FREE-RADICAL OXIDATION OF (E)-RETINOIC ACID BY PROSTAGLANDIN-H SYNTHASE

Cooxidative metabolism of all-trans (E)-retinoic acid (RA) by prostagl andin H synthase was investigated employing ram seminal vesicle micros omes (RSVM) or purified, RSVM-derived enzyme. RA was shown to undergo hydroperoxide [H2O2 or 5-phenyl-4-penten-1-yl hydroperoxide (PPHP)]- o r arachidonic acid-dependent cooxidation by microsomal prostaglandin H (PGH) synthase as evidenced by UV spectroscopic analysis of reaction mixtures. Cooxidation of RA by microsomal or purified PGH synthase, us ing PPHP as substrate, was characterized by uptake of dioxygen which w as first order with respect to enzyme concentration. Dioxygen uptake w as inhibited by the peroxidase reducing substrate 2-methoxyphenol. In addition, O-2 uptake was inhibited by the spin trap nitrosobenzene. ES R spin trapping studies, using alpha-phenyl-N-tert-butylnitrone (PBN) as the spin trap, demonstrated the formation of RA-PBN adducts, charac terized by hyperfine coupling constants of a(H) = 3.2 G and a(N) = 15. 8 G. Reverse phase HPLC analysis of reaction mixtures demonstrated the formation of 4-hydroxy-RA, 5,6-epoxy-RA, 4-oxo-RA, (13Z)-retinoic aci d, and other geometric isomers which were identified on the basis of c ochromatography with synthetic standards, UV spectroscopy, and/or mass spectrometry. Mechanisms are proposed for the hydroperoxide-dependent , PGH synthase-catalyzed oxidation of RA that are consistent with thes e results.