THE METABOLISM OF THE METHYLATED PURINES: I. THE ENZYMATIC DETERMINATION OF URINARY URIC ACID

Abstract

Myers and coworkers (1) have suggested that caffeine, theophylline, and theobromine may be oxidized in the 8 posit,ion by the animal body to give rise to methylated uric acids. Certain of these methylated derivatives react, as does uric acid with phosphotungstic acid, in an alkaline medium to form blue-colored reduction products. Since some of the methyluric acids are precipitated along iyith uric acid by both a.mmoniaca,l silver and acid silver lactate, the presence of such methylated derivatives in urine would give rise to high values for uric acid by both the direct and silver precipitation procedures. Consequently it is impossible by these methods of analysis to decide whether the increased rcduct,ion of the phosphotungstic acid reagent by a urine collected after the ingestion of caffeine or theophylline is due to extra, uric acid formed by the demethylation and oxidation of these compounds or to the presence of methyluric acids. The present paper describes a,n enzymatic procedure which will distinguish between uric acid and its methylated derivatives. The proposed method is similar in principle to that of Blauch and Koch (2) and of Bulger and Johns (3) for blood uric acid in that the enzyme uricase is used for the destruction of uric acid. Keilin and Hartree (4) have reported that uricase is a highly specific enzyme and does not oxidize any of the mono-, di-, or trimethyluric acids. A suitable aliquot of the urine is allowed to react with arsenophosphotungstic acid in an alkaline medium. The blue color which is obtained is measured in the Evelyn photoelectric calorimeter and is referred to as the total color, since the reduction is due in part to the presence of compounds other than uric acid. A second aliquot of urine is buffered at the optimum pH for uricase activity and incubated with the enzyme for 2 hours at the optimum temperature. All of the uric acid is destroyed, but the non-uric acid reducing substances including the methyluric acids are not oxidized by the uricase. The solution is then treated with sodium tungstate and