Hyperpolarized NMR Research Articles

Nuclear hyperpolarization in electron-transfer proteins: Revealing unexpected light-induced 15N signals with field-cycling magic-angle spinning NMR

The solid-state photo-CIDNP (photo-chemically induced dynamic nuclear polarization) effect allows for nuclear hyperpolarization, i.e., non-Boltzmann nuclear spin population. The effect relies on the light-induced formation of a spin-correlated radical pair (SCRP) and has been observed in various photosynthetic reaction center (RC) proteins and flavin-containing light, oxygen, voltage (LOV) proteins. Both systems exhibit strongly enhanced NMR signals originating from the electron transfer partners. Here, we present experimental data on the magnetic field dependence of the 15N solid-state photo-CIDNP effect in both phototropin LOV1 C57S from Chlamydomonas reinhardtii and the bacterial photosynthetic RC from Rhodobacter sphaeroides. Using a pneumatic field-cycling system, samples containing a frozen solution of the proteins are explored between 0.25 T and 9.4 T. Both systems yield hyperpolarized 15N NMR signals across the entire magnetic field range originating from the electron transfer moieties. Also, in both systems, hyperpolarized signals from unexpected positions are detected between 1.0 T and 2.0 T: position N-1 of the flavin in the LOV1 protein and the τ-N of the axial magnesium-coordinating histidine of the donor. A first attempt to explain the occurrence of these unexpected signals based on quantum chemical calculations is presented.

Differentiating leukemia subtypes based on metabolic signatures using hyperpolarized 13C NMR.

Leukemia is a group of blood cancers that are classified in four major classes. Within these four classes, many different subtypes exists with similar origin, genetic mutations, and level of maturity, which can make them difficult to distinguish. Despite their similarities, they might respond differently to treatment, and therefore distinguishing between them is of crucial importance. A deranged metabolic phenotype (Warburg effect) is often seen in cancer cells, leukemia cells included, and is increasingly a target for improved diagnosis and treatment. In this study, hyperpolarized 13C NMR spectroscopy was used to characterize the metabolic signatures of the six leukemia cell lines ML-1, CCRF-CEM, THP-1, MOLT-4, HL-60, and K562. This was done using [1-13C]pyruvate and [1-13C]alanine as bioprobes for downstream metabolite quantification and kinetic analysis on cultured cells with and without 2-deoxy-D-glucose treatment. The metabolic signatures of similar leukemia subtypes could be readily distinguished. This includes ML-1 and THP-1, which are of the similar M4 and M5 AML subtypes, CCRF-CEM and MOLT-4, which are of the similar T-ALL lineage at different maturation states, and HL-60 and K562, which are of the closely related M1 and M2 AML subtypes. The data presented here demonstrate the potential of hyperpolarized 13C NMR spectroscopy as a method to differentiate between leukemia subtypes of similar origin. Combining this method with bioreactor setups could potentially allow for better leukemia disease management as metabolic signatures could be acquired from a single biopsy through repeated experimentation and intervention.

A patient-derived cell model for malignant transformation in IDH-mutant glioma

Malignant transformation (MT) is commonly seen in IDH-mutant gliomas. There has been a growing research interest in revealing its underlying mechanisms and intervening prior to MT at the early stages of the transforming process. Here we established a unique pair of matched 3D cell models: 403L, derived from a low-grade glioma (LGG), and 403H, derived from a high-grade glioma (HGG), by utilizing IDH-mutant astrocytoma samples from the same patient when the tumor was diagnosed as WHO grade 2 (tumor mutational burden (TMB) of 3.96/Mb) and later as grade 4 (TMB of 70.07/Mb), respectively. Both cell models were authenticated to a patient’s sample retaining endogenous expression of IDH1 R132H. DNA methylation profiles of the parental tumors referred to LGG and HGG IDH-mutant glioma clusters. The immunopositivity of SOX2, NESTIN, GFAP, OLIG2, and beta 3-Tubulin suggested the multilineage potential of both models. 403H was more prompt to cell invasion and developed infiltrative HGG in vivo. The differentially expressed genes (DEGs) from the RNA sequencing analysis revealed the tumor invasion and aggressiveness related genes exclusively upregulated in the 403H model. Pathway analysis showcased an enrichment of genes associated with epithelial-mesenchymal transition (EMT) and Notch signaling pathways in 403H and 403L, respectively. Mass spectrometry-based targeted metabolomics and hyperpolarized (HP) 1-13C pyruvate in-cell NMR analyses demonstrated significant alterations in the TCA cycle and fatty acid metabolism. Citrate, glutamine, and 2-HG levels were significantly higher in 403H. To our knowledge, this is the first report describing the development of a matched pair of 3D patient-derived cell models representative of MT and temozolomide (TMZ)-induced hypermutator phenotype (HMP) in IDH-mutant glioma, providing insights into genetic and metabolic changes during MT/HMP. This novel in vitro model allows further investigation of the mechanisms of MT at the cellular level.Graphic

Trapping Highly Reactive Metal(H)2(η2‐H2) Species to Form Trihydride Complexes and Clusters

AbstractMetal dihydride dihydrogen‐derived intermediates can be short‐lived and difficult to detect despite their ubiquitous role in chemical transformations such as hydrogenation. Here, highly reactive [Ir(H)3(η2‐η2‐COD)(IMes)] forms by trapping [Ir(H)2(η2‐H2)(η2‐η2‐COD)(IMes)]X [X=Cl or MeO], resulting from reaction of the Ir(I) precatalyst [IrCl(η2‐η2‐COD)(IMes)] with a base and H2. This trihydride allows access to the unusual [Ir2(H)4(OMe)(IMes)2(pyridine)3]X, [Ir2(H)2(μ2‐H)2(η2‐η2‐COD)(IMes)2], [Ir3(H)9(IMes)3], [Ir4(H)12(IMes)4] and [Ir2(H)4(μ2‐H)2(IMes)2(pyridine)2]. A combination of 2D NMR, parahydrogen hyperpolarized NMR, MS, and XRD are used to characterise these reaction products and rationalise their formation via the moiety {Ir(H)3(IMes)}. Consequently, the results of these studies have implications for iridium‐catalyzed polarization transfer from parahydrogen via the well‐known precursor [IrCl(η2‐η2‐COD)(IMes)].

Rapid Protein-Ligand Affinity Determination by Photoinduced Hyperpolarized NMR.

The binding affinity determination of protein-ligand complexes is a cornerstone of drug design. State-of-the-art techniques are limited by lengthy and expensive processes. Building upon our recently introduced novel screening method utilizing photochemically induced dynamic nuclear polarization (photo-CIDNP) NMR, we provide the methodological framework to determine binding affinities within 5-15 min using 0.1 mg of protein. The accuracy of our method is demonstrated for the affinity constants of peptides binding to a PDZ domain and fragment ligands binding to the protein PIN1. The method can also be extended to measure the affinity of nonphoto-CIDNP-polarizable ligands in competition binding experiments. Finally, we demonstrate a strong correlation between the ligand-reduced signals in photo-CIDNP-based NMR fragment screening and the well-established saturation transfer difference (STD) NMR. Thus, our methodology measures protein-ligand affinities in the micro- to millimolar range in only a few minutes and informs on the binding epitope in a single-scan experiment, opening new avenues for early stage drug discovery approaches.

Polarization losses from the nonadiabatic passage of hyperpolarized solutions through metallic components

From complex-mixture analysis to in vivo molecular imaging, applications of liquid-state nuclear spin hyperpolarization have expanded widely over recent years. In most cases, hyperpolarized solutions are generated ex situ and transported from the polarization instrument to the measurement device. The sample hyperpolarization usually survives this transport, since the changes in magnetic fields that are external to the sample are typically adiabatic (slow) with respect to the internal nuclear spin dynamics. The passage of polarized samples through weakly magnetic components such as stainless steel syringe needles and ferrules is not always adiabatic, which can lead to near-complete destruction of the magnetization. To avoid this effect becoming “folklore” in the field of hyperpolarized NMR, we present a systematic investigation to highlight the problem and investigate possible solutions. Experiments were carried out on: (i) dissolution-DNP-polarized [1-13C]pyruvate with NMR detection at 1.4T, and (ii) 1.5-T-polarized H2O with NMR detection at 2.5μT. We show that the degree of adiabaticity of solutions passing through metal parts is intrinsically unpredictable, likely depending on many factors such as solution flow rate, degree of remanent ferromagnetism in the metal, and nuclear spin species. However, the magnetization destruction effects can be suppressed by application of an external field on the order of 0.1–10mT.

Hyperpolarized 1H and 13C NMR Spectroscopy in a Single Experiment for Metabolomics.

The application of NMR spectroscopy to complex mixture analysis and, in particular, to metabolomics is limited by the low sensitivity of NMR. We recently showed that dissolution dynamic nuclear polarization (d-DNP) could enhance the sensitivity of 13C NMR for complex metabolite mixtures, leading to the detection of highly sensitive 13C NMR fingerprints of complex samples such as plant extracts or urine. While such experiments provide heteronuclear spectra, which are complementary to conventional NMR, hyperpolarized 1H NMR spectra would also be highly useful, with improved limits of detection and compatibility with the existing metabolomics workflow and databases. In this technical note, we introduce an approach capable of recording both 1H and 13C hyperpolarized spectra of metabolite mixtures in a single experiment and on the same hyperpolarized sample. We investigate the analytical performance of this method in terms of sensitivity and repeatability, and then we show that it can be efficiently applied to a plant extract. Significant sensitivity enhancements in 1H NMR are reported with a repeatability suitable for metabolomics studies without compromising on the performance of hyperpolarized 13C NMR. This approach provides a way to perform both 1H and 13C hyperpolarized NMR metabolomics with unprecedented sensitivity and throughput.

Substrate interaction of graphene quantum dots in solution studied by hyperpolarized NMR

The reversible adsorption of CO2-derived species to graphene quantum dots in CO2 electroreduction reaction was characterized by nuclear spin hyperpolarized NMR. Hyperpolarization of 13C-labeled sodium bicarbonate by the dissolution dynamic nuclear polarization (DNP) technique provided a 5000-fold signal enhancement and enabled real-time and in-situ 13C NMR experiments. The R1 and R2 relaxation rates were measured using time-resolved DNP-NMR experiments, and the bound species were confirmed to be carbonic acid and its conjugates. A significant difference in the relaxation rates of bound and free species was found, indicating a reversible binding. In addition to relaxation rates, kinetic rate constants were fitted from the small-flip angle experiments and compared to theoretical calculations. The pH-dependent adsorption and desorption rate constants were found in the range of 3.5–9.5 and 0.7–2.0 s−1, with the average binding distance at 279 - 291 pm. The DNP-NMR technique was demonstrated as an effective way to study surface adsorption, with potential in electrochemistry and kinetics applications.

Modelling and correcting the impact of RF pulses for continuous monitoring of hyperpolarized NMR.

Monitoring the build-up or decay of hyperpolarization in nuclear magnetic resonance requires radio-frequency (RF) pulses to generate observable nuclear magnetization. However, the pulses also lead to a depletion of the polarization and, thus, alter the spin dynamics. To simulate the effects of RF pulses on the polarization build-up and decay, we propose a first-order rate-equation model describing the dynamics of the hyperpolarization process through a single source and a relaxation term. The model offers a direct interpretation of the measured steady-state polarization and build-up time constant. Furthermore, the rate-equation model is used to study three different methods to correct the errors introduced by RF pulses: (i)a correction ( denoting the RF pulse flip angle), which is only applicable to decays; (ii)an analytical model introduced previously in the literature; and (iii)an iterative correction approach proposed here. The three correction methods are compared using simulated data for a range of RF flip angles and RF repetition times. The correction methods are also tested on experimental data obtained with dynamic nuclear polarization (DNP) using 4-oxo-TEMPO in H glassy matrices. It is demonstrated that the analytical and iterative corrections allow us to obtain accurate build-up times and steady-state polarizations (enhancements) for RF flip angles of up to 25 during the polarization build-up process within % error when compared to data acquired with small RF flip angles ( ). For polarization decay experiments, corrections are shown to be accurate for RF flip angles of up to 12. In conclusion, the proposed iterative correction allows us to compensate for the impact of RF pulses offering an accurate estimation of polarization levels, build-up and decay time constants in hyperpolarization experiments.

In-Cell NMR Approach for Real-Time Exploration of Pathway Versatility: Substrate Mixtures in Nonengineered Yeast.

The central carbon metabolism of microbes will likely be used in future sustainable bioproduction. A sufficiently deep understanding of central metabolism would advance the control of activity and selectivity in whole-cell catalysis. Opposite to the more obvious effects of adding catalysts through genetic engineering, the modulation of cellular chemistry through effectors and substrate mixtures remains less clear. NMR spectroscopy is uniquely suited for in-cell tracking to advance mechanistic insight and to optimize pathway usage. Using a comprehensive and self-consistent library of chemical shifts, hyperpolarized NMR, and conventional NMR, we probe the versatility of cellular pathways to changes in substrate composition. Conditions for glucose influx into a minor pathway to an industrial precursor (2,3-butanediol) can thus be designed. Changes to intracellular pH can be followed concurrently, while mechanistic details for the minor pathway can be derived using an intermediate-trapping strategy. Overflow at the pyruvate level can be induced in nonengineered yeast with suitably mixed carbon sources (here glucose with auxiliary pyruvate), thus increasing glucose conversion to 2,3-butanediol by more than 600-fold. Such versatility suggests that a reassessment of canonical metabolism may be warranted using in-cell spectroscopy.

Pathways and their usage in the conversion of carbohydrates by aqueous barium hydroxide: insights from hyperpolarized and quantitative NMR

Elusive intermediates and products were visualized in the conversion of glucose in aqueous barium hydroxide. Competing pathways resembling different biochemical glycolysis pathways were observed in this manner.

Competitive Displacement Restores the Hyperpolarized 15N NMR Signal in Blood Plasma.

Hyperpolarized (HP) NMR can improve the sensitivity of conventional NMR experiments by several orders of magnitude, thereby making it feasible to detect the signal of low sensitivity nuclei such as 13C and 15N nuclei in vivo. Hyperpolarized substrates are usually administered by direct injection into the bloodstream, and interaction with serum albumin can cause rapid decay of the hyperpolarized signal due to the shortening of the spin-lattice (T1) relaxation time. Here we report that the 15N T1 of 15N labeled, partially deuterated tris(2-pyridylmethyl)amine decreases dramatically upon binding to albumin to such an extent that no HP-15 signal could be detected. We also demonstrate that the signal could be restored using a competitive displacer, iophenoxic acid, which binds stronger to albumin than tris(2-pyridylmethyl)amine. The methodology presented here eliminates the undesirable effect of albumin binding and should widen the range of hyperpolarized probes for in vivo studies.

Fine optimization of a dissolution dynamic nuclear polarization experimental setting for 13C NMR of metabolic samples.

NMR-based analysis of metabolite mixtures provides crucial information on biological systems but mostly relies on 1D H experiments for maximizing sensitivity. However, strong peak overlap of H spectra often is a limitation for the analysis of inherently complex biological mixtures. Dissolution dynamic nuclear polarization (d-DNP) improves NMR sensitivity by several orders of magnitude, which enables C NMR-based analysis of metabolites at natural abundance. We have recently demonstrated the successful introduction of d-DNP into a full untargeted metabolomics workflow applied to the study of plant metabolism. Here we describe the systematic optimization of d-DNP experimental settings for experiments at natural C abundance and show how the resolution, sensitivity, and ultimately the number of detectable signals improve as a result. We have systematically optimized the parameters involved (in a semi-automated prototype d-DNP system, from sample preparation to signal detection, aiming at providing an optimization guide for potential users of such a system, who may not be experts in instrumental development). The optimization procedure makes it possible to detect previously inaccessible protonated C signals of metabolites at natural abundance with at least 4times improved line shape and a high repeatability compared to a previously reported d-DNP-enhanced untargeted metabolomic study. This extends the application scope of hyperpolarized C NMR at natural abundance and paves the way to a more general use of DNP-hyperpolarized NMR in metabolomics studies.

Monitoring of CaCO3 Nanoscale Structuration through Real-Time Liquid Phase Transmission Electron Microscopy and Hyperpolarized NMR.

Calcium carbonate (CaCO3) is one of the most significant biominerals in nature. Living organisms are able to control its biomineralization by means of an organic matrix to tailor a myriad of hybrid functional materials. The soluble organic components are often proteins rich in acidic amino-acids such as l-aspartic acid. While several studies have demonstrated the influence of amino acids on the crystallization of calcium carbonate, nanoscopic insight of their impact on CaCO3 mineralization, in particular at the early stages, is still lacking. Herein, we implement liquid phase-transmission electron microscopy (LP-TEM) in order to visualize in real-time and at the nanoscale the prenucleation stages of CaCO3 formation. We observe that l-aspartic acid favors the formation of individual and aggregated prenucleation clusters which are found stable for several minutes before the transformation into amorphous nanoparticles. Combination with hyperpolarized solid state nuclear magnetic resonance (DNP NMR) and density functional theory (DFT) calculations allow shedding light on the underlying mechanism at the prenucleation stage. The promoting nature of l-aspartic acid with respect to prenucleation clusters is explained by specific interactions with both Ca2+ and carbonates and the stabilization of the Ca2+-CO32-/HCO3- ion pairs favoring the formation and stabilization of the CaCO3 transient precursors. The study of prenucleation stages of mineral formation by the combination of in situ LP-TEM, advanced analytical techniques (including hyperpolarized solid-state NMR), and numerical modeling allows the real-time monitoring of prenucleation species formation and evolution and the comprehension of their relative stability.

Analysis of Complex Mixtures by Chemosensing NMR Using para-Hydrogen-Induced Hyperpolarization.

ConspectusNuclear magnetic resonance (NMR) is a powerful technique for chemical analysis. The use of NMR to investigate dilute analytes in complex systems is, however, hampered by its relatively low sensitivity. An additional obstacle is represented by the NMR signal overlap. Because solutes in a complex mixture are usually not isotopically labeled, NMR studies are often limited to 1H measurements, which, because of the modest dispersion of the 1H resonances (typically ∼10 ppm), can result in challenging signal crowding. The low NMR sensitivity issue can be alleviated by nuclear spin hyperpolarization (i.e., transiently increasing the differences in nuclear spin populations), which determines large NMR signal enhancements. This has been demonstrated for hyperpolarization methods such as dynamic nuclear polarization, spin-exchange optical pumping and para-hydrogen-induced polarization (PHIP). In particular, PHIP has grown into a fast, efficient, and versatile technique since the recent discovery of non-hydrogenative routes to achieve nuclear spin hyperpolarization.For instance, signal amplification by reversible exchange (SABRE) can generate proton as well as heteronuclear spin hyperpolarization in a few seconds in compounds that are able to transiently bind to an iridium catalyst in the presence of para-hydrogen in solution. The hyperpolarization transfer catalyst acts as a chemosensor in the sense that it is selective for analytes that can coordinate to the metal center, such as nitrogen-containing aromatic heterocycles, sulfur heteroaromatic compounds, nitriles, Schiff bases, diaziridines, carboxylic acids, and amines. We have demonstrated that the signal enhancement achieved by SABRE allows rapid NMR detection and quantification of a mixture of substrates down to low-micromolar concentration. Furthermore, in the transient complex, the spin configuration of p-H2 can be easily converted to spin hyperpolarization to produce up to 1000-fold enhanced NMR hydride signals. Because the hydrides’ chemical shifts are highly sensitive to the structure of the analyte associating with the iridium complex, they can be employed as hyperpolarized “probes” to signal the presence of specific compounds in the mixture. This indirect detection of the analytes in solution provides important benefits in the case of complex systems, as hydrides resonate in a region of the 1H spectrum (at ca. −20 ppm) that is generally signal-free. The enhanced sensitivity provided by non-hydrogenative PHIP (nhPHIP), together with the absence of interference from the complex matrix (usually resonating between 0 and 10 ppm), set the detection limit for this NMR chemosensor down to sub-μM concentrations, approximately 3 orders of magnitude lower than for conventional NMR. This nhPHIP approach represents, therefore, a powerful tool for NMR analysis of dilute substrates in complex mixtures as it addresses at once the issues of signal crowding and NMR sensitivity. Importantly, being performed at high field inside the NMR spectrometer, the method allows for rapid acquisition of multiple scans, multidimensional hyperpolarized NMR spectra, in a fashion comparable to that of standard NMR measurements.In this Account, we focus on our chemosensing NMR technology, detailing its principles, advantages, and limitations and presenting a number of applications to real systems such as biofluids, beverages, and natural extracts.

LED-Based Photo-CIDNP Hyperpolarization Enables 19F MR Imaging and 19F NMR Spectroscopy of 3-Fluoro-DL-tyrosine at 0.6 T

Although 19F has high potential to serve as a background-free molecular marker in bioimaging, the molar amount of marker substance is often too small to enable 19F MR imaging or 19F NMR spectroscopy with a sufficiently high signal-to-noise ratio (SNR). Hyperpolarization methods such as parahydrogen-based hyperpolarization or dynamic nuclear polarization (DNP) can significantly improve the SNR, but require expensive and complex sample preparation and the removal of toxic catalysts and solvents. Therefore, we used the biologically compatible model of the fluorinated amino acid 3-Fluoro-DL-tyrosine with riboflavin 5ʹ-monophosphate (FMN) as a chromophore dissolved in D2O with 3.4% H2Odest., allowing to transform light energy into hyperpolarization of the 19F nucleus via photo-chemically induced dynamic nuclear polarization (photo-CIDNP). We used a low-cost high-power blue LED to illuminate the sample replacing traditionally used laser excitation, which is both potentially harmful and costly. For the first time, we present results of hyperpolarized 19F MRI and 19F NMR performed with a low-cost 0.6 T benchtop MRI system. The device allowed simultaneous dual-channel 1H/19F NMR. 19F imaging was performed with a (0.94 mm)2 in-plane resolution. This enabled the spatial resolution of different degrees of hyperpolarization within the sample. We estimated the photo-CIDNP-based 19F signal enhancement at 0.6 T to be approximately 465. FMN did not bleach out even after multiple excitations, so that the signal-to-noise ratio could be further improved by averaging hyperpolarized signals. The results show that the easy-to-use experimental setup has a high potential to serve as an efficient preclinical tool for hyperpolarization studies in bioimaging.

Comprehensive Quantitative and Calibration-Free Evaluation of Hyperpolarized Xenon–Host Interaction by Multiparametric NMR

The probing of microscopic environments by hyperpolarized xenon NMR has spurred investigations in supramolecular chemistry as well as important biosensing and molecular imaging applications. While xenon exchange with host structures at micromolar concentrations and below can be readily detected, a quantitative analysis is limited, requiring complementary experimentation by different methodologies and thus lacking completeness and compromising the validity and comparability of numerical results. Here, a new NMR measurement and data analysis approach is introduced for the comprehensive characterization of the host-xenon binding dynamics. The application of chemical exchange saturation transfer of hyperpolarized 129Xe under parametric modulation of the saturation RF amplitude and xenon gas saturation of the solution enables a delineation of exchange mechanisms and, through modeling, a numerical estimation of the various reaction rate constants (and thus magnetization exchange rate constants), the xenon affinity, and the total host molecule concentration. Only the numerical xenon solubility is additionally required for input, a quantity that has a low impact on the measurement uncertainty and is derivable from metrological data collections. Signal calibration by a reference material may thus be avoided, qualifying the method as calibration-free. For demonstration a xenon exchange with the host cucurbit[6]uril at low concentration is investigated, with the numerical results being validated by standard quantitative NMR data obtained at high concentration. The readiness to evaluate xenon exchange for the one sample at hand and in a single experimental attempt by the proposed method may allow comprehensive quantitative studies in supramolecular chemistry, biomacromolecular structure and dynamics, and sensing.

Understanding Parahydrogen Hyperpolarized Urine Spectra: The Case of Adenosine Derivatives.

Parahydrogen hyperpolarization has emerged as a promising tool for sensitivity-enhanced NMR metabolomics. It allows resolution and quantification of NMR signals of certain classes of low-abundance metabolites that would otherwise be undetectable. Applications have been implemented in pharmacokinetics and doping drug detection, demonstrating the versatility of the technique. Yet, in order for the method to be adopted by the analytical community, certain limitations have to be understood and overcome. One such question is NMR signal assignment. At present, the only reliable way to establish the identity of an analyte that gives rise to certain parahydrogen hyperpolarized NMR signals is internal standard addition, which can be laborious. Herein we show that analogously to regular NMR metabolomics, generating libraries of hyperpolarized analyte signals is a viable way to address this limitation. We present hyperpolarized spectral data of adenosines and give an early example of identifying them from a urine sample with the small library. Doing so, we verify the detectability of a class of diagnostically valuable metabolites: adenosine and its derivatives, some of which are cancer biomarkers, and some are central to cellular energy management (e.g., ATP).

Parahydrogen hyperpolarization of minimally altered urine samples for sensitivity enhanced NMR metabolomics

Removing SPE from urine sample preparation retains the majority of the urinary metabolome, giving access to the most information rich hyperpolarized NMR spectra up to date.

In vivo hyperCEST imaging: Experimental considerations for a reliable contrast.

HyperCEST contrast relies on the reduction of the solvent signal after selective saturation of the solute magnetization. The scope of this work is to outline the experimental conditions needed to obtain a reliable hyperCEST contrast in vivo, where the "solvent" signal (ie, the dissolved-phase signal) may change over time due to the increase in xenon (Xe) accumulation into tissue. Hyperpolarized 129 Xe was delivered to mice at a constant volume and rate using a mechanical ventilator, which triggered the saturation, excitation, and acquisition of the MR signal during the exhale phase of the breath cycle-either every breath or every 2, 3, or 4 breaths. Serial Z-spectra and hyperCEST images were acquired before and after a bolus injection of cucurbit[6]uril to assess possible signal fluctuations and instabilities. The intensity of the dissolved-phase Xe signal was observed to first increase immediately after the beginning of the hyperpolarized gas inhalation and NMR acquisition, and then decrease before reaching a steady-state condition. Once a steady-state dissolved-phase magnetization was established, a reliable hyperCEST contrast, exceeding 40% signal reduction, was observed. A reliable hyperCEST contrast can only be obtained after establishing a steady-state dissolved phase 129 Xe magnetization. Under stable physiological conditions, a steady-state dissolved-phase Xe magnetization is only achieved after a series of Xe inhalations and RF excitations, and it requires synchronization of the breathing rate with the MR acquisition.