Effect of gentisic acid and related compounds on bovine testicular hyaluronidase.

The inhibition of the spreading effect of hyaluronidase by the previous administration of sodium salicylate was reported by Guerra and Robles Gil (1). Subsequent investigations indicated that this phenomenon could not be explained on the basis of the direct inhibition of hyaluronidase by salicylates, since such inhibition could only be demonstrated in vitro at high salicylate concentrations (2-5). A possible explanation of this discrepancy was afforded by the report by Meyer et al. (6) of an inhibitor of hyaluronidase in the urine of individuals to whom salicylates had been administered. The active substances were identified as gentisic and gentisuric acids (metabolic products of salicylic acid). These workers also reported that synthetic gentisic acid as well as other hydroquinones (hydroquinonedicarboxylic acid, homogentisic acid, and polyporic acid) and the corresponding quinones inhibited hyaluronidase. It was postulated that the therapeutic activity of salicylates in rheumatic diseases may be a consequence of their conversion to gentisic acid. Clinical reports have varied in their conclusions with respect to the therapeutic efficacy of gentisic acid (7, 8). The reported inhibition of hyaluronidase in vitro is contrary to the results of I,owenthal and Gagnon (9) and Calesnick and Buetner (5). It is the purpose of this communication to report a series of cxperimerits indicating that the inhibition of hyaluronidase by impure gentisic acid is not due to gentisic acid and probably not due to the corresponding quinone. These conclusions are partially based on the behavior of t,he enzyme toward the hydroquinone-benzoquinone system. The preparation of pure gentisic acid was of primary iniportance. Of the various methods available at the t,ime this work was initiated, the proccdurc of Mauthner (lo), involving the persulfate oxidation of salicylic acid, seemed most suitable. In our hands, however, this method yielded a highly colored product. Since numerous recrystallizations did not remove colored contaminants, it was necessary to devise new methods of purification. Lead salt fractionation was found most suitable for purification of large quantities.