title: Effect of cholesterol enrichment on 12-hydroxyeicosatetraenoic acid metabolism by mouse peritoneal macrophages.
abstract: The metabolism of 12-hydroxyeicosatetraenoic acid (12-HETE) was investigated in mouse peritoneal macrophages enriched in cholesterol by incubation with acetylated low density lipoproteins. After incubating with labeled arachidonic acid, cholesterol-rich cells released more 12-HETE into the medium than unmodified macrophages. With time, however, 12-HETE decreased in the medium of both cell preparations suggesting re-uptake of this monohydroxyfatty acid and perhaps further metabolism. When control macrophages were incubated with radiolabeled 12-HETE for 2 hr, almost 70% of the cell-associated 12-HETE label was incorporated into phospholipids. In contrast, in cholesterol-rich cells, only 31% of the 12-HETE label was incorporated into phospholipids. Bee venom phospholipase completely hydrolyzed the label, suggesting that the monohydroxyfatty acid was esterified at the sn-2 position of the phospholipid. In cholesterol-rich cells, 69% of the 12-HETE was diverted into neutral lipids. Two major neutral lipids were identified in cholesterol-rich macrophages. One neutral lipid band which migrated with an Rf value of 0.34 contained the hydroxylated fatty acid esterified to a glyceride. The other neutral lipid band having an Rf value of 0.49 contained cholesterol and by further analysis was found to contain predominantly cholesteryl-12-HETE. The labeled fatty acids in these two neutral lipids were mostly oxidized products of 12-HETE in contrast to the native 12-HETE observed in the phospholipids. Cholesterol-rich macrophages released 25% more products of 12-HETE metabolism than control macrophages. Two major products were observed in the medium which eluted in the area of a standard di-HETE, LTB4, on high performance liquid chromatography (HPLC) analysis. We propose that the reincorporation of 12-HETE into these neutral lipids and the increased capacity for further metabolism of this biologically potent hydroxyfatty acid could be a mechanism by which the cholesterol-rich macrophage maintains its membrane function, and regulates the amount of 12-HETE in the pericellular space.
mesh_terms: D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D005990:Glycerol / Q000378:metabolism; D006868:Hydrolysis; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism*; D008055:Lipids / Q000096:biosynthesis; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D010529:Peritoneal Cavity / Q000166:cytology
chemical_list: D001095:Arachidonic Acids; D006893:Hydroxyeicosatetraenoic Acids; D008055:Lipids; D016718:Arachidonic Acid; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D002784:Cholesterol; D005990:Glycerol