Frederiks W M, Bosch K S, Kooij A
University of Amsterdam, Department of Cell Biology and Histology, The Netherlands.
J Histochem Cytochem. 1995 Jul;43(7):723-6. doi: 10.1177/43.7.7608527.
The tetrazolium salt method previously developed for the detection of xanthine oxidoreductase activity in unfixed cryostat sections has been validated for quantitative purposes. The specificity of the enzyme reaction was studied by incubating unfixed cryostat sections of rat liver in test medium containing the substrate hypoxanthine, in control medium that lacked the substrate, and in medium containing substrate and allopurinol, a specific inhibitor of xanthine oxidoreductase activity. The specific reaction rate was determined cytophotometrically by subtracting the amount of final reaction product generated in the control reaction from that formed in the test reaction. Highest specific enzyme activity in rat liver was found when the incubation medium contained 18% (w/v) polyvinyl alcohol, 100 mM phosphate buffer, pH 7.8, 0.45 mM 1-methoxyphenazine methosulfate, 5 mM tetranitro BT, and 0.5 mM hypoxanthine. Enzyme activity was present in liver parenchymal cells and in sinusoidal cells (endothelial and Kupffer cells) and was completely inhibited by allopurinol. A linear relationship was observed between the specific amount of final reaction product generated at 37 degrees C and incubation time at least up to 21 min, as well as section thickness up to 12 microns. Xanthine oxidoreductase activity, expressed as mumoles substrate converted per cm3 tissue/min, was 1.61 +/- 0.34 in pericentral areas and 1.24 +/- 0.16 in periportal areas. These values are similar to biochemical data reported in the literature. In conclusion, the tetrazolium method to detect xanthine oxidoreductase activity in unfixed cryostat sections of rat liver gives a reliable reflection of in situ activity.
先前开发的用于检测未固定低温恒温器切片中黄嘌呤氧化还原酶活性的四氮唑盐法已得到定量验证。通过将大鼠肝脏的未固定低温恒温器切片在含有底物次黄嘌呤的测试培养基中、在缺乏底物的对照培养基中以及在含有底物和黄嘌呤氧化还原酶活性特异性抑制剂别嘌呤醇的培养基中孵育，研究了酶反应的特异性。通过从测试反应中形成的最终反应产物量中减去对照反应中产生的最终反应产物量，以细胞光度法测定特异性反应速率。当孵育培养基含有18%（w/v）聚乙烯醇、100 mM磷酸盐缓冲液（pH 7.8）、0.45 mM 1-甲氧基吩嗪硫酸甲酯、5 mM四硝基BT和0.5 mM次黄嘌呤时，在大鼠肝脏中发现最高的特异性酶活性。酶活性存在于肝实质细胞和窦状细胞（内皮细胞和库普弗细胞）中，并被别嘌呤醇完全抑制。在37℃下产生的最终反应产物的特定量与孵育时间至少长达21分钟以及切片厚度高达12微米之间观察到线性关系。以每立方厘米组织每分钟转化的底物微摩尔数表示的黄嘌呤氧化还原酶活性，在中央周围区域为1.61±0.34，在门静脉周围区域为1.24±0.16。这些值与文献中报道的生化数据相似。总之，用于检测大鼠肝脏未固定低温恒温器切片中黄嘌呤氧化还原酶活性的四氮唑法能够可靠地反映原位活性。
