Document Detail


Real-time detection of 13C NMR labeling kinetics in perfused EMT6 mouse mammary tumor cells and betaHC9 mouse insulinomas.
MedLine Citation:
PMID:  15334410     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
A Mancuso; N J Beardsley; S Wehrli; S Pickup; F M Matschinsky; J D Glickson
Publication Detail:
Type:  Comparative Study; Evaluation Studies; Journal Article; Research Support, U.S. Gov't, P.H.S.; Validation Studies    
Journal Detail:
Title:  Biotechnology and bioengineering     Volume:  87     ISSN:  0006-3592     ISO Abbreviation:  Biotechnol. Bioeng.     Publication Date:  2004 Sep 
Date Detail:
Created Date:  2004-08-30     Completed Date:  2005-02-10     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  7502021     Medline TA:  Biotechnol Bioeng     Country:  United States    
Other Details:
Languages:  eng     Pagination:  835-48     Citation Subset:  IM    
Copyright Information:
Copyright 2004 Wiley Periodicals, Inc.
Affiliation:
Department of Radiology/6069, B6 Blockley Hall, 423 Guardian Drive, Philadelphia, Pennsylvania 19104-6021, USA. mancuso@rad.upenn.edu
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MeSH Terms
Descriptor/Qualifier:
Animals
Carbon Isotopes
Cell Culture Techniques / instrumentation,  methods*
Cell Line, Tumor
Cells, Immobilized / metabolism
Glucose / metabolism*
Glutamic Acid / metabolism*
Insulinoma / metabolism*
Isotope Labeling / methods
Kinetics
Magnetic Resonance Spectroscopy / instrumentation,  methods*
Mammary Neoplasms, Experimental / metabolism*
Metabolic Clearance Rate
Mice
Online Systems*
Reproducibility of Results
Sensitivity and Specificity
Grant Support
ID/Acronym/Agency:
2RO1-CA51935/CA/NCI NIH HHS; 2RO1-CA51950/CA/NCI NIH HHS
Chemical
Reg. No./Substance:
0/Carbon Isotopes; 50-99-7/Glucose; 56-86-0/Glutamic Acid

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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