Hyperpolarized 13C-Substrate Preparation

Solutions of [1-¹³C]pyruvate (Cambridge Isotope Laboratories, MA, USA) and [1,3-¹³C]AcAc (Cambridge Isotope Laboratories, MA, USA) were prepared separately for polarization as previously described^{32 (link), 43 (link)}. Briefly, neat [1-¹³C]pyruvate was mixed with OX063 (15 mM) and gadoteridol ([Gd³⁺] = 2 mM)^{32 (link)}. A [1,3-¹³C₂]AcAc solution, prepared by hydrolysis of ethyl [1,3-¹³C₂]AcAc with 5 M NaOH at 45 °C for 45 min, was mixed with an equal volume of glycerol and added OX063 (15 mM) and gadoteridol ([Gd³⁺] = 2mM)^{43 (link)}. To be polarized, the ¹³C-pyruvate (4 µL) and ¹³C-acetoacetate (36 µL) solutions were sequentially added into a polarization sample cup placed in liquid nitrogen forming separate layers of the ¹³C-enriched substrates. The frozen solutions were then loaded into a 3.35T HyperSense polarizer (Oxford Instruments, UK) and polarized for 2 h at ~1.4 K with ~94.1 GHz microwave irradiation. The polarized sample was quickly dissolved in the superheated KH buffer (4 mL). Three milliliters of the co-polarized solution was mixed with additional KH buffer (20 mL) and injected directly into the perfused heart through a polyethylene catheter. Final concentrations for both ¹³C-pyruvate and ¹³C-acetoacetate injected into the heart were 2 mM.