Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay
Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay
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DOI：
10.1152/ajpcell.00028.2004
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发表时间：
2004-10-01
影响因子：
5.5
通讯作者：
Dikalov, S
中科院分区：
文献类型：
--
作者：
Fink, B;Laude, K;Dikalov, S
最近，证明了超氧化物将二氢乙锭氧化成特异性荧光产物（氧乙锭），其与乙锭的不同之处在于其分子结构中存在额外的氧原子（Zhao H，Kalivendi S，Zhang H，Joseph J，Nithipatikom K，Vasquez-Vivar J，and Kalyanaraman B. Free Radic Biol Med 34：1359 - 1368，2003）。我们已经适应了这种新的基于HPLC的测定，以量化该产品作为一种工具，以估计在完整的组织细胞内超氧化物。使用C-18柱分离氧化乙锭和氧化乙锭，并使用荧光检测进行定量。最初的无细胞实验与超氧化钾和黄嘌呤氧化酶证实oxythidium从dihydroethidium的形成。oxythidium的形成被抑制超氧化物歧化酶，但没有过氧化氢酶，并没有发生后，加入过氧化氢，过氧亚硝酸盐，或次氯酸。在牛主动脉内皮细胞（BAEC）和小鼠动脉中，氧化还原循环药物甲萘醌使二氢乙锭形成氧乙锭的量增加9倍（对照组为0.4 nmol/mg，20 μ M甲萘醌组为3.6 nmol/mg），而聚乙二醇结合的超氧化物歧化酶（PEG-SOD）显著抑制了这种作用。血管紧张素II治疗BAEC引起oxythidium形成的两倍增加，PEG-SOD（0.5 nmol/mg）也降低了这种作用。此外，在血管紧张素II诱导的高血压和DOCA-盐高血压的小鼠的动脉中，oxythidium的形成以对应于基于细胞色素c还原估计的超氧化物产生的方式增加。使用HPLC检测氧乙硫铵代表了一种新的，方便的，定量检测超氧化物在完整的细胞和组织中的方法。
Recently, it was demonstrated that superoxide oxidizes dihydroethidium to a specific fluorescent product (oxyethidium) that differs from ethidium by the presence of an additional oxygen atom in its molecular structure (Zhao H, Kalivendi S, Zhang H, Joseph J, Nithipatikom K, Vasquez-Vivar J, and Kalyanaraman B. Free Radic Biol Med 34: 1359 - 1368, 2003). We have adapted this new HPLC-based assay to quantify this product as a tool to estimate intracellular superoxide in intact tissues. Ethidium and oxyethidium were separated using a C-18 column and quantified using fluorescence detection. Initial cell-free experiments with potassium superoxide and xanthine oxidase confirmed the formation of oxyethidium from dihydroethidium. The formation of oxyethidium was inhibited by superoxide dismutase but not catalase and did not occur upon the addition of H2O2, peroxynitrite, or hypochlorous acid. In bovine aortic endothelial cells ( BAEC) and murine aortas, the redox cycling drug menadione increased the formation of oxyethidium from dihydroethidium ninefold (0.4 nmol/mg in control vs. 3.6 nmol/mg with 20 muM menadione), and polyethylene glycolconjugated superoxide dismutase (PEG-SOD) significantly inhibited this effect. Treatment of BAEC with angiotensin II caused a twofold increase in oxyethidium formation, and this effect also was reduced by PEG-SOD (0.5 nmol/mg). In addition, in the aortas of mice with angiotensin II-induced hypertension and DOCA-salt hypertension, the formation of oxyethidium was increased in a manner corresponding to superoxide production estimated on the basis of cytochrome c reduction. Detection of oxyethidium using HPLC represents a new, convenient, quantitative method for the detection of superoxide in intact cells and tissues.