¹³C NMR of glutamate for monitoring the pentose phosphate pathway in myocardium

After administration of ¹³C-labeled glucose, the activity of the pentose phosphate pathway (PPP) is often assessed by the distribution of ¹³C in lactate. However, in some tissues, such as the well-oxygenated heart, the concentration of lactate may be too low for convenient analysis by NMR. Here, we examined ¹³C-labeled glutamate as an alternative biomarker of the PPP in the heart. Isolated rat hearts were perfused with media containing [2,3-¹³C₂]glucose and the tissue extracts were analyzed. Metabolism of [2,3-¹³C₂]glucose yields [1,2-¹³C₂]pyruvate via glycolysis and [2,3-¹³C₂]pyruvate via the PPP. Pyruvate is in exchange with lactate or is further metabolized to glutamate through pyruvate dehydrogenase and the TCA cycle. A doublet from [4,5-¹³C₂]glutamate, indicating flux through the PPP, was readily detected in ¹³C NMR of heart extracts even when the corresponding doublet from [2,3-¹³C₂]lactate was minimal. Benfotiamine, known to induce the PPP, caused an increase in production of [4,5-¹³C₂]glutamate. In rats receiving [2,3-¹³C₂]glucose, brain extracts showed well-resolved signals from both [2,3-¹³C₂]lactate and [4,5-¹³C₂]glutamate in ¹³C NMR spectra. Assessment of the PPP in the brain based on glutamate had a strong linear correlation with lactate-based assessment. In summary, ¹³C NMR analysis of glutamate enabled detection of the low PPP activity in isolated hearts. This analyte is an alternative to lactate for monitoring the PPP with the use of [2,3-¹³C₂]glucose.