10. Vitamins

Question 1

Difference between Fat Soluble Vitamins and Water Soluble Vitamins

Answer 1

Fat soluble vitamins Water soluble vitamins
Solubility in fat Soluble Not soluble
Water solubility Not soluble Soluble
Carrier proteins Present *No carrier proteins
Storage Stored in liver *No storage
Excretion Not excreted Excreted
Toxicity Hypervitaminosis may result Unlikely, since excess is excreted.
Major vitamins. A,D,E and K B and C
Deficiency Manifests only when stores are *Manifests rapidly as there
depleted. is no storage
Treatment of Single large doses may Regular dietary supply is
deficiency prevent deficiency required
Absorption along with lipids, requiring bile salts. Absorption simple

Question 2

Explain Hemorrhagic disease of newborns?

Answer 2

Hemorrhagic disease of the newborn is attributed 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.
ii. It is often advised that pre-term infants be given prophylactic doses of vitamin K (1 mg Menadione).

Question 3

Vitamin K – dependant clotting factors and importance?

Answer 3

Vitamin K is necessary for coagulation. Factors dependent on vitamin K are Factor II (pro- thrombin); Factor VII (SPCA); Factor IX (Christmas factor); Factor X (Stuart Prower factor)
b. All these factors are synthesized by the liver as inactive zymogens. They undergo post-trans- lational modification; gamma carboxylation of glutamic acid residues. These are the binding sites for calcium ions. The gamma carboxy glutamic acid (GCG) synthesis requires vitamin K as a co-factor

Question 4

Explain Functions of Vitamin E?

Answer 4

Vitamin E is the most powerful natural anti- oxidant (Chapter 20). Free radicals are continuously being generated in living systems. Their prompt inactivation is of great importance. Vitamin E is a known biological antioxidant able to quench the lipid pero- xidation chain and to protect the plasma membranes from the attack of free radicals.

ii. The free radicals would attack bio-membranes. Vitamin E protects RBC from hemolysis. By preventing the peroxidation, it keeps the structural and functional integrity of all cells.
iii. Gradual deterioration of ageing process is due to the cumulative effects of free radicals. Vitamin E also boosts immune response.
iv. It reduces the risk of atherosclerosis by reducing oxidation of LDL (Fig. 33.13; see Chapter 25 for Atherosclerosis).
v. Vitamin E can depress leukocyte oxidative bactericidal activity.

Question 5

Explain Vitamin E sparing action of Se

Answer 5

Selenium is present in glutathione peroxidase; an important enzyme that oxidizes and destroys the free radicals (Chapter 20). Selenium has been found to decrease the requirement of vitamin E and vice versa. They act synergistically to minimize lipid peroxidation.

Question 6

Explain Different Types of Rickets?

Answer 6

i. The classical vitamin D deficiency rickets can be cured by giving vitamin D in the diet.
ii. The hypophosphatemic rickets mainly result from defective renal tubular reabsorption of phosphate. Supplementation of vitamin D along with phosphate is found to be useful.
iii. 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.
iv. Renal rickets: In kidney diseases, even if vitamin D is available, calcitriol is not synthesized. These cases will respond to administration of calcitriol.
v. 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).

Question 7

Explain biochemical effects/Actions of Viatmin D

Answer 7

The sites of action are:
a. intestinal villi cells
b. bone osteoblasts
c. kidney distal tubular cells.
A. Vitamin D and Absorption of Calcium
Calcitriol promotes the absorption of calcium and phosphorus from the intestine. In the brush-border surface, calcium is absorbed passively. From the intestinal cell to blood, absorption of calcium needs energy. It is either by the sodium-calcium exchange mechanism or by pumping out the calcium-calbindin complex. Calcitriol acts like a steroid hormone. It enters the target cell and binds to a cytoplasmic receptor. The hormone-receptor complex interacts with DNA and causes derepression and conse- quent transcription of specific genes that code for Calbindin (Fig. 33.10). Due to the increased availability of calcium binding protein, the absorption of calcium is increased.
B. Effect of Vitamin D in Bone
Mineralization of the bone is increased by increasing the activity of osteoblasts (Chapter 35). Calcitriol coordinates the remodelling action of osteoclasts and osteoblasts. It produces the differentiation of osteoclast precursors from multinucleated cells of osteoblast lineage. 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.
C. 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)

Question 8

Absorption and storage of vitamin A

Answer 8

i. Beta carotene is cleaved by a di-oxygenase, to form retinal. The retinal is reduced to retinol by an NADH or NADPH dependent retinal reductase present in the intestinal mucosa. Intestine is the major site of absorption (Fig. 33.3).
ii. The absorption is along with other fats and requires bile salts. In biliary tract obstruction and steatorrhoea, vitamin A absorption is reduced.
iii. Within the mucosal cell, the retinol is re- esterified with fatty acids, incorporated into chylomicrons and transported to liver. In the liver stellate cells, vitamin is stored as retinol palmitate.

Question 9

Explain Biochemical functions of Vitamin A (retin –ol, -al, -oic acid)

Answer 9

i. Retinoic acid has a role in the regulation of gene expression and differentiation of tissues. All-trans-retinoic acid and 9-cis- retinoic acid act like steroid hormones. They bind to nuclear receptors; retinoic acid along with the receptor binds to the response elements of DNA. Retinoic acid receptors (RAR) bind all-trans-retinoic acid, while retinoic x receptors (RXR) bind to 9-cis- retinoic acid. RXRs form dimers with vitamin D-receptor also. This explains why deficiency of vitamin A impairs vitamin D function; when there is lack of 9-cis-retinoic acid to form receptor dimers, vitamin D function is not
optimal.
ii. 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. In vitamin deficiency, miscarriages are noticed in female rats while atrophy of germinal epithelium and sterility are seen in male rats.
iii. Anti-oxidant property: There is a correlation between the occurrence of epithelial cancers and vitamin A deficiency. The anticancer activity has been attributed to the natural anti- oxidant property of carotenoids. Fresh vegetables containing carotenoids were shown to reduce the incidence of cancer.
iv. Beta carotenes may be useful in preventing heart attacks. Those who were given beta carotene supplements suffered half as many heart attacks as in the group taking placebo.
v. Vitamin A is necessary for the maintenance of normal epithelium and skin

Question 10

Explain Therapeutic uses of vitamin A

Answer 10

When deficiency of vitamin A is identified, supplementation is given as capsules or injection. Therapeutic dose is generally
20-50 times higher than the RDA. All-trans-retinoic acid is used as adjuvant in the treatment of promyelocytic leukemia. It causes remission due to its effect on differentiation of cells.

Question 11

Synthesis and activation of Vitamin D-explain?

Answer 11

Vitamin D is derived either from 7-dehydro- cholesterol or ergosterol by the action of ultraviolet radiations. 7-dehydrocholesterol, an intermediate of a minor pathway of cholesterol synthesis, is available in the Malpighian layer of epidermis. In the skin, ultraviolet light (290-315 nm) breaks the bond between position 9 and 10 of the steroid ring. So, the ring B is opened, to form the provitamin, secosterol (Fig. 33.8). The cis double bond between 5th and 6th carbon atoms, is then isomerized to a trans double bond (rotation on the 6th carbon atom) to give rise to vitamin D3 or cholecalciferol (Fig. 33.8). So, vitamin D is called the “sun-shine vitamin”. Excessive exposure to sunlight does not result in vitamin D toxicity since excess previtamin D3 and D3 are destroyed by sunlight itself.
Commercially the vitamin is derived from the fungus, ergot. The ergosterol when treated with ultraviolet light, ergocalciferol or vitamin D2 is produced.

Activation of Vitamin D
i. Vitamin D is a prohormone. The cholecal- ciferol is first transported to liver, where hydroxylation at 25th position occurs, to form 25-hydroxy cholecalciferol (25-HCC).
ii. In plasma, 25-HCC is bound to “vitamin D binding protein” (VDBP), an alpha-2 globulin.
iii. In the kidney, it is further hydroxylated at the
1st position.
Thus 1,25-dihydroxy cholecalciferol (DHCC) is generated. Since it contains three hydroxyl groups at 1, 3 and 25 positions, it is also called Calcitriol (Fig. 33.9). The calcitriol thus formed is the active form of vitamin; it is a hormone

Question 12

Which are Thiamine dependant Enzymes?

Answer 12

i. 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.
ii. Alpha ketoglutarate dehydrogenase: An analogous biochemical reaction that requires TPP is the oxidative decarboxylation of alpha ketoglutarate to succinyl CoA and CO2 (See citric acid cycle, Fig.18.2).
iii. Transketolase: The second group of enzymes that use TPP as co-enzyme are the trans- ketolases, in the hexose monophosphate shunt pathway of glucose (Fig. 10.3).
iv. The main role of thiamine (TPP) is in carbo- hydrate metabolism. So, the requirement of thiamine is increased along with higher intake of carbohydrates.

Question 13

Explain Types of beri beri and lab diagnosis?

Answer 13

Beriberi: Deficiency of thiamine leads to beriberi. It is a Singhalese word, meaning “weakness”. The early symptoms are anorexia, dyspepsia, heaviness and weakness. Subjects feel weak and get easily exhausted.
B. Wet beriberi: Here cardiovascular manifes- tations are prominent. Edema of legs, face, trunk and serous cavities are the main features. Palpitation, breathlessness and distended neck veins are observed. Death occurs due to heart failure.
C. Dry beriberi: In this condition, CNS manifes- tations are the major features. Walking becomes difficult. Peripheral neuritis with sensory disturbance leads to complete paralysis.
D. Infantile beriberi: It occurs in infants born to mothers suffering from thiamine deficiency. Restlessness and sleeplessness are observed.
E. Wernicke-Korsakoff syndrome: It is also called as cerebral beriberi. Carl Wernicke in 1894 and Sergiei Sergievich Korsakoff in 1887 described the condition. Clinical features are those of encephalopathy (ophthalmoplegia, nystagmus, cerebellar ataxia) plus psychosis. It is seen only when the nutritional status is severely affected.

Biochemical Parameters
In thiamine deficiency, blood thiamine is reduced, but pyruvate, alpha ketoglutarate and lactate are increased. Erythrocyte transketolase activity is reduced; this is the earliest manifestation seen even before clinical disturbances.
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.

Question 14

Which are FAD-dependent Enzymes?

Answer 14

1. Succinate to fumarate by succinate dehydro- genase (Fig. 18.2, step 6).
2. Acyl CoA to alpha-beta unsaturated acyl CoA by acyl CoA dehydrogenase (Fig. 11.9, step 1)
3. Xanthine to uric acid by xanthine oxidase (see Fig. 39.15).
4. Pyruvate to acetyl CoA by pyruvate dehydrogenase (Fig. 9.22).
5. Alpha ketoglutarate to succinyl CoA by alpha ketoglutarate dehydrogenase (Fig.18.2, step 4)

Question 15

Explain Niacin deficiency?

Answer 15

Deficiency of niacin leads to the clinical condition called pellagra. The symptoms of pellagra are:

i. Dermatitis: In early stages, bright red erythema occurs, especially in the feet, ankles and face (Fig. 34.8A). Increased pigmentation around the neck is known as Casal’s necklace. The dermatitis is precipitated by exposure to sunlight.
ii. Diarrhea:Thediarrheamaybemildorsevere with blood and mucus. This may lead to weight loss. Nausea and vomiting may also be present.
iii. Dementia: It is frequently seen in chronic cases. Delerium is common in acute pellagra. Irritability, inability to concentrate and poor memory are more common in mild cases. Ataxia and spasticity are also seen.

Question 16

Which are NAD+ dependant enzymes?

Answer 16

1. Lactate dehydrogenase (lactate → pyruvate) (Fig. 9.14)
2. Glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate →
   1, 3-bisphosphoglycerate) (Fig. 9.10)
3. Pyruvate dehydrogenase (pyruvate → acetyl CoA) (Fig. 9.22)
4. Alpha ketoglutarate dehydrogenase (alpha keto- glutarate → succinyl CoA (Fig.18.2)
5. Beta hydroxy acyl CoA dehydrogenase (beta hydroxy acyl CoA → beta keto acyl CoA (Step 3, Fig.11.9)
6. Glutamate dehydrogenase (Glutamate → alpha keto glutarate (Fig.14.9)

Question 17

NADPH Generating Reactions?

Answer 17

1. Glucose-6-phosphate dehydrogenase in the hexose monophosphate shunt pathway (Glucose-6- phosphate → 6-phosphogluconolactone) (Fig. 10.1)
2. 6-phosphogluconate dehydrogenase in the shunt pathway (6-phosphogluconate → 3- keto-6-phos- phogluconate) (Fig. 10.1).
3. Cytoplasmic isocitrate dehydrogenase
4. Malic enzyme (malate to pyruvate).

Question 18

Causes of Niacin deficiency?

Answer 18

i.Dietary deficiency of Tryptophan

ii. Deficient synthesis: Kynureninase, an
important enzyme in the pathway of try- ptophan, is pyridoxal phosphate dependent. So conversion of tryptophan to niacin is not possible in pyridoxal deficiency.
iii. Isoniazid(INH):Itisananti-tuberculousdrug, which inhibits pyridoxal phosphate formation. Hence there is block in conversion of trypto-
+ phan to NAD .
iv. Hartnup disease: Tryptophan absorption from intestine is defective in this congenital disease. Moreover, tryptophan is excreted in urine in large quantities. This leads to lack of tryptophan and consequently deficiency of nicotinamide.
v. Carcinoidsyndrome:Thetumorutilizesmajor portion of available tryptophan for synthesis of serotonin; so tryptophan is unavailable.

Question 19

Functions of PLP?

Answer 19

The pyridoxal phosphate (PLP) acts as co-enzyme for many reactions in amino acid metabolism (Box 34.6).
1. Transamination
These reactions are catalyzed by amino trans- ferases (transaminases) which employ PLP as the co-enzyme (Fig. 14.8). For example:
Alanine + Alpha keto glutarate → Pyruvate + Glutamic acid (Enzyme Alanine transaminase).
2. Decarboxylation
All decarboxylation reactions of amino acids require PLP as co-enzyme. A few examples are given below:
i. Glutamate → GABA (gamma amino butyric acid) (Fig. 16.2). GABA is an inhibitory neurotransmitter, and hence in B6 deficiency, especially in children, convulsions may occur.
ii. Histidine → histamine, which is the mediator of allergy and anaphylaxis (Fig. 16.9).
iii. 5-hydroxytryptophan→serotonin(Fig.17.10).
iv. Cysteine → taurine (Fig. 15.22).
v. Serine → ethanol amine (Fig. 15.12).
3. Sulfur Containing Amino Acids
PLP plays an important role in methionine and cysteine metabolism. For details see Chapter 15. a. Homocysteine + Serine → Cystathionine.
(Enzyme Cystathionine synthase) (Fig.15.15) b. Cystathionine → Homoserine + Cysteine
(Enzyme Cystathionase)
Both these reactions require PLP. Hence in
vitamin B6 deficiency homocysteine in blood is increased. Homocysteine level is correlated with myocardial infarction. Therefore, pyridoxine is used to prevent homocysteinemia.
4. Heme Synthesis
ALA synthase is a PLP dependent enzyme. This is the rate limiting step in heme biosynthesis (Fig. 21.4). So, in B6 deficiency, anemia may be seen.
5. Production of Niacin
Pyridoxal phosphate is required for the synthesis of niacin from tryptophan (one vitamin is necessary for synthesis of another vitamin) (Figs17.8 and 17.9).
3-hydroxy kynurenine → 3-hydroxy anthranilic acid (Enzyme Kynureninase).
Kynureninase is a PLP dependent enzyme. Hence in vitamin B6 deficiency niacin production is less. Moreover kynurenine cannot be converted further, which is metabolized to xanthurenic acid and excreted through urine.
6. Glycogenolysis
Phosphorylase enzyme (glycogen to glucose-1- phosphate) requires PLP. In fact, more than 70% of total PLP content of the body is in muscles, where it is a part of the phosphorylase enzyme.

Question 20

How INH, cycleserine, OCPs and ethanol cause B6 deficiency?

Answer 20

Effect of Drugs on Vitamin B6
i. INH:Isonicotinicacidhydrazide(isoniazid)is an antituberculosis drug. It inhibits pyridoxal kinase; reduces the formation of PLP and causes vitamin B6 deficiency.
ii. Cycloserine: It acts as B6 antagonist.
iii. Oral contraceptives: Mild vitamin B6 defi- ciency may be seen in women taking oral
contraceptive pills.
iv. Ethanol: It is converted to acetaldehyde,
which inactivates PLP. Hence B6 deficiency neuritis is quite common in alcoholics.

Question 21

Biotin dependant carboxylation reactions?

Answer 21

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
2. Propionyl CoA carboxylase
   Propionyl CoA +CO2+ATP→
   Methyl malonyl CoA +ADP+Pi
   (Step 1, Fig. 11.11).
3. Pyruvate carboxylase
   Pyruvate + CO2 +ATP→Oxaloacetate +ADP +Pi
   (Fig. 9.24). 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.

Question 22

Biotin independent carboxylation reactions?

Answer 22

i. Carbamoyl phosphate synthetase, which is the stepping stone for urea and pyrimidine synthesis (Step 1, Fig. 14.13).
ii. Addition of CO2 to form C6 in purine ring.
iii. Malicenzyme,convertingpyruvatetomalate.

Question 23

Biotin antagonists and their applications?

Answer 23

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.
ii. 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.
iii. The affinity of avidin to biotin is greater than most of the usual antigen-antibody reactions. Therefore avidin-biotin system is commonly utilized for detection of pathogens in the ELISA test.
iv. DNA is generally labelled by radioactive nucleotides. Recently, biotin labelling of DNA is becoming more popular. Biotin is added to nucleotides, which will be incorporated into the newly synthesized DNA. The fixed biotin can be identified by reaction with avidin.

Question 24

Explain basis of FIGLU test?

Answer 24

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.

Question 25

How folate deficieny causes Macrocytic anaemia?

Answer 25

1. Reduced DNA synthesis
   In folate deficiency, THFA is reduced and thymi- dylate 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

It is the most characteristic feature of folate deficiency (Fig. 34.20). During erythropoiesis, DNA synthesis is delayed, but protein synthesis is continued. Thus hemoglobin accumulates in RBC precursors. This asynchrony or dis- sociation between the maturity of nucleus and cytoplasm is manifested as immature looking nucleus and mature eosinophilic cytoplasm in the bone marrow cells.
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. Leukopenia and thrombocytopenia are also manifested.

The peripheral blood picture in folate deficiency is described as macrocytic, and in cobalamin deficiency as megaloblastic. In B12 deficiency, there are additional neurological symptoms.

Question 26

Explain Folate trap?

Answer 26

The production of methyl THFA is an irreversible step (Fig. 14.16). Therefore, the only way for generation of free THFA is step No. 1 where methyl THFA combines with VIT B12 to form methyl cobalamine. When B12 is deficient, this reaction cannot take place. This is called the methyl folate trap, since the folate gets trapped in the form of methyl THFA which is irreversible. This leads to the associated folic acid scarcity in B12 deficiency.

Question 27

Explain Schilling test?

Answer 27

Radioactivelabelled(Cobalt-60)vitamin B12, one microgram is given orally. Simultaneously an intramuscular injection of unlabeled vitamin B12 is given, in order to saturate tissues with normal vitamin B12. So, radioactive vitamin B12 will not bind to body tissues. Therefore, the entire absorbed radioactivity will pass into the urine. The patient’s urine is then collected over the next 24 hours to assess the absorption. In a normal person, the ingested vitamin B12 will be absorbed into the body. Since the liver receptors for vitamin B12 are saturated by the injection, much of the ingested vitamin B12 will be excreted in the urine. In patients with pernicious anemia or with deficiency due to impaired absorption, less than 5% of the radioactivity is detected in urine. 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.

Question 28

Explain Forms of B12 and their importance?

Answer 28

a. Cyanocobalamin
During the isolation procedure, cyanide is added to get stable crystals. The CN group has no physiological function, it is only a laboratory artefact.
Oral preparations are in this form.

b. Hydroxy cobalamin
Injectable preparations are in this form.

c. Adenosyl cobalamin (Ado-B12)
This is the major storage form, seen in liver. It is also present as functional co-enzyme.

d. Methyl cobalamin
When the methyl group replaces adenosyl group, it is known as methyl cobalamin. This is the major form seen in blood circulation as well as in cytoplasm of cells.
The Ado-B12 and methyl B12 are the functional co-enzymes in the body.