vitamin-a2 and retinol-palmitate

The kinetics of esterification of exogenous retinol by cell membranes prepared from the crude homogenate of the frog retinal pigment epithelium was studied. The formation of retinyl palmitate from added retinol was directly assayed by high performance liquid chromatography (HPLC). A linear relationship was observed between the amount of protein (up to 2 mg) in the incubation medium and the amount of retinyl palmitate formed. At room temperature, this reaction took less than 2 hours to complete. By varying the substrate concentration in the incubation medium, the reciprocal of initial velocity of the reaction (nmol retinyl palmitate formed per hour) was plotted against the reciprocal of substrate concentration (nmol of retinol). This double-reciprocal plot shows that the apparent Km of the reaction was 10 microM with an apparent Vmax of 9.1 nmol of retinyl palmitate per hour per mg protein. When this assay was repeated in the presence of 3,4-didehydroretinol (20 microM), the kinetics of the reaction showed the pattern of that of a competitive inhibitor, suggesting that 3,4-didehydroretinol competes with retinol for the same active site for esterification. The esterification of 3,4-didehydroretinol resulted in the formation of 3,4-didehydroretinyl palmitate, which was also measured by HPLC. The amount of 3,4-didehydroretinyl palmitate formed by this reaction decreased in proportion to increased retinol concentration in the incubation mixture. This further confirms that a competition exists between the esterification of retinol and 3,4-didehydroretinol by retinal pigment epithelium of the frog.

Topics: Animals; Chromatography, High Pressure Liquid; Diterpenes; Esters; Kinetics; Pigment Epithelium of Eye; Ranidae; Retinyl Esters; Vitamin A

The membrane fraction of the retinal pigment epithelium (RPE) of the frog (Rana pipiens) catalyzed the esterification of tritiated retinol to retinyl esters. This esterification reaction was inhibited in the presence of 3,4-didehydroretinol.

Topics: Animals; Diterpenes; Esterification; In Vitro Techniques; Pigment Epithelium of Eye; Rana pipiens; Retinyl Esters; Vitamin A

HPLC (high-performance liquid chromatography) was used to analyse retinyl and 3,4-didehydroretinyl compounds in tissue extracts from goldfish and bullfrog tadpoles. Using silica columns (packed with 10-micron mu Porasil or 5-micron Ultrasphere particles) eluted with n-hexane (containing a small amount of dioxane or diethyl ether), the authentic all-trans retinyl and 3,4-didehydroretinyl palmitates, retinal and 3,4-didehydroretinal, retinol and 3,4-didehydroretinol were completely separated. Liver and eye extracts of the goldfish and bullfrog tadpoles had mainly esterified all-trans retinol and all-trans 3,4-didehydroretinol. In the liver, these vitamin A were conjugated to a number of fatty acids whereas in the eye, principally one fatty acid was used. Moreover, the relative proportions of all-trans retinol and all-trans 3,4-didehydroretinol (obtained by analysing the saponified esters) were significantly different between some of these body compartments.

Topics: Animals; Chromatography, High Pressure Liquid; Diterpenes; Esters; Eye; Goldfish; Larva; Liver; Rana catesbeiana; Retinaldehyde; Retinoids; Retinyl Esters; Vitamin A