Quantitative Analysis of Labile Metabolites in Biological Samples

生物样品中不稳定代谢物的定量分析

• 批准号: 10208099
• 负责人: G. A. Nagana Gowda
• 金额: $ 37.07万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10208099
• 关键词: Acetyl Coenzyme A; Address; Aging; Antioxidants; Area; Biochemical Reaction; Biological; Blood; Blood Cells; Cell Culture Techniques; Cell Death; Cell Line; Cell Survival; Cell physiology; Cells; Cellular Metabolic Process; Clinical Trials; Coenzyme A; Coenzymes; Cytoplasm; Development; Diabetes Mellitus; Disease; Ensure; Equilibrium; Freezing; Functional disorder; Glutathione Disulfide; Harvest; Health; Heart; Heart failure; In Situ; In Vitro; Intervention; Investigation; Ion Channel; Isotope Labeling; Kinetics; Lead; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Measures; Mediating; Metabolism; Methodological Studies; Methodology; Methods; Mitochondria; Mus; NADH; NADP; NMR Spectroscopy; Nature; Nuclear Magnetic Resonance; Obesity; Organic solvent product; Outcome; Outcome Study; Outcomes Research; Oxidation-Reduction; Oxides; Precipitation; Proteins; Protocols documentation; Regulation; Research; Research Personnel; Sampling; Science; Signal Transduction; Skeletal Myoblasts; Specimen; Time; Tissues; Translating; Translations; Variant; Whole Blood; base; cofactor; enzyme activity; experimental study; inhibitor/antagonist; interest; metabolomics; mitochondrial metabolism; novel; nutrition; nutritional supplementation; pyruvate carrier; time use; tool

PROJECT SUMMARY Coenzymes and antioxidants mediate hundreds of biochemical reactions and are fundamental to cellular and mitochondrial function. The redox coenzymes undergo reversible oxidation and reduction reactions, while the balance between oxidized and reduced forms drive important cellular functions including regulation of ion channels, cell signaling, cell survival and death. Cellular dysfunction associated with these coenzymes has been implicated in many diseases including cancer, heart failure, diabetes, obesity and aging. Given the importance of NAD metabolites and their declining levels in aging, numerous clinical trials of nutritional supplementation of the coenzymes' precursors are currently underway. Despite the immense interest and the need to determine cellular and subcellular levels of these metabolites, no reliable method exists currently for their simultaneous and comprehensive measurement. The major challenge is that these molecules are unstable and their levels are sensitive to sample harvesting, extraction and measurement conditions. As a result, errors involved with their measurement using conventional methods often outweigh biological variations and potentially lead to incorrect inferences. To overcome these challenges, and building on our preliminary studies of methodological developments using nuclear magnetic resonance (NMR) spectroscopy, in this proposal, we seek to develop methods to reliably measure the coenzymes and antioxidants in blood, cells, tissue as well as two subcellular components: mitochondria and cytoplasm. Further, we seek to develop methods to measure these coenzymes in live cells and mitochondria in real time. We will also translate the protocols and measurements to the widely used mass spectrometry platform for broader dissemination. The proposed project has three main aims: (1) Develop methods to reliably measure the coenzymes and antioxidants in blood, tissue, cells and two subcellular components: mitochondria and cytoplasm using NMR spectroscopy; (2) Develop methods to measure the coenzymes and antioxidants in live cells and mitochondria in real time using NMR spectroscopy; and (3) use NMR spectroscopy to guide the translation of the methods for analysis of the unstable (coenzymes and antioxidants) as well stable metabolites to mass spectrometry for wider applications in the metabolomics field. The outcome will provide robust methods to analyze important coenzymes and antioxidants in a broad range of biological sample types that can be used by many researchers. The new methods will also provide novel avenues for investigation of live cells and mitochondrial metabolism in real time. These developments will impact numerous areas of biomedical science.

项目摘要 辅酶和抗氧化剂介导数百种生物化学反应，是细胞和 线粒体功能氧化还原辅酶经历可逆的氧化和还原反应，而氧化还原辅酶经历可逆的氧化和还原反应。 氧化和还原形式之间的平衡驱动重要的细胞功能， 通道，细胞信号，细胞存活和死亡。与这些辅酶相关的细胞功能障碍已经被发现。 与许多疾病有关，包括癌症、心力衰竭、糖尿病、肥胖和衰老。十分重要 NAD代谢产物及其在衰老中水平的下降，许多营养补充剂的临床试验， 辅酶的前体目前正在研究中。尽管有巨大的兴趣和需要确定 这些代谢物的细胞和亚细胞水平，目前还没有可靠的方法存在的同时， 综合测量主要的挑战是这些分子不稳定， 对样品采集、提取和测量条件敏感。因此，与其相关的错误 使用常规方法的测量通常超过生物变化，并可能导致不正确的测量。 推论为了克服这些挑战，并在我们对方法论的初步研究的基础上， 发展使用核磁共振（NMR）光谱，在这个建议中，我们寻求发展 可靠地测量血液、细胞、组织以及两种亚细胞中的辅酶和抗氧化剂的方法 组成部分：线粒体和细胞质。此外，我们寻求开发测量这些辅酶的方法 在肝细胞和线粒体中的真实的时间。我们还将把协议和测量结果广泛地翻译给 使用质谱平台进行更广泛的传播。拟议项目有三个主要目标：（1） 开发可靠测量血液、组织、细胞和两种亚细胞中辅酶和抗氧化剂的方法 成分：线粒体和细胞质使用NMR光谱;（2）开发方法来测量 使用NMR光谱法在真实的时间内测定活细胞和线粒体中的辅酶和抗氧化剂;以及（3）使用 核磁共振光谱法，以指导翻译的方法，分析不稳定的（辅酶和 抗氧化剂）以及稳定的代谢物用于质谱分析，以在代谢组学领域中获得更广泛的应用。 结果将提供强大的方法来分析重要的辅酶和抗氧化剂在广泛的 许多研究人员可以使用的生物样本类型。新方法也将提供新的途径 用于真实的实时研究活细胞和线粒体代谢。这些发展将影响 许多生物医学领域。