The Gaschromatographic Determination of Fatty Acids. Composition of Serum Cholesterol Esters Separated by Thin Layer Chromatography

Abstract

A method is described for the determination of the fatty acid composition of serum cholesterol esters by gas liquid chromatography, after their separation by thin layer chromatography.Serum extracts were prepared and washed by the method of Folch et al. Thin layer plates (20×20cm) were coated with Silica Gel G (Merk, Germany) to a thickness of 500μ.A solution of total serum lipids in chloroform was applied linealy across the plate (2cm from the bottom). The plate was developed in a solvent system composed of petroleum ether (bp. 30-60°C)/diethylether, 95/5, v/v.Thin layer chromatogram of total serum lipids showed widely separeted band of cholesterol esters (Rf, 0.9) and other three bands of triglyceride (Rf, 0.3) cholesterol (Rf, 0.2) and phospholipids (origin). The developing time was about 20 minutes. After chromatography, the band of cholesterol esters was scraped onto filter paper and eluted with 40ml of chloroform/methanol, 2/1, v/v.An alkaline hydrolysis of cholesterol esters was performed with N/5-methanolic KOH for 2hr. and fatty acid was methylated by 10% BF3 methanol for 3 minutes. Methyl esters of fatty acid were chromatographed on a 25% ethylene glycol succinate polyester column at 198°C, using hydrogen flame ionization detector.The minimum amount of cholesterol esters required for this method is about 4.0mg which correspond to 3ml of human serum. The following are average values of fatty acid composition of serum cholesterol esters from young healthy women (5 cases) analyzed by this method. Myristic acid: 0.8, palmitic acid: 14.5, palmitoleic acid: 4.6, stearic acid: 2.0, oleic acid: 20.4, linoleic acid: 52.1, and arachidonic acid: 5.6.By the procedures described, cholesterol esters of serum lipids were quantitatively recoverd from thin layer plates and there were no detectable changes on their fatty acid compositions during thin layer chromatography.Triplicate determination indicate that the method gives highly reproducible results.