INTRACELLULAR FLUX ANALYSIS APPLIED TO THE EFFECT OF DISSOLVED-OXYGENON HYBRIDOMAS

Quantitative estimates of intracellular fluxes and measurements of int racellular concentrations were used to evaluate the effect of dissolve d oxygen (DO) concentration on CRL 1606 hybridoma cells in batch cultu re. The estimates of intracellular fluxes were generated by combining material balances with measurements of extracellular metabolite rates of change. Experiments were performed at DO levels of 60% and 1% air s aturation, as well as under oxygen-limited conditions. Cell extracts w ere analyzed to evaluate the effect of DO on the intracellular concent rations of the glutamate dehydrogenase reactants, as well as the redox state of the pyridine nucleotides in the cytosol and mitochondria. Th e relationship between cell density and pyridine nucleotide redox stat e was also investigated. Dissolved oxygen concentration had a signific ant effect on nitrogen metabolism and the flux through glutamate dehyd rogenase was found to reverse at low DO, favoring glutamate formation. The NAD in the cytosol and mitochondria was more reduced under low DO conditions while the cytosolic NAD was more oxidized at low DO. Cytos olic NAD was reduced at higher cell densities while the redox states o f cytosolic NADP and mitochondrial NAD did not exhibit significant var iation with cell density. These results point to the fundamental role of the intracellular oxidation/ reduction state in cell physiology and the possibility of controlling physiological processes through modula tion of the dissolved oxygen level or the oxidation/reduction potentia l of the culture.