4277
Pyruvate keto-enol tautomerization and its potential impact on apparent pyruvate T11Oxford Centre for Clinical Magnetic Resonance Research, University of Oxford, Headington, United Kingdom, 2Department of Physiology Anatomy & Genetics, University of Oxford, Oxford, United Kingdom, 3Wolfson Brain Imaging Centre, University of Cambridge, Cambridge, United Kingdom
Synopsis
Pyruvic acid is the most commonly studied metabolic agent for dynamic nuclear polarization. In aqueous solution, pyruvic acid can exist in three forms: hydrate (gem-diol), keto, and enol. The composition depends on pH. This study investigates the differences in longitudinal relaxation properties of the three tautomers with the objective of further understanding the factors contributing to the apparent T1 of pyruvic acid.
Introduction
Dynamic nuclear polarization (DNP) is a signal enhancement technique that can provide up to four orders of magnitude in 13C signal enhancement [1]. Pyruvic acid is a popular choice as a DNP agent because of its key metabolic functions, favourable chemical properties, and long T1. It is currently the only DNP agent approved for clinical studies [2].
As an alpha-keto acid, pyruvic acid in an aqueous solution can be deprotonated twice upon addition of base [3], as shown in Figure 1. At low pH, the keto form exists in rapid equilibrium with the hydrated (gem-diol) form through the addition of water. The keto form is favoured at physiological pH. Simultaneously, a chemical equilibrium exists between the keto and enol forms [4]. At high pH, a second deprotonation at the methyl group can occur, leaving a stabilized enol. This study examines the longitudinal relaxation properties of the different forms of pyruvate with the objective of further understanding the factors contributing to the apparent T1 of pyruvic acid.
Methods
An aqueous solution of 250 mM [1-13C]pyruvic acid, 250 mM [2-13C]pyruvic acid and 100 mM [13C]urea was placed in a 10 mm NMR tube inside a 1H/13C dual-tuned probe in a Varian 11.7 T vertical bore spectrometer. The pH was adjusted using 5 M NaOH and monitored using a pH probe. Thermal equilibrium 13C spectra were acquired at each pH examined (TR = 5 min, 90° tip, 2 averages, 30 kHz/24k points). An inversion recovery sequence was used to measure the T1 of each species at selected pH values (TR = 3 min, TI from 0.5 s to 120 s, 2 averages, 10 kHz/4096 points).Results
Representative 13C spectra at low, neutral, and high pH are shown in Figure 2. Enol resonances were identified at 185.7 ppm (C1 enol) and 77.1 ppm (C2 enol). Relative to the well known resonance frequencies at physiological pH, all resonances shift upfield at low and high pH. The fractional composition of the three forms of pyruvate is shown as a function of pH in Figure 3. The measured T1 for observable species at each pH value are summarized in Table 1.Discussion
As expected, the hydrate and keto forms are in rapid chemical exchange at low pH, resulting in both hydrate and keto forms exhibiting a similar T1. Approaching neutral pH, the T1 of both hydrate and keto resonances increase. The enol resonances are generally not observed in hyperpolarized experiments at physiological pH, presumably because of the short T1, but the enol form is observable above pH 6 with averaging at thermal equilibrium polarization. Keto-enol tautomerization may contribute to the apparent T1 of the keto form of pyruvic acid as the nuclei in the molecule experience more rapid T1 relaxation during the time spent in the enol form.
Under the current polarization protocol for human studies, the pyruvic acid is dissolved in hot unbuffered water for injection such that the AH111501 radical forms a precipitate that can be removed using a mechanical filter. However, the time spent at low pH may contribute a greater loss of polarization due to chemical exchange with the short-T1 hydrate species. Dissolution in strong base would not be appropriate either due to the short T1 of the enol form. Therefore, these results provide motivation for the development of a radical that can be phase separated at neutral pH to conserve polarization during the dissolution process.
Conclusion
Keto-enol tautomerization of pyruvic acid was observed above pH 6. The enol species is typically not observed in hyperpolarized 13C experiments due to its short T1 relative to the keto form, but the chemical exchange between the keto and enol forms should be taken into account in understanding the apparent pyruvate T1 at physiological pH.Acknowledgements
The authors would like to acknowledge the following sources of funding: NIHR Oxford Biomedical Research Centre (JYCL), Novo Nordisk Fellowship Programme (JJJJM), Medical Research Council (LAJY), British Heart Foundation (DJT), and Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society 098436/Z/12/B (CTR).
References
[1] Ardenkjær-Larsen JH, Fridlund B, Gram A, Hansson G, Hansson L, Lerche MH, Servin R, Thaning M, Golman K. Increase in signal-to-noise ratio of >10,000 times in liquid-state NMR. Proceedings of the National Academy of Sciences. 2003 Sep 2;100(18):10158-63.
[2] Nelson SJ, Kurhanewicz J, Vigneron DB, Larson PE, Harzstark AL, Ferrone M, van Criekinge M, Chang JW, Bok R, Park I, Reed G. Metabolic imaging of patients with prostate cancer using hyperpolarized [1-13C] pyruvate. Science Translational Medicine. 2013 Aug 14;5(198):198ra108-.
[3] Meany JE. Lactate dehydrogenase catalysis: roles of keto, hydrated, and enol pyruvate. Journal of Chemical Education. 2007 Sep;84(9):1520.
[4] Chiang Y, Kresge AJ, Pruszynski P. Keto-enol equilibria in the pyruvic acid system: determination of the keto-enol equilibrium constants of pyruvic acid and pyruvate anion and the acidity constant of pyruvate enol in aqueous solution. Journal of the American Chemical Society. 1992 Apr;114(8):3103-7.
Figures
