HEPOXILIN A(3) IS OXIDIZED BY HUMAN NEUTROPHILS INTO ITS OMEGA-HYDROXY METABOLITE BY AN ACTIVITY INDEPENDENT OF LTB4 OMEGA-HYDROXYLASE

Hepoxilin A(3)-methyl ester is taken up by intact human neutrophils wh ere it is first hydrolyzed into the free acid which is subsequently co nverted into a single major metabolite. The structure of this metaboli te was determined through mass spectral analysis of several derivative s, and through identity with an authentic compound prepared by chemica l synthesis, The metabolite was identified as omega-hydroxy-hepoxilin A(3) showing that the epoxide functionality of the parent hepoxilin is not opened during incubation with human neutrophils. All attempts to investigate hepoxilin metabolism in broken cells, despite the presence of protease inhibitors (Aproteinin, PMSF, DFP) and supplementation wi th NADPH were unsuccessful. Metabolism of hepoxilin A(3) required the intact cell, while parallel experiments with LTB4 as substrate demonst rated that this eicosanoid was metabolized into its omega-hydroxy meta bolite regardless of whether intact or broken cell preparations were u sed provided that NADPH was present in the latter. Hepoxilin metabolis m in intact cells was inhibited dose-dependently by CCCP (0.01-100 mu M), a mitochondrial uncoupler, whereas LTB4 metabolism was unaffected by CCCP. This data suggests that metabolism of hepoxilin A(3) occurs i n intact human neutrophils through omega-oxidation, is likely located in the mitochondrial compartment of the cell (inhibition by CCCP) and is carried out by an activity that is independent of the well characte rized, relatively stable microsomal LTB4 omega-hydroxylase. (C) 1997 E lsevier Science B.V.