Hyperpolarized ¹³C allows a direct measure of flux through a single enzyme-catalyzed step by NMR

¹³C NMR is a powerful tool for monitoring metabolic fluxes in vivo. The recent availability of automated dynamic nuclear polarization equipment for hyperpolarizing ¹³C nuclei now offers the potential to measure metabolic fluxes through select enzyme-catalyzed steps with substantially improved sensitivity. Here, we investigated the metabolism of hyperpolarized [1-¹³C₁]pyruvate in a widely used model for physiology and pharmacology, the perfused rat heart. Dissolved ¹³CO₂, the immediate product of the first step of the reaction catalyzed by pyruvate dehydrogenase, was observed with a temporal resolution of ≈1 s along with H¹³CO₃^-, the hydrated form of ¹³CO₂ generated catalytically by carbonic anhydrase. In hearts presented with the medium-chain fatty acid octanoate in addition to hyperpolarized [1-¹³C₁]pyruvate, production of ¹³CO₂ and H¹³CO₃^- was suppressed by ≈90%, whereas the signal from [1-¹³C ₁]lactate was enhanced. In separate experiments, it was shown that O₂ consumption and tricarboxylic acid (TCA) cycle flux were unchanged in the presence of added octanoate. Thus, the rate of appearance of ¹³CO₂ and H¹³CO₃^- from [1-¹³C₁]pyruvate does not reflect production of CO ₂ in the TCA cycle but rather reflects flux through pyruvate dehydrogenase exclusively.