In vitro and in vivo glucuronidation of 24,25-dihydroxyvitamin D3

Article abstract of DOI:10.1016/S0039-128X(99)00057-4

Glucuronidation of 24,25-dihydroxyvitamin D₃ has been investigated in in vitro and in vivo experiments. Three positional isomers of 24,25-dihydroxyvitamin D₃ monoglucuronide were synthesized from 24,25-dihydroxyprovitamin D₃

Full text of DOI:10.1016/S0039-128X(99)00057-4

Steroids 64 (1999) 715–725
In vitro and in vivo glucuronidation of 24,25-dihydroxyvitamin D3૾
Tatsuya Higashi, Maki Horike, Ryuta Kikuchi, Kazutake Shimada*
Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-0934, Japan
Received 2 December 1998; received in revised form 22 February 1999; accepted 25 February 1999
Abstract
Glucuronidation of 24,25-dihydroxyvitamin D3 has been investigated in in vitro and in vivo experiments. Three positional isomers of
24,25-dihydroxyvitamin D3 monoglucuronide were synthesized from 24,25-dihydroxyprovitamin D3 derivatives with Koenigs-Knorr
reaction and used as standard samples. In the presence of the rat liver microsomal fraction and uridine-5Ј-diphosphoglucuronic acid,
24,25-dihydroxyvitamin D3 gave 3- and 24-glucuronides as the main products in almost equal amounts, but only a small amount of the
corresponding 25-glucuronide was obtained. 24,25-Dihydroxyvitamin D3 monoglucuronide was deconjugated with rat intestine homoge-
nate, which indicated the entero-hepatic circulation of 24,25-dihydroxyvitamin D3. After the administration of 24,25-dihydroxyvitamin D3
to rats, its 3- and 24-glucuronides were identified from the bile as inferred from the in vitro experiment. However, the in vivo glucuronidation
occurred at the 24-position in preference to the 3-position, and the corresponding 25-glucuronide was not detected. These glucuronides were
identified in comparison with standard samples based on their chromatographic behavior during high-performance liquid chromatography
and data obtained from liquid chromatography-electrospray ionization-mass spectrometry, which was helpful in identifying these
compounds. © 1999 Elsevier Science Inc. All rights reserved.
Keywords: 24,25-Dihydroxyvitamin D3; Monoglucuronide; Synthesis; Rat liver microsome; Rat bile; LC-MS
1. Introduction
paper in this series, we reported that 25-hydroxyvitamin D3
3-glucuronide [25(OH)D33G] and 25(OH)D325G were ob-
tained as biliary metabolites of rats dosed with 25(OH)D3
per os (p.o.) [5], in which glucuronidation was observed not
only at the sec-hydroxy group (3G) but also at the tert-hydroxy
one (25G) in a ratio of 5:2. This glucuronidation was also
observed during an in vitro study using rat liver microsomal
fraction as an enzyme source [6]. It is inferred from our data
that 24,25(OH)2D3 is also converted to its glucuronide.
In the present paper,† we first synthesized three posi-
tional isomers of 24,25(OH)2D3 monoglucuronide as stan-
dard samples. Second, in vitro glucuronidation of
24,25(OH)2D3 was investigated, and the formed monoglu-
curonides were identified in comparison with the standard
synthetic samples based on their chromatographic behavior
during high-performance liquid chromatography (HPLC)
and data obtained from liquid chromatography-electrospray
ionization-mass spectrometry (LC-ESI-MS). Their struc-
tures were also confirmed by enzymic hydrolysis using
␤-glucuronidase. In addition, the entero-hepatic circulation
of 24,25(OH)2D3 was also investigated. Third, 24,25(OH)2D3
was administered to rats, and 24,25(OH)2D3 monoglucu-
ronides excreted into the bile were identified by similar
methods.
(24R)-24,25-Dihydroxyvitamin D3 [24,25(OH)2D3] is
one of the major metabolites of vitamin D3, which causes a
marked increase in bone volume and mechanical strength in
animals without hypercalcemia at pharmacological doses
[1,2]. Based on these criteria, the metabolite is being exam-
ined as an anti-osteoporosis medicine, and much interest is
focused on the metabolism of 24,25(OH)2D3. 24,25(OH)2D3 is
reported to be oxidized on its side chain at the C23 or C24
position by vitamin D 24-hydroxylase to give metabolites such
as 25-hydroxy-24-oxovitamin D3 [25(OH)-24-oxo-D3] and
(23S)-23,25-dihydroxy-24-oxovitamin D3 [23,25(OH)2-24-
oxo-D3] [3], but conjugates of 24,25(OH)2D3, such as gluc-
uronide or sulfate, still remain poorly understood.
Recently, 23,25(OH)2-24-oxo-D3 23-glucuronide was re-
ported as a biliary metabolite obtained from dogs dosed
with 24,25(OH)2D3, but a conjugate of 24,25(OH)2D3 has
not been obtained [4]. On the other hand, in a previous
* Corresponding author. Tel.: ϩ81-76-234-4459; fax: ϩ81-76-234-
4459.
E-mail address: shimada@dbs.p.kanazawa-u.ac.jp (K. Shimada)
૾ Part of this work was reported in a preliminary communication [7].
0039-128X/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(99)00057-4

716
T. Higashi et al. / Steroids 64 (1999) 715–725
2. Experimental
2.2.2. (20S,22␰,24R)-3␤-(tert-Butyldimethylsilyloxy)-22-
phenylsulfonylcholesta-5,7-diene-24,25-diol (4)
BuLi (2.5 M solution in hexane; 2.1 ml) was added
dropwise to a solution of 3 (375 mg, 0.660 mmol) and
(2R)-2,3-dihydroxy-3-methylbutan-1-yl p-toluenesulfonate
[12] (362 mg, 1.32 mmol) in tetrahydrofuran (THF) (10 ml)
at Ϫ50°C. The resulting mixture was stirred under argon
gas at Ϫ30°C for 2 h and quenched with saturated aq.
NH4Cl. The mixture was extracted with AcOEt, and the
organic layer was washed (brine) and then dried over an-
hydrous Na2SO4. After removal of the solvents under re-
duced pressure, the crude product thus obtained was purified
by flash column chromatography (30 x 1.0 cm i.d.) [hexane/
AcOEt (1:1 v/v)] to give compound 4 (epimers at C22; total
2.1. Materials and apparatus
24,25(OH)2D3 was obtained from Duphar (Amsterdam,
The Netherlands). (20S)-22-Phenylsulfonyl-23,24-dinor-
chola-5,7-dien-3␤-ol (1) [8,9] and (24R)-24-(tert-butyldi-
methylsilyloxy)cholesta-5,7-diene-3␤,25-diol (2) [10] were
synthesized from ergosterol in our laboratories.
Melting points were measured on a Yanagimoto melting
point apparatus (Kyoto, Japan) without correction. Optical
rotation was measured on a Horiba SEPA-300 polarimeter
(Tokyo, Japan) for solutions in CHCl3 at 24°C, and [␣]D
values are given in units of g/dl. UV spectra (in EtOH) were
obtained on a Hitachi U-2000 spectrophotometer (Tokyo).
1H-NMR spectra were obtained with a Jeol JNM-EX-270
(270 MHz) spectrometer (Tokyo). CDCl3 was used as the
solvent with tetramethylsilane as internal standard. The ab-
breviations used are s ϭ singlet, d ϭ doublet, dd ϭ doublet
of doublets, and m ϭ multiplet. Mass spectra [electron
impact (EI) or fast-atom bombardment (FAB)] were deter-
mined with Jeol JMS-SX-102A and Jeol JMS-DX-303
spectrometers, respectively. Column and flash column chro-
matographies were carried out with Silica gel 60 (70–230
mesh; E. Merck, Darmstadt, Germany) and Wakogel FC-40
(20–40 mesh; Wako, Osaka, Japan), respectively. Prepara-
tive (prep.) thin-layer chromatography (TLC) was con-
ducted with 0.5 mm pre-coated Silica gel 60 F254 (Merck).
Isolute C18 (EC) cartridges (500 mg: International Sorbent
Tech., Hengoed, UK) were obtained from Uniflex (Tokyo),
and piperidinohydroxypropyl Sephadex LH-20 (PHP-LH-
20) [11] was prepared in our laboratories. ␤-Glucuronidase
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 the enzymic hydro-
lysis.
1
369 mg, 83.4%) as a colorless foam. H-NMR of the main
epimer (less polar) ␦: 0.06 [6H, s, Si(Me)2], 0.37 (3H, s,
H-18), 0.88 (9H, s, Si-tert-Bu), 1.03 (3H, d, J ϭ 6.6 Hz,
H-21), 1.21, 1.27 (3H each, s, H-26, 27), 3.50–3.60 (2H, m,
H-3␣, 22), 3.78 (1H, m, H-24), 5.32, 5.51 (1H each, m, H-6,
7), 7.56–7.91 (5H, m, H-Ph).
2.2.3. (24R)-3␤-(tert-Butyldimethylsilyloxy)cholesta-5,7-
diene-24,25-diol (5)
Five percent Na-Hg (7.0 g) and Na2HPO4 (1.5 g) were
added to a solution of 4 (264 mg, 0.394 mmol) in THF/
MeOH (40:3 v/v, 8.6 ml), and then the mixture was stirred
under N2 gas at room temperature for 3.5 h. The resulting
suspension was poured into chilled 0.05 M sodium phos-
phate buffer (pH 7.3), and the insoluble material was re-
moved by filtration. The filtrate was extracted with AcOEt,
and the organic layer was washed (brine) and then dried
over anhydrous Na2SO4. After removal of the solvents un-
der reduced pressure, the crude product thus obtained was
purified by flash column chromatography (30 x 1.0 cm i.d.)
[hexane/AcOEt (3:2 v/v)] to give compound 5 (154 mg,
73.8%) as a colorless solid. 1H-NMR ␦: 0.07 [6H, s,
Si(Me)2], 0.62 (3H, s, H-18), 0.90 (9H, s, Si-tert-Bu), 0.94
(3H, s, H-19), 0.96 (3H, d, J ϭ 6.5 Hz, H-21), 1.17, 1.21
(3H each, s, H-26, 27), 3.33 (1H, m, H-24), 3.57 (1H, m,
H-3␣), 5.38, 5.55 (1H each, m, H-6, 7).
2.2. Syntheses of 24,25(OH)2D3 monoglucuronides
2.2.1. (20S)-3␤-(tert-Butyldimethylsilyloxy)-22-
phenylsulfonyl-23,24-dinorchola-5,7-diene (3)
2.2.4. (24R)-3␤-(tert-Butyldimethylsilyloxy)-25-
hydroxycholesta-5,7-dien-24-yl Acetate (6)
A mixture of 1 (363 mg, 0.799 mmol), tert-butyldimeth-
ylsilyl chloride (240 mg, 1.59 mmol) and imidazole (220
mg, 3.23 mmol) in N,N-dimethylformamide (4.0 ml) was
stirred at room temperature for 1 h. The resulting solution
was diluted with AcOEt, washed (H2O) and then dried over
anhydrous Na2SO4. After removal of the solvents under
reduced pressure, the crude product thus obtained was pu-
rified by column chromatography (37 x 1.8 cm i.d.)(CHCl3)
to give compound 3 (443 mg, 97.6%) as a colorless solid.
1H-NMR ␦: 0.06 [6H, s, Si(Me)2], 0.59 (3H, s, H-18), 0.89
(9H, s, Si-tert-Bu), 0.91 (3H, s, H-19), 1.23 (3H, d, J ϭ 6.3
Hz, H-21), 2.82–3.18 (2H, m, H-22), 3.56 (1H, m, H-3␣),
5.36, 5.52 (1H each, m, H-6, 7), 7.54–7.93 (5H, m, H-Ph).
A solution of 5 (50.5 mg, 95.3 ␮mol) in pyridine/Ac2O
(2:1 v/v, 1.5 ml) was stirred at room temperature for 4 h.
The resulting solution was poured into chilled H2O and
extracted with AcOEt. The organic layer was washed
(chilled 5% HCl, 5% NaHCO3, and brine) and then dried
over anhydrous Na2SO4. After removal of the solvents un-
der reduced pressure, the crude product thus obtained was
purified by column chromatography (30 x 1.0 cm i.d.)
[CHCl3/MeOH (100:1 v/v)] to give compound 6 (50.1 mg,
92.0%) as colorless needles (from hexane-CH2Cl2). mp
1
151.5–152.5°C. [␣]D Ϫ42.2° (c ϭ 0.102). H-NMR ␦: 0.06
[6H, s, Si(Me)2], 0.61 (3H, s, H-18), 0.89 (9H, s, Si-tert-
Bu), 0.93 (3H, s, H-19), 0.94 (3H, d, J ϭ 6.9 Hz, H-21),

T. Higashi et al. / Steroids 64 (1999) 715–725
717
1.20 (6H, s, H-26, 27), 2.11 (3H, s, Ac), 3.57 (1H, m, H-3␣),
4.78 (1H, dd, J ϭ 3.0, 6.9 Hz, H-24), 5.39, 5.55 (1H each,
m, H-6, 7).
the reaction mixture was stirred under argon gas at room
temperature for 2.5 h. The resulting mixture was filtered, the
filtrate was evaporated under reduced pressure and the crude
product thus obtained was purified by flash column chro-
matography (30 x 1.0 cm i.d.) [hexane/AcOEt (1:1 v/v)] to
give methyl {[(24R)-24-acetoxycholesta-5,7-dien-25-ol-3␤-
yl]-2,3,4-tri-O-acetyl-␤-D-glucopyranosid}uronate (10, 26.3
mg, 47.1%) as a colorless semi-solid. 1H-NMR ␦: 0.60 (3H,
s, H-18Ј), 0.91 (3H, s, H-19Ј), 0.94 (3H, d, J ϭ 6.6 Hz,
H-21Ј), 1.20 (6H, s, H-26Ј, 27Ј), 2.02, 2.05, 2.11 (6H, 3H,
3H, s each, 4 x Ac), 3.62 (1H, m, H-3Ј␣), 3.75 (3H, s,
COOMe), 4.02 (1H, d, J ϭ 9.2 Hz, H-5), 4.67 (1H, d, J ϭ
7.9 Hz, H-1), 4.78 (1H, dd, J ϭ 3.0, 6.6 Hz, H-24Ј), 4.98,
5.24 (1H, 2H, m each, H-2, 3, 4), 5.38, 5.56 (1H each, m,
H-6Ј, 7Ј).
2.2.5. (24R)-3␤,25-Dihydroxycholesta-5,7-dien-24-yl
Acetate (7)
A solution of 6 (50.0 mg, 87.4 ␮mol) and tetrabutylam-
monium fluoride (TBAF) (1.0 mmol) in THF (1.5 ml) was
stirred at room temperature for 2 h. The resulting solution
was diluted with AcOEt, washed (H2O) and then dried over
anhydrous Na2SO4. After removal of the solvents under
reduced pressure, the crude product thus obtained was pu-
rified by flash column chromatography (30 x 1.0 cm i.d.)
[CHCl3/MeOH (50:1 v/v)] to give compound 7 (33.3 mg,
1
83.2%) as a colorless semi-solid. H-NMR ␦: 0.62 (3H, s,
H-18), 0.94 (3H, s, H-19), 0.95 (3H, d, J ϭ 4.6 Hz, H-21),
1.20 (6H, s, H-26, 27), 2.11 (3H, s, Ac), 3.63 (1H, m, H-3␣),
4.78 (1H, dd, J ϭ 3.0, 6.9 Hz, H-24), 5.39, 5.57 (1H each,
m, H-6, 7).
Diglucuronide acetate methyl ester (11) was not sepa-
rated from decomposed compounds of Br-sugar by the pre-
vious flash column chromatography; the crude fraction con-
taining compound 11 was subjected to prep. TLC [20 x 20
cm, hexane/AcOEt (1:1 v/v), developed two times]. The
zone corresponding to Rf ca. 0.6 was extracted [CHCl3/
MeOH (5:1 v/v)] to yield compound 11 (14.5 mg, 18.4%) as
a colorless semi-solid. 1H-NMR ␦: 0.60 (3H, s, H-18Ј), 0.91
(3H, s, H-19Ј), 0.93 (3H, d, J ϭ 7.3 Hz, H-21Ј), 1.18, 1.23
(3H each, s, H-26Ј, 27Ј), 2.01, 2.02, 2.05 (21H, s each, 7 x
Ac), 3.62 (1H, m, H-3Ј␣), 3.74, 3.75 (3H each, s, COOMe),
4.00, 4.03 (1H, d, J ϭ 9.6 Hz, 2 x H-5), 4.67, 4.76 (1H, d,
J ϭ 7.6, 7.6 Hz, 2 x H-1), 4.85 (1H, m, H-24Ј), 4.99, 5.23
(2H, 4H, m each, 2 x H-2, 3, 4), 5.38, 5.56 (1H each, m,
H-6Ј, 7Ј).
2.2.6. (24R)-24-(tert-Butyldimethylsilyloxy)-25-
hydroxycholesta-5,7-dien-3␤-yl Acetate (8)
A solution of 2 (230 mg, 0.434 mmol) in pyridine/Ac2O
(2:1 v/v, 6.0 ml) was stirred at room temperature for 2.5 h.
The resulting solution was poured into chilled H2O and
extracted with AcOEt. The organic layer was washed
(chilled 5% HCl, 5% NaHCO3, and brine) and then dried
over anhydrous Na2SO4. After removal of the solvents un-
der reduced pressure, the crude product thus obtained was
purified by column chromatography [hexane/AcOEt (10:1
v/v)] to give compound 8 (213 mg, 85.8%) as a colorless
1
semi-solid. H-NMR ␦: 0.10 [3H each, s, Si(Me)2], 0.62
2.2.9. Koengs-Knorr reaction of compound 9
(3H, s, H-18), 0.92 (9H, s, Si-tert-Bu), 0.95 (3H, s, H-19),
1.15 (6H, s, H-26, 27), 2.04 (3H, s, Ac), 3.40 (1H, m, H-24),
4.70 (1H, m, H-3␣), 5.40, 5.56 (1H each, m, H-6, 7).
Freshly prepared Br-sugar (417 mg, 1.05 mmol) and
Ag2CO3 (305 mg, 1.10 mmol) were added to a solution of
9 (95.7 mg, 0.209 mmol) in CHCl3 (4.0 ml), and the reac-
tion mixture was stirred under argon gas at room tempera-
ture for 5 h. The resulting mixture was filtered, the filtrate
was evaporated under reduced pressure and the crude prod-
uct thus obtained was purified by flash column chromatog-
raphy (33 x 1.0 cm i.d.) [hexane-AcOEt (3:2 v/v)] to give
methyl {[(24R)-3␤-acetoxycholesta-5,7-dien-24-ol-25-yl]-
2,3,4-tri-O-acetyl-␤-D-glucopyranosid}uronate (13, 76.5
mg, 47.3%) as a colorless amorphous substance (from Hex-
ane-CH2Cl2). mp 180.5–182.5°C. [␣]D Ϫ38.9° (c ϭ 0.099).
1H-NMR ␦: 0.62 (3H, s, H-18Ј), 0.94 (3H, d, J ϭ 5.9 Hz,
H-21Ј), 0.95 (3H, s, H-19Ј), 1.16, 1.21 (3H each, s, H-26Ј,
27Ј), 2.02, 2.03, 2.04 (3H, 3H, 6H, s each, 4 x Ac), 3.36
(1H, m, H-24Ј), 3.75 (3H, s, COOMe), 4.03 (1H, d, J ϭ 9.6
Hz, H-5), 4.71 (1H, m, H-3Ј␣), 4.76 (1H, d, J ϭ 7.9 Hz,
H-1), 4.98, 5.25 (1H, 2H, m each, H-2, 3, 4), 5.40, 5.56 (1H
each, m, H-6Ј, 7Ј). Methyl {[(24R)-3␤-acetoxycholesta-5,7-
dien-25-ol-24-yl]-2,3,4-tri-O-acetyl-␤-D-glucopyranosid}-
uronate (12, 43.3 mg, 26.7%) was obtained from hexane/
AcOEt (1:1 v/v) fraction as colorless needles (from hexane-
2.2.7. (24R)-24,25-Dihydroxycholesta-5,7-dien-3␤-yl
acetate (9)
A solution of 8 (208 mg, 0.364 mol) and TBAF (3.5
mmol) in THF (3.5 ml) was stirred at room temperature for
30 min. The resulting solution was diluted with AcOEt,
washed (H2O) and then dried over anhydrous Na2SO4. After
removal of the solvents under reduced pressure, the crude
product thus obtained was purified by column chromatog-
raphy (30 x 1.8 cm i.d.) [hexane/AcOEt (1:1 v/v)] to give
compound 9 (155 mg, 93.1%) as a colorless semi-solid.
1H-NMR ␦: 0.63 (3H, s, H-18), 0.95 (3H, s, H-19), 0.96
(3H, d, J ϭ 5.9 Hz, H-21), 1.17, 1.22 (3H each, s, H-26, 27),
2.04 (3H, s, Ac), 3.34 (1H, m, H-24), 4.70 (1H, m, H-3␣),
5.39, 5.57 (1H each, m, H-6, 7).
2.2.8. Koengs-Knorr reaction of compound 7
Freshly prepared methyl 2,3,4-tri-O-acetyl-1-bromo-1-
deoxy-␣-D-glucopyranuronate (Br-sugar) (119 mg, 0.300
mmol) and Ag2CO3 (86.2 mg, 0.313 mmol) were added to
a solution of 7 (33.0 mg, 72.1 ␮mol) in CHCl3 (2.0 ml), and
1
CH2Cl2). mp 163.5–165°C. [␣]D Ϫ44.2° (c ϭ 0.103). H-
NMR ␦: 0.63 (3H, s, H-18Ј), 0.94 (3H, d, J ϭ 8.6 Hz,

718
T. Higashi et al. / Steroids 64 (1999) 715–725
H-21Ј), 0.95 (3H, s, H-19Ј), 1.14, 1.16 (6H, s, H-26Ј, 27Ј),
2.02, 2.03, 2.05 (3H, 3H, 6H, s each, 4 x Ac), 3.38 (1H, m,
H-24Ј), 3.75 (3H, s, COOMe), 4.06 (1H, d, J ϭ 9.6 Hz,
H-5), 4.64 (1H, d, J ϭ 7.6 Hz, H-1), 4.71 (1H, m, H-3Ј␣),
5.07, 5.24 (1H, 2H, m each, H-2, 3, 4), 5.41, 5.57 (1H each,
m, H-6Ј, 7Ј).
a Vycor filter at 0°C with argon gas bubbling through the
solution. After removal of the solvents under reduced pres-
sure, the obtained residue was dissolved in EtOH (20 ml)
and stored in the dark at 60°C for 14 h and room temper-
ature for 13.5 h under argon gas. The solvent was evapo-
rated off, and the crude product thus obtained was purified
by prep. TLC [20 x 20 cm, hexane/AcOEt (2:1 v/v); devel-
oped four times]. The zone corresponding to Rf ca. 0.6 was
extracted [CHCl3-MeOH (5:1 v/v)] to yield compound 16
2.2.10. Methyl {[(5Z,7E)-(3S,24R)-24-acetoxy-25-hydroxy-
9,10-secocholesta-5,7,10 (19)-trien-3␤-yl]-2,3,4-tri-O-
acetyl-␤-D-glucopyranosid}uronate (14)
(4.30 mg, 13.7%) as a colorless oil. UV ␭
nm: 264.0,
max
1
A solution of 10 (32.6 mg, 42.1 ␮mol) in Et2O (400 ml)
was intermittently irradiated (for 10, 5 and 4 s) with a 400
W high-pressure mercury lamp through a Vycor filter at 0°C
with argon gas bubbling through the solution. After removal
of the solvents under reduced pressure, the obtained residue
was dissolved in EtOH (20 ml) and stored in the dark at 65
for 10 h and room temperature for 15 h under argon gas. The
solvent was evaporated off, and the crude product thus
obtained was purified by prep. TLC [20 x 20 cm, hexane/
AcOEt (3:2 v/v); developed two times]. The zone corre-
sponding to Rf ca. 0.7 was extracted [CHCl3/MeOH (5:1
␭
nm: 227.0. H-NMR ␦: 0.55 (3H, s, H-18Ј), 0.91 (3H,
min
d, J ϭ 5.9 Hz, H-21Ј), 1.14, 1.15 (3H each, s, H-26Ј, 27Ј),
2.027, 2.033, 2.04, 2.07 (3H each, s, 4 x Ac), 3.53 (1H, m,
H-24Ј), 3.75 (3H, s, COOMe), 4.05 (1H, d, J ϭ 9.6 Hz,
H-5), 4.64 (1H, d, J ϭ 7.9 Hz, H-1), 4.85 (1H, d, J ϭ 1.5
Hz, H-19Ј), 4.94 (1H, m, H-3Ј), 5.07 (1H, brs, H-19Ј), 5.27
(1H, 2H, m each, H-2, 3, 4), 6.04, 6.21 (1H each, d, J ϭ
11.2 Hz, H-6Ј, 7Ј). FAB-MS m/z: 797 [MϩNa]ϩ (1.75%),
774 [M]ϩ, 43 (100%).
2.2.13. (5Z,7E)-(3S,24R)-3,25-Dihydroxy-9,10-
v/v)] to yield compound 14 (5.2 mg, 15.8%) as a colorless
secocholesta-5,7,10 (19)-trien-24-yl ␤-D-glucopyranuronic
acid (17)
1
oil. UV ␭
nm: 264.0, ␭
nm: 228.0. H-NMR ␦: 0.53
max
min
(3H, s, H-18Ј), 0.92 (3H, d, J ϭ 6.3 Hz, H-21Ј), 1.20 (6H,
s, H-26Ј, 27Ј), 1.95, 2.016, 2.021 (3H, 6H, 3H, s each, 4 x
Ac), 3.75 (3H, s, COOMe), 3.95 (1H, m, H-3Ј), 4.03 (1H, d,
J ϭ 9.2 Hz, H-5), 4.67 (1H, d, J ϭ 7.6 Hz, H-1), 4.77 (1H,
m, H-24Ј), 4.79, 5.03 (1H each, brs, H-19Ј), 5.01, 5.24 (1H,
2H, m each, H-2, 3, 4), 6.00, 6.17 (1H each, d, J ϭ 10.9 Hz,
H-6Ј, 7Ј). FAB-MS m/z: 797 [MϩNa]ϩ (2.84%), 43
(100%).
A solution of 0.1 M KOH-MeOH (0.3 ml) was added to
a solution of 16 (2.06 mg, 2.66 ␮mol) in MeOH (0.3 ml),
and the mixture was stirred at room temperature for 14 h.
The mixture was diluted with H2O and neutralized with 5%
HCl under ice-cooling. After the addition of NaCl, the
mixture was extracted with THF. The organic layer was
washed (brine) and then dried over anhydrous Na2SO4.
After removal of the solvents under reduced pressure, the
crude product thus obtained was purified by prep. TLC [8 x
20 cm, CHCl3/MeOH/H2O (70:30:4 v/v/v)]. The zone cor-
responding to Rf ca. 0.4 was extracted [CHCl3/MeOH/H2O
(70:30:4 v/v/v)] to yield compound 17 (1.32 mg, 83.8%) as
a colorless semi-solid. UV ␭max nm: 264.0, ␭min nm: 227.0.
FAB-MS m/z: 591 [M-H]Ϫ (12.5%), 153 (100%).
2.2.11. (5Z,7E)-(3S,24R)-24,25-Dihydroxy-9,10-
secocholesta-5,7,10 (19)-trien-3␤-yl ␤-D-glucopyranuronic
acid (15)
A solution of 0.1 M NaOH-MeOH (0.3 ml) was added to
a solution of 14 (3.10 mg, 4.00 ␮mol) in MeOH (0.4 ml),
and the mixture was stirred at room temperature for 24 h.
The mixture was diluted with H2O and neutralized with 5%
HCl under ice cooling. After the addition of NaCl, the
mixture was extracted with THF. The organic layer was
washed (brine) and then dried over anhydrous Na2SO4.
After removal of the solvents under reduced pressure, the
crude product thus obtained was purified by prep. TLC [10
x 20 cm, CHCl3/MeOH/H2O (70:30:4 v/v/v)]. The zone
corresponding to Rf ca. 0.3 was extracted [CHCl3/MeOH/
H2O (70:30:4 v/v/v)] to yield compound 15 (1.95 mg,
2.2.14. Methyl {[(5Z,7E)-(3S,24R)-3-acetoxy-24-hydroxy-
9,10-secocholesta-5,7,10 (19)-trien-25-yl]-2,3,4-tri-O-
acetyl-␤-D-glucopyranosid}uronate (18)
A solution of 13 (33.5 mg, 43.3 ␮mol) in Et2O (400 ml)
was intermittently irradiated (for 10, 5 and 3 s) with a 400
W high-pressure mercury lamp through a Vycor filter at 0°C
with argon gas bubbling through the solution. After removal
of the solvents under reduced pressure, the obtained residue
was dissolved in EtOH (25 ml) and stored in the dark at
60°C for 16 h and room temperature for 29 h under argon
gas. The solvent was evaporated off, and the crude product
thus obtained was purified by prep. TLC [20 x 20 cm,
hexane/AcOEt (3:2 v/v); developed three times]. The zone
corresponding to Rf ca. 0.5 was extracted [CHCl3/MeOH
(5:1 v/v)] to yield compound 18 (5.45 mg, 16.3%) as a
colorless oil. UV ␭max nm: 264.5, ␭min nm: 227.0. 1H-NMR
␦: 0.55 (3H, s, H-18Ј), 0.92 (3H, d, J ϭ 5.6 Hz, H-21Ј),
1.16, 1.21 (3H each, s, H-26Ј, 27Ј), 2.02, 2.04 (12H, s each,
84.0%) as a colorless semi-solid. UV ␭
nm: 264.0, ␭
max
min
nm: 228.0. FAB-MS m/z: 591 [M-H]Ϫ (25.3%), 12 (100%).
2.2.12. Methyl {[(5Z,7E)-(3S,24R)-3-acetoxy-25-hydroxy-
9,10-secocholesta-5,7,10 (19)-trien-24-yl]-2,3,4-tri-O-
acetyl-␤-D-glucopyranosid}uronate (16)
A solution of 12 (31.3 mg, 40.4 ␮mol) in Et2O/CH2Cl2
(400:1 v/v, 401 ml) was intermittently irradiated (for 10, 8
and 5 s) with a 400 W high-pressure mercury lamp through

T. Higashi et al. / Steroids 64 (1999) 715–725
719
4 x Ac), 3.35 (1H, m, H-24Ј), 3.75 (3H, s, COOMe), 4.02
2.5. Methylation
(1H, d, J ϭ 9.6 Hz, H-5), 4.75 (1H, d, J ϭ 9.2 Hz, H-1),
4.84 (1H, d, J ϭ 1.5 Hz, H-19Ј), 4.94 (1H, m, H-3Ј), 5.06
(1H, brs, H-19Ј), 4.99, 5.24 (1H, 2H, m each, H-2, 3, 4),
6.02, 6.21 (1H each, d, J ϭ 11.2 Hz, H-6Ј, 7Ј). FAB-MS
m/z: 797 [MϩNa]ϩ (1.75%), 774 [M]ϩ (0.84%), 43
(100%).
Freshly prepared ether solution of diazomethane (1 ml)
was added to the solution of glucuronides [ca. 100 ng in
MeOH (50 ␮l)]. The mixture was stored at room tempera-
ture for 1 h and the solvent was evaporated.
2.6. Incubation procedure for in vitro glucuronidation
2.2.15. (5Z,7E)-(3S,24R)-3,24-Dihydroxy-9,10-
secocholesta-5,7,10 (19)-trien-25-yl ␤-D-glucopyranuronic
acid (19)
The liver microsomal fraction from Wistar strain rats
(female, ca. 140 g, 7 w, Japan S.L.C., Hamamatsu, Japan)
was prepared as previously reported [6]. The assay medium
containing 24,25(OH)2D3 [10 ␮g in EtOH (10 ␮l)], uridine-
5Ј-diphosphoglucuronic acid [4 ␮M in 0.1 M Tris-HCl
buffer (Tris buffer; pH 7.2; 0.2 ml)], MgCl2 [10 ␮mol in
Tris buffer (50 ␮l)], Triton X-100 [0.5 ␮l in Tris buffer (50
␮l)], ethylenediaminetetraacetic acid.2Na [20 nmol in Tris
buffer (50 ␮l)], microsomal fraction [2 mg protein in Tris
buffer containing 0.25 M sucrose (60 ␮l)] and Tris buffer in
a total volume of 1.0 ml. The mixture was incubated in air at
37°C for 6 h. The reaction was stopped by cooling on ice.
A solution of 0.1 M KOH-MeOH (0.3 ml) was added to
a solution of 18 (2.00 mg, 2.58 ␮mol) in MeOH (0.3 ml),
and the mixture was stirred at room temperature for 16 h.
The mixture was diluted with H2O and neutralized with
5% HCl under ice-cooling. After the addition of NaCl,
the mixture was extracted with THF. The organic layer
was washed (brine) and then dried over anhydrous
Na2SO4. After removal of the solvents under reduced
pressure, the crude product thus obtained was purified by
prep. TLC [10 x 20 cm, CHCl3/MeOH/H2O (70:30:4
v/v/v)]. The zone corresponding to Rf ca. 0.4 was ex-
tracted [CHCl3/MeOH/H2O (70:30:4 v/v/v)] to yield
compound 19 (1.26 mg, 82.4%) as a colorless semi-solid.
2.7. Separation of the formed monoglucuronides from
incubation specimens
UV ␭
nm: 264.0, ␭
nm: 227.0. FAB-MS m/z: 591
min
max
An incubation specimen was mixed with EtOH (1 ml)
and subjected to centrifugation at 1500g for 10 min. The
obtained precipitate was re-suspended with Tris buffer (1
ml) and EtOH (1 ml) followed by centrifugation as above.
The combined supernatant was passed through an Isolute
C18 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 PHP-
LH-20 column chromatography (20 x 6 mm i.d.). After
washing with 90% EtOH (5 ml) and 0.1 M AcOH in 90%
[M-H]Ϫ (12.8%), 153 (100%).
2.3. HPLC analysis
HPLC was performed on a Shimadzu LC-10AT chro-
matograph (Kyoto) equipped with a Shimadzu SPD-10A
UV (265 nm) or a Shimadzu SPD-M6A photodiode array
UV (200–340 nm) detector. J’sphere ODS-H80 (4 ␮m, 150
x 4.6 mm i.d.) (YMC, Kyoto) column was used at a flow
rate of 1 ml/min under ambient conditions. The pH of the
mobile phase containing AcONH4 or NaClO4 was adjusted
with AcOH or HClO4, respectively.
EtOH (5 ml), the glucuronides 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 evaporated under N2 gas stream. The residue was re-
dissolved in EtOH and stored at Ϫ20°C until use.
2.4. LC-MS analysis
LC-MS was performed using a Finnigan MAT LCQ (San
Jose, CA, USA) connected to a JASCO PU-980 (Tokyo)
chromatograph, and ESI was used. The spray needle voltage
was 5 kV, and the heated capillary temperature was set at
200°C. The sheath gas and auxiliary gas flow rate was 70
and 20 units, respectively. The relative collision energy for
MSn was 20%. The capillary voltage was 1 or Ϫ1 V, and the
tube lens offset was 10 or Ϫ10 V in the positive- or nega-
tive-ion mode, respectively. Develosil ODS HG-5 (5 ␮m,
150 x 2.0 mm i.d.) and YMC-Pack Pro C18 (5 ␮m, 150 x
3.0 mm i.d.) columns were used at a flow rate of 0.25 and
0.4 ml/min, respectively, at 30°C.
2.8. Enzymic hydrolysis of monoglucuronides
The glucuronide [ca. 25 ng in EtOH (20 ␮l)] dissolved in
acetate buffer (0.88 ml) and ␤-glucuronidase (ca. 1500
Fishman units) in acetate buffer (0.1 ml) were separately
pre-incubated 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 x 3), and the organic layer
was evaporated under N2 gas stream. The residue was dis-
solved in EtOH and subjected to HPLC, and 24,25(OH)2D3
was identified in comparison with an authentic sample
[MeOH/H2O (5:1 v/v), tR 6.4 min].

720
T. Higashi et al. / Steroids 64 (1999) 715–725
2.9. Deconjugation of monoglucuronides with rat intestine
homogenate
min]. After neutralization with 2% NaHCO3 and dilution
with H2O, each obtained fraction was treated as previ-
ously described. The EtOH eluate from an Isolute C18
cartridge was evaporated under N2 gas stream, and a part
of the residue was subjected to HPLC, LC-MS and en-
zymic hydrolysis.
A female Wistar strain rat (140 g, 7 w, Japan S.L.C.) was
starved for 15 h before being killed. The intestine was
homogenized in ice-cold acetate buffer followed by centrif-
ugation at 1500g for 10 min. The supernatant was diluted
with the buffer to a concentration of 10 mg protein/ml and
used as an enzyme solution (1 ml). 24,25(OH)2D33G [300
ng in EtOH (40 ␮l)] was added to the solution and then
incubated at 37°C for 3 h. The reaction was stopped by the
addition of ice-cold MeOH (1 ml), and the mixture was
centrifuged at 2800g for 10 min. The supernatant was di-
luted with H2O (2 ml) and then applied to an Isolute C18
cartridge. After washing with H2O (4 ml) and 30% MeOH
(3 ml), the remaining glucuronide was eluted with 70%
MeOH (3 ml), and then the formed 24,25(OH)2D3 was
eluted with MeOH (3 ml). The obtained glucuronide frac-
tion was subjected to PHP-LH-20 column chromatography
and then treated as described in the in vitro experimental
section. The solvent of the corresponding fraction was evap-
orated under N2 gas stream, and a part of the obtained
residue was subjected to HPLC and compared with an
authentic sample.
3. Results
3.1. Syntheses of 24,25(OH)2D3 monoglucuronides
Three positional isomers of 24,25(OH)2D3 monoglucu-
ronide (15, 17, 19) were synthesized as shown in Fig. 1. In
order to synthesize these glucuronides selectively, the start-
ing materials (1, 2) were converted to 24,25(OH)2proD3 3-
and 24-acetate (9, 7), respectively. The former was sub-
jected to the Koenigs-Knorr reaction using Ag2CO3 and
Br-sugar [13], and two isomers of monoglucuronide acetate
methyl ester (G’) (12, 13) were obtained in a ratio of 3:5.
The reacted position was confirmed by the usual acetylation
using Ac2O and pyridine. Although compound 13 easily
gave the respective acetate, which was confirmed by TLC
and 1H-NMR (H-24, ␦ 4.85 ppm), the reaction did not work
on the tert-hydroxy group of compound 12; therefore com-
pounds 12 and 13 were confirmed as 24GЈ and 25GЈ, re-
spectively. Both compounds were subjected to photochem-
ical reaction followed by alkaline hydrolysis to give the
desired 24,25(OH)2D3-24G (17) and -25G (19) with a total
yield of 2.4% and 5.1%, respectively.
On the other hand, 24,25(OH)2D3 3G (15) was synthe-
sized from compound 7. The Koenigs-Knorr reaction was
carried out with the compound, and 3G’ (10) was obtained
together with 3,25-diG’ (11) in a ratio of 3:1. The photo-
chemical reaction followed by alkaline hydrolysis gave
24,25(OH)2D3 3G (15) with a total yield of 2.9%.
All the compounds synthesized here exhibited satisfac-
tory spectral data. Furthermore, the structures of glucu-
ronides were confirmed by enzymic hydrolysis using ␤-glu-
curonidase.
2.10. Bile samples from rats
Wistar strain rats (male: ca. 170 g, female: ca. 140 g,
7 w, Japan S.L.C.) were used. The rats were anaesthetized
with diethylether, and the bile duct was cannulated with a
polyethylene tube (SP 31) (Natsume, Tokyo, Japan) for the
collection of bile. All animals were starved for 15 h prior to
the administration of 24,25(OH)2D3. A suspension of
24,25(OH)2D3 (0.5 mg) in dimethylsulfoxide (0.1 ml) with
saline (0.7 ml) and Tween 80 (0.2 ml) was orally given to
each rat, and the bile was collected over a period of 24 h
following the administration.
2.11. Separation of the excreted monoglucuronides from
rat bile
A bile specimen (2 ml) was diluted with 0.5 M sodium
phosphate buffer (pH 7.0) (40 ml) and passed through an
Isolute C18 cartridge followed by PHP-LH-20 column
chromatography as described in the in vitro experimental
section. The obtained fraction containing 24,25(OH)2D3
monoglucuronides was subjected to prep. HPLC [MeCN/
0.5% AcONH4 (pH 5.0) (1:2 v/v), 24,25(OH)2D3-3G; tR
8.1–9.1 min, -24G; tR 15.2–16.2 min, -25G; tR 13.2–14.4
min]. After dilution with H2O, each obtained fraction was
applied to an Isolute C18 cartridge in the manner previ-
ously described to remove any inorganic salts, and then
the solvent was evaporated under N2 gas stream. Each
fraction was further purified by prep. HPLC [MeCN/2%
NaClO4 (pH 3.0) (2:3 v/v), 24,25(OH)2D3-3G; tR 9.2–
10.6 min, -24G; tR 13.2–14.6 min, -25G; tR 14.9–16.0
3.2. Characterization of 24,25(OH)2D3 monoglucuronides
formed in incubation specimen
The incubation specimen was pre-treated according to
the procedure shown in Fig. 2 and then subjected to UV-
HPLC analysis. As a consequence, two major peaks were
observed, which were identified as 24,25(OH)2D3-3G and
-24G in comparison with standard synthetic samples based
on their chromatographic behavior during HPLC using three
solvent systems [tR of 24,25(OH)2D3 -3G, -24G, and -25G;
MeCN/2% NaClO4 (pH 3.0) (2:3 v/v), 10.2, 15.4 and, 16.9
min, MeCN/0.5% AcONH4 (pH 5.0) (1:2 v/v), 8.1, 14.9,
and 13.8 min, MeOH/2% NaClO4 (pH 3.0) (7:3 v/v), 16.0,
24.8, and 22.3 min]. Furthermore, photodiode array UV
detection of these two metabolites showed the characteristic

T. Higashi et al. / Steroids 64 (1999) 715–725
721
Fig. 1. Syntheses of 24,25(OH)2D3 monoglucuronides.
UV absorbance (␭ ca. 230 nm, ␭
ca. 268 nm) of the
max
rates of these glucuronides from the substrate were about
7.0% and 6.5% for 24,25(OH)2D3-3G and -24G, respec-
tively. The peak corresponding to 24,25(OH)2D3 25G was
also observed in the HPLC chromatogram, but photodiode
array UV detection could not be carried out due to the
shortage of the sample. These data prompted us to use
LC-MS for the identification of the compound.
min
vitamin D structure. The recovery rates of 24,25(OH)2D3
monoglucuronides during the pre-treatment were almost
equal in three positional isomers [n ϭ 2, 24,25(OH)2D3-3G;
68.7%, -24G; 66.5% and -25G; 71.9%], and the conversion
In the previous communication [7], we used LC-atmo-
spheric pressure chemical ionization (APCI)-MS for the
characterization of 24,25(OH)2D3 monoglucuronide formed
from in vitro experiments, but it was performed by only
selected ion monitoring (SIM) in the negative-ion mode,
because of the low sensitivity of the used instrument
(Hitachi M-1000H). In order to make the identification of
monoglucuronides more reliable, the pre-treated sample
was subjected to an ion-trap LC-ESI-MSn [Develosil ODS
HG-5, MeCN/10 mM AcONH4 (1:2 v/v)] in the positive-
and negative-ion modes. As a consequence, the peaks cor-
responding to 24,25(OH)2D3-3G, -24G, and -25G were
observed in the LC-MS chromatogram in both the positive-
and negative-ion modes (Fig. 3), and the mass spectrum of
each glucuronide was identical with that of an authentic
Fig. 2. Procedure for characterization and determination of monoglucu-
ronides in in vitro and in vivo experiments.

722
T. Higashi et al. / Steroids 64 (1999) 715–725
Fig. 3. LC-ESI-MS chromatograms of 24,25(OH)2D3 monoglucuronides. a) Positive-ion mode. b) Negative-ion mode. Conditions: column, Develosil ODS
HG-5, mobile phase, MeCN/10 mM AcONH4 (1:2 v/v), detection, m/z 610 and 591 were monitored in a) and b), respectively, after m/z 500–700 was scanned.
sample. However, only a molecular-related ion in both the
positive-ion (m/z 610; [MϩNH4]ϩ) and negative-ion modes
(m/z 591; [M-H]Ϫ) was obtained, and any fragment ion was
not observed in the spectrum. Therefore, we attempted LC-
MS2 analysis using the above ion as a precursor ion. Al-
though any fragmentation was not observed in the negative-
ion mode, the following characteristic fragment ions were
detected in the positive-ion mode, and its product ion mass
spectrum of each glucuronide was identical with that of the
authentic sample (Fig. 4a and b): m/z 593 [MϩH]ϩ, 575
[593–H2O]ϩ, 557 [593–2H2O]ϩ, 417 [geninϩH]ϩ, 399
[417–H2O]ϩ, 381 [417–2H2O]ϩ, and 363 [417–3H2O]ϩ.
Furthermore, the formed 24,25(OH)2D3 25G was converted
to the methyl ester with diazomethane which gave a molec-
ular-related ion (m/z 665, [MϩAcO]Ϫ) in LC-MS [YMC-
Pack Pro C18, MeOH/10 mM AcONH4 (4:1 v/v), tR 8.3
min] operating in the negative-ion mode. Compared with
24,25(OH)2D3 25G, the intensity of a molecular-related ion
of the methyl ester was increased to about 4 times, which
was very useful in identifying a small amount of glucuro-
nide. These data showed that 24,25(OH)2D3 25G was formed
in in vitro experiments together with 24,25(OH)2D3-3G and
-24G.
In addition to these data, the structure of the formed
24,25(OH)2D3 25G was confirmed by the enzymic hydro-
lysis using ␤-glucuronidase. The hydrolyzed product gave a
peak corresponding to 24,25(OH)2D3 on the reversed-phase
HPLC, which was confirmed by co-chromatography with an
authentic sample.
3.3. Deconjugation of glucuronide in rat intestine
The entero-hepatic circulation of 24,25(OH)2D3 was in-
vestigated using 24,25(OH)2D3 3G as a model compound.

T. Higashi et al. / Steroids 64 (1999) 715–725
723
Fig. 4. Product ion mass spectra of 24,25(OH)2D3 monoglucuronides. a) Authentic sample. b) From incubation specimen. c) From rat bile. Vertical line:
relative abundance. Horizontal line: m/z. Conditions: precursor ion, m/z 610, relative collision energy, 20%, scan range, m/z 300–650, chromatographic
conditions, the same as in Fig. 3.

724
T. Higashi et al. / Steroids 64 (1999) 715–725
Table 1
Formation ratio of 24,25(OH)2D3 monoglucuronide
24,25(OH)2D3 3G 24,25(OH)2D3 24G 24,25(OH)2D3 25G
In vitro 1a (7.0%)b
0.9 (6.5%)
4.1 (122 ng/ml)
ϽϽ0.1 (Ϫ)c
Not detected
In vivo 1 (30 ng/ml)d
a The value of 3G was taken as 1.
b Conversion rate (mean of two animals).
c The value was too small to calculate.
d Yield from 1 ml of bile (mean of three animals).
corresponding to three positional isomers of 24,25(OH)2D3
monoglucuronide were analyzed using HPLC and LC-MS
and subjected to enzymic hydrolysis. First, the chromato-
graphic behavior of the putative in vivo glucuronides were
compared with that of synthetic standards [MeOH/2% Na-
ClO4 (pH 3.0) (3:1 v/v), 24,25(OH)2D3 -3G; tR 7.2 min,
-24G; tR 10.6 min, and -25G; 9.7 min]. As a consequence,
although 24,25(OH)2D3 25G was not detected, the peaks
corresponding to 24,25(OH)2D3-3G and -24G were ob-
served clearly. These two metabolites were separately
treated with ␤-glucuronidase, and the disappearance of
them and the formation of 24,25(OH)2D3 were confirmed
by HPLC. Furthermore, the LC-MSn spectra of the metab-
olites, which were obtained in the same conditions of the in
vitro experiments, were agreed with those of synthetic stan-
dards, that is, the product ion mass spectra derived from the
ion at m/z 610 showed the following characteristic ions: m/z
593, 575, 557, 417, 399, 381, and 363 (Fig. 4c). These
results led to the conclusions that the two metabolites were
24,25(OH)2D3-3G and -24G, and the corresponding 25G
was not formed. Although precise quantitative determina-
tion was not performed, ca. 30 ng of 24,25(OH)2D3 3G and
120 ng of 24,25(OH)2D3 24G were obtained from 1 ml of
bile (Table 1).
Fig. 5. Chromatograms of 24,25(OH)2D3 3G and its hydrolyzed product. a)
Incubation with rat intestine homogenate. b) Incubation with boiled rat
intestine homogenate. Upper: remaining substrate. Lower: hydrolyzed
product. Conditions: column, J’sphere ODS-H80, mobile phase, upper:
MeCN/0.5% AcONH4 (pH 5.0) (4:9 v/v), lower: MeCN/H2O (3:2 v/v).
4. Discussion
The glucuronide was incubated with rat intestine homoge-
nate, and the remaining substrate and the formed product
were monitored with UV-HPLC (Fig. 5). Compared with a
case of boiled intestine (inactivated enzyme), 68% of the
substrate disappeared and 24,25(OH)2D3 was produced with
a conversion rate of 17%. These results suggested that a part
of the glucuronides of 24,25(OH)2D3 was deconjugated by
the enzyme from bacteria in the intestine and formed
24,25(OH)2D3 is probably reabsorbed and further oxidized
to other metabolites.
It is well known that 24,25(OH)2D3 is metabolized to
25(OH)-24-oxo-D3 and then to 23,25(OH)2-24-oxo-D3 [3],
but conjugates of 24,25(OH)2D3 have been poorly investi-
gated. From this point of view, we examined in vitro glu-
curonidation of 24,25(OH)2D3 using rat liver microsomal
fraction as an enzyme source. LC-ESI-MSn was very useful
for the characterization of the formed 24,25(OH)2D3 mono-
glucuronide. Because a glucuronide has an anionic func-
tional group, it can be detected in the negative-ion mode
with strong intensity, but the positive-ion mass spectrome-
try has a greater tendency to produce fragment ions which
afford more structural information [14]. Based on this point,
the determination of their structures was performed with
LC-MS2 operating in the positive-ion mode, and the frag-
ment ions that occurred from the loss of water or sugar
molecules were observed as expected. In consequence, the
glucuronidation mainly occurs at the 3- and 24-positions of
3.4. Characterization of 24,25(OH)2D3 monoglucuronide
in rat bile
The bile specimen was subjected to solid phase extrac-
tion and anion exchange chromatography followed by two
successive prep. HPLC (Fig. 2). The obtained fractions

T. Higashi et al. / Steroids 64 (1999) 715–725
725
24,25(OH)2D3 in almost equal amounts, and a very small
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of the vitamin D-repleted rat femur by the treatment with a large dose
of 24R,25(OH)2D3. Bone 1989;10:117–23.
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HF. Human 25-hydroxyvitamin D3-24-hydroxylase, a multicatalytic
enzyme. Biochemistry 1996;35:8465–72.
[4] Shimoyamada A, Tomiyama S, Shimizu M, Yamamoto K, Kunii S,
Yamada S. In vivo metabolism of 24R,25-dihydroxyvitamin D3:
structure of its major bile metabolite. Biochim Biophys Acta 1997;
1346:147–57.
[5] Shimada K, Nakatani I, Saito K, Mitamura K. Separation and char-
acterization of monoglucuronides of vitamin D3 and 25-hydroxyvi-
tamin D3 in rat bile by high-performance liquid chromatography. Biol
Pharm Bull 1996;19:491–4.
[6] Shimada K, Kamezawa Y, Mitamura K. In vitro glucuronidation of
25-hydroxyvitamin D3 and its pro-form. Biol Pharm Bull 1997;20:
596–600.
[7] Shimada K, Higashi T, Hayashi R. Characterization of (24R)-24,25-
dihydroxyvitamin D3 monoglucuronides formed from a rat liver mi-
crosomal fraction using high-performance liquid chromatography and
liquid chromatography-mass spectrometry. Anal Commun 1998;35:
33–6.
[8] Takayama H, Ohmori M, Yamada S. Facile, stereoselective synthesis
of (24R)-24,25-dihydroxyvitamin D3 using D-glyceric acid as a chiral
synton. Tetrahedron Lett 1980;21:5027–8.
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improved synthesis of (24R)-24,25-dihydroxyvitamin D3. Synthesis
1990;1990:193–5.
[10] Kobayashi N, Higashi T, Saito K, Murayama T, Douya R, Shimada
K. Specificity of polyclonal antibodies raised against a novel 24,25-
dihydroxyvitamin D3-bovine serum albumin conjugant linked
through the C-11␣ or C-3 position. J Steroid Biochem Mol Biol
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1986;328:705–12.
As already described in the introductory section, the mono-
glucuronide of 23,25(OH)2-24-oxo-D3 was reported as a bili-
ary metabolite obtained from dogs dosed with 24,25(OH)2D3,
but that of 24,25(OH)2D3 has not been obtained [4]. However,
we proved that 24,25(OH)2D3 monoglucuronide was formed
in vitro. It is supposed that one possible reason for this dis-
crepancy is the entero-hepatic circulation of 24,25(OH)2D3.
Shimoyamada et al. collected bile from the gall bladder of dogs
given 24,25(OH)2D3 at 504 h after treatment [4]. It is inferred
that the formed 24,25(OH)2D3 monoglucuronide is deconju-
gated in the intestine as shown in our experiment (Fig. 5), and
then the oxidation of the 24,25(OH)2D3 side chain followed by
the glucuronidation of the oxidized metabolite occurs. To ob-
tain more information, an in vivo study using rats was per-
formed.
We have now shown that 24,25(OH)2D3-3G and -24G
are excreted in rat bile after oral administration of
24,25(OH)2D3 and the glucuronidation occurs at the 24-
position in preference to the 3-position. Based on these
results, it is inferred that the glucuronidation occurs on the
side chain more easily than on the A-ring. As mentioned
previously, in the in vivo study using 25(OH)D3, the glu-
curonidation occurred at 25-position with a large propor-
tion, although it is tert-hydroxy group. However, it was not
observed on 24,25(OH)2D3. These results demonstrated that
in the presence of a sec-hydroxy group on the side chain, the
glucuronidation at 25-position hardly occurs. Although the
formation ratio of 3G and 24G is different between in vivo
and in vitro experiments (Table 1), it is obvious that the
following metabolic pathway of 24,25(OH)2D3 exits, that is,
intact 24,25(OH)2D3 is conjugated as a glucuronide and
then excreted into the bile.
The presence of other conjugated vitamin D metabolites,
such as 23,25(OH)2-24-oxo-D3 23-glucuronide, in rat bile
should also be considered. Their identification and charac-
terization using LC-MSn is now in progress in our labora-
tories, details of which will be reported in the future.
[13] Shimada K, Sugaya K, Kaji H, Nakatani I, Mitamura K, Tsustumi N.
Syntheses and enzymatic hydrolysis of 25-hydroxyvitamin D mono-
glucuronides. Chem Pharm Bull 1995;43:1379–84.
Acknowledgments
[14] Bowers LD, Sanaullah. Direct measurement of steroid sulfate and
glucuronide conjugates with high-performance liquid chromatogra-
phy-mass spectrometry. J Chromatogr B 1996;687:61–8.
We thank the Ministry of Education, Science, Sport and
Culture of Japan for financial support through a Grant-in Aid.