Isotope tracing is the process of using metabolites with elements of different isotopes in order to study various aspects of metabolism, such as mechanisms and flux. Isotopes, such as 13C and 2H, are incorporated into metabolites in specific positions of the molecule; these metabolic reactions go unaffected since only the number of neutrons are affected. After feeding the isotope marked metabolites to the cells, the cells are incubated, the products are extracted, separated out using chromatography, and analyzed using nuclear magnetic resonance or mass spectrometry, in which isotopes within the metabolic products can be recognized through their difference in mass or vibrational mode. By identifying the locations of the isotopes in the products, one can track how the atoms move through the metabolic pathway and possibly infer the mechanisms involved. Isotope tracing can be used for metabolic flux analysis in order to determine the flux during certain parts of the metabolic pathway. By providing a ratio of marked and unmarked metabolites, one can calculate flux values for parts of a reaction based on the ratio of marked to unmarked products and the stoichiometry of the metabolic reactions. For such experiments, it is useful to control certain metabolites of interest (keep concentrations constant) so that responses to certain perturbations can be clearly characterized.
Austin Batugal