Biochemistry Notes 2

CARBOHYDRATE METABOLISM
Digestion, Absorption and Transport of

Carbohydrates
The dietary carbohydrate consists of:
 Polysaccharides: Starch, glycogen and cellulose
 Disaccharides: Sucrose, maltose and lactose
 Monosaccharides: Mainly glucose and fructose

Digestion in Mouth
 Salivary glands secrete α-amylase.
 Acts briefly on dietary starch and glycogen breaking some

α-(1 → 4) bonds.
 α-amylase hydrolyzes starch into dextrins.

Action of α-amylase on starch or glycogen
Digestion in Stomach
Carbohydrate digestion halts temporarily in stomach.

Digestion in Intestine
There are two phases of intestinal digestion:
1. Digestion due to pancreatic α-amylase
2. Digestion due to intestinal enzymes :
Sucrase
Maltase
Lactase
Isomaltase
Digestion due to pancreatic α-amylase
 Pancreatic α-amylase degrades dextrins further
into a mixture of :
Maltose, Isomaltose and α-limit dextrin.
 The α-limit dextrins are smaller oligosaccharides
containing 3 to 5 glucose units.

Digestion due to intestinal enzymes
The end products of carbohydrate digestion are:
• Glucose
• Fructose
• Galactose
Flow sheet of digestion of carbohydrates.
Absorption of Carbohydrates
Two mechanisms are responsible for the absorption of

monosaccharides:
1. Active transport against a concentration gradient,
from a low glucose concentration to a higher
concentration.
2. Facilitative transport, with concentration
gradient , from a higher concentration to lower conc.

Active Transport
• The transport of glucose and galactose occurs by an active

transport.
• Active transport requires:
- Energy
- A specific transport protein
- Presence of sodium ions

Transport of glucose, fructose, galactose, mannose.
Facilitative Transport
• Fructose and mannose are transported by a Na+

independent facilitative diffusion process, requiring
specific glucose transporter, GLUT-5.
• Movement of sugar in facilitative diffusion is strictly
from a higher concentration to a lower one until it
reaches an equilibrium.
Transport of Carbohydrates
 Sodium independent transporter, GLUT-2 facilitates
transport of sugars out of the mucosal cells.
 Through portal circulation transported to the liver.

Lactose Intolerance
• Intolerance to lactose (the sugar of milk).
• Due to deficiency of enzyme lactase.
• lactose undergoes bacterial fermentation with the

production of:
- H2 and CO2 gases
- acetic acid , propionic acid and butyric acid

 Abdominal cramps and flatulence results from the:
-- Accumulation of gases
-- Osmotically active products that draw water
from the intestinal cells into the lumen resulting
in diarrhoea and dehydration.
 Treatment for this disorder is simply to remove
lactose from the diet.

GLYCOLYSIS
Embden Meyerhof pathway.
Definition
Glycolysis is the sequence of reactions that
converts glucose into pyruvate in the presence
of oxygen (aerobic) or lactate in the absence of
oxygen (anaerobic) with the production of ATP.

Location
Glycolysis is found in cytosol of all cells.

Reactions Of Glycolysis
 I st phase: Energy requiring phase.
 II nd phase: Energy generating phase.

Phases of the glycolytic pathway.
Anaerobic Glycolysis
• The re-oxidation of NADH by conversion of

pyruvate to lactate by lactate dehydrogenase
• Tissues that function under hypoxic conditions
produce lactate, e.g. skeletal muscle, smooth
muscle and erythrocytes.

Regulation of Glycolysis
Glycolysis is regulated at 3 irreversible steps.These

reactions are catalysed by:
1. Hexokinase and glucokinase
2. Phosphofructokinase-I
3. Pyruvate kinase
Regulation of phosphofructokinase by fructose-2,6-
bisphosphate
Regulation of liver pyruvate kinase.
Significance of Glycolysis
 Glycolysis is the principal route for glucose metabolism

for the production of ATP molecules.
 Glycolysis provide ATP in the absence of oxygen and

allows tissues to survive anoxic episodes.
 In erythrocytes, glycolysis supplies 2,3-BPG which is

required for transport of oxygen by Hb.
 Generates precursors for biosynthetic pathway:
 Pyruvate transaminated to amino acid alanine.
 Pyruvate provides substrate acetyl-CoA for
fatty acid biosynthesis.
 Glycerol-3-phosphate, required for synthesis of
triacylglycerol is derived from glycolysis.

 It also provide pathway for the metabolism of fructose
and galactose derived from diet.
 In mammals, glucose is the only fuel that the brain uses

under non-starvation conditions and the only fuel that
red blood cells can use at all.
Glycolysis & Dental Caries
Energetic of Glycolysis
 The net reaction of aerobic glycolysis of glucose into two
molecules pyruvate generates:
– 2 molecules of NADH (2.5 x2 = 5)
– 4 molecules of ATP at subs level phosphorylation
 Two molecules of ATP per mole of glucose are consumed
 The net gain is 7 moles of ATP

 Under aerobic conditions, 7 molecules of ATP are
produced.
 In anaerobic glycolysis, on the other hand, only 2
moles of ATP are produced per molecule of
glucose.
Rapoport Lubering Cycle
• In Rapoport lubering cycle, production of ATP by
substrate phosphorylation from 1,3-BPG to 3-BPG
is bypassed in the erythrocyte.
• There is no net production of ATP when
glycolysis takes this route.

Significance of Rapoport Lubering Cycle
 It supplies 2,3-BPG required for transport of oxygen by

hemoglobin. 2,3-BPG regulates the binding and release
of oxygen from hemoglobin
 2, 3-BPG present in erythrocytes acts as a buffer.

Catabolic fates of pyruvate.
Conversion Of Pyruvate To Acetyl-CoA
• Pyruvate is converted to acetyl CoA by oxidative
decarboxylation in mitochondria.
• Irreversible reaction catalyzed by a multienzyme complex

pyruvate dehydrogenase (PDH)
• PDH requires five coenzymes :
thiamine pyrophosphate (TPP), lipoate, coenzyme-A,
FAD and NAD+.

Oxidative decarboxylation of pyruvate by the pyruvate
dehydrogenase complex.
Energetics in conversion of pyruvate to Acetyl Co A
 One molecule of NADH is produced for each molecule of

pyruvate.
 Oxidation of NADH by electron transport chain results
in synthesis of 2.5 ATP molecules

Citric Acid Cycle
Citric acid cycle or Krebs cycle or tricarboxylic acid (TCA) cycle

Definition
The citric acid cycle is a series of reactions in mitochondria
that brings about the catabolism of acetyl-CoA to CO2 and
H2O with generation of ATP.

Energetics of Citric Acid Cycle
 Three molecules of NADH and one FADH2 are

produced
 One molecule of ATP is generated at substrate level
during the conversion of succinyl-CoA to succinate.
Total 10 ATP are generated from one mole of acetyl-CoA.

Significance of Citric Acid Cycle
 Provide energy in the form of ATP.
 Final common pathway for the oxidation of

carbohydrates, lipids, and proteins.
 Amphibolic (catabolic and anabolic) process , has a
dual function.
 Pathways originate from the TCA cycle:
– Gluconeogenesis
– Transamination
– Fatty acid synthesis
– Heme synthesis.
Amphibolic role of the citric acid cycle
Regulation of Citric Acid Cycle
 Citric acid cycle is regulated at three steps by:
1. Citrate synthase
2. Isocitrate dehydrogenase
3. α-ketoglutarate dehydrogenase.
 Activities of these enzymes are dependent on the
energy status of the cycle.

 Excess of ATP, NADH and succinyl-CoA, which
signals high energy status of the cell, inhibit these
enzymes.
 High level of ADP which signals low energy

status of the cell stimulates the operation of the
cycle.
Regulation of citric acid cycle.
GLUCONEOGENESIS
Synthesis of glucose from non-carbohydrate precursors.

Location of Gluconeogenesis
 Liver is the major tissue
 During starvation, the kidney is also capable of

making glucose by gluconeogenesis.
Non-carbohydrate Precursors of gluconeogenesis
 Lactate
 Glycerol
 Glucogenic amino acids
 Propionic acid
 Intermediates of TCA
Major non-carbohydrate substrates and their entry points into
gluconeogenesis.
Conversion of propionate to succinyl-CoA.
Conversion of glycerol to dihydroxyacetone phosphate.
Pathway of Cori cycle or lactic acid cycle.
Glucose alanine cycle or Cahill cycle.
Characteristics of Gluconeogenesis
1. Glycolysis and gluconeogenesis share the same
pathway but in opposite direction.
2. Seven reversible reactions of glycolysis are used
by gluconeogenesis.
3. Involves glycolysis plus some special reactions.

Significance of Gluconeogenesis
1. Maintains blood glucose level when
carbohydrate is not available in sufficient
amounts from the diet.
2. During starvation glucose is provided to the

brain and other tissues like erythrocytes, lens,
cornea of the eye and kidney
3. Gluconeogenesis is used to clear the products of
metabolism of other tissues from blood.
 Lactate, produced by muscle and erythrocytes
 Glycerol produced by adipose tissue
 Propionyl-CoA produced by oxidation of odd

carbon number fatty acids and carbon skeleton
of some amino acids.
Regulation of Gluconeogenesis
 Gluconeogenesis regulated by four key enzymes:
1. Pyruvate carboxylase
2. Phosphoenolpyruvate carboxykinase
3. Fructose-1,6-bisphosphatase
4. Glucose-6-phosphatase
 The hormones glucagon and epinephrine
stimulate gluconeogenesis by inducing the
synthesis of the key enzymes
 Insulin inhibits the gluconeogenesis by

repressing their synthesis.
 During starvation and in diabetes mellitus,
high level of glucagon stimulates
gluconeogenesis.
 However in well-fed state, insulin suppresses

the gluconeogenesis.
Glycogen Metabolism
 Glycogen metabolism includes:
Glycogenesis
Glycogenolysis
 Glycogenesis and glycogenolysis are both

cytosolic processes.
Glycogenesis
 Glycogenesis is the pathway for the formation of
glycogen from glucose.
 This process requires energy, supplied by ATP

and uridine triphosphate (UTP).
 It occurs in muscle and liver.

• Schematic representation of glycogenesis
(mechanism of branching)
Schematic representation of glycogenesis

(mechanism of branching)
Glycogenolysis
Degradation of glycogen to glucose-6-phosphate in
muscle and to glucose in liver

Schematic representation of glycogenolysis
(mechanism of debranching)
Significance of Glycogenolysis and Glycogenesis
In liver
 Following a meal, excess glucose is removed from
the portal circulation and stored as glycogen by
glycogenesis.
 Conversely, between meals, blood glucose levels
are maintained within the normal range by release
of glucose from liver glycogen by glycogenolysis.

In muscle
 The function of muscle glycogen is to act as a
readily available source of glucose within the
muscle itself during muscle contraction.
 The muscle cannot release glucose into the blood,

because of the absence of glucose-6-phosphatase
 Muscle glycogen stores are used exclusively by

muscle
Regulation of Glycogenesis and Glycogenolysis
• The principal enzymes controlling glycogen
metabolism are:
- Glycogen phosphorylase
- Glycogen synthase
Principal enzymes are regulated reciprocally:
- Hormonal regulation
- Allosteric regulation.
Allosteric regulation of glycogenesis and glycogenolysis
Hormonal regulation
Regulation of glycogen synthesis by action of protein phosphatase-1.
Inactivation of glycogen
synthase kinase by
insulin.
Glycogen Storage Disease
 Glycogen storage disease is a group of genetic
diseases, that result from a defect in enzyme
required for either glycogen synthesis or
degradation.
 Characterized by deposition of either normal or
abnormal glycogen in the specific tissues

Pentose Phosphate Pathway
The pentose phosphate pathway is an alternative
route for the oxidation of glucose.
It is the pathway for formation of pentose

phosphate.
It is also called hexose monophosphate shunt.

Characteristics of Pentose Phosphate pathway
 A multicyclic process
 It does not generate ATP.
 Three molecules of glucose-6-phosphate give

rise to three molecules of CO2 and three
molecules of 5-carbon sugars
Location
The enzymes of pentose phosphate pathway
are present in cytosol of all cells.

Pathway divided in to two phases:
1. Phase I : Oxidative irreversible phase
2. Phase II : Non-oxidative reversible phase.

Outline of pentose phosphate pathway.
Phase I: Oxidative
Phase II
Significance of Pentose Phosphate Pathway
 The pentoses (ribose-5-phosphate) required for the
biosynthesis of nucteotide and nucleic acids RNA and
DNA are provided by pentose phosphate pathway.
 It provides a route for the interconversion of
pentoses and hexoses

 It generates NADPH which are required in
1. Biosynthesis of
Fatty acids
Cholesterol
Steroid hormones
Neurotransmitters.
2. Detoxification reactions
 In RBC, NADPH is required to maintain the
level of reduced glutathione. The reduced
glutathione protects the RBC membrane from
toxic effect of H2O2 by reducing H2O2to H2O
 NADPH also keeps iron of hemoglobin in
reduced ferrous (Fe2+) state and prevents the
formation of methemoglobin.
Role of NADPH
and glutathione
Macrophage
bactericidal
activity
Regulation of Pentose Phosphate Pathway
 Glucose-6-phosphate dehydrogenase (G-6-PD)

is the rate limiting enzyme.
 The activity of this enzyme is regulated by
cellular concentration of NADPH. NADPH
is competitive inhibitor of G-6-PD.

An increased concentration of NADPH decreases activity
of G-6-PD, for example:
– Under well-fed condition, the level of NADPH
decreases and pentose phosphate pathway is
stimulated.
– In starvation and diabetes, the level of NADPH
is high and inhibits the pathway.

Insulin enhances the pathway by inducing the
enzyme G-6-PD and 6-phosphogluconolactone
dehydrogenase.
Disorders of Pentose Phosphate Pathway
Deficiency of Glucose-6-phosphate dehydrogenase

(G-6-PD)
Glucose 6-phosphate dehydrogenase deficiency is
X linked inherited disorder, characterized by
hemolytic anemia, due to excessive hemolysis.
Most individuals are asymptomatic. However, some
individuals with G-6-PD deficiency develop hemolytic anemia
if they are exposed to drugs like antibiotic, antipyretic or
Antimalarial, e.g. primaquine ,chloroquine

A 20-year-old male suffering from malaria was treated
with chloroquine and manifested as hemolytic anemia.
Provisional diagnosis of glucose-6-phosphate
dehydrogenase (G-6-PD) deficiency was made.
Questions
a. Which reaction is catalyzed by the enzyme G-6-PD?
b. How does deficiency of G-6-PD produce hemolytic
anemia?
c. Name the pathway in which this reaction occurs.

G-6-PD deficiency and resistance to malaria
Persons with G-6-PD deficiency cannot support
growth of the malarial parasite, Plasmodium
falciparum and thus are less susceptible to malaria
than the normal person.

Wernicke-Korsakoff Syndrome
 A genetic disorder due to reduced activity of

the TPP-dependent transketolase enzyme.
 The reduced activity of transketolase is due to
reduced affinity for TPP

 In the chronic thiamine deficiency the
transketolase enzyme has a much reduced
activity leading to the Wernicke- Korsakoff
syndrome.
 The symptoms of Wernicke-Korsakoff

syndrome include weakness, mental disorder,
loss of memory, partial paralysis, etc.
Uronic Acid Pathway (Glucuronic Acid Cycle)
Definition
 A pathway in liver for the conversion of glucose to
glucuronic acid, ascorbic acid (except in humans)
and pentoses
 An alternative oxidative pathway for glucose but
does not generate ATP

Significance of Uronic Acid Pathway
 Uronic acid pathway is a source of UDP -glucuronate.
 UDP-glucuronate is a precursor in synthesis of

proteoglycans (glycosaminoglycans) and glycoproteins.
 UDP-glucuronate is involved in detoxification reactions

that occur in liver e.g.bilirubin,steroid hormones
Metabolic role of UDP-glucuronate.
 The uronic acid pathway is a source of UDP-glucose,
which is used for glycogen formation.
 The uronic acid pathway provides a mechanism
by which dietary D-xylulose can enter the central
metabolic pathway
Galactose Metabolism And Galactosemia
Galactose is derived from disaccharide, lactose
(the milk sugar) of the diet.
 It is important for the formation of:
Glycolipids
Glycoproteins
Proteoglycans
Lactose during lactation.
Galactose is readily converted in the liver to glucose.

Galactosemia
 It is an inborn error of galactose metabolism.
 Caused by deficiency of enzyme galactose-1-
phosphate uridyl transferase
 The inherited deficiencies of galactokinase and
UDPgalactose-4-epimerase also lead to minor
types of galactosemia.
 It causes a rise in galactose in blood and urine and leads
to accumulation of galactose and galactose-1-phosphate
in blood, liver, brain , kidney and eye lenses.
 In these organs, the galactose is reduced to galactitol by
the enzyme aldose reductase.

Clinical findings
The accumulation of galactitol and galactose-1-

phosphate in liver, brain and eye lenses causes:
- Liver failure (hepatomegaly followed by cirrhosis)
- Mental retardation and
- Cataract formation
Treatment:
 Galactose in milk and milk products should be
eliminated from the diet.
 Sufficient galactose for the body’s need can be
synthesized endogenously as UDP-galactose.

A 6-month-old infant was presented with elevated blood
and urine galactose.
Questions
a. Name the disease.
b. Give the biochemical steps related to the disease
and point out the metabolic defect.
c. What are the clinical manifestation of the disease

BLOOD GLUCOSE LEVEL AND ITS REGULATION
 Blood glucose level maintained within 70-100 mg/dl.
 Levels above normal range : Hyperglycemia,
Levels below normal range : Hypoglycemia.
 After the intake of a carbohydrate meal, blood
glucose level rises to 120-140 mg/dl.

Factors involved in the regulation of blood glucose
are:
1. Hormones
2. Metabolic processes
3. Renal mechanism
Two major hormones controlling blood glucose
levels are:
1. Insulin (hypoglycemic hormone)
2. Glucagon (hyperglycemic hormone).

Reciprocal control of insulin and glucagon on the
homeostasis.
Maintenance of Glucose in Fed State
(Hyperglycemic condition)
 Increased blood glucose level ,hyperglycemia

occur after each meal
 Increased level of blood glucose releases insulin
 Insulin reduces the blood glucose level in a

number of ways
Various metabolic systems affected by insulin.
Effect of glucagon on blood glucose.
Maintenance of Blood Glucose in Fasting State
(Hypoglycemic Condition)
.
Decreased level of blood glucose (hypoglycemia)
causes release of hyperglycemic hormones, e.g.
 Glucagon
 Epinephrine or adrenaline
 Glucocorticoids
 Growth hormone
 ACTH
 Thyroxin.
Glucagon
Glucagon opposes the action of insulin. It acts

primarily in the liver as follows:
 In the liver, it stimulates glycogenolysis &

inhibits glycogen synthesis
Enhances gluconeogenesis from amino acids and

lactic acids
Epinephrine or Adrenaline
 Stimulates glycogenolysis in the liver and the
muscle by stimulating glycogen phosphorylase
 In muscle due to absence of glucose-6-

phosphatase, glycogenolysis results with the
formation of lactate, whereas in the liver, glucose
is the main product, leading to increase in blood
glucose.
Glucocorticoids
 Increases Gluconeogenesis by increasing the:
 activity of enzymes of gluconeogenesis.

 protein catabolism to provide glucogenic amino acid
 hepatic uptake of amino acids.
 Inhibit utilization of glucose in extra-hepatic

tissues.
Growth hormone and anterior pituitary hormones
 Growth hormone and ACTH antagonise the

action of insulin.
 Growth hormone decreases glucose uptake in the
muscle and ACTH decreases glucose utilization

by the tissue.
Thyroxine
 Accelerates hepatic glycogenolysis with

consequent rise in blood glucose.
 It may also increase the rate of absorption of

hexoses from the intestine
Renal Control Mechanism
If the blood glucose level is raised above 180
mg/100 ml, complete tubular reabsorption of glucose
does not occur and the extra amount appears in the
urine causing glycosuria.

GLYCOSURIA
 Excretion of detectable amount of sugar in urine
is known as glycosuria.
 Glycosuria results from the rise of blood glucose
above its renal threshold level (180 mg%)

Glycosuria may be due to various reasons on the
basis of which is classified into following groups:
1. Alimentary glycosuria
2. Renal glycosuria
3. Diabetic glycosuria.
DIABETES MELLITUS
Definition
Syndrome of impaired carbohydrate,fat and protein

metabolism, caused by either:
 Lack of insulin secretion
or
 Decreased sensitivity of tissues to insulin.

Classification of Diabetes Mellitus
1. Type I diabetes mellitus or insulin dependent
diabetes mellitus (IDDM) or juvenile diabetes.
2. Type II diabètes mellites or non insulin
dépendent diabetes mellitus (NIDDM) or adult
diabetes mellitus.
Type l diabetes mellitus
Cause
Lack of insulin secretion due to destruction of pancreatic

beta cells. The destructions of beta cells may be due to:
1. Viral infection
2. Autoimmune disorder
3. Hereditary tendency of beta cell degeneration.

Onset
 At about 14 years of age and for this reason it is
called juvenile diabetes mellitus.
 Juvenile’ means teenage in Latin.

Symptoms
 It develops symptoms very abruptly with:
– Polyuria (frequent urination)
– Polydypsia (excessive thirst)
– Polyphagia (excessive hunger).

 Loss of body weight, weakness, and tiredness.
 Hyperglycemia with glycosuria and
ketoacidosis
 The patients of type-I diabetes mellitus are not
obese.
Treatment
Since patients of IDDM (type-I) fail to secrete
insulin, administration of exogenous insulin is
required.
Type II diabetes mellitus
Cause
 Decreased sensitivity of target tissues to insulin.
(insulin resistance).
 Inadequate insulin receptors on cell surfaces of target

tissues.
 This syndrome is often found in an obese person.

Onset
• After age 40 and the disorder develops gradually.
• Therefore, this syndrome is referred to as adult
onset diabetes.
Symptoms
The symptoms are developed gradually
Similar to that of type-I
Except Ketoacidosis is usually not present in type
II diabetes mellitus
Treatment
 NIDDM (type-II) can be treated in early stages

by diet control, exercise and weight reduction
 No exogenous insulin administration is required..

Drugs that increase insulin sensitivity or drugs
that cause additional release of insulin by the
pancreas may be used.
 In the later stages insulin administration is

required.
Metabolic changes occur in diabetes mellitus
Changes in levels of insulin and glucagon affect
metabolism in three tissues; liver, muscle and
adipose tissue.
The lack of insulin activity results in failure of
transfer of glucose from the blood into cells and
leads to hyperglycemia.
Elevated levels of blood glucose and ketone
bodies are the characteristic feature of untreated
diabetes mellitus.
 The body responds as it were in the fasting state
with stimulation of:
– Glycogenolysis
– Gluconeogenesis
– Lipolysis
– Proteolysis.
 Increased lipolysis leads to increased formation
of ketone bodies causing ketoacidosis.
Due to increased rate of proteolysis the amino
acids released from muscle are converted to glucose
by gluconeogenesis.
GLUCOSE TOLERANCE TEST (GTT)
 Glucose tolerance test (GTT) is a test to assess the
ability of the body to utilize glucose.
 GTT can be performed by two ways:
1. Oral GTT
2. Intravenous GTT
Types of Glucose Tolerance Curves
1. Normal glucose tolerance curve
2. Decreased glucose tolerance
3. Increased glucose tolerance.

Decreased glucose tolerance occurs in:
 Diabetes mellitus
 Certain endocrine disorders like:
– Hyperthyroidism
– Hyperpituitarism
– Hyperadrenalism (Cushing’s syndrome).

Increased glucose tolerance occurs in :
– Hypothyroidism (myxedema, cretinism)
– Hypoadrenalism (Addison’s disease)
– Hypopituitarism.
The glucose tolerance curves
Significance of GTT
 GTT is not necessary in symptomatic or in known
cases of diabetic patients
 GTT is most important in the investigation of
asymptomatic hyperglycemia or glycosuria
such as renal glycosuria & alimentary glycosuria.
 Give useful information of endocrine dysfunctions.
 It is also helpful in recognizing milder cases of diabetes.

Lipid Metabolism
Digestion and Absorption of Lipids
Dietary Lipids include :
 Neutral fat (triacylglycerol or triglycerides)
 Fatty acids
 Phospholipids
 Glycolipids
 Sterols
 Fat soluble vitamins (A, D, E and K).

Digestion in mouth and stomach
Little or no digestion occurs in the mouth or stomach.
Digestion in Small Intestine
In duodenum dietary fat is emulsified by bile salts.

Hydrolysis of dietary triacylglycerols
Emulsified triacylglycerols hydrolyzed by pancreatic lipase.

Hydrolysis of dietary phospholipids
Phospholipids are digested by pancreatic phospholipase-A2

Hydrolysis of cholesterol ester
Cholesterol esters hydrolyzed by pancreatic cholesterol
esterase which produces cholesterol plus free fatty acid.

Products of Lipid Digestion (Micelle Formation)
 Products of dietary lipid digestion are:
• Free fatty acids
• Free cholesterol
• 2-monoacylglycerol
• Lysophospholipid
 These, together with bile salts, form mixed micelles.
 Fat soluble vitamins A, D, E and K are also packaged
in these micelles and are absorbed from the micelles
along with the products of dietary lipid.

Absorption and transport of lipid from intestinal lumen.
 2-MAG: 2-monoacylglycerol;
 C: cholesterol;
 CE: cholesterol ester;
 FA: fatty acid;
 LysoPL: lysophospholipid;
 TG: triacylglycerol)
Abnormalities in Lipid Digestion and Absorption
Lipid Malabsorption
 Lipid malabsorption results in a loss of lipid as much

as 30 g/day including the fat soluble vitamins and
essential fatty acids in the feces.
 Conditions in which the feces contain large amounts

of fat and fatty acids, commonly stearic acid
therefore called steatorrhea, (Greek, steato = fat).
 Steatorrhea caused by a number of conditions. The
most common causes are:
• Bile salt deficiency occurs in liver disease or due to
obstruction in the bile duct.
• Pancreatic enzyme deficiency occurs in pancreatitis
or cystic fibrosis.
• Defective chylomicron synthesis occurs in
congenital abetalipoproteinemia.
FATTY ACID METABOLISM
 Fatty acids are amphipathic compounds containing a

long hydrocarbon chain and a terminal carboxylate
group (R-COOH).
 Fatty acids are stored in adipose tissues as

triacylglycerol also called neutral fats or triglyceride.
 Fatty acids mobilized from triacylglycerol are oxidized

to meet energy needs of a cell.
 For the utilization of stored fatty acids in the form of
triacylglycerol in adipose tissues requires three stages of
processing:
1. Mobilization of lipid (lipolysis):
In this process, triacylglycerols of adipose tissue degraded to

fatty acids and glycerol and transported to the energy
requiring tissues. Glycerol produced by lipolysis is
metabolized by liver.
2. Activation of fatty acids:
In the tissues fatty acids are activated to fatty acyl-CoA and

transported into mitochondria for degradation.
3. Oxidation of fatty acyl-CoA:
Fatty acyl-CoA degraded by oxidation into acetyl-CoA,

which is then oxidized in the citric acid cycle
1. Mobilization of Fatty Acids from Triacylglycerol
(Lipolysis)
Lipolysis generates fatty acids and glycerol.
Metabolism of glycerol
Activation and Transport of Fatty Acids into Mitochondria
Activation of fatty acid to acyl-CoA.

Transport of Acyl-CoA into Mitochondria by
Carnitine Transport System
 Activated long chain fatty acids are carried across the
inner mitochondrial membrane by carnitine, (β-hydroxy
γ-trimethyl ammonium butyrate).
 Carnitine is formed from lysine and methionine in liver
and kidney
Activation and transport of fatty acids into mitochondria
by carnitine shuttle.
• CAT-I: carnitine acyltransferase-l;
• CAT-II: carnitine acyltransferase-ll

β-Oxidation of Saturated Even Carbon Fatty Acids
 In β-oxidation two carbon units sequentially removed
beginning from the carboxyl end of the fatty acid in the
form of acetyl-CoA.
 It is called β-oxidation because oxidation of fatty acids
occurs at the β-carbon atom.
 β-oxidation pathway occurs in mitochondria.

Sequence of Reactions of β-oxidation
Acyl-CoA is degraded by a repeated sequence of four

reactions :
1. Oxidation by FAD
2. Hydration
3. Oxidation by NAD
4. Cleavage.
β-oxidation of saturated even carbon
fatty acids
Overall process of β-oxidation.
Energy yield from the β-oxidation of fatty acids
Complete β-oxidation of palmitoyl CoA (16 carbon acid)

occurs through 7 cycles of β-oxidation yielding
8 acetyl-CoA,
7 FADH2
7 NADH
Therefore the number of ATPs formed:
• 7x 1.5 = 10.5 ATPs from the 7 FADH2
• 7x 2.5= 17.5 ATPs from the 7 NADH
• 80 ATPs from 8 molecules of acetyl-CoA in TCA cycle.
• Total of 108 ATPs.

 Two high energy phosphate bonds are consumed in the
activation of palmitate, in which ATP is split into AMP
and PPi.
 Thus, the net yield from the complete oxidation of
palmitate is 108-2 =106 ATPs

Regulation of β-oxidation
 Rate limiting step in the β-oxidation pathway is the

formation of acyl-carnitine which is catalyzed by
carnitine-acyl transferase-l (CAT-I).
 CAT-I is an allosteric enzyme. Malonyl-CoA is an
inhibitor of CAT-I.
 In well-fed state due to increased level of insulin,
concentration of malonyl-CoA increases which inhibits
CAT-I and leads to decrease in fatty acid oxidation.
 In starvation, due to increased level of glucagon

concentration of malonyl-CoA decreases and stimulates
the fatty-acid oxidation
Oxidation of a Fatty Acid with an Odd Number of
Carbon Atoms
Fatty acids, having an odd number of carbon atoms,

are oxidized by the pathway β-oxidation producing
acetyl-CoA until a three-carbon, propionyl-
CoA residue remains.

 In the oxidation of odd carbon fatty acids, the
propionyl-CoA and acetyl-CoA, rather than two
molecules of acetyl-CoA are produced in the
final round of degradation.
 Propionyl-CoA is converted to succinyl-CoA

constituent of citric acid cycle.
Figure 11.11:
Conversion of
propionyl-CoA to
succinyl-CoA.
Metabolism of Ketone Bodies
Acetoacetate, β-hydroxybutyrate and acetone
are known as ketone bodies

 Acetoacetate and β-hydroxybutyrate are interconverted
by the mitochondrial enzyme β-hydroxybutyrate
dehydrogenase.
 Acetoacetate continually undergoes spontaneous

nonenzymatic decarboxylation to yield acetone
 The concentration of total ketone bodies in the blood of

well-fed condition does not normally exceed 0.2
mmol/L.
Interconversion of ketone bodies.
Ketone bodies are water soluble energy yielding
substances. Acetone is an exception, since it cannot be
metabolized and is readily exhaled through lungs.

Formation of ketone bodies.
 Liver is the only organ that synthesizes ketone bodies.

and transferred to other organs as fuel
 The synthesis of ketone bodies occurs in mitochondria

of hepatic cells
Utilization of Ketone Bodies
 The site of production of ketone bodies is the liver.
 But the liver cannot utilize ketone bodies because it

lacks particular enzyme CoA-transferase which is
required for activation of ketone bodies.
Figure 11.16: Activation and
utilization of ketone bodies.
Significance of Ketogenesis
 Ketogenesis is a mechanism that allows the liver to
oxidize increasing quantities of fatty acids.
 During deprivation of carbohydrate (starvation &
diabetes mellitus) acetoacetate and β-hydroxybutyrate
serve as alternative source of energy for extra hepatic
tissues
 In prolonged starvation 75% of the energy needs of
the brain are supplied by ketone bodies reducing its
need for glucose.
 Acetoacetate also has a regulatory role in lipid

metabolism. High levels of acetoacetate in the blood
specify an abundance of acetyl units and lead to a
decrease in the rate of lipolysis in adipose tissue.
 The formation and export of ketone bodies releases
coenzyme-A, allowing continued fatty acid oxidation.
 Ketone bodies are water soluble transportable form of

derivatives of acetyl-CoA. They do not need to be
incorporated into lipoproteins or carried by albumins as
do the other lipids.
Regulation of Ketogenesis
The ketone body formation is regulated at three levels:
1. Factors regulating lipolysis
2. Factors regulating β-oxidation of fatty acids
3. Factors regulating oxidation of acetyl-CoA

Figure 11.18:
Steps of regulation of
formation of ketone
bodies.
Disorders of Ketone Body Metabolism
Ketosis
 When the rate of formation of the ketone bodies by liver
exceeds the capacity of the peripheral tissues to use
them up, their levels begin to rise in blood.
 An increase in concentration of ketone bodies in blood is

called ketonemia and eventually leads to excretion of
ketone bodies into the urine called ketonuria.
 The overall condition (ketonemia and ketonuria) is

called ketosis.
Ketoacidosis
 The acidosis caused by over production of ketone bodies

is termed as ketoacidosis.
 Acetoacetate and β-hydroxybutyrate when present in

high concentration in blood, are buffered by HCO3– of
bicarbonate buffer. The excessive use of HCO3– depletes
the alkali reserve causing ketoacidosis.
 Ketoacidosis is seen in type I diabetes mellitus, whereas

in type II diabetes ketoacidosis is relatively rare.
Figure 11.19: Overproduction of ketone bodies in diabetes
and starvation.
Drain off oxaloacetate for glucose synthesis, slows

oxidation of acetyl-CoA by citric acid pathway, diverting
acetyl-CoA to the formation of ketone bodies.
De Novo Synthesis of Fatty Acid
DE NOVO SYNTHESIS OF FATTY ACIDS
 De novo synthesis means new synthesis.
 Fatty acid synthesis occurs mainly in the liver and to a

lesser extent, in adipose tissue, kidney and brain.
Fatty acid synthesis occurs in three phases:
1. Transport of acetyl-CoA from mitochondria to cytosol.
2. Carboxylation of acetyl-CoA to malonyl-CoA.
3. Reactions of fatty acid synthase complex.

Transport of acetyl-CoA from mitochondria to cytosol
Biosynthesis of malonyl-CoA.
Fatty acid synthase complex is a multienzyme complex
possessing 6 different enzymes and one acyl carrier
protein (ACP) molecule. The six enzymes are:
1. Malonyl/acetyl transacylase (MAT)
2. Ketoacyl synthase (KS)
3. Ketoacyl reductase (KR)
4. Hydratase (H)
5. Enoyl reductase (ER)
6. Thioesterase (TE)
Schematic diagram of fatty acid synthase multienzyme
complex showing sequence of enzymes.
1. ER: enoyl reductase;
2. H: hydratase;
3. KR: ketoacyl reductase;
4. KS: ketoacyl synthase;
5. MAT: malonyl/acetyl transacylase;
6. TE: thioesterase
ACP: acyl carrier protein;
 The fatty acid synthase (FAS) enzyme is a dimmer of
identical subunits (homodimer).
 Each subunit contains all of the six enzymes, as well as

an acyl carrier protein (ACP).
 Even though each subunit possesses all enzymes

required for fatty acid synthesis, the monomers are not
active.
 A dimmer is required for the synthesis.

 The ACP segment contains the vitamin pantothenic
acid in the form of 4-phosphopantetheine.
 4-phosphopantetheine provides the sulfhydryl (–SH)

group to which the growing fatty acid chain is attached
as it is synthesized.
 Thus, the function of the ACP in fatty acid biosynthesis

is analogous to the role of coenzyme-A in fatty acid
oxidation.
.
 Fatty acid synthase has one more sulfhydryl (–SH)
group which is furnished by a specific cysteine residue
of 3-ketoacyl synthase enzyme.
 Both –SH groups participate in fatty acid biosynthesis.

Reactions de novo fatty acid synthesis
Figure 11.23: De novo synthesis of fatty acids.
• ACP: acyl carrier protein;
• KS: ketoacyl synthase

Regulation of Fatty Acid Synthesis
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. The
acetyl-CoA carboxylase is regulated by following
mechanisms
Allosteric Mechanism
Acetyl-CoA carboxylase is an allosteric enzyme,
palmitoyl-CoA, the principle product of fatty acid
synthesis, is a feedback inhibitor of the enzyme and
citrate is an allosteric activator.
Covalent Modification of Enzyme
Acetyl-coA carboxylase is also regulated by covalent
modification. Phosphorylation, triggered by the hormones
glucagon and epinephrine inactivates the enzyme and
thereby reduces fatty acid synthesis.
Regulation of fatty acid synthesis by allosteric mechanism
and covalent modification.
TRIACYLGLYCEROL METABOLISM
Triacylglycerols are esters of the alcohol glycerol and
fatty acids. It contains a glycerol backbone to which 3-
fatty acids are esterified.
Triacylglycerol serves as the body’s major fuel
storage reserve.
 Human can store only few hundred grams of glycogen

in liver and muscle, hardly enough to supply the body’s
energy needs for 12 hours.
 In contrast, the total amount of stored triacylglycerol in

70 kg man is about 15 kg, enough to support basal
energy needs for as long as 12 weeks.
 Triacylglycerols have the highest energy content of all
stored nutrients.
 Whenever carbohydrate ingested in excess of the body’s

capacity to store glycogen, the excess is converted to
triacylglycerol and stored in adipose tissue
Biosynthesis of Triacylglycerols
The precursors for the synthesis of triacylglycerol are

fatty acyl-CoA and glycerol-3-phosphate
: Biosynthesis of triacylglycerol.
Fate of Triacylglycerol Formed in Liver and
Adipose Tissue
 The triacylglycerol stored in adipose tissue are

continually undergoing lipolysis (hydrolysis) and re-
esterification through triacylglycerol cycle
 The resultant of these two processes, lipolysis

(breakdown) and re-esterification (synthesis)
determines the level of circulating free fatty acids in the
plasma
Triacylglycerol cycle.
 The triacylglycerol stored in adipose tissue undergoes
hydrolysis by a hormone sensitive lipase to form free
fatty acids and glycerol.
 Some of the fatty acids released by lipolysis of

triacylglycerol in adipose tissue pass into the
bloodstream, and remainder are used for resynthesis of
triacylglycerol.
 Some of the fatty acids released into the blood are taken
up by several tissues, including muscle, where it is
oxidized to provide energy and some are taken up by the
liver.
 Much of the fatty acid taken up by liver is not oxidized

but is recycled to triacylglycerol and exported again into
the blood in the form of VLDL back to adipose tissue,
and reesterified into triacylglycerol
 The glycerol, released in adipose tissue, cannot be
metabolized by adipocytes because they lack glycerol
kinase.
 Rather, glycerol is transported through the blood to the

liver, which can phosphorylate it. The resulting glycerol
phosphate can be used to form triacylglycerol in the
liver or to be converted to DHAP.
Regulation of triacylglycerol metabolism
 The rate of biosynthesis and degradation of

triacylglycerols depends on the metabolic resources
and requirements of the moment.
 The rate of triacylglycerol biosynthesis is regulated

by the action of hormones.
 When the mobilization of fatty acids is required to meet
energy needs, breakdown of triacylglycerol and thus
release of fatty acids from adipose tissue is stimulated by
the hormones glucagon and epinephrine.
 Epinephrine, and glucagon, stimulates hormone
sensitive lipase by increasing c-AMP and
phosphorylation.
 Simultaneously, these hormonal signals decrease the rate
of glycolysis and increase the rate of gluconeogenesis in
the liver.
 Insulin stimulates the conversion of dietary
carbohydrates and proteins to triacylglycerol.
 In the presence of insulin, hormone sensitive lipase

is dephosphorylated and becomes inactive and
inhibits breakdown of triacylglycerol.
METABOLIC ROLE OF ADIPOSE TISSUE
 Adipose tissues store and supply fatty acids.
 Cosmetically adipose tissue is viewed as an enemy;

however its importance in energy homeostasis is
second only to that of the liver.
 There are two types of adipose tissue, white and brown,
with different roles.
 Human adipose tissue is mostly of the white type.

 White adipose tissue (WAT) is amorphous and widely
distributed in the body: under the skin, around deep
blood vessels, and in the abdominal cavity. White
adipose tissue contains specialized cells, adipocytes that
are devoted solely to the function of storing fat.
 The typical adult has 13 kg of adipose tissue. Obesity

results when this amount increases.
 The adipocytes of WAT are large (diameter 30 to70
μm), spherical cells, completely filled with a single
large lipid droplet that squeezes the mitochondria and
nucleus against the plasma membrane.
 The lipid droplet contains triacylglycerols (TAGs) and

cholesterol esters and is coated with a monolayer of
phospholipids. Specific protein perilipin and the
enzymes for synthesis and breakdown of TAGs are
associated with the surface of the droplets.
 Brown adipose tissue cells have more mitochondria and
a richer supply of capillaries than WAT cells, and it is
the cytochromes of mitochondria and the hemoglobin in
capillaries that give brown adipose tissues its
characteristic brown color.
Schematic view of adipocytes of white and brown adipose
tissues.
White Adipose Tissue Metabolism
 In adipose tissue triacylglycerol is stored in the
adipocytes.
 Like other cell types, adipocytes have an active

glycolytic metabolism, oxidize pyruvate and fatty
acids via the citric acid cycle, and carry out oxidative
phosphorylation.
 When carbohydrate intake is high, adipose tissue can
convert glucose (via pyruvate and acetyl-CoA) to fatty
acids, convert fatty acids to triacylglycerols, and store
triacylglycerols as large lipid droplets.
Synthesis and Degradation of triacylglycerol in adipose
tissue
 In adipose tissue triacylglycerol is synthesized from acyl
CoA (active form of fatty acids) and glycerol-3-
phosphate.
 When, the demand for fuel rises (between meals),

hormone sensitive lipases in adipocytes hydrolyzes
stored triacylglycerols to release free fatty acids, and
glycerol.
White adipose
tissue metabolism.
Regulation of adipose tissue metabolism
 Lipolysis and reesterification are regulated by many
nutritional, metabolic, and hormonal factors that
influence either, the rate of esterification or the rate of
lipolysis.
 Lipolysis is controlled by level of cAMP, thus the

processes which destroy or preserve cAMP have an
effect on
Regulation of lipolysis in white adipose tissue.lipolysis
Disorder of Lipid Transport and Storage
Fatty liver
 Fatty liver is the excessive accumulation of fat primarily
neutral fat, triacylglycerol in the liver.
 Liver contains about 5% fat. In pathological conditions

this may go up to 25–30% and is known as fatty liver or
fatty infiltration of liver.
 When accumulation of lipid in the liver becomes chronic,

fibrotic changes occur in cells which may finally lead to
cirrhosis and impairment of liver function.
 Fatty liver occurs in conditions in which there is an
imbalance between hepatic triacylglycerol synthesis
and the secretion of VLDL.
 Fatty liver falls into two main categories:

1. The first type is
 associated with the increased levels of plasma free fatty

acids.
 The increasing amounts of free fatty acids are taken up by the

liver and esterified to triacylglycerol, but the production of
VLDL does not keep pace with the increasing influx of free
fatty acids, allowing triacylglycerol to accumulate which in
turn causes fatty liver.
 This occurs during starvation and feeding high fat diet

2. The second type of fatty Liver is due to impairment in
the biosynthesis of plasma lipoproteins. This defect may
be due to:
 A block in apolipoprotein synthesis
 A block in the synthesis of lipoprotein from lipid
and apolipoprotein
 Defect in the synthesis of phospholipids that are
found in lipoproteins
 A failure in the secretory mechanism itself.
Factors that Cause Fatty Liver
1. High fat diet.
2. Starvation or uncontrolled diabetes mellitus
3. Alcoholism
4. High cholesterol diet
5. Use of certain chemicals

6. Dietary deficiency of:
• Lipotropic factors: Choline, Betain ,Methionine,

Lecithin
• Essential fatty acids
• Essential amino acids
• Protein deficiency , kwashiorkor

Lipotropic Factors
 The substances that prevent the accumulation of fat in

the liver are known as lipotropic factors.
 Dietary deficiency of these factors can result in fatty

liver.
 The various lipotropic agents are : choline, methionine

,betaine .
 Vitamin B12 and folic acid have also lipotropic effect, as

these are involved in the formation of methionine from
homocysteine.
Lipoprotein Metabolism And Transport Of
Lipids
 Four main types of lipoproteins are :
1.Chylomicrons
2. Very low density lipoproteins (VLDL)
3. Low density lipoproteins (LDL)
4. High density lipoproteins (HDL)

Structure of lipoprotein.
Metabolism of Lipoproteins
The pathways of lipoprotein metabolism include two

cycles, one exogenous and one endogenous; these cycles
are interconnected and both centered on the Liver.
The exogenous pathway involves metabolism of
chylomicrons
1. Dietary lipids are packaged into chylomicrons.
2. Fatty acids from triacylglycerol of chylomicrons are
released by lipoprotein lipase to adipose and muscle
tissues, during transport through capillaries.
3. Chylomicron remnants containing largely protein and
cholesterol are taken by the liver.
4. In the liver, the remnants release their cholesterol
The endogenous pathway involves metabolism of
VLDL, LDL and HDL
5. In endogenous pathway lipids synthesized or packaged in
the liver are delivered to peripheral tissues by VLDL.

6. Removal of triacylglycerol from VLDL converts VLDL
to VLDL remnants, also called intermediate density
lipoprotein IDL
7. IDL is either taken up by the liver or further removal of
triacylglycerol from IDL produces low density
lipoprotein (LDL),
8. LDL delivers cholesterol to extrahepatic tissues or
returns to the liver. Approximately 30% of LDL is
degraded in extra hepatic tissues and 70% in the liver.

9. High density lipoprotein (HDL), originates in the liver as
small, protein-rich nascent HDL particles
10. Excess cholesterol in lipoproteins and extrahepatic
tissues is transported back to the liver by HDL in reverse
cholesterol transport.
The overview of formation and transport of lipoproteins,
showing exogenous and endogenous lipoprotein metabolic
cycle.
The numbered steps are discussed in the text.
(C: cholesterol; CM: chylomicron; CMR: chylomicron

remnant; LPL: lipoprotein lipase; FFA: free fatty acid;
LDL: low density lipoprotein, IDL: intermediate
density lipoprotein)
Metabolism of chylomicrons, the exogenous pathway
Metabolism of VLDL LDL and HDL the endogenous
pathway
Metabolism of VLDL and LDL.
Formation of foam cells by macrophage by receptor
independent mechanism of LDL uptake.
Metabolism of HDL
Reverse Cholesterol Transport
 This is the process whereby excess cholesterol contained
in extra hepatic tissue is taken to the liver, by HDL for
utilization or excretion through bile.
 The LCAT esterifies the cholesterol content of HDL &

prevent it from re-entering the cells.
 Thus esterification by LCAT serves to trap cholesterol

within the lipoprotein , preventing it from deposition in
the tissues.
Significance of reverse cholesterol transport
 By reverse cholesterol transport cellular and lipoprotein
cholesterol is delivered back to the liver.
 This is important because the steroid nucleus of

cholesterol cannot be degraded; and the liver is the only
organ that can remove excess cholesterol by secreting it in
the bile for excretion in the feces.
 Reverse cholesterol transport prevents deposition of
cholesterol in tissues ( anti- atherogenic).
 An elevated HDL cholesterol (good cholesterol) level

decreases the risk of coronary heart disease.
Diagnostic Importance of Lipoproteins
The blood levels of certain lipoproteins have diagnostic
importance. The ratio of HDL cholesterol to that in the
LDL cholesterol can be used to evaluate susceptibility
to the development of heart disease. For healthy person,
LDL/HDL ratio is 3:5.

 Raised plasma LDL-cholesterol concentration is
associated with an increased risk of ischemic heart
disease.
 Whereas raised plasma concentration of HDL
cholesterol is associated with a decreased risk of
ischemic heart disease and seems to have protective
effect.
 LDL cholesterol is called bad cholesterol because
excess cholesterol is present in the form of LDL.
 HDL cholesterol is called good cholesterol

Disorders of Lipoprotein Metabolism
Disorders of lipoprotein metabolism are:
• Hyperlipoproteinemia
• Hypolipoproteinemia
• Familial Hypercholesterolemia.
Hyperlipoproteinemia
The causes of hyperlipoproteinemia are complex, and
different disease mechanisms can give rise to similar lipid

patterns. In practice lipoprotein disorders are classified as
follows:
 Primary hyperlipoproteinemia when the disorder is not

due to some other disorders.
 Secondary hyperlipoproteinemia when the disorder is

manifested due to some other disease.
Hypolipoproteinemia
Hypolipoproteinemia is also classified as:
 Primary hypolipoproteinemia is due to reduced synthesis of

protein, e.g.:
–– Abetalipoproteinemia
–– Tangier disease
 Secondary hypoliporoteinemia, e.g.:
–– Kwashiorkor in children
–– Severe malabsorption
–– Some forms of chronic liver disease.

Familial Hypercholesterolemia
 Hypercholesterolemia is a genetic disorder caused by the

mutation of LDL receptors gene. The molecular defect
of hypercholesterolemia is an absence or deficiency of
functional receptors for LDL (B-100/E).
Cholesterol Metabolism
 Cholesterol is the major sterol in human
 Cholesterol is an amphipathic lipid which can be

synthesized by most cells of the body and it is obtained
from the diet in foods of animal origin.
 It is not synthesized in plants.
 The major source of dietary cholesterol is egg yolk and

meat, particularly liver.
 An abnormality in either cholesterol metabolism or
transport through the plasma appears to be related to the
development of atherosclerosis that can lead to
myocardial infarction or stroke.
Structure of cholesterol.
De Novo Synthesis of Cholesterol
 Cholesterol is synthesized by most cells of the body.

Liver and intestine are major site of cholesterol
synthesis.
 All 27-carbon atoms of cholesterol are derived from the

acetyl-CoA.
 The reactions of cholesterol biosynthesis occurs into 5

stages
Five stages of cholesterol biosynthesis.
The first two stages take place in the cytoplasm and next
three in the endoplasmic reticulum.
1. Condensation of three molecules of acetyl-CoA to

mevalonate
2. Conversion of mevalonate to activated isoprene units
3. Polymerization of six isoprene units to form squalene
4. Cyclization of squalene to form parent steroid
nucleus lanosterol
5. Formation of cholesterol from lanosterol.

Biosynthesis of cholesterol, showing its five stages.
Energy Cost of Cholesterol Synthesis
 Cholesterol biosynthesis is a complex and energy-

expensive process.
 ATP is consumed only in the steps that convert

mevalonate to the activated isoprene units.
 Three ATP molecules are used to create each of the six

activated isoprenes required to construct squalene, for a
total cost of 18 ATP molecules.
Regulation of De Novo Synthesis of Cholesterol
 Cholesterol biosynthesis is a complex and energy-

expensive process.
 Excess cholesterol cannot be catabolized for use as fuel

and must be excreted.
 In mammals, cholesterol production is regulated by:
• Intracellular cholesterol concentration
• Supply of ATP, and
• Hormones glucagon and insulin.

 The synthesis of mevalonate by HMG-CoA reductase
is the committed step in cholesterol biosynthesis.
 Short-term regulation of the activity of existing
HMG-CoA reductase is regulated by reversible
covalent alteration, i.e. by phosphorylation and
dephosphorylation.
 Hormone, glucagon stimulates its phosphorylation,
inactivating the enzyme, and insulin promotes
dephosphorylation, activating the enzyme and favoring
cholesterol synthesis.
 In the long-term regulation, the number of molecules of
HMG-CoA reductase is increased or decreased in
response to cellular concentrations of cholesterol.
 HMG-CoA reductase in liver is inhibited by mevalonate

and by cholesterol
 Mevalonate and cholesterol repress transcription of the

HMGCoA reductase via activation of a transcription
factor, sterol regulatory element-binding protein
(SREBP) and thus decreases the cholesterol synthesis.
 Small quantities of oxysterols such as 25-
hydroxycholesterol are formed in the liver and act as
regulators of cholesterol synthesis. Oxysterols inhibits
cholesterol synthesis by stimulating proteolysis of
HMG-CoA reductase
Regulation of cholesterol synthesis.
Metabolic Fates of Cholesterol
Cholesterol has several metabolic fates.
 A small fraction of the cholesterol made in the liver is

incorporated into the membranes of the hepatocytes.
 Small quantities of oxysterols such as 25-

hydroxycholesterols are formed in the liver and act as
regulators of cholesterol synthesis. Oxysterols inhibits
cholesterol synthesis by stimulating proteolysis of HMG-
CoA reductase.
 In other tissues, cholesterol is converted into steroid
hormones in the adrenal cortex and gonads.
 Vitamin D hormone is synthesized in liver and kidney,

which regulates calcium and phosphorus metabolism.
 Most of the cholesterol is exported in one of the three

forms, as bile acids, biliary cholesterol, or cholesteryl
ester.
Metabolic fates of cholesterol.
Synthesis of bile acid and its regulation
Transport of Cholesterol
 Cholesterol is transported in body fluids in the form of
lipoprotein particles.
 Cholesterol esters that are resynthesized in the mucosal

cells, together with some unesterified cholesterol are
incorporated into chylomicrons, which transport
cholesterol and other dietary lipids from intestine
 During circulation, only about 5% of the cholesterol
ester is lost. The rest 95% of the chylomicron
cholesterol is delivered to the liver through LRP (LDL
receptor related protein) receptor in the form of
chylomicron remnants.
 Cholesterol in excess of the liver’s own needs is

exported into the blood in the form of VLDL.
 Triacylglycerols of VLDL are hydrolyzed by the action
of lipoprotein lipase. The resulting remnants, which are
rich in cholesteryl esters, are called intermediate
density lipoproteins (IDL).
 Half of the IDL are taken up by the liver for processing,

and half are converted to low density lipoprotein by the
removal of more triacylglycerol.
 Low density lipoprotein is the major carrier of

cholesterol in blood.
 The role of LDL is to transfer cholesterol to peripheral
tissues through LDL receptors and regulate de novo
cholesterol synthesis
 HDL picks up cholesterol from the peripheral tissues and

from other lipoproteins and converts it to cholesterol
esters by LCAT enzyme.
 These HDL-cholesterol esters are ultimately returned to

the liver through HDL receptor (SR-B1) for excretion,
where it is degraded or excreted in the bile.
Transport of cholesterol.
Excretion of Cholesterol
 Cholesterol is excreted in feces
 Unlike many other metabolites, cholesterol cannot be
destroyed by oxidation to CO2 and H2O, because of
absence of enzymes capable of catabolizing the
steroid ring.
 It is excreted in the bile either as cholesterol or after
conversion to bile acids.

 About 1 gm of cholesterol is eliminated from the body
per day. Roughly, half is excreted in the form of bile
acids and half is in the form of cholesterol.
 Moreover, some dietary cholesterol is excreted in feces

without being absorbed.
 Some of the cholesterol in the intestine is acted on by

intestinal bacterial enzymes and converted to neutral
sterols, coprostanol, cholestanol and excreted through
feces.
Disorder of Cholesterol Metabolism
Atherosclerosis
Atherosclerosis is the general term for hardening of

the arteries, due to formation of plaque, results in the
endothelial damage and narrowing of the lumen.
 Atherosclerosis is due to dysregulation of cholesterol
metabolism. As noted earlier, cholesterol cannot be
catabolized by animal cells. Excess cholesterol can
only be removed by excretion or by conversion to
bile salts.
 When the sum of cholesterol synthesized and cholesterol
obtained in the diet exceeds the amount required for the
synthesis of membrane, bile salts, and steroids,
pathological accumulations of cholesterol (plaques) can
obstruct blood vessels, a condition is called
atherosclerosis.
Narrowing of blood vessel due to formation of plaque.
Factors responsible for development of atherosclerosis
1. Age 8. Cigarette smoking

2. Sex
9. Diabetes mellitus
3. Genetic factor
4. Hyperlipidemia 10. Minor or soft risk factors
5. Lipoprotein(a) (LPa)
6. Level of HDL
7. Hypertension
 Age
As age advances, the elasticity of the vessel wall

decreases and formation of plaques progresses. Formation
of plaque leads to narrowing of the lumen .
 Sex
Males are affected more than females. Male sex hormone

is atherogenic or conversely that female sex hormones are
protective.
 Genetic factor
Hereditary genetic derangement of lipoprotein metabolism
leads to high cholesterol level.
 Hyperlipidemia
Increased levels of serum cholesterol , triacylglycerol, low
density lipoprotein (LDL) are associated with increased risk of
atherosclerosis
 Lipoprotein(a) (LPa)
Elevated LPa levels are associated with an increased risk of
coronary heart disease

 Level of HDL
Low level of HDL is associated with atherosclerosis. HDL
has protective effect against atherosclerosis. HDL participates in
reverse transport of cholesterol
 Hypertension
It acts probably by mechanical injury of the arterial wall due to
increased blood pressure.
 Cigarette smoking
Cigarettes smoking increase the risk due to reduced level of HDL
and accumulating carbon monoxide that may cause endothelial
cell injury.
 Diabetes mellitus
The risk is due to the coexistence of other risk factors such as
obesity, hypertension, and hyperlipidemia.
 Minor or soft risk factors
These include lack of exercise, stress, obesity, high caloric
intake, diet containing large quantities of saturated fats, use
of oral contraceptive, alcoholism etc.
The risk is due to increased LDL and decreased HDL levels.

THANK YOU
VITAMINS
Definition and classification of vitamins
 Vitamins are organic nutrients that are required in small

quantities for a variety of biochemical functions and
which generally cannot be synthesized by the body and
must, therefore, be supplied by the diet.
 Some vitamins can be synthesized by intestinal

microorganisms, but in quantities that are not sufficient to
meet our needs.
Classification of vitamins
1. Water soluble vitamins
2. Fat soluble vitamins

Water soluble vitamins
 Vitamin B complex, e.g.

– Thiamine (vitamin B1)
– Riboflavin (vitamin B2)
– Niacin (vitamin B3)
– Pantothenic acid (vitamin B5)
– Pyridoxine (vitamin B6)
– Biotin
– Folic acid
– Cobalamin (vitamin B12)
 Vitamin C or ascorbic acid.
Fat soluble vitamins
– Vitamin A or retinol
– Vitamin D or cholecalciferol
– Vitamin E or tocopherol
– Vitamin K.
Fat Soluble Vs. Water Soluble Vitamins
Fat soluble vitamins Water soluble vitamins

Function as coenzymes, Function as precursor for
hormones and coenzymes and
antioxidants antioxidants
Toxic when taken in Usually non-toxic
excessive quantities
Can be stored Not stored extensively
except vitamin B12,
Thiamine (Vitamin B1)
Thiamine consists of a pyrimidine ring attached to a thiazole

ring by methylene bridge.
Structure of thiamin.
Active Form of Thiamine
Thiamine pyrophosphate (TPP) is an active coenzyme

form of vitamin thiamine
Sources
 It is present in all natural foods but particularly good

dietary sources are unrefined cereals, meat, nuts, green
vegetables, eggs, etc.
 White bread and polished rice are very poor sources of

the vitamin thiamine.
Functions
 Thiamine is required mainly for carbohydrate

metabolism
 Thiamine pyrophosphate (TPP) is a coenzyme involved

in oxidative decarboxylation and transketolase
reactions .
1. Oxidative Decarboxylation
 TPP is a coenzyme for pyruvate dehydrogenase complex

which catalyzes the conversion of pyruvate into acetyl
CoA.
 Acetyl-CoA is a precursor for the synthesis of

neurotransmitter acetylcholine and also for the synthesis
of myelin. Thus, thiamine is required for the normal
functioning of the nervous system.
2. TPP is a coenzyme for α-ketoglutarate dehydrogenase
which catalyzes the conversion of α-ketoglutarate to
succinyl-CoA in TCA cycle
3. TPP is a coenzyme for the enzyme transketolase, in the
pentose phosphate pathway of glucose oxidation

Nutritional Requirements
Nutritional Research Council recommends daily intake

of 1.0 to 1.5 mg of thiamine for adults which is increased
with increased muscular activity, dietary carbohydrates
and in pregnancy and lactation.
Deficiency Manifestations
 The deficiency of vitamin B1 results in a condition called

beriberi.
 Deficiency of thiamine occurs in population who consume

exclusively polished rice as staple food.
 The early symptoms of thiamine deficiency are anorexia

(lack of appetite) , nausea, mental confusion, peripheral
neuritis, and muscle fatigue.
Beriberi
Beriberi is divided into 4 types, relating to the body system

involved i.e. Nervous or cardiovascular or age of patient
(infantile).
1. Dry beriberi (neuritic beriberi): affects the nervous system
2. Wet beriberi (cardiac beriberi): affects cardiovascular system
3. Infantile beriberi: affects children of malnourished mothers.
4. Cerebral beriberi : chronic alcohol consumption.

Dry beriberi (neuritic beriberi)
It develops when the diet chronically contains slightly less

than requirements. It is characterized by:
–Vomiting.
–Poor appetite
–Peripheral neuritis
–Difficulty in walking
–Tingling or loss of sensation, numbness in hands

and feet
–Severe muscular weakness and fatigue.
–Mental confusion/speech difficulties
–Dry skin
–Involuntary eye movements (nystagmus)

Wet beriberi (cardiac beriberi)
 It develops when the deficiency is more severe in which

cardiovascular system is affected in addition to
neurological symptoms.
 Wet beriberi is characterized by:
− Peripheral Edema (swelling of lower legs)
−Heart enlargement and cardiac insufficiency.
−Increased heart rate tachycardia
−It is sometimes fatal

Infantile beriberi
 Infantile beriberi is observed in breast fed

infants (between two and six months of age)
whose mothers have inadequate thiamine
intake. The breast milk of these mothers is
deficient in thiamine.
 It is characterized by
−GI disturbances such as vomiting ,diarrhea
− Cardiac dilation (enlargement of heart),
−Tachycardia (rapid heart rate)
− Convulsions,
−Edema
−In acute condition, the infant may die due to

cardiac failure.
Wernicke-Korsakoff Syndrome
 Wernicke-Korsakoff Syndrome (WKS) is a
neurological disorder caused by a deficiency of
vitamin thiamine (vitamin B1) due to chronic
alcohol consumption
 It is also known as cerebral beriberi.
 In chronic alcoholics, the nutritional deficiencies
result from either poor intake of food or
malabsorption of nutrients from intestine.
Symptoms
 Mental confusion and impaired short-term memory.
 Impaired person’s ability to learn new information or tasks.
 Ataxia (Loss of muscle coordination that can cause weakness

in limbs and leg tremor)
 Paralysis of eye muscles
 Nystagmus (Abnormal eye movements back & forth

movements of eye)
 Double vision, eyelid drooping

Antimetabolites
Thiamine can be destroyed if the diet contains thiaminase.

Thiaminase is present in raw fish and seafood.
Thiamine Assay
Whole blood or Erythrocyte transketolase (requiring TPP

as a coenzyme) activity is used as a measure of thiamine
deficiency.
Riboflavin (Vitamin B2)
Riboflavin is a yellow compound (Flavus = yellow in
Latin) consisting of a isoalloxazine ring with a ribitol
(sugar alcohol) side chain

Active Form of Riboflavin
 Flavin mononucleotide (FMN)
 Flavin adenine dinucleotide (FAD)

Structure of riboflavin and its active coenzyme forms FMN and
FAD.
Sources
 The main dietary sources of riboflavin are yeast,
germinating seeds, green leafy vegetables milk and milk
products, eggs, liver , meat etc.
 Cereals are a poor source

 The RDA for vitamin B2 is 1.3 to 1.7 mg for adults.
 It is related to protein use and increases during growth,
pregnancy, lactation and wound healing.

Functions
 Riboflavin is a precursor of coenzymes FMN and FAD, which
are required by several oxidation-reduction reactions in
metabolism.
 FMN and FAD serve as coenzymes for oxidoreductase
enzymes involved in carbohydrate, protein, lipid, nucleic acid
metabolism and electron transport chain.
 Decarboxylation of pyruvate and α-ketoglutarate requires FAD
 Fatty acyl CoA dehydrogenase requires FAD in fatty acid
oxidation
 Synthesis of active form of folate (5-methyl THF) requires
FADH2
 FAD is required to convert tryptophan to niacin (vit B3)
 Reduction of the oxidized form of glutathione (GSSG) to

its reduced form (GSH) is also FAD dependent. Thus they
are also involved in detoxification reactions
 It is needed for maintenance of mucosal epithelial &

ocular tissues.
Riboflavin deficiency (ariboflavinosis) is quite rare.
It is most commonly seen in chronic alcoholics.

The characteristic symptoms of riboflavin deficiency are:
 Cheilosis: Cracks at the angles of the mouth,
 Glossitis: Inflammation of the tongue that becomes

swollen and magenta colored
 Dermatitis: Rough and scaly skin
 Vascularization (the development of blood vessels)

of cornea. The eyes become red, itchy, watery and
sensitive to bright light.
Niacin (Vitamin B3)
Structure
Niacin is a general name for the nicotinic acid and

nicotinamide, either of which may act as a source of the
vitamin in the diet. Niacin is a simple derivative of
pyridine.
Active Form
Active forms of niacin are:
 Nicotinamide adenine dinucleotide (NAD+)
 Nicotinamide adenine dinucleotide phosphate (NADP+)

Sources
 Yeast, liver, legumes and meats are major sources of niacin.
 Limited quantities of niacin can also be obtained from the
metabolism of tryptophan. For every 60 mg of tryptophan, 1
mg equivalent of niacin can be generated.
Nutritional Requirement
 The RDA for niacin is 15 to 20 mg.
 Tryptophan can only provide 10% of the total niacin
requirement.
Functions
 Niacin is a precursor of coenzymes, nicotinamide adenine
dinucleotide (NAD+) and nicotinamide adenine
dinucleotide phosphate (NADP+).
 NAD+ and NADP+ are involved in various oxidation and
reduction reactions.
 They are, therefore involved in many metabolic pathways
of carbohydrate, lipid and protein.

 Generally, NAD+ catalyze oxidation-reduction reactions
in oxidative pathways, e.g. citric acid cycle and
glycolysis.
 Whereas NADP+ are often found in pathways concerned

with reductive synthesis, e.g. synthesis of cholesterol,
fatty acid and pentose phosphate pathways.
Deficiency Manifestation
Pellagra
 Deficiency of niacin in human causes pellagra, a disease

involving :
 Skin
 Gastrointestinal tract
 Central nervous system.

 The symptoms of pellagra are characterized by three ‘Ds’:
1. Photosensitive Dermatitis
2. Diarrhea
3. Depressive psychosis (dementia) and if not treated death.
 Untreated pellagra is fatal.

Niacin deficiency occurs in:
 Population dependent on maize (corn) or sorghum

(jowar) as the staple food.
 In maize, niacin is present in bound form niacytin.
 Sorghum content high amount of leucine. Excess of

leucine inhibit conversion of tryptophan to niacin
 Deficiency of vitamin B6 (pyridoxal phosphate) leads to
niacin deficiency as it is involved as a coenzyme in the
pathway of synthesis of niacin from tryptophan.
 Malignant carcinoid syndrome in which tryptophan is

diverted to formation of serotonin.
 In Hartnup disease, a genetic disorder in which tryptophan

absorption and trans- portation is impaired.
. The conversion of tryptophan into nicotinic acid.
Pantothenic Acid (Vitamin B5)
The name pantothenic acid is derived from the Greek word
‘pantothene,’ meaning from “everywhere” and gives an
indication of the wide distribution of the vitamin in foods.
Active form
1. Coenzyme-A (CoA-SH)
2. Acyl carrier protein (ACP).
Source
Eggs, liver, yeast, wheat germs, cereals, etc. are important
sources of pantothenic acid, although the vitamin is widely
distributed.
The RDA of pantothenic acid is not well established. A

daily intake of about 5–10 mg is advised for adults
Functions
 Pantothenic acid is a component of coenzyme-A (CoA-
SH) and acyl carrier protein (ACP).
 The thiol (-SH) group of CoA-SH and ACP acts as a
carrier of acyl groups.
 Coenzyme-A participates in reactions concerned with:
– Reactions of citric acid cycle
– Fatty acid synthesis and oxidation
– Synthesis of cholesterol
– Utilization of ketone bodies.
 ACP participate in reactions concerned with fatty acid
synthesis.
 No clear-cut case of pantothenic acid deficiency has been

reported.
 Clinical signs observed in experimentally induced
deficiencies are:
Paraesthesia (abnormal tingling sensation)
Headache
Dizziness
Gastrointestinal malfunction.
Pyridoxine (Vitamin B6)
Structure
 Vitamin B6 consists of a mixture of three different closely
related pyridine derivatives namely:
1. Pyridoxine
2. Pyridoxal
3. Pyridoxamine.
 All the three have equal vitamin activity, as they can be
interconverted in the body.

Structure of three different forms of vitamin B
Active Form of Vitamin B6
Pyridoxal phosphate (PLP) is the active form of vitamin

B6.
Sources
 Pyridoxine occurs mainly in plants, whereas pyridoxal

and pyridoxamine are present mainly in animal
products.
 Major dietary sources of vitamin B6 are yeast,

unrefined cereals, pulses, meat, poultry fish, potatoes
and vegetables.
 Dairy products and grains contribute lesser amounts.

 The RDA for vitamin B6 is 1.6 to 2.0 mg.
 Requirements increase during pregnancy and lactation

Functions
Active form of vitamin B6, pyridoxal phosphate (PLP)
acts as coenzyme in amino acid metabolism. For
example:
– Transamination
– Decarboxylation
– Nonoxidative deamination
– Trans- sulfuration
– Condensation reactions of amino acids.
 Transamination reactions
Transamination reactions are catalyzed by transaminases

and PLP acts as coenzyme converting amino acid to keto
acid, e.g. aspartate transaminase (AST) and alanine
transaminase (ALT)
 Non-oxidative deamination
Hydroxyl group containing amino acids (serine,

threonine) are non-oxidatively deaminated to α-keto acids
and ammonia, which requires PLP.
 Decarboxylation reaction
PLP acts as coenzyme in decarboxylation of some amino
acids. The amino acids are decarboxylated to corresponding
amines.
– γ-Amino butyric acid (GABA)
– Serotonin and melatonin
– Histamine
– Catecholamines (dopamine, norepinephrine and
epinephrine)
 Trans- sulfuration reaction:
PLP is a coenzyme for cystathionine synthase involved in

synthesis of cysteine from methionine In these reactions
transfer of sulfur from methionine to serine occurs to
produce cysteine.
 Condensation reactions
Pyridoxal phosphate is required for the condensation

reaction of L-glycine and succinyl CoA in the synthesis of
heme.
 Other reactions requiring PLP are:
– For niacin coenzyme (NAD+/NADP+) synthesis
from tryptophan
– For synthesis of serine from glycine
– FOR the synthesis of sphingomyelin.

 Vitamin B6 deficiency causes neurological disorders

such as depression, nervousness and irritability.
 Severe deficiency of pyridoxine causes
epileptic seizures (convulsions) in infants.
 Demyelination of nerves causes peripheral

neuropathy.
• Vitamin B6 deficiency causes hypochromic
microcytic anemia due to decreased heme
synthesis.
The commonest cause of pyridoxine deficiency is:
 Drug antagonism, e.g. isoniazide (INH), used in the

treatment of tuberculosis can combine with pyridoxal
phosphate forming an inactive derivative with pyridoxal
phosphate.
 Alcoholism: Alcoholics may be deficient owing to

metabolism of ethanol to acetaldehyde, which stimulates
hydrolysis of the phosphate of the pyridoxal phosphate
Biotin
Biotin was known formerly as vitamin H.
Structure
It consists of a tetrahydro-thiophene ring bound to an
imidazole ring and a valeric acid side chain.
Structure of biotin.
Sources
 It is widely distributed in foods.
 Liver, kidneys, vegetables and egg yolk are the

important sources of biotin.
 Biotin is also synthesized by intestinal bacteria.

Active Form of Biotin
biocytin.
A daily intake of about 150–300 mg

is recommended for adults.
Functions
Biotin is a coenzyme of carboxylase reactions, where it is a

carrier of CO2.
 Conversion of acetyl-CoA into malonyl-CoA catalyzed

by acetyl-CoA carboxylase in fatty acid synthesis.
 Conversion of pyruvate into oxaloacetate, catalyzed by

pyruvate carboxylase in gluconeogenesis .
 Conversion of propionyl-CoA to D-methyl malonyl-CoA
catalyzed by propionyl-CoA carboxylase in the pathway
of conversion of propionate to succinate.
 Catabolism of branched chain amino acid catalyzed by β-

methylcrotonyl-CoA carboxylase
 Since biotin is widely distributed in plant and animal

foods and intestinal bacterial flora supply adequate
amounts of biotin, the natural deficiency of biotin is not
well characterized in humans.
 The experimentally induced symptoms of biotin

deficiency are nausea, anorexia, glossitis, dermatitis,
alopecia (loss of hair), depression and muscular pain.
Folic Acid
Structure
 Folic acid consists of three components:
1. Pteridine ring,
2. P-amino benzoic acid (PABA) and
3. L-glutamic acid
 In a folic acid molecule, the number of glutamic acid

residues varies from one to seven. Folic acid usually has
one glutamic acid residue.
The structure and numbering of atoms of folic acid.
Active Form of Folic Acid
Tetrahydrofolate (THF) is the active form of folic acid.
Source
 Folic acid is found in green leafy vegetables, liver, yeast.
 The word folate is related to folium which means leaf in

Latin.
 The RDA of folate is 200 mg.
 Requirements increase during pregnancy and lactation.

Formation of tetrahydrofolate from folic acid.
Functions
 Tetrahydrofolate (THF) acts as a carrier of one-

carbon units. The one carbon units are:
Methyl CH3
Methylene CH2
Methenyl CH
Formyl CHO
Formimino CH = NH
 One carbon unit binds to THF through N5 or N10 or both
N5, N10 position.
 The THF coenzymes serve as acceptors or donors of one

carbon units in a variety of reactions involved in amino
acid and nucleic acid metabolism.
Five major reactions in which THF is involved are:
1. Conversion of serine to glycine:
The conversion of serine to glycine is accompanied

by the formation of N5,N10- methylene THF.
2. Synthesis of thymidylate (pyrimidine nucleotide):
The enzyme thymidylate synthase that converts

deoxyuridylate (dUMP) into thymidylate (TMP)
uses N5, N10- methylene THF as the methyl donor
for this reaction.
3. Catabolism of histidine :
Histidine in the course of its catabolism is converted into
formimino- glutamate(FIGLU). This molecule donate the
formimino group to THF to produce N 5 formimino THF.
4. Synthesis of purine:
N5-Formyl THF intermediate formedin histidine
catabolism is used in the biosynthesis of purineand
therefore in the formation of both DNA and RNA.
5. Synthesis of methionine from homocysteine:
Homocysteine is converted to methionine in presence of
N5-methyl THF, and vitamin B12.
– In this reaction the methyl group bound to cobalamin
(Vitamin B12) is transferred to homocysteine to form
methionine and the cobalamin then removes the methyl
group from N5-methyl THF to form THF.
– This step is essential for the liberation of free THF and

for its repeated use in one carbon metabolism. In B12
deficiency, conversion of N5-methyl THF to free THF is
blocked.
The combined role of vitamin B12 and folate and folate
trap.
 Megaloblastic or macrocytic anemia
 Accumulation and excretion of FIGLU in the urine
 Hyperhomocysteinemia
Neural tube defect in foetus - Spina bifida
Cobalamin (Vitamin B12)
Structure
 Vitamin B12 bears a complex corrin ring (containing

pyrrols similar to porphyrin), linked to a cobalt atom
held in the center of the corrin ring, by four coordination
bonds with the nitrogen of the pyrrole groups.
 The remaining coordination bonds of the cobalt are
linked with the nitrogen of dimethylbenzimidazole
nucleotide and sixth bond is linked to either methyl or
5'-deoxy- adenosylor hydroxy group to form
methylcobalamin, adenosylcobalamin or
hydroxycobalamin respectively
Structure of Vit B12.
(R: either
methyl
or
deoxyadenosyl
or
hydroxy group)
Active form of Vitamin B12
 Methylcobalamin
 Deoxyadenosylcobalamin.
Sources
 Dietary sources of vitamin B12 are of animal origin ;

meat, eggs, milk, dairy products, fish, poultry, etc.
 Vitamin B12 is absent in plant foods.
 Humans obtain small amounts of B12 from intestinal

flora.
Nutritional Requirements (RDA )
3 μg with higher allowances for pregnancy and

lactating women.
Absorption, Transport and Storage
• The intestinal absorption of vitamin B12 requires an
intrinsic factor (IF), a glycoprotein secreted by parietal
cells of the stomach.
• In stomach IF binds the dietary vitamin B12 to form
vitamin B12-IF complex.
• This complex binds to specific receptors on the surface of
the mucosal cells of the ileum.
• After binding to the receptor, the bound vitamin B12 is
released from the complex and enters the illeal mucosal
cells through a Ca2+ dependent process.
Functions
There are only two human enzyme systems that are

known to require vitamin B12 coenzyme.
1. Isomerization of methylmalonyl-CoA to succinyl-CoA
2. Conversion of homocysteine to methionine

Role of vit B12 in
isomerization of
methylmalonyl-CoA to
succinyl-CoA
Role of vit B12 conversion of homocysteine to methionine
This is the only mammalian reaction require both vitamins

 Pernicious anemia
 Megaloblastic anemia
 Methylmalonic aciduria
sub acute combined degeneration
 Neuropathy (sub acute combined degeneration,
SCD) : is progressive degeneration of the
posterior and lateral columns of spinal cord.
 Nerve fibers that control movement and sensation

are damaged.
Vitamin B12 Deficiency Causes
Functional Folate Deficiency: The Folate Trap
 S-adenosyl methionine forms homocysteine, which may

be remethylated by methyltetrahydrofolate catalysed by
methionine synthase, a vitamin B12 dependent enzyme.
 Impairment of methionine synthase in vitamin B12

deficiency results in the accumulation of
methyltetrahydrofolate that cannot be used and cannot be
converted to free THF.
 Thus, most of THF is irreversibly “trapped” as methyl
THF.
 There is therefore functional deficiency of folate,

secondary to the deficiency of vitamin B12.
 Folate trap creates folate deficiency and an adequate

supply of free THF is not available for the synthesis of
purine and pyrimidine bases.
Vitamin C (Ascorbic Acid)
Structure
 It is a six-carbon sugar derivative .
 Humans cannot synthesize ascorbic acid, due to lack of

the enzyme gluconolactone oxidase.
Structure of ascorbic acid.
Active Form of Ascorbic Acid
Ascorbic acid itself is an active form.
Sources
 The main dietary sources of vitamin C are leafy

vegetables and fruits, especially citrus fruits, strawberries,
tomatoes, spinach and potatoes.
 Cereals contain no vitamin C.
 Animal tissues and dairy products are very poor sources.

The recommended daily allowance is about 60–70 mg.

Additional intakes are recommended for women during
pregnancy and lactation.
Functions
 Vitamin C is the coenzyme for two groups of
hydroxylases.
–Copper containing hydroxylases
–α-ketoglutarate linked iron containing

hydroxylases
 Ascorbic acid has specific roles in the number of non-

enzymatic effects as result of its action as a reducing
agent and oxygen radical quencher.
Role of Ascorbic acid in the copper containing hydroxylases
 Dopamine β-hydroxylase is a copper containing enzyme

involved in the synthesis of the catecholamines
(norepinephrine and epinephrine), from tyrosine in the
adrenal medulla and central nervous system.
 A number of peptide hormones have a carboxy terminal

amide that is derived from a terminal glycine residue. This
glycine is hydroxylated on the α-carbon by a copper
containing enzyme, peptidylglycine hydroxylase, which
again, requires ascorbate for reduction of Cu2+.
Role of Ascorbic acid in the iron containing hydroxylases
 Proline hydroxylases and lysine hydroxylases are required for the

postsynthetic modification of procollagen to collagen.
 Aspartate β-hydroxylase is required for the postsynthetic

modification of the precursor of protein C.
 Vitamin K-dependent protease that hydrolyses activated factor V

in the blood clotting cascade.
 Trimethyllysine and γ-butyrobetain hydroxylases are required for

the synthesis of carnitine.
Non-enzymatic effects as result of its action as a reducing
agent and oxygen radical quencher
 Ascorbic acid is a water soluble antioxidant:
– It reduces oxidized vitamin E (tocopherol) to
regenerate functional vitamin E.
– Vitamin C thought to be involved in the prevention of
atherosclerosis and coronary heart disease by
preventing oxidation of LDL.

– Antioxidant property of vitamin C is also associated
with prevention of cancer by inhibiting nitrosamine
formation from naturally occurring nitrates during
digestion.
• In addition to other roles of vitamin C, it enhances the

absorption of inorganic iron. Ascorbic acid facilitates the
absorption of iron from intestine by reducing it to the
Fe++ (ferrous) state.
 Deficiency has been observed in infants receiving un-

supplemented cow’s milk and in infants receiving breast
milk from deficient mother.
 Deficiency occurs most commonly in the elderly,

especially those who do not eat fresh fruits and vegetables
and who tend to cook in frying pans, the combination of
heat and large area of food in contact with air irreversibly
oxidizes the vitamin and loses its biological activity.
 Deficiency also can occur in iron overload.

Scurvy
 Deficiency of ascorbic acid causes scurvy.
 Vitamin C is required for formation of collagen, where it is

needed for the hydroxylation of proline and lysine residues, of
protocollagen.
 Hydroxyproline and hydroxylysine are essential for the

collagen cross-linking and collagen strength and stability.
 Since vitamin C is required for normal collagen formation,

vitamin C is also involved in bone and dentin formation as well
as wound healing process.
Symptoms of scurvy
 Symptoms of scurvy are related to deficient collagen
formation. These include:
– Fragility of vascular walls causing bleeding tendency,
muscle weakness, soft spongy, swollen bleeding
gums, loosening of teeth.
– Abnormal bone development and osteoporosis. In
children bone formation is impaired.

– Poor wound healing.
– Anemia due to impaired erythropoiesis.
– Milder forms of vitamin C deficiency are more
common and manifestation of such include easy
bruising (contusion) and formation of petechiae both
due to increased capillary fragility.

Vitamin A
Structure
 Vitamin A contains a single 6-membered ring to which is

attached an 11-carbon side chain.
 Vitamin A is an alcohol (retinol), but can be converted

into an aldehyde (retinal), or acid (retinoic acid).
Structure of vitamin A, retinol.
Active Form
Vitamin A consists of three biologically active molecules

which are collectively known as retinoids.
1. Retinol: Primary alcohol (CH2OH) containing form
2. Retinal: Aldehyde (CHO) containing form
3. Retinoic acid: Carboxyl (COOH) containing form

 Each of these compounds are derived from the plant precursor
molecule, β-carotene.
 β-carotene which consists of two molecules of retinal linked

at their aldehyde ends is also referred to as the provitamin
form of vitamin A.
 The retinol and retinal are interconverted by enzyme retinal

aldehyde reductase.
 The retinoic acid is formed by oxidation of retinal. The

retinoic acid can not be reduced to either retinol or retinal
Conversion of β-carotene (provitamin) to biologically active forms of
vitamin A.
Sources
 The richest dietary sources are fish liver oils (cod liver
oil). Meat is rather low in vitamin A.
 Other good sources are milk and dairy products, dark-

green leaves, such as spinach and yellow and red fruits
and vegetables, such as carrots, tomatoes, and peaches
etc.
The RDA of vitamin A for adults is 800–1000 retinol

equivalents. (1 retinol equivalent = 1 mg retinol = 6 mg β-
carotene).
Functions of Vitamin A
 Vitamin A is required for a variety of functions such as
– Vision
– Cell differentiation and growth
– Mucus secretion
– Maintenance of epithelial cells

 Different forms of the vitamin have different functions.
– Retinal and retinol are involved in vision.
– Retinoic acid is involved in cellular differentiation

and metabolic processes.
– β-carotene is involved in antioxidant function.

Role of Vitamin A in Vision
 The role of vitamin A in vision has been known through

the studies of G Wald, who received the Nobel Prize in
1943
 The cyclic events occur in the process of vision, known

as rhodopsin cycle or Wald’s visual cycle
 Both rod and cone cells of retina contain a photoreceptor
pigment in their membrane and vitamin A is a component

of these pigments.
 Rhodopsin or visual purple, the visual pigment of rod

cells in the retina consists of 11-cis-retinal bound to
protein opsin.
Wald’s visual cycle
 When rhodopsin absorbs light, the 11-cis-retinal is
converted to all-trans retinal.
 The isomerization is associated with a conformational

change in the protein opsin.
 Conformational changes in opsin generates a nerve

impulse that is trans- mitted by the optic nerve to the brain.
 This is followed by dissociation of the all-trans retinal

from opsin
 The all-trans retinal is immediately isomerized by retinal
isomerase to 11-cis-retinal.
 This combines with opsin to regenerate rhodopsin and

complete the visual cycle.
 The conversion of all-trans retinal to 11-cis-retinal is

incomplete and therefore remaining all-trans retinal
which is not converted to 11-cis-retinal is converted to all-
trans retinol by alcohol dehydrogenase and is stored in the
liver.
 When needed, retinol re-enters the circulation and is
taken up by the retina, where it is converted back to 11-
cis-retinal which combines with opsin again to form
rhodopsin
Dark adaptation time
The time taken for regeneration of rhodopsin is known as

dark adaptation time. Dark adaptation time is increased in
vitamin A deficient individuals
Role of Vitamin A in Color Vision
 Color vision is mediated by three pigments called
porphyropsin, iodopsin and cyanopsin and are sensitive to
the three essential colours: red, green and blue
respectively.
 All these pigments consist of 11-cis-retinal bound to
protein opsin .
 When light strikes retina, it bleaches one or more of these
pigments, depending on the color quality of the light.
 The pigments are converted to all-trans retinal, and the

protein moiety opsin is released as in the case of
rhodopsin.
 This reaction gives rise to the nerve impulse that is read
out in the brain as color:
– Red if porphyropsin is split
– Green if iodopsin is split
– Blue if cyanopsin is split.
 If mixtures of the three are converted, the color read out

in the brain depends on the proportions of the three split.
Cellular Differentiation and Metabolic Effect
 Retinoic acid is an important regulator of gene

expression especially during growth and development.
 Retinoic acid is essential for normal gene expression

during embryonic development such as cell
differentiation in spermatogenesis and in the
differentiation of epithelial cells.
 Retinoic acids exert a number of metabolic effects on tissues.
These include:
– Control of biosynthesis of membrane glycoproteins
and glycosaminoglycans (mucopolysaccharide)
necessary for mucus secretion. The normal mucus
secretion maintains the epithelial surface moist and
prevents keratinization of epithelial cell.
– Control of biosynthesis of cholesterol.

Antioxidant Function
 β-carotene is an antioxidant and may play a role in

trapping free radicals in tissues.
 The antioxidant property of lipid soluble vitamin A may

account for its possible anticancer activity.
 High levels of dietary carotenoids have been associated

with a decreased risk of cardiovascular disease
 Clinically, degenerative changes in eyes and skin are

observed commonly in individuals with vitamin A
deficiency.
 Vitamin deficiency may be primary (dietary

insufficiency) or secondary.
The causes of secondary deficiency may include:
– Impaired absorption of lipids
– Failure to synthesize apo B-48 and therefore inability

to form chylomicrons into which vitamin A is
normally incorporated after absorption.
– Lack of lipase, as in pancreatitis.
– Failure in converting β - carotene to retinol; because

of an enzyme defect.
– Impaired storage in hepatic cell in liver disease.
– Failure to synthesize retinol binding proteins, thus

affecting transport to target tissues.
Effect on Vision
The earliest symptoms of vitamin A deficiency is:
– Impaired dark adaptation or night blindness (nyctalopia)
– Poor vision in dim light
– Xerophthalmia.
Night blindness (nyctalopia)
 This is characterized by loss of vision in night (in dim or

poor light) since dark adaptation time is increased.
 Prolonged deficiency of vitamin A leads to an irreversible

loss of visual cells.
 Severe vitamin A deficiency causes dryness of cornea and

conjunctiva, a clinical condition termed as xerophthalmia
(dry eyes).
 If this situation prolongs, keratinization and ulceration of
cornea takes place
 This results in destruction of cornea. The cornea becomes

totally opaque resulting in permanent loss of vision
(blindness), a clinical condition termed as kerato- malacia.
 White opaque spots develop on either side of cornea in

vitamin A deficiency are known as Bitot’s spot.
Effect on Skin and Epithelial Cells
 Vitamin A deficiency causes keratinization of epithelial

cells of skin which leads to keratosis of hair follicles,
and dry, rough and scaly skin.
 Keratinization of epithelial cells of respiratory, urinary

tract makes them susceptible to infections.
Other Symptoms of Vitamin A Deficiency
 Failure of growth in children.
 Faulty bone modelling producing thick cancellous

(spongy) bones instead of thinner and more compact ones.
 Abnormalities of reproduction, including degeneration of

the testes, abortion or the production of malformed
offspring.
Hypervitaminosis A
Excessive intakes of vitamin A lead to accumulation beyond the

capacity of intracellular binding proteins. Unbound vitamin A
causes membrane lysis and tissue damage. Symptoms of toxicity
affect:
 The central nervous system and leads to headache, nausea,

ataxia, and anorexia. These all associated with increased
cerebrospinal fluid pressure.
 The liver: hepatomegaly with histological changes and

hyperlipidemia
 Calcium homeostasis: thickening of the long bones,
hypercalcemia, and calcification of soft tissues, bone and
joint pain,
 The skin: loss of hair (alopecia), scaly and rough skin.
 In pregnant women, the hypervitaminosis A may cause
congenital malformation in growing fetus (teratogenic
effect).
Why Vitamin A is Considered as a Hormone?
 Within cells both retinol and retinoic acid function by binding

to specific receptor proteins present in the nucleus of target
tissues.
 Following binding, the receptor-vitamin complex interacts

with several genes involved in growth and differentiation and
affects expression of these genes.
Vitamin D (Cholecalciferol)
Vitamin D is also known as calciferol because of its role
in calcium metabolism and antirachitic factor because it
prevents rickets.
Vitamin D could be thought of as a hormone
rather than a vitamin
 As it can be synthesized in the body
 It is released in the circulation
 Has distinct target organs
 Action of vitamin D is similar to steroid hormones. It

binds to a receptor in the cytosol. Following binding, the
receptor vitamin complex interacts with DNA to
stimulate the synthesis of calcium binding protein.
Structure
Vitamin D is a steroid compound. The naturally produced

vitamin D3 or cholecalciferol is obtained from animal
sources in the diet, or made in the skin by the action of
ultraviolet light from sunlight on
Structure of 1,25-dihydroxycholecalciferol an active
form of vitamin D3.
The formation of vitamin D3 in the body and
vitamin D2 commercially.
Active Form of Vitamin
 Cholecalciferol is an inactive form of vitamin D.
 It needs further metabolism to produce the active form of

the vitamin. 1, 25 dihydroxycholecalciferol also known
as calcitriol is the active form of vitamin D.
Activation of vitamin D.
Sources
 Best sources are cod liver oil and often fish oils and
sunlight induced synthesis of vitamin D3 in skin.
 Egg yolk and liver are good sources.
The daily requirements of vitamin D is 200–400 IU.

Functions
 Vitamin D (Calcitriol) plays an essential role as a

hormone in the regulation of calcium and phosphorus
metabolism.
 It maintains the normal plasma level of calcium and

phosphorus by acting on intestine, kidneys and bones.
 Action of calcitriol on intestine
It increases the plasma calcium and phosphorus

concentration by stimulating the absorption of
calcium and phosphorus from the intestine.
 Action of calcitriol on kidney
It stimulates the reabsorption of calcium and

phosphorus from the kidney and decreases their
excretion.
 Action of calcitriol on bone
– Calcitriol promotes the mineralization of bones by

deposition of calcium and phosphorus.
– Calcitriol along with PTH stimulates the mobilization

of calcium and phosphorus from bone.
Sites of formation of vitamin D3 to its metabolically
active form 1,25-dihydroxycholecalciferol and its
function.
1,25-DHCC: 1,25-dihydroxycholecalciferol;
PTH: parathyroid hormone

Deficiency of vitamin D causes:
– Rickets (rachitis) in growing children and
– Osteomalacia in adults.
Rickets
 Rickets is characterized by formation of soft and pliable

bones due to poor mineralization and calcium deficiency.
 Due to softness, the weight bearing bones are bent and

deformed.
 The main features of the rickets are, a large head with

protruding forehead, pigeon chest, bow legs, (curved
legs), knock knees and abnormal curvature of the spine
(kyphosis).
Bowing of legs in rickets.
 Rachitic children are usually anemic or prone to
infections. Rickets can be fatal when severe.
 Rickets is characterized by low plasma levels of calcium

and phosphorus and high alkaline phosphatase activity.
Osteomalacia (Adult Rickets)
 Deficiency of vitamin D in adults causes osteomalacia.

This is a condition similar to that of rickets.
 Osteomalacia characterized by demineralization of

previously formed bones, Demineralization of bones
makes them soft and susceptible to fractures.
 Osteomalacia especially occurs in women who have little

exposure to sunlight, and especially after several
pregnancies
Renal Rickets (Renal Osteodystrophy)
 In chronic renal failure synthesis of calcitriol in kidney is

impaired. As a result, the deficiency of calcitriol occurs
which leads to hypocalcemia and hyperphosphatemia.
 It can be treated by oral or intravenous administration of

calcitriol (active form of vitamin D).
Vitamin D Resistant Rickets
 As the name implies, this is a disease which does not

respond to treatment with vitamin D.
 There are various possible causes of this condition and

all involve a defect in the metabolism or mechanism of
action of 1,25 dihydroxycholecalciferol.
– Due to defective vitamin D receptor
– Due to a defective 1, α-hydroxylase activity in kidney
– Due to liver disease and kidney failure as the

production of 25-hydroxycholecalciferol and 1,25
dihydroxycholecalciferol respectively will be
inefficient in the damaged tissue
Hypervitaminosis D
 High doses of vitamin D over a long period are toxic.
 The early symptoms of hypervitaminosis D include nausea,
vomiting, anorexia, increased thirst, loss of weight, etc.
 Hypercalcemia is seen due to increased bone resorption and
intestinal absorption of calcium.
 The prolonged hypercalcemia causes calcification of soft
tissues and organs such as kidney and may lead to formation
of stones in the kidneys.

Vitamin E (Tocopherol)
Structure
Vitamin E consists of eight naturally occurring tocopherols,

of which α-tocopherol is the most active form
Sources
The major dietary sources of vitamin E are fats and oils.

The richest sources are germ oil, corn oil, fish oil, eggs,
lettuce and alfalfa.
A daily consumption of about:
10 mg (15 IU) of α-tocopherol for a man
8 mg (12 IU) for α- woman is recommended.
One mg of α-tocopherol is equal to 1.5 IU.

Functions
 Vitamin E acts as a natural antioxidant by scavenging

free radicals and molecular oxygen.
 Vitamin E is important for preventing peroxidation of

polyunsaturated fatty acids in cell membranes.
 Protection of erythrocyte membrane from oxidant is the

major role of vitamin E in humans. It protects the RBCs
from hemolysis.
 Vitamin E also helps to prevent oxidation of LDL.
Oxidized LDL may be more atherogenic than native LDL
and thus vitamin E may protect against athreo- matous
coronary heart disease.
 Whether vitamin E affects human fertility is unknown.
 In animals, vitamin E is required for normal reproduction

and prevents sterility.
 The major symptom of vitamin E deficiency in human is

hemolytic anemia due to an increased red blood cell fragility.
 Another symptom of vitamin E deficiency is retro- lental

fibroplasia (RLF) observed in some premature infants of low
birth weight. Children with this defect show neuropathy.
Hypervitaminosis E
 Unlike other fat soluble vitamins such as A and D, vitamin E

does not seem to have toxic effects.
Vitamin K
 This vitamin is called an anti-hemorrhagic factor as its

deficiency produced uncontrolled hemorrhages due to
defect in blood coagulation.
 In 1929, H Dam gave the name koagulation vitamin from

the Danish word koagulation. It is now called vitamin K.
Structure
 Vitamin K1 or phylloquinone derived from plant.
 Vitamin K2 or menaquinones, produced by

microorganisms.
 Vitamin K3 or menadione is a synthetic product

Structure of vitamin K.
Sources
 Excellent sources are cabbage, cauliflower, spinach and

other green vegetables.
 Good sources include tomatoes, cheese, dairy products,

meat, egg yolk, etc.
 The vitamin is also synthesized by microorganisms in the

intestinal tract.
The suggested intake for adults is 70–140 mg/day.

Functions of Vitamin K
 Vitamin K plays an important role in blood coagulation.
 Vitamin K is required for the activation of blood clotting
factors, prothrombin (II), factor VII, IX and X.
 These blood clotting proteins are synthesized in liver in
inactive form, and are converted to active form by
vitamin K dependent carboxylation reaction.
 In this, vitamin K dependent carboxylase enzyme adds
the extra carboxyl group at  -carbon of glutamic acid
residues of inactive blood clotting factors.
Role of vitamin K in the gamma-carboxylation of glutamyl residues of inactive
proteins of blood-clotting factors.
 Vitamin K is also required for the carboxylation of
glutamic acid residues of osteocalcin, a Ca2+ binding
protein present in bone.
 Anticoagulants, Dicumarol and warfarin are structurally

similar to vitamin K and inhibit the action of vitamin K.
Vitamin K is widely distributed in nature and its production

by the intestinal micro flora ensures that dietary deficiency
does not occur.
 Vitamin K deficiency is associated with hemorrhagic

disease.
 In vitamin K deficiency, clotting time of blood is

increased. Uncontrolled hemorrhages occur on minor
injuries as a result of reduction in prothrombin and other
clotting factors.
Vitamin K deficiency, however, is found in:
 Patients with liver disease and biliary obstruction.

Biliary obstruction inhibits the entry of bile salts to the
intestine.
 In new-born infants, because the placenta does not pass

the vitamin to the fetus efficiently, and the gut is sterile
immediately after birth.
 Following antibiotic therapy that sterilizes the gut.
 In fat malabsorption, that impairs absorption of vitamin

K.
Hypervitaminosis K
Excessive doses of vitamin K produce a hemolytic anemia

(due to increased breakdown of RBCs) and jaundice (in
infants).
 The extracellular space in the tissues of multicellular
animals is filled with gel like material, called
extracellular matrix (ECM).
 ECM is also called ground substance.
 ECM often referred to as “connective tissue”, that
provide structural and biochemical support for
surrounding cells.
The extracellular matrix contains:
1. Water
2. Fibrous proteins: The main fibrous proteins that build the
extracellular matrix are collagen and elastin
3. Glycoproteins: The main glycoproteins are fibrillin,
fibronectin, and laminins
4. Proteoglycans (glycosaminoglycans, GAGs): The major
proteoglycans are, hyaluronic acid, chondroitin sulfate,
keratan sulfate, heparan sulfate and heparin
The main fibrous proteins of ECM
The main fibrous proteins of ECM are:
• Collagen
• Elastin
 Collagens are the major structural component of the
ECM. In the matrix, collagen will give the cell tensile
strength and facilitate cell-to-cell adhesion and
migration. Collagens provide scaffolding for the
attachment of laminin, proteoglycans and cell surface
receptors.
 Elastins in contrast to collagens, give elasticity to
tissues, allowing them to stretch when needed and then
return to their original state. This is useful in blood
vessels, the lungs, in skin, and the ligamentum nuchae.
These tissues contain high amounts of elastins.
Structure and Function of Collagen
 Collagen is the main protein of connective tissue and
the most abundant protein in mammals.
 It has great tensile strength and is present to some
extent in nearly all organs and serves to hold cells
together in discrete units.
Structure of Collagen
 All collagen types have triple helical structure.
 The basic structural unit of collagen is tropocollagen,

which consists of three polypeptide chains called α-
chains.
 These three polypeptide chains twisted around each

other in a triple helix forming a rope like structure,
which has great tensile strength
Right-handed collagen triple helix formed from three
left-handed α-chains.
 The three helically interwind polypeptides are of equal
length, each having about 1000 amino acids residues.
 The three polypeptide chains are held together by

hydrogen bonds between chains.
 Collagen has an unusual amino acid composition with

33% of the total residues being glycine (Gly), 10%
proline (Pro), 10% hydroxyproline (Hyp) and 1%
hydroxylysine (Hyl).
Formation of Collagen Fibrils
 Individual tropocollagen molecules spontaneously
laterally aggregate to form fibril.
 They arrange themselves under physiological conditions
into staggered, parallel and overlapping array structures.
 Each tropocollagen molecule overlaps its neighbour by a
length approximately three quarters of a molecule
 The regularity of gaps and overlaps is responsible for
the banded appearance of these fibers in connective
tissues.
 The quarter staggered, parallel, and overlapping array
structural arrangement of tropocollagen to form
collagen fiber;
 The regularity of gaps and overlaps generates the
banded appearance in the collagen fiber;
 Electron micrographic banded appearance of fibrillar
collagen.
 These fibriller arrays of tropocollagen molecules
become connected and subsequently stabilized by intra-
and intercovalent cross-links through action of copper
requiring enzyme lysyl oxidase.
Types
Functions
• It gives strength, support and shape to the
tissues
• Collagen contributes to proper alignment of
cells
• Collagen (that is exposed in blood vessels)
contributes to thrombus formation.
Disorders
Ehlers–Danlos Syndrome
• It is heterogeneous group of genetic disorders
characterized by stretchy skin and loose joints.
Osteogenesis Imperfecta (OI)
• It is a group of genetic disorders in which
biosynthesis of type I collagen is defective.
• OI is characterized by fragile bones, hence also
known as the brittle bone syndrome.
• The disorder is characterized by fragile
bones, thin skin, abnormal teeth and weak
tendons.
Epidermolysis Bullosa
• Epidermolysis bullosa is a rare heritable
disorder due to mutation in gene encoding
collagen.
• It is characterized by skin breaks and
blistering of the skin and epithelial tissue.
Structure and function of Elastin
 Elastin occurs with collagen in connective tissues.
 Elastin is a rubber-like protein, which can stretch to
several times their length and then rapidly return to their
original size and shape when the tension is released.
 Elastin is present in large amounts, particularly in tissues
that require these physical properties, e.g. lung, blood
vessels and ligaments.
Structure of Elastin
 The basic subunit of elastin fibrils is tropoelastin which
contains about 800 amino acid residues.
 Unlike collagen, elastin does not contain repeat (Gly-X-Y)
sequences.
 Although elastin and collagen contain similarly high amounts
of glycine and proline and both lack cysteine and tryptophan
elastin contains less hydroxyproline and no hydroxylysine
and glycosylated hydroxylysine.
 Elastin has very high content of alanine and other nonpolar
aliphatic residue, i.e. valine, leucine and isoleucine.
Desmosine cross-link of elastin,
formed from four lysine residues.

Plasma proteins
The major plasma proteins are:
1. Albumin
2. Globulin
3. Fibrinogen
The normal value of plasma proteins are:
 Total protein 6 to 8 gm%
 Albumin 3.5 to 6 gm%
 Globulin 2 to 3.5 gm%
 Fibrinogen 200 to 400 mg%
 A/G ratio 1.2:1 to 2.5:1

Synthesis of Plasma Proteins
 All the albumin and fibrinogen are synthesized by the

liver only
 Similarly 50 to 80% of the globulin is formed in the liver
 The remainder of the globulins are formed almost

entirely in the lymphoid tissues.
 The A/G ratio can be altered in the liver disease.

Separation of Plasma Proteins
The plasma proteins can be separated by :
- Salting out
- Electrophoresis
- Ultracentrifugation
- Immunoelectrophoresis.
Electrophoretic separation of serum proteins
Quick review of plasma protein
• Most abundant plasma protein: Albumin
• Least abundant plasma protein: α1-Globulin
• Plasma protein with fastest electrophoretic mobility: Prealbumin,
transthyretin followed by albumin.
• Plasma protein with slowest electrophoretic mobility: Gamma globulin
• Plasma protein not synthesized in liver: Immunoglobulins ( by plasma cells
of B-lymphocyte lineage)
• Plasma protein not a glycoprotein: albumin.
• Plasma protein exclusively synthesized in liver: albumin.

ALBUMIN
• Albumin is a globular protein with 585 amino acid residues
accounting for approximately 50% of plasma protein
• Albumin is synthesized exclusively by the liver.
Functions of albumins
Albumin plays a predominant role in:
 Maintenance of plasma oncotic pressure.

 Transport of metabolites which are poorly soluble in
water :
 Fatty acids
 Bilirubin
 Calcium
 Certain steroid hormones
 Copper
 Some of the plasma tryptophan
 A variety of drugs, like sulfonamides, penicillin
G, dicumarol, aspirin, and digoxin
• Buffering function: albumin has maximum buffering
capacity
• Nutritive function : Degradation of albumin provide
essential amino acids during malnutrition.

Clinical significance
Hypoalbuminaemia: Malnutrition, nephrotic syndrome, cirrhosis of
liver
Hyperalbuminaemia: Acute dehydration have no clinical significance.
Analbuminaemia (less than 1gm/l , normal= 3.5 to 6gm/dl):
Analbuminaemia is a rare hereditary abnormalities there may be no
symptoms or signs not even edema due to compensatory increase in
plasma globulin concentration.

Globulins
Globulins constitute several fractions.
 α1-globulin
 α2-globulin
 β-globulin and
 γ-globulin
Immunoglobulins (Ig)
• The immunoglobulins are γ -globulins, called
antibodies.
• Constitute about 20% of all the plasma proteins
• produced by plasma cells and by lymphocytes in
response to a variety of antigen.

Schematic structure of immunoglobulin G.
 The basic immunoglobulin is Y shaped consist of four
polypeptide chains:
– two H and
– two L chains
 The four chains are linked by disulfide bond
 L chain may be either of two types, Kappa (қ) or
Lambda (λ) but not both
 The heavy chains may be of five types and are
designated by Greek letter:
– alpha (α)
– gamma (γ),
– delta (δ),
– mu (μ) and
– epsilon (ε)
 Immunoglobulins are named as per their heavy chain
type as IgA, IgG, IgD, IgM and IgE
 The L and H chains are subdivided into variable ( towards
the carboxyl terminal end) and constant ( towards the
amino terminal end) regions.
 L chain consists of one variable (VL) and one constant

(CL) domain or region.
 Most H-chains consist of one variable (VH) and three

constant (CH-1, CH-2, and CH-3) domains.
 The hinge region between the CH-1 and CH-2 domains
confers flexibility and allows both Fab arms to move
independently , thus helping them to bind to antigenic site
Light chain type
• Light chain may be either of two types, Kappa (k) or
Lambda (l) but not both.
• In a given immunoglobulin either 2k or 2 l but not the
mixture of kappa and lambda
• Most abundant light chain in human is k

 Enzyme (papain) digestion splits the
immunoglobulin molecule into two fragments
1. Fab: Fragment for antigen binding. Located in

variable region.
2. Fc: Crystallisable fragment or fragment for
complement binding
Monomeric, dimeric, and pentameric forms of
immunoglobulins.
 Most common Ig in serum: IgG
 Least common Ig in serum: IgE
 Largest Ig: IgM
 Pentamer Ig: IgM
 Dimmer: Secretary IgA
 Ig fixes the complement: IgG and IgM
 Ig present in secretion: IgA

 Ig can cross placenta: IgG
 IgG can not cross placenta: IgG2Ig involved in primary
immune response: IgM
 Ig secondary immune response: IgG
 Ig with J chain: IgM and IgA
 Ig with secretary piece: IgA

Disorders due to changes in immunoglobulins
Quantitative changes in the amount of immunoglobulins in the
plasma and urine are known in several pathological conditions in
human.
– Multiple Myeloma
– Amyloidosis
– Bence Jones Proteins
– Cryoglobulinaemia
– Waldenstrom’s Macroglobulinaemia
Enzymes
 Enzymes are biological catalyst produced by living
tissues.
 They are proteins with the exception of few classes of

RNA molecules called ribozyme.
 They accelerate specific chemical reactions without

being consumed in the process.
 They function in aqueous solutions under very mild

conditions of temperature and pH.
 The catalytic activity of enzyme depends on their native
protein conformation. If an enzyme is denatured or
dissociated into its subunits, catalytic activity is usually
lost.
 Thus, the primary, secondary, tertiary, and quaternary

structures of protein enzymes are essential to their
catalytic activity
Enzyme Classification
 Enzymes are classified according to the type
of reaction they catalyse.
 All enzymes have formal ‘EC’ (Enzyme

Commission) number and names, and most
have trivial names.
 According to the International Union of Biochemistry
and Molecular Biology (IUBMB) system, enzymes are
classified into seven major classes
 Enzymes are classified according to the type of reaction

they catalyse.
The seven classes as per IUBMB are as follows:
1. EC-1: Oxidoreductase
2. EC-2: Transferase
3. EC-3: Hydrolase
4. EC-4: Lyase
5. EC-5: Isomerase
6. EC-6: Ligase
7. EC-7: Translocases
EC-1 Oxidoreductases
Catalyzes oxidation-reduction reactions.

Enzymes in this category include :
 Dehydrogenases
 Reductases
 Oxidases
 Peroxidases.
EC-2 Transferases
Catalyses the transfer of a group such as, amino,

carboxyl, methyl or phosphate, etc. from one
molecule to another
Enzymes in this category include :
 Amino transferase or transaminase
 Kinase: catalyzes the transfer of phosphate groups
 Transcarboxylase.
EC-3 Hydrolases
Catalyze the cleavage of C-O, C-N, C-C and some

other bonds with the addition of water.
Enzymes in this category are:
 All digestive enzymes like:

- α-amylase,
- pepsin,
- trypsin,
- chymotrypsin, etc.
 Acid phosphatase
EC-4 Lyases
Catalyze the cleavage of C-O, C-C and C-N bonds by means

other than hydrolysis or oxidation, giving rise to compound
with double bonds or catalyze the reverse reaction, by the
addition of group to a double bond.
In cases where reverse reaction is important, then
synthase, (not synthetase of group EC-6) is used
in the name.
EC-5 Isomerases
Catalyze intramolecular structural rearrangement in a
molecule. They are called epimerases, isomerases or
mutases, depending on the type of isomerism involved.

EC-6 Ligases (Synthetases)
Catalyze the joining of two molecules coupled with the
hydrolysis of ATP.
EC-7: Translocases (A new EC Class)
Translocases catalyze the movement of ions or
molecules across membranes or their separation
within membranes.
.
Examples are:
 Enzymes catalyzing the translocation of:
Hydrons (H+), inorganic cations, inorganic anions, amino
acids and peptides, and carbohydrates and their derivatives.
 Enzymes of the reaction that provided the driving

force for the translocation linked to:
Oxidoreductase reactions, hydrolysis of a nucleoside
triphosphate, hydrolysis of a diphosphate, and
decarboxylation reaction.
Zymogen OR Proenzyme
 Enzymes found in in an inactive (precursor)
form, called zymogen or Proenzyme.
 Zymogen have the prefix “pro” or suffix “ogen”.

 For example,
- prothrombin,
- proelastase
- chymotrypsinogen,
- trypsinogen,
- pepsino- gen
Cofactors (Coenzyme And Activator)
 Some enzymes require an additional non-protein
component for its activity. This additional
compo- nentis called cofactor
– Inorganic ions, called activators.
– Organic compounds, called coenzymes

 Enzymes without its cofactor is referred to as
an apoenzyme
 The complete catalytically active enzyme is
called holoenzyme.
 Apoenzyme + Cofactor = Holoenzyme.

MECHANISM OF ENZYME ACTION
Formation of an enzyme-substrate (ES) complex is
the first step in enzymatic catalysis which is
subsequently converted to product and free enzyme.

Substrate is bound through non-covalent
interactions at the active site of the enzyme.
The active site of an enzyme is the region that
binds the substrate and which contains the
specific amino acid residues.

Models for binding of substrate to enzyme
1. Lock and key model or rigid template model of

Emil Fisher.
2. Induced fit model or hand-in-glove model of

Daniel E Koshland
Lock and Key Model or Rigid Template Model
of Emil Fisher
 Enzyme is pre-shaped and the active site has a
rigid structure, complementary to that of the
substrate.
 Substrate fits into the active site in much the
same way that a key fits into a lock.

Representation of Fisher’s lock and key model.
This model explains all mechanisms but do not
explain the changes in the enzyme activity in the
presence of modulator.
Induced Fit Model or Hand-in-glove Model of
Daniel Koshland
Enzymes are flexible
Shapes of active site can be modified by the
binding of the substrate.
Substrate induces a conformational change in
enzyme.
Conformational change in enzyme induces
reciprocal changes in its bound substrate that
alters their orientation and configuration and
strains the structure of the bound substrate.
intrinsic binding energy is liberated.
Intrinsic binding energy converts substrate into

product.
Schematic representation of induced fit model of Koshland
SPECIFICITY OF ENZYME ACTION
 Ability of enzyme to discriminate between two substrates.
 Enzymes are highly specific both in the reaction catalyzed
and in their choice of substrates.
 Specificity makes it possible for number of enzymes to
co-exist in cell without interfering in each other’s actions.

Types of Specificity
1. Substrate specificity
2. Reaction specificity
3. Stereo specificity
Substrate Specificity
i. Absolute substrate specificity
ii. Relative substrate specificity
iii. Broad substrate specificity.

Absolute substrate specificity
Certain enzymes will act on only one substrate
and catalyze one reaction, e.g. Glucokinase,
lactase, urease, etc.

Relative substrate specificity
Enzyme acts on more than one substrate.
 Group specificity
 Bond specificity.
Chymotrypsin acts on several proteins by
hydrolyzing peptide bonds attached to aromatic
amino acids.
Trypsin hydrolyzes peptide linkages involving

arginine or lysine.
α-amylase, cleaves α-(1→4) glycosidic bonds
of carbohydrates.
Lipase hydrolyzes ester bonds of lipids.

Broad substrate specificity
 Enzyme acts on more than one structurally
related substrates.
 hexokinase catalyzes the phosphorylation of

more than one kind of hexoses such as glucose,
fructose and mannose.

Reaction Specificity
Enzyme is specific to a particular reaction but
not to substrate (s) and catalyzes only one type
of reaction.
Example of reaction specificity.
Stereo Specificity
 L-lactate dehydrogenase will act only on
L-lactic acid and not D-lactic acid.
 L-amino acid oxidase and D-amino acid

oxidase act only on L and D-amino acids.
 Salivary α-amylase acts on the α-1,4

glycoside linkage and is inactive on β-1,4
glycoside bond
Factors Affecting The Velocity Of
Enzyme Reaction
 Substrate concentration
 Enzyme concentration
 pH i.e. H+ ion concentration
 Temperature
 Product concentration
 Activators and coenzymes
 Time
 Physical agents
Effect of Substrate Concentration
V0 : initial velocity
Vmax : maximum velocity
Km : 1/2 Vmax = Michaelis Menten constant
[S] : substrate concentration

Effect of Enzyme Concentration
Effect of Hydrogen Ion Concentration pH
 Each enzyme has an optimum pH, i.e. a pH at which
the enzyme activity is maximum.
 Below or above this pH, enzyme activity is
decreased.
 The optimum pH differs from enzyme to enzyme.
– Pepsin = 1.2
– Trypsin = 8.0
Effect of pH on enzyme activity
Changes in pH can alter the following:
 Ionization state of the amino acids
present in the active site of the enzyme.
 The ionization state of the substrate.
 Drastic change in pH denatures enzyme

Effect of Temperature
 Enzyme catalyzed reactions show an increase in rate with
increasing temperature only within a relatively small and
low temperature range.
 Each enzyme shows the highest activity at a particular
temperature called optimum temperature.
 The activity progressively declines both above and below
this temperature.
Effect of temperature on enzyme activity
Increase in velocity is due to the increase in the
kinetic energy.
Further elevation of the temperature results in a
decrease in reaction velocity due to denaturation
of the enzyme protein.

Low temperature also decreases enzyme activity
and enzymes may be completely inactive at
temperature of 0°C and below.
The inactivity at low temperature is reversible.

Most of the body enzymes have the optimum
temperature close to 37°C to 38°C and have
progressively less activity as the temperature
rises.
Effect of Product
Accumulation of products of the reaction causes the
inhibition of enzyme activity for some enzymatic
reactions.
Effect of Activators and Co-enzymes
In absence of activators and coenzymes, enzymes
become functionally inactive.

Effect of Time
Under optimum conditions of pH and temp, time

required for an enzyme reaction is less.
The time required for the completion of an

enzyme reaction increases with changes in
temperature and pH from its optimum.
ENZYME KINETICS
 The study of enzyme reaction rates and how they change

in response to changes in experimental parameters is
known as kinetics.
 One of the key factors affecting the enzyme reaction rates

is the concentration of substrate [S].
Effect of Substrate Concentration
V0 : initial velocity
Vmax : maximum velocity
Km : 1/2 Vmax = Michaelis Menten constant
[S] : substrate concentration
Significance of Km (Michaelis Constant)
1. Km provides a amount of the substrate required for

significant catalysis to occur.
2. It is a measure of the affinity of the enzyme for its
substrate, a high Km indicates weak binding and a

low Km indicates strong binding with its substrate.
• A low Km mitochondrial form
• A high Km cytosolic form.

Significance of Vmax
 The Vmax of a reaction is an index of the catalytic
efficiency of an enzyme.
 The Vmax is useful in comparing the activity of one
enzyme with that of another.

Lineweaver-Burk plot
(Double reciprocal plot)
ENZYME INHIBITION
Any substance that can diminish the velocity of

an enzyme reaction is called inhibitor.
Two general classes of inhibitors are:
1. Reversible inhibitor
2. Irreversible inhibitor.
REVERSIBLE INHIBITOR
Reversible inhibitors bind to enzymes through
non-covalent bonds and the activity of the enzyme
is restored fully when the inhibitor is removed
from the system.

Different types of reversible inhibitors are:
i. Competitive or substrate analogue inhibitor
ii. Non-competitive inhibitor
iii. Uncompetitive inhibitor.

Competitive or Substrate Analogue Inhibitor
A competitive inhibitor is a structural analogue of
the substrate.
Chemical structure of inhibitor (I) resembles that
of substrate (S) and binds to enzyme at active
site, forming EI complex rather than ES-complex.

Diagrammatic representation of competitive inhibition
E: Enzyme; S: Substrate; I: Competitive inhibitor; P: Product

The inhibition could be overcome by increasing
substrate concentration
Enzyme kinetics of competitive inhibitor
Enzyme kinetics of competitive inhibitor
Vmax is unaltered
Km is increased
Drugs act as competitive inhibitors
 Sulphonamide
Analogue of P- aminobenzoic acid (PABA) and inhibits
the synthesis of folic acid in microorganisms.
 Isoniazide [Isonicotinic acid hydrazine (INH)]
It is an anti-tuberculosis drug, inhibits the biosynthesis of
NAD and restrict the growth of the organisms that cause
tuberculosis.
 Dicumarol
It is an anticoagulant drug structurally similar to vitamin
K. It inhibits the vitamin K activity and inhibits the
formation of prothrombin.
 Physostigmine
It inhibits acetylcholinesterase and use to treat glaucoma
and myasthenia gravis.

Drugs such an ibuprofen (anti-inflammatory drug),
statin (cholesterol lowing drug) are competitive
inhibitors of enzymes, that involved in the
prostaglandins and cholesterol synthesis
respectively.
Non-competitive Inhibitors
 No competition occurs between substrate and

inhibitor.
 Inhibitor is usually structurally different from the

substrate.
 It binds at a site on the enzyme molecule other

than the substrate-binding site.
 Noncompetitive inhibitor can bind free enzyme
(EI) or the enzyme substrate complex (EIS)
 However, EIS complex does not continue to form

product.
 Noncompetitive inhibitor lowers the concentration

of functional enzymes.
 Noncompetitive inhibition cannot be overcome by

increasing the substrate concentration
Diagrammatic representation of noncompetitive inhibition.
(E = Enzyme; S = Substrate; I = Non-competitive inhibitor; P = Product)

Examples of non-competitive inhibitors are:
– Ethanol or certain narcotic drugs are non-competitive

inhibitor of acid phosphatase.
– Trypsin inhibitors occur in soybean and raw egg

white, inhibit activity of trypsin.
– Ascaris parasites (worm) contain pepsin and trypsin

inhibitors, inhibit action of pepsin and trypsin.
For non-competitive inhibition, the Km value is unchanged while
Vmax is lowered
Uncompetitive Inhibitor
 Uncompetitive inhibitor can bind only to the
enzyme-substrate (ES) complex.
 It does not have affinity for free enzyme.
 Enzyme-substrate-inhibitor complex, ESI does
not continue to form any product.

 Uncompetitive inhibition cannot be overcome by
the addition of more substrate.
 Consequently Vmax cannot be attained, even at
high substrate concentration.

Uncompetitive inhibitor binds only to enzyme-substrate
complex.
Uncompetitive inhibitor decreases both Vmax and Km.
 The herbicide glyphosate, also known as
Roundup, is an uncompetitive inhibitor of an
enzyme in the biosynthetic pathway for aromatic
amino acids in bacteria
 Nontoxic in animals because they lack the
enzyme.
IRREVERSIBLE INHIBITOR
An irreversible inhibitor binds with an enzyme
tightly covalently and forms a stable complex.
An irreversible inhibitor cannot be released by

dilution or dialysis or simply by increasing the
concentration of substrate.
Irreversible inhibitors can be divided into three
categories:
 Substrate analogue inhibitor or affinity labels
 Group specific inhibitors
 Suicide inhibitor or mechanism based
inactivation.
In terms of enzyme kinetics, the effect of an
irreversible inhibitor is like that of the reversible
non-competitive inhibitors resulting in a decreased
in Vmax but having no effect on the Km

Substrate Analogue Irreversible
Inhibitor or Affinity Labels
 Substrate analogues or affinity labels are molecules
that are structurally similar to the substrate.
 These substrate analogues possess a highly reactive
group which is not present in the natural substrate.

The reactive group of substrate analogues
covalently reacts with amino acid residues of the
active site of the enzyme and permanently block
the active site of the enzyme
3-Bromoacetal phosphate (BAP) inhibits enzyme
phosphotriose isomerase of glycolysis.

Group Specific Irreversible Inhibitor
 These inhibitors react with specific R-groups
(side chain) of amino acid residues in the
active site of enzyme.

 Examples of group specific irreversible inhibitors:
– Di-isopropylphosphofluoride (DIPF)
– Iodoacetamide
– Heavy metals
 DIPF can inhibit an enzyme acetylcholine
esterase by covalently reacting with hydroxyl
group of a serine residue present at the active site
of the enzyme
 DIPF has also been found to inhibit trypsin,
chymotrypsin, elastase and phosphoglucomutase

Irreversible inhibition of acetylcholinesterase by a group-specific
inhibitor, diisopropylphosphofluoride (DIPF).
 Iodoacetamide and heavy metals like, Pb2+, Ag+,
Hg2+, etc. which react with sulfhydryl (-SH) group
of cysteine residues present at the active site of the
enzyme and makes them inactive.

Suicide Inhibitor or Mechanism Based inactivation
 These compounds are relatively unreactive until they bind
to the active site of a specific enzyme.
 On binding to the active site of the enzyme they carry out
the first few catalytic activities of the normal enzyme
reaction.
 Instead of being transformed into a normal product,
however, the inhibitor is converted to a very reactive
compound that combines irreversibly with the enzyme
leading to its irreversible inhibition
 These are also called mechanism based inactivation
because they utilize the normal enzyme reaction
mechanism to inactivate the enzyme

Example Suicide Inhibitor
Penicillin
Inactivates bacterial enzyme glycopeptidyl

transpeptidase involved in the formation of bacterial
cell wall.
Aspirin
Inactivates an enzyme cyclo-oxygenase required for

the synthesis of prostaglandins .
 Disulfiram (antabuse)
 Inhibits aldehyde dehydro- genaseenzyme resulting in

accumulation of acetaldehyde.
 Antabuse, or disulfiram is, a medicine for treatment of
alcohol abuse and alcohol dependence
 Antabuse is prescribed to people who want to quit
drinking.
Clinical Application of Enzyme Inhibitor
 Enzyme inhibitors have therapeutic applications.
 Most antibiotics and anticancer drugs that are used
therapeutically are either competitive inhibitor or
mechanism based suicide inhibitor.

ALLOSTERIC ENZYME
Allosteric enzyme is a regulatory enzyme.
The term allosteric derives from Greek word,

allo means other and steros means space or site.
Allosteric enzymes are those having other site in
addition to active site for binding of modulator
(regulatory metabolites).
Allosteric enzymes may be inhibited or

stimulated by their modulators
Modulators that inhibit enzyme activity are termed
negative modulators. Whereas those that increase
enzyme activity are called positive modulators.

ISOENZYME
 Isoenzymes or isozymes are multiple forms (isomers)

of the same enzyme that catalyze the same biochemical
reaction.
 Isoenzymes show different chemical and physical

properties like electrophoretic mobility and kinetic
properties.
 Only those enzymes, which are in polymeric form

demonstrate isoenzyme.
 For example:
1. Lactate dehydrogenase (LDH)
2. Creatine kinase (CK)

Lactate Dehydrogenase (LDH)
 Lactate dehydrogenase is a tetrameric enzyme that
catalyzes the oxidation of L-lactate to pyruvate.
 LDH is made up of two types of polypeptide M (muscle)
type and H (heart) type.

 LDH has five isoenzymes:
– LDH1
– LDH2
– LDH3
– LDH4
– LDH5.
 Five isoenzymes of LDH can be detected by
electrophoresis as they have different electrophoretic
mobilities.
 LDH1 is the fastest moving fraction towards the anode
and LDH5 is the slowest moving isoenzyme of LDH.
 LDH1 predominates in cells of cardiac muscle, and

erythrocytes and LDH5 is the most abundant form in
the liver and in skeletal muscle
Clinical Applications of LDH
1. Significant elevation of LDH1 and LDH2 occurs within
24 to 48 hours after myocardial infarction.
2. Predominant elevation of LDH2 and LDH3 occur in

leukaemia.
3. LDH3 elevated in malignancy of many tissues.
4. Elevation of LDH5 occurs after damage to the liver or
skeletal muscle.
Creatine Kinase (CK)
Creatine kinase isoenzymes are dimer that are made up of
two types of polypeptide chains, which may be either M
(muscle) type or B (brain) type, generating three
isoenzymes.
1 CK1 (BB) : present in the brain
2 CK2 (MB) : present only in Cardiac tissue
3 CK3 (MM) : present in skeletal muscle

Clinical Application
1. CK1 may be elevated in neonates particularly in damaged brain
or very low birth weight new-born
2. Increased level of CK2 occurs in myocardial infarction Cardiac
tissue is the only tissue which has mixed MB (CK2) isoenzyme.
3. CK-MB isoenzyme starts to increase within 4 hours after an acute
myocardial infarction (AMI) and reaches a maximum within 24 hrs.
4. Elevated levels of CK3 in serum occur in dystrophies and
myopathies.
CLINICAL SIGNIFICANCE OF ENZYMES
Certain enzymes are used:
 For the diagnosis of the disease
As therapeutic agents
As analytical reagents.

Diagnostic Use of Enzymes
The enzymes that are found in plasma can be
categorized into two major groups:
 Plasma specific enzyme
 Plasma nonspecific enzyme.

The plasma specific enzymes are:
– The enzymes involved in blood coagulation
– Ferroxidase
– Pseudocholinesterase
– Lipoprotein lipase.
These enzymes are clinically of interest when their
concentration decreases in plasma.

 The plasma nonspecific enzymes are present in very high
concentration in tissues than in the plasma.
 Estimation of plasma nonspecific enzymes is very

important for the diagnosis of several disease.
Enzymes useful for the diagnosis of diseases
Alanine transaminase (ALT)
 Alanine transaminase was known formerly as glutamate

pyruvate transaminase (GPT).
 The plasma ALT normal value for adult is 10 to 40 U/L.
 ALT level is elevated in liver diseases (viral or toxic

hepatitis), jaundice and cirrhosis of liver.
 Aspartate transaminase (AST)
 It was known formerly as glutamate oxaloacetate

transminase (GOT).
 The plasma AST normal value for adults is 10 to 30 U/L.
 Increased AST level occurs after myocardial infarction.
 It is moderately elevated in liver disease.

The plasma AST level starts increasing after 6 to 8 hours
after the onset of chest pain with peak values 18 to 24 hours
and the values fall to normal level by the fourth or fifth day.
Alkaline phosphatase (ALP)
 ALP hydrolyzes organic phosphate at alkaline pH.
 Normal serum level for adults is 3-13 KA units/dl.
 It is elevated in certain bone and liver disease.
 Very high levels may be noticed in obstructive

jaundice, bone diseases such as Paget’s disease, rickets,
osteomalacia, carcinoma of bone and
hyperparathyroidism
Acid phosphatase (ACP)
 It hydrolyzes phosphoric acid ester at pH 5 to 6.
 Normal serum value for ACP is 0.5 to 4 KA units/dL.
 Acid phosphatase enzyme is useful for the diagnosis

and prognosis of prostate cancer. ACP is therefore an
important tumor marker.
Amylase
 It catalyzes hydrolysis of starch and glycogen.
 Normal serum value is 50-120 U/L.
 The activity of serum amylase is increased in acute

pancreatitis, chronic pancreatitis, mumps and obstruction
of pancreatic duct.
Creatine kinase (CK) : Refer isoenzyme.
Lactate dehydrogenase (LDH) : Refer isoenzyme.

Enzymes as Tumor Marker
Elevated enzyme levels may signal the presence of

malignancy.
Enzyme Assays in Myocardial Infarction/Cardiac Markers
Diagnostic enzymes include:
 Creatine kinase
 Lactate dehydrogenase
 Serum aspartate aminotransferase, also called
 serum glutamate oxaloacetate transaminase.

Nonenzyme proteins includes :
 Myoglobin (Mb)
 Cardiac troponin T and I (cTnT and cTnI).

Various enzyme assays and their time course after onset
of acute myocardial infarction.
Enzyme Assays in Liver Diseases
1. Enzymes in hepatocyte damage:
 Aspartate aminotransferase
 Alanine aminotransferase.
ALT is the more liver-specific enzyme.
2. Enzymes in cholestasis:
 Alkaline phosphatase
 5’-nucleotidase
 γ-glutamyl transferase.
Enzyme Assays in Pancreatitis
 Serum Amylase
 Urine amylase
 Lipase
Use Of Enzymes In Laboratory Investigations
(Enzyme-based Assays)
Enzymes can be used as analytical laboratory reagents

Therapeutic Use of Enzymes
Some enzymes are used in the treatment of some diseases

of human being
AMINO ACIDS PEPTIDES AND PROTEINS
General Structural characteristic of Amino Acids
 All the 20 amino acids found in proteins have a carboxyl (COOH)

group and an amino (-NH2) group bound to the same carbon atom
called the α carbon
 Amino acids differ from each other in their side chains
or R-groups, attached to the α-carbon
 The 20 amino acids of proteins are often referred to as
the standard or primary or normal amino acids.
 One of the 20 amino acids, proline is an imino (-NH)
(secondary amino group) acid not an amino (-NH2)
acid as are other 19.

 All naturally occurring amino acids are optically active except
glycine (no asymmetric carbon) which is optically inactive
 All the amino acids found in proteins are exclusively of the
L-configuration.
 Two free D-amino acids D-serine and D-aspartic acid
have been found to be present in the brain tissues. D-
Alanine and D- glutamate are found in some bacterial
cell walls.
Classification Of Amino Acids
 Chemical nature of the amino acid in the solution.
 Structure of the side chain of the amino acids.
 Nature or polarity of the side chain of the amino acids.
 Nutritional requirement of amino acids.
 Metabolic product of amino acids.

Nutritionally Essential Amino Acids
Phenylalanine Tryptophan Histidine
Valine Isoleucine Arginine
Threonine Methionine Lysine
Leucine
 The mnemonics : PVT. TIM. HALL
 Arginine and Histidine nutritionally semi essential ,

synthesized at rates inadequate to support growth of children.
Essential Amino Semi-Essential Non-essential
Acids Amino Acids Amino Acids
Phenylalanine Histidine All the other amino
Valine Arginine acids
Threonine
Tryptophan
Isoleucine
Methionine
Lysine
Leucine
Amino acids coded by stop codons
 There are actually 22 rather than 20 amino acids specified

by stop codons.
 The two extra ones are selenocysteine and Pyrrolysine

Selenocysteine : 21st Amino acid
 Selenocysteine contains selenium rather than sulfur in

cysteine
 Precursor amino acid for selenocysteine is serine. It

provides the carbon skeleton of selenocysteine.
 Biosynthesis of selenocysteine requires cysteine, selenite

(SeO4), ATP, a specific tRNASec, and several enzymes.
 It is encoded by UGA codon, which is normally a stop

codon.
 Unlike hydroxyproline and hydroxylysine, selenocysteine
is incorporation in peptides during protein synthesis rather
than created through a post translational modification.
 Selenocysteine is a constituent of several human enzymes that
catalyze redox reactions.
Glutathione peroxidase,
Thioredoxin reductase and
Deiodinase
22nd Amino acid: Pyrrolysine
 Pyrrolysine (Pyl or O) encoded by UAG (normally a stop
codon)
 It is similar to lysine, but with an added pyrroline ring linked
to the end of the lysine side chain.
 Pyrrolysine was discovered in 2002 at the active site of
methyl-transferase enzyme from a methane-producing bacteria

Properties Of Amino Acids
 Genetic code specifies an amino acid.
 Amino acid not coded by genetic code are called derived

amino acid
 All naturally occurring amino acids are optically active

except glycine (no asymmetric carbon) which is optically
inactive
 Amino acid coded by stop codon are selenocysteine and

Pyrrolysine.
 Amino acid exist in three charged state, positive
negative or neutral depending on the two factors:
–Isoelectric pH of the amino acid
–pH of the surrounding media

Nonionic and zwitterionic forms of amino acid. A zwitterion can act
as either an acid (proton donor) or base (proton acceptor).
lionization of Amino Acids
Due to ionizing property of amino acids, amino acids
exert:
 Acid base behavior.
 Amphoteric properties (zwitter ion formation)
 Buffering activity.
 Molecules which carry equal number of ionizable groups
of opposite charge and as a result bear no net charge are
called zwitterions or ampholytes.
 Zwitter is a German word which means hermaphrodite.
 The net charge of an amino acid depends upon the pH of

the medium.
 At pH less than Isoelectric pH (pI) amino acid is positively
charged because ionized COO– group accepts a proton and
becomes uncharged (COOH).
 While at pH greater than Isoelectric pH (pI) it is negatively

charged as the NH3+ group loses its proton and becomes
uncharged.
 At physiological pH (7.4) carboxyl group is negatively
charged and amino group is positively charged.

PROTEIN
Definition
 Proteins are macromolecules composed of one or more
polypeptide chains, each with a characteristic sequence of
amino acids linked by peptide bonds.
 Each protein has specific and unique sequence of amino

acids that defines both its three dimensional structure and
its biologic function.
Formation of peptide bond.
Classification of Proteins
They are most conveniently classified on the basis of
1. Function
2. Molecular shape
3. Composition
4. Nutritional quality.
Structure of Proteins
Four levels of organization Protein structure
1. Primary structure
2. Secondary structure
3. Tertiary structure and
4. Quaternary structure.
PRIMARY STRUCTURE OF PROTEINS
Sequence of amino acids linked together by covalent

peptide linkage is called primary structure of the protein
Backbone of a polypeptide chain showing N-terminal,
C-terminal and variable side (R).
SECONDARY STRUCTURE OF PROTEINS
 For stability of primary structure hydrogen bonding

between the hydrogen of NH and oxygen of C=0 group
within the polypeptide chain occur which gives rise
folding and twisting of primary structure.
 Regular folding and twisting of the polypeptide chain

brought about by hydrogen bonding is called secondary
structure of protein.
The different kinds of secondary structure
are
• α-Helix (helicoidal state)
• β-Pleated sheet (stretched state)
• β-turn and loops.

Formation of hydrogen bond in α-helix. In α-helix, the CO group of
residue 1 forms hydrogen bond with the NH group of residue 4.
Schematic diagram of α-helical structure of protein
α –HELIX
 First structure elucidated by Pauling and Corey.
 Due to intramolecular hydrogen bonding backbone of

polypeptide chain is twisted by an equal amounts around
each α-carbon & forms a coil or helix.
 C=O group of each amino acid is bonded to the -NH of

the amino acid that is situated four residues ahead in the
linear sequence
 Hydrogen bonds have an essentially optimal nitrogen to
oxygen (N-0) distance of 2.8 Å.
 A complete turn of helix contains an average of 3.6 amino

acid residues and the distance it rises per turn (its pitch) is
0.54 nm
 The axial distance between adjacent amino acids is 1.5 Å
 Proteins contains only L-amino acids, for which a right
handed α- helix is more stable and only right handed α-
helices are present in protein.
 Proline will not allow the formation of α- helix. It
disrupts the conformation of the helix, producing a bend.
 Glycine because of its small size induces bends in the
helix.
 Alanine favors α- helix.
 Examples of proteins whose major secondary structure is
α- helix.
• Hemoglobin
• Myoglobin
•
Peptide bond formation with proline
β-Pleated sheet (stretched state)
 β because it was the second structure elucidated by

Pauling & Corey.
 Polypeptide chains are fully extended . β -sheet appear

“pleated” or zigzag and these structures are therefore
often called “β -pleated sheet’.
 Unlike α-helix, β-pleated sheets are composed of two or

more polypeptide chains.
 β-pleated sheet is stabilized by interchain hydrogen bonds
 The axial distance between adjacent amino acids in β-

pleated sheet is 3.5Å in contrast with 1.5Å for the α-helix.
 The arrangement of polypeptide chains in β -pleated sheet

conformation is either Parallel or Anti-parallel pleated
sheet.
 Examples of proteins whose major secondary structure is
β -pleated sheet
–Parallel β -pleated sheet : Flavodoxin
–Anti-parallel β -pleated sheet: Silk fibrion
–Both Parallel and Anti-parallel β sheet:
Carbonic anhydrase
Antiparallel
Parallel
 β-pleated sheet occurs as a principal secondary structure in
amyloid proteins found in people with amyloidosis e.g.,
Alzheimer disease
 Amyloid proteins that accumulate are misfolded proteins
derived from immunoglobulins.
 Amyloid proteins are insoluble and their accumulation in
tissues and organs disrupt normal physiological process.

Turns and bends
 Turns and bends: short segments of amino acids that join

two units of a secondary structure
 A β turn involves four amino acid residues, in which the
first residue is hydrogen bonded to the fourth, resulting in
a tight 1800 turn.
 The peptide groups of the central two residues do not
participate in any inter-residue hydrogen bonding.
Two antiparallel beta strands connected
by a bend, i.e. β-turn.
 β-turn is a space saving method of turning a corner and
cause polypeptide chains to be compact molecule
 Glycine and proline residues often occur in b-turns, the
former because it is small and flexible; the latter because
peptide bonds involving imino nitrogen of proline
assume the cis configuration, a form that is particularly
responsible to a tight turn.
 β-turns are often found near the surface of a protein, and
they constitute readily accessible sites, or epitopes, for
recognition and binding of antibodies.
Loops
Unlike bends and turns, loops are long segments of amino
acid that joins two secondary structures.

TERTIARY STRUCTURE
 The peptide chain, with its secondary structure, may be

further folded and twisted about itself forming three
dimensional arrangement of the polypeptide chain
 The three dimensional folded compact and biologically
active conformation of a protein is referred to as its
tertiary structure, e.g. myoglobin
 It indicates, in 3-dimentional space, how secondary
features –helices, sheets, bends, turns, and loops assemble
to form domains.
Figure 5.23: Schematic tertiary structure of protein.
Figure 5.24: Tertiary structure of myoglobin.
Tertiary Structure Stabilizing Forces
 Hydrogen bonds
 Hydrophobic interactions
 Van der Waals forces
 Disulfide bond
 Ionic (electrostatic) bonds or salt bridges

Quaternary Structure Of Protein
The arrangement of polypeptide subunits in three

dimensional complexes is called the quaternary structure
of the protein
Figure 5.25: Quaternary structure of polymeric protein
hemoglobin.
Quaternary Structure Stabilizing Forces
 Hydrophobic interactions
 Hydrogen bond
 Ionic bonds.
 Vander Waal’s forces

Examples of proteins having quaternary structure
 lactate dehydrogenase
 Hemoglobin
 Creatine kinase
 Insulin has two polypeptide chains which are connected
by disulphide bond.
 It does not have quaternary structure
 Insulin is the first protein in which complete sequencing
of amino acids was done by Sanger

Bonds Responsible for Protein Structure
1. Covalent bond
 Peptide bonds
 Disulphide bond
2. Noncovalent bond
 Hydrogen bond
 Hydrophobic bond or interaction
 Electrostatic or ionic bond
 Van der Waals interactions.

Covalent Bond
 Peptide bonds (–CO-NH–) : bond formed by the

condensation of the amino group of one amino acid
with the carboxyl group of another amino acid with a
removal of a water molecule
 Disulfide bond (-S-S-) : bond formed between the

sulfhydryl group (-SH) of side chain of cysteine
residues.
Noncovalent Bonds
 Hydrogen bond : formed between -NH and -CO groups
of peptide bond by sharing single hydrogen. Side chains
of some amino acids can also form hydrogen bond.
 Hydrophobic bond or interaction : formed by

interaction between nonpolar hydrophobic R groups
(side chain) of amino acids like alanine, valine, leucine,
isoleucine, methionine, phenylalanine and tryptophan.
 Electrostatic or ionic bond or salt bond : formed between
oppositely charged groups such as amino (NH3+) terminal
and carboxyl (COO–) terminal groups of the peptide and
oppositely charged R-groups of polar amino acid residues.
 Van der Waals interactions : include both an attractive and a

repulsive forces (between both polar and nonpolar side chain
of amino acid residues).
Properties of proteins
 Colloidal nature
Colloidal protein molecules exert osmotic pressure.
Colloidal osmotic pressure or oncotic pressure
exerted by protein maintain blood volume.

 Molecular weight
Albumin = 6,9000
γ-Globulin = 1,60,000.
 Solubility
globular proteins, such as, albumin have higher

solubility than elongated fibrous proteins. Moreover,
smaller molecules are more soluble than larger
molecules.
 Shape of the protein
Scleroproteins like keratin, collagen are in the form of

fibers. While soluble proteins tend to be of rounded
shape and are called globular proteins.
 Amphoteric nature
 Isoelectric pH of the protein
 Hydration of proteins
Shell-like layer of water, called the “solvation layer”
or water envelope is held around each protein particle
in an aqueous medium.
Denaturation of protein
Disorganization of primary, secondary, tertiary and
quaternary structure by breaking of Hydrogen bond,
ionic bond and hydrophobic bondwithout breakage
of any peptide linkage.
Denaturation of proteins leads to:
- Unfolding of natural coils of native protein
- Decrease in solubility and increase in perceptibility
- Loss of biological activities
- Increased digestibility
Denaturing agents
- Physical agents
- Chemical agents
- Mechanical means.
Significance of denaturation
- Digestibility of native protein is increased
- in blood analysis to eliminate the proteins of
the blood
Coagulation
Irreversible denaturation. For example boiled egg.

Biochemistry Notes 2

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Biochemistry Notes 2

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CARBOHYDRATE METABOLISM

Digestion, Absorption and Transport of

The dietary carbohydrate consists of:

 Polysaccharides: Starch, glycogen and cellulose

 Disaccharides: Sucrose, maltose and lactose

 Monosaccharides: Mainly glucose and fructose

 Salivary glands secrete α-amylase.

 Acts briefly on dietary starch and glycogen breaking some

 α-amylase hydrolyzes starch into dextrins.

Carbohydrate digestion halts temporarily in stomach.

There are two phases of intestinal digestion:

1. Digestion due to pancreatic α-amylase

2. Digestion due to intestinal enzymes :

 Pancreatic α-amylase degrades dextrins further

Maltose, Isomaltose and α-limit dextrin.

 The α-limit dextrins are smaller oligosaccharides

containing 3 to 5 glucose units.

Two mechanisms are responsible for the absorption of

1. Active transport against a concentration gradient,

from a low glucose concentration to a higher

2. Facilitative transport, with concentration

gradient , from a higher concentration to lower conc.

• The transport of glucose and galactose occurs by an active

• Active transport requires:

- A specific transport protein

- Presence of sodium ions

• Fructose and mannose are transported by a Na+

• Movement of sugar in facilitative diffusion is strictly

from a higher concentration to a lower one until it

 Sodium independent transporter, GLUT-2 facilitates

transport of sugars out of the mucosal cells.

 Through portal circulation transported to the liver.

• Intolerance to lactose (the sugar of milk).

• Due to deficiency of enzyme lactase.

• lactose undergoes bacterial fermentation with the

- H2 and CO2 gases

- acetic acid , propionic acid and butyric acid

-- Osmotically active products that draw water

from the intestinal cells into the lumen resulting

in diarrhoea and dehydration.

 Treatment for this disorder is simply to remove

lactose from the diet.

Glycolysis is the sequence of reactions that

converts glucose into pyruvate in the presence

of oxygen (aerobic) or lactate in the absence of

oxygen (anaerobic) with the production of ATP.

Glycolysis is found in cytosol of all cells.

 I st phase: Energy requiring phase.

 II nd phase: Energy generating phase.

• The re-oxidation of NADH by conversion of

• Tissues that function under hypoxic conditions

produce lactate, e.g. skeletal muscle, smooth

muscle and erythrocytes.

Glycolysis is regulated at 3 irreversible steps.These

1. Hexokinase and glucokinase

 Glycolysis is the principal route for glucose metabolism

 Glycolysis provide ATP in the absence of oxygen and

 In erythrocytes, glycolysis supplies 2,3-BPG which is

 Pyruvate transaminated to amino acid alanine.

 Pyruvate provides substrate acetyl-CoA for

fatty acid biosynthesis.

 Glycerol-3-phosphate, required for synthesis of

triacylglycerol is derived from glycolysis.

 In mammals, glucose is the only fuel that the brain uses