High-resolution 1H NMR Spectroscopy Research Articles

Study of natural mango juice spoilage and microbial contamination with Penicillium expansum by high resolution 1H NMR spectroscopy

High resolution 1H NMR has been applied to monitor the changes in the composition of natural mango juice subjected to spoilage and to microbial contamination with Penicillium expansum. A vast number of compounds undergoing changes upon these processes have been identified and their variations followed throughout time (132 h). Besides the formation of typical fermentation products (e.g. acetate, lactic acid, acetoin and isopropanol/2,3-butanediol) and the utilization of the major sugars (sucrose, glucose and fructose), there were changes in organic acids (e.g. decreases of quinic and shikimic acids with formation of 3,4,5-trihydroxycyclohexane acid in spoiled juice, and decreases of citric and malic acids in contaminated juice), amino acids (decreases of alanine, leucine, isoleucine and valine), and less abundant components such as oligosaccharides and aromatic compounds. The possibility of using these changes as early indicators of natural spoilage or P. expansum contamination is discussed.

Qualitative high field 1H-NMR spectroscopy for the characterization of endogenous metabolites in earthworms with biochemical biomarker potential

This study was designed to provide a database of the endogenous metabolites in earthworm extracts of the species, Eisenia veneta and Lumbricus terrestris by high resolution 1H-NMR spectroscopy in view of identifying biomarkers of toxicity or stress in environmental metabolomics studies. 1D and 2D NMR spectroscopic techniques enabled the identification and confirmation of the organic components in the tissue extracts of whole and segmented earthworms, dissected organs, and coelomic fluid. The extracts gave rise to characteristic 1H-NMR spectral fingerprints of the low MW metabolites contained, specific to the species of earthworm, and to the specific regions or dissected organs of the earthworms under investigation. Distinct changes in the normal biochemistry were observed upon starvation and cooling, such as markedly decreased glucose and maltose, but increased lactate, acetate, succinate, formate and acetone. Additionally, slightly decreased threonine, arginine, lysine, leucine, citrate, asparagine and glycine were observed. Furthermore, lactate could be identified as a biomarker of acute toxic stress in expressed coelomic fluid following exposure to a model ecotoxin (3-trifluoromethylaniline). This work supports the application of 1H-NMR spectroscopy for the study of changes in the normal invertebrate biochemistry in order to allow for the reliable assessment of biomarker responses following toxicity testing.

A metabolomic investigation of splanchnic metabolism using 1H NMR spectroscopy of bovine blood plasma

This is the first study introducing an NMR-based metabolomic approach to elucidate metabolism in the portal-drained viscera and liver. High-resolution 1H NMR spectroscopy was performed on blood plasma obtained from the mesenteric artery, the hepatic portal vein and the hepatic vein of steers before and after interruminal infusion of short-chain fatty acids (SCFA). Partial least squares regression discriminant analysis (PLS-DA) on 1H NMR spectra obtained for arterial and portal plasma samples revealed increases in the intensities of spectral regions that could mostly be assigned to the methyl group of propionate, β-hydroxybutyrate as well as acetate in portal plasma samples compared with arterial plasma samples. PLS-DA on portal and hepatic plasma samples showed decreases in the intensities of spectral regions corresponding to the regions where methyl and methylene groups of n-butyrate, propionate, iso-butyrate, valerate, iso-valerate as well as lactate are reported to have chemical shifts. In addition, spectral differences likely to reflect a hepatic excretion of β-hydroxybutyrate and glucose were observed between portal and hepatic samples. All findings were supported by traditional biochemical analyses. Consequently, the present study clearly demonstrated the great potential of the NMR-based metabolomic approach for elucidating splanchnic metabolism. In addition, prediction of metabolite concentrations from the 1H NMR spectra obtained on plasma samples was investigated using PLS, which in relation to use 1H NMR spectra quantitatively revealed promising results with R-values in the region of 0.87–0.98.

Characterization of the biochemical effects of 1-nitronaphthalene in rats using global metabolic profiling by NMR spectroscopy and pattern recognition

Metabolic fingerprints, in the form of patterns of high-concentration endogenous metabolites, of 1-nitronaphthalene (NN)-induced lung toxicity have been elucidated in bronchoalveolar lavage fluid (BALF), urine, blood plasma, and intact lung and liver tissue using NMR spectroscopy-based metabolic profiling. A single dose of NN (75 mg kg−1) was administered orally to Sprague–Dawley rats. BALF and lung tissue were obtained 24 h after dosing from these animals and matched control rats post-mortem. High-resolution 1H-NMR spectroscopy of BALF samples indicated that NN caused increases in concentrations of choline, amino acids (leucine, isoleucine and alanine) and lactate together with decreased concentrations of succinate, citrate, creatine, creatinine and glucose. In addition, the intact lung weights were higher in the NN-treated group (p<0.01), consistent with pulmonary oedema. The NMR-detected perturbations indicated that NN induces a perturbation in energy metabolism in both lung and liver tissue, as well as surfactant production and osmolyte levels in the lungs. As well as reporting the first NMR spectroscopic combined examination of BALF and intact lung, this study indicates that such holistic approaches to investigating mechanisms of lung toxicity may be of value in evaluating disease progression or the effects of therapeutic intervention in pulmonary conditions such as surfactant disorders or asthma.

A Metabonomic Strategy for the Detection of the Metabolic Effects of Chamomile (Matricaria recutita L.) Ingestion

A metabonomic strategy, utilizing high-resolution 1H NMR spectroscopy in conjunction with chemometric methods (discriminant analysis with orthogonal signal correction), has been applied to the study of human biological responses to chamomile tea ingestion. Daily urine samples were collected from volunteers during a 6-week period incorporating a 2-week baseline period, 2 weeks of daily chamomile tea ingestion, and a 2-week post-treatment phase. Although strong intersubject variation in metabolite profiles was observed, clear differentiation between the samples obtained before and after chamomile ingestion was achieved on the basis of increased urinary excretion of hippurate and glycine with depleted creatinine concentration. Samples obtained up to 2 weeks after daily chamomile intake formed an isolated cluster in the discriminant analysis map, from which it was inferred that the metabolic effects of chamomile ingestion were prolonged during the 2-week postdosing period. This study highlights the potential for metabonomic technology in the assessment of nutritional interventions, despite the high degree of variation from genetic and environmental sources.

Kinetics of oligomerization reactions in formaldehyde solutions: NMR experiments up to 373 K and thermodynamically consistent model

Aqueous and methanolic formaldehyde solutions are industrially important complex reacting mixtures in which formaldehyde is bound in a variety of oligomerization products. Reaction kinetics in these systems have to be taken into account in the design of reactors and separation equipment. The present paper reports on studies on reaction kinetics of aqueous and methanolic formaldehyde solutions by quantitative high-resolution on-line 1H NMR spectroscopy. The experiments were carried out at temperatures up to 373 K partially with a pressurized NMR probe and extend the temperature range in which data were available up to now. The NMR data are used to develop a reaction kinetic model, which is consistent with a physico-chemical thermodynamic equilibrium model, for formaldehyde containing mixtures. This is an important step towards developing thermodynamically consistent process models for reactions and separations with formaldehyde containing mixtures, which take reaction kinetics explicitly into account.

Metabonomics: Its Potential as a Tool in Toxicology for Safety Assessment and Data Integration

The functional genomic techniques of transcriptomics and proteomics promise unparalleled global information during the drug development process. However, if these technologies are used in isolation the large multivariate data sets produced are often difficult to interpret, and have the potential of missing key metabolic events (e.g. as a result of experimental noise in the system). To better understand the significance of these megavariate data the temporal changes in phenotype must be described. High resolution 1H NMR spectroscopy used in conjunction with pattern recognition provides one such tool for defining the dynamic phenotype of a cell, organ or organism in terms of a metabolic phenotype. In this review the benefits of this metabonomics/metabolomics approach to problems in toxicology will be discussed. One of the major benefits of this approach is its high throughput nature and cost effectiveness on a per sample basis. Using such a method the consortium for metabonomic toxicology (COMET) are currently investigating approximately 150 model liver and kidney toxins. This investigation will allow the generation of expert systems where liver and kidney toxicity can be predicted for model drug compounds, providing a new research tool in the field of drug metabolism. The review will also include how metabonomics may be used to investigate co-responses with transcripts and proteins involved in metabolism and stress responses, such as during drug induced fatty liver disease. By using data integration to combine metabolite analysis and gene expression profiling key perturbed metabolic pathways can be identified and used as a tool to investigate drug function.

Formation of an interlocked quadruplex dimer by d(GGGT).

A tetranucleotide sequence d(GGGT) has been shown to self-assemble into an interlocking quadruplex dimer. UV-melting studies indicated the existence of two species that each showed distinct quadruplex melting transitions, a low-T(m) species, Q(l), and a high-T(m) species, Q(h). Conditions were controlled to favor the formation of either Q(l) or Q(h). Q(l) and Q(h) each showed circular dichroism spectra characteristic of parallel quadruplexes. Negative ion nano-electrospray ionization mass spectrometry confirmed that Q(l) was a tetrameric complex, d(GGGT)(4), and Q(h) was an octameric complex, d(GGGT)(8). High-resolution (1)H NMR spectroscopy evidenced that d(GGGT)(4) was a C(4)-symmetric parallel tetramolecular quadruplex. The (1)H NMR spectrum of d(GGGT)(8) was consistent with a structure formed by the dimerization of a parallel, "slipped" tetramolecular quadruplex that has its diagonal strands staggered by one base. This "slippage" results in two guanine bases at the 5' end of the quadruplex being presented diagonally that are not involved in tetrads. Two such "slipped" quadruplexes dimerize via these free G-bases at the 5' ends by forming an extra G-tetrad. Each "slipped" quadruplex contributes two guanine bases to this extra G-tetrad. The formation of a novel GTGT tetrad is also observed at both the 3' ends of the interlocked quadruplex dimer.

Conformational analysis of 1,3,6,2-dioxazaphosphocanes and 1,3,6,2-dioxazasilocanes

The steric structure of 1,3,6,2-dioxazaphosphocanes and 1,3,6,2-dioxazasilocanes in solution was established by high-resolution 1H, 13C, and 31P NMR spectroscopy and PM3 calculations. It is shown that the preferred conformation of the eight-membered heterocycle is chair-chair (crown).

NMR Study on the Binding of Neuropeptide Achatin-I to Phospholipid Bilayer: The Equilibrium, Location, and Peptide Conformation

Molecular mechanism of the binding of neuropeptide achatin-I (Gly-D-Phe-Ala-Asp) to large unilamellar vesicles of zwitterionic egg-yolk phosphatidylcholine (EPC) was investigated by means of natural-abundance 13C and high-resolution (of 0.01 Hz order) 1H NMR spectroscopy. The binding equilibrium was found to be sensitive to the ionization state of the N-terminal N H 3 + group in achatin-I; the de-ionization of N H 3 + decreases the bound fraction of the peptide from ∼15% to nearly none. The electrostatic attraction between the N-terminal positive N H 3 + group and the negative P O 4 − group in the EPC headgroup plays an important role in controlling the equilibrium. Analysis of the 13C chemical shifts ( δ) of EPC showed that the binding location of the peptide within the bilayer is the polar region between the glycerol and ester groups. The binding caused upfield changes Δ δ of the 13C resonance for almost all the carbon sites in achatin-I. The changes Δ δ for the ionic Asp at the C-terminus are more than five times as large as those for the other residues. The drastic changes for Asp result from the dehydration of the ionic C O 2 − groups, which are strongly hydrated by electrostatic interactions in bulk water. The side-chain conformational equilibria of the aromatic D-Phe and ionic Asp residues were both affected by the binding, and the induced changes in the equilibria appear to reflect the peptide-lipid hydrophobic interactions.

New water-soluble analog of the fusion peptide of influenza virus hemagglutinin: synthesis and properties

A water-soluble analogue F32 of the fusion peptide from the influenza virus hemagglutinin was synthesized. It consisted of 32 aa residues and retained the ability to interact with lipid membranes; its N-terminal sequence 1-24 coincided with that of the fusion protein from hemagglutinin (strain A/PR/8/34), whereas residues 25-32 (GGGKKKKK) provided its solubility in water. The peptide induced the conductivity fluctuations in planar bilayer lipid membranes characteristic of active fusion peptides. Conditions were found using CD spectroscopy under which the structure of F32 inside detergent micelles, where it can be studied by high-resolution 1H NMR spectroscopy, is close to the structure of the peptide during its interaction with phospholipid liposomes. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.

Defining a metabolic phenotype in the brain of a transgenic mouse model of spinocerebellar ataxia 3.

Many of the spinocerebellar ataxias (SCAs) are caused by expansions of CAG trinucleotide repeats encoding abnormal stretches of polyglutamine. SCA3 or Machado-Joseph disease (MJD) is the commonest dominant inherited ataxia disease, with pathological phenotypes apparent with a CAG triplet repeat length of 61-84. In this study a mouse model of SCA3 has been examined which was produced using a human yeast artificial chromosome containing the MJD gene with a CAG triplet expansion of 84 repeats. These mice have previously been shown to possess a mild progressive cerebellar deficit. NMR-based metabolomics/metabonomics in conjunction with multivariate pattern recognition identified a number of metabolic perturbations in SCA3 mice. These changes included a consistent increase in glutamine concentration in tissue extracts of the cerebellum and cerebrum and spectra obtained from intact tissue using magic angle spinning (1)H-NMR spectroscopy. Furthermore, these profiles demonstrated metabolic abnormalities were present in the cerebrum, a region not previously implicated in SCA3. As well as an increase in glutamine both brain regions demonstrated decreases in GABA, choline, phosphocholine and lactate (representing the summation of lactate in vivo, and postmortem glycolysis of glucose and glycogen). The metabolic changes are discussed in terms of the formation of neuronal intranuclear inclusions associated with SCA3. This study suggests high-resolution (1)H-NMR spectroscopy coupled with pattern recognition may provide a rapid method for assessing the phenotype of animal models of human disease.

Selective alterations of brain osmolytes in acute liver failure: protective effect of mild hypothermia

The principal cause of mortality in patients with acute liver failure (ALF) is brain herniation resulting from intracranial hypertension caused by a progressive increase of brain water. In the present study, ex vivo high-resolution 1H-NMR spectroscopy was used to investigate the effects of ALF, with or without superimposed hypothermia, on brain organic osmolyte concentrations in relation to the severity of encephalopathy and brain edema in rats with ALF due to hepatic devascularization. In normothermic ALF rats, glutamine concentrations in frontal cortex increased more than fourfold at precoma stages, i.e. prior to the onset of severe encephalopathy, but showed no further increase at coma stages. In parallel with glutamine accumulation, the brain organic osmolytes myo-inositol and taurine were significantly decreased in frontal cortex to 63% and 67% of control values, respectively, at precoma stages ( p<0.01), and to 58% and 67%, respectively, at coma stages of encephalopathy ( p<0.01). Hypothermia, which prevented brain edema and encephalopathy in ALF rats, significantly attenuated the depletion of myo-inositol and taurine. Brain glutamine concentrations, on the other hand, did not respond to hypothermia. These findings demonstrate that experimental ALF results in selective changes in brain organic osmolytes as a function of the degree of encephalopathy which are associated with brain edema, and provides a further rationale for the continued use of hypothermia in the management of this condition.

High-resolution 1H NMR spectroscopy in a live mouse subjected to 1.5 Hz magic angle spinning.

It is demonstrated that the resolution of the (1)H NMR metabolite spectrum in a live mouse can be significantly enhanced by an ultraslow magic angle spinning of the animal combined with a modified phase-corrected magic angle turning (PHORMAT) pulse sequence. Proton NMR spectra were measured of the torso and the top part of the belly of a female BALBc mouse in a 2 T field while spinning the animal at a speed of 1.5 Hz. It was found that even in this relatively low field, with PHORMAT an isotropic spectrum is obtained with line widths that are a factor of 4.6 smaller than those obtained in a stationary mouse. It is concluded that in vivo PHORMAT has the potential to significantly increase the utility of (1)H NMR spectroscopy for biochemical and biomedical animal research.

Biochemical characterization of muscle tissue of limb girdle muscular dystrophy: an 1H and 13C NMR study.

The metabolic differences between the muscle biopsies of patients with limb girdle muscular dystrophy (LGMD) and normal controls were characterized using high-resolution 1H and 13C NMR spectroscopy. In all, 44 metabolites were unambiguously assigned in the perchloric acid extracts of skeletal muscle tissue, using 2D double quantum filtered (DQF COSY), total correlation (TOCSY), and 1H/13C heteronuclear multiple quantum coherence (HMQC) spectroscopy. The concentrations of glycolytic substrate, glucose (p=0.03), gluconeogenic amino acids, glutamine (p=0.02) and alanine (p=0.009) together with glycolytic product, lactate (p=0.04), were found to be significantly lowered in LGMD patients as compared with controls. The reduction in the concentration of glucose may be attributed to the decrease in the concentration of gluconeogenic amino acids in the degenerated muscle. Reduction in the rate of anaerobic glycolysis and lowered substrate concentration appear to be the possible reasons for the decrease in the concentration of lactate. A significant reduction in the concentration of choline in LGMD patients was also observed compared with controls. Lower concentration of choline may be the result of decreased rate of membrane turnover in LGMD patients. The data presented here provide an insight into the potentials of in-vitro NMR spectroscopy in the study of muscle metabolism.

A new dimension of 1H-NMR spectroscopy in assessment of liver graft dysfunction.

High-resolution 1H-NMR spectroscopy of serum and urine samples of an 11-year-old male living related orthotopic liver transplant recipient is reported. Serum glutamine increased to abnormal levels along with simultaneous abnormal excretion of urinary glutamine post-transplantation. High levels of glutamine in both blood and urine and concomitant reduced urea levels in urine were found to be evidence of impairment in urea cycle and compatible with persistently abnormal graft function. Thus glutamine levels in serum and urine, and urea in the urine as observed by 1H-NMR spectroscopy highlight their important roles in monitoring liver graft function; increased glutamine levels lead to brain damage, if untreated.

An integrated metabonomic investigation of acetaminophen toxicity in the mouse using NMR spectroscopy.

An integrated metabonomics study using high-resolution 1H NMR spectroscopy has been applied to investigate the biochemical composition of intact liver tissue (using magic angle spinning), liver tissue extracts, and blood plasma samples obtained from control and acetaminophen-treated mice. Principal components analysis was used to visualize similarities and differences in biochemical profiles. The time- and dose-dependent biochemical effects of acetaminophen were related to the drug toxicity, as determined using histopathology. Metabolic effects in intact liver tissue and lipid soluble liver tissue extracts from animals treated with the high dose level of acetaminophen included an increase in lipid triglycerides and monounsaturated fatty acids together with a decrease in polyunsaturated fatty acids, indicating mitochondrial malfunction with concomitant compensatory increase of peroxisomal activity. In addition, a depletion of phospholipids was observed in treated liver tissue, which suggested an inhibition of enzymes involved in phospholipid synthesis. There was also a depletion in the levels of liver glucose and glycogen. In addition, the aqueous soluble liver tissue extracts from high dose animals also revealed an increase in lactate, alanine, and other amino acids, together with a decrease in glucose. Plasma spectra showed increases in glucose, acetate, pyruvate, and lactate. These observations all provide evidence for an increased rate of glycolysis. These findings could indicate a mitochondrial inability to use pyruvate in the citric acid cycle and also reveal the impairment of fatty acid beta-oxidation in liver mitochondria of such treated mice.

Vitamin E deficiency and metabolic deficits in neuronal ceroid lipofuscinosis described by bioinformatics.

The mnd mouse, a model of neuronal ceroid lipofusinosis (NCL), has a profound vitamin E deficiency in sera and brain, associated with cerebral deterioration characteristic of NCL. In this study, the vitamin E deficiency is corrected using dietary supplementation. However, the histopathological features associated with NCL remained. With use of a bioinformatics approach based on high-resolution solid and solution state 1H-NMR spectroscopy and principal component analysis (PCA), the deficits associated with NCL are defined in terms of a metabolic phenotype. Although vitamin E supplementation reversed some of the metabolic abnormalities, in particular the concentration of phenylalanine in extracts of cerebral tissue, PCA demonstrated that metabolic deficits associated with NCL were greater than any effects produced from vitamin E supplementation. These deficits included increased glutamate and N-acetyl-L-aspartate and decreased creatine and glutamine concentrations in aqueous extracts of the cortex, as well as profound accumulation of lipid in intact cerebral tissue. This is discussed in terms of faulty production of mitochondrial-associated membranes, thought to be central to the deficits in mnd mice.

Migration Phenomena in Methacrylic Esters and Polymers Derived from Glycerol

Isomerization reactions were analyzed in methacrylic monomers and polymers derived from glycerol. It was observed that this kind of reactions takes place in similar way on both 1,3-dihydroxypropyl methacrylate and poly(1,3-dihydroxypropyl methacrylate). This polymer only could be synthesized in pure state by acid hydrolysis from precursor polymers such as poly(cis-(2-phenyl-1,3-dioxan-5-yl) methacrylate) (PCPDM) or poly(trans-(2-phenyl-1,3-dioxan-5-yl) methacrylate) (PTPDM), which were prepared by radical polymerization of the corresponding monomers. The isomerization reactions were analyzed qualitatively and quantitatively by means of high resolution 1H NMR spectroscopy. It was shown that, as a consequence of migration of the ester group in the methacrylic residue, the 1,3-dihydroxy units in poly(1,3GM) evolved to 2,3-dihydroxy units, giving a copolymer poly(1,3-dihydroxypropyl methacrylate-est-2,3-dihydroxypropyl methacrylate). The copolymer is very rich in the 2,3-dihydroxy units that are thermodynamically more favored by around 900 cal · mol−1 at room temperature. 13C NMR spectrum of a copolymer of 1,3GM (90%) and 2,3GM (10%) in D2O solution.

Synthesis, high-resolution NMR spectroscopic analysis, and single-crystal X-ray diffraction of isoxazoline tetracycles

Three isoxazoline tetracycles were obtained enantiomerically pure by intramolecular 1,3-dipolar cycloaddition. The characterization of the new compounds was performed by high-resolution 1H and 13C NMR spectroscopy. The relative configuration of the new chiral centers was determined by NOESY experiments and confirmed by single-crystal X-ray structural analysis.