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

Abstract

Oxygenation of arachidonic acid by either of the two cyclooxygenase (COX) isozymes yields the prostaglandin endoperoxide PGH 2 as the major product and the monohydroxylated 11 -hydroxyeicosatetraenoic acid (HETE) and 15-HETE as by-products of about 2-5% abundance ( 1 ). 11-HETE is exclusively of the 11 R confi guration, similar in confi guration to the fi rst oxygenation of arachidonic acid to the 11 R -peroxyl radical that will form the 9,11-endoperoxide of PGH 2 ( 2, 3 ). 15-HETE is formed as a mixture of the 15 S -and 15 R -enantiomers, in contrast to the confi guration of C15 in PGH 2 , which is strictly S ( 4 ). The HETE by-products are thought to arise from a slightly different alignment of substrate in the active site compared with when PGH 2 is formed ( 5 ), rather than resulting from incomplete or otherwise faulty catalysis. Neither 15-hydroperoxy-eicosatetraenoic acid (HPETE) nor 11-HPETE can serve as substrates for formation of PGH 2 in the cyclooxygenase reaction as demonstrated for the COX-1 enzyme ( 6, 7 ). It has not been established whether formation of the HETE by-products follows a particular biological rationale.Acetylation by aspirin (acetylsalicylic acid) of a serine residue in the oxygenase active site channel of both COX isozymes has discrete effects on the catalytic activities of the two enzymes ( 8, 9 ). Whereas COX-1 loses all oxygenase Abstract 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 identifi ed 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 Eeicosadienoic 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 specifi c 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. -Mulugeta, S., T. Suzuki, N. T. Hernandez, M. Griesser, W. E. Boeglin, and C. Schneider. Identifi cation and ab...

COX-2-dependent and -independent biosynthesis of dihydroxy-arachidonic acids in activated human leukocytes

“…This could have been due to the ability of NS-398 to inhibit leukocyte lipid body formation independent of COX-2 inhibition leading to the suppression of LOX-derived eicosanoids ( 27 ). Aspirin did not completely inhibit formation of 5,15-diHETE (0.10 ± 0.05 ng/10 6 cells, n = 6), compatible with the expected formation 5 S ,15 R -diHETE from 5 S -HETE ( 16 ). None of the differences between the means of the treatment groups were statistically signifi cant using a two-tailed unpaired t -test.…”

“…Besides COX-2, COX-1 is the only other mammalian enzyme capable of oxygenating the 11-carbon of arachidonic acid, but COX-1 does not react with 5-HETE ( 17 ). The inverse reaction (of 5-LOX with 11 R -HETE) is very ineffi cient ( 16 ) and thus unlikely to be relevant in cells. The levels of 5,11-diHETE were between 0.01 and 0.04 ng/10 6 cells.…”

“…Our interest in the biosynthesis of 5,15-diHETE stemmed from its formation as a byproduct of the oxygenation of 5 S -HETE by COX-2 in vitro ( 16 ). This fi nding implicated crossover of the 5-LOX and COX-2 pathways as an alternative biosynthetic route of 5,15-diHETE in vivo with 5-LOX forming 5 S -HETE as the fi rst step, followed by COX-2 catalysis as the second step.…”

“…The main product of the reaction is a diendoperoxide that is structurally similar to the arachidonic acid-derived endoperoxide of prostaglandin biosynthesis ( 17 ). The catalytic byproducts include 5,15-diHETE (as a ‫ف‬ 4:1 mixture of the 5 S ,15 S -and 5 S ,15 R -diastereomers) and 5 S ,11 R -diHETE ( 16 ). The diHETEs are the 5 S -hydroxy analogs of the 15-and 11-HETE byproducts of the COX-1 and COX-2 reactions with arachidonic acid ( 18,19 ).…”

Biosynthesis of hemiketal eicosanoids by cross-over of the 5-lipoxygenase and cyclooxygenase-2 pathways

“…RP-HPLC analysis of short-time incubation (<5 min) of ½1-14 C 5S-HETE 1 with recombinant COX-2 gave the diendoperoxide 2 as the main product together with 5,11-diHETE and 5,15-diHETE as by-products (Fig. 1A) (5,12). When the same incubation was extracted 30 min after the addition of ½1-14 C 5S-HETE, the diendoperoxide was no longer present, and instead two more polar peaks 3 and 4 were detected (Fig.…”