Novel Sample Formulations for Pure and Persistent Hyperpolarized Solutions via Dissolution Dynamic Nuclear Polarization

Nuclear Magnetic Resonance (NMR) spectroscopy allows one to study and analyze the structure, motions and interactions of a broad variety of molecules. However, this technique has a major inconvenience: its low sensitivity, which therefore often results in the use of highly concentrated samples in order to observe tiny signals. As recently as 2003, a new technique known as dissolution-DNP or d-DNP was invented by Ardenkjaer-Larsen et al. to overcome this drawback and to get more intense NMR signals in solution with enhancements factors larger than 10'000. This method consists essentially in mixing paramagnetic species (radicals) with samples containing the metabolites to be analyzed. These mixtures are then rapidly frozen at very low temperatures (T = 1.2 â 4.2 K) in liquid helium and, by applying a proper microwave irradiation, the polarization of the electrons can be transferred to nuclei such as 1H or 13C. This allows one to build up and store the enhanced magnetization of these nuclei and then to dissolve the samples by injecting a superheated solvent via a dissolution stick. The resulting hyperpolarized solution can be finally transferred to an NMR spectrometer where the signals can be recorded. In the first two chapters of this thesis, the principles of NMR are introduced and the theory of DNP is explained in some detail. The dissolution equipment used in our laboratory and its different parts are shown, in particular the DNP polarizer where the transfer of polarization from electrons to nuclei occurs, the microwave source that is connected to the polarizer, and the dissolution system itself, which comprises the dissolution stick and the dissolution transfer line. Another chapter is dedicated to the optimization of our DNP setup in order to achieve the highest possible polarization before the dissolution process. Several radicals are tested under carefully controlled conditions to identify the best suited for our DNP system. Furthermore, the modulation of the microwave frequency has been optimized in order to enhance the polarization transfer. Finally, a number of dissolution experiments are presented that relate to different projects that have been carried out in the course of this thesis. The extent of the polarization can be determined accurately by looking directly at the hyperpolarized NMR spectrum. Filterable polymers containing suitable radical moieties have been synthesized in order to obtain pure hyperpolarized solutions. A final chapter outlines future applications and projects that can benefit from dissolution Dynamic Nuclear Polarization

The effect of Maillard reaction products on zinc metabolism in the rat

The effect of giving Maillard reaction products (MRP) on zinc metabolism was investigated in the rat. In Expt 1, MRP were prepared by incubating casein with either glucose or lactose under controlled reaction conditions, and were quantified as either ‘early' or ‘advanced' after estimation of lysine loss and lysine destruction respectively. In Expt 2, the effect of the purified early MRP fructose-lysine (FL) on Zn metabolism was studied. The experimental diets containing 20 mg Zn/kg were given to weanling rats for 21 d. Zn balance was assessed over 9-14 d (Expt 1), or 1-14 d (Expt 2). Femur, liver, kidney and serum Zn concentrations were determined at 21 d. The major effect of the MRP in the casein-sugar mixtures was on urinary Zn excretion. The casein-glucose MRP induced up to a 6-fold increase in the quantity of Zn excreted in the urine. The magnitude of the hyperzincuria increased with the extent of the Maillard reaction. Similar dietary levels of casein-lactose MRP increased urinary Zn loss 2-fold. Free FL had no effect on urinary Zn. Faecal Zn, Zn retention, liver, femur and serum Zn were generally not influenced by giving MRP from casein-sugar mixtures or by giving free FL, although kidney Zn was decreased in rats fed on FL. It was concluded that although urinary Zn excretion can be increased by the presence of MRP in the diet, this is only a minor excretory pathway and would have little influence on overall Zn nutrition in individuals fed on a diet adequate in Z

Microwave frequency modulation to enhance Dissolution Dynamic Nuclear Polarization Dedicated to To Martial Rey, as a token of appreciation

Hyperpolarization by Dissolution Dynamic Nuclear Polarization is usually achieved by monochromatic microwave irradiation of the ESR spectrum of free radicals embedded in glasses at 1.2 K and 3.35 T. Hovav et al. (2014) have recently shown that by using frequency-modulated (rather than monochromatic) microwave irradiation one can improve DNP at 3.35 T in the temperature range 10-50 K. We show in this Letter that this is also true under Dissolution-DNP conditions at 1.2 K and 6.7 T. We demonstrate the many virtues of using frequency-modulated microwave irradiation: higher polarizations, faster build-up rates, lower radical concentrations, less paramagnetic broadening, more efficient cross-polarization, and less critical frequency adjustments. © 2014 The Authors. Published by Elsevier B.V

Long-Lived States of Magnetically Equivalent Spins Populated by Dissolution-DNP and Revealed by Enzymatic Reactions

Hyperpolarization by dissolution dynamic nuclear polarization (D-DNP) offers a way of enhancing NMR signals by up to five orders of magnitude in metabolites and other small molecules. Nevertheless, the lifetime of hyperpolarization is inexorably limited, as it decays toward thermal equilibrium with the nuclear spin-lattice relaxation time. This lifetime can be extended by storing the hyperpolarization in the form of long-lived states (LLS) that are immune to most dominant relaxation mechanisms. Levitt and co-workers have shown how LLS can be prepared for a pair of inequivalent spins by D-DNP. Here, we demonstrate that this approach can also be applied to magnetically equivalent pairs of spins such as the two protons of fumarate, which can have very long LLS lifetimes. As in the case of para-hydrogen, these hyperpolarized equivalent LLS (HELLS) are not magnetically active. However, a chemical reaction such as the enzymatic conversion of fumarate into malate can break the magnetic equivalence and reveal intense NMR signals

Electrical Neuroimaging of Music Processing in Pianists With and Without True Absolute Pitch

True absolute pitch (AP), labeling of pitches with semitone precision without a reference, is classically studied using isolated tones. However, AP is acquired and has its function within complex dynamic musical contexts. Here we examined event-related brain responses and underlying cerebral sources to endings of short expressive string quartets, investigating a homogeneous population of young highly trained pianists with half of them possessing true-AP. The pieces ended regularly or contained harmonic transgressions at closure that participants appraised. Given the millisecond precision of ERP analyses, this experimental plan allowed examining whether AP alters music processing at an early perceptual, or later cognitive level, or both, and which cerebral sources underlie differences with non-AP musicians. We also investigated the impact of AP on general auditory cognition. Remarkably, harmonic transgression sensitivity did not differ between AP and non-AP participants, and differences for auditory cognition were only marginal. The key finding of this study is the involvement of a microstate peaking around 60 ms after musical closure, characterizing AP participants. Concurring sources were estimated in secondary auditory areas, comprising the planum temporale, all transgression conditions collapsed. These results suggest that AP is not a panacea to become a proficient musician, but a rare perceptual feature

Understanding wellbeing among college music students and amateur musicians in Western Switzerland

Musical performance requires the ability to master a complex integration of highly specialized motor, cognitive, and perceptual skills developed over years of practice. It often means also being able to deal with considerable pressure within dynamic environments. Consequently, many musicians suffer from health-related problems and report a large number of physical and psychological complaints. Our research aimed to evaluate and analyze the wellbeing of two distinct groups of musicians, college music students and amateur performers in the French-speaking part of Switzerland. A total sample of 126 musicians was recruited for the study (mean age ±SD = 22.4 ± 4.5 years, 71 male). Wellbeing was assessed through the World Health Organization Quality of Life-BREF questionnaire evaluating two general measures, quality of life (QoL) and general health, and four specific dimensions: physical health, psychological health, social relationships, and environment. For both groups, respondents’ QoL was high on each measure: median scores were higher than 4 for the two general measures and higher than 70 for the four specific dimensions. Among the dimensions, respondents had the highest mean score for environment (75.0), then social relationships and physical health (74.0 and 73.8, respectively), and finally, psychological health (70.3). Differences between groups of musicians emerged in terms of overall QoL and general health, as well as the physical health dimension, where college music students scored lower than the amateur musicians; conversely, college music students scored higher than the amateurs on social relationships. Our overview of musicians’ wellbeing in Western Switzerland demonstrates that, while music making can offer some health protective effects, there is a need for greater health awareness and promotion among advanced music students. This research offers insight into musicians’ wellbeing and points to the importance of involving different actors (teachers, administrators, support staff) in facilitating healthy music making

Hyperpolarized Water to Study Protein-Ligand Interactions

The affinity between a chosen target protein and small molecules is a key aspect of drug discovery. Screening by popular NMR methods such as Water-LOGSY suffers from low sensitivity and from false positives caused by aggregated or denatured proteins. This work demonstrates that the sensitivity of Water-LOGSY can be greatly boosted by injecting hyperpolarized water into solutions of proteins and ligands. Ligand binding can be detected in a few seconds, whereas about 30 min is usually required without hyperpolarization. Hyperpolarized water also enhances proton signals of proteins at concentrations below 20 M so that one can verify in a few seconds whether the proteins remain intact or have been denatured

Cross polarization from H-1 to quadrupolar Li-6 nuclei for dissolution DNP

Cross polarization from protons to quadrupolar Li-6 nuclei is combined with dynamic nuclear polarization of protons at 1.2 K and 6.7 T using TEMPOL as a polarizing agent followed by rapid dissolution. Compared to direct Li-6 DNP without cross-polarization, a higher nuclear spin polarization P(Li-6) can be obtained in a shorter time. A double resonance H-1-Li-6 probe was designed that is equipped for Longitudinally Detected Electron Spin Resonance

Drug Screening Boosted by Hyperpolarized Long-Lived States in NMR

Transverse and longitudinal relaxation times (T1ρ and T1) have been widely exploited in NMR to probe the binding of ligands and putative drugs to target proteins. We have shown recently that long-lived states (LLS) can be more sensitive to ligand binding. LLS can be excited if the ligand comprises at least two coupled spins. Herein we broaden the scope of ligand screening by LLS to arbitrary ligands by covalent attachment of a functional group, which comprises a pair of coupled protons that are isolated from neighboring magnetic nuclei. The resulting functionalized ligands have longitudinal relaxation times T1(1H) that are sufficiently long to allow the powerful combination of LLS with dissolution dynamic nuclear polarization (D-DNP). Hyperpolarized weak “spy ligands” can be displaced by high-affinity competitors. Hyperpolarized LLS allow one to decrease both protein and ligand concentrations to micromolar levels and to significantly increase sample throughput