Water soluble vitamins

Water-soluble Vitamins
R. C. Gupta
Professor and Head
Dept. of Biochemistry
National Institute of Medical Sciences
Jaipur, India

Vitamins:
A heterogeneous group of
organic compounds
Essential for animals and
human beings
Required in very minute
quantities
EMB-RCG

Vitamins do not provide energy
But their dietary intake is essential
They perform some functions vital for
normal health, growth and reproduction
EMB-RCG

Deficiencies of vitamins produce specific
diseases
These can be cured by taking the deficient
vitamins or foods containing the vitamins
EMB-RCG

Several deficiency diseases were
discovered long before the discovery of
the vitamins
In some instances, the treatment was
discovered before the discovery of the
vitamin e.g. scurvy and beriberi
EMB-RCG

Scurvy was common in sailors going on
long voyages
It was debilitating and even fatal
EMB-RCG

James Lind cured scurvy by giving lemons
Vitamin C was discovered much later

Beriberi was common in people whose
staple diet consisted of polished rice
It was cured by giving them rice
polishings; thiamin was discovered later
EMB-RCG

The chemical natures of vitamins were not
known at the time of their discovery
Hence, they were named after the letters
of the alphabet
These names have now been largely
replaced by chemical names
EMB-RCG

Vitamins can be classified into
two groups on the basis of their
solubility:
Water-soluble vitamins
Fat-soluble vitamins
EMB-RCG

Soluble in water
Not stored in the body
Excess intake is wasteful
Excess intake doesn’t cause toxicity
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include:
Vitamin B-complex family
Vitamin C
EMB-RCG

B-complex family comprises:
Thiamin
Riboflavin
Niacin
Pantothenic acid
Pyridoxine
Biotin
Folic acid
Cobalamin
EMB-RCG

As water-soluble vitamins are not stored
in the body, they must be taken every day
Losses can occur during cooking as some
of them are heat-labile
EMB-RCG

Some water-soluble vitamins can be
synthesized by intestinal bacteria
If intestinal bacteria are destroyed, e.g. by
antibiotic therapy, extra intake is required
EMB-RCG

Thiamin (vitamin B1) is heat-stable in acidic
medium but not in basic medium
It is oxidized by mild oxidizing agents to
thiochrome which is biologically inactive
It is made up of a substituted pyrimidine
linked by a methylene bridge to substituted
thiazole
Thiamin

Thiamin forms a coenzyme, thiamin
pyrophosphate (TPP)
It is also known as thiamin diphosphate
(TDP)
Functions

Thiamin pyrophosphate
C
C
C CH
CC
CH
‒ CH2 ‒ CH2 ‒ O ‒ P ‒ O ‒ P ‒OH
S
N
N
NH2
Ι
‒ CH2 ‒ N+
ΙΙ ΙΙ
ΙΙ
O O
OHOH
Ι
CH3
H3C‒

TPP is a coenzyme for:
1. Transketolase
2. a-Keto acid dehydrogenases

Transketolase is a coenzyme in the
hexose monophosphate shunt
a-Keto acid dehydrogenases include:
• Pyruvate dehydrogenase
• a-Ketoglutarate dehydrogenase
• Branched-chain a-keto acid
dehydrogenase

Activity of a-keto acid dehydrogenases
is impaired in thiamin deficiency
This can limit the availability of energy

Sources of
thiamin
Yeast
Kidney
FishMeat
Pulses
Liver
Cereals Nuts

In cereals, thiamin is present mainly in the
outer layer of the grain
Removal of the outer layer, e.g. by milling,
causes considerable loss of thiamin

The recommended daily allowance (RDA)
for thiamin is 0.5 mg/1,000 kcal of energy
or 1-1.5 mg/day in adults
The requirement increases in alcoholics
and in hyper-metabolic states e.g. fever,
pregnancy, hyperthyroidism etc
Requirement

People consuming polished rice or refined
wheat flour are prone to thiamin deficiency
Outer layer of the grain is removed while
refining wheat flour or polishing rice
Deficiency

Refined white flour has much less
thiamin than whole wheat flour
Refined white flour Whole wheat flour

Parboiling of rice decreases the loss of
thiamin
Polished rice Parboiled rice

During parboiling, paddy is soaked in warm
water for a few hours and is, then, steam-
dried
Thiamin percolates into the deeper part of
the grain
Polishing of parboiled rice leads to a limited
loss of thiamin

Alcoholics are prone to thiamin deficiency
as alcohol impairs:
• Absorption of thiamin
• Conversion of thiamin into TPP

Deficiency of thiamin
causes beriberi which
affects:
Central nervous system
Cardiovascular system
Gastrointestinal tract
EMB-RCG

Thiamin deficiency causes peripheral
neuritis involving sensory and motor nerves
Sensory involvement leads to hyper-
aesthesia, numbness, tingling and pain
Motor involvement leads to muscular
weakness, sluggish reflexes, ataxia and
paralysis
Central nervous system

The heart muscle becomes weak resulting
in congestive heart failure
This, in turn, causes oedema and ascites
Cardiovascular system
Oedema Ascites

Involvement of gastrointestinal tract
causes:
• Anorexia
• Dyspepsia
• Constipation
Gastrointestinal tract

Oedema is a common feature if cardio-
vascular system is involved
Hence, beriberi mainly involving cardio-
vascular system is known as wet beriberi

Involvement of central nervous system
doesn’t cause oedema
Hence, beriberi mainly involving central
nervous system is known as dry beriberi
Mixed beriberi is more common in which
different systems are involved in varying
degrees

Infantile beriberi can occur when mother is
deficient in thiamin
It usually occurs between two and six
months of age
It is mainly the wet form of beriberi with
pronounced oedema

Vomiting, diarrhoea, hoarseness and
weight loss are common in infantile beriberi
The disease responds promptly to thiamin
administration

Laboratory findings in beriberi are:
• Increased pyruvic acid level in blood
• Decreased thiamin concentration in RBCs
• Decreased transketolase activity in RBCs

Riboflavin was known in the past as
vitamin B2
It is heat-stable in neutral and acidic
medium but not in basic medium
Its aqueous solution is unstable in sunlight
and ultraviolet light
Riboflavin

Chemically, riboflavin is 6,7-dimethyl-9-D-
ribityl isoalloxazine
Riboflavin can be readily reduced to
leucoriboflavin

Riboflavin (6,7-dimethyl-9-D-ribityl isoalloxazine)
H C—3
||
1
2
45
6
7
8 9
10
CH —C—C—C—CH OH2 2
H
|
OH
|
OH
|
OH
|
H
|
H
|
3
NN
N
H C—3 O
NH
O
NH
N

Functions
Riboflavin functions in the
form of two coenzymes:
Flavin
mononucleotide
(FMN)
Flavin adenine
dinucleotide
(FAD)

FMN and FAD can undergo reversible
oxidation and reduction
They participate in a number of oxidation-
reduction reactions as coenzymes
Riboflavin portion of FMN and FAD can
reversibly combine with two hydrogen
atoms

H C—3
||
CH — C — C — C — CH — O — P — OH2 2
H
|
OH
|
OH
|
OH
|
H
|
H
|
NN
N
H C—3 O
NH
O
||
|
OH
H C —3
||
H
NN
N
H
H C —3 O
NH
FMNH2
FMN
AH2
A
CH — C — C — C — CH — O — P — OH2 2
H
|
OH
|
OH
|
OH
|
H
|
H
|
O
||
|
OH
O
O
FMN
FMNH2
H
H

FMN is a:
Constituent of respiratory
chain
Constituent of microsomal
hydroxylase system
Coenzyme for L-amino
acid oxidase
EMB-RCG

HC—3
||
NN
N
HC—3 O
NH
FAD
HC—3
||
N
HC—3 O
NH
H
N
N
H
AH 2
A
N
N
N
N
NH 2
NH 2
H
|
OH
|
OH
|
OH
|
OH
|
H
|
H
|
O
||
CH— C — C — C — CH— O — P — O — P — O — CH222
OH
|
O
||
|
|
N
N
OH
OH
H
H
OH
OH
H
H
H
H
H
H
O
O
N
N
FADH 2
H
|
OH
|
OH
|
OH
|
OH
|
H
|
H
|
O
||
CH— C — C — C — CH— O — P — O — P — O — CH222
OH
|
O
||
O
O
FAD
FADH2
AH2
A
H
H

FAD is a:
Constituent of respiratory
chain
Constituent of microsomal
hydroxylase system
Coenzyme for many
enzymes
EMB-RCG

Some enzymes requiring FAD as a
coenzyme are:
• D-Amino acid oxidase
• Acyl CoA dehydrogenase
• Succinate dehydrogenase
• Glycerol-3-phosphate dehydrogenase
• Xanthine oxidase
• Sphingosine reductase

Dietary
sources of
riboflavin
Milk Dairy products Eggs
Meat Nuts Leafy vegetables
Kidney Liver

The recommended daily allowance (RDA)
for riboflavin is 0.6 mg/1,000 kcal
Or it is 2 mg/day for adults
Requirement

An isolated deficiency of riboflavin is rare
It is generally combined with other
deficiencies
Deficiency

Angular stomatitis
(fissures at the angles of mouth)
Cheilosis (cracked and swollen lips)
Glossitis
(swollen, painful, magenta-coloured tongue)
Seborrheic dermatitis (rough and scaly skin)
Corneal vascularisation
(growth of blood vessels into the cornea)
Clinical features of deficiency are:

Angular stomatitis and glossitis

Laboratory diagnosis of riboflavin deficiency
is difficult
Serum and urinary riboflavin are low in
severe deficiency
Erythrocyte riboflavin is decreased
The urinary excretion of riboflavin after a
test dose is decreased

Niacin was known in the past as anti-
pellagra factor, pellagra-preventing factor
and vitamin B3
It occurs in two forms, niacin (nicotinic
acid) and niacinamide (nicotinamide)
Niacin

Niacin and niacinamide are equally active
Niacin is converted into niacinamide in the
body

Niacin
(nicotinic acid)
Niacinamide
(nicotinamide)
N N
–COOH –CONH2

Functions
Niacin performs its functions in the
form of two coenzymes:
Nicotinamide
adenine dinucleotide
(NAD)
Nicotinamide
adenine dinucleotide
phosphate (NADP)

Nicotinamide combines with ribose and
phosphoric acid to form a nucleotide
This combines with an adenine nucleotide
to form a dinucleotide

— CONH2
CH — O — P — O — P — O — CH2 2
NN
+
N
NH2
|
N
OH*OH
HH
OHOH
HH
H H HH
OO
N
OH
|
OH
|
O O
|| ||
NAD (in NADP, —OH* is esterified with phosphoric acid)
Nicotinamide adenine dinucleotide (NAD)

— CONH2
CH — O — P — O — P — O — CH2 2
NN
+
N
NH2
|
N
OH*OH
HH
OHOH
HH
H H HH
OO
N
OH
|
OH
|
O O
|| ||
NAD (in NADP, —OH* is esterified with phosphoric acid)
OH
Ι
P
Ι
=‒ OHO
Nicotinamide adenine dinucleotide phosphate
(NADP)

NAD and NADP can undergo reversible
They can act as coenzymes for several
oxido-reductases
CH CHC — CONH2 C — CONH2
N
|
R
+
N
|
R
CH CHCH CH
CH CH2
NAD (or NADP )
+ +
NADH (or NADPH)
AH2 A
+ H
+
CH2CH
N+

NAD and NADP act as coenzymes in
many metabolic pathways such as:
• Glycolysis
• Hexose monophosphate shunt
• Citric acid cycle
• Synthesis of fatty acids and steroids
• Oxidation of fatty acids
• Oxidative deamination of amino acids

NAD generally acts as coenzyme in
catabolic pathways
NADP generally acts as coenzyme in
anabolic pathways

Some enzymes which require NAD as a
coenzyme are:
• Lactate dehydrogenase
• Isocitrate dehydrogenase
• Malate dehydrogenase
• b-Hydroxyacyl CoA dehydrogenase
• Glutamate dehydrogenase
• IMP dehydrogenase

NAD is also a
constituent of:
Respiratory
chain
Microsomal
hydroxylase
system

Examples of enzymes requiring NADP
as a coenzyme are:
• Glucose-6-phosphate dehydrogenase
• 6-Phosphogluconate dehydrogenase
• b-Ketoacyl CoA reductase
• a,b-Unsaturated acyl CoA reductase
• Squalene synthetase
• Cholesterol 7-a-hydroxylase
• Thioredoxin reductase
• Haem oxygenase

Sources of niacin
Eggs
Fish Tomatoes
Green leafy
vegetables
Milk Meat

Niacin is also synthesized in human beings
from tryptophan
It has been shown that 1 mg of niacin is
synthesized from 60 mg of tryptophan

Pyridoxal phosphate is required as a
coenzyme for synthesis of niacin
Excess of leucine inhibits the conversion
of tryptophan into niacin

The daily requirement for niacin is 6.6
mg/1,000 kcal
Or the adult requirement can be taken as
20 mg/day
Requirement

Deficiency of niacin causes pellagra
Clinical features are stomatitis, glossitis,
diarrhoea, dermatitis and dementia
If untreated, the disease can be fatal
Deficiency

Dermatitis usually affects the exposed
parts of the body

Pellagra is common in people consuming
maize and sorghum as their staple food
These two are poor in niacin and
tryptophan, and rich in leucine

Pantothenic acid was known in the past as
vitamin B5
It is heat-stable in neutral medium but not
in acidic or basic medium
It is not destroyed by oxidizing or reducing
agents
Pantothenic acid

Pantoic acid
Pantothenic acid
b-Alanine
Pantothenic acid is made up of
pantoic acid and b-alanine

Functions
• Coenzyme A
(CoA)
• Acyl carrier
protein (ACP)
Pantothenic acid
performs its
functions as a
constituent of:
Both these contain pantothenic acid in the
form of 4’-phosphopantetheine

Pantothenic acid is first phosphorylated at
C4 of the pantoic acid residue
The product is 4’-phosphopantothenic acid
This combines with cysteine to form 4’-
phosphopantothenyl cysteine

CH — C — CH — C — N — CH — CH —2 2 2 C — N — CH — CH — SH2
CH3
|
H
|
H
|
COOH
|
O
||
O
||
CH3
| ||
OHO
|
O = P — OH
4´-Phosphopantothenic acid Cysteine
4´-Phosphopantothenyl cysteine
|
OH

Decarboxylation of the cysteine residue
converts 4’-phosphopantothenyl cysteine
into 4’-phosphopantetheine
4’-Phosphopantetheine is linked with AMP
to form dephosphocoenzyme A
Ribose moiety of dephosphocoenzyme A is
phosphorylated at C3 to form coenzyme A

N
N
NH2
|
N
OH
H
O
H
H
N
O = P — OH
H
|
|
CH2
|
O = P — OH
CH C CH C N CH CH2 2 2— — — — — — — C — N — CH — CH — SH2 2
CH3
|
H
|
H
|
O
||
O
||
CH3
| |
OH
|
O
|
|
O
O
|
O = P — OH
|
O
Coenzyme A
N
NN
N
O

In ACP, 4’-phosphopantetheine is esterified
with a serine residue of the protein
The –SH group of 4’-phosphopantetheine
remains free

Coenzyme A is also represented as CoA-
SH as its terminal –SH group binds various
compounds
Coenzyme A participates in a variety of
reactions in the metabolism of carbo-
hydrates, lipids and amino acids
Role of Coenzyme A

Examples of reactions requiring
coenzyme A are:
• Oxidative decarboxylation of a-keto
acids
• Activation of fatty acids
• Activation of some amino acids

A number of coenzymes are required in
this multi-step reaction
Coenzyme A is one of them
Oxidative decarboxylation of
a-keto acids

R ‒ C ‒ COOH + CoA‒SH + NAD+
O
‖
R ‒ C ~ S‒CoA + NADH + H+ + CO2
O
‖
a-Keto acid
Acyl CoA
Oxidative decarboxylation

Pyruvate is converted into acetyl CoA by
oxidative decarboxylation
a-Ketoglutarate is converted into succinyl
CoA by oxidative decarboxylation

Before fatty acids can take part in any
reaction, they have to be converted into
their CoA derivatives
This reaction, known as activation of fatty
acids, is catalysed by acyl CoA synthetase
(thiokinase)
Activation of fatty acids

R ‒ CH2 ‒ COOH + CoA‒SH + ATP
R ‒ CH2 ‒ C ~ S‒CoA + AMP + PPi
O
‖
Fatty acid
Acyl CoA
Activation of fatty acid

Some amino acids are converted into their
CoA derivatives before they can be
metabolized
Examples are leucine, isoleucine and
valine
Activation of amino acids

An important role of CoA is to provide
active acetate (acetyl CoA)
Active acetate is required for synthesis of
fatty acids, cholesterol, ketone bodies,
acetylcholine etc

Coenzyme A also forms active succinate
(succinyl CoA)
Active succinate is required for haem
synthesis and for gluconeogenesis from
some amino acids

Pantothenic acid is a constituent of acyl
carrier protein (ACP) also
ACP is a part of the multienzyme complex
which catalyses de novo synthesis of fatty
acids
Role of acyl carrier protein

Pantothenic acid is widely distributed in
animal and plant foods
It is also synthesized by intestinal bacteria
Sources

Dietary sources of
pantothenic acid
LiverKidney Meat
Eggs
Wheat Peas
Sweet
potatoes
Yeast

The recommended daily intake is 10 mg
A smaller intake may be sufficient for
infants and children
Requirement

Deficiency of pantothenic acid has not
been reported in human beings
In animals, deficiency causes loss of
weight, loss of hair, greying of hair,
anaemia and necrosis of adrenal glands
Deficiency

Human deficiency can be produced
experimentally
It leads to neurological and gastrointestinal
disturbances

Pyridoxine was known in the past as
vitamin B6
It consists of three closely related pyridine
derivatives
These are pyridoxine, pyridoxal and
pyridoxamine
All the three are equally active as vitamins
Pyridoxine

CH OH
|
2 CHO
|
CH NH2 2
|
—CH OH2 —CH OH2 —CH OH2HO— HO— HO—
H C—3 H C—3 H C—3
N N N
Pyridoxine Pyridoxal Pyridoxamine
N N N

Pyridoxine, pyridoxal and pyridoxamine
are converted into coenzymes
The conzymes are:
• Pyridoxine phosphate
• Pyridoxal phosphate
• Pyridoxamine phosphate
Functions

Pyridoxine, pyridoxal and pyridoxamine are
phosphorylated by a common enzyme
The three coenzymes are interconvertible
The phosphate group is provided by ATP
The enzyme is pyridoxal kinase

N
HO‒
H3C‒
‒CH2OH
CH2OH
Ι
CH2OH
Ι
HO‒
H3C‒
‒CH2‒O‒P‒OH
+ ATP + ADP
‖
O
OH
Pyridoxine Pyridoxine phosphate
Pyridoxal
kinase
N
I

N
HO‒
H3C‒
‒CH2OH
CHO
I
CHO
I
HO‒
H3C‒
‒CH2‒O‒P‒OH
+ ATP + ADP
‖
O
OH
Pyridoxal Pyridoxal phosphate
Pyridoxal
kinase
N
I

N
HO‒
H3C‒
‒CH2OH
CH2NH2
I
CH2NH2
I
HO‒
H3C‒
‒CH2‒O‒P‒OH
+ ATP + ADP
‖
O
OH
Pyridoxamine Pyridoxamine phosphate
Pyridoxal
kinase
N
I

Vitamin B6 coenzymes are required mainly
in the metabolism of amino acids
Pyridoxal phosphate (PLP) can form a
Schiff base with an amino acid

Schiff base
The amino acid, thus bound, can undergo
various reactions
Ι
‖
Ι
H3C‒
HO‒ ‒CH2‒O‒ P
C‒H
R‒CH‒COOH
N
N

The amino acid bound to pyridoxal
phosphate can undergo:
• Transamination
• Deamination
• Decarboxylation
• Transulphuration
• Desulphydration

Pyridoxal phosphate is also required
in:
• Metabolism of tryptophan
• Synthesis of haem
• Cellular uptake of amino acids
• Formation of g-amino butyric acid
• Glycogenolysis

These reactions are catalysed by specific
transaminases
The amino group of an amino acid is
transferred to an a-keto acid
This results in the formation of a new
amino acid and a new a-keto acid
PLP acts as a carrier of the amino group
Transamination

Transamination reactions are important
in:
• Formation of new amino acids
• Catabolism of amino acids

 Subjects deficient in thiamin retain most of the
test dose in tissues and excrete less in urine
 Measurement of transketolase activity in
erythrocytes can confirm the diagnosis
NH2 NH2
| |
R — CH — COOH1
R — C — COOH1
R — CH — COOH2
CHO
|
|
HO—
HO—
H C—3
H C—3
N
N
—CH O — P2—
—CH O — P2—
CH NH2 2
Amino acid
Pyridoxal phosphate
Pyridoxamine phosphate
Amino acid
a-Keto acid
O
||
R — C — COOH2
a-Keto acid
O
||
N
N

Deamination
PLP acts as a coenzyme for:
• Serine deaminase
• Threonine deaminase

Decarboxylation
PLP is a coenzyme for
decarboxylases acting on:
• Glutamate
• Arginine
• Tyrosine

PLP is a coenzyme for cystathionine
synthetase and cystathionine g-lyase
These two transfer sulphur from
homocysteine to serine forming cysteine
Transulphuration

PLP is a coenzyme for cysteine
desulphydrase
This enzyme removes the sulphydryl group
from cysteine
Desulphydration

One of the intermediates in the catabolism
of tryptophan is 3-hydroxykynurenine
This is converted into 3-hydroxyanthranilic
acid by kynureninase, a PLP-dependent
enzyme
Tryptophan metabolism

When PLP is not available, 3-hydroxy-
anthranilic acid is not formed
3-Hydroxykynurenine is spontaneously
converted into xanthurenic acid
Xanthurenic acid, which is an alternate
metabolite, is excreted in urine

Tryptophan 3-Hydroxy-
kynurenine
Kynureninase
3-Hydroxy-
anthranilic acid
Excreted in
urineAcetoacetyl CoA
Xanthurenic
acid
Sponta-
neous
Urinary xanthurenic acid excretion is an
indicator of pyridoxine deficiency
PLP
In the presence of PLP In absence of PLP

One of the enzymes involved in the
synthesis of haem is d-aminolevulinic acid
synthetase
This enzyme requires PLP as a coenzyme
Synthesis of haem

Cellular uptake of L-amino acids is an
active process
This requires the participation of pyridoxal
phosphate
Cellular uptake of amino acids

Gamma-amino butyric acid (GABA) acts as
a neurotransmitter in brain
It is formed by the action of glutamate
decarboxylase on glutamate
PLP is required as a coenzyme in this
reaction
Formation of g-amino butyric acid

Phosphorylase is a key enzyme of glyco-
genolysis
Phosphorylase requires PLP as a
coenzyme
Glycogenolysis

Sources of pyridoxine include animal as
well as plant foods
Another source is bacterial synthesis in
the intestine
Sources

Dietary
sources of
pyridoxine
Leafy
vegetables
Meat
Eggs
Milk
Wheat
Corn Beans
Potato
Bananas

Pyridoxine is mainly required in the
metabolism of amino acids
Its requirement depends upon the protein
intake
An intake of 1.25 mg/100 gm of proteins
has been recommended
Requirement

Deficiency is very rare
It may sometimes occur in infants and
pregnant women
Deficiency may also occur in patients
taking isoniazid, an anti-tuberculosis drug
Isoniazid forms a complex with pyridoxal
and prevents its activation
Deficiency

Clinical features of pyridoxine deficiency
are:
• Nausea
• Vomiting
• Dermatitis
• Microcytic anaemia
• Convulsions
Convulsions are more common in children
while anaemia is more common in adults

Chronic pyridoxine deficiency may cause
hyperhomocysteinaemia
Hyperhomocysteinaemia increases the
risk of cardio-vascular diseases

Urinary excretion of xanthurenic acid is
increased in pyridoxine deficiency
Measuring xanthurenic acid excretion
Giving a test dose of tryptophan
Pyridoxine deficiency can be diagnosed by:

Biotin is also known as anti-egg white
injury factor
When raw egg white is fed to rats, they
develop some symptoms which are
relieved by biotin
Biotin

It has been shown that raw egg white
contains a protein, avidin
Avidin forms a complex with biotin
preventing its intestinal absorption
This leads to a deficiency of biotin

Avidin is inactivated by heat
Therefore, cooked eggs do not hamper
absorption of biotin
Biotin is heat-stable

Biotin is a heterocyclic sulphur-containing
monocarboxylic acid
HN
|
HC
|
H2C
NH
|
CH
|
CH — (CH2)4— COOH
O
||
C
S

Biotin is a coenzyme for carboxylases
It is also known as co-carboxylase
Biotin is firmly bound to the enzyme
‒COOH group of biotin is bonded with
e-NH2 group of a lysine residue of enzyme
Functions

Some carboxylation reactions
requiring biotin are:
Carboxylation of
pyruvate
Carboxylation of
acetyl CoA
Carboxylation of
propionyl CoA
EMB-RCG

This reaction converts pyruvate into oxalo-
acetate
Oxaloacetate is an intermediate in citric
acid cycle
This reaction is important for the normal
operation of citric acid cycle
Carboxylation of pyruvate

CH3
|
C = O
|
COOH
+ CO2 + ATP
Pyruvate
COOH
|
CH2
|
C = O
|
COOH
+ ADP + Pi
Oxaloacetate
Pyruvate
carboxylaseBiotin

Carboxylation converts acetyl CoA into
malonyl CoA
This reaction is important in fatty acid
synthesis
Carboxylation of acetyl CoA

Carboxylation of acetyl CoA
CH3— C ~ S — CoA + CO2 + ATP
CH2— C ~ S — CoA + ADP + Pi
Acetyl CoA
Malonyl CoA
Acetyl CoA
carboxylase
Biotin
O
||
O
||
COOH
|

Propionyl CoA is carboxylated to D-methyl-
malonyl CoA
This is one of the reactions in the gluco-
neogenic pathway for conversion of
propionate into glucose
Carboxylation of propionyl CoA

CH3
CH3
|
|
CH2
H — C — COOH
|
|
O = C ~ S — CoA + CO2 + ATP
O = C ~ S — CoA + ADP + Pi
Propionyl CoA
D-Methylmalonyl CoA
Propionyl CoA carboxylaseBiotin

Bacterial synthesis in the intestine provides
sufficient amounts of biotin
Dietary sources include animal foods as
well as plant foods
Sources

CauliflowerAvocado
Berries
Eggs Meat
Nuts Sources
of biotin

Biotin requirement is not known with
certainty as the intestinal bacteria meet
most of the requirement
The daily intake has been estimated to be
100 to 300 mg
Requirement

Deficiency of biotin is unknown in human
beings
Deficiency may occur in animals when
they are fed raw egg white
Deficiency

Clinical features of biotin deficiency in
animals are:
• Retarded growth
• Loss of weight
• Dermatitis
• Loss of hair
• Muscular inco-ordination
• Paralysis

Folic acid is also known as folacin or
pteroylglutamic acid
It is made up of pteridine, para-amino-
benzoic acid and glutamic acid
Folic acid

H N2 N
N
|
OH
1
2
3
4
N
5
6
7
8
N
9 10
CH — N —2 — C — N — CH
| |
H COOH
COOH
|
CH2
|
CH2
|
O
||
H
|
Pteridine para-Amino-
benzoic acid
Glutamic
acid
Pteroylgutamic acid (folic acid)

Folic acid is found in food as pteroyl
monoglutamate, pteroyl triglutamate and
pteroyl heptaglutamate
The last two are converted into pteroyl
monoglutamate in the intestinal mucosa
Folic acid is heat-stable in neutral medium

Folic acid functions as a coenzyme,
tetrahydrofolate (H4-folate or FH4)
Folate is first reduced to 7,8-dihydrofolate
(H2-folate or FH2)
7,8-Dihydrofolate is, then, reduced to
5,6,7,8-tetrahydrofolate
Functions

Dihydrofolate
Tetrahydrofolate
Folate
Dihydrofolate reductase
Dihydrofolate reductase
NADPH + H+
NADPH + H+
NADP+
NADP+

Amethopterin and aminopterin are
competitive inhibitors of dihydrofolate
reductase
They act as folic acid antagonists or folic
acid anti-metabolites

H4-Folate is a carrier of one-carbon units
The one-carbon unit may be attached to N5
or N10 of H4-folate

The one-carbon units carried by H4-
folate may be:
• Formyl (–CHO) group
• Formate (–HCOO‒) group
• Methyl (–CH3) group
• Methylene (=CH2) group
• Methenyl (=CH) group
• Formimino (–CH=NH) group

H N2 N
N
N
H
5
H
N
10
CH — N —2 — C — N — CH
| |
CHO COOH
COOH
|
CH2
|
CH2
|
O
||
H
|
H N2 N
N
N
|
CH3
5
H
N
10
CH — N —2 — C — N — CH
| |
H COOH
COOH
|
CH2
|
CH2
|
O
||
H
|
N -Methyl-H -folate
5
4
N -Formyl-H -folate (f -H -folate)
10 10
4 4
H N2 N
N
N
|
CH
||
NH
5
H
N
10
CH — N —2 — C — N — CH
| |
H COOH
COOH
|
CH2
|
CH2
|
O
||
H
|
N -Formimino-H -folate (fi -H -folate)5 5
4 4
|
OH
|
OH
|
OH

H4-Folate can:
• Receive one-carbon units from
various compounds
• Transfer one-carbon units to various
compounds

Sources of one-carbon units
Tetrahydrofolate may receive
one-carbon units from:
• Formiminoglutamic acid
• Methionine
• Choline
• Thymine
• Serine

Formiminoglutamic acid (FIGLU) is formed
in the body from histidine:
Histidine → Urocanic acid → → FIGLU
FIGLU can transfer its formimino group to
tetrahydrofolate:
FIGLU+ H4-Folate → fi5-H4-Folate + Glutamate

Methionine, choline and thymine are the
source of methyl groups
Serine can contribute its hydroxymethyl
group

Utilization of one-carbon units
The one-carbon unit carried by tetra-
hydrofolate can be used for synthesis of:
• Purines
• Serine
• Methionine
• Choline
• Thymine
• n-Formylmethionine

Synthesis of purines:
The carbon atoms two and eight of
purines are contributed by f10-H4-
folate

Synthesis of serine:
The hydroxymethyl group for the
conversion of glycine into serine is
provided by N5, N10-methylene-H4-folate

Synthesis of methionine:
The methyl group for the conversion of
homocysteine into methionine is
provided by N5-methyl-H4-folate

Synthesis of choline:
The methyl groups for the synthesis of
choline from serine are provided by N10-
methyl-H4-folate

Synthesis of thymine:
The methyl group of thymine is provided
by N5, N10-methylene-H4-folate

Synthesis of n-formylmethionine:
• The formyl unit of f10-H4-folate converts
methionine into n-formylmethionine
• n-Formylmethionine initiates protein
synthesis in prokaryotes

Green leafy vegetables are the major
source of folic acid
Folic acid is present in several other
vegetables, yeast, meat, fish, milk etc
Intestinal bacteria also synthesize folic
acid
Sources

Sources of folic acid
Green leafy
vegetables
Other
vegetables
Fish MilkMeat
Yeast

Requirement
Infants and children 100 mg/day
Adult men and women 100 mg/day
Pregnant women 300 mg/day
Lactating women 150 mg/day

Folic acid is required for the synthesis of
purines and thymine
Its deficiency impairs nucleic acid synthesis
This leads to growth failure and megalo-
blastic anaemia
Leukopenia can also occur
Deficiency

Megaloblasts in
folic acid
deficiency

Folic acid deficiency during pregnancy can
cause neural tube defects in the baby
This occurs very early in pregnancy
Folic acid supplements are recommended
from conception or even earlier

Deficiency can be diagnosed by giving a
test dose of histidine
Urinary excretion of FIGLU is measured
after giving the test dose
The excretion is increased in subjects
deficient in folic acid
Laboratory diagnosis

Vitamin B12 exists in several forms
Cyanocobalamin was the first form
discovered
It was isolated as a red crystalline
compound from liver in 1948
Cobalamin (Vitamin B12)

Vitamin B12 activity was found in
compounds in which the cyanide
group is replaced by:
• Hydroxyl group (hydroxocobalamin)
• Methyl group (methylcobalamin)
• Nitro group (nitrocobalamin)

Vitamin B12 has a complex structure
Molecular formula of cyanocobalamin is
C63H88O14N14PCo
It has four pyrrole rings with a cobalt atom
at the centre (corrin ring)
The tetrapyrrole is heavily reduced and
substituted

The cobalt atom of the corrin ring
forms co-ordination bonds with:
• Nitrogen atoms of four pyrrole rings
• Cyanide group
• 5,6-Dimethylbenzimidazole

5,6-Dimethylbenzimidazole is linked with
ribose-3-phosphate
The phosphate group of ribose-3-
phosphate is linked with the pyrrole ring D
(IV) through amino propanol

N
N
OH
H
O
H
H H
O
H C –3
H C –3
CH OH2
H N – OC – H C –2 2
HN – OC – H C – H C –2 2
|
CH2
|
CH – CH3
|
O
|
O = P – OH
NH2
|
|
CO
|
CH2
|
CH2
|
N
N
H C–3
H C–3
NH2
CH2
|
|
CO
CO
|
|
|
CH2
CH2
|
|
A
C
D BN N
– CH – CO – NH2 2
– CH – CH – CO – NH2 2 2|
CH3
|
CH3
CH3
CH3
– CH3
CH3
NH2
Co+
CN

Vitamin B12 is heat-stable in acidic and
neutral medium
It is present in food in association with
proteins

The ingested vitamin B12 is released from
proteins by gastric hydrochloric acid
Most of the vitamin binds to R-proteins
present in gastric juice and saliva
R-Proteins are synthesized by many cells,
and include transcobalamin I and trans-
cobalamin III
Absorption, transport and storage

Gastric parietal cells secrete intrinsic factor
(IF)
IF is a glycoprotein of 45 kD which can
bind vitamin B12
At low pH, affinity of vitamin B12 for R-
proteins is much higher than that for IF

Most of the vitamin binds to R-proteins in
the stomach
In duodenum, R-proteins are hydrolysed
by pancreatic proteases
Vitamin B12 released from R-proteins is
bound to IF

One IF molecule binds one molecule of
vitamin B12
A specific receptor on ileal mucosa binds
the IF-vitamin B12 complex
The vitamin is taken up by the mucosal
cells and is transferred to plasma

Most of the vitamin is bound to trans-
cobalamin II in plasma
Transcobalamin II-vitamin B12 complex is
taken up by cells which require vitamin B12
These cells take up the complex with the
help of a specific receptor

Transcobalamin II is hydrolysed in the cell
by lysosomal enzymes
Vitamin B12 is released, mostly in the form
of hydroxocobalamin
It is converted into coenzymes, and is
utilized in the cell

A significant amount of vitamin B12 is
stored in the body
In well-nourished adults, vitamin B12 stores
are about 2,000-5,000 mg
About 60% of the vitamin is stored in liver,
mostly bound to transcobalamin III

Vitamin B12 forms coenzymes known as
cobamides (B12 coenzymes)
The coenzymes are formed by replace-
ment of the cyanide or hydroxyl group
The major B12 coenzymes are methyl-
cobalamin and adenosylcobalamin
Functions

In methylcobalamin, the cyanide group is
replaced by a methyl group
In adenosylcobalamin, it is replaced by 5´-
deoxyadenosine

Methylcobalamin Adenosylcobalamin

The cobamides function as coenzymes,
mainly in the transfer of one-carbon units
They complement the function of tetra-
hydrofolate

Besides H4-folate, cobamides are also
involved in transfer of one-carbon units
An example of one such reaction is
synthesis of methionine from homocysteine
Transfer of one-carbon units

H4-FolateN5-Methyl-H4-folate
MethylcobalaminCobalamin
Methionine Homocysteine
H4-Folate released in this reaction returns
to the folate pool
It can again participate in one-carbon
transfer reactions

In cobalamin deficiency, H4-folate cannot
return to folate pool
It is trapped as methyl-H4-folate (known as
folate trap)
Thus, cobamides help by sharing a part of
the load on H4-folate

Adenosylcobalamin acts as a coenzyme
in the conversion of methylmalonyl CoA
into succinyl CoA
Methylmalonyl CoA is formed mainly from
isoleucine, valine and methionine
It is also formed from fatty acids having an
odd number of carbon atoms
Formation of succinyl CoA

CH3
|
HOOC — C — H
|
C ~ S — CoA
Methylmalonyl CoA isomeraseCobamide
L-Methylmalonyl CoA
Succinyl CoA
||
O
|
CH — C ~ S — CoA2
||
O
CH — COOH2

Succinyl CoA may be:
• Converted into glucose or
• Oxidized in citric acid cycle

In vitamin B12 deficiency, methylmalonic
acid is excreted in large amounts in urine
(methylmalonic aciduria)
Rarely, methylmalonic aciduria may be
caused by an inherited defect in
methylmalonyl CoA isomerase

Vitamin B12 cannot be synthesized by any
plant or animal
It is synthesized only by some bacteria
Animals acquire it through bacterial
synthesis in their intestines
Bacteria present in the human intestine
also synthesize vitamin B12
Sources

Dietary
sources of
vitamin B12
Eggs
Liver Kidney Meat
Cheese
Milk

Requirement
Age and sex Requirement
Infants and
children 0.2-1 µg/day
Adult men and
women 1 µg/day
Pregnant and
lactating women 1.5 µg/day

Deficiency
Deficiency of vitamin B12 is historically
associated with pernicious anaemia
The disease is also known as Addison-
Biermer anaemia
It was a fatal disease before liver was
introduced for its treatment in 1926

A prescription for pernicious anaemia in 1936

Liver was believed to have an anti-
pernicious anaemia factor (APAF)
Castle (1930) showed that stomach
produced a compound necessary for
absorption of APAF
He named this compound as the intrinsic
factor, and APAF as the extrinsic factor

The extrinsic factor later turned out to be
vitamin B12
The basic cause of pernicious anaemia
was found to be absence of intrinsic factor
Autoimmune destruction of gastric parietal
cells leads to absence of intrinsic factor

Clinical features of deficiency may take
long to develop
Hepatic stores of vitamin B12 can last
several years
The deficiency affects the haemopoietic
system and the nervous system

The characteristic haematological feature is
megaloblastic anaemia
Large and immature red cell precursors are
released into circulation
This is done to compensate ineffective
haemopoiesis

Involvement of nervous system causes
sub-acute combined degeneration (SACD)
This is degeneration of dorsal and lateral
columns of spinal cord

Postrior column
Lateral column
SACD
Anterior column
Degeraration
Normal

SACD leads to sensory as well as motor
disturbances
Numbness, tingling, sore tongue and
ataxia are common neurological features
Psychiatric abnormalities can also occur
The neuropathy is believed to be due to
accumulation of methylmalonic acid

Deficiency of vitamin B12 is not always due
to pernicious anaemia
It can also occur due to deficient intake or
decreased absorption
Such deficiency causes megaloblastic
anaemia but not neuropathy

Ascorbic acid
Ascorbic acid (vitamin C) prevents a
specific deficiency disease, scurvy
Therefore, it is also known as anti-
scorbutic factor
It is very heat-labile, specially in basic
medium

Chemically, the structure of ascorbic acid
resembles that of hexoses
Like hexoses, it can exist as L- and D-
isomers
Only L-isomer possesses vitamin activity

Ascorbic acid can be readily oxidized to
dehydroascorbic acid
L-Ascorbic acid and L-dehydroascorbic
acid possess equal vitamin activity

C = O
|
C – OH
||
C – OH
|
H – C
|
HO – C – H
|
CH OH2
H – C
C = O
|
|
|
|
HO – C – H
|
CH OH2
C = O
C = O
L-Ascorbic acid L-Dehydroascorbic acid
O O

Vitamin C is synthesized by all plants and
animals via uronic acid pathway
Exceptions are guinea pigs and primates
which require vitamin C from outside
Guinea pigs and primates lack L-gulono-
lactone oxidase
This enzyme converts L-gulonolactone into
L-ascorbic acid

From an average diet, 70 to 95% of the
ingested ascorbic acid is absorbed
However, as the intake increases, the
proportion of absorption decreases

Cells take up vitamin C with the help of
some transporters
The transporters involved in cellular
uptake are:
• Sodium-Vitamin C Transporters (SVCTs)
• Glucose Transporters (GLUTs)

SVCT1 and SVCT2 are active transport
systems for vitamin C
Transport by SVCT1 and SVCT2 is
sodium-linked
GLUT1 and GLUT3 transport vitamin C by
facilitated diffusion

SVCT1 and SVCT2 transport the reduced
form (ascorbic acid) into the cells
GLUT1 and GLUT3 transport the oxidized
form (dehydroascorbic acid) into the cells
SVCT2 transports vitamin C in all the
tissues with the exception of erythrocytes

Tissue distribution
Total amount of ascorbic acid in an
adult is 2-3 gm
It is distributed in all tissues and body
fluids
It is present in high concentrations in
the glands

The highest concentration is found in the
adrenal glands followed by other glands
The concentration in plasma is 0.5-1.5
mg/dl
The vitamin begins to appear in urine when
the plasma level exceeds 1.5 mg/dl

Functions
Ascorbic acid can undergo reversible
Hence, ascorbic acid acts as a coenzyme
in some oxidation-reduction reactions

C = O
|
C – OH
||
C – OH
|
H – C
|
HO – C – H
|
CH OH2
H – C
C = O
|
|
|
|
HO – C – H
|
CH OH2
C = O
C = O
L-Ascorbic acid
(reduced)
L-Dehydroascorbic acid
(oxidized)
O O
A AH2

Ascorbic acid is required
for the synthesis of:
• Collagen
• Carnitine
• Neurotransmitters
• Tyrosine etc

Ascorbic acid acts as a coenzyme for
prolyl hydroxylase and lysyl hydroxylase
These two hydroxylate proline and lysine
residues in the newly synthesized collagen
Hydroxylation allows collagen molecule to
mature and assume its triple helix structure

Hence, it is essential for the formation and
maintenance of:
• Matrix of bones
• Cartilages
• Dentine
• Blood vessels
• Scar tissue etc
Ascorbic acid plays an important role in
post-translational modification of collagen

Ascorbic acid is a coenzyme for e-N-
trimethyl-lysine hydroxylase and g-butyro-
betaine hydroxylase
These two are necessary for synthesis of
carnitine

Ascorbic acid is a coenzyme for dopamine
b-hydroxylase
This enzyme participates in the synthesis
of norepinephrine and epinephrine from
dopamine

Ascorbic acid is a coenzyme for para-
hydroxyphenylpyruvate hydroxylase
Thus, it participates in the catabolism of
tyrosine

Ascorbic acid is a coenzyme for peptidyl-
glycine a-amidating mono-oxygenase
This enzyme adds amide groups to several
peptide hormones
This addition greatly increases their
stability

Ascorbic acid is required for the formation
of bile acids from cholesterol
Cholesterol is converted into 7-a-hydroxy-
cholesterol for bile acid synthesis
This reaction, catalysed by cholesterol 7-a-
hydroxylase, requires ascorbic acid

Ascorbic acid is a reductant, and keeps
iron and copper in reduced state
By converting ferric ions into ferrous ions, it
helps in the intestinal absorption of iron

Ascorbic acid also acts as an anti-oxidant
Along with other anti-oxidants, it helps in
combating oxidative stress

Sources
Indian gooseberry (amla) is the richest
source of vitamin C
All citrus fruits are rich in vitamin C
Several other fruits and vegetables are
good sources

Sources
of
vitamin C
Green leafy
vegetables
CauliflowerTomatoes
Berries
Amla OrangeLemon
Kiwi

Considerable losses of vitamin C can
occur during cooking
Hence, some raw fruits and salads should
be included in the daily diet

Requirement
Age and sex RDA (ICMR, 2010)
Infants 25 mg/day
Children and adults 40 mg/day
Pregnant women 60 mg/day
Lactating women 80 mg/day

Deficiency
Deficiency of vitamin C produces scurvy
A full-blown picture of scurvy is rare these
days
Isolated signs and symptoms of vitamin C
deficiency are still seen

Signs and symptoms of scurvy
include:
• Swollen, spongy and bleeding gums
• Loosening of teeth
• Petechial haemorrhages
• Anaemia
• Retardation of skeletal growth
• Easy fracturability of bones
• Delayed union of fractures
• Delayed healing of wounds

Bleeding gums in vitamin C deficiency

Laboratory diagnosis of deficiency can be
made by ascorbic acid saturation test
After a test dose of ascorbic acid, urinary
excretion of ascorbic acid is measured
The excretion is low in subjects deficient in
vitamin C

Water soluble vitamins

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