Quantitative analysis of vitamin E, cholesterol and phospholipid fatty acids in a single aliquot of human platelets and cultured endothelial cells

Abstract

A reliable procedure is described for the joint analysis of vitamin E (tocopherols), cholesterol and phospholipids in the same minute sample of human platelets and on human cultured endothelial cells. The whole procedure is based on the extraction of total lipids, thin-layer chromatography of all compounds of interest and microcolumn purification of tocopherols and cholesterol. The combined use of butyl hydroxytoluene and ascorbic acid in the purification steps allowed a complete recovery of the tocopherols analyzed, as well as of cholesterol by high-performance liquid chromatography. The detection of these lipids was performed with fluorometric, spectrophotometric and evaporative light-scattering detectors whose respective sensitivities were compared. The fatty acid composition of phospholipid classes from the same sample, separated on the same silica gel plate, was determined by gas-liquid chromatography. The whole procedure is rapid since it requires about 4 h to analyse tocopherols and cholesterol and to prepare methylated fatty acids, 28 samples being easily completed within one working day. The evaluation of the whole membrane antioxidant status requires as little as one 25 cm2 confluent culture flask (about 0.75 x 10(6) cells) for endothelial cells or two ml of blood (3 x 10(8) platelets).