JOURNAL OF BONE A N D MINERAL RESEARCH Volume 6, Number 3, 1991 Mary Ann Liebert. Inc.. Publishers

Normocalcemia Without Hyperparathyroidism in Vitamin D-Deficient Rats UWE KOLLENKIRCHEN, JOHN FOX, and MARIAN R. WALTERS

ABSTRACT Despite numerous attempts, no reliable dietary regimen exists to achieve vitamin D deficiency (-D) in rats without attendant changes in plasma parathyroid hormone (PTH), Ca, or phosphate. This represents an important obstacle to proper investigations of the physiologic role(s) of vitamin D metabolites in the function of 1,25-dihydroxyvitamin D, [ 1,25-(OH),D,] target tissues. This paper describes the successful development of such a diet, which uses a combination of high Ca content, properly controlled Ca/P ratio, and lactose. Normal weanling rats were fed diets containing A, 0.8% Ca, 0.5% P, +D1, or -D diets containing B, 0.8% Ca and 0.5% P; C, 2.0% Ca and 1.25% P; or D, 2.0% Ca, 1.25% P, and 20% lactose. After 6 diet weeks group D rats remained normocalcemic and normophosphatemic, but diet groups B and C became hypocalcemic (6.9 f 0.8 and 7.2 + 0.4 mg/dl, respectively). Thus high dietary Ca and P was incapable of maintaining normal plasmd Ca levels in the absence of dietary lactose. The normocalcemia in group D was not maintained by elevated PTH secretion because N-terminal PTH levels were also normal (14 + 3 versus 20 + 5 pglml). In contrast, PTH levels were markedly elevated in hypocalcemic groups B and C (47 + 7 and 48 f 10 pg/ml, respectively). Plasma 25-OHD3and 1,25-(OH),D, levels were reduced to < 120 and < 12 pg/ml, respectively, in all - D groups. Thus the high-Ca diet and the use of normal weanlings did not impede the development of vitamin D deficiency. This rat model of normocalcemic vitamin D deficiency will greatly facilitate elucidation of the physiologic role(s) of vitamin D metabolites in the normal functioning of 1,25(OH),D3 targets.

INTRODUCTION HE ACTIVE METABOLITE of vitamin D,, 1,25-dihydroxyvitamin D, ( I ,25-(OH),D,], has effects in many tissues in addition to its classic targets, intestine, kidney, and bone."' For example, 1 ,25-(OH),D, receptors have been described in heart, skeletal and vascular smooth muscle cell^,^*-^^ 1ung,C4) pancreas,(6) pituitary,(6) and as well as in cells of the hematopoietic system.'') Other novel roles of 1,25-(OH),D, include inhibition of proliferation and stimulation of differentiation in several cell lines. To determine the physiologic role of 1,25-(OH)1D,and its receptors in both classical and nonclassical target tissues, traditional endocrine experiments have endeavored to ob-

T

serve changes in cellular function in the presence and absence of vitamin D, or its metabolites. These studies have been compromised in the past because vitamin D-deficient animals are invariably hypocalcemic and have marked secondary hyperparathyroidism. Furthermore, administration of vitamin D, or 1,25-(OH),D, to these animals dramatically increases plasma Ca and reduces parathyroid hormone (PTH) levels.(1o)Since the function of many of the target tissues depends critically upon plasma Ca, it is virtually impossible to determine if observed changes in function following vitamin D administration result from vitamin D metabolite action at that tissue or simply a physiologic response to the change in plasma Ca.'") Similarly, the issue of vitamin D effects is complicated by changing PTH levels. Thus a simple means of preventing hypocal~

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Department of Physiology, Tulane University School of Medicine, New Orlean\, LA 701 12.

273

~

KOLLENKIRCHEN ET AL.

274

cemia during the development of vitamin D deficiency is essential if the role of vitamin D , in its targets is to be interpreted appropriately. Several reports have described diets that were able to prevent severe hypocalcemia from developing in vitamin D-deficient rats.('*-'5' However, the use of dietary means to maintain normocalcemia in vitamin D-deficient rats has not been consistently successful, and plasma PTH is usually elevated (or unreported) in these systems. For example, Lester et al.'") reported both normocalcemia and normophosphatemia in rats fed a vitamin D-deficient diet containing whole wheat flour, whereas Underwood and DeLuca,(I6)using an identical diet, were unable to maintain normal plasma Ca and phosphate levels. They resorted to constant intravenous infusions of Ca and phosphate solutions to maintain normocalcemia and normophosphatemia when studying the effects of vitamin D on bone growth and mineralization in rats."') High-Ca, vitamin D-deficient diets supplemented with lactose have been used to maintain normocalcemia, but these frequently induce hypophosphatemia. 1 2 - 1 4 ) This paper describes the successful use of a 2% Ca and 1.25% P diet containing 20% lactose to achieve normal levels of plasma Ca, phosphate, and P T H in vitamin D-deficient rats.

MATERIALS AND METHODS Animals and diets

In preliminary studies weanling, 22-day-old male rats from vitamin D unsupplemented mothers (Holtzman colony 5 ) were housed in hanging wire cages under incandescent light. They were fed synthetic vitamin D-replete and deficient diets containing varying amounts of C a and P as outlined in Results. In these diets casein and sucrose were the protein and carbohydrate sources, respectively. In some diets sucrose (20%) was substituted with an equal amount of lactose, and some groups of rats received Ca gluconate (2070) in the drinking water. The diets were color coded to prevent errors during feeding and were purchased from Teklad (Madison, WI). After varying periods described in Results, the rats were weighed and blood samples ( 0.5 ml) for plasma Ca and phosphate analysis were obtained from ether-anesthetized animals by clipping the tip of the tail. For the major study normal weanling, 22-day-old male C D rats were obtained from Charles River Labs. Upon receipt they were randomly divided into four groups and fed one of the diets described in Table 1. After 6 diet weeks the rats were anesthetized with pentobarbital sodium (60 mglkg, ip) and exsanguinated by cardiac puncture. Plasma was collected for analysis. The proximal 6 cm of small intestine was removed and placed in ice-cold isotonic saline. Duodenal active Ca transport was measured using a modification(18'of the everted gut sac A tibia was removed and defatted by soaking for 3 days with an excess of ethanol and dichloromethane (1:l). Each bone was dried at 100°C, weighed, ashed at 55OoC overnight, and reweighed. Tibia1 ash content was expressed as a percentage of the fat-free dry bone.

-

TABLE1. Diet

Group

1

2 3 4

COMPOSITION OF THE

A B C D

Vifamin D ,

Ca

W/g)

(To)

2.2 0 0 0

(TD87092) (TD87093) (TD87094) (TD87095)

DIETS~

P

mo)

Lacfoseb

0.8 0.5 0.8 0.5 2.0 1.25 2.0 1.25

(Yo)

0 0 0 20

aTD, test diet number (Teklad, Madison, WI). hAn equal amount of sucrose was replaced with lactose.

Plasma analyses

Vitamin D3 metabolites were extracted and purified using minor modifications 1'") of established methods.'" z z ) Recoveries of 25-OHDl and 1,25-(OH),D, averaged 70-80%. 25-OHDI was quantitated by radioimmunoassay using sheep antiserum 02282."' ") The assay detection limit was 2-4 pg per tube ( - 10 pg/ml for 1 ml plasma). 1,25-(OH),D, levels were quantitated by radioreceptor assay using a calf thymus receptor prepared using an established method.'2z) The assay detection limit was 0.4 pg per tube ( - 1-2 pg/ml for 1 ml plasma). Plasma N-terminal parathyroid hormone levels were measured with a homologous rat PTH-( 1-34) radioimmunoassay using anti-rat PTH-( 1-34) antiserum G813-PTH, described previously."" 2 4 z 5 ) The assay detection limit was 6 pg rat PTH-(1-34) per ml. Plasma Calz6) and phosphate"') levels were measured using standard procedures. Statistical analysis

All data are presented as mean f SEM (standard error of the mean). The significance of differences was determined by analysis of variance and Tukey's test. ('')

RESULTS Preliminary experimenf s

To approximate the levels of dietary Ca and P necessary to maintain normal plasma parameters and to test the efficacy of dietary lactose supplementation, a preliminary set of experiments was performed. These experiments utilized the rat strain commonly used in such regimens and thought to be more suitable for the rapid development of vitamin D deficiency (Holtzman colony 5 in which pregnant mothers are fed a diet low but not deficient in vitamin D). These preliminary studies established that several strategies were unacceptable for these experiments. First, under these conditions the rats exhibited unacceptably high rates of mortality when fed vitamin D-deficient diets, particularly with C a and P contents of 0.47 and 0.3% (e.g., 31 of 90 dead by 4 diet weeks). Second, average body weights were reduced in rats fed the 0.47% Ca, 0.3% P , vitamin D-deficient diet. Increasing dietary Ca and P content and inclusion of 20% lactose in the diet increased average body

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NORMAL. Ca AND PTH IN VITAMIN D-DEFICIENT RATS weights, which normalized at dietary Ca, P, and lactose levels of 1.5, 0.9, and 20% (not shown). Third, when Ca gluconate (2%) was added to the drinking water of weanling rats fed vitamin D-deficient diets containing 0.47% Ca, and 0.3% P or 1.0% Ca and 0.6% P, they exhibited both substantially decreased body weights and severe hypophosphatemia (not shown), necessitating sacrifice after only 2 diet weeks. Although normocalcemia was not consistently achieved, these preliminary experiments suggested that a further increase in dietary Ca and P content might prove successful in producing normocalcemia in vitamin D-deficient rats. Whether dietary lactose supplementation resulted in significant physiologic changes in these animals was also evaluated. Despite vitamin D deficiency and moderate hypocalcemia (8.6 versus 10.7 mg/dl in vitamin D-replete animals), upon gross autopsy rats exhibited no deleterious effects in selected tissue weights (testis, heart, and kidney) nor in the histologic appearance of numerous tissues (brain, lung, heart, spleen, liver, adrenal, kidney, seminal vesicle, pancreas, salivary glands, lymph nodes, stomach, small intestine, testis, prostate, and urinary bladder). Unfortunately, despite the invaluable information that these early experiments provided, the limited samples available from tail bleeding were inadequate for full assessment of the Ca homeostatic endocrine system as needed to fully determine whether the vitamin D-deficient rats were normal with respect to other parameters. Additionally, although these preliminary studies in Holtnnan rats were gathered under a different protocol than in the subsequent experiment and thus d o not allow direct comparisons of possible strain differences, in the subsequent study weanlings of normal mothers were evaluated both to ensure improved survival and to establish if vitamin D deficiency could still be achieved rapidly.

11

r

A Co’cium

E. Phosphate

E 4-

h

:I7

T

T

4

+ n c. mi

a.

a

-

Diet A

Diet B

Oiet C

Diet D

A vitamin D-deficient diet that achieves normal plasma levels of Ca, phosphare, and PTH

FIG. 1. Effect of dietary Ca, P, lactose, and vitamin D, on plasma Ca, phosphate, and N-terminal PTH levels. Weanling Charles River rats were fed the diets described in Table 1 for 6 weeks before study. Values are mean f SEM, n = 5 per group. * P < 0.05; **P < 0.01; significance of difference from diet A (ANOVA and Tukey’s test).

With weanling Charles River rats and the dietary regimens described in Table I , plasma Ca levels were significantly lower in the vitamin D-depleted groups B and C than in vitamin D-replete group A (Fig. 1A). However, the rats in the high Ca, P, and lactose-supplemented, vitamin D-deficient group D were normocalcemic. Plasma phosphate levels were similar in all dietary groups (Fig. IB). PTH levels in the normocalcemic, vitamin D-deficient group D were also normal, whereas the hypocalcemic rats in groups B and C developed marked secondary hyperparathyroidism (Fig. IC). The normocalcemia, normophosphatemia, and normal PTH levels were maintained in rats fed diet D for an additional 13 weeks, whereas hypocalcemia and secondary hyperparathyroidism continued in rats fed diets B and C (data not shown). In the vitamin D-depleted groups plasma 25-OHD, levels were all similar and were reduced by at least 99.5% ( P < 0.001) versus the levels seen in vitamin D-replete diet group A (Fig. 2A). Plasma 1,25-(OH),D, levels were also substantially ( P < 0.01) lower (73-88%) in the three groups fed vitamin D-deficient diets (Fig. 2B). As with 25-

OHD,, there were no significant differences in 1,25(OH),D, levels between the groups of vitamin D-deficient rats. Thus the rats in group D were vitamin D deficient but with normal levels of plasma Ca, phosphate, and PTH. Low 25-OHD1 and 1,25-(OH),D, levels were maintained in the vitamin D-deficient groups for an additional 13 weeks, with no differences between the groups (data not shown). The vitamin D-deficient state of the rats of group D was reflected in the classical assay of 1,25-(OH),D, responsiveness, active Ca transport in the everted gut sac preparation. Thus duodenal active Ca transport was significantly lower in all three vitamin D-deficient groups when compared with the vitamin D-replete rats (Fig. 3A). There was no effect of dietary Ca, P, o r lactose on Ca transport in the vitamin D-deficient rats. Similarly, tibial mineral content was significantly reduced in the rats fed diet B, indicating the presence of vitamin D-deficiency rickets (Fig. 3B). In contrast, tibial mineral content was normal in the

276

KOLLENKIRCHEN ET AL. A. 25(OH)D3

30

4

-

.-0

E 10

a

4

0

L

5

1;, P

v,

5 1

3

+

5-a

v

v)

N

E m

:

P +

**

**

2

0 0

h 0.1

1

r

-

&

B. Tibia ash

B. 1,25(OH)2D3 6o

A. Ca transport

1 1

,i,,;,+, Diet A

Diet A

Diet B

Diet C

Diet B

Diet C

Diet 0

FIG. 2. Effect of dietary Ca, P , lactose, and vitamin D, on plasma 25-OHD3 and 1,25-(OH),D3 levels. Weanling Charles River rats were fed the diets described in Table 1 for 6 weeks before study. Values are mean * SEM, n = 5 per group. * P < 0.01; significance of difference from diet A (ANOVA and Tukey’s test).

FIG. 3. Effect of dietary Ca, P, lactose, and vitamin D1 on duodenal active Ca transport (everted gut sac technique) and tibia1 ash content. Weanling Charles River rats were fed the diets described in Table 1 for 6 weeks before study. Values are mean f SEM, n = 5 per group. **P < 0.01; significance of difference from diet A (ANOVA and Tukey’s test).

vitamin D-deficient rats with normocalcemia, normophosphatemia, and normal PTH levels (Fig. 3B). Importantly, body weight was not affected by vitamin D deficiency in any of the test groups. After 6 diet weeks the rats in groups A, B, C , and D weighed 336 f 19, 292 17, 323 + 21, and 295 f 18 g, respectively. Furthermore, in contrast to the preliminary studies with Holtzman rats, in Charles River rats there was no significant mortality associated with any diet.

lactose group also prevented the development of hypophosphatemia, a frequent complication of dietary Ca supplementation. ‘ 1 z - 1 4 ) Furthermore, these studies underscore the importance of dietary lactose in the maintenance of normocalcemia. Increasing dietary Ca t o 2% and P to 1.25% was incapable of maintaining normocalcemia in the absence of dietary lactose (Fig. 1). This effect of lactose is achieved by increased intestinal C a absorption by enhancing the passive, or diffusional, component of Ca trans-

*

DISCUSSION This paper describes the rapid (within 6 weeks) development of vitamin D deficiency in a normocalcemic, normophosphatemic, normal P T H rat model. Weanling male rats of the Charles River strain, when fed a high-Ca (2070) diet containing 20% lactose substituted for sucrose in the normal diet, did not become hypocalcemic, despite the rapid development of vitamin D deficiency. Maintenance of a Ca/P molar ratio of 1.25:l in the diet of the high Ca and

Importantly, these studies also showed that the normocalcemia can be achieved in vitamin D-deficient rats without concomitantly elevated P T H secretion, since PTH levels were normal in the normocalcemic, vitamin D-deficient rats (Fig. 1). Normal plasma C a and PTH levels were maintained in similar rats fed diet D for 19 weeks. In contrast, PTH levels were markedly elevated in the hypocalcemic animals. Normal growth was also maintained during the development of vitamin D deficiency on this diet. This is important since stimulation of growth by vitamin D administration to vitamin D-deficient animals is another vari-

211

NORMAL Ca AND PTH IN VITAMIN D-DEFICIENT RATS able that must be controlled when investigating the role of vitamin D in both novel and classic target tissues. Finally, maintenance of normocalcemia during the development of vitamin D deficiency in Charles River rats also prevented significant hypocalcemia-induced mortality. Importantly, the normocalcemic rats fed the vitamin Ddeficient diet were markedly vitamin D depleted. Although 25-OHD, was detectable in the plasma of all vitamin D-deficient rats, our radioimmunoassay for 25-OHD1 is considerably more sensitive than most other assays, such that we were able to measure levels of 50-100 pg/ml in the plasma of the vitamin D-depleted rats (Fig. 2). This contrasts with the standard competitive protein binding assay using vitamin D binding protein, which depending on the volume of plasma extracted has a detection limit of 1-5 ng/ml.(”,’4,’6,221 Thus by conventional criteria these rats are vitamin D deficient with nondetectable 25-OHD, levels. 1,25-(OH),D, was also detectable in plasma of all vitamin D-depleted rats, but levels were markedly reduced (Fig. 2). The low 25-OHD, and 1,25-(OH),DI levels were maintained in similar rats fed the vitamin D-deficient diets for an additional 13 dietary weeks. These reduced 1,25(OH),D, levels were accompanied by appropriately reduced active duodenal Ca transport in all vitamin D-deficient groups (Fig. 3). As with 25-OHD, levels, there were no differences in plasma 1,25-(OH),D, levels between the vitamin D-depleted groups. Low-Ca diets increase the metabolic clearance rate of 1,25-(OH),D, and decrease plasma 25-OHD, levels in rats.‘”) Thus it is important to note that feeding the high-Ca diet did not impede the reductions in plasma 25-OHD, or 1,25-(OH),D, levels, ensuring the rapid achievement of vitamin D deficiency in the normal rat strain studied. Finally, the decreased tibia1 mineral content seen in the vitamin D-deficient rats fed the 0.8% Ca diet was completely reversed in the normocalcemic, vitamin D-deficient rats (Fig. 3). These results support those of Underwood and DeLuca,(‘’) who reported that vitamin D is not directly necessary for bone mineralization but merely supports mineralization by maintaining normal plasma Ca and phosphate levels. In conclusion, we have demonstrated rapid development of vitamin D deficiency in a normocalcemic, normophosphatemic rat model by feeding a lactose-containing, highCa and high-P diet to normal weanling male Charles River C D rats. We have also shown that, although the rats are severely vitamin D depleted, the normocalcemia is not maintained by elevated PTH secretion. Moreover, the normocalcemic, normal PTH, vitamin D-deficient state is stable in that it was maintained throughout a 19 week test period. This rat model of normocalcemic vitamin D deficiency will prove very useful in the elucidation of the physiologic role(s) of vitamin D and its metabolites in the normal functioning of 1,25-(OH),Dl targets whose function is dependent upon extracellular Ca.

ACKNOWLEDGMENTS We thank Drs. Milan Uskokovic (Hoffman LaRoche) and Hunter Heath (Mayo Clinic) for providing vitamin D ,

metabolites and antiserum G813-PTH, respectively. Dr. Rick Fish, Dept. of Vivarial Science, Tulane Medical School, and Drs. James Blanchard and Gary Baskin, Delta Regional Primate Center, provided invaluable assistance with the autopsies. M. Bijoy Mathew and Diana Woods also provided expert technical assistance. This work was supported by a Grant-in-Aid from the American Heart Association, by USPHS-NIH Grant DK 3 1847 (Walters), and by a Grant-in-Aid from the American Heart AssociationLouisiana (Fox). Parts of this work were presented at the 73rd annual meeting of FASEB (New Orleans, March 1989) and at the Joint Meeting of the International Conference on Calcium Regulating Hormones and the American Society for Bone and Mineral Research, Montreal, Canada, September 1989, and are published as abstracts (FASEB J 3:A774, 1989, and J Bone Miner Res 4(Suppl. I):S703, 1989).

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KOLLENKIRCHEN ET AL.

278 13. Holtrop ME, Cox KA. Clark MB, Holick MF, Anast CS 1981 1,25-Dihydroxycholecalciferolstimulates osteoclasts in rat bones in the absence of parathyroid hormone. Endocrinology 108:2293-2301. 14. Holtrop ME, Cox KA, Carnes DL, Holick M F 1986 Effects of serum calcium and phosphorus o n skeletal mineralization in vitamin D-deficient rats. Am J Fhysiol 251:E234-E240. IS. Lester GE. Van der Wiel CJ, Gray TK, Talmage RV 1982 Vitamin D deficiency in rats with normal serum calcium concentrations. Proc Natl Acad Sci USA 79:4791-4794. 16. Underwood JL, Phelps ME, DeLuca H F 1984 Complex carbohydrate diets are not capable of maintaining normal plasma calcium and phosphorus levels in vitamin D-deficient rats. Proc Natl Acad Sci USA 81:2352-2353. 17. Underwood JL, DeLuca H F 1984 Vitamin D is not directly necessary for bone growth and mineralization. Am J Physiol 246:E493-E498. 18. Thomas ML, lbarra MJ 1987 Effects of ovariectomy on duodenal calcium transport in the rat: Altered ability to adapt to low-calcium diet. Proc Soc Exp Biol Med 185:8488. 19. Schachter D, Dowdle EB, Schenker H 1960 Active transport of calcium by the small intestine of the rat. Am J Physiol 198:263-268. 20. Fox J, Kollenkirchen U, Walters MR 1991 Deficiency of vitamin D metabolites directly stimulates renal 25-hydroxyvitamin D,-I-hydroxylase activity in rats. Metabolism (in press). 21. Hollis BW, Kilbo T 1988 The assay of circulating 1.25(OH),D using non-end-capped C,, silica (C,,-OH): Performance and validation. In: Norman AW, Schaefer K, Grigokit H-G von Herrath D (eds.) Vitamin D. Molecular, Cellular and Clinical Endocrinology. Walter de Gruyter, Berlin, pp. 710-719. 22. Reinhardt TA, Horst RL 1988 Simplified assays for the determination of 25-OHD, 24,25-(OH),D and 1,25-(OH)>D. In: Norman AW, Schaefer K, Grigoleit H-G, von Herrath D

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Address reprint requests to: John Fox, Ph.D. Deparlrnenl of Physiology Tulane Universiry School of Medicine 1430 Tulane Avenue New Orleans, LA 70112 Received for publication April 30, 1990; in revised form October 24, 1990; accepted November 2, 1990.