Tricarboxylic acid cycle inhibition by Li+ in the human neuroblastoma SH-SY5Y cell line: A 13C NMR isotopomer analysis

A previous model using 13C nuclear magnetic resonance isotopomer analysis provided for direct measurement of the oxidation of 13C-enriched substrates in the tricarboxylic acid cycle and/or their entry via anaplerotic pathways. This model did not allow for recycling of labeled metabolites from tricarboxylic acid cycle intermediates into the acetyl-CoA pool. An extension of this model is now presented that incorporates carbon flow from oxaloacetate or malate to acetyl-CoA. This model was examined using propionate metabolism in the heart, in which previous observations indicated that all of the propionate consumed was oxidized to CO2 and water. Application of the new isotopomer model shows that 2 mM [3-13C]propionate entered the tricarboxylic acid cycle as succinyl-CoA (an anaplerotic pathway) at a rate equal to 52% of tricarboxylic acid cycle turnover and that all of this carbon entered the acetyl-CoA pool and was oxidized. This was verified using standard biochemical analysis; from the rate (mumol.min-1.g dry wt-1) of propionate uptake (4.0 +/- 0.7), the estimated oxygen consumption (24.8 +/- 5) matched that experimentally determined (24.4 +/- 3).

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Entry of [(1,2-13C2)acetyl]- l-carnitine in liver tricarboxylic acid cycle and lipogenesis . A study by 13C NMR spectroscopy in conscious, freely moving rats

European Journal of Biochemistry ◽

10.1046/j.1432-1327.1999.00524.x ◽

1999 ◽

Vol 263 (1) ◽

pp. 287-291 ◽

Cited By ~ 18

Author(s):

Tommaso Aureli ◽

Caterina Puccetti ◽

Maria Enrica Di Cocco ◽

Arduino Arduini ◽

Rita Ricciolini ◽

...

Keyword(s):

Nmr Spectroscopy ◽

13C Nmr ◽

Tricarboxylic Acid Cycle ◽

13C Nmr Spectroscopy ◽

Tricarboxylic Acid ◽

Acid Cycle ◽

Freely Moving Rats ◽

Freely Moving

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A general model for analysis of the tricarboxylic acid cycle with use of [13C]glutamate isotopomer measurements

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1994.266.6.e1012 ◽

1994 ◽

Vol 266 (6) ◽

pp. E1012-E1022 ◽

Cited By ~ 1

Author(s):

J. A. Vogt ◽

A. J. Fischman ◽

M. Kempf ◽

Y. M. Yu ◽

R. G. Tompkins ◽

...

Keyword(s):

13C Nmr ◽

Tricarboxylic Acid Cycle ◽

Nonlinear Least Squares ◽

Tricarboxylic Acid ◽

Acetyl Coa ◽

Metabolic System ◽

Acid Cycle ◽

Nmr Data ◽

13C Nuclear Magnetic Resonance ◽

Model Equations

A generalized steady-state model was developed for determining tricarboxylic acid cycle fractional fluxes from 13C nuclear magnetic resonance (NMR) data. The model relates the measured mole fractions of [13C]glutamate isotopomers to the fractional fluxes and predicted mole fractions of isotopomers of oxaloacetate (OAA) and acetyl-CoA. This model includes cycling between OAA and fumarate. Fractional fluxes are determined by fitting the model equations to NMR parameters by use of nonlinear least squares. Although only fractional fluxes can be determined from 13C-NMR data, when they are combined with mass spectroscopic measurements, absolute values can be derived. A specific metabolic system represented by published 13C-NMR data from extracts of hearts perfused with [13C]acetate, [13C]pyruvate (PYR), and [13C]acetate plus [13C]PYR was used to test the model. The intensities of predicted 13C-NMR splitting patterns were compared with observed values, and there was excellent agreement between observed and predicted signal intensities. With this model, important physiological parameters, including the OAA-derived fraction of inflow to PYR, PYR-derived fraction of inflow to acetyl-CoA, citrate-derived fraction of inflow to OAA, and PYR-derived fraction of inflow to OAA, can be determined.

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Analysis of tricarboxylic acid cycle of the heart using 13C isotope isomers

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1990.259.3.h987 ◽

1990 ◽

Vol 259 (3) ◽

pp. H987-H995 ◽

Cited By ~ 35

Author(s):

C. R. Malloy ◽

A. D. Sherry ◽

F. M. Jeffrey

Keyword(s):

13C Nmr ◽

Nmr Spectra ◽

Tricarboxylic Acid Cycle ◽

Tricarboxylic Acid ◽

13C Nmr Spectra ◽

Acid Cycle ◽

Rat Hearts ◽

13C Nuclear Magnetic Resonance ◽

Peak Areas

13C-nuclear magnetic resonance (NMR) spectroscopy provides a new approach to the analysis of metabolic pathways, because it detects an interaction between adjacent 13C nuclei. Previous models of isotope distribution in the tricarboxylic acid cycle were designed for analysis of radioisotope data and did not consider the information provided by 13C-13C coupling. A mathematical model of the tricarboxylic acid cycle was developed that preserves all isotope isomer (isotopomer) information and yields simple relationships between 13C-NMR spectra of glutamate and metabolic parameters under steady-state conditions. With the use of relative peak areas measured from the spectra of tissues supplied with 13C-enriched substrate(s), the relative fluxes through both oxidative (acetyl-CoA utilization) and nonoxidative (anaplerotic) pathways of the tricarboxylic acid cycle can be determined. Furthermore, with the judicious selection of 13C-labeling patterns in a mixture of substrates, direct substrate competition experiments can be performed. The perchloric acid extracts of Langendorff and working rat hearts oxidizing 13C-enriched fatty acids or carbohydrates are analyzed to illustrate this approach, and the importance of measuring the fractional enrichment of the available substrate is demonstrated. The technique can of course be used with all tissues, not just heart, and is applicable to the analysis of in vivo 13C-NMR spectra.

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