1H NMR Metabolomics Reveals Contrasting Response by Male and Female Mussels Exposed to Reduced Seawater pH, Increased Temperature, and a Pathogen

Human activities are fundamentally altering the chemistry of the world's oceans. Ocean acidification (OA) is occurring against a background of warming and an increasing occurrence of disease outbreaks, posing a significant threat to marine organisms, communities, and ecosystems. In the current study, 1H NMR spectroscopy was used to investigate the response of the blue mussel, Mytilus edulis, to a 90-day exposure to reduced seawater pH and increased temperature, followed by a subsequent pathogenic challenge. Analysis of the metabolome revealed significant differences between male and female organisms. Furthermore, males and females are shown to respond differently to environmental stress. While males were significantly affected by reduced seawater pH, increased temperature, and a bacterial challenge, it was only a reduction in seawater pH that impacted females. Despite impacting males and females differently, stressors seem to act via a generalized stress response impacting both energy metabolism and osmotic balance in both sexes. This study therefore has important implications for the interpretation of metabolomic data in mussels, as well as the impact of environmental stress in marine invertebrates in general.

Unusual 1H NMR chemical shifts support (His) Cɛ1—H⋅⋅⋅O⩵C H-bond: Proposal for reaction-driven ring flip mechanism in serine protease catalysis

13C-selective NMR, combined with inhibitor perturbation experiments, shows that the Cɛ1—H proton of the catalytic histidine in resting α-lytic protease and subtilisin BPN′ resonates, when protonated, at 9.22 ppm and 9.18 ppm, respectively, which is outside the normal range for such protons and ≈0.6 to 0.8 ppm further downfield than previously reported. They also show that the previous α-lytic protease assignments [Markley, J. L., Neves, D. E., Westler, W. M., Ibanez, I. B., Porubcan, M. A. & Baillargeon, M. W. (1980) Front. Protein Chem. 10, 31–61] were to signals from inactive or denatured protein. Simulations of linewidth vs. pH demonstrate that the true signal is more difficult to detect than corresponding signals from inactive derivatives, owing to higher imidazole pKa values and larger chemical shift differences between protonated and neutral forms. A compilation and analysis of available NMR data indicates that the true Cɛ1—H signals from other serine proteases are similarly displaced downfield, with past assignments to more upfield signals probably in error. The downfield displacement of these proton resonances is shown to be consistent with an H-bond involving the histidine Cɛ1—H as donor, confirming the original hypothesis of Derewenda et al. [Derewenda, Z. S., Derewenda, U. & Kobos, P. M. (1994) J. Mol. Biol. 241, 83–93], which was based on an analysis of literature x-ray crystal structures of serine hydrolases. The invariability of this H-bond among enzymes containing Asp-His-Ser triads indicates functional importance. Here, we propose that it enables a reaction-driven imidazole ring flip mechanism, overcoming a major dilemma inherent in all previous mechanisms, namely how these enzymes catalyze both the formation and productive breakdown of tetrahedral intermediates.

Dimethylsulfoxide oxidizes glutathione in vitro and in human erythrocytes:kinetic analysis by 1H NMR

The interaction of dimethylsulfoxide (Me2SO) with glutathione was investigated under non-equilibrium conditions in solution using 1H NMR and in intact erythrocytes using 1H spin-echo NMR. In solution the reaction was observed to follow second-order kinetics (Rate = k1[glutathione][Me2SO]) at 300 K pH 7.4, ksol = 4.7 × 10-5 mol -1 L1 s-1. In intact erythrocytes the rate constant for the cellular environment, kcell, was found to be slightly larger at 8.1 × 10-5 mol-1 L1 s-1. Furthermore, the reaction of Me2SO with erythrocyte glutathione showed a biphasic dependence on the Me2SO concentration, with little oxidation of glutathione occurring until the Me2SO concentration exceeded 0.5 mol L-1. The results suggest that at lower concentrations, Me2SO can be effectively removed, most probably by reaction with glutathione, which is regenerated by glutathione reductase, although preferential reaction with other cellular components (e.g., membrane or cellular thiols) cannot be ruled out. Thus the concentrations of Me2SO that are commonly used in cryopreservation of mammalian cells (∼1.4 mol L-1) can cause oxidation of intracellular glutathione.

Metabolic signatures of Huntington's disease (HD): 1H NMR analysis of the polar metabolome in post mortem human brain

Huntington’s disease (HD) is an autosomal neurodegenerative disorder affecting approximately 5-10 persons per 100,000 worldwide. The pathophysiology of HD is not fully understood but the age of onset is known to be highly dependent on the number of CAG triplet repeats in the huntingtin gene. Using 1H NMR spectroscopy this study biochemically profiled 39 brain metabolites in post-mortem striatum (n=14) and frontal lobe (n=14) from HD sufferers and controls (n=28). Striatum metabolites were more perturbed with 15 significantly affected in HD cases, compared with only 4 in frontal lobe (P<0.05; q<0.3). The metabolite which changed most overall was urea which decreased 3.25-fold in striatum (P<0.01). Four metabolites were consistently affected in both brain regions. These included the neurotransmitter precursors tyrosine and L-phenylalanine which were significantly depleted by 1.55-1.58-fold and 1.48-1.54-fold in striatum and frontal lobe, respectively (P=0.02-0.03). They also included L-leucine which was reduced 1.54-1.69-fold (P=0.04-0.09) and myo-inositol which was increased 1.26-1.37-fold (P<0.01). Logistic regression analyses performed with MetaboAnalyst demonstrated that data obtained from striatum produced models which were profoundly more sensitive and specific than those produced from frontal lobe. The brain metabolite changes uncovered in this first 1H NMR investigation of human HD offer new insights into the disease pathophysiology. Further investigations of striatal metabolite disturbances are clearly warranted.

Photochromism of the complex between 40-(2-hydroxyethoxy)-7-hydroxyflavylium and b-cyclodextrin, studied by 1H NMR, UVeVis, continuous irradiation and circular dichroism

1st International Caparica Conference on Chromogenic and Emissive Materials

Use of chemometrics to characterize tropical wines from different vintages and grape cultivars according to the 1H NMR spectroscopy data.

2011

Metabolic fingerprint of açaí (Euterpe oleracea) berry pulp using 1H NMR spectroscopy and chemometric analysis.

Portanto, o objetivo deste trabalho foi avaliar a influência de diferentes cultivares e sazonalidade do açaí utilizando a técnica de 1H NMR para criação de perfis de impressão digital associada a métodos quimiométricos, obtidos a partir de frutos geneticamente modificados (cultivada no controle agronômico) e o comércio na cidade de Belém

Spectroscopic correlation analysis of NMR-based metabonomics in exercise science

Spectroscopic studies of complex clinical fluids have led to the application of a more holistic approach to their chemical analysis becoming more popular and widely employed. The efficient and effective interpretation of multidimensional spectroscopic data relies on many chemometric techniques and one such group of tools is represented by so-called correlation analysis methods. Typical of these techniques are two-dimensional correlation analysis and statistical total correlation spectroscopy (STOCSY). Whilst the former has largely been applied to optical spectroscopic analysis, STOCSY was developed and has been applied almost exclusively to NMR metabonomic studies. Using a 1H NMR study of human blood plasma, from subjects recovering from exhaustive exercise trials, the basic concepts and applications of these techniques are examined. Typical information from their application to NMR-based metabonomics is presented and their value in aiding interpretation of NMR data obtained from biological systems is illustrated. Major energy metabolites are identified in the NMR spectra and the dynamics of their appearance and removal from plasma during exercise recovery are illustrated and discussed. The complementary nature of two-dimensional correlation analysis and statistical total correlation spectroscopy are highlighted.

A H-1-NMR STUDY OF BILIRUBIN IX-ALPHA SOLUBILIZATION BY CHOLATE MICELLES - APPLICATION OF NUCLEAR OVERHAUSER EFFECTS

The solubilization of bilirubin IX-Alpha in aqueous solution by sodium cholate micelles has been examined by 270 MHz 1H-NMR spectroscopy. Incorporation of bilirubin into the micelles is accompanied by specific shifts of bilirubin vinyl and bridgehead protons and the C18 and C19 methyl groups of the steroid. The observed chemical shifts show a monotonic concentration dependence suggesting that changes in aggregation size are continuous. Nuclear Overhauser effects (NOE) have been shown to be a useful probe or micellization. A 4:1 cholate/bilirubin mixture has been investigated by difference NOE spectroscopy. The observation of intermolecular nuclear Overhauser effects between peripheral protons of bilirubin and cholate are diagnostic of spatially proximate groups. Inter-cholate nuclear Overhauser effects increase in magnitude upon bilirubin incorporation suggesting closer packing of steroid molecules on solubilization of the pigment. Intramolecular nuclear Overhauser effects observed for solubilized bilirubin are consistent with a compact intramolecularly hydrogen-bonded conformation resembling that determined for bilirubin in the solid state.

NMR relaxation study of disorder in the mixed system BP x GPI(1-x) and the low temperature transition from classical to quantum rotation

1H NMR spin-lattice relaxation time (T1) studies have been carried out in the temperature range 100 K to 4 K, at two Larmor frequencies 11.4 and 23.3 MHz, in the mixed system of betaine phosphate and glycine phosphite (BPxGPI(1-x)), to study the effects of disorder on the proton group dynamics. Analysis of T1 data indicates the presence of a number of inequivalent methyl groups and a gradual transition from classical reorientations to quantum tunneling rotations. At lower temperatures, microstructural disorder in the local environments of the methyl groups, result in a distribution in the activation energy (Ea) and the torsional energy gap (E01). For certain values of x, the magnetisation recovery shows biexponential behaviour at lower temperatures.

High-Pressure Nmr And Compressibility Evidence For A Phase-Transition In The Protonic Conductor (NH4)4Fe(CN)6.1.5H2o

1H NMR at high hydrostatic pressures and compressibility studies show that the protonic conductor (NH4)4Fe(CN)6·1.5H2O undergoes a phase transition around 0.45 GPa. The transition is characterized by a large hysteresis. From the NMR studies, an activation volume of 6% is obtained below the phase transition, indicating the dominance of Frenkel defects.

A 13C-NMR study of non-planar distortions in amides

The technique of 13C-NMR spectroscopy of oriented systems to problems of biological importance has been suggested and used to investigate non-planar distortions in substituted amides—models for peptides. The studies in conjunction with the proton magnetic resonance data on 5N-[13C]methyl[13C]formamide oriented in a nematic solvent provide all the direct dipolar couplings between the interacting nuclei in the system. When the 13C- and the 1H-NMR experiments are performed under non-identical conditions, 22 different direct dipolar couplings are obtained. It is demostrated that they can be used to determine unambiguously non-planar distortions around the nitrogen atom together with other geometrical data and the molecular order.

Synthesis and biological evaluation of novel 1-(4-methoxyphenethyl)-1H-benzimidazole-5-carboxylic acid derivatives and their precursors as antileukemic agents

We report here the synthesis and preliminary evaluation of novel 1-(4-methoxyphenethyl)-1H-benzimidazole-5-carboxylic acid derivatives 6(a–k) and their precursors 5(a–k) as potential chemotherapeutic agents. In each case, the structures of the compounds were determined by FTIR, 1H NMR and mass spectroscopy. Among the synthesized molecules, methyl 1-(4-methoxyphenethyl)-2-(4-fluoro-3-nitrophenyl)-1H-benzimidazole-5-carboxylate (5a) induced maximum cell death in leukemic cells with an IC50 value of 3 μM. Using FACS analysis we show that the compound 5a induces S/G2 cell cycle arrest, which was further supported by the observed down regulation of CDK2, Cyclin B1 and PCNA. The observed downregulation of proapoptotic proteins, upregulation of antiapoptotic proteins, cleavage of PARP and elevated levels of DNA strand breaks indicated the activation of apoptosis by 5a. These results suggest that 5a could be a potent anti-leukemic agent.