Noriyuki Masuda

Deconjugation of Bile Salts by Bacteroides and Clostridium

Deconjugation of bile salts by four strains of Bacteroides and four strains of Clostridium was studied by use of resting cells and cell‐free culture supernatants. Bacteroides strains yielded active cells but showed relatively low bile salt hydrolase (BSH) activity in the culture supernatants while the reverse was the case for the spore‐forming clostridial strains.

Transformation of Bile Acids by Mixed Microbial Cultures from Human Feces and Bile Acid Transforming Activities of Isolated Bacterial Strains

Microbial transformation of cholic acid and chenodeoxycholic acid by anaerobic mixed cultures of human fecal microorganisms was investigated, and the results were examined in relation to the bile acid transforming activities of 75 bacterial strains isolated from the same fecal cultures. The reactions involved in the mixed cultures were dehydrogenation and dehydroxylation of the 7α‐hydroxy group in both primary bile acids and epimerization of the 3α‐hydroxy group in all metabolic bile acids. Extensive epimerization of the 7α‐hydroxy group of chenodeoxycholic acid yielding ursodeoxycholic acid was also demonstrated by certain fecal samples. 7α‐Dehydrogenase activity was widespread among the fecal isolates (88% of 16 facultative anaerobes and 51% of 59 obligate anaerobes), and 7α‐dehydroxylase activity was revealed in one of the isolates, an unidentified gram‐positive nonsporeforming anaerobic bacterium. 3α‐Epimerization was effected by seven strains assigned to Eubacterium lentum, which were also active for 3α‐ and 7α‐dehydrogenations. No microorganism accounting for 7α‐epimerization was recovered among the isolates. Splitting of conjugated bile acid was demonstrated by the majority of obligate anaerobes but the activity was rare among facultative anaerobes.

Purification and characterization of NADP-dependent 7β-hydroxysteroid dehydrogenase from Peptostreptococcus productus strain b-52

An NADP-dependent 7 beta-hydroxysteroid dehydrogenase was purified 11.5-fold over the activity in crude cell extracts prepared from Peptostreptococcus productus strain b-52, by using Sephadex G-200 and DEAE-cellulose column chromatography. 7 beta-Dehydrogenation was the sole transformation of bile acids catalyzed by the partially purified enzyme. The enzyme preparation (spec. act. 2.781 IU per mg protein) had an optimum pH of 9.8. Lineweaver-Burk plots showed a Michaelis constant (Km) value of 0.05 mM for 3 alpha, 7 beta-dihydroxy-5 beta-cholanoic acid whereas higher values were obtained with 3 alpha,7 beta-dihydroxy-5 beta-cholanoyl glycine (0.20 mM), and 3 alpha,7 beta-dihydroxy-5 beta-cholanoyl taurine (0.26 mM). NADP but not NAD could function as an electron acceptor, and had a Km value of 0.30 mM. A molecular weight of 64000 was determined by SDS-polyacrylamide gel electrophoresis. The addition of 0.4 mM of either bile acid to the growth medium suppressed not only cell growth, but also the enzyme yield.

Effect of Neomycin on the In Vitro Transformation of Bile Acids by Mixed Microbial Flora of Human Feces

In the digestive tract of man and animals, resident microorganisms cause a series of bile acid-transforming reactions involving deconjugation, dehydroxylation, dehydrogenation and epimerization, all of which can be reproduced in in vitro cultures of mixed intestinal microorganisms. However, our knowledge of the individual microorganisms causing these reactions is incomplete, and there is still much to learn about the metabolism of bile acids by the Bacteroides, the predominant members of the normal intestinal flora. To elucidate the significance of these organisms as a whole, the transformation of bile acids by mixed fecal cultures in the presence and in the absence of neomycin which is known to facilitate the relative, although not selective, growth of the Bacteroides (5, 19) was compared. Portions of fecal specimens, homogenized with 10 volumes of a sodium phosphate buffer solution (0.02 M, pH 7.0) and freed of coarse debris by centrifugation, were inoculated into tubes of a buffered 2% peptone-yeast extract broth (11), containing 0.4 msr cholic acid (CA) or chenodeoxycholic acid (CDCA), the two primary bile acids constituting the major components of human bile. Neomycin and related antibiotics were filter-sterilized separately and added aseptically to this medium at the final concentrations recommended by Finegold et al (6). After anaerobic incubation for 1 to 7 days, the spent culture medium was extracted and assayed for bile acids by gas-liquid chromatography (GLC) in the manner described in an earlier study (11). Anaerobic incubation was carried out in an anaerobic jar which was evacuated and filled with a gas mixture of 90% nitrogen and 10% carbon dioxide (freed of oxygen by passage over heated copper gauze). The replacement was repeated three times. The jar previously contained a lump of steel wool impregnated with acidic copper sulfate. The experimental data with fecal samples from three healthy adults are summarized in Tables 1 and 2. In cultures without antibiotics, CA was completely degraded by 7a-dehydroxylation yielding the corresponding quantities of deoxycholic acid (DCA) by the 2nd and 3rd day of incubation. 7-Ketodeoxycholic acid (7KD), a 7a-dehydrogenation product, was detected early in the incubation period but disappeared later, concurrently with the increased formation of DCA through 7a-dehydroxylation. It thus appears that 7a-dehydrogenation is reversible and that the oxidation product, 7KD, is reduced to CA as an anaerobic environ-