13C NMR is a powerful non-invasive tool for monitoring intermediary metabolism in isolated cells and organs. Recently, rates of 13C enrichment of cellular glutamate (Glu) have been used to estimate TCA cycle flux (VTCA) in isolated perfused hearts and in brain in vivo. Techniques such as this could potentially be used to measure localized 02 consumption in human tissues, if one knew the extent to which sub- compartmental exchange reactions contribute to the observed kinetics. By fitting such 13C enrichment data to kinetic models of varying complexity, various investigators have found that alpha-KG : Glu exchange (VX) appears to be much faster than VTCA in mammalian brain, and comparable to VTCA in heart. The values of VX in these perfused heart studies varied considerably and there is some indication that assumptions about subcellular metabolite pool sizes may be the source of these differences. Recent experiments have shown that time-dependent changes in 13C isotopomers (as reflected by Glu multiplets) may be more sensitive to small changes in metabolite pool sizes and exchange fluxes than total 13C enrichment in Glu C4 and C3. It has also been shown that Glu is not always fully detected in intact hearts, suggesting that 13C NMR may prove useful in determining the concentration of metabolites in the cytosol. A comprehensive kinetic model of heart metabolism will be developed in this project using 13C isotopomer data obtained from 13C and 1H NMR and GC-MS to evaluate metabolite pool sizes, VTCA and VX. Three hypotheses will be tested: 1) that all 13C-enriched metabolites detected by 13C NMR are located in the cytosol; 2) that alterations in cytosolic or mitochondrial redox state will influence the rate of 13C appearance in Glu via changes in subcellular metabolite distributions and inter-compartmental metabolite exchange fluxes without changes in VTCA; and 3) that the pool size of exchanging fatty acids in post- ischemic hearts will cause 13C NMR to underestimate citric acid cycle flux during oxidation of long chain fatty acids, but not during oxidation of acetate or carbohydrates. HMQC-TOCSY will be used to measure groups of 13C isotopomers by indirect 1H detection and VTCA as determined by temporal 13C measurements will be compared with VTCA as determined using magnetization transfer techniques. The long term goal is to better understand the factors that modulate cytosolic and mitochondrial metabolite pool sizes and thereby substrate utilization in vivo so that interventions may be logically designed to alter substrate handling in post-ischemic hearts or underperfused tissue.
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