Microsomal retinoic acid metabolism. Effects of cellular retinoic acid-binding protein (type I) and C18-hydroxylation as an initial step.

Abstract This report extends our observation that cellular retinoic acid-binding protein type I (CRABP) serves as substrate for retinoic acid metabolism by testis microsomes. Retinoic acid bound to excess CRABP was metabolized at 70% of the unbound retinoic acid rate with testis microsomes and at the same rates as unbound retinoic acid with kidney and lung microsomes. Chromatography of testis, lung, kidney, and liver microsomal incubations provided two sets of metabolites each, P1 and P2. The composition of P2 was characteristic of the individual tissue. CRABP had modest quantitative affects on P2 composition, but did not affect P2 qualitatively. Retinoids bound to CRABP, isolated from a testis microsomal incubation, consisted of 50% retinoic acid, 32% P1 and 17% P2, suggesting that CRABP may bind retinoic acid metabolites in vivo. The effect of CRABP on the rate of metabolism was retinoid specific. Two major components of P2, 4-hydroxy-retinoic acid and 4-oxo-retinoic acid, when bound to CRABP were metabolized slowly, if at all, by testis microsomes, in contrast to CRABP-bound retinoic acid which had an elimination t1/2 of 40 min. Unbound retinoic acid, 4-hydroxy-retinoic acid, and 4-oxo-retinoic acid had elimination t1/2 values of 35, 40, and 9 min, respectively. Reduced metabolism of CRABP-bound C4-derivatized retinoids suggests pathways of retinoic acid metabolism besides the one initiated by C4-hydroxylation. This was corroborated by identification of 18-hydroxy-retinoic acid as a testis, lung, and liver microsomal retinoic acid metabolite. Ketoconazole inhibited the metabolism by testis microsomes of free and CRABP-bound retinoic acid with IC50 values of 2 and 0.7 microM, respectively, denoting catalysis by cytochrome P-450. These results indicate that cloistering retinoic acid in CRABP, while permitting metabolism, may operate throughout CRABP-expressing tissues as a mechanism of controlling the concentrations of free retinoic acid.