title: Real-time detection of 13C NMR labeling kinetics in perfused EMT6 mouse mammary tumor cells and betaHC9 mouse insulinomas.
abstract: 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.affiliation: Department of Radiology/6069, B6 Blockley Hall, 423 Guardian Drive, Philadelphia, Pennsylvania 19104-6021, USA. mancuso@rad.upenn.edu
authors.forename: A
authors.initials: A
authors.lastname: Mancuso
authors.forename: N J
authors.initials: NJ
authors.lastname: Beardsley
authors.forename: S
authors.initials: S
authors.lastname: Wehrli
authors.forename: S
authors.initials: S
authors.lastname: Pickup
authors.forename: F M
authors.initials: FM
authors.lastname: Matschinsky
authors.forename: J D
authors.initials: JD
authors.lastname: Glickson
mesh_terms: D000818:Animals; D002247:Carbon Isotopes; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D045744:Cell Line, Tumor; D018914:Cells, Immobilized / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D007340:Insulinoma / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000295:instrumentation / Q000379:methods*; D008325:Mammary Neoplasms, Experimental / Q000378:metabolism*; D008657:Metabolic Clearance Rate; D051379:Mice; D009862:Online Systems; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D002247:Carbon Isotopes; D018698:Glutamic Acid; D005947:Glucose