Medical Education Readiness Program

DeVry Medical International

2300 SW 145 Ave, Suite 200
Miramar, FL 33027-4150

Course: Medical Biochemistry

Instructor: Anamika Sengupta PhD, MEd
Ph. No: 754 210 8376
Email: asengupta@[Link]

Recommended Textbook: Mark’s Basic Medical biochemistry: A Clinical Approach (4th edition)
Editors: Michael Lieberman and Allan Marks
Lippincott Williams and Wilkins: Philadelphia, PA
IBSN: 978-1-60831-572-7

CONTENTS
Lecture # Lecture Title
1. DNA structure & replication
2. RNA Structure & transcription
3. Translation
4. Amino acids & protein structure
5. DNA technologies
6. Chromatin & chromosomes
7. Mendelian genetics
8. Mutation & DNA repair
9. Enzymology
10. Vitamins
11. Introduction to carbohydrates
12. Glycolysis
13. Tricarboxylic acid cycle
14. Electron transport chain
15. Glycogen metabolism & gluconeogenesis
16. Disorders of carbohydrate metabolism
17. Urea cycle & amino acid metabolism
18. Nucleotide biosynthesis and metabolism
19. Lipids & lipoproteins
20. Fatty acid biosynthesis and cholesterol metabolism
21. Carnitine shuttle & beta oxidation of fatty acids
22. Enzymes of diagnostic importance
23. Serum plasma proteins & their associated disorders
24. Hemostasis & thrombosis
25. Biochemical & molecular endocrinology
Lecture 1- DNA Structure and Replication
Learning Objectives:

• Know the composition & structure of DNA based on Watson-Crick

DNA model
• Focus on concepts of directionality, complementarity in DNA
structure
• Learn the process of DNA replication with focus on:
-Pre-replication complex
-Replication complex
-Factors involved in formation of each complex
-Replication fork
-Direction of replication of leading & lagging strand
• Know the enzymes involved in DNA replication & their functions
• Know the cyclins & CDKs that control cell cycle and thus DNA
replication
• Associate cell cycle with events of DNA replication

Nucleic Acids (DNA and RNA)

The structural unit of all nucleic acids is the nucleotide. Nucleotides are molecular compounds
composed of Carbon, Oxygen, Hydrogen, Nitrogen, and Phosphorus elements (atoms). These
ingredients are combined in a particular arrangement to yield 3 components which each nucleotide
possesses: nitrogen-containing base + pentose sugar + phosphate group

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There are 5 nitrogen-containing bases used in the construction of nucleotides: Adenine (A), Guanine
(G), Cytosine (C), Thymine (T), and Uracil (U). A sixth nitrogen base, hypoxanthine (I), does exist and is
used as a precursor to the formation of A and G. This sixth base is found in some types of RNA.

A, G and I are large 2-ring bases (Purines).

C, T, and U are smaller 1-ring bases (Pyrimidines).

There are 2 pentose sugars used in the construction of nucleotides: ribose (RNA) and deoxyribose
(DNA).

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The term “nucleotide” refers to 1 base covalently linked to 1 sugar (ribose or deoxyribose) which is
linked to a phosphate group. The term “nucleoside” refers to 1 base covalently linked to 1 sugar
(ribose or deoxyribose) without the phosphate group.

Base Nucleoside (sugar + base) Nucleotide (sugar + base

+phosphate)
Ribonucleoside Deoxyribonucleoside NMP NDP NTP
dNMP dNDP dNTP
Purine
Adenine adenosine deoxyadenosine AMP ADP ATP
dAMP dADP dATP
Guanine guanosine deoxyguanosine GMP GDP GTP
dGMP dGDP dGTP
Hypoxanthine inosine deoxyinosine IMP IDP ITP
dIMP dIDP dITP
Pyrimidines
Cytosine cytidine deoxycytidine CMP CDP CTP
dCMP dCDP dCTP
Thymine thymidine deoxythymidine TMP TDP TTP
dTMP dTDP dTTP
Uracil uridine deoxyuridine UMP UDP UTP
dUMP dUDP dUTP

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There are two chemical bonds within a nucleotide. The first bond is a glycosidic bond, a covalent bond
joining the sugar molecule with the base at the 1’ carbon position. The second bond is the ester bond,
another covalent bond, between the 5’ carbon and the oxygen from the P-group.

Nucleic acids are built by joining single nucleotides together, whereby individual nucleotides are linked
together through a phosphodiester bond. A phosphodiester bond is a covalent bond where a shared
phosphate residue connects two nucleotides at the 3’ and 5’ positions. For this bond to be formed,
energy is required. This energy is derived from cleaving the terminal two P-groups from each
nucleotide before addition to the growing nucleic acid strand.

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Under normal physiological circumstances, all DNA is double-stranded. And the reason for DNA being
double stranded is the ability of nitrogen-bases on opposite nucleic acid strands to base pair with one
another.

There are two major forces involved in base pairing: hydrogen bonding and base stacking. Hydrogen
bonds are non-covalent interactions between a hydrogen atom from one nucleotide on one nucleic
acid strand and an electronegative atom (such as nitrogen or oxygen) from another nucleotide on the
opposite nucleic acid strand.

All purines form hydrogen bonds with pyrimidines. More specifically, Adenine only base pairs with
Thymine, and Guanine only base pairs with Cytosine (this is for DNA; remember, RNA does not have
Thymine; instead it has Uracil and the base pair is Adenine with Uracil). This pairing is called “Watson-
Crick base pairing” after the discoverers of DNA structure. Non Watson-Crick base pair can occur and
will be discussed in another chapter.

Base stacking refers to hydrophobic interactions between adjacent bases in a single nucleic acid strand.

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In order to maintain the structural integrity of double stranded (ds)-DNA, the 2 complementary nucleic
acid strands need to be situated in an anti-parallel manner, meaning the 5’-end of one strand is bound
to the 3’-end of the other strand and vice-versa.

DNA Structure
DNA is composed of 2 strands wound around each other in a right-handed helical structure with the
base pairs in the middle and the sugar moieties and phosphate groups on the outside.

The sugar-phosphate backbones of the two strands are slightly offset from the center of the helix,
resulting in grooves between the two strands of different sizes. These grooves are termed the Major
Groove and the Minor Groove.

The Major groove is larger, more open, and partly exposes the hydrophobic base pairs. The minor
groove is less open, more constricted, and the hydrophobic base pairs are partially blocked by the
sugar-phosphate backbone of the DNA molecule. Interestingly, most protein-DNA interactions occur in
the major groove since the base pairs are more exposed to the external environment.

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There are actually 3 major forms or structural arrangements of DNA—known as the A-form, B-form,
and Z-form. The B-form is the most physiologically relevant, as this is the primary form found in all
cells. The A-form is a shorter, more tightly-wound form that only exists in situations where water is
scarce. And finally, the Z-form is attained at high ionic concentrations.

There are over 3 billion base pairs in the entire human genome, with extremely limited storage space
inside the cell. Not only inside the cell, but inside the nucleus of the cell, where nearly all DNA is stored
(a small amount of DNA is stored in the mitochondria of the cell). As such, DNA is packed into varying
orders of structural arrangement through protein interactions, ultimately resulting in the compact
structure known as the chromosome.

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The Cell Cycle
There are 4 basic stages to the cell cycle. Each stages has specific functions:

G 1 : During this phase, the biosynthetic activities of the cell are at a high rate. This phase is marked by
synthesis of millions of proteins that are required in S phase, mainly those needed for DNA replication.

S-phase: This phase commences when DNA synthesis begins and ends when the DNA in the cell has
effectively doubled.

G 2 : During this phase, the biosynthetic activities of the cell are again high, marked by synthesis of
millions of proteins that are required for mitosis.

Mitosis: Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus
into two identical sets in two nuclei. It is generally followed immediately by cytokinesis, which divides
the nuclei, cytoplasm, organelles, and cell membrane into two cells containing roughly equal shares of
these cellular components.

The cell cycle is a tightly-regulated process, controlled in large part by the interaction between cyclins
and cyclin-dependent kinases. The levels of cyclins fluctuate greatly depending on the phase of the cell
cycle. The graphs below list the phases of the cell cycle in which certain cylcins and CDKs can be found.

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9
DNA replication
The significant events of DNA replication are as follows:

• Origin of replication complex (ORC) binds at the origin of replication (ori). The result is
recruitment of other proteins including the MCM complex.

• DNA unwinds from histones and the MCM complex hydrolyzes H-bonds to create regions of
ssDNA. This site of separation is called the Replication Bubble.

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• Topoisomerase relaxes the DNA while single-stranded DNA binding proteins coat ssDNA.

• Primase forms RNA primers to begin DNA synthesis.

• DNA polymerase α begins synthesizing anew DNA strand. DNA polymerases δ and ε take over
and synthesize rest of DNA strand (aided by Proliferating Cell Nuclear Antigen). PCNA
increases the processivity or how long the polymerase synthesizes DNA. The DNA
Polymerases can only add nucleotides to the 3’OH group of an existing primer

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• The Leading strand is synthesized continuously in 5’ to 3’ direction towards the replication
fork.

• The lagging strand is synthesized in small fragments called Okazaki fragments in 5’ to 3’

direction but away from the fork. Multiple RNA primers are involved in the synthesis of the
lagging strand.

• RNA primers are removed by Flap endonuclease 1 and RNAse H. The

resulting gaps are filled in by DNA polymerases δ. DNA ligase then seals the DNA fragments

• Geminin binds to Cdt1 of newly synthesized DNA and prevents re-synthesis by blocking the
MCM

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 • Histones bind to new DNA packaging or condensing them to form chromosomes

During DNA replication, DNA polymerase will always move along and read the parental template
strands in the 3’-5’ direction while adding new nucleotides to the daughter strands in the 5’-3’
direction. Due to the anti-parallel nature of dsDNA, DNA polymerase must work in two different
directions during DNA replication, known as bi-directional synthesis.

Another feature of DNA synthesis is that replication is semi-conservative, meaning that replication
produces two copies of DNA, where each copy contains one of the original/parental strands and one
new daughter strand. Because DNA replication is bi-directional and because new nucleotides can only
be added to the growing daughter strands in the 5’-3’ direction, the DNA synthesis of these two
daughter strands is different. The two daughter strands are known as the Leading strand and Lagging
strand.

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DNA synthesis proceeds along the leading strand in a 5’-3’
direction, producing a continuous strand. Lagging strand
synthesis runs in the opposite direction of the leading
strand, and as such, is made in short fragments (100-500
base pairs) known as Okazaki fragments. Eventually, the
Okazaki fragments are joined together by DNA ligase.
Finally, DNA replication is considered a process of high
fidelity, where fidelity refers to how accurately DNA
polymerases can copy the DNA. DNA polymerases δ and
ε maintain high fidelity during DNA replication due to an
enzymatic activity known as 3’-5’ exonuclease
proofreading.

This activity allows DNA polymerase δ and ε to remove

incorrectly-placed, mismatched nucleotides that were inadvertently incorporated into the daughter
strands during DNA replication. Thus, this activity preserves the integrity of the DNA replication
process.

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Lecture 2- RNA Structure and Transcription
Learning Objectives:

• Identify the major types of cellular RNAs

• Know the structural and functional characteristics of different type of
RNAs
• Emphasize on the roles of different regulatory elements present in a
gene that regulates of transcription of a gene
• Explain the role of different types of RNA polymerase enzymes
• Know the different steps involved in transcription of a DNA into
mRNA
• Describe different post transcriptional events that helps in maturation
of an mRNA

DNA vs. RNA

Transcription is defined as the synthesis of a ribonucleic acid (RNA) molecule using deoxyribonucleic
acid (DNA) as a template. The genetic information stored in double-stranded DNA is transferred into a
single-stranded RNA molecule.

While both are nucleic acids, there are major differences between DNA and RNA. (1) Even though both
DNA and RNA are synthesized in the nucleus, RNA primarily resides in the cytoplasm of the cell and
DNA is retained in the nucleus. (2) DNA uses the sugar, deoxyribose, while RNA uses the sugar, ribose.
(3) DNA uses thymine in its T-A base pairing while RNA uses uracil in its U-A base pairing. (4) DNA is a
double-stranded molecule while RNA is primarily single-stranded.

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Types of RNA
There are five general classes of RNA molecules found in eukaryotic cells: ribosomal RNA (rRNA),
messenger RNA (mRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), and micro RNA (microRNA).

Ribosomal RNAs: These RNAs, together with proteins, form the ribosome which is needed for
protein synthesis.

4 eukaryotic rRNA molecules are transcribed to make the ribosome. Three are initially synthesized as a
single, large RNA transcript (polycistronic) which is then processed to form 28S, 18S, and 5.8S rRNAs.
The fourth RNA (5S) is transcribed by itself. Of these RNA pieces, the 28S, 5.8S, and 5S rRNAs associate
with ribosomal proteins to form the large ribosomal subunit. The 18S rRNA associates with different
ribosomal proteins to form the small ribosomal subunit. The large ribosomal subunit is termed the 60S
subunit whereas the small ribosomal subunit is termed the 40S subunit. These two subunits interact
form a functional 80S ribosome

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Messenger RNAs: These RNAs carry the genetic information from the nucleus to the ribosome for
protein synthesis and have been coined the “working copy of the genome.”

Transfer RNAs: These RNAs function as amino acid carriers and as recognition molecules. tRNAs
identify the mRNA nucleotide sequence and links that sequence to the amino acid sequence of
proteins. All tRNAs have 4 distinct loops: the D-loop, the anticodon loop, the variable loop, and the
TψC loop.

D-loop: the D-loop contains several modified

bases (e.g., methylated cytosine and
dihydrouridine) and acts as a recognition site for
aminoacyl-tRNA synthetases.

Anticodon loop: the anticodon loop serves to

recognize the complementary codon of an
mRNA molecule. Base pairing occurs between
these two complementary trinucleotide
sequences (codon base pairs with anticodon).

Variable loop: the variable loop provides structural stability to the tRNA molecule and is variable both
in nucleotide composition and in length.

TψC loop: the TψC loop contains the modified base pseudouridine and facilitates association with
aminoacyl-tRNA synthetases.

Finally, there is an acceptor stem for each tRNA molecule which serves as the linkage site for amino
acids. This acceptor stem is formed by base pairing between nucleotides at each end of the tRNA. The

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last three bases found at the extreme 3' end are unpaired and always have the same sequence: 5'-CCA-
3'. The amino acid is attached via an ester bond at the 3' end of the acceptor stem.

Small nuclear RNAs: These RNA’s combine with proteins to form an RNA-protein complex known as
the spliceosome. The spliceosome is located in the nucleus and is involved in the splicing (removing)
and ligating of introns and exons, respectively, from pre-mRNA molecules.

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Micro RNAs: These RNA’s serve as important regulators of gene expression. One mechanism utilized
is that microRNA’s interact with a multiprotein structure known as RISC (stands for RNA-induced
silencing complex). This RISC-microRNA complex then searches the cell looking for complementary
mRNA. Once bound to a target mRNA, the microRNA in the RISC activates a RNA-degrading function of
the RISC (known as RNAse activity) which then subsequently chews up the mRNA target.

Transcription
Requirements of transcription

• RNA polymerase
RNA polymerases are large multimeric enzymes that transcribe defined segments of DNA into RNA
with a high degree of selectivity and specificity. Eukaryotic cells possess three different RNA
polymerases termed RNA polymerase I, II, III. Each of these polymerases specializes in transcription of
different classes of RNA:

• RNA Pol I: transcribes rRNA

• RNA Pol II: transcribes mRNA (protein-coding genes), snRNA, microRNA

• RNA Pol III: transcribes tRNA

• Transcription factors

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Integral to the process of transcription are proteins known as transcription factors (TFs). These
proteins work by recognizing and binding certain nucleotide sequence motifs (regulatory DNA
sequences also called “response elements” that accept TFs are) through functional regions called
domains. TF domains bind response elements and attract other TFs, creating a complex that
eventually facilitates binding of RNA pol, thus beginning the process of transcription.

• Regulatory DNA sequences to which transcription factors & RNA polymerase

bind to
a. Promoters: Promoters are sequence-specific sites, usually located upstream of the gene that
initiates and regulate, gene transcription. To initiate transcription, transcription factors which are
specific proteins involved in initiation of transcription first bind to the promoter region of the gene. It
forms a transcription complex which the recruits the specific RNA polymerase for initiation of
transcription.

The promoters in eukaryotes have a core promoter region composed of consensus sequences known
as the TATA box and CAAT box.

The TATA box consists of a conserved sequence (5'-TATAAA-3‘) and is usually located about 30 bps
upstream of the gene of interest. It is a part of the core promoter with conserved sequences called
sequence motifs. The binding site for RNA polymerase II is found in the TATA box.

The CAAT box consists of a conserved sequence motif (5’-GGCCAATCT-3’) and is located about 70-75
bps upstream of the gene of interest.

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 b. Enhancers: There are other sequence-specific region in the DNA that also affect the level of
transcription. One region is known as the enhancer. Enhancers, like promoters, contain sequence
motifs that bind special transcription factors. Once bound these TFs increase or “enhance” the level of
transcription.

c. Silencers are yet another sequence-specific region in the DNA that affects the level of transcription.
These regions bind protein factors known as repressor proteins because they work to decrease or
repress the level of transcription for a particular gene.

Process of Transcription
Once bound, RNA polymerase itself will unwind the dsDNA and create a transcription bubble.

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RNA polymerase will then begin creating the RNA product using only ONE DNA strand as the template.
Just like DNA replication, nucleotides are added in succession to create the daughter RNA strand
through phosphodiester bond formation. The parental DNA template strand is also called the non-
coding strand while the unused non-template strand is also called the coding strand (the non-
template strand is also known as the coding strand because it has the same base sequence as the
newly-synthesized RNA transcript, although the RNA molecule has uracil instead of thymine).

As the RNA polymerase moves along the DNA template, unwinding the dsDNA as it goes along and
then zipping it up again behind it, it moves the transcription bubble along the chain, adding new
nucleotides continuously because it always proceeds in the 5’-3’ direction. This process of enzyme
movement along the template strand is known as translocation.

5’

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Transcription is completed when the RNA polymerase reaches a region of DNA known as the
terminator sequence. Once reached, this sequence of bases signals the enzyme to stop adding
nucleotides to the RNA molecule and detach from the DNA. Once the enzyme is detached, the RNA
molecule is then released as well.

Post-transcriptional Processing
In order to ensure stability and transfer of newly-synthesized mRNA’s to the cytoplasm, eukaryotic
cells have evolved post-transcriptional mechanisms to protect and assist the mRNA after it has been
created.

(1) The first post-transcriptional processing event that occurs in eukaryotic cells is the capping of the
5’-end of newly-synthesized mRNA molecules. The 5' end of all eukaryotic mRNAs are capped with a
unique 5'—>5' linkage to a 7-methylguanosine residue. The 5’-CAP provides protection from cellular
nucleases while also serving as a docking site for the translational machinery.

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(2) The second post-transcriptional processing event that occurs in eukaryotic cells is the addition of a
poly(A) tail to the 3’-end of newly-synthesized mRNA molecules. The poly(A) tail consists of multiple
adenosine residues added to the 3’-end of the mRNA molecules (~ 250 residues in total). The
adenosine residues are not encoded by the DNA but instead added by the action of a polymerase
known as poly(A) polymerase using ATP as the substrate. Polyadenylation helps protect mRNAs from
degradation by cellular nucleases and also is important for proper export from the nucleus to the
cytoplasm (export is facilitated by poly(A)-binding proteins).

3) The third post-transcriptional processing event that occurs in eukaryotic cells is known as splicing, a
process which removes introns, the intervening sequences or regions of RNA that do not code for
protein. Splicing removes these introns from the pre-mRNA and then ligates the remaining exons,( the
expressed sequences) together to form a continuous RNA molecule that can now be exported from the
nucleus and code for a functional protein.

The splicing process actually involves a large complex of cellular proteins and snRNA’s (snRNA’s: U1,
U2, U4, U5, U6), which interact to form the spliceosome. It is this multimeric RNA-protein complex that
will ligate the exons together while excising the introns from the pre-mRNA molecule.

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Alternate Splicing

The splicing process is not always precise, meaning that the spliceosome can re-connect/recombine
exons in multiple ways (but sequentially) during RNA splicing. This is known as alternative splicing.
Through alternative splicing, multiple mature mRNA’s can be produced which can be translated into
different protein isoforms. Thus, a single gene can account for multiple proteins, thereby increasing
the biodiversity of proteins within the genome.

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Lecture 3 -Translation
Learning Objectives:
• Learn the structure & role of the different components of eukaryotic
translation apparatus namely ribosomes, tRNA and mRNA
• Differentiate between the role of tRNA, mRNA and rRNA in protein
synthesis in eukaryotes
• Focus on understanding wobble hypothesis & degeneracy of the genetic
code and how they impact translation
• Learn the role of aminocyl tRNA synthetase enzyme in activation of amino
acids (AA) , aminoacylation of tRNA & proofreading
• Focus on the understanding of
 Initiation: The process and the role of different initiation
factors
 Elongation: The steps, enzymes involved and role of
elongation factors
 Termination of translation
• Calculate the ATP/GTP usage in the process of translation
• Focus on the clinical correlates related to abnormal protein folding
(amyloids, prions)

What is translation?
The process of decoding an mRNA into a polypeptide chain by the ribosome followed by
modification of the polypeptide into a functional protein.

Translation apparatus of a cell consist of 3 components

1. Ribosomes
2. tRNAs (transfer RNAs)
3. mRNA (messenger RNAs)

1. Ribosomes: Large complex cell organelles that serve as sites for translation
Structural components of a ribosome

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 • Small ribosomal subunit (40S; S=Svedberg unit): Reads the mRNA from which a protein is
synthesized; monitors complementarities between codons & anticodons.

• Large ribosomal subunit (60S): Joins amino acids (AA) to form a polypeptide chain;
facilitates peptide bond formation between AA.
The large subunit also has 3 RNA binding sites
a. A-site or Aminoacyl site: Binds to aminoacyl tRNA or a tRNA bound to an amino acid
b. P-site or peptidyl site: Binds to peptidyl tRNA or tRNA containing the growing
polypeptide chain
c. E-site or the exit site: Binds to free or uncharged tRNA (tRNA without an amino acid)
before it leaves the ribosome.

• rRNA & proteins: Ribozymes (ribonucleic acid enzymes) or catalytic RNAs are
present in the 60S; they catalyze peptide bond formation

2. tRNA: Small single stranded cytoplasmic RNA (73-93 nbp) with a folded 3-D
structure that serves as an adapter in translating the language of nucleic acids
(mRNA) into the language of proteins.

Structural features of a tRNA (Cloverleaf model)

• Amino acid arm (3’ end): Also known as the accepter stem, an amino acid is attached to
an adenosine at the extreme 3’ end by an ester linkage. Most tRNA molecules recognize
a specific AA; some amino acids are recognized by more than one tRNAs.

• 5’ end of tRNA: Contains poly guanylate (pG) residue.

• The anticodon arm: Contains the anticodon that binds to the specific codon coding for
the AA attached to the 3’end of the tRNA.

• D arm: Contains 2-3 residues of an unusual base dihydrouridine (D).

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 • TΨC arm: Contains ribothymidine (T) & pseudouridine (Ψ) which are unusual bases as
well. Note that D & TΨC arms enable folding of the tRNA molecule and are used for
orientation of the tRNA in aminoacyl tRNA synthetases.

3. mRNA: Provides the genetic code that is decoded into amino acid sequence
of specific proteins by the process of translation.

• mRNA is composed of numerous codons; each codon specifies a specific AA. The coding
sequence of an mRNA is linear, non-overlapping. AUG (coding for methionine) initiates
translation and UAA, UAG & UGA are termination codons that signal termination of
protein synthesis.
• mRNA has a 5’methyl cap & a 3’ poly A tail. The cap helps the mRNA to bind to the small
subunit of the ribosome. Reading of the mRNA during translation occurs in the 5’ to 3’
direction

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Genetic code is almost universal & degenerate
Universality of the genetic code:
The genetic code across all organisms (prokaryotes and eukaryotes) is the same (with a small
exception of the mitochondrial genetic code).That means the prokaryotic and eukaryotic amino
acids are coded by the same codons.

What is degeneracy of the genetic code?

• There are only 20 amino acids in the cell.

• The mRNA molecule codes for the sequence of amino acids in a particular protein. The
coding system is composed of triplet codons. As there are 4 bases (A, U, C, G), there can
be 64 possible codon combinations (43 = 64). So the number of codons exceeds the
number of amino acids present in a cell.
• Each amino acid can be coded by more than one codon (multiple codons) in a cell. This
makes the genetic code degenerate

Fig: Degeneracy of the genetic code

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Degeneracy to WOBBLE Hypothesis
• There are at least 20 different tRNAs in a cell, each of which recognizes and binds to a
specific amino acid (20 AA are present) and transfers them from the cytoplasm to the
ribosome during translation.
• As the genetic code is degenerate, some amino acids are coded by multiple codons (refer
to the table above).
• So each tRNA has to be able to recognize and bind to more than one codon (coding for
the particular amino acid) on the mRNA during translation.
• How is this possible?? It is explained by the Wobble hypothesis

According to the Wobble hypothesis

• During codon-anticodon pairing, the first 2 bases at the 5’ end of a particular codon (in an
mRNA) form strong specific Watson-Crick base pair with their complementary bases at
the 3’end of the anticodon (of an aminoacyl-tRNA).
• The pairing of the 3rd base of the codon “wobbles”. It pairs loosely & nonspecifically with
the 1st base at the 5’ end of the anticodon. This enables a tRNA to bind to multiple codons
differing in the 3rd base. Note (refer to the table) that multiple codons coding for a
particular AA differs in the 3rd base of their codon. This is called the wobble hypothesis
• C in the 5’ anticodon position: Recognizes
specific codons with G only
• U in the 5’ anticodon position: Binding is less
specific; U can bind to A or G
• G in the 5’ anticodon position: Binding is less
specific; G can bind to U or C
• I in the 5’ anticodon position: I can recognize 3
different bases A, U, C
• A is never found in the 5’ anticodon position
• So I, U, G are called wobble bases. The presence
of these bases at the 5’ end of an anticodon of a
tRNA enables the tRNA bind to multiple mRNA
codons differing in their 3rd base.

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Translation Takes Place in 5 Stages

Stage I - Activation of amino acids

Stage II - Initiation
Stage III - Elongation
Stage IV – Termination & ribosome recycling
Stage V – Folding and post translational modifications

Stage I

• Activation of amino acids occur when they get attached

to their specific tRNAs in the cytosol. The enzyme
aminoacyl-tRNA synthetase plays a lead role in this
process.
• Each AA has a specific aminoacyl synthetase enzyme that
can attach the AA to its specific tRNA thus activating it
• Attachment involves formation of an ester linkage
between the COOH group of the AA & the OH group on
the adenine (A) residue of the CCA sequence at the 3’
terminal end of its specific tRNA
• The reaction occurs in 2 steps and requires 2 high energy
phosphate bonds (ATPs). One ATP is used in the process
directly and the second ATP is used to regenerate ADP
from the AMP produced

Amino Acid +tRNA+2ATP Aminoacyl-tRNA +AMP+2Pi

The aminoacyl tRNA synthetase has 2 functions

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 • It activates AA by attaching it to its specific tRNA and this prepares it for incorporation
into a protein

• It has a proofreading function which ensures attachment of the correct AA to its specific
tRNA and thus placement of the correct amino acid at the specific position of a growing
polypeptide chain.

2 Step Process

1. Amino acid is activated. The aminoacyl tRNA synthetase attaches AMP to the carboxylic acid
of the amino acid. The AMP is derived from an ATP molecule
2. Amino acid is attached to the tRNA. The energy associated with the presence of the AMP
moiety is used to form a covalent bond between the carboxylic acid of the amino acid and the
3/ adenosine of the tRNA.

Stage II – Initiation (involves several steps & initiation factors)

1. Ribosome dissociates into the 2 subunits; eukaryotic initiation factor 3 (eIF3) associates
with the 40S subunit

2. Formation of Ternary complex : eIF2 and GTP binds to the tRNA charged with the
initiating AA, methionine (methionyl-tRNAMet)

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3. Formation of 43S pre-initiation complex: The ternary complex binds to 40S + eIF3; eIF5,
IF1 & eIF1a, join together to form 43S pre-initiation complex

4. Activation of mRNA: eIF4F complex (eIF4E, eIF4G, eIF4A) also called the cap binding
complex activates the mRNA; eIF4A is a helicase and removes secondary structures from
the 5’ end of mRNA getting it ready for translation. Formation and binding of the cap
binding complex to the mRNA requires hydrolysis of 1 ATP

5. 43S-preinitiation complex bind to the 5’ end of the mRNA

6. The preinitiation complex is released and scans the mRNA for an AUG codon which is the
initiation codon coding for methionine attached to the tRNA in the 43S preinitiation
complex.

7. The initiation factors are released. As the initiating AUG (AUG within the Kozak sequence)
in the mRNA is spotted, the anticodon of methionyl-tRNAMet binds to the AUG followed by
hydrolysis of eIF2 bound GTP (facilitated by eIF5); all other eIFs are released simultaneously.

8. The 60S binds to the 40S subunit placing AUG bound to methionyl-tRNAMet at the P site of
the 60S subunit.

The A site of the ribosome is occupied by the next codon in the mRNA which specifies the
next AA in the protein being translated.

Translation of the mRNA into a protein continues with methionine (AUG) as the first amino
acid

33
Regulation of initiation
Insulin stimulates protein synthesis in cells; it activates eIF4E of the cap binding complex and
thus activates mRNA for protein synthesis

Viral infections, starvation and heat shock phosphorylates eIF2 deactivating it. This prevents
formation of the ternary complex inhibiting protein synthesis.

Stage III- ELONGATION

Involves 3 steps
1. Binding of the next aminoacyl-tRNA to the A site

a. An aminoacyl-tRNA (charged with the 2nd AA in the protein) binds to an elongation factor
(eEF1A) which is bound to GTP. (Think elongation ternary complex)

b. This complex binds to the A site; hydrolysis of the GTP into GDP& Pi occurs. The GTPase
activity of eEF1A causes the eEF1A-GDP complex to dissociate from the mRNA-ribosome
complex.

2. Formation of a peptide bond

a. Round 1: Formation of a peptide bond between the COOH of the P site amino acid & the NH 2
of the A site amino acid occurs and is catalyzed by peptidyl transferase enzyme (a rRNA with
enzymatic activity) present in the 60S subunit.

b. The tRNA in the A-site now has the growing polypeptide chain & the P-site has the uncharged
tRNA.

34
3. Translocation-Movement of the mRNA along the
ribosome; an energy requiring step

a. The next elongation factor (eFF2 bound to GTP) binds to

the mRNA-ribosome complex and hydrolyzes GTP inducing
a conformational change in the ribosome.

b. This moves the mRNA and its attached tRNAs with

respect to the ribosome.

c. The uncharged (empty) tRNA moves from P to E-site.

d. The tRNA with the growing peptide moves from A to P

site.

e. Uncharged tRNA is released from the E site of the

ribosome as the next aminoacyl-tRNA occupies the A site.

Stage IV - TERMINATION

Elongation continues until a stop codon (UAA, UAG, UGA) enters the A site of the 60S ribosome.
As tRNAs specific for stop codons are unavailable in the cytoplasm, translation stops.

1. Eukaryotic release factors eRF1 & eRF3 form a complex & bind to the ribosome at the A site;
eRF3 has GTPase activity. (These release factors structurally resemble tRNAs)

2. Peptidyl transferase enzyme hydrolyzes the bond between the peptide chain and the tRNA at
the P site releasing the peptide chain; requires hydrolysis of a GTP molecule by eRF3

3. Ribosome dissociates into its subunits

35
Protein synthesis is an energy demanding process

Expenditure of ATPs/GTPs per AA during translation

4 High energy phosphate bonds (ATPs/GTPs) are required for incorporation of each AA in the
protein chain during the process of translation.

For the initiating Methionine (AUG)

2ATPs-Activation step
1GTP-Initiation step (binding of methionine to the ribosomal P site)
1ATP- Initiation step (spent in formation of the cap binding complex)
For any amino acid during elongation (2nd to the last AA, including a non-initiating
methionine present in other positions of the polypeptide )
2 ATP- Activation step
1 GTP-1st step of elongation (for attachment of the AA onto the ribosomal A site)
1 GTP - 3rd step of elongation i.e translocation

Note that the GTP hydrolyzed during the termination process is for releasing the entire
polypeptide chain from the ribosome). So it will not be counted in the ATPs/GTPs counts per AA.

Polyribosomes/Polysomes

Multiple ribosomes can read a particular mRNA and synthesize multiple copies of the same
protein simultaneously. This is called polysomes. As one ribosome moves along a mRNA
translating it, a 2nd ribosome can bind to the 5” end of the mRNA and start translation and as it
moves along the mRNA it makes space for a 3rd ribosome to get attached to the 5’ end of the
mRNA and so on………...

36
 Fig: Formation of polyribosome

Stage V: Folding & post translational modification

Chaperons or heat shock protein fold and refold the nascent (immature) polypeptide into its 3-
dimensional conformation
• Disulphide isomerases introduce disulphide bonds which stabilize the folded structure
of the proteins
• Post translational modification involves addition of various functional groups such as
methylation, glycosylation, hydroxylation, iodination etc which imparts functionality to
the proteins in the cell.

Targeting Proteins to Subcellular/Extracellular Locations

• After post translational modifications, some proteins may remain in the cell’s cytoplasm
to carry out their functions
• Some proteins may enter the mitochondria or nucleus (guided by a signal or target
sequence)
• Secretory proteins: Synthesized in the ribosomes attached to the RER (rough
endoplasmic reticulum).They are processed in the E lumen, golgi & lysosomes; released
into the cell cytoplasm and packaged into secretory vesicles. In response to a particular
stimulation they are released from the cell cytoplasm into the external environment
(blood, tissue fluid) by exocytosis.

37
CLINICAL CORRELATES

A. Ribosomopathies:

Congenital human disorders resulting from defects in ribosomal proteins/rRNA genes/other

genes involved in ribosome biogenesis.

Ribosomes are prominent drug targets of many antibacterial drugs: Antibacterial drugs often act
by interfering with the translation of bacterial proteins at different stages of the elongation cycle
thus inhibiting bacterial replication and killing off the bacteria.

B. Diseases related to misfolded proteins

• Amyloidosis: Misfolded proteins generally get degraded immediately. In some cases
misfolded protein forms insoluble aggregates called amyloids which accumulate in the
cells of different organs or extracellular space and are associated with pathology of a
group of diseases called amyloidosis

• Alzheimer’s disease is an example of localized amyloidosis caused by deposits of beta

amyloid protein deposits (amyloid plaques) in the inter-neuronal spaces of the brain
which interfere with inter neuronal communications.

C. Creutzfeldt-Jakob Disease (CJD)

• Rare, degenerative, invariably fatal brain disorder. It is a form of spongiform
encephalopathy; 300 cases/year is diagnosed with CJD in the US
• Appears in later life (60+); aggressive, death occurs within a year of diagnosis.
• May be sporadic, hereditary, or acquired
• Caused by misfolded prion proteins

38
• The normal prion protein is found throughout the body and brain. While not essential to
life, it does seem to play a role in helping neurons communicate and transport minerals.
Its amino acid chains fold mainly into a helical shape. They are harmless when structurally
normal (PrPc).

• When misfolded (PrPsc), they become infectious. Misfolded PrPsc is insoluble, resistant to
protease mediated destruction and induces transformation of other normal PrPc present
in the cell into misfolded PrPsc. Thus presence of one PrPsc induces formation of large
number of PrPsc in a chain reaction at the cellular level.
• PrPsc builds up in the brain and causes the infected brain cells to die. When the infected
cells die, prions are released into normal tissue and go on to infect more cells without any
reaction from the immune system. Eventually, large clusters of cells die leading to the
mental and behavioral symptoms of prion diseases CJD
• Transformation of PrPc to PrPsc can be sporadic, genetic (due to a dominant mutation in
the PrPc gene) or transmitted (from eating uncooked beef obtained from a cow with
bovine spongiform encephalopathy or mad cow disease).

Fig: Misfolded prions causing CJD

39
Lecture 4 - Amino Acids and Protein Structure
Learning Objectives:

• Emphasize on structural details, classification, functional

significance & pathologies associated with different classes of
Amino Acids (AA)
• Learn the role of AA as buffers; differentiate between pH, pKa &
pI
• Learn the effect of pH on the ionizable groups of different AA
• Differentiate between the types of secondary, tertiary &
quaternary structures of proteins
• Emphasize on pathologies associated with protein misfolding

Properties of Amino Acids and Proteins

Proteins are polymers of amino acids produced by living cells. A variety of proteins exist with diverse
functions, sizes, shapes, and structures. There are only twenty different amino acids composing
different proteins in different combinations. The sequence of amino acids, which ultimately
determines the characteristics of the protein, is determined by the genetic information contained in
the DNA residing in the nucleus of cells.

Each amino acid is composed of 4 distinct groups: a basic amino

group; an acidic carboxyl group, a hydrogen atom; and a
variable side chain known as the R-group. The central carbon is
known as the α-carbon, a carbon to which the 4 different
groups are attached.

40
Because the α-carbon of nearly all amino acids is considered an asymmetrical carbon atom (meaning it
is attached to 4 different groups of atoms), amino acids can form stereoisomers, which are compounds
that have the same molecular formula but the atoms are arranged differently in 3-dimensional space.

One class of stereoisomers is known as enantiomers, which are compounds that have the same
molecular formula and are mirror images of each other—they can’t be superimposed atop one
another. The amino acids are considered enantiomers because they are mirror-images of each other
yet can’t be laid directly atop the other.

One measurable property that distinguishes enantiomers from one another is that they are optically
active, meaning they can rotate polarized light. This light rotation can be measured using a device
known as a polarimeter, in which plane-polarized light is passed through a tube containing the
enantiomer (amino acid) and a device at the other end measures how the plane-polarized light is
rotated after passing through the enantiomer.

The amino acid enantiomers can thus be classified based on their optical properties, how the amino
acid enantiomers rotate plane polarized light. If the enantiomer rotates the light to the right, then the
amino acid is considered a D-amino acid (Dextrorotatory). If the enantiomer rotates light to the left,
then the amino acid is considered a L-amino acid
(Levorotatory).

41
AQll of the amino acids form stereoisomers or enantiomers except one—glycine. Glycine does not
because it does not have an asymmetrical α-carbon atom.

Each amino acid has its own specific properties based on its
unique side chain. Based on these unique side groups and
the properties they confer to each amino acid, the amino
acids can be grouped or classified into various categories:

42
Aliphatic amino acids
Aliphatic amino acids are composed of hydrocarbon chains. These hydrocarbon chains give these
amino acids a hydrophobic characteristic and thus they are often found in the interior of proteins or in
the portion of a protein that is in a cellular membrane. Glycine is sometimes grouped into this category
despite its lack of a hydrocarbon chain.

43
Aromatic amino acids

One important property of aromatic amino acids is that these amino acids absorb ultraviolet (UV) light,
which have an absorption maxima around 280 nm. UV absorption is an extremely important property
of amino acids because you can deduce how much protein you have in an unknown solution based on
the UV absorption profile. A common laboratory machine that can be used to measure UV absorption
of proteins is known as a spectrophotometer. This machine will pass a light source, at varying
wavelengths, through a protein sample. Based on how much of that light source is absorbed vs. how
much gets transmitted through, the detector of the machine will record this and give you an
absorbance reading. From this absorbance reading, you can then estimate the amount of protein in
your sample using the Beer-Lambert law.

44
Polar and charged Amino acids (Acidic and Basic amino acids)
Acidic and basic amino acids are electrically charged amino acids. These amino acids have a charge at
physiological pH of 7.4. Acidic amino acids have a negative charge while basic amino acids have a
positive charge.

Acidic: Basic:

Amino acids with uncharged side chains

These are hydrophilic amino acids that have larger electro negative atoms such as oxygen, nitrogen or
sulfur which can give these amino acids a slight negative charge. But their R groups are uncharged.
These amino acids along with the acidic and basic amino acids are usually found on regions of proteins
that interact with water such as the surface of the protein.

45
Sulfur containing amino acids
Cysteine is an important amino acid because it plays a primary role in stabilizing protein structure since
it can participate in the formation of disulfide bonds with other cysteine residues in the protein. These
are largely hydrophobic. Methionine is placed in the core of proteins.

Proline
Proline is unusual in that the side group is not only covalently bound to the α-carbon but also the
amine group. This unique structure, when incorporated into a peptide chain, gives that peptide chain
rigidity or strength. Proline would disrupt some secondary structures of proteins if not placed correctly.
For example, if proline was placed in the middle of an α-helix, that helical structure would no longer
exist. However, proline is often found as the first amino acid in an α-helix.

Amphoterism
Amino acids are amphoteric molecules, meaning they have characteristics of both acid and base due to
the presence of basic (amino) and acidic (carboxyl) groups. Therefore, amino acids, like, H 2 O, have the
capacity to donate or accept H+-ions, and, depending on the pH of the environment, amino acids can
assume a number of states.

46
The relationship between the ionization state of amino acids and the pH of the environment can be
described using the Henderson-Hasselbalch equation:

K a is the acid dissociation constant (K a tells you what proportion of the acid is ionized). In other words,
K a reveals what proportion of acid (HA) has dissociated into its conjugate base form (A-).

Due to the amphoterism of amino acids, the ionization state of amino acids will change when the pH
changes. Thus, this can be represented in a titration curve, shown below (for the amino acid alanine).

47
A simple rule can be applied in determining the ionization state of amino acids—for pH values BELOW
the pK a , the ionizable group will be protonated (retains H+ ions) whereas for pH values ABOVE the pK a ,
the ionizable group will be deprotonated (loses H+ ions). When pH = pK a , exactly half of the ionizable
groups will be protonated and half
deprotonated. It is at this point in the titration
curve that the amino acid acts as the best
buffer. The amino acid can exists as a
zwitterion, this is the point in the titration curve
in which the amino acid carries a net neutral
charge because the carboxyl group is
deprotonated (COO-) and the amino group is
protonated (NH 3 +). This midway point is known
as the isoelectric point (pI) and it is here that
the amino acid has the lowest buffering
capacity. The pI of an amino acid can be
calculating by averaging the two pKa values of
that amino acid.

Protonation & deprotonation during

titration

48
 H

For amino acids that have three ionizable groups such as aspartic acid, the pI can be calculated by
averaging the two pKas that are closest to each other. The three pKas for aspartic acid are 2.1, 3.9, and
9.8. The calculated pI (3.0) would be the average of 2.1 and 3.9 since they are closer to each other
than 3.9 and 9.8.

The pH of a solution and the pKa of the ionizable groups determine the charge of an amino
acid

49
Protein Structure
Upon completion of translation, polypeptides undergo a series of conformational changes before
assuming their final, functional state. The first level of ordered protein structure is the PRIMARY
STRUCTURE which refers to the linear structure of the protein with amino acids are linked together by
peptide bonds. The amino acid composition of the peptide chain has a large impact on its physical and
chemical properties. For example, proteins rich in aliphatic amino acids are very hydrophobic in nature
and relatively insoluble in water; therefore, these proteins are likely to be found in cell membranes.
Proteins rich in polar amino acids, like the basic or acidic amino
acids, are more water soluble; these proteins could be found in
the aqueous environment inside or outside the cell.

The SECONDARY STRUCTURE of a protein refers to the local structure of the polypeptide chain, and
this structure is determined by H-bond interactions between amino acids at different locations in the
polypeptide chain. There are 2 general types of secondary structure: the α-helix and β-pleated sheet.

50
Alzheimer’s Disease:

Caused by misfolding in beta sheets of amyloid proteins. The misfolded proteins accumulates as
plaques in the inter-neuronal spaces. Accumulation of large amount of amyloid plaques which are
resistant to protease degradation interferes in neuronal communication and eventually leads to
neuronal degeneration

Sickle cell anemia

Caused by a missense mutation that leads to replacement of glutamic acid replaced by Valine in βeta
chains of hemoglobin. This leads to misfolding of the beta chains forming large fibrils. The misfolding
affects the structure, function of beta hemoglobin, and the structure of the RBCs (making them sickle
shaped) in the patient.

TERTIARY STRUCTURE of a protein refers to the 3-dimensional, biologically-active conformation of the

protein. This 3-dimensional structure occurs when the side chain functional groups interact with one
another. These interactions include covalent disulfide bonds, H-bonds, ionic bonds (salt interactions),
and hydrophobic interactions. These interactions cause the secondary structures to fold into certain
conformations and result in a biologically-active protein. It also creates sites for ligand binding
and helps to maintain appropriate AA residues on the surface for the subcellular localization of
proteins.

The last tier of protein structure is referred to as QUATERNARY STRUCTURE, when two or more
separate peptide chains (tertiary structures) are held together by covalent or non-covalent
interactions. These peptide pieces which are linked together to yield a quaternary structure are known
as protein subunits. Multi domain enzymes, transmembrane and globular proteins are examples of
quaternary structures. Hemoglobin, the oxygen-carrying protein of the blood composed of two α-

51
polypeptide chains or subunits and two β-polypeptide chains or subunits, as well as an inorganic
prosthetic heme group is another example of quaternary structure

PROTEIN PURIFICATION

The aim of a purification process is not only removal of unwanted contaminants, but also the
concentration of the desired protein and the transfer to an environment where it is stable and
in a form ready for the intended application.

52
Under certain conditions, proteins adsorb to a variety of solid phases, preferably in a selective
manner. The adsorption principle is further explored in column chromatography. Due to their high
resolving power, different chromatography techniques have become dominant for protein purification

Chromatography
Refers to a group of separation techniques that involves a retardation of molecules with respect to the
solvent front that progresses through the material. The name literally means “color drawing” and was
originally used to describe the separation of natural pigments on filter papers by differential retardation

Column chromatography is the most common physical configuration, in which the stationary phase is
packed into a tube, a column, through which the mobile phase, the eluent, is pumped. The degree to
which the molecule adsorbs or interacts with the stationary phase will determine how fast it will be
carried by the mobile phase. Chromatographic separation of protein mixtures has become one of the
most effective and widely used means of purifying individual proteins. There are two variants of column
chromatography:

Figure. Illustrations of two classical chromatographic methods

A. Ion Exchange Chromatography. The charges of a protein are used for purification.
B. Size Exclusion Chromatography. Protein size is used for fractionation

Ion Exchange Chromatography:

Ionic interactions are the basis for purification of proteins by this process. The separation is due to
competition between proteins with different surface charges for oppositely charged groups on an ion
exchanger adsorbent. It is possible to separate proteins using either fixed positive charges on the
stationary phase, anion exchanger, or fixed negative charges, cation exchanger. Normally, proteins with
the same charge as the resin will pass through the column without adsorbing while proteins with the
opposite charge will be bound. The pH interval in which ion exchange chromatography is carried out is
restricted by the pH range in which the protein is stable. To achieve good adsorption, the pH of the buffer
chosen should be at least one pH unit above or below the isoelectric point of the analytes to be
separated.

53
Initially, molecules that do not bind or bind weakly to the stationary phase are first to wash away. Altered
conditions are needed for the elution of the molecules that bind to the stationary phase. Alteration of
pH can be done to release the protein of interest. This type of chromatography is further subdivided into
cation exchange chromatography and anion-exchange chromatography. Positively charged molecules
bind to anion exchange resins while negatively charged molecules bind to cation exchange resins. The
ionic compound consisting of the cationic species M+ and the anionic species B- can be retained by the
stationary phase.

Figure: Ion Exchange Chromatogram

A typical chromatogram from an ion exchange purification using salt gradient elution.

Size Exclusion Chromatography:

Size-exclusion chromatography (SEC), also known as molecular sieve chromatography, is a

chromatographic method in which molecules in solution are separated by their size, and in some cases
molecular weight. It is usually applied to large molecules or macromolecular complexes such as proteins
and industrial polymers. Protein size is used for fractionation.
Process: A mixture of molecules dissolved in liquid (the mobile phase) is applied to a chromatography
column which contains a solid support in the form of microscopic spheres, or “beads” (the stationary
phase).The mass of beads within the column is often referred to as the column bed. The beads act as
“traps” or “sieves” and function to filter small molecules which become temporarily trapped within the
pores.
Large sample molecules cannot or can only partially penetrate the pores, whereas smaller molecules can
access most or all pores. Thus, large molecules elute first, smaller molecules elute later, while molecules
that can access all the pores elute last from the column. Particles of different sizes will elute (filter)
through a stationary phase at different rates.

54
55
LECTURE 5 - DNA TECHNOLOGIES
Learning Objectives:
1. Define RE; summarize their role in DNA technology; Define & characterize
recognition sequences; differentiate between sticky/blunt ends
2. Define recombinant DNAs; Reflect on the role of rDNAs in gene cloning; learn
the important of plasmids vectors
3. Focus on definition & importance of cDNA in molecular biology
4. Define hybridization probes. How does it differ from ASO probes; Understand
the role of ASO probes in detection of homozygosity/ heterozygosity of gene
mutation
5. Emphasize of procedural details and the clinical importance of electrophoresis,
different blotting techniques; understand Sanger method for nucleic acid
sequencing
6. Learn the underlying principle, procedural details and clinical importance of
polymerase chain reaction (PCR). Emphasize on the role of primers in PCR.
7. Acquire a basic understanding of gene therapy

3 major technological breakthroughs in the field of DNA Technology in the

seventies were
• Discovery of restriction endonucleases: These enzymes facilitated cleaving of DNA into short
fragments which were easy to handle
• Invention of DNA cloning: Facilitated production of multiple copies of a DNA fragment of
interest that enabled creation of DNA/gene libraries
• Construction of hybridization probes: Probes enabled identification of a gene of interest from a
gene library or a complex mixture of nucleic acids

The ability to manipulate DNA molecules with precision in vitro or in vivo impacted all aspects of cell
biology studies and laid the foundation of the advanced DNA technologies that is being used in the
medical science of the 21st century

56
RESTRICTION ENDONUCLEASES (RE)
Definition: Bacterial enzymes capable of cutting a DNA molecule at strictly defined sites called
“recognition sites” by cleaving the sugar phosphate backbone of the DNA duplex leading to production
of double stranded fragments of average sizes

Fig: Function of restriction endonuclease

Function of RE in bacterial cells.

The RE protects the bacterial cell from invading viruses by chopping up and thus inactivating the viral
DNA. As it restricts the replication of a virus within a bacterial cell it is named as “restriction
endonuclease”

How are bacterial DNA protected from being cleaved by its own RE?

Bacterial restriction-methylase system protects the bacterial DNA from being chopped up by its own
RE. A bacterium labels its own DNA by methylating it in a specific pattern with the help of a specific
DNA methyl-transferase enzyme.

Thus a bacterium that makes restriction endonuclease for defense also synthesizes a specific DNA
methyl-transferase enzyme that methylates its own DNA and
protects it from being fragmented by its RE.

Fig: Bacterial-restriction-methylase system

57
Characteristics of Recognition Sites of RE

1. Substrate for RE are 4-8 nbp long double stranded palindromes called “recognition sites”.
Recognition sites may be different/same for different RE. Different RE having same recognition
sites is called isoschizomers. Examples are Sac I and Sst I

2. Recognition sites may be ambiguous or unambiguous.

3. Length of the recognition site dictates the frequency of cuts produced by the particular RE in a
random sequence of DNA. Example: Recognition site of a particular RE is 4 nbp long. This RE will
produce cuts at a distance of 44 = 256 nbp on a given DNA strand. The fragments generated will
be 256 nbp long. This applies to DNA strands where A,T,G,C % is 25% each

Note: There are 4 different bases (A,T,G, C) in a DNA , so the probability of finding a particular
base at one location on a DNA strand = ¼.The probability of finding the required base at n
locations = (¼)n ; If n = 4,6,8 (restriction sequences can be up to 8nbp long); the frequency of the
restriction site on a DNA = 4Length of restriction site

4. Restriction site for particular RE may contain restriction sites for another RE.

Patterns of cuts produced by RE

RE hydrolyzes the sugar phosphate backbone of DNA molecules at specific recognition sites leaving a
phosphate group at the 5’ end and a hydroxyl group at the 3’ends of both the strands of a duplex DNA.

Most commonly used restriction enzymes (type II) cleave within its recognition sequence generating
asymmetric or symmetric cuts with one of the 3 types of ends.

1. 5’overhangs

2. 3’ overhangs

58
 3. Blunt ends

Fig: Patterns of cuts produced by restriction endonucleases

Significance of the symmetric and asymmetric cuts by RE

The 5’ and 3’ overhangs are called “staggered/ sticky/ cohesive” ends. 2 DNA fragments flanked by
cohesive ends produced as a result of cleavage with the same RE can be easily rejoined by using the
enzyme DNA ligase even if they are obtained from 2 separate sources (like a bacteria and human).

Enzymes that cut symmetrically at precisely opposite sites in the two strands of DNA generate DNA
fragments with blunt ends without overhangs. When 2 DNA fragments with blunt ends are attempted
to be rejoined with the help of DNA ligase, yields are low. So RE that produce DNA fragments with
blunt ends are less popular for use in recombinant DNA technology & gene cloning.

DNA ligase

Enzymes of microbial origin (T4 bacteriophage) that ligate or join 2 DNA fragments by catalyzing the
formation of a phosphodiester bond between 5’ phosphate group of one strand and the 3’ hydroxyl
group of another stand of DNA.

Uses of Restriction Endonucleases

A. Production of recombinant DNA molecules

Recombinant DNA

Restriction enzymes helps in creating recombinant DNA

molecules which are engineered DNA molecules produced by
joining DNA fragments obtained from 2 different sources cleaved
by using the same restriction enzyme. Creating a recombinant
DNA with the help of RE was a major technical breakthrough that
had dramatic impact on all aspects of cell biology and gave birth
to the modern recombinant DNA technology, different types of
DNA analysis and genetic engineering.

Fig: Construction of a recombinant DNA

59
B. DNA/Gene cloning
A process of creating multiple copies of isolated DNA fragment/fragments by a combination of in vitro
or in vivo methods. Entire gene sequence/DNA fragment/specific DNA sequences can be cloned.

Steps involved in DNA/ Gene cloning

1. Isolation of the DNA from the cells of the organism that contains the desired gene/DNA
fragment
2. Purification of the isolated DNA by column chromatography
3. Fragmentation of the purified DNA by a specific restriction enzyme (the RE chosen should
not have a recognition sequence within the coding sequence of gene of interest and should
produce fragments with cohesive ends)

4. Incorporation of the DNA fragments into vectors (discussed later)

5. Introduction of the recombinant vectors into host cells ([Link] cells/yeast cells/mammalian
cells). The process of is called transformation.
6. Division of the host cells facilitates replication of the recombinant vector and thus the
inserted DNA fragment within the host cells.
7. The host cells containing the cloned DNA fragments are plated on agar medium containing
antibiotics. This allows only those host cells which contain the recombinant plasmids (with
the antibiotic resistant gene) to survive. The E. coli cells where transformation has failed will
die.
8. The multiple copies (clones) of the DNA of interest can be isolated from the E. coli cells.

Fig 5: Summary of gene cloning

60
Plasmids used as vectors DNA/gene cloning

Plasmids: Circular double stranded DNA molecules ranging in size from a few kilo bases to about 100
kb. They are present in bacterial cells and nuclei of some eukaryotic cells and are capable of replication
independent of the host cell. Most commonly used vectors in gene cloning

A plasmid vector used for gene/ DNA cloning is custom made by modifying natural plasmids (removing
unnecessary segments and adding essential sequences)

Basic structural features of a typical plasmid vector

A typical plasmid vector should possess the following components:

• Polylinker site: A site that contain recognition sequences for restriction enzymes
• Antibiotic resistant gene (ampr) for positive selection of transformed E. coli cells containing the
plasmid (the ampr gene in the plasmid will produce β lactamase which will inactivate the
antibiotic Ampicillin when it is applied to the medium such that only transformed [Link] cells will
survive in a medium containing the antibiotic ampicillin).
• Replication origin sequence (ORI) for amplification of the plasmid in any host cell
• Site for insertion of the gene of interest

Fig: Structure of a typical plasmid

61
What is a cDNA (complementary DNA)?
DNA copies of an mRNA can be synthesized by a process called reverse transcription in presence of an
enzyme called reverse transcriptase (isolated from Retroviruses). Such DNA copies of an mRNA contain
only the coding regions (exons) of that particular gene. They are called cDNA or complementary DNA

Use of cDNA: Used for expression of eukaryotic proteins in a prokaryotic cell like E. coli which lacks a
splicing machinery, cDNAs are produced by reverse transcription of the mRNA of that particular gene.
The cDNAs are then inserted into suitable expression vectors and transformed into [Link] cells where
they are expressed into the respective proteins.

Fig: Synthesis of cDNA

HYBRIDIZATION PROBES
Definition: Single or double stranded fragments of DNA/RNA (100-1000 nbp) tagged to a radioactive or
fluorescent marker that helps to detect a complementary target DNA present in a complex nucleic acid
mixture or a large gene library by complementary base pair hybridization

Synthesis of probes: A required probe can be synthesized chemically or they can be generated by PCR
or cloning methods

Uses: Molecular probes are widely used for screening a gene library in search of a particular gene of
interest. They are used for detecting nucleotide sequences by blotting methods as well as in other
molecular techniques like microarrays

62
Allele Specific Oligonucleotides probes
Short (15-20 nbp) synthetic probes linked to a radioactive/fluorescent/enzyme tag that helps to
identify difference in the DNA sequence between 2 alleles of a gene. They are used in pairs with one
probe having a sequence complementary to the normal gene and the other probe having a sequence
complementary to the mutated gene.

Example: Sickle cell anemia is produced by mutation in the codon of the 6th amino acid glutamate
(GAG) of beta hemoglobin DNA such that it changes to valine (GTG).

To test whether this mutation in present in homozygous or heterozygous condition in a given DNA
sample, an ASO probe (S) would be designed having a sequence complementary to the mutated beta
hemoglobin DNA that produces sickle cell anemia. A second ASO probe (A) would be synthesized
complementary to the normal sequence of beta hemoglobin DNA.

Detection of sickle cell anemia using ASO probes?

Segments of beta hemoglobin gene from 6 different samples are amplified separately by PCR and the
products of all 6 samples are blotted separately on a 2 membrane strips (dot blot). Alkali treatments of
the samples are done to get single stranded DNA (for hybridization). The ASO probes (A) and (S) are
applied to different blots for hybridization. The membranes are washed to remove any weakly
hybridized/unhybridized probes. Detection of hybridized ASO label is done by autoradiography which
allows reading of the genotype of the sample.

Samples 1, 4: Normal alleles as they show strong hybridization with ASO probe (A) having sequence
complementary to the normal allele but not with S

Samples 3, 5: Heterozygous carriers as they have sequences complementary to both A and S probes

Samples 2, 6: Affected with sickle cell anemia as they show strong hybridization with only the S probe.

Fig: Detection of sickle cell anemia using ASO probes

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III. ELECTROPHORESIS AND BLOTTING
What is Electrophoresis?

A procedure that enables sorting of molecules (DNA/ RNA/ proteins) based on either size (molecular
weight) or charge density (charge to mass ratio). The mixture of molecules being sorted is dispensed
into small wells in a gel matrix composed of cross linked polymers (agarose/polyacrylamide gels are
commonly used) and subjected to an electrical field.

Based on their charge to mass ratio, or their molecular weight, the molecules move through the gel at
different rates from the negative electrode (cathode) towards the positive electrode (anode). This
results in separation of the molecules in the gel matrix on the basis of size or charge density.

What is Blotting?

Following gel electrophoresis, the separated DNA/RNA/protein mixture is transferred from the gel to a
solid matrix which is a membrane (nylon membrane for nucleic acids and nitrocellulose membrane for
proteins). This is followed by identification of the DNA/RNA/protein of interest from the separated
mixture, using a specific hybridization probe. This entire process is called blotting.

Types of Blotting

Southern blotting: When a specific DNA fragment of interest present in a complex DNA mixture
(separated by gel electrophoresis) is detected with the help of a specific molecular probe by
complementary hybridization, the process is called Southern blotting.

Northern Blotting: When a specific RNA fragment of interest present in a complex RNA mixture
(separated by gel electrophoresis) is detected with the help of a specific molecular probe by
complementary hybridization, the process is called Northern blotting.

Western Blotting: When a specific protein of interest present in a complex protein mixture (separated
by polyacrylamide gel electrophoresis) is detected with the help of a specific antibody raised against
the protein of interest by antigen antibody interaction, the process is called Western blotting.

We will focus on Southern Blotting as we are dealing with DNA in particular

Steps involved in Southern blotting

1. DNA to be analyzed is digested into fragments with restriction endonuclease

2. DNA fragments are separated according to size by agarose gel electrophoresis

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 3. Denaturation of double stranded DNA fragments into single stranded fragments by NaOH
treatment of the gel
4. Transfer of the single stranded DNA fragments from the gel onto a nitrocellulose/nylon
membrane (DNA fragments when transferred to the membrane retains the same pattern of
separation as in the gel)
5. The nitrocellulose/nylon membrane is then incubated with multiple copies of a labeled
single stranded DNA probe. The probes bind to the fragment of DNA that has sequence
complementary to it on the membrane.
6. The membrane is washed to remove unbound excess probes. For an enzyme labeled probe,
the membrane is incubated with the enzyme substrate. The location of the DNA fragment
of interest on the membrane at the site of hybridization of the probe, can be detected on
an X ray film by autoradiography

Fig: Southern blotting Fig: Basic principle of electrophoresis & blotting

DNA SEQUENCING (SANGER METHOD)

The Sanger method or the chain terminator method of DNA sequencing was established by Dr.
Frederick Sanger, a British Scientist in 1974. It is widely accepted worldwide because of its practicality.

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Why do we need to know the sequence of a DNA??

DNA sequencing is a powerful molecular tool that allows molecular biologists to identify and locate
specific genes and regulatory sequences in a DNA fragment, compare homologous genes across
species, identify gene mutations etc.

Sanger method: Requirements

• Single stranded DNA (ssDNA) of unknown sequence (template)

• Labeled DNA primer

• Deoxynucleotide triphosphates (dNTPs): dATP, dCTP, dGTP, dTTP

• Dideoxynucleotide triphosphates (ddNTPs): ddATP, ddCTP, ddGTP, ddTTP or chain terminating

nucleotides (for structure of ddNTP refer the slide 6 of DNA Technology II lecture)

Methodology

• The process starts with a single stranded DNA template to the 3’ end which, a short
complementary oligonucleotide primer is annealed. The annealing of the primer facilitates
attachment of DNA polymerase enzyme which helps in synthesis of the daughter strand by
incorporation of dNTPs complementary to the template DNA strand.. The primer or dNTPs can
be attached to the radioactive/fluorescent labels to facilitate detection of the final product

• The reaction mixture containing the template DNA with the annealed primers are then divided
into 4 tubes labeled A,T,G,C

• The following reagents are added to each tube

"G" tube: all four dNTP's, ddGTP and DNA polymerase

"A" tube: all four dNTP's, ddATP and DNA polymerase

"T" tube: all four dNTP's, ddTTP and DNA polymerase

"C" tube: all four dNTP's, ddCTP and DNA polymerase

The concentration of the ddNTPs added to each tube is one-hundredth the concentration of the
normal dNTPs.

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• As the daughter strand is synthesized by incorporation of dNTPs at the 3’end of the primer
with the help of DNA polymerase, the ddNTPs (chain terminators) compete with the dNTPs for
incorporation into the daughter strand being synthesized. Occasionally one ddNTP will be
incorporated into the daughter stand leading to termination of synthesis of the new chain at
that point. This is because the ddNTPs lack a 3’OH group on its sugar moiety which prevents
formation of a phosphodiester bond between this incorporated ddNTP and the next incoming
dNTP.

• As the same chain of DNA is being synthesized over and over again several times in each tube,
the new chain will be terminated at all positions with time, due to incorporation of a ddNTP in
place of a potential dNTP, producing fragments of different lengths in the different tubes

• The contents of all four tubes are denatured into single strands which are then separated
according to size by polyacrylamide gel electrophoresis (content of each tube is run in a
different lane on the gel)

• During electrophoresis, the longer fragments in each lane (corresponding to a separate tube)
travel shorter distances and vice versa through the gel. The shorter fragments has terminated
closer to the primer attached to the 3’ end of the template
• So reading the gel from the bottom to the top across all the four lanes will give the sequence of
the newly synthesized daughter strand in 5’ to 3’ direction which is complementary to the
sequence of our template stand (sequence of interest) which is also antiparallel.

Note: With the improvement of technology, presently molecular biologists sequence a fragment of
DNA in an automatic sequencer works on the principle of the Sanger method. More DNAs can be
sequenced in a shorter period of time in a sequencer and all reactions are performed in the same
tube containing all four ddNTPs each labeled with a different colored fluorescent dye fluorescing
at different wavelengths of light which can detected with laser scanner.

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 Fig: Diagrammatic representation of DNA sequencing by Sanger method

POLYMERASE CHAIN REACTION (PCR)

Molecular technique that allows DNA from a selected region of a genome to be effectively amplified
billion fold in a cell free system (in vitro) through temperature sensitive enzymatic reactions within a
short period of time (2-3 hr)

Requirements for PCR reaction

• Template DNA: DNA extracted from cells containing the target gene for amplification by
PCR
• 2 primers (forward/reverse): The primers used in PCR are synthetic oligonucleotide
sequences complementary to a short sequence at the 3’ end of the sense and antisense
strands of the target gene present in the template DNA. They bind to the 3’ ends of the
sense and antisense strand of template DNA respectively allowing binding of DNA
polymerase enzyme for synthesis of the daughter strands during the PCR reaction.
• DNA polymerase enzyme: Required for synthesis of daughter DNA strand. They are isolated
from thermophilic bacteria and remains stable at high temperatures
• dNTPs (deoxyribonucletotide triphosphates): These are the building blocks (dATP, dGTP,
dCTP, dTTP) used by DNA polymerase to synthesize a daughter strand complementary to
the template DNA during PCR
• Buffer solutions, divalent and monovalent cations (Mg+2, K+)

Steps involved in PCR

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Amplification of a piece of DNA by PCR involves 3 temperature dependent steps which are repeated for
30-40 cycles in a thermal cycler. The steps are

• Denaturation: During this step the template DNA is expose to a high temperature of 94°-95°C
for about 1 min allowing the double stranded template DNA to melt and denature into 2
single strands. The temperature also activates the DNA polymerase enzyme
• Annealing: Single stranded primers (forward and reverse) anneal to the complementary
regions at the 3’ ends of the denatured single stranded template DNAs. The temperature
required for annealing is between 55°-70°C and time required is about 45 seconds. DNA-DNA
hydrogen bonds are formed between the primers and the template strands. Polymerase
enzyme can now bind to the primer and start synthesis of the complementary daughter
strand.
• Extension/Elongation: In this step the DNA polymerase enzyme synthesizes the new daughter
DNA strand complementary to the single stranded template by adding dNTPs in a 5’ to 3’
direction. The temperature necessary for this elongation step is 72°C which is ideal for
activity of DNA polymerase enzyme which can optimally polymerize 1000 nbp/min leading to
exponential amplification of the template DNA. These three steps are repeated for 30-40
cycles in a sequential manner in the PCR thermal cycler for billion fold amplification of the
target DNA.

Fig: Basic principles of PCR

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Verification of PCR product

On completion of the PCR reaction cycles in the thermal cycler, the PCR product is subjected to gel
electrophoresis (fig. 14) for verification of whether

• Product has been formed: If we see bands on the stained gel (ethidium bromide used for
staining), it will tell us the template DNA has been amplified to form the product. Lanes 1,2,4,5
(fig 14) shows distinct bands of products formed. Lane 3 does not show any band indicating that
PCR amplification was unsuccessful in that reaction mixture
• Product formed is of the right size: If the band on the gel has the expected size (1800 kD in this
case seen in lane 1), it indicates that the primers have amplified the target gene. If however we
see products of lower size (500 kD seen in lanes 2,4) it indicates that mispriming has occurred
(primers have fit onto a different gene on the template DNA and amplified a non-target gene)
• Used primers are very specific: If the primers are specific to the target gene we will see only
one band in the entire lane of the desired size. If we see multiple bands (lane 5) then the
primers used are amplifying others genes along with the target gene. This is not desirable and
the primers need to be redesigned.

Fig: Verification of PCR product by gel electrophoresis

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Quantitative PCR (qPCR) or Real Time PCR

A new method of PCR called real time or quantitative PCR which enabled detection as well as absolute
quantification of the initial template copy number, was a major development in the field of molecular
biology.

Clinical Use of PCR

PCR technology has become an essential research and diagnostic tool for improving human health and
quality of life. PCR technology allows a scientist to isolate genetic material even from a single cell,
amplify then and use it for several purposes.

Blood screening and detection of infections

Blood screening: Highly sensitive PCR nucleic acid amplification testing (NAT) technology is used
nowadays for screening donated blood samples for presence of infectious agents like HIV, Hepatitis B,
C etc. This advanced technology has an advantage over the traditional antibody based serological tests
done in the 1970s as it capable of detecting the presence of very small amount of microbial DNA/RNA
in blood even in the window period.

The major pathogens detected by PCR based diagnostic tests include HIV (for detection of AIDS),
hepatitis B, C (can cause liver infections leading to liver cancer), human Papillomavirus (causes cervical
cancer), Clamydus trachomatis (causes infertility in women), Mycobacterium tuberculosis

PCR and genetic testing

PCR based technologies are used for genetic testing for detection of SNPs. New PCR based tests for
diagnosis of disease predisposition, cancer screening, cancer therapy selection and pharmacogenetics
are being developed. These tests can tell whether an individual is predisposed to develop cancer later
in life, whether he has a correct metabolic pathway to metabolize a particular drug or even if the
individual produces correct target for a drug in his body.

DNA profiling/ DNA fingerprinting

With the discovery of DNA technologies like PCR, DNA profiling or DNA fingerprinting can be done with
very small amount of starting sample recovered from crime sites. DNA is extracted from a hair or a few
skin cells obtained from the crime site. Using PCR a specific regions of DNA (short tandem repeat)
locus which are inherited allelic traits and highly polymorphic, are amplified. The amplified DNAs are
separated and the pattern of STRs (small tandem repeats) are detected by electrophoresis

Pre and post implantation genetic diagnosis

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Pre-implantation genetic diagnosis (PGD): A group of tests done as a part of assisted reproductive
techniques (methods used to achieve pregnancy by artificial methods like in vitro fertilization) or high
risk pregnancies. These tests screen embryos for presence of genetic/chromosomal disorders prior to
implantation in the uterus. It allows pre-selection of chromosomally normal embryos and helps to
avoid giving birth to children with heritable and genetic disorders.

PCR has become an integral part of the pre-implantation/genetic diagnosis. After 3 days of in vitro
fertilization, when the embryo is composed of 6-8 cells (blastomeres), a single cell is isolated from the
embryo, DNA is extracted, and the sequence of interest is amplified by PCR and studied for the
presence or absence of mutations that cause genetic disorders

GENE THERAPY

What is gene therapy?

A technique for correcting defective genes that leads to disease development

What are the different approaches used for replacing a therapeutic gene at the cellular level?
• Insertion of a normal gene at a non-specific locus to perform the function of a specific non-
functional gene (most common approach) p[resent in a patient
• Replacement of the abnormal gene with its normal copy by homologous recombination
• Repair of the mutated gene through selective reverse mutation
• Altering the regulatory elements of a gene to rectify its function

Different modes of delivery of therapeutic genes

Direct delivery: Therapeutic genes are packaged in genetically modified, replication incompetent viral
vectors. The viruses containing the therapeutic gene are administered directly into the target organ.

Cell based delivery: Stem cells are isolated for the bone marrow of patients and grown in culture
medium. The stem cells are transfected with viral vectors containing the therapeutic gene. The
recombinant stem cells that incorporate the therapeutic gene in their genome by homologus
recombination are selected by antibiotic selection and injected into the target organ.

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 Fig: Different modes of delivery of therapeutic genes

Current status of gene therapy

The current status of gene therapy is still experimental although big research funds are being utilized in
the US and Europe for its improvement. In the US, gene therapy products have yet to be approved by
the FDA for commercialization.

Clinical trials of gene therapy methods started in 1990 but suffered several major setbacks. In 1999, a8
year old boy (Jesses Gelsinger) died due to multiple organ failure while participating in a gene therapy
trial for ornithine transcarboxylase deficiency. It was a result of unpredicted acute immune response.
Another setback came in 2003 when a second child participating in a French gene therapy trial
developed leukemia later on.

Factors that prevent the translation of gene therapy from bench side to bedside

• Short life of the treatment: Integration of the therapeutic DNA into the genome of target cells is
not guaranteed and the degree of acceptation or rejection varies between different target cell
types. Sometimes for longer lasting effect patients have to go through multiple rounds of gene
therapy which is expensive and considered as a major drawback associated gene therapy
• Immune response: Introduction of a foreign gene specifically through viral vectors often evokes
unpredicted acute immune response in the patient which may put the patient’s life under
potential risk. Example, the case of Jesse Gelsinger in 1999
• Virus as Vectors: Use of viral vectors like retrovirus, adenovirus, and adeno-associated virus are
common in gene therapy. These viruses are genetically manipulated before introduction into

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 patients such that they are no longer capable of replication in the host cell. But at times the
recombinant viruses mutate back to the replication competent form under suitable
environment of the host cell. This may lead to viral infection and unpredicted complications.
• Multigene disorders: Disorders like diabetes, Alzheimer’s diseases, cardiovascular diseases
which involve defect in multiple genes cannot be addressed by gene therapy which is tailored
around correction of a single gene involved in disease progression.

Success in a single gene therapy case in China

China is the only country where a particular gene therapy has been approved and licensed to treat
patients with head and neck squamous cell carcinoma.
The drug is called Gendicine. It is actually a recombinant replication incompetent engineered human
adenovirus particles carrying human p53 gene. p53 is a tumor suppressor gene present in all normal
cells which induces apoptosis of tumor cells. In a tumor cell, p53 stops DNA repair mechanisms, cuts
off anti apoptotic mechanisms and activates the immune system enabling the body to get rid of tumor
cells. Thus it is a protection the body has at cellular levels for destroying tumor cells. Combination of
Gendicine with chemotherapy and radiotherapy have successfully been able to treat patients with
head and neck squamous cell carcinoma, common in parts of China

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Lecture 6- Chromatin & Chromosomes
Learning Objectives
• Explain the structure and function of chromatin.
• Describe DNA binding proteins.
• Describe how histone modifications can affect chromatin accessibility.
• Understand the “end of replication problem” and how hTERT and
telomeres solve this.
• Focus on different chromosomal abnormalities associated with
aneuploidy, polyploidy, & chromosomal translocations

DNA Packaging
DNA exists in the nucleus of eukaryotic cells in a complex
with DNA-binding proteins. This arrangement is known as
chromatin, the combination of DNA interacting with DNA-
binding proteins.

There are two major classes of DNA-binding proteins: (1) histone proteins and (2) non-histone proteins.
Histone proteins serve as the key structural determinants for DNA packaging while non-histone DNA-
binding proteins include proteins such as transcription factors and DNA synthesis enzymes.

There are five histone proteins: H1, H2a, H2B, H3, & H4. These histones are the major protein
constituent of chromatin and their primary function is to serve as a docking and coiling site for DNA. 8
histone proteins are used to coil and wind DNA into a much shorter length: two each of H2A, H2B, H3,
& H4. These 8 histone proteins form an octameric core around which the DNA will wind itself. This
association between DNA and the octameric core of histone proteins is known as the nucleosome.
Histone H1 binds/associates with the outside of the nucleosome core to stabilize the core complex and
link adjacent cores to one another.

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The nucleosome is the fundamental unit of chromatin, where the DNA will continuously wind itself
around these histone cores for the entire length of the genome. This has often been described as
“beads along a string,” where the beads are the histone cores and the string is the linker DNA.

The nucleosomes themselves are further organized into more tightly-packed structures, called the
30nm solenoid. Solenoids are constructed by winding the nucleosome particles into a spring-shaped
structure with ~ 6 nucleosomes per turn. The solenoids can further fold onto each other to generate a
300nm filament.

Finally, the chromatin filaments are compacted into the mature chromosome, using a nuclear scaffold.
The scaffold, a structure made of protein, acts as support for the filaments and forms the core of the
chromosome. The filaments are arranged around the scaffold to form many loops.

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Genome Accessibility
There are two forms of chromatin, a lightly-packed form called euchromatin and a more densely
packed form called heterochromatin. Euchromatin is a dispersed section of chromatin that is
transcriptionally active while heterochromatin is a tightly-packed form of DNA that is transcriptionally-
inactive. DNA can assume either of these two chromatin states based on certain structure-altering
modifications. There are cellular enzymes that can directly modify histones, to relax and open up the
DNA or modify in a way that makes the histones bind more tightly to the DNA.

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One example of a histone modification is acetylation. Histone acetylation involves the attachment of
acetyl groups to lysine residues in the N-terminal tails of histone proteins. This modification changes
the net charge on the histone protein, causing it respond differently to the negatively-charged DNA.
The two enzymes responsible for this activity are known as histone acetyltransferases (HAT, adds
acetyl groups) and histone deacetyltransferases (HDAC, subtracts acetyl groups). HAT modifies
histones to open the nucleosome and make the DNA more accessible for transcription. HDAC modifies
histones to close the nucleosome and make the DNA less accessible for transcription.

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Chromosomes
When a cell prepares to divide, the chromatin threads coil and greatly condense to form the
characteristic chromosomes.

Humans have 23 pairs of homologous chromosomes

(homologous meaning the same), one set of 23
chromosomes from Mom and the other set of homologous
chromosomes from Dad. Each chromosome in the G1
phase is made of one chromatid

At the completion of DNA replication, the cell ends up with a duplicate set of identical chromatids.
These identical chromatids are now called sister chromatids. At this juncture in the cell cycle (at the
completion of S-phase), the cell has doubled the # of chromatids but note the number of
chromosomes is the same.

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At the completion of mitosis, each daughter cell will have received one complete set of chromatids.

When discussing chromosomes, ploidy refers to the number of sets of chromosomes in a cell. So, for
humans, we are known as diploid organisms because we have 46 chromosomes, two complete sets
(one from Mom and one from Dad) in all our somatic cells. Haploid refers to cells with a single set of
unpaired chromosomes. Human gametes are haploid, containing only one set of chromosomes.

In somatic cells, 22 chromosome pairs are symmetrical (known as autosomes or non-sex

chromosomes) and the last pair is potentially asymmetrical. Women are comprised of two X-
chromosomes while men are comprised of one X- and one Y-chromosome. The male-determining Y-
chromosome is missing one short arm.

Centromeres

Centromeres are the most constricted regions of chromosomes and link sister chromatids.
Centromeres are vitally important during cell division because the microtubule-based spindle fibers

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attach at a specific protein region in the centromere called the kinetochore. From this region, the
spindle fibers can pull apart the sister chromatids in preparation for cell division.

Centromeres have no defined DNA sequence (meaning, they do not code for genes in this region) but
have large amounts of repetitive DNA, present as tightly-condensed heterochromatin.

The position of the centromere differs among the chromosomes and can be described three ways:
metacentric, submetacentric, or acrocentric. In metacentric chromosomes, the centromere is located
near the center of the chromosome, dividing the arms into equal lengths. Submetacentric
chromosomes have the centromere offset from the center, yielding arms of unequal length. Finally,
Acrocentric chromosomes have the centromeres at the far end of the chromosome.

The genetic locations of genes can be described based on centromere position, as well as the
chromosome # and banding pattern (laboratory dyes can be used to stain chromosomes, resulting in
differential banding patterns corresponding to regions of heterochromatin and euchromatin). The

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nomenclature begins with the chromosome # (which chromosome the gene is found on), followed by
the chromosome arm (which arm of the chromosome the gene is found on), followed by the banding
patterns.

Telomeres
Telomeres, like centromeres, are repeated elements of heterochromatic DNA sequence placed at the
ends of chromosomes. The sequence that gets repeated thousands of times is 5’-TTAGGG-3’. These
repeated sequences are important because they protect the chromosome ends from damage and
provide a genetic cushion during DNA replication when genetic material is lost due to the “end-of-
replication problem.”

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Telomeres are needed to protect the ends of DNA because genetic material is lost at the conclusion of
each round of DNA replication since DNA polymerase is unable to fill in the gaps left by primer
degradation at the terminal 5’-ends of the newly-synthesized lagging daughter strands.

The cellular enzyme responsible for synthesizing and capping the chromosome ends with telomeric
repeats is human telomerase reverse transcriptase (hTERT). This is a ribonucleoprotein which uses its
own RNA template (5’-CCCUAACCC-3’) to make the telomeric DNA repeats.

Active telomerase is only found in fetal tissues, adult germ cells, lymphocytes, proliferative stem cells,
and cancer-causing tumor cells. Most somatic cells have negligible telomerase activity; therefore, most

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somatic cells lose portions of their telomeres during each round of DNA replication. If too much
telomeric DNA is lost in a cell’s lifespan, that cell will enter a state called senescence. A senescent cell
is one which loses its functional and replicative capacity. Thus, somatic cells have a defined number of
cell divisions before they reach senescence. This limit is known as the Hayflick Limit which is about 50-
60 cycles.

Chromosomal Abnormalities
Mutations may also be of a larger scale, involving whole chromosomes or segments of chromosomes.
These types of mutations are categorized as chromosomal abnormalities. One abnormality deals with
whole sets of chromsomes. To review, Ploidy refers to the # of sets of chromosomes. Haploid refers
to a cell that has one complete set of chromosomes (e.g., human gametes). Diploid refers to cells with
2 complete sets of chromosomes (e.g., somatic cells).

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Polyploidy refers to cells that possess an extra set or sets of chromosomes. Polyploidy is quite rare
in humans but does occur with low frequency. The only polyploid conditions that have been observed
in humans are Triploidy & Tetrapolidy.

Triploidy refers to individuals who have 3 sets of chromosomes (69 chromosomes) in each cell’s
nucleus whereas Tetraploidy refers to individuals who have 4 sets of chromosomes (92 chromosomes)
in each cell’s nucleus.

The most common cause of Triploidy is the fertilization of one egg by 2 sperm (called dispermy). The
resulting zygote receives 23 chromosomes from the egg and 23 chromosomes from each of the two
sperm cells. Tetraploidy can be caused by a mitotic failure in the early embryo in which all of the
duplicated chromosomes migrate to 1 of 2 daughter cells, or it can also result from the fusion of 2
diploid zygotes.

Aneuploidy refers to cells that contain missing or additional chromosomes (cells that do not contain
a multiple of 23 chromosomes). Usually only one chromosome is affected, but it is possible for more
than one chromosome to be missing or duplicated. The most common aneuploidies are Monosomy
and Trisomy. Monosomy indicates the presence of only 1 copy of a particular chromosome in an
otherwise diploid cell. Trisomy refers to 3 copies of one particular chromosome in an otherwise diploid
cell. The most common cause of aneuploidy is nondisjunction, the failure of chromosomes to disjoin
normally during meiosis.

The most common human disease associated with monosomy is monosomy of the X chromosome,
which leads to a disease known as Turner syndrome. This occurs in women who only possess one
sex-linked X-chromosome (instead of the normal 2). The syndrome manifests itself in a number of
ways. There are characteristic physical abnormalities, such as short stature, swelling of the limbs, a
broad chest, low hairline, low-set ears, and webbed necks. Females with Turner Syndrome typically
experience gonadal dysfunction (non-working ovaries), which results in amenorrhea (absence of

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menstrual cycle), and sterility. While usually not life-threatening, there are other concurrent health
issues frequently associated with Turner Syndrome, including congenital heart disease, hypothyroidism
(reduced hormone secretion by the thyroid), diabetes, vision problems, hearing concerns, and a
number of autoimmune diseases. Finally, some cognitive deficits are often observed, with particular
difficulties in memory, math, and depth perception.

Trisomy 21 is the most common aneuploidy condition. This trisomy produces Down syndrome, a
condition associated with intellectual disability, a characteristic facial appearance, and weak muscle
tone in infancy. All affected individuals experience cognitive delays, but the intellectual disability is
usually mild to moderate. However, people with Down syndrome often experience a gradual decline in
thinking ability (cognition) as they age, usually starting around age 50. Down syndrome is also
associated with an increased risk of developing Alzheimer’s disease.

Additionally, people with Down syndrome may have a variety of birth defects, such as a heart defect or
digestive abnormalities (blockage of the intestine). This disease can be readily identified/confirmed
using a simple human karyotype to detect the additional chromosome 21.

Chromosomal Translocations

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Translocation, defined as the interchange of genetic material between 2 nonhomologous
chromosomes, is another form of chromosomal abnormality. There are two basic types of
translocations, referred to as Reciprocal & Robertsonian. Reciprocal translocations occur when breaks
occur in 2 different chromosomes and the genetic material is mutually exchanged. The resulting
chromosomes are called derivative chromosomes. Often, the carrier of a reciprocal translocation is
unaffected because he or she has a normal complement of genetic material.

Robertsonian translocation: The short arms of 2 nonhomologous chromosomes are lost and the
remaining long arms fuse at the centromere to form a single, hybrid chromosome. Robertsonian
translocations are confined to chromosomes with acrocentric centromeres (so, chromosomes 13, 14,
15, 21, & 22) because these chromosomes have very tiny, fragile p-arms. Most individuals who have
chromosomes which have undergone Robertsonian translocation are usually unaffected since there is
essentially no gene-coding material contained within the tiny p-arms of acrocentric chromosomes.
Because the carriers of Robertsonian translocations lose no essential genetic material, they are
phenotypically normal but have only 45 chromosomes in each cell.

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Philadelphia Chromosome: One of the more studied reciprocal translocations involves
chromosomes 9 and 22. In this translocation, most of chromosome 22 is transferred to the
chromosome 9 while a small, distal portion of the q-arm of chromosome 9 is swapped onto
chromosome 22. The net effect is a smaller chromosome 22, which has been labeled the Philadelphia
chromosome. This Philadelphia chromosome is consistently found in 95% of all individuals with
chronic myelogenous leukemia (CML), a cancer of white blood cells, characterized by increased and
unregulated growth of predominantly myeloid cells in the bone marrow.

The underlying cause of this cancer is due to a new gene formed stemming from the reciprocal
translocation. A gene called Abl in chromosome 9 normally codes for a tyrosine kinase, an intracellular
protein which phosphorylates other target proteins to activate them. The gene in chromosome 22,
called Bcr, codes for a GTPase-activating protein (protein that hydrolyzes GTP). When this
translocation takes place, forming the Philadelphia chromosome, a new, chimeric gene is created =
Bcr-Abl. This new gene, when transcribed and translated, leads to a new oncogenic protein. This is
because the tyrosine kinase portion of Abl is now unable to turn itself off, and so that protein
continuously phosphorylates target proteins, activating other proteins and initiating many signaling
pathways. Specifically, this new oncogenic protein highly phosphorylates the IL-3 receptor, a signaling
receptor on myeloid leukocytes. The Bcr-Abl protein tricks the myeloid cells—these myeloid cells think
they are receiving cytokine signals for the IL-3 receptor, when in fact, the Bcr-Abl protein is providing
the positive signal. This positive signal induces these myeloid cells to undergo continuous activation
and cell division, leading to cancer of the myeloid leukocytes.

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89
Lecture 7- Mendelian Genetics
Learning Objectives:

• Understand the 3 Mendelian principles of genetics and the

exceptions
• Understand and utilize genetic probabilities to predict
outcomes of mating
• Learn the role of dosage compensation and X chromosome
inactivation in females
• Compare & contrast the properties of various types of
inheritance
• Learn how to identify inheritance patterns of genetic diseases
using a pedigree chart
Mendel and the Life of Peas
Johann (Gregor) Mendel was one of the most influential geneticists in the last 150 years. While
working as a Catholic monk in Brunn, Czech Republic, he began investigating the concept of heredity
(how traits are passed from one generation to the next, from parent to offspring).

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In one of Mendel’s first experiments, he crossed-fertilized tall and dwarf (short) pea plants to
investigate how height was inherited. The seeds that resulted from these cross-fertilizations were
sown the next year, yielding hybrids that were uniformly tall. Mendel observantly noted that the
dwarf characteristic seemed to have disappeared in the progeny of the cross, for all hybrid plants were
tall.

To explore the hereditary makeup of these tall hybrids, Mendel allowed them to undergo self-
fertilization. When he examined the progeny, he found that they consisted of both tall and dwarf
plants, in an approximate ratio of 3:1. Mendel was struck by the reappearance of the dwarf
characteristic and reasoned that these hybrids from the parental cross, in spite of their tall appearance,
must carry a latent genetic factor for dwarfness, one that was masked by the expression of another
factor for tallness. He said that the latent factor was recessive and that the expressed factor was
dominant. He also inferred that these recessive and dominant factors separated from each other
when the hybrid plants reproduced. This enabled Mendel to explain the reappearance of the dwarf
characteristic in the next generation.

Mendel performed similar experiments to study the inheritance of other various traits. In each
experiment, called a monohybrid cross (because only one trait was being studied), Mendel observed
that only one of the two contrasting characteristics appeared in the hybrids and that when these
hybrids were self-fertilized, they produced two types of progeny, each resembling one of the plants in
the original crosses. Furthermore, he found that each progeny consistently appeared in a ratio of 3:1.
Thus, each trait that Mendel studied seemed to be controlled by a heritable factor that existed in two
forms, one dominant, and the other recessive. We now know these factors to be genes and their
dominant and recessive forms are alleles.

Mendel’s Genetic Principles

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The regular numerical relationships that Mendel observed in these crosses led him to an important
conclusion: genes come in pairs. Mendel proposed that each of the parental strains that he used in his
experiments carried two identical copies of a gene (diploid) that was homozygous (refers to the
presence of identical alleles at a specified genetic locus). However, during the production of gametes,
Mendel proposed that these two copies are reduced to one; that is, the gametes that emerge from
meiosis carry a single copy of a gene (haploid). Mendel then recognized that the diploid gene number
would be restored when sperm and egg unite to form a zygote. Furthermore, he understood that if
the sperm and egg came from genetically different plants (as they did in his crosses), the hybrid zygote
would inherit two different alleles, one from the mother and one from the father, known as a
heterozygote. Mendel realized that the different alleles present in a heterozygote must coexist even
though one is dominant and the other recessive, and that each of these alleles would have an equal
chance of entering a gamete when the heterozygote reproduces.

In addition to Mendel’s scientific discoveries,

he revolutionized the study of genetics by
utilizing symbols to describe hereditary
phenomena, as shown in the diagram. In
genetics, letters are chosen to denote the
alleles of a gene, where a capital letter refers
to the dominant trait and a lowercase letter
refers to the recessive trait, and the allelic
constitution is referred to as the genotype.

In the above example, Mendel’s monohybrid

cross between tall and dwarf pea plants is
pictorially represented. As parental strains,
the tall and dwarf pea plants form the P
generation of the experiment. These parental
strains were highly inbred, meaning that their
allelic constitution was homozygous—two big
“D’s” for the tall parent and two little “d’s” for
the dwarf parent. Now, because each parent
is homozygous, they can only contribute one type of allele during gamete formation. Therefore, when
Mendel crossed these two parental strains, all plant progeny, called the “F1” (Filial) generation were of
a “Dd” genotype, yielding a tall phenotype (phenotype refers to the physical appearance of an
organism). When Mendel self-fertilized the F1 plants, gametes “D” and “d” were formed in equal
proportions, a process known as allele segregation, perhaps the most important discovery that Mendel
made. The result of crossing two F1 heterozygotes is that the two kinds of gametes, “D” and “d”, can
unite in all possible ways, producing four kinds of zygotes: “DD,” “Dd,” “dD,” and “dd.” This can be

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visually represented by drawing a Punnett Square, where you write out the alleles from each possible
gamete and then do the various zygote combinations. When you do this, you get three different
genotypes, but because of dominance, three of the genotypes have the same phenotype. Thus, in the
F2 generation, the plants are tall and dwarf, in a 3:1 ratio, 3 tall plants for every 1 dwarf plant.

From these monohybrid crossing experiments, two key genetic principles were discovered.

The principle of dominance: States that one allele may conceal the presence of another. The dominant
allele affects the phenotype of the organism. The other allele of the pair that has no noticeable effects
on the phenotype is the recessive allele.

The principle of segregation: States that two different alleles segregate from one another during the
formation of gametes (haploid). During fertilization they reunite to produce a diploid zygote.

Mendel also performed experiments with plants that differed in two traits. He crossed plants that
produced yellow, round seeds with plants that produced green, wrinkled seeds. The purpose of these
experiments was to see if the two seed traits, color and texture, were inherited independently.
Because the F 1 seeds were all yellow and round, the alleles for these two characteristics were
dominant. Mendel grew plants from
these seeds and allowed them to
self-fertilize. He then classified the
F 2 seeds and counted them by
phenotype. The four phenotypic
classes in the F 2 represented all
possible combinations of color and
texture traits. Two classes, the
yellow, round seeds and green,
wrinkled seeds resembled the
parental strains. The other two
classes green, round seeds and
yellow, wrinkled seeds showed new
combinations of traits. The four
classes had an approximate ratio of
9 yellow, round; 3 yellow, wrinkled;
3 green, round; 1 green, wrinkled.
Mendel then reasoned that each
trait was controlled by a different
gene segregating two alleles, and
the two genes were inherited
independently. This was the

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foundation for Mendel’s third principle, the principle of independent assortment which states that the
alleles of different genes segregate or assort independently of each other.

This dihybrid cross can be represented pictorially using the Punnett Square method by writing out the
various gamete combinations and then combining them into the various zygote combinations. If this is
done, you can see how Mendel arrived at the 9:3:3:1 phenotypic ratio.

Exceptions to Mendel’s principles

1. Linkage: If two genes are closely located on the same chromosomes, they are called linked gene
and they do not separate independent of each other. Mendel was lucky in that the 7 traits he used in
his land mark studies were found on 7 different chromosomes.

2. Incomplete dominance: Considered an exception to Mendelian principles. A trait regulated by 2

alleles produce 3 genotypes but shows presence of 3 phenotypes. The third phenotype may result
from incomplete expression or incomplete dominance of both alleles as neither allele is completely
dominant over the other. The result is often a blending of the two alleles producing a new phenotype.
Remember that genes which strictly follow Mendelian principles have 3 genotypes producing 2
phenotypes.

Examples

Genotype for fur color Phenotypes for fur color

1. BB (homozygous dominant) Black fur
2. ww ( homozygous recessive) White fur
3. Bw (Heterozygous, incomplete dominant) Grey fur

In humans incomplete dominance is observed for hair type. Neither straight hair nor curly hair is
dominant. The heterozygote has wavy hair.

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3. Codominance: Here again a trait is regulated by 2 alleles. Combination of the alleles produce 3
genotypes and 3 phenotypes. It is thus considered an exception to the Mendelian principles. The third
phenotype here is a result of equal expression of both the alleles (codominance).

Examples:

Genotype for fur color Phenotypes for fur color

1. BB (homozygous dominant) Black fur
2. ww ( homozygous recessive) White fur
3. Bw (Heterozygous, codominant) Black and white fur

In humans, the ABO blood type is a good example of codominance. An individual with AB blood type
has both A and B gene (both dominant gene) expressing equally to produce a distinct phenotype.

4. Multiple alleles: Here a trait is controlled by 3 or more genes. They produce > 3 genotypes and >
4 phenotypes in the population. Human ABO blood type is also an example of multiple alleles. 3 genes
that regulate the trait are A (dominant), B (dominant) and O (recessive). The genotypes and phenotype
associated with ABO blood type is shown below

Genotypes for ABO blood types (6) Phenotypes for ABO blood
types
1. AA or AO A blood type
2. BB or BO B blood type
3. AB AB blood type
OO O blood type

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5. Polygenic inheritance: This occurs when one trait is determined by more than one gene pair.
Hair, skin, and eye color are good examples.

Calculating Genetic Probabilities

The Punnett Square method is ideal for dealing with monohybrid and dihybrid crosses, but it quickly
loses its effectiveness when you deal with more than two genes. Thus, an alternative method should
be used, based on the principle of probability.

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Mendelian segregation is like a coin toss; when a heterozygote produces gametes, half contain one
allele and half contain the other. The probability that a particular gamete contains the dominant allele
is therefore ½, and the probability that it contains the recessive allele is also ½. These probabilities are
the frequencies of the two types of gametes produced by the heterozygote.

We can use frequencies to predict the outcome of other genetic crosses, such as crossing two
heterozygotes. In such a cross, the gametes will be combined randomly to produce the next
generation. Let’s suppose the cross is Aa x Aa (shown below). The chance that a zygote will be AA is
simply the probability that each of the uniting
gametes contains A, or (1/2) x (1/2) = (1/4),
since the two gametes are produced
independently. The chance for an aa
homozygote is also 1/4. The chance for an Aa
heterozygote is 1/2 because there are two
ways of creating a heterozygote—A may come
from the egg and a from the sperm, or vice
versa. Because each of these events has a
one-quarter chance of occurring,
the total probability that an offspring is
heterozygous is (1/4) + (1/4) = (1/2). We
therefore obtain the following probability
distribution of the genotypes from the mating of Aa x Aa: AA = 1/4; Aa = 1/2; aa = 1/4. By applying the
Mendelian principle of dominance, we conclude that (1/4) + (1/2) = (3/4) of the progeny will have the
dominant phenotype and 1/4 will have the recessive.

Problem 1
Let us consider a couple both heterozygous for cystic fibrosis, an autosomal recessive disease. The
defective gene, only when present in homozygous condition causes the disease. If the couple were
to have 4 children, what would be the probability of having:

1. 3 children unaffected and one affected

2. 2 unaffected and 2 affected
3. 1 unaffected and 3 affected
4. 0 unaffected and all 4 affected
5. 4 unaffected and 0 affected

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If “U” stands for unaffected and “A” stands for affected, then

The combinations for 3 children unaffected and one affected will be 4

UUUA, UUAU, UAUU, AUUU

Probability: 4 X (3/4)3 X 1/4=108/256

The combinations for 2 children unaffected and 2 affected will be 6

UUAA, AAUU, UAAU, AUUA, AUAU, UAUA

Probability: 6 X (3/4)2 X (1/4)2 =54/256

The combinations for 1 child unaffected and 3 affected will be 4

UAAA, AAAU, AUAA, AAUA

Probability: 4 X3/4 X (1/4)3 = 12/256

The combinations for 0 unaffected and all four kids affected is 1

AAAA

Probability: (1/4)4 = 1/256

The combinations for 4 children unaffected and 0 affected will be 1

UUUU

Probability: (3/4)4 =81/256

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Problem 2
A couple heterozygous for PKU (phenylketonuria, an autosomal recessive disease) has 3 children.
What is the probability of one child (out of the 3) being a boy and the boy being affected?

As both parents are heterozygous for the disease, probability for their children being affected is ¼ and
probability of being normal (unaffected) is ¾.

3 children; 1 affected (A) and 2 unaffected (U)

Number of combinations: UUA, AUU, AUA=3

Probability of having 1 affected child out of the 3: 3 X (3/4)2 X 1/4 = 27/64

The couple has 3 children. Now let us look at the probability of the couple having 2 girls and 1 boy

The total combinations of boys (B) and girls (G) that the couple can have:

BBB, GGG, GBG, BGB, BGG, GGB, GBB, BBG= 8

3 out of the 8 combinations have 2 G and 1B

So the probability of the boy having PKU is: 27/64 X 3/8

Pedigrees
The diagnosis of genetic conditions or diseases is often a difficult process. The study of these
conditions requires a great deal of careful research, including examining patients, interviewing
relatives, and sifting through vital statistics on births, deaths, and marriages. The accumulated data
can be used to build diagrams that show the relationships and disease heritability among the members
of a family. This type of diagram is known as a pedigree and is one of the most commonly used tools in
medical genetics.

In pedigrees, it is customary to represent males as squares and females as circles; a horizontal line
connecting a circle and a square represents a mating; the offspring of a mating are shown beneath the
mates, using a vertical line, starting with the first born at the left and proceeding through the birth
order to the right; individuals that have a genetic condition are indicated by coloring or shading; finally,
the family generations in a pedigree are usually denoted by Roman numerals, and particular individuals

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within a generation are referred to by regular, Arabic numbers (1st born, 2nd born, 3rd born, and so
on).

Pedigrees are one tool used by the medical profession to medically inform the doctor and patient
about the potential propensity of inheriting a genetic disease or disorder. Traits (phenotype) caused
by dominant alleles are the easiest to identify since every individual who carries the dominant allele
manifests that trait, making it possible to trace the transmission of the dominant allele through the
pedigree. Recessive traits are not so easy to identify because they may occur in individuals whose
parents are not affected. Sometimes several generations of pedigree data are required to trace the
transmission of recessive allele.

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Patterns of Inheritance
Autosomal Dominant Diseases
An autosomal dominant disease is a heritable trait located on one of the 22 non-sex chromosomes
and, by definition, manifests itself in the heterozygote. Autosomal dominant disorders are

• Equally represented among males and females; there is no greater propensity for one sex or
the other.
• When looking at a family history in a pedigree, autosomal dominant disorders are passed
vertically through generations.
• Finally, affected parents have a 50% chance of passing the disease-allele to their offspring
(assuming the afflicted parent is a heterozygote).

One example of an autosomal dominant disorder is Polydactylism, a heritable trait in which the
individual has an extra digit or digits (extra fingers or toes). This disease is not uncommon, affecting 1
in every 500 births. A collection of genes has been implicated in this disease, a family of
developmental transcription factors, specifically, transcriptional repressors—DNA-binding proteins
which shut off transcription of genes at certain key points during embryonic development. An
inactivating mutation in any one of these developmental transcriptional repressor genes can lead to
polydactylism.

Shown here is an actual pedigree taken from a family with polydactylism. Notice the properties of an
autosomal dominant disorder—equal representation in males and females; shows vertical
transmission, from parent to offspring; and the mating of an affected parent with an unaffected one
yields approximately a 50% transmission rate to the children.

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In spite of polydactylism being an autosomal dominant disorder, it exhibits incomplete penetrance
where not all individuals with a dominant mutant allele phenotypically develop the disease. The term
indicates that a dominant allele does not always “penetrate” into the phenotype of the individual.
Thus, penetrance is a measure of how frequently a gene is expressed. Polydactylism has 85%
penetrance in the population at large, meaning that only 85% of persons carrying the abnormal allele
show symptoms of the condition; the remaining 15% never develop polydactylism but can still transmit
the gene to their offspring.

Polydactylism, is also characterized by variable expressivity where there is also considerable variation
in the severity of the disorder or in the type of abnormalities present even though the genetic defect or
genotype is the same among individuals. Expressivity, as it sounds, simply refers to the degree in
which a trait is expressed. In the case of polydactylism, the extra digit may vary from a small skin tag to
a fully formed digit or may vary in terms of the number of additional digits. The molecular explanation
of incomplete penetrance and variable expressivity may not always be understood, but in most cases,
the range of phenotypes is thought to be due to influences of the environment and/or other genes
influencing one trait.

Autosomal Recessive Diseases

Caused by the inheritable trait located on one of the 22 non-sex chromosomes and, by definition,
manifests itself only in the homozygote.

• Autosomal recessive diseases, like dominant diseases, are equally represented among males
and females.
• Autosomal recessive diseases usually manifest themselves among siblings while the parents do
not possess the phenotypic disease (skips generations).
• If both parents are affected then the trait will be expressed in the next generation
• And finally, heterozygous parents have approximately 25% chance of birthing children with
phenotypic disease.

One example of an autosomal recessive disorder is Phenylketonuria, a heritable trait in which

mutations at the locus encoding the metabolic enzyme phenylalanine hydroxylase render homozygotes
unable to metabolize the amino acid phenylalanine. This inability to metabolize phenylalanine results
in a buildup of toxic metabolites that is highly destructive to the nervous system,. As toxic metabolites
build up, the central nervous system becomes progressively damaged and mental capacity is lost

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Sex-linked Diseases
X-Chromosome Inactivation
Sex-linked genes are those genes located on either the X or Y chromosome. Because only about 50-80
functional genes are known to be located on the human Y chromosome, nearly all sex-linked diseases
refer to genes located in the X chromosome, with the X chromosome possessing over 1,000 functional
genes.

Females have 2 X chromosomes and thus twice as many X-linked

genes as males who have only one X chromosome. Yet the quantity of
X-linked proteins are nearly equal in both sexes. How does this
occur? How does the body compensate for this genetic difference?
The process is called dosage compensation by which one of the two
X chromosomes in females is inactivated, thereby equalizing the
production of X-linked proteins between females and males.

So, how is X-linked inactivation in female mammals achieved? This

process is now known to occur very early in female embryonic
development. At this time, each INDIVIDUAL embryonic cell makes an
independent decision to silence one of its X chromosomes. The
chromosome to be inactivated is CHOSEN AT RANDOM. Thus, in
some cells, the X chromosome contributed by the father is inactivated
whereas in other cells the X chromosome contributed by the mother

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is inactivated. Once an X chromosome is chosen to be inactivated in a cell, it will remain inactive in ALL
descendants of that cell. As a result of the X inactivation process, all normal females have two distinct
populations of cells: 50% of her cells have an active paternally-derived X chromosome, and the other
50% of her cells have an active maternally-derived X chromosome. With these two populations of cell,
females are considered genetic mosaics for the X chromosome, meaning a genetic blend. Inactivation
ratio of maternal X: paternal X is 1:1 in a normal female. If this ratio changes, it is called skewed X
inactivation which might occur due to embryological defects

X chromosome has a locus called X inactivation center (XIC) with a gene called XIST. . In humans, the
XIST gene encodes a 17-kb transcript that apparently is NOT translated into any protein. Initially, both
X chromosomes make this XIST transcript. During the random process of X inactivation, whichever X
chromosome (maternal or paternal) has been chosen to be kept ACTIVE, the XIST gene of this X is
silenced or permanently shut off.

The X chromosome that is chosen to be INACTIVATED will keep an active XIST gene, making lots of XIST
transcripts. These XIST transcripts soon begin to migrate from their point of origin, moving along the
particular X chromosome, coating it with the XIST transcripts. This coating leads to initiate methylation
of the many genes on the X thus turning them off. Interestingly,
not ALL genes on an inactivated X chromosome are
transcriptionally silenced.

One unique feature of inactive X chromosomes is that they can be readily identified in mammalian cells
during the cell cycle. The inactive X chromosome condenses into a darkly staining
mass that is tethered to the nuclear membrane. This mass, termed a Barr body, represents a highly-
condensed, transcriptionally-inactive X
chromosome, visible during the S-phase of
the cell cycle. An important note about these
Barr bodies—the number of Barr bodies in
somatic cells is always one less than the
total number of X chromosomes. Normal
females have one Barr body in each somatic
cell while normal males have none.

With this understanding, we can now

approach X-linked diseases.

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X-linked dominant Diseases
• Manifest in all individuals who carry an X chromosome with the mutant gene. This is true for
heterozygous women and hemizygous men (“hemizygous” refers to only one copy of each X-
linked gene due to the presence of only one X chromosome).
• As females have two X chromosomes, either of which can potentially carry the disease gene,
they are about twice as commonly affected as males.
• Because this is a dominant trait, X-linked dominant disorders are passed vertically through
generations, whereby the disease phenotype is seen in generation after generation.
• Now, unlike autosomal dominant disorders, X-linked dominant disorders are transmitted in a
certain pattern depending on whether the mother or father is the disease carrier. If the father
is the one afflicted with the disease, he CAN NOT transmit the trait to his son yet will transmit
the disease to ALL of his daughters. Affected mothers are usually heterozygotes and thus have
a 50% chance of passing the disease allele to their sons or daughters.

One example of an X-linked dominant disease is Rett syndrome. This is a neurodevelopmental disorder
with varying severity, affecting ~ 1 in 10,000 females and is the second most common form of
neurodevelopmental disorder in females, after Down syndrome. Rett syndrome is seen in a much
smaller percentage of males. The gene largely responsible for this disease is the MECP2 (methyl
cytosine binding protein 2) gene. This protein is extremely important for nervous system development
and function. The MECP2 protein primarily acts as a transcriptional repressor by binding to methylated
cytosines in the genome and recruiting other proteins which serve to turn off transcription. The loss of
functional MECP2 protein in neurons leads to loss of transcriptional repression and inappropriate and
uncontrolled transcription of various genes involved in brain development.

Shown here is a pedigree from a family with Rett syndrome. The mother in the F2 generation is a
carrier for this mutated MECP2 gene located on the X chromosome, yet she has not succumbed to the
disease phenotype, in spite of this disease being X-linked dominant. This is likely due to preferential
silencing of the mutated X chromosome—a majority of the X chromosomes with the MUTATED MECP2
gene were likely inactivated whereas the majority of her body’s somatic cells are using an active,
normal X chromosome, resulting in a normal phenotype.

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 X-linked recessive diseases
• Much more common and prevalent than X-linked dominant diseases.
• Like the autosomal recessive diseases, X-linked recessive disease phenotypes are certainly
manifested in homozygous carriers only.
• Men, who are hemizygous for the X chromosome, will be affected by the disease if they
acquire an X chromosome with the mutant gene since they lack a second X chromosome. Thus,
males are more frequently affected with X-linked recessive diseases than are females.
• Females are carriers of the disease and are phenotypically normal (as they have 2 X
chromosomes). A heterozygous female will only express the disease if she has skewed X
inactivation.
• These diseases skips generations before re-appearing.
• An affected father CAN NOT transmit the trait to his son because the father has to donate a Y
chromosome. However, if the father is afflicted with the disease, then ALL of his daughters will
inherit the disease gene and become a carrier because fathers must pass their X chromosome
to their daughters.
• Women heterozygous for the affected trait (which is commonly the case), have a 50% chance to
pass the trait to their sons or daughters. For a son who receives the mutant X chromosome, in
spite this being a recessive trait, sons who receive the mutant X chromosome will display the
disease phenotype.

One example of an X-linked recessive disease is hemophilia A. Hemophilia A is the most common of
the severe bleeding disorders, affecting one in ~ 5,000 to 10,000 males worldwide. One of the most
famous cases of X-linked hemophilia A occurred in the royal families of Europe in the 20th century.
Queen Victoria of the United Kingdom carried the mutant gene for hemophilia A, transmitting it to one
son and two carrier daughters. These children then transmitted the gene to many members of the
various European royal families. Note the royal pedigree outlining hemophilia A. You can clearly see,
in this X-linked recessive disease, the predominance of males afflicted with the disease whereas the
females are the carriers who transmit the mutant X chromosome to their affected sons.

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Hemophilia A is caused by deficient or defective factor VIII, a key component of the clotting cascade.
The genetic location for factor VIII is located along the distal portion of the long arm of chromosome X.
Fibrin formation is affected, resulting in prolonged and often severe bleeding from wounds and
hemorrhages in the joints and muscles. Bruising is also frequently seen. Current treatment includes
supplementation with recombinant factor VIII.

Y-linked Diseases
Because the Y-chromosome is small and does not contain many genes, few traits are Y-linked, and Y-linked
diseases are rare. Since only males have a Y chromosome, Y-linked traits are passed only from father to son, in
every generation with no inter-chromosomal genetic recombination.

3 characteristic features

• The trait occurs in males only

• It reoccurs in all sons of affected males
• The daughters of affected men are not
only phenotypically normal but also
do not have affected offspring

Mitochondrial Inheritance
Mitochondria, are the sites of the cellular energy production in form of ATP, are thought to have
originated when a proto-eukaryotic cell formed a symbiotic relationship with a bacterial cell. Over
time, the majority of the bacterial genome was transferred to the nucleus of the proto-eukaryotic cell,
but not all. Mitochondria, contains a small circular chromosome with 13 genes that code for the very
hydrophobic proteins required in the electron transport chain, 2 ribosomal RNA genes, and 22 tRNA
genes. Mutations in these genes most often result in devastating consequences and are often lethal. A

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few mutations in the mitochondrial DNA can allow viable offspring to be born. These individuals usually
have defects in tissues, such as the brain, muscle, and heart, which require a lot of cellular energy to
perform their functions. During conception, the mitochondria from the sperm do not enter the egg.
Thus, only the mitochondria from the mother are passed to the embryo. Even though only the mother
can pass on mitochondrial DNA defects, the results can affect both male and female offspring equally.

Leigh’s Syndrome

An example of an inherited mitochondrial disorder. Most individuals with the condition do not survive
past the age of 6 years. While most people with Leigh syndrome have a mutation in nuclear DNA,
about 20 percent have a mutation in mtDNA.

Most genes mutations causing Leigh syndrome are involved in the process of energy production in
mitochondria. Mitochondria use oxygen to convert the energy from food into a form cells can use
through a process called oxidative phosphorylation. Five protein complexes, made up of several proteins
each, are involved in this process. The complexes are named complex I, complex II, complex III, complex
IV, and complex V. During oxidative phosphorylation, the protein complexes drive the production of
adenosine triphosphate (ATP), the cell's main energy source, through a step-by-step transfer of
negatively charged particles called electrons. Many of the gene mutations associated with Leigh
syndrome affect proteins in these complexes or disrupt their assembly. These mutations reduce or
eliminate the activity of one or more of these complexes, which can lead to Leigh syndrome.

Many individuals with this condition develop weakness or paralysis of the muscles that move the eyes
(ophthalmoparesis); rapid, involuntary eye movements (nystagmus); or degeneration of the nerves that
carry information from the eyes to the brain (optic atrophy). Severe breathing problems are common,
and these problems can worsen until they cause acute respiratory failure. Some affected individuals
develop hypertrophic cardiomyopathy, which is a thickening of the heart muscle that forces the heart to
work harder to pump blood. In addition, a substance called lactate can build up in the body, and
excessive amounts are often found in the blood, urine, or the fluid that surrounds and protects the brain
and spinal cord (cerebrospinal fluid) of people with Leigh syndrome.

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Lecture 8 - Mutations and DNA Repair
Learning Objectives:

• What are mutagens and how do different mutagens cause

different types of mutations?
• Identify and characterize the various types of genetic
mutations.
• Learn the clinical correlates associated with the different
types of gene mutations discussed in class.
• Identify, characterize and differentiate between different
types of DNA repair mechanisms existing at cellular levels.
Causes of Genetic Mutations
The human genome contains ~ 3 billion base pairs. To replicate this amount of genetic material
without incurring a mistake is asking a lot of the cellular replication machinery. Also, metabolic
processes within the cell can generate toxic, reactive byproducts that need to be removed lest they
cause DNA damage. Finally, environmental factors can also lead to DNA damage. If these replicative
mistakes or damaged regions in the DNA are not repaired, a mutation can result. A mutation can be
simply described as any heritable change in DNA sequence; however, mutations often have deleterious
consequences for the host organism, such as causing various diseases.

Genetic mutations can be broadly classified into spontaneous mutations and induced
mutations.

• Spontaneous mutations can occur due to mistakes in normal, cellular biosynthetic reactions.
One example would be replication errors, where an incorrect nucleotide is added during DNA
replication. Another type of spontaneous mutation would be the generation of toxic waste
products during aerobic respiration (e.g., superoxide radicals), which, if not efficiently removed,
can lead to spontaneous DNA damage.
Finally, a third type of spontaneous
mutation is due to mutagenic nucleotides.
These are naturally-occurring nucleotides

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 which undergo a spatial re-arrangement of their atoms to assume a slightly different form. This
spatial restructuring is known as a tautomeric shift, in which the H-atom is shifted from one
position in the base to another, altering the bonds between atoms, and allowing the newly-
restructured nucleotide to form a non-Watson-Crick base pair.

• Induced mutations are caused by external/ environmental agents called mutagens. There are
various types of mutagens in the environment.

(1) Ionizing radiation: This is a form of energy that is able to liberate or eject electrons from an atom or
molecule. This process, called ionization, results in the formation of two charged particles or ions: the
molecule with a net positive charge, and the free electron with a negative charge. These two species
are very reactive, with the potential to chemically modify or react with the genetic environment (DNA).
Examples of ionizing radiation include X-rays and gamma rays.

(2) Non-ionizing radiation: This type of energy does not yield charged particles or ions but can move
electrons from inner to outer orbits within an atom, causing the atom to become chemically unstable.
One example of non-ionizing radiation is
ultraviolet radiation from the sun. UV
radiation can cause the formation of
covalent bonds between adjacent
pyrimidine bases, forming pyrimidine
dimers (usually thymine), which are then

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unable to properly base pair with purines during DNA replication.

(3) Chemicals: A variety of chemicals can also induce various types of mutations within the DNA.
Examples include alkylating agents, which are agents that modify DNA by attaching alkyl groups to the
bases, ultimately interrupting correct base pairing and causing a mutation. Intercalating agents, such
as acridine dyes, can physically insert themselves between DNA bases, distorting the DNA helix and
causing frameshift mutations. Base analogs, such as 5-bromouracil or the anti-HIV drug AZT, can be
substituted for a true base during DNA replication.

(4) Biological agents: Certain biological elements can alter the genetic material and lead to mutations.
Examples include infectious microbes like viruses that can insert their genetic material into eukaryotic
DNA, causing mutations due to an ill-placed insertion.

According to the type of genetic changes induced, mutations can be

classified into
[Link]-pair substitution:
One base is replaced by another (also called a point mutation). If a purine is replaced by another
purine, or a pyrimidine replaced by another pyrimidine, this mutation is called a transition mutation.
If a purine is replaced with a pyrimidine or vice versa, this mutation is called a transversion mutation.
Base-pair substitutions can result in a change in the amino acid sequence. However, because of the
degeneracy of the genetic code, many of these base-pair substitutions do not, in fact, change the
amino acid sequence and thus have no consequence. These types of base-pair substitutions, which do
not change the amino acid sequence, are called silent substitutions or silent mutations. Non-silent

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mutations consist of two basic types: missense mutations and nonsense mutations. Missense
mutations are base-pair substitutions which result in an amino acid change while nonsense mutations
are base-pair substitutions which produce one of the three stop codons in the mRNA.

Sickle Cell disease: Results from a single base

pair [Link] is an autosomal recessive
disorder. The mutation that causes this disease is a
missense mutation—adenine is replaced by
thymine, resulting in a new codon. This new codon
codes for valine when the normal codon and amino
acid should be glutamic acid. This missense
mutation occurs in the β-globin gene of
hemoglobin. This one amino acid change results in
a fairly large deformation in the hemoglobin
protein, and the red blood cells which carry these
mutated hemoglobin proteins assume a
characteristic “sickle” shape, rendering them
ineffective.

One of the main problems with this disease is that

sickle cells are unable to provide adequate oxygen
to the rest of the body due to their abnormal
shape, which prevents these cells from accessing
various tissues. Also, these abnormal red blood
cells tend to stick to the vascular endothelium (the

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inner lining of the blood vessels). The resulting vascular obstruction produces localized hypoxemia
(lack of oxygen) and can result in painful episodes. Finally, sickled red blod cells die much more quickly
than normal red blood cells. Normal, healthy red blood cells live in the body for ~ 120 days; sickled red
blood cells only live for 10-20 days. This heightened death of the red blood cells causes anemia (lower-
than-normal red blood cell count).

[Link] or deletion:
This type of mutation consists of adding or removing 1 or more base pairs from the DNA. This type of
mutation tends to be especially harmful when the number of extra or missing bases is not a multiple of
3 because the reading frame for translation consists of tri-nucleotide codons.
If an insertion or deletion mutation is not a multiple of 3, this can alter the reading frame and then is
termed a frameshift mutation.

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Huntington Chorea: One human disease attributed to an insertion mutation is Huntington’s
disease, an autosomal domiannt disorder. This, like Alzheimer's disease, is a neurodegenerative
genetic disorder that affects muscle coordination and leads to cognitive decline and psychiatric
problems. Huntington’s disease is due to a tri-nucleotide insertion in the Huntington gene. The
Huntington gene normally codes for the Huntington protein which is involved in the transport of
vesicles in various cellular secretory pathways. The defect occurs when a CAG repeat is inserted into
the Huntington gene, producing a modified protein with an additional string of glutamines at the
amino terminus of the Huntington protein. This CAG tri-nucleotide repeat varies in length between
individuals and may change length between successive generations as the gene is passed from parent
to child. When the length of this repeated section reaches a certain threshold, it produces an altered
form of the protein, called mutant Huntington protein (mHtt). This mutant protein, while not
completely understood, is very toxic to neuronal cells in the brain, leading to apoptosis or neuronal cell
death. The greater the number of CAG repeats inserted into the DNA correlates with greater severity
of the disease and age of onset.

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Fragile X syndrome: Another tri-nt repeat disease, an X-linked dominant disorder. This genetic
disease is the most widespread and most common single-gene cause of autism and mental retardation.
It results in an array of intellectual disabilities ranging from mild to severe as well as physical
characteristics such as elongated face, large or protruding ears, hypermobile joints, and large testes.
The disease was so named because the X-chromosomes of afflicted individuals show a propensity to
break when cultured in a special medium in the laboratory.

Fragile X is caused by a tri-nucleotide repeat in a gene

called the FMR1 gene. The normal function of the
FMR1 protein is to assist mRNA export from the
nucleus to the cytoplasm. In a normal X chromosome,
the FMR1 gene contains 6-50 copies of a CGG repeat.
Those with Fragile X Syndrome have > 230 inserted
repeats, with repeats in many individuals totaling near
4,000. This large addition of CGG repeats causes a
constriction in the X-chromosome and makes it fragile
(can break easily). You can actually see this fragile
portion of the X-chromosome, these long CGG
repeats, in a standard human karyotype.

Interestingly enough, the repeats themselves are not what cause symptomatic disease. This is because
the repeats are inserted in a 5’-region of the mRNA that is not translated. What causes symptomatic
disease is a silencing of the FMR1 gene. When there are more than 230 CGG repeats in the 5’-region of
the FMR1 gene, DNA methyltransferases methylate (add a methyl group) these repeats, which sends a
signal to the cell not to transcribe this gene. Thus, the absence of this protein, due to gene silencing,
leads to disease.

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[Link]-site mutation:
These types of mutations can occur in the DNA sequence that defines the splicing sites in a pre-mRNA
molecule. Each pre-mRNA molecule has a consensus sequence that denotes the 5’-splice site and a
sequence that denotes the 3’-splice site. The spliceosome recognizes these short, consensus
sequences to properly cut and excise unwanted introns. Changing a single nucleotide at these
consensus sites alters the sequence and may prevent splicing from occurring. Alternatively, a splice-
site mutation may involve the formation of a new, unwanted splice site somewhere else in the pre-
mRNA (such as in an exon).

DNA Repair
As stated earlier, several processes can lead to mutations including ionizing and nonionizing radiation,
mutagens, and replication errors. Cells have developed several responses to the presence of this DNA
damage. Some cells will enter a state of suspended animation involving cell cycle check points and
transcriptional program activation. Some cells go into a programed cell death mode called apoptosis.
Many cells however will attempt to fix the damage through DNA repair mechanisms.

Different DNA repair mechanisms exist and are involved in the repair of different types of DNA
damage.

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1. Direct Reversal
This is most direct type of repair. As the name implies, the DNA
damage is reversed by direct enzymatic modification of the damaged
base. No nucleotides are removed from the DNA strand during this
process. This type of repair is used with base modification types of
DNA damage. An example is the damage caused by the mutagen N-
nitrosodimethylamine. N-nitrosodimethylamine will interact with the
guanine base and the result is methylation of the oxygen associated
with carbon 6 in the purine ring forming O6-methyguanine. This type
of modification changes the normal G-C base pair to a G-T base pair.
To correct this base modification, an enzyme called O6-
methylguanine methyltransferase will remove the methyl group and
thus restore the normal guanine residue.

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2. Excision Repair
There are three types of excision repair mechanisms: Base excision,
Nucleotide excision, and Mismatch. These mechanisms have three
common steps: 1. DNA repair endonucleases excise the damaged
base/nucleotides, 2. DNA polymerase fills in the gap, and 3. DNA
ligase seals the DNA strand breaks.

A. Base excision repair

Base excision repair involves DNA damage that does not cause a
distortion in the DNA strands. These types of damage include
oxidation, deamination, alkylation or methylation of the bases. This
repair mechanism begins with the repair enzymes recognizing the
modified base and cleaving the glycosidic bond holding the
nitrogenous base to the deoxyribose sugar. Next the de-based
nucleotide is removed from the DNA strand. DNA polymerase fills in
the space and DNA ligase fixes the break.

B. Nucleotide excision repair

Nucleotide excision repair involves DNA damage that distorts the DNA double helix. An example of this
type of damage is the formation of thymidine or
pyrimidine dimers. The repair enzymes recognize the
DNA distortion and will start the repair process by
cleaving the phosphodiester bonds near the DNA
damage. Approximately 24-32 nucleotides will be
removed near the DNA distortion. Next DNA polymerase
fills in the gap and DNA ligase fixes the strand breaks.

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Xeroderma pigmentosum
A rare autosomal recessive genetic disorder of DNA repair in which there is ability to repair damage
caused by ultraviolet (UV) light. The absorption of the high-energy UV -light leads to the formation of
pyrimidine dimers, namely cyclobutane-pyrimidine dimers and pyrimidine-6-4-pyrimidone
photoproducts at cellular levels. In a healthy, normal human being, the damage is first excised by
endonucleases. DNA polymerase then repairs the missing sequence, and ligase "seals" the transaction.
This process is known as nucleotide excision repair. In a patient with Xeroderma, nucleotide excision
repair enzymes are mutated so the normal repair process is impaired. UV induced damage to DNA in
epidermal cells is common in these patients. Multiple basal cell carcinomas (basaliomas) and other skin
malignancies frequently occur in the patient. All exposure to sunlight is often forbidden for the patient.

C. Mismatch repair
Mismatch repair is used to fix base mismatches that occur during DNA replication. These mismatches
result in small distortions in the DNA strands. The mechanism of repair is similar to nucleotide excision
repair where several nucleotides are removed prior to DNA polymerase filling in the gap and DNA
ligase fixing the strand break. The important step for this repair mechanism is the determination of
which strand is the parental stand and which one is the newly made daughter strand. The parental
strand is the strand the cell wants the use as a template when polymerase fills in the gap after the
nucleotide removal. To determine which strand is the parental strand, repair enzymes recognize
methylated sites on the parental strand which are absent on the daughter strand. This ensures that
the nucleotide placed in the daughter strand is recognized as the mismatched nucleotide.

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121
Lecture 9 - Enzymology

Learning Objectives:

• Describe the characteristics of enzymatic reactions from the

viewpoint of free energy, equilibrium, and kinetics.

• Describe the general structure/composition of enzymes and

factors which affect their catalytic activity.

• Describe enzyme kinetics based on the Michaelis-Menten

equation and the significance of the Michaelis constant (K m ).

• Differentiate between the mechanisms of action of different

types of enzyme inhibitors with reference to Lineweaver-Burk
plots.

Enzymes
Enzymes are organic protein molecules that act as catalysts. A catalyst is any substance that speeds up
the rate of a chemical reaction without itself being consumed or permanently changed by the reaction.
Enzymes act as catalysts by lowering the energy needed for biochemical reactions.

• Biochemical catalysts (proteins/RNA) present in cells

• They accelerate the rates of biochemical reactions at cellular levels
• Characterized by high substrate specificity
• Affected by temperature & pH
• Remain unaltered at the end of the reaction(s) and can be reused

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Factors affecting Enzyme activity

• Temperature- Active at optimal temperature. Gets denatured at higher temperatures and

remains inactive at lower temperatures

• pH- Different enzymes act at different pH. Change of pH inactivates the enzyme

• Ionic strength (salt)

• Cofactor availability

Enzyme Structure
Each enzyme found in the body is unique in that the enzyme is built to specifically interact with a
specific compound known as the substrate (can also be called the reactant, or starting material). This
selectivity of an enzyme for a particular substrate is known as the enzyme’s substrate specificity. The
specific substrate binds to the substrate binding site of the active site of the enzyme. Each enzyme
also catalyzes a specific type of reaction which is called its reaction specificity controlled by the

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catalytic site of the active site The amino acid composition and 3D arrangement of the amino acid in
the active site determines the activity of the enzyme

There are two main models for how a substrate can bind to the enzyme’s active site.

Lock and Key model: In the lock-and-key model, the substrate and enzyme’s active site possess
complementary surfaces or shapes—the substrate fits perfectly into the active site just as a key would
fit smoothly into a rigid lock. For example, glucose fits perfectly in the active site of the enzyme
glucokinase but galactose does not.

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Induced-fit model: In the induced-fit model, the substrate initially binds to the enzyme and induces a
conformational change in the active site that repositions the side chains of the amino acids to increase
or enhance the binding interactions with the substrate.

In order for a reactant to be converted to a product in a biochemical reaction, the reactant must go
through a sequence of molecular rearrangements that results in a highly-unstable transition-state
intermediate. The structure of the transition-state intermediate is partway between reactant and
product, but its free energy content is higher than either the reactant or product (this means energy
must be put into the reaction to get to this transition state intermediate; this required energy is known
as the activation energy). In order for the reaction to proceed all the way to product, this activation
energy barrier must be overcome. Enzymes serve to LOWER this activation energy barrier.

Cofactors and coenzymes

A cofactor is a non-protein chemical compound that is bound to an enzyme and is required for the
enzyme’s biological activity. Cofactors can be thus be considered "helper molecules" that assist in
biochemical reactions. A cofactor is typically inorganic in nature.

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An inactive enzyme, without the cofactor is called an apoenzyme, while the complete enzyme with
cofactor bound to the active site is a holoenzyme.

Coenzymes are loosely bound organic molecules that serve as helper molecules for enzymes. They are
mostly vitamin derivatives.

Allosteric site: A second, important site on the enzyme, separate or distinct from the catalytic/active
site is known as the allosteric site. The allosteric site is reserved for allosteric molecules. These
molecules can be activators or inhibitors that bind to the allosteric site and modify the conformation of
the enzyme, thereby affecting the enzymatic reaction.

Some enzymes are grouped into a sub-category known as isoenzymes (also called isozymes).
Isoenzymes are enzymes that act on the same substrate and catalyze the same biochemical reaction,
producing the same product. However, they differ in their amino acid sequence (mostly marginally)

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and have differential expression in different tissues. They often differ in their kinetic properties.
Examples: Glucokinase (express in the hepatocytes & pancreatic beta cells) and Hexokinase (express all
peripheral tissues)

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Enzyme Classification

Enzyme Kinetics
Enzymatic reactions occur in 3 steps. We start with an enzyme, “E,” which binds to substrate, “S” (the
reactant). This interaction is the 1st step of an enzyme-catalyzed reaction—the formation of the
enzyme-substrate complex. The 2nd step involves converting the substrate “S” in the enzyme-substrate
complex to product, “P,” with the newly-formed product still bound to the enzyme. Finally, in the 3rd
step, the product, “P,” is released from the enzyme, freeing the enzyme to find another substrate
molecule and start the reaction over again. There are double arrows drawn because this is an
equilibrium type of reaction, meaning the reaction can go either way.

The conversion of substrate into product occurs over time, defined as “v,” the velocity of the reaction,
where less product formed means LESS available enzyme and more product formed means MORE
available enzyme. This can be represented in the graph below. When the substrate concentration, [S],
is low, only a fraction of the available active sites of the enzyme molecules are occupied; thus, the rate
or velocity of the reaction is dependent on [S]. However, as [S] concentration increases, the reaction
rate increases also, eventually reaching an asymptote. The asymptotic point is known as V max , the
highest rate of the reaction when ALL active sites of ALL enzyme molecules are occupied by substrate.
The linear part of the curve is called 1st order kinetics because a change in [S] leads to a direct change
in v. The plateau part of the curve is known as zero order kinetics because a change in [S] will NOT
lead to a direct change in v.

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KINETICS of an enzyme catalyzed reaction is provided by Michealis-Menten equation

Vi = initial velocity; Vmax = maximum velocity of the reaction; Km = affinity of an enzyme for its substrate

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The kinetic parameter, K m : Known as the Michaelis-Menten constant and refers to the substrate
concentration that yields half the maximal reaction rate. Thus, K m is a numerical value for S that yields
½ V max .

K m also represents the inverse affinity of an enzyme for a particular substrate. For example, a
smaller K m represents greater affinity of the enzyme for that particular substrate (practically, this
means that less substrate is required to reach ½ V max) ; conversely, a larger K m represents lower affinity
of the enzyme for that particular substrate (this means that more substrate is required by the enzyme
to reach ½ the maximum velocity, V max ).

Lineweaver-Burk plot
This is a slightly modified enzyme kinetics graph. Inversion of the Michaelis-Menton plot gives a linear
Lineweaver-Burk Plot allowing for more accurate determination of V max etc.

This plot represents a straight line and can be fitted to a straight-line equation: y = mx + b, where “y” =
1/v, the slope, “m” = K m /V max , “x” = 1/[S], and the y-intercept, “b” = 1/V max .

Enzyme Inhibition
Enzymes are extremely important therapeutic targets, especially in treating numerous viral infections.
There are 2 general types of enzyme inhibitors: reversible inhibitors and irreversible inhibitors.
Reversible inhibitors are drugs that bind to the enzyme but can come back off. These reversible
inhibitors interact with the enzyme through non-covalent interactions such as H-bonding, ionic and

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hydrophobic interactions. Irreversible inhibitors interact with the enzyme through covalent
interactions and hence can permanently modify/inactivate the enzyme.

Reversible inhibitors can be further subdivided based on mechanism of inhibition

• Competitive inhibition: Here the inhibitor mimics the substrate in structure and COMPETES
with the substrate for binding to the enzyme’s catalytic site. Competitive inhibitors when
present in high concentrations, outcompete the enzyme substrate and bind to the active site of
the enzyme. It thus decreases the affinity of the enzyme for its substrate and prevents
formation of the enzyme-substrate complex.

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Effect of competitive inhibitor on enzyme activity- Graphical presentation

• Non-competitive inhibition: Here the inhibitor is so named because it DOES NOT COMPETE
with the substrate for binding to the enzyme’s catalytic site. Instead, the non-competitive
inhibitor binds to the enzyme at a site SEPARATE from the active site. This causes a
conformational change in the enzyme which blocks substrate binding and thus product
formation. A non-competitive inhibitor can inhibit both the first and the second step of the
enzyme catalyzed reaction leading to decrease in the velocity of the reaction. It does not affect
the affinity of the enzyme for its substrate.

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Effect of noncompetitive inhibitor on enzyme activity- Graphical presentation

Heavy metals (like lead, mercury etc) act as a non-competitive inhibitor of enzymes like
ferrochelatase, the last enzyme in heme synthesis. The heavy metals blocks the sulfhydryl side
chains of cysteine residue in the enzyme protein, preventing protein folding, leading to
inactivation of the enzyme.

Uncompetitive inhibition: This is so termed because it doesn’t fall into either the competitive
or non-competitive inhibitor categories. The inhibitors in this case binds to a site on the
enzyme ONLY AFTER the enzyme binds to its substrate. Thus, the uncompetitive inhibitor binds
only to the enzyme-substrate complex; it does not bind to free enzyme. Once bound, the
uncompetitive inhibitor prevents the product from being formed, inhibiting the second step of
an enzyme catalyzed reaction. So the presence of the inhibitor eventually reduces the reaction
velocity but increases the affinity of the enzyme for the substrate (although no product
formation occurs).

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Effect of uncompetitive inhibitor on enzyme activity- Graphical presentation

Lithium: An uncompetitive inhibitor of the enzyme inositol monophosphatase is used in the

treatment of bipolar disorder. It prevens the recycling of inositol by uncompetitive inhibition of the
enzyme inositol monophosphatase and thus slows down IP3-DAG related cell signalling pathways in
target cells like neurons.

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Lecture 10 - Vitamins
Learning Objectives:

• Describe vitamins in general

• Know each water soluble vitamin, their roles at cellular levels;
the deficiency disorders associated with each vitamin;
symptoms of these disorders.
• Know the different fat soluble vitamins, their absorption
mechanism, storage forms in the body, cellular functions,
deficiency disorders, and toxicity issues.
• Describe the role of vitamins as coenzymes in promoting
enzyme activity
• Compare and contrast the functions & deficiency disorders of
different vitamins

Most vitamins are essential nutrients meaning humans do not possess the enzymes for their synthesis and thus
the vitamins must be supplied in their diets. In fact, humans only have enzymes that allow for the synthesis of
one vitamin in sufficient quantities without dietary supplementation, vitamin D. The name vitamin comes from a
compound word developed in the early 1900’s by Kazimierz Funk, a polish biochemist. It was shown that organic
micronutrients that were “vital” for survival and prevention of the disease beriberi contained an amine. Funk
coined the term vital amine which was later shortened to vitamin.

Vitamins are organic molecules meaning they contain carbon with carbon-carbon or carbon-hydrogen bonds.
Unlike the other organic macromolecules of the body (carbohydrates, proteins, and lipids), vitamins do not
provide energy. Most act as individual units instead of polymers and function as cofactors in enzymatic reaction.
They provide specificity, proximity, and orientation in the substrate recognition site of enzymes.

Vitamins can be separated based on their solubility. In humans there are a total of 13 recognized vitamins of
which 4 are fat soluble and 9 water soluble. The solubility of the vitamins determines how the vitamin is
digested from foods that are consumed, absorbed in the GI tract, transported, and stored throughout the body.

Water Soluble Vitamins

Thiamine (B 1 )

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Thiamine is vitamin B 1 . Its active form is thiamine pyrophosphate (TPP) which is simply the
phosphorylated form of thiamine. TPP is essential in carboxylase reactions in addition to some
transferase reactions. It is also used as an important coenzyme in the biosynthesis of acetylcholine and
gamma-aminobutyric acid (GABA).

TPP is an important cofactor in the enzyme pyruvate dehydrogenase. This enzyme converts pyruvate
(made during glycolysis) into the acetyl component of acetyl-CoA. The pyrophosphate component aids
TPP binding to the enzyme. The reactive carbon, which contains a dissociable proton that upon
removal generates a carbanion, extends into the enzymes active site. The carbanion forms a covalent
bond with a keto group of the substrate while cleaving the adjacent carbon-carbon bond. In the case of
pyruvate this generates CO 2 and the acetyl (acetaldehyde).

Sources: Thiamine is synthesized by bacteria, fungi and plants. Animals must consume the vitamin
since they lack the necessary enzymes for its synthesis. Food Sources: Seeds, nuts, wheatgerm,
legumes, lean meat

Deficiency

• Deficiency in thiamine can result from inadequate intake, decreased GI absorption, reduction in
liver stores due to hepatic steatosis and fibrosis, or lack of magnesium which is essential to
incorporation of the vitamin into enzymes that use it.
• Consumption of ethanol can also lead to a thiamine deficiency. Ethanol not only effects
absorption of dietary thiamine but also blocks the formation of TPP.
• Wernicke-Korsakoff syndrome is often seen in alcoholics. Symptoms might begin as loss of
appetite and progress to depression, peripheral neuropathy and unsteadiness. Deterioration

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 beyond that point includes paralysis eye, abnormal stance and gait, deranged mental function,
amnesia like symptoms, and impairment of conceptual functions.
• Severe thiamine deficiency is called Beriberi (translates as I can’t I can’t). There are two forms
of this condition however both usually present at the same time. Effects to the peripheral
nervous system including impairment of sensory, motor, and reflex in distal more than proximal
limb segments is referred to as Dry beriberi. Effects to the cardiovascular system are referred to
as Wet beriberi.
• Thiamine is destroyed/unstable in the presence of heat, UV light, and gamma irradiation. Foods
that are frozen do not lose the vitamin.

Riboflavin (B 2 )
Riboflavin is a three ringed structure (isoalloxazine) attached to a sugar alcohol (ribitol) molecule. It is
used in the synthesis of flavin monophosphate (FMN) [addition of a phosphate to riboflavin] and flavin
adenine dinucleotide (FAD) [addition of AMP to FMN]. Proteins which contain FMN or FAD are called
flavoproteins.

FMN and FAD are cofactors in enzymes

involved in oxidation-reduction reactions
which are important in the metabolism of
carbohydrates and lipids. FMN and FAD are
able to transfer two electrons via protonation
of the isoalloxazine ring.

Sources: Food sources: Milk, meats, nuts,

legumes

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Deficiency

• A deficiency in riboflavin can occur without replenishing in diet. A riboflavin deficiency alone is
not very common. It is usually accompanied by deficiency with other vitamins. Hypothyroidism
is known to effect the formation of FMN and FAD.
• The symptoms of a deficiency are of an oral-ocular-genital syndrome: Angular cheilitis,
photophobia, and scrotal dermatitis.
• Riboflavin is heat stable and survives cooking. However, it is very susceptible to UV light
degradation.

Niacin (B 3 )

Niacin exists as two forms, nicotinic acid and nicotinamide. Nicotinic acid is classically referred to as
niacin but both forms exist and function in the body with nicotinamide more prevalent. Both forms are
converted to nicotinamide adenine dinucleotide (NAD) or its phosphorylated form nicotinamide
adenine dinucleotide phosphate (NADP).NAD and NADP are involved in oxidation/reduction reactions.

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Synthesis

Niacin is not completely essential. The liver can synthesize

niacin from the amino acid tryptophan. This conversion is
not very efficient and cannot supply adequate levels to
sustain bodily functions and thus some niacin must be
supplied by the diet. Thiamine, riboflavin, vitamin B 6 and
iron are important cofactors in the enzymatic reactions of
this process. Thus a deficiency in any of them can result in a
niacin deficiency.

Food sources: meats, nuts, legumes

Deficiency

Pellagra: Chronic deficiency leads to the condition.

Symptoms of a niacin deficiency include nausea, skin and
mouth lesions, anemia, headaches, and tiredness. The
progress of pellagra involves dermatitis, diarrhea, and
dementia (the 3Ds). If left untreated, pellagra can lead to
death.

Hartnup’s Disease is a genetic condition that leads to niacin

deficiency. It is a defect in the kidneys and intestines. The result of this condition is a difficulty in
breakdown and absorbing dietary tryptophan which leads to a reduction in the amount of niacin that
can be synthesized in the body.

Toxicity: Niacin is toxic at pharmacological doses (1.5-6 g per day). Niacin flush is a disease
characterized by itchy to tingly sensation of the skin. In severe cases, it can become very painful. Niacin
flush is the result of increased levels of nicotinic acid. High levels of nicotinamide do not show these
symptoms, however, nicotinamide can be converted to nicotinic acid. Niacin flush usually only occurs
in people taking supplements which contain high levels of nicotinic acid.

Pantothenic Acid (B 5 )

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Pantothenic acid cannot be synthesized by animals. Only
the D form is biologically active. Pantothenic acid is
required for the synthesis of Coenzyme A. It is added to
many cosmetic products but its use there is not validated.

Deficiency is very rare and would be accompanied with

other nutrient deficiencies. Toxicity has not been reported.

Food Sources: small amounts in all foods, yeast, grains, egg

yolk, liver

Fig: Structure of COASH derived from vit B5

Pyridoxine (B 6 )

Several forms of vitamin B 6 exist including pyridoxine, pyridoxal, pyridoxamine and their 5’ phosphates
forms. Pyridoxine is the most common in the diet and in supplementation form. Pyridoxal phosphate
(PLP) is the active form of the vitamin.

Food Sources: small amounts in most foods, yeast, liver, wheat germ, nuts, beans, bananas

Functions

• Pyridoxine is an important cofactor in amino acid metabolism including transamination and

deamination reactions. It is involved directly in the removal of the amine group from the amino
acids.
• Pyridoxine is also important for glycogen utilization. In the enzyme glycogen phosphorylase,
pyridoxine serves to stabilize the incoming phosphate molecule which is used during the
removal of glucose units from the glycogen molecule.
• Other roles of pyridoxine include synthesis of neurotransmitters, serotonin, and
noradrenaline, lipid synthesis, heme synthesis, niacin synthesis, and histamine synthesis.
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Deficiency

A dietary deficiency is uncommon. However, as protein intake increases, the requirement for
pyridoxine also increases because of its important in amino acid metabolism. A mild deficiency results
in irritability, nervousness, and depression. More severe cases result in neurological disease including
somnolence, confusion, and neuropathy.

Drugs such as isoniazid (TB antibiotic), cycloserine, penicillamine, and hydrocortisone interfere with
pyridoxine metabolism. This can also affect synthesis of other compounds such as niacin since
pyridoxine is important in those processes.

Toxicity

Toxicity can occur but only with supplementation and not from dietary intake. Toxic levels of
pyridoxine can lead to destruction of the dorsal root ganglia resulting in pain and numbness in
extremities.

Biotin (B 7 )

Biotin is often found as a coenzyme in carboxylase enzymes. It is important in enzymes

involved in fatty acid synthesis, isoleucine and valine synthesis, and gluconeogenesis. It is found in
cosmetic and hair products. It is thought to strengthen hair and nails but there is no scientific evidence
of this.

Food sources: Corn, soy, egg yolk, liver, kidney, tomatoes

Deficiency

Deficiency is rare since intestinal bacteria produce more than the daily requirement for biotin. Egg
whites contain a protein (avidin) that can bind to biotin making it biologically unavailable. Someone
would have to consume 2 or more raw egg whites daily for several months to develop a deficiency.

Symptoms of a biotin deficiency: Hair loss, conjunctivitis, dermatitis, and neurological symptoms.

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No toxicity has been reported for this vitamin

Folate (B 9 )

Folate is an essential vitamin for humans. Humans cannot synthesize it and thus it must be consumed.
Food Sources: Many vegetables especially dark leafy ones, yeast, liver, beer

Folate is important in singe carbon transfer reactions. These reactions include synthesis of DNA, repair
of DNA, and methylation of DNA. Folate is important in aiding rapid cell division and growth especially
in infancy and pregnancy. It is needed to produce healthy red blood cells and prevent anemia. Folate is
required for vitamin B 12 activation.

Folate is not active until it is converted to dihydrofolic acid in the liver and other derivatives such as
tetrahydrofolate. Both conversions are done by the enzyme dihydrofolate reductase which uses the
energy associated with NADPH to perform the reaction.

Deficiency: Common symptoms of deficiency in adults include diarrhea, anemia, peripheral

neuropathy, mental confusion, cognitive decline, ulcers, and behavioral disorders.

• Megaloblastic anemia: Cell division of erythroblasts is affected by severe folate deficiency

which leads to low production of RBCs (anemia) by these cells. The erythroblasts get
transformed into large hollow cells (megaloblasts) seen in the blood of the patient.

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 • Folate deficiency during pregnancy may lead to neural tube defects of the embryo. The baby
with a condition called spina bifida is born with spinal cord defects (due to failure of the neural
tube to close during development). A condition called Anencephaly (characterized by the
absence of a major portion of the brain, skull, and scalp that occurs during embryonic
development) may be caused by severe folate deficiency in the mother during pregnancy

It is suggested that folate supplementation should be taken from one month prior to
conception and through the first trimester. Since half of all pregnancies are unplanned, many
women would be unable to do so or are often unaware of the fact that they should be on folate
supplementation. For this reason, the FDA mandated that grain products be fortified with
folate. (Mandate began in 1996 with full implementation by 1998). Since the implementation of
folate fortification, spina bifida cases have decreased noticeably.

• Folate can be destroyed by heat and oxidation

Cobalamin (B 12 )
Cobalamin (vitamin B 12 ) is important for normal functioning of the brain and nervous system and for
blood formation. It is important in DNA synthesis and fatty acid synthesis.

Bacteria are the only organisms that can synthesize vitamin B 12 . Food Sources: liver, kidney, egg,
cheese (only animal products) Since the only dietary source of vitamin B 12 is animal products, people
who are vegan, must supply vitamin B 12 through supplementation.

Absorption of Vit B12:

Vitamin B 12 absorption depends on its release

from dietary sources. Antacids and acid blocking medications will reduce the amount released in the
stomach since ingested proteins which contain vitamin B 12 will not be denatured. In addition, it cannot

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be absorbed on its own. A protein called intrinsic factor (IF) is required for its GI absorption. Intrinsic
factor is produced by parietal cells in the stomach. The receptors in the GI tract only recognize IF/B 12
complexes. This binding also protects the vitamin B 12 from catabolism by gut bacteria.

Function: Vitamin B 12 functions as cofactor in some isomerase and methyltransferase reactions. It

works along with Vit B 9 in single carbon transfer reactions at cellular levels.

Deficiency: Vitamin B 12 deficiency can take years to develop. Vitamin B 12 is recycled. We have enough
vitamin B 12 to last 6 years without additional intake. Interactions with certain substances can affect
vitamin B 12 . Excessive alcohol intake decreases B 12 absorption in the GI tract by lowering the intrinsic
factor production in the stomach. Folate supplementation can masks a B 12 deficiency and thus allow
potential irreversible neurological damage.

• Pernicious anemia: Low stomach acid and insufficiency in the production of intrinsic factor can
lead to a deficiency of Vit B 12 and thus pernicious anemia. It is a type of megaloblastic anemia
caused by B12 deficiency in specific. Red blood cells don't divide normally and are too large.
They may have trouble getting out of the bone marrow. Without enough red blood cells to
carry oxygen to your body, you may feel tired and weak. Severe or long-lasting pernicious
anemia can damage the heart, brain, and other organs in the body. Pernicious anemia also can
cause other problems, such as nerve damage, neurological problems (such as memory loss),
and digestive tract problems

• FOLATE TRAP – The folate cycle is below which is dependent on Vit B 12 . Deficiency of B 12 may
trap folate in its methylated form and make it unavailable for DNA synthesis reactions

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Note: An adult individual with folate deficiency only will show symptoms of megaloblastic anemia.

Folate deficiency during early pregnancy can cause defective brain and spinal cord development in the
fetus leading to spina bifida.

Folate and cobalamine deficiency in adults will lead to anemia as well as neurological symptoms
(caused by the cobalamine deficiency in specific).

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Vitamin C (Ascorbic acid)

Vitamin C is a cofactor in several enzymatic reactions.

Collagen synthesis (3 enzymes)

In the biosynthesis of norepinephrine from dopamine.

Vitamin C also works as an antioxidant protecting the body from the formation of reactive oxygen
species. In addition, it works as a natural antihistamine.

Vitamin C is synthesized by most organisms except, bats, guinea pigs, capybaras, and most primates
including humans. Thus it must be supplied in the diet. Lack of vitamin C results in a condition called
scurvy.

Deficiency

• Scurvy: The main hallmark of scurvy is lack of collagen synthesis. Symptoms include brown
spots under skin, spongy gums, and bleeding mucous membranes. It takes 1-6 months for
symptoms to appear after no vitamin C intake.
• Smokers required increased intakes of vitamin C due to the destruction of the vitamin by
chemicals in the cigarette smoke
• Use of vitamin C to reduce the time of a cold is controversial. Vitamin C consumption at high
doses is toxic if taken over a long period of time (3000mg/day). It can act as a pro-oxidant in
that case. The body is only able to absorb 300 mg/day thus any consumption beyond that is not
necessary.

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Fat Soluble Vitamins
Vitamin A

Vitamin A exists as three forms in the human body; Retinol, Retinal, and Retinoic acid. Each form is
involved in specific reactions or processes. The storage form is retinyl esters stored in the liver

Food Sources: Liver, egg yolk, butter, milk, dark green, yellow and orange vegetables

Interconversion of the different forms in the body

Precursors of vit A:

Beta carotene: Beta carotene is the water soluble precursor found in plants. It can be split into two
retinal molecules by β-carotene dioxygenase.

Retinyl esters: The animal form of vit A obtained from animal based diet

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Retinol

Important in reproduction, is the major transport form via blood. It is also the form in which vit A is
absorbed in the intestine along with dietary fats

Retinal

Important for vision. Participates in the visual cycle in photoreceptors present in the retina. Retinal is a
structural component of
rhodopsin. Each molecule of
rhodopsin present primarily in the
retinal rod cells (helping in dim light
vision) consists of the protein opsin
attached to a 11-cis retinal
molecule. Upon irradiation by
light, the retinal component is
transformed from cis to the trans
(all trans retinal) isomer. This
isomerization of retinal results in a
conformational change to opsin
generating a nerve impulse which
travels through optic nerve to the
visual cortex in the brain and is interpreted as vision

Retinoic Acid

Important in cell differentiation and growth and embryonic development. Acts by binding to specific
transcription factors (retinoic acid receptor)

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Transport of vitamin A in the body must occur with the aid of a protein called retinol binding protein
(RBP). This protein serves two functions, when vitamin A is bound, it serves as the recognition
molecule for receptors on cells and it protects from harmful consequences that would occur from the
presence of free vitamin A. Many of the fat soluble vitamins are stored in the liver or adipose tissues.
Vitamin A is stored in the liver and the average human has a 1 year supply.

Deficiency

Night blindness: The loss of retinaldehyde from to the back of the eye (retinal pigmented epithelium)
causes night blindness. Vitamin A is important to cornea health and the loss of the vitamin to the front
of the eye results in corneal softening and opacity. In fact this is the most common form of blindness
called Xeropthalmia in the world.

Toxicity

High levels of free or unbound vitamin A in blood is toxic. Once the RBPs are occupied, free vitamin A
can act as a pro-oxidant. Normal intake of vitamin A in the diet does not pose a problem, only when
vitamin A is consumed in supplement form, it might be toxic. Toxic levels of vitamin A can result in
bone pain, hair loss, dermatitis, double vision, headaches, diarrhea, and vomiting.

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High intakes of carotene are not toxic. The body will not
convert the carotene into vitamin A fast enough to
generate a vitamin A toxicity. The only side effect is
yellowing of the skin.

Vitamin D

Vitamin D is a nonessential nutrient. It is synthesized

by the human body. Synthesis of vitamin D begins at
the skin where the power of the sun (UV light)
converts 7 -dehydrocholesterol (7-DHC) to vitamin D 3 .
This molecule is still in an inactive form. Two
hydroxylation events must occur to generate the
active form of the vitamin. The first occurs in the liver
and results in hydroxylation of carbon 25 and the
second occurs in the kidney with hydroxylation at
carbon 1. The resulting compound 1,25
dihydroxyvitamin D 3 (calcitriol) is the active
compound.

The amount of sun exposure determines the amount

of vitamin D in the human body. As little as 5 minutes
of summer sun exposure around noon is enough to
generate a week’s quantity of vitamin D. Some factors
can affect the efficiency of vitamin D production such
as darker skin or living in northern (southern) latitudes.
For example, people living north of 40° latitude have
about 3 months out of the year in which the sun is not strong enough to support adequate vitamin D

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synthesis. The active role of vitamin D is as a hormone. Vitamin D acts on the intestines, kidneys, and
bones. It is important in calcium homeostasis and thus bone maintenance.

Deficiency

Rickets: A
deficiency in
vitamin D results
in rickets in children. Bowing of the legs from inadequate
calcification of the bones is a common symptom. Weak bones,
bone pain, are some other symptoms

Osteomalacia: In adults, vitamin D deficiency leads to

osteomalacia. Similar to the results in children, osteomalacia
results in poor mineralization of the bones and results in soft,
flexible, brittle and deformed bones. This can lead to
osteoporosis.

Toxicity

Vitamin D can reach toxic levels especially in children but only

with vitamin D supplementation. The result is often an increase
in blood calcium concentration. This calcium can form in soft tissues such as the kidney and can lead to
hardening of vessels.

Vitamin E

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Vitamin E is from a family of molecules known as tocopherols. The name means to bring forth offspring
since it was discovered that rats who were feed a tocopherol free diet were unable to produce young.
There are 4 forms of tocopherols labeled alpha, beta, gamma, and delta. Each describes the location of
methyl groups on different carbons in the molecule. Only the alpha tocopherol is active in the human
body.

The main role of vitamin E is an antioxidant and is in fact the most abundant natural antioxidant. It
especially protects lipids from oxidation. This action is important for maintaining the plasma
membrane of cells.

Deficiency

Reports of neurological symptoms have been reported with a vitamin E deficiency. In infants, a vitamin
E deficiency can lead to hemolytic anemia (red blood cells lysis), thrombocytosis, and edema.

Toxicity: Recent studies suggest that vitamin E supplementation is not necessary and is in fact harmful.
Too much vitamin E may also interfere with vitamin K function.

Vitamin K

Vitamin K is a group of chemical compounds with very similar structures, only the isoprenoid units in
the side chain vary in number. Vitamin K is required for the activation of coagulation factors (factors II,
VII, IX, X and thrombin). These factors are made and stored in the liver as inactive precursors.

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Activation requires carboxylation of specific glutamic acid residues in these factors. This carboxylation
is mediated by enzymes, such as gamma glutamyl carboxylase (GGC), which uses vitamin K as its
coenzyme. Carboxylation activates, these coagulation factors. In addition to acting on coagulation
factors, vitamin K is important in activating proteins involved in bone metabolism.

Humans cannot synthesize vitamin K. It is absorbed from dietary sources like green leafy vegetables.
However, the bacteria in the human gut convert the plant form of vit K (phylloquinone) to the animal
form of vit K (menaquinone). This is the form used at cellular levels. So we depend on gut bacteria for
the usable supply of vit K supply. Newborns are given an injection of vitamin K at birth. These injections
will give time for their guts to be colonized by bacteria that can synthesize the vitamin K.

Food sources: Green leafy vegetables, vegetable oils

Deficiency: Dietary deficiencies are very rare due to the fact that gut bacteria can synthesis vitamin K
for humans. Certain drugs however can interfere with the action of vitamin K. Warfarin, a drug
designed to “thin” the blood, is one such interfering drug.

During the normal function as a cofactor, vitamin K becomes oxidized. The normal course of action is to
reset the reduced form by an enzyme called vitamin K epoxide reductase (VKOR). The drug warfarin is
designed to interfere with VKOR and thus vitamin K cannot be reset to the reduced form and be used
in another carboxylase reaction.

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Lecture 11- Introduction to Carbohydrates

Learning Objectives:
• Describe structure and nomenclature of carbohydrates
• Explain anomeric carbon, stereoisomers, epimers, enantiomers,
hemiacetal and hemiketal groups
• Differentiate between reducing & non reducing sugars, types of
cyclic sugars
• Describe various types of glycosidic bonds in di, oligo &
polysaccharides
• Identify the major carbohydrates in our body & their functions
• Learn the significance of glycoconjugates (glycolipids &
glycoproteins)
• Learn the structural differences between different homo &
heteropolysaccharides

Carbohydrates contain carbon, hydrogen and oxygen. They have a large number of hydroxyl
groups making then polar thus enabling the carbohydrates to dissolve in water. They have either
an aldehyde or a ketone group. The carbohydrates can be classified as being monosaccharides,
oligosaccharides (including disaccharides) or polysaccharides. An oligosaccharide has 2-10
monosaccharide units joined together by glycosidic bonds whereas polysaccharides can have
several hundred units of monosaccharides. Some carbohydrates can also have nitrogen,
phosphate and sulfur and some can react with lipids and proteins to form glycolipids and
glycoproteins respectively.

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Nomenclature of Carbohydrates

Glyceraldehyde and dihydroxyacetone form the basis of the related carbohydrates in the body.
All carbohydrates contain at least one asymmetrical (chiral) carbon and are, therefore, optically
active. Carbohydrates can exist in either of two conformations. This is determined by the
orientation of the hydroxyl group about the asymmetric carbon farthest from the carbonyl. The
carbohydrates that are of importance in the body exist in the D-conformation. Enantiomers of
these are the mirror image conformations and are in the L-conformation.

Monosaccharides
The number of carbons in monosaccharides determines their classification.

Classification
3 Carbons- Triose (Glyceraldehyde, Dihydroxyacetone)

4 Carbons- Tetrose (Erythrose)

5 Carbons- Pentose (Ribose, Ribulose, Xylulose)

6 Carbons- Hexose (Glucose, Galactose, Mannose, Fructose)

7 Carbons- Heptose (Sedoheptulose)

9 Carbons- Nanose (Neuraminic acid)

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Aldoses

Ketoses

Difference between Stereoisomers/ Epimers/ Enantiomers

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Stereoisomers: Have the same chemical formula. Differ in the position of OH around one or
more asymmetric C.

Epimers: Differ in the position of OH around one asymmetric C only

Enantiomers: Mirror images of each other; Not superimposable

Cyclic Sugars

The aldehyde and ketone groups of the carbohydrates with five and six carbons will
spontaneously react with alcohol groups present in adjacent carbons to produce intramolecular
hemiacetals or hemiketals, respectively resulting in the formation of five or six-membered
rings. Furanoses are six-membered rings while pyranoses are 5 membered rings. The structures
can be written in the Fischer or Haworth projections. The carbons in carbohydrates are
numbered from the carbonyl carbon, for aldoses, or the carbon nearest the carbonyl, for
ketoses.

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The rings can open and re-close, allowing rotation to occur about the carbonyl carbon yielding
two distinct configurations (α and β) of the hemiacetals and hemiketals. The carbon on which
this rotation occurs is also known as the anomeric carbon and the two isomers are called
anomers. Carbohydrates can switch between α and β anomers, a process called mutarotation.

Hemiacetal
Hemiketal

Alpha and Beta Anomers: When drawn in the Haworth projection, the α configuration places
the hydroxyl attached to the anomeric carbon below the ring compared. The β configuration
places the hydroxyl above the ring structure.

The furanose and pyranose ring structures are also described by the two conformations known
as the chair form and the boat form. The chair form is the more stable of the two. Constituents
of the ring that project above or below the plane of the ring are axial and those that project

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parallel to the plane are equatorial. In the chair conformation, the orientation of the hydroxyl
group about the anomeric carbon of α-D-glucose is axial and equatorial in β-D-glucose.

Chair form of α-D-Glucose

Disaccharides

Covalent bonds between the anomeric hydroxyl of a cyclic sugar and the hydroxyl of a second
sugar are called glycosidic bonds, and the resultant molecules are called glycosides. The linkage
of two monosaccharides to form disaccharides involves a glycosidic bond. Some important
disaccharides are sucrose, lactose and maltose.

Sucrose: It is composed of glucose and fructose through an α–(1,2)–β-glycosidic bond.

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Lactose: It is found exclusively in the milk of mammals and consists of galactose and glucose in a β–(1,4)
glycosidic bond.

Maltose: the major degradation product of starch, is composed of 2 glucose monomers in an α–(1,4)
glycosidic bond.

Reducing and non- reducing sugars

Reducing and non-reducing sugars refer to sugars
with two or more units bound together. A reducing
sugar will have a reducing end. A reducing end has an anomeric carbon free and not involved in
a glycosidic bond. The free OH of the anomeric carbon is available for chemical reactions.
Lactose and maltose are examples of reducing sugars.

Non-reducing sugars do not have a free anomeric carbon with an OH gr available. Sucrose is an
example of a non-reducing sugar. The anomeric carbons of the glucose and fructose sugars are
both involved in the glycosidic bond.

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Polysaccharides

Polysaccharides are high molecular weight polymers comprised of monomeric building blocks.
The predominant monosaccharide found in polysaccharides is D-glucose. Polysaccharides
composed of a single monosaccharide building block are known as homopolysaccharides.
Those composed of more than one type of monosaccharide are known as
heteropolysaccharides.

Homopolysaccharides

Glycogen
Glycogen is the major storage carbohydrate in animals. It is a homopolymer of glucose in α–(1,4) linkage
and is also highly branched, with α–(1,6) branch linkages occurring every 4-10 residues. It has a very
compact coiled structure. This allows large amounts of carbon energy to be stored in a small space, with
little effect on cellular osmolarity.

Starch

Starch is the major

storage carbohydrate in plant cells. Its structure is identical to glycogen, except for a much

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lower degree of branching (about every 20–30 residues). Unbranched starch is called amylose;
branched starch is called amylopectin.

Heteropolysaccharides

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Glycosaminoglycan (GAG)
Glycosaminoglycans (GAGs) are large unbranched polysaccharides and are composed of a
repeating disaccharide. One sugar in the disaccharide is an acidic sugar (contains a carboxylic
acid group) and the other is an amino sugar (contains an amide group). The amino sugar is
usually glucosamine or galactosamine and the acidic sugar is glucuronic acid or iduronic acid.
GAGs tend to have a negative charge associated with them because of the acidic sugar and the
addition of sulfate groups. These negative charges increase the
interaction of the GAG with water and the result is a gel like matrix
which supplies lubrication like properties to synovial fluid and
mucus.

Glycoproteins
Glycoproteins are proteins with some carbohydrate modifications. These carbohydrate
modifications are added to the protein in the Golgi complex of the cell. The carbohydrate is
attached to asparagine (N-linked) or serine/threonine (O-linked) amino acids. Most
glycoproteins end up on the outer surface of the cell membrane.
The addition of saccharides to proteins is important for several reasons.

1. Increase polarity and solubility of the proteins.

2. May influence the folding process.
3. Protect from proteolytic enzymes.
4. Responsible for specific biological
activities:

 Intracellular
targeting of proteins

 Cell-cell interactions,
Tissue development

 Extracellular
signaling

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Glycolipids
Glycolipids are lipid molecules with carbohydrates attached to them. Glycolipids are important
because they can provide energy, used in cellular recognition for chemicals, provide membrane
stability, and provide attachment sites for cell to cell interactions to form tissues.

Metabolism of Dietary Carbohydrates

First step in carbohydrate metabolism occurs with saliva (pH 6.8) which contains amylase. This
enzyme cleaves the alpha 1,4 linkages of dietary sugars that were just consumed.

The small intestine is the main site of carbohydrate digestion. The main enzyme is alpha
amylase secreted from the pancreas.

Complex carbs are digested to Di- and Tri-saccharides and eventually to monosaccharide units.

The resultant monosaccharides are transported across the intestinal wall to the hepatic portal
vein to travel to the liver. In the liver, carbohydrates such as galactose and fructose can enter
glycolysis or are converted to glucose for transport throughout the body.

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 Lecture 12- Glycolysis

Learning Objectives:
• Know the individual steps of glycolysis , the enzymes involved in each
step and their regulations
• Know the steps where energy is consumed and generated inn
glycolysis
• Describe the difference between aerobic and anaerobic glycolysis
• Know the regulation of the committing enzyme of glycolysis (PFK-1)
• How does PFK 2 regulate PFK-1?
• Learn the role of glucagon/ epinephrine/insulin in regulation of
glycolysis

Under aerobic conditions, the dominant product of glucose oxidation in most tissues is pyruvate. This
oxidation process is called glycolysis. It occurs in the cytoplasm of all cells. It is common pathway of
aerobic an anaerobic respiration.
When oxygen is depleted, as for instance during prolonged vigorous exercise, anaerobic glycolysis
(fermentation) occurs and the dominant glycolytic product is lactate.

Some tissues/cells, most notably the red blood cells (RBCs) and transparent cells of the lens and retina,
do not contain mitochondria and therefore, use anaerobic glycolysis as their only means to generate
energy.

Energy Derived from Glucose Oxidation

Aerobic glycolysis of glucose to pyruvate requires two equivalents of ATP to activate the process, with
the subsequent production of four equivalents of ATP (net +2 ATP) and two equivalents of NADH.

Glucose + 2 ADP + 2 NAD+ + 2 Pi  2 Pyruvate + 2 ATP + 2 NADH + 2 H+

The NADH generated during glycolysis is used to fuel mitochondrial ATP synthesis via oxidative
phosphorylation, producing either two or three equivalents of ATP depending upon whether the
glycerol phosphate shuttle or the malate-aspartate shuttle is used to transport the electrons from
cytoplasmic NADH into the mitochondria. The net yield from the oxidation of 1 mole of glucose to 2
moles of pyruvate is, therefore, either 6 or 8 moles of ATP.

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The complete oxidation of the 2 moles of pyruvate, through the TCA cycle resulting in NADH and
FADH 2 production, yields an additional 30 moles of ATP; the total yield, therefore being either 36 or 38
moles of ATP from the complete oxidation of 1 mole of glucose to CO 2 and H 2 O.

ATP (adenosine triphosphate) is the ultimate storage form for all living systems and is, itself, a
nucleotide molecule able to store energy in its phosphate bonds. ATP is constantly being broken down
into ADP to release energy (2 e-) and resynthesized almost immediately. We’ll see this in detail in the
electron transport chain.
Consider: The total quantity of ATP in the human body is about 0.1 mole (~.5 gram!) but our
metabolism consumes 100 to 150 moles of ATP daily (50 to 75 kg!); every ATP molecule is recycled
1000 to 1500 times daily.

Individual Reactions of Glycolysis

The pathway of glycolysis can be seen as consisting of 2 separate phases:

Phase I: Chemical priming phase requiring energy in the form of ATP.

In the first phase, 2 equivalents of ATP are used to convert glucose to fructose-1,6-bisphosphate (F-1,6-
BP or DP).

* The F1, 6BP reaction (Reaction 3) is a rate limiting step in that Vmax for this enzyme is somewhat
lower.

** The phosphorylation of glucose (Reaction 1) is crucial as well, since glucose 6 phosphate (G6P)
cannot leave the cell. Glucose is able to diffuse out of a cell.

Phase II: The energy-yielding phase.

In the second phase F-1,6-BP is degraded to pyruvate, with the production of 4 equivalents of ATP and
2 equivalents of NADH.

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Energy surplus comes about when more glucose enters glycolysis, especially after a
meal. This state is indicated by increased levels (concentration) of ATP, Acetyl CoA,
NADH and reduced levels of inorganic phosphate (PO4 or Pi)

Energy deficit occurs when there is a shortage of energy containing intermediates indicated by
increased levels (concentration) of ADP (less ATP), NAD+, inorganic phosphate (Pi) and reduced CoA
(as CoASH).

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Reactions of Glycolysis
Reaction 1: Hexokinase/Glucokinase
The ATP-dependent phosphorylation of glucose forms glucose-6-
phosphate (G6P) in the first reaction of glycolysis. This catalysis is
performed by tissue-specific isoenzymes known as hexokinases.
This phosphorylation reaction accomplishes two goals:
- First: Hexokinase converts nonionic glucose into an anion
(G6P) that is now trapped in the cell, since cells lack
transport systems for phosphorylated sugars.
- Second: Biologically inert glucose becomes activated into a
labile form capable of being further metabolized.

Four mammalian isozymes of hexokinase are known (Types I - IV)

[different Km for each]
Type IV isoenzyme is often referred to as glucokinase (GK).
Glucokinase is the form of the enzyme that predominates in hepatocytes. The high Km (low affinity) of
glucokinase for glucose means that this enzyme is saturated only at very high concentrations of
substrate.(e.g. post-prandial, NOT glycogenolysis)

Comparison of the activities of hexokinase and glucokinase. The Km for hexokinase is significantly
lower (0.1mM) than that of glucokinase (10mM). The low Km values indicate increased affinity of
the hexokinases (I, II, III) for glucose. This difference ensures that non-hepatic tissues will rapidly
and efficiently trap blood glucose within their cells by converting it quickly to glucose-6-
phosphate.

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After meals the postprandial blood glucose levels are high and liver glucokinase activity increases
significantly allowing the liver to form G6P, trapping EXCESS circulating glucose.
The affinity of glucokinase (HK IV) is sufficiently low so that only when normal circulating concentration
of glucose exceeds 110 mg/dL (~5mM)l liver is able to phosphorylate glucose to G6P. Excess liver G6P
will be converted to G1P and stored as glycogen. Portal blood glucose level can be as high as three
times the peripheral level.
It is the stored glycogen that allows the liver to buffer blood glucose levels during and between meals.
When blood glucose falls to very low levels, tissues such as liver (and kidney) which contain
glucokinases but are not highly dependent on glucose, do not continue to use the reduced glucose
supplies.

At the same time, tissues such as the brain, which are critically dependent on glucose, continue to
scavenge blood glucose using their high affinity (low Km) hexokinases to protect viability of those
tissues for as long as possible.

Under various conditions of glucose deficiency, such as long periods between meals, the liver is
stimulated to supply the blood with glucose through the pathway of gluconeogenesis.
The levels of glucose produced during gluconeogenesis are insufficient to fully activate glucokinase and
this “new” glucose passes out of hepatocytes and into the blood.

Regulation of Hexokinase
The regulation of hexokinase and glucokinase activities is also different. Hexokinase is allosterically
inhibited by product (G6P) accumulation, whereas glucokinase (found in the liver), is not regulated by
glucose 6 phosphate (reaction 1).

This lack of control of GK insures that liver will only accumulate glucose during times of glucose excess.
When glucose levels decline GK activity declines favoring glucose utilization by all other tissues.

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Reaction 2: Phosphohexose Isomerase
The second reaction of glycolysis is an isomerization, in which
G6P is converted to fructose-6-phosphate (F6P). The enzyme
catalyzing this reaction is phosphohexose isomerase (also known
as phosphoglucose isomerase). The reaction is freely reversible
at normal cellular concentrations of the two hexose phosphates
and thus catalyzes this interconversion during glycolytic carbon
flow and during gluconeogenesis.

Reaction 3: 6-Phosphofructo-1-Kinase (Phosphofructokinase-1, PFK-1)

The next reaction of glycolysis involves the utilization of a
second ATP to convert F6P to fructose-1,6-bisphosphate
(F-1,6-BP). This reaction is catalyzed by 6-phosphofructo-1-
kinase, better known as phosphofructokinase-1 or PFK-1.

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The PFK reaction is not readily reversible because it requires input
of a large amount of (positive) free energy (G0' = +5.4 kcal/mol) in
order for the reverse reaction to occur. Effectively this prohibits
the reversal of the reaction in [Link]-1 is considered to be the
rate-limiting enzyme of glycolysis.

A second isoenzyme, PFK-2 also up-regulates activity of PFK1 and

by conversion of F6P to F2,6BP. F2,6BP is an allosteric activator of
PFK1.

Regulation of Phosphofructokinase-1 (PFK-1)

Phosphofructokinase 1 (PFK1) is activated by the presence of ADP, AMP and a molecule called F 2,6 BP.
This enzyme is inhibited by citrate and ATP. ATP is the biggest inhibitor of this enzyme. Small levels of
activator molecules can overcome the inhibition by ATP.

Fructose 2,6 Bisphosphate levels regulate PFK1 activity

Fructose 2,6 BP activates glucose breakdown in glycolysis through allosteric modulation of PFK-1.
An increase in Fructose 2,6 BP in liver activates PFK-1 by increasing the enzyme’s affinity for Fructose 6-
P.

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At physiological concentration of ATP, PFK-1 is almost completely inactive but interaction with Fructose
2,6 BP overcomes the ATP inhibition and activates the enzyme to stimulate

Role of PFK-2 (phosphofructo kinase-2)

This is the enzyme that phosphorylates Fructose 6- phosphate to form Fructose 2,6 –Bisphosphate,
which as mentioned above, can activate PFK-1. This is the kinase part of the enzyme. PFK-2 must be
dephosphorylated for the kinase activity to be on. When PFK-2 is phosphorylated the reverse reaction
is turned on activating the phosphatase part of the enzyme lowering levels of F2,6BP

Fed State

In a fed state, Insulin is released from the pancreas and causes glucose to move into cells. In addition
to the influx of glucose, insulin will cause the dephosphorylation of PFK-2 thus activating its kinase
activity. Once activated, PFK-2 will convert Fructose 6-P to Fructose 2,6 Bisphosphate. Increased levels
of F 2,6 BP increase glycolysis rate by activating PFK-1 and inhibiting F1,6 Bisphosphotase.

Fasting State

In the fasting state, Glucagon is released. When glucagon is released it increases blood glucose levels
by mobilizing stored hepatic glucose. To prevent this glucose being used up rapidly, it needs to inhibit
glycolysis. It accomplishes this by phosphorylation of PFK-2 by Protein Kinase A though a glucagon
signaling pathway. Phosphorylation of PFK-2 enhances the activity of the phosphatase (breaks down
Fructose 2,6 BP to Fructose 6-P) and at the same time inhibits the kinase activity. This results in lower
levels of F 2,6 BP to activate PFK-1.

Note: Both Glucagon and Insulin regulate the activity of PFK-2 by either phosphorylating the enzyme
or dephosphorylating the enzyme respectively. Please do not confuse this with phosphorylation of
Fructose 6-Phosphate or dephosphorylation thereof.

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Reaction 4: Aldolase
Aldolase catalyzes the lysis of F-1,6-BP into two 3-carbon products: dihydroxyacetone phosphate
(DHAP) and glyceraldehyde-3-phosphate (GAP). The aldolase reaction proceeds readily in the reverse
direction, being utilized for both glycolysis and gluconeogenesis.

Reaction 5: Triose Phosphate Isomerase

The two products of the aldolase reaction equilibrate readily in a reaction catalyzed by triose
phosphate isomerase. Succeeding reactions of glycolysis utilize GAP as a substrate; thus, the aldolase
reaction is pulled in the glycolytic direction by mass action principals.

Reaction 6: Glyceraldehyde 3 Phosphate Dehydrogenase (GAPDH)

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The second phase of glucose catabolism features the energy-yielding glycolytic reactions that produce
ATP and NADH. In the first of these reactions, glyceraldehyde-3-P dehydrogenase (GAPDH) catalyzes
the NAD+-dependent oxidation of GAP to 1,3-bisphosphoglycerate (1,3-BPG) and NADH. The GAPDH
reaction is reversible, and the same enzyme catalyzes the reverse reaction during gluconeogenesis.
[second Pi added w/o expenditure of ATP]

Note: Heavy metals such as Hg2+ can irreversibly bind to cysteine and stop this step inhibiting
glycolysis

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Reaction 7: Phosphoglycerate Kinase
The high-energy phosphate of 1,3-BPG is used to form ATP and 3-phosphoglycerate (3PG) by the
enzyme phosphoglycerate kinase. Note that this is the only reaction of glycolysis or gluconeogenesis
that involves ATP and yet is reversible under normal cell conditions.

Effect of arsenic on glycolysis:

Arsenic behaves like inorganic phosphate. When present in the cell in high concentrations, it will bind
with GAP to produce 1-arseno-3-phosphoglycerate, which in the next step will release the arsenic to
produce 3-phosphoglycerate. Presence of arsenic will not inhibit glycolytic process but by bypassing
the ATP producing step 7, it will inhibit the production of 2 ATPs by glycolysis.

Reaction 8 and 9: Phosphoglycerate Mutase and Enolase

The remaining reactions of glycolysis are aimed at converting the relatively low energy phosphoacyl-
ester of 3-PG to a high-energy form and harvesting the phosphate as ATP. The 3-PG is first converted to
2-PG by phosphoglycerate mutase and then 2-PG conversion to phosphoenoylpyruvate (PEP) is
catalyzed by enolase

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Reaction 10: Pyruvate Kinase
The final reaction of aerobic glycolysis is catalyzed by the
highly regulated enzyme pyruvate kinase (PK). In this
strongly exergonic reaction, the high- energy phosphate
of PEP is conserved as ATP. The loss of phosphate by PEP
leads to the production of pyruvate in an unstable enol
form, which spontaneously tautomerizes to the more
stable, keto form of pyruvate. This reaction contributes a
large proportion of the free energy of hydrolysis of PEP.
In erythrocytes, the fetal PK isozyme has much greater
activity than the adult isoenzyme; as a result, fetal
erythrocytes have comparatively low concentrations of
glycolytic intermediates.

Regulation of Pyruvate Kinase

Inhibition of pyruvate kinase occurs when cells sense abundant energy levels present. Signals that
indicate high energy levels are high concentrations of ATP and acetyl-CoA. High levels of these
substances activate a kinase which phosphorylates pyruvate kinase. In this phosphorylated form,

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pyruvate kinase is inactive. Pyruvate kinase can be activated on the other hand by high levels of the
upstream glycolytic intermediate F 1,6 BP.

Overview of regulatory steps in glycolysis

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Examination of the free energy (ΔG) of the glycolytic reactions helps reveal the control points of the
entire process. Most control points in a reaction pathway will operate far from equilibrium. Reactions
at equilibrium will have ΔG near 0. In the reactions of glycolysis, three have very negative ΔG values.
These reactions are catalyzed by hexokinase, phosphofructokinase 1, and pyruvate kinase.

Other hexoses require minimal conversion.

Galactoseis converted to Gal1P and then G1P by Galactose 1 phosphate UDP transferase and then to
G6P by galactokinase. G6P can then enter glycolysis

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Fructose is converted to F1P by fructose kinase and then cleaved to glyceraldehyde and DHAP by F1P
aldolase. GAP and DHAP can enter glycolysis

Mannose is converted to M6P by hexokinase and then to F6P by phosphomannose isomerase. F6P can
continue glycolysis

Aerobic vs. Anaerobic Glycolysis

Under aerobic conditions, pyruvate in most cells is further metabolized via the TCA cycle. Under
anaerobic conditions and in erythrocytes under aerobic conditions, pyruvate is converted to lactate by
the enzyme lactate dehydrogenase (LDH), and the lactate is transported out of the cell into the
circulation.
The conversion of pyruvate to lactate, under anaerobic conditions, provides the cell with a mechanism
for the oxidation of NADH (produced during the GAPDH reaction) to NAD+. The enzyme that catalyzes
the reaction is called lactate dehydrogenase (LDH)
Pyruvate + NADH + H+ → Lactate + NAD+

Metabolism of Alcohol
Excessive NADH may be seen in ethanol/alcohol poisoning syndromes with a resulting deficit in NAD+.
The increased NADH is the result of the detoxification of the ethanol to acetaldehyde by the enzyme
alcohol dehydrogenase. The reduction of pyruvate to lactate generates oxidized NAD+ needed as
substrate for GAPDH (glycolysis reaction 6). Without sufficient NAD+ glycolysis will cease.

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Anaerobic glycolysis in muscle cells is energetically inefficient due to low yields of NADH (2 per glucose)
which produces only 5-6 ATP.
However when muscle needs large amounts of energy the rate of ATP production from anaerobic
glycolysis is approximately 100X faster than from oxidative respiration (via ETC).

During quick, rapid exertion muscle cells do not need (nor do they have time) to energize aerobic
respiration pathways due to a requirement to generate the maximum amount of ATP in the shortest
time frame. Longer term muscle contractions have time to utilize ATP from aerobic glycolysis.
During aerobic glycolysis the electrons of cytoplasmic NADH are transferred to mitochondrial carriers
of the oxidative phosphorylation pathway generating a continuous pool of cytoplasmic NAD+. Aerobic
glycolysis generates substantially more ATP per mole of glucose oxidized than anaerobic glycolysis.

Lactate Metabolism
Anaerobic glycolysis in aerobic organisms requires that oxidation of NADH occur through the reduction
of an organic substrate. In humans (all terrestrial animals) this reaction is carried out by lactate
dehydrogenase (LDH) which reduces pyruvate to lactate (lactic acid). The lactate produced during
anaerobic glycolysis diffuses from the tissues and is transported to highly aerobic tissues such as
cardiac muscle and liver where lactate can be used for energy.

Erythrocytes (normal) derive all of their ATP needs through anaerobic glycolysis. Skeletal muscle,
especially under exertion, will utilize anaerobic glycolysis until aerobic metabolism kicks in. Large
quantities of NADH are oxidized to NAD+, reducing pyruvate to lactate.

The lactate will be transported via blood to liver (heart, kidney also) where it will reoxidized to
pyruvate by LDH. This pyruvate is then oxidized in the TCA cycle, while NADH goes to ETC. If there is an
energy surplus in liver cells, pyruvate can be diverted back to glucose via gluconeogenesis. This is
known as the Cori Cycle.

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Alternative pathways

2,3 BPG Shunt

An offshoot of the phosphoglycerate kinase pathway occurs only within erythrocytes: the formation of
2,3-BPG by the enzyme bisphosphoglycerate mutase.

The synthesis of 2,3 BPG (2,3

bisphosphoglycerate) occurs only in RBCs. Some
molecules of 1,3 bissphosphoglycerate are
shunted, not all, as glycolysis is the only source of
ATPs in RBCs
2,3 BPG is critical for controlling hemoglobin
affinity for oxygen. Note that 2,3 BPGP degrades
2,3-BPG to 3-phosphoglycerate, a normal
intermediate of glycolysis. The 2,3-BPG shunt
consumes 1 ATP per cycle (e.g. each 2,3BPG). The
process is not reversible under physiological
conditions.

Importance of 2,3 BPG

2,3-BPG, in association with hydrogen ion, regulates hemoglobin’s affinity for oxygen. 2,3 BPG easily
binds to partially deoxygenated (a.k.a hydroxyhemoglobin) hemoglobin, mainly in deep tissue. Once
bound, 2,3 BPG allosterically upregulates the release of the remaining oxygen molecules bound to the
hemoglobin, thus enhancing the ability of RBCs to release oxygen near tissues that need it most.
Hydrogen ion (H+) is able to bind to the #1 carbon (C=O of 2,3 BPG) which facilitates it’s binding to

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hemoglobin.

At low pH (higher [H+]) in deep tissues, the 2,3 BPG promotes release of oxygen and formation of
hydroxyhemoglobin. Therefore the free 2,3 BPG level inside the RBCs will drop as it binds Hb. At higher
pH (lower [H+]) in the lungs, the 2,3 BPG has lower affinity for binding to hemoglobin, allowing oxygen
to bind easily, so free 2,3 BPG level increases.

Because of the low steady-state concentration of fetal 1,3-BPG, the 2,3-BPG shunt is greatly reduced in
fetal cells. 2,3-BPG loosely binds to fetal Hb ( due to the structural difference of fetal hemoglobin) .
This allows fetal erythrocytes have a higher oxygen affinity than maternal erythrocytes. Therefore,
transfer of oxygen from maternal hemoglobin to fetal hemoglobin is favored, assuring the fetal oxygen
supply. The high pH in the fetal tissue also promotes loose binding of 2, 3 BPG to fetal hemoglobin
allowing more oxygen binding to hemoglobin.

In the newborn, an erythrocyte isozyme of the M-type with comparatively low PK activity displaces the
fetal type, resulting in an accumulation of glycolytic intermediates. The increased 1,3-BPG levels
activate the 2,3-BPG shunt, producing 2,3-BPG needed to regulate oxygen binding to hemoglobin.

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Pentose Phosphate Pathway/Shunt
This is an alternative pathway for the metabolism of glucose. Its main role is as follows:
1. It generates 3-7 carbon sugars
2. It generates Ribose for nucleotide synthesis
3. It generates NADPH for anabolic reactions

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Lecture 13 -The Tricarboxylic Acid Cycle

Learning Objectives:

• Learn the function & regulation of PDH

• Outline the sequence of reactions in the TCA cycle and their
significance
• Emphasize on the reactions in the TCA cycle and their significance
• Identify the 4 oxidative enzymes involved in the TCA cycle, their
regulations and their roles
• Discuss the importance of the intermediates synthesized by the TCA
cycle. Learn the role of NADH/ NAD+ ratio in regulation of the TCA
cycle
• Learn the regulation of TCA cycle by substrate supply, presence of
allosteric effectors
• Explain anaplerotic & cataplerotic reactions

Tricarboxylic Acid (Kreb/Citric Acid Cycle)

The TCA cycle is a series of 8 enzymatic reactions which occur in the mitochondrial matrix. The
resulting products are the formation of two CO 2 molecules, 3 NADH, 1 FADH 2 , and one ATP (via GTP
formation). The products of the TCA cycle account for more then 2/3 of the ATP generated via
oxidative metabolism.

Each pyruvate (from glucose) must be converted into acetyl-CoA by the pyruvate dehydrogenase (PDH)
Complex in order for it to enter the TCA cycle. Oxidation of fatty acids, acetate, ketone bodies, and
amino acids also generate acetyl-CoA. This makes the TCA cycle and acetyl-CoA the central point or
hub in the metabolism of all energy yielding macromolecules.

Essentially unlimited numbers of acetyl-CoA can enter the cycle since oxaloacetate, used in the first
step of the cycle, is regenerated in the last step.

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 The Pyruvate Dehydrogenase Complex (PDH)
The enzyme has 3 major components:

1. Pyruvate Dehydrogenase
(PDH) [E1]
2. Dihydrolipoyl transacetylase
(DLTA) [E2]
3. Dihydrolipoyl Dehydrogenase
( DLDH) [E3]

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Acetyl groups are formed in the PDH complex via oxidative decarboxylation of pyruvate. Five reactions
occur within the three enzyme components mentioned above. The overall reaction is (pyruvate + CoA
+ NAD+  acetyl-CoA + CO 2 + NADH).

Five coenzymes are required by the PDH. These are:

Thiamine Pyrophosphate (TPP), Lipoamide, CoASH, FAD, NAD+. Four vitamins are required by the PDH.
These are: Thiamine, Pantothenic Acid, Riboflavin, Nicotinamide.

Step 1: Pyruvate dehydrogenase (E1), which contains the TPP group, decarboxylates pyruvate
generating CO 2 .
Step 2: The remaining two carbon components from pyruvate (acetyl) are transferred to the lipoamide
group (found in an oxidized state) of Dihydrolipoyl transacetylase (E2). This resets the TPP in E1 which
is now ready for another pyruvate molecule.
Step 3: The acetyl is transferred from E2 to CoA generating the acetyl-CoA and a lipoamide group in a
reduced state.
Step 4: Dihydrolipoyl Dehydrogenase (E3) uses its oxidized FAD+ group to oxidize the lipoamide group
of E2.
Step 5: The reduced FAD of E3 transfers the elections to NAD+ generating NADH.

Regulation of Pyruvate dehydrogenase

PDH is regulated by phosphorylation of its E1 enzyme. This phosphorylation occurs when the kinase
domain of pyruvate dehydrogenase is active. High NADH and acetyl-CoA levels activate the pyruvate
dehydrogenase kinase domain & phosphorylate E1 of PDH. Phosphorylated PDH is inactive.

The presence of insulin or calcium will reverse the phosphorylation of E1 by activating the pyruvate
dehydrogenase phosphatase domain and result in increased acetyl CoA production from pyruvate.

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In addition to phosphorylation, PDH is regulated by the levels of NAD+/NADH in the cell since NAD+ is a
substrate for the complex. High cellular NADH levels inhibits PDH. Thus a high NADH/ NAD+ ratio or
low NAD+/NADH ratio in the cell will keep PDH inhibited.

Reactions of the TCA Cycle

Reaction 1
Citrate Synthase combines oxaloacetate and acetate (from acetyl-CoA) to generate citrate. So, a 4
carbon molecule (oxaloacetate) is combined with a 2 carbon molecule (acetate) to form a 6 carbon
molecule (citrate). This reaction is a sequential ordered mechanism in that oxaloacetate must bind to
the enzyme first before acetyl-CoA can bind and the reaction occurs.

Reaction 2
Aconitase interconverts Citrate and Isocitrate. The name of the enzyme refers to the aconitate
intermediate that forms during this reaction. This intermediate is only present for a very brief period of
time and is never released from the enzyme.

Reaction 3
Isocitrate dehydrogenase generates α-ketoglutarate from isocitrate. This reaction is a two-step

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reaction. The first step results in the formation of NADH and the second step results in the formation
of CO 2 . When looking at the reaction note that a 6 carbon molecule (isocitrate) is converted to a 5
carbon molecule (α-ketoglutarate) and the reduction in carbon is from the production of CO 2 .

Reaction 4
α-Ketoglutarate Dehydrogenase converts α-Ketoglutarate to Succinyl-CoA. The result of this reaction
is the concurrent formation of CO 2 and NADH. Thus a 5 carbon molecule (α-Ketoglutarate) is converted
to a 4 carbon molecule (Succinyl). The enzyme α-Ketoglutarate Dehydrogenase is structurally similar to
pyruvate dehydrogenase.

Regulation of α-Ketoglutarate Dehydrogenase is same as PDH

Reaction 5
GTP is generated by Succinyl-CoA synthetase during the conversion of Succinyl-CoA to Succinate. GTP
is then later converted to ATP by nucleoside diphosphate kinase.

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Reaction 6
Succinate Dehydrogenase generates FADH 2 during the formation of Fumarate from Succinate. This
enzyme is also a component of the electron transport chain. It is the only membrane bound enzyme in
the TCA Cycle. Malonate is a structural analog of succinate and works as a competitive inhibitor of
Succinate Dehydrogenase.

Reaction 7
Fumarate is converted to Malate by Fumarase. Note: do not confuse malonate and malate.

Reaction 8
Oxaloacetate is regenerated by malate dehydrogenase from malate. During this reaction, NADH is
generated.

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Regulation of enzymes of the TCA cycle
Similar to what is observed in glycolysis, the rate determining steps have negative delta G values. These
regulatory steps are those catalyzed by enzymes namely citrate synthase (reaction 1), isocitrate
dehydrogenase (reaction 3), and α-ketoglutarate dehydrogenase (reaction 4). Regulation of these
enzymes is performed by three mechanisms, substrate availability, product inhibition, and competitive
feedback by downstream intermediates.

Regulation of Citrate synthase

The regulation of the first step in the TCA cycle is mostly controlled by its substrates and products. The
substrates of citrate synthase are usually found at concentrations low enough that the enzyme is not
saturated and thus its product, citrate, is the main controller by competitive inhibition. Citrate
competes with oxaloacetate. To some degree, citrate synthase is controlled by high levels of NADH but
not as much as other enzymes of the cycle. Competitive inhibition can also be observed by feedback of
succinyl-CoA which competes with acetyl-CoA.

Regulation of Isocitrate dehydrogenase

Isocitrate dehydrogenase is allosterically inhibited and activated. ADP will activate the enzyme while

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NADH or ATP will inactivate it. Calcium will also activate isocitrate dehydrogenase.

Regulation of α-ketoglutarate dehydrogenase

α-ketoglutarate dehydrogenase is inhibited by its products succinyl-CoA and NADH. This enzyme
similar to isocitrate dehydrogenase is activated by calcium.

Calcium is an activator of several TCA enzymes as indicated above. Calcium will activate pyruvate
dehydrogenase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase. Thus, the signal for
muscle contraction (calcium) stimulates ATP production to help fuel that contraction.

Other Inhibitors
Arsenate and fluoroacetate are exogenous enzyme inhibitors which act as “poisons” in that they will
interfere with the normal activities of the pathways.

- Arsenates include a variety of molecules containing the ionic form (AsO 4 -3) and is usually ingested in
foods, groundwater or as intentional poisons. Arsenate ion is also an electron donor (reducing agent)
and forms bonds much like phosphate ions (PO 4 -3). Arsenates interfere with pyruvate dehydrogenase
and α-ketoglutarate dehydrogenase by binding to the lipoamide group.

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 Fluoroacetate is usually found in a salt form (FCH 2 CO 2 Na). It is commonly found in pesticides and
rodenticides and also n plants such as gifblaar (Dichapetalum cymosum, a plant endemic to southern
Africa). Fluoroacetate reacts with acetyl CoA to form fluro-cetyl CoA which makes fluorocitrate through
reaction 1. Fluorocitrate binds to aconitase inhibiting it competitively. This inhibits formation cis-
aconitate (intermediate) and thus isocitrate (product of reaction 2). Result is inhibition of the TCA
cycle.

Anaplerotic and Cataplerotic Reactions

TCA intermediates are constantly being used by the cells to synthesize other molecules. Thus there is a
danger that the cycle can come to a halt if too many TCA intermediates are used for these other
reactions. To overcome this, side reactions ensure that the intermediates are replenished. Some

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tissues such as muscle and brain cells rely on these side reactions since they have inadequate levels of
TCA cycle enzymes.

Cataplerotic Reactions: When TCA intermediated are in abundant, the cell uses them to make other
compounds through reactions called cataplerotic reactions

Anaplerotic reactions: When the cell runs low in TCA intermediates they are replenished by other
reactions called anaplerotic reactions

Fig: Anaplerotic/cataplerotic reactions

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Lecture 14 - Electron Transport Chain (ETC)
Learning objectives:
• Describe the shuttle systems which bring cyt. NADH into the
mitochondrial matrix

• Outline mitochondrial electron transport system with reference to the

different complexes

• Understand the mechanism and importance of Q-cycle

• Describe the mechanism of ATP synthesis by oxidative

phosphorylation and the importance of PMF in ETC

• Emphasize on the effects of inhibitors and uncouplers on the electron

transport chain & Ox. phosphorylation

NADH can be used for a variety of reductive functions in the biochemical pathways. The reducing
potential of NADH (mitochondrial) is absolutely critical to ATP synthesis via oxidative phosphorylation.

Most of the energy needed to resynthesize ATP is obtained from oxidation of reduced NADH released
within the mitochondria during TCA. However, 2 moles of NADH (per mole of glucose) are produced in
the cytosol during glycolysis. These NADH must be transported into the mitochondria in order to
convert them to ATP.

There are two mechanisms by which this cytosolic NADH will be transported:

- Malate aspartate shuttle for NADH produces about 2.3 to 2.7 equivalents of ATP per NADH

- Glycerol phosphate shuttle produces about 1.6 to 2.3 equivalents of ATP per NADH (one third less)

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Malate aspartate shuttle

In the mitochondria, OAA is

transaminated to aspartate and
pumped out into the cytoplasm.
Glutamate acts as the amine
donor.

Aspartate converts back to

oxaloacetate in the cytoplasm
which accepts electrons from the
cyt. NADH and reduces to
malate

Malate enters the inner

mitochondrial matrix through a
specific transporter

Malate in the mitochondrial

matric gets oxidized to oxaloacetate regenerating a NADH.

Mit. Oxaloacetate keeps the shuttle going

The pool of α -ketoglutarate (as glutamate) is maintained inside mitochondria due to an antiport
system that translocates aspartate out and glutamate in. cytosolic malate dehydrogenase (MDHc).
And mitochondrial malate dehydrogenase are also involved in the

Glycerol phosphate shuttle

- Begins with two different glycerol-3-phosphate dehydrogenases (G3PDs):
- Cytosolic G3PD which produces glycerol-3-phosphate
- G3PD complex is integral mitochondrial protein that oxidizes the glycerol-3-phosphate produced by
the cytosolic enzyme. This catalytic site is on the outer surface of the inner membrane, allowing ready
access to the product of the second, or cytosolic, glycerol-3-phosphate dehydrogenase.
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The mitochondrial G3PD is coupled to an FAD-linked dehydrogenase in the inner mitochondrial
membrane. The electrons are transferred to CoQ (ubiquinone) which then transfers the electrons to
Complex 3 (III) of electron transport.
In some tissues, such as heart and skeletal muscle, mitochondrial G3PD is present in very low amounts,
and the malate aspartate shuttle is the dominant pathway for aerobic oxidation of cytosolic NADH,
producing a third more ATP per glucose.

Glycerol-3-phosphate shuttle yields 1/3 less (1.6-2.3) ATP per NADH because reducing
equivalents (pairs of electrons) are shuttled directly into CoQ in the inner mitochondrial
membrane.

Overview of ATP Synthesis and the Electron Transport Chain (ETC)

Oxidation of NADH/ FADH 2 (electron donors) permits the “liberated” electrons to remain within the
mitochondrial matrix and is transferred via intermediate electron carriers to the terminal acceptor
oxygen, reducing it to water in the mitochondrial matrix.

Coupled to the oxidation reduction steps is a transport process in which protons (H+) from the
mitochondrial matrix are translocated to the intermembrane region between the inner and outer
mitochondrial membranes. The redistribution of protons leads to formation of a proton gradient across
the mitochondrial membrane. The energy of the proton gradient is known as the chemiosmotic
potential, or proton motive force (PMF). This potential is the sum of the concentration difference of
protons across the membrane and the difference in electrical charge across the membrane.

In the presence of ADP, protons pumped out of the matrix, flow down their gradient from the
intermembrane space (IMS) back into the mitochondrial matrix. This flow of protons is facilitated by
ATP synthase (acting as proton channels) present in the inner mit. membrane. The inflow of protons

196
back into the matrix enables the ATP synthases to phosphorylate ADP to ATP (oxidative
phosphorylation).

Direct chemical analysis has shown that for every 2 electrons transferred from NADH to oxygen, 2.5 to
3 equivalents of ATP are synthesized requiring 19 to 21.9 kcal of energy.
The remaining 31.7 kcal of available energy in the proton gradient (generated by electron transport)
contains sufficient energy to drive normal ATP synthesis by ATP synthase.

ETC is found in some form in all living organisms. Anaerobic bacteria utilize a non-oxygen terminal
electron acceptor in Complex 4. Aerobic organisms absolutely require oxygen for this purpose.

ETC components
The electron transport chain works via a series of reduction-oxidation (Redox) type of reactions. The
redox reactions involve the passing of electrons from one component to the next. To understand why
electrons go from one component to the next and do not have the ability to travel backward, we must
examine the redox or midpoint potential of these components. The redox potential, which is measured
in volts or millivolts, describes the point at which a half the molecules of a particular type are reduced
and half are oxidized. Electrons are passed from compounds that have a lower redox potential to
compounds that have a higher redox potential.

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Coenzymes & Cofactors of ETC

NADH (Nicotinamide adenine dinucleotide)

Free water soluble electron carrier. It carries 2 electrons.

FMN (Flavin mononucleotide) and FADH 2 (Flavin adenine dinucleotide)

Water soluble but is protein bound electron carrier. They can carry 1 or 2
electrons.

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Coenzyme Q
Free Lipid soluble electron carrier. They can carry 1 to 2 electrons.

Iron-Sulfur clusters
Protein bound electron carrier; transfers 1 electron at a time; found as 2Fe-2S or 4Fe-4S configurations;
An electron is transferred to the sulfur which is then stabilized by iron

Cytochromes and Heme

Protein bound electron carrier; transfers 1 electron at a time; acts as an electron carrier between
complex III & IV and forms an integral part of complexes III & IV; cytochrome types are cyt b, cyt c 1 , cyt
c & cyt a & cyt a3

Heme a part of cytochrome; consists of a large porphyrin ring and Fe+3 in the center. Different
subtypes are present

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COMPONENTS OF ETC

Complex I (NADH: CoQ oxidoreductase)

Composed of NADH dehydrogenase (aka NADH reductase) with FMN as cofactor, plus 22-24 non-
heme-iron-sulfur (Fe-S) proteins in 5-7 clusters. Complex I transfers electrons from NADH to CoQ. The
transmembrane channel “pumps” 4 protons (H+) from the matrix to the intermembrane space.

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Complex II (succinate dehydrogenase or succinate: CoQ oxidoreductase)

Unlike the other complexes of ETC, complex II is not a transmembrane molecule. The succinate
dehydrogenase enzyme (a TCA enzyme) containing FAD is an integral part of complex II. It also contains
7-8 Fe-S proteins in 3 clusters. Free energy from the electron donated by FADH2 and flowing through
Complex II is insufficient to pump protons from the mit. matrix to the IMS. This explains why a pair of
electrons originating from NADH in complex I supports production of ~2.5 equivalents of ATP, while 2
electrons from succinate (FADH) support the production of only ~1.5 equivalents of ATP.

Complex III
Contains cytochrome b, cytochrome c1 and one Fe-S protein, known as the Rieske iron sulfur protein.
Cytochrome c is the smallest of the cytochromes (12,000 MW). The transmembrane channel of this
complex “pumps” protons (H+) from the matrix to the intermembrane space.
In contrast to the heme of hemoglobin and myoglobin, the heme iron of all cytochromes participates in
the cyclic redox reactions of electron transport, alternating between the oxidized (Fe+3) and reduced
(Fe+2) forms.

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The Q-cycle

A QH 2 from the Q pool comes to the Q p site of the complex III. One electron from QH 2 is transported to
cytochrome c (intermediate between complex III & IV) via Fe-S centers & Cyt.c 1 ; the process is coupled
with the pumping of 2 protons from the mit. matrix to the IMS. The 2nd electron is transferred to a
ubiquinone (Q) or semiquinone (QH.) present at Qn site of complex III via the cytochrome b L & b H

A second QH 2 coming into the Q p site goes through the same process pumping 2 protons out, and
transferring one electron to cyt.c and one electron through bL and bH generates a QH 2 at the Qn site
which goes back into the Q pool. This is called the Q cycle

Main features of the Q-Cycle:

2 QH 2 are oxidized to Q pumping 4H+ into the IMS (@2 electrons/QH2)
A QH 2 is produced at the Qn site which is recycled to the Q-pool (recycling of electrons)
2 cyt.c molecules are reduced in the process transferring one electron at a time from complex III to
complex IV

Complex IV (cytochrome oxidase)

Contains the hemeproteins cytochrome a and cytochrome a3, as well as copper-containing proteins in
which the copper undergoes a transition from Cu+1 to Cu+2 during the transfer of electrons through the
complex to molecular oxygen. Oxygen is the final (terminal) electron acceptor, with water being the
final product of oxygen reduction and more protons are pumped to the intermembrane space.
Oxygen consumption is directly proportional to rate of respiration.

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Electron transport is linked to oxidative phosphorylation

Coupled to the oxidation reduction steps is a transport process in which protons (H+) from the
mitochondrial matrix are translocated to the intermembrane region between the inner and outer
mitochondrial membranes. The energy released during electron transfer at each complex is used to
pump out protons from the mit. matrix into the IMS. The redistribution of protons leads to formation
of a proton gradient across the mitochondrial membrane. The energy of the proton gradient is known
as the chemiosmotic potential, or proton motive force (PMF). This potential is the sum of the
concentration difference of protons across the membrane and the difference in electrical charge across
the membrane.

Complex I, III, and IV (NOT II) use the passage of electrons to translocate protons from the matrix to
IMS. Large proton gradient = Proton Motive Force. PMF is used by Complex V ( ATP synthase ) to
synthesize ATP

Complex V - ATP synthase

The protons from IMS flow back into the matrix through the complex V which drives the

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phosphorylation of ADP to ATP. Therefore we get 2.5 ATP per NADH (from pumping of 10 protons) and
1.5 ATP per FADH 2 (from 6 protons).

ATP synthase is composed of 3 individual components:

- F 0 : Localized within the membrane and has been shown to physically rotate.
- F 1 : Protrudes from the inside of the inner membrane into the matrix

ATP synthase is a multiple subunit complex that binds ADP and inorganic phosphate at its catalytic site
inside the mitochondrion, using the proton gradient (1/3 fewer from FADH 2 ) to provide energy for the
reaction.

Damaged mitochondria display increased permeability to protons which allow them to “escape” from
the outer matrix. This effectively reduces, and in some cases reverses, the ATP synthase reaction. The

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ATP synthase becomes a very efficient ATP hydrolase or ATPase and quickly depletes the ATP. The cells
will have insufficient energy (ATP) and will die.
A number of drugs, especially those used to treat HIV (enzyme inhibitors), also inhibit mitochondrial
enzymes. Oxygen radicals (O-, H 2 O 2 , etc) also damage the mitochondrial matrix.

This same basic process occurs when there is no oxygen present (hypoxia). The proton flux decreases
as the electrons “pull” protons back into mitochondrial matrix. No new ATP is made while ATP
hydrolysis within the inner matrix occurs.
Without oxygen cells will lose over 80% of ATP production; a few will be generated by glycolysis.
Neurons in the CNS will begin to die within 6 minutes when there is reduced/absent oxygen from
blood. This is the basis of cardiopulmonary resuscitation (CPR).

Effects of Inhibitors & uncouplers on oxidative phosphorylation

The pathway of electron flow through the electron transport assembly and the unique properties of the
PMF, have been determined through the uses of a number of important antimetabolites.

Some of these agents are inhibitors of electron transport at specific sites in the electron transport
assembly, while others stimulate electron transport by discharging the proton gradient. A more
comprehensive list can be found in the table below.

For example, antimycin A is a specific inhibitor of cytochrome b. In the presence of antimycin A,

cytochrome b can be reduced but not oxidized. As expected, cytochrome c remains oxidized in the
presence of antimycin A, as do the downstream cytochromes a and a3 of complex IV.

Another class of antimetabolites are the uncoupling agents exemplified by 2,4-dinitrophenol (DNP).
Uncoupling agents act as lipophilic weak acids, associating with protons (ionophore) on the exterior of
mitochondria, passing through the membrane with the bound proton, and dissociating the proton on the
interior of the mitochondrion. These agents cause maximum respiratory rates but the electron transport
generates no ATP, since the translocated protons do not return to the interior through ATP synthase.

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 Name Function Site of Action Sources/Uses
Rotenone e- transport inhibitor Complex I Pesticide/jicama plant
Barbiturates e- transport inhibitor FeS to CoQ transfer/II CNS
depressant/anesthetic
Malonate Competitive inhibitor Complex II Found in fruits
Antimycin A e- transport inhibitor Complex III piscicide
Cyanide e- transport inhibitor Complex IV Dyes, foods (cassava)
Carbon Monoxide e- transport inhibitor Complex IV Combustion/carbon
oxidation
Azide e- transport inhibitor Complex IV airbags
2,4-dinitrophenol Uncoupler PMF Dyes, dietary aid
Pentachlorophenol Uncoupler PMF Pesticide/hull paints
Oligomycin ATP synthesis inhibitor Complex V Macrolide
antimicrobial

Thermogenin: Natural uncoupling components also exist. Some cells (such as brown adipose tissue)

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express proteins known as uncoupling proteins (UCP1). These uncoupling proteins dissipate the proton
motive force and this up-regulates the rate of electron transport. A faster electron transport results in
heat generation. This is the main mechanism in which the human body maintains temperature.

Effect of Cyanide on the electron transport chain

Cyanide is a chemical compound that contains monovalent combining group CN. This group consists of
a carbon atom triple- bonded to a nitrogen atom. Cyanide is considered to be toxic because it binds to
cytochrome c oxidase ie. complex IV of the electron transport chain. It binds tightly to the iron present
in the cytochromes of complex IV and inhibits the normal activity of the complex. The cytochromes of
complex IV thus cannot transport any electrons to the terminal electron acceptor, oxygen. This
eventually blocks the passage of electrons throughout the electron transport chain, inhibiting proton
pumping at the different complexes and halting ATP production. Therefore, the person is deprived of
chemical energy to perform the many numerous processes that sustain life, and the person dies.

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Effect of carbon monoxide (CO) on electron transport chain
Carbon monoxide intoxication causes impaired oxygen delivery and utilization at the cellular level. The
affinity of Hb for CO is almost 300 times higher than for oxygen. An environment in which there is 100
ppm of CO is enough to form 16 % carboxyhemoglobin. The situation is worsened since the binding of
CO to one of the heme groups of hemoglobin increases the affinity of the other three heme groups for
oxygen (co-operativity), so the delivery of oxygen to tissues is very affected. The brain and the heart,
that has a high oxygen consumption, are the most affected by CO intoxication. Myoglobin has even a
greater affinity for CO than hemoglobin. It is considered that the inhibition of Cytochrome Oxidase by
CO also plays a role in CO intoxication. CO binds to the reduced form of iron in heme groups (Fe++) in
cytochrome Oxidase

Reactive Oxygen Species (ROS)

A partial reduction of oxygen can result in reactive oxygen species (ROS) such as superoxide (O 2 ·-),
hydrogen peroxide (H 2 O 2 ), and hydroxyl radical (OH·). Many of the ROS molecules are radicals, they
have extra electrons associated with them and will readily give that electron up. The loss of the extra
electron to DNA, proteins, or lipids can result in damage. Thus, high levels of ROS in cells are very
harmful to the cell.

Cells have developed mechanisms and enzymes to neutralize the ROS molecules. Below is a list of
some enzymes used in the detoxification of ROS.

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Chapter 15 - Glycogen, Gluconeogenesis and the Pentose
Phosphate Pathway
Lecture Objectives:

• Describe the structure of glycogen, its functions and storage sites

• Outline the pathways for glycogen synthesis & degradation
• Emphasize on the hormonal regulation of glycogen synthesis and
breakdown by insulin and glucagon ratio in blood
• Identify the different glycogen storage diseases, their causes,
symptoms
• Learn how glycogenolysis and gluconeogenesis play a role in
regulation of glucose homeostasis
• Outline the pathway of gluconeogenesis and its importance

Glucose is the principle source of cellular energy. We have about 10g of glucose in the plasma and
extracellular fluid volume. We can absorb glucose 2-3 hours following a carbohydrate meal. After a
meal, if glucose levels are high, glucose is converted into glycogen. Glycogen is a polysaccharide
storage form of glucose synthesized in the liver and skeletal muscle cells.

When glucose levels fall below normal, blood glucose is replenished by breaking down liver glycogen to
glucose, to prevent hypoglycemia. Severe hyperglycemia can lead to confusion, disorientation and
coma. Hypoglycemic shock (a blood glucose concentration below 2.5mmol/L (45mg/dL) leads to coma.

Total glycogen in skeletal muscle is greater than in the liver, however the liver contains more glycogen
per gram of tissue.

Liver: 75g Glucose

Skeletal muscles: 250g Glucose
Blood and ECF 10g Glucose

During sleep or starvation, glucose levels have to be maintained at homeostatic range (80-100mg/dL).
Assuming that hepatic glycogen stores are depleted, the liver can synthesize glucose from protein, by
gluconeogenesis. Amino acids from muscle are used to synthesize glucose. The two most commonly
used amino acids for this purpose are Alanine and Glutamine.

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The processes that help to maintain normal blood glucose levels (80-100mg/dL of blood) are:

Glycogenesis: Making glycogen from excess glucose in blood

Glycogenolysis: Breaking down glycogen to produce glucose during hypoglycemia

Gluconeogenesis: Making new glucose from mostly AA during starvation

Glycogenesis
Excess glucose present in the blood to be converted to glycogen. Glycogen is a polymer of Glucose1-P
formed from Glucose 6-P. It is a highly branched molecule with lots of non-reducing ends.

For glycogen synthesis to start a central protein, glycogenin, is required. This forms the core of a
glycogen molecule. Glycogenin is an enzyme also known as Glycosyltransferase. It attaches the C-1 of
the first UDP-glucose to a Tyrosine residue on it. Glycogenin thereafter catalyzes the formation of a
‘Glycogen Primer” by joining 4-7 glucose units via glycosidic bonds. Once the primer is formed,
glycogen synthase adds glucose units to the non-reducing end of the glycogen primer. Glycogenin
remains attached to the reducing end of the starting glycogen chain

Glycogen is a highly branched molecule. The glucose units in a glycogen chains are linked by alpha 1.4
linkages and branch points are linked by alpha 1,6 glycosidic linkages. A glycogenin molecule remains
attached to the reducing end of a glycogen polymer.

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STEPS in Glycogenesis
1. G6P is converted to G1P by phosphoglucomutase.

2. Next, UDP glucose pyrophosphorylase then adds the UMP component of a UTP molecule to the
phosphate on C1 of glucose resulting in UDP-glucose. This makes the glucose unit “activated”
for incorporation into the growing glycogen molecule.

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 3. Finally, Glycogen synthase, the main enzyme involved in glycogen synthesis will add the
activated glucose unit to the growing glycogen chain. During this reaction, a highly reactive
oxonium ion intermediate is formed which attacks the non-reducing end of the glycogen chain.
The glycogen synthase is activated by insulin

In addition to growing linear chains, glycogen becomes branched. The branching enzyme (Amylo 1,4-
1,6-transferase) will transfer the last 7 glucose units from the end of a growing chain to what will
become the branch point on that chain. This transfer will only occur if the growing chain is at least 11

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glucose units long and will transfer that chain no closer than 4 units from another branch point. This
branch point forms an α 1,6 linkage.

Overview of glycogen synthesis

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Glycogenolysis
The process begins with the binding of glucagon to its receptor on hepatocytes (GCCR receptors).
Glucagon is secreted by the alpha cells of the Islets of Langerhans in response to low blood glucose
levels. Glucagon binding to its receptor stimulates the breakdown of glycogen to glucose by activation
of the main enzyme involved in the pathway called glycogen phosphorylase.

1. Active glycogen phosphorylase removes single glucose units from alpha 1,4 linkages in the
glycogen molecule. Glycogen Phosphorylase works by adding a phosphate to C1 of the non-
reducing end glucose unit. This phosphorylation breaks the α 1,4 glycosidic bond that held that
particular glucose unit to the glycogen molecule.
Glycogen phosphorylase uses Vitamin B6 (the pyridoxal 5- phosphate form of B6) as its
coenzyme .

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 2. The next reaction is a reverse reaction of glycogenesis. Phosphoglucomutase converts the G1P
to G6P.

3. In the liver (not in the muscle cells), glucose phosphatase removes the phosphate from G6P to
generate glucose. This glucose is released in blood resulting in an increase in blood glucose
level.

4. Debranching: Glycogen’s highly branched structure allows for a lot of glucose storage. To fully utilize
that storage and get the glucose from the glycogen, glycogen has to be debranched. Debranching
occurs via a debranching enzyme [α(1,4) transglycosylase] and a glucosidase.

A glycogen branch has glucose units removed from it until it gets to within four glucose units from the
branch point. The debranching enzyme then removes the 3 units on the reducing end of that branch
and transfers them to the end of a longer glycogen chain where they can be removed by glycogen
phosphorylase. The single glucose unit that remained at the branch point is then removed by the
enzyme glucosidase.

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 Regulation of Glycogen Metabolism
Glycogen metabolism is controlled through the phosphorylation (kinases) and dephosphorylation
(phosphatases) of glycogen synthase and glycogen phosphorylase. This overall control is mediated
through hormones namely insulin, glucagon, and epinephrine.

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Regulation of Glycogenesis
Glycogen synthase can be activated or inhibited by various mechanisms. Glycogen synthase a (GS-a) is
the active form and is favored by increased G6P and insulin stimulation.

Insulin binding activates IRS-1 products which are then able to upregulate Protein Phosphatase 1 (PP1)
which dephosphorylates glycogen synthase to the more active dephosphorylated GS -a form.

The ongoing synthesis of glycogen by GS-a continues as long as there is sufficient G6P (energy surplus).

Low cyclic AMP (cAMP) levels (due to reduced glucagon secretion) also favor GS-a through
inhibition/inactivation of PKA which prevents activation of protein phosphatase 1 inhibitor (PP1-I).

Inevitably as G6P decreases (energy deficit) the phosphorylated GS-b form predominates and
glycogenolysis (due to increased glucagon) is favored while glycogenesis is reduced.

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Regulation of Glycogenolysis
In the liver, both glucagon and epinephrine can stimulate glycogenolysis. In the skeletal muscle
however, glucagon does not stimulate glycogenolysis as there are no receptors for glucagon on skeletal
muscle cells. However, Epinephrine can stimulate glycogenolysis in skeletal muscle via beta receptors.
Note that glycogenolysis in skeletal muscle does not contribute to blood glucose as it ensures the
supply of glucose for glycolysis in skeletal muscle. Skeletal muscle cells lack glucose 6-phosphatase,
thus ensuring that G6P cannot be converted back to glucose and leave the muscle cells. The glucose 6-
P is thus committed to glycolysis in muscle cells.

Under glucagon (during fasting) and epinephrine (stress) cAMP levels are increased in ce4lls like the
hepatocytes. Increased cAMP activates PKA which then activates phosphorylase kinase. Phosphorylase
kinase phosphorylates glycogen phosphorylase activating it to its “a” form. Phosphorylase kinase also
leads to glycogen synthase kinase 3 (GSK-3) activation which then phosphorylates and turns off
glycogen synthase to its inactive “GS-b”. Glucagon also activates protein phosphatase 1 inhibitors (PP1-
I), which keeps the dephosphorylation cascade off.

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 Glycogen Storage Disorders
Type/Name Enzyme(s)/Proteins Affected Organ Primary Manifestation

Type 0 Glycogen Synthase Liver Hypoglycemia,

hyperketonia, early death
Type Ia: GSD Ia or Glucose 6-phosphatase Liver Hepatomegaly, kidney
Van Gierke’s [Ia and Ib comprise 80%+ failure, thrombocyte
of all GSDs] dysfunction
Type Ib Microsomal G6P Liver Similar to Ia with
translocase neutropenia and increased
infections
Type Ic Microsomal P Liver Similar to Ia
transporter
Type II: Pompe’s Lysosomal acid Skeletal and - Infantile form = fatal by 2
glucosidase Cardiac Muscle yrs
- Juvenile form = myopathy
- Adult form = muscular
dystrophy-like
Type III: Cori’s Debranching enzyme Liver and Muscle Infant hepatomegaly,
and Forbe’s myopathy
Type IV: Branching enzyme Liver and Muscle Hepatomegaly,
Anderson’s splenomegaly, & liver
cirrhosis
Type V: McArdle’s Muscle phosphorylase Skeletal Muscle Exercise-induced cramps and
pain myoglobinuria

Type VI: Her’s Liver phosphorylase Liver hepatomegaly

Type VII: Tarui’s PFK-1 Muscle and Similar to V with hemolytic

RBC’s anemia
Type VIIb, VIII or Phosphorylase kinase Liver, muscle, Similar to VI
Type IX and leukocytes

Type X PKA Liver, Muscle hepatomegaly

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Gluconeogenesis
As mentioned earlier, in time of fasting or starvation, the blood glucose level needs to be maintained.
When hepatic glycogen stores are depleted, amino acids from muscle proteins are mobilized and
converted to glucose through Gluconeogenesis. Many glycolytic enzymes are used in this process but
gluconeogenesis is not quite the complete reversal of glycolysis. The three irreversible steps in
glycolysis are bypassed by three unique enzymes involved in gluconeogenesis.

1st BYPASS reaction - 2 step reaction

Catalyzed by Pyruvate carboxylase- Rate limiting enzyme of gluconeogenesis

Gluconeogenesis begins with the conversion of pyruvate to oxaloacetate. Besides glucose, glucogenic
amino acids like alanine can also produce pyruvate. Carboxylation of pyruvate generate oxaloacetate.
The addition of CO 2 to pyruvate is accomplished by the enzyme pyruvate carboxylase. This is an ATP
dependent reaction. Biotin serves as a coenzyme of pyruvate carboxylase and carries the activated
bicarbonate

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Phosphoenolpyruvate is formed from oxaloacetate by the enzyme PEP carboxykinase. This enzyme
uses GTP as a phosphate donor. In the reaction CO 2 is liberated from oxaloacetate. This reaction of
gluconeogenesis is “reversing” the pyruvate kinase step of glycolysis.

2nd BYPASS reaction

Fructose 6-phosphate is formed from fructose 1,6 bisphosphate by the enzyme fructose 1,6
bisphosphatase. This is because the glycolytic reaction performed by phosphofructo kinase 1 is
irreversible.

3rd BYPASS reaction

Glucose is made from glucose 6-phosphate by the enzyme glucose 6-phosphatase. This is a “reverse”
of the glycolytic reaction done by hexokinase. This is the same reaction that occurs in liver cells during
glycogenolysis to liberate glucose from glycogen stores.

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Regulation of Gluconeogenesis
There are two major control points of gluconeogenesis.

The first is regulation of pyruvate carboxylase which is involved in the conversion of pyruvate to
oxaloacetate. Pyruvate carboxylase is under the opposite control of pyruvate dehydrogenase of
glycolysis. Where pyruvate dehydrogenase is stimulated by ADP and inhibited by acetyl-CoA, pyruvate
carboxylase is inhibited by ADP and stimulated by acetyl-CoA. High fructose 2,6 bisphosphate levels
down regulate the enzyme pyruvate carboxylase ( F-2,6 BP turns on PFK -1 and thus glycolysis).

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Dynamics of glucose utilization at cellular levels

Primary source of glucose in fed state: Dietary glucose

Primary source of glucose during fasting (6-7 hours after feeding): Glycogenolysis occurring in the liver
Primary source of glucose during starvation (after 24hrs of fasting): Gluconeogenesis

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Lecture 16 - Disorders of Carbohydrate Metabolism
Learning Objectives
• Identify hypoglycemia, and hyperglycemia from blood glucose values
• Describe insulin production, insulin release & its mechanism of action at cellular
levels (insulin cascade)
• Focus on tissue specific & metabolic effects of insulin & insulin resistance
• Describe glucagon response pathway & its cross talk with epinephrine
• Differentiate between Type I & Type II diabetes in terms of causes, symptoms,
effects at cellular levels, important diagnostics and discussed treatments
• Learn why diabetic ketoacidosis is related to type I & not type II
• Emphasize on the significance of HbA 1C

Normal Fasting Blood Glucose Level (BGL) is generally less than 90 mg/dL (5 mM). It is characterized by
a lot of individual variation.

Hyperglycemia: BGL is 140 mg/dL (7.8 mM) or greater

• Symptoms: Frequent urination, Thirst, Headaches, Blurred vision, Nausea, Tired

Hypoglycemia: BGL is 60 mg/dL (3.3 mM) or lower

• Symptoms: Confusion, Shakiness, Dizziness, Tired, Tachycardia

The two pancreatic hormones, Insulin and Glucagon are the main glucose regulatory hormones. Insulin
is secreted by the pancreatic beta- cells in response to high blood glucose levels whereas glucagon is
secreted by the pancreatic alpha cells in response to low or subnormal blood glucose levels.

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Insulin
Mechanism of Insulin Release
Glucose enters the beta Islet cells via GLUT 2 transporters, the expression of which is not insulin
dependent. Glucose then undergoes glycolysis, generating ATP. The ATP levels rise, closing the ATP-
dependent potassium channels (K+ATP). This results in depolarization of the beta Islet cells. This is
followed by opening of the voltage-gated calcium channels, allowing calcium to flow into the cell. This
stimulates calcium-induced release of storage vesicles through exocytosis.

Insulin Production
The mature functional insulin molecule is made up of
two peptides held together by disulfide bonds. The two
peptides that make up this protein are translated from
the same mRNA transcript and thus post-translational
processing of the new peptide occurs. The immature
insulin peptide is called pre-proinsulin and consists of a

225
signal peptide sequence and chains A, B, and C. The signal peptide sequence is required to target the
newly made peptide to the endoplasmic reticulum (ER) of the cell where the processing of the insulin
peptide occurs. Once the peptide is in the ER, the signal sequence is removed via endopeptidase
cleavage. The resulting molecule is called proinsulin. In addition to the removal of the signal sequence,
the reducing environment of the ER causes two disulfide bonds to form between chains A and B.
Finally, an endopeptidase cleaves chain C from chains A and B forming the fully functional insulin
molecule.

Since the C-peptide is made in equal molar proportions as mature insulin molecules by beta cells,
measuring the ratios of C-peptide to insulin in diabetics provide an indication on the levels of insulin
made by beta cells versus the amount of insulin coming from exogenous sources such as insulin
injections (which do not contain the chain C peptide). C-peptide helps to access beta cell functionality
in diabetics

Insulin is released in 2 Phases:

1. First phase is release of insulin stored in the

beta cells
2. Release of the insulin being synthesized in
response to glucose increase

Insulin Receptors
The receptor is a tetramer consisting of 2 alpha and 2 beta subunits. The extracellular face (alpha
subunits) of the insulin receptor is bound to a transmembrane Tyrosine Kinase (beta subunit) which in
turn activates a signal transduction cascade which results in several cellular responses.

One response is the translocation of the insulin dependent GLUT 4 glucose transporter.

1. Insulin binds to the alpha subunit of the receptor at the cell membrane.
2. The binding of insulin results in a conformational change of the beta subunit. This results in
activation of the kinase domain which autophosphorylates its tyrosine residues.
3. The phosphorylated tyrosines become binding sites for Insulin Receptor Substrates (IRS) such as
IRS1 which in turn are phosphorylated.
4. Once IRS1 is phosphorylated, it becomes the binding site for other proteins such as PI3 Kinase
which, now that it has been recruited near the membrane, can interact with its substrate PIP 2
to generate PIP 3 .
5. The presence of PIP 3 at the cellular membrane will recruit Protein Kinase B (AKT) to the
membrane where it is then activated by PDK1 (Phosphoinositide-dependent kinase 1).

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 6. Activation of PKB leads to additional signal responses, one of which is the translocation of the
glucose transporters like GLUT4 to the cell membrane.
7. GLUT4 is then used by the cell to transport glucose into the cytoplasm.

The binding of insulin to its receptor also results in the following:

1. An increase in the transport of potassium into cells and thus it can be used to treat
hyperkalemia.
2. An increase in gene expression such as GLUT
3. An increase in glycogenesis
4. It inhibits glycogenolysis
5. An increase in amino acid transport into cells and protein synthesis
6. Inhibits apoptosis
7. Increase Lipid synthesis and decrease lipid
degradation

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Glucagon and Stress Hormones
Glucagon and the stress hormones such as epinephrine, work to raise blood glucose levels. Glucagon
binds to the glucagon receptor which is found only in liver cells. Muscles do not have glucagon
receptors. Epinephrine (stress hormone) bind to adrenoreceptors in muscle and liver to activate
different responses.

1. The binding of glucagon or epinephrine to its receptor results in activation of specific G-

proteins.
2. These G-proteins activate adenylate cyclase which converts ATP to cyclic AMP (cAMP).
3. Increased level of cAMP leads to activation of Protein Kinase A (PKA).
4. PKA is then used to phosphorylate many different kinases which leads to:
a. Increased Gluconeogensis (in the liver)
b. Decreased glycolysis (in the liver)
c. Increased Glycogenolysis
d. Decreased Glycogenesis
e. Increased lipolysis

High levels of epinephrine can also stimulate the release of additional glucagon from the alpha cells of
the pancreas.

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 Diabetes
Type I: Absolute deficiency of insulin

Type II: Relative decrease in insulin

Gestational: Occurs during pregnancy

Type I Diabetes mellitus: (IDDM)

Caused by relative or absolute deficiency of the hormone insulin. When the diabetes is autoimmune in
nature it is genetic. However environmental causes can lead to diabetes type I as well. In type 1
diabetes beta cells are destroyed (diabetes type Ia) due to different reasons which leads to deficiency
or total lack of insulin. The beta cells can also become non responsive to changes in blood glucose
levels and stop producing insulin (diabetes type Ib)

Causes:

• Genetic:
Diabetes is a polygenic disorder. Defect in numerous genes can lead to the disease. In the
autoimmune type, defects occur in one or more of the HLA genes present on chromosome 6. Defects
in HLA genes like IDDMI, HLA-DR, HLA-DQ are strongly linked to diabetes type I. These defective genes
produce defective MHC proteins which are the underlying cause of autoimmunity associated with

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diabetes type I. Autoantibodies are often produced against endogenous proteins of localized in beta
cells like insulin, tyrosine phosphate , GAD etc leading to destruction of beta cell population in the
patient

Example: Autoantibodies produced against glutamic acid decarboxylase (GAD) which are proteins
expressed by beta cells population might lead to beta cell
damage and onset of type I diabetes.

• Environmental:
Studies have shown that in identical twins having the same genetic material, if one twin is diabetic,
there is only a 30-50% chance that the second twin may get diabetes.

Some evidences point to the fact that exposure to parasitic infections might often decrease the risk of
developing type I diabetes.

Some drugs have been shown to destroy the beta Islet cells including the rodenticide Pyrinuron and
the chemotherapeutic drug Streptozotocin.

Other causes of diabetes type I may include trauma, pancreatitis, or

tumors of the pancreas.

Clinical Symptoms of Diabetes Type I

Rapid weight loss

Ketosis and acidosis (through fatty acid degradation)

Polyphagia, Polydipsia and Polyuria

Neurological symptoms such as confusion, loss of consciousness

Lab Findings: Hyperglycemia, renal glycosuria

Renal and Microvascular complications

Ketoacidosis associated with Type I diabetes

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In diabetes Type I, patients cannot utilize blood glucose due to lack of insulin

Cellular glucose levels low; Blood glucose levels are high

Β-oxidation of fatty acids provide cellular ATP

Some amino acids are broken down to yield acetyl-CoA

Acetyl CoA levels are high due to excess fatty acid oxidation

The excess acetyl-CoA produced will overwhelm the TCA cycle

Acetyl-CoA is converted to Ketone Bodies: Acetoacetate, Hydroxybutyrate, Acetone

Ketones are acids and will contribute H+ to the blood, thus lowering the blood pH; this may result in
metabolic acidosis more specifically ketoacidosis (blood pH below 7.35).

Symptoms of ketoacidosis

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Treatment of Type I Diabetes mellitus (IDDM)
Diet: Reduce Carbohydrate intake

INSULIN: Injectable

Porcine Insulin: Differs from human insulin by 1 amino acid

Bovine Insulin: Differs from Human Insulin by 2 amino acids

These can give rise to allergic reactions

Human Insulin: (Humulin)

Made by genetic engineering using E Coli

Rapid-acting: Usually taken before a meal to cover the blood glucose elevation from eating. This type
of insulin is used with longer-acting insulin.

Short-acting: Usually taken about 30 minutes before a meal to cover the blood glucose elevation from
eating. This type of insulin is used with longer-acting insulin.

Intermediate-acting: Covers the blood glucose elevations when rapid-acting insulins stop
working. This type of insulin is often combined with rapid- or short-acting insulin and is usually taken
twice a day.

Long-acting: This type of insulin is often combined, when needed, with rapid- or short-acting
insulin. It lowers blood glucose levels when rapid-acting insulins stop working. It is taken once or
twice a day.

Note: An overdose of insulin can cause hypoglycemic shock (blood sugar below 40mg/dL)

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 Peak
Onset Duration
(time
Brand (length of (how
Type period
Name time before long
when
insulin insulin
insulin is
reaches works
most
bloodstream) for)
effective)

Humalog 10 - 30 30
(lispro) minutes 3-5
Rapid-acting minutes -
Novolog hours
3 hours
(Aspart)

Regular 30 minutes - 1 2 - 5 Up to 12
Short-acting
(R) hour hours hours

Intermediate- 4 - 12 Up to 24
NPH (N) 1.5 - 4 hours
acting hours hours

Minimal
Long-acting Glargine 0.8 - 4 hours Up to 24
peak
Detemir hours

TYPE II DIABETES
Characterized by presence of insulin but insulin not being effective at cellular levels (insulin resistance).
Type II diabetics exhibit slow or no effects of insulin on glucose metabolism due to defects in the
insulin receptors or downstream signaling cascade molecules involved in glucose metabolism in
particular

Problems with the insulin receptor may be due to

• Mutant insulin receptor

• Autoantibodies produced against insulin receptors
• Diminished receptor production due to increased insulin levels in blood. High ligand
concentration downregulating the receptor expression
• Defects in insulin signaling pathway (defective downstream signaling molecule)

In most cases, diabetes type II patients have high insulin production by pancreatic beta cells since the
body is trying to compensate for the reduced effect of insulin at cellular levels

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However, the effect of insulin on fat and protein metabolism in a diabetes type II patient remains
active. Hyperinsulinemia in these patients often lead to high fat and protein synthesis.

Symptoms:

• Higher than normal blood glucose levels (hyperglycemia)

• Higher insulin levels (hyperinsulinemia)
• Polydipsia, polyuria, polyphagia
• High risk with obesity; low HDL, high triglycerides, high LDL
• High blood pressure
• Not prone to ketoacidosis (as insulin turns on fatty acid synthesis and switches off fatty acid
oxidation)
• Symptoms of metabolic syndrome

Late complications of type II diabetes is eventual destruction of beta cells as they are overworked. This
eventually leads to low insulin levels in the patient (cusp of diabetes type I and II)

Note: Weight gain can lead to insulin resistance or vice versa.

Treatment of Type II Diabetes mellitus

Biguanides: Metformin

Mode of Action:
a. Inhibits gluconeogenesis
b. Decreases absorption of glucose from the GIT ( gastro intestinal tract) causing GIT
discomfort

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 c. Increases glucose uptake into skeletal muscle
d. Inhibits glycogenolysis

Biguanides like metformin help to maintain the blood glucose levels of a type II diabetic patient at the
homeostatic level (90-100mg/dL). Biguanides are less likely cause hypoglycemic shock in diabetic type
II patients. However, an overdose may lead to hypoglycemia.

Side Effects: Lactic acidosis characterized by abdominal cramps. Only an extremely high dosage of
metformin can cause severe metabolic acidosis

Gestational diabetes
Diabetes during pregnancy

Fetus overproduces insulin resulting in large baby (Growth Hormone Activity of Insulin)

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Diagnostic tests for diabetes
Random blood sugar test: A blood sample will be taken at a random time. Regardless of the timing of
the last meal, a random blood sugar level of 200 mg/dL or 11.1 mmol/L or higher is suggestive
diabetes.
Fasting blood sugar test: A blood sample will be taken after an overnight fast. A fasting blood sugar
level less than 100 mg/dL (5.6 mmol/L) is normal. A fasting blood sugar level from 100 to 125 mg/dL
(5.6 to 6.9 mmol/L) is considered prediabetes. If it's 126 mg/dL (7 mmol/L) or higher on two separate
tests, the patient has diabetes.

Oral glucose tolerance test: For this test, overnight fasting is required. The first step is to measure the
fasting blood glucose level. Then the patient is allowed to drink a sugary liquid, and thereafter the
blood glucose levels are tested periodically for the next two hours. A blood glucose level less than 140
mg/dL (7.8 mmol/L) is normal. A reading of more than 200 mg/dL (11.1 mmol/L) after two hours is
indicative of diabetes. A reading between 140 and 199 mg/dL (7.8 mmol/L and 11.0 mmol/L) indicates
pre-diabetic conditions.

Blood test for autoantibodies

Quantification of levels of Hemoglobin A

1c
• In the presence of transition metals (Fe, Cu), glucose is auto-oxidized generating ROS
• Oxidized glucose can attach to lysine and valine residues of proteins (non-enzymatic glycolation
event) present in the blood
• Hemoglobin, albumin and collagen are examples of proteins that can be glycolated by presence
of high levels of oxidized glucose in blood
• Measurement of Hemoglobin A 1c (most stable form of glycosylated hemoglobin) , can give an
indication of average glycemia over a period of about 3 months preceding the measurement.
• Given as a percentage of total hemoglobin; Normal Hb 1c (< 5.6%), Pre diabetic (5.7-6.4%),
Diabetic (>6.4%);Goal for diabetic patients is <7%

Urine Tests: To look for the presence of a byproduct of excess fat oxidation namely ketones. Glucose levels in
urine is also measured.

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Chapter 17- Urea Cycle and Amino Acid Metabolism
Learning Objectives:

1. Describe how nitrogen is removed from amino acids (AA) in terms of:
• Toxicity of ammonia (hyperammonemia, its cause and effects)
• Transamination reactions
• Role of glutamate & α-ketoglutarate
• Role of Alanine in carrying muscle nitrogen via blood
2. Learn the reactions of urea cycle and the enzymes involved
3. Know the different fates of AA (SAM, hormones etc)
4. Explain the importance of nitrogen balance
5. Emphasize on the causes & symptoms to differentiate between the disorders
related to AA metabolism

Amino acids are used for protein synthesis. Excess dietary amino acids are converted to metabolites for
cellular uses like the TCA cycle intermediates. Glucogenic amino acids and ketogenic amino acids are
mostly used for the purpose. Cells can use these metabolites for energy production directly or via
production of glucose through gluconeogenesis.

The C-skeleton of AA is used to make the above mentioned metabolites. The amino group (via
ammonia) is converted to urea and excreted out of the body through urine.

The amino group or nitrogen of amino acids is eliminated

• By aquatic organisms as ammonia (Ammonotelic)
• By birds & reptiles as uric acid (Uricotelic)

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 • By humans as urea (Ureotelic)

Why is Ammonia Disposed?

Ammonia is toxic and excess accumulation of ammonia in blood (hyperammonemia) causes
encephalopathy or neuronal degeneration (discussed later)

Aminotransferases are enzymes that play the lead role in disposal of nitrogen from
amino acids as ammonia
Aminotransferases (transaminases) are capable of removing the amino group from most amino acids
and producing the corresponding α -keto acid. They use vitamin B 6 as a cofactor which is required to
stabilize the amine group during the transfer. The amino acids which is the NH 2 donor & substrate of
the enzyme binds to the active site of the enzyme. NH 2 is removed from the amino acid and
transferred via PLP (vit B6) to α-ketoglutarate or oxaloacetate which act as acceptors of NH 2 group.
The end products of the reactions are glutamate and a ketoacid

Several fates of the Glutamate formed by transamination reactions

In Liver:

Glutamate produced in the liver due to transamination of amino acids undergo a second
transamination donating its NH 2 group to oxaloacetate. The end products produced is aspartate
which l enters the urea cycle. The reaction will regenerate alpha ketoglutarate which then can be
reused for other transamination reactions occurring in the liver.

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 In

Peripheral tissues:

Glutamate formed in peripheral tissue due to transamination reactions undergoes the following
reactions:

1. Glutamate will convert to glutamine which is the transport form of nitrogen through blood. The
enzyme involved is glutamine synthetase. It requires an ATP
2. Glutamine is released in blood and transported to the liver through transporter proteins. In the
liver the glutamine reconverts to glutamate by the enzyme glutaminase (expressed only in liver,
kidneys, neurons)

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 3. Glutamate undergoes deamination to release ammonium; enzyme involved is glutamate
dehydrogenase (GDH )which uses NADP/ NAD+ as a cofactor

4. Ammonium formed enters urea cycle

Role of alanine in transport of amino acid nitrogen

Alanine is another major carrier of amino acid nitrogen via blood to the liver for urea formation.
Muscle primarily export amino acid nitrogen as alanine via blood to the liver. The cycle is also known as
the Cahill cycle which has similarities with the Cori cycle occurring in the liver

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The Urea Cycle
• Occurs in the liver. The cycle combines two amino groups (one from ammonia and the other
from aspartate) with carbon from bicarbonate; Requires ATPs

• Energy spent is recovered in the oxidation of the carbon skeletons left after deamination of the
original amino acid

• Involves 5 reactions, 2 are mitochondrial and the remaining 3 occur in the cytosol.

Step 1

Ammonia is combined with bicarbonate to yield carbamoyl phosphate. This step is necessary for the
urea cycle to occur even though technically it is not in the cyclic portion of the pathway. This is an
irreversible step and the rate limiting step of the cycle. Two ATP are required for this step to occur.

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 Step 2

The carbamoyl portion of carbamoyl phosphate is transferred to ornithine to make citrulline by

orinithine transcarbamoylase. Note that ornithine & citrulline are non-standard AA which are not
involved in protein synthesis

Step 3

Argininosuccinate synthetase performs a two-step reaction in which AMP is first added to citrulline
and then aspartate displaces the AMP moiety to form argininosuccinate.

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 Step 4

Argininosuccinase splits argininosuccinate into

fumarate and arginine.

Step 5

Arginine is hydrolyzed by arginase to regenerate ornithine and yield urea.

Regulation of the Urea Cycle

Most enzymes in the cycle are regulated by substrate concentrations.

Carbamoyl phosphate synthetase is also allosterically activated by N-acetylglutamate. During protein

degradation, amino acids that are release are converted into glutamate. Recall that glutamate
dehydrogenase releases the amine group to form ammonia to begin nitrogen excretion. In addition,
the increased levels of glutamate activate N-acetylglutamate synthase which combines the acetyl
moiety from acetyl-CoA with glutamate to form N-acetylglutamate. The N-acetylglutamate activates
carbamoyl phosphate synthetase stimulating the urea cycle.

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Excreting urea
Liver produces and releases urea into blood. Kidneys filter urea out of blood and excretes it with urine.

• Liver disease are characterized by high blood NH 3 (hyperammonemia). This is because of over
accumulation of ammonium in liver due to the slow urea cycle and subsequent release of
ammonium into blood and its conversion into ammonia. High ammonia can cross blood brain
barrier leading to ammonia accumulation in the neurons which
a. Depletes neurons of their alpha- ketoglutarate reserves leading to slow TCA and low ATP
production
b. Lowers glutamate level in neurons causing low GABA levels (all glutamate changes to
glutamine).
c. Result is severe neuronal dysfunction called hepatic encephalopathy

• Kidney disease is characterized by high blood urea as kidneys are unable to filter urea from
blood and excrete it out through urine

Nitrogen Balance
There is a careful balance between ingestion and excretion of nitrogen.
Average Diet: Protein content > amount required ‘Neutral N balance’
Growing Child: N excreted < consumed ‘Positive N balance’

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Malnutrition: Proteins are degraded and more N is lost ‘Negative N balance’

Uses of Amino Acids

Amino acids can be used in energy generating pathway. Amino acids that can be converted into
glucose precursors such as pyruvate, α-ketoglutarate, succinyl-CoA, fumarate, or oxaloacetate are
called glucogenic. While other amino acids can be converted into fatty acids or ketone bodies are
called ketogenic. Five amino acids are glucogenic and ketogenic; Isoleucine, phenylalanine, threonine,
tryptophan, and tyrosine.

Methionine is converted into S-adenosylmethionine (SAM) in an ATP dependent reaction. SAM is an

important methyl group donor in over 40 metabolic reactions.

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Tyrosine is used to make Melanin, Dopamine, Norepinephrine, and Epinephrine

Tryptophan is used to make serotonin.

Glutamate is used to make gamma-aminobutyric acid (GABA).

Histidine is used to make histamine.

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Inherited diseases of Amino Acid Metabolism
[Link] (PKU)

Mutations in the phenylalanine hydroxylase (PAH) gene leads to either the total absence of PAH
(classical PKU) or quantitative deficiency of PAH ( variant form of PKU). This prevents the conversion
of phenylalanine to tyrosine at cellular levels. Phenylalanine accumulates and is converted to toxic
phenylpyruvate and phenyllactate. Low tyrosine levels leads to mental retardation, very light skin and
neurological symptoms like increased frequency of seizures etc

Treatment:

Early detection and restrict phenylalanine in the diet and supplement with tyrosine. Babies born with
classical PKU are prescribed with low phenylalanine containing formula and are often given tyrosine
supplementation.

Individuals with variant type of PKU (characterized by last expression) must also be aware of foods
and beverages containing artificial sweetener aspartame. The degradation of aspartame results in
increased levels of phenylalanine.

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2. Alkaptonuria (Black Urine Disease)

This is also an inherited defect in the Phe-Tyr pathway and is due to a deficiency of the enzyme that
catalyzes oxidation of homogentisic acid, an intermediate in the catabolism of Tyr and Phe.

Homogentisic acid accumulates and is excreted in the urine. It becomes oxidized to a black pigment,
imparting a dark color to the urine. It also deposits in cartilage (leading to arthritis like symptoms)
and heart valves (leading to cardiac symptoms). May precipitate as crystal sin the kidney. The current
treatment is Vitamin C.

Treatments: Phenylalanine and tyrosine restricted diet. Vit C helps to bring down levels of
homogentisic acid

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3. Cystinuria

This is an autosomal recessive disorder of the intestinal absorption and proximal tubular reabsorption
of dibasic amino acids. Because of transport deficiency, cysteine which is normally reabsorbed in the
proximal renal tubule remains in the glomerular filtrate .The cysteine spontaneously oxidizes to its
disulfide form, cystine, which is very insoluble and tends to
precipitate as stones in the urinary tract, kidney stones, bladder etc.
Stones do not show up on X ray. Abdominal CT and 24 hr urine
collection needs to be done

Treatment: Restrict dietary intake of methionine (precursor of

cysteine). Plenty of fluid intake is recommended.

[Link] Syrup Urine Disease (MSUD)

Isoleucine, Leucine and Valine are branched amino acids that accumulate in MSUD. This is a
metabolic disorder of branched chain amino acids. The urine of affected persons smells like maple
syrup. Due to the deficiency of Branched Chain α Keto acid Dehydrogenase Complex (BCKDC). This
leads to an increase in branched chain amino acids. The compound responsible for the odor is
SOTOLON.

The condition leads to acute metabolic decompensation and metabolic crisis. Anabolic and catabolic
pathways at cellular levels gets affected.

Treatments: Dietary restriction of branched chain amino acids; insulin and glucose injections.
Thiamine supplementation helps for some forms of the disease.

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Chapter 18 - Nucleotide Synthesis and Metabolism
Lecture Objectives:
Compare & contrast the biosynthesis of purine & pyrimidine nucleotides in terms of:
• Salvage & De novo pathways
• Requirements of the pathways
• Control steps
• Regulatory elements
• End products
• Fate of the end products

Learn the cause, symptoms & effects of disorders associated with purine & pyrimidine
metabolism (gout, LNS, SCID, orotic aciduria)

Emphasize on the role of folate cycle in DNA synthesis; learn the clinical significance of
methotrexate & 5-flurouracil

Nucleotides contain a pentose sugar, a nitrogenous base and a phosphate. Purines have two rings
whereas pyrimidines have one.

Nucleotides used for:

1. Precursors of DNA and RNA

2. Components of coenzymes (NADH , NADPH, FMN and CoA)
3. Synthesis of the energy-yielding molecules ATP and GTP
4. Cellular messengers (cAMP)

*** All purine & pyrimidine bases are synthesized in the ribo form and then converted into deoxy ribo
form

Common requirements for synthesis of both purine & pyrimidine nucleotides

1. 5-Phosphoribosyl 1-pyrophosphate (PRPP)
Synthesized from ribose-5 phosphate by the enzyme PRPP synthetase. The enzyme
requires ATP & Mg2+ (cofactor). It is activated by free Pi and inhibited by purine/ pyrimidine
ribonucleotides

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Fig: Synthesis of PRPP

2. Ribonucleotide Reductase (RR)

Converts the bases from the ribo to the deoxy ribo form for DNA synthesis. Acts on ribonucleotide
diphosphates only.

The ribose forms undergo a reduction reaction resulting in the removal of the hydroxyl group in the 2’
position of the ribose sugar forming deoxyribose sugar. Ribonucleotide reductase (RR) requires NADPH
for the reduction reaction. After ribonucleotide reductase has performed its reaction, it must be reset
to its reduced form for another reaction to occur. A series of reduction-oxidation reactions involving
the proteins thioredoxin and thioredoxin reductase are responsible for this reset. The oxidation of
NADPH is the facilitator for the RR mediated reduction.

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Fig: Conversion of ribonucleotide to a deoxyribonucleotide

De Novo Pathway-Purine nucleotide biosynthesis

Ribose-5-phosphate is converted to phosphoribose pyrophosphate (PRPP) by an enzyme called ribose

PRPP synthetase or phosphate diphosphokinase. The ring of purines begins to be synthesized on PRPP.
The basic steps involve first, the addition of glutamine’s amine group to PRPP by a glutamine
amidotansferase. This is the rate limiting step. Next an entire glycine amino acid is added followed by
the addition of a carbon from a formate molecule that is associated with tetrahydrofolate (folate is a B
vitamin). Another glutamine amine group is added and the first ring is closed in an ATP dependent
reaction. Bicarbonate is used to add a carbon molecule forming the second ring in an ATP dependent
reaction. Next an aspartate is added via the amine group to the growing second ring. The enzyme
adeylosuccinate lyase removes fumarate and thus leaves the amine group of the aspartate in the purine
ring. The last atom of the purine ring, a carbon, comes from the formate molecule associated with
another tetrahydrofolate. The second ring then closes. The resulting nitrogenous base is called
hypoxanthine and since the nitrogenous base is attached to a phosphate sugar, the nucleotide is called
inosine monophosphate (IMP).

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IMP to AMP/GMP conversion
For conversion of IMP to AMP aspartate & GTP are used. Fumarate is released. Enzymes involved are
adenylosuccinate synthetase and adenylosuccinate lyase. Conversion of IMP to GMP involves
dehydrogenation followed by addition of a glutamine (amide group donor). Enzymes involved are IMP
dehydrogenase and Xanthosine monophosphate. Requires an ATP

253
AMP/ GMP formed can be used for RNA/DNA synthesis & also in energy metabolism
of a cell

254
 RIBONUCLEOTIDE REDUCTASE (RR)-Regulation and structure

Ribonucleotide reductase is a tetrameric enzyme consisting of two heterodimers. The heterodimers

consist of two proteins. Subunit 2 contains the active site where NTP (ADP, CDP, UDP, and GDP) are
converted to dNTPs. The subunit 1 contains two allosteric control sites.

• The primary regulatory site (activity-determining site). Only when an ATP is bound to the
primary site, ribonucleotide reductase is active. If dATP or any other nucleotide is bound to the
primary site, ribonucleotide reductase gets inactive.
• Substrate specificity site: The substrate specificity of the enzyme shifts based on specific
effector bound to this site
The enzyme requires thioredoxin and NADPH for its activity

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 Regulation of Purine nucleotide biosynthesis

• Formation of PRPP by PRPP synthetase (Ribose phosphate pyrophospho-kinase) is inhibited by

ADP / GDP. Activated by Pi
• Addition of first nitrogen by glutamine amido phosphoribosyl transferase (GPRT) is inhibited by
ATP, ADP, AMP, GTP, GDP , GMP & stimulated by PRPP
• GTP/ ATP: Control production of AMP & GMP respectively
• IMP to AMP (adenylosuccinate synthase) is inhibited by AMP
• IMP to GMP (IMP dehydrogenase) is inhibited by GMP
• ATP/ GTP produced at the end can be used in earlier steps ( see fig) as the synthesis proceeds

Purine salvage pathways

Excess purines and pyrimidines are recovered from both dietary and in vivo sources via a “salvage”
process. Recovery of dietary nucleotides cannot provide all the nucleic acid needs of the body but do
form a pool of extrahepatic nucleotides for DNA/RNA synthesis. Salvage reactions take place mainly
in liver but lymphocytes also depend on this mechanism.

In either pathway a series of phosphorylase enzymes dephosphorylate the nucleotides to release the
purine and pyrimidine bases which then go through a series of reactions to reform the nucleotides. .
The major reactions involved are:

1. AMP is formed from adenine & PRPP by Adenine phosphoribosyltransferase (APRT)

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2. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) salvages IMP & GMP
• Hypoxanthine + PRPP forms IMP
• Guanine + PRPP forms GMP

3. Adenosine can be deaminated to form Inosine; Adenosine can be phosphorylated to AMP;

AMP can be deaminated to IMP; IMP can form GMP

Fig: purine salvage pathways

PURINE METABOLISM
Excess accumulation of purine in the cell due to overactive de novo / salvage pathways or due to dietary
consumption, calls for catabolism of purines.

AMP is converted to IMP by AMP deaminase. IMP would then be processed to inosine by a
dephosphorylation reaction.

Adenosine would be converted to inosine by the enzyme Adenosine deaminase (ADA). Further
degradation of inosine occurs by the action of purine nucleoside phosphorylate (PNP) which breaks
the glycosidic bond between the sugar and the nitrogenous base forming ribose 1-phosphate (a precursor
to form PRPP) and hypoxanthine.

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Guanine nucleotides are converted to guanosine which is also acted on by PNP forming ribose 1-
phosphate and the free nitrogenous base guanine.

The ultimate degradation product of either ATP or GTP (hypoxanthine and guanine) and their
derivatives is uric acid. The enzyme that helps in oxidation of hypoxanthine and guanine to uric acid in a
2-step process is xanthine oxidase.

Decreased activity of the enzyme HGPRT involved in the salvage pathway leads to
Increased accumulation of hypoxanthine & guanine.
High xanthine production (by xanthine oxidase).
High URIC ACID production (by xanthine oxidase) leading to a condition called gout

Clinical presentation of gout:

High URIC ACID levels lead to impaired uric acid excretion through urine
Excess uric acid often deposits as insoluble uric acid crystals in the joints & big toe
Deposits of uric acid crystals can also occur in kidneys & ureter

SYMPTOMS: Warmth, pain swelling, tenderness in joints/big toe; pain starts at night; limited mobility
of the effected joint; peeling & itching of the skin

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Allopurinol (Xanthine oxidase inhibitor): Treatment for gout
• Allopurinol is a hypoxanthine analog & a competitive inhibitor of Xanthine oxidase
• Xanthine oxidase converts allopurinol into alloxanthine (oxypurinol) which also can bind to &
inactivate xanthine oxidase.
Net Effect:
• Production of xanthine & uric acid decreases in a patient with gout
• Allopurinol( hypoxanthine analogue) reacts with PRPP to produce an unstable intermediate;
overtime makes the cell deficient in PRPP; inhibits de novo synthesis of purines

Severe or total loss of HGPRT activity: Lesch-Nyhan syndrome (LNS)

• Rare inherited X-linked recessive disorder. Caused by mutations in the HGPRT gene (X-linked
gene).There are over 60 known mutations in the HPRT gene located on the X chromosome, all of
which can cause this disease
• Called juvenile gout; Severe HGPRT deficiency causes a build-up of uric acid in all body fluids
• Results in both hyperuricemia and hyperuricosuria, severe gout & kidney problems
• Neurological signs: Poor muscle control & moderate intellectual disability: Accumulation of the
purine metabolites will interfere with dopamine synthesis in the CNS manifesting as retardation
and a compulsive self-mutilation, characterized by lip & finger biting (horror autotoxicus).These
complications usually appear in the first year of life
• Death usually occurs before age 20 and affected individuals do not reproduce. These patients
also have kidney stones, nephropathy, and severe gout at an early age

Note: Allopurinol can treat the gout like symptoms but not the neurological symptoms of LNS

Severe combined Immunodeficiency syndrome (SCID)

Deficiency of ADA allows deoxyadenosine (dATP) to reach levels that are 50-fold higher than normal.
dATP levels are especially high in lymphocytes, which have abundant amounts of the salvage enzymes,
including nucleoside kinases, due to their need for large amounts of nucleotides during an immune
response.

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Accumulated dATP is toxic to B and T lymphocytes through inhibition of ribonucleotide reductase,
preventing other dNTPs from being produced. This stops DNA synthesis leading to severe combined
immune deficiency (SCID) which is usually fatal in infancy unless special measures are taken.

SCID is a group of very rare, life-threatening diseases that are present at birth. The disease causes the
child to have very little or no immune system. As a result, the child's body is unable to fight off
infections. This disease process is also known as the "boy in the bubble" syndrome, because living in the
normal environment can prove fatal to these children.

Approximately one in 100,000 children are born with this inherited disease. These children become very
ill with illnesses such as pneumonia, meningitis, and chickenpox and can die within the first year of their
life. With new types of treatments like gene therapy, children with SCID can now be successfully
treated.

De Novo Synthesis of Pyrimidine nucleotides

Unlike purine synthesis, pyrimidine synthesis does not occur on the PRPP sugar. Also, only three
different types of molecules are used as atom donors for the generic ring synthesis, these include
glutamine, aspartate, and bicarbonate. First
the pyrimidine ring is made and then the ring
is added to PRPP. Pyrimidine synthesis
begins with a reaction similar to the one
observed in the Urea Cycle in which the
amine from glutamine is combined with

260
carbon of bicarbonate and a phosphate from ATP to form carbamoyl phosphate. Next the amino acid
aspartate is added in its entirety. The ring is then closed by dihydroorotase. An irreversible oxidation
step occurs by dihydroorotate dehydrogenase to form orotate (orotic acid). Now that the pyrimidine
ring has formed, PRPP is added forming orotidine monophosphate (OMP). The carboxyl group on the
orotate rings is removed in a decarboxylation step yielding UMP. Uridine is a glycosylated pyrimidine-
analog containing uracil attached to a ribose ring (or more specifically, a ribofuranose) via a β-N 1 -
glycosidic [Link] to other nucleotides, UMP is converted to its di and tri phosphate forms by
uridine specific kinases.

Fates of UDP

• UDP can be converted to UTP & CTP; used for RNA synthesis
• UDP can be converted to dUDP by the enzyme RR
• CTP via CDP can be converted into dCDP
• dUDP via dUMP is used as substrate for DNA synthesis ***
• dCDP can be used for DNA synthesis

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Disorders associated with pyrimidine biosynthesis

Orotic aciduria
Deficiencies in either orotate phosphoribosyl transferase (OPT) or OMP decarboxylase result in over
accumulation of orotic acid manifesting as orotic aciduria. Symptoms are orotic acid in the urine,
retarded growth, megaloblastic severe anemia and leukopenia.

The disorder can be easily treated with uridine and/or cytidine which will increase UDP production via
the action of nucleoside kinases. High UDP/UTP production then inhibits CPS-II, thus attenuating orotic
acid production in the long run.

Regulation of Pyrimidine synthesis

• Step 1 is the rate limiting step of pyrimidine biosynthesis and is catalyzed by carbamoyl
phosphate synthetase II (CPSII). CPSII is highly regulated. It is inhibited by UTP, UDP. It is
activated by presence of high levels of PRPP and ATP.

262
 • High levels of UMP inactivate orotate monophosphate decarboxylase (product inhibition).

Conversion of UMP to dTMP and dTTP

• UMP is the first pyrimidine base synthesized. UMP via UDP is converted to dUMP by the enzyme
RR.
• Thymidylate synthase enzyme thereafter transfers a methyl group from a folate derivative
(methylenetetrahydrofolate) to the uridine ring generating dTMP. This methylation reaction is
the rate limiting step in DNA synthesis. The methyl donor for the rate limiting reaction is folate
(vit B9; FH2 & FH4). The main enzyme involved in the folate cycle is dihydrofolate reductase.

• The product dTMP is then phosphorylated by specific kinases including thymidine kinase to
generate the dTDP and dTTP forms.

• Methylenetetrahydrofolate is regenerated from dihydrofolate in a two-step reaction.

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Clinical Correlate

• 5-Flurouracil is an inhibitor of the enzyme thymidylate synthase. Its presence inhibit DNA
synthesis by inhibiting the methyl transfer to U to form a T
• Methotrexate, a competitive inhibitor of the enzyme dihydrofolate reductase inhibits the
folate cycle;
Both chemicals inhibit DNA synthesis at cellular levels

**Methotrexate & 5’ fluorouracil are used in cancer chemotherapy for inhibiting cell
proliferation

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Salvage pathway of pyrimidine synthesis

Pyrimidine catabolism contributes to cellular energy metabolism

Pyrimidines are catabolized to yield alanine and β-aminoisobutyrate. β-aminoisobutyrate is converted
to malonyl-CoA & methylmalonyl-CoA.

• Malonyl-CoA is a precursor of fatty acid synthesis in hepatocytes.

• Methylmalonyl-CoA is converted to succinyl-CoA which is a Vit B 12 dependent process. Succinyl
CoA can enter mit. TCA cycle producing NADH/FADH2 which then produces cellular ATP via
electron transport chain in the mitochondria. Thus, catabolism of pyrimidine nucleotides
contributes to the energy metabolism of the cell.

265
Overview of Nucleic acid metabolism

An overview

266
Lecture 19- LIPIDS AND LIPOPROTEINS
Learning Objectives:

• Learn the function of lipids with special emphasis on:

-Saturated & unsaturated fatty acids (FA): Differences
-PUFAs, omega 3/ 6 FAs
-Trans fats: Adverse health effects
-Structural lipids: Structural & functional differences
-Storage lipids: Mobilization & storage of triacylglycerol

• Compare /contrast the pathologies related to abnormal

accumulation of membrane lipids
• Lipoproteins: Compare /contrast the structure & function of
different types.
• Apolipoproteins: Focus on important functions & the
associated pathologies

LIPIDS: CHARACTERISTIC FEATURES

LIPIDS are a diverse group of biological compounds present in our body which are
• Insoluble in water (all lipids); soluble in organic solvents
• Principal stored form of energy in our body (storage lipids)
• Structural elements of biological membranes (structural lipids)
• Involved in multiple functions in our body as enzyme cofactors, hormones, intracellular
messengers, chaperones, emulsifying agents etc
• Most lipids are composed of fatty acids and their derivatives

FATTY ACIDS (FA)

Carboxylic acids with long hydrocarbon chains (4-36C long). Principally of 2 types

A. Saturated FA: The hydrocarbon chain is unbranched with no double bonds; solid at room temp (RT);
unhealthy; found in meats, chips, dairy; examples are myristic acid, palmitic acid

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B. Unsaturated FA:

i. Monounsaturated FA: The hydrocarbon chain contains one double (C9 & C10; ∆9); waxy consistency at
RT (Palmitoleic acid; Oleic acid)

ii. Polyunsaturated FA/PUFA: The hydrocarbon chain contains multiple double bonds in cis
configuration; Oily consistency at RT; linolenic & arachidonic acids

Sources of unsaturated FA: Nuts, olive oils, vegetable oils, avocado etc

TRANS FATS

Oils with high PUFA content (vegetable oils, olive oils etc) spoil easily and become rancid due to oxidative
cleavage of the double bonds into aldehydes and carboxylic acids of shorter chain lengths which are
volatile. Thus these oils have shorter shelf life. To improve the shelf life and increase their stability at
room temperatures vegetable oils are often subjected to partial hydrogenation. This industrial process
helps aims to convert cis double bonds of unsaturated fatty acids into single bonds which raises the

268
melting temperature of the oils. Hydrogenated oils are thus solid at room temperature. The process of
hydrogenation often leads to undesirable conversion of some cis double bonds into trans double bonds
producing trans fatty acids. Due to this hydrogenated vegetable oils have higher trans-fat.
Overconsumption of trans fat increases risk of heart attacks by increasing LDL levels and lowering HDL
levels in blood

Heating and reheating of vegetable oils also lead to production of trans-fat

PUFAs with health benefits

ω-3 fatty acids: PUFAs with one of the double bonds between C3 and C4 (from the ω carbon , the C most
distant from the COOH group of the hydrocarbon chain of FA)
Example: Linolenic acid

ω-6 fatty acids: PUFAs with one of the double bonds between C6 and C7 (from the ω carbon)
Example: Arachidonic acid, linoleic acid

Too much ω-6 in the diet can be harmful. Aim should be cut down on ω-6 and increase consumption of
ω-3.
A healthy dietary balance of ω-6 : ω-3 (1:1 to 1:4 ) is considered to be cardioprotective.
The imbalance between omega-3 and omega-6 fatty acids may contribute to obesity, depression,
dyslexia, hyperactivity

Mediterranean diet rich in fish & veggies has a good balance of ω-3 & ω-6

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Storage lipid: Triacylglycerol

• Simplest lipids which are fatty acid esters of glycerol. They are also referred to as triglycerides
or fats
• Composition: Consist of 3 fatty acid chains esterified to a single glycerol molecule. The 3 fatty
acid molecules may be similar or of different types.
• Triacylglycerols are non- polar, hydrophobic & insoluble in water. They are stored in human
adipocytes (under the skin as fat droplets).Oxidation of triacylglycerols yields very high energy

Fig: structure of triacylglycerol

Fig: Triacylglycerol molecule stored as fats in adipocytes

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Mobilization of triacylglycerol from adipocytes into blood

Triacylglycerols are mobilized and released into blood as fatty acid and glycerol when blood glucose
levels are low .Decrease in insulin and increase in glucagon during fasting activates the cAMP, protein
kinase A (PKA) mediated cascade in the adipocytes. This phosphorylates and activates the enzyme
“hormone sensitive lipase” present in adipocytes. The activated lipase breaks down triacylglycerol into
glycerol and fatty acids which are released in the blood. The fatty acid molecules bind to albumin and
are carried to the muscle and liver tissues where they are oxidized to produce energy via acetyl CoA. The
glycerol goes to the liver and enters gluconeogenic pathways to produce glucose.

Note: Hormone sensitive lipase mobilizes triglycerides (stored in adipocytes) as fatty acid and glycerol
into blood for energy production during hypoglycemic conditions (under high glucagon/
epinephrine/cortisol or low insulin).

Lipoprotein lipase (LPL) breakdown triglycerides present in lipoproteins like chylomicron/VLDL and
releases them for storage in the adipocytes. LPL is activated by high levels of insulin. It recognizes and
binds to Apo C-II on the surface of chylomicrons/VLDLs for further action. ApoC-II acts as a cofactor of
LPL and enables it to break the triglycerides of chylomicrons, VLDL into fatty acid and glycerol for
release into target tissues.

Fig: Mobilization of triacylglycerols

Structural Lipids: Phospholipids and glycolipids

The lipid bilayer: It is the central architectural feature of biological membranes that serve as a barrier for
entry of ions, polar molecules into the cell. It is composed of membrane lipids namely phospholipids and
glycolipids (complex lipids) which are amphiphatic in nature

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TYPES OF PHOSPHOLIPIDS

A. Glycerophospholipids or phosphoglycerides

• Composed of 2 fatty acid molecules attached by ester linkages to C1 and C2 of a glycerol

molecule
• Presence of a head group composed of a highly charged group like choline/ethanolamine
attached to C3 of glycerol by a phosphodiestor bond.
• The polar head group has negative charge at pH 7 due to the phosphate group
• Fatty acids can be of a wide variety (generally C18 saturated FA or C18, C20 unsaturated FA at
C2)
Note: Plasmalogens is a phosphoglyceride present in plasma membrane of cardiac cells where
one of the two FA chains is linked to glycerol by an ether linkage

Fig: Structure of a phospholipid

B. Sphingolipids

• Composed of a long chain amino alcohol called sphingosine (pink) in place of glycerol
• Sphingosine is attached to one long chain fatty acid at C2 (yellow) by an amide linkage
(ceramide)
• C1 of sphingosine is attached to a polar head (X) by mostly a phosphodiestor linkage

• Sphingomyelin is a sphingolipid with its polar head group composed of phosphocholine or

phosphoethanolamine. It is abundant in the plasma membrane of animal cells and myelin

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 sheath of neurons.

Fig: Structure of sphingolipids

Fig: Structure of a sphingolipid

TYPES OF GLYCOLIPIDS

A. Glycosphingolipids: Structure is same as sphingolipids with different head groups

• Contain one or more sugars (mono/ disaccharides) as the head group attached to the C1 of
sphingosine molecule
• Present in the outer face of plasma membranes of neural/non neural tissues
• Also called neutral glycolipids with no charge at pH 7

Cerebrosides (containing single sugar molecule glucose/galactose) and gobosides (containing 2 or more
sugar molecules) are examples of glycosphingolipid

B. Gangliosides:
• Contain oligosaccharide molecules as their polar head group with residues of sialic acid (N-
acetylneuroaminic acid) imparting a net negative charge at pH 7
• Present in nerve tissue of the brain
Note: Glycosphingolipids a

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 Fig: Structure of glycolipids Ganglioside

Function of glycolipids
• 60 different sphingolipids are found in cellular membranes
• Many are prominent in the plasma membrane of neurons (gangliosides); participate in
conduction of nerve impulses
• Carbohydrate moiety of glycolipids define human blood groups
• Gangliosides are used as stem cell markers

CLINICAL CORRELATES
Membrane lipids undergo constant metabolic turnover. Their rate of synthesis is counterbalanced by
the rate of breakdown. The breakdown of the membrane lipid is catalyzed by hydrolytic enzymes present
in the lysosome. Each hydrolysable bond is cleaved by a specific hydrolytic enzyme. Secretion of
defective hydrolytic enzymes or reduced production of these enzymes lead to accumulation of the
partial breakdown products of the membrane lipids in various tissues. Such conditions may lead to
serious pathologies.

A. Niemann-Pick disease
• Rare metabolic disorder characterized by autosomal recessive inheritance. common in
Jewish populations originating from eastern/ central Europe
• Patients produce a defective form of the lysosomal enzyme sphingomylinase which under
normal conditions breaks down sphingomyelin by cleaving its phosphocholine head
group.
• The defective enzyme is unable to break down sphingomyelin molecules
(sphingomyelinase); metabolic turnover rate is affected, sphingomyelin accumulates in
tissues principally the brain and also liver, spleen, lungs etc

Symptoms
• Enlarged liver, spleen due to abnormal accumulation of membrane lipids;
• Mental retardation due to degeneration of the brain tissue, seizures, often slurred speech
• Lack of muscle coordination, dystonia (difficulty in moving limbs), paralysis of the eye.

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 Types:
• Type A (neurological type): Severe form; expresses in infantile stage; characterized by
hepatosplenomegaly retarded growth, progressive degeneration of the nervous system;
death within 18 months-2 years

• Type B (non-neurological type): Not very severe form; expresses in the preteen years.
Reduced production of sphingomyelinase; about 10% normal levels of sphingomyelinase
is present; little or no neurological involvement; other health complications like
enlarged liver, spleen, respiratory problems, etc exist; elevated cholesterol and other
fats in blood.

Fig: Characteristics of Neimann-pick disease

B. Tay-Sachs Disease
• More common type of lipid storage disorder; characterized by autosomal recessive inheritance
• Caused by lack or inactivity of the lysosomal enzyme hexasominidase (HEXA) which breaks
down gangliosides (GM2 type) UNDER NORMAL CONDITIONS
(Note: Gangliosides are made and biodegraded rapidly in early life as the brain develops)
• Deficiency or inactivity of HEXA leads to accumulation of GM2 gangliosides in the nerve cells of
the brain, spinal cord, to some extent spleen, leading to malfunction of these organs due to
degeneration at tissue level.

Symptoms

• The classic type is characterized by infantile onset. The child may be healthy during birth but
may show symptoms by 3-6 months of age; patients die by the age of 3
• Symptoms include growth retardation, difficulty swallowing, seizures, decreased mental skills
and muscle tone (strength); deafness, blindness, paralysis
• A rare form can develop in late childhood or early adulthood
• Important diagnostic feature: Fundoscopy typically shows a grey-white area around the retinal
fovea centralis, due to lipid-laden ganglion cells, leaving a central 'cherry-red' spot.

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 Fig: Tay-Sachs disease

LIPOPROTEINS
Definition: Plasma lipoproteins are large macromolecular spherical complexes of lipids and specific
proteins called apoproteins. They have a hydrophobic lipid core composed of triacylglycerol,
cholesterol, cholesterol esters and phospholipids. The hydrophilic surface is composed of amino acid
side chains of the different types of apoproteins and polar heads of phospholipids.

Function: Plasma lipoproteins help to transport these lipids (insoluble in water) from the tissue of
origin (liver) or sites of absorption (intestinal epithelium) to tissues where they are either stored
(adipocytes) or used for energy production.

Fig: Structural features of lipoprotein particles

Different classes of lipoproteins: Different combinations of lipids & apoprotein produce lipoprotein
particles of different densities namely

• Chylomicrons (least dense due to low protein content, largest in size)

• Very Low Density Lipoproteins (VLDL)

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 • Low Density Lipoproteins (LDL)
• High Density Lipoproteins (HDL; most dense due to high protein content and smallest in size)

Note: IDL or intermediate density lipoprotein is not a major class of lipoproteins. It is an

intermediate form between VLDL and LDL.
Principal types of apoproteins associated with different classes of lipoproteins

Note: ApoB48 is a truncated version of ApoB100. Its molecular weight is 48% of the molecular weight of
ApoB100. It is produced from the same gene as ApoB100, in the intestinal epithelium. Its mRNA
undergoes RNA editing (insertion of a stop codon at a certain position) in the intestinal epithelium but
not in the liver cells which accounts for its truncation.

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A. Chylomicrons- structure, function and metabolism

• Largest but least dense lipoprotein particles synthesized in the endoplasmic reticulum of
the epithelial cells lining the small intestine
• They transport dietary fats absorbed in the small intestine to target tissues namely the
adipose tissue, heart & skeletal muscles, lactating mammary tissues for usage or storage
• They principally transport dietary fats as triacylglycerol. Chylomicron also contain some
cholesterol esters.
• ApoB48 is the principal apoprotein composing chylomicrons. They acquire ApoC-II and
ApoE (from HDL) as they move through the lymphatic system and blood.
• ApoC-II activates lipoprotein lipase enzyme which cleaves triacylglycerols into fatty acids
and glycerol and unloads them into the target tissues for storage/oxidation
• The chylomicron remnant (depleted of triacylglycerols) containing cholesterol
compounds, ApoB-48 & ApoE, move to the liver
• ApoE binds to receptors on the liver cells mediating receptor mediated endocytic
uptake of these remnant chylomicrons. The remnants release their cholesterol esters in
the liver cells and are degraded in the lysosomes.

B. Very Low density lipoproteins (VLDL) - Structure, function and metabolism

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 • Produced in the liver hepatocytes. Excess dietary fat, carbohydrates and excess
endogenous cholesterol (synthesized by liver cells) are converted into triacylglycerol and
packaged into VLDLs
• VLDLs particles are rich in triacylglycerol, cholesterol, cholesterol esters (produced in the
liver). They also contain apoB-100, apoC, apoE
• VLDLs transport triacylglycerol via blood from the liver to the muscles & adipose tissues
for either storage or oxidation. Release of triacylglycerol as free fatty acid is catalyzed by
lipoprotein lipase activated by apoC.
• The remnant VLDLs are rich in cholesterol and called intermediate density lipoproteins
(IDLs). Some IDLs are converted into low density lipoproteins (LDLs) after they release free
fatty acids. Some are removed from circulation by receptor mediated endocytic uptake
(apoE mediated) into the hepatocytes.

C. Low Density lipoproteins (LDLs): structure, function and metabolism

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 • Produced from VLDLs; very high in cholesterol and cholesterol ester content
• ApoB-100 is the major apoprotein present in LDLs
• Transport cholesterol to extra-hepatic tissues that require cholesterol and express LDL receptors.
• LDL receptors: Synthesized in ER and golgi and present in clathrin-coated pits on the plasma
membrane of extra-hepatic cells ( example, hepatocytes, steroid hormone synthesizing cells) in
many copies; receptors are recycled for reuse by these cells; LDL receptor synthesis is regulated
by the intracellular cholesterol content of the cell. Low intracellular cholesterol content increases
LDL receptor synthesis. The receptors bind to LDL (by recognizing the ApoB-100) in circulation
and transport them through receptor mediated endocytosis via endosomes into the cell. The
cholesterol of LDLs is used for various cell functions like steroid hormone synthesis

Fig: LDL uptake at the cellular level through LDL receptors

Note: Administration of statin drugs reduce total cholesterol levels in a patient suffering from
hypercholesterolemia by the following methods
a. It inhibits endogenous cholesterol synthesis reducing intracellular cholesterol levels in cells
that require cholesterol.
b. Low intracellular cholesterol levels up regulate LDLR production by these cells (as they require
cholesterol for different functions). The result is rapid clearance of LDL from blood due to
increased LDL uptake by these cells.

D. High Density lipoproteins (HDLs): Structure, function and metabolism

• Synthesized in the liver/ intestine in the immature form (nascent HDL) composed of ApoA1 and
small amounts of cholesterol. Nascent HDL is protein rich

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 • While in circulation the nascent HDL receives cholesterol and phospholipids from chylomicrons,
VLDLs, LDLs. The transfer is catalyzed by enzymes like PLPT (phospholipid transfer protein), LCAT
(lecithin cholesterol acyltransferase) present in the blood.
• HDL also collects cholesterol from peripheral tissues and foam cells (using ABCG transporter
proteins).
• The cholesterol received from various sources is converted into cholesterol ester (reaction
catalyzed by LCAT )and packed into the core of the maturing HDL particle (HDL3)
• The maturing HDL also receives ApoC, ApoE, ApoA from the other types of lipoproteins.
• The cholesterol rich mature HDL then returns to the liver where it unloads its cholesterol content
into the hepatocytes
• The liver hepatocytes use some of this cholesterol to synthesize bile while the excess is excreted
through bile.
• So HDL helps in reverse transport of excess cholesterol (floating in blood as lipoproteins and
present in peripheral tissues/foam cells etc), to the liver for excretion/use. Thus HDL is thus
called the good cholesterol. High HDL levels indicate that the body is trying to get rid of excess
cholesterol.

Fig: HDL formation and metabolism

CLINICAL CORRELATES
A. Role of lipids and lipoproteins in Coronary Heart Disease (CHD)

• CHD is a leading cause of death and disabilities in America. 13 million people have CHD
• Risk factor for CHD is elevated LDL levels (>4.1 mM/L) and low HDL levels (< 0.9mM/L) in blood.
Ratio of HDL: LDL below 0.3 is considered a risk factor for CHD

• The HDL: LDL ratio is affected by

a. High saturated fat content (myristic, palmitic acid in dairy, beef, poultry) in diet increases,
down-regulating LDL receptor mRNA & decreasing receptor mediated catabolism of LDLs,
raising LDL concentrations in the blood and thus the risk for CHD

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 b. High consumption of trans fatty acids increases plasma LDL levels & lowers plasma HDL levels,
increasing risks for CHD
c. Genotypes like apoE4 allele (associated with increased absorption rates of dietary
cholesterol), increase plasma LDL levels increasing risk for CHD

Note: PUFAs increase LDL clearance from blood. Diets low in cholesterol and saturated fats are
recommended to prevent CHD

B. Clinical relevance of apoproteins (also called apolipoproteins)

a. ApoA (A1): Major apoprotein component of HDL. Deficiency is associated with HDL
deficiencies. It also plays a protective role against Alzheimer’s disease.

b. ApoB: Main apoprotein present in chylomicrons and LDLs. High levels are associated with
heart diseases. Non-fasting ApoB/ApoA1 ratio is often used for estimation of the risk of acute
myocardial infarction & ischemic strokes. Higher the ratio greater is the risk

Abetalipoproteinemia
• A rare autosomal recessive disorder caused by a deficiency of apoB48 and apoB100
• Formation of chylomicrons, VLDL and LDL are affected
• Interferes with the normal absorption of fat and fat-soluble vitamins at cellular levels from
dietary sources.
• Plasma levels of LDL and VLDL are low to absent. This hinders transport of cholesterol and
triacylglycerols from liver to the different tissues

Clinical features: Symptoms develop in the first few months of life. Growth retardation, weight loss,
steatorrhea (fatty, foul smelling stool) are the initial symptoms. Impaired nerve functions, progressive
ataxia (due to spinocerebellar degeneration), and degeneration of the retina occur. Characterized by
early death (3 years of age)

Cause: Mutations in the MTTP gene cause abetalipoproteinemia

Diagnostic feature: Acanthocytes (red cells with long, spiky projections) seen in the blood

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Familial defective apo B100
Autosomal dominant disorder. Caused by a mutant apoB-100 protein which mainly prevents LDL
particles from binding to their receptors (LDLR) on the peripheral cells (liver cells, steroid hormone
producing cells etc). Receptor mediated uptake of LDL by peripheral tissues is inhibited. This results in
high plasma LDL levels and high total cholesterol levels in a patient.

Clinical presentation similar to familial hypercholesterolemia. About 4% of patients with clinical familial
hypercholesterolemia may have familial defective apoB-100

Fig: Lipoproteins active during FED state

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Lecture 20 - FATTY ACID BIOSYNTHESIS & CHOLESTEROL
METABOLISM
Learning Objectives:

• Develop a clear idea about synthesis of palmitic acid. With

emphasize on:
-Fatty acid synthase complex
-Steps involved in production of palmitic acid
-Reaction types involved in each step of FA synthesis
-Activation of acetyl CoA to malonyl CoA, and its regulation
-Role of citrate & malate shuttle
-Regulation of FA synthesis by insulin, glucagon, ATP, AMP,
citrate
• Learn the clinical features of pyruvate carboxylase deficiency
disease
• Emphasize primarily on the clinical & biological
• importance of rate limiting step of intracellular cholesterol
biosynthesis
• Focus on short & long term regulation of cholesterol
biosynthesis; know the mechanism of action of statin drugs
• Learn the role of “cholesterol oxidation” in atherosclerosis

Biosynthesis of Fatty Acids

• Occurs in the cytoplasm. A 16C long palmitic acid which is the primary fatty acid is
synthesized by the process.
• Modification of palmitic acid leads to production of other long/ short chain saturated &
unsaturated fatty acids.
• The enzymes that catalyze synthesis of fatty acids are collectively referred to as the fatty
acid synthase (FAS) complex

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 • 2 major variants, FAS-I (vertebrates), FAS-II (plant/bacteria)

FAS-I

• Composed of a single multifunctional polypeptide chain with seven active sites present in
separate domains or subunits. The multiple domains function as distinct but linked enzymes
that catalyze distinct reactions.
• Contains 2 carrier proteins as part of the complex: The acyl carrier protein (ACP) that acts as a
shuttle carrying reaction intermediates from one active site to another of the FAS complex; β-
ketoacyl synthase (K-SH) a condensing enzyme is also a part of FAS. During FA biosynthesis, the
growing FA chain alternates between K-SH and ACP.

Fig: Structure of the fatty acid synthase complex in mammals. The individual enzymes are
colored and the matrix structure is in grey.

General characteristics of fatty acid synthesis

• Palmitic acid is synthesized 2 carbons at a time from acetyl CoA in the cell cytoplasm
• Synthesis begins from the methyl end and proceeds toward the carboxylic acid end of the FA
chain.
• During the step-wise 2-carbon extensions, acetyl CoA is first activated to malonyl CoA; malonyl
CoA extends the FA chain by 2C in each step
• Extension of the FA by every 2-carbons involves repeated cycles of
o Condensation
o Reduction
o Dehydration
o Reduction
• Synthesis of a 16C long palmitic acid, 7 cycles of these reactions occur adding 14 C from the
methyl end
• C15 & C16 are added directly by acetyl CoA (without conversion into malonyl CoA).

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The overall reaction in fatty acid synthesis occurs in 2 steps

I. Formation of Malonyl-CoA

7 Acetyl-CoA + 7 CO 2 + 7 ATP 7 malonyl-CoA + 7 ADP + 7 P i

II. Seven cycles of condensations/dehydrations/ reductions

Acetyl-CoA + 7 malonyl-CoA + 14 NADPH + 14 H+ Palmitic acid + 7 CO 2 + 8 CoA + 14 NADP+ + 6

H2O

Note: Note 1 H 2 O is used up to hydrolyze to release the mature palmitic acid (hydrolysis of the
thioester bond linking the mature palmitic acid to the last enzyme involved in the biosynthesis).

NADPH serves as electron donor in the reactions that involve substrate reduction.

Details of Step 1

• Malonyl CoA is formed from acetyl CoA by carboxylation reaction catalyzed by the enzyme
acetyl CoA carboxylase
• It is an irreversible reaction that occurs in the cytoplasm and requires 1 ATP molecules per
molecule of malonyl CoA formed.
• It is the committed step of FA biosynthesis. FA synthesis is regulated at this step as well
• Acetyl CoA carboxylase is a complex with 2 active sites. As with other carboxylation
reactions (e.g., Pyruvate Carboxylase), the enzyme prosthetic group is biotin.
• ATP-dependent carboxylation of the biotin, carried out at one active site, is followed by
transfer of the carboxyl group to acetyl-CoA at a second active site.
• The overall reaction, can be summarized as:

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Regulation of Acetyl CoA carboxylase

Acetyl CoA carboxylase is under allosteric regulation.

• Citrate is a positive effector. Citrate plays an important role in diverting cellular metabolism
from consumption (oxidation) to storage. When concentrations of ATP and acetyl CoA are high
in cytoplasm, citrate is transported out of the mitochondria where it acts as a precursor of
acetyl CoA as well as it activates acetyl CoA carboxylase enzyme for formation of malonyl CoA.
Citrate also inhibits the activity of phosphofructokinase-1 thus reducing the flow of C through
glycolysis
• Palmitoyl CoA/palmitic acid is a negative effector (due to end product inhibition).

Details of step II

Catalyzed by FAS complex with the different enzymes (catalyzing the different steps) present as
different domains of the complex

• Attachment: Acetyl CoA and malonyl CoA attach to separate Acyl carrier proteins (ACP) of
the FAS complex (remember that acetyl CoA is involved in this step of the first cycle only;
the other 6 cycles involve only malonyl CoA)
• Condensation: Acetyl CoA-ACP donates its acetyl group to the malonyl CoA-ACP liberating
CO 2 and CoA; enzyme involved is β-ketoacyl ACP synthase
• Reduction: The keto group at the end of the condensed molecule (acetyl CoA & malonyl
CoA) is reduced to OH; enzyme involved is β-ketoacyl ACP reductase. NADPH serves as an
electron donor in the reduction step.
• Dehydration: The OH group is removed H 2 O as by the action of the enzyme β-hydroacyl ACP
dehydratase; leads to formation of a double bond.
• Reduction: The double bond is removed; reaction is catalyzed by enoyl ACP reductase;
requires a second NADPH; the product butyryl ACP
• The butyryl ACP enters the next cycle at the condensation step; condenses with another
malonyl CoA-ACP and goes through a series of reduction, dehydration, reduction to add the
next 2 C to the fatty acid chain at the methyl terminal end.
• This continues for 7 cycles using 7 malonyl CoA, 14 NADPH to produce the complete 16 C
long primary FA, palmitic acid.

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Fig: Reactions involved in FA synthesis

The Citrate & Malate shuttle: For transport of acetyl CoA from Mit. to cytoplasm

The citrate shuttle: Acetyl CoA in eukaryotes is formed in the mitochondria from FA oxidation,
pyruvate oxidation and catabolism of amino acids

• Mitochondrial (Mit) inner membrane being impermeable to acetyl CoA, direct transfers across
the membrane is not possible.
• Indirect transfer through the citrate shuttle occurs. Mit. acetyl CoA enters the citric acid cycle,
reacts with oxaloacetate to form citrate (enzyme: citrate synthase) within Mit.
• Citrate can now cross the inner & outer Mit membrane (and be available in the cytoplasm), into
the cytoplasm where it is cleaved into acetyl CoA & oxaloacetate by citrate lyase enzyme;
acetyl-CoA in cytoplasm is used for FA synthesis.

Fig: The Citrate Shuttle

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Malate Shuttle

• The oxaloacetate in the cytoplasm can be reduced to malate (catalyzed by malate

dehydrogenase) which can return to the mitochondrial matrix.
• Or the cytosolic malate can be oxidized by malic enzymes to generate cytosolic NADPH and
pyruvate; the pyruvate then returns to the [Link].
• Pyruvate in [Link] converted back to oxaloacetate (enzyme pyruvate carboxylase) which
enters the Krebs cycle.
• Deficiency of the enzyme pyruvate carboxylase enzyme can lead to serious medical
condition

Fig: Citrate and Malate shuttle

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How does a body handle high a carbohydrate rich meal??

Hormonal Regulation of FA Synthesis

Three hormonal signals determine the state of FA synthesis through covalent phosphorylation of acetyl
CoA carboxylase, the rate-limiting enzyme. Acetyl CoA carboxylase is inhibited/ inactivated by
phosphorylation.

• Glucagon /epinephrine inhibit FA biosynthesis in the hepatocytes under hypoglycemic

conditions (fasting):

Process: Hypoglycemia (due to fasting/anxiety/stress) induces release of glucagon/ epinephrine from the
pancreatic alpha cells/ adrenal medulla. These hormones act on liver hepatocytes (principle site for FA
synthesis) through G protein coupled receptors. Stimulation of G protein coupled receptors increase cAMP
levels (2nd messenger) in the hepatocytes. High cAMP levels activate PKA (protein kinase A). PKA
phosphorylates and thus activates AMP activated protein kinases. AMP activated protein kinases
phosphorylates acetyl CoA carboxylase inactivating it. This inhibits malonyl CoA production from acetyl CoA
in the cytosol turning off FA biosynthesis in the hepatocytes

• Insulin is lipogenic (anti-lipolytic) and stimulates FA biosynthesis in the hepatocytes in

hyperglycemic states (fed state/insulin resistance)

Process: Insulin levels are high in fed state or in a patient suffering from insulin resistance (high glucose and high
insulin levels due to malfunction of insulin receptors). In both cases, insulin acts on hepatocytes through insulin-
receptor tyrosine kinase cascade which activates hepatic protein phosphatases (HPP-1) and phosphodiesterases.
Phosphodiesterases reduce cAMP to AMP thus decreasing cAMP levels in the hepatocytes. Low cAMP levels turns
off PKA (turning off phosphorylation cascade). Hepatic protein phosphatases dephosphorylates and thus activates

290
acetyl CoA carboxylase enzyme present in the hepatocyte cell cytoplasm. The activated enzyme increases malonyl
CoA production from acetyl CoA (provided by citrate) in the hepatocyte cytoplasm. Presence of abundant malonyl
CoA, turns on palmitic acid biosynthesis in the hepatocytes

Fig: Regulation of acetyl CoA carboxylase by insulin & glucagon

A CLINICAL CORRELATE

Pyruvate carboxylase deficiency disease

Pyruvate carboxylase is a mitochondrial enzyme (biotin dependent) required for conversion of

pyruvate to oxaloacetate and thus initiation of gluconeogenesis. It is also essential for maintenance of
mitochondrial oxaloacetate levels required for TCA cycle and citric shuttle (essential for FA synthesis).

Pyruvate carboxylase deficiency affects several metabolic pathways at cellular levels.

A. Due to absence of the enzyme, pyruvate cannot produce oxaloacetate and is shunted to
alternative pathways that produce lactic acid and alanine.

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B. The lack of oxaloacetate prevents gluconeogenesis and urea cycle function.
C. The production of citrate is limited also due to lack of oxaloacetate which inhibits TCA cycle
D. Abundant acetyl-CoA (as it cannot enter TCA cycle via citrate due to lack of oxaloacetate) is
shunted to produce ketone bodies.
E. Because cells cannot use TCA cycle to produce energy, energy is extracted from glucose exclusively
through glycolysis. This causes glucose to be degraded at a very high rate, resulting in a glucose
deficit, thereby compounding problems
F. Aspartic acid, which is derived from oxaloacetate, is required for the urea cycle. A decrease in
aspartic acid production reduces ammonia disposal and leads to increased serum ammonia levels.
G. Aspartate deficiency slows nucleotide biosynthesis.

The disease caused by deficiency of pyruvate carboxylase enzyme is called pyruvate carboxylase
deficiency disease which is an inherited disorder classified into 3 distinct types

North America (type A): Characterized by lactic, pyruvate + alanine acidemia. Severe mental,
psychomotor & developmental retardation.
Cause: Point mutation in the gene produces inactive pyruvate carboxylase enzyme with amino acid
substitutions.

France & UK (type B): Characterized by early onset, severe respiratory distress, increased serum
lactate, ammonia, citrulline, proline and lysine; intracellular redox disturbance: Usually do not survive
beyond 3 months of age.
Cause: Nonsense mutation in the gene leads to Pyruvate carboxylase deficiency

Benign Type (type C): Mild type characterized by preservation of motor and mental abilities, mild
episodes of metabolic acidosis with elevated lactate, pyruvate, alanine, b-hydroxybutyrate,
acetoacetate, lysine, proline. Occurs due to quantitative deficiency of the enzyme.

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Synthesis of long chain FAs and unsaturated fats

• Palmitate & stearate: Precursors of oleate & palmitoleate (most common monounsaturated FAs
found in animal tissue)
• Double bonds are introduced by oxidative reactions catalyzed by fatty acyl-CoA desaturase,
NADPH, cytochrome b5 located in ER
9
• Mammalian hepatocytes can readily introduce double bonds up to ∆ position but not between
C10 and the methyl terminal end of FAs.
9,12,15
• Thus mammals cannot synthesize linoleate {18:2 (∆9.12) or α-linolenate {18:3 (∆ ); they are
essential fatty acids (obtained from dietary sources like vegetable oils) required for synthesis of
other important FAs.
• Arachidonate is a PUFA which is a metabolite of linoleic acid and thus is synthesized in our
body

Fig: Synthesis of long chain FAs and unsaturated FAs from palmitic acid

CHOLESTEROL METABOLISM

FREE CHOLESTEROL

• The Human body contains 140 gm (approx) of free cholesterol

• Free cholesterol is absorbed from dietary sources
• Cholesterol is also endogenously synthesized by all nucleated cells (0.5-1 gm/day); 50%
synthesis occurs in the liver cells cytoplasm
• 40-60% of free cholesterol is found in the plasma membrane of cells where it interacts with the
membrane phospholipids and sphingolipids and modulates affecting membrane properties. It
also manipulates the behavior and function of proteins found in the cell membrane (example,
ion channels, receptors etc)
• It is the mother lipid from which all steroid hormones are synthesized

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 • It helps in synthesis of bile which helps in emulsification of fats in the small intestine,
absorption of fat soluble vitamins and serves as a vehicle for excretion of excess cholesterol.

CHOLESTEROL ESTERS

• Preferred form of transport of cholesterol through blood plasma

• Formed by ester linkage between OH group of cholesterol and COOH group of long chain fatty
acids
• Sites of esterification include intestinal mucosal cells (esterification of dietary cholesterol) and
liver cells ( esterification of endogenous cholesterol)
• (endogenous cholesterol)
• Packaged in secreted lipoprotein particles (chylomicrons, VLDL, LDL) for transport

Enzymes involved in esterification of cholesterol

LCAT (lecithin cholesterol acyltransferase)

• Enzyme present in blood plasma which catalyzes formation of cholesterol esters present in HDL
from lecithin (phosphatidylcholine) and free cholesterol
• Helps in reverse transport of cholesterol in form of cholesterol ester to the liver by HDL

ACAT (acylCoA: cholesterol acyltransferase):2 isoforms express in humans

ACAT1: Present in all tissues. Produces cholesterol esters for intracellular storage

ACAT2: Expresses in liver and intestinal mucosal cells; produces cholesterol esters that are packaged
into VLDL and chylomicrons for transport

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Note: ACAT1 also expresses highly in differentiating monocyte-macrophages which are associated with
atherosclerotic lesions in human aorta (Chang, 2006).ACAT2 influences diet induced
hypercholesterolemia, a risk factor for atherosclerotic lesions.

CHOLESTEROL BIOSYNTHESIS

• Cholesterol biosynthesis occurs in the cell cytoplasm. It is synthesized from acetyl CoA produced
in the mitochondria by oxidation of pyruvate, fatty acids etc and is made available in the cytosol
by the citrate shuttle.
• NADPH is an electron donor in the process
• The process of cholesterol synthesis produces many intermediates like the geranyl and farnesyl
pyrophosphates which produce prenylated proteins with functional importance in the cell.
Examples are
a. Rho/Ras: Small GTPase proteins involved in cellular signal transduction
b. Dolichol: Used for synthesis of oligosaccharide chains of glycoproteins
c. Ubiquinone/Coenzyme Q: Hydrogen carrier of electron transport chain
d. Heme a: A constituent of respiratory chain with farnesyl side chain

Regulation of cholesterol biosynthesis

• The formation of mevalonate from HMG-CoA is the rate limiting step that regulates
endogenous biosynthesis of cholesterol
• HMG-CoA reductase is the rate limiting enzyme that serves as a control point regulating the
biosynthetic pathway

Short term regulation or hormonal regulation of cholesterol biosynthesis

• HMG-CoA reductase is inhibited by phosphorylation and activated by dephosphorylation

• Phosphorylation of HMG-CoA reductase is regulated by the enzyme AMP-activated protein
kinase

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• This protein kinase is active in phosphorylated state which is regulated directly by cellular levels
of cAMP
• High cellular cAMP levels (corresponding to low cellular ATP levels) activate AMP-activated
protein kinase by phosphorylation which inhibits HMG-CoA reductase (through
phosphorylation) and turns off endogenous cholesterol biosynthesis
• Cellular cAMP levels are regulated by hormones like glucagon, epinephrine and insulin
• Glucagon and epinephrine increase levels of cellular cAMP and thus reduce endogenous
cholesterol biosynthesis through phosphorylation of HMG-CoA
• Insulin decreases cellular cAMP levels and activates protein phosphatases stimulating
cholesterol biosynthesis by activating HMG-CoA reductase through phosphatase mediated
dephosphorylation
• Fed state or insulin resistant diabetes (type II)-Promotes endogenous cholesterol biosynthesis
• Fasting state or when the body is under stress-Glucagon or epinephrine inhibits cholesterol
biosynthesis

Fig: regulation of endogenous cholesterol biosynthesis by insulin and glucagon

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Long term regulation of endogenous cholesterol biosynthesis

Long term regulation involves turning cholesterol biosynthesis off or on by formation and
degradation of HMG-CoA. The main role players are high intracellular cholesterol levels and a
regulatory protein SREBP (steroid regulatory element binding protein).

• Cholesterol: High levels of intracellular cholesterol turns off endogenous cholesterol

biosynthesis in the particular cell by stimulating proteolytic degradation of HMG-CoA reductase
by binding to the sterol sensing domain of the enzyme.

• SREBP (sterol regulatory element binding protein): Low intracellular cholesterol levels in cells
that synthesize cholesterol, promote release of the N- terminal end of the precursor SREBP
protein (attached ER membrane) into the cytoplasm (Site one protease, or S1P mediated).

The N-terminal end released acts as mature SERBP and a transcription factor. It diffuses into
the nucleus and increases transcription rate of HMG-CoA reductase gene producing more HMG-
CoA. This promotes endogenous cholesterol biosynthesis in the respective cells

Fig: Regulation of cholesterol biosynthesis by SREBP

Statin drugs! Are they good for you?

• Statin drugs (Lovastatin, Lipitor etc) inhibit cholesterol synthesis, lower levels of blood
cholesterol and the risk of cardiovascular diseases
• Statin drugs (obtained from fungal sources) act as competitive inhibitors and regulate
cholesterol synthesis by inhibiting the enzyme HMG-CoA-reductase.

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 Effect of statin on total cholesterol levels in patients with hyperlipidemia:
• The immediate effect of administration of statin drugs is inhibition of endogenous cholesterol
biosynthesis (turning off HMG-CoA reductase)
• Continuous administration of statin drugs decreases levels of intracellular cholesterol in cells like
hepatocytes/steroid hormone producing cells (which require constant supply of cholesterol for
synthesis of bile or steroid hormones). To meet their cholesterol demands, these cells upregulate
LDL receptor synthesis which helps in endocytic uptake of LDL from blood reducing blood LDL
levels.

Net effect: Low serum total cholesterol levels; High HDL/LDL ratio

Side effects associated with long term Statin use: Long term inhibition of endogenous cholesterol biosynthesis
depletes the cellular prenoid/isoprenoids (produced from the intermediates of cholesterol biosynthesis
pathway). This leads to multiple side effects.

OXIDATION OF CHOLESTEROL

• Cholesterol is oxidized by enzymatic and non-enzymatic pathways

• The enzymatic pathways: Catalyzed by cytochrome P450 enzymes; leads to synthesis of bile
acids and occurs in the liver hepatocytes.
• The non-enzymatic pathways (autoxidation): Free radical mediated; oxidize cholesterol to
hydroperoxides and oxysterols
• Membrane cholesterol as well as cholesterol rich lipoprotein particle, LDL are susceptible to
oxidation by free radicals (hydroxyl radicals, superoxide radicals etc). The process is called lipid
peroxidation and causes damage in a chain reaction.
• The presence of a double bond in ∆ 5-6 position of its sterol nucleus makes cholesterol more
susceptible to oxidation.

A CLINICAL CORRELATE

ATHEROSCLEROSIS

• Atherosclerosis is a major cause of morbidity and mortality due to cardiovascular diseases

across the world.

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• It involves plaque formation in arterial walls and narrowing of the arterial passage, restricting
blood flow to the heart.
• Oxidation of LDL cholesterol is one of the important contributors of atherogenesis.
• The first step of atherogenesis is the passage of plasma LDL from the arterial lumen into the
arterial wall where it gets trapped in the ECM (extracellular matrix) of the sub-endothelial
spaces in the vessel wall.
• The trapped LDL is subjected to oxidative modifications by various cellular and biochemical
mediators: ROS (Reactive Oxygen Species), & various enzymes.
• Oxidized LDL (OxLDL) induces inflammatory response in the vessel wall which recruits
monocytes. OxLDL also helps to differentiate monocytes into macrophages.
• These macrophages engulf OxLDL particles (considering them as foreign bodies). This converts
the differentiating macrophages into lipid loaded cells called the foam cells
• Accumulation of foam cells in the arteries followed by their calcification leads to formation of
plaques in the arterial wall (hallmark of atherosclerosis), which limits blood flow to the heart.

Fig: Mechanism of
atherosclerotic plaque
formation.

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Lecture 21- CARNITINE SHUTTLE & BETA OXIDATION OF
FATTY ACIDS
Learning Objectives:

• Learn β oxidation of fatty acids (FAs) with emphasis on

o Differences between oxidation of short , medium long and very long chain
FAs
o Role of carnitine in β oxidation
o Different steps of carnitine shuttle
o Regulation of the entry of FAs into the mitochondria
o Different stages of β oxidation; the 4 different reactions involved in stage I
o Overall reaction of β oxidation, calculation of ATP production
• Differentiate between the different disorders involving carnitine shuttle
• Learn how oxidation of unsaturated & odd chain FAs are different from β
oxidation of long chain FAs
• Learn how to calculate the total ATPs/GTPs produced by oxidation of even/ odd
chain FAs
• Focus on the role of peroxisomes in oxidation of fatty acids. How are the
peroxisome related to disorders of fatty acid oxidation?

BETA OXIDATION OF FATTY ACIDS (FAs)

General characteristics of fatty acid oxidation

• Oxidation of long chain fatty acids (FAs) provides 80% of energy supply to mammalian heart and
liver tissues under all physiological conditions; all cells can oxidize FAs except neurons and RBCs.
• Oxidation of FA takes place in the mitochondria although they are synthesized in the cell
cytoplasm.
• Not all FAs use the same set of enzymes for catabolism; FAs of different chain lengths use
slightly different sets of enzymes
• Short chain FAs (< 6C long) and medium chain FAs (6-12C long) can enter the mitochondria
passively for oxidation
• Majority of dietary or storage FAs are long chain (14-24C long) and these have to be shuttled
across mitochondrial membranes into the matrix via the carnitine shuttle
• Very long chain FAs (>24C long) are oxidized in the peroxisomes prior until they attain a chain
length of 24C for mitochondrial oxidation. Branch chain fatty acids are also oxidized in
peroxisomes. Peroxisomal oxidation is predominant in plants.

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 • The end product, acetyl CoA, can either enter the TCA cycle to be oxidized to carbon dioxide or
the acetyl CoA may be converted into ketone bodies under certain conditions for use as fuel
when glucose is unavailable
• The electrons removed during the oxidation of FA drive ATP synthesis through the respiratory
chain

What is carnitine?? How is it important in FA oxidation

Carnitine is an ammonium containing compound (β hydroxy-ϒ-trimethyl ammonium butyrate)

biosynthesized in the body from methionine and lysine amino acids in liver & kidney cells. It is required
in the body for transport of long chain fatty acids into the mitochondria for oxidation and supply of
metabolic energy

Carnitine is stored abundantly in muscles. They are also obtained from animal based food. L-carnitine is
the active form in our body while D-carnitine is the inactive form.

Transportation of FAs into the mitochondria

The Carnitine Shuttle: Transports the long chain FAs from cytoplasm into the mitochondrial matrix for
oxidation. It involves III steps

• Step I: Activation of fatty acids for catabolism

• Transfer of FAs to cytosolic CoA (CoASH) catalyzed by the enzyme acyl CoA synthetase or
thiokinase
• High energy thioester linkage is formed between COOH of FA and thiol group of CoASH, yield
fatty-acyl CoA
• Occurs in 2 steps in the cytosolic face of the outer mitochondrial membrane and requires 2ATP
molecules (1 ATP is used in the reaction and the other is used to regenerate ATP from the AMP
produced). It is the only ATP requiring step in oxidation of FAs
• There are different Acyl CoA synthetases for activation of different chain lengths of fatty acids

Fig: Step I

• Step II: The fatty acyl-CoA exchanges its CoA with carnitine to form fatty acyl-carnitine; occurs in
the cytosolic face of the mitochondrial membrane
• Rate limiting step; catalyzed by carnitine palmitoyl transferase-I (CPT-I) present in the outer
mitochondrial membrane

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 • The fatty acyl-carnitine crosses the outer mitochondrial membrane and moves into the inter-
membrane space.(note: this esterification reaction with carnitine can occur either on the
cytosolic face or in the inter-membrane space of the mitochondria)
• Carnitine-acylcarnitine transporter protein present in the inner mitochondrial membrane then
transfers the fatty acyl-carnitine into the mitochondrial matrix across the inner membrane

Fig: Step II

• Step III: In the mitochondrial matrix, the carnitine of fatty acyl-carnitine ester is exchanged
with intra-mitochondrial CoA, forming fatty acyl-CoA again

The transfer is catalyzed by the enzyme carnitine pamitoyl transferase-II (CPT-II) located on the
inner mitochondrial membrane

Free carnitine released in the mitochondrial matrix gets back to the cytoplasmic pool to be
reused

Fig: Step III

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 Fig: The carnitine shuttle

REGULATION OF THE CARNITINE SHUTTLE

• The entry of fatty acid into the mitochondria for oxidation is highly regulated

• Malonyl CoA inhibits CPT-I. This is the reason why newly synthesized FAs present in the
cytoplasm of hepatocytes (when FA synthesis is on) cannot be oxidized simultaneously in the
mitochondria. The high levels of malonyl-CoA inhibits CPT-I and prevents entry of the newly
synthesized FAs into the mitochondria of hepatocytes for oxidation.

Fig: Inhibition of CPT-I by High levels of malonyl CoA

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 • FA oxidation in hepatocyte mitochondria is strictly regulated by insulin and glucagon in fed and
fasting states. It is done by activation/ inactivation of acetyl CoA carboxylase enzyme through
dephosphorylation/dephosphorylation (mechanism described in details in regulation of fatty
acid biosynthesis in chapter 20). Acetyl CoA carboxylase regulates malonyl CoA production in
the hepatocytes, which in turn controls FA entry in the mitochondria by turning CPT-I on/ off.

Example: When the cell has low energy/ATP, phosphorylation of acetyl CoA carboxylase (via
glucagon) will turn off malonyl-CoA production from acetyl CoA. Low levels of malonyl-CoA in
the hepatocyte cytoplasm will turn on CPT-I, promoting entry of FA for oxidation into the
mitochondria.
When cellular energy is high, insulin will induce opposite effects.

CLINICAL CORRELATES ASSOCIATED WITH THE CARNITINE SHUTTLE

A. Systemic primary carnitine deficiency

• Autosomal recessive disease; occurs due to mutation in the SLC22A5 gene (on chromosome 5)
which produces a defective carnitine transporter protein that hinders reabsorption of carnitine
by kidney tubules; results in decreased levels of plasma carnitine and high levels of urine
carnitine.
• Characterized by childhood onset (3 months - 2 years)
• Symptoms: Chronic muscle weakness, hypoketotic hypoglycemia, liver dysfunction, poor
feeding, myopathy of skeletal & heart muscles
• Treatment: L- carnitine supplement

B. Carnitine palmitoyl transferase-I (CPT-I) deficiency

• Rare autosomal recessive metabolic disorder; occurs due to deficiency of CPT-1 enzyme caused
by mutation in the CPT1 gene
• Affected individual may present with lower levels of CPT-1 or total absence of CPT-1 enzyme;
long chain FAs thus cannot be transported into the mitochondria via carnitine shuttle for
oxidation.
• Symptoms: Patient will present with high plasma carnitine levels, hypoketotic hypoglycemia
during fasting, viral infections or illness; patient is at risk for nervous failure, seizure, coma,
sudden death
• Can be diagnosed in early childhood

C. Carnitine palmitoyl transferase - II (CPT-II)

• Caused by mutation in CPT2 gene; presents in 3 forms

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 • Adult form: Highly variable onset age; attacks characterized by severe hypoglycemia, low blood
carnitine, cardiomyopathy, and muscle damage is induced by exercise, fasting, viral infections,
high fat diet. Otherwise patient is normal
• Severe Infantile form: Onset age <1 year or sometimes later. Characterized by hypoketotic
hypoglycemia, seizures, loss of consciousness, hepatomegaly and cardiomegaly; sudden death
may occur
• Fatal neonatal form: Occurs hours to days after birth. Fatal due to cardiac, respiratory or liver
failure; leads to death
• Treatment for the adult form: Avoid fasting, strenuous exercise; include small and medium
chain FAs in diet; avoid long & very long chain FAs in diet; carnitine supplement recommended.

Oxidation of fatty acids in the mitochondria

Occurs in 3 stages

• Stage 1 (β-oxidation): Successive removal of 2C units from the COOH end of the fatty acyl chain
in form of acetyl-CoA (16C palmitic acid undergoes 7 rounds of oxidation to produce 8 acetyl-
CoA molecules)
• Formation of each acetyl-CoA molecule involves removal of 4H ions by dehydrogenase enzymes
producing reduced electron carriers NADH & FADH 2
• Stage II: The acetyl-CoA produced enters the TCA cycle in the mitochondria to get oxidized to
CO2
• Stage III: NADH & FADH 2 produced through TCA cycle and β-oxidation donate electrons to
mitochondrial respiratory chain leading to production of ATP

Fig: Oxidation of FAs

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Stage I or β-oxidation involves 4 different reactions
1. Dehydrogenation of fatty acyl-CoA: Involves formation of a double bond between C2 & C3
producing trans ∆2-enoyl-CoA; FAD is the electron acceptor and leads to production of FADH 2

The Acyl-CoA dehrydrogenase (CAD) enzyme catalyzing this step exists as several isoenzymes acting on
FAs of different chain lengths

• VLCAD: Specific for very long chain FAs

• LCAD: Specific for long chain FAs
• MCAD: Specific for medium chain FAs
• SCAD: Specific for short chain FAs
2
2. Hydration (adding an H 2 O molecule to the double bond of trans ∆ -enoyl-CoA): Catalyzed by enoyl-
CoA hydratase; product formed is 3-hydroxyacyl CoA

3. Dehydrogenation of hydroxyacyl-CoA to β-ketoacyl CoA: catalyzed by β-hydroxyacyl-CoA

dehydrogenase; NAD+ is the electron acceptor producing an NADH

4. Release of acetyl CoA: β-ketoacyl CoASH reacts with a free CoA to give off an acetyl-CoA molecule
from its COOH end; catalyzed by acyl-CoA acyltransferase enzyme (thiolase)

The last 3 enzymes form a complex called TFP (tri-functional protein) present in the mitochondria

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Fig: Steps involved in beta oxidation

Energy production by beta oxidation

Stage I: Overall reaction

Palmitoyl-CoA + 7 CoA + 7 NAD+ + 7 FAD + 7 H 2 O 7 H+ + 7 FADH 2 + 7 NADH + 8 acetyl-CoA

Energy production by NADH and FADH2 in stage I:

• ATP produced /FADH 2 =1.5
• ATP produced /NADH = 2.5
• Total ATP produced per cycle = 4.0; 7 cycles will produce 7X4=28ATPs
• 2 ATP were spent to allow entry of the palmitic acid molecule into the cytoplasm
• So net ATPs produced by stage I is 26

Stages II and III: The 8 acetyl CoA molecules produced by stage I enter the TCA cycle and electron
transport chain in stages II and III to produce ATPs
• Each acetyl CoA will produce 3 NADH and 1 FADH 2 and I GTP when oxidized to CO 2 by the TCA
cycle.
• Each NADH produces 2.5 ATP through electron transport chain. So total ATPs produced from
NADH molecules will be 2.5X3=7.5 ATPs
• Each FADH2 produces 1.5 ATPs through the electron transport chain.
• So total energy phosphate bonds produced per acetyl CoA ( through stage II and III ) is
7.5+1.5+1=10
• 8 acetyl CoA will produce 8X10 = 80 high energy phosphate bonds

Total ATPs/ GTPs (stages I, II, III): 26 + 80 =106

β oxidation of an odd chain FA (17C)

Odd chain fatty acid produces propionyl-CoA containing the last 3

carbons, as the end product (instead of acetyl CoA). Propionyl
CoA through methylmalonyl-CoA is converted into succinyl-CoA &
channeled into TCA cycle. Requires Vit B 12 & propionyl-CoA
carboxylase enzyme for this extra step

The total energy (ATP/GTPs) produced by oxidation of odd chain

fatty acid (example: 17C long):

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 • A 17C FA will undergo 7 cycles of beta oxidation yielding 28ATPs
• Will also produce 7 acetyl CoA + I propionyl CoA.
• Each acetyl CoA produces 10 ATPs/GTPs (via TCA cycle and electron transport chain). So total
ATPs/GTPs produced by 7 acetyl CoA is 10X7=70 ATPs/GTPs
• The propionyl CoA produces 1 GTP, 1 FADH2 and 1 NADH (through TCA cycle). So number of
ATPs/ GTPs produced is 5.
• ATP used: Propionyl CoA also uses 1ATP to convert into succinyl CoA via methylmalonyl CoA.
• 2 ATPs are used for activation of the FA for oxidation

Total ATPs/ GTPS produced by oxidation of I molecule of 17C long odd chain FA: 28+70+5-3=100
ATPs/ GTPs

Beta oxidation of unsaturated fatty acids

Occurs in the mitochondrial matrix; but involves 3 additional enzymes, namely
• 3,2 enoyl-CoA isomerase
• 2, 4 dienoyl-CoA reductase
• 3,5-2,4 dienoyl-CoA isomerase
These enzymes get rid of intermediates with sterical problems like wrong stereoisomers formed during
β oxidation of unsaturated FAs

Peroxisomal oxidation of very long chain fatty acids

Peroxisomes are structures derived from the ER. They contain >40 enzymes which play a role in
the catabolism of very long chain fatty acids, branched chain fatty acids, D-amino acids,
and polyamines, and biosynthesis of plasmalogens. They also contain approximately 10% of the total
activity of two enzymes in the pentose phosphate pathway, which is important for energy metabolism.
They help in production of reactive oxygen species (ROS).

Very long chain and branched chain FAs are shortened in the peroxisomes prior to β oxidation in the
mitochondria of humans (in plants the entire process occurs in the peroxisomes). ABCD transporter
protein helps in transport of these FAs into the peroxisomes

Peroxisomal Beta Oxidation

Beta oxidation of fatty acids occurs in the peroxisomes (a eukaryotic organelle) in addition to the
mitochondria, though in a modified form. Peroxisomal oxidation has advantages over mitochondrial
oxidation - fatty acids can diffuse across the peroxisomal membrane. After very long chain fatty acids
are partly degraded, they are attached to carnitine for transport to mitochondria. Deficiency in the
ability to metabolize very long chain fatty acids in peroxisomes is linked to the disease X-
Adrenoleukodystrophy, in which myelin is destroyed by the unmetabolized fatty acids.

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Differences in Peroxisomal beta-Oxidation

• 1. In the acyl-CoA oxidase reaction, electrons from FADH2 are passed directly to O2 (creating
H2O2) instead of to the ETS. This results in loss of two ATPs compared to mitochondrial
oxidation.
• 2. Activities for enoyl-CoA hydratase and 3-L-hydroxyacyl-CoA are present on the same
polypeptide.
• 3. Peroxisomal thiolase prefers longer chain fatty acids and has little activity on acyl-CoAs with
chains shorter than 8 carbons.
• 4. Peroxisomes contain both a carnitine acetyl transferase and a carnitine acyl transferase,
reflecting the need to move these peroxisomal breakdown products into the mitochondrion.

CLINICAL CORRELATES

A. Inherited diseases related to defective beta oxidation

Acyl CoA dehydrogenase deficiencies (CAD): characterized by autosomal recessive inheritance.
Can be categorized into 4 types
• SCAD deficiency: May express during childhood or middle age. Features are metabolic
acidosis, seizures, developmental delay, myopathy; no hypoglycemia
• MCAD deficiency: Common in Caucasians(1:10000); fatal hypoglycemia after fasting or
stress; muscle weakness, exercise intolerance, fatty liver, cerebral edema etc
• LCAD deficiency: Very rare; overlaps with VLCAD deficiency
• VLCAD deficiency: Unable to oxidize very long FAs; characterized by non-ketotic
hypoglycemia, cardiomyopathy, skeletal myopathy, hepatic dysfunction, sudden death
in infancy
B. Zellweger’s Syndrome:
• Autosomal recessive disorder caused by mutation in the PEX family of genes that
produces peroxisomes. So these patients lack peroxisomes.
• Patients exhibit cerebrohepatorenal syndrome characterized by
specific facial features, polycystic kidneys, liver & CNS
dysfunction, destruction of adrenal glands
• High serum levels of iron + copper, acidic serum and CSF;
• There is no cure for this disease
• The infantile form of Zellweger’s is called infantile Refsum
disease (IRD)

Is characterized by hypomyelination (especially cerebral); craniofacial

abnormalities, hepatomegaly, progressive adrenal dysfunction.

Relation between FA oxidation ketogenesis, insulin and glucose levels during starvation

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Coordination between carbohydrate and lipid metabolism in the hepatocytes

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Lecture 22 -Enzymes of Diagnostic Importance
Learning Objectives:
• Learn the importance of enzymes as diagnostic markers. Focus on important
factors that increase diagnostic precision when using enzymes as diagnostic
markers
• Learn the principles underlying methods used for analysis of enzyme activity
and quantification (with reference to ELISA)
• Learn the importance of specific enzymes in diagnosis of
o Liver diseases
o Pancreatic diseases
o Cardiac abnormalities
o Bone diseases
o Muscular dystrophies
o Pancreatic cancer

What are enzymes??

Chemical substances which act as biological catalysts and augment the rate of specific metabolic and
biochemical reaction within the body. Most enzymes are protein in nature with a few exceptions
(peptidyl transferase is rRNA present in the ribosome with catalytic activity).

Localization: Under normal conditions majority of the enzymes occur predominantly in different
cellular compartments (cytoplasm or mitochondria, ribosomes etc) within cells of specific organs (sites
of synthesis or action).

The concentration/activity of most enzymes in the blood plasma/serum is normally very low. Some
enzymes are absent in the blood plasma/serum under normal conditions.

Conditions of abnormal release of enzymes in blood plasma/serum

• Abnormal proliferation of cells

• An increase in the rate of cell turnover or damage
• Constitutive enzyme synthesis by the cells of origin
• Reduced clearance from plasma

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Note: Decrease in enzyme concentration in the blood plasma/serum can also occur in response to
decreased synthesis of the enzymes (congenital condition)

Clinical or diagnostic significance of enzymes

Measurement of plasma/ serum concentration of enzyme or its activity helps in:

• Diagnosis of various diseases: As the concentration and activity of certain enzymes change in
the blood plasma/serum in response to abnormal or diseased conditions, the enzyme levels
provide valuable information about the cell/ tissue of origin
• Prognosis of various diseases: Checking the levels of marker enzymes in a patient’s blood at
regular intervals can provide hints about certain aspects of his/her medical condition namely
the progression of the particular disease, and the effects of medication, which contribute
towards prognosis.

Clinical diagnosis of diseases using enzyme markers

Involves multiple steps

• Checking for presence/absence of the marker enzyme(s) in blood plasma/serum: Enables

diagnosis of a disease(s).

• Checking for change in concentration (increase or decrease) of the marker enzyme(s) in

plasma/serum/ over a period of time: Enables diagnosis of a disease and enables a physician
to monitor the progression of a disease(s)

• Change in enzyme localization: Enables detection of abnormal deposits of enzyme(s) at a

foreign site due to leakage from cell of origin

• Pattern of enzyme clearance: Enzymes released into blood are eventually cleared by the kidney
(n case of smaller molecules) or destroyed by blood proteases. Abnormal accumulation of
enzymes in blood plasma/serum may be a consequence of impaired clearance.

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 Steps taken for diagnostic precision
• Estimation of more than one marker enzyme: Elevated plasma/serum levels of a specific
enzyme that serves as a marker of myocytes damage may be indicative of cardiac muscle
damage but not necessarily of the conditions which lead to the damage (cardiac
arrest/oxidative stress/ ischemia). Estimation of an array of enzymes which serve as markers of
these specific conditions can together lead to a complete diagnosis.

• Estimation of isoenzyme activities: Different isoforms of a specific enzyme often has tissue
specific expressions. In addition to measuring the total activity of a particular enzyme in the
blood plasma/serum of a patient, estimation of concentration/activity of the different tissue
specific isoenzymes are necessary
Isoenzymes: Enzymes with similar function but slightly different structure and localization

• Serial time dependent estimation of marker enzymes: Rate of change of plasma enzyme
concentration/activity reflects a balance between the rate of secretion of the enzyme from the
cell of origin and the rate of clearance from the blood. A persistently raised enzyme
concentration/ activity in blood determined through serial time dependent estimation of
enzyme(s) markers may be suggestive of either a chronic condition or impaired clearance

Factors affecting selection of marker enzymes for diagnosis of a specific disease

• Enzyme distribution in various tissues

• Half-life of the enzyme in bloodstream
• Intracellular distribution of enzymes
• Type of suspected cell damage
• Availability of assays and feasibility of enzyme measurement

Factors that affect enzyme activity

Plasma enzyme activities must be interpreted in relation to the sex and age-matched reference
ranges of the issuing laboratory.

1. Age: Levels of some enzymes vary with age of the patient.

Example: Plasma aspartate transaminase activity is moderately higher during the neonatal
period than in adults; plasma alkaline phosphatase activity of bony origin is higher in children
than in adults and peaks during the pubertal bone growth spurt before falling to adult levels.

2. Gender: Plasma γ-glutamyl transferase activity is higher in men than in women. Acid
phosphatase levels are higher in males

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 3. Physiological conditions (pregnancy): Plasma alkaline phosphatase activity rises during the last
trimester of pregnancy because of the presence of the placental isoenzyme: several enzymes,
such as the transaminases and creatine kinase rise moderately in plasma during and
immediately after labor.

4. General health of the patient: Physical exercise, food intake and medications also affect
plasma/serum level of enzymes.

Principle underlying quantitative analysis of enzyme concentration /activity

The concentration of the active enzyme present in a plasma/serum sample is very low. Measurement
of the enzyme activity is thus proportional to the enzyme concentration.

A serum/plasma sample containing small amounts of enzyme X (the limiting factor) is incubated with
its specific substrate (provided in excess). The rate of the reaction which depends entirely on the
concentration of enzyme X in the plasma/serum, is measured from the amount of substrate consumed
or product formed after a limited amount of time (fixed time method).

The photometric analysis of enzyme concentration (proportional to enzyme activity) is conducted

following Beer-Lambert’s law.

Enzyme Linked Immuno Sorbent Assay (ELISA)

A sensitive technique used in research and diagnostic laboratories for quantifying hormones, enzymes,
neurotransmitters, etc present in micro/nano quantities in blood/secretions/body fluids/ tissues.
Based on the antigen-antibody reaction.

• The target protein is being quantified is the antigen

• Primary antibody raised specifically against the antigen (target protein) is used for
quantification.
OR
• Secondary antibody raised against the primary antibody which is specific to the antigen (target
molecule) may be used for quantification
• The primary or secondary antibody used, is labeled with signal enzymes (as tags)
• Substrate specific to the signal enzyme is added; enzyme substrate reaction is allowed to occur
• The color of the product formed (proportional to the quantity of the antigen ) is quantified by
spectrophotometric analysis.

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 enzyme

antibody

Antigen
Fig: An antibody attached to an antigen Fig: Principle of ELISA

Types of ELISA

Direct: Antigen of interest (present in blood serum sample) is immobilized on the inner surface of
coated ELISA well plates; Enzyme labeled primary antibody raised against the specific antigen you
intend to quantify is added to the plates. Enzyme labeled primary antibody binds to its specific antigen
attached to the well plates. Substrate specific to the signal enzyme is added. The color of the product
formed is measured by spectrophotometry.

Indirect: Antigen of your interest (present in blood serum sample) is immobilized on the inner surface
of coated ELISA well plates; primary antibody (not labeled) raised against the antigen you intend to
quantify is added to the plates. Unlabeled primary antibody binds to the immobilized antigen. Next
enzyme labeled secondary antibody (raised against the specific primary antibody) is added to the ELISA
plates. The secondary antibody binds the primary antibody-antigen complex. Substrate specific to the
signal enzyme is added. The color of the product formed is measured using a spectrophotometer.

Example: Indirect ELISA is used commonly in diagnosis of a specific infections which requires
quantification of specific antibody titers present in a patient’s blood. Example: Diagnosis of hepatitis C
infection in a patient

The ELISA well plates in this case will be coated with the immobilized viral (Hep C) coat protein. If the
patient has Hep C infection there will be anti Hep C primary antibodies (1°Ab) in the patient’s serum
(antigen for quantification in this case). During the process of ELISA, when the patient’s serum is added
to the coated ELISA plates, patient’s anti Hep C 1°Ab will recognize and bind to the Hep C viral coat
proteins immobilized on the well plate wall. Washing the well plates will remove unbound serum
components.
Next enzyme labeled secondary antibodies (raised against anti- Hep C primary antibodies) are added to
the ELISA plates. 2°Ab will binds the primary antibody-Hep C-viral coat protein complex. Substrate
specific to the signal enzyme (attached to the 2°Ab) is added. Formation of color (using
spectrophotometry) will confirm the presence of Hep C infection and the quantity of 1°Ab will

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determine the stage of infection. If the patient does not have the infection, the complex and thus color
development will not occur

Sandwich: The process is same as indirect ELISA. The only difference is the ELISA wells here are coated
with a general antibody called the capture antibody to which the target antigen present in the serum
sample binds. The detection method is same as indirect ELISA. The antigen of interest is thus
sandwiched between the capture antibody and the primary and labeled secondary antibody complex.
Sandwich ELISA is very sensitive (up to 2 to 5 times more sensitive than direct or indirect ELISA).
Either monoclonal or polyclonal antibodies can be used as the capture and detection antibodies in the
sandwich ELISA systems. Monoclonal antibodies (recognize a single epitope) and allows fine detection
and quantification of small differences between antigens. A polyclonal is often used as the capture
antibody to pull down as much of the antigen as possible.

The antigen to be measured must contain at least two antigenic epitope capable of binding to antibody

Fig: The 3 common types of ELISA

MARKER ENZYMES OF DIAGNOSTIC SIGNIFICANCE

Enzymes of the liver

1. Aspartate transaminase (AST) and Alanine transaminase (ALT)

Characteristics:

• Localization: ALT located in cytosol of hepatocytes exclusively; AST present in cytosol and
mitochondria of hepatocytes. It also expresses in muscle cells (skeletal/cardiac muscles)
• Activity: The activity of ALT in hepatocytes is very high when compared to AST

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 • Normal functions: ALT catalyzes conversion of alanine to pyruvate by deamination; AST
catalyzes conversion of aspartate to oxaloacetate by deamination. The amine group of
alanine/ aspartate transferred to oxoglutarate molecules (in both reactions) leads to
formation of glutamate which thereafter enters the urea cycle. ALT, AST can catalyze
formation of aspartate and alanine back again from oxaloacetate and pyruvate
• Normal levels in blood: Normal levels: AST levels: <31-35 U/L; ALT:45 U/L

Diagnostic importance: When liver cells are damaged due to infections, inflammation etc (hepatitis,
liver cirrhosis) abnormal amounts of these 2 enzymes are released in the bloodstream. The levels of
ALT and AST serve as important markers of impaired liver function and play an important role in
assessing and monitoring the degree of hepatocyte damage in the liver.

Common symptoms associated with liver diseases: Fatigue, nausea, vomiting, abdominal pain that
increases after eating, yellowing of the skin, nails, elevated bilirubin levels in blood etc

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Note: Elevation in serum ALT is considered to be relatively specific for liver disease. AST is elevated in
response to liver diseases as well as other types of tissue damage, (skeletal/ cardiac muscle damage,
muscle necrosis and renal disorders).

Example: In acute viral hepatitis there is a 100-1000 times increase in both ALT and AST but ALT level
is increased more than that of AST

AST: ALT ratio: The AST: ALT ratio is typically less than 1. In case of liver diseases, the ratio falls much
below 1 due to significant increase in ALT levels when compared to AST. The ration increases
significantly above 1 when AST is increasing without much increase in ALT. This occurs in conditions
that involve skeletal/cardiac muscle damage (as AST is present in muscles but not ALT). However the
exact mechanism of alteration in the ratio of AST/ALT in progression of different types of liver disease
is unclear and the correlation with and the accuracy in predicting the severity of different kinds of
liver diseases (degree of fibrosis, presence of cirrhosis) are controversial.

Conditions under which plasma/serum ALT and AST activities increase significantly

Acute viral or toxic hepatitis (both ALT and AST)

Alcoholic/ non-alcoholic liver cirrhosis (AST, ALT)

Cholestatic jaundice (ALT, AST)

Myocardial infarction (AST)

Skeletal muscle disease (AST)

After trauma or surgery (AST)

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2. Alkaline phosphatase (ALP)
A group of enzymes that dephosphorylates proteins, nucleic acids and alkaloids at a higher pH. This
enzyme plays an important role in the mineralization and thus growth and development of bones and
teeth.
Localization: Has wide distribution in the body (intestinal wall, renal tubules and placenta, lactating
breasts).
ALP, present in high concentration in the osteoblasts of bone and in the cells of the hepatobiliary tract;
higher amounts in growing children, 3rd trimester of pregnancy, and in the elderly

Condition under which plasma ALP activity is high

Physiological: There is a gradual increase in the proportion of liver ALP with age. In elderly the
plasma/ bone isoenzyme activity may increase slightly.

Pathological: Bone diseases, Rickets (osteomalacia), secondary hyperparathyroidism, liver diseases

ALP as a significant marker of Cholestasis

Cholestasis: Impaired flow of bile from liver to the duodenum. May be obstructive (gall stones,
tumors blocking the bile duct) or metabolic (impaired synthesis of bile in the liver). Cholestasis may
be classified into intrahepatic and extra-hepatic

Symptoms of cholestasis

• Clay-colored or white stools

• Dark urine
• Inability to digest certain foods
• Itching
• Nausea or vomiting
• Pain in the right upper part of the abdomen
• Yellow skin or eyes

Significant elevation of plasma ALP levels is an important marker of cholestasis

ALP as marker of bone diseases, such as

Paget's disease: Disrupts the body's normal bone recycling process (old bone tissue replaced with new
bone tissue). Over time, the affected bones may become fragile. Paget's disease most commonly
occurs in the pelvic bone, skull, spine and legs. The risk of Paget's disease of bone increases with age.
The risk increases if there is a family history of the diseases.

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Symptoms and Complication: Bone pain, weak and broken bones, bone deformities, hearing loss and
pinched nerves in the spine.

Causes: Viral infection in the bone cells that may be present for many years before problems appear.
Hereditary factors seem to influence susceptibility to the disease.

Osteomalacia: Osteomalacia refers to a softening of the bones, often caused by a vitamin D deficiency.
Osteomalacia involves a defect in the bone-building process. The soft bones are more likely to bow and
fracture.

Symptoms: Signs of osteomalacia may be apparent in X-ray pictures or other diagnostic tests.

As the disease progresses, patient may experience bone pain and muscle weakness. The dull, aching
pain most commonly affects the lower back, pelvis, hips, legs and ribs. The pain may be worse at night.

3. GAMMA GLUTAMYL TRANSFERASE

• Expresses in cells of liver, kidney, pancreas & prostrate; involved in glutathione metabolism;
catalyzes transfer of glutamyl group from donors like glutathione to acceptor molecules like
peptides/peptide-like compounds and transports the glutamyl peptides into the cell
• Higher levels in males (55 U/L) ; lower in females (38 U/L)

Clinical significance

• The GGT test is sometimes used to help detect liver disease and bile duct obstructions. It is
usually ordered in conjunction with ALT, AST, ALP, and bilirubin.
• GGT can be used to screen for chronic alcohol abuse (it will be elevated in about 75% of
chronic drinkers). Used to monitor alcohol use and/or abuse in people receiving treatment
for alcoholism or alcoholic hepatitis

PANCREATIC ENZYMES

Trypsin, pancreatic α amylases, lipases

• Enzymes secreted from the exocrine pancreas

• They help in digestion of proteins, carbohydrates and lipids in the alkaline environment within
the duodenum and jejunum (small intestine).
• Pancreatic enzymes are released in their inactive forms (zymogens). They are normally only
activated after they reach the small intestine. An enzyme, enterokinase, cleaves a peptide
from trypsinogen, forming the active enzyme trypsin. Trypsin then activates the other
enzymes.

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Fig: Activation of pancreatic zymogens

• Pancreatitis: A clinical condition caused by inappropriate activation of trypsinogen to trypsin

in the pancreas. The active trypsin induces auto-digestion or digests/destroys the pancreatic
tissue (acinar cells) and triggers an inflammatory response in the pancreas.
• Causes: Inappropriate activation of trypsinogen into trypsin in the pancreas. Pancreatic acinar
cells synthesize and secrete a trypsin inhibitor that acts as a safeguard against trypsin
activation within the pancreas. Genetic mutations that decrease the activity of the pancreatic
trypsin inhibitor, increases the risk for pancreatitis.
Another protective mechanism is the autolysis (self-digestion) of trypsin. Mutations in the
gene that codes for trypsinogen could result in premature activation of trypsinogen to trypsin
or even make the trypsin resistant to autolysis.

Amylase and Lipase as Markers of Acute pancreatitis

Elevated plasma levels of amylase and lipase (5-10 times), a consequence of damage of acinar cells of
the pancreas by unwanted activation of trypsin in the pancreas serve as markers of pancreatitis

• Normal values: Amylase-28-100 U/L; Lipase-40-200 U/L

• Plasma levels of lipase peak first in response to pancreatitis. Amylase levels peak later
• Amylase is a small molecule that can undergo glomerular filtration. Excessive amounts present
in blood correspond to high amylase levels in the urine.
• Elevated plasma levels and low urine levels of amylase is suggestive of severe glomerular
impairment
• Lipase passing through the glomerulus is reabsorbed in the kidney tubules and thus does not
appear in the urine
• Chronic pancreatitis leading to pancreatic pseudocysts is characterized by very high plasma
amylase levels (in the thousands) and not lipase.

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 • Pancreatic pseudocysts : Fluid-filled cavities arising from the pancreas and surrounded by a wall
of fibrous or inflammatory tissue, but lacking an epithelial cover

Fig: Pancreatic pseudocysts

General symptoms of pancreatitis

Upper abdominal pain that radiates to the back; pain that feels worse after eating;
Nausea; vomiting; tenderness when touching the stomach; losing weight without trying;
Oily, smelly stools (steatorrhea)

Enzymes that serve as markers of heart diseases

1. Creatine kinase (CK)

• Expresses highly in the skeletal and smooth muscles

• Function: Catalyzes reversible phosphorylation of creatine into phosphocreatine in presence of
ATP. Creatine-phosphocreatine system serves as an energy reservoir for rapid regeneration of
ATP in the muscles. Phosphocreatine supplies inorganic phosphate and phosphorylates ADP to
ATP when necessary
• Creatine + ATP Phosphocreatine +ADP
• Very low levels of CK is present in blood plasma/serum under normal conditions

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Fig: Distribution of serum CK

Isoenzymes of CK

CK protein is composed of 2 subunits designated as the M subunit (for muscle) and the B subunit (for
brain). Different combinations of M, B subunits form 3 isoenzymes of CK

CK-BB or CK-1: Expresses principally in the brain and to some extent in smooth muscles of GI & genital
tracts; levels increase after brain damage and malignancy.

CK-MB or CK-2: Expresses principally in the cardiac muscles; less in skeletal muscles; serum levels
increase significantly in response to myocyte damage , cardiomyopathy, myocardial infarction

CK- MM or CK-3: Predominant expression in skeletal muscles; expresses in heart muscles in small
quantities; High levels of CK-MM in blood is indicative of skeletal muscle damage. It will be high in
people with muscular dystrophies (Duchene muscular dystrophy and Becker muscular dystrophy, viral
myositis etc) which leads to weakening and damage to skeletal muscles

Fig: 3 isoenzymes of CK

CK-MB (CK2) as a marker for myocardial infarction

• CK-MB levels rise 3 to 6 hours after a heart attack or myocardial infarction due to ischemic
cardiomyopathy (disease of the heart muscles)

• If there is no further damage to the heart muscle, the level peaks at 12 to 24 hours and returns
to pre-infarction level by 48 hours.

• The levels of total CK and CK-MB in the patient’s blood/serum are used as diagnostic markers of
cardiomyopathy, the underlying causing of myocardial infarction, ischemia etc.

2. Troponin (not an enzyme)

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 • Troponins are protein molecules that are part of cardiac and skeletal muscle. Smooth muscle
cells do not contain troponins. Troponin is a complex of three regulatory proteins
(troponin C, troponin I, and troponin T) that plays an integral role in muscle contraction.
• Troponin T and I specific to cardiomyocytes. They are absent in blood under normal conditions

• Clinical significance: Troponin I, T are released in the blood in response to myocyte damage
which may be caused by MI, severe pulmonary embolism causing acute right heart overload,
heart failure, myocarditis. etc
• After myocyte injury, troponin is released in blood from the damaged myocytes in 2–4 hr, peaks
between 12-24 hr and persists for up to 7 days
• Troponin T, I levels in blood are measured to evaluate patients with chest pain to see if they
have had a heart attack and associated cardiomyopathy. They are thus superior markers for
myocardial infarction (MI)

Fig: Time dependent expression of important markers of MI

Important Note: Myoglobin (oxygen binding protein present in cardiac and skeletal muscles)
peaks in blood within 3-4 hrs of MI; CK-MB and cardiac troponin peaks around 12-24 hrs of
infarction; Ck-MB levels fall rapidly thereafter; troponin peaks persists longer and is detectable
in blood upto 7 days post infarction.

3. Lactate dehydrogenase (LDH)

Widely distributed in the body; present in all cells with high concentration in cardiac/skeletal muscle
cells

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 • Glycolytic enzyme that catalyzes conversion of L-lactate to pyruvate under hypoxic condition
Lactate + NAD+ Pyruvate + NADH

• Composed of 4 peptide chains of 2 types, H and M types

• Isoenzymes of LDH
LDH1 (HHHH) and LDH2 (HHHM): Predominates in cardiac muscles, kidney, erythrocytes
LDH4 (HMMM), LDH5 (MMMM): Predominates in skeletal muscle, liver

LDH3 (HHMM), LDH2 and

HDH 4: Predominates in lungs, spleen, endocrine glands

• Clinical Significance: During illness or injury, the LDH levels (4-10 fold) may be elevated in
blood/serum. High levels of LDH in the blood indicate acute or chronic cell damage which may
be a consequence of
• Heart diseases (MI)
• Liver diseases
• Muscle diseases
• Blood diseases (megaloblastic anemia/leukemia)
• Kidney diseases
• Malignancy

ACID PHOSPHATASE

• Lysosomal enzyme that hydrolyses organic phosphates from different molecules during
digestion; acts in an acidic pH; present in the lysosome and functions when the lysosomes fuse
with the endosomes.
• Present in high concentration primarily in the male prostate gland (100 times more acid
phosphatase). So the level of the enzyme is higher in males when compared to females

• Different forms of acid phosphatase are present in small quantities in bone, liver, spleen,
kidney, red blood cells and platelets

• Clinical significance: Acute increase in acid phosphatase levels is a marker for metastatic
prostate cancer

• Bone diseases, lysosomal diseases (Gaucher’s disease), sickle cell anemia, all moderately
increase the serum levels of acid phosphatase

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Lecture 23- Serum/Plasma Proteins & their Associated
Disorders
Learning Objectives:

• Learn the composition of plasma, role of plasma proteins as

acute phase reactants.
• Emphasize plasma protein electrophoresis & immune fixation
electrophoresis.
• Evaluate the role of albumin in maintenance of colloidal
osmotic pressure.
• Emphasize on role of alpha 1 antitrypsin in development of
emphysema.
• Differentiate between the role of haptoglobin & hemopexin.
Learn the role of transferrin in iron metabolism
• Evaluate the role of ceruloplasmin in Cu metabolism;
differentiate between Wilson’s & Menke’s syndrome.
• Compare/contrast multiple myeloma versus Waldenstrom’s
macroglubulinemia in terms of:
-causes
-Prevalence & symptoms
-Diagnostic features & genetic testing

Composition of blood plasma

Plasma is composed of 92% water, organic components (glucose, fats, vitamins), inorganic components
(Na+, Mg+2, K+, Ca+2, bicarbonates, phosphates, dissolved oxygen, dissolved carbon dioxide), other
organic compounds (secretions, enzymes, metabolites) and dissolved proteins (albumin, globulins,
fibrinogen, clotting factors).

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Fig: Composition of blood

When blood is centrifuged in the presence of an anticoagulant, the cellular components of blood (RBCs,
WBCs, platelets) settle at the bottom (pellet) occupying 40-50% of the total blood volume. The liquid
part separates as yellowish fluid called plasma.

If blood is allowed to coagulate, the clear straw yellow fluid that separates from the blood cells is called
serum

SERUM = PLASMA-COAGULATION FACTORS

Characteristics of plasma/ serum proteins

• Plasma and serum contain the same proteins except fibrinogen (present in plasma but absent in
serum)
• Contain simple & conjugated proteins (glycoproteins, lipoproteins)

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 • Most of these proteins are synthesized in the liver (except gamma globulins)
• Total serum protein content: 6-8 gm/dL (with slight variations)

• The total plasma/serum protein content of an individual is influenced by

a. Water balance in the body
b. Nutritional status
c. Hepatic and renal functions
d. Metabolic disorders in the body

Note: Quantification of total plasma/serum protein content is clinically significant

Hyperproteinemia: Increase of total protein concentration. These increases can be relative or

absolute.

Relative increase of plasma proteins is related to water levels of the body. Dehydration, or loss of
water, causes all fractions of protein to be increased by the same percentage. A dehydrated patient
commonly displays hyperproteinemia. Dehydration may be caused by decreased fluid intake or by
increased water loss

Absolute increases are due to increased protein synthesis.

Immune system disorders, on the other hand, are extra-hepatic and are able to significantly elevate
total protein. Multiple myeloma is a malignancy resulting in overproduction of gamma globulins. An
absolute increase in the serum total protein is a common finding.

Hypoproteinemia may be due to increased protein loss or to low protein intake. Starvation or
malabsorption disorders will decrease amount of amino acids available for protein synthesis.
Accelerated protein loss may be due to diseases such as nephrotic syndrome where albumin is lost
through damaged tubules or glomeruli. Hypoproteinemia may also be due to blood loss that occurs as
the result of a traumatic injury. In this case, water is replaced more rapidly than the proteins. Serum
protein levels also decrease in patients with extensive burns.

Acute Phase Reactants (APRs)

APRs are those plasma proteins that elevate or decline in response to inflammation, which may be
caused by infections, surgery or trauma, conditions like myocardial infarction, and tumor growth. It is
assumed that all acute phase reactants play a part in host defense.

If the level of a plasma protein increases in response to inflammatory condition, it is called positive
acute phase reactant (majority of plasma proteins)

If its levels decrease, it is called negative acute phase reactant (albumin, prealbumin and transferrin)

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 Since the acute phase reaction is associated with many diseases, measurements of APRs have clinical
significance

ELECTROPHORETIC SEPARATION OF PLASMA PROTEINS

What is electrophoresis: A technique of separating proteins/ nucleic acids present in a mixture on the
basis of their size & charge density in an electrical field.

Principle underlying electrophoretic separation of protein

Proteins are amphoteric molecules; can be +/- charged due to the ionizable amine, carboxyl groups, the
acidic/basic R groups; charge depends on the pH of the medium & pI (isoelectric point) of the particular
protein.

Mobility of proteins in an electrical field is dependent on its charge. Negatively charged proteins move
toward the anode (+ electrode) & positively charged proteins toward the cathode (- electrode).

In order to make the separation uniform and one-directional (from cathode towards anode), a buffer
with a pH of 8.4-8.8 which is higher than the pI of all proteins is used. At this pH, all the proteins in the
mixture will be deprotonated and attain a net negative charge and move from the cathode (top) toward
the anode (bottom) at different rates, enabling uniform separation in an electric field

Fig: Electrophoresis

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Electrophoretic separation of plasma proteins

Electrophoresis enables separation of the plasma proteins into five/six major fractions or bands based
on their movement in an electrical field on the basis of their charge density (charge per unit surface
area) and molecular weight. The separation is done in an alkaline pH of 8.4-8.8 (reasons described
above). Staining the gel containing the separated plasma proteins with Coomassie blue, enables
visualization of the separated proteins as individual bands

The albumin fraction migrate the farthest from the cathode and closest to the anode (+) due to its
strong negatively charge and its low molecular weight (66000D).

Scanning of the gel with the help of a densitometer, gives a quantitative analysis of each protein
fraction

Fig: Gel with separated plasma proteins

Densitometric analysis of the separated plasma proteins

Albumin: Major protein component; forms the largest peak closest to the + electrode (anode)

α1 and α2 globulins, β globulin, γ globulin (closest to the – electrode) comprise smaller fractions of
the total serum proteins

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FUNCTIONAL IMPORTANCE OF INDIVIDUAL PLASMA/SERUM PROTEINS

1. ALBUMIN- Characteristics

• Major protein constituting about 60% of the total protein content (3.4-4.7g/dL); synthesized by
liver @ 12g/day
• Composed of single polypeptide chain (585 AA long; MW of 66000 D)
• Most hydrophilic protein in plasma; provides 80% of colloid osmotic pressure; helps maintain
blood volume and fluid distribution
• Half- life 17-20 days; avoids renal excretion due to size and net negative charge at pH 7.2 of
blood (pI=4.5)
• Crosses the vascular endothelium; also present in interstitial fluid and lymph

Functions of albumin

• Serves as a source of amino acid at tissue level

• Buffering function: High plasma concentration & histidine rich constitution enables it to
maintain acid-base balance in the plasma (buffering action)
• Serves as binding protein for free fatty acids, Ca+2, bilirubin, Cu+2, drugs
• Unbound drugs are pharmacologically active. In patients with severe hypoalbuminemia,
otherwise desirable plasma concentration of drug might be in the toxic range
• Regulation of colloidal osmotic pressure or oncotic pressure
Normal hydrostatic pressure of blood (33 torr) forces fluid from the capillaries into the
interstitial spaces at the arterial end of the blood vessels. Colloid osmotic pressure of plasma
proteins (ability of these proteins to attract water & electrolytes on their surfaces) pulls the
fluid back into the capillaries at the venous end. Albumin being the most hydrophilic plasma
protein provides 80% of the colloid osmotic pressure or oncotic pressure for fluid
reabsorption.

EDEMA: When albumin concentration in plasma/serum drops below 2 g/dL

(hypoalbuminemia), lack of colloidal osmotic pressure leads to fluid build-up in the interstitial
spaces leading to edema

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 Albumin is considered a negative acute phase reactant protein. During inflammatory
processes and chronic inflammatory disorders, albumin levels decrease. Hypoalbuminemia
may occur due to
Decrease in albumin synthesis or Increase in catabolism of albumin may occur due to
1. Liver cirrhosis
2. Malnutrition
3. Malabsorption
4. Nephrotic syndrome
5. Burns

Hyperalbuminemia: High albumin concentration in plasma/serum; occurs subsequent to

fluid depletion

2. GLOBULINS- Characteristics
• Molecular Weight: 90000-1300000D
• Types : Electrophoretically separable into α 1 , α 2 , β and γ globulins
• Synthesis: α and β globulins in liver; γ globulin (antibodies) in the reticulo-endothelial
cells (plasma cells, B cells)

A. Clinically Important α 1 globulins:

α 1 antitrypsin,
α 1 acid glycoprotein
α 1 fetoprotein

i. α 1 -Antitrypsin (α 1 AT)
• Synthesized in the hepatocytes within the liver; an acute phase reactant
• Principle inhibitor of serine protease enzymes in plasma; plays a major role in blood
clotting cascade and immune responses
• Inhibitor of trypsin & elastase (prevents breakdown of connective tissue by elastase in
inflamed areas)
• 75 genetic variants of the enzyme are known; underlying cause of inherited metabolic
disorders of the lung and liver

Neonatal hepatitis: The Z allele, (a genetic variant of the antitrypsin gene with a point
mutation) in the homozygous condition leads to neonatal hepatitis. The mutant gene
codes for the defective form of polymerized α 1 AT, which cannot be secreted from the
endoplasmic reticulum of the hepatocytes; the protein accumulates in the hepatocytes
leading to the gradual destruction of the hepatocytes. The consequence is neonatal
jaundice.
A person can be born with Z alleles or develop the mutation during his/her lifetime which
will cause hepatitis for the same reason.

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Emphysema: A consequence of α 1 AT deficiency

Under conditions: Elastase secreted from WBCs like neutrophils in high concentrations in the
lung for protecting the lung from microbes (bacteria). Antitrypsin neutralizes the elastase when
required so that they do not damage the elastic tissue within the lungs thus providing
protection to the lung
Under low levels of α antitrypsin:
1
Neutrophil elastase secreted in the lungs remain active; elastic lung tissue ( alveolar walls) gets
damaged by elastolytic action of elastase; consequence is emphysema

Fig: Emphysema

Smoke increases chances of emphysema: Smoke that enters the lungs due to chain smoking
oxidizes methionine residues of the α1 antitrypsin molecules into methionine sulfoxide,
inactivating the capacity of α1 antitrypsin to neutralize elastase. Thus smoking by itself can cause
chronic bronchitis and emphysema. Smoking combined with low levels of α1 antitrypsin in blood
may definitely increase chances of developing emphysema

B. Clinically important α globulins

2
• Haptoglobin

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• Ceruloplasmin
• α Macroglobulin
2
i. HAPTOGLOBIN
• Glycoprotein synthesized by the liver; an acute phase reactant
• Function: Destruction of RBCs (hemolysis) releases hemoglobin (Hb) into the plasma where it is
converted from tetramers into dimers; haptoglobin binds to the excess Hb-dimers present in
blood plasma under such conditions; the Hb-haptoglobin complex is taken up by hepatocytes
and macrophages for further degradation of Hb. Globin & iron of Hb are recycled; heme is
metabolized into conjugated bilirubin between liver and macrophages and excreted along with
bile from the liver

• Serum haptoglobin levels are decreased under all hemolytic conditions: High concentration of
Hb (consequence of rupture of RBCs) is released in the plasma in patients suffering from severe
hemolytic anemia. All haptoglobin molecules bind to the Hb-dimers trying to clear the blood for
macrophage/liver mediated excretion through bile. Due to saturation of haptoglobin molecules
are with Hb dimers, a blood test in these patients will reveal low free haptoglobin levels (a
diagnostic marker for severe hemolytic anemia).

Fig: Removal of Hb-dimers from blood by haptoglobin during severe hemolytic anemia

Hemoglobinuria: Occurs during severe intravascular hemolysis.

Hemoglobin (Hb) appears in urine when

A. Plasma concentration of Hb is very high (>60 µmol/L), a consequence of severe intravascular

hemolysis (damage of RBCs mediated by oxidative stress, infection etc).The high plasma Hb

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 concentration saturates plasma haptoglobin. The kidneys tries to remove excess Hb from blood
by filtration. So Hb appears in urine. As the kidneys are working hard in this process they get
exhausted. Long term effects will be precipitation of Hb in kidney tubules leading to
compromised kidney function

B. Low plasma haptoglobin levels (liver disease): Hb released in blood remains unbound;
undergoes glomerular filtration, appears in urine and leading to severe kidney damage

ii. CERULOPLASMIN
• Blue colored copper containing α 2 globulin present in plasma/serum; glycoprotein in nature
synthesized by the liver;
• Transporter of 90% of blood copper content; it has enzymatic activities as well
• Each molecule binds to 6 atoms of copper; due to tight binding, the copper in ceruloplasmin is
not readily exchangeable
• It exhibits copper dependent oxidase activity and helps to oxidize Fe+2 to Fe+3 promoting
transport of Fe+3 in the plasma with the help of transferrin

Note: Albumin plays an essential role in copper transport in the human body as well; 10% of
plasma copper is bound to albumin which is readily exchangeable at the tissue level.

Copper homeostasis: Copper is a trace metal which is required at cellular levels for neuronal functions,
collagen synthesis in the bone, electron transport chain etc. The level of copper in the blood is
regulated very carefully as over accumulation of copper (a redox active metal) can lead to generation
of free radicals which are toxic.

The dietary copper absorbed by intestinal enterocytes makes its way to the liver hepatocyte via the
portal circulation. An ATPase called ATP7A present in the enterocyte membrane helps release dietary
copper in the portal circulation. When copper gets to the hepatocytes, a 2nd ATPase molecule ATP7B
helps to bind copper to apoceruloplasmin (inactive) converting it to active ceruloplasmin. The copper
bound to ceruloplasmin is then distributed from the liver via blood to different organs.

WILSON’S DISEASE

• Autosomal recessive inherited disorder of copper metabolism

• Occurs due to a mutation in ATP7B gene located on chromosome 13 which leads to low expression
of the gene product (a P-type ATPase protein) primarily involved in copper hemostasis in the liver
hepatocytes.

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• This interferes with the copper uptake in the liver hepatocytes, increased apoceruloplasmin
levels, low ceruloplasmin levels and toxic build-up of copper in the hepatocytes (due to
decreased excretion through the biliary canaliculi)
• Excess copper from hepatocytes leaks out in the blood and deposits in the brain, kidneys, eyes
and lungs;
• Copper deposits in cornea produce Kayser-Fleischer rings (characteristic feature); other
symptoms include hepatitis, liver cirrhosis, neurological symptoms

Fig: Copper metabolism in the hepatocytes

Fig: Wilson’s disease: Symptoms

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MENKE’s syndrome
• Genetic disorder caused by a defect in the ATP7A gene which inhibits distribution of copper from
the intestinal enterocytes to other parts via blood
• Leads to deficiency of copper in the brain and other parts of the body; copper builds up in the
small intestine and kidneys.
• Affects structure of bone, skin, hair, blood vessels; interferes with nerve tissue function.
• Patients have kinky hair so it is also called kinky hair syndrome

C. Clinically Important β-Globulins

Transferrin
Hemopexin
Complement protein
C-reactive protein

i. TRANSFERRIN (TF)

• β1 globulin; MW of 76000D; glycoprotein synthesized by the liver; negative acute phase

protein; exists in 20 polymorphic forms
• Plays a central role in iron metabolism in our body. Plasma concentration is 300 mg/dL;
transports Fe @ 300 g /dL representing total Fe binding capacity of plasma
• Each molecule of TF transfers two Fe+3 ions from site of release (liver, gut etc) to sites where Fe
is used (bone marrow, for RBC production).
• TF levels are high with low iron saturation in iron deficiency anemia; TF levels are normal to low
in types of anemia characterized by defective Fe incorporation in Hb/ RBC

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Note: 2 types of plasma membrane bound transferrin receptors TfR1, TfR2 on the surface of target cells.
The holoenzyme (Tf+iron) binds to these receptors and are internalized by receptor mediated
endocytosis. The clathrin coated endosomes fuse with the lysosome where the acidic pH dissociates Fe
from the receptor protein. The dissociated Fe is released into the cytoplasm of the target cell. The apoTf
is not degraded in the lysosome but is recycled. It is returned back to the plasma membrane bound to its
receptor, dissociates from its receptor in the plasma, picks up more iron and delivers it to cells that
require iron.

[Link]

• Beta globulin synthesized in the liver; MW 57000-80000 D

• Low levels at birth; reaches adult levels (0.5-1.0 g/L) in the first year
• Binds to free heme produced by the breakdown of Hb
• Transports heme to the liver for conversion into bilirubin
• Plasma levels decrease in hemolytic anemia at birth

Note: you should have prior knowledge of C-reactive proteins and complement proteins from
immunology

[Link] GLOBULINS or IMMUNOGLOBULINS

(Structure, function etc covered in Immunology)

IMMUNOPROLIFERATIVE DISORDERS
Immune proliferative disorders are also called gammopathies. They result from hyper proliferation of
mature antibody secreting neoplastic plasma cells or plasmacytoid lymphocytes. These disorders are
characterized by excessive secretion of antibodies or immunoglobulins in the blood plasma/serum by
the neoplastic plasma cells. Thus they are also called hypergammaglobulinemia.

Most common types of gammopathies are shown in the diagram below

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Monoclonal gammopathies

• Occur due to neoplastic expansion of a single type of plasma cells

• Result in abnormal secretion of a particular type of antibody in blood of the patient
• Diagnosis: serum electrophoresis of the patient exhibits a prominent increase in a particular
gamma globulin fraction
• Examples: Multiple Myeloma& Waldenstroms macroglobulinemia

Polyclonal gammopathies

• Occur due to neoplastic expansion of multiple plasma cell lines

• They occur secondary to other disorders like asthma, inflammatory disorders etc

MULTIPLE MYELOMA (MM)

• Most common type of hematological cancer; occurs due to proliferation of a single clone of
plasma cells resulting in abnormal production of a single type of antibodies
• IgG myeloma is most prevalent followed by IgA, IgD types. IgE type is very rare
• Occasionally the neoplastic plasma cells secrete only the light chain component of the
immunoglobulin in excess. This type is called the light chain myeloma
• 4/100,000 of Western Europeans and North Americans are diagnosed per year; it is twice as
common in black Americans than white Americans
• Median age of onset, 60 years; environmental factors affect the development of this disease;
men and women are equally affected
• Survival after diagnosis is variable between 3-10 years

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 Fig: neoplastic plasma cell line producing abnormal quantities of antibodies

Symptoms

• Bone pain (typically the back & thorax)

• Weakness, fatigue & malaise, weight loss initially
• Anemia
• Prone to infections
• Hypercalcemia
• Renal failure/ kidney stones
• Spinal cord compression
• Pathological fractures
• Neuropathies

Clinical Manifestations of Multiple Myeloma

Lytic bone disease

The abnormal proliferation of neoplastic plasma cells in the bone marrow prevents growth of osteoblasts
(normal bone cells) preventing new bone growth but increases the activity of osteoclasts (which break
down old or worn-out bone under normal conditions).This leads to bone loss with no new bone
formation and is the underlying cause of soft spots or osteolytic bone lesions visible by X ray. These
lesions cause bone pain and lead to rapid decrease in bone strength. It also causes hypercalcemia due to
excessive bone destruction

Hematological abnormalities
It is a cancer of the blood; abnormal proliferation of malignant cells interfere with the production of
other normal blood cells leading to leukopenia (low WBC count), anemia (low RBC count) &
thrombocytopenia (low platelet count). Roulex formation (sticking of RBC) is a characteristic
hematological finding.
+2
Kidney defects: High immunoglobulin & Ca levels in blood overstress the kidneys leading to kidney
dysfunction and kidney stones.

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Fig: Osteolytic lesions visible in X ray

Diagnostic Features of Multiple Myeloma

Blood tests
• Total serum protein content: Increases abnormally due to increase in gamma globulin content;
albumin levels are slightly reduced; A/G ratio is lowered

• Plasma /Serum electrophoresis: Exhibits an abnormal M-spike due to abnormal amount of Ig

present in serum/plasma produced by the malignant plasma cells. This spike is called
paraprotein/M protein

Fig: Gel electrophoresis and densitometry of patient’s plasma/serum proteins

• Urine tests/urine electrophoresis: In some patients abnormal amounts of free light chains of Ig
(kappa & lamda) are produced (by themselves) in addition to the M protein (Ig)

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 As light chains have small molecular weight, they are excreted by the kidney through urine. Urine
electrophoresis of these patients will show presence of Bence-Jones protein (excess Ig light
chains in urine); prolonged presence of Bence-Jones protein in the urine can imply renal damage.
The Ig light chain assay needs to be confirmed through both plasma and urine electrophoresis

• Immune-fixation electrophoresis (IFE): Aims at identification of the specific type of Ig secreted

in excess by the neoplastic plasma cells in the patient’s serum. The gel containing the separated
protein from the patient’s plasma/serum is incubated with labeled antibodies specific to different
classes of gamma globulins (IgG/IgA/IgD/IgE) that may be overproduced in multiple myeloma.
The antibody binding helps to confirm the identity of the M spike (whether the M-spike is
composed of IgG, IgA, IgD or IgE)

Fig: Immuno fixation following urine and plasma electrophoresis

• Marrow aspiration and biopsy: Collection & laboratory analysis of bone marrow samples from
different bones will show abnormally high % (>30%) of plasma cells in the bone marrow
• Genetic tests: cytogenetic testing is done to identify the chromosomal abnormalities that caused
the disease. It also gives an idea about how aggressive the disease is. Translocations
(chromosomes 14, 16, 20) or deletion (in the p arm of chromosome 17) are often the cause of
multiple myeloma.
• Approximately 15% of patients with MM exhibit translocation between chromosomes 14 and 16
or chromosomes 14 and 20. MM caused by deletion of chromosome 17p is characterized as high
risk. This type of MM is aggressive often shortens life expectancy of patients. The high risk type
is treated with more aggressive therapy

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 MM patient
Fig: Bone marrow biopsy of a normal and MM patient

WALDENSTROM’S MACROGLOBULINEMIA (WM)

• It is a clonal disease of virgin B lymphocytes and classified as monoclonal gammopathy. It is

characterized by excess secretion of IgM in the plasma /serum of the patients by the neoplastic
B lymphocytes.
• It is an indolent lymphoma (spreads slowly) and expresses at later stages of life (60-70 years of
age)
• It is not very common

Fig: An IgM pentamer

SYMPTOMS

• Weakness, fatigue & dizziness

• Nose & gum bleeding, bruising
• Headaches, blurry vision
• Prone to infections
• Anemia
• Numbness & tingling of hands, feet in cold weather
• Enlarged lymph nodes in neck, groin, underarms

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 • Heart problems
• Neurological problems

CLINICAL MANIFESTATIONS

• Hyperviscosity syndrome: Excess production and secretion of IgM which is a pentamer with
multiple subunits, leads to increase in serum viscosity. This is called hyperviscosity syndrome and
leads to circulatory impairment
• Anemia: The malignant neoplastic lymphoplasmacytic cells infiltrate back into the bone marrow
and destroy RBCs. Consequence is severe anemia
• Cryoglobulinemia: Cryoglobulins are single (type I) or mixed (type II, II) immunoglobulin that
undergo reversible precipitation at low temperatures (form gels below 37°C) which dissolve at
high temperatures. IgM can form type I or type II cryoglobulins which may lead to hyperviscosity
of plasma in cold weather causing spasms in capillaries, pain, cyanosis, tingling of fingers, toes in
the patient.

DIAGNOSTIC FEATURES
• Complete blood cell count: RBC, WBC and platelet counts in patients’ blood will be low due to
bone marrow infiltration of the malignant lymphoplasmacytic cells and destruction of normal
blood cell population
•
• Serum protein electrophoresis & immune fixation: Characterized by detection of the M -SPIKE
due to presence of excess IgM in the serum of patients; use of IgM specific antibody will
confirm the spike to be IgM

• Viscosity testing of the patients’ blood (Cryocrit): Measures the level of cryoglobulin in the
blood plasma/serum of the patient; serum sample of patient placed in cold (4°C for 72 hrs),
precipitation of cyroglobulins is checked; serum is then exposed to a temp of 37°C to check for
dissolution of cryoglobulins

• Bone marrow biopsy: Will exhibit overpopulation of neoplastic B cells

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Lecture 24- Hemostasis and Thrombosis
Learning Objectives:

• Distinguish between hemostasis & thrombosis

• Differentiate between primary, secondary & tertiary
hemostasis
• Emphasize on Von-Willibrand’s factor, its role in platelet plug
formation
• Learn the steps & the significance of the extrinsic/intrinsic/
common pathways of the coagulation cascade; tertiary
hemostasis
• Learn the role of protein C, S, thrombomodulin, antithrombin
III
• Emphasize on the role of Vit K in coagulation
• Know the role of warfarin & heparin in blood coagulation
• Compare/ contrast PT, PTT, bleeding time
• Differentiate between presentation of hemophilia, DIC, vWD

Hemostasis: Normal physiological process; comprises a series of regulated processes that maintain
blood in a fluid, clot-free state in normal vessels while rapidly forming a localized hemostatic plug
(clot) at the site of vascular injury to prevent significant blood loss.

Thrombosis: The pathologic counterpart of hemostasis; it involves the formation of blood clot
(thrombus) within intact vessels. It occurs when the physiological regulators of hemostasis are
inactivated or turned off under certain conditions.

MECHANISM OF HEMOSTASIS

• Divided into 3 steps:

A. Primary hemostasis: Vasoconstriction + formation of platelet plug

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 B. Secondary hemostasis: Activation of the coagulation cascades leading to formation of fibrin
meshwork for stabilization of the plug
C. Tertiary hemostasis: Plasmin mediated breakdown of the clot
• Hemostasis is rapid, localized and a reversible process

• The role players of hemostatic process are

A. Endothelial cells in the blood vessel wall
B. Blood platelets
C. Blood clotting factors

Fig: Overview of primary and secondary hemostasis

[Link] hemostasis: Aims at formation of platelet plug that stops the blood flow
temporarily
Associated events are

[Link]: Mediated by thromboxane A 2 (T x A 2 ) secreted from the damaged endothelium of

the blood vessel wall and the membrane of the platelets. It restricts flow of blood to the site of vascular
damage. T x A 2 also plays a role in activation of the blood platelets for adhesion to the vessel wall at the
site of injury.

2. Aggregation + Adhesion of Platelets: Factor VIII + Von Willebrand factor (vWF) play the most
important roles in facilitating platelet aggregation and adhesion to the vessel wall. vWF is a glycoprotein
secreted by the damaged endothelium; factor VIII is also secreted by the endothelium. Factor VIII bound
to vWF (vWF complex) serves as a bridge and anchors the aggregated platelets to the exposed collagen
on damaged vessel wall

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3. Release of arachidonic acid from membrane of the aggregated platelet: vWF activates
phospholipase A2 which releases arachidonic acid, the precursor of TxA2, from membrane
phospholipids.

4. Induction for more platelets to aggregate at the site of injury: Mediated by TxA2 produced from the
arachidonic acid and ADP

5. Binding of thrombin to aggregated platelets: Leads to activation of G protein, PKC, PLC,

+2
DAG and IP3 induced release of Ca and ADP in the aggregated platelets at the site of injury.

6. Activation of myosin light chain kinase enzyme (MLCK) in the membrane of aggregated platelets
induces change in shape of the aggregated platelets which allows aggregation of more platelets at the
site of injury

7. Platelet Plug formed: This entire process leads to the formation of a platelet plug that temporarily
blocks the cut end of the blood vessel inhibiting blood flow temporarily. Fibrinogen acts as a glue in this
process and hydroxytryptamine (serotonin) has a role to play in the process

Fig: Overview of primary hemostasis

Use of “Aspirin” as a blood thinner

Aspirin acts as a blood thinner as well as an analgesic (pain/ fever reducer).It acts as a blood thinner by decreasing
the production of thromboxane (TxA2) from blood platelets which prevents activation and tendency of platelets
to stick or aggregate, keeping the blood flowing smoothly. Aspirin irreversibly inactivates cyclooxygenase (COX)
enzymes required for the production of thromboxane and prostaglandins from cellular arachidonate. The drug’s

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effect as an analgesic is due to ability to also lower prostaglandin production from arachidonate in specific, as
prostaglandin in involved in inflammatory pathways which leads to pain and fever.

Von Willebrands factor (vWF)

• Released by damaged endothelium in the vessel wall in response to vascular injury.
• vWF binds to factor VIII released from the damaged endothelium to form the vWF complex.
• vWF complex enables binding of activated platelets to the exposed collagen in the vascular wall
at the site of injury
• Binding to vWF also prevents degradation of factor VIII by blood proteases
• Release of factor VIII from vWF is initiated by thrombin making it available for the intrinsic
pathway

Fig: The vWF complex

CLINICAL CORRELATES

a. Thrombocytopenia or Low platelet count: Patients with platelet counts below 40,000/µl of blood may
develop spontaneous hemorrhaging due to insufficient platelet plug formation

b. Von Willibrand disease (vWD): Clinically heterogeneous hemorrhagic disease that occurs due to
deficiency/dysfunction of Von Willibrand’s factor (vWF); vWF gene located on chromosome 12 (tip of p
arm)

i. vWD-I: Most common hereditary coagulation abnormality; occurs due to quantitative

deficiency of vWF (20-50% of normal levels). May be asymptomatic or exhibit mild symptoms

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 ii. vWD-II: Associated with structural abnormality of vWF protein that affects vWF and factor VIII
interaction.

iii. vWD-III: Rare disease that occurs due to mutation of vWF gene and total lack of vWF. Patient
exhibits severe bleeding disorder.
B. Secondary Hemostasis – Involves stabilization of the platelet plug by formation of a
fibrin meshwork around it

• Permanently seals the ruptured blood vessel

• Acts through activation of 2 coagulation cascades namely the extrinsic and intrinsic coagulation
cascade
• Each cascade is a series of reactions that progress through conversion of multiple coagulation
factors (produced by the liver & released as zymogens or inactive enzymes in blood) into active
serine protease enzymes.
• The activated coagulation factor involved in one activates the coagulation factor involved in the
next reaction.
• Extrinsic pathway initiates the generation of active factor Xa, the central element of the 2
cascades; intrinsic pathway amplifies generation of Xa.
• Xa converts prothrombin to thrombin which then converts fibrinogen into fibrin ( through the
common pathway)

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Note: Factor IV is calcium, factor VI is unknown

i. Extrinsic cascade
Characteristics:

• It is a shortcut process
• Is completed in 12-15 sec
• It initiates generation of Xa
• By itself, can only form a small fibrin clot ; it is a quick patch process

Associated events

• Vascular injury releases tissue factor (factor III/ thromboplastin); source is damaged sub-
endothelial tissues
+2
• Tissue factor in presence of Ca activates VII to VIIa
• VIIa converts inactive factor X into its active form Xa
• Xa can generate small amounts of thrombin from prothrombin directly
• This initial thrombin generated is required for activation of factor V to Va (promotes formation
of prothrombinase complex of the common pathway

Fig: Extrinsic cascade

ii. Intrinsic Cascade

Initiated by contact with blood
• With exposed collagen of injured blood vessel
• With negatively charged surfaces (glass, plastic, fabric)

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 • Thrombin generated by extrinsic pathway plays a role in initiation (through activation of factors
XI, VIII and V)

Characteristics

• Slower process when compared to extrinsic cascade

• Amplifies production of Xa
• Amplifies production of large amounts of thrombin. One molecule of Xa can generate 1000
molecules of thrombin

Associated Events

• Presence of thrombin + damaged vessel leads to:

• Activation of inactive factor XII into active XIIa (facilitated by kininogen/kallikrein)
• XIIa catalyzes the activation of XI into XIa
+2
• XIa catalyzes conversion of inactive factor IX into IXa (Ca dependent)
+2
• IXa and factor VIIIa in presence of Ca binds to the membrane phospholipids of aggregated
platelets to form the tenase complex
• Binding of inactive factor X to the tenase complex activates it into Xa (amplifying production of
Xa)

Note: The kininogen- kallikrein system: High molecular weight kininogen (HMWK) is a 626 amino acid
long protein that acts as a cofactor in contact activation process. It activates kallikrein and factor XII
which is necessary for activation of factor XI, the precursor of bradykinin (a potent vasodilator that
induces smooth muscle contraction).The genes responsible for production of kininogen is located on
Chromosome 3

Fig: Intrinsic cascade

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iii. The Common pathway
• Xa generated by both pathways
+2
• Prothrombinase complex: Va in presence of Ca binds to the negatively charged phospholipids
on platelet/ monocyte/endothelial cell membrane at the site of injury.

• Xa binds to membrane bound Va to form the prothrombinase complex

+2
• Xa catalyzes release of a γ-carboxyglutamate fragment (strong Ca chelator) from the N-terminal
end of inactive prothrombin (factor II) enabling its binding to platelet membrane & catalyzing its
conversion to active thrombin

• Thrombin catalyzes conversion of soluble fibrinogen into soluble fibrin monomers. These
monomers polymerize into insoluble fibrin polymers catalyzed by factor XIIIa (transglutaminase
enzyme) activated by thrombin. Insoluble fibrin polymer forms a network around the platelet
plug

Fig: Common pathway

Note: The extrinsic and intrinsic cascades occur simultaneously

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 Fig: The complete clotting cascade

C. Tertiary hemostasis: Involves

Clot retraction: Repair process; initiated by contractile protein of platelets in the clot; involves retraction
of the edges of the wound to prevent further hemorrhage

Fibrinolysis: Plasmin mediated breakdown of clot

Events:

• Liver produces inactive plasminogen

• Conversion of inactive plasminogen into active plasmin is promoted by tissue plasminogen
activator (tPA, released by damaged endothelium, a few days after injury (release suppressed
initially). Urokinase a serine protease enzyme present in blood also plays a role in activating
plasminogen to plasmin
• Plasmin degrades fibrin into fibrin degradation products (FDP)
• Plasmin also promotes release of more tPA and urokinase
• FDPs also compete with thrombin and prevent further conversion of
fibrinogen into fibrin
• Plasmin is eventually inactivated by α antiplasmin/α macroglobulin
2 2
Clinical correlate: Administration of tPA can be a treatment option for life threatening thrombotic
disorders like stroke, myocardial infarction due to its ability to dissolve clots. However it is
associated with many side effects

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Fig: Mechanism of tertiary hemostasis

Note: Red arrows: signify inhibition; Blue arrows: signify activation; Black arrows: signify conversion

**Streptokinase is also a fibrinolytic drug that activates plasminogen into plasmin. It is administered
to dissolve existing intravascular clots

CLINICAL CORRELATE

HEMOPHILIA

• A group of X- linked recessive disorders that occur due to deficiency of specific coagulation
factors and abnormal platelet function.
• Occurs in males; females are mostly asymptomatic genotypic carriers. Rare phenotypic
expression occurs in females

Types: Hemophilia A- Deficiency of factor VIII

Hemophilia B- Deficiency of factor IX (christmas disease)

Symptoms: Both types bleed with similar frequency; bleeding can occur anywhere. Internal bleeding in
knees, ankles, joints and also organs/ tissue is the major concern in hemophilia. Symptoms include
excessive bleeding from cuts and injuries, appearance of large, deep bruises, pain, swelling, tightness of
joints, nose bleeding without any reason. Hemophilia can be categorized into mild, moderate and severe
categories.
• Abnormal bleeding occurs in response to injury/trauma or surgery in patients with mild
hemophilia
• Abnormal bleeding occurs in response to injury or surgery in moderate hemophilia

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 • Abnormal bleeding occurs spontaneously and at an early age in severe hemophilia
• Frequency of occurrence hemophilia A and B is 1/5000 individuals

Late complications: Can develop in patients with hemophilia A &B

• Joint destruction due to hemarthrosis (bleeding in joint spaces) often leads to a number of
orthopedic abnormalities
• Development of inhibitor antibodies: Patients with severe hemophilia are often supplemented
with infusion of coagulation factors VIII or IX. 30% of such patients over time often develop
inhibitor antibodies against these coagulation factors which destroys the infused coagulation
factors (antigen-antibody reaction) making the specific treatment ineffective.

Physiological Regulators of Coagulation

A. Protein C, Protein S and thrombomodulin

B. Antithrombin III (serpin)

A. Role of protein C, S & thrombomodulin in regulation of hemostasis

Protein C:
Protein C is a 62-kD, trypsin serine protease enzyme synthesized in the liver. It circulates in the blood
as an inactive zymogen at a concentration of 4 μg/mL. It helps to turn off fibrin production by
inactivating factors VIIIa and Va and thus serves as a major regulator of coagulation.

Mechanism of action of protein C:

• Thrombin activates protein C (PC) to its active form aPC).
• Thrombin binds to the proteoglycan thrombomodulin, an integral component of the
endothelial membrane and cleaves the activation peptide of PC thus activating it to aPC.
• aPC with its cofactor, protein S (PS) converts factors Va and VIIIa into their inactive zymogens
preventing formation of the prothrombinase and tenase complexes of the intrinsic and extrinsic
cascades
• The net effect is inhibition of the large scale activation of factor X, termination of fibrin
production thus turning off the coagulation cascade

355
Fig: Mechanism of action of protein C (PC)

Deficiency of protein C

• Disturbs delicate balance between pro-coagulants and anticoagulants in blood

• Protein C deficiency may lead to thrombotic disorders

B. Regulation of Coagulation by Antithrombin III and Heparin

Antithrombin III (serpin):

Another important physiological regulator of the coagulation cascade. Antithrombin is a plasma
glycoprotein synthesized by the liver. It is capable of neutralizing thrombin, the role player in the process
of hemostasis and thus turns off the hemostatic mechanism. Antithrombin has arginine containing active
site which binds to serine containing site of thrombin neutralizing it. It can also directly inhibit the activity
of factors IXa, Xa, XIa, XIIa, plasmin and kallikrein.

Heparan sulphate present on the surface of blood vessel endothelium serves as a physiological
activator of antithrombin III. It potentiates the activity of antithrombin III by binding to lysyl residues
on antithrombin/ serpin, inducing allosteric alteration in its structure and improving its interaction with
thrombin.

The mechanism of action of heparin as an anticoagulant is similar to the mechanism of action of

antithrombin & heparin sulphate.

356
Fig: Mechanism of action of heparin

Heparin: Mimics antithrombin III; blocks activation of several factors involved in the intrinsic cascade
turning the cascade off; it prevents formation of Xa & thrombin and inactivates any available thrombin
& Xa

Note: Heparin injection is not used to dissolve existing clots but to prevent formation of future clots
and also to prevent existing clots from increasing in size. Heparin is used for short term
anticoagulant therapy.

Role of vitamin K in blood coagulation

• Vit K is a fat soluble vitamin that serves as a cofactor of the enzyme gamma glutamyl carboxylase
(GGC), produced by the liver.

• GGC is involved in post translational modification and thus activation of coagulation factors II,
VII, IX, X.

• GGC catalyzes carboxylation of some glutamic acid residues (gamma carboxylation) on these
coagulation factor proteins enabling their binding to Ca+2 and membrane phospholipids of
platelets (a requirement for functionality of these factors).

• Vit K acts as a cofactor of GGC in this gamma carboxylation process and as a consequence gets
oxidized from vit K (hydroquinone) into vit K -2,3 epoxide which is an inactive form

357
 • The enzyme, Vit K epoxide reductase (VKOR), reduces Vit K epoxide back into the active form (Vit
K hydroquinone) so that it can be reused for promoting further gamma carboxylation.

Note: Long term use of antibiotics to get rid of stomach infections reduce intestinal bacteria. This
might reduce Vit K levels in the body which might slow down the hemostatic cascade leading to
bleeding disorders

Fig: Role of Vitamin K in mediating hemostasis

NON PHYSIOLOGICAL/THERAPEUTIC REGULATORS OF COAGULATION

• Calcium chelators (for in vitro use only): EDTA (ethylene diaminetetra acetic acid),
citrate/oxaloacetate are commonly used. They chelate Ca+2 lowering its availability in blood
and hindering the activity of certain coagulation factors; lack of Ca+2 prevents formation of
tenase/ prothrombinase complexes. They are added to blood samples in diagnostic
laboratories to inhibit coagulation of the blood

• Heparin: Available as sodium/potassium/lithium/ammonium salts Heparin injections as

anticoagulants are administered to patients with arterial /venous thrombosis to prevent
development of future clots. It is considered as a short term therapy. They are not administered
to dissolve the existing clots.

• Warfarin ( Coumadin): Prescribed to patients who have a high risk of thrombosis (clotting);
prevents development of future clots and lowers the chances of embolism (occurs when part of
a blood clot breaks off and blocks the blood supply to one of the major organs). It is used as a
long term anticoagulant therapy to prevent formation of clots in major organs.

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 Mechanism of action of Warfarin

Warfarin acts as an anticoagulant by depleting vit K reserves in our body. It prohibits the action
of Vit K epoxide reductase (VKOR) and thus the conversion of Vit K epoxide back to Vit K
hydroquinone (active form). Vit K cannot be reused which results in gradual depletion of available
stores of Vit K. Functional impairment of clotting factors occurs due to under-decarboxylation by
Vit K which slows down or impairs the coagulation cascade of the patient with thrombotic
disorder.

Fig: Mechanism of action of Warfarin

Note: Some rodenticides (have active anticoagulants like warfarin, bromodiolone, difethiolone
etc) act by inhibiting coagulatory cascades in rodents.

High levels of rodenticides in blood of humans (due to careless handling or ingestion of such
pesticides) may be life threatening as they have the same effect on VKOR and thus deplete Vit K
levels.

CLINICAL CORRELATES

• Coagulation factors are synthesized in the liver. Severe liver diseases lower production of the
clotting factors and thrombocytes (liver & kidneys produced a glycoprotein hormone thrombopoietin
which stimulates production of platelets from megakaryocytes which are bone marrow cells). This might
lead to thrombocytopenia and slowing of the coagulation cascades. The result may be
hemorrhagic tendencies.

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 • Vit K deficiency (due to malabsorption) or impaired Vit K metabolism and thus partial/total non-
carboxylation of the blood factors II, VII, IX and X This leads to delayed clotting and
uncontrolled internal hemorrhage (bleeding).

• Disseminated intravascular coagulopathy (DIC): Pathological process that occurs due to

dysregulation between coagulation and fibrinolysis. Leads to widespread activation of the blood
coagulation cascades that results in formation of small clots in blood vessels throughout the
body. Long term effects are multiple organ damage (due to compromised blood supply to the
organs) and low levels of coagulation factors in blood (due to over consumption) which may lead
to severe bleeding problems. DIC generally occur secondary to other diseases.

TESTS USED FOR DETERMINING THE EFFICIENCY OF BLOOD COAGULATION

I. Prothrombin Time (PT):

• A test that measures efficiency of the extrinsic and common cascades; detects deficiencies in
clotting factors namely I,II, V, VII,X, and fibrinogen

• Ordering PT: For patients with symptoms of bleeding disorders; before surgical procedures; to
monitor effectiveness of anticoagulant therapies (warfarin therapy)

• Normal value of PT: 11-15 sec

• Prolonged PT: May be a consequence of liver disease, Vit K deficiency, vWD, hemophilia or
administration of blood thinners

Fig: Procedure of PT test

II. Partial Thromboplastin Time (PTT):

• Measures efficiency of intrinsic and common cascades of coagulation.

• Detects deficiencies of factors XII, XI, IX, VIII, pre-kellikrein

• Ordering PTT: For patients with unexplained bleeding/bruising, thromboembolism; symptoms

of chronic liver diseases; when switching anticoagulant therapies in patients (heparin to
warfarin)

360
 • Normal value of PTT: 25-37 sec

• High PTT: May be indicative of 30-40% deficiency of the above mentioned clotting factors;
vWD; bleeding disorders (hemophilia); liver disease; Vit K deficiency; presence of lupus
anticoagulant

Fig: Procedure of PTT test

III. Bleeding Time:

• Standard test used to assess platelet function.

• Procedure: Standard incision is made in the finger tip of the patient. The time taken to stop
bleeding (which is the time taken for formation of platelet plug) is recorded as the bleeding
time.
• Normal value: 2-5 min (Duke’s method); 3-9 min (Ivy method)
• Prolonged bleeding time: Indicative of thrombocytopenia, platelet dysfunction, blood vessel
defect, platelet aggregation defect, Von Willibrand’s disease

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Lecture 25- Molecular Biochemical Endocrinology
Learning Objectives:

• Concentrate on synthesis & secretion of

protein/peptide/amine hormones from different endocrine
organs
• Focus principally on the clinical correlates related to the
synthesis/secretion of these hormones
• Emphasize on the general pathway of synthesis of steroid
hormone from cholesterol
• Learn why & how specific zones of the adrenal cortex
synthesizes specific cortical steroids
• Focus on the clinical correlates like
-Classical/non-classical CAH
-PCOS
• Differentiate between steroid biosynthesis in the ovarian,
testicular & adrenal cortical cells
ENDOCRINOLOGY

The central theme of endocrinology is mainly centered on hormones. It relates to

• Synthesis & secretion of hormones
• Physiological action of hormones
• Feedback effect of hormones
• Inactivation and degradation of hormones

Definition of hormones

Chemical substances produced in minute quantities by endocrine (ductless) glands in our body. They
are released in blood & carried via blood to their target organs where they initiate receptor mediated
functions.

362
Fig: Release of hormones by endocrine cells into the blood

Properties of hormones

• May be amines/peptide/proteins/glycoprotein/steroid in nature; they circulate in the plasma in

very low concentrations
• Rate of hormone production by endocrine glands are controlled by homeostatic feedback
mechanisms
• Receptor mediated function at the level of target cells; receptors may be bound to plasma
membrane/nuclear membrane/cytoplasmic membrane of the target cells

• Duration of physiological effect depends on rate of binding to transport proteins, inactivation of

the hormones, & rate of elimination

Fig: Receptor mediated action of hormones

363
Hormones produced by different endocrine glands

GLANDS HORMONES PRODUCED & THEIR NATURE

Hypothalamus Oxytocin-Peptide
Antidiuretic hormone (ADH)-Peptide
Releasing & inhibiting factors (acts on pituitary)
Pituitary Anterior Posterior
1. Anterior Growth hormone (GH) -Protein Oxytocin
(adenohypophysis) Prolactin-Protein ADH
2. Posterior Follicle stimulating hormone (FSH)-Glycoprotein
(neurohypophysis) Luteinizing hormone (LH)- Glycoprotein
Thyroid stimulating hormone (TSH)- Glycoprotein
Adenocorticotrophic hormone (ACTH)-Peptide
Melanocyte stimulating hormone (MSH)-Peptide

Thyroid T3 (Triiodothyronine)-Amine
T4 (Thyroxine)- Amine
Calcitonin- Peptide
Parathyroid Parathyroid hormones- Peptide

Adrenal Medulla Cortex (steroids)

1. Medulla Epinephrine -Amine Glucocorticoids
2. Cortex Norepinephrine-Amine Mineralocorticoids
Sex corticoids
Pancreas Insulin - Protein
Glucagon- Protein
Somatostatin- Peptide
Gonads Testes Ovary
1. Testes (male) Teatosterone- Steroid Estrogen - Steroid
2. Ovary (female) Progesterone- Steroid
Pineal Melatonin- Amine
Thymus Thymosin- Peptide

364
 Fig: Different endocrine glands

ENDOCRINE AXES
Much of the endocrine system is organized into endocrine axes. Each axis involves the hypothalamus,
pituitary and the peripheral endocrine glands. The endocrine axes are driven by feedback loops, and
physiologically driven negative feedback loops.

Feedback loops: The predominant mode of a closed feedback loop among endocrine glands is
endocrine driven negative feedback loop. In a negative-feedback loop, "hormone A" acts on one or
more target organs to induce a change (either a decrease or increase) in circulating levels of
"component B," and the change in component B then inhibits secretion of hormone A. Negative-
feedback loops confer stability by keeping a physiological parameter (e.g., blood glucose) within a
normal range.

Physiological response-driven feedback loop: In the response-driven configuration, secretion of a

hormone is stimulated or inhibited by a change in the level of a specific extracellular parameter (e.g.,
an increase in blood glucose stimulates insulin secretion). Altered hormone levels lead to changes in
the physiology of target organs (e.g., decreased hepatic gluconeogenesis, increased uptake of glucose
by muscle) that directly regulate the parameter (i.e., blood glucose) in question. The change in the
parameter (i.e., decreased blood glucose) then inhibits further secretion of the hormone (i.e., insulin
secretion drops as blood glucose falls).

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SYNTHESIS OF HORMONES

Peptide/Protein hormones

• Protein/ peptide hormones require transcription of specific genes followed by post

transcriptional modifications (excision of introns)
• Translation of the mRNA occurs in the ribosomes of the rough endoplasmic reticulum (RER)
• Post-translational modifications (conversion of a pro-hormone to active hormone) occurs in the
golgi
• Stored in secretory vesicles and released when necessary

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A. Synthesis & Secretion of Posterior Pituitary Hormones
• The hypothalamus and the posterior pituitary (neurohypophysis) form a neurosecretory
system composed of neurosecretory cells
• The cell bodies of these neurosecretory cells are located in well-defined clusters (called nuclei)
in certain parts of the hypothalamus (paraventricular nuclei PVN, supraoptic nuclei SON)
• The axons of the neurosecretory cells terminate on the capillaries in the posterior pituitary
• Oxytocin and vasopressin which are hormones secreted from the posterior pituitary are
synthesized in the cell body of the neurosecretory cells and thus in the hypothalamus Both the
hormones are released from the posterior pituitary or neurohypophysis in response to specific
stimulation

Oxytocin-Synthesis & Release

• Hypothalamic magnocellular neurons of the PVN, SON synthesize inactive form of oxytocin
called pro-oxyphysin (109 amino acid long) in response to sensory input like stretch.
• Pro-oxyphysin (oxytocin + neurophysin-1, a carrier protein) undergoes structural modification
(fragmentation/disulphide bond formation) in the RER and golgi, & packaged into secretory
vesicles
• As the vesicles travel down the axon of the magnocellular neurons to the neurophypophysis,
mature oxytocin (a nonapeptide) is separated neurophysin-1.
• Opening of voltage gated Ca+2 channels, influx of Ca+2 & subsequent membrane depolarization
at the axon terminals (in response to the sensory input) releases oxytocin from
neurophypophysis into the capillary beds to be carried to the target organs (uterus mammary
glands etc) via blood

Fig: Synthesis release and action of oxytocin on its target organs

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Vasopression or antidiuretic hormone (ADH) - Synthesis and release

• Synthesized as pre-prohormone in the cell bodies of hypothalamic SON neurons

• Processed into active nonapeptide (9 amino acids) & stored in the post. pituitary; high plasma
osmolality (water deprivation) stimulates ADH release in blood
• Antidiuresis: ADH binds to cellular receptors on the distal convoluted tubule & collecting duct of
kidneys; induces synthesis of aquaporin channels increasing cell permeability to water;
facilitates tubular reabsorption of water
• ADH maintains water content and thus tonicity of body fluids.

Fig: ADH synthesis, release and action Fig: Polydipsia associated with DI

CLINICAL CORRELATE: Diabetes insipidus (DI)

DI is of 2 types

1. Central diabetes insipidus: Lack of ADH production/secretion by hypothalamus or pituitary

Causes: Head injury, infections, loss of blood supply to the gland, surgery, tumor.

2. Nephrogenic diabetes insipidus: Failure of the kidneys to respond to ADH.

Causes: Use of specific drugs, hypercalcemia, kidney disease

Symptoms of DI: Excessive thirst (polydipsia), excessive urination (polyuria)

B. Synthesis & secretion of pancreatic hormones

INSULIN

• Insulin is a polypeptide secreted by the pancreatic β cells; the gene that encodes insulin is
located in the p arm of chromosome 11
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• Synthesized as a pre-proinsulin (inactive) in the ribosomes of RER of β cells; removal of the
N terminal signal sequence converts pre-proinsulin to pro-insulin (inactive) in the lumen of
the RER
• Pro-insulin attains proper conformation (folding through interchain disulphide bonds
formation) & is transported to the golgi
• The pro-insulin is released from golgi complex into the cytoplasm in storage vesicles where
protease enzyme cleaves the additional C chain of pro-insulin
• The active insulin is a dimer, composed of A & B chains (51 amino acids) held together by
two interchain disulphide bonds (the B chain has an additional intrachain disulphide bond).
It is stored in cytoplasmic storage vesicles in β cells

Insulin Secretion: High plasma glucose concentration is the primary stimulus in human

• Glucose enters β cells through GLUT2 transporters (expressed on β cell membrane)

• Glucose undergoes phosphorylation by glucokinase enzyme, enters glycolysis/TCA cycle
within the β cells
• Results in high intracellular ATP/ADP ratio leading to closure of ATP sensitive K+ channels,
membrane depolarization & subsequent opening of voltage gated Ca+2 channels
• Elevated Ca+2 influx into β cells is responsible for activation of exocytosis of insulin from the
storage vesicles within the β cells into the blood vessels surrounding these cell

Fig: Insulin synthesis and secretion by pancreatic beta cells

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 Fig: Mechanism of insulin release

CLINICAL CORRELATES

i. Diabetes Type I:

Type 1A: Results from autoimmune destruction of insulin producing beta cells in the islets; Occurs in
genetically susceptible subjects; may be triggered by environmental agents.

C-peptide: Cleaved off during maturation of insulin and secreted in equimolar amounts as insulin in
blood. Helps to determine the functionality of beta cells. It helps to access a person’s endogenous
insulin producing capability. C peptide levels in a patient with type 1A diabetes, will be low and thus
indicative of the small size of the functional pancreatic beta cell population in the patient. C peptide
levels in patients with type II diabetes will be normal to high as Type II is associated with insulin
insensitivity anf high levels of insulin in blood

Type 1B: Does not involve autoimmune destruction of β cells; β cells are non-responsive and does not
produce less insulin; idiopathic (cause unknown) in nature; 5-10% in Caucasian population

Note: C peptide levels in patients with type 1B will be very low to absent as beta cells (although
present) are non-responsive and thus not making/ releasing insulin

ii. Maturity Onset Diabetes of the Young (MODY)

Patients have a mutation in the gene that produces pancreatic enzyme glucokinase. It is a genetic
condition (autosomal dominant). Patients produce glucokinase with reduced activity and much higher
k m . Characterized by early onset age (<25 years). Metabolism of glucose in β cells is affected, &

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secretion of insulin occurs only in response to much higher glucose levels than normal individuals;
MODY can occur due to mutations in other genes (HNF)

iii. Neonatal Diabetes:

Inherited disorder that develops within 3 months of birth; may be transient or permanent; permanent
form occurs due to a mutation in the KCNJ11 gene which encodes for a subunit of the K ATP channel in
many tissues including the pancreas. The mutant K ATP channel fails to close in response to high
ATP/ADP ration in the β cells. This prevents depolarization and blocks subsequent activation and
opening of Ca+2channels and Ca+2 influx in the β cells .The result is inhibition of insulin release from the
vesicle into blood.

C. Synthesis & secretion of thyroid hormones

Role of TSH

Synthesis of the thyroid hormones is under the control of the hypothalamo-pituitary-thyroid axis.
Thyroid stimulating hormone (TSH) released from pituitary (in response to hypothalamic TRH)
regulates

• Synthesis of specific proteins required for thyroid hormone synthesis, storage & release
• Uptake of iodide into the thyroid gland
• Uptake of nutrients by the thyroid gland

Thyroglobulin: A glycoprotein synthesized in the ribosomes attached to the rough endoplasmic

reticulum (RER) in the follicular epithelial cells; secreted into the lumen of the follicles where it is
stored as colloid. Thyroglobulin is composed of multiple tyrosine residues.

Fig: Thyroid follicles

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The tyrosine residues required for thyroid hormone synthesis is supplied by thyroglobulin. Iodination
of tyrosine residues & coupling of iodotyrosine residues lead to production of active thyroid hormones
T3 (triiodothyronine) and T4 (thyroxine)

Iodide uptake by thyroid

Iodide (I-) required for thyroid hormone synthesis is readily absorbed from blood; dietary sources
(iodized salt, seafood) supply the iodide required for the synthesis of thyroid hormone. I- enters the
thyroid epithelial cells through a Na+/I- symporter (NIS) located in the basolateral membrane of the
follicular epithelial cells. I- is released into the colloid through a chloride/iodide transport protein
(pendrin) present at the apical membrane of the epithelial cells and oxidized into I+ (iodine) by the
enzyme thyroid peroxidase (TPO) in presence of H 2 O 2

Organification of Tg

Occurs in the follicular lumen. I+ binds to C3/C5 of the tyrosine residues of Tg forming
monoiodotyrosine (MIT) or diiodotyrosine (DIT) residues followed by coupling of MIT and DIT residues.
These iodotyrosine residues are stored in the lumen attached to thyroglobulin until a stimulus is
received for secretion into blood as mature hormones.

Fig: Transport of iodide into the follicular epithelium and organification of tyrosine residues

Release of thyroid hormones

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TSH regulates the release of thyroid hormones from the thyroid gland into the blood. As T3 & T4 are
synthesized in the colloidal space which has no direct access to the bloodstream, release of T3 & T4
involve endocytic uptake of the hormones in vesicles back into the thyroid epithelial cells at the apical
surface. Proteolytic digestion and cleavage of Tg containing MIT & DIT occur when vesicles fuse with
phagolysosomes. This leads to formation of mature T3 and T4 which are then released into circulation
through the basolateral membrane of the follicular epithelium.

CLINICAL CORRELATES

i. Radioactive iodine therapy

Radioactive iodine I131 is administered to patients with certain types of thyroid cancer. As the thyroid
gland absorbs almost all the iodine present in the blood, the therapeutic I131 concentrates in the
thyroid gland. The β rays emitted from I131 destroy cancerous thyroid cells and abnormal growths in
the thyroid

ii. Congenital Hypothyroidism (CH)

• Common metabolic-endocrine disorder; occurs in 1 out of 2000-4000 newborns

• 15-20% CH cases occur due to defects in T3/T4 biosynthesis (dyshormonogenesis)
• General characteristic is enlargement of the thyroid gland (goiter)

Causes of congenital hypothyroidism

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AR=Autosomal Recessive; AD= Autosomal Dominant

Note: Iodine deficiency is the most common cause of hypothyroidism related goiter

D. Synthesis and secretion of adrenal medullary hormones

Adrenal medulla: Central, extremely vascular part of the adrenal gland is called the medulla. It is
composed of cells called phoechromocytes/chromafin cells which are modified neuronal cells. These
cells synthesize & secrete 2 hormones namely epinephrine & norepinephrine, collectively called
catecholamines. Epinephrine is produced in large amounts, norepinephrine & dopamine to a lesser
extent, in response to sympathetic neuronal stimulation.

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Synthesis of catecholamines:

Catecholamines are hormones derived from amino acid tyrosine. Synthesis involves 4 cytosolic steps

• Hydroxylation of tyrosine to L-dihydrophenylalanine (L-DOPA). Enzyme that catalyzes the

step is tyrosine hydroxylase
• Decarboxylation of L-DOPA to dopamine. Enzyme involved is DOPA-decarboxylase
• Hydroxylation of dopamine to norepinephrine. Enzyme involved is dopamine β-hydroxylase
• Methylation of norepinephrine to epinephrine. Enzyme involved is phenylethanolamine N-
methyltransferase

Fig: Synthesis of catecholamines

Release of catecholamines:

Release occurs in response to sympathetic nerve stimulation of the adrenal medulla. Binding of
acetylcholine (released by sympathetic nerve terminals) to cholinergic receptors on the surface of the
chromaffin cells leads to depolarization of the cells. This activates voltage gated Ca+2 channels leading
to Ca+2 influx. High Ca+2 conc. induce exocytosis of catecholamines from the secretory vesicles
underlying the synaptic membranes. 50-60% of the hormones binds to albumin with low affinity in
circulation; have short half-life of 10 sec-1.7 min. They exert their physiological effects through G-
protein coupled receptors at target cells.

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Fig: Release of catecholamines

CLINICAL CORRELATE

Pheochromocytoma:
Tumors in the adrenal medullary cells. The condition leads to overproduction of catecholamines.
Tumors can develop at any age

Symptoms: Sustained hypertension, sweating, headache, palpitation, high blood pressure; can be life
threatening, high concentration of VMA (vanylmandelic acid), a product produced by breakdown of
catecholamines, in the urine of the patient

STEROID HORMONES - BIOSYNTHESIS & SECRETION

General characteristics associated with biosynthesis of steroid hormones

Unlike protein hormones which are produced as gene products, steroid hormones are produced by
modification of cholesterol in specific cells; Cholesterol can be synthesized from acetyl CoA or obtained
from high density lipoprotein particles (LDL) present in blood

In the first step of the process, cholesterol present in the cytoplasm of a steroid hormone- producing
cell (adrenal, ovarian or testicular cells) is carried into the mitochondrial matrix by steroid Acute
Regulatory Protein (STAR).

Cytochrome P 450 scc enzyme (CYP11A1) present in the inner mitochondrial membrane then converts the
cholesterol into pregnenolone which is released into the cytoplasm.

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Conversion of cholesterol to pregnenolone in the inner mitochondrial matrix is the rate limiting step
in the process of steroid hormone biosynthesis.

Cells lacking STAR/CYP11A1 are incapable of steroid hormone biosynthesis; deficiency of these
enzymes in the steroid producing cells leads to different disorders.

Fig: Structure of cholesterol

Secretion of steroid hormones: Steroid hormones are synthesized in response to specific stimulus and
being lypophilic they diffuse through the plasma membrane in to the blood post synthesis. Unlike
protein or peptide hormones they are not stored in the cells synthesizing them.

General pathway of steroid hormone biosynthesis

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 A. BIOSYNTHESIS AND SECRETION OF ADRENAL STEROIDS

Adrenal cortex: Composed of 3 distinct zones producing 3 distinct categories of steroid hormones
from cholesterol. Synthesis of adrenal hormones are under the control of pituitary secretion of
ACTH( adrenal corticosteroid hormone). Secretion of pituitary ACTH is pulsatile (high in the
morning)

• Zona glomerulosa: Produces mineralocorticoid, aldosterone

• Zona fasiculata: Produces principally glucocorticoids like cortisol & small quantities of
adrenal androgens
• Zona reticularis: Produces principally adrenal androgen & small quantities of
glucocorticoids. DHEA is the principle adrenal steroid produced.

Compartmentalization of steroid hormone secretion by different regions of adrenal cortex

• Cells in the zona glomerulosa: Lack the enzyme 17α-hydroxylase. Thus these cells can only
convert pregnenolone to progesterone. These cells also lack 11β-hydroxylase but possess
aldosterone synthase enzyme which converts 11-deoxycoticosterone to aldosterone.

• Cells of Zona fasciculata: Contain enzymes 17α-hydroxylase which convert progesterone to

17-OH-progesterone & via intermediate steps produce principally cortisol (the main
glucocorticoid produced by human adrenal cortex)
11β-hydroxylase is also present in these cells which converts deoxycorticosterone
producing little amount of corticosterone ( but not aldosterone). Presence of 17,20 lyase
activity in these cells helps in production of some adrenal androgens (DHEA,
androstenedione) as well.
• Cells of zona reticularis: contains 17α-hydroxylase and 17, 20 lyase which produces
principally DHEA and androstenedione from cholesterol. Testosterone is not produced in
these cells due to lack of 17 beta HSD.

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 Fig: Different layers of the adrenal medulla

CLINICAL CORRELATES

Congenital Adrenal Hyperplasia (CAH)

Autosomal recessive disorder; develops due to deficiency or absence in the enzyme 21-
hydroxylase caused by mutations in the gene CYP21A2 producing the enzyme. Cortisol,
aldosterone production stops; excess production of adrenal androgens by adrenal cortex
occurs.
Often significantly low blood cortisol levels by negative feedback effect stimulate excess
production of pit ACTH which further increases adrenal androgen production.

2 forms:

• Classical CAH: Most severe form; deficiency of 21-hydroxylase is presented during neonatal
period or early infancy;
• Non classical CAH: Late onset of 21-hydroxylase deficiency

Clinical Presentation of CAH

• Females with the classical form (neonatal onset): Ambiguous genitalia (clitoral enlargement,
labial fusion). High plasma levels of hydroxyprogesterone; mostly exhibit salt-losing
tendency (salt wasting)
• Females with non-classical CAH (late onset): Hirsutism (excess male-like facial hair growth)
& menstrual irregularity, acne, high plasma levels of DHEA
• Males with classical salt-losing form: Failure to thrive (due to severe dehydration,
hyponatremia & hyperkalemia). Death within 7 to 14 days of birth
• Males with the non-classic form (non-salt-losing): Early virilization (pubic hair, growth spurt,
adult body odor)

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 Hormone replacement therapy might be a treatment option for patients with classical forms of
CAH

Fig: Clitoromegaly in an infant with CAH ACTH stimulating production of adrenal androgens

B. Synthesis of testicular steroids: Testosterone

Synthesis of Testosterone
Leydig cells present in the interstitial spaces between the seminiferous tubules of the male testes
produce 95% of principle male androgen, testosterone from cholesterol synthesized from acetyl CoA or
acquired from LDL. They are rich in LH receptors and synthesize and secrete testosterone in response
to hypothalamic LH stimulation.

Synthesis involves 5 steps

• Conversion of cholesterol to pregnenolone: Occurs in the mitochondria and catalyzed by
P450scc (CYP11A1); rate limiting step induced by LH
• Hydroxylation of pregnenolone to 17α-OH-pregnenolone; Occurs in SER & catalyzed by 17α-
hydroxylase
• Production of dehydroepiandrosterone (DHEA) followed by androstenediol; Occurs in SER:
Catalyzed by 17, 20 lyase.
• Conversion of androstenediol to testosterone; catalyzed by 3 β-HSD
• Pregnenolone can also be converted to progesterone and then testosterone via
androstenedione catalyzed by 17 β-HSD

Note: Leydig cells can produce testosterone from cholesterol as they have 17 β-HSD (adrenal gland lack
this enzyme)

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Fig: Synthesis of testosterone from cholesterol in Leydig cells

C. Synthesis of ovarian steroids: Estrogen and progesterone

Histology of the ovarian follicles

• Oocyte: Immature ovum

• Granulosa cells: Cells surrounding the oocyte. These expresses FSH receptors
• Thecal cells: Thin layer of cells which encloses the granulosa cells and express LH receptors.
There are 2 layers of theca, the theca externa & theca interna.

Fig: Histology of ovarian follicle

• Synthesis of ovarian steroids begins in the thecal cells. Cholesterol is converted to

pregnenolone (Cyt P 450 scc or CYP11A1). Pregnenolone follows the same steps of general steroid
synthesis to produce DHEA & androstenedione. This occurs in response to LH stimulation.

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 • Granulosa cells produce aromatase in response to pituitary FSH. Androstenedione, diffuses
into the neighbouring granulosa cells where aromatase converts androstenedione into estrone
and then estradiol. Some amount of testosterone produced in the thecal cells also diffuse into
the granulosa cells to be converted into estradiol by aromatase.

• Prior to ovulation: Granulosa cells aromatize the thecal androgens into estrogen; a FSH
mediated activity.
• After ovulation: Granulosa cells turn into granulosa lutein cells which produce & secrete
progesterone into the bloodstream (LH mediated) from cholesterol via pregnenolone.

Fig: 2 cell theory of estrogen synthesis in the ovary

CLINICAL CORRELATES

Polycystic Ovarian Syndrome (PCOS)

i. Most common anovulatory infertility in female. Characterized by enlarged cystic ovaries.
Defective follicular maturation and ovulation, irregular periods, excessive hair growth, acne,
obesity. The patients are at risk of having high blood pressure, insulin resistant diabetes

Causes:

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• Elevated LH secretion from pituitary stimulates ovarian thecal cells to produce excess
androstenedione. Androstenedione is converted to testosterone (catalyzed by 17 beta
HSD) in the granulosa cells and not estradiol due to lack of aromatase P450 enzyme
(caused by insufficient FSH production). This increases total testosterone levels in blood
of the patients (underlying cause of hirsutism, acne, obesity) and decreases estradiol
production (cause of defective follicular maturation and anovulation).

• Decreased hepatic synthesis of SHBG (sex hormone binding globulin; binds to

testosterone in circulation and decreases into bioavailability) increases bioavailability of
testosterone or circulating androgens.

• Some of the circulating testosterone is converted to DHT (dihydrotestosterone, the

most potent form of intracellular testosterone) in target tissues

• Hyper-insulinemia may be one of the underlying causes of high androgen production.

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