Category Archives: dDNP

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.

Hyperpolarized 13C MRI: Path to Clinical Translation in Oncology #DNPNMR

Kurhanewicz, John, Daniel B. Vigneron, Jan Henrik Ardenkjaer-Larsen, James A. Bankson, Kevin Brindle, Charles H. Cunningham, Ferdia A. Gallagher, et al. “Hyperpolarized 13C MRI: Path to Clinical Translation in Oncology.” Neoplasia 21, no. 1 (January 2019): 1–16.

https://doi.org/10.1016/j.neo.2018.09.006

This white paper discusses prospects for advancing hyperpolarization technology to better understand cancer metabolism, identify current obstacles to HP (hyperpolarized) 13C magnetic resonance imaging’s (MRI’s) widespread clinical use, and provide recommendations for overcoming them. Since the publication of the first NIH white paper on hyperpolarized 13C MRI in 2011, preclinical studies involving [1-13C]pyruvate as well a number of other 13C labeled metabolic substrates have demonstrated this technology\’s capacity to provide unique metabolic information. A dose-ranging study of HP [1-13C]pyruvate in patients with prostate cancer established safety and feasibility of this technique. Additional studies are ongoing in prostate, brain, breast, liver, cervical, and ovarian cancer. Technology for generating and delivering hyperpolarized agents has evolved, and new MR data acquisition sequences and improved MRI hardware have been developed. It will be important to continue investigation and development of existing and new probes in animal models. Improved polarization technology, efficient radiofrequency coils, and reliable pulse sequences are all important objectives to enable exploration of the technology in healthy control subjects and patient populations. It will be critical to determine how HP 13C MRI might fill existing needs in current clinical research and practice, and complement existing metabolic imaging modalities. Financial sponsorship and integration of academia, industry, and government efforts will be important factors in translating the technology for clinical research in oncology. This white paper is intended to provide recommendations with this goal in mind.

First hyperpolarized [2-13C]pyruvate MR studies of human brain metabolism

Chung, Brian T., Hsin-Yu Chen, Jeremy Gordon, Daniele Mammoli, Renuka Sriram, Adam W. Autry, Lydia M. Le Page, et al. “First Hyperpolarized [2-13C]Pyruvate MR Studies of Human Brain Metabolism.” Journal of Magnetic Resonance 309 (December 2019): 106617.

https://doi.org/10.1016/j.jmr.2019.106617.

We developed methods for the preparation of hyperpolarized (HP) sterile [2-13C]pyruvate to test its feasibility in first-ever human NMR studies following FDA-IND & IRB approval. Spectral results using this MR stable-isotope imaging approach demonstrated the feasibility of investigating human cerebral energy metabolism by measuring the dynamic conversion of HP [2-13C]pyruvate to [2-13C]lactate and [5- 13C]glutamate in the brain of four healthy volunteers. Metabolite kinetics, signal-tonoise (SNR) and area-under-curve (AUC) ratios, and calculated [2-13C]pyruvate to [2- 13C]lactate conversion rates (kPL) were measured and showed similar but not identical inter-subject values. The kPL measurements were equivalent with prior human HP [1-13C]pyruvate measurements.

[NMR] PhD scholarship in Hyperpolarization of Water for Angiography and Perfusion #DNPNMR

We have an open position as PhD student.

Application deadline is soon: Nov 15, 2019!

The PhD project is part of a EU FET Open funded consortium targeting alternatives to conventional Gadolinium based MR contrast agents for angiography and perfusion imaging. The consortium pursues several approaches, but this PhD project specifically aims to develop and study hyperpolarized water by dissolution Dynamic Nuclear Polarization for perfusion and angiography. Hyperpolarized water can provide a signal that is several orders of magnitude stronger than the tissue signal, and could enable superior angiograph and perfusion imaging. The key idea in the project is to study formulations of the hyperpolarized water that can extend the relaxation time of the water protons after injection into the blood stream, e.g. through micro-emulsions. This would shift hyperpolarized water from being an invasive, intra-arterial method to becoming equivalent in use to conventional MR contrast agents as a non-invasive, intravenous agent.

Online application at: https://www.dtu.dk/Om-DTU/Job-og-karriere/Ledige-stillinger/job?id=03a3248e-556b-47de-bfe0-92ac0c9906f0

Best regards, Jan

Jan Henrik Ardenkjær-Larsen

Professor and Section Head

Center for Magnetic Resonance

Technical University of Denmark 

Department of Health Technology

Ørsted Plads, bldg 349, office 126

DK – 2800 Kgs. Lyngby

Phone +45 45253918

Mobile +45 40272775

jhar@elektro.dtu.dk

hypermag.dtu.dk

cmr.elektro.dtu.dk

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The influence of Ho3+ doping on 13C DNP in the presence of BDPA #DNPNMR

Khattri, Ram B., Ali A. Sirusi, Eul Hyun Suh, Zoltan Kovacs, and Matthew E. Merritt. “The Influence of Ho3+ Doping on 13C DNP in the Presence of BDPA.” Physical Chemistry Chemical Physics 21, no. 34 (2019): 18629–35.

https://doi.org/10.1039/C9CP03717A

Polarization transfer from unpaired electron radicals to nuclear spins at low-temperature is achieved using microwave irradiation by a process broadly termed dynamic nuclear polarization (DNP). The resulting signal enhancement can easily exceed factors of 104 when paired with cryogenic cooling of the sample. Dissolution-DNP couples low temperature polarization methods with a rapid dissolution step, resulting in a highly polarized solution that can be used for metabolically sensitive magnetic resonance imaging (MRI). Hyperpolarized [1-13C]pyruvate is a powerful metabolic imaging agent for investigation of in vitro and in vivo cellular metabolism by means of NMR spectroscopy and MRI. Radicals (trityl OX063 and BDPA) with narrower EPR linewidths typically produce higher nuclear polarizations when carbon-13 is the target nucleus. Increased solid-state polarization is observed when narrow line radicals are doped with lanthanide ions such as Gd3+, Ho3+, Dy3+, and Tb3+. Earlier results have demonstrated an incongruence between DNP experiments with trityl and BDPA, where the optimal concentrations for polarization transfer are disparate despite similar electron spin resonance linewidths. Here, the effects of Ho-DOTA on the solid-state polarization of [1-13C]pyruvic acid were compared for 3.35 T (1.4 K) and 5 T (1.2 K) systems using BDPA as a radical. Multiple concentrations of BDPA were doped with variable concentrations of Ho-DOTA (0, 0.2, 0.5, 1, and 2 mM), and dissolved in 1 : 1 (v/v) of [1-13C] pyruvic acid/sulfolane mixture. Our results reveal that addition of small amounts of Ho-DOTA in the sample preparation increases the solid-state polarization for [1-13C] pyruvic acid, with the optimum Ho-DOTA concentration of 0.2 mM. Without Ho-DOTA doping, the optimum BDPA concentration found for 3.35 T (1.4 K) is 40 mM, and for 5 T (1.2 K) system it is about 60 mM. In both systems, inclusion of Ho-DOTA in the 13C DNP sample leads to a change in the breadth (ΔDNP) of the extrema between the P(+) and P(−) frequencies in microwave spectra. At no combination of BDPA and Ho3+ did polarizations reach those achievable with trityl. Simplified analysis of increased polarization as a function of decreased electron T1e used to explain results in trityl are insufficient to describe DNP with BDPA.

Identification of Intracellular and Extracellular Metabolites in Cancer Cells Using 13C Hyperpolarized Ultrafast Laplace NMR #DNPNMR

Zhang, Guannan, Susanna Ahola, Mathilde H. Lerche, Ville-Veikko Telkki, and Christian Hilty. “Identification of Intracellular and Extracellular Metabolites in Cancer Cells Using 13C Hyperpolarized Ultrafast Laplace NMR.” Analytical Chemistry 90, no. 18 (September 18, 2018): 11131–37.

https://doi.org/10.1021/acs.analchem.8b03096

Ultrafast Laplace NMR (UF-LNMR), which is based on the spatial encoding of multidimensional data, enables one to carry out 2D relaxation and diffusion measurements in a single-scan. Besides reducing the experiment time to a fraction, it significantly facilitates the use of nuclear spin hyperpolarization to boost experimental sensitivity, because the time consuming polarization step does not need to be repeated. Here we demonstrate the usability of hyperpolarized UF-LNMR in the context of cell metabolism, by investigating the conversion of pyruvate to lactate in the cultures of mouse 4T1 cancer cells. We show that 13C ultrafast diffusion – T2 relaxation correlation measurements, with the sensitivity enhanced by several orders of magnitude by dissolution dynamic nuclear polarization (DDNP), allows the determination of the extra- vs. intracellular location of metabolites due to their significantly different values of diffusion coefficients and T2 relaxation times. Under the current conditions, pyruvate located predominantly in the extracellular pool, while lactate remained primarily intracellular. Contrary to the small flip angle diffusion methods reported in the literature, the UFLNMR method does not require several scans with varying gradient strength, and it provides a combined diffusion and T2 contrast. Furthermore, the ultrafast concept can be extended to various other multidimensional LNMR experiments, which will provide detailed information about the dynamics and exchange processes of cell metabolites.

Metabolic Measurements of Nonpermeating Compounds in Live Cells Using Hyperpolarized NMR

Liu, Mengxiao, and Christian Hilty. “Metabolic Measurements of Nonpermeating Compounds in Live Cells Using Hyperpolarized NMR.” Analytical Chemistry 90, no. 2 (January 16, 2018): 1217–22.

https://doi.org/10.1021/acs.analchem.7b03901

Hyperpolarization by dissolution dynamic nuclear polarization (D-DNP) has emerged as a technique for enhancing NMR signals by several orders of magnitude, thereby facilitating the characterization of metabolic pathways both in vivo and in vitro. Following the introduction of an externally hyperpolarized compound, real-time NMR enables the measurement of metabolic flux in the corresponding pathway. Spin relaxation however limits the maximum experimental time and prevents the use of this method with compounds exhibiting slow membrane transport rates. Here, we demonstrate that electroporation can serve as a method for membrane permeabilization for use with D-DNP in cell cultures. An electroporation apparatus hyphenated with stopped flow sample injection permits the introduction of the hyperpolarized metabolite within 3 s after the electrical pulse. In yeast cells that do not readily take up pyruvate, the addition of the electroporation pulse to the D-DNP experiment increases the signals of the downstream metabolic products CO2 and HCO3 -, which otherwise are near the detection limit, by 8.2 and 8.6-fold. Modeling of the time dependence of these signals then permits the determination of the respective kinetic rate constants. The observed conversion rate from pyruvate to CO2 normalized for cell density was found to increase by a factor of 12 due to the alleviation of the membrane transport limitation. Using electroporation therefore extends the applicability of D-DNP to in vitro studies to a wider range of metabolites, and at the same time reduces the influence of membrane transport on the observed conversion rates.

Chiral Recognition by Dissolution DNP NMR Spectroscopy of 13C-Labeled dl-Methionine

Monteagudo, Eva, Albert Virgili, Teodor Parella, and Míriam Pérez-Trujillo. “Chiral Recognition by Dissolution DNP NMR Spectroscopy of 13C-Labeled Dl-Methionine.” Analytical Chemistry 89, no. 9 (May 2, 2017): 4939–44.

https://doi.org/10.1021/acs.analchem.7b00156

A method based on d-DNP NMR spectroscopy to study chiral recognition is described for the first time. The enantiodifferentiation of a racemic metabolite in a millimolar aqueous solution using a chiral solvating agent was performed. Hyperpolarized 13C-labeled DL-methionine enantiomers were differently observed with a single-scan 13C NMR experiment, while the chiral auxiliary at thermal equilibrium remained unobserved. The method developed entails a step forward in the chiral recognition of small molecules by NMR spectroscopy, opening new possibilities in situations where the sensitivity is limited; e.g. when a low concentration of analyte is available or when the measurement of an insensitive nucleus, like 13C, is required. The advantages and current limitations of the developed method, as well as, future perspectives are discussed.

Characterization of Chemical Exchange Using Relaxation Dispersion of Hyperpolarized Nuclear Spins #DNPNMR

Liu, Mengxiao, Yaewon Kim, and Christian Hilty. “Characterization of Chemical Exchange Using Relaxation Dispersion of Hyperpolarized Nuclear Spins.” Analytical Chemistry 89, no. 17 (September 5, 2017): 9154–58.

https://doi.org/10.1021/acs.analchem.7b01896

Chemical exchange phenomena are ubiquitous in macromolecules, which undergo conformational change or ligand complexation. NMR relaxation dispersion (RD) spectroscopy based on a Carr-Purcell-Meiboom-Gill pulse sequence is widely applied to identify the exchange and measure the life time of intermediate states on the millisecond time scale. Advances in hyperpolarization methods improve the applicability of NMR spectroscopy when rapid acquisitions or low concentrations are required, through an increase in signal strength by several orders of magnitude. Here, we demonstrate the measurement of chemical exchange from a single aliquot of a ligand hyperpolarized by dissolution dynamic nuclear polarization (D-DNP). Transverse relaxation rates are measured simultaneously at different pulsing delays by dual-channel 19F NMR spectroscopy. This two-point measurement is shown to allow the determination of the exchange term in the relaxation rate expression. For the ligand 4-(trifluoromethyl)benzene-1-carboximidamide binding to the protein trypsin, the exchange term is found to be equal within error limits in neutral and acidic environments from D-DNP NMR spectroscopy, corresponding to a pre-equilibrium of trypsin deprotonation. This finding illustrates the capability for determination of binding mechanisms using D-DNP RD. Taking advantage of hyperpolarization, the ligand concentration in the exchange measurements can reach on the order of tens of μM and protein concentration can be below 1 μM, i.e. conditions typically accessible in drug discovery.

Highly Repeatable Dissolution Dynamic Nuclear Polarization for Heteronuclear NMR Metabolomics #DNPNMR

Bornet, Aurélien, Mickaël Maucourt, Catherine Deborde, Daniel Jacob, Jonas Milani, Basile Vuichoud, Xiao Ji, et al. “Highly Repeatable Dissolution Dynamic Nuclear Polarization for Heteronuclear NMR Metabolomics.” Analytical Chemistry 88, no. 12 (June 21, 2016): 6179–83. 

https://doi.org/10.1021/acs.analchem.6b01094

At natural 13C abundance, metabolomics based on heteronuclear NMR is limited by sensitivity. We have recently demonstrated how hyperpolarization by dissolution dynamic nuclear polarization (D-DNP) assisted by cross-polarization (CP) provides a reliable way of enhancing the sensitivity of heteronuclear NMR in dilute mixtures of metabolites. In this Technical Note, we evaluate the precision of this experimental approach, a critical point for applications to metabolomics. The higher the repeatability, the greater the likelihood that one can detect small biologically relevant differences between samples. The average repeatability of our state-of-the-art D-DNP NMR equipment for samples of metabolomic relevance (20 mg dry weight tomato extracts) is 3.6% for signals above the limit of quantification (LOQ), and 6.4% when all the signals above the limit of detection (LOD) are taken into account. This first report on the repeatability of D-DNP highlights the compatibility of the technique with the requirements of metabolomics, and confirms its potential as an analytical tool for such applications.

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