Haramaki N, Han D, Handelman G J, Tritschler H J, Packer L
Department of Molecular and Cell Biology, University of California at Berkeley 94720-3200, USA.
Free Radic Biol Med. 1997;22(3):535-42. doi: 10.1016/s0891-5849(96)00400-5.
In cellular, tissue, and organismal systems, exogenously supplied alpha-lipoic acid (thioctic acid) has a variety of significant effects, including direct radical scavenging, redox modulation of cell metabolism, and potential to inhibit oxidatively-induced injury. Because reduction of lipoate to dihydrolipoate is a crucial step in many of these processes, we investigated mechanisms of its reduction. The mitochondrial NADH-dependent dihydrolipoamide dehydrogenase exhibits a marked preference for R(+)-lipoate, whereas NADPH-dependent glutathione reductase shows slightly greater activity toward the S(-)-lipoate stereoisomer. Rat liver mitochondria also reduced exogenous lipoic acid. The rate of reduction was stimulated by substrates which increased the NADH content of the mitochondria, and was inhibited by methoxyindole-2-carboxylic acid, a dihydrolipoamide dehydrogenase inhibitor. In rat liver cytosol, NADPH-dependent reduction was greater than NADH, and lipoate reduction was inhibited by glutathione disulfide. In rat heart, kidney, and brain whole cell-soluble fractions, NADH contributed more to reduction (70-90%) than NADPH, whereas with liver, NADH and NADPH were about equally active. An intact organ, the isolated perfused rat heart, reduced R-lipoate six to eight times more rapidly than S-lipoate, consistent with high mitochondrial dihydrolipoamide dehydrogenase activity and results with isolated cardiac mitochondria. On the other hand, erythrocytes, which lack mitochondria, somewhat more actively reduced S- than R-lipoate. These results demonstrate differing stereospecific reduction by intact cells and tissues. Thus, mechanisms of reduction of alpha-lipoate are highly tissue-specific and effects of exogenously supplied alpha-lipoate are determined by tissue glutathione reductase and dihydrolipoamide dehydrogenase activity.
在细胞、组织和机体系统中，外源性提供的α-硫辛酸（硫辛酸）具有多种显著作用，包括直接清除自由基、对细胞代谢进行氧化还原调节以及抑制氧化诱导损伤的潜力。由于硫辛酸还原为二氢硫辛酸是许多这些过程中的关键步骤，我们研究了其还原机制。线粒体依赖NADH的二氢硫辛酰胺脱氢酶对R(+)-硫辛酸表现出明显的偏好，而依赖NADPH的谷胱甘肽还原酶对S(-)-硫辛酸立体异构体的活性略高。大鼠肝脏线粒体也能还原外源性硫辛酸。还原速率受到增加线粒体NADH含量的底物的刺激，并受到二氢硫辛酰胺脱氢酶抑制剂甲氧基吲哚-2-羧酸的抑制。在大鼠肝脏胞质溶胶中，依赖NADPH的还原作用大于依赖NADH的还原作用，并且硫辛酸的还原受到谷胱甘肽二硫化物的抑制。在大鼠心脏、肾脏和脑的全细胞可溶性组分中，NADH对还原作用的贡献（70 - 90%）大于NADPH，而在肝脏中，NADH和NADPH的活性大致相当。一个完整的器官，即离体灌注的大鼠心脏，还原R-硫辛酸的速度比S-硫辛酸快6至8倍，这与线粒体中二氢硫辛酰胺脱氢酶的高活性以及离体心脏线粒体的结果一致。另一方面，缺乏线粒体的红细胞还原S-硫辛酸比R-硫辛酸更活跃一些。这些结果表明完整细胞和组织的立体特异性还原存在差异。因此，α-硫辛酸的还原机制具有高度的组织特异性，外源性提供的α-硫辛酸的作用取决于组织中的谷胱甘肽还原酶和二氢硫辛酰胺脱氢酶活性。
