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You are here: Home / Key Scientific Articles / Parallel labeling experiments with [1,2-(13)C]glucose and [U-(13)C]glutamine provide new insights into CHO
cell metabolism.
Ahn WS, Antoniewicz MR.
Metab Eng. 2013 Jan;15:34-47.
Department of Chemical and Biomolecular Engineering, Metabolic Engineering and Systems Biology Laboratory, University of Delaware, 150 Academy St,
Newark, DE 19716, USA.
Abstract
We applied a parallel labeling strategy using two isotopic tracers, [1,2-(13)C]glucose and [U-(13)C]glutamine, to determine metabolic fluxes in Chinese
hamster ovary (CHO) cells. CHO cells were grown in parallel cultures over a period of six days with glucose and glutamine feeding. On days 2 and 5,
isotopic tracers were introduced and (13)C-labeling of intracellular metabolites was measured by gas chromatography-mass spectrometry (GC-MS).
Metabolites in glycolysis pathway reached isotopic steady state for [1,2-(13)C]glucose within 1.5h, and metabolites in the TCA cycle reached isotopic steady
state for [U-(13)C]glutamine within 3h. Combined analysis of multiple data sets produced detailed flux maps at two key metabolic phases, exponential
growth phase (day 2) and early stationary phase (day 5). Flux results revealed significant rewiring of intracellular metabolism in the transition from growth to
non-growth, including changes in oxidative pentose phosphate pathway, anaplerosis, amino acid metabolism, and fatty acid biosynthesis. At the growth
phase, de novo fatty acid biosynthesis correlated well with the lipid requirements for cell growth. However, surprisingly, at the non-growth phase the fatty
acid biosynthesis flux remained high even though no new lipids were needed for cell growth. Additionally, we identified a discrepancy in the estimated TCA
cycle flux obtained using traditional stoichiometric flux balancing and (13)C-metabolic flux analysis. Our results suggested that CHO cells produced
additional metabolites from glucose that were not captured in previous metabolic models. Follow-up experiments with [U-(13)C]glucose confirmed that
additional metabolites were accumulating in the medium that became M+3 and M+6 labeled.
Copyright © 2012 Elsevier Inc. All rights reserved.
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