This project involves the development of mathematical models to estimate the flux through different pathways using isotopomer analysis and 13C NMR data. Three programs are under development: tcaSIM, a simulation of the citric acid cycle and related pathways which generates 13C isotopomer data for use in the design of experiments; tcaCALC, a model which estimates relative pathway fluxes from NMR spectra obtained at metabolic and isotopic steady state; and tcaFLUX, a kinetic analysis of the citric acid cycle which allows measurement of absolute flux from systems at metabolic, but not isotopic, steady state. There has been much progress in the development of the kinetic analysis. It has been applied to multiplet data and fractional enrichment data collected from intact hearts and after freeze-clamping. A number of different substrate groups were examined. The model can be used to estimate citric acid cycle activity, the exchange between citric acid cycle intermediates and amino acids (which will include transportation between different cellular compartments), the contribution of labeled substrate to acetyl-CoA, and anaplerosis. The citric acid cycle rate determined by the model was used to estimate oxygen consumption; the values obtained were similar to those measured directly. It has been found that the use of multiplet data along with standard fractional carbon enrichments in this analysis is beneficial, reducing the correlation between parameters that otherwise exists. There has also been success with the application of data available from GCMS to isotopomer analysis. A Finnegan GCQ bench-top GCMS with tandem mass spectrometry capability is in use. This allows the investigator to acquire, in addition to standard full-scan mass spectra, mass spectra of parent ions which have been fragmented after inducing collisions with the carrier gas. The mass spectra of these fragments give additional information on the isotopomers present in the metabolite mixture. Using the methyl-8 derivative of glutamate isolated from hearts perfused under a variety of conditions, such data was analyzed in a similar fashion to NMR multiplet data to determine substrate utilization and anaplerosis. The results were very similar to those obtained using NMR data, indicating that this technique will be useful as a more-sensitive, complementary approach to NMR. (Core 3) REPORT PERIOD: (09/01/97-08/31/98)
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