1H Magic Angle Spinning NMR Research Articles

Topographical Variation in Metabolic Signatures of Human Gastrointestinal Biopsies Revealed by High-Resolution Magic-Angle Spinning 1H NMR Spectroscopy

Individual and topographical variation in the metabolic profiles of multiple human gastrointestinal tract (GIT) biopsies have been characterized using high-resolution magic-angle spinning (HRMAS) 1H NMR spectroscopy and pattern recognition. Samples from antrum, duodenum, jejunum, ileum, and transverse colon were obtained from 8 male and 8 female participants. Each gut region generated a highly characteristic metabolic profile consistent with the varying structural and functional properties of the tissue at different longitudinal levels of the gut. The antral (stomach) mucosa contained higher levels of choline, glycogen, phosphorylethanolamine, and taurine than other gut regions. The spatially close regions of the duodenum and jejunum were equivalent in terms of their gross biochemical composition with high levels of choline, glutathione, glycerophosphocholine (GPC), and lipids relative to other gut regions. The ileal mucosa showed poor discrimination from the duodenum and jejunum tissues and generated strong amino acids signatures but had relative low GPC signals. The colon (large intestine) was high in acetate, glutamate, inositols, and lactate and low in creatine, GPC, and taurine compared to the small intestine. These longitudinal metabolic variations in the human GIT could be attributed to functional variations in energy metabolism, osmoregulation, gut microbial activity, and oxidative protection. This work indicates that 1H HRMAS NMR studies may be of value in analyzing local metabolic variation due to pathological processes in gut biopsies.

High-Resolution Solid-State NMR Studies of Poly(vinyl phosphonic acid) Proton-Conducting Polymer: Molecular Structure and Proton Dynamics

The structure and the local proton mobility of poly(vinyl phosphonic acid) were studied by solid-state NMR under fast magic-angle spinning. At elevated temperatures, the signature of the hydrogen-bonded P-OH protons is observed in 1H magic-angle spinning (MAS) NMR as a single resonance at 10.5 ppm. Both 1H double-quantum NMR and variable-temperature experiments demonstrate that P-OH protons are mobile and thus able to contribute to proton conductivity. Below room temperature, two different types of hydrogen-bonded P-OH resonances are observed at 10.5 and 15 ppm, and 1H double-quantum NMR demonstrates that these protons are immobile on the NMR time scale. By means of first-principles calculations of a model polymer, we have assigned the additional hydrogen-bonded species at lower temperatures to phosphonic acid anhydride and charged anhydride. Also, in the 31P MAS NMR spectrum, two distinct resonances appear, arising from "normal" phosphonic acid and phosphonic acid anhydride. 31P double-quantum NMR experiments reveal that there is no phase segregation between normal and phosphonic acid anhydride and the condensation reaction occurs randomly throughout the system. The formation of acid anhydride leads to a decrease in proton conductivity through two mechanisms, (1) decrease in the number of charge carriers and (2) blockage of charge transport pathways through immobilization of charge carriers together with a hindered reorientation of the anhydride group. Our results provide strong evidence for these mechanisms by demonstrating that the conductivity is greatly influenced by the presence of phosphonic acid anhydride.

A magic angle spinning NMR study of xerogels functionalized by 3-mercaptopropyl groups

1H magic angle spinning NMR spectroscopy was used to study xerogels containing the 3-mercaptopropyl group. These xerogels were synthesized using tetraethoxysilane, 1,2-bis(triethoxysilyl)ethane, and 1,4-bis(triethoxysilyl)benzene as structuring agents. The assignment of the NMR signals observed showed the presence of thiol groups introduced during syntheses and organic bridges in the frame of polysilsesquioxane samples. An analysis of the 1H magic angle spinning NMR spectra also showed the presence of small amounts of alcohols, water participating in H-bonding, and nonhydrolyzed alkoxyl groups in the xerogels. In several instances, the structural units of T n and Q m types present in the xerogels were identified. The 1H magic angle spinning NMR spectroscopy combined with 13C and 29Si solid-state NMR spectroscopy allows the composition of xerogels and the nature of the structural units they contain to be identified more thoroughly and reliably.

NMR Characterization of Phosphonic Acid Capped SnO2 Nanoparticles

Phosphonic acid capped SnO2 nanoparticles with diameters less than 5 nm were synthesized and characterized with multinuclear solution and solid-state magic angle spinning (MAS) NMR. Two types of phosphonic acid ligands were used to derivatize the SnO2 surface, producing (i) water soluble SnO2 nanoparticles capped with 2-carboxyethanephosphonic acid (CEPA) and (ii) insoluble SnO2 nanoparticles capped with phenylphosphonic acid (PPA). Multiple surface environments were observed with 31P solution and solid-state MAS NMR for both capping agents. The 31P resonances of derivatized SnO2 nanoparticles display isotropic chemical shifts that are more shielded compared to the native phosphonic acids. This observation is indicative of a strong interaction between the phosphonic acid group and the SnO2 surface. 1H MAS NMR spectra display a complete absence of the acidic protons of the phosphonic acid groups, strongly supporting the formation of P−O−Sn linkages. 1H → 31P cross polarization (CP) build-up behavior confir...

Multinuclear Solid-State NMR Spectroscopy of Doped Lanthanum Fluoride Nanoparticles

Multinuclear solid-state NMR spectroscopy and powder X-ray diffraction (XRD) experiments are applied to comprehensively characterize a series of pure and lanthanide-doped LaF3 nanoparticles (NPs) that are capped with di-n-octadectyldithiophosphate ligands (Ln3+ = diamagnetic Y3+ and Sc3+ and paramagnetic Yb3+ ions), as well as correlated bulk microcrystalline materials (LaF3, YF3, and ScF3). Solid-state 139La and 19F NMR spectroscopy of bulk LaF3 and the LaF3 NPs reveal that the inorganic core of the NP retains the LaF3 structure at the molecular level; however, inhomogeneous broadening of the NMR powder patterns arises from distributions of 139La and 19F NMR interactions, confirming a gradual change in the La and F site environments from the NP core to the surface. 139La and 19F NMR experiments also indicate that low levels (5 and 10 mol %) of Ln3+ doping do not significantly change the LaF3 structure in the NP core. Similar doping levels of paramagnetic Yb3+ ions severely broaden 19F resonances, but only marginally effect 139La powder patterns, suggesting that the dopant ions are uniformly distributed throughout the NP core and occupy vacant La sites. Measurements of 139La T1 and T2 relaxation constants are seen to vary between the bulk material and NPs and between samples with diamagnetic and paramagnetic dopants. 45Sc NMR experiments confirm that the dopants are integrated into the La sites of the LaF3 core. Solid-state 1H and 31P magic-angle spinning (MAS) NMR spectra aid in probing the nature of the capping ligands and their interactions at the NP surface. 31P cross-polarization (CP)/MAS NMR experiments identify not only the dithiophosphate head groups but also thiophosphate and phosphate species which may form during NP synthesis. Finally, 19F-31P CP/MAS and 1H MAS experiments confirm that ligands are coordinated to the NP surface.

Study the effects of mechanical activation on Li–N–H systems with 1H and 6Li solid-state NMR

To gain insight into the effects of mechanical activation (MA) on the hydrogen desorption of the lithium amide (LiNH 2) and lithium hydride (LiH) mixture, LiNH 2 and LiH + LiNH 2 were mechanically activated by high-energy ball milling. The formed products were studied with in situ 1H and 6Li nuclear magic angle spinning (MAS) magnetic resonance (NMR) spectroscopy from ambient temperature to 180 °C. Up-field chemical shift was observed in 6Li MAS NMR spectra with increased milling time, indicating that average local electronic structure around Li nuclei was modified during MA. 1H MAS NMR was used to dynamically probe ammonia release from the activated LiNH 2 at temperature as low as 50 °C. In the case of activated LiH + LiNH 2 mixtures, the 1H MAS NMR results implied that MA enhanced the dehydrogenation reaction of LiNH 2 + LiH = Li 2NH + H 2.

Filler Distribution and Domain Size of Elastomer Compounds by Solid-State NMR and AFM

Abstract Solid-state NMR methods were used to characterize the filler distribution in rubber blends and domain size of thermoplastic vulcanizates (TPV). Correlation between filler content and magic angle spinning (MAS) 13C and 1H NMR signal linewidth was established, and filler content in each component of the rubber blends was determined. In IIR/BR and BIMS/BR blends, carbon black enriches in BR phase. Comparison of the carbon black enrichment in both blends suggests that BIMS has a slightly stronger interaction with fillers than does IIR. Results obtained from AFM analysis were compared with NMR observations. Spin diffusion NMR was demonstrated to be useful for quantifying the amount of interfacial component in TPV. Its advantages — ease of quantification and high sensitivity for smaller domains — make it a complementary approach to microscopic techniques for elastomer morphology characterization.

Solid-State NMR Study of Ibuprofen Confined in MCM-41 Material

Ibuprofen (an anti-inflammatory drug that is a crystalline solid at ambient temperature) has been encapsulated in MCM-41 silica matrices with different pore diameters (35 and 116 A). Its behavior has been investigated by magic angle spinning (MAS) 1H, 13C, and 29Si solid-state NMR spectroscopy at ambient and low temperature. This study reveals an original physical state of the drug in such materials. At ambient temperature, ibuprofen is not in a solid state (crystalline or amorphous) and is extremely mobile inside the pores, with higher mobility in the largest pores (116 A). The interaction between ibuprofen and the silica surface is weak, which favors fast drug release from this material in a simulated intestinal or gastric fluid. The quasi-liquid behavior of ibuprofen allows the use of NMR pulse sequences issued from solution-state NMR, such as the INEPT sequence, to characterize these solid-state samples. The solid-state MAS NMR study shows that the proton of the carboxylic acid group of ibuprofen is i...

Metabolic Profiling of Liver from Hypercholesterolemic Pigs Fed Rye or Wheat Fiber and from Normal Pigs. High-Resolution Magic Angle Spinning 1H NMR Spectroscopic Study

This study presents the first application of a high-resolution magic angle spinning 1H NMR approach to elucidate the metabolic effects of a hypercholesterolemic condition and two high-fiber diets based on rye and wheat bread, respectively, in intact pig liver biopsy samples. Standard 1D and spin-echo 1H spectra were acquired on a total of 20 biopsy samples, and 2D total correlation spectroscopy experiments were carried out on selected samples for assignment of the observed resonances. Principal component analyses and partial least-squares regression discriminant analysis revealed differences in the hepatic lipid content and choline-containing compounds between normal and hypercholesterolemic pigs. In addition, the results demonstrated that the liver metabolite profile of hypercholesterolemic pigs fed a high-fiber rye bread differed from that of pigs fed high-fiber wheat bread with respect to both the lipoprotein fractions and the choline-containing compounds. These findings suggest that earlier reports on high-fiber rye diet-induced effects on plasma HDL/LDL content partially can be ascribed to effects on liver cholesterol metabolism and that the hepatic phospholipase pathways of phosphatidylcholine breakdown are affected by the high-fiber rye diet.

Evidence for Specificity in Lipid-Rhodopsin Interactions

The interaction of bovine rhodopsin with poly- and monounsaturated lipids was studied by (1)H MAS NMR with magnetization transfer from rhodopsin to lipid. Experiments were conducted on bovine rod outer segment (ROS) disks and on recombinant membranes containing lipids with polyunsaturated, docosahexaenoyl (DHA) chains. Poly- and monounsaturated lipids interact specifically with different sites on the rhodopsin surface. Rates of magnetization transfer from protein to DHA are lipid headgroup-dependent and increased in the sequence PC < PS < PE. Boundary lipids are in fast exchange with the lipid matrix on a time scale of milliseconds or shorter. All rhodopsin photointermediates transferred magnetization preferentially to DHA-containing lipids, but highest rates were observed for Meta-III rhodopsin. The experiments show clearly that the surface of rhodopsin has sites for specific interaction with lipids. Current theories of lipid-protein interaction do not account for such surface heterogeneity.

Base-induced Brönsted Acid Sites on Dealuminated HY Zeolite: A Solid-state NMR Spectroscopy Study

Using trimethylphosphine (TMP) and d 5-pyridine(deuterated pyridine) as the basic probe molecules, the concentrations of Brönsted acid sites on both HY zeolite and dealuminated HY zeolite have been quantitatively determined using solid-state 1H and 31P magic-angle spinning (MAS) NMR. After adsorption of the probe molecules, the concentration of Brönsted acid sites on the dealuminated HY zeolite increases by about 25%, whereas that in the parent HY sample remains almost unchanged. The increase in the concentration of Brönsted acid sites is due to the appearance of base-induced Brönsted acid sites in the dealuminated HY zeolite. The terminal SiOH in the vicinity of the aluminum atom is “induced” to form a bridging hydroxyl group (SiOHAl) in the presence of the basic probe molecules. The mechanism of formation of the induced Brönsted acid sites has also been discussed.

Direct Solid-State NMR Spectroscopic Evidence for the NH4AlF4 Crystalline Phase Derived from Zeolite HY Dealuminated with Ammonium Hexafluorosilicate

Multinuclear (1)H, (19)F, and (27)Al MAS (magic angle spinning) and corresponding 2D HETCOR (heteronuclear correlation) NMR spectroscopy, in combination with powder XRD measurements, provide the direct evidence for the NH(4)AlF(4) crystalline phase, which was formed from zeolite HY dealuminated with an aqueous (NH(4))(2)SiF(6) solution at 80 degrees C. The NH(4)AlF(4) crystalline phase exhibits a characteristic second-order quadrupolar-induced (27)Al NMR line shape spreading from 0 to -90 ppm (in a magnetic field of 11.7 T) and two (19)F resonances at -151 and -166 ppm in the (19)F NMR spectrum. An( 27)Al quadrupolar coupling constant (C(Q)) of 9.5 MHz and an asymmetry parameter (eta) of 0.1 were identified, for the first time, for the NH(4)AlF(4) crystalline phase observed. On the basis of the (19)F{(27)Al} TRAPDOR (transfer population in double resonance) NMR results, the (19)F resonances at -151 and -166 ppm are therefore assigned to (19)F spins associated with the fluorines in the terminal Al-F and the bridging Al-F-Al groups, respectively.

A combined1H-NMR spectroscopy- and mass spectrometry-based metabolomic study of the PPAR-α null mutant mouse defines profound systemic changes in metabolism linked to the metabolic syndrome

The mobilization of triacylglycerides from storage in adipocytes to the liver is a vital response to the fasting state in mammalian metabolism. This is accompanied by a rapid translational activation of genes encoding mitochondrial, microsomal, and peroxisomal beta-oxidation in the liver, in part under the regulation of peroxisome proliferator-activated receptor-alpha (PPAR-alpha). A failure to express PPAR-alpha results in profound metabolic perturbations in muscle tissue as well as the liver. These changes represent a number of deficits that accompany diabetes, dyslipidemia, and the metabolic syndrome. In this study, the metabolic role of PPAR-alpha has been investigated in heart, skeletal muscle, liver, and adipose tissue of PPAR-alpha null mice at 1 mo of age using metabolomics. To maximize the coverage of the metabolome in these tissues, (1)H-NMR spectroscopy, magic angle spinning (1)H-NMR spectroscopy, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry were used to examine metabolites in aqueous tissue extracts and intact tissue. The data were analyzed by the multivariate approaches of principal components analysis and partial least squares. Across all tissues, there was a profound decrease in glucose and a number of amino acids, including glutamine and alanine, and an increase in lactate, demonstrating that a failure to express PPAR-alpha results in perturbations in glycolysis, the citric acid cycle, and gluconeogenesis. Furthermore, despite PPAR-alpha being weakly expressed in adipose tissue, a profound metabolic perturbation was detected in this tissue.

Integrated Metabonomic Analysis of Bromobenzene-Induced Hepatotoxicity: Novel Induction of 5-Oxoprolinosis

We present here a definitive metabonomic analysis in order to detect novel biomarker and metabolite information, implicating specific putative protein targets in the toxicological mechanism of bromobenzene-induced centrilobular hepatic necrosis. Male Han-Wistar rats were dosed with bromobenzene (1.5 g/kg, n = 25) and blood plasma, urine and liver samples were collected for NMR and magic angle spinning (MAS) NMR spectroscopy at various time-points postdose, with histopathology and clinical pathology performed in parallel. Liver samples were analyzed by 600 MHz 1H MAS NMR techniques and the resultant spectra were correlated to sequential 1H NMR measurements in urine and blood plasma using pattern recognition methods. 1D 1H NMR spectra were data-reduced and analyzed using principal components analysis (PCA) to show the time-dependent biochemical variations induced by bromobenzene toxicity. In addition to a holistic view of the effect of hepatic toxicity on the metabolome, a number of putative protein targets of bromobenzene and its metabolites were identified including those enzymes of the glutathione cycle, exemplified by the presence of a novel biomarker, 5-oxoproline, in liver tissue, blood plasma, and urine. As such, this work establishes the importance of metabonomics technology in resolving the mechanistic complexity of drug toxicity as well as the benefits of frontloading this approach in drug safety evaluation and biomarker discovery.

The Diversity of the Liquid Ordered (L o) Phase of Phosphatidylcholine/Cholesterol Membranes: A Variable Temperature Multinuclear Solid-State NMR and X-Ray Diffraction Study

To investigate the properties of a pure liquid ordered (L o) phase in a model membrane system, a series of saturated phosphatidylcholines combined with cholesterol were examined by variable temperature multinuclear ( 1H, 2H, 13C, 31P) solid-state NMR spectroscopy and x-ray scattering. Compositions with cholesterol concentrations ≥40 mol %, well within the L o phase region, are shown to exhibit changes in properties as a function of temperature and cholesterol content. The 2H-NMR data of both cholesterol and phospholipids were used to more accurately map the L o phase boundary. It has been established that the gel-L o phase coexistence extends to 60 mol % cholesterol and a modified phase diagram is presented. Combined 1H-, 2H-, 13C-NMR, and x-ray scattering data indicate that there are large changes within the L o phase region, in particular, 1H-magic angle spinning NMR and wide-angle x-ray scattering were used to examine the in-plane intermolecular spacing, which approaches that of a fluid L α phase at high temperature and high cholesterol concentrations. Although it is well known for cholesterol to broaden the gel-to-fluid transition temperature, we have observed, from the 13C magic angle spinning NMR data, that the glycerol region can still undergo a “melting”, though this is broadened with increasing cholesterol content and changes with phospholipid chain length. Also from 2H-NMR order parameter data it was observed that the effect of temperature on chain length became smaller with increasing cholesterol content. Finally, from the cholesterol order parameter, it has been previously suggested that it is possible to determine the degree to which cholesterol associates with different phospholipids. However, we have found that by taking into account the relative temperature above the phase boundary this relationship may not be correct.

Characterization of the Liquid-Ordered State by Proton MAS NMR

We investigated if magic angle spinning (MAS) 1H NMR can be used as a tool for detection of liquid-ordered domains (rafts) in membranes. In experiments with the lipids SOPC, DOPC, DPPC, and cholesterol we demonstrated that 1H MAS NMR spectra of liquid-ordered domains (l o) are distinctly different from liquid-disordered (l d) and solid-ordered (s o) membrane regions. At a MAS frequency of 10 kHz the methylene proton resonance of hydrocarbon chains in the l d phase has a linewidth of ∼50 Hz. The corresponding linewidth is ∼1 kHz for the l o phase and several kHz for the s o phase. According to results of 1H NMR dipolar echo spectroscopy, the broadening of MAS resonances in the l o phase results from an increase in effective strength of intramolecular proton dipolar interactions between adjacent methylene groups, most likely because of a lower probability of gauche/ trans isomerization in l o. In spectra recorded as a function of temperature, the onset of l o domain (raft) formation is seen as a sudden onset of line broadening. Formation of small domains yielded homogenously broadened resonance lines, whereas large l o domains (diameter >0.3 μm) in an l d environment resulted in superposition of the narrow resonances of the l d phase and the much broader resonances of l o. 1H MAS NMR may be applied to detection of rafts in cell membranes.

High-Resolution Magic Angle Spinning 1H NMR Spectroscopy and Reverse Transcription-PCR Analysis of Apoptosis in a Rat Glioma

The functional genomic approaches of transcriptomics, proteomics and metabolomics aim to measure the mRNA, protein or metabolite complement of a cell, tissue or organism. In this study we have investigated the compatibility of transcriptional analysis, using Reverse Transcription (RT)-PCR, and metabolite analysis, by high-resolution magic angle spinning (HRMAS) 1H NMR spectroscopy, in BT4C rat glioma following the induction of programmed cell death. The metabolite and transcriptional changes that accompanied apoptosis were examined at 0, 4 and 8 days of ganciclovir/thymidine kinase gene therapy. Despite the high spinning speeds employed during HRMAS 1H NMR spectroscopy of one-half of the tumor samples, RT-PCR analysis of the pro-apoptotic transcripts Bcl-2, BAK-1, caspase-9 and FAS was possible, producing similar results to those detected in the unspun half of the tumors. Furthermore, the expression of FAS was inversely correlated with some of the key metabolic changes across the time period examined including the increases CH=CH and CH=CHCH2 lipid resonances which accompany apoptosis. This study demonstrates how combined transcriptomic and metabolomic studies of tumors can be used to understand the molecular events that accompany well documented metabolic perturbations during cell death processes.

Quantification of homonuclear dipolar coupling networks from magic-angle spinning 1H NMR

Numerical simulations of magic-angle spinning (MAS) spectra of dipolar-coupled nuclear spins have been used to assess different approaches to the quantification of dipolar couplings from 1H solid-state NMR. Exploiting the translational symmetry of periodic spin systems allows extended networks with ‘realistic’ numbers of spins to be considered. The experimentally accessible parameter is shown to be the root-sum-square of the dipolar couplings to a given spin. The effectiveness of either fitting the resulting spinning sideband spectra to small spin system models, or using analyses based on moment expansions, has been examined. Fitting of the spinning sideband pattern is found to be considerably more robust with respect to experimental noise than frequency domain moment analysis. The influence of the MAS rate and system geometry on robustness of the quantification is analysed and discussed.

Metabolite identification in human liver needle biopsies by high-resolution magic angle spinning1H NMR spectroscopy

High-resolution magic angle spinning (HR-MAS) 1H NMR spectroscopy of intact human liver needle biopsies has not been previously reported. HR-MAS NMR spectra collected on 17 specimens with tissue amounts between approximately 0.5 and 12 mg showed very good spectral resolution and signal-to-noise ratios. One-dimensional 1H spectra revealed many intense signals corresponding to cellular metabolites. In addition, some high molecular weight metabolites, such as glycogen and mobile fatty acids, could be observed in some spectra. Resonance assignments for 22 metabolites were obtained by combining the analysis of three different types of 1D 1H spectral editing, such as T2 filtering or the nuclear Overhauser effect and 2D TOCSY and 13C-HSQC spectra. Biochemical stability of the liver tissue during up to 16 h of magic angle spinning at 277 K was studied. Biochemical trends corresponding to the different pathologies were observed, involving free fragments of lipids among other metabolites. NMR signal intensity ratios can be useful for discrimination among non-pathological, hepatitis C affected and cirrhotic liver tissues. Overall, this work demonstrates the applicability of HR-MAS NMR spectroscopy to the biochemical characterization of needle biopsies of the human liver.

Gel-phase HR-MAS 1H NMR spectroscopy as a probe for solid-tethered diimide rotaxanes and catenanes

The design and kinetically-controlled construction of a series of solid-tethered supramolecular systems utilising crown ether–naphthalene diimide host–guest chemistry are described. Functionalised polystyrene beads (ArgoGel-OH™) were utilised as the gel-phase solid support for the assembly of an appended diimide-crown catenane, and for a porphyrin-stoppered diimide-crown rotaxane. The structures of the resulting solid-tethered systems were probed using gel-phase high-resolution magic-angle spinning (HR-MAS) 1H NMR spectroscopy. The advantages of using this spectroscopic tool in conjunction with optical microscopy to probe solid-tethered supramolecular systems are discussed.