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Evaluation of High Resolution Magic-Angle Coil Spinning NMR Spectroscopy for Metabolic Profiling of Nanoliter Tissue Biopsies
Abstract
High-resolution magic-angle sample spinning (HR-MAS) (1)H NMR spectroscopy of tissue biopsies combined with chemometric techniques has emerged as a valuable methodology for disease diagnosis and environmental assessments. However, the tissue mass required for such experiments is of the order of 10 mg, and this can compromise the metabolic evaluation because of tissue heterogeneity. Tissue availability is often a limitation for clinical studies due to histopathological requirements, which are currently the gold standard for diagnosis, for example, in the case of tumors. Here, we introduce the use of a rotating micro-NMR detector that optimizes the coil filling factor such that mass-limited samples can be measured. We show the results for measuring nanoliter volume tissue biopsies using a commercial HR-MAS probe for the first time. The method has been tested with bovine muscle and human gastric mucosal tumor tissue samples. The gain in mass sensitivity is approximate up to 17-fold, and the adequate spectral resolution (3 Hz) allows the measurement of the metabolite profiles in nanoliter volume samples, thereby limiting the ambiguity resulting from heterogeneous tissues; thus, the approach presents diagnostic potential for studies by metabonomics of mass-limited biopsies.
... However, the optimal spectral resolution acquired from these µMAS probes is highly insufficient for metabolic investigations. It was not until 2012; a µMAS probe had emerged and demonstrated the possibility of metabolic profiling with µg tissues [19]. This mini-review highlights the different strategies and progress of the development of µMAS NMR spectroscopy towards µg-scale biospecimens. ...
... The main advantage of MACS is its versatility; it can readily adapt to different NMR probes (liquid or MAS) at various fields. The teams of Wong and Nicholson have carried out an evaluation of the original MACS for metabolic profiling with 500 µg of animal and human tissues [19] (Figure 2a). Despite the seven-fold sensitivity enhancement by MACS, the spectral resolution is visibly poor (0.1 ppm at best, or 40 Hz at 9.4 T) as compared to that obtained from HR-MAS with mg tissues; however, a few important metabolites (i.e., lipids, lactate, choline, creatine) were identified. ...
Analysis of microscopic specimens has emerged as a useful analytical application in metabolomics because of its capacity for characterizing a highly homogenous sample with a specific interest. The undeviating analysis helps to unfold the hidden activities in a bulk specimen and contributes to the understanding of the fundamental metabolisms in life. In NMR spectroscopy, micro(µ)-probe technology is well-established and -adopted to the microscopic level of biofluids. However, this is quite the contrary with specimens such as tissue, cell and organism. This is due to the substantial difficulty of developing a sufficient µ-size magic-angle spinning (MAS) probe for sub-milligram specimens with the capability of high-quality data acquisition. It was not until 2012; a µMAS probe had emerged and shown promises to µg analysis; since, a continuous advancement has been made striving for the possibility of µMAS to be an effective NMR spectroscopic analysis. Herein, the mini-review highlights the progress of µMAS development—from an impossible scenario to an attainable solution—and describes a few demonstrative metabolic profiling studies. The review will also discuss the current challenges in µMAS NMR analysis and its potential to metabolomics.
... One approach to solve these problems is the use of the emerging micro-NMR technology, high-resolution magic-angle coil spinning (HR-MACS), to generate highresolution spectra from a small amount of samples at a low spinning rate [15]. A secondary tuned circuit and a simple and robust rotor insert were designed to fit inside a standard MAS sample rotor, thus converting the MAS probe into a micro-MAS probe for analysis of small-volume samples [16,17]. Alan et al. applied HR-MACS for a high-sensitivity and high-resolution 1 H micro-NMR-based metabolomics study on yeast cells. ...
Metabolism is a fundamental process that underlies human health and diseases. Nuclear magnetic resonance (NMR) techniques offer a powerful approach to identify metabolic processes and track the flux of metabolites at the molecular level in living systems. An in vitro study through in-cell NMR tracks metabolites in real time and investigates protein structures and dynamics in a state close to their most natural environment. This technique characterizes metabolites and proteins involved in metabolic pathways in prokaryotic and eukaryotic cells. In vivo magnetic resonance spectroscopy (MRS) enables whole-organism metabolic monitoring by visualizing the spatial distribution of metabolites and targeted proteins. One limitation of these NMR techniques is the sensitivity, for which a possible improved approach is through isotopic enrichment or hyperpolarization methods, including dynamic nuclear polarization (DNP) and parahydrogen-induced polarization (PHIP). DNP involves the transfer of high polarization from electronic spins of radicals to surrounding nuclear spins for signal enhancements, allowing the detection of low-abundance metabolites and real-time monitoring of metabolic activities. PHIP enables the transfer of nuclear spin polarization from parahydrogen to other nuclei for signal enhancements, particularly in proton NMR, and has been applied in studies of enzymatic reactions and cell signaling. This review provides an overview of in-cell NMR, in vivo MRS, and hyperpolarization techniques, highlighting their applications in metabolic studies and discussing challenges and future perspectives.
Graphical abstract
... HR-MAS NMR spectroscopy has been explored in a number of studies on various cancer tissues [32][33][34][35][36][37][38] and gallstones, 39 among others. Recent studies have tested HR-MAS coil spinning NMR spectroscopy for the use of smaller tissue samples, 40 and combined slice localization with HR-MAS NMR spectroscopy for improved spatial resolution of metabolites. 41 While innovations in HR-MAS strive to achieve spatially localized NMR spectroscopic metabolomics, typical sample sizes of 10-15 mg of tissue will inevitably contain heterogeneous regions of various cell types, microenvironments and histologies in the same sample. ...
Nuclear magnetic resonance (NMR) spectroscopy and matrix assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) are both commonly used to detect large numbers of metabolites and lipids in metabolomic and lipidomic studies. We have demonstrated a new workflow, highlighting the benefits of both techniques to obtain metabolomic and lipidomic data, which has realized for the first time the combination of these two complementary and powerful technologies. NMR spectroscopy is frequently used to obtain quantitative metabolite information from cells and tissues. Lipid detection is also possible with NMR spectroscopy, with changes being visible across entire classes of molecules. Meanwhile, MALDI MSI provides relative measures of metabolite and lipid concentrations, mapping spatial information of many specific metabolite and lipid molecules across cells or tissues. We have used these two complementary techniques in combination to obtain metabolomic and lipidomic measurements from triple‐negative human breast cancer cells and tumor xenograft models. We have emphasized critical experimental procedures that ensured the success of achieving NMR spectroscopy and MALDI MSI in a combined workflow from the same sample. Our data show that several metabolites phospholipid species were differentially distributed in viable and necrotic regions of breast tumor xenografts. This study emphasizes the power of combined NMR spectroscopy – MALDI imaging to advance metabolomic and lipidomic studies.
... Importantly, biomarkers identified using tissue can be translated to biomarkers detection in the relatively easily accessible biofluids such as blood and urine. Technological advancements in NMR have reduced the amount of tissue needed to as little as a few nanoliters, which is beneficial for analysis of mass limited samples (Wong et al. 2012). ...
Nuclear magnetic resonance (NMR) spectroscopy is a major analytical method used in the growing field of metabolomics. Although NMR is relatively less sensitive than mass spectrometry, this analytical platform has numerous characteristics including its high reproducibility and quantitative abilities, its nonselective and noninvasive nature, and the ability to identify unknown metabolites in complex mixtures and trace the downstream products of isotope labeled substrates ex vivo, in vivo, or in vitro. Metabolomic analysis of highly complex biological mixtures has benefitted from the advances in both NMR data acquisition and analysis methods. Although metabolomics applications span a wide range of disciplines, a majority has focused on understanding, preventing, diagnosing, and managing human diseases. This chapter describes NMR-based methods relevant to the rapidly expanding metabolomics field.
... Attempts have been made to achieve greater sample homogeneity using microdissection and analysis of smaller samples, 10 but this has limited applicability with lower sensitivity and routine and automated operation is usually not possible. An alternative approach is magic-angle coil spinning NMR spectroscopy, 11 where small samples (∼600 ng) are inserted into an inner sample holder with a closewound radiofrequency (RF) coil that also spins at the magic angle such that the NMR signal is picked up inductively in the main static receiver coil. This provides excellent filling factors and hence much improved sensitivity, but the approach still suffers from a lack of flexibility in operation and automation. ...
... Moreover, these large rotors suffer from temperature gradients due to frictional heating even at moderate (2-5 kHz) MAS rates, resulting in additional broadening in temperature-dependent peaks. A great advance for NMR-based metabolomics for mass/volume-limited samples is the introduction of the inductively coupled high-resolution magic angle coil spinning method (HRMACS) [9][10][11][12][13][14]. In the innovative work, the tiny secondary coil is in close proximity to, and is spun with the sample holder. ...
Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical technique and has been widely used in metabolomics. However, the intrinsic low sensitivity of NMR prevents its applications to systems with limited sample availabilities. In this study, a new experimental approach is presented to analyze mass-scarce samples in limited volumes of less than 300 nL with simple handling. The sample is loaded into the glass capillary, and this capillary is then inserted into a Kel-F rotor. The experimental performance of the capillary-inserted rotor (capillary-insert) is investigated on an isotropic solution of sucrose by the use of a high-resolution micro-sized magic angle spinning (HRuMAS) probe. The acquired NMR signal's sensitivity to a given sample amount is comparable or even higher in comparison to that recorded by the standard solution NMR probe. More importantly, this capillary-insert coupled with the HRuMAS probe allows in-depth studies of heterogeneous samples as the MAS removes the line broadening caused by the heterogeneity. The NMR analyses of mass-limited cultured neurospheres have been demonstrated, resulting in high quality spectra where numerous metabolites are unambiguously identified.
... Note, that the temperature rise due to spinning is a complex function of many parameters as discussed above, and detailed comparison with conventional micro solenoid coils is above the scope of this work. Furthermore, negligible residual susceptibility artefacts were observed for high-resolution MACS experiments making these coils another instrumental option for HRMAS [23] on excised tissues and biopsies [24]. Most importantly, we do observe a RF field compression inside the volume between the two flat resonators. ...
Magic Angle Coil Spinning (MACS) aids improving the intrinsically low NMR sensitivity of heterogeneous microscopic samples. We report on the design and testing of a new type of monolithic 2D MACS resonators to overcome known limitations of conventional micro coils. The resonators’ conductors were printed on dielectric substrate and tuned without utilizing lumped element capacitors. Self-resonance conditions have been computed by a hybrid FEM-MoM technique. Preliminary results reported here indicate robust mechanical stability, reduced eddy currents heating and negligible susceptibility effects. The gain in is in agreement with the NMR sensitivity enhancement according to the principle of reciprocity. A sensitivity enhancement larger than 3 has been achieved in a monolithic micro resonator inside a standard 4 mm rotor at 500 MHz. These 2D resonators could offer higher performance micro-detection and ease of use of heterogeneous microscopic substances such as biomedical samples, microscopic specimens and thin film materials.
... Importantly, the tissue can be recovered after NMR experiments and can be used for other studies such as proteomic and genomic analysis. Current technological advancements in HR-MAS NMR enable analysis of as little as a few nano-grams of tissue [101]. Such capabilities, combined with minimal sample preparation and fast data acquisition, promises metabolic profiling of biopsied tissue for translation to clinical applications. ...
The field of metabolomics continues to witness rapid growth driven by fundamental studies, methods development, and applications in a number of disciplines that include biomedical science, plant and nutrition sciences, drug development, energy and environmental sciences, toxicology, etc. NMR spectroscopy is one of the two most widely used analytical platforms in the metabolomics field, along with mass spectrometry (MS). NMR's excellent reproducibility and quantitative accuracy, its ability to identify structures of unknown metabolites, its capacity to generate metabolite profiles using intact bio-specimens with no need for separation, and its capabilities for tracing metabolic pathways using isotope labeled substrates offer unique strengths for metabolomics applications. However, NMR's limited sensitivity and resolution continue to pose a major challenge and have restricted both the number and the quantitative accuracy of metabolites analyzed by NMR. Further, the analysis of highly complex biological samples has increased the demand for new methods with improved detection, better unknown identification, and more accurate quantitation of larger numbers of metabolites. Recent efforts have contributed significant improvements in these areas, and have thereby enhanced the pool of routinely quantifiable metabolites. Additionally, efforts focused on combining NMR and MS promise opportunities to exploit the combined strength of the two analytical platforms for direct comparison of the metabolite data, unknown identification and reliable biomarker discovery that continue to challenge the metabolomics field. This article presents our perspectives on the emerging trends in NMR-based metabolomics and NMR's continuing role in the field with an emphasis on recent and ongoing research from our laboratory.
... Besides the gain in sensitivity, another main advantage of the inductively coupled coil is that it can be readily coupled to any form of NMR probes such as the goniometer probe used in single-crystal NMR experiment. Inductively coupled coils have been successfully applied in various NMR applications to small-size samples such as in microfluidic devices [54] and in standard magic-angle spinning (MAS) probes for micro-size biopsy [55,56] and solids with expensive isotopic enrichment, 43 Ca [57]. ...
Since the 1990s, we have witnessed a steady progress in solid-state 17O NMR spectroscopy for biomolecules. Up until today, majority of the reported studies are considered as preliminary works, but significant, for laying the foundations of 17O NMR applications to biological solids. This has taken over 20 years with nearly 100 studies in methodology developments and NMR assessments. Currently, the field is entering an exciting stage where small biomolecules can routinely be characterized and new area of research can be exploited, such as investigations of macromolecules and multi-dimensional correlation experiments. This review provides a summary of the important contributions and millstones in the field, including the methodologies for probing the dilute 17O-spin in biomolecules and the investigations of the nuclear properties in relationships to the oxygen local surroundings.
... In cases where sample size is limited (such as neuron cells), analysis of fewer cells-in a smaller volume-would ease the sample preparation and may improve the high-throughput efficiency (e.g., coupling with micro-fluidic devices for cell separation techniques). One promising approach for volume-, or mass-, limited bio-specimens is the uses of a high-resolution magic-angle coil spinning (HR-MACS) ( Wong et al., 2012Wong et al., , 2013. HR-MACS, as with the original MACS experiment ( Sakellariou et al., 2007), utilizes a secondary tuned circuit and a simple and robust rotor insert, designed to fit inside a standard MAS sample rotor to convert the MAS probe into a μMAS probe without any probe modification. ...
The low sensitivity and thus need for large sample volume is one of the major drawbacks of Nuclear Magnetic Resonance (NMR) spectroscopy. This is especially problematic for performing rich metabolic profiling of scarce samples such as whole cells or living organisms. This study evaluates a ¹H HR-MAS approach for metabolic profiling of small volumes (250 nl) of whole cells. We have applied an emerging micro-NMR technology, high-resolution magic-angle coil spinning (HR-MACS), to study whole Saccharomyces cervisiae cells. We find that high-resolution high-sensitivity spectra can be obtained with only 19 million cells and, as a demonstration of the metabolic profiling potential, we perform two independent metabolomics studies identifying the significant metabolites associated with osmotic stress and aging.
... In the case of type 1 diabetes, a metabolic dysregulation of lipid and amino-acid metabolism was found to precede NMR spectroscopy and mass spectrometry are the main techniques that are used for the metabolic profiling of biofluids (for example, urine, blood plasma, amniotic fluid and cerebrospinal fluid) and tissues in the form of extracts or intact biopsies 94,95 . New solid-state NMR methods allow analysis of samples of less than 1 mg, thereby allowing detailed studies of tissue heterogeneity, such as between tumour tissue and tumour margins 96 . ...
Metabolic phenotyping involves the comprehensive analysis of biological fluids or tissue samples. This analysis allows biochemical classification of a person's physiological or pathological states that relate to disease diagnosis or prognosis at the individual level and to disease risk factors at the population level. These approaches are currently being implemented in hospital environments and in regional phenotyping centres worldwide. The ultimate aim of such work is to generate information on patient biology using techniques such as patient stratification to better inform clinicians on factors that will enhance diagnosis or the choice of therapy. There have been many reports of direct applications of metabolic phenotyping in a clinical setting.
... However, wellestablished synchronization techniques for total suppression or separation of the spinning sidebands [28] can be employed in order to separate the information contained in the central, isotropic peak of the spectrum [28,29] thus making slow speed NMR a useful tool for samples where high spinning speeds are prohibited. It has recently been demonstrated that the use of hand-wound MACS shows great potential for metabolic profiling in tissue biopsies [30]. To evaluate the wirebonded MACS inserts for metabolomic NMR spectroscopy, we have acquired 1 H NMR spectra of early stage Drosophila pupae, seeFigure 5. To preserve the tissue integrity from the centrifugal forces generated from the sample spinning, we performed slow spinning speed experiments. ...
This article describes the development and testing of the first automatically microfabricated probes to be used in conjunction with the magic angle coil spinning (MACS) NMR technique. NMR spectroscopy is a versatile technique for a large range of applications, but its intrinsically low sensitivity poses significant difficulties in analyzing mass- and volume-limited samples. The combination of microfabrication technology and MACS addresses several well-known NMR issues in a concerted manner for the first time: (i) reproducible wafer-scale fabrication of the first-in-kind on-chip LC microresonator for inductive coupling of the NMR signal and reliable exploitation of MACS capabilities; (ii) improving the sensitivity and the spectral resolution by simultaneous spinning the detection microcoil together with the sample at the "magic angle" of 54.74° with respect to the direction of the magnetic field (magic angle spinning - MAS), accompanied by the wireless signal transmission between the microcoil and the primary circuit of the NMR spectrometer; (iii) given the high spinning rates (tens of kHz) involved in the MAS methodology, the microfabricated inserts exhibit a clear kinematic advantage over their previously demonstrated counterparts due to the inherent capability to produce small radius cylindrical geometries, thus tremendously reducing the mechanical stress and tearing forces on the sample. In order to demonstrate the versatility of the microfabrication technology, we have designed MACS probes for various Larmor frequencies (194, 500 and 700 MHz) testing several samples such as water, Drosophila pupae, adamantane solid and LiCl at different magic angle spinning speeds.
Nuclear Magnetic Resonance (NMR) spectroscopy is one of the two major analytical platforms in the field of metabolomics, the other being mass spectrometry (MS). NMR is less sensitive than MS and hence it detects a relatively small number of metabolites. However, NMR exhibits numerous unique characteristics including its high reproducibility and non-destructive nature, its ability to identify unknown metabolites definitively, and its capabilities to obtain absolute concentrations of all detected metabolites, sometimes even without an internal standard. These characteristics outweigh the relatively low sensitivity and resolution of NMR in metabolomics applications. Since biological mixtures are highly complex, increased demand for new methods to improve detection, better identify unknown metabolites, and provide more accurate quantitation continues unabated. Technological and methodological advances to date have helped to improve the resolution and sensitivity and detection of a larger number of metabolite signals. Efforts focused on measuring unknown metabolite signals have resulted in the identification and quantitation of an expanded pool of metabolites including labile metabolites such as cellular redox coenzymes, energy coenzymes, and antioxidants. This chapter describes quantitative NMR methods in metabolomics with an emphasis on recent methodological developments, while highlighting the benefits and challenges of NMR-based metabolomics.
Nuclear magnetic resonance (NMR) spectroscopy has played an integral role in medical and environmental metabolic research. However, smaller biological entities, such as eggs and small tissue samples, are becoming increasingly important to better understand toxicity, biological growth/development, and diseases. Unfortunately, their small sizes make them difficult to study using conventional 5 mm NMR probes due to limited sensitivity. The use of microcoil NMR holds great potential for the analysis of such samples, where the coil can be designed to match the sample size to significantly improve NMR mass sensitivity and the filling factor. Here, we compare the potential of planar and Helmholtz microcoil designs to execute complex experiments for the analysis of intact, mass-limited biological samples. The planar coil offers the advantage of an open access design, potentially allowing flow systems to be incorporated and varying sample sizes to be studied; however, its relatively inhomogeneous B1 field leads to reduced NMR performance. The Helmholtz microcoil overcomes this drawback with its symmetrical design, improving B1 homogeneity across the sample but with the caveat that the size and shape of the sample is limited to the spacing between the two parallel coils. The line shape, sensitivity, and RF performance are compared on both coils using standard samples and biological samples. This study found that the Helmholtz microcoil used here considerably outperforms the planar coil in multipulse experiments and has great potential to study complex biological samples in the 50-200 nL range.
NMR spectroscopy has been applied to cells and tissues analysis since its beginnings, as early as 1950. We have attempted to gather here in a didactic fashion the broad diversity of data and ideas that emerged from NMR investigations on living cells. Covering a large proportion of the periodic table, NMR spectroscopy permits scrutiny of a great variety of atomic nuclei in all living organisms non-invasively. It has thus provided quantitative information on cellular atoms and their chemical environment, dynamics, or interactions. We will show that NMR studies have generated valuable knowledge on a vast array of cellular molecules and events, from water, salts, metabolites, cell walls, proteins, nucleic acids, drugs and drug targets, to pH, redox equilibria and chemical reactions. The characterization of such a multitude of objects at the atomic scale has thus shaped our mental representation of cellular life at multiple levels, together with major techniques like mass-spectrometry or microscopies.
NMR studies on cells has accompanied the developments of MRI and metabolomics, and various subfields have flourished, coined with appealing names: fluxomics, foodomics, MRI and MRS (i.e. imaging and localized spectroscopy of living tissues, respectively), whole-cell NMR, on-cell ligand-based NMR, systems NMR, cellular structural biology, in-cell NMR… All these have not grown separately, but rather by reinforcing each other like a braided trunk. Hence, we try here to provide an analytical account of a large ensemble of intricately linked approaches, whose integration has been and will be key to their success.
We present extensive overviews, firstly on the various types of information provided by NMR in a cellular environment (the “why”, oriented towards a broad readership), and secondly on the employed NMR techniques and setups (the “how”, where we discuss the past, current and future methods). Each subsection is constructed as a historical anthology, showing how the intrinsic properties of NMR spectroscopy and its developments structured the accessible knowledge on cellular phenomena. Using this systematic approach, we sought i) to make this review accessible to the broadest audience and ii) to highlight some early techniques that may find renewed interest. Finally, we present a brief discussion on what may be potential and desirable developments in the context of integrative studies in biology.
Current microcoil probe technology has emerged as a significant advancement in NMR applications to biofluids research. It has continued to excel as a hyphenated tool with other prominent microdevices, opening many new possibilities in multiple omics fields. However, this does not hold for biological samples such as intact tissue or organisms, due to the considerable challenges of incorporating the microcoil in a magic‐angle spinning (MAS) probe without relinquishing the high‐resolution spectral data. Not until 2012 did a microcoil MAS probe show promise in profiling the metabolome in a submilligram tissue biopsy with spectral resolution on par with conventional high‐resolution MAS (HR‐MAS) NMR. This result subsequently triggered a great interest in the possibility of NMR analysis with microgram tissues and striving toward the probe development of “high‐resolution” capable microcoil MAS NMR spectroscopy. This review gives an overview of the issues and challenges in the probe development and summarizes the advancements toward metabolomics. Microcoil probe technology has emerged as a significant NMR application for biofluids and biosolids. However, this does not hold for biotissues due to the challenges in incorporating a microcoil under a magic‐angle spinning (MAS) condition without relinquishing the high‐resolution spectral data. This review provides a comprehensive basis for advancing microcoil MAS with “high‐resolution” capability and bridging to metabolomics.
The use of miniaturized NMR receiver coils is an effective approach for improving detection sensitivity in studies using MRS and MRI. By optimizing the filling factor (the fraction of the coil occupied by the sample), and by increasing the RF magnetic field produced per unit current, the sensitivity gain offered by NMR microcoils is particularly interesting when small volumes or regions of interest are investigated. For in vivo studies, millimetric or sub-millimetric microcoils can be deployed in tissues to access regions of interest located at a certain depth. In this study, the implementation and application of a tissue-implantable NMR microcoil with a detection volume of 850 nL is described. The RF magnetic field generated by the microcoil was evaluated using a finite element method simulation and experimentally determined by high spatial resolution MRI acquisitions. The performance of the microcoil in terms of spectral resolution and limit of detection was measured at 7 T in vitro and in vivo in rodent brains. These performances were compared with those of a conventional external detection coil. Proton MR spectra were acquired in the cortex of rat brain. The concentrations of main metabolites were quantified and compared with reference values from the literature. In vitro and in vivo results obtained with the implantable microcoil showed a gain in sensitivity greater than 50 compared with detection using an external coil. In vivo proton spectra of diagnostic value were obtained from brain regions of a few hundred nanoliters. The similarities between spectra obtained with the implanted microcoil and those obtained with the external NMR coil highlight the minimally invasive nature of the coil implantation procedure. These implantable microcoils represent new tools for probing tissue metabolism in very small healthy or diseased regions using MRS.
In this chapter, we summarize data preprocessing and data analysis strategies used for analysis of NMR data for metabolomics studies. Metabolomics consists of the analysis of the low molecular weight compounds in cells, tissues, or biological fluids, and has been used to reveal biomarkers for early disease detection and diagnosis, to monitor interventions, and to provide information on pathway perturbations to inform mechanisms and identifying targets. Metabolic profiling (also termed metabotyping) involves the analysis of hundreds to thousands of molecules using mainly state-of-the-art mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy technologies. While NMR is less sensitive than mass spectrometry, NMR does provide a wealth of complex and information rich metabolite data. NMR data together with the use of conventional statistics, modeling methods, and bioinformatics tools reveals biomarker and mechanistic information. A typical NMR spectrum, with up to 64k data points, of a complex biological fluid or an extract of cells and tissues consists of thousands of sharp signals that are mainly derived from small molecules. In addition, a number of advanced NMR spectroscopic methods are available for extracting information on high molecular weight compounds such as lipids or lipoproteins. There are numerous data preprocessing, data reduction, and analysis methods developed and evolving in the field of NMR metabolomics. Our goal is to provide an extensive summary of NMR data preprocessing and analysis strategies by providing examples and open source and commercially available analysis software and bioinformatics tools.
The magic angle coil spinning (MACS) technique has been introduced as a very promising extension for solid state NMR detection, demonstrating sensitivity enhancements by a factor of 14 from the very first time it has been reported. The main beneficiary of this technique is the scientific community dealing with mass- and volume-limited, rare, or expensive samples. However, more than a decade after the first report on MACS, there is a very limited number of groups who have continued to develop the technique, let alone it being widely adopted by practitioners. This might be due to several drawbacks associated with the MACS technology until now, including spectral linewidth,heating due to eddy currents, and imprecise manufacturing. Here, we report a device overcoming all these remaining issues, therefore achieving: 1) spectral resolution of approx. 0.01 ppm and normalized limit of detection of approx. 13 nmol\√s calculated using the anomeric proton of sucrose at 3 kHz MAS frequency; 2) limited temperature increase inside the MACS insert of only 5 °C at 5 kHz MAS frequency in an 11.74 T magnetic field, rendering MACS suitable to study live biological samples. The wafer-scale fabrication process yields MACS inserts with reproducible properties, readily available to be used on a large scale in bio-chemistry labs. To illustrate the potential of these devices for metabolomic studies, we further report on: 3) ultra-fine ¹H-¹H and ¹³C-¹³C J-couplings resolved within 10 minutes for a 340 mM uniformly ¹³C-labeled glucose sample; and 4) single zebrafish embryo measurements through ¹H-¹H COSY within 4.5 hours, opening the gate for the single embryo NMR studies.
In this article, we review the state of the field of high resolution magic angle spinning MRS (HRMAS MRS)‐based cancer metabolomics since its beginning in 2004; discuss the concept of cancer metabolomic fields, where metabolomic profiles measured from histologically benign tissues reflect patient cancer status; and report our HRMAS MRS metabolomic results, which characterize metabolomic fields in prostatectomy‐removed cancerous prostates. Three‐dimensional mapping of cancer lesions throughout each prostate enabled multiple benign tissue samples per organ to be classified based on distance from and extent of the closest cancer lesion as well as the Gleason score (GS) of the entire prostate. Cross‐validated partial least squares‐discriminant analysis separations were achieved between cancer and benign tissue, and between cancer tissue from prostates with high (≥4 + 3) and low (≤3 + 4) GS. Metabolomic field effects enabled histologically benign tissue adjacent to cancer to distinguish the GS and extent of the cancer lesion itself. Benign samples close to either low GS cancer or extensive cancer lesions could be distinguished from those far from cancer. Furthermore, a successfully cross‐validated multivariate model for three benign tissue groups with varying distances from cancer lesions within one prostate indicates the scale of prostate cancer metabolomic fields. While these findings could, at present, be potentially useful in the prostate cancer clinic for analysis of biopsy or surgical specimens to complement current diagnostics, the confirmation of metabolomic fields should encourage further examination of cancer fields and can also enhance understanding of the metabolomic characteristics of cancer in myriad organ systems. Our results together with the success of HRMAS MRS‐based cancer metabolomics presented in our literature review demonstrate the potential of cancer metabolomics to provide supplementary information for cancer diagnosis, staging, and patient prognostication. Building upon the concept of cancer metabolomic field effects, we report a semi‐quantitative, three‐dimensional method of mapping cancer lesions and HRMAS MRS‐scanned samples, which enabled the distance‐dependent existence of metabolomic fields and particularities regarding pathological features of the closest cancer to be identified in prostates, using histologically benign tissue.
Since the beginning of high-resolution magic-angle spinning (HR-MAS) NMR spectroscopy in 1990s, we have witnessed tremendous instrumentation and methodological advancements in the HR-MAS NMR technique for semisolids. With HR-MAS, it is now possible to acquire reliable high-quality spectra in a routine and high-throughput fashion, and it has become a well-integrated metabolic screening tool for ex vivo biospecimens such as tissue biopsies, cells and organisms for NMR-based metabolomics research. This chapter provides the basic principles of HR-MAS and describes a few recent noteworthy developments that could strengthen the role of HR-MAS as a frontline NMR technique for metabolomics.
In order to study metabolic processes in animal models of diseases and in patients, microdialysis probes have evolved as powerful tools that are minimally invasive. However, analyses of microdialysate, performed remotely, do not provide real-time monitoring of microdialysate composition. Microdialysate solutions can theoretically be analyzed online inside a preclicinal or clinical MRI scanner using MRS techniques. Due to low NMR sensitivity, acquisitions of real-time NMR spectra on very small solution volumes (μL) with low metabolite concentrations (mM range) represent a major issue. To address this challenge we introduce the approach of combining a microdialysis probe with a custom-built magnetic resonance microprobe that allows for online metabolic analysis (¹H and ¹³C) with high sensitivity under continuous flow conditions. This system is mounted inside an MRI scanner and allows performing simultaneously MRI experiments and rapid MRS metabolic analysis of the microdialysate. The feasibility of this approach is demonstrated by analyzing extracellular brain cancer cells (glioma) in vitro and brain metabolites in an animal model in vivo. We expect that our approach is readily translatable into clinical settings and can be used for a better and precise understanding of diseases linked to metabolic dysfunction.
This study evaluates the performances of a recently introduced 1 mm HRμMAS NMR probe for metabolic profiling of nanoliter samples. It examines essential NMR criteria such as spectral data qualities and repeatability, and experimental practicality. The report also discusses the difficulties related to the sample preparation in HRμMAS experiments and considers possible solutions and improvements.
A prototype 1 mm High-Resolution micro-Magic Angle Spinning (HRμMAS) probe is described. High quality (1)H NMR spectra were obtained from 490 μg of heterogeneous biospecimens, offering a rich-metabolite profiling. The results demonstrate the potential of HRμMAS as a new NMR analytical tool in metabolomics.
NMR spectroscopy is one of the most utilized analytical platforms for undertaking 'metabolic phenotyping' studies, whereby changes in body fluid and tissue metabolite profiles are used for diagnostic and prognostic purposes, monitoring disease progression, and even examining drug metabolism and treatment response. This permits rapid stratification of patient subgroups based on objective assessment of biochemical information that can be in turn used to inform the clinical decision-making process. NMR spectroscopy is a particularly advantageous platform as it is rapid, nondestructive, inherently quantitative, and highly reproducible. The purpose of this article is to provide the reader with an insight into how NMR spectroscopy is currently being used to study key aspects of human metabolism and metabolic phenotyping and explores how this information can be translated into clinical practice. Moreover, practical aspects of experimental design, data analysis, and technological requirements relevant to NMR spectroscopy-based phenotyping are considered.
Sixty six years after its discovery, magnetic resonance deserves the qualification of a mature technique. This paper presents the state of the art of the instrumentation in NMR and MRI. Superconducting magnets are at the heart of the NMR instruments and their performances towards high magnetic fields target high resolution and high precision applications. On the other hand, miniature systems could answer to questions that are not currently answered like the analysis outside the laboratory and in the field, chemical analysis on microfluidic chips, or systems for dedicated organ imaging. Here are presented the use of permanent magnets and specialized detectors as novel paths towards miniaturization, signal enhancement methods by hyperpolarization in order to show their potential towards coupled applications offering high sensitivity information, and finally, innovative applications that could have interest in cancer biomedicine.
The field of metabolomics continues to make a major impact on numerous disciplines, including the biomedical sciences, food and plant sciences, environmental science, and others. Nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry are the two primary analytical tools used for the analysis of complex mixtures. NMR spectroscopy represents an unparalleled tool for metabolite detection and identification because of its ability to visualize metabolic profiles in intact biofluids and biopsied tissue quantitatively and reproducibly and with high throughput. The challenges in analyzing highly complex biological matrices are also driving NMR capabilities to new levels. As a result, NMR-based metabolomics has seen tremendous growth in instrumentation and methods development, as well as a growing range of applications in areas including disease diagnostics, systems biology, personalized medicine, and many others. This review focuses on a number of exciting recent developments in the field of NMR-based metabolomics.
Even though microcoils improve the sensitivity of NMR measurement of tiny samples, magnetic-field inhomogeneity due to the bulk susceptibility effect of the coil material can cause serious resonance-line broadening. Here, we propose to fabricate the microcoil using a thin, hollow copper capillary instead of a wire and fill paramagnetic liquid inside the capillary, so as to cancel the diamagnetic contribution of the copper. Susceptibility cancellation is demonstrated using aqueous solution of NiSO4. In addition, the paramagnetic liquid serves as coolant when it is circulated through the copper capillary, effectively transferring the heat generated by radiofrequency pulses.
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Applications of NMR for metabolomics and metabolic profiling continue to grow rapidly as does the refinement of methods for the measurement, analysis and interpretation of complex data sets. Metabolomics (metabonomics) is a set of global measurements performed on biological samples with the goal of quantifying as many metabolites as possible and evaluating changes in metabolite levels as a result of an applied stress. Metabolic profiling experiments follow a more limited set of metabolites often through specific pathways. NMR is also well suited for metabolite fingerprinting, which involves the comprehensive and simultaneous analysis of a wide variety of compounds. For the purpose of this Review, we do not distinguish between metabolomics and metabonomics, and have elected to use the term metabolomics throughout. Figure 1 illustrates the rapid growth in publications on topics that include the keywords NMR and metabolomics/metabonomics since the year 2000. Due to space limitations, this Review covers only papers published between 2011 and the first half of 2014.
Magic angle spinning nuclear magnetic resonance (MAS-NMR) spectroscopy offers a convenient means for the rapid determination of metabolic profiles from intact malignant tissues with high resolution. The implementation of MAS-NMR spectroscopy requires minimal sample processing, hence being compatible with complementary histological or biochemical analyses. The metabolites routinely detected in (1)H MAS-NMR spectra can simultaneously inform on many of the metabolic alterations that characterize malignant cells, including altered choline metabolism and the so-called Warburg effect. Clinical MAS-NMR profiles have been attributed with diagnostic or prognostic value, correlating to disease subtype, tumor stage/grade, response to chemotherapy, and patient survival. Herein, the scientific rationale behind MAS-NMR and its utility for translational cancer research and patient stratification is summarized. Moreover, a basic protocol for the analysis of tumor samples by MAS-NMR spectroscopy is detailed.
Analysis of model organisms, such as the submillimeter-size Caenorhabditis elegans, plays a central role in understanding biological functions across species and in characterizing phenotypes associated with genetic mutations. In recent years, metabolic phenotyping studies of C. elegans based on (1)H high-resolution magic-angle spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy have relied on the observation of large populations of nematodes, requiring labor-intensive sample preparation that considerably limits high-throughput characterization of C. elegans. In this work, we open new platforms for metabolic phenotyping of C. elegans mutants. We determine rich metabolic profiles (31 metabolites identified) from samples of 12 individuals using a (1)H NMR microprobe featuring high-resolution magic-angle coil spinning (HR-MACS), a simple conversion of a standard HR-MAS probe to μHR-MAS. In addition, we characterize the metabolic variations between two different strains of C. elegans (wild-type vs slcf-1 mutant). We also acquire a NMR spectrum of a single C. elegans worm at 23.5 T. This study represents the first example of a metabolomic investigation carried out on a small number of submillimeter-size organisms, demonstrating the potential of NMR microtechnologies for metabolomics screening of small model organisms.
In this contribution, the latest developments in solid state NMR are presented in the field of organic–inorganic (O/I) materials (or hybrid materials). Such materials involve mineral and organic (including polymeric and biological) components, and can exhibit complex O/I interfaces. Hybrids are currently a major topic of research in nanoscience, and solid state NMR is obviously a pertinent spectroscopic tool of investigation. Its versatility allows the detailed description of the structure and texture of such complex materials. The article is divided in two main parts: in the first one, recent NMR methodological/instrumental developments are presented in connection with hybrid materials. In the second part, an exhaustive overview of the major classes of O/I materials and their NMR characterization is presented.
Metabolomic profiles of tissues could greatly contribute to advancements in personalized medicine but are influenced by differences in adopted pre-analytical procedures; non homogeneous pre- and post-excision ischemia times are potential sources of variability. In this study we monitored the impact of ischemia on the metabolic profiles, acquired with high-resolution magic-angle-spinning 1H NMR, of 162 human liver samples collected during and up to six hours after routine surgery. The profiles changed significantly as a function of intraoperative warm ischemia (WI) and post-resection cold ischemia (CI) time, with significant variations in the concentration of the same sixteen metabolites. Therefore a tight control of the pre-analytical phase is essential for reliable metabolomic analyses of liver diseases. The NMR profiles provide a reliable "fingerprint" of ischemia and have predictive value: the best-performing predictive models are found to discriminate extreme time points of CI (0' vs. 360 ') in the training set with cross validation accuracy ~90%; samples in the validation cohort can discriminate short (≤60') from long (≥180') CI with an accuracy of ~80%. For WI, the corresponding figures are 95.6 % and 92% respectively. Therefore, ischemia NMR profiles might become a tool for tissue quality control in biobanks.
Cancer is not only a complex genetic disease, but also a disease of dysregulated bioenergetic metabolism. With improved technological advancements, the focus has shifted from changes in an individual biochemical pathway or metabolite toward changes in the context of the global network of metabolic pathways in a cell, tissue or organism. This global approach allows identifying changes in the pattern of metabolite expression in addition to changes in individual metabolite or pathway. Such a metabolomics approach promises a better understanding of tumor biology and identification of potential biomarkers with applications as diagnostic, prognostic and therapeutic targets. In this review, we discuss various techniques used in metabolomics and analysis of the data generated and its specific uses in cancer research including novel biomarker identification, development of more sensitive and specific diagnostic methods, monitoring of currently used cancer therapeutics to evaluate the prognostic outcome with a given therapy and evaluating novel therapeutic strategies.
Metabolomics offers a revolutionary framework for phenotyping individuals at a molecular level that is needed for new breakthroughs in cell biology and personalized medicine. Separation science plays several critical roles in metabolomics for reliable quantification and improved identification of metabolites in complex sample mixtures. Separation efficiency and peak capacity are two important parameters for determining the performance of a separation in terms of the total number of unique compounds that are baseline resolved prior to detection. Higher peak capacity within a wider separation window reduces solute coelution that can complicate relative quantification or spectral matching for unknown identification. The use of a wider bore/thicker nonpolar stationary phase film as the second dimension column enhances separation performance by increasing sample loadability that is important when quantifying low abundance metabolites in complex biological samples.
The Magic-Angle Coil Spinning (MACS) resonator allows a simple approach for nanoliter Nuclear Magnetic Resonance (NMR) detections with enhanced sensitivity and high-resolution under sample Magic-Angle Spinning (MAS). Currently, the spectral resolution acquired with MACS is not efficient for detailed characterization of semi-solids like biopsies, where sub-hertz resolution is necessary. Here, we describe the two sources of line broadening from MACS, sample temperature gradient and anisotropic magnetic susceptibility, and present a refined High-Resolution Magic-Angle Coil Spinning (HR-MACS) resonator that improves the spectral resolution. We demonstrate with the high quality HR-MACS NMR spectra of micro-nematodes and tissue biopsy, and illustrate its potential for NMR-based metabolomics of nanoliter tissue samples.
Recent reports on microcoils are reviewed. The first part of the review includes a discussion of how the geometries of the sample and coil affect the NMR signal intensity. In addition to derivation of the well-known result that the signal intensity increases as the coil size decreases, the prediction that dilution of a small sample with magnetically inert matter leads to better sensitivity if a tiny coil is not available is given. The second part of the review focuses on the issues specific to solid-state NMR. They include realization of magic-angle spinning (MAS) using a microcoil and harnessing of such strong pulses that are feasible only with a microcoil. Two strategies for microcoil MAS, the piggyback method and magic-angle coil spinning (MACS), are reviewed. In addition, MAS of flat, disk-shaped samples is discussed in the context of solid-state NMR of small-volume samples. Strong RF irradiation, which has been exploited in wide-line spectral excitation, multiple-quantum MAS (MQMAS), and dipolar decoupling experiments, has been accompanied by new challenges regarding the Bloch-Siegert effect, the minimum time resolution of the spectrometer, and the time scale of pulse transient effects. For a possible solution to the latter problem, recent reports on active compensation of pulse transients are described.
High-resolution magic angle spinning (HR MAS) magnetic resonance spectroscopy (MRS) is a high throughput technology which has a high degree of reproducibility. Its non-destructive nature allows specimens to be evaluated by microscopy after spectral analysis, making direct comparisons to morphologic characteristics feasible. The type of information obtained from cancer tissue using HR MAS MRS depends on the study details, from collection and storage to extraction of information from the resulting spectra. HR MAS spectral explorations for clinically relevant issues are performed on single metabolite levels, or by utilizing multivariate tools ranging from unsupervised pattern recognition to supervised neural networks. HR MAS MRS is also feasible in small endoscopic biopsies from patients with Barrett's esophagus. It is important to establish a method of classification involving multivariate analysis, providing a fast, automated, robust and objective analysis of the HR MAS MRS.
Cell culture media conditioned by human foreskin fibroblasts (HFFs) provide a complex supplement of protein and metabolic factors that support in vitro proliferation of human embryonic stem cells (hESCs). However, the conditioning process is variable with different media batches often exhibiting differing capacities to maintain hESCs in culture. While recent studies have examined the protein complement of conditioned culture media, detailed information regarding the metabolic component of this media is lacking.
Using a (1)H-Nuclear Magnetic Resonance ((1)H-NMR) metabonomics approach, 32 metabolites and small compounds were identified and quantified in media conditioned by passage 11 HFFs (CMp11). A number of metabolites were secreted by HFFs with significantly higher concentration of lactate, alanine, and formate detected in CMp11 compared to non-conditioned media. In contrast, levels of tryptophan, folate and niacinamide were depleted in CMp11 indicating the utilisation of these metabolites by HFFs. Multivariate statistical analysis of the (1)H-NMR data revealed marked age-related differences in the metabolic profile of CMp11 collected from HFFs every 24 h over 72 h. Additionally, the metabolic profile of CMp11 was altered following freezing at -20°C for 2 weeks. CM derived from passage 18 HFFs (CMp18) was found to be ineffective at supporting hESCs in an undifferentiated state beyond 5 days culture. Multivariate statistical comparison of CMp11 and CMp18 metabolic profiles enabled rapid and clear discrimination between the two media with CMp18 containing lower concentrations of lactate and alanine as well as higher concentrations of glucose and glutamine.
(1)H-NMR-based metabonomics offers a rapid and accurate method of characterising hESC conditioning media and is a valuable tool for monitoring, controlling and optimising hESC culture media preparation.
We evaluated the relationship between infection by proteocephalid cestodes and the sex and weight classes of tucunaré (Cichla piquiti) captured between August 1999 and June 2001 in the Volta Grande Reservoir, Minas Gerais, Brazil. A total of 96 fish, 75.9 ± 9.3% males and 88.9 ± 6.4% females, were parasitized by Proteocephalus macrophallus and P. microscopicus, with total mean intensities of 76.6 ± 23.9 and 145.2 ± 36.7, respectively, during this period. In the majority of the months analysed, males showed 71.4-100% prevalence of parasitism and females 80-100%. Although there was no significant difference, females showed a higher mean intensity of infection (145.2 ± 36.7) than males (76.6 ± 23.9). Fish weighing 300-800 g showed a higher mean abundance of parasites (P < 0.05) compared with the biggest specimens weighing 801-2750 g. Analysing both males and females together, the greatest mean intensities of infection were found in October and December (P < 0.05) independent of the year, which coincides with the months of highest rainfall. These results show that fish living in reservoirs may be more susceptible to intermediate hosts than those that live in rivers.
HRMAS NMR is considered a valuable technique to obtain detailed metabolic profile of unprocessed tissues. To properly interpret the HRMAS metabolomic results, detailed information of the actual state of the sample inside the rotor is needed. MRM (Magnetic Resonance Microscopy) was applied for obtaining structural and spatially localized metabolic information of the samples inside the HRMAS rotors. The tissue was observed stuck to the rotor wall under the effect of HRMAS spinning. MRM spectroscopy showed a transference of metabolites from the tissue to the medium. The sample shape and the metabolite transfer after HRMAS indicated that tissue had undergone alterations and it can not be strictly considered as intact. This must be considered when HRMAS is used for metabolic tissue characterization, and it is expected to be highly dependent on the manipulation of the sample. The localized spectroscopic information of MRM reveals the biochemical compartmentalization on tissue samples hidden in the HRMAS spectrum.
Metabolic profiling, metabolomic and metabonomic studies require robust study protocols for any large-scale comparisons and evaluations. Detailed methods for solution-state NMR spectroscopy have been summarized in an earlier protocol. This protocol details the analysis of intact tissue samples by means of high-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy and we provide a detailed description of sample collection, preparation and analysis. Described here are (1)H NMR spectroscopic techniques such as the standard one-dimensional, relaxation-edited, diffusion-edited and two-dimensional J-resolved pulse experiments, as well as one-dimensional (31)P NMR spectroscopy. These are used to monitor different groups of metabolites, e.g., sugars, amino acids and osmolytes as well as larger molecules such as lipids, non-invasively. Through the use of NMR-based diffusion coefficient and relaxation times measurements, information on molecular compartmentation and mobility can be gleaned. The NMR methods are often combined with statistical analysis for further metabonomics analysis and biomarker identification. The standard acquisition time per sample is 8-10 min for a simple one-dimensional (1)H NMR spectrum, giving access to metabolite information while retaining tissue integrity and hence allowing direct comparison with histopathology and MRI/MRS findings or the evaluation together with biofluid metabolic-profiling data.
There is a growing need for fast, highly sensitive and quantitative technologies to detect and profile unaltered cells in biological samples. Technologies in current clinical use are often time consuming, expensive, or require considerable sample sizes. Here, we report a diagnostic magnetic resonance (DMR) sensor that combines a miniaturized NMR probe with targeted magnetic nanoparticles for detection and molecular profiling of cancer cells. The sensor measures the transverse relaxation rate of water molecules in biological samples in which target cells of interest are labeled with magnetic nanoparticles. We achieved remarkable sensitivity improvements over our prior DMR prototypes by synthesizing new nanoparticles with higher transverse relaxivity and by optimizing assay protocols. We detected as few as 2 cancer cells in 1-microL sample volumes of unprocessed fine-needle aspirates of tumors and profiled the expression of several cellular markers in <15 min.
The Human Metabolome Database (HMDB, http://www.hmdb.ca) is a richly annotated resource that is designed to address the broad needs of biochemists, clinical chemists, physicians, medical geneticists, nutritionists and members of the metabolomics community. Since its first release in 2007, the HMDB has been used to facilitate the research for nearly 100 published studies in metabolomics, clinical biochemistry and systems biology. The most recent release of HMDB (version 2.0) has been significantly expanded and enhanced over the previous release (version 1.0). In particular, the number of fully annotated metabolite entries has grown from 2180 to more than 6800 (a 300% increase), while the number of metabolites with biofluid or tissue concentration data has grown by a factor of five (from 883 to 4413). Similarly, the number of purified compounds with reference to NMR, LC-MS and GC-MS spectra has more than doubled (from 380 to more than 790 compounds). In addition to this significant expansion in database size, many new database searching tools and new data content has been added or enhanced. These include better algorithms for spectral searching and matching, more powerful chemical substructure searches, faster text searching software, as well as dedicated pathway searching tools and customized, clickable metabolic maps. Changes to the user-interface have also been implemented to accommodate future expansion and to make database navigation much easier. These improvements should make the HMDB much more useful to a much wider community of users.
We describe a method that directly relates tissue neuropathological analysis to medical imaging. Presently, only indirect and often tenuous relationships are made between imaging (such as MRI or x-ray computed tomography) and neuropathology. We present a biochemistry-based, quantitative neuropathological method that can help to precisely quantify information provided by in vivo proton magnetic resonance spectroscopy (1HMRS), an emerging medical imaging technique. This method, high resolution magic angle spinning (HRMAS) 1HMRS, is rapid and requires only small amounts of unprocessed samples. Unlike chemical extraction or other forms of tissue processing, this method analyzes tissue directly, thus minimizing artifacts. We demonstrate the utility of this method by assessing neuronal damage using multiple tissue samples from differently affected brain regions in a case of Pick disease, a human neurodegenerative disorder. Among different regions, we found an excellent correlation between neuronal loss shown by traditional neurohistopathology and decrease of the neuronal marker N-acetylaspartate measured by HRMAS 1HMRS. This result demonstrates for the first time, to our knowledge, a direct, quantitative link between a decrease in N-acetylaspartate and neuronal loss in a human neurodegenerative disease. As a quantitative method, HRMAS 1HMRS has potential applications in experimental and clinical neuropathologic investigations. It should also provide a rational basis for the interpretation of in vivo 1HMRS studies of human neurological disorders.
Assigning functions to every gene in a living organism is the next challenge for functional genomics. In fact, 85–90% of the 19,000 genes of the nematode Caenorhabditis elegans genome do not produce any visible phenotype when inactivated, which hampers determining their function, especially when they do not belong to previously characterized gene families. We used ¹H high-resolution magic angle spinning NMR spectroscopy (¹H HRMAS-NMR) to reveal the latent phenotype associated to superoxide dismutase (sod-1) and catalase (ctl-1) C. elegans mutations, both involved in the elimination of radical oxidative species. These two silent mutations are significantly discriminated from the wild-type strain and from each other. We identify a metabotype significantly associated with these mutations involving a general reduction of fatty acyl resonances from triglycerides, unsaturated lipids being known targets of free radicals. This work opens up perspectives for the use of ¹H HRMAS-NMR as a molecular phenotyping device for model organisms. Because it is amenable to high throughput and is shown to be highly informative, this approach may rapidly lead to a functional and integrated metabonomic mapping of the C. elegans genome at the systems biology level.
• functional genomics
• metabolic profiling
• metabonomics
• nuclear magnetic resonance
Various issues affecting nuclear magnetic resonance probe resolution are discussed, with emphasis on high-resolution or magic angle spinning (MAS) in this part. Symmetric positioning of chip capacitors at the magic angles addresses the largest B0 perturbation with ultra-low-inductance decoupling coils, and symmetric spinner drive designs with silicon-nitride coilforms are shown to be extremely beneficial in reducing spinning-, decoupling-, and variable temperature-induced thermal gradients. Other factors discussed include MAS sample cell design, high frequency (HF) coils, centrifugation, shimming, B1 homogeneity, B0 stability, and geometric compensation for variable angle spinning resolution. ©1998 John Wiley & Sons, Inc. Concepts Magn Reson 10: 239–260, 1998
High-resolution, proton nuclear magnetic resonance (NMR) spectra of 5-nanoliter samples have been obtained with much higher mass sensitivity [signal-to-noise ratio (S/N) per micromole] than with traditional methods. Arginine and sucrose show a mean sensitivity enhancement of 130 compared to 278-microliter samples run in a 5-millimeter tube in a conventional, commercial probe. This can reduce data acquisition time by a factor of >16,000 or reduce the needed sample mass by a factor of about 130. A linewidth of 0.6 hertz was achieved on a 360-megahertz spectrometer by matching the magnetic susceptibility of the medium that surrounds the detection cell to of the copper coil. For sucrose, the limit of detection (defined at S/N = 3) was 19 nanograms (56 picomoles) for a 1-minute data acquisition. This technique should prove useful with mass-limited samples and for use as a detector in capillary separations.
ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
Sensitivity in solid-state nuclear magnetic resonance can be improved by the use of rotating microcoils (1). The connection to the rest of the electronics is performed through resonant inductive coupling. This mode of detection of nuclear induction has major advantages as it provides a wireless way to obtain extremely high radio-frequency amplitudes per unit current under sample rotation and thus improved sensitivity for size-limited samples. We review the circuit electronics and discuss experimental optimization of the probe and coil parameters. We also present alternative geometries for coupling between rotating coils and we explicitly calculate the signal enhancement. Two practical cases are presented, namely a standard magic angle sample spinning probe and a double sample rotation probe. We conclude with a theoretical discussion about the limits of detection attainable with coil miniaturization. © 2011 Wiley Periodicals, Inc. Concepts Magn Reson Part A 38A: 33–51, 2011.
The application of nuclear magnetic resonance (NMR) to systems of limited quantity has stimulated the use of micro-coils (diameter <1 mm). One method recently proposed for the union of micro-coils with Magic Angle sample Spinning (MAS), involves the integration of a tuned micro-coil circuit within standard MAS rotors inductively coupled to the MAS probe coil, termed “magic-angle coil spinning” (MACS). The spinning of conductive materials results in the creation of circulating Foucault (eddy) currents, which generate heat. We report the first data acquired with a 4 mm MACS system and spinning up to 10 kHz. The need to spin faster necessitates improved methods to control heating. We propose an approximate solution to calculate the power losses (heat) from the eddy currents for a solenoidal coil, in order to provide insight into the functional dependencies of Foucault currents. Experimental tests of the dependencies reveal conditions which result in reduced sample heating and negligible temperature distributions over the sample volume.
Increasing the sensitivity and throughput of NMR-based metabolomics is critical for the continued growth of this field. In this paper the application of micro-coil NMR probe technology was evaluated for this purpose. The most commonly used biofluids in metabolomics are urine and serum. In this study we examine different sample limited conditions and compare the detection sensitivity of the micro-coil with a standard 5 mm NMR probe. Sample concentration is evaluated as a means to leverage the greatly improved mass sensitivity of the micro-coil probes. With very small sample volumes, the sensitivity of the micro-coil probe does indeed provide a significant advantage over the standard probe. Concentrating the samples does improve the signal detection, but the benefits do not follow the expected linear increase and are both matrix and metabolite specific. Absolute quantitation will be affected by concentration, but an analysis of relative concentrations is still possible. The choice of the micro-coil probe over a standard tube based probe will depend upon a number of factors including number of samples and initial volume but this study demonstrates the feasibility of high-throughput metabolomics with the micro-probe platform.
High-resolution magic-angle spinning has become a valuable tool to study biologic samples; however, there are only a few high-resolution magic-angle spinning methodologies that have been explored specifically to deal with small semisolid biomaterials such as tissues. Here, we report a novel high-resolution (1)H NMR spectroscopic approach using magic-angle coil spinning. We demonstrate its potential for intact tissues by combining a resonant microcoil with a large-volume commercial magic-angle spinning rotor, where the microcoil offers incredible sensitivity and the large-volume magic-angle spinning rotor provides stable slow magic-angle spinning. This approach provides high-resolution spectra with high sensitivity and the preservation of living biologic specimens for an accurate chemical analysis.
Current clinical strategy for staging and prognostication of colorectal cancer (CRC) relies mainly upon the TNM or Duke system. This clinicopathological stage is a crude prognostic guide because it reflects in part the delay in diagnosis in the case of an advanced cancer and gives little insight into the biological characteristics of the tumor. We hypothesized that global metabolic profiling (metabonomics/metabolomics) of colon mucosae would define metabolic signatures that not only discriminate malignant from normal mucosae, but also could distinguish the anatomical and clinicopathological characteristics of CRC. We applied both high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) and gas chromatography mass spectrometry (GC/MS) to analyze metabolites in biopsied colorectal tumors and their matched normal mucosae obtained from 31 CRC patients. Orthogonal partial least-squares discriminant analysis (OPLS-DA) models generated from metabolic profiles obtained by both analytical approaches could robustly discriminate normal from malignant samples (Q(2) > 0.50, Receiver Operator Characteristic (ROC) AUC >0.95, using 7-fold cross validation). A total of 31 marker metabolites were identified using the two analytical platforms. The majority of these metabolites were associated with expected metabolic perturbations in CRC including elevated tissue hypoxia, glycolysis, nucleotide biosynthesis, lipid metabolism, inflammation and steroid metabolism. OPLS-DA models showed that the metabolite profiles obtained via HR-MAS NMR could further differentiate colon from rectal cancers (Q(2)> 0.60, ROC AUC = 1.00, using 7-fold cross validation). These data suggest that metabolic profiling of CRC mucosae could provide new phenotypic biomarkers for CRC management.
High resolution magic angle spinning (HRMAS) has become an extremely versatile tool to study heterogeneous systems. HRMAS relies on magic angle spinning of the sample to average out to zero magnetic susceptibility differences in the sample and to obtain resonance linewidths approaching those of liquid state NMR. Shimming such samples therefore becomes an important issue. By analyzing the different sources of magnetic field perturbations present in a sample under MAS conditions, we propose a simple protocol to obtain optimum shim settings in HRMAS. In the case of aqueous samples, we show that the lock level cannot be used as a reliable indicator of the quality of the shims at high spinning speeds. This effect is explained by the presence of temperature gradients imparted by the sample rotation.
We report the construction of a dual-channel microcoil nuclear magnetic resonance probehead allowing magic-angle spinning for mass-limited samples. With coils down to 235 mum inner diameter, this allows high-resolution solid-state NMR spectra to be obtained for amounts of materials of a few nanoliters. This is demonstrated by the carbon-13 spectrum of a tripeptide and a single silk rod, prepared from the silk gland of the Bombyx mori silkworm. Furthermore, the microcoil allows for radio frequency field strengths well beyond current probe technology, aiding in getting the highest possible resolution by efficiently decoupling the observed nuclei from the abundantly present proton nuclei.
Nuclear magnetic resonance (NMR) can probe the local structure and dynamic properties of liquids and solids, making it one of the most powerful and versatile analytical methods available today. However, its intrinsically low sensitivity precludes NMR analysis of very small samples-as frequently used when studying isotopically labelled biological molecules or advanced materials, or as preferred when conducting high-throughput screening of biological samples or 'lab-on-a-chip' studies. The sensitivity of NMR has been improved by using static micro-coils, alternative detection schemes and pre-polarization approaches. But these strategies cannot be easily used in NMR experiments involving the fast sample spinning essential for obtaining well-resolved spectra from non-liquid samples. Here we demonstrate that inductive coupling allows wireless transmission of radio-frequency pulses and the reception of NMR signals under fast spinning of both detector coil and sample. This enables NMR measurements characterized by an optimal filling factor, very high radio-frequency field amplitudes and enhanced sensitivity that increases with decreasing sample volume. Signals obtained for nanolitre-sized samples of organic powders and biological tissue increase by almost one order of magnitude (or, equivalently, are acquired two orders of magnitude faster), compared to standard NMR measurements. Our approach also offers optimal sensitivity when studying samples that need to be confined inside multiple safety barriers, such as radioactive materials. In principle, the co-rotation of a micrometre-sized detector coil with the sample and the use of inductive coupling (techniques that are at the heart of our method) should enable highly sensitive NMR measurements on any mass-limited sample that requires fast mechanical rotation to obtain well-resolved spectra. The method is easy to implement on a commercial NMR set-up and exhibits improved performance with miniaturization, and we accordingly expect that it will facilitate the development of novel solid-state NMR methodologies and find wide use in high-throughput chemical and biomedical analysis.
High-resolution magnetic field probes based on pulsed liquid-state NMR are presented. Static field measurements with an error of 10 nanotesla or less at 3 tesla are readily obtained in 100 ms. The further ability to measure dynamic magnetic fields results from using small ( approximately 1 microL) droplets of MR-active liquid surrounded by susceptibility-matched materials. The consequent high field homogeneity allows free induction decay signals lasting 100 ms or more to be readily achieved. The small droplet dimensions allow the magnetic field to be measured even in the presence of large gradients. Highly sensitive detection yields sufficient SNR to follow the relevant field evolution without signal averaging and at bandwidths up to hundreds of kHz. Transient, nonreproducible effects and drifts are thus readily monitored. The typical application of k-space trajectory mapping has been demonstrated. Potential further applications include characterization, tuning, and maintenance of gradient systems as well as the mapping of the static field distribution of MRI magnets. Connection of the probes to a standard MR spectrometer is similar to that used for imaging coils.
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