Convenient Non-Chromatographic Assays for the Microbial Deconjugation and 7α-OH Bioconversion of Taurocholate

Abstract

We described two convenient assay methods to estimate bile acid deconjugation and bile acid bioconversion at the 7α-OH position by individual microorganisms grown in media containing taurocholic acid. The methods are based on (i) a selective chemical assay for taurine conjugates previously described and (ii) the use of a cell-free preparation of 7α-hydroxysteroid dehydrogenase from Escherichia coli to directly quantify 7α-OH groups. These non-chromatographic approaches have been applied to the study of three model strains of intestinal organisms, E. coli, Bacteriodes fragilis , and Clostridium perfringens , grown in standard media in the presence of purified tritiated taurocholate. Assay results were confirmed by thin-layer chromatography solvent systems designed to separate conjugated from unconjugated bile acid and unmodified cholic acid nucleus from 7α-OH bioconversion product(s) (primarily 3α,12α dihydroxy, 7-keto-cholanoic acid). In addition, 7α-hydroxysteroid dehydrogenase activity was demonstrated in cell-free extracts of all three organisms. Of the three organisms, only C. perfringens was demonstrated to (i) deconjugate taurocholic acid, (ii) contain 3α-hydroxysteroid dehydrogenase activity, (iii) convert cholic acid into at least five labeled metabolites visible on thin-layer chromatography, and (iv) catalyze significant tritium exchange with water in the medium.