Hyperpolarization of [1-13C]Ketoisocaproate-d2 by Reversible Exchange with Parahydrogen Enables Profiling of Branched-Chain-Amino-Acid Metabolism in Cellulo and in Vivo.

Hyperpolarized 1 3C magnetic resonance imaging (MRI) is the only method to image metabolic fluxes in real time, non-invasively, and in vivo. To date, however, most studies have used [1-1 3C]pyruvate and dynamic nuclear polarization (dDNP). Here, we establish efficient hyperpolarization (HP) of protio and partially-deuterated [1-1 3C]ketoisocaproate (KIC) using Spin-Lock-Induced-Crossing-Signal Amplification by Reversible Exchange (SLIC-SABRE), a high-throughput, uncomplex and low-cost method based on parahydrogen. We demonstrate 13C polarization up to ≈28% and T1 relaxation times > 200 s at 1 T in methanol-d4. A rapid purification procedure allowed us to obtain biocompatible formulations with ≈11% 13C polarization at the time of injection, sufficient for in cellulo and in vivo studies. We found that branched-chain-amino-acid transaminase (BCAT) activity leads to HP [1-1 3C]leucine formation exclusively in BCAT1-high MDA-MB231 breast-cancer cells, but not in BCAT1-low MCF7 or PyB6 cells. In a proof-of-concept in vivo experiment, 13C magnetic resonance spectroscopy imaging of the healthy mouse brain detected [1-13C]leucine formation after intravenous injection of SABRE [1-13C]KIC-d2. Our results demonstrate that [1-13C]KIC-d2 provides a sensitive readout of BCAT1-dependent metabolism and that SLIC-SABRE can rapidly generate this probe for 13C MRI, extending accessible parahydrogen-based hyperpolarization to amino-acid pathways relevant to cancer biology and chemoresistance.