MASS SPECTROMETRIC QUANITIFICATION OF MARKERS FOR PROTEIN OXIDATION

脂蛋白氧化与糖尿病的发病机制有关 动脉硬化。然而，生理上相关的通路 介导氧化损伤的机制尚未确定。三 可能的机制是酪氨酸基、羟基和氧化还原。 活性金属离子。酪氨酸基形式的O，O‘-Dityroine交联剂 在蛋白质中。高活性的羟基自由基氧化 苯丙氨酸残基为o-酪氨酸和m-酪氨酸。金属离子 通过鲜为人知的途径氧化低密度脂蛋白。 探讨酪氨酸基、羟基自由基及 金属离子在动脉粥样硬化中的作用，我们研制出了一种高度敏感和 测定O，O‘-二氨基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， 采用稳定同位素稀释气相色谱-质谱法。 我们发现在低密度脂蛋白中有选择性地产生O，O‘-二硫酪氨酸 用酪氨酸根氧化的。S显露脂肪条纹的分析 氧化产物的形态相似；与正常主动脉相比 组织中，O，O‘-二氢酪氨酸选择性增加，而NO O-酪氨酸的变化。一种选择性增加的检测方法 从血管病变中分离的低密度脂蛋白中的O，O‘-二氢酪氨酸是一致的 假设人类动脉粥样硬化中的氧化损伤是 部分由酪氨酸基调节。相比之下，这些观察结果 不支持自由金属离子作为低密度脂蛋白的催化剂 动脉壁的氧化作用。

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.