From the Department of Hygienic Sciences, Kobe
Pharmaceutical University, Kobe 658-8558, Japan, the
§ Faculty of Pharmaceutical Sciences, Nagasaki University,
Nagasaki 852-8521, Japan, the ¶ Department of Environmental
Medicine, Research Institute, Osaka Medical Center for Maternal and
Child Health, Osaka 594-1101, Japan, the
Chugai Pharmaceutical
Co. Ltd., Tokyo 171-8301, Japan, and ** Women and Infants'
Hospital, Brown University, Providence, Rhode Island 02905
Received for publication, April 18, 2002, and in revised form, September 18, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
22-Oxacalcitriol (OCT) is an analog of
calcitriol, characterized by potent differentiation-inducing activity
and low calcemic liability. The metabolism of OCT has been studied and
its polar metabolites, such as 24/26-hydroxylated-OCT and
hexanor-1 The hormonal form of vitamin D3,
1 Recently, the metabolism of OCT has been studied in primary parathyroid
cells (17) and keratinocytes (18) as well as osteosarcoma, hepatoma,
and keratin cell lines (19). In all these systems, OCT is degraded into
hydroxylated and side-chain truncated metabolites, 1 There are multiple aims of this study as follows: to identify the less
polar metabolites of OCT including 3-epi-OCT in a rat osteosarcoma cells (UMR 106 cells); to investigate the further metabolism of OCT metabolites; to compare the production rate of OCT
metabolites in UMR 106, human colon carcinoma cells (Caco-2) and
porcine kidney cells (LLC-PK1); and to examine the
biological activity of OCT metabolites. Our findings provide clear
evidence that OCT is metabolized to at least three, and possibly five, less polar metabolites through two novel pathways, namely the C-3
epimerization and C-25 dehydration pathways in addition to the
well-known C-23/C-24/C-26 hydroxylation pathways in target cells.
Materials--
OCT and its putative metabolite
24-ene-22-oxa-1 Cell Culture--
UMR 106, Caco-2, and LLC-PK1cells
were obtained from the American Type Culture Collection (ATCC,
Manassas, VA). UMR 106 cells were maintained in Dulbecco's modified
Eagle's medium (DMEM) containing 10% fetal calf serum (FCS). Caco-2
cells were maintained in Eagle's Medium containing 10% FCS and 1%
non-essential amino acids. LLC-PK1 cells were maintained in
Medium 199 containing 5% FCS. All culture media contained penicillin
(100 IU/ml) and streptomycin (100 µg/ml). Cells were cultured at
37 °C in a humidified atmosphere of 5% CO2 in air, and
the medium was changed every three days. In the experiments described
below, OCT or 3-epi-OCT was added to culture medium in
ethanolic solution, the final ethanol concentration in the medium never
exceeding 0.1% (v/v).
Generation of OCT and 3-epi-OCT Metabolites--
For structure
assignments, 10 plates of UMR 106 cells cultured in 150-mm culture
dishes were used. Monolayers were washed with 10 ml of
phosphate-buffered saline without calcium and magnesium (PBS( Purification of OCT and 3-epi-OCT Metabolites by HPLC--
Lipid
extraction was performed according to the method of Bligh and Dyer (25)
as modified by Makin et al. (26). Lipid was extracted from
cells and media with methanol and dichloromethane. The organic layer
was evaporated to dryness under a stream of nitrogen, redissolved in
hexane/2-propyl alcohol/methanol (HIM), (88:10:2, v/v/v), and subjected
to purification by HPLC. Analytical HPLC of metabolites was carried out
using a model 600 pump and a model 996 photodiode array detector
(Waters Associates, Milford, MA). Separation of metabolites was
initially achieved using a Zorbax SIL column (4.6 × 250 mm,
Dupont Instruments, Wilmington, DE) eluted with HIM (88:10:2) at a flow
rate of 1.0 ml/min. Samples with peaks representing a typical
chromophore based on the vitamin D cis-triene structure
( 1H NMR and LC-MS Analyses--
The 500-MHz
1H NMR spectra of the metabolites of OCT or
3-epi-OCT were measured on a Varian VXR-500 (1H:
499.9 MHz). Purified metabolites (2-5 µg) were dissolved in 40 µl
of CDCl3 with a very small amount of CHCl3
(7.24 ppm, used as an internal standard for 1H NMR
spectroscopy) and transferred into a nanoprobe. Two-dimensional spectra
were obtained as described previously (27). LC-MS analysis was carried
out on a QUATTROII (Micromass, Manchester, UK) equipped with an
electrospray ionization (ESI) source in the positive ion mode. The HPLC
system consisting of a MAGIC2002 Micro-LC full system (Michrom Bio
Resources, Inc.) and a Develosil ODS-HG-5 column (2.0 × 150 mm,
NOMURA CHEMICAL, Tokyo, Japan) was used. As the mobile phase, methanol,
10 mM ammonium acetate (50:50, v/v; A) and methanol, 10 mM ammonium acetate (98:2, v/v; B) were used, and a linear
gradient elution was run from an A/B ratio of 65:35 to 20:80, at a flow
rate of 0.2 ml/min. The column temperature was maintained at 40 °C.
Mass spectra were obtained by averaging each peak and subtracting the background.
VDR and DBP Binding Assays--
Displacement of
1 Transfection and Luciferase Activity Assay--
The human
osteosarcoma cell line MG-63 (ATCC) was maintained in Dulbecco's
modified Eagle's medium supplemented with penicillin (100 IU/ml),
streptomycin (100 µg/ml), and 10% dextran-coated charcoal-treated
FCS. Cells (2 × 105) were suspended in 2 ml of the
medium and transfected with 0.5 µg of luciferase reporter plasmid
(pGVB2 vector, Toyo Ink Co., Ltd., Tokyo, Japan) carrying a human
osteocalcin gene promoter ( Anti-proliferative Activity Assay and Cell Surface Antigen
Expression Analysis--
The human promyelocytic leukemia cells
(HL-60) were kindly provided by Dr. M. Inaba of Osaka City University
Medical School. The cells were maintained in RPMI 1640 medium (Nissui
Pharmaceutical Co., Tokyo, Japan) supplemented with 10% dextran-coated
charcoal-treated FCS and kanamycin (0.06 mg/ml) at 37 °C in a
humidified atmosphere of 5% CO2 in air. For
synchronization at the S phase, cells (4 × 106) were
cultured in 30 ml of RPMI 1640 medium for 24 h and subsequently cultured for 16 h in RPMI 1640 medium supplemented with 2.5 mM thymidine. After washing with PBS( Statistics--
Values were calculated as means ± S.E.
Significance levels were determined by Student's t test.
Metabolism of OCT in UMR 106 Cells
Incubation of 10 µM OCT with UMR 106 cells for
48 h resulted in the formation of 5 metabolites (Fig.
1A), all of which demonstrated the typical vitamin D chromophore (,20-dihydroxyvitamin D3
(1
,20(OH)2D3), have been identified. In
contrast, little is known about the less polar metabolites of OCT,
which have been found in relatively large amounts. In this study, the
in vitro metabolism of OCT was studied in UMR 106, Caco-2,
and LLC-PK1 cells to identify the less polar metabolites
and to assess their biological activity. OCT was initially metabolized
to three less polar metabolites, 3-epi-OCT and two
dehydrates, 25-dehydroxy- 25-ene-22-oxa-1
(OH)D3
(25-ene-22-oxa-1
(OH)D3) and
25-dehydroxy-24-ene-22-oxa-1
(OH)D3 (24-ene-22-oxa-1
(OH)D3). We also observed further
metabolites, the two C-3 epimers of the C-25 dehydrates,
25-ene-3-epi-22-oxa-1
(OH)D3 and
24-ene-3-epi-22-oxa-1
(OH)D3. The structures
of these metabolites were successfully assigned by 1H NMR
and LC-MS analyses. The three cell lines differ in their ability to
metabolize OCT through the C-3 epimerization or the C-25 dehydration
pathway. The biological activity of the OCT metabolites assessed by a
luciferase reporter gene transcriptional activation system, binding
assays for the vitamin D receptor (VDR) and vitamin D-binding protein
(DBP), and assays for regulatory activities of cell
differentiation and proliferation was found to be lower than that of
OCT. Thus, both the C-3 epimerization and C-25 dehydration may work to
reduce the biological activity of OCT.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,25-dihydroxyvitamin D3
(1
,25(OH)2D3 or
calcitriol)1 plays a crucial
role in the regulation of calcium metabolism but it also regulates cell
growth and differentiation in a variety of normal and malignant cells
(1-3). Despite the potency of 1
,25(OH)2D3 as a negative growth regulator of colon (4, 5), breast (6, 7), and
prostate cancer cells (8), its hypercalcemic properties have precluded
its therapeutic use. To overcome this problem, vitamin D analogs with
enhanced differentiation/anti-proliferative and reduced hypercalcemic
properties have been synthesized. OCT is a synthetic analog, which has
an oxygen atom at position 22, and has received government approval for
use as an agent for the treatment of secondary hyperparathyroidism and
psoriasis in Japan. OCT is rapidly cleared from the circulation due to
its extremely low affinity for DBP (9, 10), and binds the chicken
vitamin D receptor (VDR) with an approximately 8-fold lower affinity
than 1
,25(OH)2D3 (11). However, OCT inhibits
growth of psoriatic fibroblasts and enhances the immune response more
effectively than 1
,25(OH)2D3 (12, 13). In
contrast, OCT has reduced calcemic effects both in terms of mobilizing
calcium from bone and in stimulating intestinal calcium transport in
vitamin D-deficient and normal rats (14, 15). Recently, Kato and
co-workers (16) reported that OCT induced interaction of the VDR with a
transcriptional factor TIF-2, but not with other transcriptional
factors such as SRC-1 and AIB-1, while
1
,25(OH)2D3 induced interactions of the VDR
with all of the three co-factors tested. These factors such as
metabolic clearance, tissue-specific distribution, cellular uptake,
intracellular metabolism, and transcriptional regulation could
contribute to differences in biological activity.
,20(OH)2D3 and
hexanor-20-oxo-1
-hydroxyvitamin D3
(20-oxo-1
(OH)D3). However, despite relatively large
amounts of products from OCT (19), the structures and properties of the
less polar metabolites have not yet been clarified. In the case of
1
,25(OH)2D3, one of these less polar
metabolites has been identified as
3-epi-1
,25(OH)2D3, in which a
hydroxyl group at C-3 of the A-ring is epimerized from the
to the
position (20, 21). The C-3 epimerization of 1
,25(OH)2D3 occurs in vitro
(20-22) and in vivo (23), and is a highly
tissue-specific/cell differentiation-dependent process (21). 3-epi-1
,25(OH)2D3 retains
24% binding affinity to VDR (24), and exhibits significant activity in
reducing growth and promoting differentiation of Caco-2 cells (21).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(OH)D3 were synthesized by Kubodera
et al. of Chugai Pharmaceutical Co., Ltd., Japan. The
synthesis and unambiguous stereochemical assignment of
24-ene-22-oxa-1
(OH)D3 will be described
elsewhere.2
3-epi-OCT and
3-epi-1
,25(OH)2D3 were
synthesized by Hatakeyama et al. of Nagasaki University,
Japan. 1
,25(OH)2D3 and 25-hydroxyvitamin D3 (25(OH)D3) were obtained from Solvay-Duphar
Co. (Weesp, The Netherlands).
1
,25(OH)2[26,27-methyl-3H]D3
(6.6 TBq/mmol) and 25(OH) [23,24(n)-3H]D3
(3.0 TBq/mmol) were purchased from Amersham Biosciences. Culture media
and antibiotics were purchased from Invitrogen. Deuterized chloroform
(CDCl3, 99.8%, NMR analytical grade) was purchased from
EURISO-TOP (Gif-Sur-Yvette, France). Organic solvents of HPLC grade
were obtained from Wako Pure Chemical Industries, Ltd.
)) and
then incubated in 10 ml of media containing 1% bovine serum albumin,
in the presence of 10 µM OCT or 3-epi-OCT for
48 h at 37 °C. The no-cell control consisted of 10 ml of the
medium and 10 µM OCT or 3-epi-OCT was
incubated for the same length of time. For measurement of amounts of
OCT and 3-epi-OCT metabolites, three plates each of UMR 106, LLC-PK1, and Caco-2 cells cultured in 150-mm culture dishes
were used. For time-course studies, the cells were incubated with 5 µM substrates for periods of time ranging from 1 to
48 h. For dose-response studies, the cells were incubated with
increasing amounts (0.1-10 µM) of substrates for 24 h.
max, 265 nm,
min, 228 nm) were collected and evaporated to dryness, then redissolved in HIM (88:10:2). These
were further purified on the same HPLC system using a Zorbax CN column
(4.6 × 250 mm, Dupont Instruments) eluted with HIM (88:10:2) at a
flow rate of 0.9 ml/min. After further purification with the same
previous HPLC system, the metabolites were pure enough for LC-MS and
1H NMR analyses. Concentrations of the metabolites in
ethanol were determined spectrophotometrically using a molar extinction
coefficient,
265 of 18,000.
,25(OH)2[3H]D3 from
calf-thymus cytosol receptors (Yamasa Co. Ltd., Chiba, Japan) by OCT
and its metabolites was determined as described elsewhere (19).
Solutions containing 500 µl of the calf thymus cytosol receptor
prepared with phosphate buffer (0.3 M KCl, 0.05 M K2HPO4, 0.05 M
KH2PO4, pH 7.4) were mixed with increasing
amounts of 1
,25(OH)2D3 (0.0078-64 pg/tube), OCT (0.5-1024 pg/tube), 3-epi-OCT (32-16384 pg/tube),
25-ene-22-oxa-1
(OH)D3 (8-32768 pg/tube) or
24-ene-22-oxa-1
(OH)D3 (8-32768 pg/tube) in 20 µl
ethanol, and the samples were incubated for 1 h at 20 °C. Next,
34 fmol of 1
,25(OH)2[3H]D3 in
25 µl of ethanol was added, and the samples were incubated for 1 h at 20 °C. The addition of 200 µl of dextran/charcoal (0.05% dextran T-150/0.5% Norit A Charcoal Decolorizing Neutral) in freshly prepared phosphate buffer (0.05 M
Na2HPO4, 0.05 M
NaH2PO4, pH 7.4) was used to separate the bound
and free forms of
1
,25(OH)2[3H]D3. The assay
tubes were incubated on ice for 10 min and centrifuged at 3000 rpm for
10 min at 4 °C. Each supernatant was collected in 500 µl and
transferred into a scintillation vial to measure radioactivity.
Competitive displacement of 25(OH) [3H]D3
from vitamin D-deficient rat serum DBP by OCT and its metabolites was
determined under equilibrium ligand-binding conditions (19). A total of
82 fmol of 25(OH) [3H]D3 in 50 µl of
ethanol was mixed with increasing amounts of 25(OH)D3
(0.003125-16 ng/tube), 1
,25(OH)2D3
(0.24-4000 ng/tube) or OCT metabolites (0.06-1000 ng/tube) in 100 µl of ethanol. Next, 1 ml of vitamin D-deficient rat serum diluted
1:70,000 with freshly prepared barbital acetate buffer (3.5 mM acetic acid, 3.5 mM sodium barbiturate, 0.13 M NaCl, 0.1% ovalbumin, pH 8.6) was added, and the samples
were incubated on ice for 1 h. To separate the bound and free
forms of 25(OH) [3H]D3, 500 µl of
dextran/charcoal (0.025% dextran T-150/0.25% Norit A charcoal
decolorizing neutral) in freshly prepared barbital acetate buffer was
used. The assay tubes were vortexed and centrifuged at 3000 rpm for 10 min at 4 °C. Following centrifugation, 1.0 ml of each supernatant
was collected and transferred into a scintillation vial to measure radioactivity.
848/+10) including the VDRE (28) or a rat
CYP24 gene promoter (
291/+9) including the two VDREs (29),
and 0.25 µg of the pRL-CMV vector (pGVB2 vector, Toyo Ink Co., Ltd.)
as an internal control. The transfection agent used was Tfx-50 reagent
(Promega Corp. Madison, WI). The cells were incubated with
10
9 or 10
8 M
1
,25(OH)2D3 or OCT compounds for 48 h.
The luciferase activity of the cell lysates was measured with a
luciferase assay system (Toyo Ink Co., Ltd.). Transactivation measured
by luciferase activity was standardized with the luciferase activity of
the same cells determined by the Sea Pansy luciferase assay system as a
control (Toyo Ink Co., Ltd.) (30). Each set of experiments was repeated at least three times.
) twice, the cells
were cultured in normal medium for 10 h and then in 2.5 mM thymidine medium for 16 h. The cells thus obtained
were used for flow cytometry. The cells (105 cells/well)
were placed in 24-well tissue culture plates and cultured for 3 days
with OCT or its metabolites (10
9, 10
8
M). Each group of cells was washed with PBS(
) and
resuspended in PBS(
) containing 0.2% Triton X-100 and 100 µg of
RNase, then incubated at 37 °C for 1 h. Cells were washed with
PBS(
) and incubated with 0.5 ml of DNA-staining solution containing
propidium iodide (50 µg/ml) at 4 °C for 20 min. The cells were
analyzed with a flow cytometer equipped with an argon laser (488 nm,
Becton Dickinson FACScanTM), and cell cycle distribution
was analyzed by ModiFiT LT (Verity). For analysis of cell surface CD11b
antigen expression, HL-60 cells (105 cells/well) were
cultured for 3 days with OCT or 3-epi-OCT metabolites (10
9, 10
8 M) under the same
conditions as for flow cytometry. Each group of cells was washed with
PBS(
), and the cells (2 × 105 cells) were
resuspended in 100 µl of diluent solution containing 1% bovine serum
albumin and 1% sodium azide. Then the cells were incubated with 10 µl of human monoclonal fluorescein isothiocyanate-conjugated CD11b
antibody (Sigma) for 30 min at room temperature. The cells were washed
once with diluent solution and then fixed in 300 µl of PBS(
)
containing 2% paraformaldehyde. Fluorescence was detected by a Becton
Dickinson FACScanTM at an excitation wavelength of 490 nm
and an emission wavelength of 520 nm. Results were recorded as the mean
fluorescence index, which is the product of the percent fluorescence
and the mean fluorescence intensity, with 104 cells being
counted per treatment. Each set of experiments was repeated at least
three times.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
max of 265 nm,
min of 228 nm; data not shown). These metabolites were
not formed when OCT was incubated with medium in the absence of cells,
or when cells were incubated with medium alone (data not shown). 2 of 5 metabolites, labeled Metabolites 4 and 5, were polar metabolites of OCT
and identical to the previously identified
1
,20(OH)2D3 and 24-hydroxy-OCT (24(OH)OCT),
respectively (19). Three other less polar metabolites, labeled
Metabolites 1, 2, and 3 have not been isolated and identified to date
and were thus purified by extensive rechromatography twice on Zorbax CN
and Zorbax SIL for structure assignments.
View larger version (35K):
[in a new window]
Fig. 1.
HPLC chromatograms of the lipid extracts from
UMR 106 cells incubated with 10 µM
OCT or 3-epi-OCT for 48 h. A, OCT
metabolites; B, 3-epi-OCT metabolites. HPLC
analysis was performed using a Zorbax-SIL column (4.6 × 250 mm)
eluted with HIM 88:10:2 at a flow rate of 1.0 ml/min. Metabolites 1, 2, and 3 were identified as 25-ene-22-oxa-1 (OH)D3, and
24-ene-22-oxa-1
(OH)D3, and 3-epi-OCT,
respectively. Metabolites 1' and 2' were identified as
25-ene-3-epi-22-oxa-1
(OH)D3, and
24-ene-3-epi-22-oxa-1
(OH)D3,
respectively.
Identification of Less Polar Metabolites of OCT (Metabolites 1, 2, and 3)
OCT Metabolite 1--
The 1H chemical shifts and
coupling constants assigned by one-dimensional and two-dimensional COSY
and NOESY spectra of Metabolite 1 are summarized in Table
I. The most pronounced differences between the 1H NMR spectra of Metabolite 1 and OCT were
found in the 1.6-1.8 ppm and 4.6-4.8 ppm regions. The disappearance
of one of the singlets from the methyl protons in position 26 or 27 (observed at 1.21 and 1.23 ppm in OCT) and the appearance of two
singlets at 4.70 and 4.74 ppm, which were assigned to protons of the
exo-methylene group, were observed. These findings indicate that
dehydration took place at the C-25 hydroxyl group. The resonances from
H-24 (2.23 ppm) were shifted downfield compared with those of OCT (1.71 ppm). In the two-dimensional COSY spectrum, cross-peaks of the neighboring protons were detected. Connectivity of the H-23 and 23'
(3.33 and 3.63 ppm) to the H-24 resonance (2.23 ppm) was observed. The
other cross-peaks observed in two-dimensional COSY analysis are shown
in Fig. 2A. Metabolite 1 was
assigned to 25-ene-22-oxa-1(OH)D3, which has an OCT-like
structure with an additional exo-methylene group at the end of the
side-chain as a result of dehydration at the C-25 hydroxyl group of
OCT. In the LC-MS spectrum of Metabolite 1, [M+NH4]+ was observed at
m/z 418.6 and indicated a reduction of 18 mass units from OCT (Table I). Metabolite 1 gave peaks at
m/z 401.6 [M+H]+ and 423.7 [M+Na]+. Therefore, the LC-MS spectrum supports the
assignment of Metabolite 1 structure by 1H NMR
analysis.
|
|
OCT Metabolite 2--
The 1H NMR spectrum of
Metabolite 2 also showed an unchanged structure of ring A, containing
the triene system (Table I). The resonance from H-24 (5.32 ppm) was
changed in contrast with that of OCT (1.71 ppm). The intensity of the
H-24 signal was found to be only half of the expected value. Two
singlets from the methyl protons in positions 26 and 27 (1.64 and 1.71 ppm) were shifted downfield compared with those of OCT (1.21 and 1.23 ppm). The resonances from H-23 and 23' (3.79 and 4.01 ppm) were also
shifted downfield. These findings indicate that an olefin group was
introduced between C-24 and C-25, and only one proton atom at H-24
remained in Metabolite 2. In the two-dimensional COSY spectrum,
cross-peaks showing connectivity between the neighboring protons were
clearly observed (Fig. 2B). The signal at 1.71 ppm was
assigned to the proton of trans position against C-23
(namely, C-26 position) because of the presence of cross-peaks with
H-23' and 24 that demonstrate long range coupling. The signal at 1.64 ppm was also assigned to the proton of cis position against
C-23 (namely, C-27 position). The LC-MS spectrum of Metabolite 2 in
principle showed the same features as the spectrum of Metabolite 1;
[M+NH4]+ was observed at
m/z 418.6, suggesting a reduction of 18 mass units from OCT (Table I). Consequently, Metabolite 2 was assigned to
24-ene-22-oxa-1(OH)D3, a dehydrate of OCT in which a
double bond was introduced between the C-24 and C-25 positions by
dehydration of the C-25 hydroxyl group of OCT, as was seen in
Metabolite 1. 1H NMR and LC-MS spectra of synthesized
24-ene-22-oxa-1
(OH)D3 were completely congruent with
those of Metabolite 2. Metabolite 2 co-migrated with authentic
24-ene-22-oxa-1
(OH)D3 on Zorbax SIL chromatography.
OCT Metabolite 3-- Metabolite 3 was inferred to be 3-epi-OCT by co-migration with the authentic standard in HPLC analysis. To confirm this interpretation, 1H NMR and LC-MS spectral analyses were performed. In the 1H NMR spectrum of Metabolite 3, the signals of H-3 (4.03 ppm) and H-1 (4.31 ppm) were observed in the same positions as for authentic 3-epi-OCT, shifted upfield compared with OCT (Table I). These findings suggest that both protons in positions 1 and 3 tended to be in axial arrangement. In the LC-MS spectra of OCT and 3-epi-OCT, [M+NH4]+ was observed at m/z 436.7. In the spectrum of Metabolite 3, [M+NH4]+ was also observed at m/z 436.7 and the other ions, 419.6 [M+H]+ and 441.7 [M+Na]+ showed identical patterns to the spectra of OCT and 3-epi-OCT respectively (Table I). It was indicated that the structure of Metabolite 3 is a diastereomer or a geometric isomer of OCT. Based on the findings of HPLC, 1H NMR, and LC-MS analyses, Metabolite 3 was assigned as 3-epi-OCT, which has a changed configuration of a hydroxyl group at C-3 of the A-ring.
Metabolism of 3-epi-OCT in UMR 106 Cells
Incubation of UMR 106 cells with 10 µM
3-epi-OCT for 48 h resulted in the formation of 4 metabolites (Fig. 1B). 2 of 4 metabolites, labeled
Metabolites 3' and 4' were corresponded to the C-3 epimers of
1,20(OH)2D3 and 24(OH)OCT, respectively. Two
other less polar metabolites, labeled Metabolites 1' and 2' were
purified for structure assignments by 1H NMR spectroscopy
and LC-MS analyses.
Identification of Less Polar Metabolites of 3-epi-OCT (Metabolites 1' and 2')
The proton chemical shifts assigned by one- and two-dimensional
COSY and NOESY spectra of purified Metabolites 1' and 2' are summarized
in Table I. Except for the chemical shifts of H-1 and 3, the resonances
from all protons of Metabolites 1' and 2' matched those of Metabolites
1 and 2, respectively. The chemical shifts of H-1 and 3 of Metabolites
1' and 2' were observed at the same upfield position as
3-epi-OCT compared with OCT. In the LC-MS spectra of
Metabolites 1' and 2', [M+NH4]+ was also
observed at m/z 418.6, and the other ions, 401.6 [M+H]+ and 423.7 [M+Na]+ showed identical
patterns to the spectra of Metabolites 1 and 2 (Table I). From the
findings of 1H NMR and LC-MS analyses, Metabolites 1' and
2' were assigned as
25-ene-3-epi-22-oxa-1(OH)D3 and
24-ene-3-epi-22-oxa-1
(OH)D3, respectively. In
this experiment, OCT was not detected as a metabolite of
3-epi-OCT. This finding suggests that C-3 epimerization
occurs unidirectionally as previously reported (20, 31).
Production Rates of OCT Metabolites in UMR 106, Caco-2 and LLC-PK1 Cells
Because of a lack of authentic compounds for
25-ene-22-oxa-1(OH)D3 and 24(OH)OCT, the amounts of
these metabolites were measured by Zorbax SIL HPLC using the
metabolites purified from UMR 106 cell culture as the standard
compounds. The same less polar metabolites of OCT were generated in all
cell lines tested, although there were differences existing in the
amounts of products formed among cell types (Fig.
3). The major metabolite found in cell
cultures of Caco-2 and LLC-PK1 was 24(OH)OCT, whereas
25-ene-22-oxa-1
(OH)D3 appeared to be more prevalent in
UMR 106 cells. Interestingly, in UMR 106 cells the production ratio of
25-ene-22-oxa-1
(OH)D3 to
24-ene-22-oxa-1
(OH)D3 was 2:1; however, in Caco-2 and
LLC-PK1 cells, the production ratios of
25-ene-22-oxa-1
(OH)D3 to
24-ene-22-oxa-1
(OH)D3 were 1:4 and 1:11,
respectively.
|
Dose Response and Time Course Studies of Metabolism of OCT and 3-epi-OCT in UMR 106 Cells
25-Ene-22-oxa-1(OH)D3,
24-ene-22-oxa-1
(OH)D3, 24(OH)OCT, and
3-epi-OCT were produced in a dose-dependent
manner in up to 10 µM OCT as shown in Fig.
4A. When UMR 106 cells were
incubated with 0.1 µM OCT, 24(OH)OCT was predominantly
produced. However, when the cells were incubated with 1-10
µM OCT, the major metabolite was
25-ene-22-oxa-1
(OH)D3. Interestingly, the production
ratio of 25-ene-22-oxa-1
(OH)D3 to
24-ene-22-oxa-1
(OH)D3 was 2:1 at any concentration
of OCT. In a time course study, 25-ene-22-oxa-1
(OH)D3 and 24(OH)OCT were first detected approximately 1 h after the incubation was begun, and continued to increase up to the end of the
incubation period (Fig. 4B).
24-Ene-22-oxa-1
(OH)D3 and 3-epi-OCT were
first apparent at 3 h of incubation.
24-Ene-22-oxa-1
(OH)D3 continued to increase up to the
end of the incubation period, whereas 3-epi-OCT gradually
increased and reached only 30% of the amount of
25-ene-22-oxa-1
(OH)D3 by the end of the incubation period.
|
We also examined dose response and time course studies of
3-epi-OCT metabolism in UMR 106 cells. In a dose response
study, two dehydrates were produced in a dose-dependent
manner in up to 10 µM 3-epi-OCT (Fig.
4C). At any concentration, the ratio of
25-ene-3-epi-22-oxa-1(OH)D3 to
24-ene-3-epi-22-oxa-1
(OH)D3 was exactly 2:1.
In a time course study, the amounts of the two dehydrates continued to
increase up to the end of the incubation period (Fig.
4D).
VDR and DBP Binding Properties of OCT and Its Less Polar Metabolites
All of the metabolites tested had a lower binding affinity for VDR
than OCT (Fig. 5A). Relative
binding affinities calculated for 50% displacement of
1,25(OH)2[3H]D3 were only
0.13, 0.30, and 0.47% of OCT for 25-ene-22-oxa-1
(OH)D3, 24-ene-22-oxa-1
(OH)D3, and 3-epi-OCT,
respectively. Fig. 5B shows the rat plasma DBP binding assay
with the metabolites in comparison with
1
,25(OH)2D3 and OCT. All of the metabolites
tested had an extremely low binding affinity for DBP, like OCT.
|
Target Gene Activation by OCT and its Less Polar Metabolites
The transcription-inducing activities of OCT metabolites on a
human osteocalcin gene promoter in transfected MG-63 cells are shown in
Fig. 6A. At 108
M, the transcriptional activities were about 8, 24, and
33% of OCT for 25-ene-22-oxa-1
(OH)D3,
24-ene-22-oxa-1
(OH)D3, and 3-epi-OCT, respectively. Similarly, the transcription-inducing activities of OCT
metabolites on a rat CYP24 gene promoter in transfected MG-63 cells are shown in Fig. 6B. At 10
8
M, the transcription-inducing activities were about 2, 11, and 15% of OCT for 25-ene-22-oxa-1
(OH)D3,
24-ene-22-oxa-1
(OH)D3, and 3-epi-OCT,
respectively. When we examined the transcriptional activities of the
metabolites on a human osteocalcin gene promoter and a rat
CYP24 gene promoter in transfected UMR 106 cells, we observed the same induction levels for the two genes as observed in
transfected MG-63 cells (data not shown). Thus,
24-ene-22-oxa-1
(OH)D3 and 3-epi-OCT were
found to be less active than OCT, with potencies between one-third and
one-tenth in terms of the activation of vitamin D target genes.
|
Anti-proliferative and Differentiation-inducing Activities of OCT and Its Less Polar Metabolites
At 108 M, no metabolites of OCT showed
significant activity of arresting the cell cycle at
G0-G1 phase as compared with
1
,25(OH)2D3 and OCT (Fig.
7A). Three OCT metabolites had
little inducing effect on cell surface CD11b antigen expression in a
human promyelocytic leukemia cell line, HL-60 (Fig. 7B). At
10
8 M, the biological activities were only 8, 8, and 12% of OCT for 25-ene-22-oxa-1
(OH)D3,
24-ene-22-oxa-1
(OH)D3, and 3-epi-OCT, respectively.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We were able to detect several distinct peaks ascribed to less
polar metabolites of OCT in sufficient quantities to assign structures
using 1H NMR and LC-MS techniques. The formation of these
less polar metabolites was shown to be an enzymatic process, because
the metabolites were not observed in no-cell controls incubated with OCT. The novel less polar metabolites identified include two dehydrates (25-ene-22-oxa-1(OH)D3 and
24-ene-22-oxa-1
(OH)D3) and an A-ring diastereomer
(3-epi-OCT). In addition, we demonstrated that the 3-epi-OCT was also converted into two dehydrates
(25-ene-3-epi-OCT and 24-ene-3-epi-OCT). These
findings clearly indicate that UMR 106 cells are able to metabolize OCT
to its less polar metabolites both via the C-25 dehydration
and C-3 epimerization pathway (Fig. 8).
To the best of our knowledge, this is the first definite structural assignment of the C-25 dehydrates of OCT. Siu-Caldera et al.
(22) studied the metabolism of 1
,25(OH)2D3
in UMR 106 and ROS 17/2.8 cells and found a peak corresponding to a
less polar metabolite (denoted as M1). Due to the insufficient
quantity, its definite structural assignment was not established. If
this metabolite M1 is one of the two dehydrates of
1
,25(OH)2D3, namely
25-ene-1
-hydroxyvitamin D3 or 24-ene-1
-hydroxyvitamin
D3, then C-25 dehydration would likely be a common
metabolic pathway of both 1
,25(OH)2D3 and synthetic analogs.
|
Two possible metabolic routes from OCT to dehydrates can be considered;
one via the direct C-25 dehydration, and the other via the C-24 and
C-26 hydroxylation. The latter route via the CYP24 metabolic pathway
seems unlikely because the dehydration of hydroxyl groups at positions
C-24/C-25 or C-25/C-26 has not been found in the metabolism of steroid
hormones. It is generally accepted that 24(OH)OCT is metabolized to
24-oxo-OCT, and that 26-hydroxy-OCT (26(OH)OCT) is likely to be
metabolized to 23,26-dihydroxy-OCT. Therefore, both 24(OH)OCT and
26(OH)OCT seem unlikely to be precursors of the two OCT dehydrates. The
former route is more plausible. Both the
24-ene-22-oxa-1(OH)D3 and the
25-ene-22-oxa-1
(OH)D3 can be formed from OCT by
enzymatic C-25 dehydroxylation followed by dehydrogenation at the
positions C-24 and C-26, respectively. If the latter oxidation occurs
non-enzymatically, then the production of
24-ene-22-oxa-1
(OH)D3 is expected to be greater than
that of 25-ene-22-oxa-1
(OH)D3 on the basis of the
chemical reaction. In this study, unexpectedly, the production of
25-ene-22-oxa-1
(OH)D3 was greater than that of
24-ene-22-oxa-1
(OH)D3 in UMR 106 cells. Therefore, the
C-25 dehydration process of OCT is suspected to be under strict
cell-specific control, or further metabolism of the dehydrates may
differ with cell line. The findings of the biological studies here with
two dehydrates of OCT demonstrated that their biological activities are
considerably lower than OCT. Thus, it appears that like the C-23/C-24
hydroxylation pathways, the C-25 dehydration pathway contributes to
reducing the high potency of OCT.
In this study, we also have shown that OCT is converted into its C-3
epimer. The C-3 epimerization is a unique and important pathway because
the resulting C-3 epimer may be metabolized under the same way as the
parent OCT. Reddy et al. (31) recently demonstrated that
3-epi-1,25(OH)2D3 was further
metabolized via the C-23/C-24 oxidation pathways as
1
,25(OH)2D3. The C-3 epimerization is not specific to 1
,25(OH)2D3 and OCT. Higashi
et al. identified
3-epi-24,25(OH)2D3-24-glucuronide in
rat bile (32) and 3-epi-24,25(OH)2D3
in rat plasma (33). More recently, we reported a definite structural
assignment for 3-epi-24,25(OH)2D3
isolated from a UMR 106 cell culture (27). In addition, Reddy et
al. (34) demonstrated that synthetic vitamin D analogs,
1
,25(OH)2-16-ene-23-yne-vitamin D3 and
1
,25(OH)2-16-ene-23-yne-20-epi-vitamin D3 are metabolized to their respective C-3 epimers in UMR
106 cells. These findings clearly indicate that most of the vitamin D
derivatives are metabolized through the C-3 epimerization pathway in vitro and in vivo. It is also interesting to
note that the rate of C-3 epimerization varies depending upon the
structure of vitamin D derivatives. We observed that the rate of C-3
epimerization of 1
,25(OH)2D3 was about
3-fold higher than that of OCT in UMR 106 cells. In addition, the rate
of C-3 epimerization of 1
,25(OH)2D3 was
about 2-fold higher than that of 24,25(OH)2D3
(27). Recently, Reddy et al. (34) reported that the rate of
C-3 epimerization of
1
,25(OH)2-16-ene-23-yne-D3 was 10-fold lower
than that of its C-20 epimer. These findings imply that the enzyme(s)
responsible for C-3 epimerization can recognize structural differences
not only in the A-ring but also in the side-chain of vitamin D
derivatives. The C-3 epimerization pathway has been shown to be present
in a variety of normal and malignant cells. However, its contribution to the metabolism of vitamin D appears to be relatively low compared with the C-23/C-24 oxidation pathways, except for specific cell lines
(e.g. UMR 106 cells). Rat osteosarcoma ROS 17/2.8 cells, in
which the C-24 oxidation pathway is not active, has been shown to
metabolize 1
,25(OH)2D3 via the C-3
epimerization pathway (22). In contrast, the perfused rat kidney and
human promyelocytic leukemia cell line HL-60, in which the C-23/C-24
hydroxylation pathways are highly expressed, do not metabolize
1
,25(OH)2D3 to via the C-3 epimerization
pathway (22, 35). These findings imply that C-3 epimerization pathway
is cell-selective and contributes to the metabolism of vitamin D in
concert with the C-23/C-24 hydroxylation pathways. It is interesting to
note that 3-epi-1
,25(OH)2D3 was almost equipotent to 1
,25(OH)2D3 in
suppressing parathyroid hormone secretion in bovine parathyroid cells
(36) and in inhibiting keratinocyte proliferation (24, 37), and more
potent than 1
,25(OH)2D3 in inducing HL-60
cell apoptosis (38). In addition, a high metabolic stability of
3-epi-1
,25(OH)2D3 in target cells has been proposed by Reddy et al. (30). Thus, the C-3
epimerization pathway appears to play an important role not only in the
regulation of intracellular concentrations of
1
,25(OH)2D3 and its analogs, but also in the
formation of metabolite(s) with a different biological activity profile.
In summary, we present evidence that a novel C-25 dehydration pathway
is involved in the metabolism of OCT. Furthermore, we also demonstrated
that OCT is metabolized to 3-epi-OCT, and that the resulting
3-epi-OCT is further metabolized to two dehydrates. In UMR
106 cells, OCT is predominantly metabolized via the C-25 dehydration
pathway. On the other hand, in Caco-2 and LLC-PK1 cells,
OCT is predominately metabolized via the C-23/C-24 hydroxylation pathways. The interplay of these metabolic pathways may be important in
the regulation of OCT metabolism and its biological functions in its
target cells.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Yoshiyasu Kubo and Toyoko Sakurai for excellent technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a grants-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan, a grant for cooperative research administered by the Japan Private School Promotion Foundation, and a grant-in-aid from the Ministry of Health and Welfare of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Hygienic
Sciences, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan. Tel.: 81-78-441-7563; Fax: 81-78-441-7565; E-mail: t-okano@kobepharma-u.ac.jp.
Published, JBC Papers in Press, November 1, 2002, DOI 10.1074/jbc.M203773200
2 N. Kubodera, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
1, 25(OH)2D3, 1
,25-dihydroxyvitamin
D3;
OCT, 22-oxacalcitriol (22-oxa-1
,25-dihydroxyvitamin
D3);
1
, 20(OH)2D3,
hexanor-1
,20-dihydroxyvitamin D3;
24-ene-22-oxa-1
(OH)D3, 25-dehydroxy-24-ene-22-oxa-1
-hydroxyvitamin D3;
VDR, vitamin D receptor;
DBP, vitamin D-binding protein
(Gc-globulin);
HPLC, high performance liquid chromatography;
LC-MS, liquid chromatography-mass spectrometry;
PBS, phosphate-buffered
saline;
FCS, fetal calf serum.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Abe, E., Miyaura, C., Sakagami, H., Takeda, M., Konno, K., Yamazaki, T., Yoshiki, S., and Suda, T. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4990-4995[Abstract] |
2. | Colston, K. W., Colston, J. M., and Feldman, D. (1981) Endocrinology 108, 1083-1086[Abstract] |
3. | Suda, T., Takahashi, N., and Martin, T. J. (1995) Endocrinol. Rev. 4, 266-279 |
4. | Eisman, J. A., Barkla, D. H., and Tutton, P. J. M. (1987) Cancer Res. 47, 21-25[Abstract] |
5. | Eisman, J. A., Koga, M., Sutherland, R. L., Barkla, D. H., and Tutton, P. J. M. (1989) Proc. Soc. Exp. Med. 191, 221-226 |
6. | Colston, K. W., Berger, U., and Coombes, R. C. (1989) Lancet 1, 188-191[Medline] [Order article via Infotrieve] |
7. | Iino, Y., Yoshida, M., Sugamata, N., Maemura, M., Ohwada, S., Yokoe, T., Ishikita, T., Horiuchi, R., and Morishita, Y. (1992) Breast Cancer Res. Treat. 22, 133-140[Medline] [Order article via Infotrieve] |
8. | Peehl, D. M., Skowronski, R. J., Leung, G. K., Wong, S. T., Stamey, T. A., and Feldman, D. (1994) Cancer Res. 54, 805-810[Abstract] |
9. | Kobayashi, T., Tsugawa, N., Okano, T., Masuda, S., Takeuchi, A., Kubodera, N., and Nishii, Y. (1994) J. Biochem. 155, 373-380 |
10. | Kobayashi, T., Okano, T., Tsugawa, N., Masuda, S., Takeuchi, A., and Nishii, Y. (1991) Cont. Nephrol. 91, 129-133 |
11. | Okano, T., Tsugawa, N., Masuda, S., Takeuchi, A., Kobayashi, T., and Nishii, Y. (1989) J. Nutr. Sci. Vitam. 35, 529-533 |
12. | Morimoto, S., Imanaka, S., Koh, E., Shiraishi, T., Nabata, T., Kitano, S., Miyashita, Y., Nishii, Y., and Ogihara, T. (1989) Biochem. Int. 19, 1143-1149[Medline] [Order article via Infotrieve] |
13. | Abe, J., Takita, Y., Nakano, T., Miyaura, U., Suda, T., and Nishii, Y. (1989) Endocrinology 124, 2645-2647[Abstract] |
14. | Tsugawa, N., Okano, T., Masuda, S., Kobayashi, T., Kubodera, N., Sato, K., and Nishii, Y. (1994) J. Bone Miner. Metab. 12, S13-S17 |
15. | Finch, J. L., Brown, A. J., Kubodera, N., Nishii, Y., and Slatopolsky, E. (1993) Kidney Int. 43, 561-566[Medline] [Order article via Infotrieve] |
16. |
Takeyama, K.,
Masuhiro, Y.,
Fuse, H.,
Endoh, H.,
Murayama, A.,
Kitanaka, S.,
Suzawa, M.,
Yanagisawa, J.,
and Kato, S.
(1999)
Mol. Cell. Biol.
19,
1049-1055 |
17. | Brown, A. J., Berkoben, M., Ritter, C., Kubodera, N., Nishii, Y., and Slatopolsky, E. (1992) Biochem. Biophys. Res. Commun. 189, 759-764[Medline] [Order article via Infotrieve] |
18. | Bikle, D. D., Abe-Hashimoto, J., Su, M. J., Felt, S., Gibson, D. F. C., and Pillai, S. (1995) J. Invest. Dermatol. 105, 693-698[Abstract] |
19. |
Masuda, S.,
Byford, V.,
Kremer, R.,
Makin, H. L. J.,
Kubodera, N.,
Nishii, Y.,
Okazaki, A.,
Okano, T.,
Kobayashi, T.,
and Jones, G.
(1996)
J. Biol. Chem.
271,
8700-8708 |
20. | Masuda, S., Kamao, M., Schroeder, N. J., Makin, H. L. J., Jones, G., Kremer, R., Rhim, J., and Okano, T. (2000) Biol. Pharmacol. Bull. 23, 133-139 |
21. | Bischof, M. G., Sui-Caldera, M. -L., Weiskopf, A., Vouros, P., Cross, H. S., Peterlok, M., and Reddy, G. S. (1998) Exp. Cell Res. 241, 194-201[CrossRef][Medline] [Order article via Infotrieve] |
22. | Siu-Caldera, M. -L., Sekimoto, H., Weiskopf, A., Vouros, P., Muralidharan, K. R., Okamura, W. H., Bishop, J., Norman, A. W., Uskokovic, M. R., Schuster, I., and Reddy, G. S. (1999) Bone 24, 457-463[CrossRef][Medline] [Order article via Infotrieve] |
23. | Sekimoto, H., Sui-Caldera, M. -L., Weiskopf, A., Vouros, P., Muralidharan, K. R., Okamura, W. H., Uskokovic, M. R., and Reddy, G. S. (1999) FEBS Lett. 448, 278-282[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Norman, A. W.,
Bouillon, R.,
Farach-Carson, M. C.,
Bishop, J. E.,
Zhou, L. X.,
Nemere, I.,
Zhao, J.,
Muralidharan, K. R.,
and Okamura, W. H.
(1993)
J. Biol. Chem.
268,
20022-20030 |
25. | Bligh, E. G., and Dyer, W. J. (1957) Can. J. Biochem. 37, 911-917 |
26. | Makin, G., Lohnes, D., Byford, V., Ray, R., and Jones, G. (1989) Biochem. J. 262, 173-180[Medline] [Order article via Infotrieve] |
27. | Kamao, M., Tatematsu, S., Reddy, G. S., Hatakeyama, S., Sugiura, M., Ohashi, N., Kubodera, N., and Okano, T. (2001) J. Nutr. Sci. Vitam. 47, 108-115 |
28. |
Ozono, K.,
Liao, J.,
Kerner, S. A.,
Scoot, R. A.,
and Pike, J. W.
(1990)
J. Biol. Chem.
265,
21881-21888 |
29. |
Ohyama, Y.,
Ozono, K.,
Uchida, M.,
Yoshimura, M.,
Shinki, T.,
Suda, T.,
and Yamamoto, O.
(1996)
J. Biol. Chem.
271,
30381-30385 |
30. | Lorenz, W. W., McCann, R. O., Longiaru, M., and Cormier, M. J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4438-4442[Abstract] |
31. | Reddy, G. S., Muralidharan, K. R., Okamura, W. H., Tserng, K. Y., and McLane, J. A. (2001) Steroids 66, 441-450[CrossRef][Medline] [Order article via Infotrieve] |
32. | Higashi, T., Kikuchi, R., Miura, K., Shimada, K., Hiyamizu, H., Ooi, K., Iwabuchi, Y., Hatakeyama, S., and Kubodera, N. (1999) Biol. Pharmacol. Bull. 22, 767-769 |
33. | Higashi, T., Ogasawara, A., and Shimada, K. (2000) Anal. Sci. 16, 477-482 |
34. | Reddy, G. S., Rao, D. S., Siu-Caldera, M. -L., Astecker, N., Weiskopf, A., Vouros, P., Sasso, G. J., Percy, S., Manchand, P. S., and Uskokovic, M. R. (2000) Arch. Biochem. Biophys. 383, 197-205[CrossRef][Medline] [Order article via Infotrieve] |
35. | Rao, D. S., Campbell, M. J., Koeffler, H. P., Ishizuka, S., Uskokovic, M. R., Spagnuolo, P., and Reddy, G. S. (2001) Steroids 66, 423-431[CrossRef][Medline] [Order article via Infotrieve] |
36. | Brown, A. J., Ritter, C., Slatopolsky, E., Muralidharan, K. R., Okamura, W. H., and Reddy, G. S. (1999) J. Cell. Biochem. 73, 106-113[CrossRef][Medline] [Order article via Infotrieve] |
37. | Schuster, I., Astecker, N., Egger, H., Herzig, G., Reddy, G. S., Schmid, J., and Vorisek, G. (1997) in Vitamin D, chemistry, biology and clinical applications of the steroid hormone (Norman, A. W. , Bouillon, R. , and Thomasset, M., eds) , pp. 551-558, University of California, Riverside, CA |
38. | Nakagawa, K., Sowa, Y., Kurobe, M., Ozono, K., Siu-Caldera, M. -L., Reddy, G. S., Uskokovic, M. R., and Okano, T. (2001) Steroids 66, 327-337[CrossRef][Medline] [Order article via Infotrieve] |