Towards single cell ICP-MS normalized quantitative experiments using certified selenized yeast.

In the search for a normalized procedure to replicate and compare single cell-inductively coupled plasma-mass spectrometry (SC-ICP-MS) experiments, SELM-1, a certified reference material containing selenium enriched yeast cells has been used. Selenium concentrations (both, intra- and extracellular) have been measured using either sequential or simultaneous procedures. Regarding quantitative results, the sequential procedure involving cell washing followed by freeze drying of the washed material and intracellular Se quantification using SC-ICP-MS provided best results. In this case, intracellular Se accounted for 1304 ± 48 mg kg-1 (corresponding to 64% of the certified Se content). The average mass of Se per yeast cell was 41.6 fg Se with a dispersion of 1.6-279 fg Se/cell. In the isolated extracellular Se fraction, the Se concentration accounted for 412 ± 48 mg kg-1 (about 21% of the total Se). Thus, the sequential procedure provided a total Se recovery of about 85% with respect to the certified value. The direct dilution and simultaneous measurement of intra- and extracellular Se by SC-ICP-MS provided results of 1024 ± 42 mg kg-1 for intracellular and 316 ± 30 mg kg-1 for extracellular Se representing a total recovery of about 66%. In both cases, an initial thorough characterization of the cell density per solid weighed material was conducted by flow cytometry and the cell integrity ensured using confocal microscopy. These results clearly demonstrated that with appropriate sample preparation, SC-ICP-MS is a unique tool, which is capable of providing quantitative information about intracellular and extracellular Se. In addition, SELM-1 seems the ideal tool to enable data normalization at the single cell level to replicate, benchmark, and improve new SC-ICP-MS studies by using the same material for data validation.

Addressing the presence of biogenic selenium nanoparticles in yeast cells: analytical strategies based on ICP-TQ-MS.

Several organisms have demonstrated the ability of synthesising biogenic selenium-containing nanoparticles. Such particles from biological sources have attracted great attention due to several proven activities as antioxidants or antimicrobial agents. However, little is known in terms of size (distribution), shapes, chemical composition and number/amount/concentration of these particles. Therefore, in this work, we proposed the use of complementary analytical strategies that enabled the detection and characterization of selenium-containing nanoparticles in selenized yeast (Saccharomyces cerevisiae). The first strategy to address the intracellular presence of Se within yeast cells, involves the use of single cell ICP-TQ-MS (inductively coupled plasma-mass spectrometry). For this aim, selenium and phosphorous (as constitutive element) were measured as oxides (80Se16O+ and 31P16O+, resp.) in the triple-quadrupole mode. Then, a simple and fast cell lysis by mechanical disruption is conducted (approx. 30 min) in order to prove the presence of selenium-containing nanoparticles (SeNPs). The lysate is analysed by single particle ICP-TQ-MS and, complementarily, by liquid chromatography coupled to ICP-TQ-MS to cover a wider range of particle sizes. One of the samples revealed the presence of dispersed SeNPs with sizes between a few nm and up to 250 nm also confirmed by transmission electron microscopy (TEM) in the form of elemental selenium. The analysis of the certified reference material SELM-1 showed the presence of spherical SeNPs of 4 to 7 nm diameter. These biogenic particles, at least partially, were made of elemental selenium as well. The whole study reveals the excellent capabilities of "single" event ICP-MS methodologies in combination with HPLC-based strategies for a complete characterization of nanoparticulated material in biological samples.

Quantification of nitrite and nitrate in seawater by triethyloxonium tetrafluoroborate derivatization-headspace SPME GC-MS.

Triethyloxonium tetrafluoroborate derivatization combined with direct headspace (HS) or SPME-gas chromatography-mass spectrometry (GC-MS) is proposed here for the simultaneous determination of nitrite and nitrate in seawater at micromolar level after conversion to their corresponding volatile ethyl-esters (EtO-NO and EtO-NO(2)). Isotopically enriched nitrite [(15)N] and nitrate [(15)N] are employed as internal standards and for quantification purposes. HS-GC-MS provided instrumental detection limits of 0.07 µM NO(2)(-) and 2 µM NO(3)(-). Validation of the methodology was achieved by determination of nitrite and nitrate in MOOS-1 (Seawater Certified Reference Material for Nutrients, NRC Canada), yielding results in excellent agreement with certified values. All critical aspects connected with the potential inter-conversion between nitrite and nitrate (less than 10%) were evaluated and corrected for by the use of the isotopically enriched internal standard.

Full-field transmission x-ray microscopy for bio-imaging.

A full-field hard-x-ray microscope at SSRL has successfully imaged samples of biological and environmental origin at 40 nm resolution. Phase contrast imaging of trabeculae from a female mouse tibia, loaded in vivo to study the effects of weight-bearing on bone structure, revealed a complex network of osteocytes and canaliculi. Imaging of cordgrass roots exposed to mercury revealed nanoparticles with strong absorption contrast. 3D tomography of yeast cells grown in selenium rich media showed internal structure.

Unsaturated fatty acids isolated from human lipoproteins activate protein phosphatase type 2Cbeta and induce apoptosis in endothelial cells.

Activity of serine/threonine protein phosphatase type 2C is known to be stimulated by certain unsaturated fatty acids and this enzyme dephosphorylates Bad, thus acting on apoptosis. This prompted us to investigate endothelial cell death. Here, we present evidence for the presence of protein phosphatase type 2Cbeta (PP2Cbeta) in human umbilical vein endothelial cells (HUVECs) and report on colocalization of PP2Cbeta and Bad in the cytosol of endothelial cells. Lipophilic compounds that stimulated PP2Cbeta activity in vitro were found to induce cell death of HUVECs. Lipoproteins did neither influence PP2Cbeta activity nor affect cell behaviour. Lipoproteins treated with the lipoprotein lipase, however, stimulated the activity of PP2Cbeta at least 10-fold concomitantly triggering cell death. Analytical methods revealed that both effects - stimulation of PP2Cbeta and apoptosis - were caused by free fatty acids liberated from VLDL, LDL and HDL with oleic acid and linoleic acid as major constituents. The results provide novel insights in endothelial apoptosis and suggest that PP2Cbeta participates in the development and progress of atherosclerosis.

Identification of selenomethionine in selenized yeast using two-dimensional liquid chromatography-mass spectrometry based proteomic analysis.

Selenium-enriched yeast has been commonly used as a nutritional supplement. Here we describe a protocol used to investigate the metabolic fate of inorganic selenium in yeast. We provide definitive, mass spectrometry based evidence for the non-specific incorporation of selenomethionine in the yeast proteome involving the replacement of about 30% of all methionine with selenomethionine.

Automated in-tube solid-phase microextraction coupled with liquid chromatography-electrospray ionization mass spectrometry for the determination of selected benzodiazepines.

A simple, rapid, and sensitive method, which allowed us to simultaneously determine seven benzodiazepines (diazepam, nordiazepam, temazepam, oxazepam, 7-aminoflunitrazepam, N-desmethylflunitrazepam, and clonazepam) in buffer solution and in urine and serum samples, was investigated by automated in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). In-tube SPME, in which the analytes were extracted from the sample directly into an open tubular capillary column by repeated draw/eject cycles of sample solution, is an extraction technique for organic compounds in aqueous samples. The separation of benzodiazepines was carried out under ion-suppressed reversed-phase conditions by using methanol/50mM ammonium acetate in water (60:40) as a mobile phase with a Supelco LC-18 column. The optimal extraction condition was 10 draw/eject cycles of 30 mL of sample in 100mM Tris-HCl (pH 8.5) at a flow rate of 0.3 mL/min using a piece of 60-cm length Supelco-Q plot capillary column as the extraction capillary. The quantitative study was explored by operating in selected-ion monitoring (SIM) mode. The calibration curves were linear in the range from 0.5 ng/mL or 2 ng/mL to 500 ng/mL. The detection limits were from 0.02 ng/mL to 2 ng/mL. At the optimized capillary and fragmentor voltages, the characteristic ions for each compound clearly showed up in the spectra and it is possible to use the LC-MS to identify these compounds. The method was applied to the analysis of biological samples without interfering peaks. However, the recoveries for some of the compounds in serum samples need to be further improved.

Speciation of dimethylarsinic acid and monomethylarsonic acid by solid-phase microextraction-gas chromatography-ion trap mass spectrometry.

A solid-phase microextraction (SPME) method has been developed to determine two methylated arsenic species in human urine samples by GC-MS. The direct extraction of the methyl arsenic compounds by SPME after thioglycol methylate derivatization was studied. Direct extraction with SPME was suitable for the determination of trace levels of dimethylarsinic acid (DMA) and monomethylarsonic acid (MMA) in urine samples. Four different commercial SPME fibers were tested for the extraction of methyl arsenic compounds, and the best results were obtained using the polydimethylsiloxane coating. The extraction and desorption time profiles of DMA and MMA were determined. The detection limits for DMA and MMA using the SPME-GC-MS method were 0.12 and 0.29 ng/ml, respectively. The method is linear in the 1 to 200 ng/ml range.

Electrospray mass spectrometry of trimethyllead and triethyllead with in-tube solid phase microextraction sample introduction

A study of positive ionization electrospray mass spectrometry (ES-MS) was performed on trimethyllead (TML) and triethyllead (TEL). The system consisted of in-tube solid phase microextraction (SPME) coupled directly to an electrospray mass spectrometer. Fragmentation patterns of compounds were observed by applying different fragmentation voltages. High voltages produced sufficient fragmentation to elucidate the dissociation of the trialkyllead compounds. Electrospray mass spectrometry has been shown to be a suitable detection system for organolead speciation. Applying fragmentation energy programming, it might be possible to obtain in parallel the molecular and atomic signals of lead compounds. Copyright 1999 John Wiley & Sons, Ltd.

Simultaneous determination of dimethylarsinic acid and monomethylarsonic acid after derivatization with thioglycol methylate by gas chromatography/mass spectrometry.

A gas chromatography/mass spectrometry (GC/MS) method has been developed to determine two methylated arsenic species in human urine samples. The yield of derivatization for dimethylarsinic acid (DMA) and monomethylarsonic acid (MMA) using thioglycol methylate (TGM) was measured. The detection limit for the derivatized DMA and MMA using the GC/MS method are 0.95 and 0.8 ng cm-3, respectively. This simple and rapid method has good precision and accuracy. Fragmentation routes of derivatized MMA and DMA are suggested on accurate mass measurements.

[A statistical epidemiologic study of arterial pressure in schoolchildren in Harghita County to establish normal values]. / Studiu statistico-epidemiologic al tensiunii arteriale la scolari în vederea stabilirii valorilor normale pentru judetul Harghita.

The epidemiological study was organized in schools in urban and rural areas. The study is readily reproducible and can be carried out longitudinally in the course of several years. Blood pressure was determined in children ranging in age from 7 to 14 years, and included 1838 children from urban areas--954 boys and 884 girls, and 1622 from rural areas--838 boys and 784 girls, per total 3460 children. Recording was done in the classroom in a quiet, friendly atmosphere, eliminating all psychical factors that might influence pressure, and was never taken before or after physical education. The pressure gage was applied on the right arm the cuff being adjusted to a child's arm. Pressure was determined thrice by two doctors independently, according to the Korotkov method, phase IV representing diastolic pressure. The data were processed statistically calculating the mean and standard deviation per determinations, per age, and finally calculating the proportions (individual values in the range of initial variations that correspond to percentages of 5, 25, 50, 75, 90, 95) of the total population analyzed. The tables give the results of systolic and diastolic pressure values in children in urban and in rural areas, as well as percentile curves of the systolic and diastolic pressure per sex. On the basis of the results children were considered to be candidates to high blood pressure whose systolic and/or diastolic tension in terms of age and sex was situated at percentage 95 or higher.