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The biotransformation of hyodeoxycholic acid by Pseudomonas sp. NCIB 10590 under anaerobic conditions
J. steroid Biochem. Vol. 19, No. 3. pp. 1355-1362. Printed in Great Britain. All rights reserved
1983 Copyright
c
0022-4731/83 $3.00 + 0.00 1983 Pergamon Press Lrd
THE BIOTRANSFORMATION OF HYODEOXYCHOLIC BY PSEUDOMOiVAS SP. NCIB 10590 UNDER ANAEROBIC CONDITIONS
ACID
R. W. OWEN* and R. F. BILTON Department of Chemistry and Bi~hemistry, Liverpool Polytechnic, Liverpool L3 3AF, England (Received 17 May 1982) Summary-The bacterial degradation of hyodeoxycholic acid under anaerobic conditions was studied. The major acidic product has been identified as 6@-hydroxy-3-oxochol-4-ene-24-oic acid whilst the major neutral product has been identified as 6a-hydroxyandrosta-l,4-diene-3,17-dione. The minor acidic products were 3,6-dioxochola-1,4-diene-24-oic acid, 3-oxochol-5-ene-24-oic acid, 3-oxochol-4-ene-24-oic acid, 3-ox~hoia-~,~diene-24-oic acid and ~-hydroxy-3~xochola-1 &diene-24-oic acid and the minor neutral products were androst-4-ene-3,17-dione, androst-4-ene-3,6,1 %trione, androsta- I ,4-diene-3,6,17trione, androsta- 1,4-diene-3,17-dione, 17p-hydroxyandrosta- 1,4-diene-3-one and 6a-hydroxyandrost-4ene-3,17-dione. Evidence is presented which suggests that under aerobic conditions, one pathway of hyodeoxycholic acid metabolism exists whilst under anaerobic conditions an extra biotransformation pathway becomes operative involving the induction of a 6a-dehydroxylase enzyme. A biochemical pathway of hvodeoxvcholic acid metabolism by bacteria under anaerobic conditions is discussed incorporating a scheme involving such an enzym;.
INTRODUCTION
The aerobic degradation of bile acids by bacteria has been studied in detail [l-8]. It has been revealed that the mode of action initially involves oxidation of the 3a-hydroxyl group to a ketone group. Subsequently after nuclearsteroid dehydrogenation of the A-ring to give C,, unsaturated steroids the side-chain is cleaved sequentially to give unsaturated CZ2and C,, metabolites. Under aerobic conditions C,, (androstane) derivatives can be further degraded via aromatic secosteroids to give non-steroidal products [9, IO]. Several studies [8, 1l] have also revealed that oxidation of the B-ring of bile acids carrying a hydroxyl group at C, can also be carried out by bacteria under aerobic conditions thus giving rise to unsaturated bile acids possessing either 4,6-diene-3-one or 1,4,6-trien3-one structures. It is apparent that oxidation of the A-ring precedes oxidation of the B-ring because bile acids with only an oxidised B-ring were not isolated. The low yield of AB-ring unsaturated bile acids produced under aerobic conditions can probably be attributed to the specific requirements for induction of the bacterial enzyme 7ar-dehydroxylase. Bacteria which are able to synthesise this enzyme can remove the hydroxyl group at C, of bile acids [12, 131. Strict anaerobic bacteria of the human intestine in fact have such a potent 7a-dehydroxylase system that the faecal bile acids are almost exclusively deoxycholic
*Present address and to whom requests for reprints should be sent: Public Health Laboratory Service, Centre for Applied Microbiology and Research Bacterial Metabolism Research Laboratory, Porton Down, Salisbury, Wiltshire SP4 OJG, England.
acid (DCA) and lithocholic acid (LA) which are produced by 7a-dehydroxylation of cholic acid (CA) and chenodeoxycholic acid (CDCA) respectively [ 141. Studies on a semi-purified 7a-dehydroxylase from Bacteroides spp. have revealed that for induction to occur strict anaerobiosis is required [15]. This obser-
vation has been compounded by studies involving the degradation of bile acids by Pseudomonas sp. NCIB 10590. Under aerobic conditions, minimal induction of 7u-dehydroxyiase occurs which is reflected by the low yield of AB-ring unsaturated steroids [4,6]. Under anaerobic conditions the induction of the enzyme is such that the major degradative products of CA [16] and CDCA [17] possess a 4,6-dienone structure. The aerobic bacterial metabolism of hyodeoxycholic acid (HDC) has also been studied IS]. The results showed that the degradative pattern is similar to that of other bile acids and that the 6a-hydroxyl group remains intact or else is oxidised to a ketone group. In a study of the anaerobic metabolism of HDC by Pseudomonas sp. NCIB 10590 we present evidence for the operation of an additional pathway which involves a 6u-dehydroxylation step. EXPERIMENTAL HDC, Sa-cholestane and 1,4-androstadiene-3,17dione were obtained from Koch Light Laboratories (Colnbrook, Bucks). General reagents were of “Analar” grade and obtained from BDH and all solvents were redistilled before use. Infra red (i.r.) spectra were determined from KBr discs on a PerkinElmer 457 spectrophotometer. Ultraviolet (u.v.) spectra were determined for solutions in methanol on a
1355
Pye-Unicam SP. 1800 spectrophotometer. Nuclear magnetic resonance (NMR) proton spectra were recorded on a Jeol 220 spectrometer operating at 720 MHz at 3O.C from solutions in deuterated chloroform. Mass spectra were obtained using a DuPont 2 l-491 series mass spectrometer either by direct inlet or by combined CC-MS using a Varian aerograph 2700 gas chromatngraph. Data reduction was performed with a Dupont 2 I-0948 data system. Analysis by gas liquid chromatography (GLC) was performed at 260, C using 3% OV-17 on 80/100 mesh acid washed silanised “supelcoport” in 1.5 M x 3 mm silanised glass columns obtained from Phase Separations (Queensferry, U.K.). Retention times were measured relative to the internal standard Scc-cholestane with a flow rate of 30 ml min ’ nitrogen through the column and 30 ml min _’ H, to the flame ionisation detector in a Hewlett-Packard HP 5470 instrument. Analysis by thin layer chromatography (TLC) was performed on 0.25 mm layers of Kiesel gel GF 254 (obtained from E. Merck, Darmstadt. West Germany) in metha~oi-dichioromethane (1: 19, v/v) and the mobilities were measured relative to androsta- I ,4-diene-3,17-dione. Products containing a 4-ene-3-one or a I ,4-diene-3-one chromophore were detected under U.V. light (254 nm) and all components were finally visualised as characteristically coioured spots by spraying the plates with freshly prepared an~saldehyd~ reagent [lS] and heating in an oven at I 10°C for IO min. As an aid to tentative identification of steroids by TLC oxidation was performed by overspotting with a solution of Jones chromic reagent [Is)] diluted I:4 with acetone. Acetyiation was performed by overspotting with acetyf chloride. Reduction was achieved by overspotting with potassium borohydride reagent (I g KBH,, t ml 2N NaOH, 8 ml H&If. Acidic steroids were methylated with ethereal diazomethane priar to analysis. Trimethylsilyl (TMS) ethers were prepared using h&-trimethylsityl acetamide. The biotransformation medium used in this study contained g/I distilled water: sodium hyodeoxycholate, 1.0: K,HPO,; 0.7; KH?PO+ 0.3; KNOI. 1.0; MgS0,‘7Hz0, 0.1; FeS0,.7H,O, 0.0025; ZnS0,.7Hz0, 0.0025; MnSO,‘4H,O. 0.0025; final pH, 7.2. Solutions of sodium hyodeoxycholate, mineral salts, trace elements and magnesium sulphate were autoclaved separately before mixing. Shakefask cultures were grown on a LH Engineering orbital incubator at 28°C. Cells obtained from 4 x 1L shake-flask cultures of Pxudomoncrs sp. NCIB 10590 by centrifugation at 10,000s on a MSE Mistral 4L centrifuge were used to inoculate 4 1 of the culture medium. This gave a final. cell density of I x 10’ cellsml-’ (OD of I .O at 540 nm). The culture medium was steamed for 30 min and cooled immediately before inoculation. The bottle top of the culture was loosened prior to incubation at 28°C for 6 weeks under 9OU,:,Hz: IO’?<:CO?. The
course of the fermentation and OD,,,, was foIIo\~ced ;LI waeekly intervals. After 6 weeks incubation the tbr.mentation was terminated hy direct extraction of the’ metabotites into an equal volume (twice) of cth>l acetate. After drying over anhydrous MgSO, the solvent was removed in ~YXYJO at 30 C to yield I .4 g of a tarry residue. The residue was taken up in warm dichloromethane (20 ml) and the mixture was sepnrated by preparative thin layer chromatography into a series of fractions from which steroids 2--13 were crystailised. .4nr/rost-4-c~nc-.?, IF’-&no (2). Recrystallisation ot 2 from methanoljdichloromethane gave white powdery crystals (2 mg) m.p. 164 C. This compound was found to be identical to authentic androst-4-ene-3.17” dione in its TLC, GLC and spectroscopic properties. ~77rfrosr-4-enr-.?,6,, IT-rricezr (3). Recrystallisatj~~n of 3 from methatroljdichloromethane gave yellow crystals (5.5 mg) m.p. 218-220 C. p’,,,.,* 1730 (17-ketone), 169 I (6-ketone), 1665 (3-ketone) and IhlOcm ’ (C,-C, double bond); i,,,;,, 248 nm (< 14.380); M ’ 300 (92”,,. C,,H,,O, requires M I, 300) and n?Itz I37 (4ene-3.6-dione. X4”,,): GLC R, 1.65: TLC R, I.?. after oxidation R, I .7. after acetylation R, 1.2 and after reduction R, 0.59. .4rl(/ro.~~(1-l,$-~iirnc~-.~.h,I7-trionr (4). Recrystallisation of 4 from methanol&dichloromethane gave yellow needles (3 mg) m.p. 208-210 ‘C. This compound was found to be identical to androsta1.4-diene-3.6, I?-trione described by Tenneson et N/.[5]. A ndros ta - 1,4-dienc -3. I 7-dione Recrystal(5). lisation of 5 from methanol/dichloromethane gave white prisms (19 mg) m.p. 136--l 37 C. This compound was found to be identical to authentic androsta- 1,4-diene-3,17-dione in its TLC, GLC and spectroscopic properties. 17~~-I~~,dr~ro.~~unrtro.vta-1,4-die~~~-?-onr (6). Recrystallisation of 6 from methanol/dichloromethane gave white needles (2 mg) m.p. 167-168 ‘C. C’,,,,, 3480 (hydroxyl), 1655 (17-ketone), 1655 (3-ketone), 1615 and 1595 cm. ’ (Cl -C1 and C,--C, double bonds); i.,,,,, 242 nm (i 15,220); 6 0.82. 1.24 f6H, 5. l%CH, and 19-CH,), 2.30-2.58 (2H. m. 16CH,). 3.64 (IH. t, J =9 Hz, 17-H), 6.06 (1H, s. 4H), 6.22 (1H. d, showing further splitting- J = ION:, 2-H) and 7.03 (1H. d, J = IO HI. I-H); M’ 2X6 (IS”,,, C,,,H,,O, requires M + 286). it~ji’ 122 ( I .4-diene-3-one, 1OO’,‘,,) and mjc 268 (M ‘-H,O, S’;). GLC K, 1.7: TLC R, 0.68, after oxidation R, 1.OO,after acetylation R, I .U3 and after reduction R, 0.68. &. -~l?~ilro.~~(lrtdro,~t -4cw -3, I7-cliot?r (7). Recrystallisation of 7 from methanol~dichl(~r~?methane gave white crystals (IO mg) m.p. 233’ C. This compound was identical to hr-hydroxyandrost-4-ene-3,17-dionc described by Tenneson et td.[5]. &3(-Iz~~hs~~ - 1,4-mzrlrtlstudirnP-3, I 7-dionr (8). Recrystallisation of 8 from methanol/dichloromethane gave white crystals (28mg) m.p. 240-243 C. This compound was identical in its properties to 6~-hy-
1357
Bacterial degradation of hyodeoxycholic acid droxyandrosta1,4-diene-3,17-dione described by Tenneson et a1.[5]. Methyl 3,6-dioxochola-1,4-diene-24-oate (9). Isolated as a slightly impure solid (8 mg) A,,, 250 nm (5 14,210). M+ 398 (84x, C25H,,0, requires M+ 398) m/e 135 (1,4-diene-3,6-dione, 100%) and m/e 283 (M+-105, 6C-side chain, 53%). GLC R, 15.4; TLC R, 1.23, after oxidation R, 1.23, after acetylation 1.23 and after reduction R, 0,52. Methyl 3-oxochol-5-ene-24-oate (10). Isolated as a crystalline solid from methanol/dichloromethane (70 mg). V,,,,, 1745 (carboxyl), 1702 (3-ketone) and 1670cm-’ (C,& double bond). M+ 386 (34x, C25H3803 requires M+ 386) m/e 124 (5-ene-3-one, 100%) and m/e 271 (M+-115, 6C side-chain, 11%). GLC R, 8.21; TLC R, 1.26, after oxidation R, 1.26, after acetylation Rr 1.26 and after reduction R, 0.92. Methyl 3-oxochol-4-ene-24-oate (11). Recrystallation of 11 from methanol/dichloromethane gave spikey crystals (63 mg) m.p. 141-143°C (free acid). V,,,,, 1746 (carboxyl), 1676 (3-ketone) and 1620 cm-’ (C,-C, double bond). i,,, 241 nm (5 12,700); M+ 386 (65”/, C,,H,,O, requires M+ 386) m/e 124 (Cene3-one, 79”/,) and m/e 271 (M+-I 15, 6C-side chain, 5%); GLC R, 6.20; TLC R, 1.21, after oxidation RI 1.21, after acetylation R, 1.21 and after reduction R, 1.21. Methyl 3-oxochola-1,4-diene-24-oate (12). Recrystallisation of 12 from methanol/dichloromethane gave white crystals (14mg). V,,, 1745 (carboxyl) 1665 (3-ketone) 1610 and 1598cm-’ (C,-C2 and C6C, double bonds); ,l,,, 245 nm; 6 0.79, 1.21 (6H, s, 18-CH, and 19-CH,), 0.93 (3H, d, J = 6 Hz, 21CH,), 3.67 (3H, s, 24-OCH,), 6.24 (IH, d, J = 10 Hz, 2-h), 6.50 (lH, s, 4H) and 7.01 (lH, d, J = 10 Hz); M+ 384 (lo%, C25H3603 requires M+ 384) m/e 122 (1,4-diene-3-one, 100%) and m/e 269 (M+-115, 6C-side chain, 14%). GLC R,8.70; TLC R, 1.18, after oxidation R, 1.18, after acetylation R, 1.18 and after reduction R, 1.18. Methyl &-hydroxy-3-oxochol-4-ene-24-oate (13). Recrystallisation of 13 from methanol-dichloromethane gave white prisms (233 mg) mp 184185°C. VIll&X3465 (hydroxyl), 1722 (carboxyl), 1666 (3-ketone) and 1654 (C4C, double bond; A,,,,, 242 nm; 6 0.69, 1.18 (6H, s, 18-CH, and 19-CH,) 0.92 (3H, d, J = 6 Hz, 21-CH,), 3.67 (3 H, 2, 24-OCH,), 4.35 (lH, M, 6H), and 6.16 (lH, s, 4-H). M+ 402 (100% C,,H,,O, requires M + 402) m/e 139, 140 (4-ene-3-one-6-01, 44 and 27%) m/e 269 (M +-133, 6C-side chain + 6-OH, 17%) and m/e 384 (M+-18, 6-OH, 32%). GLC R, 10.1; TLC R, 0.65, after oxidation R, 1.32, after acetylation R, 1.27 and after reduction R, 0.65. Methyl &-hydroxy-3-oxochola-1,4-diene-24-oate (14). Recrystallisation from methanol/dichloromethane gave white crystals (44 mg) m.p. 142-144°C (free acid); V,,,,, 3415 (hydroxyl), 1736 (carboxyl), 1668 (3-ketone), 1624 and 1610cm-’ (C,&, and C,-C, double bonds). i.,,, 245 nm. 6 0.72, 1.20 (6H,
s, 18-CH, and 19-CH,), 0.90 (3H, d, J = 6Hz, 21-CH,), 3.66 (3H, s, 24-OCH,), 4.46 (lH, m, 6-OH), 6.23 (lH, d, showing further splitting, J = 10 Hz, 2-H), 6.46 (lH, s, 4-H), 6.98 (lH, d, J = 10 Hz, 1-H). M+ 400 (90x, C,,H,,04 requires M + 400) m/e 138 (1,4-diene-3-one-6-01, 100%) m/e 267 (MC-133, 6C-side chain +6-OH) and m/e 382 (M+-18, 6-OH, 26%). GLC R, 10.4; TLC R, 0.49, after oxidation R, 1.23, after acetylation R, 1.19 and after reduction R, 0.49. RESULTS
The degradation of HDC (1) under aerobic conditions by Pseudomonas sp. NCIB 10590 has been observed by Tenneson et aZ.[5] who reported the production of C-22 (bisnor) acidic and C-19 (androstane) intermediates. A limited study of HDC metabolism under anaerobic conditions has also been carried out with an Escherichia coli. strain [20]. However, the anaerobic transformation of HDC by bacteria has not been studied in detail. A 4-l anaerobic fermentation of sodium hyodeoxycholate by Pseudomonas sp. NCIB 10590 enabled the isolation of thirteen metabolites from the fermentation beer. The assigned structures are shown in Figs 1 and 2. The major neutral metabolite (8) and three (4, 5 and 6) of the minor neutral metabolites gave i.r. spectra typical of 1,4-diene-3-one A-ring steroids (e.g.
o& oq!p o
(2)
(3)
oJ3p o& // & / J3P // & (5)
0 (4)
0
?H
0
0
(6)
bH (7)
0
0
O’H
(8) Fig. 1. Neutral metabolites isolated after the anaerobic degradation of hyodeoxycholic acid by pseudomonas sp. NCIB 10590.
R. W. OWEN and R. F.
R-CH3CHCH2CH2COOH
o@ J4? R
R
o& od+ (I II
(121
dH
dH
(13)
(141
Fig. 2. Acidic metabolites isolated after the anaerobic degradation of hyodeoxycholic acid by pseudomonas sp.
NCIB 10590.
i.r. spectrum of (6))1655. 1615 and 1595cm ‘, x/?-unsaturated ketone). The U.V. spectra of 5, 6 and 8 supported this observation (i,,,, 244 nm, di-[Isubstituted Q-Unsaturated ketone in a sixmembered ring, double bond exocyclic) while the U.V. 250 nm di-/I-substituted z. spectrum of 4 (I,,,, p-unsaturated ketone in a six-membered ring, double bond exocyclic, extended by a carbonyl group at C,) was typical of a 1,4-diene-3,6-dione structure [21]. Further spectroscopic study of compounds 4 and 8 revealed that they were identical to androsta-I ,4diene-3,6,17-trione and ha-hydroxyandrostaI .4diene-3,17-dione respectively which have been described in detail by Tenneson ef a/.[5]. Compounds 5 and 6 were found to be identical in their spectroscopic and mass spectral properties to authentic androsta- 1,4-diene-3,17-dione and I 7/Ihydroxyandrosta1,4-diene-3,17-dione respectively. This showed that the 6a-hydroxyl group of HDC had been lost during anaerobic fermentation to these products. Metabolites 2, 3 and 7 gave i.r. spectra typical of 4-ene-3-one A-ring steroids (e.g. i.r. spectrum of 2-1666 and 1616 cm-‘, a$-unsaturated ketone). The U.V. spectra of 2 and 7 supported this observation (&,,,, 242 nm, di+‘-substituted a. b-unsaturated ketone in a six-membered ring. double bond exocyclic) [2l] whilst the U.V. spectrum of 3 (I.,,,,,, 248 nm was typical for a 4-ene-3,6-dione structure [2l].
BILTON
Further study of 2 and 7 including mass spectral analyses revealed that 2 was identical to authentrc androst-4-ene-3.17-dione whilst 7 was identical to br-hydroxyandrost-4-ene-3,17-dione described by Tenneson CI ul. [5]. Reactions on TLC revealed that compound 3 could not be oxidised or acetylated but was readily reduced suggesting the absence of hydroxyl groups and the presence of free ketone groups. This was contirmed by the i.r. spectrum which displayed two peaks in the free carbonyl group region (1730 and 1691 cm corresponding to a l7-ketone group and a 6-ketone group respectively) with none in the hydroxyl group region. Sufficient of compound 3 was not available for PMR analysis; however the mass spectrum revealed a molecular ion at m/c> 300 (M +. 92”,,) and an intense ion at m/e 137 (84’3;,) confirming a 4-ene-3,6-dione structure [22]. Compound 3 is therefore assigned the structure androst-4-en-3.6. I7trione. One major (13) and five minor (9-12, 14) acidic metabolites were isolated. Structural analyses were carried out on the methyl ester derivatives. Compounds 9, 12 and 14 gave i.r. spectra typical of I .4-diene-3-one A-ring steroids. The U.V. spectra 01 12 and 14 supported this observation (i,,,, 244nm) whilst the U.V. spectrum of 9 (j .“,.,,. 250 nm) was typical for a 1,4-diene-3,6-dione structure (21). TLC reactions revealed that the oxidation product of 14 corresponded in colour (orange) and R, to 9 whilst 9 and 12 could not be oxidised. The reduction product of 9 was slightly less polar than 14 on TLC using potassium borohydride as reducing agent; however reduction with sodium borohydride gave two reduction products, one of which was identical to the potassium borohydride product. the other corresponding in colour (mauveegrey) and R, to 14 on TLC. 12 and 14 could not be reduced. Compound 14 was easily acetylated but 9 and 12 could not be acetylated on TLC. This indicated that compound 9 did not possess a hydroxyl group only a free ketone group, compound 14 possessed a hydroxyl group but not an unconjugated ketone group whilst compound 12 did not possess either. This was confirmed by the i.r. spectra. The position and stereochemistry of the hydroxyl group carried by compound 14 was assigned from the PMR spectrum. In addition to 3 vinylic protons in the range 6.2336.98 6 corroborating a I .4-diene-3-one A-ring structure, 14 gave rise to a broad multiplet centred at 4.46 6 corresponding to one proton. The dihedral (karplus angles) for the 6[I-proton and the two protons at C, are about 60 and 180 C at C,. On this basis the hydroxyl group in compound I4 was assigned the 6r-configuration [23]. The mass spectrum of 14 revealed an intense molecular ion at m/e 400 (M +, 90”,,), an intense ion (base peak) at m/e 138 (lOOo/,); an intense ion at m:‘r 282 (M +- 18,26%, 6a -hydroxyl group) and an intense ion at m/c 267 (M ‘-133. 42”,J due to the loss of the
Bacterial degradation of hyodeoxycholic acid free bile acid side-chain water (6-OH).
(6C) and the elements
of
The mass spectrum of 9 gave an intense molecular ion at m/e 398 (M+ 84x), an intense ion (base peak) at m/e 135 ( 100°/O) which corresponded to a 1,4-diene-3,6-dione structure and an intense ion at m/e 283 (M+-115, 53%) and to the loss of the full bile acid side chain (6C). The mass spectrum of 12 gave a low intensity molecular ion at m/e 384 (M+, IO%), an intense ion at m/e 122 (100%) and a low intensity ion at m/e 269 (M+ 115, 6C-side chain, 14%). On the basis of the above data compound 9 was assigned the structure 3,6-dioxochola-1,4-diene-24oic acid, compound 12 was assigned the structure 3-oxo-chola-1,4-diene-24-oic acid and compound 14 was assigned the structure 6~-hydroxy-3-oxo~hola1,4-diene-24-oic acid. Of the three remaining acidic intermediates, 11 and 13 gave i.r. spectra typical of 4-ene-3-one, A-ring steroids. The U.V. spectra (La,, 242nm) supported this observation. Further support came from the PMR spectrum of 13; one vinylic proton at 6.16 6. Reactions on TLC revealed that 13 possessed a hydroxyl group but not a free ketone group. This was confirmed by the i.r. spectrum which displayed a peak at 3465 cm-’ in the hydroxyl group region. However 11 did not appear to possess either a hydroxyl group or a free ketone group by its reactions on TLC. This was confined by the i.r. spectrum. The position and stereochemistry of the hydroxyl group in 13 was elucidated from the PMR spectrum. In addition to one vinylic proton at 6.16 6 (corroborating a 4-ene-3-one structure), 13 gave rise to a broad multiplet centred at 4.35 6 typical of a G/J-proton (23). On this basis the hydroxyl group in 13 was assigned the &-position. The mass spectrum of 13 displayed a base peak molecular ion at m/e 402 (M+, 100x), low intensity ions at m/e 139 and 140 (44% 27%) corresponding to a 4-ene-3-one-6-01 structure, a low intensity ion at m/e 384 (M+-18, 32% 6u-hydroxyl group) and a low intensity ion at m/e 269 (M+-133, 6C side-chain + 6OH, 17%).
1359
The mass spectrum of 11 displayed an intense molecular ion at m/e 386 (M+, 65’/J, an intense ion at m/e 124 (790/,) typicai of a 4-ene-3-one, A-ring steroid and a low intensity ion at m/e 271 (5%) due to the loss of the full bile acid side-chain (6C). On the basis of the above data compound 11 and compound 13 were assigned the structures 3-0x0chol-4-ene-24-oic acid and 6a -hydroxy-3-oxochol-5ene-24oic acid respectively. The mass spectrum of the remaining acidic metabolite 10 gave an intense (base peak) ion at m/e 124 (100%) which is typical for 4-ene-fone, A-ring steroids. However this was not confirmed by the U.V. and i.r. spectra. Compound 10 could not be oxidised or acetylated, but could be reduced to a compound identical in colour (blue) and R,A,, to 3/I-hydroxy3-ox~hol-5-ene-24-oic acid. Furthermore the reduced product could be oxidised to a compound identical in colour (rust-orange) and Rf,D,, to 3-oxochol-4-ene-24-oic acid. The mass spectrum gave, in addition to the base peak at m/e 124, a low intensity molecular ion at m/e 386 (M +, 34%) and a low intensity ion at m/e 271 (M+-1 IS, 6C side-chain, 11%). On the basis of the above data compound IO has been tentatively assigned the structure 3-oxochol-5-ene-24-oic acid. The yield of the steroidal metabolites isolated is listed in Table 1. DISCUSSION
The results show that a resting cell suspension of Pseudomonas sp. NCIB 10590 is capable of extensive degradation of HDC under anaerobic conditions. Tenneson et al.]51 reported that the aerobic metabolites produced from HDC by Pseadomu~as sp. NCIB 10590 were predominantly 1,Cdienone steroids retaining the hydroxyl group at C,. Under anaerobic conditions the products again were dominated by 1,4-dienone steroids; however, in addition another class of compounds were present at significant levels. The second class of compounds were also mainly 1,4-dienone steroids but they had
Table 1. Yield of metabolites with respect to starting material after 6 weeks anaerobic incubation Metabolite Number L
3 4 6
8 9 10 11 12 13 14
Metabolite Androst-4-en-3,17-dione Androst-4-en-3,6,17-trione Androsta- 1,4-dien-3,6,17-trione Androsta-1,4-dien-3,17-dione 17@-Hydroxyandrosta-1,4dien-3-one 6a-Hydroxyandrost-4-en-3,17-dione ~-Hydroxyandros~1,4-dien-3,17-dione 3,6-Dioxochola-l,4-diene-24-oic acid 3-Oxochol-5-ene-24-oic acid 3-Oxochol-4-ene-24-oic acid 3-Oxochola- 1,4-diene-24-oic acid 6x-Hydroxy-3-oxochol-4ene-24-oic acid 6a-H~drox~-3-oxochoia-l,~iene-Z~oic acid
Yield WJ 0.04
0.11 0.06 0.48 0.04 0.19 0.54 0.20 1.75 1.58 0.33 5.83 1.09
I.360
K.
W.
OWEN
and R. F.
lost the 6x-hydroxyl group present on the substrate molecule. The presence of this class was explained by the isolation of 3-oxochol-5-ene-24-oic acid 10 as an initial intermediate from the fermentation beer. This indicated that the hydroxyl group of HDC was removed fairly early during the anaerobic fermentation and was confirmed by further time course experiments. It is probable that the production of 10 is a result of 6x-dehydroxylation which would involve the removal of the 6a-hydroxyl group and the 5fl-hydrogen atom in a manner analogous to the production of 3~,12a-dihydroxy-5fl-choI-6-ene-24-oic acid after dehydroxylation at C, of cholic acid [24]. The isolation of 3-oxochol-4-ene-24-oic acid 11 from the culture medium suggests that 10 is isomerised at C, in a manner analogous to the reaction carried out by the Pseudomonad during the metabolism of cholesterol [25]. Side chain degradation and nuclearsteroid dehydrogenation at C,& would explain the presence of androsta-l,4-diene-3,17-dione 5 and l7fl-hydroxyandrosta1,4-diene-3-one 6 in the medium. Comparing the anaerobic metabolism of HDC with aerobic metabolism by the Pseudomonad it is apparent that apart from the induction of a 6x-dehydroxylase enzyme the degradative pattern is similar under both conditions in that the majority of the metabolites contain an unsaturated A-ring. However whilst under aerobic conditions the acidic metabolites are dominated by Cz2 steroids, under anaerobic conditions the predominant acidic metabolites are C,, steroids. Thus the pathway involving steroids retaining the substituent at C, (hydroxyl or ketone group) of the parent bile acid appears to be almost identical to that operating under aerobic conditions [5]. The evidence suggests strongly that 3a- hydroxysteroid dehydrogenation precedes nuclear steroid dehydrogenation of the A-ring and that an unsaturated A-ring is a prerequisite for induction of bile acid side-chain degrading enzymes. The mechanism of side-chain cleavage probably occurs by /I-oxidation from a C, acyl-CoA derivative via a CZ1 metabolite through to a C,, androstane compound. However the mechanism may be slightly different in the case of HDC degradation (anaerobic) because Czz steroids were not isolated. This however may be due to retardation of metabolism due to the lack of molecular oxygen which aids the rapid dissimilation of bile acids by bacteria. The second pathway of HDC degradation which becomes operative under anaerobic conditions also appears to proceed in similar manner to that described for other bile acids [7].
Scheme I. Proposed pathway of hyodeoxycholic acid degradation by Pseudomonas sp NCIB 10590 under anaerobic conditions. which also operates during aerobic metabolism 5. Compounds isolated during this study are identified by arabic numerals.
BILTO\
(13)dH
(14) O’H
.$&!fQq 47 $!$?!!_qJ dH
o&
O;l
R2
-/
R2
0
0 (9)
0
OH(8)
OH17)
OH
R, R,
R3
=
Cl-l,
.CH.CH,.CH,.COOH
R,
=
CH,
.CH.COOH
R,
=--OH,-OH
or
=
0
Bacterial
degradation
of hyodeoxycholic
acid
1361
the induction of a 6a-dehydroxylase enzyme enables a second pathway to become operative. On the basis of these results we propose the following pathways (Schemes 1 and 2) for the degradation of HDC by Pseudomonas sp. NCIB 10590 under anaerobic
conditions.
Acknowledgements-R. W. Owen was in receipt of a Liverpool Education Authority Research Assistantship during this study. We are grateful to M. H. Thompson of the PHLS Centre for Applied Microbiology and Research, Bacterial Metabolism Research Laboratory, Porton Down, Wiltshire SP4 OJG, England for the mass spectral analyses and to the Cancer Research Campaign for providing the necessary equipment for such analyses. REFERENCES
Scheme 2. Additional pathway of hyodeoxycholic acid degradation by Pseudomonas sp NCIB 10590 operating only under anaerobic conditions. Compounds isolated during this study
are identified
by arabic
numerals.
Thus in conclusion Pseudomonas sp. NCIB 10590 is capable of extensive degradation of HDC under anaerobic conditions. The mechanism of degradation is similar to that under aerobic conditions; however
S., Fujii T., Saburi Y., and Eguchi T.: I. Hayakawa Microbiological degradation of cholic acid. Nature 179 (4558) (1957) 537-538. 2. ?enn&m G. E., Bilton R. F. and Mason A. N.: The degradation of lithocholic acid by Pseudomonas Sp. NCIB 10590. Febbs Letters 91 (1) (1978) 14G-143. 3. Tenneson M. E., Bilton R. F. and Mason A. N.: The degradation of taurocholic acid and glycocholic acid by Pseudomonas Sp. NCIB 10590. Biochem. Sot. Trans. 6 (1978) 975-977. 4. Tenneson M. E., Baty J. D., Bilton R. F. and Mason A. N.: The degradation of chenodeoxycholic acid by Pserrdomonus Sp. NCIB 10590. J. steroid Biochem. IO (1979) 31 I-316. 5. Tenneson M. E., Baty J. D., Bilton R. F. and Mason A. N.: The degradation of hyodeoxycholic acid by Pseudomonas Sp. NCIB 10590. J. steroid Biochem. 11 (1979) 1227-1232. 6. Tenneson M. E., Baty J. D., Bilton R. F. and Mason A. N.: The degradation of cholic acid by Pseudomonas Sp. NCIB 10590. Biochem. J. 184 (1979) 613-618. I Bilton R. F., Mason A. N. and Tenneson M. E.: Microbial degradation of deoxycholic acid by Pseudomonas Sp. NCIB 10590. Characterisation of products and a postulated pathway. Tetrahedron 37 (1981) 2509-2513. 8 Severina L. O., Torgov 1. V., Skrjabin G. K., Wulfson N. S., Zaretskii V. I. and Papcrnaja 1. B.: The enzymatic transformation of cholic acid by the culture Mycobacterium mucosum 12 10. Tetrahedron 25 ( 1969) 485-491. of steroid 9. Sih C. J. and Wang K. C.: Mechanism oxidation by microorganisms. II-Isolation and characterisation of 3act-H-4a3’-Propionic acid -7@methylhexahydro-I ,5_indanedione. J. Am. Chem. Sot. 85 (1963) 2135-2137. of steroids by 10. Miclo A. and Germain P.: Bioconversion mutants of Nocordia restrictus. C.R. Sot. Biol. 172 (1978) 534541. Il. Severina L. O., Torgov I. V.. Skrjabin G. K.. Wulfson N. S., Zaretskii V. I. and Papernaja 1. B.: Transformation of cholic acid by the culture Mycobacrerium mucosum N 12 10. Tetrahedron 24 (1968)2 145-2 153. of bile salts 12. Hill M. J. and Drasar B. S.: Degradation by human intestinal bacteria. Cur 9 (1968) 22-27. T. and Norman A.: Parameters in 13. Midvedt 7cc-dehydroxylation of bile acids by anaerobic Lactobacilli. Acta Pathol. Microbial. Stand. 72 (1968) 3 13-329. 14. Danielson H., Eneroth P., Hellstrom K., Lindstedt S. and Sjovall J.: On the turnover and excretory products of cholic and chenodeoxycholic acid in man. Bile acids and steroids 134. J. biol. Chem. 238 (7) (1963) 2299-2304.
1362
R. W. OWEN and R. F. Bi~rol\i
15. Aries V. C. and Hill M. J.: Degradation of steroids by intestinal bacteria. II-Enzymes catalysing the oxidoreduction of the 3~(-, 7r- and 12%-hydroxyl groups in cholic acid. and the dehydroxylatjon of the 7-hydroxyi group. Bi~ctfim. hinphys. Aria 202 (1970) 535-543. 16. Owen R. W.: The degradation ofcholic acid by Pseudomonas Sp. NCIB 10590 under anaerobic conditions. PhD Thesis, Council for National Academic Awards (1980) 5473. 17. Owen R. W. The biotransformation of chenodeoxycholic acid by ~~~dorn~~~~ Sp. NCIB 10590 under anaerobic conditions. PhD Thesis, Council for National Academic Awards (1980) 46-54. 18. Kritchevsky D., Martak D. S. and Rothblat G. II.: Anisaldehyde reagent for steroids. An&t. Biochem. 5 (1963)388-392. 19. Bowers A., Halsall T. G., Jones E. R. H. and Lemin A. J.: Triterpenes and related compounds (XVIII): ~iucidation of the structure of polyporenic acid C. J. them. Sot. (1953) 2555-2557.
20. Tenneson M. E., Owen A. W. and Mason A. N. The anaerobic side-chain cleavage of bile acids by k\cherichfu co/i isolated from human faeces. B&hem. SW. Truns. 5 (1977) 1758%1760. of steroids. Cilenz. 21. Dorfman L.: UitravioIet absorption Ret’. 53 (1953) 47-144. 22. Zaretskii Z. V.: Muss Sp~cfrometr~ ofS/eroid.s. Chap. 3 Wiley, New York. (1976). 23. Bridgeman J. E., Cherry P. C., Clegg A. S., Evans J. M.. Jones E. R. H., Kasal A., Meakins G. D., Morisawa Y., Richards E. E., and Woodgate P. D.: Proton magnetic resonance spectra of ketones, alcohols and acetates in the androstane, pregnane and oestrane series. 1. them. Sac. (C) (1970) 250-257. 24. Samuelson 9.: Bile acids and steroids: On the mechanism of the biological formation of deoxycholic acid from cholic acid. J. hiol. Chem. 235 (1960) 361-366. 25. Owen R. W.: The Metabolism of Sterols I-The degradation of cholesterol by Pseudomonus Sp. NCIB 10590 under anaerobic conditions. PhD Thesis, Council for National Academic Awards (1980) 92-100.
1983 Copyright
c
0022-4731/83 $3.00 + 0.00 1983 Pergamon Press Lrd
THE BIOTRANSFORMATION OF HYODEOXYCHOLIC BY PSEUDOMOiVAS SP. NCIB 10590 UNDER ANAEROBIC CONDITIONS
ACID
R. W. OWEN* and R. F. BILTON Department of Chemistry and Bi~hemistry, Liverpool Polytechnic, Liverpool L3 3AF, England (Received 17 May 1982) Summary-The bacterial degradation of hyodeoxycholic acid under anaerobic conditions was studied. The major acidic product has been identified as 6@-hydroxy-3-oxochol-4-ene-24-oic acid whilst the major neutral product has been identified as 6a-hydroxyandrosta-l,4-diene-3,17-dione. The minor acidic products were 3,6-dioxochola-1,4-diene-24-oic acid, 3-oxochol-5-ene-24-oic acid, 3-oxochol-4-ene-24-oic acid, 3-ox~hoia-~,~diene-24-oic acid and ~-hydroxy-3~xochola-1 &diene-24-oic acid and the minor neutral products were androst-4-ene-3,17-dione, androst-4-ene-3,6,1 %trione, androsta- I ,4-diene-3,6,17trione, androsta- 1,4-diene-3,17-dione, 17p-hydroxyandrosta- 1,4-diene-3-one and 6a-hydroxyandrost-4ene-3,17-dione. Evidence is presented which suggests that under aerobic conditions, one pathway of hyodeoxycholic acid metabolism exists whilst under anaerobic conditions an extra biotransformation pathway becomes operative involving the induction of a 6a-dehydroxylase enzyme. A biochemical pathway of hvodeoxvcholic acid metabolism by bacteria under anaerobic conditions is discussed incorporating a scheme involving such an enzym;.
INTRODUCTION
The aerobic degradation of bile acids by bacteria has been studied in detail [l-8]. It has been revealed that the mode of action initially involves oxidation of the 3a-hydroxyl group to a ketone group. Subsequently after nuclearsteroid dehydrogenation of the A-ring to give C,, unsaturated steroids the side-chain is cleaved sequentially to give unsaturated CZ2and C,, metabolites. Under aerobic conditions C,, (androstane) derivatives can be further degraded via aromatic secosteroids to give non-steroidal products [9, IO]. Several studies [8, 1l] have also revealed that oxidation of the B-ring of bile acids carrying a hydroxyl group at C, can also be carried out by bacteria under aerobic conditions thus giving rise to unsaturated bile acids possessing either 4,6-diene-3-one or 1,4,6-trien3-one structures. It is apparent that oxidation of the A-ring precedes oxidation of the B-ring because bile acids with only an oxidised B-ring were not isolated. The low yield of AB-ring unsaturated bile acids produced under aerobic conditions can probably be attributed to the specific requirements for induction of the bacterial enzyme 7ar-dehydroxylase. Bacteria which are able to synthesise this enzyme can remove the hydroxyl group at C, of bile acids [12, 131. Strict anaerobic bacteria of the human intestine in fact have such a potent 7a-dehydroxylase system that the faecal bile acids are almost exclusively deoxycholic
*Present address and to whom requests for reprints should be sent: Public Health Laboratory Service, Centre for Applied Microbiology and Research Bacterial Metabolism Research Laboratory, Porton Down, Salisbury, Wiltshire SP4 OJG, England.
acid (DCA) and lithocholic acid (LA) which are produced by 7a-dehydroxylation of cholic acid (CA) and chenodeoxycholic acid (CDCA) respectively [ 141. Studies on a semi-purified 7a-dehydroxylase from Bacteroides spp. have revealed that for induction to occur strict anaerobiosis is required [15]. This obser-
vation has been compounded by studies involving the degradation of bile acids by Pseudomonas sp. NCIB 10590. Under aerobic conditions, minimal induction of 7u-dehydroxyiase occurs which is reflected by the low yield of AB-ring unsaturated steroids [4,6]. Under anaerobic conditions the induction of the enzyme is such that the major degradative products of CA [16] and CDCA [17] possess a 4,6-dienone structure. The aerobic bacterial metabolism of hyodeoxycholic acid (HDC) has also been studied IS]. The results showed that the degradative pattern is similar to that of other bile acids and that the 6a-hydroxyl group remains intact or else is oxidised to a ketone group. In a study of the anaerobic metabolism of HDC by Pseudomonas sp. NCIB 10590 we present evidence for the operation of an additional pathway which involves a 6u-dehydroxylation step. EXPERIMENTAL HDC, Sa-cholestane and 1,4-androstadiene-3,17dione were obtained from Koch Light Laboratories (Colnbrook, Bucks). General reagents were of “Analar” grade and obtained from BDH and all solvents were redistilled before use. Infra red (i.r.) spectra were determined from KBr discs on a PerkinElmer 457 spectrophotometer. Ultraviolet (u.v.) spectra were determined for solutions in methanol on a
1355
Pye-Unicam SP. 1800 spectrophotometer. Nuclear magnetic resonance (NMR) proton spectra were recorded on a Jeol 220 spectrometer operating at 720 MHz at 3O.C from solutions in deuterated chloroform. Mass spectra were obtained using a DuPont 2 l-491 series mass spectrometer either by direct inlet or by combined CC-MS using a Varian aerograph 2700 gas chromatngraph. Data reduction was performed with a Dupont 2 I-0948 data system. Analysis by gas liquid chromatography (GLC) was performed at 260, C using 3% OV-17 on 80/100 mesh acid washed silanised “supelcoport” in 1.5 M x 3 mm silanised glass columns obtained from Phase Separations (Queensferry, U.K.). Retention times were measured relative to the internal standard Scc-cholestane with a flow rate of 30 ml min ’ nitrogen through the column and 30 ml min _’ H, to the flame ionisation detector in a Hewlett-Packard HP 5470 instrument. Analysis by thin layer chromatography (TLC) was performed on 0.25 mm layers of Kiesel gel GF 254 (obtained from E. Merck, Darmstadt. West Germany) in metha~oi-dichioromethane (1: 19, v/v) and the mobilities were measured relative to androsta- I ,4-diene-3,17-dione. Products containing a 4-ene-3-one or a I ,4-diene-3-one chromophore were detected under U.V. light (254 nm) and all components were finally visualised as characteristically coioured spots by spraying the plates with freshly prepared an~saldehyd~ reagent [lS] and heating in an oven at I 10°C for IO min. As an aid to tentative identification of steroids by TLC oxidation was performed by overspotting with a solution of Jones chromic reagent [Is)] diluted I:4 with acetone. Acetyiation was performed by overspotting with acetyf chloride. Reduction was achieved by overspotting with potassium borohydride reagent (I g KBH,, t ml 2N NaOH, 8 ml H&If. Acidic steroids were methylated with ethereal diazomethane priar to analysis. Trimethylsilyl (TMS) ethers were prepared using h&-trimethylsityl acetamide. The biotransformation medium used in this study contained g/I distilled water: sodium hyodeoxycholate, 1.0: K,HPO,; 0.7; KH?PO+ 0.3; KNOI. 1.0; MgS0,‘7Hz0, 0.1; FeS0,.7H,O, 0.0025; ZnS0,.7Hz0, 0.0025; MnSO,‘4H,O. 0.0025; final pH, 7.2. Solutions of sodium hyodeoxycholate, mineral salts, trace elements and magnesium sulphate were autoclaved separately before mixing. Shakefask cultures were grown on a LH Engineering orbital incubator at 28°C. Cells obtained from 4 x 1L shake-flask cultures of Pxudomoncrs sp. NCIB 10590 by centrifugation at 10,000s on a MSE Mistral 4L centrifuge were used to inoculate 4 1 of the culture medium. This gave a final. cell density of I x 10’ cellsml-’ (OD of I .O at 540 nm). The culture medium was steamed for 30 min and cooled immediately before inoculation. The bottle top of the culture was loosened prior to incubation at 28°C for 6 weeks under 9OU,:,Hz: IO’?<:CO?. The
course of the fermentation and OD,,,, was foIIo\~ced ;LI waeekly intervals. After 6 weeks incubation the tbr.mentation was terminated hy direct extraction of the’ metabotites into an equal volume (twice) of cth>l acetate. After drying over anhydrous MgSO, the solvent was removed in ~YXYJO at 30 C to yield I .4 g of a tarry residue. The residue was taken up in warm dichloromethane (20 ml) and the mixture was sepnrated by preparative thin layer chromatography into a series of fractions from which steroids 2--13 were crystailised. .4nr/rost-4-c~nc-.?, IF’-&no (2). Recrystallisation ot 2 from methanoljdichloromethane gave white powdery crystals (2 mg) m.p. 164 C. This compound was found to be identical to authentic androst-4-ene-3.17” dione in its TLC, GLC and spectroscopic properties. ~77rfrosr-4-enr-.?,6,, IT-rricezr (3). Recrystallisatj~~n of 3 from methatroljdichloromethane gave yellow crystals (5.5 mg) m.p. 218-220 C. p’,,,.,* 1730 (17-ketone), 169 I (6-ketone), 1665 (3-ketone) and IhlOcm ’ (C,-C, double bond); i,,,;,, 248 nm (< 14.380); M ’ 300 (92”,,. C,,H,,O, requires M I, 300) and n?Itz I37 (4ene-3.6-dione. X4”,,): GLC R, 1.65: TLC R, I.?. after oxidation R, I .7. after acetylation R, 1.2 and after reduction R, 0.59. .4rl(/ro.~~(1-l,$-~iirnc~-.~.h,I7-trionr (4). Recrystallisation of 4 from methanol&dichloromethane gave yellow needles (3 mg) m.p. 208-210 ‘C. This compound was found to be identical to androsta1.4-diene-3.6, I?-trione described by Tenneson et N/.[5]. A ndros ta - 1,4-dienc -3. I 7-dione Recrystal(5). lisation of 5 from methanol/dichloromethane gave white prisms (19 mg) m.p. 136--l 37 C. This compound was found to be identical to authentic androsta- 1,4-diene-3,17-dione in its TLC, GLC and spectroscopic properties. 17~~-I~~,dr~ro.~~unrtro.vta-1,4-die~~~-?-onr (6). Recrystallisation of 6 from methanol/dichloromethane gave white needles (2 mg) m.p. 167-168 ‘C. C’,,,,, 3480 (hydroxyl), 1655 (17-ketone), 1655 (3-ketone), 1615 and 1595 cm. ’ (Cl -C1 and C,--C, double bonds); i.,,,,, 242 nm (i 15,220); 6 0.82. 1.24 f6H, 5. l%CH, and 19-CH,), 2.30-2.58 (2H. m. 16CH,). 3.64 (IH. t, J =9 Hz, 17-H), 6.06 (1H, s. 4H), 6.22 (1H. d, showing further splitting- J = ION:, 2-H) and 7.03 (1H. d, J = IO HI. I-H); M’ 2X6 (IS”,,, C,,,H,,O, requires M + 286). it~ji’ 122 ( I .4-diene-3-one, 1OO’,‘,,) and mjc 268 (M ‘-H,O, S’;). GLC K, 1.7: TLC R, 0.68, after oxidation R, 1.OO,after acetylation R, I .U3 and after reduction R, 0.68. &. -~l?~ilro.~~(lrtdro,~t -4cw -3, I7-cliot?r (7). Recrystallisation of 7 from methanol~dichl(~r~?methane gave white crystals (IO mg) m.p. 233’ C. This compound was identical to hr-hydroxyandrost-4-ene-3,17-dionc described by Tenneson et td.[5]. &3(-Iz~~hs~~ - 1,4-mzrlrtlstudirnP-3, I 7-dionr (8). Recrystallisation of 8 from methanol/dichloromethane gave white crystals (28mg) m.p. 240-243 C. This compound was identical in its properties to 6~-hy-
1357
Bacterial degradation of hyodeoxycholic acid droxyandrosta1,4-diene-3,17-dione described by Tenneson et a1.[5]. Methyl 3,6-dioxochola-1,4-diene-24-oate (9). Isolated as a slightly impure solid (8 mg) A,,, 250 nm (5 14,210). M+ 398 (84x, C25H,,0, requires M+ 398) m/e 135 (1,4-diene-3,6-dione, 100%) and m/e 283 (M+-105, 6C-side chain, 53%). GLC R, 15.4; TLC R, 1.23, after oxidation R, 1.23, after acetylation 1.23 and after reduction R, 0,52. Methyl 3-oxochol-5-ene-24-oate (10). Isolated as a crystalline solid from methanol/dichloromethane (70 mg). V,,,,, 1745 (carboxyl), 1702 (3-ketone) and 1670cm-’ (C,& double bond). M+ 386 (34x, C25H3803 requires M+ 386) m/e 124 (5-ene-3-one, 100%) and m/e 271 (M+-115, 6C side-chain, 11%). GLC R, 8.21; TLC R, 1.26, after oxidation R, 1.26, after acetylation Rr 1.26 and after reduction R, 0.92. Methyl 3-oxochol-4-ene-24-oate (11). Recrystallation of 11 from methanol/dichloromethane gave spikey crystals (63 mg) m.p. 141-143°C (free acid). V,,,,, 1746 (carboxyl), 1676 (3-ketone) and 1620 cm-’ (C,-C, double bond). i,,, 241 nm (5 12,700); M+ 386 (65”/, C,,H,,O, requires M+ 386) m/e 124 (Cene3-one, 79”/,) and m/e 271 (M+-I 15, 6C-side chain, 5%); GLC R, 6.20; TLC R, 1.21, after oxidation RI 1.21, after acetylation R, 1.21 and after reduction R, 1.21. Methyl 3-oxochola-1,4-diene-24-oate (12). Recrystallisation of 12 from methanol/dichloromethane gave white crystals (14mg). V,,, 1745 (carboxyl) 1665 (3-ketone) 1610 and 1598cm-’ (C,-C2 and C6C, double bonds); ,l,,, 245 nm; 6 0.79, 1.21 (6H, s, 18-CH, and 19-CH,), 0.93 (3H, d, J = 6 Hz, 21CH,), 3.67 (3H, s, 24-OCH,), 6.24 (IH, d, J = 10 Hz, 2-h), 6.50 (lH, s, 4H) and 7.01 (lH, d, J = 10 Hz); M+ 384 (lo%, C25H3603 requires M+ 384) m/e 122 (1,4-diene-3-one, 100%) and m/e 269 (M+-115, 6C-side chain, 14%). GLC R,8.70; TLC R, 1.18, after oxidation R, 1.18, after acetylation R, 1.18 and after reduction R, 1.18. Methyl &-hydroxy-3-oxochol-4-ene-24-oate (13). Recrystallisation of 13 from methanol-dichloromethane gave white prisms (233 mg) mp 184185°C. VIll&X3465 (hydroxyl), 1722 (carboxyl), 1666 (3-ketone) and 1654 (C4C, double bond; A,,,,, 242 nm; 6 0.69, 1.18 (6H, s, 18-CH, and 19-CH,) 0.92 (3H, d, J = 6 Hz, 21-CH,), 3.67 (3 H, 2, 24-OCH,), 4.35 (lH, M, 6H), and 6.16 (lH, s, 4-H). M+ 402 (100% C,,H,,O, requires M + 402) m/e 139, 140 (4-ene-3-one-6-01, 44 and 27%) m/e 269 (M +-133, 6C-side chain + 6-OH, 17%) and m/e 384 (M+-18, 6-OH, 32%). GLC R, 10.1; TLC R, 0.65, after oxidation R, 1.32, after acetylation R, 1.27 and after reduction R, 0.65. Methyl &-hydroxy-3-oxochola-1,4-diene-24-oate (14). Recrystallisation from methanol/dichloromethane gave white crystals (44 mg) m.p. 142-144°C (free acid); V,,,,, 3415 (hydroxyl), 1736 (carboxyl), 1668 (3-ketone), 1624 and 1610cm-’ (C,&, and C,-C, double bonds). i.,,, 245 nm. 6 0.72, 1.20 (6H,
s, 18-CH, and 19-CH,), 0.90 (3H, d, J = 6Hz, 21-CH,), 3.66 (3H, s, 24-OCH,), 4.46 (lH, m, 6-OH), 6.23 (lH, d, showing further splitting, J = 10 Hz, 2-H), 6.46 (lH, s, 4-H), 6.98 (lH, d, J = 10 Hz, 1-H). M+ 400 (90x, C,,H,,04 requires M + 400) m/e 138 (1,4-diene-3-one-6-01, 100%) m/e 267 (MC-133, 6C-side chain +6-OH) and m/e 382 (M+-18, 6-OH, 26%). GLC R, 10.4; TLC R, 0.49, after oxidation R, 1.23, after acetylation R, 1.19 and after reduction R, 0.49. RESULTS
The degradation of HDC (1) under aerobic conditions by Pseudomonas sp. NCIB 10590 has been observed by Tenneson et aZ.[5] who reported the production of C-22 (bisnor) acidic and C-19 (androstane) intermediates. A limited study of HDC metabolism under anaerobic conditions has also been carried out with an Escherichia coli. strain [20]. However, the anaerobic transformation of HDC by bacteria has not been studied in detail. A 4-l anaerobic fermentation of sodium hyodeoxycholate by Pseudomonas sp. NCIB 10590 enabled the isolation of thirteen metabolites from the fermentation beer. The assigned structures are shown in Figs 1 and 2. The major neutral metabolite (8) and three (4, 5 and 6) of the minor neutral metabolites gave i.r. spectra typical of 1,4-diene-3-one A-ring steroids (e.g.
o& oq!p o
(2)
(3)
oJ3p o& // & / J3P // & (5)
0 (4)
0
?H
0
0
(6)
bH (7)
0
0
O’H
(8) Fig. 1. Neutral metabolites isolated after the anaerobic degradation of hyodeoxycholic acid by pseudomonas sp. NCIB 10590.
R. W. OWEN and R. F.
R-CH3CHCH2CH2COOH
o@ J4? R
R
o& od+ (I II
(121
dH
dH
(13)
(141
Fig. 2. Acidic metabolites isolated after the anaerobic degradation of hyodeoxycholic acid by pseudomonas sp.
NCIB 10590.
i.r. spectrum of (6))1655. 1615 and 1595cm ‘, x/?-unsaturated ketone). The U.V. spectra of 5, 6 and 8 supported this observation (i,,,, 244 nm, di-[Isubstituted Q-Unsaturated ketone in a sixmembered ring, double bond exocyclic) while the U.V. 250 nm di-/I-substituted z. spectrum of 4 (I,,,, p-unsaturated ketone in a six-membered ring, double bond exocyclic, extended by a carbonyl group at C,) was typical of a 1,4-diene-3,6-dione structure [21]. Further spectroscopic study of compounds 4 and 8 revealed that they were identical to androsta-I ,4diene-3,6,17-trione and ha-hydroxyandrostaI .4diene-3,17-dione respectively which have been described in detail by Tenneson ef a/.[5]. Compounds 5 and 6 were found to be identical in their spectroscopic and mass spectral properties to authentic androsta- 1,4-diene-3,17-dione and I 7/Ihydroxyandrosta1,4-diene-3,17-dione respectively. This showed that the 6a-hydroxyl group of HDC had been lost during anaerobic fermentation to these products. Metabolites 2, 3 and 7 gave i.r. spectra typical of 4-ene-3-one A-ring steroids (e.g. i.r. spectrum of 2-1666 and 1616 cm-‘, a$-unsaturated ketone). The U.V. spectra of 2 and 7 supported this observation (&,,,, 242 nm, di+‘-substituted a. b-unsaturated ketone in a six-membered ring. double bond exocyclic) [2l] whilst the U.V. spectrum of 3 (I.,,,,,, 248 nm was typical for a 4-ene-3,6-dione structure [2l].
BILTON
Further study of 2 and 7 including mass spectral analyses revealed that 2 was identical to authentrc androst-4-ene-3.17-dione whilst 7 was identical to br-hydroxyandrost-4-ene-3,17-dione described by Tenneson CI ul. [5]. Reactions on TLC revealed that compound 3 could not be oxidised or acetylated but was readily reduced suggesting the absence of hydroxyl groups and the presence of free ketone groups. This was contirmed by the i.r. spectrum which displayed two peaks in the free carbonyl group region (1730 and 1691 cm corresponding to a l7-ketone group and a 6-ketone group respectively) with none in the hydroxyl group region. Sufficient of compound 3 was not available for PMR analysis; however the mass spectrum revealed a molecular ion at m/c> 300 (M +. 92”,,) and an intense ion at m/e 137 (84’3;,) confirming a 4-ene-3,6-dione structure [22]. Compound 3 is therefore assigned the structure androst-4-en-3.6. I7trione. One major (13) and five minor (9-12, 14) acidic metabolites were isolated. Structural analyses were carried out on the methyl ester derivatives. Compounds 9, 12 and 14 gave i.r. spectra typical of I .4-diene-3-one A-ring steroids. The U.V. spectra 01 12 and 14 supported this observation (i,,,, 244nm) whilst the U.V. spectrum of 9 (j .“,.,,. 250 nm) was typical for a 1,4-diene-3,6-dione structure (21). TLC reactions revealed that the oxidation product of 14 corresponded in colour (orange) and R, to 9 whilst 9 and 12 could not be oxidised. The reduction product of 9 was slightly less polar than 14 on TLC using potassium borohydride as reducing agent; however reduction with sodium borohydride gave two reduction products, one of which was identical to the potassium borohydride product. the other corresponding in colour (mauveegrey) and R, to 14 on TLC. 12 and 14 could not be reduced. Compound 14 was easily acetylated but 9 and 12 could not be acetylated on TLC. This indicated that compound 9 did not possess a hydroxyl group only a free ketone group, compound 14 possessed a hydroxyl group but not an unconjugated ketone group whilst compound 12 did not possess either. This was confirmed by the i.r. spectra. The position and stereochemistry of the hydroxyl group carried by compound 14 was assigned from the PMR spectrum. In addition to 3 vinylic protons in the range 6.2336.98 6 corroborating a I .4-diene-3-one A-ring structure, 14 gave rise to a broad multiplet centred at 4.46 6 corresponding to one proton. The dihedral (karplus angles) for the 6[I-proton and the two protons at C, are about 60 and 180 C at C,. On this basis the hydroxyl group in compound I4 was assigned the 6r-configuration [23]. The mass spectrum of 14 revealed an intense molecular ion at m/e 400 (M +, 90”,,), an intense ion (base peak) at m/e 138 (lOOo/,); an intense ion at m:‘r 282 (M +- 18,26%, 6a -hydroxyl group) and an intense ion at m/c 267 (M ‘-133. 42”,J due to the loss of the
Bacterial degradation of hyodeoxycholic acid free bile acid side-chain water (6-OH).
(6C) and the elements
of
The mass spectrum of 9 gave an intense molecular ion at m/e 398 (M+ 84x), an intense ion (base peak) at m/e 135 ( 100°/O) which corresponded to a 1,4-diene-3,6-dione structure and an intense ion at m/e 283 (M+-115, 53%) and to the loss of the full bile acid side chain (6C). The mass spectrum of 12 gave a low intensity molecular ion at m/e 384 (M+, IO%), an intense ion at m/e 122 (100%) and a low intensity ion at m/e 269 (M+ 115, 6C-side chain, 14%). On the basis of the above data compound 9 was assigned the structure 3,6-dioxochola-1,4-diene-24oic acid, compound 12 was assigned the structure 3-oxo-chola-1,4-diene-24-oic acid and compound 14 was assigned the structure 6~-hydroxy-3-oxo~hola1,4-diene-24-oic acid. Of the three remaining acidic intermediates, 11 and 13 gave i.r. spectra typical of 4-ene-3-one, A-ring steroids. The U.V. spectra (La,, 242nm) supported this observation. Further support came from the PMR spectrum of 13; one vinylic proton at 6.16 6. Reactions on TLC revealed that 13 possessed a hydroxyl group but not a free ketone group. This was confirmed by the i.r. spectrum which displayed a peak at 3465 cm-’ in the hydroxyl group region. However 11 did not appear to possess either a hydroxyl group or a free ketone group by its reactions on TLC. This was confined by the i.r. spectrum. The position and stereochemistry of the hydroxyl group in 13 was elucidated from the PMR spectrum. In addition to one vinylic proton at 6.16 6 (corroborating a 4-ene-3-one structure), 13 gave rise to a broad multiplet centred at 4.35 6 typical of a G/J-proton (23). On this basis the hydroxyl group in 13 was assigned the &-position. The mass spectrum of 13 displayed a base peak molecular ion at m/e 402 (M+, 100x), low intensity ions at m/e 139 and 140 (44% 27%) corresponding to a 4-ene-3-one-6-01 structure, a low intensity ion at m/e 384 (M+-18, 32% 6u-hydroxyl group) and a low intensity ion at m/e 269 (M+-133, 6C side-chain + 6OH, 17%).
1359
The mass spectrum of 11 displayed an intense molecular ion at m/e 386 (M+, 65’/J, an intense ion at m/e 124 (790/,) typicai of a 4-ene-3-one, A-ring steroid and a low intensity ion at m/e 271 (5%) due to the loss of the full bile acid side-chain (6C). On the basis of the above data compound 11 and compound 13 were assigned the structures 3-0x0chol-4-ene-24-oic acid and 6a -hydroxy-3-oxochol-5ene-24oic acid respectively. The mass spectrum of the remaining acidic metabolite 10 gave an intense (base peak) ion at m/e 124 (100%) which is typical for 4-ene-fone, A-ring steroids. However this was not confirmed by the U.V. and i.r. spectra. Compound 10 could not be oxidised or acetylated, but could be reduced to a compound identical in colour (blue) and R,A,, to 3/I-hydroxy3-ox~hol-5-ene-24-oic acid. Furthermore the reduced product could be oxidised to a compound identical in colour (rust-orange) and Rf,D,, to 3-oxochol-4-ene-24-oic acid. The mass spectrum gave, in addition to the base peak at m/e 124, a low intensity molecular ion at m/e 386 (M +, 34%) and a low intensity ion at m/e 271 (M+-1 IS, 6C side-chain, 11%). On the basis of the above data compound IO has been tentatively assigned the structure 3-oxochol-5-ene-24-oic acid. The yield of the steroidal metabolites isolated is listed in Table 1. DISCUSSION
The results show that a resting cell suspension of Pseudomonas sp. NCIB 10590 is capable of extensive degradation of HDC under anaerobic conditions. Tenneson et al.]51 reported that the aerobic metabolites produced from HDC by Pseadomu~as sp. NCIB 10590 were predominantly 1,Cdienone steroids retaining the hydroxyl group at C,. Under anaerobic conditions the products again were dominated by 1,4-dienone steroids; however, in addition another class of compounds were present at significant levels. The second class of compounds were also mainly 1,4-dienone steroids but they had
Table 1. Yield of metabolites with respect to starting material after 6 weeks anaerobic incubation Metabolite Number L
3 4 6
8 9 10 11 12 13 14
Metabolite Androst-4-en-3,17-dione Androst-4-en-3,6,17-trione Androsta- 1,4-dien-3,6,17-trione Androsta-1,4-dien-3,17-dione 17@-Hydroxyandrosta-1,4dien-3-one 6a-Hydroxyandrost-4-en-3,17-dione ~-Hydroxyandros~1,4-dien-3,17-dione 3,6-Dioxochola-l,4-diene-24-oic acid 3-Oxochol-5-ene-24-oic acid 3-Oxochol-4-ene-24-oic acid 3-Oxochola- 1,4-diene-24-oic acid 6x-Hydroxy-3-oxochol-4ene-24-oic acid 6a-H~drox~-3-oxochoia-l,~iene-Z~oic acid
Yield WJ 0.04
0.11 0.06 0.48 0.04 0.19 0.54 0.20 1.75 1.58 0.33 5.83 1.09
I.360
K.
W.
OWEN
and R. F.
lost the 6x-hydroxyl group present on the substrate molecule. The presence of this class was explained by the isolation of 3-oxochol-5-ene-24-oic acid 10 as an initial intermediate from the fermentation beer. This indicated that the hydroxyl group of HDC was removed fairly early during the anaerobic fermentation and was confirmed by further time course experiments. It is probable that the production of 10 is a result of 6x-dehydroxylation which would involve the removal of the 6a-hydroxyl group and the 5fl-hydrogen atom in a manner analogous to the production of 3~,12a-dihydroxy-5fl-choI-6-ene-24-oic acid after dehydroxylation at C, of cholic acid [24]. The isolation of 3-oxochol-4-ene-24-oic acid 11 from the culture medium suggests that 10 is isomerised at C, in a manner analogous to the reaction carried out by the Pseudomonad during the metabolism of cholesterol [25]. Side chain degradation and nuclearsteroid dehydrogenation at C,& would explain the presence of androsta-l,4-diene-3,17-dione 5 and l7fl-hydroxyandrosta1,4-diene-3-one 6 in the medium. Comparing the anaerobic metabolism of HDC with aerobic metabolism by the Pseudomonad it is apparent that apart from the induction of a 6x-dehydroxylase enzyme the degradative pattern is similar under both conditions in that the majority of the metabolites contain an unsaturated A-ring. However whilst under aerobic conditions the acidic metabolites are dominated by Cz2 steroids, under anaerobic conditions the predominant acidic metabolites are C,, steroids. Thus the pathway involving steroids retaining the substituent at C, (hydroxyl or ketone group) of the parent bile acid appears to be almost identical to that operating under aerobic conditions [5]. The evidence suggests strongly that 3a- hydroxysteroid dehydrogenation precedes nuclear steroid dehydrogenation of the A-ring and that an unsaturated A-ring is a prerequisite for induction of bile acid side-chain degrading enzymes. The mechanism of side-chain cleavage probably occurs by /I-oxidation from a C, acyl-CoA derivative via a CZ1 metabolite through to a C,, androstane compound. However the mechanism may be slightly different in the case of HDC degradation (anaerobic) because Czz steroids were not isolated. This however may be due to retardation of metabolism due to the lack of molecular oxygen which aids the rapid dissimilation of bile acids by bacteria. The second pathway of HDC degradation which becomes operative under anaerobic conditions also appears to proceed in similar manner to that described for other bile acids [7].
Scheme I. Proposed pathway of hyodeoxycholic acid degradation by Pseudomonas sp NCIB 10590 under anaerobic conditions. which also operates during aerobic metabolism 5. Compounds isolated during this study are identified by arabic numerals.
BILTO\
(13)dH
(14) O’H
.$&!fQq 47 $!$?!!_qJ dH
o&
O;l
R2
-/
R2
0
0 (9)
0
OH(8)
OH17)
OH
R, R,
R3
=
Cl-l,
.CH.CH,.CH,.COOH
R,
=
CH,
.CH.COOH
R,
=--OH,-OH
or
=
0
Bacterial
degradation
of hyodeoxycholic
acid
1361
the induction of a 6a-dehydroxylase enzyme enables a second pathway to become operative. On the basis of these results we propose the following pathways (Schemes 1 and 2) for the degradation of HDC by Pseudomonas sp. NCIB 10590 under anaerobic
conditions.
Acknowledgements-R. W. Owen was in receipt of a Liverpool Education Authority Research Assistantship during this study. We are grateful to M. H. Thompson of the PHLS Centre for Applied Microbiology and Research, Bacterial Metabolism Research Laboratory, Porton Down, Wiltshire SP4 OJG, England for the mass spectral analyses and to the Cancer Research Campaign for providing the necessary equipment for such analyses. REFERENCES
Scheme 2. Additional pathway of hyodeoxycholic acid degradation by Pseudomonas sp NCIB 10590 operating only under anaerobic conditions. Compounds isolated during this study
are identified
by arabic
numerals.
Thus in conclusion Pseudomonas sp. NCIB 10590 is capable of extensive degradation of HDC under anaerobic conditions. The mechanism of degradation is similar to that under aerobic conditions; however
S., Fujii T., Saburi Y., and Eguchi T.: I. Hayakawa Microbiological degradation of cholic acid. Nature 179 (4558) (1957) 537-538. 2. ?enn&m G. E., Bilton R. F. and Mason A. N.: The degradation of lithocholic acid by Pseudomonas Sp. NCIB 10590. Febbs Letters 91 (1) (1978) 14G-143. 3. Tenneson M. E., Bilton R. F. and Mason A. N.: The degradation of taurocholic acid and glycocholic acid by Pseudomonas Sp. NCIB 10590. Biochem. Sot. Trans. 6 (1978) 975-977. 4. Tenneson M. E., Baty J. D., Bilton R. F. and Mason A. N.: The degradation of chenodeoxycholic acid by Pserrdomonus Sp. NCIB 10590. J. steroid Biochem. IO (1979) 31 I-316. 5. Tenneson M. E., Baty J. D., Bilton R. F. and Mason A. N.: The degradation of hyodeoxycholic acid by Pseudomonas Sp. NCIB 10590. J. steroid Biochem. 11 (1979) 1227-1232. 6. Tenneson M. E., Baty J. D., Bilton R. F. and Mason A. N.: The degradation of cholic acid by Pseudomonas Sp. NCIB 10590. Biochem. J. 184 (1979) 613-618. I Bilton R. F., Mason A. N. and Tenneson M. E.: Microbial degradation of deoxycholic acid by Pseudomonas Sp. NCIB 10590. Characterisation of products and a postulated pathway. Tetrahedron 37 (1981) 2509-2513. 8 Severina L. O., Torgov 1. V., Skrjabin G. K., Wulfson N. S., Zaretskii V. I. and Papcrnaja 1. B.: The enzymatic transformation of cholic acid by the culture Mycobacterium mucosum 12 10. Tetrahedron 25 ( 1969) 485-491. of steroid 9. Sih C. J. and Wang K. C.: Mechanism oxidation by microorganisms. II-Isolation and characterisation of 3act-H-4a3’-Propionic acid -7@methylhexahydro-I ,5_indanedione. J. Am. Chem. Sot. 85 (1963) 2135-2137. of steroids by 10. Miclo A. and Germain P.: Bioconversion mutants of Nocordia restrictus. C.R. Sot. Biol. 172 (1978) 534541. Il. Severina L. O., Torgov I. V.. Skrjabin G. K.. Wulfson N. S., Zaretskii V. I. and Papernaja 1. B.: Transformation of cholic acid by the culture Mycobacrerium mucosum N 12 10. Tetrahedron 24 (1968)2 145-2 153. of bile salts 12. Hill M. J. and Drasar B. S.: Degradation by human intestinal bacteria. Cur 9 (1968) 22-27. T. and Norman A.: Parameters in 13. Midvedt 7cc-dehydroxylation of bile acids by anaerobic Lactobacilli. Acta Pathol. Microbial. Stand. 72 (1968) 3 13-329. 14. Danielson H., Eneroth P., Hellstrom K., Lindstedt S. and Sjovall J.: On the turnover and excretory products of cholic and chenodeoxycholic acid in man. Bile acids and steroids 134. J. biol. Chem. 238 (7) (1963) 2299-2304.
1362
R. W. OWEN and R. F. Bi~rol\i
15. Aries V. C. and Hill M. J.: Degradation of steroids by intestinal bacteria. II-Enzymes catalysing the oxidoreduction of the 3~(-, 7r- and 12%-hydroxyl groups in cholic acid. and the dehydroxylatjon of the 7-hydroxyi group. Bi~ctfim. hinphys. Aria 202 (1970) 535-543. 16. Owen R. W.: The degradation ofcholic acid by Pseudomonas Sp. NCIB 10590 under anaerobic conditions. PhD Thesis, Council for National Academic Awards (1980) 5473. 17. Owen R. W. The biotransformation of chenodeoxycholic acid by ~~~dorn~~~~ Sp. NCIB 10590 under anaerobic conditions. PhD Thesis, Council for National Academic Awards (1980) 46-54. 18. Kritchevsky D., Martak D. S. and Rothblat G. II.: Anisaldehyde reagent for steroids. An&t. Biochem. 5 (1963)388-392. 19. Bowers A., Halsall T. G., Jones E. R. H. and Lemin A. J.: Triterpenes and related compounds (XVIII): ~iucidation of the structure of polyporenic acid C. J. them. Sot. (1953) 2555-2557.
20. Tenneson M. E., Owen A. W. and Mason A. N. The anaerobic side-chain cleavage of bile acids by k\cherichfu co/i isolated from human faeces. B&hem. SW. Truns. 5 (1977) 1758%1760. of steroids. Cilenz. 21. Dorfman L.: UitravioIet absorption Ret’. 53 (1953) 47-144. 22. Zaretskii Z. V.: Muss Sp~cfrometr~ ofS/eroid.s. Chap. 3 Wiley, New York. (1976). 23. Bridgeman J. E., Cherry P. C., Clegg A. S., Evans J. M.. Jones E. R. H., Kasal A., Meakins G. D., Morisawa Y., Richards E. E., and Woodgate P. D.: Proton magnetic resonance spectra of ketones, alcohols and acetates in the androstane, pregnane and oestrane series. 1. them. Sac. (C) (1970) 250-257. 24. Samuelson 9.: Bile acids and steroids: On the mechanism of the biological formation of deoxycholic acid from cholic acid. J. hiol. Chem. 235 (1960) 361-366. 25. Owen R. W.: The Metabolism of Sterols I-The degradation of cholesterol by Pseudomonus Sp. NCIB 10590 under anaerobic conditions. PhD Thesis, Council for National Academic Awards (1980) 92-100.