HR-MAS NMR Research Articles

Phosphine-Stabilized Ruthenium Nanoparticles: The Effect of the Nature of the Ligand in Catalysis

Various ligands not forming monometallic complexes were used for Ru nanoparticle stabilization, enabling the control of size, shape, and electronic properties. HRMAS NMR spectroscopy allowed us to study surface-bound molecules, evidencing ligand hydrogenation and decomposition of THFduring the RuNP synthesis. Catalysis studies underscore the importance of the nature of the ligands. The RuNPs were tested in the hydrogenation of aromatics, showing very high activities (TOF > 60 000 h–1, 40 bar, 393 K). A pronounced ligand effect was found, and dialkylaryl phosphine ligands gave the fastest catalyst.

Effect of enzymatic treatment of different starch sources on the in vitro rate and extent of starch digestion.

Gelatinized wheat, potato and waxy maize starches were treated enzymatically in order to increase the degree of branching of the amylopectin fraction and thereby change the starch degradation profile towards a higher proportion of slowly digestible starch (SDS). The materials were characterized by single-pulse 1H HR-MAS NMR spectroscopy and in vitro digestion profile according to the Englyst procedure. Using various concentrations and incubation times with branching enzyme (EC 2.4.1.18) without or with additional treatment with the hydrolytic enzymes; β-amylase (EC 3.2.1.2), α-glucosidase (EC 3.2.1.20), or amyloglucosidase (EC 3.2.1.3) the proportion of α-(1–6) linkages was increased by up to a factor of 4.1, 5 and 5.8 in waxy maize, wheat and potato starches, respectively. The proportion of SDS was significantly increased when using hydrolytic enzymes after treatment with branching enzyme but it was only for waxy maize that the proportion of α-(1–6) bonds and the in vitro digestion profile was significantly correlated.

Analysis of cancer tissues by means of spectroscopic methods.

The personalized approach in cancer treatment stimulates the search for new analytical techniques, including spectroscopic methods such as Raman spectroscopy, mass spectrometry MALDI (matrix-assisted laser desorption/ionization) imaging and high-resolution magic angle spinning nuclear magnetic resonance (HR MAS NMR). The purpose of these studies is determination of metabolic profiles of cancer tissues, and their application in diagnostics and therapy of cancers. The review is mainly focused on application of HR MAS NMR technique. Qualitative and quantitative analysis of metabolites by means of this method is described for breast cancer tissues. In the near future HR MAS NMR in vitro studies of metabolic profiles combined with in vivo studies using MRI scanners may be applied as a new diagnostic tool.

Authenticity study of Phyllanthus species by NMR and FT-IR Techniques coupled with chemometric methods

The importance of medicinal plants and their use in industrial applications is increasing worldwide, especially in Brazil. Phyllanthus species, popularly known as quebra-pedras in Brazil, are used in folk medicine for treating urinary infections and renal calculus. This paper reports an authenticity study, based on herbal drugs from Phyllanthus species, involving commercial and authentic samples using spectroscopic techniques: FT-IR, 1H HR-MAS NMR and 1H NMR in solution, combined with chemometric analysis. The spectroscopic techniques evaluated, coupled with chemometric methods, have great potential in the investigation of complex matrices. Furthermore, several metabolites were identified by the NMR techniques.

Redox and thiol–ene cross-linking of mercapto poly(ε-caprolactone) for the preparation of reversible degradable elastomeric materials

A novel thiol-functionalized PCL (PCL-HDT) was synthesized following a convenient two-step procedure. Taking advantage of the pendant thiol group, degradable elastomeric materials have been prepared from PCL-HDT by redox or thiol–ene reaction. Elastomers were characterized by HRMAS NMR spectroscopy to confirm the formation of disulfide or thioether cross-links. The thermal and mechanical properties of elastomers have been assessed by DSC, DMA and tensile tests. Disulfide containing elastomers (EMSS) and thioether containing elastomers (EMTE) exhibited improved mechanical properties with ultimate strains up to 220%. The stability of the mechanical properties at temperatures close to body temperature was confirmed by DMA with G′ ≈ 200 MPa and G′′ ≈ 15 MPa. Finally, the reversibility of the disulfide formation and breaking has been evaluated, and confirmed the potential of these degradable elastomers as biomaterials.

Radiation-Grafting of Cotton-g-DMAEMA for Biomedical Applications

ABSTRACTThis work covers the design of stimuli-responsive membranes and their ever-expanding range of use. Stimuli-responsive membranes that change their physicochemical properties in response to changes in their environment were synthesized for biomedical application. Responsive cotton-g-[2-(dimethylamino) ethyl methacrylate] membranes were obtained using γ-rays by mutual irradiation (direct method). The effect of absorbed dose, dose rate, and monomer concentration on the grafting yield was determined. The grafted samples were verified by the FTIR-ATR, 1H and 13C HRMAS NMR and 13C CPMAS NMR spectroscopies; thermal properties were analyzed by TGA and DSC and the stimuli-responsive behavior was studied by DSC.

Investigation of Different Apple Cultivars by High Resolution Magic Angle Spinning NMR. A Feasibility Study

(1)H HR-MAS NMR spectroscopy was applied to apple tissue samples deriving from 3 different cultivars. The NMR data were statistically evaluated by analysis of variance (ANOVA), principal component analysis (PCA), and partial least-squares-discriminant analysis (PLS-DA). The intra-apple variability of the compounds was found to be significantly lower than the inter-apple variability within one cultivar. A clear separation of the three different apple cultivars could be obtained by multivariate analysis. Direct comparison of the NMR spectra obtained from apple tissue (with HR-MAS) and juice (with liquid-state HR NMR) showed distinct differences in some metabolites, which are probably due to changes induced by juice preparation. This preliminary study demonstrates the feasibility of (1)H HR-MAS NMR in combination with multivariate analysis as a tool for future chemometric studies applied to intact fruit tissues, e.g. for investigating compositional changes due to physiological disorders, specific growth or storage conditions.

Lipid composition and deposition during grain filling in intact barley ( Hordeum vulgare) mutant grains as studied by 1H HR MAS NMR

Conventional barley has an excellent nutritional value, but a lower content of lipids than oat. Nevertheless, barley lipids deserve a focused interest due to their fatty acid composition and their vitamin E composition. In this study, 1H HR MAS NMR was used to monitor the deposition of lipids during grain filling of conventional barley, and of two barley endosperm mutants with increased lipid content. The lipids in the mutants are primarily synthesized between days 9 and 13, whereas the lipid biosynthesis in the conventional barley took place ten days later. GC analysis on barley flour lipids showed that the mutants and the control exhibit comparable relative levels of the major fatty acids: C16:0, C18:1 and C18:2 despite the higher content of lipids in the mutants. Differences in the minor fatty acid composition between control and mutants were also observed. The degree of unsaturation was found to be increasing during grain filling for both conventional barley and the two mutants.

Application of HR‐MAS NMR spectroscopy for studying chemotype variations of Withania somnifera (L.) Dunal

Withania somnifera (L.) Dunal (Solanaceae), commonly known as Ashwagandha, is one of the most valued Indian medicinal plants with a number of pharmaceutical and nutraceutical applications. Metabolic profiling has been performed by HR-MAS NMR spectroscopy on fresh leaf and root tissue specimens from four chemotypes of W. somnifera. The HR-MAS NMR spectroscopy of lyophilized defatted leaf tissue specimens clearly distinguishes resonances of medicinally important secondary metabolites (withaferin A and withanone) and its distinctive quantitative variability among the chemotypes. A total of 41 metabolites were identified from both the leaf and root tissues of the chemotypes. The presence of methanol in leaf and root tissues of W. somnifera was detected by HR-MAS NMR spectroscopy. Multivariate principal component analysis (PCA) on HR-MAS (1) H NMR spectra of leaves revealed clear variations in primary metabolites among the chemotypes. The results of the present study demonstrated an efficient method, which can be utilized for metabolite profiling of primary and secondary metabolites in medicinally important plants.

A metabolomic and systems biology perspective on the brain of the Fragile X syndrome mouse model

Fragile X syndrome (FXS) is the first cause of inherited intellectual disability, due to the silencing of the X-linked Fragile X Mental Retardation 1 gene encoding the RNA-binding protein FMRP. While extensive studies have focused on the cellular and molecular basis of FXS, neither human Fragile X patients nor the mouse model of FXS--the Fmr1-null mouse--have been profiled systematically at the metabolic and neurochemical level to provide a complementary perspective on the current, yet scattered, knowledge of FXS. Using proton high-resolution magic angle spinning nuclear magnetic resonance ((1)H HR-MAS NMR)-based metabolic profiling, we have identified a metabolic signature and biomarkers associated with FXS in various brain regions of Fmr1-deficient mice. Our study highlights for the first time that Fmr1 gene inactivation has profound, albeit coordinated consequences in brain metabolism leading to alterations in: (1) neurotransmitter levels, (2) osmoregulation, (3) energy metabolism, and (4) oxidative stress response. To functionally connect Fmr1-deficiency to its metabolic biomarkers, we derived a functional interaction network based on the existing knowledge (literature and databases) and show that the FXS metabolic response is initiated by distinct mRNA targets and proteins interacting with FMRP, and then relayed by numerous regulatory proteins. This novel "integrated metabolome and interactome mapping" (iMIM) approach advantageously unifies novel metabolic findings with previously unrelated knowledge and highlights the contribution of novel cellular pathways to the pathophysiology of FXS. These metabolomic and integrative systems biology strategies will contribute to the development of potential drug targets and novel therapeutic interventions, which will eventually benefit FXS patients.

HRMAS-nuclear magnetic resonance spectroscopy characterization of tomato “flavor varieties” from Almería (Spain)

1H HRMAS NMR spectroscopy has been applied to investigate differences at a metabolic level between “flavor varieties” of high quality tomatoes marketed as Protected Geographical Indication from Almería (Spain). The varieties studied include the non-hybrid Raf tomato, one of the most tasteful commercial tomatoes that is traditionally grown in this Spanish region, together with other two hybrid varieties of commercial names Rambo and Zayno. Standard quality parameters were poorly reliable for establishing specific correlations with a given variety. In contrast, 1H HRMAS NMR spectroscopy, in combination with principal component analysis (PCA) and assigned signal analysis (ASA) provided a clear differentiation between varieties as a function of the ripening process and revealed the existence of variety-dependent relationships between external appearance and metabolic content. γ-Aminobutyric acid (GABA) is envisioned as a good marker for monitoring the ripening process. The content in other taste-relevant metabolites (fructose and organic acids) proved to be variety-dependent. The results show a preferential accumulation of citric acid and fructose in Rambo and Raf varieties, respectively. Raf fruits are characterized by high fructose to glucose and fructose to citric acid ratios. The content of malic acid during ripening also remains high for Raf tomato. These combined features are responsible of the unique characteristic and exceptional taste of this traditional line of tomato.

Metabolic Profiling of Lung Granuloma in Mycobacterium tuberculosis Infected Guinea Pigs: Ex vivo 1H Magic Angle Spinning NMR Studies

A crucial and distinctive feature of tuberculosis infection is that Mycobacterium tuberculosis (Mtb) resides in granulomatous lesion at various stages of disease development and necrosis, an aspect that is little understood. We used a novel approach, applying high resolution magic angle spinning nuclear magnetic resonance spectroscopy (HRMAS NMR) directly to infected tissues, allowing us to study the development of tuberculosis granulomas in guinea pigs in an untargeted manner. Significant up-regulation of lactate, alanine, acetate, glutamate, oxidized and the reduced form of glutathione, aspartate, creatine, phosphocholine, glycerophosphocholine, betaine, trimethylamine N-oxide, myo-inositol, scyllo-inositol, and dihydroxyacetone was clearly visualized and was identified as the infection progressed. Concomitantly, phosphatidylcholine was down-regulated. Principal component analysis of NMR data revealed clear group separation between infected and uninfected tissues. These metabolites are suggestive of utilization of alternate energy sources by the infiltrating cells that generate much of the metabolites in the increasingly necrotic and hypoxic developing granuloma through the glycolytic, pentose phosphate, and tricarboxylic acid pathways. The most relevant changes seen are, surprisingly, very similar to metabolic changes seen in cancer during tumor development.

Characterization of the Effects of Silver Nanoparticles on Liver Cell Using HR-MAS NMR Spectroscopy

AgNPs (silver nanoparticles) has been widely used for the commercial products, which have antimicrobial agent, medical devices, food industry and cosmetics. Despite, AgNPs have been reported as toxic to the mammalian cell, lung, liver, brain and other organs and many researchers have investigated the toxicity of AgNPs. In this study, we investigated toxicity of the AgNPs to the liver cell using metabolomics based on HRMAS NMR (High Resolution Magic Angle Spinning Nuclear Magnetic Resonance) technics, which could apply to the intact tissues or cells, to avoid the sample destruction. Target profiling and multivariative statistical analysis were performed to analyze the 1D 1 H spectrum. The results show that the concentrations of many metabolites were affected by the AgNPs in the liver cell. The concentrations of glutathione (GSH), lactate, taurine, and glycine were decreased and most of amino acids, choline analogues, and pyruvate were increased by the AgNPs. Moreover, the levels of the metabolites were recovered upto similar level of metabolites in the normal cell by the pre-treatment of NAC, external antioxidant. The results suggest that the depletion of the GSH by the AgNPs might induce the conversion of lactate and taurine to the pyruvate.

DRIFT and HR MAS NMR characterization of humic substances from a soil treated with different organic and mineral fertilizers

In this study, using DRIFT and HR MAS NMR, we analyzed the humic substances isolated from a soil treated, over 40 years, with different organic, mineral and organic plus mineral treatments and cultivated with maize as the main crop. As expected, the structure of humic substances was very complex but by combining both techniques (DRIFT and HR MAS NMR) additional information was obtained on aromatic and aliphatic components, the most recalcitrant parts of these macromolecules. In so doing we wanted to investigate the relationship between HS structure and long-term management practices. An elevated content of lignin, aminoacids, peptides and proteins was observed mainly for farmyard manure treatments with respect to mineral or liquid manure amendments; this supports how the different management practices have greatly influenced the humification process of cultivated soils.

EPA or DHA Supplementation Increases Triacylglycerol, but not Phospholipid, Levels in Isolated Rat Cardiomyocytes

It is well recognized that a high dietary intake of long-chain polyunsaturated fatty acids (LC-PUFA) has profound benefits on health and prevention of chronic diseases. In particular, in recent years there has been a dramatic surge of interest in the health effects of n-3 LC-PUFA derived from fish, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. Notwithstanding, the metabolic fate and the effects of these fatty acids once inside the cell has seldom been comprehensively investigated. Using cultured neonatal rat cardiomyocytes as model system we have investigated for the first time, by means of high-resolution magic-angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy in combination with gas chromatography (GC), the modification occurring in the cell lipid environment after EPA and DHA supplementation. The most important difference between control and n-3 LC-PUFA-supplemented cardiomyocytes highlighted by HR-MAS NMR spectroscopy is the increase of signals from mobile lipids, identified as triacylglycerols (TAG). The observed increase of mobile TAG is a metabolic response to n-3 LC-PUFA supplementation, which leads to an increased lipid storage. The sequestration of mobile lipids in lipid bodies provides a deposit of stored energy that can be accessed in a regulated fashion according to metabolic need. Interestingly, while n-3 LC-PUFA supplementation to neonatal rat cardiomyocytes causes a huge variation in the cell lipid environment, it does not induce detectable modifications in water-soluble metabolites, suggesting negligible interference with normal metabolic processes.

Serum and Urine Metabolite Profiling Reveals Potential Biomarkers of Human Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a common malignancy in the world with high morbidity and mortality rate. Identification of novel biomarkers in HCC remains impeded primarily because of the heterogeneity of the disease in clinical presentations as well as the pathophysiological variations derived from underlying conditions such as cirrhosis and steatohepatitis. The aim of this study is to search for potential metabolite biomarkers of human HCC using serum and urine metabolomics approach. Sera and urine samples were collected from patients with HCC (n = 82), benign liver tumor patients (n = 24), and healthy controls (n = 71). Metabolite profiling was performed by gas chromatography time-of-flight mass spectrometry and ultra performance liquid chromatography-quadrupole time of flight mass spectrometry in conjunction with univariate and multivariate statistical analyses. Forty three serum metabolites and 31 urinary metabolites were identified in HCC patients involving several key metabolic pathways such as bile acids, free fatty acids, glycolysis, urea cycle, and methionine metabolism. Differentially expressed metabolites in HCC subjects, such as bile acids, histidine, and inosine are of great statistical significance and high fold changes, which warrant further validation as potential biomarkers for HCC. However, alterations of several bile acids seem to be affected by the condition of liver cirrhosis and hepatitis. Quantitative measurement and comparison of seven bile acids among benign liver tumor patients with liver cirrhosis and hepatitis, HCC patients with liver cirrhosis and hepatitis, HCC patients without liver cirrhosis and hepatitis, and healthy controls revealed that the abnormal levels of glycochenodeoxycholic acid, glycocholic acid, taurocholic acid, and chenodeoxycholic acid are associated with liver cirrhosis and hepatitis. HCC patients with alpha fetoprotein values lower than 20 ng/ml was successfully differentiated from healthy controls with an accuracy of 100% using a panel of metabolite markers. Our work shows that metabolomic profiling approach is a promising screening tool for the diagnosis and stratification of HCC patients.

The degradation characteristics of microbial biomass in soil

Soil microbial biomass is a primary source of soil organic carbon (SOC) and therefore plays a fundamental role in carbon and nitrogen cycling. However, little is known about the fate and transformations of microbial biomass in soil. Here we employ HR-MAS NMR spectroscopy to monitor 13C and 15N labeled soil microbial biomass and leachate degradation over time. As expected, there is a rapid loss of carbohydrate structures. However, diffusion edited HR-MAS NMR data reveals that macromolecular carbohydrates are more resistant to degradation and are found in the leachate. Aromatic components survive as dissolved species in the leachate while aliphatic components persist in both the biomass and leachate. Dissolved protein and peptidoglycan accumulate in the leachate and recalcitrant amide nitrogen and lipoprotein persists in both the degraded biomass and leachate. Cross-peaks that appear in 1H– 15N HR-MAS NMR spectra after degradation suggest that specific peptides are either selectively preserved or used for the synthesis of unknown structures. The overall degradation pathways reported here are similar to that of decomposing plant material degraded under similar conditions suggesting that the difference between recalcitrant carbon from different sources is negligible after decomposition.

High Resolution Magic Angle Spinning NMR Applied to the Analysis of Organic Compounds Bound to Solid Supports

In situ structural characterization of organic compounds attached to solid supports can be achieved by high-resolution magic angle spinning NMR (HRMAS NMR), a technique that provides solution-like spectra for resin-bound molecules. This review outlines the principles of the technique, the influence of the solid support on data quality, and NMR experiments that are useful for obtaining valuable information. The review describes, with multiple examples mainly from the last 7 years, how HRMAS NMR has been applied to monitor solid-phase reactions, elucidate reaction products and quantify compound loading on a solid support. Other applications, such as conformational analysis of immobilized compounds and investigation of molecular interactions with compounds in solution, are also discussed.

Guanidine-based polycarbonate hydrogels: from metal-free ring-opening polymerization to reversible self-assembling properties

Bio-inspired stabilization of aliphatic polycarbonate-based hydrogels has been carried out by the metal-free ring-opening co-polymerization (ROP) of 6-membered cyclic carbonates containing, respectively, protected guanidine and carboxylic acid functions. Polyethylene glycol (PEG) bound to methylcarboxy trimethylene carbonate at each extremity was used as the cross-linker, and the copolymerizations were performed in CH2Cl2 for 24 h in the presence of 1,8-diazabicyclo[5.4.0]-undec-7-ene (DBU) and N-(3,5-trifluoromethyl)phenyl-N′-cyclohexylthiourea (TU), the catalyst and co-catalyst, respectively. Well-defined hydrogels of various compositions and presenting a high gel fraction were obtained. HR-MAS NMR has been successfully employed to validate our purification technique as well as to assess the selective de-protection of the guanidine and carboxylic acid functions. Evidence of self-assembling properties has been attested by differential scanning calorimetry (DSC), HR-MAS NMR analysis and swelling test experiments in aqueous buffered solutions at pH 4 and 8.

Metabolomic Characterization of Ovarian Epithelial Carcinomas by HRMAS-NMR Spectroscopy

Objectives. The objectives of the present study are to determine if a metabolomic study by HRMAS-NMR can (i) discriminate between different histological types of epithelial ovarian carcinomas and healthy ovarian tissue, (ii) generate statistical models capable of classifying borderline tumors and (iii) establish a potential relationship with patient's survival or response to chemotherapy. Methods. 36 human epithelial ovarian tumor biopsies and 3 healthy ovarian tissues were studied using 1H HRMAS NMR spectroscopy and multivariate statistical analysis. Results. The results presented in this study demonstrate that the three histological types of epithelial ovarian carcinomas present an effective metabolic pattern difference. Furthermore, a metabolic signature specific of serous (N-acetyl-aspartate) and mucinous (N-acetyl-lysine) carcinomas was found. The statistical models generated in this study are able to predict borderline tumors characterized by an intermediate metabolic pattern similar to the normal ovarian tissue. Finally and importantly, the statistical model of serous carcinomas provided good predictions of both patient's survival rates and the patient's response to chemotherapy. Conclusions. Despite the small number of samples used in this study, the results indicate that metabolomic analysis of intact tissues by HRMAS-NMR is a promising technique which might be applicable to the therapeutic management of patients.