R. Katayama et al.

(%)
100Conversion

80
6040200(%)
100Conversion(A) Aerobic culture

Day 0Day 1Day 2Day 3(B) Anaerobic culture

6040200Day 0Day 1Day 2604020(%)
10080

Day 3Day 0Day 1Day 2Day 3Day 4Day 3Day 4(B) Anaerobic culture

806040200Day 4(A) Aerobic culture

800Day 4Conversion

Conversion(%)
100Day 0Day 1Day 2

Fig. 2. Conversion of taurocholic acid (TCA) to taurine-conjugated 3α,12α-dihydroxy-7-oxo-5β-cholanoic acid (T-3α12α7=O) by Escherichia coli. Changes
in percentage composition of individual bile acids werefollowed during the course of (A) aerobic and (B) anaerobic incubation for 4 days. , TCA; , T-3α12α7=O;, Unknown.

Fig. 3. Conversion of glycocholic acid (GCA) to glycine-conjugated 3α,12α-dihydroxy-7-oxo-5β-cholanoic acid (G-3α12α7=O) by Escherichia coli. Changes
in percentage composition of individual bile acids werefollowed during the course of (A) aerobic and (B) anaerobic incubation for 4 days. , GCA; , G-3α12α7=O;, Unknown.

to ββ was detected only in the nonamidate fraction,and a peak corresponding to βα only in the glycineconjugate fraction. No more than two internal standard peaks were detected in each fraction. Thesegas chromatograms demonstrate that the bile acidsin the analytical samples were separated completely by PHP GEL column chromatography. This wasalso confirmed in the experiments with GCA andfree CA.
Figure 2 shows the time course of conversion of TCA by E. coli. In aerobic culture, TCA
was dehydrogenated to T-3 α12 α7= O, and theconversion proceeded almost linearly at a rate ofabout 15% a day (or 0.15 µmol/day under our experimental conditions). Small amounts of unidentified compounds were formed. However, there waslittle dehydrogenation of TCA in anaerobic culture.
Figure 3 shows the conversion of GCA byE. coli. GCA was dehydrogenated to the corre-sponding glycine-conjugated 7-oxo-bile acid. The
conversion rate was almost the same as that of
TCA in aerobic culture, but GCA was slightly more
susceptible to the reaction than TCA, especially inanaerobic culture.
Figure 4 shows the conversion of free CA by E.
coli. In this case, the reaction occurred much more
quickly than with TCA or GCA, with 80% conversion after 1 day in aerobic culture and about 50%
conversion after 2 days in anaerobic culture.
Medium cultured for 4 days without CA
was filtered through a Millipore filter (MILLEXGV0.22) and the filtrate was incubated aerobicallyfor 1 to 4 days with 1 mM GCA without addition ofcofactors. No oxidation was detected at any timeof incubation (data not shown), suggesting that noenzyme reaction took place in the medium.
These results suggest that conjugated bile acids aretaken up by E. coli as conjugate forms, dehydroge-66

Dehydrogenation of conjugated bile acid

Conversion(%)
100806040200(%)
100

Conversion

The difference in rates between the present report
and the previous report presumably represents differences in substrate penetration into living cells.
It is well known that E. coli possesses nohydrolase activity that will hydrolyze conjugatedbile acids (Drasar and Hill, 1966; Midtvedt andNorman, 1967; Imamura et al., 1979; Chikai et al.,
1987; Kayahara et al., 1994; Uchida et al., 1999).
Accordingly, in the present experiments, no deconjugation of TCA and GCA was detected. E.
coli possesses 7α-hydroxysteroid dehydrogenase(7α-HSDH) (Aries et al., 1969; Macdonald et al.,
1973; Prabha et al., 1989) as a non-inducible intracellular enzyme that requires NAD + as a cofactor (Macdonald et al., 1973; Prabha et al., 1989).
Disruption of E. coli during culture might release 7α-HSDH into the medium, but the present experiments ruled out the possibility that released enzyme
activity catalyzes the conversion, since the filtrateprepared from medium cultured for 4 days wascompletely ineffective in 7α-dehydrogenation ofGCA.
If conjugated bile acids are taken up by E.
coli, the corresponding conjugated 7-oxo-bile acids
are formed and excreted into the culture medium.
However, it remains unclear how conjugated bile
acids are take up by E. coli. Bile acids may crossthe cell membrane by ionic or nonionic diffusion,as demonstrated with intestinal bile acid absorption(Dietschy, 1968). Nonionized bile acids may penetrate the membrane easily, but conjugated bile acids
are unlikely to pass through lipid bilayers because
their pKa values are low (taurine-conjugated, < 1.5;
glycine-conjugated, 3.5–5.2) and they are ionizedat the pH of the present culture medium. The pH
in our experiments increased to about 9 in aerobiccultures, whereas it decreased to around 6.5 in anaerobic cultures (Ogura et al., 2003). However, therate of dehydrogenation of conjugated bile acids,
presumably reflecting the rate of influx of substrate,
was greatest in aerobic culture and least in anaerobic culture.
These observations suggest that E. coli possesses an influx mechanism for conjugated bileacids. Mallonee and Hylemon (1996) reported thata bile acid transporter in Eubacterium sp. strain(A) Aerobic culture

Day 0Day 1Day 2Day 3Day 4Day 3Day 4(B) Anaerobic culture

806040200Day 0Day 1Day 2Fig. 4. Conversion of cholic acid (CA) to 3α,12αdihydroxy-7-oxo-5β-cholanoic acid (3α12α7= O) byEscherichia coli. Changes in percentage composition ofindividual bile acids were followed during the course of(A) aerobic and (B) anaerobic incubation for 4 days. ,CA; , 3α12α7=O; , Unknown.

nated as conjugate forms and excreted from the cell
as conjugate forms.
Discussion
The present experiments demonstrated that E. coli
dehydrogenates taurine- or glycine-conjugatedCA to the corresponding 3α12 α7=O without deconjugation. These reactions occurred only underaerobic conditions, and the conversion rates werelower than for free CA. TCA and GCA were about
60% dehydrogenated after 4 days (Fig. 1), whereas
free CA was about 80% dehydrogenated after 1 day
and almost completely dehydrogenate after 4 days
(Ogura et al., 2003).
Haslewood and Haslewood (1976) showed that
7α-hydroxysteroid dehydrogenase prepared from E.
coli oxidizes both conjugated and free 7α-hydroxylbile acids at the same rate in an in vitro system.

67

R. Katayama et al.

VPI 12708 showed a much higher activity forfree bile acids than for glycocholic acid or 7-oxocholic acid. Conversely, Thanassi et al. (1997)
demonstrated that E. coli possesses a mechanism
to actively pump out TCA. T-3α12α7=O was not
examined in that report, but it is presumed that thisoxo bile acid is excreted by the same mechanism.
It is not yet known whether this efflux mechanism
is also responsible for influx of bile acids, but if
this efflux mechanism is a rate-limiting step, influxof conjugated bile acids will be restricted under anaerobic conditions.
As shown in the present paper, conjugated bile
acids are dehydrogenated by E. coli without deconjugation. On the other hand, 7α-dehydroxylationof bile acids by intestinal bacteria is considered
to take place after deconjugation (Aries and Hill,1970), and 7α-dehydroxylating bacteria show muchhigher activities when cultured with bacteria possessing deconjugation activity than when cultured
alone (Hirano and Masuda, 1982; Narushima et al.,2002); nevertheless, this dehydroxylation reaction
has also been suggested to occur without deconjugation in vivo (Hepner et al., 1972).

chromatography. Steroids 1982;39:631–641.
4 Chikai T, Nakao H, Uchida K. Deconjugation ofbile acids by human intestinal bacteria implanted in
germ-free rats. Lipids 1987;22:669–671.
5 Dietschy JM. Mechanisms for the intestinal absorption of bile acids. J Lipid Res 1968;9:297–309.
6 Drasar BS, Hill MJ. The deconjugation of bile salts
by human intestinal bacteria. Lancet 1966;I:1237–1238.
7 Fieser LF, Rajagopalan S. Selective oxidation withN-bromosuccinimide. I. Cholic acid. J Am Chem
Soc 1949;71:3935–3938.
8 Groh H, Schade K, Hörhold-Schubert C. Steroid
metabolism with intestinal microorganisms. J Basic
Microbiol 1993;33:59–72.
9 Haslewood ES, Haslewood GAD. The specificity
of a 7α-hydroxy steroid dehydrogenase from Escherichia coli. Biochem J 1976;157:207–210.
10 Hepner GW, Hofmann AF, Thomas PJ. Metabolismof steroid and amino acid moieties of conjugatedbile acids in man. J Clin Invest 1972;51:1889–1897.
11 Hirano S, Masuda N. Enhancement of the 7α-dehydroxylase activity of a gram-positive intestinal
anaerobe by Bacteroides and its significance in the7-dehydroxylation of ursodeoxycholic acid. J LipidRes 1982;23:1152–1158.
12 Hylemon PB, Harder J. Biotransformation of monoterpenes, bile acids, and other isoprenoids in anaerobic ecosystems. FEMS Microbiol Rev 1999;22:475–488.
13 Imamura T, Sakamoto N, Tamaki M, Hirano S.
Transformation of bile acids by members of Enterobacteriaceae. Nippon Saikingaku Zasshi 1979;34:513–520 (in Japanese).
14 Kayahara T, Tamura T, Amuro Y, Higashino K,Igimi H, Uchida K. Δ22-β-Muricholic acid in monoassociated rats and conventional rats. Lipids 1994;
29:289–296.
15 Macdona ld I A, Wi l l ia ms CN, Ma hony DE .
7α-Hydroxysteroid dehydrogenase from Escherichia
coli B: preliminary studies. Biochim Biophys Acta
1973;309:243–253.
16 Macdonald IA, Bokkenheuser VD, Winter J, McLernon
AM, Mosbach EH. Degradation of steroids in thehuman gut. J Lipid Res 1983;24:675–700.
17 Mallonee DH, Hylemon PB. Sequencing and expression of a gene encoding a bile acid transporter fromEubacterium sp. Strain VPI 12708. J Bacteriol
1996;178:7053–7058.
18 Midtvedt T, Norman A. Bile acid transformations
by microbial strains belonging to genera found inintestinal contents. Acta Pathol Microbiol Scand
1967;71;629–638.

Acknowledgments: The authors thank Professor Hisao
Ito, Division of Organ Pathology, Department of Microbiology and Pathology, School of Medicine, Tottori
University Faculty of Medicine, and Professor Yoshito
Irizawa, Division of Legal Medicine, Department ofSocial Medicine, School of Medicine, Tottori University
Faculty of Medicine, for reviewing this manuscript.
References
1 Aries V, Crowther JS, Drasar BS, Hill MJ. Degradation of bile salts by human intestinal bacteria. Gut
1969;10:575–576
2 Aries V, Hill MJ. Degradation of steroids by intestinal bacteria. II. Enzymes catalysing the oxidoreduction of the 3α-, 7α- and 12α-hydroxyl groups
in cholic acid, and the dehydroxylation of the7-hydroxyl group. Biochim Biophys Acta 1970;
202:535–543.
3 Arimoto K, Adachi K, Yamaga N. 7β,12β-Dihydroxy-5β-cholan-24-oic acid as an internal standard
for quantitative determination of bile acids by gas68

Dehydrogenation of conjugated bile acid

19 Narushima S, Itoh K, Kuruma K, Uchida K. Bile
acid transformation in gnotobiotic mice associatedwith human intestinal flora. Bioscience Microflora
2002;21:145–153.
20 Ogura Y, Yamaga N, Kido Y, Katayama R, Yamada
K, Uchida K. Aerobic and anaerobic biotransformation of bile acids by Escherichia coli (I). Bioscience
Microflora 2003;22:133–137.
21 Prabha V, Gupta M, Gupta KG. Kinetic properties of
7α-hydroxysteroid dehydrogenase from Escherichia
coli 080. Can J Microbiol 1989;35:1076–1080.
22 Thanassi DG, Cheng LW, Nikaido H. Active effluxof bile salts by Escherichia coli. J Bacteriol 1997;
179:2512–2518.
23 Uchida K, Satoh T, Narushima S, Itoh K, Takase H,Kuruma K, et al. Transformation of bile acids andsterols by Clostridia (fusiform bacteria) in Wistar
rats. Lipids 1999;34:269–273.

24 Yamaga N, Adachi K, Kohara H, Shimizu K. Internal standards for quantitative gas chromatographyof individual bile acids after group separation of bileacids in urine. J Chromatogr 1987;422:25–32.
25 Yamaga N, Nagano Y, Yamada K. An examinationof alkaline hydrolyzing conditions of conjugatedbile acids with carbonyl groups. Yonago Acta Med
1997;40:73–77.
26 Yamaga N, Ikebuchi J, Kohara H, Ogura Y, Yamada
K. Analysis of bile acids in urine specimens fromhealthy humans: Determination of several bile acids
with β-hydroxyl and carbonyl groups. J Biochem
1996;119:725–730.
Received September 2, 2004; accepted September 30, 2004
Corresponding author: Yoshio Ogura, PhD69