NMR STUDIES OF CELLULAR METABOLISM

细胞代谢的核磁共振研究

• 批准号: 6106703
• 负责人: Robert E London
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6106703
• 关键词: acetone; cellular respiration; cytochrome P450; enzyme activity; fluorine; glutathione; glycerol kinase; halogenation; intracellular; laboratory rat; nonsteroidal antiinflammatory agent; nuclear magnetic resonance spectroscopy; phenols; pyruvate kinase; quinones

Two lines of investigation of the metabolism of fluorinated compounds and the toxicological behavior of phenolic compounds for which adducts are formed as a result of the formation of quinone methide intermediates have recently converged in a study of the toxicity of 4-trifluoromethyl phenol (4-TFMP). This compound was initially used as a control in studies of cresol metabolism, since it is generally assumed that trifluoromethyl groups are metabolically inert. However, 4-TFMP was found to exhibit cytotoxicity similar to cresol in studies of percision-cut rat liver slices. Analysis of the glutathione adduct indicates formation of a difluoro-quinone methide intermediate as a result of spontaneous (non-enzymatic) defluorination. This behavior is somewhat analogous to proposals for the reaction of trifluridine with thymidylate synthetase. The development of an artificial liver represents a long standing goal of toxicology as well as medicine. Attempts to utilize various bioreactor models have had only moderate success, with systems being relatively short lived and showing poor expression of drug metabolizing enzymes such as cytochrome P450. However, recent advances in the development of stem cell technology have brought this goal considerably closer to reality. We have recently been working with Prof. Lola Reid and Dr. Jeffrey Macdonald at the University of North Carolina to utilize NMR as a non-invasive tool for assessing various metabolic and flow parameters in several hepatic bioreactor model systems. Dr. Reid?s group has recently developed an antibody screening method that will be used to negatively sort human hepatic progenitors by fluorescent activated cell sorting, allowing enrichment of hepatic progenitor populations. We also recently have been able to demonstrate the feasibility of NMR monitoring of fluorinated xenobiotic metabolism, as well as a number of other metabolic parameters in a commercially available bioreactor. Preliminary NMR studies utilizing a miniature bioreactor that mimics a human liver lobule have also been performed. A short project involving the metabolism of propionate by E. coli was recently completed. We were able to observe NMR resonances arising from peptide components of the cell wall. This approach may prove useful for evaluating cell wall turnover and for monitoring the effects of antibiotics which interfere with this process.

代谢的两条研究路线 含氟化合物与酚类化合物的毒理学行为 作为形成加合物的结果的化合物 醌甲基化物中间体的形成最近 在一项关于4-三氟甲基苯酚毒性的研究中， （4-TFMP）。这种化合物最初被用作研究中的对照 甲酚代谢，因为通常认为， 三氟甲基是代谢惰性的。然而，4-TFMP 的研究中发现， 切取的大鼠肝脏切片。谷胱甘肽加合物的分析 表明形成了二氟醌甲基化物中间体作为 自发（非酶促）脱氨的结果。这种行为 有点类似于三氟尿苷反应的建议， 胸苷酸合成酶 人工肝的发展 代表了毒理学和医学的长期目标。 利用各种生物反应器模型的尝试仅 中等成功，系统寿命相对较短， 显示药物代谢酶的表达差， 细胞色素P450然而，最近在开发 干细胞技术使这一目标更加接近 现实我们最近一直在与洛拉里德教授和 博士北卡罗来纳州大学的杰弗里·麦克唐纳利用 核磁共振作为一种非侵入性的工具，用于评估各种代谢和 几种肝脏生物反应器模型系统中的流动参数。博士 里德s小组最近开发了一种抗体筛选方法 它将被用来对人类肝祖细胞进行负分选， 荧光激活细胞分选，允许富集肝脏 祖先群体我们最近还能够 证明核磁共振监测氟化的可行性 异生物质代谢，以及许多其他代谢 在可商购获得的生物反应器中的参数。初步 利用模拟人类的微型生物反应器进行NMR研究 肝小叶也已进行。 一个简短的项目， E.大肠杆菌最近完成。 我们能够观察到肽产生的NMR共振， 细胞壁的组成部分。这种方法可以证明是有用的， 评估细胞壁周转和监测的影响， 抗生素会干扰这一过程。