CARBON-13 NUCLEAR MAGNETIC RESONANCE STUDIES OF CARDIAC METABOLISM (MATHEMATICAL MODELING, NMR)

The last decade has witnessed the increasing use of Nuclear Magnetic Resonance (NMR) techniques for following the metabolic fate of compounds specifically labeled with ('13)C. The goals of the present study are: (1) to develop reliable quantitative procedures for measuring the ('13)C enrichment of specific carbon sites in compounds enriched by the metabolism of ('13)C-labeled substrates in rat heart, and (2) to use these quantitative measurements of fractional ('13)C enrichment within the context of a mathematical flux model describing the carbon flow through the TCA cycle and ancillary pathways, as a means for obtaining unknown flux parameters.; Rat hearts have been perfused in vitro with various combinations of glucose, acetate, pyruvate, and propionate to achieve steady state flux conditions, followed by perfusion with the same substrates labeled with ('13)C in specific carbon sites. The hearts were frozen at different times after addition of ('13)C-labeled substrates and neutralized perchloric acid extracts were used to obtain high resolution proton-decoupled ('13)C NMR spectra at 90.55 MHz. The fractional ('13)C enrichment (F.E.) of individual carbon sites in different metabolites was calculated from the area of the resolved resonances after correction for saturation and nuclear Overhauser effects. These F.E. measurements by ('13)C NMR were validated by the analysis of ('13)C-('1)H scalar coupling patterns observed in ('1)H NMR spectra of the extracted metabolites.; A mathematical flux model of the TCA cycle and ancillary transamination reactions was constructed to describe the formation of all the isotopic isomers of the TCA cycle and associated metabolites arising from metabolism of a variety of ('13)C-labeled substrates. The F.E. kinetics were used with the FACSIMILE program to solve for unknown flux parameters by means of non-linear least sqaures analysis of the more than 200 simultaneous differential equations used to describe the reactions. An alternative to using the F.E. kinetics was explored by calculation of the steady state labeling patterns in the TCA cycle using equations derived from the input fluxes (unknown) and input F.E.'s (measured) of the starting substrates while systematically varying the input fluxes using the oxygen consumption conservation equation.; The results obtained from perfusion of hearts with glucose plus either 2-('13)C acetate or 3-('13)C pyruvate are similar to those obtained by previous investigators using ('14)C-labeled substrates. (Abstract shortened with permission of author.)...