Multi-compound polarization by DNP allows simultaneous assessment of multiple enzymatic activities in vivo

Abstract

Methods for the simultaneous polarization of multiple 13C-enriched metabolites were developed to probe several enzymatic pathways and other physiologic properties in vivo, using a single intravenous bolus. A new method for polarization of 13C sodium bicarbonate suitable for use in patients was developed, and the co-polarization of 13C sodium bicarbonate and [1-(13)C] pyruvate in the same sample was achieved, resulting in high solution-state polarizations (15.7% and 17.6%, respectively) and long spin-lattice relaxation times (T1) (46.7 s and 47.7 s respectively at 3 T). Consistent with chemical shift anisotropy dominating the T1 relaxation of carbonyls, T1 values for 13C bicarbonate and [1-(13)C] pyruvate were even longer at 3 T (49.7s and 67.3s, respectively). Co-polarized 13C bicarbonate and [1-(13)C] pyruvate were injected into normal mice and a murine prostate tumor model at 3T. Rapid equilibration of injected hyperpolarized 13C sodium bicarbonate with 13C CO2 allowed calculation of pH on a voxel by voxel basis, and simultaneous assessment of pyruvate metabolism with cellular uptake and conversion of [1-(13)C] pyruvate to its metabolic products. Initial studies in a Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model demonstrated higher levels of hyperpolarized lactate and lower pH within tumor, relative to surrounding benign tissues and to the abdominal viscera of normal controls. There was no significant difference observed in the tumor lactate/pyruvate ratio obtained after the injection of co-polarized 13C bicarbonate and [1-(13)C] pyruvate or polarized [1-(13)C] pyruvate alone. The technique was extended to polarize four 13C labelled substrates potentially providing information on pH, metabolism, necrosis and perfusion, namely [1-(13)C]pyruvic acid, 13C sodium bicarbonate, [1,4-(13)C]fumaric acid, and 13C urea with high levels of solution polarization (17.5%, 10.3%, 15.6% and 11.6%, respectively) and spin-lattice relaxation values similar to those recorded for the individual metabolites. These studies demonstrated the feasibility of simultaneously measuring in vivo pH and tumor metabolism using nontoxic, endogenous species, and the potential to extend the multi-polarization approach to include up to four hyperpolarized probes providing multiple metabolic and physiologic measures in a single MR acquisition.

Figure 1

Dynamic hyperpolarized spectrum for a co-polarized solution of [1-¹³C] pyruvate and ¹³C sodium bicarbonate, with data collected at 11.7T. The final solution pH was 7.8–7.9. The mono-exponential decay for bicarbonate and pyruvate peaks were used to calculate the T₁’s.

Figure 2

In vivo data collected at 3T. A T₂ weighted anatomic image (A), pH image of the same slice demonstrating the distribution of calculated pH (B), spectra for the corresponding voxels shown in the T2 weighted image (C) and the lactate/pyruvate ratio image (D) are shown for a wildtype mouse. Following dissolution, 300 µL of a solution with 55 mM pre-polarized ¹³C bicarbonate and 10 mM ¹³C pyruvate were injected intravenously. Data were acquired with a resolution of 1 cm³. The pH for individual voxels was calculated using the Henderson-Hasselbalch equation and pK_a of 6.17 at 37° C. The peak ratios of observed ¹³C lactate to ¹³C pyruvate are shown beneath the corresponding spectra. Regions of the CO₂ resonance have been scaled up to demonstrate the signal-to-noise achieved in each voxel.

Figure 3

In vivo data collected at 3T. A T₂ weighted anatomic image (A), pH image of the same slice demonstrating the distribution of pH calculated (B), spectra for the corresponding voxels shown in the T2 weighted image (C) and the Lactate/Pyruvate ratio image (D) are shown for a TRAMP tumor bearing mouse. The tumor is visible in the upper half of the slice. Following dissolution, 300 µL of a solution 55 mM in pre-polarized ¹³C bicarbonate and 10 mM in ¹³C pyruvate were injected intravenously. Data was acquired with a resolution of 1 cm³. The pH for individual voxels was calculated using the Henderson-Hasselbalch equation and pK_a of 6.17 at 37° C. The peak ratios of observed ¹³C lactate to ¹³C pyruvate are also shown beneath the corresponding spectra. Regions of the CO₂ resonance have been scaled up to demonstrate the signal-to-noise achieved in each voxel.

Figure 4

(a) Multi-compound polarization of four ¹³C biomolecules of interest: fumarate, pyruvate, urea, and bicarbonate in vitro. These data were obtained in a 10 mm NMR tube 60s following dissolution at 11.7T. Levels of polarization achieved were similar to that obtained for the individual compounds, performed using an optimized microwave frequency. T₁’s in solution were similar to those obtained for the separate species. Standard deviations are reported (N=3). (b) Multi-compound polarization in vivo performed in a TRAMP mouse model at 3T.