Hypersense dissolution dnp system

Twenty-six mg of [1-¹³-C]-pyruvic acid (Cambridge Isotopes)
containing 1.5 mM Gadoteridol (Bracco Diagnostics) and 15 mM OX063 (GE
Healthcare) were polarized using a HyperSense dissolution DNP system (Oxford
Instruments). The polarized substrate was rapidly dissolved in 4 mL of 80mM
NaOH, 50 mM NaCl, 0.1 g/L EDTA and 40 mM Trizma pH 7.6 (Sigma Aldrich) to yield
a neutral, isotonic, 37°C solution of 80mM hyperpolarized
[1-¹³C] pyruvate. Two hundred μL of the hyperpolarized
pyruvate solution was administered to the animals via a tail vein catheter.
Imaging was performed on a 7T Biospec MR system with a 1H/13C dual-tuned
volume coil (Bruker Biospin MRI) and a custom built 13C surface coil. Anatomic
imaging was performed using a fast spin-echo (FSE) sequence. Dynamic 13C data
was acquired using a previously described radial multi-band frequency encoding
sequence^{65 (link)} and a
constrained reconstruction algorithm^{66 (link)}. The constrained reconstruction results were normalized
and relative signal curves for hyperpolarized pyruvate and lactate were
generated as well as apparent relative metabolic conversion rate maps which were
co-registered with the anatomic images.

Hyperpolarized [1-¹³C]-pyruvate was prepared using a HyperSense dissolution DNP system (Oxford Instruments). 26mg aliquots of [1-¹³C]-pyruvic acid (Cambridge Isotopes) containing 1.5mM Gadoteridol (Bracco Diagnostics) and 15mM OX63 (GE Healthcare) were cooled to 1.45K in a 3.35T magnetic field, and irradiated at approximately 94.13 GHz for 45min or until solid-state polarization levels reached a plateau. The frozen substrate was then dissolved in 4mL of 180°C distilled water containing 80mM NaOH, 50 mM NaCl, 0.1 g/L EDTA, and 80mM Trizma pre-set crystals (pH 7.6), resulting in a 37°C isotonic solution containing 80mM HP [1-¹³C]-pyruvate. Once dissolution was complete, 200uL of the solution was drawn and administered to animals via tail-vein catheter.

The sodium salt of [U-¹³C]αKB (Cambridge Isotopes Laboratories, Andover, MA) was dissolved to a concentration of 3.4M in 50/50 glycerol/water (vol/vol), and mixed with trityl radical OX063 (15mM) (Oxford Instruments, Abingdon, UK) as well as Gd-DOTA (1.0mM) (Guerbet, Roissy, France). For each experiment, ~60mg of this mixture was polarized using a HyperSense dissolution DNP system (Oxford Instruments) and rapidly dissolved in 1X phosphate buffered saline (PBS) to yield 40mM solutions of HP [U-¹³C]αKB. For comparison, [1,2-¹³C]pyruvic acid was also polarized using similar methods to also yield 40mM solutions of HP [1,2-¹³C]pyruvate (with similar pH, close to neutral). Although only the C₁ resonances of both molecules were directly relevant to this study, doubly labeled pyruvate was used to produce a similar appearance of the NMR spectra as [U-¹³C]αKB, which is the form of ¹³C-labeled αKB that was readily available commercially. Hyperpolarized C₃ and C₄ resonances of αKB were not observable due to their very short T₁ relaxation times due to directly attached protons.