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Q.1- Discuss the steps of activation of vitamin D, and explain the reason for the fact that Vitamin D is toxic in excess, but excessive exposure to sunlight does not lead to vitamin D toxicity.

Answer- Vitamin D3 (cholecalciferol) can be synthesized by humans in the skin upon exposure to ultraviolet-B (UVB) radiation from sunlight, or it can be obtained from the diet. When exposure to UVB radiation is insufficient for the synthesis of adequate amounts of vitamin D3 in the skin, adequate intake of vitamin D from the diet is essential for health. Plants synthesize ergosterol, which is converted to vitamin D2 (Ergocalciferol) by ultraviolet light.

In response to ultraviolet radiation of the skin, a photochemical cleavage results in the formation of vitamin D from 7-dehydrocholesterol. Cutaneous production of vitamin D is decreased by melanin and high solar protection factor sunblocks, which effectively impair skin penetration of ultraviolet light. The increased use of sunblocks in North America and Western Europe and a reduction in the magnitude of solar exposure of the general population over the past several decades has led to an increased reliance on dietary sources of vitamin D. In the United States and Canada, these sources largely consist of fortified cereals and dairy products, in addition to fish oils and egg yolks. Vitamin D from plant sources is in the form of vitamin D2, whereas that from animal sources is vitamin D3. These two forms have equivalent biologic activity and are activated equally well by the vitamin D hydroxylases in humans.

Activation of Vitamin D

Vitamin D itself is biologically inactive, and it must be metabolized to its biologically active forms. After it is consumed in the diet or synthesized in the epidermis of skin, vitamin D enters the circulation and is transported to the liver. In the liver, vitamin D is hydroxylated to form 25-hydroxyvitamin D (calcidiol; 25-hydroxyvitamin D, the major circulating form of vitamin D. The serum 25-hydroxyvitamin D concentration a useful indicator of vitamin D nutritional status. In the kidney, the 25-hydroxyvitamin D31-hydroxylase enzyme catalyzes a second hydroxylation of 25-hydroxyvitamin D, resulting in the formation of 1,25-dihydroxyvitamin D (calcitriol, 1alpha,25-dihydroxyvitamin D]—the most potent form of vitamin D. Most of the physiological effects of vitamin D in the body are related to the activity of 1,25-dihydroxyvitamin D (Figure-1)

Figure-1- showing the steps of activation of vitamin D in the body.

Sunlight exposure can provide most people with their entire vitamin D requirement. Serum vitamin D concentrations following exposure to 1 minimal erythemal dose of simulated sunlight (the amount required to cause a slight pinkness of the skin) is equivalent to ingesting approximately 20,000 IU of vitamin D2

Although excess dietary vitamin D is toxic, excessive exposure to sunlight does not lead to vitamin D toxicity, because there is a limited capacity to form the precursor, 7 dehydrocholesterol, and prolonged exposure of previtamin D to sunlight leads to formation of inactive compounds. Vitamin D toxicity (hypervitaminosis D) induces abnormally high serum calcium levels (hypercalcemia), which could result in bone loss, kidney stones, and calcification of organs like the heart and kidneys if untreated over a long period of time.

Q.2- Why is vitamin D considered a hormone? Describe the mechanism of action of vitamin D.

Answer- Vitamin D is actually a hormone since –

1) Structurally it has a cyclopentano perhydrophenanthrene nucleus, like a steroid hormone.

2) Its mechanism of action resembles that of hormones (Figure-2).

Figure-2- Showing the mechanism of action of a steroid hormone. Vitamin D has a similar action. Click the link below to see the animations of mechanism of action of a steroid hormone.

3) Like hormones it is required only in small amount.

4) Like hormones, the formation of the active form is subjected to feed back inhibition.

5) Like hormones, vitamin D has specific target organs like intestine, bone and kidneys.

6) Like hormones, it is produced in one organ and  acts upon distant organs for its  functions.

Mechanisms of Action

Most if not all actions of vitamin D are mediated through a nuclear transcription factor known as the vitamin D receptor (VDR). Upon entering the nucleus of a cell, 1,25-dihydroxyvitamin D associates with the VDR and promotes its association with the retinoic acid X receptor (RXR). In the presence of 1,25-dihydroxyvitamin D the VDR/RXR complex binds small sequences of DNA known as vitamin D response elements (VDREs) and initiates a cascade of molecular interactions that modulate the transcription of specific genes (Figure-3). More than 50 genes in tissues throughout the body are known to be regulated by 1,25-dihydroxyvitamin D.









Figure-3- showing the mechanism of action of vitamin D

Q.3- Justify the statement that “Vitamin D metabolism is both regulated by and regulates calcium homeostasis”. What are the other functions performed by vitamin D?

Answer- The main function of vitamin D is to maintain calcium homeostasis, and in turn, vitamin D metabolism is regulated by factors that respond to plasma concentrations of calcium and phosphate.

Maintenance of serum calcium levels within a narrow range is vital for normal functioning of the nervous system, as well as for bone growth and maintenance of bone density.

Flow chart- showing role of vitamin D in the absorption of calcium from gut.

The parathyroid glands sense the serum calcium level, and secrete parathyroid hormone (PTH) if it becomes too low, for example, when dietary calcium intake is inadequate. PTH stimulates the activity of the 1-hydroxylase enzyme in the kidney, resulting in increased production of calcitriol, the biologically active form of vitamin D3. Increased calcitriol production restores normal serum calcium levels in three different ways:

1) by activating the vitamin D-dependent transport system in the small intestine, increasing the absorption of dietary calcium(Flow chart),

2) by increasing the mobilization of calcium from bone into the circulation; and

3) by increasing the reabsorption of calcium by the kidneys (Figure-4).

PTH is also required to increase calcium mobilization from bone and calcium reabsorption by the kidneys.

When there is adequate calcium concentration, Calcitriol acts to reduce its own synthesis by inducing the 24-hydroxylase and repressing the 1-hydroxylase in the kidney. 

Other functions of vitamin D

In addition, calcitriol is involved in insulin secretion, synthesis and secretion of parathyroid and thyroid hormones, inhibition of production of interleukin by activated T-lymphocytes and of immunoglobulin by activated B-lymphocytes, differentiation of monocyte precursor cells, and modulation of cell proliferation. In most of these actions, it acts like a steroid hormone, binding to nuclear receptors and enhancing gene expression, although it also has rapid effects on calcium transporters in the intestinal mucosa.


 Figure-4- showing the role of vitamin D in maintaining calcium homeostasis.

Q.4 – Discuss the causes, clinical manifestations, laboratory diagnosis and treatment of vitamin D deficiency in children.


Rickets is a disease of growing bone that is unique to children and adolescents. It is caused by a failure of osteoid to calcify in a growing person. Failure of osteoid to calcify in adults is called osteomalacia. Vitamin D deficiency rickets occurs when the metabolites of vitamin D are deficient. Less commonly, a dietary deficiency of calcium or phosphorus may also produce rickets.


In the vitamin D deficiency state, hypocalcemia develops, that stimulates excess parathyroid hormone, which acts to stimulate renal phosphorus loss, further reducing deposition of calcium in the bone. Excess parathyroid hormone also produces changes in the bone similar to those occurring in hyperparathyroidism. Early in the course of rickets, the calcium concentration in the serum decreases. After the parathyroid response, the calcium concentration usually returns to the reference range, though phosphorus levels remain low. Alkaline phosphatase, which is produced by overactive osteoblast cells, leaks to the extracellular fluids so that its concentration rises to anywhere from moderate elevation to very high levels.


Vitamin D deficiency rickets does not occur in formula-fed infants because formula and milk sold in the United States contains 400 IU of vitamin D per liter. Except in pediatric patients with chronic malabsorption syndromes or end-stage renal disease, nearly all cases of rickets occur in breastfed infants who have dark skin and receive no vitamin D supplementation.

Causes of vitamin D deficiency

The clinical syndrome of vitamin D deficiency can be a result of-

a) deficient production of vitamin D in the skin,

b) lack of dietary intake,

c) accelerated losses of vitamin D,

d) impaired vitamin D activation or

e)  resistance to the biologic effects of 1,25(OH)2D.

The elderly people are particularly at risk for vitamin D deficiency, since both the efficiency of vitamin D synthesis in the skin and the absorption of vitamin D from the intestine decline with age.

Similarly, intestinal malabsorption of dietary fats leads to vitamin D deficiency. This is further exacerbated in the presence of terminal ileal disease, which results in impaired enterohepatic circulation of vitamin D metabolites. In addition to intestinal diseases, accelerated inactivation of vitamin D metabolites can be seen with drugs that induce hepatic cytochrome P450 mixed function oxidases, such as barbiturates, phenytoin, and rifampin.

Impaired 25-hydroxylation, associated with severe liver disease or isoniazid, is an infrequent cause of vitamin D deficiency. Impaired 1-hydroxylation is prevalent in the population with profound renal dysfunction and a decrease in functional renal mass. Thus, therapeutic interventions should be considered in patients whose creatinine clearance is <0.5 mL/s (30 mL/min).

Mutations in the renal -1-hydroxylase are the basis for the genetic disorder, pseudo-vitamin D–deficiency rickets. This autosomal recessive disorder presents with the syndrome of vitamin D deficiency in the first year of life. Patients present with growth retardation, rickets, and hypocalcemic seizures. Serum 1,25(OH)2D levels are low, despite normal 25(OH)D levels and elevated PTH levels. Treatment with vitamin D metabolites that do not require 1-hydroxylation results in disease remission, although lifelong therapy is required.

A second autosomal recessive disorder, hereditary vitamin D–resistant rickets, a consequence of vitamin D receptor mutations, is a greater therapeutic challenge. These patients present in a similar fashion during the first year of life, but alopecia often accompanies the disorder, demonstrating a functional role of the VDR ( Vitamin D receptor) in postnatal hair regeneration. Serum levels of 1,25(OH)2D are dramatically elevated in these individuals, both because of increased production due to stimulation of 1-hydroxylase activity as a consequence of secondary hyperparathyroidism and because of impaired inactivation, since induction of the 24-hydroxylase by 1,25(OH)2D requires an intact VDR. Since the receptor mutation results in hormone resistance, daily calcium and phosphorus infusions may be required to bypass the defect in intestinal mineral ion absorption.

Occasionally, deficiency severe enough to cause maternal Osteomalacia results in rickets with metaphyseal lesions in neonates.

Summary of Causes of Impaired Vitamin D action

  • Vitamin D deficiency
    • Impaired cutaneous production
    • Dietary absence
    • Malabsorption
  • Accelerated loss of vitamin D
    • Impaired enterohepatic circulation
  • Impaired 25-hydroxylation  
  • Impaired 1-hydroxylation
    • Hypoparathyroidism  
    • Renal failure
    • 1-hydroxylase mutation
  • X-linked hypophosphatemic rickets
  • Target organ resistance
    • Vitamin D receptor mutation
  • Phenytoin therapy
  • Liver disease
    •  Isoniazid therapy

    Clinical Manifestations

    • Generalized muscular hypotonia of an unknown mechanism is observed in most patients with clinical signs of rickets.
    • Craniotabes (softening of the entire skull) manifests early in infants with vitamin D deficiency, although this feature may be normal in infants, especially for those born prematurely.
    • If rickets occurs at a later age, thickening of the skull develops. This produces frontal bossing and delays the closure of the anterior fontanelle. In the long bones, laying down of uncalcified osteoid at the metaphases leads to spreading of those areas, producing knobby deformity, which is visualized on radiography as cupping and flaring of the metaphyses.
    • Weight bearing produces deformities such as bowlegs and knock-knees(Figure-5)

    Figure-5 -showing bending of bones( bow legs).

    In the chest, knobby deformities results in the rachitic rosary along the costochondral junctions (Figure-6)

    Figure-6- showing rachitic rosary along the costochondral junctions.

    The weakened ribs pulled by muscles also produce flaring over the diaphragm, which is known as Harrison groove.

     Figure-7-showing Harrison groove and pot belly

    The sternum may be pulled into a pigeon-breast deformity (Figure-7).

    Figure-8- showing chest deformity in Rickets

    In more severe instances in children older than 2 years, vertebral softening leads to Kyphoscoliosis (Figure-8)

     Figure-9-Showing Kyphoscoliosis

    The ends of the long bones demonstrate that same knobby thickening.

    At the ankle, palpation of the tibial malleolus gives the impression of a double epiphysis (Marfan sign).

    Because the softened long bones may bend, they may fracture one side of the cortex (ie, greenstick fracture).

    Figure-10-Showing the clinical findings in Rickets

    Regardless of the cause, the clinical manifestations of vitamin D deficiency are largely a consequence of impaired intestinal calcium absorption. Mild to moderate vitamin D deficiency is asymptomatic, whereas long-standing vitamin D deficiency results in hypocalcemia accompanied by secondary hyperparathyroidism, impaired mineralization of the skeleton (osteopenia on x-ray or decreased bone mineral density), and proximal myopathy. In the absence of an intercurrent illness, the hypocalcemia associated with long-standing vitamin D deficiency rarely presents with acute symptoms of hypocalcemia, such as numbness, tingling, or seizures. However, the concurrent development of hypomagnesemia, which impairs parathyroid function, or the administration of potent bisphosphonates, which impair bone resorption, can lead to acute symptomatic hypocalcemia in vitamin D–deficient individuals.

    Laboratory Investigations

    • Early on in the course of rickets, the calcium (ionized fraction) is low; however it is often within the reference range at the time of diagnosis as parathyroid hormone levels increase.
    • Calcitriol levels maybe normal or elevated because of increased parathyroid activity. Because levels of serum 25(OH)D reflect body stores of vitamin D and correlate with symptoms and signs of vitamin D deficiency better than levels of other vitamin D metabolites, 25(OH)D (D2+D3) measurement is generally considered the best way to diagnose deficiency. Goal 25(OH)D levels are 30 to 40 ng/mL (about 75 to 100 nmol/L); whether levels above this may be beneficial remains uncertain.
    • If the diagnosis is unclear, serum levels of 1,25(OH)2D and urinary Ca concentration can be measured. In severe deficiency, serum 1,25(OH)2D is abnormally low, usually undetectable. Urinary Ca is low in all forms of the deficiency except those associated with acidosis.
    • The phosphorus level is invariably low for age unless recent partial treatment or recent exposure to sunlight has occurred.
    • Alkaline phosphatase levels are elevated.
    • A generalized aminoaciduria occurs from the parathyroid activity; aminoaciduria does not occur in familial hypophosphatemia rickets (FHR).
    • PTH levels are also measured to confirm the diagnosis, because in vitamin D deficiency PTH level is high.

    Imaging Studies

    Bone changes, seen on x‑rays, precede clinical signs. In rickets, changes are most evident at the lower ends of the radius and ulna. The diaphyseal ends lose their sharp, clear outline; they are cup-shaped and show a spotty or fringy rarefaction. Later, because the ends of the radius and ulna have become noncalcified and radiolucent, the distance between them and the metacarpal bones appears increased. The bone matrix elsewhere also becomes more radiolucent. Characteristic deformities result from the bones bending at the cartilage-shaft junction because the shaft is weak. As healing begins, a thin white line of calcification appears at the epiphysis, becoming denser and thicker as calcification proceeds. Later, the bone matrix becomes calcified and opacified at the subperiosteal level.


    • Correction of Ca and P deficiencies
    • Supplemental vitamin D

    Ca deficiency (which is common) and P deficiency should be corrected. As long as Ca and P intake is adequate, adults with Osteomalacia and children with uncomplicated rickets can be cured by giving vitamin D 40 μg (1600 IU) po once/day. Serum 25(OH)D and 1,25(OH)2D begin to increase within 1 or 2 days. Serum Ca and phosphate increase and serum alkaline phosphatase decreases within about 10 days. During the 3rd wk, enough Ca and P are deposited in bones to be visible on x‑rays. After about 1 mo, the dose can usually be reduced gradually to the usual maintenance level of 10 to 15 μg (400 to 600 IU) once/day. If tetany is present, vitamin D should be supplemented with IV Ca salts for up to 1 wk. 

    Vitamin D Toxicity Usually, vitamin D toxicity results from taking excessive amounts. Marked hypercalcemia commonly causes symptoms. Diagnosis is typically based on elevated blood levels of 25(OH)D. Treatment consists of stopping vitamin D, restricting dietary Ca, restoring intravascular volume deficits, and, if toxicity is severe, giving corticosteroids or for children with increased skin pigmentation.


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    Q.1- Out of Retinol or Retinoic acid – Which form is required for the gene expression and tissue differentiation and what is the mechanism of regulation of gene expression?

    Answer- Retinoic acid (RA) and its isomers act as hormones to affect gene expression and thereby influence numerous physiological processes.

    All- trans retinoic acid and 9-cis retinoic acid have active role in growth, development and tissue differentiation. They have different actions in different tissues.

    Mechanism of regulation of gene expression

    Like vitamin D, retinoic acid interacts with nuclear receptors that bind to control elements which are specific regions on the DNA to regulate the expression of specific genes.

    There are two families of nuclear retinoid receptors (RAR) that bind to All- trans retinoic acid or 9-cis retinoic acid and RXR that bind to 9-cis retinoic acid and to some of the other physiologically active retinoids. RXR can form active dimer with RAR and the receptors for calcitriol, thyroid hormone and the receptors for long chain fatty acid derivatives. The result is that a large number of genes are sensitive to control by retinoic acid.

    All-trans-RA and 9-cis-RA are transported to the nucleus of the cell bound to cytoplasmic retinoic acid-binding proteins. Within the nucleus, all-trans-RA binds to retinoic acid receptors (RAR) and 9-cis-RA binds to retinoid receptors (RXR). RAR and RXR form RAR/RXR heterodimer, which bind to regulatory regions of the chromosome called retinoic acid response elements (RARE). Binding of all-trans-RA and 9-cis-RA to RAR and RXR respectively allows the complex to regulate the rate of gene transcription. Nuclear Receptors in the Gonads increase gene expression and maintain reproductive tissues while nuclear receptors in epithelial cells regulate cell differentiation. Through the stimulation and inhibition of transcription of specific genes, retinoic acid plays a major role in cellular differentiation, the specialization of cells for highly specific physiological roles. Many of the physiological effects attributed to vitamin A appear to result from its role in cellular differentiation(Figure-1)











    Figure-1- showing the role of retinoic acid in gene expression.

    Q.2- What is visual cycle? What is the role of vitamin A in visual cycle?

    Answer- In the retina, retinaldehyde functions as the prosthetic group of the light-sensitive opsin proteins, forming rhodopsin (in rods) and iodopsin (in cones). Any one cone cell contains only one type of opsin, and is sensitive to only one color. In the pigment epithelium of the retina, all-trans-retinol is isomerized to 11-cis-retinol and oxidized to 11-cis-retinaldehyde. This reacts with a lysine residue in opsin, forming the holoprotein rhodopsin.

    As shown in Figure-2, the absorption of light by rhodopsin causes isomerization of the retinaldehyde from 11-cis to all-trans, and a conformational change in opsin. This results in the release of retinaldehyde from the protein, and the initiation of a nerve impulse. The formation of the initial excited form of rhodopsin, bathorhodopsin, occurs within picoseconds of illumination. There is then a series of conformational changes leading to the formation of metarhodopsin II, which initiates a guanine nucleotide amplification cascade and then a nerve impulse. The nerve impulse generated by the optic nerve is conveyed to the brain where it can be interpreted as vision.  The final step is hydrolysis to release all-trans-retinaldehyde and opsin. The key to initiation of the visual cycle is the availability of 11-cis-retinaldehyde, and hence vitamin A. By a series of reactions all trans retinol is converted to 11-cis- retinal which reassociates with opsin to form Rhodopsin. In deficiency, both the time taken to adapt to darkness and the ability to see in poor light are impaired.












    Figure-2- showing the role of vitamin A in visual cycle

    Vitamin A deficiency and vision

    Vitamin A deficiency (VAD) among children in developing nations is the leading preventable cause of blindness. The earliest evidence of vitamin A deficiency is impaired adaptation to darkness (nyctalopia), which can lead to night blindness.

    • Bitot spots – Areas of abnormal squamous cell proliferation and keratinization of the conjunctiva can be seen in young children with VAD.
    • Xerophthalmia results from keratinization of the conjunctiva.
    • Keratomalacia- In advanced deficiency; the cornea becomes hazy and can develop erosions, which can lead to its destruction (Keratomalacia).
    • Blindness due to retinal injury – Vitamin A has a major role in photo transduction.. VAD leads to a lack of visual pigments; this reduces the absorption of various wavelengths of light, resulting in blindness. 

     Q.3- Why is vitamin A commonly called Anti-infective vitamin?


    Why do the children deficient in vitamin A get more prone to respiratory and gastrointestinal infections?

    Answer- Vitamin A is commonly known as the anti-infective vitamin, because it is required for normal functioning of the immune system, and even mild deficiency leads to increased susceptibility to infectious diseases.

    1) Vitamin A and retinoic acid (RA) play a central role in the development and differentiation of white blood cells, such as lymphocytes, which play critical role in the immune response. Activation of T-lymphocytes, the major regulatory cells of the immune system, appears to require all-trans-RA binding of RAR.

    2) The skin and mucosal cells (cells that line the airways, digestive tract, and urinary tract) function as a barrier and form the body’s first line of defense against infections. Retinol and its metabolites are required to maintain the integrity and functioning of these cells. Keratinizations of mucous membranes in vitamin A deficiency add up to the risk to infections.

    3) The onset of infection reduces blood retinol levels very rapidly. This phenomenon is generally believed to be related to decreased synthesis of retinol binding protein (RBP) by the liver, since it is a negative ‘Acute phase protein’, that results in decreased circulatory concentration of the vitamin with further deterioration of the immune system. In this manner, infection stimulates a vicious cycle.

    4) Vitamin A also plays a role in iron utilization, humoral immunity, T cell–mediated immunity, natural killer cell activity, and phagocytosis.

    All the components of immune system are affected in deficiency. Hence in brief it can be said that the pathogenic invasion is enhanced and immune system is weakened in vitamin A deficiency leading to increased susceptibility to infections.

    Q.4- How does Zinc deficiency affect the functioning of vitamin A?

    Answer- Zinc deficiency is thought to interfere with vitamin A metabolism in several ways: (1) zinc deficiency results in decreased synthesis of retinol binding protein (RBP), which transports retinol through the circulation to tissues (e.g., the retina) and also protects the organism against potential toxicity of retinol; (2) zinc deficiency results in decreased activity of the enzyme that releases retinol from its storage form, retinyl palmitate, in the liver; and (3) zinc is required for the enzyme that converts retinol into retinal.  Thus zinc deficiency precipitates vitamin A deficiency producing a variety of symptoms.


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    Q.1- Discuss the important functions of Vitamin B6.

    Answer- Vitamin B6, also called pyridoxine, is one of eight water-soluble B vitamins. There are six forms of vitamin B6: pyridoxal (PL), pyridoxine (PN), pyridoxamine (PM)-(figure-1), and their phosphate derivatives: pyridoxal 5′-phosphate (PLP), pyridoxine 5′-phosphate (PNP), and pridoxamine 5′-phospate (PNP). PLP is the active coenzyme form, and has the most importance in human metabolism. In the body, pyridoxine is found primarily in the liver and muscles. Pyridoxine is utilized by the liver to synthesize pyridoxal phosphate (PLP), the active coenzyme form.













    Figure 1- Showing forms of B6- a) Pyridoxine ,  b) Pyridoxal and  c) Pyridoxamine.

    Vitamin B6 serves as a coenzyme of approximately 100 enzymes that catalyze essential chemical reactions in the human body.

    1) It plays an important role in protein, carbohydrate and lipid metabolism. Pyridoxal phosphate is a coenzyme for many enzymes involved in amino acid metabolism, especially transamination, deamination and decarboxylation reactions. It is also the cofactor of glycogen phosphorylase, where the phosphate group is catalytically important. Some 80% of the body’s total vitamin B6 is pyridoxal phosphate in muscle, mostly associated with glycogen phosphorylase. This is not available in deficiency, but is released in starvation, when glycogen reserves become depleted, and is then available, especially in liver and kidney, to meet increased requirement for gluconeogenesis from amino acids.

    2) It is also required for the synthesis of sphigomyelin and other Sphingolipids

    3) Its major function is the production of serotonin from the amino acid tryptophan in the brain and other the synthesis of the neurotransmitters, dopamine, norepinephrine and gamma-aminobutyric acid (GABA) and so it has a role in the regulation of mental processes and mood. These neurotransmitters are produced by decarboxylation where B6 acts as a coenzyme for the respective enzymes.

    4) Furthermore, it is involved in the conversion of tryptophan to the vitamin niacin.

    5) It is required for the formation of hemoglobin and the growth of red blood cells It acts as coenzyme for the enzyme ALA synthase, in the first step of haem biosynthesis. Deficiency leads to Sideroblastic anemia

    6) It promotes  absorption of vitamin B12

    7) It is required for the production of prostaglandins and hydrochloric acid in the gastrointestinal tract,

    8) It is required for the sodium-potassium balance, and in histamine metabolism.

    9) As part of the vitamin B-complex it may also be involved in the down regulation of the homocysteine blood level. The enzymes Cystathionine β synthase and Cystathionine lyase require vitamin B6 as coenzyme. Deficiency leads to homocysteinemia

    10) Vitamin B6 also plays a role in the improvement of the immune system.

    11) In addition, B6 is important in steroid hormone action. Pyridoxal phosphate removes the hormone receptor complex from DNA binding, terminating the action of the hormones. In vitamin B6 deficiency, there is increased sensitivity to the actions of low concentrations of estrogens, androgens, cortisol, and vitamin D.

    12) Vitamin B6 promotes iron excretion and this has been used as a rationale for treatment in iron storage diseases.

    13) Vitamin B6 may be helpful in some women with premenstrual dysphoric disorder, also known as premenstrual syndrome (PMS), and may be useful in some cases of gestational diabetes and for protection against metabolic imbalances associated with the use of some oral contraceptives.

     Q.2- What are the important functions of niacin in the body?

    Answer- The term niacin refers to both nicotinic acid and its amide derivative, nicotinamide (niacinamide)- Figure-2








    Figure-2- Showing structure of Nicotinic acid and Nicotinamide

    Both are used to form the coenzymes nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+). Niacin is a member of the water-soluble B- vitamin complex. The amino acid tryptophan can be converted to nicotinic acid in humans; therefore niacin is not really a vitamin provided that an adequate dietary supply of tryptophan is available. Some 60 mg of tryptophan is equivalent to 1 mg of dietary niacin.

    Redox reactions

    As many as 200 enzymes require the niacin coenzymes, NAD+ and NADP+, mainly to accept or donate electrons for redox reactions. NAD+ functions most often in energy producing reactions involving the degradation (catabolism) of carbohydrates, fats, proteins, and alcohol. NADP+ functions more often in biosynthetic (anabolic) reactions, such as in the synthesis of all macromolecules, including fatty acids and cholesterol. Some of the important enzymes requiring NAD+ and NADP+ are as follows-

    NAD+ dependent enzymes

    1)     Glyceraldehyde-3-phosphae dehydrogenase

    2)     Pyruvate dehydrogenase complex

    3)     Mitochondrial isocitrate dehydrogenase

    4)     Alpha keto glutarate dehydrogenase complex

    5)     Malate dehydrogenase

    6)     Lactate dehydrogenase

    7)     Beta hydroxy acyl co A dehydrogenase

    8)    Cytosolic glycerol-3-phosphate dehydrogenase

    NADPH dependent enzymes

    1)      HMG co A reductase

    2)     Enoyl reductase

    3)     Keto acyl reductase

    4)     Dihydrofolate reductase

    5)     Met hemoglobin reductase

    6)     Ribonucleotide reductase

    Non-redox reactions

    The niacin coenzyme, NAD+, is the substrate (reactant) for two classes of enzymes (mono-ADP-ribosyltransferases and poly-ADP-ribose polymerase) that separate the niacin moiety from NAD+ and transfer ADP-ribose to proteins.

    1) Mono-ADP-ribosyltransferases enzymes were first discovered in certain bacteria, where they were found to produce toxins, such as cholera and diphtheria. These enzymes and their products, ADP-ribosylated proteins, have also been found in the cells of mammals and are thought to play a role in cell signaling by affecting G-protein activity. G-proteins are proteins that bind guanosine-5′-triphosphate (GTP) and act as intermediaries in a number of cell-signaling pathways.

    2) Poly-ADP-ribose polymerases (PARPs) are enzymes that catalyze the transfer of many ADP-ribose units from NAD+ to acceptor proteins. PARPs appear to function in DNA repair and stress responses, cell signaling, transcription, regulation or apoptosis, chromatin structure, and cell differentiation, suggesting a possible role for NAD+ in cancer prevention. At least five different PARPs have been identified, and although their functions are not yet well understood, their existence indicates a potential for considerable consumption of NAD+.

    3) A third class of enzymes (ADP-ribosyl cyclase) catalyzes the formation of cyclic ADP-ribose, a molecule that works within cells to provoke the release of calcium ions from internal storage sites and probably also plays a role in cell signaling.

    Other functions of niacin in the body

    • High doses of nicotinic acid (1.5-4 g/day) can reduce total and low-density lipoprotein cholesterol and triacylglycerols and increase high-density lipoprotein cholesterol in patients at risk of cardiovascular disease.
    • Type 1 diabetes mellitus results from the autoimmune destruction of insulin-secreting b-cells in the pancreas. There is evidence that nicotinamide may delay or prevent the development of diabetes.



















    Flow chart- showing the synthesis and role of NAD in the redox and non redox reactions

    Q.3- Discuss the role of niacin as a lipid lowering drug. What is the cause of hot flushes observed in patients on niacin therapy and how can these be treated?

    Answer- Nicotinic acid, or niacin, has been used as a lipid-modifying agent for decades. It was previously shown to reduce the flux of nonesterified fatty acids (NEFAs) to the liver, resulting in reduced hepatic TG synthesis and VLDL secretion. Recently a receptor for nicotinic acid called GPR109A has been discovered; it is expressed in adipocytes and, when activated, suppresses the release of NEFA by adipose. Niacin reduces plasma triglyceride and LDL-C levels and raises the plasma concentration of HDL-C. Niacin is also the only currently available lipid-lowering drug that significantly reduces plasma levels of Lp (a). If properly prescribed and monitored, niacin is a safe and effective lipid-lowering agent.

    The most frequent side effect of niacin is cutaneous flushing, which is mediated by activation of the same receptor GPR109A in the skin, leading to local generation of prostaglandin D2 production. Flushing can be reduced by formulations that slow the absorption and by taking aspirin prior to dosing.

    Q.4- What is the significance of Pantothenic acid in the body?

    Answer- Pantothenic acid, also known as vitamin B5, is essential to all forms of life.








    Figure 3- Showing structure of Pantothenic acid

    Functions of Pantothenic acid

    Coenzyme A

    Pantothenic acid is a component of coenzyme A (CoASH) (Figure-4) , an essential coenzyme in a variety of reactions.













    Figure-4 -showing the structure of Co enzyme A

    CoASH is required for chemical reactions that generate energy from food (fat, carbohydrates, and proteins). The synthesis of essential fats, cholesterol, and steroid hormones requires CoA, as does the synthesis of the neurotransmitter, acetylcholine, and the hormone, melatonin. Heme, a component of hemoglobin, requires a CoA-containing compound for its synthesis. Metabolism of a number of drugs and toxins by the liver requires CoA.

    Coenzyme A was named for its role in acetylation reactions. Most acetylated proteins in the body have been modified by the addition of an acetate group that was donated by CoA. Protein acetylation affects the 3-dimensional structure of proteins, potentially altering their function. For example, acetylation reactions can alter the activity of peptide hormones. Protein acetylation appears to play a role in cell division and DNA replication and also affects gene expression by facilitating the transcription of mRNA. Additionally, a number of proteins are modified by the attachment of long-chain fatty acids donated by CoA. These modifications are known as protein acylation and appear to play a central role in cell signaling.

    Acyl-carrier protein

    The acyl-carrier protein requires Pantothenic acid in the form of 4′-phosphopantetheine for its activity as an enzyme. The pantetheine moiety is formed after combination of pantothenate with cysteine, which provides the –SH prosthetic group of CoA and ACP. Both CoA and the acyl-carrier protein are required for the synthesis of fatty acids. 


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    Q.1-What do you know about one carbon metabolism?


    Discuss the structure and coenzyme role of folic acid?


    Folic acid participates in the transfer of single carbon units. Folate-dependent single-carbon transfer reactions are important in amino acid metabolism and in pathways leading to biosynthesis of DNA, RNA, membrane lipids, and neurotransmitters.

    Structure of folic acid- Folic acid is a composite molecule, being made up of three parts: a pteridine ring system (6-methylpterin), para-amino benzoic acid, and Glutamic acid (Figure-1).

    Figure-1- Structure and Reduction of folic acid

    The glutamic acid doesn’t participate in the coenzyme functions of folic acid. Instead, folic acid in the interior of the cell may contain a “chain” of three to eight  glutamic acid residues, which serves as a negatively charged “handle” to keep the coenzyme inside cells and/or bound to the appropriate enzymes. The pteridine portion of the coenzyme and the p-amino benzoic acid portion participate directly in the metabolic reactions of folate.

    Reduction of folic acid-To carry out the transfer of 1-carbon units, NADPH must reduce folic acid twice in the cell. The pyrazine ring of the 6-methylpterin is reduced at each of the two N-C double bonds by folate reductase enzyme.

    The resulting 5, 6, 7, 8-tetrahydrofolate is the acceptor of 1-carbon groups (Figure-1)

    Forms of folic acid as one carbon carrier-Tetrahydrofolate can carry one-carbon fragments attached to N-5 (formyl, formimino, or methyl groups), N-10 (formyl) or bridging N-5–N-10 (methylene or methenyl groups).

    Sources of one carbon fragments– The major point of entry for one-carbon fragments into substituted folates is methylene-tetrahydrofolate, which is formed by the reaction of glycine, serine, and choline with tetrahydrofolate. Serine is the most important source of substituted folates for biosynthetic reactions, and the activity of serine hydroxy methyltransferase is regulated by the state of folate substitution and the availability of folate. The reaction is reversible, and in liver it can form serine from glycine as a substrate for gluconeogenesis. One carbon fragments are also produced from the metabolism of Tryptophan and Histidine.

    Utilization of one carbon fragments- Methylene-, methenyl-, and 10-formyl-tetrahydrofolates are inter convertible (Figure-2)

    The N5,N10-methylene-tetrahydrofolate can either donate its single-carbon group directly, be oxidized by NADP to the methenyl form, or be reduced by NADH to the methyl form (See figure). Depending on the biosynthetic pathway involved, any of these species can donate the 1-carbon group to an acceptor. The methylene form donates its methyl group during the biosynthesis of thymidine nucleotides for DNA synthesis, the methenyl form donates its group as a Formyl group during purine biosynthesis, and the methyl form is the donor of the methyl group to sulfur during methionine formation. When one-carbon folates are not required, the oxidation of formyl-tetrahydrofolate to yield carbon dioxide provides a means of maintaining a pool of free folate.



    Figure-2- showing the conversions of one-carbon units on Tetrahydrofolate.

    Transfer of Methyl Group

    Although Tetrahydrofolate can carry a methyl group at N-5, the methyl group’s transfer potential is insufficient for most biosynthetic reactions.

     S-Adenosylmethionine (adoMet) is more commonly used for methyl group transfers. It is synthesized from ATP and methionine by the action of methionine Adenosyl transferase (See figure-3).

    Methyl transfer from S-AdoMet is highly favored chemically and metabolically. First, transfer of the methyl group relieves a positive charge on the Sulfur of S-AdoMet. Secondly, the bond between the Sulfur and the 5′ carbon of the adenosine is rapidly hydrolyzed, leaving homocysteine and free adenosine.


    Figure- 3-showing the synthesis of S- Adenosyl Methionine and remethylation of Homocysteine to form Methionine.  

    Homocysteine itself is converted to methionine by the transfer of a methyl group from N5-methyl-tetrahydrofolate to homocysteine, regenerating methionine. The methyl group of N5-methyl-tetrahydrofolate is derived from serine, originally, so the net effect of this pathway is to move methyl groups from serine to a variety of acceptors, including homocysteine, nucleic acid bases, membrane lipids, and protein side chains.

    Q.2-What is “folate trap” or “methyl group trap”?


    Why does vitamin B12 deficiency precipitate folic deficiency also?


    What is the reason that folic and vitamin B12 deficiencies always coexist?


    If megaloblastic anemia is treated by giving only folic acid the neurological symptoms of B12 deficiency worsen, why is it so?

    Answer- The conversion of 5,10-methylene-THF into 5-methyl-THF, which is catalyzed by MTHFR (5,10-methylenetetrahydrofolate reductase), is irreversible. The only way to make further use of 5-methyl-THF and to maintain the folate cycle consists in the vitamin-B12-dependent remethylation of homocysteine to methionine (regenerating THF). The methyl group transfer is therefore greatly dependent on 5-methyl-THF and the availability of vitamin-B12. In humans, this is the only known direct link of the metabolism of two vitamins; folic acid and vitamin-B12 both need each other ( See figure 4 below).


    Figure-4- showing the interdependence of folic acid and vitamin B12 and the methylation cycle.

    In cases of vitamin-B12 deficiency, it is possible that, in spite of sufficient availability of folates (and 5-methyl-THF), an intracellular deficiency of biologically active THF arises. This situation is called a ‘folate trap’ (or methyl group trap) because, on one hand, the concentration of 5-methyl-THF continues to rise and on the other hand, due to it being prevented from releasing methyl groups, a ‘metabolic dead-end situation’ develops, which leads to the inevitable blockage of the methylation cycle. The co-factors for the C1-transfers decrease and replication as well as the cell division rate are reduced. Hence, the principal problem is the decreasing activity of methionine synthase under vitamin-B12 deficiency with secondary disorders affecting the folate metabolism and insufficient de-novo synthesis of purines and pyrimidines. There is therefore functional deficiency of folate, secondary to the deficiency of vitamin B12.

    The deficiency in active folic acids first affects the quickly dividing and highly proliferating hematopoiesis cells in the bone marrow and can even lead to pancytopenia.

    Clinically, there is no difference between vitamin-B12 deficiency anaemia and folic acid deficiency anaemia. If such anaemia is treated with vitamin-B12, the blockage is immediately stopped and the blood count quickly normalizes. However, if the anaemia is exclusively treated with folic acid, it is simply converted to dihydrofolate and THF.

    Long-term therapy using high doses of folic acid could therefore conceal the real cause i.e. pernicious (vitamin B12-deficiency) anaemia for a long time. The serum folate continues to rise (congestion of non-regenerated 5-methyl-THF) while the intracellular folate concentration (erythrocytes) drops. This situation interrupts the methylation cycle with numerous cell processes, among them the synthesis of myelin, the nerve fiber lining, being blocked due to a deficiency of methyl groups. A long undetected (causal) vitamin-B12-deficiency can therefore result in serious neurological damage.
    Exclusive folic acid therapy can thus lead to neurological damage or even cause serious  damage progression.

     Q.3-What are folate antagonists? What is their importance in the clinical field?

    Answer- Folate antagonists were originally developed as antileukemic agents, but are now being used and/or investigated in the treatment of a wide range of cancerous and non-cancerous diseases. The clinical importance of some of the folate antagonists is as follows-

    A) Folate antagonists used in non cancerous diseases

    1) Sulfanilamide is the simplest of the sulfa drugs, used as antibacterial agents. The similarity of sulfanilamide to p-amino benzoic acid is shown in Figure-5. Because its shape is similar to that of p-aminobenzoic acid, sulfanilamide inhibits the growth of bacteria by interfering with their ability to use p-aminobenzoic acid to synthesize folic acid. Sulfa drugs were the first antimetabolites to be used in the treatment of infectious disease. Because humans don’t make folic acid, sulfanilamide is not toxic to humans in the doses that inhibit bacteria. This ability to inhibit bacteria while sparing humans made them useful in preventing or treating various infections.

    Figure-5-Showing the structural similarity of Sulfanilamide and p-Amino benzoic acid

    2) Trimethoprim and Pyrimethamine

    The one-carbon fragment of methylene-tetrahydrofolate is reduced to a methyl group with release of dihydrofolate, which is then reduced back to tetrahydrofolate by dihydrofolate reductase. The dihydrofolate reductases of some bacteria and parasites differ from the human enzyme; inhibitors of these enzymes can be used as antibacterial drugs (eg, trimethoprim) and Antimalarial drugs (eg, pyrimethamine).

    B) Folate antagonists used as anticancer drugs

    a) Inhibitors of dihydrofolate reductase

    1) Methotrexate, an analog of 10-methyl-tetrahydrofolate, inhibits dihydrofolate reductase and has been exploited as an anti-cancer

    2) Aminopterin is also an inhibitor of DHFR enzyme and is used as an anticancer drug.

    b) Inhibitors of Thymidylate synthase

    The methylation of deoxyuridine monophosphate (dUMP) to thymidine monophosphate (TMP), catalyzed by thymidylate synthase, is essential for the synthesis of DNA(Figure-6). Thymidylate synthase and dihydrofolate reductase are especially active in tissues with a high rate of cell division.



    Figure-6- showing the action of thymidylate synthase. Thymidylate is synthesized by the Methylation of uridylate (dUMP) in a reaction catalyzed by the enzyme Thymidylate synthase. This reaction requires a methyl donor and a source of reducing equivalents, which are both provided by N5, N10-methylene THF). For this reaction to continue, the regeneration of THF from Dihydro folate (DHF) must occur.


    1) The antagonist 5-fluorouracil acts to indirectly inhibit the enzyme thymidylate synthase. The primary effect of TS inhibition by 5-FU is a depletion of dTMP and dTTP levels, resulting in inhibition of DNA synthesis and “thymine-less death.” Thymine-less death occurs when a cell can still synthesize RNA and protein, but is unable to make DNA resulting in cell overgrowth, and later, death.

    2) Thymitaq (TM), or Nolatrexed dichloride (also referred to as AG337), is a noncompetitive inhibitor, of Thymidylate Synthase.

    All these inhibitors are used as anticancer drugs.

    Trimetrexate, Lometrexol and Pemetrexed are also some of the upcoming folate antagonists used in cancer chemotherapy.

     Q.4-Why do the cancer patients on Methotrexate therapy develop glossitis, oral ulcers and diarrhea? How can these symptoms be treated?

     Answer- Methotrexate, an analog of 10-methyl-tetrahydrofolate, inhibits dihydrofolate reductase and has been exploited as an anti-cancer drug.  Methotrexate blocks the cell’s ability to regenerate THF, leading to inhibition of these biosynthetic pathways. The lack of nucleotides prevents DNA synthesis, and these cancer cells cannot divide without DNA synthesis.

    Unfortunately, the effects of Methotrexate are nonspecific and other rapidly dividing cells such as epithelial cells in the oral cavity, intestine, skin, and blood cells are also inhibited. This leads to the side effects associated with methotrexate (and other cancer chemotherapy drugs) such as mouth sores, low white blood cell counts, stomach upset, hair loss, skin rashes, and itching. Less frequent adverse effects include reversible increases in transaminases and hypersensitivity-like pulmonary syndrome. Chronic low-dose methotrexate can cause hepatic fibrosis.

    Leucovorin (LV) is a form of folic acid that can help “rescue” or reverse the toxic effects of methotrexate. LV is not a folate antagonist per se, but the folate derivative 5-formyltetrahydrofolate, After transport through the membrane,LV is metabolized to 5,10-MTHF, thus increasing the availability of 5,10-MTHF. Through this mechanism, it modulates the actions of many folate antagonists.LV can also enhance the antitumor activity of some folate antagonists.

    N5-formyl THF is normally administered 24 hours following treatment with methotrexate; it can be converted to THF by these normal cells by bypassing the block caused by methotrexate. Therefore, these normal cells can synthesize deoxy thymidine and carry out DNA synthesis.





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    Q.1- Justify the statement – ‘Biotin Is a Coenzyme of Carboxylase Enzymes’.

    Answer- Biotin functions to transfer carbon dioxide in a small number of carboxylation reactions Acetyl-CoA, Pyruvate, Propionyl-CoA, and Methylcrotonyl-CoA carboxylases). Biotin is attached at the active site of carboxylases.

    A holocarboxylase synthetase catalyzes the transfer of biotin onto a lysine residue of the apo enzyme to form the biocytin residue of the holoenzyme. Bicarbonate as a source of CO2 is required in the initial reaction for the carboxylation (figure -1).




    Figure- 1-(A) showing the attachment of biotin to the enzyme (B) showing the general reaction for biotin dependent carboxylases

    The reactive intermediate is 1 N carboxy biocytin, formed from bicarbonate in an ATP-dependent reaction. The carboxy group is then transferred to the substrate for carboxylation.

    1) Role of Biotin in carboxylation reactions

    Each Biotin dependent carboxylase catalyzes an essential metabolic reaction;

    Acetyl-CoA carboxylase (ACC) catalyzes the binding of bicarbonate to acetyl-CoA to form malonyl-CoA (Figure-2). Malonyl-CoA is required for the synthesis of fatty acids. The former is crucial in cytosolic fatty acid synthesis, and the latter functions in regulating mitochondrial fatty acid oxidation.


    Figure-2 Shows carboxylation of Acetyl co A to form Malonyl co A,the first and the rate limiting step in fatty acid synthesis

    Pyruvate carboxylase is a critical enzyme in gluconeogenesis—the formation of glucose from sources other than carbohydrates, for example, amino acids. Oxaloacetate formed from pyruvate can be utilized in many other ways depending upon the need of the cell (Figure-3)




    Figure-3 showing the carboxylation of pyruvate to Oxaloacetate , the first step of gluconeogenesis.

    Propionyl-CoA carboxylase catalyzes essential steps in the metabolism of certain amino acids, cholesterol, and odd chain fatty acids (fatty acids with an odd number of carbon molecules). 



    Figure- 4-showing the fate of Propionyl co A

    Propionyl co A is converted first to D- Methyl malonyl co A  and then to its L isomer, ultimately to succinyl co A for complete utilization in the TCA cycle (Figure-4).

    Anaplerotic reactions catalyzed by biotin dependent pyruvate carboxylase (PC) and Propionyl-coenzyme A carboxylase (PCC) regenerate oxaloacetate for the citric acid cycle

    Methylcrotonyl-CoA carboxylase catalyzes an essential step in the catabolism of leucine, an essential amino acid.

    2) Role of Biotin in cell cycle regulation

    Biotin also has a role in regulation of the cell cycle, acting to biotinylate key nuclear proteins such as histones and other proteins.

    Histone biotinylation

    Histones are proteins that bind to DNA and package it into compact structures to form nucleosomes—integral structural components of chromosomes. The compact packaging of DNA must be relaxed somewhat for DNA replication and transcription to occur. Modification of histones through the attachment of acetyl or methyl groups (acetylation or methylation) has been shown to affect the structure of histones, thereby affecting replication and transcription of DNA. Mounting evidences indicate that biotinylation of histones plays a role in regulating DNA replication and transcription as well as cellular proliferation and other cellular responses (Figure-5)



    Figure-5- showing the role of biotin in the body. Holocarboxylase synthetase (HCS) catalyzes biotinylation of the apoenzyme while Biotinidase catalyzes the release of biotin from histones and from the peptide products of carboxylase breakdown.

    Although the major role of biotin is as a coenzyme with carboxylase enzymes as mentioned above, Biotin also plays a special role in enabling the body to use blood glucose as a major source of energy for body fluids. It also activates protein/amino acid metabolism in the hair roots and fingernail cells. Due to its beneficial effects for hair, skin and nails, biotin is also known as the “beauty vitamin”.

    Q.2-What is egg white injury ?


    Raw egg whites contain Avidin, a glycoprotein that strongly binds with biotin and prevents its absorption. once a biotin-avidin complex forms, the bond is essentially irreversible; the biotin-avidin complex is not broken during passage of the food bolus through the stomach and intestines. As a result, biotin is not liberated from food, and the biotin-avidin complex is lost in the feces. Thus, the ingestion of large quantities of raw egg white over a long period can result in a biotin deficiency.

    Cooking egg white denatures avidin, rendering it susceptible to digestion and therefore unable to prevent the absorption of dietary biotin. 

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    Q. 1- What is the active form of thiamine? Explain the coenzyme role of thiamine.

    Answer- Thiamine is also known as vitamin B1. Thiamine occurs in the human body as free thiamine and in various phosphorylated forms: thiamine monophosphate (TMP), thiamine triphosphate (TTP), and thiamine  pyrophosphate (TPP), which is also known as thiamine diphosphate. Thiamine pyro phosphate is the active form of Thiamine. It is rapidly converted to its active form, in the brain and liver by a specific enzyme, TPP synthetase. The synthesis of TPP from free thiamine also requires the presence of magnesium and adenosine triphosphate (ATP)-(Figure-1)









    Figure-1- reaction showing the  activation of thiamine

    Coenzyme functions

    Thiamin has a central role in energy-yielding metabolism, and especially the metabolism of carbohydrates. Pyruvate dehydrogenase, Alpha-ketoglutarate dehydrogenase, and branched chain ketoacids (BCKA) dehydrogenase each comprise a different enzyme complex found within mitochondria. They catalyze the decarboxylation of pyruvate, Alpha-ketoglutarate, and branched-chain amino acids to form acetyl-coenzyme A , succinyl-coenzyme A, and derivatives of branched chain amino acids, respectively; all products play critical roles in the production of energy from food. In addition to the thiamine coenzyme (TPP), each dehydrogenase complex requires a niacin-containing coenzyme (NAD), a riboflavin-containing coenzyme (FAD), and lipoic acid.

    Transketolase catalyzes critical reactions in pentose phosphate pathway. One of the most important intermediates of this pathway is ribose-5-phosphate, a phosphorylated 5-carbon sugar required for the synthesis of the high-energy ribonucleotides, ATP and guanosine triphosphate (GTP). It is also required for the synthesis of the nucleic acids, DNA and RNA, and the niacin-containing coenzyme NADPH, which is essential for a number of biosynthetic reactions. Because transketolase decreases early in thiamine deficiency, measurement of its activity in red blood cells has been used to assess thiamine nutritional status.

    Certain non-coenzyme functions of thiamine are important for nervous tissues and muscles.  It has an important role in the metabolism of neurotransmitters like acetylcholine, adrenaline, and serotonin. Its triphosphate form (TTP) in particular plays a role in the conduction of nerve impulses. TTP phosphorylates, and so activates, a chloride channel in the nerve membrane.

     Q.2- Why is it said that the requirement of thiamine increases with the increasing carbohydrate load ?

    Answer- Because thiamine is required for enzymes involved in glucose metabolism such as Pyruvate dehydrogenase complex, Alpha keto glutarate dehydrogenase complex and transketolase, so its requirement also increases with the increasing carbohydrate load for the proper functioning of these enzymes. The severe thiamine deficiency disease known as Beriberi is the result of a diet that is carbohydrate rich and thiamine deficient. In subjects on a relatively high carbohydrate diet, this results in increased plasma concentrations of lactate and pyruvate which may cause life-threatening lactic acidosis.

     Q.3- What is the cause of lactic acidosis in thiamine deficiency?

    Answer- Lactate, a product of anaerobic glucose metabolism, is generated from pyruvate with lactate dehydrogenase as a catalyst. Pyruvate is normally aerobically metabolized to CO2 and H2 O in the mitochondrion. Initially pyruvate is converted to Acetyl co A with pyruvate dehydrogenase complex acting as a catalyst requiring thiamine, niacin, riboflavin, pantothenic acid and lipoic acid as coenzymes. Acetyl co A is completely oxidized in the Krebs cycle.

    Normally, pyruvate is in a state of equilibrium with lactate and under condition like thiamine deficiency, when PDH complex becomes less active, the equilibrium is shifted towards production of lactate. Lactate is cleared from blood, primarily by the liver, with the kidneys (10-20%) and skeletal muscles to a lesser degree.  Lactic acidosis results from an increase in blood lactate levels when lactate production exceeds consumption and body buffer systems become overburdened. (See Figure-2)

    Acute thiamine deficiency, an uncommon cause of hemodynamic instability in Western countries, is manifested by acute heart failure and neurological deficits. Severe metabolic acidosis is one of its least recognized features. Empiric treatment with thiamine is initiated immediately. Delaying thiamine administration in patients with deficiency can cause severe life-threatening metabolic acidosis and affect recovery.















    Figure-2- showing the role of thiamine in the conversion of pyruvate to Acetyl co A and in the conversion of Alpha keto glutarate to Succinyl co A. The block in the conversion of Pyruvate to Acetyl co A due to non availability of thiamine leads to lactic acidosis.

     Q.4-Explain how a deficiency of thiamine in the diet will produce serious effects on health?


    Describe the link in chemical and metabolic terms between polished rice and sudden cardiac failure.


    thiamine deficiency (causing beriberi) is most common among people subsisting on white rice or highly refined carbohydrates in developing countries and among alcoholics. Symptoms include diffuse polyneuropathy, high-output heart failure, and Wernicke-Korsakoff syndrome.


    Primary thiamine deficiency is caused by inadequate intake of thiamine. It is commonly due to a diet of highly refined carbohydrates (eg, polished rice, white flour, and white sugar). It also develops when intake of other nutrients is inadequate or it can occur with other B vitamin deficiencies.

    Secondary thiamine deficiency is caused by increased demand (eg, due to hyperthyroidism, pregnancy, lactation, strenuous exercise, or fever), impaired absorption (eg, due to prolonged diarrhea), or impaired metabolism (eg, due to hepatic insufficiency). In alcoholics, many mechanisms contribute to thiamine deficiency; they include decreased intake, impaired absorption and use, increased demand, and possibly an apoenzyme defect.


    Deficiency causes degeneration of peripheral nerves, thalamus, mammillary bodies, and cerebellum. Cerebral blood flow is markedly reduced, and vascular resistance is increased.

    The heart may become dilated; muscle fibers become swollen, fragmented, and vacuolized, with interstitial spaces dilated by fluid. Vasodilation occurs and can result in edema in the feet and legs. Arteriovenous shunting of blood increases. Eventually, high-output heart failure may occur.

    Symptoms and Signs

    Early symptoms are nonspecific: fatigue, irritability, poor memory, sleep disturbances, precordial pain, anorexia, and abdominal discomfort.

    Dry beriberi refers to peripheral neurologic deficits due to thiamine deficiency. These deficits are bilateral and roughly symmetric, occurring in a stocking-glove distribution. They affect predominantly the lower extremities, beginning with paresthesias in the toes, burning in the feet (particularly severe at night), muscle cramps in the calves, pains in the legs, and plantar dysesthesias. Calf muscle tenderness, difficulty rising from a squatting position, and decreased vibratory sensation in the toes are early signs. Muscle wasting occurs. Continued deficiency worsens polyneuropathy, which can eventually affect the arms.

    Wernicke-Korsakoff syndrome, which combines Wernicke’s encephalopathy and Korsakoff’s psychosis, occurs in some alcoholics who do not consume foods fortified with thiamine. Wernicke’s encephalopathy consists of psychomotor slowing or apathy, nystagmus, ataxia, ophthalmoplegia, impaired consciousness, and, if untreated, coma and death. It probably results from severe acute deficiency superimposed on chronic deficiency. Korsakoff’s psychosis consists of mental confusion, dysphonia, and confabulation with impaired memory of recent events. It probably results from chronic deficiency and may develop after repeated episodes of Wernicke’s encephalopathy.

    Cardiovascular (wet) beriberi is myocardial disease due to thiamine deficiency. The first effects are vasodilation, tachycardia, a wide pulse pressure, sweating, warm skin, and lactic acidosis. Later, heart failure develops, causing orthopnea and pulmonary and peripheral edema. Vasodilation can continue, sometimes resulting in shock.

    Infantile beriberi occurs in infants (usually by age 3 to 4 wk) who are breastfed by thiamine-deficient mothers. Heart failure (which may occur suddenly), aphonia, and absent deep tendon reflexes are characteristic.

    Because thiamine is necessary for glucose metabolism, glucose infusions may precipitate or worsen symptoms of deficiency in thiamine-deficient people.


    • Favorable response to thiamine

    Diagnosis is usually based on a favorable response to treatment with thiamine in a patient with symptoms or signs of deficiency. Similar bilateral lower extremity polyneuropathies due to other disorders (eg, diabetes, alcoholism, vitamin B12 deficiency, heavy metal poisoning) do not respond to thiamine.

    • In conjunction with whole blood or erythrocyte transketolase activity a thiamine loading test (preloading and post loading, ) is the best indicator of thiamine deficiency. An increase of more than 15% in enzyme activity is a definitive marker of deficiency.
    • If laboratory confirmation is needed, blood thiamine, pyruvate, alpha-ketoglutarate, and lactate can be measured. Also, urinary excretion of thiamine and its metabolites can be measured. A rise in blood lactate level is indicative of thiamine deficiency.


    Supplemental thiamine, with dose based on clinical manifestations


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