Hepoxilin A 3 is oxidized by human neutrophils into its ω-hydroxy metabolite by an activity independent of LTB 4ω-hydroxylase

Abstract

Hepoxilin A 3-methyl ester is taken up by intact human neutrophils where it is first hydrolyzed into the free acid which is subsequently converted into a single major metabolite. The structure of this metabolite was determined through mass spectral analysis of several derivatives, and through identity with an authentic compound prepared by chemical synthesis. The metabolite was identified as ω-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 with NADPH were unsuccessful. Metabolism of hepoxilin A 3 required the intact cell, while parallel experiments with LTB 4 as substrate demonstrated that this eicosanoid was metabolized into its ω-hydroxy metabolite regardless of whether intact or broken cell preparations were used provided that NADPH was present in the latter. Hepoxilin metabolism in intact cells was inhibited dose-dependently by CCCP (0.01–100 μM), a mitochondrial uncoupler, whereas LTB 4 metabolism was unaffected by CCCP. This data suggests that metabolism of hepoxilin A 3 occurs in intact human neutrophils through ω-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 characterized, relatively stable microsomal LTB 4 ω-hydroxylase.