Modeling enrichment kinetics from dynamic 13C-NMR spectra: theoretical analysis and practical considerations.
Measurements of oxidative metabolism in the heart from dynamic 13C nuclear magnetic resonance (NMR) spectroscopy rely on 13C turnover in the NMR-detectable glutamate pool. A kinetic model was developed for the analysis of isotope turnover to determine tricarboxylic acid cycle flux (VTCA) and the interconversion rate between alpha-ketoglutarate and glutamate (F1) by fitting the model to NMR data of glutamate enrichment. The results of data fitting are highly reproducible when the noise level is within 10%, making this model applicable to single or grouped experiments. The values for VTCA and F1 were unchanged whether obtained from least-squares fitting of the model to mean experimental enrichment data with standard deviations in the cost function (VTCA = 10.52 mumol.min-1.g dry wt-1, F1 = 10.67 mumol.min-1.g dry wt-1) or to the individual enrichment values for each heart with the NMR noise level in the cost function (VTCA = 10.67 mumol.min-1.g dry wt-1, F1 = 10.18 mumol.min-1.g dry wt-1). Computer simulation and theoretical analysis indicate that glutamate enrichment kinetics are insensitive to the fractional enrichment of acetyl-CoA and changes in small intermediate pools (< 1 mumol/g dry wt). Therefore, high-resolution NMR analysis of tissue extracts and biochemical assays for intermediates at low concentrations are unnecessary. However, a high correlation between VTCA and F1 exists, as anticipated from competition for alpha-ketoglutarate, which indicates the utility of introducing independent experimental constraints into the data fitting for accurate quantification.
Yu
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Nuclear Magnetic Resonance Center, Massachusetts General Hospital, Boston, USA.
Alpert
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Lewandowski
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D000085:Acetates / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002247:Carbon Isotopes; D002952:Citric Acid Cycle; D018698:Glutamic Acid / Q000378:metabolism; D006019:Glycolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008433:Mathematics; D008954:Models, Biological; D009206:Myocardium / Q000378:metabolism*
D000085:Acetates; D002247:Carbon Isotopes; D018698:Glutamic Acid; D000255:Adenosine Triphosphate