FREE AND ESTERIFIED OXYSTEROL - FORMATION DURING COPPER-OXIDATION OF LOW-DENSITY-LIPOPROTEIN AND UPTAKE BY MACROPHAGES

We have defined the lipid composition of copper-oxidized LDL (Cu-oxLDL ) and a macrophage-foam cell model generated by the uptake of this mod ified lipoprotein. An HPLC method previously developed by our group fo r the measurement of lipid oxidation products of LDL was extended to p ermit the analysis of an array of 7-ketocholesteryl esters. Gas chroma tography was used for the quantitation of oxysterols (free and esterif ied) in Cu-oxLDL and their subsequent uptake by macrophages. LDL (1.0 mg protein/ml) was oxidized using Cu(II) (20 mu m) for up to 48 h at 3 7 degrees C. Resident mouse peritoneal macrophages were incubated with 24 h Cu-oxLDL (50 mu g/ml) for 24 h. In 24 h Cu-oxLDL, cholesterol co mprised approximately 50% of total sterols, 7-ketocholesterol comprise d approximately 30%, with five other oxysterols comprising the remaind er (7 alpha- and 7 beta-hydroxycholesterol, cholesterol alpha- and bet a-epoxides, and 6 beta-hydroxycholesterol). Macrophages that were incu bated with 24 h Cu-oxLDL displayed a profile of oxysterols remarkably similar to that of 24 h Cu-oxLDL itself. The majority of cholesteryl e sters and 7-ketocholesteryl esters in Cu-oxLDL and in Cu-oxLDL-loaded macrophages contained fatty acyl chains which are presumed oxidized. T his work represents a comprehensive survey of free and esterified oxys terols in Cu-oxLDL and Cu-oxLDL-loaded macrophages and provides a basi s for exploring how oxysterols are metabolized by macrophages and auth entic human foam cells, and how, in turn, these oxysterols influence c ellular metabolism.