Innovations in Generation and Analysis of 2D [13C,1H] COSY NMR Spectra for Metabolic Flux Analysis Purposes

Winden, Wouter van; Schipper, Dick; Verheijen, P.J.T.; Heijnen, Joseph J.

doi:10.1006/mben.2001.0193

Cited by 59 publications

(23 citation statements)

References 34 publications

(46 reference statements)

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“…Assuming that the labeling biomass follows first-order wash-out kinetics, the GC-MS and NMR data of the amino acid isotopomer composition (Additional file 1) were corrected for the deviation from the isotopic steady state at the time of harvesting [20,55,56], using an isotopic nonsteady-state correction [57,58]. …”

Section: Methodsmentioning

confidence: 99%

A systematic investigation of Escherichia coli central carbon metabolism in response to superoxide stress

et al. 2010

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BackgroundThe cellular responses of bacteria to superoxide stress can be used to model adaptation to severe environmental changes. Superoxide stress promotes the excessive production of reactive oxygen species (ROS) that have detrimental effects on cell metabolic and other physiological activities. To antagonize such effects, the cell needs to regulate a range of metabolic reactions in a coordinated way, so that coherent metabolic responses are generated by the cellular metabolic reaction network as a whole. In the present study, we have used a quantitative metabolic flux analysis approach, together with measurement of gene expression and activity of key enzymes, to investigate changes in central carbon metabolism that occur in Escherichia coli in response to paraquat-induced superoxide stress. The cellular regulatory mechanisms involved in the observed global flux changes are discussed.ResultsFlux analysis based on nuclear magnetic resonance (NMR) and mass spectroscopy (MS) measurements and computation provided quantitative results on the metabolic fluxes redistribution of the E. coli central carbon network under paraquat-induced oxidative stress. The metabolic fluxes of the glycolytic pathway were redirected to the pentose phosphate pathway (PP pathway). The production of acetate increased significantly, the fluxes associated with the TCA cycle decreased, and the fluxes in the glyoxylate shunt increased in response to oxidative stress. These global flux changes resulted in an increased ratio of NADPH:NADH and in the accumulation of α-ketoglutarate.ConclusionsMetabolic flux analysis provided a quantitative and global picture of responses of the E. coli central carbon metabolic network to oxidative stress. Systematic adjustments of cellular physiological state clearly occurred in response to changes in metabolic fluxes induced by oxidative stress. Quantitative flux analysis therefore could reveal the physiological state of the cell at the systems level and is a useful complement to molecular systems approaches, such as proteomics and transcription analyses.

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Section: Methodsmentioning

confidence: 99%

A systematic investigation of Escherichia coli central carbon metabolism in response to superoxide stress

et al. 2010

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“…From the isotopomer data collected at 6.8 d and 12.9 d, we calculated the steady state isotopomer abundances (at t = ∞), by using methods published previously [34], and validated that the isotopomer abundances at the end of passage 2 (12.9 d) were 98 ± 2% of their steady state value. Fig.…”

Section: Mass Isotopomer Abundancesmentioning

confidence: 95%

Global metabolic effects of glycerol kinase overexpression in rat hepatoma cells

Sriram

Rahib

et al. 2008

Molecular Genetics and Metabolism

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Glycerol kinase has several diverse activities in mammalian cells. Glycerol kinase deficiency is a complex, single-gene, inborn error of metabolism wherein no genotype-phenotype correlation has been established. Since glycerol kinase has been suggested to exhibit additional activities than glycerol phosphorylation, expression level perturbation in this enzyme may affect cellular physiology globally. To investigate this possibility, we conducted metabolic investigations of wild type and two glycerol kinase-overexpressing H4IIE rat hepatoma cell lines constructed in this study. The glycerol kinase-overexpressing cell lines exhibited a significantly higher consumption of carbon sources per cell, suggesting excess carbon expenditure. Furthermore, we quantified intracellular metabolic fluxes by employing stable isotope ( 13 C) labeling with a mathematically designed substrate mixture, gas chromatography-mass spectrometry, and comprehensive isotopomer balancing. This flux analysis revealed that the pentose phosphate pathway flux in the glycerol kinase-overexpressing cell lines was two-fold higher than that in the wild type, in addition to subtler flux changes in other pathways of carbohydrate metabolism. Furthermore, the activity and transcript level of the lipogenic enzyme glucose-6-phosphate dehydrogenase, the rate-limiting enzyme of the pentose phosphate pathway, were also about two-fold higher than that of the wild type; these data corroborate the flux analysis results. This study shows that glycerol kinase affects carbon metabolism globally, possibly through its additional functions, and highlights glycerol kinase's multifaceted role in cellular physiology.

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“…To quantify overlapping multiplets (a-amino acid and LVA peaks) on the HSQC spectra, a peak deconvolution software was written based on a spectral model originally proposed by van Winden et al (2001). Additionally, 2-D spectra were acquired that were J-scaled along the F1 dimension, by incorporating pulse sequences described by Willker et al (1997) and Brown (1984) into the HSQC pulse sequence.…”

Section: Nmr Spectroscopy and Calculation Of Bondomer Abundancesmentioning

confidence: 99%

Flux quantification in central carbon metabolism of Catharanthus roseus hairy roots by 13C labeling and comprehensive bondomer balancing

2007

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Innovations in Generation and Analysis of 2D [13C,1H] COSY NMR Spectra for Metabolic Flux Analysis Purposes

Cited by 59 publications

References 34 publications

A systematic investigation of Escherichia coli central carbon metabolism in response to superoxide stress

A systematic investigation of Escherichia coli central carbon metabolism in response to superoxide stress

Global metabolic effects of glycerol kinase overexpression in rat hepatoma cells

Flux quantification in central carbon metabolism of Catharanthus roseus hairy roots by 13C labeling and comprehensive bondomer balancing

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