Studies on Enzymatic Formation and Destruction of Uric Acid in Mammalian Blood

Abstract

The colorimetric estimation of uric acid by a phosphotungstic acid reagent is made more specific by the use of uricase. The uricase preparation used consists of ox-kidney which has been acetone-dehydrated, benzene-defatted, air-dried, and finely ground. In the studies described here we used the direct colorimetric method 1 that has been in use in this laboratory for a number of years. The cyanide solution described in this method is replaced by a solution of 2.5% sodium cyanide in 25% urea, and the tubes are kept at room temperature for 3 hours instead of being heated to allow the color to develop. Two-cc quantities of blood are incubated for 2 hours at 40° to 48°C. with 50-mg samples of uricase, after which the blood proteins as well as the uricase are precipitated with tungstic acid. The uric acid color-value of this filtrate is determined and subtracted from the value obtained on a filtrate from untreated blood—the difference represents true uric acid.

The average value obtained on 25 samples of human blood before uricase action was 3.0 mg% (varying from 1.9 to 4.6) and the average value of these same samples treated with uricase was 0.8 mg% (from 0.7 to 1.1). The true uric-acid value for these samples, then, is 2.2 mg % (varying from 1.1 to 3.8). Recoveries on 15 samples of human blood to which uric acid had been added in quantities of from 2 to 16 mg % averaged 92% (varying from 82 to 100%). The uric acid added to blood was quantitatively destroyed by uricase, leaving the same non-uric acid color-value as that of the original blood.

14 typical sample of dog blood gave the following results: The uric-acid color-value on the untreated blood was 2.1 mg % and on the uricase-treated blood, 1.8 mg %, giving 0.3 mg as the true uric-acid value. The true uric-acid value obtained on 10 samples of dog Mood ranged from 0 to 0.5 mg %. Uric acid added to dog blood is recovered with the same accuracy as with human blood, and is quantitatively destroyed when uricase is allowed to act on it, showing that there is nothing in dog blood to prevent the detection of uric acid by this method.