ESTERIFICATION OF OXYSTEROLS IN HUMAN SERUM - EFFECTS ON DISTRIBUTIONAND CELLULAR UPTAKE

Oxysterols, oxidized derivatives of cholesterol, may enter the circula tion as contamination of cholesterol-containing food, arise through pe roxidation of lipoproteins, or be generated by enzymatic reactions. Th ey are found in serum associated either with lipoproteins or with albu min. In these studies, 25-hydroxycholesterol (25OHC) was used as a mod el oxysterol of oxysterols with serum components and their delivery to cultured cells. 25OHC added in vitro to fresh human serum was readily esterified during incubation at 37 degrees C, most likely by serum le cithin:cholesterol acyltransferase (LCAT) as it was blocked by known i nhibitors of LCAT. The 25OHC-esters formed were identified as monoeste rs by comparing their elution on high performance liquid chromatograph y and thin-layer chromatography with that of chemically synthesized 25 OHC mono- and diesters. Esterification doubled the percentage of 25OHC associated with lipoproteins, concomitantly decreasing the amount ass ociated with albumin. Whereas unesterified 25OHC readily transferred b etween isolated lipoproteins, 25OHC-esters remained associated with do nor lipoproteins unless human lipoprotein-deficient serum was added. T hat cholesteryl ester transfer protein (CETP) mediated transfer of 25O HC-esters was demonstrated by the ineffectiveness of rat lipoprotein-d eficient serum as well as by the ability of IC-4, an anti-CETP monoclo nal antibody, to suppress the transfer. Esterification of 25OHC in ser um limited its entry into human erythrocytes and fibroblasts (GM 3468A cells) in vitro. Up-regulation of fibroblast low density lipoprotein (LDL)-receptors enhanced the uptake of esterified 25OHC from medium, b ut did little to enhance the total uptake of 25OHC. Thus, esterificati on of oxysterols in serum shifts their distribution away from albumin into LDL and high density lipoprotein (HDL) and limits their availabil ity to cells in culture.