Identification of L-ascorbic acid in the urine of normal and chloretone-treated rats.

About 10 years ago, King, Longenecker, and their associates made the interesting observation that the urine of rats subsisting on a milk diet which was supplemented with a non-saponifiable fraction from oat oil, grass leaf oil, or alfalfa leaf oil (2) or with certain terpenes (3) or hypnotics, particularly chloretone (4), contained considerable quantities of a reducing substance. This reducing substance could be oxidized rapidly by 2, 6dichlorophenolindophenol. Urine from rats treated with the non-saponifiable material from oats (2), or with cl-carvone, isophorone (3), or phenobarbital (4), had an antiscorbutic potency for guinea pigs corresponding approximately to the ascorbic acid content of the urine as measured by indophenol titration. Inasmuch as no other naturally occurring substance is known to have an equally high antiscorbutic potency, the reducing substance in the urine was presumably L-ascorbic acid. A number of other investigators have confirmed the effect of chloretone on the increase of reducing substances in the urine of rats (5-8). Cattle (9-11) and sheep (12) have been reported to give a similar response to chloretone administration. Preliminary to other studies in this laboratory it was considered desirable to isolate and characterize the principal reducing substance present in the urine of chloretone-treated rats. At the same time a procedure was worked out for the isolation, in pure form, of a 2,4-dinitrophenylhydrazine derivative of L-dehydroascorbic acid from materials containing relatively small quantities of ascorbic acid. Inasmuch as the precise structures of this derivative and its possible isomers (13) are not known, as well as for brevity of presentation, this derivative will be referred to subsequently in this paper as Compound I. The isopropylidene derivative prepared from Compound I will be referred to as Compound II.