JOURNAL OF BONE AND MINERAL RESEARCH

Volume 9, Number 12, 1994

Mary Ann Liebert, Inc., Publishers

Bone-Forming Ability of 24R ,25Dihydroxyvitamin D, in the

Hypophosphatemic Mouse

TOMOO YAMATE,' HIROYUKI TANAKA,' YUMIKO NAGAI,* HIDEYUKI YAMATO,*

NOBUYUKI TANIGUCHI,' TOSHITAKA NAKAMURA,3 and YOSHIKI SEINO'

ABSTRACT

To determine whether 24R,25-dihydroxyvitamin D, [24R,25(OH),D3] exerts unique biologic effects on bone, we

examined the effects of the vitamin D metabolites, 24R,25(OH),D3 and la,25-dihydroxyvitamin D,
[la,25(OH),D3], on the hypophosphatemic (Hyp) mouse, a model for X-linked hypophosphatemic rickets in
humans. The Hyp mice were administered 1-10,000 pg/kg/day of 24R,25(OH),D3, 0.01-10 pg/kg/day of
la,25(OH),D3, or vehicle alone, given daily for 28 days by intraperitoneal injection. 24R,25(OH),D3 at doses of
1-1000 pg/kg/day had dose-dependent effects in increasing bone size, dry bone weight, and bone mineral
content without causing hypercalcemia. la,25(OH),D3 a t doses of 1 or 10 pglkglday, which we considered to
have activity similar to that of 1000 pg/kg/day of 24R,25(OH),D3 with respect to cell differentiation activity,
caused severe bone resorption and hypercalcemia. At 0.1 pg/kg/day, la,25(OH),D3 increased bone size,
similarly to a dose of 1000 pg/kg/day of 24R,25(OH),D3, without significantly affecting dry bone weight o r bone
mineral content, as did 1000 pg/kg/day of 24R,25(OH),D3. These findings suggest that 24R,25(OH),D3 exerts
unique activity in the Hyp mouse rather than merely mimicking the activity of la,25(OH),D3.

INTRODUCTION ovariectomized dogs.'20' It is interesting that 24R,25(OH),D3

had these effects on bone formation without inducing hypercal-

T HE BIOLOGIC ROLE played by 24R,25-dihydroxyvitamin D,

[24R,25(OH),D3] is a matter of continuing controversy,"'
although it has been established that another vitamin D metabo-
lite, la-25-dihydroxyvitamin D, [ la,25(OH),D3], facilitates
the intestinal absorption of calcium and phosphorus and en-
hances renal mineral reabsorption.'2.3' However, recently it was
reported that 24R,25(OH),D3, alone or together with
la,25(OH),D3, has physiologic effects, especially in bone,'4'
on calcium and phosphorus homeo~tasis.'~' Some reports have
suggested that 24R,25(OH),D3 is important and necessary for
chondrocyte function, demonstrating that it increases the syn-
thesis of DNA'" and pr~teoglycan'~' and increases alkaline
phosphatase activity."," Takigawa et al.""' demonstrated that
24R, 25(OH),D, induced chondrocyte differentiation. More-
over, some investigators have shown that 24R,25(OH),D3
increases bone formation'' ' - I y ) and prevents bone atrophy in
cemia even when given at high d o s e ~ . ( ' ~ - ' ~ '
The hypophosphatemic (Hyp) mouse is a model for familial
X-linked hypophosphatemic rickets in humans.'") It manifests
hypophosphatemia, elevated serum alkaline phosphatase activ-
ity, reduced renal phosphate reabsorption, and abnormal vita-
min D metab~lism.'~*-*~' Bes'ides these biochemical abnormal-
ities, the mutant animals display growth retardation resulting
from rickets and skeletal deformities associated with histologic
evidence of osteomalacia. Besides the abnormal regulation of
la-hydroxylase activity in Hyp mice,'25,26' Seino et aI.'22'
demonstrated elevated vitamin D catabolism, which was later
found to be caused by increased renal 24-hydroxylase activi-
tY .(27-2y' These findings indicate that 24R,25(OH),D3, may
play some physiologic role in Hyp mice, and this prompted us to
examine the effects of this vitamin D metabolite on this mutant
strain. Glorieux et a ~ " ~reported
' that in Hyp mice, the subcu-

'Department of Pediatrics, Okayama University Medical School, Okayama. Japan.

'Kureha Chemical Industry. Tokyo, Japan.
'Department of Orthopedic Surgery, Faculty of Medicine. University of Occupational and Environmental Health, Kitakyushu, Japan.

1967
1968
taneous injection of 24R,25(OH),D3 at a dose of 0.5 pg/kg/day
for 18 days was not effective in improving bone mineralization.
However, Nakamura et a1.“s.16)found that the oral administra-
tion of 24R,25(OH),D3 at a dose of 500 pg/kg/day for 2 years
increased bone mineral content and the mechanical strength of
bone in vitamin D-replete rats. In light of these findings, we
decided to use very high doses of 24R,25(OH),D3 (1-10,OOO
kg/kg/day) to examine the effect of this agent.
MATERIALS AND METHODS
Animals
Inbred Hyp mice were initially provided by the Jackson
Laboratory (Bar Harbor, ME). We then established a homozy-
gous Hyp strain: the males were all C57BL16J Hyp/Y, and the
females were all C57BL/6J Hyp/Hyp. For more than 4 years
since then we have raised only the homozygous Hyp mice in our
experimental animal facility, and we have not had any pheno-
type other than homozygous Hyp mice. The 4-week-old male
Hyp mice (CSBL/6JHyp/Y) and control male mice (C57BL/
6J+/Y) of the same age, purchased from Japan SLC (Shizuoka,
Japan), were used in these experiments. The animals were
housed in plastic cages under a 12 h light and I2 h dark cycle and
were maintained throughout the experimental period on Oriental
MF (Oriental Yeast Co., Ltd., Tokyo, Japan), containing 1.2%
calcium, 0.91% phosphorus, and 80 IU/lOO g of vitamin D.
Food and tap water were available ad libitum to all mice.
Experimental protocol
A total of 157 Hyp mice and 1 13 control mice, all 4 weeks old,
were divided into I0 groups: 5 groups of animals were adrninis-
tered doses of 1, 10, 100, 1000, and 10,OOO pg/kg/day,
respectively, of 24R,2S(OH),D3; 4 groups were administered
doses of 0.01, 0.1, 1, and 10 kg/kg/day, respectively, of
la,25(OH),D3; and 1 group was administered the vitamin D
solvent alone (0.75% ethanol and 0.1% Tween 80). The
24R,25(OH),D3 was donated by Kureha Chemical Industry
Co., Ltd. (Tokyo, Japan), and la,25(OH),D3 was purchased
from Roussel Uclaf (Paris, France). All animals were treated
with 0.1 ml intraperitoneal injections of the vitamin D metabo-
lites or the solvent every day for 28 days. The mortality rate of
the animals during the experimental period was less than 1%,
which was not different from their natural mortality rate. All
mice except those in the groups administered I and 10 pg/kg/day
of la,25(OH),D3 (which were all killed by exsanguination in
the second week of the experiment because they were dying at
this time) were killed at the end of the experiment (12 h after the
last injection) after exsanguination by cardiac puncture. Before
exsanguination, fresh urine was collected with Pasteur pipettes.
The tibiae and femora were then obtained for study.
Analytic procedure
Assay of serum calcium and phosphorus: Serum calcium
and phosphorus concentrations were determined by the or-
thocresolfthalein complexone (OCPC) method using Wako kit
YAMATE ET AL.
272-21801 (Wako Pure Chemical Industries, Ltd., Osaka Japan)
and the p-methylaminophenol reduction method using Wako kit
270-49801, respectively.
Assay ofvitamin D metabolites: The serum levels of three
vitamin D metabolites, la,25(OH),D, 24R,25(OH),D, and
25-hydroxyvitamin D, [25(OH)D,], were assayed in four
groups of animals: those administered vehicle, t h a e adminis-
tered 0.1 pg/kg/day of 1a,25(OH),D3, and those administered
10 and 1000 pg/kg/day of 24R,25(OH),D3. For the assay,
0.3-0.5 ml serum from two mice in the same group were used.
Vitamin D metabolites were extracted by the dropwise addition
of acetonitrile. The extract was then applied directly to a
C 18/OH cartridge (Analytichem International, Harbor City,
CA) to separate the two fractions containing either
la,25(OH),D3 or 24R,25(OH),D3 and 25(OH)D3.‘”~”2.”4’ The
residues containing 24R,25(OH),D3 and 25(OH)D, were ap-
plied to a Gilson high-performance liquid chromatography
(HPLC) system to obtain genuine 24R,25(OH),D3 and
25(OH)D, separated residues. The residues containing
la,25(OH),D3 were also applied to the same HPLC system to
obtain a genuine separated residue.‘”) The radioreceptor assay
for la,25(OH),D was conducted with a receptor for
la,25(OH),D3 obtained from bovine mammary t i ~ s u e . ” ~The ’
competitive binding assay for 25(OH)D, and 24R,25(OH),D3
was performed similarly with a vitamin D binding protein
obtained from the serum of normal rats.‘”)
Assay of urinary cAMP and creatinine: Urinary cAMP
was determined by radioimmunoassay using a Yamasa kit
(Yamasa Shoyu Company, Ltd., Chiba Japan). Creatinine
concentration was determined by the Jaffe method using Wako
kit 277-1050.
Bone analysis: The lengths of the tibia and femur in all
animals were measured with a micrometer. Then, with the
specimen laid directly on soft x-ray film, radiographs were taken
at 25 kVp, 20 mA, for 2 minutes with a soft x-ray apparatus
(Type SRO-MSO;Sofron, Tokyo, Japan). The femur specimens
were defatted in acetone for 7 days and then dried with a vacuum
drier overnight, after which they were weighed. The dried tissue
was then baked at 600°C for 48 h in an electric furnace (Model
FA-21 ;Yamato, Tokyo, Japan). The bone ash thus obtained was
weighed and dissolved in 3 N HCI; the calcium concentration
was measured by the OCPC method, phosphorus by the method
of Fiske and Subbarow, and magnesium by the xylydyl blue
method using Wako kit 274-83901.
Statistical analysis
Differences between the treatment groups and control groups
were tested for significance using one-way analysis of variance.
RESULTS
In Hyp mice, 24R,25(OH),D3 did not induce hypercalcemia
of over 10 mg/dl at any dose except that of 10,000 pglkglday,
whereas la,25(OH),D3 induced hypercalcemia at doses of 1
and 10 pg/kg/day (Table 1). Hypercalcemia of more than I0
mg/dl was observed in four groups of control mice: those that
had received 10,000 pg/kg/day of 24R,25(OH),D3 and those
that had received 0. I , 1, and 10 pg/kg/day of 1a,25(OH),D3.
BONE-FORMING ABILITY OF 24,25(OH),D, 1969

TABLE1. SERUM CALCIUM AND PHOSPHORUS LEVELS”

24R. 250, ( p g l k g ) la,250, (pglkgl

Vehicle I I0 Id Id 104 10-2 10-1 I I0
Hyp mouse
Ca, mg/dl 7.0 6.2 9.7’ 8.8’ 7.4 12.5’ 7. I 8.7 12.0’ 14.3’
(SEW (0.3) (0.2) (0.4) (0.2) (0.5) (1.0) (0.3) (0.5) (0.4) (1.2)
P, mg/dl 5.0 7.2‘ 6.2 5.8 8.2‘ 11.8’ 8.3‘ 9.4d 9.2d 7.9‘
(SEW (0.2) (1.1) (0.9) (0.4) (0.6) (0.5) (0.7) (0.7) (0.4) (0.5)
Control mouse
Ca, mg/dl 9.1 8.7 9.5 10.0‘ 8.8 13.7’ 9.3 11.3d 15.2’ 17.2’
(SEW (1.6) (0.5) (0.6) (0.3) (0.4) (0.8) (0.2) (0.5) (0.4) (0.4)
P, mg/dl 12.2 11.3 10.I 9.8 11.2 9.4 12.1 11.6 8.1 8.0
(SEW (1.3) (1.0) (0.8) (1.0) (0.8) (0.5) (1.1) (1.0) (0.4) (0.7)
“Data are presented as mean ? standard error of the mean (SEM). Serum calcium and phophorus concentrations were determined
by the orthocrestolfthalein complex (OCPC) method and the p-methylaminophenol reduction method, respectively. Indicated values
are significantly different from vehicle group.
’ p < 0.001.
‘ p < 0.05.
dp < 0.01,

Serum phosphorus levels were increased in all experimental
groups of Hyp mice treated with 24R,25(OH),D3 or
la,25(OH),D3.
Serum 24R,25(OH),D3, levels in the groups administered 10
or lo00 pg/kg/day of 24R,25(OH),D3 were about 10 or 100
times greater, respectively, than those in the group administered
vehicle alone, but the serum 24R,25(OH),D3 levels of those
animals administered 0.1 pgikglday of la,25(OH),D3 were
approximately one-half those in the animals administered vehi-
cle alone (Fig. IA). The serum la,25(OH),D, levels in the
group administered 0.1 pg/kg/day of la,25(OH),D3 were about
1.5 times greater than those in the group administered vehicle
alone, but the serum la,25(OH),D3 levels in the group admin-
istered lo00 pg/kg/day of 24R,25(OH),D3 were significantly
less than those in the group administered vehicle alone (Fig.
IB). The 25(OH)D, level did not differ significantly among the
four groups (Fig. IC).
The urinary cAMP/creatinine ratio was significantly reduced
compared with controls in the groups administered both vitamin
D metabolites, being reduced to a greater extent in the group
administered la,25(OH),D, than in that administered
24R,25(OH),D3 (Fig. 2).
The bones of all animals that were treated with 1 or 10
pg/kg/day of la,25(OH),D, were so brittle that we could not
extract them intact. The length of the tibia in the Hyp mice
increased dose dependently with the administration of
24R,25(OH),D3. The length of the tibia in Hyp mice treated
with 0.1 pg/kg/day of la,25(OH),D3 was similar to that in
animals treated with 10oO pglkglday of 24R,25(OH),D3. Not
only the length but also the shape of the tibia was improved by
the administration of the vitamin D metabolites, as demonstrated
by the clear appearance of the tibial tubercles (Fig. 3). In the Hyp
mice, the length of the femur was also increased dose depen-
dently by the administration of the vitamin D metabolites (Table
2); 24R,25(OH),D3 had no effect on bone formation in control
mice.
Radiographic examination showed a marked increase in bone
density, especially in the tibial metaphysis, in Hyp mice treated
with 1000 pg/kg/day of 24R,25(OH),D3, but not in those
treated with 0.1 pg/kg/day of la,25(OH),D3 (Fig. 4).
Dry bone weight, ash weight, and the ratio of ash to dry bone
weight in Hyp mice (right femur) were all increased significantly
by the administration of 1000 and 10,OOO pg/kg/day of
24R,25(OH),D3, but they were not increased significantly by
the administration of 0. I pg/kg/day of la,25(OH),D3. In these
three groups, the calcium and phosphorus content in bone ash
was also increased. However, the increase in the groups treated
with 24R,25(OH),D3 was greater than that in the group treated
with la,25(OH),D3. The ratio of calcium to phosphorus in bone
ash was equally improved in these three groups (Table 3).
DISCUSSION
The administration of 24R,25(OH),D3, even in large doses,
has been reported to be free of any adverse effects, including
hypercaIcemia.(”) We administered 24R,25(OH),D3, in mas-
sive doses to Hyp mice, a model for X-linked hypophosphatemic
rickets in humans. The agent was administered daily for 28 days
and induced increases in bone size, dry bone weight, and bone
mineral content and normalized bone shape, the ratio of ash to
dry bone weight, and the ratio of calcium to phosphorus in the
bone. These effects of 24R,25(OH),D3 were dose dependent.
Importantly, no sign of toxicity, including hypercalcemia, was
observed in any animals administered less than 10oO pglkglday
of 24R.25(OH),D3 during the experimental period. However, 1
or 10 pg/kg/day of la,25(OH),D3, which we considered to have
activity similar to that of 1000 pg/kg/day of 24R,25(OH),D3
with respect to cell differentiati~n,‘~~.~’)
induced severe bone
resorption and marked hypercalcemia. At a dose of 0.1 pg/kg/
day, la,25(OH),D3 also increased bone length and normalized
bone shape similarly to a dose of 1OOO pg/kg/day of
1970 YAMATE ET AL.

P<O.Ol

= @Serum 24R,25(OH)2D level I Pc0.05

(nrnol/rng cr)
5
5 10' 80
T

-f 70
60
Y 10'
0 50
8g 10'
40
30
a 20
2:
$ 10' 10
0
8 Vehicle 103pg/kg/day lO-'pg/kg/day

1 24R, 25m l a , 25b

@ Serum 1a,25(OH)2D level (mean f SEM)

=
1 *O
FIG. 2. Urinary cAMP/Cr (creatinine) ratio in Hyp mice.
Data are presented as mean 2 SEM. Urinary CAMPwas deter-
-f 60
mined by radioimmunoassay. Creatinine concentration was
-
Q) determined by the Jaffe method.
Q
F
0 40
5d pg/kg/day of 24R,25(OH),D3, shown by radiography, was
F
compatible with the chemical analysis of the bone. At least in
20 this experiment, we could not find any points on the two
8 dose-response curves of 24R,25(OH),D3 and 1a,25(OH),D3 at
which the biochemical and skeletal effects of the two vitamin D
0 '

25. a Serum 25(OH)D level

metabolites intersected. These findings suggest that the effects
of 24R,25(OH),D3 did not merely mimic those of
la,25(OH),D3. We therefore believe that the action of the two
=5 20. vitamin D metabolites is qualitatively different.
- We confirmed that a massive dose of 24R,25(OH),D3 re-
-5
Q)
15. duced the serum level of la,25(OH),D3 in both Hyp and control
mice (data not shown). Matsumoto et al. ,'38' who reported the
s same result in calcium-deficient rats, suggested that 24R,25
P
E
lo' (OH),D, enhances the degradation of 1a,25(OH),D3. In this
experiment, however, we were unable to elucidate the mecha-
i 5. nism underlying this phenomenon. Norman et al.'39,40' demon-
strated that 24R,25(OH),D3 acted as an allosteric effector and
0.
Vehicle lo
10' 2[pg/kg/c 1 diminished the affinity of la,25(OH),D3 for its receptor. Yam-
24R.25Dx ln.25D1
*: pe0.05
ato et demonstrated that 24R,25(OH),D3 had a specific
**: P'O.01 inhibitory effect on the formation and function of osteoclastic
***: p<o.o01 cells stimulated by la,25(OH),D3. 24R,25(OH),D3 may act by
mean2SEM
diminishing the effects of la,25(OH),D3. We also found that
FIG. 1. Concentration of vitamin D metabolites in Hyp mice. la,25(OH),D, reduced serum 24R,25(OH),D3 concentrations
Data are presented as mean ? SEM. For assay, 0 . 3 4 . 5 ml in Hyp mice. Further study is essential to elucidate this interac-
serum from two mice in the same group was used. Vitamin D tion of vitamin D metabolites.
metabolites were extracted with acetonitrile, and the extract was The urinary cAMP/Cr ratio in the group administered 0.1
applied to a C 18/OH cartridge to separate the two fractions. The pg/kg/day of la,25(OH),D, was reduced to a greater extent
residues were applied to a Gilson HPLC system to obtain than that in the group administered 1000 p,g/kg/day of 24R3,25
genuine 24R,25(OH),D3-, 25(OH),D,-, and la,25(OH),D3-
(OH),D,. This suggests that hyperparathyroidism was not rele-
separated residues. 24R,25(OH),D3 and 25(OH)D, were as-
sayed by competitive protein binding assay. la,25(OH),D3 was vant to the experimental findings.
assayed by radioreceptor assay. Glorieux et al.'30' demonstrated that 24R,25(OH),D3 admin-
istered with phosphorus did not improve bone mineralization in
Hyp mice, but bone mineralization was corrected by
24R,25(OH),D3; however, it did not significantly increased dry la,25(OH),D,.'42' However, the dose of 24R,25(OH),D3 they
bone weight, ash or the ratio of ash to dry bone weight, and it did used in their experimental protocol was 0.5 pg/kg/day, far less
not increased calcium, phosphorus, or magnesium content in the than the doses we used in this experiment (I-10,OOOpglkgl
bone in Hyp mice to the same extent as lo00 pg/kg/day of day).
24R,25(OH),D3. The marked increase in bone density, particu- Nakamura et a1."','6' showed that 24R,25(OH),D3 had
larly in the tibia1 metaphysis, in Hyp mice treated with 1000 bone-forming ability (increase in bone mineral density and
BONE-FORMING ABILITY OF 24,25(OH),D, 1971

FIG. 3. Right tibia. The bones of all animals treated with I or 10 pg/kg/day of la,25(OH),D3 were so brittle that they could not
be extracted intact.

mechanical strength) in normal Wistar rats. In our experiments
here, we found that 24R,25(OH),D3 had no definite effects on
bone formation in control mice. This discrepancy in findings
may be a result of differences in the experimental period. Our
experimental period (4 weeks) was much shorter than theirs (2
years), and it is possible, therefore, that the bone-forming ability
of 24R,25(OH),D3 in control mice may not have been mani-
fested in the shorter period. The effects of this agent may have
been more apparent in the Hyp mice, in which 24K,25(OH),D3
plays a significant role. These results were obtained only by the
intraperitoneal injection of vitamin D metabolites. However, we
cannot exclude the possibility that results may have been
different if the vitamin D metabolites had been given by a
different route.
It is thought that 24R,25(OH),D3 acts after being converted
into la, 24R, 25(OH),D, by renal la-hydr~xylase.'~"Further
studies are essential to elucidate the bone-forming effects of la,
24R, 25(OH),D, and other vitamin D metabolites derived from
24R. 25(OH),D,.
In conclusion, 24R,25(OH),D3 at doses of 1-1ooO pg/kg/day
had dose-dependent effects in increasing bone formation and
bone mineralization without inducing hypercalcemia in Hyp

OF RIGHTFEMORA
TABLE2. LENGTHS AND TIBIAE"

Hyp mouse

24R, 2 5 4 (pglkg) la,2 5 4 ( ~ d k g )

Control,
vehicle Vehicle 1 10 lo2 Id 104 l o- , 10-1

Femur, mm 14.89' 10.15 10.55 10.70' 10.70' 11.61' 12.03' 10.74' 1 1 .26d
(0.14) (0.16) (0.18) (0.18) (0.18) (0.22) (0.22) (0.19) (0.26)
Tibia, mm 17.58' 11.94 12.71' 12.80d 12.61 13.56" 14.16' 12.34 13.56'
(0.07) (0.23) (0.18) (0.18) (0.09) (0.24) (0.16) (0.20) (0.25)
aData are presented as mean ? SEM. Lengths of the tibia and femur were measured with a micrometer. The bones of all animals
treated with 1 and 10 p.g/kg/day of la, 25 (OH),D, were so brittle that they could not be extracted intact. Indicated values are
significantly different from Hyp mouse vehicle group values.
bp < 0.001.
'p < 0.05.
dp < 0.01.
1972 YAMATE ET AL.

Hyp mouse

FIG. 4. Radiography of tibia1 metaphysis. The right tibiae were laid directly on soft x-ray film and radiographs were taken at 25
kVp, 20 mA, for 2 minutes with a soft x-ray apparatus.

TABLE3. DRY BONEWEIGHT, ASH WEIGHT, RATIOOF ASHTO DRY BONEWEIGHT, AND BONEMINERAL CONTENT
AND RATIOOF CALCIUM TO PHOSPHORUS) IN THE RIGHTFEMUR^
(CALCIUM, PHOSPHORUS, MAGNESIUM,

Hyp mouse

24R, 2503 ( P g l k g )
Control,
vehicle Vehicle 1 10 Id Id 104 lop2 lo-'

Dry bone weight, mg 38.9' 19.3 21.1 22.8 23.2' 28.8* 27.6d 21.1 23.3
(2.2) (1.5) (0.7) (1.7) (0.7) (1.6) (1.6) (0.8) (1.5)
Ash, mg 22.6b 8.6 9. I 10.0 9.8 14.7d 15.1d 8.5 10.8
(1 .O) (1.0) (0.7) (1.0) (0.5) (1.0) (0.4) (0.5) (0.8)
AsWdry bone weight, % 53.3' 44.0 43.1 45.0 42.2 50.9' 51.8' 40.4 46.2
(1.2) (2.3) (1.9) (1.5) (1.2) (1.0) (0.5) (2.3) (1.0)
Bone Ca content, mg 8.53* 2.19 3.05' 3.45* 3.4@ 4.93' 5.68' 2.82 4.12d
(0.41) (0.28) (0.11) (0.22) (0.13) (0.19) (0.26) (0.14) (0.41)
Bone P content, mg 4.6Ib I .59 1.73' 1.95 1.92 2.87' 2.70d 1.68 2.26'
(0.23) (0.15) (0.06) (0.15) (0.06) (0.20) (0.21) (0.07) (0.20)
Bone Mg content, mg 0.23d 0.10 0. I 1 0.12 0.12 0.16d 0.18' 0.09 0.14
(0.02) (0.01) (0.00) (0.01) (0.01) (0.01) (0.00) (0.01) (0.02)
Ca/P 1 .90d 1.54 1.76 1.78 1.81' 1.82' 1.93' 1.68 1.82'
(0.02) (0.09) (0.04) (0.04) (0.04) (0.03) (0.02) (0.05) (0.03)

"Data are presented as mean ? SEM. After being defatted in acetone for 7 days, femur specimens were dried with a vacuum drier
overnight and then weighed. The dry tissues was then baked at 600°C for 48 h in an electric furnace. Bone ash thus obtained was
weighed and dissolved in 3 N HCI; the concentration of calcium was measured by the orthocresolfthalein complex (OCPC) method,
phosphorus by the method of Fiske and Subbarow, and magnesium by the xylydyl blue method. Indicated values are significantly
different from values in Hyp mouse vehicle group.
bp < 0.001.
' p < 0.05.
dp < 0.01.

mice. We suggest that 24R,25(OH),D3 possesses a unique We thank Professor Anthony W. Norman for his constructive
bone-forming activity rather than merely mimicking the effects suggestions and comments. We also thank Rumi Abe-Nojima
of la,25(OH),D3. for her technical assistance.

REFERENCES
ACKNOWLEDGMENTS
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This study was supported in part by grants from the Ministry of 24,25-dihydroxyvitamin D, administration on parameters of
of Health and Welfare and the Ministry of Education of Japan. calcium metabolism. J Clin Endocrinol Metab 69:467-469.
BONE-FORMING ABILITY OF 24,25(OH),D,
2. Norman AW, Roth J , Orci L 1982The vitamin Dendocrine system:
Steroid metabolism, hormone receptors, and biological response
(calcium binding proteins). Endocr Rev 3:331-366.
3 . Haussler MR 1986 Vitamin D receptors: Nature and function. Annu
Rev Nutr 6527-562.
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