Formation of a novel dihydroxy acid from arachidonic acid by lipoxygenase-catalyzed double oxygenation in rat mononuclear cells and human leukocytes

Abstract

Elicited rat peritoneal mononuclear cells converted arachidonic acid to a new dihydroxy acid, 5(S), 15(S)-dihydroxy-6,13-trans-8,11-cis-eicosatetraenoic acid (5,15-DiHETE). In this system, the amount of 5,15-DiHETE formed was about 20% that of leukotriene B4. The structure of the compound was determined by ultraviolet and mass spectrometric analysis, and comparison to a reference compound prepared by incubation of synthetic 5(R,S)-hydroxy- or hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5(R,S)-HETE or HPETE) with soybean lipoxygenase (linoleate:oxygen oxidoreductase, EC 1.13.11.12). Cell incubations performed under an atmosphere of 18O2 demonstrated that both hydroxyl groups in the cell product derived from molecular oxygen and that the oxygen atoms were from different oxygen molecules. Steric analysis indicated that each hydroxyl group had the S-configuration. The structural data thus indicate that 5,15-DiHETE is formed by an enzymatic double oxygenation of arachidonic acid catalyzed by both C-5 and C-15 lipoxygenases. Incubations with with [3H 8]5 (S)-HETE and [3H8]15(S)-HETE revealed that both compounds could be converted to the product. When [3H8]5(S), 15(S)-DiHPETE was added to cells, the majority of the substrate was reduced to 5,15-DiHETE. Leukocytes obtained from three human donors with peripheral blood eosinophilia also synthesized 5,15-DiHETE. Formation of the compound occurred in both eosinophils and neutrophils from these donors.