In Vivo Microdialysis Studies of Oxidative Stress

氧化应激的体内微透析研究

• 批准号: 6779087
• 负责人: CRAIG E LUNTE
• 金额: $ 21.07万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6779087
• 关键词: DNA damage; analytical chemistry; antioxidants; biomarker; capillary electrophoresis; catecholamines; chromatography; cytoprotection; electrochemistry; fluorescent dye /probe; free radical oxygen; glutathione; ischemia; laboratory rat; lipid peroxides; method development; microdialysis; neurotransmitter transport; nucleoside analog; oxidative stress; reperfusion; xanthine oxidase

DESCRIPTION (provided by applicant): The focus of this proposal is the study of oxidative stress in vivo using microdialysis sampling. Oxidative stress produces reactive oxygen species and ultimately free radicals that cause damage to DNA and other biomolecules. We propose to study the physiology of oxidative stress using in vivo microdialysis sampling at specific tissue sites during ischemia and reperfusion in the heart and brain. Microdialysis will be used to monitor oxygen free radicals (OFRs), an endogenous antioxidant that may serve as a protective agent (glutathione), and biomarkers for oxidative DNA damage (8-hydroxydeoxyguanosine) and lipid peroxidation (malondialdehye). In order to carry out the proposed studies, it will be necessary to first develop a suite of analytical methods to determine each of the target analytes in microdialysis samples. Microdialysis sampling results in samples of a few microliters or less. Therefore, methods based on capillary electrophoresis (CE), micellar electrokinetic chromatography (MEKC), and capillary electrokinetic chromatography (CEC) will be developed. The modified nucleosides and antioxidants are charged and electroactive. OFRs will be stabilized using radical trapping agents introduced through the microdialysis probe that produce electroactive products. All of these analytes will be detected using electrochemical methods. Malondialdehyde will be derivatized to a fluorescent analyte and determined by laser induced fluorescence. The role of OFR production will then be studied in vivo during ischemia and reperfusion in the heart and brain using microdialysis sampling and the newly developed methods of analysis. Studies will be aimed at elucidating the role of various proposed mechanisms of OFR formation during ischemia and reperfusion. In particular, the role xanthine oxidase plays in the production of OFRs in the brain will be investigated. OFR formation as a function of catecholamine release will be studied in both the heart and brain. Microdialysis will be used to locally administer inhibitors of xanthine oxidase and of catecholamine release and uptake in vivo to determine the role of these pathways.

描述（由申请人提供）：该建议的重点是使用微透析采样的体内氧化应激研究。氧化应激会产生活性氧，并最终导致DNA和其他生物分子损害的自由基。我们建议使用在缺血期间在特定组织部位进行体内微透析采样的氧化应激的生理，并在心脏和大脑中再灌注。微透析将用于监测可能用作保护剂（谷胱甘肽）的内源性抗氧化剂（OFRS），以及用于氧化DNA损伤的生物标志物（8-羟基脱氧氧）和脂质过多（脂肪氧）（丙二氧化）。为了进行拟议的研究，有必要首先开发一套分析方法，以确定微透析样品中的每个目标分析物。微透析采样会导致几种或更少的样品。因此，将开发基于毛细管电泳（CE），胶束电动色谱法（MEKC）和毛细管电动色谱法（CEC）的方法。修饰的核苷和抗氧化剂是充电和电活性的。通过通过产生电活性产物的微透析探针引入的自由基捕获剂将稳定OFR。所有这些分析物将使用电化学方法检测到。丙二醛将被衍生于荧光分析物，并通过激光诱导的荧光确定。然后，将使用微透析采样和新开发的分析方法在缺血期间在体内研究OFR产生的作用。研究将旨在阐明缺血和再灌注过程中OFR形成各种机制的作用。特别是，将研究黄嘌呤氧化酶在大脑中OFRS产生中的作用。将在心脏和大脑中研究OFR形成随儿茶酚胺释放的函数。微透析将用于局部施用黄嘌呤氧化酶的抑制剂以及体内的儿茶酚胺释放和摄取的抑制剂，以确定这些途径的作用。