THE ENZYMATIC CLEAVAGE OF d-CAROTENE INTO VITAMIN A BY SOLUBLE ENZYMES OF RAT LIVER AND INTESTINE* BY JAMES ALLEN OLSONt

That 13-carotene can serve as a precursor of vitamin A in mammals was demonstrated almost 40 years ago." 2 In spite of innumerable nutritional and chemical studies on this reaction, however, neither the pathway nor the mechanism of vitamin A formation has been clarified.3-5 With respect to the pathway of vitamin A formation, two major hypotheses have been suggested: (1) carotene is cleaved at the central 15-15' double bond to yield two molecules of vitamin A, and (2) carotene is cleaved peripherally to yield one molecule of vitamin A via a series of 13-apo-carotenals.4 Although f3-apo-carotenals have been found in nature,6 are highly effective biologically, and are converted to vitamin A in the mammal,4 they do not accumulate during the cleavage of ,8-carotene in vivo.7 The cleavage of ,8-carotene to vitamin A seemingly proceeds by an oxidative reaction. In isolated sections of intestine oxygen is required for the conversion of carotene to vitamin A,8 a requirement which correlates better with the cleavage reaction than with the absorption of carotene into gut slices.9 Furthermore, oxygen'8 is incorporated into vitamin A in the liver of carotene-treated animals when 0218 gas is used but not when H2018 is employed.'0 Purely chemical studies also accord with an oxidative cleavage mechanism."' 12 Although the intestine seems to be the major organ in the rat for carotene cleavage into vitamin A,7 1" the liver also catalyzes this reaction.'4 Interestingly, bile salts are necessary for the uptake and cleavage of carotene by gut slices,7 but are not required for cleavage in the liver.'4 In the present report an enzyme has been found in the supernatant solution of rat liver and intestine which converts 13-carotene into retinal and retinol as its sole products. Oxygen is required for the reaction, and the immediate product is retinal. The enzyme is inhibited by sulfhydryl binding reagents and by ferrous-ion chelating agents. The enzyme has been tentatively designated as 1-carotene 15-15' oxygenase. Materials and Methods.-Preparation of radioactive #-carotene: Sodium acetate J-C'4 was added to growing cultures of Phycomyces blakesleeanus, and the synthesized ,B-carotene was extracted and purified as previously described.'5 16 The three times recrystallized compound was greater than 98% pure, as judged by two-dimensional chromatography on silica gel thin-layer plates, and had a specific activity of 6560 cpm per Mg. The labeled ,B-carotene was stored in hexane in the presence of a-tocopherol in a red glass container in the cold. Just prior to each experiment a suitable quantity was purified through a small column of 6% water-deactivated alumina, dried in the dark under nitrogen, dissolved in 0.05 ml of 20% Tween 40 in distilled acetone, and made up to a suitable volume with 0.15 M tris-hydroxymethyl-aminomethane (Tris) buffer, pH 8.0. Preparation of organ homogenates: Male or female rats of the Wistar strain weighing 150-300 gm were anesthetized with ether, opened by midline incision, and the liver or kidneys were quickly removed and washed in saline. The upper half of the intestine was also removed, cut longitudinally to expose the mucosal surface, and washed in cold saline. Each tissue was minced on a cold Petri plate and homogenized with a loose-fitting, motor-driven glass homogenizer in 5 vol of cold 0.15