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Per-deuteration does not affect metabolic rates of hyperpolarized pyruvate in the isolated heart
Alexander Funk1, Chalermchai Khemtong1, Nesmine Maptue1, Dean Sherry1,2, and Craig Malloy1,3

1UT Southwestern Medical Center, Dallas, TX, United States, 2UT Dallas, Richardson, TX, United States, 3Veterans Affairs North Texas Healthcare System, Dallas, TX, United States

Synopsis

The effects of deuteration in pyruvate on conversion to alanine and lactate, and flux into the TCA cycle were investigated by 13C NMR of tissue extracts from isolated hearts and studies of hyperpolarized [1-13C1, 3,3,3-2H3]pyruvate. No kinetic isotope effects were noted, but substantial exchange of water protons with deuterium was observed in alanine but not lactate or glutamate. Deuteration of pyruvate had little effect on metabolism in heart tissue, and the data suggests that formation of a Schiff base in alanine transaminase may occur without net interconversion of pyruvate and alanine.

Introduction

In recent years, deuteration of metabolites for hyperpolarization (HP) experiments has become increasingly popular. Since the 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 can influence fluxes in some metabolic pathways. The addition of the 2H label introduces further isotopomers that allows observation of more additional biochemical information, including the exchange processes between pyruvate, lactate (via LDH) and alanine (via ALT) as well as the kinetic isotope effects into pyruvate dehydrogenase (via PDH) (Figure 1). The 2H isotopomers are distinguishable due to chemical shift isotope effects in the 13C NMR spectrum. The conversion of pyruvate to alanine has shown a de-deuteration reaction in previous experiments of isolated cells, which is also expected here.[5]

Methods

Three set of isolated rat heart perfusions were performed to investigate the behavior of per-deuteration on metabolism. Set 1 contained a 1:1 mixture of [2-13C1,3,3,3-2H3]pyruvate and [U-13C3]pyruvate as an internal control and were perfused for 30 minutes to steady state. A control set contained [2-13C1]pyruvate. Set 2 were perfused with just [2-13C1,3,3,3-2H3]pyruvate for 3 or 6 minutes respectively. Set 3 were injected with hyperpolarized [1-13C1,3,3,3-2H3]pyruvate or [1-13C1]pyruvate as a control. Set 1 and 2 were analyzed as tissue extracts by 13C{1H} NMR, while set 3 were obtained directly over time after injection.

Results

The first set of perfusions showed no difference in the relative rates between metabolites derived from [2-13C1,3,3,3-2H3]pyruvate and [U-13C3]pyruvate, indicating no kinetic isotope effect is present in the metabolism of pyruvate to lactate, alanine or glutamate (Table 1). However, a substantial de-deuteration is observed for alanine but not lactate (Figure 2). The second set of perfusions further highlighted the de-deuteration of alanine. By supplying the heart with [2-13C1,3,3,3-2H3]pyruvate on the time scale of a hyperpolarization experiment, it was possible to observe the rate of de-deuteration between 3 and 6 minutes. In fact, the stage of de-deuteration of alanine at 6 minutes is almost identical to the distribution at 30 minutes, indicating that the de-deuteration happens mostly during the HP time course. A final set of experiments was performed to investigate the direct effect of per-deuteration on metabolism by injection of hyperpolarized [1-13C1,3,3,3-2H3]pyruvate. Even though the C1 resonances observed in the HP experiment is 3 bonds away, there is a small chemical shift isotope effect (Figure 3). The alanine resonance is broadened and actually shows a small shift change over time, due to the distribution of 2H isotopomers changing with the de-deuteration reaction happening in real time. Furthermore, the lactate to bicarbonate ratio was unaffected compared to a control, indicating no change in PDH flux and therefore, confirming the lack of kinetic isotope effects that were observed in the tissue extract experiments.

Discussion

Previous studies using per-deuterated glucose found significant kinetic isotope effects into alanine and glutamate. Since pyruvate is the product of glycolysis, our finding that there is little kinetic isotope effect of deuteration of exogenous pyruvate is somewhat unexpected. However, earlier studies have reported compartmentation of glycolytic metabolism separate from metabolism of exogenous pyruvate in the cytosol.[5,6] The de-deuteration of the metabolites observed here allowed insight into the mechanism of ALT. Traditionally; it is assumed that there is a linear mechanism from pyruvate to alanine, via a Schiff base intermediate. These data suggest, however, that the Schiff base reaction allows multiple de-deuteration per conversion of pyruvate to alanine (Figure 4). Additionally, it is believed that the availability of just one iso-enzyme of ALT in heart tissue allows the de-deuterated species to be limited to alanine and not exchange back to pyruvate and lactate.[4,7]

Conclusions

Overall, per-deuteration of pyruvate does not affect the relative rates of metabolism in isolated hearts, and, therefore, HP experiments with per-deuterated pyruvate are a valid alternative. However, de-deuteration can complicate analysis and broaden resonances in hyperpolarization experiments and other tissue types might experience

Acknowledgements

This project was supported by NIH P41EB015908.

References

(1) Jeffrey, F. M.; Rajagopal, A.; Malloy, C. R.; Sherry, A. D. 13C-NMR: A Simple yet Comprehensive Method for Analysis of Intermediary Metabolism. Trends Biochem. Sci. 1991, 16 (1), 5–10.

(2) Ardenkjær-Larsen, J. H.; Fridlund, B.; Gram, A.; Hansson, G.; Hansson, L.; Lerche, M. H.; Servin, R.; Thaning, M.; Golman, K. Increase in Signal-to-Noise Ratio of > 10,000 Times in Liquid-State NMR. PNAS 2003, 100 (18), 10158–10163.

(3) Vuichoud, B.; Milani, J.; Bornet, A.; Melzi, R.; Jannin, S.; Bodenhausen, G. Hyperpolarization of Deuterated Metabolites via Remote Cross-Polarization and Dissolution Dynamic Nuclear Polarization. J. Phys. Chem. B 2014, 118 (5), 1411–1415.

(4) Barb, A. W.; Hekmatyar, S. K.; Glushka, J. N.; Prestegard, J. H. Probing Alanine Transaminase Catalysis with Hyperpolarized 13CD3-Pyruvate. Journal of Magnetic Resonance 2013, 228, 59–65.

(5) Funk, A. M.; Anderson, B. L.; Wen, X.; Hever, T.; Khemtong, C.; Kovacs, Z.; Sherry, A. D.; Malloy, C. R. The Rate of Lactate Production from Glucose in Hearts Is Not Altered by Per-Deuteration of Glucose. Journal of Magnetic Resonance 2017, 284 (Supplement C), 86–93.

(6) Mallet, R. T. Pyruvate: Metabolic Protector of Cardiac Performance. Proceedings of the Society for Experimental Biology and Medicine 223 (2), 136–148.

(7) Taegtmeyer, H.; Peterson, M. B.; Ragavan, V. V.; Ferguson, A. G.; Lesch, M. De Novo Alanine Synthesis in Isolated Oxygen-Deprived Rabbit Myocardium. J. Biol. Chem. 1977, 252 (14), 5010–5018.

Figures

Figure 1: Illustration of the behavior of the 2H and 13C labels of [2-13C1,3,3,3-2H3]pyruvate.

Figure 2: 13C NMR spectrum of tissue extracts pefused with [2-13C1,3,3,3-2H3]pyruvate and [U-13C3]pyruvate highlighting a) lactate and b) alanine C2 resonance.

Figure 3: a) Stack of 13C NMR spectra acquired of isolated hearts injected with hyperpolarized [1-13C1,3,3,3-2H3]pyruvate showing a) the C1 resonances of lactate, alanine and bicarbonate as well as b) their chemical shift behavior over time.

Figure 4: Proposed mechanism for conversion of pyruvate to alanine in alanine transaminase (ALT). a) with the original linear mechanism, and b) with side reaction involving the Schiff base

Table 1: Overview of the relative concentrations of isotopomers of lactate, alanine and glutamate obtained through 13C and 1H NMR spectra of tissue extracts.

Proc. Intl. Soc. Mag. Reson. Med. 27 (2019)
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