Compiled Metabolism

LECTURE NOTES ON METABOLISM
OUTLINE
DIGESTION AND ASORPTION OF FOOD
METABOLISM OF CARBOHYDRATES
LIPID METABOLISM
PROTEIN METABOLISM I
NUCLEOTIDE METABOLISM
MOLECULAR BIOLOGY TOOLS
COMPILED, EDITED AND

ARRANGED
BY
OLUKA AUSTEN (AUSTEN-BEST)
NB: THIS COMPILATION DOES NOT INCLUDE THE FOLLOWING;
PROTEIN AND AMINO ACID METAB II (WHICH DOES NOT HAVE A SOFT COPY) AND
NUCLEIC ACID METBOLISM (WHICH IS ON A SEPERATE PDF)
1|Page
COMPILED AND ARRANGED BY AUSTEN-BEST
STUDY LIKE THERE IS NO TOMORROW BECAUSE IF YOU KEEP

PUTTING OFF YOUR STUDIES FOR TOMORROW, YOU WILL
PROBABLY BE TOO LATE.
OLUKA AUSTEN (AUSTEN-BEST)
2|Page
DIGESTION AND ABSORBTION OF FOOD

BY
Prof I E Ezeagu
Department of Medical Biochemistry
UNEC
DIGESTION OF FOOD:
Definition:
Many of the inorganic components of food are in the form of large insoluble molecules
which have to be broken down into simpler compounds before they can pass through the
mucous membrane of the alimentary canal into the blood and lymph.
breaking- down process is termed ‘digestion’,

passage of the digested nutrients through the mucous membrane ‘absorption’
Alimentary Canal
The alimentary canal is a tube extending from mouth to anus. It islined with mucous
membrane. Its function is the ingestion, comminution, digestion and absorption of food
and elimination of solid waste material.
• The various parts are:
mouth,
pharynx,
esophagus,
stomach,
small and
Large intestine.
Digestion in the mouth

Mainly mechanical: Mastication
Mix food with saliva which acts as a lubricant.
Saliva is secreted into the mouth by three pairs of salivary glands:
the parotids, which are sited in front of each ear,
the sub-maxillary glands, which lie on each side of the lower jaw;
and the sublingual glands, which are underneath the tongue.
Saliva is about 99% water, the remaining 1% consisting of mucin, inorganic salts
and other enzymes – α-amylase and the complex lysozyme
3|Page
Ptyalin
Hydrolyses starch /glycogen to maltose and dextrins at the α-1,4-glycosidic bonds to

form maltose. Starch and glycogen require another enzyme to hydrolyse the α-1,6-
glycosidic bonds to ensure full hydrolysis. Ptyalin seems to have a stronger action on
cooked starch than on raw carbohydrates but this only occurs in vitro. In the mouth there is
usually no difference in its action. Some anions acts as activation of ptyalin e.g: Cl-, Br-, I-,
NO- PO43-. Cl- and HCO-3Its activity is decreased by low pH
Salivary lipase
This is a fat digesting enzyme, salivary (lingual) lipase. Hydrolyses about 30% of
total oil (fat) ingested. Also hydrolyses dietary triacylglycerols (triglycerides) (TAG) to
glycerol and fatty acids or 2’monoacyglycerol.
DIGESTION IN THE STOMACH

Gastric juices:
Presence of
enzyme precursor:- pepsinogen and in small quantities pro-rennin and lipase

Free HCl
Inorganic chloride
lactic acid
phosphates
HCl
Produced by the oxyntic cells
Activates pepsinogen to active enzyme, pepsin and the pro-rennin to yield rennin
Lowers pH to 1.5-2.0
Hydrolysis of sucrose to glucose and fructose
Splits the nucleoproteins of the food into nucleic acid and protein
Pepsin
An endopeptidase
Hydrolysis of proteins to peptones and some few amino acids
Pepsin attacks the peptide bonds at the phenyl group (NH3 of tyrosine and phenylalanine)
of amino acids to yield phenolic acids
It activates other peptidases such as trypsinogen to trypsin, chemotrypsinogen to chymotrypsin -
- to their active states
Rennin
Precursor pro-rennin activated by intestinal enterokinsase
Found in suckling mammals
Clots or curdles milk
4|Page
Conversion of milk protein (soluble caseinogen) by H+ to the insoluble Ca salt of casein in

the presence of Ca2+ ions so forming curd.
DIGESTION IN THE DUODENUM

Pancreatic juice secreted by the pancrease; produced by the acini cells of the pancrease
Contains trypsin as trypsinogen (zymogen)
Zymogen: Secreted in its inactive form trypsinogen to prevent auto-digestion since

it acts on protein molecules – to avoid autocatalysis- attacking the pancrease.
Trypsinogen is activated to the enzyme trypsin by enterokinase
Trypsin:
An endopeptidase and a serine protease.
It has the amino acid aspartate at its active site,
Attacks all proteins:
Preferentially action on peptide linkages involving the carboxyl group of either basic
amino acids - lysine (and/or arginine) and phenylalanine or tyrosin) residues.
Involved in the activation of many digestive enzymes from their inactive to their active state:
Proelastase to elastase,
Chymotrypsinogen to chymotrypsin,
Procarboxylase to carboxylase.
Its pH is about 7.5 – 8.8 due to the presence of NaHCO3.

Chymotrypsin:
An endopeptidase, preferentially acting on the carboxyl linkages of phenylalanine or
tyrosine,
specific for peptide bonds containing uncharged amino acid residues such as aromatic amino acids
Activated by active trypsin
Elastase:
Has broad specificity
Attacks bonds next to small amino acid residues such as glycine, alanine and serine.
Hydrolyses fibrous proteins
5|Page
Collagenase
hydrolyzes collagen
Pancreatic Amylase – amylopsin
hydrolyzes branching polysaccs to form maltose and higher oligosaccs containing α-1,4-
linkages, isomaltose (α -1,6-linkages) and small amounts of glucose.
The amylase finishes the work begun by ptyalin and thus all starch and glycogen are
converted to maltose.
Note:α -1,6 branches requires further hydrolysis by α-dextrinase or isomaltase

Pancreatic Lipase
glyceride hydrolyzing enzyme otherwise called carboxyesterase
hydrolyzes fats to fatty acids and glycerol
Hydrolyzing activity increases with:
Mol.wt. of the component fatty acid

Extent of unsaturation
The number of fatty acids in the molecule of the glyceride
Quantity of the ingested fat, its nature, pH and motility of the bowel
The resulting mixture usually consists of undigested fat (TAGs),
diglycerides, monoglycerides.
Bile
Secreted by the liver, stored in the gall bladder;
Contains a number of bile acids:- Sodium glycocholate, sodium taurocholate, also: bile
pigments, bilirubin and biliverdin, some mucin, cholesterol.
Bile acids are end products of cholesterol breakdown and thus a major rout of
elimination of cholesterol from the body, via the feces.
The bile salts reduces the surface tension at fat-water interfaces,
Makes emulsification of fats a much simpler process- enabling lipase to work more rapidly.
If fat droplets are small enough, they can be absorbed directly.
Clinical Significance of Bile Acids

Their synthesis and subsequent excretion in the faeces represent the only significant
mechanism for the elimination of excess cholesterol.
Bile acids and phospholipids solubilize cholesterol in the bile, thereby preventing the
precipitation of cholesterol in the gall bladder.
6|Page
They facilitate the digestion of dietary TAG by acting as emulsifying agents that render fats
accessible to pancreatic lipase.
They facilitate the intestinal absorption of fat soluble vitamins
DIGESTION IN THE SMALL INTESTINE

The intestinal juice secreted by the glands of Brunner and Lieberkūhn
Contains Digestion-Completing Enzymes:.

The major site of CHO digestion is in the Small intestine,
Another α-amylase –like enzyme called amylopsin cleave the remainder of the α-1,4-
glucosidic linkages
Any residual α-1,6-glucosidic linkages are split by an α-1,6-glucosidase, which also is active in the
small intestine.
Carboxypeptidase: an exopeptidase , attacks the terminal amino acids possessing the free
carboxyl group; thus resulting in the stepwise liberation of amino acids from the carboxyl
end of protein molecule
Aminopeptidase : an exopeptidase, attack peptide bonds at the terminal amino acids of

polypeptides and oligopeptides to releasing free amino acid.
The resulting dipeptides and tripeptides are hydrolysed by dipeptidases and

tripeptidases of various specificity, and completes digestion of proteins to free amino
acids.
Disaccharidases and oligosaccharidases: α-glucosidase (maltase) removes single glucose

residues from α - (1-4) linked oligosaccharides and disaccharides, starting from non-
reducing ends.
Lactase: lactose to glucose + galactose
Sucrase: hydrolyse sucrose to glucose + fructose
Maltase: maltose to glucose
Phospholipase:
Attacks phospholipids to produce glycerol, fatty acids, phosphoric acid, and bases such as
choline.
Lecithin: oleic, palmitic, choline & P
Cephalin: oleic, palmitic, aminoethanol
7|Page
Cephalin: oleic, palmitic, serine
Cholesterol Esterase; in the pancreatic juice hydrolyses cholesterol esters, bile acids as co-factors
Digestion of Nucleo-protein
Like polysaccharides and proteins, nucleic acids are high- mol wt. polymers made
up of monomeric units, nucleotides. Nucleic acids may be therefore be referred to also as
polynucleotides. These nucleotides may be ultimately hydrolyzed into simpler subunits.
A nitrogen-containing ring compound with basic properties,
A pentose sugar
Phosphoric acid.
An intermediate product of hydrolysis is nucleoside which is a combination of the N-base

with the pentose sugar. The nucleic acids DNA and RNA are hydrolysed by the
polynucleotidases – deoxyribonuclease (DNase) and ribonuclease (RNase) respectively.
The enzymes catalyze the cleavage of the ester bonds between the sugar and phosphoric
acid in the nucleic acids. The end products are the component nucleotides. Nucleosidases
attack the linkage between the sugar and nitrogenous bases, liberating the free purines and
pyrimidinesand the nucleosides.Phosphotases complete the hydrolysis of the nucleosides
by separating the orthophosphoric acid from the ribose or deoxyribose.
Final Products of Digestion

Carbohydrates: converted to monosaccharides: glucose, fructose ,galactose, pentoses, etc.
Proteins : amino acids
Fats and oil: fatty acids and glycerol
Nucleic acid: Phosphate, N-Bases Ribose/Deoxyribose
Digestion in the Large Intestine
In man no digestion takes place, except intestinal putrefaction and fermentation

Absorption of water takes place, and the semiliquid intestinal contents gradually
become more solid. During this period considerable bacterial activity occurs. By
fermentation/putrefaction, the bacteria release various gases:- CO2, methane, hydrogen,
nitrogen, and H2S as well as acetic, lactic and butyric acids
Some nutritional benefits is derived from bacterial activity in the synthesis of certain
vitamins, particularly vitamins K, B12 and possibly other members of the B- complex, which
are made available to the body.
(Refer Probiotic and Prebiotics in nutrition)
Defects in Digestion and Absorption

GIT diseases may cause decreased synthesis and release of secretions needed for
nutrient digestion.
8|Page
Any deficiency in the enzymes for digestion of the carboseg. Disaccharidase

deficiency, will lead to the presence of osmotically active substances in the
GIT which causes diarrhoea and other diseases
Lactase Deficiency (Lactose Intolerance)

Intolerance to lactose, the sugar of milk. This is difference from intolerance to
milk resulting from a sensitivity to milk protein, usually to the β-lactoglobulin
Signs include abdominal cramps, diarrhea and flatulence. They are attributed
to accumulation of lactose.
There are three types:

Inherited lactase deficiency: rare, symptoms develop very soon after birth. The feeding of a
lactose-free diet results in disappearance of the symptoms. Occurrence of lactose in the urine is
a prominent feature of this syndrome.
Secondary low-lactase activity – digestion of lactose is limited even in normal humans,
intolerance to milk is not uncommon as a consequence of intestinal disease, eg.,
kwashiorkor, colitis and gastroenteritis, tropical and non –tropical sprue.
Primary low-lactase activity: relatively common, particularly among non -whites, represents a
gradual decline in activity of lactase in susceptible individuals.
Sucrase Deficiency: Inherited deficiency of the disaccharidases,sucrase and isomaltase.

These two deficiencies coexist because sucrase and isomaltase occur together as a
complex enzyme. Symptoms occur in early childhood and are same as above.
Disacchariduria: increase in the excretion of disaccharides, observed in some patients

with disaccharidase deficiency. As much as 300mg or more of disaccharide may be
excreted in the urine of these people and in patients with intestinal damage (e.g. sprue – a
chronic malabsorption disorder associated with glossitis, indigestion, weakness anaemia
and steatorrhoea (passage of pale bulky greasy stool).
Inflammatory Bowel Diseases

ulcerative colitis and crohn’s disease (also called regional enteritis),
characterized by acute relapsing or chronic inflammation of various segments of the GIT,
especially intestines.
Chronic Pancreatitis
Inflammation of the pancrease due to long time excessive use of alcohol, gallstones, viral
infection, liver diseases;
pancreatic tissue becomes dysfunctional.
Gastroesophageal Reflux Disease
9|Page
Regorging of stomach content
10 | P a g e
Absorption of Digested Nutrients

Prof I.E. Ezeagu
Dept. of Medical Biochemistry
UNEC
Absorption of Digested Nutrients
The main organ in the monogastric mammal for absorption of dietary nutrients is the
small intestine. This part of the tract is specially adapted to absorption because its inner
surface area is increased by folding and the presence of villi.The primary function of the
bio-membrane is to allow movement of all compounds necessary for the normal
function of the cell across its membrane barrier.
Transport Systems
Absorption of a nutrient from the lumen of the intestine can take place at
least by 4 ways:
1 .Passive transport involving Simple diffusion:
Metabolites of low molecular weights move or diffuse across the membrane.
The process depends on a concentration gradient across the membrane and of course,
this gradient disappears as diffusion occurs.
Not an important mechanism of transport across bio-membranes since the rate is too
slow and no selectivity is permitted
Facilitated Diffusion:
Similar to simple diffusion, concentration gradient is required and does not involve an
expenditure of energy.
However, differs in that:
The membrane contains component called a carrier or permease which catalyzes
the process, that is, speeds up the rate of diffusion much more than is predicted
from simple diffusion
The diffusion is stereospecific, and
The rate of penetration of the metabolite approaches a limiting value with
increasing concentration on one side of the membrane.
Pinocytosis:
Also called “cell drinking” , in which cells have the capacity to engulf large
molecules in solution or suspension.
Such a process is particularly important in many newborn suckled mammals, in which

immunoglobulins present in colostrum are absorbed intact.
Also in the small intestine where cells engulf fat droplets
11 | P a g e
4. Active Transport
Similar to diffusion except that the metabolite moves across the membrane against a
concentration gradient which requires an expenditure of metabolic energy;
the metabolite moves from an area of low conc to one of higher conc.
There are 3 major types of active transport systems in animal tissues:
The Na+ and K+ pump which utilizes metabolic energy transport 3Na+ out of the
cell and 2K+ into it
Active-transport system for glucose and other sugars and

Active transport systems for amino acids.
Of these the Na+ and K+ pump appears to be most active and of universal
occurrence, it is present in the membrane of nearly all animal cells. Moreover, much
evidence suggests that Na+-K+ pump is also essential for the operation of the pumps for
glucose and amino acids.The Na+K+ ATPase pumps Na+ out of the cell to create an inward-
directed Na+ gradient at the expense of ATP. The inward-directed Na+ gradient so generated
“pulls” glucose into the cell by means of a passive carrier which has two binding sites, one
for Na+ and the other for glucose. (symport – transport of 2 mols in the same direction)
Absorption of Fat
Great majority of absorbed fat appears in the form of chylomicrons. Chylomicrons
are synthesized in the intestinal wall, and they contain largely triacylglycerol, cholesterol
(both free and esterified), phospholipid, Small (0.5%) but important amount of protein are
also present.In the form of mixed micelles, medium and short chain fatty acids can be
absorbed rapidly from the lumen of the intestine directly into the portal blood stream. The
entry of these fatty acids is Na+ dependent and takes place against a concentration gradient.
12 | P a g e
CARBOHYDRATE METABOLISM OVERVIEW

BIC 203
LECTURE 1
Learning Objectives:
By the end of this session you should be able to:

Outline the two stages of glycolysis.
Describe the steps of glycolysis between glucose and pyruvate and recognize all the
intermediates and enzymes and the cofactors that participate in the reactions.
Mention ATP-generating reactions.
Illustrate the regulation of glycolysis.
Carbohydrate Metabolism –Overview

Glucose is the major fuel of most organisms. Major pathways of CHO
metabolism either begin or end with glucose. Other monosaccharides can easily be
converted to glucose or its metabolic intermediates.Energy generation is a major
function of carbohydrate/glucose metabolism
Major Pathways of CHO Metabolism

CHO Metabolism in Mammalian Cells can be classified into:
Glycolysis: Oxidation of glucose to pyruvate (aerobic state) or lactate
(anaerobic state)
Krebs cycle orTricarboxylic Acid cycle: After oxidation of pyruvate to acetyl CoA,
acetyl CoA enters the Krebs cycle for the aim of production of ATP.
13 | P a g e
Hexose monophosphate shunt (HMP) :Enables cells to produce ribose-5-phosphate

and NADPH.
Glycogenesis: Synthesis of glycogen from glucose, when glucose levels are high.
Glycogenolysis: Degradation of glycogen to glucose when glucose in short supply.
Gluconeogenesis: Formation of glucose from noncarbohydrate sources.
Electron Transport Chain: The NADH and succinate generated in the citric acid cycle
are oxidized, providing energy to power ATP synthase in the mitochondrion.
Glycolysis: (Embden-Meyerhof Pathway)from the Greek glyk-, sweet, and

lysis, splitting
Glycolysis :Refers to the sequence of rxns by which glucose is broken down to give
pyruvate and energy.
Occurs in the cell cytosol
May be aerobic [Glucose Pyruvate] summary of Rxn = Glucose + 2ADP + 2Pi +

2NAD+2 pyruvate + 2ATP + 2NADH + 2H+ + 2H2O
OR
Anaerobic [Glucose Lactate] or Ethanol & acetic acid. (Alcoholic Fermentation; alcohol +
CO2))
Summary of Rxn = Glucose + 2ADP + 2Pi ----- 2 Lactate + 2ATP + 2H2O
ANAEROBIC GLYCOLYSIS
Occurs in cells that lack mitochondria – eg RBCs.
Under hypoxic conditions eg skeletal muscle during strenuous exercise
Glucose converted to lactate
Energy is produced
Alcoholic Fermentation
Occurs in microorganisms eg yeast, bacteria
Glucose is converted to alcohol and CO2
Reactions are basically anaerobic
Energy is also produced
14 | P a g e
REACTIONS OF GLYCOLYSIS (10 STEPS)
Note: The irreversible Reactions (1, 3 and 10 )
DIFFERENCES BETWEEN GLUCOKINASE AND HEXOKINASE
Glycolysis: Energy-Investment
In reactions 1-5 of glycolysis,
Energy is required to add phosphate groups to glucose.
Glucose is converted to two three-carbon molecules.
15 | P a g e
Glycolysis: Energy-Production
In reactions 6-10 of glycolysis, energy is generated;
Sugar phosphates are cleaved to triose phosphates.
Four ATP molecules are produced.
GLYCOLYSIS: REACTIONS 6-10
Glycolysis: Overall Reaction
16 | P a g e
In glycolysis,
Two ATP add phosphate to glucose and fructose-6-phosphate.
Four ATP are formed in energy-generation by direct transfers of phosphate groups to
four ADP.
There is a net gain of 2 ATP and 2 NADH.
C6H12O6 + 2ADP + 2Pi + 2NAD+2C3H3O3- + 2ATP + 2NADH + 4H+
LEARNING CHECK
In glycolysis, what compounds provide phosphate groups for the production of
ATP and at what steps?
In reaction 7, phosphate groups from two 1,3-bisphosphoglycerate molecules
are transferred to ADP to form two ATP.
In reaction 10, phosphate groups from two phosphoenolpyruvate molecules are

used to form two more ATP.
ATP Generating Points In Glycolysis
What is “substrate–level phosphorylation”?
substrate Level phosphorylation; Is the synthesis of ATP coupled to

the conversion of a substrate to a product
ADP + Pi → ATP
This is different from the synthesis of ATP in the Electron Transport Chain
(ETC) – by oxidative phosphorylation.
REGULATION OF GLYCOLYSIS
17 | P a g e
Rate of glycolysis is controlled primarily by allosteric regulation of the 3 key

enzymes (irreversible steps), hexokinase, PFK-1, and pyruvate kinase.
PFK-I is the major regulatory enzyme of glycolysis. In the liver only, PFK-1 is
activated by fructose-2,6-diphosphate (F-2,6-DP).
PFK-II, the enzyme that synthesize the activator F-2,6-DP, is itself a regulatory enzyme.
It is inhibited by citrate & ATP and by phosphorylation. The reverse reaction is
catalyzed by fructose-2,6-diphosphatase(F-2,6-DPase).
Hormones also regulate glycolysis e.g., glucagon inhibits glycolysis by repressing the
synthesis of F-2,6-DP. Insulin promotes glycolysis by stimulating the synthesis of F-
2,6-DP.
GLYCOLYSIS – SUMMARY
In summary, a single glucose molecule in produces a total of 2 molecules of pyruvate, 2

molecules of ATP, 2 molecules of NADH
2 ATP molecules are used in steps 1 & 3, 2 ATP molecules are generated in step 7 and
2 molecules in step 10. This gives a total of 4 ATP molecules produced
If you subtract the 2 ATP molecules used in steps 1 & 3 from the 4 generated from
steps 7&10, you end up with a net total of 2 ATP molecules produced.
18 | P a g e
THE FATE OF PYRUVATE

Lecture 2
The Fate of Pyruvate:

The fate of pyruvate from glycolysis depends on:
The organism involved
The tissue in question (type of cell)
The redox state of the tissue (O2 availability)
The Fate of Pyruvate (junction Mol)
19 | P a g e
Occurs in anaerobic microorganisms such as yeast.
Decarboxylatespyruvate to acetaldehyde, which is reduced to ethanol.
Regenerates NAD+ to continue glycolysis.

NADH is reused to regenerate NAD+
ALCOHOLIC FERMENTATION
20 | P a g e
Occurs in cells without mitochondria eg RBCs

Also during inadequate O2 supply eg skeletal muscle during strenuous exercise
Pyruvate is converted/reduced to lactate
NADH produced in G3P-DH2ase (rxn 6 in glycolysis) is used as reducing agent
The NAD+ produced ensures continuity of glycolysis
Rxn is catalyzed by lactate DH2ase and is reversible.
2ATP is produced, 2NADH produced are reused
Pyruvate is reduced to lactate.
NADH oxidizes to NAD+ allowing glycolysis to continue.

O O lactate
|| || dehydrogenase
CH3—C—C—O- + NADH + H+
pyruvate
OH O
| ||
CH3—CH—C—O- + NAD+
lactate
21 | P a g e
Lactate in Muscles
During strenuous exercise,
Oxygen in the muscles is depleted.
Anaerobic conditions are produced.
Lactate accumulates.
C6H12O6 + 2ADP + 2Pi 2CH3–CH–COO- + 2ATP
glucose lactate
Muscles tire and become painful.
After exercise, a person breathes heavily to repay the oxygen debt and reform pyruvate in
the liver.
LEARNING CHECK
Discuss briefly lactic acidosis
LACTIC ACIDOSIS
Arises in the skeletal muscle during strenuous exercise
Under this condition, NADH production from glycolysis and TCA cycle rxns far exceeds the
oxidative capacity of the ETChain
NADH/NAD+ ratio is high and favours lactate formation from pyruvate
Lactate accumulates in the muscle causing a drop in intracellular pH
Causes cramps/stiffness in the muscle (lactic acidosis)
There is increased blood lactate, and decreased bicarbonate and low pH
The Cori cycle: processing lactate
made during anaerobic exercise
Cori Cycle
Serves to prevent Lactic acidosis

Lactate is produced (high conc) in the muscle during strenuous exercise from glucose
Lactate moves to liver via blood
Is converted to glucose by gluconeogenesis in the liver
Glucose moves through blood back to muscle to complete the cycle
22 | P a g e
Pyruvate formed in the cytosol is transported into the mitochondrion
Here it is converted to acetyl-CoA by the enzyme pyruvate DH2ase
Five (5) coenzymes participate in the rxn – CoA, NAD, FAD, Lipoic acid and TPP,
with Mg2+ as cofactor
Acetyl-CoA may be used in the TCA cycle or in lipogenesis
Rxn is irreversible – reason glucose is not synthesized from fat (Acetyl-CoA)
Pyruvate Dehydrogenase complex

Pyruvate DH2ase is a complex enzyme
Has 3 components – E1, E2, E3
– Pyruvate dehydrogenase – decarboxylation of pyruvate using TPP
23 | P a g e
– Dihydrolipoyltransacetylase – transfer of 2-C fragment to CoA using lipoic acid
– Dihydrolipoyl dehydrogenase – reoxidation of lipoic acid using the NAD-FAD

system
Mechanism of action of Pyruvate DH2ase complex
Summary
Under aerobic conditions (oxygen present),
Three-carbon pyruvate is decarboxylated.
Two-carbon acetyl CoA and CO2 are produced.
O O pyruvate || ||
dehydrogenase
CH3—C—C—O- + HS—CoA + NAD+

pyruvate
O
||
CH3—C—S—CoA + CO2 + NADH

acetyl CoA
Learning Check
Discuss the 3 possible fates of Pyruvate
24 | P a g e
Pathways for Pyruvate
25 | P a g e
FATE OF ACETYL-CoA
BIC 203 (Lecture 3)
Learning Check
What vitamins are required for acetyl CoA production from pyruvate?
What is the major role of coenzyme A in catabolic reactions?
Major sources/uses of Acetyl-CoA
Acetyl-CoA
Acetyl coenzyme A (acetyl-CoA) is an important molecule in metabolism - used in many
biochemical reactions
Its main function is to convey the carbonatoms within the acetyl group to the TCA cycle to be
oxidized for energy production.
The acetyl group is further oxidized to carbon dioxide and water, with the energy thus released
captured in the form of 11 ATP and 1 GTP molecules per acetyl group that enters the cycle.
Acetyl-CoA is also an important component in the biogenic synthesis of the neurotransmitter
acetylcholine
The Krebs cycle
26 | P a g e
Also known as citric acid cycle

Occurs in matrix of mitochondria
Series of redox reactions
2 decarboxylation reactions release CO2
Reduced coenzymes (NADH and FADH2) are the most important outcome
One molecule of ATP generated by substrate-level phosphorylation
The Krebs Cycle

The Eight reactions of the Krebs cycle
The TCA cycle/Krebs cycle
27 | P a g e
THE TCA CYCLE - FEATURES

Is another sequence of 8 rxns
Occurs in the mitochondria
Acetyl-CoA is completely oxidized to CO2 and H2O
Acetyl-CoA could arise from glycolysis, beta-oxidation of FAs, Amino acid catabolism,
ketone body metabolism
Energy is yielded in form of 3NADH, FADH2, and GTP/ATP (12 ATP total)
NADH and FADH2 go into the ETChainrxns
GTP/ATP produced in the TCA cycle is by substrate level phosphorylation
ENERGY YIELDING REACTIONS OF THE TCA CYCLE

1. ISOCITRATE DEHYDROGENASE RXN (3)
2. α-KETOGLUTARATE DEHYDROGENASE RXN (4)
28 | P a g e
3. SUCCINYL-COA SYNTHETASE RXN (5)
GTP/ATP synthesis is by substrate level phosphorylation

4. SUCCINATE DEHYDROGENASE RXN (6)
5. MALATE DEHYDROGENASE RXN (8)
29 | P a g e
Energy yield of TCA Cycle (1 cycle)
ENERGY YIELD OF TCA CYCLE (2 CYCLES)

A molecule of glucose yields 2 pyruvate mols
A molecule of glucose yields 2 acetyl-CoA mols

2 acetyl-CoA mols take 2 turns in TCA cycle
30 | P a g e
This yields 24 ATP molecules
Total energy yield per glucose mol in aerobic respiration
REGULATION OF TCA CYCLE
Major Functions of TCA cycle

Serves as a common pathway for metabolism of CHOs, lipids and proteins (as acetyl-CoA)
Liberates much energy from the above fuel mols (NADH, FADH2, GTP/ATP)
Facilitates the reactions of the ETChain by its location in the mitochondrial matrix
Provides intermediates for biosynthesis eg alpha- Kg, OAA, Succinyl-CoA etc
31 | P a g e
The Electron Transport Chain (ETC)

Lecture 04
FATE of NADH, FADH2 - ATP Synthesis

ATP synthesis from NADH and FADH2 occurs in the mitochondria
Specifically at the inner mitochondrial membrane
Through electron transport & oxidative phosphorylation
NADH = 3ATP
FADH2 = 2ATP
FATE of NADH, FADH2 - ATP Synthesis
THE MITOCHONDRIA
32 | P a g e
The Mitochondrion is one of the cell organelles

“Power house” of the cell
Major function is energy (ATP) production
Derives energy from fuel mols (CHOs, lipids & amino acid) via oxidation of NADH and
FADH2
Site for rxns of the ETChain and oxidative phosphorylation
Also location for TCA cycle and FA oxidation rxns
The Electron Transport Chain (ETC)

The ETC is the pathway in which NADH and FADH2 are reoxidized with concomitant
generation of energy (ATP) from ADP+ Pi
The process occurs in the inner mitochondrial membrane
The chain involves the following components:
COMPONENTS OF ETC
NAD – functions as cofactor for the dehydrogenases that utilize NADH
FMN and FAD – act as prosthetic groups for flavin linked dehydrogenases.
Coenzyme Q (Ubiquinone) – transfers electrons to the cytochromes
Cytochromes (a, b and c) – transfer electrons to molecular O2 to form H2O
The overall role of these components is to transfer electrons from NADH and FADH2 to
molecular oxygen
33 | P a g e
ORGANIZATION OF THE ETC
The ETC is organized into Complexes I, II, III and IV;CoQ and Cyt C are not part
of the complexes
A. Complex I (NADH-Dehydrogenase)
It transfers electrons from NADH to CoQ – carries out the oxidation of NADH and reduction
of CoQ:
NADH NAD+ + e + H+
Serves as link b/w the ETChain and glycolysis, TCA cycle and FA oxidation – pathways that
generate NADH
Contains 30 polypeptides, FMN, Fe-S clusters, all involved in electron transfer process
Electrons move from complex I to complex III through CoQ
Complex II (Succinate Degydrogenase)
Succinate DH2ase is the only TCA cycle enzyme that is an integral membrane protein of the
inner mitochondrial membrane.
Made up of 4 subunits, Fe-S clusters, and FAD which is converted to FADH2 when succinate
is converted to fumarate (Rxn 6 of TCA cycle)
FADH2 passes electrons to CoQ through the Fe-S clusters
Free energy change of reaction is not sufficient to drive proton transport across inner
mitochondria memb – no proton translocation.
COMPLEX III (Ubiquinol Dehydrogenase)

Also called or UbiquinolReductase
Composed of the enzyme protein, Fe- S, Cyt b and Cyt C1
Reduced CoQ passes electron to Cyt C via Cyt b and Cyt C 1
As in complex I, passage of electrons through complex III is accompanied by proton transport
across the inner mitochondrial membrane – proton translocation
Complex IV (Cytochrome C Oxidase)
Contains Cytochromes aa3, 2copper atoms
Accepts electrons from cyt C
Passes electrons to O2 to form H2O
34 | P a g e
O2 and Cytochrome C Oxidase are the final destination of electrons derived from NADH and
FADH2
Complex IV also drives proton transport across the inner mitochondrial membrane –
proton translocation
ATP synthesized in Complexes I,III& IV
OXIDATIVE PHOSPHORYLATION
Is the synthesis of ATP from the energy derived from electron transport in the ETChain
As electrons pass from one member of the chain to the next, they lose much of their free
energy.
In the course these rxns, protons move from the matrix side to the intermembrane space.
Part of this energy is captured and stored by the synthesis of ATP from ADP + Pi
The remainder of the energy is lost as heat
The Actions of The Three Proton Pumps and ATP Synthase in the Inner Membrane
of Mitochondria
LEARNING CHECK
What is Oxidative Phosphorylation?
Transfer of e from NADH to O2 yields 3ATP while FADH2 to O2 yields 2ATP explain?
Oxidative Phosphorylation – 3ATP from NADH
35 | P a g e
Electron transfer in the ETChain is an oxidative process

Oxidative phosphorylation is d/4 the phosphorylation of ADP to ATP which is linked to the
electron transfer process.
Transfer of e from NADH to O2 yields 3ATP

This is b/cos the electrons pass through complexes I, III and IV, each complex yielding
1ATP
Oxidative Phosphorylation – 2ATP from FADH2
Transfer of e- from FADH2 to O2 yields 2ATP

This is b/cos the electrons pass through complexes II, III and IV
But there is no ATP synthesis at complex II
b/cos there is no proton translocation here
For FADH2, ATP is synthesized at complexes III and IV only
The energy derived from electron transport in complex II is not sufficient to drive proton
transport across the membrane
Other alternate entries
Mechanism of Oxidative Phosphorylation

Many models /hypothesis have been proposed to account for the mechanism of electron transport
and oxidative phosphorylation and ATPsynthesis
Prominent among these are:
The Chemical hypothesis (by E C Slater)
The Conformational coupling model (P D Boyer)
The Chemiosmotic hypothesis (Peter Mitchell)
36 | P a g e
THE CHEMIOSMOTIC HYPOTHESIS
Proton pump across the inner mitochondrial membrane and ATP synthesis
THE CHEMIOSMOTIC HYPOTHESIS

Is the most acceptable of the hypothesis and proposed that:
As electrons move along the ETChain, part of the energy is used to drive protons
from the matrix into the intermembrane space
The process creates electrochemical potential/gradient
The gradient has potential energy – proton motive force (PMF)
Proton pump/translocation creates pH gradient and electrochemical gradient

across the inner mitochondrial membran
The electrochemical and pH gradients tend to attract protons back into the matrix
from the inter-membrane space.
The flow of protons down this gradient drives the synthesis of ATP via the ATP synthase
channel.
37 | P a g e
The rxn ADP + Pi → ATP is catalyzed by ATP synthase

The electrochemical gradient across the membrane also drives the uptake of ADP and
Pi into the mitochondrial matrix and export of ATP to the cytosol
Electron transport is coupled to oxidative phosphorylation
INHIBITORS OF ETCHAIN
These substances inhibit electron transport at different complexes
Inhibitors of complex I: Rotenone, amobarbital, ptericidin, demerol
Inhibitors of complex II: Carboxin and 2-thenoyltrifluoroacetone
Inhibitors of complex III: Antimycin A (antibiotic)
Inhibitors of complex IV: Hydrogen cyanide, hydrogen sulphide, Sodium Azide and carbon
monoxide
By this mechanism, these chemicals kill microoganisms and are used as antibiotics.
INHIBITORS OF ETCHAIN- Hydrogen Cyanide
38 | P a g e
UNCOUPLING AGENTS
These are substances that cause the collapse of the proton gradient across the inner
mitochondrial membrane
Electron transport continues but ATP synthesis does not occur
Energy produced from electron transport is dissipated as heat
Egs include: 2,4-dinitrophenol, dicumaroletc
Some uncouplers/inhibitors specifically inhibit ATP synthesis by binding to the active site of
ATP synthase enzyme
The binding closes the proton channel of the ATP synthase and prevents re-entry of protons
into the matrix
The pH and electrochemical gradients cannot be dissipated
Electron transport is halted, indicating that electron transport and oxidative phosphorylation are
tightly coupled processes.
Egoligomycin (antibiotic).COUPLING OF ELECTRON TRANSPORT AND ATP
SYNTHESIS
39 | P a g e
40 | P a g e
PENTOSE PHOSPHATE PATHWAY

Lecture 04
Objectives
To understand the function of the pentose phosphate pathway in production of NADPH and
ribose precursors for nucleic acid synthesis.
To relate defects in the pentose phosphate pathway to disease conditions.
To understand the role of NADPH in elimination of oxidants.
Also known as:

Pentose shunt
Hexose monophosphate shunt (HMP)
Phosphogluconate pathway
It is an alternative pathway for Complete Glucose Oxidation
Site: It occurs in the cytoplasm of all cells except muscle, and nonlactating mammary
gland (low activity).
THE PENTOSE PATHWAY IS A SHUNT

The pathway begins with the glycolytic intermediate glucose-6-P.
It reconnects with glycolysis because two of the end products of the pentose pathway are
glyceraldehyde 3-P and fructose 6-P; two intermediates further down in the glycolytic
pathway.
It is for this reason that the pentose pathway is often referred to as a shunt.
Pentose pathway is a shunt
NADPH + H+is formed from two separate reactions.

The glucose 6-phosphate DH (G6PD) reaction is the rate limiting step and is essentially
irreversible.
Cells have a greater need for NADPH than ribose 5-phosphate.
41 | P a g e
42 | P a g e
The enzyme is highly specific for NADP+; the Km for NAD+ is

1000 greater than for NADP+.
43 | P a g e
THE NONOXIDATIVE PHASE OF THE PENTOSE PATHWAY
Transketolase (TPP) and transaldolase are the link back to glycolysis.

Glyceraldehyde 3-phosphate
Fructose 6-phosphate
44 | P a g e
What does the pentose phosphate pathway achieve?

Complete Glucose Oxidation.
Production of ribose residues for nucleotide and nucleic acid synthesis [ribose 5-
phosphate]
-DNA
-RNA
Various cofactors (CoA, FAD, SAM, NAD+/NADP+)
Generation of reducing potential in the form of NADPH to be used in anabolic
reactions requiring electrons.
mainly used for reductive syntheses of fatty acids, steroids, amino acids via glutamate
dehydrogenase; and
production of reduced glutathione in erythrocytes and other cells.
Glutathione and Erythrocytes

GSH is extremely important particularly in the highly oxidizing environment of the red blood
cell.
Mature RBCs have no mitochondria and are totally dependent on NADPH from the pentose
phosphate pathway to regenerate GSH from GSSG via glutathione reductase.
In fact, as much as 10% of glucose consumption, by erythrocytes, is mediated by the pentose
pathway
The reduced form of glutathione serves as a sulfhydryl buffer.
It maintains cysteine residues in hemoglobin and other proteins in a reduced state.
GSH is essential for normal RBC structure and keeping hemoglobin in Fe ++ state.
Reduced glutathione also detoxifies peroxides.
2GSH + ROOH GSSG + H2O + ROH
45 | P a g e
Cells with low levels of GSH are susceptible to hemolysis.

Individuals with reduced GSH are subject to hemolysis.
This is often clinically seen as black urine under certain conditions.

Conditions for hemolytic anemia related G6PD deficiency.
The ingestion of oxidative agents that generate peroxides or reactive oxygen species
(ROS).
– Antimalarials - pamaquine
– purine glycoside from fava beans.
Individules with G6PD deficiency cannot produce sufficient GSH to cope with the
ROS.
Proteins become cross linked leading to Heinz body formation and cell lysis.
Glucose-6-phosphate Dehydrogenase (G6PD) Deficiency (favism)

causes Hemolytic Anemia
Mutations present in some populations causes a deficiency in glucose 6-phosphate
dehydrogenase, with consequent impairment of NADPH production.
Detoxification of H2O2 is inhibited, and cellular damage results - lipid peroxidation leads to
erythrocyte membrane breakdown and hemolytic anemia.
Most G6PD-deficient individuals are asymptomatic - only in combination with certain
environmental factors (sulfa antibiotics, herbicides, some antimalarials) do clinical
manifestations occur.
NB: *toxic ingredient of fava beans
LEARNING CHECK
Discuss G6PD deficiency (favism) and Hemolytic Anemia.
Glucose-6-P DH2ase (G6PD) deficiency

Is a genetic disorder arising from the mutation of the gene coding for the enzyme
Is sex linked, occurring more in males
The enzyme is very important in the RBCs- It protects the RBCs against oxidative
stress/attack from oxidants/reactive oxygen species (ROS) egH 2O2.
NADPH produced by the enzyme is used to reduce oxidized glutathione (GSSG) to the
reduced form (GSH) – catalyzed by glutathione reductase
46 | P a g e
Reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) and

removal of oxidants by glutathione reductase (GSSGR) and glutathione peroxidase
(GSHPX) respectively (SOD = Superoxide dismutase; H2O2 = Hydrogen peroxide)
In G6PD deficiency (favism), the RBC lacks NADPH.
Glutathione remains in the oxidized form and
There is accumulation of oxidants eg H2O2 which is toxic to RBCs.
Lysis of RBCs occurs, resulting in hemolyticanemia
Sufferers are to abstain from fava beans, sulphonamides, aspirin, primaquine, etc
Drugs that can precipitate this reaction:

Antimalarial agents
Sulfonamides (antibiotic)
Aspirin
Nonsteroidal, antiinflammatory drugs (nsaids)
Nitrofurantoin
Quinidine
Quinine
Exposure to certain chemicals
LEARNING CHECK
HMP Shunt is inactive in muscles discuss
Individuals with G6PD deficiency must not eat Fava beans why?
Individials with favism present with black/dark urine why?
47 | P a g e
GLUCONEOGENESIS
Lecture 06
Learning Objectives :
Chemical steps of gluconeogenesis; associate enzymes
Precursors that can enter gluconeogenesis;
Relationship of glycolysis to gluconeogenesis; shared enzymes, irreversible steps
Liver as the primary gluconeogenesis organ; Cori Cycle, Alanine Cycle.
GLUCONEOGENESIS
Definition: Is the synthesis of Glucose from non-carbohydrate sources
It is one of the two main mechanisms the body uses to keep blood glucose levels from
dropping too low.
This process occurs during periods of fasting, starvation, or intense exercise.
Site: Liver & Kidney
Cell compartment: occurs partly in the cytoplasm and partly in the mitochondria (depending on
the substrate)
FUNCTIONS OF GLUCONEOGENESIS
To meet the glucose need of the body when dietary sources are not available (especially for the
brain and RBCs)
To supply intermediates of TCA cycle
To clear the lactate produced by the muscle and the RBCs
To remove glycerol produced in the adipose tissue or absorbed from intestine
To maintain normal blood glucose level
48 | P a g e
Irreversible Glycolytic Steps Bypassed

G l y c o l y s i s G l u c o n e o g e n e s i s
1. H e x o k i n a s by Glucose-6-phosphatase
2. P h o s p h o f r u c t o k i n a s e - 1 by Fructose 1,6-bisphosphatase
3. P y r u v a t e k i n a s e ( P y r K by Pyruvate Carboxylase and
P h o s ph o en o lpy ru v at e ca rb o xy ki na s e
49 | P a g e
Gluconeogenesis as it takes place in the mammalian liver.
By pass 1:
Glucose-6-Phosphatase catalyzes:
glucose-6-phosphate + H2O glucose + Pi

This is primarily a function of the liver to buffer blood glucose levels
Glucose-6-Phosphatase is NOT present in brain and muscle!
By pass 2:
50 | P a g e
Fructose-1,6-bisphosphatase catalyzes:
fructose-1,6-bisP + H2O fructose-6-P + Pi.
By pass 3:
51 | P a g e
SUBSTRATES OF GLUCONEOGENESIS
Glucogenic Amino Acids
Glycerol
Pyruvate
Lactate
Intermediates of TCA Cycle
Also propionyl CoA derived from odd chain fatty acid.
52 | P a g e
Intermediates in citric acid cycle can be used for gluconeogenesis through oxaloacetate
The source of pyruvate and oxaloacetate for gluconeogenesis during fasting or

carbohydrate starvation is mainly amino acid catabolism.
Some amino acids are catabolized to pyruvate, oxaloacetate, or precursors of these.

Muscle proteins may break down to supply amino acids. These are transported to
liver where they are deaminated and converted to gluconeogenesis inputs.
53 | P a g e
Glycerol, derived from hydrolysis of triacylglycerols in fat cells, is also a significant input
to gluconeogenesis.
GLUCOGENIC AMINO ACIDS
54 | P a g e
Liver is the major source of blood glucose from gluconeogenesis

Is the primary gluconeogenic organ
Produces glucose for export to brain, muscle, RBC’s
Uses many small metabolites and amino acids to feed gluconeogenesis
Liver function is highly sensitive to insulin & glucagon
+
Matrix
55 | P a g e
Lactate and glucose shuttle between active muscle/RBC and liver

Liver gluconeogenesis supplies the blood glucose for use by muscle, RBC’s and brain
(120 g/day)
Note: the brain fully oxidizes glucose, so it does not funnel back lactate
The liver can also use the amino acid Alanine similarly to Lactate
Following transamination to pyruvate, gluconeogenesis allows the liver to convert
it to glucose for secretion into the blood
56 | P a g e
Regulation of gluconeogenesis
Pyruvate can go “up” or “down” depending upon energy needs
.
To prevent the waste of a futile cycle, Glycolysis & Gluconeogenesis are reciprocally
regulated.
F-1,6-Bisphosphatase is the most
Important control site in Gluconeogenesis.
57 | P a g e
Reciprocal regulation by ATP/AMP

AMP inhibits Fructose-1,6-bisphosphatase but activates Phosphofructokinase o
ATP inhibits Phosphofructokinase but activate Fructose-1,6-bisphosphatase In
high ATP/AMP ratio: stimulate gluconeogenesis
In low ATP/AMP ratio: stimulate glycolysis
Reciprocal regulation by fructose-2,6-bisphosphate:

Fructose-2,6-bisphosphate stimulates Glycolysis.
Fructose-2,6-bisphosphate activates Phosphofructokinase-1.
Fructose-2,6-bisphosphate inhibits Fructose-1,6-bisphosphatase.
Reciprocal regulation by hormones

Phosphofructokinase-1 (PFK-1)
Induced in feeding by insulin
Repressed in starvation by glucagon
Fructose-1,6-bisphosphase
Repressed in feeding by insulin
Induced in starvation by glucagon
So:
Insulin activates glycolysis but inhibits gluconeogenesis;
Glucagon activates gluconeogenesis but inhibits glycolysis.
58 | P a g e
Summary
Purpose- alternative source of glucose rather than dietary.
Carbohydrates or glycogen breakdown
Primary precursers are lactate, pyruvate, glycerol, part of fatty acids and certain amino
acids (glucogenic)
3 essentially irreversible steps of glycolysis are bypassed.
Regulated via pyruvate carboxylase, fructose 1,6 bisphosphatase, and
phosphofructokinase-2/fructosebisphosphatase-2
PATHWAYS IN GLUCONEOGENESIS
59 | P a g e
60 | P a g e
METABOLISM OF GLYCOGEN
Lecture 07
The relationships among 4 common metabolic pathways that involve glucose
GLUCOSE ANABOLISM
Glucose storage: glycogenesis
Polysaccharide that is the only stored carbohydrate in humans
Insulin stimulates hepatocytes and skeletal muscle cells to synthesize glycogen
Glucose release: glycogenolysis
Glycogen stored in hepatocytes broken down into glucose and release into blood
Sources of blood glucose in fed, fasting, and starved states:
GLYCOGEN METABOLISM
61 | P a g e
GLYCOGENESIS AND GLYCOGENOLYSIS
Glycogen Function
In liver – The synthesis and breakdown of glycogen is regulated to maintain blood glucose
levels.
In muscle - The synthesis and breakdown of glycogen is regulated to meet the energy
requirements of the muscle cells.
Metabolic Regulation of Mammalian Glycogen Levels
Glycogen reserves are the mostimmediately available large source ofmetabolic energy for
mammals.
Storage and utilization are underdietary and hormonal control.
62 | P a g e
Regulation of glycogen metabolism
Regulating site for glycogen synthesis Glycogen synthase

Regulating site for glycogen catabolism Glycogen phosphorylase
Glycogen Phosphorylase
AMP activates Phosphorylase
ATP & glucose-6-phosphate inhibit Phosphorylase
Thus glycogen breakdown is inhibited when ATP and glucose-6-phosphate are
plentiful.
Glycogen Synthase
Activated by glucose-6-P (opposite of effect on Phosphorylase).
Thus Glycogen Synthase is active when high blood glucose leads to elevated intracellular glucose-6-P.
Regulation by hormones
Glucagon and epinephrine:
– Inhibit glycogen synthase
– Activate glycogen phosphorylase
– Increase glycogen catabolism and increase blood glucose
Insulin:
– Inhibit glycogen phosphorylase
– Activate glycogen synthase
– Increase glycogen synthesis and decrease blood glucose
Hormonal Control of Carbohydrate Metabolism

Enzymes control the metabolism of carbohydrates, but...
Several hormones also affect Carb. metabolism
Insulin o
Glucagon
o Epinephrine
63 | P a g e
Insulin
High levels of glucose induce releaseof insulin from β-cells of islets ofLangerhan in the
pancreas.
Insulin is polypeptide hormone.
Detected by receptors at surfaceof muscle cells.
Increases glycogenesis in muscle.
Insulin reducesglucose in the blood and stimulates conversion of glucose to fats,

proteins, ribulose 5-phosphate and glycogen; inhibits the conversion of fats, proteins,
glycogen and ribulose 5-phosphate to glucose
GLUCAGON
During low glucose levels

Acts primarily in liver.
A polypeptide hormone produced in α-cells of the islets of Langerhanof the pancreas.
Receptors on surface of liver cells.
Stimulates glycogen breakdown & inhibits glycogenesis.
Glucagon also blocks glycolysis & stimulates gluconeogenesis.
64 | P a g e
Glucagon is a 29 amino acid peptide hormone formed and released from the a cells of the
islets of Langerhans, in the pancreas.
Glucagon is a hormone that opposes the action of insulin
Mainly in the liver.
EPINEPHRINE A.K.A. ADRENALINE

Epinephrine - low glucose levels
Acts primarily on skeletal muscle.
Receptors on surface of cells.
Stimulates glycogen breakdown & inhibits glycogenesis.
Glucagon and epinephrine both stimulate intracellular pathway via increasing levels of
cAMP.
Epinephrine & glucagon have opposite effects to insulin.

Act to increase glucose in the blood.
Stimulate conversion of fats, glycogen and pyruvate to glucose
Inhibit conversion of glucose to fats, glycogen and pyruvate
65 | P a g e
cAMP Cascade
A cyclic AMPcascade is usedby both epinephrineand glucagon.
A cascade is amechanism in whichenzymes activate other enzymes sequentially usually leading
to an amplification of an initial signal
Epinephrine/Glucagon Cascade
Regulating Glycogen Metabolism
Glycogen Storage Diseases:
A family of serious, although not necessarily fatal diseases caused by mutations in

the enzymes involving in glycogen storage and breakdown.
66 | P a g e
FRUCTOSE AND GALACTOSE

METABOLISM
Lecture 08
FRUCTOSE METABOLISM
Fructose is metabolized in the liver

Entry into cells does not require insulin
In muscles, fructose just enters glycolysis
Or could be made into glycogen if insulin stimulus is available!
F6P G6P G1P UDP-glucose Glycogen
FRUCTOSE METABOLISM IN THE LIVER

In other tissues Fru is converted to F6P by hexokinase
In the liver, Fru is converted to F-1-P by fructokinase - a different enzyme altogether
Aldolase B converts F1P to DHAP + Glyceraldehyde
67 | P a g e
Glyceraldehyde is converted to G3P by a kinase

DHAP is also converted to G3P by triose isomerase
2 G3P mols go into glycolysis (note: 2ATP used)
Full pathway produces 4 ATP (gross) as in the use of glucose
DISORDERS OF FRUCTOSE METABOLISM

Essential fructosuria – caused by deficiency of fructokinase.Characterized by excretion of
large amounts of fructose in urine.
Hereditary Fructose Intolerance – caused by deficiency of Aldolase B
GALACTOSE METABOLISM
The first enzyme to act on galactose is galactokinase. This converts galactose into galactose-
1-phosphate.
68 | P a g e
UDP-galactose-4-epimerase produces UDP-glucose.
69 | P a g e
Phosphoglucomutasecatalyzes the reversible reaction:
glucose-1-phosphate glucose-6-phosphate
Galactose is converted to Galactose-1-Phosphate by galactokinase in the liver.
An initial UDP-glucose (facilitator molecule) exchanges glucose with Galactose to give UDP-Gal
+ Glu-1- P, catalyzed by Uridyltransferase.
Glu-1-P is released and converted to G6P by phosphoglucomutase which goes into glycolysis
UDP-Gal is converted to UDP-Glu by epimerase
UDP-Glu reacts with another Gal-1-P and liberates Glu-1-P which goes into glycolysis again.
The cycle continues and this way Gal enters glycolysis
GALACTOSEMIA
Is a hereditary disorder caused by deficiency of Uridyltransferase
Common in infants
Accumulation of galactose occurs to toxic levels
Galactose is converted to galactitol by galactosereductase, using NADPH
This occurs more in the lens of the eye and the neural tissue
GALACTOSEMIA & GALACTOSURIA

In the eye, this causes osmotic swelling and damage to the lens
Cataract results
If untreated could result in blindness
Neurological disorders in the neural tissue eg mental retardation
Galactosemia is managed by avoiding galactose in diet
Galactosuria results from deficiency of galactokinase
70 | P a g e
DIABETES MELLITUS
Occurs when an individual either doesn’t make enough, or is unable to utilize, the hormone
insulin to regulate blood glucose levels
Epidemic
Sixth leading cause of death in the United States
o Number of people with diabetes is rising annually
Change in Blood Glucose After Eating a High-Carbohydrate Meal
HYPOGLYCEMIA
A blood glucose level that is too low (usually below 70 mg/dl)
Signs and symptoms
Hunger
Nervousness
Dizziness
Light-headed
Eating or drinking carbohydrate rich foods
Relieves symptoms
71 | P a g e
Raises blood glucose
FASTING HYPOGLYCEMIA
Occurs in the morning after an overnight fast

Occurs during long stretches between meals or after exercise
May be caused by
Medications
Illness
Drinking too much alcohol
Certain tumors
Hormone imbalances
FORMS OF DIABETES
A. Type 1
Usually begins in childhood or early adulthood
5–10% of diabetics
Immune system destroys beta cells of the pancreas, No insulin produced
Common symptoms of elevated blood sugar

Polydipsia
Polyuria
Polyphagia
Require insulin and frequent blood glucose monitoring
B. Type 2
Overweight individuals develop this form frequently
90–95% of diabetics
Can go undiagnosed
Damages vital organs without individual being aware of it
Polycystic ovary syndrome
Hormonal imbalance in women
Have higher incidence of insulin resistance and hyperinsulinemia
Increased risk of developing type 2 diabetes
FORMS OF DIABETES
Prediabetes
Impaired glucose tolerance
Fasting blood sugar between 100 mg/dl and 126 mg/dl
High risk of developing diabetes and heart disease
DIABETES
Long-term damage from diabetes
Nerve damage
72 | P a g e
Leg and foot amputations

Eye diseases
Blindness
Slowing of onset of complications
Control level of blood glucose through
Diet
Insulin or oral medication
Monitoring blood glucose
Regular healthcare visits
Slowing of onset of complications
Control level of blood glucose through

Diet
Insulin or oral medication
Monitoring blood glucose
Regular health carevisits
Quick Review
Diabetes involves inadequate regulation of blood glucose levels
Type 1 diabetes – inadequate production of insulin

Type 2 diabetes – insulin resistance
Chronic high blood glucose can damage vital organs. Diabetics need medications
and/or insulin to manage blood glucose. High-fiber diet and routine exercise play a key
role in managing and preventing diabetes. Polycystic ovary syndrome increases the risk
of developing type 2 diabetes
73 | P a g e
LIPID METABOLISM
LIPID TRANSPORT; The role of serum albumin and lipoprotein
FATTY LIVER
DEGRADATION OF LIPIDS; Hepatic beta oxidation of fatty acids
INTRODUCTION
Fatty acids play critical roles in mammalian energy metabolism. Moreover, they are important
substrates for the synthesis of membrane phospholipids and biologically active compounds
like eicosanoids and leukotrienes
Because of their low solubility in aqueous solutions such as blood plasma and interstitial fluid,
fatty acids are in need of binding proteins to increase their concentration in vascular and
interstitial compartments. Albumin acts as main fatty acid binding protein in extracellular
fluids
The metabolism of lipids frequently requires that a particular lipid be transported in the blood
between different organs.
Free fatty acids are transported by serum albumin, whereas the neutral lipids (triacylglycerol and
cholesteryl esters) are transported by lipoproteins.
LIPOPROTEINS
Lipoproteins are molecular complexes of lipids and specific proteins called apolipoproteins.
A lipoprotein is a biochemical assembly whose purpose is to
transport hydrophobic lipid (a.k.a. fat) molecules in water, as in blood or extracellular fluid.
They have a single-layer phospholipid and cholesterol outer shell, with
the hydrophilic portions oriented outward toward the surrounding water
and lipophilic portions of each molecule oriented inwards toward the lipids molecules within
the particles.
Apolipoproteins are embedded in the membrane, both stabilising the complex and giving it
functional identity determining its fate. Thus the complex serves to emulsify the fats.
Many enzymes, transporters, structural proteins, antigens, adhesions, and toxins are
lipoproteins.
Examples include the plasma lipoprotein particles classified
as HDL, LDL, IDL, VLDL and chylomicrons lipoproteins, according to density / size (an
inverse relationship), compared with the surrounding plasma water. These complex protein
capsules enable fats to be carried in all extracellular water, including the blood stream (an
example of emulsification), subgroups of which are primary drivers / modulators
of atherosclerosis,the transmembrane proteins of mitochondrion, chloroplast, and
bacterial lipoproteins.
All lipoproteins consist of a hydrophilic shell and a hydrophobic core.
The hydrophilic shell contains proteins, phospholipids and unesterified cholesterol-
amphipathic molecules that interact favorably with both the aqueous environment and
the inner core.
The hydrophobic core contains the neutral lipids-triacylglycerol (TAG) and cholesterylesters
(CE) which are insoluble in water.
APOLIPOPROTEINS
Apolipoproteins are proteins that bind lipids (oil-soluble substances such as fat
and cholesterol) to form lipoproteins. They transport the lipids through
the lymphatic and circulatory systems.
The lipid components of lipoproteins are insoluble in water. However, because of their detergent-
like (amphipathic) properties, apolipoproteins and other amphipathic molecules (such as
phospholipids) can surround the lipids, creating the lipoprotein particle that is itself water-
soluble, and can thus be carried through water-based circulation (i.e., blood, lymph).
Apolipoproteins have four major functions including;
74 | P a g e
serving a structural role,

acting as ligands(recognition sites) for lipoprotein receptors,
guiding the formation of lipoproteins,
serving as activators or inhibitors of enzymes involved in the metabolism of lipoproteins.
Apolipoproteins thus play a crucial role in lipoprotein metabolism
THE APOLIPOPROTEINS
Apolipoprotein A-I: Apo A-I is synthesized in the liver and intestine and is the major
structural protein of HDL accounting for approximately 70% of HDL protein.
Apo A-I is an activator of lecithin: cholesterol acyltransferase (LCAT), an enzyme that
converts free cholesterol into cholesteryl ester in reverse cholesterol transport.
Apolipoprotein B-48: Apo B-48 is synthesized in the intestine and is the major structural
protein of chylomicrons and chylomicron remnants.
Apolipoprotein B-100: Apo B-100 is synthesized in the liver and is the major structural
component of VLDL, IDL, and LDL. There is a single molecule of Apo B-100 per VLDL,
IDL, and LDL particle. Apo B-100 is a ligand for the LDL receptor and therefore plays an
important role in the clearance of lipoprotein particles.
Apo C-II is a co-factor for lipoprotein lipase (LPL) and thus stimulates triglyceride
hydrolysis.
Apo-D: involved in reverse cholesterol transport
Apo-E: is recognised by a receptor in the liver
CLASSES OF LIPOPROTEINS
The four major classes of lipoprotein particles in the human serum include:
A. Chylomicrons:
-Largest & Least Dense of the lipoproteins and do not migrate in an electric field
-They are formed in the smooth endoplasmic reticulum of the intestinal mucosa cells from dietary
lipids, primarily triacylglycerol (TAGs),cholesteryl esters (CE) and phospholipids
-Apoprotein B-48 is unique to chylomicrons. It is termed B-48 because it contains only 48% of
the entire apo B protein. During apo B-48 movement from the ER to the Golgi, it is loaded
with lipid. This forms secretory vesicles that fuse with the plasma membrane and are
released into the lymphatic system
-Once in the plasma, chylomicrons receive apo E (for liver recognition) and apo C-II (for
activation of lipoprotein lipase) thus making the chylomicrons functional.
-The TAGs in chylomicrons are cleaved by lipoprotein lipase, once activated by apo C-II,
predominantly in the capillaries of adipose and muscle tissues
-FFAs can directly enter adipose or muscle cells for storage or energy, or can be transported on
albumin to other parts of the body. Most cells can oxidize FFAs to produce energy.
Adipocytes can re-esterify FFAs to produce TAGs.
-Liberated glycerol is returned to the liver, where it is almost exclusively used to produce
glycerol-3-phosphate, which can enter glycolysis or gluconeogenesis.
75 | P a g e
-Chylomicron remnants - cholesterol esters, phospholipids, and apolipoproteins - are taken up by

the liver via recognition of apo E, where they are hydrolyzed and recycled.Apo C-II is
returned to HDL.
B. VLDL: Very Low Density Lipoprotein

-VLDLs are produced in the liver and are composed primarily of TAG, which they deliver to
peripheral tissues. VLDLs contain apo B-100 when secreted, but must pick up apo C-II and
apo E from circulating HDL.
The function of VLDLs is to carry triacylglycerol from the liver to the peripheral tissues
where lipoprotein lipase degrades the lipids.
-As TAG is removed from the VLDL, the particle receives cholesteryl esters from HDL. This
process is accomplished by cholesteryl ester transfer protein.
-As VLDLs pass through the circulation, lipoprotein lipase liberates FFAs and the VLDL becomes
denser. apo C-II and E are returned to HDL, while apo B-100 is retained which is
recognized by receptors on peripheral tissues and the liver. Remaining TAGs are transferred
to HDL in exchange for cholesterol esters.
• -As they lose triglyceride, the proportion of cholesterol increases and they become denser;
they become a low-density lipoprotein (LDL)
C. LDL; Low Density Lipoprotein

The primary function of LDLs is to deliver cholesterol to extra hepatic tissues,where they are taken
up by receptor-mediated endocytosis;and their contents are degraded in the lysosomes.
-They have much less TAGs, but more cholesterol, than VLDLs. They are normally taken up
(endocytosed) by cells across the body. However, when LDL levels are too high
(hypercholesterolemia), LDL gets deposited in blood vessel walls.
The endocytosed cholesterol inhibits HMG-CoA reductase and decreased synthesis of LDL
receptors
-They do through recognition of apo B-100 or apo E (but not apo B-48) by LDL receptors on the
plasma membrane.
Excess cholesterol is esterified for storage by acyl CoA:cholesterolacyltransferase
(ACAT).
The higher the LDL, the higher the risk of cardiovascular problems.
D. HDL; High Density Lipoprotein

-HDLs are synthesized by the Liver and intestine is approximately 50% protein.
-They serve as a circulating reservoir of apo C-II and apo E for transfer to other lipoproteins.
Newly synthesized chylomicrons and VLDL particles obtain some of their apoproteins from
HDL reservoir following secretion.
-HDL is important in moving cholesterol from extrahepatic tissues to the liver (reverse
cholesterol transport).
Elevated plasma levels of HDL are associated with decreased incidence of coronary
atherosclerosis. Cholesterol is taken up from the surface of cells by HDL, esterified to
cholesterol esters and ultimately returned to the liver either by uptake of HDL particles by
the liver or by the transfer of cholesterol esters to VLDL and chylomicron remnants,
followed by remnant uptake.
-This makes them candidate molecules to mop up cholesterol from developing or existing
atheromas on vessel walls.
-Important proteins associated with reverse cholesterol transport;
(a) Lecithin Cholesterol Acyl Transferase (LCAT)-is a plasma enzyme that esterifies HDL
cholesterol. The fatty acid used for esterification comes from lecithin (phosphatidyl
choline).LCAT is activated by apo-A1, which is associated with HDL.(b)Cholesterol Ester
Transfer Protein (apo-D) is associated with HDL and facilitates the transfer of cholesterol
esters to VLDL and chylomicron remnants,in exchange for triacylglycerol.
Lipoprotein (a)
76 | P a g e
Lp(a), is nearly identical to LDL but is larger and denser than LDL, and when present in large
quantities is a risk factor for heart disease. In addition,Lp (a) contains one molecule of Apo
(a) for every molecule of Apo-B100
- Levels appear to be predominantly mediated by genetics, though lifestyle is also involved.
Lp(a) is similar to plasminogen, and it is possible that it contributes to heart attacks by binding
to plasminogen activators and slowing down the breakdown of blood clots.
Abnormalities of Lipoprotein Metabolism

Type 1;Hyperlipoproteinemia
The metabolic defect is the deficiency of lipoprotein lipase.
There is accumulation of chylomicron in the plasma which leads to elevated
chylomicrons. TAG is also elevated but cholesterol level is normal
Deposition of lipids in the subcutaneous tissues leads to eruptive xanthomas which
are plaques containing triglycerides and cholesterol.
There is also hepatomegaly and abdominal pain.
TYPE IIA: PRIMARY CHOLESTEROLEMIA
Caused by LDL-receptor defect or defective binding of apo-B100 to the receptor or receptor
– LDL complex is not internalized.
LDL is elevated as well as cholesterol but the TAG is normal.
This type of hyperlipidemia carries an increased risk of atherosclerosis and coronary artery
disease.
TYPE IIB-HYPERLIPOPROTEINEMIA
Caused by increases in apo-B and apo-C11,leading to elevated level of LDL and VLDL.
TAG and cholesterol are also slightly elevated
Deposition of these lipids in the cornea leads to corneal arcus,indicating
hypercholesterolemia.
TYPE III-Hyperlipoproteinemia
The metabolic defect is abnormal apo-E and increase in apo-C11.
VLDL and chylomicron are elevated as well as cholesterol but TAG is increased
only slightly.
There is an increased risk of vascular disease.
TYPE IV: FAMILIAL ENDOGENOUS HYPERLIPOPROTEINEMIA
This results from overproduction of VLDL and increased apo-C11
Plasma is milky and cloudy
This type of hyperlipoproteinemia is associated with diabetes mellitus, ischemic heart
disease and obesity.
TYPE V:HYPERLIPOPROTEINEMIA
Elevated chylomicrons and VLDLs due to unknown cause
Cholesterol is normal but TAG is increased.
Creamy layer remains over milky plasma after 24
hours. Common features include ischemic heart disease.
FATTY LIVER DISEASE

Fatty liver disease refers to the deposition of excessive triglycerides in the hepatocytes
and can range from fatty liver alone (steatosis) to fatty liver associated with inflammation
(steatohepatitis).
Steatohepatitis may lead to cirrhosis and ultimately hepato-cellular carcinoma may result.
Fatty liver may be associated with alcohol abuse (alcohol-related fatty liver) or occur
without a history of alcohol use (non-alcohol related fatty liver disease) which may occur in
conditions such as diabetes mellitus or obesity.
CAUSES OF FATTY LIVER

The increased levels of plasma free fatty acids resulting from mobilization of fat from
adipose tissue or increased synthesis
77 | P a g e
The hydrolysis of lipoprotein or chylomicron triacylglycerol by lipoprotein lipase in extra

hepatic tissues.
Increasing amounts of free fatty acids are taken up by the liver and esterified.
The production of plasma lipoprotein does not keep pace with the influx of free
fatty acids allowing triacylglycerol to accumulate.
During starvation and the feeding of high-fat diets, the quantity of triacylglycerol
present in the liver is significantly increased
Uncontrolled diabetes mellitus cause fatty appearance and enlargement of the liver.
Relative insulin deficiency allows increased activity of the hormone sensitive lipase
leading to breakdown of fat in adipose tissue and subsequent transport to the liver as
non-esterified fatty acid (NEFA) carried by albumin.
Toxic injury to the liver as obtained in poisoning by carbon tetrachloride, chloroform,
phosphorus, lead, arsenic and ethionine (α- amino-γ-ethyl-mercaptobutyric acid) lead to
decreased synthesis of VLDL and thus, limits the export of FFAs and TAG from the
liver to peripheral sites.
Alcoholism leads to fat accumulation in the liver, hyperlipidemia, and ultimately
cirrhosis. Metabolism of of ethanol generates acetaldehyde in a reaction that leads to
excess production of NADH.
The NADH generated in turn inhibits the formation of oxaloacetate from malate in the
TCA cycle. Thus, the oxidation of acetyl-CoA is impaired. Accumulation of acetyl Co-A
favors fatty acid synthesis and since fatty acid is not oxidized; it accumulates in the liver
resulting in fatty liver.
Deficiency of lipotropic factors. The substances that prevent the accumulation of fat in the
liver are known as lipotropic factor. The phenomenon is said to be lipotropism. Choline,
methionine, lecithin, omega-3-fatty acids etc are said to have lipotropic activity.
The deficiency of essential fatty acids depresses the synthesis of phospholipids
and, therefore, cholesterol is involved in esterification causing fatty livers
Clinical Relevance
Fatty liver is a common cause of liver enzyme derangements in diabetic patients. It is also a
cause of enlarged liver (hepatomegaly) in these patients. Non alcoholic steatohepatitis
(NASH) can lead to liver cirrhosis.
HEPATIC BETA-OXIDATION OF FATTY ACIDS

Fatty acid β-oxidation is a multi step process by which fatty acids are broken down by various
tissues to produce energy
Most fatty acid oxidation occurs in the mitochondria.
On being released from chylomicrons or very-low-density lipoproteins (VLDLs) by lipoprotein
lipase in the capillary beds in the body, free fatty acids primarily enter the cell via fatty acid
transporters on the cell surface. Fatty acid transporters include fatty acid translocase
(FAT/CD36), tissue specific fatty acid transport proteins (FATP), and plasma membrane
bound fatty acid binding protein (FABPpm).
The uptake and oxidation of fatty acids by cells can be divided into three major stages:
Activation
Transfer of fatty acids into mitochondria
β-oxidation of fatty acids
ACTIVATION
Fatty acids are converted to fatty acyl-coA immediately upon entry into the cell
This traps the fatty acids in the cell and generates a high energy thioester bond with coA-SH.
The reaction is catalysed by fatty acyl-CoA synthetase (thiokinase) and is driven by the
hydrolysis of pyrophosphate.
78 | P a g e
Fatty acid + HS – CoA+ ATP Fatty acyl CoA + AMP + PPi

Fatty acids activation occurs in the cytosol
TRANSFER OF FATTY ACIDS INTO MITOCHONDRIA

Long-chain acyl-CoA cannot pass through the inner mitochondrial membrane, but its
metabolic product, acylcarnitine
Fatty acid having a chain length of C12 or greater, enter the carnitine shuttle,which consists of
two enzymes
carnitine acyl transferase 1 (CAT-1)
carnitine acyl transferase 2 (CAT-2)
and one translocase-carnitine translocase.
Carnitine-acyl transferase 1 (CAT-1) is associated with the outer surface of the inner
mitochondrial membrane.It transfers the fatty acid from fatty CoA to carnitine to form
fatty acyl carnitine. and is a major site of regulation of mitochondrial fatty acid uptake
Carnitine translocase transports fatty acyl carnitine into the mitochondria and transports free
carnitine back out of the mitochondria.
Carnitine-acyl transferase II (CAT-II) is associated with the inner surface of the inner
mitochondrial membrane. It converts the long-chain acylcarnitine back to long-chain acyl-
CoA
β-oxidation of fatty acids

This involves the sequential removal of two carbon fragments from the carboxyl end of the
fatty acids molecule.
Each cycle of oxidation involves four reactions and generates one molecule each of acetyl-
CoA, NADH and FADH2.
This pathway is called β-oxidation because the two oxidation steps that generate FADH2 and
NADH both involve the β-carbon atom.
First step-oxidation(dehydrogenation)
Fatty acyl-CoA is acted upon by an enzyme acyl-CoA dehydrogenase which is FAD
dependent.
Fatty acylCoA undergoes dehydrogenation and forms a trans-double bond at the α and β
carbons to form trans-Δ2-enoyl-CoA
The electrons which were removed from the fatty acyl-CoA chain are transferred to FAD which
gets reduced to FADH2. This FADH2 immediately via the Electron Transport System gets
converted to ATP molecules.
HYDRATION
-Second step– HYDRATION
Enoyl-CoA hydratase catalyzes this reaction where water is added. Hydration occurs at the
double bond resulting in the formation of β-hydroxyacyl-CoA.
79 | P a g e
C. OXIDATION
-Third step– β-hydroxyacyl-CoA undergoes dehydrogenation to form β-ketoacyl-CoA in the
presence of β-hydroxyacyl-CoA dehydrogenase. The electrons available as a result of
dehydrogenation are accepted by NAD+ to form NADH + H+ which immediately exchanges
these electrons with oxygen in the Electron Transport System to form ATP molecules.
THIOLYTIC CLEAVAGE
Fourth step– This reaction is called thiolysis as acyl-CoA acetyltransferase (also known
as thiolase) in the presence of CoA-SH causes the cleavage of β-ketoacyl-CoA to form
acetyl CoA and the thioester of the original fatty acid with two carbons less.
This cleavage occurs as the β carbon ketone group is a good target for nucleophilic attack by the
thiol (-SH) group of the coenzyme A.
The new fatty acyl-CoA (n-2 carbons) formed again participates in the β-oxidation cycle to form
a new fatty acyl-CoA with two carbons less (n-4 carbons) and a new molecule of acetyl
CoA. This process continues till the entire fatty acid is converted into acetyl CoA molecules.
Acetyl CoA formed from the above steps now enters the Kreb’s cycle to get oxidized to
CO2 and H2O.
CALCULATIONS FOR THE ENERGETICS OF FATTY

ACID OXIDATION
How many Acetyl CoA units will be formed from fatty acids with different chain lengths?
Since the Acetyl group of the acetyl CoA is formed by two carbons, we should divide the
number of carbons in the acyl group between two.
Miristic acid (14 carbons): 14 carbons /2 = 7 Acetyl CoA
Palmitic acid (16 carbons): 16 carbons/2 = 8 Acetyl CoA
In order to be completed degraded to Acetyl CoA, the fatty acid in form of acyl CoA should
experiment several “rounds” in the Beta-oxidation process. For each round, an Acetyl CoA
is released and, a NADH and a FADH2 are produced.
We know already how many acetyls CoA are formed from each fatty acid: n/2, where n is the
number of carbons.
Now, how many rounds are necessary for converting all the fatty acid to acetyl CoA?
Since in the last round we already obtain two Acetyl CoA, the number of necessary rounds is
(n/2) -1
Observe: Miristic acid (14 carbons)
1st round:
Produce one acyl CoA of 12 carbons and one Acetyl CoA + NAD H.H+ + FADH2
2nd round:
80 | P a g e
3rd round:
4th round:
5th round:
6th round:
But the acyl CoA of 2 carbons is already an Acetyl CoA, so it is the last round!
So, after 6 rounds, all the Miristic acid has been converted to Acetyl CoA.
Then, what we have obtained as a result of the beta-oxidation of Miristic acid?
7 acetyl CoA and 6 NADH + 6 FADH2
The acetyl CoA units are oxidized up to CO2 and H2O in the Krebs Cycle.
In terms of ATP, the yielding depends on the kind of yielding that is used for reduced
cofactors:
If during your course it is considered that each NADH.H+ yields 2.5 ATP and each FADH2
yields 1.5 ATP, then
7 acetyl CoA x 10 ATP/AcetylCoA in the Krebs Cycle: 70 ATP
6 NADH x 2.5 ATP/NADH = 15 ATP
6 FADH2 x 1.5 ATP/FADH2 = 9 ATP
Minus 2 ATP used in the activation (Miristic acid to miristyl CoA)
70+11+9-2 = 92 ATP
If during your course it is considered that each NADH yields 3 ATP and each FADH2 yields 2
ATP, then
7 acetyl CoA x 12 ATP/AcetylCoA in the Krebs Cycle: 84 ATP
6 NADH x 3 ATP/NADH = 18 ATP
6 FADH2 x 2 ATP/FADH2 = 12 ATP
Minus 2 ATP used in the activation (Miristic acid to Miristyl coA)
84+18+12-2 = 112 ATP
How to calculate the energetic balance of any fatty acid?
Step 1.- Number of Carbons/2 = Number of Acetyl CoA formed.
Step 2.- Number of rounds in the Beta-oxidation necessary for converting the whole fatty acid to
Acetyl Co A units: Number of Acetyl CoA minus 1 [(n/2)-1]
Step 3 (Option a) If you consider that each NADH yields 2.5 ATP and each FADH2 yields
1.5 ATP then multiply the number of rounds times 4 and multiply the number of Acetyl
CoA times 1o
OR
Step 3 (Option b).- If you consider that each NADH yields 3 ATP and each FADH2 yields 2
ATP then multiply the number of rounds times 5 and multiply the number of Acetyl CoA
times 12.
Step 4.- Take two ATP that were used for the activation of the Fatty Acid
Example:
Fatty acid with 12 Carbons
– Option (a):
Step 1: 12/2 = 6 Acetyl CoA

Step 2: 6-1 = 5 rounds
81 | P a g e
Step 3 (Option a): (5 x 4) + (6 x10)
Step 4 = -2
Total: 78 ATP
– Option (b):
Step 1: 12/2 = 6 Acetyl CoA
Step 2: 6-1 = 5 rounds
Step 3 (Option b): (5 x 5) + (6 x12)
Step 4 = -2
Total: 95 ATP
ASSIGNMENT
I would like now that the students calculates the energetic balance of the total oxidation of a
fatty acid with 18 carbons. Assume that the oxidation of each NADH.H+ yields 3 ATP and
that each FADH2 yields 2 ATP.
Energetics of β-oxidation
Each turn of the degradation cycle yields a mole of acetyl-CoA and produces 1 mole of FADH2 and
one NADH.As the system requires an input of energy in form of 1 ATP only in the initial
activation step, the energy production during each turn of the cycle is appreciable .
The overall reaction for the degradation of a long –chain fatty acid such as palmitate can be
written as follows;
•
Each β-oxidation cycle can be represented as following:
C(n)Acyl-CoA + CoA-SH + FAD + NAD+ + H2O → C(n-2)Acyl CoA + Acetyl
CoA + FADH2 + NADH + H+
Complete oxidation of Palmitoyl CoA can be represented as following:

(Equation A)
PalmitoylCoA + 8CoASH + 7FAD + 7NAD++ 7H2O → 8Acetyl CoA +
7FADH2+ 7NADH + 7H+
The re-oxidation of FADH2 and NADH via the mitochondrial electron transport chain yields 2
and 3 molecules of ATP respectively. The energy yield from the oxidation of a molecule of
palmitate in the β-oxidation pathway can be calculated as shown below;
ENERGETICS OF Β-OXIDATION OF PALMITATE

Palmitic acid is a 16-carbon fatty acid and will take 7 turns in β-oxidation reactions;
The activation to palmitoyl-CoA involves the breaking of 2 high energy bonds of ATP. This is
equivalent to the use of 2 ATP molecules,i.e -2ATP
From Palmitoyl-CoA, we have 8 molecules of acetyl-CoA in 7 turns of reaction that yielded
7NADH and 7FADH2
7NADH (7x3) =21ATP(produced)
7FADH2 (7x2) =14ATP(produced)
In the TCA cycle, each acetyl-CoA molecule will yield 12 mol of ATP. For the 8 acetyl-CoA
molecules produced in the β-oxidation of palmitate (8x12)=96ATP produced
Total energy (ATP) yield from 1 molecule of palmitate=(-2+21+14+96)ATP=(131-2)=129
ATP(net gain)
Therefore, the total energy yield is 129 molecules of ATP for oxidation of palmitate
METABOLISM OF ODD- CHAIN FATTY ACIDS (Metabolism

of propionate)
82 | P a g e
Fatty acids with an odd number of carbon atoms are oxidized by the pathway of β-oxidation,
producing acetylCoA, until a three-carbon (propionyl-CoA) residue remains.
This compound is converted to succinyl-CoA, a constituent of the citric acid cycle . Hence, the
propionyl residue from an odd-chain fatty acid is the only part of a fatty acid that is
glucogenic.
Saturated fatty acids undergo β-oxidation but unsaturated fatty acids have a slight variation in the
pathway. β-oxidation pathway for unsaturated fatty acids includes two additional enzymes
isomerase and reductase.
Let us consider an example of monounsaturated fatty acid such as oleic acid
Oxidation of monounsaturated fatty acids (For example oleic acid)
•
Oleoyl CoA undergoes three cycles of β-oxidation like normal saturated fatty acids to yield 3
molecules of acetyl CoA and results in the formation of 12-carbon fatty acyl-CoA with a cis
double bond now between carbon 3 and 4. This product is known as cis-Δ3-Dodecenoyl-CoA.
The above product formed has a cis double bond and cannot further participate in β-oxidation.
Thus by the action of Δ3,Δ2-enoyl-CoA isomerase, cis-Δ3-Dodecenoyl-CoA is converted to
trans-Δ2-Dodecenoyl-CoA. This is the significance of the isomerase enzyme in the β-
oxidation of unsaturated fatty acids.
trans-Δ2-Dodecenoyl-CoA now is acted upon by the enzymes of β-oxidation pathway in five
continuous cycles to yield another 6 molecules of acetyl CoA.
The acetyl-CoA molecules now enter the Kreb’s cycle.
Regulation of fatty acid oxidation:

The carnitine shuttle is a rate limiting step in the oxidation of fatty acids in the mitochondria
and thus fatty acid oxidation can be regulated at this step.
This is achieved by controlling the rate at which fatty acids enter the mitochondria
Malonyl CoA, an intermediate of fatty acid synthesis present in the cytosol is an inhibitor of
carnitineacyltransferase I. This indicates that when fatty acid synthesis is in progress,
oxidation of fatty acid cannot occur at the same time as the carnitine shuttle is impaired
by inhibition of carnitineacyltransferase I.
Fatty acid oxidation is also regulated by the acetyl CoA to CoA ratio: as the ratio increases, the
CoA-requiring thiolase (the enzyme participating in β-oxidation) reaction decreases.
When [NADH]/[NAD+] ratio increases, the enzyme β-hydroxyacyl-CoA dehydrogenase is

inhibited.
Positive Effectors: Starvation and a general low-energy level
– Low insulin and high glucagon (i.e. low insulin:glucagon ratio)
– ADP
Negative Effectors: General indicators of sufficient energy in the cell:
– High insulin and low glucagon
– High ATP
READ UP CARNITINE CYCLE DEFECTS FROM THE NOTE

83 | P a g e
Biosynthesis of Lipids
Mrs Uzoamaka Okoli
What you expected to know
Biosynthesis of Fatty Acids and Eicosanoids
Metabolism of Triacylglycerol
Metabolism of Membrane phospholipids
Cholesterol Synthesis
Fatty Acid Synthesis
Fatty acid degradation and synthesis
are relatively simple processes that are
essentially the reverse of each other
84 | P a g e
THE DIFFERENCES BETWEEN FATTY ACID BIOSYNTHESIS AND BREAKDOWN

Intermediates in synthesis are linked to -SH groups of acyl carrier proteins (as
compared to -SH groups of CoA
Synthesis in cytosol; breakdown in mitochondria o
Enzymes of synthesis are one polypeptide
o Biosynthesis uses NADPH/NADP+; breakdown uses NADH/NAD+
What are the sources of ACoA in cytosol?

Amino acid degradation produces cytosolic acetyl-CoA
FA oxidation produces mitochondrial acetyl-CoA
Glycolysis yields cytosolic pyruvate which is converted to acetyl-CoA in
mitochondria
Citrate-malate-pyruvate shuttle provides cytosolic acetate units and reducing
equivalents for fatty acid synthesis
Formation of Malonyl- CoA
Bicarbonate as a source of CO2 is required in the initial reaction for the carboxylation of
acetyl-CoA to malonyl-CoA in the presence of ATP and acetyl-CoA carboxylase.
This reaction is first catalyzed and committed step of FA synthesis
The reaction takes place in two steps: (1) carboxylation of biotin involving ATP and (2)
transfer of the carboxyl to acetyl-CoA to form malonyl-CoA.
Acetyl-coA carboxylase
Acetyl-CoA carboxylase has a requirement for the vitamin biotin
85 | P a g e
The enzyme is a multienzyme protein containing a variable number of identical

subunits, each containing biotin, biotin carboxylase, biotin carboxyl carrier
protein, and transcarboxylase
Step 1 Condensation
The first reaction in the formation of a fatty acid chain is condensation of the
activated acetyl and malonyl groups to form acetoacetyl-ACP,. In this reaction, catalyzed by
-ketoacyl-ACP synthase (KS), the acetyl group is transferred from the Cys -SH group of the
enzyme to the malonyl group on the OSH of ACP, becoming the methyl-terminal two-carbon
unit of the new acetoacetyl group.
Step 2 Reduction of the Carbonyl Group

The acetoacetyl-ACP formed in the condensation step now undergoes reduction
of the carbonyl group at C-3 to form D-- hydroxybutyryl-ACP. This reaction is catalyzed
by - ketoacyl-ACP reductase (KR) and the electron donor is NADPH.
Step 3 Dehydration The elements of water are now removed from C-2 and C-3 of D--
hydroxybutyryl-ACP to yield a double bond in the product, trans-2- butenoyl-ACP. The
enzyme that catalyzes this dehydration is - hydroxyacyl-ACP dehydratase (HD).
86 | P a g e
Step 4 Reduction of the Double Bond

Finally, the double bond of trans-2-butenoyl-ACP is reduced (saturated) to form butyryl-
ACP by the action of enoyl-ACP reductase (ER); again, NADPH is the electron donor
87 | P a g e
Palmitate and stearate serve as precursors of the two most common monounsaturated
fatty acids of animal tissues: palmitoleate, 16:1(9), and oleate, 18:1(9); both of these fatty
acids have a single cis double bond between C-9 and C-10
.The double bond is introduced into the fatty acid chain by an oxidative reaction catalyzed
by fatty acyl–CoA desaturase, a mixed-function oxidase
88 | P a g e
FATTY ACID SYNTHESIS REGULATION

The reaction catalyzed by acetyl-CoA carboxylase is the rate-limiting step in the
biosynthesis of fatty acids, and this enzyme is an important site of regulation.
Palmitoyl-CoA, the principal product of fatty acid synthesis, is a feedback inhibitor of the
enzyme; citrate is an allosteric activator
Citrate plays a central role in diverting cellular metabolism from the consumption
(oxidation) of metabolic fuel to the storage of fuel as fatty acids
89 | P a g e
KETOGENESIS, KETONE BODIES AND KETOACIDOSIS

Mrs Uzoamaka Okoli
WHAT YOU ARE EXPECTED TO KNOW
Ketone bodies
Their functions as fuel to extrahepatic tissues
Ketone body synthesis –Ketogenesis
3 ways Ketogenesis can be regulated
What can cause ketoacidosis
Basics to remember
Fatty acids are degraded by the sequential removal of two-carbon units.
FA are degraded by oxidation at the β carbon
Fatty acids are linked to coenzyme A there are oxidized.
ATP drives the formation of thioester linkage between the carboxyl group of a fatty acid
and the sulfhydryl group of CoA (CoA thioseter).
The activation reaction occurs on the outer mitochondrial membrane where it is
catalyzed by acetyl CoA synthase (Fatty acid thiokinase)
Carbon Atoms of degraded Amino acids emerge in major metabolic intermediates.
The strategy of amino acids degradation is to form major metabolic intermediates that can be
converted to glucose or oxidized by the citric acid cycle
90 | P a g e
Infact, the carbon skeletons, of the diverse set of 20 amino acid are funneled into only seven
molecules: Pyruate, Acetyl CoA, Aceto – acetatyl CoA, α-Ketoglutarate, Succinyl CoA,
Fumarate and Oxaloacetate.
Amino acids that are degraded to acetyl CoA or acetoacetyl CoA are termed Ketogenic because
they give rise to Ketone bodies
In contrast, aminoacids that are degraded to Pyruvate
of the basic set of twenty amino acids, only leucine,and lysine are purely ketogenic (
isoleucine phenylalanine, tryptophan and tyrosine are ketgenic or glucogenic and the
remaining 14 aminoacids are termed purely glucogenic.
Acetyl CoA, NADH, and FADH2 Are Generated
in Each Round of Fatty Acid Oxidation
A saturated acyl CoA is degraded by a recurring
sequence of four reactions:
oxidation by flavin adenine dinucleotide(FAD),
hydration,
oxidation by NAD+,
and thiolysis by CoA.
The fatty acyl chain is shortened
by two carbon atoms as a result of these reactions,
and FADH2, NADH, and acetyl CoA are generated.
Because oxidation is on the β carbon, this series of
reactions is called the β-oxidation pathway.
KETONE BODIES
KETOGENESIS
Simply the formation or synthesis of ketone bodies
Two acetyl CoA molecules formed in β-oxidation condense with one another to
form acetoacetyl-CoA by a reversal of the thiolase reaction.
FORMATION OF KETONE BODIES
91 | P a g e
Enzymes catalyzing these reactions are

3-ketothiolase,
hydroxymethylglutaryl CoA synthase,
hydroxymethylglutaryl CoA cleavage enzyme,
and β-hydroxybutyrate dehydrogenase.
Acetoacetate spontaneously decarboxylates to form acetone
KETONE BODIES ARE A MAJOR FUEL IN EXTRAHEPATIC TISSUES
Acetoacetate and 3-hydroxybutyrate are normal fuels of respiration and are
quantitatively important as sources of energy.
Indeed, heart muscle and the renal cortex use acetoacetate in preference to glucose.
In contrast, glucose is the major fuel for the brain and red blood cells in well-nourished
people on a balanced diet.
However, the brain adapts to the utilization of acetoacetate during starvation and diabetes
REGULATION OF KETOGENESIS
Ketogenesis is regulated at three crucial steps:
(1)control of free fatty acid mobilization from adipose tissue;
the activity of carnitine palmitoyltransferase-I in liver, which determines the
proportion of the fatty acid flux that is oxidized rather than esterified;
and (3) partition of acetyl-CoA between the pathway of ketogenesis and the citric acid cycle.
Carnitine Carries Long-Chain Activated Fatty Acids into the Mitochondrial Matrix
Fatty acids are activated on the outer mitochondrial membrane, whereas they are oxidized in
the mitochondrial matrix.
A special transport mechanism is needed to carry long-chain acyl CoA molecules across the
inner mitochondrial membrane.
Activated long-chain fatty acids are transported across the membrane by conjugating them
to carnitine, a zwitterionic alcohol
Acyl group is transferred from the sulphur atom of CoA

to the hydroxyl group of Carnitine
Catalyzed by Carnitine Acyl transferase 1 Locatated in the cytosolic face of the inner
mitochondrial matrix
92 | P a g e
Acyl carnitine is then shuttled across the inner mitochondrial membrane by a translocase
The acyl group is traacnsferred back to CoA on the matrix side of the membraane.
This reaction, which is catalyzed by carnitine acyltransferase II (carnitine palmitoyl
transferase II)
Finally, the translocase returns carnitine to the cytosolic side in exchange for an incoming
acyl carnitine
Ketoacidosis
Ketoacidosis is a metabolic state associated with high concentrations of ketone bodies, formed by
the breakdown of fatty acids and the deamination of amino acids. Ketoacidosis Results From Prolonged
Ketosis. Higher than normal quantities of ketone bodies present in the blood or urine constitute
ketonemia (hyperketonemia) or ketonuria, respectively. The overall condition is called ketosis.
Acetoacetic and 3-hydroxybutyric acids are both moderately strong acids and are buffered when present
in blood or other tissues. However, their continual excretion in quantity progressively depletes the alkali
reserve, causing ketoacidosis. This may be fatal in uncontrolled diabetes mellitus
In diabetic ketoacidosis, a high concentration of ketone bodies is usually accompanied by

insulin deficiency, hyperglycemia, and dehydration. Particularly in type 1 diabetics the lack of
insulin in the bloodstream prevents glucose absorption, thereby inhibiting the production of
oxaloacetate through reduced levels of pyruvate, and can cause unchecked ketone body production
(through fatty acid metabolism) potentially leading to dangerous glucose and ketone levels in the
blood. Hyperglycemia results in glucose overloading the kidneys and spilling into the urine
(transport maximum for glucose is exceeded). Dehydration results following the osmotic movement
of water into urine (Osmotic diuresis), exacerbating the acidosis.
In alcoholic ketoacidosis, alcohol causes dehydration and blocks the first step of gluconeogenesis
by depleting oxaloacetate.The body is unable to synthesize enough glucose to meet its needs, thus creating
an energy crisis resulting in fatty acid metabolism, and ketone body formation.
And also in starvation- Ketosis is mild in starvation but severe in diabetes mellitus
93 | P a g e
ESSENTIAL FATTY ACID

They are said to be essential because they are not produced in the body of animals, but are needed by
the body for our survival.
These are polyunsaturated fatty acids (PUFA)
linoleic acid (18c, ω-6, 2= bonds), LA
αlinolenic acid (18c, ω-3, 3= bonds) ALA
Arachidonic acid (20c, ω-6, 4= bonds) AA
In human arachidonic acid is produced from linoleic acid hence not classified as essential in
humans. Human are not able to incorporate double bond between 3 and 4 or 6 and 7 carbon atoms of
a fatty acid. Hence they require dietary source of linoleic and linolenic acid
Omega 3 FA are broken down in the body into Eicosapentanoic acid (EPA) and Docosahexanoic
acid (DHA). Omega 6 FA are broken down in the body into Arachidonic acid
Omega 3 FA(ALA)
SOURCE: includes fish oil, salmon, sardines ,codliver oil, olive oil, nuts etc
functions includes
Anti-inflammatory agent,
gene expression,
cell signaling,
improves bone,
brain and CVS health,
immunity.
Deficiency causes skin changes(eczema,flaky skin),slow healing.sterility,arthritis,heart diseases,
Omega 6 FA(Linoleic Acid)

SOURCE includes vegetable oil,corn oil,soybean oil,sun flower oil,egg,
nuts FUNCTIONS include
inflammatory agent,
eicosanoid synthesis
DEFICIENCY causes lack of growth, poor wound healing ,vison loss,learning
difficulty,mood changes.
CHOLESTEROL
Steroid from which other steroids are formed
27C compound widely distributed in the body
Light yellow crystalline solid soluble in chlorofoam and other fat solvents.
Most important animal
Found mainly in euchariots as cholesteryl esters but absent in prokaryotes.
Present in tissues and plasma lipoproteins as free cholesterol.
94 | P a g e
Eliminated from the body in the bile as cholesterol or bile salts.
FUNCTIONS
An important component of cell membranes and modulate fluidity
Helps insulate nerve fibres since it is a poor conductor
Involved in fmxn of bile acid and salts used in fat absorption
Involved in synthesis of steroids eg. Glucocorticoids, androgen and estrogen
Involved in synthesis of vitamin D3 from 7dehydrocholesterol
Forms cholesterol esters via esterification of the OH grp at C3.
STRUCTURE
Has a cyclopetanoperhydrophenanthrene ring structure
Made up of 3 cyclohexane rings (A,B,C)and a cyclopentane ring (D)
A total of 27 carbon atoms
OH grp at the 3rd position
Double bond between C5 and C6.
An 8 carbon side chain attached at C17.
Cholesterol Degradation Products

Humans cannot metabolise cholesterol ring structure to Co2 and H2O.
Rather the steroid nucleus is eliminated by conversion to bile salts\bile acid
Bile acids also serve as solubilizing agent to enhance its excretion in faeces.
Others are converted to other products like steroids e,g glucocorticoids
BILE
Consist of phosphatidylcholine or lecithin and conjugated bile salt/acid.
Either stored in gall bladder or secreted into the duodenum from the liver for digestion.
They serve as emulsifying agents for digestion of dietary fats(TAG) and other complex lipids.
95 | P a g e
BILE ACID
Bile acid is a 24 carbon cpds synthesized in the liver from cholesterol
It has an alpha oriented hydroxyl group at position 7
They are the major excreted form of cholesterol
SYNTHESIS
The reaction of its synthesis is divided into 3 stages
Hydroxylation at C7 and C12 catalysed by microsomal hydroxylase.

7 α hydroxylation is the 1st committed step by 7 α hydroxylase
Followed by 12 α hydroxylation by 12 α hydroxylase(occurs only in production of
cholic acid). NADPH and O2are required for this reaction. vitamin C acts as a cofactor
Removal of 3 carbon units to make it 24 carbon unit.

Here the side chain hydroxylated at C26 is oxidised to an acid containing COOH gp.
This is followed by removal of propionic acid (3 carbon units)
The pathway is divided into 2
One forming cholic acid and the other forming chenodeoxycholic acid [Primary bile acid]
Conjugation of the side chain COOH grp with either glycine or

taurine. This forms the conjugated primary bile acids
Glycocholic and taurocholic acid from cholic acid
Glycochenodeoxycholic acid and taurocheno- deoxycholic acid from chenodeoxycholic acid.
Note that reduction of the double bond in the ring system uses NADPH.
The conjugated bile acids are excreted through bile
SECONDARY BILE ACID/BILE SALT

Secondary bile acids are produced by action of intestinal bacteria on 10 bile acids.
This is by deconjugation and 7 α dehydroxylation.
The 20 bile acids are deoxycholic acid from cholic acid and lithocholic acid from
chenodeoxycholic acid
STEROID HORMONES
Cholesterol is the precursor of all classes of steroid hormone.
Glucocorticoids (cortisol)
Mineralocorticoids (Aldosterone)
Sex hormones (androgens,estrogen,progesterone)
These are carried by plasma proteins from site of production to site of action viz PP,CBG,SHBG
SYNTHESIS
Synthesis involves shortening the hydrocarbon chain of cholesterol and
hydroxylation of the steroid nucleus.
The rate limiting reaction is conversion of cholesterol to pregnenolone
This is in the presence of CYP P450 oxidase enz , desmolase and NADPH
96 | P a g e
Pregnenolone is converted to progesterone

Progesterone is converted to either the aldosterone, cortisol or sex hormones
These involves other steps controlled by other enzymes
Deficency of any of this enzymes gives rise to some genetic diseases.
LDL CHOLESTEROL AND ATHEROSCLEROSIS

Atherosclerosis was derived from 2 Greek words; athero-artery and sclerosis- hardening
Here oxidized LDL cholesterol particles are deposited in the wall of arteries.
Plasma LDL is mainly regulated via the apo B-LDL receptor pathway
But a small part are degraded by non specific uptake by macrophages.
This process is accelerated by free radical oxidative damage of LDL.
When macrophages are overloaded with cholesterol they form foam cells
Foam cells form the hall mark of atherosclerotic plaques.
The smooth muscles cells migrate from the media to the intima of the vessel walls.
Here they proliferate and deposit ECM components like collagen
Thus converting the fatty streak into a mature fibrofatty atheroma.
Growth factors from platelets and macrophages eg;PDGF are involved
The plaque leads to narrowing of the vessel lumen
The blood flows here becomes turbulent with tendency of clot formation.
This occludes vessels leading to ischemia of tissues they supply.
Subsequently infarction or death due to reduced oxygen supply eg; MI
This process is reversible if checked early , if not it becomes irreversible.
This is by reducing plasma lipids esp LDL-chol and increasing HDL
RISK FACTORS FOR ATHEROSCLEROSIS

Serum cholesterol >240mg/dl
LDL-cholesterol >160mg/dl
HDL-cholesterol <60mg/dl
Ratio of apo B:A, of 1.4 and above
Lp[a] >30mg/dl especially with increase LDL
Serum TAG >150mg/dl
Homocysteine level >15umol/l
Increase hormone sensitive C-reactive protein
97 | P a g e
Other predisposing factors
Obesity (truncal)
Sedentary life style
Hypertension and DM
Cigarette smoking and alcohol
Stress (type A personality)
Increase age
Gender (males more than females)
Post menopausal oestrogen deficiency
High carbohydrate intake
Infection like chlamydia pneumonia
PREVENTION
Involves reduction of body’s total and LDL –cholesterol.
Increasing HDL –cholesterol.
This is by reducing dietary cholesterol by using vegetable oils which contains PUFA
Increase intake of green leafy vegetables.
Increase exercise.
Use of agents that will increase excretion of bile acids while decreasing reabsorption.
DISORDERS OF LIPID METABOLISM

These are known as lipid storage diseases.
They include
sphingolipidoses,
leukodystrophies and
demyelinating diseases
SPHINGOLIPIDOSES
Sphingolipids are catabolised by a grp of lysosomal hydrolytic enzymes
Absence of this enzymes leads to sphingolipid deposition disease.

These includes:
NIEMANN PICK DISEASE

Here there is lack of sphingomyelinase.
Leads to accumulation of sphingomyelin and cholesterol in brain, liver and
spleen. Results in mental retardation ,hepatosplenomegally, neurological deficits.
And cherry red spot in macular of the
retina. Death occurs by 2-3 years of age.
GAUCHER’S DISEASE
Deficiency of beta-glucocerebrosidase.
Leads to accumulation of glucocerebroside in liver, spleen and brain.
98 | P a g e
Cerebroside filled cells in the bones (Gaucher cells).

Causes hepatosplenomegally, erosion of long bones,neurological deficits and mental
retardation.
TAY SACH’S DISEASE

Deficiency of hexosaminidase A
Leads to deposition of GM2 gangliosides in brain and macular of retina.

Causes cherry red spot, blindness, mental retardation, muscular weakness.
Death occurs at age 3-4 years.
SANDHOFF’S DISEASE
Absence of hexosaminidase A and B.
Leads to deposition of globosides and GM2 gangliosides in the retina and brain.
Causes cherry red spot, mental retardation and neurological deficit
Symptoms similar to Tay sach’s but are rapidly progressing.
FABER’S DISEASE
Here there is deficiency of ceramidase .
Leads to accumulation of ceramide.
Characterised by dermatitis , hoarseness, skeletal deformation, mental retardation and tissue

granulomas.
FABRY’S DISEASE
Deficiency of α-galactosidase
Leads to accumulation of ceramide trisaccharides(globosides) in the skin and kidney.

Presents with renal failure , skin rash, telangiectasis , pain in the lower extremeties.
LEUKODYSTROPHIES
Metachromatic leukodystrophy:
Absence of arysulfatase A.
Leads to accumulation of sulfogalactosyl ceramides(sulfatides) in white matter of brain.

Presents with neurological deficit, optic atrophy and speech difficulty.
OTHERS
Zellweger syndrome
Defect in peroxisomal beta-oxidation of VLCFA due to absence of peroxisome
biogenesis.
99 | P a g e
Leads to accumulation of VLCFA in blood and tissues.
MULTIPLE SCLEROSIS
A demyelinating disease characterised by loss of phospholipids eg;
sphingophospholipids and ethanolamine plasmalogen from white matter of the
brain and CNS.
Hence the white matter resembles the grey matter
100 | P a g e
EICOSANOIDS
Eicosanoids:
Compounds containing a 20-carbon core
Comprise:
prostaglandins
thromboxanes
leukotrienes
lipoxins
hydroxyeicosatetraenoic acids (HETEs)

hepoxilins
Eicosanoid biosynthesis
In polyunsaturated fatty acid metabolism, especially metabolism of linoleic and arachidonic acid:
101 | P a g e
Main sites of eicosanoid biosynthesis

Endothelial cells
Leukocytes
Platelets
Kidney
102 | P a g e
Unlike histamine, eicosanoids are NOT synthesized in advance and stored in granules – when needed,
they can be produced very quickly from arachidonate released from membranes
Main steps of eicosanoid biosynthesis
1) Activation of phospholipase A2 (PLA2)
2) Release of arachidonate from membrane phospholipids by PLA2
3) Eicosanoid synthesis: COX or LO pathway + subsequent cell-specific modifications by

synthases / isomerases (conversion of the precursor PGH2 to another prostanoid, conversion
of LTA4…)
103 | P a g e
Eicosanoid biosynthesis
In almost all cell types (except for red blood cells)
3 pathways:
A) cyclooxygenase (COX) – produces prostaglandins and thromboxanes
B) lipoxygenase (LO) – produces leukotrienes, lipoxins, 12- and 15-HETEs, and

hepoxilins
C) cytochrome P450s (monooxygenases) – produce the other HETEs (20-HETE);

principal pathway in the proximal tubules
Cyclooxygenase (COX) pathway
Prostaglandin H synthase, present as two isoenzymes (PGHS-1/COX-1, PGHS-2/COX-2),

each possessing two activities:
cyclooxygenase – catalyzes addition of two molecules of O2 to the arachidonic acid

molecule, forming PGG2
hydroperoxidase – converts the hydroperoxy function of PGG2 to an OH group (of

PGH2)
The enzyme is also capable of self-catalyzed destruction!
Mostly, a given cell type produces 1 type of prostanoids: platelets produce almost exclusively
thromboxanes, vascular endothelial cells prostacyclins, heart muscle makes PGI2, PGE2,
PGF2
104 | P a g e
PRODUCTS OF COX PATHWAY
Platelets contain thromboxane synthase producing TXA2, TXB2
Vascular endothelial cells contain prostacyclin synthase which converts PGH 2 to prostacyclin PGI2
105 | P a g e
106 | P a g e
Peptidoleukotriene biosynthesis:
107 | P a g e
108 | P a g e
STRUCTURAL FEATURES
Prostaglandins – cyclopentane ring

Thromboxanes – six-membered oxygen-containing ring
Leukotrienes – 3 conjugated double bonds + one more unconjugated
Lipoxins – conjugated trihydroxytetraenes
PROSTAGLANDIN NOMENCLATURE
The three classes A, E, F (third letter) are distinguished on the basis of the functional groups about
the cyclopentane ring
The subscript numerals refer to the number of double bonds in the side chains
The subscript refers to the configuration of the 9–OH group (projects down from the plane of
the ring)
109 | P a g e
Eicosanoids, like hormones, display profound effects at extremely low concentrations

They have a very short half-life; thus, they act in an autocrine or paracrine manner (unlike
hormones)
Biological effects depend not only on the particular eicosanoid but also on the local
availability of receptors that it can bind to
In general, eicosanoids mediate:
inflammatory response, notably as it involves the joints (rheumatoid arthritis), skin

(psoriasis), and eyes
production of pain and fever
regulation of blood pressure
regulation of blood clotting
regulation of renal function
control of several reproductive functions, such as the induction of labor
Mechanisms of action
Via the G protein-coupled receptors:
a) G s stimulate adenylate cyclase (AC)
b) G i inhibit adenylate cyclase
110 | P a g e
Effects of prostaglandins
Mediate inflammation:
cause vasodilation redness, heat (PGE1, PGE2, PGD2, PGI2)
increase vascular permeability swelling (PGE2, PGD2, PGI2)

Regulate pain and fever (PGE2)
PGE2, PGF2 stimulate uterine muscle contractions during labor
Prostaglandins of the PGE series inhibit gastric acid secretions (synthetic analogs are used to treat
gastric ulcers)
Regulate blood pressure: vasodilator prostaglandins PGE, PGA, and PGI2 lower systemic arterial
pressure
Regulate platelet aggregation: PGI2 = potent inhibitor of platelet aggregation
PGE2 inhibits reabsorption of Na+ and water in the collecting duct. PGI2:
vasodilatation and regulation of glomerular filtration rate.
Biological role of thromboxanes
Thromboxanes are synthesized by platelets and, in general, cause vasoconstriction and

platelet aggregation
TXA2 is also produced in the kidney (by podocytes and other cells) where it causes
vasoconstriction and mediates the response to ANGII
Thus, both thromboxanes and prostaglandins (PGI2) regulate coagulation.
In Eskimos, higher intake of eicosapentaenoic acid and group 3 prosta-noids may be
responsible for low incidence of heart diseases and prolonged clotting times since TXA3
111 | P a g e
is a weaker aggregator than TXA2 and both PG3 and TXA3 inhibit arachidonate
release and TXA2 formation
Biological role of leukotrienes

LTs are produced mainly in leukocytes that also express receptors for LTs
Leukotrienes are very potent constrictors of the bronchial airway muscles: (LTC4, LTD4, and LTE4 =
the slow-reacting substance of anaphylaxis)
They increase vascular permeability
They cause attraction (LTB4) and activation of leukocytes (primarily eosinophils and
monocytes), promote diapedesis (increase expression of integrins on the leukocyte
surface), enhance phagocytosis
They regulate vasoconstriction
BUT:
Overproduction of LTB4 was demonstrated in:
Crohn's disease
rheumatoid arthritis
psoriasis
cystic fibrosis
Leukotrienes are also suspected of participating in atherosclerosis development
112 | P a g e
Excessive bronchoconstriction can be found in some forms of asthma
Lipoxins
Lipoxins are produced mainly by leukocytes and platelets stimulated by cytokines (IL-4, TGF-
β):
a) 5-lipoxygenase (5-LO) of neutrophils produces leukotriene LTA4 which enters

platelets where it is converted by 15-LO to LXA4 or LXB4
b) 15-LO of epithelial cells and monocytes forms 15-HPETE which becomes a

substrate of 5-LO and epoxid hydrolase of leukocytes
…transcellular biosynthesis
Main products: LXA4, LXB4
Biological roles of lipoxins
Unlike pro-inflammatory eicosanoids, lipoxins attenuate the inflammation and appear to

facilitate the resolution of the acute inflammatory response
Hypothesis: in the first phase of the inflammatory response, leukotrienes are produced (e.g.
LTB4) → then, the level of PGs rises and PGs „switch“ the syntheses from leukotriene
production to the pathway which, in the 2nd phase, produces lipoxins promoting the
resolution of inflammation
Therefore, potential therapeutic use of LXs in the treatment of inflammatory diseases

(glomerulonephritis, asthma) is being extensively studied
Effects of LXs mediating the resolution of inflammation

LXs inhibit chemotaxis of neutrophils and eosinophils and diapedesis
Inhibit formation of ROS (neutrophils, lymphocytes) and ONOO- (neutrophils)
Inhibit production of specific cytokines by leukocytes
Stimulate non-inflammatory phagocytosis (of apoptotic neutrophils…)
Antagonize LT receptors
Affect not only the cells of the myeloid line:
113 | P a g e
inhibit the contraction of the bronchial smooth muscle

inhibit production of cytokines by the cells of colon, fibroblasts…
inhibit the interaction between leukocytes and endothelial cells
Mediators of different phases of inflammation
Biological effects of HETEs
5-HETE participates in host defense against bacterial infection (chemotaxis and degranulation of
neutrophils and eosinophils)
20-HETE causes vasoconstriction (by its effect on the smooth muscle of vessels); in kidney, it
regulates Na+ excretion, diuresis, and blood pressure
12- a 15-HETE are produced in kidney and participate in the regulation of the renin-angiotensin
system (probably mediate feed-back inhibition of renin; 12-HETE also mediates secretion
of aldosteron induced by ANGII)
Biological roles of hepoxilins

HXA3 stimulates glucose-induced insulin secretion by pancreatic β cells
Under oxidative stress, HXA3 formation is stimulated and HXA3 upregulates the expression of
glutathione peroxidase…compensatory defense response to protect cell viability?
In vitro, stable analogs of HXA3 induce apoptosis of tumour cells and inhibit tumour growth
114 | P a g e
115 | P a g e
Metabolism of Proteins and amino acids I

Outline
Fate of dietary proteins
Essential and nonessential amino acids (nutritionally)
Amino acid transport
Catabolism of amino acids
Role of glutamate in metabolism of amino acid nitrogen
Urea cycle and the regulations
Nitrogen balance
In born errors of amino acid metabolism
Introduction
Proteins are about most abundant cellular molecule, they play important role in almost all biological
processes. Protein digestion begins in the stomach, and the primary proteolytic enzyme is pepsin a
nonspecific protease, with maximal activity at pH 2. Enzymatic hydrolysis of proteins which yields
free amino acids occurs before catabolism of these amino acids can take place.Dietary proteins are
hydrolysed to free amino acids by action of gastric, pancreatic and intestinal proteases and free amino
acid liberated are absorbed into blood stream and transported to the liver through hepatic portal
vein.The liver also synthesizes the major plasma proteins (eg, albumin) and deaminates amino acids
that are in excess of requirements, forming urea, which is transported to the kidney and excreted
Protein turnover: continuous degradation and resynthesize of cellular proteins occur in all forms of
life, each day humansturns 1% to 2% of their total protein.High protein degradation occurs in tissues
undergoing structural rearrangement, e.g uterine tissue during pregnancy, skeletal muscles in
starvation, tadpole tail tissue during metamorphosis. Ornithine decarboxylase is one of the enzymes
with the shortest half-lifethis participate in synthesis of polyamines-important cellular cations useful
in cell differentiation and growth.Haemoglobin is limited by life of red cell, while lens and crystallin
by life of the organism.About 75 % of liberated AAs are reutilized; the remaining excess AAs not
incorporated into protein are rapidly degraded; after deamination (carbon skeleton of amino acids will
be discussed in detail) amino nitrogen is excreted as urea, and the carbon skeletons that remain after
transamination are:
1.oxidized to CO2 via the citric acid cycle
form glucose (gluconeogenesis),
form ketone bodies.
Several amino acids are also the precursors of other compounds, eg, purines, pyrimidines, hormones
suchasepinephrine and thyroxine, and neurotransmitters.
116 | P a g e
Fig. 1: fate of dietary protein, tissue protein and Amino acid metabolism showing major pathways and
end products
Essential and nonessential amino acids

Amino acid deficiency can result if nutritionally essential AAs are absent in diet, this presents with
cases like kwashiorkor and marasmus (energy deficiency inclusive here). Nutritionally essential and
nutritionally nonessential amino acids, all the 20 amino acids are essential for health(therefore, are
better described as nutritionally essential and nonessential AAs), of these 20 AAs, 8 must be present
in diet, this could be best described as nutritionally essential, the other 12 are nutritionally
nonessential (Table 1)
HILL VATT MP(you can use this acronym to remember nutritionally essential AAs); H &A are only
nutritionally essential in children as they cannot be produced at rate which the body needed them, but
are nutritionally nonessential in adults
Table 1: Nutritionally (essential and nonessential amino acids)
Nutritionally Essential Nutritionally Nonessential
*Arginine Alanine
*Histidine Asparagine
Isoleucine Aspartate
117 | P a g e
Leucine Cysteine
Lysine Glutamate
Methionine Glutamine
Phenylalanine Glycine
Threonine Proline
Tryptophan Serine
Valine tyrosine
* Nutritionally ‘semiessential’ synthesised at rates inadequate to support growth of children
Catabolism of amino acids

First step involved in catabolism of AAs is the removal of α-amino group, about 85% of amino
nitrogen is excreted as urea. The two (2) main reactions involved are (i) Transamination, and (ii)
Oxidative deamination
Transamination reactions involve transfer of α-amino group of an amino acid to a keto-acid (mostly α-
keto glutarate to form a new AA (mostly glutamate). Transamination reactions are mostly catalysed
by enzymes called transaminases or aminotransferases. All aminotransferases require pyridoxal
phosphate (a derivative or functional form of vit B6). Most amino acids used to synthesize or as
precursors for amino acid derivatives are obtained from the diet or protein turnover.Transfer of amino
group also occurs during degradation. Transamination is the most reaction involving free amino acids,
threonine, lysine, hydroxyproline and proline do not involve in transamination
reactions.Aminotransferase (transaminase) reactions form pyruvate from alanine, oxaloacetate from
aspartate, and α-ketoglutarate from glutamate. Because these reactions are reversible, the cycle also
serves as a source of carbon skeletons for the synthesis of these amino acids (essential AAs can be
formed from their keto acids when they are therapeutically administered).
Pyridoxal phosphate (PLP) is a cofactor for aminotransferase, amino group transfer involves enzyme
associated intermediate derived from PLP, the active site of the resting aminotransferase contains PLP
covalently attached to anε-amino group of lysine residue of the enzyme. The linkage – CH =N – is
called a Schiff base, the carbon originates from aldehyde group of PLP while nitrogen originates from
lysine residue.
Assignment: 1.Discuss how aminotransferases utilize pyridoxal phosphate to transfer amino group
(use structures to support your answer) important terms to consider include: Schiff base, aldimine,
ketimine, epsilon (ε)- amino group of lysine residue.
2. Write briefly on amino acid transport
118 | P a g e
Role of glutamate in metabolism of amino acid nitrogen

(i). The amino groups of most amino acids are funnelled to glutamate by means of transamination
with α-ketoglutarate,
L-glutamate is unique because it is the only amino acid thatundergoes oxidative deamination at an
appreciable rate in mammalian tissues with liberation of amino group as free ammonia. This reaction
is catalysed by glutamate dehydrogenase (GDH) (the enzyme uses both NAD+ and NADP+ as
coenzyme)
The sequential action of transamination (resulting in the collection of amino groups from other amino
acids onto α-KG to produce glutamate and subsequent oxidative deamination of that glutamate (
regenerating α-KG) provide a pathway whereby the amino groups of most amino acids can be
released as free ammonia.
GDH is an allosteric enzyme: is regulated by ADP, GDP activate the enzyme for energy need towards
glutamate degradation and ATP and GTP indicates availability of energy and activates glutamate
synthesis.
119 | P a g e
Fig. 2: L-glutamate dehydrogenase catalysed oxidative deamination of glutamate
(ii). L-glutamate is involved in synthesis of L-glutamine by glutamine synthetase, glutamine serves

as a storage form of ammonia, during oxidative deamination of glutamate α-KG activates enzyme
glutamine synthetase to produce glutamine and avoid accumulation of toxic ammonia, ammonia is
then transported to liver in form of glutamine.
Figure 3
120 | P a g e
Glutamate is involved in synthesis of N-acetylglutamate (reaction catalysed by N-acetyl

glutamate synthase), the allosteric activator of the rate determining enzyme of urea
synthesis (carbamoyl phosphate synthetase I)
l-Glutamate acetyl-CoA N-
acetyl-l-glutamate CoASH
Transamination of oxaloacetate by glutamate aminotransferase forms aspartate which is the donor

of the second nitrogen for the urea synthesis.
Figure 4
α-KG also undergoes reductive amination to form glutamate (catalysed by Glutamate

dehydrogenase)
Figure 5: Reductive amination
Most of the non-essential amino acids are formed by transamination of their α-keto acids using
amino nitrogen of glutamateeg alanine
121 | P a g e
Figure 6
Alanine is involved in transporting amino group from muscle to liver which involves
glutamate formation from α-KG then making the amino group available for urea synthesis
Non-oxidative deamination
The amino acid of serine and threonine can directly be deaminated by serine and threonine
dehydratases respectively; here PLP is also a prosthetic group; transamination is the most common
reaction involving free amino acids; an obligate amino and α-keto acid pair in all of these reactions is
glutamate and a ketoglutarate.
This means that amino group transfer between alanine and aspartate would have to occur via coupled
reactions, with glutamate intermediate (Figure 7). The equilibrium constant for aminotransferases is
close to one so that the reactions are freely reversible
Fig. 7
The main destination of glutamine and alanine in the blood is the liver (Figure 8). Here ammonia
is released by alanine aminotransferase, glutaminase, and glutamate dehydrogenase
122 | P a g e
Fig. 8: glutamine and alanine transport from muscle to liver
Urea biosynthesis occurs in four stages:
transamination,
oxidative deamination of glutamate,
ammonia transport,
reactions of the urea cycle
123 | P a g e
Fig. 9: The flow of nitrogen in amino acid catabolism
Alanine-pyruvate aminotransferase (alanine aminotransferase)and glutamate-α-ketoglutarate

aminotransferase(glutamate aminotransferase) catalyze the transfer of amino groups to
pyruvate (forming alanine) or to α-ketoglutarate (forming glutamate)
The formation of ammoniafrom α-amino groups thus occurs mainly via the α-amino nitrogen
of L-glutamate, the δ-amino group of ornithine—but not the ε-aminogroup of lysine
124 | P a g e
Fig. 10: Alanine amino transferase Glutamate amino transferase
Urea biosynthesis
Nitrogen atoms of urea come from free ammonia and Aspartate, The urea cycle starts and finishes
with ornithine, the carbons in original and final ornithine are same, the carbon and oxygen of urea is
derived from CO2, urea is produced in liver and transported in blood to the kidney for excretion
Summary
Ammonia condenses with bicarbonate to form carbamoyl phosphate, this reacts with ornithine to form
citrulline, these two reactions occur in mitochondria matrix, aspartate (donor of the second nitrogen)
reacts with citrulline to form argininosuccinate (at expense of ATP), this is then cleaved to form
arginine and fumarate, arginine is then hydrolysed to urea and ornithine, these occur in cytosol
(Figure: 11)
The first reaction is not really part of the cycle but contributes to it.
Synthesis of urea requires five enzymes

Reaction 1
Carbamoyl phosphate synthetase I (CPS I)
125 | P a g e
The enzyme catalyses the condensation of ammonium ion and bicarbonate to form carbamoyl
phosphate (at the expense of 2 ATP) 1 ATP activates bicarbonate while the other donates phosphate
group of carbamoyl phosphate
CPS I occurs in mitochondria matrix and uses free ammonia as nitrogen donor (depends on N-
acetylglutamate (NAG) for activity). CPS I catalyses the rate limiting step in urea biosynthesis and it
is allosterically regulated by NAG. CPS II occurs in cytosol, this uses amide group from glutamine
and participates in pyrimidine biosynthesis, not affected by NAG
Reaction 2
Formation of citrulline
Citrulline formation is catalysed by ornithine transcarbamoylase(ornithine carbamoyl transferase) in
mitochondrial matrix by combination of carbamoyl phosphate and ornithine, thereafter transported to
cytosol by special membrane carrier system
Reaction 3
Argininosuccinatesynthetase catalyses the condensation of citrulline with aspartate to form
argininosuccinate (the α-amino group of aspartate provides the second nitrogen of urea.Here ATP is
hydrolysed (ATP = AMP + PPi) to provide energy needed for the reaction. PPi is an inhibitor of the
step
Reaction 4
Argininosuccinate is cleaved by argininosuccinatelyase (argininosuccinase) to arginine and fumarate,
here with exception of α-amino group of aspartate the molecule is released as fumarate, arginine
formed here becomes the precursor of urea.Fumarate may enter mitochondria and metabolised to
oxaloacetate by fumarase and malate DH of the TCA cycle and become transaminated and can enter
another turn of urea cycle as aspartate.
Reaction 5
Cleavage of arginine
Arginase catalyses the hydrolytic cleavage of arginine to ornithine and urea, ornithine therefore enters
the mitochondria for another round of cycle.
The inner mitochondrial membrane contains a citrulline/ornithine exchange transporter
Land animals and humans excrete urea, they are known to be ureotelic, birds are uricotelic as they are
known to excrete uric acid, while some species of fish excrete ammonia and are referred to as
ammonotelic.The nitrogen-containing groups that contribute to the formation of urea are shaded.
Reactions 1 and 2 occur in the matrix of liver mitochondria, while reactions 3 , 4 , and 5 occur in liver
cytosol.
CO2 (as bicarbonate), ammonium ion, ornithine, and citrulline
enter the mitochondrial matrix via specific carriers present in the inner membrane of liver
mitochondria.
126 | P a g e
Fig. 11: Biosynthesis of urea: Urea cycle: reactions and intermediates of urea biosynthesis.
Regulation
The urea biosynthesis is regulated by an allosteric effector and enzyme
induction CPSI requires activator NAG
NAG is formed from acetyl-CoA and glutamate, a reaction catalysed by N-acetyl glutamate
synthetase which is activated by arginine.
Induction of urea cycle enzymes occur when delivery of ammonia or amino acids to liver rises,
concentration of intermediates also regulates activity through mass action High protein diet and
starvation result in induction of urea cycle
Urea is transported in blood to the kidney where it is filtered and excreted in
urine Stoichiometry
NH3 + CO2+ Aspartate + 3ATP Urea + fumarate + 2ADP + AMP + PPi + 2Pi + 3H2O
All defects in urea synthesis result in ammonia intoxication.

Common to all urea biosynthesis disorders include vomiting, avoidance of high-protein foods,
intermittent ataxia, irritability, lethargy, and mental retardation.
Hyperammonemia Type 1. A consequence of carbamoyl phosphate synthetase I deficiency
Hyperammonemia Type 2. A deficiency of ornithine transcarbamoylase produces X-chromosome
linked deficiency.
Citrullinemiais an autosomal recessive inheritance: This is rare disorder, plasma and cerebrospinal
fluid citrulline levels are elevated and 1–2 g of citrulline are excreted daily, this results from the
deficiency of argininosuccinatesynthetase (In citrullinemia condition feeding arginine and benzoate
promotes nitrogen excretion).
Argininosuccinic aciduria
127 | P a g e
A rare disease characterized by elevated levels of argininosuccinate in blood, cerebrospinal fluid, and
urine is associated with friable, tufted hair (trichorrhexisnodosa). Both early-onset and late-onset
types are known, the metabolic defect isthe absence of argininosuccinase (argininosuccinatelyase).
Diagnosis by measurement of erythrocyte argininosuccinase activity can be performed on umbilical
cord blood or amniotic fluid cells.
Hyperargininemia
Here, elevation of blood and cerebrospinal fluid of arginine occur, low erythrocyte levels of arginase
and a urinary amino acid pattern resembling that of lysine-cystinuria. This pattern may reflect
competition by arginine with lysine and cystine for reabsorption in the renal tubule. A low-protein
diet lowers plasma ammonia levels and abolishes lysine-cystinuria
Gene therapy for rectification of defects in the enzymes of the urea biosynthesis is an area of active
investigation.
Encouraging preliminary results have been obtained, for example, in animal models using an
adenoviral vector to treat citrullinemia.
Nitrogen balance
A healthy adult eating different and plenty diet is said to be in nitrogen balance. Here, nitrogen intake
equals nitrogen excreted daily, resulting in no net change in the amount of body nitrogen.
Positive nitrogen balance occurs when excess is ingested over excreted this occurs in growing
children, who are increasing their body weight and incorporating more amino acids into proteins than
they break down, this also occurs during pregnancy and re-feeding after starvation.
Negative nitrogen, here excreted nitrogen exceeds intake; this occurs in surgery, advanced cancer, in
malnutrition and during starvation
In starvation carbon chains of amino acids from proteins are needed for gluconeogenesis; ammonia
released from amino acids is excreted mostly as urea, these are not reincorporated into protein. A diet
deficient in an essential amino acid also leads to a negative nitrogen balance, since the body proteins
are degraded to provide the deficient essential amino acid and may also exist in senescence.
Catabolism of carbon skeleton of amino acids

Transamination is the first reaction in catabolism of AAs(exception are proline, hydroxyproline,
lysine and threonine) the carbon skeleton are degraded into amphibolic intermediates (fig 12). The
carbon skeleton of amino acids are mobilised into 7 molecules, which include pyruvate, oxaloacetate,
α-KG, fumarate, acetyl-CoA, acetoacetyl CoA, or succinyl- CoA
Amino acids can be classified into glucogenic and ketogenic according to their metabolic end
products, in this Leucine and lysine are purely ketogenic because their catabolism yields acetyl- CoA
and acetoacetyl CoA
14 amino acids are purely glucogenic, and only 4 are both ketogenic and glucogenic
Table 2: classification of amino acids based on degradation

Purely glucogenic Purely ketogeic Glucogenic/ketogenic
Alanine Methionine Leucine Isoleucine
Arginine Proline Lysine Phenylalanine

Aspartate Serine Tryptophan
Cysteine Threonine Tyrosine
Valine Glutamate
Histidine Glycine
128 | P a g e
Asparagine Glutamine
Fig. 12: Amphibolic intermediates formed from catabolism carbon skeleton of AAs
Amino acid converted to pyruvate: 6 amino acids form pyruvate: these include glycine, serine,
alanine, hydroxyproline, cysteine, threonine
Degradation of AAs
Alanine
Alanine is converted to pyruvate in a transamination reaction with α-ketoglutarate catalysed by
alanine aminotransferase, may be because of the central role of alanine in metabolism, no metabolic defect is
known about its catabolism.
129 | P a g e
Fig. 13:
Cystine and cysteine

Cystine is first reduced to cysteine by cystinereductase, two different pathways then
convertcysteine to pyruvate. There are numerousabnormalities of cysteine metabolism.
Cystine, ornithine arginine and lysine (COAL) are excreted in cystine-lysinuria (cystinuria),
a defect in renal reabsorption of these amino acids. Apart fromcystine calculi, cystinuria is
benign. The mixed disulfide ofl-cysteine and l-homocysteine excreted bycystinuric patients is
more soluble than cystine and reduces formation of cystine calculi.
Several metabolic defects result in vitamin B6-responsive or vitamin B6-unresponsive
homocystinurias. These include a deficiency in the reaction catalyzed by cystathionine β-
synthase
Serine homocysteine cystathionine H2 O
Consequences include osteoporosis and mental retardation. Defective carrier-mediated

transport of cystine results in cystinosis (cystine storage disease) with deposition of cystine
crystals in tissues and early mortality from acute renal failure. Epidemiologic and other data
link plasma homocysteine levels to cardiovascular risk, but the role of homocysteine as a
causal cardiovascular risk factor remains controversial.
Cysteine
130 | P a g e
3-mercaptopyruvate pathwayCatabolism of L-cysteine via the cysteine sulfinate pathway

Here transamination of cysteine yields
thiopyruvate and loss of H2S results in pyruvate
Fig 14: degradation of cysteine
Catabolism of glycine
The glycine cleavage complexof liver mitochondria splits glycine to CO2 and NH4
+
and forms N5, N10-methylene tetrahydrofolate.
+
Glycine H4folate NAD CO2 NH3 5,10-CH2-
+
H4folate NADH H
The glycine cleavage system (or complex) consists of three enzymes and an “H-protein” that
has a covalently attached dihydrolipoyl moiety. The enzymes included (1) glycine
dehydrogenase (decarboxylating), (2) an ammonia-forming aminomethyltransferase, and (3)
dihydrolipoamide dehydrogenase (H4folate, tetrahydrofolate).
In nonketotic hyperglycinemia, a rare inborn error of glycine degradation presently known

only in Finland, glycine accumulates in all body tissues including the central nervous system.
The defect in primary hyperoxaluriais the failure to catabolize glyoxylate formed by the
deamination of glycine. Subsequent oxidation of glyoxylate to oxalate results in urolithiasis,
nephrocalcinosis, and early mortality from renal failure or hypertension.Glycinuriaresults
from a defect in renal tubular reabsorption.
Serine
Catabolism of serine continues with that of glycine after the first reaction which converts serine to
glycine, the reaction is catalyzed by glycine hydroxymethyltransferase.
Threonine
Threonine aldolase cleaves threonine to glycine and acetaldehyde, the oxidation of
acetaldehyde to acetate by aldehyde DH is followed by formation of acetyl-CoA catalysed by
acetate thiokinase, the glycine then follows its pathway to form CO2 and NH3
131 | P a g e
Fig. 15: Intermediates in the conversion ofthreonine to glycine and acetyl-CoA.
Tryptophan
Metabolism of tryptophan is complex, many branch points are involved
The first reaction of tryptophan metabolism is catalysed by tryptophan dioxygenase (or tryptophan
pyrrolase or tryptophan oxygenase (heme containing enzyme), which produces N-formylkynurenine,
further into the reaction glutaryl CoA, picolinic, nicotinate mononucleotide are produced down the
pathway, because kynurenine hydroxylase is inhibited by estrogen, this makes women to be
susceptible to pellagra, disease produced by niacin deficiency (take time to study this pathway)
132 | P a g e
Serotonine and melatonine are tryptophan derivatives (read about them)
Fig. 16 tryptophan degradation
Enzymes indicated by number are

tryptophan oxygenase,
kynurenineformamidase,
kynurenine hydroxylase,
kynureninase,
aminotransferase,
3hydroxyanthranilate oxidase,
spontaneousnonenzymatic reaction,
picolinate carboxylase,
quinolinatephosphoribosyltransferase,
aldehyde dehydrogenase, and
complex series of reactions
Proline
133 | P a g e
The catabolism of proline takes place in mitochondria. Since proline does not participate in
transamination,its α-amino nitrogen is retained throughout a two-stage oxidation to
glutamate. Oxidation to 1-pyrroline-5-carboxylate is catalyzed byproline dehydrogenase,
followed by oxidation to glutamate that is catalyzed by 1-pyrroline-5-carboxylate
dehydrogenase (or glutamate- -semialdehde dehydrogenase). There are two metabolic
disorders of proline catabolism; this is inherited as autosomal recessive traits, both are
consistent with a normal adult life. The metabolic block in type I hyperprolinemiais at proline
dehydrogenase. The metabolic block in type II hyperprolinemiais at 1-pyrroline-5-
carboxylate dehydrogenase, which also participates in the catabolism of arginine, ornithine,
and hydroxyproline (see below). Since proline and hydroxyproline catabolism are affected,
both 1-pyrroline-5-carboxylate and 1-pyrroline- 3-hydroxy-5-carboxylate are excreted.
Fig. 17 Proline degradation
134 | P a g e
Fig. 18:
Histidine
Catabolism of histidine proceeds viaurocanate, 4-imidazolone-5-propionate, and N-
formiminoglutamate
(Figlu).Formimino group transfer to tetrahydrofolate forms glutamate, subsequently α-
ketoglutarate.
Disorders of histidine of catabolism include histidinemia and urocanicaciduria associated
with impaired histidase.Catabolism of histidine proceeds via urocanate, 4-imidazolone- 5-
propionate, and N-formiminoglutamate (Figlu). Formimino group transfer to tetrahydrofolate
forms glutamate, then -ketoglutarate (Figure 18). In folic acid deficiency, transfer of the
formimino group is impaired, and Figlu is excreted. Excretion of Figlu following a dose of
histidine thus can be used to detect folic acid deficiency. Benign disorders of histidine
catabolism include histidinemiaand urocanic aciduria associated with impaired histidase.
135 | P a g e
Arginine and Ornithine.

Arginine is converted toornithine, glutamate γ-semialdehyde, and then α-ketoglutarate
Mutations in ornithine-aminotransferase elevate plasma and urinaryornithine and cause
gyrate atrophy of the retina.Treatment involves restricting dietary arginine.
In hyperornithinemia-hyperammonemia syndrome, a defective mitochondrial ornithine-
citrulline antiporter impairs transport of ornithine into mitochondria for use in urea synthesis.
Fig. 19:
Aspartate and asparagine are converted to oxaloacetate, utilize during gluconeogenesis or oxidation
through TCA, these reactions are similar to that of glutamine and glutamate but here α-ketoglutarate,
no metabolic defects associate with metabolism of these Aas
Catabolism to amphibolic intermediates of L-asparagine(top) and of L-glutamine (bottom).(ALA, L-

alanine; PYR, pyruvate.)
136 | P a g e
Fig. 20: Phenylalanine degradation and tyrosine
Tyrosine is the first product of phenylalanine degradationTyrosine is synthesized by

hydroxylation of phenylalanine, three quarters of ingested phenylalanine is hydroxylated to
tyrosine by phenylalanine hydroxylase (this depends on tetrahydrobiopterin) (Figure 20)
Further degradation of tyrosine results in formation of fumarate and acetoacetate. First
tyrosine is transaminated to p-hydroxyphenylpyruvate by tyrosine aminotransferase (the
enzyme is inducible by glucocorticoids and dietary tyrosine). P-hydroxylphenylpyruvate
oxidase produces homogentisic acid (in a complex reaction that involves decarboxylation,
oxidation, migration of carbon side chain and hydroxylation) (one of the reactions require
ascorbic acid for activity). The aromatic ring is next cleaved by homogentisate oxidase (iron
containing enzyme) to maleylacetoacetate, this isomerises from cis to trans to form
fumarylacetoacetate by maleylacetoacetate isomerase (may require glutathione for activity)
Fumarate can be utilized in TCA cycle for energy or for gluconeogenesis, acetoacetate can be
used as acetyl CoA, for lipid synthesis or energy
The probable metabolic defect in type I tyrosinemia (tyrosinosis) is at fumarylacetoacetate

hydrolase (fumarylacetoacetate)
Therapy employs a diet low in tyrosine and phenylalanine. Untreated acute and chronic
tyrosinosis leads to death from liver failure. Alternate metabolites of tyrosine are also
excreted in type II tyrosinemia(Richner-Hanhart syndrome),a defect in
tyrosineaminotransferase, and in neonataltyrosinemia, due to lowered activity of p-
hydroxyphenylpyruvate hydroxylase, therapy employs a diet low in protein.
137 | P a g e
The metabolic defect in alkaptonuriais a defective homogentisate oxidase, the urine darkens
on exposure
to air due to oxidation of excreted homogentisate. Late in the disease, there is arthritis and
connective tissue pigmentation(ochronosis) due to oxidation of homogentisate to
benzoquinone acetate, which polymerizes and binds to connective tissue.
Hyperphenylalaninemiasarise from defects in phenylalaninehydroxylase, (type I, classic

phenylketonuria
(PKU), frequency 1 in 10,000 births), in dihydrobiopterin reductase (types II and III), or in
dihydrobiopterin biosynthesis (types IV and V) (see Figure 27–12). Alternative catabolites
are excreted (Figure 29–14). A diet low in phenylalanine can prevent the mental retardation
of PKU. DNA probes facilitate prenatal diagnosis of defects in phenylalanine hydroxylase or
dihydrobiopterin reductase.
Fig. 21:
138 | P a g e
Fig.:
Methionine metabolism
First in methionine metabolism is production of S-adenosylmethionine (SAM), here methionine is
condensed with ATP in the presence of methionine adenosyl transferase, the reaction proceeds to
form propionyl CoA, with rearrangement through methyl malonyl CoA results in succinyl CoA
139 | P a g e
Lysine degradation
The first six reactions of l-lysine catabolism in human liverform crotonyl-CoA, which is then
degraded to acetyl-CoA by the reactions of fatty acid catabolism .
Reactions 1 and 2 convert the Schiff base formed between α-ketoglutarate and the ε-amino
group of lysine to l-α-aminoadipate-δ-semialdehde. Reactions 1 and2 both are catalyzed by a
single bifunctional enzyme, aminoadipatesemialdehde synthase, whose N-terminal and C-
terminal domains contain lysine-α-ketoglutarate reductase and saccharopine dehydrogenase
activity, respectively. Reduction of l-α-aminoadipate-δ-semialdehde to l-α- aminoadipate
(reaction 3) is followed by transamination to α-ketoadipate (reaction 4). Conversion to the
thioester glutaryl- CoA (reaction 5) is followed by the decarboxylation of glutaryl- CoA to
crotonyl-CoA (reaction 6). Subsequent reactions are those of fatty acid catabolism.
Hyperlysinemia can result from a metabolic defect in either the first or second activity of the
bifunctional enzyme aminoadipatesemialdehde synthase, but this is accompanied by elevated
levels of blood saccharopine only if the defect involves the second activity. A metabolic
defect at reaction 6 results in an inherited metabolic disease that is associated with striatal and
140 | P a g e
cortical degeneration, and is characterized by elevated concentrations of glutarateand its

metabolites glutaconateand 3-hydroxyglutarate. The challenge in management of these
metabolic defects is to restrict dietary intake of l-lysine
without producing malnutrition.
Table 3: some in born errors associated with amino acids
141 | P a g e
NUCLEIC ACID METABOLISM

BY
Dr. NDUBISI A.C
DEPT. OF MED. BIOCHEMISTRY
Nucleic acid Metabolism
Nucleotides play a variety of important roles in all cells. They are the precursors of DNA and
RNA. They are the essential carriers of chemical energy-a role primarily plays by ATP and
to some extent GTP.
They are components of the cofactors NAD,FAD,S-adenosylmethionine, and coenzyme A, as
well as of activated biosynthetic intermediates such as UDP-glucose and CDP-diacylgylcerol.
Some such as cAMP and c-GMP are also cellular second messenger.
Types of Pathway for nucleotide synthesis
De novo pathway
Salvage pathway
De novo synthesis
This begins with their metabolic precursors: amino acids, ribose-5-phosphate, co2 and HN3.
Salvage Pathway.
This recycle the free bases and nucleosides
released from nucleic acid breakdown.
Both types of pathways are important in cellular metabolism.
De novo synthesis of Purines.

STEP 1
In the first committed step of the pathway, an amino group donated by glutamine is attached at
c1 of PRPP.
The resulting s-phosphoribosylamine is highly unstable, with a half life of 30 sec. at a pH of
7.5. The purine ring is subsequently built on the structure.
STEP 2
The addition of 3 atoms from glycin. An ATP is consumed to activate the glycine carboxyl grp (in
the form of an acyl phosphate) for this condensation reaction.
STEP 3
The added glycin amino grp is then formulated by N10- formyltetrahydrofolate.
STEP 4
Ntrogen is contributed by glutamine.
STEP 5
Dehydration and ring closure yield the five membered imidazole ring of the purine nucleus, as
5-iminoimidazole ribonucleotide.
At this point, three of the six atoms needed for the second ring in the purine structure are in
place. STEP 6
Carboxyl group is added.
This carboxylation is unusual in that it does not contain biotin but instead it uses the
bicarbonate generally present in acqeous solution.
Rearrangement.
STEP8&9
Aspartate donates its amino group in two steps.
STEP 10
The final carbon contribution by N10 formyl tetrahydrofolate.
A second ring closure takes place to yield the second fused ring of purine nucleus.
The first intermediate to have a complete purine ring is inosinate (IMP)
142 | P a g e
THE REACTION PATHWAY
143 | P a g e
144 | P a g e
Biosynthesis of AMP and GMP from IMP

Conversion of inosinate to adenylate requires the insertion of an amino group derived from
aspartate; this takes place in two reactions similar to the used to introduce N-1 of purine ring
(Step 8 & 9).
A key difference is that GTP rather than ATP is the source of the high –energy phosphate in
synthesizing adenylosuccinate.
Guaninylate is formed by the addition of an amino group derived from glutamine. ATP is
cleared to AMP and PPi in the final step.
Purine Nucleotide Biosynthesis is regulated by feedback inhibition.
Three major feedback mechanisms cooperate in regulatory the overall rate of de novo purine
nucleotide synthesis and the relation rates of formation of the two end products, adenylate
and guanylate.
The first mechanism is exerted on the first reaction that is unique to purine synthesis- transfer of
amino group to PRPP to form 5-phosphoribosyllamine. This reaction is catalyzed by the
atmospheric enzyme glutamine-PRPP aminotransferase, which is inhibited by the end
product IMP, AMP and GMP. These same nucleotide also inhibits the synthesis of PRPP
from ribose phosphate by ribose phosphate pyrophosphate kinase. AMP and GMP act
synergistically in this concerted inhibition.
Thus, whenever either AMP or GMP accumulates to excess, the first step in the biosynthesis
from PRPP is partially inhibited.
145 | P a g e
In the second control mechanism, exerted at a later stage, an excess of GMP in the cell inhibits
formation of xanthylate from inosinate by IMP dehydrogenase, without affecting the
formation of AMP-Conversely, an accumulation of adenylate inhibits formation of
ademylosuccinate synthetase, without affecting the biosynthesis of GMP.
In the third mechanism, GTP is required in the conversion of IMP to GMP, a reciprocal
arrangement that tends to balance the synthesis of two ribonucleotides.
DIAGRAM
Adenosuccinate
Inosinate Adenylate(AMP)
Xanthine Guanylate(GMP)
SALVAGE PATHWAY FOR PURINES

Purine that result from the normal turnover of cellular nucleic acids, or that are obtained from the
diet and not degraded, can be reconverted into nucleoside triphosphates and used by the body. This is
referred to as the “salvage pathway” for purines.
Conversion of purine bases to nucleotides.
Two enzymes are involved: adenine phosphoribosyl transferase (APRT) and hypoxanthine-
guanine
phosphoribosyltranferase (HPRT). Both enzymes use PRPP as their source of ribose-5-phosphate
group. The release of pyrophosphate makes these reactions irreversible.
• Diagram
PRPP PPi
Hypoxanthine IMP
HP-GP ribosyltransferase
PRPP PPi
Guanine GMP
HG-GP ribosyltransferase
PRPP PPi
Adenine AMP
Adenine P-ribosyltransferase
Regulation of purine nucleotide synthesis

The committed step in de novo synthesis is the reaction catalysed by amidotransferase is
inhibited by AMP and GMP.
Availability of PRPP is another important regulatory factor.
The activity of PRPP synthetase is regulated by negative modfiers; purines and pyrimidine
nucleotides.
DEGRADATION OF PURINE NUCLEOTIDE

Degradation of dietary nucleic acids occurs in the small intestine where a family of pancreatic
enzymes hydrolyses the nucleotides to nucleosides and free bases.
Inside cells, purines nucleotides are sequentially degraded by specific enzymes, with uric acid as
the end products of this pathway (mammals other than Primates oxidize uric acid further to
allantoin, which in some animals other than mammals may be further degraded to urea or
ammonia).
146 | P a g e
Degradation of dietary nucleic acid in the small intestine

Ribonucleases and dioxyribonucleases secreated by the pancrease hydrolyse RNA and DNA
primarily to Oligonucleotides.
Oligonucleotides are further hydolysed by pancreatic phosphodiesterases producing a mixture of 3’- and
5’ mononuleotides. A family of nucloetidaeses removes the phosphate groups hydrolytically releasing
nucleosides that may be absorved by intestinal mucosa cells, or be furhther degraded to free bases
before uptake.
Note: dietray purines and pyrimidines are not used to a large extent for the synthesis of tissue
nucleoic acid. Instead the dietray purines are generally converted touric acid by intestinal
mucosa cells.Most of the uric acid enters the blood and is eventually excreted in the urine.
For this reasons individual with a tendency towards gout should be careful about consuming
food such as organ meat, sardine or dry beans which contains high amount of nucleic acid.
The remainder of the dietary purines are metabolized by intestinal flora.
DISEASES ASSOCIATED WITH PURINE DEGRADATION

1. GOUT;
This is a disorder characterized by high level of uric acid in the blood as a result of over
production or under excretion of uric acid.
Hyperuricemia: Results in the deposition of crystals of soduim urates- the end product of
purine metabolism- in the tissue especially the kidneys and joints causing acute and
progressing to chronic gouty athritis (NB Hyperuricemia does not always lead to gout but
gout usually procceed to hyperuricemia)
The deposition of needle shaped monosodium urate crytsals initiates an inflamatory process
involving the infiltration of granulocides that phagosotize the urate crystals. This process
generates oxygen metabolites that damage tissue resulting in the release of lysosomal
enzyme that evoke an inflamatory response. In addition lactate production in the synovial
tissues increases, resulting in a decrease in PH that foster further deposition of urate crystals.
Types of gouts
Primary gouts: Over production of uric acid may occur because of an inherited
abnormality in the enzyme of purine metabolism. This is defined as primary gout.
Example :
X-linked mutations in the PRPP synthethase gene, Lesch –Nyhan syndrome which also
causes hyper uricemia as a result of the decreased salvage of hypoxanthine and guanine
basis.
Lesch –Nyhan syndrome: This is an X-linked reccessive inherited disorder associated with a
visually complete deficiency of hypo xanthine guanine phospho ribosyl transferase and
therefore the inability to salvage hypoxanthine and guanine. The enzyme deficiency results
in the increase level of PRPP and decreased IMP and GMP, causing increasd de novo purine
synthesis.This results in excessive production of uric acid plus characteristics nuerologic
features including self motilation and involuntary movement.
B. Secondary hyper uricemia: This form of gout is caused by a variety of disorder and
lifestyles for example in patients with chronic renal insufficiency, those undergoing
chemotherapy, those with myeloproliferative disorders, and those who consume excess
amount of alcohol or purine rich food. Gout can also be an adverse effect of seemingly
unrelated metabolic diseases, such as von Gierke diseaes or fructose intolerance.
TREATMENT OF GOUT
Colchincine : This is used in treating acute attack of gout. It decreases the movement
of granulocides in to the affected areas.
ANTI INFLAMMATORY DRUGS: Examples
Aspirin: This relieves pain
Probenecid (or Sulfinpyrazone): This prevents the deposition of urate crystals.
Allopurinol: It is an inhibitor of uric acid synthesis. It is more toxic and it is reserve
for those patient whose hyperuricemia is as a result of over production of urates.
Allopurinol is converted in the body to Oxypurinol which inhibits xanthine oxidase
resulting in the accumulation of hypo xanthine and xanthine. These compounds are
more soluble than uric acid and therefore less lightly to initiate an inflamatory response.
147 | P a g e
Adenosine deaminase deficiency: Adenosine deaminase(ADA) is expressed in the

cytosol of all cells, but in humans, lymphocytes have the highest activity of these
enzyme. A deficiency of ADA results in an accumulation of adenosine, which is
converted to its ribonucleotide or deoxyribonucleotide forms by cellular kinases.
As dATP levels rise, ribonucleotide reductase is inhibited, thus preventing the production of all
deoxyribose-containing nucleotides. Consequently, cells cannot make DNA and divide. In it’s most
severe form, this autosomal recessive disorder causes severe combined immunodeficiency
disease(SCID), involving a lack of both T cells and B cells. Children with this disorder usually die
at the age of 2
SYNTHESIS OF PYRIMIDINE NUCLEOTIDES

DE NOVO SYNTHESIS OF PYRIMIDINE:
De novo pyrimidine nucleotide biosynthesis proceeds in a somewhat different manner from
purine nucleotide synthesis, the six-membered pyrimidine ring is made first and then attached
to the ribose-5 phosphate.
Sources of atoms in the pyrimidine ring are glutamine, co2, and aspartate
Required in the process is carbomoyl phosphate also an intermediate in urea cycle.
Note the carbamoylphosphate required in urea synthesis is made in mitochondria by
carbomoyl phosphate synthetase 1,
whereas the carbamoyl phosphate required by pyrimidine biosynthesis is made in cytosol by a
different form of enzyme carbamoyl phosphate synthetase 11.
STEPS:
STEP 1:Synthesis of carbamoyl phosphate
Carbamoylphosphate is synthesized from glutamine and co2 catabolysed by carbamoyl phosphate
synthetase II (CPS II).
CPS II is inhibited by UTP (the end-product of this pathway, which can be converted into the
other pyrimidine nucleotides). It is activated by ATP and PRPP.
STEP 2: Rate limiting step.

The second step in pyrimidine synthesis is the formation of carbamoylaspartate, catalyzed
by aspartate transcarbamoylase.
C2 and N3 are derived from carbomoyl phosphate and the rest are from aspartate.
STEP 3 : Formation of pyrimidine ring

The pyrimidine ring is then closed hydrolytically by dihydroorotase, giving you
dihydroorotate. Here 3rd nitrogen and c4 are joined.
STEP 4:Oxidation.
The resulting dihydroorotate is oxidized to produce orotic acid (orotate) by the enzye
dihydroorotate dehydrogense. Hydrogen atoms are removed from c5 and c6 positions.
The enzyme that produces orotate, dihydrooratate dehydrogenase is located inside
mitochondria.
All other reactions in pyrimidine biosynthesis are cytosolic.
STEP 5 Formation of OMP.

The completed pyrimidine ring is converted to the nucleotide orotidine 5’-monophosphate in the
second stage of pyrimidine nucleotide synthesis. PRPP is again the ribose5-phosphate
donor.
The enzyme orotate phosphoribosyltransferase produces OMP and releases pyrophosphate,
thereby making the reaction irreversible.
STEP 6: Decarboxylation reaction

148 | P a g e
OMP, the parent pyrimidine mononucleotide is converted to uridine monophosphate(UMP) by

Orotidylate decarboxylase which removes the carboxyl group. C7 of the OMP is removed.
Orotate phosphoriboxyltrasnferase and orotidylate decarboxylase are also domains of a single
polypeptide chain called UMP synthase. Deficiency of this bifunctional enzyme can cause
orotic aciduria.
Synthesis of Triphosphates.
UMP is phosphorylated to form UDP with the help of ATP. The enzyme is nucleoside mono
phosphate kinase(UMP kinase). Next the UDP is phosphorylated to UTP(Uridine
triphosphate) with the help of ATP. The enzyme is nucleoside diphosphate kinase.
Formation of CTP
UTP is converted to CTP by adding an amino group from glutamine catalysed by CTP
synthetase. It needs ATP.
Structural pathway of De novo synthesis of Pyrimidine
Breaking the pathway into two
Synthesis of thymidine monophosphate from dUMP.

dUMP is converted to dTMP by thymidylate synthase which uses N5, N10-Methylene
tetrahydrofolate as the source the methyl group.
149 | P a g e
Inhibitors of this enzyme can be used in the treatment of cancer eg 5-fluorouracil.
Salvage pathway
This pathway helps in salvaging the pyrimidine bases however only few pyrimidine bases are
salvaged in human cells.
Uridine and Cytidine can be salvaged by uridine-cytidine kinase.
Deoxycytidine and thymidine can be salvaged by deoxycytidine kinase and thymidine kinase
respectively.
Each of these enzymes catalyses the phosphorylation of a nucleoside(s) making use of ATP
and forming UMP, CMP, dCMP and TMP.
Regulation of pyrimidine synthesis

The first two enzymes of pyrimidine nucleotide biosynthesis are sensitive to allosteric regulation,
and the first three and the last two enzymes of the pathway are regulated at genetic level by
apparently coordinate repression and depression.
-Cabamoyl phosphate synthetase II is inhibited by UTP and purine nucleotide but activated by
PRPP.
-Aspartate trancarbamolase is inhibited by CTP and activated by ATP.
Degradation of pyrimidine nucleotides
Unlike the purine rings, which are not cleaved in human cells, the pyrimidine ring can be
opened and degraded to highly soluble structures, such as β-alanine and β-
aminoisobutyrate which are precursors of acetylCOA and succinylCOA, respectively.
THANK YOU.
150 | P a g e
MOLECULAR BIOLOGY TOOLS

BIC 201
Introduction
The successful development of methods that enables the study of macromolecules has been one
of the major driving forces in the field of molecular biology in the last several decades, as
well as one of its greatest triumphs.
DNA SEQUENCING
DNA sequencing is the process of determining the precise order of the nucleotide within a
DNA molecule. It includes any method or technology that is used to determine the mode
of the four bases (A,T,G,C) in a strand of DNA.
Dna sequencing methods
There are two main methods of DNA Sequencing.
The Sanger method: older classified chain termination method.
High- through put (HTS) sequencing: newer methods that can process a large number of
DNA molecules quickly. Such methods are collectively called HTS or Next- generation
sequencing (NGS) methods.
POLYMERASE CHAIN REACTION (PCR)

This is a powerful method developed by Kary Mullus in 1983 for amplifying a particular
segment of DNA. This procedure is carried out in vitro. PCR used the enzyme DNA
polymerase that directs the synthesis of DNA from deoxynucleotide substrates on a single
stranded DNA template.
Nested PCR
RT-PCR or Reverse Transcriptase PCR
Real Time PCR
Gradient PCR
Multiplex PCR
AFLP PCR
Applications of PCR
DNA –bases phylogeny or functional analysis of genes.
DNA amplification
Diagnosis of hereditary disease.
Identification of genetic finger prints of forensic samples
Paternity testing and detection
Analysis of allelic sequence variations
Detection of mutation
Assay for the presence of pathogens
Mutagenesis or modification of DNA (GMO`s, Transgenic/ Genetic engineering)
Cloning of genomic DNA or cDNA etc…
PCR PROCEDURE
RESTRICTION ENZYMES (RE)
restriction endonuclease cleave single or double strands of DNA sequences at specific
site known as recognition sites (RS) that are usually palindromic.
Recognition Sequences and Cutting Sites of Selected Restriction Endonucleases
AluI A G ↓ C T
BamHI G ↓ G A T C C
BglII A ↓ G A T C T
Nomencleature of RE
151 | P a g e
BIOTECHNOLOGY
Biotechnology is a technique that utilizes biological systems, living organism or parts of it
to develop or create products.
Applications include;
Biopharmaceuticals
Genetherapy
Pharmacogenomics
Genetically Engineered
Insulin Gene testing
etc
GENOMIC LIBRARY
Genomic library is a collection of the total genomic DNA in a single organism.
Types of Genomic Libraries:
Nuclear and organelle genomic library.
Applications of Genomic Library:
1. It helps in the determination of the complete genome sequence of a given organism.
2. It serves as a source of genomic sequence for generation of transgenic animals through
genetic engineering.
3. It helps in the study of the function of regulatory sequences in vitro.
4. It helps in the study of genetic mutations in cancer tissues.
5. Genomic library helps in identification of the novel pharmaceutical important genes.
Procedure for constructing genomic DNA
RECOMBINANT DNA TECHNOLOGY (rDNA)

Recombinant DNA are DNA sequence that result from the use of laboratory method to bring
together genetic material from multiple sources, creating sequence that would not otherwise be found
in biological organisms.
Steps in molecular cloning / rDNA technology
Choice of host organism and cloning
vector Preparation of vector DNA
Preparation of DNA to be cloned
Creation of recombinant DNA with DNA ligase
Introduction of recombinant DNA into the host
organism Selection of organisms containing vector
sequence
Screening for clones with desired DNA insert and biological properties
PROBES
A hybridization probe is a fragment of DNA or RNA of variable length (usually 100–1000 bases
long) which can be radioactively labelled. It can then be used in DNA or RNA samples to detect
the presence of nucleotide sequences (the DNA target) that are complementary to the sequence in
the probe.
Types include; DNA Probes, RNA Probes, cDNA Probes, Synthetic oligonucleotides as probes.
BLOTTING
A technique that entails immobilization of proteins or nucleic acids on a solid membrane
support, and then detection using a specific antibody or probe of complementary nucleic
acid sequence, blotting significantly increases the potential for identification and
characterization of proteins and nucleic acids.
1) Southern blotting
Southern blotting is named after Edward M. Southern. This method is used for analysis of DNA
sequences.
152 | P a g e
Applications of Southern blot

It is used in the technique called RFLP (Restriction fragment length polymorphism) mapping /
DNA finger printing.
Also used in phylogenetic analysis (identification of structurally related gene).
To identify the gene arrangements.
2) Western blotting
Western blotting is named after W. Neal Burnette. This method is used for detection and analysis
of protein in a given sample.
Applications of western blot
Used in clinical purposes.
Used to detect specific protein in low quantity.
Iii) Used to quantifying a gene product.
3) Northern blotting
This method is used to analysis and detection of RNA in a sample.
Applications of northern blot
Used in DNA screening or identification of a particular gene
Useful in cDNA cloning because the size of a specific mRNA can be compared with the size of
cloned cDNA
VECTORS
A vector is a DNA molecule that carries a foreign DNA into a host cell where it replicates
and produces many copies of itself as well as the foreign DNA or gene.
Plasmids: This is an extra chromosomal circular DNA molecule that automatically replicated itself inside
a bacterial cell. It has a cloning capacity of 100-10,000 bps or 0.1 – 10 kilo base.
Phages : This is a derivative of bacteriophage lambda. It is a linear DNA molecule whose
region can be replaced with a foreign DNA molecule without disruption of it s life cycle.
It can clone up to 20 kilo bases
Cosmids : Another vector designed especially for cloning large DNA fragments. Cosmids behave both
as plasmids and as phages. They have room for large inserts (35–50 kb).
Bacterial artificial chromosome (BAC): These are base of bacteria mini F plasmids with cloning
capacity limit of upto 75- 300kb.
Yeast artificial chromosome (YAC): These contain telomerase, origin of replication and a selectable
marker for identification in yeast cells with limit of 100- 1000kb.
GENE THERAPY
Gene therapy is an experimental technique that uses genes to treat or prevent disease. In medicine,
gene therapy is the therapeutic delivery of nucleic acid into a patient’s cell as a drug to treat diseases.
The new DNA usually contains a functioning gene to correct the effects of a disease causing
mutation. Though it has had limited success in treating human disease, gene therapy may be a
promising treatment option for some genetic diseases, including muscular dystrophy and cystic
fibrosis. The gene is preferably introduced using a vector.
Types of gene therapy
Somatic
germ line gene therapy Techniques for
carrying out gene therapy.
Gene augmentation
therapy Gene inhibition
therapy
Killing of specific cells
Challenges of gene therapy
Delivering the gene to the right place and switching it on.
153 | P a g e
Avoiding immune response

Making sure the new gene doesn’t disrupt the function of other genes
The cost of gene therapy
SCREENING FOR GENETIC DISEASES/ DISEASE GENE IDENTIFICATION

Disease gene identification is a process by which scientists identify the mutant genotypes responsible
for an inherited genetic disorder.
Significance
Knowledge of which genes (when non-functional) cause which disorders will simplify
diagnosis of patients and provide insights into the functional characteristics of the mutation. The
advent of modern-day high-throughput sequencing technologies combined with insights
provided from the growing field of genomics is resulting in more rapid disease gene
identification, thus allowing scientists to identify more complex mutations.
Detection techniques
Pre-genomics techniques
Loss of heterozygosity (LOH)
Post-genomics techniques
Identity by descent mapping
Homozygosity/autozygosity mapping
Genome-wide knockdown studies
Whole exome sequencing
MONOCLONAL ANTIBODIES
Monoclonal antibody (MAb) is a single type of antibody that is directed against a specific antigenic
determinant (epitope). It was a dream of scientists to produce MAbs for different antigens. This is
produced using successfully hybridize antibody—producing B-lymphocytes with myeloma cells in
vitro to create a hybridoma. The production of mAb is termed hybridoma technology.
Production of Monoclonal Antibodies:

The establishment of hybridomas and production of MAbs involves the following
Immunization
Cell fusion
Selection of hybridomas
Screening the products
Cloning and propagation
Characterization and storage
154 | P a g e

Compiled Metabolism

Uploaded by

Document Information

Original Description:

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Compiled Metabolism

Uploaded by

Copyright:

Available Formats

LECTURE NOTES ON METABOLISM

DIGESTION AND ASORPTION OF FOOD

MOLECULAR BIOLOGY TOOLS

COMPILED, EDITED AND

STUDY LIKE THERE IS NO TOMORROW BECAUSE IF YOU KEEP

OLUKA AUSTEN (AUSTEN-BEST)

DIGESTION AND ABSORBTION OF FOOD

breaking- down process is termed ‘digestion’,

Digestion in the mouth

and the sublingual glands, which are underneath the tongue.

Hydrolyses starch /glycogen to maltose and dextrins at the α-1,4-glycosidic bonds to

DIGESTION IN THE STOMACH

enzyme precursor:- pepsinogen and in small quantities pro-rennin and lipase

Conversion of milk protein (soluble caseinogen) by H+ to the insoluble Ca salt of casein in

DIGESTION IN THE DUODENUM

Zymogen: Secreted in its inactive form trypsinogen to prevent auto-digestion since

Its pH is about 7.5 – 8.8 due to the presence of NaHCO3.

Hydrolyses fibrous proteins

Note:α -1,6 branches requires further hydrolysis by α-dextrinase or isomaltase

Mol.wt. of the component fatty acid

If fat droplets are small enough, they can be absorbed directly.

Clinical Significance of Bile Acids

DIGESTION IN THE SMALL INTESTINE

Contains Digestion-Completing Enzymes:.

Aminopeptidase : an exopeptidase, attack peptide bonds at the terminal amino acids of

The resulting dipeptides and tripeptides are hydrolysed by dipeptidases and

Disaccharidases and oligosaccharidases: α-glucosidase (maltase) removes single glucose

Maltase: maltose to glucose

Cephalin: oleic, palmitic, serine

An intermediate product of hydrolysis is nucleoside which is a combination of the N-base

Final Products of Digestion

Digestion in the Large Intestine

In man no digestion takes place, except intestinal putrefaction and fermentation

(Refer Probiotic and Prebiotics in nutrition)

Defects in Digestion and Absorption

Any deficiency in the enzymes for digestion of the carboseg. Disaccharidase

Lactase Deficiency (Lactose Intolerance)

There are three types:

Sucrase Deficiency: Inherited deficiency of the disaccharidases,sucrase and isomaltase.

Disacchariduria: increase in the excretion of disaccharides, observed in some patients

Inflammatory Bowel Diseases

Gastroesophageal Reflux Disease

Regorging of stomach content

Absorption of Digested Nutrients

Such a process is particularly important in many newborn suckled mammals, in which

Active-transport system for glucose and other sugars and

CARBOHYDRATE METABOLISM OVERVIEW

By the end of this session you should be able to:

Carbohydrate Metabolism –Overview

Major Pathways of CHO Metabolism

Hexose monophosphate shunt (HMP) :Enables cells to produce ribose-5-phosphate

Glycolysis: (Embden-Meyerhof Pathway)from the Greek glyk-, sweet, and

Occurs in the cell cytosol

May be aerobic [Glucose Pyruvate] summary of Rxn = Glucose + 2ADP + 2Pi +

Summary of Rxn = Glucose + 2ADP + 2Pi ----- 2 Lactate + 2ATP + 2H2O

REACTIONS OF GLYCOLYSIS (10 STEPS)

Note: The irreversible Reactions (1, 3 and 10 )

DIFFERENCES BETWEEN GLUCOKINASE AND HEXOKINASE

GLYCOLYSIS: REACTIONS 6-10

Glycolysis: Overall Reaction

In reaction 10, phosphate groups from two phosphoenolpyruvate molecules are

ATP Generating Points In Glycolysis

What is “substrate–level phosphorylation”?