Plasma Ca influences vitamin D metabolite as rats develop vitamin D deficiency UWE KOLLENKIRCHEN, Department of Physiology,
MARIAN R. WALTERS, AND JOHN FOX Tulane University School of Medicine, New Orleans, Louisiana
KOLLENKIRCHEN, UWE, MARIAN R. WALTERS,AND JOHN FOX. Plasma Ca influences vitamin D metabolite levels as rats develop vitamin D deficiency. Am. J. Physiol. 260 (Endocrinol. Metab. 23): E447-E452,1991.-The hypocalcemia that accompanies vitamin D deficiency is a major obstacle to proper interpretation of the role(s) of vitamin D metabolites in Casensitive tissues. This paper describes the development and complete characterization of a dietary regimen with which normocalcemia was maintained in rats throughout the development of vitamin D deficiency. Normal weanling rats were fed diets containing 0.8% Ca, 0.5% P, and vitamin D3 (group A), or vitamin D-deficient diets containing 0.8% Ca and 0.5% P (group B); 2.0% Ca and 1.25% P (group C); or 2.0% Ca, 1.25% P, and 20% lactose (group D) for 19 wk. Group D rats were normocalcemic and normophosphatemic with normal parathyroid hormone (PTH) levels throughout the study. In contrast, from 4-19 diet wk, groups B and C were hypocalcemic with elevated PTH. Initially, plasma 25-hydroxyvitamin D3 [ 25( OH)D,] levels decreased most rapidly, and 1,25-dihydroxyvitamin D3 [ 1,25( OH)gDB] levels decreased least rapidly in group B rats, such that plasma 25(OH)D3 levels were reduced to 200300 pg/ml before a decrease in l,25(OH)2D3 levels was observed. However, vitamin D metabolite levels were similar in groups B, C, and D from 4-19 wk. Duodenal active Ca transport mirrored changes in plasma l,25(OH)2DS levels and was abolished after 10 wk. The results also suggested that vitamin D may not be necessary for normal bone mineralization since tibia mineral content and plasma alkaline phosphatase levels were similar in normocalcemic groups A and D throughout the study. dietary calcium; amino-terminal parathyroid hormone; 1,25dihydroxyvitamin D,; 25-hydroxyvitamin D,; active calcium absorption; bone mineral content
THE PHYSIOLOGICALLY ACTIVE metabolite ofvitaminDS, 1,25dihydroxyvitamin D3 [1,25(OH)2D& is the major stimulator of intestinal Ca absorption. This steroid hormone also plays an important role in bone mineral mobilization and renal tubular Ca reabsorption (9, 20). However, 1,25(OH)zD3 receptors have also been described in tissues other than these “classical” 1,25(OH)2D3 targets, e.g., heart (25, 29, 32), testis (31), lung (29), pancreas (21), skin (5, 12), as well as in cells of the hematopoetic system (22). 1,25(OH)2D3 increases Ca uptake by rat cardiac myocytes (30) and inhibits proliferation and stimulates differentiation in several cell lines (12, 22). Because Ca is an important element in the regulation of cell function, it has been suggested that 1,25(OH)ZD3 may play a role in intracellular Ca homeostasis (28). Studies of the physiological role of 1,25(OH)2D3 and 0193~1849/91
levels
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its receptors have been compromised in the past because vitamin D-deficient animals are invariably hypocalcemic and have marked secondary hyperparathyroidism (6, l416). Administration of 1,25(OH)2D3 to these animals dramatically increases plasma Ca and reduces parathyroid hormone (PTH) levels (6). Because the function of many of the target tissues described above depends critically on plasma Ca and because PTH has also been shown to have major effects on the function of the cardiovascular system (18), it is virtually impossible to determine if observed changes in function after vitamin D administration result from vitamin D metabolite action at that tissue or simply a physiological response to changes in plasma Ca and/or PTH (16). Thus a simple means of preventing hypocalcemia and secondary hyperparathyroidism during the development of vitamin D deficiency is essential if the role of vitamin D3 in its targets is to be interpreted appropriately. For example, because the spermatogenesis cycle in rats takes 6-8 wk (31), it is important for appropriate study of the effects of vitamin D deficiency on testicular function that the vitamin D-depleted animals are not only normocalcemic and vitamin D-deficient at the time of semen analysis but that they were also normocalcemic and vitamin Ddeficient throughout the preceding 6-8 wk. Dietary Ca supplementation has been reported to prevent severe hypocalcemia in vitamin D-deficient rats (14, l5), but these dietary regimens frequently result in hypophosphatemia, and plasma PTH is usually elevated (or unreported) in these systems. Furthermore, the long-term effects of these diets on plasma Ca, phosphate, and PTH have not been reported. Thus extensive studies were undertaken to develop a reliable model of vitamin D deficiency unaccompanied by changes in other parameters of Ca metabolism in rats by varying the dietary Ca and P contents. Furthermore, it was also important to establish that these parameters were sustained throughout the dietary period, allowing testing of vitamin D effects both early and late during the development of vitamin D deficiency. This ensures that physiological effects measured at any given time would reflect the results of a constant and sustained hormonal and metabolic milieu. These studies also permitted a determination of whether changes in dietary Ca intake and plasma Ca levels influence 1) the rate of development of vitamin D-deficiency, 2) the rate of loss of active duodenal Ca transport [an important index of 1,25(OH)2D3 action], and 3) bone mineral homeostasis
0 1991 the American
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METHODS
Normal weanling male CD (Sprague-Dawley) rats were obtained from Charles River Labs. On receipt, the rats were randomly divided into four groups and housed in hanging wire cages under incandescent light and were fed one of the four diets described in Table 1. The diets were purchased from Teklad, Madison, WI, and were color coded to reduce the possibility of errors in feeding. T he day after receipt, 12 rats fed the vitamin D-replete diet (diet A) were anesthetized with pentobarbital sodium (60 mg/kg, ip) and were exsanguinated by cardiac puncture. Subsequently, five rats from each dietary group were similarly exsanguinated after 1-19 diet wk. In all cases, plasma was collected for analysis. The proximal 6 cm of small intestine was removed and placed in ice-cold isoto nit saline . Duodenal active Ca tran .sport was m.easured us1 ng the everted gut sac techn .ique (26). A tibia was removed and defatted by soaking in excess dichloromethane-methanol (1:l) for 3 days. After drying at 100°C and weighing, the dry bones were ashed overnight at 550°C and reweighed. Tibia mineral content was expressed as a percentage of the fat-free dry weight (6, 7). Plasma analyses. Before assay, vitamin D3 metabolites were extracted from plasma and purified using minor modifications (8) of established methods (13, 23). Recoveries of 25(OH)D3 and l,25(OH)2D3 averaged 70-80%. 25(OH)Ds was quantitated by radioimmunoassay (6, 7) using sheep antiserum 02282. The detection limit was 1.5-2 pg/tube (6-10 pg/ml for 1 ml plasma). l,25(OH)ZD3 level s were quantitated by radioreceptor assay using a calf thymus receptor prepared using an established method (23). The assay detection limit was 0.4 pg/tube (l-2 pg/ml for 1 ml plasma). Plasma NH2-terminal parathyroid hormone (PTH) levels were measured with a homologous rat PTH-( l34) radioimmunoassay with goat antiserum G813-PTH, which was described previously (6, 7). The assay detection limit was 6 pg rat PTH-(1-34)/ml. Plasma alkaline phosphatase activity was measured by quantitating the hydrolysis of p-nitrophenol phosphate to p-nitrophenol using a kit (Medical Analysis Systems, Camarillo, CA). Plasma Ca levels were measured using methylthymol blue (lo), and plasma phosphate was measured by the method of Chen et al. (4). Statistical analysis. All data are presented as means t SE. For statistical analysis, undetectable values were arbitrarily assigned the assay detection limit. The significance of differences between control (group A) and 1. Composition of diets
TABLE Group
A B c
D
Diet
TD87092 TD87093 TD87094 TD87095
Vitamin Dt3, IWE:
Ca, %
P, %
Lactose, 5%
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0.5 0.5 1.25 1.25
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treatment (groups B, C, and D) were determined at each time point by analysis of variance (ANOVA) and Dunnett’s test. When variances were unequal (estimated by F max or Bartlett’s test), the data were log-transformed before ANOVA. Nonparametric tests (H test of Kruskal and Wallis and U test of Mann and Whitney) were used if log-transformed data still showed unequal variances. Results were analyzed using commercial software (Quickstat; CCP-Soft, Marburg, FRG). RESULTS
Plasma Ca, phosphate, and PTH. Plasma Ca levels in rats fed the 2% Ca lactose-supplemented vitamin Ddeficient diet (diet D) did not differ significantly from control (vitamin D-replete diet, group A) at any time during the study (Fig. IA). In contrast, rats fed both the 0.8 and 2.0% Ca vitamin D-deficient diets (groups B and C, respectively) first exhibited a significant (P < 0.05) decrease in plasma Ca levels from control after 4 diet wk. A further large decrease was observed in both of these groups from 4 to 6 diet wk. However, plasma Ca levels were consistently lower in group B than in group C (Fig. IA). Plasma phosphate concentrations were variable but similar among the diet groups at all time points. There were no significant differences in plasma phosphate levels between normocalcemic groups A and D at any time point (Fig. 1B). The only significant difference from the control (group A) was seen in groups B and C at Loeeh 15 (Fig. 1B). Similarly, there were no significant differences in plasma NHa-terminal PTH levels between normocalcemic groups (A and D) throughout the 19-wk experimental period (Fig. 1C). In contrast, the hypocalcemic vitamin D-deficient rats (groups B and C) exhibited significantly elevated plasma PTH levels by 6 diet wk, with levels continuing to increase thereafter. Plasma vitamin D metabolite levels. Plasma 25(OH)D3 levels decreased rapidly in all rats fed vitamin D-deficient diets and were significantly (P < 0.01) reduced from vitamin D-replete controls (group A) after 1 diet wk (Fig. 2A). 25(OH)Da levels decreased most rapidly in rats fed the 0.8% Ca vitamin D-deficient diet (group B) such that plasma levels were significantly lower than observed in rats fed the 2% Ca lactose-supplemented diet (group D) after both 1 (P < 0.01) and 2 (P < 0.05) diet wk. From diet weeh 4 until the end of the study, plasma 25( OH)Ds levels continued to decrease, achieving levels of 30 pg/ml by 19 diet wk, with no significant differences between the vitamin D-deficient groups (Fig. 2A). In contrast, plasma 25(OH)Ds levels were stable in the control vitamin D-replete group throughout the study. Plasma l,25(OH)ZD3 levels decreased with time in all dietary groups (Fig 2B). This age-related decrease in l,25(OH)2D3 levels in normal rats is similar to that observed in previous reports (1). Levels decreased most rapidly in rats fed the 2% Ca lactose-supplemented vitamin D-deficient diet (group D), such that l,25(OH)2Ds levels were significantly reduced in these animals after 2 diet wk as compared with both control rats (P < 0.01) and vitamin D-depleted rats fed the 0.8% Ca diet (group
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Diet week
of dietary Ca, P, lactose, and vitamin D3 on plasma Ca, phosphate, and NHz-terminal parathyroid hormone (PTH) levels in rats. Weanling male rats were fed diets described in Table 1 for O19 wk. Values are means t SE, n = 5 rats/group. Missing SE bars are within symbols. *P < 0.05; **P < 0.01; significance of difference from rats fed diet A.
2. Effect of dietary Ca, P, lactose, 25hydroxyvitamin DC3 [25(OH)D:J and active [ L25WU,D:,] 1evels and duodenal serosal-to-mucosal ratios. Weanling male in Table 1 for O-19 wk. Values are means SE bars are within symbols. *P < 0.05; difference from rats fed diet A.
B; P < 0.01). From 4 diet wk until the end of the study, l,25(OH)2D3 levels were significantly (P < 0.05) lower in all vitamin D-depleted groups than in controls, with no significant differences being seen between the vitamin D-depleted groups (Fig. 2B). Duodenal active Ca transport. Like plasma 1,25 ( OH)2D3 levels, active Ca transport [ serosal-to-mucosal (S/M) ratio] also decreased with time in all dietary groups (Fig. 2C). This age-related decrease in Ca transport is also similar to that reported previously (2). Again, Ca transport decreased most rapidly in rats fed the highCa lactose-supplemented diet (group D) and was significantly (P < 0.05) reduced from group A after 2 diet wk. Active Ca transport was abolished (S/M ratios not sig-
nificantly different from 1.0) in all vitamin D-depleted groups from 10 diet wk until the end of the study (Fig. 2C). There were no significant differences in Ca transport between the vitamin D-depleted groups at any time point. Bone mineral parameters. Tibia mineral content increased with aging in all dietary groups (Fig. 3A). Initially, tibia mineral content increased most rapidly in the rats fed both 2% Ca diets (groups C and D) such that after 2 diet wk the mineral content was significantly (P < 0.05) higher than in the vitamin D-replete control rats (group A). The rate of increase of tibia mineral content then slowed in these two groups; at 4 diet wk, tibia mineral content was not significantly different
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and vitamin D3 on plasma 1,25dihydroxyvitamin DS Ca transport in rats. S/M, rats were fed diets described t SE, n = 5/group. Missing **p < 0.01; significance of
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Diet week FIG. 4. Effect of dietary Ca, P, lactose, and vitamin D:, on growth rate in rats. Weanling male rats were fed diets described in Table 1 for O-19 wk. Values are means t SE, n = 5 rats/group. Missing SE bars are within symbols. *P < 0.05; significance of difference from rats fed diet A.
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FIG. 3. Effect of dietary Ca, P, lactose, and vitamin D3 on tibia mineral content and plasma alkaline phosphatase levels in rats. Weanling male rats were fed diets described in Table 1 for O-19 wk. Values are means t SE, n = 5 rats/group. Missing SE bars are within symbols. “P < 0.05; **P < 0.01; significance of difference from rats fed diet A.
among the four groups. Apart from the initial increase at l-2 wk, tibia mineral content in the normocalcemic vitamin D-depleted rats (group D) was not significantly different from that of the vitamin D-replete rats. However, at lo-19 wk, tibia mineral content was significantly (P < 0.01) lower in the nonlactose-supplemented vitamin D-deficient rats (groups B and C) than in vitamin Dreplete controls (Fig. 3A). Plasma alkaline phosphatase levels decreased with time in all dietary groups (Fig. 3B). There were no significant differences in alkaline phosphatase levels between the two normocalcemic groups (A and D) at any time point (Fig. 3B). In contrast, levels were significantly higher in the hypocalcemic hyperparathyroid rats (groups B and C) compared with group A rats from lo19 wk. Body weight. Growth was similar in all dietary groups. No significant differences in body weight were observed at any time point between control rats (diet A) and rats fed diets C and D. However, body weights in rats fed diet B were significantly (P < 0.05) lower than in the control group A after 15 and 19 diet wk (Fig. 4). DISCUSSION
These studies have convincingly demonstrated that severe vitamin D deficiency can be successfully achieved
in rats without concurrent changes in the plasma levels of Ca, phosphate, or NH2-terminal PTH. This important advance has been achieved by feeding normal Charles River CD weanling rats a vitamin D-deficient 2.0% Ca, 1.25% P diet, containing 20% lactose (diet 0). With this experimental model, plasma Ca, phosphate, and PTH levels were not significantly different from vitamin Dreplete animals (group A) at any time during the 19-wk study period. Dietary lactose was important in the maintenance of the normocalcemia, since simply increasing dietary Ca and P to the levels used in diet D resulted in a degree of hypocalcemia little different from that seen in rats fed the 0.8% Ca, 0.5% P, vitamin D-deficient diet (diet B). Lactose is known to enhance passive intestinal Ca absorption (3), although the mechanism by which lactose exerts this effect is obscure. Dietary lactose, with or without high Ca, has been used by others to increase plasma Ca levels in vitamin D-deficient rats, but, in those studies, normocalcemia was either not achieved or was accompanied by significant hypophosphatemia (14, 15, 19, 24). Thus this simple and now well-characterized dietary regimen will be an invaluable tool in studying the effects of vitamin D deficiency in the absence of changes in plasma Ca, phosphate, and PTH on biological processes and, in particular, on those that take prolonged periods to occur, e.g., spermatogenesis. While the initial goal of these studies was to develop a normocalcemic rat model of vitamin D deficiency, they also provided considerable additional important- information. First, the experimental design permitted analysis of the quantitative and temporal relationships between changes in plasma 25(OH)D3 and l,25(OH)zD3 levels during the development of vitamin D deficiency in a classic animal model. Thus, in rats fed the 0.8% Ca diet (diet B), it was not until 25(OH)D3 levels had decreased by >95% (i.e., to 200-300 pg/ml) that plasma l,25(OH)2D3 levels decreased below that seen in vitamin D-replete controls (week 4, Fig. 2). This is important because a frequently used (14, 16, 23) definition of vitamin D deficiency is undetectable 25(OH)D3 levels (cl-5 rig/ml by competitive protein binding assay) and hypocalcemia. Thus, by conventional criteria, the animals fed diet B were vitamin D-deficient after 4 diet wk, whereas,
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at that time, plasma l,25(OH)zD3 was only starting to decrease from control levels (37 vs. 74 pg/ml, respectively). It was not until after 6-10 diet wk that plasma l,25(OH)2D3 began to approach undetectable (C 1 pg/ ml) levels in this vitamin D-deficient group. Thus plasma 25(OH)D3 must be decreased to extremely low levels before a major impact on plasma l,25(OH)zD3 levels is observed in the face of a hypocalcemic stimulus to l,25(OH)2D3 synthesis. This is an extremely important distinction since, using conventional criteria, such rats may be “vitamin D deficient” but not l,25(OH)2D3 deficient. Second, these studies also permitted a direct determination of the effects of dietary and plasma Ca on the initial rates of changes in vitamin D metabolite levels during the development of l,25(OH)2D3 deficiency in rats. Plasma l,25(OH)2D3 levels decreased most rapidly, and 25(OH)D3 levels decreased least rapidly in rats fed the 2.0% Ca lactose-supplemented vitamin D-deficient diet (diet 0). Conversely, plasma l,25(OH)2D3 was maintained at a higher level for longer, and plasma 25(OH)D3 levels decreased most rapidly in rats fed the 0.8% Ca vitamin D-deficient diet (diet B). Thus the lower dietary Ca intake was associated with an accelerated initial rate of fall in plasma 25(OH)D3 levels. This more rapid decrease in 25(OH)D3 levels in rats fed diet B most likely is a result of the higher conversion to l,25(OH)2D3. A l,25(0H)2D3-induced stimulation of the side-chain oxidation of 25(OH)D3 (17) may also play a role, since l,25(OH)2D3 has been shown to increase the metabolic clearance rate of 25(OH)D3 in rats (11). The more rapid decrease in plasma l,25(OH)2D3 levels in rats fed diet D is an expected consequence of normal adaptative responses to lactose-enhanced passive Ca absorption. Thus the lactose-enhanced Ca influx resulted in decreased renal l,25(OH)2D3 synthesis and consequent decreased duodenal active Ca transport (Fig. 2C). However, despite these early differences in vitamin D metabolite levels, by 4 diet wk, plasma 25(OH)D3 levels were similar in all vitamin D-depleted groups and thereafter decreased at about the same rate in all groups, achieving levels of -30 pg/ml by 19 diet wk. Thus, for practical purposes, maintenance of normocalcemia did not markedly impede the rate of development of vitamin D deficiency but did prevent the development of hypocalcemia. The changes in plasma l,25(OH)2D3 levels were mirrored by similar changes in active duodenal Ca transport. Thus l,25(OH)2D3 levels and Ca transport decreased most rapidly in rats fed the 2% Ca lactose-supplemented diet (diet 0). However, as was seen with 1,25(OH)zDs levels, the initial differences between the vitamin Ddepleted groups disappeared by 6 diet wk, and active Ca was abolished in all vitamin D-deficient groups by 10 diet wk. Third, these studies also permitted a determination of the role of vitamin D in bone mineralization. It has been unclear whether the osteopenia that results from vitamin D deficiency is a consequence of the requirement of vitamin D for normal bone formation or only because vitamin D deficiency also results in hypocalcemia and secondary hyperparathyroidism, which prevent normal bone mineralization. While bone histology would be nec-
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essary to confirm that mineralization is normal at the cellular level, these studies have provided evidence that vitamin D is not essential for normal bone formation. Apart from a small increase over the first 2 wk (presumably related to the higher Ca and P intake), rats with marked vitamin D deficiency, but with normal plasma Ca, phosphate, and PTH levels, had completely normal mineral levels in the tibia throughout the 19-wk study period (Fig. 3). Conversely, bone mineral content was reduced in rats that became hypocalcemic and hyperparathyroid during the development of vitamin D deficiency. The differences in bone mineral content were mirrored by similar changes in plasma alkaline phosphatase levels (Fig. 3), a biochemical index of osteoblastic activity and bone turnover. Alkaline phosphatase activity was entirely normal in both normocalcemic groups at all time points but was elevated in the hypocalcemic vitamin Ddeficient rats. These results support those of Underwood and DeLuca (27), who reported that vitamin D is not directly necessary for bone growth and mineralization but only supports mineralization by maintaining normal plasma Ca and phosphate levels. In conclusion, we have successfully developed and fully characterized a normocalcemic rat model of vitamin D deficiency by feeding a high Ca high P lactose-containing vitamin D-deficient diet to normal weanling male rats. Despite severe vitamin D depletion in this group, the normocalcemia was not a result of elevated PTH secretion and was maintained throughout the 19-wk duration of the study. Thus this rat model will be important for proper investigation of the role(s) of vitamin D metabolites in the normal functioning of l,25(OH)zD3 targets, particularly those in which the function depends on extracellular Ca. These studies also showed that the initial rate of development of vitamin D depletion was reduced in the normocalcemic rats, but, for practical purposes, this was inconsequential, since after 6 diet wk no differences in vitamin D metabolite levels were seen between groups. Finally, these studies provided further evidence that vitamin D is not directly required for normal bone mineralization. We thank Drs. Milan R. Uskokovic (Hoffman LaRoche) and Hunter Heath for providing vitamin D3 metabolites and antiserum G813-PTH, respectively. Bijoy Mathew, Diana Woods, and Lamont Tillery also provided expert technical assistance. This work was supported by a grant-in-aid from the American Heart Association, by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-31847 (to M. R. Walters), and by a grantin-aid from the American Heart Association of Louisiana (to J. Fox). Parts of this work have been presented at the 73rd annual meeting of FASEB in New Orleans in March 1989, at the Joint Meeting of the International Conference on Ca Regulating Hormones, and the Am. Sot. Bone Miner. Res. in Montreal, Canada in September 1989 and have been published as abstracts (FASEB J. 3: A774,1989 and J. Bone Miner. Res. 4, SuppZ. 1: S703, 1989). Address for reprint requests: J. Fox, Dept. of Physiology, Tulane Univ. School of Medicine, 1430 Tulane Ave., New Orleans, LA 70112. Received
18 June
1990; accepted
in final
form
30 October
1990.
REFERENCES 1. ARMBRECHT, H.J., L. R. FORTE, AND B. P. HALLORAN. age and dietary calcium on renal 25(OH)D metabolism, 1,25(OH)2D3, and PTH. Am. J. Physiol. 246 (Endocrinol.
Effect of serum Metab.
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9): E266-E270, 1984. 2. ARMBRECHT, H. J., T. V. ZENSER, M. E. H. BRUNS, AND B. B. DAVIS. Effect of age on intestinal calcium absorption and adaptation to dietary calcium. Am. J. Physiol. 236 (Endocrinol. Metab. Gastrointest. Physiol. 5): E769-E774, 1979. 3. BRONNER, F. Calcium absorption. In: Physiology of the GastrointestinaZ Tract (2nd ed.), edited by L. R. Johnson. New York: Raven, 1987, p. 1419-1435. 4. CHEN, P. S. JR., T. Y. TORIBARA, AND H. WARNER. Microdetermination of phosphorus. Anal. Chem. 28: 1756-1758, 1956. 5. COLSTON, K., M. HIRST, AND D. FELDMAN. Organ distribution of the cytoplasmic 1,25-dihydroxycholecalciferol receptor in various mouse tissues. Endocrinology 107: 1916-1922, 1980. 6. FOX, J. Verapamil induces PTH resistance but increases duodenal calcium absorption in rats. Am. J. Physiol. 255 (Endocrinol. Metab. 18): E702-E707,1988. 7. Fox, J., AND C. P. DELLA-SANTINA. Oral verapamil and Ca and vitamin D metabolism in rats: effect of dietary Ca. Am. J. Physiol. 257 (Endocrinol. Metab. 20): E632-E638, 1989. 8. Fox, J., U. KOLLENKIRCHEN, AND M. R. WALTERS. Deficiency of vitamin D metabolites directly stimulates renal 25-hydroxyvitamin D&hydroxylase activity in rats. Metab. Clin. Exp. In press. 9. FRASER, D. R. Regulation of the metabolism of vitamin D. Physiol. Reu. 60: 551-613, 1980. 10. GINDLER, E. M., AND J. D. KING. Rapid calorimetric determination of calcium in biologic fluids with methylthymol blue. Am. J. Clin. Pathol. 58: 376-382, 1972. 11. HALLORAN, B. P., D. D. BIKLE, M. J. LEVENS, M. E. CASTRO, R. K. GLOBUS, AND E. HOLTON. Chronic 1,25-dihydroxyvitamin D, administration in the rat reduces the serum concentration of 25hydroxyvitamin D by increasing metabolic clearance rate. J. CZin. Invest. 78: 622-628, 1986. 12. HOLICK, M. F. Vitamin D photobiology: recent advances in the biochemistry and some clinical applications. In: Vitamin D. Chemical, Biochemical and Clinical Update, edited by A. W. Norman, K. Schaefer, H.-G. Grigoleit, and D. von Herrath. Berlin: de Gruyter, 1985, p. 219-228. 13. HOLLIS, B. W., AND T. KILBO. The assay of circulating 1,25(OH),D using non-end-capped C18 silica (&-OH): performance and validation. In: Vitamin D. Molecular, Cellular and Clinical Endocrinology, edited by A. W. Norman, K. Schaefer, H.-G. Grigoleit and D. von Herrath. Berlin: de Gruyter, 1988, p. 710-719. 14. HOLTROP, M. E., K. A. Cox, D. L. CARNES, AND M. F. HOLICK. Effects of serum calcium and phosphorus on skeletal mineralization in vitamin D-deficient rats. Am. J. Physiol. 251 (Endocrinol. Metab. 14): E234-E240, 1986. 15. HOWARD, G. A., AND D. J. BAYLINK. Matrix formation and osteoid maturation in vitamin D-deficient rats made normocalcemic by dietary means. Miner. Electrolyte Metab. 3: 44-50, 1980. 16. KWIECINSKI, G. G., G. I. PETRIE, AND H. F. DELUCA. Vitamin D is necessary for reproductive functions of the male rat. J. Nutr. 119: 741-744,1989. 17. LOHNES, D., AND G. JONES. Side chain metabolism of vitamin D3 in osteosarcoma cell line UMR-106. Characterization of products.
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J. Biol. Chem. 262: 14394-14401, 1987. 18. MASSRY, S. G., K. ISEKI, AND V. M. CAMPESE. Serum calcium, parathyroid hormone, and blood pressure. Am. J. Nephrol. 6, Suppl.. 1: 19-28, 1986. 19. MILLER, S. C., M. A. MILLER, AND T. H. OMURA. Dietary lactose improves endochondrial growth and bone development and mineralization in rats fed a vitamin D-deficient diet. J. Nutr. 118: 7277, 1988. 20. NORMAN, A. W., J. ROTH, AND L. ORCI. The vitamin D endocrine system: steroid metabolism, hormone receptors, and biological response. Endocrinol. Reu. 3: 331-366, 1982. 21. PIKE, J., L. GOOZE, AND M. HAUSSLER. Biochemical evidence for 1,25-dihydroxyvitamin D receptor macromolecules in parathyroid, pancreatic, pituitary and placental tissues. Life Sci. 26: 407-414, 1980. 22. REICHEL H., H. P. KOEFFLER, R. BARBERS, R. MUNKER, AND A. W. NORMAN. 1,25-dihydroxyvitamin D3 and the hematopoetic system. In: Vitamin D. Chemical, Biochemical and Clinical Update, edited by A. W. Norman, K. Schaefer, H.-G. Grigoleit, and D. von Herrath. Berlin: de Gruyter, 1985, p. 167-176. 23. REINHARDT, T. A., AND R. L. HORST. Simplified assays for the determination of 25-OHD, 24,25-(OH),D and 1,25-(OH)2D. In: Vitamin D. Molecular, Cellular and Clinical Endocrinology, edited by A. W. Norman, K. Schaefer, H.-G. Grigoleit and D. von Herrath. Berlin: de Gruyter, 1988, p. 720-726. 24. SCHAAFSMA, G., W. J. VISSER, P. R. DEKKER, AND M. VAN SCHAIK. Effect of dietary calcium supplementation with lactose on bone in vitamin D-deficient rats. Bone NY 8: 357-362, 1987. 25. SIMPSON, R. U., G. A. THOMAS, AND A. J. ARNOLD. Identification of 1,25-dihydroxyvitamin D3 receptors and activities in muscle. J. BioZ. Chem. 260: 8882-8891, 1985. 26. THOMAS, M. L., AND M. J. IBARRA. Effects of ovariectomy on duodenal calcium transport in the rat: altered ability to adapt to low-calcium diet. Proc. Sot. Exp. Biol. Med. 185: 84-88, 1987. 27. UNDERWOOD, J. L., AND H. F. DELUCA. Vitamin D is not directly necessary for bone growth and mineralization. Am. J. Physiol. 246 (Endocrinol. Metab. 9): E493-E498, 1984. 28. WALTERS, M. R., D. L. CUNEO, AND A. P. JAMISON. Possible significance of new target tissues for 1,25-dihydroxyvitamin D3. J. Steroid Biochem. 19: 913-920, 1983. 29. WALTERS, M. R., G. HEBERT, T. T. ILENCHUK, P. C. RIGGLE, AND W. C. CLAYCOMB. Further evidence for 1,25-dihydroxyvitamin D effects in new targets: heart and lung. Excerpta Med. Int. Cong. Ser. 735: 479-484, 1987. 30. WALTERS, M. R., T. T. ILENCHUK, AND W. C. CLAYCOMB. 1,25dihydroxyvitamin DS stimulates 4”Ca2+ uptake by cultured adult rat ventricular cardiac muscle cells. J. BioL. Chem. 262: 2536-2541, 1987. 31. WALTERS, M. R., B. C. OSMUNDSON, AND R. C. CARTER. 1,25dihydroxyvitamin D and the testis. Ann. NY Acad. Sci. 513: 482485, 1988. 32. WALTERS, M. R., D. C. WICKER, AND P. C. RIGGLE. 1,25-dihydroxyvitamin D3 receptors identified in the rat heart. J. MOL. Cell. Cardiol. 18: 67-72, 1986.
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MARIAN R. WALTERS, AND JOHN FOX Tulane University School of Medicine, New Orleans, Louisiana
KOLLENKIRCHEN, UWE, MARIAN R. WALTERS,AND JOHN FOX. Plasma Ca influences vitamin D metabolite levels as rats develop vitamin D deficiency. Am. J. Physiol. 260 (Endocrinol. Metab. 23): E447-E452,1991.-The hypocalcemia that accompanies vitamin D deficiency is a major obstacle to proper interpretation of the role(s) of vitamin D metabolites in Casensitive tissues. This paper describes the development and complete characterization of a dietary regimen with which normocalcemia was maintained in rats throughout the development of vitamin D deficiency. Normal weanling rats were fed diets containing 0.8% Ca, 0.5% P, and vitamin D3 (group A), or vitamin D-deficient diets containing 0.8% Ca and 0.5% P (group B); 2.0% Ca and 1.25% P (group C); or 2.0% Ca, 1.25% P, and 20% lactose (group D) for 19 wk. Group D rats were normocalcemic and normophosphatemic with normal parathyroid hormone (PTH) levels throughout the study. In contrast, from 4-19 diet wk, groups B and C were hypocalcemic with elevated PTH. Initially, plasma 25-hydroxyvitamin D3 [ 25( OH)D,] levels decreased most rapidly, and 1,25-dihydroxyvitamin D3 [ 1,25( OH)gDB] levels decreased least rapidly in group B rats, such that plasma 25(OH)D3 levels were reduced to 200300 pg/ml before a decrease in l,25(OH)2D3 levels was observed. However, vitamin D metabolite levels were similar in groups B, C, and D from 4-19 wk. Duodenal active Ca transport mirrored changes in plasma l,25(OH)2DS levels and was abolished after 10 wk. The results also suggested that vitamin D may not be necessary for normal bone mineralization since tibia mineral content and plasma alkaline phosphatase levels were similar in normocalcemic groups A and D throughout the study. dietary calcium; amino-terminal parathyroid hormone; 1,25dihydroxyvitamin D,; 25-hydroxyvitamin D,; active calcium absorption; bone mineral content
THE PHYSIOLOGICALLY ACTIVE metabolite ofvitaminDS, 1,25dihydroxyvitamin D3 [1,25(OH)2D& is the major stimulator of intestinal Ca absorption. This steroid hormone also plays an important role in bone mineral mobilization and renal tubular Ca reabsorption (9, 20). However, 1,25(OH)zD3 receptors have also been described in tissues other than these “classical” 1,25(OH)2D3 targets, e.g., heart (25, 29, 32), testis (31), lung (29), pancreas (21), skin (5, 12), as well as in cells of the hematopoetic system (22). 1,25(OH)2D3 increases Ca uptake by rat cardiac myocytes (30) and inhibits proliferation and stimulates differentiation in several cell lines (12, 22). Because Ca is an important element in the regulation of cell function, it has been suggested that 1,25(OH)ZD3 may play a role in intracellular Ca homeostasis (28). Studies of the physiological role of 1,25(OH)2D3 and 0193~1849/91
levels
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its receptors have been compromised in the past because vitamin D-deficient animals are invariably hypocalcemic and have marked secondary hyperparathyroidism (6, l416). Administration of 1,25(OH)2D3 to these animals dramatically increases plasma Ca and reduces parathyroid hormone (PTH) levels (6). Because the function of many of the target tissues described above depends critically on plasma Ca and because PTH has also been shown to have major effects on the function of the cardiovascular system (18), it is virtually impossible to determine if observed changes in function after vitamin D administration result from vitamin D metabolite action at that tissue or simply a physiological response to changes in plasma Ca and/or PTH (16). Thus a simple means of preventing hypocalcemia and secondary hyperparathyroidism during the development of vitamin D deficiency is essential if the role of vitamin D3 in its targets is to be interpreted appropriately. For example, because the spermatogenesis cycle in rats takes 6-8 wk (31), it is important for appropriate study of the effects of vitamin D deficiency on testicular function that the vitamin D-depleted animals are not only normocalcemic and vitamin D-deficient at the time of semen analysis but that they were also normocalcemic and vitamin Ddeficient throughout the preceding 6-8 wk. Dietary Ca supplementation has been reported to prevent severe hypocalcemia in vitamin D-deficient rats (14, l5), but these dietary regimens frequently result in hypophosphatemia, and plasma PTH is usually elevated (or unreported) in these systems. Furthermore, the long-term effects of these diets on plasma Ca, phosphate, and PTH have not been reported. Thus extensive studies were undertaken to develop a reliable model of vitamin D deficiency unaccompanied by changes in other parameters of Ca metabolism in rats by varying the dietary Ca and P contents. Furthermore, it was also important to establish that these parameters were sustained throughout the dietary period, allowing testing of vitamin D effects both early and late during the development of vitamin D deficiency. This ensures that physiological effects measured at any given time would reflect the results of a constant and sustained hormonal and metabolic milieu. These studies also permitted a determination of whether changes in dietary Ca intake and plasma Ca levels influence 1) the rate of development of vitamin D-deficiency, 2) the rate of loss of active duodenal Ca transport [an important index of 1,25(OH)2D3 action], and 3) bone mineral homeostasis
0 1991 the American
Physiological
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E448
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[it has been suggested that vitamin tial for normal bone mineralization
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(1% a7)1.
METHODS
Normal weanling male CD (Sprague-Dawley) rats were obtained from Charles River Labs. On receipt, the rats were randomly divided into four groups and housed in hanging wire cages under incandescent light and were fed one of the four diets described in Table 1. The diets were purchased from Teklad, Madison, WI, and were color coded to reduce the possibility of errors in feeding. T he day after receipt, 12 rats fed the vitamin D-replete diet (diet A) were anesthetized with pentobarbital sodium (60 mg/kg, ip) and were exsanguinated by cardiac puncture. Subsequently, five rats from each dietary group were similarly exsanguinated after 1-19 diet wk. In all cases, plasma was collected for analysis. The proximal 6 cm of small intestine was removed and placed in ice-cold isoto nit saline . Duodenal active Ca tran .sport was m.easured us1 ng the everted gut sac techn .ique (26). A tibia was removed and defatted by soaking in excess dichloromethane-methanol (1:l) for 3 days. After drying at 100°C and weighing, the dry bones were ashed overnight at 550°C and reweighed. Tibia mineral content was expressed as a percentage of the fat-free dry weight (6, 7). Plasma analyses. Before assay, vitamin D3 metabolites were extracted from plasma and purified using minor modifications (8) of established methods (13, 23). Recoveries of 25(OH)D3 and l,25(OH)2D3 averaged 70-80%. 25(OH)Ds was quantitated by radioimmunoassay (6, 7) using sheep antiserum 02282. The detection limit was 1.5-2 pg/tube (6-10 pg/ml for 1 ml plasma). l,25(OH)ZD3 level s were quantitated by radioreceptor assay using a calf thymus receptor prepared using an established method (23). The assay detection limit was 0.4 pg/tube (l-2 pg/ml for 1 ml plasma). Plasma NH2-terminal parathyroid hormone (PTH) levels were measured with a homologous rat PTH-( l34) radioimmunoassay with goat antiserum G813-PTH, which was described previously (6, 7). The assay detection limit was 6 pg rat PTH-(1-34)/ml. Plasma alkaline phosphatase activity was measured by quantitating the hydrolysis of p-nitrophenol phosphate to p-nitrophenol using a kit (Medical Analysis Systems, Camarillo, CA). Plasma Ca levels were measured using methylthymol blue (lo), and plasma phosphate was measured by the method of Chen et al. (4). Statistical analysis. All data are presented as means t SE. For statistical analysis, undetectable values were arbitrarily assigned the assay detection limit. The significance of differences between control (group A) and 1. Composition of diets
TABLE Group
A B c
D
Diet
TD87092 TD87093 TD87094 TD87095
Vitamin Dt3, IWE:
Ca, %
P, %
Lactose, 5%
2.2 0 0 0
0.8 0.8 2.0 2.0
0.5 0.5 1.25 1.25
0 0 0 20
TD, test diet no. (Teklad, Madison, sucrose was replaced with lactose.
WI).
In
D, an equal amount
of
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treatment (groups B, C, and D) were determined at each time point by analysis of variance (ANOVA) and Dunnett’s test. When variances were unequal (estimated by F max or Bartlett’s test), the data were log-transformed before ANOVA. Nonparametric tests (H test of Kruskal and Wallis and U test of Mann and Whitney) were used if log-transformed data still showed unequal variances. Results were analyzed using commercial software (Quickstat; CCP-Soft, Marburg, FRG). RESULTS
Plasma Ca, phosphate, and PTH. Plasma Ca levels in rats fed the 2% Ca lactose-supplemented vitamin Ddeficient diet (diet D) did not differ significantly from control (vitamin D-replete diet, group A) at any time during the study (Fig. IA). In contrast, rats fed both the 0.8 and 2.0% Ca vitamin D-deficient diets (groups B and C, respectively) first exhibited a significant (P < 0.05) decrease in plasma Ca levels from control after 4 diet wk. A further large decrease was observed in both of these groups from 4 to 6 diet wk. However, plasma Ca levels were consistently lower in group B than in group C (Fig. IA). Plasma phosphate concentrations were variable but similar among the diet groups at all time points. There were no significant differences in plasma phosphate levels between normocalcemic groups A and D at any time point (Fig. 1B). The only significant difference from the control (group A) was seen in groups B and C at Loeeh 15 (Fig. 1B). Similarly, there were no significant differences in plasma NHa-terminal PTH levels between normocalcemic groups (A and D) throughout the 19-wk experimental period (Fig. 1C). In contrast, the hypocalcemic vitamin D-deficient rats (groups B and C) exhibited significantly elevated plasma PTH levels by 6 diet wk, with levels continuing to increase thereafter. Plasma vitamin D metabolite levels. Plasma 25(OH)D3 levels decreased rapidly in all rats fed vitamin D-deficient diets and were significantly (P < 0.01) reduced from vitamin D-replete controls (group A) after 1 diet wk (Fig. 2A). 25(OH)Da levels decreased most rapidly in rats fed the 0.8% Ca vitamin D-deficient diet (group B) such that plasma levels were significantly lower than observed in rats fed the 2% Ca lactose-supplemented diet (group D) after both 1 (P < 0.01) and 2 (P < 0.05) diet wk. From diet weeh 4 until the end of the study, plasma 25( OH)Ds levels continued to decrease, achieving levels of 30 pg/ml by 19 diet wk, with no significant differences between the vitamin D-deficient groups (Fig. 2A). In contrast, plasma 25(OH)Ds levels were stable in the control vitamin D-replete group throughout the study. Plasma l,25(OH)ZD3 levels decreased with time in all dietary groups (Fig 2B). This age-related decrease in l,25(OH)2D3 levels in normal rats is similar to that observed in previous reports (1). Levels decreased most rapidly in rats fed the 2% Ca lactose-supplemented vitamin D-deficient diet (group D), such that l,25(OH)2Ds levels were significantly reduced in these animals after 2 diet wk as compared with both control rats (P < 0.01) and vitamin D-depleted rats fed the 0.8% Ca diet (group
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Diet week
of dietary Ca, P, lactose, and vitamin D3 on plasma Ca, phosphate, and NHz-terminal parathyroid hormone (PTH) levels in rats. Weanling male rats were fed diets described in Table 1 for O19 wk. Values are means t SE, n = 5 rats/group. Missing SE bars are within symbols. *P < 0.05; **P < 0.01; significance of difference from rats fed diet A.
2. Effect of dietary Ca, P, lactose, 25hydroxyvitamin DC3 [25(OH)D:J and active [ L25WU,D:,] 1evels and duodenal serosal-to-mucosal ratios. Weanling male in Table 1 for O-19 wk. Values are means SE bars are within symbols. *P < 0.05; difference from rats fed diet A.
B; P < 0.01). From 4 diet wk until the end of the study, l,25(OH)2D3 levels were significantly (P < 0.05) lower in all vitamin D-depleted groups than in controls, with no significant differences being seen between the vitamin D-depleted groups (Fig. 2B). Duodenal active Ca transport. Like plasma 1,25 ( OH)2D3 levels, active Ca transport [ serosal-to-mucosal (S/M) ratio] also decreased with time in all dietary groups (Fig. 2C). This age-related decrease in Ca transport is also similar to that reported previously (2). Again, Ca transport decreased most rapidly in rats fed the highCa lactose-supplemented diet (group D) and was significantly (P < 0.05) reduced from group A after 2 diet wk. Active Ca transport was abolished (S/M ratios not sig-
nificantly different from 1.0) in all vitamin D-depleted groups from 10 diet wk until the end of the study (Fig. 2C). There were no significant differences in Ca transport between the vitamin D-depleted groups at any time point. Bone mineral parameters. Tibia mineral content increased with aging in all dietary groups (Fig. 3A). Initially, tibia mineral content increased most rapidly in the rats fed both 2% Ca diets (groups C and D) such that after 2 diet wk the mineral content was significantly (P < 0.05) higher than in the vitamin D-replete control rats (group A). The rate of increase of tibia mineral content then slowed in these two groups; at 4 diet wk, tibia mineral content was not significantly different
FIG.
FIG.
and vitamin D3 on plasma 1,25dihydroxyvitamin DS Ca transport in rats. S/M, rats were fed diets described t SE, n = 5/group. Missing **p < 0.01; significance of
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Diet week FIG. 4. Effect of dietary Ca, P, lactose, and vitamin D:, on growth rate in rats. Weanling male rats were fed diets described in Table 1 for O-19 wk. Values are means t SE, n = 5 rats/group. Missing SE bars are within symbols. *P < 0.05; significance of difference from rats fed diet A.
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FIG. 3. Effect of dietary Ca, P, lactose, and vitamin D3 on tibia mineral content and plasma alkaline phosphatase levels in rats. Weanling male rats were fed diets described in Table 1 for O-19 wk. Values are means t SE, n = 5 rats/group. Missing SE bars are within symbols. “P < 0.05; **P < 0.01; significance of difference from rats fed diet A.
among the four groups. Apart from the initial increase at l-2 wk, tibia mineral content in the normocalcemic vitamin D-depleted rats (group D) was not significantly different from that of the vitamin D-replete rats. However, at lo-19 wk, tibia mineral content was significantly (P < 0.01) lower in the nonlactose-supplemented vitamin D-deficient rats (groups B and C) than in vitamin Dreplete controls (Fig. 3A). Plasma alkaline phosphatase levels decreased with time in all dietary groups (Fig. 3B). There were no significant differences in alkaline phosphatase levels between the two normocalcemic groups (A and D) at any time point (Fig. 3B). In contrast, levels were significantly higher in the hypocalcemic hyperparathyroid rats (groups B and C) compared with group A rats from lo19 wk. Body weight. Growth was similar in all dietary groups. No significant differences in body weight were observed at any time point between control rats (diet A) and rats fed diets C and D. However, body weights in rats fed diet B were significantly (P < 0.05) lower than in the control group A after 15 and 19 diet wk (Fig. 4). DISCUSSION
These studies have convincingly demonstrated that severe vitamin D deficiency can be successfully achieved
in rats without concurrent changes in the plasma levels of Ca, phosphate, or NH2-terminal PTH. This important advance has been achieved by feeding normal Charles River CD weanling rats a vitamin D-deficient 2.0% Ca, 1.25% P diet, containing 20% lactose (diet 0). With this experimental model, plasma Ca, phosphate, and PTH levels were not significantly different from vitamin Dreplete animals (group A) at any time during the 19-wk study period. Dietary lactose was important in the maintenance of the normocalcemia, since simply increasing dietary Ca and P to the levels used in diet D resulted in a degree of hypocalcemia little different from that seen in rats fed the 0.8% Ca, 0.5% P, vitamin D-deficient diet (diet B). Lactose is known to enhance passive intestinal Ca absorption (3), although the mechanism by which lactose exerts this effect is obscure. Dietary lactose, with or without high Ca, has been used by others to increase plasma Ca levels in vitamin D-deficient rats, but, in those studies, normocalcemia was either not achieved or was accompanied by significant hypophosphatemia (14, 15, 19, 24). Thus this simple and now well-characterized dietary regimen will be an invaluable tool in studying the effects of vitamin D deficiency in the absence of changes in plasma Ca, phosphate, and PTH on biological processes and, in particular, on those that take prolonged periods to occur, e.g., spermatogenesis. While the initial goal of these studies was to develop a normocalcemic rat model of vitamin D deficiency, they also provided considerable additional important- information. First, the experimental design permitted analysis of the quantitative and temporal relationships between changes in plasma 25(OH)D3 and l,25(OH)zD3 levels during the development of vitamin D deficiency in a classic animal model. Thus, in rats fed the 0.8% Ca diet (diet B), it was not until 25(OH)D3 levels had decreased by >95% (i.e., to 200-300 pg/ml) that plasma l,25(OH)2D3 levels decreased below that seen in vitamin D-replete controls (week 4, Fig. 2). This is important because a frequently used (14, 16, 23) definition of vitamin D deficiency is undetectable 25(OH)D3 levels (cl-5 rig/ml by competitive protein binding assay) and hypocalcemia. Thus, by conventional criteria, the animals fed diet B were vitamin D-deficient after 4 diet wk, whereas,
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at that time, plasma l,25(OH)zD3 was only starting to decrease from control levels (37 vs. 74 pg/ml, respectively). It was not until after 6-10 diet wk that plasma l,25(OH)2D3 began to approach undetectable (C 1 pg/ ml) levels in this vitamin D-deficient group. Thus plasma 25(OH)D3 must be decreased to extremely low levels before a major impact on plasma l,25(OH)zD3 levels is observed in the face of a hypocalcemic stimulus to l,25(OH)2D3 synthesis. This is an extremely important distinction since, using conventional criteria, such rats may be “vitamin D deficient” but not l,25(OH)2D3 deficient. Second, these studies also permitted a direct determination of the effects of dietary and plasma Ca on the initial rates of changes in vitamin D metabolite levels during the development of l,25(OH)2D3 deficiency in rats. Plasma l,25(OH)2D3 levels decreased most rapidly, and 25(OH)D3 levels decreased least rapidly in rats fed the 2.0% Ca lactose-supplemented vitamin D-deficient diet (diet 0). Conversely, plasma l,25(OH)2D3 was maintained at a higher level for longer, and plasma 25(OH)D3 levels decreased most rapidly in rats fed the 0.8% Ca vitamin D-deficient diet (diet B). Thus the lower dietary Ca intake was associated with an accelerated initial rate of fall in plasma 25(OH)D3 levels. This more rapid decrease in 25(OH)D3 levels in rats fed diet B most likely is a result of the higher conversion to l,25(OH)2D3. A l,25(0H)2D3-induced stimulation of the side-chain oxidation of 25(OH)D3 (17) may also play a role, since l,25(OH)2D3 has been shown to increase the metabolic clearance rate of 25(OH)D3 in rats (11). The more rapid decrease in plasma l,25(OH)2D3 levels in rats fed diet D is an expected consequence of normal adaptative responses to lactose-enhanced passive Ca absorption. Thus the lactose-enhanced Ca influx resulted in decreased renal l,25(OH)2D3 synthesis and consequent decreased duodenal active Ca transport (Fig. 2C). However, despite these early differences in vitamin D metabolite levels, by 4 diet wk, plasma 25(OH)D3 levels were similar in all vitamin D-depleted groups and thereafter decreased at about the same rate in all groups, achieving levels of -30 pg/ml by 19 diet wk. Thus, for practical purposes, maintenance of normocalcemia did not markedly impede the rate of development of vitamin D deficiency but did prevent the development of hypocalcemia. The changes in plasma l,25(OH)2D3 levels were mirrored by similar changes in active duodenal Ca transport. Thus l,25(OH)2D3 levels and Ca transport decreased most rapidly in rats fed the 2% Ca lactose-supplemented diet (diet 0). However, as was seen with 1,25(OH)zDs levels, the initial differences between the vitamin Ddepleted groups disappeared by 6 diet wk, and active Ca was abolished in all vitamin D-deficient groups by 10 diet wk. Third, these studies also permitted a determination of the role of vitamin D in bone mineralization. It has been unclear whether the osteopenia that results from vitamin D deficiency is a consequence of the requirement of vitamin D for normal bone formation or only because vitamin D deficiency also results in hypocalcemia and secondary hyperparathyroidism, which prevent normal bone mineralization. While bone histology would be nec-
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essary to confirm that mineralization is normal at the cellular level, these studies have provided evidence that vitamin D is not essential for normal bone formation. Apart from a small increase over the first 2 wk (presumably related to the higher Ca and P intake), rats with marked vitamin D deficiency, but with normal plasma Ca, phosphate, and PTH levels, had completely normal mineral levels in the tibia throughout the 19-wk study period (Fig. 3). Conversely, bone mineral content was reduced in rats that became hypocalcemic and hyperparathyroid during the development of vitamin D deficiency. The differences in bone mineral content were mirrored by similar changes in plasma alkaline phosphatase levels (Fig. 3), a biochemical index of osteoblastic activity and bone turnover. Alkaline phosphatase activity was entirely normal in both normocalcemic groups at all time points but was elevated in the hypocalcemic vitamin Ddeficient rats. These results support those of Underwood and DeLuca (27), who reported that vitamin D is not directly necessary for bone growth and mineralization but only supports mineralization by maintaining normal plasma Ca and phosphate levels. In conclusion, we have successfully developed and fully characterized a normocalcemic rat model of vitamin D deficiency by feeding a high Ca high P lactose-containing vitamin D-deficient diet to normal weanling male rats. Despite severe vitamin D depletion in this group, the normocalcemia was not a result of elevated PTH secretion and was maintained throughout the 19-wk duration of the study. Thus this rat model will be important for proper investigation of the role(s) of vitamin D metabolites in the normal functioning of l,25(OH)zD3 targets, particularly those in which the function depends on extracellular Ca. These studies also showed that the initial rate of development of vitamin D depletion was reduced in the normocalcemic rats, but, for practical purposes, this was inconsequential, since after 6 diet wk no differences in vitamin D metabolite levels were seen between groups. Finally, these studies provided further evidence that vitamin D is not directly required for normal bone mineralization. We thank Drs. Milan R. Uskokovic (Hoffman LaRoche) and Hunter Heath for providing vitamin D3 metabolites and antiserum G813-PTH, respectively. Bijoy Mathew, Diana Woods, and Lamont Tillery also provided expert technical assistance. This work was supported by a grant-in-aid from the American Heart Association, by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-31847 (to M. R. Walters), and by a grantin-aid from the American Heart Association of Louisiana (to J. Fox). Parts of this work have been presented at the 73rd annual meeting of FASEB in New Orleans in March 1989, at the Joint Meeting of the International Conference on Ca Regulating Hormones, and the Am. Sot. Bone Miner. Res. in Montreal, Canada in September 1989 and have been published as abstracts (FASEB J. 3: A774,1989 and J. Bone Miner. Res. 4, SuppZ. 1: S703, 1989). Address for reprint requests: J. Fox, Dept. of Physiology, Tulane Univ. School of Medicine, 1430 Tulane Ave., New Orleans, LA 70112. Received
18 June
1990; accepted
in final
form
30 October
1990.
REFERENCES 1. ARMBRECHT, H.J., L. R. FORTE, AND B. P. HALLORAN. age and dietary calcium on renal 25(OH)D metabolism, 1,25(OH)2D3, and PTH. Am. J. Physiol. 246 (Endocrinol.
Effect of serum Metab.
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