Determination of the Interaction of Arsenic and Human Serum Albumin by Online Microdialysis Coupled to LC with Hydride Generation Atomic Fluorescence Spectroscopy

Study on the stoichiometry and affinity of the arsenicals bound to HSA is an important step toward a better understanding of arsenic toxic effects. After incubation of As^III or As^V with HSA at the physiological conditions (pH 7.43 and 37 °C), the free arsenicals and arsenic-HSA complexes were separated and detected by the combined techniques of microdialysis and liquid chromatography with hydride generation atomic fluorescence spectroscopy (MD–LC–HGAFS). The decrease of As^III peak response rather than As^V indicated that HSA reacted with As^III but not As^V. The binding plots indicated that the binding between HSA and As^III was in Scatchard pattern when the concentration ratios of As^III to HSA were ≤1:1. The strong binding sites (n ₁) were 1.6 and the stability constant (K ₁) was 1.54 × 10⁶ M⁻¹. When the concentration ratios of As^III to HSA were >1:1, the binding was in Plasvento pattern with the stability constant K ₂ ≅ 0 and no specific binding of As^III with HSA. On the contrary, As^V did not show binding with HSA. The results showed that As^III reacted with HSA more readily than As^V, which provides a chemical basis for arsenic toxicity.

     

HPLC-ICP-MS method development to monitor arsenic speciation changes by human gut microbiota

Inorganic arsenic (iAs) has been classified as a type 1 carcinogen and has also been linked to several noncancerous health effects. Prior to 1995, the As^V methylation pathway was generally considered to be a detoxification pathway, but cellular and animal studies involving MMA^III (mono metyl arsonous acid) and DMA^III (dimethyl arsinous acid) have indicated that their toxicities meet or exceed that of iAs, suggesting an activation process. In addition, thiolated arsenic metabolites were observed in urine after oral exposure of inorganic arsenic in some studies, for which the toxicological profile was not yet fully characterized in human cells. Studies have revealed that microorganisms from the gut environment are important contributors to arsenic speciation changes. This presystemic metabolism necessitates the development of protocols that enable the detection of not only inorganic arsenic species, but also pentavalent and trivalent methylated, thiolated arsenicals in a gastrointestinal environment. We aim to study the biotransformation of arsenic (As) using a Simulator of the Human Intestinal Microbial Ecosystem (SHIME). To be able to analyze the arsenicals resulting from biotransformation reactions occurring in this system, a method using liquid chromatography hyphenated to an inductively coupled plasma mass spectrometer (HPLC‐ICP‐MS) was developed. A Hamilton PRP‐X100 anion exchange column was used. The method allowed separation, identification and quantification of As^III(arsenite), As^V(arsenate), DMA^V(dimethylarsinicacid), MMA^V(monomethylarsonicacid) and MMMTA (monomethylmonothioarsenate). Attempts to optimize the same method for also separating MMA^III and DMA^III did not succeed. These compounds could be successfully separated using a method based on the use of a Zorbax C₁₈ column. The properties of the column, buffer strength, pH and polar nature of mobile phase were monitored and changed to optimize the developed methods. Linearity, sensitivity, precision, accuracy and resolution of both methods were checked. The combination of the two methods allowed successful quantification of arsenic species in suspensions sampled in vitro from the SHIME reactor or in vivo from the human colon and feces. Copyright © 2011 John Wiley & Sons, Ltd.     

Competitive Binding of Tolmetin to β-Cyclodextrin and Human Serum Albumin: <Superscript>1</Superscript>H NMR and Fluorescence Spectroscopy Studies

The host–guest interaction of tolmetin (TOL) with β-cyclodextrin (β-CD) and the influence of human serum albumin (HSA) on the formation of the inclusion complex were studied by 1D and 2D NMR spectroscopy. The TOL/β-CD inclusion complex formed at a molar ratio of 1:1 with a binding constant value of 2164.5 L·mol^?1. Data analysis showed that the addition of 10 μmol·L^?1 of HSA weakened the strength of TOL binding to β-CD (K _a = 1493 L·mol^?1). The interaction of TOL with HSA in the absence and presence of β-CD was studied by analyzing the fluorescence quenching data. The Stern–Volmer quenching constants and the binding constants are found to be smaller in the presence of β-CD, suggesting that β-CD hinders the strong interaction of TOL with HSA by complex formation. Additionally, the presence of β-CD does not induce conformational and microenvironmental changes on HSA.     

Chromatographic Framework to Determine the Memantine Binding Mechanism on Human Serum Albumin Surface

In this work, the interaction of memantine with human serum albumin (HSA) immobilized on porous silica particles was studied using a biochromatographic approach. The determination of the enthalpy change at different pH values suggested that the protonated group in the memantine–HSA complex exhibits a heat protonation with a magnitude around 65 kJ mol^?1. This value agrees with the protonation of a guanidinium group, and confirmed that an arginine group may become protonated in the memantine–HSA complex formation. The thermodynamic data showed that memantine–HSA binding, for low temperature (<293 K), is dominated by a positive entropy change. This result suggests that dehydration at the binding interface and charge–charge interactions contribute to the memantine–HSA complex formation. Above 293 K, the thermodynamic data ΔH and ΔS became negative due to van der Waals interactions and hydrogen bonding which are engaged at the complex interface. The temperature dependence of the free energy of binding is weak because of the enthalpy–entropy compensation caused by a large heat capacity change, ΔC _p = ? 3.79 kJ mol^?1 K^?1 at pH = 7. These results were used to determine the potential binding site of this drug on HSA.     

Simple and selective determination of arsenite and arsenate by electrospray ionization mass spectrometry

Arsenic pollution of public water supplies has been reported in various regions of the world. Recently, some cancer patients are treated with arsenite (As^III); most Japanese people consume seafoods containing large amounts of negligibly toxic arsenic compounds. Some of these arsenic species are metabolized, but some remain intact. For the determination of toxic As^III, a simple, rapid and sensitive method has been developed using electrospray ionization mass spectrometry (ESI-MS). As^III was reacted with a chelating agent, pyrrolidinedithiocarbamate (PDC, C₄H₈NCSS^-) and tripyrrolidinedithiocarbamate-arsine, As(PDC)₃, extracted with methyl isobutyl ketone (MIBK). A 1 μL aliquot of MIBK layer was directly injected into ESI-MS instrument without chromatographic separation, and was detected within 1 min. Arsenate (As^V) was reduced to As^III with thiosulfate, and then the total inorganic As was quantified as As^III. This method was validated for the analysis of urine samples. The limit of detection of As was 0.22 μg L⁻¹ using 10 μL of sample solution, and it is far below the permissible limit of As in drinking water, 10 μg L⁻¹, recommended by the WHO. Results were obtained in < 10 min with a linear calibration range of 1-100 μg L⁻¹. Several organic arsenic compounds in urine did not interfere with As^III detection, and the inorganic As in the reference materials SRM 2670a and 1643e were quantified after the reduction of As^V to As^III.     

Thermodynamic study of indoxylsulfate interaction with human serum albumin and competitive binding with p-cresylsulfate

Indoxylsulfate (IS) and p-cresylsulfate (PCS) are natural compounds endowed with toxicity. These molecules are harmful to the environment and removed by the procedure of dialysis. Knowledge of their interaction with biologic compounds such as proteins and particularly human serum albumin (HSA) is limited. This study was therefore designed to determine the thermodynamic parameters of the interaction of IS with HSA and in competition with PCS. Results showed that IS binding is moderate (K _a = 1,750 ± 39 M^?1). The interaction is mainly electrostatic (?H° = ?36.2 ± 1.7 kJ mol^?1) and yields a modification of conformation upon binding (?_conf S° < 0). The thermodynamic parameters obtained at different temperatures show an enthalpy–entropy compensation process. Competition with PCS reveals that affinity for IS decreased by 36 %, with profound modification of the binding forces involved and a release of PCS from the binding site.     

Computational and experimental study on the interaction of three novel rare earth complexes containing 2,9-dimethyl-1,10-phenanthroline with human serum albumin

In this paper, several rare earth [terbium(III), ytterbium(III) and yttrium(III)] complexes containing 2,9-dimethyl-1,10-phenanthroline (Me₂Phen) were successfully synthesized and characterized by means of elemental analysis (CHN), infrared spectroscopy (FT-IR), UV–vis absorption spectroscopy and ¹HNMR. To explore the potential medicinal value of these complexes (MMe₂Phen), their binding interactions with human serum albumin (HSA) were investigated through UV–vis and fluorescence spectroscopies and also molecular docking examinations. The thermodynamic parameters, binding forces and Förster resonance distance between these complexes and Trp-214 of HSA were estimated from the analysis of fluorescence measurements. The values of estimated binding constants (K_b) ranging for the formation of MMe₂Phen:HSA complex were in the order of 10⁵ M^?1. The thermodynamic parameters determined by van’t Hoff analysis of K_b (ΔH°?<?0 and ΔS°?<?0) clearly indicate the major rules of hydrogen bonds and van der Waals interactions in the formation process of MMe₂Phen:HSA. The values of Stern–Volmer constant and the evaluation of dynamic quenching constant at various temperatures provided good evidences for static quenching mechanism. Furthermore, the results of molecular docking calculation and competitive binding experiments represent the binding of these complexes to site 3 of HSA located in subdomain IB, containing both polar and apolar residues. The consistency of computational and experimental results, according to the binding sites and the order of binding affinities (TbMe₂Phen?>?YbMe₂Phen?>?YMe₂Phen), supports the accuracy of docking calculation.     

Molecular Origin of the Hydrolytic Activity and Fixed Regioselectivity of a ZrIV‐Substituted Polyoxotungstate as Artificial Protease

A multitechnique approach has been applied in order to identify the thermodynamic and kinetic parameters related to the regioselective hydrolysis of human serum albumin (HSA) promoted by the Wells–Dawson polyoxometalate (POM), K₁₅H[Zr(α₂‐P₂W₁₇O₆₁)₂]. Isothermal titration calorimetry (ITC) studies indicate that up to four POM molecules interact with HSA. While the first interaction site is characterized by a 1:1 binding and an affinity constant of 2×10⁸ M ^?1, the three remaining sites are characterized by a lower global affinity constant of 7×10⁵ M ^?1. The higher affinity constant at the first site is in accordance with a high quenching constant of 2.2×10⁸ M ^?1 obtained for fluorescence quenching of the Trp214 residue located in the only positively charged cleft of HSA, in the presence of K₁₅H[Zr(α₂‐P₂W₁₇O₆₁)₂]. In addition, Eu^III luminescence experiments with an Eu^III‐substituted POM analogue have shown the replacement of water molecules in the first coordination sphere of Eu^III due to binding of the metal ion to amino acid side chain residues of HSA. All three interaction studies are in accordance with a stronger POM dominated binding at the positive cleft on the one hand, and interaction mainly governed by metal anchoring at the three remaining positions, on the other hand. Hydrolysis experiments in the presence of K₁₅H[Zr(α₂‐P₂W₁₇O₆₁)₂] have demonstrated regioselective cleavage of HSA at the Arg114?Leu115, Ala257?Asp258, Lys313?Asp314 or Cys392?Glu393 peptide bonds. This is in agreement with the interaction studies as the Arg114?Leu115 peptide bond is located in the positive cleft of HSA and the three remaining peptide bonds are each located near an upstream acidic residue, which can be expected to coordinate to the metal ion. A detailed kinetic study has evidenced the formation of additional fragments upon prolonged reaction times. Edman degradation of the additional reaction products has shown that these fragments result from further hydrolysis at the initially observed cleavage positions, indicating a fixed selectivity for K₁₅H[Zr(α₂‐P₂W₁₇O₆₁)₂].     

Determination of arsenic and antimony in seawater by voltammetric and chronopotentiometric stripping using a vibrated gold microwire electrode

The oxidation potentials of As⁰/As^III and Sb⁰/Sb^III on the gold electrode are very close to each other due to their similar chemistry. Arsenic concentration in seawater is low (10–20 nM), Sb occurring at ∼0.1 time that of As. Methods are shown here for the electroanalytical speciation of inorganic arsenic and inorganic antimony in seawater using a solid gold microwire electrode. Anodic stripping voltammetry (ASV) and chronopotentiometry (ASC) are used at pH ≤ 2 and pH 8, using a vibrating gold microwire electrode. Under vibrations, the diffusion layer size at a 5 μm diameter wire is 0.7 μm. The detection limits for the As^III and Sb^III are below 0.1 nM using 2 min and 10 min deposition times respectively. As^III and Sb^III can be determined in acidic conditions (after addition of hydrazine) or at neutral pH. In the latter case, oxidation of As⁰ to As^III was found to proceed through a transient As^III species. Adsorption of this species on the gold electrode at potentials where Sb^III diffused away is used for selective deposition of As^III. Addition of EDTA removes the interfering effect of manganese when analysing As^III. Imposition of a desorption step for Sb^III analysis is required. Total inorganic arsenic (iAs = As^V + As^III) can be determined without interference from Sb nor mono-methyl arsenious acid (MMA) at 1.6 < pH < 2 using E_dep = −1 V. Total inorganic antimony (iSb = Sb^V + Sb^III) is determined at pH 1 using E_dep = −1.8 V without interference by As.     

A new 1-D chain based on the trivacant monocapped Keggin arsenomolybdate and the copper complex linker: synthesis,crystal structure,and ESI-MS analyses

A new organic–inorganic hybrid (H₂en)₂[[Cu(en)₂]As^IIIAs^VMo^VI₉O₃₄]·6H₂O (1), containing a 1-D helical chain based on the trivacant monocapped Keggin arsenomolybdate and the copper complex linker {[Cu(en)₂][As^IIIAs^VMo^VI₉O₃₄]}_n^4n? (en = ethylenediamine), has been synthesized and characterized by IR spectra, TG analyses, single-crystal X-ray diffraction, and high-resolution electrospray ionization mass spectrometry (ESI-MS). Large voids are observed and a 1-D chain containing repeated (H₂O)₈ water units from lattice water molecules is formed along the a axis in the crystal structure. The high-resolution ESI-MS shows that the intact framework [Cu(en)₂][As^IIIAs^VMo^VI₉O₃₄]^4? exists in solution.     

Solution Chemistry of Arsenic Anions in the Presence of Metal Cations

The interaction of arsenic(V) and arsenic(III) oxyanions with metal cations was investigated by potentiometry under temperature and ionic strength conditions approaching those prevailing in natural waters. The selection includes the major metal cations and some other ions of high environmental relevance. Ionic pairs [M(As^VO₄)]^?, [M(HAs^VO₄)] and [M(H₂As^IIIO₃)]⁺ formation is suggested for all +2 metal cations, based on the potentiometric results. These ion-pairs between arsenic anions and other metal cations are hardly ever mentioned or taken into account when arsenic speciation in natural waters is considered. These results provide the basis for studying arsenic speciation in natural aquatic systems, on which environmental fate, bioavailability and toxicity of the element depend. Some extrapolations to the conditions of the natural waters are presented as well as some insights into the adsorption process onto hydrous oxides.     

Interaction Between Ginkgolic Acid and Human Serum Albumin by Spectroscopy and Molecular Modeling Methods

The interaction of ginkgolic acid (15:1, GA) with human serum albumin (HSA) was investigated by FT–IR, CD and fluorescence spectroscopic methods as well as molecular modeling. FT–IR and CD spectroscopic showed that complexation with the drug alters the protein’s conformation by a major reduction of α-helix from 54 % (free HSA) to 46–31 % (drug–complex), inducing a partial protein destabilization. Fluorescence emission spectra demonstrated that the fluorescence quenching of HSA by GA was by a static quenching process with binding constants on the order of 10⁵ L·mol^?1. The thermodynamic parameters (ΔH = ?28.26 kJ·mol^?1, ΔS = 11.55 J·mol^?1·K^?1) indicate that hydrophobic forces play a leading role in the formation of the GA–HSA complex. The ratio of GA and HSA in the complex is 1:1 and the binding distance between them was calculated as 2.2 nm based on the Förster theory, which indicates that the energy transfer from the tryptophan residue in HSA to GA occurs with high probability. On the other hand, molecular docking studies reveal that GA binds to Site II of HSA (sub-domain IIIA), and it also shows that several amino acids participate in drug–protein complexation, which is stabilized by H-bonding.     

Mechanistic Features of the TiO2 Heterogeneous Photocatalysis of Arsenic and Uranyl Nitrate in Aqueous Suspensions Studied by the Stopped‐Flow Technique

The dynamics of the transfer of electrons stored in TiO₂ nanoparticles to As^III, As^V, and uranyl nitrate in water was investigated by using the stopped‐flow technique. Suspensions of TiO₂ nanoparticles with stored trapped electrons (e_trap^?) were mixed with solutions of acceptor species to evaluate the reactivity by following the temporal evolution of e_trap^? by the decrease in the absorbance at λ=600 nm. The results indicate that As^V and As^III cannot be reduced by e_trap^? under the reaction conditions. In addition, it was observed that the presence of As^V and As^III strongly modified the reaction rate between O₂ and e_trap^?: an increase in the rate was observed if As^V was present and a decrease in the rate was observed in the presence of As^III. In contrast with the As system, U^VI was observed to react easily with e_trap^? and U^IV formation was observed spectroscopically at λ=650 nm. The possible competence of U^VI and NO₃^? for their reduction by e_trap^? was analyzed. The inhibition of the U^VI photocatalytic reduction by O₂ could be attributed to the fast oxidation of U^V and/or U^IV.     

Chemical analysis of main arsenic species in mixtures of As and AsF6

Known analytical techniques are not applicable to the accurate and precise determination of As^V and total arsenic (As_t) in the mixtures of As^III and AsF₆⁻. For this reason, an accurate and precise analytical procedure for determination of the content of As^V and As_t in the range of 5-10 mg of As with a relative standard deviation (R.S.D.) smaller than 0.4% was developed. The results were proved by the determination of As^III by titration with KBrO₃ and gravimetric determination of AsF₆⁻ species.     

A feasibility study on oxidation state of arsenic in cut tobacco,mainstream cigarette smoke and cigarette ash by X-ray absorption spectroscopy

This work describes the application of synchrotron-based X-ray Absorption Near-Edge Structure spectroscopy to study the oxidation state of arsenic in cigarette mainstream smoke, cut tobacco and cigarette ash. The level of arsenic in the total particulate matter of the smoke is approximately 1 ppm for the standard research reference cigarette 2R4F and its replacement 3R4F. Smoke particulate samples collected by a conventional glass-fiber membrane (commercially known as Cambridge filter pad) and a jet-impaction method were analyzed and compared. In addition smoke particulate samples were aged either at ambient temperature or at 195 K. X-ray Absorption Near-Edge Structure spectroscopy results revealed that the cut tobacco powder and cigarette ash contained almost exclusively As^V. The smoke particulate samples however contained a mixture of As^III and As^V. The As^V in the smoke particulate was reduced to As^III upon aging. Stabilizing the smoke particulate matter at 195 K by solid CO₂ slowed down this aging reaction and revealed a higher percentage of As^V. This behavior is consistent with the redox properties of the arsenic species and the smoke particulate matrix.     

Human Saliva-Based Quantitative Monitoring of Clarithromycin by Flow Injection Chemiluminescence Analysis: A Pharmacokinetic Study

Human saliva quantitative monitoring of clarithromycin (CLA) by chemiluminescence (CL) with flow injection analysis was proposed for the first time, which was based on the quenching effect of CLA on luminol–bovine serum albumin (BSA) CL system with a linear range from 7.5?×?10^?4 to 2.0 ng/ml. This proposed approach, offering a maximum sample throughput of 100 h^?1, was successfully applied to the quantitative monitoring of CLA levels in human saliva during 24 h after a single oral dose of 250 mg intake, with recoveries of 95.2～109.0 % and relative standard deviations lower than 6.5 % (N?=?7). Results showed that CLA reached maximum concentration of 2.28?±?0.02 μg/ml at approximately 3 h, and the total elimination ratio was 99.6 % in 24 h. The pharmacokinetic parameters including absorption rate constant (0.058?±?0.006 h^?1), elimination rate constant (0.149?±?0.009 h^?1) and elimination half-life time (4.66?±?0.08 h) were obtained. A comparison of human saliva and urine monitoring was also given. The mechanism study of BSA–CLA interaction revealed the binding of CLA to BSA is an entropy driven and spontaneous process through hydrophobic interaction, with binding constant K _BSA–CLA of 4.78?×?10⁶ l/mol and the number of binding sites n of 0.82 by flow injection–chemiluminescence model. Molecular docking analysis further showed CLA might be in subdomain IIA of BSA, with K _BSA–CLA of 6.82?×?10⁵ l/mol and ΔG of ?33.28 kJ/mol.     

Molecular dynamics and energetic perceptions of substrate recognition by thymidylate kinase

Plasmodium deoxyguanylate pathways are an attractive area of investigation for future metabolic and drug discovery studies due to their unusual substrate specificities. We investigated the energetic contribution to thymidylate kinase substrate binding, and the forces underlying ligand recognition. The binding constant varied from 8 × 10⁴ M^?1 at 290 K to 6 × 10⁴ M^?1 at 310 K for dGMP, and from 16 × 10⁴ M^?1 at 290 K to 4 × 10⁴ M^?1 at 310 K for TMP. ΔC _p was estimated as ?1.75 kJ mol^?1 K^?1 for TMP and +2 kJ mol^?1 K^?1 for dGMP. In comparison with TMP, the binding of dGMP to PfTMK produced less favorable enthalpy change, positive or favorable entropic contribution at lower temperature, positive heat capacity change, negative $ \Updelta S_{\text{HE}}^{^\circ } $ , positive ΔS _other, higher total solvent-exposed surface area and more or less rigid body binding. These changes indicate unfavorable conditions for proper binding and lower conformational changes, and suboptimal structural reordering during dGMP binding.     

Excretion patterns of arsenic and its metabolites in human saliva and urine after ingestion of Chinese seaweed

There are no reports in scientific literature on arsenic species in human saliva after seaweed exposure. The present article reports for the first time the regular excretion patterns of arsenic in the saliva of volunteers with one-time ingestion of Chinese seaweed. Total arsenic and speciation analyses were carried out by high-performance liquid chromatography–inductively coupled plasma–mass spectrometry (HPLC-ICP-MS). Results show that the excretion time of total arsenic in saliva is a trifle earlier than that in urine, total arsenic in human saliva also shows a regular excretion pattern like that in urine within 72 h after exposure to seaweed. For speciation analysis, four species, including the major dimethylarsinic acid (DMA) species, were detected in urine prior to seaweed intake. Six species were detected in urine after seaweed ingestion, including DMA, methylarsonic acid (MMA), oxo-dimethylarsinoylethanol (oxo-DMAE), thio-dimethlyarsenoacetate (thio-DMAA), arsenite (As^III) and arsenate (As^V). In saliva samples, three species were found before seaweed ingestion, with the major peak identified as As^III. After consumption, the kinds of arsenic metabolites in saliva were less than those in urine. The major species was inorganic arsenic (iAs As^III+As^V), followed by DMA, MMA and a trace amount of oxo-DMAE. Taken together, the present study suggests that saliva assay can be used as a potential tool for understanding the regular excretion pattern of total arsenic after seaweed ingestion. Whether or not it’s an efficient tool for assessing arsenic metabolites in humans exposed to seaweed requires further investigation.     

Speciation of arsenic in ground water samples: A comparative study of CE-UV, HG-AAS and LC-ICP-MS

The performance of capillary electrophoresis-ultraviolet detector (CE-UV), hydride generation-atomic absorption spectrometry (HG-AAS) and liquid chromatography-inductively coupled plasma mass spectrometry (LC-ICP-MS) have been compared for the speciation of arsenic (As) in groundwater samples. Two inorganic As species, arsenite (As^III), arsenate (As^V) and one organo species dimethyl arsenic acid (DMA) were mainly considered for this study as these are known to be predominant in water. Under optimal analytical conditions, limits of detection (LD) ranging from 0.10 (As^III, AsT) to 0.19 (DMA) μg/l for HG-AAS, 100 (As^III, DMA) to 500 (As^V) μg/l for CE-UV and 0.1 (DMA, MMA) to 0.2 (As^III, As^V) μg/l for LC-ICP-MS, allowed the determination of the above three species present in these samples. Results obtained by all the three methods are well correlated (r² = 0.996*** for total As) with the precision of <5% R.S.D. except CE-UV. The effect of interfering ions (e.g. Fe²⁺, Fe³⁺, SO₄²⁻ and Cl⁻) commonly found in ground water on separation and estimation of As species were studied and corrected for. Spike recovery was tested and found to be 80-110% at 0.5 μg/l As standard except CE-UV where only 50% of the analyte was recovered. Comparison of these results shows that LC-ICP-MS is the best choice for routine analysis of As species in ground water samples.