In Vivo Metabolic Imaging of [1-13 C]Pyruvate-d3 Hyperpolarized By Reversible Exchange With Parahydrogen.

Metabolic magnetic resonance imaging (MRI) using hyperpolarized (HP) pyruvate is becoming a non-invasive technique for diagnosing, staging, and monitoring response to treatment in cancer and other diseases. The clinically established method for producing HP pyruvate, dissolution dynamic nuclear polarization, however, is rather complex and slow. Signal Amplification By Reversible Exchange (SABRE) is an ultra-fast and low-cost method based on fast chemical exchange. Here, for the first time, we demonstrate not only in vivo utility, but also metabolic MRI with SABRE. We present a novel routine to produce aqueous HP [1-13 C]pyruvate-d3 for injection in 6 minutes. The injected solution was sterile, non-toxic, pH neutral and contained ≈30 mM [1-13 C]pyruvate-d3 polarized to ≈11 % (residual 250 mM methanol and 20 µM catalyst). It was obtained by rapid solvent evaporation and metal filtering, which we detail in this manuscript. This achievement makes HP pyruvate MRI available to a wide biomedical community for fast metabolic imaging of living organisms.

Real-Time Pyruvate Chemical Conversion Monitoring Enabled by PHIP.

In recent years, parahydrogen-induced polarization side arm hydrogenation (PHIP-SAH) has been applied to hyperpolarize [1-13C]pyruvate and map its metabolic conversion to [1-13C]lactate in cancer cells. Developing on our recent MINERVA pulse sequence protocol, in which we have achieved 27% [1-13C]pyruvate carbon polarization, we demonstrate the hyperpolarization of [1,2-13C]pyruvate (∼7% polarization on each 13C spin) via PHIP-SAH. By altering a single parameter in the pulse sequence, MINERVA enables the signal enhancement of C1 and/or C2 in [1,2-13C]pyruvate with the opposite phase, which allows for the simultaneous monitoring of different chemical reactions with enhanced spectral contrast or for the same reaction via different carbon sites. We first demonstrate the ability to monitor the same enzymatic pyruvate to lactate conversion at 7T in an aqueous solution, in vitro, and in-cell (HeLa cells) via different carbon sites. In a second set of experiments, we use the C1 and C2 carbon positions as spectral probes for simultaneous chemical reactions: the production of acetate, carbon dioxide, bicarbonate, and carbonate by reacting [1,2-13C]pyruvate with H2O2 at a high temperature (55 °C). Importantly, we detect and characterize the intermediate 2-hydroperoxy-2-hydroxypropanoate in real time and at high temperature.

Metabolic Tumor Imaging with Rapidly Signal-Enhanced 1-13 C-Pyruvate-d3.

The metabolism of malignant cells differs significantly from that of healthy cells and thus, it is possible to perform metabolic imaging to reveal not only the exact location of a tumor, but also intratumoral areas of high metabolic activity. Herein, we demonstrate the feasibility of metabolic tumor imaging using signal-enhanced 1-13 C-pyruvate-d3 , which is rapidly enhanced via para-hydrogen, and thus, the signal is amplified by several orders of magnitudes in less than a minute. Using as a model, human melanoma xenografts injected with signal-enhanced 1-13 C-pyruvate-d3, we show that the conversion of pyruvate into lactate can be monitored along with its kinetics, which could pave the way for rapidly detecting and monitoring changes in tumor metabolism.

A Field-Independent Method for the Rapid Generation of Hyperpolarized [1-13 C]Pyruvate in Clean Water Solutions for Biomedical Applications.

Hyperpolarization methods in magnetic resonance enhance the signals by several orders of magnitude, opening new windows for real-time investigations of dynamic processes in vitro and in vivo. Here, we propose a field-independent para-hydrogen-based pulsed method to produce rapidly hyperpolarized 13 C-labeled substrates. We demonstrate the method by polarizing the carboxylic carbon of the pyruvate moiety in a purposely designed precursor to 24 % at ≈22 mT. Following a fast purification procedure, we measure 8 % polarization on free [1-13 C]pyruvate in clean water solutions at physiological conditions at 7 T. The enhanced signals allow real-time monitoring of the pyruvate-lactate conversion in cancer cells, demonstrating the potential of the method for biomedical applications in combination with existing or developing magnetic resonance technologies.

Signal-enhanced real-time magnetic resonance of enzymatic reactions at millitesla fields.

The phenomenon of nuclear magnetic resonance (NMR) is widely applied in biomedical and biological science to study structures and dynamics of proteins and their reactions. Despite its impact, NMR is an inherently insensitive phenomenon and has driven the field to construct spectrometers with increasingly higher magnetic fields leading to more detection sensitivity. Here, we are demonstrating that enzymatic reactions can be followed in real-time at millitesla fields, three orders of magnitude lower than the field of state-of-the-art NMR spectrometers. This requires signal-enhancing samples via hyperpolarization. Within seconds, we have enhanced the signals of 2-13C-pyruvate, an important metabolite to probe cancer metabolism, in 22 mM concentrations (up to 10.1% ± 0.1% polarization) and show that such a large signal allows for the real-time detection of enzymatic conversion of pyruvate to lactate at 24 mT. This development paves the pathways for biological studies in portable and affordable NMR systems with a potential for medical diagnostics.

Production of highly concentrated and hyperpolarized metabolites within seconds in high and low magnetic fields.

Hyperpolarized metabolites are very attractive contrast agents for in vivo magnetic resonance imaging studies enabling early diagnosis of cancer, for example. Real-time production of concentrated solutions of metabolites is a desired goal that will enable new applications such as the continuous investigation of metabolic changes. To this end, we are introducing two NMR experiments that allow us to deliver high levels of polarization at high concentrations (50 mM) of an acetate precursor (55% 13C polarization) and acetate (17% 13C polarization) utilizing 83% para-state enriched hydrogen within seconds at high magnetic field (7 T). Furthermore, we have translated these experiments to a portable low-field spectrometer with a permanent magnet operating at 1 T. The presented developments pave the way for a rapid and affordable production of hyperpolarized metabolites that can be implemented in e.g. metabolomics labs and for medical diagnosis.

Pulsed Magnetic Resonance to Signal-Enhance Metabolites within Seconds by utilizing para-Hydrogen.

Diseases such as Alzheimer's and cancer have been linked to metabolic dysfunctions, and further understanding of metabolic pathways raises hope to develop cures for such diseases. To broaden the knowledge of metabolisms in vitro and in vivo, methods are desirable for direct probing of metabolic function. Here, we are introducing a pulsed nuclear magnetic resonance (NMR) approach to generate hyperpolarized metabolites within seconds, which act as metabolism probes. Hyperpolarization represents a magnetic resonance technique to enhance signals by over 10 000-fold. We accomplished an efficient metabolite hyperpolarization by developing an isotopic labeling strategy for generating precursors containing a favorable nuclear spin system to add para-hydrogen and convert its two-spin longitudinal order into enhanced metabolite signals. The transfer is performed by an invented NMR experiment and 20 000-fold signal enhancements are achieved. Our technique provides a fast way of generating hyperpolarized metabolites by using para-hydrogen directly in a high magnetic field without the need for field cycling.