Fig. 3

Spatial and temporal compartmentalization of cellular metabolism may bias the estimation of whole-cell level fluxes. a Consider the case of a metabolite synthesized from two nutrients in media: A and B. Let us assume that feeding the cells with an isotopic form of B leads to an isotopic steady-state in which a small fraction of the intracellular metabolite pool is labeled. In this case, 13C-MFA would infer that the relative contribution of nutrient B to producing the metabolite is smaller than that of A. However, this might not be the case when considering spatial (b) and temporal (c) compartmentalization of metabolic activities. b Consider the case where the metabolite is synthesized mostly from nutrient B in mitochondria and at a lower rate from nutrient A in the cytosol. If the metabolite pool size is markedly larger in the cytosol, feeding cells with labeled nutrient B would lead to a small fraction of the whole-cell total metabolite pool to be isotopically labeled. c Consider the case where in a certain cell cycle phase (e.g., G2/M) the metabolite is rapidly synthesized and mostly from nutrient B, while in other phases (G1/S) it is slowly produced and mostly from A. now, if the metabolite pool size is markedly larger in G1/S, feeding a population of cells (homogenous in terms of cell cycle phase) with labeled nutrient B would lead to a small fraction of the total metabolite pool to be labeled