Development of analysis for all biologically relevant derivatives of NO

由NO产生的活性氮氧化物产生各种生物分子的硝化加合物。我们建立了一系列氨基酸和核酸碱基的硝化加合物的鉴定和定量方法，如3-硝基-L-酪氨酸、硝基-L-色氨酸和8-硝基鸟氨酸。该分析将高效液相色谱系统与电化学(EC)分析相结合。Pronase消化样品中的每一种硝基化合物经反相高效液相色谱分离后，首先在-900 mV下进行电化学还原，然后在300 mV下用分析池进行检测。3-硝基酪氨酸的检出限可达10^~(-15)~(-15)摩尔。在感染流感病毒的小鼠的支气管肺泡灌洗液中，蛋白结合的3-硝基酪氨酸增加；而在诱导型一氧化氮合酶缺陷的小鼠中，蛋白结合的3-硝基酪氨酸没有被鉴定出来。同样，在哮喘和慢性支气管炎患者的痰中也检测到相当数量的3-硝基酪氨酸。然而，在小鼠和人类样本中，硝基色氨酸的水平都低于检测限值。此外，我们用抗3-硝基鸟嘌呤抗体进行的免疫组织化学分析清楚地显示，在病毒感染的小鼠肺的支气管上皮细胞中形成了3-硝基鸟苷。这是第一次证明在生物系统中内源形成的硝化核苷酸碱基。这些方法有助于我们更好地了解生物相关性和生物硝化的机理。

Reactive nitrogen oxides derived from NO produce nitration adducts of various biological molecules. We have developed a series of methods for identification and quantification of nitrated adducts of amino acids and nucleic acid bases, such as 3-nitro-L-tyrosine, nitro-L-tryptophan, and 8-nitroguanine. The assay couples an HPLC system with electrochemical (EC) analysis. Each nitrated compound in Pronase-digested samples, being separated by reverse-phase HPLC, was first reduced electrochemically at -900 mV, followed by detection with an analytical cell at +300 mV. Detection limit for 3-nitrotyrosine reached 10^<-15>mol. Increased protein-bound 3-nitrotyrosine was demonstrated in bronchoalveolar lavage from mice infected with influenza virus; whereas protein-bound 3-nitrotyrosine was not identified with mice deficient in inducible NO synthase. Similarly, appreciable amount of 3-nitrotyrosine was detected in sputum from patients with bronchial asthma and chronic bronchitis. The level of nitrotryptophan was, however, below detection limit in both murine and human samples. Also, our immunohistochemical analysis with a specific anti-3-nitroguanine antibody clearly showed formation of 3-nitroguanosine in the bronchial epithelial cells of the virus-infected mouse lungs. This is the first demonstration of a nitrated nucleotide base formed endogenously in biological systems. The present methods may help us to better understand the biological relevance and the mechanism of biological nitration.