Measurement of dilute 29Si species in solution using a large volume coil and DEFT NMR

A large-sample-volume nuclear magnetic resonance (NMR) spectroscopy probehead has been developed for the detection and characterization of low concentrations of 29Si species in aqueous solution. The approach described entails the use of a large-diameter radio frequency solenoid coil that permits substantially larger sample volumes to be investigated at moderate magnetic field strengths, compared with conventional NMR probehead configurations. In addition, difficulties presented by long 29Si T1 relaxation times have been circumvented by using the DEFT NMR pulse sequence, which permits more rapid signal averaging. Through a combination of these hardware and methodological improvements, high-resolution 29Si NMR spectra have been obtained at 4.2 T (29Si resonance frequency = 36.8 MHz) for an 800 microM solution of 96% 29Si-enriched silicic acid, H4SiO4 (pH approximately 8), with a signal-to-noise ratio of 16 and a line width of 31 Hz after 3 h of total measurement time.     

Method for determining molar concentrations of metabolites in complex solutions from two-dimensional 1H-13C NMR spectra

One-dimensional (1D) (1)H nuclear magnetic resonance (NMR) spectroscopy is used extensively for high-throughput analysis of metabolites in biological fluids and tissue extracts. Typically, such spectra are treated as multivariate statistical objects rather than as collections of quantifiable metabolites. We report here a two-dimensional (2D) (1)H-(13)C NMR strategy (fast metabolite quantification, FMQ, by NMR) for identifying and quantifying the approximately 40 most abundant metabolites in biological samples. To validate this technique, we prepared mixtures of synthetic compounds and extracts from Arabidopsis thaliana, Saccharomyces cerevisiae, and Medicago sativa. We show that accurate (technical error 2.7%) molar concentrations can be determined in 12 min using our quantitative 2D (1)H-(13)C NMR strategy. In contrast, traditional 1D (1)H NMR analysis resulted in 16.2% technical error under nearly ideal conditions. We propose FMQ by NMR as a practical alternative to 1D (1)H NMR for metabolomics studies in which 50-mg (extract dry weight) samples can be obtained.     

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.     

Determination of the primary structures of heparin- and heparan sulfate-derived oligosaccharides using band-selective homonuclear-decoupled two-dimensional 1H NMR experiments

Band-selective homonuclear-decoupled (BASHD) two-dimensional NMR experiments are applied to the assignment of 1H NMR spectra of oligosaccharides, using as an example a heparin-derived hexasaccharide. The anomeric (H1) region of the 1H NMR spectrum is band-selected in the F1 dimension. With the increased resolution that results from less truncation of interferograms in the t1 dimension, finer digital resolution in the F1 dimension, and collapse of multiplets to singlets in the F1 dimension, cross-peaks to the anomeric protons of the two iduronic acid residues, which overlap in normal two-dimensional total correlation spectroscopy (TOCSY) and rotating frame Overhauser enhancement spectroscopy (ROESY) spectra of the hexasaccharide, are resolved in BASHD-TOCSY and BASHD-ROESY spectra, leading to an unequivocal assignment of the 1H NMR spectrum of the hexasaccharide. Incorporation of the water attenuation by transverse relaxation method for the complete and selective elimination of the water resonance into two-dimensional BASHD experiments makes it possible to observe oligosaccharide resonances at the frequency of the water resonance, as demonstrated with the observation of cross-peaks to resonances at the frequency of the water resonance in BASHD-TOCSY spectra of a second heparin-derived hexasaccharide.     

Metabolic assessment of human liver transplants from biopsy samples at the donor and recipient stages using high-resolution magic angle spinning 1H NMR spectroscopy

This work presents the first application of high-resolution magic angle spinning (HR-MAS) 1H NMR spectroscopy to human liver biopsy samples, allowing a determination of their metabolic profiles before removal from donors, during cold perfusion, and after implantation into recipients. The assignment of peaks observed in the 1H HR-MAS NMR spectra was aided by the use of two-dimensional J-resolved, TOCSY and 1H-13C HMQC spectra. The spectra were dominated by resonances from triglycerides, phospholipids, and glycogen and from a variety of small molecules including glycerophosphocholine (GPC), glucose, lactate, creatine, acetate, amino acids, and nucleoside-related compounds such as uridine and adenosine. In agreement with histological data obtained on the same biopsies, two of the six livers were found to contain high amounts of triglycerides by NMR spectroscopy, which also indicated that these tissues contained a higher degree of unsaturated lipids and a lower proportion of phospholipids and low molecular weight compounds. Additionally, proton T2 relaxation times indicated two populations of lipids, a higher mobility triglyceride fraction and a lower mobility phospholipid fraction, the proportions of which changed according to the degree of fat content. GPC was found to decrease from the pretransplant to the posttransplant biopsy of all livers except for one with a histologically confirmed high lipid content, and this might represent a biomarker of liver function posttransplantation. NMR signals produced by the liver preservation solution were clearly detected in the cold perfusion stage biopsies of all livers but remained in the posttransplant spectra of only the two livers with a high lipid content and were prominent mainly in the graft that later developed primary graft dysfunction. This study has shown biochemical differences between livers used for transplants that can be related to the degree and type of lipid composition. This technology might therefore provide a novel screening approach for donor organ quality and a means to assess function in the recipient after transplantation.     

Between-person comparison of metabolite fitting for NMR-based quantitative metabolomics

Nuclear magnetic resonance (NMR) spectroscopy is widely used as an analytical platform for metabolomics. Many studies make use of 1D spectra, which have the advantages of relative simplicity and rapid acquisition times. The spectral data can then be analyzed either with a chemometric workflow or by an initial deconvolution or fitting step to generate a list of identified metabolites and associated sample concentrations. Various software tools exist to simplify the fitting process, but at least for 1D spectra, this still requires a degree of skilled operator input. It is of critical importance that we know how much person-to-person variability affects the results, in order to be able to judge between different studies. Here we tested a commercially available software package (Chenomx' NMR Suite) for fitting metabolites to a set of NMR spectra of yeast extracts and compared the output of five different people for both metabolite identification and quantitation. An initial comparison showed good agreement for a restricted set of common metabolites with characteristic well-resolved resonances but wide divergence in the overall identities and number of compounds fitted; refitting according to an agreed set of metabolites and spectral processing approach increased the total number of metabolites fitted but did not dramatically increase the quality of the metabolites that could be fitted without prior knowledge about peak identity. Hence, robust peak assignments are required in advance of manual deconvolution, when the widest range of metabolites is desired. However, very low concentration metabolites still had high coefficients of variation even with shared information on peak assignment. Overall, the effect of the person was less than the experimental group (in this case, sampling method) for almost all of the metabolites.     

Hierarchical Alignment and Full Resolution Pattern Recognition of 2D NMR Spectra: Application to Nematode Chemical Ecology

Nuclear magnetic resonance (NMR) is the most widely used nondestructive technique in analytical chemistry. In recent years, it has been applied to metabolic profiling due to its high reproducibility, capacity for relative and absolute quantification, atomic resolution, and ability to detect a broad range of compounds in an untargeted manner. While one-dimensional (1D) (1)H NMR experiments are popular in metabolic profiling due to their simplicity and fast acquisition times, two-dimensional (2D) NMR spectra offer increased spectral resolution as well as atomic correlations, which aid in the assignment of known small molecules and the structural elucidation of novel compounds. Given the small number of statistical analysis methods for 2D NMR spectra, we developed a new approach for the analysis, information recovery, and display of 2D NMR spectral data. We present a native 2D peak alignment algorithm we term HATS, for hierarchical alignment of two-dimensional spectra, enabling pattern recognition (PR) using full-resolution spectra. Principle component analysis (PCA) and partial least squares (PLS) regression of full resolution total correlation spectroscopy (TOCSY) spectra greatly aid the assignment and interpretation of statistical pattern recognition results by producing back-scaled loading plots that look like traditional TOCSY spectra but incorporate qualitative and quantitative biological information of the resonances. The HATS-PR methodology is demonstrated here using multiple 2D TOCSY spectra of the exudates from two nematode species: Pristionchus pacificus and Panagrellus redivivus. We show the utility of this integrated approach with the rapid, semiautomated assignment of small molecules differentiating the two species and the identification of spectral regions suggesting the presence of species-specific compounds. These results demonstrate that the combination of 2D NMR spectra with full-resolution statistical analysis provides a platform for chemical and biological studies in cellular biochemistry, metabolomics, and chemical ecology.     

Reactions in polyethoxysilane coatings using solution and solid-state29Si NMR spectroscopy

Solution and solid-state²⁹Si nuclear magnetic resonance (NMR) spectroscopy was used to follow the prehydrolysis and subsequent film formation of a polyethoxysilane mixture. Significant differences in intensity of the Q₂ resonances in the cross-polarization and dipolar decoupled spectra suggest a mobile phase is present in the cured film. Incorporation of solvent in the film is observed directly by¹³C cross polarization magic-angle-spin (CP MAS) NMR. The NMR results support the conclusions reached from a parallel chemical study.[/p]     

Determination of residue-specific acid dissociation constants for peptides by band-selective homonuclear-decoupled (1)H NMR

Acid dissociation constants of side-chain acidic groups of amino acid residues in peptides can be determined by 1H NMR, provided resonances can be resolved for carbon-bonded reporter protons located near the acidic group. We report here that the increased resolution of the band-selective homonuclear-decoupled (BASHD) TOCSY experiment greatly extends the range of application of the NMR method for determination of residue-specific, side-chain acid dissociation constants of peptides that contain multiple residues of the same amino acid. Chemical shift-pH titration curves are obtained from cross-peaks for reporter protons in BASHD-TOCSY spectra measured as a function of pH. The method is based on using sequence-dependent differences in the chemical shifts of resonances for the backbone CalphaH protons and the increased resolution in BASHD-TOCSY spectra from collapse of CalphaH multiplets to singlets in the F1 dimension to resolve resonances for the side-chain reporter protons. Application of the method is demonstrated by determination of residue-specific pKA values for each of the side-chain ammonium groups of the six lysine residues in the hexadecapeptide Ac-SRGKAKVKAKVKDQTK-NH2. Chemical shift-pH titration curves were obtained for the lysine side-chain CepsilonH2 reporter protons from their resolved CalphaH-CepsilonH2 TOCSY cross-peaks in BASHD-TOCSY spectra. Relative acidities of the six ammonium groups were also determined from the residue specific chemical shift-pH titration data by a pH-independent method, and calculation of fractional concentrations of protonation microspecies using the residue-specific pKAs is also described.     

Quantitative 1H NMR spectroscopy of blood plasma metabolites

The absolute quantification of blood plasma metabolites by proton NMR spectroscopy is complicated by the presence of a baseline and broad resonances originating from serum macromolecules and lipoproteins. A method for spectral simplification of proton NMR spectra of blood plasma is presented. Serum macromolecules and metabolites are completely separated by utilizing the large difference in translational diffusion coefficients in combination with diffusion-sensitized proton NMR spectroscopy. The concentration of blood plasma metabolites can be quantified by using formate as an internal concentration reference. The results are compared with those obtained with ultrafiltration, a traditional method for separating macromolecules and metabolites, and demonstrate an excellent correlation between the two methods. The general nature of diffusion-sensitized NMR spectroscopy allows application on a wide range of biological fluids.     

NMR detection with multiple solenoidal microcoils for continuous-flow capillary electrophoresis

Nuclear magnetic resonance (NMR) spectroscopy represents a promising on-line detector for capillary electrophoresis (CE). The inherent poor sensitivity of NMR mandates the use of NMR probes with the highest mass sensitivity, such as those containing solenoidal microcoils, for CE/NMR hyphenation. However, electrophoretic current degrades the resolution of NMR spectra obtained from solenoidal coils. A new method to avoid microcoil NMR spectral degradation during continuous-flow CE is demonstrated using a unique multiple solenoidal coil NMR probe. The electrophoretic flow from a single separation capillary is split into multiple outlets, each possessing its own NMR detection coil. While the CE electrophoretic flow is directed through one outlet, stopped-flow, high-resolution NMR spectra are obtained from the coil at the other outlet. The electrophoretic flow and NMR measurements are cycled between the outlets to allow a continuous CE separation with "stopped-flow" detection. As a new approach for improving multiple coil probe performance, the magnetic field homogeneity is automatically adjusted (via the shim coils of the magnet) for the active coil. The multiple microcoil CE/NMR coupling has been used to analyze a <3 nmole mixture of amines while obtaining between 1 and 2 Hz line width, demonstrating the ability to avoid electrophoretic current-induced line broadening.     

Targeted profiling: quantitative analysis of 1H NMR metabolomics data

Extracting meaningful information from complex spectroscopic data of metabolite mixtures is an area of active research in the emerging field of "metabolomics", which combines metabolism, spectroscopy, and multivariate statistical analysis (pattern recognition) methods. Chemometric analysis and comparison of 1H NMR1 spectra is commonly hampered by intersample peak position and line width variation due to matrix effects (pH, ionic strength, etc.). Here a novel method for mixture analysis is presented, defined as "targeted profiling". Individual NMR resonances of interest are mathematically modeled from pure compound spectra. This database is then interrogated to identify and quantify metabolites in complex spectra of mixtures, such as biofluids. The technique is validated against a traditional "spectral binning" analysis on the basis of sensitivity to water suppression (presaturation, NOESY-presaturation, WET, and CPMG), relaxation effects, and NMR spectral acquisition times (3, 4, 5, and 6 s/scan) using PCA pattern recognition analysis. In addition, a quantitative validation is performed against various metabolites at physiological concentrations (9 microM-8 mM). "Targeted profiling" is highly stable in PCA-based pattern recognition, insensitive to water suppression, relaxation times (within the ranges examined), and scaling factors; hence, direct comparison of data acquired under varying conditions is made possible. In particular, analysis of metabolites at low concentration and overlapping regions are well suited to this analysis. We discuss how targeted profiling can be applied for mixture analysis and examine the effect of various acquisition parameters on the accuracy of quantification.     

Structural and spatially resolved studies on the hardening of a commercial resin-modified glass-ionomer cement

A commercial photopolymerizable resin-modified glass-ionomer (Fuji II LC) was studied using a variety of nuclear magnetic resonance (NMR) techniques. ¹H and ¹⁹F stray-field imaging (STRAFI) enabled to follow the acid–base reaction kinetics in self-cured (SC) samples. Gelation and maturation processes with 25 min and 40 h average time constants, respectively, were distinguished. In self- & photo-cured (SPC) samples, two processes were also observed, which occurred with 2 s and 47 s average time constants. ¹H, ²⁷Al and ²⁹Si magic angle spinning (MAS) NMR, ¹³C cross-polarization (CP)/MAS NMR and ²⁷Al multiple quanta (MQ)MAS NMR spectroscopy were used to obtain structural information on the glass and cements that were either SC or SPC. The presence of methacrylate groups was identified in the solid component. Unreacted hydroxyl ethylmethacrylate (HEMA) was detected in self-cured cement. ²⁷Al data showed that approximately 28% and 20% of Al is leached out from glass particles in SC and SPC samples, respectively. The upfield shift detected in ²⁹Si MAS NMR spectra of the cements is consistent with a decrease in the number of Al species in the second coordination sphere of the silicon structures. Scanning electron microscopy (SEM) showed existence of 3D shrinkage of the cement matrix in photo-cured cements.     

Capillary HPLC-NMR Coupling: High-Resolution (1)H NMR Spectroscopy in the Nanoliter Scale

Coupling HPLC and NMR is one of the most powerful techniques for simultaneous separation and structural elucidation of unknown compounds in mixtures. To date, however, minimizing the detection volume, as is required when coupling NMR with miniaturized separation techniques, has been accompanied by a dramatic loss in resolution of the NMR spectra. Here, we report on the coupling of gradient capillary HPLC with on-column, high-resolution NMR detection. On-line stopped-flow and static (1)H NMR spectra were acquired with capillary columns of 75-315 μm i.d. With detection over a length of 1.2 cm, cell volumes cover a range of 50-900 nL. An on-line-detected NMR separation of dansylated amino acids was carried out in a 315 μm i.d. fused silica capillary packed to a length of 12 cm with C(18) stationary phase. The low solvent consumption makes the use of fully deuterated solvents economically feasible. NMR spectra with resolution on the order of 3 Hz were obtained using a 50 nL detection cell to measure 1.1 nmol of dansylated γ-aminobutyric acid under static conditions in a 75 μm i.d. capillary.     

Robust algorithms for automated chemical shift calibration of 1D 1H NMR spectra of blood serum

In biofluid NMR spectroscopy, the frequency of each resonance is typically calibrated by addition of a reference compound such as 3-(trimethylsilyl)-propionic acid- d 4 (TSP) to the sample. However biofluids such as serum cannot be referenced to TSP, due to shifts resonance caused by binding to macromolecules in solution. In order to overcome this limitation we have developed algorithms, based on analysis of derivative spectra, to locate and calibrate (1)H NMR spectra to the alpha-glucose anomeric doublet. We successfully used these algorithms to calibrate 77 serum (1)H NMR spectra and demonstrate the greater reproducibility of the calculated chemical-shift corrections ( r = 0.97) than those generated by manual alignment ( r = 0.8-0.88). Hence we show that these algorithms provide robust and reproducible methods of calibrating (1)H NMR of serum, plasma, or any biofluid in which glucose is abundant. Precise automated calibration of complex biofluid NMR spectra is an important tool in large-scale metabonomic or metabolomic studies, where hundreds or even thousands of spectra may be analyzed in high-resolution by pattern recognition analysis.     

Effect of treated filler loading on the photopolymerization inhibition and shrinkage of a dimethacrylate matrix

This study shows how treated filler loading influences the photopolymerization of a dimethacrylate comonomer mixture, regarding, in particular, shrinkage and inhibition under atmospheric oxygen, present in oral environment. Bis-GMA/TEGDMA (75/25 wt.%) resins were loaded with hybrid filler (Ba aluminosilicate glass and pyrogenic silica), treated with γ-methacryloxy(propyl)trimethoxysilane, at 0–50 wt.% and light cured over a total of 30 s (45 mW/cm²). Degree of double-bond conversion (DC), obtained using FTIR, decreased with filler content. ¹H MAS spectra (293–340 K) and STRAFI images (293 K) were obtained as a function of irradiation time and filler concentration. ¹H signals of unreacted methacrylate groups were more intense for higher loaded resins and resonances from –CH₂SiO₂(OH) (T²) and –CH₂SiO₃– (T³) units, also observed by ²⁹Si NMR, were resolved and suggest the presence of T²–resin bonds. 1D images show a reduction on polymerization contraction and reaction inhibition at the composite resin surface with filler loading. 2D resin images present a highly mobile surface layer, hence with lower DC.     

Application of nonselective 1D (1)H-(31)P inverse NMR spectroscopy to the screening of solutions for the presence of organophosphorus compounds related to the chemical weapons convention

1D nonselective (1)H-(31)P HSQMBC, HSQC, and (31)P decoupled HSQC NMR experiments were applied to the screening of original OPCW proficiency test samples for the presence of organophosphorus (OP) compounds related to the Chemical Weapons Convention. The HSQC and HSQMBC spectra are compared to 1D (1)H NMR spectra with WET solvent suppression and (31)P[(1)H] spectra of the same samples. The 1D nonselective HSQC and HSQMBC experiments are shown to be the most sensitive NMR experiments to selectively screen samples for the presence of organophosphorus(OP) compounds. These experiments are at least three times more sensitive than the (31)P[(1)H] NMR experiment and allow the determination of the number of OP compounds present in the sample and their alkyl group bound to the phosphorus atom. Samples spiked at the 5-10 ppm level can be screened within an hour for the presence of OP compounds, whereas for the (31)P[(1)H] experiments, an overnight acquisition is necessary. The sensitivity of the experiments decreases in the order (31)P decoupled HSQC, HSQMBC, and HSQC. For the different alkyl groups, the sensitivity of these experiments decreases in the order methyl approximately isopropyl > ethyl > propyl.     

Statistical total correlation spectroscopy: an exploratory approach for latent biomarker identification from metabolic 1H NMR data sets

We describe here the implementation of the statistical total correlation spectroscopy (STOCSY) analysis method for aiding the identification of potential biomarker molecules in metabonomic studies based on NMR spectroscopic data. STOCSY takes advantage of the multicollinearity of the intensity variables in a set of spectra (in this case 1H NMR spectra) to generate a pseudo-two-dimensional NMR spectrum that displays the correlation among the intensities of the various peaks across the whole sample. This method is not limited to the usual connectivities that are deducible from more standard two-dimensional NMR spectroscopic methods, such as TOCSY. Moreover, two or more molecules involved in the same pathway can also present high intermolecular correlations because of biological covariance or can even be anticorrelated. This combination of STOCSY with supervised pattern recognition and particularly orthogonal projection on latent structure-discriminant analysis (O-PLS-DA) offers a new powerful framework for analysis of metabonomic data. In a first step O-PLS-DA extracts the part of NMR spectra related to discrimination. This information is then cross-combined with the STOCSY results to help identify the molecules responsible for the metabolic variation. To illustrate the applicability of the method, it has been applied to 1H NMR spectra of urine from a metabonomic study of a model of insulin resistance based on the administration of a carbohydrate diet to three different mice strains (C57BL/6Oxjr, BALB/cOxjr, and 129S6/SvEvOxjr) in which a series of metabolites of biological importance can be conclusively assigned and identified by use of the STOCSY approach.     

Structural studies of geopolymers by <Superscript>29</Superscript>Si and <Superscript>27</Superscript>Al MAS-NMR

A systematic study of geopolymers by ²⁹Si and ²⁷Al MAS NMR has been carried out in an attempt to understand polymer structural details. ²⁷Al MAS NMR data shows that transient aluminium species are formed during the reaction of metakaolin with NaOH. Interaction of silicate anions with the aluminium sites of metakaolin was evident during the synthesis of geopolymers as observed from low field shift of ²⁹Si MAS NMR resonance lines of silicate centres. As the reaction progresses, the coordination of aluminium (IV, V and VI) in metakaolin changes almost completely to IV. ²⁹Si MAS NMR of selected compositions of the ternary system of sodium silicate, metakaolin and aqueous alkali reveals that geopolymerisation occurs in a distinct compositional region. At high alkalinity [> 30% (mol/mol) overall Na²O content], connectivity of silicate anions is reduced, consistent with poor polymerisation. At low alkalinity [<10% (mol/mol) overall Na²O content], a clear ²⁹Si NMR resonance line due to unconverted metakaolin is observed. NMR spectra were recorded from a series of samples with a fixed Na²O content (20 mol%) and varied SiO²/Al²O³ ratio to observe aluminium substitution in the cross-linked silicon tetrahedra of polymer network. Aluminium insertion into the silicate network is confirmed from the observed ²⁹Si NMR shift as a function of Si/Al ratio. The identification of the presence or absence of metakaolin in the cured geopolymer product is not possible even by ²⁹Si NMR as the signal from metakaolin is indistinguishable from a broad ²⁹Si NMR peak consisting of many resonance lines from the network of cross-linked silicon/aluminium tetrahedra. In an attempt to identify metakaolin signal, we prepared geopolymers with higher SiO²/Al²O³ molar ratios. Since aluminium substitutions in the silicate tetrahedral network are decreased, this results in better-resolved ²⁹Si NMR lines. The ²⁹Si NMR signal due to metakaolin is then distinguishable in the spectra of cured products in a series of samples with 3 to 11 mol% metakaolin. These results indicate that a geopolymer structure is a network of silicon/aluminium tetrahedra with some presence of unreacted metakaolin. The silicon/aluminium tetrahedra might have connectivity ranging from 1 to 4.