Contribution of exogenous substrates to acetyl coenzyme A: measurement by carbon-13 NMR under non-steady-state conditions

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Abstract
A method is presented for the rapid determination of substrate selection in a manner that is not restricted to conditions of metabolic and isotopic steady state. Competition between several substrates can be assessed directly and continuously in a single experiment, allowing the effect of interventions to be studied. It is shown that a single proton-decoupled 13C NMR spectrum of glutamate provides a direct measure of the contribution of exogenous 13C-labeled substrates to acetyl-CoA without measurement of oxygen consumption and that steady-state conditions need not apply. Two sets of experiments were performed: one in which a metabolic steady state but a non-steady-state 13C distribution was achieved and another in which both metabolism and labeling were not at steady state. In the first group, isolated rats hearts were supplied with [1,2-13C]lactate, and unlabeled glucose. 13C NMR spectra of extracts from hearts perfused under identical conditions for 5 or 30 min were compared. In spite of significant differences in the spectra, the measured contributions of acetate, lactate, and unlabeled sources to acetyl-CoA were the same. In the second set of experiments, the same group of labeled substrates was used in a regional ischemia model in isolated rabbit hearts to show regional differences in substrate utilization under both metabolic and isotopic non steady state. This enzyme probe of substrate selection was also demonstrated in intact hearts where excellent time resolution (3 min) of substrate selection was feasible. Since the basis of the method involves measurement of two resonance areas (C4 and C3) and the relative multiplet components of the carbon 4 resonance, simple J-modulated editing schemes allow substrate selection to be measured even when 13C-13C coupling cannot be resolved, as would be expected in vivo. The time resolution of these measurements may not be limited by technical contraints but by the rate of carbon flux in the citric acid cycle. Although this technique is demonstrated for the heart, it is applicable to all tissues.