In vitro Formation of Deoxycholic and Lithocholic Acid by Human Intestinal Microorganisms. ∗ †

Elimination of the 7-hydroxysubstituent of the bile acids by intestinal microorganisms is a common reaction occurring in many species (1) and has been reproduced in vitro by a mixture of anaerobic intestinal microorganisms from the rat(2). Samuelsson has demonstrated that the reaction mechanism is a dehydration to a Δ⁶-derivative, followed by reduction (3).

Human bile contains 3 main bile acids, cholic, chenodeoxycholic and deoxycholic acid, with a mean ratio of 1.1:1.0:0.6(4). Cholic and chenodeoxycholic acids are formed directly from cholesterol in the liver (1). Due to the action of microbial enzymes the 7-hydroxyl group of cholic acid is removed and the “secondary” bile acid deoxycholic acid is formed(5). Contrary to the rapid transformation of cholic acid, chenodeoxycholic acid is transformed slowly during the enterohepatic circulation. Three days after administration of chenodeoxycholic acid-24-C¹⁴ only 3% of the isotope in the human bile was recovered as lithocholic acid(6). Although definitive proof is lacking, investigation so far indicates that deoxycholic and lithocholic acids are not metabolized by the human liver to a significant extent(6,7). The different concentration of deoxycholic and lithocholic acids in human bile, therefore, might be explained by a difference in ability of the intestinal flora to remove the 7-hydroxyl group from cholic and chenodeoxycholic acid or by a difference in accessibility for absorption of the “secondary” bile acids.

The aim of the present investigation was to study the formation of deoxycholic and lithocholic acid in subcultures of mixed human intestinal microorganisms.

Experimental. The 24-C¹⁴-labeled cholic, deoxycholic and chenodeoxycholic acids were prepared according to Bergström et al.(8).

Labeled bile acids as sodium salts dissolved in 80% ethanol were added to thick walled centrifuge tubes (inside diameter 13 mm, height 100 mm). After evaporation, the residue was taken up in 4 ml of culture medium. The bile acid concentration was always 0.2 mM/l broth. The tubes were plugged with cotton and autoclaved for 20 minutes. The first subculture was made by inoculating one loop of human feces into each experimental tube and into a tube without bile acid. Subsequent serial stock subcultures of mixed microorganisms were made in medium free of bile acids. One drop from a serial stock subculturc was inoculated into the respective set of experimental tubes containing the bile acids. For aerobic incubation the tubes were cotton-stoppered.