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，对多种血管床氧化损伤有效，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引起的血管内皮功能障碍。硫辛酸是否可以免除一氧化氮，从而减轻内皮功能障碍是一个没有答案的问题，具有重要的临床意义。我们已经证明，培养的人内皮细胞吸收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，这与细胞产生一氧化氮的增加有关。在这项提议中，我们试图证实这些新的结果，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。在第一个目的中，我们将评估硫辛酸在内皮细胞培养中的抗氧化作用。氧化应激将由与细胞具有生物学相关性的物质引发，包括H202和最低氧化的人低密度脂蛋白（LDL）。硫辛酸对细胞的保护作用将通过基于荧光的细胞内氧化应激测定、脂质过氧化测定（f2 -异前列腺素）和细胞抗氧化剂测定来评估。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增强内皮细胞一氧化氮生成的能力。我们将重点关注细胞内二氢硫辛酸是否直接或通过谷胱甘肽起作用，是否与抗坏血酸协同作用，以及是否预防或逆转氧化LDL导致的受损一氧化氮生成。表明硫辛酸增强内皮细胞一氧化氮的生成和作用，将成为一项小型双盲、安慰剂对照临床试验的基础，以证明硫辛酸能够逆转一氧化氮依赖性内皮功能障碍（目的3）。由于高胆固醇血症而导致氧化应激增加的受试者将在服用硫辛酸或安慰剂8周前后研究血浆和尿液f2 -异前列腺素和血流介导的肱动脉扩张的变化。尽管硫辛酸被广泛使用，但这些研究对于建立该药物临床使用的生物学基础和基本原理是必要的。

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.