1H HR-MAS Research Articles

Changes in mouse brain metabolism following a convulsive dose of soman: A proton HRMAS NMR study

Soman, an irreversible organophosphorus cholinesterase inhibitor, induces status epilepticus and, in sensitive brain areas, seizure-related brain damage (e.g. brain edema and neuronal loss). The brain metabolic disturbances associated with these events are ill known. In the present study, we thus evaluated these changes in a murine model of soman-induced status epilepticus up to 7 days after intoxication. Mice, protected by HI-6 and atropine methyl nitrate, were poisoned with soman (172 microg/kg) and then sacrificed at set time points, from 1 h to 7 days. Brain biopsies from the piriform cortex (Pir) and cerebellum (Cer) were analyzed by 1H HRMAS NMR spectroscopy. Spectra were then analyzed using both a supervised multivariate analysis and the QUEST procedure of jMRUI for the quantification of 17 metabolites. The multivariate analysis clearly showed the metabolic differences between a damaged structure (Pir) and a structure with less prominent changes (cerebellum) and helped to globally assess the time course of metabolic changes. Analysis of the individual metabolites showed that the major changes took place in the piriform cortex but that cerebellum was not change-free. The most prominent changes in the former were an early (1-4 h) increase in alanine and acetate, a delayed increase in lactate, glycerophosphocholine and glutamine as well as a delayed decrease in myo-inositol and N-acetylaspartate. A week after poisoning, some metabolic disturbances were still present. Further research will be necessary to clarify what could be the involvement of these metabolites in physiological processes and how they might become useful surrogate markers of brain damage and repair.

Influence of dialkyne structure on the properties of new click-gels based on hyaluronic acid

Hydrogels have been widely used in tissue engineering as a support for tissue formation and/or to deliver drug locally. A novel procedure for the in situ rapid chemical gelation of aqueous solutions of hyaluronan (HA) was employed. HA was functionalised with an arm bearing a terminal azido group (HAAA). When HAAA was mixed with a series of dialkyne reagents of different length, a 1,3-dipolar cycloaddition (“click-chemistry”) reaction took place in the presence of catalytic amount of Cu(I) resulting in fast gelation at room temperature. The resulting gels were characterised in terms of degree of cross-linking by 1H HR-MAS NMR. The kinetic of gelation and the determination of elastic moduli as well as the degree of swelling and the controlled release of a model drug, were studied as a function of chemical nature of the dialkyne group, catalyst concentration, HAAA concentration and temperature. All these variables allowed the swelling ratio and the extent of release of a drug, doxorubicin, entrapped within the gel, to be modulated. In all cases the kinetic of release reached the stationary state within 150 h. The height of the plateau was dependent on the overall (chemical and topological) degree of cross-linking.

Longitudinal Profiling of Articular Cartilage Degradation in Osteoarthritis by High-Resolution Magic Angle Spinning 1H NMR Spectroscopy: Experimental Study in the Meniscectomized Guinea Pig Model

This study assessed the 1H HRMAS NMR spectroscopic profile of articular cartilage in both physiological and osteoarthitic situations. One-dimensional and two-dimensional 1H HRMAS NMR spectra were obtained from the tibial plateau cartilage of healthy and operated (unilateral medial meniscectomy and sham surgery) guinea pigs at different stages of disease, over a 6-month period. The major osteoarthritis-induced 1H HRMAS NMR changes were an increase of the N-acetyl peak of proteoglycans (at day 20 after meniscectomy) and a decrease after day 60 as the pathology evolved. These proteoglycan changes revealed by 1H HRMAS NMR analysis were validated by proteoglycan biochemistry assays. 1H HRMAS NMR analysis also evidenced a sharp increase in methylene resonances of chondrocyte membrane lipids from day 90 as a marker of apoptosis. There was an increase of the mobile methyl group of collagen at day 120, which was associated with collagen breakdown. 1H HRMAS NMR analysis provided a multifactorial and sequential picture of cartilage degradation at the extracellular matrix and chondrocyte levels.

High resolution magic angle spinning 1H NMR of childhood brain and nervous system tumours

BackgroundBrain and nervous system tumours are the most common solid cancers in children. Molecular characterisation of these tumours is important for providing novel biomarkers of disease and identifying molecular pathways which may provide putative targets for new therapies. 1H magic angle spinning NMR spectroscopy (1H HR-MAS) is a powerful tool for determining metabolite profiles from small pieces of intact tissue and could potentially provide important molecular information.MethodsForty tissue samples from 29 children with glial and primitive neuro-ectodermal tumours were analysed using HR-MAS (600 MHz Varian gHX nanoprobe). Tumour spectra were fitted to a library of individual metabolite spectra to provide metabolite values. These values were then used in a two tailed t-test and multi-variate analysis employing a principal component analysis and a linear discriminant analysis. Classification accuracy was estimated using a leave-one-out analysis and B632+ bootstrapping.ResultsGlial tumours had significantly (two tailed t-test p < 0.05) higher creatine and glutamine and lower taurine, phosphoethanolamine, phosphorylcholine and choline compared with primitive neuro-ectodermal tumours. Classification accuracy was 90%. Medulloblastomas (n = 9) had significantly (two tailed t-test p < 0.05) higher creatine, glutamine, phosphorylcholine, glycine and scyllo-inositol than neuroblastomas (n = 7), classification accuracy was 94%. Supratentorial primitive neuro-ectodermal tumours had metabolite profiles in keeping with other primitive neuro-ectodermal tumours whilst ependymomas (n = 2) had metabolite profiles intermediate between pilocytic astrocytomas (n = 10) and primitive neuro-ectodermal tumours.ConclusionHR-MAS identified key differences in the metabolite profiles of childhood brain and nervous system improving the molecular characterisation of these tumours. Further investigation of the underlying molecular pathways is required to assess their potential as targets for new agents.

Characterization of Organic Molecules Attached to Gold Nanoparticle Surface Using High Resolution Magic Angle Spinning 1H NMR

Structural elucidation of molecules attached to nanoparticle surface holds key to the successful chemical modifications of nanomaterial surface. In this investigation, we effectively optimized 1H HRMAS NMR conditions and applied one- and two-dimensional techniques to fully characterize ligand structures on surfaces of gold nanoparticles (GNPs). We found that there are significant differences in detection sensitivity depending on the distance between the surface of GNP and protons in the ligand molecule, with the loss of sensitivity for protons closer to the nanoparticles. Furthermore, NMR spectra of aromatic protons in ligands attached to GNP seem to have a broad base compared with aliphatic ligands, indicating some degree of potential π−π stacking effects. Our results demonstrate that 1H HRMAS NMR is an irreplaceable method for fully characterizing nanoparticle surface-bound molecules.

Bulk Carbohydrate Grain Filling of Barley β‐Glucan Mutants Studied by 1H HR MAS NMR

ABSTRACTTemporal and genotypic differences in bulk carbohydrate accumulation in three barley genotypes differing in the content of mixed linkage β‐(1→3),(1→4)‐D‐glucan (β‐glucan) and starch were investigated using proton high‐resolution, magic angle spinning, nuclear magnetic resonance (1H HR MAS NMR) during grain filling. For the first time, 1H HR MAS NMR spectra of flour from immature barley seeds are analyzed. Spectral assignments are made using two‐dimensional (2D) NMR methods. Both α‐ and β‐glucan biosynthesis were characterized by inspection of the spectra as well as by calibration to the reference methods for starch and β‐glucan content. Starch was quantified with very good calibrations to the α‐(1→4) peak (5.29–5.40 ppm) and the region 3.67–3.83 ppm covering starch glycopyranosidic protons from H5 and H6. In contrast, the spectral inspection of the β‐anomeric region 4.45–4.85 ppm showed unexpected lack of intensity in the high β‐glucan mutant lys5f at seed maturity, resulting in poor calibration to reference β‐glucan content. We hypothesize that the lack of β‐glucan signal in lys5f indicates partial immobilization of the β‐glucan that appears to be either genotypic dependent or water/β‐glucan ratio dependent.

The effect of single and repeated UVB radiation on rabbit lens

In our previous investigations, a significant cumulative effect of ultraviolet radiation (UVR) on the corneal and aqueous humour metabolic profiles was revealed. The purpose of the present study was to monitor the alterations in the rabbit lenses under the same experimental design and thereby supplement and complete prior findings. Albino rabbit eyes were exposed to single (312 nm, 3.12 J/cm2) or repeated (312 nm, 3 x 1.04 J/cm2) UVB irradiations of the same overall doses. Lenticular samples were analysed by high resolution magic angle spinning proton nuclear magnetic resonance (HR-MAS 1H NMR) spectroscopy. Special grouping patterns between the UVB-irradiated and untreated control samples were evaluated using principal component analysis (PCA). Percentage alterations in the lenticular metabolite concentrations from UVR-B exposed rabbits were calculated relative to the levels in the control group. UVB irradiation of the albino rabbit lenses resulted in a significant decrease in the concentrations of antioxidants (glutathione), osmolytes (taurine, myoinositol) and amino acids (alanine), and a concomitant elevation in the contents of a sugar-related compound, sorbitol. Repeated UVR-B exposure of the rabbit eye had a stronger effect on the lenticular metabolic profile than a single irradiation of the same overall dose. This study reveals the cumulative effect of repeated UVB irradiations, and shows that even a 48-hour interval between subsequent UVR-B exposures is not sufficient for the healing processes to restore lenticular integrity.

Monitoring the thermodynamically-controlled formation of diimide-based resin-attached rotaxanes by gel-phase HR MAS1H NMR spectroscopy

The thermodynamically controlled self-assembly of rotaxane and pseudorotaxane systems consisting of (i) a naphthodiimide thread unit terminated at one end with a pyridine ligand, and covalently linked at the other to a gel-phase polystyrene resin support, (ii) a dinaphtho-crown ether shuttle unit, and (iii) a ruthenium carbonyl metalloporphyrin stopper unit, is investigated by high resolution magic angle spinning proton (HR MAS 1H) NMR spectroscopy. The effects of variable concentration of the solution-phase components, the temperature, and added Li+ and Na+ ions are described, and the limitations of the technique are addressed. The dynamic behaviour is compared directly to the solution-phase analogues, where a bulky stopper group is substituted for the polystyrene resin bead.

Starch Granule Hydration—A MAS NMR Investigation

Starch is the most important energy resource in human diet, and starch is used extensively as a food ingredient to manipulate the quality of our food. In both applications, starch functionality is intimately related to its hydration level. This paper aims at elucidating the starch granule hydration by investigating genotype-specific differences for native wheat, maize, and potato starches by 1H high-resolution (HR) magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. The preparations as analyzed in D2O suspensions at room temperature provided NMR spectra with large differences in signal-to-noise (S/N) ratio ranging over several orders of magnitude. It was possible to assign a wide range of components including anomeric α-1,4 and α-1,6-protons from reducing and non-reducing ends, respectively. We utilized the effect that only mobile protons (e.g, dissolved or partially hydrated) are observed using 1H HR-MAS spectroscopy, whereas immobile protons (e.g., in water-inaccessible regions) of the starch granule are not observed due to strong homonuclear interactions to verify the hypothesis that the variations in signal intensities between the different starches are caused by genotype-specific variations in assembly of the starch granules and that the signal intensity, thus, indicates the extent of accessible granule hydration surfaces. Moreover, events taking place during thermal starch granule hydration (gelatinization) were investigated for ten representative starches. NMR spectra of suspended samples were acquired at 30, 45 and 70 °C and again after cooling at 30 °C. A substantial increase in NMR signal intensity occurs above the gelatinization temperature due to extensive proton mobilization in the starch granule assembly. The relative integrated spectral intensities at 30 °C before and after gelatinization at 70 °C showed differences in gain factors between 4 and 193. Also, 31P MAS NMR spectra displayed a similar significant intensity gain upon gelatinization. The results showed that the phosphate groups in the starch granule are mobilized concomitantly with the protons and thus deeply “buried” in the immobile (water inaccessible) domains.

NMR and X-Ray Studies of Starches Derived from Tropical Fruit Seed Gelatinization Process

Gelatinization of the starch obtained from fruit seed such as mango and cumbaru was investigated applying an analytic methodology developed in the polymer nuclear magnetic resonance (NMR) laboratory at IMA/UFRJ, employing high resolution nuclear magnetic resonance spectroscopy in the solid state for hydrogen nucleus, using 1H HR-MAS pulse sequence. The results showed that NMR can be used instead of some techniques normally applied to study the gelatinization process, because 1H HR-MAS and relaxation time allow the evaluation of this process at the molecular level. NMR was also able to indicate the best gelatinization conditions. To start this study the glass transition (Tg) determination was a first step, because the Tg of seed starches are different than the Tg of starches derived from cereal, for example. The 1H HR-MAS NMR showed some useful information on the fruit seed starches gelatinization process. X-ray measurements were also used to support the data obtained from NMR technique. It was verified, from X-ray, that mango starch exhibits crystallinity in the A form and cumbaru showed a predominant amorphous phase. The use of 1H HR-MAS was shown to be a new, powerful method to follow the gelatinization process, because this process can be understood at the molecular level.

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

A novel chemotaxonomical method based on 1D 1H 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.

Synthesis of new functionalized polymers and their use as stabilizers of Pd, Pt, and Rh nanoparticles. Preliminary catalytic studies

AbstractPolymers I‐X with functionalized main chain were synthesized from commercial polymers containing reactive groups, anhydride (for Gantrez), or carboxylate (for polyacrylates) moieties, to be used as stabilizers of metallic nanoparticles. 1H HRMAS, 13C MAS NMR, IR, and GPC techniques were applied to characterize the new polymers. Metal transition nanoparticles stabilized with these polymers were prepared in organic solvents (Pd and Pt) and also in water/ethanol medium (Rh), under hydrogen atmosphere. TEM analysis revealed the formation of small nanoparticles, in the range ∼ 3–20 nm, depending on the solvent, metal precursor, metal concentration, and polymer nature. Preliminary catalytic applications, CC couplings using palladium and hydrogenations using platinum and rhodium systems in organic and also aqueous medium, were tested. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007

Solid State NMR Study of Couma utilis Seeds

This article studies the seed of sorva (Couma utilis) that is a native fruit of Amazons State, in Brazil. Sorva seed components were analyzed by 1H and 13C using solid state high resolution nuclear magnetic resonance (NMR) spectroscopy, because they are important nuclei to be used to evaluate plant seeds. High resolution 1H NMR (1H HRMAS) with magic angle sample spinning (MAS), and 13C NMR using MAS and particularly 13C NMR with cross-polarization (CP) and MAS are techniques that allow the evaluation of the seed in detail. The variable contact–time (VCT) experiment is another useful technique that can be used to measure a proton spin-lattice relaxation time in the rotating frame, which is a parameter that gives response on the intermolecular interaction and is related to the sample spatial homogeneity. From the NMR results it was found that the sorva seeds are mainly constituted of polysaccharides, glycoproteins, and triacylglycerides.

Chemometric analysis applied in 1H HR-MAS NMR and FT-IR data for chemotaxonomic distinction of intact lichen samples

This paper describes the potentiality of chemometric analysis applied in 1H HR-MAS NMR and FT-IR data for lichen chemotaxonomic investigations. Lichens present a difficult morphologic differentiation and the chemical analyses are frequently employed for their taxonomic classification, mainly due to the secondary metabolites to be relatively constant for these organisms. The lichen chemotaxonomic classification is usually carried out by color reactions, chromatography, fluorescence and mass spectrometry analysis, where the identification is obtained by one or more techniques. There are some papers which use the carbohydrate content in chemotaxonomy investigation. However, the majority of these techniques involve laborious and time consuming sample pre-treatment. This work focuses on application of 1H high resolution magic angle spinning – nuclear magnetic resonance (HR-MAS NMR) and Fourier transform infrared (FT-IR) associated with chemometric analysis to intact samples. In comparison to other traditional techniques, 1H HR-MAS NMR and FT-IR allied with chemometrics provided a fast and economic method for lichen chemotaxonomy. Both methods were useful for lichen analysis and permitted the satisfactory distinction among families, genera and species, although better results were achieved for FT-IR data.

Strategies for data analyses in a high resolution 1H NMR based metabolomics study of a mouse model of Batten disease

Using an NMR based approach, employing both solution state and high resolution magic angle spinning (HR MAS) H-1 NMR spectroscopy, in conjunction with an array of statistical methods, we report cerebral metabolic deficits in a mouse model of Batten disease (Cln3 null mutant mice). Batten disease is the most common progressive neurodegenerative disorder of childhood and is caused by mutations in the Cln3 gene. In particular, brain tissue from Cln3 mice was characterised by increased concentrations of glutamine, myo-inositol, scyllo-mositol, aspartate and lactate, alongside decreased concentrations of N-acetyl-L-aspartate (NAA), N-acetyl-L-glutamate (NAG), gamma-amino butyric acid (GABA), glutamate and creatine. Accompanying changes in lipid deposition were also detected in intact cortical tissue by HR MAS 1H NMR spectroscopy. To realise the true potential of metabolomic datasets necessitates a comprehensive analysis of the data, such that useful biological information can be extracted and used to generate hypotheses which can be further tested and refined. We found that using a combination of univariate and multivariate analyses, a maximal number of metabolic deficits were successfully identified. In particular the complementary nature of the statistical approaches allowed the definition of changes which were relative, absolute or simply a change in variance, allowing a greater understanding of the disease processes detected.

Pre-selection of potential molecular markers for the geographic origin of dried beef by HR-MAS NMR spectroscopy

The complexity of meat composition and the requirement of high analytical rates to prevent frauds and, in general, to assess food quality are stimuli to find analytical methods able to detect appropriate indicators of a given quality. We present here an evaluation of 1D 1H HR-MAS NMR spectroscopy for the selection of potential molecular markers of one specific quality, geographic origin. Dried beef samples of certified origin were tested, and preliminary results on a limited sample set confirmed the good aptitude of this method for rapid food analysis, demonstrated earlier on dairy products. Fat content as well as specific metabolites, probably linked to feeding system, are shown to be good candidates for markers of origin. These results are in agreement with recent studies on the relative impact of genetic and dietary conditions on the metabolic profile of animal specimens. 1H HR-MAS is thus an interesting tool for pre-screening potential markers, that can complement standard chemical and biochemical analysis.

New unsaturated derivatives of Xanthan gum: Synthesis and characterization

Unsaturated Xanthan derivatives, which can be used for the development of biodegradable three-dimensional networks having hydrogel properties, were prepared by esterification under various conditions. Xanthan gum derivatives with different degrees of substitution were obtained by reaction with an unsaturated organic acid (acrylic acid) or with acid reactive derivatives (acryloyl chloride, maleic anhydride). The presence of acrylate and maleate groups in the modified structure of Xanthan gum was detected by ATR–FTIR, 1H liquid NMR and 1H HRMAS NMR spectroscopies. The degree of substitution as determined by 1H NMR could be controlled by varying the chemical nature of functionalisation agent, reaction time and temperature.The results proved that this polysaccharide can be modified by esterification with acids or unsaturated acid derivatives for further synthesis of hydrogels. Maleic anhydride presents a higher reactivity as compared to acrylic acid and acryloyl chloride.

NMR Studies on the interaction between (−)-epigallocatechin gallate and cyclodextrins, free and bonded to silica gels

The interaction between (−)-epigallocatechin-3-gallate (EGCG) and β- or γ-cyclodextrin (CD), in free solution and bonded to silica beads, has been studied by 1H HR-MAS NMR spectroscopy. The chromatographic retardation of EGCG on columns packed with CD-silica beads was shown to be due to the interaction of EGCG with the CD ligands because no nonspecific interaction with the silica gel could be observed. EGCG forms a tighter complex with β-CD than with γ-CD and NMR data obtained from hydroxy protons together with mm2 calculations suggest that for β-CD intermolecular hydrogen bonding, in addition to hydrophobic interaction, stabilizes the complex.

Poly(vinyl alcohol)-graft-poly(ethylene glycol) resins and their use in solid-phase synthesis and supported TEMPO catalysis

New poly(vinyl alcohol)-graft-poly(ethylene glycol) (PVA-g-PEG) resins with various PEG chain lengths, which have high loadings and good swelling both in water and organic solvents, have been prepared via an anionic polymerization of ethylene oxide onto PVA beads and applied in solid-phase synthesis, supported TEMPO catalysis and in HR-MAS 1H NMR spectral analysis.

Postnatal Biochemical Changes in Rat Lens: An Important Factor in Cataract Models

Purpose: Administration of several cataractogenic agents is effective during the first 21 days of rat lens development, a period of the highest sensitivity of the tissue. Thus, cataract formation and lens maturation affect the biochemical profile of rat lens simultaneously and might be difficult to evaluate separately. The purpose of this study was to use high-resolution magic angle spinning proton nuclear magnetic resonance (HR-MAS 1H NMR) to investigate exclusively the effect of maturation on the metabolic profile of rat lens. Methods: Albino Sprague-Dawley rats (n = 15) were divided into five groups of three animals and sacrificed at designated times (7, 14, 20, 30, and 60 days). The lenses were dissected, frozen, and thereafter analyzed with HR-MAS 1H NMR spectroscopy. Special grouping patterns among the tissue samples of different age and the relative percentage changes in particular metabolite concentrations were evaluated using appropriate statistical methods (principal components, one-way ANOVA). Results: Time-dependent alterations in the metabolic profile of the rat lens tissue were dominated by a significant increase in taurine, hypo-taurine, and myoinositol concentrations at the age of 30 days. Contents of the energy metabolites and amino acids were nearly constant between the ages of 14 and 30 days, showing a significant decrease in the 60-day-old rat lenses. Conclusions: HR-MAS 1H NMR spectroscopy showed its suitability to assess the natural alterations in the metabolic profile of maturing rat lens. The results can be used in future cataract research designed to evaluate the metabolic effect of different cataractogenic agents during this postnatal period.