IN VIVO DETECTION OF FREE RADICALS USING NMR

该项目将确定一种新的核磁共振方法用于检测活组织中自由基活性的可行性。该方法将采用一种新的自由基自旋陷阱（DEPMPO），最近开发的电子自旋共振（ESR）光谱的使用。与其他硝酮自旋陷阱不同，DEPMPO含有一个磷原子，与其反应位点紧密结合，这使我们能够使用磷核磁共振光谱（31 P-NMR）进行检测。在初步的实验中，我们表明，DBPMPO与特定的自由基的反应导致可区分的化学位移的磷光谱。此外，我们已经发现，这些位移代表的DEPMPO自由基反应，这是非常稳定的生物溶液中的“还原”产品。31 P-NMR检测还原的DEPMPO-自由基加合物的能力使得可以测量自由基活性在感兴趣的组织中的积累。这大大提高了该方法在体内应用的潜在灵敏度。我们假设自旋阱31 P-NMR技术将使我们能够测量活组织中的自由基活性，具有足够的灵敏度来检测并可能成像局部炎症区域，如感染，缺血再灌注，关节炎，炎症性肺病和各种自身免疫性疾病可能发生的区域。因此，这项技术可能会对医疗保健产生重大影响。具体目标是：1）确定DEPMPO与常见的氧基和碳基自由基体外反应产生的特征31 P-NMR谱。这些实验涉及生物系统中常见的自由基的产生，分析它们与DEPMPO形成加合物的反应，用ESR和31 P-NMR分析加合物形成和衰变的动力学，以及用质谱法测定它们的组成。2)确定使用31 P-NMR检测体外细胞系统中DEPMPO捕获的自由基的能力。我们将测试31 P-NMR检测胡敏素中性粒细胞激活产生的细胞外自由基的能力。第二个系列将研究因对乙酰氨基酚过量而导致细胞内自由基产生而经历氧化应激的分离肝细胞。3)确定在局部炎症和缺血再灌注损伤模型中使用31 P-NMR在体内检测DEPMPO捕获的自由基的能力。在这些实验中，我们将测试31 P-NMR检测腹膜局部炎症和离体心脏缺血-再灌注的能力。虽然拟议的实验中心围绕一个单一的现有的自由基陷阱，我们设想的发展，在未来的研究中，新的核磁共振敏感的探针，检测其他类型的自由基，探针使用其他核磁共振敏感的同位素，并划分到特定的细胞器或器官。

This project will determine the feasibility of a new NMR methodology for detecting free radical activity in living tissue. The approach will employ the use of a novel free radical spin trap (DEPMPO), recently developed for electron spin resonance (ESR) spectroscopy. Unlike other nitrone spin traps, DEPMPO contains a phosphorus atom, bound closely to its reactive site, which has enabled us to use phosphorus-nuclear magnetic resonance spectroscopy (31P-NMR) for detection. In preliminary experiments we demonstrate that reactions of DBPMPO with specific free radicals result in distinguishable chemical shifts in the phosphorus spectrum. Furthermore, we have found that these shifts represent the "reduced" products of DEPMPO-free radical reactions, which are extremely stable in biological solution. The ability of 31P-NMR to detect the reduced DEPMPO-radical adducts makes it possible to measure the accumulation of free radical activity in the tissue of interest. This greatly enhances the potential sensitivity of the approach for in vivo applications. We hypothesize that spin-trap 31P-NMR technology will allow us to measure free radical activity in living tissue, with sufficient sensitivity to detect and possibly image localized areas of inflammation, as might occur with infection, ischemia-reperfusion, arthritis, inflammatory lung disease and various autoimmune diseases. Therefore, this technology could have a major impact on health care. The specific aims are: 1) To define the characteristic 31P-NMR spectra which occurs from in vitro reactions of DEPMPO with common oxygen- and carbon- based free radicals These experiments involve the generation of free radicals commonly seen in biological systems, analysis of their reactions to form adducts with DEPMPO, the kinetics of adduct formation and decay with both ESR and 31P-NMR, and the determination of their composition by mass spectrometry. 2) To determine the abilIty to detect DEPMPO trapped free radicals for in vitro cell systems using 31P-NMR. We will test the ability of 31P-NMR to detect free radicals generated extracellularly by activation of humin neutrophils. A second series will study isolated hepatocytes undergoing oxidant stress from acetaminophen overdose, resulting in intracellular free radical production. 3) To determine the ability to detect DEPMPO-trapped free radicals in vivo using 31P-NMR In models of localized Inflammation and Ischemia-reperfusion Injury. In these experiments we will test the ability of 31P-NMR to detect localized inflammation in the peritoneum and ischemia-reperfusion in the isolated heart. Although the proposed experiments center around a single existing free radical trap, we envision the development, in future studies, of new NMR-sensitive probes which detect other types of radicals, probes which use other NMR-sensitive isotopes and which compartmentalize to specific organelles or organs.