Internal dose of vanadium in rats following repeated exposure to vanadyl sulfate and sodium orthovanadate via drinking water.

Vanadium is a ubiquitous environmental contaminant that exists in multiple oxidation states. Humans are exposed to vanadyl (V4+) and vanadate (V5+) from dietary supplements, food, and drinking water and hence there is a concern for adverse human health. The current investigation is aimed at identifying vanadium oxidation states in vitro and in vivo and internal concentrations following exposure of rats to vanadyl sulfate (V4+) or sodium metavanadate (V5+) via drinking water for 14 d. Investigations in simulated gastric and intestinal fluids showed that V4+ was stable in gastric fluid while V5+ was stable in intestinal fluid. Analysis of rodent plasma showed that the only vanadium present was V4+, regardless of the exposed compound suggesting conversion of V5+ to V4+ in vivo and/or instability of V5+ species in biological matrices. Plasma, blood, and liver concentrations of total vanadium, after normalizing for vanadium dose consumed, were higher in male and female rats following exposure to V5+ than to V4+. Following exposure to either V4+ or V5+, the total vanadium concentration in plasma was 2- to 3-fold higher than in blood suggesting plasma as a better matrix than blood for measuring vanadium in future work. Liver to blood ratios were 4-7 demonstrating significant tissue retention following exposure to both compounds. In conclusion, these data point to potential differences in absorption and disposition properties of V4+ and V5+ salts and may explain the higher sensitivity in rats following drinking water exposure to V5+ than V4+ and highlights the importance of internal dose determination in toxicology studies.

Arsenic exposure and incidence of type 2 diabetes in Southwestern American Indians.

Association of urinary arsenic concentration with incident diabetes was examined in American Indians from Arizona who have a high prevalence of type 2 diabetes and were screened for diabetes between 1982 and 2007. The population resides where drinking water contains arsenic at concentrations above federally recommended limits. A total of 150 nondiabetic subjects aged ≥25 years who subsequently developed type 2 diabetes were matched by year of examination and sex to 150 controls who remained nondiabetic for ≥10 years. Total urinary arsenic concentration, adjusted for urinary creatinine level, ranged from 6.6 µg/L to 123.1 µg/L, and inorganic arsenic concentration ranged from 0.1 µg/L to 36.0 µg/L. In logistic regression models adjusted for age, sex, body mass index, and urinary creatinine level, the odds ratios for incident diabetes were 1.11 (95% confidence interval (CI): 0.79, 1.57) and 1.16 (95% CI: 0.89, 1.53) for a 2-fold increase in total arsenic and inorganic arsenic, respectively. Categorical analyses suggested a positive relationship between quartiles of inorganic arsenic and incident diabetes (P = 0.056); post-hoc comparison of quartiles 2-4 with quartile 1 revealed 2-fold higher odds of diabetes in the upper quartiles (OR = 2.14, 95% CI: 1.19, 3.85). Modestly elevated exposure to inorganic arsenic may predict type 2 diabetes in American Indians. Larger studies that include measures of speciated arsenic are required for confirmation.

Quantitation of Total Vanadium in Rodent Plasma and Urine by Inductively Coupled Plasma - Mass Spectrometry (ICP-MS).

Human exposure to vanadium (V) is anticipated because it is a drinking water contaminant. Due to limited data on soluble V salts, the National Toxicology Program is investigating the toxicity in rodents following drinking water exposure. Measurement of internal V dose allows for interpretation of toxicology data. The objective of this study was to develop and validate an inductively coupled plasma-mass spectrometric method to quantitate total V in rat plasma. The method was linear (r ≥ 0.99) from 5.00 - 1,000 ng V/mL. Intra- and inter-day relative error (% RE) and relative standard deviation (% RSD) of spiked plasma samples were 8.5% - 15.6% RE and ≤ 1.8% RSD and 7.3% - 11.7% RE and ≤ 3.1% RSD, respectively. The limit of detection was 0.268 ng V/mL plasma and absolute percent recovery was 113%. Standards up to 7,500 ng V/mL plasma were diluted into the validated range (5.6% RE, 0.9% RSD). V in extracted plasma samples over 15 days at ambient and refrigerated conditions was from 97.7 - 126% of day 0. Determined plasma V concentrations after three freeze-thaw cycles and after frozen storage for up to 63 days ranged from 100 - 106% and 100 - 122% of day 0, respectively. The method was extended to rat urine (accuracy and precision -2.0 - 0.3% RE and <0.6% RSD, respectively for same linear range). These data demonstrate that the method is suitable to quantitate V in rat plasma and urine.

Validation and application of a method for the determination of total chromium in rat tissues by inductively coupled plasma mass spectrometry.

The validation of a method for the determination of chromium (Cr) in F-344/N rat tissues by inductively coupled plasma-mass spectrometry is described. Samples were analyzed after a rapid, open-vessel microwave digestion procedure. Performance of the method was evaluated using kidney tissue across a concentration range of 0.50-5.00 microg Cr/g tissue. Data for method linearity, accuracy, precision, digest stability, and storage stability are presented along with limits of detection and quantitation data. Data from a method cross-validation for B6C3F1 mouse kidney tissue are also presented. After validation, the method was applied to analyze samples collected in support of two chronic toxicity and carcinogenesis studies conducted by the National Toxicology Program.

Characterization of Zinc Carbonate Basic as a Source of Zinc in a Rodent Study Investigating the Effects of Dietary Deficiency or Excess.

Zinc deficiency and excess can result in adverse health outcomes. There is conflicting evidence regarding whether excess or deficient zinc in the diet can contribute to carcinogenicity. The objective of this study was to characterize zinc carbonate basic for use as a source of dietary zinc in a rodent toxicity and carcinogenicity study investigating the effects of zinc deficiency and excess. Because of the complex chemistries of zinc carbonate basic compounds, inconsistent nomenclature, and literature and reference spectra gaps, it was necessary to employ multiple analytical techniques, including Karl Fischer titration, combustion analysis, inductively coupled plasma-optical emission spectrometry, X-ray diffraction, infrared spectroscopy, X-ray fluorescence spectrometry, and thermogravimetric analysis to characterize the test article. Based on the collective evidence and through the process of elimination, the test article was found to be composed mainly of zinc carbonate basic with zinc oxide as a minor component. The zinc content was determined to be 56.6% (w/w) with heavy metals such as arsenic, cadmium, mercury and lead below the limit of quantitation of less than or equal to 0.01%. The test material was stable at ambient temperature. Based on the work described in this manuscript, the test article was suitable for use as a source of zinc in studies of deficiency and excess in the diet.

Immobilized pH gradient isoelectric focusing as a first-dimension separation in shotgun proteomics.

Shotgun proteomics, where a tryptic digest of a complex proteome sample is directly analyzed by either single dimensional or multidimensional liquid chromatography tandem mass spectrometry, has gained acceptance in the proteomics community at large and is widely used in core facilities. Here we review the development in our laboratory of an alternative first-dimension separation technique for shotgun proteomics, immobilized pH gradient isoelectric focusing (IPG-IEF). The key advantages of the technology over other multidimensional separation formats (simplicity, high resolution, and high sensitivity) are discussed. The concept of using peptide pI to filter large shotgun proteomics datasets generated by the IPG-IEF technique to minimize false positives and negatives is also introduced. Finally, an account of the comparison of the technique with the established gold standard for multidimensional separation of peptides, strong cation exchange chromatography, is presented, along with the prospects for the use of peptide pI along with accurate mass measurement for the identification of peptides.

Development of an analytical method for assessment of silver nanoparticle content in biological matrices by inductively coupled plasma mass spectrometry.

Silver nanoparticles (AgNPs) are a broad class of synthetic nanoparticles that are utilized in a wide variety of consumer products as antimicrobial agents. Despite their widespread use, a detailed understanding of their toxicological characteristics and biological and environmental hazards is not available. To support research into the biodistribution and toxicology of AgNPs, it is necessary to develop a suitable method for the assessment of AgNP content in biological samples. Two methods were developed and validated to analyze citrate-coated AgNP content that utilize acid digestion of rodent feces and liver tissue samples, and a third method was developed for the dilution and direct analysis of rodent urine samples. Following sample preparation, the silver content of each sample was determined by inductively coupled plasma mass spectrometry (ICP-MS) to quantify the silver and AgNP levels present. Analysis of rat feces matrix yielded analytical recoveries ranging from 82 to 93 %. Liver tissue spiked with a formulation of AgNPs over a range of concentrations yielded analytical recoveries between 88 and 90 %, providing acceptable accuracy results. The analysis of silver in urine samples exhibited recovery values ranging from 80 to 85 % for AgNP formulations and 62-84 % for standard silver ion solutions. All determinations exhibited a high degree of analytical precision. The results obtained here suggest that matrix interference plays a minimal role in AgNP recovery in feces and liver tissue, while the urine matrix can exhibit a significant effect on the determination of silver content.

Development, validation, and application of an ultra-performance liquid chromatography-sector field inductively coupled plasma mass spectrometry method for simultaneous determination of six organotin compounds in human serum.

Organotin compounds (OTCs) are heavily employed by industry for a wide variety of applications, including the production of plastics and as biocides. Reports of environmental prevalence, differential toxicity between OTCs, and poorly characterized human exposure have fueled the demand for sensitive, selective speciation methods. The objective of this investigation was to develop and validate a rapid, sensitive, and selective analytical method for the simultaneous determination of a suite of organotin compounds, including butyl (mono-, di-, and tri-substituted) and phenyl (mono-, di-, and tri-substituted) species in human serum. The analytical method utilized ultra-performance liquid chromatography (UPLC) coupled with sector field inductively coupled plasma mass spectrometry (SF-ICP-MS). The small (sub-2 µm) particle size of the UPLC column stationary phase and the sensitivity of the SF-ICP-MS enabled separation and sensitive determination of the analyte suite with a runtime of approximately 3 min. Validation activities included demonstration of method linearity over the concentration range of approximately 0.250-13.661 ng mL(-1), depending on the species; intraday precision of less than 21%, interday precision of less than 18%, intraday accuracy of -5.3% to 19%, and interday accuracy of -14% to 15% for all species; specificity, and matrix impact. In addition, sensitivity, and analyte stability under different storage scenarios were evaluated. Analyte stability was found to be limited for most species in freezer, refrigerator, and freeze-thaw conditions. The validated method was then applied for the determination of the OTCs in human serum samples from women participating in the Snart-Foraeldre/MiljØ (Soon-Parents/Environment) Study. The concentration of each OTC ranged from below the experimental limit of quantitation to 10.929 ng tin (Sn) mL(-1) serum. Speciation values were confirmed by a total Sn analysis.

Analysis of human serum and whole blood for mineral content by ICP-MS and ICP-OES: development of a mineralomics method.

Minerals are inorganic compounds that are essential to the support of a variety of biological functions. Understanding the range and variability of the content of these minerals in biological samples can provide insight into the relationships between mineral content and the health of individuals. In particular, abnormal mineral content may serve as an indicator of illness. The development of robust, reliable analytical methods for the determination of the mineral content of biological samples is essential to developing biological models for understanding the relationship between minerals and illnesses. This paper describes a method for the analysis of the mineral content of small volumes of serum and whole blood samples from healthy individuals. Interday and intraday precision for the mineral content of the blood (250 µL) and serum (250 µL) samples was measured for eight essential minerals--sodium (Na), calcium (Ca), magnesium (Mg), potassium (K), iron (Fe), zinc (Zn), copper (Cu), and selenium (Se)--by plasma spectrometric methods and ranged from 0.635 to 10.1% relative standard deviation (RSD) for serum and 0.348-5.98% for whole blood. A comparison of the determined ranges for ten serum samples and six whole blood samples provided good agreement with literature reference ranges. The results demonstrate that the digestion and analysis methods can be used to reliably measure the content of these minerals and potentially of other minerals.

Analysis of the DNA damage produced by a platinum-acridine antitumor agent and its effects in NCI-H460 lung cancer cells.

High-performance liquid chromatography in conjunction with electrospray mass spectrometry (LC-ESMS) was used to structurally characterize the adducts formed by the platinum-acridine agent [PtCl(en)(N-(2-(acridin-9-ylamino)ethyl)-N-methylpropionimidamide)](NO(3))(2) (compound 1) in cell-free DNA. Compound 1 forms monofunctional adducts exclusively with guanine, based on the fragments identified in enzymatic digests (dG*, dGMP*, dApG*, and dTpG*, where the asterisk denotes bound drug). The time course of accumulation and DNA adduct formation of compound 1 and the clinical drug cisplatin in NCI-H460 lung cancer cells at physiologically relevant drug concentrations (0.1 µM) was studied by inductively-coupled plasma mass spectrometry (ICP-MS). Compound 1 accumulates rapidly in cells and reaches intracellular levels of up to 60-fold higher than those determined for cisplatin. The hybrid agent shows unusually high DNA binding levels: while cisplatin adducts form at a maximum frequency of 5 adducts per 10(6) nucleotides, compound 1 produces 25 adducts per 10(6) nucleotides after only 3 h of continuous incubation with the lung cancer cells. The high overall levels of compound 1 in the cells and in cellular DNA over the entire 12-h treatment period translate into a rapid decrease in cell viability. Possible implications of these findings for the mechanism of action of compound 1 and the agent's potential to overcome tumor resistance to cisplatin are discussed.

Validation of a Method for the Determination of Total Chromium in Rat Feces by Inductively Coupled Plasma Optical Emission Spectrometry.

The validation of a method for the determination of total chromium in Fischer-344 rat feces by inductively coupled plasma optical emission spectrometry following a rapid, atmospheric-pressure microwave digestion is described. The performance of the method was evaluated over the concentration range of 5.00 to 200 µg Cr/g feces. Data for method linearity, accuracy, precision, digest stability, and storage stability are presented along with limit of detection and limit of quantitation data. Data from a cross-validation method for B6C3F1 mouse feces are also presented. Following validation, the method was applied to analyze samples collected in support of two chronic toxicological investigations.

Calculation of the isoelectric point of tryptic peptides in the pH 3.5-4.5 range based on adjacent amino acid effects.

Current algorithms for the calculation of peptide or protein pI, based on the charge associated with individual amino acids, can calculate pI values to within +/-0.2 pI units. Here, we present a new pI calculation algorithm that takes into account the effect of adjacent amino acids on the pI value. The algorithm accounts for the effect of adjacent amino acids+/-3 residues away from a charged aspartic or glutamic acid, as well as effects on the free C terminus, and applies a correction term to the corresponding pK values. The correction increments are derived from a 5000-peptide training set using a genetic optimization approach. The accuracy of the new pI values obtained with this method approaches the error associated with the manufacture of the IPG strip (<+/-0.03 pI units). The approach is demonstrated for cytosolic cell extracts derived from the breast-cancer cell line DU4475, and from membrane preparations from human lung-tissue samples. One potential application of a more highly accurate pI calculation is data filtering of MS/MS outputs that will allow for more complex database searches including gene finding, and validation, and detection of coding single-nucleotide polymorphisms in their expressed form.

A comparison of immobilized pH gradient isoelectric focusing and strong-cation-exchange chromatography as a first dimension in shotgun proteomics.

Recently, we have developed a high-resolution two-dimensional separation strategy for the analysis of complex peptide mixtures. This methodology employs isoelectric focusing of peptides on immobilized pH gradient (IPG) gels in the first dimension, followed by reversed-phase chromatography in the second dimension, and subsequent tandem mass spectrometry analysis. The traditional approach to this mixture problem employs strong-cation-exchange (SCX) chromatography in the first dimension. Here, we present a direct comparison of these two first-dimensional techniques using complex protein samples derived from the testis of Rattus norvegicus. It was found that the use of immobilized pH gradients (narrow range pH 3.5-4.5) for peptide separation in the first dimension yielded 13% more protein identifications than the optimized off-line SCX approach (employing the entire pI range of the sample). In addition, the IPG technique allows for a much more efficient use on mass spectrometer analysis time. Separation of a tryptic digest derived from a rat testis sample on a narrow range pH gradient (over the 3.5-4.5 pH range) yielded 7626 and 2750 peptides and proteins, respectively. Peptide and protein identification was performed with high confidence using SEQUEST in combination with a data filtering program employing pI and statistical based functions to remove false-positives from the data.