ABSTRACT
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.
ABSTRACT
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 (â¼7% 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.
Subject(s)
Hydrogen Peroxide , Pyruvic Acid , Humans , Pyruvic Acid/metabolism , HeLa Cells , Hydrogenation , Lactic AcidABSTRACT
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.
Subject(s)
Neoplasms , Pyruvic Acid , Humans , Pyruvic Acid/metabolism , Hydrogen , Magnetic Resonance Imaging/methods , Carbon IsotopesABSTRACT
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.
ABSTRACT
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 1â Hz) are reported. The lifetimes of these states are an order of magnitude longer than the longitudinal relaxation times and up to fourâ 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.
Subject(s)
Magnetic Resonance Imaging , Molecular Probes , Electrons , Magnetic Resonance SpectroscopyABSTRACT
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 ≈22â mT. Following a fast purification procedure, we measure 8 % polarization on free [1-13 C]pyruvate in clean water solutions at physiological conditions at 7â 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.
Subject(s)
Magnetic Resonance Imaging , Pyruvic Acid , Carbon Isotopes , Hydrogen , Magnetic Resonance Imaging/methods , Magnetic Resonance Spectroscopy/methods , WaterABSTRACT
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.
Subject(s)
Magnetic Resonance Spectroscopy , Animals , Computer Simulation , Magnetic Resonance Imaging , Male , Metabolome , Mice, Inbred C57BLABSTRACT
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.
Subject(s)
Amyloid beta-Peptides/chemistry , Humans , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Protein FoldingABSTRACT
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.
ABSTRACT
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.
Subject(s)
Dendrimers/chemistry , Copper/chemistry , Macromolecular Substances/chemistry , Magnetic Resonance SpectroscopyABSTRACT
Quantification of dipolar couplings in biological solids is important for the understanding of dynamic processes. Under Magic Angle Spinning (MAS), order parameters are normally obtained by recoupling of anisotropic interactions involving the application of radio frequency pulses. We have recently shown that amide backbone order parameters can be estimated accurately in a spin-echo experiment in case the rotor spinning angle is slightly mis-calibrated. In this work, we apply this method to determine methyl order parameters in a deuterated sample of the SH3 domain of chicken α-spectrin in which the methyl containing side chains valine and leucine are selectively protonated.
Subject(s)
Anisotropy , Nuclear Magnetic Resonance, Biomolecular/methods , Animals , Chickens , Deuterium , Leucine/chemistry , Proteins/chemistry , Spectrin/chemistry , Valine/chemistryABSTRACT
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.
Subject(s)
Chemistry Techniques, Analytical/methods , Contrast Media/chemical synthesis , Magnetic Fields , Acetates/chemistry , Acetates/metabolism , Contrast Media/chemistry , Hydrogen/chemistry , Magnetic Resonance Imaging/instrumentationABSTRACT
Magic-angle spinning (MAS) is an essential ingredient in a wide variety of solid-state NMR experiments. The standard procedures to adjust the rotor angle are not highly accurate, resulting in a slight misadjustment of the rotor from the magic angle ( θRL=tan-12 ) on the order of a few millidegrees. This small missetting has no significant impact on the overall spectral resolution, but is sufficient to reintroduce anisotropic interactions. Shown here is that site-specific 1 H-15 N dipolar couplings can be accurately measured in a heavily deuterated protein. This method can be applied at arbitrarily high MAS frequencies, since neither rotor synchronization nor particularly high radiofrequency field strengths are required. The off-MAS method allows the quantification of order parameters for very dynamic residues, which often escape an analysis using existing methods.
Subject(s)
Carbon Isotopes/analysis , Deuterium/chemistry , Magnetic Resonance Spectroscopy/methods , Nitrogen Isotopes/analysis , Nuclear Magnetic Resonance, Biomolecular/methods , Spectrin/chemistry , src Homology Domains , Animals , Anisotropy , ChickensABSTRACT
Nuclear spin singlet states are silent states in nuclear magnetic resonance (NMR). However, they can be probed indirectly and offer great potential for the development of contrast agents for magnetic resonance imaging (MRI). Introduced here are two novel concepts: Firstly, the bimodal NMR/fluorescence properties of 13 C2 -tetraphenylethylene. It possesses a long-lived singlet state in organic solvents, and it shortens upon the addition of water. This simultaneously increases the aggregation-induced emission (AIE) of the molecule, resulting in a substantial enhancement of fluorescence. Secondly, introduced is a bimolecular switch for singlet states based on 3-2 H-coumarin containing an isolated proton. Upon UV-light exposure, a dimer forms, leading to a coupling between two previously isolated protons. A nuclear spin singlet state can now be populated. Excitation with a wavelength of 254â nm results in partial ring cleavage of the molecule back to its monomer.
ABSTRACT
We describe the synthesis and characterisation of open fullerene (1) and its reduced form (2) in which CH4 and NH3 are encapsulated, respectively. The 1 Hâ NMR resonance of endohedral NH3 is broadened by scalar coupling to the quadrupolar 14 N nucleus, which relaxes rapidly. This broadening is absent for small satellite peaks, which are attributed to natural abundance 15 N. The influence of the scalar relaxation mechanism on the linewidth of the 1 H ammonia resonance is probed by variable temperature NMR. A rotational correlation time of τc =1.5â ps. is determined for endohedral NH3 , and of τc =57±5â ps. for the open fullerene, indicating free rotation of the encapsulated molecule. IR spectroscopy of NH3 @2 at 5â K identifies three vibrations of NH3 (ν1 , ν3 and ν4 ) redshifted in comparison with free NH3 , and temperature dependence of the IR peak intensity indicates the presence of a large number of excited translational/ rotational states. Variable temperature 1 Hâ NMR spectra indicate that endohedral CH4 is also able to rotate freely at 223â K, on the NMR timescale. Inelastic neutron scattering (INS) spectra of CH4 @1 show both rotational and translational modes of CH4 . Energy of the first excited rotational state (J=1) of CH4 @1 is significantly lower than that of free CH4 .
ABSTRACT
Self-assembling processes occur in a variety of compounds such as peptides, proteins and DNA. These processes have been linked to pathologies and have as well been exploited for designing responsive contrast agents for disease detection. Novel methods to investigate and detect self-assembly therefore hold promise to obtain more insights into disease progression or open pathways to the design of novel self-assembling materials. In this article we are introducing nuclear singlet states to probe self-assembly in the dipeptide isoleucine-phenylalanine (IF) as a thermoresponsive on/off switch for nuclear magnetic resonance (NMR). We have investigated the relaxation and singlet state properties of the ß-protons of phenylalanine in the IF dipeptide in aqueous solutions. At IF concentrations of 2 wt% and above 308 K, a long lived nuclear singlet state, as compared to the longitudinal relaxation, was observed. At 308 K the dipeptide starts forming a gel and no singlet state is accessible at lower temperatures. Upon heating, the gel disassembles and an isotropic liquid forms making the singlet state accessible again. This demonstrates the thermoresponsive on-off character of the nuclear spin singlet state in the IF dipeptide.
ABSTRACT
Hyperpolarization techniques are key to extending the capabilities of MRI for the investigation of structural, functional and metabolic processes inâ vivo. Recent heterogeneous catalyst development has produced high polarization in water using parahydrogen with biologically relevant contrast agents. A heterogeneous ligand-stabilized Rh catalyst is introduced that is capable of achieving 15 N polarization of 12.2±2.7 % by hydrogenation of neurine into a choline derivative. This is the highest 15 N polarization of any parahydrogen method in water to date. Notably, this was performed using a deuterated quaternary amine with an exceptionally long spin-lattice relaxation time (T1 ) of 21.0±0.4â min. These results open the door to the possibility of 15 N inâ vivo imaging using nontoxic similar model systems because of the biocompatibility of the production media and the stability of the heterogeneous catalyst using parahydrogen-induced polarization (PHIP) as the hyperpolarization method.
Subject(s)
Choline/chemistry , Hydrogen/chemistry , Metal Nanoparticles/chemistry , Rhodium/chemistry , Water/chemistry , Amines/chemistry , Catalysis , Deuterium/chemistry , Hydrogenation , Nitrogen Isotopes/chemistryABSTRACT
In this paper we report a methodology for calculating the inelastic neutron scattering spectrum of homonuclear diatomic molecules confined within nano-cavities of spherical symmetry. The method is based on the expansion of the confining potential into multipoles of the coupled rotational and translational angular variables. The Hamiltonian and the INS transition probabilities are evaluated analytically. The method affords a fast and computationally inexpensive way to simulate the inelastic neutron scattering spectrum of molecular hydrogen confined in fullerene cages. The potential energy surface is effectively parametrized in terms of few physical parameters comprising an harmonic term, anharmonic corrections and translation-rotation couplings. The parameters are refined by matching the simulations against the experiments and the excitation modes are identified for transfer energies up to 215 meV.
ABSTRACT
The fine structure of the rotational ground state of molecular ortho-hydrogen confined inside the fullerene cage C60 is investigated by inelastic neutron scattering (INS). The INS line corresponding to transitions between the three sub-levels comprising the ortho ground state to the non-degenerate para ground state was studied as a function of temperature down to 60 mK in neutron energy gain. The experiments show that at ambient pressure the three ortho sub-levels are split into a low energy non-degenerate level and a high energy doubly degenerate level separated by 0.135 ± 0.010 meV. This observation is consistent with hydrogen molecules being located at sites with axial symmetry superseding the icosahedral symmetry of isolated rigid C60 cages in the solid phase. To gain insight into the role of inter-cage interactions in determining the symmetry breaking potential, the effects of hydrostatic pressure on the fine structure of the line was also investigated. The analysis of the INS spectra shows that the potential and the energy levels of H2 are sensitive to the orientation of neighbouring cages, consistent with the low-temperature crystalline phase of C60.
ABSTRACT
Long-lived nuclear spin states have a relaxation time much longer than the longitudinal relaxation time T1. Long-lived states extend significantly the time scales that may be probed with magnetic resonance, with possible applications to transport and binding studies, and to hyperpolarised imaging. Rapidly rotating methyl groups in solution may support a long-lived state, consisting of a population imbalance between states of different spin exchange symmetries. Here, we expand the formalism for describing the behaviour of long-lived nuclear spin states in methyl groups, with special attention to the hyperpolarisation effects observed in (13)CH3 groups upon rapidly converting a material with low-barrier methyl rotation from the cryogenic solid state to a room-temperature solution [M. Icker and S. Berger, J. Magn. Reson. 219, 1 (2012)]. We analyse the relaxation properties of methyl long-lived states using semi-classical relaxation theory. Numerical simulations are supplemented with a spherical-tensor analysis, which captures the essential properties of methyl long-lived states.