Using multiple tracers for 13C metabolic flux analysis.

(13)C-Metabolic flux analysis ((13)C-MFA) is a powerful technique for quantifying intracellular metabolic fluxes in living cells. These in vivo fluxes provide important information on the physiology of cells in culture, which can be used for metabolic engineering purposes and serve as inputs for systems biology modeling. The (13)C-MFA technique consists of several steps: (1) selecting appropriate tracers for a given system of interest, (2) performing isotopic labeling experiments, (3) measuring isotopic labeling distributions in metabolic products, (4) estimating metabolic fluxes using least-squares regression, and (5) evaluating the goodness of fit and computing confidence intervals for estimated fluxes. In this chapter, we provide guidelines for performing (13)C-MFA studies using multiple isotopic tracers, a technique that is especially useful for elucidating fluxes in complex biological systems where multiple carbon sources are present. Here, as an example, we describe key steps and decision points for designing (13)C-MFA studies for microbes grown on mixtures of glucose and xylose. The general concepts described in this chapter are applicable to many other biological systems. For example, the same procedures can be applied to design (13)C-MFA studies in mammalian cells, which are generally grown in complex media containing multiple substrates such as glucose and amino acids.