Leishmania donovani: Metabolite mapping of promastigotes using proton nuclear magnetic resonance spectroscopy

Proton nuclear magnetic resonance spectroscopy was used for studying the intracellular metabolite profile of promastigotes of Leishmania donovani. The major intracellular metabolites observed in the promastigotes were acetate, alanine, succinate, glycine, -glycerophosphorylcholine, acetoacetate, arginine and ethanol. A comparative study of the intracellular metabolite profile of promastigotes of different strains of L. donovani showed that, all the major intracellular metabolites were present in promastigotes of different strains. A quantitative estimation of metabolites showed a strain specific (Finger print) metabolite profile which can be used for strain/species identification/differentiation.     

Online coupling of enantioselective capillary gas chromatography with proton nuclear magnetic resonance spectroscopy

The hyphenation of enantioselective capillary gas chromatography and mass spectrometry is not always sufficient to distinguish between structural isomers, thus requiring peak identification by NMR spectroscopy. Here the first online coupling of enantioselective capillary gas chromatography with proton nuclear resonance spectroscopy is described for the unfunctionalized chiral alkane 2,4‐dimethylhexane resolved on octakis(6‐O‐methyl‐2,3‐di‐O‐pentyl)‐γ‐cyclodextrin at 60°C. NMR allows constitutional and configurational isomers (diastereomers and enantiomers) to be distinguished. Enantiomers display identical spectra at different retention times, which enable an indirect identification of these unfunctionalized alkanes. The presented method is still at an early development stage, and will require instrumental optimization in the future. Chirality 2010. © 2010 Wiley‐Liss, Inc.     

Metabolic differentiation of Arabidopsis treated with methyl jasmonate using nuclear magnetic resonance spectroscopy

NMR spectroscopy combined with principal component analysis was applied to Arabidopsis thaliana treated with methyl jasmonate in order to obtain macroscopic metabolic changes caused by the treatment. As the first step several chromatographic and NMR spectroscopic techniques were utilized to identify metabolites of Arabidopsis. Sephadex LH-20 showed a high efficiency in the separation of phenolic metabolites in the plant. For identification of minor metabolites two-dimensional J-resolved NMR technique was directly applied to the plant extract and results in a number of elucidation of the metabolites of which signals overlap in ¹H NMR spectra. The chemical structure of the identified metabolites were confirmed by various two-dimensional NMR spectroscopy including correlated spectroscopy, heteronuclear single quantum coherence, and heternuclear multiple bond correlation. As next step, a statistical approach, principal component analysis based on projected J-resolved NMR spectra was performed for metabolic alteration of methyl jasmonate-treated Arabidopsis. The results show that methyl jasmonate caused an increase of flavonoids, fumaric acid, sinapoyl malate, sinigrin, tryptophan, valine, threonine, and alanine and a decrease of malic acid, feruloyl malate, glutamine, and carbohydrates after 24 h treatment.     

Grade classification of neuroepithelial tumors using high-resolution magic-angle spinning proton nuclear magnetic resonance spectroscopy and pattern recognition

Clinical data have shown that survival rates vary considerably among brain tumor patients, according to the type and grade of the tumor. Metabolite profiles of intact tumor tissues measured with high-resolution magic-angle spinning proton nuclear magnetic resonance spectroscopy (HRMAS (1)H NMRS) can provide important information on tumor biology and metabolism. These metabolic fingerprints can then be used for tumor classification and grading, with great potential value for tumor diagnosis. We studied the metabolic characteristics of 30 neuroepithelial tumor biopsies, including two astrocytomas (grade I), 12 astrocytomas (grade II), eight anaplastic astrocytomas (grade III), three glioblastomas (grade IV) and five medulloblastomas (grade IV) from 30 patients using HRMAS (1)H NMRS. The results were correlated with pathological features using multivariate data analysis, including principal component analysis (PCA). There were significant differences in the levels of N-acetyl-aspartate (NAA), creatine, myo-inositol, glycine and lactate between tumors of different grades (P<0.05). There were also significant differences in the ratios of NAA/creatine, lactate/creatine, myo-inositol/creatine, glycine/creatine, scyllo-inositol/creatine and alanine/creatine (P<0.05). A soft independent modeling of class analogy model produced a predictive accuracy of 87% for high-grade (grade III-IV) brain tumors with a sensitivity of 87% and a specificity of 93%. HRMAS (1)H NMR spectroscopy in conjunction with pattern recognition thus provides a potentially useful tool for the rapid and accurate classification of human brain tumor grades.     

Chemical proteomics from a nuclear magnetic resonance spectroscopy perspective

Proteomics is the study of the protein complement of a genome and employs a number of newly emerging tools. One such tool is chemical proteomics, which is a branch of proteomics devoted to the exploration of protein function using both in vitro and in vivo chemical probes. Chemical proteomics aims to define protein function and mechanism at the level of directly observed protein–ligand interactions, whereas chemical genomics aims to define the biological role of a protein using chemical knockouts and observing phenotypic changes. Chemical proteomics is therefore traditional mechanistic biochemistry performed in a systems-based manner, using either activity- or affinity-based probes that target proteins related by chemical reactivities or by binding site shape/properties, respectively. Systems are groups of proteins related by metabolic pathway, regulatory pathway or binding to the same ligand. Studies can be based on two main types of proteome samples: pooled proteins (1 mixture of N proteins) or isolated proteins in a given system and studied in parallel (N single protein samples). Although the field of chemical proteomics originated with the use of covalent labeling strategies such as isotope-coded affinity tagging, it is expanding to include chemical probes that bind proteins noncovalently, and to include more methods for observing protein–ligand interactions. This review presents an emerging role for nuclear magnetic resonance spectroscopy in chemical proteomics, both in vitro and in vivo. Applications include: functional proteomics using cofactor fingerprinting to assign proteins to gene families; gene family-based structural characterizations of protein–ligand complexes; gene family-focused design of drug leads; and chemical proteomic probes using nuclear magnetic resonance SOLVE and studies of protein–ligand interactions in vivo.     

Assessment of alterations in X-ray irradiation-induced DNA damage of glioma cells by using proton nuclear magnetic resonance spectroscopy

Glioma is one of the most common types of brain tumors. DNA damage is closely associated with glioma cell apoptosis induced by X-ray irradiation. Alterations of metabolites in glioma can be detected noninvasively by proton nuclear magnetic resonance (1H NMR) spectroscopy. To noninvasively explore the micro mechanism in X-ray irradiation-induced apoptosis, the relationship between metabolites and DNA damage in glioma cells was investigated. Three glioma cell lines (C6, U87 and U251) were randomly designated as control (0 Gy) and treatment groups (1, 5, 10, 15 Gy). After X-ray exposure, each group was separated into four parts: (i) to detect metabolites by 1H NMR spectroscopy; (ii) to make cell colonies; (iii) to detect cell cycle distribution and apoptosis rate by flow cytometry; and (iv) to measure DNA damage by comet assay. The metabolite ratios of lactate/creatine and succinate/creatine decreased (lactate/creatine: C6, 22.17–66.27%; U87, 15.93–44.56%; U251, 26.27–74.48%. succinate/creatine: C6, 14.41–48.35%; U87, 22.03–70.62%; U251, 17.33–60.06%) and choline/creatine increased (C6, 52.22–389.68%; U87, 56.15–82.36%; U251, 31.87–278.62%) in the treatment groups compared with the control group (each P < 0.05), which linearly depended on DNA damage. An increasing dose of X-ray irradiation increased numbers of apoptotic cells (P < 0.01), and the DNA damage parameters were dose-dependent (P < 0.05). The colony-forming rate declined (P < 0.01) and the percentage of cells at G1 stage increased when exposed to 1 Gy X-ray (three cell lines, P < 0.05). Metabolite alterations detected by 1H NMR spectroscopy can be used to determine DNA damage induced by X-ray irradiation. 1H NMR spectroscopy is a noninvasive method to predict DNA damage of glioma cell at the micro level.     

Resolution and sensitivity of high field nuclear magnetic resonance spectroscopy

NMR frequency assignments are usually considered a prerequisite for the analysis of NOESY spectra, in turn required for the calculation of biomolecular structures. In contrast, as we propose here, relatively high numbers of unambiguous NOE identities can be consistently achieved in an automated manner by relying only on grouping resonances into connected spin systems. To achieve this goal, we have developed for proteins two protocols, SPI and BACUS, based on Bayesian inference. SPI (Grishaev and Llinás, 2002c) produces a list of the (1)H resonance frequencies from homo- and hetero-nuclear multidimensional spectra, grouped into effective spin systems. BACUS automatically establishes probabilistic identities of NOESY cross-peaks in terms of the chemical shifts provided by SPI. BACUS requires neither assignment of resonances nor an initial structural model. It successfully copes with chemical shift overlap and does so without cycling through 3D structure calculations. The method exploits the self-consistency of the NOESY graph by taking advantage of a network of J- as well as NOE-connected "reporter" protons sorted via SPI. BACUS was validated by tests on experimental NOESY data recorded for the col 2 and kringle 2 domains.     

Rapid determination of sugar content in biomass hydrolysates using nuclear magnetic resonance spectroscopy

Large populations of potential cellulosic biomass feedstocks are currently being screened for fuel and chemical applications. The monomeric sugar content, released through hydrolysis, is of particular importance and is currently measured with time‐consuming HPLC methods. A method for sugar detection is presented here that employs ¹H NMR spectra regressed against primary HPLC sugar concentration data to build partial least squares (PLS) models. The PLS2 model is able to predict concentrations of both major sugar components, like glucose and xylose, and minor sugars, such as arabinose and mannose, in biomass hydrolysates. The model was built with 65 samples from a variety of different biomass species and covers a wide range of sugar concentrations. Model predictions were validated with a set of 15 samples which were all within error of both HPLC and NMR integration measurements. The data collection time for these NMR measurements is less than 20 min, offering a significant improvement to the 1 h acquisition time that is required for HPLC. Biotechnol. Bioeng. 2013; 110: 721–728. © 2012 Wiley Periodicals, Inc.     

Complex glycosaminoglycans: profiling substitution patterns by two-dimensional nuclear magnetic resonance spectroscopy

Biological and pharmacological interactions of heparin and structurally related glycosaminoglycans (GAGs) such as heparan sulfate (HS) involve complex sequences of variously sulfated uronic acid and aminosugar residues. Due to their structural microheterogeneity, these sequences are usually characterized in statistical terms, by high-performance liquid chromatographic analysis of fragments obtained by enzymatic or chemical degradation. Nuclear magnetic resonance (NMR) spectroscopy is also currently used for structural characterization of GAGs. However, the use of monodimensional NMR analysis of complex GAGs is often limited by severe signal overlap that does not allow reliable quantitative measurements. Using magnetically equivalent signals, the higher resolution achieved by two-dimensional NMR methods could be also exploited for quantitative applications. In this work, heteronuclear single quantum coherence (HSQC) spectroscopy has been evaluated to determine variously substituted monosaccharide components of HS and HS mimics obtained by chemical modification of the Escherichia coli K5 polysaccharide (K5-PS) structurally related to the common biosynthetic precursor of heparin and HS. Heparin was used as a model for assessing the influence of 1H-13C spin-spin couplings on "volumes" of the corresponding signals. For major signals, the HSQC approach permitted quantification of additional structural features both in heparins and in a typical HS. The method was applied to profile the substitution patterns of K5-PS derivatives involving different degrees of N,O-sulfation and N-acetylation, including O-sulfated heparosans bearing free amino groups.     

The identification of metal-binding ligand residues in metalloproteins using nuclear magnetic resonance spectroscopy.

The identification of metal-binding ligands in metalloproteins is an important step in gaining detailed information regarding the environment of the active site. Traditionally, techniques such as 13Cd-substitution for the active metal followed by isotope-filtered NMR techniques have been used to this end. However, for medium to high molecular weight proteins (>20 kDa), these experiments may not be beneficial due to extensive 1H spectral overlap. Here, we describe an alternative approach, where metal-binding ligands such as histidine and cysteine are specifically 15N backbone labeled, excess EDTA is added and changes to (1H-15N) HSQC spectra are followed. Under these conditions, the amide groups of all 15N labeled histidine and cysteine residues, which were either ligands or residues close to the active site, were identified unambiguously for metallo-beta-lactamase from Bacteroides fragilis.     

Intra-abdominal fat burden discriminated in vivo using proton magnetic resonance spectroscopy

Objective: To assess proton magnetic resonance spectroscopy (¹H‐MRS) as a means to distinguish among mice with disparate intra‐abdominal body fat compositions, and to measure changes in intra‐abdominal fat burden during weight loss and regain. Research Methods and Procedures: Intra‐abdominal fat burden was analyzed as a ratio of integrated areas under the curves of fat to water ¹H‐MRS signals collected from a region of interest standardized across B6.V‐Lep^ob, C57BL/6, and A‐ZIP/F mice that exhibited various genotypically related body fat compositions, ranging from obese (B6.V‐Lep^ob) to minimal body fat (A‐ZIP/F). ¹H‐MRS analysis of fat burden was compared with intra‐abdominal fat volume and with a single cross‐sectional intra‐abdominal fat area calculated from segmented magnetic resonance images. Similar measurements were made from obese B6.V‐Lep^ob mice before, during, and after they were induced to lose weight by leptin administration. Results: Relative amounts of intra‐abdominal fat analyzed by ¹H‐MRS differed significantly according to body composition and genotype of the three strains of mice (p < 0.05). Intra‐abdominal fat assessed by ¹H‐MRS correlated with both intra‐abdominal fat volume (r = 0.88, p < 0.001) and body weight (r = 0.82, p < 0.001) among, but not within, all three genotypes. During weight loss and regain, there was a significant overall pattern of changes in intra‐abdominal fat quantity that occurred, which was reflected by ¹H‐MRS (p = 0.006). Discussion: Results support the use of localized ¹H‐MRS for assessing differences in intra‐abdominal fat. Refinements in ¹H‐MRS voxel region of interest size and location as well as instrument precision may result in improved correlations within certain body compositions.     

A proton nuclear magnetic resonance study of the interaction of cadmium with human erythrocytes

The binding of Cd²⁺ by molecules in the intracellular region of human erythrocytes has been studied by ¹H-NMR spectroscopy. From changes in spin-echo Fourier transform NMR spectra for both intact and hemolyzed erythrocytes to which CdCl₂ was added, direct evidence was obtained for the binding of Cd²⁺ by intracellular glutathione and hemoglobin. Time-courses were measured by ¹H-NMR for the uptake of Cd²⁺ by intact erythrocytes in saline/glucose solution and in whole blood. In both cases, the uptake, as indicated by changes in the ¹H-NMR spectrum for intracellular glutathione, plateaus after about 30 min. The effectiveness of the disodium salt of EDTA and of various thiol-chelating agents for releasing glutathione from its Cd²⁺ complexes in hemolyzed erythrocytes was also studied. EDTA was found to be more effective than thiols, and dithiols more effective than monothiols.     

A proton nuclear magnetic resonance study of gamma-glutamyl-amino acid cyclotransferase in human erythrocytes

Spin-echo NMR spectroscopy was shown to be a reliable technique for the monitoring of the in situ cleavage of gamma-Glu-Ala by gamma-glutamyl-amino acid cyclotransferase in whole erythrocytes and hemolysates. Of particular importance was the difference in chemical shifts between peptide resonances and those of the constituent amino acids. Using lysates of varying dilution, it was shown that the specific activity of the enzyme was not concentration-dependent, thus suggesting a lack of cytosolic low-molecular-weight-effectors or enzyme dissociation. Furthermore, the initial velocities of the reaction as a function of substrate concentration obeyed Michaelis-Menten kinetics with a Km = 2.0 +/- 0.3 mmol/l and Vmax = 137 +/- 7 mmol/h/l of cell water in 1H2O medium. Similar analysis in 2H2O medium revealed a solvent kinetic isotope effect of 1.9 +/- 0.4 at low substrate concentrations. The implications of this observation for the mechanism of the reaction are discussed. Cleavage of the peptide by a suspension of intact erythrocytes was at a rate 300 times less than the corresponding lysate flux, thus indicating the rate limitation by transport in the coupled system.     

Observations of aerobic, growing escherichia coli metabolism using an on-line nuclear magnetic resonance spectroscopy system

An experimental system has been constructed which enables on-line measurements of phosphorus-31 ((31)P) nuclear magnetic resonance (NMR) spectra for growing bacterial suspensions under anaerobic or aerobic conditions. A sample stream from a laboratory bioreactor is circulated to the NMR sample chamber in a gas exchange system which permits maintenance of aerobic conditions for high-cell-density cultures. (31)P NMR spectra with resolution comparable with those obtained traditionally using dense, concentrated, nongrowing cell suspensions can be obtained at cell densities above 25 g/L with acquisition times ranging from 14 to 3 minutes which decline as cell density increases. This system has been employed to characterize the changes in intracellular state of a stationary phase culture which is subjected to a transition from aerobic to anaerobic conditions. Both intracellular NTP level and cytoplasmic pH are substantially lower under anaerobic conditions. Also, the system has been employed to observe the response of a growing culture to external addition of acetate. Cells are able to maintain pH difference across the cytoplasmic membrane at extracellular acetate concentrations of 5 and 10 g/L. However, acetate concentrations of 20 g/L cause collapse of the transmembrane DeltapH and sharp reduction of the growth rate of the culture. The experimental configuration described should also permit NMR observations of many other types of microbial cultures and of other nuclei. (c) 1993 John Wiley & Sons, Inc.     

Aluminum-mediated metabolic changes in rat serum and urine: a proton nuclear magnetic resonance study

The toxic effects of Al(3+) have been studied in 90-days AlCl(3) orally treated male albino rats (n = 7) using (1)H NMR spectroscopy-based metabolic profile of rat serum and urine, serum enzyme tests, behavioral impairment, and histopathology of kidney and liver. Metabolic profile of 90-days Al(3+)-treated rat sera showed significantly elevated levels of alanine, glutamine, beta-hydroxy-butyrate, and acetoacetate and significantly decreased level of acetone when compared with that of control rats. However, metabolic profile of 90-days Al(3+)-treated rat urine showed significantly decreased levels of citrate, creatinine, allantoin, trans-aconitate, and succinate and significantly increased level of acetate when compared to control rats. The overall perturbations observed in the metabolic profile of serum and urine demonstrate the impairment in the tricarboxylic acid cycle, liver and kidney metabolism, which was further reinstated by clinical chemistry and histopathological observations. Moreover, "in vivo" behavioral impairment has also been observed as the indication of aluminum neurotoxicity.     

Lipid profiles of breast cancer cell lines: proton nuclear magnetic resonance spectroscopy

Nuclear magnetic resonance spectroscopy was performed on breast cancer cell lines MCF-7, MDA-MB231 and T47D. Proton spectra showed discrepancies of lipid quantity in the different lines. The high resolution lines of lipids were not as intense in the membrane preparations. These results show the potential of NMR spectroscopy to study the involvement of membrane lipids in proliferation, metastasis or drug resistance process.     

Examination of primary metabolic pathways in a murine hybridoma with carbon-13 nuclear magnetic resonance spectroscopy

Primary metabolism of a murine hybridoma was probed with (13)C nuclear magnetic resonance (NMR) spectroscopy. Cells cultured in a hollow fiber bioreactor were serially infused with [1-(13)C] glucose, [2-(13)C] glucose, and [3-(13)C] glutamine. In vivo spectroscopy of the culture was used in conjunction with off-line spectroscopy of the medium to determine the intracellular concentration of several metabolic intermediates and to determine fluxes for primary metabolic pathways. Intracellular concentrations of pyruvate and alanine were very high relative to levels observed in normal quiescent mammalian cells. Estimates made from labeling patterns in lactate indicate that 76% of pyruvate is derived directly from glycolysis; some is also derived from the malate shunt, the pyruvate/melate shuttle associated with lipid synthesis and the pentose phosphate pathway. The rate of formation of pyruvate from the pentose phosphate pathway was estimated to be 4% of that from glycolysis; This value is a lower limit and the actual value may be higher. Incorporation of pyruvate into the tricarboxylic acid (TCA) cycle appears to occur through only pyruvate dehydrogenase; no pyruvate carboxylase activity was detected. The malate shunt rate was approximately equal to the rate of glutamine uptake. The rate of incorporation of glucosederived acetyl-CoA into lipids was 4% of the glucose uptake rate. The TCA cycle rate between isocitrate and alpha-ketoglutarate was 110% of the glutamine uptake rate. (c) 1994 John Wiley & Sons, Inc.     

A proton nuclear magnetic resonance study of the interaction of mercury with intact human erythrocytes

The binding of mercuric ion (Hg(II)) by small molecules in the intracellular region of intact human erythrocytes has been studied by ¹H-NMR spectroscopy. HgCl₂ added to intact erythrocytes in saline-glucose suspension is found to cross the membrane and reach an equilibrium distribution among the molecules of the erythrocyte within 4 min. In the intracellular region Hg(II) reacts with GSH and hemoglobin to form the ternary mixed-ligand complex GSH-Hg(II)-hemoglobin. The analogous complex with ergothioneine is formed after all the GSH is complexed. ¹H-NMR spectra show that the GSH-Hg(II)-hemoglobin complex also forms in simpler solutions containing HgCl₂, GSH and hemoglobin, whereas the complex Hg(GSH)₂ predominates in solutions of GSH and HgCl₂. The lifetime of the GSH in the GSH-Hg(II)-hemoglobin complex is shown to be less than 30 s, which provides direct evidence for the first time that Hg(II) complexes in biological systems are quite labile, even though their thermodynamic stability is large. The effectiveness of eight sulfhydryl-containing ligands, some of which have been used as antidotes for Hg(II) poisoning, for releasing GSH from its Hg(II) complex in hemolyzed erythrocytes was also studied. Dithiol ligands were found to be more effective than monothiols, with dithioerythritol the most effective of the dithiols.     

Determination of the three-dimensional solution structure of barnase using nuclear magnetic resonance spectroscopy

The solution conformation of the ribonuclease barnase has been determined by using 1H nuclear magnetic resonance (NMR) spectroscopy. The 20 structures were calculated by using 853 interproton distance restraints obtained from analyses of two-dimensional nuclear Overhauser spectra, 72 phi and 53 chi 1 torsion angle restraints, and 17 hydrogen-bond distance restraints. The calculated structures contain two alpha-helices (residues 6-18 and 26-34) and a five-stranded antiparallel beta-sheet (residues 50-55, 70-75, 85-91, 94-101, and 105-108). The core of the protein is formed by the packing of one of the alpha-helices (residues 6-18) onto the beta-sheet. The average RMS deviation between the calculated structures and the mean structure is 1.11 A for the backbone atoms and 1.75 A for all atoms. The protein is least well-defined in the N-terminal region and in three large loops. When these regions are excluded, the average RMS deviation between the calculated structures and the mean structure for residues 5-34, 50-56, 71-76, 85-109 is 0.62 A for the backbone atoms and 1.0 A for all atoms. The NMR-derived structure has been compared with the crystal structure of barnase [Mauguen et al. (1982) Nature (London) 297, 162-164].     

A proton nuclear magnetic resonance study of the binding of methylmercury in human erythrocytes

The binding of methylmercury, CH₃Hg(II), by small molecules in the intracellular region of human erythrocytes has been studied by ¹H-NMR spectroscopy. To suppress or completely eliminate interfering resonances from the much more abundant hemoglobin protons, spectra were measured by a technique based on the transfer of saturation throughout the envelope of hemoglobin resonances following a selective presaturation pulse or by the spin-echo Fourier transform method. With these techniques, ¹H-NMR spectra were measured for the more abundant intracellular small molecules, including glycine, alanine, creatine, lactic acid, ergothioneine and glutathione, in both intact and hemolyzed erythrocytes to which CH₃Hg(II) had been added. The results for intact erythrocytes indicate that part of the CH₃Hg(II) is complexed by intracellular glutathione. These results also indicate that exchange of CH₃Hg(II) among glutathione molecules is fast, with the average lifetime of a CH₃Hg(II)-glutathione complex estimated to be less than 0.01 s. From exchange-averaged chemical shifts of the resonance for the proton on the α-carbon of the cysteine residue of glutathione, it is shown that, in hemolyzed erythrocytes, the sulfhydryl group of glutathione binds CH₃Hg(II) more strongly than the sulfhydryl groups of hemoglobin.