Metabonomics studies of intact hepatic and renal cortical tissues from diabetic db/db mice using high-resolution magic-angle spinning 1H NMR spectroscopy

A metabonomics approach based on high-resolution magic-angle spinning (HRMAS) ¹H NMR spectroscopy was applied to investigate the metabolite composition in intact hepatic tissues and renal cortical tissues from db/db mice of 8 weeks old, an animal model of type 2 diabetes mellitus. Compared to the control group, the hepatic tissues of diabetic mice have elevated levels of triglyceride and bile acid and declined levels of trimethylamine-N-oxide, phosphocholine, glycerophosphocholine, and choline. The biochemical changes are less obvious in renal cortical tissues of diabetic mice. The WET_CPMG pulse sequence was selected for our metabonomics study after the quality and reproducibility of the spectra obtained from the NOEPR, NOEPR_CPMG, and WET_CPMG pulse sequences were analyzed together with principal component analysis. The influence of line-broadening factor of exponential window function for spectral manipulation on class separation was paid attention to for the first time, and an optimal value was obtained under our experimental conditions. These studies show the efficiency of HRMAS ¹H NMR spectroscopy for tissue metabonomics study in combination with multivariate statistical analysis, which may help to explore the etiological factor of diabetes mellitus from a new perspective.

1H‐HRMAS NMR study of cold smoked Atlantic salmon (Salmo salar) treated with E‐beam

¹H High resolution magic angle spinning (HRMAS) NMR spectroscopic data in combination with principal components analysis and analysis of variance were used to differentiate between irradiated and non‐irradiated cold‐smoked Atlantic salmon (Salmo salar). NMR profiling was obtained, with a spectral acquisition time of less than 8 min, from a small sample size of intact white salmon muscle, by non‐destructive analysis that includes a very simple and rapid sample preparation step. Results obtained enable the use of creatine, trimethylamine oxide and the sum of phosphorylcholine and glycerophosphorylcholine as diagnostic compounds to detect irradiation treatment. This study shows the potential of ¹H‐HRMAS to be a rapid method for investigating compositional changes due to food processing as well as to confirm the presence or absence of some bioactive compounds in irradiated samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Characterization of free,restricted, and entrapped water environments in poly(N‐isopropyl acrylamide) hydrogels via 1H HRMAS PFG NMR spectroscopy

Different water environments in poly(N‐isopropyl acrylamide) (PNIPAAm) hydrogels are identified and characterized using ¹H high resolution magic angle spinning (HRMAS) nuclear magnetic resonance (NMR). Local water environments corresponding to a “free” highly mobile species, along with waters showing restricted dynamics are resolved in these swollen hydro‐gels. For photo‐initiated polymerized PNIPAAm gels, an additional entrapped water species is observed. Spin–spin R₂ relaxation experiments support the argument of reduced mobility in the restricted and entrapped water species. By combining pulse field gradient techniques with HRMAS NMR it is possible to directly measure the self‐diffusion rate for these different water environments. The behavior of the heterogeneous water environments through the lower critical solution temperature transition is described. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2014 , 52, 1521–1527     

1H‐HRMAS NMR study of smoked Atlantic salmon (Salmo salar)

High‐resolution magic angle spinning (HRMAS) NMR spectroscopic data of smoked Atlantic salmon (Salmo salar) were fully assigned by combination of one‐ and two‐dimensional‐HRMAS experiments. Complete representative spectra, obtained after few minutes of analysis time, revealed a large number of minor and major compounds in the sample. The methodology is limited by the low sensitivity of NMR, and therefore HRMAS only enables the determination of the most relevant components. These were fatty acids (FAs), carbohydrates, nucleoside derivatives, osmolytes, amino acids, dipeptides and organic acids. For the first time, spectra were resolved sufficiently to allow semiquantitative determination in intact muscle of the highly polyunsaturated FA 22:6 ω‐3. Additionally, the feasibility of ¹H‐HRMAS NMR metabolite profiling was tested to identify some bioactive compounds during storage. This profiling was carried out by the non‐destructive and direct analysis (i.e. without requiring sample preparation and multiple step procedures) of intact salmon muscle. The proposed procedure can be applied to a large number of samples with high throughput due to the short time of analysis and quick evaluation of the data. Copyright © 2010 John Wiley & Sons, Ltd.     

Study of metabonomic profiles of human esophageal carcinoma by use of high-resolution magic-angle spinning 1H NMR spectroscopy and multivariate data analysis

Esophageal carcinoma (EC) is one of the most common malignant tumors. EC survival has remained disappointingly low because of the high malignancy of esophageal cancer and the lack of obvious clinical symptoms at an early stage. Early diagnosis is often difficult because the small tumor nodules are frequently missed. Metabonomics based on high-resolution magic-angle spinning (HRMAS) NMR has been popular for tumor detection because it is highly sensitive, provides rich biochemical information and requires no sample pretreatment. ¹H HRMAS spectra of non-involved adjacent esophageal tissues and of well differentiated and moderately differentiated esophageal carcinoma tumors were recorded and analyzed by use of multivariate and statistical analysis techniques. Moderately differentiated EC tumors were found to have increased total choline, alanine, and glutamate and reduced creatine, myo-inositol, and taurine compared with non-involved adjacent tissues. Moreover, clear differences between the metabonomic profiles of EC tissues enabled tumor differentiation. Furthermore, the integral Gly/MI ratio for samples of different tissue types were statistically significantly different; this was sufficient both for distinguishing non-involved tissues from esophageal carcinoma and for classification of well differentiated and moderately differentiated EC tumors.

The Effect of Blood Contained in the Samples on the Metabolomic Profile of Mouse Brain Tissue: A Study by NMR Spectroscopy

(1) Recently, metabolic profiling of the tissue in the native state or extracts of its metabolites has become increasingly important in the field of metabolomics. An important factor, in this case, is the presence of blood in a tissue sample, which can potentially lead to a change in the concentration of tissue metabolites and, as a result, distortion of experimental data and their interpretation. (2) In this paper, the metabolomic profiling based on NMR spectroscopy was performed to determine the effect of blood contained in the studied samples of brain tissue on their metabolomic profile. We used 13 male laboratory CD-1^® IGS mice for this study. The animals were divided into two groups. The first group of animals (n = 7) was subjected to the perfusion procedure, and the second group of animals (n = 6) was not perfused. The brain tissues of the animals were homogenized, and the metabolite fraction was extracted with a water/methanol/chloroform solution. Samples were studied by high-frequency ¹H-NMR spectroscopy with subsequent statistical data analysis. The group comparison was performed with the use of the Student’s test. We identified 36 metabolites in the brain tissue with the use of NMR spectroscopy. (3) For the major set of studied metabolites, no significant differences were found in the brain tissue metabolite concentrations in the native state and after the blood removal procedure. (4) Thus, it was shown that the presence of blood does not have a significant effect on the metabolomic profile of the brain in animals without pathologies.     

Metabolite assignment of ultrafiltered synovial fluid extracted from knee joints of reactive arthritis patients using high resolution NMR spectroscopy

Currently, there are no reliable biomarkers available that can aid early differential diagnosis of reactive arthritis (ReA) from other inflammatory joint diseases. Metabolic profiling of synovial fluid (SF)—obtained from joints affected in ReA—holds great promise in this regard and will further aid monitoring treatment and improving our understanding about disease mechanism. As a first step in this direction, we report here the metabolite specific assignment of ¹H and ¹³C resonances detected in the NMR spectra of SF samples extracted from human patients with established ReA. The metabolite characterization has been carried out on both normal and ultrafiltered (deproteinized) SF samples of eight ReA patients (n = 8) using high-resolution (800 MHz) ¹H and ¹H─¹³C NMR spectroscopy methods such as one-dimensional ¹H CPMG and two-dimensional J-resolved¹H NMR and homonuclear ¹H─¹H TOCSY and heteronuclear¹H─¹³C HSQC correlation spectra. Compared with normal SF samples, several distinctive ¹H NMR signals were identified and assigned to metabolites in the ¹H NMR spectra of ultrafiltered SF samples. Overall, we assigned 53 metabolites in normal filtered SF and 64 metabolites in filtered pooled SF sample compared with nonfiltered SF samples for which only 48 metabolites (including lipid/membrane metabolites as well) have been identified. The established NMR characterization of SF metabolites will serve to guide future metabolomics studies aiming to identify/evaluate the SF-based metabolic biomarkers of diagnostic/prognostic potential or seeking biochemical insights into disease mechanisms in a clinical perspective.     

Identification of metabolites in coriander seeds (Coriandrum Sativum L.) aided by ultrahigh resolution total correlation NMR spectroscopy

NMR is a fast method for obtaining a holistic snapshot of the metabolome and also offers quantitative information without separating the compounds present in a complex mixture. Identification of the metabolites present in a plant extract sample is a crucial step for all plant metabolomics studies. In the present work, we used various two dimensional (2D) NMR methods such as J-resolved NMR, total correlation spectroscopy (TOCSY), and heteronuclear single quantum coherence sensitivity enhanced NMR spectroscopy for the identification of 36 common metabolites present in Coriandrum sativum L. seed extract. The identified metabolites belong to the following classes: organic acids, amino acids, and carbohydrates. ¹H NMR spectra of such complex mixtures in general display tremendous signal overlap due to the presence of a large number of metabolites with closely resonating multiplet signals. This signal overlapping leads to ambiguity in an assignment, and hence, identification of metabolites becomes tedious or impossible in many cases. Therefore, the utility of pure-shift proton spectrum along the indirect (F₁) dimension of the F₁-PSYCHE-TOCSY spectrum is demonstrated for overcoming ambiguity in assignment of metabolites in crowded spectral regions from Coriandrum sativum L. seed extract sample. Because pure-shift NMR methods yield ultrahigh resolution spectrum (i.e., a singlet peak per chemical site) along one or more dimensions, such spectra provide better identification of metabolites compared with regular 2D TOCSY where signal overlap and peak distortions lead to ambiguity in the assignment. Nine metabolites were unambiguously assigned by pure-shift F₁-PSYCHE-TOCSY spectrum, which was unresolved in regular TOCSY spectrum.     

Metabolomics of meat exudate: Its potential to evaluate beef meat conservation and aging

In this study we analyzed the exudate of beef to evaluate its potential as non invasive sampling for nuclear magnetic resonance (NMR) based metabolomic analysis of meat samples. Exudate, as the natural juice from raw meat, is an easy to obtain matrix that it is usually collected in small amounts in commercial meat packages. Although meat exudate could provide complete and homogeneous metabolic information about the whole meat piece, this sample has been poorly studied. Exudates from 48 beef samples of different breeds, cattle and storage times have been studied by ¹H NMR spectroscopy. The liquid exudate spectra were compared with those obtained by High Resolution Magic Angle Spinning (HRMAS) of the original meat pieces. The close correlation found between both spectra (>95% of coincident peaks in both registers; Spearman correlation coefficient = 0.945) lead us to propose the exudate as an excellent alternative analytical matrix with a view to apply meat metabolomics. 60 metabolites could be identified through the analysis of mono and bidimensional exudate spectra, 23 of them for the first time in NMR meat studies. The application of chemometric tools to analyze exudate dataset has revealed significant metabolite variations associated with meat aging. Hence, NMR based metabolomics have made it possible both to classify meat samples according to their storage time through Principal Component Analysis (PCA), and to predict that storage time through Partial Least Squares (PLS) regression.     

HRMAS NMR spectroscopy combined with chemometrics as an alternative analytical tool to control cigarette authenticity

In this paper, we present for the first time the use of high-resolution magic angle spinning nuclear magnetic resonance (HRMAS NMR) spectroscopy combined with chemometrics as an alternative tool for the characterization of tobacco products from different commercial international brands as well as for the identification of counterfeits. Although cigarette filling is a very complex chemical mixture, we were able to discriminate between dark, bright, and additive-free cigarette blends belonging to six different filter-cigarette brands, commercially available, using an approach for which no extraction procedure is required. Second, we focused our study on a specific worldwide-distributed brand for which established counterfeits were available. We discriminated those from their genuine counterparts with 100 % accuracy using unsupervised multivariate statistical analysis. The counterfeits that we analyzed showed a higher amount of nicotine and solanesol and a lower content of sugars, all endogenous tobacco leaf metabolites. This preliminary study demonstrates the great potential of HRMAS NMR spectroscopy to help in controlling cigarette authenticity.

Coacervation of Elastin‐Like Recombinamer Microgels

The investigation of the coacervation (self‐aggregation) behavior of biomicrogels which can potentially be used as drug carriers is an important topic, because self‐aggregation can not only cause loss of activity, but also toxicity and immunogenicity. To study this effect microgels from elastin‐like recombinamer are synthesized using miniemulsion technique. The existence of coacervation for such microgels, at different concentrations and temperatures, is studied and proved by cryo‐field emission scanning clectron microscopy (cryo‐FESEM), cryo‐transmission electron microscopy (cryo‐TEM), and by a novel ¹H high‐resolution magic angle sample spinning (HRMAS), nuclear magnetic resonance (NMR) spectroscopy, and relaxometry methods. The findings by ¹H HRMAS NMR spectroscopy and relaxometry show simultaneous processes of volume phase temperature transition and coacervation with different sensitivity for hydrophobic and hydrophilic amino acid side‐chains in the microgel. The coacervation process is more evidential by the behavior of glycine α‐CH₂, ¹H NMR peak as compared to the proline β‐CH₂.

     

Discrimination of allied species within the genus Turbinaria (Fucales, Phaeophyceae) using HRMAS NMR spectroscopy

A novel chemotaxonomical method based on 1D (1)H HRMAS NMR spectroscopy is being tested for taxonomical purposes. This powerful technique allowed us to discriminate between specimens belonging to two sister species of Turbinaria, which are difficult to tell apart using only morphological characters. Based on spectra analysis, the results allowed us to successfully group the specimens according to their species. Thus, the efficiency of HRMAS NMR spectroscopy for the discrimination of algal species and for the pre-screening of potential chemomarkers is demonstrated.     

A metabonomics investigation of multiple sclerosis by nuclear magnetic resonance

Multiple sclerosis (MS) is a nervous system disease that affects the fatty myelin sheaths around the axons of the brain and spinal cord, leading to demyelination and a broad range of signs and symptoms. MS can be difficult to diagnose because its signs and symptoms may be similar to other medical problems. To find out which metabolites in serum are effective for the diagnosis of MS, we utilized metabolic profiling using proton nuclear magnetic resonance spectroscopy (¹H‐NMR). Random forest (RF) was used to classify the MS patients and healthy subjects. Atomic absorption spectroscopy was used to measure the serum levels of selenium. The results showed that the levels of selenium were lower in the MS group, when compared with the control group. RF was used to identify the metabolites that caused selenium changes in people with MS by building a correlation model between these metabolites and serum levels of selenium. For the external test set, the obtained classification model showed a 93% correct classification of MS and healthy subjects. The regression model of levels of selenium and metabolites showed the correlation (R²) value of 0.88 for the external test set. The results indicate the suitability of NMR as a screen for identifying MS patients and healthy subjects. A novel model with good prediction outcomes was constructed between serum levels of selenium and NMR data. Copyright © 2012 John Wiley & Sons, Ltd.     

Resolving the structure of ligands bound to the surface of superparamagnetic iron oxide nanoparticles by high-resolution magic-angle spinning NMR spectroscopy

A major challenge in magnetic nanoparticle synthesis and (bio)functionalization concerns the precise characterization of the nanoparticle surface ligands. We report the first analytical NMR investigation of organic ligands stably anchored on the surface of superparamagnetic nanoparticles (MNPs) through the development of a new experimental application of high-resolution magic-angle spinning (HRMAS). The conceptual advance here is that the HRMAS technique, already being used for MAS NMR analysis of gels and semisolid matrixes, enables the fine-structure-resolved characterization of even complex organic molecules bound to paramagnetic nanocrystals, such as nanosized iron oxides, by strongly decreasing the effects of paramagnetic disturbances. This method led to detail-rich, well-resolved (1)H NMR spectra, often with highly structured first-order couplings, essential in the interpretation of the data. This HRMAS application was first evaluated and optimized using simple ligands widely used as surfactants in MNP synthesis and conjugation. Next, the methodology was assessed through the structure determination of complex molecular architectures, such as those involved in MNP3 and MNP4. The comparison with conventional probes evidences that HRMAS makes it possible to work with considerably higher concentrations, thus avoiding the loss of structural information. Consistent 2D homonuclear (1)H- (1)H and (1)H- (13)C heteronuclear single-quantum coherence correlation spectra were also obtained, providing reliable elements on proton signal assignments and carbon characterization and opening the way to (13)C NMR determination. Notably, combining the experimental evidence from HRMAS (1)H NMR and diffusion-ordered spectroscopy performed on the hybrid nanoparticle dispersion confirmed that the ligands were tightly bound to the particle surface when they were dispersed in a ligand-free solvent, while they rapidly exchanged when an excess of free ligand was present in solution. In addition to HRMAS NMR, matrix-assisted laser desorption ionization time-of-flight MS analysis of modified MNPs proved very valuable in ligand mass identification, thus giving a sound support to NMR characterization achievements.     

Stimuli-Responsive Polymers in Solution Investigated by NMR and Infrared Spectroscopy

Summary: Temperature-induced and solvent composition-induced phase separation in solutions of poly(N-isopropylmethacrylamide) (PIPMAm) and other thermoresponsive polymers as studied by NMR and infrared (IR) spectroscopy is discussed. The fraction p of phase-separated units (units with significantly reduced mobility) and subsequently, e.g., thermodynamic parameters characterizing the coil-globule phase transition induced by temperature, were determined from reduced integrated intensities in high-resolution ¹H NMR spectra. This approach can be especially useful in investigations of phase separation in solutions of binary polymer systems. Information on behaviour of water during temperature-induced phase transition was obtained from measurements of ¹H NMR relaxation times of HDO molecules. NMR and IR spectroscopy were used to investigate PIPMAm solutions in water/ethanol (D₂O/EtOH) mixtures where the phase separation can be induced by solvent composition (cononsolvency). Some differences in globular-like structures induced by temperature and solvent composition were revealed by these methods.     

Full 1H and 13C NMR spectral assignment of conjugated valerolactone metabolites isolated from urine of black tea consumers by means of SPE-prepLC–MS–LC–MS-NMR

The health benefits of black tea have been linked to polyphenol metabolites that target specific modes of action in the human body. A major bottleneck in unravelling the underlying mechanisms is the preparative isolation of these metabolites, which hampers their structural elucidation and assessment of in vitro bioactivity. A solid phase extraction (SPE)-preparative liquid chromatography (prepLC)–MS–LC–MS-NMR workflow was implemented for preparative isolation of conjugated valerolactone metabolites of catechin-based polyphenols from urine of black tea consumers. First, the urine was cleaned and preconcentrated using an SPE method. Subsequently, the clean urine concentrate was injected on a preparative LC column, and conjugated valerolactones were obtained by MS-guided collection. Reconstituted fractions were further separated on an analytical LC column, and valerolactone fractions were collected in an MS-guided manner. These were reconstituted in methanol-d₄ and identified and quantified using 1D and 2D homo- and hetereonuclear NMR experiments (at a field strength of 14.1 T), in combination with mass spectrometry. This resulted in the full spectral ¹H and ¹³C NMR assignments of five conjugated valerolactones. These metabolites were collected in quantities of 8–160 μg and purities of 70–91%. The SPE-prepLC–MS–LC–MS-NMR workflow is suitable for isolating metabolites that occur at sub-μM concentrations in a complex biofluid such as urine. The workflow also provides an alternative for cumbersome and expensive de novo synthesis of tea metabolites for testing in bioactivity assays or for use as authentic analytical standards for quantification by mass spectrometry.     

NMR spectroscopy evaluation of direct relationship between soils and molecular composition of red wines from Aglianico grapes

¹H NMR spectroscopy was employed to investigate the molecular quality of Aglianico red wines from the Campania region of Italy. The wines were obtained from three different Aglianico vineyards characterized by different microclimatic and pedological properties. In order to reach an objective evaluation of “terroir” influence on wine quality, grapes were subjected to the same winemaking procedures. The careful subtraction of water and ethanol signals from NMR spectra allowed to statistically recognize the metabolites to be employed in multivariate statistical methods: Principal Component Analysis (PCA), Discriminant Analysis (DA) and Hierarchical Clustering Analysis (HCA). The three wines were differentiated from each other by six metabolites: α-hydroxyisobutyrate, lactic acid, succinic acid, glycerol, α-fructose and β-d-glucuronic acid. All multivariate analyses confirmed that the differentiation among the wines were related to micro-climate, and carbonate, clay, and organic matter content of soils. Additionally, the wine discrimination ability of NMR spectroscopy combined with chemometric methods, was proved when commercial Aglianico wines, deriving from different soils, were shown to be statistically different from the studied wines. Our findings indicate that multivariate statistical elaboration of NMR spectra of wines is a fast and accurate method to evaluate the molecular quality of wines, underlining the objective relation with terroir.     

Carbon-13 Nuclear Magnetic Resonance Spectroscopy Study of the Glucose Stress of Human Lenses

The sorbitol theory in diabetic cataractogenesis was based on sorbitol accumulation under glucose stress. Sorbitol accumulation was examined by ¹³C nuclear magnetic resonance spectroscopy (NMR) for the first time in matched human lenses incubated in 5.5 mM and 35.5 mM C-1 ¹³C-enriched glucose up to 28 hours. The results showed that sorbitol and lactate in human tens can be detected at 35.5 mM, but not in 5.5 mM glucose solution. The glycolysis metabolic pathway of human lenses may be quite different from that of animals. The accumulation of metabolites can be traced and quantified by the intensities of ¹³C NMR peaks. Therefore, ¹³C NMR spectroscopy can be used as a valuable tool to investigate human lens carbohydrate metabolism non-interventionally.     

Metabolic Profiling of Human Colorectal Cancer Using High Resolution 1H Nuclear Magnetic Resonance Spectroscopy

Colorectal cancer (CRC) is the third commonest malignancy cancer worldwide. Clear understandings of global metabolic profiling of the normal mucosa and cancer tissues are vitally important to aid optimizing the clinical management strategy and understanding CRC biology. We studied metabolic characteristics of 20 CRC and 20 distant normal mucosa tissues extracts from 20 patients using high resolution ¹H NMR spectroscopy in conjunction with multivariate analyses, such as principal component analysis (PCA). Compared with distant normal mucosa tissues, lactate, taurine, ornithine and polyamine were present at significantly higher levels in CRC tissue extracts whereas myo‐inositol was present at significantly lower level. Two metabolites ratios such as myo‐inositol/taurine and myo‐inositol/(ornithine+polyamine) appear to be the most valuable biomarkers for the differentiation CRC from normal mucosa tissues. Our data suggested that HR ¹H NMR spectroscopy combined with multivariate analyses is a potentially useful technology for detecting malignant changes in the normal mucosa tissues, the technique may be further exploited for future CRC biomarker research or identification of targets for therapeutic manipulations.     

Determination of Pentacyclic Triterpenes from Betula utilis by High-Performance Liquid Chromatography and High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy

A rapid and reliable method was developed and validated for determining betulin and betulinic acid in bark in Betula utilis by high-resolution magic angle spinning ¹H nuclear magnetic resonance (HR-MAS NMR) spectroscopy. HR-MAS NMR spectroscopy clearly distinguished the resonances of betulin and betulinic acid in the bark of all accessions of B. utilis. The concentrations of betulin and betulinic acid were calculated and added to the spectra. The determination of the targeted metabolites in chloroform extract of bark of each accession of B. utilis was performed by high-performance liquid chromatography (HPLC). Quantitatively, betulin was present at higher concentrations than betulinic acid in all accessions. The HR-MAS NMR and HPLC results showed that betulin and betulinic acid varied significantly among accessions of B. utilis. Principal component analysis of the NMR and HPLC results provided classification into three metabolic groups in which the betulin concentration was high, moderate, or low. The results show that HR-MAS NMR is rapid for fingerprinting of betulin and betulinic acid in the bark of B. utilis, while minimizing the drawbacks associated with solvent extraction.