7P-Dehydroxylation of ursodeoxycholic acid by whole cells and cell extracts of the intestinal anaerobic bacterium, Eubacterium species V.P.I.
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1 7PDehydroxylation of ursodeoxycholic acid by whole cells and cell extracts of the intestinal anaerobic bacterium, Eubacterium species V.P.I B. A. White, R. J. Fricke, and P. B. Hylemon' Department of Microbiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA Abstract Whole cells and cell extracts of Eubacterium species V. P. I dehydroxylated [3H]ursodeoxycholic acid or [ "C]chenodeoxycholic forming lithocholic acid. 7BDehydroxylation specific activity was 146 and 386 nmol hr' mg protein' for cell extracts and whole cells, respectively. 7a or 78Dehydroxylation activity was detected only in whole cells or cell extracts prepared from cultures grown in the presence of cholic acid. The addition of NAD' (0.5 mm) to anaerobically dialyzed cell extracts stimulated 78 and 7adehydroxylation activity by 5 and 40fold, respectively. The level of 78dehydroxylation specific activity was approximately 3 to 5fold lower than 7 adehydroxylation in whole cells and 3fold lower in cell extracts. Substrate saturation kinetics for ursodeoxycholic acid and chenodeoxycholic acid were hyperbolic and showed substrate inhibition at concentrations above 200 pm. The apparent K,,, values for ursodeoxycholic and chenodeoxycholic acid were 14.5 pm and 49 pm, respectively. Both 7a and 78dehydroxylase activities were inactivated (60% to 70%) by heating for 6 min at 45OC. Moreover, both activities coeluted from a anaerobic BioGel A 1.5M column as a single peak at approximately 114,000 (M,).M These data show that this intestinal anaerobic bacterium has both 7a and 78dehydroxylase activities which may be catalyzed by the same enzyme.white, B. A., R. J. Fricke, and P. B. Hylemon. 78Dehydroxylation of ursodeoxycholic acid by whole cells and cell extracts of the intestinal anaerobic bacterium, Eubacterium species V. P. I J. Lipid Res Supplementary key words substrate saturation kinetics induction of 7afldehydroxylase pathways of ursodeoxycholic acid metabolism The administration of selected bile acids as therapeutic agents for the dissolution of cholesterol gallstones has been practiced since the early 1970's (1). Chenodeoxycholic acid was the first bile acid found to induce cholesterol gallstone dissolution (2); however, severe hepatotoxicity has been associated with the feeding of this bile acid to several species of laboratory animals (35). Recently, ursodeoxycholic acid, the 76epimer of cheno deoxycholic acid, has been reported to be equally efficient in the dissolution of cholesterol gallstones (6), but does not appear to induce the hepatotoxicity seen during chenodeoxycholic acid treatment (7). This difference has been attributed to the decreased microbial formation and circulation of lithocholic acid in the bile of ursodeoxycholic acidtreated animals as compared to the large amounts of lithocholic acid found in chenodeoxycholic acidtreated animals. In vivo studies of chenodeoxycholic acid and ursodeoxycholic acid metabolism during gallstone dissolution therapy have reported the epimerization of the 7hydroxy group of the bile acid nucleus (811). The epimerization is carried out by the intestinal microbial flora and is believed to proceed through a 7ketolithocholic acid intermediate. In this regard, Macdonald, Hutchison, and Forrest (12) recently reported that pure cultures of Clostridium absonum could epimerize chenodeoxycholic acid to ursodeoxycholic acid and the biotransformation proceeded through a 7ketolithocholic acid intermediate. Studies by Fedorowski et al. (13), using washed fecal suspension, demonstrated the 76dehydroxylation of ur Abbreviations: Systematic names of bile acids referred to in the text by their trivial names are as follows: cholic aicd, 3a,7a,l2atrihydroxy 5flcholan24oic acid; chenodeoxycholic acid, 3a,7adihydroxy5/3 cholan24oic acid; ursodeoxycholic acid, 3a,7fldihydroxy5flcholan 24oic acid; deoxycholic acid, 3a,l2udihydroxy5flcholan24oic acid; 7ketolithocholic acid, 3ahydroxy7 keto5flcholan24oic acid; lithocholic acid, 3ahydroxy5flcholan24oic acid; NAD+, nicotinamide adenine dinucleotide; NADH, nicotinamide adenine dinucleotide, reduced form; NADP+, nicotinamide adenine dinucleotide phosphate; NADPH, nicotinamide adenine dinucleotide phosphate, reduced form; FAD, flavin adenine dinucleotide; FADH2, flavin adenine dinucleotide, reduced form; FMN, flavin mononucleotide; FMNH2, flavin mononucleotide, reduced form. ' Reprint requests should be sent to Dr. P. B. Hylemon, Department of Microbiology, Virginia Commonwealth UniversityMedical College of Virginia, Box 678MCV Station, Richmond, VA Journal of Lipid Research Volume 23,
2 important tool for answering two major questions vital to the interpretation of the in vivo studies of bile acid metabolism during gallstone dissolution therapy. First, does the 7Bdehydroxylation of ursodeoxycholic acid proceed via the direct removal of the 7Bhydroxy group or, alternatively, must ursodeoxycholic acid first be epimerized to chenodeoxycholic acid which is then 7adehydroxylated? Second, if ursodeoxycholic acid is directly 7/3dehydroxylated, is it catalyzed by the same enzyme that carries out the 7adehydroxylation of primary bile acids? MATERIALS AND METHODS HOURS HOURS Fig. 1. Induction of 7a and 7fidehydroxylase activities in cultures of Eubacterium sp. V.P.I Cholic Acid (large arrows, A) or ursodeoxycholic acid (large arrows, B) was added to parallel growing cultures (circles). Culture samples (small arrows) were taken and the whole cell activities of 7adehydroxylase (squares) and 78dehydroxylase (triangles) were determined. sodeoxycholic acid without detectable accumulation of a 7ketolithocholic acid intermediate. However, it is difficult in these studies to distinguish between the direct removal of the 7Bhydroxy group and a rapid epimerization of ursodeoxycholic acid to chenodeoxycholic acid which can be 7adehydroxylated by the intestinal microflora. Certain intestinal anaerobic bacteria (141 6) are known to be capable of the 7adehydroxylation of primary bile acids; however, there is no direct evidence that the microbial flora of man can 7Bdehydroxylate bile acids. We have previously reported that Eubacterium sp. V. P. I has a cholic acid inducible 7adehydroxylase (17, 18). This organism represents a potentially TABLE 1. 7Dehydroxylation of chenodeoxycholic acid and ursodeoxycholic acid by whole cell suspensions of Eubacterium sp. V.P.I Chenodeoxycholic Acid" Uninduced 24 Inducedh 980 Ursodeoxycholic Acid" a Units are nmoles of lithocholic acid formed per hr per mg whole cell protein. Cultures were induced with colic acid as described in Fig. 1. Growth of Bacteria Characteristics, growth conditions, and media for the anaerobic culturing of Eubacterium sp. V. P. I have been described previously (1 8). Bacterial cultures, in 1 or 2 liter volumes, were induced to synthesize 7adehydroxylase by the addition of 0.1 mm sodium cholate at hourly intervals during logarithmic growth (17). Cell extracts were prepared anaerobically by disruption with a chilled French Pressure cell (19). The 105,000 g supernatant fluid was immediately dialyzed (4 C) against one liter of anaerobic 50 mm sodium phosphate buffer (ph 6.8) containing 12% glycerol (vol/vol) and 5 mm glutathione for 18 hr with one change of buffer. Protein concentrations were determined by the method of Kalb and Bernlohr (20) or that of Lowry et al. (21). Quantitative enzyme assay for 7deydroxylase Enzymatic 7dehydroxylation of [ ''C]carboxyllabeled chenodeoxycholic acid or [ 1 1,123H (N)]ursodeoxycholic acid was followed by measuring the rate of lithocholic acid formation using a radiochromatographic assay procedure (22). Unless otherwise noted, the standard whole cell reaction mixture contained (1.O ml total volume): 50 mm sodium phosphate buffer (ph 6.8) containing 10% glycerol (vol/vol), 100 pm radiolabeled bile acid substrate, and whole cells (12 mg of protein). The standard cell extract reaction mixture contained (1.O ml total volume): 25 mm sodium acetatemorpholinopropanesulfonate (MOPS) buffer (ph 7.5), 100 pm radiolabeled bile acid substrate, 0.5 mm NAD+, and cell extract (1 2 mg of protein). Assays were initiated by the addition of protein and were incubated anaerobically (37 C) under an argon atmosphere for 2 min. Enzyme activity was terminated by acidification. The acidified mixtures were extracted and the bile acid products were quantitated as described previously (1 9). The products of 7dehydrox 146 Journal of Lipid Research Volume 23, 1982
3 ~ ~ ~ ylation were characterized and identified as described previously (22). Reduced flavin nucleotides were prepared as described by Feighner and Hylemon (23). was linear with time up to 4 min and the rate was linear with protein ( mg of protein). PLBiochemicals Inc. and flavin nucleotides and Torula yeast glucose6phosphate dehydrogenase were from Sigma Chemical Co. Bile acids and bile salts were obtained from Calbiochem. All other chemicals were the highest grade commercially available. Anaerobic gel filtration column chromatography Anaerobic gel filtration chromatography of induced cell extracts was carried out on a column (1.6 X 73 cm) of BioGel A 1.5 M at 4 C (19). The column was washed with anaerobic 50 mm sodium acetatemops buffer (ph 7.5) containing 12% glycerol (vol/vol) and 5 mm glutathione. Protein was eluted with the same buffer and fractions (3.5 ml) were collected by hand in stoppered tubes under an argon atmosphere. The column was calibrated with NADH:flavin oxidoreductase (260,000) from Eubacterium sp. V. P. I (24) catalase (240,000), aldolase (1 58,000), bovine serum albumin (68,000), and blue dextran Chemicals and enzymes [ 24'4C]Chenodeoxycholic acid (50 mci/mmol) and [ 11, 123 H (N)]ursodeoxycholic(37 Ci/mmol) acid were purchased from New England Nuclear. [''C]7Ketolith ocholic acid was synthesized enzymatically from [''C]chen~de~xy~holic acid using 7ahydroxysteroid dehydrogenase (25). Radiolabled bile acids were 99% pure as determined by thinlayer chromatography. Molecular weight standards for column chromatography were Combithek Calibration Proteins I1 from Boehringer Mannheim. The pyridine nucleotides were purchased from RESULTS Induction of chenodeoxycholic acid and ursodeoxycholic acid 7a and 78dehydroxylation The time course relationship between cell growth and the induction of both 7a and 78dehydroxylation activities measured in whole cell suspensions is shown in Fig. 1. When cholic acid (panel A), was added to logarithmic cultures of Eubacterium sp. V. P. I as an inducer, 7a and 78dehydroxylation activities increased 40 and!%fold, respectively. Although both activities increased at approximately the same rate, maximal 78dehydroxylation activity was 3fold lower than 7adehydroxylation activity in whole cell suspensions (Table 1). In contrast, ursodeoxycholic acid did not induce significant amounts of 7a or 78dehydroxylation activities (Fig. 1B). Cofactor requirements for the 7dehydroxylation of chenodeoxycholic acid and ursodeoxycholic acid The effects of various pyridine and flavin nucleotides on the specific activities of 7a and 78dehydroxylation were determined with either chenodeoxycholic acid or TABLE 2. Cofactor requirements for the 7dehydroxylation of chenodeoxycholic acid and ursodeoxycholic acid by anaerobically dialyzed cell extracts of Eub~cterIum sp. V.P.I Enzyme Specific Activity' Chenodeoxycholic Ursodeoxycholic Cofactors (mm) Acid Acid No additions 139 f f 24 NAD+ (0.5) 503 f f 22 NAD' (0.5) FADHz (0.2) 521 f f 85 NAD+ (0.5) FMNHz (0.2) 478 f f 50 NAD' (0.5) FAD (0.2) 626 f f 89 NAD' (0.5) FMN (0.2) 569 f f 45 NAD+ (0.5) + FADHZ (0.2) + NADH (0.5) 231 f f 25 NADP' (0.5) 218 f NADH (0.5) 34 f 7 29 f 5 NADPH (0.5) 80 f & 18 FADHz (0.2) 189 & f 9 FMNHZ (0.2) 224 f FAD (0.2) 244 f f 3 FMN (0.2) 181 f f 7 Units are nmoles lithocholic acid formed per hr per mg protein. White, Fricke, and Hylemon of ursodeoxycholic acid 147
4 .. C..... _.. \ r'., ' Fig. 2. 7BDehydroxylation of ursodeoxycholic acid (U) to lithocholic acid (L) by cell extracts of Eubacfenum sp. V.P.I Lane 1, uninduced cell extract containing 0.5 mm NAD+ and 0.2 mm FADH2; lane 2, induced cell extract containing 0.5 mm NAD' and 0.2 mm FADH2; lane 3, induced cell extract containing 0.5 mm NAD+; lane 4, induced cell extract containing 0.2 mm FADH2; lane 5, induced cell extract, no additions; lane 6, ursodeoxycholic acid standard; lane 7, lithocholic acid standard. The reaction mixtures were terminated after 5 min and the bile acid products were extracted and chromatographed on thinlayer plates. Bile acid products were detected by spraying thinlayer plates with phosphomolybdic acid and heating for 1015 min at 18OOC. ursodeoxycholic acid as the substrate. As shown in Table 2, the specific activity of 78dehydroxylase was approximately 3 to 5fold lower than 7adehydroxylase in dialyzed cell extracts. NAD+ was the only cofactor that significantly stimulated the activities of 7a and 78dehydroxylation when added alone to reaction mixtures. NADH inhibited both 7a and 78dehydroxylation activities. When reaction mixtures containing ursodeoxycholic acid were chromatographed on thinlayer plates, neither 7ketolithocholic acid nor chenodeoxycholic acid was detected (Fig. 2). In addition, Eubacterium sp. V.P.I does not possess detectable 78hydroxysteroid dehydrogenase activity when assayed by methods described previously (25). Moreover, no bile acid products were detected when ['4C]7ketolithocholic acid was added as the substrate in our standard cell extract 7dehydroxylase assay. Saturation kinetics Saturation kinetics for both 7a and 70dehydroxylation activities were determined for NAD+, chenodeoxycholic acid, and ursodeoxycholic acid. The saturation curve for NAD+ with ursodeoxycholic acid (Fig. 3) as the substrate was hyperbolic and double reciprocal plots yielded an apparent K, value of 0.6 pm. The substrate saturation kinetics for chenodeoxycholic acid (Fig. 4) showed substrate inhibition at concentrations above 200 pm. The apparent K, was calculated to be 49 pm. Kinetics for ursodeoxycholic acid (Fig. 5) were amost identical to the kinetics for chenodeoxycholic acid, but showed a somewhat lower apparent K,,, of 14.5 pm. 148 Journal of Lipid Research Volume 23, 1982
5 1 0 I IO NAD'O I I I I I I I I I I 1 O ; Fig. 3. The effect of NAD+ concentration on 7fbdehydroxylation activity using ursodeoxycholic acid (100 PM) as the substrate. The K, value was determined by a double reciprocal plot (inset) which was drawn by linear regression analysis. Assays contained 1 mg of cell extract protein and all other standard assay components described in the Materials and Methods Section. The assays also contained an NADPHregenerating system consisting of 0.1 mm NADPH, 5 mm glucose6phosphate,and 2.5 units of NADPHspecificglucose6phosphate dehydrogenase (GPP DH). The G6PDH system was employed to inhibit the activity of NADP'dependent 7ahydroxysteroid dehydrogenase which is a competing enzyme for primary bile acid substrates in extracts of this bacterium. The G6P DH system did not inhibit 7adehydroxylation activity at standard assay concentrations of NAD' and primary bile acid I CHENODEOXYCHOLIC ACID (pm 1 Fig. 4. The effect of chenodeoxycholic acid concentration on 7adehydroxylation activity in the presence of 1 mm NAD'. The K,,, value was determined by a double reciprocal plot (inset) which was drawn from linear regression analysis. Assay conditions were as described in Fig. 3. White, Fricke, and Hylemon 7&Dehydroxylation of deoxycholic acid 149
6 03e a I ET.: i l l 1 I 1 6 I t I IOOO/URSODEOXY CHOLIC ACID (a) I 60 URSODEOXYCHOLIC ACID (pm) Fig. 5. The effect of ursodeoxycholic acid concentration on 78dehydroxylation activity in the presence of 1 mm NAD+. The K, value was determined by a double reciprocal plot (inset) which was drawn from linear regression analysis. Assay conditions were as described in Fig. 3. Thermal inactivation of chenodeoxycholic acid and ursodeoxycholic acid 7dehydroxylation activity The data in Fig. 6 shows the effect of heating cell extracts on 7a and 78dehydroxylase activities. 7a and 78dehydroxylation activities were assayed using stan Anaerobic gel filtration chromatography dard conditions. Approximately, 60% to 70% of the initial 7a and 7fl dehydroxylation activity was lost when cell extracts were heated at 45 C for 6 min. Furthermore, both 7a and 78dehydroxylase activities decayed at approximately the Same rates Cell extracts containing 7a and 78dehydroxylation activities were chromatographed anaerobically on a Bio o 1 0 ; I I I I I PREINCUBATION (min) at 45OC Fig. 6. The effect of heating cell extracts on 7a (circles) and 78 (triangles) dehydroxylation activities. Cell extracts were heated for time periods of up to 6 min at 45OC. 150 Journal of Lipid Research Volume 23, 1982
7 P a ' ' Fig. 7. Anaerobic BioGel A 1.5M gel filtration chromatography of cell extracts containing 7a (triangles) and 78 (squares) dehydroxylation activities. Enzyme activities were determined as described in Materials and Methods. Gel A 1.5M gel filtration column as described in Materials and Methods. Both 7a and 7Bdehydroxylase activities coeluted as a single peak (Fig. 7), with an estimated relative molecular weight (Mr) of 1 14,000. Interestingly, 70dehydroxylation activity of these fractions was 2 to 3fold higher than 7adehydroxylation activity. The basis of this result is not known. All other experiments showed 7adehydroxylase activities 3 to 5 fold higher than 70dehydroxylase activities. DISCUSSION Several lines of evidence in this study demonstrate that Eubacterium sp. V.P.I is capable of the direct 70dehydroxylation of ursodeoxycholic acid. First, Eubacterium sp. V.P.I does not have any detectable 70hydroxysteroid dehydrogenase activity when assayed spectrophotometrically or by thinlayer chromatography techniques. Secondly, the formation of 7ketolithocholic acid or chenodeoxycholic acid from ursodeoxycholic acid was not detected under our standard assay conditions. Moreover, no bile acid products were detected when ['4C]7ketolithocholic acid was used as the substrate under the conditions required for 7a and 78dehydroxylation. bacterium. In addition, both activities, in dialyzed cell extracts, required the addition of NAD+ for maximal activities and were inhibited by NADH (Table 2). Thermal inactivation showed that the two activities decayed at approximately the same rate when heated at 45 C. Finally, both 7a and 70dehydroxylation activities coeluted in a single peak during gel filtration chromatography. However, further purification of 7a and 70dehydroxylase and the isolation of suitable 7dehydroxylase negative mutant strains of bacteria will be necessary to confirm the relationship between 7a and 78dehydroxylation in this bacterium. When the in vivo studies of the metabolism of chenodeoxycholic acid and ursodeoxycholic acid during gall Ckrtridium almnlwl d" it CHENODEOXYCHOLIC ACID 7KETOLITHOCHOLIC ACID.. I t URSODEOXYCHOLIC ACID gortridiumrp Evbvtrlum w. LITHOCHOLIC ACID LITHOCHOLIC ACID Both 7a and 7fLdehydroxylase activities appear to EuMriu" V.P.I. IZMB be carried out by the same enzyme complex. For ex Fig. 8. Pathways for the microbial biotransformation of chenodeoxyamp1e, both 7a and 78dehydroxy1ation activities were cholic acid and ursodeoxycholic acid during cholesterol gallstone disrapidly and specifically induced by cholic acid in this solution therapy. White, Fricke, and Hylemon 7flDehydroxylation of udeoxycholic acid 151
8 stone dissolution therapy are viewed in relationship to the in vitro data presented in this paper, additional interpretations are possible. In this regard, there appear to be two possible pathways for the formation of lithocholic acid during cholesterol gallstone dissolution therapy with either chenodeoxycholic acid or ursodeoxycholic acid (Fig. 8). First, lithocholic acid can be formed by the direct microbial 7dehydroxylation of either chenodeoxycholic acid or ursodeoxycholic acid. Alternatively, either of the 7hydroxy bile acids can be converted to its 7 epimer through a 7ketolithocholic acid intermediate (1 2) and the epimerized bile acid can then be 7dehydroxylated to lithocholic acid. Finally, the slower rate of 7pdehydroxylation in this bacterium is consistent with the in vivo studies of Fedorowski et al. (13) which showed that the rate of 7dehydroxylation of ursodeoxycholic acid was approximately 5fold slower than chenodeoxycholic acid. However, the in vivo rates of 7dehydroxylation of ursodeoxycholic acid and chenodeoxycholic acid are likely to vary as a function of the composition of the intestinal microflora and bile acid concentration (Figs. 4 and 5). In this regard, Hirano and coworkers (26) recently reported the isolation of several new strains of 7adehydroxylating intestinal bacteria that appear distinct from the Eubacterium species used in the current investigation. In summary, this report represents the first documentation of the direct 7&dehydroxylation of ursodeoxycholic acid in both whole cells and cell extracts of an intestinal anaerobic bacterium. These studies may be of practical significance in the treatment of cholesterol gallstones by chenodeoxycholic acid therapy if an inhibitor of 7a and 7pdehydroxylase is discovered. Such an inhibitor would prevent the formation of lithocholic acid by intestinal bacteria and may increase the effectiveness of these bile acids in the dissolution of cholesterol gallstones. I We would like to thank Dr. Thomas L. Glass for his help in the preparation of this manuscript. This investigation was supported by Grant CA from the National Cancer Institute, DHHS and by Public Health Service Research Grant AM from the National Institute of Arthritis, Metabolism and Digestive Diseases. Manuscript received 29 April 1987 and in revised form 70 August REFERENCES 1. Hofmann, A. F The medical treatment of cholesterol gallstones: major advance in preventive gastroenterology. Am. J. Med Thistle, J. L., and A. F. Hofmann, Efficacy and specificity of chenodeoxycholic acid therapy for dissolving gallstones. N. Engl. J. Med Dyrska, H., G. Salen, G. Zaki, T. Chen, and E. H. Mosbach Hepatic toxicity in the rhesus monkey treated with chenodeoxycholic acid for six months: biochemical and ultrastructure studies. Gastroenterology Palmer, R. H Bile acids, liver injury and liver disease. Arch. Intern. Med Morrissey, K. P., C. K. McSherry, R. L. Swarm, W. H. Nieman, and J. E. Deitrick Toxicity of chenodeoxycholic acid in the nonhuman primate. Surgery. 77: Makino, I., K. Shinazaki, K. Yoshino, et al Dissolution of cholesterol gallstones by ursodeoxycholic acid. Jpn. J. Gastroenterol Stiehl, A., P. Czygan, B. Kommerell, H. J. Weiss, and K. H. Holtmiiller Ursodeoxycholic acid versus chenodeoxycholic acid: comparison of their effects on bile acid lipid composition in patients with cholesterol gallstones. Gastroenterology. 75: Salen, G., G. S. Tint, B. Eleav, N. Deering, and E. H. Mosbach Increased formation of ursodeoxycholic acid in patients treated with chenodeoxycholic acid. J. Clin. Invest Fedorowski, T., G. Salen, A. Colallilo, G. S. Tint, E. H. Mosbach, and J. C. Hall Metabolism of ursodeoxycholic acid in man. Gastroenterology. 73: Higgashi, S., T. Setoguchi, and T. Katsuki Conversion of 7ketolithocholic acid by human intestinal anaerobic microorganisms: interchangeability of chenodeoxycholic and ursodeoxycholic acid. Jpn. J. Gastroenterology Yahiro, K., T. Setoguchi, and T. Katsuki Effect of cecum and appendix on 7adehydroxylation and 78epimerization of chenodeoxycholic acid in the rabbit. J. Lipid Res. 21: Macdonald, I. A,, D. M. Hutchison, and T. P. Forrest Formation or urso and ursodeoxycholic acids from primary bile acids by Clostridium absonum. J. Lipid Res. 22: Fedorowski, T., G. Salen, G. S. Tint, and E. H. Mosbach Transformation of chenodeoxycholic acid and ursodeoxycholic acid by human intestinal bacteria. Gastroenterology. 77: Stellwag, E. J., and P. B. Hylemon Characterization of 7adehydroxylase in Clostridium leptum. Am. J. Clin. Nutr. 31: Aries, V., and M. J. Hill Degradation of steroids by intestinal bacteria. 11. Enzymes catalyzing the oxidoreduction of the 3a, 7a, and 12ahydroxyl groups of cholic acid and the dehydroxylation of the 7ahydroxyl group. Biochim. Biophys. Acta Ferrari, A., and L. Beretta Activity on bile acids of a Clostridium bifermentans cellfree extract. FEBS Lett. 75: Hylemon, P. B., A. F. Cacciapuoti, B. A. White, T. R. Whitehead, and R. J. Fricke aDehydroxylation of cholic acid by cell extracts of Eubacterium species V.P.I Am. J. Clin. Nutr White, B. A,, R. L. Lipsky, R. J. Fricke, and P. B. Hylemon Bile acid induction specificity of 7adehydroxylase activity in an intestinal Eubacterium species. Steroids White, B. A., A. F. Cacciapuoti, R. J. Fricke, T. R. Whitehead, E. H. Mosbach, and P. B. Hylemon Cofactor requirements for 7adehydroxylation of cholic and cheno 152 Journal of Lipid Research Volume 23, 1982
9 deoxycholic acid in cell extracts of the intestinal anaerobic bacterium, Eubacterium species of V.P.I J. Lipid Res Kalb, V. F., and R. W. Bernlohr, Jr A new spectrophotometric assay for protein in cell extracts. Anal. Biochem Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. 3. Biol. Chem Stellwag, E. J., and P. B. Hylemon aDehydroxylation of cholic acid and chenodeoxycholic acid by Clostridium leptum. J. Lipid Res Feighner, S. D., and P. B. Hylemon Characterization of a corticosticosteroid 21 dehydroxylase from the in testinal anaerobic bacterium, Eubacterium lentum. J. Lipid Res. 21: Lipsky, R. H., and P. B. Hylemon Characterization of an NADH:flavin oxidoreductase induced by cholic acid in a 7adehydroxylating intestinal Eubacterium species. Biochim. Biophys. Acta Hylemon, P. B., and J. A. Sherrod Multiple forms of 7ahydroxysteroid dehydrogenase in selected strains of Bacteroides fragilis. 1. Bactm'ol Hirano, S., R. Nakama, M. Tamaki, N. Masuda, and H. Oda Isolation and characterization of thirteen intestinal microorganisms capable of 7adehydroxylating bile acids. Appl. Environ. Microbiol. 41: White, Fricke, and Hylemon 7flDehydroxylation of udmxycholic acid 153
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