A novel method for the separation and identification of bile acids and phospholipids of bile on thin-layer chromatograms.

Cholesterol gallstone disease, so common in the western world, ‘has consider&le medical and economic importance. Under normal physiological conditions, cholesterol is held in solution in the bile. If the solubility of cholesterol in bile is changed, cholesterol crystals will precipitate and lead to the formation of gallstones. The solubility of cholesterol in bile depends on three major lipid components of bile: conjugated bile salts, phospholipids and cholesterol’. Knowledge of bile lipid composition is important in our understanding of the mechanism of gallstone formation. Paper chromatography2 and silicic acid column chromatography3 have been used for the analysis of the composition of bile lipids. However, the latter technique has been found less satisfactory for routine bile analysis or to experiment on small animals because of the limited quantity of the bile samples. Although thin-layer chromatographic separation of bile lipids4 can be performed using three successive solvent systems, it takes as long as 8 h and does not delineate the free bile acids one from the other. Since bile contains a variety of compounds, different plates and different solvent systems must be used for the separation of each class of compounds. Various solvent systems have been proposed to achieve the separation of bile acids and their conjugates5*6 on thin-layer chromatograms. Our method successfully accompiishes the separation of severai ciasses of biie iipids on a singie piate which makes a direct comparison possible between different bile lipid components such as cholesterol, free and conjugated bile acids, lecithin and lysolecithin. The solvent system and the spray reagent used in our technique, not previously described, are satisfactory for a routine check of bile lipid composition and may be used for the skparation of individual bile acids, both free and conjugated, in biological fluids on the same plate.