Characterization of new conjugated metabolites in bile of rats administered 24,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3

Article abstract of DOI:10.1016/S0039-128X(00)00087-8

The characterization of new conjugated vitamin D metabolites in rat bile was performed using HPLC, liquid chromatography/tandem mass spectrometry combined derivatization, and GC-MS. After the administration of 24,25- dihydroxyvitamin D₃ to rats

Full text of DOI:10.1016/S0039-128X(00)00087-8

Steroids 65 (2000) 281–294
Characterization of new conjugated metabolites in bile of rats
administered 24,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3
Tatsuya Higashia, Kanako Miuraa, Ryuta Kikuchia, Kazutake Shimadaa,*, Hiroko Hiyamizub,
Hidenori Ooib, Yoshiharu Iwabuchib, Susumi Hatakeyamab, Noboru Kuboderac
aFaculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-0934, Japan
bFaculty of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyou-machi, Nagasaki 852-8521, Japan
cChugai Pharmaceutical Co., 1-9 Kyoubashi 2-chome, Chuo-ku, Tokyo 104-8301, Japan
Received 15 November 1999; received in revised form 30 December 1999; accepted 6 January 2000
Abstract
The characterization of new conjugated vitamin D metabolites in rat bile was performed using HPLC, liquid chromatography/tandem
mass spectrometry combined derivatization, and GC-MS. After the administration of 24,25-dihydroxyvitamin D3 to rats, 23,25-dihydroxy-
24-oxovitamin D3 23-glucuronide, 3-epi-24,25-dihydroxyvitamin D3 24-glucuronide, and 24,25-dihydroxyvitamin D3 3-sulfate were
obtained as new biliary metabolites together with 24,25-dihydroxyvitamin D3 3- and 24-glucuronides. The above metabolites, except
24,25-dihydroxyvitamin D3 3-glucuronide, were obtained from rats dosed with 25-hydroxyvitamin D3. 23,25-Dihydroxyvitamin D3
23-glucuronide was also obtained from the bile of rats administered 25-hydroxyvitamin D3 in addition to its 3-glucuronide, 25-glucuronide,
and 3-sulfate. Thus, it was found that 24,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 were directly conjugated as glucuronide and
sulfate, whereas at the C-23 position, they were hydroxylated and then conjugated. Furthermore, we found that the C-3 epimerization acts
as one of the important pathways in vitamin D metabolism. © 2000 Elsevier Science Inc. All rights reserved.
Keywords: 24,25-Dihydroxyvitamin D3; 25-Hydroxyvitamin D3; Rat bile; Conjugated metabolite; Liquid chromatography tandem mass spectrometry;
3-Epi-24,25-dihydroxyvitamin D3 24-glucuronide
1. Introduction
olite to be excreted [4]. A study of the metabolism of
vitamin D is expected to be helpful in understanding the
physiological role of the metabolites and in developing new
drugs.
24,25(OH)2D3 is reported to be further oxidized on its
side-chain at the 23- or 24-position by CYP24 to give
metabolites such as 25-hydroxy-24-oxovitamin D3
[25(OH)-24-oxo-D3] and (23S)-23,25-dihydroxy-24-oxovi-
tamin D3 [23,25(OH)2-24-oxo-D3] [1]. Thus, the phase I
reactions in vitamin D metabolism, particularly oxidation of
the side-chain, have been investigated thoroughly, but little
is known regarding the conjugates of vitamin D metabolites,
such as the glucuronides or sulfates [5].
In previous papers in this series, we reported that mono-
glucuronides of 24,25(OH)2D3 [6] or 25(OH)D3 [7] were
obtained as biliary metabolites of rats dosed with
24,25(OH)2D3 or 25(OH)D3, respectively, per os (p.o.). On
the contrary, Shimoyamada et al. reported that in dogs
dosed with 24,25(OH)2D3, 23,25(OH)2-24-oxo-D3 23-gluc-
uronide (23G) was excreted into the bile [8]. These data
In the vitamin D-supplemented state, 25-hydroxyvitamin
D3 [25(OH)D3], a major circulating metabolite, is metabo-
lized to various side-chain dihydroxylated metabolites.
Among these metabolites, (24R)-24,25-dihydroxyvitamin
D3 [24,25(OH)2D3] and (23S)-23,25-dihydroxyvitamin D3
[23,25(OH)2D3] are reported to be formed by the same
enzyme, 25-hydroxyvitamin D-24-hydroxylase (CYP24)
[1]. The physiological roles of the side-chain dihydroxy-
lated metabolites still remain poorly understood. For exam-
ple, 24,25(OH)2D3 at pharmacological doses was reported
to cause a marked increase in bone volume [2] and mechan-
ical strength [3] in animals without hypercalcemia, and it is
expected to be an anti-osteoporotic drug. On the other hand,
it has been argued that 24,25(OH)2D3 is a catabolic metab-
* Corresponding author. Tel.: ϩ81-76-234-4459; fax: ϩ81-76-234-
4459.
E-mail address: shimada@dbs.p.kanazawa-u.ac.jp (K. Shimada)
0039-128X/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(00)00087-8

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T. Higashi et al. / Steroids 65 (2000) 281–294
indicate that the dosed compounds are either directly con-
jugated as glucuronides or oxidized and then conjugated.
In the present paper and in a preliminary communication
[9], we studied other conjugated vitamin D metabolites in
rat bile after the administration of 24,25(OH)2D3 or
25(OH)D3, and new metabolites (peaks A, B, C, and D in
Fig. 1) were isolated. The new metabolites were identified
using HPLC with photodiode array UV detection, liquid
chromatography (LC)/multistage tandem mass spectrometry
(MSn, n ϭ 2 or 3), and gas chromatography/MS (GC/MS).
Fishman units) in acetate buffer (0.1 ml) were preincubated
separately at 37°C for 20 min. The two solutions were
mixed and incubated at 37°C for 3 h. The reaction mixture
was extracted with AcOEt (1 ml ϫ 3), and the organic layer
was evaporated under a N2 gas stream. The residue was
dissolved in EtOH and subjected to HPLC.
2.4. Acidic solvolysis of sulfate
The sulfate (ca. 25 ng) was dissolved in AcOEt (1 ml)
containing 1 ␮l of 0.5 M H2SO4. This solution was stored at
room temperature for 1 h, washed with H2O, and the solvent
was evaporated. The residue was dissolved in EtOH and
subjected to HPLC.
2. Materials and methods
2.1. Materials
2.5. Derivatization for LC/MSn analysis
24,25(OH)2D3 and 25(OH)D3 were obtained from
Duphar (Amsterdam, The Netherlands) and Wako (Osaka,
Japan), respectively. (24S)-24,25-Dihydroxyvitamin D3
[24S,25(OH)2D3] and 23,25(OH)2-24-oxo-D3 were donated
by Kureha Chemical Co. (Tokyo, Japan). 24,25(OH)2D3 G
[24,25(OH)2D3-3G, -24G, and -25G], 25(OH)D3 3-sulfate
(3S), 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), and pip-
eridinohydroxypropyl Sephadex LH-20 (PHP-LH-20) were
the same as those used in previous papers [6,10]. Isolute
C18 (EC) cartridges (500 mg; International Sorbent Tech.,
Hengoed, UK) were obtained from Uniflex (Tokyo). ␤-Glu-
curonidase, originating from Escherichia coli (Type IX-A),
was obtained from Sigma (St. Louis, MO, USA). An acetate
buffer [0.1 M AcONa-AcOH (pH 5.0)] was used for enzy-
mic hydrolysis. All other reagents and solvents were of
analytical grade.
2.5.1. Reaction with PTAD
Steroids (ca. 100 ng) were dissolved in AcOEt (50 ␮l)
containing PTAD (5 ␮g) and kept at room temperature for
1.5 h. MeOH (50 ␮l) was added to decompose excess
reagent, and the solvent was evaporated under a N2 gas
stream.
2.5.2. Acetylation
Steroids (ca. 100 ng) were dissolved in pyridine/Ac2O
(2:1 v/v, 50 ␮l) and stored at room temperature for 1.5 h.
MeOH (50 ␮l) was added to the mixture, and the solvent
was evaporated under a N2 gas stream.
2.6. LC/MS analysis
LC/MS was performed using a Finnigan MAT (San Jose,
CA, USA) LCQ connected to a JASCO (Tokyo) PU-980
chromatograph, and the following conditions were used.
MS/MS was performed in the trap with helium gas as the
collision gas, and the relative collision energy was set at
20%.
2.2. HPLC analysis
HPLC was performed on a Shimadzu (Kyoto, Japan)
LC-10AT chromatograph equipped with a Shimadzu SPD-
10A UV detector (265 nm) or a Shimadzu SPD-M6A pho-
todiode array UV (200–340 nm) detector. A J’sphere ODS
H-80 [4 ␮m, 150 ϫ 4.6 mm inner diameter (i.d.); YMC,
Kyoto)] and a Develosil 60-5 (5 ␮m, 250 ϫ 4.6 mm i.d.;
Nomura Chemical, Seto, Japan) were used in reversed-
phase and normal-phase HPLC, respectively, at a flow rate
of 1 ml/min at 30°C. The pH of the mobile phase containing
AcONH4 or NaClO4 was adjusted with AcOH or HClO4,
respectively.
The photodiode array UV detection of each compound
was performed using a J’sphere ODS H-80 column and the
following mobile phases: peaks A, B, and D, MeCN/2%
NaClO4 (pH 3.0) (2:3 v/v); peak C, MeCN/2% NaClO4 (pH
3.0) (7:13 v/v).
2.7. Electrospray ionization (ESI)
The spray needle voltage was 5 kV, and the heated
capillary temperature, the sheath gas flow rate, and the
auxiliary gas flow rate were set at 200°C, 70 units, and 20
units, respectively. The capillary voltage was 1 or Ϫ1 V,
and the tube lens offset was 10 or Ϫ10 V in the positive or
the negative-ion mode, respectively. A Develosil ODS
HG-5 (5 ␮m, 150 ϫ 2.0 mm i.d.) and a YMC Pack Pro C18
(5 ␮m, 150 ϫ 3.0 mm i.d.) columns were used at a flow rate
of 0.3 and 0.4 ml/min, respectively, at 30°C. For the
ESI-MS analysis of each compound, the following columns
and mobile phases were used: 24,25(OH)2D3 24G, peaks A,
B, and D, Develosil ODS HG-5 and MeCN/10 mM Ac-
ONH4 (1:2 v/v); peak C, Develosil ODS HG-5 and
MeCN/10 mM AcONH4 (2:3 v/v); 25(OH)D3 3S-PTAD
adduct, YMC Pack Pro C18 and MeOH/10 mM AcONH4
2.3. Enzymic hydrolysis of glucuronide
The glucuronide [ca. 25 ng in EtOH (20 ␮l)] dissolved in
acetate buffer (0.88 ml) and ␤-glucuronidase (ca. 1500

T. Higashi et al. / Steroids 65 (2000) 281–294
283
Fig. 1. Representative chromatograms and UV spectra of conjugated metabolites from rat bile. (a), Female rat administered 24,25(OH)2D3; (b), male rat
administered 24,25(OH)2D3; (c), female rat administered 25(OH)D3; (d), male rat administered 25(OH)D3. Conditions: column, J’sphere ODS-H80; mobile
phase, MeCN/0.5% AcONH4 (pH 5.0) (1:2 v/v).
(1:1 v/v); peaks A-, B-, and D-PTAD adducts, YMC Pack
Pro C18 and MeOH/10 mM AcONH4 (7:3 v/v); peak C-
PTAD adduct, Develosil ODS HG-5 and MeCN/10 mM
AcONH4 (2:3 v/v); peaks A- and B-acetates, YMC Pack Pro
C18 and MeCN/10 mM AcONH4 (3:1 v/v); peak C-acetate,
Develosil ODS HG-5 and MeCN/10 mM AcONH4 (2:3

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T. Higashi et al. / Steroids 65 (2000) 281–294
v/v); peak D-acetate, YMC Pack pro C18 and MeOH/10
mM AcONH4 (13:2 v/v).
cartridge. After washing with H2O (5 ml), the steroids were
eluted with EtOH (5 ml), and H2O (0.56 ml) was added to
the eluate. The entire sample was applied to a column (20 ϫ
6 mm i.d.) of PHP-LH-20. After washing with 90% EtOH (5
ml) and 0.1 M AcOH in 90% EtOH (5 ml), the conjugated
metabolites were eluted with 0.1 M AcONH4 in 90% EtOH
(5 ml). The eluate was diluted with H2O (7 ml) and then
applied to an Isolute C18 cartridge to remove AcONH4.
After washing with H2O (5 ml), the desired compounds
were eluted with EtOH (5 ml), which was subsequently
evaporated under a N2 gas stream.
2.8. Atmospheric pressure chemical ionization (APCI)
The source current was 5 ␮A, and the heated capillary
temperature and the sheath gas flow rate were set at 225°C
and 80 units, respectively, with a vaporizer temperature of
350°C. The capillary voltage and the tube lens offset were
1 and 10 V, respectively. A J’sphere ODS H-80 column was
used at a flow rate of 1 ml/min at 30°C. MeOH/H2O (3:1
v/v) was used as the mobile phase.
2.13. Analysis of bile samples from rats dosed with
24,25(OH)2D3
2.9. Derivatization for GC/MS analysis:
trimethylsilylation
The above residue was redissolved in EtOH and sub-
jected to prep. HPLC [J’sphere ODS-H80, MeCN/0.5%
AcONH4 (pH 5.0) (1:2 v/v); peak A, retention time (tR)
5.5–6.8 min; peak B, tR 12.5–14.0 min; peak C, tR 27.9–
30.1 min]. After dilution with H2O, each fraction obtained
was applied to an Isolute C18 cartridge in the manner
described above to remove any inorganic salts and then
evaporated. Each fraction was further purified by HPLC
[J’sphere ODS-H80, MeCN/2% NaClO4 (pH 3.0) (2:3 v/v);
peak A, tR 8.3–9.3 min; peak B, tR 13.1–14.3 min;
MeCN/2% NaClO4 (pH 3.0) (7:13 v/v); peak C, tR 14.6–
16.1 min]. After neutralization with 2% NaHCO3 and dilu-
tion with H2O, each fraction obtained was treated as de-
scribed above. The EtOH eluate was evaporated, and part of
the resulting residue was subjected to HPLC and LC/MS.
After hydrolysis, the residue obtained was subjected to
GC/MS.
The genin of metabolites obtained by enzymic hydrolysis
was purified by preparative (prep.) HPLC. The solvent was
evaporated, and the residue was dissolved in pyridine (50
␮l). N,O-bis(trimethylsilyl)trifluoroacetamide, containing
2% trimethylsilyl chloride (50 ␮l), was added to this solu-
tion and stored at 60°C for 1 h. The solvent and excess
reagent were evaporated, the residue was dissolved in hex-
ane (100 ␮l), and an aliquot was subjected to GC/MS.
2.10. GC/MS analysis
GC/MS was performed with a Finnigan MAT GCQ,
using EI ionization. A Rtx-5MS capillary column (25 m ϫ
0.25 mm i.d., 0.25 ␮m df) was inserted into the ion source
through a heated (275°C) transfer line. The temperature
programmable injector was used and set at 60°C for the first
minute and then programmed successively at a rate of
180°C/min–250°C, where it was held for 30 min. The flow
rate of the helium carrier gas was set at 40 cm/s. The column
oven temperature was maintained at 60°C for 5 min and
then successively programmed at a rate of 40°C/min to
290°C, where it was held for 30 min. The ionization energy
was set at 70 eV.
2.14. Analysis of bile samples from rats dosed with
25(OH)D3
The above residue was redissolved in EtOH and sub-
jected to prep. HPLC [J’sphere ODS-H80, MeCN/0.5%
AcONH4 (pH 5.0) (1:1 v/v); 25(OH)D3 3S, tR 8.3–9.3 min;
MeCN/0.5% AcONH4 (pH 5.0) (1:2 v/v); 24,25(OH)2D3
24G, tR 14.8–15.9 min; peak A, tR 5.5–6.8 min; peak B, tR
12.5–13.7 min; peak C, tR 27.9–29.7 min; peak D, tR 7.3–
8.2 min]. After dilution with H2O, each fraction obtained
was applied to an Isolute C18 cartridge in the manner
described above to remove any inorganic salts and then
evaporated. Each fraction was further purified by HPLC
[J’sphere ODS-H80, MeCN/2% NaClO4 (pH 3.0) (1:1 v/v);
25(OH)D3 3S, tR 6.9–7.7 min; MeCN/2% NaClO4 (pH 3.0)
(2:3 v/v); 24,25(OH)2D3 24G, tR 14.5–15.5 min; peak A, tR
8.3–9.3 min; peak B, tR 13.1–14.3 min; peak D, tR 9.1–9.8
min; MeCN/2% NaClO4 (pH 3.0) (7:13 v/v); peak C, tR
14.6–16.1 min]. After neutralization with 2% NaHCO3 and
dilution with H2O, each fraction obtained was treated as
described previously. The EtOH eluate was evaporated, and
part of the resulting residue was subjected to HPLC and
1
2.11. H-Nuclear magnetic resonance (1H-NMR) of genin
of peak B
1H-NMR spectra of the genin of peak B was obtained
with a JEOL (Tokyo) alpha (600 MHz) spectrometer at
27°C, and CDCl3 was used as the solvent.
2.12. Separation of the conjugated vitamin D metabolites
from rat bile
Bile was collected from Wistar strain rats (male, ca.
170 g; female, ca. 140 g, 7 w, Japan S.L.C., Hamamatsu,
Japan) dosed with 24,25(OH)2D3 (0.5 mg) or 25(OH)D3
(0.5 mg) as described in a previous paper [6]. The bile
specimen (2 ml) was diluted with 0.5 M sodium phosphate
buffer (pH 7.0; 40 ml) and passed through an Isolute C18

T. Higashi et al. / Steroids 65 (2000) 281–294
285
LC/MS. After hydrolysis, the residue obtained was sub-
jected to GC/MS.
as a colorless oil by column chromatography [hexane/
23
AcOEt (2:1 v/v)] : [␣]D Ϫ1.2° (c 2.49, CHCl3); IR (neat)
1
3543, 1616, 1088 cmϪ1; H-NMR ␦: 1.64 (q, J ϭ 7.5 Hz,
2.15. Synthesis of 3-epi-24,25(OH)2D3
2H), 1.73 (br s, 1H), 2.06 (br s, 1H), 2.11–2.27 (m, 2H),
2.34–2.53 (m, 2H), 3.80 (s, 3H), 4.14 (t, J ϭ 2.1 Hz, 2H),
4.51 (s, 2H), 4.99 (dd, J ϭ 1.5, 10.2 Hz, 1H), 5.06 (dd, J ϭ
1.5, 16.8 Hz, 1H), 5.83 (ddt, J ϭ 10.2, 16.8, 6.9 Hz, 1H),
6.87 (d, J ϭ 8.7 Hz, 2H), 7.27 (d, J ϭ 8.7 Hz, 2H).
2.15.1. General
Optical rotations were measured with a JASCO DIP-140
polarimeter. Infrared (IR) spectra were obtained using a
1
JASCO FT/IR-5300 spectrophotometer. H-NMR spectra
were recorded on a VARIAN (Palo Alto, CA, U.S.A.)
Gemini-300 (300 MHz) or a JEOL FX-200 (200 MHz)
spectrometers using CDCl3 as a solvent. Chemical shifts are
reported in parts per million (ppm) downfield from tetra-
methylsilane or are calibrated from CHCl3. The abbrevia-
tions used are: s, singlet; d, doublet; t, triplet; hept, heptet;
m, multiplet; and br, broad. HRMS were recorded on a
JEOL JMS-AX-500 instruments. All reactions were carried
out under an atmosphere of Ar. All extracts were dried over
MgSO4 and evaporated under reduced pressure with a ro-
tary evaporator. Chromatographic purification was carried
out with Merck (Darmstadt, Germany) silica gel 60 (70–
230 mesh).
2.15.4. (5R)-5-(t-butyldimethylsilyl)oxy-1-(4-
methoxybenzyl)oxynona-8-en-2-yne (4)
A mixture of crude 3 (1.60 g), t-butyldimethylsilyl chlo-
ride (1.43 g, 9.48 mmol), and imidazole (1.29 g, 19.0 mmol)
in N,N-dimethylformamide (DMF; 10 ml) was stirred at
room temperature for 3 h. The reaction mixture was diluted
with AcOEt, washed with H2O, dried, evaporated, and chro-
matographed. Elution with AcOEt-hexane (20:1 v/v) gave 4
24
(2.03 g, 82% from 2) as a colorless oil: [␣]D ϩ20.3° (c
1.63, CHCl3); IR (neat) 1612, 1513, 1300, 1250, 1176, 1082
1
cmϪ1; H-NMR ␦: 0.08 (s, 3H), 0.09 (s, 3H), 0.90 (s, 9H),
1.57–1.81 (m, 2H), 2.04–2.22 (m, 2H), 2.37–2.42 (m, 2H),
3.78–3.84 (m, 1H), 3.81 (s, 3H), 4.13 (t, J ϭ 2.4 Hz, 2H),
4.52 (s, 1H), 4.97 (dd, J ϭ 1.8, 10.2 Hz, 1H), 5.03 (dd, J ϭ
1.8, 17.1 Hz, 1H), 5.84 (ddt, J ϭ 10.2, 17.1, 6.6 Hz, 1H),
6.88 (d, J ϭ 8.7 Hz, 2H), 7.28 (d, J ϭ 8.7 Hz, 2H).
2.15.2. (5S)-5,6-Epoxy-1-(4-methoxybenzyl)oxy-2-hexyne
(2)
BuLi in hexane (1.56 M, 12.2 ml) was added to a stirred
solution of 4-methoxybenzyl propargyl ether (3.86 g, 18.5
mmol) in tetrahydrofuran (THF; 58 ml) at Ϫ78°C, and the
mixture was stirred at Ϫ78°C for 1 h. (S)-(ϩ)-Epichloro-
hydrin (1) (0.9 ml, 11.6 mmol) and BF3 ⅐ Et2O (2.1 ml, 18.5
mmol) were added, and the mixture was stirred at Ϫ78°C
for 1.5 h. The reaction was quenched with saturated NaCl,
and the mixture was extracted with Et2O. The extract was
washed with H2O, dried, and the solvent was evaporated.
The resulting residue was dissolved in THF (58 ml) and
treated with 60% NaH dispersed in oil (740 mg, 18.5 mmol)
at Ϫ10°C. After being stirred at Ϫ10°C for 1 h, the reaction
mixture was diluted with Et2O, washed with H2O, dried,
evaporated, and chromatographed. Elution with hexane/
2.15.5. (5R)-5-(t-butyldimethylsilyl)oxynona-8-en-2-yn-1-ol
(5)
2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ; 971
mg, 4.27 mmol) was added to a stirred solution of 4 (793
mg, 2.04 mmol) in CH2Cl2/H2O (20:1 v/v, 10.5 ml) at room
temperature. After 3 h, the reaction mixture was diluted
with CH2Cl2 and filtered through Celite. The filtrate was
washed with saturated NaHCO3, dried, and the solvent was
evaporated. The resulting residue was dissolved in MeOH
(8 ml) and cooled in an ice bath. NaBH4 (38.4 mg, 1.01
mmol) was added to this solution, and the mixture was
stirred at 0°C for 10 min. The reaction mixture was diluted
with CH2Cl2, washed with saturated NaCl, dried, evapo-
rated, and chromatographed. Elution with hexane/AcOEt
24
Et2O (3:1 v/v) gave 2 (2.50 g, 92%) as a colorless oil: [␣]D
22
ϩ12.5° (c 2.37, CHCl3); IR (neat) 1612, 1513, 1248, 1071
(20:1 v/v) gave 5 (429 mg, 79%) as a colorless oil: [␣]D
1
1
cmϪ1; H-NMR ␦: 2.50–2.90 (m, 4H), 3.11 (m, 1H), 3.80
ϩ21.2° (c 2.8, CHCl3); H-NMR ␦: 0.06 (s, 3H), 0.08 (s,
(s, 3H), 4.13 (t, J ϭ 2.1 Hz, 2H), 4.51 (s, 2H), 6.87 (d, J ϭ
8.4 Hz, 2H), 7.28 (d, J ϭ 8.4 Hz, 2H).
3H), 0.89 (s, 9H), 1.52–1.76 (m, 3H), 1.98–2.22 (m, 2H),
2.34–2.38 (m, 2H), 3.80 (ddt, J ϭ 4.5, 6.6, 12.3 Hz, 1H),
4.25 (dt, J ϭ 6.3, 2.1 Hz, 2H), 4.96 (dd, J ϭ 2.1, 10.2 Hz,
1H), 5.03 (dd, J ϭ 2.1, 17.1 Hz, 1H), 5.82 (ddt, J ϭ 10.2,
17.1, 6.6 Hz, 1H).
2.15.3. (5R)-1-(4-Methoxybenzyl)oxynona-8-en-2-yn-5-ol
(3)
Allylmagnesium chloride in THF (0.41 M, 38.4 ml) was
added dropwise to a stirred solution of 2 (1.48 g, 6.32
mmol) in THF (45 ml) at Ϫ78°C, and the mixture was
allowed to warm to room temperature. After 40 min, the
reaction was quenched with saturated NH4Cl, and the mix-
ture was extracted with AcOEt. The extract was washed
with H2O, dried, and the solvent was evaporated to give 3
(1.60 g) as a pale yellow oil, which was used for the next
reaction without purification. The pure sample was obtained
2.15.6. (2Z)-(5R)-5-(t-Butyldimethylsilyl)oxy-3-iodonona-
2,8-dien-1-ol (6)
Seventy percent [(MeOCH2CH2O)2AlH2]Na in toluene
(2.24 g, 7.22 mmol) was dissolved in Et2O (20 ml), and the
resulting solution was cooled in an ice bath. Compound 5
(807 mg, 3.01 mmol) in Et2O (10 ml) was added to this
solution at 0°C, and the mixture was stirred at room tem-
perature for 6 h. The reaction mixture was treated with

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T. Higashi et al. / Steroids 65 (2000) 281–294
AcOEt (0.42 ml) with cooling in an ice bath to destroy
excess hydride and then cooled to Ϫ50°C. After addition of
I2 (1.15 g, 4.53 mmol), the mixture was stirred at Ϫ50°C for
10 min and then allowed to warm to room temperature.
After 1.5 h, the reaction was quenched by the addition of
2% Na2S2O3, and the mixture was extracted with Et2O. The
extract was washed with H2O and saturated NaHCO3, dried,
evaporated, and chromatographed. Elution with hexane/
Et2O (10:1 v/v) gave 6 (773 mg, 65%) as a pale yellow oil:
2.15.9. (2Z)-(5R)-[2-[5-(t-Butyldimethylsilyl)oxy-2-
methylenecyclohexylidene]ethyl]diphenylphosphine oxide
(9)
BuLi in hexane (1.53 M, 447 ␮l) was added to an
ice-cooled solution of diphenylphosphine (119 ␮l, 0.690
mmol) in THF (2.3 ml), and the mixture was stirred at 0°C
for 10 min. The mixture was cooled to Ϫ50°C, and a
solution of crude 8 (44 mg) in THF (1 ml) was added. After
being stirred at Ϫ50°C for 1 h, the reaction was quenched
by the addition of H2O; the mixture was allowed to warm to
room temperature, and then CHCl3 (6.9 ml) and 30% H2O2
(5.1 ml) were added. After being stirred for 30 min, the
mixture was diluted with CHCl3, washed with saturated
Na2S2O3 and H2O, dried, evaporated, and chromato-
graphed. Elution with AcOEt/hexane (2:1 v/v) gave 9 (56
24
[␣]D Ϫ3.9° (c 2.37, CHCl3); IR (neat) 3338, 1643, 1463,
1
1365, 1254, 1029 cmϪ1; H-NMR ␦: 0.06 (s, 3H), 0.07 (s,
3H), 0.87 (s, 9H), 1.44–1.68 (m, 3H), 2.10 (br q, J ϭ 8.1
Hz, 2H), 2.58 (dd, J ϭ 5.7, 14.1 Hz, 1H), 2.67 (dd, J ϭ 6.6,
14.1 Hz, 1H), 3.96 (q, J ϭ 5.7 Hz, 1H), 4.18 (br d, J ϭ 4.5
Hz, 2H), 4.61 (dd, J ϭ 1.8, 9.3 Hz, 1H), 5.03 (dd, J ϭ 1.8,
15.6 Hz, 1H), 5.80 (ddt, J ϭ 9.3, 15.6, 3.0 Hz, 1H), 5.89 (t,
J ϭ 5.7 Hz, 1H).
24
mg, 69% from 7) as a colorless crystalline solid: [␣]D
Ϫ20.7° (c 1.20, CHCl3); IR (neat) 1631, 1254, 1190, 1095
cmϪ1; 1H-NMR ␦: 0.00 (s, 6H), 0.84 (s, 9H), 1.41–1.82 (m,
4H), 2.05–2.40 (m, 3H), 3.20 (dddd, J ϭ 1.5, 6.3, 15.3, 21.3
Hz, 1H), 3.36 (dt, J ϭ 9.3, 15.3 Hz, 1H), 3.52 (hept, J ϭ 3.9
Hz, 1H), 4.68 (s, 1H), 4.92 (s, 1H), 5.35 (td, J ϭ 6.9, 7.8 Hz,
1H), 7.41–7.54 (m, 6H), 7.65–7.75 (m, 4H); HRMS calcd
for C27H37O2SiP [Mϩ]: 452.2307, found 452.2301.
2.15.7. (2Z)-(5R)-2-[5-(t-Butyldimethylsilyl)oxy-2-
methylenecyclohexylidene]ethanol (7)
After Ar gas was introduced into a solution of 6 (181 mg,
0.457 mmol) in MeCN (25 ml) for 15 min, Pd(PPh3)4 (53
mg, 45.9 ␮mol) and triethylamine (77 ␮l, 0.552 mmol) were
added, and the resulting mixture was heated at reflux for 4 h.
The solvent was removed, followed by purification by col-
umn chromatography. Elution with hexane/Et2O (2:1 v/v)
gave 7 (82 mg, 67%) as a pale yellow oil: [␣]D24 Ϫ40.6° (c
2.79, CHCl3); IR (neat) 3344, 1639, 1466, 1371, 1254,
2.15.10. (5Z,7E)-(3R,24R)-3,24,25-Tri(t-butyldimethyl-
silyl)oxy-9,10-secocholesta-5,7,10(19)-triene (11)
A solution of 9 (53 mg, 0.117 mmol) in THF (1.5 ml) was
cooled to Ϫ78°C and treated with 1.52 M BuLi in hexane (80
␮l, 0.119 mmol). The resulting deep red solution was stirred at
Ϫ78°C for 20 min, and a solution of (1R,3aR,7aR)-1-[(1R,4R)-
4,5-di(t-butyldimethylsilyl)oxy-1,5-dimethylhexyl]-7a-methyl-
octahydro-4H-inden-4-one (10) [11] (13 mg, 24.8 ␮mol) in
THF (1.2 ml) was added. After being stirred at Ϫ78°C for 1 h,
the reaction mixture was allowed to warm to Ϫ50°C, quenched
with saturated NH4Cl, and extracted with AcOEt. The extract
was washed with H2O and saturated NaCl, dried, evaporated,
and chromatographed. Elution with hexane/AcOEt (30:1 v/v)
gave 11 (19 mg, 99%) as a colorless viscous oil: [␣]D20 Ϫ6.0°
(c 0.975, CHCl3); IR (neat) 1466, 1369, 1252, 1165, 1093,
1
1095, 1011 cmϪ1; H-NMR ␦: 0.05 (s, 3H), 0.06 (s, 3H),
0.88 (s, 9H), 1.47 (br s, 1H), 1.52–1.64 (m, 1H), 1.82–1.92
(m, 1H), 2.07 (td, J ϭ 1.5, 13.5 Hz, 1H), 2.20 (dd, J ϭ 8.7,
13.5 Hz, 1H), 2.37 (dd, J ϭ 5.4, 13.2 Hz, 1H), 2.41 (dd, J ϭ
5.4, 13.2 Hz, 1H), 3.83 (hept, J ϭ 3.9 Hz, 1H), 4.10–4.30
(m, 2H), 5.44 (t, J ϭ 6.9 Hz, 1H).
2.15.8. (2Z)-(5R)-2-[5-(t-Butyldimethylsilyl)oxy-2-
methylenecyclohexylidene]-1-chloroethane (8)
Dimethyl sulfide (100 ␮l, 1.61 mmol) was added to an
ice-cooled solution of N-chlorosuccinimide (181 mg, 1.36
mmol) in CH2Cl2 (4.4 ml), and the mixture was stirred at
0°C for 40 min. Part of the resulting mixture (1.12 ml) was
added to a solution of 7 (47 mg, 0.175 mmol) in CH2Cl2
(0.57 ml) at Ϫ20°C, and the reaction mixture was allowed
to warm to room temperature. After being stirred for 1 h, the
reaction mixture was diluted with hexane, washed with H2O
and saturated NaCl, dried, and the solvent was evaporated.
The residue was taken up with AcOEt/hexane (10:1 v/v) and
filtered through silica gel. Evaporation of the filtrate gave 8
(44 mg) as a pale yellow oil, which was used for the next
1
1041 cmϪ1; H-NMR ␦: 0.03, 0.05, 0.06, 0.07, 0.08 (each s,
18H), 0.54 (s, 3H), 0.85 (s, 9H), 0.88 (s, 9H), 0.89 (s, 9H), 0.92
(d, J ϭ 5.4 Hz), 1.11 (s, 3H), 1.18 (s, 3H), [1.26–1.35 (m),
1.41–1.56 (m), 1.61–1.68 (m), 20H], 1.89–2.48 (m, 7H), 2.83
(dd, J ϭ 0.3, 15.3 Hz, 1H), 3.22 (br s, 1H), 3.78 (m, 1H), 4.82
(s, 1H), 5.03 (s, 1H), 6.05 (d, J ϭ 11.1 Hz, 1H), 6.18 (d, J ϭ
11.1 Hz, 1H).
2.15.11. (5Z,7E)-(3R,24R)-9,10-Secocholesta-5,7,10(19)-
triene-3,24,25-triol [3-epi-24,25(OH)2D3] (12)
Bu4NF in THF (1 M, 105 ␮l) was added to a stirred solution
of 11 (16 mg, 21.1 ␮mol) in THF (0.5 ml) at room tempera-
ture. After being stirred at Ϫ78°C for 4 days, the reaction
mixture was diluted with AcOEt, washed with H2O and satu-
rated NaCl, dried, evaporated, and chromatographed. Elution
with AcOEt/hexane (5:1 v/v) gave 12 (7 mg, 79%) as a col-
1
reaction without further purification: H-NMR ␦: 0.06 (s,
6H), 0.83 (s, 9H), 1.56–1.69 (m, 1H), 1.82–1.92 (m, 1H),
1.82–1.95 (m, 1H), 2.39 (br s, 1H), 2.43 (br s, 1H), 3.86 (q,
J ϭ 3.9 Hz, 1H), 4.19 (dd, J ϭ 6.3, 8.0 Hz, 2H), 4.84 (s,
1H), 5.02 (s, 1H), 5.46 (t, J ϭ 7.8 Hz, 1H).
23
orless crystalline solid: [␣]D ϩ29.3° (c 0.345, CHCl3); IR

T. Higashi et al. / Steroids 65 (2000) 281–294
287
1
(neat) 3367, 1643, 1442, 1377, 1219, 1157, 1051 cmϪ1; H-
NMR ␦: 0.56 (s, 3H), 0.94 (d, J ϭ 6.3 Hz, 3H), 1.17 (s, 3H),
1.22 (s, 3H), 1.12–1.74 (m, 17H), 1.89–2.02 (m, 7H), 2.10–
2.20 (m, 2H), 2.27 (dd, J ϭ 9.0, 12.9 Hz, 1H), 2.41 (dt, J ϭ
6.0, 13.5 Hz, 1H), 2.58 (dd, J ϭ 3.9, 12.9 Hz, 1H), 2.68 (br t,
J ϭ 7.8 Hz, 1H), 2.82 (dd, J ϭ 3.3, 12.0 Hz, 1H), 3.33 (dd, J ϭ
6.6, 4.8 Hz, 1H), 3.89 (br tt, J ϭ 4.0, 8.4 Hz, 1H), 4.84 (d, J ϭ
2.1 Hz, 1H), 5.06 (br s, 1H), 6.04 (d, J ϭ 11.1 Hz, 1H), 6.24
(d, J ϭ 11.1 Hz, 1H); HRMS calcd for C27H44O3 [Mϩ]:
416.3290, found 416.3270.
[MϪH]Ϫ in the positive- and the negative-ion modes, re-
spectively. These data indicated that peak A was the mono-
glucuronide of 23,25(OH)2-24-oxo-D3. The MS/MS, using
the ion at m/z 624 as a precursor ion, showed a satisfactory
product ion mass spectrum and supported the above struc-
ture: m/z 607 [MϩH]ϩ, 589 [607ϪH2O]ϩ, 431
[geninϩH]ϩ, 413 [431ϪH2O]ϩ, and 395 [431–2H2O]ϩ, but
the conjugation position (3, 23 or 25) could not be deter-
mined. We have reported that the PTAD adducts of conju-
gated vitamin D metabolites are cleaved at the C-6–7 bond
of the vitamin D skeleton and provide a product ion with
conjugation position indicated by a positive-ESI-MS/
MS/MS [10]. That is, in the case where the conjugation
position is at the A-ring, a characteristic product ion at m/z
474 for the glucuronide or 378 for the sulfate is observed,
whereas in the case where conjugation is at the side-chain,
only a corresponding ion at m/z 298 is observed. Thus, peak
A was converted to the PTAD adduct and subjected to
LC/ESI-MS/MS/MS using m/z 799 [MϩNH4]ϩ and m/z
782 [MϩH]ϩ as precursor ions with 20% relative collision
energy. The product ion mass spectrum of the peak A
derivative (tR 4.5 min) showed m/z 298, but not 474. This
result indicated that peak A had a glucuronic acid at the
side-chain, but there were still two possible conjugation
positions, that is, at the 23- or 25-positions. The mass
spectrum of acetylated peak A (tR 3.9 min) afforded evi-
dence to indicate the conjugation position. The spectrum
showed the molecular-related ions at m/z 792 [MϩNH4]ϩ
and 773 [MϪH]Ϫ in the positive- and the negative-ion
modes, respectively, indicating the presence of four reactive
sec- and one non-reactive tert-hydroxy groups in peak A.
These results showed that peak A was 23,25(OH)2-24-
oxo-D3 23G.
3. Results
3.1. Search for conjugated vitamin D metabolites in rat
bile
The bile specimens were subjected to solid phase extrac-
tion, followed by anion exchange chromatography, and then
UV-HPLC. Four new major peaks were observed in the chro-
matogram from female rats administered 24,25(OH)2D3 (Fig.
1a), and one of them was 24,25(OH)2D3 24G, which has
previously been reported [6]. Fractions containing the other
three peaks, which we called peaks A, B, and C, were collected
and further purified by prep. HPLC. The obtained fractions
were subjected to photodiode array UV-HPLC. As a result, all
three fractions showed typical UV absorption spectra (␭
:
max
268 nm, ␭min: 230 nm) of the vitamin D-triene structure. Peak
C was scarcely observed in the chromatogram from male rats
(Fig. 1b). Incidentally, 24,25(OH)2D3 3G was also isolated by
a previously described method [6], and its elution position is
shown by an arrow in Fig. 1.
On the other hand, a new metabolite showing the typical
UV absorption spectra of vitamin D was obtained from rats
dosed with 25(OH)D3, and called peak D (Fig. 1c and d).
Furthermore, fractions having the same tR as authentic
25(OH)D3 3S and 24,25(OH)2D3 24G, and peaks A, B, and
C as above were also collected. Two major peaks in Fig. 1c
(tR 20–24 min) were not vitamin D compound as deter-
mined by their UV spectra.
3.3. Identification of the peak B metabolite as 3-epi-
24,25(OH)2D3 24G
All of the ESI-MSn spectral data for peak B completely
agreed with those of 24,25(OH)2D3-24G or -25G, but its tR
(10.6 min) was different from those of the two glucuronides.
Enzymic hydrolysis of the peak B metabolite with ␤-glu-
curonidase gave a new peak, which eluted closely with
24,25(OH)2D3 [J’sphere ODS H-80, MeCN/H2O (3:2 v/v),
the genin of peak B tR 10.7 min, 24,25(OH)2D3 tR 10.9 min;
Develosil 60–5, hexane/i-PrOH (22:3 v/v), the genin of
peak B tR 13.0 min, 24,25(OH)2D3 tR 12.9 min]. These data
indicated that the metabolite was a monoglucuronide of
dihydroxyvitamin D3. Furthermore, the GC/MS mass spec-
trum of the TMS-genin derivative completely agreed with
that of TMS-24,25(OH)2D3 (Fig. 2), but their tR values were
much different [the genin of peak B derivative tR 21.9 min;
24,25(OH)2D3 derivative tR 19.5 min]. Based on these re-
sults, we guessed that the genin of peak B was a stereoiso-
mer of 24,25(OH)2D3, such as 24S,25(OH)2D3, which was
obtained from incubation of 24,25(OH)2D3 with the chick
kidney homogenate [12]. However, TMS-24S,25(OH)2D3
3.2. Identification of the peak A metabolite as
23,25(OH)2-24-oxo-D3 23G
Peak A completely disappeared and a new single peak,
which co-eluted with 23,25(OH)2-24-oxo-D3 [J’sphere
ODS H-80, MeCN/H2O (3:2 v/v), tR 7.8 min; Develosil
60–5, hexane/i-PrOH (22:3 v/v), tR 9.2 min], emerged after
treatment with ␤-glucuronidase. Furthermore, GC/MS data
for the trimethylsilyl (TMS)-derivatized genin and
23,25(OH)2-24-oxo-D3 completely agreed: tR (21.4 min),
m/z 646 [M]ϩ, 541 [MϪTMSOHϪCH3]ϩ, 487 [cleavage of
C-23–24 bond]ϩ, 397 [487ϪTMSOH]ϩ, 307 [487–2TM-
SOH]ϩ, and 131 [C(CH3)2OTMS]ϩ (Fig. 2). In the LC/
ESI-MS mass spectra of the intact peak A (tR 5.1 min), the
base ion peak was observed at m/z 624 [MϩNH4]ϩ and 605

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T. Higashi et al. / Steroids 65 (2000) 281–294
Fig. 2. GC/MS spectra of TMS derivatives of pyro-genins of peaks A, B, and D.
(tR 19.7 min) and the TMS-genin of peak B did not co-
migrate.
It is well-known that the reaction of vitamin D com-
pounds with PTAD gives 2-epimers, 6S- and 6R-adducts,
because the reagent attacks at the s-cis diene of vitamin D
from the ␤- and ␣-sides, respectively [13]. It is also obvious
that the 6S-adduct forms preferentially to the 6R-one in the
case of compounds with no hydroxy group at the 1␣-posi-
tion. The PTAD adduct of the genin of the peak B metab-
olite showed two equivalent peaks (tR 5.7 and 6.2 min) in

T. Higashi et al. / Steroids 65 (2000) 281–294
289
LC/APCI-MS monitoring at m/z 574 [MϩHϪH2O]ϩ,
3.4. Identification of the peak C metabolite as
24,25(OH)2D3 3S
which were much different from the data from
24,25(OH)2D3 [tR 5.0 (6R) and 7.4 min (6S) with a ratio of
ca. 1:5] (Fig. 3). This result suggested that the conformation
of the A-ring of the genin was different from that of the
usual vitamin D compounds. We obtained ca. 7 ␮g of the
genin from seven rats in the manner described above, and
1H-NMR spectral data suggested that the genin was 3-epi-
24,25(OH)2D3. That is, except for the chemical shift of a
proton at the 3-position [the genin, 3.89 ppm;
24,25(OH)2D3, 3.94 ppm], a clear difference between the
two compounds could not be observed. Although the A-ring
of 24,25(OH)2D3 exists essentially in the ␣-chair form, that
of its 3-epimer is inferred to exist in the ␤-chair form
because the A-ring is stabilized when the 3-hydroxy group
is in the equatorial position. Therefore, it was inferred that
the difference between 24,25(OH)2D3 and the genin in the
reaction with PTAD was caused by the different conforma-
tions.
Peak C hardly changed when treated with ␤-glucuron-
idase. This metabolite was surmised not to be a glucuro-
nide but to have an acidic functional group, such as a
sulfuric group, because it was retained on an anion ex-
change gel, PHP-LH-20. In LC/ESI-MS, the peak C me-
tabolite (tR 4.8 min) gave an ion only at m/z 495 [MϪH]Ϫ
in the negative-ion mode, which indicated that its molec-
ular weight was larger than 24,25(OH)2D3 by 80 mass
units. Based on these data, we inferred that this com-
pound was a sulfate of 24,25(OH)2D3. The genin of peak
C obtained by solvolysis with sulfuric acid co-migrated
with 24,25(OH)2D3 on the reversed- and normal-phase
HPLC. Incidentally, we have reported that sulfates of
vitamin D metabolites are poorly hydrolyzed by treat-
ment with sulfatase [17], and peak C showed the same.
The structure of peak C was also analyzed by LC/ESI-
MSn combined with some derivatization. After acetyla-
tion (tR 12.6 min), the molecular weight of the compound
increased by 42–538, which indicated that this metabolite
had one pri- or sec-hydroxy group. The PTAD adduct of
peak C (tR 3.1 min) showed molecular-related ions at
m/z 689 [MϩNH4]ϩ and 670 [MϪH]Ϫ in the positive-
and the negative-ion modes, respectively. Its produc-
t ion mass spectrum, using m/z 689 and then 654
[MϩHϪH2O]ϩ as precursor ions, showed an ion at m/z
378, which indicates that the sulfated position was a
3-hydroxy group. In addition, the following characteristic
fragment ions were observed: m/z 636 [654ϪH2O]ϩ, 574
[654ϪSO3]ϩ. These results showed that peak C was
24,25(OH)2D3 3S.
To identify the genin more reliably, we synthesized
3-epi-24,25(OH)2D3 based on the Lythgoe coupling [14]
of the A-ring fragment 9 with the C/D-ring fragment 10
(Fig. 4). The synthesis of the A-ring fragment 9 followed
a method we had previously developed [15,16]. Reaction
of S-(ϩ)-epichlorohydrin (1) with lithium acetylide of
p-methoxybenzyl propargyl ether followed by base-treat-
ment gave epoxide 2. Sequential epoxide-opening of 2
with allylmagnesium chloride and silylation gave silyl
ether 4, which was then subjected to oxidative deprotec-
tion to give alcohol 5. Hydroalumination of 5 with so-
dium bismethoxyethoxyaluminum hydride, followed by
treatment of the resulting alkenylaluminum complex with
iodine, gave (Z)-iodoalkene 6, which, upon palladium-
catalyzed cyclization, afforded dienol 7. The dienol 7 was
then converted to the required A-ring fragment 9 through
chlorination, reaction of 8 with lithium diphenylphos-
phide, and oxidation. According to the Hoffman La
Roche procedure [14], the A-ring fragment 9 was allowed
to react with the C/D-ring fragment 10 to give triene
11 almost quantitatively. Finally, desilylation of 11
with tetrabutylammonium fluoride furnished 3-epi-
3.5. Identification of the peak D metabolite as
23,25(OH)2D3 23G
The results from LC/ESI-MS/MS (tR 6.5 min) and enzymic
hydrolysis indicated that peak D was a monoglucuronide of a
dihydroxyvitamin D3. The GC/MS spectrum of the TMS-
genin derivatives (tR 16.1 min) showed a series of fragment
ions at m/z 487 [cleavage of C-23–24 bond]ϩ, 397 [487ϪTM-
SOH]ϩ, and 307 [487–2TMSOH]ϩ, which are peculiar to
compounds having a hydroxy group at the 23-position [18],
together with the following ions: m/z 632 [M]ϩ, 542 [M-
TMSOH]ϩ, 527 [542-CH3]ϩ, 452 [632–2TMSOH]ϩ, 362
[632–3TMSOH]ϩ, and 131 [(CH3)2COTMS]ϩ (Fig. 2).
On the basis of these data and the reported metabolic pathway
of 25(OH)D3, the genin of peak D was identified as
23,25(OH)2D3. The conjugation position was determined to be
at the 23-position by LC/ESI-MSn after derivatization [PTAD-
adduct (tR 6.3 min) and acetate (tR 6.8 min)] as above. From
1
24,25(OH)2D3 (12). The H-NMR spectrum of the syn-
thetic 3-epi-24,25(OH)2D3 gave a broad triplet of triplets
peak of H-3 at 3.89 ppm, which agreed with that of the
genin of the biliary metabolite. Furthermore, the behavior
of the synthetic compound and the genin in LC/APCI-MS
after derivatization with PTAD and in GC/MS com-
pletely agreed. From these results, the genin was identi-
fied as 3-epi-24,25(OH)2D3.
On the other hand, the conjugation position was deter-
mined from the ESI-MSn spectral data for the PTAD (tR 7.2
min) and the acetylated (tR 5.7 min) derivatives as above.
As a result, peak B was identified as 3-epi-24,25(OH)2D3
24G.
the results stated above, peak
23,25(OH)2D3 23G.
D was identified as

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T. Higashi et al. / Steroids 65 (2000) 281–294
Fig. 3. LC/APCI-MS chromatograms of PTAD adducts of 24,25(OH)2D3, 3-epi-24,25(OH)2D3 and genin of peak B. (a), PTAD adducts of 24,25(OH)2D3
and 3-epi-24,25(OH)2D3; (b), PTAD adduct of genin of peak B. Monitoring ion: m/z 574 [MϩHϪH2O]ϩ.
3.6. Identification of 25(OH)D3 3S and other metabolites
tained from LC/ESI-MSn. The putative 25(OH)D3 3S was
co-eluted with an authentic sample [J’sphere ODS H-80,
MeOH/2% NaClO4 (pH 3.0) (4:1 v/v), tR 9.8 min], and its
PTAD adduct (tR 7.7 min) also gave the same product
ions as the authentic sample in positive-ESI-MS/MS/MS:
25(OH)D3 3S in bile from rats dosed with 25(OH)D3
was identified by comparison with the standard sample
based on their chromatographic behavior and data ob-

T. Higashi et al. / Steroids 65 (2000) 281–294
291
Fig. 4. Synthesis of 3-epi-24,25(OH)2D3. Reagents (yield): i, CHϵCCH2OMPM, BuLi, BF3⅐Et2O then NaH (92%); ii, CH2ϭCHCH2MgCl; iii, TBSCl,
imidazole (82% from 2); iv, DDQ then NaBH4 (79%); v, [(MeOCH2CH2O)2AlH2]Na then I2 (65%); vi, Pd(PPh3)4, Et3N (67%); vii, N-chlorosuccinimide,
Me2S; viii, Ph2PH, BuLi then H2O2 (69% from 7); ix, BuLi (99%); x, Bu4NF (79%).
m/z 638 [MϩHϪH2O]ϩ and 378 [cleavage of C-6–7
bond]ϩ.
The other metabolites [24,25(OH)2D3-3G, -24G,
25(OH)D3-3G, and -25G] were also identified by compari-
son with standard samples as previously reported [6,7].
ing the determination of their structures, had not been done.
We have identified the monoglucuronides of 24,25(OH)2D3
[6] and 25(OH)D3 [7] from rat bile dosed with each genin,
but there are many unsolved questions, such as the relation-
ship between side-chain oxidation and the phase II reaction.
In these respects, we studied the conjugated metabolites
formed after the administration of 24,25(OH)2D3 or
25(OH)D3 and found the metabolic pathway shown in Fig.
5. The structures of these metabolites were determined us-
ing LC/MSn and GC/MS, and the former method combined
with the derivatization reported previously [10] was very
useful in determining the conjugation position. Incidentally,
monoglucuronides of 25(OH)D3 and 24,25(OH)2D3 were
isolated by a method we had previously reported [6,7]. The
approximate yields of each compound are also shown in
4. Discussion
It has been accepted that vitamin D compounds are
excreted after successive oxidation of the side-chain, which
leads to the formation of polar compounds, such as C-23
carboxylic acid [1,19]. On the contrary, a few reports sug-
gested that vitamin D metabolites were excreted into the
bile as conjugates [20], but a detailed investigation, includ-

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T. Higashi et al. / Steroids 65 (2000) 281–294
Fig. 5. Metabolic pathways of 25(OH)D3 and 24,25(OH)2D3. Approximate yield from 1 ml of bile from rats administered 25(OH)D3 (a) and 24,25(OH)2D3
(b) (mean of 2 or more animals); (c), from only female rats.
Fig. 5, which were determined by comparison with known
amounts of the synthetic standards co-eluted or eluted
closely in HPLC. Glucuronidation of dihydroxylated me-
tabolites occurred mainly at the sec-hydroxy group on the
side-chain; on the other hand, sulfation was observed only at
the 3-hydroxy group.
As mentioned in the introduction, 24,25(OH)2D3 was
reported to be metabolized to 23,25(OH)2-24-oxo-D3 23G
and then excreted into the bile in dogs [8]. The present study
showed that a similar metabolism occurred in rats; more-
over, the yield of 23,25(OH)2-24-oxo-D3 23G was several
times that of 24,25(OH)2D3-3G or -24G reported previously
[6]. We also searched for the monoglucuronide of 25(OH)-
24-oxo-D3, the intermediate of 23,25(OH)2-24-oxo-D3 from
24,25(OH)2D3, in the bile using LC/MS (monitoring: m/z
590 [MϪH]Ϫ), but this metabolite could not be found.
[23]. Recently, 3-epi-1,25(OH)2D3 was isolated from the
serum of rats treated with 1,25(OH)2D3 [24]. To our knowl-
edge, the present paper is the first to report the isolation and
characterization of a 3␣-hydroxyvitamin D metabolite with
no hydroxy group at the 1␣-position. In the case of
1,25(OH)2D3, it has been reported that the reaction proceeds
from 3␤ toward 3␣ unidirectionally and is enzymatic, but
not inhibited by a P450 inhibitor, such as ketoconazole
[21,23]. In other steroids, 3-epi-digoxigenin is known as a
metabolite of digoxin and is formed via the 3-oxo form by
oxidoreductase [25]. Although the mechanism of the for-
mation of 3-epi-24,25(OH)2D3 is not clear, this epimeriza-
tion is one of the important pathways in vitamin D metab-
olism because the yield of 3-epi-24,25(OH)2D3 24G was
much larger than that of 24,25(OH)2D3-3G or -24G [6].
From the bile of rats administered 25(OH)D3,
24,25(OH)2D3 24G, 23,25(OH)2-24-oxo-D3 23G, and
3-epi-24,25(OH)2D3 24G were obtained together with
25(OH)D3-3G and -25G. It is not surprising that the above
metabolites were formed because 25(OH)D3 metabolized to
24,25(OH)2D3 in the kidney. We did not search for
24,25(OH)2D3 3G in this bile because its amount was in-
ferred to be very small. We also isolated 23,25(OH)2D3 23G
The genin of peak
B was identified as 3-epi-
24,25(OH)2D3 by comparison with a synthetic standard in
1H-NMR and various MS spectral data. Epimerization of
the 3-hydroxy group of vitamin D metabolites has been
reported for 1␣,25-dihydroxyvitamin D3 [1,25(OH)2D3] in-
cubated in several cell lines, such as human keratinocytes
[21], colon adenocarcinoma [22], and rat osteosarcoma

T. Higashi et al. / Steroids 65 (2000) 281–294
293
from the bile, in a slightly smaller yield than that of
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Part of this work was supported by grants from the
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1

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