Hyperpolarization of [1‐ 13 C]Ketoisocaproate‐d 2 by Reversible Exchange with Parahydrogen Enables Profiling of Branched‐Chain‐Amino‐Acid Metabolism in Cellulo and in Vivo

ABSTRACT Hyperpolarized 1 3 C 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 3 C]pyruvate and dynamic nuclear polarization (dDNP). Here, we establish efficient hyperpolarization (HP) of protio and partially‐deuterated [1‐ 1 3 C]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 13 C polarization up to ≈28% and T 1 relaxation times > 200 s at 1 T in methanol‐d 4 . A rapid purification procedure allowed us to obtain biocompatible formulations with ≈11% 13 C 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 3 C]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, 13 C magnetic resonance spectroscopy imaging of the healthy mouse brain detected [1‐ 13 C]leucine formation after intravenous injection of SABRE [1‐ 13 C]KIC‐d 2 . Our results demonstrate that [1‐ 13 C]KIC‐d 2 provides a sensitive readout of BCAT1‐dependent metabolism and that SLIC‐SABRE can rapidly generate this probe for 13 C MRI, extending accessible parahydrogen‐based hyperpolarization to amino‐acid pathways relevant to cancer biology and chemoresistance.