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General Information info

Manuscript title Dynamics and control of the central carbon metabolism in Hepatoma cells.
PubMed ID 20426867
Journal BMC Systems Biology
Year 2010
Authors Klaus Maier, Ute Hofmann, Mathias Reuss and Klaus Mauch
Affiliations Institute of Biochemical Engineering, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany
Keywords Hepatoma Cells, in vivo Dynamics, Dynamic Model, Control
Full text article Downloadarticle Maier_2010.pdf
Project name not specified

Experiment Description info

Organism Homo sapiens
Strain HepG2 (ATCC number HB-8065)
Data type metabolites at steady-state
Data units (mmol/L)
Execution date not specified

Experimental Details info

Temperature (0C) 37.0
pH not specified
Carbon source glucose,
Culture mode batch
Process condition aerobic
Dilution rate (h-1)
Working volume (L) not specified
Biomass concentration (g/L) not specified
Medium composition

Alanyl-glutamine-free William's medium E (PAN Biotech GmbH) that was supplemented with penicillin (100 U/mL), streptomycin (100 mg/mL), and Gibco Insulin-Transferrin-Selenium (100X) supplement (Invitrogen, Karlsruhe, Germany).

General protocol information Sampling method: Removal of cell culture medium by suction (5 seconds).

Quenching procedure: hot air

Extraction technique: hot water

Sample analyzing method: GC-MS, LC-MS, HPLC-UV/RI

Methods description - Notes

Extra- and intracellular samples were collected in triplicate directly before and after the stimulus, as well as 1, 2, 5, 10, 30, 60, 120, and 180 min after glucose deprivation. The sampling approach and the processing of the samples were done as previously described [1].
The concentrations of Lanine, serine, glucose, lactate, pyruvate, fumarate, malate, cis-aconitate, isocitrate, and citrate were determined by GC-MS as described before [1,2]. After glucose deprivation, extracellular glucose concentrations were determined in 10 μl of diluted (1:9 v/v) medium samples, the intracellular glucose concentrations before and after perturbation were determined in 5 and 50 μl of cell extract, respectively. Phosphoenolpyruvate, 3-phosphoglycerate, dihydroxyacetonphosphate, fructose-1,6-bisphosphate, glucose-6-phosphate, 6-phosphogluconate, sedoheptulose-7-phosphate, ribose-5-phosphate, and ribulose-5-phosphate were determined by LC-MS-MS as described by Hofmann [1] with the following modifications: HPLC separation was performed at 20 °C on a Synergi Hydro-RP column (150 × 2 mm, 4 μm; Phenomenex, Aschaffenburg, Germany) at a flow rate of 0.2 ml/min. Nucleotide analysis was performed by reversed phase ion pair high performance liquid chromatography. The HPLC system (Agilent Technologies, Waldbronn, Germany) consisted of an Agilent 1200 series autosampler, an Agilent 1200 series Binary Pump SL, an Agilent 1200 series thermostatted column compartment, and an Agilent 1200 series diode array detector set at 260 and 340 nm. The nucleotides were separated and quantified on an RP-C-18 column that was combined with a guard column (Supelcosil LC-18-T; 15 cm × 4.6 mm, 3 μm packing and Supelguard LC-18-T replacement cartridges, 2 cm; Supelco, Bellefonte, USA) at a flow rate of 1 ml/min.

[1] Hofmann U, Maier K, Niebel A, Vacun G, Reuss M, Mauch K. Identification of metabolic fluxes in hepatic cells from transient 13C-labeling experiments: Part I. Experimental observations. Biotechnol Bioeng 2008, 100:344-354.
[2] Maier K, Hofmann U, Bauer A, Niebel A, Vacun G, Reuss M, Mauch K. Quantification of statin effects on hepatic cholesterol synthesis by transient (13)C-flux analysis. Metab Eng 2009, 11:292-309.

Data file
Downloadmetabolites KIMODATAID58_v0.xlsx
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Entered by Administrator KiMoSysFirst name: Administrator
Affiliation: INESC-ID/IST
Interests: mathematical modeling, accessible data, use of data

Created 2013-06-06 13:47:09 UTC

Updated 2014-06-12 22:42:09 UTC

Version 0

Status (reviewed) 2013-12-06 17:17:38 UTC

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