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On‐line monitoring of the Krebs cycle in isolated heart mitochondria by 13C NMR spectroscopy

When isolated rat heart mitochondria were incubated with [3‐13C]pyruvate in the presence of oxygen and aspartate or malate, the spin label 13C was incorporated into the components of the Krebs cycle, which we monitored in situ by 13C NMR spectroscopy. In the spectra 21 isotopomers of citrate, 2‐ketoglutarate, glutamate, succinate, fumarate, malate and aspartate were conclusively found and the presence of ten more isotopomers could be inferred from metabolic reasons. Chemical shifts, δ, carbon–carbon coupling constants, J, and spin–lattice relaxation rates, T1, of these compounds were measured in suspensions of isolated mitochondria during respiration. It is demonstrated that the 13C signal of Tris buffer can be used to follow the respirational change in pH. It is shown by comparison of the spectra of a complete suspension and its fluid and solid components, that the observed signals are entirely due to the supernatant solution, and not to the mitochondrial matrix. By supplying different cosubstrates it was possible to emphasize various parts of the Krebs cycle. Mitochondria incubated with aspartate released only labelled glutamate and aspartate, whereas the cosubstrate malate allowed the observation of all the metabolites mentioned above. The appearance of multiplet signals indicated recycling of metabolites and can be understood in terms of known metabolic pathways and trans‐membrane transport. The spectroscopic and biochemical information which is necessary in order to evaluate series of carbon spectra recorded during stimulus experiments performed with suspensions of isolated mitochondria is presented.