Category Archives: dDNP

Glyoxalase activity in human erythrocytes and mouse lymphoma, liver and brain probed with hyperpolarized 13C-methylglyoxal #DNPNMR #dDNP

Shishmarev, Dmitry, Philip W. Kuchel, Guilhem Pagès, Alan J. Wright, Richard L. Hesketh, Felix Kreis, and Kevin M. Brindle. “Glyoxalase Activity in Human Erythrocytes and Mouse Lymphoma, Liver and Brain Probed with Hyperpolarized 13C-Methylglyoxal.” Communications Biology 1, no. 1 (December 2018): 232.

https://doi.org/10.1038/s42003-018-0241-1

Methylglyoxal is a faulty metabolite. It is a ubiquitous by-product of glucose and amino acid metabolism that spontaneously reacts with proximal amino groups in proteins and nucleic acids, leading to impairment of their function. The glyoxalase pathway evolved early in phylogeny to bring about rapid catabolism of methylglyoxal, and an understanding of the role of methylglyoxal and the glyoxalases in many diseases is beginning to emerge. Metabolic processing of methylglyoxal is very rapid in vivo and thus notoriously difficult to detect and quantify. Here we show that 13C nuclei in labeled methylglyoxal can be hyperpolarized using dynamic nuclear polarization, providing 13C nuclear magnetic resonance signal enhancements in the solution state close to 5,000-fold. We demonstrate the applications of this probe of metabolism for kinetic characterization of the glyoxalase system in isolated cells as well as mouse brain, liver and lymphoma in vivo.

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.

Simultaneous T1 and T2 mapping of hyperpolarized 13C compounds using the bSSFP sequence #DNPNMR

Milshteyn, Eugene, Galen D. Reed, Jeremy W. Gordon, Cornelius von Morze, Peng Cao, Shuyu Tang, Andrew P. Leynes, Peder E.Z. Larson, and Daniel B. Vigneron. “Simultaneous T1 and T2 Mapping of Hyperpolarized 13C Compounds Using the BSSFP Sequence.” Journal of Magnetic Resonance 312 (March 2020): 106691.

https://doi.org/10.1016/j.jmr.2020.106691

As in conventional 1H MRI, T1 and T2 relaxation times of hyperpolarized (HP) 13C nuclei can provide important biomedical information. Two new approaches were developed for simultaneous T1 and T2 mapping of HP 13C probes based on balanced steady state free precession (bSSFP) acquisitions: a method based on sequential T1 and T2 mapping modules, and a model-based joint T1/T2 approach analogous to MR fingerprinting. These new methods were tested in simulations, HP 13C phantoms, and in vivo in normal Sprague-Dawley rats. Non-localized T1 values, low flip angle EPI T1 maps, bSSFP T2 maps, and Bloch-Siegert B1 maps were also acquired for comparison. T1 and T2 maps acquired using both approaches were in good agreement with both literature values and data from comparative acquisitions. Multiple HP 13C compounds were successfully mapped, with their relaxation time parameters measured within heart, liver, kidneys, and vasculature in one acquisition for the first time.

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.

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.

[NMR] PhD Position in Dissolution DNP at the University of Vienna #DNPNMR

Dear colleagues,

a 4-year Ph.D. position at the institute of biological chemistry at the University of Vienna is available in the field of dissolution DNP with a special focus on biomineralization.

A project description can be found below. I would like to invite interested students to send an application including a letter of motivation (German or English) directly to:

dennis.kurzbach@univie.ac.at.

With kind regards,

Dennis Kurzbach

Project Description:

The DNP group of the University Vienna (www.Vienna-DNP.at) is looking for a motivated student to carry out a PhD thesis project centered around novel (hyperpolarization) tools based on NMR spectroscopy for the characterization of biomineralization processes.

Our lab is working on combining dissolution dynamic nuclear polarization (DDNP) [1], a technique to improve signal intensities in NMR spectra, with time-resolved detection of biomolecular NMR spectra on milliseconds to minutes time-scales. Our lab is equipped with 8 state-of-the-art NMR devices and integrated into a fully equipped biochemistry facility.

The project is centered around the understanding of biomineralization processes, i.e. the ability of living organisms to produce solid phases, and aims at the development of time-resolved models that describe the mineralization events at an atomistic level of resolution to obtain a better understanding of the biological function of the underlying molecular interactions. In other words, an idea shell be developed of how biominerals form with time and of how individual atoms act at different points in time.

The interdisciplinary PhD project combines biochemical and biophysical research of medicinal and societal relevance with cutting-edge instrumentational and methodological developments of the NMR technology.

The successful candidate holds a relevant degree in physics, chemistry, biochemistry or a similar field. She/he will work on the development of NMR techniques such as ultrafast measurements in combination with signal-improved NMR to characterize the interaction of designer peptides with calcium phosphates and silicates. These experiments allow determining the state of a protein in less than 1 s, which will eventually allow one to develop a model of the mineral formation to guide future developments in materials design and fundamental biophysics.

The University of Vienna aims at increasing the employment of women in both managing and academic positions and therefore invites applications from qualified female candidates.

The University of Vienna offers

  • a dynamic research location with well-established research funding provisions
  • attractive working conditions in a city with a high quality of life
  • comprehensive advice and support in relation to finding accommodation, change of schools and dual career
  • wide range of support services offered by central service
  • institutions Funding Notes

References

[1] Dennis Kurzbach, Sami Jannin: Dissolution Dynamic Nuclear

Polarization Methodology and Instrumentation, eMagRes, 2018, 7, DOI:

10.1002/9780470034590.emrstm1563.

___________________________________

Ass.-Prof. Dr. Dennis Kurzbach

University of Vienna

Faculty of Chemistry

Institute of Biological Chemistry

Währinger Str. 38

1070 Vienna

Austria

www.Vienna-DNP.at

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In vivo melanoma imaging based on dynamic nuclear polarization enhancement in melanin pigment of living mice using in vivo dynamic nuclear polarization magnetic resonance imaging #DNPNMR

Hyodo, Fuminori, Tatsuya Naganuma, Hinako Eto, Masaharu Murata, Hideo Utsumi, and Masayuki Matsuo. “In Vivo Melanoma Imaging Based on Dynamic Nuclear Polarization Enhancement in Melanin Pigment of Living Mice Using in Vivo Dynamic Nuclear Polarization Magnetic Resonance Imaging.” Free Radical Biology and Medicine 134 (April 2019): 99–105.

https://doi.org/10.1016/j.freeradbiomed.2019.01.002

Melanin is a pigment that includes free radicals and is widely distributed in living animals. Malignant melanoma is one of the most progressive tumors in humans with increasing incidence worldwide, and has shown resistance to chemotherapy, resulting in high mortality at the metastatic stage. In general, melanoma involves the abnormal accumulation of melanin pigment produced by malignant melanocytes. Electron paramagnetic resonance (EPR) spectroscopy and imaging is a powerful technique to directly visualize melanomas using endogenous free radicals in the melanin pigment. Because melanin radicals have a large linewidth, the low spatial resolution of EPR imaging results in blurred images and a lack of anatomical information. Dynamic nuclear polarization (DNP)-MRI is a noninvasive imaging method to obtain the spatio-temporal information of free radicals with MRI anatomical resolution. Proton signals in tissues, including free radicals, can be dramatically enhanced by EPR irradiation at the resonance frequency of the free radical prior to applying the MRI pulse sequence. However, the DNP effects of free radicals in the pigment of living organisms is unclear. Therefore, if endogenous free radicals in melanin pigment could be utilized as a bio-probe for DNP-MRI, this will be an advantage for the specific enhancement of melanoma tissues and might allow the separate noninvasive visualization of melanoma tissues without the need for probe administration. Here, we report that biological melanin pigment induced a in vivo DNP effect by interacting with water molecules. In addition, we demonstrated in vivo melanoma imaging based on the DNP effects of endogenous free radicals in the melanin pigment of living mice.

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