Real-Time Metabolic Magnetic Resonance Spectroscopy of Pancreatic and Colon Cancer Tumor-Xenografts with Parahydrogen Hyperpolarized 1-13C Pyruvate-d3.

Parahydrogen-induced polarization (PHIP) is an emerging technique to enhance the signal of stable isotope metabolic contrast agents for Magnetic Resonance (MR). The objective of this study is to continue establishing 1-13C-pyruvate-d3, signal-enhanced via PHIP, as a hyperpolarized contrast agent, obtained in seconds, to monitor metabolism in human cancer. Our focus was on human pancreatic and colon tumor xenografts. 1-13C-vinylpyruvate-d6 was hydrogenated using parahydrogen. Thereafter, the polarization of the protons was transferred to 13C. Following a workup procedure, the free hyperpolarized 1-13C-pyruvate-d3 was obtained in clean aqueous solution. After injection into animals bearing either pancreatic or colon cancer xenografts, slice-selective MR spectra were acquired and analyzed to determine rate constants of metabolic conversion into lactate and alanine. 1-13C-pyruvate-d3 proved to follow the increased metabolic rate to lactate and alanine in the tumor xenografts.

Hyperpolarization of 15N-Pyridinium by Using Parahydrogen Enables Access to Reactive Oxygen Sensors and Pilot In Vivo Studies.

Magnetic resonance with hyperpolarized contrast agents is one of the most powerful and noninvasive imaging platforms capable for investigating in vivo metabolism. While most of the utilized hyperpolarized agents are based on 13C nuclei, a milestone advance in this area is the emergence of 15N hyperpolarized contrast agents. Currently, the reported 15N hyperpolarized agents mainly utilize the dissolution dynamic nuclear polarization (d-DNP) protocol. The parahydrogen enhanced 15N probes have proven to be elusive and have been tested almost exclusively in organic solvents. Herein, we designed a reaction based reactive oxygen sensor 15N-boronobenzyl-2-styrylpyridinium (15N-BBSP) which can be hyperpolarized with para-hydrogen. Reactive oxygen species plays a vital role as one of the essential intracellular signalling molecules. Disturbance of the H2O2 level usually represents a hallmark of pathophysiological conditions. This H2O2 probe exhibited rapid responsiveness toward H2O2 and offered spectrally resolvable chemical shifts. We also provide strategies to bring the newly developed probe from the organic reaction solution into a biocompatible injection buffer and demonstrate the feasibility of in vivo 15N signal detection. The present work manifests its great potential not only for reaction based reactive sensing probes but also promises to serve as a platform to develop other contrast agents.

Molecular precursors to produce para-hydrogen enhanced metabolites at any field.

Enhancing magnetic resonance signal via hyperpolarization techniques enables the real-time detection of metabolic transformations even in vivo. The use of para-hydrogen to enhance 13 C-enriched metabolites has opened a rapid pathway for the production of hyperpolarized metabolites, which usually requires specialized equipment. Metabolite precursors that can be hyperpolarized and converted into metabolites at any given field would open up opportunities for many labs to make use of this technology because already existing hardware could be used. We report here on the complete synthesis and hyperpolarization of suitable precursor molecules of the side-arm hydrogenation approach. The better accessibility to such side-arms promises that the para-hydrogen approach can be implemented in every lab with existing two channel NMR spectrometers for 1 H and 13 C independent of the magnetic field.

Real-time cell metabolism assessed repeatedly on the same cells via para-hydrogen induced polarization.

Signal-enhanced or hyperpolarized nuclear magnetic resonance (NMR) spectroscopy stands out as a unique tool to monitor real-time enzymatic reactions in living cells. The singlet state of para-hydrogen is thereby one source of spin order that can be converted into largely enhanced signals of e.g. metabolites. Here, we have investigated a parahydrogen-induced polarization (PHIP) approach as a biological assay for in vitro cellular metabolic characterization. Here, we demonstrate the possibility to perform consecutive measurements yielding metabolic information on the same sample. We observed a strongly reduced pyruvate-to-lactate conversion rate (flux) of a Hodgkin's lymphoma cancer cell line L1236 treated with FK866, an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT) affecting the amount of NAD+ and thus NADH in cells. In the consecutive measurement the flux was recovered by NADH to the same amount as in the single-measurement-per-sample and provides a promising new analytical tool for continuous real-time studies combinable with bioreactors and lab-on-a-chip devices in the future.

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.

In vivo singlet state filtered nuclear magnetic resonance: towards monitoring toxic responses inside living organisms.

In line with recent paradigm shifts in toxicity testing, in vivo nuclear magnetic resonance (NMR) is a powerful tool for studying the biological impacts and perturbations caused by toxicants in living organisms. However, despite the excellent molecular insights that can be obtained through this technique, in vivo NMR applications are hampered by considerable experimental challenges such as poor line shape and spectral overlap. Here, we demonstrate the application of singlet-filtered NMR to target specific metabolites and facilitate the study of metabolite fluxes in living Daphnia magna, an aquatic keystone species and model organism. Informed by mathematical simulations and experiments on ex vivo organisms, singlet state NMR is used to monitor the flux of metabolites such as d-glucose and serine in living D. magna, during the environmentally relevant processes of anoxic stress and reduced food availability. Overall, singlet state NMR is shown to have significant future potential for studying metabolic processes in vivo.

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.

Hyperpolarization of 15N in an amino acid derivative.

Hyperpolarization is a nuclear magnetic resonance (NMR) technique which can be used to significantly enhance the signal in NMR experiments. In recent years, the possibility to enhance the NMR signal of heteronuclei by the use of para-hydrogen induced polarization (PHIP) has gained attention, especially in the area of possible applications in magnetic resonance imaging (MRI). Herein we introduce a way to synthesize a fully deuterated, 15N labelled amino acid derivative and the possibility to polarize the 15N by means of hydrogenation with para-hydrogen to a polarization level of 0.18%. The longevity of the polarization with a longitudinal relaxation time of more than a minute can allow for the observation of dynamic processes and metabolic imaging in vivo. In addition, we observe the phenomenon of proton-deuterium exchange with a homogeneous catalyst leading to signal enhanced allyl moeities in the precursor.

Decoupling of Spin Decoherence Paths near Zero Magnetic Field.

We demonstrate a method to quantify and manipulate nuclear spin decoherence mechanisms that are active in zero to ultralow magnetic fields. These include (i) nonadiabatic switching of spin quantization axis due to residual background fields and (ii) scalar pathways due to through-bond couplings between 1H and heteronuclear spin species, such as 2H used partially as an isotopic substitute for 1H. Under conditions of free evolution, scalar relaxation due to 2H can significantly limit nuclear spin polarization lifetimes and thus the scope of magnetic resonance procedures near zero field. It is shown that robust trains of pulsed dc magnetic fields that apply π flip angles to one or multiple spin species may switch the effective symmetry of the nuclear spin Hamiltonian, imposing decoupled or coupled dynamic regimes on demand. The method should broaden the spectrum of hyperpolarized biomedical contrast-agent compounds and hyperpolarization procedures that are used near zero field.

Bimodal Fluorescence/Magnetic Resonance Molecular Probes with Extended Spin Lifetimes.

Bimodal molecular probes combining nuclear magnetic resonance (NMR) and fluorescence have been widely studied in basic science, as well as clinical research. The investigation of spin phenomena holds promise to broaden the scope of available probes allowing deeper insights into physiological processes. Herein, a class of molecules with a bimodal character with respect to fluorescence and nuclear spin singlet states is introduced. Singlet states are NMR silent but can be probed indirectly. Symmetric, perdeuterated molecules, in which the singlet states can be populated by vanishingly small electron-mediated couplings (below 1 Hz) are reported. The lifetimes of these states are an order of magnitude longer than the longitudinal relaxation times and up to four minutes at 7 T. Moreover, these molecules show either aggregation induced emission (AIE) or aggregation caused quenching (ACQ) with respect to their fluorescence. In the latter case, the existence of excited dimers, which are proposed to use in a switchable manner in combination with the quenching of nuclear spin singlet states, is observed.

Exotic nuclear spin behavior in dendritic macromolecules.

Dendrimers are a class of branched, highly symmetric macromolecules that have been shown to be useful for a vast number of different applications. Potential uses as fluorescence sensors, in catalysis and perhaps most importantly in medical applications as drug delivery systems or cytotoxica have been proposed. Herein we report on an exotic behaviour of the nuclear spins in a dendritic macromolecule in the presence of different paramagnetic ions. We show that the stability of the long lived nuclear singlet state, is affected by the presence of Cu(II), whereas other ions did not have any influence at all. This effect could not be observed in the case of a simple tripeptide, in which the nuclear singlet stability was influenced by all investigated paramagnetic ions, a potentially useful effect in the development of Cu(II) selective probes. By adding a fluorescent marker to our molecule we could show that the nuclear singlet multimer (NUSIMER) is taken up by living cells. Furthermore we were able to show that nuclear singlet state NMR can be used to investigate the NUSIMER in the presence of living cells, showing that an application in in vivo NMR can be feasible.

Early Divergence in Misfolding Pathways of Amyloid-Beta Peptides.

The amyloid cascade hypothesis proposes that amyloid-beta (Aß) aggregation is the initial triggering event in Alzheimer's disease. Here, we utilize NMR spectroscopy and monitor the structural dynamics of two variants of Aß, Aß40 and Aß42, as a function of temperature. Despite having identical amino acid sequence except for the two additional C-terminal residues, Aß42 has higher aggregation propensity than Aß40. As revealed by the NMR data on dynamics, including backbone chemical shifts, intra-methyl cross-correlated relaxation rates and glycine-based singlet-states, the C-terminal region of Aß, especially the G33-L34-M35 segment, plays a particular role in the early steps of temperature-induced Aß aggregation. In Aß42, the distinct dynamical behaviour of C-terminal residues at higher temperatures is accompanied with marked changes in the backbone dynamics of residues V24-K28. The distinctive role of the C-terminal region of Aß42 in the initiation of aggregation defines a target for the rational design of Aß42 aggregation inhibitors.

Spontaneous Enhancement of Magnetic Resonance Signals Using a RASER.

Nuclear magnetic resonance is usually drastically limited by its intrinsically low sensitivity: Only a few spins contribute to the overall signal. To overcome this limitation, hyperpolarization methods were developed that increase signals several times beyond the normal/thermally polarized signals. The ideal case would be a universal approach that can signal enhance the complete sample of interest in solution to increase detection sensitivity. Here, we introduce a combination of para-hydrogen enhanced magnetic resonance with the phenomenon of the RASER: Large signals of para-hydrogen enhanced molecules interact with the magnetic resonance coil in a way that the signal is spontaneously converted into an in-phase signal. These molecules directly interact with other compounds via dipolar couplings and enhance their signal. We demonstrate that this is not only possible for solvent molecules but also for an amino acid.

High field parahydrogen induced polarization of succinate and phospholactate.

The signal enhancement provided by the hyperpolarization of nuclear spins of metabolites is a promising technique for diagnostic magnetic resonance imaging (MRI). To date, most 13C-contrast agents are hyperpolarized utilizing a complex or cost-intensive polarizer. Recently, the in situ parahydrogen-induced 13C hyperpolarization was demonstrated. Hydrogenation, spin order transfer (SOT) by a pulsed NMR sequence, in vivo administration, and detection was achieved within the magnet bore of a 7 Tesla MRI system. So far, the hyperpolarization of the xenobiotic molecule 1-13C-hydroxyethylpropionate (HEP) and the biomolecule 1-13C-succinate (SUC) through the PH-INEPT+ sequence and a SOT scheme proposed by Goldman et al., respectively, was shown. Here, we investigate further the hyperpolarization of SUC at 7 Tesla and study the performance of two additional SOT sequences. Moreover, we present first results of the hyperpolarization at high magnetic field of 1-13C-phospholactate (PLAC), a derivate to obtain the metabolite lactate, employing the PH-INEPT+ sequence. For SUC and PLAC, 13C polarizations of about 1-2% were achieved within seconds and with minimal equipment. Effects that potentially may explain loss of 13C polarization have been identified, i.e. low hydrogenation yield, fast T1/T2 relaxation and the rarely considered 13C isotope labeling effect.

Nuclear hyperpolarization of (1-13C)-pyruvate in aqueous solution by proton-relayed side-arm hydrogenation.

We employ Parahydrogen Induced Polarization with Side-Arm Hydrogenation (PHIP-SAH) to polarize (1-13C)-pyruvate. We introduce a new method called proton-relayed side-arm hydrogenation (PR-SAH) in which an intermediate proton is used to transfer polarization from the side-arm to the 13C-labelled site of the pyruvate before hydrolysis. This significantly reduces the cost and effort needed to prepare the precursor for radio-frequency transfer experiments while still maintaining acceptable polarization transfer efficiency. Experimentally we have attained on average 4.33% 13C polarization in an aqueous solution of (1-13C)-pyruvate after about 10 seconds of cleavage and extraction. PR-SAH is a promising pulsed NMR method for hyperpolarizing 13C-labelled metabolites in solution, conducted entirely in high magnetic field.

Localized singlet-filtered MRS in vivo.

MR is a prominent technology to investigate diseases, with millions of clinical procedures performed every year. Metabolic dysfunction is one common aspect associated with many diseases. Thus, understanding and monitoring metabolic changes is essential to develop cures for many illnesses, including for example cancer and neurodegeneration. MR methodologies are especially suited to study endogenous metabolites and processes within an organism in vivo, which has led to many insights about physiological functions. Advancing metabolic MR techniques is therefore key to further understand physiological processes. Here, we introduce an approach based on nuclear spin singlet states to specifically filter metabolic signals and particularly show that singlet-filtered glutamate can be observed distinctly in the hippocampus of a living mouse in vivo. This development opens opportunities to make use of the singlet spin phenomenon in vivo and besides its use as a filter to provide scope for new contrast agents.

Singlet-filtered NMR spectroscopy.

Selectively studying parts of proteins and metabolites in tissue with nuclear magnetic resonance promises new insights into molecular structures or diagnostic approaches. Nuclear spin singlet states allow the selection of signals from chemical moieties of interest in proteins or metabolites while suppressing background signal. This selection process is based on the electron-mediated coupling between two nuclear spins and their difference in resonance frequency. We introduce a generalized and versatile pulsed NMR experiment that allows populating singlet states on a broad scale of coupling patterns. This approach allowed us to filter signals from proton pairs in the Alzheimer's disease-related b-amyloid 40 peptide and in metabolites in brain matter. In particular, for glutamine/glutamate, we have discovered a long-lived state in tissue without the typically required singlet sustaining by radiofrequency irradiation. We believe that these findings will open up new opportunities to study metabolites with a view on future in vivo applications.