Synthesis of 5-oxo-6,8,11,14-eicosatetraenoic acid and identification of novel omega-oxidized metabolites in the mouse macrophage

The metabolism of arachidonic acid by the 5-lipoxygenase pathway not only l eads to the formation of leukotrienes but also to the biologically active e icosanoid 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE). The synthesis of 5-oxo-ETE was investigated in the elicited peritoneal macrophage and the formation of 5-hydroxyeicosatetraenoic acid (5-HETE) as well as 5-oxo-ETE was quantitated using stable isotope dilution tandem mass spectrometry. The metabolism of 5-oxo-ETE in these same cells led to the formation of a seri es of novel less lipophilic metabolites oxidized near the methyl terminus t hat were structurally characterized using electrospray LC/MS and LC/MS/MS. Five novel metabolites of 5-oxo-ETE were identified including 5,18-diHETE, 5,19-diHETE, 5-oxo-19-HETrE, 5-oxo-18-HETrE, and 5,19-diHETrE. These metabo lites corresponded to omega -1 and omega -2 oxidation of 5-oxo-ETE presumab ly formed by a specific cytochrome P450. There was no evidence for the form ation of omega -oxidation (20-hydroxy metabolites), which are known product s of metabolism of 5-oxo-ETE in other cell types. None of the metabolites w ere found to elevate intracellular calcium release, suggesting that this me tabolic pathway may result in inactivation of 5-oxo-ETE. This is the first report of the biosynthesis of 5-oxo-ETE by tissue resident cell outside of the blood and the formation of novel omega -1 and omega -2 oxidation of thi s eicosanoid.