Synthetic access to arsenic-containing phosphatidylcholines

We wish to disclose the first synthesis of 1-O-hexadecanoyl-2-O-((15-(dimethylarsinoyl)pentadecanoyl)oxy)-sn-glycero-3-phosphocholine, which belongs to the group of arsenic-containing phosphatidylcholines (AsPCs), recently discovered in herring caviar. The synthesized product will serve as a model compound to study biological and toxicological properties of arsenolipids in food.     

Human metabolism of arsenolipids present in cod liver

We report results from the first investigation of the human metabolism of arsenic-containing lipids (arsenolipids), significant arsenic constituents of some seafood products. Two male volunteers ingested canned cod liver and the arsenic metabolites in their urine were monitored by high-performance liquid chromatography inductively coupled plasma mass spectrometry over a 66-h period. Volunteer A consumed 85 g (wet mass) of cod liver containing a total of approximately 120 μg arsenic, 77% of which was present as arsenolipids, and volunteer B consumed 85 g (wet mass) of cod liver, 25% of which was present as arsenolipids, together with 20 g of cod liver oil, containing a total of about 180 μg arsenic. The structures of the arsenolipids are currently unknown, whereas the majority of the non-lipid arsenic in the cod liver was identified as arsenobetaine, which was excreted unchanged. The arsenolipids were rapidly metabolised to water-soluble compounds and excreted in the urine; peak arsenic concentrations were recorded between 7 and 15 h (volunteer A) and between 6.5 and 15 h (volunteer B), and by the end of the experiment about 90% of the ingested arsenic had been accounted for in the urine for both volunteers. The major arsenolipid metabolite was dimethylarsinate (DMA), constituting 73% (volunteer A) or 41% (volunteer B) of the total urinary arsenic, and most of the remaining arsenolipid-derived arsenic, constituting about 10% (volunteer A) and 5% (volunteer B), comprised four novel arsenic-containing fatty acids, namely oxo-dimethylarsenopropanoic acid, thio-dimethylarsenopropanoic acid, oxo-dimethylarsenobutanoic acid, and thio-dimethylarsenobutanoic acid. Unchanged arsenobetaine (15% for volunteer A and 51% for volunteer B) made up the remaining urinary arsenic together with trace quantities of other, mostly unknown, arsenicals. In a second experiment (volunteer A only), performed with pure cod liver oil, which contains only arsenolipids, DMA and the same four arsenic fatty acids were excreted in the urine. The study shows that arsenolipids in cod liver are bioavailable, and that they are quickly biotransformed to several water-soluble arsenicals, the structures of which suggest that the native arsenolipids contain a dimethylarsine oxide moiety.     

A 2‐O‐Methylriboside Unknown Outside the RNA World Contains Arsenic

Lipid‐soluble arsenic compounds, also called arsenolipids, are ubiquitous marine natural products of currently unknown origin and function. In our search for clues about the possible biological roles of these compounds, we investigated arsenic metabolism in the unicellular green alga Dunaliella tertiolecta, and discovered an arsenolipid fundamentally different from all those previously identified; namely, a phytyl 5‐dimethylarsinoyl‐2‐O ‐methyl‐ribofuranoside. The discovery is of particular interest because 2‐O ‐methylribosides have, until now, only been found in RNA. We briefly discuss the significance of the new lipid in biosynthesis and arsenic biogeochemical cycling.     

Direct measurement of lipid-soluble arsenic species in biological samples with HPLC-ICPMS

Lipid-soluble arsenicals (arsenolipids) occur in a wide range of biological samples where they may play a key role in the biosynthesis of organoarsenic compounds from inorganic arsenic. The study of these compounds has been hindered, however, by the lack of a suitable analytical technique able to separate and measure the various lipid species. As a source of arsenolipids, we used 10 crude fish oils from various regions of the world. Total arsenic analyses on the fish oils, performed with ICPMS following acid digestion with microwave-assisted heating, gave concentrations from 4.3 to 10.5 mg As kg(-1). All of the arsenic was soluble in non-polar solvents such as hexane. Analysis of the fish oils for arsenolipids was performed by normal phase HPLC-ICPMS with various mixtures of organic solvents as mobile phases. Inherent problems of instability associated with the introduction of organic solvents to the plasma were overcome by the use of reduced column flow, a chilled spray chamber, and the addition of oxygen directly to the plasma. All ten fish oils appeared to contain the same 4-6 major arsenolipids, but in varying amounts depending on the origin of the fish. Further chromatography with both normal phase and reversed-phase conditions on some of the oils indicated the presence of many more minor arsenolipids. Quantification was achieved by external calibration against triphenylarsine oxide or triphenylarsine sulfide, and the sum of species following HPLC of the oils matched well the total arsenic results (92-107%). The method was applied to samples of food supplements (fish oil capsules) and a packaged food product (cod liver) whereby arsenolipids were measured and found to be significant arsenic constituents. This study represents the first attempt to directly measure intact arsenolipids and, with appropriate sample preparation, may be suitable for quantitative measurement of these arsenicals in a range of biological samples, including foodstuffs.     

Detection of arsenic-containing hydrocarbons in canned cod liver tissue

Arsenic is a metalloid well known to be potentially toxic depending of its species. Lipid-soluble arsenicals (arsenolipids) are present in a wide range of biological samples in which they could play a role in the biosynthesis of organoarsenic compounds from inorganic arsenic compounds. Arsenolipids have recently attracted considerable interest. In order to gain deeper insights into the impact of arsenolipids new analytical approaches for reliable determination of this class of arsenic-containing hydrocarbons in various matrices are needed.High concentrations of arsenolipids were found in seafood which served as sample material in this study. We report the investigation of three arsenolipids found in canned cod liver from which they were extracted and purified by solid phase extraction (SPE) using a silica gel column and ethyl acetate/methanol as eluent. Analytical studies were conducted by means of gas chromatography coupled with ICP-MS, MIP-AES and EI-qMS and by TOF-MS. The results obtained by GC-ICP-MS and GC-MIP-AES showed the existence of numerous arsenic compounds in the SPE fractions collected. Three major peaks were found within a retention time window between 10 and 25 min. The presence of arsenic compounds in the fish tissue could be confirmed using GC-EI-qMS analysis. Corresponding information of the molecular weights of the major arsenic species were provided by TOF-MS which allows highly accurate mass determinations. The results showed the presence of the arsenic-containing hydrocarbons with the following molecular formulas: C₁₇H₃₇AsO (calculated for [M+H]⁺ 333.2133; found 333.2136; Δm = 0.90 ppm); C₁₉H₄₁AsO (calculated for [M+H]⁺ 361.2446; found 361.2446; Δm = 0.00 ppm); C₂₃H₃₇AsO (calculated for [M+H]⁺ 405.2133; found 405.2145; Δm = 2.96 ppm). Suggestions for the corresponding structures are discussed.     

Cationic vesicles consisting of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and phosphatidylcholines and their interaction with erythrocyte membrane

We studied the formation and stability of vesicles consisting of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and phosphatidylcholines by electron spin resonance (ESR) analysis and observation of their hemolytic activities. In contrast with previous findings on dimethyldialkylammoniums, DOTAP formed vesicles at 37 degrees C with phosphatidylcholines containing either saturated acyl chains such as dimyristoylphosphatidylcholine (DMPC) or unsaturated acyl chains such as dilinoleoylphosphatidylcholine (DLPC). Phosphatidylcholines made the bilayer more rigid and significantly reduced the hemolytic activity of DOTAP. In the presence of equimolar concentration of DOTAP and phosphatidylcholines, formation of tightly aggregated structures of several erythrocytes was observed, as previously reported for the vesicles containing dimethyldipalmitylammonium. These findings indicate that DOTAP vesicles were stabilized by phosphatidylcholines with either saturated acyl chains or unsaturated acyl chains, and the interaction with the lipid bilayer of biological membranes as cationic vesicles became prominent with minimal membrane damage by DOTAP monomers.     

Fatty acid composition and volatile compounds of caviar from farmed white sturgeon (Acipenser transmontanus)

The present study was conducted to characterize caviar obtained from farmed white sturgeons (Acipenser transmontanus) subjected to different dietary treatments. Twenty caviar samples from fish fed two experimental diets containing different dietary lipid sources have been analysed for chemical composition, fatty acids and flavour volatile compounds. Fatty acid make up of caviar was only minimally influenced by dietary fatty acid composition. Irrespective of dietary treatments, palmitic acid (16:0) and oleic acid (OA, 18:1 n-9) were the most abundant fatty acid followed by docosahexaenoic acid (DHA, 22:6 n-3) and eicopentaenoic (EPA, 20:5 n-3). Thirty-three volatile compounds were isolated using simultaneous distillation-extraction (SDE) and identified by GC-MS. The largest group of volatiles were represented by aldehydes with 20 compounds, representing the 60% of the total volatiles. n-Alkanals, 2-alkenals and 2,4-alkadienals are largely the main responsible for a wide range of flavours in caviar from farmed white surgeon.     

p‐Azidophenylarsenoxide: An Arsenical “Bait” for the In Situ Capture and Identification of Cellular Arsenic‐Binding Proteins

Identification of arsenic‐binding proteins is important for understanding arsenic health effects and for developing arsenic‐based therapeutics. We report here a strategy for the capture and identification of arsenic‐binding proteins in living cells. We designed an azide‐labeled arsenical, p‐azidophenylarsenoxide (PAzPAO), to serve bio‐orthogonal functions: the trivalent arsenical group binds to cellular proteins in situ, and the azide group facilitates click chemistry with dibenzylcyclooctyne. The selective and efficient capture of arsenic‐binding proteins enables subsequent enrichment and identification by shotgun proteomics. Applications of the technique are demonstrated using the A549 human lung carcinoma cells and two in vitro model systems. The technique enables the capture and identification of 48 arsenic‐binding proteins in A549 cells incubated with PAzPAO. Among the identified proteins are a series of antioxidant proteins (e.g., thioredoxin, peroxiredoxin, peroxide reductase, glutathione reductase, and protein disulfide isomerase) and glyceraldehyde‐3‐phosphate dehydrogenase. Identification of these functional proteins, along with studies of arsenic binding and enzymatic inhibition, points to these proteins as potential molecular targets that play important roles in arsenic‐induced health effects and in cancer treatment.     

One‐Dimensional Arsenic Allotropes: Polymerization of Yellow Arsenic Inside Single‐Wall Carbon Nanotubes

The pnictogen nanomaterials, including phosphorene and arsenene, display remarkable electronic and chemical properties. Yet, the structural diversity of these main group elements is still poorly explored. Here we fill single‐wall carbon nanotubes with elemental arsenic from the vapor phase. Using electron microscopy, we find chains of highly reactive As₄ molecules as well as two new one‐dimensional allotropes of arsenic: a single‐stranded zig‐zag chain and a double‐stranded zig‐zag ladder. These linear structures are important intermediates between the gas‐phase clusters of arsenic and the extended sheets of arsenene. Raman spectroscopy indicates weak electronic interaction between the arsenic and the nanotubes which implies that the formation of the new allotropes is driven primarily by the geometry of the confinement. The relative stabilities of the new arsenic structures are estimated computationally. Band‐gap calculations predict that the insulating As₄ chains become semiconducting, once converted to the zig‐zag ladder, and form a fully metallic allotrope of arsenic as the zig‐zag chain.     

Speciation and Determination of Trace Amount of Inorganic Arsenic in Water,Environmental and Biological Samples

A new speciation and preconcentration method based on dispersive liquid‐liquid microextraction has been developed for trace amounts of As(III) and As(V) in urine and water samples. At pH 4, As(III) is complexed with ammoniumpyrrolidine dithiocarbamate and extracted into 1‐Hexyl‐3‐methylimidazolium hexafluorophosphate, as an ionic liquid (IL) and As(III) is determined by electrothermal atomic absorption spectrometery (ETAAS). Arsenic(V) in the mixing solution containing As(III) and As(V) was reduced by using KI and ascorbic acid in HCl solution and then the procedure was applied to determination of total arsenic. Arsenic(V) was calculated as the difference between the total arsenic content and As(III) content. The effect of various parameters on the recovery of the arsenic ions has been studied. Under the optimum conditions, the enrichment factor 135 was obtained. The proposed method was successfully applied to the determination of trace amounts of As(III) and As(V) in water and biological samples.     

Size exclusion chromatography coupled to electrospray ionization mass spectrometry for analysis and quantitative characterization of arsenic interactions with peptides and proteins

Arsenic‐binding proteins are of toxicological importance since enzymatic activities can be blocked by arsenic interactions. In the present work, a novel methodology based on size exclusion chromatography coupled to electrospray ionization mass spectrometry (SEC‐ESI‐MS) was developed with special emphasis to preserve the intact proteins and their arsenic bindings. The eluent composition of 25 mM Tris/HCl, pH 7.5, with the addition of 100‐mM NaCl optimized for SEC with UV detection provided the highest SEC separation efficiency, but was not compatible with the ESI‐MS because of the non‐volatility of the buffer substance and of the salt additive. In order to find the best compromise between chromatographic separation and ionization of the arsenic‐binding proteins, buffer type and concentration, pH value, portion of organic solvent in the SEC eluent as well as the flow rate were varied. In the optimized procedure five different arsenic‐binding peptides and proteins (glutathione, oxytocin, aprotinin, α‐lactalbumin, thioredoxin) covering a molar mass range of 0.3–14 kDa could be analyzed using 75% 10‐mM ammonium formate, pH 5.0/25% acetonitrile (v : v) as eluent and a turbo ion spray source operated at 300 °C and 5.5 kV. A complete differentiation of all peptides and proteins involved in the arsenic‐binding studies as well as of their arsenic‐bound forms has become feasible by means of the extracted ion chromatograms (XIC) of the mass spectrometric detection. The new method offered the possibility to estimate equilibrium constants for the reaction of phenylarsine oxide with different thiol‐containing biomolecules by means of the XIC peak areas of reactants and products. Limits of detection in the range of 2–10 µM were obtained by SEC‐ESI‐MS for the individual proteins. Copyright © 2009 John Wiley & Sons, Ltd.     

Clicking the Arsenic–Carbon Triple Bond: An Entry into a New Class of Arsenic Heterocycles

Arsaalkynes can undergo regioselective and quantitative [3+2] cycloaddition reactions with organic azides to give hitherto unknown 3H‐1,2,3,4‐triazaarsole derivatives. The reaction product was obtained as a white, air‐ and moisture‐stable solid, and the presence of a planar, five‐membered arsenic heterocycle was unambiguously verified by means of X‐ray crystallography. DFT calculations gave insight into the electronic structure of these novel compounds compared to tetrazoles and triazaphospholes. The coordination chemistry towards Re^I was investigated and compared with the structurally related phosphorus‐containing ligand. These preliminary investigations pave the way for a new class of arsenic heterocycles and fill the gap between the azaarsoles already known.     

Speciation and determination of bioavailable arsenic species in soil samples by one‐step solvent extraction and high‐performance liquid chromatography with inductively coupled plasma mass spectrometry

A new analytical method was developed to determine the bioavailable arsenic species (arsenite, arsenate, monomethylarsonic acid, and dimethylarsonic acid) in soil samples using high‐performance liquid chromatography with inductively coupled plasma mass spectrometry. Bioavailable arsenic was extracted with ammonium phosphate buffer by a simplified one‐step solvent extraction procedure. To estimate the effect of variables on arsenic extraction, a two‐level Plackett–Burman factorial design was conducted to screen the significant factors that were further investigated by a separate univariate approach. The optimum conditions were confirmed by compromising the stability of arsenic species and the extraction efficiency. The concentration of arsenic species was determined in method blank and soil‐certified reference materials both spiked with standard solutions of arsenic species. All the target arsenic species were stable during the whole extraction procedure. Furthermore, the proposed method was applied to release bioavailable arsenic from contaminated soil samples, showing that the major arsenic species in soil samples were inorganic arsenic: arsenite and arsenate, of which the latter was dominant.     

Study on the Aggregation and Deaggregation Behaviors of Phosphatidylcholines

Phosphatdylcholines and their analogs, the functional building block of the membrane, are recently found to mediate multiple physiological processes and exhibit a broad range of desirable pharmacological effects, which involve hydrophobic lipophilic interactions (HLI) between the phospholipid and the cell membrane. The HLI behavior of phosphatJdylcholines (Ln) and their analogues 1, 2-diacyl-sn-glycerol-3-phosphoric acid bromoethyl ester (Pn), have been investigated in MeOH-H20 binary systems of different volume fractions (designated as Φ) of the organic component, by employing α-nephthylethyl lauryl ether (Np-12) as fluorescent probe. A very interesting observation is that the Ln possesses double character, i. e., it behaves both as an aggregator and as a deaggregator. The effects of the structure and the environment on the coaggregation and deaggregtion are also discussed.     

Nitrite‐Selective Electrode Based On Cobalt(II) tert‐Butyl‐Salophen Ionophore

A series of polymeric nitrite‐selective electrodes containing a new lipophilic ionophore Co(II) tert‐butyl‐salophen is reported. The stability of Co(II) ionophores within a PVC‐based membrane was investigated by leaching experiments. Different membrane compositions were explored in order to reach the lowest possible limit of detection for a PVC‐based nitrite selective polymeric membrane electrode containing this ionophore. The optimal electrode showed a limit of detection of 2×10^?6 M and exhibited four orders of magnitude of discrimination over nitrate, chloride and bromide. The electrodes were evaluated in undiluted human urine and attest to the robustness of the ionophore.     

Speciation of Arsenic by Potentiometric Stripping Analysis Using Gold(III) Solution as Chemical Reoxidant and a Wall‐Jet Flow Cell

A simple procedure is described for the potentiometric stripping of arsenic with a wall‐jet cell by means of potentiostatic co‐deposition of gold and arsenic at a glassy‐carbon electrode and subsequent chemical stripping with Au(III). Optimum medium containing 160 mg L^?1 of Au(III) in HCl 0.1 M, where it is possible to speciate As(III) and As(V). As(V) was electrodeposited directly without prior chemical reduction at working electrode. As(III) was first determined at an electrodeposition potential of ?0.1 V. Afterwards, total arsenic was determined by an electrodeposition potential of ?0.7 V, from the area of peak obtained of the differential stripping potentiogram by using the standard addition method. The original As(V) concentration in the sample was calculated by difference. The possibilities of the optimized method were demonstrated by determinations of As(III), As(V) and total arsenic in samples of polluted water.     

Qualitative and quantitative characterization of the arsenic‐binding behaviour of sulfur‐containing peptides and proteins by the coupling of reversed phase liquid chromatography to electrospray ionization mass spectrometry

Phenylarsenic‐substituted cysteine‐containing peptides and proteins were completely differentiated from their unbound original forms by the coupling of reversed phase liquid chromatography with electrospray ionization mass spectrometry. The analysis of biomolecules possessing structure‐stabilizing disulfide bridges after reduction provides new insights into requirements concerning the accessibility of cysteine residues for reducing agents as well as for arsenic compounds in a spatial protein structure. Complementary binding studies performed using direct ESI‐MS without chromatographic coupling in different solvent systems demonstrated that more than one binding site were activated for aprotinin and lysozyme in denaturing solvents because of a stronger defolding. From the intensities of the different charge states occurring in the mass spectra as well as from the LC elution behaviour, it can be deduced that the folding state of the arsenic‐bound protein species resembles the native, oxidized conformation. In contrast, although the milk protein α‐lactalbumin has several disulfide bridges, only one phenylarsenic moiety was bound under strongly denaturing conditions. Because of the charge state distribution in the ESI mass spectra, a conformational change to a molten globule structure is assumed. For the second considered milk protein ß‐lactoglobulin, a noncovalent interaction with phenylarsine oxide was detected. In general, smaller apparent binding constants for the condensation reactions of the biomolecules with phenylarsine oxide leading to covalent arsenic–sulfur bindings were determined from direct injection ESI‐MS measurements than from LC‐ESI‐MS coupling. The following order of binding affinities for one phenylarsenic group can be assumed from both ESI‐MS and LC‐ESI‐MS: nonapeptide vasopressin > nonapeptide vasotocin > lysozyme > aprotinin > α‐lactalbumin > thioredoxin. Kinetic investigations by LC‐ESI‐MS yielded a partial reaction order of 2 for vasopressin, Lys and α‐lactalbumin and corresponding half‐lives of 0.93, 2.56 and 123.5 min, respectively. Copyright © 2012 John Wiley & Sons, Ltd.     

Highly Sensitive Voltammetric Speciation and Determination of Inorganic Arsenic in Water and Alloy Samples Using Ammonium 2‐Amino‐1‐Cyclopentene‐1‐Dithiocarboxylate

A simple, fast, reproducible (2.5% RSD at 3.0 μg/L), and sensitive method is described for quantifying As(III) (0.3 μg/L detection limit, 0.5–440 μg/L dynamic range). Anodic stripping voltammetry (ASV) is performed after accumulating arsenic at a mercury film electrode at ?0.350 V vs. Ag/AgCl (saturated KCl) for 20 s in 0.2 M HCl containing 8 μM ammonium 2‐amino‐1‐cyclopentene‐1‐dithiocarboxylate (AACD), without oxygen removal. This is the first report of using AACD in ASV and in electrochemical quantification of As(III). Total arsenic is determined after sodium‐sulfite‐reduction of As(V) to As(III). Interferences are minimal. Method validation involved water and metal alloy samples.     

Feasibility of Separation and Quantification of Inorganic Arsenic Species Using Ion-Exchange Membranes and Laser-Induced Breakdown Spectroscopy

Analytical methods for the determination of inorganic arsenic species have attracted much attention due to the high toxicity of these compounds and related legislative regulations for food. A new method for the separation and quantitation of As(III) and As(V) was developed using ion-exchange membranes and laser-induced breakdown spectroscopy (LIBS). Using the anion-exchange polymer membrane, As(V) was selectively collected on the membrane, and As(III) was filtered through the membrane. The separated As(V) on the membrane was directly determined by LIBS. The As(III) in the filtrate was subsequently oxidized to As(V) and collected by the membrane for LIBS analysis. The detection limit for As(V) was estimated to be 10?mg/kg. The recovery efficiencies for the arsenic species as standards were in the range of 97–99%. This method was applied for the analysis As-spiked water certified reference materials, and the results showed that the recovery for As(V) was 98.9%. This new speciation method is cost-effective, simple, and low labor-intensive for the quantitation of inorganic arsenic.     

Authentication of Kalix (N.E. Sweden) vendace caviar using inductively coupled plasma-based analytical techniques: evaluation of different approaches

Different analytical approaches for origin differentiation between vendace and whitefish caviars from brackish- and freshwaters were tested using inductively coupled plasma double focusing sector field mass spectrometry (ICP-SFMS) and multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS). These approaches involve identifying differences in elemental concentrations or sample-specific isotopic composition (Sr and Os) variations. Concentrations of 72 elements were determined by ICP-SFMS following microwave-assisted digestion in vendace and whitefish caviar samples from Sweden (from both brackish and freshwater), Finland and USA, as well as in unprocessed vendace roe and salt used in caviar production. This data set allows identification of elements whose contents in caviar can be affected by salt addition as well as by contamination during production and packaging. Long-term method reproducibility was assessed for all analytes based on replicate caviar preparations/analyses and variations in element concentrations in caviar from different harvests were evaluated. The greatest utility for differentiation was demonstrated for elements with varying concentrations between brackish and freshwaters (e.g. As, Br, Sr). Elemental ratios, specifically Sr/Ca, Sr/Mg and Sr/Ba, are especially useful for authentication of vendace caviar processed from brackish water roe, due to the significant differences between caviar from different sources, limited between-harvest variations and relatively high concentrations in samples, allowing precise determination by modern analytical instrumentation. Variations in the ⁸⁷Sr/⁸⁶Sr ratio for vendace caviar from different harvests (on the order of 0.05-0.1%) is at least 10-fold less than differences between caviar processed from brackish and freshwater roe. Hence, Sr isotope ratio measurements (either by ICP-SFMS or by MC-ICP-MS) have great potential for origin differentiation. On the contrary, it was impossible to differentiate between Swedish caviar processed from brackish water roe and Finnish freshwater caviar based solely on ¹⁸⁷Os/¹⁸⁸Os ratios.