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Quantitative Analysis of Labile Metabolites in Biological Samples

生物样品中不稳定代谢物的定量分析

基本信息

批准号：
10621182
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
项目类别：
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/10621182
关键词：
Acetyl Coenzyme A Address Aging Antioxidants Area Biochemical Reaction Biological Blood 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 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 Precipitation Proteins Protocols documentation Reaction Regulation Research Research Personnel Sampling Science Signal Transduction Skeletal Myoblasts Starvation Time Tissues Translating Translations Variant Whole Blood cofactor enzyme activity experimental study inhibitor interest metabolomics mitochondrial metabolism novel nutrition nutritional supplementation oxidation pyruvate carrier time use tool

项目摘要

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.

项目总结辅酶和抗氧化剂调节数百种生化反应，是细胞和线粒体功能。氧化还原辅酶经历可逆氧化和还原反应，而氧化和还原形式之间的平衡驱动重要的细胞功能，包括调节离子通道、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与许多疾病有关，包括癌症、心力衰竭、糖尿病、肥胖和衰老。鉴于这一重要性 NAD代谢物及其在衰老过程中水平的下降，许多营养补充的临床试验辅酶的前体目前正在进行中。尽管有极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来测定它们的同时和综合测评。主要的挑战是这些分子是不稳定的，它们的水平对样品的采集、提取和测量条件敏感。因此，与他们的使用传统方法进行的测量经常超过生物变化，并可能导致不正确的结果推论。为了克服这些挑战，并在我们对方法学的初步研究的基础上发展利用核磁共振(核磁共振)波谱，在这个建议中，我们寻求开发方法可靠地测定血液、细胞、组织以及两种亚细胞中的辅酶和抗氧化剂成分：线粒体和细胞质。此外，我们还寻求开发测量这些辅酶的方法。在活细胞和线粒体中进行实时检测。我们还将把协议和测量转换为广泛的利用质谱学平台进行更广泛的传播。拟议的项目有三个主要目标：(1) 开发可靠的方法来测量血液、组织、细胞和两个亚细胞中的辅酶和抗氧化剂成分：线粒体和细胞质的核磁共振波谱；(2)建立测量方法使用核磁共振光谱实时检测活细胞和线粒体中的辅酶和抗氧化剂；以及(3)使用核磁共振光谱学指导不稳定(辅酶和辅酶)分析方法的翻译抗氧化剂)以及稳定的代谢物到质谱学，以便在代谢组学领域得到更广泛的应用。结果将提供强有力的方法来分析广泛范围内的重要辅酶和抗氧化剂可供多名研究人员使用的生物样本类型。新的方法也将提供新的途径用于实时研究活细胞和线粒体代谢。这些事态发展将对生物医学的众多领域。