Category Archives: Dissolution

Stable isotope resolved metabolomics classification of prostate cancer cells using hyperpolarized NMR data #DNPNMR

Frahm, Anne Birk, Pernille Rose Jensen, Jan Henrik Ardenkjær-Larsen, Demet Yigit, and Mathilde Hauge Lerche. “Stable Isotope Resolved Metabolomics Classification of Prostate Cancer Cells Using Hyperpolarized NMR Data.” Journal of Magnetic Resonance 316 (July 2020): 106750.

https://doi.org/10.1016/j.jmr.2020.106750

Metabolic fingerprinting is a strong tool for characterization of biological phenotypes. Classification with machine learning is a critical component in the discrimination of molecular determinants. Cellular activity can be traced using stable isotope labelling of metabolites from which information on cellular pathways may be obtained. Nuclear magnetic resonance (NMR) spectroscopy is, due to its ability to trace labelling in specific atom positions, a method of choice for such metabolic activity measurements. In this study, we used hyperpolarization in the form of dissolution Dynamic Nuclear Polarization (dDNP) NMR to measure signal enhanced isotope labelled metabolites reporting on pathway activity from four different prostate cancer cell lines. The spectra have a high signal-to-noise, with less than 30 signals reporting on 10 metabolic reactions. This allows easy extraction and straightforward interpretation of spectral data. Four metabolite signals selected using a Random Forest algorithm allowed a classification with Support Vector Machines between aggressive and indolent cancer cells with 96.9% accuracy, -corresponding to 31 out of 32 samples. This demonstrates that the information contained in the few features measured with dDNP NMR, is sufficient and robust for performing binary classification based on the metabolic activity of cultured prostate cancer cells.

Use of dissolved hyperpolarized species in NMR: Practical considerations #DNPNMR

Berthault, Patrick, Céline Boutin, Charlotte Martineau-Corcos, and Guillaume Carret. “Use of Dissolved Hyperpolarized Species in NMR: Practical Considerations.” Progress in Nuclear Magnetic Resonance Spectroscopy 118–119 (June 2020): 74–90.

https://doi.org/10.1016/j.pnmrs.2020.03.002

Hyperpolarization techniques that can transiently boost nuclear spin polarization are generally carried out at low temperature – as in the case of dynamic nuclear polarization – or at high temperature in the gaseous state – as in the case of optically pumped noble gases. This review aims at describing the various issues and challenges that have been encountered during dissolution of hyperpolarized species, and solutions to these problems that have been or are currently proposed in the literature. During the transport of molecules from the polarizer to the NMR detection region, and when the hyperpolarized species or a precursor of hyperpolarization (e.g. parahydrogen) is introduced into the solution of interest, several obstacles need to be overcome to keep a high level of final magnetization. The choice of the magnetic field, the design of the dissolution setup, and ways to isolate hyperpolarized compounds from relaxation agents will be presented. Due to the non-equilibrium character of the hyperpolarization, new NMR pulse sequences that perform better than the classical ones will be described. Finally, three applications in the field of biology will be briefly mentioned.

Catalytic cycle of carbohydrate dehydration by Lewis acids: structures and rates from synergism of conventional and DNP NMR #DNPNMR #DDNP

Jensen, Pernille Rose, and Sebastian Meier. “Catalytic Cycle of Carbohydrate Dehydration by Lewis Acids: Structures and Rates from Synergism of Conventional and DNP NMR.” Chemical Communications 56, no. 46 (June 9, 2020): 6245–48.

https://doi.org/10.1039/D0CC01756F

Lewis acids play key roles in many chemical reactions. Structural and functional (kinetic) detail in Lewis acid catalysed fructose conversion are derived herein by the combined use of conventional and dissolution dynamic nuclear polarization (D-DNP) NMR. Structural information obtained with D-DNP NMR was used to identify conditions that stabilize an elusive initial intermediate and to determine its chemical structure. Carbohydrate dehydration through this intermediate had been predicted computationally. Complementary kinetic NMR assays yielded rate constants spanning three orders of magnitude for the three biggest energy barriers in the catalytic cycle.

Gadolinium Effect at High-Magnetic-Field DNP: 70% 13C Polarization of [U-13C] Glucose Using Trityl #DNPNMR

Capozzi, Andrea, Saket Patel, W. Thomas Wenckebach, Magnus Karlsson, Mathilde H. Lerche, and Jan Henrik Ardenkjær-Larsen. “Gadolinium Effect at High-Magnetic-Field DNP: 70% 13C Polarization of [U-13C] Glucose Using Trityl.” The Journal of Physical Chemistry Letters 10, no. 12 (June 20, 2019): 3420–25.

https://doi.org/10.1021/acs.jpclett.9b01306.

We show that the trityl electron spin resonance (ESR) features, crucial for an efficient dynamic nuclear polarization (DNP) process, are sample-composition-dependent. Working at 6.7 T and 1.1 K with a generally applicable DNP sample solvent mixture such as water/glycerol plus trityl, the addition of Gd3+ leads to a dramatic increase in [U-13C] glucose polarization from 37 ± 4% to 69 ± 3%. This is the highest value reported to date and is comparable to what can be achieved on pyruvic acid. Moreover, performing ESR measurements under actual DNP conditions, we provide experimental evidence that gadolinium doping not only shortens the trityl electron spin−lattice relaxation time but also modifies the radical g-tensor. The latter yielded a considerable narrowing of the ESR spectrum line width. Finally, in the frame of the spin temperature theory, we discuss how these two phenomena affect the DNP performance.

Hyperpolarized water through dissolution dynamic nuclear polarization with UV-generated radicals #DNPNMR

Pinon, Arthur C., Andrea Capozzi, and Jan Henrik Ardenkjær-Larsen. “Hyperpolarized Water through Dissolution Dynamic Nuclear Polarization with UV-Generated Radicals.” Communications Chemistry 3, no. 1 (December 2020): 57.

https://doi.org/10.1038/s42004-020-0301-6

In recent years, hyperpolarization of water protons via dissolution Dynamic Nuclear Polarization (dDNP) has attracted increasing interest in the magnetic resonance community. Hyperpolarized water may provide an alternative to Gd-based contrast agents for angiographic and perfusion Magnetic Resonance Imaging (MRI) examinations, and it may report on chemical and biochemical reactions and proton exchange while perfoming Nuclear Magnetic Resonance (NMR) investigations. However, hyperpolarizing water protons is challenging. The main reason is the presence of radicals, required to create the hyperpolarized nuclear spin state. Indeed, the radicals will also be the main source of relaxation during the dissolution and transfer to the NMR or MRI system. In this work, we report water magnetizations otherwise requiring a field of 10,000 T at room temperature on a sample of pure water, by employing dDNP via UV-generated, labile radicals. We demonstrate the potential of our methodology by acquiring a 15N spectrum from natural abundance urea with a single scan, after spontaneous magnetization transfer from water protons to nitrogen nuclei.

Dissolution dynamic nuclear polarization NMR studies of enzyme kinetics: setting up differential equations for fitting to spectral time courses #DNPNMR

Kuchel, Philip W., and Dmitry Shishmarev. “Dissolution Dynamic Nuclear Polarization NMR Studies of Enzyme Kinetics: Setting up Differential Equations for Fitting to Spectral Time Courses.” Journal of Magnetic Resonance Open, March 2020, 100001.

https://doi.org/10.1016/j.jmro.2020.100001

Dissolution dynamic nuclear polarization (dDNP) provides strikingly increased sensitivity for detecting NMR-receptive nuclei in molecules that are substrates of enzymes and membrane transport proteins. This paves the way for studying the kinetics of many such catalysed reactions on previously unattainable short time scales (seconds). Remarkably, this can also be carried out not only in vitro, but in whole cells, tissues, and even in vivo. The information obtained from the emergent NMR time courses is a sequence of spectral-peak intensities (integrals) as a function of time. Typically, for 13C NMR studies, these consist of a series of spectra acquired every 1 s for a total time span of ~3 min.

Dissolution Dynamic Nuclear Polarization Methodology and Instrumentation #DNPNMR

Kurzbach, Dennis, and Sami Jannin. “Dissolution Dynamic Nuclear Polarization Methodology and Instrumentation,” 7:16, 2018. 

https://onlinelibrary.wiley.com/doi/abs/10.1002/9780470034590.emrstm1563

Dissolution dynamic nuclear polarization (d-DNP) denotes a method to enhance signals in nuclear magnetic resonance (NMR) spectroscopy by hyperpolarization of nuclear spins at cryogenic temperature in the solid state prior to a rapid dissolution, transfer of a sample to a conventional NMR spectrometer, and detection at ambient temperatures in the liquid state. The purpose of this chapter is to review the general methodology behind d-DNP, and instrumentational aspects going from basic tasks, such as sample preparation, over operational aspects, such as the use of crosspolarization techniques, magnetic tunnels, and the removal of polarization agents, to future perspectives, such as the long-distance transport of hyperpolarized substrates.

Creating a clinical platform for carbon‐13 studies using the sodium‐23 and proton resonances #DNPNMR

Grist, James T., Esben S.S. Hansen, Juan D. Sánchez‐Heredia, Mary A. McLean, Rasmus Tougaard, Frank Riemer, Rolf F. Schulte, et al. “Creating a Clinical Platform for Carbon‐13 Studies Using the Sodium‐23 and Proton Resonances.” Magnetic Resonance in Medicine, March 13, 2020, mrm.28238.

https://doi.org/10.1002/mrm.28238

Purpose: Calibration of hyperpolarized 13C-MRI is limited by the low signal from endogenous carbon-containing molecules and consequently requires 13C-enriched external phantoms. This study investigated the feasibility of using either 23Na-MRI or 1H-MRI to calibrate the 13C excitation.

Methods: Commercial 13C-coils were used to estimate the transmit gain and center frequency for 13C and 23Na resonances. Simulations of the transmit B1 profile of a Helmholtz loop were performed. Noise correlation was measured for both nuclei. A retrospective analysis of human data assessing the use of the 1H resonance to predict [1-13C]pyruvate center frequency was also performed. In vivo experiments were undertaken in the lower limbs of 6 pigs following injection of hyperpolarized 13C-pyruvate.

Results: The difference in center frequencies and transmit gain between tissue 23Na and [1-13C]pyruvate was reproducible, with a mean scale factor of 1.05179 ± 0.00001 and 10.4 ± 0.2 dB, respectively. Utilizing the 1H water peak, it was possible to retrospectively predict the 13C-pyruvate center frequency with a standard deviation of only 11 Hz sufficient for spectral–spatial excitation-based studies.

Conclusion: We demonstrate the feasibility of using the 23Na and 1H resonances to calibrate the 13C transmit B1 using commercially available 13C-coils. The method provides a simple approach for in vivo calibration and could improve clinical workflow.

Creating a clinical platform for carbon‐13 studies using the sodium‐23 and proton resonances #DNPNMR

Grist, James T., Esben S.S. Hansen, Juan D. Sánchez‐Heredia, Mary A. McLean, Rasmus Tougaard, Frank Riemer, Rolf F. Schulte, et al. “Creating a Clinical Platform for Carbon‐13 Studies Using the Sodium‐23 and Proton Resonances.” Magnetic Resonance in Medicine, March 13, 2020, mrm.28238.

https://doi.org/10.1002/mrm.28238

Purpose: Calibration of hyperpolarized 13C-MRI is limited by the low signal from endogenous carbon-containing molecules and consequently requires 13C-enriched external phantoms. This study investigated the feasibility of using either 23Na-MRI or 1H-MRI to calibrate the 13C excitation.

Methods: Commercial 13C-coils were used to estimate the transmit gain and center frequency for 13C and 23Na resonances. Simulations of the transmit B1 profile of a Helmholtz loop were performed. Noise correlation was measured for both nuclei. A retrospective analysis of human data assessing the use of the 1H resonance to predict [1-13C]pyruvate center frequency was also performed. In vivo experiments were undertaken in the lower limbs of 6 pigs following injection of hyperpolarized 13C-pyruvate.

Results: The difference in center frequencies and transmit gain between tissue 23Na and [1-13C]pyruvate was reproducible, with a mean scale factor of 1.05179 ± 0.00001 and 10.4 ± 0.2 dB, respectively. Utilizing the 1H water peak, it was possible to retrospectively predict the 13C-pyruvate center frequency with a standard deviation of only 11 Hz sufficient for spectral–spatial excitation-based studies.

Conclusion: We demonstrate the feasibility of using the 23Na and 1H resonances to calibrate the 13C transmit B1 using commercially available 13C-coils. The method provides a simple approach for in vivo calibration and could improve clinical workflow.

NMR-based metabolomics and fluxomics: developments and future prospects #DNPNMR

Giraudeau, Patrick. “NMR-Based Metabolomics and Fluxomics: Developments and Future Prospects.” The Analyst 145, no. 7 (2020): 2457–72.

https://doi.org/10.1039/D0AN00142B.

NMR spectroscopy is an essential analytical technique in metabolomics and fluxomics workflows, owing to its high structural elucidation capabilities combined with its intrinsic quantitative nature. However, routine NMR “omic” analytical methods suffer from several drawbacks that may have limited their use as a method of choice, in particular when compared to another widely used technique, mass spectrometry. This review describes, in a critical and perspective discussion, how some of the most recent developments emerging from the NMR community could act as real game changers for metabolomics and fluxomics in the near future. Advanced developments to make NMR metabolomics more resolutive, more sensitive and more accessible are described, as well as new approaches to improve the identification of biomarkers. We hope that this review will convince a broad end-user community of the increasing role of NMR in the “omic” world at the beginning of the 2020s.

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