Lodge J K, Traber M G, Packer L
Department of Molecular and Cell Biology, University of California at Berkeley, 94720-3200, USA.
Free Radic Biol Med. 1998 Aug;25(3):287-97. doi: 10.1016/s0891-5849(98)00048-3.
Mono-thiols can act either as pro- or anti-oxidants during metal-catalyzed low density lipoprotein (LDL) peroxidation, however investigation of the role of vicinal thiols has been neglected. Therefore dihydrolipoic acid (DHLA), a vicinal dithiol, and lipoic acid, its oxidized form, were used to investigate Cu2+-mediated LDL peroxidation. We demonstrate here that DHLA inhibited Cu2+-dependent LDL peroxidation by chelating copper. DHLA (0-20 microM) increased lag-times of conjugated diene formation in LDL (100 microg/ml) oxidized with 5 microM Cu2+ in a concentration dependent manner, and this effect was saturated after 5 microM DHLA; enough to chelate all of the added Cu2+. In a similar fashion DHLA prevented LDL-mediated reduction of Cu2+ to Cu+. Lipoic acid had no effect in these systems. DHLA alone also reduced Cu2+, however this was inhibited when DHLA was in excess of the copper concentration. Hence there is complex formation between the two species. Copper:DHLA complex formation was further investigated and found to be dependent upon pH and the presence of oxygen. At low pH (<6), or in the absence of oxygen, the complex is stable, presumably due to vicinal thiol chelation. As the pH is increased, the carboxylate group also participates in copper chelation, this results in a less stable complex which is susceptible to oxidation, and copper is eventually released. Electron spin resonance studies demonstrate the formation of hydroxyl, but not superoxide, radicals during Cu2+-catalyzed DHLA oxidation. Thus in our LDL experiments at physiological pH, DHLA is able to either reductively inactivate Cu2+ when Cu2+ is in excess, or effectively chelate Cu2+ when DHLA is in excess. The Cu2+:DHLA complex eventually undergoes copper-catalyzed oxidation, copper is released and LDL peroxidation proceeds. DHLA, thus, has both pro- and antioxidant properties depending upon the ratio of Cu2+:DHLA and the pH. These results provide an additional mechanism of thiol-mediated formation of radicals and metal chelation.
在金属催化的低密度脂蛋白（LDL）过氧化过程中，单硫醇既可以作为促氧化剂，也可以作为抗氧化剂，然而，邻位硫醇作用的研究却被忽视了。因此，使用二氢硫辛酸（DHLA），一种邻位二硫醇，及其氧化形式硫辛酸，来研究Cu2+介导的LDL过氧化。我们在此证明，DHLA通过螯合铜来抑制Cu2+依赖性的LDL过氧化。DHLA（0 - 20 microM）以浓度依赖性方式增加了在5 microM Cu2+存在下氧化的LDL（100 microg/ml）中共轭二烯形成的延迟时间，并且在5 microM DHLA后这种效应达到饱和；这足以螯合所有添加的Cu2+。以类似的方式，DHLA阻止了LDL介导的Cu2+还原为Cu+。硫辛酸在这些系统中没有作用。单独的DHLA也能还原Cu2+，然而当DHLA超过铜浓度时这种还原作用会受到抑制。因此，这两种物质之间形成了复合物。对铜：DHLA复合物的形成进行了进一步研究，发现其依赖于pH值和氧气的存在。在低pH值（<6）或无氧条件下，该复合物是稳定的，推测是由于邻位硫醇螯合作用。随着pH值升高，羧基也参与铜螯合，这导致形成一种不太稳定的复合物，该复合物易被氧化，最终铜被释放。电子自旋共振研究表明，在Cu2+催化的DHLA氧化过程中形成了羟基自由基，但没有超氧自由基。因此，在我们的生理pH值LDL实验中，当Cu2+过量时，DHLA能够通过还原使Cu2+失活，或者当DHLA过量时有效地螯合Cu2+。Cu2+：DHLA复合物最终会经历铜催化的氧化，铜被释放，LDL过氧化继续进行。因此，DHLA根据Cu2+：DHLA的比例和pH值具有促氧化和抗氧化特性。这些结果提供了硫醇介导的自由基形成和金属螯合的另一种机制。
