438 Biochimica et Biophysica A cta, 385 (1975) 438--442

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA Report BBA 21406 THE FUNCTIONAL METABOLISM OF VITAMIN D IN CHICKS FED LOW-CALCIUM AND LOW-PHOSPHORUS DIETS

SAMUEL EDELSTEIN, ARIE HARELL, ARIE BAR and SHMUEL HURWITZ Department o f Endocrinology, Municipal Governmental Medical Centre, Ichilov Hospital, Tel Aviv-Jaffo and the Institute of Animal Sciences, Agricultural Research Organisation, The Volcani Centre, Bet Dagan (Israel)

(Received February 4th, 1975)

Summary Radioactively labelled cholecalciferol was administered continuously to chicks that were fed normal, low-calcium and low-phosphorus diets. It has been possible to show t h a t under such steady state conditions with regard to cholecalciferol, and mineral restriction, the animal reacts by increased accumulation of 1,25-dihydroxycholecalciferol in the intestinal and the kidney cell, which was associated in the intestine with an increased calcium-binding activity. A similar accumulation of 1,25-dihydroxycholecalciferol in bone was not noticed. It is proposed that the intestine and the kidney, but not bone, are the main target organs for cholecalciferol in the maintenance of calcium homeostasis, and that both calcium and phosphorus play a role in the regulation of the formation and subsequent function of 1,25-dihydroxycholecalciferol.

Vitamin D has long been known to be intimately associated with calcium homeostasis. Insight into its mode of action has been recently gained by the discoveries concerning its conversion into 25-hydroxycholecalciferol in the liver and then into the metabolically active metabolite 1,25-dihydroxycholecalciferol in the kidney [1 ]. The specific localization of this latter metabolite in the nucleus of the target cell, and the involvement of RNA and protein synthesis in its intestinal action [2,3], support the idea t h a t 1,25dihydroxycholecalciferol is a steroid hormone. The intestine, kidney and bone are the main tissues associated with calcium homeostasis, and are also believed to be target organs for vitamin D action. Nicolaysen et al. [4], in 1953, showed that intestinal calcium absorption increased in animals placed on a low-calcium diet. This could be demonstrated only in vitamin D-fed animals. More recently this adaptation phenomenon

439 has been found to be associated with a corresponding increase in calciumbinding protein [5]. Moreover, phosphorus deficiency has been f o u n d to result in a similar increase in the calcium-absorptive capacity and calciumbinding protein [5]. Under such conditions of adaptation to low-mineral intake, more 1,25-dihydroxycholecalciferol was produced by the kidney. Therefore, this metabolite has been implicated as the principal stimulant in the adaptation process. Most of the information is, however, derived from observations made either on isolated organs or on animals following a single injection of radioactively labelled vitamin, rather than on animals in a steady state with regard to their vitamin D status. Furthermore, opinions differ with regard to the factors responsible for the regulation of 1,25-dihydroxycholecalciferol formation under such conditions [6--8]. The skeleton is k n o w n to respond to calcium restriction b y increased resorption, and the kidney b y increased calcium reabsorption. In neither case has the role of vitamin D as mediator of those processes been demonstrated. In the present study, metabolites of cholecalciferol were measured in the three target tissues, intestine, kidney and bone, under conditions o. J o t h calcium or phosphorus restriction. Since chemical methods for measurements of these metabolites are n o t readily available, radioactively labelled cholecalciferol was administered continuously to the animals. This procedure ensured steady state conditions of measurable cholecalciferol metabolites. Day-old chicks (Light cross) were fed for three weeks on a vitamin Dfree diet [9] and were injected at three-day intervals with 0.75 ug of [1,2 -~ H, 4-14C]cholecalciferol. During the first ten days of feeding, the diet was adequate with regard to all nutrients except for vitamin D. On the 11th day, the birds were divided into three groups and were fed, respectively: (a) the same diet (1.1% Ca, 0.7% P); (b) low-calcium diet (0.2% Ca, 0.7% P); and (c) low-phosphorus diet (1.1% Ca, 0.3%P). On the 20th day, the birds were killed and kidney, bone, intestinal mucosa and its nuclear fraction were analysed for cholecalciferol metabolites. Intestinal calcium-binding activity, bone ash, and plasma concentrations of calcium and p h o s p h o r u s were also estimated. As expected (Table I), bone ash was reduced b o t h in birds fed either the low-calcium or the low-phosphorus diets. The low-phosporus treatment resulted in a marked h y p o p h o s p h a t e m i a and slight hypercalcemia. The lowcalcium treatment resulted in hypocalcemia and a slight hypophosphatemia. The lipid extract, prepared from intestinal mucosa cells (Fig. 1), contained, in normal birds, 0.06 pmol of 1,25-dihydroxycholecalciferol per g of tissue (31%); but, in birds fed the low-calcium or the low-phosphorus diet, there was some three-fold increase, up to 1.98 pmol per g of tissue (71%) and 2.10 pmol per g of tissue (72%) respectively. The isolated nuclear fraction of these cells contained 90--100% 1,25-dihydroxycholecalciferol; its concentration in these cells was markedly larger in birds fed the low-calcium and the low-phosphorus diets, compared to those fed the normal diet (Table II). These results correlate well with the intestinal calcium-binding protein as determined b y calcium-binding activity measurements (Table II). Such

440 TABLE I PLASMA CALCIUM, PLASMA INORGANIC PHOSPHORUS AND BONE ASH OF CHICKS FED NORMAL, LOW-CALCIUM AND LOW-PHOSPHORUS DIETS H e p a r i n i s e d b l o o d s a m p l e s w e r e o b t a i n e d f r o m five b i r d s o f e a c h g r o u p w h i c h w e r e t h e n k i l l e d b y dislocation of the neck. Plasma calcium was d e t e r m i n e d by direct E D T A titration under ultraviolet light w i t h C a l c e i n as a n i n d i c a t o r , P l a s m a i n o r g a n i c p h o s p h o r u s w a s d e t e r m i n e d i n t r i c h l o r o a c e t t c a c i d f i l t r a t e s a c c o r d i n g to G o m o r i [ 2 0 ] . B o n e s w e r e c l e a n e d of a d h e r i n g t i s s u e , d r i e d a t 1 0 5 ° C a n d a s h e d at 6 0 0 ° C f o r 6 h. V a l u e s g i v e n in t h e t a b l e a r e t h e m e a n s -+ S.E. Diet

Dietary minerals (%)

Normal Low-Ca Low-P

Plasma minerals (rag/100 ml)

Bone ash (% o f d r y w t )

Ca

P

Ca

P

1.1 0.2 1.1

0.7 0.7 0.3

1 0 . 0 -+ 0 . 2 8.9 -+ 0.3 1 2 . 0 + 0.6

6.6 -+ 0.1 5.2 -+ 0 . 2 1,9 + 0.1

36.6 -+ 0 . 5 27.9 -+ 0.1 26.1 + 0 . 2

05

2.0

15

1.0

o

0.5

E o.

2.0 Intestine

iiiili

1.5

il~ii ii)i i!i ~i

1.0

0,5

o t

2

3

NORMAL

4

5

DIET

1

2

3

LOW-Ca

4

5

DIET

1

2

3

LOW- P

4

5

DIET

Fig.1. T h e d i s t r i b u t i o n o f c h o l e c a l c i f e r o l m e t a b o l i t e s in i n t e s t i n e , k i d n e y a n d b o n e o f c h i c k s f e d n o r m a l , l o w - c a l c i u m a n d l o w - p h o s p h o r u s diets. L i p i d s e x t r a c t e d f r o m t h e t i s s u e s w e r e s u b j e c t e d t o thin-layer chromatographic analysis [12] for the estimation of choleealciferol metabolites. The amount o f 1 - h y d r o x y l a t e d m e t a b o l i t e w a s c a l c u l a t e d f r o m t h e 5H/14C s p e c i f i c r a d i o a c t i v i t y r a t i o s [ 1 5 ] . 1. P o l a r m e t a b o l i t e s ; 2. 1 , 2 5 - d i h y d r o x y c h o l e c a l c i f e r o l ; 3. 2 4 , 2 5 - d i h y d r o x y c h o l e c a l c i f e r o l l 4. 2 5 - d i h y d r o x y c h o l e c a l c f f e r o l ; 5. h y d r o x y c h o l e c a l c i f e r o l .

correlation between 1,25-dihydroxycholecalciferol accumulation in the intestine and calcium transport was reported for rats fed a vitamin D-deficient and low-phosphorus diet, and dosed daffy with radioactively labelled 25hydroxycholecalciferol [ 1 0 ] . Only trace amounts of 1,25-dihydroxycholecalciferol were detected in bones obtained from the control group of birds {Fig. 1). Somewhat more 1,25-dihydroxycholecalciferol was found in bones obtained from the birds

441 T A B L E II NUCLEAR 1,25-DIHYDROXYCHOLECALCIFEROL CONTENT AND CALCIUM-BINDING A C T I V I T Y O F I N T E S T I N A L M U C O S A C E L L S O B T A I N E D F R O M B I R D S F E D N O R M A L , LOWCALCIUM AND LOW-PHOSPHORUS DIETS I n t e s t i n a l m u c o s a cells f r o m five b i r d s of e a c h g r o u p w e r e s c r a p e d , p o o l e d , m i n c e d w i t h scissors and divided into t h r e e p o r t i o n s . T h e first p o r t i o n was u s e d f o r a n a l y s i s o f c h o l e c a l c i f e r o l m e t a b o l i t e s as described in Fig. 1. T h e s e c o n d p o r t i o n was s u b j e c t e d t o calcium-binding activity analysis b y t h e C h e l e x assay [ 2 1 ] , a n d the third p o r t i o n for t h e p r e p a r a t i o n o f p u r i f i e d n u c l e i [ 2 2 ] a n d analysis o f cholecalciferol m e t a b o l i t e s . Diet

Normal Low-Ca Low-P

Nuclear 1,25-dihydroxycholecalciferol

Calcinm-binding activity

(pmol/g m u c o s a [ _ _

(% of normal)

0.38 0.73 0.78

100 132 * 151

* I n a similar e x p e r i m e n t , d u o d e n a l calcium-binding a c t i v i t y , w h i c h r e s p o n d s m a x i m a l l y to t r e a t m e n t , was 1 8 2 % a n d 1 7 2 % of c o n t r o l s in c h i c k s f e d t h e l o w - c a l c i u m and the l o w - p h o s p h o r u s diet, r e s p e c t i v e l y . T h e a p p a r e n t l o w e r r e s p o n s e o b s e r v e d in the p r e s e n t e x p e r i m e n t m a y b e d u e t o t h e f a c t t h a t m u c o s a f r o m t h e entire length o f t h e i n t e s t i n e was t a k e n for analysis.

fed the low-calcium and the low-phosphorus diets, as compared to the controls. In the kidneys, only trace amounts of 1,25-dihydroxycholecalciferol were f o u n d when taken from the control group of birds; but, in those fed the low-calcium diet, there was an increase in 1,25-dihydroxycholecalciferol content which was even more pronounced in the kidneys obtained from the group of birds fed the low-phosphorus diet (Fig. 1). In plasma obtained from all three groups of birds, only minute amounts of 1,2 5-dihydroxycholecalciferol could be detected, and no differences in its level could be established. This finding was predictable, considering the fact that even in the rachitic chick, following a single dose of cholecalciferol, only 1--2% of the total antirachitic activity of plasma is due to 1,25-dihydroxycholecalciferol [11]. In the rat, on the other hand, higher proportions of 1,25-dihydroxycholecalciferol may be present [12]. At our present state of knowledge, 1,25-dihydroxycholecalciferol is regarded as the active principle of vitamin D. We suggest that b o t h calcium and phosphorus play a role in the regulation of its formation and subsequent function, and that the intestine is the most important target organ. If the availability of dietary calcium or phosphorus is restricted, the animal reacts by increased accumulation of 1,25-dihydroxycholecalciferol in the intestinal cell which results in an increased absorption efficiency of these minerals [4,5, 10,131. The similarity in the responses of b o t h vitamin D metabolite pattern and intestinal calcium-binding protein to calcium and phosphorus deficiency seems to suggest a c o m m o n mechanism for the induction of the regulatory changes at the level of the intestinal cell. This does not eliminate the possibility of differences b e t w e e n the treatments in the events leading to the adaptation p h e n o m e n o n as previously suggested [ 1 9 ] . The increased level of 1,25-dihydroxycholecalciferol in the kidney in response to the dietary mineral restrictions points to a possible importance of this tissue as a target organ for vitamin D, presumably in the reabsorption of calcium which is associated with the presence of a vitamin D-dependent calcium-binding protein [14].

442

With regard to bone, only small amounts of 1,25-dihydroxycholecalciferol were recovered under either normal conditions or mineral restriction. Furthermore, the amount of 1,25-dihydroxycholecalciferol in bone, relative to other cholecalciferol metabolites was low (2--9% in bone, compared with 31--72% in intestine}. This suggests that bone can not be regarded as a major target organ for 1,25-dihydroxycholecalciferol in relation to calcium homeostasis. Other studies showed that, following a single injection of cholecalciferol to rachitic birds, some 35% of the total metabolites of cholecalciferol found in bone was 1,25-dihydroxycholecalciferol [ 1 5 ] . Under such conditions, however, there is a sudden and rapid rise in the blood level of 1,25-dihydroxycholecalciferol. Since several tissues in the body, including bone and muscle, contain high-affinity binding proteins for cholecalciferol type of steroids [16,17 ], 1,25-dihydroxycholecalciferol will be then taken up, whereas under normal physiological conditions this metabolite is hardly detectable in these tissues. This, however, does not necessarily exclude the possibility that vitamin D play a role in bone metabolism [ 1 8 ] . This work was supported by the Wellcome Trust and the Israel Ministry of Health. We are grateful to Mrs Roni Sapir and Mrs Marcella Cotter for their skilled technical assistance. References

1 2 3 4 5

Fraser, D.R. a n d K o d i c e k , E, (1970) Nature 228, 764--766 Emtage, J.S., Lawson, D.E.M. and Kodicek, E. (1973) Nature 246, 100--101 Emtage, J.S., Lawson, D.E.M. and Kodicek, E. (1974) Biochem. J. 140, 239--247 Nicolaysen, R., Eag-Larsen, N, and Maim, O.J. (1953) Physiol. Rev. 33, 424--444 Wasserman, R.H. and Corradino, R.A., (1973) in V i t a m i n s and H o r m o n e s (Harris, R.S., M u n s o n , P.L., Diczfalusy, E. a n d Glover, J., eds), Vol. 31, pp, 43--103, A c a d e m i c Press, New Y o r k 6 Fraser, D.R. and Kodicek, E. (1973) Nat. New Biol. 241, 163--166 7 Larkins, R,G., Coiston, K.W., Galante, L.S., MacAuley, S.J., Evans, I.M.A. and M a c l n t y r e , I. (1973) Lancet, £, 289--291 8 DeLuca, H.F. (1974) Biochem. Soc. Spec. Publ. 3, 5--26 9 Httrwitz, S, and Bar, A. (1972) Am. J. Physiol. 222, 761--767 10 Tanaka, Y., Frank, H. and DeLuca, H.F. (1973) Science 181, 564--566 11 Lawson, D.E.M., Wilson, P.W. and Kodicek, E. (1969) Biochem. J. 115, 269--277 12 Lawson, D.E.M., Bell, P.A,, Pelc, B,, Wilson, P.W. and Kodicek, E. (1971) Biochem. J, 121, 673--682 13 Httrwitz, S., Bar, A. and Cohen, I. (1973) Am. J. Physiol. 225, 150---154 14 Taylor, A.N. and Wasserman, R.H. (1972) Am. J. Physiol. 223, 110---114 15 Weber, J.C., Pons, V. and Kodicek, E. (1971) Biochem. J. 125, 147--153 16 Edelstein, S. (1974) Biochem. Soe. Spec. Publ. 3, 43--54 17 Edelstein, S. (1975) in V i t a m i n s and H o r m o n e s (Harris, R.S., Munson, P.L., Diczfalusy, E. and Glovcr, J., eds), Vol. 32, A c a d e m i c Press, New York, in the press 18 Reynolds, J.J. (1974) Biochem. Soc. Spec. Publ. 3, 91--102 19 Bar, A. and Wasserman, R.H. (1973) Biochem. Biophys. Res. Commun. 54, 191--196. 20 Gomori, G. (1942) J. Lab. Clin. Med. 27, 955---960 21 Wasserman, R,H. and Taylor, A.N. (1966) Science 152, 791--793 22 Lawson, D.E.M., Wilson, P.W., Barker, D.C. and Kodicek, E. (1969) Biochem. J. 115, 262--268

The functional metabolism of vitamin D in chicks fed low-calcium and low-phosphorus diets.

438 Biochimica et Biophysica A cta, 385 (1975) 438--442 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA Report...
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