Project Summary/Abstract Despite extensive knowledge of metabolic pathways, understanding the basic metabolic mechanisms of prominent diseases is limited by the ability to grasp the regulation of complex metabolic systems. Genetic gain/loss of function approaches have provided a wealth of information about how metabolism can be regulated, but have not been very successful at identifying the cause of complex disease. Ideally, top-down approaches would be used to examine the function of metabolism using quantitative approaches, then to guide gain/loss of function studies. The scientific community is moving quickly to stable isotopes because of high information yield, convenience, and the capacity to translate methods between human subjects, rodent models and cell preparations. To accomplish this goal, it is essential to extend earlier concepts using computational methods, and to integrate mass spectrometry (MS) with nuclear magnetic resonance (NMR). However, in order for the approaches to be embraced by the molecular physiologist/geneticist or clinical scientist interested in disease physiology, they must be flexible, reliable and easy-to-use methods relevant in a wide range of conditions. In this TR&D project we will perform cell, rodent and computational studies to develop translation methods to address these needs. First, we will integrate tracer flux approaches and metabolomics to identify target enzymes in the regulation of metabolism or its dysregulation during disease. Secondly, we will leverage the specificity of NMR isotopomer analysis and the sensitivity of mass isotopomer analysis to measure flux with high confidence on milligram scale samples. Finally, we will develop and distribute a free, open source software platform that simulates NMR and MS data based on flux/tracer input and calculates flux from experimental data in a single, easy to use graphical interface complete. Accomplishing these aims will provide the scientific community with new tools that will guide tactical studies in pharmacology, genetics and clinical science.