Labile Photo-Induced Free Radical in α-Ketoglutaric Acid: a Universal Endogenous Polarizing Agent for In Vivo Hyperpolarized 13 C Magnetic Resonance

Abstract

Hyperpolarized (HP) ¹³ C magnetic resonance enables non-invasive probing of metabolism in vivo. To date, only ¹³ C-molecules hyperpolarized with persistent trityl radicals have been injected in humans. We show here that the free radical photo-induced in alpha-ketoglutaric acid (α-KG) can be used to hyperpolarize photo-inactive ¹³ C-molecules such as [1-¹³ C]lactate. α-KG is an endogenous molecule with an exceptionally high radical yield under photo-irradiation, up to 50 %, and its breakdown product, succinic acid, is also endogenous. This radical precursor therefore exhibits an excellent safety profile for translation to human studies. The labile nature of the radical means that no filtration is required prior to injection while also offering the opportunity to extend the ¹³ C relaxation time in frozen HP ¹³ C-molecules for storage and transport. The potential for in vivo metabolic studies is demonstrated in the rat liver following the injection of a physiological dose of HP [1-¹³ C]lactate.

Keywords: dynamic nuclear polarization; imaging agents; keto acids; metabolism; photochemistry.

Figure 1

Production of photo‐induced radicals in a solution of 80:20 lactic acid:water (w:w) and α‐KG. A. Radical concentration as a function of the photo‐irradiation time for 75 mM (green circles) and 300 mM (yellow diamonds) of α‐KG. The build‐up time constant was 66 s for the sample containing 300 mM α‐KG and 31 s for the 75 mM sample. The dashed lines show the exponential fit used to extract the radical build‐up times. B. Normalized X‐band ESR dispersion and absorption spectra for the photo‐generated radical in the 300 mM α‐KG sample at the 75 s time point.

Figure 2

¹³C MR spectra of diluted (3:50 v:v in H₂O) photo‐irradiated (blue) and non‐irradiated (orange) 80:20 [1‐¹³C]lactic acid:water (w:w) solution containing 300 mM [1‐¹³C]α‐KG. The [1‐¹³C]lactic acid resonance is the large peak at 183.2 ppm (natural abundance lactic acid C2 and C3 doublets are visible at 69.6 and 21 ppm, respectively). The [1‐¹³C]α‐KG resonance is at 170.8 ppm. Three peaks from lactic acid impurities were detected within the 177–180 ppm region. The inset highlights the ¹³CO₂ peak (127.6 ppm) that was only detected in the photo‐irradiated sample.

Figure 3

Microwave DNP spectrum of a photo‐irradiated 80:20 [1‐¹³C]lactic acid:water (w:w) solution containing 300 mM [1‐¹³C]α‐KG. The measurements were performed at 7 T and 1.35 K with (blue dots) and without (red diamonds) microwave frequency modulation. Microwave frequency was modulated with an amplitude of 52 MHz and a rate of 1.5 kHz.

Figure 4

¹³C polarization measured inside the polarizer and following dissolution. A) Solid‐state ¹³C polarization build‐up recorded in a photo‐irradiated 80:20 [1‐¹³C]lactic acid:water (w:w) sample containing 300 mM [1‐¹³C]α‐KG. B) Liquid‐state ¹³C polarization measured at 14.1 T. The first HP ¹³C spectrum recorded after sample transfer is displayed in the inset together with the thermally polarized ¹³C spectrum used to determine the liquid‐state ¹³C polarization. The integrals of the two peaks are 3.0±0.1 and 1.6±0.1 for the HP and thermally polarized signals, respectively.

Figure 5

HP ¹³C MR experiments in the rat liver in vivo. A. Sum of the ¹³C spectra acquired with a ¹³C surface coil placed on top of the rat liver region between 34 and 74 s following injection of a 1 mL 42 mM [1‐¹³C]lactate bolus via the tail vein. The inset spectrum highlights the area of interest containing the lactate metabolites.¹³C signals (numbered 1 to 6) from the injected [1‐¹³C]lactate (183.7 ppm–#1) and its metabolic products ([1‐¹³C]pyruvate (171 ppm–#5), [1‐¹³C]pyruvate hydrate (179 ppm–#2), [1‐¹³C]alanine (176.7 ppm–#3), [1‐¹³C]aspartate (175.2 ppm–#4), and [¹³C]bicarbonate (161 ppm–#6) were detected in all experiments. The [1‐³C]malate (180.6 ppm) signal should also be present but is obscured by the shoulder of the [1‐¹³C]lactate peak. B. Metabolic pathways showing the flow of ¹³C label from [1‐¹³C]lactate into the TCA cycle and gluconeogenesis pathway in the liver. ¹³C labelling from the injected lactate is first converted to C1 of pyruvate. The ¹³C labels highlighted in grey are the ones that can be expected to be observed in the HP ¹³C spectra. Enzymes are abbreviated in bold font: lactate dehydrogenase (LDH), alanine aminotransferase (ALT), pyruvate dehydrogenase (PDH), carbonic anhydrase (CA), pyruvate carboxylase (PC), malate dehydrogenase (MDH), fumarate hydratase (FH), aspartate aminotransferase (AST), and phosphoenolpyruvate carboxykinase (PEPCK).