Measurements of energy regulation and mitochondrial function are critically important for understanding cell metabolism in normal and diseased states. In vivo magnetic resonance spectroscopy (MRS) of hyperpolarized 13C-labeled substrates can provide unique non-invasive assessments of these key metabolic processes, with high spatial and temporal resolution. Pyruvate (Pyr) is an endogenous substance playing a central role in catabolic and anabolic metabolism. In the cytosol, Pyr can be reduced to lactate (Lac) as the end product of glycolysis (GLY) or amidated to alanine (Ala). In mitochondria, it is converted to form acetyl CoA and CO2 as the first step towards oxidative phosphorylation (OXPHOS) or directly enter the tricarboxylic acid cycle (TCA) via oxaloacetate. The flux of these metabolites provides critical information related to key metabolic processes in normal and disease states, and goal of this research proposal is to develop advanced new metabolic imaging tools to enhance the in vivo assessment of GLY, OXPHOS, and mitochondrial function. Applications include diagnosis and treatment assessment for cancers, cardiac pathologies, metabolic diseases, and neurodegenerative and neuropathological disorders. The key objectives of the proposed research are 1) improved sensitivity using indirect 1H-13C detection methods, and 2) the development of quantitative tools for data interpretation using asymmetries in the Pyr spectral peaks.