A colorimetric assay for xanthine oxidase in rat liver homogenates.

In studying the oxidation of xanthine manometrically by using liver homogenate systems (l), certain irregularities in the method have become evident to the authors. Occasionally an oxygen consumption has been observed after 2 to 3 hours of incubation which is greatly in excess of the theoretical requirement for the complete oxidation of the xanthine substrate. In certain experiments the measurements have to be continued this long or longer because of low xanthine oxidase activity in some animals. This excess oxygen uptake has been found to be inhibited by aureomycin and toluene, indicating that bacterial growth in the system may be the cause of this effect. In many experiments a large endogenous respiration is oft,en encountered which entails extensive incubation of the homogenate system before linear oxidation of the xanthine substrate is attained. Furthermore, such manometric data are generally difficult or impossible to interpret. In addition, a more direct chemical method would negate errors in the results which often attend a high endogenous respiration. For these reasons, it appeared important to develop a simpler and more direct assay for liver xanthine oxidase which would overcome the effects of these variations in the manometric technique. Assays for xanthine oxidase in homogenate systems have involved several criteria. Axelrod and Elvehjem (I) have described the manometric method for the oxidation of xanthine in the Warburg respirometer at 37”. Figge and Strong (2) have employed methylene blue as hydrogen acceptor in the Thunberg method, after dialysis, which is essentially a modification of the assay used by Green and Dixon (3). Van Meter and Oleson (4) have used 2-amino-4-hydroxy-6-pteridinecarboxaldehyde to inhibit xanthine oxidase in the endogenous respiration. Kalkar (5) has employed the spectrophotometric determination of the enzymatic oxidation of hypoxanthine or xanthine to uric acid which results in a marked increase in the absorption