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177 results on '"Malloy, C. R."'

5. TCA cycle kinetics in the rat heart by analysis of [sup.13]C isotopomers using indirect [sup.1]H[[sup.13]C] detection

Author

CARVALHO, R. A., ZHAO, P., WIEGERS, C. B., JEFFREY, F. M. H., MALLOY, C. R., and SHERRY, A. D.

Subjects
Glutamate -- Research, Cardiology -- Research, Biological sciences
Abstract

This study was designed to test the hypothesis that indirect [sup.1H][[sup.13]C] detection of tricarboxylic acid (TCA) cycle intermediates using heteronuclear multiple quantum correlation-total correlation spectroscopy (HMQC-TOCSY) nuclear magnetic resonance (NMR) spectroscopy provides additional [sup.13]C isotopomer information that better describes the kinetic exchanges that occur between intracellular compartments than direct [sup.13]C NMR detection. NMR data were collected on extracts of rat hearts perfused at various times with combinations of [2-[sup.13]C]acetate, propionate, the transaminase inhibitor aminooxyacetate, and [sup.13]C multiplet areas derived from spectra of tissue glutamate were fit to a standard kinetic model of the TCA cycle. Although the two NMR methods detect different populations of [sup.13]C isotopomers, similar values were found for TCA cycle and exchange fluxes by analyzing the two data sets. Perfusion of hearts with unlabeled propionate in addition to [2-[sup.13]C] acetate resulted in an increase in the pool size of all four-carbon TCA cycle intermediates. This allowed the addition of isotopomer data from aspartate and malate in addition to the more abundant glutamate. This study illustrates that metabolic inhibitors can provide new insights into metabolic transport processes in intact tissues. [sup.13]C isotopomers; aminooxyacetate; metabolism; NMR; glutamate

Published
2001

33. Evaluation of carbon flux and substrate selection through alternate pathways involving the citric acid cycle of the heart by 13C NMR spectroscopy.

Author

Malloy, C R, Sherry, A D, and Jeffrey, F M

Abstract

A previous 13C NMR technique (Malloy, C. R., Sherry, A.D., and Jeffrey, F.M.H. (1987) FEBS Lett. 212, 58-62) for measuring the relative flux of molecules through the oxidative versus anaplerotic pathways involving the citric acid cycle of the rat heart has been extended to include a complete analysis of the entire glutamate 13C spectrum. Although still simple in practice, this more sophisticated model allows an evaluation of 13C fractional enrichment of molecules entering both the oxidative and anaplerotic pathways under steady-state conditions. The method was used to analyze 13C NMR spectra of intact hearts or their acid extracts during utilization of 13C-enriched pyruvate, propionate, acetate, or various combinations thereof. [2-13C]Pyruvate was used to prove that steady-state flux of pyruvate through pyruvate carboxylase is significant during co-perfusion of pyruvate and acetate, and we demonstrate for the first time that a nine-line 13C multiplet may be detected in an intact, beating heart. Acetate or pyruvate alone provided about 86% of the acetyl-CoA; in combination, about 65% of the acetyl-CoA was derived from acetate, about 30% was derived from pyruvate, and the remainder from endogenous sources. Propionate reduced the contribution of exogenous acetate to acetyl-CoA to 77% and also reduced the oxidation of endogenous substrates. Equations are presented which allow this same analysis on multiply labeled substrates, making this technique extremely powerful for the evaluation of substrate selection and relative metabolic flux through anaplerotic and oxidative pathways in the intact heart.

Published
1988
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35. Propionate metabolism in the rat heart by 13C n.m.r. spectroscopy

Author

Sherry, A D, Malloy, C R, Roby, R E, Rajagopal, A, and Jeffrey, F M

Abstract

High-resolution 13C n.m.r. spectroscopy has been used to examine propionate metabolism in the perfused rat heart. A number of tricarboxylic acid (TCA) cycle intermediates are observable by 13C n.m.r. in hearts perfused with mixtures of pyruvate and propionate. When the enriched 13C-labelled nucleus originates with pyruvate, the resonances of the intermediates appear as multiplets due to formation of multiply-enriched 13C-labelled isotopomers, whereas when the 13C-labelled nucleus originates with propionate, these same intermediates appear as singlets in the 13C spectrum since entry of propionate into the TCA cycle occurs via succinyl-CoA. An analysis of the isotopomer populations in hearts perfused with [3-13C]pyruvate plus unlabelled propionate indicates that about 27% of the total pyruvate pool available to the heart is derived directly from unlabelled propionate. This was substantiated by perfusing a heart for 2 h with [3-13C]propionate as the only available exogenous substrate. Under these conditions, all of the propionate consumed by the heart, as measured by conventional chemical analysis, ultimately entered the oxidative pathway as [2-13C] or [3-13C]pyruvate. This is consistent with entry of propionate into the TCA cycle intermediate pools as succinyl-CoA and concomitant disposal of malate to pyruvate via the malic enzyme. 13C resonances arising from enriched methylmalonate and propionylcarnitine are also detected in hearts perfused with [3-13C] or [1-13C]propionate which suggests that 13C n.m.r. may be useful as a non-invasive probe in vivo of metabolic abnormalities involving the propionate pathway, such as methylmalonic aciduria or propionic acidaemia.

Published
1988
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