Tertov V V, Kaplun V V, Dvoryantsev S N, Orekhov A N
Institute of Experimental Cardiology, Russian Academy of Science, Moscow.
Biochem Biophys Res Commun. 1995 Sep 14;214(2):608-13. doi: 10.1006/bbrc.1995.2329.
It has been generally accepted that oxidized low density lipoprotein (LDL) plays an important role in atherogenesis. However, oxidized LDL was not detected in patients' blood and the extent of LDL oxidation in vivo is unknown. We have suggested that LDL oxidation may lead to a formation of covalent links between lipids and apolipoprotein B. LDL were oxidized by copper ions, 2,2'-azobis-(2-aminopropane hydrochloride), sodium hypochlorite or by incubation with macrophages. Oxidized LDL were delipidated by repeated extraction with organic solvents. After mild alkaline hydrolysis protein-bound sterols were identified colorimetrically and by high-performance liquid chromatography. Protein-bound phospholipid residues were detected by nuclear magnetic resonance and colorimetric determination of phosphate. Using radiolabeled lipids it was also shown that free and esterified cholesterol, phospholipids, as well as triglyceride and free fatty acid residues can form covalent bonds with apolipoprotein B. The ability of lipids to bind to apolipoprotein B correlates with the degree of unsaturation of their fatty acids and depends on the nature of polar head of phospholipids. When LDL were oxidized with copper ions, the content of protein-bound lipids increased gradually up to 24 h of incubation, while the levels of conjugated dienes, hydroperoxides and thiobarbituric acid-reactive substances changed in varying manners. It has been demonstrated that the content of protein-bound sterols in multiple-modified desialylated LDL of patients with coronary atherosclerosis is higher than that in native LDL. Our results suggest that the level of protein-bound lipids may be a marker of LDL oxidation and can be used to evaluate the association of lipoprotein oxidation and atherogenesis.
氧化型低密度脂蛋白（LDL）在动脉粥样硬化形成过程中起重要作用，这一点已被广泛接受。然而，在患者血液中未检测到氧化型LDL，且体内LDL氧化程度尚不清楚。我们曾提出，LDL氧化可能导致脂质与载脂蛋白B之间形成共价连接。LDL通过铜离子、2,2'-偶氮二（2-氨基丙烷盐酸盐）、次氯酸钠氧化，或与巨噬细胞共同孵育进行氧化。氧化型LDL通过用有机溶剂反复萃取进行脱脂。轻度碱水解后，采用比色法和高效液相色谱法鉴定与蛋白质结合的甾醇。通过核磁共振和磷酸盐比色测定法检测与蛋白质结合的磷脂残基。使用放射性标记的脂质还表明，游离和酯化胆固醇、磷脂以及甘油三酯和游离脂肪酸残基可与载脂蛋白B形成共价键。脂质与载脂蛋白B结合的能力与其脂肪酸的不饱和度相关，并取决于磷脂极性头部的性质。当LDL用铜离子氧化时，与蛋白质结合的脂质含量在孵育24小时内逐渐增加，而共轭二烯、氢过氧化物和硫代巴比妥酸反应性物质的水平则以不同方式变化。已证明，冠状动脉粥样硬化患者的多次修饰去唾液酸LDL中与蛋白质结合的甾醇含量高于天然LDL。我们的结果表明，与蛋白质结合的脂质水平可能是LDL氧化的标志物，可用于评估脂蛋白氧化与动脉粥样硬化形成之间的关联。
