AN IMPROVED METHOD FOR QUANTIFICATION OF CHOLESTEROL AND CHOLESTERYL ESTERS IN HUMAN MONOCYTE-DERIVED MACROPHAGES BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH IDENTIFICATION OF UNASSIGNED CHOLESTERYL ESTER SPECIES BY MEANS OF SECONDARY-ION MASS-SPECTROMETRY

The measurement of cholesteryl esters in human monocyte-derived macrop hages using previously described high performance liquid chromatograph y methods is hampered by the presence in these cells of large amounts of triglycerides. We present a simple reversed phase high performance liquid chromatography protocol for quantification of cholesterol and c holesteryl esters in human monocyte/macrophages or other triglyceride- rich cells. Our method requires only lipid extraction and hydrolysis o f triglycerides using a solution of ethanolic potassium hydroxide and is of sufficient sensitivity to allow measurement in 10(5) cells. Use of this protocol led to the isolation of eight previously unassigned c holesteryl ester peaks comprising 16% of the total cholesteryl ester c ontent of human monocyte-derived macrophages. Using time-of-flight sec ondary ion mass spectrometry and synthesized authentic standards, seve n of these peaks were found to comprise cholesterol esterified with po lyunsaturated n-3 (omega 3) (cholesteryl eicosapentaenoate, docosatrie noate, docosapentaenoate, and docosahexaenoate) and n-6 (omega 6) (cho lesteryl docosatetraenoate, eicosadienoate, and eicosatrienoate) fatty acids. The remaining peak was shown to be the cholesteryl eater of n- 7 (omega 7) palmitoleic acid by comparison with a commercially availab le standard. The identification of all the cholesteryl esters in chole sterol-loaded human monocyte-derived macrophages will assist future st udies of lipid metabolism in these cells.