THE DEGRADATION OF CHENODEOXYCHOLIC ACID BY PSEUDOMONAS Spp. N.C.I.B. 10590 M. E. TnNNasoNt, J. D. BATY,* R. F. BILTONand A. N. MASON Department of Chemistry and Biochemistry, Liverpool Polytechnic, England and *Department of Biochemical Medicine, University of Dundee, Scotland (Received 7 Jury 1978) SUMMARY The microbial degradation of chenodeoxycholic acid by Pseudomonasspp. N.C.I.B. 10590 has been studied. Two major products have been isolated and identified as 7a-hydroxy-Wandrostadiene-3,17dione and 7x-hydroxy-3-oxo-l,4-pregnadiene-2O-carboxylic acid. Two minor products were isolated and evidence is given for the following structures: 1.46androstatriene-3,17-dione and 7z-hydroxy-3-0x0-4pregnene-20-carboxylic acid. A possible pathway of chenodeoxycholic acid degradation is suggested.

INTRODUCTION

The microbial degradation of bile acids has been implicated in the aetiology of colon [l] and breast [2] cancer and is potentially of importance in the commercial production of physiologically active steroids [3]. Reports on the microbial degradation of chenodeoxycholic acid have been mainly limited to observations of dehydroxylation and oxidation and subsequent reduction of hydroxyl groups. The production of lithocholic acid from chenodeoxycholic acid by 7a-dehydroxylation has been reported with many species of Bacteroides, Bijdobacteria. Clostridia, Streptococci and Veillonelia isolated from human faeces [4,5] and with Lactobacillae spp. [6] and Bacteroides Fragilis [7]. A series of investigations with a selection of bacteria isolated from the rat intestine [6,8,9] has shown that many such bacteria have the ability to oxidise and reduce the 3a and 7a hydroxyl groups. Isolated products include 7a-hydroxy-3-oxo-5/I-choIan-24-oic acid [8], 3a-hydroxy-7-oxo-S/Lcholan-24oic acid [6-93, 3,7dioxo-SB-cholan-24oic acid [6,8] and 3/?,7adihydroxy-5)!I-cholan-24-oic acid [8,9]. Oxidation through both a 3-0~0 and 7-0~0 derivative followed by reduction back to chenodeoxycholic acid has also been observed [6]. A strain of Escherichia coli isolated from a faecal sample of a colon cancer patient has the ability to degrade chenodeoxycholic acid under anaerobic conditions [lo]. This is the only reported observation of side-chain cleavage of chenodeoxycholic acid. In this paper we present evidence for the structure and configuration of the two main side-chain cleavage products (2 and 4) isolated during the aerobic degradation of chenodeoxycholic acid by Pseudomonas spp. N.C.I.B. 10590. Two minor components have been t Present address : International Development Laboratory, E. R. Squibb & Sons Ltd., Reeds Lane, Moreton, Merseyside, England.

isolated and their structures are discuseed. Th: &!ation of these metabolites has enabled the postulation of a pathway of chenodeoxycholic acid degradation.

EXPERIMENTAL Chenodeoxycholic acid, Sa-cholestane and 1,4androstadiene-3,17-dione were obtained from Koch Light. General reagents were of Analar grade and

obtained from B.D.H. and all solvents were redistilled before use. Melting points were determined using a Kofler hotstage apparatus and are uncorrected. Elemental analyses were determined by the Butterworth Microanalytical Consultancy. Infra red (I.R.) spectra were determined from KBr discs on a Perkin-Elmer 457 spectrophotometer. Ultraviolet (U.V.) spectra were determined for solutions in methanol on a PyeUnicarn SP 1800 spectrophotometer. Nuclear magnetic resonance (n.m.r.) proton spectra were recorded on a Varian HA 100 spectrometer from solutions in deuterated chloroform. Mass spectra were recorded on an A.E.I. MS 12 spectrometer. Analysis by gas chromatography was performed at 260” using 3% OV-17 on 80/100 mesh “Supelcoport” in a 1.5 m x 3mm column obtained from Phase Separations. Retention times were measured relative to 5a-cholestane with a flow rate of 30ml min- ’ nitrogen in a Hewlett-Packard HP 5470 instrument. Analysis by thin layer chromatography (t.1.c.) was performed on 0.25mm layers of Kieselgel GFzs4, obtained from Merck, in methanoldichloromethane (1: 9, V/V) and the mobilities were measured relative to l&androstadiene-3,17dione. Products containing a 4-en-3-one or a l&hen-3-one structure were detected under U.V. light; other products were detected by their colour with anisaldehyde reagent [ll]. Purification was achieved by preparative t.1.c. on Kieselgel GFw as above. 311

312

M. E. TENNEWN,J. D. BATY, R. F. BILTONand A. N. MASON

Oxidation was performed on t.1.c. by overspotting with a solution of Jones’ chromic reagent [12] diluted 1:4 with acetone. Acetylation was performed by overspotting with acetyl chloride. Reduction was performed by overspotting with potassium borohydride reagent. Acidic steroids were methylated with diazomethane. The culture medium consisted of sodium chenodeoxycholate (l.Og); K2HPOb (1.6g), KH2P04 (0.4g), KN03 (1.0 g) (mineral salts); FeSO, . 7H20 (2.5 mg), ZnS04. 7Hz0 (2.5 mg), MnS04 .4H20 (2.5 mg) (trace elements); MgS04.7Hz0 (0.1 g) and distilled water to I 1. (pH 7.2). Solutions of sodium chenodeoxycholate, mineral salts, trace elements and magnesium sulphate were autoclaved separately before mixing Shake-flask cultures were grown on an L.H. Engineering orbital incubator and the ten litre culture was grown in an L.H. Engineering fermenter with constant aeration of 2 litres min-’ and stirring at 200 rev/min- ‘. The incubation temperature was 28°C at all times. The ceils obtained from a one litre shake-flask culture of PseudomoMs spp. N.C.I.B. 10590 by centrifugation at 10,OOOgon an MSE Mistral 4L centrifuge were used to inoculate 101 of the culture medium. The course of the transformation was followed by sampling the culture at 1 h intervals. The cell density was calculated from the absorbance of the culture at 54Onm. Filtration of the samples through a 0.45pm Millipore filter enabled the direct determination of the absorbance at 252 mn of the steroidal mixture. Extraction of the samples with ethyl acetate followed by t.1.c. and gas chromatography analysis enabled determinations of the relative concentrations of chenodeoxycholic acid, the major acidic and the major neutral steroids. When the absorbance at 252nm reached a maximum, after 14 h, the culture was terminated by direct extraction of the metabolites into ethyl acetate (3 x 1 litre). After drying over MgS04 the solvent was removed under reduced pressure at 50°C to yield 3.7g of a tarry residue. The residue was then taken up in warm dichloromethane (20 ml), which on cooling gave a white precipitate of unchanged chenodeoxycholic acid (200 mg). The remaining mixture was separated by preparative t.1.c. into a series of fractions from which steroids 2. 3, 4 and 5 were crystallized. 7sc-hydroxy-l,4-androstadiene-3,17-dione (2). Recrystallisation of 2 from methanol/dichloromethane yielded white prisms (22 mg) m.p. 238-239°C. (Found: C, 75.87; H, 7.90. C1sH2,0J requires: C, 76.00; H, 8.ooo/,). v,,, 3325 (hydroxyl), 1738 (17-ketone), 1655 (fketone), 1612 and 1598cm-’ (C1-C2 and Cb-Cs double bonds); LX 244 nm (el4,850); 60.90, 1.22 (6H, s, 18-CHJ and 19-CHJ), 2.50-2.70 (2H, m, 16-CH2), 4.10 (1 H, s, 7/?-H), 6.08 (lH, s, slight ‘splitting, 4-H), 6.17 (lH, d, showing further splitting, .J = 10 H& 2-H) and 7.01 (lH, d, J = 10 HZ, 1-H); M+300 (C~~HUGJ requires M +300), m/u 121 (I&dien-fone), m/e 150 (1,4-dien-3-one-7-01) and m/e 282 (M+-H,O). GLC RF

3.0; t.1.c. R, 0.76, after oxidation RF 1.02, after acetylation RF 1.01 and after reduction RF 0.46. ’ 1,4,6-androstatriene-3,17-dione (3). Recrystallisation of 3 from methanol/dichloromethane yielded white needles (7 mg) m.p. 212-214°C. vmax1735 (lFketone), 1648 (3-ketone), 1622, 1602 and 1576 cm-’ (Ci-C&-Cs and C6-C, double bonds); A,,,,, 224, 256 and 300nm (~12,800, 12,120 and 13,850); M+ 282 (Ci9HZZ02 requires M+ 282) and m/e 134 (1,4,6trien-3-one). GLC RF 1.5; t.1.c. RF 0.98, after oxidation RF 0.98, after acetylation R, 0.98 and after reduction RF 0.86. Methyl 7x-hydroxy-3-oxo-l&pregnadiene-20-oate (methyl ester of 4). Recrystalhsation of methyl 4 from methanol/dichloromethane yielded white prisms (16mg) m.p. 282-284°C (Found: C, 74.11; H, 8.61. C23H3204 requires: C, 74.20; H, 8.60%). v,,, 3532 (hydroxyl), 1722(carboxyl), 1652(3-ketone), 1610 and 16OOcm-’ (C,CI and C4C5 double bonds); &,,, 244nm (~14,835); 60.79, 1.19 (6H, s, 18-CHs and 19-CH3), 1.22 (3H, d, J = 6Hz, 21-CH3), 3.68 (3H, s, 22-0CH3), 4.08 (lH, s, 7/I-H), 6.06 (lH, s, slight splitting, 4-H) 6.16 (lH, d, showing further splitting, .J = lOHz, 2-H) and 7.00 (lH, d, J = lOHz, 1-H): M+ 372 (C23H320h requires M+ 372), m/e 121 (1,4-dien-3-one), m/e 150 (l&dien-3-one-7-ol), m/e 267 (M+-side-chain + H20) and m/e 354 (M+-H*O). GLC RF 7.5; t.1.c. RF 0.80, after oxidation R, 1.04. after acetylation R, 1.09 and after reduction R, 0.80. Methyl 7r-hydroxy-3-oxo-4-pregnene-20-oate (methyl ester of 5). Recrystallisation of methyl 5 from methanol/dichloromethane yielded white prisms (5 mg) m.p. 275-277°C. v,,, 3535 (hydroxyl), 1725 (carboxyl), 1647 (3-ketone) and 1610 (Cd-C5 double bond); A,,,,, 241 nm (~15,290); a.79, 1.22 (6H, s, ‘18-CH3 and 19-CH& 1.24 (3H, d, J = 6 Hz, 21-CHS), 3.69 (3H, s, 22-OCH& 4.05 (lH, s, 7/I-H) and 6.15 (lH, s, 4-H); M+ 374 (C2JH3404 requires M+ 374), mJe 124 (4-en-3-one), m/e 152 (4-en-3-one-7-ol), m/e 269 (M+-side-chain + H20) and m/e 356 (M+-H20). GLC RF 6.5; t.1.c. RF 0.86, after oxidation RF 1.08, after acetylation RF 1.12 and after reduction RF 0.86. Phenolic compounds. A crude mixture of compounds remained after crystallisation of the steroidal metabolites. A,,,,, 218 and 275 nm (methanol), 220 and 298 nm (NaOH methanol). GLC two main metabolites RF 0.2 and 0.8; t.1.c. two main metabolites RF 0.79 and 0.70; other metabohtes RF 0.83 and 0.42. RESULTS

Pseudomonas spp. N.C.I.B. 10590 grew rapidly on sodium chenodeoxycholate, in a mineral salts medium. The course of the transformation was followed by measurement of the increase in both cell density and the concentration of l&dien-3-oxo steroids (Fig. 1). The growth of the Pseudomonas species on sodium chenodeoxycholate showed a typical lag, log and stationary phase, while the concentration of 1,4-dien-3-oxo steroids in the medium

Degradation of chenodeoxychohc

I

0

I

I

I

I

4

I

I

I

I 6

I

I

Time

I

I 12

I

I

I

I I 16

313

acid

11

11 20

11

1 3

(hard

Fig. I. The relationship between cell density (O--O) and concentration of l,4-dien-3-oxo steroid (A-A) during the oxidation of chenodeoxycholic acid (I) by Pseudowonas spp. N.C.I.B. 10590.

showed a maximum after 14 h. The concentration of chenodeoxycholic acid was shown to decrease with time, w’hereas the concentration of the major acidic and neutral 1,4dien3-oxo steroids reached a maximum after 13 h and 16 h respectively (Fig. 2). The metabolites isolated after 14 h transformation of chenodeoxycholic acid (1) are shown in Fig. 3. The major neutral compound (2) was isolated as a crystalline solid the mass spectrum of which shows a’ molecular ion at m/e 300 and intense ions at m/e 121 and 122, suggesting a steroidal l&lien-3-one A ring structure [13]. Confirmation of this structure is pro-

vided by the infra red spectrum (1655, 1612 and 1598cm-‘, all-unsaturated ketone), the U.V. spectrum (i,,,,, 244 nm. di-/I-substituted a/I-unsaturated ketone, double bond exocyclic)[14] and by the nuclear magnetic resonance proton spectrum (three vinylic protons in the range 6.08-7.016). Compound 2 is easily oxidised, acetylated and reduced suggesting the presence of both a hydroxyl and a ketone group. This is confirmed by the I.R. spectrum which contains a peak at 3325 cm- ’ characteristic of hydroxyl groups and a peak at 1738cm-’ characteristic of a ketone gronp in a five-membered ring. An intense ion at m/e

0-6r

Time

hcurs)

Fig. 2. The relationship between the concentration of chenodeoxycholic acid (1) (M), the major neutral (2) (A-A) and acidic (4) (B-m) V-dien-3-oxo steroids during the degradation of chenodeoxycholic acid by Pseudomonas spp. N.C.I.B. 10590. ’

314

M. E. TENNESON,J. D. BATY,R. F. BILTONand A. N. MASON

suggesting the presence of a ketone group. This is confirmed by the I.R. spectrum which contains a peak at 1735 cm-’ characteristic of a ketone group in a fivemembered ring. Compound 3 is, therefore, assigned the structure androsta-1,4,6-triene-3,17-dione. The major acidic steroid (4) was isolated as a crystalline solid. The methyl ester of 4 shows a molecular ion at m/e 372 and intense ions at m/e 121 and 122 in the mass spectrum, suggesting a steroidal l,rldien3-one A ring structure. Confirmation of this structure is provided as before by the I.R., the U.V. and the n.m.r. proton spectra. Compound 4 is easily oxidised and acetylated but resists reduction suggesting the presence of a hydroxyl group. This is confirmed as before by the I.R. spectrum. An intense ion at m/e 150 in the mass spectrum of methyl 4 suggests the presence of a steroidal 1,4dien-3-one-7-01 structure. The n.m.r. spectrum of methyl 4 shows a broad single COOH peak centred at 4.08 6 and on this basis the hydroxyl group of 4 is assigned the 7a configuration. Confirmation for this assignment is provided by the presence of an intense ion at m/e 354 in the mass spectrum. An intense ion at m/e 267 in the mass spectrum of (5) methyl 4 corresponds to loss of the side-chain from (4) Cr,. Compound 4 is therefore assigned the structure 7a-hydroxy-3-oxo-l,4-pregnadiene-20-carboxylic acid. Fig. 3. Metabolites isolated from the degradation of chenodeoxycholic acid by Pseudomonas spp. N.C.I.B. 10590. A minor acidic compound (5) was isolated, the methyl ester of which shows a molecular ion at m/e 374 and an intense ion at mfe 124 in the mass spec150 in the mass spectrum of 2 suggests the presence trum, suggesting a steroidal 4-en-3-one A ring strucof a steroidal 1,4dien-3-one-7-01 structure [13]. The ture [13]. Confirmation of this structure is provided nuclear magnetic proton spectrum of 2 shows a broad by the infra red spectrum (1647 and 161Ocm-‘, absingle peak centred at 4.106. The ‘I/I-proton bisects unsaturated ketone), the U.V. spectrum (J.,,, 241 nm, the dihedral angle between the two protons at C,; di-/l-substituted afi-unsaturated ketone, double bond presumably the coupling constant is too small to exocyclic) [14] and by the n.m.r. proton spectrum resolve the signal into a triplet. This observation (one vinylic proton at 6.156). Compound 5 is easily accords with the positions and patterns for protons oxidised and acetylated but resists reduction suggestat C, in 5a steroids [15]. On this basis the hydroxyl ing the presence of a hydroxyl group. This is congroup of 2 is assigned the 7a configuration. Further firmed by the I.R. spectrum. An intense ion at m/e support for this assignment is provided by the obser152 in the mass spectrum of methyl 5 suggests the vation [16] that loss of the elements of water from presence of a steroidal 4-en-3-one-7-01 structure [13]. the molecular ion in the mass spectrometer occurs The hydroxyl group is assigned the 70: configuration more readily with axial hydroxyl groups than with from n.m.r. proton spectrum. Again a broad single equatorial hydroxyl groups. With 2 an intense ion peak centred at 4.056 is shown. Intense ions at m/e was observed at m/e 282 in the mass spectrum indicat356 and m/e 269 in the mass spectrum, confirmed ing the presence of a 7a axial hydroxyl group. the loss of a 7a hydroxyl group and the side-chain Compound 2 is therefore assigned the structure 7a- from Cr,. Compound 5 is therefore assigned the hydroxy-1,4-androstadiene_3,17dione. structure 7a-hydroxy-3-oxo-4-pregnene-20carboxylic A minor neutral compound (3) was isolated, the acid. mass spectrum of which shows a molecular ion at The yield of the steroidal metabolites isolated is m/e 282 and an intense ion at m/e 134 suggesting listed in table one. a steroidal 1,4,6-trien-3-one A ring structure 1133. A crude mixture of compounds was left after the Confirmation of this structure is provided by the I.R. removal of the steroidal metabolites. This mixture contained two main metabolites both of which gave spectrum (1648, 1622, 1602 and 1576cm-’ a/?,$unsaturated ketone) and by the U.V. spectrum (A,,,, very short retention times on gas chromatography suggesting that they are low mol. wt. compounds. The 224, 256 and 300 nm, di+substituted a&unsaturated U.V. spectrum of the mixture (&,., 218 and 275 nm) ketone, double bond exocyclic, extended by a double which showed a bathochromic shift in alkaline solubond at C,-C,) [14]. Compound 3 resists both oxitions (II,., 229 and 298 mn) indicated the presence dation and acetylation but does not correspond to the of phenolic compounds. oxidation product of 2. However, 3 is easily reduced

Degradation of chenodeoxycholic DISCUSSION

The isoiation and identification of stttroidal metabolites 2, 3, 4 and 5 during the degradation of chenodeoxycholic acid by Pseudomonas’ spp. N.C.I.B. 10590 is the first recorded incidence of the microbial sidechain cleavage of chenodeoxycholic acid under aerobic conditions. The microbial degradation of bile acids has been studied for many years and the majority of metabolites isolated showing side-chain cleavage are 4-e&30x0 steroids [17]. The results obtained for the degradation of bile acids by Pse~omo~ N.C.I.B. 10590 show a predominance of l&lien-3-oxo-steroids in the metabolites isolated [l&20] (Table 1). All products containing a double bond in the A ring possess a ketone group at C3 [lx 201 and it is, therefore, suggested that 3a-hydroxystero~ dehydrogenation precedes the dehydrogenation of ring A. Dehydrogenation between C, and C1 presumably occurs after dehydrogenation between C4 and Cs, since no products containing a single double bond at C,-Cz have been isolated [ 17,201. The products isolated and identified from the degradation of deoxycholic acid by Pseudomonas N.C.I.B. 10590 exhibit a ratio of J-en-3-oxo to lkdien-3-oxo steroids from 1: 1 (CX), 1:4 (Cz2) to i:25 (C,s)[20], suggesting that dehydrogenation between C, and C2 may occur at any stage during bile acid degradation. It seems likely that dehydration between C, and C7 may occur at any stage during chenodeoxycholic acid degradation. This is indicated by the isolation of 4-Gdien-3-oxo steroids

during the degradation of cholic acid [17.21,22]. Metabolites possessing a C& double bond (3) could possibly have been produced during chemical isolation, since a facile dehydration can occur in mild acid [23]. However, great care was taken to avoid this and 3 was detected after extraction of the bacterial culture and its concentration did not increase during the separative procedures. It has been postulated [17] that for side-chain cleavage to occur the bile acid must first be transformed to at least a 4-en-3-oxo steroid. This hypothesis is supported by the fact that all side-chain cleavage products isolated so far are such metabolites. The mechanism of side-chain cleavage probably occurs by &oxidation from a Cz4 bile acid through a C,i metabolite to a C,9 androstane. This is illustrated in the degradation of chenodeoxycholic acid (1) (C,,) by

315

acid

Pseudomonas spp. N.C.I.B. 10590 where compound 4 (C,,) is produced in the medium before compound 2 (C,,) (Fig. 2). Nuclear ring fission of bile acids has hen reported previously. Cholic acid and lithocholic acid have been degraded to non-steroidal products [24,25]. l,~Androstadiene-3;17dione has been shown to be degraded by 9a-hydroxylation, followed by aromatisation of the steroid A ring and fission of the C,-C,, bond to give a 9,10-secosteroid. The steroid is then degraded to non-steroidal products [26]. It seems likely that the non-steroidal products produced from chenodeoxycholic acid are phenolic in nature. With the above facts taken into consideration a degradative pathway for chenodeoxycholic acid is proposed (Scheme 1). Compounds 2 and 4 have been produced under anaerobic conditions by an E. coli stain isolated from a faecal sample of a colon cancer patient [lo]. Since chenodeoxycholic acid is a primary bile acid in man and is fed in relatively large quantities to certain patients suffering from gallstones [27J, a study on the carcinogenicity of compounds 2 and 4 would be of value. IS these conipounds are carcinogenic this will add support to the hypothesis that the degradation

-

m COOH

0s

‘-an

;

0 ’ [~I

COOH

*-on

Table 1. Yield of metabolites with respect to starting material after 14 h incubation Metabolite

Yield (“/,I

20.5 7z-Hydroxy-I&androstadiene-3.1%dione (2) 1,4,~Androstatri~e-3.17-dione (3) 3.5 7z-Hydroxy-3-oxo- i.~pregnadiene-2~arboxyiic 10.5 acid (44) 7z-Hydroxy-3-oxo+pregnene-2O-carboxylic acid(5) 5.5

Scheme 1. Proposed microbial degradative pathway of chenodeoxycholic acid. Previously reported metaboiites are showti completely enclosed and suggested metabolites are shown in square brackets.

M. E. TEN-N,

316

J. D. BATY, R. F. BILTONand A. N. MACON

of bile acids by bacteria is implicated in the aetiology of colon cancer Cl].

Acknowledgements-We should like to thank Dr. R. J. Abraham of the Organic Chemistry Department, Liverpool University for the nuclear magnetic resonance proton spectra. The work was financially supported by the Science Research Council.

REFERENCES

1. Hill M. J.: The role of colon anaerobes in the metabolism of bile acids and steroids, and its relation to colon cancer. Cancer 36 (1975) 2387-2400. 2. Drasar B. S. and Irving D.: Environmental factors and cancer of the colon and breast. Br. J. Cancer 27 (1973) 167-172. 3. Appleweig N.: Will there be enough steroids? Chemical Week, July 10th (1974) 31-36. 4. Hill M. J. and Drasar B. S.: Degradation of bile salts by human intestinal bacteria. Gut 9 (1968) 22-27. 5. Aries V. C. and Hill M. J.: Degradation of steroids by intestinal bacteria. II. Enzymes catalysing the oxidoreduction of 32-. 72-. and 12r-hydroxyl groups in cholic acid, and the dehydroxylation of the ‘I-hydroxyl group. Biochifn. biophys. Acta 202 (1970) 535-543. 6. Midtvedt T. and Norman A.: Parameters in 7cldehydroxylation of bile acids by anaerobic Lactobacilli. Acta

Pathol.

Microbial.

Scand. 72 (1968) 313-329.

I. Edenharder R., Stubenrauch S. and Slemrova J.: Transformation of chenodeoxycholic acid by saccharolytic Bacteroides species. Zbl. Bakt. Hyg., I. Abt., Orig. B 162 (1976) 506-519.

8. Midtvedt T. and Norman A.: Bile acid transformations by intestinal-type bacteria. Acta Pathol. Microbial. Stand.

71 (1967) 629-638.

9. Dickinson A. B.. Gustafsson B. E. and Norman A.: Determination of bile and conversion potencies of intestinal bacteria by screening in oitro and subsequent establishment in germ free rats. Actu Pathol. Microbial. Stand.

79 (1971) 691-698.

IO. Tenneson M. E., Owen R. W. and Mason A. N.: The anaerobic side-chain cleavage of bile acids by Escherichia co/i isolated from human faeces. Biochem. Sot. Trans. 5 (1977) 1758-1760. 11. Kritchevsky D., Martak D. S. and Rothblat G. H.: Anisaldehyde reagent for steroids. Analyt. Biochem. 5 (1963) 388-392.

12. Bowers A., Halsall T. G., Jones E. R. H. and Lemin A. J.: The chemistry of the triterpenes and related compounds. XVIII. Elucidation of the structure of polyporenic acid C. J. Chem. Sot. (1953) 2555-2557.

13. Budzikiewicz H.: Steroids. In Biochemical Applications in Mass spectrometry (Edited by G. R. Wall&). WileyInterscience, New York (1972) DD. 251-289. 14. Dorfman L.: Ultraviolet ibso;&on of steroids. Chem. Reo. 53 (1953) 47-144’. 15. 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. J. Chem. Sot. C. (1970)-26257. 16. Zietz E. and Sniteller G.: Localisation of functional groups in steroids by mass spectrometry XI. 3,12,17/Itrihydroxyandrostanes, 12,17/fdihydroxyandrostan-3ones, 3,12dihydroxyandrostan-17-ones, and 12-hydroxyandrostane-3,17diones. Tetrahedron 30 (1974) 585-596.

17. Hayakawa S.: Microbiological transformation of bile acids. Adu. Lipid Res. 11 (1973) 143192. 18. Barnes P. J., ‘Baty J. D., siltoh R. F. and Mason A. N.: Degradation of deoxycholic acid by Pseudomonas species NClB 10590. Tetrahedron 32 (1976) 89-93. 19. Tenneson M. E., Bilton R. F. and Mason A. N.: A scheme for the microbial degradation of lithocholic acid involving testosterone as an intermediate. Biothem. Sot.

Trans. 6 (1978) 428-430.

20. Tenneson M. E.: The microbial oxidation of steroids. Ph.D.-Thesis, Council for National Academic Awards (1977). 21. Severina L. 0, Torgov I. V., Skrjabin G. K., Wulfson N. S., Zaretskii V. I. and Papernaja I. B.: Transformation of cholic acid by the culture Mycobacterium N1210.

Tetrahedron

24 (1968) 21452153.

22. Severina L. O., Torgov I. V., Skrjabin G. K., Wulfson N. S., Zaretskii V. I. and Papernaja I. B.: The enzymatic transformation of cholic acid by the culture Mycobacterium 485-491.

mucosum

1210.

Tetrahedron

25

(1969)

23. Hasegawa K.: The metabolism of bile acids. I. The metabolism of cholic acid by Aspergillus cinnamomeus. II. The metabolism of 3,7dihydroxy-12-oxocholanic acid by Aspergillus cinnamomeus. Hiroshima J. Med. Sci. 8 (1959) 277-283. 24. Hayakawa S., Kanematsu Y. and Fujiwara T.: 12adehydroxylation of cholic acid by Arthrobacter simplex.

Nature

214 (1967) 52&521.

25. Hayakawa S., Kanematsu Y. and Fujiwara T.: Degradation of bile acids by Arthrobactrr simplex. Biochem. J. 115 (1969) 249-256. 26 Sih C. J. and Whitlock H. W.: Biochemistry of steroids. Ann. Rev. Biochem.

37 (1968) 661-694.

27. Pederson L. and Bremmelgaard A.: Hepatic morphology and bile acid composition of bile and urine during chenodeoxycholic acid therapy for radiolucent gallstones. Scand. J. Gastroent. 11 (1976) 385-391.