Pentose phosphate pathway (PPP) plays an important role in cellular metabolism, e.g. for biosynthesis of RNA and DNA ribose moieties and generation of NADPH cofactors. However, due to the complexity of the pathway, including cycling and reversible reactions, quantification of PPP fluxes remains challenging using 13C-metabolic flux analysis (13C-MFA). Here, we estimated PPP metabolism in CHO cell culture using multiple isotopic tracers and mass spectrometry. To validate the metabolic model, we designed several 13C-glucose tracer mixtures and compared the result with the traditional [1,2-13C]glucose tracer. Importantly, we demonstrate loss of C3 fragment in the non-oxidative PPP of CHO cells. In addition, to quantify metabolic fluxes of PPP, we introduced key measurements in the glycolysis-related metabolites for 13C-MFA. In the end, we achieved consistent results for oxidative PPP and acquired narrow confidence intervals and good flux observability of transaldolase and transketolase reactions.

Pentose phosphate pathway metabolism of CHO cells elucidated using multiple isotopic tracers and mass spectrometry

245th National Meeting (2013)

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