Identification and absolute configuration of dihydroxy-arachidonic acids formed by oxygenation of 5S-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 11R-hydroxyeicosatetraenoic acid (HETE), 15R-HETE, and 15S-HETE as by-products. Acetylation of COX-2 by aspirin abrogates prostaglandin synthesis and triggers formation of 15R-HETE as the sole product of oxygenation of arachidonic acid. Here, we investigated the formation of by-products of the transformation of 5S-HETE by native COX-2 and by aspirin-acetylated COX-2 using HPLC-ultraviolet, GC-MS, and LC-MS analysis. 5S,15S- dihydroxy (di)HETE, 5S,15R-diHETE, and 5S,11R-diHETE were identified as by-products of native COX-2, in addition to the previously described di-endoperoxide (5S,15S-dihydroxy-9S,11R,8S,12S-diperoxy-6E,13E-eicosadienoic acid) as the major oxygenation product. 5S,15R-diHETE was the only product formed by aspirin-acetylated 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 5S-HETE, and their formation was attenuated in the presence of the COX-2 specific inhibitor, NS-398. Aspirin-treated CT26 cells gave 5,15-diHETE as the most prominent product formed from 5S-HETE. 5S,15S-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 cross-over of the 5-LOX and COX-2 pathways as an additional biosynthetic route.
Mulugeta
Surafel
S
Division of Clinical Pharmacology, Department of Pharmacology, Vanderbilt University Medical School, Nashville, TN 37232, USA.
Suzuki
Takashi
T
Hernandez
Noemi Tejera
NT
Griesser
Markus
M
Boeglin
William E
WE
Schneider
Claus
C
D000107:Acetylation / Q000187:drug effects; D000818:Animals; D001241:Aspirin / Q000737:chemistry / Q000494:pharmacology*; D002460:Cell Line; D002942:Circular Dichroism; D051546:Cyclooxygenase 2 / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D010100:Oxygen / Q000378:metabolism*; D013237:Stereoisomerism
D006893:Hydroxyeicosatetraenoic Acids; D051546:Cyclooxygenase 2; D001241:Aspirin; D010100:Oxygen