ESTERIFICATION OF OXYSTEROLS BY HUMAN PLASMA LECITHIN-CHOLESTEROL ACYLTRANSFERASE

In the present study, lecithin-cholesterol acyltransferase (LCAT) cata lyzed esterification of oxysterols was investigated by using discoidal bilayer particles (DBP) containing various oxysterols, phosphatidylch olines, and apolipoprotein A-I. The esterified oxysterols were analyze d by high pressure liquid chromatography, gas chromatography, and mass spectrometry. LCAT esterified all oxysterols tested that are known to be present in human plasma. The esterification yields in almost all c ases were relatively high, often as high as the yield of cholesterol e sterification. When DBP preparations containing 27-hydroxycholesterol and various phosphatidylcholines were used for the LCAT reaction, both monoesters and diesters were produced. The mass spectrometry analysis showed that the monoester was produced by the esterification of the 3 beta-hydroxyl group and not the 27-hydroxyl group. The diesters were apparently produced by the esterification of the 27-hydroxyl group onl y after the esterification of the 3 beta-hydroxyl group. Phosphatidylc holine containing a saturated acyl group at sn-1 position and an unsat urated acyl group at sn-2 position gave generally high esterification yield. The esterification of various oxysterols was compared by using DBP containing dioleoyl-phosphatidylcholine and individual oxysterols. All oxysterols produced 3 beta-oleoyl monoesters, Unlike 27-hydroxych olesterol, 25-hydroxycholesterol, 7 alpha-hydroxycholesterol, 7 beta-h ydroxycholesterol, or cholestanetriol did not produce diesters. Variou s factors influencing the formation of the monoesters and diesters fro m 27-hydroxycholesterol were investigated. When dioleoyl-phosphatidylc holine was used as the acyl donor, prolonged dialysis of DBP preparati ons and increase in the ratio of the enzyme concentration to substrate particle concentration increased the diester formation. Significant a mounts of diesters were also produced by using 1-palmitoyl-2-oleoyl-ph osphatidylcholine and other phosphatidylcholines as the acyl donors. B y analyzing the conditions of monoester and diester formation, a schem e for the LCAT reaction pathway was proposed.