A Method for Estimation of Both Bile Salts and Cholesterol in Small Amounts of Bile

Abstract

In experimental and human studies on the chemistry of the bile, it often happens that only very small amounts of bile are available in the gall bladder, as the contents of the diseased viscus are frequently very scanty. It is in these very ones that analysis of the bile may be the most important. The following method enables one to determine both the bile acids and cholesterol in a single cubic centimeter of bile.

Following the suggestion of Dr. F. C. Koch the marked affinity of petroleum ether for bile salts was utilized. The principle is that petroleum ether has the power of making a quantitative separation of the bile acids and cholesterol in alcoholic solutions. The procedure is as follows:

One cc. of bile is diluted with 6 cc. of alcohol, brought to a boil and filtered to deproteinize. The filtrate is then shaken in a separatory funnel with 30 cc. of petroleum ether and extracted 3 times. The alcoholic fraction is then warmed to drive out the petroleum ether and used for the bile acid determination. The ether fraction (about 100 cc.) containing the cholesterol is placed in an ordinary 37° incubator and will evaporate to dryness in 30 to 45 minutes, as its boiling point is 25° to 65°C. It is then dissolved in chloroform, made up to 5 cc. and used for an ordinary colorimetric cholesterol estimation. The pigments remain in the alcoholic fraction.

Thirty-five samples of bile, normal and abnormal from dogs and humans have been studied for controls of this technique. In no case could any test for bile acid be obtained in the ether fraction or for cholesterol in the alcohol fraction.

In 2 cases difficulty in separating the layers in the funnel occurred. This in each case was overcome by the addition of 2 drops of water.