Jerlich A, Fabjan J S, Tschabuschnig S, Smirnova A V, Horakova L, Hayn M, Auer H, Guttenberger H, Leis H J, Tatzber F, Waeg G, Schaur R J
Institute of Biochemistry, University of Graz, Austria.
Free Radic Biol Med. 1998 May;24(7-8):1139-48. doi: 10.1016/s0891-5849(97)00439-5.
The aim of this study was to further clarify which part of human low density lipoprotein (LDL) is attacked by the MPO/H2O2/Cl- -system and which reactive oxygen species is responsible for the attack. Therefore the influence of this system on the modification of the lipid and protein moiety of LDL was studied in vitro. Using the monochlorodimedone assay it was found that HOCl is produced in micromolar quantities in the absence of LDL and is rapidly consumed by LDL in a concentration dependent manner. The consumption of HOCl was reflected in the formation of HOCl-specific epitopes on apo B-100 as determined by an antibody raised against HOCl-modified LDL. The absorbency at 234 nm was applied to measure continuously the extent of modification of LDL. The general kinetic pattern of the absorbency measurement consisted of a lag phase where no LDL modification was observed, followed by a rapid increase of absorbency and a plateau phase. Finally the absorbency decreased due to LDL precipitation. Time dependent absorption spectra indicated that this kinetic pattern is mainly caused by light scattering due to particle aggregation rather than by a specific absorption at 234 nm due to conjugated diene formation. In agreement with this finding a low rate of thiobarbituric acid reactive substances (TBArS) formation was observed after a lag phase. The aggregation of LDL occurs most likely by modification of apo B-100, which was determined fluorimetrically in terms of LDL-tryptophan destruction in presence of the MPO/H2O2/Cl(-)-system. The kinetic course of tryptophan fluorescence generally consisted of a rapid decrease leveling off into a low plateau phase. Gas chromatographic determinations of linoleic acid in LDL in presence of the MPO system showed that this polyunsaturated fatty acid (PUFA) is easily attacked by HOCl. Consistent with this finding NMR spectra of HOCl modified LDL indicated a complete disappearance of bis-allylic methylene groups. Since lipid peroxidation products only partially account for this loss of PUFAs, other reactions of HOCl with unsaturated lipids--probably chlorohydrin formation--must be involved. Summarizing, although the rate of lipid peroxidation is low, both the lipid and the protein moiety of LDL are readily modified by the MPO system. It appears that the immediate consequence of apo B-100 modification is its aggregation. It is concluded that MPO, which has been detected in atherosclerotic lesions, is able to contribute to the modification of LDL into a form recognizable for uncontrolled uptake by macrophages.
本研究的目的是进一步阐明人类低密度脂蛋白（LDL）的哪一部分受到MPO/H2O2/Cl-系统的攻击，以及哪种活性氧物质对此攻击负责。因此，在体外研究了该系统对LDL脂质和蛋白质部分修饰的影响。使用一氯二甲基酮测定法发现，在没有LDL的情况下会产生微摩尔量的次氯酸（HOCl），并且LDL会以浓度依赖的方式迅速消耗它。通过针对HOCl修饰的LDL产生的抗体测定，HOCl的消耗反映在载脂蛋白B-100上HOCl特异性表位的形成上。使用234nm处的吸光度连续测量LDL的修饰程度。吸光度测量的一般动力学模式包括一个滞后阶段，在此阶段未观察到LDL修饰，随后吸光度迅速增加并进入平稳阶段。最后，由于LDL沉淀，吸光度下降。时间依赖性吸收光谱表明，这种动力学模式主要是由颗粒聚集引起的光散射所致，而不是由共轭二烯形成导致的234nm处的特定吸收所致。与此发现一致，在滞后阶段后观察到硫代巴比妥酸反应性物质（TBArS）的形成速率较低。LDL的聚集最有可能是由载脂蛋白B-100的修饰引起的，这是通过在MPO/H2O2/Cl(-)系统存在下对LDL-色氨酸破坏进行荧光测定确定的。色氨酸荧光的动力学过程通常包括快速下降，然后平稳进入低平稳阶段。在MPO系统存在下对LDL中亚油酸的气相色谱测定表明，这种多不饱和脂肪酸（PUFA）很容易受到HOCl的攻击。与此发现一致，HOCl修饰的LDL的核磁共振光谱表明双烯丙基亚甲基完全消失。由于脂质过氧化产物仅部分解释了PUFA的这种损失，因此HOCl与不饱和脂质的其他反应——可能是氯醇的形成——一定也参与其中。总之，尽管脂质过氧化速率较低，但LDL的脂质和蛋白质部分都很容易被MPO系统修饰。似乎载脂蛋白B-100修饰的直接后果是其聚集。得出的结论是，在动脉粥样硬化病变中检测到的MPO能够促使LDL被修饰成一种可被巨噬细胞无节制摄取识别的形式。
