Nuclear Magnetic Resonance (NMR) Spectroscopy has a high diagnostic value and allows for the detection of metabolites and characterization of metabolism in vivo [1–4]. However, it also suffers from low sensitivity, and the tracking of metabolic kinetics in vivo is not possible with standard approaches that require a large number of averages to obtain signals above the noise level. To overcome this challenge, hyperpolarization techniques were introduced enhancing NMR signals by over four orders of magnitude . Such large signal enhancements enable the study of metabolic real-time kinetic events in vivo. In order to do this, heteronuclei, like 13C are typically used, since they have the advantage of a longer relaxation time T1 than protons, which allows for longer traceability of the metabolite in vivo – for 13C typically up to several minutes .
There are different approaches to achieve hyperpolarization. Of those, dissolution Dynamic Nuclear Polarization (DNP) is the most prominent hyperpolarization technique for studying metabolism in vivo. DNP uses electron polarization to enhance the signals of close nuclear spins [7–9]. It has been used in clinical trials and produces highly polarized metabolites in high quantities [10–15]. However, it is a slow technique, taking tens of minutes to hours to produce hyperpolarized molecules, which appears to be a significant barrier to translation into clinical practice which was tried to circumvent by polarizing up to four samples at the same time [16, 17].
Para-hydrogen (pH2) induced polarization (PHIP) is a comparably new technique in preclinical applications and offers the possibility to prepare hyperpolarized metabolites in a much shorter amount of time. In pH2 nearly all hydrogen molecules occupy the same spin state, offering a high degree of spin order [18–21]. This spin order is transferred to the molecule of interest by a method, which is called Para-Hydrogen Induced Polarization by means of Side-Arm Hydrogenation (PHIP-SAH) [22–32]. The general workflow is depicted in Fig. 1.
A precursor, in which the molecule of interest is linked to an unsaturated side-arm via a labile bond, is hydrogenated using pH2 and a catalyst in acetone. Afterwards, the spin order from the hydrogen nuclei is transferred to the 13C of interest using a series of carefully timed radiofrequency pulses called the MINERVA (Maximizing Insensitive Nuclei Enhancement Reached Via para-hydrogen Amplification) sequence . After the transfer, the side-arm is cleaved by addition of a basic water solution. After a rapid evaporation of the acetone, the remaining aqueous solution is worked-up by addition of a buffer to adjust the pH to physiological conditions and filtration of the hydrogenation catalyst – leading to a clean solution of the hyperpolarized metabolite in water, which can then be injected into an organism. The whole procedure from the start of hydrogenation to the injection requires typically about one minute. Once injected, the hyperpolarized spin label is visible in the NMR spectra and can be used for the localization and characterization of disease or the investigation of metabolism in vivo. [34–39] The spectra of the metabolites can either be measured over the whole body [40, 41] or selectively over a region of interest, for example a single organ [42, 43]. However, usually a choice has to be made as to which region should be observed, because the allowed injection volume and therefore the sensitivity of the experiment is limited especially in preclinical studies performed on rodents. Multiple injections would provide a way to work around this limitation. In vivo studies with DNP in rodents (mostly rats) demonstrated that two successive injections can be used to study the same region of interest, as to get two data points from one animal [44–46]. Studying large cohorts however appears to be out of reach even with multiple samples being enhanced at the same time.
This work demonstrates the possibility to study two different organs of the same animal with PHIP, using two injections of hyperpolarized 1-13C-pyruvate. We recorded spectra of the region of the brain and the liver of mice within less than half an hour and analyzed them with regard to the conversion of pyruvate to lactate.
This study demonstrates that the high throughput of hyperpolarized metabolites achieved with para-hydrogen enables extensive studies of complex biological systems. With this, metabolism in different organs of the same animal can be studied simultaneously, opening possibilities to study organ crosstalk and reduce the necessary number of animals by accessing more information from each one. The method is minimally invasive, the animals can be woken up again and used for further studies, enabling also longitudinal characterization of metabolism and disease progression.