THE RABBIT 15-LIPOXYGENASE PREFERENTIALLY OXYGENATES LDL CHOLESTEROL ESTERS, AND THIS REACTION DOES NOT REQUIRE VITAMIN-E

The oxidation of low density lipoprotein (LDL) by mammalian 15-lipoxyg enases (15-LOX) was implicated in early atherogenesis, We investigated the molecular mechanism of 15-LOX/LDL interaction and found that duri ng short term incubations, LDL cholesterol esters are oxygenated prefe rentially by the enzyme. Even when the LDL particle was loaded with fr ee linoleic acid, cholesteryl linoleate constituted the major LOX subs trate. In contrast, only small amounts of free oxygenated fatty acid i somers were detected, and re-esterification of oxidized fatty acids in to the LDL ester lipid fraction was ruled out. When LDL was depleted f rom alpha-tocopherol, specific oxygenation of the cholesterol esters w as not prevented, and the product pattern was not altered. Similar res ults were obtained at low (LDL/LOX ratio of 1:1) and high LOX loading (LDL/LOX ratio of 1:10) of the LDL particle. During long term incubati ons (up to 24 h), a less specific product pattern was observed. Howeve r, when the hydroperoxy lipids formed by the 15-LOX were immediately r educed by the phospholipid hydroperoxide glutathione peroxidase, when the reaction was carried out with vitamin E-depleted LDL, or when the assay sample was diluted, the specific pattern of oxygenation products was retained over a long period of time. These data suggest that mamm alian 15-LOX preferentially oxidize LDL cholesterol esters, forming a specific pattern of oxygenation products. During long term incubations , free radical-mediated secondary reactions, which lead to a more unsp ecific product pattern, may become increasingly important. These secon dary reactions appear to be suppressed when the hydroperoxy lipids for med are immediately reduced, when alpha-tocopherol-depleted LDL was us ed, or when the incubation sample was diluted. It may be concluded tha t 15-LOX-initiated LDL oxidation constitutes a dual-type oxygenase rea ction with an initial enzymatic and a subsequent nonenzymatic phase. T he biological relevance of this dual-type reaction for atherogenesis w ill be discussed.