IN VIVO DETECTION OF FREE RADICALS USING NMR

该项目将确定一种新的核磁共振方法的可行性，用于检测活组织中的自由基活性。该方法将采用一种新型的自由基自旋阱（DEPMPO），最近开发用于电子自旋共振（ESR）光谱。与其他硝基自旋陷阱不同，DEPMPO含有一个磷原子，与它的反应位点紧密结合，这使我们能够使用磷核磁共振波谱（31P-NMR）进行检测。在初步实验中，我们证明了DBPMPO与特定自由基的反应会导致磷光谱中明显的化学位移。此外，我们发现这些位移代表了depmpo自由基反应的“还原”产物，在生物溶液中非常稳定。31P-NMR检测还原的depmpo自由基加合物的能力使得测量感兴趣组织中自由基活性的积累成为可能。这大大提高了该方法在体内应用的潜在灵敏度。我们假设自旋陷阱31P-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。因此，这项技术可能会对医疗保健产生重大影响。具体目的是：1)确定DEPMPO与常见氧基自由基和碳基自由基体外反应的特征31P-NMR谱。实验包括生物体系中常见的自由基的生成，与DEPMPO形成加合物的反应分析，用ESR和31P-NMR分析加合物的形成和衰变动力学，以及用质谱法测定其组成。2)利用31P-NMR检测体外细胞系统中DEPMPO捕获自由基的能力。我们将测试31P-NMR检测由人类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产生氧化应激的分离肝细胞，导致细胞内自由基的产生。3)在局部炎症和缺血再灌注损伤模型中，利用31P-NMR检测体内depmpo捕获自由基的能力。在这些实验中，我们将测试31P-NMR检测腹膜局部炎症和离体心脏缺血再灌注的能力。虽然提出的实验围绕着一个单一的现有自由基陷阱，但我们设想在未来的研究中开发新的核磁共振敏感探针，用于检测其他类型的自由基，使用其他核磁共振敏感同位素的探针，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.