This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. A two-compartment description (1) of brain metabolism embodied in current models of glutamate - glutamine cycling, is now well accepted, and is the basis for the metabolic models now used routinely by several groups to determine the rates of glutamate neurotransmitter release in animals and humans in vivo. Briefly, compartmentation in the brain is thought to involve astrocytes and neurons. Astrocytes take up the glutamate released from neurons, but have higher glutamine levels together with higher glutamine synthetase and pyruvate carboxylase activity. The """"""""glutamine-glutamate"""""""" cycle is completed by glutamine uptake by neuronal cells, which contain the majority of glutamate and have higher glutaminase activity. The high pyruvate carboxylase activity in glia is thought to maintain citric acid cycle intermediates and allow the flow of carbon from astrocytes to neurons. In contrast, the main fate of glucose in neurons is thought to be oxidation via pyruvate dehydrogenase. Despite general agreement on the role of metabolic compartmentation in the CNS, current metabolic models used for quantifying glutamate cycling make significant assumptions, some of whose correctness remain unresolved. 13C NMR offers a unique opportunity to test the detailed assumptions of different compartmentation models in vivo, and while there has been considerable progress, much of the information available from these experiments - particularly isotopomer labeling kinetics - remains unexploited at this time. Isotopomer analysis allows the investigator to determine not only the enrichment kinetics of a particular carbon position, but also that of its immediate neighbors in the same molecule. Four possible labeling patterns can arise for each C position, and it is often possible to distinguish all four of these so-called multiplets, and hence from one carbon four measurements can be made, not just one. Each measurement represents a distinct group of isotopomers, and hence four mathematically distinct variables that can improve the estimation of pathway fluxes by providing additional constraints to the model. That such measurements are possible has been demonstrated, both in animals and human subjects;it remains to provide new models to take advantage of this additional information. In summary, the challenge is to understand all the available information about distribution of 13C in the primate brain. Specifically, we need to be able to interpret site-specific enrichment information and multiplet data, and any combination of the two. The Research Resource at Southwestern has developed the mathematical tools to do just that. These tools need to be implemented at Vanderbilt to serve our scientific goals. The technical goal of this research project is to acquire proton-decoupled 13C NMR spectra of glutamate and other neurotransmitters in the human brain during infusion of [1,6-13C] glucose, and to exploit the additional information available from models that incorporate isotopomer information into the analysis. These analyses will be compared with the results obtained using traditional two-compartment models. A high priority is to acquire data with sufficient chemical shift resolution that 13C_13C spin-spin coupling is easily resolved in neurotransmitters such as glutamate and gamma-aminobutyric acid. The biological goal is to measure flux in the citric acid cycle and the rate of anaplerotic reactions, including neurotransmitter synthesis, in the brain. The Vanderbilt group will focus on pulse sequence development and data acquisition;the UTSWMC group will be responsible for analysis of individual spectra and data sets, and for fitting the data to the two-compartment model of brain metabolism already under development in the lab. At UT Southwestern, the general two-compartment model will be refined depending on the data obtained routinely at Vanderbilt. Initially, it is anticipated that spin-coupled multiplet data will be available from glutamate. Depending on the availability of other data such as enrichment in gamma aminobutyric acid, aspartate or glutamine, the model will be refined. The primary responsibility of the UT Southwestern group will be to perform detailed analysis of 13C spectra provided by Vanderbilt, to assess and validate the assumptions of the two compartment model. In particular, incorporation of isotopomer kinetic analyses will provide unique opportunities for cross validation of models of label flow within and between compartments. These data will initially be used to assess the performance of the model of Oz et al in rhesus monkey studies, and subsequently in humans. Throughout this process, careful attention will be given to the suitability of the model. Discrepancies between data and fitted time curves may require re-evaluation of the model. Steps will be taken to determine whether all model parameters are identifiable. The effect of error in the data will also be checked, and confidence limits on estimated parameters determined. The model can be adapted to create different versions that fit with the various hypotheses regarding cerebral compartmentation and test whether these can be distinguished experimentally.
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