• Vitamin B7 Biotin
• Vitamin B9 Folic acid
• Vitamin B12 Cyanocobalamin

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Dr. Ashish Agravatt

MBBS; M.D. Biochemistry

Biotin

• 1. Acetyl CoA carboxylase

This enzyme adds CO2 to acetyl CoA to form malonyl CoA. This is the rate limiting reaction in biosynthesis of fatty acids

Acetyl CoA +CO2+ATP→ Malonyl CoA + ADP+Pi

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• 2. Propionyl CoA carboxylase

Propionyl CoA +CO2+ATP→Methyl malonylCoA

+ADP+Pi

3. Pyruvate carboxylase

• Pyruvate + CO2 +ATP→Oxaloacetate +ADP +Pi
• This is important in two aspects. One, it provides the oxaloacetate, which is the catalyst for TCA cycle. Second, it is an important enzyme in the gluconeogenic pathway.

�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�

i. Avidin, a protein present in egg white has

great affinity to biotin. Hence intake of raw

(unboiled) egg may cause biotin deficiency.

Biotin was originally named as anti-egg- white injury-factor. Avidin is heat labile, and boiling of egg will neutralize the inhibitory activity. One molecule of avidin can combine with four molecules of biotin. It is curious that egg white contains avidin and egg yolk contains biotin.

• Requirement of Biotin

About 200-300 mg will meet the daily requirements.

• Sources of Biotin
• Normal bacterial flora of the gut will provide adequate quantities of biotin. Moreover, it is distributed ubiquitously in plant and animal tissues. Liver, yeast, peanut, soybean, milk and egg yolk are rich sources.

Biotin Deficiency:

• Biotin deficiency is very rare because it is widely found in most of the foods we eat
• However, deficiency can occur and cause dermatitis, alopecia, neurological abnormalities, lethargy, low blood sugar, high blood ammonia, acidosis and hallucinations.

Alopecia

Folates

The role of folate within the body

• The various form of folate function as a single-carbon donor-acceptors in a variety of biosynthetic reactions as shown below:
• Synthesis of methionine. By donation of methyle group from N-5-methyl-tetrahydrofolate and requires vitamin B₁₂ as a coenzyme.
• Pyrimidine synthesis which is a rate limiting step in DNA synthesis.
• Purine synthesis.
• Conversion of serine into glycin.
• Histidine catabolism. 

Chemistry of Folic Acid�

• The Latin word folium means leaf of vegetable.

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• Folic acid is abundant in vegetables. It is composed of three constituents. The pteridine group linked with para amino benzoic acid (PABA)) is called pteroic acid. It is then attached to glutamic acid to form pteroyl glutamic acid or folic acid

Co-enzyme Functions of Folic Acid�

• A. The folic acid is first reduced to 7,8-dihydrofolic acid and further reduced to 5,6,7,8-tetrahydrofolic acid (THFA)
• Both reactions are catalyzed by NADPH dependent folate reductase.

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• B. The THFA is the carrier of one-carbon groups.
• One carbon compound is an organic molecule

that contains only a single carbon atom. The

following groups are one carbon compounds:

• i. Formyl (-CHO) ii. Formimino (-CH=NH)

iii. Methenyl (-CH=) iv. Methylene (-CH2–)

v. Hydroxymethyl (-CH2OH) vi. Methyl (-CH3).

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• Methyl group in N5-methyl THFA is used for synthesis of active methionine, which takes

part in transmethylation reaction transmethylation reactions are required

for synthesis of choline, epinephrine,

creatine,etc.

Causes for Folate Deficiency�

• Folic acid deficiency is very common in India, and is perhaps the most commonly seen vitamin deficiency.

i. Pregnancy: Folate deficiency is commonly

seen in pregnancy, where requirement is increased.

ii. Defective absorption: In sprue, celiac

disease, gluten induced enteropathy, resection

of jejunum and short-circuiting of jejunum ingastroileostomy, absorption is defective.

• iii. Drugs: In the diet, folacins are mainly in

polyglutamate form. Gastrointestinal

enzymesin the gut remove the glutamate

residues and only the mono-glutamate form

of folic acid is absorbed. Anticonvulsant

drugs (hydantoin, dilantin, phenytoin,

phenobarbitone) will inhibit the intestinal

enzyme, so that folate absorption is

reduced.

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• iv. Hemolytic anemias: As requirement of folic acid becomes more, deficiency is manifested.
• v. Dietary deficiency: Absence of vegetables in food for prolonged periods may lead to deficiency.
• vi. Folate trap:

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Deficiency Manifestations�

• 1. Reduced DNA synthesis
• In folate deficiency, THFA is reduced and thymidylate synthase enzyme is inhibited. Hence dUMP is not converted to dTMP. So dTTP is not available for DNA synthesis. Thus cell division is arrested. Very rapidly dividing cells in bone marrow and intestinal mucosa are therefore most seriously

affected.

2. Macrocytic Anemia�

• i. It is the most characteristic feature of folate

deficiency.During erythropoiesis,DNA synthesis is delayed, but protein synthesis is continued. Thus hemoglobin accumulates in

RBC precursors. This asynchrony or dissociation between the maturity of nucleus and cytoplasm is manifested as immature looking nucleus and mature eosinophilic cytoplasm in the bone marrow cells.

• ii. Reticulocytosis is often seen. These abnormal RBCs are rapidly destroyed in spleen. This hemolysis leads to the reduction of lifespan of RBC. Reduced generation and increased destruction of RBCs result in anemia.
• iii. Leukopenia and thrombocytopenia are also seen

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3. Homocysteinemia�

• Folic acid deficiency may cause increased homocysteine levels in blood. Plasma homocysteine levels above 15 micromoles / L is known to increase the risk of coronary artery diseases. Providing adequate doses of pyridoxine, B12 and folic acid may lower the homocysteine levels.

4. Birth Defects�

• Folic acid deficiency during pregnancy may lead to homocysteinemia and neural tube defects in the fetus. Folic acid prevents birth defects (fetal malformations such as spina bifida). So, supplementation of folic acid from early pregnancy is a must to prevent neural tube defects in the child

What are Neural Tube Defects?

Anencephaly

Encephalocele

What are Neural Tube Defects?

Spina Bifida

Spina Bifida

• The damage that occurs may lead to muscle weakness, paralysis, and loss of bowel and bladder control.
• Hydrocephalus also occurs frequently in these babies.

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5. Cancer�

• Folic acid is beneficial in prevention of cancer.

Folate deficiency contributes to the etiology of bronchial carcinoma and cervical carcinoma.

What is the Recommended Daily Value?

• Adults (14-years and older) 400 mcg/d

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• Pregnancy (all ages) 600 mcg/d

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• Breastfeeding (all ages) 500 mcg/d

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• Previously affected pregnancy 4000 mcg/d

VITAMIN b 12 ( Cobalamin)

The Vitamin B₁₂ Family

• Cyanocobalamin – CN-B₁₂
• Hydroxycobalamin – OH-B₁₂
• 5’-Adenosylcobalamin- AS-B₁₂
• Methylcobalamin- CH₃-B₁₂

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Inactive Active

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Daily vitamin B₁₂ requirement

• The only source available to man is dietary.
• The main dietary source is liver, kidney, red meat, eggs, shellfish and dairy products.
• Normal mixed diet contains 5-30 μg /day.

• Vitamin B₁₂ is relatively stable and little is lost

during cooking.

•   Typical daily losses of vitamin B₁₂ are between 1-4 μg.
• The vitamin is lost mainly in urine and faeces.
• Since normally there is no consumption of vitamin B₁₂ within the body, the daily requirement matches daily losses.

Vitamin B₁₂

• Synthesized by bacteria and stored in animal body
• Commercially available as CN B₁₂, OH B₁₂, CH3 B₁₂
• Stored in the liver as the Transcobalamin I
• Absorbed only in the presence of the intrinsic factor (a glycoprotein released by parietal cells)
• Transported to tissues via transcobalamin II
• Transcobalamin I is the storage form
• Present in foods such as liver, fish, eggs, milk
• Absent in vegetables and fruits
• None in Vegan Vegetarian diet

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Vitamin B_12�Biochemical Reactions

• Coenzyme in DNA and Serotonin synthessis
• Synthesis of Purines, Pyrimidines, NA
• Synthesis of RBC and Proteins
• Maintains Myeline sheath of Nerve cells
• 3 Carbon Fatty Acid Metabolism
• Methylation Reactioms
• Homocysteine to Methionine
• Methyl melonyl CoA to Succinyl CoA
• Tetrahydrofolate to Methyl Tetrahydro Folate
• SAM-e (S-Adenosyl Methionine) –powerful mood elevator

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The role of vitamin B₁₂ within the body

Vitamin B₁₂ is required as coenzyme for two metabolic reaction: 

•      (1) Isomerization of L-methylmalonyl CoA to succinyl CoA. This is important substrate in Hb synthesis.

•      (2) Methylation of homocystine to methionine. This step is important in intracellular synthesis of folate coenzyme. 

COBALAMIN REACTIONS

N�H

N�H

CH₃

THF

Homocysteine

Methionine

Methyl����Cobalamin

Methylmalonyl �CoA

Succinyl CoA

Adenosyl�Cobalamin

Causes of B₁₂ deficiency

• Pernicious anemia (autoimmune gastritis against parietal cells - loss of intrinsic factor)
• Rarely due dietary deficiency
• Drugs : OCP, Trimethoprim, Methotrexate,

Phenytoin, Theophyllin

• Intestinal parasites - D.latum Gastrectomy, Chronic gastritis, PPI, H₂ Blocker
• Old age, Poor dietary Intake, Hypochlorhydria
• Malabsorption syndromes

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Deficiency Manifestations�

• i. Folate trap: Vitamin B12 deficiency causes

simultaneous folate deficiency due to the folate trap. Therefore all the manifestations of folate deficiency are also seen

• ii. Megaloblastic anemia: In the peripheral

blood, megaloblasts and immature RBCs are

observed

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• Abnormal homocysteine level: In vitamin

B12 deficiency, step No. 2 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

• Demyelination: In vitamin B12 deficiency,

step 3 is also suppressed due to the non-availability of active methionine.

• Therefore methylation of phosphatidyl ethanolamine to phosphatidyl choline is not adequate. This leads to deficient formation of myelin sheaths of nerves, demyelination and neurological lesions.

• Subacute combined degeneration: Damage to nervous system is seen in B12 deficiency (but not in folate deficiency). There Idemyelination 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

is associated with vitamin B12 deficiency

Diagnosis of B₁₂ deficiency

• Homocysteine levels (N < 13 μmols/ l)
• Methyl Malonic Acid (MMA) levels
• Serum B₁₂ levels (N = 200 - 600 pg/ml)
• IF Antibodies
• Schilling test

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The day we attempt learning new things, we start realizing how inadequate our knowledge is !

THAN Q