TOOLS FOR COMPREHENSIVE ANALYSIS OF METABOLIC FLUXES IN VIVO

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Despite recent advances in molecular biology that have provided a fuller understanding of the transcriptional basis of disease, technology that allows the ultimate end product of transcription, metabolic flux, to be measured in vivo is very limited. The importance of measuring flux is highlighted by a number of examples in animal models where changes in enzyme expression do not match changes in flux through the enzyme. Thus, if the metabolic basis of disease is to be explicitly understood, techniques which measure in vivo fluxes must be further developed so that they can be easily utilized by basic and clinical scientists. NMR isotopomer analysis of small molecule metabolites is well suited to make these measurements because chemical shift and spin-spin coupling allow the isotopomer populations to be very well defined. Knowledge of isotopic distributions in metabolites is tantamount to knowing the flux through the biochemical pathways that generated the isotopomers. The continued focus of Core I is to address technology development for NMR based metabolic analysis with an emphasis on improved sensitivity of NMR isotopomer analysis of in vivo metabolism. The specific aims are: 1. Multiplexing metabolic flux. This aim seeks to expand the number of metabolic pathways measured in a single in vivo experiment by incorporating additional13C tracers of hepatic fluxes into the techniques developed in the previous funding cycle. This aim will improve the experimental efficiency of the NMR isotopomer method (compared to other methods of isotope detection) by making simultaneous measurements of multiple hepatic fluxes. 2. Mathematical analysis of tracer data from complex systems. Our aim is to more fully refine the mathematical models that describe hepatic fluxes with respect to parameter sensitivity, experimental design and extend an existing kinetic model from a simple onecompartment tissue model to a more realistic two-compartment model. This will enhance our capabilities to use 13C NMR multiplet data for analysis of liver and brain metabolism as we move toward more in vivo work on our new high field human scanners. 3. Improve NMR detection of 2H and 13C for metabolic studies. To increase the applicability of our metabolic flux measurements, it is essential that we continue to increase the sensitivity of both the 2H and 13C NMR experiments, the backbone of this methodology. We will extend 2H NMR and JHSQC experiments to 18.8T to take advantage of the increased sensitivity and dispersion of the higher magnetic field and develop the necessary methods to take maximum advantage of new micro-solenoidal probes that offer higher sensitivity for sample limited cases. Finally, we will test the suitability of new high temperature superconducting NMR probes for metabolic measurements.