ISOTOPE DILUTION MASS SPECT QUANTIFICATION OF 3 NITROTYROSINE IN PROTEINS

反应性氮物种的氧化损伤已被牵连 在动脉粥样硬化和其他炎症的发病机制中 疾病。对组织损伤的机制知之甚少， 然而，因为有毒的中间体是短暂的。上一次传入 体外研究表明，3-硝基酪氨酸代表一种 活性氮物种氧化蛋白质的特异性标志物。 因此，对这种硝化芳香氨基酸的检测可以作为 体内氮素物种对组织损伤的指标。在这里我们 描述一种高度灵敏和具体的分析方法 测定游离和蛋白质结合的3-硝基酪氨酸。化验结果 涉及蛋白质的酸解，通过以下方法分离3-硝基酪氨酸 离子交换层析和3-硝基酪氨酸还原为 3-氨基酪氨酸与连二亚硫酸盐。还原的氨基酸然后是 转化为其正丙基，全七氟丁酰衍生物和 同位素稀释气相色谱负离子定量 化学电离质谱仪。阿托莫尔水平 用这种方法可以重复性地测定3-硝基酪氨酸。 用稳定同位素测定组织中3-硝基酪氨酸水平 稀释气相色谱/质谱仪应提供 探索活性氮物种对环境影响的有力工具 体内的氧化反应。

Oxidative damage by reactive nitrogen species has been implicated in the pathogenesis of atherosclerosis and other inflammatory diseases. The mechanisms of tissue damage are poorly understood, however, because the toxic intermediates are short-lived. Previous in vitro studies have suggested that 3-nitrotyrosine represents a specific marker of protein oxidation by reactive nitrogen species. The detection of this nitrated aromatic amino acid may thus serve as an indicator of tissue injury by nitrogen species in vivo. Here we describe a highly sensitive and specific analytical method for quantifying free and protein-bound 3-nitrotyrosine. The assay involves acid hydrolysis of proteins, isolation of 3-nitrotyrosine by ion exchange chromatography, and reduction of 3-nitrotyrosine to 3-aminotyrosine with dithionite. The reduced amino acid is then converted to its n-propyl, per-heptafluorobutyryl derivative and quantified by isotope dilution gas chromatography negative-ion chemical ionization mass spectrometry. Attomole levels of 3-nitrotyrosine can be reproducibly measured in this manner. Quantifying 3-nitrotyrosine levels of tissues by stable isotope dilution gas chromatography/mass spectrometry should provide a powerful tool for exploring the impact of reactive nitrogen species on oxidative reactions in vivo.