Quantitative Isotopomer Rates in Real‐Time Metabolism of Cells Determined by NMR Methods

Tracer‐based metabolism is becoming increasingly important for studying metabolic mechanisms in cells. NMR spectroscopy offers several approaches to measure label incorporation in metabolites, including 13C‐ and 1H‐detected spectra. The latter are generally more sensitive, but quantification depends on the proton–carbon 1JCH coupling constant, which varies significantly between different metabolites. It is therefore not possible to have one experiment optimised for all metabolites, and quantification of 1H‐edited spectra such as HSQCs requires precise knowledge of coupling constants. Increasing interest in tracer‐based and metabolic flux analysis requires robust analyses with reasonably small acquisition times. Herein, we compare 13C‐filtered and 13C‐edited methods for quantification and show the applicability of the methods for real‐time NMR spectroscopy of cancer‐cell metabolism, in which label incorporations are subject to constant flux. We find an approach using a double filter to be most suitable and sufficiently robust to reliably obtain 13C incorporations from difference spectra. This is demonstrated for JJN3 multiple myeloma cells processing glucose over 24 h. The proposed method is equally well suited for calculating the level of label incorporation in labelled cell extracts in the context of metabolic flux analysis. Filtering out the flux: The quantification of 1H‐detected 13C‐edited NMR spectra depends on a coupling constant that varies significantly between metabolites. Therefore, we have compared 13C‐filtered and 13C‐edited quantification methods and show their ability to robustly quantify 13C‐label incorporation in real‐time for cancer‐cell metabolism, in which label incorporation is subject to constant flux.