Metabolic classification of South American Ilex species by NMR-based metabolomics

The genus Ilex to which mate (Ilex paraguariensis) belongs, consists of more than 500 species. A wide range of metabolites including saponins and phenylpropanoids has been reported from Ilex species. However, despite the previous works on the Ilex metabolites, the metabolic similarities between species which can be used for chemotaxonomy of the species are not clear yet. In this study, nuclear magnetic resonance (NMR) spectroscopy-based metabolomics was applied to the classification of 11 South American Ilex species, namely, Ilex argentina, Ilex brasiliensis, Ilex brevicuspis, Ilex dumosa var. dumosa, I. dumosa var. guaranina, Ilex integerrima, Ilex microdonta, I. paraguariensis var. paraguariensis, Ilex pseudobuxus, Ilex taubertiana, and Ilex theezans. ¹H NMR combined with principal component analysis (PCA), partial least square-discriminant analysis (PLS-DA) and hierarchical cluster analysis (HCA) showed a clear separation between species and resulted in four groups based on metabolomic similarities. The signal congestion of ¹H NMR spectra was overcome by the implementation of two-dimensional (2D)-J-resolved and heteronuclear single quantum coherence (HSQC). From the results obtained by 1D- and 2D-NMR-based metabolomics it was concluded that species included in group A (I. paraguariensis) were metabolically characterized by a higher amount of xanthines, and phenolics including phenylpropanoids and flavonoids; group B (I. dumosa var. dumosa and I. dumosa var. guaranina) with oleanane type saponins; group C (I. brasiliensis, I. integerrima, I. pseudobuxus and I. theezans) with arbutin and dicaffeoylquinic acids, and group D (I. argentina, I. brevicuspis, I. microdonta and I. taubertiana) with the highest level of ursane-type saponins. Clear metabolomic discrimination of Ilex species and varieties in this study makes the chemotaxonomic classification of Ilex species possible.     

NMR-based metabolomics at work in phytochemistry

Metabolomics is defined as both the qualitative and quantitative analysis of all metabolites in an organism unraveling correlation with other OMICs data. Many of the technologies used in metabolomics have method-specific advantages and drawbacks in terms of diversity of metabolites detected, sensitivity, or resolution. In this paper, the potential of NMR spectrometry applied to metabolomics is reviewed using examples of Nicotiana tabacum and Catharanthus roseus.     

Metabolic fingerprinting of wild type and transgenic tobacco plants by 1H NMR and multivariate analysis technique

The metabolomic analysis of wild type and constitutive salicylic acid producing tobacco plants (CSA tobacco, Nicotiana tabacum 'Samsun' NN) plants overexpressing salicylate biosynthetic genes was carried out by 1H NMR spectrometry and multivariate analysis techniques. The principle component analysis (PCA) of the 1H NMR spectra showed a clear discrimination between those samples by PC1 and PC2. The discrimination of non-inoculated, TMV-virus inoculated, and systemic leaves or veins could also be obtained by PCA analysis. Major peaks in 1H NMR spectra contributing to the discrimination were assigned as those of chlorogenic acid, malic acid, and sugars. This method allows an efficient differentiation between wild type and transgenic plants without any pre-purification steps.     

Metabolic discrimination of sucrose starvation from<Emphasis Type="Italic">Arabidopsis</Emphasis> cell suspension by<Superscript>1</Superscript>H NMR based metabolomics

Whole cell extracts ofArabidopsis cell cultures maintained on various sucrose concentrations (0,3, and 6%) were analyzed by¹H NMR spectroscopy to determine the comprehensive metabolic change in these cultures during sucrose starvation. The amount of sucrose, glucose, and fructose in the cells decreased to almost nothing after 12 h of culture in medium without sucrose. In contrast, the total free amino acid content of the cells increased as the culture proceeded. Among the free amino acids, phenylalanine and malic acid increased the most, followed by asparagine and alanine, whereas glutamic acid did not change significantly. These results are in agreement with previous studies using HPLC.¹H NMR spectroscopy enabled measurement of changes in the sugar and free amino acid content of whole cell extracts without fractionation and complicated sample preparation. These results indicate that comprehensive metabolic changes in the cells can be determined by a simple, rapid method using whole cell extracts and¹H NMR spectroscopy.     

Metabolic phenotyping of genetically modified mice: An NMR metabonomic approach

Monocyte chemoattractant protein-1 (MCP-1) plays a relevant role in macrophage migration but recent findings suggest an additional role in lipid and glucose metabolism. We report the use of ¹H NMR spectroscopy as a useful complementary method to assess the metabolic function of this gene in a comparative strategy. This metabonomic analysis was rapid, simple, quantitative and reproducible, and revealed a suggestive relationship between the expression of the MCP-1 gene and hepatic glucose and taurine concentrations. This approach should be considered in genetically modified mice when a metabolic alteration is suspected, or in routine assessment of metabolic phenotype.     

Sequential Main-Chain Proton and Carbon Resonance Assignments for Wild-Type Yeast Iso-1 Ferrocytochrome c and Identification of Secondary Structural Elements

Yeast iso-1 cytochrome c is a naturally occurring protein that possesses an unusually reactive Cysl02 that imbues iso-1 with a complicated solution chemistry which includes spontaneous dimerization and poorly characterized redox reactions. For this reason previous studies of this typical member of the c-type cytochromes have been relegated to variant proteins in which the 102 position has been mutated, with most common changes involving serine and threonine. However, we have determined sequential ¹H resonance assignments for the wild-type native protein because it is the actual participant in yeast mitochondrial electron transfer processes and because the wild-type native protein should be the fundamental assignment basis. In addition to ¹H resonance assignments for 97 of 106 amino acids, we have also provided an extensive database of long-range NOEs. Comparison of these NOEs and a chemical shift index based upon -H resonances has lead to identification of solution secondary structural elements that are consistent with the solid-state crystal structure. Although there is currently no efficient expression system that would facilitate isotope labeling of iso-1 cytochrome c, we tried to assess the usefulness of future heteronuclear experiments by using natural-abundance ¹H/¹³C HMQC experiments to unambiguously assign 35 -C resonances.     

Metabolomic analysis of methyl jasmonate treated Brassica rapa leaves by 2-dimensional NMR spectroscopy

The metabolomic analysis of Brassica rapa leaves treated with methyl jasmonate was performed using 2-dimensional J-resolved NMR spectroscopy combined with multivariate data analysis. The principal component analysis of the J-resolved NMR spectra showed discrimination between control and methyl jasmonate treated plants by principal components 1 and 2. While the level of glucose, sucrose and amino acids showed a decrease after methyl jasmonate treatment, hydroxycinnamates and glucosinolate were highly increased. Methyl jasmonate treatment resulted in a long-term accumulation of indole glucosinolate and indole-3-acetic acid, lasting up to 14 days after treatment. Malate conjugated hydroxycinnamates also exhibited an increase until 14 days after methyl jasmonate treatment, these compounds might play an important role in plant defence responses mediated by methyl jasmonate.     

Evidence of Metyrapone Reduction by two Mycobacterium strains shown by 1H NMR

In situ 1H NMR monitoring of metyrapone incubations with resting-cells of two strains of Mycobacterium, Mycobacterium aurum MO1 and Mycobacterium sp. RP1, showed the biotransformation of this compound, and more precisely the carbonyl-reduction of metyrapone into the corresponding alcohol, metyrapol. This reduction produced both enantiomers. The use of inhibitors allowed us to show the multiple enzymatic activities involved in this biotransformation including carbonyl reductase (EC 1.1.1.1.84) from the short-chain dehydrogenase superfamily and aldehyde reductase (EC 1.1.1.2) from the aldo-keto reductase superfamily.     

Comparison of the diastereoisomeric excess of uridine,inosine and adenosine cyanohydrins determined by HPLC-DAD and 1H NMR.

The separation of the diastereoisomers of the nucleoside derivatives of uridine, inosine and adenosine was performed by HPLC using chiral and no chiral columns, it was observed with the no chiral columns the resolution was good enough to determine diastereoisomeric excess. These methods were compared with ¹H NMR, and no significant differences were observed between the three techniques. Diastereoisomeric uridine (3a), inosine (3b) and adenosine (4c) cyanohydrins were resolved by ¹H nuclear magnetic resonance (¹H NMR), chiral normal phase-high-performance liquid chromatography-diode array detector (NP-HPLC-DAD) and reversed phase (RP-HPLC-DAD); these methods allowed the assesment of the percent diastereoisomeric excess (% de) of the nucleosidic cyanohydrins of 3a (4, 6 and 4), 3b (10, 8 and 6) and 4c (4, 4 and 4). To the best of our knowledge, there are no reports using analytical techniques for the separation of the epimers of 3a, 3b and 4c.     

Assessment of energy metabolism in the developing brain following aglycemic hypoxia by1H and31P NMR

The role played by external calcium and calcium channels in the recovery from aglycaemic hypoxia in cortical brain slices from 10-day old rats was investigated by¹H and³¹P NMR. 30 minutes of aglycaemic hypoxia significantly decreased the levels of phosphocreatine (PCr), ATP, lactate and intracellular pH (pH_i). After a 30 minute recovery period there was incomplete recovery of PCr and ATP with lactate increasing by 50% with pH_i normal. When the aglycemic hypoxia was carried out in media which had no added calcium (≈10 μM) the PCr and ATP recovery was significantly greater. Application of diltiazem or verapamil but not nifedipine significantly improved the recovery from the aglycemic hypoxia. These data suggest that calcium influx through L-type voltagegated calcium channels is involved in the ischemic damage in neonatal brain which manifests itself as a decrease in the energy state and an increase in lactate. Dedication This article is dedicated to our friend and colleague Herman Bachelard. We wish to thank him for his comradeship, advice and support over many years. Our hope for him is a long and fruiful retirement and that he will remain active in the neurosciences for many years, even though the establishment has blown for “full time”.     

On using oscillating time-dependent restraints in MD simulation

The use of time-dependent restraints in molecular simulation in order to generate a conformational ensemble for molecules that is in accordance with measured ensemble averages for particular observable quantities is investigated. Using a model system consisting of liquid butane and the cyclic peptide antamanide the reproduction of particular average ³ J-coupling constant values in a molecular dynamics simulation is analysed. It is shown that the multiple-valuedness and the sizeable gradients of the Karplus curve relating ³ J-coupling constants measured in NMR experiments to the corresponding torsional-angle values cause severe problems when trying to restrain a ³ J-coupling constant to a value close to the extrema of the Karplus curve. The introduction of a factor oscillating with time into the restraining penalty function alleviates this problem and enhances the restrained conformational sampling.     

High-resolution magic angle spinning 1H NMR analysis of whole cells of Thalassiosira pseudonana (Bacillariophyceae): Broad range analysis of metabolic composition and nutritional value

Chemical composition of the microalga Thalassiosira pseudonana Hasle & Heimdalwas studied with different proton nuclearmagnetic resonance (¹H NMR)techniques, and by comparing NMR spectrafrom extraction samples with a spectrumfrom a sample of whole cells we show thathigh-resolution magic angle spinning (HRMAS) ¹H NMR can be used for broadrange analysis of metabolic composition inmicroalgal whole cells. Signals fromimportant metabolites such aspolyunsaturated fatty acids (PUFAs)eicosapentaenoic (EPA) and docosahexaenoic(DHA) acids were seen in a ¹H NMRspectrum of lipophilic extract, andpossibly also signals from the carotenoidfucoxanthin. In a spectrum of hydrophilicextract we assigned signals to amino acidssuch as glutamine (Gln) and glutamic acid(Glu), carbohydrate and ATP. These findingswere compared to a spectrum of HR MAS¹H NMR analysis of whole cells, whereit was possible to find signals coincidentwith the different metabolites seen inspectra of the extraction samples. Sincethe position of resonance peaks in a NMRspectrum depends on the chemicalsurroundings of each atom at the time ofanalysis some peak shift differencesbetween extract and whole cell samplespectra may occur, but signal shifts werenot significantly different between theanalyses here. In addition, application ofHR MAS highly increased spectral resolutionin the complex whole cell sample. Wetherefore suggest that HR MAS ¹H NMRanalysis is a suitable analysis tool tostudy metabolic composition directly onwhole cells of microalgae, making itpossible to study a broad range ofmetabolites simultaneously without tediousextraction procedures.     

Synthesis and structural analysis of five novel oligosaccharides prepared by glucosyltransfer from beta-D-glucose 1-phosphate to isokestose and nystose using Thermoanaerobacter brockii kojibiose phosphorylase

Five novel oligosaccharides (tetra-, penta- and hexa-saccharides) were synthesized by glucosyltransfer from beta-D-glucose 1-phosphate to isokestose (O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-alpha-D-glucopyranoside) or nystose (O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-alpha-D-glucopyranoside) using Thermoanaerobacter brockii kojibiose phosphorylase. The oligosaccharides were identified as 2(2-alpha-D-glucopyranosyl)(m)isokestose; [O-alpha-D-glucopyranosyl-(1-->2)](m)-O-[beta-D-fructofuranosyl-(2-->1)](2)-alpha-D-glucopyranoside: m=1, 2, and 3, and 2(2-alpha-D-glucopyranosyl)(n)nystose; [O-alpha-D-glucopyranosyl-(1-->2)](n)-O-[beta-D-fructofuranosyl-(2-->1)](3)-alpha-D-glucopyranoside: n=1 and 2 using gas liquid chromatography analysis of the methyl derivatives, and MALDI-TOF-MS and NMR measurements of the newly formed oligosaccharides. 1H, 13C NMR signals of each saccharide were assigned using 2D-NMR techniques, including COSY, HSQC, HSQC-TOCSY, HMBC, CH(2)-selected E-HSQC, and CH(2)-selected E-HSQC-TOCSY.     

The solution conformation of sialyl-α(2→6)-lactose studied by modern NMR techniques and Monte Carlo simulations

Summary We present a comprehensive strategy for detailed characterization of the solution conformations of oligosaccharides by NMR spectroscopy and force-field calculations. Our experimental strategy generates a number of interglycosidic spatial constraints that is sufficiently large to allow us to determine glycosidic linkage conformations with a precision heretofore unachievable. In addition to the commonly used {¹H,¹H} NOE contacts between aliphatic protons, our constraints are: (a) homonuclear NOEs of hydroxyl protons in H₂O to other protons in the oligosaccharide, (b) heteronuclear {¹H,¹³C} NOEs, (c) isotope effects of O¹H/O²H hydroxyl groups on¹³C chemical shifts, and (d) long-range heteronuclear scalar coupling across glycosidic bonds.We have used this approach to study the trisaccharide sialyl-(26)-lactose in aqueous solution. The experimentally determined geometrical constraints were compared to results obtained from force-field calculations based on Metropolis Monte Carlo simulations. The molecule was found to exist in 2 families of conformers. The preferred conformations of the (26)-linkage of the trisaccharide are best described by an equilibrium of 2 conformers with angles at –60° or 180° and of the 3 staggered rotamers of the angle with a predominantgt conformer. Three intramolecular hydrogen bonds, involving the hydroxyl protons on C8 and C7 of the sialic acid residue and on C3 of the reducing-end glucose residue, contribute significantly to the conformational stability of the trisaccharide in aqueous solution. Supplementary material available from the corresponding author: Table containing values for the dihedral angles , , , , and for bond angles , for the six lowest-energy conformations of sialyl-(26)-lactose (1 page).     

Interaction of local anesthetics with small phospholipid vesicles investigated by proton NMR spectroscopy

The effects of the local anesthetics tetracaine, procaine (both charged at pH 6), and benzocaine (uncharged) on phospholipid liposomes have been investigated by 500 MHz ¹H NMR Spectroscopy. All the drugs reverse the Pr³⁺ induced shifts of phospholipid resonances in the same sequence as they are shifted by addition of Pr³⁺: choline POCH₂- > choline-CH₂N > choline-N(CH₃)₃ > glycerol > glycerol > acyl C₂ > acyl C₃. The drug effects result from incorporation of positive charges (tetracaine and procaine) and from the induction of a conformational change of the phospholipid head group via an action on the lipid glycerol backbone (benzocaine). From titration experiments with tetracaine on liposomes containing Pr³⁺ inside and outside is derived that the drug passes the bilayer by transverse diffusion. Tetracaine partitions outside/inside at a ratio of 21. Changes in linewidths of the drug resonances when incorporated into the liposomes allow the conclusion that the tetracaine molecule is located in an elongated way between the lipid acyl chains with its nitrogen group near the glycerol backbone. Benzocaine, showing strong effects on the line shapes of the protons on C₂ and C₃ of the lipid acyl chains is also located near the glycerol backbone, the region with the strongest hydrophobic forces.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 30), Cardiology.     

Metabolic Discrimination of Catharanthus roseus Calli According to Their Relative Locations Using 1H-NMR and Principal Component Analysis

Creating a plant-cell suspension culture involves first transferring the callus into liquid media, but there are no objective criteria for selecting the location of the callus to be transferred. In this study, inner and outer cells of Catharanthus roseus with various elicitors in solid-state cultures were differentiated by ¹H NMR (nuclear magnetic resonance) spectrometry and principal component analysis (PCA). It was found that the samples of various elicitors and relative locations could be separated in PCA-derived score plots. Especially, there was a clear separation between nontreated samples and those cotreated with silver nitrate and methyl jasmonate. Loading-plot analysis was therefore applied to data obtained from nontreated samples and those cotreated with silver nitrate and methyl jasmonate to determine the separation of major metabolites on score plots. The levels of valine, lactic acid, threonine, alanine, arginine, acetic acid, malic acid, succinic acid, citric acid, asparagine, choline, lactose, fumaric acid, phenylalanine, tryptophan, and formic acid were higher in the inner callus than in the outer callus, whereas 2-oxoglutaric acid, oxalacetic acid, sucrose, and glucose dominated in the outer callus. The results obtained in this study suggest that inner and outer calli can be differentiated by ¹H-NMR-based metabolomic analysis.     

Water uptake and oil distribution during imbibition of seeds of western white pine (<Emphasis Type=Italic>Pinus monticola</Emphasis> Dougl. ex D. Don) monitored in vivo using magnetic resonance imaging

Dry or fully imbibed seeds of western white pine (Pinus monticola Dougl. ex D. Don) were studied using high-resolution magnetic resonance imaging (MRI). Analyses of the dry seed revealed many of the gross anatomical features of seed structure. Furthermore, the non-invasive nature of MRI allowed for a study of the dynamics of water and oil distribution during in situ imbibition of a single seed with time-lapse chemical shift selective MRI. During soaking of the dry seed, water penetrated through the seed coat and megagametophyte. The cotyledons of the embryo (located in the chalazal end of the seed) were the first to show hydration followed by the hypocotyl and later the radicle. After penetrating the seed coat, water in the micropylar end of the seed likely also contributed to further hydration of the embryo; however, the micropyle itself did not appear to be a site for water entry into the seed. A model that describes the kinetics of the earlier stages of imbibition is proposed. Non-viable pine seeds captured with MRI displayed atypical imbibition kinetics and were distinguished by their rapid and uncontrolled water uptake. The potential of MR microimaging for detailed studies of water uptake and distribution during the soaking, moist chilling (stratification), and germination of conifer seeds is discussed.Electronic Supplementary Material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.     

Isolation and some properties of ciguatoxin

Ciguatoxin, the principal toxin of ciguatera produced by a toxic dinoflagellate and accumulated through the food chain in the viscera of moray eels, was purified. The probable molecular formula C₆₀H₈₆O₁₉ was obtained for the first time by high resolution mass spectrometry. ¹H NMR spectra suggested the presence of a primary hydroxyl group suitable for preparing an antigen necessary to develop an enzyme immunoassay. author for correspondence     

Equilibrium of the Cis-Trans Isomerisation of the Peptide Bond with N-alkyl Amino Acids Measured by 2D NMR

Summary The conformationalcis-trans equilibrium around the peptide bond in model tripeptides has been determined by 2D NMR methods (HOHAHA, ROESY). The study was limited to three different N-substituted amino acids in position 2, namely Pro (proline), Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), and N-MePhe (N-methylphenylalanine). In all cases the amino acid in position 1 was tyrosine and in position 3, phenylalanine. The results of our studies show that thecis-trans ratio depends mostly on the configuration of the amino acids forming the peptide bond undergoing thecis-trans isomerisation. The amino acid following the sequence (in position 3) does not have much influence on thecis-trans isomerisation, indicating that there is no interaction of the side chains between these amino acids. The model peptides with the L-Tyr-L-AA-(L-or D-)Phe (where AA is N-substituted amino acid) chiralities give 80–100% more of thecis form in comparison to the corresponding peptides with the D-Tyr-L-AA-(L-or D-)Phe chiralities. These results indicate that the incorporation of N-substituted amino acids in small peptides with the same chirality as the precedent amino acid involved in the peptide bound undergoing thecis/trans isomerisation moves the equilibrium to a significant amount of thecis form.     

Quantitative determination of metabolites in human urine by <Superscript>1</Superscript>H NMR spectroscopy

Conditions for registration of urinary ¹H NMR spectra have been optimized in order to achieve maximal accuracy of quantitative analysis. Urinary samples from patients with acute pancreatitis have been investigated and spectral data of identified urinary metabolites and results of their quantitative determination are given. Employment of ¹H NMR spectra is perspective for the development of new laboratory diagnostic methods.