MASS SPECTROMETRIC QUANITIFICATION OF MARKERS FOR PROTEIN OXIDATION

脂蛋白氧化与糖尿病的发病机制有关， 动脉粥样硬化 然而，生理相关的途径 介导的氧化损伤尚未被确定。 三 可能的机制是酪氨酰自由基、羟基自由基和氧化还原 活性金属离子 酪氨酸自由基形成o，o '-二酪氨酸交联 在蛋白质中。 高活性羟基自由基氧化 苯丙氨酸残基转化为邻酪氨酸和间酪氨酸。 金属离子 氧化低密度脂蛋白（LDL）的途径知之甚少。 探讨酪氨酰自由基、羟自由基、 金属离子在动脉粥样硬化，我们开发了一个高度敏感， 用于测量O，O ′-二酪氨酸水平的定量方法， 蛋白质、脂蛋白和组织中的o-酪氨酸和m-酪氨酸， 使用稳定同位素稀释气相色谱-质谱法。 我们发现，o，o ′-二酪氨酸在LDL中选择性产生， 用酪氨酰自由基氧化。 脂肪条纹病的诊断分析 氧化产物的模式相似;与正常主动脉相比 组织中o，o ′-二酪氨酸选择性升高， o-酪氨酸的变化。 检测到选择性增加 从血管病变中分离的LDL中的o，o '-二酪氨酸与 假设人类动脉粥样硬化中的氧化损伤是 部分由酪氨酰自由基介导。 相反，这些观察 不支持游离金属离子作为LDL催化剂的作用 动脉壁的氧化

Lipoprotein oxidation has been implicated in the pathogenesis of atherosclerosis. However, the physiologically relevant pathways mediating oxidative damage have not yet been identified. Three potential mechanisms are tyrosyl radical, hydroxyl radical, and redox active metal ions. Tyrosyl radical forms o,o'-dityrosine cross-links in proteins. The highly reactive hydroxyl radical oxidizes phenylalanine residues to o-tyrosine and m-tyrosine. Metal ions oxidize low density lipoprotein (LDL) by poorly understood pathways. To explore the involvement of tyrosyl radical, hydroxyl radical, and metal ions in atherosclerosis, we developed a highly sensitive and quantitative method for measuring levels of o,o'-dityrosine, o-tyrosine, and m-tyrosine in proteins, lipoproteins, and tissue, using stable isotope dilution gas chromatography-mass spectrometry. We showed that o,o'-dityrosine was selectively produced in LDL oxidized with tyrosyl radical. Analysis of fatty streak s reveale d a similar pattern of oxidation products; compared with normal aortic tissue, there was a selective increse in o,o'-dityrosine with no change in o-tyrosine. The detectin of a selective increase of o,o'-dityrosine in LDL isolated from vascular lesions is consistent with the hypothesis that oxidative damage in human atherosclerosis is mediated in part by tyrosyl radical. In contrast, these observations do not support a role for free metal ions as catalysts of LDL oxidation in the artery wall.