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Metabolic-flux analysis of hybridoma cells under oxidative and reductive stress using mass balances

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Abstract

Hybridoma cells were grown at steady state under both reductiveand oxidative stress and the intracellular fluxes weredetermined by mass-balancing techniques. By decreasing the dissolved oxygen pressure (pO2) in the bioreactor, the reduced formof nicotinamide adenine nucleotide (NADH) was enhanced relativeto the oxidized form (NAD+). Oxidative stress, as a resultof which the NAP(P)+/NAD(P)H-ratio increases, was generatedby both the enhancement of the pO2 to 100% air saturationand by the addition of the artificial electron acceptorphenazine methosulphate (PMS) to the culture medium. It wasfound that fluxes of dehydrogenase reactions by which NAD(P)H isproduced decreased under hypoxic conditions. For example, thedegradation rates of arginine, isoleucine, lysine and theglutamate dehydrogenase flux were significantly lower at oxygenlimitation, and increased at higher pO2 levels and when PMSwas added to the culture medium. In contrast, the prolinesynthesis reaction, which requires NADPH, decreased under PMSstress. The flux of the NADH-requiring lactate dehydrogenase reaction also strongly decreased from 19 to 3,4 pmol/cell/day,under oxygen limitation and under PMS stress, respectively. Thedata show that metabolic-flux balancing can be used to determinehow mammalian respond to oxidative and reduction stress.

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Author notes

  1. Hendrik P.J. Bonarius

    Present address: Department of Cell Biology, Novo Nordisk Ltd., HAB 1.094, Niels Steensenvej 1, 2820, Gentofte, Denmark)

  2. José H.M. Houtman

    Present address: Bio R&D, Fort Dodge Animal Health Holland, Weesp, The Netherlands

Authors and Affiliations

  1. Department PRF-Biotechnology, Hoffman-La Roche Ltd., Bldg. 66/112, CH-4002, Basel, Switzerland

    Hendrik P.J. Bonarius

  2. Department PRF-Biotechnology, Hoffman-La Roche Ltd., Bldg. 66/112, CH-4002, Basel, Switzerland

    José H.M. Houtman & Georg Schmid

  3. Department of Food Science, Food and Bioengineering Group, Wageningen Agricultural University, P.O. Box 8129, 6700 EV, Wageningen, The Netherlands

    Cornelis D. de Gooijer & Johannes Tramper

Authors
  1. Hendrik P.J. Bonarius
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  2. José H.M. Houtman
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  3. Georg Schmid
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  4. Cornelis D. de Gooijer
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  5. Johannes Tramper
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Bonarius, H.P., Houtman, J.H., Schmid, G. et al. Metabolic-flux analysis of hybridoma cells under oxidative and reductive stress using mass balances. Cytotechnology 32, 97–107 (2000). https://doi.org/10.1023/A:1008142218103

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