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

人们普遍认为氧化型低密度脂蛋白(OxLDL)参与了动脉粥样硬化的形成。我们开发了一种使用单抗检测OxLDL的灵敏方法，从而证明了循环血浆中OxLDL的存在。最近，被广泛用作模型的铜诱导的低密度脂蛋白氧化是否反映了体内氧化低密度脂蛋白的特征的问题出现了。体内氧化低密度脂蛋白的另一种模型被称为最小修饰低密度脂蛋白(MM-LDL)，它是在较温和的条件下制备的。在本研究中，我们对各种修饰的低密度脂蛋白进行了表征。MM-低密度脂蛋白的制备方法是由Berliner等人描述的。其中低密度脂蛋白与含FeS_4的PBS在4℃条件下透析4天。与铜诱导的氧化低密度脂蛋白相比，MM-低密度脂蛋白含有1/4的TBARS，但两种修饰的低密度脂蛋白中共轭双烯的数量和对抗氧化型磷脂酰胆碱(OxPC)抗体的反应性几乎相同。当从修饰低密度脂蛋白中提取的总脂类中含有含醛OxPC时，MM-LDL中含醛OxPC的含量是铜诱导的OxLDL的10倍。从这些结果可以清楚地看出，氧化修饰的低密度脂蛋白中的氧化产物和修饰结构可以根据治疗的不同而不同。这项研究的下一步是将体内存在的oxLDL与这些修饰的低密度脂蛋白模型进行比较。为了做到这一点，必须改进从血浆中分离微量OxLDL的程序和测定氧化产物的灵敏方法。低至10pmoL的含醛OxPC可以作为荧光衍生物被检测到，然而，使用ESI/MS技术实现了更灵敏的分析。离子交换高效液相色谱分离和密度梯度超速离心法均未成功分离出血浆中的OxLDL。为此，我们目前正在尝试一种免疫沉淀程序。

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.