MHz Proton Nuclear Magnetic Resonance Research Articles

Metabolomic approach to profile functional and metabolic changes in heart failure.

BackgroundHeart failure (HF) is characterized by a series of adaptive changes in energy metabolism. The use of metabolomics enables the parallel assessment of a wide range of metabolites. In this study, we appraised whether metabolic changes correlate with HF severity, assessed as an impairment of functional contractility, and attempted to interpret the role of metabolic changes in determining systolic dysfunction.MethodsA 500 MHz proton nuclear magnetic resonance (1H-NMR)-based analysis was performed on blood samples from three groups of individuals: 9 control subjects (Group A), 9 HF patients with mild to moderate impairment of left ventricle ejection fraction (LVEF: 41.9 ± 4.0 %; Group B), and 15 HF patients with severe LVEF impairment (25.3 ± 10.3 %; Group C). In order to create a descriptive model of HF, a supervised orthogonal projection on latent structures discriminant analysis (OPLS-DA) was applied using speckle tracking-derived longitudinal strain rate as the Y-variable in the multivariate analysis.ResultsOPLS-DA identified three metabolic clusters related to the studied groups achieving good values for R2 [R2(X) = 0.64; R2(Y) = 0.59] and Q2 (0.39). The most important metabolites implicated in the clustering were 2-hydroxybutyrate, glycine, methylmalonate, and myo-inositol.ConclusionsThe results demonstrate the suitability of metabolomics in combination with functional evaluation techniques in HF staging. This innovative tool should facilitate investigation of perturbed metabolic pathways in HF and their correlation with the impairment of myocardial function.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0661-3) contains supplementary material, which is available to authorized users.

Analytical Procedures Used in the Control of Bacterial Glycoconjugate Vaccines

Bacterial glycoconjugate vaccines consist of capsular polysaccharide chemically conjugated to a carrier protein. Vaccines against Haemophilus influenzae type b (Hib) have proven effective in preventing invasive infection by Hib in young children. Because the stability of the conjugate is essential for vaccine efficacy, physicochemical analyses are necessary to ensure the integrity of these vaccines. Analytical methods used in conjugate vaccine development and production vary among manufacturers due to different components and conjugation chemistries. In the recent past, control authorities have focused on chemical analyses and conventional immunodiffusion and enzyme-linked immunosorbent assays to evaluate the protein and polysaccharide components of glycoconjugate vaccines. We have developed one-dimensional 500 MHz proton nuclear magnetic resonance (NMR) spectroscopy to determine the identity and purity of the polysaccharide component. The NMR spectrum of polyribosyl ribitol phosphate (PRP) was consistent with the structure of the repeating unit, and any trace contaminants were readily detected. We have also developed gel-permeation chromatography to assess the structure and stability of two conjugate vaccines. The characteristic peak height relationship for each manufacturer's product was consistent, with no detectable changes when stored according to manufacturer's specifications. Vaccine stability was determined by examining samples subjected to high temperatures for prolonged periods of time, and aggregation and degradation of vaccines was detected using this method. These physicochemical analyses are essential because there is no suitable animal model that correlates with immunogenicity in man. Therefore, there is an urgent need to develop new analytical procedures and biological correlates of activity in order to control Hib vaccines and develop new glycoconjugate vaccines against other pathogenic bacteria. In this study, NMR and fast-protein liquid chromatography (FPLC) analyses were used to determine the identity, purity, and stability of two Hib conjugate vaccines licensed for use in the UK. Our results demonstrate that physicochemical methods can be used to evaluate the manufacturing consistency and stability of protein-polysaccharide conjugate vaccines.

1H and 31P NMR Lipidome of Ethanol‐Induced Fatty Liver

Hepatic steatosis (fatty liver), an early and reversible stage of alcoholic liver disease, is characterized by triglyceride deposition in hepatocytes, which can advance to steatohepatitis, fibrosis, cirrhosis, and ultimately to hepatocellular carcinoma. In the present work, we studied altered plasma and hepatic lipid metabolome (lipidome) to understand the mechanisms and lipid pattern of early-stage alcohol-induced-fatty liver. Male Fischer 344 rats were fed 5% alcohol in a Lieber-DeCarli diet. Control rats were pair-fed an equivalent amount of maltose-dextrin. After 1 month, animals were killed and plasma collected. Livers were excised for morphological, immunohistochemical, and biochemical studies. The lipids from plasma and livers were extracted with methyl-tert-butyl ether and analyzed by 750/800 MHz proton nuclear magnetic resonance (¹H NMR) and phosphorus (³¹P) NMR spectroscopy on a 600 MHz spectrometer. The NMR data were then subjected to multivariate statistical analysis. Hematoxylin and Eosin and Oil Red O stained liver sections showed significant fatty infiltration. Immunohistochemical analysis of liver sections from ethanol-fed rats showed no inflammation (absence of CD3 positive cells) or oxidative stress (absence of malondialdehyde reactivity or 4-hydroxynonenal positive staining). Cluster analysis and principal component analysis of ¹H NMR data of lipid extracts of both plasma and livers showed a significant difference in the lipid metabolome of ethanol-fed versus control rats. ³¹P NMR data of liver lipid extracts showed significant changes in phospholipids similar to ¹H NMR data. ¹H NMR data of plasma and liver reflected several changes, while comparison of ¹H NMR and ³¹P NMR data offered a correlation among the phospholipids. Our results show that alcohol consumption alters metabolism of cholesterol, triglycerides, and phospholipids that could contribute to the development of fatty liver. These studies also indicate that fatty liver precedes oxidative stress and inflammation. The similarities observed in plasma and liver lipid profiles offer a potential methodology for detecting early-stage alcohol-induced fatty liver disease by analyzing the plasma lipid profile.

O.601 Ultrasound examination in thyrolingual duct cysts diagnostic

The historical development of polyglycolic acid (PGA) and polylactic acid (PLA) polymers and copolymers for use in surgery is set down. Details of the synthesis of PGA and PLA polymers from their cyclic diesters are described, as well as a series of glycolide/lactide copolymers. The reactions were followed by time sampling techniques. The resulting samples were characterized by gel permeation chromatography (g.p.c.), differential scanning calorimetry (d.s.c.), thermogravimetric analysis (t.g.a.) and 220 MHz proton nuclear magnetic resonance spectroscopy (n.m.r.). Details of these analytical technicques are given. The respective reactivity ratios of glycolide and lactide are elucidated. The effect of 60Co γ radiation on the molecular properties of PGA is also shown.

O.603 A new method of resorbable osteosynthesis in craniosynostos

The historical development of polyglycolic acid (PGA) and polylactic acid (PLA) polymers and copolymers for use in surgery is set down. Details of the synthesis of PGA and PLA polymers from their cyclic diesters are described, as well as a series of glycolide/lactide copolymers. The reactions were followed by time sampling techniques. The resulting samples were characterized by gel permeation chromatography (g.p.c.), differential scanning calorimetry (d.s.c.), thermogravimetric analysis (t.g.a.) and 220 MHz proton nuclear magnetic resonance spectroscopy (n.m.r.). Details of these analytical technicques are given. The respective reactivity ratios of glycolide and lactide are elucidated. The effect of 60Co γ radiation on the molecular properties of PGA is also shown.

Development of Terahertz FU CW Gyrotron Series for DNP

The new gyrotron series at the Research Center for Development of Far Infrared Region, University of Fukui (FU), the so-called FU CW (continuous-wave) gyrotron series, is being developed for the application to high-power terahertz technologies. The second gyrotron of the series, FU CW II gyrotron with an 8 T liquid He-free superconducting magnet, has been constructed and the operation test was successfully carried out. It will be used for enhancing the sensitivity of 600 MHz proton nuclear magnetic resonance by the use of dynamic nuclear polarization (DNP). The designed operation mode of the gyrotron is TE2,6 at the second harmonic. The corresponding designed frequency is 394.6 GHz. The real operation frequency is 394.3 GHz at the TE0,6 mode, because of the fabrication error of the cavity diameter. The operation is in complete CW at an output power of around 30 W or higher at the TE0,6 cavity mode. There are many other operation modes at the fundamental and the second harmonics. Typical output power of the fundamental and the second harmonics is higher than 100 and 20 W, respectively. The third gyrotron, FU CW III gyrotron with a 20 T superconducting magnet, has already been constructed and the operation test has been almost completed. The highest frequency is around 980 GHz at the second harmonic operation. This gyrotron demonstrates the possibility of an application to higher-frequency DNP of up to 1 GHz.

Functional identification ofp-cumate operons in the terpene-degradingRhodococcussp. strain T104

We identified a p-cumate degrading gene cluster (designated cmt) with a novel organization in our genomic studies of the terpene-degrading Rhodococcus sp. strain T104. The mutant strain SN140, isolated for the inability to grow on p-cumate, accumulates 2,3-dihydroxy-p-cumate as identified by liquid chromatography-mass spectrometry and 300 MHz proton nuclear magnetic resonance spectroscopy. Reverse transcriptase polymerase chain reaction experiments showed that the cmt genes are operonic and induced specifically by growth on p-cumate. This report is the first example of identifying the genes for p-cumate degradation in a gram-positive organism based on functional data.

Sequence Analysis of Polyarylate (U-Polymer) and Its Polyestercarbonate Using 1H and 13C NMR

The dyad and triad-level sequence analyses of polyarylate; U-Polymer, which is an aromatic copolyester of bisphenol-A and 50/50 isophthalic/terephthalic acid, and of polyestercarbonate prepared from U-Polymer and bisphenol-A polycarbonate were reported in a solvent system of o-chlorophenol/deuterated chloroform mixture (75/25 v/v) at 80 °C using 600 MHz proton nuclear magnetic resonance (1H NMR). The well-resolved proton peaks were observed, which made the detailed sequence analysis possible. The methyl proton peaks of the bisphenol-A units were split into the dyad sequence in the chain. The aromatic proton peaks of the isophthalic and terephthalic units were split into the triad sequence in the chain. These peaks could be assigned by a comparison with the spectra of homopolymers and the model copolymers. It was confirmed that the sequence distribution in U-Polymer and the polyestercarbonate are random and obey Bernoullian statistics, and the dyad and triad information obtained coincides with each other by a comparison of the number-average sequence lengths from the dyad and triad sequences. 13C NMR peaks of the aliphatic quaternary carbons of bisphenol-A units were also split into the dyad sequence in the chain. It was confirmed that the solvent system of the 75/25 (v/v) mixture of o-chlorophenol /deuterated chloroform has the potentiality for the detailed sequence analysis of polyarylate and polyestercarbonate.

Position isomer separation of non-peripheral substituted zinc dibenzo-di(3,4-pyrido)porphyrazines

The cyclotetramerisation products from the 1,8-diazabicyclo[5,4,0]undec-7-ene catalysed reaction of a 1:1 mol mixture of 3,6-didecylphthalonitrile and 3,4-dicynopyridine in the presence of zinc chloride have been isolated by chromatography. The fractions comprise zinc octadecylphthalocyanine and compounds in which one, two, three and four pyridinoid rings replace the didecylbenzenoid moiety. The zinc bis(1,4-didecylbenzo)bis(3,4-pyrido)porphyrazine fraction was separated further into four sub-fractions and the components have been characterised by 400 MHz proton nuclear magnetic resonance spectroscopy, visible region spectroscopy, fluorescence spectroscopy and by cyclic voltammetry.

Nondestructive quantitative determination of docosahexaenoic acid and n−3 fatty acids in fish oils by high‐resolution 1H nuclear magnetic resonance spectroscopy

AbstractBy using a 500 MHz proton nuclear magnetic resonance (1H NMR) spectrometer we have developed a quantitative method for determining the contents of docosahexaenoic acid (DHA) in fish oils (mg/g), the molar proportions (mol%) of DHA to all other fatty acids composing the fish oils, and the molar proportions of total n‐3 fatty acids to all other non‐n‐3 fatty acids in the fish oils. After examining the suitability of ethylene glycol dimethyl ether (EGDM), methanol, and 1,4‐dioxane as internal standards, experimental conditions were optimized by mainly using EGDM as an internal standard. By setting the pulse repetition time at 30 s, five times longer than the longest T1 of the 1H NMR signals of fish oils, good reproductibility of data and analytical times less than 10 min were achieved. The use of the internal standard also allowed us to quantify DHA on a weight basis (mg/g). Verification of the method was carried out in an interlaboratory study between Japan and Norway on bonito, tuna, and salmon oils. The relative errors in the 1H NMR data between Japan and Norway were 0.57–5.29% for quantification of DHA, 0.7–2.09% for the molar proportion of DHA, and 0.1–1.41% for the molar proportion of total n‐3 fatty acids. Good agreement was observed between the 1H NMR data and those obtained by gas chromatography (GC). The sample preparation before 1H NMR measurements required only two steps: sample weighing and preparation of an internal standard solution. Based on the high reproducibility, simplicity of the procedure, and clarity of principle, the proposed 1H NMR method was judged to be a promising alternative to the GC method in quantification of DHA and n‐3 fatty acids in fish oils.

Structural heterogeneity in the core oligosaccharide of the S-layer glycoprotein from Aneurinibacillus thermoaerophilus DSM 10155.

The surface layer glycoprotein of Aneurinibacillus thermoaerophilus DSM 10155 has a total carbohydrate content of 15% (by mass), consisting of O-linked oligosaccharide chains. After proteolytic digestion of the S-layer glycoprotein byPronase E and subsequent purification of the digestion products by gel permeation chromatography, chromatofocusing and high-performance liquid chromatography two glycopeptide pools A and B with identical glycans and the repeating unit structure -->4)-alpha-l-Rha p -(1-->3)-beta-d- glycero -d- manno -Hep p -(1--> (Kosma et al., 1995b, Glycobiology, 5, 791-796) were obtained. Combined evidence from modified Edman-degradation in combination with liquid chromatography electrospray mass-spectrometry and nuclear magnetic resonance spectroscopy revealed that both glycopeptides contain equal amounts of the complete core structure alpha-l-Rha p -(1-->3)-alpha-l-Rha p -(1-->3)-beta-d-Gal p NAc-(1-->O)-Thr/Ser and the truncated forms alpha-l-Rha p -(1-->3)-beta-d-Gal p NAc-(1-->O)-Thr/Ser and beta-d-Gal p NAc-(1-->O)-Thr/Ser. All glycopeptides possessed the novel linkage types beta-d-Gal p NAc-(1-->O)-Thr/Ser. The different cores were substituted with varying numbers of disaccharide repeating units. By 300 MHz proton nuclear magnetic resonance spectroscopy the complete carbohydrate core structure of the fluorescently labeled glyco-peptide B was determined after Smith-degradation of its glycan chain. The NMR data confirmed and complemented the results of the mass spectroscopy experiments. Based on the S-layer glycopeptide structure, a pathway for its biosynthesis is suggested.

1H-NMR Profile of Glycosaminoglycans in Human Urine

A study was performed on human urinary glycosaminoglycans (GAGs) by one- and two-dimensional 500MHz proton nuclear magnetic resonance (1H-NMR) spectroscopy. Urinary GAGs were separated by the precipitation methods with hexadecyl pyridinium and with 85% ethanol, and purified by the β-elimination reaction in alkaline medium containing sodium borohydride to remove proteins and/or peptides. The spectrum of each purified GAG sample from healthy human urine which included chondroitin sulfates (ChS), dermatan sulfate (DS) and heparan sulfate (HS) showed the characteristic signal(s) corresponding to each GAG chain. The assignments and integrations of the structure specific signals in the spectrum made it possible to perform qualitative and quantitative analyses on human urinary GAG mixtures. Furthermore, analytical results of urinary GAGs in children, adults and the aged indicate that the present method is applicable to investigate the effect of aging on excretion of urinary GAGs.

Detection and determination of dilute, low molecular weight organic compounds in water by 500 MHz proton nuclear magnetic resonance spectroscopy

The feasibility of using high-field proton NMR spectroscopy to analyze aqueous solutions of organic solutes at micromolar concentrations has been evaluated. N-Nitrosodimethylamine (NDMA) and benzene served as model compounds. The WATR (water attenuation by transverse relaxation) method of solvent suppression was optimized to permit detection of the protons of analytes in the submicromolar concentration regime. All data were collected in blocks to permit quantitative estimation of detection limits and experimental error

The Dewar valence isomer of the (6-4) photoadduct of thymidylyl-(3′–5′)-thymidine monophosphate: Formation, alkaline lability and conformations properties

The formation of the Dewar valence isomer of the pyrimidine(6-4)pyrimidone photoadduct of thymidylyl-(3′–5′)-thymidine monophosphate (TpT) was investigated under different irradiation conditions. This photoproduct was generated on exposure of TpT to far-UV radiation. However, no detectable amount of the Dewar isomer or its precursor (pyrimidine(6-4)pyrimidone photoadduct) was observed following acetone photosensitization of TpT. The Dewar valence isomer was much more unstable than the pyrimidine(6-4)pyrimidone photoproduct when treated with hot piperidine. A detailed conformational analysis of the TpT Dewar isomer photoproduct is reported as inferred from extensive one- and two-dimensional 300 and 620 MHz proton nuclear magnetic resonance ( 1H NMR) measurements and molecular mechanics calculations.

Termini of Salmonella flagellin are disordered and become organized upon polymerization into flagellar filament

The terminal regions of Salmonella flagellin are essential for polymerization to form the flagellar filament. It has recently been suggested, on the basis of results from circular dichroism spectroscopy and scanning calorimetry, that these regions are disordered in solution. We report here direct evidence for disorder and mobility in the terminal regions of flagellin using 400 MHz proton nuclear magnetic resonance (n.m.r.) spectroscopy. Comparison of the n.m.r. spectra of monomeric and polymeric flagellin shows that the terminal regions become organized when polymerized to form the filament.

Purification and chemical characterisation of the inhibitor of lipid peroxidation from intestinal mucosa

The antioxidant previously isolated from intestinal mucosa has been subjected to further purification and identification. Although this inhibitor moved as a single spot on thin-layer chromatography in a number of different solvent systems, it proved to be a mixture of free carboxylic acids whose relative composition was similar in different batches. Detailed studies involving the use of high-pressure liquid chromatography, combined gas chromatography-mass spectrometry, high-field 360 MHz proton nuclear magnetic resonance spectroscopy, fast atom bombardment mass spectrometry and other techniques established that the inhibitor was a mixture of carboxylic acids of the following identity and relative composition (the major components comprising 92% of the total fatty acids): palmitic acid, 14.8%; palmitoleic acid, 3.6%; stearic acid, 7.0%; oleic acid, 21.0%; linoleic acid, 27.6% arachidonic acid, 18.0%. Mixtures of authentic fatty acids of the same relative concentration showed inhibition of peroxidation, comparable with the purified inhibitor from intestinal mucosa. A study of the inhibitory activity of the components of the mixture using malonaldehyde estimation, diene conjugation and arachidonic acid estimation showed that the inhibitory activity was due to palmitoleic and oleic acids only, the latter being the major component.

N-acetylglucosaminyltransferase substrates prepared from glycoproteins by hydrazinolysis of the asparagine-N-acetylglucosamine linkage. Purification and structural determination of oligosaccharides with mannose andN-acetylglucosamine at the non-reducing termini

Sixteen asparagine-linked oligosaccharides ranging in size from (Man)2(GlcNAc)2 (Fuc)1 to (GlcNAc)6(Man)3(GlcNAc)2 were obtained from human α1-acid glycoprotein and fibrinogen, hen ovomucoid and ovalbumin, and bovine fetuin, fibrin and thyroglobulin by hydrazinolysis, mild acid hydrolysis and glycosidase treatment. The oligosaccharides hadN-acetylglucosamine at the reducing termini and mannose andN-acetylglucosamine residues at the non-reducing termini and were prepared for use asN-acetylglucosaminyltransferase substrates. Purification of the oligosaccharides involved gel filtration and high performance liquid chromatography on reverse phase and amine-bonded silica columns. Structures were determined by 360 MHz and 500 MHz proton nuclear magnetic resonance spectroscopy, fast atom bombardment-mass spectrometry and methylation analysis. Several of these oligosaccharides have not previously been well characterized.

ChemInform Abstract: Enzymatic Synthesis of 2,3‐O‐Isopropylidene‐sn‐glycerol, a Chiral Building Block for Platelet‐Activating Factor.

An enzymatic synthesis of 2, 3-O-isopropylidene-sn-glycerol (10), the synthetic key intermediate for platelet-activating factor, was achieved. Several 1, 3-di-O-acyl-2-benzylglycerols (5a-d) were synthesized from dihydroxyacetone dimer (2), and subjected to enzyme-catalyzed asymmetric hydrolysis. The optical purities of the mono-hydrolyzed products (6) were determined from the 400 MHz proton nuclear magnetic resonance spectra after conversion of 6 to the esters of (-)α-methoxy-α-trifluoromethylphenylacetic acid. Upon hydrogenolysis of the benzyl ether, followed by protection of diol and hydrolysis of the acetate, (-)-6a afforded 10.

Role of the N-terminal part of the coat protein in the assembly of cowpea chlorotic mottle virus: A 500 MHz proton nuclear magnetic resonance study and structural calculations

The interaction of the oligonucleotides (Ap) 8A and (A-T) 5 with empty capsids of the coat protein of cowpea chlorotic mottle virus (CCMV) has been studied with 500 MHz 1H nuclear magnetic resonance. It is found that these oligonucleotides specifically bind to the arginine and lysine residues of the N-terminal arm of the protein. Upon this binding, immobilization of part of the N-terminal arm occurs. In addition, secondary structure predictions and energy calculations have been performed on the N-terminal arm. These calculations were carried out as a function of the charges on the arginine and lysine side-chains. For free coat protein, where the arginine and lysine side-chains are charged, the arm is found in a random-coil conformation. In the neutralized state, as for the coat protein in the virus, the arm adopts an α-helical conformation. The results support a previously published model for the assembly of CCMV, in which a random-coil to α-helix conformational transition, induced by neutralizing the arginine and lysine side-chains, plays an essential role.

NUCLEAR MAGNETIC RESONANCE STUDIES OF THE INTERACTION OF Ca~(2+) WITH SIALIC ACID~*

The interaction of Ca~(2+) with sialic acid has been investigated by 500 MHz proton nuclear magnetic resonance spectroscopy. The changes of chemical shifts were observed for all sialic acid protons at different Ca~(2+) concentrations, and these spectral observations were also accompanied by changes in the J-coupling constants between the H_7 and H_8 protons and between the H_8 and H'_9 potens. Analysis of these Ca~(2+)-induced changes indicated that three oxygen atoms among (OH)_7, (OH)_8 and (OH)_9 of sialie acid formed a geometrical space suitable for Ca~(2+) coordination As a consequence, sialic acid provided a high-affinity binding site for Ca~(2+), the binding constant was about 104.72m~(-1).