Separation and analysis of oxidized low-density lipoprotein in vivo using immunological technique.

利用氧化磷脂酰胆碱识别抗体(DLH3)建立了一种灵敏的检测氧化低密度脂蛋白(OxLDL)的方法，证实了人循环血浆中存在氧化低密度脂蛋白(OxLDL)。氧化低密度脂蛋白被认为是动脉粥样硬化的危险因素，是一种异质修饰颗粒的混合物。体内存在的氧化低密度脂蛋白的确切性质尚未阐明。在本研究中，我们试图从人血浆中分离出氧化低密度脂蛋白，并分析其修饰结构。阐明体内氧化低密度脂蛋白本身是了解氧化低密度脂蛋白在体内代谢行为和临床意义的关键一步。此前，基于DLH3抗体的夹心ELISA法估计，人血浆中大约每10000个低密度脂蛋白颗粒中就有一个氧化低密度脂蛋白颗粒。为了分析如此微量的物质，我们引入了电喷雾电喷雾质谱(ESI-LC/MS/MS)技术。人低密度脂蛋白经丙烯醛或4-羟基壬烯醛(HNE)处理后，经胰酶消化后，进行LC/MS/MS分析。我们成功地检测和鉴定了含有丙烯醛修饰的赖氨酸或HNE修饰的组氨酸的载脂蛋白B的胰酶片段。这些典型的修饰结构是蛋白质氧化修饰的良好标志，对于体内OxLDL的分析是有用的。我们还成功地用MS/MS检测了氧化的磷脂和氧化的胆固醇，很可能需要从血浆中有效的浓度步长来分析血浆氧化低密度脂蛋白。在初步试验中，使用OxLDL吸收透析膜分离血浆中的OxLDL，并鉴定了含有丙烯醛修饰的赖氨酸或HNE修饰的组氨酸的apoB的胰酶片段。修改为

We have proved the occurrence of oxidized low-density lipoprotein (OxLDL) in human circulating plasma through establishing a sensitive method to measure OxLDL using a monoclonal atnibdy (DLH3) recognizing oxidized phosphatidylcholine. OxLDL, known for a risk factor for atherosclerosis, is a mixture of heterogeneously modified particles. The precise nature of OxLDL present in vivo has not yet been elucidated. In this study we tried to separate OxLDL from human plasma and analyze its modified structures. Elucidation of in vivo OxDL itself should be a crucial step for understanding metabolic behavior and clinical significance of OxLDL in vivo.It was estimated previously, based on the sandwich ELISA using DLH3 antibody, that there is approximately one OxLDL particle in 10000 LDL particles in human plasma. To analyze such minute amount of materials, we introduced ESI-LC/MS/MS technique. Human LDL pre-treated either with acrolein or 4-hydroxynonenal (HNE) was trypsin-digensted, then the samole was subjected to LC/MS/MS analysis. We successfully detected and identified tryptic fragments of apoB containing acrolein-modified lysine or HNE-modified histidine. These typical modified structures are good markers for oxidative modification of proteins, and they are useful for analysis of OxLDL in vivo. We also successfully detected oxidized phospholipids and oxidized cholesterols using MS/MS. It is very likely that effective concentration step of OxLDL from plasma is needed to analyze plasma OxLDL. Separation of OxLDL in plasma using an OxLDL-absorbing dialysis membrane in a preliminary trial, that and identified tryptic fragments of apoB containing acrolein-modified lysine or HNE-modified histidine. modified with