Rapid 13C Hyperpolarization of the TCA Cycle Intermediate α-Ketoglutarate via SABRE-SHEATH

alpha-Ketoglutarate is a key biomolecule involved in a number of metabolic pathways-most notably the TCA cycle. Abnormal alpha-ketoglutarate metabolism has also been linked with cancer. Here, isotopic labeling was employed to synthesize [1-C-13,5-C-12,D-4]alpha- ketoglutarate with the future goal of utilizing its [1-C-13]-hyperpolarized state for real-time metabolic imaging of alpha-ketoglutarate analytes and its downstream metabolites in vivo. The signal amplification by reversible exchange in shield enables alignment transfer to heteronuclei (SABRE-SHEATH) hyperpolarization technique was used to create 9.7% [1-C-13] polarization in 1 minute in this isotopologue. The efficient C-13 hyperpolarization, which utilizes parahydrogen as the source of nuclear spin order, is also supported by favorable relaxation dynamics at 0.4 mu T field (the optimal polarization transfer field): the exponential C-13 polarization buildup constant T-b is 11.0 +/- 0.4 s whereas the C-13 polarization decay constant T-1 is 18.5 +/- 0.7 s. An even higher C-13 polarization value of 17.3% was achieved using natural abundance alpha-ketoglutarate disodium salt, with overall similar relaxation dynamics at 0.4 mu T field, indicating that substrate deuteration leads only to a slight increase (similar to 1.2-fold) in the relaxation rates for C-13 nuclei separated by three chemical bonds. Instead, the gain in polarization (natural abundance versus [1 -C-13]-labeled) is rationalized through the smaller heat capacity of the spin bath comprising available C-13 spins that must be hyperpolarized by the same number of parahydrogen present in each sample, in line with previous N-15 SABRE-SHEATH studies. Remarkably, the C-2 carbon was not hyperpolarized in both alpha-ketoglutarate isotopologues studied; this observation is in sharp contrast with previously reported SABRE SHEATH pyruvate studies, indicating that the catalyst-binding dynamics of C-2 in alpha-ketoglutarate differ from that in pyruvate. We also demonstrate that C-13 spectroscopic characterization of alpha-ketoglutarate and pyruvate analytes can be performed at natural C-13 abundance with an estimated detection limit of 80 micromolar concentration x *%P-13C. All in all, the fundamental studies reported here enable a wide range of research communities with a new hyperpolarized contrast agent potentially useful for metabolic imaging of brain function, cancer, and other metabolically challenging diseases.

Automated summary

Alpha-ketoglutarate is a vital biomolecule involved in various metabolic pathways, and abnormal metabolism of alpha-ketoglutarate is linked to cancer. This study successfully synthesized highly polarized alpha-ketoglutarate using isotopic labeling, which could potentially be used as a contrast agent for real-time metabolic imaging.