Yonago Acta medica 2007;50:33–40

Biotransformation of Bile Acids by Bacteroides sp. Strain
T-40 Isolated from Human Microﬂora
Yoshio Ogura, Tsuyoshi Takei, Takao Suzuki*, Nobuo Yamaga, Kikuji Itoh†, Kazuo
Yamada and Kiyohisa Uchida
Division of Medical Biochemistry, Department of Pathophysiological and Therapeutic Science, School of Medicine, Tottori University Faculty of Medicine and *Division of Functional
Radiation Science, Research Center for BioScience and Technology, Tottori University, Yonago
683-8503, and †Laboratory of Veterinary Public Health, Graduate School of Agricultural andLife Sciences, The University of Tokyo, Tokyo 113-8657 JapanThe effects of Bacteroides sp. strain T-40 isolated from human feces on the biotransformation of bile acids were examined in an anaerobic culture system. Bacteroides sp. T-40
oxidized cholic acid (CA) and chenodeoxycholic acid (CDCA) to 3 ,12 -dihydroxy-7oxo-5 -cholanoic acid and 3 -hydroxy-7-oxo-5 -cholanoic acid, and reduced these oxobile acids to CA and CDCA, respectively. However, the reduction activities were lower
than the oxidation activities. Hyocholic acid was dehydrogenated, but to a lesser extent
than CA or CDCA. On the other hand, -muricholic acid, which has a hydroxyl groupat the position of 7 , was not dehydrogenated. Glycocholic acid was converted to free 3,12 -dihydroxy-7-oxo-5 -cholanoic acid but any glycine conjugated 7-oxo product was
not detected. These data indicate that Bacteroides sp. T-40 possesses bile acid hydrolase
and 7 -hydroxysteroid dehydrogenase, by which conjugated bile acids are initially deconjugated, and then undergo oxidization of the 7 -hydroxy group.
Key words: Bacteroides; bile acid; biotransformation; dehydrogenase; hydrolase

man intestine as shown in Fig. 1. The ﬁrst step,oxidation of the 7 -hydroxy group of CDCA, iscarried out by 7 -hydroxysteroid dehydrogenase(7 -HSDH), and the second step, reduction of the
7-oxo group of 7=O-LCA, by 7 -hydroxysteroid
dehydrogenase (7 -HSDH). In a previous paper,
we reported the oxidation/reduction characteristics of 7 -hydroxyl bile acids by Eschericha coli,a facultative anaerobe, under aerobic and anaerobic conditions (Ogura et al., 2005). Meanwhile,

Ursodeoxycholic acid (UDCA), which is present
in small amounts in human bile and intestine, isa useful therapeutic agent for dissolution of cholesterol gallstones (Sugata and Shimizu, 1974)
and for treatment and prevention of hepatic diseases (Angulo et al., 1999). It is speculated that
UDCA is generated from chenodeoxycholic acid
(CDCA) through two-step reaction processes via
3 -hydroxy-7-oxo-5 -cholanoic acid (7=O-LCA)(by microbial biotransformation reactions) in hu-Abbreviations: CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; GCA, glycocholic acid; GLC,gas-liquid chromatography; G- , glycine-conjugated 7 ,12 -dihydroxy-5 -cholanoic acid; HCA, hyocholic acid;
HSDH, hydroxysteroid dehydrogenase; LCA, lithocholic acid; -MCA, -muricholic acid; -MCA, -muricholic acid;
Me-DMES, methyl ester dimethylethylsilyl ether; MPYG, modiﬁed peptone yeast extract glucose; 7=O-DCA, 3 ,12-dihydroxy-7-oxo-5 -cholanoic acid (7-oxodeoxycholic acid); 7=O-LCA, 3 -hydroxy-7-oxo-5 -cholanoic acid (7-oxolithocholic acid); PHP GEL, piperidinohydroxypropyl dextran gel; T- , taurine-conjugated 7 ,12 -dihydroxy-5 -cholanoicacid; UDCA, ursodeoxycholic acid; , 7 ,12 -dihydroxy-5 -cholanoic acid33

Y. Ogura et al.

Fig. 1. Reaction sequences of chenodeoxycholic acid (CDCA), 3 -hydroxy-7-oxo-5 -cholanoic acid (7=O-LCA) andursodeoxycholic acid (UDCA). 7 -HSDH, 7 -hydroxysteroid dehydrogenase; 7 -HSDH, 7 -hydroxysteroid dehydrogenase.

one strain of Bacteroides (Bacteroides sp. strainT-40) was isolated from healthy human feces, inwhich a relatively high amount of UDCA wasdetected, and this bacterium was found to havethe highest 7 -HSDH activity among bacteria isolated from the feces. In the present experiments,
we examined the effect of Bacteroides sp. T-40,
an obligate anaerobe, on bile acid biotransformation (the ﬁrst step for production of UDCA) in ananaerobic culture system.

(Arimoto et al., 1982; Yamaga et al., 1987), andused as internal standards for analysis of bile acids by capillary gas-liquid chromatography (GLC).
These bile acids were estimated to be more than
96% pure so far examined by capillary GLC. Dimethylethylimidazole was purchased from Tokyo
Kasei Kogyo, Tokyo, Japan. Piperidinohydroxypropyl dextran gel (PHP GEL) was purchasedfrom Shimadzu, Kyoto, Japan. The other reagentsand solvents of analytical grade were obtainedfrom Wako Pure Chemical Industries, Osaka,
Japan. If not otherwise stated, the solvents weredistilled once before use.

Materials and Methods
Chemicals

Bacteroiological procedures

CA, CDCA, hyocholic acid (HCA), and glycocholic acid (GCA) were purchased from Sigma
Chemical, St. Louis, MO. -Muricholic acid( -MCA) and -muricholic acid ( -MCA) weresynthesized according to the methods reported byIida et al. (1989). 3 ,12 -Dihydroxy-7-oxo-5 cholanoic acid (7=O-DCA) and 7=O-LCA wereprepared by the oxidation of CA and CDCA
with N-bromosuccinimide (Fieser and Rajagopalan, 1949), respectively. 7 ,12 -Dihydroxy-5 cholanoic acid ( ), glycine-conjugated 7 ,12 dihydroxy-5 -cholanoic acid (G- ), and taurineconjugated 7 ,12 -dihydroxy-5 -cholanoic acid(T- ) were synthesized as described previously

Eighty-four strains (E. coli: 4, Enterococcus: 12,Bacteroidaceae 38, Bifidobacterium: 8, Eubacterium: 5, Clostridium: 1 and unidentiﬁed anaerobes: 6) were isolated from a human fecal sample
using 3 non-selective and 7 selective agar plates(Narushima et al., 2006) and characterized on
bases of colony form, Gram stain, morphology,
growth in air and spore formation (Mitsuoka etal., 1965). Each strain was cultured anaerobicallyfor 3 days in a modified peptone yeast extract
glucose (MPYG) medium (Hirano et al., 1981;
Takahashi and Morotomi, 1994) containing CAand the bile acids transformed from CA were examined by thin-layer chromatography (Van den34

Biotransformation of bile acid by Bacteroides sp.

chromatography to separate bile acids into free,glycine-conjugate and taurine-conjugate fractions(Yamaga et al., 1987). Glycine-conjugate fraction
was hydrolyzed in alkaline solution (Yamaga etal., 1997). The hydrolysate and free fraction wereacidiﬁed with diluted hydrochloric acid, and thebile acids were extracted with diethylether. The
extracted bile acids were converted into methylester dimethylethylsilyl ether (Me-DMES) derivatives as described previously (Yamaga et al., 1987,1996). The methyl ester of 7=O-LCA was converted to its methoxime derivative as described by
Horning et al. (1968), to separately determine thiscompound and CDCA by GLC.

Ende et al., 1982) and GLC (Ogura et al., 2003).
The colony (Bacteroides sp. T-40) which showed
the highest production of 7=O-DCA was selectedfor the present experiment. Strain T-40 was finally identiﬁed by approximately 1,500 bp of the16S rDNA gene according to methods publishedpreviously (Miyamoto and Itoh, 2000). Although
this sequence suggested strong similarity for thespecies in Bacteroides cluster, the sequence homology between strain T-40 and type strains in
the Bacteroides cluster was less than 95%. E.
coli K-12 which possesses 7 -HSDH (Ogura etal., 2003) was obtained from the American Type
Culture Collection, Manassas, VA. Both bacteria,
the strain T-40 and E. coli K-12, were precultured
anaerobically in MPYG medium (Hirano et al.,1981; Takahashi and Morotomi, 1994) at 37˚Cfor 1 day by the gas-pack method (BBL Gas Pak
Anaerobic System; Becton Dikinson, Sparks,MD). Aliquots of the growth of the strain T-40 (8107/10 µL) and K-12 (3 107/10 µL) were added
to 10 mL of MPYG medium containing 0.5 mMbile acid and cultured anaerobically at 37˚C for 4days.

Capillary GLC
An aliquot of the bile acid derivatives dissolved
in n-hexane was injected into a gas chromatograph (Model G14A; Shimadzu) equipped with aflame ionization detector, a solventless injector,
and a computerized data system (Model C-R4A;Shimadzu). A Hicap CBP-1 capillary column(25 m 0.25 mm I.D.; Shimadzu) was used. Thecolumn temperature was maintained at 285˚C andhelium was used as the carrier gas.

Analytical methods
Bile acids were extracted from the medium aspreviously described (Ogura et al., 2003). A portion of the cultured medium, 100 µL, was treatedwith 8 volumes of ethanol by warming for 10 minand then filtered. The filtrate was evaporated todryness under a stream of nitrogen and the residue was hydrolyzed in alkaline solution. Afterhydrolysis, the reaction mixture was acidified
with diluted hydrochloric acid and bile acids wereextracted with diethylether. In the PHP GELanalysis, prior to the analysis of bile acids, ,G- and T- as internal standards were added
to 100 µL of the culture medium. The analytical sample with the three internal standards wastreated with 8 volumes of ethanol by warming for
10 min and then ﬁltered. The ﬁltrate was evaporated to dryness under a stream of nitrogen andthe residue was subjected to PHP GEL column

Experiments with cell-free enzyme preparation
Crude 7 -HSDH was prepared from Bacteroides
sp. T-40 according to the method of Macdonald etal. (1973). The enzyme activity was assayed forbile acid transformation by a slight modiﬁcationof the method reported by Macdonald et al. (1974,1975). The reaction was performed at 37˚C for
10 min with 7 mM NAD+, 0.2 M glycine-NaOH
buffer (pH 9.5), 0–4 mM bile acid and 45 µL of
the crude enzyme preparation in ﬁnal volume of 1mL. One unit of enzyme activity of 7 -dehydrogenation was deﬁned as the amount of enzyme required to yield 1 nmole of NADH per min under
the conditions described above.

35