Comprehensive Biochemistry for Dentistry Textbook for Dental Students

326 11 Vitamins

• Anterograde amnesia (loss of ability to recall latest past events and generate new
memories, old memories remain intact)

• Retrograde amnesia (loss of ability to recall old memories, new memories can be
generated)

• Confusion
• Change in personality

Wernicke-Korsakoff Syndrome
It is a neurological condition in which clinical manifestations are mixed.

11.5.4  Vitamin B2

H istory
• In 1926, Goldberger and his coworkers observed a dietary factor to treat

pellagra.
• In 1932, Warburg and Christian discovered a yellow pigment from tissues that

was riboflavin.
• In 1933, Kuhn isolated riboflavin in pure form.
• In 1935, Paul Karrer described chemical structure of riboflavin.

Riboflavin is a deep yellow-colored compound and signifies “ribose” and
“flavin.”

It contains “ribitol” which is a sugar alcohol obtained by reduction of ribose.
It contains flavin nucleus, and its name is derived from the Latin word “fla-
vus” which means “yellow.”

Chemical Structure
1. Forms of Vitamin B2 (Riboflavin)

Riboflavin exists in animal- and plant-based sources in protein-bound form.
Active Forms of Riboflavin

• FMN (flavin mononucleotide also called as riboflavin-5′-phosphate)
• FAD (flavin adenine dinucleotide)

Around 80–90% of riboflavin in diet exists in FMN and FAD forms.
Certain enzymes act in the presence of these coenzymes. They are

called holoenzymes (flavoproteins), for example, succinate dehydroge-
nase, monoamine oxidase, etc.
Riboflavin acts as precursor in synthesis of FMN and FAD.
2 . Structure
• Riboflavin is a deep yellow-colored water soluble organic compound.
• Chemically, riboflavin is 7,8-dimethyl-10-ribityl-isoalloxazine.
• It contains a flavin nucleus and d-ribitol.
• Flavin nucleus is 6,7-dimethyl-isoalloxazine. It is made up of three hetero-
cyclic rings linked together.

11.5  Water Soluble Vitamins 327

7α - Methyl CH2 – CHOH – CHOH – CHOH – CH2OH
Group N9 N1

H3C O
7
A C
6 BC

H3C C NH
O
N Benzene
6α - Methyl Pyrimidine Ring
Group Ring Azine
Ring

[6,7 – Dimethyl – 9 – D – Ribityl iso – Alloxane]
Riboflavin

[Vitamin B2]

Fig. 11.13  Vitamin B2

• Flavin nucleus is linked to d-ribitol (ribose alcohol). The d-ribitol has an
open chain configuration. Its first carbon is attached to ninth position on the
flavin nucleus as in Fig. 11.13.

Riboflavin is a yellowish-orange water soluble vitamin which is
thermostable.

On exposure to UV rays, riboflavin is converted into lumiflavin which gives
greenish-yellow fluorescence.

Dietary Sources of Riboflavin
1 . Animal Sources

• Good sources are eggs, liver, kidneys, and lean meat (low-fat, high-protein
contents).

• Milk and cheese.
2. Plant Sources

Good sources are whole cereals, green leafy vegetables, pulses, and nuts.
Bacteria in colon synthesize free form of riboflavin. It is absorbed through
intestinal mucosa.

328 11 Vitamins

Recommended Dietary Allowance of Riboflavin
RDA for Infants and Children

• 0.5 mg to 1 mg/day

RDA for Adults

• 1.5 mg/day

A bsorption, Transport, and Storage of Riboflavin
Site and Process of Absorption

• Dietary riboflavin is covalently bound to flavoproteins.
• In the stomach

–– Hydrochloric acid causes hydrolysis of flavoproteins. It releases FMN and
FAD.

• In the intestine
–– Flavin adenine dinucleotide is converted into flavin mononucleotide by the
action of FAD pyrophosphatase enzyme.
–– Flavin mononucleotide is converted into free riboflavin by FMN phosphatase
enzyme.

• Free riboflavin is absorbed through mucosa of small intestine by the follow-
ing processes:
–– At low intraluminal concentration, riboflavin is absorbed by “carrier-­
mediated” process which is pH-dependent and Na+ independent.
–– At high intraluminal concentration, thiamine is absorbed by passive
diffusion.

• Within enterocytes
–– Free riboflavin undergoes phosphorylation into flavin mononucleotide.
Reaction is catalyzed by flavokinase enzyme. It is ATP-dependent process.
–– FMN is transported across cells (transcellular) from mucosa to serosa.

• At serosa end of the small intestine, transcellular about 90% of FMN is dephos-
phorylated by alkaline phosphatase enzyme into riboflavin.

• Riboflavin enters hepatic portal vein and reaches the liver.
• Within the liver

–– Riboflavin is converted into FMN and FAD.
–– They are released into blood circulation.

Transport of Riboflavin

• In Plasma
–– Riboflavin is bound to plasma proteins like albumin and globulin. It is trans-
ported by blood circulation in bound form.
–– Plasma contains free riboflavin.

11.5  Water Soluble Vitamins 329

• Tissue Level

–– Free riboflavin can easily pass through cell membranes.

–– Its transportation occurs by passive diffusion at high concentration.

–– Its transportation occurs by carrier-mediated process.
–– Calcium and calmodulin (calcium-modulated protein) act as carriers of

riboflavin across cell membrane.

Absorption Inhibitors
Divalent metal ions as iron, zinc, copper, and manganese bind the riboflavin in
intestinal lumen. They inhibit mucosal absorption of riboflavin.

Alcohol inhibits mucosal absorption of riboflavin.

Storage

• Riboflavin is stored in the liver, heart, and kidneys.
• FAD is the predominant (80–90%) storage form of riboflavin.
• FMN is another storage form of riboflavin.

Excretion

• Primarily, riboflavin and its metabolites (hydroxymethylriboflavin, lumiflavin,
carboxymethylflavin) are excreted in urine.

• Small amount of riboflavin is excreted in stools (derived from unabsorbed ribo-
flavin produced by bacteria).

• Riboflavin is also excreted in milk.

Functions of Riboflavin
• Riboflavin is necessary for synthesis of coenzymes (FMN and FAD).
• FMN and FAD act as coenzymes in oxidation-reduction reactions involved in

various metabolic pathways. FMN and FAD accept two hydrogen atoms in the
reaction, and they are reduced to FMNH2 and FADH2.
• FMN acts as coenzyme in oxidation deamination of L-amino acids into ammonia
catalyzed by L-amino acid oxidase enzyme.
• FMN acts as coenzyme in electron transport chain with NADH dehydrogenase
enzyme in complex I.
• FAD acts as coenzyme in conversion of xanthine into hypoxanthine catalyzed by
xanthine oxidase enzyme in purine metabolism.
• FAD acts as coenzyme in conversion of succinic acid into fumaric acid catalyzed
by succinate dehydrogenase enzyme.
• FAD acts as coenzyme in conversion of glycine into glyoxylate catalyzed by
glycine oxidase enzyme.
• FAD acts as coenzyme in conversion of acyl CoA into alpha, beta-unsaturated
acyl CoA catalyzed by acyl CoA dehydrogenase enzyme.
• FAD acts as coenzyme in the conversion of α-ketoglutarate into succinyl CoA
catabolized by α-ketoglutarate dehydrogenase enzyme.

330 11 Vitamins

Deficiency Disorders of Riboflavin

Predisposing Factors to Riboflavin Deficiency
Riboflavin deficiency is rare in nature. The following factors predispose to its
deficiency:

Vegetarian Diet

• Deficiency of riboflavin is endemic. It is found in population who is strictly veg-
etarian and consumes little milk and milk products (vegans).

• Sportsmen who are dependent on vegetarian diet.

Poverty

• Population in slums and remote settings

Physiological Condition

• Pregnant women
• Lactating women

Chronic Alcoholics

• It is associated with poor intake of diet.

Diuretic

• Loop diuretics for prolonged period (furosemide).

Clinical Manifestations
Angular Cheilitis

• It is the inflammation of mucous membrane on the angle of the mouth.
• It is characterized by difficulty in mouth opening and cracking of corners of the

mouth.

Glossitis

• It is the inflammation of the mucosa of the tongue and papillary atrophy.
• It is manifested as redness of the tongue, burning sensation, and loss of taste.

11.5  Water Soluble Vitamins 331

Seborrheic Dermatitis

• It is the inflammation over the scalp.
• It is characterized by redness and dandruff over the scalp.
• Skin rash (redness and itch) appears on the skin of the face.

Redness and burning sensation in eyes
Normocytic and hypochromic or normochromic anemia
Anorexia (loss of appetite), nausea, and vomiting
Loss of body weight and fatigue

Peripheral Neuropathy

• It is the damage to peripheral nerves.
• It is characterized by pain, burning sensation, and tingling.

Deficiency manifestations of riboflavin are called ariboflavinosis.

Therapeutic Applications of Riboflavin

• Role in alleviating (tingling, burning sensations) in carpal tunnel syndrome
(compression on median nerve that passes through carpal tunnel in the
wrist)

• Role in alleviating symptoms in angular cheilitis and glossitis
• Role in management of seborrheic dermatitis and eczema
• Role in migraine treatment

11.5.5  Nicotinic Acid

History
• In 1867, nicotinic acid was prepared from nicotine through oxidation process.
• In 1915, Goldberger observed that pellagra could be produced in healthy persons

by feeding them on diet deficient of a particular factor, which was abundant in
milk and meat. He named the factor as pellagra-preventing factor (P-P factor).
It was nicotinic acid.
• In 1937, C. A. Elvehjem described the structure of nicotinic acid. He isolated P-P
factor from liver extract. He named it as vitamin B3 (due to its sequence in series
of vitamin B complex).

Chemical Structure
1 . Forms and Structure of Nicotinic Acid

It exists in three forms:
• Niacin (nicotinic acid)

–– Chemically, it is pyridine-3-carboxylic acid.

332 11 Vitamins

Fig. 11.14  Nicotinic acid 5 4
(Niacin)
3 C–OH
6 O

2

N

• Nicotinamide (niacinamide)
–– Chemically, it is acid amide, pyridine-3-carboxylic amide.

• Inositol hexanicotinate (hexaniacinate)
–– Chemically, it contains six molecules of nicotinic acid attached to one
molecule of inositol in the center.

Nicotinamide exists in body tissues in metabolically active forms:
• Nicotinamide Adenine Dinucleotide (NAD+)

–– It is composed of nicotinamide + adenine + d-ribose sugar + two mole-
cules of phosphoric acid.

–– Nicotinamide adenine dinucleotide is also called diphosphopyridine
nucleotide (DPN).

• Nicotinamide Adenine Dinucleotide Phosphate (NADP+)
–– It is composed of nicotinamide + adenine + d-ribose sugar + two mole-
cules of phosphoric acid.
–– It has an additional phosphoric acid residue which is attached to C2 of
d-ribose sugar.
–– Nicotinamide adenine dinucleotide phosphate. It is also called triphos-
phopyridine nucleotide (TPN) as in Fig. 11.14.

Nitrogen atom in nicotinamide contains one unit positive charge. So they are
designated with + sign as suffix.
Nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide
phosphate act as coenzymes.

D ietary Sources of Nicotinic Acid
1. Animal Sources

• Good sources are the liver, kidney, meat, and fish.
2 . Plant Sources

• Rich sources are whole cereals (bran) and yeast.
• Good sources are nuts, peas, beans, lentils, and green vegetables.
• Poor sources are milk, eggs, and fruits.

Recommended Dietary Allowance of Nicotinic Acid
RDA for Infants and Children

• It is around 5 mg/day.

11.5  Water Soluble Vitamins 333

RDA for Adults
• It is around 15–20 mg/day.

RDA in Pregnancy and Lactation

• It is around 20–25 mg/day.

Absorption, Transport, and Storage of Nicotinic Acid
Absorption

• Nicotinic acid and nicotinamide are absorbed from mucosa of small intestine.

Excretion

• Nicotinic acid and nicotinamide are excreted in urine.
• They are metabolized in the liver. Enzyme nicotinamide methyltransferase in the

liver transfers methyl group from active methionine to nicotinamide. Thus,
metabolite (N′-methyl nicotinamide) is excreted in urine.

Functions of Nicotinic Acid
Metabolically active forms, NAD+ and NADP+, act as coenzymes in metabolic

reactions in the body. They serve as coenzyme with holoenzymes in oxidation-­

reduction reactions.

Hydrogen and Electron Transfer

• NAD+ and NADP+ are involved in hydrogen and electron transfer reactions.
• Both coenzymes accept hydrogen atom from metabolite. They are reduced and

metabolite is oxidized.
• At time of reduction of NAD+ and NADP+, each coenzyme accepts one hydrogen

atom and one electron from another hydrogen atom of metabolite.
• NAD+ and NADP+ are reduced into NADH and NADPH.
• H+ from the metabolite is released in tissues.

Lactate dehydrogenase

Lactic acid Pyruvic acid

NAD+ NADH + H+

Lactate dehydrogenase Lactic acid
Pyruvic acid

NADH + H+ NAD+

334 11 Vitamins

Enzymes Dependent on NAD+

• NAD+ acts as coenzyme in the conversion of ethanol into acetaldehyde catalyzed
by alcohol dehydrogenase enzyme.

• NAD+ acts as coenzyme in conversion of malate into oxaloacetic acid catalyzed
by malate dehydrogenase enzyme.

• NAD+ acts as coenzyme in the conversion of pyruvic acid into acetyl CoA cata-
lyzed by pyruvate dehydrogenase enzyme.

Enzymes Dependent on NADP+

• NADP+ acts as coenzyme in the conversion of glucose-6-phosphate into
6-­phosphogluconolactone catalyzed by glucose-6-phosphate dehydrogenase
enzyme.

D eficiency Disorders of Nicotinic Acid
Niacin deficiency is caused by the following factors:

Deficiency of Tryptophan

• Tryptophan is necessary for biosynthesis of niacin.
• Population who has maize as staple diet suffers from niacin deficiency. The

maize protein (zein) lacks tryptophan amino acid. It leads to niacin
deficiency.
• Population who has sorghum as staple diet suffers from niacin deficiency.
Leucine amino acid in sorghum inhibits quinolinate phosphoribosyltransfer-
ase (QPRT) enzyme. It is responsible de novo synthesis of NAD+ coenzyme.

Administration of Antitubercular Drug (Isoniazid)

• Oral administration of isoniazid is responsible for diminished synthesis of pyri-
doxine. It causes tryptophan deficiency and it affects niacin level in the body.

Congenital Disorder

• Absorption of dietary tryptophan (nonpolar amino acids) is impaired in congeni-
tal disorder called Hartnup disease (autosomal recessive trait).

Its deficiency results in a disorder called pellagra (pelle means skin and agra
means rough).

Pellagra is characterized by three clinical manifestations:

Dermatitis

• It is a skin disorder.

11.5  Water Soluble Vitamins 335

• It is characterized by redness and scales on the face, feet, and ankles.
• Exposure to sunlight aggravates the condition.

Diarrhea

• It is characterized by water loose stools.
• Stools are mucus and or blood stained.
• Nausea, vomiting, and loss of weight are common manifestations with diarrhea.

Dementia

• It is characterized by loss of concentration, weak memory, and irritability.

• Behavior change.
• Delirium (disturbed state of mind with restlessness, incoherent speech, and

illusion (distorted perception) in advanced condition).

These cardinal manifestations of pellagra are called 3Ds.

Therapeutic Application

• It is helpful in the management of dyslipidemia (high serum level of LDL
and/or triglycerides).

• It is helpful in prophylaxis of atherosclerosis and coronary artery disease.
• It has a preventive role in nonalcoholic fatty liver disease in rats.
• Topical application of 1-methyl nicotinamide is helpful in the treatment of

rosacea (condition with progressive facial dermatitis).

11.5.6  Pantothenic Acid

H istory
• In 1938, R. J. Williams and R. W. Truesdail isolated pantothenic acid.
• In 1945, Lipmann discerned coenzyme A.

Pantothenic acid is derived from the Greek work “pantos” whose meaning is
“everywhere.” Pantothenic acid obtains its name owing to its abundance in cells
of plants and animals.

Pantothenic acid is also called as vitamin B5.

C hemical Structure and Forms
1 . Forms of Pantothenic Acid

Free Form
• Pantothenic acid is found in acid form.
• It is soluble in water.
• It is thermolabile.

336 11 Vitamins

Bioactive Form
• Bioactive form is called coenzyme A (co-acetylase).
• It is present in all body tissues.
• Coenzyme A is also found in bound state with proteins.
2 . Structure of Pantothenic acid
• It is composed of beta-alanine and pantoic acid (dihydroxy dimethyl butyric acid).
• Both units are linked together by peptide bond.
3. Structure of Coenzyme A
• Pantothenic acid has two linkages on either side.
• On one side, pantothenic acid is attached to adenosine-3-phosphate through a

pyrophosphate linkage.
• On other side, pantothenic acid is attached to beta-mercaptoethanolamine

(thiolethanolamine) through a peptide linkage.
• Coenzyme A is represented as CoA-SH.
• Terminal thiol group (-SH) of beta-mercaptoethanolamine is the reactive

site of coenzyme A as in Fig. 11.15.

Biosynthesis of Coenzyme A
Human, plant, and animal tissues can synthesize coenzyme A. Its synthesis follows
an elaborate biochemical process as:

• Phosphorylation
–– Pantothenic acid undergoes phosphorylation in the presence of ATP to form
4′-phsophopantothenic acid. Reaction is catalyzed by pantothenate kinase
enzyme.

• Synthesis
–– Cysteine is linked to 4′-phsophopantothenic acid in the presence of ATP and
enzyme phosphopantetheinyl synthetase to form 4′-phsophopantothenyl
cysteine.

• Decarboxylation
–– 4′-Phsophopantothenyl cysteine undergoes decarboxylation by phosphopan-
tetheinyl cysteine decarboxylase enzyme to form 4′-phsophopantetheine.

• Adenylation (AMPylation)
–– 4′-Phsophopantetheine undergoes adenylation (addition of AMP to protein
molecule) to form dephospho-CoA.

• Phosphorylation
–– Dephospho-CoA undergoes phosphorylation to form coenzyme A.

Fig. 11.15  Vitamin B3 CH3 OH

–
– ––

––
–
–
O∝CHH2––CCHβ–3–Cγ H––CO–– NH––CH2––CH2––C––OH
O

Pantoic acid β-Alanine

11.5  Water Soluble Vitamins 337

Dietary Sources
Pantothenic acid is widely found in animals, plants, bacteria, and food.

Animal Sources

• Good sources are liver, kidneys, eggs, and yeasts.
• Average sources are chicken, milk, and fishes.

Plant Sources

• Good sources are cereals, legumes, and vegetables.

Royal jelly is the richest source of pantothenic acid.
Royal jelly is also a rich source of biotin and pyridoxine.

Recommended Dietary Allowance of Pantothenic Acid
Requirement of pantothenic acid among humans is uncertain.

RDA for Infants and Children

• It is about 1–4 mg/day.

RDA for Adults

• It is about 5–10 mg/day.

E xcretion
• Pantothenic acid is excreted in urine, sweat, and milk.
• Urinary excretion of pantothenic acid is between 2 and 4 mg/day.

Functions of Pantothenic Acid
Pantothenic acid in biologically active form serves multiple roles in human metabo-
lism as:

Synthesis of Acetyl CoA

• Coenzyme A links with acetate and forms acetyl CoA. It serves the following
functions:
–– There is a high-energy sulfur bond in acetyl CoA.
–– Acetyl CoA is involved in the synthesis of oxaloacetate formation in citric
acid cycle.
–– Acetyl CoA is involved in cholesterol formation.
–– Acetyl CoA is involved in acetylcholine synthesis.
–– Acetyl CoA is involved in synthesis of ketone bodies.

338 11 Vitamins

Synthesis of Succinyl CoA

• α-Ketoglutarate undergoes oxidative decarboxylation to form succinyl CoA
(active succinate). It serves the following functions:
–– Succinyl CoA is involved in the synthesis of heme.
–– Succinyl CoA is involved in utilization of ketone bodies by extrahepatic
tissues.

Beta Oxidation of Fatty Acids
Acyl synthase (thiokinase) is necessary for beta oxidation of fatty acids.

De Novo Synthesis of Fatty Acids
Pantothenic acid is a necessary component of acyl carrier protein which is involved
in de novo synthesis of fatty acids in extramitochondrial tissues.

Synthesis of Adrenocortical Hormones
Pantothenic acid is required for the formation of acetyl CoA and cholesterol. These
compounds are necessary for the synthesis of adrenocortical hormones in adrenal
cortex.

D eficiency Disorders of Pantothenic Acid
Deficiency of pantothenic acid has not been observed among humans owing to its
abundance in plants and animals which constitute dietary sources of humans.

Deficiency Manifestations in Experimental Animals

• Dryness of the skin, thinning and loss of hair, and dermatitis
• Diarrhea, loss of appetite, and symptoms of gastritis
• Fatty liver
• Demyelination of peripheral nerves

Deficiency Manifestations in Human Volunteers
Deficiency of pantothenic acid was induced by the use of omega-methyl pantothe-
nate (antagonist) to human volunteers. The following manifestations were
observed:

• Burning feet syndrome
–– Tingling or burning sensation in the feet
–– Numbness in feet
–– Fatigue

• Loss of appetite, nausea, vomiting, and hyperacidity
• Loss of concentration, confusion, and irritability

11.5  Water Soluble Vitamins 339

11.5.7  V itamin B6

H istory
• In 1930, Rudolf Peters induced skin lesions (rat acrodynia) in rats by feeding a

diet rich in thiamine and riboflavin but deficient of other nutrients.
• In 1934, Paul Gyorgy treated skin lesions in rats through a diet enriched with a

factor which he called vitamin B6.
• In 1938, Samuel Lepkovsky extracted and crystallized vitamin B6.
• In 1938, Leslie Harris and Karl Folkers showed pyridine derivative nature of

vitamin B6

C hemical Structure
Forms and Structure of Vitamin B6
Vitamin B6 exists in three forms:

• Chemically, vitamin B6 is 2-methyl-3-hydroxy-4,5-dihydroxymethyl
pyridine.

• Pyridoxine (pyridoxol) is a stable form.
• Another two forms are:

–– An aldehyde (pyridoxal)
–– An amine (pyridoxamine) as in Fig. 11.16.
• These forms are water soluble.
• They are damaged on exposure to UV light.

Vitamin B6 exists in two biologically functional forms:

• Pyridoxal phosphate
• Pyridoxamine phosphate

They act as coenzyme in metabolic pathways.

CH2OH Alcoholic group O Aldehyde group CH2.NH2 Amine group
C–H

4 4 HO 4

HO 3 5 CH2OH HO 5 CH2OH 3 5 CH2OH

H3C 2 6 H3C 6 H3C 2 6

1 N N1
Pyridoxal Pyridoxamine
Pyridoxine (pyridoxal)
[Vitamin B6]

Fig. 11.16  Forms of Vitamin B6

340 11 Vitamins

Pyridoxal phosphate is synthesized in the brain, liver, and kidneys through phos-
phorylation as follows:

Pyridoxal kinase

Pyridoxine Pyridoxal phosphate

ATP ADP + Pi

D ietary Sources of Vitamin B6
Vitamin B6 is produced by microbes and plants. It is also synthesized in human
colon by bacteria.

Dietary sources contain pyridoxine as the main form of vitamin.

1 . Animal Sources
• Good sources are the liver, kidneys, eggs, meat, and fish.
• Poor source is milk.

2 . Plant Sources
• Rich sources are yeast and whole cereals (bran).
• Good sources are nuts, pea, beans, lentils, and vegetables.

R ecommended Dietary Allowance of Vitamin B6
RDA for Infants and Children

• It is around 0.2–0.5 mg/day.

RDA for Adults

• It is around 1.5–2 mg/day.

RDA in Pregnancy and Lactation

• It is 2.5 mg/day.

RDA of pyridoxine is dependent on dietary intake of protein per day.
Absorption, Transport, and Storage of Vitamin B6
Absorption

• Dietary pyridoxine is absorbed through intestinal mucosa.
• After absorption, it enters enterohepatic circulation and reaches the liver.
• Pyridoxine undergoes phosphorylation by pyridoxal kinase and dephosphoryla-

tion by alkaline phosphatase enzymes.

Transport

• Pyridoxal-P in blood circulation binds to albumin, and it is distributed to body
tissues.

11.5  Water Soluble Vitamins 341

Storage

• Pyridoxine is stored mainly in muscles (70%).
• It is also stored in the liver (20%).
• Small amount of pyridoxine is stored in body tissues as pyridoxal-P.

Excretion

• Pyridoxine is excreted in urine.
• Metabolite of pyridoxine and pyridoxamine is 4-pyridoxic acid. It is excreted in

urine.

Functions of Vitamin B6
It acts as coenzyme in many metabolic reactions in the body as:

Arachidonic Acid Synthesis

• Arachidonic acid is synthesized from linoleic acid through action of desaturase
enzyme. Pyridoxal-P acts as coenzyme in the reaction.

CoA-SH Synthesis
• Pyridoxal-P acts as coenzyme in synthesis of CoA-SH from pantothenic acid.

Decarboxylation

• Amino acids undergo decarboxylation catalyzed by decarboxylase enzyme in
presence of pyridoxal-P as coenzyme.

• Histidine into histamine and CO2.
• Glutamic acid into gamma-aminobutyric acid (GABA) and CO2.

Glycogenolysis

• Glycogen is converted into glucose-1-phosphate catalyzed by phosphorylase
enzyme in the presence of pyridoxal-P as coenzyme.

Heme Synthesis

• Alpha-amino beta-keto adipic acid undergoes decarboxylation to produce
D-aminolevulinic acid (ALA). It is necessary for the formation of tetrapyrrole
rings in heme synthesis. It requires ALA synthase enzyme and pyridoxal phos-
phate as coenzyme.

342 11 Vitamins

Non-oxidative Deamination (Hydroxyl Group Containing Amino Acids)

• Hydroxyl group containing amino acids like serine and threonine undergoes non-
oxidative deamination by deaminase enzyme and pyridoxal-P as coenzyme.

Non-oxidative Deamination (Sulfur Containing Amino Acid)

• Sulfur containing amino acid like cysteine undergoes non-oxidative deamination
by desulfhydrase enzyme and pyridoxal-P as coenzyme.

Niacin Synthesis

• Kynureninase enzyme converts 3-hydroxy kynurenine into 3-hydroxy anthra-
nilic acid which is necessary for niacin synthesis.

• The activity of kynureninase enzyme is dependent of pyridoxal-P as coenzyme.
• In deficiency of pyridoxal-P, the 3-hydroxy kynurenine is converted into xanth-

urenic acid, and synthesis of niacin is impaired.

Sphingomyelin Synthesis

• Pyridoxal-P is necessary as coenzyme in synthesis of sphingomyelin.

Transamination

• Pyridoxal-P is necessary as coenzyme for catalytic activity of aminotransferases
(SGPT and SGOT).

Transsulfuration

• Pyridoxal-P is necessary as coenzyme in transsulfuration metabolic pathway. It
involves transfer of sulfhydryl group from homocysteine to cysteine (reverse
transsulfuration pathway in humans) and cysteine to homocysteine (direct trans-
sulfuration pathway in bacteria).

D eficiency Disorders of Vitamin B6

Factors Predisposing to Deficiency of Vitamin B6
Administration of Antitubercular Drug (INH)

• Isonicotinic acid hydrazide (INH) is an antitubercular drug.
• INH inhibits enzyme pyridoxal kinase which is necessary for phosphorylation of

pyridoxine.
• Another view is that INH complexes with pyridoxine to form hydrazone. It is

responsible incomplete phosphorylation of pyridoxine.

11.5  Water Soluble Vitamins 343

• Administration of INH in tubercular patients produces symptoms similar to defi-
ciency of pyridoxal-P.

• Severity of manifestations is dose dependent.
• Supplementation of pyridoxine helps to overcome deficiency symptoms.

The Use of Oral Contraceptives
Prolonged use of oral contraceptives results in deficiency of pyridoxal-P.

Chronic Alcoholics

• Ethyl alcohol is converted into acetaldehyde by alcohol dehydrogenase enzyme.
Acetaldehyde inhibits coenzyme activity of pyridoxal-P.

• It results in decrease in serum pyridoxal-P level in chronic alcoholics.

Malnutrition

• Dietary deficiency of vitamin B6 is common in poor population especially slum
dwellers.

• Population who consume polished rice as staple diet suffers from deficiency of
vitamin B6.

Anemia

• Pyridoxal-P deficiency results in diminished synthesis of heme. Serum iron

level is normal in the body. However, utilization of iron in erythropoiesis is

impaired.
• Pyridoxal-P deficiency causes microcytic hypochromic anemia. It is also

called sideroblastic anemia (characterized by abnormal erythroblasts
having a ring of iron granules loaded in mitochondria encircling the
nucleus).

Peripheral Neuritis

• Pyridoxal-P deficiency causes decreased synthesis of sphingomyelin. It results in
demyelination of nerves and inflammation of peripheral nerves.

• It is characterized by neuralgia, numbness, and burning sensation in extremities.

Dermatitis

• Pyridoxal-P deficiency causes decreased synthesis of niacin.
• It results in redness and scales on the skin.

344 11 Vitamins

Convulsions in Infants

• Pyridoxal-P deficiency results in decreased synthesis of neurotransmitters like
GABA, serotonin, and adrenaline.

• The activity of pyridoxal-P-dependent glutamic acid decarboxylase enzyme is
decreased. Thus, synthesis of GABA is diminished. It leads convulsions in infants.

11.5.8  Biotin

H istory
• In 1927, Boas found that rats that were fed upon large amount of raw egg white

suffered from symptoms of dermatitis and retarded growth and termed the disor-
der as egg-white injury. Later on, it was observed that fed based on cooked eggs
did not produce similar symptoms.
• In 1934, Lease and Parsons observed egg-white injury in chickens.
• In 1936, Kogl and Jonnis extracted a compound from yolk of dried egg and
called it biotin.
• In 1942, Du Vigneaud and colleagues described the structure of biotin.
• Biotin was termed as anti-egg white vitamin.
• Biotin is also called vitamin B7 or vitamin H (H for German words “haar and
haut” representing the hair and skin).
• Biotin is a sulfur-rich vitamin B complex.
• Factor responsible for egg-white injury was discovered as avidin
(glycoprotein).
• Avidin is a basic protein which binds with biotin to render it unavailable for
mucosal absorption.

C hemical Structure
1. Forms of Biotin

Biotin exists in two forms:
• Free Form

–– Biotin is a free form.
• Bound Form

–– Biocytin is a bound form of biotin with protein in tissues.
–– Biocytin is linked to lysine moiety by amide linkage.
Depending on Source
• Alpha-Biotin
–– It is found in egg yolk.
• Beta-Biotin
–– It is found in the liver.
2. Structure of Biotin
• Chemically, it is hexahydro-2-oxo-1-thieno-3-4-imidazole-4-valeric acid.
• Biotin is a heterocyclic monocarboxylic acid containing sulfur atom.
• Biotin is composed of imidazole ring and thiophene ring linked together.
• Valeric acid side chain is attached to thiophene ring as in Fig. 11.17.

11.5  Water Soluble Vitamins 345

CO2 O NH
Binding site C 3
2
HN CH
1 3 Imidazole ring
2 CH CH2 CH2 CH2
Thiophene HC 4
ring H2C 5

C OH
O
1S

Site for binding
with lysine

Fig. 11.17 Biotin

Dietary Sources of Biotin
1 . Animal Sources

• Rich sources are egg yolk, liver, meat, kidneys, and milk.
2 . Plant Sources

• Good sources are nuts, legumes, and almonds.

R ecommended Dietary Allowance of Biotin
RDA of biotin for adults is around 100–300 μg/day.

Absorption, Transport, and Storage of Vitamin
Absorption
Transportation
Excretion

F unctions of Biotin
Biotin acts as coenzyme with carboxylase enzyme. This enzyme catalyzes reactions
involved in carbon dioxide fixation. Biotin serves role in the following metabolic
reactions:

Role in Gluconeogenesis

• Pyruvic acid is converted into oxaloacetic acid by pyruvate carboxylase enzyme
in gluconeogenesis.. Pyruvate carboxylase activity is dependent on biotin as
coenzyme.

346 11 Vitamins

Role in Fatty Acid Synthesis

• Acetyl CoA is converted into malonyl CoA in fatty acid synthesis by acetyl CoA
carboxylase enzyme. Its activity is biotin dependent.

Carboxylation of Propionyl CoA

• Propionyl CoA is a metabolite in isoleucine, leucine, and valine amino acid
catabolism.

• It is the end product in oxidation of odd-chain fatty acids.
• Propionyl CoA undergoes carboxylation into methylmalonyl CoA by propionyl

CoA enzyme which is biotin dependent.

D eficiency Disorders of Biotin
Biotin deficiency in humans is rare owing to its wide presence in plant and animals
dietary sources. Colonic bacteria also produce biotin.

However, biotin deficiency might arise due to following factors:

Prolonged Administration of Broad-Spectrum Antibiotics

• Administration of broad-spectrum antibiotics for prolonged period results in
damage of colonic bacteria.

Intake of Raw Eggs

• Intake of raw eggs (15–20 eggs per day) for prolonged period inhibits absorption
of dietary biotin. Avidin in egg white binds with biotin to form complex. Mucosal
absorption of biotin is inhibited.

Biotin Deficiency Manifestations

• Anorexia, loss of weight, nausea, and vomiting are common GIT
manifestations.

• Glossitis is the inflammation of tongue. It is associated with burning sensation
and loss of taste.

• Anemia is microcytic and hypochromic type.
• Fatigue, irritability, and depression are common CNS manifestations.

11.5.9  F olic Acid

History
• In 1931, Lucy Willis demonstrated that anemia in pregnancy could be treated

with an extract from brewer’s yeast.

11.5  Water Soluble Vitamins 347

• In 1941, H. K. Mitchell, E. E. Snell, and R. J. Williams isolated folic acid from
leaves of spinach.

• In 1943, B. Stokstad described chemical structure of folic acid.

Folate

• Folate is vitamin B9.
• It derives its name from the Latin word folium meaning leaf. It is found in

green leafy vegetables.
• Folate refers to water soluble, naturally occurring heterogeneous com-

pounds belonging to family of vitamin B complex.
• Folate is polyglutamates.

Folic Acid

• Folic acid is a synthetic form of folate. It is used for food fortification and
supplements.

• Folic acid has better stability in food and better digestibility in alimentary
canal than folate.

• Folic acid is monoglutamate.

F orms and Chemical Structure
Forms of Folic Acid
Depending on number of glutamate residues attached to pteridine-PABA, it has the
following congeners:

• Monoglutamate (one glutamic acid residue)
• Polyglutamate (three to seven glutamic acid residues)
• Tetrahydrofolate (reduced folate)

–– It is a biological active variant of folate. It acts as coenzyme.
–– Four hydrogen atoms are attached to pteridine residue at positions 5, 6, 7, and 8.
–– Folic acid is reduced to 7,8-dihydrofolate by folate reductase enzyme. It is

again reduced to tetrahydrofolate by folate reductase. NADPH acts as reduc-
ing equivalent to donate hydrogen atoms to folate. Ascorbic acid acts as
cofactor in the reduction reaction as in Fig. 11.19.
• 5-Formyl Tetrahydrofolate (f5 FH4)
–– In 1948, it was found in liver extract and observed as growth factor for
Leuconostoc citrovorum and termed as citrovorum factor.
–– It is also called folinic acid or leucovorin.
–– It is a reduced folate with a formyl group at position 5 of pteroyl complex.
–– It is biological active form of folate and is used as an adjuvant with chemo-
therapeutic drugs like 5-fluorouracil and methotrexate in treatment of cancer.
It is also used in management of megaloblastic anemia.
–– It was extracted.

348 11 Vitamins

• 10-Formyl Tetrahydrofolate (f10 FH4)
–– It is called rhizopterin or Streptococcus lactis R factor (SLR).
–– It was extracted from the liver.
–– It contains formyl group at position 10 of pteroyl complex.

Structure of Folic Acid

• Folic acid derives its name from leaves in which (green leafy vegetables) it is
found in abundance.

• Folic acid is called pteroyl glutamic acid, vitamin B9, or folate.
• It is a water soluble heterogeneous compound.
• Chemically, folate is pteroyl glutamic acid (PGA). It is comprised of three

components:
–– Pteridine nucleus (made up of pyrimidine and pyrazine rings)
–– Para-amino benzoic acid (pteridine nucleus  +  para-amino benzoic

acid = pteroyl complex).
–– Glutamic acid (it exists in one to seven residues) as in Fig. 11.18.

D ietary Sources of Folic Acid
Animal Sources

• Rich sources are the liver, kidneys, yeast, and meat.
• Good source is milk.

Plant Sources

• Sources are vegetables like spinach, cauliflower, peas, asparagus, broccoli, pea-
nuts, tomato juice, banana, papaya, and citrus fruit.

H2N N 8N COOH
HN
2 7 9 10 CH2
3 CH2
6C CH2 NH C H CH2
O N CH
5
Para-amino COOH
OH benzoic acid
Glutamic
Pteridine nucleus acid

Pteroic acid

Fig. 11.18  Structure of Folic acid

11.5  Water Soluble Vitamins 349

Recommended Dietary Allowance of Folic Acid
RDA for Infants and Children

• It is 50 μg in infants and 100–300 μg for children.

RDA for Adults

• It is 400 μg for adults.

RDA in Pregnancy and Lactation

• Its RDA is 600 μg in pregnancy and 500 μg in lactation.

Absorption, Transport, and Storage of Folic Acid
Absorption

• Folate exists in polyglutamate form in natural foods. It is stable for limited
days. Preparation of food can destroy its biological activity. However, food forti-
fication is performed by addition of folic acid whose shelf life is higher than
folate and can be active for months.

• Polyglutamate is unable to pass through intestinal mucosa. It is cleavaged by
folate conjugase enzyme into monoglutamate. Folate conjugase is found in
mucosa of duodenum and jejunum.

• Proton-coupled folate transporter is present in brush border surface of duode-
num and jejunum. It supports proton and folate ions through intestinal epithe-
lium. Its activity is pH-dependent with optimum activity and is seen at pH of 5.5.

• Within Enterocytes
–– Small amount of monoglutamates is reduced into dihydrofolate and tetrahy-
drofolate by folate reductase enzyme.
–– Tetrahydrofolate undergoes methylation to form 5-methyl THF inside
enterocytes.
–– The 5-methyltetrahydrofolate escapes through the basolateral surface of
enterocytes and enters the liver through the hepatic portal vein.

Transportation

• The liver stores about 20% of 5-methyl THF, while the remaining 80% is
released into blood circulation.

• Within blood circulation
–– 5-methyl THF is present in circulation in three states:
Unbound form (30% of folates)
Bound form with albumin (66% of folates)
Bound with high affinity binders (3% of folates)

Excretion of folic acid

• About 2 μg of folate is excreted in urine.
• About 20% of dietary folate remains unabsorbed. It is excreted in stools.
• Trace amount of folate is excreted in saliva.

350 11 Vitamins

Functions of Folic Acid
1. T etrahydrofolate serves as highly adaptable coenzyme in various metabolic reac-

tions in which it helps to transfer one-carbon unit. This is called folate-mediated
one-carbon metabolism.

Definition
One-carbon metabolism is the transfer of one-carbon unit from a donor
molecule to an acceptor molecule with the help of tetrahydrofolate as a
coenzyme.

One-carbon metabolism is responsible for the formation of different compounds
in the body.

Tetrahydrofolate is significantly involved in one-carbon metabolism.
Metabolically active tetrahydrofolate contains a reduced pteroyl complex with poly-
glutamates. The polyglutamates have three to seven glutamate residues linked
through γ-peptide bonds.

Occurrence

• One-carbon metabolism occurs in the cytoplasm and mitochondria.

Types of One-Carbon Units
One-carbon units which take part in metabolism are enlisted as follows:

Methyl unit (–CH3)
Hydroxymethyl unit (CH2OH)
Methylene unit (〓CH2)
Methenyl unit (–CH〓)
Formyl unit (–CH〓O)
Formimino unit (–CH〓NH)
One-carbon unit is linked covalently with tetrahydrofolate coenzyme either at N5
or N10, or both these positions in pteroyl complex are occupied by one-carbon units
as in Fig. 11.19.
Therefore, THF acts as an acceptor and a donor of one-carbon units in vari-
ous metabolic reactions.

Fig. 11.19  Showing inter- NH
convertibility of one-car-
N H
bon unit carriers
H2N 2 8 H
7

3 4 6 CH2 NH R
N 5H

OH N H

[5, 6, 7, 8, –Tetrahydrofolate]

11.5  Water Soluble Vitamins 351

Sources of One-Carbon Units
The following compounds provide one-carbon units. They enter one-carbon pool of
the body.

• Choline and Betaine
–– Choline is a nitrogenous compound and a nutrient. It was extracted from the
bile of pig and ox. Chemically, choline is N,N,N-trimethylammonium com-
pound. It is a structural component of phospholipids like lecithin. It is present
as phosphatidylcholine. Choline can donate three methyl units to one-­
carbon pool of the body.
–– Betaine is N-methylated amino acid. Chemically, betaine is N,N,N-­
trimethylglycine (derivative of glycine).It was discovered in beet plant (roots

have rich concentration of sucrose). Betaine is a metabolite of choline and is
synthesized in the liver through choline oxidation. It can donate methyl unit.
–– Methyl units from choline and betaine are oxidized into hydroxymethyl
units. They are further oxidized in the presence of NADP into formyl
group. It is accepted by THF to form 5,10-formyl THF.
• Histidine provides formimino group, and THF acts as acceptor to form N5-
formimino tetrahydrofolate.
• Tryptophan provides formyl group, and THF acts as acceptor to form N10-
formyl THF.
• Conversion of serine to glycine provides hydroxymethyl group which is con-
verted into methylene group, and THF acts as acceptor to form N5,N10-­
methylene THF.
• Oxidative deamination of glycine provides methylene group, and THF
accepts it to form methylene THF.

Utilization of One-Carbon Moiety
One-carbon units are used for biosynthesis of compounds in the body.
Tetrahydrofolate serves as coenzyme in these reactions.

Role of N5,N10-Methylenetetrahydrofolate
It is the most important compound, one-carbon metabolism.
Synthesis of N5-Methyltetrahydrofolate

N5,N10-methylenetetrahydrofolate undergoes reduction in the presence of NADH

to form N5-methyltetrahydrofolate. Reaction is catalyzed by methylenetetrahydro-

folate reductase.
Biosynthesis of Thymidine
N5,N10-methylenetetrahydrofolate acts as donor of one-carbon unit. It is
essential for methylation reaction in which 2-deoxy-uridine-5-monophosphate

(dUMP) is converted into 2-deoxythymidine-5-monophosphate (dTMP). Reaction
is catalyzed by thymidylate synthase enzyme. Therefore, N5,N10-­
methylenetetrahydrofolate is essential for biosynthesis of thymidine (deoxy-­
ribosyl-­thymine) or thymine-deoxy-riboside.
Biosynthesis of Glycine from Serine

352 11 Vitamins

N5,N10-methylenetetrahydrofolate has a role in synthesis of glycine from serine. Beta
carbon of serine carries hydroxymethyl group which is cleavaged by THF-­dependent
serine hydroxymethyl transferase enzyme into glycine and 5,10-methylene THF.
Oxidative Deamination of Glycine
Glycine is deaminated by multienzyme complex in the presence of pyridoxal phos-
phate. There are formation of NH3 and CO2 and release of methylene which is car-
ried by THF.

Role of 10-Formyl Tetrahydrofolate (f10 THF)
It can donate formyl group to metabolic reactions. It is synthesized from
5,10-m­ ethenyl THF through the action of methenyl THF cyclohydrolase.
Purine Biosynthesis De Novo
10-Formyl tetrahydrofolate donates formyl groups in purine biosynthesis. Formyl
groups provide C2 and C8 of purine ring.
Synthesis of Formyl-Methionyl-tRNA
10-Formyl tetrahydrofolate donates formyl group in the formylation process of methi-
onine. Formyl-methionyl-tRNA is essential for initiation of translation in prokaryotes
Conversion of Glycine into Serine
10-Formyl tetrahydrofolate donates formyl group to glycine and helps in biosynthe-
sis of serine.

Role of Methenyl THF
5,10-Methenyl THF acts as donor and acceptor of methenyl unit in metabolic reac-
tions. It is synthesized from 5,10-methylene THF via the action of methylene THF
dehydrogenase enzyme. It is also produced during breakdown of histidine (forma-
tion of 5-formimino THF which is converted into 5,10-methylene THF by
formiminotransferase-­cyclodeaminase enzyme).

Methenyl THF and 5-formyl THF are found in cells. However, they do not
act as coenzymes.

Role of 5-Methyl THF
It is synthesized from N5,N10-methylenetetrahydrofolate by reduction. THF can
also accept methyl group from serine.
Conversion of Homocysteine into Methionine
5-Methyl THF provides its methyl unit to cobalamin and forms methylcobalamin. It
in turn transfers methyl group to homocysteine, and it is converted into
methionine.

Generally, deficiency of cobalamin is followed by insufficiency of folic acid. It
is due to failure of methyl group transfer to cobalamin and regeneration of THF as
in Fig. 11.20. This phenomenon is called as folate trap.

2. Folic acid is necessary for biosynthesis of DNA (de novo synthesis of thymine
and purine).

3. Folic acid is essential for proliferation of cells.
4. Folic acid is necessary for erythropoiesis.

11.5  Water Soluble Vitamins 353
N5, N10-Methylene THF

REDUCTASE ENZYME

N5-Methyl
THF

Vit B12

TETRAHYDROFOLATEMETHYL HOMOCYSTEINEHOMOCYSTEINE METHYL TRANSFERAS
COBALAMIN

ONE-CARBON METABOLISM

METHIONINE
Fig. 11.20  Showing conversion of homocysteine into methionine

Deficiency Disorders of Folic Acid
Deficiency of folic acid is generally found in the following conditions:
Poor Dietary Intake
• Dietary deficiency of folate is very common in developing countries. It is more

prevalent in vegetarians whose diet lacks vegetable and fruits.

354 11 Vitamins

Pregnancy

• Requirement of folic acid is increased in pregnancy and lactation. Therefore,
lack of folate supplementation results in folate deficiency.

Malabsorption Syndrome

• It is an inflammatory condition of intestinal mucosa owing to multiple factors. It
causes impairment in the absorption of dietary folates.

Drug Induced

• Drugs like phenobarbitone, phenytoin sodium inhibit absorption of dietary
folates.

D eficiency Manifestations
Megaloblastic Anemia

• Folic acid is necessary for DNA synthesis. Its deficiency results in impaired
DNA replication and megaloblastic anemia.

Impaired DNA Synthesis

• Folic acid is necessary for biosynthesis of purine and pyrimidine (dTMP). Its
deficiency results in decreased synthesis of DNA.

Neural Tube Defects

• They are birth defects in the spinal cord and brain. They are attributed to defi-
ciency of folate in mother. Neural tube defect is manifested as spina bifida (defect
in the spine) and anencephaly (little brain formation).

Homocysteinemia

• Folate deficiency results in increased plasma level of homocysteine (normal
4–10 mmol/L). Homocysteinemia is implicated in inflammation of blood vessels
and coronary artery disease.

Folate Therapy

• 1 mg of folate is administered orally for management of megaloblastic anemia.
Folate supplements are co-administered with vitamin B12 supplements.

11.5  Water Soluble Vitamins 355

11.5.10  Vitamin B12

H istory
• In 1934, William Murphy and George Minot discovered the effectiveness of liver

therapy in treatment of pernicious anemia.
• In 1964, D Hodgkin proposed structure of cobalamin.
• In 1965, Robert Woodward synthesized cobalamin.

Forms and Chemical Structure of Vit. B12
Forms of Vitamin B12

• Hydroxocobalamin
Hydroxocobalamin is a type of vitamin B12 which is found in nature. It is synthe-
sized by bacteria.

• Cyanocobalamin
Cyanocobalamin is a synthetic analog of hydroxocobalamin. It contains a cya-
nide group. It is more stable than hydroxocobalamin. It is easily crystalized.

• Methylcobalamin
It is a form of cobalamin in which cyanide group is replaced with methyl group.
It is metabolically active analog of cyanocobalamin

• Cobamide
It is a highly active form of cobalamin in the body. It contains adenosyl group
linked to central cobalt atom via C → CO bond. It lacks cyanide ligand.

Cobamide exists in four forms:
1. Dimethyl-benzimidazole cobamide (DBC)
2. Benzimidazole cobamide (BC)
3. Adenyl cobamide (AC)
4. Methyl cobamide (MC)

Chemical Structure

• Vitamin B12 is water soluble essential micronutrient. It is called cobalamin.
• It is octahedral in shape. It has a stable cobalt-carbon bonded chemical structure.

• Cobalamin is made up of three components:
–– Corrin ring

–– Corrin ring has a tetrapyrrole structure. Four rings are numbered as I, II, III,

and IV. These rings are linked together by methylene bridges except ring I and

IV, which are linked directly without methylene bridge.
–– Cobalt atom

–– Cobalt is a heavy metal. It is positioned in the center of tetrapyrrole nucleus.

Cobalt atom is linked through coordinative bonding nitrogen atom in each

pyrrole ring. This is corrin ring.
–– DBI ring

356 11 Vitamins

HOH HH H
N–C–C
HH C–C–C–N

H2C H HO H

A

HO H H3C C–H H H H
N HH
N H–C–H C–C–N
H–N–C–C

H D CO N B H OH
HOH H
HH H

H–N–C–C–C C–C–C–N
HH
H–C–H N H HOH

H C

H–C–H CH O
R
H2N – C – H2C – H2C CH A,B,C,D Pyrrole I
O rings
H3C CH H
ON CH3 N
O
P Nucleus

Phosphate CH3
Group CH3 Dimethyl – Benzimidazole

OO N
OH

C C [Cynocabalamin]
H H Vitamin B12

H C
C

HO – H2C O H

Ribose

Fig. 11.21  Vitamin B12

–– The nitrogen atom of 5,6-dimethyl benzimidazole is linked to C5 of ribose
sugar, and it is termed as DBI ring.

–– DBI ring is attached to cobalt atom through its nitrogen atom and propionic
acid of IV pyrrole ring through phosphate bonding as in Fig. 11.21.

D ietary Sources of Folic Acid
Animal Sources

• Richest source is the liver.
• Good sources are meat, fish, and eggs.
• Milk and milk products contain negligible amount of cobalamin.

11.5  Water Soluble Vitamins 357

Plant Sources

• Vegetables and fruits do not contain vitamin B12.

Microflora in human colon synthesizes cobalamin. It is unavailable for
absorption and is excreted in feces.

Microflora in human small intestine also produces cobalamin. Important
organisms are Klebsiella species and Pseudomonas.

Recommended Dietary Allowance of Cobalamin
• RDA of cobalamin for adults is 2 μg/day.
• RDA of cobalamin in pregnancy and lactation is 2.5 μg/day.
• RDA of cobalamin for children is 0.5–1.2 μg/day.

A bsorption, Transport, and Storage of Folic Acid

Absorption
Release of Dietary Cobalamin in Stomach

• Dietary cobalamin is associated with protein in natural food sources. After intake
of food, hydrochloric acid in stomach helps to denature proteins and release free
cobalamin.

Formation of Haptocorrin-Vitamin B12 Complex in the Stomach

• Free cobalamin is liable to destruction by HCL.  It complexes with a protein
called haptocorrin or R-protein. It is synthesized by salivary glands in the mouth.
However, low pH of the stomach favors formation of haptocorrin-vitamin B12
complex in stomach. Haptocorrin-B12 complex passes through the pylorus and
enters the proximal part of the duodenum.

Formation of Intrinsic Factor-Vitamin B12 Complex in the Duodenum

• Duodenum contains pancreatic protease. It is secreted by the pancreas. This
enzyme partly hydrolyzes the haptocorrin-vitamin B12 complex. Vitamin B12 is
released in the duodenum. Alkaline pH in duodenum favors association of intrinsic
factor of castle with vitamin B12 to form intrinsic factor-vitamin B12 complex. This
complex moves toward distal portion of the duodenum and reaches the ileum.

Receptor-Mediated Absorption

• Brush border epithelium of ileum contains cubilin and megalin receptors. They

facilitate absorption of intrinsic factor-vitamin B12 complex through intestinal
epithelium. Vitamin B12 is internalized and IF is released at cell surface.

358 11 Vitamins

Within Enterocytes

• Vitamin B12 attaches to transcobalamin-II within enterocytes. Complex is called
holo-transcobalamin-II. It enters the hepatic portal vein and reaches the liver.

Transport

• Cobalamin is transported in circulation in methylcobalamin form. It remains
associated with transcobalamin-II.

Storage of Vitamin B12

• Human body can store nearly 3000 μg cobalamin.
• The liver can store about 60% of total cobalamin, while the remaining 30% of

cobalamin is stored in muscles.
• Maximum liver storage of cobalamin is 4 mg.

Excretion

• Cyanocobalamin is excreted up to trace amount of 0.3 μg/day.

F unctions of Vitamin B12
Conversion of Homocysteine into Methionine

• Methionine synthase enzyme catalyzes conversion of 5-methyltetrahydrofolate
into tetrahydrofolate. Enzyme requires cobalamin as cofactor for its activity.

• In the reaction, methyl unit is accepted by cobalamin to form methylcobalamin.
Methyl group is in active form and transferred to homocysteine to form methionine.

Conversion of Ribonucleotides into Deoxyribonucleotides

• Ribonucleotides are reduced into deoxyribonucleotides by ribonucleotides
reductase enzyme. It is an iron-dependent enzyme. Reaction occurs in the pres-
ence of vitamin B12.

• Ribonucleotide reductase acts on ribonucleoside 5′-diphosphates (ADP, UDP,
GDP, and CDP). Reduction occurs at 2’C of ribose sugar to form deoxyribonu-
cleoside 5′-diphosphates.

• In the reaction, NADPH provides hydrogen atoms for reductive synthesis of
deoxyribonucleotides.

Conversion of Deoxyuridine Monophosphate into Deoxythymidine
Monophosphate

• Deoxyuridine monophosphate (dUMP) is converted into deoxythymidine mono-
phosphate (dTMP) by thymidylate synthetase enzyme. The deoxythymidine
monophosphate is a monomeric structural component of DNA.

11.5  Water Soluble Vitamins 359

• Thymidylate synthetase is a cobalamin-dependent enzyme.

Thymidylate synthetase

5,10-methylenetetrahydrofolate + dUMP dihydrofolate + dTMP

• Cobalamin-dependent enzyme that catalyzed reductive methylation is an impor-
tant de novo pathway for the synthesis of deoxyribonucleotides.

Conversion of Methyl Malonyl CoA into Succinyl CoA

• Methyl malonyl CoA is a metabolite which is produced in beta oxidation of odd-­
chain fatty acids like propionic acid. Methyl malonyl CoA is converted into suc-
cinyl CoA by methyl malonyl CoA mutase enzyme in the presence of vitamin B12.

D eficiency Disorders of Vitamin B12

Megaloblastic Anemia
Definition
Megaloblastic anemia is characterized by reduced count of erythrocytes with
appearance of megaloblasts in blood circulation.
Megaloblastic anemia is a type of macrocytic anemia.

Etiology

• Deficiency of folic acid
• Deficiency of vitamin B12

Pathogenesis of Megaloblastic Anemia

• Deficiency of either folic acid or vitamin B12 or both decreases the synthesis of
DNA in proliferating proerythroblasts (megaloblasts). It is due to the following
factors:
–– Diminished de novo synthesis of purine (folate deficiency).
–– Diminished conversion of dUMP into dTMP (folate deficiency). This leads to
reduction in dTTP formation in nuclear sap.
–– Reduced conversion of ribonucleotides into deoxyribonucleotides by reduc-
tase enzyme (cobalamin deficiency).
–– Reduced conversion of 5-methyl THF into THF, called folate trap (cobalamin
deficiency).

• Decreased DNA synthesis at S-phase manifests as:
–– Delayed unwinding of DNA strands and replication fork formation.
–– dUTP is incorporated into newly synthesizing DNA strands,
–– Ligation of Okazaki segments is delayed.
–– Failure to repair DNA strands and continuous fragmentation of DNA strands.
–– Prolongation of S-phase and delayed resting phase of megaloblasts.
–– Nucleus of megaloblast appears immature with open chromatin.

360 11 Vitamins

• However, synthesis of protein and RNA is normal in megaloblasts. Maturation of
cytoplasm is normal with adequate concentration of hemoglobin.

• Nuclear-cytoplasmic asynchrony:
–– It is the disproportionate nuclear to cytoplasmic ratio in proliferation
megaloblasts.
–– Maturation of cytoplasm continues normally, while nucleus remains imma-
ture. These megaloblasts have larger cytoplasmic volume in comparison to
normal megaloblasts. They have large size than normal.
–– Megaloblasts undergo macrocytosis.

Clinical Manifestations

• Pale color of the skin, nails, and conjunctiva.
• Inflamed and smooth appearance of the tongue owing to loss of papillae (glos-

sitis) and altered taste sensation.
• Loss of appetite.
• Loss of body weight.
• Muscular weakness.
• Patient suffers from diarrhea, steatorrhea (clay-colored stool due to the presence

of fat), abdominal distension, nausea, and vomiting.
• Difficulty in breathing and dizziness (dyspnea).
• Decrease memory, concentration, and restlessness.
• Increased predisposition to infections.

Hematological Manifestations

• Decrease in RBC count below 1 million per cubic millimeter. Anisocytosis (vari-
ation in size of RBC) and poikilocytosis (variation in shape of RBC) are well
marked in peripheral blood smear.

• Decrease in Hb concentration below 12 g%.
• ↑ in diameter of RBC (8.2 μm), while normal is 7.2 μm.
• ↑ in mean corpuscular volume to 100–160 μm3, while normal is between 77 and

94 μm3.
• ↑ in mean corpuscular hemoglobin to 50 pg, while normal is 28–32 pg.
• ↑ in reticulocyte count >4% while normal count is around 1%.
• ↓ in platelet count.
• The presence of hypersegmented neutrophils (neutrophil with five or more lobes

in nucleus, while normally three to four lobes are present in a nucleus) is a char-
acteristic feature of megaloblastic anemia.
• Increased hemolysis of RBC in the liver, spleen, and bone marrow.

Biochemical Manifestations

• Serum cobalamin concentration is <300 pg/mL.
• Fecal cobalamin concentration increases.

11.5  Water Soluble Vitamins 361

• Urinary cobalamin level is decreased owing to impaired absorption of cobalamin
from small intestine.

• Serum homocysteine and serum methylmalonic acid levels are increased.
• Increased serum indirect bilirubin level.

Bone Marrow
Aspiration from the bone marrow shows the following manifestations:

• Hypoxia in the bone marrow stimulates erythropoiesis. The bone marrow shows

hyperplasia.
• Presence of 70% proerythroblasts and early normoblasts (normal is 30%).
• Presence of 30% late normoblasts (normal is 70%).

It is termed megaloblastic hyperplasia of the bone marrow.

Macrocytic Anemia
Definition
Macrocytic anemia is a type of anemia characterized by presence of increased
size of erythrocytes in blood circulation.

Normal diameter of RBC is 7.2 μ, and normal mean corpuscular volume (MCV)
is between 80 and 90 μm3. In macrocytosis, the size of erythrocytes is increased to
8.4 μ, and their MCV is increased between 90 and 160 μm3.

Etiology

• Deficiency of folate or cobalamin or both
• Chronic alcoholism
• Liver disease

Types of Macrocytic Anemia
Megaloblastic Anemia

• Megaloblastic anemia is due to deficiency of folic acid or cobalamin. This type
of macrocytic anemia is associated with impaired synthesis of DNA.

Non-Megaloblastic Macrocytic Anemia

• This type of macrocytic anemia is caused by liver disease. Cell membranes of
erythrocytes have larger surface area than their volume. Under electron micro-
scope, erythrocytes appear as thin and bell shaped. They are termed as
leptocytes.

Alcohol-Induced Macrocytic Anemia

• Prolonged alcohol consumption might have deleterious effect on the bone mar-
row. The erythrocytes have round shape and larger size than normal.

362 11 Vitamins

Pernicious Anemia (Addison Anemia)
It is characterized by reduced count of erythrocytes along with the presence of meg-
aloblasts in blood circulation and hyperplasia of the bone marrow.

The word “pernicious” means fatal/injurious/harmful.

Historical Aspect

• In 1855, a physician named Thomas Addison described pernicious anemia. He
was uncertain about the cause of anemia and called it idiopathic anemia.

• In 1872, a physician in Germany, Anton Biermer, coined the term pernicious
anemia, owing to severity of disease and lack of proper treatment.

• Studies of G. H. Whipple, G. R. Minot, and P. Murphy proved that a diet rich in
liver to patients could cure pernicious anemia. In 1934, they were awarded with
Nobel Prize.

Etiology

• Primarily, secretion of intrinsic factor of castle is decreased in the stomach.
• Secondarily, absorption of dietary vitamin B12 is impaired. It leads to its defi-

ciency in plasma.

Pathogenesis

• Autoimmunity is directed against gastric mucosa and parietal cells. There is
depletion of intrinsic factor which leads to impaired absorption and deficiency of
vitamin B12.

• Synthesis of DNA is impaired. It leads to pernicious anemia (a type of megalo-
blastic anemia).

Clinical Manifestation of Pernicious Anemia
These are the same as seen in megaloblastic anemia.

• Intrinsic factor (IF) of castle is a glycoprotein. It is secreted by parietal (oxyn-
tic) cells of gastric mucosa. It helps in the absorption of dietary vitamin B12.

• Due to atrophic gastritis (Ch. inflammation of gastric mucosa with loss of
glandular activity), parietal cells are damaged and antibodies are produced
against IF.

• It leads to either decrease or absence of IF in the stomach. It results in defi-
ciency of vitamin B12.

Folate Trap

• Deficiency of cobalamin is responsible for folate trap. Therefore, deficiency of
cobalamin is associated with folate insufficiency.

11.5  Water Soluble Vitamins 363

Increased Plasma Homocysteine Level

• Deficient cobalamin causes rise in plasma homocysteine level. It is due to
impaired methylation of homocysteine. It is excreted in urine and the condition
is called homocystinuria.

Impaired Myelin Synthesis

• Impaired methylation of homocysteine is responsible for deficiency of methio-
nine and S-adenosyl methionine (SAM). Therefore, methylation of phosphati-
dylethanolamine to form phosphatidylcholine does not occur. Choline deficiency
is manifested as impaired synthesis of myelin. Demyelination is the cause of
neurological lesions of CNS.

Achlorhydria

• Gastric atrophy results in damage of parietal cells. The secretion of HCl is either
reduced or absent. It affects digestion of food.

Congenital Pernicious Anemia
It is prevalent in neonates. Its occurrence is limited. It is an inherited trait and is
characterized deficiency of intrinsic factor in new born babies. Intrinsic factor is
helpful in absorption of dietary cobalamin.

High-Energy Facts

• Isoprene/isoprenoid is five-carbon skeleton-based unsaturated branched-­
chain hydrocarbon with molecular formula (C5H8) as CH2〓C(CH3)–
CH〓CH2. It is a colorless volatile liquid produced by conifers and citrus
plants. The liquid has strong aroma. Its polymeric compounds are constitu-
ent of natural rubber. Biologically important compounds like carotene, reti-
nol, tocopherols, lanosterol, and squalene are derived from isoprene units.

• Terpene and terpenoid are volatile unsaturated hydrocarbons found in
essential oils of conifers and citrus plants. These are derived from isoprene
units. Terpenes have multiples of isoprene units as (C5H8)n. Based on
number of isoprene units, terpenes are classified as:

• Hemiterpenes, monoterpenes, diterpenes, and tetraterpenes contain single,
two, four, and eight isoprene units.

• Physical Manifestation of Bleeding
–– Petechiae
–– Purpura
–– Ecchymosis (Bruise)
–– Hematoma

364 11 Vitamins

Suggested Readings

Epstein JB, Gorsky M (1999) Topical application of vitamin a to oral leukoplakia—a clinical case
series. Cancer 86(6):921–927

Garcia NM, Hildebolt CF, Miley D, Dixon DA, Couture RA, Anderson Spearie CL, Langenwalter
EM, Shanon WD, Deych E, Mueller C, Civitelli R (2011) One-year effects of vitamin D and
calcium supplementation on chronic periodontitis. J Periodontol 82(10):25–32

Gorsky M, Epstein JB (2002) The effect of retinoids on premalignant oral lesions—focus on topi-
cal therapy. Cancer 95(6):1258–1264

Gupta A (2017) Role of vitamin B12 and folic acid in nutritional anemia. In: Nutritional anemia in
preschool children. Springer-Nature, Singapore

Institute of Medicine, Food and Nutrition Board (2001) Dietary reference intakes for vitamin a,
vitamin k, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel,
silicon, vanadium, and zinc. National Academy Press, Washington, DC

Lodi G, Sardella A, Bez C, Demarosi F, Carrassi A (2002) Systematic review of randomized trials
for the treatment of oral leukoplakia. J Dent Educ 66(8):896–902

National Institutes of Health. Vitamin A: A fact sheet. 2017. https://ods.od.nih.gov/factsheets/
VitaminA-HealthProfessional/

National Institutes of Health-Office of Dietary Supplements. Nutrient recommendations: dietary
reference intake. U. S. Department of Health and Human Services, Bethesda. 2017. https://ods.
od.nih.gov/Health_Information/Dietary_Reference_Intakes.aspx

National Institutes of Health-Office of Dietary Supplements. Vitamin A.  U. S.  Department
of Health and Human Services, Bethesda. 2017. https://ods.od.nih.gov/factsheets/
VitaminA-HealthProfessional/

National Institutes of Health-Office of Dietary Supplements. Vitamin D.  U. S.  Department
of Health and Human Services, Bethesda. 2017. https://ods.od.nih.gov/factsheets/
VitaminD-HealthProfessional/

Piattelli A, Fioroni M, Santinelli A, Rubini C (1999) Bcl-2 expression and apoptotic bodies in
13-cis-retinoic acid (isotretinoin)-topically treated oral leukoplakia: a pilot study. Oral Oncol
35(3):314–320

Stone WL, Krishnan K, Campbell SE, Qui M, Whaley SG, Yang H (2004) Tocopherols and the
treatment of colon cancer. Ann N Y Acad Sci 1031:223–233

World Health Organization (1999) Thiamine deficiency and its preventive and control in major
emergencies. WHO, Geneva

Part III
Metabolism

Digestion and Absorption of Proteins 12

Dietary proteins are building elements of the living body. Proteins are primarily
necessary for generation and regeneration of body tissues. Proteins have higher
impact on growth than lipids and carbohydrates. 1 g of protein provides 4 calories
of energy.

A healthy adult person should consume around 0.8 g/kg body weight of protein
per day. It is around 560 g in adult male who has 70 kg of body weight. Its daily
requirement increases during pregnancy and lactation. Children in age group
between 1 and 5 years require 30–40 g of protein daily. Old-aged persons, convales-
cents, and persons suffering from chronic diseases like cirrhosis and renal failure
require higher quantity of protein. The carbohydrates are important component of
human diet.

12.1 D ietary Sources

1 . Plant Sources
• Cereals, pulses, peas, beans.
• Nuts like walnuts, almonds, cashew, and Brazil nuts are enriched with high-­
quality of proteins.
• Spirulina is the richest protein source. Its dry weight contains around
60–70% of proteins. It contains almost all essential amino acids.
• Seeds from plants like flax, pumpkin, hemp, and sesame are rich in protein,
PUFA, and minerals.

2 . Animal Sources
• Milk and milk products are good source of calcium and protein. Milk contains
whey and casein proteins.
• Egg contains 6–8 g of protein.
• Fish and liver are sources of protein.
• Pork, beef, and chicken provide high-quality amino acids.

© Springer Nature Singapore Pte Ltd. 2019 367
A. Gupta, Comprehensive Biochemistry for Dentistry,
https://doi.org/10.1007/978-981-13-1035-5_12

368 12  Digestion and Absorption of Proteins

12.2 Digestion of Proteins

12.2.1 D igestion in Oral Cavity

• Proteolytic enzymes in oral cavity are absent. Protein digestion does not start in
oral cavity. Cooking of food brings about denaturation of proteins. They are mas-
ticated in the mouth and swallowed as bolus.

12.2.2 Digestion in Stomach

• Gastric juice contains important proteolytic enzymes. They are present in inac-
tive form called as “zymogen.”

• Proteolytic enzymes in stomach are as follows:
–– Pepsinogen
–– Rennin
–– Gelatinase

• Pepsinogen is an inactive proteolytic enzyme secreted by chief cells of gastric
mucosa.

• It is converted into pepsin by HCl in the stomach. The activated pepsin can cata-
lyze conversion of pepsinogen into pepsin, called as “autocatalysis.”

HCl

Pepsinogen Pepsin
(Zymogen) (Active form)

Pepsin (Autocatalysis)

Pepsinogen Pepsin

(Optimum pH 2.0)

• Optimum pH for catalytic activity of pepsin is 2.0. It is maintained by secretion
of hydrochloric acid.

Pepsin Action on Proteins
• Pepsin is an endopeptidase. It can cleavage peptide bonds formed between car-

boxylic groups of aromatic amino acids like phenylalanine, tyrosine, and trypto-
phan with amino groups of another amino acids.
• It liberates proteoses and peptones.

Pepsin

Proteins Proteoses + Peptones

• Pepsin cannot digest keratin protein, fibroins from silk, and mucoprotein.
• Pepsin is denatured at pH >5.0.

12.2  Digestion of Proteins 369

Pepsin Action on Casein of Milk
• Casein is a phosphorus-containing protein of milk. It is present in soluble form

(caseinogen) in milk.
• Pepsin is the chief milk protein-digesting enzyme in humans. Pepsin acts on

soluble casein and converts it into soluble paracasein and whey protein.
• In the presence of calcium ions, soluble paracasein is precipitated into calcium

paracaseinate (insoluble). It is called as “milk curdling.”

Pepsin in adults or Rennin in infants

Casein Paracasein + Whey Protein (Proteose)

(Soluble) (Soluble)

Paracasein + Calcium ions Calcium Paracaseinate
(Soluble) (Insoluble coagulum or curd)

Action of Rennin on Milk
• Rennin is also named as “chymosin.” It is a proteolytic enzyme. It is present in

the stomach of infants. It is absent in adult humans. It is also found in “proren-
nin” form in the stomach of calves.
• Optimum pH for rennin activity is 2.0.
• Rennin acts on casein protein in milk and converts it into calcium paracaseinate.
Its activity is similar to pepsin.

Action of Gelatinase enzyme
• It cleavages gelatin protein in diet into polypeptides. Gelatin is used in bakery to

prepare jellies, cakes, desserts, and other products. It has gelling action. It is
digested in the stomach by gelatinase enzyme.

12.2.3 D igestion in the Small Intestine

• The duodenum receives pancreatic juice and intestinal juice. They contain many
proteolytic enzymes.
Proteolytic Enzymes in Pancreatic Juice
–– Trypsin
–– Chymotrypsin
–– Carboxypeptidase
–– Elastase
–– Collagenase

370 12  Digestion and Absorption of Proteins

Proteolytic Enzymes in Intestinal Juice
–– Enterokinase
–– Aminopeptidase
–– Tripeptidase and dipeptidase

Action of Proteolytic Enzymes
1. Trypsin

• It is the main proteolytic enzyme in pancreatic juice. It is secreted by acinar
cells of the pancreas in “trypsinogen” form.

• Trypsinogen is activated by an enzyme called as “enterokinase.” This enzyme
is secreted by intestinal glands.

• Trypsin also catalyzes conversion of trypsinogen into trypsin, and the process
is called as autocatalysis.

Enterokinase/Ca++

Trypsinogen Trypsin

Trypsin (autocatalysis)

• The optimum pH for activity of trypsin is 7.8.
• Trypsin activates chymotrypsinogen into chymotrypsin.
• Trypsin is an endopeptidase. It can hydrolyze into proteoses and peptones

into polypeptides, tripeptides, and dipeptides.
• Trypsin also has mild proteolytic activity on casein protein at pH 7.8.
• Trypsin can activate fibrinogen and proelastase into fibrin and elastase.
2 . Chymotrypsin
• It is secreted by the pancreas in chymotrypsinogen form. It is activated by

trypsin into chymotrypsin. It is a powerful protein-splitting enzyme.
• The optimum pH for activity of chymotrypsin is 7.8.
• Chymotrypsin is an endopeptidase. It can hydrolyze proteins into polypep-

tides, tripeptides, and dipeptides.
• It has mild hydrolytic action on casein protein.

Trypsin and Chymotrypsin

Native Proteins Polypeptides + Tri and Dipeptides

3 . Carboxypeptidase
• Carboxypeptidase is secreted by the pancreas in “pro-carboxypeptidase”
form. It is activated by trypsin.
• Carboxypeptidase is an exopeptidase. It hydrolyzes terminal peptide bond at
the carboxy-terminal end of polypeptides and tripeptides. It liberates amino
acids.

12.2  Digestion of Proteins 371

• Pancreatic juice contains two types of carboxypeptidases as A and
B. Carboxypeptidase A is a zinc-containing metalloprotein.

• The optimum pH for carboxypeptidases A and B is 7.8.
• Carboxypeptidase A splits peptide bond at C-terminal involving aromatic

amino acids like phenylalanine, tyrosine, and tryptophan.
• Carboxypeptidase B splits peptide bond at C-terminal involving basic amino

acids like arginine and lysine.
• Both carboxypeptidases cannot split dipeptides.

Carboxypeptidase

Polypeptides Amino acids

4 . Elastase
• Elastase is secreted by the pancreas in “proelastase” form. It is activated by
trypsin.
• The optimum pH for its activity is 7.8.
• It splits elastin protein. It hydrolyzes peptide bonds linked to carboxylic group
of neutral aliphatic amino acids like glycine, valine, and alanine.
• It liberates peptides.

5. Collagenase
• Collagenase is secreted by the pancreas.
• The optimum pH for its activity is 7.8.
• It splits collagen protein. It liberates peptides.

6. Enterokinase
• Enterokinase is also called as “enteropeptidase.” It is secreted by intestinal
glands. It is localized in brush border surface of intestinal mucosa.
• It is an endopeptidase.
• It activates trypsinogen into trypsin in the presence of calcium ions.

7 . Aminopeptidase
• Aminopeptidase is secreted by intestinal glands in the duodenum and jeju-
num. It is an exopeptidase.
• The optimum pH for its activity is 8.0.
• It cleavages terminal peptide bonds at amino-terminal end of polypeptides
and tripeptides.
• It cannot split dipeptides.
• It liberates amino acids.

8 . Tripeptidase and Dipeptidase
• These enzymes split tripeptides and dipeptides. They liberate free amino
acids in the lumen of the intestine.

Mixture of aminopeptidase, tripeptidase, and dipeptidase in intestinal juice is
called as “Erepsin.” It digests polypeptides, tripeptides, and dipeptides into
free amino acids.

372 12  Digestion and Absorption of Proteins

12.3 A bsorption of Amino Acids

Dietary proteins are digested into constituent amino acids in the lumen of the intes-
tine. Amino acids are absorbed from the mucosa of the small intestine. They enter
the hepatic portal vein and reach the liver. A small fraction of tri- and dipeptides
remain undigested in the lumen of the intestine. They can enter through brush bor-
der surface of mucosal cells of the small intestine. Within the enterocytes, they are
hydrolyzed by tri- and dipeptidases into amino acids.

12.4 M echanism of Absorption

Based on the isomeric form of amino acids, there are two mechanisms involved in
the absorption of amino acids. They are as follows:

12.4.1 P assive Diffusion of Amino Acids

• This mechanism involves diffusion of amino acids through the mucosa of the
small intestine.

• This mechanism favors concentration gradient.
• d-amino acids are absorbed by passive diffusion. It is a slow absorption

process.

12.4.2 A ctive Transport of Amino Acids

• This mechanism involves active transportation of amino acids through the
mucosa of small intestine.

• l-amino acids are absorbed by active transport.
• Active transport is dependent on energy. The ATP is utilized in the transport of

amino acids.
• The process requires a protein carrier and sodium ions.
• Carrier molecule is located on the outer surface of microvilli of the small intes-

tine. It links with l-amino acid and sodium ion and passes through the intestinal
membrane. It is a co-transport method.
• Carrier moves to inner surface of microvilli and dissociates from l-amino acid
and sodium ion.
• Sodium ion is pumped out by sodium pump.
• Amino acids pass through enterocytes and enter portal circulation.

12.5  Clinical Significance 373

12.4.3 I nfluence of Meister Cycle (Gamma-Glutamyl Cycle)
in Amino Acid Absorption

Glutathione is a tripeptide. It is a γ-glutamyl-cysteinyl-glycine molecule. According
to Meister, glutathione is actively involved in the translocation of active group of
l-amino acids except l-proline amino acid. It transports amino acids into tissues of
the kidneys, seminal vesicle, brain, and small intestine.

The process repeats in a cyclic pathway in which glutathione is regenerated. A
brief detail about the Meister cycle has been given below:

• Glutathione interacts with l-amino acid in the presence of sodium ion, and
gamma-glutamyl-amino acid complex is formed. The reaction is catalyzed by
gamma-glutamyl transferase enzyme.

Gamma-Glutamyl transferase

Glutathione + L-amino acid γ-glutamyl-amino acid complex + Cysteinyl-Glycine

• γ-Glutamyl-amino acid complex is broken into L-amino acid and 5-Oxo-Proline
compound by gamma-glutamyl cyclo-transferase enzyme. L-amino acid is
absorbed through the membrane.

Gamma-Glutamyl Cyclo-transferase

γ-Glutamyl-Amino acid complex L-amino acid + 5-Oxo-Proline

• 5-Oxo-Proline is converted into L-Glutamic acid by 5-Oxo-Prolinase enzyme. It
combines with cysteinyl-glycine to form glutathione.

5-Oxo-Prolinase

5-Oxo-Proline L-Glutamic acid

L-Glutamic acid + Cysteinyl-Glycine Glutathione

12.5 C linical Significance

1. Allergy to Dietary Proteins
• Dietary proteins may induce immunological response in selected individuals.
Generally, dietary proteins are digested into amino acids which are not
antigenic.
• For a protein to become antigenic, it must be an oligopeptide of minimum 6–7
amino acid residues. These peptides cannot be absorbed through intestinal
mucosa.

374 12  Digestion and Absorption of Proteins

• These antigenic peptides pass in between the mucosal cells (paracellular) and
enter blood circulation, for example, gluten-induced antibodies and antibod-
ies to the colostrum.

2. Protein-Losing Enteropathy
• In the condition, there is excessive loss of plasma proteins through the intes-
tinal mucosa. It is due to inflammation of gut mucosa. The plasma protein
level is decreased.

3. Oxoprolinuria
• It is a clinical condition characterized by excessive excretion of pyrogluta-
mate in urine. It is called as pyroglutamate aciduria.
• It is due to deficiency of 5-oxoprolinase enzyme.

• Whey protein is the mixture of globular proteins found in whey. These
proteins are alpha-lactalbumin, beta-lactoglobulin, and immunoglobulins.
–– Whey is the liquid that is left after the milk has been coagulated. It
contains lactose, minerals, and whey proteins. Cow’s milk contains
80% casein and 20% whey proteins, while human breast milk has 60%
whey proteins and 40% casein.

• Rennin is also called as “chymosin.” It is a proteolytic enzyme secreted by
mucosa of abomasum in ruminants. It acts on casein. Rennin is also
secreted by chief cells of stomach in infants. Its secretion is high in early
infancy, and it is replaced by pepsin at the end of infancy. In adult humans,
rennin is absent. Rennin coagulates or curdles milk.
–– Renin is a proteolytic enzyme secreted by cells of the juxtaglomeru-
lar apparatus of kidneys. Renin converts angiotensinogen into angio-
tensin I.

• Endopeptidase is an enzyme that cleavages peptide bond within protein
molecule. It cannot split terminal peptide bonds, for example, trypsin, chy-
motrypsin, elastase, and pepsin.
–– Exopeptidase is an enzyme that cleavages terminal peptide bonds
either from amino- or carboxy-terminal.

• Carboxypeptidase is an enzyme that cleavages peptide bond at carboxy-­
terminal of a polypeptide chain.

• Aminopeptidase is an enzyme that cleavages peptide bond at amino-­
terminal of a polypeptide chain.

Suggested Readings

Baron DN (1982) A short textbook of chemical pathology, 4th edn. Wiley, New York
Conn EE, Stump PK (1969) Outline of biochemistry, 2nd edn. Wiley, New Delhi
Harper HA (1979) Review of physiological chemistry, 17th edn. Lange Medical, New York
Kleiner IS, Orten JM (1966) Biochemistry, 7th edn. Mosby, St. Louis

Suggested Readings 375

Latner AL, Cantarow A, Trumper M (1975) Clinical biochemistry, 7th edn. W.  B. Saunders,
Philadelphia

Mazur A, Harrow B (1971) Textbook of biochemistry, 10th edn. W. B. Saunders, Philadelphia
Murray RK et al (1999) Harper’s biochemistry. Lange Medical, New York
Murray RK et al (2003) Harper’s illustrated biochemistry, 26th edn. Lange Medical, New York
Oser BL (ed) (1965) Hawk’s: physiological chemistry, 14th edn. Mc-Graw Hill, New York
Robins RJ, Davies DD (1981) The role of glutathione in amino-acid absorption. Lack of cor-

relation between glutathione turnover and amino-acid absorption by the yeast Candida uti-
lis. Biochem J 194(1):63–70 Available at: h­ttp://https://www.ncbi.nlm.nih.gov/pmc/articles/
PMC1162717/?page=1

13Metabolism of Proteins and Amino Acids

Proteins are integral component of human diet. They represent about 12  kg dry
weight of the body in a healthy person of 70 kg weight. Proteins are composed of
20 amino acids.

Dietary proteins are hydrolyzed by digestive enzymes in the alimentary canal.
Constituent amino acids are absorbed through intestinal mucosa. Free amino acids
are transported to the liver through the hepatic portal vein. They enter blood circula-
tion and are distributed to tissues. Tissue proteins undergo proteolysis to release free
amino acids which enter blood circulation.

Fats and carbohydrates have definitive storage in body tissues. Unfortunately, the
body lacks storage proteins to supply amino acids as is performed by tissue triglyc-
erides and glycogen in the liver and skeletal muscles.

Amino acids are either supplemented by food or provided by breakdown of tis-
sue proteins or synthesized in body tissues.

Surplus amino acids in the body are catabolized in different phases. Initially,
amino group is either transaminated or deaminated that results into formation of
alpha-keto acids and ammonia. In second phase, ammonia is transformed into urea
and excreted. Carbon skeleton of alpha-keto acid is metabolized into intermediate
metabolites. They generate calories.

13.1 A mino Acid Pool

Definition
Amino acid Pool is defined as the Labile store of free amino acids from different
sources together constitutes amino acid pool.

Cells, blood circulation, and extracellular fluid contain free amino acids which
together constitute amino acid pool.

© Springer Nature Singapore Pte Ltd. 2019 377
A. Gupta, Comprehensive Biochemistry for Dentistry,
https://doi.org/10.1007/978-981-13-1035-5_13