Quantitative Analysis of Labile Metabolites in Biological Samples

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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.