C-13-NMR spectroscopic evaluation of the citric acid cycle flux in conditions of high aspartate transaminase activity in glucose-perfused rat hearts

A new mathematical model, based on the observation of C-13-NMR spectra of t wo principal metabolites (glutamate and aspartate), was constructed to dete rmine the citric acid cycle flux in the case of high aspartate transaminase activity leading to the formation of large amounts of labeled aspartate an d glutamate. In this model, the labeling of glutamate and aspartate carbons by chemical and isotopic exchange with the citric acid cycle are considere d to be interdependent. With [U-C-13]Glc or [1,2-C-13]acetate as a substrat e, all glutamate and aspartate carbons can be labeled. The isotopic transfo rmations of 32 glutamate isotopomers into 16 aspartate isotopomers or vice versa were studied using matrix operations; the results were compiled in tw o matrices. We showed how the flux constants of the citric acid cycle and t he C-13-enrichment of acetyl-CoA can be deduced from C-13-NMR spectra of gl utamate and/or aspartate. The citric acid cycle flux in beating Wistar rat hearts, aerobically perfused with [U-C-13]glucose in the absence of insulin , was investigated by C-13-NMR spectroscopy. Surprisingly, aspartate instea d of glutamate was found to be the most abundantly-labeled metabolite, indi cating that aspartate transaminase (which catalyses the reversible reaction : (glutamate + oxaloacetate <----> 2-oxoglutarate + aspartate) is highly ac tive in the absence of insulin. The amount of aspartate was about two times larger than glutamate. The quantities of glutamate (G(0)) or aspartate (A( 0)) were approximately the same for all hearts and remained constant during perfusion: G(0) = (0.74 +/- 0.03) mu mol/g; A(0) = (1.49 +/- 0.05) mu mol/ g. The flux constants, i.e., the fraction of glutamate and aspartate in exc hange with the citric acid cycle, were about 1.45 min(-1) and 0.72 min(-1), respectively; the flux of this cycle is about (1.07 +/- 0.02) mu mol min(- 1) g(-1). Excellent agreement between the computed and experimental data wa s obtained, showing that: i) in the absence of insulin, only 41% of acetyl- CoA is formed from glucose while the rest is derived from endogenous substr ates; and ii) the exchange between aspartate and oxaloacetate or between gl utamate and 2-oxoglutarate is fast in comparison with the biological transf ormation of intermediate compounds by the citric acid cycle. (C) Societe fr ancaise de biochimie et biologie moleculaire / Elsevier, Paris.