Frederiks W M, Marx F
Laboratory of Cell Biology and Histology, University of Amsterdam, Academic Medical Centre, The Netherlands.
J Histochem Cytochem. 1993 May;41(5):667-70. doi: 10.1177/41.5.8468447.
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.
黄嘌呤氧化还原酶以两种功能不同的形式存在。在正常情况下，该酶的大部分以依赖NAD(+)的脱氢酶形式存在，产生NADH和尿酸盐。在各种（病理）生理条件下，脱氢酶可转化为依赖氧的氧化酶形式，产生氧自由基和/或过氧化氢以及尿酸盐。四氮唑盐用于显示该酶脱氢酶和氧化酶形式的总活性。我们基于半透膜技术开发了一种仅在未固定的低温恒温切片中使用铈检测氧化酶形式的方法。孵育介质含有次黄嘌呤作为底物、铈离子和叠氮化钠以抑制过氧化氢酶和过氧化物酶活性。在第二步反应中，二氨基联苯胺在钴离子存在下通过过氢氧化铈的分解而聚合。在泌乳牛乳腺腺泡的乳滴中发现大量最终反应产物，而分泌乳汁的上皮细胞几乎不含任何最终反应产物。在大鼠十二指肠中，在肠上皮细胞和杯状细胞的细胞质中发现酶活性，但在黏液中未发现。在无底物或有底物和黄嘌呤氧化酶特异性抑制剂别嘌呤醇存在的情况下进行的对照反应在两种组织中均完全呈阴性，但十二指肠固有层中的多形核白细胞除外。这些细胞中的阳性非特异性反应是由髓过氧化物酶活性引起的。我们得出结论，本方法对黄嘌呤氧化酶活性的检测具有特异性。此外，在本程序中省略组织的化学固定可防止脱氢酶形式转化为氧化酶形式。
