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Vanadium (V) is a trace mineral whose biological activity, role as a micronutrient, and pharmacotherapeutic applications remain unknown. Over the last years, interest in V has increased due to its potential use as an antidiabetic agent mediated by its ability to improve glycemic metabolism. However, some toxicological aspects limit its potential therapeutic application. The present study aims to evaluate the effect of the co-treatment with copper (Cu) and bis(maltolato)oxovanadium(IV) (BMOV) as a possible strategy to reduce the toxicity of BMOV. Treating hepatic cells with BMOV reduced cell viability under the present conditions, but cell viability was corrected when cells were co-incubated with BMOV and Cu. Additionally, the effect of these two minerals on nuclear and mitochondrial DNA was evaluated. Co-treatment with both metals reduced the nuclear damage caused by BMOV. Moreover, treatment with these two metals simultaneously tended to reduce the ND1/ND4 deletion of the mitochondrial DNA produced with the treatment using BMOV alone. In conclusion, these results showed that combining Cu and V could effectively reduce the toxicity associated with V and enhance its potential therapeutic applications.

Background: The objective of this study was to evaluate the accumulation of ions in blood and organs caused by Ti metal particles in a mandibular defect in rats, together with a description of the local reaction of oral tissues to these titanium alloy debris. Methods: Twenty Sprague-Dawley rats were randomly distributed into three groups: an experimental group with a mandibular bone defect filled with metallic debris obtained by implantoplasty; a positive control group; and a negative control group. Thirty days after surgery, the rats were euthanized and perilesional tissue surrounding the mandibular defect was removed, together with the lungs, spleen, liver and brain. Two blood samples were collected: immediately before surgery and before euthanasia. The perilesional tissue was histologically analyzed using hematoxylin-eosin staining, and titanium, aluminum and vanadium ion concentrations in blood and organs were measured by TQ-ICP-MS. Descriptive and bivariate analyses of the data were performed. Results: All rats with implanted metal debris showed metal particles and a bone fracture callus on the osseous defect. The metal particles were surrounded by a foreign body reaction characterized by the presence of histiocytes and multinucleated giant cells. The experimental group had a significant higher concentration of Ti ions in all studied organs except lung tissue (p < 0.05). In addition, there were more V ions in the brain in the experimental group (p = 0.008). Conclusions: Although further studies are required to confirm the clinical relevance of these results, Ti metal particles in the jaw might increase the concentration of metal ions in vital organs and induce a foreign body reaction. This article is protected by copyright. All rights reserved. Keywords: Ion release; Ti6Al4V; implantoplasty; metal particles; peri-implantitis; titanium.

Streptomycetes are important biotechnological bacteria that produce several clinically bioactive compounds. They have a complex development, including hyphae differentiation and sporulation. Cytosolic copper is a well-known modulator of differentiation and secondary metabolism. The interruption of the Streptomyces coelicolor SCO2730 (copper chaperone, SCO2730::Tn5062 mutant) blocks SCO2730 and reduces SCO2731 (P-type ATPase copper export) expressions, decreasing copper export and increasing cytosolic copper. This mutation triggers the expression of 13 secondary metabolite clusters, including cryptic pathways, during the whole developmental cycle, skipping the vegetative, non-productive stage. As a proof of concept, here, we tested whether the knockdown of the SCO2730/31 orthologue expression can enhance secondary metabolism in streptomycetes. We created a SCO2730/31 consensus antisense mRNA from the sequences of seven key streptomycetes, which helped to increase the cytosolic copper in S. coelicolor, albeit to a lower level than in the SCO2730::Tn5062 mutant. This antisense mRNA affected the production of at least six secondary metabolites (CDA, 2-methylisoborneol, undecylprodigiosin, tetrahydroxynaphtalene, α-actinorhodin, ε-actinorhodin) in the S. coelicolor, and five (phenanthroviridin, alkylresorcinol, chloramphenicol, pikromycin, jadomycin G) in the S. venezuelae; it also helped to alter the S. albus metabolome. The SCO2730/31 consensus antisense mRNA designed here constitutes a tool for the knockdown of SCO2730/31 expression and for the enhancement of Streptomyces’ secondary metabolism.

A systematic investigation on the cellular uptake, intracellular dissolution, and in vitro biological effects of ultra-small (

Drought stress in plants causes differential expression of numerous genes. One of these differentially expressed genes in rice is a specific amidohydrolase. We characterized this amidohydrolase gene on the rice chromosome 12 as the first plant guanine deaminase (OsGDA1). The biochemical activity of GDA is known from tea and coffee plants where its catalytic product, xanthine, is the precursor for theine and caffeine. However, no plant gene that is coding for GDA is known so far. Recombinant OsGDA1 converted guanine to xanthine in vitro. Measurement of guanine and xanthine contents in the OsGDA1 knockout (KO) line and in the wild type Tainung 67 rice plants also suggested GDA activity in vivo. The content of cellular xanthine is important because of its catabolic products allantoin, ureides, and urea which play roles in water and nitrogen stress tolerance among others. The identification of OsGDA1 fills a critical gap in the S‐adenosyl‐methionine (SAM) to xanthine pathway. SAM is converted to S‐adenosyl‐homocysteine (SAH) and finally to xanthine. SAH is a potent inhibitor of DNA methyltransferases, the reduction of which leads to increased DNA methylation and gene silencing in Arabidopsis. We report that the OsGDA1 KO line exhibited a decrease in SAM, SAH and adenosine and an increase in rice genome methylation. The OsGDA1 protein phylogeny combined with mutational protein destabilization analysis suggested artificial selection for null mutants, which could affect genome methylation as in the KO line. Limited information on genes that may affect epigenetics indirectly requires deeper insights into such a role and effect of purine catabolism and related genetic networks.

Cisplatin is a chemotherapeutic drug widely used in the treatment of several tumours, but this chemotherapy presents problems in terms of side-effects and patient resistance. The detection and determination of cisplatin-induced adducts and the relationship with the physiological or clinical effects of this drug under different repair conditions could be a good measure to assess patient's response to such chemotherapy. A new methodological approach to detect and quantify cisplatin adducts by use of high-performance liquid chromatography with inductively coupled plasma mass-spectrometric detection (HPLC–ICP-MS) and isotope-dilution analysis (IDA), is evaluated for its application in vivo, under different repair conditions. This analysis is combined with the use of the Comet assay, which detects DNA strand-breaks, and the w/w+ SMART assay, which monitors induction of somatic mutation and recombination in Drosophila melanogaster in vivo under different conditions of nucleotide-excision repair. Results show that (i) cisplatin induces in Drosophila several adducts not detected in mammals. The two most abundant cisplatin-induced adducts, identified by electrospray-mass spectrometry as G monoadduct and G–G intrastrand cross-links, were quantified individually; (ii) cisplatin induces higher levels of G monoadducts and G–G cross-links in NER-proficient than in NER-deficient cells; (iii) the level of adducts correlates with their biological consequences, both in terms of DNA strand-breaks (tail-moment values), and of somatic mutation and recombination (frequency of mosaic eyes and clones in 104 cells), when the repair status is considered. This work demonstrates the validity and potential of the adduct detection and quantification methodology in vivo, and its use to correlate adducts with their genetic consequences.

Quantitative proteomics and absolute determination of proteins are topics of fast growing interest, since only the quantity of proteins or changes in their abundance reflect the status and extent of changes of a given biological system. Quantification of the desired proteins has been carried out by molecule specific MS techniques, but relative quantifications are commonplace so far even resorting to stable isotope labelling techniques such as ICAT and SILAC. In the last decade the idea of using element-selective mass spectrometric detection (e.g. ICP-MS instruments) to achieve absolute quantification has been realised and ICP-MS stands now as a new tool in the field of quantitative proteomics. In this review the emerging role of ICP-MS in protein and proteomic analysis is highlighted. The potential of ICP-MS methods and strategies for screening multiple heteroatoms (e.g. S, P, Se, metals) in proteins and their mixtures and extraordinary capabilities to tackle the problem of absolute protein quantifications, via heteroatom determinations, are discussed and illustrated. New avenues are also open derived from the use of ICP-MS for precise isotope abundance measurements in polyisotopic heteroatoms. The "heteroatom (isotope)-tagged proteomics" concept is focused on the use of naturally present element tags and also extended to any protein by resorting to bioconjugation reactions (i.e. labelling sought proteins and peptides with ICP-MS detectable heteroatoms). A major point of this review is displaying the possibilities of using a "hard" ion source, the ICP, to complement well-established "soft" ion sources for mass spectrometry to tackle present proteomic analysis.

Aims: To study the effects of the selenium enrichment protocols in yeast at various points in the cell cycle, total selenium accumulation and the forms of selenium incorporated. Methods and Results: The use of selenized yeast as enriched selenium supplements in human nutrition has become a topic of increasing interest over the last decade. Four enrichment procedures have been evaluated using sodium selenite as the selenium source: enrichment during the growth phase; enrichment at the non-growth phase, both of these at different selenium levels; enrichment by seeding in a fermentable carbon source (glucose); Se-enrichment with a non-fermentable carbon source (glycerol). A nitric acid digestion of the yeast samples prepared under different conditions has been performed in order to evaluate the total selenium incorporated into the yeast cells. Also, an enzymatic digestion of the yeast samples with pepsin has been carried out as an initial step to begin the process of determining which of the different possible selenium species are formed. The cell count evaluations of the selenium-enriched yeast showed that the growth phase, seeding and the use of YEPG media is influenced by the addition of Se, while the non-growth phase is not. Total selenium incorporation studies showed that seeding the yeast permits more accumulation of selenium. Speciation studies of the enriched yeast showed that the growth phase increases the formation of L-Se-methionine. Conclusions: When the aim of enriching yeast with selenium is the formation of L-Se-methionine, the best enrichment procedure is using the growth phase with small concentrations of sodium selenite. Significance and Impact of the Study: The use of selenium supplements is widespread and most of the supplements use selenium-enriched yeast in their formulation. Studies made on supplements do not have the appropriate Se-species for optimal absorption in the human body. This study presents and compares methods for the best selenium yeast enrichment that could ultimately be used in selenium supplement formulations.

The extraction/pre-concentration capabilities of the liquid ion exchanger 7-(4-ethyl-1-methyloctyl)-8-hydroxyquinoline (Kelex-100) immobilized on octadecyl silica have been evaluated for the analysis of trace metals in sea-water by quadruple ICP–MS (ICP–QMS) and the results are compared with those directly obtained by double focusing ICP–MS (ICP–SMS), after a simple 1+4 dilution of the sea-water sample. The retention capabilities of the different materials (Kelex-100 retained on Amberlite XAD-7 and Bondapack C-18 (BC-18)) were evaluated by using copper as test element and ICP–QMS for detection. Kelex-100 supported onto BC-18 provided the best performance and was evaluated, both on-line and off-line, for the pre-concentration of different trace elements present in sea-water. Using the selected “off-line” procedure, satisfactory recoveries (95–104%) and adequate detection limits in the ng l −1 range (V, 10; Ni, 30; Mn, 70; Cu, 170; Zn, 150; Cd, 7; Pb, 10 and U, 1 ng l −1 ) were obtained. Such multi-elemental trace metal determinations in sea-water were also attempted directly, after a simple 1+4 dilution of the sample and calibration with internal standards (IS) using different mass resolutions to avoid polyatomic interferences when necessary. The analytical performance characteristics and limitations of both approaches for multi-elemental determinations in coastal sea-water are compared.

A radiofrequency glow discharge (rf-GD) has been used as an ion source for mass spectrometry using gas chromatography as the sample introduction technique for the speciation of seleno amino acids. The use of helium as the plasma gas allowed the monitoring of selenium's major isotope (80Se), interfered by polyatomic species when using Ar as the plasma gas. Optimization of the power and pressure of the source was undertaken to achieve the best sensitivity for selenium determination and detection limits of about 100 pg (as Se) have been obtained for the derivatized Se-methionine. Studying the cell pressure and power on the fragmentation patterns obtained for Se-methionine and Se-ethionine has also been undertaken to characterize the source. No significant variations were observed on the mass spectra under different operating conditions, although noticeable variation of the fragment intensities was observed. The system has been applied to the determination of Se-methionine in selenium-enriched yeast after enzymatic extraction of the corresponding species using Proteinase K to cleave the peptide bonds. The results obtained showed a Se-methionine content of about 78% in the enriched yeast.

Two different approaches for the derivatisation of selenomethionine and detection by gas chromatography-inductively coupled plasma mass spectrometry (GC-ICP-MS) are compared and applied for the determination of the amino acid in pharmaceutical preparations used as selenium nutritional supplements. The first derivatisation is accomplished in two steps, starting with the esterification of the carboxylic acid group of the seleno-amino acid using propan-2-ol, followed by the acylation of the amino group with trifluoroacetic acid anhydride. Alternatively, the ethyl chloroformate–ethanol method was carried out for the simultaneous esterification and acylation steps catalysed by a pyridine solution. The derivatives were finally extracted into chloroform and injected into the gas chromatograph. The different parameters affecting the efficiency of both derivatisation approaches were optimised, as well as the instrumental operating conditions. Standard GC-MS chromatograms and mass spectra for both derivatisation procedures were also obtained to confirm the structure of the corresponding derivatised amino acids. The propan-2-ol–trifluoroacetic acid anhydride procedure was finally selected and was used to determine selenomethionine in a parenteral solution by GC-ICP-MS. The advantages and disadvantages of both procedures are discussed.

The analytical potential of complementary chromatographic separations such as size exclusion (SEC) plus anion-exchange chromatography coupled with inductively coupled plasma time-of-flight mass spectrometry (ICP-TOF-MS), was evaluated for the multi-elemental speciation of trace elements associated with methallothionein-like proteins (MLPs) in mussel hepatopancreas. The initial separation of the proteins is performed by 'off-line' SEC, collecting fractions which are then analysed for the trace elements by ICP-TOF-MS in small sample volumes. Further purification is accomplished by the separation of the different proteins contained in the above obtained MLP fraction on a Mono Q (HR 5/5) anion-exchange column [fast protein liquid chromatography (FPLC)], using an ammonium acetate gradient at a physiological pH of 7.4 as the mobile phase. The separated proteins are detected 'on-line' by UV absorption at 254 and 280 nm. Specific detection (16 metals) with on-line ICP-TOF-MS was also performed. This hybrid approach was applied to investigate the metal binding patterns of the proteins expressed by mussels which had been previously exposed to 16 trace metals in an aquarium tank for 1 and 2 weeks. The exceptional characteristics of ICP-TOF-MS for quasi-simultaneous monitoring of many trace elements in transient signals are illustrated by this 'on-line' coupling to FPLC separation, paving the way to virtually simultaneous multi-elemental speciation of trace metals in biological material.

The use of an inductively coupled plasma mass spectrometer (ICP-MS) with a time-of-flight (TOF) analyser is illustrated for the simultaneous determination of seven elements forming hydrides (As, Bi, Ge, Hg, Sb, Se and Sn) using flow injection (FI) as the sample introduction system. The chemical conditions for an adequate simultaneous volatile species generation as well as the plasma parameters for simultaneous determination were evaluated. The compromise hydride generation conditions were: 1% NaBH4 and 0.7 M HNO3 final concentration, while an Ar carrier gas flow rate of 1.1 l min−1 provided optimum results for the transport of the volatile species to the ICP-MS. Analytical figures of merit were evaluated under these compromise conditions and compared with those obtained by continuous nebulisation of the same elements in the ICP-MS(TOF). Detection limits in the low pg ml−1 region, linearity over more than five orders of magnitude and repeatability of 2–5% were achieved with the proposed methodology. Application of such a strategy was demonstrated by the simultaneous multi-elemental determination of As, Bi, Ge, Hg, Sb, Se and Sn in spiked urine samples with recoveries better than 80%. For Se, accurate results were only obtained by avoiding volatile species generation, demonstrating the presence of selenium species other than Se(IV) in human urine. The accuracy of the method was also checked by analysing a certified urine sample (NIST SRM 2670) by FI-ICP-MS(TOF) with satisfactory results.

The design, construction and performance characteristics of an easily removable interface to couple a gas chromatograph (GC) to an inductively coupled plasma mass spectrometer (ICP-MS) for trace element speciation is described. The interface is based on the use of a heated metallic tube where the last part of the capillary GC column is inserted. This metallic tube is connected to a metallic T-piece where a flow of argon carrier gas is introduced perpendicularly and externally to the metallic tube, creating a high velocity flow of intermediate sheath gas, which prevents condensation of the analytes eluting from the column on the walls of the T-piece or on the connection tubing to the ICP-MS torch. A flexible non-heated PTFE tube is used to connect the exit of the T-piece to the ICP-MS torch. This new interface has been applied to the separation and detection of different organometallic compounds of Se, S, Pb, Hg and Sn.

The analytical potential of fast protein liquid chromatography (FPLC) coupled with a double focusing inductively coupled plasma mass spectrometer (DF-ICP-MS) was evaluated for the multielemental speciation of trace elements in human serum. The separation of the main serum proteins was performed in a MonoQ (HR5/5) anion exchange column using an ammonium acetate gradient (0-0.5 mol l –1 ) at the physiological pH of 7.4 (0.05 mol l –1 TRIS-HCl buffer) as the mobile phase. Proteins were first detected on-line spectrophotometrically at 295 nm and specific detection of metals was also carried out on-line by using a double focusing ICP-MS working at both low (m/Δm=300) and medium resolution (m/Δm=3000) in order to avoid possible polyatomic interferences. The use of variable resolution to carry out multielement speciation studies allowed the detection of Ca, Sr, Fe, Cu, Zn, Se, Mn, Cr, Pb, Al and Sn in different serum samples and introduces a new dimension to this area of investigation. From the multielemental study it was observed that detectable levels of Cr, Al, Pb and Sn were present in uraemic sera while those elements could not be detected in healthy subjects.

A rapid and fairly accurate semiquantitative method for the determination of trace amounts of metals in water samples of low and high salinity was successfully applied to a double focusing inductively coupled plasma mass spectrometer. The method was used at both low (m/ Δm=300) and medium (m/Δm=3000) resolution, establishing the corresponding molar response curve in both modes. The ionisation conditions (Tionandne) were calculated through the Saha equation by adjusting the molar response of a multi-element solution to a third order polynomial equation by least squares. The validation of the proposed methodology was performed by elemental analysis of a Riverine Water reference material (SLRS-3) from the National Research Council of Canada. Results obtained were in good agreement with the certified values, working at low resolution when no polyatomic interferences were found and at medium resolution when necessary. Matrix effects were also evaluated using sea-water samples, after a 5-fold dilution. An increase in the analytical response for all the elements, used as internal standards, in comparison with the corresponding curve in Milli-Q water was observed. However, once the ionisation conditions had been calculated on the same saline matrix, recoveries of most of the elements under study were satisfactory for semiquantitative analysis.

A semiquantitative procedure for the determination of trace metals in natural waters and sediments by ICP-MS was developed. The method is based on the establishment of the instrumental molar response curveversus mass using a multi-elemental solution containing elements from Mg to Bi with different ionisation potentials. The ionisation conditions in the plasma (electron density, ne, and ionisation temperature, Tion) were determined by least-squares fitting, using the Saha equation, by adjusting the molar response curve to a third-order polynomial. The effect of matrix constituents, such as Na+, K+, Ca2+, Mg2+, Cl– and SO42–, on both the shape of the molar response curve and on the plasma ionisation conditions was evaluated. It was observed that the ionisation degrees calculated in the plasma did not change significantly in the presence of the matrix constituents. However, the shape of the molar response curve was affected by high concentrations of matrix components and this effect was corrected by adding internal standards to the samples. The semiquantitative procedure was applied to the analysis of local natural waters and the results were compared with those obtained by external calibration and standard additions. Validation was performed by analysis of riverine water (SLRS-3) and sediment (PACS-1 and MESS-2) reference materials from the National Research Council of Canada with satisfactory results.

For the analysis of biological and environmental materials by inductively coupled plasma mass spectrometry (ICP-MS), several quantification procedures can be used depending on the precision and accuracy required. Semiquantitative methods based on the molar response curve were compared with conventional external calibration and standard additions for the analysis of waters and sewage sludges. For the analysis of biological materials, where higher quality data were required, isotope dilution analysis using enriched isotopes was applied. It was observed that the molar sensitivity for different elements in ICP-MS was a simple function of the mass of the isotopes measured after normalization for ionization efficiency which could be fitted to a third-order polynomial equation. Element ionization adjustments for the third-order polynomial, using the Saha equation, allowed the calculation of the plasma ionization temperature and electron density. For the determination of trace metals in waters and sewage sludges, the samples were spiked with different internal standards, ionization corrections were performed and the results obtained agreed with those obtained by external calibration and standard additions within a factor of 2 but, on average, the agreement was within 20%. The determination of molybdenum in biological reference materials was performed by isotope dilution analysis taking into account possible sources of error in the measurements by ICP-MS such as mass discrimination, detector dead time, isobaric interferences and random error propagation. © 1997 by John Wiley & Sons, Ltd.

Vitamin E supplements are either isolated from plants sources or prepared synthetically. Isolation from plants includes eight different tocopherol structures. Vitamin E synthesis includes seven different stereoisomers, which involves the use of several catalysts that may lead to trace element contamination in the vitamin. The use of ICP-MS is an ideal technique for detecting these trace elements. However, the oily nature of the samples requires the development of a sample preparation methodology. This study was done upon the request of synthetic vitamin E manufacturers to test the trace metal purity of their samples. In this work, the comparison of an acid microwave digestion and emulsion preparation is discussed. Cromium, nickel, tin and lead were found in the synthetic vitamin E analyzed and 200, 60, 9 and 45 ppb were the concentrations found respectively for these elements. Digesting the samples gives slightly lower detection limits compared to the emulsion preparation.

An alternative method for the determination of trace levels of fluoride in drinking and sea-water samples is presented. It is based on the formation of the aluminium monofluoride complex in excess of Al3+ and separation of the two species formed (AlF2+ and Al3+) in a small (5 cm long, CG2) ion exchange guard column. The final determination is accomplished by both ICP-MS specific detection and post column derivatisation with fluorimetric detection. Fundamental studies on the formation kinetics of the complex, ion chromatographic separation and optimum aluminium concentration were carried out using spectrofluorimetric detection by post-column reaction of the species with 8-hydroxyquinoline-5-sulfonic acid in a micellar medium of cetyltrimethylammonium bromide. Fluorimetric detection showed good detection limits, but interferences from cations such as Mg2+ and Zn2+ required the use of the longer CS2 ion exchange column. Iron interfered in relatively large amounts but adding EDTA to the sample solution eliminated the interference. A similar separation methodology was applied using ICP-MS detection for the indirect determination of fluoride, by monitoring aluminium at mass 27. In this case, a detection limit of 0.1 ng ml–1 was obtained using 0.45 M HNO3 as eluent and no interference caused by high concentrations of iron was observed. The proposed method was applied to the determination of very low levels of fluoride in natural waters.