Synthesis of the 3-sulfates of S-acyl glutathione conjugated bile acids and their biotransformation by a rat liver cytosolic fraction

Article abstract of DOI:10.1016/j.chemphyslip.2012.02.001

The 3-sulfates of the S-acyl glutathione (GSH) conjugates of five natural bile acids (cholic, chenodeoxycholic, deoxycholic, ursodeoxycholic, and lithocholic) were synthesized as reference standards in order to investigate their possible formation by a ra

Full text of DOI:10.1016/j.chemphyslip.2012.02.001

Chemistry and Physics of Lipids 165 (2012) 261–269
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Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
Synthesis of the 3-sulfates of S-acyl glutathione conjugated bile acids and their
biotransformation by a rat liver cytosolic fraction
Kuniko Mitamuraa, Naohiro Horia, Shiori Minoa, Takashi Iidab, Alan F. Hofmannc, Shigeo Ikegawaa,∗
a Faculty of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-osaka 577-8502, Japan
b Department of Chemistry, College of Humanities and Sciences, Nihon University, Sakurajosui, Setagaya-ku, Tokyo 156-8550, Japan
c Department of Medicine, University of California, San Diego, La Jolla, CA 92093-063, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The 3-sulfates of the S-acyl glutathione (GSH) conjugates of five natural bile acids (cholic, chenodeoxy-
cholic, deoxycholic, ursodeoxycholic, and lithocholic) were synthesized as reference standards in order
to investigate their possible formation by a rat liver cytosolic fraction. Their structures were confirmed by
proton nuclear magnetic resonance, as well as by means of electrospray ionization-linear ion-trap mass
spectrometry with negative-ion detection. Upon collision-induced dissociation, structurally informative
product ions were observed. Using a triple-stage quadrupole instrument, selected reaction monitoring
analyses by monitoring characteristic transition ions allowed the achievement of a highly sensitive and
specific assay. This method was used to determine whether the 3-sulfates of the bile acid–GSH conju-
gates (BA–GSH) were formed when BA–GSH were incubated with a rat liver cytosolic fraction to which
3ꢀ-phosphoadenosine 5ꢀ-phosphosulfate had been added. The S-acyl linkage was rapidly hydrolyzed to
form the unconjugated bile acid. A little sulfation of the GSH conjugates occurred, but greater sulfation at
C-3 of the liberated bile acid occurred. Sulfation was proportional to the hydrophobicity of the unconju-
gated bile acid. Thus GSH conjugates of bile acids as well as their C-3 sulfates if formed in vivo are rapidly
hydrolyzed by cytosolic enzymes.
Received 6 December 2011
Received in revised form 31 January 2012
Accepted 3 February 2012
Available online 10 February 2012
Keywords:
Bile acid sulfation
Bile acid–glutathione conjugate
Collision-induced dissociation
Selected reaction monitoring
© 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
the intestine and return to the liver where they are efficiently taken
up by the hepatocyte by a variety of mechanisms (Hofmann et al.,
In mammals, bile acids (BAs) are synthesized in pericentral hep-
atocytes from cholesterol and then N-acylamidated (conjugated)
with glycine or taurine in peroxisomes. BAs are then secreted into
the biliary tract via canalicular BA transporters such as the bile salt
export pump and multidrug resistance protein 2 (Alrefai and Gill,
2007). BAs are efficiently absorbed in the distal intestine with the
result that a recycling pool of BA occurs.
2010; Ridlon et al., 2006). On entering the hepatocyte, the carboxyl
group is activated by conversion to its acyl-adenylate derivative
that in turn is converted to its acyl-coenzyme A (CoA) thioester
derivative (Ikegawa et al., 1999; Kelley and Vessey, 1994; Mano
et al., 2001). The acyl-CoA thioester then reacts with the amino
group of taurine or glycine (in peroxisomes) to form conjugated
(N-acylamidated) BAs which join the recycling conjugated BAs.
Based on analyses of bile samples from numerous species, con-
jugation of C24 BA is predominantly N-acylamidation with glycine
or taurine in health (Hofmann et al., 2010). In healthy verte-
brates, glucuronides [either at C-3 (ethereal) or C-24 (ester)],
N-acetylglucosaminides, or S-acyl conjugates with glutathione
(GSH) or N-acetylcysteine (NAC) of endogenous BAs are present
in only trace proportions in bile. Nonetheless, our laboratory has
identified trace concentrations of GSH conjugates of BA in rats
(Mitamura et al., 2011a) and infants, but not in adults (Mitamura
et al., 2011b). Sulfation, i.e. esterification with the sulfate moiety,
is a major mode of conjugation for lithocholate in man, with the
result that about half of the lithocholyl amidates in bile are sulfated
(Rossi et al., 1987). Nonetheless, sulfated amidates have never been
shown to be major biliary BAs in any species in health. In patients
with cholestasis, however, sulfated (and amidated) BAs are formed
BAs undergo bacterial deconjugation in the distal intestine, and
a fraction of the unconjugated BAs thus formed are absorbed from
Abbreviations:
BAs, bile acids; CoA, coenzyme A; GSH, glutathione;
NAC, N-acetylcysteine; LC, liquid chromatography; ESI, electrospray ioniza-
tion; MS, mass spectrometry; CA-GSH, S-(cholyl)glutathione; CDCA-GSH, S-
(chenodeoxycholyl) glutathione; DCA-GSH, S-(deoxycholyl) glutathione; LCA-GSH,
S-(lithocholyl)glutathione; UDCA-GSH, S-(ursodeoxcholyl)glutathione; CA, cholic
acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; UDCA, ursodeoxy-
cholic acid; LCA, lithocholic acid; PAPS, 3ꢀ-phosphoadenosine 5ꢀ-phosphosulfate;
1H NMR, proton-nuclear magnetic resonance; CID, collision-induced dissociation;
HPLC, high-performance liquid chromatography; RP, reversed-phase; SRM, selected
reaction monitoring; IS, internal standard.
∗
Corresponding author. Tel.: +81 6 6721 2332; fax: +81 6 6730 1394.
E-mail address: ikegawa@phar.kindai.ac.jp (S. Ikegawa).
0009-3084/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2012.02.001

262
K. Mitamura et al. / Chemistry and Physics of Lipids 165 (2012) 261–269
in greatly increased amounts and are eliminated mostly in urine
(Raedsch et al., 1981; Hedenborg et al., 1985).
structures of BA–GSH 3-sulfates were as follows: ion source volt-
age, −4 kV; capillary temperature, 270 ◦C; capillary voltage, −20 V;
sheath gas (nitrogen gas) flow rate, 50 arbitrary units; auxiliary gas
(nitrogen gas) flow rate, 5 arbitrary units; tube lens offset voltage,
−100 V. For tandem MS (MS/MS) analysis, helium gas was used as
the collision gas and the normalized collision energy was set at 35%.
The ionization conditions of the second system for the analytical
studies on the sulfation of BA–GSH were as follows: spray voltage,
−3 kV; sheath gas (nitrogen gas) pressure, 35 arbitrary units; aux-
iliary gas flow rate, 5 arbitrary units; capillary temperature, 350 ◦C.
For collision-induced dissociation (CID), argon gas was used as the
collision gas at a pressure of 1.5 mTorr. The LC separations were
conducted on a reversed-phase (RP) semi-micro column, TSKgel
ODS-100 V (5 ␮m, 150 mm × 2.0 mm I.D.) from Tosoh Co. (Tokyo,
Japan) by a linear gradient elution: 30% solvent B (acetonitrile used
for first system and acetonitrile–methanol, 3:7, v/v used for sec-
ond system) to 80% B against solvent A (5 mM ammonium acetate
buffer, pH 6.0) over 30 min at a flow rate of 200 ␮L/min.
In previous work from this laboratory, we have described the
synthesis of S-acyl conjugates of BAs with NAC as well as their cor-
responding sulfates (Mitamura et al., 2009, 2011c). When these
thioesters were incubated with a cytosolic fraction of rat liver,
the NAC conjugates were hydrolyzed and sulfation of the liberated
unconjugated BA occurred (Mitamura et al., 2011c).
In the present paper, we have pursued a similar experimen-
tal approach with GSH conjugates of the five BAs predominating
in human bile (cholic, deoxycholic, chenodeoxycholic, ursodeoxy-
cholic, lithocholic). The GSH conjugates of these BAs as well as their
corresponding C-3 sulfates were synthesized. The biotransforma-
tion of the synthetic BA–GSH conjugates by a rat liver cytosolic
fraction was assessed by liquid chromatography (LC)/electrospray
ionization (ESI)-mass spectrometry (MS).
2. Experimental
2.4. Synthesis of CA-, CDCA-, DCA-, UDCA-, and LCA-GSH
2.1. Materials
3-sulfates (1a–1e)
S-(Cholyl)glutathione (CA-GSH), S-(chenodeoxycholyl)glutat-
hione (CDCA-GSH), S-(deoxycholyl)glutathione (DCA-GSH), S-
(ursodeoxycholyl)glutathione (UDCA-GSH), S-(lithocholyl)glutat-
hione (LCA-GSH) and S-([2,2,4,4,-2H4]-deoxycholyl)glutathione
(d4-DCA-GSH) were synthesized in our laboratories by the method
previously reported (Mitamura et al., 2007, 2011a). Cholic acid (CA),
chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), ursodeoxy-
cholic acid (UDCA), lithocholic acid (LCA), and their 3-sulfates
were stock samples in our laboratories. 3ꢀ-Phosphoadenosine-5ꢀ-
phosphosulfate (PAPS, lithium salt hydrate) was obtained from
Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile and ammonium
acetate of HPLC grade were purchased from Nacalai Tesque Inc.
(Kyoto, Japan), and distilled water of HPLC grade was purchased
from Wako Pure Chemical Industries Ltd. (Tokyo, Japan). An Oasis®
HLB cartridge (adsorbent weight, 60 mg and 1 g) was provided by
Waters Co. (Milford, MA, USA) and was successively conditioned by
washings with methanol and water prior to use. All other chemi-
cals and solvents were analytical grade and obtained from Nacalai
Tesque Inc. Water from a Millipore water filtration system (Milli
Q UV Plus) was used to prepare the aqueous solutions described
below.
2.4.1. General method
To a solution of 3-sulfates of CA, CDCA, DCA, UDCA, LCA
(120–280 mg) in dioxane (2 mL) was added two equimolar
amounts of p-nitrophenol (2 equimolar) and 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride, and the mix-
ture was stirred at room temperature for 6–8 h. After dilution
of the reaction mixture with water, the resulting solution was
loaded onto an Oasis® HLB cartridge (adsorbent weight, 1 g). Water
(20 mL) was used to elute nonhydrophobic reaction constituents,
after which the p-nitrophenyl esters were eluted with methanol
(20 mL). After evaporation of methanol in vacuo, the crude prod-
ucts which without purification were treated with two equimolar
amounts of GSH (reduced form) and trimethylamine (200 ␮L)
in N,Nꢀ-dimethylformamide (10 mL) with continuous stirring for
6–8 h at room temperature under a stream of N2 gas. After dilu-
tion of the reaction mixture with water (20 mL), the resulting
solution was loaded onto an Oasis® HLB cartridge (1 g of solid
phase). After washing with water (20 mL), GSH conjugate was
eluted with methanol (20 mL) and the solvent was evaporated in
vacuo. The products were purified by preparative RP-HPLC with
UV detection at 215 nm on a Cosmosil 5C18-AR-II column (5 ␮m,
250 mm × 20 mm I.D.; Nacalai Tesque Inc.) by a linear gradient elu-
tion: 30% solvent B (acetonitrile) to 100% B against solvent A (0.1%
trifluoroacetic acid) over 30 min at a flow rate of 4 mL/min. The frac-
tion containing desirable product was collected and lyophilized to
dryness.
2.2. Apparatus
Proton-nuclear magnetic resonance (1H NMR) spectra were
recorded on a JNM-ECP700 (700.12 MHz, JEOL Ltd., Tokyo, Japan)
or a JNM-ECA500 (500.16 MHz, JEOL Ltd.) with CD3OD containing
tetramethylsilane as the solvent; chemical shifts were expressed
as ı ppm relative to tetramethylsilane. The following abbreviations
are used: s, singlet; d, doublet; m, multiplet. Preparative high-
performance liquid chromatography (HPLC) was carried out with a
Shimadzu LC-10A VP (Shimadzu Co., Kyoto, Japan) equipped with
a UV detector (SPD-20A; Shimadzu Co.).
2.4.2. S-(3-Sulfooxycholyl)glutathione (CA-GSH 3-sulfate; 1a)
This compound 1a was obtained from CA 3-sulfate (120 mg)
as colorless amorphous solids with isolated yield 5.5 mg (3.2%).
1H NMR ı: 0.70 (3H, s, 18-CH3), 0.91 (3H, s, 19-CH3), 1.05 (3H,
d, J = 6.8 Hz, 21-CH3), 2.19–2.35 (2H, m, Glu-␤,␤ꢀ-H2), 2.47–2.55
(3H, m, Glu-␥,␥ꢀ-H2 and 23-H), 2.72–2.77 (1H, m, 23-Hꢀ), 3.23–3.26
and 3.42–3.45 (2H, m, Cys-␤,␤ꢀ-H2), 3.84–3.87 (1H, m, 7␤-H),
3.94–3.96 (3H, m, Gly-␣,␣ꢀ-H2 and 12␤-H), 4.01–4.04 (1H, m, Glu-
␣-H), 4.18–4.22 (1H, m, 3␤-H), 4.40–4.70 (1H, m, Cys-␣-H). ESI–MS:
2.3. LC/ESI-MS
The LC/ESI-MS analyses were carried out using two analyti-
cal systems. The first consisted of a Finnigan LTQ linear ion-trap
mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA)
equipped with an ESI source and coupled to a Paradigm MS4 pump
(Michrom Bioresources Inc., Auburn, CA, USA) and an autosam-
pler (HTC PAL, CTC Analytics, Zwingen, Switzerland). The second
was a Finnigan TSQ Quantum Access triple-stage quadrupole mass
spectrometer equipped with a LC-2010CHT system (Shimadzu Co.).
For the first system, the ionization conditions for verifying the
m/z 778.3 [M+H]+, m/z 776.3 [M−H]−, m/z 387.8 [M−2H]2−
.
2.4.3. S-(3-Sulfooxychenodeoxycholyl)glutathione (CDCA-GSH
3-sulfate; 1b)
This compound 1b was obtained from CDCA 3-sulfate (180 mg)
as colorless amorphous solids with isolated yield 7.5 mg (2.8%).
1H NMR ı: 0.68 (3H, s, 18-CH3), 0.92 (3H, s, 19-CH3), 0.95 (3H,
d, J = 6.7 Hz, 21-CH3), 2.14–2.22 (2H, m, Glu-␤,␤ꢀ-H2), 2.49–2.55

K. Mitamura et al. / Chemistry and Physics of Lipids 165 (2012) 261–269
263
NH2
O
COOH
R3
R3
NH
C OH
O
C S
O
HN
O
COOH
R1
R2
R1
R2
H
H
CA: R1 = OH, R2 = α-OH, R3 = OH
CDCA: R1= OH, R2 = α-OH, R3 = H
DCA: R1 = OH, R2=H, R3 = OH
UDCA: R1 = OH, R2 = β-OH, R3 = H
CA-GSH 3-sulfate (1a): R1 = OSO3H, R2 = α-OH, R3 = OH
CDCA-GSH 3-sulfate (1b): R1 = OSO3H, R2 = α-OH, R3 = H
DCA-GSH 3-sulfate (1c): R1= OSO3H, R2 =H, R3 = OH
UDCA-GSH 3-sulfate (1d): R1 = OSO3H, R2 = β-OH, R3 = H
LCA-GSH 3-sulfate (1e): R1 = OSO3H, R2 = R3 = H
CA-GSH: R1 = OH, R2 = α-OH, R3 = OH
CDCA-GSH: R1= OH, R2 = α-OH, R3 = H
DCA-GSH: R1 = OH, R2=H, R3 = OH
UDCA-GSH: R1 = OH, R2 = β-OH, R3 = H
LCA-GSH: R1 = OH, R2 = R3 = H
LCA: R1 =OH, R2 = R3 = H
CA 3-sulfate: R1 = OSO3H, R2 = α-OH, R3 = OH
CDCA 3-sulfate: R1= OSO3H, R2 = α-OH, R3 = H
DCA 3-sulfate: R1= OSO3H, R2 =H, R3 = OH
UDCA 3-sulfate: R1 = OSO3H, R2 = β-OH, R3 = H
LCA 3-sulfate: R1 = OSO3H, R2 = R3 = H
NH2
O
COOH
R2
HO
NH
C O
O
NO2
C S
O
D
D
HN
O
COOH
HO3SO
R1
HO
H
H
D
D
[2,2,4,4-d4]-DCA-GSH (IS)
CA-PNP 3-sulfate: R1 = α-OH, R2 = OH
CDCA-PNP 3-sulfate: R1 = α-OH, R2 = H
DCA-PNP 3-sulfate: R1 =H, R2 = OH
UDCA-PNP 3-sulfate: R1 = β-OH, R2 = H
LCA-PNP 3-sulfate: R1 = R2 = H
Fig. 1. Chemical structures of BA–GSH 3-sulfates and their related compounds.
(3H, m, Glu-␥,␥ꢀ-H2 and 23-H), 2.60–2.65 (1H, m, 23-Hꢀ), 3.11–3.15
and 3.41–3.44 (2H, m, Cys-␤,␤ꢀ-H2), 3.77–3.80 (1H, m, 7␤-H),
3.89–3.93 (3H, m, Gly-␣,␣ꢀ-H2 and Glu-␣-H), 4.11–4.15 (1H, m, 3␤-
H), 4.57–4.60 (1H, m, Cys-␣-H). ESI–MS: m/z 762.3 [M+H]+, m/z
3.40–3.43 (2H, m, Cys-␤,␤ꢀ-H2), 3.46–3.50 (1H, m, 7␣-H), 3.91 (2H,
s, Gly-␣,␣ꢀ-H2), 4.02 (1H, t, J = 6.3 Hz, Glu-␣-H), 4.20–4.25 (1H, m,
3␤-H), 4.58–4.60 (1H, m, Cys-␣-H). ESI–MS: m/z 762.3 [M+H]+, m/z
760.3 [M−H]−, m/z 379.9 [M−2H]2−
.
760.3 [M−H]−, m/z 379.9 [M−2H]2−
.
2.4.6. S-(3-Sulfooxylithocholyl)glutathione (LCA-GSH 3-sulfate;
1e)
2.4.4. S-(3-Sulfooxydeoxycholyl)glutathione (DCA-GSH 3-sulfate;
1c)
This compound 1e was obtained from LCA 3-sulfate (200 mg)
as colorless amorphous solids with isolated yield 23.7 mg (8.9%).
1H NMR ı: 0.67 (3H, s, 18-CH3), 0.94 (3H, s, 19-CH3), 0.94 (3H, d,
J = 6.4 Hz, 21-CH3), 2.12–2.21 (2H, m, Glu-␤,␤ꢀ-H2), 2.49–2.54 (3H,
m, Glu-␥,␥ꢀ-H2 and 23-H), 2.59–2.64 (1H, m, 23-Hꢀ), 3.11–3.15 and
3.39–3.44 (2H, m, Cys-␤,␤ꢀ-H2), 3.91 (2H, s, Gly-␣,␣ꢀ-H2), 3.96 (1H,
t, J = 6.4 Hz, Glu-␣-H), 4.24–4.30 (1H, m, 3␤-H), 4.57–4.60 (1H, m,
Cys-␣-H). ESI–MS: m/z 746.3 [M+H]+, m/z 744.3 [M−H]−, m/z 371.9
This compound 1c was obtained from DCA 3-sulfate (230 mg)
as colorless amorphous solids with isolated yield 7.4 mg (2.2%).
1H NMR ı: 0.70 (3H, s, 18-CH3), 0.92 (3H, s, 19-CH3), 1.01 (3H,
d, J = 6.8 Hz, 21-CH3), 2.19–2.28 (2H, m, Glu-␤,␤ꢀ-H2), 2.52–2.58
(3H, m, Glu-␥,␥ꢀ-H2 and 23-H), 2.64–2.68 (1H, m, 23-Hꢀ), 3.17–3.20
and 3.34–3.40 (2H, m, Cys-␤,␤ꢀ-H2), 3.63–3.66 (1H, m, 12␤-H),
3.95–3.98 (3H, m, Gly-␣,␣ꢀ-H2 and Glu-␣-H), 4.31–4.37 (1H, m, 3␤-
H), 4.59–4.64 (1H, m, Cys-␣-H). ESI–MS: m/z 762.3 [M+H]+, m/z
[M−2H]2−
.
760.3 [M−H]−, m/z 379.9 [M−2H]2−
.
2.5. Preparation of rat liver cytosolic fraction
2.4.5. S-(3-Sulfooxyursodeoxycholyl)glutathione (UDCA-GSH
3-sulfate; 1d)
Animal studies were approved by the Institutional Animal
Care and Use Committee of Kinki University. Male Wistar rats
(230–250 g), given a commercial pellet diet and water ad libitum,
were used. Animals were fasted overnight and then killed. The
liver (10 g wet weight) was minced and homogenized in 3 vol-
umes of ice-cold 10 mM phosphate buffered saline (pH 7.4) by 10
This compound 1d was obtained from UDCA 3-sulfate (180 mg)
as colorless amorphous solids with isolated yield 54.8 mg (24.0%).
1H NMR ı: 0.69 (3H, s, 18-CH3), 0.94 (3H, s, 19-CH3), 0.94 (3H, d,
J = 6.6 Hz, 21-CH3), 2.19–2.25 (2H, m, Glu-␤,␤ꢀ-H2), 2.49–2.55 (3H,
m, Glu-␥,␥ꢀ-H2 and 23-H), 2.60–2.65 (1H, m, 23-Hꢀ), 3.12–3.16 and

Fig. 2. Negative-ion (A) ESI-MS spectrum of LCA-GSH 3-sulfate and its MS/MS spectra obtained by CID of (B) [M−H]− and (C) [M−2H]2− ions. Proposed major m/z values of fragment ions are depicted.

K. Mitamura et al. / Chemistry and Physics of Lipids 165 (2012) 261–269
265
Table 1
Observed ions of BA–GSH 3-sulfates on negative-ion ESI-MSn.
m/z
CA-GSH 3-sulfate
CDCA-GSH 3-sulfate
DCA-GSH 3-sulfate
UDCA-GSH 3-sulfate
LCA-GSH 3-sulfate
MS
[M−H]−
776.3 (63)
387.9 (100)
760.3 (50)
379.9 (100)
760.3 (45)
379.9 (100)
760.3 (34)
379.9 (100)
744.3 (29)
371.8 (100)
[M−2H]2−
MS/MSa
[M−H–H2O]−
758.3 (7)
696.3 (100)
678.3 (4)
N.D.
647.3 (2)
567.4 (4)
N.D.
464.3 (<1)
306.1 (5)
288.0 (<1)
272.1 (2)
254.1 (<1)
503.3 (3)
742.3 (9)
680.3 (100)
662.3 (3)
646.3 (<1)
631.3 (4)
551.3 (6)
517.4 (<1)
448.4 (2)
306.1 (10)
288.1 (1)
272.1 (3)
254.1 (2)
487.3 (4)
742.3 (10)
680.3 (100)
662.4 (3)
646.6 (<1)
631.4 (2)
551.4 (5)
517.4 (<1)
448.4 (1)
306.1 (8)
288.1 (<1)
272.2 (2)
254.1 (1)
487.3 (4)
742.4 (17)
680.4 (100)
662.4 (3)
N.D.
631.4 (6)
551.4 (9)
517.4 (1)
448.4 (3)
306.2 (17)
288.2 (3)
272.2 (5)
254.1 (3)
487.4 (6)
726.3 (18)
664.3 (100)
646.3 (2)
N.D.
615.4 (3)
535.4 (9)
501.5 (2)
432.4 (3)
306.1 (18)
288.1 (2)
272.1 (5)
254.1 (4)
471.3 (6)
[M−H–SO3]−
[M−H–SO3–H2O]−
[M−H–SO3–NH2–H2O]−
[M−H–pyroGlu]−
[M−H–pyroGlu–SO3]−
[M−H–pyroGlu–SO3–NH2–H2O]−
[M−H–pyroGlu–SO3–Gly–CO]−
[GSH–H]−
[GSH–H–H2O]−
[GSH–H–SH2]−
[GSH–H–SH2–H2O]−
[M−H–273]−
MS/MSb
[M−H–pyroGlu]−
[M−H–pyroGlu–H2O]−
[M−H–pyroGlu–Gly–CO]−
[M−H–GSH]−
N.D.
631.3 (3)
613.3 (2)
528.3 (5)
453.3 (76)
370.8 (7)
362.8 (3)
353.7 (9)
315.2 (14)
306.1 (33)
288.1 (8)
272.1 (30)
254.1 (14)
210.1 (3)
143.0 (3)
298.2 (6)
581.4 (3)
487.3 (100)
471.3 (16)
631.3 (3)
N.D.
631.4 (3)
613.4 (2)
528.4 (6)
453.3 (78)
370.8 (7)
362.8 (4)
353.8 (10)
315.2 (16)
306.1 (34)
288.2 (9)
272.1 (31)
254.1 (16)
210.1 (2)
N.D.
615.3 (3)
597.3 (3)
512.3 (5)
437.3 (79)
362.7 (9)
354.7 (5)
345.8 (11)
N.D.
306.1 (36)
288.1 (8)
272.1 (32)
254.1 (15)
210.1 (2)
N.D.
629.4 (2)
544.3 (5)
469.3 (71)
378.7 (6)
370.7 (5)
361.8 (8)
323.2 (13)
306.1 (5)
288.1 (8)
272.1 (31)
254.1 (14)
210.0 (3)
143.0 (2)
N.D.
528.4 (5)
453.3 (73)
370.8 (7)
362.7 (4)
353.8 (10)
315.2 (15)
306.1 (33)
288.1 (8)
272.2 (32)
254.1 (15)
210.1 (2)
N.D.
[M−2H–H2O]2−
[M−2H–NH2–H2O]2−
[M−2H–NH2–2H2O]2−
[M−2H–pyroGlu]2−
[GSH–H]−
[GSH–H–H2O]−
[GSH–H–SH2]−
[GSH–H–SH2–H2O]−
[GSH–H–SH2–H2O–CO2]−
[GSH–2H–H2O]2−
[M−H–pyroGlu–NH2–H2O]−
[M−H–179]−
298.2 (6)
N.D.
487.3 (100)
471.3 (17)
298.3 (7)
581.4 (3)
487.3 (100)
471.4 (16)
N.D.
N.D.
503.0 (100)
N.D.
565.3 (3)
471.3 (100)
455.3 (17)
[M−H–273]−
[M−H–289]−
Values in parenthesis represent relative intensity. N.D., not detectable.
[M−H]− ions of each BA–GSH 3-sulfate were used as precursor ion.
a
b
[M−2H]2− ions of each BA–GSH 3-sulfate were used as precursor ion.
Table 2
MS/MS optimized conditions for quantitative determination of target analytes by the triple-quadrupole analyzer.
Analyte
Tube lens offset voltage (V)
Collision energy (V)
Transition ions for SRM
Precursor ion (m/z)
Product Ion (m/z)
CA-GSH 3-sulfate
CDCA-GSH 3-sulfate
DCA-GSH 3-sulfate
UDCA-GSH 3-sulfate
LCA-GSH 3-sulfate
−90
−115
−87
35
19
30
21
38
19
36
20
39
18
31
29
30
29
30
42
47
41
41
44
25
27
35
26
26
29
[M−H]− (776.3)
[GSH–H]− (305.9)
[M−H–GSH]− (469.1)
[M−H–SO4]− (680.3)
[M−H–273]− (486.7)
[GSH–H]− (306.0)
[M−H–273]− (487.1)
[GSH–H]− (305.7)
[M−H–273]− (487.1)
[GSH–H]− (305.9)
[M−H–273]− (470.9)
[GSH–H]− (306.0)
[GSH–H]− (305.6)
[GSH–H]− (305.7)
[GSH–H]− (306.0)
[GSH–H]− (305.9)
[HSO4]− (97.0)
[M−2H]2− (387.8)
[M−H]− (760.3)
−114
−126
−92
[M−2H]2− (379.9)
[M−H]− (760.3)
[M−2H]2− (379.8)
[M−H]− (760.0)
−125
−92
[M−2H]2− (379.9)
[M−H]− (744.3)
−118
−91
[M−2H]2− (371.7)
[M−H]− (696.3)
CA-GSH
−106
−107
−113
−110
−113
−120
−115
−124
−120
−120
−70
CDCA-GSH
DCA-GSH
UDCA-GSH
LCA-GSH
CA 3-sulfate
CDCA 3-sulfate
DCA 3-sulfate
UDCA 3-sulfate
LCA 3-sulfate
CA
[M−H]− (680.3)
[M−H]− (680.3)
[M−H]− (680.3)
[M−H]− (664.3)
[M−H]− (487.0)
[M−H]− (471.3)
[HSO4]− (97.0)
[M−H]− (471.3)
[HSO4]− (97.0)
[M−H]− (471.3)
[HSO4]− (97.0)
[M−H]− (455.3)
[HSO4]− (97.0)
[M−H+CH3COOH]− (467.3)
[M−H+CH3COOH]− (451.3)
[M−H]− (391.3)
[M−H]− (406.8)
CDCA
DCA
UDCA
LCA
−75
[M−H]− (390.7)
−117
−70
[M−H–CO2]− (344.8)
[M−H]− (390.7)
[M−H+CH3COOH]− (451.3)
[M−H+CH3COOH]− (435.3)
[M−H]− (684.3)
−78
[M−H]− (375.3)
d4-DCA-GSH
−112
[GSH–H]− (306.1)

266
K. Mitamura et al. / Chemistry and Physics of Lipids 165 (2012) 261–269
100
50
100
(A)
LCA-GSH 3-Sulfate
(B)
LCA-GSH 3-Sulfate
LCA-GSH 3-Sulfate
m/z 744.3 [M-H]-
m/z 744.3 [M-H]-
50
ꢀ 305.9 [GSH-H]-
ꢀ 305.9 [GSH-H]-
0
0
100
100
LCA-GSH 3-Sulfate
m/z 371.7 [M-2H]2-
ꢀ 470.9 [M-H-273]-
m/z 371.7 [M-2H]2-
ꢀ 470.9 [M-H-273]-
50
50
0
0
100
100
LCA-GSH
LCA-GSH
m/z 664.3 [M-H]-
m/z 664.3 [M-H]-
50
50
ꢀ 305.9 [GSH-H]-
→ 305.9 [GSH-H]-
0
0
100
100
LCA 3-Sulfate
LCA 3-Sulfate
m/z 455.3 [M-H]-
ꢀ 97.0[HSO4]-
m/z 455.3 [M-H]-
ꢀ 97.0[HSO4]-
50
50
0
0
100
100
LCA
LCA
m/z 435.3 [M-H+CH3COOH]-
ꢀ 375.3 [M-H]-
m/z 435.3 [M-H+CH3COOH]-
ꢀ 375.3 [M-H]-
50
50
0
0
100
100
d4-DCA-GSH
(IS)
d4-DCA-GSH
(IS)
m/z 684.3 [M-H]-
ꢀ 306.1[GSH-H]-
m/z 684.3 [M-H]-
ꢀ 306.1[GSH-H]-
50
0
50
0
10
20
30
10
20
30
Time (min)
Time (min)
Fig. 3. Typical extracted ion chromatograms of (A) reference LA-GSH 3-sulfate, LCA-GSH, LCA 3-sulfate, and LCA and (B) incubation mixture obtained at 0.5 min incubation of
LCA-GSH with a rat liver cytosolic fraction by means of SRM mode of TSQ mass spectrometer. Chromatographic and mass spectrometric conditions are detailed in Section 2.
rapid strokes using a motor-driven Teflon-glass homogenizer. The
homogenate was centrifuged for 10 min at 600 × g in the R19A rotor
of a Hitachi himac CR 21 centrifuge (Hitachi, Tokyo, Japan), fol-
lowed by further centrifugation of the supernatant at 9000 × g for
10 min. The supernatant was centrifuged at 105,000 × g for 1 h. The
resulting supernatant was used as a cytosolic fraction. The whole
procedure was carried out at 0–4 ◦C.
The protein concentration was determined by the method of
Smith et al. (1985) with a Pierce® BCA Protein Assay Kit (Thermo
Fisher Scientific) using bovine serum albumin as standard.
protected group with alkali (Goto et al., 1979). This method is
not adequate for obtaining our target compounds because of the
sensitivity to alkali of the thioester bond present in the BA–GSH
conjugates. Accordingly, coupling the carboxyl group of BA 3-
sulfates with the thiol group of GSH by the activated ester method
was attempted. Each BA 3-sulfate was converted to the correspond-
ing p-nitrophenyl ester, and the activated ester thus obtained was
used to form the GSH conjugate. As the results, pure samples of the
BA–GSH 3-sulfates were obtained after purification by preparative
HPLC.
2.6. Incubation of BA–GSH with rat liver cytosolic fraction
3.2. Structure confirmation of BA–GSH 3-sulfates
Rat liver cytosolic fraction (0.5 mg protein) was dispersed
in 0.05 M phosphate buffer (pH 5.5) (980 ␮L) containing MgCl2
(2 ␮mol) and PAPS (200 nmol) and pre-incubated at 37 ◦C for 5 min.
The reaction was started by the addition of a solution of CA-GSH,
CDCA-GSH, DCA-GSH, UDCA-GSH, and LCA-GSH (each 200 nmol) in
methanol (20 ␮L). After 0.5, 5, 10, 15, 30, and 60 min of incubation,
a 100-␮L aliquot was collected from the incubation mixture and
the reaction(s) was terminated by the addition of acetic acid (4 ␮L)
and acetonitrile (200 ␮L). After addition of d4-DCA-GSH (100 ng)
in methanol (10 ␮L) as an internal standard (IS) and dilution with
water (1 mL), the resulting mixture was passed through an Oasis®
HLB (adsorbent weight, 60 mg) cartridge. After washing with water
(1.5 mL), BAs were then eluted with methanol (1.5 mL), followed
by evaporation of the solvent under a gentle stream of N2 gas
at room temperature, 10-␮L aliquot of the re-dissolved solution
of 30% acetonitrile (100 ␮L) were subjected to an LC/ESI–MS/MS
analysis in the negative-ion mode. The transitions (precursor and
product ions) for each analyte were monitored as shown in Table 2.
The structure of the products was confirmed by means of 1H
NMR and LC/ESI-MS. In the 1H NMR spectra, the following pro-
ton signals were assigned: angular H3-18, H3-19 (each s) and
H3-21 methyl protons in a higher field region of 0.67–1.05 ppm,
H2-23 methylene protons (each m) at 2.47–2.77 ppm, and H-3␤,
H-7 and/or H-12␤ methyne protons (occurring at 3.46–4.37 ppm)
geminally attached to oxygen-containing functions in the aglycone
moieties, and H2-␤,␤ꢀ in glutamic acid moieties at 2.14–2.35 ppm
(m), H2-␥,␥ꢀ in glutamic acid moieties at 2.47–2.58 ppm (m), H2-
␤,␤ꢀ in cysteine moieties at 3.11–3.26 and 3.34–3.45 ppm (each m),
H2-␣,␣ꢀ in glycine moieties (each s) at 3.89–3.98 ppm (m), H-␣ in
glutamic acid moieties at 3.89–4.04 ppm (m), and H-␣ in cysteine
moieties at 4.40–4.70 ppm (m).
The formation of major product ions of the sulfates under
negative-ion ESI–MS/MS on the LTQ mass spectrometer was
also interpreted, and fragmentation pathways are proposed
for those BA–GSH 3-sulfates. The typical negative-ion ESI–MS
and CID spectra of the double conjugate of LCA are shown in
Fig. 2. In the full-scan mode, the spectra of all the conjugates
showed doubly charged [M−2H]2− ion as the base peak along
3. Results and discussion
3.1. Chemical synthesis of BA–GSH 3-sulfates
with
a
moderate deprotonated molecule [M−H]−, indicating
the covalently condensed product of BA 3-sulfate and GSH.
The delocalization of the negative charge onto the carboxylic
acid and sulfuric acid groups is responsible for stability of the
[M−2H]2− ion. Further MS/MS experiments at the normalized
collision energy of 35% were performed. Under the CID con-
dition, singly charged [M−H]− ion underwent characteristic
fragmentation patterns: the fragmentation of the sulfates afforded
the same type of ions (Table 1). Thus, the sulfates showed the
[M−H–SO3]− ion as the base peak with weak [M−H–H2O]−,
Our initial efforts were directed to the chemical synthesis of
the 3-sulfates of BA–GSH as reference compounds (Fig. 1), as syn-
thetic reference compounds are necessary to develop a sensitive
selected reaction monitoring (SRM) method that would be valid
for complex biological matrices. The synthetic method for prepa-
ration of BA 3-sulfates in the literature involved the protection
of 7- and 12-hydroxy groups with a formyloxy group, sulfation
of the C-3 hydroxy group, followed by final deprotection of the

K. Mitamura et al. / Chemistry and Physics of Lipids 165 (2012) 261–269
267
100
50
(A)
(B)
100
50
m/z 744.3 [M-H]- ꢀ 664.3 [M-H-SO3]-
m/z 744.3 [M-H]- ꢀ 664.3 [M-H-SO3]-
0
100
0
100
50
0
50
0
m/z 371.9 [M-2H]2- ꢀ 471.1 [M-H-273]-
m/z 371.9 [M-2H]2- ꢀ 471.1 [M-H-273]-
0
10
20
30
0
10
20
30
Time (min)
Time (min)
471.2
664.2
100
50
0
(D)
100
50
0
(C)
437.3
272.1
306.1
362.5
254.2
455.2
726.3
744.1
800
513.3
329.4
646.2
566.3
306.0
272.1
300
212.4
370.1
535.2
128.0
161.1
231.1
529.4
471.2
700.7
592.6 615.0
408.7
355.1
482.5
200
400
500
m/z
600
700
100
200
300
400
500
600
700
800
m/z
Fig. 4. LC/ESI-MS/MS analysis of (A) reference LCA-GSH 3-sulfate and (B) the incubation mixture obtained at 0.5 min incubation of LCA-GSH with rat liver cytsolic fraction by
means of linear ion-trap mass spectrometer. Extracted ion chromatograms were obtained by monitoring the characteristic transitions of [M−H]− at m/z 744.3 → [M−H−SO3]−
at 664.3 (upper) and [M−2H]2− at m/z 371.9 → [M−H−273]− at m/z 471.1 (lower). Product ion spectra were obtained by CID of (C) [M−H]− ion at m/z 744.3 and (D) [M−2H]2−
ion at m/z 371.9 of the peak associated with retention time at 6 min. Mobile phase used was 30% solvent B (acetonitrile) to 80% B against solvent A (5 mM ammonium acetate
buffer, pH 6.0) over 30 min at a flow rate of 200 ␮L/min.
[M−H–SO3-H2O]−, [M−H–SO3–NH2–H2O]− ions. The ions
[M−H–pyroglutamic acid]−, [M−H–pyroglutamic acid–SO3]−,
[M−H–pyroglutamic acid–SO3–NH2–H2O]−, [M−H–pyroglutamic
acid–SO3–Glycine–CO]−, present in all the CID spectra, are formed
by the loss of GSH and sulfuric acid groups. Similarly, the CID
spectra of [M−H]− ion is characterized by a cleavage of the S–CH2
bond, leading to a loss of 273 Da and the formation of correspond-
ing negative ion at m/z 471, 487, and 503. The weak [GSH−H]−
ion at m/z 306 is formed by the loss of steroid moiety. As a result
of the localization of the negative charge at the GSH moiety, the
frequently occurring fragments (m/z 288, 272, 254) are formed by
the loss of H2O, H2S, and CO2 molecules in various combinations
from the GSH fragment ion.
from the GSH moiety. These fragment ions are common in tandem
mass spectra of GSH conjugates of low molecular weight substances
(Dieckhaus et al., 2005). These spectral data were consistent with
the structures of BA–GSH 3-sulfates. In addition to negative-ion
mode, the positive ESI was also investigated, but the obtained
results were found less satisfying than in the negative-ion mode
(data not shown).
3.3. Sulfation of BA–GSH with rat liver cytosolic fraction
In order to investigate the sulfation of BA–GSH, the amount of
remaining BA–GSH and the BA–GSH 3-sulfates that were formed
were determined by means of SRM mode of triple-stage quadrupole
(TSQ) mass spectrometer. Unconjugated BAs and BA 3-sulfates
were also determined, because the thioester bond of the GSH conju-
gate might be hydrolyzed, generating the unconjugated BA moiety
(and its sulfate) during the incubation. Tube lens offset voltage and
collision energy of each BA and their conjugates under negative-
ion ESI-MS and MS/MS were optimized by directly injecting the
standard solution into the mass spectrometer. BA–GSH 3-sulfates
CID of doubly charged [M−2H]2− ion generated [M−H–273]−
ion as the base peak with moderate [M−H–GSH]− ion and with-
out ion characteristic of sulfated conjugate, which give rise to the
ion formed by the loss of SO3 (80 Da). The fragmentation pattern
showed the ions formed by the loss of glutamic acid and glycine,
ions derived from the GSH, and ions by the neutral loss of 289 Da
(Table 1). The latter ions are mostly charge-induced fragmentation
Table 3
Biotransformation of LCA-GSH and DCA-GSH by a rat liver cytosol containing PAPS. Results shown as percent of the sum of substrate and its metabolites.
Compound
%
0.5 min
5 min
10 min
15 min
30 min
60 min
LCA-GSH
LCA-GSH 3-sulfate
LCA
86.98
0.02
13.0
0.00
11.47
N.D.
88.45
0.08
7.72
N.D.
92.15
0.13
5.56
N.D.
94.25
0.19
2.66
N.D.
96.94
0.40
1.23
N.D.
98.03
0.74
LCA 3-sulfate
DCA-GSH
DCA-GSH 3-sulfate
DCA
88.66
0.01
11.31
0.02
0.19
N.D.
99.62
0.19
0.02
N.D.
99.65
0.33
N.D
N.D.
N.D.
99.12
0.88
N.D.
N.D.
98.32
1.68
N.D.
99.56
0.44
DCA 3-sulfate
N.D., not detectable.

268
K. Mitamura et al. / Chemistry and Physics of Lipids 165 (2012) 261–269
20
18
16
14
12
10
8
3.0
(A)
(B)
2.5
2.0
1.5
1.0
0.5
0.0
6
4
2
0
0
10
20
30
40
50
60
0
10
20
30
40
50
60
Time (min)
Time (min)
10
8
60
50
40
30
20
10
0
(C)
(D)
6
4
2
0
0
10
20
30
40
50
60
0
10
20
30
40
50
60
Time (min)
Time (min)
Fig. 5. Time course for (A) hydrolysis of BA–GSH, (B) formation of BA–GSH 3-sulfates, (C) liberation of unconjugated BA, and (D) appearance of BA 3-sulfates by incubation
with rat liver cytosolic fraction. A 100 nmol each of BA–GSH was separately incubated with rat liver cytosolic fraction (0.5 mg protein) in the presence of PAPS (100 nmol)
at 37 ◦C. Values represent the average of triplicate incubations. CA and its derivatives, open circle; CDCA and its derivatives, open triangle; DCA and its derivatives, filled
triangle; UDCA and its derivatives, open square; LCA and its derivatives, filled circle.
generated similar ions using linear ion-trap mass spectrometer.
But the most abundant product ions were different by mass spec-
trometer. The most abundant transitions that could be used for
the monitoring ion are listed in Table 2. The chromatographic
conditions, especially the composition of mobile phase, were
optimized through several trials. When using a TSKgel ODS-
100V column and a linear gradient elution of 30% solvent B
(acetonitrile-methanol, 3:7, v/v) to 80% B against solvent A (5 mM
ammonium acetate–acetic acid buffer, pH 6.0) over 30 min at a
flow rate of 200 ␮L/min, satisfactory chromatographic separation of
NH2
NH2
O
O
COOH
COOH
R2
R2
NH
NH
C S
O
C S
O
Sulfotransferase
HN
O
HN
O
COOH
COOH
HO
R1
BA-GSH
HO3SO
R1
BA-GSH 3-sulfate
H
H
PAPS
PAP
Carboxylesterase
Carboxylesterase
GSH
GSH
R2
R2
C OH
O
C OH
O
Sulfotransferase
HO
R1
BA
HO3SO
R1
BA 3-sulfate
H
H
PAPS
PAP
CA, R1=α-OH, R2=OH; CDCA, R1=α-OH, R2=H; DCA, R1=H, R2=OH; UDCA, R1=β-OH, R2=H; LCA, R1=R2=H
Fig. 6. Sulfation of LCA-GSH by a rat liver cytosolic fraction.

K. Mitamura et al. / Chemistry and Physics of Lipids 165 (2012) 261–269
269
BA–GSH, BA–GSH 3-sulfate, BA 3-sulfate, and unconjugated BA was
achieved.
conjugates underwent rapid hydrolysis. The liberated unconju-
gated BAs then underwent sulfation at C-3 with hydrophobic BAs
undergoing greater sulfation than hydrophilic BAs (Fig. 5). Further
work is required to define whether the 3-sulfates of the GSH con-
jugates of BAs are present in rodent urine and bile and whether
such formation of the sulfate conjugates of BA–GSH is an important
excretion route for BA–GSH conjugates.
Fig. 3A shows a typical extracted ion chromatogram of LCA-GSH
and its related compounds. Calibration graphs were then con-
structed by plotting the peak-area ratio of each compound to that
of [2,2,4,4-d4]-DCA-GSH (IS) versus the weights of each BA. The
response was linear in the range of 0.05–100 ng.
To investigate the substrate specificity, individual BA–GSH
(100 nmol, [10 ␮M]) were incubated with a rat liver cytosolic frac-
tion (0.5 mg protein) at 37 ◦C for 60 min. A 100-␮L aliquot was
collected from the incubation mixture at appropriate times. After
the addition of IS and solid phase extraction, BA–GSH, BA–GSH
3-sulfates, along with unconjugated BAs and BA 3-sulfates were
determined by the method described above. Fig. 3B shows a typ-
ical extracted ion chromatogram of the incubation mixture at
0.5 min incubation of LCA-GSH. The chromatogram clearly revealed
the appearance of LCA-GSH 3-sulfate, LCA, and LCA 3-sulfate, and
remaining LCA-GSH. The identification of the peak of LCA-GSH 3-
sulfate was carried out by comparison of retention time and CID
spectrum with those of reference compound by means of linear
ion-trap mass spectrometer. Although coexistence of several ions
originating from endogenous compounds were observed in the
product ion spectra (Fig. 4C and 4D) obtained by CID of [M−H]− and
[M−2H]2− of the peak, the CID spectra were identical with those of
the reference compounds (Fig. 2) with respect to the ion species and
the relative abundances of the product ion. As shown in Table 3 and
Fig. 5A, the BA–GSH proportion decreased rapidly with complete
biotransformation after 30 min incubation. In contrast, the enzy-
matic production of BA–GSH 3-sulfates was extremely limited; it
reached a peak in 0.5 min and then decreased in a time dependent
manner (Fig. 5B). LCA, which is a monohydroxy BA and therefore
the most lipophilic of the BAs examined, was most effectively sul-
fated to form LCA-GSH 3-sulfate with the conversion rate of 0.02%
at 0.5 min. CDCA-GSH, DCA-GSH, and UDCA-GSH were metabolized
into their corresponding 3-sulfates at a more moderate rate. The tri-
hydroxy CA-GSH, which is more hydrophilic than the other BA–GSH
tested, showed feeble formation of its 3-sulfate.
Acknowledgements
The authors acknowledge support for this work by a Grant-
in-Aid for Scientific Research (C) for 2009–2011 (To S.I., Grant
21590184) and 2011–2013 (To. K.M., Grant 23590209) from the
Japan Society for the Promotion of Science, and Culture of Japan, and
High-Tech Research Center Project for Private Universities: match-
ing fund subsidy from the Ministry of Education, Culture, Sports,
Science and Technology (2007–2011).
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In a previous paper (Mitamura et al., 2009), we demonstrated
that BA-NACs were easily hydrolyzed to form unconjugated BAs
by carboxyl esterases, which are widely distributed throughout
the body, with high levels occurring in the liver, kidneys, testes,
lungs, and plasma. Furthermore, we showed that BA-NAC 3-sulfates
were formed from BA-NAC conjugates when incubated with a rat
liver cytosolic fraction, albeit to a very limited extent, and were
labile, as the thioester bond of the NAC conjugates underwent rapid
hydrolysis (Mitamura et al., 2011c). As shown in Fig. 5C and D,
both unconjugated BAs and BA 3-sulfates were generated during
the incubation. The BA–GSH conjugates underwent sulfation at C-
3, but the fraction formed was small and transient. Unconjugated
BAs were generated, and these in turn were sulfated. The BA 3-
sulfates could be formed by either hydrolysis of the thioester bond
of the sulfated GSH conjugates or by sulfation of the unconjugated
BAs (Fig. 6). The latter route appears to be the dominant one. The
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4. Conclusion
The 3-sulfates of the GSH conjugates of the five BAs present
in human bile were synthesized and their MS and NMR prop-
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the BA–GSH conjugates were incubated with a rat liver cytoso-
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lic fraction, albeit to
a very limited extent. Moreover, the
BA–GSH conjugates were labile, as the thioester bond of the GSH