Observations on the oxidation of homogentisic acid in urine.

Homogentisic acid, which is considered to be an intermediate in the metabolism of tyrosine and phenylalanine, is excreted in a number of different conditions. The most widely studied of these, which is caused by a genetic inability to oxidize homogentisic acid, was termed alcaptonuria, owing to the characteristic darkening of the urine on standing. It has also been shown (Sealock & Silberstein, 1940; Rogers & Gardner, 1949) that, normally, ascorbic acid plays some part in the metabolismofhomogentisicacid, since the latter has been claimed to be present in the urine of scorbutic subjects and of premature infants (Levine, 1949). In both these latter cases, but not in congenital alcaptonuria, the administration of ascorbic acid stops the excretion of homogentisic acid. Dietary amino-acid imbalance may also interfere with the normal metabolism of tyrosine and phenylalanine and result in excretion of homogentisic acid (Glynn, Himsworth & Neuberger, 1945; Neuberger & Webster, 1947). Homogentisic acid is the reduced form of 1:4-benzoquinone-2-acetic acid, both together constituting an oxidation-reduction system. The high positive redox potential of benzoquinoneacetic acid, however, makes it appear extremely unlikely that it could exist for any appreciable length oftime in a biological medium. It is therefore surprising that Fishberg (1948) reported its presence in urines from a case of enterogenous cyanosis, from scorbutic animals and from cases of rheumatic fever. The last observation was of special interest since it suggested a possible relationship of rheumatic fever to a vitamin C disturbance, a relationship for which some evidence already existed (Rinehart, 1938). Since the methods for detecting the presence of benzoquinoneacetic acid depend largely on its oxidizing properties, special care must be taken to avoid the development of oxidizing substances in urine after its collection. This could arise, for instance, as a result of the growth of nitrite-forming bacteria. The nitrite so produced would not only give several of the tests used by Fishberg (1948) to demonstrate benzoquinoneacetic acid, but might oxidize any homogentisic acid present to the quinoneandthus account forherunexpected findings. Itwas therefore decided to investigate the action of oxidants on homogentisic acid in urine; and secondly, in the light of these results, to re-investigate Fishberg's claims to have found benzoquinoneacetic acid in urines from rheumatic fever cases.