Systematic comparison of local approaches for isotopically nonstationary metabolic flux analysis

Quantification of reaction fluxes of metabolic networks can help us understand how the integration of different metabolic pathways determine cellular functions. Yet, intracellular fluxes cannot be measured directly but are estimated with metabolic flux analysis (MFA) that relies on the patterns of isotope labeling of metabolites in the network. For metabolic systems, typical for plants, where all potentially labeled atoms effectively have only one source atom pool, only isotopically nonstationary MFA can provide information about intracellular fluxes. There are several global approaches that implement MFA for an entire metabolic network and estimate, at once, a steady-state flux distribution for all reactions with identifiable fluxes in the network. In contrast, local approaches deal with estimation of fluxes for a subset of reactions, with smaller data demand for flux estimation. Here we present a systematic comparative review and benchmarking of the existing local approaches for isotopically nonstationary MFA. The comparison is conducted with respect to the required data and underlying computational problems solved on a synthetic network example. Furthermore, we benchmark the performance of these approaches in estimating fluxes for a subset of reactions using data obtained from the simulation of nitrogen fluxes in the Arabidopsis thaliana core metabolism. The findings pinpoint practical aspects that need to be considered when applying local approaches for flux estimation in large-scale plant metabolic networks.