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-lipoxygenases (15-LOX) was implicated in early atherogenesis. We investigated the molecular mechanism of 15-LOX/LDL interaction and found that during short term incubations, LDL cholesterol esters are oxygenated preferentially by the enzyme. Even when the LDL particle was loaded with free linoleic acid, cholesteryl linoleate constituted the major LOX substrate. In contrast, only small amounts of free oxygenated fatty acid isomers were detected, and re-esterification of oxidized fatty acids into the LDL ester lipid fraction was ruled out. When LDL was depleted from alpha-tocopherol, specific oxygenation of the cholesterol esters was not prevented, and the product pattern was not altered. Similar results 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 incubations (up to 24 h), a less specific product pattern was observed. However, when the hydroperoxy lipids formed by the 15-LOX were immediately reduced 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 mammalian 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 unspecific product pattern, may become increasingly important. These secondary reactions appear to be suppressed when the hydroperoxy lipids formed are immediately reduced, when alpha-tocopherol-depleted LDL was used, or when the incubation sample was diluted. It may be concluded that 15-LOX-initiated LDL oxidation constitutes a dual-type oxygenase reaction with an initial enzymatic and a subsequent nonenzymatic phase. The biological relevance of this dual-type reaction for atherogenesis will be discussed.
Institute of Biochemistry, University Clinics Charité, Humboldt University, Hessische Str. 3-4, D-10115 Berlin, Federal Republic of Germany.
J
J
Belkner
H
H
Stender
H
H
Kühn
D000818:Animals; D001093:Arachidonate 15-Lipoxygenase / Q000378:metabolism*; D002788:Cholesterol Esters / Q000378:metabolism*; D008078:Cholesterol, LDL / Q000378:metabolism*; D004951:Esterification; D005227:Fatty Acids / Q000378:metabolism; D006801:Humans; D006880:Hydroxy Acids / Q000378:metabolism; D015227:Lipid Peroxidation; D010100:Oxygen / Q000378:metabolism; D011817:Rabbits; D013379:Substrate Specificity; D014810:Vitamin E / Q000378:metabolism*
D002788:Cholesterol Esters; D008078:Cholesterol, LDL; D005227:Fatty Acids; D006880:Hydroxy Acids; D014810:Vitamin E; C021976:cholesteryl linoleate; D001093:Arachidonate 15-Lipoxygenase; D010100:Oxygen