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

Abstract

The measurement of cholesteryl esters in human monocyte-derived macrophages using previously described high performance liquid chromatography 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 cholesteryl esters in human monocyte/macrophages or other triglyceride-rich cells. Our method requires only lipid extraction and hydrolysis of 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 cholesteryl ester peaks comprising 16% of the total cholesteryl ester content of human monocyte-derived macrophages. Using time-of-light secondary ion mass spectrometry and synthesized authentic standards, seven of these peaks were found to comprise cholesterol esterified with polyunsaturated n-3 (omega 3) (cholesteryl eicosapentaenoate, docosatrienoate, docosapentaenoate, and docosahexaenoate) and n-6 (omega 6) (cholesteryl docosatetraenoate, eicosadienoate, and eicosatrienoate) fatty acids. The remaining peak was shown to be the cholesteryl ester of n-7 (omega 7) palmitoleic acid by comparison with a commercially available standard. The identification of all the cholesteryl esters in cholesterol-loaded human monocyte-derived macrophages will assist future studies of lipid metabolism in these cells.