ASSAY OF BETA-CAROTENE 15,15'-DIOXYGENASE ACTIVITY BY REVERSE-PHASE HIGH-PRESSURE LIQUID-CHROMATOGRAPHY

beta-Carotene 15,15'-dioxygenase catalyzes the conversion of beta-caro tene into two molecules of retinal. Although this enzyme reaction is t he first step in providing animals with vitamin A, there is little kno wledge 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 beta-carotene dioxygenase. Al l-trans-beta-carotene solubilized in aqueous solution with Tween 40 wa s incubated with an enzyme preparation of rat tissue at 37 degrees C f or 30 min. Then, the reaction was stopped with a formaldehyde treatmen t followed by addition of acetonitrile. After centrifugation, the supe rnatant was directly subjected to high-pressure liquid chromatographic analysis of retinal. This assay method did not involve any vigorous e xtraction or concentration procedure. All-trans- and 13-cis-retinals, major geometric isomers found in the reaction mixture, were coeluted a t 7.5 min, but they were well separated from other possible metabolite s of beta-carotene such as retinoic acid, retinol, and apocarotenals. Moreover, the recovery of retinal reached more than 93% and the detect ion limit of standard retinal was 0.2 pmol/0.2 mi of reaction mixture. Enzyme activities of rat tissues were 694, 180, 16, 8, and approx 1 p mol retinal/mg protein/h in the intestine, liver, brain, lung, and kid ney homogenates, respectively. (C) 1996 Academic Press, Inc.