Yonago Acta medica 2004;47:63–69

Dehydrogenation of Conjugated Cholic Acid by Escherichia
coli
Rie Katayama, Yoshio Ogura, Nobuo Yamaga, Koji Kimura* 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 Japan
7α-Dehydrogenation of taurocholic acid and glycocholic acid by Escherichia coli (E. coli)
was examined in aerobic and anaerobic culture conditions. Bile acids in the culture
medium of E. coli were extracted, separated into free, glycine-conjugate and taurineconjugate fractions by piperidinohydroxypropyl dextran gel column chromatography,hydrolyzed in alkali and analyzed by gas-liquid chromatography. Both conjugated cholic
acids were dehydrogenated to the corresponding 3α,12α-dihydroxy-7-oxo-5β-cholanoic
acid without deconjugation and no deconjugation of both conjugated cholic acid wasdetected in aerobic cultures. But there was little transformation in anaerobic cultures.
These data suggest that conjugated cholic acids are taken up by E. coli in an aerobic culture as conjugate forms, dehydrogenated without deconjugation and excreted from thecell as conjugate forms.
Key words: dehydrogenation; Escherichia coli; glycocholic acid; taurocholic acid

Primary bile acids synthesized from cholesterolin the liver are conjugated mainly with taurine or
glycine, and excreted with bile into the duodenum.
Intestinal microorganisms are well known to transform sterols and bile acids in human and animal gut(Macdonald et al., 1983; Groh et al., 1993; Hylemonand Harder, 1999). In the gut, these primary conjugated bile acids undergo various transformations
by intestinal microorganisms. The major reactions
are deconjugation, oxidation of hydroxyl groups(dehydrogenation), reduction of oxo groups, and dehydroxylation.

Escherichia coli (E. coli) has no deconjugation activity (Drasar and Hill, 1966; Midtvedt andNorman, 1967; Imamura et al., 1979; Chikai et al.,
1987; Kayahara et al., 1994; Uchida et al., 1999),
but has dehydrogenation activity that forms 7-oxobile acids (Macdonald et al., 1973; Heslewood andHaslewood, 1976; Imamura et al., 1979). In a previous paper, we reported that E. coli dehydrogenates
the 7α -hydroxyl group of both unconjugatedcholic acid and taurocholic acid (TCA) (Oguraet al., 2003). In addition, we found that TCA is
dehydrogenated by E. coli to taurine-conjugatedAbbreviations: CA, cholic acid; GCA, glycocholic acid; GLC, capillary gas-liquid chromatography; G-βα, glycine-conjugated 7β,12α-dihydroxy-5β-cholanoic acid; 7α-HSDH, 7α-hydroxysteroid dehydrogenase; Me-DMES,methyl ester dimethylethylsilyl ether; PHP GEL, piperidinohydroxypropyl dextran gel; TCA, taurocholic acid; Tαβ, taurine-conjugated 7α,12β-dihydroxy-5β-cholanoic acid; T-3α12α7=O, taurine-conjugated 3α,12α-dihydroxy-7oxo-5β-cholanoic acid; 3α12α7=O, 3α,12α-dihydroxy-7-oxo-5β-cholanoic acid; ββ, 7β,12β-dihydroxy-5β-cholanoicacid

63

R. Katayama et al.

Table 1. Relative retention time of bile acid derivatives
Derivative

Retention time Relative retention
(min)
time

Internal standard

7β,12β-Dihydroxy-5β-cholanoic acid Me-DMES
7β,12α-Dihydroxy-5β-cholanoic acid Me-DMES
7α,12β-Dihydroxy-5β-cholanoic acid Me-DMES

Substrate and product

Cholic acid Me-DMES
3α,12α-Dihydroxy-7-oxo-5β-cholanoic acid Me-DMES
Unknown

12.61
11.62
9.82

1.08
1.00
0.85

1.00
0.92
0.78

1.28
1.18
1.00

1.59
1.72
1.90

1.46
1.58
1.76

1.88
2.03
2.25

Me-DMES, methyl ester dimethylethylsilyl ether.

3α,12α-dihydroxy-7-oxo-5β -cholanoic acid (T-3α12α7=O) without deconjugation.
In the present experiments, we examined in detail the dehydrogenation of glycine- and taurineconjugated cholic acids by E. coli.

Bacteriological procedures
E. coli K-12 was obtained from the American Type
Culture Collection (Manassas, VA). Brain-heart
infusion (BHI; Becton Dickinson, Sparks, MD)
was used for all cultures. E. coli was precultured
aerobically in BHI medium at 37˚C for 1 day.
Aliquots (3 × 107/10 µL) of E. coli were added to2 mL of BHI medium containing 1 mM bile acids,and the bacteria were cultured at 37˚C for 4 days.
Aerobic culture was performed by incubation in athermostat, and anaerobic culture was performedby the gas-pack method (BBL GasPak Pouch System; Becton Dickinson).

Material and Methods
Chemicals
Glycocholic acid (GCA), TCA and cholic acid (CA)were purchased from Sigma Chemicals Co., St.
Louis, MO. 3α,12α-Dihydroxy-7-oxo-5β-cholanoicacid (3α12α7=O) was prepared by the oxidation ofCA wit h N-bromosucci n i m ide ( Fieser a ndRajagopalan, 1949). 7β,12β-Dihydroxy-5β-cholanoic acid (ββ), glycine-conjugated 7β,12 α-dihydroxy-5β-cholanoic acid (G-βα) and taurine-conjugated 7α,12β-dihydroxy-5β-cholanoic acid (T-αβ)were synthesized as described previously (Arimotoet al., 1982; Yamaga et al., 1987) and used as internal standards for analysis of bile acids by capillarygas-liquid chromatography (GLC). Piperidinohydroxypropyl dextran gel (PHP GEL) was purchased from Shimadzu, Kyoto, Japan and dimethylethylsilylimidazole from Tokyo Kasei Kogyo Co.
Ltd., Tokyo, Japan. The other reagents and solvents, of analytical grade, were obtained from
Wako Pure Chemical Industries, Osaka, Japan. If
not otherwise stated, the solvents were distilled
once before use.

Analytical methods
Prior to analysis of bile acids, ββ, G-βα and T-αβ asinternal standards were added to 100 µL of the culture medium. The analytical sample with the threeinternal standards was treated with 8 volumes ofethanol at 80˚C for 10 min, and filtered. The filtrate was evaporated to dryness under a stream ofnitrogen and the residue was subjected to PHPGEL column chromatography to separate bile acids
into nonamidate (free), glycine-conjugate andtaurine-conjugate fractions (Yamaga et al., 1987).
Glycine- and taurine-conjugate fractions were hydrolyzed individually in alkaline solution (Yamagaet al., 1997). The hydrolysates and nonamidate
fraction were acidified with dilute hydrochloric
acid, and the bile acids were extracted with ethyl
ether. The extracted bile acids were converted into64

Dehydrogenation of conjugated bile acid

Nonamidate fractionGlycine-conjugate fraction

Taurine-conjugate fraction

��

��
9.834

19.938

11.616

12.605��

3�12�7=O

min

26.4

20.78622.47019.3

13.408

12.1

5

26.4

19.3

12.910

min

12.1

5

26.4

19.3

13.96312.1

510.95018.452CA

min

Fig. 1. Gas chromatograms of bile acids in nonamidate, glycine- and taurine-conjugate fractions after a piperidinohydroxypropyl dextran gel (PHP GEL) column of bile acids. The medium cultured with taurocholic acid (TCA) for
4 days added three internal standard compounds, 7β,12β-dihydroxy-5β-cholanoic acid (ββ), glycine-conjugated
7β,12α-dihydroxy-5β-cholanoic acid (G-βα) and taurine-conjugated 7α,12β-dihydroxy-5β-cholanoic acid (T-αβ).
The sample was fractionated by PHP GEL column. Each fraction was hydrolyzed, and the bile acid extracts wereconverted into Me-DMES derivatives. See Table 1 for gas chromatographic condition. CA, cholic acid; 3α12α7=O,3α,12α-dihydroxy-7-oxo-5β-cholanoic acid.

column temperature was maintained at 285˚C andhelium was used as the carrier gas.
Relative retention times of bile acid derivatives in the present GLC analysis are given in Table
1.

methyl ester dimethylethylsilyl ether (Me-DMES)
derivatives as described previously (Yamaga et al.,
1987; Yamaga et al., 1996). Briefly, bile acids were
methylated with diazomethane, and then left in 70µL of dimethylethylsilylimidazole in an air-tightvessel at room temperature overnight.

Results

Capillary gas liquid chromatography

Figure 1 shows gas chromatograms of the bile acidsin the nonamidate, glycine- and taurine-conjugate
fractions obtained by PHP GEL column chromatography of the medium after culture with TCA for
4 days. Peaks corresponding to CA, 3α12 α7=Oand αβ (internal standard) were detected in thetaurine-conjugate fraction. A peak corresponding

An aliquot of the bile acid derivatives dissolved inn-hexane was injected into a gas-liquid chromatograph (model GC-14A; Shimadzu) equipped witha flame 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. The65