DOMESTICANIMAL ENDOCRINOLOGY
Vol 7(3)425-435, 1990
EFFECTS OF GROWTH HORMONE ADMINISTRATION ON VITAMIN D METABOLISM AND VITAMIN D RECEPTORS IN THE PIG J.P. Goff, *,1 T. J. Caperna,** and N. C. Steele** *US Department of Agriculture, Agncultural Research Serv=ce, National Animal Disease Center, Metabolic D=seases and Immunology Research Un=t P.O. Box 70, Ames, IA 50010 and **US Department of Agnculture, Agricultural Research Serv=ce, Non-Ruminant An=mal Nutnt~on Laboratory, Beltswlle, MD 20705 Rece,ved January 4, 1990
ABSTRACT Twelve 36-kg pigs were given either 100 IJg/kg porcine pituitary growth hormone CoGH) or placebo injections datly for 33 days Serum was obtained weekly for analysis of minerals and vttamin D metabohtes On day 34, the pigs were sacrificed and renal and duodenal tissue were obtained for analysis of vitamin D receptor content (VDR) Animals treated with pGH grew faster and had a htgher rate of bone accretion than did control animals Serum concentrations of 1,25-dihydroxyvitamin D (1,25-(OH)2D) were significantly higher m pGH-treated pigs than in control pigs at all time points following initiation of treatment, with the greatest difference observed at day 28 (42 4 + 4 9 pg/ml in controls vs 65 4 +_ 4 7 pg/ml in pGH-treated pigs) Serum 24,25-dihydroxyvitamin D tended to be lower in pGH-treated pigs than in control pigs, being significantly lower on day 21 of the experiment (3 22 + 52 vs 6 73 -+ 1 22 ng/ml, respectively) Serum concentrations of 25-hydroxyvltamin D and calcium were unaffected by pGH treatment K~dneys of control ptgs contained significantly more unoccupied vitamin D receptors than &d kidneys from pGH-treated pigs ('73 3 + 4 3 vs 58 3 + 4 1 fmoles/ mg protein) Duodemd tissue unoccupmd vitamin D receptor content was similar in both pGH-treated (245 + 17 9 fmoles/mg protein) and control (263 + 21 8 fmoles/ mg protein) pigs Duodenal occupmd vitamin D receptor concentration was similar in both pGH-treated (6 8 + 75 fmoles/mg protein) and control pigs (5 32 + 77 fmoles/ m 8 protein) These data indicate that pGH treatment increases circulating levels of 1,25-(OH)2D and increases small intestinal mass, thereby increasing intestinal calcmm absorption capablliues necessary to support a h~gher rate of bone accretion INTRODUCTION In dogs a n d humans, g r o w t h h o r m o n e administration m c r e a s e s the rates o f intestinal c a l c i u m a b s o r p t i o n , b o n e c a l c m m accretion, a n d c a l c i u m r e t e n t t o n ( 1 , 2 ) S m c e c a l c i u m m e t a b o l i s m is greatly influenced b y the c i r c u l a t i n g conc e n t r a t i o n o f 1,25-dihydroxTvitamln D ( 1 , 2 5 - ( O H ) 2 D ) , it has b e e n s u g g e s t e d that i n c r e a s e d c a l c i u m r e t e n t i o n d u r i n g g r o w t h h o r m o n e a d m i n i s t r a t i o n m a y b e effected b y i n c r e a s e d 1,25-(OH)2D c o n c e n t r a t i o n s (3) In s u p p o r t o f this c o n c e p t , a c r o m e g a l y is associated w i t h i n c r e a s e d s e r u m c o n c e n t r a t i o n s o f 1,25(OH)2D (4) a n d h y p o p h y s e c t o m y w i t h r e d u c e d s e r u m c o n c e n t r a t i o n s o f 1,25(OH)2D ( 5 ) . H o w e v e r , t w o r e c e n t studies have s h o w n that l o n g - t e r m g r o w t h h o r m o n e t r e a t m e n t o f deficient c h i l d r e n was not associated w i t h a n y increase Copyright © 1990 by DOMENDO, INC
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in serum 1,25-(OH)2D concentration, although net calcium absorption did increase during therapy (6,7) Intestinal calcium absorption is altered by certain physiological states such as age (8), pregnancy (9), and lactation (10) In addition to alterations in serum concentrations of 1,25-(OH)2D, these physiologic states seem to be associated with alterations in the number of receptors for 1,25-(OH)2D (VDR) in the tissues (11,12) The objective of the present study was to determine if the increased intestinal calcium absorption associated with chronic growth hormone administration is mediated by changes in vitamin D metabolism, tissue VDR concentration, or both MATERIALS AND METHODS A n i m a l s . Twelve crossbred (Duroc X York )< Landrace) barrows were housed individually in pens maintained at 22 C Pigs were fed a 91% calcium, 94% phosphorus, corn-soybean meal-skim milk based diet (13) Pigs were restrictively fed to 85% of calculated caloric voluntary intake (14) beginning 1 wk before the start of the trial and continuing throughout the study Pigs were fed once dally at 0800 hr and all feed was consumed by 1200-1300 hr One group of six pigs was injected with porcine pituitary growth hormone (pGH, USDA-B1, 100 pg/kg BWt), and the control group was injected with diluent (50 mM carbonate buffer in 15 M NaCI, pH 9 5) Pigs were injected daily at the time of feeding Feed and pGH dosage were adjusted weekly according to weight gain Pigs were treated for 33 days and killed on day 34 Blood was withdrawn by venipuncture at 0700 hr (prior to feeding and treatment) on days 0, 7, 14, 21, and 28 for analysis of mineral and vitamin D metabohte concentrations At slaughter, pigs were stunned by electric shock, exsanguinated, and internal organs and left femur and humerus were immediately removed and weighed Analyses. Serum concentrations of 25-hydroxyvitamin D (25-OHD), 24,25dihydroxyvitamm D (24,25-(OH)zD), and 1,25-(OH)zD were determined by the method of Reinhardt and Horst (15) Serum calcium and magnesium concentrations were determined by atomic absorption spectrophotometry (16) Phosphorus (17) and hydroxyprohne, an index of relative bone resorption activity (18), were determined colorimetrically Kidney and duodenum were obtained at slaughter for analysis of their vitamin D receptor content A 7-cm section of duodenum obtained 10 cm distal to the pylorus was obtained from each pig and flushed with 10 ml of 10 mM Trisbuffered saline containing 50 IU aprotinin/ml (pH 7 4, 4 C) All subsequent steps were performed at 4 C The mucosa was scraped from the serosa and placed in 10 mM Tris, 1mM dithiothrettol, 1 5 mM ethylene diamme tetraacetlc acid (EDTA) buffer (pH 7 4) containing 200 lag/ml soybean trypsin inhibitor (TEDI), mixed vigorously, and centrifuged at 3000 g for 5 mm Addition of soybean trypsin inhibitor stabilizes the receptor protein during the assay procedure (19) The supernatant was discarded, and the pellet was resuspended in four volumes (weight volume) of 600 mM KCI, 50 mM Tris, 1 5 mM EDTA, 5mM dithiothreitol buffer (pH 7 4) containing 200 lag/ml soybean trypsin inhibitor (KTEDI) The mucosa was then homogenized using a Brinkman Instruments Polytron (Westbury, N'Y), and the homogenate was centrifuged at 229,000 X g for 20 min in a Beckman ultracentrifuge Homogenization of the cells in a high salt buffer extracts the VDR protein from the cell nucleus into the cytosol (20,21) The supernatant (containing the VDR) was collected and
GROWTH HORMONE EFFECTS ON VITAMIN D METABOLISM
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assayed for unoccupied VDR Unoccupied VDR was estimated by adding 500 gl of the cell cytosol (diluted in KTEDI buffer to 0 5-1 0 mg protem/ml) to each of three 12 X 75 mm borosilicate glass tubes contammg 3 6 nM 1,25dihydroxy[26,27-methyl-3H]vitamin D 3 (pH]-1,25-(OH)2D3) [150,000 cpm/tube, specific activity ---- 88 Ci/mmole 1,25-(OH)2D3], and to three tubes containing 100 ng radtomert 1,25-(OH)2D 3 (150-fold excess) in addttaon to [3H]-l,25(OH)2D 3 m order to determine total and nonspectfic bmding of [3H]-l,25(OH)2D 3 m the samples After incubation at 4C for 18 hr, the receptor bound [3H]-l,25-(OH)2D 3 was separated from free pH]-l,25-(OH)2D 3 by hydroxylapatire (22) Specific binding was estimated by the difference between total bindmg and that obtained in the presence of excess radioinert 1,25-(OH)2D Protein content of the cytosol preparation was determined (23), and receptor assay results are expressed as fmoles VDR/mg protein in the cytosol preparation Renal tissues (a transverse section through one pole of the kidney that included cortex only) were handled similarly except that the tissue was minced finely immediately after collection Duodenal occupied VDR concentration was estimated by assaying the cytosol preparation (undiluted) for 1,25-(OH)2D usmg a modification of the assay used for 1,25-(OH)2D analysis of serum Briefly, the 1,25-(OH)2D in the CH3CN soluble hpids was partially purified by sequential C-18 and silica Sep-pak chromatography The 1,25-(OH)2D isolated from the silica Sep-pak was subjected to an additional HPLC purification step utilizing a Zorbax Sil column developed in 9/1 hexane/lsopropanol before quantttation by competltwe radioreceptor binding assay (15) Renal cytosol preparations contained too little 1,25-(OH)2D to be accurately measured (< 2 fmoles/mg protein) RESULTS Serum calcium concentrations in control and pGH-treated pigs did not differ throughout the course of the experiment (Figure l-A) Serum magnesium concentration (Figure l-B) was statlStlcly higher and phosphorus (Figure 1C) lower m control pigs than in pGH-treated pigs on day 14, however, since these differences were present at the start of the experiment, the effect of pGH on plasma magnesium and phosphorus was negligible Serum hydroxyproline concentrations were numerically higher tn pGH-treated pigs than in control pigs, however, these differences were not significant as the pGH-treated pigs entered the experiment with higher serum concentrations of hydroxyprohne (Figure l-D) Serum 1,25-(OH)2D concentration was higher in pGH-treated pigs than in control pigs at all time points following initiation of treatment Over the 4 wk of the experiment, serum 1,25-(OH)2D concentration decreased from 85 0 -+ 13 2 pg/ml to 42 4 + 4 9 pg/ml in control pigs Serum 1,25-(OH)2D concentration of pGH-treated p~gs exhibited a smaller decline over the course of the e x p e r t m e n t m f r o m 86 3 + 8 6 pg/ml to 65 4 _+ 4 7 pg/ml (Figure 2A) Serum 24,25-(OH)2D concentration was numerically lower in pGH-treated pigs than in control pigs, being significantly lower on day 21 of the experiment (Figure 2-B) Serum concentrations of 25-OHD were not significantly affected by pGH treatment Serum concentrations of 25-OHD were between 10 and 15 ng/ml through day 21 of the experiment On day 28, serum 25-OHD concentrations in both groups increased to about 27 ng/ml (Figure 2-C), perhaps due to batch variation in vttamm D supplementation of the diet fed to both groups of pigs during that time
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Control pGH-Treated VDR content (fmoles/mg protein) Kidney Unoccupied 73 3 +- 4 3 58 3 + 4 I b Duodenum Unoccupied 263 0 -+ 21 8 245 0 +_ 17 9 Occupied 5 3 +- 0 7 7 6 8 -+ 0 7 5 % Occupied 201 _+ 0 2 8 2 7 3 + 031 Tissue Weights Kidney (g) 223 0 -+ 12 358 0 + 17 b Kidney (% b o d y w t ) 0 3 9 7 -+ 0 0 1 6 0 5 4 6 -+ 0 0 1 6 b Small intestine (g) 1154 0 +- 45 1628 0 -+ 62 b Small intestine (% b o d y w t ) 2 0 6 -+ 0 0 6 2 4 9 +- 0 l0 b • Barrows were injected daily with either bicarbonate buffer (control) or 100 tag pGH/kg (pGHtreated) for 33 days ( n = 6 pigs/group) b Significantly different from controls (p< 05)
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Ptgs treated w t t h e x o g e n o u s p G H g r e w faster and had larger organs and bones than did c o n t r o l pigs as seen in previous s m d m s ( 1 3 , 2 4 ) By the e n d o f the e x p e r i m e n t , the h u m e r u s and f e m u r o f pGH-treated pigs w e r e a b o u t 18% heavier than m c o n t r o l ptgs (Table 2) Caperua et a l ( 2 5 ) d e m o n s t r a t e d that the c a l c m m c o n t e n t (rag c a l c i u m / g f e m u r dry w e i g h t ) o f femurs from pGH-treated and c o n t r o l pigs w e r e similar Since the pGH-treated and control
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GOFF, CAPERNA AND STEELE T,~LE 2 INFLUENCEOF PORCINE GROWTH HORMONE ( p G H ) ADMINISTRATIONON GROWTH CHARACTERISTICS,AND TISSUE MEASUREMENTSIN SW1NI~a Control
Growth Measurements I m t t a l w e l g h t (kg) F m a l w e t g h t (kg) Average dally gain ( g / d ) Bone M e a s u r e m e n t s F e m u r (g) F e m u r (% b o d y w t ) H u m e r u s (g) H u m e r u s (% b o d y w t )
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• Barrows w e r e m l e c t e d daffy w i t h e i t h e r b i c a r b o n a t e buffer ( c o n t r o l ) or 100 lag p G H / k g (pGHtreated) for 33 days (nffi6 p i g s / g r o u p ) b Significantly different f r o m c o n t r o l s ( p < 0 5 )
pigs consumed identical amounts of dlet/kg body weight, these data indicate that the pGH-treated pigs absorbed calcium from the dmt and deposited that calcium into bone at a greater rate than did control pigs Pigs treated with pGH had higher serum 1,25-(OH)2D concentrations than did control pigs within 1 wk of the mitiauon of treatment and continuing throughout the experiment The higher serum 1,25-(OH)zD concentration of pGH-treated pigs enables them to absorb more of the dietary calcium, permitting formation of bone with normal calcium content The increased serum concentrauons of 1,25-(OH)2D may be the result of a direct effect of pGH on renal 25-hydroxyvitamin D 1-hydroxylase as suggested by Gray (26), or may be a response of the calcium homeostatic system to the increased rate of bone growth induced by pGH The faster rate of bone calcium accretion in pGH-treated pigs w oul d be e x p e c t e d to remove calcium from the extracellular fluid calcium pool at a faster rate, stimulating secretion of parathyroid hormone Parathyroid hormone enhances renal production of 1,25-(OH)2D (and decreases synthesis of 24,25(OH)2D) increasing the rate of entry of calcium into the extracellular fluid calcium pool as a result of enhanced intestinal calcium absorption The results of this study are supported by earlier observauons of elevated 1,25-(OH)zD levels in acromegaly (4) and restorauon of 1,25-(OH)2D production when growth hormone is given to hypopitmtary rats (6,27) In addition to circulating 1,25-(OH)2D concentration, the responsiveness of 1,25-(OH)2D target tissues to 1,25-(OH)2D ss dependent on the amount of the vitamin D receptor protein present m the tissue (28) If growth hormone treatment increased tissue VDR concentration, tissues from growth hormonetreated animals w o u l d exhibit a greater response to a given level of 1,25(OH)2D than w o u l d tissues from untreated animals Yeh e t a l (29) determined that hypophysectomy of vitamin D-deficmnt rats resulted m a reduction m intestinal u n o c c u p i e d VDR However, m the same study they also observed that m vitamin D replete rats, hypophysectomy led to an increase in intestinal u n o c c u p i e d VDR, but because intestinal mucosa was thinner m hypophysectomlzed rats, the total amount of u n o c c u p i e d VDR per segment of intestine was r ed u ced by hypophysectomy In the present study, pGH treatment of vitamin D replete pigs failed to increase intestinal o c c u p i e d or u n o c c u p i e d VDR concentration and actually decreased kidney u n o c c u p i e d VDR concentration The higher serum 1,25-(OH)zD m the pGH-treated pigs may have been responsible for the slightly higher level of occupi ed VDR in the d u o d e n u m of those pigs O c c u p i e d VDR represented 2 73 + 31% of total receptor in the pGH.treated pigs and 2 01 _ 28% in the control pigs Perhaps only very
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small c h a n g e s m o c c u p i e d r e c e p t o r status are necessary for i n c r e a s e d activation o f 1,25-(OH)2D driven p r o c e s s e s O u r data suggest that p G H t r e a t m e n t increases c a l c i u m a b s o r p t i o n b y increasing the mass o f the intestine a n d increasing s e r u m 1,25-(OH)2D c o n c e n t r a t i o n s It d o e s not a p p e a r to increase the r e s p o n s i v e n e s s o f individual intestinal cells to 1,25-(OH)2D s t i m u l a u o n b y m c r e a s m g cell VDR Renal tissue f r o m p G H - t r e a t e d pigs is significantly l o w e r in VDR suggesting a d i m i n i s h e d c a p a c i t y o f individual renal cells to r e s p o n d to 1,25-(OH)2D H o w e v e r , this effect m a y have b e e n c o m p e n s a t e d for b y the increased mass o f renal tissue in the p G H - t r e a t e d animals Presumably, alteration in renal VDR c o n c e n t r a t i o n m i g h t have effects o n renal c a l c i u m t r a n s p o r t and f e e d b a c k i n h i b i t i o n o f renal p r o d u c t i o n o f 1 , 2 5 - ( O H ) z D It has b e e n o b s e r v e d that t r e a t m e n t o f rats w i t h 1,25-(OH)2D 3 results in u p r e g n l a u o n o f intestinal and renal VDR ( 3 0 , 3 1 ) Since s e r u m 1,25-(OH)2D c o n c e n t r a t i o n s m the p G H - t r e a t e d pigs in this s t u d y w e r e h i g h e r t h a n in controls, an increase in VDR in p G H - t r e a t e d p i g tissues m i g h t have b e e n e x p e c t e d as a result o f h o m o l o g o u s u p r e g n l a u o n b y 1 , 2 5 - ( O H ) 2D Recently, w e have o b s e r v e d that in rats i n c r e a s e d s e r u m levels o f 1,25-(OH)2D i n d u c e d b y dietary c a l c i u m stress also failed to u p r e g u l a t e intestinal VDR and, as in the p r e s e n t e x p e r i m e n t , r e s u l t e d in a r e d u c t i o n in renal VDR ( 3 2 ) These data s u g g e s t e d that p a r a t h y r o i d h o r m o n e m i g h t m o d u l a t e VDR c o n t e n t o f cells We can s p e c u l a t e that the a c c e l e r a t e d b o n e a c c r e t i o n i n d u c e d b y p G H t r e a t m e n t m a y have increased p a r a t h y r o i d h o r m o n e s e c r e t i o n in the pigs Perhaps p a r a t h y r o i d h o r m o n e is a m o d u l a t i n g f a c t o r r e s p o n s i b l e for the r e d u c e d level o f VDR in the renal tissue o f p G H - t r e a t e d pigs In s u m m a r y , t r e a t m e n t o f pigs w i t h p G H increases the rate o f b o n e a c c r e t i o n To m e e t the c a l c i u m d e m a n d s o f b o n e growth, net r e t e n t i o n o f dietary c a l c i u m m u s t b e i n c r e a s e d T h e results o f this s t u d y indicate that p G H - t r e a t m e n t is a c c o m p a n i e d b y an increase in c i r c u l a t i n g levels o f 1,25-(OH)2D and an increase in small intestinal mass, w h i c h h k e l y increases intestinal c a l c m m a b s o r p u o n c a p a b f l i u e s G r o w t h h o r m o n e t r e a t m e n t did not alter the c o n c e n t r a u o n o f VDR an intestinal tissue, and thus w o u l d not b e e x p e c t e d to increase the responsaveness o f individual epithelaal cells to s t t m u l a u o n b y 1,25-(OH)2D ACKNOWLEDGEMENTS/FOOTNOTES The authors wish to thank C Hauber, D Gavelek, C Evock, and D Komarek for their technical expertise and diligence, whmh allowed compleuon of this project, and L Oppedal for preparation of the manuscript tTo whom correspondence should be addressed No endorsements are hereto imphed REFERENCES 1 Henneman PH, Forbes AP, Moldawar M, Dempsey EF Effects of human growth hormone m man J Chn Invest 39 1223-1228, 1960 2 Heaney RP, Harris WH, CoclanJ, Wemberg EH Growth hormone effect on skeletal renewal in adult dog 2 Mineral Kaneuc studms Calctlied T~ssue 10 14-19, 1972 3 Brown DJ, Spanos E, Maclntyre I Role of pituitary hormones m regulating renal vitamin D metabolism in man Br Med J 280 177-180, 1980 4 Eslraldsen PC, Lund B, Soresen OH, Lund B, Bishop JE, Norman AW Acromegaly and vitamin D metabolism J Clin Endocrmol Metab 49 484-486, 1979 5 Spanos E, Barrett D, MacIntyre I, Pike JW, Safihan EF, Haussler MR Effect of growth hormone on vitamin D metabohsm Nature 273 246-247, 1978 6 Germer JM, Hot'st RL, Broadus AE, Rasmussen H, Genel M Parathyroid function and vitamin D metabolism during human growth hormone replacement J Chn Endocrinol Metab 49 185-188, 1979
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7 Chipman JJ, Zerwekh J, Nicar M, Marks J, Pak CYC Effect of growth hormone administration reciprocal changes in serum 1,25-Dihydroxyvitamm D and intestinal calcium absorption J Chn Endocrlnol Metab 51 321-324, 1980 8 Horst RL, DeLuca HF, Jorgensen NA The effect of age on calcium absorption and accumulation of 1,25-dlhydroxyvitamln D3 in intestinal mucosa of rats Metab Bone Dis Relat Res 1 29-33, 1978 9 Halloran BP, DeLuca HF Calcium transport in small intestine during pregnancy and lactation Am J Physlol 239 E64-69, 1980 10 Boass A, Toverud SU, Pike JW, Haussler MR Calcium metabolism during lactation Enhanced intestinal calcium absorption in vitamin D-deprived hypocalcemlc rats Endocrinology 109 900-907, 1981 11 Horst RL, Remhardt TA Changes in intestinal 1,25-dlhydroxyvitamm D receptor during aging, gestation and pregnancy in rats Proc of Seventh Workshop on Vitamin D, Rancho Mirage, CA In Norman AW, Schaefer K, Grigolelt HG, Herrath, DV (eds) Vitamin D Molecular, Cellular and Clinical Endocrinology, Walter de Gruyter Co, Berlin, pp 229-232, 1988 12 GoffJP, Horst RL, Remhardt TA Duodenum and colon 1,25-dlhydroxyvltamm D receptor concentration ts increased during lactation in the rat Seventh Workshop on Vitamin D, Rancho Mirage, CA In Norman AW, Schaefer K, Grlgoleit HG, Herrath, DV (eds) Vitamm D Molecular, Cellular and Clinical Endocrinology, Walter de Gruyter Co, Berlin, pp 246-249, 1988 13 Campbell RH, Steele NC, Caperna TJ, McMurtry JP, Solomon MB, Mitchell AD Interrelationships between energy intake and endogenous porcine growth hormone administration on the performance, body composluon and protein and energy metabolism of growing pigs weighing 25 to 55 kilograms live weight J Anim Sci 66 1643-1651, 1988 14 Agricultural Research Council (ARC) The Nutrient Requirements of Pigs Commonwealth Agricultural Research Bureau, The Royal Society, London, pp 307-325, 1981 15 Reinhardt TA, Horst RL Slmphfied assay for the determination of 25-OH D, 24,25(OH)2D, and 1,25-(OH)2D Seventh Workshop on Vitamin D, Rancho Mirage, CA In Norman AW, Schaefer K, Grigolelt HG, Herrath, DV (eds) Vitamin D Molecular, Cellular and Clinical Endocrinology, Walter de Gruyter C o , Berlin, pp 720-724, 1988 16 Periran-Elmer Corp Analytical methods for atomic absorption spectrophotometry Perlan-Elmer, Norwalk CT, 1965 17 Parekh AC, Jung DH Serum inorganic phosphorus determlnauon using p-phenylenedlamlne as a reducing agent Cllnica Chimlca Acta 27 373-378, 1970 18 Dabev D, Struck H Mmrohter determination of free hydroxyprohne in blood serum Biochemical medicine 5 17-21, 1971 19 Hlrst MA, Feldman D Cleavage of the rat intestinal 1,25-dihydroxyvltamin D3 receptor by an endogenous protease to a form with defective DNA binding Arch B10chem Blophys 250 153-161, 1986 20 Feldman D, McCaln TA, Hirst MA, Chen TL, Colston KW Characterization of a cytoplasmic receptor-like binder for 1,25-dthydroxycholecalciferol in rat intestinal mucosa J Biol Chem 254 10378-10384, 1979 21 Walters MR, Hunziker W, Norman AW Unoccupied 1,25-dlhydroxyvltamln D3 receptors nuclear/cytosol ratio depends on ionic strength J Blol Chem 255 67996805, 1980 22 Wecksler WR, Norman AW An hydroxylapatite batch assay for the quantltation of 1,25-dihydroxyvitamin D3-receptor complexes Anal Biochem 92 314-323, 1979 23 Bradford MM A rapid and sensitive method for the quanutauon of microgram quantities of protein utilizing the principle of protein-dye binding Anal Blochem 72 248-256, 1976 24 Etherton "I'D, Wiggins JP, Chung CS, Evock CM, Rebhun JF, Walton PE Stimulation of pig growth performance by porcine growth hormone and growth hormonereleasing factor J Atom Sci 63 1389-1398, 1986 25 Caperna TJ, Campbell RG, Steele NC Interrelationships of exogenous porcine growth hormone administration and feed intake level affecting various tissue levels of iron, copper, zinc and bone calcium of growing pigs J Atom Sci 67 654-663, 1989
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26 Gray RW, Garthwalte TL Activation of renal 1 25-dihydroxyvitamin D3 synthesis by phosphate deprivation evidence for a role for growth hormone Endocrinology 116 189-193, 1985. 27. Fontaine O, Pavlovitch H, Balsan S 25.Hydroxycholecalciferol metabolism in hypophysectomized rats Endocrinology 102 1822-1828, 1978 28. Chen TL, Hauschka PV, Cabrales S, Feldman D The effects of 1,25.dihydroxyvitamin D3 and dexamethasone on rat osteoblast-like primary cell cultures Receptor occupancy and functional expression patterns for three different bioresponses Endocrinology 118 250-259, 1986 29 Yeh JK, Aloia yF, Vaswani AN, Semla H Effect of hypophysectomy on the occupied and unoccupied binding sites for 1,25.dihydroxyvitamin D3 in rat intestine Bone 7 49-53, 1986 30 Reinhardt TA, Horst RL, Engstrom GW, Atkins KB Ketoconazole potentiates 1,25(OH)2D3°directed upregulation of 1,25-(OH)2D3-receptors in rat intestine and bone Proc of Seventh Workshop on Vitamin D, Rancho Mirage, CA In Norman AW, Schaefer K, Grigoleit HG, Herrath, DV (eds) Vitamin D Molecular, Cellular and Clinical Endocrinology, Walter de Gruyter Co, Berlin, pp 233-236, 1986 31 Costa EM, Feldman D Homologous up-regulation of the 1,25-(OH)2 vitamin D3 receptor in rats Biochem Biophys Res Commun 137 742-747, 1986 32 Goff JP, Reinhardt TA, Beckman MJ, Horst RL Contrasting effects of exogenous 1,25-(OH)=D versus endogenous 1,25-(OH)=D, induced by dietary calcium restriction, on vitamin D receptors Endocrinology 126 1031-1035, 1990
Vol 7(3)425-435, 1990
EFFECTS OF GROWTH HORMONE ADMINISTRATION ON VITAMIN D METABOLISM AND VITAMIN D RECEPTORS IN THE PIG J.P. Goff, *,1 T. J. Caperna,** and N. C. Steele** *US Department of Agriculture, Agncultural Research Serv=ce, National Animal Disease Center, Metabolic D=seases and Immunology Research Un=t P.O. Box 70, Ames, IA 50010 and **US Department of Agnculture, Agricultural Research Serv=ce, Non-Ruminant An=mal Nutnt~on Laboratory, Beltswlle, MD 20705 Rece,ved January 4, 1990
ABSTRACT Twelve 36-kg pigs were given either 100 IJg/kg porcine pituitary growth hormone CoGH) or placebo injections datly for 33 days Serum was obtained weekly for analysis of minerals and vttamin D metabohtes On day 34, the pigs were sacrificed and renal and duodenal tissue were obtained for analysis of vitamin D receptor content (VDR) Animals treated with pGH grew faster and had a htgher rate of bone accretion than did control animals Serum concentrations of 1,25-dihydroxyvitamin D (1,25-(OH)2D) were significantly higher m pGH-treated pigs than in control pigs at all time points following initiation of treatment, with the greatest difference observed at day 28 (42 4 + 4 9 pg/ml in controls vs 65 4 +_ 4 7 pg/ml in pGH-treated pigs) Serum 24,25-dihydroxyvitamin D tended to be lower in pGH-treated pigs than in control pigs, being significantly lower on day 21 of the experiment (3 22 + 52 vs 6 73 -+ 1 22 ng/ml, respectively) Serum concentrations of 25-hydroxyvltamin D and calcium were unaffected by pGH treatment K~dneys of control ptgs contained significantly more unoccupied vitamin D receptors than &d kidneys from pGH-treated pigs ('73 3 + 4 3 vs 58 3 + 4 1 fmoles/ mg protein) Duodemd tissue unoccupmd vitamin D receptor content was similar in both pGH-treated (245 + 17 9 fmoles/mg protein) and control (263 + 21 8 fmoles/ mg protein) pigs Duodenal occupmd vitamin D receptor concentration was similar in both pGH-treated (6 8 + 75 fmoles/mg protein) and control pigs (5 32 + 77 fmoles/ m 8 protein) These data indicate that pGH treatment increases circulating levels of 1,25-(OH)2D and increases small intestinal mass, thereby increasing intestinal calcmm absorption capablliues necessary to support a h~gher rate of bone accretion INTRODUCTION In dogs a n d humans, g r o w t h h o r m o n e administration m c r e a s e s the rates o f intestinal c a l c i u m a b s o r p t i o n , b o n e c a l c m m accretion, a n d c a l c i u m r e t e n t t o n ( 1 , 2 ) S m c e c a l c i u m m e t a b o l i s m is greatly influenced b y the c i r c u l a t i n g conc e n t r a t i o n o f 1,25-dihydroxTvitamln D ( 1 , 2 5 - ( O H ) 2 D ) , it has b e e n s u g g e s t e d that i n c r e a s e d c a l c i u m r e t e n t i o n d u r i n g g r o w t h h o r m o n e a d m i n i s t r a t i o n m a y b e effected b y i n c r e a s e d 1,25-(OH)2D c o n c e n t r a t i o n s (3) In s u p p o r t o f this c o n c e p t , a c r o m e g a l y is associated w i t h i n c r e a s e d s e r u m c o n c e n t r a t i o n s o f 1,25(OH)2D (4) a n d h y p o p h y s e c t o m y w i t h r e d u c e d s e r u m c o n c e n t r a t i o n s o f 1,25(OH)2D ( 5 ) . H o w e v e r , t w o r e c e n t studies have s h o w n that l o n g - t e r m g r o w t h h o r m o n e t r e a t m e n t o f deficient c h i l d r e n was not associated w i t h a n y increase Copyright © 1990 by DOMENDO, INC
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in serum 1,25-(OH)2D concentration, although net calcium absorption did increase during therapy (6,7) Intestinal calcium absorption is altered by certain physiological states such as age (8), pregnancy (9), and lactation (10) In addition to alterations in serum concentrations of 1,25-(OH)2D, these physiologic states seem to be associated with alterations in the number of receptors for 1,25-(OH)2D (VDR) in the tissues (11,12) The objective of the present study was to determine if the increased intestinal calcium absorption associated with chronic growth hormone administration is mediated by changes in vitamin D metabolism, tissue VDR concentration, or both MATERIALS AND METHODS A n i m a l s . Twelve crossbred (Duroc X York )< Landrace) barrows were housed individually in pens maintained at 22 C Pigs were fed a 91% calcium, 94% phosphorus, corn-soybean meal-skim milk based diet (13) Pigs were restrictively fed to 85% of calculated caloric voluntary intake (14) beginning 1 wk before the start of the trial and continuing throughout the study Pigs were fed once dally at 0800 hr and all feed was consumed by 1200-1300 hr One group of six pigs was injected with porcine pituitary growth hormone (pGH, USDA-B1, 100 pg/kg BWt), and the control group was injected with diluent (50 mM carbonate buffer in 15 M NaCI, pH 9 5) Pigs were injected daily at the time of feeding Feed and pGH dosage were adjusted weekly according to weight gain Pigs were treated for 33 days and killed on day 34 Blood was withdrawn by venipuncture at 0700 hr (prior to feeding and treatment) on days 0, 7, 14, 21, and 28 for analysis of mineral and vitamin D metabohte concentrations At slaughter, pigs were stunned by electric shock, exsanguinated, and internal organs and left femur and humerus were immediately removed and weighed Analyses. Serum concentrations of 25-hydroxyvitamin D (25-OHD), 24,25dihydroxyvitamm D (24,25-(OH)zD), and 1,25-(OH)zD were determined by the method of Reinhardt and Horst (15) Serum calcium and magnesium concentrations were determined by atomic absorption spectrophotometry (16) Phosphorus (17) and hydroxyprohne, an index of relative bone resorption activity (18), were determined colorimetrically Kidney and duodenum were obtained at slaughter for analysis of their vitamin D receptor content A 7-cm section of duodenum obtained 10 cm distal to the pylorus was obtained from each pig and flushed with 10 ml of 10 mM Trisbuffered saline containing 50 IU aprotinin/ml (pH 7 4, 4 C) All subsequent steps were performed at 4 C The mucosa was scraped from the serosa and placed in 10 mM Tris, 1mM dithiothrettol, 1 5 mM ethylene diamme tetraacetlc acid (EDTA) buffer (pH 7 4) containing 200 lag/ml soybean trypsin inhibitor (TEDI), mixed vigorously, and centrifuged at 3000 g for 5 mm Addition of soybean trypsin inhibitor stabilizes the receptor protein during the assay procedure (19) The supernatant was discarded, and the pellet was resuspended in four volumes (weight volume) of 600 mM KCI, 50 mM Tris, 1 5 mM EDTA, 5mM dithiothreitol buffer (pH 7 4) containing 200 lag/ml soybean trypsin inhibitor (KTEDI) The mucosa was then homogenized using a Brinkman Instruments Polytron (Westbury, N'Y), and the homogenate was centrifuged at 229,000 X g for 20 min in a Beckman ultracentrifuge Homogenization of the cells in a high salt buffer extracts the VDR protein from the cell nucleus into the cytosol (20,21) The supernatant (containing the VDR) was collected and
GROWTH HORMONE EFFECTS ON VITAMIN D METABOLISM
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assayed for unoccupied VDR Unoccupied VDR was estimated by adding 500 gl of the cell cytosol (diluted in KTEDI buffer to 0 5-1 0 mg protem/ml) to each of three 12 X 75 mm borosilicate glass tubes contammg 3 6 nM 1,25dihydroxy[26,27-methyl-3H]vitamin D 3 (pH]-1,25-(OH)2D3) [150,000 cpm/tube, specific activity ---- 88 Ci/mmole 1,25-(OH)2D3], and to three tubes containing 100 ng radtomert 1,25-(OH)2D 3 (150-fold excess) in addttaon to [3H]-l,25(OH)2D 3 m order to determine total and nonspectfic bmding of [3H]-l,25(OH)2D 3 m the samples After incubation at 4C for 18 hr, the receptor bound [3H]-l,25-(OH)2D 3 was separated from free pH]-l,25-(OH)2D 3 by hydroxylapatire (22) Specific binding was estimated by the difference between total bindmg and that obtained in the presence of excess radioinert 1,25-(OH)2D Protein content of the cytosol preparation was determined (23), and receptor assay results are expressed as fmoles VDR/mg protein in the cytosol preparation Renal tissues (a transverse section through one pole of the kidney that included cortex only) were handled similarly except that the tissue was minced finely immediately after collection Duodenal occupied VDR concentration was estimated by assaying the cytosol preparation (undiluted) for 1,25-(OH)2D usmg a modification of the assay used for 1,25-(OH)2D analysis of serum Briefly, the 1,25-(OH)2D in the CH3CN soluble hpids was partially purified by sequential C-18 and silica Sep-pak chromatography The 1,25-(OH)2D isolated from the silica Sep-pak was subjected to an additional HPLC purification step utilizing a Zorbax Sil column developed in 9/1 hexane/lsopropanol before quantttation by competltwe radioreceptor binding assay (15) Renal cytosol preparations contained too little 1,25-(OH)2D to be accurately measured (< 2 fmoles/mg protein) RESULTS Serum calcium concentrations in control and pGH-treated pigs did not differ throughout the course of the experiment (Figure l-A) Serum magnesium concentration (Figure l-B) was statlStlcly higher and phosphorus (Figure 1C) lower m control pigs than in pGH-treated pigs on day 14, however, since these differences were present at the start of the experiment, the effect of pGH on plasma magnesium and phosphorus was negligible Serum hydroxyproline concentrations were numerically higher tn pGH-treated pigs than in control pigs, however, these differences were not significant as the pGH-treated pigs entered the experiment with higher serum concentrations of hydroxyprohne (Figure l-D) Serum 1,25-(OH)2D concentration was higher in pGH-treated pigs than in control pigs at all time points following initiation of treatment Over the 4 wk of the experiment, serum 1,25-(OH)2D concentration decreased from 85 0 -+ 13 2 pg/ml to 42 4 + 4 9 pg/ml in control pigs Serum 1,25-(OH)2D concentration of pGH-treated p~gs exhibited a smaller decline over the course of the e x p e r t m e n t m f r o m 86 3 + 8 6 pg/ml to 65 4 _+ 4 7 pg/ml (Figure 2A) Serum 24,25-(OH)2D concentration was numerically lower in pGH-treated pigs than in control pigs, being significantly lower on day 21 of the experiment (Figure 2-B) Serum concentrations of 25-OHD were not significantly affected by pGH treatment Serum concentrations of 25-OHD were between 10 and 15 ng/ml through day 21 of the experiment On day 28, serum 25-OHD concentrations in both groups increased to about 27 ng/ml (Figure 2-C), perhaps due to batch variation in vttamm D supplementation of the diet fed to both groups of pigs during that time
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Control pGH-Treated VDR content (fmoles/mg protein) Kidney Unoccupied 73 3 +- 4 3 58 3 + 4 I b Duodenum Unoccupied 263 0 -+ 21 8 245 0 +_ 17 9 Occupied 5 3 +- 0 7 7 6 8 -+ 0 7 5 % Occupied 201 _+ 0 2 8 2 7 3 + 031 Tissue Weights Kidney (g) 223 0 -+ 12 358 0 + 17 b Kidney (% b o d y w t ) 0 3 9 7 -+ 0 0 1 6 0 5 4 6 -+ 0 0 1 6 b Small intestine (g) 1154 0 +- 45 1628 0 -+ 62 b Small intestine (% b o d y w t ) 2 0 6 -+ 0 0 6 2 4 9 +- 0 l0 b • Barrows were injected daily with either bicarbonate buffer (control) or 100 tag pGH/kg (pGHtreated) for 33 days ( n = 6 pigs/group) b Significantly different from controls (p< 05)
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Ptgs treated w t t h e x o g e n o u s p G H g r e w faster and had larger organs and bones than did c o n t r o l pigs as seen in previous s m d m s ( 1 3 , 2 4 ) By the e n d o f the e x p e r i m e n t , the h u m e r u s and f e m u r o f pGH-treated pigs w e r e a b o u t 18% heavier than m c o n t r o l ptgs (Table 2) Caperua et a l ( 2 5 ) d e m o n s t r a t e d that the c a l c m m c o n t e n t (rag c a l c i u m / g f e m u r dry w e i g h t ) o f femurs from pGH-treated and c o n t r o l pigs w e r e similar Since the pGH-treated and control
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GOFF, CAPERNA AND STEELE T,~LE 2 INFLUENCEOF PORCINE GROWTH HORMONE ( p G H ) ADMINISTRATIONON GROWTH CHARACTERISTICS,AND TISSUE MEASUREMENTSIN SW1NI~a Control
Growth Measurements I m t t a l w e l g h t (kg) F m a l w e t g h t (kg) Average dally gain ( g / d ) Bone M e a s u r e m e n t s F e m u r (g) F e m u r (% b o d y w t ) H u m e r u s (g) H u m e r u s (% b o d y w t )
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pigs consumed identical amounts of dlet/kg body weight, these data indicate that the pGH-treated pigs absorbed calcium from the dmt and deposited that calcium into bone at a greater rate than did control pigs Pigs treated with pGH had higher serum 1,25-(OH)2D concentrations than did control pigs within 1 wk of the mitiauon of treatment and continuing throughout the experiment The higher serum 1,25-(OH)zD concentration of pGH-treated pigs enables them to absorb more of the dietary calcium, permitting formation of bone with normal calcium content The increased serum concentrauons of 1,25-(OH)2D may be the result of a direct effect of pGH on renal 25-hydroxyvitamin D 1-hydroxylase as suggested by Gray (26), or may be a response of the calcium homeostatic system to the increased rate of bone growth induced by pGH The faster rate of bone calcium accretion in pGH-treated pigs w oul d be e x p e c t e d to remove calcium from the extracellular fluid calcium pool at a faster rate, stimulating secretion of parathyroid hormone Parathyroid hormone enhances renal production of 1,25-(OH)2D (and decreases synthesis of 24,25(OH)2D) increasing the rate of entry of calcium into the extracellular fluid calcium pool as a result of enhanced intestinal calcium absorption The results of this study are supported by earlier observauons of elevated 1,25-(OH)zD levels in acromegaly (4) and restorauon of 1,25-(OH)2D production when growth hormone is given to hypopitmtary rats (6,27) In addition to circulating 1,25-(OH)2D concentration, the responsiveness of 1,25-(OH)2D target tissues to 1,25-(OH)2D ss dependent on the amount of the vitamin D receptor protein present m the tissue (28) If growth hormone treatment increased tissue VDR concentration, tissues from growth hormonetreated animals w o u l d exhibit a greater response to a given level of 1,25(OH)2D than w o u l d tissues from untreated animals Yeh e t a l (29) determined that hypophysectomy of vitamin D-deficmnt rats resulted m a reduction m intestinal u n o c c u p i e d VDR However, m the same study they also observed that m vitamin D replete rats, hypophysectomy led to an increase in intestinal u n o c c u p i e d VDR, but because intestinal mucosa was thinner m hypophysectomlzed rats, the total amount of u n o c c u p i e d VDR per segment of intestine was r ed u ced by hypophysectomy In the present study, pGH treatment of vitamin D replete pigs failed to increase intestinal o c c u p i e d or u n o c c u p i e d VDR concentration and actually decreased kidney u n o c c u p i e d VDR concentration The higher serum 1,25-(OH)zD m the pGH-treated pigs may have been responsible for the slightly higher level of occupi ed VDR in the d u o d e n u m of those pigs O c c u p i e d VDR represented 2 73 + 31% of total receptor in the pGH.treated pigs and 2 01 _ 28% in the control pigs Perhaps only very
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small c h a n g e s m o c c u p i e d r e c e p t o r status are necessary for i n c r e a s e d activation o f 1,25-(OH)2D driven p r o c e s s e s O u r data suggest that p G H t r e a t m e n t increases c a l c i u m a b s o r p t i o n b y increasing the mass o f the intestine a n d increasing s e r u m 1,25-(OH)2D c o n c e n t r a t i o n s It d o e s not a p p e a r to increase the r e s p o n s i v e n e s s o f individual intestinal cells to 1,25-(OH)2D s t i m u l a u o n b y m c r e a s m g cell VDR Renal tissue f r o m p G H - t r e a t e d pigs is significantly l o w e r in VDR suggesting a d i m i n i s h e d c a p a c i t y o f individual renal cells to r e s p o n d to 1,25-(OH)2D H o w e v e r , this effect m a y have b e e n c o m p e n s a t e d for b y the increased mass o f renal tissue in the p G H - t r e a t e d animals Presumably, alteration in renal VDR c o n c e n t r a t i o n m i g h t have effects o n renal c a l c i u m t r a n s p o r t and f e e d b a c k i n h i b i t i o n o f renal p r o d u c t i o n o f 1 , 2 5 - ( O H ) z D It has b e e n o b s e r v e d that t r e a t m e n t o f rats w i t h 1,25-(OH)2D 3 results in u p r e g n l a u o n o f intestinal and renal VDR ( 3 0 , 3 1 ) Since s e r u m 1,25-(OH)2D c o n c e n t r a t i o n s m the p G H - t r e a t e d pigs in this s t u d y w e r e h i g h e r t h a n in controls, an increase in VDR in p G H - t r e a t e d p i g tissues m i g h t have b e e n e x p e c t e d as a result o f h o m o l o g o u s u p r e g n l a u o n b y 1 , 2 5 - ( O H ) 2D Recently, w e have o b s e r v e d that in rats i n c r e a s e d s e r u m levels o f 1,25-(OH)2D i n d u c e d b y dietary c a l c i u m stress also failed to u p r e g u l a t e intestinal VDR and, as in the p r e s e n t e x p e r i m e n t , r e s u l t e d in a r e d u c t i o n in renal VDR ( 3 2 ) These data s u g g e s t e d that p a r a t h y r o i d h o r m o n e m i g h t m o d u l a t e VDR c o n t e n t o f cells We can s p e c u l a t e that the a c c e l e r a t e d b o n e a c c r e t i o n i n d u c e d b y p G H t r e a t m e n t m a y have increased p a r a t h y r o i d h o r m o n e s e c r e t i o n in the pigs Perhaps p a r a t h y r o i d h o r m o n e is a m o d u l a t i n g f a c t o r r e s p o n s i b l e for the r e d u c e d level o f VDR in the renal tissue o f p G H - t r e a t e d pigs In s u m m a r y , t r e a t m e n t o f pigs w i t h p G H increases the rate o f b o n e a c c r e t i o n To m e e t the c a l c i u m d e m a n d s o f b o n e growth, net r e t e n t i o n o f dietary c a l c i u m m u s t b e i n c r e a s e d T h e results o f this s t u d y indicate that p G H - t r e a t m e n t is a c c o m p a n i e d b y an increase in c i r c u l a t i n g levels o f 1,25-(OH)2D and an increase in small intestinal mass, w h i c h h k e l y increases intestinal c a l c m m a b s o r p u o n c a p a b f l i u e s G r o w t h h o r m o n e t r e a t m e n t did not alter the c o n c e n t r a u o n o f VDR an intestinal tissue, and thus w o u l d not b e e x p e c t e d to increase the responsaveness o f individual epithelaal cells to s t t m u l a u o n b y 1,25-(OH)2D ACKNOWLEDGEMENTS/FOOTNOTES The authors wish to thank C Hauber, D Gavelek, C Evock, and D Komarek for their technical expertise and diligence, whmh allowed compleuon of this project, and L Oppedal for preparation of the manuscript tTo whom correspondence should be addressed No endorsements are hereto imphed REFERENCES 1 Henneman PH, Forbes AP, Moldawar M, Dempsey EF Effects of human growth hormone m man J Chn Invest 39 1223-1228, 1960 2 Heaney RP, Harris WH, CoclanJ, Wemberg EH Growth hormone effect on skeletal renewal in adult dog 2 Mineral Kaneuc studms Calctlied T~ssue 10 14-19, 1972 3 Brown DJ, Spanos E, Maclntyre I Role of pituitary hormones m regulating renal vitamin D metabolism in man Br Med J 280 177-180, 1980 4 Eslraldsen PC, Lund B, Soresen OH, Lund B, Bishop JE, Norman AW Acromegaly and vitamin D metabolism J Clin Endocrmol Metab 49 484-486, 1979 5 Spanos E, Barrett D, MacIntyre I, Pike JW, Safihan EF, Haussler MR Effect of growth hormone on vitamin D metabohsm Nature 273 246-247, 1978 6 Germer JM, Hot'st RL, Broadus AE, Rasmussen H, Genel M Parathyroid function and vitamin D metabolism during human growth hormone replacement J Chn Endocrinol Metab 49 185-188, 1979
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