Quantitative 1H NMR Spectroscopy Research Articles

1H NMR Based Metabolomics in Human Sepsis and Healthy Serum.

Early diagnosis is essential but challenging in severe sepsis. Quantifying and comparing metabolite concentrations in serum has been suggested as a new diagnostic tool. Here we used proton nuclear magnetic resonance spectroscopy (1H NMR) based metabolomics to analyze the possible differences in metabolite concentrations between sera taken from septic patients and healthy controls, as well as between sera of surviving and non-surviving sepsis patients. We took serum samples from 44 sepsis patients when the first sepsis induced organ dysfunction was found. Serum samples were also collected from 14 age and gender matched healthy controls. The samples were analyzed by quantitative 1H NMR spectroscopy for non-lipid metabolites. We found that the serum levels of glucose, glycine, 3-hydroxybutyrate, creatinine and glycoprotein acetyls (mostly alpha-1-acid glycoprotein, AGP) were significantly (p < 0.05) higher in sepsis compared to healthy sera, whereas citrate and histidine were significantly (p < 0.05) lower in sepsis patients compared to healthy controls. We found statistically significantly higher serum lactate and citrate concentrations in non-survivors compared to 30-day survivors. According to our study, 3-hydroxybutyrate, citrate, glycine, histidine, and AGP are candidates for further studies to enable identification of phenotype association in the early stages of sepsis.

Quantitative 1H-NMR spectroscopy as an efficient method for identification and quantification of PVC, ABS and PA microparticles.

As an extension of the new, size-independent, fast and easy quantitative 1H-NMR (qNMR) spectroscopy as an alternative method for microplastic (MP) analysis we herein present the possibility to analyze three environmentally highly relevant MP particles by qNMR spectroscopy. The investigations cover polyvinyl chloride (PVC) powder with a size <50 μm, acrylonitrile butadiene styrene (ABS) granules with a size distribution of 100-300 μm, and polyamide (PA) fibres with a length of approx. 500 μm and a diameter of approx. 20-30 μm. For quantification, the integration method or the peak-fitting method combined with the calibration curve method was used. Linearity above 0.99R2, precision of 99.1-99.9% and accuracy of 95.6-110% for all three polymer types demonstrate the high analytical potential of the method. Moreover, the limit of detection (40-84 μg mL-1) for all polymer types is in the lower environmentally relevant range.

Analysis of urinary metabolic alteration in type 2 diabetic rats treated with metformin using the metabolomics of quantitative spectral deconvolution 1H NMR spectroscopy

Metformin has been used clinically as the first-choice drug to treat type 2 diabetes patients. It is not metabolized in liver and the residue which is excreted unchanged in the urine. Identification and quantification of metformin in the urine can be used as a unique marker for the metformin treated diabetic patients. The aim of our study was to identify residual metformin in the urine of type 2 diabetic rats using quantitative spectral deconvolution of 1H NMR method. Additionally, quantitative assessment of residual metformin and other identified metabolites in the urine, as well as the evaluation of metabolic alteration due to metformin treatment of diabetic condition were also carried out. Partial least square discriminant analysis (PLS-DA) and statistical analysis were used to discriminate the metformin treated type 2 diabetic, the lean, the obese, and the type 2 diabetic rat groups. Furthermore, in order to evaluate the statistical difference of metabolite levels between the groups, ANOVA test was performed. The presence of metformin in the urine of the metformin treated diabetic rats from the 1H NMR spectral analysis and its identity was confirmed by 2D NMR spectroscopy experiments including the 2D 1H J-resolved (JRES), and 1H heteronuclear multiple correlation (HMBC). Based on the PLS-DA model and statistical analysis, it was established that metformin treatment significantly influenced the improvement of glucose and 1-methylnicotinamide metabolism; increase the growth and activity of gut microbiota as indicated by the elevation of trimethylamine (TMA), phenylacetylglycine (PAG) and indoxylsulfate levels; as well as the suppression of tricarboxylic acid (TCA) cycle metabolism as shown by the decreased levels of citrate, 2-oxoglutarate, succinate, and fumarate. This study provided new approach to the quantitative analysis of metformin in urine and further exploration on the effect of metformin treatment on the type 2 diabetic rats revealed useful findings on its metabolic states.

Quantitative analysis of PET microplastics in environmental model samples using quantitative 1H-NMR spectroscopy: validation of an optimized and consistent sample clean-up method.

Identification and quantification of microplastics (MP) in environmental samples is crucial for understanding the risk and distribution of MP in the environment. Currently, quantification of MP particles in environmental samples and the comparability of different matrices is a major research topic. Research also focusses on sample preparation, since environmental samples must be free of inorganic and organic matrix components for the MP analysis. Therefore, we would like to propose a new method that allows the comparison of the results of MP analysis from different environmental matrices and gives a MP concentration in mass of MP particles per gram of environmental sample. This is possible by developing and validating an optimized and consistent sample preparation scheme for quantitative analysis of MP particles in environmental model samples in conjunction with quantitative 1H-NMR spectroscopy (qNMR). We evaluated for the first time the effects of different environmental matrices on identification and quantification of polyethylene terephthalate (PET) fibers using the qNMR method. Furthermore, high recovery rates were obtained from spiked environmental model samples (without matrix ~ 90%, sediment ~ 97%, freshwater ~ 94%, aquatic biofilm ~ 95%, and invertebrate matrix ~ 72%), demonstrating the high analytical potential of the method. Graphical abstract.

Correction to: Quantitative analysis of PET microplastics in environmental model samples using quantitative 1H-NMR spectroscopy: validation of an optimized and consistent sample clean-up method.

The original version of this article contained a mistake.

Limits of thiol chemistry revealed by quantitative analysis of mixed layers of thiolated-PEG ligands grafted onto gold nanoparticles

HypothesisThe functionalization of gold nanoparticles is commonly based on the use of thiol groups for the anchoring of organic ligands. To functionalize gold nanoparticles with mixed layers in defined proportions, different thiolated ligands are often used and assumed to graft equally on the surface. This assumption is however generally not verified and a quantitative investigation of the grafting density of mixed organic layers of thiolated ligands is therefore required. ExperimentsGold nanoparticles were exposed to solutions containing various proportions of two PEG ligands containing a thiol group at one extremity and a methoxy, carboxylate, or alkyne group at the other. A systematic study was performed on the resulting particles in order to quantify the composition of the PEG layer by quantitative 1H NMR spectroscopy. FindingsOur results showed that the grafting of the PEG ligands with either a carboxylate or an alkyne group is strongly hindered in the presence of the methylated PEG ligands, despite the use of identical thiol anchoring groups. This is the first report on the quantification of mixed layers of PEGylated ligands on gold nanoparticles that demonstrates the severe limits of thiol chemistry for the functionalization of gold nanoparticles with mixed monolayers.

Preparation of DESIGNER extracts of red clover (Trifolium pratense L.) by centrifugal partition chromatography

Starting with an isoflavone-rich red clover extract (RCE), this study expands on the DESIGNER approach to Deplete and Enrich Select Ingredients to Generate Normalized Extract Resources using countercurrent separation (CCS) methodology. A hydrostatic CCS (also known as centrifugal partition chromatography, CPC) technique was used to enrich and deplete selected bioactive isoflavones of RCE extracts.In order to efficiently prepare large enough DESIGNER extracts from RCE for biological testing including in vivo assays, it was necessary to choose a balance between resolution and a loading capacity of at least 1 g per separation for the selected solvent system (SS). Adding 3 mL of DMSO to the sample containing equal amounts of upper and lower phases of hexanes-ethyl acetate-methanol-water (HEMWat 5.5/4.5/5/5, v/v) allowed 1 g of RCE to be dissolved in the sample without disrupting the chromatographic resolution of the target isoflavones. CPC experiments using other solubility modifiers, acetone and acetonitrile indicated that these modifiers increase solubility significantly, even better than DMSO, but the separation of target compounds was sufficiently disturbed to be unacceptable for producing the desired DESIGNER extracts.The preparation of DESIGNER extracts was achieved with two sequential CPC separations. The first produced a biochanin A enriched fraction (93.60% w/w) with only small amounts of other isoflavones: 2.30% w/w prunetin, 1.17% w/w formononetin, and 0.12% w/w irilone. Gravimetric investigations of this step demonstrated the high efficiency of CCS technology for full and unbiased sample recovery, confirmed experimentally to be 99.80%. A formononetin enriched fraction from this first separation was re-chromatographed on a more polar HEMWat (4/6/4/6, v/v) SS to produce a formononetin enriched DESIGNER fraction of 94.70% w/w purity. The presence of the minor (iso)flavonoids: 3.16% w/w pseudobaptigenin, 0.39% w/w kaempferol, and 0.31% w/w genistein was also monitored in these fractions. Chromatographic fractions, combined fractions, and DESIGNER extracts were analyzed with quantitative 1H NMR (qHNMR) spectroscopy which provided purity information, quantitation, and structural identification of the components.

Functional Metabolome Analysis of Penicillium roqueforti by Means of Differential Off-Line LC-NMR.

UPLC-TOF/MS profiling, followed by the recently reported differential off-line LC-NMR (DOLC-NMR) and quantitative 1H NMR spectroscopy (qHNMR), led to the differential qualitative analysis and accurate quantitation of l-tryptophan-induced metabolome alterations of Penicillium roqueforti, which is typically used in making blue-mold cheese. Among the 24 metabolites identified, two tetrapeptides, namely, d-Phe-l-Val-d-Val-l-Tyr and d-Phe-l-Val-d-Val-l-Phe, as well as cis-bis(methylthio)silvatin, are reported for the first time as metabolites of P. roqueforti. Antimicrobial activity tests showed strong effects of the catabolic l-tryptophan metabolites 3-hydroxyanthranilic acid, anthranilic acid, and 3-indolacetic acid against Saccharomyces cerevisiae, with IC50 values between 15.6 and 24.0 μg/mL, while roquefortine C and cis-bis(methylthio)silvatin inhibited the growth of Gram-negative Escherichia coli and Gram-positive Bacillus subtilis with IC50 values between 30.0 and 62.5 μg/mL.

Quantifying Ligand Exchange on InP Using an Atomically Precise Cluster Platform

The surface chemistry of a colloidal nanoparticle is intrinsic to both its structure and function. It is therefore necessary to characterize the surfaces of colloidal materials to rationally underpin any synthetic, catalytic, or transformative mechanisms they enable. Here we characterize the surface properties of colloidal InP clusters and quantum dots by examining the binding of traditional stabilizing ligands including carboxylates, phosphonates, and thiolates. By using the In37P20X51 (X = carboxylate) cluster species as an ideally monodisperse and well-defined starting scaffold, we quantify surface-exchange equilibria. Using quantitative 1H and 31P NMR spectroscopy, we show that 1:1 metathesis-type binding models are insufficient to fully describe the surface dynamics. In particular, for the case of the highly reversible carboxylate ligand exchange, a more detailed isotherm approach using a two-site, competitive model is necessary. This model is used to deconvolute L- and X-type binding modalities. We additionally quantify the reversible and irreversible ligand-exchange reactions observed in the thiolateand phosphonate systems.

The first application of quantitative 1H NMR spectroscopy as a simple and fast method of identification and quantification of microplastic particles (PE, PET, and PS).

Microplastic (0.001-5mm) is a serious problem for the environment and is globally distributed. It has been detected in marine and limnic waters as well as in organisms. Until now, microplastic (MP) particles in environmental samples are mainly identified by Fourier transform infrared (FTIR) or Raman spectroscopy. Usually, for quantitative detection, time-consuming counting of MP particles in the sample is described. Therefore, a great need for research in the field of size-independent quantitative analysis of MP particles is evident. We present herein the application of quantitative 1H NMR spectroscopy (qNMR) as a new method for the qualitative and quantitative analysis of MP in solution. Polyethylene (PE) granules with a size distribution of < 300μm, polyethylene terephthalate (PET) fibers with a length of approx. 500μm, and polystyrene (PS) beads with a size distribution of 0.5-1mm were qualitatively and quantitatively analyzed as prototypical MP particles in model samples using a calibration curve method. As internal standard, the residual proton signal of the deuterated solvent was used. For all polymer types, linearity of the method is > 0.994 R2, and the precision is in the range of 99.4-99.9%. The limit of detection (LOD) is in the range of 19-21μg/mL and the limit of the quantification (LOQ) is in the range of 74-85mg/mL, so the LOD and LOQ are observed in an environmentally relevant size. In this work, we therefore show that size-independent qualitative and quantitative determination of microplastic particles in model samples using qNMR is possible. Graphical abstract Working flow for the first application of qNMR as a simple and fast method of identification and quantification of microplastic (MP) particles (PE, PET, PS). 338 × 190mm (96 × 96DPI).

Effect of Formulation on the Binding Efficiency and Selectivity of Precipitation Molecularly Imprinted Polymers.

This study investigated the effect of feed formulation: the template:functional monomer (T:fM) and functional monomer:crosslinker (fM:X) ratios as well as the initiator concentration, on the binding performance and selectivity of caffeine (CAF) and theophylline (THP) imprinted polymers obtained by precipitation polymerisation in acetonitrile at 60 °C using methacrylic acid (MAA) and ethylene glycol dimethacrylate (EGDMA) as functional monomer and crosslinker, respectively. Template incorporation, monitored by quantitative 1H-NMR spectroscopy, ranged from 8 to 77% and was found to be more favourable at both high and low T:fM ratios, low fM:X ratio and high initiator concentration. The resulting T:fM ratio in most MIPs were found to be lower than their feed ratios. Incorporation of THP into the polymers was observed to be consistently higher than CAF and, for most MIPs, the observed binding capacities represent less than 10% of the incorporated template. Improved imprinting factors were obtained from molecularly imprinted polymers (MIPs) with high crosslinker content, i.e., fM:X ratio of 1:10, and high initiator concentration, i.e., initiator:total monomer (I:tM) ratio of 1:5, while T:fM ratio (1:2 to 1:8) was found not to influence binding capacities and imprinting factors (IF). The NIPs showed no preference for either CAF or THP in competitive selectivity studies while MIPs were observed to bind preferentially to their template with THP displaying higher selectivity (72–94%) than CAF (63–84%). Template selectivity was observed to increase with increasing initiator concentration, with MIPs from I:tM ratio of 1:5 shown to be the most selective towards CAF (84%) and THP (93%). The fM:X ratio only showed minimal effect on MIP selectivity. Overall, for the MIP systems under study, template incorporation, binding capacity, imprinting factor and selectivity are enhanced at a faster rate of polymerisation using an I:tM ratio of 1:5. Polymer particles obtained were between 66 to 140 nm, with MIPs generally smaller than their NIP counterparts, and have been observed to decrease with increasing T:fM and fM:X ratios and increase with increasing initiator concentration.

The Role of Ionic Liquid Breakdown in the Electrochemical Metallization of VO2: An NMR Study of Gating Mechanisms and VO2 Reduction.

Metallization of initially insulating VO2 via ionic liquid electrolytes, otherwise known as electrolyte gating, has recently been a topic of much interest for possible applications such as Mott transistors and memory devices. It is clear that the metallization takes place electrochemically, and, in particular, there has previously been extensive evidence for the removal of small amounts of oxygen during ionic liquid gating. Hydrogen intercalation has also been proposed, but the source of the hydrogen has remained unclear. In this work, solid-state magic angle spinning NMR spectroscopy (1H, 2H, 17O, and 51V) is used to investigate the thermal metal-insulator transition in VO2, before progressing to catalytically hydrogenated VO2 and electrochemically metallized VO2. In these experiments electrochemical metallization of bulk VO2 particles is shown to be associated with intercalation of hydrogen, the degree of which can be measured with quantitative 1H NMR spectroscopy. Possible sources of the hydrogen are explored, and by using a selectively deuterated ionic liquid, it is revealed that the hydrogenation is due to deprotonation of the ionic liquid; specifically, for the commonly used dialkylimidazolium-based ionic liquids, it is the "carbene" proton that is responsible. Increasing the temperature of the electrochemistry is shown to increase the degree of hydrogenation, forming first a less hydrogenated metallic orthorhombic phase then a more hydrogenated insulating Curie-Weiss paramagnetic orthorhombic phase, both of which were also observed for catalytically hydrogenated VO2. The NMR results are supported by magnetic susceptibility measurements, which corroborate the degree of Pauli and Curie-Weiss paramagnetism. Finally, NMR spectroscopy is used to identify the presence of hydrogen in an electrolyte gated thin film of VO2, suggesting that electrolyte breakdown, proton intercalation, and reactions with decomposition products within the electrolyte should not be ignored when interpreting the electronic and structural changes observed in electrochemical gating experiments.

Wild Thymus capitatus Hoff. Et Link. Chemical Composition, Antioxidant and Antimicrobial Activities of the Essential Oil

The composition of the essential oil (EO) obtained by hydrodistillation from the leaves of Thymus capitatus Hoff. et Link. growing wild in Sythonia (Greece) was analysed by GC-FID, GC-MS and quantitative 1H NMR spectroscopy. Thirty nine compounds were identified, representing approximately 99.0% of the oil. Major component determined by GC-FID was carvacrol (81.8%), followed by linalool (3.5%) and (E)-caryophyllene (3.5%). Abundance of carvacrol determined by quantitative 1H NMR spectroscopy was 74.9% and was in reasonable agreement with GC-FID result. The oil was tested for the antioxidant activity in DPPH assay and showed a dose dependent free radical scavenging activity with EC50 value of 1.03 μg/ml. Antimicrobial effect of the EO was tested against Gram negative, Gram positive bacteria, and fungi. The tested EO showed significant activity against fungi, especially S. cerevisiae. The most sensitive bacteria were E. coli ATCC 25922, E. fecalis and S. epidermidis. Human pathogen Burkholderia cepacia, ATCC 25416 was used in the investigation of T. capitatus for the first time.

Quantitative ¹H-NMR Spectroscopy for Profiling Primary Metabolites in Mulberry Leaves.

The primary metabolites in aqueous extract of mulberry (Morus alba L.) leaves were characterized by using proton nuclear magnetic resonance (1H-NMR) spectroscopy. With the convenience of resonance assignment, GABA together with the other 10 primary metabolites was simultaneously identified and quantified in one 1H-NMR spectrum. In this study, external calibration curves for metabolites were employed to calculate the concentrations of interests. The proposed quantitative approach was demonstrated with good linearity (r2 ranged in the interval of 0.9965–0.9999), precision, repeatability, stability (RSD values in the ranges of 0.35–4.89%, 0.77–7.13% and 0.28–2.33%, respectively) and accuracy (recovery rates from 89.2% to 118.5%). The established 1H-NMR method was then successfully applied to quantify 11 primary metabolites in mulberry leaves from different geographical regions within a rapid analysis time and a simple sample preparation procedure.

Authentic compound-free strategy for simultaneous determination of primary coumarins in Peucedani Radix using offline high performance liquid chromatography–nuclear magnetic resonance spectroscopy–tandem mass spectrometry

Herein, a strategy is proposed for the simultaneous determination of primary coumarins in Peucedani Radix (Chinese name: Qianhu). The methodology consists of three consecutive steps: 1) Semi-preparative LC in combination with a home-made automated fraction collection module to fragment the universal metabolome standard into ten fractions (Frs. I–X); 2) LC–accurate MS/MS and quantitative 1H NMR spectroscopy conducted in parallel to acquire the qualitative and quantitative data of each fraction; 3) Robust identification and quantification of components by use of LC coupled to multiple reaction monitoring. In this final step, the most significant fractions (Frs. III–X) were pooled to serve as the pseudo-mixed standard solution. Meticulous online parameter optimization was performed to obtain the optimal parameters, including ion transitions and collision energies. Concerns were particularly paid onto pursuing the parameters being capable of monitoring regio-specific isomers, notably praeruptorin E vs. 3′-isovaleryl-4′-angeloylkhellactone. The quantitative performance of the method was validated according to diverse assays. Eleven primary coumarins (1–11) were unambiguously identified and absolutely quantified, even though no external reference compound was used. Above all, the integrated strategy not only provides a feasible pipeline for the quality assessment of Peucedani Radix, but more importantly, shows the potential for authentic compound-free quantitative evaluation of traditional Chinese medicines.

High Value-Added Biomolecules from Beach Waste of Marine Origin-Screening for Potential Candidates among Seagrass of the Cymodoceaceae Family

Background: Detrital leaves from seagrass of the Cymodoceaceae family were assayed for biologically active molecules that have commercial as well as societal benefits. Objective: We focused on L-chiro-inositol, a very rare natural occurring cyclitol, and chicoric acid, a polyphenolic compound, in which both applications were found in the nutraceutical segment. Method: Six species of seagrass belonging to the genera Syringodium, Cymodocea and Halodule were collected from their native habitat. The L-chiro-inositol content of the crude aqueous extracts prepared from different batches of Cymodocea nodosa flotsam was measured by quantitative 1H-NMR spectroscopy. High concentrations were found with values ranging from 22.2 to 35.0 mg.g-1 plant dw. The presence of L-chiro-inositol in the tropical species C. rotundata, C. serrulata, Syringodium isoetifolium, and Halodule pinifolia was also characterized by qualitative NMR. The chicoric acid content of crude aqueous methanolic extracts prepared from C. rotundata, C. serrulata, S. isoetifolium, and Halodule pinifolia was determined by quantitative HPLC-DAD. The values found ranged from 0.39 to 6.15 mg.g-1 dry weight. Chicoric acid was unambiguously identified as the major phenolic in S. isoetifolium, and Halodule pinifolia, while it was found in mixture with flavonol derivatives in C. rotundata and C. serrulata. Flavonol derivatives are also of interest for their bioactivity. Result: Considering the demonstrated therapeutic applications of L-chiro-inositol and chicoric acid, their high value on the nutraceutical market, and their rare occurrence in the plant kingdom, their presence in Syringodium, Cymodocea and Halodule detrital leaves makes this abundant biomass of interest for dietary and pharmaceutical applications. Conclusion: These results show that there is a real potential for harvesting beachcast Cymodoceacea.

Absolute quantification of poly(dl-lactide-co-glycolide) in microspheres using quantitative 1H NMR spectroscopy

The complex nature and the manufacturing process of poly(dl-lactide-co-glycolide) (PLGA), a key component of PLGA-based microspheres, have made the quantification of this copolymer difficult. The main purpose of the current study was to investigate the potential of three different methods for the quantitative analysis of the PLGA content of clinical products. In this regard, leuprorelin acetate microspheres from different vendors were chosen as templates to validate quantitative 1H nuclear magnetic resonance (qHNMR) spectroscopy, size exclusion chromatography (SEC), and high-performance liquid chromatography (HPLC) methods qHNMR proved to be an excellent and rapid PLGA quantification method compared to the other two. The recovery value was 99.12% and the linearity correlation coefficient was 0.9999. The results obtained from the qHNMR method were found to match the data provided by the vendor, suggesting that qHNMR can be utilized as a reliable quality control and inspection tool for PLGA-based clinical products.

Establishment of an analytical method for accurate purity evaluations of acylcarnitines by using quantitative 1H NMR spectroscopy

Recently, it has become possible to examine metabolism abnormalities by detecting increases in specific acylcarnitines in blood tests of newborn babies using tandem mass spectrometer. However, acylcarnitine standard solutions with metrological traceability to the International System of Units (SI) for accurate calibration of tandem mass spectrometer are not yet available worldwide. In this study, we examined a quantitative 1H NMR procedure for obtaining accurate and SI-traceable purity evaluations of acylcarnitines having different numbers of carbon atoms as each raw material for their standard solutions. In particular, the solvent composition and measurement temperature were optimized to reduce the influence of water signal overlapping on analyte signals. It was found that, when the signal of that 1H which directly bound to the asymmetric carbon of the acylcarnitine is the target signal, it was possible to reduce overlapping of the water signal on the target signal by using deuterium oxide as a solvent. On the other hand, in the case of an acylcarnitine that was poorly soluble in deuterium oxide, it was possible to reduce overlapping of the water signal on the target signal by adding an appropriate amount of deuterium oxide to methanol-d 4 in which the acylcarnitine had high solubility. At this time, the optimum mixing volume ratio of methanol-d 4/deuterium would be 80:20. The overlapping of the water signal could be also reduced when the measurement temperature was 15 °C to 40 °C. When the measurement temperature was an around room temperature (in this study, 25 °C), fine shimming could be performed easily. Therefore, the optimum temperature would be 25 °C, because fine shimming was essential to quantify any signal area accurately. Finally, this study confirmed that accurate values with SI traceability could be obtained at about 1 % or less expanded uncertainty for five kinds of acylcarnitines.

Concentration Measurement of Amino Acid in Aqueous Solution by Quantitative 1H NMR Spectroscopy with Internal Standard Method.

We describe a procedure to determine concentrations of amino acid standard solutions by quantitative NMR spectroscopy using an internal standard. The measurement samples were prepared by solvent exchange to remove any intense solvent signal in the 1H NMR spectra. The method was demonstrated on valine aqueous solutions of different concentrations. The accuracy of the measured concentrations that fell well within the range of the expanded uncertainty is also discussed. All of the results are in good agreement with the preparation values. We believe that this approach should be useful to determine the concentrations of standard solutions whose solute components are difficult to weigh because of extremely small amount or hygroscopicity.

Impact of Azithromycin on the Quorum Sensing-Controlled Proteome of Pseudomonas aeruginosa.

The macrolide antibiotic, azithromycin (AZM), has been reported to improve the clinical outcome of cystic fibrosis patients, many of whom are chronically-infected with Pseudomonas aeruginosa. However, the highest clinically-achievable concentrations of this drug are well-below the minimum inhibitory concentration for P. aeruginosa, raising the question of why AZM exhibits therapeutic activity. One possibility that has been raised by earlier studies is that AZM inhibits quorum sensing (QS) by P. aeruginosa. To explicitly test this hypothesis the changes brought about by AZM treatment need to be compared with those associated with specific QS mutants grown alongside in the same growth medium, but this has not been done. In this work, we used quantitative 2D-difference gel electrophoresis and 1H-NMR spectroscopy footprint analysis to examine whether a range of clinically-relevant AZM concentrations elicited proteomic and metabolomic changes in wild-type cultures that were similar to those seen in cultures of defined QS mutants. Consistent with earlier reports, over half of the AZM-induced spot changes on the 2D gels were found to affect QS-regulated proteins. However, AZM modulated very few protein spots overall (compared with QS) and collectively, these modulated proteins comprised only a small fraction (12–13%) of the global QS regulon. We conclude that AZM perturbs a sub-regulon of the QS system but does not block QS per se. Reinforcing this notion, we further show that AZM is capable of attenuating virulence factor production in another Gram-negative species that secretes copious quantities of exoenzymes (Serratia marcescens), even in the absence of a functional QS system.