METABOLIC FLUX DETERMINATION IN C6 GLIOMA-CELLS USING C-13 DISTRIBUTION UPON [1-C-13]GLUCOSE INCUBATION

A mathematical model of mammalian cell intermediary metabolism is pres ented. It describes the distribution of the carbon-13 isotope (C-13) a t the different carbon positions of metabolites in cells fed with C-13 -enriched substrates. The model allows the determination of fluxes thr ough different metabolic pathways from C-13- and H-1-NMR spectroscopy and mass spectrometry data. The considered metabolic network includes glycolysis, gluconeogenesis, the citric acid cycle and a number of rea ctions corresponding to protein or fatty acid metabolism. The model wa s used for calculating metabolic fluxes in a rat tumor cell line, the C6 glioma, incubated with [1-C-13]glucose. After evolution to metaboli c and isotopic steady states, the intracellular metabolites were extra cted with perchloric acid. The specific enrichments of glutamate, aspa rtate and alanine carbons were determined from C-13-, H-1-NMR spectros copy, or mass spectrometry data. Taking into account the rate of gluco se consumption and of lactate formation, determined from the evolution of glucose and lactate contents in the cell medium, and knowing the a ctivity of the hexose monophosphate shunt, it was possible to estimate the absolute values of all the considered fluxes. From the analysis t he following results were obtained. (a) Glucose accounts for about 78% of the pyruvate and 57% of the CoASAc. (b) A metabolic channelling oc curs at the citric acid cycle level; it favours the conversion of carb ons 2, 3, 4, and 5 of 2-oxoglutarate into carbons 1, 2, 3, and 4 of ox aloacetate, respectively. The percentage of channelled metabolites amo unts to 39%. (c) The pyruvate carboxylase activity and the efflux from the citric acid cycle are estimated to be very low, suggesting a lack of glutamine production in C6 cells. The results emphasize different metabolic characteristics of C6 cells when compared to astrocytes. the ir normal counterpart.