Premium
Examination of primary metabolic pathways in a murine hybridoma with carbon‐13 nuclear magnetic resonance spectroscopy
Author(s) -
Mancuso Anthony,
Sharfstein Susan T.,
Tucker Sean N.,
Clark Douglas S.,
Blanch Harvey W.
Publication year - 1994
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260440504
Subject(s) - citric acid cycle , pyruvate carboxylase , glutamine , biochemistry , pentose phosphate pathway , glycolysis , metabolism , pyruvate decarboxylation , nuclear magnetic resonance spectroscopy , chemistry , metabolic pathway , biology , enzyme , stereochemistry , amino acid
Primary metabolism of a murine hybridoma was probed with 13 C nuclear magnetic resonance (NMR) spectroscopy. Cells cultured in a hollow fiber bioreactor were serially infused with [1− 13 C] glucose, [2− 13 C] glucose, and [3− 13 C] glutamine. In vivo spectroscopy of the culture was used in conjunction with off‐line spectroscopy of the medium to determine 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 levels 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/melate shuttle associated with lipid synthesis and the pentose phosphate pathway. The rate of formation of pyruvate from the pentose phosphate pathway was estimated to be 4% of that from glycolysis; This value is a lower limit and the actual value may be higher. Incorporation 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 to the rate of glutamine uptake. The rate of incorporation of glucosederived acetyl‐CoA into lipids was 4% of the glucose uptake rate. The TCA cycle rate between isocitrate and α‐ketoglutarate was 110% of the glutamine uptake rate. © 1994 John Wiley & Sons, Inc.