BIOTRANSFORMATION OF ALL-TRANS-RETINOL AND ALL-TRANS-RETINAL TO ALL-TRANS-RETINOIC ACID IN RAT CONCEPTAL HOMOGENATES

Catalysis of the oxidation of all-trans-retinol (vitamin A(1)) or of a ll-trans-retinal to all-trans-retinoic acid (all-trans-RA) by rat conc eptal enzymes was investigated during organogenesis. products of the r eaction were identified and quantified with HPLC by comparing their el ution times with those of authentic standard retinoids. Under the incu bation and assay conditions utilized, all-trans-retinol and all-trans- retinal were converted to readily detectable quantities of all-trans-R A. Rat conceptal homogenates from gestational days 10.5, 11.5 and 12.5 each exhibited enzymatic activity for oxidation of all-trans-retinol and all-trans-retinal to all-trans-RA. Enzymatic catalysis was verifie d by showing that: (1) both reactions were coenzyme dependent; (2) the rates of reactions increased as concentrations of conceptal protein i ncreased; (3) both reactions were abolished by heating the tissue homo genates (100 degrees, 5 min); and (4) both reactions exhibited substra te saturation. Under the same experimental conditions, formation of al l-trans-RA from all-trans-retinol was much slower than from all-trans- retinal, suggesting that oxidation of all-trans-retinol to all-trans-r etinal was the rate-limiting step for biotransformation of all-trans-r etinol to all-trans-RA in embryonic tissues. When NAD or NADP were rep laced by NADH or NADPH, the rate of oxidation of all-trans-retinol was reduced markedly, indicating that the reaction was catalyzed primaril y by an NAD/NADP-dependent dehydrogenase(s). Carbon monoxide (CO:O-2 = 90:10) did not inhibit the reaction. NAD appeared to be a more effect ive cofactor than NADP in catalyzing oxidation of all-trans-retinal to all-trans-RA. When NAD was omitted, formation of all-trans-RA from al l-trans-retinal was reduced by approximately 55%. Replacing NAD by NAD H or NADPH also reduced the conversion of all-trans-retinal to all-tra ns-RA by about 60%. These observations suggested at least two pathways for the generation of all-trans-RA From all-trans-retinal in embryos: oxidation catalyzed by an NAD/NADP-dependent dehydrogenase(s) and oxi dation catalyzed by an oxidase(s) that did not require NAD, NADH, NADP or NADPH. Conversion of all-trans-retinol to all-trans-RA was inhibit ed strongly by low concentrations of citral, but not by high concentra tions of sodium azide, 4-methylpyrazole, or metyrapone. Similarly, oxi dation of all-trans-retinal was inhibited strongly by citral but not b y metyrapone. Our studies suggested that: (I) biotransformation of all -trans-retinol to all-trans-RA in embryos was catalyzed by an NAD/NADP -dependent retinol dehydrogenase(s); (2) biotransformation of all-tran s-retinal to all-trans-RA in embryos was catalyzed by an NAD/NADP-depe ndent retinal dehydrogenase(s) and a retinal oxidase(s); and (3) oxida tion of all-trans-retinol to all-trans-retinal was the rate-limiting s tep in biotransformation of all-trans-retinol to all-trans-RA.