Calcif. Tiss. Res. 18,289--295 (1975) 9 by Springer-Verlag 1975
The Metabolism of Labeled Parathyroid Hormone V I . Effects o f V i t a m i n D S t a t u s M. W . N e u m a n , W . F. N e u m a n , a n d K . L a n e Department of Radiation Biology and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, New York Received June 21, accepted October 20, 1974 Labeled 125I-PTH with full biological activity was utilized to investigate the interrelationship between vitamin D and parathyroid hormone (PTH) function. The distribution of labeled PTH was studied in rats and chickens on diets containing various amounts of vitamin D and calcium. The effects of vitamin D deficiency were found to be minor. There was a slightly delayed (10-30%) blood clearance of the hormone and a smaller deposition in the liver (2030%) in the D-deficient animals. Deposition in bone and kidney appeared to be essentially normal. I t is concluded that any failure of PTH to evoke a bone response in the deficient animal is not due to the failure of binding of hormone at the target organ. Key words: Vitamin D - - Parathyroid - - Metabolism - - Rat - - Chicken.
Introduction The concept t h a t t h e r e is a n i n t e r a c t i o n b e t w e e n v i t a m i n D a n d p a r a t h y r o i d h o r m o n e has e x i s t e d for some t i m e [13]. I n 1958, i t was first conclusively shown [6] t h a t d i e t a r y v i t a m i n D is r e q u i r e d for m o b i l i z a t i o n of calcium from bone b y p a r a t h y r o i d hormone. I t has since been d e m o n s t r a t e d , however, t h a t t h e renal effects of p a r a t h y r o i d h o r m o n e do n o t require t h e presence of v i t a m i n D [12]. R e c e n t evidence i n d i c a t e s t h a t p a r a t h y r o i d h o r m o n e s t i m u l a t e s t h e h y d r o x y l a t i o n of v i t a m i n D to t h e 1 , 2 5 - d i h y d r o x y m e t a b o l i t e [4, 5]. W h e t h e r t h e v i t a m i n e x e r t s a n influence on t h e m e t a b o l i s m of p a r a t h y r o i d hormone, is a question t h a t has n o t been answered. The recent p r e p a r a t i o n of a r a d i o i s o t o p e - l a b e l e d p a r a t h y r o i d h o r m o n e (125IP T H ) which a p p e a r s to h a v e full biological a c t i v i t y [8, 14] has p e r m i t t e d a new e x p e r i m e n t a l a p p r o a c h to this old p r o b l e m . The o b j e c t of t h i s p a p e r was to det e r m i n e w h e t h e r or n o t t h e d i s t r i b u t i o n a n d m e t a b o l i s m of P T H is affected b y v i t a m i n D. R a t s a n d chickens were p l a c e d on different d i e t a r y regimens of v i t a m i n D a n d calcium. The u p t a k e of 125I-PTtt in t h e t a r g e t organs (liver, k i d n e y , a n d bone [9]) was o b s e r v e d in b o t h species. I n chickens, t h e d i s t r i b u t i o n of labeled m e t a b o l i t e s in these organs [9] was also e x a m i n e d c h r o m a t o g r a p h i c a l l y . W i t h i n t h e limits of t h e s t u d y t h e d i s t r i b u t i o n a n d m e t a b o l i s m of p a r a t h y r o i d h o r m o n e was affected v e r y little b y v i t a m i n D deficiency. For reprints: W. F. I~euman, Department of Radiation Biology and Biophysics, University of Rochester, Rochester, New York 14642, USA. 4 Calcif.Tiss. Res.
290
M . W . Neuman et al.
Methods
Preliminary Experiments with Rats Weanling, Sprague-Dawley rats obtained from Blue Spruce Farms (Altamont, N . Y . ) were randomly divided into three groups of 10 animals each: controls, vitamin D-deficient and D-deficient plus calcium. The control diet had the following composition (wt. % ) : casein (18, corn starch (25), dextrose (46), peanut oil (8), salt mixture (1), vitamin mixture (1), CaHPO 4 (0.9), CaCO 3 (0.2), and contained 1700 I.U. of vitamin D3/kg. The deficient diet was identical except lacking in vitamin D. The calcium supplement diet was identical to the deficient diet except t h a t dextrose was reduced 4% to accommodate the addition of 4% CaCO a. These diets were obtained from Biological Research Products, Chagrin Falls, Ohio, Catalog No. 170735. After 2 weeks on the diets the drinking water was supplemented with KI, 0.05 mM to facilitate the clearance of radioiodide in the distribution experiments. After 5 weeks, the animals were injected intravenously with 125I-PTH and killed 30 min later. The labeled hormone was prepared by controlled voltage electrolysis as previously described [8, 14]. Such labeled hormone has been shown [8, 14] to exhibit undiminished biological activity in four assay systems: CO 2 production by mitochondria, calcium mobilization by surving embryonic bones in culture, serum calcium elevation in parathyroidectomized rats and the adenyl cyclase assay. The injection was adjusted to body weight, 50 ~zl/100 g. The fluid injected was heat denatured (50 ~ 2 h) rat plasma 1 : 1 containing 10 EzCi/ml of 125I-PTH equivalent to 1 ~g/ml of native bovine hormone. Blood samples were collected in heparinized capillary tubes and, after centrifugation, the plasma was examined for radioactivity. Tissue samples taken were: kidney cortex, tibia, liver, spleen, intestine, and sartorius muscle. I n t a c t tibia were placed in well counters. Soft tissues were homogenized ill buffer and a n aliquot t a k e n for radioactivity assay as previously described [9].
Studies with Chickens Two-day-old White Leghorn chickens were placed on a vitamin D-free diet (Cornell Rachitogenic Diet, normal calcium (1.2%) and phosphate (0.65%) content, No. TD-68-366 from General Biochemicals Corp.) for 7 weeks. At this time they weighed 200 g, which is less t h a n half the normal weight. Half the chickens (deficient) were continued on the diet without treatment, while each of the other half (vitamin D-repleted) received 0.3 ~zg of vitamin D 3 (crystalline vitamin Da, Schwartz Mann, Orangeburg, N. Y.) in 0.2 ml of sesame oil administered by stomach tube for 7 days. Four chickens from b o t h groups were then given freshly prepared biologically active 125I-PTH (0.2 ml, 6.6 EzCi, 2 izg in 0.01 M HC1 containing 0.5 mg albumin [8, 14]) by wing vein, without anesthesia. Five minutes post-injection the chickens were killed by decapitation. Blood was collected, and samples of liver, kidney, and bone (femurs) were quickly t a k e n and after weighing, fast frozen for subsequent analysis. Based on previous experience [9] the metabolism of the hormone is nearly complete in 60 min. Because the results of the preliminary studies in rats showed only minor differences at 30 rain, it seemed i m p o r t a n t to examine deposition in target organs a t 5 rain post-injection. The most recent extraction procedures [10] were employed. The frozen tissues were homogenized in the gel filtration buffer (3 M guanidine-HC1; 2.3 M formic acid and, after clarification and addition of markers, subjected to gel filtration on P-10 Biogel columns as described previously [8]). Recovery of total radioactivity in the tissues as delivered to the chromatography columns were 95.4 :~- 0.74%, 97.3 • 2.01% and 99.0 ~: 1.76% for the kidney, liver and bone, respectively (mean ~_ S.E.M.).
Results
Preliminary Experiments with Rats T h e e f f e c t s of t h e t h r e e d i e t a r y r e g i m e n s o n t h e r a t s a r e g i v e n i n T a b l e 1. T h e l a c k of v i t a m i n D c a u s e d a m a r k e d i m p a i r m e n t i n g r o w t h w h i c h w a s p a r t i a l l y
Effects of Vitamin D Status on Metabolism of Labeled PTH
291
Table 1. Effects of diets on rats Diet
q- Vit. D
-- Vit. D
+ Ca, -- Vit. D
Body wt. (g) Kidney wt. (% BW) Tibia wt. ( % BW) Serum Ca (mM) Serum Pi (mM)
163 ~ 0.72 • 0.25 • 2.68 ~ 3.81 ~
127 ~ 3.4 a 0.77 • 0.01 0.27 :~ 0.01 1.50 4- 0.5 a 2.84 -4-0.01 a
144 • 8.8 0.73 :~ 0.02 0.28 :~ 0.01 2.93 • 0.06 b 0.84 • 0.01 a
2.2 0.02 0.01 0.04 0.03
Results given as mean ~ S. E.M. (n = 6). a Significantly differs from control value, p < 0.001. b Significantly differs from control value, p < 0.01.
Table 2. Tissue uptake of total radioactivity in rats Diet
-4-Vit. D
-- Vit. D
-- Vit. D, A- Ca
Plasma Kidney cortex Tibia Liver Spleen Intestine Muscle
4.06 • 0.21 18.7 ==0.6 0.97 -4-0.04 3.71 -- 0.14 1.62 ! 0.08 0.82 • 0.03 0.27 -V 0.02
4.51 :t: 0.19 14.0 -4-0.8a 1.02 -4-0.06 3.53 • 0.27 1.47 • 0.12 0.70 • 0.05 0.26 • 0.01
4.67 • 0.21 13.5 • a 1.12 • 0.04 b 4.40 :~ 0.25 1.64 ~_ 0.12 0.77 ~ 0.03 0.28 ~- 0.02
Results given as mean per cent dose cpm/g :L S. E.M. (n = 6). a Significantly different from control p value
The Metabolism of Labeled Parathyroid Hormone V I . Effects o f V i t a m i n D S t a t u s M. W . N e u m a n , W . F. N e u m a n , a n d K . L a n e Department of Radiation Biology and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, New York Received June 21, accepted October 20, 1974 Labeled 125I-PTH with full biological activity was utilized to investigate the interrelationship between vitamin D and parathyroid hormone (PTH) function. The distribution of labeled PTH was studied in rats and chickens on diets containing various amounts of vitamin D and calcium. The effects of vitamin D deficiency were found to be minor. There was a slightly delayed (10-30%) blood clearance of the hormone and a smaller deposition in the liver (2030%) in the D-deficient animals. Deposition in bone and kidney appeared to be essentially normal. I t is concluded that any failure of PTH to evoke a bone response in the deficient animal is not due to the failure of binding of hormone at the target organ. Key words: Vitamin D - - Parathyroid - - Metabolism - - Rat - - Chicken.
Introduction The concept t h a t t h e r e is a n i n t e r a c t i o n b e t w e e n v i t a m i n D a n d p a r a t h y r o i d h o r m o n e has e x i s t e d for some t i m e [13]. I n 1958, i t was first conclusively shown [6] t h a t d i e t a r y v i t a m i n D is r e q u i r e d for m o b i l i z a t i o n of calcium from bone b y p a r a t h y r o i d hormone. I t has since been d e m o n s t r a t e d , however, t h a t t h e renal effects of p a r a t h y r o i d h o r m o n e do n o t require t h e presence of v i t a m i n D [12]. R e c e n t evidence i n d i c a t e s t h a t p a r a t h y r o i d h o r m o n e s t i m u l a t e s t h e h y d r o x y l a t i o n of v i t a m i n D to t h e 1 , 2 5 - d i h y d r o x y m e t a b o l i t e [4, 5]. W h e t h e r t h e v i t a m i n e x e r t s a n influence on t h e m e t a b o l i s m of p a r a t h y r o i d hormone, is a question t h a t has n o t been answered. The recent p r e p a r a t i o n of a r a d i o i s o t o p e - l a b e l e d p a r a t h y r o i d h o r m o n e (125IP T H ) which a p p e a r s to h a v e full biological a c t i v i t y [8, 14] has p e r m i t t e d a new e x p e r i m e n t a l a p p r o a c h to this old p r o b l e m . The o b j e c t of t h i s p a p e r was to det e r m i n e w h e t h e r or n o t t h e d i s t r i b u t i o n a n d m e t a b o l i s m of P T H is affected b y v i t a m i n D. R a t s a n d chickens were p l a c e d on different d i e t a r y regimens of v i t a m i n D a n d calcium. The u p t a k e of 125I-PTtt in t h e t a r g e t organs (liver, k i d n e y , a n d bone [9]) was o b s e r v e d in b o t h species. I n chickens, t h e d i s t r i b u t i o n of labeled m e t a b o l i t e s in these organs [9] was also e x a m i n e d c h r o m a t o g r a p h i c a l l y . W i t h i n t h e limits of t h e s t u d y t h e d i s t r i b u t i o n a n d m e t a b o l i s m of p a r a t h y r o i d h o r m o n e was affected v e r y little b y v i t a m i n D deficiency. For reprints: W. F. I~euman, Department of Radiation Biology and Biophysics, University of Rochester, Rochester, New York 14642, USA. 4 Calcif.Tiss. Res.
290
M . W . Neuman et al.
Methods
Preliminary Experiments with Rats Weanling, Sprague-Dawley rats obtained from Blue Spruce Farms (Altamont, N . Y . ) were randomly divided into three groups of 10 animals each: controls, vitamin D-deficient and D-deficient plus calcium. The control diet had the following composition (wt. % ) : casein (18, corn starch (25), dextrose (46), peanut oil (8), salt mixture (1), vitamin mixture (1), CaHPO 4 (0.9), CaCO 3 (0.2), and contained 1700 I.U. of vitamin D3/kg. The deficient diet was identical except lacking in vitamin D. The calcium supplement diet was identical to the deficient diet except t h a t dextrose was reduced 4% to accommodate the addition of 4% CaCO a. These diets were obtained from Biological Research Products, Chagrin Falls, Ohio, Catalog No. 170735. After 2 weeks on the diets the drinking water was supplemented with KI, 0.05 mM to facilitate the clearance of radioiodide in the distribution experiments. After 5 weeks, the animals were injected intravenously with 125I-PTH and killed 30 min later. The labeled hormone was prepared by controlled voltage electrolysis as previously described [8, 14]. Such labeled hormone has been shown [8, 14] to exhibit undiminished biological activity in four assay systems: CO 2 production by mitochondria, calcium mobilization by surving embryonic bones in culture, serum calcium elevation in parathyroidectomized rats and the adenyl cyclase assay. The injection was adjusted to body weight, 50 ~zl/100 g. The fluid injected was heat denatured (50 ~ 2 h) rat plasma 1 : 1 containing 10 EzCi/ml of 125I-PTH equivalent to 1 ~g/ml of native bovine hormone. Blood samples were collected in heparinized capillary tubes and, after centrifugation, the plasma was examined for radioactivity. Tissue samples taken were: kidney cortex, tibia, liver, spleen, intestine, and sartorius muscle. I n t a c t tibia were placed in well counters. Soft tissues were homogenized ill buffer and a n aliquot t a k e n for radioactivity assay as previously described [9].
Studies with Chickens Two-day-old White Leghorn chickens were placed on a vitamin D-free diet (Cornell Rachitogenic Diet, normal calcium (1.2%) and phosphate (0.65%) content, No. TD-68-366 from General Biochemicals Corp.) for 7 weeks. At this time they weighed 200 g, which is less t h a n half the normal weight. Half the chickens (deficient) were continued on the diet without treatment, while each of the other half (vitamin D-repleted) received 0.3 ~zg of vitamin D 3 (crystalline vitamin Da, Schwartz Mann, Orangeburg, N. Y.) in 0.2 ml of sesame oil administered by stomach tube for 7 days. Four chickens from b o t h groups were then given freshly prepared biologically active 125I-PTH (0.2 ml, 6.6 EzCi, 2 izg in 0.01 M HC1 containing 0.5 mg albumin [8, 14]) by wing vein, without anesthesia. Five minutes post-injection the chickens were killed by decapitation. Blood was collected, and samples of liver, kidney, and bone (femurs) were quickly t a k e n and after weighing, fast frozen for subsequent analysis. Based on previous experience [9] the metabolism of the hormone is nearly complete in 60 min. Because the results of the preliminary studies in rats showed only minor differences at 30 rain, it seemed i m p o r t a n t to examine deposition in target organs a t 5 rain post-injection. The most recent extraction procedures [10] were employed. The frozen tissues were homogenized in the gel filtration buffer (3 M guanidine-HC1; 2.3 M formic acid and, after clarification and addition of markers, subjected to gel filtration on P-10 Biogel columns as described previously [8]). Recovery of total radioactivity in the tissues as delivered to the chromatography columns were 95.4 :~- 0.74%, 97.3 • 2.01% and 99.0 ~: 1.76% for the kidney, liver and bone, respectively (mean ~_ S.E.M.).
Results
Preliminary Experiments with Rats T h e e f f e c t s of t h e t h r e e d i e t a r y r e g i m e n s o n t h e r a t s a r e g i v e n i n T a b l e 1. T h e l a c k of v i t a m i n D c a u s e d a m a r k e d i m p a i r m e n t i n g r o w t h w h i c h w a s p a r t i a l l y
Effects of Vitamin D Status on Metabolism of Labeled PTH
291
Table 1. Effects of diets on rats Diet
q- Vit. D
-- Vit. D
+ Ca, -- Vit. D
Body wt. (g) Kidney wt. (% BW) Tibia wt. ( % BW) Serum Ca (mM) Serum Pi (mM)
163 ~ 0.72 • 0.25 • 2.68 ~ 3.81 ~
127 ~ 3.4 a 0.77 • 0.01 0.27 :~ 0.01 1.50 4- 0.5 a 2.84 -4-0.01 a
144 • 8.8 0.73 :~ 0.02 0.28 :~ 0.01 2.93 • 0.06 b 0.84 • 0.01 a
2.2 0.02 0.01 0.04 0.03
Results given as mean ~ S. E.M. (n = 6). a Significantly differs from control value, p < 0.001. b Significantly differs from control value, p < 0.01.
Table 2. Tissue uptake of total radioactivity in rats Diet
-4-Vit. D
-- Vit. D
-- Vit. D, A- Ca
Plasma Kidney cortex Tibia Liver Spleen Intestine Muscle
4.06 • 0.21 18.7 ==0.6 0.97 -4-0.04 3.71 -- 0.14 1.62 ! 0.08 0.82 • 0.03 0.27 -V 0.02
4.51 :t: 0.19 14.0 -4-0.8a 1.02 -4-0.06 3.53 • 0.27 1.47 • 0.12 0.70 • 0.05 0.26 • 0.01
4.67 • 0.21 13.5 • a 1.12 • 0.04 b 4.40 :~ 0.25 1.64 ~_ 0.12 0.77 ~ 0.03 0.28 ~- 0.02
Results given as mean per cent dose cpm/g :L S. E.M. (n = 6). a Significantly different from control p value
