Isolation and identification of 5,6-epoxyretinoic acid: a biologically active metabolite of retinoic acid

Abstract

A highly biologically active metabolite of retinoic acid (8III) has been isolated in pure form from intestinal mucosa of vitamin A deficient rats given [3H]retinoic acid. This metabolite has been positively identified as 5, 6-epoxyretinoic acid based on the ultraviolet absorption spectrum and mass spectrum of its methylated derivative. This identification was confirmed by cochromatography of the methylated metabolite and synthetic methyl 5, 6-epoxyretinoate on reverse-phase and straight-phase high-pressure liquid chromatography. The 5, 6-epoxyretinoic acid is a true in vivo generated metabolite of retinoic acid and not an artifact of the isolation procedure. In addition, 5, 8-oxyretinoic acid previously isolated in this laboratory from intestinal mucosa was probably generated from 5, 6-epoxyretinoic acid by the acidic conditions used in the extraction and isolation of the 5, 8-oxyretinoic acid.

HPLC-MS/MS analysis of the products generated from all-trans-retinoic acid using recombinant human CYP26A

“…As earlier attempts to identify the products of CYP26A have lacked physico-chemical approaches, the application of LC-MS and tandem MS conditions to detect and characterize these highly labile retinoids has provided an additional level of confidence to the identification work. While many of the hydroxylations we ascribe to CYP26A have been previously reported in various biological systems (7)(8)(9)(10)(11)(12)(13)(14), this is the first demonstration that all such metabolites can be produced by CYP26A alone. Furthermore, the gene dose and time course experiments indicate that atRA can be repeatedly hydroxylated to produce a series of increasingly water-soluble products, thereby giving credence to the theory that CYP26A is a catabolic system for protecting the cell from further stimulation by a potently biologically active signaling molecule.…”

“…The emergence of a family of inducible retinoic acid-metabolizing cytochrome P450 proteins (P450-RAIs or CYP26s) with high specificity for atRA across species simply reinforces the importance of maintaining a tight regulation of the levels of this highly potent ligand inside all cells (2)(3)(4)(5)(6). Over the past two decades, several catabolic steps have been suggested for reducing the biological activity of atRA, including: a ) oxidation at the 4 position of the ␤ -ionone ring (7)(8)(9); b ) oxidation at C-18 (9, 10); c ) 5,6-epoxidation (11)(12)(13)(14); and d ) glucuronidation (15)(16)(17)(18)(19).…”

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

“…5,6-Epoxy-RA has been identified as a major in vivo metabolite of atRA (as well as retinol and RAc) in rodents (12,(41)(42)(43). We have recently identified 5,6-epoxy-RA as a major in vivo metabolite of 13-cis RA in skin, 2 and we have detected the glucuronide of 5,6-epoxy-RA in human bile.…”

Regulation of germ cell meiosis in the fetal ovary

“…There are conflicting reports as to whether the metabolites produced during RA oxidation are biologically active; in vitro several oxidative metabolites have been shown capable of binding RARs and possess pharmacological activity (Idres et al, 2002). Further, differentiation and proliferation was stimulated by some metabolites in spermatogonia in vitamin A-deficient mice (Gaemers et al, 1996) and in epithelial differentiation of intestinal cells in vivo (McCormick et al, 1978). In contrast to these adult tissues described above, it seems that RA oxidative metabolites are superfluous to mouse embryonic development; genetic ablation of the gene encoding the RA-producing enzyme RALDH2 rescued the affects of Cyp26a1 loss of function in mice (Niederreither et al, 2002).…”