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NMR Spectroscopy Studies of Hybridoma Metabolism in a Simple Membrane Reactor
Author(s) -
Fernandez Erik J.,
Mancuso Anthony,
Clark Douglas S.
Publication year - 1988
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1002/btpr.5420040308
Subject(s) - chemistry , alanine , intracellular , nuclear magnetic resonance spectroscopy , extracellular , nmr spectra database , metabolism , glutamine , intracellular ph , substrate (aquarium) , membrane , oxygen , in vivo , chromatography , biochemistry , amino acid , spectral line , biology , stereochemistry , organic chemistry , ecology , physics , microbiology and biotechnology , astronomy
Nuclear magnetic resonance (NMR) was used to examine the metabolism of hybridoma cells in a simple immobilized‐cell membrane reactor. Although the reactor was not suitable for maintaining viable cells over extended periods, several types of experiments were performed. Phosphorylated and carbonaceous metabolites were observed on‐line with 31 P and 13 C NMR. Complementary information about glutamine metabolism was provided by 15 N NMR spectra of concentrated supernatants. Extracellular contributions to NMR spectra were measured in a second vessel containing only medium. 3I P NMR measurements demonstrated that the perfused cells were stable to changes in the concentration of dissolved oxygen and to a brief interruption in medium recirculation. In tracer experiments utilizing [I‐ 13 C]D‐glucose as a substrate, lactate and alanine were detected in vivo, along with some as of yet unassigned compounds. When cells were exposed to a high initial concentration of [I‐ 13 C]D‐glucose, lactate and alanine appeared immediately and attained their maximum recorded levels at similar times. When cells were subjected to a step change in the glucose concentration from 2–10 mM, intracellular lactate and nucleoside triphosphates (NTP) increased sharply, but alanine formation was not evident until nearly five hours later. In both cases, the intracellular concentrations of labeled lactate and alanine were estimated directly from the 13 C NMR spectra.