Hypersense polarizer

Unless otherwise noted, all chemicals and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as received. A mixture of pyridine (6.2 M), 2,6-lutidine (4.3 M), 2-picoline (5.0 M), nicotinamide (2.7 M) or 2,4,6-collidine (3.78 M) and BDPA radical (40 mM) dissolved in 50 μL of DMSO-sulfolane (1:1 v/v) was polarized in a HyperSense polarizer (3.35 T, Oxford Instruments Molecular Biotools, UK) according to the manufacturer's procedures. The polarization was carried out at ~1.05 K with 94.055 GHz microwave irradiation for 2 h. The dissolution liquid of HP agents in distilled water (4 mL) from the polarizer was rapidly mixed with a pre-determined volume of hydrochloric acid or sodium hydroxide in a 10-mm NMR tube to achieve the desired pH. ¹⁵N-NMR spectra were acquired at room temperature (~23 °C) on a 400-MHz spectrometer using a 5 degree flip angle with a repetition time (TR) of 5 s. All ¹⁵N peaks were externally referenced with ¹⁵N-nitrobenzene (372 ppm). ¹⁵N MRI were acquired on a 9.4 T Agilent vertical bore microimager (Agilent, USA). MR images and spectra were processed using ImageJ (NIH, USA) and ACD/SpecManager (ACD Labs, Canada). The T₁, signal enhancement and pH titration experiments were investigated with the natural abundant ¹⁵N compounds. ¹⁵N-labeled pyridine was used in T₁ measurement in plasma and CSI experiments.

[2-¹³C] pyruvic acid (Cambridge Isotope Laboratories, United States) was doped with 15 mM trityl radical (OX063; tris[8-carboxyl-2,2,6,6-tetra-[2-(1-hydroxyethyl)]-benzo-(1,2-d:4,5-d)-bis-(1,3)-dithiole-4-yl]-methyl sodium salt; Oxford Instruments, United Kingdom) and 1 mM ProHance. This sample was inserted into a HyperSense polarizer (Oxford Instruments, United Kingdom). The frozen sample was hyperpolarized at 1.4 K by applying microwave irradiation (94.116 GHz at 100 mW) until steady state polarization was achieved (~1–1.5 h).
The sample was dissolved using 4 ml of hot 20 mM PBS (pH 7.4) and rapidly transferred to a 14 T NMR magnet. 3 ml of the dissolved sample was mixed with 20 ml of Krebs–Henseleit electrolytes and injected directly into the perfused liver (nominal final concentration of HP pyruvate is 4 mM). Injection of HP substrate as a bolus was achieved with the use of long tubing running along the length of the glass perfusion column and terminating immediately above the catheter. Substrate enters the liver via the catheter placed in the portal vein. NMR signal acquisition was started prior to injection.

Hyperpolarization of neat ¹³C₁-pyruvic acid (Sigma Isotec) and 15 mM OX-063 Trityl radical (Oxford Instruments) was conducted at 95 GHz with 50 mW microwave powers in a Hypersense polarizer (Oxford Instruments) operating at 1.4 K. In the multi-bolus experiments the solution was stored at ∼1 T in the fringe field; signal lifetimes measured from the decay of the multi-bolus envelope revealed for this in vitro sample a T₁ = 62.89±6.71 s. In vivo hyperpolarized experiments were conducted by injecting 60 mM ¹³C₁-pyruvate neutralized to pH≈7.6, either in single or multi-bolus volumes as indicated in the text.