EXAMINATION OF PRIMARY METABOLIC PATHWAYS IN A MURINE HYBRIDOMA WITH C-13 NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY

Primary metabolism of a murine hybridoma was probed with C-13 nuclear magnetic resonance (NMR) spectroscopy. Cells cultured in a hollow fibe r bioreactor were serially infused with [1-C-13] glucose, [2-C-13] glu cose, and [3-C-13] glutamine. In vivo spectroscopy of the culture was used in conjunction with off-line spectroscopy of the medium to determ ine the intracellular concentration of several metabolic intermediates and to determine fluxes for primary metabolic pathways. Intracellular concentrations of pyruvate and alanine were very high relative to lev els observed in normal quiescent mammalian cells. Estimates made from labeling patterns in lactate indicate that 76% of pyruvate is derived directly from glycolysis; some is also derived from the malate shunt, the pyruvate/malate shuttle associated with lipid synthesis and the pe ntose phosphate pathway. The rate of formation of pyruvate from the pe ntose phosphate pathway was estimated to be 4% of that from glycolysis ; this value is a lower limit and the actual value may be higher. Inco rporation of pyruvate into the tricarboxylic acid (TCA) cycle appears to occur through only pyruvate dehydrogenase; no pyruvate carboxylase activity was detected. The malate shunt rate was approximately equal t o the rate of glutamine uptake. The rate of incorporation of glucose-d erived acetyl-CoA into lipids was 4% of the glucose uptake rate. The T CA cycle rate between isocitrate and alpha-ketoglutarate was 110% of t he glutamine uptake rate. (C) 1994 John Wiley and Sons, Inc.