Introduction

Stable isotopes are widely used to investigate metabolism. Since many applications are restricted by the poor sensitivity of ¹³C and the low concentration of metabolic products, there is strong interest in hyperpolarization methods which temporarily increase ¹³C polarization by 10,000-fold or more. In recent years, deuteration of metabolites for hyperpolarization experiments has become increasingly popular. Since T₁ decay of ¹³C polarization limits the duration of data acquisition, incorporation of ²H prolongs the T₁ of ¹³C in some molecules, enabling a longer period of observation[1-2]. In particular, perdeuterated glucose and perdeuterated pyruvate have been of interest [3,4].However, ²H significantly influences fluxes in some metabolic pathways. The effect of perdeuteration of glucose on the relative fluxes of glycolysis has been examined. No kinetic isotope effect was observed from glucose to lactate, but a slowing of relative flux was observed into alanine and glutamate. In addition, a wealth of new information was introduced into the system by the formation of deuterated isotopomers, which due to chemical shift isotope effects and extensive quadrupolar couplings allowed additional insight into pyruvate compartmentation and proton-deuteron displacement in alanine transaminase[4]. The effect of deuteration on pyruvate metabolism has not been examined in intact tissues. Here, we investigated the relative rates of metabolism of [2-¹³C₁,3,3,3-²H₃]pyruvate through alanine aminotransferase, pyruvate dehydrogenase and lactate dehydrogenase in the isolate rat heart.

Methods

Metabolism of deuterated metabolites were investigated in perfused rat hearts in two studies. A 1:1 mixture of [U-¹³C₃]pyruvate and [2-¹³C₁, 3,3,3-²H₃]pyruvate (n=3) or [U-¹³C₃]pyruvate as a control experiment were used (n=3) . Tissue extracts were analyzed by ¹H, ²H and ¹³C NMR spectroscopy. The exensive coupling of the ¹³C resonances to the quadrupolar deuterium nuclei produces complicated multiplet spectra in glutamate. Therefore, the ¹³C label was chosen to be in C2 position, to avoid direct coupling and enable a simple analysis of glutamate C5. However, a through-bond chemical shift isotope effect can be expected on the C2 position of lactate and alanine, allowing analysis of the ¹³C and ²H labeling in the intracellular pyruvate pool.

Results & Discussion

In this experimental design, [U-¹³C₃]pyruvate was used as an internal control and was present in all experiments. The effects of deuteration on metabolism of [2-¹³C₁]pyruvate were examined. The labeling pattern of alanine and lactate were both used to monitor the labeling of intracellular pyruvate. The proton-decoupled ¹³C NMR spectrum of the control hearts behaved as expected and showed the 1:1 ratio of singlet (from [2-¹³C₁]pyruvate) and quartets (from [U-¹³C₃]pyruvate) in the C2 of both lactate and alanine. A total of seven ¹³C-labelled isotopomers can be expected in the deuterated experiment (Figure 1). In the ¹³C NMR spectrum derived from the hearts treated with deuterated pyruvate, the C2 of both lactate and alanine show four singlets of varying intensity and also the quartet derived from [U-¹³C₃]pyruvate (Figure 2). The four singlets represent the four possible species of [2-¹³C₁,3,3,3-²H₃]pyruvate, where one, two or three ²H nuclei are replaced by protons and are shifted according to the number of neighboring ²H nuclei. The distribution of species was calculated as previously reported[4]. The resulting distribution between all possible isotopomers for lactate and alanine is shown in Table 1, confirming the deuterium exchange with solvent protons in alanine aminotransferase. The contribution of [2-¹³C₁]pyruvate relative to [U-¹³C₃]pyruvate in the intracellular pyruvate pool was not sensitive to deuteration. However, in the presence of deuterium, the contribution of endogenous glycogen to the intracellular pyruvate pool was increased with a range from 0.19 to 0.32 in lactate and from 0.14 to 0.25 in alanine. The analysis of glutamate was performed at the C5 position (singlet vs doublet) and followed the 1:1 ratio observed in lactate and alanine, meaning PDH flux is not inhibited by the deuteration. Interestingly, the chemical shift isotope effect is often regarded as going upfield. Here, the shift effect has a different sign and is observed downfield (Figure 3), which is likely due to the carbonyl group in glutamate C5.[5]

Conclusion

The potential use of perdeuterated pyruvate in hyperpolarization experiments was investigated. Similar to the effects seen when using perdeuterated glucose, the exchange between alanine and pyruvate through alanine aminotransferase introduces exchange of ²H with solvent ¹H. However, little effect was observed on relative fluxes into lactate and alanine. The glutamate C5 analysis showed an unusual chemical shift isotope effect, but it follows the same pattern as lactate and alanine. Overall, there is no observable kinetic isotope effect in lactate, alanine and glutmate, confirming the validity for the use of deuterated pyruvate for hyperpolarization experiments.

Acknowledgements

This project was supported by NIH P41EB015908 and R37HL034557.