Identification and absolute configuration of dihydroxy-arachidonic acids formed by oxygenation of 5 S-HETE by native and aspirin-acetylated COX-2

Biosynthesis of the prostaglandin endoperoxide by the cyclooxygenase (COX) enzymes is accompanied by formation of a small amount of 11 R- hydroxyeicosatetraenoic acid (HETE), 15 R-HETE, and 15 S-HETE as by-products. Acetylation of COX-2 by aspirin abrogates prostaglandin synthesis and triggers formation of 15 R-HETE as the sole product of oxygenation of arachidonic acid. Here, we investigated the formation of by-products of the transformation of 5 S-HETE by native COX-2 and by aspirin-acetylated COX-2 using HPLC-ultraviolet, GC-MS, and LC-MS analysis. 5 S,15 S-dihydroxy (di)HETE, 5 S,15 R-diHETE, and 5 S,11 R-diHETE were identified as by-products of native COX-2, in addition to the previously described di-endoperoxide (5 S,15 S-dihydroxy-9 S,11 R,8 S,12 S-diperoxy-6 E,13 E-eicosadienoic acid) as the major oxygenation product. 5 S,15 R-diHETE was the only product formed by aspirinacetylated COX-2. Both 5,15-diHETE and 5,11-diHETE were detected in CT26 mouse colon carcinoma cells as well as in lipopolysaccharide-activated RAW264.7 cells incubated with 5 S-HETE, and their formation was attenuated in the presence of the COX-2 specific inhibitor, NS-398. Aspirintreated CT26 cells gave 5,15-diHETE as the most prominent product formed from 5 S-HETE. 5 S,15 S-diHETE has been described as a product of the cross-over of 5-lipoxygenase (5-LOX) and 15-LOX activities in elicited rat mononuclear cells and human leukocytes, and our studies implicate crossover of the 5-LOX and COX-2 pathways as an additional biosynthetic route.