This project involves the development of mathematical models for the measurement of relative and absolute flux rates through the citric acid cycle and related pathways, using 13C NMR data and isotopomer analysis. A kinetic model which allows the measurement of absolute citric acid cycle flux, based on standard chemical kinetics, is under investigation. This model is similar to that of Chance et al (J. Biol. Chem. 258:13785, 1983), which is based on measurement of fractional enrichments in glutamate. This work extends Chance's analysis to take advantage of 13C NMR multiplet information. Oxygen consumption estimated by the model was compared with that measured experimentally. In one series of hearts, perfused with unlabeled glucose and [2-13C]acetate in identical fashion to the earlier work of Chance et al, 13C NMR data was collected after freeze-clamping hearts at different time intervals. Twenty-four hearts (three at each time point) were used to obtain the time-course of the change in glutamate multiplets. This data was fitted by weighted non-linear least square analysis to the kinetic model. The citric acid cycle flux was estimated as 11.9 1 0.6 mmoles/min/g dry weight. This predicts an oxygen consumption rate of 24.3, which closely matched that actually measured (24.9 1 3.5). In a second series of experiments, time-resolved 13C NMR spectra were collected from hearts perfused inside a magnet, using unenriched glucose and either 13C-enriched acetate or pyruvate as substrates. Oxygen consumption measurements were made at the same time. From the NMR spectra, the change in glutamate multiplets with time were obtained. Estimates of the citric acid cycle flux (mmoles/min/g dry weight) obtained from the model were 11.6 1 1.6 with enriched acetate (n=5) and 6.9 1 1.1 with enriched pyruvate (n=6). The oxygen consumption (23.8 1 3.1 and 17.6 1 2.8) estimated from these flux rates matched that measured directly (24.7 1 1.8 and 17.0 1 4.0, acetate- and pyruvate-enriched substrates, respectively).