ISOTOPE DILUTION MASS SPECTROMETRIC QUANTIIFCATION OF 3 NITROTYROSEINE

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。对组织损伤的机制了解得很差， 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 体内含氮物种对组织损伤的抑制作用。在这里我们 描述一种高度灵敏的ANS特定分析方法 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。3-硝基酪氨酸的阿托摩尔水平可以 以这种方式可重复测量。3-硝基酪氨酸的定量测定 用稳定同位素稀释气相色谱/质量测定组织水平 光谱分析应该为解释这种影响提供一个强有力的工具 活性氮物种对体内氧化读数的影响。

Oxidative damage by reactive nitrogen species has been implicated in the pathogenesis of atherosclerosis and other inflammatory diseases. The mechanims of tissue damage are poorly understoond, 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 indciator of tissue injury by nitrogen species in vivo. Here we describe a highly sensitive ans 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 quanitfied by isotope dilution gas chromagtography negative-io n 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 chromtogrpahy/mass spectrometry should provide a powerful tool for exploriong the impact of reactive nitrogen species on oxidative readtions in vivo.