The metabolism of primary, 7-oxo, and 7P-hydroxy bile acids by Clostridium absonum

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1 The metabolism of primary, 7-oxo, and 7P-hydroxy bile aids by Clostridium absonum J. Derek Sutherland and Ian A. Madonald Departments of Mediine and Biohemistry, Dalhousie University, Halifax, Nova Sotia, Canada B3H H7 Abstrat Clostridium absonum was shown to metabolie primary bile aids to give rise to both 7-oxo bile aids and 78- hydroxy (urso) bile aids. At relatively low redox potential (Eh) values, high yields of urso bile aids were ahieved (6-75%). If, however, the Eh value of the ulture was allowed to rise above approximately -1 mv, the 7-oxo bile aid would tend to predominate (more than 75%) and the death phase was aelerated. Growth of C. absonum in sterile graduated ylinders instead of in onventional Erlenmeyer flasks was effetive in delaying the rise in Eh value with time (whih appears largely due to diffusion of atmospheri oxygen into the medium) and in preserving a higher viable ount of organisms. It is proposed that the formation of exess amounts of 7-XO bile aid is a manifestation of oxygen toxiity and that it ould be mediated by an inreasing intraellular NADP:NADPH ratio. Additionally, the reation: primary bile aid e oxo bile aid P urso bile aid was shown to be partially reversible. When the organisms were grown with [2- C]henodeoxyholi,-holi, or -7-keto-lithoholi aid, this reation ould be learly demonstrated. The addition of an equimolar onentration of deoxyholi aid (whih itself is not metabolied) effetively enhaned the rate of bioonversion of holate and 7-keto-lithoholi, but not henodeoxyholate (whose rate of bioonversion was the fastest of the three). When the organisms were grown with urso bile aids (ursoholi or ursodeoxyholi) or with 7-keto-deoxyholi aid, very little metabolism ourred unless deoxyholi aid was added whih indued formation of primary and keto bile aids. In all ases, formation of oxo bile aid from primary or urso bile aid ourred as the Eh value of the medium rose with time and ould thus be delayed by the use of a ylinder instead of a flask for growing the ulture.!i These results were rationalied by demonstrating that indution of 7a- and 78- hydroxysteroid dehydrogenase is strongly mediated by henodeoxyholi and deoxyholi aids, weakly mediated by holi and 7-keto-lithoholi aids, and ineffetive with 7-keto-deoxyholi, ursoholi, and ursodeoxyholi aids.-sutherland, J. D., and I. A. Madonald. The metabolism of primary, 7- oxo, and 7B-hydroxy bile aids by Clostridium absonum. J. Lipid Res Supplementary key words aids urso bile aids hydroxysteroid dehydrogenase keto bile The possible metaboli fate of the 7a-hydroxyl groups of primary bile aids in the human intestine inludes: 7a-OH dehydroxylation, oxidation (to the ketone), and epimeriation (to the 7P-OH group). A number of feal Clostridia and Eubaterium isolates have been shown as ative for 7a-OH dehydroxylation (1-), while faultatives suh as E. oli (5) and anaerobes suh as B. fragilis (6, 7) and C. sfiorosfihaeroides-like organisms (8) an oxidie 7a-OH groups (but not 7B-OH groups) to the orresponding ketone. It is now well established that the third reation, 7a-OH epimeriation, an our in mixed feal ultures (9-1 l), and very reently, human feal isolates responsible for this reation have been desribed as leithinase-lipase-negative Clostridia (1 2). In a reent report, we have shown that a soil isolate, C. absonum a perfringens-like organism (13), an onvert both primary bile aids into urso (7B-OH) bile aids and 7-oxo bile aids (1). Additionally, we have shown the presene of 7a- and 7P-hydroxysteroid dehydrogenases in ell-free preparations of C. absonum (15), strongly supporting the hypothesis that 7a-OH epimeriation proeeds through a 7-oxo intermediate. It is the purpose of this ommuniation to report further study on the mehanism by whih primary bile aids an be onverted to urso-bile aids and vie versa in C. absonum. Materials MATERIALS AND METHODS Clostridium absonum strain VPI #695 (1, 15) was used throughout the study. Brain heart infusion (BHI) Abbreviations: BHI, brain heart infusion; TLC, thin-layer hromatography; Eh, redox potential; HSDH, hydroxysteroid dehydrogenase; NAD, niotinamide adenine dinuleotide; NADP, niotinamide adenine dinuleotide phosphate; UC, ursoholi aid (3a-, 78-, 12atrihydroxy-5~-holan-2-oi aid); 7K-DC, 7-keto-deoxyholi aid (3a-, 12o-dihydroxy-7-keto-5~-holan-2-oi aid); CDC, henodeoxyholi aid (3a-, 7a-dihydroxy-5fl-holan-2-oi aid); UDC, ursodeoxyholi aid (3a-, 7@-dihydroxy-5~-holan-2-oi aid); 7K-LC, 7-keto-lithoholi aid (3a-hydroxy-7-keto-5~-holan-2-oi aid); DC, deoxyholi aid (3a-, 12a-dihydroxy-5~-holan-2-oi aid); ATCC, Amerian Type Culture Colletion; VPI, Virginia Polytehni Institute. To whom reprint requests sould be addressed. Downloaded from by guest, on Deember 1, Journal of Lipid Researh Volume 23, 1982

2 broth and ooked meat broth were produts of Difo Laboratories, Detroit, MI. Choli aid was from J. T. Baker Chemials, Phillipsburg, N J; deoxyholi aid (DC) was from Calbiohemials, Los Angeles, CA; henodeoxyholi (CDC) and ursodeoxyholi (UDC) aids were from Sigma Chemials, St. Louis, MO; 7-ketodeoxyholi aid (7K-DC) and 7-keto-lithoholi aid (7K-LC) were from Steraloids, Wiltshire, NH; ursoholi aid (UC) was kindly donated by Drs. R. A. DiPietro and A. F. Hofmann of the Division of Gastroenterology, Shool of Mediine, University of California, San Diego; labeled [2- C]holi aid and CDC were produts of New England Nulear, Lahine, Quebe. Labeled intermediates, [2-IC]7KLC and 7K-DC, and labeled urso-bile aids, [2-1C]UDC and UC, were made by growing C. absonum in the presene of [2-1C]CDC or holi aid in 1-ml BHI ultures (eah at a onentration of 2 lop M) and extrating the spent baterial medium as desribed before (1 ). Ether, methanol, hloroform, aeti aid, and parahydroxybenaldehyde (Komarowsky s reagent) were from Canadian Laboratories, Montreal, Quebe. Liquid sintillation fluid (atalogue #R ) was from British Drug House, Montreal, Quebe. Commerial sheep s blood plates were a produt of Beton Dikinson, Cokeysville, MD. Growth of C. absonum Time ourse studies were performed with 5-ml ultures whih were either grown in 125-ml flasks or 5- ml graduated ylinders at 37OC and outside an anaerobi hamber. Cultures were inoulated with 5 ml of a 1- ml overnight starter ulture. Cultures ontained 2 M 2-1C-labeled primary or 7-oxo bile aid, or 5 M 2-1C-labeled urso bile aid with and without the simultaneous addition of 2 * M DC (unlabeled). Approximately.1 pci of label was used per study. Sampling volumes of 3.5 ml were withdrawn at various time intervals. The absorbane at 66 nm of eah ulture sample was measured in a Bekman DB-GT spetrophotometer equipped with a Bekman 1 inh reorder. Cultures were then froen at -2 C until extration. Estimation of Eh values Redox potential (Eh) values were estimated by an Orion Eh probe using a.5% ysteine standard whih gave an Eh value of +25 mv. Identifiation of produts Produts of 7a-OH oxidation and epimeriation of holi aid and CDC were identified by their R, values on TLC plates (1, 15), their olor and fluoresene harateristis under a UV lamp after spraying with Komarowsky s reagent (16), as well as the ability of ommerial standards to o-hromatograph with the C. absonum produt in question. Extration, thin-layer hromatography elution, and ounting proedures were performed as desribed previously (1 ). Viable ount estimations Samples of C. absonum broth ultures (at t = 2 and 72 hr) were diluted serially into ie-old BHI broth and.1-ml aliquots were spread over blood agar plates that were inubated anaerobially in a Gas Pak system (BBL). Colonies were ounted after 2 hr inubation. Effet of various bile aid induers on 7a- and 7& HSDH ativities Ten ml of starter ulture was added to 1 ml of BHI broth ontaining - lop M bile aid induer as before (15). Cells were harvested at 6 hr by entrifuging at 6 g for 2 min at OC. The pellet was resuspended in 3 ml of.1 M sodium phosphate buffer ph 7. ontaining lop3 M EDTA and lop3 M dithiothreitol. The bateria were lysed in a Frenh pressure ell and the ell-free preparation was entrifuged at 6 g for 2 min at C. The supernatant fluid was deanted from the pellet. A muopolysaharide-like substane that tended to float on the supernatant fluid was generally avoided in transferring the ell-free preparation. Cellfree preparations were immediately assayed. The formation of NAD(P)H was followed at 3 nm and 25 C using a Bekman DB-GT grating spetrophotometer and 1 inh Bekman reorder. Eah assay uvette (1. ml) ontained 1. mm NAD(P),.3 M glyine/naoh buffer, ph 1.5 or ph 9.5, 1.- M bile aid, and 1-5 p1 of enyme solution. RESULTS Time ourse studies with primary bile aids (Figs. 1 and 2), 7-oxo bile aids (Figs. 3 and ) and urso bile aids (Figs. 5 and 6) are presented. These reations were performed in singlet. However, repeated experiments on two seleted time ourse urves revealed that the metaboli urves were reproduible within a 1% differene margin. The addition of DC to the system was shown to be effetive in promoting the formation of UC from holi aid when the ultures were grown in flasks (ompare Fig. lb to Fig. la), and hastened the rate of formation of UC but did not affet the final yield when the ultures were grown in ylinders (ompare Fig. Id to Fig. IC). However, DC had only a marginal effet on inreasing the UDC yield from CDC (Fig. 2b ompared to Fig. 2a, and Fig. 2d ompared to Fig. 2). Growth of the organisms in a ylinder instead Downloaded from by guest, on Deember 1, 218 Sutherland and Madonald Metabolism of 7-oxo and 7&hydroxy bile aids by C. absonum 727

3 ' TIME Ihrsl Fig. 1. Time ourse urve for the degradation of 2. M [2- ''C]holi aid by a 5-ml ulture of C. absonum A) grown in a flask without DC, B) grown in a flask in the presene of 2. M DC, C) grown in a ylinder without DC, and D) grown in a ylinder with 2-1- M DC. Symbols: W -W, holi aid; A-A, UC; -, 7K-DC; -, absorbane of ulture at 66 nm. of a flask was effetive in sustaining a higher level of UC (giving as muh as a 6% yield) from holi aid (Fig. Id ompared to lb). When CDC was a substrate, growth of the organisms in a ylinder was dramatially effetive in maintaining the level of UDC after 8 hr (Fig. 2 and 2d). When the organisms were grown in a flask, most of the UDC generated in the first 8 hr was rapidly onverted to 7K-LC and ould not thereafter be reovered (Fig. 2a and 2b). The onversion of UDC to 7K-LC was atually more thorough in the presene of DC than in its absene. The explanation for this observation is not yet lear. Similar results were obtained in Fig. 3 whih shows the metabolism of 7K-DC. The addition of DC to the flask was shown to be effetive in promoting the formation of higher levels of UC (ompare Fig. 3b with 3a). However, growth of the organisms in the ylinder plus the addition of DC was most effetive in generating UC. When the substrate was 7K-LC (Fig. ), the piture was similar, exept that the addition of DC did not play a large role in inreasing the yield of UDC (Fig. b ompared to Fig. a). Again, it was only when the organisms were grown in a ylinder that a high yield of UDC was maintained in the ulture (up to about 6 hr, Fig. ). When the organisms were fed UC (Fig. 5) or UDC (Fig. 6) and grown in flasks, the results were in marked ontrast with those for the primary or oxo substrates (Figs. 1-); very little metabolism ourred (Figs. 5a and 6a). The ells ould be indued to metabolie UC or UDC by the addition of DC to the ulture whih was effetive when the ratio of DC:urso bile aid was :l (Figs. 5b and 6b), or greater. However, the main metaboli produts were oxo bile aids (7K-DC and 7K- LC). Only minor amounts of holi aid and CDC were produed, and they leveled out to about 3% and 67, respetively. When the same systems were studied using a ylinder (instead of a flask), the yields of holi aid and CDC ould be enhaned to about 12% and 15%, respetively (Figs. 5 and 6). Moreover, the use of a ylinder was instrumental in delaying the metabolism of most of the UC and UDC to 7K-DC and 7K-LC. A further addition of DC to the ylinder ultures whih gave a final DC onentration of - M and a ratio of DC:UDC or DC:UC of 8:l was also performed. Very similar results to the :l (DC:urso bile aid) systems were obtained giving no improved yield of primary bile aid (results not shown). In Fig. 7, the Klett units (absorbane at 66 nm), ph values, and Eh values of C. absonum ultures are represented for ultures with and without 2 - M CDC. As seen in Fig. 7a and 7b, ultures in ylinders (triangles) E - u..2 : o. u 6% o TIME Ihrsl Fig. 2. Time ourse urve for the degradation of 2- low M [2- IC]CDC by a ulture of C. absonum. Sequene A-D idential to Fig. 1. Symbols: - m, CDC; A - A, UDC; -, 7K- LC, -, absorbane of ulture at 66 nm. Downloaded from by guest, on Deember 1, Journal of Lipid Researh Volume 23, 1982

4 I- Ip E 1 Q 8.8 a Y. u Iy. VI TIME (hrsl Fig. 3. Time ourse urve for the degradation of 2 - M [2- lc17k-dc by a 5-ml ulture of. absonum A) grown in a flask without DC, B) grown in a flask with 2. M DC, ) grown in a ylinder with 2 M DC. Symbols as in Fig. 1. grew similarly to those in flasks (squares), but higher absorbane values between 12 and 8 hr in the former suggest that the ells in the ylinder may have survived in greater numbers. This observation was onfirmed by the viable ount data whih showed that at 2 hr there were about 3.2 X 1' organisms/ml in the flask ulture (no bile aid added) ompared to 3.9 X 1' organisms/ ml in a ylinder ulture. At 72 hr there were 3.8 X lo and 2.2 X lo7 organisms/ml, respetively. Differenes in the bile aid degradation patterns between flask ul I.2 L I- 2.2 e.o TIME (hrsl E D.n t -2 Y m Iy m m Fig.. Time ourse urve for the degradation of 2. IO-* M [2- IC]7K-LC by a 5-ml ulture of C. absonum. Sequene A-C as in Fig. 3; symbols as in Fig. 2. 'p - P 1.u. A& E. C I- 6.6 V :. VI TIME Ihrsl Fig. 5. Time ourse urve for the degradation of 5. M [2- I~CIUC by a 5-ml ulture of C. absonum A) grown in a flask without DC, B) grown in a flask in the presene of 2. M DC, C) grown in a ylinder in the presene of 2 M DC. Symbols as in Fig. 1. tures and ylinder ultures annot, however, be explained on the basis of viable ell differenes sine muh of the metaboli ativity ours in the first 12 hr when there are no measurable differenes in absorbane values at 66 nm. When the ph value was studied with respet to time of ulture, the ph initially dropped (regardless of whether the ulture was in a flask or ylinder or whether it ontained bile aid or not) and then reovered (Fig..n 2 1.n 8.8 I- Iy VI. V e o. : 1 m 8.8," 2.2 : o TIME (hrs) Fig. 6. Time ourse urve for the degradation of 5- M [2- IC]UDC by a 5-ml ulture of C. absonum. Sequene A-C idential to Fig. 5; symbols as in Fig. 2. Downloaded from by guest, on Deember 1, 218 Sutherland and Madonald Metabolism of 7-~ and 78-hydroxy bile aids by C. absonum 729

5 a d a -2 5 (Control) (CDC) TIME ihr5) Fig. 7. Time ourse urves of C. absonum ultures showing the effet of time on A) absorbane values at 66 nm without CDC in the medium, B) absorbane values at 66 nm with CDC in the medium, C) ph values without CDC present, D) ph values with CDC present, E) Eh values without CDC present, and F) Eh values with CDC present. Symbols: W - W, flask ultures; A - A, ylinder ultures. 7 and 7d). The rise bak to neutrality was slow when ylinders were used and not muh affeted by the addition of bile aid. But the rise bak to neutrality was relatively rapid in a flask and never reovered to neutrality when CDC was added. Similar results were found when other bile aids were tested (results not shown) inluding 7K- LC (2 * M), a mixture of holi aid and DC (eah 2 M), and a mixture of 7K-DC and DC (eah 2 M). However, when 5 - M UC was added, the ph urves were similar to those of the ontrol system (no bile aid) (Fig. 7). When the Redox potential was studied (Fig. 7e and 7, the Eh value was shown to drop from about -1 mv (ahieved by autolaving medium) to around -25 mv by the growth of the organism (all systems). The primary differene between the flask and ylinder ultures was that the ylinder was effetive in retarding the rise in Eh with time as the ulture aged. Fig. 8 shows 7-HSDH ativities measured against a) holi aid (ph 1.5), 6) UDC (ph 9.5), ) CDC (ph 1.5) (all with NADP), and d ) holi aid (ph 9.5) with NAD. The induer bile aid at M is represented on the absissa. As an be seen here, both CDC and DC are effetive in induing all enyme systems; 7K-LC and holi aid work at an intermediate level, while 7K-DC and the urso-bile aids are ineffetive. Note that the ativity against UDC (7p-HSDH) is totally absent in the ontrol and requires an induer, while a finite amount of 7a-HSDH is present without an induer. DISCUSSION This work, in effet, represents an extension of a previous study (1) and further demonstrates that the epimeriation of bile aids by. absonum an our via an oxidation-redution mehanism whih is mediated by 7- oxo bile aids. The reation sheme: primary (7a-OH) bile aid P 7-keto bile aid P urso (7P-OH) bile aid appears to be partially reversible under appropriate onditions. Two features of the C. absonum ulture appear to be most important in determining the outome of the mirobial degradation of the starting bile aid: a) the ability of the initial bile aid to indue the bile aid metaboli system of the ulture or the presene of an added induer (i.e., DC), and b) the shape of the fermentation vessel. a) As established earlier, DC has the unusual property of induing the 7a- and 7P-HSDH systems (15), yet itself is not atabolied. Thus DC provides a useful tool in probing the metabolism of bile aids suh as 7K- DC and urso bile aids whih (in ontrast to CDC, holi, Downloaded from by guest, on Deember 1, 218 Cholate I NADP I ph 1.5 UDC I NADP I ph 9.5 Cholate I NAD / ph 9.5 CDC I NADP I ph 1.5 $ 1 n = o 3o-129 3a k 3o-7o-12a 3a-12a-7k 3-7B 39-7B-12~1- No a (DC) (CDC) I7K-LC) (Choli) (7K-DC) IUDC) (UC) Addition Fig. 8. Levels of HSDH ativity indued by the addition of various bile aids to the ulture as designated. 73 Journal of Lipid Researh Volume 23, 1982

6 and 7K-LC) would otherwise not reat or reat very poorly sine they are substrates, but non-induers (or at best, poor induers) of the 7a- and 78-HSDH systems (1). 6) In an earlier study (1) it was unlear to us why the oxo-intermediate should aumulate and the viable ell ount should drop rapidly in the aging ulture. The urrent study suggests that the ells are able to generate and maintain a low Eh value in their ulture for only the first 6-9 hr (probably by C2 evolution (1)) after whih they are at the mery of the rate of oxygen diffusion into the system, and the prodution of 7-oxo bile aid may be one manifestation of oxygen poisoning whih, in a flask, auses the abrupt death of most of the organisms. A ylinder restrits oxygen diffusion and delays the death of the organisms and the formation of 7- oxo bile aid. We have also shown that layering.5 m of olive oil on a ulture in a flask or growing the organisms in a flask in a Gas Pak System is equally effetive in preventing formation of 7-oxo bile aids2 We proposed earlier (1 ) that an inreasing intraellular ratio of NADP/NADPH due to other metaboli ativities just prior to the death phase of the ells might give rise to greater amounts of 7-oxo bile aid. Our urrent data are still onsistent with this hypothesis. The reason why formation of 7K-LC in the CDC ultures in flasks is more prominent than formation of 7K-DC in the holi system is not yet lear, but may relate to rate differenes in the metabolism of the two primary bile aids (ompare Fig. lb with Fig. 2b). Additionally, there appears to be an anaerobi equilibrium (Eh value < -1 mv) and an aerobi (Eh value > -1 mv) equilibrium. When a difuntional bile aid (CDC, 7K-LC, or UDC) was substrate and suffiient induer was present, there is at equilibrium about 75% UDC, 2% CDC, and 5% 7K-LC (ompare Figs. 2d,, and 6) if the ulture stays suffiiently anaerobi. As the ulture beomes more aerobi (Eh value rising above -1 mv or so), the equilibrium shifts to give about 87% 7K-LC, 8% UDC, and 5% CDC (ompare Figs. 2b, b, and 6b). (The primary differene between the flask and ylinder experiments was that, in the former, the ulture beame aerobi faster.) A similar relationship may exist for the trifuntional bile aids, exept that the reation is markedly slower even in the presene of DC induer. (This may simply relate to differenes in enyme kinetis, e.g., K,,, value for C. absonum 7a-HSDH is onsiderably higher for holi than CDC (1 5).) In fat, in some ases, the reation may not atually reah anaerobi equilibrium before the aerobi phase ommenes. It is, however, partiularly Sutherland, J. D., and I. A. Madonald. Unpublished observation. diffiult to drive the reation when UC is the starting substrate (Fig. 5a-). Lastly, there was a orrelation between the relative ability of a given bile aid to indue 7a- and 7b-HSDH (Fig. 8) and the ability of C. absonum to transform the bile aid to other produts (Figs. 1-6). Urso bile aids and 7K-DC were very poor induers and they required the presene of DC; holi and 7K-LC were intermediate induers and their transformation by C. absonum was marginally enhaned by the presene of DC in the ulture. CDC was a good induer and its metabolism ould be only very slightly enhaned by DC addition. The diffiulty in foring the reverse reation (partiularly with UC) even in the presene of DC may be beause urso bile aids behave as repressors. (In an earlier ommuniation (1 5), we demonstrated that UDC does indeed behave as a repressor.) The relationship between primary, 7-oxo, and urso bile aids has also been studied using mixed feal flora and difuntional bile aids (9, 11). However, neither Hayase and oworkers (13) nor the authors of this report2 have sueeded in isolating C. absonum from human fees, thus suggesting that one or more of the other speies of feal bateria may be responsible for the in vivo formation of UDC (17). The reent report by Edenharder and Knafli (12) suggests that the organisms normally responsible for 7-epimeriation in the human intestine may be leithinase-negative/lipase-negative Clostridia (12). Epimeriation by these organisms is also evidently mediated by a 7-oxo derivative and, similarly to C. absonum, UDC is not metabolied (no induer added). Further omparisons between C. absonum and the human feal lostridial isolates must await further studies. The ability of C. absonum to epimerie bile aids in the presene of mixed feal ultures is urrently under study.l Manusript reeived 13 August 1981, in revised form 17 November 1981, and in re-revised form 5 February REFERENCES 1. White, B. A., R. L. Lipsky, R. F. Frike, and P. B. Hylemon Bile aid indution speifiity of 7a-dehydroxylase ativity in an intestinal Eubaterium speies. Steroids. 35: White, B. A., A. F. Caiapuoti, R. J. Frike, T. R. Whitehead, E. H. Mosbah, and P. B. Hylemon Cofator requirements for 7a-dehydroxylation of holi and henodeoxyholi aid in ell extrats of the intestinal anaerobi baterium, Eubaterium speies VPI J. Lipid Res Stellwag, E. J., and P. B. Hylemon a-Dehydroxylation of holi aid and henodeoxyholi aid by Clostridium leptum. J. Lipid Res. 2: Downloaded from by guest, on Deember 1, 218 Sutherland and Madonald Metabolism of 7-oxo and 7&-hydroxy bile aids by C. absonum 731

7 . Hirano, S., R. Nakama, M. Tamaki, N. Masuda, and H. Oda Isolation and harateriation of thirteen intestinal miro-organisms apable of 7a-dehydroxylating bile aids. Appl. Environ. Mirobiol. 1: Madonald, I. A,, C. N. Williams, D. E. Mahony a-Hydroxysteroid dehydrogenase from Esherihia oli: preliminary studies. Biohim. Biophys. Ata. 39: Madonald, I. A., C. N. Williams, D. E. Mahony, and W. M. Christie NAD- and NADP-dependent 7ahydroxysteroid dehydrogenases from Bateroides fragilis. Biohim. Biophys. Ata Hylemon, P. B., and J. A. Sherrod Multiple forms of 7a-hydroxysteroid dehydrogenase in seleted strains of Bateroides fraelis. J. Bateriol. 122: Mahony, D. E., C. E. Meier, I. A. Madonald, and L. V. Holdeman Bile salt degradation by nonfermentative lostridia. Appl. Environ. Mirobiol Fedorowski, T., G. Salen, G. S. Tint, and E. H. Mosbah Transformation of henodeoxyholi aid and ursodeoxyholi aid by human intestinal bateria. Gastroenterology. 77: Serva, R. P., H. Fromm, G. L. Carlson, L. Mendelow, and S. Ceryak Intraoloni onversion in man of henodeoxyholi aid (CDC) to ursodeoxyholi aid (UDC) with and without formation of 7-keto-lithoholi aid as an intermediate. Gastroenterology. 78: 1252 (Abstrat). 11. Hirano, S., N. Masuda, and H. Oda In vitro trans- formation of henodeoxyholi aid and ursodeoxyholi aid by human intestinal flora, with partiular referene to the mutual onversion between the two bile aids. J. Lipid Res. 22: Edenharder, R., and T. Knafli Epimeriation of henodeoxyholi aid to ursodeoxyholi aid by human intestinal leithinase-lipase-negative Clostridia. J. Lipid Res Hayase, M., N. Mitsui, K. Tamai, S. Nakamura, and S. Nishida Isolation of Clostridium absonum and its ultural and biohemial properties. Infet. Immun Madonald, I. A., D. M. Huthison, and T. P. Forrest Formation of urso- and ursodeoxy-holi aids from primary bile aids by Clostridium absonum. J. Lipid Res. 22: Madonald, I. A., and P. D. Roah Bile salt indution of 7a- and 7@-hydroxysteroid dehydrogenases in Clostridium absonum. Biohim. Biophys. Ata Madonald, I. A Detetion of bile salts with Komarowsky s reagent and group speifi dehydrogenases. J. Chromatogr. 136: Salen, G., T. Fedorowski, A. Colallilo, G. S. Tint, and E. H. Mosbah The metabolism of ursodeoxyholi aid in man. In Bile Aid Metabolism in Health and Disease. G. Paumgartner and A. Stiehl, editors. MTP Press Ltd., Lanaster, England Downloaded from by guest, on Deember 1, Journal of Lipid Researh Volume 23, 1982