Use of a single<sup>13</sup>C NMR resonance of glutamate for measuring oxygen consumption in tissue

Jeffrey, F. M H; Reshetov, Alexander; Storey, Charles; Carvalho, Rui A.; Sherry, A. Dean; Malloy, Craig R.

doi:10.1152/ajpendo.1999.277.6.e1111

Cited by 33 publications

(57 citation statements)

References 27 publications

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“…Although the model currently allows the use of [1,[6][7][8][9][10][11][12][13] C 2 ]glucose only as enriched substrate, it can be extended to simultaneously analyze mixtures of substrates with appropriately different labeling patterns. This will help determine the extent to which different substrates are used in different compartments of the brain.…”

Section: Discussionmentioning

confidence: 99%

“…The time courses of 13 C multiplets used in this study were presented in a report on the metabolism of [1,[6][7][8][9][10][11][12][13] C 2 ]glucose in the rat brain. 4 The measurements from glutamine and glutamate were analyzed.…”

Section: Introductionmentioning

confidence: 99%

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confidence: 99%

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Modeling of Brain Metabolism and Pyruvate Compartmentation Using ¹³C NMR in Vivo: Caution Required

Jeffrey

Marin‐Valencia

Good

et al. 2013

J Cereb Blood Flow Metab

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Two variants of a widely used two-compartment model were prepared for fitting the time course of [1,[6][7][8][9][10][11][12][13] C 2 ]glucose metabolism in rat brain. Features common to most models were included, but in one model the enrichment of the substrates entering the glia and neuronal citric acid cycles was allowed to differ. Furthermore, the models included the capacity to analyze multiplets arising from 13 C spin-spin coupling, known to improve parameter estimates in heart. Data analyzed were from a literature report providing time courses of [1,6-13 C 2 ]glucose metabolism. Four analyses were used, two comparing the effect of different pyruvate enrichment in glia and neurons, and two for determining the effect of multiplets present in the data. When fit independently, the enrichment in glial pyruvate was less than in neurons. In the absence of multiplets, fit quality and parameter values were typical of those in the literature, whereas the multiplet curves were not modeled well. This prompted the use of robust statistical analysis (the Kolmogorov-Smirnov test of goodness of fit) to determine whether individual curves were modeled appropriately. At least 50% of the curves in each experiment were considered poorly fit. It was concluded that the model does not include all metabolic features required to analyze the data.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Modeling of Brain Metabolism and Pyruvate Compartmentation Using ¹³C NMR in Vivo: Caution Required

Jeffrey

Marin‐Valencia

Good

et al. 2013

J Cereb Blood Flow Metab

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“…Metabolic Fate of [1,[6][7][8][9][10][11][12][13] C 2 ]Glucose in DB-1 Cells- Fig. 2 shows the entire metabolic network that is included in the current analysis (see under "Experimental Procedures" for details).…”

Section: P Nmr-a Typicalmentioning

confidence: 99%

“…Both types of analysis require validated mathematical models, which have been formulated for the heart (5-10), liver (11-13), brain (14 -16), and tumors (17). Only the heart model has been validated by comparison between model-derived and experimental parameters (6,7). Modeling of tumor metabolic fluxes has been limited to steady-state analysis with dynamic measurements of glucose uptake and lactate production subsequently added to convert to absolute fluxes; none of these models has been validated.…”

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Bonded Cumomer Analysis of Human Melanoma Metabolism Monitored by 13C NMR Spectroscopy of Perfused Tumor Cells

Shestov¹,

Mancuso²,

Lee³

et al. 2016

Journal of Biological Chemistry

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A network model for the determination of tumor metabolic fluxes from 13 C NMR kinetic isotopomer data has been developed and validated with perfused human DB-1 melanoma cells carrying the BRAF V600E mutation, which promotes oxidative metabolism. The model generated in the bonded cumomer formalism describes key pathways of tumor intermediary metabolism and yields dynamic curves for positional isotopic enrichment and spin-spin multiplets. Cells attached to microcarrier beads were perfused with 26 mM [1,6-13 C 2 ]glucose under normoxic conditions at 37°C and monitored by 13 C NMR spectroscopy. Excellent agreement between model-predicted and experimentally measured values of the rates of oxygen and glucose consumption, lactate production, and glutamate pool size validated the model. ATP production by glycolytic and oxidative metabolism were compared under hyperglycemic normoxic conditions; 51% of the energy came from oxidative phosphorylation and 49% came from glycolysis. Even though the rate of glutamine uptake was ϳ50% of the tricarboxylic acid cycle flux, the rate of ATP production from glutamine was essentially zero (no glutaminolysis). De novo fatty acid production was ϳ6% of the tricarboxylic acid cycle flux. The oxidative pentose phosphate pathway flux was 3.6% of glycolysis, and three non-oxidative pentose phosphate pathway exchange fluxes were calculated. Mass spectrometry was then used to compare fluxes through various pathways under hyperglycemic (26 mM) and euglycemic (5 mM) conditions. Under euglycemic conditions glutamine uptake doubled, but ATP production from glutamine did not significantly change. A new parameter measuring the Warburg effect (the ratio of lactate production flux to pyruvate influx through the mitochondrial pyruvate carrier) was calculated to be 21, close to upper limit of oxidative metabolism.Cancer cells exhibit metabolic patterns that differ significantly from those of terminally differentiated adult tissues. They require rapid production of both energy and biosynthetic precursors to sustain a high rate of proliferation. The network of biochemical pathways underlying these processes is complex. Most of the individual enzymatic conversions involved in this network have probably already been identified, but a comprehensive and quantitative description of the fluxes involved is lacking. Methods to produce such descriptions will likely be very useful in the design of new therapeutics that specifically target the metabolic abnormalities of cancer as well as in clinical implementation of non-invasive methods for detection of cancer and its therapeutic response.The kinetics of 13 C isotope labeling as monitored by NMR spectroscopy has been used to measure flux through various metabolic pathways of perfused isolated cells and organs and in vivo (1-4). Kinetic data provide estimates of absolute flux, whereas steady-state data measure relative flux. Both types of analysis require validated mathematical models, which have been formulated for the heart (5-10), liver (11-13), brain (14 -16), a...

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Real‐time detection of ¹³C NMR labeling kinetics in perfused EMT6 mouse mammary tumor cells and βHC9 mouse insulinomas

Mancuso

Beardsley

Wehrli

et al. 2004

Biotech & Bioengineering

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A method was developed for obtaining high signal-to-noise 13C NMR spectra of intracellular compounds in metabolically active cultured cells. The method allows TCA cycle labeling kinetics to be determined in real time without significant oxygen transport limitations. Cells were immobilized on the surface of nonporous microcarriers that were either uncoated or coated with polypeptides and used in a 12-cm3 packed bed. The methods were tested with two EMT6 mouse mammary tumor cell lines, one strongly adherent and the other moderately adherent, and a weakly adherent mouse insulinoma line (betaHC9). For both EMT6 lines, NTP and oxygen consumption measurements indicated that the number of cells in the spectrometer ranged from 6 x 10(8) to 1 x 10(9). During infusion of [1-13C]glucose, labeling in C-4 glutamate (indicative of flux into the first half of the TCA cycle) could be detected with 15-min resolution. However, labeling for C-3 and C-2 glutamate (indicative of complete TCA cycle activity) was fivefold lower and difficult to quantify. To increase TCA cycle labeling, cells were infused with medium containing [1,6-13C2]glucose. A 2.5-fold increase was observed in C-4 glutamate labeling and C-3 and C-2 glutamate labeling could be monitored with 30-min resolution. Citrate synthase activity was indirectly detected in real time, as [3,4-13C2]glutamate was formed from [2-13C]oxaloacetate and [2-13C]acetate (of acetyl-CoA). Cell mass levels observed with betaHC9 cells were somewhat lower. However, the 13C S/N was sufficient to allow real-time monitoring of the response of intracellular metabolite labeling to a step change in glucose and a combined glutamine/serum pulse.

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Use of a single¹³C NMR resonance of glutamate for measuring oxygen consumption in tissue

Cited by 33 publications

References 27 publications

Modeling of Brain Metabolism and Pyruvate Compartmentation Using ¹³C NMR in Vivo: Caution Required

Modeling of Brain Metabolism and Pyruvate Compartmentation Using ¹³C NMR in Vivo: Caution Required

Bonded Cumomer Analysis of Human Melanoma Metabolism Monitored by 13C NMR Spectroscopy of Perfused Tumor Cells

Real‐time detection of ¹³C NMR labeling kinetics in perfused EMT6 mouse mammary tumor cells and βHC9 mouse insulinomas

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Use of a single13C NMR resonance of glutamate for measuring oxygen consumption in tissue

Cited by 33 publications

References 27 publications

Modeling of Brain Metabolism and Pyruvate Compartmentation Using 13C NMR in Vivo: Caution Required

Modeling of Brain Metabolism and Pyruvate Compartmentation Using 13C NMR in Vivo: Caution Required

Bonded Cumomer Analysis of Human Melanoma Metabolism Monitored by 13C NMR Spectroscopy of Perfused Tumor Cells

Real‐time detection of 13C NMR labeling kinetics in perfused EMT6 mouse mammary tumor cells and βHC9 mouse insulinomas

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Resources

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Use of a single¹³C NMR resonance of glutamate for measuring oxygen consumption in tissue

Modeling of Brain Metabolism and Pyruvate Compartmentation Using ¹³C NMR in Vivo: Caution Required

Modeling of Brain Metabolism and Pyruvate Compartmentation Using ¹³C NMR in Vivo: Caution Required

Real‐time detection of ¹³C NMR labeling kinetics in perfused EMT6 mouse mammary tumor cells and βHC9 mouse insulinomas