Abstract
The fractional 13C enrichments in specific carbon sites of key metabolites in perchloric acid extracts of perfused rat hearts were measured as a function of time after addition of 13C labeled substrates. Accurate quantitation of 13C fractional enrichments in glutamate, aspartate, and alanine, which are in exchange with their respective α-ketoacids, is made possible by the relative large pool size of these metabolites. A detailed mathematical flux model of the citric acid cycle and ancillary reactions has been constructed with the FACSIMILE program and used to solve unknown flux parameters by optimization techniques using nonlinear least squares analysis of the simultaneous differential equations required to describe the reactions. Parameters calculated from the quantitative analysis of the resonance line splitting caused by 13C-labeling of one or more adjacent carbon atoms in the same molecule were found to be in excellent agreement with the corresponding parameters derived from the mathematical model. The quality of this agreement serves as an important cross-check for the appropriateness of the model’s topology as well as the general validity of the calculated flux parameters. The present results adumbrate the practicality of 13C NMR when used in conjunction with mathematical modeling for the assessment of metabolic networks and the measurements of metabolic flux parameters in living systems.
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References
Andres A, Satrustegui J, Machado A (1980) Development of NADPH-producing pathways in rat heart. Biochem J 186: 799–803
Bailey IA, Gadian DG, Mathews PM, Radda GK, Seeley PJ (1981) Studies of metabolism in the isolated perfused rat heart using 13C NMR. FEBS Lett 123: 315–318
Bergmeyer UH (1974) Methods of enzymatic analysis. Academic Press New York
Chance EM, Seeholzer SH, Kobayashi K, Williamson JR (1983) Mathematical analysis of isotope labeling in the citric acid cycle with applications to 13C NMR studies in perfused rat hearts. J Biol Chem 258: 13785–13794
Chance EM, Curtis AR, Jones IP, Kirby CR (1977) FACSIMILE: A computer program for flow and chemistry simulation and general initial value problems. Rep No R8775. United Kingdom Atomic Energy Authority HM Stationery Office London
Chua B, Kao R, Rannels DE, Morgan HE (1978) Hormonal and metabolic control of proteolysis. Biochem Soc Symp 48: 1–15
Clore GM, Chance EM (1978) The mechanism of reaction of fully reduced membrane-bound cytochrome oxidase with oxygen at 176K. Biochem J 173: 799–810
Davis EJ, Lin RC, Chao D (1972) Sources and disposition of aerobically generated intermediates in heart muscle. In: Mehlman MA, Hansen R (eds) Energy Metabolism and Regulation of Metabolic Processes in Mitochondria. New York, Academic Press pp 211–238
Davis EJ, Quastel JH (1964) The effects of short chain fatty acids and starvation on the metabolism of glucose and lactate by the perfused guinea pig heart. Canad. J Biochem 42: 1605–1621
Frenkel R (1972) Bovine heart malic enzyme. J Biol Chem 247: 5569–5572
Harris RK, Newman RH (1976) Choice of pulse spacings for accurate T1 and NOE measurements in NMR spectroscopy. J Magn Res 24: 449–456
Lin RC, Davis EJ (1974) Malic enzymes of rabbit heart mitochondria. J Biol Chem 249: 3867–3875
London RE, Kollman VH, Matwiyoff NA (1975a) The quantitative analysis of carbon-carbon coupling in the 13C nuclear magnetic resonance spectra of molecules biosynthesized from 13C enriched precusors. J Am Chem Soc 97: 3565–3573
London RE, Matwiyoff NA, Kollman VH (1975b) Differential nuclear overhauser enhancement for 13C siglet and 13C-13C multiplet resonances of a carboxyl carbon. J Magn Res 18: 555–557
Nagel WO, Dauchy RT, Sauer LA (1980) Mitochondrial malic enzymes. J Biol Chem 255: 3849–3854
Neurohr KJ, Barrett EJ, Shulman RG (1983) in vivo carbon-13 nuclear magnetic resonance studies of heart metabolism. Proc Natl Acad Sci USA 80: 1603–1607
Nuutinen EM, Hiltunen JK, Hassinen IE (1981) The glutamate dehydrogenase system and the redox state of mitochondrial free nicotinamide adenine dinucleotide in myocardium. FEBS Lett 128:356–360
Pearce FJ, Deleeuw G, Forster J, Williamson JR, Tutwiler GF (1979) Inhibition of fatty acid oxidation in normal and hypoxic perfused rat hearts by 2-Tetradecylglycidic acid. J Mol Cell Cardiol 11: 893–915
Peuhkurinen KJ (1982)Accumulation and disposal of tricarboxylic acid cycle intermediates during propionate oxidation in the isolated perfused rat heart. Biochem. Biophys. Acta 721: 124–134
Randle PJ, Tubbs PK (1979) Carbohydrate and fatty acid metabolism. In: Berne, RM, Spherelakis N, Geiger SR (eds) Handbook of Physiology, The Cardiovascular System. Bethesda: pp 804–844
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© 1985 Martinus Nijhoff Publishers, Dordrecht
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Williamson, J.R., Seeholzer, S.H., Chance, E.M. (1985). Flux analysis of 13C NMR metabolite enrichments in perfused rat hearts using FACSIMILE. In: Sideman, S., Beyar, R. (eds) Simulation and Imaging of the Cardiac System. Developments in Cardiovascular Medicine, vol 43. Springer, Dordrecht. https://doi.org/10.1007/978-94-009-4992-8_28
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DOI: https://doi.org/10.1007/978-94-009-4992-8_28
Publisher Name: Springer, Dordrecht
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