Identification of 12-lipoxygenase products in the gills of carp, Cyprinus carpio

Abstract

SUMMARY: A lipoxygenase was found in the crude enzyme solution from the gills of carp, Cyprinus carpio. It oxidized arachidonic acid (AA) more efficiently than linoleic acid, eicosapentaenoic acid and docosahexaenoic acid. Lipoxygenase activity was constantly detected in the gill microsomes and was found to be optimum at pH 7.2. It was not stimulated by reduced nicotinamide adenine dinucleotide phosphate and was not inhibited by SKF525A, a cytochrome P450 inhibitor. The oxygenated products extracted from the reaction mixtures of crude enzyme solution and AA were purified by reverse-phase and straight-phase high-performance liquid chromatography (HPLC). The major metabolite was identified as 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) by ultraviolet spectrophotometry, gas chromatography-mass spectrometry and chiral phase HPLC. In addition, 12-hydroxyeicosapentaenoic acid (12-HEPE) and 13-hydroxyoctadecadienoic acid (13-HODD) were also found in the reaction products as minor components. Similar results were obtained by the analysis of the reaction products of AA and carp gill microsomes. These results confirm the presence of 12-lipoxygenase in carp gill microsomes.