In Vivo Microdialysis Studies of Oxidative Stress

描述(申请人提供)：本提案的重点是利用微透析取样研究体内氧化应激。氧化应激产生活性氧物种，最终产生自由基，对DNA和其他生物分子造成损害。我们建议在心脏和大脑的缺血和再灌流过程中，利用体内特定组织部位的微透析取样来研究氧化应激的生理学。微透析将用于监测氧自由基(OFRs)，一种可用作保护剂的内源性抗氧化剂(谷胱甘肽)，以及氧化DNA损伤(8-羟基脱氧鸟苷)和脂质过氧化(丙二醛)的生物标志物。为了开展拟议的研究，有必要首先开发一套分析方法来确定微透析样品中的每一种目标分析物。微渗析取样的结果是样品只有几微升或更少。因此，毛细管电泳法(CE)、胶束电动毛细管色谱(MEKC)和毛细管电动毛细管电泳法(CEC)将成为未来的研究热点。修饰的核苷和抗氧化剂带电并具有电活性。将使用通过产生电活性产品的微透析探头引入的自由基捕捉剂来稳定OFRs。所有这些分析物都将使用电化学方法进行检测。丙二醛将衍生成荧光分析物，并用激光诱导荧光法进行测定。然后，将利用微透析取样和新开发的分析方法，在体内研究心脏和大脑缺血和再灌流期间氧自由基产生的作用。研究的目的是阐明各种已提出的氧自由基形成机制在缺血和再灌流过程中的作用。特别是，黄嘌呤氧化酶在大脑产生OFRs中所起的作用将被研究。我们将在心脏和大脑中研究氧自由基的形成与儿茶酚胺释放的关系。微透析法将被用来在体内局部给药黄嘌呤氧化酶和儿茶酚胺释放和摄取的抑制剂，以确定这些途径的作用。

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.