Ox063

[1-¹³C]pyruvate was polarized by dissolution dynamic nuclear polarization (DNP) as described in previous studies³⁶ . Briefly, pure [1-¹³C]pyruvic acid ( ≥ 99% enriched, Sigma ISOTEC, Miamisburg, OH) was mixed with 15 mM trityl radical OX063 and 1.0 mM Gd-DOTA (Guerbet, Roissy, France), and then polarized in a commercial HyperSense polarizer (Oxford Instruments, Tubney Woods, UK) operating at 3.35 Tesla, 94.1 GHz, and 1.3 K, for a period of approximately one hour. The polarized sample was rapidly dissolved in NaOH/Tris buffer to attain a neutral, room temperature 80 mM HP [1-¹³C]pyruvate solution, which was rapidly transported to the MRI scanner for injection.

27 mg [1,5-¹³C₂,3,6,6,6-D₄]zymonic acid [14 (link)], 1.7 mg Ox063 trityl radical (GE Healthcare, Chicago, IL, USA) and 24 µL DMSO were vortexed for 35 min. The mixture was added to a DNP sample cup and frozen in liquid nitrogen. 25 µL of a sample, containing 10 M ¹³C-urea in glycerol, 30 mM Ox063 and 1.5 mM DOTAREM (Guerbet, Villepinte, France) was subsequently added on top of the frozen layer and also frozen in liquid nitrogen. The sample cup was then polarized at 1.2 K for three hours by irradiation with a microwave frequency of 94.169 GHz and a power of 100 mW using a HyperSense® DNP Polarizer (Oxford Instruments, Abingdon, UK). Dissolution was performed with 2.99 ml D₂O containing 80 mM TRIS, 0.3 mM EDTA and 50 mM NaOH, resulting in solutions containing 50 mM hyperpolarized zymonic acid and 100 mM urea with a pH 6.7 ± 0.4.