Modified structure and biological activities for oxidized LDL present in vivo searching for a better model for the physiological oxidized LDL

体内氧化低密度脂蛋白的修饰结构和生物活性寻找更好的生理氧化低密度脂蛋白模型

基本信息

批准号：
13672303
负责人：
ITABE Hiroyuki
金额：
$ 2.3万
依托单位：
Teikyo University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

It is widely believed that oxidized LDL (OxLDL) is involved in atherogenesis. We have demonstrated the presence of OxLDL in circulating plasma by developing a sensitive method of measuring OxLDL using a monoclonal antibody. Recently, a question arises whether copper-induced oxidation of LDL, which has been widely utilized as a model, reflects the characteristics of OxLDL in vivo. An alternative model for in vivo OxLDL is called minimally modified LDL (MM-LDL) which is prepared under milder conditions. In the present study, various modified LDLs were characterized.MM-LDL was prepared by the method described by Berliner et al. , in which LDL was dialyzed against PBS containing FeS04 at 4 ℃ for 4 days. While the MM-LDL contained a quarter as much of TBARS compared to copper-induced OxLDL, the amount of conjugated dienes and reactivity to anti-oxidized phosphatidylcholine (OxPC) antibody in the both modified LDLs were almost the same. When aldehyde-containing OxPC in the total lipids extracted from the modified LDLs, 10-fold larger amount of aldehyde-containing OxPC was present in MM-LDL than copper-induced OxLDL. From these results, it is clear that oxidized products and modified structures in the oxidatively modified LDL can be varied depending on the treatment.The next step for this study is to compare the oxLDL present in vivo with these modified LDL models. In order to do this, a procedure to isolate minute amount of OxLDL from plasma and sensitive methods to determine oxidation products must be improved. As low as 10 pmol of aldehyde-containing OxPC can be detected as fluorescent derivatives, however, even more sensitive analysis was achieved using ESI/MS procedure. Isolation of OxLDL from plasma was not successful so far by ion-exchange HPLC or density gradient ultracentrifugation. We are currently trying an immuno-precipitaion procedure for this purpose.

人们普遍认为，氧化的LDL（OXLDL）参与动脉粥样硬化。我们通过开发一种使用单克隆抗体测量OXLDL的敏感方法来证明了循环血浆中OXLDL的存在。最近，出现了一个问题，它是否已被广泛用作模型的LDL氧化反映了体内OxLDL的特征。体内OXLDL的替代模型称为在米勒条件下制备的最小修饰的LDL（MM-LDL）。在本研究中，表征了各种修饰的LDL。mm-LDL是通过Berliner等人描述的方法制备的。，其中LDL与含有FES04的PBS透析为4天4天。与铜诱导的OXLDL相比，MM-LDL含有四分之一的TBAR，但在两个修饰的LDL中，共轭字典和对抗氧化磷脂酰胆碱（OXPC）抗体的反应性的量几乎相同。当从修饰的LDL中提取的总脂质中含醛的OXPC时，MM-LDL中的含醛的OXPC比铜诱导的OXLDL大10倍。从这些结果中可以明显看出，氧化的产物和氧化修饰的LDL中的修饰结构可以根据处理而变化。该研究的下一步是将体内存在的OXLDL与这些修饰的LDL模型进行比较。为此，必须改善一种将微量OXLDL与等离子体和灵敏方法分离的程序，以确定氧化产物。可以将含醛的OXPC的低至10 pmol的OXPC检测为荧光衍生物，但是，使用ESI/MS程序可以实现更敏感的分析。通过离子交换HPLC或密度梯度超速离心，从血浆中分离出OXLDL并未成功。目前，我们正在为此目的尝试采用免疫培训程序。