 G2-M (prior to mitosis).
 M phase (prior to anaphase/cytokinesis).
2) Arrests cell if conditions not appropriate.
 Cyclin-dependent kinases:
1) Constitutive and inactive
 Always present in cells but inactive.
 Depend on cyclins to activate.
 Central components of cell cycle control.
2) Kinase enzymes (lead to phosphorylation of other proteins).
 Cyclins:
1) Regulatory proteins that control cell cycle events by activating CDKs.
2) Phase specific ―multiple subtypes of cyclins for each phase‖.
 Cyclin-CDK complexes:
1) Phosphorylate other proteins to coordinate cell cycle progression.
2) Must be activated and inactivated at appropriate times for cell cycle to
progress.

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 G1-S Checkpoint:
1) During G1 phase  Cdk activity is suppressed.
2) Mitogens activate Cdk  entry into S phase:
 ↑ G1 cyclin levels  ↑ Cdk activity.
3) Cyclin-Cdk complexes activate E2F proteins.
 E2F are transcription factors that bind to DNA promoter regions.
 Activate genes for S phase.
4) E2F normally inhibited:
 Inhibited by E2F binding to retinoblastoma proteins (Rb).
 Inhibition released by G1-S-Cdk phosphorylation of Rb.
5) Rb regulates cell growth ―Tumor suppressor‖.

 DNA damage can arrest cell division:

1) Allows for repair, prevents development of mutant cells/cancer.
2) DNA damage initiates signaling pathways to arrest cell cycle.
3) ATM pathway: Activated by double strand breaks:
 ATM: Ataxia Telangiectasia Mutated.
 ATM gene mutation  Ataxia Telangiectasia.
4) ATR pathway: Single stranded breaks.
5) Both pathways lead to phosphorylation of proteins (P53 protein)  cause
cell cycle/growth arrest.
 P53 Protein:
1) Major target of ATM/ATR systems.
2) Phosphorylated after DNA damage.
 Prevents p53 breakdown.
 Increases levels/activity.
3) P53 induces transcription of p21 protein:
4) P21 binds to Cdks  inhibits Cdk activity.
5) Blocks cell progression through cell cycle.
6) P53/p21 = tumor suppressors.

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 Tumor suppressors:
1) P53 induces p21, which inhibits CDKs  hypophosphorylation
(activation) of Rb  inhibition of G1-S progression.
2) Mutations in tumor suppressor genes can result in unrestrained cell division
(eg, Li-Fraumeni syndrome).

RETINOBLASTOMA
 Rare childhood eye malignancy.
 Mutations in RB1gene which codes for Rb protein.
 Abnormal Rb  unregulated cell growth (via E2F).

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LI-FRAUMENI SYNDROME
 Syndrome of multiple malignancies at an early age ―Sarcoma, Breast,
Leukemia, Adrenal Gland‖ ―SBLA‖ cancer syndrome.
 Mutation in tumor suppressor gene TP53 which codes for p53 protein.
 Mutation: Cycle not arrested to allow for DNA repair  accumulation of
damage  malignancy.

CELL TYPES:

Permanent
 Remain in G0, regenerate from stem cells.
 Neurons, skeletal and cardiac muscle, RBCs.
Stable (quiescent)
 Enter G1 from G0 when stimulated.
 Hepatocytes, lymphocytes.
Labile
 Never go to G0, divide rapidly with a short G1.
 Most affected by chemotherapy.
 Bone marrow, gut epithelium, skin, hair follicles, and germ cells.

CANCER DRUGS & CELL CYCLE

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ENDOPLASMIC RETICULUM

 Found in all eukaryotic cells.

 Folded membrane of sacs/tubules.
 Continuous with nuclear membrane.
 Site of synthesis of proteins and lipids.

ROUGH ENDOPLASMIC RETICULUM:

 Surface of ER covered with ribosomes  gives granular or ―rough‖ appearance.

 Site of synthesis of secretory (exported) proteins.
 Abundant in cell that secrete proteins:
1) Goblet cells of intestines (mucus).
2) Plasma cells (antibodies).
3) Pancreatic beta cells (insulin).
 Membrane bound ribosomes:
1) Found in RER.
2) Produce proteins mostly for secretion from cell.
 Protein hormones, digestive enzymes.
 Free ribosomes:
1) Found ―free‖ in cytosol.
2) Produce proteins mostly used by cell.
 Synthesis of cytosolic and organellar proteins.
 Nissl Bodies
1) Rough endoplasmic reticulum in neurons.
2) Synthesize neurotransmitters.

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 Post-translational modification of proteins by RER:

1) Eg, N-linked oligosaccharide addition to proteins inside the RER.
2) Most of these proteins are targeted for export to the Golgi apparatus.
3) These proteins pass from the RER to the Golgi apparatus within COP II
coated transport vesicles.
4) The Golgi apparatus sorts and distributes proteins to the cell membrane
organelles, and secretory granules.

SMOOTH ENDOPLASMIC R ETICULUM

 Portions of ER without ribosomes.

 Important for lipid/steroid synthesis  Adrenal glands and gonads.
 Also detoxification of drugs and toxins.
 Lots of SER found in hepatocytes:
1) Synthesis of cholesterol/lipoproteins.
2) Contains many detoxification enzymes as cytochrome P450 family of
enzymes.
 Sarcoplasmic reticulum = SER in myocytes  Stores calcium for muscle
contraction.

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GOLGI APPARATUS
 Proteins leave ER in vesicles  transported to Golgi  Fuse with Golgi membrane 
empty their contents.
 Cis Golgi network: Vesicles come into cis face from RER.
 Trans Golgi network: Vesicles leave from trans face.
 Proteins sorted/shipped by adding signal sequences.
 Golgi function:
 Distribution center for proteins and lipids from the ER to the vesicles and
plasma membrane.
 Modifies N-oligosaccharides on asparagine.
 Adds O-oligosaccharides on serine and threonine.
 Adds mannose-6-phosphate to proteins for trafficking to lysosomes.

 N-linked Oligosaccharides:
 Synthesized in endoplasmic reticulum.
 Sugars added to asparagine (extra N molecule).
 Modified in Golgi apparatus (trimmed, sugars added).
 O-linked Oligosaccharides:
 Occurs in Golgi apparatus.
 Sugars added to serine/threonine (extra O molecule).
 Example: Mucins heavily O-glycosylated.
 Mannose-6-Phosphate:
 Added to proteins destined for lysosomes.
o Acid hydrolase enzymes.
o Added to N-linked oligosaccharides.
 Triggers packaging in trans-Golgi  lysosomes.
 Process disrupted/abnormal in I-cell disease.

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 I-cell Disease ―Inclusion Cell Disease‖

 Rare autosomal recessive metabolic disorder.
 Lysosomal storage disease (mucolipidosis).
 Onset in 1st year of life:
 Growth failure.
 Coarse facial features.
 Hypotonia/Motor delay.
 Failure of processing in Golgi apparatus:
 Mannose-6-phosphate NOT found on lysosome proteins.
 Deficiency: N-acetylglucosaminyl-1-phosphotransferase.
 Phosphate not added to mannose due to missing enzyme.
 Result: enzymes secreted outside of cell.
 Hydrolases missing from lysosomes.
 Can be detected in blood/urine (outside cell).
 Lysosomes contain inclusions of undigested glycosaminoglycans and
glycolipids.

ENDOSOMES
 Membrane-bound compartments in cells.
 Formed by endocytosis:
1) Invagination of plasma membrane to surround molecules.
2) Pinching off of membrane to form enclosed structure.
 Endosomes are sorting centers for material from outside the cell or from the Golgi,
sending it to lysosomes for destruction or back to the membrane/Golgi for further
use.
 Receptor-mediated endocytosis:
1) Cells take up specific molecules (ligands) that bind receptors.\
2) Receptors often located in coated pits.
 Pinocytosis:
1) Cells ingest droplets of liquid from extracellular space.
 Phagocytosis:
1) Cells extend pseudopods.
2) Encircle particles.
3) Important part of immune defense.
4) Macrophages, Neutrophils = professional phagocytes.

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LYSOSOMES
 Acidic (pH~4.8).
 Contain many acid hydrolase enzymes (40+types):
 These enzymes require acidic environment.
 Break down cellular waste, fats, carbohydrates, proteins.
 Generate simple compounds.
 Returned to cytoplasm to be used by cell.

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PEROXISOME
 Membrane-enclosed organelle involved in:
1) Catabolism of very-long-chain fatty acids (through β-oxidation).
 β-oxidation can occur in mitochondria but also peroxisomes.
 Peroxisomes preferentially oxidize longer fatty acids.
2) α-oxidation (strictly peroxisomal process).
3) Catabolism branched-chain fatty acids like phytanic acid (through α-
oxidation), amino acids, and ethanol.
4) Synthesis of cholesterol, bile acids, and plasmalogens (important membrane
phospholipid, especially in white matter of brain).
 Peroxisomal disorders commonly lead to neurologic diseases due to
deficits in synthesis of plasmalogens, important phospholipids in myelin.

PEROXISOMAL DISEASES:
If a peroxisomal disorder is suspected, plasma concentration of VLCFA
should be measured. VLCFA levels are elevated in nearly all peroxisomal
disorders.

1) Zellweger syndrome: ―cerebro-hepato-renal syndrome‖

 Autosomal recessive disorder
 Caused by mutations in genes that encode peroxins, proteins
required for the normal assembly of peroxisomes ―mutated PEX
genes‖  ↓ peroxisomes synthesis.
 Hypomyelination  newborns presents with hypotonia, seizures,
apnea, or inability to eat.
 Craniofacial dysmorphism.
 Hepatomegaly, renal cysts ―The highest concentration of peroxisomes
is in the liver and kidney‖.
 Early death.
2) Refsum disease:
 Autosomal recessive disorder of α-oxidation → phytanic acid not
metabolized to pristanic acid.
 Defect in peroxisomal alpha oxidation  accumulation of phytanic
acid within the body leads to neurologic disturbances.
 Scaly skin, ataxia, cataracts/night blindness, shortening of 4th
toe, epiphyseal dysplasia.

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PROTEASOME
 Barrel-shaped protein complex that degrades
Degradation of proteins and
damaged or ubiquitin-tagged proteins.
polypeptides occurs in proteasomes
 Defects in the ubiquitin-proteasome system have
and lysosomes.
been implicated in neurodegenerative disorders
such as Parkinson's and Alzheimer’s diseases. Proteasomes mainly degrade
nuclear and cytoplasmic proteins.
 Proteasomes function to degrade unneeded or
improperly folded intracellular proteins to small Lysosomes degrade cellular
polypeptides or to amino acids. organelles and extracellular proteins.
 Proteasomes also function to degrade viral
proteins for expression on MHC Class I
molecules for recognition by T lymphocytes.

 Ubiquitin is a protein found in all eukaryotic cells that undergoes ATP-dependent

attachment to other proteins, labeling them for degradation.
 Ubiquitination plays an important role in many cell functions, including antigen
processing, muscle wasting, cell cycle regulation, DNA repair, and disposal of
misfolded proteins and regulatory enzymes.

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PROTEASOMES AND DISEASES:

CYSTIC FIBROSIS:
 Deletion of phenylalanine from position 508 (∆F508) in the CFTR (cystic
fibrosis transmembrane regulator) gene  CFTR protein fails to fold
correctly  activates ubiquitin ligases that mark the protein for
degradation in proteasomes.
 CFTR codes for chloride channels which fluidify the mucous of the
exocrine glands.
 Without these channels  mucous plugs  obstruct & induce
inflammation  fibrosis of the ostium of the glands.

AR PARKINSONISM:
 Together, the Parkin, PINK1 and DJ-1 genes code for a protein
complex that promotes the degradation of misfolded proteins via the
ubiquitin-proteasome system.
 Mutations in Parkin, PINK1, and DJ-1 are each associated with
autosomal recessive forms of Parkinson's disease that have an early age
of onset (< 50 years).

CELL TRAFFICKING

SECRETORY PATHWAY
 Begins with translation of mRNA in cytosol  protein enters endoplasmic
reticulum lumen  transferred to Golgi  exits Golgi in vesicle  exocytosis at
plasma membrane  secretion.

SIGNAL SEQUENCES
 Found on proteins undergoing synthesis (translation).
 Used to pull free ribosomes to ER membrane  creates rough ER.
 Leads to proteins entry into ER lumen.
 Many will ultimately be secreted (via secretory pathway).
 Short peptides (proteins) found on N-terminal of protein.
 Directs protein-ribosome to endoplasmic reticulum.

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SIGNAL RECOGNITION PARTICLE (SRP)

 Ribonucleoproteins found in cytosol.
 Complex particle with many proteins and RNA which recognize signal
sequences.
 Moves proteins from cytosol to ER.
 SRP Receptor: Found on ER membrane, binds SRPs.
 Protein translocated through pore into ER lumen.

Vesicular trafficking proteins

COATED VESICLES
 Vesicles with protein coat on surface.
 Formed from specialized portions of membranes.
 Different coats in different forms of traffic.
 Important for secretory pathway.
 Also important in transport from cell surface.
 Three well-characterized coats: • Clathrin • COPI • COPII

CLATHRIN-COATED VESICLES
 Transport between:
 Plasma membrane and Golgi.
 Plasma membrane  endosomes (receptor mediated endocytosis).
 Major vesicle: receptor-mediated endocytosis:
 Uptake of extracellular component into vesicle.
 Receptors found in ―clathrin-coated pits‖.
 LDL-receptor, Growth factor receptors.

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COPI AND COPII VESICLES

1) COPI:
 Golgi  Golgi (retrograde);
 cis-Golgi  ER.
2) COPII:
 ER  cis-Golgi (anterograde).
―Two (COPII) steps forward (anterograde); one (COPI) step back
(retrograde).‖

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CYTOSKELETAL ELEMENT S
 A network of protein fibers within the cytoplasm that supports cell structure, cell and
organelle movement, and cell division.

 TYPE OF FILAMENT, PREDOMINANT FUNCTION AND

EXAMPLES:
1) Microfilaments: (actin filaments)
 Function: Muscle contraction, cytokinesis.
 Example: Actin, microvilli ―Extensions of intestinal cell membranes‖
2) Intermediate filaments:
 Function: Maintain cell structure & shape.
 Example: Vimentin, desmin, cytokeratin, lamins, glial fibrillary acid
proteins (GFAP), neuroflaments.
 Often used as tumor markers by immunohistochemical staining.
3) Microtubules:
 Function: Movement, cell division
 Example: Cilia, flagella, mitotic spindle, axonal trafficking, centrioles.

 IMMUNOHISTOCHEMICAL STAINS FOR INTERMEDIATE

FILAMENTS

STAIN CELL TYPE IDENTIFIES

Vimentin Mesenchymal tissue Mesenchymal tumors (eg, sarcoma),

―Cells/tissue derived from but also many other tumors (eg,
mesoderm in embryo‖ endometrial carcinoma, renal cell
(eg, fibroblasts, endothelial carcinoma, meningioma)
cells, macrophages).
DesMin Muscle Muscle tumors (eg,
rhabdomyosarcoma, leiomyoma,
lieomyosarcoma)
Cytokeratin Epithelial cells Epithelial tumors (eg, squamous cell
carcinoma)
GFAP NeuroGlia (eg, astrocytes, Astrocytoma, Glioblastoma
Schwann cells,
oligodendrocytes)
Neurofilaments Neurons Neuronal tumors (eg,
neuroblastoma, medulloblastoma,
retinoblastoma)
Lamins Forms nuclear envelope
• Separates nucleus from
cytoplasm.

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 NB: Z-disks in sarcomeres:

o Contain vimentin and desmin.

 MICROTUBULES

 Polymers of alpha and beta tubulin: ―Heterodimer‖ units: one alpha, one beta.
 Each dimer has 2 GTP bound:
 Alpha GTP: part of structure & not giving energy.
 Beta GTP: can be hydrolyzed giving energy.
 Grow from a centrosome near nucleus:
 Microtubules grow from it in a star like pattern.
 Microtubules have a (-) end toward the centerosome and (+) end away from
the centrosome.
 Negative end Near Nucleus
Positive end Points to Periphery
 Dynamic instability of microtubules:
 Grows slowly, collapses quickly.
 Molecular motor proteins:
1) Transport cellular cargo toward opposite ends of microtubule tracks.
 Organelles (mitochondria) and secretory vesicles.
2) Dynein—retrograde to microtubule (+  -).
 Towards the nucleus/cell body.
3) Kinesin—anterograde to microtubule (-  +).
 Away from nucleus/cell body.
 Important for axonal transport (toward terminal).

 Incorporated into flagella, cilia, and mitotic spindles.

 Also involved in slow axoplasmic transport in neurons.
 Drugs that act on microtubules (Microtubules Get Constructed Very Poorly):
1) Mebendazole (antihelminthic)
2) Griseofulvin (antifungal)
3) Colchicine (antigout):
 Prevent microtubule assembly.
 Disrupts chemotaxis, generation of cytokines, phagocytosis.
4) Vincristine/Vvinblastine (anticancer)  ↓ polymerization of microtubules.
5) Paclitaxel (anticancer)  enhance polymerization – block breakdown.

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CILIA STRUCTURE
 Motility structures.
 Built from microtubules and dynein.
 Microtubules/proteins in cilia formed into an a structure called ―axoneme‖.
 Structures arranged in special pattern (―9 x 2‖):
 9 doublet microtubules in ring.
 Surround a pair (―2‖) microtubules (arrows in A).
 Basal body (base of cilium below cell membrane) consists of 9 microtubule triplets
(arrow in B) with no central microtubules.
 Axonemal dynein:
 Forms bridges between microtubules 9 doublets.
 Activated dynein  pulls on neighboring doublets  movement of the cilia.
 Requires ATP (―microtubule dependent ATPase‖).
 Sliding of doublets  bending of cilia/flagella.
 Gap junctions enable coordinated ciliary movement.
 Kartagener syndrome (1° ciliary dyskinesia):
 Immotile cilia due to a dynein arm defect.
 Results in ↓ male and female fertility due to immotile sperm and
dysfunctional fallopian tube cilia, respectively.
 ↑ Risk of ectopic pregnancy.
 Can cause bronchiectasis, recurrent sinusitis, chronic ear infections,
conductive hearing loss, and situs inversus (eg, dextrocardia on CXR C).

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SODIUM-POTASSIUM PUMP
 Na+-K+ ATPase is located in the plasma membrane with ATP site on cytosolic side.
 For each ATP consumed, 3Na+ go out of the cell (pump phosphorylated) and 2K+
come into the cell (pump dephosphorylated). (Pumpkin = pump K+ in.)
 Ouabain inhibits by binding to K+ site.
 Cardiac glycosides (digoxin and digitoxin) directly inhibit the Na+-K+
ATPase, which leads to indirect inhibition of Na+/Ca2+ exchange  ↑
[Ca2+]i  ↑ cardiac contractility.
 Plasma membrane is an asymmetric lipid bilayer containing cholesterol,
phospholipids, sphingolipids, glycolipids, and proteins.

THE NUCLEOLUS:

 Round, dense, basophilic (dark-staining) body that can be identified within the
nucleus on standard light microscopy.
 The site of ribosomal subunit maturation and assembly.
 RNA polymerase I synthesizes the vast majority of rRNA from within the nucleolus.
 As cells become more differentiated, their growth slows and they require fewer
numbers of ribosomes for protein production.
 In contrast, malignant cells with high metabolic activity usually have a large number
of active rRNA genes and prominent nucleoli.

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VITAMINS

a) A, D, E, K.
b) Absorption dependent on gut and pancreas.
c) Toxicity more common than for water-soluble vitamins because fat-soluble
vitamins accumulate in fat.
d) Malabsorption syndromes with steatorrhea, such as cystic fibrosis and celiac
disease, or mineral oil intake can cause fat-soluble vitamin deficiencies.

a) All wash out easily from body except B12 and B9 (folate).
b) B12 stored in liver for ~ 3–4 years.
c) B9 stored in liver for ~ 3–4 months.
d) B-complex deficiencies often result in dermatitis,
glossitis, and diarrhea.
e) Can be coenzymes (eg, ascorbic acid) or precursors to
organic cofactors (eg, FAD, NAD+).

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FUNCTION  Antioxidant.
 Constituent of visual pigments (retinal).
 Essential for normal differentiation of epithelial cells into specialized
tissue (pancreatic cells, mucus-secreting cells).
 Prevents squamous metaplasia.
 Used to treat measles and acute promyelocytic leukemia (APL).
Forms  Topically for wrinkles and Acne.
 Oral isotretinoin to treat severe cystic acne.
 All-trans retinoic acid to treat acute promyelocytic leukemia.

Storage  Stored in the liver, mainly in the perisinusoidal stellate (Ito) cells.
 These stores are often sufficient enough to last around 6 months.
 Liver content of vit A is very low in newborn but increases rapidly with
intake of colostrum, breast milk and fortified formula.

DEFICIENCY  Eye:
 Night blindness (nyctalopia).
 Corneal degeneration (keratomalacia).
 Bitot spots on conjunctiva.
 Skin:
 Dry, scaly skin (xerosis cutis).
 Immunosuppression:

EXCESS  Acute toxicity: (after the ingestion of a single high dose of vitamin A)
 Nausea, vomiting, vertigo, and blurred vision.
 Chronic toxicity: (after long-term ingestion of high doses of vitamin A)
 Alopecia, dry skin (eg, scaliness).
 Hepatic toxicity and enlargement.
 Arthralgias.
 Pseudotumor cerebri.
 Teratogenic:
 Cleft palate, cardiac abnormalities.
 Microcephaly.
 Fetal death (especially in the first trimester of pregnancy).
 Therefore a ⊝ pregnancy test and two forms of contraception are
required before isotretinoin (vitamin A derivative) is prescribed.
Isotretinoin is teratogenic.

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 FUNCTION:
 Cofactor for several dehydrogenase enzyme reactions:
i. Pyruvate dehydrogenase (links glycolysis to TCA cycle).
ii. α-ketoglutarate dehydrogenase (TCA cycle).
iii. Branched-chain ketoacid dehydrogenase.
iv. Transketolase (HMP shunt).
 DEFICIENCY:
 Causes: alcoholic or malnourished patients.
 Diagnosis
i. ↑ RBC Transketolase activity following vitamin B1 administration.
 Wernicke-Korsakoff syndrome: Chronic thiamine (B1) deficiency leads to the
diminished ability of cerebral cells to utilize glucose.
i. Wernicke-encephalopathy:
1. Acute syndrome.
2. Classic triad  Confusion, ophthalmoplegia, ataxia.
ii. Korsakoff syndrome:
1. Chronic neurological condition.
2. Amnesia (retrograde or antegrade) or memory loss ―permanent‖.
3. Confabulation.
4. Personality change.
iii. Damage to medial dorsal nucleus of thalamus, mammillary bodies.
 Dry beriberi
i. Polyneuritis + myelin degeneration  toe drop, wrist drop, foot drop,
areflexia.
ii. Symmetrical muscle wasting.
 Wet beriberi
i. Peripheral vasodilatation  edema.
ii. High- cardiac output heart failure (dilated cardiomyopathy).

Think ATP: α-ketoglutarate

dehydrogenase, Transketolase, and
Dry  nerves.
Pyruvate dehydrogenase.
Wet  Heart. Spell beriberi as Ber1Ber1 to
remember vitamin B1.
[Cite your source here.]

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 Treatment:
i. Immediate thiamin supplementation.
ii. Give thiamin before glucose:
1. Thiamine use by the body is maximal in states of accelerated carbohydrate
metabolism because it acts as a cofactor for the enzyme Transketolase in
the pentose phosphate pathway as well as the enzymes a-ketoglutarate
dehydrogenase and pyruvate dehydrogenase, both of which require
thiamine as a cofactor.
2. So, giving glucose (carbohydrate) to a patient with ↓thiamin will
exacerbate the condition.

 FUNCTION
 Component of flavins FAD and FMN. 2B 2C 2ATP
 In electron transport chain:
i. FMN is a component of complex I.
ii. FAD is a component of complex II.
 In Kreb’s cycle:
i. FAD is a cofactor for succinate dehydrogenase, which is an enzyme that
mediates the conversion of succinate into fumarate.
 DEFICIENCY
 Cheilosis (inflammation of lips, scaling and fissures
at the corners of the mouth).
 Corneal vascularization.
 Dx of B2 deficiency:
 Erythrocyte gluthatione reductase assay ―uses
FAD as a cofactor‖.
 Urinary riboflavin excretion.

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KQB: B2 can be degraded by light “phototherapy”; so in patients with neonatal jaundice

treated with phototherapy should be supplemented with riboflavin to prevent its
deficiency secondary to degradation by phototherapy.

1. Synthesis requires vitamins B2 and B6.

2. Derived from tryptophan.
 FUNCTION
 Constituent of NAD+, NADP+ (used in redox reactions).
 Used to treat dyslipidemia:
i. ↓ VLDL & ↓LDL
3B 3D 3ATP
ii. ↑ HDL.
 DEFICIENCY
 Glossitis.
 Pellagra("rough skin" in Italian vernacular):
i. Causes:
1. ↓Intake:
a. In developing countries  Corn-based
diet (niacin in com occurs in a bound,
unabsorbable form). NAD derived from
b. In developed countries, it is primarily seen Niacin
in patients with impaired nutritional intake
(eg, alcoholism, chronic illness).
2. ↓ Absorption:
a. Hartnup disease.
3. ↑ Metabolism:
a. Malignant carcinoid syndrome (↑ tryptophan
metabolism).
4. Isoniazid (↓ vitamin B6).

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ii. Symptoms of pallegra

1. Diarrhea.
2. Dementia (also hallucinations).
3. Dermatitis:
a. C3/C4 dermatome circumferential ―broad collar‖ rash [Casal
necklace]
b. Hyperpigmentation of sun exposed limbs A).
c. Characterized by rough, thick, scaly skin.
The 3 D’s of B3.
 EXCESS Deficiency of vitamin B3 
 Facial flushing (induced by prostaglandin, not pellagra.
histamine; can avoid by taking aspirin with niacin), Excess of vitamin B3 
 Hyperglycemia. podagra.
 Hyperuricemia.

Hartnup disease:
 Autosomal recessive.
 Deficiency of neutral amino acid (eg, tryptophan) transporters in proximal renal
tubular cells and on enterocytes  neutral aminoaciduria and ↓ absorption from the
gut  ↓tryptophan  ↓ niacin  pellagra-like symptoms.
 The main laboratory finding in Hartnup disease is aminoaciduria, restricted to the
neutral amino acids (alanine, serine, threonine, valine, leucine, isoleucine,
phenylalanine, tyrosine, tryptophan, and histidine).
 The urinary excretion of proline, hydroxyproline, and arginine remains unchanged,
and this important finding differentiates Hartnup disease from other causes of

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FUNCTION  Essential component of coenzyme A (CoA, a cofactor for acyl

transfers, OAA citrate).
 Fatty acid synthase. B5 is ―pento‖thenic acid.

DEFICIENCY  Dermatitis,
 Enteritis.
 Alopecia.
 Adrenal insufficiency.
 Burning feet syndrome: TCA will be interrupted due to deficiency of
CoA>>>leading to weakness and burning feet.

FUNCTION  Converted to the active form ―pyridoxal phosphate (PLP)‖, a

cofactor used in:
 Transamination (eg, ALT and AST), any increase in AST &
ALT is accompanied by ↓B6.
 Decarboxylation reactions.
 Glycogen phosphorylase (glycogenolysis).
 Synthesis of cystathionine, heme, niacin, histamine, and
neurotransmitters including serotonin, epinephrine, norepinephrine
(NE), dopamine, and GABA.

DEFICIENCY  Causes:
 Alcoholic hepatitis (↑ALT & AST).
 Isoniazid
i. Forms an inactive derivative with pyridoxal phosphate.
ii. Dietary supplementation with B6 is, thus, an adjunct to
isoniazid treatment.
 Oral contraceptives.
 C/P:
 CNS: Convulsions, hyperirritability (↓GABA), peripheral
neuropathy.
Blood: Sideroblastic anemia due to impaired hemoglobin
synthesis and iron excess.

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FUNCTION  CO2 carrier hence cofactor for carboxylation enzymes (which add a 1-
carbon group):
 Pyruvate carboxylase: pyruvate (3C)  oxaloacetate (4C).
i. In gluconeogenesis; so, ↓biotin  fasting hypoglycemia.
 Acetyl-CoA carboxylase: acetyl-CoA (2C)  malonyl-CoA (3C)
i. In FA synthesis.
 Propionyl-CoA carboxylase: propionyl-CoA (3C) 
methylmalonyl-CoA (4C)
i. In odd chain fatty acid.

DEFICIENCY  Dermatitis, alopecia, enteritis.

 ↑ Pyruvate  converted to lactic acid  metabolic acidosis.
 Caused by
 Excessive ingestion of raw egg whites.
―Avidin ―egg-white protein‖ avidly binds biotin.‖
 Antibiotic use.

B5 & B7 are absorbed in the small and large intestine via the sodium-dependent
multivitamin transporter. Vit B6 by passive diffusion.

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FUNCTION  Converted to tetrahydrofolic acid (THF) ―the biological active form‖, a

coenzyme for 1-carbon transfer/methylation reactions.
 DNA and RNA synthesis:
 ↓Folate  ↓dTMP  ↓ DNA synthesis  promotes
megaloblastosis and erythroid precursor cell apoptosis.
 Because thymidine supplementation can moderately increase
dTMP levels, it can reduce erythroid precursor cell apoptosis.

Metabolism  Found in leafy green vegetables.

 Absorbed in jejunum. Folate from foliage.
 Small reserve pool stored primarily in the liver.

DEFICIENCY  Most common vitamin deficiency in the United States.

 Macrocytic, megaloblastic anemia;
 Hypersegmented polymorphonuclear cells (PMNs) due to
inability of cells (including red cell precursors) to make DNA and,
therefore, they cannot divide.
 Glossitis.
 No neurologic symptoms (as opposed to vitamin B12 deficiency).
 Labs:
i. ↑ Homocysteine.
ii. Normal methylmalonic acid levels.
 Causes of deficiency:
 Alcoholism.
 Pregnancy (↑demand). Supplemental maternal folic acid at least 1
month prior to conception and during early pregnancy to ↓ risk of
neural tube defects.
 Several drugs (eg, phenytoin, sulfonamides, methotrexate).

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UW: High doses of folate may

antagonize phenytoin thereby
precipitating seizures in a select
group of patients.

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FUNCTION  Cofactor for methionine synthase (transfers CH3 groups as

methylcobalamin) and methylmalonyl-CoA mutase.
 Important for DNA synthesis.

Metabolism  Found in animal products.

 Synthesized only by microorganisms.
 Very large reserve pool (several years) stored primarily in the liver.

Deficiency  Caused by:

 Malabsorption (eg, sprue, enteritis, Diphyllobothrium latum).
 Lack of intrinsic factor (pernicious anemia, gastric bypass
surgery). Anti-intrinsic factor antibodies diagnostic for pernicious
anemia.
 Absence of terminal ileum (surgical resection, eg, for Crohn
disease)
 Insufficient intake (eg, veganism).
 Presentations:
 Macrocytic, megaloblastic anemia: Hypersegmented PMNs.
 Subacute combined degeneration of the following:
i. Dorsal columns  loss of proprioception/vibration,
Romberg sign.
ii. Lateral corticospinal tract  spastic muscle weakness,
hyperreflexia,
 Paresthesias.
 Associated with ↑ serum homocysteine and methylmalonic
acid levels, along with 2° folate deficiency.
i. Prolonged deficiency  irreversible nerve damage.

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Secondary folate deficiency:

 Due to B12 deficiency, which make folate to be trapped in methy-THF
form.
Primary folate deficiency:
 Due to THF deficiency  ↓ thymedylate synthase; which normally
convert dUMP  dTMP. ↑ dUMP incorporation into DNA cuases
apoptosis.

NB: usage of folate alone in B12 deficiency can worse demyelination and cause
abnormal myelin synthesis by depleting the concentration of unmethylated cobalamin
available for methylmalonyl-CoA processing  ↑MMA  toxic to the myelin  ↑SCD.

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 Found in fruits and vegetables.

Vitamin C deficiency causes sCurvy
 FUNCTION due to a Collagen synthesis defect.
 Antioxidant.
 Facilitates iron absorption by reducing it to Fe2+ state.
 Necessary for hydroxylation of proline and lysine in collagen synthesis.
 Necessary for dopamine β-hydroxylase, which converts dopamine to NE.
 Ancillary treatment for methemoglobinemia by reducing Fe3+ to Fe2+.
 DEFICIENCY

 Scurvy—swollen gums, bruising, petechiae, hemarthrosis (due to fragile

capillaries by ↓collagen cross linking, ↑bleeding time + normal PT), anemia, poor
wound healing, perifollicular and subperiosteal hemorrhages, ―corkscrew‖ hair.

 Infantile scurvey:
i. Vit C is destroyed by excessive heating.
ii. Child with bottle fed formula whose mother boil it excessively.
iii. C/P: easy bruising, gum bleeding doesn’t occur until teeth eruption.
 Weakened immune response.

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 EXCESS vitamin C:
 Nausea, vomiting, diarrhea, fatigue, calcium oxalate nephrolithiasis.
 Can ↑ risk of iron toxicity in predisposed individuals (eg, those with transfusions,
hereditary hemochromatosis).

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Forms  D2 = ergocalciferol—ingested from plants.

 D3 = cholecalciferol—consumed in milk, formed in sun-exposed skin
(stratum basale).
 25-OH D3 = storage form.
 1,25-(OH)2 D3 (calcitriol) = active form.

FUNCTION  ↑ Intestinal absorption of calcium and phosphate.

 ↑ Bone mineralization at low levels.
 ↑ Bone resorption at higher levels.

Regulation  ↑ PTH, ↓ Ca2+, ↓ PO43–  ↑ 1,25-(OH)2D3 production.

 1,25-(OH)2D3 feedback inhibits its own production.
 ↑ PTH  ↑ Ca2+ reabsorption and ↓ PO43– reabsorption in the
kidney.

DEFICIENCY  Rickets in children (deformity, such as genu varum ―bow legs‖ A)

 Osteomalacia in adults (bone pain and muscle weakness),
 Hypocalcemic tetany.
 All exclusively breastfed infants should receive vitamin D
supplementation to prevent rickets:
o Infants are generally shielded from direct sunlight due to sunburn
risk.
o In addition infants with dark skin pigmentation require more
sunlight exposure to produce adequate vitamin D. Melanin is a
natural sunblock and prevents ultraviolet rays from reaching the
skin for vitamin D synthesis.
 Deficiency is exacerbated by low sun exposure, pigmented skin,
prematurity.

EXCESS  Hypercalcemia, hypercalciuria, loss of appetite, stupor.

 Seen in granulomatous disease (↑ activation of vitamin D by
epithelioid macrophages).

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All vitamins and trace minerals are present in adequate amounts in breast milk
except vitamins D and K. Vitamin K is given parenterally at birth to prevent
hemorrhagic disease in the newborn.

FUNCTION  Antioxidant (protects RBCs and membranes from free radical

damage).

DEFICIENCY  The cells that are most susceptible include neurons with long axons
(due to large membrane surface areas) and erythrocytes (due to high
oxygen exposure).
 Hemolytic anemia, acanthocytosis (fragile RBCs)
 Muscle weakness.
 Posterior column and spinocerebellar tract demyelination.
Neurologic presentation may appear similar to vitamin B12 deficiency,
but without megaloblastic anemia, hypersegmented neutrophils, or ↑
serum methylmalonic acid levels.

Excess  High-dose supplementation may alter metabolism of vitamin K 

enhanced anticoagulant effects of warfarin.
 Risk of enterocolitis in infants.

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Activation  In the intestine: by microorganisms.

 In the liver: by epoxide reductase to the reduced form.
 Synthesized by intestinal flora.

FUNCTION  Cofactor for the γ-carboxylation of glutamic acid residues on various

proteins required for blood clotting  introduce a binding site for
calcium which is required for providing a bridge for binding to
platelets.
 Necessary for the maturation of clotting factors II, VII, IX, X,
and proteins C and S. Warfarin inhibits vitamin K–dependent
synthesis of these factors and proteins.
K is for Koagulation.
DEFICIENCY  Bleeding tendency with ↓ PT and ↓ aPTT but normal bleeding time
(neonates have
sterile intestines and are unable to synthesize vitamin K).
 Can also occur after prolonged use of broad-spectrum antibiotics.
 Not in breast milk; neonates are given vitamin K injection at birth to
prevent hemorrhagic disease of the newborn.

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FUNCTION  Essential for the activity of 100+ enzymes.

 Important in the formation of zinc fingers (transcription factor motif).
DEFICIENCY  Delayed wound healing, suppressed immunity.
 Hypogonadism.
 ↓ Adult hair (axillary, facial, pubic).
 Dysgeusia, anosmia.
 Acrodermatitis enteropathica A.
 May predispose to alcoholic cirrhosis.

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1. KWASHIORKOR
 Protein malnutrition resulting in skin lesions, edema due to ↓ plasma oncotic
pressure, liver malfunction (fatty change due to ↓ apolipoprotein synthesis).
 Clinical picture is small child with swollen abdomen.
Kwashiorkor results from protein deficient MEALS:
Malnutrition
Edema
Anemia
Liver (fatty)
Skin lesions (hyperkeratosis/ hyperpigmentation, dyspigmentation)

2. MARASMUS
 Malnutrition not causing edema.
 Diet is deficient in calories but no nutrients are entirely absent.
Marasmus results in Muscle wasting.

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ETHANOL METABOLISM

 FOMEpizole:
 Inhibits alcohol dehydrogenase and is an antidote For Overdoses of Methanol
or Ethylene glycol.
 Disulfram:
 Inhibits acetaldehyde dehydrogenase (acetaldehyde accumulates, contributing
to
hangover symptoms), discouraging drinking.
 NAD+ is the limiting reagent.
 Alcohol dehydrogenase operates via zero-order kinetics and uses thiamin as a
cofactor; so chronic alcoholism is the most common cause of thiamin deficiency.
 Ethanol metabolism ↑ NADH/NAD+ ratio in liver, causing:
 Pyruvate  lactate (lactic acidosis)
 Oxaloacetate  malate (prevents gluconeogenesis  fasting hypoglycemia)
 Dihydroxyacetone phosphate  glycerol-3-phosphate (combines with fatty acids to
make triglycerides  hepatosteatosis).

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 End result is clinical picture seen in chronic alcoholism.

 Additionally, ↑ NADH/NAD+ ratio disfavors TCA production of NADH  ↑
utilization of acetyl-CoA for ketogenesis (↑ ketoacidosis) and lipogenesis (↑
hepatosteatosis).

3 year old girl brought unconscious after ingesting unknown quantity of ethanol. Her serum
blood glucose concentration is 30mg/dl. What is the mechanism of hypoglycemia?
Increasing reduction of pyruvate to lactate (due to ↑ NADH from alcohol metabolism)
not by inhibiting pyruvate dehydrogenase (which occur due to B1 def in prolonged user of
alcohol).

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DIETARY ENERGY
 The result of metabolism of 1 gram of the following:
 Protein or carbohydrate  4 Calories.
 Fat  9 Calories.
 Ethanol  7 Calories.

ENZYME KINETICS:

 ∆G ―the change in free energy‖:

 The amount of free energy (G) produced or
consumed by a chemical reaction.
 Calculated as Gproduct – Gsubsbstrate
 ∆G = 0  the reaction at equilibrium ―reversible‖
 -ve ∆G:
 The reaction favors product formation.
 Energy released ―Spontaneous.‖
 Irreversible.
 +ve ∆G:
 The reaction favors substrate formation.
 Energy required ―non-spontaneous‖.

 Enzymes as Catalysts:
 For the reaction to proceed, there has to be an
input of energy to overcome this barrier; this is
known as the energy of activation (∆G++).
 This need for energy affects the rate of the
reaction; that is, the greater (∆G++), the slower
the reaction.
 Enzymes will ↓∆G++  less energy is required
to start the reaction, and therefore the rate of the
reaction is increased.
 Enzymes do not affect the (∆G) of the reaction.
 Enzymes have high specificity for their
substrates (affinity)

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 Most enzymatic reactions follow a hyperbolic curve (ie, Michaelis-Menten kinetics);

however, enzymatic reactions that exhibit a sigmoid curve usually indicate
cooperative kinetics (eg, hemoglobin).

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 Vmax is the maximal reaction rate when the substrate is saturating.

 Directly proportional to the enzyme concentration ―number of enzyme
available‖.
 Gene induction (↑ expression)  ↑ number of enzymes  ↑ Vmax
 Gene repression (↓ expression)  ↓ number of enzymes  ↓ Vmax
 Receptors upregulation ↑ Vmax. . Receptors Downregulation ↓ Vmax
 Irreversible non-competitive ―allosteric‖ inhibitors ↓ Vmax

 Km is the substrate concentration at which the rate is

half maximal (1/2 Vmax).
 Inversely related to the affinity of the
enzyme for its substrate
(high affinity, low Km).
 Changing the shape of the enzymes
 ↓ affinity.
 Phosphorylation and
dephosphorylation change the
affinity.
 Potentiation = sensitization =
activation = ↑ affinity.
 Reversible competitive inhibitors 
↑ Km. ―↑substrate concentration‖
NB: There is a difference between ACTIVATION &
INDUCTION. Activation  ↑Affinity BUT Induction ↑
number of enzymes.

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 ↑ y-intercept, ↓ Vmax.
 The further to the right the x-intercept (i.e. closer to zero), the greater the Km and the
lower the affinity.
 Reversible competitive inhibitors cross each other competitively, whereas
noncompetitive inhibitors do not.

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METABOLISM SITES

Mitochondria  Fatty acid oxidation (β-oxidation), acetylCoA production.

 TCA cycle, oxidative phosphorylation.
 Ketogenesis.
Cytoplasm  Glycolysis, HMP shunt.
 Synthesis of steroids (SER), proteins (ribosomes, RER), fatty
acids, cholesterol, and nucleotides.
Both  Heme synthesis, Urea cycle, Gluconeogenesis. HUGs take two (ie,
both).

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ENZYME TERMINOLOGY
An enzyme’s name often describes its function. For example, glucokinase is an enzyme that
catalyzes the phosphorylation of glucose using a molecule of ATP. The following are
commonly used enzyme descriptors.
 Kinase:
 Catalyzes transfer of a phosphate group from a high-energy molecule (usually
ATP) to a substrate (eg, phosphofructokinase).
 Phosphorylase:
 Adds inorganic phosphate onto substrate without using ATP (eg, glycogen
phosphorylase).
 Phosphatase:
 Removes phosphate group from substrate (eg, fructose-1,6-bisphosphatase).
 Dehydrogenase:
 Catalyzes oxidation-reduction reactions (eg, pyruvate dehydrogenase).
 Hydroxylase:
 Adds hydroxyl group (-OH) onto substrate (eg, tyrosine hydroxylase).
 Carboxylase:
 Transfers CO2 groups with the help of biotin (eg, pyruvate carboxylase).
 Mutase:
 Relocates a functional group within a molecule (eg, vitamin B12–dependent
methylmalonyl-CoA mutase).
 Synthase/synthetase:
 Combines 2 molecules into 1 (condensation reaction) either using an energy
source (synthase, eg, glycogen synthase) or not (synthetase, eg, PRPP
synthetase).

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RATE-DETERMINING ENZYMES OF METABOLIC

PROCESSES

PROCESS ENZYME REGULATORS

Glycolysis Phosphofructokinase-1 AMP ⊕, fructose-2,6-bisphosphate ⊕
(PFK-1) ATP ⊝, citrate ⊝
Gluconeogenesis Fructose-1,6- Citrate ⊕
bisphosphatase AMP ⊝, fructose-2,6-bisphosphate ⊝
TCA cycle Isocitrate dehydrogenase ADP ⊕
ATP ⊝, NADH ⊝
Glycogenesis Glycogen synthase Glucose-6-phosphate ⊕, insulin ⊕,
cortisol ⊕
Epinephrine ⊝, glucagon ⊝
HMP shunt Glucose-6-phosphate NADP+ ⊕
dehydrogenase (G6PD) NADPH ⊝
De novo Carbamoyl phosphate ATP ⊕, PRPP ⊕
pyrimidine synthetase II UTP ⊝
synthesis
De novo purine Glutamine- AMP ⊝, inosine monophosphate (IMP)
synthesis phosphoribosylpyrophospha ⊝,
te (PRPP) amidotransferase GMP ⊝
Urea cycle Carbamoyl phosphate N-acetylglutamate ⊕
synthetase I
Fatty acid Acetyl-CoA carboxylase Insulin ⊕, citrate ⊕
synthesis (ACC) Glucagon ⊝, palmitoyl-CoA ⊝
Fatty acid Carnitine acyltransferase I Malonyl-CoA ⊝
oxidation
Ketogenesis HMG-CoA synthase
Cholesterol HMG-CoA reductase Insulin ⊕, thyroxine ⊕
synthesis Glucagon ⊝, cholesterol ⊝

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ACTIVATED CARRIERS

CARRIER MOLECULE CARRIED IN ACTIVATED FORM

ATP Phosphoryl groups
NADH, NADPH, FADH2 Electrons
CoA, lipoamide Acyl groups
Biotin CO2
Tetrahydrofolates 1-carbon units
S-adenosylmethionine (SAM) CH3 groups
TPP Aldehydes

UNIVERSAL ELECTRON ACCEPTORS

 Nicotinamides (NAD+ from vitamin B3, NADP+) and flavin nucleotides (FAD+ from
vitamin B2).
 NAD+ is generally used in catabolic processes to carry reducing equivalents away as
NADH.
 NADPH:
 NADPH is a product of the HMP shunt.
 NADPH is used in:
 Anabolic processes (steroid and fatty acid synthesis).
 Respiratory burst.
 Cytochrome P-450 system.
 Glutathione reductase.

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GLUCOSE ABSORPTION IN THE INTESTINE

 By sodium glucose symporter ―SGLUT1‖
1. It is secondary active transport.
2. SGLUT2 is present in the kidney.
3. SGLUT1 transport glucose along with galactose not fructose.
4. Fructose is transported by GLUT5 which is transported by diffusion.

GLUCOSE UPTAKE

 GLUT-2 has a higher KM for glucose (lower affinity) than all the other transporters.
WHY?
 Imagine what would happen if the hepatocytes had a low Km ―high affinity‖
transporter  The liver would then remove so much glucose that there would
be little left for the peripheral tissues.

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GLYCOLYSIS (GLUCOSE  PYRUVATE)

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 Net glycolysis (cytoplasm):

1. Glucose + 2 Pi + 2 ADP + 2 NAD+  2 pyruvate + 2 ATP + 2 NADH + 2 H+ +
2H2O.
2. The produced ATP through glycolysis from substrate level phosphorylation.
3. Equation not balanced chemically, and exact balanced equation depends on
ionization state of reactants and products.

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 Phosphorylation of glucose to yield glucose-6-phosphate serves as the 1st

committed step of glycolysis (also serves as the 1st step of glycogen synthesis
in the liver).
 Reaction is catalyzed by either hexokinase or glucokinase, depending on the
tissue.
 At low glucose concentrations, hexokinase sequesters glucose in the tissue.
 At high glucose concentrations, excess glucose is stored in the liver.

 Heterozygous mutations of the glucokinase gene cause a decrease in beta cell

metabolism of glucose, less ATP formation, and diminished insulin secretion. This produces a
type of maturity-onset diabetes of the young, which is characterized by mild, nonprogressive
hyperglycemia that often worsens with pregnancy-induced insulin resistance.
 Homozygous mutations lead to fetal growth retardation and severe hyperglycemia at
birth.

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 FBPase-2 (fructose bisphosphatase-2) and PFK-2 (phosphofructokinase-2) are the

same bifunctional enzyme whose function is reversed by phosphorylation by protein
kinase A.
 Fasting state: ↑ glucagon  ↑ cAMP  ↑ protein kinase A  ↑ FBPase-2, ↓ PFK-
2, less glycolysis, more gluconeogenesis.
 Fed state: ↑ insulin  ↓ cAMP  ↓ protein kinase A  ↓ FBPase-2, ↑ PFK-2, more
glycolysis, less gluconeogenesis.

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 In red blood cells, no mitochondria  anaerobic glycolysis represents the only

pathway for ATP production, yielding a net 2 ATP/glucose.

 Through substrate level phosphorylation in glycolysis by 2 enzymes:

 Phosphoglycerate kinase.
 Pyruvate kinase.
 2,3-BPG binds to the β-chains of HbA and decreases its affinity for oxygen 
rightward shift in the oxygen dissociation curve. This occurs to allow unloading of
oxygen in tissues.

 Familial erythrocytosis:
 Due to a β-globin mutation that decrease the positive charge (histidine &
lysine) of the binding site  ↓ 2,3-BPG binding  ↑ hemoglobin oxygen
affinity.

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 Pathophysiology:
1. The RBC has no mitochondria and is totally dependent on anaerobic glycolysis
for ATP. In pyruvate kinase deficiency, the decrease in ATP causes the
erythrocyte to lose its characteristic biconcave shape and signals its destruction
in the spleen.
2. In addition, decreased ion pumping by Na+/K+-ATPase results in loss of ion
balance and causes osmotic fragility, leading to swelling and lysis.
 Epidemiology:
1. Second most common genetic deficiency that causes a hemolytic anemia
(glucose 6-phosphate dehydrogenase, G6PDH, is the most common).
 Characteristics include:
1. Chronic hemolysis.
2. ↑ 2,3-BPG  ↓ oxygen affinity of HbA.
3. Absence of Heinz bodies (Heinz bodies are more characteristic of G6PDH
deficiency)

 Mitochondrial irreversible enzyme complex

Arsenic inhibits lipoic acid
linking glycolysis and TCA cycle.
↓PDH.
 Differentially regulated in fed/fasting states
(active in fed state). Arsenic poisoning clinical findings:
1. Insulin activate it by dephosphorylation. vomiting, rice-water stools, garlic
2. Glucagon inhibit it by phosphorylation. breath, QT prolongation.
 Reaction: pyruvate + NAD+ + CoA  acetyl- Imagine a vampire (pigmentary
coA + CO2 + NADH. skin changes, skin cancer),
 The complex contains 3 enzymes that require 5 vomiting and having diarrhea,
cofactors: running away from a cutie (QT
―Tender Loving Care For Nancy‖ prolongation) with garlic breath.
1. Thiamine pyrophosphate (B1)
2. Lipoic acid
3. CoA (B5; pantothenic acid)
4. FAD (B2, riboflavin)
5. NAD+ (B3, niacin)
 Activated by:
1. ↑ NAD+/NADH ratio
2. ↑ ADP
3. ↑ Ca2+
 The complex is similar to the α-ketoglutarate dehydrogenase complex (same
cofactors, similar substrate and action), which converts α-ketoglutarate  succinyl-
CoA (TCA cycle).

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 Causes a buildup of pyruvate that gets shunted to lactate (via LDH) and alanine (via
ALT). X-linked.
 FINDINGS: ―one of the most common neurodegenerative disorders‖
1. Neurologic defects.
2. Lactic acidosis.
3. ↑ Serum alanine starting in infancy.
 TREATMENT:
1. ↑ Intake of ketogenic nutrients (eg, high fat content or ↑ lysine and leucine).
Lysine and Leucine—the onLy pureLy ketogenic amino acids.
 NB: Amino acid catabolism following removal of the amino group
results in formation of intermediates that are either glucogenic
(producing intermediates of the critic acid cycle or pyruvate) or
ketogenic (producing acetoacetate or its precursors).

UW: Glycerol can aggravate lactic acidosis in patients with PDH deficiency because it
can be converted to dihyroxyacetone phosphate, which in turn can form pyruvate and
subsequently lactate.
K: Hypoglycemia  ↓glycolysis  ↓ATP  ↓Na/K+ ATPase pump  Na stays in
the cell  cell depolarization  Arrhythmia & seizures & ↑sympathetic & CNS
symptoms.

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 Under anaerobic glycolysis; NADH give electron to pyruvate to form lactate and
regenerate NAD, NAD is required to convert glyceraldehyde 3 phosphate to 1,3
biphosphoglycerate in glycolysis.
 Lactate dehydrogenase (LDH): Requires zinc. Bidirectional enzyme:
 Converts pyruvate to lactate in anaerobic condition most classically in the
muscle.
 Converts lactate to pyruvate in the liver where lactate, usually generated by
working skeletal muscles, is taken up from the blood and converted to
pyruvate for gluconeogenesis ―Cori cycle.‖
 In liver failure, liver can’t recycle lactate  lactic acidosis.

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Case: 9 month old girl has tonic-clonic seizure, she has failure to thrive and developmental
delay since birth. She is 3rd percentile for length and weight. Physical examination shows
microcephaly, tachypnea and hypotonia. Laboratory study show hypoglycemia,
hypoalaninemia and lactic acidosis. After intravenous glucose is administered, her serum
glucose concentration returns to within the reference range, deficiency of which enzyme?
Pyruvate carboxylase is a mitochondria enzyme requiring biotin. It is activated by
acetyl-coA (from beta- oxidation). The product oxaloacetate (OAA), a citric acid
cycle intermediate, cannot leave the mitochondria but is reduced to malate that can
leave via a Malate shuffle. In the cytoplasm malate is reduced to (OAA).
 Pyruvate carboxylase deficiency:
 Lactic acidosis.
 CNS damage by lactic acidosis.
 Hypoglycemia due to ↓gluconeogenesis.

TCA CYCLE (KREBS CYCLE)

Citrate Is Krebs’ Starting Substrate For Making Oxaloacetate

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 Pyruvate  acetyl-CoA produces 1 NADH, 1 CO2.

 The TCA cycle produces 3 NADH, 1 FADH2, 2 CO2, 1 GTP per acetyl-CoA = 10
ATP/ acetyl-CoA (2× everything per glucose).
 TCA cycle reactions occur in the mitochondria.
 4 dehydrogenases giving NADH except succinate DH giving FADH2
 The rate limiting step:
 Isocitrate dehydrogenase (ICDH): mention its regulation?
 α-ketoglutarate dehydrogenase complex requires the same cofactors as the pyruvate
dehydrogenase complex (B1, B2, B3, B5, lipoic acid).
 Mention the 2 reactions producing Co2!!!
 What will happen if there is excess energy in the cell?
 ↑ATP  inhibit ICDH  ↑Citrate  citrate shuttle  FA synthesis.
 What is the reaction through which there is substrate level phosphorylation in Kreb’s
cycle?
 Succinyl CoA  succinate; producing GTP.

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ELECTRON TRANSPORT CHAIN AND OXIDATIVE

PHOSPHORYLATION

 As NADH can’t pass through the thick mitochondrial membrane; NADH electrons
from glycolysis enter mitochondria via the malate-aspartate or glycerol-3-
phosphate shuttle.

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 The primary goal of the ETC is the generation of a proton gradient between the
matrix and the intermembrane space of the mitochondria. ATP synthase uses this
gradient to phosphorylate ADP to ATP.
 NADH donates electrons to protein complex I, through coenzyme Q to complex III,
and via cytochrome c to complex IV.
 CoQ: is lipid soluble from cholesterol synthesis pathway. Statins will ↓CoQ
 statins will affect highly aerobic tissues like muscles ―myositis‖
 UW: statins decrease levels of CoQ by decreasing farnesyl PP.
 FADH2 electrons are transferred to complex II bypassing complex I.
 NADH uses all 3 proton pumps (complex I, III, IV) but FADH2 uses only
complex III, IV  lower energy level than NADH.
 The final electron acceptor is molecular oxygen (O2), which is converted to water
H2O.
 Effects of hypoxia on ETC:
 The electron on the Cu+ on the cytochrome a3 can’t go anywhere  the
electrons stop flowing  no H+ gradient  no ATP synthesis.
 ↑NADH, ↓NAD+  LDH works to regenerate NAD  lactic acidosis.
 NAD+ produced by LDH is used by G3P DH to produce ATP.
 These are energetically favorable reactions, such that free energy is liberated at each
step. This energy is used to pump protons (H+) out of the mitochondrial matrix into
the intermembrane space.

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ATP PRODUCED VIA ATP SYNTHASE

 1 NADH  2.5 ATP.
 1 FADH2  1.5 ATP.

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OXIDATIVE PHOSPHORYLATION POISONS

 Effects of ETC inhibition:

 No electron current  no H+ gradient  no ATP produced.
 ↑ NADH (FADH2).
 ↓ O2 consumption  ↑PO2  respiratory depression.
 Electron transport inhibitors:
 Directly inhibitors, causing a ↓ proton gradient and block of ATP synthesis:
 Rotenone: complex one inhibitor.
 ―An-3-mycin‖ (antimycin) A: complex 3 inhibitor.
 CO/CN: complex 4 inhibitors (4 letters).
 Others:
 Barbiturates  ↓complex I.
 Doxrubicin  ↓CoQ  affect heart ―need high ATP‖ 
DCM.
 ATP synthase inhibitors: {Oligomycin}
 Causing an ↑ proton gradient.
 No ATP is produced because electron transport stops.
 Uncoupling agents:
 ↑ Permeability of membrane  bypass F0, ↓ proton gradient  no
ATP.
 ATP synthesis stops, but electron transport continues. Produces heat.
 ↓ ATP  ↑ PFK-1, ICDH  ↑ metabolic rate causing:
 ↑Body temperature.
 ↑Co2 production by TCA & ↑O2 consumption by ETC  ↑
respiratory rate  respiratory alkalosis which is rapidly
compensated by metabolic acidosis. But when too much
uncouplers  no ATP  ↓neuronal transmission in phrenic
nerve  respiratory depression ―combined respiratory &
metabolic acidosis‖.
 Examples of uncoupling agents:
 2,4-Dinitrophenol (used illicitly for weight loss),
 Aspirin (fevers often occur after aspirin overdose),
 Thermogenin in brown fat.

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 Cyanide poisoning:
o Binds to a variety of iron-containing enzymes, the most important of which is
the cytochrome a-a3 complex. Minute amounts of cyanide can inhibit aerobic
metabolism and rapidly result in death.
o Clinical picture:
 Rapidly-developing cutaneous flushing,
 Tachypnea, headache, and tachycardia, often accompanied by
nausea/vomiting, confusion, and weakness.
 Respiratory distress and cardiac dysfunction may follow.
o Laboratory studies:
 Severe lactic acidosis.
 ↓ Difference between arterial and venous O2 content (i.e the venous
blood is still highly oxygenated).
o Treatment:
 Nitrites. Methemoglobin can bind and sequester cyanide in the blood,
thereby keeping the poison away from mitochondrial.

 Sodium thiosulfate: it combines with cyanide to form the less-toxic

thiocyanate, which is excreted in the urine.

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 Carbon monoxide poisoning:

o Overview:
 Colorless, odorless, nonirritant gas that is generated as a byproduct of
incomplete hydrocarbon combustion.
 Carbon monoxide emission from automobiles and faulty home heaters can
result in carbon monoxide poisoning in poorly ventilated spaces.
o Pathophysiology:
 CO has 220 times more affinity for hemoglobin than does O2  formation
of carboxyhemoglobin.
 Carbon monoxide ↓ the O2 content of the blood by occupying oxygen
binding sites.
 ↓ Release of oxygen from hemoglobin in tissues by altering hemoglobin
conformation into the relaxed form that has a very high affinity for oxygen.
This results in a leftward shift of the oxygen dissociation curve and tissue
hypoxia via deficient unloading of oxygen.
o Treatment:
 100% or hyperbaric oxygen.

ATP PRODUCTION
 Aerobic metabolism of glucose produces:
1. 32 net ATP via Malate-aspartate shuttle (heart and liver).
2. 30 net ATP via glycerol-3-phosphate shuttle (muscle).
 Anaerobic glycolysis produces only 2 net ATP per glucose molecule.
 Arsenic causes glycolysis to produce zero net ATP.

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GLYCOGEN SYNTHESIS & GLYCOGENOLYSIS:

 The liver stores glycogen to maintain blood glucose during a short fast (12-18
HOURS).
 Muscle stores glycogen to provide an additional source of fuel during exercise.

 Linear glycogen is made by α-1,4 glycosidic bonds by glycogen synthase and is

broken by glycogen phosphorylase.
 Branches are made by α-1,6 glycosidic bonds by branching enzyme and is broken
by debranching enzyme.

1. Glucose should be activated first to UDP-glucose by glycogen synthase.

2. Branching enzyme removes polysaccharides of seven glucoses in length and creates
branch points by synthesizing α-1,6 glycosidic bonds.

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 By glycogen phosphorylase which breaks the α-1,4 glycosidic bonds, releasing the
individual glucose units as glucose-1-phosphate (G1P) ―trapped in the cell‖.
 ROLE OF GLYCOGENOLYS IS:
1. In muscle, G-l-P is converted to glucose 6-phosphate for glycolysis for
energy production.
2. In the liver, it is converted to glucose by glucose 6-phosphatase and released
into circulation.
 STEPS OF GLYCOGENOLYSIS:
1. Glycogen phosphorylase liberates glucose-1-phosphate residues off branched
glycogen until 4 glucose units remain on a branch ―limit dextrin‖.
2. Then 4-α-d-glucanotransferase (debranching enzyme) moves 3 molecules of
glucose-1-phosphate from the branch to the linkage.
3. Then α-1,6-glucosidase (debranching enzyme) cleaves off the last residue,
liberating glucose.
4. Note: A small amount of glycogen is degraded in lysosomes by α-1,4-
glucosidase (acid maltase).
 ―Limit dextrin‖ refers to the one to four residues remaining on a branch after
glycogen phosphorylase has already shortened it

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 Periodic acid–Schiff (PAS) stain identifies glycogen and is useful in identifying these
diseases.
 Very Poor Carbohydrate Metabolism. ―Von Gierk (I), Pompe (II), Cori (III),
McArdle (V)‖
 Types I, II, III, and V are autosomal recessive.
 Glycogen storage diseases that affect liver typically present with hypoglycemia and
lactic acidosis. Symptoms usually do not start until age 3-4 months, when
glycogenolysis is needed to sustain blood glucose during longer feeding intervals.

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VON GIERKE DISEASE (TYPE I)

 Pathogenesis & findings:

 ↓ Glucose-6-phosphatase 
 Impaired gluconeogenesis and glycogenolysis  severe fasting
hypoglycemia.
 ↑ G6P 
o Trapped in the liver and kidney  ↑osmolarity 
hepatomegaly & renomegaly.
o Inhibits lactate to pyruvate conversion  ↑↑ lactic acid.
o More substrate for HMP, more bases formed 
Hyperuricemia.
o ↑Acetyl CoA  hyperlipidemia & ketosis.
 Short stature doll-like facies, protruding abdomen emaciated extremities.
 Treatment:
 Frequent oral glucose/corn starch; avoidance of fructose and galactose.
POMPE DISEASE (TYPE II)
 ↓ Lysosomal acid α-1,4-glucosidase with α-1,6-glucosidase activity (acid maltase).
 Cardiac and skeletal muscle are particularly susceptible, as the ballooning lysosomes
interfere with contractile function.
 Cardiomegaly, hypertrophic cardiomyopathy, hypotonia, exercise intolerance, and
systemic findings lead to early death. ―PomPe trashes the PumP (1,4) (heart, liver,
and muscle)‖
 Muscle biopsy will show accumulation of glycogen in lysosomes.
CORI DISEASE (TYPE III)
 Debranching enzyme (α-1,6-glucosidase)  Gluconeogenesis is intact.
 Milder form of von Gierke (type I) with normal blood lactate levels.
 Accumulation of limit dextrin–like structures in cytosol
o ―Short outer branches & Single glucose residue at branch.‖

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MCARDLE DISEASE (TYPE V)

 Skeletal muscle glycogen phosphorylase (Myophosphorylase).
 Blood glucose levels typically unaffected. McArdle = Muscle
 Pathogenesis & findings:
 During exercise, affected individuals are unable to efficiently mobilize glucose
through glycogenolysis and cannot produce sufficient energy to maintain
muscle activity. This leads to painful muscle cramps.
 Some of the myocytes die from insufficient energy and release myoglobin,
which is cleared by the kidneys, leading to myoglobinuria, or dark urine
after exercise.
 Hallmark is a flat venous lactate curve with normal rise in ammonia
levels during exercise.
 ―Second-wind phenomenon‖
 Patient’s better tolerance for aerobic exercise such as walking and
cycling after approximately 10 minutes.
 Due to combination of increased blood flow and the ability of the
muscle to find alternative sources of energy, like fatty acids and
proteins.

HERS DISEASE (TYPE VI)

 ↓ Hepatic glycogen phosphorylase.
 Mild fasting hypoglycemia ―gluconeogenesis is not affected here‖
 Hepatomegaly.

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 Activation of glycogen phosphorylase:

 Phosphorylation dependent activation:
 Through epinephrine and glucagon induced increase in cyclic AMP.
 Non-phosphorylation dependent activation by:
 Through ↑ in intracellular Ca concentration (during muscle
contraction)
 AMP under extreme conditions.

Calcium is responsible for synchronization of skeletal

muscle contraction and glycogen breakdown.

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 Epinephrine:
 In the liver 
 ↑ Glycogenolysis and gluconeogenesis.
 In the skeletal muscle 
 ↓ Glucose uptake by the skeletal muscle ―no release of glucose from
muscles‖.
 ↑ Alanine release from the skeletal muscle, which serve as a source of
gluconeogenesis in the liver.
 In adipose tissue 
 ↑ Breakdown of TG, thereby increasing the circulating free fatty acids
and glycerol that can be utilized as gluconeogenic substrate.

 Glucagon ↑ Gluconeogenesis in liver only; muscle do not have glucagon receptor.

 Insulin inhibits glucagon secretion, but glucagon increases insulin secretion.

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GLUCONEOGENESIS

 Source of pyruvate for gluconeogenesis:

 Alanine by ALT enzyme using B6 as a cofactor & all other amino acids
except lysine and leucine which are strictly ketogenic.
 Lactate ―from RBCs and anaerobic glycolysis in muscles‖ by LDH  Cori’s
cycle.
 Fatty acids:
 Odd-chain fatty acids yield 1 propionyl-CoA during metabolism,
which can enter the TCA cycle (as succinyl-CoA), undergo
gluconeogenesis, and serve as a glucose source.
 Even-chain fatty acids cannot produce new glucose, since they yield
only acetyl-CoA equivalents.
 Site of gluconeogenesis:
 Occurs primarily in liver.
 Muscle cannot participate in gluconeogenesis because it lacks glucose-6-
phosphatase.
 Also found in kidney, intestinal epithelium.

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 Function of gluconeogenesis:
 Serves to maintain euglycemia during fasting.
Deficiency of the key gluconeogenic enzymes causes fasting hypoglycemia.

Pathway Produces Fresh Glucose.

Enzyme Reaction Control
Pyruvate carboxylase In mitochondria. Requires ATP, Biotin,
(PC) Pyruvate  oxaloacetate. Co2. Activated by acetyl-
CoA.
Phosphoenolpyruvate In cytosol. Requires GTP.
carboxykinase Oxaloacetate  Induced by Glucagon,
(PEPCK) phosphoenolpyruvate. epinephrine, cortisol.
Fructose-1,6- In cytosol. Citrate ⊕, AMP ⊝,
bisphosphatase Fructose-1,6-bisphosphate  fructose 2,6-
―the rate limiting step‖ fructose-6-phosphate. bisphosphate ⊝.
―opposite to PFK-1‖
Glucose-6- In ER.
phosphatase Glucose-6-phosphate  glucose.
 Gluconeogenesis requires high energy; explain!!
 ATP ↑ pyruvate carboxylase.
 GTP ↑ PEPCK.
 Where is this energy come from?
 From Beta-oxidation of fatty acids which produce also acetyl CoA which
activate PC.
 Why there is hyperglycemia with Cushing disease?
 ↑ Cortisol  ↑ PEPCK  ↑ gluconeogenesis  ↑ glucose.

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 Function:
 Activates PFK-1 (increasing glycolysis).
 Inhibits fructose 1,6-bisphosphatase (decreasing gluconeogenesis).
 Regulation:
 Increased by insulin  ↑ PFK-2  ↑ Fructose 2,6-bisphosphate.
 Decreased by glucagon  ↑ fructose 2,6-bisphosphatase  ↓ Fructose 2,6-
bisphosphate.

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HMP SHUNT (PENTOSE PHOSPHATE PATHWAY)

 A pathway that takes glucose 6-phosphate (G-6-P), produced by glycolysis, and
converts it through a different series of reactions to ribose-5-P while generating
NADPH in the process.

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 NADPH is used in:

 Anabolic processes (steroid and fatty acid
synthesis).
 In WBCs: for respiratory burst
(Phagocytosis and the oxygen dependent
pathway of microbial killing).
 In RBCs: to ↓ H2O2 build up by
Glutathione reductase, avoid hemolysis.
 In liver cells: FA synthesis, cholesterol
synthesis, Cytochrome P-450 system.
 Ribose-5-P is used in:
 Nucleotide synthesis and glycolytic
intermediates.
 2 distinct phases (oxidative and nonoxidative).
 No ATP is used or produced.
 Sites of HMP shunt:
 Cytoplasm of lactating mammary glands,
liver, adrenal cortex (sites of fatty acid or
steroid synthesis), RBCs.

 Glutathione peroxidase requires selenium which

is absorbed with vitamin E. so in vit E deficiency 
hemolytic anemia.

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 XLR; most common human enzyme deficiency; more

prevalent among African Americans. Heinz bodies—denatured
 NADPH is necessary to keep glutathione reduced, Hemoglobin precipitates
which in turn detoxifies free radicals and peroxides.
within RBCs due to
 ↓ NADPH in RBCs leads to hemolytic anemia due to
poor RBC defense against oxidizing agents (eg, fava oxidative stress.
beans, sulfonamides, primaquine, antituberculosis Bite cells—result from the
drugs).
phagocytic removal of
 Infection (most common cause) can also precipitate
hemolysis; inflammatory response produces free Heinz bodies by splenic
radicals that diffuse into RBCs, causing oxidative
damage.
 ↑ Malarial resistance.
 UW: Glutathione reductase deficiency causes a
similar clinical picture and is pathophysiologically
similar to G6PD deficiency.

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DISORDERS OF FRUCTOSE METABOLISM

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 Involves a defect in fructokinase. Autosomal recessive.

 A benign, asymptomatic condition, because of the following:
1. Fructose is not trapped in cells.
2. Hexokinase takes over the role of fructose metabolism, converting dietary
fructose into fructose-6-phosphate  enter glycolysis pathway.
 Symptoms: fructose appears in blood and urine. (fructokinase deficiency is kinder)
 Disorders of fructose metabolism cause milder symptoms than analogous disorders of
galactose metabolism.
 Dx: Copper reduction test detects reducing sugar in urine (unmetabolised fructose)

 Pathogenesis:
 Autosomal recessive deficiency of aldolase B  ↑Fructose-1-phosphate,
causing:
 ↑cell osmolarity  CNS & Liver & kidney damage.
 ↓ Available phosphate  inhibition of glycogenolysis and
gluconeogenesis  life threatening hypoglycemia.
 Symptoms: (Present following consumption of fruit, juice, or honey)
“vomiting and hypoglycemia about 20-30 minutes after fructose ingestion”.
 Liver: jaundice, cirrhosis, hypoglycemia.
 CNS: lethargy, vomiting.
 Kidney: proximal RTA, Fanconi syndrome.
 Hyperuricemia: due to phosphate trapping  ↓available phosphate for
purines recycling  purines are degraded to uric acids.
 NO CATARACT.
 Investigations:
 Urine dipstick will be ⊝ (tests for glucose only); reducing sugar can be
detected in the urine (nonspecific test for inborn errors of carbohydrate
metabolism).
 Treatment:
 ↓ Intake of both fructose and sucrose (glucose + fructose).

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DISORDERS OF GALACTOSE METABOLISM

 Galactose is the 1st food we have; so galactose deficiencies symptoms appear early
(neonates).
 Fructose deficiencies appear later when the child eats honey.
 Fructose is NOT aldose sugar like (glucose & galactose)  not reduced in lens 
no cataract.

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 Hereditary deficiency of galactokinase. Autosomal recessive.

 Galactitol accumulates if galactose is present in diet  infantile cataract.
 Relatively mild condition (hexokinase also can do its job). Galactokinase defciency is
kinder (benign condition).
 Symptoms:
 Galactose appears in blood (galactosemia) and urine (galactosuria);
 Infantile cataracts  present as failure to track objects or to develop a social
smile.

 Autosomal recessive.
 Pathogenesis:
 Absence of galactose-1-phosphate uridyltransferase 
 ↑ Galactitol, which accumulates in the lens of the eye  infantile cataract.
 ↑Galactose 1-P  osmotic damage  CNS & Liver & kidney damage.
 Symptoms: Develop when infant begins feeding (lactose present in breast milk and
routine formula)
 Infantile cataracts.
 Liver: jaundice, cirrhosis, hypoglycemia.
 CNS: lethargy, vomiting.
 Kidney: proximal RTA, Fanconi syndrome.
 Hyperuricemia: due to phosphate trapping  ↓available phosphate for
purines recycling  purines are degraded to uric acids.
 Can predispose to E coli sepsis in neonates.
 Treatment:
 Exclude galactose and lactose (galactose + glucose) from diet.
 Feed with soy based infant formula which result in regression of cataracts
and improvement in renal and liver function; soy milk consist of sucrose,
which is metabolized to glucose and fructose.

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Fructose enter the glycolysis in the middle of glycolysis bypassing the ATP requiring step & bypass
phosphofructokinase; one of the key enzymes involved in regulating the rate of glycolysis.
Galactose enter the glycolysis in the beginning. So fructose makes more energy and faster than
galactose.

SORBITOL

 An alternative method of trapping glucose in the cell is to convert it to its alcohol

counterpart, called sorbitol, via aldose reductase.
 Some tissues then convert sorbitol to fructose using sorbitol dehydrogenase; tissues
with an insufficient amount/activity of this enzyme are at risk for intracellular sorbitol
accumulation, causing osmotic damage (eg, cataracts, retinopathy, and peripheral
neuropathy seen with chronic hyperglycemia in diabetes).
 High blood levels of galactose also result in conversion to the osmotically active
galactitol via aldose reductase.

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LACTASE DEFCIENCY
 Insufficient lactase enzyme  dietary lactose
intolerance.
 Lactase functions on the brush border to digest
lactose (in human and cow milk) into glucose and
galactose.
 Types:
 Primary: age-dependent decline after
childhood (absence of lactase-persistent
allele), common in people of Asian,
African, or Native American descent.
 Secondary: loss of brush border due to
gastroenteritis (eg, rotavirus), autoimmune
disease, etc.
 Congenital lactase deficiency: rare, due to
defective gene.
 Investigations:
 ↓ Stool PH.
 Lactose hydrogen breathe test  ↑ hydrogen content.
 Intestinal biopsy reveals normal mucosa in patients with hereditary lactose
intolerance.
 FINDINGS:
 Bloating, cramps, flatulence, osmotic diarrhea.
 TREATMENT:
 Avoid dairy products or add lactase pills to diet; lactose-free milk.

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FATTY ACID METABOLISM

 Unsaturated fatty acids: have double bonds  ↑ risk of atherosclerosis.

 Essential fatty acids: Linolenic & Linoleic fatty acids  should be provided by food as
they form a precursor for arachidonic acid (prostaglandins & thromboxane &
prostacyclins & leukotriens).

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 Fatty acids are synthesized in the cytosol.

 The precursor of FA is acetyl-CoA, which is generated in the Malate shuttle 
mitochondria. transport OA in
 Citrate shuttle is used to transfer acetyl-CoA outside the gluconeogenesis.
mitochondria for FA synthesis.
Citrate shuttle  FA
 Insulin promotes several steps in the conversion of glucose
synthesis.
to acetyl-CoA by:
1) Induction of glucokinase.
2) Dephosphorylation of PFK-2/PFK-1.
3) Dephosphorylation of pyruvate dehydrogenase.
4) Activation of acetyl-CoA carboxylase by dephosphorylation.
5) Induction of fatty acid synthase.
 In addition to acetyl-CoA, the major substrates for this reaction are NADPH and ATP.
 What is the rate-limiting step for FA synthesis?
 Acetyl-CoA carboxylase; Mention its regulation???

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 Fatty acids are converted to acetyl-CoA in the mitochondria in a process known as β-

oxidation.
 This occurs in liver, muscle, and adipose tissue.
 Neither erythrocytes (which lack mitochondria) nor brain cells (fatty acids do not
easily cross the blood-brain barrier) can use fatty acids for energy, so these cells
depend on glucose even during periods of fasting.
 This pathway involves three major steps:
1. Activation of free fatty acids.
2. Transport into mitochondria.
3. Oxidation.

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1. Activation (needs ATP)

o Fatty acids greater than two carbons long must be converted to their acyl-CoA
form in order to be transported and oxidized.
o This is accomplished by fatty acyl-CoA synthetase on the outer mitochondrial
membrane.
o Shorter fatty acids pass directly into the mitochondria and are activated in the matrix.
2. Transport
o Fatty acyl-CoAs cannot cross the inner mitochondrial
membrane.
o They first must be modified by a molecule of carnitine.
This is catalyzed by carnitine palmitoyltransferase 1 (CPT
1, also known as carnitine acyltransferase-1), an
enzyme in the outer mitochondrial membrane.
o Fatty acyl carnitine is then shuttled across the inner
mitochondrial membrane, and carnitine acyltransferase-2
(also known as carnitine palmitoyltransferase-2) transfers
the fatty acyl group back to a CoA in the mitochondrial
matrix.

3. Oxidation
o Removes acetyl-CoA groups one at a time from FA acyl-CoA.
o In the process, one FADH2 and one NADH are produced in addition to acetyl-CoA.
FADH 2 and NADH are then oxidized in the electron transport chain to provide ATP.

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 Deficiency of the plasma membrane carnitine transporter, leading to urinary wasting of

carnitine.
 Inherited defect in transport of LCFAs into the mitochondria  toxic accumulation.
 Cardiac and skeletal myocytes cannot generate ATP from fatty acids 
1) ↓ ATP for SK MS  hypotonia & weakness.
2) ↓ ATP for cardiac muscle  cardiomyopathy.
 Liver is unable to synthesize ketone bodies when glucose levels are low  hypoketotic
hypoglycemia.
 Muscle biopsy shows a very low carnitine content.

 Clinical picture as McArdle disease except 2 differences:

1) The onset of weakness & muscle aches:
 Myopathic CAT/CPT deficiency  late with progressive exercise.
 McArdle  early with second wind phenomenon.
2) Muscle biopsy:
 Myopathic CAT/CPT deficiency  ↑ TG.
 McArdle  ↑ glycogen.

 Pathogenesis & C/P:

1) Acyl-CoA dehydrogenase deficiency  ↓ Ability to break down fatty acids into
acetyl-CoA 
 No ATP produced for gluconeogenesis & glycogenolysis  fasting
hypoglycemia.
 No Acetyl-CoA is produced for Ketone body production  hypoketosis.
 Causes vomiting, lethargy, seizures, coma, liver dysfunction.
 Can lead to sudden infant death ―as the infants begin to sleep longer
periods at night‖
2) No beta oxidation for FA 
 Accumulation of medium -chain fatty acids in the liver  hepatomegaly.
 Accumulation of fatty acyl carnitines in the blood.
 FAs will use another way for oxidation (like omega oxidation) resulting
in
o Dicarboxylic academia ―marker for MCAD deficiency‖.
 Treat by
1) Avoid fasting by frequent high carbohydrate meals.

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 Fatty acid synthesis requires transport of citrate from mitochondria to cytosol.

 Long-chain fatty acid (LCFA) degradation requires carnitine-dependent transport into
the mitochondrial matrix. ―SYtrate‖ = SYnthesis. CARnitine = Carriage of fatty acids.
 NB: Acetyl-CoA from FA, can’t form glucose by gluconeogenesis as its 2 carbons are
lost in TCA as CO2  FA can’t make glucose.

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Propionyl-CoA is converted to succinyl-CoA, a citric acid cycle intermediate, in the

two-step propionic acid pathway.
Because this extra succinyl-CoA can form malate and enter the cytoplasm and
gluconeogenesis, odd-carbon fatty acids represent an exception to the rule that fatty
acids cannot be converted to glucose
in humans.
Propionyl-CoA is derived from V O M I T pathways.
 UW: Propionic acidemia:
1) Congenital deficiency of propionyl CoA carboxylase  Propionyl CoA
accumulates  Propionic acidemia.
2) Clinically characterized by poor feeding, vomiting, hypotonia, lethargy,
dehydration, and an anion gap acidosis.
 UW: Methylmalonic acidemia:
1) Autosomal recessive complete or partial deficiency of the enzyme
methylmalonyl-CoA mutase.
2) Clinically:
 ↑ Methylmalonic acid  metabolic acidosis.
 Hypoglycemia.
 The presence of hypoglycemia produces ketones, resulting in a further
anion gap metabolic acidosis.
 Finally, organic acids also directly inhibit the urea cycle, leading to
hyperammonemia.
3) Diagnosis is confirmed by:
 Presence of elevated urine methylmalonic acid and propionic acid.

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 Site of production (ketogenesis):

 In the liver, fatty acids and amino acids are metabolized to acetoacetate and
β-hydroxybutyrate to be used as a fuel.
 Site of use (ketogenolysis):
 Extrahepatic tissues:
 Cardiac and skeletal muscles and renal cortex metabolize
acetoacetate and 3-hydroxybutyrate to acetyl-CoA  TCA  ATP.
 Brain doesn’t use ketones except after prolonged starvation.
 2 cells can’t utilize ketone bodies:
 RBCs  lacks mitochondria and therefore cannot use ketones.
 Liver cells  lacks succinyl-CoA acetoacetyl-CoA transferase
(thiophorase).
 Pathogenesis:
 In prolonged starvation and diabetic ketoacidosis, oxaloacetate is depleted
for gluconeogenesis.
 In alcoholism, excess NADH shunts oxaloacetate to malate.
 Both processes cause a buildup of acetyl-CoA, which shunts glucose and FFA
toward the production of ketone bodies.
 Ketone bodies:
 Acetone, acetoacetate, β-hydroxybutyrate.
 Acetone is not used as fuel but it makes breath smells like fruity odor.
 Urine test for ketones can detect acetoacetate, but not β-hydroxybutyrate.
 In alcoholics ↑↑↑ 3-hydroxybutyrate more than acetoacetate.

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 Signs of ketoacidosis
 Acetone on breath
 CNS depression and coma.
 Decreased plasma bicarbonate.
 Polydipsia, polyuria, polyphagia (exacerbated by hyperglycemia and osmotic
diuresis).
 Depletion of K+ ―by osmotic diuresis‖ (may be masked by mild
hyperkalemia).
 NB: HMG-CoA lyase for ketone production. HMG-CoA reductase for cholesterol
synthesis.

FED STATE (AFTER A MEAL)

 Glycolysis and aerobic respiration.  Glycogen reserves depleted
after day 1.
 Insulin stimulates storage of lipids, proteins, and
 RBCs lack mitochondria
glycogen.
and therefore cannot use
FASTING (BETWEEN MEALS) ketones.
 FA is used by skeletal
 Priorities are to supply sufficient glucose to the brain and
muscle and heart, it cannot
RBCs and to preserve protein.
cross BBB, so brain use
 Hepatic glycogenolysis (major); hepatic only ketone bodies.
gluconeogenesis, adipose release of FFA (minor).
 Glucagon and epinephrine stimulate use of fuel reserves.

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STARVATION DAYS 1–3

 Blood glucose levels maintained by:
1) Hepatic glycogenolysis
2) Adipose release of FFA
3) Muscle and liver, which shift fuel use from glucose to FFA
4) Hepatic gluconeogenesis from
 Peripheral tissue lactate
 Alanine.
 Adipose tissue glycerol.
 PropionylCoA (from odd-chain FFA—the only triacylglycerol components
that contribute to gluconeogenesis).

STARVATION AFTER DAY 3

 Adipose stores (ketone bodies become the main source of energy for the brain).
 After these are depleted, vital protein degradation accelerates, leading to organ failure
and death.
 Amount of excess stores determines survival time.

 UW: during prolonged starvation, glycerol kinase is responsible in providing substrate

for gluconeogenesis. Also hormone sensitive lipase is responsible during starvation for
increase in both gluconeogenesis and fatty acid oxidation, by degrading TGs into FA
and glycerol.

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 Triglycerides (TGs) are the storage form of fatty acids.

 Stored in the liver and adipose tissue.
 Formed of Glycerol + 3 FA.
 Glycerol comes from DHAP.
 FA from acetyl-coA coming from excess dietary glucose.
 The liver sends triglycerides to adipose tissue as very low-density
lipoproteins (VLDL).
 The liver makes TG from 2 sources (glucose & glycerol) but
adipose tissue makes it from one source (DHAP).
 Adipose tissue, which lacks glycerol kinase, cannot generate
glycerol 3-phosphate from glycerol.
 In the liver, triacylglycerol is incorporated into very-low-density lipoprotein (VLDL),
entering blood.

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CHOLESTEROL SYNTHESIS

UW: Bile acids are formed from cholesterol by 7-α-hydroxylase enzyme. ↓ Activity of this
enzyme  cholesterol gall stones.

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LIPID TRANSPORT, KEY ENZYMES

 Cholesterol and triglycerides are transported in the blood as lipoproteins.

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 Pancreatic lipase—degradation of dietary triglycerides (TGs) in small intestine.

 Lipoprotein lipase (LPL):
 Degradation of TGs circulating in chylomicrons and VLDLs.
 Found on vascular endothelial surface.
 Induced by insulin. Activated by Apo CII.
 Hepatic TG lipase (HL):
 Degradation of TGs remaining in IDL.
 Hormone-sensitive lipase:
 Degradation of TGs stored in adipocytes.

 When blood sugar is low, glucagon signals the adipocytes to activate hormone-sensitive
lipase, and to convert triglycerides into free fatty acids.
 Causes of hyperlipidemia in DM:
1. ↓Insulin  ↓GLUT4  glucose intracellular  ↓acetyl CoA ↓fat storage.
2. ↓Insulin  ↓LPL  ↑chylomicrons & VLDL.

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 LCAT:
 Catalyzes esterification of 2⁄3 of plasma cholesterol  removes cholesterol fatty
streaks.
 Activated by Apo A-I.
 Cholesterol ester transfer protein (CETP):
 Mediates transfer of cholesterol esters to other lipoprotein particles.

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 Lipoproteins are composed of varying proportions of cholesterol,

TGs, and phospholipids.
 LDL and HDL carry the most cholesterol.
 LDL transports cholesterol from liver to tissues. UW: lipoprotien synthesis
 HDL transports cholesterol from periphery to liver. occur in SER.

 Needed to maintain cell membrane integrity and synthesize bile acid, steroids, and
vitamin D.

 Delivers dietary TGs to peripheral tissues.

 Delivers cholesterol to liver in the form of chylomicron remnants, which are mostly
depleted of their TGs.
 Secreted by intestinal epithelial cells.

 Delivers hepatic TGs to peripheral tissue.

 Secreted by liver.

 Formed in the degradation of VLDL.

 Delivers TGs and cholesterol to liver.

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 Delivers hepatic cholesterol to peripheral tissues.

 Formed by hepatic lipase modification of IDL in the liver and peripheral tissue.
 Taken up by target cells via receptor-mediated endocytosis.
 Most of the cholesterol measured in the blood is associated with LDL.

 Mediates reverse cholesterol transport from periphery to liver (picks up cholesterol

from fatty streaks).
 Acts as a repository for apolipoproteins C and E (which are needed for chylomicron
and VLDL metabolism).
 Secreted from both liver and intestine.
 Alcohol ↑ synthesis.

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 Autosomal recessive. Deficiency in ApoB48, ApoB100.

 ↓Apo B48  ↓ dietary fat absorption  absent chylomicrons, VLDL, LDL.
 Affected infants present with severe fat malabsorption, steatorrhea, and failure to
thrive.
 Later manifestations include retinitis pigmentosa, spinocerebellar degeneration due to
vitamin E deficiency, progressive ataxia, and acanthocytosis.
 Treatment: restriction of long-chain fatty acids, large doses of oral vitamin E.

TYPE PATHOGENESIS ↑ BLOOD CLINICAL

LEVEL
I— Lipoprotein lipase Chylomicrons, Recurrent pancreatitis,
Hyperchylomicronemia or apolipoprotein TG, hepatosplenomegaly, and
(AR) C-II deficiency. cholesterol eruptive/pruritic xanthomas (no
↑ risk for atherosclerosis).
Creamy layer in supernatant
(milky appearance of blood.)
II—Familial Absent or defective IIa: LDL, Heterozygotes (1:500) have
hypercholesterolemia LDL receptors, or cholesterol cholesterol ≈ 300mg/dL;
(AD) defective ApoB- IIb: LDL, homozygotes (very rare) have
100. cholesterol, cholesterol ≈ 700+ mg/dL.
VLDL Accelerated atherosclerosis
(may have MI before age 20),
tendon (Achilles) xanthomas,
and corneal arcus.
III— Defective ApoE. Chylomicrons, Premature atherosclerosis,
Dysbetalipoproteinemia VLDL. tuberoeruptive xanthomas,
(AR) xanthoma striatum palmare.
IV— Hepatic VLDL, TG Hypertriglyceridemia (> 1000
Hypertriglyceridemia overproduction of mg/dL) can cause acute
(AD) VLDL. pancreatitis. Related to insulin
resistance.

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TTT of homozygous familial hypercholesterolemia:

 Mipomersen “new drug”: antisense RNA against Apo B100.
UW: xanthomas:
 Associated with hyperlipidemia or lymphoproliferative malignancies.
 Can be divided clinically into 5 categories:
1. Eruptive xanthomas: yellow palques that abruptly appear when plasma TGs and
lipid increases.
2. Tuberous and tendibous: tendinous often found on Achilles tendon and the
extensor tendons of the fingers.
3. Plane xanthomas: linear lesions in skin folds that are strongly associated with
primary biliary cirhhosis.
4. Xanthelasma: soft eyelid or periorbital plaques with no associated lipid
abnormalities in 50% of affected individuals.

SPHINGOLIPIDS:
 Sphingolipids are important components of cellular membranes.
 They are particularly enriched in nerve tissue.
 They have a structure similar to phospholipids, except that they are built on a
molecule of serine, rather than glycerol.
 Ceramide is the parent of sphingolipids. It is made of serine + 2 FA.

 Each is caused by a deficiency in one of the many lysosomal enzymes.

 Results in an accumulation of abnormal metabolic products.
 All are AR except Fabry disease & Hunter syndrome which are XLR.
 ↑ Incidence of Tay-Sachs, Niemann-Pick, and some forms of Gaucher disease in
Ashkenazi Jews.

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SPHINGOLIPIDOSES

Disease Findings Enzyme deficiencies Accumulated

substrate
Tay-Sachs disease Progressive 1 HeXosaminidase A GM2 ganglioside
neurodegeneration, (―TAy-SaX‖)
developmental delay,
―cherry-red‖ spot on
macula A, lysosomes
with onion skin, no
hepatosplenomegaly
(vs Niemann-Pick).
Fabry disease Early: Triad of 2 α-galactosidase A Ceramide
episodic peripheral trihexoside
neuropathy,
angiokeratomas B,
and hypohidrosis.
Late: progressive
renal failure,
cardiovascular
disease.
Metachromatic Central and peripheral 3 Arylsulfatase A Cerebroside sulfate
leukodystrophy demyelination with
ataxia, dementia.
Krabbe disease Peripheral 4 Galactocerebroside,
neuropathy, Galactocerebrosidase psychosine
destruction of
oligodendrocytes,
developmental delay,
optic atrophy, globoid
cells.
Gaucher disease Most common. 5 Glucocerebrosidase Glucocerebroside
Hepatosplenomegaly, (β-glucosidase); treat
pancytopenia, with recombinant
osteoporosis, glucocerebrosidase
avascular necrosis of
femur, bone crises,
Gaucher cells C
(lipid-laden
macrophages
resembling crumpled
tissue paper)

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Niemann-Pick Progressive 6 Sphingomyelinase Sphingomyelin.

disease neurodegeneration, No man picks
hepatosplenomegaly, (Niemann-Pick) his
foam cells. nose with his
(lipid-laden sphinger
macrophages) D, (sphingomyelinase).
―cherry-red‖ spot on
macula A .

UW: C/P of Fabry disease:

 The earliest manifestations are hypohidrosis, acroparesthesia and angiokeratomas.

 Acroparesthesia is episodic, often debilitating, burning neuropathic pain in the
extremities.
 Angiokeratomas are punctuate, dark red, non-blanching macules and papules that
classically occur between the umbilicus and the knees.
 Without enzyme replacement therapy, progressive renal insufficiency leading to
renal failure and death may occur.

MUCOPOLYSACCHARIDOSES

Hurler Developmental delay, α-l- Heparan sulfate, dermatan

syndrome gargoylism, airway iduronidase sulfate
obstruction, corneal
clouding, HSM.
Hunter Mild Hurler + aggressive Iduronate Heparan sulfate, dermatan
syndrome behavior, no corneal sulfatase sulfate.
clouding. Hunters see clearly (no
corneal clouding) and
aggressively aim for the X
(X-linked recessive).

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AMINO ACIDS
 Only L-amino acids are found in proteins.
 Substituting a D for an L amino group  "Proteolytic enzymes"(endogenous
protease) prefer L-configuration (natural) rather than D-configuration
(unnatural).
 so in a peptide chain to break the bond between two amino acids (L-
configurated) peptidases quickly recognize it and will lead to peptide
degradation hence short half-life (e.g. endogenous Opioid peptide).
 But if we replace either or both of the susceptible amino acids in the peptide
chain with D-configurated aa  the endogenous peptidases/proteases will be
unable to cleave the bond b/w the amino acids that will lead to decrease or
prevent peptide degradation hence the LONGER half-life.
 Essential amino acids: need to be supplied in the diet.
 Glucogenic: methionine (Met), histidine (His), valine (Val).
I met his valentine, she is so sweet (glucogenic).
 Glucogenic/Ketogenic: isoleucine (Ile), phenylalanine (Phe), threonine (Thr),
tryptophan (Trp).
 Ketogenic: leucine (Leu), lysine (Lys).
 UW: Asparagine:
 Non-essential amino acid that is
produced from oxaloacetate in a
transamination reaction.
 Oxaloacetate is first converted to
aspartate using glutamate to provide
amino group, then the enzyme
asparagine synthase transfers the
additional nitrogen molecule from
glutamine to aspartate to produce
asparagine.
 Asparagine becomes essential for rapidly dividing tumor cell.
 Asparagine is catabolized initially to aspartate by the enzyme
asparaginase.
 In rapidly dividing leukemic cells, the synthesis of asparagine is
impaired, so these cells survive by collecting asparagine from
circulating plasma.
 L-asparaginase work as an antineoplastic agent by lowering
circulating asparagine levels.

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 Acidic:
 Aspartic acid (Asp) and glutamic acid (Glu).
 Negatively charged at body pH.
 Basic:
 Histidine (His), lysine (Lys), arginine (Arg).
 Arg is most basic.
 His has no charge at body pH.
His lys (lies) are basic.
 Arg and His are required during periods of growth.
 Arg and Lys are ↑ in histones, which bind negatively charged DNA.

UW: Protein digestion:

Trypsinogen is activated to trypsin by duodenal enteropeptidase. Trypsin is essential for

protein digestion and absorption in two ways. It degrades complex peptides to dipeptides
and amino acids, and it activates other proteases such as carboxypeptidase, elastase and
chymotrypsin.

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 During the catabolism of proteins, amino groups are transferred to α-

ketoglutarate to form glutamate. Glutamate is then processed in the liver to
form urea, the primary disposal form of nitrogen in humans. Free ammonia is
also excreted into the urine by the kidney for regulation of acid-base status.
 Alanine is the major amino acid responsible for transferring nitrogen to the
liver for disposal.
 Glutamine:
 Produced by most body tissues and is catabolized primarily by the gut
and kidney for maintenance of cellular metabolism and acid-base
regulation, respectively.
 Transports ammonia from peripheral tissues to the kidney.
 Almost all aminotransferase enzymes use α-ketoglutarate as the amino group
acceptor.

 Amino acid catabolism results in the formation of common metabolites (eg,

pyruvate, acetylCoA), which serve as metabolic fuels.
 Excess nitrogen generated by this process is converted to urea and excreted by
the kidneys.
 Ordinarily, Careless Crappers Are Also Frivolous About Urination.

Urea cycle is an ATP dependent process, in fasting when we cannot break fatty acid we
break protein to have glucose and ATP. The break down product of protein is ammonia
which is converted into urea, when we have low ATP, urea cycle stops and we have
accumulation of ammonia.

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UW: Remember that urea synthesis is a cyclic process:

Ammonium ion, CO2, ATP and aspartate are consumed.
No net loss or gain of ornithine, citrulline, argininosuccinate or arginine.
The molecule N-acetylglutamate serves as a regulator of the urea cycle
through allosteric activation of carbamoyl phosphate synthetase I.
UW: Citrullination:
Tissue inflammation causes arginine residues in proteins such as vimentin to
be enzymatically converted into citrulline through a process called
citrullination.
This can significantly alter the shape of these proteins, which can then serve
as antigens and generate an immune response.
In RA, this immune response is exaggerated, resulting in high titers of anti-
CCP antibodies that are not usually present in other inflammatory conditions.
Thus, anti-CCP antibodies have a high specificity (95%-98%) for RA.

 Most common urea cycle disorder. X-linked recessive (vs other urea cycle
enzyme deficiencies, which are autosomal recessive).
 Interferes with the body’s ability to eliminate ammonia.
 Often evident in the first few days of life, but may present later.
 Excess carbamoyl phosphate is converted to orotic acid (part of the
pyrimidine synthesis pathway).
 Findings:
1) ↑ Orotic acid in blood and urine,
2) ↓ BUN, symptoms of hyperammonemia.
3) No megaloblastic anemia (vs orotic aciduria).

N-acetyl glutamate synthase deficiency:

 Required cofactor for carbamoyl phosphate synthetase I.

 Absence of N-acetylglutamate causes hyperammonemia.
 Presents in neonates as poorly regulated respiration and body temperature, poor
feeding, developmental delay, intellectual disability (identical to presentation of
carbamoyl phosphate synthetase I deficiency).

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 Present as progressive development of spastic diplegia, abnormal movements,

and growth delay in the setting of elevated arginine levels.
 UW: Arginase deficiency is likely underdiagnosed because the spasticity seen
commonly in this disorder may simply be attributed to cerebral palsy.
 Unlike other urea disorders, patients with arginase deficiency have mild or no
hyperammonemia.
 Treatment:
 Low-protein diet that is devoid of arginine.
 Administration of a synthetic protein made of essential amino acids
usually results in a dramatic decrease in plasma arginine concentration
and an improvement in neurologic abnormalities.

 Can be acquired (eg, liver disease) or hereditary (eg, urea cycle enzyme
deficiencies).
 Results in excess NH3, which depletes α-ketoglutarate, leading to inhibition
of TCA cycle.
 Symptoms:
1) Tremor (asterixis), slurring of speech, somnolence,
vomiting, cerebral edema, blurring of vision.
 Treatment:
1) Limit protein in diet.
2) May be given to ↓ ammonia levels:
 Lactulose to acidify the GI tract and trap NH4+
for excretion.
 Antibiotics (eg, rifaximin) to ↓ colonic ammoniagenic bacteria.
 Benzoate, phenylacetate, or phenylbutyrate react with glycine
or glutamine, forming products that are renally excreted.

Oxindole is a tryptophan derivative formed by bacteria in the gut and normally cleared by
the liver. It causes sedation, muscle weakness, hypotension and coma. Elevated levels of
oxindole have been found in patients with hepatic encephalopathy.
Burr bodies: ↑ammonia is toxic to the RBCs cell membrane causing these bodies which is
similar to acanthocytes.

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When ammonia levels rise acutely, astrocyte and neuron function are affected.
Within the brain, astrocytes and neurons interact to regulate the metabolism of
glutamate, glutamine, and ammonia in a process known as the glutamate-
glutamine cycle.
Glutamate released by neurons during neurotransmission is taken up by
astrocytes and converted to glutamine, a non-neuroactive compound.
Glutamine is then released by astrocytes and taken up by neurons, where it is
either converted back to glutamate for use as a neurotransmitter or
transaminated into α-ketoglutarate for use in the Krebs cycle.
Ammonia toxicity results in part from depletion of glutamate and alpha-
ketoglutarate in the brain during the process of ammonia detoxification.
Hyperammonemia increases the conversion of glutamate into glutamine by
glutamine synthetase within astrocytes.
The resulting increase in glutamine leads to hyperosmolarity and mitochondrial
dysfunction, causing astrocytic swelling and impairment. Increased glutamine
formation also decreases total brain glutamate stores, impairing excitatory
neurotransmission (mediated by NMDA. AMPA, and kainate receptors) and
neuronal energy production.
In addition, ammonia can be detoxified to glutamate via glutamate
dehydrogenase, depleting a-ketoglutarate and further impairing energy
metabolism in the brain.

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 Lactate is converted to pyruvate in the liver using NAD+ as a cofactor.

 Case: 28 years old patient after severe exercise comes with tachycardia and tachypnea
then had alcohol then died; Why?
 Severe exercise causes lactic acidosis due to anaerobic glycolysis.
 Alcohol consumes NAD+  ↓NAD+  ↓ conversion of lactate to pyruvate
 severe lactic acidosis.
Why in a patient with liver cell failure, there is metabolic acidosis?

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 UW: Norepinephrine and dopamine are produced in the central as well as the
peripheral nervous system, whereas epinephrine is predominantly produced in the
adrenal medulla.
 In the adrenal medulla, norepinephrine is rapidly converted to epinephrine by
phenylethanolamine-N-methyltransferase (PNMT).
 Following pituitary resection, ACTH secretion and subsequent cortisol production
would be low. The result would be decreased PNMT activity and reduced
conversion of norepinephrine to epinephrine.
 UW: COMT converts epinephrine to metanephrine and norepinephrine to
normetanephrine.
 MAO converts metanephrine and normetanephrine to vanillylmandelic acid.

 Autosomal recessive. Incidence ≈ 1:10,000.

 Screening occurs 2–3 days after birth (normal at birth because of maternal
enzyme during fetal life).
 Causes:
1) ↓ Phenylalanine hydroxylase.
2) ↓ Tetrahydrobiopterin (BH4) cofactor (malignant PKU). Deficiency of
dihydrobiopterin reductase, the enzyme responsible for reduction of
BH4 to BH2, is the most common cause for a deficiency of BH4.
 Pathogenesis:
1) Tyrosine becomes essential.
2) ↑ Phenylalanine  excess phenyl ketones in urine (phenylacetate,
phenyllactate, and phenylpyruvate.)
 Findings:
1) Intellectual disability, growth retardation, seizures.
2) ↑ Phenylalanine  ↑ Phenylacetate & Phenylactate  musty body
odor, eczema.
3) ↓ Melanin  Hypopigmentation involving the skin (fair skin), hair,
eyes, and catecholaminergic brain nuclei (substanctia negra, locus
ceruleus and vagal nucleus dorsalis).
 Treatment:
1) ↓ Phenylalanine and ↑ tyrosine in diet.
2) Tetrahydrobiopterin supplementation.
3) Avoid the artificial sweetener aspartame, which contains
phenylalanine.

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 Maternal PKU—lack of proper dietary therapy during pregnancy. Findings

in infant: microcephaly, intellectual disability, growth retardation, congenital
heart defects.
 Disorder of aromatic amino acid metabolism  musty body odor.

↓BH4  ↑phenylalanine & ↓serotonin as a neurotransmitter in the brain which needs BH4 as

a cofactor for synthesis. “MALIGNANT PKU”

 Blocked degradation of branched amino acids (Isoleucine, Leucine, Valine)

due to ↓ branched-chain α-ketoacid dehydrogenase (B1).
I Love Vermont maple syrup from maple trees (with B1ranches).

 Autosomal recessive.
 Causes ↑ α-ketoacids in the blood, especially those of leucine.
 Findings:
1) ↑ Leucine causes severe CNS defects, intellectual disability, and death.
2) Vomiting, poor feeding.
3) ↑ Isoleucine in urine  urine smells like maple syrup/burnt sugar.
 Treatment:
1) Restriction of isoleucine, leucine, valine in diet, and
Thiamine supplementation.

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 Congenital deficiency of homogentisate oxidase in the

degradative pathway of tyrosine to fumarate  pigment-
forming homogentisic acid accumulates in tissue A.
 Autosomal recessive. Usually benign.
 Findings:
1) Bluish-black connective tissue, ear cartilage, and
sclerae (ochronosis).
2) Urine turns black on prolonged exposure to air.
3) May have debilitating arthralgias (homogentisic acid toxic to cartilage).

 Types (all autosomal recessive):

1) Cystathionine synthase deficiency:
 Treatment: ↓ methionine, ↑ cysteine, ↑ B6, B12, and folate in diet.
2) ↓ Affinity of cystathionine synthase for pyridoxal phosphate:
 Treatment: ↑↑ B6 and ↑ cysteine in diet.
3) Methionine synthase (Homocysteine methyltransferase) deficiency:
 Treatment: ↑ methionine in diet.
 All forms result in excess homocysteine.
 Findings: HOMOCYstinuria:
1) ↑↑ Homocysteine in urine,
2) Osteoporosis,
3) Marfanoid habitus,
4) Ocular changes (downward and inward lens subluxation),
5) Cardiovascular effects (thrombosis and atherosclerosis ↑ stroke and MI),
6) kYphosis, intellectual disability.

UW: Individuals with complete cystathionine synthase deficiency can develop

premature acute coronary syndrome.
UW: Homocysteine is converted to methionine using methylcobalamin and methyl
tetrahydrofolate.

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 Autosomal recessive. Common (1:7000).

 Cystine is made of 2 cysteines connected by a disulfide
bond.
 Hereditary defect of renal PCT and intestinal amino acid
transporter that prevents reabsorption of Cystine,
Ornithine, Lysine, and Arginine (COLA).
 Excess cystine in the urine can lead to recurrent
precipitation of hexagonal cystine stones A.
 Treatment:
1. Urinary alkalinization (eg, potassium citrate, acetazolamide).
2. Chelating agents (eg, penicillamine) ↑ solubility of cystine stones.
3. Good hydration.
 Diagnosis:
1. Urinary cyanide-nitroprusside test is diagnostic.

HEMOGLOBIN :
 Fetal hemoglobin (HbF)
 Production begins around 8 weeks gestation and replaces all embryonic
hemoglobin by 14 weeks gestation, when erythropoiesis in the fetal liver and
spleen is established.
 Production declines at birth, and HbF comprises ~60-80% of all hemoglobin in a
term newborn.
 HbF is gradually replaced by adult hemoglobin (HbA, α2β2) during the first 6
months of life, after which HbA composes the vast majority of adult hemoglobin.
 High oxygen affinity as HbF binds to 2,3-bisphosphoglycerate poorly.
 Patients with homozygotic β-thalassemia (β -thalassemia major) are asymptomatic
at birth due to the presence of γ-globins and HbF. Switching to HbA production
and the cessation of γ-globins synthesis precipitates the symptoms of β-
thalassemia.
 Hemoglobin A2 (α2δ2)
 Normal hemoglobin variant that makes up 2% - 3% of hemoglobin in a healthy
adult.
 Functionally similar to HbA.
 Patients with beta-thalassemia major have impaired beta globin production,
resulting in an excess of alpha globin chains (eg. HbA2, HbF) and no HbA.

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 Hemoglobin H (β4) and hemoglobin Barts (γ4)

 Presents in Alpha-thalassemia that results from a shortage of alpha globin chains.
 Have a very high oxygen affinity and cannot release oxygen, resulting in tissue
hypoxia.
 Hemoglobin H disease manifests as chronic hemolytic anemia.
 Hemoglobin Barts is incompatible with life (eg, hydrops fetalis) as normal fetal
and adult hemoglobin cannot be produced.

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 Hemoglobin S (HbS)

Substitution Glutamate (-ve)  valine (nonpolar) in the beta globin chain  ↓
the negative charge on the HbS molecule, which causes HbS to move more
slowly toward the anode.
 hemoglobin C (HbC)
 Substitution glutamate (-ve)  lysine (+ve) in the beta globin chain.
 Typically asymptomatic and often have mild hemolytic anemia and
splenomegaly.

 Both HbC and HbS result from missense mutations.

 Frameshift mutations involving the alpha globin genes can cause alpha thalassemia,
which results in the production of beta tetramers (hemoglobin H) that migrate further
than HbA during electrophoresis.

 In SCD, (glutamate  valine)  alteration of a region on the β-globin surface that

interacts with a complementary site on another Hb molecule  hydrophobic
interactions that occur cause aggregation of Hb molecules (under anoxic conditions)
and subsequent erythrocyte sickling (distortion and inflexibility), which is promoted by
low oxygen levels, increased acidity, or dehydration.
 In HbC, glu is replaced by a basic polar (positively charged) lysine. Because lys is
charged (although it has opposite polarity to glu), hydrophobic interactions between
Hb molecules do not occur.

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 Sickling in SCD:
o Pathogenesis:
 < Glutamate (hydrophilic)  valine (hydrophobic) >  alteration of a
hydrophobic portion of the β globin chain that fits into a complementary site on
the globin chain of another hemoglobin molecule.
 As a result, hemoglobin molecules aggregate under anoxic conditions.
 After polymerization, HbS initially forms a gel and then a meshwork of fibrous
polymers causing the red blood cells to distort into an abnormal sickle shape.
o Predisposing factors:
 Low oxygen levels.
 Increased acidity.
 Low blood volume (dehydration).
o Site:
 Organs in which blood moves slowly (eg, spleen, liver) are predisposed to lower
oxygen levels or acidity.
 Organs with particularly high metabolic demands (eg brain, muscles, and placenta)
promote sickling by extracting more oxygen from the blood (oxygen unloading).
o Clinically: (sickling  aggregation  vasoocclusive crisis)
 In lungs  acute chest syndrome.
 In spleen  abdominal pain.
 In bone  bone pain.
o NB: HbS does not polymerize when fetal hemoglobin (HbF) is present, so patients
with sickle cell anemia often do not have symptoms until the HbF fraction decreases a
few months after delivery. Some patients with HbS may have fewer clinical
manifestations because they produce larger amounts of HbF as adults.

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 Left shift 
↑hemoglobin O2
affinity means that
O2 is relatively less
available to tissues.
 Rt shift  ↓affinity …..

 UW: Anemia severe enough to cause lactic acidosis will result in lower blood pH,
shifting the hemoglobin curve to the right. Similarly, hypoventilation causes increased
CO2 retention and respiratory acidosis that shifts the curve to the right.
 UW: Strenuous exercise will cause increased tissue oxidative phosphorylation,
increased tissue CO2 levels, and decreased tissue PH. This results in a shift of the
dissociation curve to the right and decreased hemoglobin O2 affinity.
 UW: High-oxygen-affinity hemoglobins have reduced ability to release oxygen within
the peripheral tissues, leading to renal hypoxia, increased erythropoietin synthesis,
and compensatory erythrocytosis.

 In hemoglobin (4 heme groups) after binding to 1 oxygen molecule, the oxygen affinity
of other heme molecules increases; this heme-heme interaction is responsible for the
characteristic sigmoid shape of the oxygen-hemoglobin dissociation curve.
 Myoglobin has only a single heme group and so does not experience heme-heme
interactions; therefore, its oxygen-dissociation curve is hyperbolic.

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 Heme synthesis occurs partly in the mitochondria and partly in the cytoplasm of
erythrocytes. Mitochondria are necessary for the first and the final 3 steps.
 Maturing erythrocytes lose their ability to synthesize heme when they
lose their mitochondria, which are necessary for the first and final 3
steps of heme synthesis.
 Principal sites of heme synthesis:
1. Erythrocyte precursor cells (located in the bone marrow).
 Erythrocyte precursors divide a number of times before finally losing
their nuclei and mitochondria and forming mature red blood cells that
survive for about 120 days (4 months). When erythrocytes lose their
mitochondria, they lose the ability to generate heme and therefore
hemoglobin.
2. Hepatocytes (use heme in microsomal cytochrome P450 system).

ACUTE INTERMITTENT PORPHYRIA (AIP)

 Autosomal dominant, late onset.
 Porphobilinogen Deaminase
Factors precipitating acute attacks of
(Hydroxymethylbilane Synthase)
Deficiency. porphyrias:
 Episodic, variable expression. Cytochrome P450 inducers
 CNS: (Barbiturates, Anti epileptics, EtOH)  ↑
 Anxiety, confusion, paranoia. use of heme in microsomal cytochrome
 Paralysis (Motor, sensory or p450  ↓ hepatic concentration of
autonomic neuropathy) heme  ↑ ALA synthase.
 Weakness.
Enzyme deficiencies of the early steps in
 GIT:
 Acute abdominal pain, often porphyrin synthesis cause
resulting in multiple laparoscopies neuropsychiatric manifestations
(scars on abdomen). without photosensitivity, while late step
 No photosensitivity. derangements lead to photosensitivity.
 Port-wine urine in some patients.
 Never give barbiturates.
 Dx  ↑ 5-aminolevulinic acid and porphobilinogen during acute attacks.

PORPHYRIA CUTANEA TARDA

 Most common porphyria, Autosomal dominant, late onset.
 Photosensitivity, inflammation, blistering, shearing of skin
in areas exposed to sunlight.
 Hyperpigmentation.
 Exacerbated by alcohol.
 Red-brown to deep-red urine.

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VITAMIN B6 DEFICIENCY:
 ↓ ALA synthase  the iron isn’t incorporated in
heme synthesis  deposition around mitochondria
 ringed sideroblasts  sideroblastic anemia.
 Associated with isoniazid therapy for tuberculosis.
 Also lead to dermatitis, stomatitis, neuropathy, and
confusion.

LEAD POISONING
 ↓ ALA dehydrase and ferrochelatase (heme
synthase).
 Produce a microcytic sideroblastic anemia with ringed
sideroblasts in the bone marrow.
 Clinically:
 Young children living in old house.
 Via inhalation and ingestion of lead-
based paint, dust or chips due to normal
crawling and mouthing behaviors. The incomplete blood-brain-barrier
in children is vulnerable to the neurotoxic effects of lead, which
include long-standing behavioral problems and developmental delay
or regression.
 Coarse basophilic stippling of erythrocytes.
 Headache, nausea, memory loss.
 Abdominal pain, diarrhea (lead colic).
 Lead lines in gums.
 Lead deposits in abdomen and epiphyses of bone seen on radiograph.
 Neuropathy (claw hand, wrist-drop).
 Dx:
 Increased urinary ALA.
 Increased free erythrocyte protoporphyrin.

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 USMLE ENDPOINT SERIES BY DR AHMED SHEBL Biochemistry

HORMONES:

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 USMLE ENDPOINT SERIES BY DR AHMED SHEBL Biochemistry

G-PROTEIN-COUPLED RECEPTORS

 G-protein-coupled membrane-bound receptors contain three major domains:

 Extracellular (responsible for ligand binding).
 Transmembrane (consisting of several alpha-helical regions each composed
of approximately 20 hydrophobic amino acids).
 Intracellular (coupled with G-proteins).

Protein kinase A is primarily

responsible for the
intracellular effects of the G-
protein / adenylate cyclase
second messenger system.
Some hormone receptors
that use this mechanism
include the TSH, glucagon,
PTH, and beta-adrenergic
receptors.

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 USMLE ENDPOINT SERIES BY DR AHMED SHEBL Biochemistry

RAS-MAP KINASE PATHWAY:

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 USMLE ENDPOINT SERIES BY DR AHMED SHEBL Biochemistry

 The Ras-MAP kinase pathway starts with a growth factor ligand binding to the
receptor tyrosine kinase, causing auto-phosphorylation of the receptor.
 Phosphotyrosine produced in this reaction interacts with a number of proteins (such as
SH2-domain proteins and SOS protein), leading to Ras activation.
 Ras is a G-protein that exists in active and inactive forms. Inactive Ras contains GDP,
while the active form is bound to GTP. Activated Ras begins a phosphorylation
cascade starting with activation of Raf kinase.
 This cascade results in the activation of MAP (mitogen-activated protein) Kinase,
which enters the nucleus to influence gene transcription.
 The Ras protein exists in a balance between its active and inactive forms. Inactive
(GDP-containing) Ras is activated by a signal originating from the receptor tyrosine
kinase Active (GTP-containing). Ras is inactivated by GAP (GTPase-activating
protein), which induces the hydrolysis of GTP into GDP. Mutation of Ras can lead
to an inability to split GTP; the resultant permanently activated Ras stimulates cell
proliferation and can lead to cancer.

UW: 30 AA coiled in an alpha-helical structure with repeated leucine every

7th position  leucine zipper (transcription factor which is a DNA-binding
protein).

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 USMLE ENDPOINT SERIES BY DR AHMED SHEBL Biochemistry

 Unlike the other counterregulatory (ie, insulin-opposing ) hormone receptors, cortisol

receptors are located within the cytoplasm and translocate to the nucleus after
binding to their substrate In the nucleus, the cortisol-receptor complex binds to
hormone-responsive DNA elements, altering gene transcription to enhance hepatic
glucose production and limit peripheral glucose utilization.

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 USMLE ENDPOINT SERIES BY DR AHMED SHEBL Biochemistry

 The PI3K/Akt/mTOR pathway is an intracellular signaling pathway that is important

for cellular proliferation. This pathway is typically activated when a growth factor
binds to its receptor tyrosine kinase, causing auto-phosphorylation of specific tyrosine
residues within the receptor.
 These phosphotyrosine residues activate phosphoinositide 3-kinase (PI3K), which
then phosphorylates PIP2 found in the plasma membrane to PIP3.
 This leads to activation of a protein called Akt (or protein kinase B), a
serine/threonine-specific protein kinase.
 Subsequently, Akt activates mTOR (mammalian target of rapamycin), which
translocates to the nucleus to induce genes involved in cell survival, anti-apoptosis,
and angiogenesis. mTOR activation is inhibited by PTEN (phosphatase and tensin
homolog), a tumor suppressor protein that removes the phosphate group from PIP3.
 The PI3K/Akt/mTOR pathway is highly active in many cancer cells as a result of
mutations causing increased activity of PI3K or Akt or loss of function of PTEN.
 Mutations involving certain growth factor receptors (eg, epidermal growth factor) can
also enhance activity. Several drugs targeting this pathway (eg, mTOR inhibitors
including rapamycin [sirolimus]) have shown benefit in treating certain cancers.

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 presentation similar to Marfan syndrome with pectus deformity, tall stature, arm: height ratio, upper:
lower body segment ratio, arachnodactyly, joint hyperlaxity, skin hyperelasticity, scoliosis.

Premutation (50-200 repeats) Ž tremor, ataxia, 1° ovarian insufficiency. Full mutation (>200 repeats)
postpubertal macroorchidism (enlarged testes), long face with large jaw, large everted ears, autism, mitral
valve prolapse, hypermobile joints. Self mutilation is common and can be confused with Lesch-Nyhan
syndrome.

Polyunsaturated fatty acids that cannot be synthesized in the body and must be provided in the diet
(eg, nuts and seeds, plant oils, seafood). Linoleic acid (omega-6) is metabolized to arachidonic acid,
which serves as the precursor to leukotrienes and prostaglandins. Linolenic acid (omega-3) and its
metabolites have cardioprotective and antihyperlipidemic effects

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  Describe wobble hypothesis:

- The hypothesis state that the first 2 nucleotide positions on mRNA require traditional base pairing,
while the third “wobble” nucleotide position undergo less stringent (non-traditional) base pairing.

 X-linked adrenoleukodystrophy: defective transport of fatty acid, present in childhood with adrenal
insufficiency, testicular abnormalities.

 Discuss the radio-immunoassay technique (QID 12299)

- Lab technique that use specific antibodies and known quantity of radiolabeled antigen to determine
the amount of antigen present in unknown sample.
- Then specific antibodies against a known antigen are attached to assay plate, next; a fixed quantity
of radiolabeled antigen & varying amount of unlabeled antigen are added to the plate.
- The system then is washed to remove the unbound antigens and radioactivity is measured.

 Describe the term (genetic disequilibrium):

- Genetic disequilibrium means that 2 alleles on different loci inherited together in the same gamete
more or less often than expected, mostly due to physical proximity.
- Hardy-Weinberg principle (2pq) not applicable when alleles at different loci

 Clinical picture of propionic & methylmalonic academia:

1. AR deficiency of propionyl CoA carboxylase (need biotin) or thiophorase
2. Accumulation of propionic acid / methylmalonic acid → severe metabolic acidosis
3. ↑↑ overall metabolic rate → ↑↑ glucose utilization → toxic inhibiton of gluconeogenesis by organic acid
→ hypoglycemia
4. hypoglycemia → ↑↑ free fatty acid metabolism → ketone body formation
5. organic acids → ↓↓ urea cycle → hyperammonemia
6. Presented 1 – 2 weeks after birth by lethargy, vomiting, hypotonia. The difference between them is
presence of urine methylmalonic acid

 Mention metabolites that are transformed into propionic acid during their metabolism:
- Pyrimidines: thymidine, uracil.
- Essential amino acid: Valine, isoleucine (leucine → succinyl CoA), Methionine, Threonine.
- Lipids: cholesterol, odd chain fatty acid.

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