CIinical Science (1981) 61,471476
47 1
Plasma levels of vitamin D metabolites in diphosphonatetreated rats
U. T R E C H S E L , C . M. T A Y L O R , J . A . E I S M A N , J.-P. B O N J O U R A N D
H. FLEISCH
Department of Pathophysiology. University of Berne, Murlenstrasse, Berne, Switzerland
(Received 22 December 1980/25 March 1981; accepted 21 April 1981)
Summary
1. Protein-binding assays have been used to
measure plasma 1,25-dihydroxy-vitamin D 1,25(OH),Dl as well as 25-hydroxy-vitamin D
[25-(OH)D1 in rats given 10 mg of phosphorus
(P) day-' kg-' as ethane-1-hydroxy-1,l-diphosphonate (EHDP).
2. In control animals given a normal
laboratory chow plasma 25-(0H)D and 1,25(OH),D were about 40 nmol/l and 300 pmol/l
respectively.
3. EHDP produced a decrease of plasma
1,25-(OH),D to below 50 pmol/l in 2 days.
4. Both in control and in EHDP-treated rats
plasma 1,25-(OH),D increased when dietary
calcium (Ca) was restricted to 0-1%, or dietary P
to 0.2%, indicating that the well-known
stimulatory effect of Ca or P deprivation was at
least partially effective in EHDP-treated rats.
5. In response to an increase of the oral supply
of vitamin D, to 65 nmol/day the plasma level of
25-(0H)D rose in both control and EHDP
groups. Plasma 1,25-(OH),D was not increased
above the normal value in control rats. In
EHDP-treated rats, however, plasma 1,25(OH),D rose to a level equal to that in controls,
suggesting that the effect of EHDP on plasma
1,25-(OH),D can be overcome at high precursor
concentration.
Key words: calcium, 1,25-dihydroxy-vitamin D,
diphosphonate, 25-hydroxy-vitamin D, phosphonate, vitamin D.
Correspondence: Dr U. Trechsel, Department of
Pathophysiology, University of Berne, Murtenstrasse
35,3010 Berne, Switzerland.
0143-5221/81/10047146%01.50/1
Abbreviations: 1,25-(OH),D, 1,25-dihydroxyvitamin D; 25-(OH)D, 25-hydroxy-vitamin D;
EHDP, ethane-1-hydroxy-1,I-diphosphonate.
Introduction
In recent years diphosphonates have been
recognized to be useful therapeutic agents in the
management of certain disorders of bone
metabolism, especially in Paget's disease [ 1-31.
Diphosphonates are potent inhibitors of the
formation and dissolution of calcium phosphate
crystals in vitro [4, 51 and of bone resorption in
vivo [4]. One of the compounds, ethane-lhydroxy-1,Ldiphosphonate (EHDP), when given
in large doses to animals, has been shown to
inhibit bone mineralization [6-91 and to decrease
the intestinal absorption of calcium [7, 9-111.
The latter effect is due to decreased l-hydroxylation of 25-hydroxy-vitamin D [25-(OH)D] in
the kidney [ 12-14]. 1,25-Dihydroxy-vitamin D
[ 1,25-(OH),D] is the most potent known metabolite of vitamin D in promoting calcium absorption in the gut [15, 161, as well as bone
resorption [ 17, 181, and is generally considered to
be a major hormonal form of the vitamin which
plays an important role in mineral homeostasis.
Evidence accumulated by several authors suggests that the influence of EHDP on vitamin D
metabolism is a homeostatic consequence of
the effect of the diphosphonate on bone
mineralization rather than a direct effect of the
drug on the 25-(OH)D-hydroxylating enzymes
[9, 12, 141. Therefore, EHDP-treated animals
represent an interesting model for the investigation of the regulation of vitamin D
metabolism. In the studies reported in the
@ 1981 The Biochemical Society and the Medical Research Society
472
U.Trechsel et al.
literature the effect of EHDP on vitamin D
metabolism was assessed either by measuring the
renal I-hydroxylase of chick kidneys in vitro [13,
141 or the appearance of dihydroxy derivatives in
plasma of vitamin D-deficient animals after a
single injection of radioactively labelled vitamin
D, [12, 141 or 25-(OH)D, [13].No information is
yet available on the circulating levels of vitamin
D metabolites in diphosphonate-treated rats.
Therefore we have used protein-binding assays to
measure plasma levels of 25-(0H)D and 1,25(OH),D in EHDP-treated animals. We have
studied the time-course of the effect of EHDP on
plasma 1,25-(OH),D. Furthermore we have
studied whether the effects of EHDP on plasma
1,25-(OH),D can be influenced by dietary
calcium (Ca) and phosphate (P) as well as by the
supply of vitamin D.
Methods
Animal experiments
The experiments were performed with male
Wistar rats bred in this institute. EHDP (kindly
provided by Procter and Gamble Co., Cincinnati,
OH, U.S.A.) was dissolved in distilled water, the
solution adjusted to pH 7.4 with NaOH and
injected subcutaneously (s.c.) at a dose of 10 mg
of P day-' kg-' in a volume of 2 ml/kg body wt.
Vitamin D, supplements were given orally in 0.1
ml of arachis oil. Blood for the determination of
vitamin D metabolites was obtained by aortic
puncture 24 h after the last injection of EHDP.
The animals were anaesthetized with an intraperitoneal (i.p.) injection of 30-40 mg of pentobarbital
sodium
(Nembutal;
Abbott
Laboratories, North Chicago, IL, U.S.A.)/kg.
Heparin (kindly provided by F. Hoffmann-La
Roche and Co., Basle) was used to prevent
coagulation and plasma obtained by centrifugation was frozen and kept at -2OOC until
analyses of vitamin D metabolites.
Time-course of effect of EHDP on plasma
2 5 - ( 0 H ) D and 1,25-(OH)2D
Rats weighing 191 f 14 g (mean k SD) were
given a commercial diet containing 0.9% Ca/
0.7% P and 39 nmol of vitamin D,/kg ad libitum.
They were treated with EHDP for 1, 2, 3, 4 or 7
days. Controls received an equivalent volume of
NaCl solution (150 mmol/l: saline) for either 1 or
7 days.
Influence of dietary Ca and P on the effect of
EHDP on plasma 1,25-(OH),D
Rats weighing 180 15 g (mean f SD) were
given one of three diets containing either 1.0%
Ca/l.O% P, 1.0% Ca/0.2% P or 0.2% Ca/l.O%
P for 2 weeks ad libitum. The diets were made by
adding calcium gluconate and a mixture of
K,HPO,/KH,PO,
(3:7) to a diet containing
0.14% Ca/O.22% P (Altromin C-1034, Altromin
GmbH, Lage, West Germany). Since the diet was
vitamin D-poor the rats were supplemented with
1 5 nmol of vitamin D, every 2 days. After a 7
day equilibration period the animals were treated
with EHDP for 7 days. Control animals received
an equivalent volume of saline. The diet during
EHDP-treatment was the same as the one given
during the equilibration period.
Influence of vitamin D intake on the effect of
EHDP on plasma 2 5 - ( 0 H ) D and 1,25-(OH),D
Rats were housed individually and pair-fed.
After an equilibration period the animals were
given EHDP for 7 days. Control rats received
saline.
Experiment I . Rats weighing 134 2 5 g
(mean SD) received 20 g of diet containing
0.9% Ca/0.7% P and about 0.6 nmol of vitamin
D,/day. The equilibration period was 4 days.
During the experimental period half the animals
received a supplement of 65 nmol of vitamin
D,/day.
Experiment II. Rats weighing 169 i-6 g
(mean f SD) received 30 g of a vitamin D-poor
diet containing 1.O% C a / l .O% P per day. During
the 5 day equilibration period all animals were
given 0.6 nmol of vitamin D,/day orally. During
the experimental period they received daily
supplements of 0.6, 6.5 or 65 nmol of vitamin
D,.
Determination of vitamin D metabolites
2S-(OH)D. 25-(0H)D was measured by a
modification of the method of Preece et al. [191.
The only modification was the use of 0.1%
gelatin instead of 1.0% albumin to prevent the
loss of 25-[3H]-(OH)D from solution. Plasma
samples of 0.1 ml were used. 25-(0H)-[26,27,HID, (The Radiochemical Centre, Amersham,
Bucks., U.K.; specific radioactivity 7.3 Ci/mmol)
was used as a tracer.
1,25-(OH),D. 1,25-(OH),D was extracted
with chloroform/methanol by the method used
for 25-(0H)D [19]. The extract was processed
and 1,25-(OH),D was assayed as described
previously 1201. The sensitivity of the assay was 6
fmol/assay tube. Plasma samples of 1-2 ml
were used, resulting in an assay sensitivity of
30-60 pmol/l. The assay procedures did not
discriminate between vitamin D, and D, metabolites. However, since the vitamin D supply of
Vitamin D metabolites in rats
473
and P intakes. Lowering dietary Ca or P
produced an increase in plasma 1,25-(OH),D in
control animals. EHDP-treated rats also responded to C a or P restriction with an increase of
plasma 1,25-(OH),D, but the values remained
the rats was mostly vitamin D,, vitamin D,
metabolites were probably negligible.
1,25-[3H]-(OH),D3 was produced biologically
as described by Boyle et al. [211. The substrate
25-(OH)-[23,24-3H,lD3 (specific radioactivity 78
Ci/mmol) was provided by F. Hoffman-La
Roche Inc., Nutley, NJ, U.S.A. 1,25-(OH),D3
was supplied by F. Hoffmann-La Roche & Co.
AG., Basle, Switzerland.
Results are expressed as means k SEM. Significance of the difference between groups was
determined by Student’s two-tailed t-test for
unpaired values or by Wilcoxon’s test for
unpaired values.
I:
50
-I
20
1°i
Results
Time-course of the effect of EHDP on plasma
25-(0H)D and 1,25-(OH),D
As shown in Fig. 1, EHDP at a dose of 10 mg
of P day-’ kg-l, during the first 3 days of
treatment, produced a progressive decrease of
plasma 25-(0H)D which then remained constant
for the rest of the experimental period. Plasma
1,25-(OH),D was already decreased after 1 day
of treatment and fell to values below 50 pmol/l
thereafter. When the limit of detection of plasma
1,25-(OH),D in the assay was decreased to 13
pmol/l by pooling plasma of EHDP-treated rats,
plasma 1,25-(OH),D was found to be 32 pmol/l
on day 4 and 20 pmol/l on day 7 (means of two
plasma pools for each day from groups of six
rats).
40 4 i
300
I . .
I
0
I
1
I
2
I
3
FIG. 1. Effect of EHDP on plasma 25-(0H)D and
1,25-(OH),D. EHDP was given daily in a dose of 10
mg of P/kg subcutaneously. Values are means k SEM.
n = 4 for EHDP-treated (0)and n = 3 for control (A)
animals. Significance of differences: *P < 0.05 by
Student’s t-test for unpaired values.
TABLE1. Plasma levels of 1,25(0H),D in rats on direrent Ca
and P intakes
Plasma levels of 1,25(OH),D are given as pmol/l. AU animals received
1.5 nmol of vitamin D, orally every 2 days. Rats were treated with
EHDP for 7 days. Significance of differences between groups was
evaluated with Wilcoxon’s rank test for unpaired values: ‘P < 0.005 vs
control group on the same diet; bP < 0.005, cP = 0.05 vs control group
on 1.1% Ca/l.O% P; *P<0.02, eP=0.05 vs EHDP on 1.1%
Ca/l.O% P.
Dietary Ca and P
Means
1
7
Time of treatment (days)
Influence of dietary Ca and P on the effect of
EHDP on plasma 1,25-(OH),D
Table 1 shows the plasma 1,25-(OH),D in
EHDP-treated and control rats on different Ca
1.1% Ca/l.O% P
I
4
P
.
0.1% Ca/l.O% P
Control
EHDP
Control
210
469
466
436
303
328
113
134
123
119
158
560
887
901
915
881
793
1257
369
5101’
93gb
EHDP
1.1%Ca/0.2% P
Control
EHDP
212
206
134
155
295
721
596
966
668
424
443
37 1
252
427
215
133
577
200’*d
636E
5246a*C
U. Trechsel et al.
474
TABLE2. InJuence of the intake of vitamin D on the effecf of
EHDP on plasma 2 5 - ( 0 H ) D and 1,25-(OH),D
Numbers o f animals are shown in parentheses. Significance of
differences: *P < 0.01 vs control; t P < 0.05; $P < 0.01 vs control
0.6 nmol of vitamin D,/day by Student's f-test for unpaired values.
Group
Expt. I
Control
EHDP
Control
EHDP
Supply of
vitamin D,
(nmol/day)
25-(OH)D
(nmolll)
1,25-(OH),D
(pmol/l)
0.6
0.6
65
65
33.8 ? 3.0
15.6 f 2.4'
153.5 f 32.3
169.8 t 21.5
205.5 i- 33.4
<58
93.0 f 23.6t
95.7 f 8.4
0.6
0.6
6.5
6.5
65
65
75.9 f 6.1
46.5 f 2.3*
97.6 f 8.5
55.4 t 8.4'
258.7 f 19.6
2 8 3 . 5 t 31.0
186.2 f 22.1
<4I
176.7 t 30.9
<4 1
83.2 f 4.5$
81.5 f 6.6
Expt. I1
Control
EHDP
Control
EHDP
Control
EHDP
below those observed in control animals on a
high-Ca/high-P diet.
Influence of vitamin D intake on the effect of
EHDP on plasma 2 5 - ( 0 H ) D and 1,25-(OH),D
The results of two separate experiments are
presented in Table 2. Plasma 25-(0H)D
increased with increasing supply of vitamin D
both in control and in EHDP-treated animals.
EHDP produced a decrease of plasma 25-(0H)D
in animals receiving 0.6 or 6 . 5 nmol of vitamin
D,/day, but had no effect on this in animals given
65 nmol of vitamin D,/day. In control rats
increasing dietary vitamin D, to 65 nmol/day
produced a decrease of plasma 1,25-(OH),D.
EHDP-treated animals responded to a supplementation with 65 nmol of vitamin D,/day with
an increase of plasma 1,25-(OH),D to a level
equal to that of corresponding control animals.
Discussion
Our results demonstrate that the circulating levels
of 1,25-(OH),D can be decreased to very low
values with large doses of EHDP in rats
maintained on ordinary, vitamin D-containing,
laboratory food. This finding is important when
considering previous investigations in which
EHDP was used as a tool to produce 1,25(OH),D deficiency in rats without vitamin D
depletion [22, 231, since before the present study
no information was available on the degree of
1,25-(OH),D deficiency obtained with EHDP
treatment.
A decrease of plasma 25-(0H)D was also
observed in our experiments, except at high
vitamin D intake. Previous work did not provide
conclusive evidence for an interference of EHDP
with the formation of 25-(0H)D [12, 241.
Nevertheless further work is needed to rule out
the possibility that EHDP might influence either
the hepatic vitamin D-25-hydroxylase directly or
the supply of vitamin D to this enzyme. Alternatively EHDP might increase the disappearance of 25-(0H)D from blood.
The well known stimulatory effect of a low-Ca
or a low-P diet on plasma 1,25-(OH),D in rats
[25], which was observed in the control animals,
was also present in EHDP-treated rats, although
in all the groups EHDP-treated rats had much
lower plasma 1,25-(OH),D than corresponding
control animals. These results show that the
homeostatic mechanisms which mediate the
response of plasma 1,25-(OH),D to dietary Ca or
P restriction are at least partially operative in
EHDP-treated rats. This should be considered
when EHDP is used to produce 1,25-(OH),D
deficiency in experiments involving low-mineral
diets.
We can only speculate about the mechanism
by which EHDP influences plasma 1,25-(OH),D.
The inhibitory effect of the drug on 25-(0H)DI-hydroxylation has been shown to depend on the
animal's vitamin D status [12], as well as on
dietary calcium [ 141. These results suggest that
EHDP influences I-hydroxylation indirectly. The
absence of an effect of EHDP on the 25(0H)D-I-hydroxylase in primary chick kidney
cell culture is consistent with this hypothesis [261.
Parathyroid hormone is unlikely to be responsible
for the diphosphonate-induced decrease of
Vitamin D metabolites in rats
plasma 1,25-(OH),D, since EHDP decreased
intestinal calcium absorption in thyroparathyroidectomized rats and since exogenous
parathyroid hormone did not reverse this effect
[221. One factor which might be involved is the
increase of plasma calcium observed in rats given
large doses of EHDP 191.
It is interesting to compare the relationship
between plasma 25-(0H)D and plasma 1,25(OH),D in control and EHDP-treated animals. In
control rats increasing plasma 25-(0H)D did not
lead to a rise in plasma 1,25-(OH),D, indicating
that changes in plasma 1,25(OH),D concentration were controlled by factors other than
25-(OH)D. At high plasma levels of 25-(0H)D
there was on the contrary a decrease in plasma
1,25-(OH),D. These results are consistent with
earlier findings in rats [271 and in human patients
with hypervitaminosis D [281. In EHDP-treated
animals, however, plasma 1,25-(OH),D increased
to the level observed in control animals at high
plasma levels of 25-(0H)D. The finding that in
EHDP-treated rats the inhibition of l-hydroxylation can be overcome by increasing the plasma
concentration of the substrate 25-(OH)D gives
rise to the question whether plasma 1,25-(OH),D
might be normalized with exogenous vitamin D
also in other situations in which it has been found
to be decreased, such as in chronic renal failure
[291.
Acknowledgments
Part of this work has been presented at the 4th
International Workshop on Phosphate and Other
Minerals, Strasbourg, 1979 [(Abstract) (1979)
Mineral Electrolyte Metabolism, 2, 2721. We
thank Mrs R. Bichsel, Miss A. Monod, E.
Oldenberg and C. Steiner for their skilled
technical assistance, Mrs C. Stieger for assistance
with the illustrations, Mrs B. Gyger, B. Meier and
N. Bore1 for preparing the manuscript. This work
was supported by the Swiss National Science
Foundation (3.725.76), by the F. Hoffmann-La
Roche and Co. AG, Basle, Switzerland, by the
Procter and Gamble Company, U.S.A., and by
the Ausbildungs- und Forderungsfonds der
Arbeitsgemeinschaft fur Osteosynthese (AO),
Switzerland.
References
111 ALTMAN, R.D.,
JOHNSTON, C.C., KHAIRI, M.R.A.,
WELLMANN,H., SERAFINI,A.N. & SANKEY,R.R. (1973)
Influence of disodium etidronate on clinical and laboratory
manifestations of Paget's disease of bone (osteitis deformans).
New England Journal of Medicine, 289,1379-1384.
R., PRESTON,C., WALTON,R.J. &
121 RUSSELL,R.G.G., SMITH,
WOODS,
C.G. (1974) Diphosphonates in Paget's disease.
Lancef,i, 894-898.
131 MEUNIER,
P.J., CHAPUY,M.C., ALEXANDRE,
C., BRESSOT,C.,
475
EDOUARD,C., VIGNON, E., MATHIEU,L. & TRECHSEL,U.
(1979) Effects of disodium dichloromethylene diphosphonate
on Paget's disease of bone. Lancet, ii, 489-492.
141 FLEISCH,H., RUSSELL, R.G.G. & FRANCIS,M.D. (1969)
Diphosphonates inhibit hydroxyapatite dissolution in uitro,
and bone resorption in tissue culture and in uiuo. Science, 165,
1262-1264.
151 FRANCIS,M.D., RUSSELL,R.G.G. & FLEISCH, H. (1969)
Diphosphonates inhibit formation of calcium phosphate
crystals in uitro and pathological calcification in uiuo. Science,
165,1264-1266.
161 KING,W.R., FRANCIS,
M.D. & MICHAEL,W.R. (1971) Effect
of disodium ethane-I-hydroxy-l,l-diphosphonateon bone formation. Clinical Orfhopaedics and Related Research, 78,
25 1-270.
I71 GASSER,A.B., MORGAN,D.B., FLEISCH,H.A. & RICHELLE,
L.J. (1972) The influence of two diphosphonates on calcium
metabolism in the rat. Clinical Science, 43,3 1 4 5 .
181 SCHENK,R., MERZ,W.A., MOHLBAUER,
R., RUSSELL,R.G.G.
& FLEISCH,H. (1973) Effect of ethane-I-hydroxy-l,l-diphosphonate (EHDP) and dichloromethylenediphosphonate
(CI,MDP) on the calcification and resorption of cartilage and
bone in the tibia1 epiphysis and metaphysis of rats. Calcified
Tissue Research, 11,196-214.
R., BONJOUR,
J.-P., RUSSELL,R.G.G.
191 TRECHSEL,U., SCHENK,
& FLEISCH,
H. (1977) Relation between bone mineralization,
C a absorption, and plasma C a in phosphonate-treated rats.
American Journal of Physiology, 232, ~ 2 9 8 - ~ 3 0 5 .
1101 MORGAN,
D.B., BONJOUR,
J.-P., GASSER,A.B., O'BRIEN,K. &
FLEISCH,H. (1971) The influence of a diphosphonate on the
intestinal absorption of calcium. Israel Journal of Medical
Sciences, 7,384-386.
II 1 1 BONJOUR,J.-P., RUSSELL,R.G.G., MORGAN,D.B. & FLEISCH,
H.A. (1 972) Intestinal calcium absorption, Ca-binding protein,
and Ca-ATPase in diphosphonate-treated rats. American
Journal ofPhysiology, 224,1011-1017.
1121 HILL, L.F., LUMB,G.A., MAWER,E.B. & STANBURY,
S.W.
on of the biosynthesis of 1,25dihydroxycholecalciferol in rats treated with a diphosphonate.
Clinical Science, 44,335-347.
I131 BAXTER,L.A., DELUCA,H.F., BONJOUR,J.-P. & FLEISCH,H.
(1974) Inhibition of vitamin D metabolism by ethane-lhydroxy-l,l-diphosphonate. Archives of Biochemisfry and
Biophysics, 164,655-662.
1141 TAYLOR,C.M., MAWER,E.B. & REEVE,A. (1975)The effects
of a diphosphonate and dietary calcium on the metabolism of
vitamin D, (cholecalciferol) in the chick. Clinical Science and
Molecular Medicine, 49,39 1-400.
(151 BOYLE,I.T., MIRAVET,L., GRAY, R.W., HOLICK,M.F. &
DELUCA,H.F. (1972) The response of intestinal calcium
transport to 25-hydroxy- and 1,25-dihydroxy-vitamin D in
nephrectomized rats. Endocrinology, 90,605-608.
1161 WONG,R.G., NORMAN,
A.W., REDDY,C.R. & COBURN,
J.W.
(1972) Biologic effects of 1,25-dihydroxycholecalciferol (a
highly active vitamin D metabolite) in acutely uremic rats.
Journal of Clinical Inuesfigation,51,1287-1291.
[I71 RAISZ,L.G., TRUMMEL,
C.L., HOLICK,M.F. & DELUCA,H.F.
(1972) 1,25-Dihydroxycholecalciferol: a potent stimulator of
bone resorption in tissue culture. Science, 115,768-769.
J.J., HOLICK,M.F. & DELUCA,H.F. (1973) The
1181 REYNOLDS,
role of vitamin D metabolites in bone resorption. Calcified
Tissue Research, 12,295-301.
1191 PREECE, M.A., O'RIORDAN,J.L.H., LAWSON,D.E.M. &
KODICEK,E. (1974) A competitive protein-binding assay for
25-hydroxycholecalciferol and 25-hydroxyergocalciferol in
serum. Clinica Chimica Acta, 54,235-242.
1201 EISMAN,J.A., HAMSTRA,A.J., KREAM,B.E. & DELUCA,H.F.
(1976) A sensitive, precise, and convenient method for
determination of 1,25-dihydroxyvitamin D in human plasma.
Archives of Biochemistry and Biophysics, 176,235-243.
1211 BOYLE,I.T., OMDAHL,J.L., GRAY,R.W. & DELUCA,H.F.
(1973) The biological activity and metabolism of 24,25dihydroxyvitamin D,. Journal of Biological Chemisfry, 248,
4 174-4 180.
U. (1977) Calcium
1221 BONJOUR,
J.-P., FLEISCH,H. & TRECHSEL,
absorption in diphosphonate-treated rats: effect of parathyroid
function, dietary calcium and phosphorus. Journal of Physiology (London), 264,125-139.
416
U.Trechsel et al.
1231 RIZZOLI,R., FLEISCH,
H. & BONJOUR,J.-P. (1977) Role of
1271 HUGHES, M.R., BAYLINK, D.J., GONNERMAN,W.A.,
1.25-dihydroxyvitamin D, on intestinal phosphate absorption
TOVERUD,S.U., RAMP, W.K. & HAUSSLER,M.R. (1977)
in rats with a normal vitamin D supply. Jourrra/ of Clinical
Influence of dietary vitamin D, on the circulating conInvestigation, 60,639-647.
centration of its active metabolites in the chick and rat.
1241 VON HERRATH,
D., SCHAEFER,
K., BONJOUR,
J.-P. & FLEISCH,
Endocrinology, 100,799-806.
H. (1972) Influence of disodium ethane-I-hydroxy-l,l-diphos1281 HUGHES,R.H., BAYLINK,D.J., JONES, P.G. & HAUSSLER,
phonate on vitamin D metabolism in rats. PJugers Archiu,
M.R. (1976) Radioligand receptor assay for 25-hydroxy336,249-262.
vitamin DJD, and l,25-dihydroxyvitamin DJD,. Journal of
1251 HUGHES, M.R., BRUMBAUGH,P.F., HAUSSLER, M.R.,
Clinical Investigation, 58,6 1-70.
J.E.& BAYLINK,
D.J. (1975) Regulation ofserum
WERGEDAL,
1.25-dihydroxyvitamin D, by calcium and phosphate in the
1291 HAUSSLER, M.R., BAYLINK, D.J., HUGHES, M.R.,
rat. Science, 190,578-580.
BRUMBAUGH,
P.F., WERGEDAL,J.E., SHEN, F.H., NIELSEN,
1261 TRECHSEL,U., BONJOUR, J.-P., & FLEISCH,
H. (1979)
R.L., COUNTS,S.J., BURSAC,K.M. & MCCAIN,R.A. (1976)
Regulation of the metabolism of 25-hydroxyvitamin D, in
The assay of 1.25-dihydroxyvitamin D,: physiologic and
primary cultures of chick kidney cells. Journal of Clinical
pathologic modulation of circulating hormone levels. Clinical
Investigation, 64,206-21 7.
Endocrinology, 5,151s-165s.