Determination of the proportion of cholesteryl ester in plasma.

Abstract

In normal human plasma the proportion of choles­ terol present as cholesteryl ester is very constant, but in certain clinical conditions, such as liver disease, a reduction may occur. Measurement of plasma cholesteryl ester is usually achieved indirectly as the difference between total cholesterol and free choles­ terol, which inevitably leads to a larger error in its determination. Plasma lipids in human and experi­ mental schistosomiasis (Owen and Gillett, 1978)show small but significant decreases in the percentage of cholesterol esterified. The measurement of such changes is the subject of this note. Thin-layer chromatography was used to separate free cholesterol and cholesteryl ester fractions followed by hydrolysis of both under identical conditions without prior elution from the silica gel. The non-saponifiable material was then extracted into hexane, and ali­ quots were taken for colorimetric estimation. Plasma lipids were extracted and washed accord­ ing to the procedure of Folch, and the neutral lipids were separated on 0·25 mm layers of silica gel G (Merck) using hexane-ether-acetic acid 90:20:1 (by vol) as the developing solvent. Areas correspond­ ing to free cholesterol and cholesteryl ester were located by exposure to iodine vapour and transferred to tubes (125 x 50 mm) fitted with Teflon-lined screw caps (Kimble Products, Ill., USA). Three millilitres of 1M ethanolic KOH was added and, after mixing, the contents were heated at 80°C for one hour. Hexane (5 ml) was added followed by 2% (w/v) Na2S04 (5 ml), the contents were mixed, and aliquots of the hexane phase were taken for cholesterol estimation. In preliminary experiments, recovery and extent of hydrolysis were examined under varying conditions by adding 7- 3H-cholesteryl oleate to the tubes. Determination of the percentage hydrolysis of the labelled cholesteryl ester at different temperatures (40°C, 6Q c C, and 80 c C) and for varying time inter