Assay of β-Carotene 15,15′-Dioxygenase Activity by Reverse-Phase High-Pressure Liquid Chromatography

Abstract

β-Carotene 15,15′-dioxygenase catalyzes the conversion of β-carotene into two molecules of retinal. Although this enzyme reaction is the first step in providing animals with vitamin A, there is little knowledge about its regulation in mammals. In order to facilitate studies on this enzyme, we have developed a rapid and simple assay method for the measurement of retinal formation by β-carotene dioxygenase. All-trans-β-carotene solubilized in aqueous solution with Tween 40 was incubated with an enzyme preparation of rat tissue at 37°C for 30 min. Then, the reaction was stopped with a formaldehyde treatment followed by addition of acetonitrile. After centrifugation, the supernatant was directly subjected to high-pressure liquid chromatographic analysis of retinal. This assay method did not involve any vigorous extraction or concentration procedure. All-trans- and 13-cis-retinals, major geometric isomers found in the reaction mixture, were coeluted at 7.5 min, but they were well separated from other possible metabolites of β-carotene such as retinoic acid, retinol, and apocarotenals. Moreover, the recovery of retinal reached more than 93% and the detection limit of standard retinal was 0.2 pmol/0.2 ml of reaction mixture. Enzyme activities of rat tissues were 694, 180, 16, 8, and approx 1 pmol retinal/mg protein/h in the intestine, liver, brain, lung, and kidney homogenates, respectively.