Dynamic nuclear magnetic resonance (NMR) spectroscopy of presteady state, carbon-13 (13C) entry into the NMR detectable glutamate pool will be developed and applied to metabolic evaluations of the functional state of whole hearts. Long term objectives are: l) to characterize presteady state changes in I3C NMR spectra of intact hearts as a non-destructive probe of carbon flux through pathways of intermediary metabolism, and 2) to take advantage of the unique information provided by I3C NMR to elucidate the mechanisms of metabolic support of cardiac performance. Achievement of these goals requires: l) date interpretation of presteady state changes in 13C spectra, and 2) identification of metabolic correlates to physiological function. At present, 13C NMR is not sufficiently developed to unambiguously correlate the rate of 13C appearance in the NMR detectable glutamate pool with mitochondrial activity. Preliminary work suggests that 13C labeling and recycling of glutamate may reflect different rate limiting components of the tricarboxylic acid (TCA) cycle under different physiological conditions. Shifts in both metabolic flux and tissue metabolite pools occur in response to changes in physiological function, but these metabolic events are not well characterized in the intact myocardium. Recent findings that dynamic changes in 13C spectra from intact hearts are closely related to mechanical work and respiratory activity indicate that 13C NMR is sensitive to such shifts in the metabolic support of cardiac performance. Thus, we propose to combine I3C NMR of isolated rabbit hearts and in vitro biochemical analysis with separate radiotracer analysis of metabolite oxidation and efflux from isolated mitochondria. This proposal is specifically aimed at identifying how the balance between alpha- ketoglutarate oxidation by the TCA cycle and the efflux of alpha- ketoglutarate for conversion to cytosolic glutamate determines the kinetics of 13C entry into glutamate. With systematically defined, kinetic parameters for glutamate labeling, we will apply 13C NMR to measure metabolic rates at different workloads in intact hearts. A kinetic model will be applied for data analysis and will he further developed as an accurate model of 13C turnover within the TCA cycle. Proposed experiments will generate information, on the response of carbon flux to work load, not currently available with other techniques or protocols. Ultimate applications will include use of 13C NMR spectroscopy to evaluate biochemical activity in hearts without need of tissue biopsy sampling.
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