An Update on MR Spectroscopy in Cancer Management: Advances in Instrumentation, Acquisition, and Analysis.

MR spectroscopy (MRS) is a noninvasive imaging method enabling chemical and molecular profiling of tissues in a localized, multiplexed, and nonionizing manner. As metabolic reprogramming is a hallmark of cancer, MRS provides valuable metabolic and molecular information for cancer diagnosis, prognosis, treatment monitoring, and patient management. This review provides an update on the use of MRS for clinical cancer management. The first section includes an overview of the principles of MRS, current methods, and conventional metabolites of interest. The remainder of the review is focused on three key areas: advances in instrumentation, specifically ultrahigh-field-strength MRI scanners and hybrid systems; emerging methods for acquisition, including deuterium imaging, hyperpolarized carbon 13 MRI and MRS, chemical exchange saturation transfer, diffusion-weighted MRS, MR fingerprinting, and fast acquisition; and analysis aided by artificial intelligence. The review concludes with future recommendations to facilitate routine use of MRS in cancer management. Keywords: MR Spectroscopy, Spectroscopic Imaging, Molecular Imaging in Oncology, Metabolic Reprogramming, Clinical Cancer Management © RSNA, 2024.

Solid-state nuclear magnetic resonance in the structural study of polyglutamine aggregation.

The aggregation of proteins into amyloid-like fibrils is seen in many neurodegenerative diseases. Recent years have seen much progress in our understanding of these misfolded protein inclusions, thanks to advances in techniques such as solid-state nuclear magnetic resonance (ssNMR) spectroscopy and cryogenic electron microscopy (cryo-EM). However, multiple repeat-expansion-related disorders have presented special challenges to structural elucidation. This review discusses the special role of ssNMR analysis in the study of protein aggregates associated with CAG repeat expansion disorders. In these diseases, the misfolding and aggregation affect mutant proteins with expanded polyglutamine segments. The most common disorder, Huntington's disease (HD), is connected to the mutation of the huntingtin protein. Since the discovery of the genetic causes for HD in the 1990s, steady progress in our understanding of the role of protein aggregation has depended on the integrative and interdisciplinary use of multiple types of structural techniques. The heterogeneous and dynamic features of polyQ protein fibrils, and in particular those formed by huntingtin N-terminal fragments, have made these aggregates into challenging targets for structural analysis. ssNMR has offered unique insights into many aspects of these amyloid-like aggregates. These include the atomic-level structure of the polyglutamine core, but also measurements of dynamics and solvent accessibility of the non-core flanking domains of these fibrils' fuzzy coats. The obtained structural insights shed new light on pathogenic mechanisms behind this and other protein misfolding diseases.

NMR methods to detect fluoride binding and transport by membrane proteins.

Solid-state nuclear magnetic resonance (NMR) methods can probe the motions of membrane proteins in liposomes at the atomic level, and propel the understanding of biomolecular processes for which static structures cannot provide a satisfactory description. High-resolution crystallography snapshots have provided a structural basis for fluoride channels. NMR is a powerful tool to build upon these snapshots and depict a dynamic picture of fluoride channels in native-like lipid bilayers. In this contribution, we discuss solid-state and solution NMR experiments to detect fluoride binding and transport by fluoride channels. Ongoing developments in membrane protein sample preparation and ssNMR methodology, particularly in using 1H, 19F and 13C-detection schemes, offer additional opportunities to study structure and functional aspects of fluoride channels.

GABA in the anterior cingulate cortex mediates the association of white matter hyperintensities with executive function: a magnetic resonance spectroscopy study.

White matter hyperintensities (WMH) and gamma-aminobutyric acid (GABA) are associated with executive function. Multiple studies suggested cortical alterations mediate WMH-related cognitive decline. The aim of this study was to investigate the crucial role of cortical GABA in the WMH patients. In the 87 WMH patients (46 mild and 41 moderate to severe) examined in this study, GABA levels in the anterior cingulate cortex (ACC) and posterior cingulate cortex (PCC) assessed by the Meshcher-Garwood point resolved spectroscopy (MEGA-PRESS) sequence, WMH volume and executive function were compared between the two groups. Partial correlation and mediation analyses were carried out to examine the GABA levels in mediating the association between WMH volume and executive function. Patients with moderate to severe WMH had lower GABA+/Cr in the ACC (p = 0.034) and worse executive function (p = 0.004) than mild WMH patients. In all WMH cases, the GABA+/Cr levels in the ACC mediated the negative correlation between WMH and executive function (ab: effect = -0.020, BootSE = 0.010, 95% CI: -0.042 to -0.004). This finding suggested GABA+/Cr levels in the ACC might serve as a protective factor or potential target for preventing the occurrence and progression of executive function decline in WMH people.

Analytical Framework to Understand the Origins of Methyl Side-Chain Dynamics in Protein Assemblies.

Side-chain motions play an important role in understanding protein structure, dynamics, protein-protein, and protein-ligand interactions. However, our understanding of protein side-chain dynamics is currently limited by the lack of analytical tools. Here, we present a novel analytical framework employing experimental nuclear magnetic resonance (NMR) relaxation measurements at atomic resolution combined with molecular dynamics (MD) simulation to characterize with a high level of detail the methyl side-chain dynamics in insoluble protein assemblies, using amyloid fibrils formed by the prion HET-s. We use MD simulation to interpret experimental results, where rotameric hops, including methyl group rotation and χ1/χ2 rotations, cannot be completely described with a single correlation time but rather sample a broad distribution of correlation times, resulting from continuously changing local structure in the fibril. Backbone motion similarly samples a broad range of correlation times, from ∼100 ps to µs, although resulting from mostly different dynamic processes; nonetheless, we find that the backbone is not fully decoupled from the side-chain motion, where changes in side-chain dynamics influence backbone motion and vice versa. While the complexity of side-chain motion in protein assemblies makes it very challenging to obtain perfect agreement between experiment and simulation, our analytical framework improves the interpretation of experimental dynamics measurements for complex protein assemblies.

Molecular imaging of tumor metabolism: Insight from pyruvate- and glucose-based deuterium MRI studies.

Cancer diagnosis by metabolic MRI proposes to follow the fate of glycolytic precursors such as pyruvate or glucose, and their in vivo conversion into lactate. This study compares the 2H MRI outlooks afforded by these metabolites when targeting a pancreatic cancer model. Exogenously injected [3,3',3″-2H3]-pyruvate was visible only briefly; it generated a deuterated lactate signal throughout the body that faded after ~5 min, showing a minor concentration bias at the rims of the tumors. [6,6'-2H2]-glucose by contrast originated a lactate signal that localized clearly within the tumors, persisting for over an hour. Investigations alternating deuterated and nondeuterated glucose injections revealed correlations between the lactate generation and the glucose available at the tumor, evidencing a continuous and avid glucose consumption generating well-localized lactate signatures as driven by the Warburg effect. This is by contrast to the transient and more promiscuous pyruvate-to-lactate transformation, which seemed subject to transporter and kinetics effects. The consequences of these observations within metabolic MRI are briefly discussed.

[Application of qNMR in Standard Materials Used for the Crude Drugs].

Crude drugs and Kampo formulations derived from natural materials such as plants, animals, and minerals are multicomponent medicines that contain numerous chemical constituents. Quantitative determination of characteristic constituents for quality control is crucial for the standardization and quality assurance of natural medicines. Quantitative assays to determine marker compound contents are commonly performed using HPLC systems. In order to achieve accurate quantitative determination, it is essential to use standard materials with well-defined purities corresponding to the target analytes. Many marker compounds used as standard materials must be purified and isolated from natural products while ensuring sufficient purity. However, the composition of impurities in the standard material differs among different batches due to differences in the raw materials and their extraction, separation, and purification processes. Therefore, controlling the purity of standard materials derived from natural products is more complex than that of synthetic substances. Quantitative NMR (qNMR), which has become widely used as an absolute quantitative method for low-molecule organic compounds, makes it possible to solve these issues. qNMR has been introduced into the crude drug section of the Japanese Pharmacopoeia (JP) for evaluating the purity of standard materials used for the assay. This review outlines an example of quantitative determination using relative molar sensitivity (RMS) based on qNMR adopted in the JP and introduces the latest efforts toward the application of qNMR to standard materials used for crude drugs in this context.

[The Applications of qNMR in Drug Quality Control].

NMR is well known as one of the most important methods for elucidating the structure of organic compounds. Furthermore, it has recently been recognized as a powerful tool for quantitative analysis. The quantitative NMR (qNMR) has become an official analytical method described in detail in the Japanese Pharmacopoeia. And today, it is widely applied in drug development. The qNMR method offers many new advantages over traditional and conventional quantitative analysis methods. For example, this method requires only a few milligrams of the analyte and allows absolute quantitation of the analyte without using a qualified reference standard as a control sample. Then, it can be easily applied to most chemicals without expending significant time and resources on method development. In addition, residual solvent can be determined using qNMR methods. The peak area of an NMR spectrum is directly proportional to the number of protons contributing to the resonance. Based on this principle, the residual solvent can be determined by counting the signal corresponding to the residual solvent in the sample solution. We have applied qNMR as an alternative to GC. Thus, qNMR is an innovative and promising analytical technique that is expected to make significant progress in the future. Recently, the analytical research and quality control departments have been working together to expand this technology to a wide range of areas in the pharmaceutical industry.

[Route to the Adoption of Quantitative NMR in the Japanese Pharmacopoeia].

Quantitative NMR (qNMR) is employed to determine the purity of reagents used as standards for HPLC quantification in the Japanese Pharmacopoeia (JP) and has become recognized as a new absolute quantification method in various fields such as pharmaceuticals, foods, and food additives. This report outlines how and why qNMR has been adopted as an official method in the JP and introduces its progression from JP16 to JP18. The results of a survey of companies in the Japan Pharmaceutical Manufacturers' Association regarding how and when to use qNMR from development to manufacturing stages are introduced. The issues involved in the expansion of the use of qNMR in the field of chemical pharmaceuticals in 2017 are discussed and how these were resolved.

[Standardization and Practical Application of Quantitative NMR (qNMR)].

In Japan, quantitative NMR (qNMR) has already been recognized as a standard method for determining the purity of quantitative samples not only in the Japanese Pharmacopoeia and the Japanese Standards and Specifications for Food Additives but also in the Japanese Industrial Standard (JIS K 0138: 2018). However, since there was no consensus on the establishment of a standard method, the international standardization of qNMR was initiated based on a proposal from Japan. After three years of discussion among experts, International Organization for Standardization/Technical Committee on Food (ISO/TC34) published ISO 24583: 2022 "Quantitative nuclear magnetic resonance spectroscopy-Purity determination of organic compounds used for foods and food products-General requirements for 1H-NMR internal standard method." Publication of this standard has resulted in an internationally agreed upon set of requirements for purity determination using qNMR. New technologies emerge from the cycle of basic research, practical use, and standardization, and qNMR is no exception. A novel chromatographic quantification method based on relative molar sensitivity (RMS) is now being put into practical use. The RMS of an analyte with respect to a different reference substance can be determined by using qNMR to accurately determine the molar ratio and then introducing it into the chromatographic system. This method uses the RMS determined by combining qNMR and chromatography instead of the analyte's reference material to determine its content in sample. This method has been adopted in the Japanese Pharmacopoeia, and the development of a general rule in the Japanese Agricultural Standards (JAS) is also under consideration.

[High Purity Solvents for qNMR Measurement].

Quantitative NMR (qNMR) has been adopted by documentary standards, including the Japanese Pharmacopoeia (JP), United States Pharmacopoeia (USP), and International Organization for Standardization (ISO), owing to its reliability and efficiency. Note that qNMR can be used for quantifying target components using the signal integration ratio of an analyte to a reference. In qNMR, a modern NMR instrument with high resolution and sensitivity is used to record reliable spectra. This instrument can detect small signals from impurities in a solvent, which may result in inaccurate signal integration in the spectrum. In this study, we investigated the influence of solvent quality on qNMR accuracy focusing on organic impurities, water content, and deuteration ratio. If signals from organic impurities and signals from the analyte overlap, the duplication of signal integration will directly affect the qNMR analytical result. To examine overlapping, we performed blank solvent tests. Additionally, a high water content and low deuteration ratio affect the detection sensitivity, thus reducing the signal-to-noise (S/N) ratio of the target. Thus, these factors must be considered to obtain accurate qNMR results.

Improved Detection of Target Metabolites in Brain Tumors with Intermediate TE, High SNR, and High Bandwidth Spin-Echo Sequence at 5T.

BACKGROUND AND PURPOSE: Due to high chemical shift displacement, challenges emerge at ultra-high fields when measuring metabolites using 1H-MRS. Our goal was to investigate how well the high SNR and high bandwidth spin-echo (HISE) technique perform at 5T for detecting target metabolites in brain tumors. MATERIALS AND METHODS: Twenty-six subjects suspected of having brain tumors were enrolled. HISE and point-resolved spectroscopy (PRESS) single-voxel spectroscopy scans were collected with a 5T clinical scanner with an intermediate TE (TE = 144 ms). The main metabolites, including total NAA, Cr, and total Cho, were accessed and compared between HISE and PRESS using a paired Student t test, with full width at half maximum and SNR as covariates. The detection rate of specific metabolites, including lactate, alanine, and lipid, and subjective spectral quality were accessed and compared between HISE and PRESS. RESULTS: Twenty-three pathologically confirmed brain tumors were included. Only the full width at half maximum for total NAA was significantly lower with HISE than with PRESS (P < .05). HISE showed a significantly higher SNR for total NAA, Cr, and total Cho compared with PRESS (P < .05). Lactate was detected in 21 of the 23 cases using HISE, but in only 4 cases using PRESS. HISE detected alanine in 8 of 9 meningiomas, whereas PRESS detected alanine in just 3 meningiomas. PRESS found lipid in more cases than HISE, while HISE outperformed PRESS in terms of subjective spectral quality. CONCLUSIONS: HISE outperformed the clinical standard PRESS technique in detecting target metabolites of brain tumors at 5T, particularly lactate and alanine.

Proton magnetic resonance spectroscopy of N-acetyl aspartate in first depressive episode and chronic major depressive disorder: A systematic review and meta-analysis.

N-acetyl aspartate (NAA) is a marker of neuronal integrity and metabolism. Deficiency in neuronal plasticity and hypometabolism are implicated in Major Depressive Disorder (MDD) pathophysiology. To test if cerebral NAA concentrations decrease progressively over the MDD course, we conducted a pre-registered meta-analysis of Proton Magnetic Resonance Spectroscopy (1H-MRS) studies comparing NAA concentrations in chronic MDD (n = 1308) and first episode of depression (n = 242) patients to healthy controls (HC, n = 1242). Sixty-two studies were meta-analyzed using a random-effect model for each brain region. NAA concentrations were significantly reduced in chronic MDD compared to HC within the frontal lobe (Hedges' g = -0.330; p = 0.018), the occipital lobe (Hedges' g = -0.677; p = 0.007), thalamus (Hedges' g = -0.673; p = 0.016), and frontal (Hedges' g = -0.471; p = 0.034) and periventricular white matter (Hedges' g = -0.478; p = 0.047). We highlighted a gap of knowledge regarding NAA levels in first episode of depression patients. Sensitivity analyses indicated that antidepressant treatment may reverse NAA alterations in the frontal lobe. We highlighted field strength and correction for voxel grey matter as moderators of NAA levels detection. Future studies should assess NAA alterations in the early stages of the illness and their longitudinal progression.

Perspective: use and reuse of NMR-based metabolomics data: what works and what remains challenging.

BACKGROUND: The National Cancer Institute issued a Request for Information (RFI; NOT-CA-23-007) in October 2022, soliciting input on using and reusing metabolomics data. This RFI aimed to gather input on best practices for metabolomics data storage, management, and use/reuse. AIM OF REVIEW: The nuclear magnetic resonance (NMR) Interest Group within the Metabolomics Association of North America (MANA) prepared a set of recommendations regarding the deposition, archiving, use, and reuse of NMR-based and, to a lesser extent, mass spectrometry (MS)-based metabolomics datasets. These recommendations were built on the collective experiences of metabolomics researchers within MANA who are generating, handling, and analyzing diverse metabolomics datasets spanning experimental (sample handling and preparation, NMR/MS metabolomics data acquisition, processing, and spectral analyses) to computational (automation of spectral processing, univariate and multivariate statistical analysis, metabolite prediction and identification, multi-omics data integration, etc.) studies. KEY SCIENTIFIC CONCEPTS OF REVIEW: We provide a synopsis of our collective view regarding the use and reuse of metabolomics data and articulate several recommendations regarding best practices, which are aimed at encouraging researchers to strengthen efforts toward maximizing the utility of metabolomics data, multi-omics data integration, and enhancing the overall scientific impact of metabolomics studies.

A simple 1H (12C/13C) filtered experiment to quantify and trace isotope enrichment in complex environmental and biological samples.

Nuclear magnetic resonance (NMR) based 13C tracing has broad applications across medical and environmental research. As many biological and environmental samples are heterogeneous, they experience considerable spectral overlap and relatively low signal. Here a 1D 1H-12C/13C is introduced that uses "in-phase/opposite-phase" encoding to simultaneously detect and discriminate both protons attached to 12C and 13C at full 1H sensitivity in every scan. Unlike traditional approaches that focus on the 12C/13C satellite ratios in a 1H spectrum, this approach creates separate sub-spectra for the 12C and 13C bound protons. These spectra can be used for both quantitative and qualitative analysis of complex samples with significant spectral overlap. Due to the presence of the 13C dipole, faster relaxation of the 1H-13C pairs results in slight underestimation compared to the 1H-12C pairs. However, this is easily compensated for, by collecting an additional reference spectrum, from which the absolute percentage of 13C can be calculated by difference. When combined with the result, 12C and 13C percent enrichment in both 1H-12C and 1H-13C fractions are obtained. As the approach uses isotope filtered 1H NMR for detection, it retains nearly the same sensitivity as a standard 1H spectrum. Here, a proof-of-concept is performed using simple mixtures of 12C and 13C glucose, followed by suspended algal cells with varying 12C /13C ratios representing a complex mixture. The results consistently return 12C/13C ratios that deviate less than 1 % on average from the expected. Finally, the sequence was used to monitor and quantify 13C% enrichment in Daphnia magna neonates which were fed a 13C diet over 1 week. The approach helped reveal how the organisms utilized the 12C lipids they are born with vs. the 13C lipids they assimilate from their diet during growth. Given the experiments simplicity, versatility, and sensitivity, we anticipate it should find broad application in a wide range of tracer studies, such as fluxomics, with applications spanning various disciplines.

Identification of potential serum biomarkers associated with HbA1c levels in Indian type 2 diabetic subjects using NMR-based metabolomics.

BACKGROUND: The prevalence of type 2 diabetes mellitus (T2DM), a progressive metabolic disorder characterized by chronic hyperglycemia and the development of insulin resistance, has increased globally, with worrying statistics coming from children, adolescents, and young adults from developing countries like India. Here, we investigated unique circulating metabolic signatures associated with prediabetes and T2DM in an Indian cohort using NMR-based metabolomics. MATERIALS AND METHODS: The study subjects included healthy volunteers (N = 101), prediabetic subjects (N = 75), and T2DM patients (N = 108). Serum metabolic profiling was performed using 1H NMR spectroscopy and major perturbed metabolites were identified by multivariate analysis and receiver operating characteristic (ROC) modules. RESULTS: Of the 36 aqueous abundant metabolites, 24 showed a statistically significant difference between healthy volunteers, prediabetics, and established T2DM subjects. On performing multivariate ROC curve analysis with 5 commonly dysregulated metabolites (namely, glucose, pyroglutamate, o-phosphocholine, serine, and methionine) in prediabetes and T2DM, AUC values obtained were 0.96 (95 % confidence interval (CI) = 0.93, 0.98) for T2DM; and 0.88 (95 % CI = 0.81, 0.93) for prediabetic subjects, respectively. CONCLUSION: We propose that the identified metabolite panel can be used in the future as a biomarker for clinical diagnosis, patient surveillance, and for predicting individuals at risk for developing diabetes.

LC-HRMS and NMR studies for the characterization of degradation impurities of ubrogepant along with the in silico approaches for the prediction of degradation and toxicity.

Ubrogepant is the first oral calcitonin gene-related peptide (CGRP) receptor antagonist which is used for the acute treatment of migraine in adults. The present study employs liquid chromatography-high resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance spectroscopy (NMR) techniques for the identification and characterization of degradation impurities of ubrogepant. The forced degradation study of ubrogepant was performed as per the International Council for Harmonisation (ICH) Q1A and Q1B guidelines. The in silico degradation profile of ubrogepant was predicted by Zeneth. It was observed that ubrogepant was labile to acidic hydrolysis, basic hydrolysis, and oxidative degradation conditions (H2O2), although it was stable in neutral hydrolysis and photolytic (UV light and visible light) conditions. Eight degradation impurities were formed, which were separated on reversed-phase HPLC with a gradient program on an InertSustain C8 column (4.6 × 250 mm, 5 µm) using 10 mM ammonium formate (pH unadjusted) and acetonitrile as the mobile phase. The structures of all the degradation impurities were characterized using the exact masses obtained from the HRMS/MS. Further, NMR studies were conducted on two major degradation impurities (UB-4 and UB-7). A plausible mechanism was proposed to support the structures of all the degradation impurities of UBR. In silico toxicity and mutagenicity assessment were done by DEREK Nexus, SARAH Nexus, and ProTox-II.

The developmental trajectory of 1H-MRS brain metabolites from childhood to adulthood.

Human brain development is ongoing throughout childhood, with for example, myelination of nerve fibers and refinement of synaptic connections continuing until early adulthood. 1H-Magnetic Resonance Spectroscopy (1H-MRS) can be used to quantify the concentrations of endogenous metabolites (e.g. glutamate and γ -aminobutyric acid (GABA)) in the human brain in vivo and so can provide valuable, tractable insight into the biochemical processes that support postnatal neurodevelopment. This can feasibly provide new insight into and aid the management of neurodevelopmental disorders by providing chemical markers of atypical development. This study aims to characterize the normative developmental trajectory of various brain metabolites, as measured by 1H-MRS from a midline posterior parietal voxel. We find significant non-linear trajectories for GABA+ (GABA plus macromolecules), Glx (glutamate + glutamine), total choline (tCho) and total creatine (tCr) concentrations. Glx and GABA+ concentrations steeply decrease across childhood, with more stable trajectories across early adulthood. tCr and tCho concentrations increase from childhood to early adulthood. Total N-acetyl aspartate (tNAA) and Myo-Inositol (mI) concentrations are relatively stable across development. Trajectories likely reflect fundamental neurodevelopmental processes (including local circuit refinement) which occur from childhood to early adulthood and can be associated with cognitive development; we find GABA+ concentrations significantly positively correlate with recognition memory scores.

Xplor-NIH: Better parameters and protocols for NMR protein structure determination.

The present work describes an update to the protein covalent geometry and atomic radii parameters in the Xplor-NIH biomolecular structure determination package. In combination with an improved treatment of selected non-bonded interactions between atoms three bonds apart, such as those involving methyl hydrogens, and a previously developed term that affects the system's gyration volume, the new parameters are tested using structure calculations on 30 proteins with restraints derived from nuclear magnetic resonance data. Using modern structure validation criteria, including several formally adopted by the Protein Data Bank, and a clear measure of structural accuracy, the results show superior performance relative to previous Xplor-NIH implementations. Additionally, the Xplor-NIH structures compare favorably against originally determined NMR models.

Elucidation of the Mechanisms of Inter-domain Coupling in the Monomeric State of Enzyme I by High-pressure NMR.

The catalytic cycle of Enzyme I (EI), a phosphotransferase enzyme responsible for converting phosphoenolpyruvate (PEP) into pyruvate, is characterized by a series of local and global conformational rearrangements. This multistep process includes a monomer-to-dimer transition, followed by an open-to-closed rearrangement of the dimeric complex upon PEP binding. In the present study, we investigate the thermodynamics of EI dimerization using a range of high-pressure solution NMR techniques complemented by SAXS experiments. 1H-15N TROSY and 1H-13C methyl TROSY NMR spectra combined with 15N relaxation measurements revealed that a native-like engineered variant of full-length EI fully dissociates into stable monomeric state above 1.5 kbar. Conformational ensembles of EI monomeric state were generated via a recently developed protocol combining coarse-grained molecular simulations with experimental backbone residual dipolar coupling measurements. Analysis of the structural ensembles provided detailed insights into the molecular mechanisms driving formation of the catalytically competent dimeric state, and reveals that each step of EI catalytical cycle is associated with a significant reduction in either inter- or intra-domain conformational entropy. Altogether, this study completes a large body work conducted by our group on EI and establishes a comprehensive structural and dynamical description of the catalytic cycle of this prototypical multidomain, oligomeric enzyme.