ARCHIVES
OF BIOCHEMISTRY
Vol. 282, No. 2, November
AND
BIOPHYSICS
1, pp. 326-332,199O
Binding Properties of Plasma Vitamin D-Binding Protein and Intestinal 1,25-Dihydroxyvitamin D3 Receptor in Piglets with Pseudo-Vitamin D-Deficiency Rickets, Type I: Treatment Effects with Pharmacological Doses of Vitamin D3 R. Kaune, B. Schroeder, and J. Harmeyer’ Department
of Physiology,
Received February
School of Veterinary
Medicine, D-3000 Hannover
22,1990, and in revised form June 20,199O
The effective treatment of the rachitic symptoms of pseudo-vitamin D-deficiency rickets, type I (PVDRI) by massive doses of vitamin D3 was examined. For this purpose, the affinities and the maximum binding capacities (B,,,) of the plasma vitamin D-binding protein (DBP) and of the intestinal 1,25-dihydroxyvitamin Da (1,25-(OH),D,) receptor for vitamin D3, 25-hydroxyvitamin Da (25-OHD3) and 1,25-(OH)zD3, were investigated in normal piglets and in rachitic piglets that suffered from PVDRI. The piglets were 5 to 10 weeks old and of both sexes. The B,,, of plasma DBP for 25OHD3 was 6.77 f 0.45 PM for PVDRI piglets and 7.30 t 0.4 1 FM for control piglets and showed no differences between the two groups. Equilibrium association constants (IL,) of DBP for 25-OHD3 were 4.3 X 10’ Me1 for PVDRI piglets and 4.0 X 10’ Me1 for controls and showed also no differences between the two groups. Similarly the K, of DBP for 1,25-(OH)zD3 was also the same for rachitic and control piglets (1.45 X 10’ and 1.54 X lo7 M-l, respectively). Due to the lower circulating concentration of 1,25-(OH)2D3 in the plasma of rachitic piglets compared to that of controls its free metabolite index was significantly lower in racbitic (0.42 * 0.05 X 10m5) than in control piglets (3.63 * 0.30 nuclear recepX 10m5). The Kd and B,,, of the intestinal tor for 1,25-(OH)zD3 of rachitic and control piglets were 0.315 0.05 and 0.33 f 0.05 nM and 674 4 103 and 719 + 123 fmol/mg protein, respectively, and were also not different between the two groups of piglets. It was concluded from these observations that the rachitic symptoms of PVDRI piglets resulted solely from the 1 To whom correspondence should be addressed at Department of Physiology, School of Veterinary Medicine, Bischofsholer Damm 15, D-3000 Hannover 1, FRG. 326
1. FRG
lower free 1,25-(OH)aDa concentration in plasma compared to that of normal piglets. The relative affinities of the intestinal 1,25-(OH)zD3 receptor for vitamin D3 and 25-OHDa were also measured. It was found that 50% displacement of 1,25-(OH)zD3 from the intestinal receptor of PVDRI and control piglets required a 220,000and 245,000-fold excess of the free concentration of vitamin Da, respectively, and a 20- to 42- and 23- to 71-fold excess of the free concentration of 25OHDa, respectively. Treatment of PVDRI piglets with an effective dose of 7.5 X lo4 IU of vitamin D3 led to a transient 1073-fold excess of the free concentration of vitamin D3 and a 59-fold excess of the free concentration of 25-OHD3 over free 1,25-(OH)aDa in plasma. Since a 59-fold excess of free 25-OHD3 in plasma of treated piglets could cause at least 50% displacement of 1,25-(OH)aD3 from the intestinal 1,25-(OH)zD3 receptor it is suggested that after administration of pharmacological amounts of vitamin D3 to PVDRI piglets both 25-OHDa and 1,25-(OH)2D3 acted upon the intestinal 1,25-(OH)zD3 receptor. The two metabolites probably exerted a coordinate effect in healing the rachitic symptoms. 0 1990 Academic Press, Inc.
1,25-(OH)zD3, probably the most active metabolite of vitamin DB, is known to influence intestinal and renal handling of calcium and phosphate metabolism of bone (1). In normal subjects production of 1,25-(OH)zD3 from 25%OHD3 occurs mainly in the kidney since a severe disturbance of calcium homeostasis develops in nephrectomized patients and in patients with renal dystrophy (2). A pathogenetic condition similar to nephrectomy is present in pseudo-vitamin D-deficiency rickets, type I 0003.9861/90
$3.00
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
EFFECTS
OF VITAMIN
(PVDRI)” of humans and pigs, a disease in which renal production of 1,25-(OH)zD, is absent (3, 4). Treatment of affected patients with physiological doses of either 1,25-(OH)2D:j or lol-hydroxyvitamin D3 (approximately 1 pg/day) results in prompt healing of the osteomalacic lesions (5). But treatment with pharmacological amounts of 25-OHD3 or vitamin D3 also reverses development of rachitic symptoms (6). The surprising therapeutic effects of 25-OHD3 and vitamin D3 have been attributed to extrarenal formation of 1,25-(OH)zD, (6, 7) and to a direct effect of circulating 25-OHD3 (8). It is of interest to note that effective treatment of PVDRI piglets with physiological amounts of 1,25-(OH)2Dz requires daily administration of the metabolite whereas treatment with pharmacological amounts of vitamin D:, (104-5 X lo4 IU/animal) requires treatment only at 4to 6-week intervals to maintain normocalcemia and to prevent the development of rickets. It has been shown in own previous studies in which PVDRI piglets were treated with single intramuscular injections of 104-5 X lo4 IU of vitamin D3 that the treatment resulted in a transient approximately fourfold increase in plasma 1,25-(OH)2D3 concentration 2 days after treatment (6). The 1,25-(OH)zDCi concentration, however, returned to subnormal values within 3 to 8 days. On the other hand the plasma 25-OHD3 concentration peaked 7 days after treatment and was maintained above 400 nM for at least 3 weeks after treatment. From these observations about the time courses of vitamin D metabolites in plasma in association with the long lasting effects of vitamin D3 treatment, which is also known from human patients suffering from PVDRI, the question arose whether the therapeutic effect of vitamin D3 resulted from the transient increase in plasma 1,25-(OH),D3 alone or whether it could also be caused by a direct effect of vitamin D3 or 25-OHDCi upon the intestinal 1,25-(OH),D3 receptor. The biological activity of 1,25-(OH&D3 and possibly that of other vitamin D metabolites depends on its free concentration rather than on the total concentration in plasma (9). Besides being bound to DBP vitamin Dz and its metabolites in plasma are bound to plasma proteins (e.g., albumin and lipoproteins) (10). The molar concentration of DBP in plasma (approximately lop6 M) is approximately 10 to 50 times higher than that of 25-OHD3 and about 5000 times higher than that of 1,25-(OH)zD3. The free portions of 25-OHD3 and 1,25-(OH)2D3 in plasma are only about 0.03% and l%, respectively, of their total plasma concentrations (11,12). For evaluation of the therapeutic effect of pharmacological doses of vitamin D, we have calculated the free z Abbreviations used: DBP, vitamin D-binding protein; PVDRI, pseudo-vitamin D-deficiency rickets, type I; 2.5.OHDa, 25.hydroxyvitamin D,; 1,25-(OH),D,, 1,25-dihydroxyvitamin D,; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; BSA, bovine serum albumin.
D:, TREATMENT
IN PIGS
327
concentrations of vitamin D3, 25-OHD3, and 1,25(OH)2D3 in plasma after treatment with vitamin D3 from measurements of the relative binding affinities of DBP for these metabolites and from measurements of the relative binding affinities of the intestinal 1,25-(OH)zD, receptor for these metabolites. PVDRI piglets were used for the study. The mode of inheritance, pathogenesis of symptoms, and etiology of the disease in PVDRI piglets are similar to those found in PVDRI humans and have previously been described (4, 13). The results showed that the long lasting therapeutic effect of massive doses of vitamin D, could be exerted by a direct interaction of 25OHD3 with the intestinal 1,25-(OH)zDs receptor. MATERIALS
AND METHODS
Animals. PVDRI piglets, 5 to 10 weeks old and 4 to 9 kg body weight, of both sexes were used. Nonrachitic normal piglets of the same age served as controls. The animals were crossbreeds of German Landrace and Gijttinger Miniature Pig. They were fed after weaning (4 to 6 weeks postpartum) a commercial pig starter diet with 0.9% calcium, 0.65% phosphorus, and 50 pg vitamin D,/kg. Blood samples were taken by venipuncture of the cranial vena cava with NH,heparinate-covered syringes. Plasma was obtained by centrifugation at 35OOg and was stored at -20°C. For repeated blood samplings after treatment, the animals were equipped with catheters placed into the internal jugular veins. Single injections of 7.5 X lo4 IU of vitamin D3 in water soluble form (Sanofi/ Ceva GmbH, Duesseldorf, FRG) were intramuscularly administered to the piglets. Analytical procedures. Concentrations of vitamin D.?, 25-OHDn, and 1,25-(OH)2D, in plasma were determined as previously described (6). In short, vitamin DB and the metabolites were extracted with methanol/methylene chloride (2/l, v/v) and fractionated on columns with Lipidex 5000 (United Technologies, Frankfurt, FRG) with different mixtures of n-hexane/chloroform. The fractions were dried and isolated by high pressure liquid chromatography (HPLC), on normal phase columns with isopropanolln-hexane, and subsequently on reversed phase columns with methanol/water. Vitamin D3 and 25OHD, were quantified from the uv absorbance at 265 nm, and 1,25(OH),Ds was separated with normal phase HPLC and quantified with a radioimmunoassay using an antiserum from sheep (6,14). Plasma calcium was measured as a complex with o-cresolphthaleincomplexone and the activity of alkaline phosphatase (EC 3.1.3.1) by splittingp-nitrophenylphosphate according to the procedure provided by Boehringer (Mannheim, FRG). Protein was determined with Coomassie blue using a kit from Bio-Rad (Munich, FRG) and bovine yglobulin as a standard. Preparation of the nuclear calcitriol receptor. Piglets were killed by stunning and bleeding. About 150 cm of the proximal part of the small intestine (duodenum and proximal jejunum) was immediately removed and rinsed with ice cold saline (0.9% NaCl, w/v). The tissue was opened along the mesenteric border and stored at -70°C. Receptor preparation followed the methods described for chicks by Simpson and DeLuca (15) and Walters et al. (16) and was carried out at 4°C. Thawed intestines were vibrated for 10 min in 3 vol of 50 mM Tris/ HCl, pH 7.4, with 1.5 mM EDTA, 0.2 mM PMSF, and 5 mM DTT (PTED buffer) using a vibro mixer (type El, Chemap AG, Mannedorf, Switzerland). After removal of fibrous material, the cell suspension was centrifuged for 10 min at 15OOg and the pellet was washed two times with 3 vol of PTED. The final pellet was resuspended with 3 vol of PTED and homogenized by three 20-s bursts (20,000 U/min) of an Ultra Turrax homogenizer (Ika, Staufen, Breisgau, FRG). The result-
328
KAUNE,
SCHROEDER,
ing homogenate was centrifuged at 4OOOg for 10 min. The crude nuclear pellet was washed two times by resuspension in 2 vol of PTED buffer. The receptor was extracted from the nuclear pellet by addition of 3 vol of a PTED buffer with 300 mM KC1 (KPTED buffer) and gentle stirring for 60 min. After centrifugation at 40,OOOgfor 60 min aliquots of the supernatant were frozen at -20°C. The nuclear preparation contained a protein concentration of 2.4 to 4.4 mg/ml. Competitive receptor binding assay. Equilibrium dissociation constants (I&) and maximum binding capacities (R,,,) were assayed in triplicate at 4°C. Nuclear extracts were thawed and diluted with cold KPTED containing 1% BSA to a final concentration of 0.25-0.5 mg protein/ml. This yielded 15 to 35% specific radioligand binding. Labeled and unlabeled 1,25-(OH),D, were dissolved in ethanol. Amounts of 250 ~1 of receptor solution with 60 to 125 pg protein were mixed with a constant amount of [26,27-methyl-3H]1,25-(OH),D, (100 to 150 Bq; sp act 6.67 TBq/mmol) and with increasing amounts of unlabeled 1,25-(OH),D3 (ranging from 0 to 1200 fmol) to yield a final volume of 280 ~1. After 18 to 24 h of incubation at 4°C the unbound sterol was removed by addition of 50 ~1 of dextran-coated charcoal (3 g Norit A + 0.3 g dextran T 70 in 100 ml KPTED, equilibrated for at least 2 h at 4°C). The mixture was centrifuged after 15 min at 2500s for 10 min and exactly 10 min later 250 bl of supernatant was transferred into counting vials with 5 ml liquid scintillator (Miniria 20, Zinsser, Frankfurt, FRG) and counted in a Packard Tri Carb liquid scintillation counter. The total radioactivity of the assay solution was determined after addition of KPTED buffer instead of dextran-coated charcoal solution. Specific receptor-ligand binding was calculated after subtraction of nonspecific binding, which was obtained by addition of 300. to 500. fold excesses of unlabeled hormone. Scatchard analysis was used to calculate Kd and B,,, Competitive protein binding assay of plasma. Binding capacities and affinities for vitamin D metabolites in plasma were measured with a competitive protein-binding assay according to the method of Bothe et al. (17). The procedure was similar to that used for measurement of receptor binding except that diluted plasma (1:8,000-1:16,000) was used instead of diluted receptor homogenate and that [26,27-methyl“H]25-OHDj (sp act 762 GBq/mmol) was used instead of labeled 1,25(OH)*Da. KPTED buffer was replaced by 60 mM sodium phosphate buffer, pH 7.4, and BSA by 2 g gelatine + 400 ~1 Brij-35 (30%) in 1 liter of buffer. 25.OHD, was added in amounts of 0 to 25 pmol/tube. B mali and Kd for 25.OHDa were calculated from Scatchard analysis. The Kd of 1,25-(OH)2D3 or vitamin D, binding were estimated from 50% inhibition (I&, values) of the [aH]25-OHD, binding using the Cheng and Prusoff formula (18):
Kd =
IGO 1 + concentration of [3H]25-OHD3 Kd of 25-OHDB
The ICsO was obtained from the linear regression of: logit (P) = log [P/(100-P)] against log C, where P represents the percentage binding of [3H]25-OHDs (binding in the absence of 1,25-(OH)zD3 and vitamin D, = 100%) and C represents the concentration of 1,25-(OH)zD3 or vitamin D,. The association constants (KJ were obtained as reciprocals of the equilibrium dissociation constants (K, = K;‘). of DBP DBP and free metabolite index. The plasma concentration was calculated from the B,,, for 25.OHD, in diluted plasma (19). The free metabolite index defined as the molar ratio of the metabolite and DBP (20) was used to estimate the changes of free metabolite concentrations in the course of vitamin Da treatment. By definition the free metabolite index appeared to be a suitable measure to express changes of free metabolite concentrations under different conditions. The index cannot, however, be used to compare different metabolites with different affinities for DBP. For comparison of different metabolites
AND
HARMEYER TABLE
Binding
Properties
I
of Plasma Vitamin D-Binding Protein 1,25-(0H)sDe Receptor from PVDRI and Control Piglets (means +- SEM)
(DBP) and Intestinal (Rachitic)
Control piglets Calcium (mM) Alkaline phosphatase (U/liter) Total vitamin Da (nM) Total 25.OHD3 fnM) Total 1,25-(OH)*Da (PM) DBP (PM) K. for 25.OHDa (Mm’) K, for 1,25-(OH)?D3 (M-l) Free index vitamin Dg (X103) Free index 25.OHD3 (X103) Free index 1,25-(OH),Da (X105) B,,, intestinal receptor [fmol/mg protein] Kd intestinal receptor (nM)
2.81 425 15.29 39.25 259.60 7.30 4.0 1.54 2.16 5.48 3.63
+ f f + k * x x + -t +
0.03 50 2.04 11.74 44.13 0.41 IO8 lo7 0.34 0.48 0.30
719 k 123 0.33 + 0.05
PVDRI piglets 1.33 * 0.05* 1302 + 147* 13.66 + 2.91 38.00 t 4.79 27.75 k 4.49* 6.77 rt 0.45 4.3 x lo8 1.45 x 10’ 2.04 k 0.36 5.77 f 0.74 0.42 f 0.05* 674 ? 103 0.31 t 0.05
Note. The plasma values are means of six samples each from six control and two rachitic piglets. The receptor data are means of nine control and nine rachitic piglets. DBP values for 25.OHD, and 1,25(OH)*D3 are means of three control and two rachitic piglets. *P < 0.01. the total concentrations of 25.OHDa and 1,25-(OH),Da, the DBP concentration in plasma and the K,, values of the two metabolites were used according to the formula of Bouillon et al. (21): [“free”]
=
[total] K,. [DBP]
This method, however, also does not give true values of the free plasma concentrations of the metabolites, since binding to albumin is not taken into account,. But it can be assumed that the greatest portion of the metabolites is bound to DBP and that the ratio of binding of both metabolites to DBP and to albumin is similar (9). Therefore an estimate of the proportion of free 25-OHDa and free 1,25-(OH)zD, can be obtained by this method. Chemicals. Analytical solvents were purchased from Merck (Darmstadt, FRG) and were of “chromatographical grade.” The solvents used for extraction and fractionation were of quality grade “for residue analysis.” Vitamin D, was from Serva (Heidelberg, FRG) and Vitamin D3 metabolites were kindly provided by Hoffmann La Roche (Basel, Switzerland). Labeled metabolites were from Amersham Buchler (Braunschweig, FRG). All metabolites were routinely purified and controlled by HPLC. BSA, Brij-35, Norit A and DTT were from Serva, PMSF from Sigma (Deisenhofen, FRG), and dextran T 70 from Pharmacia (Uppsala, Sweden). The other chemicals were at least of analytical grade.
RESULTS
Binding of vitamin D3 and vitamin D metabolites to DBP. The concentrations of calcium and 1,25(OH)*Ds in plasma of rachitic piglets were significantly lower than those of control piglets. The activities of alkaline phosphatase in plasma were significantly higher. The concentrations of vitamin D, and 25-OHD3 were not affected by the PVDRI condition (Table I). Scatch-
EFFECTS Control
piglet
OF VITAMIN
Rachltic
Ds TREATMENT
329
IN PIGS
piglet
30
.25 n .20 L 2
.:5 I
.lO -
.05 r
.l
2
.3
.4
5
85 IllHI
.6
.7
OL 0
.I
.2
3
.4
3
.6
85 inH1
FIG. 1. Scatchard plots of 25.OHD, binding to DBP of diluted plasma (1:8600) from a rachitic and a control piglet. The DBP content in plasma was 7.9 FM for the control and 5.7 pM for the rachitic piglet. Means of R,,, and K, are in Table I.
FIG. 2. Typical binding curves of vitamin Ds and vitamin olites by the intestinal 1,25-(OH),Ds receptor. Fifty percent hound to the receptor (control ment of [3H]1,25-(OH)2D3 quired a 44.fold excess of 25OHD3 and a 245,000-fold vitamin Ds. n , 1,25-(OH)2D3; 0, 25.OHDs; 0, vitamin D,.
ard analyses of specific 25-OHD3 binding to DBP at different ligand concentrations yielded a linear relationship with similar KG’s of about 4.0 X lOR M-’ for both rachitic and control piglets (Fig. 1, Table I). The K, of DBP for 1,25-(OH),D3 was also unaffected by the PVDRI condition (Table I). The DBP levels in plasma of rachitic piglets tended to be smaller than those of control piglets with the difference not being statistically significant. The free metabolite indices for vitamin D3 and 25-OHD, were the same for both groups of piglets. The free metabolite index of 1,25-(OH)zD3 of rachitic piglets, however, was considerably lower than that of control piglets due to the significantly lower total 1,25-(OH)zD:j concentration in plasma of PVDRI piglets (Table I).
Specificity of ligand binding to plasma DBP. The aflinities of plasma DBP for vitamin D3 and 1,25(OH)*Dc3 were not different between rachitic and control piglets. In control plasma 50% displacement of [3H]25OHD3 bound to DBP occurred with a 43-fold excess of vitamin D3 and with a 78-fold excess of 1,25-(OH),D3 (Fig. 3). From the 50% competition concentrations (I&) K,‘s for DBP of 1.70 X lo7 M-’ (vitamin Dg, Fig. 3) and of 1.54 X lo7 M-’ (1,25-(OH)2D3, Table I) were calculated. Experiments with PVDRI plasma yielded 50% displacement of [3H]25-OHD3 bound to DBP with 39-fold excess of vitamin D3 (K, = 1.80 X lo7 M-r) and with 56-fold excess of 1,25-(OH)zD3 (K, = 1.45 X lo7 Mm ‘, Table I).
Specificity of ligand binding to the intestinal 1,25(OH),D, receptor. The capacities (B,,,) and affinities (&) of the intestinal nuclear receptor for 1,25-(OH)zD3 were measured in animals different from those used for the DBP studies. B,,, and Kd were not different between rachitic and control piglets (Table I). The mean calcium concentrations and the activities of alkaline phosphatase in plasma of these animals were 3.0 k 0.1 mM and 406 + 36 U/liter for controls and 2.1 + 0.2 mM and 2098 * 187 U/liter for the PVDRI piglets, respectively. Total 1,25-(OH),D:, concentrations in plasma were 480 +- 139 pM for the control piglets and 101 + 14 pM for the rachitic piglets. In experiments with control piglets 50% displacement of [3H]1,25-(OH)2Dzi bound to the receptor by 25-OHD3 or vitamin D3 occurred with a 23- to 71-fold or >245,000-fold excess, respectively (IL = 3, Fig. 2). In experiments with PVDRI piglets 50% displacement of [3H]1,25-(OH)zD3 bound to the receptor by 25-OHD3 or vitamin D3 was obtained with a 20- to 42-fold or >220,000-fold excess, respectively (n = 2).
kJ
a
-10
100
1000 Compound
10000 [pg/tubej
100000
D metabdisplacepiglet) reexcess of
1000000
FIG. 3. Typical binding curves of vitamin D3 and vitamin D metabelites to DBP in diluted plasma. Fifty percent displacement of [sH]25OHDs bound to DBP (control plasma) required a 43-fold excess of vitamin D:, and a 7%fold excess of 1,25-(OH)2D3. a, 1,25-(OH),D:,; 0, 25-OHD,; 0, vitamin D3.
KAUNE, 5-
A
o! EOO-
0
SCHROEDER,
i
600;
. 1 .25-(OH);D3 /
r” IOOl7l 0 E 0
(
-2
-1
0
1
2
3
4
5
6
DOYS FIG. 4. Concentrations of calcium (A), vitamin D3 and 25.OHD3 (B), and 1,25-(OH)2D3 (C) of two PVDRI piglets from 2 days before to 6 days after im treatment with 7.5 X lo4 IU of vitamin D,.
Concentrations of vitamin D3 and metabotites in PVDRI plasma after treatment with pharmacological doses of vitamin D3. Administration of 7.5 X lo4 IU of vitamin D3 to rachitic piglets led to a 50% increase in the plasma calcium concentration about 3 days after treatment with persisting high values for 7 days after treatment (Fig. 4A). The activities of the alkaline phosphatase in plasma gradually declined after the vitamin D3 treatment (data not shown). In addition, the piglets showed healing of clinical symptoms of rickets such as disappearance of muscle weakness of bone and joint pain and resumed appetite. The plasma concentration of vitamin D3 transiently rose after treatment from 34-40 to
AND
HARMEYER
300-500 nM and returned to normal values 5 days after treatment (Fig. 4B). The level of 25-OHD, rose from 5070 to about 700 nM and remained at this high concentration until the end of the experimental period at 6 days postinjection (Fig. 4B). The time course of 1,25-(OH)zD3 paralleled that of 25-OHD3. Its concentration rose from subnormal values (20-70 PM) to supranormal values (215-400 PM) and stayed high for at least 1 week (Fig. 4C). Interaction of uitamin D3 and 25-OHD, with the intestinal receptor after treatment with vitamin D3. With the results obtained from the above measurements the following calculations were carried out. With the given DBP concentrations, the free metabolite indices of vitamin DX, 25OHD3, and 1,25-(OH)zD3 of rachitic piglets before and after treatment with vitamin D, were calculated (Table II). It was found that the significant rise of 1,25-(OH)zD3 in plasma resulted in a maximal free metabolite index in PVDRI piglets greater than that present in untreated control piglets (Tables I and II). Comparing the fractional rises of the free metabolite indices of vitamin D3, 25OHD3 and ~,~EJ-(OH)~D~ after treatment with vitamin D3 it was found that these were highest for vitamin D, and lowest for 1,25-(OH)2D3 (Table II). From the DBP concentration in plasma and from the measured affinities of DBP it was calculated that the “fractional free concentrations” of vitamin D,, 25 OHD3, and 1,25-(OH)aD3 in plasma after treatment with vitamin D3 were 0.87, 0.034, and 1.018% for vitamin D8, 25-OHD3, and 1,25-(OH)2D3, respectively. Unspecific binding of the metabolites of about 10 to 20% to albumin (12) was ignored. This corresponded to maximal “free concentrations” of the three compounds in plasma of 4.4 nM, 0.24 nM, and 4.1 pM, respectively. The values demonstrate that the “free concentration” of vitamin DS was 1073-fold in excess of free 1,25-(OH)2D, in plasma and that the free 25-OHD3 concentration was 59-fold in excess of free 1,25-(OH),D3. Comparing these ratios with those obtained for 50% displacement of 1,25-(OH),D, from the intestinal receptor (see above) it can be seen that the free 25OHD3 concentration in plasma allowed more than 50% displacement of 1,25-(OH)zD3 from the receptor. DISCUSSION The results reported in this study demonstrate that the therapeutic effect of a pharmacological amount of vitamin D, in healing the symptoms of PVDRI can be attributed not only to the transient rise of the plasma 1,25-(OH)aD3 concentration but also to a sustained increase in the concentration of 25-OHD, in plasma. In the experiments reported here the 1,25-(OH)2D3 concentration in plasma remained high for at least 6 days (Fig. 4C). This was much longer than had been observed in a previous study (6) where the 1,25-(OH),D3 concentra-
EFFECTS
OF VITAMIN
D, TREATMENT TABLE
Free
331
IN PIGS
II
Metabolite Indices of Vitamin D3 and Its Metabolites of Two PVDRI Piglets before and after Treatment with 7.5 X lo4 IU of Vitamin D, (Maximum Values) Piglet 2
Piglet I
Basal DBP (fiiv) Free vitamin D, Index (X10”) Free 25-OHD:, Index (Xl@) Free 1,25-(OH)1D:, Index (X105)
Treatment
Basal treatment
9.52 0.46
Treatment
Basal treatment
6.88
6.65 2.91
Basal
48.9 108 4.40
tion in plasma already declined to subnormal concentrations 3 to 8 days after treatment. The reason for the difference in the time course of plasma ~,~EY-(OH)~D, is probably the higher dose of vitamin D3 which was used in the present study (average of 3.5 X lo4 IU vs 7.5 X lo4 IU). But with the lower vitamin D3 dose used in the previous study the therapeutic effect also lasted for at least 4 weeks. It is of interest to note that 1,25-(OH)zD, concentrations in plasma of the rachitic piglets are sometimes near physiological concentrations (100 PM). Nevertheless rachitic symptoms developed. Near physiologic 1,25(OH),D:, concentrations in plasma have also been reported from PVDRI-affected children and young persons (22, 23). Since no differences in binding affinities and capacities of plasma DBP and intestinal 1,25(OH)zD3 receptor between control and rachitic piglets could be identified there is no indication to assume that factors other than defective production of 1,25-(OH),D3 contributed to the development of rachitic symptoms. This view is supported by the observation that physiological amounts of 1,25-(OH)zDx can heal the rachitic lesions (5). The suggestion that the binding properties of intestinal 1,25-(OH),D3 receptor and plasma DPB might have been altered by changes in 1,25-(OH),D3 metabolism proved to be wrong. This agrees with similar observations made by Hunziker et al. (24) with chicks. The authors also found no effect of vitamin D depletion or repletion upon the properties of the intestinal 1,25(OH),D3 receptor. For the binding properties of DBP similar findings have been reported by Rojanasathit and Haddad (25) who worked with vitamin D-deprived rats. In that study B,,, and K, of plasma DBP were unaffected by withdrawal of dietary vitamin D. The concentrations and affinities of DBP for vitamin D metabolites which we found in our study were in the same range than those reported for men, rats, and chimpanzees (12, 26). Kd values of the intestinal 1,25-
16.8
3.72
11.3
7.44
9.6
0.68
74.7 105 5.94
20.1 14.1 8.7
(OH)zD3 receptor of PVDRI and normal piglets agreed with those reported for rabbits, rats, chicks, and men (27-30). The B,,, values of the piglets were slightly higher than those reported from other species. This might have been caused by more complete extraction, or better stability of the porcine receptor during incubation or by its higher concentration in the mucosal tissue compared to those of other species. Clayton et al. (31) found a higher affinity and a lower capacity of the porcine 1,25(OH)zD3 receptor than is reported here. These authors incubated the receptor at 23°C rather than at 4°C and the Scatchard plot was not linear, indicating more than one binding site. Displacement studies which have been carried out with intestinal receptor preparations or with DBP from pigs and other species using vitamin D3, 25-OHD3, and 1,25-(OH)zD3 yielded the same sequence of binding as that reported here (27, 29, 31-33), except that in our study binding of 1,25-(OH),D3 and vitamin D3 to DBP was not significantly different. The relatively large affinity of 25-OHD, to the porcine intestinal 1,25(OH)2D3 receptor probably also provides an explanation for reported observations that intestinal calcium absorption could be induced by high concentrations of 25OHD3 in nephrectomized rats (34). Vitamin D3 and its metabolites in plasma are not exclusively bound to DBP. In human plasma 85% of 1,25(OH)2D3 and 88% of 25-OHD3 were found to be bound to DBP. Only 1 and 0.03%, respectively, of these metabolites are reported to be free. The remaining portions were bound to albumin (11, 12, 35). If partition of the compounds in pig’s plasma is similar to that reported for human plasma, the “free concentration” of 1,25(OH)zD3 has been overestimated by about 30% in our experiments and that of 25-OHD3 by about 10%. This would mean that the degree of interaction of 25-OHD, with 1,25-(OH),D3 at the intestinal receptor level after vitamin D, treatment was probably greater than documented by the data. In any case, the results which are
332
KAUNE,
SCHROEDER,
presented in this study reveal that the treatment effect of pharmacological doses of vitamin D, might be brought about by a combined action of 25OHD, and 1,25(OH)2D3 upon the intestinal 1,25-(OH),D3 receptor. On the other hand the treatment effect of vitamin D, appears not to be an effect of 25OHD3 alone as has been suggested earlier by DeLuca (8). The time course of 1,25-(OH)ZD3 in plasma of vitamin D3-treated PVDRI piglets disclosed the presence of highly active extrarenal 1-hydroxylases. The assumption that the rise of 1,25-(OH)zD3 in plasma after treatment came from extrarenal sources is supported by the observation that the conversion of 25-OHD3 to 1,25(OH)zD3 by renal cortex homogenates of rachitic piglets was completely absent even in the presence of 25-OHD, concentrations similar to those present in the plasma of vitamin D,-treated piglets (13). In addition, significant increases in 1,25-(OH),D3 in plasma after treatment with massive doses of vitamin D, have also been observed in normal nephrectomized pigs (36, 37). Finally, since in PVDRI piglets cellular expression of the renal vitamin D 1-hydroxylases is inhibited, it appears unlikely that the vitamin D-dependent enzyme is present in other organs or tissues. Our studies, however, provide no information about how 1,25-(OH)zD3 production is controlled and how extrarenal 1-hydroxylases operate. In PVDRI piglets 1,25-(OH)2D3 is probably formed after administration of massive doses of vitamin D, and, when non-vitamin Ds-treated, by one or more unspecific steroid-I-hydroxylases. Such unspecific 1-hydroxylases probably possess different kinetics compared to the vitamin D-dependent 1-hydroxylase and it is likely that they also contribute to 1,25-(OH),D3 in normal subjects after administration of pharmacological amounts of vitamin D. Among other tissues extrarenal production of 1,25(OH)2D3 has been found in immune cells such as macrophages (38) whose concentration may vary with the immune status of the individual. Therefore, it might be tempting to speculate whether the toxicity of high doses of vitamin D also varies with the immune status. REFERENCES 1. Norman,
A. W. (1987) J. N&r.
2. Gray, R., Boyle, I., and DeLuca, 1234.
117,797-807. H. F. (1971) Science 172, 12322
3. Silver, J., Landau, H., Bab, I., Shvil, Y., Friedlaender, M. M., Rubinger, D., and Popovtzer, M. M. (1985) Isr. J. Med. Sci. 21, 5356. 4. Harmeyer, J., Grabe, C. V., and Winkler, I. (1982) Exp. Biol. Med. 7,117-125. 5. Harmeyer, J., and Kaune, R. (1990) Advances in Animal Breeding and Genetics, Vol. 5, pp. 111-130, Paul Parey, Hamburg/Berlin. 6. Kaune, R., and Harmeyer,
J. (1987) Acta Endocrinol.
(Copenha-
gen)115,345-352. 7. Dusso, A., Lopez-Hilker, Kidney Int. 34,368-375.
S., Rapp, N., and Slatopolsky,
E. (1988)
AND
HARMEYER
8. DeLuca, H. F. (1988) FASEB J. 2,224-236. 9. Bikle, D. D., and Gee, E. (1988) in Vitamin D. Molecular, Cellular and Clinical Endocrinology, pp. 7033709, Walter de Gruyter, Berlin. 10. Haddad, J. G. (1987) in Bone and Mineral Research V, pp. 281~ 308, Elsevier, Amsterdam/New York/Oxford. 11. Bouillon, 451-453.
R., and Van Baelen,
H. (1981) Calcif. Tissue Int. 33,
12. Bikle, D. D., Siiteri, P. K., Ryzen, E., Haddad, J. G., and Gee, E. (1985) J. Clin. Endocrinol. Metab. 61,969-975. 13. Winkler, I., Schreiner, F., and Harmeyer, J. (1986) C&if. Tissue Int. 38,87-94. 14. Clemens, T. L., Hendy, G. N., Papapoulos, S. E., Fraher, L. J., Care, A. D., and O’Riordan, (1979) Clin. Endocrinol. 11,225-234. 15. Simpson, R. U., and DeLuca, H. F. (1982) Proc. Natl. Acad. Sci. USA 79, 16-20. 16. Walters, M. R., Hunziker, W., and Norman, A. W. (1980) J. Biol. Chem. 255,6799-6805. 17. Bothe, V., Schmidt-Gayk, H., Armbruster, F.-P., and Mayer, E. (1984) Arztl. Lab. 30, 151-156. 18. Cheng, Y.-C., and Prusoff, W. H. (1973) Biochem. Pharmacol. 22, 3099-3108. 19. Woloszczuk, W. (1985) Clin. Chem. Acta 145,27-35. 20. Bouillon, R., Van Asche, F. A., and Van Baelen, H. (1981) J. Clin. Inuest. 67,589-596. 21. Bouillon, R., Van Baelen, H., and De Moor, P. (1977) J. Clin. Endocrinol. Metab. 45, 679-684. 22. Kruse, K. (1985) Monatsschr. Kinderheilkd. 133,346-352. 23. Balsan, S., Garabedian, M., Lieberherr, M., Gueris, J., and A. Ulmann (1979) Vitamin D. Basic Research and its Clinical Application, pp. 114331149, Walter de Gruyter, Berlin, New York. 24. Hunziker, W., Walters, M. R., Bishop, J. E., and Norman, A. W. (1982) J. Clin. Inuest. 69,826-834. 25. Rojanasathit, S., and Haddad, J. G. (1977) Endocrinology 100, 6422647. 26. Bouillon, R., Van Baelen, H., and De Moor, P. (1980) J. Steroid Biochem. 13,1029-1034. 27. Duncan, W. E., AW, T. C., Walsh, P. G., and Haddad, J. G. (1983) Anal. Biochem. 132,209-214. 28. Yeh, J. K., Aloia, J. F.. Vasvani, A. N., and Semla, H. (1986) Bone 7,49-53. 29. Wecksler, W. R., Mason, R. S., and Norman, A. W. (1979) J. C/in. Endocrinol. Metab. 48, 715-717. 30. Nakada, M., and DeLuca, H. F. (1985) Arch. Biochem. Biophys. 238,129-134. 31. Clayton, J., Guilland-Cumming, D. F., Kanis, J. A., and Russel, R. G. G. (1982) in Vitamin D. Chemical, Biochemical and Clinical Endocrinology of Calcium Metabolism. pp. 821-823, Walter de Gruyter, Berlin. 32. Link, R. P., and DeLuca, H. F. (1988) Steroids 41,583~598. 33. Brumbaugh, P. F., and Haussler, M. R. (1974) J. Biol. Chem. 249, 1251-1257. 34. Boyle, I. T., Miravet, L., Gray, R. W., Holick, M. F., and DeLuca, H. F. (1972) Endocrinology 90,605-608. 35. Bikle, D. D., Gee, E., Halloran, B., Kowalski, M. A., Ryzen, E., and Haddad, J. G. (1986) J. Clin. Endocrinol. Metab. 63,954-959. 36. Littledike, E. T., and Horst, R. L. (1982) Endocrinology 111, 2008-2013. 37. Horst, R. L., Littledike, E. T., Gray, R. W., and Napoli, J. L. (1981) J. Clin. Invest. 67, 274-280. 38. Rigby, W. F. C. (1988) Zmmunol. Today 9,54-58.
OF BIOCHEMISTRY
Vol. 282, No. 2, November
AND
BIOPHYSICS
1, pp. 326-332,199O
Binding Properties of Plasma Vitamin D-Binding Protein and Intestinal 1,25-Dihydroxyvitamin D3 Receptor in Piglets with Pseudo-Vitamin D-Deficiency Rickets, Type I: Treatment Effects with Pharmacological Doses of Vitamin D3 R. Kaune, B. Schroeder, and J. Harmeyer’ Department
of Physiology,
Received February
School of Veterinary
Medicine, D-3000 Hannover
22,1990, and in revised form June 20,199O
The effective treatment of the rachitic symptoms of pseudo-vitamin D-deficiency rickets, type I (PVDRI) by massive doses of vitamin D3 was examined. For this purpose, the affinities and the maximum binding capacities (B,,,) of the plasma vitamin D-binding protein (DBP) and of the intestinal 1,25-dihydroxyvitamin Da (1,25-(OH),D,) receptor for vitamin D3, 25-hydroxyvitamin Da (25-OHD3) and 1,25-(OH)zD3, were investigated in normal piglets and in rachitic piglets that suffered from PVDRI. The piglets were 5 to 10 weeks old and of both sexes. The B,,, of plasma DBP for 25OHD3 was 6.77 f 0.45 PM for PVDRI piglets and 7.30 t 0.4 1 FM for control piglets and showed no differences between the two groups. Equilibrium association constants (IL,) of DBP for 25-OHD3 were 4.3 X 10’ Me1 for PVDRI piglets and 4.0 X 10’ Me1 for controls and showed also no differences between the two groups. Similarly the K, of DBP for 1,25-(OH)zD3 was also the same for rachitic and control piglets (1.45 X 10’ and 1.54 X lo7 M-l, respectively). Due to the lower circulating concentration of 1,25-(OH)2D3 in the plasma of rachitic piglets compared to that of controls its free metabolite index was significantly lower in racbitic (0.42 * 0.05 X 10m5) than in control piglets (3.63 * 0.30 nuclear recepX 10m5). The Kd and B,,, of the intestinal tor for 1,25-(OH)zD3 of rachitic and control piglets were 0.315 0.05 and 0.33 f 0.05 nM and 674 4 103 and 719 + 123 fmol/mg protein, respectively, and were also not different between the two groups of piglets. It was concluded from these observations that the rachitic symptoms of PVDRI piglets resulted solely from the 1 To whom correspondence should be addressed at Department of Physiology, School of Veterinary Medicine, Bischofsholer Damm 15, D-3000 Hannover 1, FRG. 326
1. FRG
lower free 1,25-(OH)aDa concentration in plasma compared to that of normal piglets. The relative affinities of the intestinal 1,25-(OH)zD3 receptor for vitamin D3 and 25-OHDa were also measured. It was found that 50% displacement of 1,25-(OH)zD3 from the intestinal receptor of PVDRI and control piglets required a 220,000and 245,000-fold excess of the free concentration of vitamin Da, respectively, and a 20- to 42- and 23- to 71-fold excess of the free concentration of 25OHDa, respectively. Treatment of PVDRI piglets with an effective dose of 7.5 X lo4 IU of vitamin D3 led to a transient 1073-fold excess of the free concentration of vitamin D3 and a 59-fold excess of the free concentration of 25-OHD3 over free 1,25-(OH)aDa in plasma. Since a 59-fold excess of free 25-OHD3 in plasma of treated piglets could cause at least 50% displacement of 1,25-(OH)aD3 from the intestinal 1,25-(OH)zD3 receptor it is suggested that after administration of pharmacological amounts of vitamin D3 to PVDRI piglets both 25-OHDa and 1,25-(OH)2D3 acted upon the intestinal 1,25-(OH)zD3 receptor. The two metabolites probably exerted a coordinate effect in healing the rachitic symptoms. 0 1990 Academic Press, Inc.
1,25-(OH)zD3, probably the most active metabolite of vitamin DB, is known to influence intestinal and renal handling of calcium and phosphate metabolism of bone (1). In normal subjects production of 1,25-(OH)zD3 from 25%OHD3 occurs mainly in the kidney since a severe disturbance of calcium homeostasis develops in nephrectomized patients and in patients with renal dystrophy (2). A pathogenetic condition similar to nephrectomy is present in pseudo-vitamin D-deficiency rickets, type I 0003.9861/90
$3.00
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
EFFECTS
OF VITAMIN
(PVDRI)” of humans and pigs, a disease in which renal production of 1,25-(OH)zD, is absent (3, 4). Treatment of affected patients with physiological doses of either 1,25-(OH)2D:j or lol-hydroxyvitamin D3 (approximately 1 pg/day) results in prompt healing of the osteomalacic lesions (5). But treatment with pharmacological amounts of 25-OHD3 or vitamin D3 also reverses development of rachitic symptoms (6). The surprising therapeutic effects of 25-OHD3 and vitamin D3 have been attributed to extrarenal formation of 1,25-(OH)zD, (6, 7) and to a direct effect of circulating 25-OHD3 (8). It is of interest to note that effective treatment of PVDRI piglets with physiological amounts of 1,25-(OH)2Dz requires daily administration of the metabolite whereas treatment with pharmacological amounts of vitamin D:, (104-5 X lo4 IU/animal) requires treatment only at 4to 6-week intervals to maintain normocalcemia and to prevent the development of rickets. It has been shown in own previous studies in which PVDRI piglets were treated with single intramuscular injections of 104-5 X lo4 IU of vitamin D3 that the treatment resulted in a transient approximately fourfold increase in plasma 1,25-(OH)2D3 concentration 2 days after treatment (6). The 1,25-(OH)zDCi concentration, however, returned to subnormal values within 3 to 8 days. On the other hand the plasma 25-OHD3 concentration peaked 7 days after treatment and was maintained above 400 nM for at least 3 weeks after treatment. From these observations about the time courses of vitamin D metabolites in plasma in association with the long lasting effects of vitamin D3 treatment, which is also known from human patients suffering from PVDRI, the question arose whether the therapeutic effect of vitamin D3 resulted from the transient increase in plasma 1,25-(OH),D3 alone or whether it could also be caused by a direct effect of vitamin D3 or 25-OHDCi upon the intestinal 1,25-(OH),D3 receptor. The biological activity of 1,25-(OH&D3 and possibly that of other vitamin D metabolites depends on its free concentration rather than on the total concentration in plasma (9). Besides being bound to DBP vitamin Dz and its metabolites in plasma are bound to plasma proteins (e.g., albumin and lipoproteins) (10). The molar concentration of DBP in plasma (approximately lop6 M) is approximately 10 to 50 times higher than that of 25-OHD3 and about 5000 times higher than that of 1,25-(OH)zD3. The free portions of 25-OHD3 and 1,25-(OH)2D3 in plasma are only about 0.03% and l%, respectively, of their total plasma concentrations (11,12). For evaluation of the therapeutic effect of pharmacological doses of vitamin D, we have calculated the free z Abbreviations used: DBP, vitamin D-binding protein; PVDRI, pseudo-vitamin D-deficiency rickets, type I; 2.5.OHDa, 25.hydroxyvitamin D,; 1,25-(OH),D,, 1,25-dihydroxyvitamin D,; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; BSA, bovine serum albumin.
D:, TREATMENT
IN PIGS
327
concentrations of vitamin D3, 25-OHD3, and 1,25(OH)2D3 in plasma after treatment with vitamin D3 from measurements of the relative binding affinities of DBP for these metabolites and from measurements of the relative binding affinities of the intestinal 1,25-(OH)zD, receptor for these metabolites. PVDRI piglets were used for the study. The mode of inheritance, pathogenesis of symptoms, and etiology of the disease in PVDRI piglets are similar to those found in PVDRI humans and have previously been described (4, 13). The results showed that the long lasting therapeutic effect of massive doses of vitamin D, could be exerted by a direct interaction of 25OHD3 with the intestinal 1,25-(OH)zDs receptor. MATERIALS
AND METHODS
Animals. PVDRI piglets, 5 to 10 weeks old and 4 to 9 kg body weight, of both sexes were used. Nonrachitic normal piglets of the same age served as controls. The animals were crossbreeds of German Landrace and Gijttinger Miniature Pig. They were fed after weaning (4 to 6 weeks postpartum) a commercial pig starter diet with 0.9% calcium, 0.65% phosphorus, and 50 pg vitamin D,/kg. Blood samples were taken by venipuncture of the cranial vena cava with NH,heparinate-covered syringes. Plasma was obtained by centrifugation at 35OOg and was stored at -20°C. For repeated blood samplings after treatment, the animals were equipped with catheters placed into the internal jugular veins. Single injections of 7.5 X lo4 IU of vitamin D3 in water soluble form (Sanofi/ Ceva GmbH, Duesseldorf, FRG) were intramuscularly administered to the piglets. Analytical procedures. Concentrations of vitamin D.?, 25-OHDn, and 1,25-(OH)2D, in plasma were determined as previously described (6). In short, vitamin DB and the metabolites were extracted with methanol/methylene chloride (2/l, v/v) and fractionated on columns with Lipidex 5000 (United Technologies, Frankfurt, FRG) with different mixtures of n-hexane/chloroform. The fractions were dried and isolated by high pressure liquid chromatography (HPLC), on normal phase columns with isopropanolln-hexane, and subsequently on reversed phase columns with methanol/water. Vitamin D3 and 25OHD, were quantified from the uv absorbance at 265 nm, and 1,25(OH),Ds was separated with normal phase HPLC and quantified with a radioimmunoassay using an antiserum from sheep (6,14). Plasma calcium was measured as a complex with o-cresolphthaleincomplexone and the activity of alkaline phosphatase (EC 3.1.3.1) by splittingp-nitrophenylphosphate according to the procedure provided by Boehringer (Mannheim, FRG). Protein was determined with Coomassie blue using a kit from Bio-Rad (Munich, FRG) and bovine yglobulin as a standard. Preparation of the nuclear calcitriol receptor. Piglets were killed by stunning and bleeding. About 150 cm of the proximal part of the small intestine (duodenum and proximal jejunum) was immediately removed and rinsed with ice cold saline (0.9% NaCl, w/v). The tissue was opened along the mesenteric border and stored at -70°C. Receptor preparation followed the methods described for chicks by Simpson and DeLuca (15) and Walters et al. (16) and was carried out at 4°C. Thawed intestines were vibrated for 10 min in 3 vol of 50 mM Tris/ HCl, pH 7.4, with 1.5 mM EDTA, 0.2 mM PMSF, and 5 mM DTT (PTED buffer) using a vibro mixer (type El, Chemap AG, Mannedorf, Switzerland). After removal of fibrous material, the cell suspension was centrifuged for 10 min at 15OOg and the pellet was washed two times with 3 vol of PTED. The final pellet was resuspended with 3 vol of PTED and homogenized by three 20-s bursts (20,000 U/min) of an Ultra Turrax homogenizer (Ika, Staufen, Breisgau, FRG). The result-
328
KAUNE,
SCHROEDER,
ing homogenate was centrifuged at 4OOOg for 10 min. The crude nuclear pellet was washed two times by resuspension in 2 vol of PTED buffer. The receptor was extracted from the nuclear pellet by addition of 3 vol of a PTED buffer with 300 mM KC1 (KPTED buffer) and gentle stirring for 60 min. After centrifugation at 40,OOOgfor 60 min aliquots of the supernatant were frozen at -20°C. The nuclear preparation contained a protein concentration of 2.4 to 4.4 mg/ml. Competitive receptor binding assay. Equilibrium dissociation constants (I&) and maximum binding capacities (R,,,) were assayed in triplicate at 4°C. Nuclear extracts were thawed and diluted with cold KPTED containing 1% BSA to a final concentration of 0.25-0.5 mg protein/ml. This yielded 15 to 35% specific radioligand binding. Labeled and unlabeled 1,25-(OH),D, were dissolved in ethanol. Amounts of 250 ~1 of receptor solution with 60 to 125 pg protein were mixed with a constant amount of [26,27-methyl-3H]1,25-(OH),D, (100 to 150 Bq; sp act 6.67 TBq/mmol) and with increasing amounts of unlabeled 1,25-(OH),D3 (ranging from 0 to 1200 fmol) to yield a final volume of 280 ~1. After 18 to 24 h of incubation at 4°C the unbound sterol was removed by addition of 50 ~1 of dextran-coated charcoal (3 g Norit A + 0.3 g dextran T 70 in 100 ml KPTED, equilibrated for at least 2 h at 4°C). The mixture was centrifuged after 15 min at 2500s for 10 min and exactly 10 min later 250 bl of supernatant was transferred into counting vials with 5 ml liquid scintillator (Miniria 20, Zinsser, Frankfurt, FRG) and counted in a Packard Tri Carb liquid scintillation counter. The total radioactivity of the assay solution was determined after addition of KPTED buffer instead of dextran-coated charcoal solution. Specific receptor-ligand binding was calculated after subtraction of nonspecific binding, which was obtained by addition of 300. to 500. fold excesses of unlabeled hormone. Scatchard analysis was used to calculate Kd and B,,, Competitive protein binding assay of plasma. Binding capacities and affinities for vitamin D metabolites in plasma were measured with a competitive protein-binding assay according to the method of Bothe et al. (17). The procedure was similar to that used for measurement of receptor binding except that diluted plasma (1:8,000-1:16,000) was used instead of diluted receptor homogenate and that [26,27-methyl“H]25-OHDj (sp act 762 GBq/mmol) was used instead of labeled 1,25(OH)*Da. KPTED buffer was replaced by 60 mM sodium phosphate buffer, pH 7.4, and BSA by 2 g gelatine + 400 ~1 Brij-35 (30%) in 1 liter of buffer. 25.OHD, was added in amounts of 0 to 25 pmol/tube. B mali and Kd for 25.OHDa were calculated from Scatchard analysis. The Kd of 1,25-(OH)2D3 or vitamin D, binding were estimated from 50% inhibition (I&, values) of the [aH]25-OHD, binding using the Cheng and Prusoff formula (18):
Kd =
IGO 1 + concentration of [3H]25-OHD3 Kd of 25-OHDB
The ICsO was obtained from the linear regression of: logit (P) = log [P/(100-P)] against log C, where P represents the percentage binding of [3H]25-OHDs (binding in the absence of 1,25-(OH)zD3 and vitamin D, = 100%) and C represents the concentration of 1,25-(OH)zD3 or vitamin D,. The association constants (KJ were obtained as reciprocals of the equilibrium dissociation constants (K, = K;‘). of DBP DBP and free metabolite index. The plasma concentration was calculated from the B,,, for 25.OHD, in diluted plasma (19). The free metabolite index defined as the molar ratio of the metabolite and DBP (20) was used to estimate the changes of free metabolite concentrations in the course of vitamin Da treatment. By definition the free metabolite index appeared to be a suitable measure to express changes of free metabolite concentrations under different conditions. The index cannot, however, be used to compare different metabolites with different affinities for DBP. For comparison of different metabolites
AND
HARMEYER TABLE
Binding
Properties
I
of Plasma Vitamin D-Binding Protein 1,25-(0H)sDe Receptor from PVDRI and Control Piglets (means +- SEM)
(DBP) and Intestinal (Rachitic)
Control piglets Calcium (mM) Alkaline phosphatase (U/liter) Total vitamin Da (nM) Total 25.OHD3 fnM) Total 1,25-(OH)*Da (PM) DBP (PM) K. for 25.OHDa (Mm’) K, for 1,25-(OH)?D3 (M-l) Free index vitamin Dg (X103) Free index 25.OHD3 (X103) Free index 1,25-(OH),Da (X105) B,,, intestinal receptor [fmol/mg protein] Kd intestinal receptor (nM)
2.81 425 15.29 39.25 259.60 7.30 4.0 1.54 2.16 5.48 3.63
+ f f + k * x x + -t +
0.03 50 2.04 11.74 44.13 0.41 IO8 lo7 0.34 0.48 0.30
719 k 123 0.33 + 0.05
PVDRI piglets 1.33 * 0.05* 1302 + 147* 13.66 + 2.91 38.00 t 4.79 27.75 k 4.49* 6.77 rt 0.45 4.3 x lo8 1.45 x 10’ 2.04 k 0.36 5.77 f 0.74 0.42 f 0.05* 674 ? 103 0.31 t 0.05
Note. The plasma values are means of six samples each from six control and two rachitic piglets. The receptor data are means of nine control and nine rachitic piglets. DBP values for 25.OHD, and 1,25(OH)*D3 are means of three control and two rachitic piglets. *P < 0.01. the total concentrations of 25.OHDa and 1,25-(OH),Da, the DBP concentration in plasma and the K,, values of the two metabolites were used according to the formula of Bouillon et al. (21): [“free”]
=
[total] K,. [DBP]
This method, however, also does not give true values of the free plasma concentrations of the metabolites, since binding to albumin is not taken into account,. But it can be assumed that the greatest portion of the metabolites is bound to DBP and that the ratio of binding of both metabolites to DBP and to albumin is similar (9). Therefore an estimate of the proportion of free 25-OHDa and free 1,25-(OH)zD, can be obtained by this method. Chemicals. Analytical solvents were purchased from Merck (Darmstadt, FRG) and were of “chromatographical grade.” The solvents used for extraction and fractionation were of quality grade “for residue analysis.” Vitamin D, was from Serva (Heidelberg, FRG) and Vitamin D3 metabolites were kindly provided by Hoffmann La Roche (Basel, Switzerland). Labeled metabolites were from Amersham Buchler (Braunschweig, FRG). All metabolites were routinely purified and controlled by HPLC. BSA, Brij-35, Norit A and DTT were from Serva, PMSF from Sigma (Deisenhofen, FRG), and dextran T 70 from Pharmacia (Uppsala, Sweden). The other chemicals were at least of analytical grade.
RESULTS
Binding of vitamin D3 and vitamin D metabolites to DBP. The concentrations of calcium and 1,25(OH)*Ds in plasma of rachitic piglets were significantly lower than those of control piglets. The activities of alkaline phosphatase in plasma were significantly higher. The concentrations of vitamin D, and 25-OHD3 were not affected by the PVDRI condition (Table I). Scatch-
EFFECTS Control
piglet
OF VITAMIN
Rachltic
Ds TREATMENT
329
IN PIGS
piglet
30
.25 n .20 L 2
.:5 I
.lO -
.05 r
.l
2
.3
.4
5
85 IllHI
.6
.7
OL 0
.I
.2
3
.4
3
.6
85 inH1
FIG. 1. Scatchard plots of 25.OHD, binding to DBP of diluted plasma (1:8600) from a rachitic and a control piglet. The DBP content in plasma was 7.9 FM for the control and 5.7 pM for the rachitic piglet. Means of R,,, and K, are in Table I.
FIG. 2. Typical binding curves of vitamin Ds and vitamin olites by the intestinal 1,25-(OH),Ds receptor. Fifty percent hound to the receptor (control ment of [3H]1,25-(OH)2D3 quired a 44.fold excess of 25OHD3 and a 245,000-fold vitamin Ds. n , 1,25-(OH)2D3; 0, 25.OHDs; 0, vitamin D,.
ard analyses of specific 25-OHD3 binding to DBP at different ligand concentrations yielded a linear relationship with similar KG’s of about 4.0 X lOR M-’ for both rachitic and control piglets (Fig. 1, Table I). The K, of DBP for 1,25-(OH),D3 was also unaffected by the PVDRI condition (Table I). The DBP levels in plasma of rachitic piglets tended to be smaller than those of control piglets with the difference not being statistically significant. The free metabolite indices for vitamin D3 and 25-OHD, were the same for both groups of piglets. The free metabolite index of 1,25-(OH)zD3 of rachitic piglets, however, was considerably lower than that of control piglets due to the significantly lower total 1,25-(OH)zD:j concentration in plasma of PVDRI piglets (Table I).
Specificity of ligand binding to plasma DBP. The aflinities of plasma DBP for vitamin D3 and 1,25(OH)*Dc3 were not different between rachitic and control piglets. In control plasma 50% displacement of [3H]25OHD3 bound to DBP occurred with a 43-fold excess of vitamin D3 and with a 78-fold excess of 1,25-(OH),D3 (Fig. 3). From the 50% competition concentrations (I&) K,‘s for DBP of 1.70 X lo7 M-’ (vitamin Dg, Fig. 3) and of 1.54 X lo7 M-’ (1,25-(OH)2D3, Table I) were calculated. Experiments with PVDRI plasma yielded 50% displacement of [3H]25-OHD3 bound to DBP with 39-fold excess of vitamin D3 (K, = 1.80 X lo7 M-r) and with 56-fold excess of 1,25-(OH)zD3 (K, = 1.45 X lo7 Mm ‘, Table I).
Specificity of ligand binding to the intestinal 1,25(OH),D, receptor. The capacities (B,,,) and affinities (&) of the intestinal nuclear receptor for 1,25-(OH)zD3 were measured in animals different from those used for the DBP studies. B,,, and Kd were not different between rachitic and control piglets (Table I). The mean calcium concentrations and the activities of alkaline phosphatase in plasma of these animals were 3.0 k 0.1 mM and 406 + 36 U/liter for controls and 2.1 + 0.2 mM and 2098 * 187 U/liter for the PVDRI piglets, respectively. Total 1,25-(OH),D:, concentrations in plasma were 480 +- 139 pM for the control piglets and 101 + 14 pM for the rachitic piglets. In experiments with control piglets 50% displacement of [3H]1,25-(OH)2Dzi bound to the receptor by 25-OHD3 or vitamin D3 occurred with a 23- to 71-fold or >245,000-fold excess, respectively (IL = 3, Fig. 2). In experiments with PVDRI piglets 50% displacement of [3H]1,25-(OH)zD3 bound to the receptor by 25-OHD3 or vitamin D3 was obtained with a 20- to 42-fold or >220,000-fold excess, respectively (n = 2).
kJ
a
-10
100
1000 Compound
10000 [pg/tubej
100000
D metabdisplacepiglet) reexcess of
1000000
FIG. 3. Typical binding curves of vitamin D3 and vitamin D metabelites to DBP in diluted plasma. Fifty percent displacement of [sH]25OHDs bound to DBP (control plasma) required a 43-fold excess of vitamin D:, and a 7%fold excess of 1,25-(OH)2D3. a, 1,25-(OH),D:,; 0, 25-OHD,; 0, vitamin D3.
KAUNE, 5-
A
o! EOO-
0
SCHROEDER,
i
600;
. 1 .25-(OH);D3 /
r” IOOl7l 0 E 0
(
-2
-1
0
1
2
3
4
5
6
DOYS FIG. 4. Concentrations of calcium (A), vitamin D3 and 25.OHD3 (B), and 1,25-(OH)2D3 (C) of two PVDRI piglets from 2 days before to 6 days after im treatment with 7.5 X lo4 IU of vitamin D,.
Concentrations of vitamin D3 and metabotites in PVDRI plasma after treatment with pharmacological doses of vitamin D3. Administration of 7.5 X lo4 IU of vitamin D3 to rachitic piglets led to a 50% increase in the plasma calcium concentration about 3 days after treatment with persisting high values for 7 days after treatment (Fig. 4A). The activities of the alkaline phosphatase in plasma gradually declined after the vitamin D3 treatment (data not shown). In addition, the piglets showed healing of clinical symptoms of rickets such as disappearance of muscle weakness of bone and joint pain and resumed appetite. The plasma concentration of vitamin D3 transiently rose after treatment from 34-40 to
AND
HARMEYER
300-500 nM and returned to normal values 5 days after treatment (Fig. 4B). The level of 25-OHD, rose from 5070 to about 700 nM and remained at this high concentration until the end of the experimental period at 6 days postinjection (Fig. 4B). The time course of 1,25-(OH)zD3 paralleled that of 25-OHD3. Its concentration rose from subnormal values (20-70 PM) to supranormal values (215-400 PM) and stayed high for at least 1 week (Fig. 4C). Interaction of uitamin D3 and 25-OHD, with the intestinal receptor after treatment with vitamin D3. With the results obtained from the above measurements the following calculations were carried out. With the given DBP concentrations, the free metabolite indices of vitamin DX, 25OHD3, and 1,25-(OH)zD3 of rachitic piglets before and after treatment with vitamin D, were calculated (Table II). It was found that the significant rise of 1,25-(OH)zD3 in plasma resulted in a maximal free metabolite index in PVDRI piglets greater than that present in untreated control piglets (Tables I and II). Comparing the fractional rises of the free metabolite indices of vitamin D3, 25OHD3 and ~,~EJ-(OH)~D~ after treatment with vitamin D3 it was found that these were highest for vitamin D, and lowest for 1,25-(OH)2D3 (Table II). From the DBP concentration in plasma and from the measured affinities of DBP it was calculated that the “fractional free concentrations” of vitamin D,, 25 OHD3, and 1,25-(OH)aD3 in plasma after treatment with vitamin D3 were 0.87, 0.034, and 1.018% for vitamin D8, 25-OHD3, and 1,25-(OH)2D3, respectively. Unspecific binding of the metabolites of about 10 to 20% to albumin (12) was ignored. This corresponded to maximal “free concentrations” of the three compounds in plasma of 4.4 nM, 0.24 nM, and 4.1 pM, respectively. The values demonstrate that the “free concentration” of vitamin DS was 1073-fold in excess of free 1,25-(OH)2D, in plasma and that the free 25-OHD3 concentration was 59-fold in excess of free 1,25-(OH),D3. Comparing these ratios with those obtained for 50% displacement of 1,25-(OH),D, from the intestinal receptor (see above) it can be seen that the free 25OHD3 concentration in plasma allowed more than 50% displacement of 1,25-(OH)zD3 from the receptor. DISCUSSION The results reported in this study demonstrate that the therapeutic effect of a pharmacological amount of vitamin D, in healing the symptoms of PVDRI can be attributed not only to the transient rise of the plasma 1,25-(OH)aD3 concentration but also to a sustained increase in the concentration of 25-OHD, in plasma. In the experiments reported here the 1,25-(OH)2D3 concentration in plasma remained high for at least 6 days (Fig. 4C). This was much longer than had been observed in a previous study (6) where the 1,25-(OH),D3 concentra-
EFFECTS
OF VITAMIN
D, TREATMENT TABLE
Free
331
IN PIGS
II
Metabolite Indices of Vitamin D3 and Its Metabolites of Two PVDRI Piglets before and after Treatment with 7.5 X lo4 IU of Vitamin D, (Maximum Values) Piglet 2
Piglet I
Basal DBP (fiiv) Free vitamin D, Index (X10”) Free 25-OHD:, Index (Xl@) Free 1,25-(OH)1D:, Index (X105)
Treatment
Basal treatment
9.52 0.46
Treatment
Basal treatment
6.88
6.65 2.91
Basal
48.9 108 4.40
tion in plasma already declined to subnormal concentrations 3 to 8 days after treatment. The reason for the difference in the time course of plasma ~,~EY-(OH)~D, is probably the higher dose of vitamin D3 which was used in the present study (average of 3.5 X lo4 IU vs 7.5 X lo4 IU). But with the lower vitamin D3 dose used in the previous study the therapeutic effect also lasted for at least 4 weeks. It is of interest to note that 1,25-(OH)zD, concentrations in plasma of the rachitic piglets are sometimes near physiological concentrations (100 PM). Nevertheless rachitic symptoms developed. Near physiologic 1,25(OH),D:, concentrations in plasma have also been reported from PVDRI-affected children and young persons (22, 23). Since no differences in binding affinities and capacities of plasma DBP and intestinal 1,25(OH)zD3 receptor between control and rachitic piglets could be identified there is no indication to assume that factors other than defective production of 1,25-(OH),D3 contributed to the development of rachitic symptoms. This view is supported by the observation that physiological amounts of 1,25-(OH)zDx can heal the rachitic lesions (5). The suggestion that the binding properties of intestinal 1,25-(OH),D3 receptor and plasma DPB might have been altered by changes in 1,25-(OH),D3 metabolism proved to be wrong. This agrees with similar observations made by Hunziker et al. (24) with chicks. The authors also found no effect of vitamin D depletion or repletion upon the properties of the intestinal 1,25(OH),D3 receptor. For the binding properties of DBP similar findings have been reported by Rojanasathit and Haddad (25) who worked with vitamin D-deprived rats. In that study B,,, and K, of plasma DBP were unaffected by withdrawal of dietary vitamin D. The concentrations and affinities of DBP for vitamin D metabolites which we found in our study were in the same range than those reported for men, rats, and chimpanzees (12, 26). Kd values of the intestinal 1,25-
16.8
3.72
11.3
7.44
9.6
0.68
74.7 105 5.94
20.1 14.1 8.7
(OH)zD3 receptor of PVDRI and normal piglets agreed with those reported for rabbits, rats, chicks, and men (27-30). The B,,, values of the piglets were slightly higher than those reported from other species. This might have been caused by more complete extraction, or better stability of the porcine receptor during incubation or by its higher concentration in the mucosal tissue compared to those of other species. Clayton et al. (31) found a higher affinity and a lower capacity of the porcine 1,25(OH)zD3 receptor than is reported here. These authors incubated the receptor at 23°C rather than at 4°C and the Scatchard plot was not linear, indicating more than one binding site. Displacement studies which have been carried out with intestinal receptor preparations or with DBP from pigs and other species using vitamin D3, 25-OHD3, and 1,25-(OH)zD3 yielded the same sequence of binding as that reported here (27, 29, 31-33), except that in our study binding of 1,25-(OH),D3 and vitamin D3 to DBP was not significantly different. The relatively large affinity of 25-OHD, to the porcine intestinal 1,25(OH)2D3 receptor probably also provides an explanation for reported observations that intestinal calcium absorption could be induced by high concentrations of 25OHD3 in nephrectomized rats (34). Vitamin D3 and its metabolites in plasma are not exclusively bound to DBP. In human plasma 85% of 1,25(OH)2D3 and 88% of 25-OHD3 were found to be bound to DBP. Only 1 and 0.03%, respectively, of these metabolites are reported to be free. The remaining portions were bound to albumin (11, 12, 35). If partition of the compounds in pig’s plasma is similar to that reported for human plasma, the “free concentration” of 1,25(OH)zD3 has been overestimated by about 30% in our experiments and that of 25-OHD3 by about 10%. This would mean that the degree of interaction of 25-OHD, with 1,25-(OH),D3 at the intestinal receptor level after vitamin D, treatment was probably greater than documented by the data. In any case, the results which are
332
KAUNE,
SCHROEDER,
presented in this study reveal that the treatment effect of pharmacological doses of vitamin D, might be brought about by a combined action of 25OHD, and 1,25(OH)2D3 upon the intestinal 1,25-(OH),D3 receptor. On the other hand the treatment effect of vitamin D, appears not to be an effect of 25OHD3 alone as has been suggested earlier by DeLuca (8). The time course of 1,25-(OH)ZD3 in plasma of vitamin D3-treated PVDRI piglets disclosed the presence of highly active extrarenal 1-hydroxylases. The assumption that the rise of 1,25-(OH)zD3 in plasma after treatment came from extrarenal sources is supported by the observation that the conversion of 25-OHD3 to 1,25(OH)zD3 by renal cortex homogenates of rachitic piglets was completely absent even in the presence of 25-OHD, concentrations similar to those present in the plasma of vitamin D,-treated piglets (13). In addition, significant increases in 1,25-(OH),D3 in plasma after treatment with massive doses of vitamin D, have also been observed in normal nephrectomized pigs (36, 37). Finally, since in PVDRI piglets cellular expression of the renal vitamin D 1-hydroxylases is inhibited, it appears unlikely that the vitamin D-dependent enzyme is present in other organs or tissues. Our studies, however, provide no information about how 1,25-(OH)zD3 production is controlled and how extrarenal 1-hydroxylases operate. In PVDRI piglets 1,25-(OH)2D3 is probably formed after administration of massive doses of vitamin D, and, when non-vitamin Ds-treated, by one or more unspecific steroid-I-hydroxylases. Such unspecific 1-hydroxylases probably possess different kinetics compared to the vitamin D-dependent 1-hydroxylase and it is likely that they also contribute to 1,25-(OH),D3 in normal subjects after administration of pharmacological amounts of vitamin D. Among other tissues extrarenal production of 1,25(OH)2D3 has been found in immune cells such as macrophages (38) whose concentration may vary with the immune status of the individual. Therefore, it might be tempting to speculate whether the toxicity of high doses of vitamin D also varies with the immune status. REFERENCES 1. Norman,
A. W. (1987) J. N&r.
2. Gray, R., Boyle, I., and DeLuca, 1234.
117,797-807. H. F. (1971) Science 172, 12322
3. Silver, J., Landau, H., Bab, I., Shvil, Y., Friedlaender, M. M., Rubinger, D., and Popovtzer, M. M. (1985) Isr. J. Med. Sci. 21, 5356. 4. Harmeyer, J., Grabe, C. V., and Winkler, I. (1982) Exp. Biol. Med. 7,117-125. 5. Harmeyer, J., and Kaune, R. (1990) Advances in Animal Breeding and Genetics, Vol. 5, pp. 111-130, Paul Parey, Hamburg/Berlin. 6. Kaune, R., and Harmeyer,
J. (1987) Acta Endocrinol.
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S., Rapp, N., and Slatopolsky,
E. (1988)
AND
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8. DeLuca, H. F. (1988) FASEB J. 2,224-236. 9. Bikle, D. D., and Gee, E. (1988) in Vitamin D. Molecular, Cellular and Clinical Endocrinology, pp. 7033709, Walter de Gruyter, Berlin. 10. Haddad, J. G. (1987) in Bone and Mineral Research V, pp. 281~ 308, Elsevier, Amsterdam/New York/Oxford. 11. Bouillon, 451-453.
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12. Bikle, D. D., Siiteri, P. K., Ryzen, E., Haddad, J. G., and Gee, E. (1985) J. Clin. Endocrinol. Metab. 61,969-975. 13. Winkler, I., Schreiner, F., and Harmeyer, J. (1986) C&if. Tissue Int. 38,87-94. 14. Clemens, T. L., Hendy, G. N., Papapoulos, S. E., Fraher, L. J., Care, A. D., and O’Riordan, (1979) Clin. Endocrinol. 11,225-234. 15. Simpson, R. U., and DeLuca, H. F. (1982) Proc. Natl. Acad. Sci. USA 79, 16-20. 16. Walters, M. R., Hunziker, W., and Norman, A. W. (1980) J. Biol. Chem. 255,6799-6805. 17. Bothe, V., Schmidt-Gayk, H., Armbruster, F.-P., and Mayer, E. (1984) Arztl. Lab. 30, 151-156. 18. Cheng, Y.-C., and Prusoff, W. H. (1973) Biochem. Pharmacol. 22, 3099-3108. 19. Woloszczuk, W. (1985) Clin. Chem. Acta 145,27-35. 20. Bouillon, R., Van Asche, F. A., and Van Baelen, H. (1981) J. Clin. Inuest. 67,589-596. 21. Bouillon, R., Van Baelen, H., and De Moor, P. (1977) J. Clin. Endocrinol. Metab. 45, 679-684. 22. Kruse, K. (1985) Monatsschr. Kinderheilkd. 133,346-352. 23. Balsan, S., Garabedian, M., Lieberherr, M., Gueris, J., and A. Ulmann (1979) Vitamin D. Basic Research and its Clinical Application, pp. 114331149, Walter de Gruyter, Berlin, New York. 24. Hunziker, W., Walters, M. R., Bishop, J. E., and Norman, A. W. (1982) J. Clin. Inuest. 69,826-834. 25. Rojanasathit, S., and Haddad, J. G. (1977) Endocrinology 100, 6422647. 26. Bouillon, R., Van Baelen, H., and De Moor, P. (1980) J. Steroid Biochem. 13,1029-1034. 27. Duncan, W. E., AW, T. C., Walsh, P. G., and Haddad, J. G. (1983) Anal. Biochem. 132,209-214. 28. Yeh, J. K., Aloia, J. F.. Vasvani, A. N., and Semla, H. (1986) Bone 7,49-53. 29. Wecksler, W. R., Mason, R. S., and Norman, A. W. (1979) J. C/in. Endocrinol. Metab. 48, 715-717. 30. Nakada, M., and DeLuca, H. F. (1985) Arch. Biochem. Biophys. 238,129-134. 31. Clayton, J., Guilland-Cumming, D. F., Kanis, J. A., and Russel, R. G. G. (1982) in Vitamin D. Chemical, Biochemical and Clinical Endocrinology of Calcium Metabolism. pp. 821-823, Walter de Gruyter, Berlin. 32. Link, R. P., and DeLuca, H. F. (1988) Steroids 41,583~598. 33. Brumbaugh, P. F., and Haussler, M. R. (1974) J. Biol. Chem. 249, 1251-1257. 34. Boyle, I. T., Miravet, L., Gray, R. W., Holick, M. F., and DeLuca, H. F. (1972) Endocrinology 90,605-608. 35. Bikle, D. D., Gee, E., Halloran, B., Kowalski, M. A., Ryzen, E., and Haddad, J. G. (1986) J. Clin. Endocrinol. Metab. 63,954-959. 36. Littledike, E. T., and Horst, R. L. (1982) Endocrinology 111, 2008-2013. 37. Horst, R. L., Littledike, E. T., Gray, R. W., and Napoli, J. L. (1981) J. Clin. Invest. 67, 274-280. 38. Rigby, W. F. C. (1988) Zmmunol. Today 9,54-58.
