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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.

氧化型低密度脂蛋白（OxLDL）参与动脉粥样硬化的形成。我们已经证明了存在的OxLDL循环血浆中开发一种灵敏的方法，测量OxLDL使用单克隆抗体。最近，出现了一个问题，是否铜诱导的氧化LDL，这已被广泛用作模型，反映了OxLDL在体内的特点。体内OxLDL的另一种模型称为最小修饰的LDL（MM-LDL），其在较温和的条件下制备。在本研究中，对各种修饰的LDL进行了表征。通过Berliner等人描述的方法制备MM-LDL，其中LDL在4 ℃下对含有FeS 〇 4的PBS透析4天。虽然MM-LDL含有的TBARS是铜诱导的OxLDL的四分之一，但两种修饰LDL中共轭二烯的量和对抗氧化磷脂酰胆碱（OxPC）抗体的反应性几乎相同。当从修饰的LDL中提取的总脂质中含有OxPC时，MM-LDL中含有OxPC的量是铜诱导的OxLDL的10倍。从这些结果可以清楚地看出，氧化修饰LDL中的氧化产物和修饰结构可以根据治疗而变化。本研究的下一步是将体内存在的oxLDL与这些修饰LDL模型进行比较。为了做到这一点，从血浆中分离出微量OxLDL的程序和测定氧化产物的灵敏方法必须改进。低至10 pmol的含环糊精的OxPC可以作为荧光衍生物被检测到，然而，使用ESI/MS程序实现了甚至更灵敏的分析。迄今为止，通过离子交换HPLC或密度梯度超离心从血浆中分离OxLDL并不成功。我们目前正在尝试一种免疫沉淀法。