HR-MAS NMR Research Articles

Synthesis and characterization of a linker for primary amines used in the solid phase organic synthesis of spermidine

A linker resin for the synthesis of functionalized spermidine in good yield is described, along with its characterization by infrared (IR), 13C solid-state nuclear magnetic resonance with cross polarization and magic angle spinning (13C CPMAS NMR) and ¹H high resolution magic angle spinning nuclear magnetic resonance (¹H HRMAS NMR. This linker has been regenerated after cleavage of spermidine and re-used without loss of efficiency.

H HRMAS NMR Derived Bio-markers Related to Tumor Grade, Tumor Cell Fraction, and Cell Proliferation in Prostate Tissue Samples

A high-resolution magic angle spinning NMR spectroscopic approach is presented for evaluating the occurrence, amount and aggressiveness of cancer in human prostate tissue samples. Using this technique, key metabolites in malignant and non-malignant samples (n = 149) were identified, and patterns of their relative abundance were analyzed by multivariate statistical methods. Ratios of various metabolites – including (glycerophophorylcholine + phosphorylcholine)/creatine, myo-inositol/scyllo-inositol, scyllo-inositol/creatine, choline/creatine, and citrate/creatine – correlated with: i) for non-malignant tissue samples, the distance to the nearest tumor and its Gleason score and; ii) the fraction of tumor cells present in the sample; and iii) tumor cell proliferation (Ki67 labelling index). This NMR-based approach allows the extraction of information that could be useful for developing novel diagnostic methods for prostate cancer.

A Model of Phospholipid Biosynthesis in Tumor in Response to an Anticancer Agent in Vivo

SummaryWe study, in this paper, a model for the core of the system of the Glycerophospholipid metabolism in the murine cells. It comprises the simple and enzymatic reactions of PhosphatidylEthanolamine and the PhosphatidylCholine . The model’s general structure is taken from a number of books and articles. We translate this model into a set of ordinary differential equations (ODEs), to propose a quantitative explanation of the experimental experiences and the observed results. In order to make it usable as a basis for simulations and mathematical analysis we need to make precise the various constants present in the equations but which are usually not directly accessible in the literature. In a first step we considered experimental data of rat’s liver cells obtained by NMR spectroscopy: given the values of metabolite concentrations we find appropriate parameter values which allow us to describe the system with ODEs. We have then performed several analyses using the developed model such as stability analysis. A first interesting result is the global stability of the system which was observed by simulation and then proved by mathematical arguments. A second important result is that we observe on the diagrams that the steady state for normal cells is precisely a singular point of order two, whereas tumoral cells present different characteristics; this fact has been proved for PhosphatidylEthanolamine N-Methyl transferase (PEMT), an enzyme which seems to be identified for the first time as a crucial element in the tumoral process. In a second step we applied our model to experimental data of proton HRMAS NMR spectroscopy for solid B16 melanoma and Lewis lung (3LL) 3LL carcinoma cells treated by Chloroethyl Nitrosourea (CENU). We performed a complete comparative analysis of parameters in order to learn the predictive statements to explain increases and decreases which one can observe in concentrations.

Proton HR-MAS MR spectroscopy of oral squamous cell carcinoma tissues: an ex vivo study to identify malignancy induced metabolic fingerprints

Oral squamous cell carcinoma (SCC) represents more than 90% of all head and neck cancers as reported by Hermans (Cancer Imaging, 5(Spec No A), S52–S57, 2005), which draws attention of investigative research for novel predictive metabolic biomarkers to understand the malignancy induced biochemical perturbations occurring at molecular level. In the present work, proton HR-MAS NMR spectroscopic studies have been performed on resected human oral SCC tumor tissues, its neighboring margins and bed tissues (n = 159), obtained from 36 patients (n = 27 training set; n = 9 unknown test set), for the identification of metabolic fingerprints. The proton NMR spectra were then subjected to chemometric unsupervised PCA and supervised OSC-filtered PCA and PLS-DA multivariate analysis. Application of PLS-DA on orthogonally signal corrected training data-set (n = 120 tissue specimens; 27 patients) allowed >95% correct classification of malignant tissues from benign samples with >98% specificity and sensitivity. The OSC-PLS-DA model thus constructed was used to predict the class membership of unknown tissue specimens (n = 39) obtained from nine patients. These tissue samples were correctly predicted in its respective histological categories with 97.4% diagnostic accuracy. The regression coefficients obtained from OSC-filtered PLS-DA model indicated that malignant tissues had higher levels of glutamate, choline, phosphocholine, lactate, acetate, taurine, glycine, leucine, lysine, isoleucine and alanine, and lower levels of creatine and PUFA, representing altered metabolic processes (lipidogenesis, protein synthesis, and volume regulation) during tumor progression. Thus proton HR-MAS MR spectroscopy could efficiently identify the metabolic perturbations of malignant tumor from non-malignant bed and margins tissue specimens, which may be helpful in understanding the extent of tumor penetration in neighboring tissues.

LC/ESI-MS n and 1H HR-MAS NMR analytical methods as useful taxonomical tools within the genus Cystoseira C. Agardh (Fucales; Phaeophyceae)

Species of the genus Cystoseira are particularly hard to discriminate, due to the complexity of their morphology, which can be influenced by their phenological state and ecological parameters. Our study emphasized on the relevance of two kinds of analytical tools, (1) LC/ESI-MS n and (2) 1H HR-MAS NMR, also called in vivo NMR, to identify Cystoseira specimens at the specific level and discuss their taxonomy. For these analyses, samples were collected at several locations in Brittany (France), where Cystoseira baccata, C. foeniculacea, C. humilis, C. nodicaulis and C. tamariscifolia were previously reported. To validate our chemical procedure, the sequence of the ITS2 has been obtained for each species to investigate their phylogenetic relationships at a molecular level. Our study highlighted the consistency of the two physico-chemical methods, compared to “classical” molecular approach, in studying taxonomy within the genus Cystoseira. Especially, LC/ESI-MS n and phylogenetic analyses converged into the discrimination of two taxonomical groups among the 5 species. The occurrence of some specific signals in the 1H HR-MAS NMR spectra and/or some characteristic chemical compounds during LC/ESI-MS n analysis could be regarded as discriminating factors. LC/ESI-MS n and 1H HR-MAS NMR turned out to be two relevant and innovative techniques to discriminate taxonomically this complex genus.

Hydrogel Network Formation Revised: High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance as a Powerful Tool for Measuring Absolute Hydrogel Cross-Link Efficiencies

In the present work, high-resolution magic angle spinning (hr-MAS) NMR spectroscopy is applied as a straightforward nondestructive technique to quantify unreacted methacrylamide functionalities in cross-linked gelatin hydrogels. By adjusting several cross-linking parameters including the ultraviolet (UV) irradiation time and the photo-initiator concentration, the cross-linking degree can be easily varied. Remarkably, under all experimental conditions typically applied for hydrogel development, no more than 40% of the methacrylamide moieties present have reacted. The hr-MAS based approach to determine the cross-linking efficiency is shown to provide an innovative and more convenient alternative to the well-established classical techniques. In addition, the results obtained are in good correlation with mechanical analysis data.

High-Resolution 1H Magic Angle Spinning NMR Spectroscopy of Intact Arctic Char (Salvelinus Alpinus) Muscle. Quantitative Analysis of n−3 Fatty Acids, EPA and DHA

The lipid and small metabolite profiles from intact muscles of Arctic char were investigated using (1)H high-resolution magic angle spinning ((1)H HR-MAS) NMR spectroscopy. Not only the total n-3 fatty acid content but also the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) contents of the muscle were obtained from the (1)H HR-MAS NMR spectra without pretreatment of the tissue or lipophilic extraction. A number of small metabolites could also be observed, where creatine/phosphocreatine, anserine and taurine were the most abundant. Thus, the use of (1)H HR-MAS NMR led to simplified analysis techniques that can give direct information on the nutritional value of the fish.

Nuclear Magnetic Resonance (NMR) Study of the Effect of Cisplatin on the Metabolic Profile of MG-63 Osteosarcoma Cells

In the present study, (1)H HRMAS NMR spectroscopy was used to assess the changes in the intracellular metabolic profile of MG-63 human osteosarcoma (OS) cells induced by the chemotherapy agent cisplatin (CDDP) at different times of exposure. Multivariate analysis was applied to the cells spectra, enabling consistent variation patterns to be detected and drug-specific metabolic effects to be identified. Statistical recoupling of variables (SRV) analysis and spectral integration enabled the most relevant spectral changes to be evaluated, revealing significant time-dependent alterations in lipids, choline-containing compounds, some amino acids, polyalcohols, and nitrogenated bases. The metabolic relevance of these compounds in the response of MG-63 cells to CDDP treatment is discussed.

Photo-Cross-Linked Poly(dl-lactide)-Based Networks. Structural Characterization by HR-MAS NMR Spectroscopy and Hydrolytic Degradation Behavior

To date, biodegradable networks and particularly their kinetic chain lengths have been characterized by analysis of their degradation products in solution. We characterize the network itself by NMR analysis in the solvent-swollen state under magic angle spinning conditions. The networks were prepared by photoinitiated cross-linking of poly(dl-lactide)−dimethacrylate macromers (5 kg/mol) in the presence of an unreactive diluent. Using diffusion filtering and 2D correlation spectroscopy techniques, all network components are identified. By quantification of network-bound photoinitiator fragments, an average kinetic chain length of 9 ± 2 methacrylate units is determined. The PDLLA macromer solution was also used with a dye to prepare computer-designed structures by stereolithography. For these networks structures, the average kinetic chain length is 24 ± 4 methacrylate units. In all cases the calculated molecular weights of the polymethacrylate chains after degradation are maximally 8.8 kg/mol, which is far below the threshold for renal clearance. Upon incubation in phosphate buffered saline at 37 °C, the networks show a similar mass loss profile in time as linear high-molecular-weight PDLLA (HMW PDLLA). The mechanical properties are preserved longer for the PDLLA networks than for HMW PDLLA. The initial tensile strength of 47 ± 2 MPa does not decrease significantly for the first 15 weeks, while HMW PDLLA lost 85 ± 5% of its strength within 5 weeks. The physical properties, kinetic chain length, and degradation profile of these photo-cross-linked PDLLA networks make them most suited materials for orthopedic applications and use in (bone) tissue engineering.

Polymer supported calixarene derivative useful for solid-phase synthesis application

A calix[4]arene derivative has been anchored to carboxyl CPG and TentaGel supports by an easily cleavable ester bond and DMT groups allow a simple loading evaluation via UV–vis spectroscopy. The loading of the calixarene on TentaGel resin has also been estimated by HR-MAS NMR experiments. The potential of the polymer supported calixarenes ( 9 and 10) in solid phase synthesis has been tested by condensation of four thymine nucleotide units onto the upper rim of the calix[4]arene skeleton.

Monitoring the allyl ester deprotection by HR MAS NMR in BAL‐solid phase peptide synthesis

The backbone amide linker strategy, in which the growing peptide chain is anchored to a solid support via a backbone amide nitrogen, has proven to be successful for the synthesis of cyclic peptides. Optimisation of the reaction conditions for the synthesis of c(Gly-Trp-βAla-Phe) could be accomplished by the help of high resolution magic angle spinning (HR MAS) NMR and the results are presented here. Signal vanishing of HR MAS NMR resonances were encountered and proven to be originated from interchain aggregations of peptide chains.

Magnetic Resonance Microscopy Contribution to Interpret High‐Resolution Magic Angle Spinning Metabolomic Data of Human Tumor Tissue

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.

Molecular effects of diethanolamine exposure on Calanus finmarchicus (Crustacea: Copepoda)

Alkanolamines are surface-active chemicals used in a wide range of industrial, agricultural and pharmaceutical applications and products. Of particular interest is the use of alkanolamines such as diethanolamine (DEA) in the removal of CO 2 from natural gas and for CO 2 capture following fossil fuel combustion. Despite this widespread use, relatively little is known about the ecotoxicological impacts of these compounds. In an attempt to assess the potential effects of alkanolamines in the marine environment, a key species in the North Atlantic, the planktonic copepod Calanus finmarchicus, was studied for molecular effects following sublethal exposure to DEA. DEA-induced alterations in transcriptome and metabolome profiling were assessed using a suppression subtractive hybridization (SSH) gene library method and high resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR), respectively. Effects were observed on transcription of genes reportedly involved in lipid metabolism, antioxidant systems, metal binding, and amino acid and protein catabolism. These effects were accompanied by altered expression of fatty acid derivates, amino acids (threonine, methionine, glutamine, arginine, alanine and leucine) and cholines (choline, phosphocholine and glycerophosphocholine). Together, SSH and HR-MAS NMR offer complementary screening tools for the assessment of molecular responses of C. finmarchicus to DEA and can be used in the study of other chemicals and organisms. Concentration–response and time–response relationships between DEA exposure and single gene transcription were investigated using quantitative PCR. Specific relationships were found between DEA exposure and the transcription of genes involved in protein catabolism (ubiquitin-specific protease-7), metal ion homeostasis (ferritin) and defence against oxidative stress (γ-glutamylcysteine synthase, glutathione synthase and Cu/Zn-superoxide dismutase). At the lowest alkanolamine concentration used in these experiments, which corresponded to 0.5% of the LC 50 concentration, no transcriptional effects were observed, giving information regarding the lower molecular effect level. Finally, similar transcription patterns were observed for a number of different genes following exposure to DEA, which indicates analogous mechanisms of toxicity and response.

Metabolic and histopathological alterations of Jatropha mosaic begomovirus-infected Jatropha curcas L. by HR-MAS NMR spectroscopy and magnetic resonance imaging

Alterations in the anatomical structures, sap translocation and metabolic profiles in Jatropha curcas L. (Euphorbiaceae), infected with Jatropha mosaic virus (JMV) have been investigated using MRI and HR-MAS NMR spectroscopy. The contrast of MRI images distinguishes abnormalities in anatomical structures of infected and healthy stem. The HR-MAS NMR spectroscopic analysis indicated that viral infection significantly affected the plant metabolism. Higher accumulation of TCA cycle intermediates, such as citrate and malate, in JMV-infected plants suggested a higher rate of respiration. The respiration rate was more than twofold as compared to healthy ones. The viral stress also significantly increases the concentrations of alanine, arginine, glutamine, valine, GABA and choline as compared to healthy ones. Microscopic examination revealed severe hyperplasia caused by JMV with a considerable reduction in the size of stem cells. Lower concentration of glucose and sucrose in viral-infected stem tissues indicates decreased translocation of photosynthates from leaves to stem due to hyperplasia caused by JMV. The MR images distinguished stele, cortical and pith regions of JMV-infected and healthy stems. Contrast of T(1)- and T(2)-weighted images showed significant differences in the spatial distribution of water, lipids and macromolecules in virus-infected and healthy stem tissues. The results demonstrated the value of MRI and HR-MAS NMR spectroscopy in studying viral infection and metabolic shift in plants. The present methodology may help in better understanding the metabolic alterations during biotic stress in other plant species of agricultural and commercial importance.

HR MAS NMR, powder XRD and Raman spectroscopy study of inclusion phenomena in βCD nanosponges

Three different samples of β cyclodextrin nanosponges (CDNS) are prepared from β cyclodextrin (βCD) and pyromellitic dianhydride (PMA). CDNS are cross-linked, nanoporous materials whose pore size can be modulated by suitable choice of the CD/PMA molar ratio. In the presence of aqueous solutions they can swell giving rise to gel-like behavior. The Raman spectra of dry and water treated CDNS are described, with emphasis on the group vibration modes in the low frequency part of spectrum, sensitive to molecular environment and cross-linking degree, and on O–H/C–H vibration modes of dry/swollen CDNS, in turn providing information on the hydration dynamics. Powder X-ray diffraction data indicate low crystallinity and the presence of bulk water within the 3D polymer network. High resolution magic angle spinning (HR MAS) NMR spectroscopy is successfully used for investigation of swollen CDNS. The NMR signals of bulk and “bound” water indicate two different states of water molecules inside the gel. Probe solute fluorescein is used to spot on the diffusion properties inside the gel. In one case the diffusion coefficient of fluorescein measured in CDNS results one order of magnitude higher than that in D2O. The acceleration effect uncovered indicates that the motion of fluorescein inside the porous gel is driven by both hydrodynamic and electrostatic factors.

A model of phospholipid biosynthesis in tumor in response to an anticancer agent in vivo.

We study, in this paper, a model for the core of the system of the Glycerophospholipid metabolism in the murine cells. It comprises the simple and enzymatic reactions of PhosphatidylEthanolamine and the PhosphatidylCholine. The model's general structure is taken from a number of books and articles. We translate this model into a set of ordinary differential equations (ODEs), to propose a quantitative explanation of the experimental experiences and the observed results. In order to make it usable as a basis for simulations and mathematical analysis we need to make precise the various constants present in the equations but which are usually not directly accessible in the literature. In a first step we considered experimental data of rat's liver cells obtained by NMR spectroscopy: given the values of metabolite concentrations we find appropriate parameter values which allow us to describe the system with ODEs. We have then performed several analyses using the developed model such as stability analysis. A first interesting result is the global stability of the system which was observed by simulation and then proved by mathematical arguments. A second important result is that we observe on the diagrams that the steady state for normal cells is precisely a singular point of order two, whereas tumoral cells present different characteristics; this fact has been proved for PhosphatidylEthanolamine N-Methyl transferase (PEMT), an enzyme which seems to be identified for the first time as a crucial element in the tumoral process. In a second step we applied our model to experimental data of proton HRMAS NMR spectroscopy for solid B16 melanoma and Lewis lung (3LL) 3LL carcinoma cells treated by Chloroethyl Nitrosourea (CENU). We performed a complete comparative analysis of parameters in order to learn the predictive statements to explain increases and decreases which one can observe in concentrations.

Study of the suitability of HRMAS NMR for metabolic profiling of tomatoes: Application to tissue differentiation and fruit ripening

The feasibility of HRMAS NMR as an efficient technique for metabolic studies of tomato fruit and tomato tissues is described. A good-taste tomato variety from Almería (Spain) (commercial name Zayno) was chosen for this study. Fruits of three different ripening stages (green, turning and red) were selected, based on their external appearance. NMR data of whole fruits were obtained through 1H HRMAS NMR spectra of tomato purée. Flesh, peel and seeds from mature red fruits were separately analysed. 1H HRMAS NMR spectra showed resolutions similar to that of solution 1H NMR with the advantages of minimal sample manipulation and possibility of analysing polar and non-polar compounds simultaneously. Seeds could be clearly distinguished by visual comparison of the spectra. Tryacylglycerols, in which linoleic (47%) and oleic (28%) acyl chains preponderate, were the major components of seeds. By contrast, signals of fructose, glucose, citric acid, and the amino acids GABA, glutamine and glutamate dominated the spectra of flesh, peel and tomato purée. Principal components analysis, performed on the covariance matrix, revealed a clear separation between peel and flesh. Apart form the presence of the cutin biopolymer, peel showed a specific accumulation of sugar and sugar moieties related to pectin degradation, as well as glycosylated metabolites. Chemometric analysis also allowed the metabolic trajectory from green to red mature stages to be visualised. However, sample heterogeneity in the analysis of the whole fruit proved to be an important issue. The source of such heterogeneity was assigned to the presence of tissues of different hardness in the small amount of sample used. The presence of two clusters of samples at the turning stage indicated that the levels of certain metabolites could diverge significantly in fruits with almost identical appearance.

Synthesis, characterization and primary evaluation of the synthetic efficiency of supported vinyltins and allyltins

Supported vinyltins and allyltins grafted to an insoluble cross-linked polystyrene matrix were prepared using methods usually employed in solution, like hydrostannylation of alkynes, transmetallation of a tin halide with organomagnesium or organozinc reagents, and substitution of an allyl halide by a supported stannylanion or SN2′ substitution of a supported β-stannylacrolein acetal by cyanocopper reagents in the presence of boron trifluoride etherate. The insoluble grafted organotin reagents were analysed by HRMAS NMR, allowing an unambiguous assignment of their isomeric distribution or the identification of side products. When involved in Stille cross-coupling reactions (vinyltins) or in addition on aldehydes (allyltins), these supported reagents exhibit similar reactivity and similar stereoselectivity when compared to the tributyltin analogues, with the advantage to prevent problems due to the contamination by tin residues.

Detection of Local Prostate Metabolites by Hrmas Nmr Spectroscopy: A Comparative Study of Human and Rat Prostate Tissues

The use of magnetic resonance spectroscopy (MRS) for the detection of in-vivo metabolic perturbations is increasing in popularity in Prostate Cancer (PCa) research on both humans and rodent models. However, there are distinct metabolic differences between species and prostate areas; a fact making general conclusions about PCa difficult. Here, we use High Resolution Magic Angle Spinning Nuclear Magnetic Resonance (HRMAS NMR) spectroscopy to provide tissue specific identification of metabolites and their relative ratios; information useful in providing insight into the biochemical pathways of the prostate. As our NMR-based approach reveals, human and rat prostate tissues have different metabolic signatures as reflected in numerous key metabolites, including citrate and choline compounds, but also aspartate, lysine, taurine, glutamate, glutamine, creatine and inositol. In general, distribution of these metabolites is not only highly dependent on the species (human versus rat), but also on the location (lobe/zone) in the prostate tissue and the sample pathology; an observation making HRMAS NMR of intact tissue samples a promising method for extracting differences and common features in various experimental prostate cancer models.

Further studies into the feasibility of using aqueous alum solutions in the destruction of VX

The destruction of phosphorus based chemical warfare agents using aqueous buffer mixtures of aluminum sul- fate (alum) and sodium aluminate is pursued. The production of VX (O-ethyl-S-(2-(diisopropylamino)ethyl)-methylphos- phonothiolate) hydrolysis products ethyl methyl phosphonic acid (EMPA), an aluminum complex of EMPA, and S-(2- (diisopropylamino)ethyl)methylphosphonothiolate (EA-2192) is characterized in acidic and basic alum buffers. The study employs 31 P high resolution magic angle nuclear magnetic resonance spectroscopy (HRMAS NMR) for both quantitative and qualitative analysis. The acidic buffer results in a slower reaction rate and is less effective than the basic buffer.