A histochemical procedure for light microscopic demonstration of xanthine oxidase activity in unfixed cryostat sections using cerium ions and a semipermeable membrane technique.

Xanthine oxidoreductase exists in two functionally distinct forms. Under normal conditions, the larger part of the enzyme occurs as an NAD(+)-dependent dehydrogenase form which produces NADH and urate. The dehydrogenase can be transformed under various (patho)physiological conditions to an oxygen-dependent oxidase form which produces oxygen radicals and/or hydrogen peroxide and urate. Tetrazolium salts are used to demonstrate the total activity of both the dehydrogenase and the oxidase form of the enzyme. We have developed a procedure to detect the oxidase form only in unfixed cryostat sections with the use of cerium on the basis of the semipermeable membrane technique. The incubation medium contained hypoxanthine as substrate, cerium ions, and sodium azide to inhibit catalase and peroxidase activity. In a second-step reaction, diaminobenzidine was polymerized in the presence of cobalt ions by decomposition of cerium perhydroxide. Large amounts of final reaction product were found in milk droplets in the acini of lactating bovine mammary gland, whereas milk-secreting epithelial cells contained hardly any final reaction product. In rat duodenum, enzyme activity was found in the cytoplasm of enterocytes and goblet cells but not in the mucus. Control reactions performed in the absence of substrate or in the presence of substrate and allopurinol, a specific inhibitor of xanthine oxidase, were completely negative in both tissues, with the exception of polymorphonuclear leukocytes in the lamina propria of duodenum. The positive nonspecific reaction in these cells was caused by myeloperoxidase activity. We conclude that the present method is specific for the detection of xanthine oxidase activity. Moreover, conversion of the dehydrogenase form into the oxidase form can be prevented by omission of chemical fixation of the tissue in the present procedure.