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

本研究利用氧化型磷脂酰胆碱单克隆抗体（DLH_3）建立了一种测定氧化型低密度脂蛋白（OxLDL）的灵敏方法，证实了人循环血浆中存在OxLDL。已知OxLDL是动脉粥样硬化的危险因素，是异质修饰颗粒的混合物。OxLDL在体内的确切性质尚未阐明。本研究尝试从人血浆中分离氧化低密度脂蛋白并分析其修饰结构。阐明体内OxDL本身应该是理解体内OxLDL代谢行为和临床意义的关键步骤，先前基于使用DLH 3抗体的夹心ELISA估计，在人血浆中大约10000个LDL颗粒中有一个OxLDL颗粒。为了分析如此微量的物质，我们引入了ESI-LC/MS/MS技术。用丙烯醛或4-羟基壬烯醛（HNE）预处理人LDL，胰蛋白酶消化后，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apoB的胰蛋白酶片段。这些典型的修饰结构是蛋白质氧化修饰的良好标记，它们可用于体内OxLDL的分析。我们还成功地检测了氧化磷脂和氧化胆固醇使用MS/MS。这是很可能的，需要有效的浓度从血浆中的步骤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的胰蛋白酶片段。改性

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