Purified [1-13C]α-Ketoisocaproate Spin-Polarized Up to 39% with Parahydrogen Enables Real-Time Metabolism Studies in Cancer Cells

Abstract

Collecting real-time kinetic information about metabolism and downstream metabolites is an emerging field to study living cells and complex systems. Via hyperpolarization techniques in magnetic resonance (MR), signals of metabolites can be enhanced by orders of magnitude. Currently, the main constraints of hyperpolarization techniques are (1) achievable degree of polarization, (2) speed of polarization procedures, (3) availability of metabolites for biological or clinical utility, and/or (4) removal of undesired components (i.e., catalysts, radicals, solvents, etc.), with mainly [1-13C]pyruvate being investigated due to its high metabolic relevance and promising clinical translation. Parahydrogen-induced polarization (PHIP), as an advancing technique, uses parahydrogen (pH2), a spin isomer of hydrogen gas, to enhance signal intensities in a matter of seconds to minutes. Here, we introduce an efficient gas/liquid reaction to synthesize a precursor of [1-13C]α-ketoisocaproate-3,3-d3 (αKIC), namely vinyl-d3 [1-13C]α-ketoisocaproic acid-3,3-d2 (VαKIC). αKIC is one of the primary substrates of branched-chain amino acid aminotransferase (BCAT), which is relevant in tumors and neurodegenerative diseases. Hydrogenation of VαKIC with pH2 achieved up to 75% 13C spin polarization directly after spin order transfer and up to 39% polarization of αKIC (4.1 mM) in a biocompatible solution. These high levels of polarization enabled us to perform in vitro studies in human cancer cells and observe the production of the downstream metabolite leucine in real-time. We thereby expand the toolbox of rapidly generated hyperpolarized metabolites and foresee that our synthesis will be suitable to obtain other relevant precursors feasible for hyperpolarization using PHIP-SAH.