Enzymatic conversion of all-trans--carotene to retinal by a partially purified enzyme from rabbit and rat intestinal mucosa was demonstrated. The enzymatic product was characterized based on the following evidence: (t) The product gave rise to its O-ethyloxime by treatment with Oethylhydroxylamine with an absorption maximum at 363 nm in ethanol characteristic of authentic retinal O-ethyloxime. Highpressure liquid chromatography (HPLC) of this derivative yielded a sharp peak with a retention time of 7.99 min corresponding to the authentic compound. The enzyme blank and boiled enzyme blank failed to show any significant HPLC peaks corresponding to retinal O-ethyloxime, retinal, or retinol. (it) The mass spectrum of the O-ethyloxime of the enzymatic product was identical to that of authentic retinal O-ethyloxime (m/z 327: 45%, Mt and m/z 282: 100%, Methoxy). (Wi) The specific activity of the enzymaticaily formed ["4CIretinal O-ethyloxime remained constant even after repeated crystallization. (iv) The enzymatic product exhibited an absorption maximum at 370 nm in light petroleum characteristic of authentic retinal. Furthermore, it was reduced by horse liver alcohol dehydrogenase to retinol with an absorption maximum at 326 nm in light petroleum. This retinol was enzymatically esterified to retinyl palmitate by rat pancreatic esterase with a retention time of 10 min on HPLC corresponding to authentic retinyl palmitate. Thus, the enzymatic product of f8-carotene cleavage by the partially purified intestinal enzyme was unequivocally confirmed to be retinal.It is well established that P-carotene is the precursor of vitamin A and its conversion into vitamin A in vivo has been unequivocally demonstrated in rats, pigs, and humans (1-7).t Subsequently, the in vitro enzymatic conversion of 83-carotene to retinal by an enzyme preparation from rat intestine and liver was independently shown by Goodman et al. (8) and Olson and Hayaishi (9). This was extended by a number of workers (10-20) using different species as the source ofthe enzyme. However, a more recent report (21) claimed that it could not duplicate the original work (8, 9, 15) and hence raised the possibility that retinal may not be the true product of (3-carotene conversion to vitamin A in vitro, although it did not question the validity of the formation of vitamin A from ,(-carotene in vivo.In view of this controversy, we decided to systematically repeat the work using the intestinal mucosa from rabbit and rat as the source of the (3-carotene cleavage (BCC) enzyme activity. It will be demonstrated conclusively in the present communication that retinal is, in fact, the product of BCC enzyme using (i) a method of synthesizing the O-ethyloxime of the enzymatic product as its derivative and subsequent separation, identification, and quantitation by high-pressure liquid chromatography (HPLC); (ii) unequivocal identification of the retinal O-ethyloxime derivative by its characteristic absorption spectrum, its mass spectrum, and its repeated crystallization to c...
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