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Parallel labeling experiments with [1,2-13C]glucose and [U-13C]glutamine provide new insights into CHO cell metabolism

Paper ID Volume ID Publish Year Pages File Format Full-Text
31647 44827 2013 14 PDF Available
Title
Parallel labeling experiments with [1,2-13C]glucose and [U-13C]glutamine provide new insights into CHO cell metabolism
Abstract

We applied a parallel labeling strategy using two isotopic tracers, [1,2-13C]glucose and [U-13C]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 13C-labeling of intracellular metabolites was measured by gas chromatography–mass spectrometry (GC–MS). Metabolites in glycolysis pathway reached isotopic steady state for [1,2-13C]glucose within 1.5 h, and metabolites in the TCA cycle reached isotopic steady state for [U-13C]glutamine within 3 h. 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 13C-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-13C]glucose confirmed that additional metabolites were accumulating in the medium that became M+3 and M+6 labeled.

► Metabolic fluxes in CHO cells were studied at growth phase and stationary phase. ► Parallel labeling experiments performed with [1,2-13C]glucose and [U-13C]glutamine. ► The new method for combined 13C-MFA was used to calculate fluxes. ► Fluxes changed for pentose phosphate pathway, anaplerosis, and amino acid metabolism. ► Fatty acid biosynthesis was active at growth phase and also at stationary phase.

Keywords
ACL, ATP citrate lyase; AT, aminotransferase; CHO, Chinese hamster ovary; MFA, metabolic flux analysis; EMU, elementary metabolite unit; GC–MS, gas chromatography–mass spectrometry; GDH, glutamate dehydrogenase; IDH, isocitrate dehydrogenase; ME, malic en
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Parallel labeling experiments with [1,2-13C]glucose and [U-13C]glutamine provide new insights into CHO cell metabolism
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Publisher
Database: Elsevier - ScienceDirect
Journal: Metabolic Engineering - Volume 15, January 2013, Pages 34–47
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us