NMR Methods for Quantitative Isotopomer Rates in Real-Time Metabolism of Cells

Tracer-based metabolism is becoming increasingly important to study metabolic mechanisms in cells. NMR offers several approaches to measure label incorporation in metabolites, including 13 C and 1 H-detected spectra. The latter are generally more sensitive but quantification depends on the proton carbon 1 J CH coupling constant which varies significantly between different metabolites. It is therefore not possible to have one experiment optimised for all metabolites and quantification of 1 H-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. Here we compare 13 C-filtered and 13 C-edited methods for quantification with a special focus towards application in real-time NMR of cancer cells under near-physiological conditions. We find an approach using a double-filter most suitable and sufficiently robust to reliably obtain 13 C-incorporations from difference spectra. This is demonstrated for JJN3 multiple myeloma cells processing glucose over 24h.