Gold-Promoted Biocompatible Selenium Arylation of Small Molecules, Peptides and Proteins.

A low pKa (5.2), high polarizable volume (3.8 Å), and proneness to oxidation under ambient conditions make selenocysteine (Sec, U) a unique, natural reactive handle present in most organisms across all domains of life. Sec modification still has untapped potential for site-selective protein modification and probing. Herein we demonstrate the use of a cyclometalated gold(III) compound, [Au(bnpy)Cl2 ], in the arylation of diselenides of biological significance, with a scope covering small molecule models, peptides, and proteins using a combination of multinuclear NMR (including 77 Se NMR), and LC-MS. Diphenyl diselenide (Ph-Se)2 and selenocystine, (Sec)2 , were used for reaction optimization. This approach allowed us to demonstrate that an excess of diselenide (Au/Se-Se) and an increasing water percentage in the reaction media enhance both the conversion and kinetics of the C-Se coupling reaction, a combination that makes the reaction biocompatible. The C-Se coupling reaction was also shown to happen for the diselenide analogue of the cyclic peptide vasopressin ((Se-Se)-AVP), and the Bos taurus glutathione peroxidase (GPx1) enzyme in ammonium acetate (2 mM, pH=7.0). The reaction mechanism, studied by DFT revealed a redox-based mechanism where the C-Se coupling is enabled by the reductive elimination of the cyclometalated Au(III) species into Au(I).

Effect of graded levels of selenium supplementation as selenite on expression of selenosugars, selenocysteine, and other selenometabolites in rat liver.

Using high pressure liquid chromatography (HPLC) coupled with selenium-specific inductively coupled plasma mass spectrometry (ICP-MS) and molecule specific (Orbitrap MS/MS) detection, we previously found that far more selenium (Se) is present as selenosugar (seleno-N-acetyl galactosamine) in Se-adequate turkey liver than is present as selenocysteine (Sec) in true selenoproteins, and that selenosugars account for half of the Se in high-Se turkey liver. To expand these observations to mammals, we studied Se metabolism in rats fed graded levels of selenite from 0 to 5 µg Se/g for 4 wk. In Se-adequate (0.24 µg Se/g) rats, 43% of liver Se was present as Sec, 32% was present as selenosugars, and 22% as inorganic Se bound to protein. In liver of rats fed 5 µg Se/g as selenite, the quantity of Sec remained at the Se-adequate plateau (11% of total Se), 22% was present as low molecular weight (LMW) selenosugars with substantial additional selenosugars linked to protein, but 64% was present as inorganic Se bound to protein. No selenomethionine was found at any level of selenite supplementation. Below the Se requirement, Se is preferentially incorporated into Sec-selenoproteins. Above the dietary Se requirement, selenosugars become by far the major LMW water soluble Se species in liver, and levels of selenosugar-decorated proteins are far higher than Sec-selenoproteins, making these selenosugar-decorated proteins the major Se-containing protein species in liver with high Se supplementation. This accumulation of selenosugars linked to cysteines on proteins or the build-up of inorganic Se bound to protein may underlie Se toxicity at the molecular level.

Selenomethionine supplementation and expression of selenosugars, selenocysteine, and other selenometabolites in rat liver.

Selenomethionine (SeMet) as a methionine analog can be incorporated into protein. In turkeys, we recently found that selenium (Se) as selenite is not metabolized to SeMet but rather to selenosugars (seleno-N-acetyl galactosamine) bound to protein as well as to selenocysteine (Sec) in selenoproteins. To characterize the metabolism of SeMet, we fed rats graded levels of SeMet from 0 to 5 µg Se/g in a Se-deficient diet for 4 wk, and investigated the fate and accumulation of liver Se using high pressure liquid chromatography (HPLC) coupled with Se-specific inductively coupled plasma mass spectrometry (ICP-MS) and molecule specific (Orbitrap MS/MS) detection. Up to 0.24 µg Se/g (Se requirement for maximal glutathione peroxidase activity), Sec accounted for ∼40% of total liver Se whereas SeMet only accounted for 3-11%. Analysis of water-soluble extracts found negligible low molecular weight (LMW) Se species in rats fed 0 and 0.08 µg Se/g, including no SeMet. At 0.24 µg Se/g and above, SeMet accounted for only 10% of LMW Se species, whereas methyl- and glutathionyl-selenosugars accounted for 70% of LMW Se species. Above the Se requirement, SeMet was ∼30% of the proteinaceous amino acids, whereas Sec levels fell to 5% in rats fed 5 µg Se/g as SeMet. Last, considerably less inorganic Se was bound to liver protein with high SeMet as compared to selenite in a parallel study. SeMet is efficiently metabolized and mixes with the common Se metabolite pool, where Se is preferentially incorporated into Sec and Sec-selenoproteins until selenoproteins plateau; with high SeMet intake, Se is increasingly accumulated as LMW selenosugars and as selenosugar-decorated proteins.

Biodegradation of saturate fraction of crude oil and production of signature carboxylic acids.

Bacteria degrading large portion of saturated hydrocarbons are important for crude oil bioremediation. This study investigates Novosphingobium sp. S1, Gordonia amicalis S2 and Gordonia terrae S5 capability of degrading wide range of saturated hydrocarbons from Congo Bilondo crude oil and discusses the degradation pathway. A parallel analytical approach combining GC-MS and LC-HRMS enabled characterization of saturated hydrocarbons and comprehensive determination of carboxylic acid metabolites produced during biodegradation, respectively. Results showed that the three strains could efficiently degrade the n-alkanes (C10-C28) as well as methyl-substituted alkanes (C11-C26). The series of mono-, hydroxy- and dicarboxylic acids identified in this study confirmed the active biodegradation of the saturate fraction and suggest their degradation was via the bi-terminal oxidation pathway. This is the first study linking these bacterial species to bi-terminal oxidation of the saturated hydrocarbons. The study highlights the potential application of the bacterial strains in the bioremediation of crude oil contaminated sites. Additionally, while carboxylic acids is indicated as a suitable and valuable metabolic biomarker, its application is considered feasible and cost effective for rapid monitoring and evaluation of hydrocarbon biodegradation.

Nontargeted Screening for Flavonoids in Salicornia Plant by Reversed-Phase Liquid Chromatography-Electrospray Orbitrap Data-Dependent MS2/MS3.

The Salicornia genus has great potential in agrifood industries because of its nutritional benefits related to its high content of antioxidant compounds, including flavonoids. A nontargeted method based on reversed-phase liquid chromatography-electrospray orbitrap data-dependent MS2/MS3 and the fragment ion search (FISh) strategy was developed to screen flavonoids in Salicornia plants. An extensive study of fragmentation of a set of flavonoid standards allowed for the definition of 15 characteristic fragment ions for flagging flavonoids in the plant matrix. The nontargeted analysis was applied to Salicornia europaea species and allowed for the annotation of 25 candidate flavonoids, including 14 that had not been reported previously. Structural prediction of two unreported flavonoids and their isomeric forms was based on an advanced data processing method using an in silico approach and in-house databases compiling flavonoid-specific chemical substitution. Finally, the method developed allowed for the optimization of extraction yields of flavonoids from the plant matrix.

Relationships between Molecular Characteristics of Novel Organic Selenium Compounds and the Formation of Sulfur Compounds in Selenium Biofortified Kale Sprouts.

Due to problems with selenium deficiency in humans, the search for new organic molecules containing this element in plant biofortification process is highly required. Selenium organic esters evaluated in this study (E-NS-4, E-NS-17, E-NS-71, EDA-11, and EDA-117) are based mostly on benzoselenoate scaffolds, with some additional halogen atoms and various functional groups in the aliphatic side chain of different length, while one compound contains a phenylpiperazine moiety (WA-4b). In our previous study, the biofortification of kale sprouts with organoselenium compounds (at the concentrations of 15 mg/L in the culture fluid) strongly enhanced the synthesis of glucosinolates and isothiocyanates. Thus, the study aimed to discover the relationships between molecular characteristics of the organoselenium compounds used and the amount of sulfur phytochemicals in kale sprouts. The statistical partial least square model with eigenvalues equaled 3.98 and 1.03 for the first and second latent components, respectively, which explained 83.5% of variance in the predictive parameters, and 78.6% of response parameter variance was applied to reveal the existence of the correlation structure between molecular descriptors of selenium compounds as predictive parameters and biochemical features of studied sprouts as response parameters (correlation coefficients for parameters in PLS model in the range-0.521 ÷ 1.000). This study supported the conclusion that future biofortifiers composed of organic compounds should simultaneously contain nitryl groups, which may facilitate the production of plant-based sulfur compounds, as well as organoselenium moieties, which may influence the production of low molecular weight selenium metabolites. In the case of the new chemical compounds, environmental aspects should also be evaluated.

Effects of the fermentation process on the selenite metabolism and selenium incorporation and speciation in a probiotic Bifidobacterium longum.

The influence of the fermentation process on selenite metabolism by a probiotic Bifidobacterium longum DD98 and its consequent enrichment in selenium (Se) were studied. The effects of sodium selenite (Na2SeO3) concentration (18-400 µg/ml), feeding time (12, 16, and 24 h), and fermentation stage (secondary and tertiary fermentation) were evaluated by measuring (i) the total Se content and its distribution between the water-soluble metabolome fraction and the water-insoluble fraction; (ii) the total concentrations of the two principal Se compounds produced: selenomethionine (SeMet) and γ-glutamyl-selenomethionine (γ-Glu-SeMet), and (iii) the speciation of Se in the metabolite fraction. The results revealed that the fermentation process notably changed the Se incorporation into metabolites (γ-Glu-SeMet and free SeMet) and proteins (bound-SeMet) in B. longum DD98. In particular, the production of SeMet was negatively correlated to that of γ-Glu-SeMet when no red precipitate was seen in the bacteria. The study offers a tool for the control of the optimization of the fermentation process towards the desired molecular speciation of the incorporated Se and hence contributes to the production of Se-enriched probiotics with good qualities and bioactivities.

Identification of γ-Glutamyl-Selenomethionine as the Principal Selenium Metabolite in a Selenium-Enriched Probiotic, Bifidobacterium longum, by Two-Dimensional HPLC-ICP MS and HPLC-ESI Orbitrap MS.

Selenium (Se)-enriched probiotics are potential sources of organic Se in the human diet, but their application in food is debated because most selenized probiotics and their metabolites are not well-characterized. We analyzed a Se-enriched probiotic, Bifidobacterium longum DD98, to unveil its Se metabolite profiles by two-dimensional high-performance liquid chromatography inductively coupled plasma mass spectrometry (HPLC-ICP MS) and HPLC-electrospray ionization Orbitrap MS. A major Se metabolite was identified as gamma-glutamyl-selenomethionine (γ-Glu-SeMet), which accounted for 42.5 ± 3.4% of water-soluble Se. Most of the remaining Se was present as SeMet (35.2 ± 0.6%) in a free or protein-bound form. In addition, 11 minor Se metabolites were identified, eight of which had not been reported before in probiotics. Six of the identified compounds contained γ-Glu-SeMet as the core structure, constituting a γ-Glu-SeMet family. This study demonstrates the presence of γ-Glu-SeMet in a probiotic, showing a different selenite metabolite pathway from that of Se-enriched yeast, and it offers an alternative and potentially attractive source of organic Se for food and feed supplementation.

Speciation of metals in indigenous plants growing in post-mining areas: Dihydroxynicotianamine identified as the most abundant Cu and Zn ligand in Hypericum laricifolium.

Ag, As, Cu, Pb and Zn were found to be the principal metallic contaminants of a post-mining area of Peru (Hualgayoc, Cajamarca). Study of metal distribution amongst roots, stems, and leaves of four indigenous hypertolerant plant species, Arenaria digyna, Puya sp., Hypericum laricifolium, Nicotiana thyrsiflora indicated significant translocation of Zn (0.6 < TF ≤ 10.0) and Cu (0.4 < TF ≤ 6.5) into aerial plant organs and substantial water-extractable fraction (20-60%) of these metals, except for A. digyna (root and stems). A study of the metal speciation by ultrahigh-performance size-exclusion (fast-SEC) and hydrophilic ion interaction (HILIC) liquid chromatography with dual ICP (inductively coupled plasma) and electrospray (ESI) Orbitrap MS detection revealed the presence of nicotianamine and deoxymugineic acid copper and zinc complexes in roots, stem and leaves of N. thyrsiflora and Puya sp., and nicotianamine alone in A. digyna. A previously unreported compound, dihydroxy-nicotianamine was identified as the most abundant Cu and Zn ligand in H. laricifolium. The presence of arsenobetaine and an arsenosugar was confirmed by ESI MS. Ag and Pb were hardly translocated to leaves and were found as high molecular species; one of the Pb-containing species co-eluted in fast-SEC-ICP MS with rhamnogalacturonan-II-Pb complex commonly found in in the walls of plants.

Characterization and Quantification of Selenoprotein P: Challenges to Mass Spectrometry.

Selenoprotein P (SELENOP) is an emerging marker of the nutritional status of selenium and of various diseases, however, its chemical characteristics still need to be investigated and methods for its accurate quantitation improved. SELENOP is unique among selenoproteins, as it contains multiple genetically encoded SeCys residues, whereas all the other characterized selenoproteins contain just one. SELENOP occurs in the form of multiple isoforms, truncated species and post-translationally modified variants which are relatively poorly characterized. The accurate quantification of SELENOP is contingent on the availability of specific primary standards and reference methods. Before recombinant SELENOP becomes available to be used as a primary standard, careful investigation of the characteristics of the SELENOP measured by electrospray MS and strict control of the recoveries at the various steps of the analytical procedures are strongly recommended. This review critically discusses the state-of-the-art of analytical approaches to the characterization and quantification of SELENOP. While immunoassays remain the standard for the determination of human and animal health status, because of their speed and simplicity, mass spectrometry techniques offer many attractive and complementary features that are highlighted and critically evaluated.

Selenoproteome Expression Studied by Non-Radioactive Isotopic Selenium-Labeling in Human Cell Lines.

Selenoproteins, in which the selenium atom is present in the rare amino acid selenocysteine, are vital components of cell homeostasis, antioxidant defense, and cell signaling in mammals. The expression of the selenoproteome, composed of 25 selenoprotein genes, is strongly controlled by the selenium status of the body, which is a corollary of selenium availability in the food diet. Here, we present an alternative strategy for the use of the radioactive 75Se isotope in order to characterize the selenoproteome regulation based on (i) the selective labeling of the cellular selenocompounds with non-radioactive selenium isotopes (76Se, 77Se) and (ii) the detection of the isotopic enrichment of the selenoproteins using size-exclusion chromatography followed by inductively coupled plasma mass spectrometry detection. The reliability of our strategy is further confirmed by western blots with distinct selenoprotein-specific antibodies. Using our strategy, we characterized the hierarchy of the selenoproteome regulation in dose-response and kinetic experiments.

Identification and determination of selenocysteine, selenosugar, and other selenometabolites in turkey liver.

Liver and other tissues accumulate selenium (Se) when animals are supplemented with high dietary Se as inorganic Se. To further study selenometabolites in Se-deficient, Se-adequate, and high-Se liver, turkey poults were fed 0, 0.4, and 5 µg Se g-1 diet as Na2SeO3 (Se(iv)) in a Se-deficient (0.005 µg Se g-1) diet for 28 days, and the effects of Se status determined using HPLC-ICP-MS and HPLC-ESI-MS/MS. No selenomethionine (SeMet) was detected in liver in turkeys fed either this true Se-deficient diet or supplemented with inorganic Se, showing that turkeys cannot synthesize SeMet de novo from inorganic Se. Selenocysteine (Sec) was also below the level of detection in Se-deficient liver, as expected in animals with negligible selenoprotein levels. Sec content in high Se liver only doubled as compared to Se-adequate liver, indicating that the 6-fold increase in liver Se was not due to increases in selenoproteins. What increased dramatically in high Se liver were low molecular weight (MW) selenometabolites: glutathione-, cysteine- and methyl-conjugates of the selenosugar, seleno-N-acetyl galactosamine (SeGalNac). Substantial Se in Se-adequate liver was present as selenosugars decorating general proteins via mixed-disulfide bonds. In high-Se liver, these "selenosugar-decorated" proteins comprised ∼50% of the Se in the water-soluble fraction, in addition to low MW selenometabolites. In summary, more Se is present as the selenosugar moiety in Se-adequate liver, mostly decorating general proteins, than is present as Sec in selenoproteins. With high Se supplementation, increased selenosugar formation occurs, further increasing selenosugar-decorated proteins, but also increasing selenosugar linked to low MW thiols.

Does selenium fortification of kale and kohlrabi sprouts change significantly their biochemical and cytotoxic properties?

BACKGROUND: The sprouts of Brassica vegetables are known from their nutritional and chemopreventive values. Moreover, sprouts fortification with some trace elements, like selenium, may increase their importance in human diet. Thus, the aim of our study was to examine if selenium enrichment of kale and kohlrabi sprouts may influence their biochemical properties (phenolic acids and L-tryptophan content, antioxidant potential) or cytotoxic activity. Additional aim of the study was to evaluate the profile of selenium compounds and to describe the multidimensional interactions between the mentioned parameters. METHODS: Selenium content in the sprouts was evaluated by double-channel atomic fluorescence spectrometer AFS-230 with the flow hydride-generation system. Separation of selenium species in water soluble fraction was performed by size-exclusion LC-ICP-MS. The identification and quantification of phenolic acids and L-tryptophan was performed by HPLC. For antioxidant activity DPPH and FRAP methods were used. Cytotoxic activity of the sprouts extracts on a panel of human metastatic carcinoma cells was evaluated by MTT test. RESULTS: Selenium content in the fortified sprouts was several orders of magnitude higher than in the unfortified ones. Only small percentage of supplemented selenium (ca. 10 %) was incorporated into the sprouts as seleno-L-methionine, while the other detected selenium species remained unidentified. Selenium fortification differently stimulated the production of phenolic acids (sinapic, chlorogenic, isochlorogenic and caffeic acid) in the tested sprouts, depending on the particular species, selenium dose and the investigated compound. PCA analysis revealed strong correlation between antioxidant parameters and phenolic acids and L-tryptophan, while Se correlated only with caffeic acid. The sprouts extracts (≥1 mg/mL) showed cytotoxic potency to all the studied cancer cell lines (SW480, SW620, HepG2, SiHa), regardless the selenium supplementation. CONCLUSION: Se-fortified kale and kohlrabi sprouts are good candidates for functional food ingredients. Moreover, these results indicate that the sprouts enriched with sodium selenite show higher nutritional value, without significant changes in their cytotoxic activity.

A chemical speciation insight into the palladium(ii) uptake and metabolism by Sinapis alba. Exposure to Pd induces the synthesis of a Pd-histidine complex.

Palladium is recognized as a technologically critical element (TCE) because of its massive use in automobile exhaust gas catalytic converters. The release of Pd into the environment in the form of nanoparticles of various size and chemical composition requires an understanding of their metabolism by leaving organisms. We provide here for the first time a chemical speciation insight into the identity of the ligands produced or used by a plant Sinapis alba L. exposed in hydropony to Pd nanoparticles and soluble Pd (nitrate). The analytical method developed was based on the concept of 2D HPLC with parallel inductively coupled plasma mass spectrometry (ICP MS) and electrospray MS detection. Size exclusion chromatography - ICP MS of the plant extracts showed no difference between the speciation of Pd after the exposure to nanoparticles and after that to Pd2+ which indicated the reactivity and dissolution of Pd nanoparticles. A comparative investigation of the Pd speciation in a control plant extract spiked with Pd2+ and of an extract of a plant having metabolized palladium indicated the response of the Sinapis alba by the formation of a Pd-histidine complex. The complex was identified via Orbitrap MS; the HPLC-MS chromatogram produced two peaks at m/z 415.0341 each corresponding to a Pd-His2 complex. An investigation by ion-mobility MS revealed a difference in their collision cross section indicating that the complexes present varied in terms of spatial conformation. A number of other Pd complexes with different ligands (including nicotianamine) circulating in the plant were detected but these ligands were already observed in a control plant and their concentrations were not affected by the exposure to Pd.

Selenized Plant Oil Is an Efficient Source of Selenium for Selenoprotein Biosynthesis in Human Cell Lines.

Selenium is an essential trace element which is incorporated in the form of a rare amino acid, the selenocysteine, into an important group of proteins, the selenoproteins. Among the twenty-five selenoprotein genes identified to date, several have important cellular functions in antioxidant defense, cell signaling and redox homeostasis. Many selenoproteins are regulated by the availability of selenium which mostly occurs in the form of water-soluble molecules, either organic (selenomethionine, selenocysteine, and selenoproteins) or inorganic (selenate or selenite). Recently, a mixture of selenitriglycerides, obtained by the reaction of selenite with sunflower oil at high temperature, referred to as Selol, was proposed as a novel non-toxic, highly bioavailable and active antioxidant and antineoplastic agent. Free selenite is not present in the final product since the two phases (water soluble and oil) are separated and the residual water-soluble selenite discarded. Here we compare the assimilation of selenium as Selol, selenite and selenate by various cancerous (LNCaP) or immortalized (HEK293 and PNT1A) cell lines. An approach combining analytical chemistry, molecular biology and biochemistry demonstrated that selenium from Selol was efficiently incorporated in selenoproteins in human cell lines, and thus produced the first ever evidence of the bioavailability of selenium from selenized lipids.

Selenium-regulated hierarchy of human selenoproteome in cancerous and immortalized cells lines.

BACKGROUND: Selenoproteins (25 genes in human) co-translationally incorporate selenocysteine using a UGA codon, normally used as a stop signal. The human selenoproteome is primarily regulated by selenium bioavailability with a tissue-specific hierarchy. METHODS: We investigated the hierarchy of selenoprotein expression in response to selenium concentration variation in four cell lines originating from kidney (HEK293, immortalized), prostate (LNCaP, cancer), skin (HaCaT, immortalized) and liver (HepG2, cancer), using complementary analytical methods. We performed (i) enzymatic activity, (ii) RT-qPCR, (iii) immuno-detection, (iv) selenium-specific mass spectrometric detection after non-radioactive 76Se labeling of selenoproteins, and (v) luciferase-based reporter constructs in various cell extracts. RESULTS: We characterized cell-line specific alterations of the selenoproteome in response to selenium variation that, in most of the cases, resulted from a translational control of gene expression. We established that UGA-selenocysteine recoding efficiency, which depends on the nature of the SECIS element, dictates the response to selenium variation. CONCLUSIONS: We characterized that selenoprotein hierarchy is cell-line specific with conserved features. This analysis should be done prior to any experiments in a novel cell line. GENERAL SIGNIFICANCE: We reported a strategy based on complementary methods to evaluate selenoproteome regulation in human cells in culture.

Comparison of analytical methods using enzymatic activity, immunoaffinity and selenium-specific mass spectrometric detection for the quantitation of glutathione peroxidase 1.

Glutathione peroxidase 1 (Gpx1), one of the most responsive selenoproteins to the variation of selenium concentration, is often used to evaluate "selenium status" at a cellular or organismal level. The four major types of analytical methodologies to quantify Gpx1 were revisited. They include (i) an enzymatic assay, (ii, iii) polyacrylamide gel electrophoresis (PAGE) with (ii) western blot detection of protein or (iii) inductively coupled plasma mass spectrometry (ICP MS) detection of selenium, and (iv) size-exclusion chromatography with ICP MS detection. Each of the four methods was optimized for the quantification of Gpx1 with maximum sensitivity. The methods based on the enzymatic and immunodetection offer a much higher sensitivity but their accuracy is compromised by the limited selectivity and limited dynamic range. The advantages, drawbacks and sources of error of each technique are critically discussed and the need for the cross-validation of the results using the different techniques to assure the quality assurance of quantitative analysis is emphasized.

Identification and determination of selenohomolanthionine - The major selenium compound in Torula yeast.

Torula yeast (Candida utilis) was found to metabolize selenium in a totally different way to Brewer's yeast (S. cerevisiae) leading to the biosynthesis of selenohomolanthionine (SeHLan), a major selenium compound accounting for 60-80% of the total selenium. The identity of SeHLan was confirmed by retention time matching in hydrophilic ion interaction chromatography (HILIC) with inductively coupled plasma mass spectrometric detection (ICP MS) using a custom synthesized standard molecule and by HILIC - Orbitrap MS and MS-MS fragmentation. Selenohomolanthionine escapes the current assays for the organic character of Se-rich yeast based on the protein-bound selenomethionine determination. A HILIC - ICP MS method was developed for the quantitative determination of selenohomolanthionine in yeast supplements with a detection limit of 146ng/g.

Speciation of Selenium in Selenium-Enriched Sunflower Oil by High-Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry/Electrospray-Orbitrap Tandem Mass Spectrometry.

The reaction of sunflower oil with selenite produces a complex mixture of selenitriglycerides with antioxidant and anticancer properties. To obtain insight into the identity and characteristics of the species formed, an analytical approach based on the combination of high-performance liquid chromatography (HPLC) with (78)Se-specific selenium detection by inductively coupled plasma mass spectrometry (ICP MS) and high-resolution (100 000), high mass accuracy (<1 ppm) molecule-specific detection by electrospray-Orbitrap MS(3) was developed. For the first time, a non-aqueous mobile phase gradient was used in reversed-phase HPLC-ICP MS for the separation of a complex mixture of selenospecies and a mathematical correction of the background signal was developed. The identical chromatographic conditions served for the sample introduction into electrospray MS. Two types of samples were analyzed: sunflower oil dissolved in isopropanol and methanol extract of the oil containing 65% selenium. HPLC-ICP MS showed 14 peaks, 11 of which could also be detected in the methanol extract. Isotopic patterns corresponding to molecules with one or two selenium atoms could be attributed by Orbitrap MS at the retention times corresponding to the HPLC-ICP MS peak apexes. Structural data for these species were acquired by MS(2) and MS(3) fragmentation of protonated or sodiated ions using high-energy collisional dissociation (HCD). A total of 11 selenium-containing triglycerol derivatives resulting from the oxidation of one or two double bonds of linoleic acid and analogous derivatives of glycerol-mixed linoleate(s)/oleinate(s) have been identified for the first time. The presence of these species was confirmed by the targeted analysis in the total oil isopropanol solution. Their identification corroborated the predicted elution order in reversed-phase chromatography: LLL (glycerol trilinoleate), LLO (glycerol dilinoleate-oleinate), LOO (glycerol linoleate-dioleinate), OOO (glycerol trioleinate), of which the extrapolation allowed for the prediction of the identity [glycerol dioleinate-stearate (OOS) and glycerol oleinate-distearate (OSS)] of the nonpolar species detected by ICP MS in the oil but not detected by electrospray MS.

Insights into the nature of uranium target proteins within zebrafish gills after chronic and acute waterborne exposures.

New data on the nature of the protein targets of uranium (U) within zebrafish gills were collected after waterborne exposure, with the aim of a better understanding of U toxicity mechanisms. Some common characteristics of the U protein target binding properties were found, such as their role in the regulation of other essential metals and their phosphorus content. In total, 21 potential protein targets, including hemoglobin, are identified and discussed in terms of the literature.