Influence of DNP Polarizing Agents on Biochemical Processes: TEMPOL in Transient Ischemic Stroke

Abstract

Hyperpolarization of ¹³C by dissolution dynamic nuclear polarization (dDNP) boosts the sensitivity of magnetic resonance spectroscopy (MRS), making possible the monitoring in vivo and in real time of the biochemical reactions of exogenously infused ¹³C-labeled metabolic tracers. The preparation of a hyperpolarized substrate requires the use of free radicals as polarizing agents. Although added at very low doses, these radicals are not biologically inert. Here, we demonstrate that the presence of the nitroxyl radical TEMPOL influences significantly the cerebral metabolic readouts of a hyperpolarized [1-¹³C] lactate bolus injection in a mouse model of ischemic stroke with reperfusion. Thus, the choice of the polarizing agent in the design of dDNP hyperpolarized MRS experiments is of great importance and should be taken into account to prevent or to consider significant effects that could act as confounding factors.

Keywords: Hyperpolarization; MRS; TEMPOL; dDNP; lactate; stroke.

Figure 1

(A–B) Representative dynamic cerebral ¹³C MRS acquired after a bolus infusion of [1-¹³C] lactate (lb = 20 Hz). The summed signal from the first 120 s post-infusion is plotted in blue. The vertical scale was normalized to the height of the summed HP lactate peak. In both groups (A–B), the HP [1-¹³C] lactate (183.5 ppm) was converted into [1-¹³C] pyruvate (171.1 ppm), [1-¹³C] alanine (176.7 ppm), and [¹³C] bicarbonate (161.2 ppm). The signal observed at 177.7 ppm (*), partially overlapping with the alanine peak at 176.7 ppm, is an impurity from the stock lactate solution. Representative axial T₂W images of the brain acquired at 1 and 2 h post-reperfusion. Images were acquired with a fast spin–echo multislice sequence (voxel size: 0.07 × 0.07 × 1 mm³, 4 averages). In both groups (A–B), the striatal lesion was slightly visible at 1 h post-reperfusion and clearly contrasted at 2 h post-reperfusion (white arrows).

Figure 2

Simplified schematic of the cerebral [1-¹³C] lactate metabolism. [1-¹³C] lactate can cross the blood–brain barrier (BBB) via monocarboxylate transporters (MCTs). The intracellular [1-¹³C] lactate and [1-¹³C] pyruvate pools are exchanged via lactate dehydrogenase (LDH). [1-¹³C] pyruvate is either converted into [1-¹³C] alanine by alanine aminotransferase (ALT) or transported into the mitochondria via mitochondrial pyruvate carriers (MPCs) and then oxidized by pyruvate dehydrogenase (PDH), producing ¹³CO₂ remaining in equilibrium with [¹³C] bicarbonate via carbonic anhydrase (CA).

Figure 3

Metabolite ratios measured following injection of HP [1-¹³C] lactate. Data are displayed as the mean ± standard deviation and overlaid with individual data points (black diamonds). Pyruvate-to-lactate ratio (PLR, A), alanine-to-lactate ratio (ALR, B), bicarbonate-to-lactate ratio (BLR, C), alanine-to-pyruvate ratio (APR, D), and bicarbonate-to-pyruvate ratio (BPR, E). The PLR was significantly lower in the MCAO group compared to the MCAO+TEMPOL group (A). A lower alanine labeling (ALR) was observed in the MCAO+TEMPOL group compared to the MCAO group (B). No changes were observed in the bicarbonate-to-lactate ratio (BLR, C).

Figure 4

Schematic representations of the chemical reactions potentially involved in the interpretation of the measured ¹³C NMR signals, with or without TEMPOL.