Cytotoxicity of Superoxide and Nitric Oxide (NO)

1. 测定了人多形核白细胞中超氧化物、一氧化氮（NO）和ONOO^-的生成。超氧化物细胞色素c还原和MCLA化学发光方法。将亚硝酸盐还原为硝酸盐后，采用Griess法测定NO、NO末端根、亚硝酸盐+硝酸盐的含量。对已有报道的HPA-NO_2-HPLC法进行了改进，建立了稳定的ONOO^-测定方法。利用这些方法，我们研究了这些活性分子在各种疾病中的作用，我们发现在多形核白细胞介导的损伤中，当白细胞的目标是心肌细胞时，可能的细胞毒性分子是ONOO^-，而当白细胞的目标是内皮细胞时，超氧化物是主要的细胞毒性分子。超氧化物、一氧化氮和一氧化氮的实时测量-我们还对来自离体器官的上述分子进行了实时测量，以探讨其细胞毒性机制，从而进一步揭示缺血再灌注损伤的潜在机制。在应用的几种方法中，我们发现氧肌红蛋白法适用于该目的。证明ONOO^-介导的损伤为了证明ONOO^-在肝硬化、胃溃疡和肠癌等各种疾病中介导的组织损伤，测量了ONOO^-和酪氨酸残基蛋白的反应产物硝基酪氨酸。同时测定血浆和组织中亚硝酸盐+硝酸盐浓度。使用硝基酪氨酸单克隆抗体对切除或活检时获得的组织样本进行免疫组织学染色。溃疡组织中硝基酪氨酸和亚硝酸盐浓度显著升高。这种增加与溃疡的严重程度相关。相比之下，这些水平在癌症中心仍然很低，在癌症边缘部分略有升高。硝基酪氨酸染色法在病变部位有部分阳性染色，但特异性有待进一步提高。少

1. MEASUREMENT OF SUPEROXIDE,NITRIC OXIDE (NO) and ONOO^-The generation of superoxide, NO and ONOO^- from human polymorphonuclear leukocytes were measured. For superoxide cytochrome c reduction and MCLA chemiluminescence methods were used. For NO,end uprodct of NO,nitrite+nitrate was measured by Griess method after reducing nitrite to nitrate. For ONOO^- we established a stable method by modifying the previously reported HPA-NO_2-HPLC method. Using these methods, the roles of these active molecules in various diseases were investigated and we found that the plausible cytotoxic molecules in polymorphonuclear leukocytes-mediated injury appears to be ONOO^- when the target of leukocytes is cardiac myocytes but superoxide is the major cytotoxic molecule when the target of leukocytes is endothelial cells.2. REAL-TIME MEASUREMENT OF SUPEROXIDE,NO and ONOO^-The real-time measurement of above mentioned molecules from isolated organs were also performed to investigate the cytotoxic mechanisms u … More nderlying the ischemia-reperfusion injury. Among several methods applied, we found that the oxymyoglobin method was applicable for that purpose.3. DEMONSTRATION OF ONOO^--MEDIATED INJURYTo demonstrate ONOO^--mediated tissue damage in various diseases such as liver cirrhosis, stomach ulcer and intestinal cancer, nitrotyrosine which is the reaction product of ONOO^- and tyrosine residues of protein was measured. Plasma and tissue concentration of nitrite+nitrate were also measured. The immunohistological staining using nitrotyrosine monoclonal antibodies was also performed for tissue samples obtained at the time of resection or through biopsy. The nitrotyrosine and nitrite+nitrate concentrations were significantly elevated in ulcer tissue. The increase correlated with severity ulcer. In contrast these levels remained low in the center of cancer and slightly elevated in the marginal portion of cancer. The nitrotyrosine staining method gave some positive staining in diseased portion but the specificity was shown to need further improvement. Less