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

Hybridoma cells were grown at steady state under both reductive and oxidative stress and the intracellular fluxes were determined by mass-balancing techniques. By decreasing the dissolved oxygen pressure (pO(2)) in the bioreactor, the reduced form of nicotinamide adenine nucleotide (NADH) was enhanced relative to the oxidized form (NAD(+)). Oxidative stress, as a result of which the NAP(P)(+)/NAD(P)H-ratio increases, was generated by both the enhancement of the pO(2) to 100% air saturation and by the addition of the artificial electron acceptor phenazine methosulphate (PMS) to the culture medium. It was found that fluxes of dehydrogenase reactions by which NAD(P)H is produced decreased under hypoxic conditions. For example, the degradation rates of arginine, isoleucine, lysine and the glutamate dehydrogenase flux were significantly lower at oxygen limitation, and increased at higher pO(2) levels and when PMS was added to the culture medium. In contrast, the proline synthesis reaction, which requires NADPH, decreased under PMS stress. 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. The data show that metabolic-flux balancing can be used to determine how mammalian respond to oxidative and reduction stress.