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VITAMINS
Classification of vitamins

VITAMIN A
1. What is the highest yielding source of Vit.A? Pon May 2009
2. Describe the chemistry, dietary sources, daily requirement, biochemical
role and deficiency manifestations of Vitamin A. Pon May 2011
3. Discuss Vit.A under the following headings. Pon May 2010
a. Sources
b. Precursor
c. Functions
d. Deficiency
4. Name the fat soluble vitamins. Describe the sources, daily requirements,
functions and deficiency manifesta-tions of Vit.A
5. What is the recommended dietary allowance for Vit.A. Pon May 2007
Fat soluble vitamins:
are A,D,E,K. Alf the fat soluble vitamins are isoprenoid compounds, since they
are made up of one ormore of five carbon units namely isoprene units The term
vitamers represents the chemically similar substances that possess qualitatively
similar vitamin activity. Some examples of vitamers are:
i. Retinol, retinal and retinoic acid are vitamers of vitamin A.
ii. Pyridoxine, pyridoxal and pyridoxamine are vitamers of vitamin B..
Forms of Vit.A:
1. All the compounds with vitamin A activity are referred to as retinoids. They are
poly isoprenoid compounds having a beta-ionone ring system.
2. Vitamin A is a term reserved to designate any com- pound possessing the
biological activity of retinol
Broad classification:
1. Animal sources:

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1. Vit.A1 refers to all forms of animal sources of Vit.A except one type available
in fish.
2. Vit A2 refers to a less potent form present in fresh water fish and in fish oils.
It has an extra double bond in the ring.
2. Plant source:
1. The , , and carotenes and cryptoxanthin are quantitatively the most
important provitamin A carotenoids.
2. The pro-vitamin, beta-carotene is present in plant tissues. It is cleaved in
intestine to two molecules of retinal but this conversion is inefficient.
Although it would appear that one molecule of -carotene should yield two of
retinol, this is not so in practice; 6 g of -carotene is equivalent to 1 g of
preformed retinol.
Forms of Vit.A1:
1. Retinol is Vit.A alcohol. It contains b-ionine ring and two side chains called
isoprenoid units.
2. Retinal is Vit A aldehyde, It is required for the visual cycle and enables
rhodopsin to mediate vision. Retinal is reduced to retinol by retinal reductase
and retinal can also be oxidized to retinoic acid. Retinal exists in isomeric forms
due to double bond side chains. All- trans variety is called Vit A1 is most
common and Vi.T A2 has extra double bond in the ring. 11-cis - retinal is the
biologically important compound.
3. Retinyl ester is the transport form from intestines to liver as part of
chylomicron.
4. Retinol palmitate is the storage form in liver.
5. Vit. A acid: retinoic acid; The physiologically most important acid derivative,
functions at the gene level as a ligand for specific nuclear transcription factors;
thus, regulate many genes involved in fundamental biologic activities of the cell.
It is not reversible to other forms.
Absorption and transport:
1. Dietary retinyl esters are hydrolyzed by pancreatic or intestinal brush border
hydrolases in the intestines. Retinol is released. Similarly, B-carotene s
hydrolyzed by b-carotene 15-15 di oxygenase of intestinal cells to release 2
moles of retinal, which is again reduced to retinol. The intestinal activity of
carotene dioxygenase is low, so that a relatively large proportion of ingested
-carotene may appear in the circulation unchanged.
2. The absorption is along with other fats and requires bile salts. In biliary tract
obstruction and steatorrhoea, vitamin A absorption is reduced.
3. Retinol enters mucosal cells where it is re esterified into retinylester and
incorporated into chylomicrons and transported via lymph. Then they are taken
up by liver and stored as retinol palmitate.
4. Transport from Liver to Tissues
1. When needed retinol is released from liver and transported by plasma
retinol binding protein. The retinol-RBP complex binds to specific
receptors on retina and other cells and release into cells.
2. The retinol-RBP complex binds to specific receptors on the retina, skin,
gonads and other tissues. The RBP does not enter in the cell. Inside the

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cytoplasm of cells, vitamin A binds to cellular retinoic acid binding protein


(CRBP) and finally to DNA
1. In the cells a cellular retinol-binding protein carries it to DNA to exert its
function.
Biochemical functions:
1. Vision:
a. Rods and cones: Retina has rods and cones; rods are involved in dim light
vision and cones in bright light and color vision.
b. Walds visual cycle:
i. Rhodopsin is present in the photoreceptor cells of the retina.
Rhodopsin consists of opsin a protein and 11-cis - retinal
ii. When light falls on retina, 11-cis retinal isomerizes to all-trans-retinal.
This leads to conformational changes in opsin and generate nerve
impulse.
iii. Part of the all-trans-retinal is immediately isomerized to 11-cis retinal
and combines again with opsin to form rhodopsin. The cycle is
repeated. Generation of nerve impulse: when light falls on retina, a

2.

3.

4.
5.

visual cascade is trigerred. Photon is absorbed by rhodopsin and


metarhodopsin II is produced which in turn activates transducin
involving GTP (guanine triphosphate) . The activated transducin
activates cyclic AMP phosphodiesterase. This enzyme degrades cyclic
AMP in the rod cells and closes the Na channel resulting in
hyperpolarization and impulse transmission to brain.
Color vision is governed by phorphyropsin (red), iodopsin (green) and cyanopsin
(blue). These are retinal-opsin complexes. Different color in the light bleaches
different pigments and send a mix of signals proportionately to brain for color
perception.
Dark adaptation: bright light depletes stores of rhodopsin; so there is a delay in
adapting to dim light from bright light; tis is calle adaptation time which is increased
in Vit A deficiency.
Dim light vision: Rods are responsible for this vision and is present in greater
proportion in nocturnal animals.
Regulation of gene expression
1. Retinol and retinoic acid function like a steroid hormone in the regulation
of gene expression and regulate protein synthesis, cell growth and
differentiation.
2. Retinoic acid binds to nuclear receptors. Retinoic acid receptors (RAR)
bind all-trans-retinoic acid, while retinoic x receptors (RXR) bind to 9-cis
retinoic acid. In combination, these nuclear receptors are capable of
transcribing and regulating hundreds of genes.
3. RXRs form dimers with vitamin D-receptor also. This explains why
deficiency of vitamin A impairs vitamin D function.

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6. Vit.A is essential for the integrity of epithelium; retinyl phosphate is essential for
mucus formation to maintain moist surface. This is due to the fact that retinol and

retinoic acid are required to prevent keratin synthesis.

7. Retinyl phosphate is also necessary for synthesis of glycoproteins essential for


growth.
8. Retinol and retinoic acid are necessary for synthesis of transferrin.
9. Vit.A promotes immune response.
10. Cholesterol synthesis and Gluconeogenesis indirectly require Vit A
11. Retinol is necessary for the reproductive system. Retinol acts like a steroid hormone
in controlling the expression of certain genes. This may account for the requirement
of vitamin A for normal reproduction.
12. Anti-oxidant property:
a. There is a correlation between the occurrence of epithelial cancers and
vitamin A deficiency. The anticancer activity has been attributed to the
natural antioxidant property of carotenoids. Fresh vegetables containing
carotenoids were shown to reduce the incidence of cancer.
b. Beta carotenes may be useful in preventing heart attacks.
13. Cholesterol synthesis requires vitamin A. Mevalonate, an intermediate in the
cholesterol biosynthesis, is diverted for the synthesis of coenzyme Q in vitamin A
deficiency. The discovery of coenzyme Q was originally made in vitamin A deficient
animals
14. Retinoic acid is used in the treatment of acne and soriasis.
15. Retinol and retinoic acid are involved in the synthesis of transferrin, the iron
transport protein.
RDA:
1. Equivalents:
a. 1 IU of Vit.A = 0.3 g of retinol
b. 1 retiol equivalent is = 1 g of retinol or 6 g of beta carotene
2. Daily requirements:
a. Children = 400-600 g / day
b. Men = 700-1000

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c. Women = 750
d. Pregnancy 1000
Deficiency manifestations:
1. Causes of deficiency:
a. Decreased intake
b. Lack of bile
c. Lack of RBP:
i. Cirrhosis
ii. Severe Protein deficiency and inadequate RBP
iii. Loss of RBP in nephrosis
2. Ocular changes:
a. Nyctalopia or night blindness: difficulty of vision in dark and prolonged
dark adaptation time.
b. Xeropthalmia: dry, wrinkled and scaly conjunctiva; dry and cloudy cornea
without lusture and moisture
c. Bitots spots: increased thickness of conjunctiva in either side of cornea.
d. Keratomalacia: softening of cornea leading to corneal ulceration,
perforation and loss of vision.
e. India: 40% of blindness in children is preventable as they are caused by
malnutrition.
3. Skin and mucous membrane:
a. Dry, thick and scaly skin called phrynoderma
b. All epithelial membranes becomes brittle and atrophic.
c. Renal calculi due to epithelial shedding in urinary tract forming a nidus
for calculi.
d. Drying and crackling of skin leads to skin infections
4. The reproductive system is adversely affected in vitamin A deficiency.
Degeneration of germinal epithelium leads to sterility in males
Assessment of deficiency:
1. Prolonged dark adaptation time
2. Serum RBP level
3. Serum Vit.A level (Normal is 25 to 50 g /dl)
Dietary sources:
1. Animal sources: milk, butter, cream, cheese, egg yolk, fish liver oil and liver.
2. Vegetable sources: carrot, papaya, mango, pumpkins, DGLV (dark green leafy
vegetables)
Hypervitaminosis. A
1. Excessive intake can lead to toxicity since the vitamin is stored.
2. Anorexia, irritability, headache, peeling of skin, drowsiness and vomiting.
3. Increased intra cranial pressure (pseudo tumor cerebri)
4. Enlargement of liver is also seen in children. Higher concentration of retinol
increases lysosomal enzymes, leading to cellular death.
5. Swelling and pain in bones
6. In pregnancy it can lead to congenital defects in fetus.
Hypercarotenemia:
It can result from persistent excessive consumption of foods rich in carotenoids.

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The skin becomes yellow, but no staining of sclera as in jaundice is observed.


VITAMIN D (CHOLECALCIFEROL)
1. Name the active form of Vitamin D. How is it synthesized in the body? Add a note
on its functions, requirements and deficiency manifestations. April 1996
2. Write an essay on Vitamin D, indicating its chemical nature, formation, biochemical
roles, physiological functions, deficiency manifestations, sources and
requirements. April 1995
3. How is Vit.D converted converted to its active form? Pon May 2010
4. What is the manifestation of vit.D deficiency in the body? Pon May 2009
5. Describe the formation and functions of calcitriol. Pon May 2014
6. What is the vitamin which could be deficient in people who are not exposed to
sunlight? Explain the mechanism. Pon Apr 2000

Forms of Vit.D:
1. Vitamin D is a fat soluble vitamin. lt resembles sterols in structure and functions
like a hormone
2. Vitamin D2 (or ergocalciferol) is from plants and vitamin D3 (or cholecalciferol) is
from animal sources. Ergocalciferol and cholecalciferol are referred to as
provitamins. During the course of cholesterol biosynthesis, 7-dehydrocholesterol
is formed as an intermediate. On exposure to sunlight, both 7dehydrocholesterol is converted to cholecalciferol in the skin by the action of
ultraviolet radiations. Commercially the vitamin is derived from the fungus,
ergot. The ergosterol when treated with ultraviolet light, ergocalciferol or vitamin
D2 is produced.Vitamin D is called the sun-shine vitamin. There is no
Vit.D1.
Absorption, transport and storage:
Vit D is absorbed in the small intestine by the help of bile. It enters through
lymph into blood circulation and is carried by alpha 2 globulin. Liver and other
tissues store Vit.D.
Activation of Vitamin D:
Liver:
The cholecalciferol is first transported to liver, where hydroxylation at 25th
position occurs, to form 25-hydroxy cholecalciferol (calcidiol). The hepatic 25hydroxylase is a microsomal monooxygenase. It requires cytochrome P-450 and
NADPH. 25-HCC is the major storage form.
Plasma: 25-HCC is bound to "vitamin D binding protein" (VDBP), an alpha-2 globulin.
Kidney: In kidney, it is further hydroxylated at the 1st position to form 1,25-dihydroxy
cholecalciferol. Since it contains three hydroxyl groups at 1, 3 and 25 positions, it is
also called Calcitriol. It is the active form of vitamin.D
Biochemical Effects of Vitamin D: The sites of action are:
a. GIT-intestinal villi cells
b. bone osteoblasts
c. kidney distal tubular cells.
GIT:
1. Calcitriol promotes the absorption of calcium and phosphorus from the
intestine. Calcitriol binds with a cytosolic receptor to form a calcitriol-

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receptor complex. This complex then approaches the nucleus and interacts
with a specific DNA leading to the synthesis of a specific calcium binding
protein. Calcium uptake is enhanced by an increased by this protein called
Calbindin.
2. Thus, the mechanism of action of 1,25- diOH-D3 is typical of steroid
hormones.
Effect of Vitamin D in Bone:
1. In the osteoblasts of bone, calcitriol stimulates calcium uptake for deposition
as calcium phosphate
2. Calcitriol stimulates osteoblasts which secrete alkaline phosphatase. Due to
this enzyme, the local concentration of phosphate is increased. The ionic
product of calcium and phosphorus increases, leading to mineralization.
Along with PTH it mobilize Ca and phosphates from the bone to maintain
their plasma level also.
Effect of Vitamin D in Renal Tubules:
Calcitriol increases the reabsorption of calcium and phosphorus by renal tubules,
therefore both minerals are conserved (PTH conserves only calcium) bone.
Kidney converts 25, Hydroxy cholecalciferol to 24,25 - DHCC an inactive form
instead of
1-25- DHCC when Vit D is available in plenty so that its biological action is kept
optimum levels.
Regulation of Vit.D:
1. 1,25 DHCC synthesis is regulated by plasma levels of Calcium and phosphate
and calcitriol itself.
2. Low Plasma level of calcium enhances the parathyroid hormone secretion which
in turn activates 1-hydroxylase. Low plasma phosphate directly activates 1
hydroxylase.
3. Hypercalcemia decreases calcitriol. Low dietary calcium and hypocalcemia
increase the rate of production of 1,25-DHCC.
4. When calcitriol concentration is adequate, 24-hydroxylase in kidney acts leading
to the synthesis of a less important compound 24, 25-DHCC. In this way, to
maintain the homeostasis of calcium, synthesis of 24,25-DHCC is also important
Vit D is a hormone - justification:
1. Vit.D3 is synthesized in the skin by UV rays of sunlight.
2. Calcitrol is produced in kidneys.
3. Calcitrol has target organs like intestine, bone and kidneys.
4. Like a steroid it binds to a receptor in the cytosol and stimulates the synthesis of
calcium binding proteins.
5. Actinomycin D is able to inhibit the action of Calcitrol suggesting that the action
of calcitrol is at DNA transcription.
6. Like hormone Calcitrol has a feed back regulatory system for its synthesis.
7. It combines with other hormone like PTH and Calcitonin to regulate Ca x P levels
in plasma.
8. Like a hormone it has half life of 10 hours.
Causes for Vitamin D Deficiency
1. Deficiency of vitamin D can occur in people who are not exposed to sunlight

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2.
3.
4.
5.
6.

properly, e.g. inhabitants of northern latitudes, in winter months, in people who


are bedridden for long periods, or those who cover the whole body (purdah).
Nutritional deficiency of calcium or phosphate may also produce similar clinical
picture.
Malabsorption of vitamin (obstructive jaundice and steatorrhea).
High phytate content in diet may also reduce the absorption of vitamin.
Abnormality of vitamin D activation. Liver and renal diseases may retard the
hydroxylation reactions.
Deficient renal absorption of phosphates.

Deficiency manifestations:
1. The deficiency diseases are rickets in children and osteomalacia in adults.
2. Clinical Features of Rickets
a. Rickets is seen in children. There is insufficient mineralization of bone.
Bones become soft and pliable. The bone growth is markedly affected.
b. Plasma calcium and phosphorus are low normal with alkaline
phosphatase (bone isoenzyme) being markedly elevated.
c. The classical features of rickets are bone deformities. Weight bearing
bones are bent. Continued action of muscles also cause bone
malformations. The clinical manifestations include bow legs, knock-knee,
rickety rosary, bossing of frontal bones, and pigeon chest.
d. An enlargement of the epiphysis at the lower end of ribs and
costochondral junction leads to beading of ribs or rickety rosary.
e. Harrison's sulcus is a transverse depression passing outwards from the
costal cartilage to axilla. This is due to the indentation of lower ribs at the
site of the attachment of diaphragm.
3. Clinical Features of Osteomalacia
a. The term is derived from Greek "osteon" = bone; and "malakia" =
softness. The bones are softened due to insufficient mineralization and
increased osteoporosis. Patients are more prone to get fractures.
b. The abnormalities in biochemical parameters are a slightly lower serum
calcium, and a low serum phosphate.
c. It may be noted that vitamin D deficiency never produces severe
hypocalcemia. Tetany will not be manifested.
d. Serum alkaline phosphatase, bone isoenzyme, is markedly increased.
4. Different Types of Rickets
a. Nutritional rickets: The classical vitamin D deficiency rickets can be cured
by giving vitamin D in the diet.
b. Hypophosphatemic rickets: mainly result from defective renal tubular
reabsorption of phosphate. Supplementation of vitamin D along with
phosphate is found to be useful.
c. Vitamin D resistant rickets: is found to be associated with Fanconi
syndrome, where the renal tubular reabsorption of bicarbonate,
phosphate, glucose and amino acids are also deficient.
d. Renal rickets: In kidney diseases, even if vitamin D is available, calcitriol
is not synthesized. These cases will respond to administration of

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calcitriol.
e. Vitamin DDependent Rickets: End organ refractoriness to 1,25-DHCC will
also lead to rickets. Either a decrease in the number of cytosolic receptor
or a structurally abnormal receptor is noticed. The bone disease has been
found to respond to megadoses of calcitriol (35 mg/day).
5. Other actions:
a. Recent research has proved that most tissues possess vitamin D receptor
and several tissues have the enzymes to generate calcitriol. 1,25-DHCC
has been found to have a modulatory effect on immunohematopoietic
system. Therapeutic doses given to children with rickets have been found
to correct the anemia, hypocellularity of the bone marrow and increased
susceptibility to infection. It has also been found to reduce the risk of
cancer and coronary vascular disease. Beneficial effects have been
observed in patients with AIDS.
Requirement of Vitamin D
a. Children = 10 microgram (400 IU)/day
b. Adults = 5 microgram (200 IU)/day
c. Pregnancy, lactation = 10 microgram/day
d. Above the age of 60 = 600 IU per day.
Sources of Vitamin D
1. Exposure to sunlight produces cholecalciferol. Moreover fish liver oil, fish and
egg yolk are good sources of the vitamin.
2. Milk contains moderate quantity of the vitamin.
Hypervitaminosis D
1. Doses above 1500 units per day for very long periods may cause toxicity.
Symptoms include weakness, polyuria, intense thirst, difficulty in speaking,
hypertension and weight loss.
2. Hypercalcemia leads to calcification of soft tissues, (metastatic calcification,
otherwise called calcinosis), especially in vascular and renal tissues.
3. Although vitamin D is toxic in higher doses, excessive exposure to sunlight
does not result in vitamin D toxicity, because excess D3 is destroyed by
sunlight itself.
VITAMIN E
1. Vitamin E (tocopherol) is a naturally occurring antioxidant. lt is essential for
normal reproduction in many animals, hence known as anti-sterility vitamin. Vitamin
E is described as a 'vitamin in search of a disease. This is due to the lack of any
specific vitamin E deficiency disease in humans
2. Naturally occurring vitamin E exists in eight chemical forms (alpha-, beta-, gamma-,
and delta-tocopherol and alpha-, beta-, gamma-, and delta-tocotrienol) that have
varying levels of biological activity. Alphatoco-pherol is the only form that is
recognized to meet human requirements.
3. Chemistry: Ther are derivatives of 6-hydroxy chromane (tocol) ring with isoprenoid
side chains. The anti oxidant property is due to chromane ring.
4. Absorption: bile is necessary and is absorbed from small intestine. Transported by
incorporation with VLDL and LDL and stored in liver, muscle and adipose tissue.

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5. Normal plasma level is less than 1 mg/dl


6. Biochemical functions:
1. it has anti oxidant property and prevents the non enzymatic oxidation of
various cell components.
2. It maintains the integrity of cell membrane and cell structure.
3. It prevents peroxidation of polyunsaturated fatty acids. Selinium and Vit.E
act synergistically to minimize lipid peroxidation.
4. It prevents haemolysis of RBCs by H2O2
5. It preserves germinal epithelium of gonads and thus prevents sterility.
6. It enhances the action of ala synthase and increases synthesis of heme.
7. It stabilizes coenzyme A and is involved in electron transport chain.
8. It prevents the oxidation of vit.A and carotenes.
9. It helps storage of creatine in muscles.
10. It is required for intestinal absorption of amino acids.
11. It is requird for nucleic acid synthesis.
12. It protects live from damage by carbon tetra chloride.
13. It plays a role in prevention of cataract.
14. It prevents oxidation of LDL and thus protects heart. It reduces the risk of
atherosclerosis by reducing oxidation of LDL
15. Vitamin E also boosts immune response. Vitamin E can depress leukocyte
oxidative bactericidal activity.
7. RDA: 10 mg for man and 8 mg for women/day
8. Sources: Nuts, seeds, and vegetable oils are among the best sources of alphatocopherol, and significant amounts are available in green leafy vegetables and
fortified cereals, soybean, canola, corn, and other vegetable oils.
9. Deficiency symptoms:
1. Sterility in animals but no significant symptoms in humans.
2. Increased fragility of RBCs, minor neurological symptoms may be observed in
human.
10. Toxicity: nil found; however two clinical trials have found an increased risk of
hemorrhagic stroke in participants taking alpha-tocopherol.
VITAMIN K
1.
2.
3.
4.

What is the role of Vit.K in maintaining body haemostasis? Pon May 2009
What are the biological functions of Vit.K? Pon May 2012
State the function of Vit. K & E. Pon May 2009
Name the Vit.K dependant coagulation factors. What is the role of Vit.K on
their activities? Pon Nov 2007; May 2003
5. Symptoms of Vit.K deficiency are met with more due to liver dysfunction
than due to lack of the vitamin. Explain why this is so. Pon apr 2002
1. Vitamin K is a fat-soluble vitamin. The "K" is derived from the German word
"koagulation. There are two naturally occurring forms of vitamin K.
a. Plants synthesize phylloquinone, which is also known as vitamin K1.
b. Bacteria synthesize vitamin K2, designated as menaquinone.
c. K3 is a synthetic form.

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2. Chemistry: they are napthoquinone derivatives with isoprenoid side chain.


3. Absorption: Vit K provided by diet or bacterial synthesis is absorbed in association
with chylomicrons with the help of bile salts. It is transported with LDL and stored in
liver.
4. Biochemical functions:
a. Vitamin K is necessary for coagulation. It brings about posttranslational
modification clotting factors 2,7,9 and 10 produced as zymogen in liver.
b. Vit. K acts as coenzyme for the carboxylation of glutamic acid by
enzyme carboxylase. Gamma carboxyglutamate is formed which is critical
to the calcium-binding function of these proteins. They are negatively
charged (COO-) and they combine with positively charged calcium ions
(Ca2+) to form a complex. The complex (eg. prothrombin - Ca) binds to the
phospholipids on the membrane surface of the platelets. This leads to the
increased conversion of prothrombin to thrombin.
a. The formation of y-carboxyglutamate is inhibited by dicumarol, an
anticoagulant and warfarin, a synthetic analogue and they can inhibit
vitamin K action.

5.
6.
7.

8.

a. The mineral-binding capacity of osteocalcin in bone also requires vitamin Kdependent gamma-carboxylation of glutamic acid residues.
b. Vit.K is also involved in ETC and oxidative phosphorylation.
RDA: adult: 70-140 microgram/day
Sources: Cabbage, cauliflower, tomatoes, spinach, alfa-alfa, green vegetables, egg
yolk, meat, liver, cheese and milk products.
Causes for Deficiency of Vitamin K
a. In normal adults dietary deficiency seldom occurs since the intestinal
bacterial synthesis is sufficient to meet the needs of the body. However
deficiency can occur in conditions of malabsorption of lipids. This can result
from obstructive jaundice, chronic pancreatitis, sprue, etc.
b. Prolonged antibiotic therapy and gastrointestinal infections with diarrhoea
will destroy the bacterial flora and can also lead to vitamin K deficiency.
Deficiency symptoms:
a. Overt vitamin K deficiency results in impaired blood clotting, usually
demonstrated by laboratory tests that measure clotting time.
b. Hemorrhagic disease of the newborn is due to vitamin K deficiency. The
newborns, especially the premature infants have relative vitamin K
deficiency. This is due to lack of hepatic stores, limited oral intake (breast
milk has very low levels, 15 mg/liter) and absence of intestinal bacterial
flora. It is often advised that pre-term infants be given prophylactic doses of
vitamin K (1 mg Menadione).
c. In children and adults, Vitamin K deficiency may be manifested as bruising
tendency, ecchymotic patches, mucous membrane hemorrhage, posttraumatic bleeding and internal bleeding.
d. Prolongation of prothrombin time and delayed clotting time are characteristic
of vitamin K deficiency. Measurement of prothrombin time (PT) is taken as an
index of liver function.
e. Warfarin and dicoumarol will competitively inhibit the gamma carboxylation

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system due to structural similarity with vitamin K. Hence they are widely
used as anticoagulants for therapeutic purposes.
9. Daily Requirement of Vitamin K
a. Recommended daily allowance is 50-100 mg/day. This is usually available in
a normal diet.
10. Sources of Vitamin K:
a. Green leafy vegetables are good dietary sources. Even if the diet does not
contain the vitamin, intestinal bacterial synthesis will meet the daily
requirements, as long as absorption is normal.
11. Toxicity:
a. There is no known toxicity associated with high doses of the phylloquinone
(vitamin K1) or menaquinone (vitamin K2) forms of vitamin K. The same is
not true for synthetic menadione (vitamin K3) and its derivatives.
b. Menadione can interfere with the function of glutathione, one of the body's
natural anti-oxidants, resulting in oxidative damage to cell membranes.
Menadione given by injection has induced liver toxicity, jaundice, and
hemolytic anemia.
12. Antagonists: Heparin and bihydroxy-coumarin, salicylates and dicumarol are
antagonists vit.K.
NIACIN
1. Write in detail the sources, daily requirement, coenzyme formation and
functions and deficiency manifestations of Vitamin Niacin.
2. Manifestation of pellagra. Pon May 2009
3. Name the amino acid, which forms niacin. What is the neuro transmitter
synthesized from that amino acid? Pon Dec 2001
4. How many mg of tryptophan are required to form 1 mg of niacin? Pon May
2009
1. Niacin or nicotinic acid is also known as pellagra preventive (P.P.) factor of Coldberg
2. Niacin is a water-soluble vitamin, which is also known as vitamin B3. Niacin is a
pyridine derivative. Structurally, it is pyridine 3 carboxylic acid.
3. It is not strictly a vitamin since it can be synthesized in the body from the essential
amino acid tryptophan.
4. The amide form of niacin is known as niacinamide or nicotinamide. Two compounds,
nicotinic acid and nicotinamide, have the biologic activity of niacin.
5. Co-enzyme Formation of Niacin:
a. Dietary nicotinamide, niacin and tryptophan, all can contribute to the
synthesis of the coenzyme NAD and NADP
b. Niacin is converted to its co-enzyme forms, viz. Nicotinamide adenine
dinucleotide (NAD+) and Nicotinamide adenine dinucleotide phosphate
(NADP+).
c. The niacin is attached to a ribose phosphate by Phosphoribosyl
pyrophosphate (PRPP) to form a mononucleotide. It is then attached to AMP
by ATP, to form the dinucleotide. Glutamine donates amide group. The
nitrogen atom of niacinamide contains one positive charge. The structure is

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abbreviated as NAD+. In the case of NADP+, one more phosphoric acid is


attached to the ribose of the AMP
Tryptaphan Niacin

Nicotinamide
PRPP > PPi

Nicotinate mononucleotide
ATP> PPi
Desamido NAD+
Glutamine>Glutamic acid
ATP> AMP+ PPi
NAD+
ATPADP+pi
NADP+
d. Other sources of these coenzymes: NAD can also be synthesized from the
dietary amino acid tryptophan via the kynurenine pathway. Tryptophan
produces quinolinate which then forms nicotinate mononucleotide and,
ultimately, NAD+ and NADP+. 60 mg of tryptophan would yield 1 mg of
Niacin.
Biochemical functions:
1. The coenzvmes NAD+ and NADP+ are involved in a variety of oxidationreduction reactions. They accept hydride ion (hydrogen atom and one
electron :H-) and undergo reduction in the pyridine ring.
2. A large number of enzymes (about 40) belonging to the class oxidoreductases
are dependent on NAD+ or NADP+.
3. NAD+ Dependent Enzymes:
4. Lactate dehydrogenase (lactate pyruvate)
5. Glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate 1,
3-bisphosphoglycerate)
6. Pyruvate dehydrogenase (pyruvate acetyl CoA)
7. Alpha ketoglutarate dehydrogenase (alpha ketoglutarate succinyl CoA
8. Beta hydroxy acyl CoA dehydrogenase (beta hydroxy acyl CoA beta keto acyl
CoA
9. Glutamate dehydrogenase (Glutamate alpha keto glutarate
NADPH dependant reactions:
1. Keto acyl ACP dehydrogenase (Beta keto acyl ACP beta hydroxy acyl ACP)
2. Alpha, beta unsaturated acyl ACP acyl ACP
3. HMG CoA reductase (HMG CoA mevalonate
4. Met-hemoglobin hemoglobin
5. Folate reductase (Folate dihydrofolate tetrahydrofolate)
6. Phenyl alanine hydroxylase (Phenylalanine tyrosine)

260

7. NADH produced is oxidized in the electron transport chain to generate ATP.


One NADH molecule is oxidized in the respiratory chain to generate 3
molecules of ATP
8. NADPH is also important for many biosynthetic reactions as it donates
reducing equivalents
RDA: The daily requirement of niacin for an adult is 15-20 mg and for children, around
10-15 mg.
Deficiency manifestations:
Causes of deficiency:
1. Tryptophan deficiency in diet; eg. Stable food containing sorghum and maize
where tryptophan is not metabolically available to body.
Deficiency symptoms:
1. Pyridoxal deficiency leads to inability to convert tryptophan to niacin.
2. Hartnup disease where congenital defect of absorption of tryptophan from
intestine is present.
3. Carcinoid syndrome where tumor tissue utilizes large amount of tryptophan
for serotonin synthe sis.
Dietary sources: rich sources are dried yeast, rice polishing, liver, peanut, whole
cereals, legumes, meat and fish.
RDA: normally 20 mg/day; additional 5 mg for pregnancy and lactation.
Therapeutic uses:
Pharmacologic doses of nicotinic acid, but not nicotinamide, have been known to
reduce serum cholesterol by reducing acetyl CoA pool. Niacin is commonly prescribed
with other lipid-lowering medications.
Deficency symptoms:
1. Niacin deficiency is mostly seen among people whose staple diet is corn or
maize. Niacin present in maize is unavailable to the body as it is in bound
form. Further, tryptophan content is low in maize.
2. Pellagra:
a. Niacin deficiency leads to the disease called pellagra characterized by
3 Ds: diarrhea, dermatitis and de mentia.
b. Dermatitis consists of bright erythema in feet, ankles and face;
pigmentation around the neck is called Casals necklace. The
dermatitis is precipitated by exposure to sunlight.
c. Symptoms related to the digestive system include a bright red
tongue, vomiting, and diarrhea.
d. Dementia occurs in chronic cases and ataxia and spasticity could also
occur.
e. Neurologic symptoms also include headache, apathy, fatigue,
depression, disorientation, and memory loss. If untreated, pellagra is
ultimately fatal.
Therapeutic uses of niacin:
1. Niacin inhibits lipolysis in the adipose tissue and decreases the circulatory
free fatty acids.
2. Triacylglycerol synthesis in the decreased.

261

3. The serum levels of low-density lipoproteins (LDL), very low-density


lipoproteins (VLDL), triacylglycerol and cholesterol are lowered. Hence niacin
is used in the treatment of hyperlipoproteinemia type II b (elevation of LDL
and VLDL).
Toxicity:
1. Glycogen and fat reserves of skeletal and cardiac muscle are depleted.
2. There is a tendency for the increased levels of glucose and uric acid in the
circulation.
ASCORBIC ACID
3. Write the sources, requirements, deficiency manifestations,
biochemical actions of Vit.C Pon apr 2000
4. Vit C plays a role in post translational modification- justfy. Pon Dec
2003
5. Explain the role of ascorbic acid in post translational modification.
Pon May 2007
Chemistry:
1. Ascorbic acid is a water soluble vitamin; destroyed by heat, alkali and on
storage. 70%is lost while cook-ing; inactivated if cooked in copper vessel.
2. Chemistry: It is a hexose and closely resemble monosaccharide; It has enolic
hydroxyl group ; it is acidic and a strong reducing agent; L-ascorbic acid and
dehydroascorbic acid are active forms; D-ascorbic acid is an inactive form.
3. Plants and animals can synthesis ascorbic acid from glucose via uronic acid
pathway. Man cannot synthesis due to absence of L-gluonolactone oxidase.
Metabolism:
1. It is rapidly absorbed through intestine and excreted by kidneys and not
stored in significant amounts.
2. On oxidation, dehydroascorbic acid is formed which is further oxidized to
oxalic acid.
3. It is partly excreted as ascorbic acid and partly as oxalic acid.
4. Blood levels are 0.4 to 1.5 mg/dl of plasma and 25 mg/dl of WBC.
5. High levels observed around healing tissues.
Biological role:
1. A reversible conversion from ascorbic to dehydro ascorbic acid makes it a
redox system.
2. It is essential for hydroxylation of proline and lysine for the subsequent
formation of collagen, osteoid, intracellular cement in blood capillaries.
3. Role in Post-translational Modifications: The hydroxylation of proline
and lysine residues of collagen is a post-translational modification taking
place intracellularly. Prolyl hydroxylase and lysyl hydroxylase are both
dioxygenases using molecular oxygen. The enzyme also contains ferrous iron
at its active site and requires a reducing agent like ascorbic acid to preserve
the iron in the reduced ferrous state. So, vitamin C deficiency leads to poor
hydroxylation. It is the major biochemical defect in scurvy.

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4. It is essential for hydroxylation of tryptophan to 5-hydroxy tryptophan for the


formation of serotonin.
5. It helps in oxidation of parahydroxy phenyl pyruvate to homogentisic acid.
6. It enhances iron absorption from the intestines by reducing ferric form to
ferrous form.
7. It helps in reconversion of met-hemoglobin to hemoglobin.
8. It helps maturation of RBC by involving in folate reductase that converts folic
acid to tetra folic acid.
9. Its presence in adrenal cortex indicates that it is necessary for the synthesis
of steroids.
10. It helps bile acid synthesis form cholesterol.
11. Promotes phagocyic activity of WBCs.
12. It has antioxidant property and may have a role in cancer prevention. Eg.
Aniline dye induced bladder cancer.
13. It may reduce the incidence of cataracts.
Deficiency manifestations:
1. Scurvy: spongy and sore gums, loose teeth, anemia, swollen joints, fragile
blood vessels, immune incompetence, delayed wound healing, hemorrhage,
sluggish function of adrenal cortex etc. most symptoms are due to impaired
collagen formation and reduced antioxidant properties.
2. Barlow's disease is infantile form of scurvy
3. Petichiae and echymosis or hematoma due to fragile capillaries.
4. Hemorrhage in conjunctiva, retina; haematuria and malena.
5. Bones are fragile, break easily and bleeding into joints.
6. Anemia is microcytic hypo chromic. Anemia is due to:
7. Loss of blood by hemorrhage
8. Decreased iron absorption
9. Decreased tetrahydrofolic acid
10. Accumulation of met-hemoglobin.
Dietary sources: amla (700 mg/100 gm); guava 9 300 mg/100 gm) lemon and leafy
vegetables.
Requirement: 75 mg/day; pregnancy , lactation and ageing 100 mg/day
Therapeutic uses:
1. Adjuvant therapy in infections, TB; wound healing, burns etc.
2. Clinical dose is 500 mg per day
3. Mega doses: unsubstantiated view that mega doses may prevent infection
and ageing.
Toxicity of Vitamin C
Since it is a water soluble substance, excess vitamin C is excreted, and not
accumulated in the body. However, more than 2000 mg of vitamin C daily for a long
time can cause iron overload, because vitamin C helps in absorption of iron.
VIT. B 12

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1. What are the coenzyme forms of Vit.B.12? Give a reaction where each is
used? Pon May 2010
2. What are the abnormal metabolites found in the urine of patients with B12
deficiency? Explain how folate is trapped in B12 deficiency and what is its
significance? Pon Nov 2007
3. A patient has been diagnosed as having megaloblastic anemia.
a. Which vitamin deficiency can cause anemia?
b. What metabolites in urine you will find in this patient?
c. What is the step in one carbon metabolism that is affected in this
case?
d. What is the step in propionic acid metabolism that is defective in
this case?
4. What are the abnormal metabolites found in urine of patients with B12
deficiency ? Explain how folate is trapped in B12 deficiency and what is its
consequences. Pon Nov 2007
5. Name the coenzymes of cyanocobalamin Pon Dec 2001
6. Write briefly on the absorption of vit. B12 Pon May 2004
Chemistry of B12: Synonyms are cobalamin, extrinsic factor (EF) of Castle. Watersoluble, heat stable and red in color. It is a unique vitamin, synthesized by only
microorganisms and not by animals and plants.
Structure:
1. It contains 4 pyrole rings and a cobalt atom and the structure is called corin
ring; Corin is linked with benzimidazole ring and now called cobalamin. The
corin ring is similar to the tetrapyrrole ring structure of heme
2. Cyanide is added to the R position to be called cyano cobalamin. It is a
laboratory compound.
3. When hydroxyl group is added to R position, hydroxy cobalamin is obtained
which is B12.
4. Inside the cells theses groups are removed and deoxy adenosyl cobalamin is
formed which is the storage form.
5. When methyl group replaces adenosyl group, methyl cobalamin is formed
which is the circulating form. Adenosyl cobalamin and methyl cobalamin are
the functional coenzymes.
Absorption of B12:
1. Dietary source of B12 is called extrinsic factor of Castle. Vitamin B12 is
absorbed from Ileum; for its absorption it requires:
2. Presence of HCl, and
3. Intrinsic factor (IF) of Castle, a constituent of normal gastric juice.
4. Cobalophilin: A binding protein secreted in the saliva.
5. Cobalophilin: Gastric acid and pepsin release the vitamin B12 from protein
binding in food and make it available to bind to cobalophilin, a binding
protein secreted in the saliva. In the duodenum, cobalophilin is hydrolyzed,
releasing the vitamin for binding to intrinsic factor.

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6. Intrinc factor: The parietal cells of stomach secretes a special protein called
intrinsic factor of Castle (lF). lt is a glycoprotein (8-15% carbohydrate) with a
molecular weight around 50,000. Intrinsic factor is resistant to proteolytic
digestive enzymes. lF generally forms a dimer, binds strongly with 1 or 2
moles of vitamin B12. This binding protects vitamin B12 against its uptake
and use by bacteria.
Mechanism absorption:
1. Vitamin B12 is absorbed from the distal third of the ileum via specific binding
site (receptors) that binds the B12-IF complex. The removal of B12 from
intrinsic factor (IF) in presence of Ca++ ions and a releasing factor (RF)
secreted by duodenum take place and B12 enters the ileal mucosal cells for
absorption into the circulation.
2. It is also shown that a small amount, about 1 to 3% may be absorbed by
simple diffusion.
3. Transport: In the mucosal cells, B12 is converted to methyl cobalamin . lt is
then transported in the circulation in a bound form to proteins namely
transcobalamins (TC-I & TC-ll).

4. Storage: B12 is an exception that it is stored. Methylcobalamin which is in


excess is taken up by the liver, converted to deoxyadenosyl B12 and stored
in this form.
5. Clinical aspects:
6. In pancreatic insufficiency, the cobalophilin-bound vitamin B12 may not be
split and the complex is excreted in faeces resulting to development of
vitamin B12 deficiency.

7. Atrophy of fundus of stomach and a lack of free HCl (achlorhydria) is usually


associated with pernicious anaemia, caused by B12 deficiency.
Functions:
1. Vit B12 is used in the synthesis of methionine from homocysteine. In this

reaction THF is released from N5-methyl THF. B12 coenzyme accepts methyl
group from methyl THFA to form methyl cobalamin. B12 deficiency leads to
accumulation of N5-methyl THF and this is called folate trap. B12 helps
release of THF from folate trap and B12 deficiency folic acid deficiency also
manifests.

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2. The metabolism of odd chain fatty acids, valine, isoleucine, methionine and
threonine leads to the production of methyl malonyl CoA. Methyl melonyl
CoA is isomerized to succinyl CoA in the presence of B12 by methyl malonyl
CoA mutase (containing AdoB12). In B12 deficiency, methyl malonyl CoA is
excreted in urine (methyl malonic aciduria).
Methyl malonyl CoA mutase +Ado B12
Fatty acids ---Methylmelonyl CoA---------------Succinyl CoA (odd no. of carbon
atoms)
FOLATE TRAP:
1. In vitamin B12 deficiency, increased folate levels are observed in plasma.
The activity of the enzyme homocysteine methyltransferase is low in B12
deficiency. As a result, the only major pathway for the conversion of N5methyl THF to tetrahydrofolate is blocked and body THF pool is reduced.
Essentially almost the entire body folate becomes trapped as N5-methyl THF.
This is known as folate trap or methyl frap. ln this manner, B12 deficiency
results in decreased folate coenzymes that leads to reduced nucleotide and
DNA synthesis. Although the tissue folate levels are adequate or high, there
is a functional folate deficiency due to the lack of THF pool. The outcome is
the development of megaloblastic anemia. Administration of the amino acid
methionine has been shown to partially correct the symptoms of B12
deficiencies. This is due to the fact that the formation of N5-methyl THF is
inhibited by S-adenosylmethionine. A combined therapy of vitamin B12 and
folic acid is generally employed to treat the patients with megaloblastic
anemia.
Causes of B12 deficiency:
1. Vitamin B12 is found only in foods of animal origin, and no plant sources.
Strict vegetarians (vegans) are at risk of developing B12 deficiency.
2. Only source is curd and milk which may not be available poor people.
3. Partial or total gastrectomy-these individuals become intrinsic factor
deficient
4. Pernicious anemia: It is manifested usually in persons over 40 years. It is
an autoimmune disease. Intrinsic factor deficiency can occur due to
autoimmune destruction of IF; this will lead to malabsorption of B12. It is
characterized by low hemoglobin levels decreased number of erythrocytes
and neurological manifestations.
5. Atrophy of gastric epithelium can also occur in iron deficiency anemia
leading lack of IF.
6. Relative deficiency of B12 in pregnancy
7. Fish tapeworm consumes B12 and infection produces B12 deficiency.
Deficiency manifestations:
1. Vitamin B12 deficiency causes simultaneous folate deficiency due to the
folate trap. Therefore all the manifestations of folate deficiency are also seen
2. In vitamin B12 deficiency convertion of homocystine to methionine is
blocked, so that homocysteine is accumulated, leading to homocystinuria.
Homocysteine level in blood has a positive correlation with myocardial

266

3.
4.
5.

6.

7.

infarction. So, B12 and folic acid are protective against ischemic heart
disease.
B12 deficiency results in decreased folate coenzymes that leads to reduced
nucleotide and DNA svnthesis causing megaloblastic anemia.
Demyelination:
In vitamin B12 deficiency, due to the non-availability of active methionine
methylation of phosphatidyl ethanolamine to phosphatidyl choline is not
adequate. This leads to deficient formation of myelin sheaths of nerves,
resulting in demyelination and neurological lesions.
Subacute combined degeneration: Damage to nervous system is seen in B12
deficiency (but not in folate deficiency). There is demyelination affecting
cerebral cortex as well as dorsal column and pyramidal tract of spinal cord.
Since sensory and motor tracts are affected, it is named as combined
degeneration. Symmetrical paresthesia of extremities, alterations of tendon
and deep senses and reflexes, loss of position sense, unsteadiness in gait,
positive Romberg's sign (falling when eyes are closed) and positive
Babinski's sign (extensor plantar reflex) are seen.
Achlorhydria: Absence of acid in gastric juice due to atrophic gastritis, is

associated with vitamin B12 deficiency.


Assessment of deficiency:
1. Serum B12 estimation by radio-immuno-assay or by ELISA.
2. Schillings test:
3. The test is for assessing weather B12 absorption from the gut is normal. It is
done in two stages.
4. Stage I: Radioactive labelled (Cobalt-60) vitamin B12, one microgram is
given orally. Simultaneously an intramuscular injection of unlabeled vitamin
B12 is given to saturate tissues with normal vitamin B12 and radioactive
vitamin B12 will not bind to body tissues. Therefore, the entire absorbed
radioactivity will pass into the urine. The patients urine is then collected
over the next 24 hours to assess the absorption.
5. In patients with pernicious anemia or with deficiency due to impaired
absorption, less than 5% of the radioactivity is detected in urine.
6. Stage II: If an abnormality is found, the test is repeated, with radioactive
vitamin plus intrinsic factor given orally, and urine is collected for 24 hours.
In pernicious anemia, there is lack of intrinsic factor, so that the first test is
abnormal; but the second test is normal.
7. In B2 deficiency, methyl melonic acid is excreted in urine.
8. Megaloblasts appear in peripheral smear.
9. Homocystine is not converted to methionine and ther will be homocystinuria.
Requirement: 1-2 microgram/day; 2microgram/day in pregnancy.
Treatment of deficiency: injectable B12 along with folic acid is ideal; dose is 1001000 microgram of B12 IM.
Dietary sources: Liver is rich in B12; curd is good source due to its lactobacillus
content; vegetables do not supply B12 and for vegetarians intestinal bacteria are the
only source of B12 supply.
Answer to Q:
1. A patient has been diagnosed as having megaloblastic anemia.

267

2. Which vitamin deficiency can cause anemia?


Pernicious anemia arises when vitamin B12 deficiency impairs the metabolism of
folic acid, leading to functional folate deficiency that disturbs erythropoiesis,
causing immature precursors of erythrocytes to be released into the circulation
(megaloblastic anemia).
3. What metabolites in urine you will find in this patient?
1. Methylmalonyl CoA mutase, leucine aminomutase, and methionine synthase
are vitamin B12 dependent enzymes. Methylmalonyl CoA is formed as an
intermediate in the catabolism of valine and by the carboxylation of
propionyl CoA arising in the catabolism of isoleucine, cholesterol, and, rarely,
fatty acids with an odd number of carbon atoms or directly from propionate,
a major product of microbial fermentation in the rumen.
2. It undergoes a vitamin B12 dependent rearrangement to succinyl CoA,
catalyzed by methylmalonyl CoA mutase. The activity of this enzyme is
greatly reduced in vitamin B12 deficiency, leading to an accumulation of
methylmalonyl CoA and urinary excretion of methylmalonic acid.
3. In vitamin B12 deficiency, conversion of homocystine to methionine is
4.
5.

6.

4. What
a.

b.

blocked, so that homocysteine is accumulated, leading to homocystinuria.


B12 deficiency leads to folate deficiency which leads to excretion of flowing
metabolites:
Histidine load test or FIGLU excretion test: Histidine is normally metabolised
to formimino glutamic acid (FIGLU) from which formimino group is removed
by THFA. Therefore in folate deficiency, FIGLU is excreted in urine.
AICAR excretion: In the purine nucleotide synthesis, the last step is the
addition of C2 with the help of N10-formyl THFA. When this is blocked, the
precursor, amino imidazole carboxamide ribosyl-5-phosphate (AICAR)
accumulates and is excreted in urine.
is the step in one carbon metabolism that is affected in this case?
Tetrahydrofolate serves as an acceptor or donor of one carbon units (formyl,
methyl etc.) in a variety of reactions involving amino acid and nucleotide
metabolism.
Tetrahydrofolate is mostly trapped as Ns-methyl THF in which form it is
present in the circulation. Vitamin B12 is needed for the conversion of N 5methyl THF to THF, in a reaction wherein homocysteine is converted to
methionine. This step is essential for the liberation of free THF and for its
repeated use in one carbon metabolism. In B12 deficiency, conversion of Nsmethyl THF to THF is blocked.
is the step in propionic acid metabolism that is defective in this

5. What
case?
a. Propionyl-CoA is a coenzyme A derivative of propionic acid (odd chain fatty
acid). The b-oxidation of saturated fatty acids containing odd number of
carbon atoms leads to a 3 carbon fragment, Propionyl-CoA which is
converted to succinyl CoA as follows:
b. PropionylC oA is carboxylated in the presence of ATP, CO2 and vitamin biofin
to D-methylmalonyl CoA.

268

c. Methylmalonyl CoA racemase converts the methylmalonyl CoA to L-form.


This reaction (D -+ L) is essential for the entry of this compound into the
metabolic reactions of the body.
d. The next enzyme, methylmalonyl CoA mutase, is dependent on vitamin B12
(deoxyadenosyl cobalamin). lt catalyses the conversion of methylmalonyl
CoA (a branched compound) to succinyl CoA (a straight chain compound),
which can enter citric acid cycle.
e. In B12 deficiency, there is an accumulation of methylmalonic acid in body,
followed by its increased excretion in urine. This causes severe metabolic
acidosis, damages the central neryous system and retards the growth. lt is
often fatal in the early years of life, a condition called Methylmalonie
acidemia

FOLIC ACID
1. Name 3 groups carried by folic acid. Pon may 2007
2. The elevated excretionof formimino glutamate (FIGLU) occurs in
which condition? Give the reaction blocked. Pon Nov 2006
Chemistry: It contains 3 groups: pteridine group; para amino benzoic acid and
glutamic acid. Chemically it is pteroylglutamic acid.
Absorbtion: is mainly from jejunum; transported by beta globulins and taken by liver;
not stored in tissues.
Co-enzyme function:
1. Folic acid is first converted to 7,8 dihydro folic acid and again into 5,6,7,8
tetrahydro folic acid by NADPH dependant folate reductase.
2. THFA is a one carbon group carrier. THF serves as an acceptor or donor of one
carbon units in a variety of reactions involving amino acid and nucleotide
metabolism. Except methyl group others are carried by tetrahydrofolic acid.
They are contributed by amino acids. One carbon groups are:
i. Formyl group
ii. Formimino group
iii. Methenyl group

269
iv.
Hydroxymethyl group
v. Methylene group
vi. Methyl group
3. Many important compounds are synthesizedi n one carbon metabolism.
4. Purines which are incorporated into DNA and RNA.
5. Pyrimidine nucleotide-deoxythymidylic acid, involved in the synthesis of DNA.
6. Clycine, serine, ethanolamine and choline are produced.
7. N-Formylmethionine, the initiator of protein biosynthesis is formed.
8. B12 coenzyme accepts methyl group from methyl THFA to form methyl
cobalamin. In B12 deficiency this transfer cannot occur and hence THFA (Folic
acid) cannot be regenerated. So folic acid deficiency coexists with B12
deficiency a condition called folate trap.
9. Methyl group in N5-methyl THFA is used for synthesis of methionine which takes
part in transmethylation reactions. This reaction is necessary for synthesis of
Choline, epinephrine, creatine, etc.
Causes of folate deficiency:
1. Relative deficiency occurs in pregnancy
2. Defective absorption in malabsorption syndromes like sprue, celiac disease,
gluten induced enteropathy, jejunal resection, gastroileostomy etc.
3. Anticonvulsants like hydantoin, phenobarbitone etc interfere with
gastrointestinal enzymes and reduce the conversion of polyglutamate form of
folate into monoglutamate form which can be absorbed.
4. Relative deficiency occurs in hemolytic anemia as the demand is more.
5. Diet poor in folate.
6. Folate trap.
Deficiency manifestations:
1. Deficiency affects DNA synthesis as thymidylate synthase enzyme is inhibited.
2. Macrocytic anemia as DNA synthesis is affected but hemoglobin synthesis
continues; macrocytes and reticulocytes are seen in peripheral smear. Increased
haemolysis, leucopenia and thrombocytopenia are other manifestations.
3. Plasma homocysteine level is increased.
4. Neural tube defects in fetus occurs due to folate deficiency in pregnancy.
5. Folate deficiency may be a factor for bronchial and cervical carcinoma
Assessment:
1. Normal blood level is 20 nanogram/ml
2. In histidine load test FIGLU is excreted in urine.
The elevated excretion of formimino glutamate (FIGLU) occurs in which
condition? Give the reaction blocked. Pon Nov 2006
1. Ans: In the metabolism of the amino acid histidine there is folic acid dependent
step at the point where formimino-glutamic acid (Figlu) is converted to
glutamic acid. In folic acid deficient patients, this reaction cannot be carried out,
as a result, figlu accumulates in the blood and excreted in urine. Figlu
excretion in urine is an index of folic acid deficiency. When a loading dose of

270

histidine is given, the excretion of Figlu in urine is increased further (Histidine


loading test).
2. Peripheral smear shows magaloblasts
3. Urinary excretion of amino imidazole carboxamide ribosyl-5-phoaphate (AICAR)
RDA: 200 microgram/day 400 in pregnancy and 300 in lactation.
Therapeutic doses:
1. 1 mg/day oral. In microcytic anemia folic acid alone if given will precipitate
neurologi-cal manifestations of B12. So combined therapy is necessary.
2. Folic acid can partially reverse the hematologic abnormalities of B12 deficiency
and, therefore, can mask a cobalamin deficiency. Thus, therapy of megaloblastic
anemia is often initiated with folic acid and vitamin B12 until the cause of the
anemia can be determined.
Folate antagonists:
1. Competitive inhibition of enzyme that incorporates PABA into dihydropteroic acid
by sulphona mides is used for its antibacterial effects.
2. Pyrimethamine is antifolate agent used as an antimalarial drug.
3. Aminopterin and Amethopterin are are powerful inhibitors of folate reductase
and THFA generation. Thus these drugs decrease the DNA formation and cell
division. They are widely used as anticancer drugs, especially for leukemias and
choriocarcinomas. Folinic acid (citrovorum factor) is given to rescue the patient
from toxicity of methotrexate used in cancer therapy.
PANTOTHENIC ACID
Chemistry: it consists of two components, pantoic acid and b-alanine, held together by
peptide linkage. lt has important metabolic role as coenzyme A.
Coenzyme A formation: Pantothena is first phosphorylated to and then Cysteine is
added. Steps as follows:

271

Biochemical functions:
1. The functions of pantothenic acid are exerted through coenzyme A or CoA
which is a central molecule involved in all the metabolisms (carbohydrate,
lipid and protein).
2. When bound to acetyl unit, it is called acetyl CoA. With succinate, succinyl
CoA is formed. HMG CoA & Acyl CoA are other derivatives.
3. -SH group is the active site where acyl groups are carried. Therefore the coenzyme A is sometimes abbreviated as CoA-SH
4. Examples of enzymes involved the participation of coenzyme A:
Pyruvate dehydrogenase
Pyruvate + CoA------------------------------------Acetyl CoA
a-Ketoglutarate dehydrogenase
a-Ketoglutarate + CoA----------------------Succiny CoA
Thiokinase
Fatty acid +CoA-----------------------------------Acyl CoA
5. Examples of group transfer by CoA:
a. Acetyl coA + Choline > Acetyl Choline +CoA
b. Acetyl CoA + Oxalo acetate > Citrate + CoA
c. Succinyl CoA + Acetoacetate > Acetoacetyl CoA + Succinate
6. Role of Acetyl CoA in metabolic integration:
a. Carbohydrates, amino acid and fatty acids metabolism lead to Acetyl
CoA formation. This in turn enters :
i. TCA cycle to release energy,
ii. Fatty acid metabolism to form triacyl glycerol,
iii. Cholesterol synthesis and its further leading to formation of
Vit. D and steroids,
iv. Ketone pathways to release energy and
7. Detoxification reactions.
8. Pantothenic acid itself is component of fatty acid synthase enzyme complex
which is involved in fatty acid synthesis.
RDA: 5-10 mg/day
Sources: egg, liver, meat, yeast, milk etc.

272

Deficiency symptoms: usually no symptoms occur due to wide availability of vitamins


in natural sources; Gopalan's burning feet syndrome is reported to be one disease due
to its deficiency.
BIOTIN
Chemistry:
1. Biotin is a water-soluble vitamin that is generally classified as a B-complex
vitamin.
2. Biotin is required by all organisms but can be synthesized only by bacteria,
yeasts, molds, algae, and some plant species
3. Biotin a heterocyclic sulfur containing monocarboxylic acid. It consists of imidazole and thiophene ring fused together with valeric acid side chain. Biotin is
covalently bound to -amino group of lysine to form biocytin in the enzymes.
Biocytin nray be regarded as the coenzyme of biotin.
Coenzyme Functions:
1. Carbohydrate: Biotin serves as a carrier of CO2 in the following carboxylation
reactions:
2. Pyruvate carboxylase converts pyruvate to oxaloacetate in gluconeogenesis.
Biotin- enzyme complex reacts with CO2 in the presence of ATP to form
carboxybiotin enzyme complex. This transfers CO2 to pyruvate to form
oxaloacetate required for citric acid cycle.
3. Fatty acid synthesis: Acetyl-CoA carboxylase I and II catalyze the binding of
bicarbonate to acetyl- CoA to form malonyl-CoA. Malonyl-CoA is required for the
synthesis of fatty acids.
4. Propionyl-CoA carboxylase catalyzes essential steps in the metabolism of
certain amino acids, cholesterol, and odd chain fatty acids. Propionyl CoA is
converted to methyl malonyl CoA in the presence of biotin.
5. Methylcrotonyl-CoA carboxylase catalyzes an essential step in the
catabolism of leucine, an essen tial amino acid. In this reaction b-Propionyl CoA
is converted to b-methyl glutaconyl CoA .
6. Histone biotinylation: Biotinylation of histones plays a role in regulating DNA
replication and transcription as well as cellular proliferation and other cellular
responses.
7. Biotin-Independent Carboxylation Reactions
8. Carbamoyl phosphate synthetase, which is the stepping stone for urea and
pyrimidine synthesis.
9. Addition of CO2 to form C6 in purine ring.
10. Malic enzyme, converting pyruvate to malate.
Biotin Antagonists
1. Avidin, a protein present in egg white has great affinity to biotin. Hence intake of
raw(unboiled) egg may cause biotin deficiency. Avidin is heat labile, and boiling
of egg will neutralize the inhibitory activity. One molecule of avidin can combine
with four molecules of biotin.
2. It is curious that egg white contains avidin and egg yolk contains biotin.

273

3. Avidin-biotin system is commonly utilized for detection of pathogens in the ELISA


test
RDA: 100-300 mg/day in addition to contributions by intestinal bacteria.
Sources: liver, kidney, egg yolk, milk, tomatoes and grains.
Deficiency
1. Although overt biotin deficiency is very rare, the human requirement for dietary
biotin has been demonstrated in two different situations: prolonged intravenous
feeding (parenteral) without bio tin supplementation .
2. Evidence of deficiency:
3. High excretion of 3-hydroxyisovaleric acid that reflects decreased activity of the
biotin- dependent enzyme, methylcrotonyl-coa carboxylase;
4. Reduced urinary excretion of biotin;
5. Propionyl-coa carboxylase activity in peripheral blood lymphocytes
6. Signs and symptoms
7. Anemia, loss of appetite, nausea, dermatitis, glossiti etc.
8. Hair loss and a scaly red rash around the eyes, nose, mouth, and genital area.
9. Neurologic symptoms in adults have included depression, lethargy,
hallucination, and numb ness and tingling of the extremities.
PYRIDOXINE
1. What are the sources and biochemical functions of pyridoxine?
2. Why does the dietary requirement of pyridoxin increase with high
protein diet? Pon May 2010
3. Functions of pyridoxin with two examples. Pon May 2013
4. Describe the sources, daily requirement, metabolic functions and
deficiency manifestations of Pyridoxine. Pon May 2014
Chemistry:
1. Vit.B6 collectively represent three compounds namely pyridoxine, pyridoxal and
pyridoxamine . They are pyridine derivatives. Pyridoxine is an alcohol, pyridoxal
is an aldehyde and pyridoxamine is an amine.
2. Coenzyme is pyridoxal phosphate. It is synthesized from all three forms. It is
synthesized by pyridoxal kinase, utilizing ATP.
Coenzyme functions:
1. It acts in many reactions of amino acid metabolism.
2. Synthesis of certain specialized products such as serotonin, histamine & niacin.
3. There is evidence that requirement of Vitamin B6 is increased due to increased
dietary protein intake, as it is involved as coenzyme in many metabolic reactions
of amino acid metabolism. Pyridoxal phosphate participates in reactions like
transamination, decarboxylation, deamination, transsulfuration & condensation
reactions of aminoacids.
4. Transamination: Eg. transaminase converts aminoacids to keto acids with PPL
acting as coenzyme. The keto acids enter the citric acid cycle and get oxidized
to generate energy.
Alanine + Alpha keto glutarate Pyruvate + Glutamic acid (Enzyme Alanine
transaminase).

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5. Decarboxylation: examples:
a. Tryptophan is first converted to 5-hydroxy tryptophan and then
decarboxylated to serotonin in the presence of PLP.
b. Histidine is converted to histamine in the presence of PLP.
c. Glutamate is converted to Gama amino butyric acid by similar
decarboxylase reaction with PLP.
d. Catecholamines are synthesized from tyrosine by decarboxylase and PLP.
6. Sulfur Containing Amino Acids (Transulfuration): PLP plays an important role
in methionine and cysteine metabolism:
a. Homocysteine + Serine Cystathionine. (Enzyme Cystathionine
synthase)
b. Cystathionine Homoserine + Cysteine (Enzyme Cystathionase)
7. In heme synthesis: PLP is required for the condensation of succinyl CoA and
glycine to form delta amino levulinic acid (ALA). In B6 deficiency, anemia may
be seen.
8. In deamination reactions dehydratase enzyme converts serine to pyruvate in
the presence of PLP.
9. Pyridoxal phosphate is required for the synthesis of niacin from tryptophan
i.e: Coenzyme for kynureninase. This is instance in which one vitamin
synthesizes another vitamin.
10.Hydroxymethyltransferase converts serine to a-ketoglutarate with PLP.
11.Other important functions of PLP:
a. Synthesis of sphingolipid and myelin formation.
b. Absorption of amino acid from intestines
c. Formation of Coenzyme A
d. Boosting immune functions
e. Preventing urinary stone formation
f. Utilization of unsaturated fatty acids.
RDA:
2-2.2 mg/day 2.5 in pregnancy and lactation and elderly.
Sources:
Egg yolk, fish, milk, meat,; wheat, corn, cabbage, roots and tubers. Rich sources
are yeast, rice polishing, wheat germs, cereals, legumes (pulses), oil seeds, egg,
milk, meat, fish and green leafy vegetables.
Deficiency:
1. Severe deficiency of vitamin B6 is uncommon.
2. Neurological:
3. In the early 1950s, seizures were observed in infants as a result of severe
vitamin B6 deficiency caused by an error in the manufacture of infant formula.
Abnormal electroencephalogram (EEG) patterns have been noted in some
studies of vitamin B6 deficiency.
4. Convulsions in infants: have been attributed to pyridoxine deficiency. It is
related to lowered activity of Glutamic acid decarboxylase, for which pyridoxal P
is a coenzyme. As a result there occurs lowering of -amino butyric acid (GABA)
in the brain which causes convulsions.

275

5. Other neurologic symptoms noted in severe vitamin B6 deficiency include


irritability, depression, and confusion; additional symptoms include inflammation
of the tongue, sores or ulcers of the mouth, and ulcers of the skin at the corners
of the mouth.
6. Hypo chromic microcytic anemia.
7. Deficiency state is indicated by increased excretion of xanthurenic acid in urine
8. Drug induced deficiency occurs in Isoniazid, d-pencillamine therapy. B6
deficiency has been observed in humans during the treatment of tuberculosis
with high doses of tuberculostatic drug Isonicotinic acid hydrazide or Isoniazid
(INH).It is believed that isoniazid forms a hydrazone complex with pyridoxine,
resulting in incomplete activation of the vitamin. During penicillamine treatment,
the drug reacts with pyridoxal-P to form inactive thiazolidine derivative.
9. Mild vitamin B6 deficiency may be seen in women taking oral contraceptive pills.
10. Deficiency of B6 will also affect tryptophan metabolism. Since niacin is produced
from tryptophan, B6 deficiency in turn leads to niacin deficiency which is
manifested as pellagra.
11. Ethanol: It is converted to acetaldehyde, which inactivates PLP. Hence B6
deficiency neuritis is quite common in alcoholics.
Therapeutic uses of Vitamin B6:
1. Vit B6 is used in morning sickness, radiation sickness, muscular dystrophies,
treatment of hyperoxaluria, and recurring oxalate stones of kidney, and mild
forms of pyridoxine deficiency have been reported to occur sometimes in women
taking oral contraceptives containing oestradiol.
2. Dutch researchers and reported in neurology that a higher intake of vitamin B6
may decrease the risk of Parkinsons disease.
Toxicity:
Excess use of vitamin 86 Q.5 g/day) in the women of premenstruasl yndromei s
associated with sensory neuropathy. Some workers have suggestedth at vitamin
86 more than 200 mg/day may cause neurological damage.
THIAMINE (VITAMIN B1)
1. Metabolic role thiamine in the body. Pon May 2009
2. Measurement of whole blood or erythrocyte transketolase activity is used
as a biochemical marker to assess beriberi. What is the basis of this? Pon
Nov 2006
3. Which enzyme in RBC is measured in thiamine deficiency? Name the other
enzymes that require thiamine for their activity. Pon Dec 2002
4. Give the Co enzyme forms of thiamine. Pon May 2005
5. Write a note on beriberi. Pon Nov 2010
6. What are the metabolic roles of thiamine in the body? Pon May 2009
Synonyms: Antiberiberi factor, antineuritic vitamin, aneurin.
Coenzyme: thiamine pyrophosphate (TPP)
Chemistry:

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Thiamine contains a substituted pyrimidine ring connected to a substituted


thiazole ring by means of methylene bridge. It contains sulphur (sulphur
containing vitamin). The vitamin is then converted to its active co-enzyme form
by additionof two phosphate groups, with the help of ATP. It is catalyzed by
thiamine pyrophosphotransferase.
Biosynthesis:
Synthesised by plants, yeasts and bacteria. Not synthesised by human beings,
hence should be supplied in diet. Intestinal bacterial flora can synthesise the
vitamin.
Metabolic roles:
1. The main role of thiamine (TPP) is in carbohydrate metabolism. So, the
requirement of thiamine is increased along with higher intake of carbohydrates

2. Pyruvate dehydrogenase: The co-enzyme form is thiamine pyrophosphate

3.

(TPP). It is used in oxidative decarboxylation of alpha keto acids, e.g. pyruvate


dehydrogenase catalyzes the breakdown of pyruvate, to acetyl CoA and carbon
dioxide.
Alpha ketoglutarate dehydrogenase: Another reaction that requires TPP is
the oxidative decarboxylation of alpha ketoglutarate to succinyl CoA and CO2.

4. Transketolase: The second group of enzymes that use TPP as co-enzyme are
the transketolases, in the hexose monophosphate shunt pathway of glucose.

5. The branched chain a-keto acid dehydrogenase (decarboxylase) catalyses the


oxidative
decarboxylation of branched chain amino acids (valine, leucine and
isoleucine) to the respective keto acids. This enzyme also requires TPP.
6. B1 is also required in amino acid Tryptophan metabolism for the activity of the
enzyme Tryptophan pyrrolase.

277

7. TPP plays an important role in the transmission of nerve impulse. lt is believed


that TPP is required for acetylcholine synthesis and the ion translocation of
neural tissue.
Deficiency Manifestations of Thiamine:
1. In thiamine deficiency, the activity of dehydrogenase-catalyzed reactionsvis are
decreased, resulting in a decreased production of ATP leading to impaired
cellular function. This can result in three distinct syndromes:
2. A chronic peripheral neuritis, beriberi, which may or may not be associated with
heart failure and edema;
3. Acute pernicious (fulminating) beriberi (shoshin beriberi), in which heart failure
and metabolic abnormalities predominate, without peripheral neuritis;
4. Wernicke encephalopathy with Korsakoff psychosis, which is associated
especially with alcohol and narcotic abuse.
5. Beriberi: This is a severe thiamine-deficiency syndrome found in areas where
polished rice is the major component of the diet. It is characterised by the
following manifestations:
6. CV: These include palpitation, dyspnoea, cardiac hypertrophy and dilatation,
which may progress to congestive cardiac failure.
7. Neurological manifestations:
8. These are predominantly those of ascending, symmetrical, peripheral
polyneuritis.
9. Polyneuritis common in chronic alcoholics. Alcohol utilization needs large doses
of thiamine. Alcohol inhibits intestinal absorption of thiamine, leading to
thiamine deficiency. Polyneuritis may also be associated with pregnancy and old
age. Thiamine deficiency may cause impairment of conversion of pyruvate to
acetyl CoA. This results in increased plasma concentration of pyruvate and
lactate, leading to lactic acidosis.
10. Wernickes encephalopathy: This is seen primarily in association with chronic
alcoholism and is due to dietary insufficiency or impaired intestinal absorption of
the vitamin. It is characterized by ophthalmoplegia, nystagmus, cerebellar
ataxia.
11. Some alcoholics develop Wernicke- Korsakoff psychosis syndrom characterized
by apathy, loss of memory, ataxia, and a rhythmic to-and-fro motion of the
eyeballs (nystagmus).
12. The neurologic consequences of Wernicke's syndrome are treatable with
thiamine supplementation.
13. GI symptoms: Amongst these, anorexia is an early symptom. There may be
gastric atony, with diminished gastric motility and nausea; fever and vomiting
occur in advanced stages.
14. Dry beriberi: When it is not associated with oedema.
15. Wet beriberi: Oedema is associated. It is probably in part to congestive cardiac
failure and in part to protein malnutrition (Low plasma albumin).
16. Infantile beriberi: It occurs in infants born to mothers suffering from thiamine
deficiency. Signs of infantile beriberi include restlessness and sleeplessness
tachycardia, vomiting, convulsions, and, if not treated, death.
Biochemical Parameters

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1. In thiamine deficiency, blood thiamine is reduced, but pyruvate, alpha


ketoglutarate and lactate are increased.
2. Erythrocyte transketolase activity is reduced with accumulation of pentose
sugars; this is the earliest manifestation seen even before clinical disturbances.
Measurement of RBC transketolase activity is a reliable diagnostic test to assess
thiamine deficiency.
Thiamine antagonists:
Pyrithiamine and oxythiamine are the two important antimetabolites of
thiamine.
Recommended Daily Allowance of Thiamine:
It depends on calorie intake (0.5 mg/1000 calories). Requirement is 1-1.5
mg/day. Thiamine is useful in the treatment of beriberi, alcoholic polyneuritis,
neuritis of pregnancy and neuritis of old age.
Sources
Aleurone layer of cereals (food grains) is a rich source of thiamine. Therefore
whole wheat flour and unpolished hand-pound rice have better nutritive value
than completely polished refined foods. When the grains are polished, aleurone
layer is usually removed. Yeast is also a very good source. Thiamine is partially
destroyed by heat.
RIBOFLAVIN
Chemistry:
1. Riboflavin was the first B complex component to be isolated in a pure state. This
vitamin is synthesized by green plants and micro-organisms.
2. Riboflavin has a dimethyl isoalloxazine ring to which a ribitol is attached. Ribitol
is the alcohol of ribose sugar. Riboflavin is converted to its active co-enzyme
forms (FMN and FAD) with the help of ATP. Riboflavin is heat stable.
3. Riboflavin is stable to heat but sensitive to light. When exposed to ultra-violet
rays of sunlight, it is converted to lumiflavin which exhibits vellow fluorescence.
Coenzymes:
1. Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are the two
coenzyme forms of riboflavin. The ribitol (5 carbon) is linked to a phosphatein
FMN.
FAD:
1. FAD is formed from FMN by the transfer of an AMP moiety from ATP.
2. During the oxidation process, FAD accepts two hydrogen atoms from substrate.
In turn, FAD is reduced to FADH2.
FAD dependent reactions:
1. Carbohydrate metabolism
a. Pyruvate dehydrogenase complex: Pyruvate -- -Acety CoA
b. a-Ketoglutadrate dehydrogenase cosmplex: a-Ketoglutarate - -> Succinyl
CoA
c. Succinate dehydrogenase: Succinate-- -> Fumarate
2. Lipid metabollsm:
a. Acyl CoA dehydrogenase: AcylC oA- a, b-Unsaturated acyl CoA

279

3. Protein metabolisn
a. Glycine oxidase: Glycine Glyoxylate + NH3
b. D Amino acid oxidase: D-Amino acid a-Keto acid + NH3
4. Purine metabolism
a. Xanthine oxidase: Xanthine - Uric acid
5. FADH2 when oxidized in the electron transport chain will generate 1 1/2 ATP
molecules
FMN dependent reactions:
1. L-Amino acid oxidase: L-Amino acid- ------ a -Keto acid + NHo
2. In the respiratory chain, the NADH dehydrogenase contains FMN. The electrons
are transported in the following manner
NAD+ ----- FMN ----- CoQ
Riboflavin Deficiency
1. Causes:
Natural deficiency of riboflavin in man is uncommon, because riboflavin is
synthesized by the intestinal flora. Riboflavin deficiency usually
accompanies other deficiency diseases such as beriberi, pellagra and
kwashiorkor.
2. Manifestations: Symptoms are confined to skin and mucous membranes.
a. Glossitis
b. Magenta colored tongue
c. Cheilosis (Greek, cheilos = lip)
d. Angular stomatitis (inflammation at the corners of mouth).
e. Circumcorneal vascularization.
f. Proliferation of the bulbar conjunctival capillaries is the earliest sign of
riboflavin deficiency.
Dietary Sources of Riboflavin
1. Animal sources: liver, egg, whole milk and fish
2. Vegetable sources: dried yeast, whole cereals, legumes and green leafy
vegetables.
Daily Requirement
1. Riboflavin is concerned mainly in the metabolism of carbohydrates and
requirement is related to calorie intake. Adults on sedentary work require about
1.5 mg per day. During pregnancy, lactation and old age, additional 0.2 to 0.4
mg /day are required.
ONE-CARBON METABOLISM
1. One-carbon (1C) groups play a pivotal role in donating carbon atoms for synthesis of
different types of compounds.
2. One carbon groups are:
a. Formyl group
b. Formimino group
c. Methanyl group
d. Hydroxymethyl group
e. Mrthylene group
f. Methyl group

280

3. Except mehyl group others are carried by tetrahydrofolic acid. They are contributed
by by amino ac-ids
4. Co2 is also a one carbon unit as it participates in carboxylation but not accepted by
many as a one arbon unit.
Formation of one carbon units:
1. The formate released from glycine and tryptophan metabolism combines with
THF to form N10- formyl THF.
2. Histidine contributes formimino fragment to produce N5-formimino THF
3. When serine is converted into glycine N5-N10 methylene THF is formed.
4. Choline and betain contribute to the formation of N5-methyl THF. Choline and
betaine are donors of hydroxy methyl groups. As serine is converted to choline,
3 one-carbon units are used up. During the conversion of choline to glycine,
these methyl groups are recovered. Hence, this pathway is called the "salvage
pathway" for one-carbon units.
Utilization of one carbon moiety:
One carbon units are used to synthesise the following compounds:
1. C2 of purine
2. Formylation of Methionyl t RNA
3. C8 of purine
4. Glycine
5. Pyrimidine nucleotide
Role of methionine and B12:
1. Active methionine is a methyl donor; after release of methyl group it
becomes homocysteine. Reconversion of homocysteine to methionine using
N5-methyl THF is a reaction which is catalyzed by a B12-containing

methyltransferase
2. B12 coenzyme accepts methyl group from methyl THFA to form methyl
cobalamin. In B12 deficiency this transfer cannot occur and hence THFA
(Folic acid) cannot be regenerated. So folic acid deficiency coexists with B12
deficiency a condition called folate trap.
3. Most of the one carbon moities are metabolically interconvertible and
catalysed by an NADP-dependant hydroxymethyl dehydrogenases

281

VITAMIN K
1.
2.
3.
4.

What is the role of Vit.K in maintaining body haemostasis? Pon May 2009
What are the biological functions of Vit.K? Pon May 2012
State the function of Vit. K & E. Pon May 2009
Name the Vit.K dependant coagulation factors. What is the role of Vit.K on
their activities? Pon Nov 2007; May 2003
5. Symptoms of Vit.K deficiency are met with more due to liver dysfunction
than due to lack of the vitamin. Explain why this is so. Pon apr 2002
1. Vitamin K is a fat-soluble vitamin. The "K" is derived from the German word
"koagulation. There are two naturally occurring forms of vitamin K.

282

a. Plants synthesize phylloquinone, which is also known as vitamin K1.


b. Bacteria synthesize vitamin K2, designated as menaquinone.
c. K3 is a synthetic form.
2. Chemistry: they are napthoquinone derivatives with isoprenoid side chain.
3. Absorption: Vit K provided by diet or bacterial synthesis is absorbed in association
with chylomicrons with the help of bile salts. It is transported with LDL and stored in
liver.
4. Biochemical functions:
a. Vitamin K is necessary for coagulation. It brings about posttranslational
modification clotting factors 2,7,9 and 10 produced as zymogen in liver.
b. Vit. K acts as coenzyme for the carboxylation of glutamic acid by
enzyme carboxylase. Gamma carboxyglutamate is formed which is critical
to the calcium-binding function of these proteins. They are negatively
charged (COO-) and they combine with positively charged calcium ions
(Ca2+) to form a complex. The complex (eg. prothrombin - Ca) binds to the
phospholipids on the membrane surface of the platelets. This leads to the
increased conversion of prothrombin to thrombin.

5.
6.
7.

8.

b. The formation of y-carboxyglutamate is inhibited by dicumarol, an


anticoagulant and warfarin, a synthetic analogue and they can inhibit
vitamin K action.
c. The mineral-binding capacity of osteocalcin in bone also requires vitamin Kdependent gamma-carboxylation of glutamic acid residues.
d. Vit.K is also involved in ETC and oxidative phosphorylation.
RDA: adult: 70-140 microgram/day
Sources: Cabbage, cauliflower, tomatoes, spinach, alfa-alfa, green vegetables, egg
yolk, meat, liver, cheese and milk products.
Causes for Deficiency of Vitamin K
a. In normal adults dietary deficiency seldom occurs since the intestinal
bacterial synthesis is sufficient to meet the needs of the body. However
deficiency can occur in conditions of malabsorption of lipids. This can result
from obstructive jaundice, chronic pancreatitis, sprue, etc.
b. Prolonged antibiotic therapy and gastrointestinal infections with diarrhoea
will destroy the bacterial flora and can also lead to vitamin K deficiency.
Deficiency symptoms:
a. Overt vitamin K deficiency results in impaired blood clotting, usually
demonstrated by laboratory tests that measure clotting time.
b. Hemorrhagic disease of the newborn is due to vitamin K deficiency. The
newborns, especially the premature infants have relative vitamin K
deficiency. This is due to lack of hepatic stores, limited oral intake (breast
milk has very low levels, 15 mg/liter) and absence of intestinal bacterial
flora. It is often advised that pre-term infants be given prophylactic doses of
vitamin K (1 mg Menadione).
c. In children and adults, Vitamin K deficiency may be manifested as bruising
tendency, ecchymotic patches, mucous membrane hemorrhage, posttraumatic bleeding and internal bleeding.
d. Prolongation of prothrombin time and delayed clotting time are characteristic

283

of vitamin K deficiency. Measurement of prothrombin time (PT) is taken as an


index of liver function.
e. Warfarin and dicoumarol will competitively inhibit the gamma carboxylation
system due to structural similarity with vitamin K. Hence they are widely
used as anticoagulants for therapeutic purposes.
9. Daily Requirement of Vitamin K
a. Recommended daily allowance is 50-100 mg/day. This is usually available in
a normal diet.
10. Sources of Vitamin K:
a. Green leafy vegetables are good dietary sources. Even if the diet does not
contain the vitamin, intestinal bacterial synthesis will meet the daily
requirements, as long as absorption is normal.
11. Toxicity:
a. There is no known toxicity associated with high doses of the phylloquinone
(vitamin K1) or menaquinone (vitamin K2) forms of vitamin K. The same is
not true for synthetic menadione (vitamin K3) and its derivatives.
b. Menadione can interfere with the function of glutathione, one of the body's
natural anti-oxidants, resulting in oxidative damage to cell membranes.
Menadione given by injection has induced liver toxicity, jaundice, and
hemolytic anemia.
12. Antagonists: Heparin and bihydroxy-coumarin, salicylates and dicumarol are
antagonists vit.K.

NIACIN
1.
2.
3.
4.

Write in detail the sources, daily requirement, coenzyme formation and


functions and deficiency manifestations of Vitamin Niacin.
Manifestation of pellagra. Pon May 2009
Name the amino acid, which forms niacin. What is the neuro transmitter
synthesized from that amino acid? Pon Dec 2001
How many mg of tryptophan are required to form 1 mg of niacin? Pon May
2009

1. Niacin is a water-soluble vitamin, which is also known as nicotinic acid or vitamin


B3. Niacin is a pyridine derivative. Structurally, it is pyridine3 carboxylic acid.
2. It is not strictly a vitamin since it can be synthesized in the body from the essential
amino acid tryptophan.
3. The amide form of niacin is known as niacinamide or nicotinamide. Two compounds,
nicotinic acid and nicotinamide, have the biologic activity of niacin;
4. In addition to its synthesis from dietary niacin, NAD can be synthesized from the

284

dietary amino acid tryptophan via the kynurenine pathway. Tryptophan produces
quinolinate which then forms nicotinate mononucleotide and, ultimately, NAD+ and
NADP+. 60 mg of tryptophan would yield 1 mg of Niacin.
5. Co-enzyme Formation of Niacin:
a. Niacin is converted to its co-enzyme forms, viz. Nicotinamide adenine
dinucleotide (NAD+) and Nicotinamide adenine dinucleotide phosphate
(NADP+).
b. The niacin is attached to a ribose phosphate to form a mononucleotide. It is
then attached to AMP, to form the dinucleotide. The nitrogen atom of
niacinamide contains one positive charge. The structure is abbreviated as
NAD+. In the case of NADP+, one more phosphoric acid is attached to the
ribose of the AMP
Tryptaphan- Niacin

Nicotinic acid
PRPP > PPi

Nicotinate mononucleotide
ATP> PPi
Desamido NAD+
Glutamine>Glutamic acid
ATP> AMP+ PPi
NAD+
A
TPADP+pi
NADP+
Biochemical functions:
1. The coenzvmes NAD+ and NADP+ are involved in a variety of oxidationreduction reactions. They accept hydride ion (hydrogen atom and one
electron :H-) and undergo reduction in the pyridine ring.
2. A large number of enzymes (about 40) belonging to the class oxidoreductases
are dependent on NAD+ or NADP+.
3. NAD+ Dependent Enzymes:
1. Lactate dehydrogenase (lactate pyruvate)
2. Glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3phosphate 1, 3-bisphosphoglycerate)
3. Pyruvate dehydrogenase (pyruvate acetyl CoA)
4. Alpha ketoglutarate dehydrogenase (alpha ketoglutarate succinyl CoA
5. Beta hydroxy acyl CoA dehydrogenase (beta hydroxy acyl CoA beta
keto acyl CoA
6. Glutamate dehydrogenase (Glutamate alpha keto glutarate
4. NADPH dependant reactions:
1. Keto acyl ACP dehydrogenase (Beta keto acyl ACP beta hydroxy acyl
ACP)
2. Alpha, beta unsaturated acyl ACP acyl ACP
3. HMG CoA reductase (HMG CoA mevalonate
4. Met-hemoglobin hemoglobin
5. Folate reductase (Folate dihydrofolate tetrahydrofolate)

285

6. Phenyl alanine hydroxylase (Phenylalanine tyrosine)


5. NADH produced is oxidized in the electron transport chain to generate ATP. One
NADH molecule is oxidized in the respiratory chain to generate 3 molecules of
ATP
6. NADPH is also important for many biosynthetic reactions as it donates reducing
equivalents
RDA: The daily requirement of niacin for an adult is 15-20 mg and for children, around
10-15 mg.
Deficiency manifestations:
1. Causes of deficiency:
a. Tryptophan deficiency in diet; eg. Stable food containing sorghum and
maize where tryptophan is not metabolically available to body.
b. Pyridoxal deficiency leads to inability to convert tryptophan to niacin.
c. Hartnup disease where congenital defect of absorption of tryptophan
from intestine is present.
d. Carcinoid syndrome where tumor tissue utilizes large amount of
tryptophan for serotonin synthe sis.
e. Dietary sources: rich sources are dried yeast, rice polishing, liver, peanut,
whole cereals, legumes, meat and fish.
f. RDA: normally 20 mg/day; additional 5 mg for pregnancy and lactation.
g. Therapeutic uses: Pharmacologic doses of nicotinic acid, but not
nicotinamide, have been known to reduce serum cholesterol by reducing
acetyl CoA pool. Niacin is commonly prescribed with other lipid-lowering
medications.
2. Deficency symptoms:
a. Niacin deficiency is mostly seen among people whose staple diet is corn
or maize. Niacin present in maize is unavailable to the body as it is in
bound form. Further, tryptophan content is low in maize.
b. Pellagra:
i. Niacin deficiency leads to the disease called pellagra
characterized by 3 Ds: diarrhea, dermatitis and de mentia.
ii. Dermatitis consists of bright erythema in feet, ankles and face;
pigmentation around the neck is called Casals necklace. The
dermatitis is precipitated by exposure to sunlight.
iii. Symptoms related to the digestive system include a bright red
tongue, vomiting, and diarrhea.
iv. Dementia occurs in chronic cases and ataxia and spasticity could
also occur.
v. Neurologic symptoms also include headache, apathy, fatigue,
depression, disorientation, and memory loss. If untreated, pellagra
is ultimately fatal.
Therapeutic uses of niacin:
1. Niacin inhibits lipolysis in the adipose tissue and decreases the circulatory free
fatty acids.
2. Triacylglycerol synthesis in the decreased.
3. The serum levels of low-density lipoproteins (LDL), very low-density lipoproteins

286

(VLDL), triacylglycerol and cholesterol are lowered. Hence niacin is used in the
treatment of hyperlipoproteinemia type II b (elevation of LDL and VLDL).
Toxicity:
4. Glycogen and fat reserves of skeletal and cardiac muscle are depleted.
3. There is a tendency for the increased levels of glucose and uric acid in the
circulation.
ASCORBIC ACID
1. Write the sources, requirements, deficiency manifestations, biochemical
actions of Vit.C Pon apr 2000
2. Vit C plays a role in post translational modification- justfy. Pon Dec 2003
3. Explain the role of ascorbic acid in post translational modification. Pon
May 2007

Chemistry:
1. Ascorbic acid is a water soluble vitamin; destroyed by heat, alkali and on
storage. 70%is lost while cook-ing; inactivated if cooked in copper vessel.
2. Chemistry: It is a hexose and closely resemble monosaccharide; It has enolic
hydroxyl group ; it is acidic and a strong reducing agent; L-ascorbic acid and
dehydroascorbic acid are active forms; D-ascorbic acid is an inactive form.
3. Plants and animals can synthesis ascorbic acid from glucose via uronic acid
pathway. Man cannot synthesis due to absence of L-gluonolactone oxidase.
Metabolism:
1. It is rapidly absorbed through intestine and excreted by kidneys and not stored
in significant amounts.
2. On oxidation, dehydroascorbic acid is formed which is further oxidized to oxalic
acid.
3. It is partly excreted as ascorbic acid and partly as oxalic acid.
4. Blood levels are 0.4 to 1.5 mg/dl of plasma and 25 mg/dl of WBC.
5. High levels observed around healing tissues.
Biological role:
0. A reversible conversion from ascorbic to dehydro ascorbic acid makes it a redox
system.
1. It is essential for hydroxylation of proline and lysine for the subsequent
formation of collagen, osteoid, intracellular cement in blood capillaries.
2. Role in Post-translational Modifications: The hydroxylation of proline and
lysine residues of collagen is a post-translational modification taking place
intracellularly. Prolyl hydroxylase and lysyl hydroxylase are both dioxygenases
using molecular oxygen. The enzyme also contains ferrous iron at its active site
and requires a reducing agent like ascorbic acid to preserve the iron in the
reduced ferrous state. So, vitamin C deficiency leads to poor hydroxylation. It is
the major biochemical defect in scurvy.
3. It is essential for hydroxylation of tryptophan to 5-hydroxy tryptophan for the
formation of serotonin.
4. It helps in oxidation of parahydroxy phenyl pyruvate to homogentisic acid.

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5. It enhances iron absorption from the intestines by reducing ferric form to ferrous
form.
6. It helps in reconversion of met-hemoglobin to hemoglobin.
7. It helps maturation of RBC by involving in folate reductase that converts folic
acid to tetra folic acid.
8. Its presence in adrenal cortex indicates that it is necessary for the synthesis of
steroids.
9. It helps bile acid synthesis form cholesterol.
10. Promotes phagocyic activity of WBCs.
11. It has antioxidant property and may have a role in cancer prevention. Eg. Aniline
dye induced bladder cancer.
12. It may reduce the incidence of cataracts.
Deficiency manifestations:
1. Scurvy: spongy and sore gums, loose teeth, anemia, swollen joints, fragile blood
vessels, immune incompetence, delayed wound healing, hemorrhage, sluggish
function of adrenal cortex etc. most symptoms are due to impaired collagen
formation and reduced antioxidant properties.
2.
3.
4.
5.
6.

Barlow's disease is infantile form of scurvy


Petichiae and echymosis or hematoma due to fragile capillaries.
Hemorrhage in conjunctiva, retina; haematuria and malena.
Bones are fragile, break easily and bleeding into joints.
Anemia is microcytic hypo chromic. Anemia is due to:
a. Loss of blood by hemorrhage
b. Decreased iron absorption
c. Decreased tetrahydrofolic acid
d. Accumulation of met-hemoglobin.
Dietary sources: amla (700 mg/100 gm); guava 9 300 mg/100 gm) lemon and leafy
vegetables.
Requirement: 75 mg/day; pregnancy , lactation and ageing 100 mg/day
Therapeutic uses:
a. Adjuvant therapy in infections, TB; wound healing, burns etc.
b. Clinical dose is 500 mg per day
c. Mega doses: unsubstantiated view that mega doses may prevent
infection and ageing.
Toxicity of Vitamin C
a. Since it is a water soluble substance, excess vitamin C is excreted, and
not accumulated in the body. However, more than 2000 mg of vitamin C
daily for a long time can cause iron overload, because vitamin C helps in
absorption of iron.
VIT. B 12
1. What are the coenzyme forms of Vit.B.12? Give a reaction where each is
used? Pon May 2010
2. What are the abnormal metabolites found in the urine of patients with B12
deficiency? Explain how folate is trapped in B12 deficiency and what is its
significance? Pon Nov 2007

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3. A patient has been diagnosed as having megaloblastic anemia.


a. Which vitamin deficiency can cause anemia?
b. What metabolites in urine you will find in this patient?
c. What is the step in one carbon metabolism that is affected in this
case?
d. What is the step in propionic acid metabolism that is defective in
this case?
4. What are the abnormal metabolites found in urine of patients with B12
deficiency ? Explain how folate is trapped in B12 deficiency and what is its
consequences. Pon Nov 2007
5. Name the coenzymes of cyanocobalamin Pon Dec 2001
6. Write briefly on the absorption of vit. B12 Pon May 2004
Chemistry of B12:
1. Synonyms are cobalamin, extrinsic factor (EF) of Castle. Water-soluble, heat stable
and red in color. It is a unique vitamin, synthesized by only microorganisms and not
by animals and plants.
2. Structure:
a. It contains 4 pyrole rings and a cobalt atom and the structure is called corin
ring; Corin is linked with benzimidazole ring and now called cobalamin. The
corin ring is similar to the tetrapyrrole ring structure of heme
a. Cyanide is added to the R position to be called cyano cobalamin. It is a
laboratory compound.
b. When hydroxyl group is added to R position, hydroxy cobalamin is obtained
which is B12.
c. Inside the cells theses groups are removed and deoxy adenosyl cobalamin is
formed which is the storage form.
d. When methyl group replaces adenosyl group, methyl cobalamin is formed
which is the circulating form. Adenosyl cobalamin and methyl cobalamin are
the functional coenzymes.
Absorption of B12:
1. Dietary source of B12 is called extrinsic factor of Castle. Vitamin B12 is absorbed
from Ileum; for its absorption it requires:
a. Presence of HCl, and
b. Intrinsic factor (IF) of Castle, a constituent of normal gastric juice.
a. Cobalophilin: A binding protein secreted in the saliva.
2. Cobalophilin: Gastric acid and pepsin release the vitamin B12 from protein binding
in food and make it available to bind to cobalophilin, a binding protein secreted in
the saliva. In the duodenum, cobalophilin is hydrolyzed, releasing the vitamin for
binding to intrinsic factor.
3. Intrinc factor: The parietal cells of stomach secretes a special protein called
intrinsic factor of Castle (lF). lt is a glycoprotein (8-15% carbohydrate) with a
molecular weight around 50,000. Intrinsic factor is resistant to proteolytic digestive
enzymes. lF generally forms a dimer, binds strongly with 1 or 2 moles of vitamin
B12. This binding protects vitamin B12 against its uptake and use by bacteria.
4. Mechanism absorption: Vitamin B12 is absorbed from the distal third of the ileum

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5.
6.

7.
8.

via specific binding site (receptors) that binds the B12-IF complex. The removal of
B12 from intrinsic factor (IF) in presence of Ca++ ions and a releasing factor (RF)
secreted by duodenum take place and B12 enters the ileal mucosal cells for
absorption into the circulation.
It is also shown that a small amount, about 1 to 3% may be absorbed by simple
diffusion.
Transport: In the mucosal cells, B12 is converted to methyl cobalamin . lt is then
transported in the circulation in a bound form to proteins namely transcobalamins
(TC-I & TC-ll).
Storage: B12 is an exception that it is stored. Methylcobalamin which is in excess is
taken up by the liver, converted to deoxyadenosyl B12 and stored in this form.
Clinical aspects:
a. In pancreatic insufficiency, the cobalophilin-bound vitamin B12 may not be
split and the complex is excreted in faeces resulting to development of
vitamin B12 deficiency.
b. Atrophy of fundus of stomach and a lack of free HCl (achlorhydria) is usually
associated with pernicious anaemia, caused by B12 deficiency.

Functions:
1. Vit B12 is used in the synthesis of methionine from homocysteine. In this
reaction THF is released from N5-methyl THF. B12 coenzyme accepts methyl
group from methyl THFA to form methyl cobalamin. B12 deficiency leads to
accumulation of N5-methyl THF and this is called folate trap. B12 helps release
of THF from folate trap and B12 deficiency folic acid deficiency also manifests.

2. The metabolism of odd chain fatty acids,


valine, isoleucine, methionine and threonine leads to the production of methyl
malonyl CoA. Methyl melonyl CoA is isomerized to succinyl CoA in the presence
of B12 by methyl malonyl CoA mutase (containing AdoB12). In B12 deficiency,
methyl malonyl CoA is excreted in urine (methyl malonic aciduria).
Methyl malonyl CoA mutase +Ado B12
Fatty acids -------------Methylmelonyl CoA--------------------------Succinyl CoA
(odd no. of carbon atoms)
3. Folate trap:
a. In vitamin B12 deficiency, increased folate levels are observed in plasma.
The activity of the enzyme homocysteine methyltransferase is low in B12
deficiency. As a result, the only major pathway for the conversion of N5methyl THF to tetrahydrofolate is blocked and body THF pool is reduced.
Essentially almost the entire body folate becomes trapped as N5-methyl
THF. This is known as folate trap or methyl frap. ln this manner, B12
deficiency results in decreased folate coenzymes that leads to reduced
nucleotide and DNA synthesis. Although the tissue folate levels are
adequate or high, there is a functional folate deficiency due to the lack of

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THF pool. The outcome is the development of megaloblastic anemia.


Administration of the amino acid methionine has been shown to partially
correct the symptoms of B12 deficiencies. This is due to the fact that the
formation of N5-methyl THF is inhibited by S-adenosylmethionine. A
combined therapy of vitamin B12 and folic acid is generally employed to
treat the patients with megaloblastic anemia.
Causes of B12 deficiency:
1. Vitamin B12 is found only in foods of animal origin, and no plant sources. Strict
vegetarians (vegans) are at risk of developing B12 deficiency.
2. Only source is curd and milk which may not be available poor people.
3. Partial or total gastrectomy-these individuals become intrinsic factor deficient
4. Pernicious anemia: It is manifested usually in persons over 40 years. It is an
autoimmune disease. Intrinsic factor deficiency can occur due to autoimmune
destruction of IF; this will lead to malabsorption of B12. It is characterized by
low hemoglobin levels decreased number of erythrocytes and neurological
manifestations.
5. Atrophy of gastric epithelium can also occur in iron deficiency anemia leading
lack of IF.
6. Relative deficiency of B12 in pregnancy
7. Fish tapeworm consumes B12 and infection produces B12 deficiency.
Deficiency manifestations:
1. Vitamin B12 deficiency causes simultaneous folate deficiency due to the folate
trap. Therefore all the manifestations of folate deficiency are also seen
2. In vitamin B12 deficiency convertion of homocystine to methionine is blocked, so
that homocysteine is accumulated, leading to homocystinuria. Homocysteine
level in blood has a positive correlation with myocardial infarction. So, B12 and
folic acid are protective against ischemic heart disease.
3. B12 deficiency results in decreased folate coenzymes that leads to reduced
nucleotide and DNA svnthesis causing megaloblastic anemia.
4. Demyelination:
a. In vitamin B12 deficiency, due to the non-availability of active methionine
methylation of phosphatidyl ethanolamine to phosphatidyl choline is not
adequate. This leads to deficient formation of myelin sheaths of nerves,
resulting in demyelination and neurological lesions.
b. Subacute combined degeneration: Damage to nervous system is seen in
B12 deficiency (but not in folate deficiency). There is demyelination
affecting cerebral cortex as well as dorsal column and pyramidal tract of
spinal cord. Since sensory and motor tracts are affected, it is named as
combined degeneration. Symmetrical paresthesia of extremities,
alterations of tendon and deep senses and reflexes, loss of position
sense, unsteadiness in gait, positive Romberg's sign (falling when eyes
are closed) and positive Babinski's sign (extensor plantar reflex) are seen.
5. Achlorhydria: Absence of acid in gastric juice due to atrophic gastritis, is
associated with vitamin B12 deficiency.
Assessment of deficiency:
1. Serum B12 estimation by radio-immuno-assay or by ELISA.

291

2. Schillings test:
a. The test is for assessing weather B12 absorption from the gut is normal.
It is done in two stages.
b. Stage I: Radioactive labelled (Cobalt-60) vitamin B12, one microgram is
given orally. Simultaneously an intramuscular injection of unlabeled
vitamin B12 is given to saturate tissues with normal vitamin B12 and
radioactive vitamin B12 will not bind to body tissues. Therefore, the
entire absorbed radioactivity will pass into the urine. The patients urine
is then collected over the next 24 hours to assess the absorption.
c. In patients with pernicious anemia or with deficiency due to impaired
absorption, less than 5% of the radioactivity is detected in urine.
d. Stage II: If an abnormality is found, the test is repeated, with radioactive
vitamin plus intrinsic factor given orally, and urine is collected for 24
hours. In pernicious anemia, there is lack of intrinsic factor, so that the
first test is abnormal; but the second test is normal.
3. In B2 deficiency, methyl melonic acid is excreted in urine.
4. Megaloblasts appear in peripheral smear.
5. Homocystine is not converted to methionine and ther will be homocystinuria.
Requirement: 1-2 microgram/day; 2microgram/day in pregnancy.
Treatment of deficiency: injectable B12 along with folic acid is ideal; dose is 1001000 microgram of B12 IM.
Dietary sources: Liver is rich in B12; curd is good source due to its lactobacillus
content; vegetables do not supply B12 and for vegetarians intestinal bacteria are the
only source of B12 supply.
Answer to Q: A patient has been diagnosed as having megaloblastic anemia.
1. Which vitamin deficiency can cause anemia?
Pernicious anemia arises when vitamin B12 deficiency impairs the metabolism of
folic acid, leading to functional folate deficiency that disturbs erythropoiesis,
causing immature precursors of erythrocytes to be released into the circulation
(megaloblastic anemia).
2. What metabolites in urine you will find in this patient?
a. Methylmalonyl CoA mutase, leucine aminomutase, and methionine synthase
are vitamin B12 dependent enzymes. Methylmalonyl CoA is formed as an
intermediate in the catabolism of valine and by the carboxylation of
propionyl CoA arising in the catabolism of isoleucine, cholesterol, and, rarely,
fatty acids with an odd number of carbon atoms or directly from propionate,
a major product of microbial fermentation in the rumen.
b. It undergoes a vitamin B12 dependent rearrangement to succinyl CoA,
catalyzed by methylmalonyl CoA mutase. The activity of this enzyme is
greatly reduced in vitamin B12 deficiency, leading to an accumulation of
methylmalonyl CoA and urinary excretion of methylmalonic acid.
c. In vitamin B12 deficiency, conversion of homocystine to methionine is
blocked, so that homocysteine is accumulated, leading to homocystinuria.
d. B12 deficiency leads to folate deficiency which leads to excretion of flowing
metabolites:
i. Histidine load test or FIGLU excretion test: Histidine is normally

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metabolised to formimino glutamic acid (FIGLU) from which formimino


group is removed by THFA. Therefore in folate deficiency, FIGLU is
excreted in urine.
ii. AICAR excretion: In the purine nucleotide synthesis, the last step is the
addition of C2 with the help of N10-formyl THFA. When this is blocked,
the precursor, amino imidazole carboxamide ribosyl-5-phosphate
(AICAR) accumulates and is excreted in urine.
3. What is the step in one carbon metabolism that is affected in this case?
a. Tetrahydrofolate serves as an acceptor or donor of one carbon units (formyl,
methyl etc.) in a variety of reactions involving amino acid and nucleotide
metabolism.
b. Tetrahydrofolate is mostly trapped as Ns-methyl THF in which form it is
present in the circulation. Vitamin B12 is needed for the conversion of N 5methyl THF to THF, in a reaction wherein homocysteine is converted to
methionine. This step is essential for the liberation of free THF and for its
repeated use in one carbon metabolism. In B12 deficiency, conversion of Nsmethyl THF to THF is blocked.
4. What is the step in propionic acid metabolism that is defective in this
case?
Propionyl-CoA is a coenzyme A derivative of propionic acid (odd chain fatty acid).
The b-oxidation of saturated fatty acids containing odd number of carbon atoms
leads to a 3 carbon fragment, Propionyl-CoA which is converted tosuccinyl CoA as
follows:
a. PropionylC oA is carboxylatedi n the presence of ATP, CO2 and vitamin
biofin to D-methylmalonyl CoA.
b. Methylmalonyl CoA racemase converts the methylmalonyl CoA to L-form.
This reaction (D -+ L) is essential for the entry of this compound into the
metabolic reactions of the body.
c. The next enzyme, methylmalonyl CoA mutase, is dependent on vitamin
812 (deoxyadenosyl cobalamin). lt catalyses the conversion of
methylmalonyl CoA (a branched compound) to succinyl CoA (a straight
chain compound), which can enter citric acid cycle.
d. In B12 deficiency, there is an accumulation of methylmalonic acid in
body, followed by its increased excretion in urine. This causes severe
metabolic acidosis, damages the central neryous system and retards the
growth. lt is often fatal in the early years of life, a condition called
Methylmalonie acidemia

FOLIC ACID
1. Name 3 groups carried by folic acid. Pon may 2007
2. The elevated excretionof formimino glutamate (FIGLU) occurs in which
condition? Give the reaction blocked. Pon Nov 2006
Chemistry: It contains 3 groups: pteridine group; para amino benzoic acid and
glutamic acid. Chemically it is pteroylglutamic acid.

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Absorbtion: is mainly from jejunum; transported by beta globulins and taken by liver;
not stored in tissues.
Co-enzyme function:
1. Folic acid is first converted to 7,8 dihydro folic acid and again into 5,6,7,8
tetrahydro folic acid by NADPH dependant folate reductase.

2. THFA is a one carbon group carrier. THF serves as an acceptor or donor of one
carbon units in a variety of reactions involving amino acid and nucleotide
metabolism. Except methyl group others are carried by tetrahydrofolic acid.
They are contributed by amino acids. One carbon groups are:
a. Formyl group
b. Formimino group
c. Methenyl group
d. Hydroxymethyl group
e. Methylene group
f. Methyl group
3. Many important compounds are synthesizedi n one carbon metabolism.
1. Purines which are incorporated into DNA and RNA.
2. Pyrimidine nucleotide-deoxythymidylic acid, involved in the synthesis of
DNA.
3. Clycine, serine, ethanolamine and choline are produced.
4. N-Formylmethionine, the initiator of protein biosynthesis is formed.
4. B12 coenzyme accepts methyl group from methyl THFA to form methyl
cobalamin. In B12 deficiency this transfer cannot occur and hence THFA (Folic
acid) cannot be regenerated. So folic acid deficiency coexists with B12
deficiency a condition called folate trap.
5. Methyl group in N5-methyl THFA is used for synthesis of methionine which takes
part in transmethylation reactions. This reaction is necessary for synthesis of
Choline, epinephrine, creatine, etc.
Causes of folate deficiency:
1. Relative deficiency occurs in pregnancy
2. Defective absorption in malabsorption syndromes like sprue, celiac disease,

294
gluten induced enteropathy, jejunal resection, gastroileostomy etc.
3. Anticonvulsants like hydantoin, phenobarbitone etc interfere with
gastrointestinal enzymes and reduce the conversion of polyglutamate form of
folate into monoglutamate form which can be absorbed.
4. Relative deficiency occurs in hemolytic anemia as the demand is more.
5. Diet poor in folate.
6. Folate trap.
Deficiency manifestations:
1. Deficiency affects DNA synthesis as thymidylate synthase enzyme is inhibited.
2. Macrocytic anemia as DNA synthesis is affected but hemoglobin synthesis
continues; macrocytes and reticulocytes are seen in peripheral smear. Increased
haemolysis, leucopenia and thrombocytopenia are other manifestations.
3. Plasma homocysteine level is increased.
4. Neural tube defects in fetus occurs due to folate deficiency in pregnancy.
5. Folate deficiency may be a factor for bronchial and cervical carcinoma
Assessment:
1. Normal blood level is 20 nanogram/ml
2. In histidine load test FIGLU is excreted in urine.
a. The elevated excretionof formimino glutamate (FIGLU) occurs in
which condition? Give the reaction blocked. Pon Nov 2006
b. Ans: In the metabolism of the amino acid histidine there is folic acid
dependent step at the point where formimino-glutamic acid (Figlu) is
converted to glutamic acid. In folic acid deficient patients, this reaction
cannot be carried out, as a result, figlu accumulates in the blood and
excreted in urine. Figlu excretion in urine is an index of folic acid
deficiency. When a loading dose of histidine is given, the excretion of
Figlu in urine is increased further (Histidine loading test).

3. Peripheral smear shows magaloblasts


4. Urinary excretion of amino imidazole carboxamide ribosyl-5-phoaphate (AICAR)
RDA: 200 microgram/day 400 in pregnancy and 300 in lactation.
Therapeutic doses:
1. 1 mg/day oral. In microcytic anemia folic acid alone if given will precipitate
neurologi-cal manifestations of B12. So combined therapy is necessary.
2. Folic acid can partially reverse the hematologic abnormalities of B12 deficiency
and, therefore, can mask a cobalamin deficiency. Thus, therapy of megaloblastic
anemia is often initiated with folic acid and vitamin B12 until the cause of the
anemia can be determined.
Folate antagonists:
1. Competitive inhibition of enzyme that incorporates PABA into dihydropteroic acid
by sulphona mides is used for its antibacterial effects.

295

2. Pyrimethamine is antifolate agent used as an antimalarial drug.


3. Aminopterin and Amethopterin are are powerful inhibitors of folate reductase
and THFA generation. Thus these drugs decrease the DNA formation and cell
division. They are widely used as anticancer drugs, especially for leukemias and
choriocarcinomas. Folinic acid (citrovorum factor) is given to rescue the patient
from toxicity of methotrexate used in cancer therapy.
PANTOTHENIC ACID
Chemistry: it consists of two components, pantoic acid and b-alanine, held together by
peptide linkage. lt has important metabolic role as coenzyme A.
Coenzyme A formation: Pantothena is first phosphorylated to and then Cysteine is
added. Steps as follows:

Biochemical functions:
1. The functions of pantothenic acid are exerted through coenzyme A or CoA which
is a central molecule involved in all the metabolisms (carbohydrate, lipid and
protein).
2. When bound to acetyl unit, it is called acetyl CoA. With succinate, succinyl CoA is
formed. HMG CoA & Acyl CoA are other derivatives.
3. -SH group is the active site where acyl groups are carried. Therefore the coenzyme A is sometimes abbreviated as CoA-SH
4. Examples of enzymes involved the participation of coenzyme A:
Pyruvate dehydrogenase
1. Pyruvate + CoA------------------------------------Acetyl CoA
a-Ketoglutarate dehydrogenase

296

2. a-Ketoglutarate + CoA----------------------Succiny CoA


Thiokinase
3. Fatty acid +CoA-----------------------------------Acyl CoA
5. Examples of group transfer by CoA:
a. Acetyl coA + Choline > Acetyl Choline +CoA
b. Acetyl CoA + Oxalo acetate > Citrate + CoA
c. Succinyl CoA + Acetoacetate > Acetoacetyl CoA + Succinate
6. Role of Acetyl CoA in metabolic integration:
a. Carbohydrates, amino acid and fatty acids metabolism lead to Acetyl CoA
formation. This in turn enters :
i. TCA cycle to release energy,
ii. Fatty acid metabolism to form triacyl glycerol,
iii. Cholesterol synthesis and its further leading to formation of Vit. D
and steroids,
iv. Ketone pathways to release energy and
v. Detoxification reactions.
b. Pantothenic acid itself is component of fatty acid synthase enzyme
complex which is involved in fatty acid synthesis.
RDA: 5-10 mg/day
Sources: egg, liver, meat, yeast, milk etc.
Deficiency symptoms: usually no symptoms occur due to wide availability of vitamins
in natural sources; Gopalan's burning feet syndrome is reported to be one disease due
to its deficiency.
BIOTIN
Chemistry:
1. Biotin is a water-soluble vitamin that is generally classified as a B-complex vitamin.
2. Biotin is required by all organisms but can be synthesized only by bacteria, yeasts,
molds, algae, and some plant species
3. Biotin a heterocyclic sulfur containing monocarboxylic acid. It consists of imida-zole
and thiophene ring fused together with valeric acid side chain. Biotin is covalently
bound to -amino group of lysine to form biocytin in the enzymes. Biocytin nray be
regarded as the coenzyme of biotin.
Coenzyme Functions:
i. Carbohydrate: Biotin serves as a carrier of CO2 in the following carboxylation
reactions:
ii. Pyruvate carboxylase converts pyruvate to oxaloacetate in
gluconeogenesis. Biotin- enzyme complex reacts with CO2 in the presence of
ATP to form carboxybiotin enzyme complex. This transfers CO2 to pyruvate
to form oxaloacetate required for citric acid cycle.
iii. Fatty acid synthesis: Acetyl-CoA carboxylase I and II catalyze the binding
of bicarbonate to acetyl- CoA to form malonyl-CoA. Malonyl-CoA is required
for the synthesis of fatty acids.
iv. Propionyl-CoA carboxylase catalyzes essential steps in the metabolism of

297

certain amino acids, cholesterol, and odd chain fatty acids. Propionyl CoA is
converted to methyl malonyl CoA in the presence of biotin.
v. Methylcrotonyl-CoA carboxylase catalyzes an essential step in the
catabolism of leucine, an essen tial amino acid. In this reaction b-Propionyl
CoA is converted to b-methyl glutaconyl CoA .
vi. Histone biotinylation: Biotinylation of histones plays a role in regulating
DNA replication and transcription as well as cellular proliferation and other
cellular responses.
vii. Biotin-Independent Carboxylation Reactions
i. Carbamoyl phosphate synthetase, which is the stepping stone for
urea and pyrimidine synthesis.
ii. Addition of CO2 to form C6 in purine ring.
iii. Malic enzyme, converting pyruvate to malate.
Biotin Antagonists
1. Avidin, a protein present in egg white has great affinity to biotin. Hence intake of
raw(unboiled) egg may cause biotin deficiency. Avidin is heat labile, and boiling
of egg will neutralize the inhibitory activity. One molecule of avidin can combine
with four molecules of biotin.
2. It is curious that egg white contains avidin and egg yolk contains biotin.
3. Avidin-biotin system is commonly utilized for detection of pathogens in the ELISA
test
RDA: 100-300 mg/day in addition to contributions by intestinal bacteria.
Sources: liver, kidney, egg yolk, milk, tomatoes and grains.
Deficiency
1. Although overt biotin deficiency is very rare, the human requirement for dietary
biotin has been demonstrated in two different situations: prolonged intravenous
feeding (parenteral) without bio tin supplementation .
2. Evidence of deficiency:
a. High excretion of 3-hydroxyisovaleric acid that reflects decreased activity of
the biotin- dependent enzyme, methylcrotonyl-coa carboxylase;
b. Reduced urinary excretion of biotin;
c. Propionyl-coa carboxylase activity in peripheral blood lymphocytes
3. Signs and symptoms
a. Anemia, loss of appetite, nausea, dermatitis, glossiti etc.
b. Hair loss and a scaly red rash around the eyes, nose, mouth, and genital
area.
c. Neurologic symptoms in adults have included depression, lethargy,
hallucination, and numb ness and tingling of the extremities.
PYRIDOXINE
1. What are the sources and biochemical functions of pyridoxine?
2. Why does the dietary requirement of pyridoxin increase with high protein
diet? Pon May 2010
3. Functions of pyridoxin with two examples. Pon May 2013
4. Describe the sources, daily requirement, metabolic functions and
deficiency manifestations of Pyridoxine. Pon May 2014

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Chemistry:
1. Vit.B6 collectively represent three compounds namely pyridoxine, pyridoxal
and pyridoxamine . They are pyridine derivatives. Pyridoxine is an alcohol,
pyridoxal is an aldehyde and pyridoxamine is an amine.
2. Coenzyme is pyridoxal phosphate. It is synthesized from all three forms. It is
synthesized by pyridoxal kinase, utilizing ATP.
Coenzyme functions:
1. It acts in many reactions of amino acid metabolism.
a. Synthesis of certain specialized products such as serotonin, histamine &
niacin.
b. There is evidence that requirement of Vitamin B6 is increased due to
increased dietary protein intake, as it is involved as coenzyme in many
metabolic reactions of amino acid metabolism. Pyridoxal phosphate
participates in reactions like transamination, decarboxylation,
deamination, transsulfuration & condensation reactions of aminoacids.
2. Transamination: Eg. transaminase converts aminoacids to keto acids with
PPL acting as coenzyme. The keto acids enter the citric acid cycle and get
oxidized to generate energy.
Alanine + Alpha keto glutarate Pyruvate + Glutamic acid (Enzyme Alanine
transaminase).
3. Decarboxylation: examples:
a. Tryptophan is first converted to 5-hydroxy tryptophan and then
decarboxylated to serotonin in the presence of PLP.
b. Histidine is converted to histamine in the presence of PLP.
c. Glutamate is converted to Gama amino butyric acid by similar
decarboxylase reaction with PLP.
d. Catecholamines are synthesized from tyrosine by decarboxylase
and PLP.
4. Sulfur Containing Amino Acids (Transulfuration): PLP plays an important
role in methionine and cysteine metabolism:
a. Homocysteine + Serine Cystathionine. (Enzyme Cystathionine
synthase)
b. Cystathionine Homoserine + Cysteine (Enzyme Cystathionase)
5. In heme synthesis: PLP is required for the condensation of succinyl CoA
and glycine to form delta amino levulinic acid (ALA). In B6 deficiency, anemia
may be seen.
6. In deamination reactions dehydratase enzyme converts serine to pyruvate
in the presence of PLP.
7. Pyridoxal phosphate is required for the synthesis of niacin from
tryptophan i.e: Coenzyme for kynureninase. This is instance in which one
vitamin synthesizes another vitamin.
8. Hydroxymethyltransferase converts serine to a-ketoglutarate with PLP.
9. Other important functions of PLP:
i. Synthesis of sphingolipid and myelin formation.
ii. Absorption of amino acid from intestines
iii. Formation of Coenzyme A

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iv.
Boosting immune functions
v. Preventing urinary stone formation
vi. Utilization of unsaturated fatty acids.
RDA:
2-2.2 mg/day 2.5 in pregnancy and lactation and elderly.
Sources:
Egg yolk, fish, milk, meat,; wheat, corn, cabbage, roots and tubers. Rich sources
are yeast, rice polishing, wheat germs, cereals, legumes (pulses), oil seeds, egg,
milk, meat, fish and green leafy vegetables.
Deficiency:
1. Severe deficiency of vitamin B6 is uncommon.
2. Neurological:
a. In the early 1950s, seizures were observed in infants as a result of severe
vitamin B6 deficiency caused by an error in the manufacture of infant
formula. Abnormal electroencephalogram (EEG) patterns have been
noted in some studies of vitamin B6 deficiency.
b. Convulsions in infants: have been attributed to pyridoxine deficiency. It is
related to lowered activity of Glutamic acid decarboxylase, for which
pyridoxal P is a coenzyme. As a result there occurs lowering of -amino
butyric acid (GABA) in the brain which causes convulsions.
c. Other neurologic symptoms noted in severe vitamin B6 deficiency include
irritability, depression, and confusion; additional symptoms include
inflammation of the tongue, sores or ulcers of the mouth, and ulcers of
the skin at the corners of the mouth.
3. Hypo chromic microcytic anemia.
4. Deficiency state is indicated by increased excretion of xanthurenic acid in urine
5. Drug induced deficiency occurs in Isoniazid, d-pencillamine therapy. B6
deficiency has been observed in humans during the treatment of tuberculosis
with high doses of tuberculostatic drug Isonicotinic acid hydrazide or Isoniazid
(INH).It is believed that isoniazid forms a hydrazone complex with pyridoxine,
resulting in incomplete activation of the vitamin. During penicillamine treatment,
the drug reacts with pyridoxal-P to form inactive thiazolidine derivative.
6. Mild vitamin B6 deficiency may be seen in women taking oral contraceptive pills.
7. Deficiency of B6 will also affect tryptophan metabolism. Since niacin is produced
from tryptophan, B6 deficiency in turn leads to niacin deficiency which is
manifested as pellagra.
8. Ethanol: It is converted to acetaldehyde, which inactivates PLP. Hence B6
deficiency neuritis is quite common in alcoholics.
Therapeutic uses of Vitamin B6:
1. Vit B6 is used in morning sickness, radiation sickness, muscular dystrophies,
treatment of hyperoxaluria, and recurring oxalate stones of kidney, and mild
forms of pyridoxine deficiency have been reported to occur sometimes in women
taking oral contraceptives containing oestradiol.
2. Dutch researchers and reported in neurology that a higher intake of vitamin B6
may decrease the risk of Parkinsons disease.
Toxicity:
Excess use of vitamin 86 Q.5 g/day) in the women of premenstruasl yndromei s

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associated with sensory neuropathy. Some workers have suggestedth at vitamin


86 more than 200 mg/day may cause neurological damage.
THIAMINE (VITAMIN B1)
1. Metabolic role thiamine in the body. Pon May 2009
2. Measurement of whole blood or erythrocyte transketolase activity is
used as a biochemical marker to assess beriberi. What is the basis of
this? Pon Nov 2006
3. Which enzyme in RBC is measured in thiamine deficiency? Name the
other enzymes that require thiamine for their activity. Pon Dec 2002
4. Give the Co enzyme forms of thiamine. Pon May 2005
5. Write a note on beriberi. Pon Nov 2010
6. What are the metabolic roles of thiamine in the body? Pon May 2009
Synonyms: Antiberiberi factor, antineuritic vitamin, aneurin.
Coenzyme: thiamine pyrophosphate (TPP)
Chemistry:
Thiamine contains a substituted pyrimidine ring connected to a substituted
thiazole ring by means of methylene bridge. It contains sulphur (sulphur
containing vitamin). The vitamin is then converted to its active co-enzyme form
by additionof two phosphate groups, with the help of ATP. It is catalyzed by
thiamine pyrophosphotransferase.
Biosynthesis:
Synthesised by plants, yeasts and bacteria. Not synthesised by human beings,
hence should be supplied in diet. Intestinal bacterial flora can synthesise the
vitamin.
Metabolic roles:
2. The main role of thiamine (TPP) is in carbohydrate metabolism. So, the
requirement of thiamine is increased along with higher intake of carbohydrates
3. Pyruvate dehydrogenase: The co-enzyme form is thiamine pyrophosphate
(TPP). It is used in oxidative decarboxylation of alpha keto acids, e.g. pyruvate
dehydrogenase catalyzes the breakdown of pyruvate, to acetyl CoA and carbon

dioxide.
4. Alpha ketoglutarate dehydrogenase: Another reaction that requires TPP is
the oxidative decarboxylation of alpha ketoglutarate to succinyl CoA and CO2.

5. Transketolase: The second group of enzymes that use TPP as co-enzyme are
the transketolases, in the hexose monophosphate shunt pathway of glucose.

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6. The branched chain a-keto acid dehydrogenase (decarboxylase) catalyses the


oxidative
decarboxylation of branched chain amino acids (valine, leucine and
isoleucine) to the respective keto acids. This enzyme also requires TPP.
7. B1 is also required in amino acid Tryptophan metabolism for the activity of the
enzyme Tryptophan pyrrolase.
8. TPP plays an important role in the transmission of nerve impulse. lt is believed
that TPP is required for acetylcholine synthesis and the ion translocation of
neural tissue.
Deficiency Manifestations of Thiamine:
1. In thiamine deficiency, the activity of dehydrogenase-catalyzed reactionsvis are
decreased, resulting in a decreased production of ATP leading to impaired
cellular function. This can result in three distinct syndromes:
a. A chronic peripheral neuritis, beriberi, which may or may not be
associated with heart failure and edema;
b. Acute pernicious (fulminating) beriberi (shoshin beriberi), in which heart
failure and metabolic abnormalities predominate, without peripheral
neuritis;
c. Wernicke encephalopathy with Korsakoff psychosis, which is associated
especially with alcohol and narcotic abuse.
2. Beriberi: This is a severe thiamine-deficiency syndrome found in areas where
polished rice is the major component of the diet. It is characterised by the
following manifestations:
a. CV: These include palpitation, dyspnoea, cardiac hypertrophy and
dilatation, which may progress to congestive cardiac failure.
b. Neurological manifestations:
i. These are predominantly those of ascending, symmetrical,
peripheral polyneuritis.
ii. Polyneuritis common in chronic alcoholics. Alcohol utilization
needs large doses of thiamine. Alcohol inhibits intestinal
absorption of thiamine, leading to thiamine deficiency. Polyneuritis
may also be associated with pregnancy and old age. Thiamine
deficiency may cause impairment of conversion of pyruvate to
acetyl CoA. This results in increased plasma concentration of
pyruvate and lactate, leading to lactic acidosis.
iii. Wernickes encephalopathy: This is seen primarily in association
with chronic alcoholism and is due to dietary insufficiency or
impaired intestinal absorption of the vitamin. It is characterized by
ophthalmoplegia, nystagmus, cerebellar ataxia.
iv. Some alcoholics develop Wernicke- Korsakoff psychosis syndrom
characterized by apathy, loss of memory, ataxia, and a rhythmic
to-and-fro motion of the eyeballs (nystagmus).
v. The neurologic consequences of Wernicke's syndrome are

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treatable with thiamine supplementation.
c. GI symptoms: Amongst these, anorexia is an early symptom. There may
be gastric atony, with diminished gastric motility and nausea; fever and
vomiting occur in advanced stages.
d. Dry beriberi: When it is not associated with oedema.
e. Wet beriberi: Oedema is associated. It is probably in part to congestive
cardiac failure and in part to protein malnutrition (Low plasma albumin).
f. Infantile beriberi: It occurs in infants born to mothers suffering from
thiamine deficiency. Signs of infantile beriberi include restlessness and
sleeplessness tachycardia, vomiting, convulsions, and, if not treated,
death.
Biochemical Parameters
1. In thiamine deficiency, blood thiamine is reduced, but pyruvate, alpha
ketoglutarate and lactate are increased.
2. Erythrocyte transketolase activity is reduced with accumulation of pentose
sugars; this is the earliest manifestation seen even before clinical disturbances.
Measurement of RBC transketolase activity is a reliable diagnostic test to assess
thiamine deficiency.
Thiamine antagonists:
Pyrithiamine and oxythiamine are the two important antimetabolites of
thiamine.
Recommended Daily Allowance of Thiamine:
It depends on calorie intake (0.5 mg/1000 calories). Requirement is 1-1.5
mg/day. Thiamine is useful in the treatment of beriberi, alcoholic polyneuritis,
neuritis of pregnancy and neuritis of old age.
Sources
Aleurone layer of cereals (food grains) is a rich source of thiamine. Therefore
whole wheat flour and unpolished hand-pound rice have better nutritive value
than completely polished refined foods. When the grains are polished, aleurone
layer is usually removed. Yeast is also a very good source. Thiamine is partially
destroyed by heat.
RIBOFLAVIN
Chemistry:
1. Riboflavin was the first B complex component to be isolated in a pure state. This
vitamin is synthesized by green plants and micro-organisms.
2. Riboflavin has a dimethyl isoalloxazine ring to which a ribitol is attached. Ribitol
is the alcohol of ribose sugar. Riboflavin is converted to its active co-enzyme
forms (FMN and FAD) with the help of ATP. Riboflavin is heat stable.
3. Riboflavin is stable to heat but sensitive to light. When exposed to ultra-violet
rays of sunlight, it is converted to lumiflavin which exhibits vellow fluorescence.
Coenzymes:
1. Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are the two
coenzyme forms of riboflavin. The ribitol (5 carbon) is linked to a phosphatein
FMN.
2. FAD:

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a. FAD is formed from FMN by the transfer of an AMP moiety from ATP.
b. During the oxidation process, FAD accepts two hydrogen atoms from
substrate. In turn, FAD is reduced to FADH2.
c. FAD dependent reactions:
i. Carbohydrate metabolism
1. Pyruvate dehydrogenase complex: Pyruvate -- -Acety
CoA
2. a-Ketoglutadrate dehydrogenase cosmplex: aKetoglutarate - -> Succinyl CoA
3. Succinate dehydrogenase: Succinate-- -> Fumarate
ii. Lipid metabollsm:
1. Acyl CoA dehydrogenase: AcylC oA- a, b-Unsaturated
acyl CoA
iii. Protein metabolisn
1. Glycine oxidase: Glycine Glyoxylate + NH3
2. D Amino acid oxidase: D-Amino acid a-Keto acid + NH3
iv. Purine metabolism
1. Xanthine oxidase: Xanthine - Uric acid
v. FADH2 when oxidized in the electron transport chain will generate
1 1/2 ATP molecules
d. FMN dependent reactions:
i. L-Amino acid oxidase: L-Amino acid- ------ a -Keto acid + NHo
ii. In the respiratory chain, the NADH dehydrogenase contains FMN.
The electrons are transported in the following manner
NAD+ ----- FMN ----- CoQ
Riboflavin Deficiency
i. Causes: Natural deficiency of riboflavin in man is uncommon, because riboflavin is
synthesized by the intestinal flora. Riboflavin deficiency usually accompanies
other deficiency diseases such as beriberi, pellagra and kwashiorkor.
ii. Manifestations: Symptoms are confined to skin and mucous membranes.
i. Glossitis
ii. Magenta colored tongue
iii. Cheilosis (Greek, cheilos = lip)
iv. Angular stomatitis (inflammation at the corners of mouth).
v. Circumcorneal vascularization.
vi. Proliferation of the bulbar conjunctival capillaries is the earliest sign of
riboflavin deficiency.
Dietary Sources of Riboflavin
i. Animal sources: liver, egg, whole milk and fish
ii. Vegetable sources: dried yeast, whole cereals, legumes and green leafy
vegetables.
Daily Requirement
i. Riboflavin is concerned mainly in the metabolism of carbohydrates and
requirement is related to calorie intake. Adults on sedentary work require
about 1.5 mg per day. During pregnancy, lactation and old age, additional 0.2
to 0.4 mg /day are required.

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ONE-CARBON METABOLISM
1. One-carbon (1C) groups play a pivotal role in donating carbon atoms for synthesis of
different types of compounds.
2. One carbon groups are:
1. Formyl group
2. Formimino group
3. Methanyl group
4. Hydroxymethyl group
5. Mrthylene group
6. Methyl group
3. Except mehyl group others are carried by tetrahydrofolic acid. They are contributed
by by amino ac-ids
4. Co2 is also a one carbon unit as it participates in carboxylation but not accepted by
many as a one arbon unit.
5. Formation of one carbon units:
1. The formate released from glycine and tryptophan metabolism combines with
THF to form N10- formyl THF.
2. Histidine contributes formimino fragment to produce N5-formimino THF
3. When serine is converted into glycine N5-N10 methylene THF is formed.
4. Choline and betain contribute to the formation of N5-methyl THF. Choline and
betaine are donors of hydroxy methyl groups. As serine is converted to
choline, 3 one-carbon units are used up. During the conversion of choline to
glycine, these methyl groups are recovered. Hence, this pathway is called the
"salvage pathway" for one-carbon units.
6. Utilization of one carbon moiety: One carbon units are used to synthesise the
following compounds:
1. C2 of purine
2. Formylation of Methionyl t RNA
3. C8 of purine
4. Glycine
5. Pyrimidine nucleotide
7. Role of methionine and B12:
1. Active methionine is a methyl donor; after release of methyl group it becomes
homocysteine. Reconversion of homocysteine to methionine using N5-methyl
THF is a reaction which is catalyzed by a B12-containing methyltransferase
2. B12 coenzyme accepts methyl group from methyl THFA to form methyl
cobalamin. In B12 deficiency this transfer cannot occur and hence THFA (Folic
acid) cannot be regenerated. So folic acid deficiency coexists with B12
deficiency a condition called folate trap.
8. Most of the one carbon moities are metabolically interconvertible and catalysed by
an NADP-dependant hydroxymethyl dehydrogenases

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