Development of a liquid chromatographic time-of-flight mass spectrometric method for the determination of unlabelled and deuterium-labelled alpha-tocopherol in blood components

A method is described for the analysis of deuterated and undeuterated alpha-tocopherol in blood components using liquid chromatography coupled to an orthogonal acceleration time-of-flight (TOF) mass spectrometer. Optimal ionisation conditions for undeuterated (d0) and tri- and hexadeuterated (d3 or d6) alpha-tocopherol standards were found with negative ion mode electrospray ionisation. Each species produced an isotopically resolved single ion of exact mass. Calibration curves of pure standards were linear in the range tested (0-1.5 muM, 0-15 pmol injected). For quantification of d0 and d6 in blood components following a standard solvent extraction, a stable-isotope-labelled internal standard (d3-alpha-tocopherol) was employed. To counter matrix ion suppression effects, standard response curves were generated following identical solvent extraction procedures to those of the samples. Within-day and between-day precision were determined for quantification of d0- and d6-labelled alpha-tocopherol in each blood component and both averaged 3-10%. Accuracy was assessed by comparison with a standard high-performance liquid chromatography (HPLC) method, achieving good correlation (r(2) = 0.94), and by spiking with known concentrations of alpha-tocopherol (98% accuracy). Limits of detection and quantification were determined to be 5 and 50 fmol injected, respectively. The assay was used to measure the appearance and disappearance of deuterium-labelled alpha-tocopherol in human blood components following deuterium-labelled (d6) RRR-alpha-tocopheryl acetate ingestion. The new LC/TOFMS method was found to be sensitive, required small sample volumes, was reproducible and robust, and was capable of high throughput when large numbers of samples were generated. Copyright (C) 2003 John Wiley Sons, Ltd.

Proteomic analysis of the site specificity of glycation and carboxymethylation of ribonuclease

Proteomic analysis using electrospray liquid chromatography-mass spectrometry (ESI-LC-MS) has been used to compare the sites of glycation (Amadori adduct formation) and carboxymethylation of RNase and to assess the role of the Amadori adduct in the formation of the advanced glycation end-product (AGE), N-is an element of-(carboxymethyl)lysine (CIVIL). RNase (13.7 mg/mL, 1 mM) was incubated with glucose (0.4 M) at 37 degreesC for 14 days in phosphate buffer (0.2 M, pH 7.4) under air. On the basis of ESI-LC-MS of tryptic peptides, the major sites of glycation of RNase were, in order, K41, K7, K1, and K37. Three of these, in order, K41, K7, and K37 were also the major sites of CIVIL formation. In other experiments, RNase was incubated under anaerobic conditions (1 mM DTPA, N-2 purged) to form Amadori-modified protein, which was then incubated under aerobic conditions to allow AGE formation. Again, the major sites of glycation were, in order, K41, K7, K1, and K37 and the major sites of carboxymethylation were K41, K7, and K37. RNase was also incubated with 1-5 mM glyoxal, substantially more than is formed by autoxidation of glucose under experimental conditions, but there was only trace modification of lysine residues, primarily at K41. We conclude the following: (1) that the primary route to formation of CIVIL is by autoxidation of Amadori adducts on protein, rather than by glyoxal generated on autoxidation of glucose; and (2) that carboxymethylation, like glycation, is a site-specific modification of protein affected by neighboring amino acids and bound ligands, such as phosphate or phosphorylated compounds. Even when the overall extent of protein modification is low, localization of a high proportion of the modifications at a few reactive sites might have important implications for understanding losses in protein functionality in aging and diabetes and also for the design of AGE inhibitors.

Compact cosmic ray detector for unattended atmospheric ionisation monitoring

Two vertical cosmic ray telescopes for atmospheric cosmic ray ionization event detection are compared. Counter A, designed for low power remote use, was deployed in the Welsh mountains; its event rate increased with altitude as expected from atmospheric cosmic ray absorption. Independently, Counter B’s event rate was found to vary with incoming particle acceptance angle. Simultaneous colocated comparison of both telescopes exposed to atmospheric ionization showed a linear relationship between their event rates.

Synthesis and structural studies of a bis(carbamoyl methyl) sulfoxide complex of uranyl nitrate

A new tri-functional ligand iBu2NCOCH2SOCH2CONiBu2 was prepared and characterized. The coordination chemistry of this ligand with uranyl nitrate was studied with IR, 1H NMR, electrospray mass-spectrometry, thermogravimetry, and elemental analysis. The structure of [UO2(NO3)2(iBu2NCOCH2SOCH2CONiBu2)] was determined by single-crystal X-ray diffraction. The uranium(VI) ion is surrounded by eight oxygens in a hexagonal bipyramidal geometry. Four oxygens from two nitrates and two oxygens from the ligand form a planar hexagon. The ligand is a bidentate chelate, bonding through sulfoxo and one of the carbamoyl groups to uranyl nitrate.

LC-MS & DFT investigations of Cryptolepis sanginolenta root extracts

The roots of Crytolepis sanguinolenta, a medicinally important ethanobotanical source of the antimalarial cryptolepine, were soxhlet extracted in anaerobic conditions, using hexane then ethanol. Samples of each extract were fractioned using flash chromatography and preparative TLC and compound identity was established using gradient HPLC-positive ion electrospray mass spectrometry. The use of argon depressed the formation of quindoline and hydroxycrytolepine. In addition to known compounds such as cryptolepine, several as yet unidentified compounds remain to be characterised in this root extract.

A study of the atmospherically relevant reaction between molecular chlorine and dimethylsulfide (DMS): establishing the reaction intermediate and measurement of absolute photoionization cross-sections

In a study using UV photoelectron spectroscopy (PES) of the atmospherically relevant reaction CH3SCH3 + Cl2 → CH3SCH2Cl + HCl bands associated with a reaction intermediate have been observed. These have been assigned to ionization of the covalently bound molecule (CH3)2SCl2 on the basis of the intensity of the observed bands as a function of reaction time, molecular orbital calculations of vertical ionization energies and evidence from infrared spectroscopy. A method has also been developed, with the flow-tube/PE spectrometer combination used, to measure photoionization cross-sections of the reagents and products at the photon energy utilized and this has allowed the photoionization cross-section of the intermediate to be estimated. This work augments an earlier study in which the rate constant of the reaction between CH3SCH3 (DMS) and Cl2 has been measured at room temperature.

Electromagnetic atmosphere-plasma coupling: the global atmospheric electric circuit

A description is given of the global atmospheric electric circuit operating between the Earth’s surface and the ionosphere. Attention is drawn to the huge range of horizontal and vertical spatial scales, ranging from 10−9 m to 1012 m, concerned with the many important processes at work. A similarly enormous range of time scales is involved from 10−6 s to 109 s, in the physical effects and different phenomena that need to be considered. The current flowing in the global circuit is generated by disturbed weather such as thunderstorms and electrified rain/shower clouds, mostly occurring over the Earth’s land surface. The profile of electrical conductivity up through the atmosphere, determined mainly by galactic cosmic ray ionization, is a crucial parameter of the circuit. Model simulation results on the variation of the ionospheric potential, ∼250 kV positive with respect to the Earth’s potential, following lightning discharges and sprites are summarized. Experimental results comparing global circuit variations with the neutron rate recorded at Climax, Colorado, are then discussed. Within the return (load) part of the circuit in the fair weather regions remote from the generators, charge layers exist on the upper and lower edges of extensive layer clouds; new experimental evidence for these charge layers is also reviewed. Finally, some directions for future research in the subject are suggested.

Mass spectrometry imaging of glucosinolates in arabidopsis flowers and siliques

Glucosinolates are multi-functional plant secondary metabolites which play a vital role in plant defence and are, as dietary compounds, important to human health and livestock well-being. Knowledge of the tissue-specific regulation of their biosynthesis and accumulation is essential for plant breeding programs. Here, we report that in Arabidopsis thaliana, glucosinolates are accumulated differentially in specific cells of reproductive organs. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI), distribution patterns of three selected compounds, 4-methylsulfinylbutyl (glucoraphanin), indol-3-ylmethyl (glucobrassicin), and 4-benzoyloxybutyl glucosinolates, were mapped in the tissues of whole flower buds, sepals and siliques. The results show that tissue localization patterns of aliphatic glucosinolate glucoraphanin and 4-benzoyloxybutyl glucosinolate were similar, but indole glucosinolate glucobrassicin had different localisation, indicating a possible difference in function. The high resolution images obtained by a complementary approach, cryo-SEM Energy Dispersive X-ray analysis (cryo-SEM-EDX), confirmed increased concentration of sulphur in areas with elevated amounts of glucosinolates, and allowed identifying the cell types implicated in accumulation of glucosinolates. High concentration of sulphur was found in S-cells adjacent to the phloem in pedicels and siliques, indicating the presence of glucosinolates. Moreover, both MALDI MSI and cryo-SEM-EDX analyses indicated accumulation of glucosinolates in cells on the outer surface of the sepals, suggesting that a layer of glucosinolate-accumulating epidermal cells protects the whole of the developing flower, in addition to the S-cells, which protect the phloem. This research demonstrates the high potential of MALDI MSI for understanding the cell-specific compartmentation of plant metabolites and its regulation.

Steric effects on uranyl complexation: synthetic, structural, and theoretical studies of carbamoyl pyrazole compounds of the uranyl(VI) ion

New bifunctional pyrazole based ligands of the type [C3HR2N2CONR'] (where R = H or CH3; R' = CH3, C2H5, or (C3H7)-C-i) were prepared and characterized. The coordination chemistry of these ligands with uranyl nitrate and uranyl bis(dibenzoyl methanate) was studied with infrared (IR), H-1 NMR, electrospray-mass spectrometry (ES-MS), elemental analysis, and single crystal X-ray diffraction methods. The structure of compound [UO2(NO3)(2)(C3H3N2CON{C2H5}(2))] (2) shows that the uranium(VI) ion is surrounded by one nitrogen atom and seven oxygen atoms in a hexagonal bipyramidal geometry with the ligand acting as a bidentate chelating ligand and bonds through both the carbamoyl oxygen and pyrazolyl nitrogen atoms. In the structure of [UO2(NO3)(2)(H2O)(2)(C5H7N2CON {C2H5}(2))(2)], (5) the pyrazole figand acts as a second sphere ligand and hydrogen bonds to the water molecules through carbamoyl oxygen and pyrazolyl nitrogen atoms. The structure of [UO2(DBM)(2)C3H3N2CON{C2H5}(2)] (8) (where DBM = C6H5COCHCOC6H5) shows that the pyrazole ligand acts as a monodentate ligand and bonds through the carbamoyl oxygen to the uranyl group. The ES-MS spectra of 2 and 8 show that the ligand is similarly bonded to the metal ion in solution. Ab initio quantum chemical studies show that the steric effect plays the key role in complexation behavior.

Energetic charged particles above thunderclouds

The French government has committed to launch the satellite TARANIS to study transient coupling processes between the Earth’s atmosphere and near-Earth space. The prime objective of TARANIS is to detect energetic charged particles and hard radiation emanating from thunderclouds. The British Nobel prize winner C.T.R. Wilson predicted lightning discharges from the top of thunderclouds into space almost a century ago. However, new experiments have only recently confirmed energetic discharge processes which transfer energy from the top of thunderclouds into the upper atmosphere and near-Earth space; they are now denoted as transient luminous events, terrestrial gamma-ray flashes and relativistic electron beams. This meeting report builds on the current state of scientific knowledge on the physics of plasmas in the laboratory and naturally occurring plasmas in the Earth’s atmosphere to propose areas of future research. The report specifically reflects presentations delivered by the members of a novel Franco-British collaboration during a meeting at the French Embassy in London held in November 2011. The scientific subjects of the report tackle ionization processes leading to electrical discharge processes, observations of transient luminous events, electromagnetic emissions, energetic charged particles and their impact on the Earth’s atmosphere. The importance of future research in this area for science and society, and towards spacecraft protection, is emphasized.

MALDI MS profiling of post-DRE urine samples highlights the potential of b-Microseminoprotein as a marker for prostatic diseases

BACKGROUND. To use spectra acquired by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) from pre- and post-digital rectal examination (DRE) urine samples to search for discriminating peaks that can adequately distinguish between benign and malignant prostate conditions, and identify the peaks’ underlying biomolecules. METHODS. Twenty-five participants with prostate cancer (PCa) and 27 participants with a variety of benign prostatic conditions as confirmed by a 10-core tissue biopsy were included. Pre- and post-DRE urine samples were prepared for MALDI MS profiling using an automated clean-up procedure. Following mass spectra collection and processing, peak mass and intensity were extracted and subjected to statistical analysis to identify peaks capable of distinguishing between benign and cancer. Logistic regression was used to combine markers to create a sensitive and specific test. RESULTS. A peak at m/z 10,760 was identified as b-microseminoprotein (b-MSMB) and found to be statistically lower in urine from PCa participants using the peak’s average areas. By combining serum prostate-specific antigen (PSA) levels with MALDI MS-measured b-MSMB levels, optimum threshold values obtained from Receiver Operator characteristics curves gave an increased sensitivity of 96% at a specificity of 26%. CONCLUSIONS. These results demonstrate that with a simple sample clean-up followed by MALDI MS profiling, significant differences of MSMB abundance were found in post-DRE urine samples. In combination with PSA serum levels, obtained from a classic clinical assay led to high classification accuracy for PCa in the studied sample set. Our results need to be validated in a larger multicenter prospective randomized clinical trial.

Detection of lower tropospheric responses to solar energetic particles at midlatitudes

Solar energetic particles (SEPs) occasionally contribute additional atmospheric ionization beyond that arising from the usual galactic cosmic ray background. During an SEP event associated with a solar flare on April 11, 2013, the vertical ionization rate profile obtained using a balloon-borne detector showed enhanced ionization with a 26% increase at 20 km, over Reading, United Kingdom. Fluctuations in atmospheric electrical parameters were also detected at the surface, beneath the balloon’s trajectory. As no coincident changes in geomagnetism occurred, the electrical fluctuations are very likely to be associated with increased ionization, as observed by the balloon measurements. The lack of response of surface neutron monitors during this event indicates that energetic particles that are not detected at the surface by neutron monitors can nevertheless enter and influence the atmosphere’s weather-generating regions.

Gut permeability in autism spectrum disorders

Objective To test whether gut permeability is increased in autism spectrum disorders (ASD) by evaluating gut permeability in a population-derived cohort of children with ASD compared with age- and intelligence quotient-matched controls without ASD but with special educational needs (SEN). Patients and Methods One hundred thirty-three children aged 10–14 years, 103 with ASD and 30 with SEN, were given an oral test dose of mannitol and lactulose and urine collected for 6 hr. Gut permeability was assessed by measuring the urine lactulose/mannitol (L/M) recovery ratio by electrospray mass spectrometry-mass spectrometry. The ASD group was subcategorized for comparison into those without (n = 83) and with (n = 20) regression. Results There was no significant difference in L/M recovery ratio (mean (95% confidence interval)) between the groups with ASD: 0.015 (0.013–0.018), and SEN: 0.014 (0.009–0.019), nor in lactulose, mannitol, or creatinine recovery. No significant differences were observed in any parameter for the regressed versus non-regressed ASD groups. Results were consistent with previously published normal ranges. Eleven children (9/103 = 8.7% ASD and 2/30 = 6.7% SEN) had L/M recovery ratio > 0.03 (the accepted normal range cut-off), of whom two (one ASD and one SEN) had more definitely pathological L/M recovery ratios > 0.04. Conclusion There is no statistically significant group difference in small intestine permeability in a population cohort-derived group of children with ASD compared with a control group with SEN. Of the two children (one ASD and one SEN) with an L/M recovery ratio of > 0.04, one had undiagnosed asymptomatic celiac disease (ASD) and the other (SEN) past extensive surgery for gastroschisis.

Sondrestrom and EISCAT radar observations of poleward-moving auroral forms

During many magnetospheric substorms, the auroral oval near midnight is observed to expand poleward in association with strong negative perturbations measured by local ground magnetometers. We show Sondrestrom and EISCAT incoherent scatter radar measurements during three such events. In each of the events, enhanced ionization produced by the precipitation moved northward by several degrees of latitude within 10–20 min. The electric fields measured during the three events were significantly different. In one event the electric field was southward everywhere within the precipitation region. In the other two events a reversal in the meridional component of the field was observed. In one case the reversal occurred within the precipitation region, while in the other case the reversal was at the poleward boundary of the precipitation. The westward electrojet that produces the negative H-perturbation in the ground magnetic field has Hall and Pedersen components to varying degrees. In one case the Hall component was eastward and the Pedersen component was westward, but the net magnetic H-deflection on the ground was negative. Simultaneous EISCAT measurements made near the dawn meridian during one of the events show that the polar cap boundary moved northward at the same time as the aurora expanded northward at Sondrestrom. Most of the differences in the electrodynamic configuration in the three events can be accounted for in terms of the location at which the measurements were made relative to the center of the auroral bulge.

Comparisons between EISCAT observations and model calculations of the high latitude ionosphere

Calculations using a numerical model of the convection dominated high latitude ionosphere are compared with observations made by EISCAT as part of the UK-POLAR Special Programme. The data used were for 24–25 October 1984, which was characterized by an unusually steady IMF, with Bz < 0 and By > 0; in the calculations it was assumed that a steady IMF implies steady convection conditions. Using the electric field models of Heppner and Maynard (1983) appropriate to By > 0 and precipitation data taken from Spiroet al. (1982), we calculated the velocities and electron densities appropriate to the EISCAT observations. Many of the general features of the velocity data were reproduced by the model. In particular, the phasing of the change from eastward to westward flow in the vicinity of the Harang discontinuity, flows near the dayside throat and a region of slow flow at higher latitudes near dusk were well reproduced. In the afternoon sector modelled velocity values were significantly less than those observed. Electron density calculations showed good agreement with EISCAT observations near the F-peak, but compared poorly with observations near 211 km. In both cases, the greatest disagreement occurred in the early part of the observations, where the convection pattern was poorly known and showed some evidence of long term temporal change. Possible causes for the disagreement between observations and calculations are discussed and shown to raise interesting and, as yet, unresolved questions concerning the interpretation of the data. For the data set used, the late afternoon dip in electron density observed near the F-peak and interpreted as the signature of the mid-latitude trough is well reproduced by the calculations. Calculations indicate that it does not arise from long residence times of plasma on the nightside, but is the signature of a gap between two major ionization sources, viz. photoionization and particle precipitation.