Quantitative in vivo nuclear magnetic resonance studies of hybridoma metabolism

Abstract

Carbon-13 nuclear magnetic resonance (NMR) spectroscopy was used to study the metabolism of a murine hybridoma cell line at two feed glutamine concentrations, 4.0 and 1.7 mM. Carbon-13 labeling patterns were used in conjunction with nutrient uptake rates to calculate the metabolic fluxes through the glycolytic pathway, the pentose shunt, the malate shunt, lipid biosynthesis, and the tricarboxylic acid (TCA) cycle. Decreasing the feed glutamine concentration significantly decreased glutamine uptake but had little effect on glucose metabolism. A significant incrase in antibody productivity occurred upon decreasing the feed glutamine level. The increased antibody productivity in concert with decreased glutamine uptake and no apparent change in glucolytic metabolism suggests that antibody production was not energy limited. Metabolic flux calculations indicate that (1) approximately 92% of the glucose consumed proceeds directly through glycolysis with 8% channeled through the pentose shunt; (2) lipid biosynthesis appears to be greater than malate shunt activity; and (3) considerable exchange occurs between TCA cycle intermediates and amino acid metabolic pools, leading to substantial loss of (13)C label from the TCA cycle. These results illustrate that (13)NMR spectroscopy is a powerfulf tool in the calculation of metabolic fluxes, particularly for exchange pathways where no net flux occurs. (c) 1994 John Wiley & Sons, Inc.

Effect of reversible reactions on isotope label redistribution

“…This analysis was applied to literature data of metabolite carbon labeling that resulted from supplying either 13 C-or 14 C-enriched substrates to several cell types growing under various conditions. The specific activities of metabolite carbon atoms taken from rat epididymal adipose tissue, goosefish islet cells, Corynebacterium glutamicum, and Escherichia coli supplied with either [2-14 C]glucose or [1-13 C]glucose demonstrate how reversibility is present in the pentose phosphate pathway and the extents of reversibility can be estimated from labeled carbon data sets.Keywords : pentose-phosphate pathway ; metabolic flux ; isotope-exchange reaction; mathematical modeling;13 C enrichment.An important parameter in metabolic pathway analysis is the Mancuso et al, 1994;Marx et al, 1996 ; Park et al, 1997;Sharfstein et al, 1994; Vallino and Stephanopoulos, 1993, flux of carbon through the pathway. Accurate determination of metabolic fluxes in vivo is a critical step in elucidating flux con-1994a, b; Wiechert et al, 1997a, b ;Zupke and Stephanopoulos, 1995).…”

Reduction of Proteinuria through Podocyte Alkalinization

“…Higher lactate production rates in disease models are the results of higher use of glucose than the control model based on the assumption that lactate generated by cultured cells was only derived from glucose. Our assumption was largely supported by NMR experiments with hybridoma cells (43,44). The amount of lactate derived from glutamine was not determined in our studies.…”

Effects of clonal variation on growth, metabolism, and productivity in response to trophic factor stimulation: a study of Chinese hamster ovary cells producing a recombinant monoclonal antibody

“…Much of the early work on industrially relevant mammalian cell culture systems, particularly hybridoma cells, focused on understanding metabolic behavior (Bonarius et al 1996;Leno et al 1992;Mancuso et al 1994Mancuso et al , 1998Ozturk and Palsson 1991;Savinell and Palsson 1992;Sharfstein et al 1994;Xie and Wang 1994;Zupke and Stephanopoulos 1995). However, it became apparent that unlike microbial systems, there was not a direct link between metabolic behavior and productivity .…”