On the determination of xanthine oxidase activity in animal tissues.

Studies, undertaken in this laboratory to evaluate the effect, of riboflavin antagonists on mammalian flavoenzymes, have necessitated the investigation of the xanthine oxidase activity of mouse tissue. In general, little difficulty has been encountered in the manometric determination of xanthine oxidase (1) in tissues of high enzymatic activity, e.g. liver and small intestine, although irregularities similar to those reported by Litwack et al. (2) were occasionally observed. However, the manometric determination of xanthine oxidase in tissues of low activity showed deficiencies of precision, mainly due to a very high endogeneous respiration complicated with the same irregularities mentioned above. Furthermore, manometric values for xanthine oxidase lack specificity in that they represent the summation of uricase and xanthine oxidase activities of the tissue being analyzed. Since an evaluation of our studies for chemotherapeutic purposes was dependent not only on changes in xanthine oxidase activity in isolated tissue, but, on the effect, of such changes on the quantitative distribution of the enzyme throughout the organism as a whole, this lack of specificity was undesirable. Xanthine oxidase has been measured non-manometrically by spectrophotometric (3), modified Thunberg, (4, 5), and calorimetric (2) procedures. All these methods were found to be inadequate for our purposes, since they were either not adaptable to routine or large scale use or were suitable only for the analyses of tissues of high enzymatic activity. The procedure outlined in this communication overcomes many of the defects reviewed above. Uricase activity can be readily distinguished from xanthine oxidase activity. The sensitivity of this method is such that tissues of low activity are as easily analyzed as more active tissues. Furthermore, the procedure is adaptable to large scale and routine use and requires a minimum of equipment. Finally, prolonged preincubation to decompose endogeneous purine is unnecessary, and hence the danger of bacterial contamination is minimized.