Sobal G, Menzel J, Sinzinger H
Department of Nuclear Medicine, Institut of Immunology, Austria.
Prostaglandins Leukot Essent Fatty Acids. 2000 Oct;63(4):177-86. doi: 10.1054/plef.2000.0204.
It is well established that oxidative modification of low-density lipoprotein (LDL) plays a causal role in human atherogenesis and the risk of atherosclerosis is increased in patients with diabetes mellitus. To examine the influence of different agents which may influence LDL-glycation and oxidation, experiments including glycation with glucose, glucose 6-phosphate, metal chelators (EDTA) and antioxidants (BHT) were performed. The influence of time dependence on the glycation process and the alteration of the electrophoretic mobility of LDL under diverse glycation and/or oxidation conditions was also investigated. The formation of conjugated dienes and levels of lipid peroxides in these different LDL-modifications were estimated. The copper-induced oxidation of LDL in vitro was determined by measurement of thiobarbituric acid reactive substances (TBARS) and expressed as nmol MDA/mg of LDL protein. We found that glycated LDL is more prone to oxidation than native LDL. Using native LDL, the maximal oxidation effect was found to reach a value of 49.72 nmol MDA/mg protein after 8 h. The maximum oxidation of the 31 days, glycated LDL with glucose was 71.76 nmol MDA/mg protein amounting to 144.33% of the value found for native LDL. In the case of glucose 6-phosphate glycation, the maximum oxidation under the same conditions amounted to 173.77% of the value found for native LDL. To measure the extent of glycation, fluorescence of advanced glycation end products (AGEs) was determined (370 nm excitation and 440 nm emission). The most potent glycation agent was glucose 6-phosphate leading to the formation of very high amounts of AGEs. This process was promoted in the absence of EDTA, which prevents the oxidative cleavage of modified Amadori products (ketoamines) to AGEs. We therefore conclude that both processes, glycation and oxidation, result in the modification of LDL. The lower the glycation-rate (+/- EDTA) as measured by relative fluorescence units RFU (generation of AGEs), the lower the additional oxidation rate after glycation as measured by TBARS (generation of MDA equivalents). Glycation and/or oxidation change the electrophoretic mobility of LDL.
低密度脂蛋白（LDL）的氧化修饰在人类动脉粥样硬化形成过程中起因果作用，且糖尿病患者患动脉粥样硬化的风险会增加，这一点已得到充分证实。为研究可能影响LDL糖基化和氧化的不同因素的作用，我们进行了包括用葡萄糖、6-磷酸葡萄糖、金属螯合剂（EDTA）和抗氧化剂（BHT）进行糖基化的实验。我们还研究了时间依赖性对糖基化过程的影响以及在不同糖基化和/或氧化条件下LDL电泳迁移率的变化。我们估算了这些不同LDL修饰中共轭二烯的形成和脂质过氧化物的水平。体外铜诱导的LDL氧化通过硫代巴比妥酸反应性物质（TBARS）的测定来确定，并以每毫克LDL蛋白中nmol丙二醛（MDA）表示。我们发现糖基化的LDL比天然LDL更容易氧化。对于天然LDL，8小时后最大氧化效应达到49.72 nmol MDA/mg蛋白。用葡萄糖进行31天糖基化的LDL的最大氧化量为71.76 nmol MDA/mg蛋白，相当于天然LDL值的144.33%。在6-磷酸葡萄糖糖基化的情况下，相同条件下的最大氧化量相当于天然LDL值的173.77%。为测定糖基化程度，我们测定了晚期糖基化终产物（AGEs）的荧光（激发波长370 nm，发射波长440 nm）。最有效的糖基化剂是6-磷酸葡萄糖，它会导致大量AGEs的形成。在没有EDTA的情况下，这一过程会加速，因为EDTA可防止修饰的阿马多里产物（酮胺）氧化裂解为AGEs。因此，我们得出结论，糖基化和氧化这两个过程都会导致LDL的修饰。通过相对荧光单位RFU（AGEs的产生）测量的糖基化速率（±EDTA）越低，通过TBARS（MDA当量的产生）测量的糖基化后额外氧化速率就越低。糖基化和/或氧化会改变LDL的电泳迁移率。
