In vitro formation of retinoic acid from retinal in rat liver

Abstract

Enzymatic conversion of retinal to retinoic acid in rat liver cytosol was detected using a rapid and sensitive assay based on high pressure liquid chromatography (HPLC). This retinal oxidase assay system did not require extraction steps or any other manipulation of the sample mixture once the sample vial was sealed for incubation. The product (retinoic acid) and the reactant (retinal) were separated by HPLC in 14.0 min with a sensitivity of 15 and 40 pmol per injection for retinoic acid and retinal, respectively. Enzymatic activity was observed to be linear with protein concentration (0-2.4 mg/mL) and time (0-30 min) and displayed a broad pH maximum of 7.7-9.7. The enzyme exhibited Michaelis-Menten single-substrate kinetics with an apparent Km of 0.25 mM. The average specific activity in nine normal rats was 35.6 +/- 3.3 nmol retinoic acid formed/h per mg protein. Incubation of the enzyme with zinc did not affect the rate of retinoic acid synthesis. Dithiothreitol inhibited the reaction. Both NAD and NADH stimulated retinoic acid formation. Formation of retinol was also observed when these pyridine nucleotides were added to the reaction mixture, indicating the presence of retinal reductase activity. The results of kinetic studies suggest that NADH may act indirectly to stimulate retinoic acid formation.

Characterization of the Cytochrome P450 CYP2J4: Expression in Rat Small Intestine and Role in Retinoic Acid Biotransformation from Retinal

“…In view of the known participation of other pathways, the extent of the contribution of P450s to the overall physiological conversion of retinals to retinoic acids is currently unknown. Cytosolic enzymes such as NADdependent dehydrogenases also contribute to retinoic acid formation in reactions with K m values which are reported to range from <1 to 250 µM (19,20). Clearly further studies are required to resolve physiological relevance.…”