Determination of Metabolic Flux Ratios From 13C-Experiments and Gas Chromatography-Mass Spectrometry Data

Nanchen, Annik; Fuhrer, Tobias; Sauer, Uwe

doi:10.1007/978-1-59745-244-1_11

Cited by 173 publications

(183 citation statements)

References 11 publications

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“…A correction matrix was generated for each metabolite using an in-house algorithm adapted from ref. 32. Integration values for a given metabolite were multiplied by the corresponding correction matrix to remove the abundances of naturally occurring isotopes that mask the labeling provided by exogenous 13 C-substrates.…”

Section: Mass Isotopomer Distribution Analysismentioning

confidence: 99%

Serine Deprivation Enhances Antineoplastic Activity of Biguanides

et al. 2014

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Metformin, a biguanide widely used in the treatment of type II diabetes, clearly exhibits antineoplastic activity in experimental models and has been reported to reduce cancer incidence in diabetics. There are ongoing clinical trials to evaluate its antitumor properties, which may relate to its fundamental activity as an inhibitor of oxidative phosphorylation. Here, we show that serine withdrawal increases the antineoplastic effects of phenformin (a potent biguanide structurally related to metformin). Serine synthesis was not inhibited by biguanides. Instead, metabolic studies indicated a requirement for serine to allow cells to compensate for biguanide-induced decrease in oxidative phosphorylation by upregulating glycolysis. Furthermore, serine deprivation modified the impact of metformin on the relative abundance of metabolites within the citric acid cycle. In mice, a serine-deficient diet reduced serine levels in tumors and significantly enhanced the tumor growth-inhibitory actions of biguanide treatment. Our results define a dietary manipulation that can enhance the efficacy of biguanides as antineoplastic agents that target cancer cell energy metabolism. Cancer Res; 74(24); 7521-33. Ó2014 AACR.

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Section: Mass Isotopomer Distribution Analysismentioning

confidence: 99%

Serine Deprivation Enhances Antineoplastic Activity of Biguanides

et al. 2014

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“…GC-MS ion fragments were corrected for natural 13 C abundance using GC/MS-Solution version 2.50SU3 from Shimadzu. For each fragment, the intensities were corrected for the natural abundance of each element using matrix-based probabilistic methods as described (11)(12)(13). The 13 C/( 12 C ϩ 13 C) ratios were used to calculate isotopic labeling proportion.…”

Section: Gas Chromatography/mass Spectrometrymentioning

confidence: 99%

“…The 13 C/( 12 C ϩ 13 C) ratios were used to calculate isotopic labeling proportion. For labeled hexoses, a fragmentation series of m/z 242 and 314 represents the [1,[2][3][4][5][6][7][8][9][10][11][12][13] C]glucose origin, and m/z 217 and 314 represent the [4-13 C]mannose origin. Glucose contribution to mannose was calculated as an average of spectra intensity ratios at m244/(m242 ϩ m244) and m316/(m314 ϩ m316).…”

Section: Gas Chromatography/mass Spectrometrymentioning

confidence: 99%

“…Because both PMM2 and MPI compete for Man-6-P, we hypothesized that the PMM2/MPI ratio partially determined the amount of mannose toward glycosylation. We labeled nine different cell lines with [6-3 H]glucose and [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]mannose and plotted the mannose contribution in N-glycans against the PMM2/MPI ratio ( Fig. 1).…”

Section: Pmm2/mpi Ratio Determines the Contribution Of Mannose To N-gmentioning

confidence: 99%

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Phosphomannose Isomerase Inhibitors Improve N-Glycosylation in Selected Phosphomannomutase-deficient Fibroblasts

Sharma

Ichikawa

et al. 2011

Journal of Biological Chemistry

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“…For each fragment, these data comprised mass intensities for the base isotopomer (without any heavy isotopes, Mϩ0) and isotopomers with increasing unit masses (up to Mϩ6) relative to that of Mϩ0. These mass distributions were normalized by dividing by the sum of Mϩ0 to Mϩ6 and corrected for naturally occurring heavy isotopes of the elements H, N, O, Si, S, and (in moieties from the derivatizing reagent) C, using matrix-based probabilistic methods as described previously (37,49), implemented in Microsoft Excel. Data were also corrected for carryover of unlabeled inoculum (37).…”

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confidence: 99%

Deletion of Genes Encoding Cytochrome Oxidases and Quinol Monooxygenase Blocks the Aerobic-Anaerobic Shift in Escherichia coli K-12 MG1655

Portnoy

Scott

Lewis

et al. 2010

Appl Environ Microbiol

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The constitutive activation of the anoxic redox control transcriptional regulator (ArcA) in Escherichia coli during aerobic growth, with the consequent production of a strain that exhibits anaerobic physiology even in the presence of air, is reported in this work. Removal of three terminal cytochrome oxidase genes (cydAB, cyoABCD, and cbdAB) and a quinol monooxygenase gene (ygiN) from the E. coli K-12 MG1655 genome resulted in the activation of ArcA aerobically. These mutations resulted in reduction of the oxygen uptake rate by nearly 98% and production of D-lactate as a sole by-product under oxic and anoxic conditions. The knockout strain exhibited nearly identical physiological behaviors under both conditions, suggesting that the mutations resulted in significant metabolic and regulatory perturbations. In order to fully understand the physiology of this mutant and to identify underlying metabolic and regulatory reasons that prevent the transition from an aerobic to an anaerobic phenotype, we utilized whole-genome transcriptome analysis, 13 C tracing experiments, and physiological characterization. Our analysis showed that the deletions resulted in the activation of anaerobic respiration under oxic conditions and a consequential shift in the content of the quinone pool from ubiquinones to menaquinones. An increase in menaquinone concentration resulted in the activation of ArcA. The activation of the ArcB/ArcA regulatory system led to a major shift in the metabolic flux distribution through the central metabolism of the mutant strain. Flux analysis indicated that the mutant strain had undetectable fluxes around the tricarboxylic acid (TCA) cycle and elevated flux through glycolysis and anaplerotic input to oxaloacetate. Flux and transcriptomics data were highly correlated and showed similar patterns.Escherichia coli has been studied extensively with respect to its physiology, genetics, and metabolism. One of the unique features of its metabolism is the ability to support robust growth under both oxic and anoxic conditions (38). During aerobic growth, when oxygen is used as a terminal electron acceptor, E. coli divides rapidly and produces carbon dioxide and acetate as major growth by-products (38), representing an efficient form of energy metabolism. In the absence of oxygen, E. coli and other microorganisms rely on anaerobic respiration and fermentation in order to oxidize substrates, recycle electron carriers, and produce ATP (38). This metabolic versatility of E. coli allows it to survive and thrive over a wide range of conditions.Since the ability to produce a number of reduced by-products, such as organic acids and ethanol, is of importance in the field of metabolic engineering, the majority of metabolic engineering designs rely on anaerobic conditions (15,30,31). It has also been shown that E. coli strains developed for overproduction of commodity chemicals can further be improved using adaptive evolution strategies (15). Adaptive evolution is often performed through a series of dilutions allowing cells to...

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Determination of Metabolic Flux Ratios From 13C-Experiments and Gas Chromatography-Mass Spectrometry Data

Cited by 173 publications

References 11 publications

Serine Deprivation Enhances Antineoplastic Activity of Biguanides

Serine Deprivation Enhances Antineoplastic Activity of Biguanides

Phosphomannose Isomerase Inhibitors Improve N-Glycosylation in Selected Phosphomannomutase-deficient Fibroblasts

Deletion of Genes Encoding Cytochrome Oxidases and Quinol Monooxygenase Blocks the Aerobic-Anaerobic Shift in Escherichia coli K-12 MG1655

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