A Rapid Method for Isolation of Mixed Conjugated Bile Acids From Bile. ∗

Abstract

In the course of studies on the comparative biochemistry of cholesterol metabolism, it became necessary to isolate conjugated bile acids from the bile of several species of animals. Published procedures are based on the use of either single-stage extraction or counter-current distribution(1). The former method does not readily yield a pure product, while the latter requires elaborate equipment. In this communication, a simple adsorption technic for the rapid isolation of the compounds is presented.

Method. The sample of bile is mixed with 20 volumes of absolute ethanol, and the precipitated salts and proteins are removed by nitration. The filtrate and washings are evaporated to dryness in vacuo, with temperature kept below 40°. The brown, gummy residue is taken up in a small volume of methanol, the solution filtered if necessary and applied to an alumina column. (The amount of alumina should be at least 60 times that of the residue.) The column is washed with methanol, and the methanol is collected in fractions. The bile pigments are completely retained on the top of the column, while bile acids, neutral steroids, and fats wash through rapidly. Occasionally the retention of bile pigments is not complete, and when this occurs, larger quantities of alumina in a wide column of high flow rate should be used. The first fraction collected often has a yellow tinge which, however, does not interfere with the subsequent steps. Each fraction is evaporated to a small volume, and excess anhydrous ether is added. The conjugated bile acids precipitate out as a white, flocculent powder, while the neutral steroids, fats, phospholipids, and unconjugated bile acids (if present) remain in solution. (Failure to employ anhydrous ether may result in a gummy, hygroscopic precipitate of bile acids.)