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

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.