Figure 3. Experimental confirmation of differences in predictions between uFBA and FBA models.
(a) TCA metabolites and pathways in the RBC metabolic model are shown, including changes in metabolite levels and metabolites found to be isotopically labeled after addition of fully labeled 13C citrate. Cofactor producing reactions are shown, while other cofactors and reaction names are omitted. Concentrations are shown as μmol/L of bag or mmol/L of bag. Time spans 0–45 days for insets. (b) uFBA (blue arrow) predicts that the depletion of intracellular malate produces extracellular malate and fumarate, while driving lactate production. Extracellular citrate is used to produce glutamate and lactate. Fluxes shown in μM. (c) FBA (red arrow) predicts extracellular citrate is used only to produce malate and fumarate and that MDH proceeds in the opposite direction than in uFBA. (d) uFBA and FBA predicted fluxes were integrated with a 13C MFA “forward” tracer simulation to simulate how labeled citrate would accumulate across the first two metabolic states. The three intracellular metabolites outside of citrate that were observed to be labeled and were absolutely quantified are shown. As fully labeled citrate is used, the only labeled versions of lactate and glutamate are shown. The unlabeled fraction of the metabolite (not shown) is the remainder. For malate, there is a small percentage of m + 3 labeling (see Fig. S11), and the remainder is unlabeled. uFBA predicted more correct labeling patterns than FBA. This is quantitatively corroborated by the residual sum of squares (RSS) for each. Abbreviations: oaa: oxaloacetate; akg: alpha-ketoglutarate; pep: phosphoenolpyruvate; MDH: malate dehydrogenase; PYK: pyruvate kinase; ICDH: isocitrate dehydrogenase. Vertical lines on metabolite time profiles denote the time intervals of the three metabolic states.