II. REVIEW OF LITERATURE
A. HISTORICAL NOTE
B. VITAMIN D METABOLISM
C. MECHANISMS OF ACTION OF 1, 25 (OH)2 D
D. BIOLOGICAL ACTIONS OF VITAMIN D
E . ROLE OF VITAMIN D IN PREGNANCY
F. ROLE OF VITAMIN D IN LACTATION
G. EFFECTS OF HYPOVITAMINOSIS D DURING PREGNANCY ON REPRODUCTIVE FUNCTION
III. MATERIALS & METHODS
Growth retardation is one of the well-known features of severe vitamin D deficiency in infants and young animals. It is generally assumed that skeletal growth retardation is due to sub-optimal concentrations of calcium and phosphorus in the extracellular fluids. The soft tissue growth retardation seen in severe vitamin D deficiency is attributed to anorexia induced by hypocalcemia. However, based upon their investigations in the rat, Steenbock and Herting (1955) proposed that vitamin D may have widespread effects on organic metabolism, of which growth is one manifestation. Recently nuclear receptor sites for l,25, dihydroxy vitamin D3 ( 1,25 (OH) 2 D3] have been reported in such diverse tissues as intestine, bone, skeletal muscle, cardiac muscle, mammary tissue, skin, testis, ovary, pancreas and parathyroid gland (Haddad and Birge, 1975; stumpf et al.,1979; Reichel et al., 1989). Even in the fetus, placenta, yolk sac , kidneys, bones and skin have nuclear receptor sites for 1,25 (OH)2D3 (Haussler, 1986). These observations suggest that vitamin D, may have a more diverse physiological role than hitherto believed to be. Moreover, it has been observed that 1,25(OH)2D3 promotes calcium binding protein synthesis in not only intestinal mucosa (a known target organ) but also in many of the tissues named above (Mayer et al., 1984; Clemens et al., l985). Based on these reports, Haussler et al. (1985) have proposed that vitamin D may have a fundamental role in the regulation of cellular growth and differentiation.
It is generally accepted that daily administration of 100 IU of vitamin D is sufficient to prevent rickets in infants. However , clinical observations suggest that optimum growth may not occur when vitamin D intake of the infant is 100 IU per day. Stearns et al (1936) compared the length of infants on vitamin D supplements varying from 60-130 to 1800 IU per day. Optimum growth was shown by infants receiving 340-600 IU of vitamin D per day. Both the lower and higher doses decreased growth significantly. These findings were subsequently confirmed by many workers (Slyker et al .,1937; Jeans and Stearns, 1938; Greer et al., 1981).
Severe vitamin D deficiency in pregnant women is known since long to produce congenital rickets (Maxwell- et al.,1939; Liu et al.,1940; snapper, 1956). However the possible role of vitamin D in reproduction including intrauterine growth of the fetus has attracted attention only recently. In chicks, vitamin D seems to be essential for proper egg hatchability (Henry and Norman, 1978) and for normal embryo development (Sunde et al.,1978).
Halloran and De Luca (1980a) have reported decreased fertility, decreased litter size and greater incidence of neonatal deaths in vitamin D deficient female rats. Administration of toxic dose of vitamin D (20, 000 IU per day) was also found to impede fertilization , if given before mating or produce degeneration and resorption of the implanted blastocyst if given in early pregnancy (Nebel and Ornstein, 1966). Toxic doses of vitamin D in pregnant rats also produced growth retardation in the fetus (Ornoy et al 1968) as well as structural alteration in the placenta (Nebel &Ornoy, 1971).
All these experimental studies on the effects of vitamin D on reproductive function have been performed either on vitamin D deficient animals where the results are clouded by the concurrent maternal malnutrition because of anorexia or in pregnant rats on toxic doses of vitamin D. Effect of administration of vitamin D in moderately high but non-toxic doses in pregnancy has not been studied. The apparent benefits of such a therapy on intrauterine and neonatal growth have been demonstrated in a few clinical studies by the author (Marya et a1., 1981a ; Marya et al, 1981B) and others (Brooke et al, 1980; Maxwell et a1., 1981). Studied by the author were conducted in Hindu women of Haryana, who in non-pregnant state, do not show any evidence of overt or occult vitamin D deficiency (Marya et al, 1981b ). Administration of 600,000 units of vitamin D, in 7th and 8th months of pregnancy led to birth of infants with significantly greater birth weight and increase in certain other anthropometric measurements such as length, head circumference and skinfold thickness (Marya et al, 1981b). Administration of 1200 IU of vitamin D, per day, throughout the third trimester also improved the fetal birth weight but to a lesser extent (Marya et a1., 1981a). Brooke et al. (1980) administered 1000 IU of vitamin D, per day to Asian immigrants in the U.K. during the third trimester of pregnancy and observed a significant decrease in the incidence of low birth weight babies. Although, there was no significant difference between the mean birth weight in the supplemented and non- supplemented groups but a follow up study revealed significantly greater weight and height of babies from the supplemented group at the age of 9 months and 12 months, even though neither the mothers nor the babies received any vitamin D supplements postnatal (Brooke et al., 1981) . The clinical studies suggest that administration of moderately high doses of vitamin D during pregnancy not only improves the intrauterine growth of the fetus but also continues to confer the beneficial effect on the growth of the baby during the first year of life . However community nutritional studies are somewhat handicapped in that even when the subjects are taken from the same socio-economic strata of the society, the environmental and nutritional conditions cannot be rigidly controlled. These difficulties assume greater importance in the studies on vitamin D where subtle differences in the solar exposure, cutaneous pigmentation and manner of dress may produce important effects on the cutaneous production of vitamin D. Hence confirmation of the results obtained in human subjects by experimental studies in the rat was considered imperative. This study was designed to elucidate the effects of vitamin D supplementation during pregnancy on the skeletal and soft tissue growth in the rat pups.
II. REVIEW OF LITERATURE
A. HISTORICAL NOTE
Like most of the vitamins, the discovery of vitamin D was a consequence of the knowledge of its deficiency disorders. The first clinical description of rickets appeared in 1650 (Olson & De Luca, 1973) after the widespread appearance of rickets in northern Europe due to industrialization. The incidence of rickets reached serious proportions with the development of urbanized industrial population. Smoky skies coupled with relatively indoor life necessitated by this environment drastically reduced the solar exposure of the people, thereby curtailing the chief source of vitamin D. Cod-liver oil was recognized as a therapeutic measure for curing rickets in 1811 (Olson & De Luca, 1973). By the beginning of 20th century, the relation between dietary deficiency and many diseases such as beriberi and scurvy was demonstrated and the term vitamin was introduced by Funk in 1912 . In a series of publications, Mellenby ( 1918 a, 1918 b, 1919) demonstrated that rickets is a deficiency disease and it could be cured by cod-liver oil- or butter fat but attributed the cure to "fat soluble A.” Huldscginsky in, 1920, provided the experimental proof of the curative effects of U.V. radiation on rickets (Olson & De Luca, 1973). In 1925, McCollum had the honor of naming the fourth discovered vitamin as vitamin D ( McCollem et al, 1925). In 1924, Steenbock showed that phytosterol and ergosterol became rich in vitamin D after U.V. radiation (Steenbock, 1924; Steenbock & Black, 1925). Subsequently, vitamin D was crystallized from irradiated ergosterol and the compound was named calciferol (Askew et al, 1930; Windaus, 1932 ). In 1935, Windaus et al. determined the chemical structure of calciferol, and 7- dehydrochlesterol was shown to be a provitamin D. After this, except for official adoption of the name vitamin D2, for ergocalciferol and vitamin D3, for cholecalciferol (Patterson, 1952), the research activity on vitamin D almost came to stand still. Only in late sixties, the role of the liver and the kidney in vitamin D metabolism was elucidated. Since then, there has been a spurt of research activity on vitamin D and every year hundreds of papers appear in literature on various aspects of vitamin D metabolism.
B. VITAMIN D METABOLISM
(i) Cutaneous Production of Vitamin D
Diet is a very poor source of vitamin D. Vitamin D does not occur in vegetable kingdom. In non-vegetarian diet, egg and fish liver oil are the only important sources of vitamin D. Cow’s milk is a poor source. However, cutaneous synthesis of vitamin is an important source of vitamin D. Most of the vitamin D synthesis occurs in the actively growing layers of the epidermis (strata spongiosum and basale) by exposure to sunlight(Holick et al., 1980). Radiation energies between 290 and 320 nm are most effective (McLaughlin et al., 1982). 7-dehydro-cholesterol present in the epidermis acts as a provitamin D. Ultraviolet radiation produces a cleavage of B ring thereby forming previtamin D, (9,10 -secosteroid). Previtamin D undergoes a temperature dependent isomerization to form vitamin D3 (also called cholecalciferol), taking 2-3 days for completion of the process . The unique thermally regulated synthesis of vitamin D3 ensures a gradual release of the vitamin from the epidermis into circulation. This concept is confirmed by the observation that subjects exposed to whole body U.V. radiation have a significant increase in the circulating concentrations of vitamin D3, about 6-9 hours after the exposure that reaches a peak 24-48 hours after the exposure, before gradually returning to baseline by 7 days (Adam et al., 1982). Once vitamin D is formed, vitamin D-binding protein in the dermal capillary circulation helps to translocate the vitamin from blood-less epidermal tissue into circulation.
Melanin pigment present in the epidermis interferes with the synthesis of vitamin D by absorbing U.V. radiation. The view of Loomis (1967) that skin pigmentation is evolved for the control of vitamin D synthesis in the skin is supported by excessive cutaneous melanin seen in populations exposed to greater U.V. radiation. Moreover, it has been observed that when surgically excised skins from Blacks and Caucasians were exposed to solar radiation, greater amount of vitamin D was produced in the latter (Holick et al., 1981). However, now it is clear that melanin is only one of the many factors that regulate photosynthesis of vitamin D in the skin. Cutaneous production of vitamin D seems to be under an autoregulatory control. Excessive exposure of even Caucasian skin to sunlight does not cause vitamin D intoxication. Continuous exposure to UV radiation depletes cutaneous provitamin D, but does not increase production of previtamin D. Holick et al. ( 1981) have reported the effect of exposure of skin for different durations to sunlight. During the first 10-15 minutes of exposure, approximately 15% of provitamin D changed to previtamin D. After one hour of exposure, 40 % provitamin D was depleted but only 15 % increase in previtamin D was observed. The remaining 25% of photolyzed provitamin D was accounted for by the presence of inactive isomers, tachysterol and lumisterol (Fig. 1). Further solar exposure depleted the stores of provitamin D in the epidermis, but the concentration of previtamin D3 or vitamin D3 did not increase. Vitamin D3 being heat-sensitive, may also be photodegraded to 5,6-trans-vitamin D3 and suprasterol (Web et al, 1986).
Because of the complex mechanism of vitamin D3 production in the epidermis, the amount of solar exposure required for providing vitamin D adequate for the body’s requirements varies in different individuals and under different conditions. The photosynthesis of vitamin D3 depends upon (i) the surface area of the skin exposed to sunlight, (ii) the time of the day of exposure (UV radiation is most intense between 11 AM and 2 PM) , (iii) the amount of melanin pigment present in the epidermis, (iv) latitude (UV radiation is most intense at the equator), (v) season (in winter less UV radiation reaches the surface of the earth, (vi) Environmental pollution such as smoke, fog and dust prevents UV radiation from reaching the earth. However, prolonged exposure to sunlight does not necessarily mean greater production of vitamin D since as mentioned earlier, solar radiation can isomerize previtamin D3 to inactive, isomers, tachysterol and lumisterol as well as produce photodegradation of vitamin D3 (Holick, 1986).
Vitamin D-binding protein has no affinity for tachysterol or lumisterol and hence translocation of these isomers into circulation does not occur. These products are sloughed off during natural turnover of skin. Patients with uremia seem to be unable to produce vitamin D in the skin. It is believed that one or more substances present in the skin of a patient with chronic renal failure act like melanin and absorb UV radiation (Holick, 1986)).
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Fig.1. Vitamin D metabolism.
(ii) Hepatic Metabolism of vitamin D
Vitamin D3, synthesized in the skin, enters the circulation bound to vitamin D-binding protein. Dietary vitamin D2 or D3 enters the circulation through lymphatic system. Subsequently, both vitamins D2 and D3 are metabolized similarly.
In the liver, vitamin D is metabolized by vitamin D-25-hydroxylase to form 25-hydroxyvitamin D [25(OH) D] (now also known as calcidiol). The enzyme is located in the mitochondrial and microsomal fractions of the hepatocytes (Ponchon & DeLuca 1969; De Luca, 1984). Although there are few reports of the presence of extrahepatic vitamin D-25-hydroxylase in the chick and the rat (Tucker et al., 1973; Olson et al., 1976), the liver seems to be the only site of 25 (OH) D synthesis in humans. The reserve capacity of vitamin D-25-hydroxylase in the liver is substantial. Severe parenchymal damage is required to lower the level of plasma 25(OH) D (Long et al., 1976). The enzyme vitamin D-25-hydroxylase does not seem to be tightly regulated since circulating levels of 25(OH) D vary with the amount of dietary intake of vitamin D or with the degree of solar exposure (Holick et al., 1986). Decreased plasma 25(OH) D levels are observed in patients with nephrotic syndrome having proteinuria greater than 4g/day, due to renal loss of vitamin D tagged to vitamin D-binding protein (Pietrek & Kokot, 197).
(iii) Renal Metabolism of Vitamin D
As early as 1833, Lucas recognized the association between chronic renal disease and bony lesions resembling rickets. Observations of similarity in bony lesions in patients of nutritional rickets and those with chronic renal failure, led Liu and Chu (1943) to propose that uremia interferes with the action of vitamin D. It was only in 1970 that Fraser& Kodicek demonstrated the intimate relation between the kidney and vitamin D metabolism. These workers demonstrated that homogenates of chick kidney could metabolize 25(OH) D to a biological active metabolite. It was also shown that physiological concentrations of 25(OH) D could not stimulate intestinal calcium transport in anephric rat (Boyle et al., 1972). Fraser & Kodicek (1970) identified the active metabolite as 1, 25-dihydroxycholecalficerol [ 1,25 (OH)2 D3] ( now also known as calcitriol). The renal 25 (OH)-1- alpha-hydroxylase is located in the proximal convoluted tubules ( Suda &Kurokova, 1983) . It is now accepted that 1, 25-dihydroxy metabolites of vitamin D2 or D 3 are the biologically active forms of vitamin D2 and D3 respectively. These metabolites are 10 times more active than vitamin D2 or D3 in healing rickets or stimulating intestinal calcium absorption (De Luca, 1984).
The activity of renal 25-OH-1-alpha hydroxylase appears to be tightly regulated since plasma 1, 25 (OH) 2 D3 concentration remains constant over a wide range of substrate 25 (OH) D3. Parathormone (PTH) seems to play a crucial role in the synthesis of calcitriol since it was found that hypocalcemic vitamin D deficient rats could metabolize calcidiol to calcitriol more effectively than normocalcemic vitamin D replete rats (Boyle et al., 1971). But, when vitamin D- deficient hypocalcemic rats were thyroparathyroidectomized, the difference was lost (Garabedian et al., 1972). However, according to Holick et al. ( 1986 ), PTH may not be absolutely essential for the synthesis of 1, 25 (OH)2 D, since patients with hypoparathyroidism often have low-normal concentrations of calcitriol. Under certain physiological conditions, factors other than PTH may regulate 1, 25 (OH)2 D synthesis. In pregnanc and lactation, growth hormone, estrogens and prolactin seem to enhance renal production of 1,25 (OH)2 D directly or indirectly (Baksi &Kenny, 1977; Boass et al., 1977) .
(iv) Alternate Renal Metabolic Pathways for 25 (OH) D
When vitamin D nutrition and circulating plasma concentrations of calcium and phosphorus are normal, 25 (OH) D is metabolized into a variety of products (Fig. 1), by hydroxylation at C 24, 25 and 26 to form 1,25 (OH)2 D, 24,25 (OH)2 D and 25,26 (OH)2 D (Holick et al., 1986). The plasma concentrations of each of 24, 25 (OH)2 D and 25,26 (OH)2D are 50-100 times the concentration of 1,25 (OH)2 D. The metabolites other than 1, 25 (OH)2 D have no biological activity. Production of 25 (OH) D is uncontrolled. Its plasma concentrations vary directly with the dietary intake/ cutaneous production of vitamin D. When plasma concentration of 1, 25 (OH)2D is adequate, remaining 25(OH) D is converted to 24,25 (OH)2 D or 25,26 (OH)2 D. Renal 25 (OH)-1-alpha-hydroxylase converts two inert metabolites mentioned above to 1,24,25 trihydroxy cholecalciferol [1,24,25 (OH)3 D] and 1,25,26 trihydroxy cholecalciferol [1,25,26 (OH)3D]. The trihydroxy metabolites again have no biological activity (De Luca, 1984).
(v) Extrarenal Metabolism of 25(OH)D
Initially, kidney was believed to be the only site of 1, 25 (OH)2 D synthesis. Twenty-four hours after injection of 3H- 25(OH)D, 3H-1,25 (OH)2 D could be detected in the blood and tissues of vitamin D deficient rats but not in vitamin D deficient rats that had undergone bilateral nephrectomy before receiving radioactive 25 (OH) D (Gray et al., 1971). Later it was discovered that bilateral nephrectomy reduced, but did not abolish the conversion of 25(OH) D to 1, 25 (OH)2 D ( Weisman et al., 1978 b). In vitro studies have confirmed that placenta is the one of the sites for 1, 25 (OH)2 D synthesis in pregnancy (Whitsett et al., 1981). In addition, in vitro, a wide variety of cultured cells from normal human bone, and osteosarcoma have a capacity to convert 25 (OH) D to 1, 25 (OH)2 D (Turner et al., 1980; Howard et al., 1981; Howard et al, 1982). These observations also help to explain why hypercalcemia occurs in some patients of sarcoidosis, tuberculosis, silicosis, Hodgkin’s disease and non-Hodgkin lymphoma. Such patients have been shown to have elevated plasma levels of 1, 25 (OH)2 D (Gkonon et al., 1984; Breslau et al., 1984; Davies et al., 1985). To date, 25-OH-1-α-hydroxylase has been reported in many cells and tissues including prostate, breast, colon, lung, pancreatic β cells, monocytes, and parathyroid cells. However, the extrarenally produced 1, 25(OH)2D primarily serves as an autocrine/paracrine factor with cell-specific functions [(Fig. 2) (Dusso et al., 2005)].
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Fig.2. Renal and extrarenal production of 1,25-dihydroxyvitamin D. [After Dusso et al (2005)[.
C. MECHANISMS OF ACTION OF 1, 25 (OH)2 D
Vitamin D receptor (VDR) was discovered in 1968 (Haussler et al., 1968). In 1980s, VDR was found to be widely distributed in different tissues of the body such as gonads, stomach, epidermis, pituitary gland, pancreas, breast, parathyroid gland, thymus, cardiac muscle, skeletal muscle, placenta etc. (Stumpf et al., 1979; Mayer et al., 1984). Initially no physiologic significance was attached to such reports; only intestine, bone and kidney continued to be recognized as the target tissues of 1, 25 (25)2 D. Subsequently, reports indicated calcium-binding protein synthesis in many of these tissues, including brain (Mayer et al., 1984). In vitro, 1, 25 (OH)2 D was found to inhibit proliferation of human fibroblast cells and keratinocytes, increase TSH synthesis, inhibit PTH synthesis (Amento et al., 1984; Smith et al., 1986).
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Fig. 3. Mechanism of genomic (left) and non-genomic (right) actions of vitamin D3. (Adapted from Sergio et al., 2003).
(i) VDR and transcription regulation
The genomic action of 1, 25(OH)2 D3 is well described. Most of the pleiotropic and long-term actions of 1, 25(OH)2 D3 are mediated through genomic actions. 1, 25(OH)2D3, in concert with vitamin D binding protein (DBP), is transported to the nucleus, where it binds to the vitamin D receptor (VDR). The VDR then complexes with retinoid X receptor (RXR), forming a heterodimer, which then binds the vitamin D-responsive element (VDRE) located in the promoter region of the gene. This association recruits either co-activators or co-suppressor molecules (depending on the tissue type). This triple complex then binds to the transcription machinery ( Sergio et al., 2003 ;Fig. 3) .
(ii) Non-Genomic action of 1,25 (OH) D
A variety of hormones, those serve as ligands for nuclear hormone receptors, also exert biological actions that do not require gene regulation. They seem to act through cell membrane receptor rather than nuclear receptors.1, 25 (OH) 2 D3 has been shown to have rapid effects in selected cells through membrane receptors.The proposed mechanism of non-genomic action of 1, 25 (OH)2 D3 is shown in Fig. 3.
D. BIOLOGICAL ACTIONS OF VITAMIN D
(i) Classical actions
The classical actions of vitamin D on the intestine, bone and kidney are concerned with calcium homeostasis.
In the enterocytes of the small intestine, the genomic action of 1,25 (OH)2 D3 results in greater production of not only calcium binding protein, calbindin, but also alkaline phosphatase and a brush border protein (Mayer et al., 1984; Wasserman et al., 1984, Bickle & Munson, 1985).The net result is greater absorption of dietary calcium and phosphates. The mechanisms by which 1, 25(OH)2 D3 regulates transcellular calcium transport are best understood in the intestine. Here 1, 25(OH)2D3 stimulates calcium entry across the brush border membrane into the cell, transport of calcium through the cell, and removal of calcium from the cell at the basolateral membrane. Entry at the brush border membrane occurs down a steep electrochemical gradient. The molecular mechanism of 1, 25 (OH)2 D3 as a stimulator of intestinal phosphate absorption remains unknown, despite many efforts by the investigators (Jones et al., 1998), but a cytosolic calcium binding protein calbindin-D9K seems to be involved (Anderson et al 1998).
Bone and muscle accumulate about 60% of injected dose of vitamin D (De Luca, 1976). Though gross skeletal abnormalities have been observed in vitamin D deficient animals, no direct effect of 1, 25 (OH)2 D3 on the process of ossification has been observed. 1, 25 (OH)2 D3 does not seem to be essential for ossification of bone. When plasma calcium and phosphate levels were maintained at normal range in vitamin D deficient rats by dietary manipulation, the skeletal histology was found to be normal (Holtrop et al., 1986). However, in cultured rat osteosarcoma cells, 1, 25 (OH)2 D3 stimulates the synthesis of osteocalcin , the bone derived protein, in a dose dependent manner (Price, 1984). In patients with postmenopausal osteoporosis, 1, 25 (OH)2 D3 administration has been shown to increase circulating osteocalcin levels (Zerwekh et al., 1985).
Mobilization of calcium from the bone is another well-known function of vitamin D, especially when administered in pharmacologic doses. At physiologic concentrations, 1, 25 (OH)2 D3 acts in concert with parathormone to stimulate osteoclastic activity (Garabedian et al., 1974). At pharmacologic concentrations, it was found to stimulate osteoclastic activity by inducing stem cells to differentiate into osteoclast cells (Haussler, 1986). Exposure to human peripheral monocytes that possess receptors for 1, 25 (OH)2 D3 results in their differentiation into multinucleated giant cells capable of mobilizing calcium from bone chips (Gray & Cohen, 1985).
The most important endocrine effect of 1, 25(OH)2D3 in the kidney is a tight control of its own homeostasis through simultaneous suppression of 1-α-hydroxylase and stimulation of 24-hydroxylase.
In the kidneys, 1, 25 (OH)2 D increases reabsorption of calcium in the distal tubules through a cytosolic transport protein calbindin-D28K (Anderson et al., 1998). However, 1, 25(OH)2 D3 involvement in the renal handling of calcium and phosphate continues to be controversial due to the simultaneous effects of 1, 25(OH)2D3 on plasma PTH and on intestinal calcium and phosphate absorption, which affect the filter load of both ions .
(ii) Non-classic actions of vitamin D
1. Role of Vitamin D Hormone in the Parathyroid Gland
Perhaps the most well-established non-classic function of 1, 25(OH)2 D3 is in the parathyroid gland. Specific localization of 1, 25(OH)2 D3 in the parathyroid gland (Stumpf et al., 1979) and the presence of VDR (Henry & Newman, 1975) strongly suggested that 1,25(OH)2 D3 may have a direct action through its receptor in the parathyroid glands. Moreover, PTH secretion by isolated parathyroid glands or cells could be suppressed by the direct administration of 1, 25(OH)2 D3 (De Luca, 1976). Likewise, the vitamin D appears to be involved with the development of both primary and secondary hyperparathyroidism (Laundry et al., 2011). The specific mechanism by which vitamin D interacts with the parathyroid gland to bring about observed effects is not yet fully understood. But, these observations have implication on prospects of possible medical treatment of hyperparathyroidism (Hellman et al.,1999).
2. Role of Vitamin D Hormone in Skin
Hosomi et al. (1978) provided evidence for the first time that 1, 25(OH)2 D3 induces keratinocyte differentiation.. Exactly how important this differentiation effect of 1, 25(OH)2 D3 is in vivo is difficult to assess. Certainly, vitamin D-deficient animals do not have a problem with keratinocyte differentiation. Thus hyperproliferation of the keratinocyte and failure to differentiate is not found in vitamin D-deficient animals (De Luca, 1971). The differentiation of the keratinocyte is associated with an inhibition of proliferation. This inhibition of proliferation has been utilized in the treatment of hyperproliferative diseases of skin as, for example, psoriasis. Both 1, 25(OH)2 D3 and analogs can be used as a significant therapy against psoriasis with as many as 70% patients responding to this treatment (Parez et al., 1986). However, exactly how 1, 25-(OH)2 D3 induces differentiation of the keratinocyte and inhibits proliferation remains to be investigated. It has been proposed that the keratinocyte functions in a paracrine fashion in which 1, 25 (OH)2 D3 is produced by the keratinocyte itself to stimulate differentiation of the keratinocyte (Bickle et al., 1986).
3. Role of Vitamin D in the Immune System
The presence of the VDR in activated T lymphocytes was reported by Provvedine et al. (1987). These results suggested a role for 1, 25-(OH)2 D3 in the immune system, but the role is just now beginning to be defined. VDR have been reported in thymus, a repository of immature lymphocytes, as well. Vitamin D deficiency markedly reduces the ability of mouse to develop delayed hypersensitivity reaction (Young et al., 1983). These results suggest that T-helper cell lymphocyte is vitamin D responsive, but both immunostimulation and immunosuppression can be found in in vivo conditions. Currently, there is no evidence that B-lymphocyte-mediated immunity is influenced by 1, 25 (OH)2 D3 (Jones et al., 1998).
The most dramatic results obtained to date in the immune system are those found in experimental autoimmune encephalomyelitis (EAE) that can be induced in mice. Administration of 1, 25 (OH)2 D3 suppressed the development of the disease in experimental animals(Fig 4) (Cantora et al., 1996). Current results strongly suggest that 1, 25 (OH)2 D3 or its analogues function by stimulation TH-2 T-helper cells to produce transforming growth factor-β1 and IL-4.
Of some interest is the idea that immunomodulation action of vitamin D might be useful in the management of transplant rejection (Jones et al., 1998). The possible use of vitamin D in the treatment of autoimmune diseases such as diabetes mellitus, rheumatoid arthritis is being investigated.
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Fig. 4. Effect of administration of 1,25 (OH) 2 D3 on the development of induced experimental autoimmune encephalitis in rats (After Cantorna et al., 1996)).
4. Role of vitamin D in Insulin Secretion.
The presence of a VDR in the cells of islets of Langerhans is now well accepted, but it is unclear as to what, if any, role vitamin D plays in the functioning of the islet cells. Initial results revealed that vitamin D-deficient rats were unable to respond to a glucose challenge by secreting appropriate amounts of insulin, which could be corrected by the administration of 1, 25(OH)2 D3 (Cherotow et al., 1983). Other studies suggested that the effect of 1, 25 (OH)2 D3 was mediated by the action of vitamin D in raising plasma calcium concentration (Cherotow et al., 1986). Moreover, onset of experimental diabetes can be delayed by administration of 1, 25 (OH)2 D3 (Methian et al., 1992). Thus, a role of vitamin D on islet insulin release is most likely; either direct or indirectly through its effect on plasma calcium concentrations. Therefore, the relationship between vitamin D and diabetes is certainly worthy of further investigations.
5. Role of Vitamin D in Reproduction
Initially, during the course of producing vitamin D-deficient rats, came the observation that female reproduction is markedly diminished in vitamin D deficiency (Halloran & De Luca, 1980). An 80 % reduction in fertility was found and could not be corrected by correcting the hypocalcemia (Kwiecinski et al., 1989 a ). This defect, therefore, is quite clearly one related to an absence of the vitamin D molecule. The infertility brought about by vitamin D deficiency in the female rat can be easily corrected by the administration of 1, 25-(OH)2D3 (Kwiecinski et al., 1989 b)). All such reports suggested that the ovary is a target of vitamin D action. More over the observations that ovarian cells contain VDR (Dokos et al., 1983) and 1, 25(OH)2 D3 accumulates in the ovarian cells (Stumpf et al., 1979) lends further support to the view.
In the case of male reproduction, vitamin D deficiency also reduces the effectiveness of the male (Kwiesinki et al., 1989 ). A significant reduction found in sperm count in vitamin D rats (Sood et al., 1992) could be reversed by vitamin D repletion (Sood et al., 1995 ). However, the diminished male fertility can also be corrected by merely providing additional calcium, raising plasma calcium concentration which in turn restores fertility (Kwiecinski et al 1989). In chicks, vitamin D seems to be essential for proper egg hatchability (Henry et al., 1978) and for normal embryo development (Sunde et al., 1978).
Severe vitamin D deficiency in human pregnancy is known since long to produce congenital rickets (Maxwell et al., 1939; Liu et al., 1940). The apparent benefits of vitamin D supplementation on intrauterine and neonatal growth of the fetus were initially demonstrated in Asian women by Brooke et al (1980 ) and by Marya and his colleagues (Marya et al., 1981; Marya et al 1984; Puri et al., 1989). Brooke et al. administered 1000 IU of vitamin D, per day to Asian immigrants in the U.K. during the third trimester of pregnancy and observed a significant decrease in the incidence of low birth weight babies. Although, there was no significant difference between the mean birth weight in the supplemented and non- supplemented groups but a follow up study revealed significantly greater weight and height of babies from the supplemented group at the age of 9 months and 12 months, even though neither the mothers nor the babies received any vitamin D supplements postnatal (Brooke et al., 1980). Studies by Marya and his colleagues (Marya et al., 1981; Marya et al 1984; Puri et al., 1989) were conducted in Hindu women of Haryana (India). Administration of 600,000 units of vitamin D, in 7th and 8th months of pregnancy led to birth of infants with significantly greater birth weight and increase in certain other anthropometric measurements such as length, head circumference and skinfold thickness . Administration of 1200 IU. of vitamin D, per day, throughout the third trimester also improved the fetal birth weight but to a lesser extent . The clinical studies suggest that administration of moderately high doses of vitamin D during pregnancy not only improves the intrauterine growth of the fetus but continues to confer the beneficial effect on the growth of the baby during the first year of life a1so.
The clinical and experimental studies led to a large number of studies on the effects of vitamin D supplementation during pregnancy on maternal and fetal outcomes. The rationality of such studies was the reports that women all over the world suffered from vitamin D deficiency during pregnancy (Dawood et al 2007; Sachan et al., 2005). Maternal vitamin D deficiency has been found to affect postnatal head and linear growth (Brunvand et al., 1996). Hollis et al. (2011) conducted a double blind trial on the possible benefits of vitamin D supplementation during pregnancy. It was concluded that vitamin D supplementation of 4000 IU/d for pregnant women is safe and most effective in achieving sufficiency in all women and their neonates regardless of race, whereas the currently suggested requirement is comparatively ineffective at achieving adequate circulating 25(OH)D concentrations, especially in African Americans.
De-Regil et al. (2012) conducted a Cochrane review of six randomized controlled trials in 1023 women. The results showed that the provision of vitamin D supplements during pregnancy improves the women’s vitamin D levels, as measured by 25-hydroxyvitamin D levels, at term. However, the clinical significance of this finding is yet to be determined as there is no evidence that vitamin D supplementation prevents pre-eclampsia, gestational diabetes or impaired glucose tolerance. Data from three trials involving 463 women show a trend for women who receive vitamin D supplementation during pregnancy to less frequently have a baby with a birth weight below 2500 grams than those women receiving no treatment or placebo, although the statistical significance was border line. The number of trials and outcomes reported are too limited, and in general are of low quality, to draw conclusions on the usefulness and safety of this intervention as a part of routine antenatal care. Further rigorous randomized trials are required to evaluate the role of vitamin D supplementation in pregnancy (De Regil et al, 2012).
E. ROLE OF VITAMIN D IN PREGNANCY
The study of vitamin D metabolism in pregnancy and lactation was prompted by the observations of osteomalacia in pregnant and lactating women in India and China (Maxwell and Miles, 1925; Ford et al. 1973). The requirement of the fetus for calcium during gestation and of the neonate during lactation puts considerable demand on the mother. Now, it is being realized that even Caucasian women tend to go into biochemical osteomalacia during pregnancy and lactation and need to be supplemented with vitamin D (Dawood and Wagner, 2007, Sachan et al., 2005;Watney and Rudd, 1974) .
(I) Intestinal Calcium Absorption in Pregnancy
Greater calcium demands of the body during pregnancy and lactation can be met with by increasing the dietary calcium intake, or by increasing efficiency of intestinal calcium absorption mechanism or decreasing calcium losses in the urine. In rats as well as humans, increased appetite for food has been observed in pregnancy (Cripps and Williams, 1975, Toverud & Boass, 1979). But in women residing in developing countries, financial constraints limit the actual increase in intake of food. Obviously, increased efficiency of intestinal calcium absorption remains the chief mechanism of increasing availability of calcium to the mother. A marked increase in the efficiency intestinal calcium absorption in later months of pregnancy has been observed in humans (Heaney &Skillman, 1971) and sheep (Braithwaite et al. , 1970). It has been attributed to vitamin D as well as some other factors. Halloran et al. (1980 b) estimated intestinal calcium transport ratio (serosal Ca++/ mucosal Ca++ ratio) in vitamin D-replete and vitamin D-deficient pregnant rats. The ratio was 6.0 in vitamin D-replete pregnant rats as compared to 3.0 in vitamin D-replete non-pregnant control rats. Surprisingly, the ratio was 3.5 in vitamin D-deficient pregnant rats as compared to 2.0 in vitamin D-deficient non-pregnant rats. In rats on a fixed dietary intake of vitamin D, the plasma concentrations of 25 (OH) D and 24, 25 (OH)2 D decreased while that of 1,25 (OH)2 D3 and PTH increased during later days of pregnancy (Reitz et al., 1977; Halloran et al.,1979; Pitkin et al., 1979;Bouillon and De Moor, 1973). Such studies are cited to explain PTH-mediated increase in synthesis of 1, 25 (OH)2 D3 during pregnancy leading to increased efficiency of intestinal calcium absorption. However, some workers have failed to observe increased plasma concentration of PTH during pregnancy (Wieland et al., 1980; Whitehead et al., 1981; Gillette et al., 1982). It has been suggested that during pregnancy, increased plasma levels of growth hormone, prolactin and placental lactogen stimulate 25 (OH) 1-alpha-hydroxylase activity in the kidney (Tanaka et al., 1976; Spanos et al., 1976; Baksi & Kenny, 1977; Baksi et al., 1978; Tanaka et al., 1978). Prolactin has been shown to increase intestinal calcium absorption not only in vitamin D-replete but also vitamin D –deficient animals (Mainoya, 1975 a, b; Pahuja and Deluca, 1981. The mechanism by which prolactin increases intestinal calcium is not exactly clear. Possibly, the effect of prolactin on calcium transport is mediated through intestinal mucosal hypertrophy. Prolactin is a tropic hormone for mammary glands. Similar action in the intestinal mucosa is quite likely (Harding & Cairnie, 1975; Maionoya, 1978). In pregnancy an increase in villus height, absorptive cell number and tissue weight has been reported (Cripps & Williams, 1975; Burdett & Ruk, 1979).
Renal conservation of calcium is another mechanism which may be utilized by the body for improving the availability of calcium to the fetus. However, due to increased GFR in later months of pregnancy, the excretion of many urinary constituents such as amino acids, glucose and calcium increases (Howarth et al., 1977; Marya et al., 1987; Maikranz et al., 1989;). Calcium conservation during pregnancy may be observed in in those with vitamin D-deficiency when the affected women tend to develop hypocalciuria ( Marya et al., 1987). An association between hypocalciuria and pregnancy-induced hypertension has also been reported (Donovan et al., 2009).
(II) Bone Metabolism in Pregnancy
The effect of pregnancy on bone metabolism is not entirely clear. It has been suggested that minerals accumulate in the bone during pregnancy in anticipation of calcium demands during lactation (Denzie et al, 1955; Heaney & Skillman, 1971). In vitamin D-deficient rat, there is roughly 25% increase in femoral bone mineral content by the end of pregnancy. In vitamin D-replete rat, however no change in mineral content could be demonstrated during pregnancy (Halloran % DeLuca, 1980 C). Many studies have suggested that in the rat under normal dietary conditions of vitamin D, calcium and phosphate intake, there is no change in bone mineral content during pregnancy. Under such conditions, the calcium requirements of the fetus and neonate are met with by increased intestinal calcium absorption (Naismith, 1966; Miller et al., 1982). Under conditions of dietary restriction of calcium, bone mineral is sacrificed to support fetal demands (Rasmussen, 1977 a, 1977 b). Many workers have investigated the changes in bone mineral content in human pregnancy but results remain inconclusive (Gambaccini et al. 1995; Yamaga et al., 1996).