Metabolic flux analysis of hybridoma cells in different culture media using mass balances

Abstract

The estn. of the intracellular fluxes of mammalian cells using only the mass balances of the relevant metabolites is not possible because the set of linear equations defined by these mass balances is undetd. Either addnl. exptl. flux data or addnl. theor. constraints are required to find 1 unique flux distribution out of the soln. space that is bound by the mass balances. Here, a method is developed using the latter approach. The uptake and prodn. rates of amino acids, glucose, lactate, O2, CO2, NH4, MAB, and the intracellular amino acid pools were detd. for 2 different steady states. The cellular compn. (total protein and protein compn., total lipids and fatty acid distribution, total carbohydrates, DNA, and RNA) was measured to calc. the requirements for biosynthesis. It was essential to det. the uptake/prodn. rates of NH3 and either CO2 or O2. In mammalian cells these are cometabolites of cyclic metabolic pathways. The flux distribution that is found using the Euclidean min. norm as the addnl. theor. constraint and taking either the CO2 or the NAD(P)H mass balance into account is shown to be in agreement with the measured O2 and CO2 metabolic rates. The metabolic fluxes in hybridoma cells in continuous culture at a sp. growth rate of 0.83/day are estd. for a medium with (optimal medium) and without (suboptimal medium) Primatone RL, an enzymic hydrolyzate of animal tissue that causes a >2-fold increase in cell d. It is concluded that (i) the majority of the consumed glucose (>90%) is channeled through the pentose phosphate pathway in rapidly proliferating cells. (Ii) Pyruvate oxidn. and tricarboxylic acid (TCA) cycle activity are relatively low, i.e., 8% of the glucose uptake in suboptimal and 14% in optimal medium, resp. Under both conditions, only a small fraction of pyruvate is further oxidized to CO2. (Iii) The flux from glutamate to alpha-ketoglutarate (catalyzed by glutamate dehydrogenase) is almost zero in medium with and even slightly reversed in medium without Primatone RL. Almost all glutamate enters the TCA cycle due to the action of transaminases. (Iv) Transhydrogenation played a significant role in hybridoma cells under these exptl. conditions. NADPH was produced at relatively high rates (11 * 10-12 to 13 * 10-12 mol/cell-day) compared to other fluxes in both culture media. [on SciFinder (R)