Mass spectrometric quantification of markers for protein oxidation by tyrosyl radical, copper, and hydroxyl radical in low density lipoprotein isolated from human atherosclerotic plaques
Mass spectrometric quantification of markers for protein oxidation by tyrosyl radical, copper, and hydroxyl radical in low density lipoprotein isolated from human atherosclerotic plaques
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DOI：
10.1074/jbc.272.6.3520
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发表时间：
1997-02-07
影响因子：
4.8
通讯作者：
Heinecke, JW
中科院分区：
文献类型：
--
作者：
Leeuwenburgh, C;Rasmussen, JE;Heinecke, JW
脂蛋白氧化与动脉粥样硬化的发病机制有关。酪氨酰自由基、羟基自由基和氧化还原活性金属离子是氧化损伤的三种机制，酪氨酰自由基在蛋白质中形成o，o '-二酪氨酸交联，高活性的羟基自由基氧化苯丙氨酸残基形成o-酪氨酸和m-酪氨酸，金属离子氧化低密度脂蛋白（LDL）的机制尚不清楚。为了探讨酪氨酰自由基、羟自由基和金属离子在动脉粥样硬化中的作用，我们建立了一种高灵敏度和定量的方法，用稳定同位素稀释气相色谱-质谱法测定蛋白质、脂蛋白和组织中的o，o ′-二酪氨酸、o-酪氨酸和m-酪氨酸水平。我们发现，o，o ′-二酪氨酸选择性地在酪氨酰自由基氧化的LDL中产生，当LDL被羟基自由基氧化时，α-酪氨酸和α，o ′-二酪氨酸都是主要产物，在铜氧化的EDL中仅形成o-酪氨酸，在三种不同的氧化体系中观察到类似的氧化产物谱，将这些发现应用于从人动脉粥样硬化病变中分离的LDL，我们检测到o，o ′-二酪氨酸增加100倍，与循环LDL中的o ′-二酪氨酸水平相比，在从动脉粥样硬化组织中分离的LDL中，o-酪氨酸和m-酪氨酸水平没有升高，脂肪条纹的分析揭示了类似的氧化产物模式;与正常主动脉组织相比，o，o ′-二酪氨酸选择性增加，而o-酪氨酸无变化。检测到o，从血管病变中分离的LDL中的o ′-二酪氨酸与人动脉粥样硬化中的氧化损伤部分由酪氨酰自由基介导的假设一致。相反，这些观察结果不支持游离金属离子作为动脉壁中LDL氧化的催化剂的作用。
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, Both a-tyrosine and a,o'-dityrosine were major products when LDL was oxidized with hydroxyl radical, Only o-tyrosine was formed in EDL oxidized with copper, Similar profiles of oxidation products were observed in bovine serum albumin oxidized with the three different systems, Applying these findings to LDL isolated from human atherosclerotic lesions, we detected a 100-fold increase in o,o'-dityrosine levels compared to those in circulating LDL, In striking contrast, levels of o-tyrosine and m-tyrosine were not elevated in LDL isolated from atherosclerotic tissue, Analysis of fatty streaks revealed a similar pattern of oxidation products; compared with normal aortic tissue, there was a selective increase in o,o'-dityrosine with no change in o-tyrosine, The detection 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.