Studies with clearing factor. 3. Application of column and thin layer chromatography for separation of lipid hydrolysis products by clearing factor.

Abstract

Many researchers have employed column and thin layer chromatography for the separation of lipids. Qualitative thin layer chro-matographic separation of cholesterol esters, triglycerides, fatty acids, cholesterol, di- and monoglycerides, and phospholipids on silica gel plates of normal plasma and of patients with lipid disorders has been reported(1). Also, separation of 1,3- and 1,2-diglycerides and α- and β-monoglycerides by thin layer chromatography, after pancreatic lipase digestion of fats, has been demonstrated(2). Silicic acid column chromatography was used for a similar purpose by Borgstrom(3), Fillerup and Mead(4), and Hirsch and Ahr-ens(5). In this report, the digestion products of the action of Clearing Factor on coconut oil were studied. The presence of α-, β-mono-glycerides and 1,2- and 1,3-diglycerides intermediates were indicated by various thin layer and column chromatographic procedures. The free fatty acids were the major end product of hydrolysis. Among several separation methods of complex lipid mixtures, column chromatography in combination with thin layer chromatography produced the best information and is recommended. Materials and methods. Post-heparin Clearing Factor was prepared as reported previously (10). Tripalmitin, triolein, and cholesterol were purchased from Mann Biochemical Laboratories. Monoolein, monopal-mitin, 1,2- and 1,3-dipalmitin were supplied by Dr. J. Hirsch. Conditions of hydrolysis of Ediol with Clearing Factor was the same as previously reported(9,10). Five ml aliquots were taken at zero time, 1 hr, 3 hr, and overnight from the incubation mixtures. Each aliquot was extracted with 50 ml of Bloor solution (3 parts ethanol:1 part diethyl ether). Thin layer plates were (20 × 20 cm glass) coated with a 225-250 layer of silica gel G and air dried(6). The plates were then activated for one hour at 160° and placed in a desiccator.