Hepoxilin A3 is oxidized by human neutrophils into its omega-hydroxy metabolite by an activity independent of LTB4 omega-hydroxylase.
Hepoxilin A3-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 omega-hydroxy-hepoxilin A3 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 A3 required the intact cell, while parallel experiments with LTB4 as substrate demonstrated that this eicosanoid was metabolized into its omega-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 microM), a mitochondrial uncoupler, whereas LTB4 metabolism was unaffected by CCCP. This data suggests that metabolism of hepoxilin A3 occurs in 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 characterized, relatively stable microsomal LTB4 omega-hydroxylase.
Reynaud
D
D
Research Institute, Hospital for Sick Children, Division of Neurosciences, Toronto, Ont., Canada.
Rounova
O
O
Demin
P M
PM
Pivnitsky
K K
KK
Pace-Asciak
C R
CR
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002855:Chromatography, Thin Layer; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D000072470:Cytochrome P450 Family 4; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D006868:Hydrolysis; D007975:Leukotriene B4 / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009243:NAD / Q000494:pharmacology; D009249:NADP / Q000378:metabolism / Q000494:pharmacology; D009504:Neutrophils / Q000201:enzymology*; D010084:Oxidation-Reduction
D009243:NAD; D007975:Leukotriene B4; D009249:NADP; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; C038243:8-hydroxy-11,12-epoxyeicosa-5,9,14-trienoic acid; D000255:Adenosine Triphosphate; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D000072470:Cytochrome P450 Family 4; C040780:leukotriene B4 20-hydroxylase; D015126:8,11,14-Eicosatrienoic Acid