131 436 .THS- t.” “it. May LifiRARY Michigan State University This is to certify that the thesis entitled THE INFLUENCE OF GESTATIONAL DIETARY CALCIUM ON SERUM l,25-DIHYDROXYCHOLECALCIFEROL IN SOWS AND THEIR BABY PIGS presented by Wuryastuti) Hastari has been accepted towards fulfillment of the requirements for Master Large Animal Clinical degree in Sciences Mair/«L O v Major pro essor Date {/2 2/:9/ 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution ———_ —-i v , 77—_ , .v— , i 7 7,, -— MSU LIBRARIES .—:_. RETURNING MATERIALS: Piace in book drop to remove this checkout from your record. FINES wil] be charged if book is returned after the date stamped beiow. THE INFLUENCE OF GESTATIONAL DIETARY CALCIUM ON SERUM 1,25-DIHYDROXYCHOLECALCIFEROL IN SOWS AND THEIR BABY PIGS BY Wuryastuti Hastari A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Large Animal Clinical Sciences 1987 ABSTRACT THE INFLUENCE OF GESTATIONAL DIETARY CALCIUM 0N SERUM 1,25-DIHYDROXYCHOLECALCIFEROL IN SOUS AND THEIR BABY PIGS By Wuryastuti Bastari Fifteen Yorkshire and crossbred sows were allotted to three groups of 5 sows, equalized for parity and fed corn-soy diets contain- ing 0.5, 0.8 and 1.1% calcium, respectively, during gestation and lac- tation. Sera for 1,25-dihydroxycholecalciferol and mineral analyses (Ca, Mg, P, Cu and Zn) were obtained at 15 and 45 days of gestation, at parturition and at weaning. At parturition, colostrum samples for 1,25(0H)2D3 analysis were collected and 5 piglets of each litter were randomly selected for study. Serum samples were obtained from these pigs at birth, at 10 and 21 days of age for 1,25(0H)2D3 assays and to determine the relationships between maternal and neonatal minerals or 1,25(OH)2D3 status. In sows, serum 1,25(OH)203 was significantly affected by dietary calcium within 15 days of initiating diets. During gestation and lactation, serum 1,25(OH)203 correlated negatively (r - - 0.88; p < 0.05) with serum calcium. Serum calcium was positively correlated (p < 0.05) with dietary calcium at days 15 and 45 of gestation and . at farrowing. Serum magnesium was inversely related to serum calcium (r - -- 0.49; p < 0.05) during gestation and early lactation. In baby pigs, the mean serum 1,25(0H)ZD3 at birth was not affected by treatment but, by 10 days of age, the baby pig serum 1,25(OH)2D3 correlated (r - - 0.62; p < 0.05) with maternal serum calcium. Serum calcium and phosphorus increased significantly (p < 0.05) as maternal dietary calcium increased. The mean colostrum and serum 1,25(0H)2- D3 were significantly correlated (r - 0.90; p < 0.05) at parturition. This study indicates that 1,25(0H)2D3 production is quickly affected by changes in dietary calcium, but these changes did not have an in utero influence on the 1,25(OH)2D3 status of fetal pigs. Dedicated with love to my mother, my father, and my husband, R. Wasito ii ACKNOWLEDGEMENTS I wish to express my sincere appreciation to Dr. H. D. Stowe, my major professor, for his consideration, encouragement and guidance dur- ing my course of study. My appreciation is also extended to Dr. E. R. Miller, Dr. S. D. Sleight and Dr. M. H. Zile, members of my guidance committee, for their help in reviewing this manuscript. Special thanks go to the Government of the Republic of Indo- nesia, Yayasan Beasiswa Supersemar and the Rockefeller Foundation for their financial support. I am also deeply grateful to Dr. E. R. Miller, Dr. L. Johnston and Dr. C. Horvath for their technical assistance, to Dr. K. Refsal for allowing me to use the endocrinology laboratory facilities for portions of the assays and to Dr. T. H. Herdt for his assistance and training in computer analysis. My thanks also go to Anne House, Joanne Klug, and Marge Pestka for their assistance in the laboratory work, to DuWain Simon, Paul Matzat and Raymond Kramer for their assistance with the research ani- mals and to Shirley Eisenhauer for typing this thesis. Finally, my deepest appreciation to my husband, Dr. R. Wasito, ‘for his remarkable assistance, but most of all for his love, concern and understanding throughout the study. iii TABLE OF CONTENTS Page LIST OF TABLES ................................................ vi LIST OF FIGURES ............................................... vii INTRODUCTION .................................................. 1 LITERATURE REVIEW ............................................. 4 Introduction ........................................... 4 Historical ............................................. 5 Metabolism ............................................. 6 Function ............................................... 10 Vitamin D Deficiency ................................... 13 Vitamin D Toxicity ..................................... 14 MATERIALS AND METHODS ......................................... 16 Animals ................................................ 16 Blood Sampling ......................................... 16 Analytical Procedures .................................. 17 Vitamin D Analyses ................................ l7 Minerals Analyses ................................. 20 Statistical analyses ................................... 21 RESULTS ....................................................... 23 1,25-dihydroxycholecalciferol .......................... 23 Serum Calcium .......................................... 24 Serum Magnesium ........................................ 24 Serum Phosphorus ....................................... 24 Serum Copper ........................................... 25 Serum Zinc ............................................. 25 iv TABLE OF CONTENTS cont... DISCUSSION .................................................... CONCLUSIONS ................................................... BIBLIOGRAPHY .................................................. Page 35 40 42 TABLE LIST OF TABLES composition 0f diets00......OOOOOOOOOOOOOOOOOOOOOO0.0... Effect of dietary calcium on sow serum and colostrum l,25(0H)2D3ooooooo-oooo-oooooo-o Effect of maternal dietary calcium on baby p18 serum 1,25(0H)2D3ooeoeeeeeoeoeeoeeoooeooeeesoeo Effect of maternal dietary calcium on serum calcium in sows and their baby pigs-ooooo-o-o-oooo Effect of maternal dietary calcium on serum magnesium in sows and their baby pigs-oooo-ooooo-o Effect of maternal dietary calcium on serum phosphorus in sows and their baby pigs-co -------- . vi PAGE 18 26 30 32 33 34 LIST OF FIGURES Figure Page 1 1,25 dihydroxyvitamin D standard curve.................. 22 2 Effect of dietary calcium on sow serum 1,25(OH)2D3 ...... 27 3 Interrelationships between serum 1,25(OH)2D3 con- centrations and dietary calcium intake in sows.......... 28 4 Interrelationships between serum 1,25(OH)2D3 and serum calcium concentrations in sows.................... 29 5 Effect of maternal dietary calcium on baby pig serum 1025(0H)2D3eeeeeeoeoee00000000000000.0000oooeeeoeo 31 vii INTRODUCTION Rickets, as a consequence of vitamin D deficiency, is rarely found in modern swine operations in the United States. However, sub- clinical rickets may still be a problem in confinement rearing facili- ties in which the sows and piglets receive little ultraviolet irradia- tion, a more important factor for producing endogenous cholecalciferol than dietary vitamin D ingestion (Haddad, 1973). Besides that, the low plasma concentration of 25-hydroxycholceca1ciferol (25-(0H)D3) at birth (Horst and Littledike, 1982) and the rapid growth rate of the pig may predispose to neonatal rickets, a condition of vitamin D deficiency associated with abnormal skeletal growth and myopathy (Smith and Stern, 1967). Realization that infants of vitamin D-deficient mothers have a great risk of developing rickets led researchers through a series of studies on finding the interrelationship between maternal and neonatal vitamin D status. Results of previous work with cattle (Goff at al., 1982), sheep (Barlet g; 51., 1978; Ross g; 31., 1979), rats (Noff and Edelstein, 1978) and humans (Hillman and Haddad, 1974) indicated that, at birth, there was a high correlation between maternal and neonatal plasma concentration of 25-(OH)D3. However, neither the maternal and neonatal relationship nor the normal value of 1,25 dihydroxycholecalci- ferol (1,25(OH)2D3) in other domestic animals has been defined. ‘ It is generally accepted that l,25(OH)2D3 is the most active metabolite of vitamin D3 (DeLuca, 1981; Holick g; 31., 1972b; Lawson g; 51., 1971). Studies in normal animals have documented that dietary l 2 calcium (Ca) and phosphorus (P), parathyroid hormone (PTH) and the plasma concentration of 1,25(OH)2D3 are important factors in regu- lating renal synthesis of 1,25(OH)2D3 (Boyle gt a1., 1971; Garabedian g; 51., 1972; Galante g; a1., 1973). However, factors which enhance the production of 1,25(OH)2D3 in various physiological states, such as gestation and lactation, have not yet been fully charac- terized. In a previous paper, Spanos g5 31. (1978) explained that there is no correlation between plasma concentration of l,25(OH)2D3 and plasma concentration of PTH and Ca during pregnancy and lactation in hu- mans. A possible causal relationship between elevated prolactin and l,25(OH)2D3 has been suggested by Spanos £3,51. (1976) who demon- strated that injection of ovine prolactin into chicks can increase cir- culating concentration of 1,25(OH)2D3. Studies in pregnant sows, on the other hand, have demonstrated that parenteral cholecalciferol treat- ment in the sows 20 days prepartum was an effective way of supplementing baby pigs with 25-(OH)D3 (via placental transport) and cholecalciferol (via the sow's milk), but not 1,25(OH)2D3 (Goff g; 31., 1984). We therefore decided to study the influence of gestational die- tary Ca on serum concentration of l,25(OH)2D3 in sows and their baby pigs by feeding different concentrations of dietary Ca to the sows dur- ing gestation and lactation. Many studies have indicated that the concentration of 1,25 (OH)ZD3 in blood varies in different disease states such as parathy- ‘ roid gland disorders (Broadus g; 31., 1980), sarcoidosis (Bell g; 51., 1979) or certain bone diseases (Rasmussen gt a1., 1980). Because changes in circulating l,25(OH)2D3 are of physiopathological impor- tance in these diseases of Ca metabolism, the development of an assay 3 that is specific for and sensitive to 1,25(OH)2D3 is really re- quired. Several different assay techniques have been reported pre- viously (Hughes 3; a1., 1976; Horst a; a1., 1981; Dokoh g; 31., 1981; Manolagas g§_§1., 1983). However, the methods frequently involve dif- ficult extractions and require relatively large amounts of sample. In 1984, Reinhardt and co-workers developed a new assay for 1,25(OH)2D3 which eliminated the use of HPLC for extraction and only needed 1 ml of serum. The assay was designed to quantitate 1,25(OH)2D3 in serum from.human subjects as well as experimental animals. However, we found the assay was capable of determining the concentration of 1,25(OH)2- D3 in colostrum. The objectives of this research were: (1) to measure the serum concentration of 1,25(OH)2D3 and Ca during gestation and lactation of sows fed a practical diet containing 0.5, 0.8 or 1.1% Ca during ges- tation and lactation; (2) to measure the serum concentration of 1,25 (OH)2D3 in neonatal pigs born to the above sows; (3) to determine the inter-relationships between maternal and neonatal 1,25(OH)2D3 status in pigs; (4) to determine the inter-relationships between serum concentration of Ca and 1,25(OH)2D3 during gestation and lactation in sows; and (5) to determine the influence of gestational dietary Ca on serum concentration of P, magnesium (Mg), copper (Cu) and zinc (Zn) in sows and their baby pigs. LITERATURE REVIEW INTRODUCTION Vitamin D is the fat-soluble vitamin which is able to cure rick- ets (Hess,g§ 51., 1929). Two major forms of vitamin D are Vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Both forms are considered to have the same activity biologically or nutritionally in most species except for chicks and New werld monkeys which utilize D3 more effectively than D2 (Hunt at 51., 1967; Steenbock g; 51., 1932; Holick g; 51., 1976). Structurally, vitamin D2 and D3 have been identified as 9,10 secoderivatives of ergosterol (Askew g; 31., 1930) and 7 dehydrocholes- terol, respectively (Windaus 3; a1., 1936). The former is produced.by the ultraviolet light irradiation of ergosterol synthesized in plants. In terms of antirachitic properties, partial irradiation of ergosterol with ultraviolet light has been considered to be better than complete irradiation (Steenbock a; 11., 1932). Vitamin D3 is generated primar- ily in the malpighian layer of the epidermis by the action of 280-305 nm wavelength of ultraviolet light on 7 dehydrocholesterol (pro-vitamin D3, an intermediate in cholesterol synthesis (Okano g; 31., 1977). The ability of the body to convert pro-vitamin D3 to vitamin D3 does not decrease with age (Mawer g; 51., 1982). Therefore, demonstration of a dietary requirement of vitamin D is unlikely in the presence of suffi- cient sun light. HISTORICAL The importance of sunlight for the normal skeletal growth had been speculated since ancient times (Hess g; 51., 1929). The soft skulls of Persians, who always wore turbans and covered most of their bodies, were used as evidence to support the above speculation (Hess g; a1., 1929). The earliest description of bone disease considered to be rickets was by two British doctors, Glisson and Whistler, in the middle of the seventeenth century (Orgler, 1953; Hess gt 51., 1929). By the 1900's, the lesions of rickets were already described (Mellanby, 1919). In 1922, McCollum and his co-workers found that a substance in codliver oil, which has antirachitic properties, was actu- ally different from vitamin A_in its stability to heat and aeration. He then named the substance vitamin D (McCollum, 1912). The structure of vitamin D from irradiation of food was deter- mined almost simultaneously by Askew g; a1. (1930) and Windaus g; 51. (1932) who called it ergocalciferol (vitamin Dz). Several years af- ter that, Windaus g; 51. (1936) also succeeded in identifying the struc- ture of vitamin D3 or cholecalciferol (Windaus g3 g1., 1936). Studies on the mode of action of vitamin D led to the informa- tion that there is a lag time between the administration of vitamin D and the initiation of the physiological action (Carlsson, 1952). The next observation led to one possible explanation for the time leg, that vitamin D may have to be further metabolized before it becomes active (Deana, 1976). In 1968, Blunt g; 51. succeeded in isolating the pure form of one of the metabolites of vitamin D3 and identified it as 25- (OH)D3. The biological activity of this compound was found to be 2 to 5 times greater than that of vitamin D3 in healing rickets in rats. 6 At that time, it was thought to be the most active form of vitamin D3. However, Haussler 3; 31. (1968) reported that 25-(OH)D3 was metabo- lized to a more polar compound. This metabolite could be obtained from the nuclear fraction of the chicken intestine and it had biological ac- tivity at least 13 times greater than cholecalciferol (Haussler 3; 31., 1968). In 1972, that more polar compound was identified as 1,25(OH)2- D3 (Holick.3; 31., 1972b). The fact that nephrectomized rats were un- able to synthesize 1,25(OH)2D3 was used by Fraser and Kodicek in England as evidence that the kidney is the site of production of 1,25- (OH)2D3 (Lawson 3; 31., 1971). Currently, l,25(OH)2D3, the most active form of vitamin D3, has been considered as a vitamin and a hor- mone (Holick 3; 31., 1972b; Norman 3; 31., 1977). METABOLISM Following its synthesis in the skin, the endogenous vitamin D3 is transported to the liver by alpha 2—globulin of serum. Vitamin D2 or supplemental vitamin D3 in the diet is absorbed primarily in the distal part of the small intestine (jejunum and ileum). Bile salts are necessary for this absorption and chylomicra are responsible in trans- ferring the absorbed vitamins D to the liver (Avioli and Haddad, 1973). Studies on the absorption of radioactive vitamin D3 in rats have revealed that about 72‘ of the vitamin D3 recovered in thoracic duct lymph was associated.with chylomicra while the remaining 283 was carried by alpha globulin (Dueland 3; 31., 1982). To function, vitamin D3 must be activated metabolically through several sequential steps to a hormonal substance. The initial step of activation occurs in the liver in which vitamin D3 is hydroxylated at 7 the C-25 position of the side chain to form 25—(OH)D3, the major cir- culating form of vitamin D3 (Holick and Clark, 1978). Although the liver is believed to be the major site of 25-hydroxylation in most spe- cies, Tucker 3; 31. (1973) demonstrated that homogenates of kidney and small intestine from chicks are also capable of producing 25-(OH)D3. This finding has been supported by Olsen 3; 31. (1976) who found that hydroxylation of vitamin D3 at C-25 still occurs in hepatectomized animals. More recent studies indicate that other organs such as lung, pituitary glands, ovaries, adrenal glands and testes of rabbits and bovine have shown significant 25 hydroxylase activity (Ichikawa 3; 31., 1983; Henry and Norman, 1984). The conversion of vitamin D3 to 25- (OH)D3 requires magnesium ions, NADPH, molecular oxygen, cytoplasmic protein and cytochrome P 450 (Bhattacharyya and DeLuca, 1974a; Yoon and DeLuca, 1980). In vivo and in vitro experiments in rats suggested that 25-hy- droxylase was regulated by a product feedback mechanism in order to pre- vent toxicity due to over-administration of vitamin D3. Therefore, only limited amounts of vitamin D3 can be hydroxylated to 25-(OH)D3 (Horsting and DeLuca, 1969). These data have been supported by Bhattacharyya and DeLuca (1974b) who demonstrated that hepatic vitamin D3 25-hydroxylation in chicks decreased twenty-four hours following the administration of physiological doses (20 IU) of vitamin D3. How- ever, additional investigation in rats did not support this concept ~ (Rojanasathit and Haddad, 1976). Furthermore, Clark and Potts (1977) have recently shown in the vitamin D-depleted rats, which received dif- ferent concentrations of dietary vitamin D3 for either a week or three weeks, that the plasma concentration of 25-(OH)D3 correlated with an 8 increase in vitamin D3 intake irrespective of the dose of vitamin D3 administered. Whether or not 25-hydroxylation of vitamin D3 is sub- ject to feedback regulation remains debatable. After 25-hydroxylation, the 25-(OH)D3 rapidly leaves the liver by binding to the plasma transport protein and goes to the mitochondria of renal tubule cells to be hydroxylated at either C-l or C-24 position to form 1,25(OH)2D3 or 24,25(OH)2D3 (Holick and Clark, 1978; Henry and Norman, 1984). In states of hypervitaminosis D, 25-(OH)D3 can also be hydroxylated at 6-23 to yield 23,25(OH)2D3 or at C-26 to yield 25,26(OH)2D3 (Napoli 3; 31., 1982; Napoli 3; 31., 1981). Hydroxylation at 1 alpha position is the very important step in vitamin D3 metabolism to generate the most active form of vitamin D3 which has the capability of stimulating either intestinal calcium trans- port or bone calcium mobilization (Boyle 3; 31., 1972; Holick 35 31., 1972b; Wong 3; 31., 1972). The l-hydroxylation step is rate limiting and requires reduced nucleotide (NADPH), molecular oxygen and cytochrome P450 for enzymatic activity (Ghazarian and DeLuca, 1974). The work of Boyle 3; 31. (1971) has introduced the concept that serum Ca concentrations regulate the hydroxylation of 25-(OH)D3 at the 1 alpha position. It has been known for some time that hypocalcemic conditions stimulate the production and release of PTH from parathyroid glands. It is now realized that this hormone acts in the kidney to sti- mulate the activity of 25-hydroxycholeca1ferol-1-hydroxylase and cause ~ an increased production of 1,25(OH)2D3. However, under normocalce- mic or hypercalcemic conditions, secretion of the PTH is minimal, the activity of 25~hydroxycholeca1ciferol-1-hydroxy1ase is also minimal and another metabolite of vitamin D3, identified as 24,25(OH)2D3. 9 becomes predominant in circulation. The 24-hydroxylated metabolites are rapidly metabolized to 1,24,25(OH)3D3 and excreted (Holick 3; 31., 1972a; Rasmussen.3§ 31., 1972; Fraser and Kodicek, 1973; Garabedian.3g 31., 1978). The role of P depletion in the synthesis of 1,25(OH)2D3 was studied following the discovery that thyroparathyroidectomized animals maintained on a low P and high Ca diet can still produce 1,25(OH)2D3. Therefore, it was believed that low P diets controlled production of 1,25(OH)2D3, even in the absence of PTH (Tanaka and DeLuca, 1973; Hughes.3§,31., 1975; Haussler 3; 31., 1977). Other in vive investiga- tions did not support this concept (Sommerville 3; 31., 1978; Norman 3; 31., 1977). These experiments provided evidence that P deprivation was much less effective in stimulating 1-hydroxylase activity than Ca depri- vation. The degree of stimulation of 1-hydroxylase activity by low Ca diets is 10 times greater than the stimulation by low P diets (Rader 3; 31., 1979; Harrison 3;,31., 1982). Hormones other than PTH have also been implicated in the regula- tion of the 25-OH-D3-l alpha-hydroxylase since many data indicate that, during gestation and lactation, there are remarkable shifts in concentra- tions of serum Ca without detectable changes in circulating PTH (Drake 3; 31., 1979; Pitkin,3§ 31., 1979). Using the Japanese quail, it was demon- strated that both ovariectomy and anti-estrogen administration caused a reduction in renal production of 1,25(OH)2D3 (Baksi and Kenny, ‘1976a). It has also been found that hypophysectomized rats have low sera concentrations of 1,25(OH)2D3 and high serum concentration of 24,25(OH)203. These conditions were reversed by the administration 10 of growth hormones for several days (Gray, 1981; Pahuja and DeLuca, 1981; Spencer and Tobiassen, 1981). Extrarenal 1,25(OH)2D3 synthesis is thought to occur because nephrectomized pregnant rats have been reported to be able to synthesize both 1,25(0H)2D3 and 24,25(OH)2D3 (Weisman 3; 31., 1979). Pre- vious work with humans indicated that placenta, decidua, bone and fetal kidney are possible sites of 1,25(OH)2D3 synthesis (Whitsett 3; 31., 1981; Delvin 3; 31., 1985; Howard 3; 31., 1981). FUNCTION In terms of physiological function, vitamin D3 and/or its meta- bolites are known to be responsible for maintaining Ca homeostasis which is important for normal calcification of bone, normal neuromuscular ac- tivity and normal muscle contraction (Norman, 1968; Tanaka and DeLuca, 1971; Omdhal and DeLuca, 1973; DeLuca, 1981). To carry out that function, vitamin D3 and/or its metabolites have three major effects. First, they initiate intestinal Ca and P ab- sorption. This effect was first proven.by Nicolaysen 3; 31. (1953) who demonstrated that intestinal Ca transport in vitamin D-deficient rats was impaired. The process of intestinal absorption of Ca has been investi- gated in vivo using various techniques and all findings point to 1,25- (OH)2D3 as the active metabolite (Schachter, 1963; Wesserman 3; 31., 1961; Martin and DeLuca, 1969). Additional investigation indicated that ‘ l,25(OH)2D3 accelerates intestinal Ca absorption from all segments of the small intestine and from colon (Harrison and Harrison, 1969). Stu- dies using thyroparathyroidectomized dogs have demonstrated that the ef- fect on Ca absorption can be obtained solely by 1,25(OH)2D3 (Schachter and Rosen, 1959). 11 The ultimate expression of vitamin D3 action is thought to be at the intestinal brush-border membrane where the vitamin, in some way, increases diffusional-permeability of the microvilli to allow the entry of Ca ions into the cell. This perhaps can be manifested as a change in membrane structure and/or an effect on Ca carriersynthesis (Wasserman and Corradino, 1971; Omdhal and DeLuca, 1973; DeLuca and Schnoes, 1976). The work of Steele 33,31. (1975) established that P transport can accompany the translocation of Ca. However, it is now apparent that vitamin D also stimulates P transport in the intestine in a manner which is different from the Ca process (Haussler, 1974). A second effect of vitamin D3 is to bring about the mobiliza- tion of Ca from previously formed.bone to contribute to the Ca and P04 pool of the plasma (DeLuca, 1981). In vitro studies, using bone organ culture, have indicated that PTH is not involved in the process of Ca reabsorption from bone (Raisz,3§ 31., 1972). However, conflicting re- sults have been obtained concerning the necessity of PTH in the action of l,25(OH)2D3 on bone resorption in vivo (Garabedian 3; 31., 1974; Reynolds 3; 31., 1976). From the studies of Tanaka and his co-workers, it is known that a form of vitamin D3 other than 1,25(OH)2D3 may also participate directly in the process of the Ca mobilization from bone (Haussler and Rasmussen, 1972; Wezeman, 1976; Bordier 3;,31., 1977). This finding has also been supported by Reynolds 3; 31. (1973) who demon- strated that, besides l,25-dihydroxycholecalciferol, 25-(OH)D3 is also 'a potent stimulator of bone mineral resorption. In terms of the mechanism of action by which vitamin D3 promotes the mineral mobilization from bone, Wong 35,31. (1977) suggested that 1,25(OH)ZD3 induces bone resorption by activating osteoclasts and 12 inhibiting osteoblasts. In another investigation, Brommage and Newman (1979) explained that 1,25(OH)2D3 stimulates the production of mineral 'solubilizer" in the bone. This solubilizer reportedly binds to the surface of bone materials, increases their solubility and increases the flux from bone to the blood. In addition to the effects in the intestine and the bone, vitamin D3 is also believed to have a role in renal tubular mineral reabsorp- tion (DeLuca, 1981). Studies on the effect of vitamin D3 on renal Ca reabsorption indicated that 1,25(OH)2D3 indeed improved renal reab- sorption of this ion (Harris 3; 31., 1976). However, since the experi- ments were done on animals with intact parathyroid glands (Gran, 1960) and since 99% of the filtered Ca is reabsorbed, even in vitamin D defi- ciency (Taylor and Wasserman, 1972), the direct action of vitamin D3 on renal Ca reabsorption remains an enigma. Regarding the possible role of vitamin D3 on P04 reabsorption, Harrison and Harrison (1941) provided evidence that vitamin D3 en- hanced P04 reabsorption. Since PTH also causes phosphaturia, it is likely that this effect is mediated through suppression of PTH secretion by hypercalcemia rather than by a direct effect of 1,25(OH)2D3 on the kidney (Puschett 3; 31., 1972; Popovtzer 3; 31., 1974). From the fore- going, it is clear that kidney, bone and small intestine are target tis- sues of vitamin D3 in which the most potent metabolite of vitamin D3, 1,25(OH)2D3, acts by binding to specific, high-affinity receptors lo- ‘cated in the cytoplasm of target cells. In recent years, however, such receptors have been also identified in many other organs and tissues; among these are human leukocytes (Provvedini 35 31., 1983; Matsui 33 31., 1985), pancreas (Ishida 3; 31., 13 1983; Clark,3§ 31., 1981), skin (Hosomi 3; 31., 1983), pituitary (Tornquist and Allardt, 1986), heart (Walters 3t 31., 1986) and adrenal (Clark 3; 31., 1986). Even though the exact role of l,25(OH)2D3 in diverse target organs has not been fully elucidated, it has been sug- gested that l,25(OH)2D3 may influence the proliferation of mono- cytes and T-lymphocytes (Provvideni 3; 31., 1983; Matsui 3;,31., 1985), differentiation of epidermal cells (Hosomi 3; 31., 1983), secretion of insulin from pancreatic B cells (Ishida 3; 31., 1983; Clark 3; 31., 1981) and production of thyroid stimulating hormone from pituitary cells (Tornquist and Allardt, 1986). VITAMIN D DEFICIENCY Low or absence of vitamin D in the animal body is a classic cause of rickets. Other factors such as dietary lack of Ca or P or both also play an important role in the development of this disease (Chick,3§ 31., 1923; Harrison and Harrison, 1975). High levels of dietary iron can also induce P deficiency with subsequent development of rickets (Brock and Diamond, 1934). Based on these facts, rickets is a nutritional defi- ciency disease which is characterized by failure of adequate deposition of Ca and P in the bone. Rickets is often a climate-related disorder, too. Previous information indicated that it is limited largely to tem- perate latitudes (Orr 3; 31., 1923). Rickets is considered to be a di- sease in young animals, in contrast to its counterpart called osteoma- lacia in adults (Nordin, 1960). Stiffness of the forelegs is the first general sign of rickets in most animals. Nervousness and tetany may also appear quite early in ani- mals with low blood Ca rickets. Swollen knee and hock joints, shortening 14 of muscles and tendons of the rear legs and scoliosis or curvature of the spine appear as the disease progresses in severity. All of these signs make animals unable to stand or walk comfortably for any period of time. In addition, enlargement of the costochondral junction can be easily pal- pated through the skin and is especially noticeable inside the thorax at the time of postmortem. These conditions persist even after healing. On the basis of the blood composition, the rachitic animals develop low blood Ca or low blood P or low blood concentrations of both Ca and P (Malherbe, 1956; Groth, 1958). VITAMIN D TOXICITY It is generally known that administration of large doses of vita- min D produces significant toxic effects. Deleterious effect is actually due to hypercalcemia resulting from excessive intestinal absorption of Ca and mobilization of Ca from bone, which then causes calcification of the arterial walls, intestinal walls and many other soft tissues such as heart, kidneys and lungs (Stanbury .5, 31., 1930; Hughes 33; 31., 1975; Davie and Lawson, 1980). Poisoning by vitamin D in dairy cows has re- sulted from prolonged prepartum administration of high doses of vitamin D3 (30,000,000 IU/day) in order to prevent parturient paresis (Capen.3§ 31., 1966). Enzootic calcinosis is a disease in cattle and other grazing ani- mals manifested by widespread mineralization of soft tissues, especially ‘ in the cardiovascular and pulmonary systems. These signs are very simi- lar to those of vitamin D toxicity. The disease has been reported from many countries under various names, but the characterization of the di- sease is known to be the same in all cases. Early experimental work has 15 indicated that enzootic calcinosis is associated with ingestion of plants belonging to the family Solanaceae (Worker and Carrilo, 1967; Gill 33 31., 1976; Singh 33 31., 1976). Additional investigations have deter- ined that the plant ($313333 33133331133) contains an active substance that mimics the action of 1,25(OH)2D3. It is effective in inducing calcium-binding protein (Ca-BP) synthesis and intestinal absorption of Ca and P in anephric, as well as in diabetic rats (Walling and Kimberg, 1975; Schneider 33 31. , 1975). However, unlike the precursor of vitamin D3, the active principle of 5313333 33133331133 is water soluble which makes it rapidly absorbed and excreted (O'Donnell and Smith, 1973). There are other data indicating that vitamin D3 sterols are also present in plant leaves of 9333333 3133333, 123333113 313333333, [133133 333133 and 13133333 3131333333. However, studies using HPLC and mass spectrophotometry have demonstrated that their vitamin D3 activities are considerably less than 3313333 33133333133 (Krook 33 31. , 1975; Peterlik 33 31. , 1977; Morris and Levack, 1982; Horst 33 31. , 1984). Calcinosis in young pigs and young sheep can also be induced by giving these animals large doses of vitamin D3 (Penn, 1970; Clegg and Hollands, 1976; Quarterman, 1964). MATERIALS AND METHODS ANIMALS. Fifteen sows, consisting of purebred'Ybrkahire, Yorkshire/Duroc crossbreds and Yorkshire/Landrace crossbreds, approximately averaging 175 kg in body weight, served as experimental animals. The sows were allot- ted to three groups of 5 sows equalized for parity, and the groups were assigned to diets containing 0.5%, 0.8t and 1.18 of calcium throughout gestation and lactation. The composition of the diets is presented in Table 1. These sows were housed in an environmentally regulated, complete confinement, gestation facility with slotted concrete floors and were tied with neck collars and chains. The sows were fed 2.5 kg of grain/day and tap water ad libitum. At approximately 14 days before farrowing, the sows were moved into a facility equipped with metal farrowing stalls. The sows farrowed during November 1985. The baby pigs were separated from the sows immediately after birth to obtain a presuckle blood sample. Each baby pig was weighed and had its tail docked, ears notched and needle teeth clipped. The baby pigs were also given parenteral antibiotic and iron dextran. Five piglets of each litter were identified by random selection to be followed on experi- ment through weaning. W Blood samples were collected into vacutainer1 tubes from the anterior vena cave of the sows at 15 days and 45 days of gestation, at 1Becton Dickinson, Rutherford, New Jersey 16 17 parturition and at weaning. Colostrum samples were obtained by manual milking during parturition. Blood samples from baby pigs were obtained by vacutainer1 tubes from the anterior vena cava at birth (colostrum deprived) and at 10 and 21 days of age. For serum preparation, the blood samples were allowed to clot at room temperature (15 - 25°C) for at least 3 hours. All blood samples were centrifuged for 15 minutes at 760 x g. Each serum sample was with- drawn.with a Pasteur pipette and placed into a 5 or 10 ml plastic vial. All serum and colostrum samples were frozen until assays were conducted. W W The 1,25(OH)2D3 in serum was quantified using a commercial radio receptor assayz. For sample extraction, 1.0 m1 of each serum sample was pipetted into a 12x75 mm borosilicate glass tube. Fifty pl of ethanol buffer containing 1700 disintegrations per minute (DPM) [3H] 1,25(OH)2D3 were added to each serum sample and also to a scintilla- tion vial containing 5 m1 scintillation liquid3 for calculating recov- eries. One ml acetonitrile was added to each sample and each tube was then vortexed for 5 seconds and centrifuged for 10 minutes at 760 x g. While the samples were being centrifuged, Sep-Pak C-184 columns were 2 Immune Nuclear Corporation, Stillwater, Minnesota. 3 Research Product International Corp., Mount Prospect, Illinois. 4 Waters Associates, Millford, Massachusetts. 18 Table 1. Composition of diets in this experiment t Calcium Ingredient 0.5t 0.8% 1.13 Ground shelled corn ’ 71.75 71.00 70.25 Soybean meal (44%) 14.50 14.50 14.50 Wheat bran 10.00 10.00 10.00 Calcium carbonate 0.50 1.25 2.00 Mono-dicalcium phosphate 1.50 1.50 1.50 Reg. T.M. salt 0.50 0.50 0.50 MSU VTM premix* 0.60 0.60 0.60 Vit. E-Se premix** 0.50 0.50 0.50 Choline chloride (60%) 0.15 0.15 0.15 100.00 100.00 100.00 * Supplied the following per kg of diet: vitamin A, 3960 IU; vita- min D3, 792 IU; riboflavin, 3.96 mg; d-pantothenic acid, 15.84 mg; niacin, 21.12 mg; vitamin B-12, 24 ug; vitamin K, 2.64 mg; choline chloride, 152.1 mg; zinc, 90 mg; iron, 71.3 mg; manganese, 4.5 mg; copper, 12 mg; iodine, 0.53 mg. ** Supplied 10 IU of vitamin E and 0.15 mg of selenium per kg of ~ diet. 19 prepared by washing with 5 m1 acetonitrile followed by two 5 m1 washes with distilled.water. After centrifugation, the supernatant was poured off into another 12x75 mm glass tube containing 0.5 m1 of 0.4 M potassium phosphate (pH 10.5) and vortexed for 5 seconds. This extract was then applied with a Pasteur pipette into the washed Sep-Pak C-18 columns. Excess salt and pigments were removed from the Sep-Pak C-l8 columns by washing twice with distilled water and the interfering polar lipids were removed by washing with methanol:distilled water (70:30). The purified vitamin D metabo- lites were then eluted with 5 m1 acetonitrile and the eluates were evap- orated to dryness using a vacuum evaporators. After the eluates had dried, each sample was reconstituted with 5 m1 of hexane:isopropanol (98:2), mixed well by vortexing and applied to a Sep-Pak Silica4 column. These columns were prepared before use by washing with 5 m1 of isopropanol follofied.by 2 washes of 5 ml of hexane: isopropanol (98:2). Each eluate tube was rinsed with an additional 5 m1 of hexane:isopropanol (98:2), vortexed for 5 seconds, and the rinse was applied to the Sep-Pak silica column. The 25-(OH)D3 and 24,25(OH)2- D3 were removed from the Sep-Pak silica column by washing with 5 ml of hexane:isopropanol (96:4). The purified 1,25(OH)ZD3 was eluted from the silica column with 5 ml of hexane:isopropanol (70:30) and dried in a vacuum evaporator. The dried samples containing the 1,25(OH)2D3 fraction were im: mediately reconstituted with 200 ml ethanol bufferz. From this volume, 50 ul of purified sample were used to determine the recovery sample and two 50 ml aliquots were used for radio receptor assay. 5 Haake Buchler Instruments, Inc., Saddle Brook, New Jersey. 20 For assay, 50 ul of each 1,25(OH)2D3 standard2 (25, 50, 100, 200 and 400 pg/tube) and samples were added to the 12x75 mm borosilicate glass tubes. Four hundred ul of calf thymus receptor in phosphate- 2 were added to each standard and sample. potassium chloride buffer These mixtures were then vortexed for 5 seconds and incubated in a 15- 20°C water bath for 60 seconds. At the end of this period, 11,500- 13,500 DPM of [3H] 1,25(ou)21)32 in 50 ul ethanol buffer were added to each tube. The tubes were mixed well and incubated again for 60 minutes at 15-20°C. Following this incubation, the tubes were placed in an ice bath for 10 minutes to cool and then each tube received 100 ul charcoal suspensionz. The tubes were vortexed for 5 seconds and placed for 20 minutes in an ice bath. After cooling, the tubes were vor- texed again for 5 seconds and centrifuged for 15 minutes at 4°C at 1800 x g. Following the centrifugation, the supernatant was poured off into a scintillation vial containing 5 ml scintillation f1uid3. The vial of supernate and scintillation fluid was mixed well by hand and 6 then placed in a beta scintillation counter . Data was expressed as mean counts per minute. 111W Determinations of Ca, Mg, Cu and Zn concentrations in the blood serum were made by atomic absorption spectrophotometry7. In prepara- tion for Ca and Mg quantification, each serum sample was pipetted into a 10 ml acid-washed flask and diluted 1:50 with 0.1% lanthanum chloride. 6 Model LS-800, Beckman Instruments, Inc., Fullerton, California. 7 Model 5000, Perkin-Elmer, Norwalk, Connecticut. 21 For Cu and Zn quantification, each sample was diluted 1:1 and 1:5, re- spectively, with 1‘ hydrochloric acid in an acid-washed glass tube. Cal- cium standards used in this analysis had Ca concentrations of 1.0, 2.0 and 3.0 ug/ml, whereas the Mg, Cu and Zn standards contained 0.5, 1.0 and 2.0 ug/ml of the respective element. The standards and diluted specimens were then aspirated into an air-acetylene flame. Calcium was read at 422.7 nm, Mg at 285.2 nm, Cu at 324.7 nm and Zn at 213.9 nm. The inorganic phosphorus (Pi) analyses were conducted in the Clin- ical Pathology Laboratory of the Veterinary Clinical Center at Michigan State University, using the Flexigem Centrifugal Analyzere. Ten ul of each serum sample was pipetted into a cuvette and mixed well with 700 ul inorganic phosphorus UV reagent which contained 210 mM sulfuric acid and 0.40 mM ammonium molybdate. Serum inorganic phosphorus concentrations 8 were determined directly with Flexigem spectrophotometer . Absorption ratios were established by reading absorptions at 340 nm and 380 nm. W The effects of dietary Ca treatments on serum 1,25(OH)2D3 and minerals were evaluated by mixed design analysis of variance. One way analysis of variance and Dunnett's test were used to determine the signi- ficant differences between treatment means at each time. The interrela- tionships between maternal and neonatal concentration of minerals or 1,25(OH)2D3 were determined by using correlation analysis. In this study, a difference was considered significant at the level of P < 0.05. All statistical analyses were performed by an IBM 4381 computer using the 8A89 program. 8 Electro Nucleonics, Inc., Fairfield, New Jersey. 9 Statistical Analytical System, Cary, North Carolina. 22 1.25 DIHYDROXYVITAMIN 0 STANDARD CURVE 9C)" 80 - 70 r- 60 '- (BIBo)% so >— 4C)’ 30 r— 20" IO - 0 1 1 1 1 1L 25 5O IOO 200 400 Picogramslml Fig 1. e—-—e standard curve established by kit manufacturer. A-—--‘ typical standard curve derived from using the kit. RESULTS Win]. The effect of dietary calcium on sow serum.and colostrum 1,25 (OH)ZD3 are presented in Table 2 and Figure 2. Within 15 days of initiating the different dietary Ca treatments, serum concentrations of l,25(OH)2D3 in sows were negatively correlated (b - -.0.49; p < 0.05) with dietary Ca (Fig. 3). As pregnancy progressed, serum 1,25(OH)2D3 concentrations in all groups of the sows tended to increase until the time of farrowing. By weaning, the mean serum concentrations of 1,25(OH)2D3 in all groups of the sows had decreased toward normal concentrations, even though the group means were still significantly dif- ferent from each other (p < 0.01). At parturition, there was a signifi- cant correlation (r - 0.90; p < 0.05) between serum and colostrum concen- trations of 1,25(OH)2D3 and a significant negative correlation (b - -0.40; p < 0.05) between dietary Ca and colostrum 1,25(OH)2D3 concen- trations. The effect of maternal dietary Ca on baby pig serum 1,25(OH)2- D3 are presented in Table 3 and Figure 5. The mean serum concentra- tions of 1,25(OH)2D3 in the precolostrum piglets from the three groups were not significantly different, not correlated with maternal serum concentrations of 1,25(OH)2D3, and tended to be inversely re- lated to maternal dietary Ca. The mean serum 1,25(OH)2D3 of the three groups of pigs at 10 days of age were more than twofold higher than the concentrations at birth, were significantly different (p < 0.01), were positively correlated (r - 0.59; p < 0.05) with maternal 1,25- (OH)2D3 concentrations and were negatively correlated (r - -0.62; p < 0.05) with maternal serum Ca. The mean concentrations of 1,25(OH)2D3 23 24 of the groups of 21-day-old pigs were approximately 80-85t of the values observed 11 days before and did not correlate with maternal serum 1,25- (OH)2D3 concentrations. Serum_salcius The effect of maternal dietary Ca on serum Ca in sows and their baby pigs are presented in Table 4. Serum Ca concentrations were affected by (p < 0.05) and positively correlated with (r - 0.52; p < 0.05) dietary Ca at days 15 and 45 of gestation and at farrowing, but only tended to be affected at weaning time. During gestation and lacta- tion, serum Ca correlated negatively (r - -0.88; P < 0.05) with serum 1,25(OH)2D3 (Fig. 4)). Serum Ca in the baby pigs increased significantly (p < 0.05) as maternal dietary Ca increased at birth and at 10 and 21 days of age. There was negative correlation (r - -0.55; p < 0.05) between neonatal serum Ca and maternal serum 1, 25(OH)2D3 concentrations. W The effect of maternal dietary Ca on serum Mg in sows and.their baby pigs is presented in Table 5. Serum Mg in sows correlated nega- tively (r - - 0.49; p < 0.05) with serum Ca during gestation and lacta- tion except at weaning and did not correlate with serum 1,25(OH)2D3 concentrations. In the baby pigs, serum Mg tended to be inversely re- lated to serum Ca and P at birth, but not at 10 days and 21 days of age. We The effect of maternal dietary Ca on serum P in sows and their baby pigs is presented in Table 6. No significant difference (p > 0.05) was observed in the mean serum concentrations of P in all groups 25 of the sows at any of the sampling periods during feeding. Serum P tended to be directly related to serum Ca and did not correlate with serum 1,25(OH)2D3 concentrations. In the baby pigs, serum P increased significantly (p < 0.05) as maternal dietary Ca increased, and related directly to serum Ca concentrations. SIIHI_£QRRQI Because of insufficient serum samples from the baby pigs, serum Cu concentrations were only determined on sows' serum. There were no significient overall Ca treatment effects on serum Cu in the sows throughout the experiment. Wins In sows, serum Zn only tended to be inversely related to dietary Ca at day 15 of pregnancy. In the baby pigs, serum Zn was not influenced by maternal dietary Ca. ‘J 26 msoum Houucou n .Amo.o v my moose Houucoo souu uaououuwo hapaoowuwcofim a 2mm + mcooa one mmsHo> « dam.m H on.mm 0H.n H ¢¢.no ovm.n H ~¢.mb m abhmm Amcficomsv ooa omo.H H nm.nH oo.a H on.bH o~m.a H mm.- m fidHumOHoo omw.~ H mm.mm bH.H H mm.v0H omH.n H mm.¢NH m Eamon Anewuwusuuomv «Ha mom.~ H mm.mn em.a H vm.hb mac.n H om.>oa m Eamon mo ovo.a H or.nn o~.H H mm.o> mach.a H mn.~m m Hanan ma “nanny H.H no.o m.c msoum\: magnum noon to mafia Rwy sswoaoo manpown .Aaa\mav nomflmovmm.a assumoaoo use sauna 30m :0 aswoaoo manpowu no uoouum .m oases 1,25tom203 (pg/ml) 27 120 r 100 . 80' BO , O . 8 - farrowing 1 1 40' is 45 135 140 D a y Figure 2. Effect of dietary calcium on sow serum 1,25(OH)2D3. (pg lml) 1,25(OH)2 03 28 100 ' b:-O.49 p < 0.05 80 r .50. 4O ’ 2° ‘6‘? ‘ ole . 13 Ca Intake (°/o) Figure 3. Interrelationships between serum 1,25(OH)2- D3 concentrations and dietary calcium intake in sows. 29 140’ ' e . . : ':.0088 ::: 112C)» . I ID‘KGD.CN5 E \ U a 100’ V 09 . O 80 N E o 00> In N ,: so. 20 so 160 720 {40 Calcium (ug/ml) Figure 4. Interrelationships between serum 1,25- (OH)2D3 and serum Ca concentrations in sows. moose Houusoo n “no.0 v my moose Houucoo Bonn ucououuwu haucooauwamfim o Sum + mcooa one mosao> a 30 nah.H H mm.mm Ho.a H m~.mo owm.a H mh.mm mN Hm omm.m H Nh.Hh n~.N H mm.¢m mow.m H mm.NHH mm 0H «5.0 H ou.mn nv.H H h¢.nv svw.a H ma.¢v mN o Amhoov H.H no.o m.o msoum\s moo mmwm anon va asflofloo manpowo Hocuouo: .Afisxoac nauamocm~.a sane» and scan so agendas sausage Assamese no somuum .n manna 31 140» 2 120+ E ‘s g: 100» V a, OOr N A :t O 50' V no N ,: 40’ 2° 5 to is is Age (days) Figure 5. Effect of maternal dietary calcium on baby pig serum 1,25(OH)2D3. 32 Table 4. Effect of maternal dietary calcium on serum calcium (pg/ml) in sows and their baby pigs. Maternal dietary calcium (8) Observation n/group 0.5 0.8b 1.1 period Sows (days on feed) 15 5 95.00 i 8.45“ 102.50 i 5.95* 123.75 i 2.39“ 45 5 88.75 i 4.30“ 100.00 3 5.00 118.75 2 4.30“ 114 5 83.75 i 2.40“ 93.75 1 2.40 103.75 3: 5.54“ (parturition) 140 5 93.75 i 3.75 101.25 x 3.75 103.75 1 3.75 (weaning) Baby pigs (days of age) 0 25 94.42 i 2.71“ 96.32 1 2.97* 97.05 t 2.37“ 10 25 80.12 i 1.82“ 87.27 i 1.35 102.80 1 1.98“ 21 25 97.05 i 2.37“ 102.80 i 1.98 104.90 1 2.88“ * Values are means 1 SEM ‘ Significantly different from control group (p < 0.05) b Control group 33 Table 5. Effect of maternal dietary calcium on serum magnesium (pg/ml) in sows and their baby pigs. Maternal dietary calcium (8) Observation n/group 0.5 0.8b 1.1 period Sows days on feed 15 5 23.75 1 2.40“ 20.00 1 0.01* 18.75 1 1.25“ 45 5 22.50 1 1.44“ 20.00 _+_ 0.01 18.25 1 2.40“ 114 5 23.75 1 2.408 21.25 1 1.25 17.50 1 2.50a (parturition) 140 5 27.50 1 2.50 26.25 1 3.75 25.00 1 2.04 (weaning) Baby pigs days of age 0 25 23.12 1 1.79 21.20 1 1.67* 19.10 1 0.71 10 25 22.89 1 1.34 23.97 1 1.37 22.30 1 0.99 21 25 22.35 1 0.92 22.17 1 0.88 20.80 1 0.44 * Values are means 1 SEM a Significantly different from control group (p b Control group < 0.05) 34 Table 6. Effect of maternal dietary calcium on serum phosphorus (mg/ml) in sows and their baby pigs. Maternal dietary calcium (%) Observation n/group 0.5 0.8b 1.1 period Sows days on feed 15 5 61.50 1 2.02 65.00 1 2.34* 66.75 1 4.30 45 5 58.25 1 5.17 62.00 1 8.97 62.25 1 1.44 114 5 63.00 1 0.41 71.75 1 2.59 74.00 1 8.40 (parturition) 140 5 74.75 1 2.14 82.00 1 5.84 83.75 1 4.03 (weaning) Baby pigs days of age 0 25 82.80 1 3.80“ 75.18 1 3.48* 72.85 1 3.14“ 10 25 84.27 1 2.83“ 70.51 1 1.78 78.53 1 2.83“ 21 25 78.54 1 3.34“ 84.12 1 2.85 87.90 1 1.90“ * Values are means 1 SEM a Significantly different from control group (p < 0.05) b Control group DISCUSSION Studies in normal non-pregnant humans or animals have documented that dietary Ca deprivation stimulates the renal synthesis of 1,25 (OH)ZD3 and increases its serum concentration. These changes pre- sumably are due to hypocalcemic stimulation of the parathyroids (Boyle g; 51., 1971). During the mammalian reproductive cycle, pregnancy and lactation have been associated with alterations in maternal skeletal metabolism, Ca economy and its hormonal regulation. In pregnancy, the influx of Ca into the fetus for normal bone mineralization and development causes a significant decrease in the plasma concentration of Ca (Pitkin, 1979). In lactation, Ca movement from the plasma into the milk results in simi- lar reduction in the concentration of calcium in the plasma (Tovered g; 51., 1976). For this Ca to be available to the fetus or to the milk, maternal absorption of Ca has to be increased. The principal regulatory factor in the absorption of Ca from the gut is vitamin D in its active form, 1,25(OH)203 (Halloran g; gl., 1979). A reduction in the serum Ca concentration, accompanied by eleva- tion in the serum concentration of 1,25(OH)2D3, was noted as early as two weeks post conception in sows fed a normal Ca diet. Further re- duction in serum Ca and elevation in serum 1,25(OH)2D3 concentrations were observed as pregnancy progressed. A significant negative correla- tion between the circulating levels of Ca and 1,25(OH)2D3 was also observed in this study, which indicates that Ca also regulates the pro- duction of 1,25(OH)2D3 during pregnancy. This corroborates a pre- vious report in rats (Halloran g; g1., 1979). 35 36 The significant differences in serum 1,25(OH)203 concentration among the groups of the sows within 15 days of initiating diets demon- strated that the production of 1,25(OH)2D3 was quickly affected by dietary Ca. These observations demonstrate how sensitive 1,25(OH)2D3 synthesis is to modest alterations in dietary Ca. Similar results were reported in normal non-pregnant cows (Blum g; g1., 1983) in which the increase in serum Ca of only 0.07 mmol/l (by CaC12 infusion over 24 hours) and a decrease in serum Ca of only 0.11 mmol/l (by Ca deprivation) were sufficient to respectively lower and raise plasma 1,25(OH)2D3 levels. As previously mentioned, extrarenal production of 1,25(OH)2D3 was elucidated by experiments involving bilateral nephrectomized preg- nant rats in which synthesis of 1,25(OH)ZD3 was not abolished (Heisman g; 31., 1979). Further investigations by Weisman and his co- workers (1979) have demonstrated that cultured explants of human decidua and placenta in vitro can synthesize 1,25(OH)ZD3 from 25-(OH)D3. They also found that the 25-(OH)DB-l alpha-hydroxylase activity of the placental cells was significantly higher than that activity of the renal cells. Gray and his colleagues (1979) have provided evidence that homo- genates of fetal rabbit and pig kidneys also synthesize 1,25(OH)2D3 in vitro when the cells were incubated with 25-(OH)D3 in serum-free media. All these findings suggest that the site of 1,25(OH)2D3 pro- duction during getation, besides the maternal kidneys, could be the feto- placental unit. Our observation that the serum concentration of 1,25(OH)2D3 in all groups of the sows increased gradually throughout gestation might re- late to the increased size of the fetoplacental unit as pregnancy 37 progressed. The reduction in the serum concentration of 1,25(0H)2D3 after farrowing helps to confirm the fact that placenta and fetal kidneys are both involved in raising the maternal serum l,25(OH)2D3 during pregnancy. However, Pitkin 33.11. (1979) indicated that both prolactin and placental lactogen are responsible for an elevation in the maternal plasma 1,25(OH)ZD3 during pregnancy. These investigators also sug- gested that reduction in the levels of plasma 1,25(OH)2D3 after deli- very was due to the reduction in the levels of prolactin and placental lactogen. These variables were not investigated in this study. There was no significant difference in concentration of 1.25- (OH)2D3 between groups of baby pigs at birth. This suggests that serum 1,25(OH)203 of neonatal pigs was unaffected, in utero, by the dam's Ca intake. While we could not find any interrelationships between serum 1.25(OH)203 of the sows and neonatal piglets, a high correla- tion between maternal and neonatal plasma concentrations of l.25(OH)2- D3 was demonstrated in sheep (Ross 5; 31., 1979). humans (Steichen g; 31.. 1980) and in cattle (Goff 1; a1.. 1982). It is possible that this discrepancy is due to differences in placentation among species. It has also been suggested by Noff and Edelstein (1978) that 1,25(OH)2D3 might be esterified by the rat fetus as a means of protection from high maternal concentrations of 1,25(OH)203. A significant correlation was found between maternal serum concen- tration of 1,25(OH)203 and neonatal piglets serum concentrations of ~Ca which suggests that maternal 1,25(OH)2D3 may affect the placental transport of Ca. Similar results have been demonstrated in cows by Goff :5 nl- (1982) who postulated that the ability of the placenta to regulate 38 serum Ca and P concentrations in the fetus is partially dependent on the maternal 1,25(OH)2D3 status. In all groups of baby pigs, the serum concentration of 1,25(OH)2- D3 was two to three times greater at 10 days of age than at birth. This can be attributed to absorption of 1,25(OH)2D3 from the sow's milk, which is essentially the sole source of nutrition for the baby pigs during their first 10 days of life. Another possibility is that the re- nal 25-(OH)D3-1 alpha-hydroxylase activity in the baby pigs increases as pigs age, and it will respond to the level of Ca. In this study we found that the amount of 1,25(OH)2D3 in colos- trum varied inversely with the amount of dietary Ca intake of the dam and was positively correlated (r - 0.90; p < 0.05) with serum concentration of 1,25(OH)ZD3. This finding supports the previous observation that the concentration of vitamin D in the circulation has a direct bearing on that in milk (Hollis 11 gl., 1983) and expands our knowledge about the colostrum concentration of 1,25(OH)2D3 in sows which had not been evaluated before. In previous studies, Gray 2; £1. (1979) have reported an inverse relationship between serum P and 1,25(OH)2D3 in humans. However, in this porcine study, serum P was not significantly affected by dietary Ca intake and was not correlated with serum concentration of 1,25(OH)2- D3. It has been explained by Gray (1981) that low dietary Ca stimu- lates the production of PTH and 1,25(OH)2D3 which mobilize both Ca and P from bone. Further action by PTH increases urinary P, consequently serum P concentration does not change. While the mechanism responsible for maintenance of nearly normal levels of P during Ca loading is not 39 apparent, the alterations in the concentrations of P clearly do not in- fluence serum concentrations of 1,25(OH)2D3. The serum Mg concentrations were significantly affected by dietary Ca treatments (p < 0.05). The fall in serum Mg during Ca loading and the increase in serum Mg level during Ca deprivation are believed to be con- sequences of the alterations in serum PTH concentrations (Bethune g; 31., 1968). According to Meintzer and Steenbock (1954), vitamin D status may have an effect on Mg absorption; however, these investigators explained that the effect of vitamin D on Mg absorption is not as direct as it is on Ca. The vitamin D effect on Mg may be due to its effect on the rates of accretion or mobilization of bone salt. In this study, we could not demonstrate any correlation between 1,25(OH)2D3 and Mg concentrations and conclude that maternal dietary Ca does not have an in utero influence on the status of Mg of the fetal pigs. It has been demonstrated that excess dietary Ca has a reducing ef- fect upon the utilization of Zn in animals fed a corn-soy diet (Tucker and Salmon, 1955; Hoekstra, 1964). A significant effect of dietary Ca treatment on serum Zn concentration was not observed in this study. It is possible that the concentration of Ca in the diet was not high or low enough to produce any significant effect on serum Zn, or Zn supplementa- tion in the diet was sufficient for maintenance of normal Zn concentra- tion during Ca treatments. There were no significant effects of dietary Ca treatments on serum concentration of Cu. It is believed that Ca does not have direct effect on Cu status but may do so indirectly via the alterations in Zn metabol- ism. CONCLUSIONS The competitive binding radioreceptor assay for 1,25(OH)2D3 used in this study was found very satisfactory for swine serum and colos- trum. Using this assay, serum 1,25(OH)2D3 of normally fed, multi- parous sows can be expected to approximate 70 pg/ml at the time of breed- ing, to increase during gestation to about 104 pg/ml by parturition and to decline during lactation to approximately 63 pg/ml by weaning. Colos- trum of these sows is expected to contain approximately 17 pg of 1,25- (OH)2D3/ml. Serum 1,25(OH)2D3 concentrations (pg/ml) of baby pigs from normally-fed sows on a corn-soy based diet can be expected to approximate 43 pg/ml prior to suckling, 94 pg/ml at 10 days of age and 68 pg/ml at 3 weeks of age. In the pregnant sows, serum 1,25(OH)2D3 concentrations re- sponded quickly and inversely to modest changes in dietary Ca. The nega- tive correlation between dietary Ca and 1,25(0H)2D3 persisted through gestation and early lactation. Based upon the serum 1,25(OH)2D3 data of newborn pigs, this parameter was unaffected by the dam's dietary Ca intake, but was especially influenced by the 1,25(OH)2D3 content of the colostrum. It was concluded that the 1,25(OH)2D3 status of the sows does not have an in utero influence on the fetal 1,25(OH)2D3 status. . Of the other nutrients (Mg, P, Cu, Zn) observed in this study, ma- ternal dietary Ca changes caused significant alterations on the maternal serum Mg concentrations during gestation and early lactation. These 40 41 changes did not have any correlation with serum 1,25(OH)2D3 concen- trations. In baby pigs, serum Ca and P increased significantly as ma- ternal dietary Ca increased. However, the correlation between serum Ca and P concentrations in baby pigs and maternal serum 1,25(OH)2D3 was only observed at the time of birth. BIBLIOGRAPHY BIBLIOGRAPHY Askew, F.A., Baurdillom, R.B.. Bruce, H.M.: The distillation of vitamin D. Proc. R. Soc. London 107(1930): 76-90. Avioli, L.V., and Haddad, J.G.: Vitamin D: current concepts. Metabolism 22 (1973): 507-531. Baksi, S.N., and Kenny, A.D.: Effect of antiestrogen on renal metabolism of 25-hydroxyvitamin D3 in vitro in female quail. J. Pharmacol. 18 (1976): 234. Baksi, S.N, and Kenny, A.D.: Ovarian influence on 25-hydroxyvitamin D3 metabolism in Japanese quail. Federation Proc. 35 (1976): 662. Barlet, J.P., Argemi, B., Davicco, M., and Lafaivre, J.: Plasma concen- tration of 2S-hydroxyvitamin D in pregnant and lactating ewes and foetal and newborn lambs. J. Endrocrinol. 79 (1978): 149-150. Bell, N.H., Stein, P.H., Panther, E., Sinha, T.K., and DeLuca, H.F.: Evidence that increased circulating 1,25-dihydroxyvitamin D is the probable cause for abnormal calcium metabolism in sarcoidosis. J. Clin. Invest. 64 (1979): 218. Bethune, J.E., Turpin, R.A., and Inoue, H.: Effect of parathyroid hor- mone extract on divalent ion excretion in man. J. Clin. Endocri- nol. Metab. 28 (1968): 673. Bhattacharyya, M.H., and DeLuca, H.F.: subcellular location of rat liver calciferol-25-hydroxylase. Arch. Biochem. Biophys. 160 (1974a): 58-62. Bhattacharyya, M.M., and DeLuca, H.F.: The regulation of rat liver cal- ciferol-25-hydroxylase. J. Biol. Chem. 248 (1974b): 2969-2973. Blum, J.W., Trechsel, U., Born, W., Tobler, P.H., Taylor, C.M., Binswanger, U., and Fischer, J.A.: Rapidity of plasma 1,25- dihydroxyvitamin D responses to hypo- and hypercalcemia in steers. Endocrinol. 113 (1983): 523-526. Blunt, J.W., DeLuca, H.F., and Schnoes, H.K.: 25-Hydroxycholecalciferol. A biologically active metabolite of vitamin D3. Biochem. 7 (1968): 3317-3322. - Bordier, P.J., Ryckwaert, A., and Marie, P.: Vitamin D metabolites and bone mineralization in man; Vitamin D. Biochemical, chemical and clinical aspects related to calcium metabolism. Am. J. Physiol. 226 (1977): 897-911. 42 43 Boyle, I.T., Gray, R.W., and DeLuca, H.F.: Regulation by calcium of in vivo synthesis of l,25-dihydroxycholecalciferol and 21,25-dihy- droxycholecalciferol. Proc. Natl. Acad. Sci. USA 68 (1971): 2131- 2134. Boyle, I.T., Miravet, L., and Gray, R.W.: The response of intestinal calcium transport to 25-hydroxy and 1,25-dihydroxy vitamin D in nephrectomized rats. Endocrinology 90 (1972): 605-608. Broadus, A.E., Horst, R.L., Lang, R., Littledike, E.T., Rasmussen, H.: The importance of circulating 1,25-dihydroxyvitamin D in the , pathogenesis of hypercalciuria and renal stone formation in pri- mary hyperparathyroidism. N. Engl. J. Med. 302 (1980): 421. Brock, J.F., and Diamond, L.K.: Rickets in rats by iron feeding. J. Pediat. 4 (1934): 442. Brommage, R., and Newman, W.F.: Mechanism of mobilization of bone min- eral by 1,25-dihydroxyvitamin D3. Am. J. Physiol. 237 (1979): 113-120. Capen, C.C., Cole, C.R., and Hibbs, J.W.: The pathology of hypervita- minosis D in cattle. Pathol. Vet. 3 (1966): 350-378. Carlsson, A.: Tracer experiments on the effect of vitamin D on the ske- letal metabolism of calcium and phosphorus. Acta Physiol. Scand. 26 (1952): 212-220. Chick, H., Dalzell, E.J., and Hume E.M.: Studies of rickets in Vienna 1919-1922. Medical Research Council Special Reports No. 77 (1923). Clark, M.B., and Potts, J.T., Jr.: 25-Hydroxyvitamin D3 regulation. Calcif. Tissues Res. 22 (1977): 29-34. Clark, S.A., Stumpf, W.E., and Sar, M.: Effect of 1,25-dihydroxyvitamin D3 on insulin secretion. Diabetes 30 (1981): 382-386. Clark, S.A., Stumpf, W.E., Bishop, C.W., DeLuca, H.F., Park, D.H., and Job, T.H.: The adrenal: a new target organ of the calciotropic hormone 1,25-dihydroxyvitamin D3. Cell Tissue Res. 243 (1986): 299-302. ‘ Clegg, F.G., and Hollands, J.G.: Cervical scoliosis and kidney lesions in sheep following dosage with vitamin D. Vet. Rec. 98 (1976): 144-146. Davie, M., and Lawson, D.E.M.: Assessment of plasma 25-hydroxyvitamin D response to ultraviolet irradiation over a controlled area in young and elderly subjects. Clin. Sci. 58 (1980): 235-242. Delvin, E.E., Arabian, A., Glorieux, F.H.. and Mamer, O.A.: In vitro metabolism of 25-hydroxycholecalciferol by isolated cells from hwman decidua. J. Clin. Endocrinol. Metab. 60 (1985): 880-885. 44 DeLuca, H.F., and Schnoes, M.R.: Metabolism and mechanism of action of vitamin D. Annu. Rev. Biochem. 45 (1976): 631-666. Deluca, H.F.: The vitamin D system: A view from basic science to the clinic. Clin. Biochem. 14 (1981): 213-222. Dokoh, 8., Pike, J.W., Chandler, J.S., Mancini, J. M., and Haussler, M.R.: An improved radiorecptor assay for 1,25-dihydroxyvitamin D in plasma. Anal. Biochem. 116 (1981): 211. Drake, T.S., Kaplan, R.A., and Lewis, T.A.: The physiologic hyperpara- thyroidism of pregnancy. Obstet. Gynecol. 53 (1979): 746. Dueland, S., Pederson, J.I., Helgerud, P., and Drevon, O.A.: Transport of vitamin D from rat intestine. Evidence for transfer of vitamin D from chylomicra to alpha globulins. J. Biol. Chem. 275 (1982 : 146-150. Fraser, D.R., and Kodicek, E.: Regulation of 25-hydroxycholecalciferol- l-hydrolase activity in kidney by parathyroid hormone. Nature 241 (1973): 163-166. Galante, L., Colston, K.W., Evans, I.M.A., Byfield, P.G.H., Matthews, E.W., and MacIntyre, I.: The regulation of vitamin D metabolism. Nature 244 (1973): 438-440. Carabedian, M. Corvol, M.T., and Bailly Du Bois, M.: The biological ac- tivity of 24,25-dihydroxycholecalciferol on cultured chondrocytes and its in vitro production in cartilage and calvarium. Excerpta Medica 421 (1978): 1673-1676. Garabedian, M., Holick, M.F., DeLuca, H.F., and Boyle, I.T.: Control of 25-hydroxycholeca1ciferol metabolism by parathyroid glands. Proc. Natl. Acad. Sci. USA 69 (1972): 1673-1676. Garabedian, M., Tanaka, Y., and Holick, M.F.: Response to intestinal calcium transport and bone calcium mobilization to 1,25-dihydoxy- vitamin D in thyroparathyroidectomized rats. Endocrinology 94 (1974): 022-1027. Ghazarian, J.G., and DeLuca, H.F.: 25-Hydroxycho1ecalciferol-l-hydro- lase, a specific requirement for NADPH and a hemoprotein compo- nent in chick kidney mitochondria. Arch. Biochem. Biophys. 160 (1974): 63-72. Gill, B.S., Singh, M., and Chopra, A.K.: Enzootic calcinosis in sheep: Clinical signs and pathology. Am.J. Vet. Res. 37 (1976): 545-552. Goff, J.P., Horst, R.L., and Littledike, E.T.: Effect of the maternal vitamin D status at parturition on the vitamin D status of the neonatal calf. J. Nutr. 112 (1982): 1387-1393. Goff, J.P., Horst, R.L., and Littledike, E.T.: Effect of sow vitamin D status at parturition on the vitamin D status of neonatal piglets. J. Nutr. 114 (1984): 163-169. 45 Gran, F.C.: The retention of parenterally injected calcium in rachitic dogs. Acta Physiol. Scand. 50 (1960): 132-139. Gray, R.W.: Control of plasma 1,25-(OH) -vitamin D3 concentrations by calcium and phosphorus in the rats: Effects of hypophysectomy. Calcif. Tiss. Int. 33 (1981): 485-488. Gray, T.K., Lester, G.E., and Lorenc, R.S.: Evidence for extrarenal l-alpha-hydroxylation of 25-hydroxyvitamin D3 in pregnancy. Science 204 (1979): 1311. Groth, A.H., Jr.: The comparative histopathology of rickets and an osteodystrophy in immature Iowa swine. Am. J. Vet. Res. 19 (1958): 409-416. Haddad, J.G.: Natural and synthetic sources of circulating 25-hydroxy- vitamin D in Man. Nature 244 (1973): 515. Halloran, R.P., Holick, M.F., and DeLuca, H.F.: Vitamin D metabolism during pregnancy and lactation in the rat. Proc. Natl. Acad. Sci. USA 76 (1979): 5549-5553. Harris, C.A., Sutton, R.A.L., and Seeley, J.F.: Effect of 1,25-(OH) - vitamin D3 on renal electrolyte handling in the vitamin D defi- cient rat: dissociation of calcium and sodium excretion. Clin. Res. 24 (1976): 685. Harrison, H.C.. and Harrison, H.E.: Calcium transport by rat colon in vitro. Am. J. Physiol. 217 (1969): 121-125. Harrison H.E., and Harrison, H.C.: Rickets then and now. J. Pediatr. 87 (1975): 1144-1151. Harrison, H.E., and Harrison, H.C.: The renal excretion of inorganic phosphate in relation to the action of vitamin D and parathyroid hormone. J. Clin. Invest. 20 (1941): 47-55. Harrison, J.E., Hitchman, J.W., Jones, 0., Tam, C.S., and Heersche, J.N.M.: Plasma vitamin D metabolite levels in phosphorus defi- cient rats during the development of vitamin D deficient rickets. Metabolism 31 (1982): 1121-1127. Haussler, M., Hughes, M., Baylink, D., Littledyke, E.T., Cork, D., and Pitt, M.: Influence of phosphate depletion on the biosynthesis and circulating level of 1 alpha 25-dihydroxyvitamin D. In: Phosphate metabolism. S.G. Massry and E. Ritz, ed. Plenum Press, New York (1977): 233-250. Haussler, M.R., and Rasmussen, H.: The metabolism of vitamin D3 in the chick. J. Biol. Chem. 247 (1972): 2328-2335. Haussler, M.R., Myrtle, J.P., and Norman, A.W.: The association of a metabolite of vitamin D with intestinal mucosa chromatin in vivo. J. Biol. Chem. 2 3 (1968): 4055-4064. 46 Haussler, M.R.: Vitamin D: mode of action and biomedical applications. Nutr. Rev. 32 (1974): 257-266. Henry, H.L., and Norman, A.W.: Vitamin D: Metabolism and Biological ac- tions. Ann. Rev. Nutr. 4 (1984): 493-520. Hess, A.F., Weinstock, M., and Helman, F.D.: The antirachitic value of irradiated phytosterol and cholesterol 1. J. Biol. Chem. 63 (1929): 305-308. Hillman, L.S., and Haddad, J.G.: Human perinatal vitamin D metabolism. I. 25-hydroxyvitamin D in maternal and cord blood. J. Pediatr. 84 (1974): 742-749. Hoekstra, H.C.: Observation on mineral interrelationships. Fed. Proc. 23 (1964): 1068. Holick, M.F., and Clark, M.B.: The photobiogenesis and metabolism of vitamin D. Federation Proc. 37 (1978): 2567-2574. Holick, M.F., Baxter, L.A., Schranfrogel, P.K., Tavela, T.E., and DeLuca, H.F.: Metabolism and biological activity of 24,25-dihydroxyvita- min D3 in the chick. J. Biol. Chem. 251 (1976): 397-402. Holick, M.F., Garabedian, M., and DeLuca, H.F.: 1,25-Dihydroxycho1ecal- ciferol: metabolite of vitamin D active on bone in anephric rats. Science 176 (1972b): 1146-1147. Holick, M.F., Schnoes, H.K., and DeLuca, H.F.: Isolation and identifica- tion of 24,25-dihydroxycholecalciferol, a metabolite of vitamin D3 made in the kidney. Biochem. 11 (1972a): 4251-4255. Hollis, B.W., Lambert, P.W., and Horst, R.L.: Factors affecting the antirachitic sterol content of native milk. Br. J. Nutr. 49 (1983): 475-480. Horst, R.L., and Littledike, E.T.: Comparison of plasma concentrations of vitamin D and its metabolites in young and aged domestic ani- mals. Comp. Biochem. Physiol. 73 (1982): 485-489. Horst, R.L., Littledike, E.T., Riley, J.L., and Apoli, J.L.: Quantita- tion of vitamin D and its metabolites and their plasma concentra- tions in five species of animals. Anal. Biochem. 116 (1981): 189-192. Horst, R.L., Reinhardt, T.A., Russell, J.R., and Napoli, J.L.: The iso- lation and identification of vitamin D2 and D3 from Medicago sativa (Alfalfa plant). Arch. Biochem. Biophys. 231 (1984): 67- 71. Horsting, M., and Deluca, H.F.: In vitro production of 25-hydroxychole- calciferol. Biochem. Biophys. Res. Commun. 36 (1969): 250-256. 47 Hosomi, J., Hosoi, J., Abe, E., Suds, T., and Kuroki, T.: Regulation of terminal differentiation of cultured mouse epidermal cells by 1,25-dyhydroxyvitamin D3. Endocrinology 113 (1983): 1950- 1956. Howard, G.A., Turner, R.T., Sherrard, D.J., and Baylink, D.J.: Human bone cells in culture metabolize 25-hydroxyvitamin D3 to 1,25- dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. J. Biol. Chem. 256 (1981): 7738-7740. Hughes, M.R., Baylink, D.J., Jones, P.G., and Haussler, M.R.: Radioli- gand receptor assay for 25-hydroxyvitamin D /D and 1,25-dihy- droxyvitamin D2/D3. J. Clin. Invest. 58 (1376 61-70. Hughes, M.R., Brumbaugh, P.F., Haussler, M.R., Wergedal, J.E., and Baylink, D.J.: Regulation of serum 1,25-dihydroxyvitamin D3 by calcium and phosphate in the rat. Science 190 (1975): 578-580. Hunt, R. D. Garcia, F. G. and Hegsted, D.H.: A comparison of vitamin and D3 in New World primates. I. Production and regression of osteodystrophia fibrosa. Lab. Anim. Sci. 17 (1967): 222-234. Ichikawa, Y., Hiwatashi, A., and Nishi, Y.: Tissue and subcellular dis- tributions of cholecalficerol-25-hydroxy1ase: cytochrome P450D25 linked monooxygenase system. Comp. Biochem. Physiol. 75B (1983): 479-488. Ishida, H., Seino, S., Seino, Y., Tsuda, R., Takemura, J., Nishi, S., Ishizuka, 8., and Imura, H.: Effect of 1,25-dihydroxyvitamin D3 on pancreatic B and D cell function. Life Sciences 30 (1983): 1779-1786. Krook, L., Wasserman, R.H., Shilvely, J.N., Tashjian, A.H., Jr., Brokken, T.D., and Morton, J.F.: Hypercalcemia and calcinosis in Florida horses: implications of the shrub, Cestrum diurnum, as the causa- tive agent. Cornell Vet. 65 (1975): 25-26. Lawson, D.E.M., Fraser, D.R., Kodicek, 3., Morris, H.R., and Williams, D.H.: Identification of 1,25-dihydroxycholecalciferol, a new kid- ney hormone controlling calcium metabolism. Nature 230 (1971): 228-230. Malherbe, W.D.: Some observations on rickets and allied bone disease in South African domestic animals. Ann. N. Y. Acad. Sci. 64 (1956): 128-146. Manolagas, S.C., Culler, F.L., Howard, J.E., Brinkman, A.S., and Deftos, L.J.: The cytoreceptor assay for 1,25-dihydroxyvitamin D and its application to clinical studies. J. Clin. Endocrinol. Metab. 56 (1983): 751-764. Martin, D.L., and DeLuca, H.F.: Calcium transport and the role of vita- min D. Arch. Biochem. Biophys. 134 (1969): 139-148. 48 Matsui, T., Nakao, Y., Koisumi, T., Nakagawa, T., and Fujita, T.: 1,25- dihydroxyvitamin D3 regulates proliferation of activated T-lym- phocyte subsets. Life Sciences 37 (1985): 95-101. Mawer, E.B.: Endocrinology of calcium metabolism. Clin. Res. 30 (1982): 271-295. McCollum, E.V., Simmonds, N., Becker, J.E., and Shipley, P.G.: Studies on experimental rickets. XXI. An experimental demonstration of the existence of a vitamin which promotes calcium deposition. J. Biol. Chem. 53 (1922): 293-312. Meintzer, R.B., and Steenbock, H.: Vitamin D and magnesium absorption. J. Nutr. 84 (1954): 285. Mellanby, E.: An experimental investigation on rickets. Lancet 1 (1919): 407-412. Morris, K.M.L., and Levack, V.M.: Evidence for aqueous soluble vitamin D-like substance in the calcinogenic plant, Irigeggm F1avescens. Life Sci. 30 (1982): 1255-1262. Napoli, J.L., Okita, R.T., Masters, B.S., and Horst, R.L.: Identifica- tion of 25,26-dihydroxyvitamin D3 as a rat renal 25-hydroxy- vitamin D3 metabolite. Biochemistry 20 (1981): 6230-6235. Napoli, J.L., Pramanik, B.C., Partridge, J.J., Uskokovic, M.R., and Horst, R.L.: 23S 25-dihydroxyvitamin D3 as a circulating metabo- lite of vitamin D3. J. Biol. Chem. 257 (1982): 9634-9639. Nicolaysen, R., Eeg Larsen, N., and Malm, O.J.: Physiology of calcium metabolism. Physiol. Rev. 33 (1953): 424-444. Noff, D., and Edelstein, 8.: Vitamin D and its hydroxylated metabolites in the rat: placental and lacteal transport, subsequent metabolic pathways and tissue distribution. Hormone Res. 9 (1978): 292-300. Nordin. B.E.C.: Osteomalacia, osteoporosis and calcium deficiency. Clin. Orthop. 17 (1960): 235-257. Norman, A.W.: The mode of action of vitamin D. Biol. Rev. 43 (1968): 97-137. Norman, A.W., Friedlander, E.J., and Henry, H.: Interrelationship be- tween the key elements of the vitamin D endocrine system: 25-OH- D3-l-hydroxy1ase, serum calcium and phosphorus levels, intes- tinal 1, 25-(OH)2D3, and intestinal calcium binding protein. In: Phosphate metabolism. S.G. Massry and E. Ritz, (eds.) Plenum Press, New York (1977): 211-231. O'Donnell, J.M., and Smith, M.W.: Vitamin D-like action of Solanum mala- coxylon on calcium transport by rat intestine. Nature 244 (1973): 357-358. 49 Okano, T., Yasumura, M., and Mizuno, K.: Photochemical conversion of 7- dehydrocholestrol into vitamin D3 in rat skin. J. Nutr. Sci. Vitaminol. 23 (1977): 165-168. Olson, E.B., Knutson, J.C., and Bhattacharyya, M.R.: The effect of hepa- tectomy on the synthesis of 25-hydroxyvitamin D3. J. Clin. Invest. 57 (1976): 1213-1220. Omdahl, J.L., and DeLuca, H.F.: Regulation of vitamin D metabolism and function. Physiol. Rev. 53 (1973): 327-372. Orgler, H.W.: The history of rickets. In: Vitamins and Hormones. R. Nicolaysen and N. Beg-Larsen, eds. Academic Press, New York, Vol. 11 (1953): 29-60. Orr, W.J., Holt, L.E., Wilkins, L., and Boone, F.H.: The calcium and phosphorus metabolism in rickets, with special reference to ultra- violet ray therapy. Am. J. Dis. Child. 26 (1923): 362-372. Pahuja, D.N., and DeLuca, H.F.: Role of the hypophysis in the regulation of vitamin D metabolism. Mol. Cell. Endocrinol. 23 (1981): 345- 350. Penn, G.B.: Calciphylactic syndrome in pigs. Vet. Rec. 86 (1970): 718- 721. Peterlik, M., Regal, D.S., and Kohler, H.: Evidence for a 1,25-dihy- droxyvitamin D-like activity in Irisetum Elevescens: possible cause for calcinosis in grazing animals. Biochem. Biophys. Res. Commun. 77 (1975): 775-781. Pitkin, R.M., Reynolds, W.A., Williams, G.A., and Hargis, G.K.: Calcium metabolism in normal pregnancy: a longitudinal study. Am. J. Obstet. Gynecol. 133 (1979): 781. Popovtzer, M.M., Robinette, J.B., DeLuca, H.F., and Holick, M.F.: The acute effect of 25-hydroxycholecalciferol on renal handling of phosphorus-evidence for a parathyroid hormone-dependent mechanism. J. Clin. Invest. 53 (1974): 913-921. Provvedini, D.M., Tsoukas, C.D., Deftos, L.J., and Manolagas, S.C.: 1,25-dihydroxyvitamin D3 receptors in human leukocytes. Science 221 (1983): 1181-1183. Puschett, J.B., Moranz, J., and Kurnich, W.S.: Evidence for a direct ac- tion of cholecalciferol and 25-hydroxyca1ciferol on the renal transport of phosphate, sodium, and calcium. J. Clin. Invest. 51 (1972): 373-385. Quarterman, J.: The distribution of vitamin D between the blood and the liver in the pig, and observations on the pathology of vitamin D toxicity. Br. J. Nutr. 18 (1964): 65-77. 50 Rader, J.L., Baylink, D.J., and Hughes, M.R.: Calcium and phosphorus deficiency in rats: Effects on PTH and 1,25-dihydroxyvitamin D3. Am. J. Physiol. 236E (1979): 118-122. Raisz, L.G., Trummel, C.L., and Holick, M.F.: l,25-dihydroxycholeca1ci- ferol: a potent stimulator of bone resorption in tissue culture. Science 175 (1972): 768-769. Rasmussen, H., Baron, R., Broadus, A., Defronzo, R., Lang, R., and Horst, R.L.: 1,25(OH)2D3 is not the only metabolite involved in the pathogenesis of osteomalacia. Am. J. Med. 69 (1980): 360-368. Rasmussen, H., Wong, M., and Bikle, D.: Hormonal control of the renal conversion of 25-hydroxycholecalcifero1 to 1,25-dihydroxychole- calciferol. J. Clin. Invest. 51 (1972): 2502-2504. Reinhardt, T.A., Horst, R.L., Orf, J.W., and Hollis, B.W.: A microassay for 1,25-dihydroxyvitamin D not requiring high performance liquid chromatography: application to clinical studies. J. Clin. Endo- crinol. Metab. 58 (1984): 91-98. Reynolds, J.J., Holick, M.F., and DeLuca, H.F.: The role of vitamin D metabolites in bone resorption. Calcif. Tiss. Res. 12 (1973): 295-301. Reynolds, J.J., Pavlovitch, H., and Balsan, S.: 1,25-dihydroxycholecal- ciferol increases bone resorption in thyroparathyroidectomized mice. Calcif. Tiss. Res. 21 (1976): 207-212. Rojanasathit, S., and Haddad, J.G.: Hepatic accumulation of vitamin D and 25-hydroxyvitamin D3. Biochem. Biophys. Acta 421 (1976): 12-21. Ross, R., Care, A.D., Taylor, C.M., Pele, B., and Sommerville, B.: The transplacental movement of metabolites of vitamin D in the sheep. In: Vitamin D: Basic Research and Its Clinical Application. Walter de Gruyter, Berlin, West Germany (1979): 341-344. Schachter, D., and Rosen, S.M.: Active transport of Ca45 by the small intestine and its dependence on vitamin D. Am. J. Physiol. 196 (1959): 357-362. Schachter, D.: Vitamin D and the active transport of calcium by the small intestine. In: The transfer of calcium and strontium across biological membrane. R.H. Wasserman, ed. Academic Press, New York (1963): 197-210. Schneider, L.E., Omdahl, J., and Schedl, H.P.: Effects of vitamin D and its metabolites on calcium transport in the diabetic rat. Endo- crinology 99 (1975): 793-799. Singh, 0., Gill, B.S., and Randhawa, N.S.: Enzootic calcinosis in sheep: soil-plant-animal relationship. Am. J. Vet. Res. 37 (1976): 553- 556. 51 Smith, R., and Stern, G.: Myopathy, osteomalacia. and hyperparathyroid- ism. Brain 90 (1967): 593-602. Sommerville, B.A., Fox, J., and Care, A.D.: The in vitro metabolism of 25-hydroxycholecalciferol by pig kidney: effect of low dietary levels of calcium and phosphorus. Br. J. Nutr. 40 (1978): 159- 162. Spanos, E., Barrett, D., MacIntyre, 1., Pike, J.W., Safilian, E.F., and Haussler, M.R.: Effect of growth hormone on vitamin D metabolism. Nature 273 (1978): 246. Spanos, E., Pike, J.W., Haussler, M.R., Colston, K.W., Evans, I.M.A.. Goldner, A.M., McCain, T.A., and MacIntyre, I.: Circulating 1,25- dihydroxyvitamin D in the chicken: enhancement by injection of prolactin and during egg laying. Life Sciences 19 (1976): 1751- 1756. Spencer, E.M., and Tobiassen, 0.: The mechanism of action of growth hor- mone on vitamin D metabolism in the rat. Endocrinology 108 (1981): 1064-1070. Stanbury, S.W., Mawer, E.E., Taylor, C.M., and de Silva, P.: The skin, vitamin D and the control of its 25-hydroxylation: an attempted integration. Miner. Electrolyte Metab. 3 (1980): 51-60. Steele, T.H., Engle, J.E., Tanaka, Y., Lorenc, R.S., Dudgeon, R.L., and DeLuca, H.F.: Phosphatemic action of 1,25-dihydroxyvitamin D3. Am. J. Physiol. 229 (1975): 489-495. Steenbock, H., Kletzien, S.H.F., and Halphin, J.G.: The reaction of the chicken to irradiated ergosterol and irradiated yeast as con- trasted with the natural vitamin D in fish liver oils. J. Biol. Chem. 97 (1932): 249-264. Steichen, J.J., Tsang, R., Gratton, T., Hamstra, A., and DeLuca, H.F.: Vitamin D homeostasis in the perinatal period: 1,25-dihydroxy- vitamin D in maternal, cord and neonatal b1ood.- N. Eng. J. Med. 302 (1980): 315-319. ' Tanaka, Y., and DeLuca, H.F.: Bone mineralization activity of 1,25-dihy- droxycholecalciferol, a metabolite of vitamin D. Arch. Biochem. Biophys. 146 (1971): 574-578. Tanaka, Y., and DeLuca, H.F.: The control of 25-hydroxyvitamin D meta- bolism by inorganic phosphorus. Arch. Biochem. Biophys. 154 (1973): 566-574. ‘. Taylor, A.N., and Wasserman, R.H.: Vitamin D-induced calcium-binding protein: comparative aspects in kidney and intestine. Am. J. Physiol. 223 (1972): 110-114. 52 Tornquist, K., and Allardt, C.L.: Effect of 1,25(0H)2D3 on the TSH secretion from rat pituitary cells. Horm. Metabol. Res. 18 (1986): 69. Tovered, S.V., Harper, C., and Munson, P.L.: Calcium metabolism during lactation. Endocrinology 99 (1976): 371-378. Tucker, G., III, Gagnon, R.E., and Haussler, M.R.: Vitamin D3-25- hydroxylase: tissue occurrence and apparent lack of regulation. Arch. Biochem. Biophys. 155 (1973): 47-57. Tucker, H.F., and Salmon, W.D.: Parakeratosis or zinc deficiency disease in the pig. Proc. Soc. Exp. Biol. Med. 88 (1955): 613. Walling, M.W., and Kimberg, D.V.: Action of Solanum malacoxylgg on in- testine of anephric rats. Gastroenterology 69 (1975): 200-205. Walters, M.R., Wicker, D.C., and Riggle, P.G.: 1,25-dihydroxyvitamin D receptors identified in the rat heart. J. Mol. Cell Cardiol. 18 (1986): 67-72. Wasserman, R.H., Kallfelz, F.A., and Comar, C.L.: Active transport of calcium by rat duodenum in vivo. Science 133 (1961): 883-884. Wasserman, R.H., and Corradino, R.A.: Metabolic role of vitamins A and D. Ann. Rev. Biochem. 40 (1971): 501-532. Weisman, Y., Harell, A., Edelstein, S., David, M., and Spirer, 2.: 1,25- dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in vitro synthesis by human decidua and placenta. Nature 281 (1979): 317- 319. Wezeman, F.H., 25-hydroxyvitamin D3: autoradiographic evidence of sites of action in epiphyseal cartilage and bone. Science 194 (1976): 1069-1071. Whitsett, J.A., Ho, M., Tsang, R.C., Norman, E.J., and Adams, H.C.: Syn- thesis of 1,25-dihydroxyvitamin D by human placenta in vitro. J. Clin. Endocrinol. Metab. 53 (1381): 484-488. Windaus, A., Lettre, H., and Schenck, P.: 7-Dehydrocho1esterol. Anal. Chem. 11 (1936): 98-108. Windaus, A., Linsert, 0., Luttringhaus, A., and Weidlich, G.: Crystal- line vitamin D2. Ann. Biochem. 492 (1932): 226-241. Wong, C.L., Luben, R., and Cohn, D.V.: l,25-dihydroxycholecalciferol and parathormone: effects in isolated osteoclast-like and osteoblast- 1ike cells. Science 197 (1977): 663-665. Wong, R.C., Myrtle, J.P., and Tsai, H.C.: Studies on calciferol metabol- ism. V. The occurrence and biological activity of 1,25-dihy- droxyvitamin D3 in bone. J. Biol. Chem. 247 (1972): 5728-5735. 53 Worker, N.A., and Carrilo, B.J.: "Enteque Seco", calcification and wast- ing in grazing animals in the Argentine. Nature 215 (1967): 72- 74. Yoon, P.S., and DeLuca, H.F.: Resolution and reconstitution of soluble components of rat liver microsomal vitamin D -25-hydroxylase. Arch. Biochem. Biophys. 203 (1980): 529-541. ..|.AI ‘8 :1il Ill 1 I 1 III I" ‘llul...f|!I.