Biosynthesis and metabolism of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid by human umbilical vein endothelial cells.

Incubation of cultured human umbilical vein endothelial cells with [1-14C]arachidonic acid, followed by reverse-phase high-pressure liquid chromatography analysis, results in the appearance of two principal radioactive products besides 6-keto-prostaglandin F1 alpha. The first peak is 12-L-hydroxy-5,8,10-heptadecatrienoic acid, a hydrolysis product of the prostaglandin endoperoxide. The second peak was esterified, converted to the trimethylsilyl ether derivative, and analyzed by gas chromatography-mass spectrometry and shown to be the lipoxygenase product 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE). Incubation of the 15-HETE precursor 15(S)-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) with endothelial cells results in the formation of four distinct UV absorbing peaks. UV and gas chromatography-mass spectrometry analysis showed these peaks to be 8,15(S)-dihydroxy-5,8,11,13-eicosatetraenoic acids (8,15-diHETE) differing only in their hydroxyl configuration and cis trans double-bond geometry. Formation of 8,15-diHETE molecules suggests the prior formation of the unstable epoxide molecule 14(S),15(S)-trans-oxido-5,8-Z-14,15-leukotriene A4 or an attack at C-10 of 15-HPETE by an enzyme with mechanistic features in common with a 12-lipoxygenase. The observation that endothelial cells can synthesize both 15-HETE and 8,15-diHETE molecules suggests that this cell type contains both a 15-lipoxygenase and a system that can synthesize 14,15-leukotriene A4.