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 LDL (OxLDL) 与动脉粥样硬化有关。我们通过开发一种使用单克隆抗体测量 OxLDL 的灵敏方法，证明了循环血浆中存在 OxLDL。最近，出现了铜是否会诱导 LDL 氧化的问题。已被广泛用作模型，反映了体内 OxLDL 的特征。体内 OxLDL 的另一种模型称为最小修饰 LDL (MM-LDL)，它是在较温和的条件下制备的。本研究中，对各种修饰的LDL进行了表征。MM-LDL是按照Berliner等人描述的方法制备的，其中LDL在4℃下用含有FeSO4的PBS透析4天，而MM-LDL的含量是其四分之一。 TBARS 与铜诱导的 OxLDL 相比，两种修饰的 LDL 中的共轭二烯含量和抗氧化磷脂酰胆碱 (OxPC) 抗体的反应性几乎相同同样，当从修饰的LDL中提取的总脂质中含有醛的OxPC时，MM-LDL中含有醛的OxPC的量比铜诱导的OxLDL多10倍。从这些结果可以清楚地看出，氧化产物。氧化修饰的 LDL 中的修饰结构可能会根据治疗的不同而有所不同。本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，必须改进从血浆中分离微量 OxLDL 的程序以及测定氧化产物的灵敏方法，可以将低至 10 pmol 的含醛 OxPC 作为荧光衍生物进行检测，然而，甚至可以实现更灵敏的分析。迄今为止，通过离子交换 HPLC 或密度梯度超速离心法从血浆中分离 OxLDL 尚未成功，我们目前正在尝试采用免疫沉淀法。