Free Radical Oxidation of (E)-Retinoic Acid by Prostaglandin H Synthase

Abstract

Cooxidative metabolism of all-trans (E)-retinoic acid (RA) by prostaglandin H synthase was investigated employing ram seminal vesicle microsomes (RSVM) or purified, RSVM-derived enzyme. RA was shown to undergo hydroperoxide [H2O2 or 5-phenyl-4-penten-1-yl hydroperoxide (PPHP)]- or 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, using PPHP as substrate, was characterized by uptake of dioxygen which was first order with respect to enzyme concentration. Dioxygen uptake was inhibited by the peroxidase reducing substrate 2-methoxyphenol. In addition, O2 uptake was inhibited by the spin trap nitrosobenzene. ESR spin trapping studies, using alpha-phenyl-N-tert-butylnitrone (PBN) as the spin trap, demonstrated the formation of RA-PBN adducts, characterized by hyperfine coupling constants of alpha H = 3.2 G and alpha 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 acid, and other geometric isomers which were identified on the basis of cochromatography 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 these results.

4-Hydroxyretinoic Acid, a Novel Substrate for Human Liver Microsomal UDP-glucuronosyltransferase(s) and Recombinant UGT2B7

“…Experimental results from in vitro and in vivo systems indicate that atRA oxidative metabolism involves both cytochrome P450 and prostaglandin H synthase-mediated processes. We have previously demonstrated that atRA and 13-cis-RA are hydroxylated at the C-4 position by prostaglandin H synthase (33,38,39). Recent reports suggest that the generation of 4-OH-and 18-OH-RA relies on a cytochrome P450-dependent process.…”

“…9 was subsequently purified by recrystallization from diethyl ether/hexane (81% yield). The purity of the latter was verified by analytical reverse phase HPLC analysis and UV spectroscopy as described previously (33).…”

Arachidonic acid‐mediated cooxidation of all‐trans‐retinoic acid in microsomal fractions from human liver

“…CYP‐dependent 4‐hydroxylation in human liver is an established pathway for the termination of the physiological and pharmacological actions of ATRA. Studies undertaken in ram seminal vesicles and with the purified enzyme have also implicated PGHS in the biotransformation of ATRA and its geometric isomer 13Z ‐RA ( Samokyszyn & Marnett 1987 ; Samokyszyn et al ., 1995 ). The present study examined the contributions of CYP‐ and PGHS‐mediated pathways of ATRA oxidation in human hepatic microsomes.…”

“…A range of lipophilic chemicals, including naturally occurring retinoids, possess readily oxidizable atoms and can act as reducing substrates for the peroxidase activity of PGHS ( Markey et al ., 1987 ). Thus, ATRA and 13Z ‐RA undergo hydrogen atom abstraction from the carbon atom at the 4‐position of the β‐ionone ring system to generate carbon centred radicals ( Samokyszyn et al ., 1995 ). The 4‐position is relatively reactive because it is a secondary allylic carbon that is fully conjugated with the unsaturated side chain.…”

“…The 4‐position is relatively reactive because it is a secondary allylic carbon that is fully conjugated with the unsaturated side chain. This radical at C‐4 combines with a molecule of oxygen to form peroxyl radicals ( Samokyszyn et al ., 1995 ). Such radicals are known to generate epoxides, which is consistent with the formation of the 5,6‐ and 5,8‐epoxides from ATRA in micellar systems containing haematin.…”