Antioxidant Functions of Lipoic Acid

描述(由申请人提供)：硫辛酸已被推广为一种抗氧化剂，在各种条件下有效，其中包括糖尿病和动脉粥样硬化。这种损伤的早期表现是由于一氧化氮生成不足或过度破坏导致的血管内皮功能障碍。硫辛酸是否可以减少一氧化氮，从而减轻内皮功能障碍，这是一个具有重要临床意义的悬而未决的问题。我们已经证明，培养的人内皮细胞摄取硫辛酸并将其还原为二氢硫辛酸。后者以浓度依赖的方式保护细胞免受内部和外部氧化应激的影响，这与细胞产生一氧化氮的增加有关。在这项提案中，我们试图确认这些新的结果，阐明它们发生的机制，并确定它们是否可能与临床动脉粥样硬化有关。在第一个目标中，我们将评估硫辛酸在培养的内皮细胞中的抗氧化效果。氧化应激将由对细胞具有生物相关性的物质启动，包括H202和最低限度氧化的人类低密度脂蛋白(LDL)。硫辛酸的细胞保护作用将通过基于荧光的细胞内氧化应激分析、通过测量F2-异前列腺素的脂质过氧化以及通过检测细胞抗氧化剂来评估。在第二个目标中，我们将确定硫辛酸的这些抗氧化作用是否以及如何解释其促进内皮一氧化氮生成的能力。我们将集中于细胞内二氢硫辛酸是直接作用还是通过谷胱甘肽作用，它是否与抗坏血酸协同作用，以及它是否防止或逆转由于氧化型低密度脂蛋白造成的一氧化氮生成受损。显示硫辛酸增强内皮一氧化氮的生成和作用将构成其逆转一氧化氮依赖的内皮功能障碍能力的小型双盲、安慰剂对照临床试验的基础(目标3)。在服用硫辛酸或安慰剂8周之前和之后，将对因高胆固醇血症而增加氧化应激的受试者进行研究，以观察血浆和尿液中F2-异前列腺素的变化以及血流介导的臂动脉扩张。尽管硫辛酸被广泛使用，但这些研究对于建立该制剂临床使用的生物学基础和理论基础是必要的。

DESCRIPTION (provided by applicant): Lipoic acid has been promoted as an antioxidant effective in a variety of conditions in which there is oxidant damage to the vascular bed, including diabetes mellitus and atherosclerosis. An early manifestation of such damage is dysfunction of the vascular endothelium due to deficient generation or excessive destruction of nitric oxide. Whether lipoic acid can spare nitric oxide and thus lessen endothelial dysfunction is an unanswered question with important clinical implications. We have shown that cultured human endothelial cells take up lipoic acid and reduce it to dihydrolipoic acid. The latter protects the cells in a concentration dependent manner against both internal and external oxidant stresses, and this is associated with an increase in nitric oxide generation by the cells. In this proposal we seek to confirm these novel results, to clarify the mechanism by which they occur, and to determine whether they might be relevant to clinical atherosclerosis. In the first aim we will assess the antioxidant efficacy of lipoic acid in endothelial cells in culture. Oxidant stress will be initiated by agents with biological relevance for the cells, including H202 and minimally oxidized human low density lipoprotein (LDL). Cell protection by lipoic acid will be assessed using a fluorescence-based assay of intracellular oxidant stress, by measuring lipid peroxidation as F2-isoprostanes, and by assay of cellular antioxidants. In the second aim, we will determine whether and how these antioxidant effects of lipoic acid can explain its ability to enhance endothelial nitric oxide generation. We will focus on whether intracellular dihydrolipoic acid acts directly or through glutathione, whether it is synergistic with ascorbic acid, and whether it prevents or reverses impaired nitric oxide generation due to oxidized LDL. Showing that lipoic acid enhances endothelial nitric oxide generation and action will form the basis for a small double-blind, placebo-controlled clinical trial of its ability to reverse nitric oxide-dependent endothelial dysfunction (aim 3). Subjects with increased oxidant stress due to hypercholesterolemia will be studied before and after taking lipoic acid or placebo for 8 weeks with regard to changes in plasma and urinary F2-isoprostanes and flow-mediated brachial artery dilation. Despite the widespread use of lipoic acid, these studies are necessary to establish the biological basis and rationale for the clinical use of the agent.