QUANTITATIVE-ANALYSIS OF VITAMIN-E, CHOLESTEROL AND PHOSPHOLIPID FATTY-ACIDS IN A SINGLE ALIQUOT OF HUMAN PLATELETS AND CULTURED ENDOTHELIAL-CELLS

A reliable procedure is described for the joint analysis of vitamin E (tocopherots), 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 chro matography of all compounds of interest and microcolumn purification o f tocopherols and cholesterol. The combined use of butyl hydroxytoluen e and ascorbic acid in the purification steps allowed a complete recov ery of the tocopherols analyzed, as well as of cholesterol by high-per formance liquid chromatography. The detection of these lipids was perf ormed with fluorometric, spectrophotometric and evaporative light-scat tering detectors whose respective sensitivities were compared. The fat ty acid composition of phospholipid classes from the same sample, sepa rated on the same silica gel plate, was determined by gas-liquid chrom atography. The whole procedure is rapid since it requires about 4 h to analyse tocopherols and cholesterol and to prepare methylated fatty a cids, 28 samples being easily completed within one working day. The ev aluation of the whole membrane antioxidant status requires as little a s one 25 cm(2) confluent culture flask (about 0.75x10(6) cells) for en dothelial cells or two mi of blood (3x10(8) platelets). (C) 1997 Elsev ier Science B.V.