7~Dehydroxylat ion of cholic acid and chenodeoxycholic acid by Clostridium leptum

The rate of 7a-dehydroxylation of primary bile acids was quantitatively measured radiochromatographically in anaerobically washed whole cell suspensions of Clostridium leptum. The pH optimum for the 7a-dehydroxylation of both cholic and chenodeoxycholic acid was 6.5-7.0. Substrate saturation curves were observed for the 7a-dehydroxylation of cholic and chenodeoxycholic acid. However, cholic acid whole cell (0.37 pM) and V (0.20 pmol hr-' mg protein-') values differed significantly from chenodeoxycholic acid whole cell (0.18 pM) and V (0.050 pmol-I hr-' mg protein-'). 7a-Dehydroxylation activity was not detected using glycineand taurine-conjugated primary bile acids, ursodeoxycholic acid, cholic acid methyl ester, or hyocholic acid as substrates. Substrate competition experiments showed that cholic acid 7a-dehydroxylation was reduced by increasing concentrations of chenodeoxycholic acid; however, chenodeoxycholic acid 7a-dehydroxylation activity was unaffected by increasing concentrations of cholic acid. A 10fold increase in cholic acid 7a-dehydroxylation activity occurred during the transition from logarithmic to stationary phase growth whether cells were cultured in the presence or absence of sodium cholate. In the same culture, a similar increase in chenodeoxycholic acid 7a-dehydroxylation was detected only in cells cultured in the presence of sodium cholate. These results indicate the possible existence of two independent systems for 7a-dehydroxylation in C. Leptum. Stellwag, E. J., and P. B. Hylemon. 7a-Dehydroxylation of cholic acid and chenodeoxycholic acid by Clostridium leptum. J . Lipid RQJ. 1979. 20: 325-333. Supplementary key words bile acids . substrate specificity . inhibitors The final composition of bile acids in biliary bile of man is dependent upon the combined action of liver biosynthetic enzymes as well as intestinal bacterial enzymes that degrade bile acids (1). Known microbial biotransformations include deconjugation of glycine or taurine conjugated bile acids to yield free bile acids (2-6), dehydroxylation at the C7 hydroxy group of the steroid nucleus (2, 7-1 l) , oxidation of the hydroxy groups at Cs, C,, and C12 (2,4, 11 14) and reduction of the ketone moieties to either a or /3 hydroxy groups (2, 11). Quantitatively, the most important bacterial modification of the primary bile acids cholic acid and chenodeoxycholic acid is 7-a-dehydroxylation which results in the formation of the secondary bile acids deoxycholic acid and lithocholic acid, respectively (2). 7a-Dehydroxylation of primary bile acids markedly alters the physical characteristics as well as the physiological effects of the bile acid molecule. Chemically, there is an alteration of the critical micellar concentration and a decrease in the solubility of secondary bile acids in aqueous solutions relative to their primary bile acid (15). Physiologically, it has been reported that oral administration of deoxycholic acid specifically suppresses the hepatic biosynthesis of chenodeoxycholic acid (16); however, in other studies, a decrease in the biosynthesis of both cholic and chenodeoxycholic acid has been reported (1719). Furthermore, deoxycholic acid has been reported to be capable of inducing the secretion of water and electrolytes from the small and large intestine via an apparent effect on adenylate cyclase activity (20). Moreover, secondary bile acids have been implicated as promoters of primary chemical carcinogens in laboratory animal studies (21). The proposed reaction mechanism for 7a-dehydroxylation of cholic acid is presented in Fig. 1. As elucidated by Samuelsson (15), the initial step in 7adehydroxylation occurs via a diaxial trans elimination of the 7a-hydroxy group and the 6p hydrogen atom. The proposed A 6 intermediate thus generated is subsequently reduced by trans hydrogenation at the 6/3 and 7a positions to yield deoxycholic acid. Abbreviations: Systematic names of bile acids referred to in the text by their trivial names are as follows: cholic acid, 3a,7a,12atrihydroxy-5P-cholanoic acid; glycocholic acid, 3a,7a,12a-trihydroxy-5P-cholanoyl glycine; taurocholic acid, 3a,7a, 12a-trihydroxy5P-cholanoyl taurine; cholic acid methyl ester, 3a,7a, I2a-trihydroxy-5P-cholanoyl methyl ester; hyocholic acid, 3a,6a,7a-trihydroxy-5P-cholanoic acid; chenodeoxycholic acid, 3a,7a-dihydroxy5P-cholanoic acid; ursodeoxycholic acid, 3a,7P-dihydroxy-5p-cholanoic acid; and lithocholic acid, 3a-monohydroxy-5~-cholanoic acid. DCCD, dicyclohexylcarbodiimide. ' Present address: Department of Physiological Chemistry, University of Wisconsin, Madison, Wisconsin. * Reprint requests should be sent to Dr. Phil Hylemon, Department of Microbiology, Virginia Commonwealth University, Box 847, Richmond, VA 23298. Journal of Lipid Research Volume 20, 1979 325 by gest, on O cber 7, 2017 w w w .j.org D ow nladed fom CHOLIC ACID PROPOSED d INTERMEDIATE DEOXYCHOLIC ACID Fig. 1. cholic acid, after Samuelsson ( 1 5). Proposed reaction mechanism tor 7a-dehydroxylation of Despite the importance of 7a-dehydroxylation in bile acid metabolism, published information regarding the characteristics of this steroid biotransformation reaction is conflicting and incomplete (2). Midvedt and Norman (4) and Dickinson, Gustaffson, and Norman (22) reported a limited distribution of 7a-dehydroxylation activity in bacteria isolated from intestinal contents. In contrast, Aries and Hill (1 1) reported that 7a-dehydroxylase is widespread in most species o f the predominant human intestinal microflora. The explanation for this discrepancy is not yet clear. In this communication we report the characterization of 7a-dehydroxylation activity in whole cells of Clostridium leptum using both cholic acid and chenodeoxycholic acid as substrate. We also provide preliminary evidence for the existence of two independent systems for 7a-dehydroxylation of the two primary bile acids and report levels of 7a-dehydroxylation activities in fecal samples from normal individuals. MATERIALS AND METHODS The organism now known as Clostridium leptum V.P.I. 10900 was originally isolated from a human fecal sample and was tentatively identified as a species ofBacteroides (2). However, after additional characterization studies by Drs. Holdeman and Moore at the Virginia Polytechnic Institute and State University, Anaerobe Laboratory, this bacterium was reclassified as a strain of Clostridium leptum. Stock cultures were maintained in chopped meat medium as described by Holdeman and Moore (23). The C. leptum V.P.I. 10900 utilized for in vitro characterization of 7a-dehydroxylation activity was cultured anaerobically under N2 in modified (made without salts solution) peptone-yeast extract medium containing 2 g/1 glucose and 0.1 mM sodium cholate essentially as described by Holdeman and Moore (23). Quantitative assay for 7a-dehydroxylation in C. leptum Enzymatic 7a-dehydroxylation of "C-carboxyllabeled cholic or chenodeoxycholic acid by whole cell suspensions of C. leptum V.P.I. 10900 was followed by measuring the rate of biotransformation to deoxycholic and lithocholic acids, respectively. Cells of C. leptum were harvested from a 1-liter stationary-phase culture (4 hr post-exponential) by centrifugation ( 13,700 g for 10 min at 25°C). The cell sediments were washed in 300 ml of 50 mM potassium phosphate buffer (pH 6.5) that had been made anaerobic by boiling for 10 min and cooling to 37°C under continuous flushing with argon gas previously passed through hot (35OOC) copper filings essentially as described previously (25). The standard reaction mixtures (1 .O ml) contained in final concentration: 2.04 p M [24-14C]cholic acid or 0.5 p M [24-14C]chenodeoxycholic acid (0.2 pCiIreaction mixture), 50 mM potassium phosphate buffer (pH 6.5), and an appropriate sample of whole cell suspension. Reaction mixtures were incubated anaerobically (37°C) in 1 x 8.5 cm test tubes equipped with rubber serum caps. The reaction mixtures were constantly flushed with argon gas via gas intake and exit needles for up to 30 min. Substrate conversion rates were linear up to 60 min. Enzymatic activity was terminated by the addition of 1 .O ml of 0.5 N HCl directly to the reaction mixtures (final pH 2.0). The acidified reaction mixtures were extracted and chrornatographed as described previously (6). The regions of the chromatogram corresponding to labeled substrate and product were located by use of a radiochromatogram scanner. These regions were scraped into scintillation vials containing Triton X1 00-based scintillation fluid and counted in a Beckman LS-350 liquid scintillation counter (5). A unit of enzyme activity was defined as the amount of enzyme required for the formation of 1 pmol of secondary bile acid formed per hr per mg whole cell protein under standard assay conditions. Reaction velocity was directly proportional to protein concentration over a range of 0.21.5 mg/nil. Protein concentration was tneasured by the method of Lowry et al. (24) after alkali solubilization ( 1 N NaOH) o f whole cells. Quantitative assays of fecal 7a-dehydroxylation activity 7a-Dehydroxylation of [24-14C]cholic acid and [24-14C]chenodeoxycholic acid by washed fecal suspensions was performed as follows. Approximately 5 g (wet weight) of freshly voided feces was suspended in 300 ml of anaerobically prepared potassium phosphate buffer (pH 6.5). The suspension was centrifuged (13,700g for 10 min at 25°C) and the top 2-3 mm of fecal sediment was removed with a spatula, suspended in 300 ml of anaerobic buffer, and centrifuged (13,700 g for 10 min at 25°C). Again, the top 2-3 mm of fecal sedi326 Journal of Lipid Research Volume 20, 1979 by gest, on O cber 7, 2017 w w w .j.org D ow nladed fom ment was removed and suspended in anaerobic potassium phosphate buffer (pH 6.5) to a turbidity of 280-300 Klett units (No. 66 filter). Aliquots of this fecal suspension were assayed for 7a-dehydroxylation activity essentially as described above. Protein concentration was determined on these aliquots. To determine the levels of viable 7a-dehydroxylating intestinal bacteria, serial 10-fold dilutions were carried out on these same fecal samples as described by Holdeman and Moore (23). Aliquots (0.5 ml) of each dilution were inoculated into peptone-yeast glucose medium (4.5 ml) containing 4 nmol of labeled cholic acid (0.2 pCi/tube). After 72 hr of incubation the bile acids were extracted with ethyl acetate and bile acid products were identified by thin-layer chromatography as described below. Identification of 'Icy-dehydroxylation products Products generated using cholic acid or chenodeoxycholic acid as substrates were isolated from C. leptum whole cell reaction mixtures and identified by thin-layer chromatography (Baker-flex Silica gel 1B-2, J. T. Baker Chemical C., Phillipsburg, NJ). Steroids were chromatographed in solvent systems S1, S6, and Slz as described by Eneroth (26). Reaction products were chromatographed separately and as mixtures with authentic bile acid standards. Bile arid 7a-dehydroxylation products and known standards were treated individually with specific 3a-, 7a-, and 1 Pa-hydroxysteroid dehydrogenases. The enzymatic conversion product generated by steroid dehydrogenase treatment was then chromatographed separately and as a mixture with known bile acid standards in solvent systems S1, s g , and slz to confirm identification. Chemicals and enzymes [24-14C]Cholic (50mCi/mmol) and [24-14C]chenodeoxycholic acids (50 mCi/mmol) were purchased from New England Nuclear, Boston, MA. [24-14C]Lithocholic acid (50 mCi/mmol) was obtained from Amersham Searle Corp., Arlington Heights, IL. The labeled lithocholic acid was purified by thin-layer chromatography prior to use. 3a-Hydroxysteroid dehydrogenase was obtained from Worthington Biochemicals, Freehold, NJ. 7a-Hydroxysteroid dehydrogenase and l Pa-hydroxysteroid dehydrogenase were isolated as described previously (13, 25). Ursodeoxycholic acid, hyocholic acid, and cholic acid methyl ester were purchased from Steraloids, Wilton, NH. All other bile acids and bile salts were obtained from Calbiochem, San Diego, CA. DicycloTABLE 1. Relative mobilities of bile acid standards and identification of secondary bile acids generated by Cloctrzdium I@um Mobility of standard (cmj Bile acid standards Scu, i f f , 12a sa, 1% 3a, 7cu 3 a 7a, 12a. %keto'' 12a, 3-keto" 3-keto' 3a, 1201, 7-keto" 3cu, 7-keto" 3cu, 7a. 12-keto" Sa, 12-keto" Product zrlrntzfimtzon Product of cliolic acid 7a-dehydroxylation Untreated Derivativc of treatment" Derivative of treatmentd Derivative of treatment' 7cu-dehydroxylation Unti-eated Derivative of treatment" Derivativr of treatmentd Drribativr of treatment" I'roduct of chenodeoxycholic arid 7.0 3.0 6.8 0.2 1 1.00 0.17 1.00 3.16 1.00 0.94 2.83 0.91 1.73 4.66 2.00 0.72 2.20 1.00 1.52 4.40 2.00 1.97 5.33 2.38 0.90 1.50 0.66 1.35 5.50 1.17 0.54 1.50 0.63 1.47 4.06 1.77 0.97 3.12 1.00 1.50 4.40 1.97 0.98 3.14 0.98 1.48 4.06 1.79 1.74 4.66 1.98 2.00 3.32 2.36 1.71 4.70 1.98 1.72 4.66 1.98 I ' Described by Eneroth (26). " Mobility ot deoxycholic acid (R,j and cholic acids (Rc). Derived by treatment with Sa-hydroxysteroid dehydrogenase. Derived by treatment with 7a-hydroxysteroid dehydrogenase. Drrived by treatment with 12a-hydroxysteroid dehydrogenase. hexylcarbodiimide (DCCD) and acriflavin were purchased from Sigma Chemical Co., St. Louis, MO.