Methods for Measuring Exchangeable Protons in Glycosaminoglycans.

Recent NMR studies of the exchangeable protons of GAGs in aqueous solution, including those of the amide, sulfamate, and hydroxyl moieties, have demonstrated potential for the detection of intramolecular hydrogen bonds providing insights into secondary structure preferences. GAG amide protons are observable by NMR over wide pH and temperature ranges; however, specific solution conditions are required to reduce the exchange rate of the sulfamate and hydroxyl protons and allow their detection by NMR. Building on the vast body of knowledge on detection of hydrogen bonds in peptides and proteins, a variety of methods can be used to identify hydrogen bonds in GAGs including temperature coefficient measurements, evaluation of chemical shift differences between oligo- and monosaccharides, and relative exchange rates measured through line shape analysis and EXSY spectra. Emerging strategies to allow direct detection of hydrogen bonds through heteronuclear couplings offer promise for the future. Molecular dynamic simulations are important in this effort both to predict and confirm hydrogen bond donors and acceptors.

Absorptive transport of amino acids by the rat colon.

The capacity of the colon to absorb microbially produced amino acids (AAs) and the underlying mechanisms of AA transport are incompletely defined. We measured the profile of 16 fecal AAs along the rat ceco-colonic axis and compared unidirectional absorptive AA fluxes across mucosal tissues isolated from the rat jejunum, cecum, and proximal colon using an Ussing chamber approach, in conjunction with 1H-NMR and ultra-performance liquid chromatography-mass spectrometry chemical analyses. Passage of stool from cecum to midcolon was associated with segment-specific changes in fecal AA composition and a decrease in total AA content. Simultaneous measurement of up to 16 AA fluxes under native luminal conditions, with correction for endogenous AA release, demonstrated absorptive transfer of AAs across the cecum and proximal colon at rates comparable (30-80%) to those across the jejunum, with significant Na+-dependent and H+-stimulated components. Expression profiling of 30 major AA transporter genes by quantitative PCR revealed comparatively high levels of transcripts for 20 AA transporters in the cecum and/or colon, with the levels of 12 exceeding those in the small intestine. Our results suggest a more detailed model of major apical and basolateral AA transporters in rat colonocytes and provide evidence for a previously unappreciated transfer of AAs across the colonic epithelium that could link the prodigious metabolic capacities of the luminal microbiota, the colonocytes, and the body tissues.NEW & NOTEWORTHY This study provides evidence for a previously unappreciated transfer of microbially generated amino acids across the colonic epithelium under physiological conditions that could link the prodigious metabolic capacities of the luminal microbiota, the colonocytes, and the body tissues. The segment-specific expression of at least 20 amino acid transporter genes along the colon provides a detailed mechanistic basis for uniport, heteroexchange, Na+-cotransport, and H+-cotransport components of colonic amino acid absorption.

TDCIPP exposure affects Artemia franciscana growth and osmoregulation.

Environmental monitoring has demonstrated widespread occurrence of the flame-retardant tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), raising concerns about the impact on aquatic life. Using 1H NMR and GC-MS metabolomics and 20-day body length experiments, we have determined that exposure to TDCIPP affects Artemia franciscana. The LC50 for a 48 h TDCIPP exposure was determined to be 37.1 ±â€¯1.3 µM. Acute exposure (48 h) to 20.0 µM did not affect A. franciscana body length but did elicit a metabolic change. Chronic exposure to 0.50 µM TDCIPP caused decreased body length in A. franciscana exposed for 20 days and elicited a metabolic response. Principal component analysis revealed variance between acute and chronic exposure along PC1 (36.4%) and between control and TDCIPP along PC2 (17.4%). One-way ANOVA indicated that 19 metabolites were significantly affected by TDCIPP exposure; namely metabolites of the osmolyte class, including betaine, phosphocholine, gadusol, taurine, glycerol and trehalose - metabolites that are essential osmoprotectants in extremophile species. Other pathways that may be perturbed by TDCIPP exposure include one carbon, glycine, serine, threonine, and glycerophospholipid metabolism.

Investigation of the Amide Proton Solvent Exchange Properties of Glycosaminoglycan Oligosaccharides.

One-dimensional 1H NMR experiments were conducted for aqueous solutions of glycosaminoglycan oligosaccharides to measure the amide proton temperature coefficients and activation energy barriers for solvent exchange and evaluate the effect of pH on the solvent exchange properties. A library of mono- and oligosaccharides was prepared by enzymatic depolymerization of amide-containing polysaccharides and by chemical modification of heparin and heparan sulfate saccharides including members that contain a 3- O-sulfated glucosamine residue. The systematic evaluation of this saccharide library facilitated assessment of the effects of structural characteristics, such as size, sulfation number and site, and glycosidic linkage, on amide proton solvent exchange rates. Charge repulsion by neighboring negatively charged sulfate and carboxylate groups was found to have a significant impact on the catalysis of amide proton solvent exchange by hydroxide. This observation leads to the conclusion that solvent exchange rates must be interpreted within the context of a given chemical environment. On their own, slow exchange rates do not conclusively establish the involvement of a labile proton in a hydrogen bond, and additional supporting experimental evidence such as reduced temperature coefficients is required.

Metabolite biomarkers of chlorothalonil exposure in earthworms, coelomic fluid, and coelomocytes.

Earthworm (Eisenia fetida) metabolomics is a useful indicator of toxicant exposure. Extracts of whole earthworms are most commonly used to measure metabolic perturbations, in addition to coelomic fluid which has been used on a more limited basis. Coelomocytes are free moving cells found within earthworm coelomic fluid, and the potential of this compartment has not been evaluated for its utility in earthworm metabolomics. In this study, earthworms were exposed to 18.5 and 37.0 mg/kg chlorothalonil, a commonly used fungicide that targets glutathione. The metabolic impacts of a 14-day chlorothalonil exposure were assessed using 1H NMR and targeted LC-MS measurements of earthworm, coelomic fluid, and coelomocyte extracts. Coelomic fluid was identified as the most sensitive matrix for measuring the effects of chlorothalonil exposure, where an increase in glutamine levels was the only biomarker observed at both doses. At the high dose, multiblocked-orthogonal partial least squares-discriminant analysis (MB-OPLS-DA) supported increased N-acetylserine and ophthalmic acid levels as additional biomarkers of exposure in coelomic fluid. These perturbations may indicate increased oxidative stress, although no changes in glutathione were observed in any matrix.

Evaluating sub-lethal stress from Roundup® exposure in Artemia franciscana using 1H NMR and GC-MS.

Global salinization trends present an urgent need for methods to monitor aquatic ecosystem health and characterize known and emerging stressors for water bodies that are becoming increasingly saline. Environmental metabolomics methods that combine quantitative measurements of metabolite levels and multivariate statistical analysis are powerful tools for ascertaining biological impacts and identifying potential biomarkers of exposure. We propose the use of the saltwater aquatic crustacean, Artemia franciscana, as a model organism for environmental metabolomics in saltwater ecosystems. Artemia are a good choice for ecotoxicity assays and metabolomics analysis because they have a short life cycle, their hemolymph is rich in metabolites and they tolerate a wide salinity range. In this work we explore the potential of Artemia franciscana for environmental metabolomics through exposure to the broad-spectrum herbicide, glyphosate. The LC50 for a 48 h exposure of Roundup® was determined to be 237 ± 23 ppm glyphosate in the Roundup® formulation. Artemia cysts were hatched and exposed to sub-lethal glyphosate concentrations of 1.00, 10.0, 50.0, or 100 ppm glyphosate in Roundup®. We profiled 48 h old Artemia extracts using 1H NMR and GC-MS. Dose-dependent metabolic perturbation was evident for several metabolites using univariate and multivariate analyses. Metabolites significantly affected by Roundup® exposure included aspartate, formate, betaine, glucose, tyrosine, phenylalanine, gadusol, and isopropylamine. Biochemical pathway analysis with the KEGG database suggests impairment of carbohydrate and energy metabolism, folate-mediated one-carbon metabolism, Artemia molting and development, and microbial metabolism.

1H NMR characterization of chitin tetrasaccharide in binary H2O:DMSO solution: Evidence for anomeric end-effect propagation.

Chitin oligosaccharides, composed of homogeneous ß(1 → 4)-linked N-acetyl-D-glucosamine (GlcNAc) sequences, is a well-known elicitor of plant immune defense, and also occur as structural elements of chitosan and nodulation (Nod) factor. Detailed microstructure characterization is required for understanding the function mode of these bioactive molecules. Herein, experimental conditions for detection and elucidation of the 1H NMR resonances of amide groups in chitin oligosaccharides are presented. The binary mixture of 70% H2O: 30% DMSO­d6 was found to be the optimal solvent for amide proton measurements in homogeneous GlcNAc sequences, facilitating differentiation of the local chemical microenvironments of all four amide groups of the chitin tetrasaccharide. Experimental evidence that anomeric end-effect triggers amide proton resonance differentiation at the adjacent residue has potential to provide important insights into the solution structure of chitin and other amino sugars containing GlcNAc sequences.

Synthesis and Structure Reassignment of Malylglutamate, a Recently Discovered Earthworm Metabolite.

Malylglutamate, a newly identified metabolite in earthworms, was synthesized using a traditional peptide coupling approach for assembling the amide from protected malate and glutamate precursors. The proposed structure (1) and a diastereomer were synthesized, but their NMR spectra did not match the natural sample. Further analysis of the natural sample using HMBC spectroscopy suggested an alternative attachment of the malyl moiety, and ß-malylglutamate (2) diastereomers were synthesized, L,L-2 and D,D-2. NMR spectra were an excellent match with the natural sample, and chiral-phase chromatography was employed to identify (-)-ß-l-malyl-l-glutamate (2) as the isomer native to Eisenia fetida.

Quantification of punicalagins in commercial preparations and pomegranate cultivars, by liquid chromatography-mass spectrometry.

BACKGROUND: Pomegranate (Punica granatum L.) - a delicious fruit once used in Ayurvedic medicine - is now largely known for the antioxidant properties of its juice, which has also been considered to have health benefits against diseases such as cancer and cardiovascular diseases. These beneficial effects are associated with the fruit's high content of polyphenolic compounds. High demand and lower production levels drive pomegranate prices up, which leads to the possibility of pomegranate products being adulterated, diluted or substituted. To ensure the presence of pomegranate in various preparations labeled as containing pomegranate, a simple method was developed to screen and quantify the specific punicalagins by mass spectrometry. RESULTS: The present method was used to analyze several pure and mixed beverages from the US market, and also to quantify punicalagins in the juice of 14 pomegranate cultivars. Punicalagins were detected in all cultivars, with higher concentrations in whole fruit juices compared with aril juices. Amongst the 20 commercial beverages, punicalagins were not detected in four preparations. CONCLUSION: The liquid chromatographic-mass spectrometric method presented herein enables an easy and rapid quantification of the specific punicalagins. The latter was detected in all cultivar samples, thus supporting that punicalagin is a suitable marker of these 14 pomegranate cultivars in commercial juices. Absence of the specific marker in four commercial preparations shows the necessity of having simple and rapid methods to evaluate the presence of pomegranate in preparations. © 2019 Society of Chemical Industry.

Metabolic Profiling of Chloroacetanilide Herbicides in Earthworm Coelomic Fluid Using 1H NMR and GC-MS.

Earthworms ( Eisenia fetida) are vital members of the soil environment. Because of their sensitivity to many contaminants, monitoring earthworm metabolism may be a useful indicator of environmental stressors. Here, metabolic profiles of exposure to five chloroacetanilide herbicides and one enantiomer (acetochlor, alachlor, butachlor, racemic metolachlor, S-metolachlor, and propachlor) are observed in earthworm coelomic fluid using proton nuclear magnetic resonance spectroscopy (NMR) and gas chromatography-mass spectrometry (GC-MS). Multiblocked-orthogonal partial least-squares-discriminant analysis (MB-OPLS-DA) and univariate analysis were used to identify metabolic perturbations in carnitine biosynthesis, carbohydrate metabolism, lipid metabolism, nitrogen metabolism, and the tricarboxylic acid cycle. Intriguingly, stereospecific metabolic responses were observed between racemic metolachlor and S-metolachlor exposed worms. These findings support the utility of coelomic fluid in monitoring metabolic perturbations induced by chloroacetanilide herbicides in nontarget organisms and reveal specificity in the metabolic impacts of herbicide analogues in earthworms.

1H NMR-Based Identification of Intestinally Absorbed Metabolites by Ussing Chamber Analysis of the Rat Cecum.

The large intestine (cecum and colon) is a complex biochemical factory of vital importance to human health. It plays a major role in digestion and absorption by salvaging nutrients from polysaccharides via fermentation initiated by the bacteria that comprise the gut microbiome. We hypothesize that the intestinal epithelium absorbs a limited number of luminal metabolites with bioactive potential while actively excluding those with toxic effects. To explore this concept, we combined 1H NMR detection with Ussing chamber measurements of absorptive transport by rat cecum. Numerous metabolites transported across the epithelium can be measured simultaneously by 1H NMR, a universal detector of organic compounds, alleviating the need for fluorescent or radiolabeled compounds. Our results demonstrate the utility of this approach to delineate the repertoire of fecal solutes that are selectively absorbed by the cecum and to determine their transport rates.

Rice SUB1A constrains remodelling of the transcriptome and metabolome during submergence to facilitate post-submergence recovery.

The rice (Oryza sativa L.) ethylene-responsive transcription factor gene SUB1A-1 confers tolerance to prolonged, complete submergence by limiting underwater elongation growth. Upon desubmergence, SUB1A-1 genotypes rapidly recover photosynthetic function and recommence development towards flowering. The underpinnings of the transition from stress amelioration to the return to homeostasis are not well known. Here, transcriptomic and metabolomic analyses were conducted to identify mechanisms by which SUB1A improves physiological function over the 24 hr following a sublethal submergence event. Evaluation of near-isogenic genotypes after submergence and over a day of reaeration demonstrated that SUB1A transiently constrains the remodelling of cellular activities associated with growth. SUB1A influenced the abundance of ca. 1,400 transcripts and had a continued impact on metabolite content, particularly free amino acids, glucose, and sucrose, throughout the recovery period. SUB1A promoted recovery of metabolic homeostasis but had limited influence on mRNAs associated with growth processes and photosynthesis. The involvement of low energy sensing during submergence and recovery was supported by dynamics in trehalose-6-phosphate and mRNAs encoding key enzymes and signalling proteins, which were modulated by SUB1A. This study provides new evidence of convergent signalling pathways critical to the rapidly reversible management of carbon and nitrogen metabolism in submergence resilient rice.

1H NMR Metabolic Profiling of Earthworm (Eisenia fetida) Coelomic Fluid, Coelomocytes, and Tissue: Identification of a New Metabolite-Malylglutamate.

Earthworm metabolism is recognized as a useful tool for monitoring environmental insults and measuring ecotoxicity, yet extensive earthworm metabolic profiling using 1H nuclear magnetic resonance (NMR) spectroscopy has been limited in scope. This study aims to expand the embedded metabolic material in earthworm coelomic fluid, coelomocytes, and tissue to aid systems toxicology research. Fifty-nine metabolites within Eisenia fetida were identified, with 47 detected in coelomic fluid, 41 in coelomocytes, and 54 in whole-worm samples and tissue extracts. The newly detected but known metabolites 2-aminobutyrate, nicotinurate, Nδ,Nδ,Nδ-trimethylornithine, and trigonelline are reported along with a novel compound, malylglutamate, elucidated using 2D NMR and high-resolution MS/MS. We postulate that malylglutamate acts as a glutamate/malate store, chelator, and anionic osmolyte and helps to provide electrolyte balance.

Separation of ten phosphorylated mono-and disaccharides using HILIC and ion-pairing interactions.

Phosphorylated carbohydrates are indispensable cogs in several key metabolic wheels for all forms of life. Here, a straightforward liquid chromatography method coupled to mass spectrometry detection was developed for phosphorylated sugars. For separation of the targeted compounds, hydrophilic interaction chromatography (HILIC) was used with a bridged-ethylene hybrid amide column under alkaline conditions using triethylamine as a mobile phase modifier. Methylphosphonic acid was added to the aqueous mobile phase to reduce the tailing of compounds containing phosphate groups, which are known to interact with stainless steel components of the separation system. Under alkaline conditions and addition of methylphosphonic acid, the retention behavior can be attributed to both conventional HILIC mechanisms as well as ion-pairing interactions in the mobile phase. This hypothesis is supported by comparing the retention behavior of phosphorylated sugars and unmodified sugars. The HILIC method resolved eight biologically important phosphorylated sugars and thereby enables simultaneous detection and quantification of these compounds: fructose-1,6-bisphosphate, glucose-1-phosphate, glucose-6-phosphate, lactose-1-phosphate, mannose-6-phosphate, ribose-5-phosphate, sucrose-6-phosphate, and threhalose-6-phosphate. Fructose-1-phosphate and fructose-6-phosphate were not resolved but quantification of total fructose-phosphate is possible.

Screening enoxaparin tetrasaccharide SEC fractions for 3-O-sulfo-N-sulfoglucosamine residues using [(1)H,(15)N] HSQC NMR.

Heparin and heparan sulfate (HS) are important in mediating a variety of biological processes through binding to myriad different proteins. Specific structural elements along the polysaccharide chains are essential for high affinity protein binding, such as the 3-O-sulfated N-sulfoglucosamine (GlcNS3S) residue, a relatively rare modification essential for heparin's anticoagulant activity. The isolation of 3-O-sulfated oligosaccharides from complex mixtures is challenging because of their low abundance. Although methods such as affinity chromatography are useful in isolating oligosaccharides that bind specific proteins with high affinity, other important 3-O-sulfated oligosaccharides may easily be overlooked. Screening preparative-scale size-exclusion chromatography (SEC) fractions of heparin or HS digests using [(1)H,(15)N] HSQC NMR allows the identification of fractions containing 3-O-sulfated oligosaccharides through the unique (1)H and (15)N chemical shifts of the GlcNS3S residue. Those SEC fractions containing 3-O-sulfated oligosaccharides can then be isolated using strong anion-exchange (SAX)-HPLC. Compared with the results obtained by pooling the fractions comprising a given SEC peak, SAX-HPLC analysis of individual SEC fractions produces a less complicated chromatogram in which the 3-O-sulfated oligosaccharides are enriched relative to more abundant components. The utility of this approach is demonstrated for tetrasaccharide SEC fractions of the low molecular weight heparin drug enoxaparin facilitating the isolation and characterization of an unsaturated 3-O-sulfated tetrasaccharide containing a portion of the antithrombin-III binding sequence.

Solution-State (17)O†Quadrupole Central-Transition NMR Spectroscopy in the Active Site of Tryptophan Synthase.

Oxygen is an essential participant in the acid-base chemistry that takes place within many enzyme active sites, yet has remained virtually silent as a probe in NMR spectroscopy. Here, we demonstrate the first use of solution-state (17)O quadrupole central-transition NMR spectroscopy to characterize enzymatic intermediates under conditions of active catalysis. In the 143 kDa pyridoxal-5'-phosphate-dependent enzyme tryptophan synthase, reactions of the α-aminoacrylate intermediate with the nucleophiles indoline and 2-aminophenol correlate with an upfield shift of the substrate carboxylate oxygen resonances. First principles calculations suggest that the increased shieldings for these quinonoid intermediates result from the net increase in the charge density of the substrate-cofactor π-bonding network, particularly at the adjacent α-carbon site.

Metabolic Impacts of Using Nitrogen and Copper-Regulated Promoters to Regulate Gene Expression in Neurospora crassa.

The filamentous fungus Neurospora crassa is a long-studied eukaryotic microbial system amenable to heterologous expression of native and foreign proteins. However, relatively few highly tunable promoters have been developed for this species. In this study, we compare the tcu-1 and nit-6 promoters for controlled expression of a GFP reporter gene in N. crassa. Although the copper-regulated tcu-1 has been previously characterized, this is the first investigation exploring nitrogen-controlled nit-6 for expression of heterologous genes in N. crassa. We determined that fragments corresponding to 1.5-kb fragments upstream of the tcu-1 and nit-6 open reading frames are needed for optimal repression and expression of GFP mRNA and protein. nit-6 was repressed using concentrations of glutamine from 2 to 20 mM and induced in medium containing 0.5-20 mM nitrate as the nitrogen source. Highest levels of expression were achieved within 3 hr of induction for each promoter and GFP mRNA could not be detected within 1 hr after transfer to repressing conditions using the nit-6 promoter. We also performed metabolic profiling experiments using proton NMR to identify changes in metabolite levels under inducing and repressing conditions for each promoter. The results demonstrate that conditions used to regulate tcu-1 do not significantly change the primary metabolome and that the differences between inducing and repressing conditions for nit-6 can be accounted for by growth under nitrate or glutamine as a nitrogen source. Our findings demonstrate that nit-6 is a tunable promoter that joins tcu-1 as a choice for regulation of gene expression in N. crassa.