Oxygenation of 5,8,11-eicosatrienoic acid by prostaglandin endoperoxide synthase and by cytochrome P450 monooxygenase: structure and mechanism of formation of major metabolites.
Incubation of 5,8,11-[1-14C]eicosatrienoic acid with prostaglandin endoperoxide synthase of ram vesicular gland microsomes led to formation of a number of polar metabolites. Four major compounds were characterized by chemical and physical methods and found to be: (11R)-hydroxy-5,8,12-eicosatrienoic acid, 8,9,11-trihydroxy-5,12-eicosadienoic acid (two diastereoisomers), and 8,9-epoxy-11-hydroxy-5,12-eicosadienoic acid. On the basis of previous studies on the mechanism of prostaglandin biosynthesis it seemed likely that the initial step of conversion of 5,8,11-eicosatrienoic acid consisted of removal of the pro-S hydrogen from C-13. The resulting carbon-centered radical was apparently attacked by dioxygen at C-13 to provide a (13R)-(hydro)peroxy derivative, which served as the precursor of (13R)-hydroxyeicosatrienoic acid. Alternatively, attack by dioxygen occurred at C-11 to produce an (11R)-peroxy radical. This intermediate was further converted to (11R)-hydroxyeicosatrienoic acid by reduction, into two 8,9,11-trihydroxy-5,12-eicosadienoic acids by successive cyclization, oxygenation, and reduction, and into the epoxy-hydroxy acid by cyclization and intramolecular epoxidation. The relative abundance of (13R)-hydroxy-5,8,11-eicosatrienoic acid, (11R)-hydroxy-5,8,12-eicosatrienoic acid, and the epoxy alcohol plus the two 8,9,11-triols was 51, 9, and 40%, respectively. The oxygenation at C-13 and C-11 of 5,8,11-eicosatrienoic acid was inhibited by 90% in the presence of diclofenac, an inhibitor of prostaglandin endoperoxide synthase. The two diastereomeric 8,9,11-trihydroxy acids and the epoxy-hydroxy acid are novel oxylipins and their formation provides independent chemical evidence for the existence of an 11-peroxy radical intermediate in prostaglandin endoperoxide synthase catalysis. Oxygenation of 5,8,11-eicosatrienoic acid by cytochrome P450 from liver microsomes of cynomolgus monkeys and phenobarbital-treated rats was also investigated. The metabolites formed included 19- and 20-hydroxyeicosatrienoic acid, 8,9- and 11,12-dihydroxyeicosadienoic acids (formed by enzymatic hydrolysis of the corresponding epoxides), and (12R)-hydroxy-5,8,10-hydroxyeicosatrienoic acid.
Department of Pharmaceutical Biosciences, Uppsala University Biomedical Center, Sweden.
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D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D015777:Eicosanoids / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D008297:Male; D008861:Microsomes / Q000378:metabolism; D010100:Oxygen / Q000737:chemistry; D010105:Oxygenases / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D012756:Sheep
D015777:Eicosanoids; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; D011451:Prostaglandin-Endoperoxide Synthases; D015126:8,11,14-Eicosatrienoic Acid; C010944:mead acid; D010100:Oxygen