Recapitulation of human pathophysiology and identification of forensic biomarkers in a translational model of chlorine inhalation injury.

Chlorine gas (Cl2) has been repeatedly used as a chemical weapon, first in World War I and most recently in Syria. Life-threatening Cl2 exposures frequently occur in domestic and occupational environments, and in transportation accidents. Modeling the human etiology of Cl2-induced acute lung injury (ALI), forensic biomarkers, and targeted countermeasures development have been hampered by inadequate large animal models. The objective of this study was to develop a translational model of Cl2-induced ALI in swine to understand toxico-pathophysiology and evaluate whether it is suitable for screening potential medical countermeasures and to identify biomarkers useful for forensic analysis. Specific pathogen-free Yorkshire swine (30-40 kg) of either sex were exposed to Cl2 (≤240 ppm for 1 h) or filtered air under anesthesia and controlled mechanical ventilation. Exposure to Cl2 resulted in severe hypoxia and hypoxemia, increased airway resistance and peak inspiratory pressure, and decreased dynamic lung compliance. Cl2 exposure resulted in increased total leucocyte and neutrophil counts in bronchoalveolar lavage fluid, vascular leakage, and pulmonary edema compared with the air-exposed group. The model recapitulated all three key histopathological features of human ALI, such as neutrophilic alveolitis, deposition of hyaline membranes, and formation of microthrombi. Free and lipid-bound 2-chlorofatty acids and chlorotyrosine-modified proteins (3-chloro-l-tyrosine and 3,5-dichloro-l-tyrosine) were detected in plasma and lung tissue after Cl2 exposure. In this study, we developed a translational swine model that recapitulates key features of human Cl2 inhalation injury and is suitable for testing medical countermeasures, and validated chlorinated fatty acids and protein adducts as biomarkers of Cl2 inhalation.NEW & NOTEWORTHY We established a swine model of chlorine gas-induced acute lung injury that exhibits several features of human acute lung injury and is suitable for screening potential medical countermeasures. We validated chlorinated fatty acids and protein adducts in plasma and lung samples as forensic biomarkers of chlorine inhalation.

Establishing Population Values for Chlorine Exposure in the United States (2015-2016) Using 2 Chlorine Biomarkers, 3-Chlorotyrosine and 3,5-Dichlorotyrosine.

BACKGROUND: In the United States, 12 million short tons of chlorine are manufactured and transported each year. Due to the volume of this volatile chemical, large- and small-scale chemical exposures occur frequently. To diagnose and treat potentially exposed individuals, reference range values for confirmatory biomarkers are required to differentiate between normal and abnormal exposure levels. METHODS: Serum surplus samples (n = 1780) from the National Health and Nutrition Examination Survey (NHANES) 2015-2016 were measured for 2 chlorine biomarkers, 3-chlorotyrosine (Cl-Tyr) and 3,5-dichlorotyrosine (Cl2-Tyr), by liquid chromatography coupled to a triple quadrupole mass spectrometer. We evaluated demographic factors associated with elevated biomarker levels. RESULTS: Participant samples were analyzed for the chlorine biomarkers Cl-Tyr and Cl2-Tyr. In the unweighted analysis of these samples, 1349 (75.8%) were under the limit of detection (< LOD) of 2.50 ng/mL for Cl-Tyr and 1773 (99.6%) were < LOD for Cl2-Tyr. Samples within the method reportable range were 2.50 to 35.6 ng/mL for Cl-Tyr and 2.69 to 11.2 ng/mL for Cl2-Tyr. Since only 7 of the 1780 participants had detectable Cl2-Tyr, statistical analysis was limited to Cl-Tyr. Of the demographic characteristics examined, age, body mass index (BMI), estimated glomerular filtration rate (eGFR), and sex exhibited statistically significant differences in the weighted prevalence of detectable Cl-Tyr. CONCLUSIONS: This is the first reported set of Cl-Tyr and Cl2-Tyr population values for the United States. This population range coupled with NHANES demographic information could help healthcare professionals distinguish between normal and abnormal chlorine biomarker levels in an emergency. With this information, an inference could be made when determining acute chlorine exposure in individuals.

A pilot PT scheme for external assessment of laboratory performance in testing synthetic opioid compounds in urine, plasma, and whole blood.

A proficiency testing (PT) scheme was prepared for laboratories engaged in bioanalytical testing for synthetic opioid compounds in urine, plasma, and whole blood. Samples were prepared using compounds included in the Opioid Certified Reference Material Kit (Opioid CRM Kit) developed by the U.S. Centers for Disease Control and Prevention. Laboratories received samples during a 2-year project with each year consisting of two PT events 6 months apart. In the first year (pilot test), participants included 10 public health laboratories throughout the United States. In the second year, the group of laboratories expanded to include clinical and forensic drug testing laboratories, and 12 additional participating labs joined the program. In Year 1, overall detection percentages for the compounds present in the PT samples were 95.5% in Event 1% and 97.2% in Event 2. There were 31 apparent false positives reported in Event 1 and four apparent false positives reported in Event 2. Carryover or contamination in laboratory analytical systems were found to be the most significant causes of the false positive results, and none of the laboratories that reported false positives in Event 1 did so in Event 2. In Year 2, overall detection percentages for the compounds present in the PT samples were 89.5% in Event 3% and 94.8% in Event 4. There was one apparent false positive reported in Event 3 and three apparent false positives reported in Event 4. Improvements in drug detection between the two PT events in each year demonstrated the benefit of PT schemes in identifying and addressing potential deficiencies in laboratory systems.

An experimental, computational, and uncertainty analysis study of the rates of iodoalkane trapping by DABCO in solution phase organic media.

NMR spectroscopy was used to measure the rates of the first and second substitution reactions between iodoalkane (R = Me, 1-butyl) and DABCO in methanol, acetonitrile and DMSO. Most of the reactions were recorded at three different temperatures, which permitted calculation of the activation parameters from Eyring and Arrhenius plots. Additionally, the reaction rate and heat of reaction for 1-iodobutane + DABCO in acetonitrile and DMSO were also measured using calorimetry. To help interpret experimental results, ab initio calculations were performed on the reactant, product, and transition state entities to understand structures, reaction enthalpies and activation parameters. Markov chain Monte Carlo statistical sampling was used to determine a distribution of kinetic rates with respect to the uncertainties in measured concentrations and correlations between parameters imposed by a kinetics model. The reactions with 1-iodobutane are found to be slower in all cases compared to reactions under similar conditions for iodomethane. This is due to steric crowding around the reaction centre for the larger butyl group compared to methyl which results in a larger activation energy for the reaction.

Confirmation of PNNL Quantitative Infrared Cross-Sections for Isobutane.

The Pacific Northwest National Laboratory (PNNL) gas-phase database is a compilation of quantitative experimental (5, 25, and 50 °C) infrared spectra of ca. 500 molecules, designed for in situ, standoff or remote sensing of gases and vapors at or near atmospheric pressure. The data are characterized by calibration on both the wavenumber and intensity axes. Recent papers have called into question the PNNL intensity values for isobutane, [2-methylpropane, HC(CH3)3], suggesting discrepancies of 30-40%. In this study, we remeasure and re-examine the intensity values of isobutane using both similar and alternate methods to those used to generate the original PNNL database spectra. Indirect confirmation from literature data of homologous molecules and direct confirmation from new results confirm that for many band integrals across the isobutane spectrum, the original PNNL data are indeed accurate to within the reported 3% experimental uncertainty.

Combining spectroscopic techniques to determine the optical constants of powdered lactose.

A method for deriving the optical constants (n/k) of organic powdered materials using pressed pellets in the mid-infrared spectral range is introduced that combines variable angle spectroscopic ellipsometry and transmission spectroscopy. The approach is applied to anhydrous lactose, in which three different forms of pellets were pressed and measured: a pure lactose pellet and a mixed lactose/potassium bromide (KBr) pellet with a large analyte percentage were used for ellipsometric measurements, and a KBr transmission pellet with only a small analyte percentage was used for transmission measurements. The transmittance data provide an initial set of oscillators and improve the spectral fitting of weak absorption features (k<0.01). Ellipsometric data for the pure and mixed pellets are then fit simultaneously to derive the final n/k values for lactose from 6000-400cm-1. An alternative method just using the ellipsometric data from the mixed pellet and the transmittance data is also presented and shows good agreement with the multi-sample analysis, providing a simpler method for powders that do not press easily into pure pellets. Finally, the derived optical constants were used to model the reflectance data, demonstrating a good match with the measured reflectance spectra if non-idealities are included.

Development of a clinical assay to measure chlorinated tyrosine in hair and tissue samples using a mouse chlorine inhalation exposure model.

Chlorine is a toxic industrial chemical with a history of use as a chemical weapon. Chlorine is also produced, stored, and transported in bulk making it a high-priority pulmonary threat in the USA. Due to the high reactivity of chlorine, few biomarkers exist to identify exposure in clinical and environmental samples. Our laboratory evaluates acute chlorine exposure in clinical samples by measuring 3-chlorotyrosine (Cl-Tyr) and 3,5-dichlorotyrosine (Cl2-Tyr) using liquid chromatography tandem mass spectrometry (LC-MS/MS). Individuals can have elevated biomarker levels due to their environment and chronic health conditions, but levels are significantly lower in individuals exposed to chlorine. Historically these biomarkers have been evaluated in serum, plasma, blood, and bronchoalveolar lavage (BAL) fluid. We report the expansion into hair and lung tissue samples using our newly developed tissue homogenization protocol which fits seamlessly with our current chlorinated tyrosine quantitative assay. Furthermore, we have updated the chlorinated tyrosine assay to improve throughput and ruggedness and reduce sample volume requirements. The improved assay was used to measure chlorinated tyrosine levels in 198 mice exposed to either chlorine gas or air. From this animal study, we compared Cl-Tyr and Cl2-Tyr levels among three matrices (i.e., lung, hair, and blood) and found that hair had the most abundant chlorine exposure biomarkers. Furthermore, we captured the first timeline of each analyte in the lung, hair, and blood samples. In mice exposed to chlorine gas, both Cl-Tyr and Cl2-Tyr were present in blood and lung samples up to 24 h and up to 30 days in hair samples.

Absolute Band Intensity of the Iodine Monochloride Fundamental Mode for Infrared Sensing and Quantitative Analysis.

Iodine monochloride (ICl) is a potential off-gas product of molten salt reactors; monitoring this heteronuclear diatomic molecule is of great interest for both environmental and safety purposes. In this paper, we investigate the possibility of infrared monitoring of ICl by measuring the far-infrared absorption cross section of its fundamental band near 381 cm-1. We have performed quantitative studies of the neat gas in a 20 cm cell at 25, 35, 50, and 70 °C at multiple pressures up to ∼9 Torr and investigated the temperature and pressure dependencies of the band's infrared cross section. Quantitative measurements were problematic due to sample adhesion to the cell walls and windows as well as reactions/possible hydrolysis of ICl to form HCl gas. Effects were mitigated by measuring only the neat gas, using short measurement times, and subtracting out the partial pressure of the HCl(g). The integrated band strength is shown to be temperature independent and was found to be equal to 9.1 × 10-19 (cm2/molecule) cm-1. As expected, the temperature dependence of the band profile showed only a small effect over this limited temperature range. We have also investigated using the absorption data along with inverse least squares multivariate methods for the quantitative monitoring of ICl effluent concentrations under different scenarios using infrared (standoff) sensing and compare these results with traditional Beer's law (univariate) techniques.

Infrared Optical Constants from Pressed Pellets of Powders: I. Improved n and k Values of (NH4)2SO4 from Single-Angle Reflectance.

In combination with other parameters, the real, n(v∼), and imaginary, k(v∼), components of the complex refractive index, n^ = n + ik, can be used to simulate the optical properties of a material in different forms, e.g., its infrared spectra. Ultimately, such n/k values can be used to generate a database of synthetic reflectance spectra for the different morphologies to which experimental data can be compared. But obtaining reliable values of the optical constants n/k for solid materials is challenging due to the lack of optical quality specimens, usually crystals, large enough to measure. An alternative to crystals is to press the powder into a uniform disk. We have produced pellets from ammonium sulfate, (NH4)2SO4, powder and derived the pellets' n and k values via single-angle reflectance using a specular reflectance device in combination with a Fourier transform infrared spectrometer. The single-angle technique measures amplitude of light reflected from the material as a function of wavelength over a wide spectral domain; the optical constants are determined from the reflectance data using the Kramers-Kronig relationship. We investigate several parameters associated with the pellets and pellet formation and their effects upon delivering the most reliable n/k values. Parameters studied include pellet diameter, mass, and density (void space), drying, grinding, sieving, and particle size in the pellet formation, as well as pressing pressure and duration. Of these parameters, using size-selected mixtures of dried, small (<50 µm) particles and pressing at ≥10 tons for at least 30 min were found key to forming highly reflective samples. Comparison of two sets of previous literature n(v∼) and k(v∼) values obtained from crystalline (NH4)2SO4 both as crystal reflectance as well as extinction spectra of aerosols measured in a flow tube shows reasonable agreement, but suggests the present values, as confirmed from two independent techniques, represent a substantial improvement for n/k values for (NH4)2SO4, also demonstrating promise to measure the optical constants of other materials.

Improved Infrared Optical Constants from Pressed Pellets: II. Ellipsometric n and k Values for Ammonium Sulfate with Variability Analysis.

Infrared reflectance analysis is facilitated via the comparison of spectra recorded in situ to a databank of actual or synthetic infrared reflectance spectra. It has recently been shown that reference spectra corresponding to the many different morphological forms of the same chemical can be generated synthetically using the imaginary, k, and real, n, components of the complex refractive index, n∼ = n + ik. One method to obtain the n and k vectors is infrared ellipsometry, which measures the changes in amplitude, tan Ψ, and phase, Δ, of polarized light reflected from the sample both as a function of wavenumber and angle of incidence. The method requires specularly reflected light, so best results are usually obtained with polished planar samples of large surface area. Due to the difficulties of obtaining such samples, however, we investigate the possibility of pressing powders of neat materials and obtaining the corresponding optical constants from the pellets. In this paper, variability in the sample pellet and preparation method is investigated, as is variability in the fitting procedure for the derived optical constants. The n/k vectors are derived from the measured ellipsometric parameters, tan ψ and Δ, as they are fit by an oscillator model which yield n(ν∼) and k(ν∼) vectors as a function of wavenumber, ν∼. Construction of the oscillator model is not automatic and depends on significant input from the analyst as well as the sample's physical characteristics. For pellet pressing, the experimental variability was found to be minimized for size-selected powdered samples as gauged by the minimal variance in ψ and Δ for three different pellets; similarly, the analytical precision for multiple measurements of the same pellet was also quite good, suggesting that a pressed pellet is a viable sample preparation method. Experimental variabilities were comparatively small; the greatest variability came in the analytic fitting procedure with differences in the k-peak values up to 10% for only the sharpest bands arising from four different fits to the same data set. The final ellipsometric n/k data are compared to literature values obtained from crystalline ammonium sulfate ((NH4)2SO4) samples as well as single-angle reflectance measurements that also used pressed pellets. Comparison with the previous literature values shows generally good agreement, although larger k-values are observed for the independent sets of data derived from pressed pellets. These data are suggested as an improved set of optical constants for (NH4)2SO4.

A quantitative method to detect human exposure to sulfur and nitrogen mustards via protein adducts.

Sulfur and nitrogen mustards are internationally banned vesicants listed as Schedule 1 chemical agents in the Chemical Weapons Convention. These compounds are highly reactive electrophiles that form stable adducts to a variety of available amino acid residues on proteins upon exposure. We present a quantitative exposure assay that simultaneously measures agent specific protein adducts to cysteine for sulfur mustard (HD) and three nitrogen mustards (HN1, HN2, and HN3). Proteinase K was added to a serum or plasma sample to digest protein adducts and form the target analyte, the blister agent bound to the tripeptide cysteine-proline-phenylalanine (CPF). The mustard adducted-tripeptide was purified by solid phase extraction and analyzed using isotope dilution LC-MS/MS. Product ion structures were identified using high-resolution product ion scan data for HD-CPF, HN1-CPF, HN2-CPF, and HN3-CPF. Thorough matrix comparison, analyte recovery, ruggedness, and stability studies were incorporated during method validation to produce a robust method. The method demonstrated long term-stability, precision (RSD < 15%), and intra- and inter-day accuracies > 85% across the reportable range of 3.00-200 ng/mL for each analyte. Compared to previously published assays, this method quantitates both sulfur and nitrogen mustard exposure biomarkers, requires only 10 µL of sample volume, and can use either a liquid sample or dried sample spot.

Mass Spectral Detection of Diethoxyphospho-Tyrosine Adducts on Proteins from HEK293 Cells Using Monoclonal Antibody depY for Enrichment.

Chronic illness from exposure to organophosphorus toxicants is hypothesized to involve modification of unknown proteins. Tyrosine in proteins that have no active site serine readily reacts with organophosphorus toxicants. We developed a monoclonal antibody, depY, that specifically recognizes diethoxyphospho-tyrosine in proteins and peptides, independent of the surrounding amino acid sequence. Our goal in the current study was to identify diethoxyphosphorylated proteins in human HEK293 cell lysate treated with chlorpyrifos oxon. Cell lysates treated with chlorpyrifos oxon were recognized by depY antibody in ELISA and capillary electrophoresis based Western blot. Tryptic peptides were analyzed by liquid chromatography tandem mass spectrometry. Liquid chromatography tandem mass spectrometry identified 116 diethoxyphospho-tyrosine peptides from 73 proteins in immunopurified samples, but found only 15 diethoxyphospho-tyrosine peptides from 12 proteins when the same sample was not immunopurified on depY. The most abundant proteins in the cell lysate, histone H4, heat shock 70 kDa protein 1A/1B, heat shock protein HSP 90 ß, and α-enolase, were represented by several diethoxyphospho-tyrosine peptides. It was concluded that use of immobilized depY improved the number of diethoxyphospho-tyrosine peptides identified in a complex mixture. The mass spectrometry results confirmed the specificity of depY for diethoxyphospho-tyrosine peptides independent of the context of the modified tyrosine, which means depY could be used to analyze modified proteins in any species. Use of the depY antibody could lead to an understanding of chronic illness from organophosphorus pesticide exposure.

Methods for quantitative infrared directional-hemispherical and diffuse reflectance measurements using an FTIR and a commercial integrating sphere.

We have developed methods to measure the directional-hemispherical (ρ) and diffuse (ρd) reflectances of powders, liquids, and disks of powders and solid materials using a commercially available, matte gold-coated integrating sphere and Fourier transform infrared spectrometer. To determine how well the sphere and protocols produce quantitative reflectance data, measurements were made of three diffuse and two specular standards prepared by the National Institute of Standards and Technology (NIST), LabSphere Infragold and Spectralon standards, hand-loaded sulfur and talc powder samples, and water. Relative to the NIST measurements of the NIST standards, our directional hemispherical reflectance values are within ±4% for four of the standards and within ±7% for a low reflectance diffuse standard. For the three diffuse reflectance NIST standards, our diffuse reflectance values are within ±5% of the NIST values. For the two specular NIST standards, our diffuse reflectance values are an order of magnitude larger than those of NIST, pointing to a systematic error in the manner in which diffuse reflectance measurements are made for specular samples using our methods and sphere. Sources of uncertainty are discussed in the paper.

Use of Hupresin To Capture Red Blood Cell Acetylcholinesterase for Detection of Soman Exposure.

Toxicity from acute exposure to nerve agents and organophosphorus toxicants is due to irreversible inhibition of acetylcholinesterase (AChE) in the nervous system. AChE in red blood cells is a surrogate for AChE in the nervous system. Previously we developed an immunopurification method to enrich red blood cell AChE (RBC AChE) as a biomarker of exposure. The goal of the present work was to provide an alternative RBC AChE enrichment strategy, by binding RBC AChE to Hupresin affinity gel. AChE was solubilized from frozen RBC by addition of 1% Triton X-100. Insoluble debris was removed by centrifugation. The red, but not viscous, RBC AChE solution was loaded on a Hupresin affinity column. Hemoglobin and other proteins were washed off with 3 M NaCl, while retaining AChE bound to Hupresin. Denatured AChE was eluted with 1% trifluoroacetic acid. The same protocol was used for 20 mL of RBC AChE inhibited with a soman model compound. The acid denatured protein was digested with pepsin and analyzed by liquid chromatography tandem mass spectrometry on a 6600 Triple-TOF mass spectrometer. A targeted method identified the aged soman adduct on serine 203 in peptide FGESAGAAS. It was concluded that Hupresin can be used to enrich soman-inhibited AChE solubilized from 8 mL of frozen human erythrocytes, yielding a quantity sufficient for detecting soman exposure.

Monoclonal Antibody That Recognizes Diethoxyphosphotyrosine-Modified Proteins and Peptides Independent of Surrounding Amino Acids.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are irreversibly inhibited by organophosphorus pesticides through formation of a covalent bond with the active site serine. Proteins that have no active site serine, for example albumin, are covalently modified on tyrosine and lysine. Chronic illness from pesticide exposure is not explained by inhibition of AChE and BChE. Our goal was to produce a monoclonal antibody that recognizes proteins diethoxyphosphorylated on tyrosine. Diethoxyphosphate-tyrosine adducts for 13 peptides were synthesized. The diethoxyphosphorylated (OP) peptides cross-linked to four different carrier proteins were used to immunize, boost, and screen mice. Monoclonal antibodies were produced with hybridoma technology. Monoclonal antibody depY was purified and characterized by ELISA, western blotting, Biacore, and Octet technology to determine binding affinity and binding specificity. DepY recognized diethoxyphosphotyrosine independent of the amino acid sequence around the modified tyrosine and independent of the identity of the carrier protein or peptide. It had an IC50 of 3 × 10-9 M in a competition assay with OP tubulin. Kd values measured by Biacore and OctetRED96 were 10-8 M for OP-peptides and 1 × 10-12 M for OP-proteins. The limit of detection measured on western blots hybridized with 0.14 µg/mL of depY was 0.025 µg of human albumin conjugated to YGGFL-OP. DepY was specific for diethoxyphosphotyrosine (chlorpyrifos oxon adduct) as it failed to recognize diethoxyphospholysine, phosphoserine, phosphotyrosine, phosphothreonine, dimethoxyphosphotyrosine (dichlorvos adduct), dimethoxyphosphoserine, monomethoxyphosphotyrosine (aged dichlorvos adduct), and cresylphosphoserine. In conclusion, a monoclonal antibody that specifically recognizes diethoxyphosphotyrosine adducts has been developed. The depY monoclonal antibody could be useful for identifying new biomarkers of OP exposure.

Immunopurification of Acetylcholinesterase from Red Blood Cells for Detection of Nerve Agent Exposure.

Nerve agents and organophosphorus pesticides make a covalent bond with the active site serine of acetylcholinesterase (AChE), resulting in inhibition of AChE activity and toxic symptoms. AChE in red blood cells (RBCs) serves as a surrogate for AChE in the nervous system. Mass spectrometry analysis of adducts on RBC AChE could provide evidence of exposure. Our goal was to develop a method of immunopurifying human RBC AChE in quantities adequate for detecting exposure by mass spectrometry. For this purpose, we immobilized 3 commercially available anti-human acetylcholinesterase monoclonal antibodies (AE-1, AE-2, and HR2) plus 3 new monoclonal antibodies. The monoclonal antibodies were characterized for binding affinity, epitope mapping by pairing analysis, and nucleotide and amino acid sequences. AChE was solubilized from frozen RBCs with 1% (v/v) Triton X-100. A 16 mL sample containing 5.8 µg of RBC AChE was treated with a quantity of soman model compound that inhibited 50% of the AChE activity. Native and soman-inhibited RBC AChE samples were immunopurified on antibody-Sepharose beads. The immunopurified RBC AChE was digested with pepsin and analyzed by liquid chromatography tandem mass spectrometry on a 6600 Triple-TOF mass spectrometer. The aged soman-modified PheGlyGluSerAlaGlyAlaAlaSer (FGESAGAAS) peptide was detected using a targeted analysis method. It was concluded that all 6 monoclonal antibodies could be used to immunopurify RBC AChE and that exposure to nerve agents could be detected as adducts on the active site serine of RBC AChE.

Pore-Engineered Metal-Organic Frameworks with Excellent Adsorption of Water and Fluorocarbon Refrigerant for Cooling Applications.

Metal-organic frameworks (MOFs) have shown promising behavior for adsorption cooling applications. Using organic ligands with 1, 2, and 3 phenylene rings, we construct moisture-stable Ni-MOF-74 members with adjustable pore apertures, which exhibit excellent sorption capabilities toward water and fluorocarbon R134a. To our knowledge, this is the first report of adsorption isotherms of fluorocarbon R134a in MOFs. The adsorption patterns for these materials differ significantly and are attributed to variances in their hydrophobic/hydrophilic pore character associated with differences in pore size.

Bridging the Gap between Sample Collection and Laboratory Analysis: Using Dried Blood Spots to Identify Human Exposure to Chemical Agents.

Public health response to large scale chemical emergencies presents logistical challenges for sample collection, transport, and analysis. Diagnostic methods used to identify and determine exposure to chemical warfare agents, toxins, and poisons traditionally involve blood collection by phlebotomists, cold transport of biomedical samples, and costly sample preparation techniques. Use of dried blood spots, which consist of dried blood on an FDA-approved substrate, can increase analyte stability, decrease infection hazard for those handling samples, greatly reduce the cost of shipping/storing samples by removing the need for refrigeration and cold chain transportation, and be self-prepared by potentially exposed individuals using a simple finger prick and blood spot compatible paper. Our laboratory has developed clinical assays to detect human exposures to nerve agents through the analysis of specific protein adducts and metabolites, for which a simple extraction from a dried blood spot is sufficient for removing matrix interferents and attaining sensitivities on par with traditional sampling methods. The use of dried blood spots can bridge the gap between the laboratory and the field allowing for large scale sample collection with minimal impact on hospital resources while maintaining sensitivity, specificity, traceability, and quality requirements for both clinical and forensic applications.

Naturally Occurring Genetic Variants of Human Acetylcholinesterase and Butyrylcholinesterase and Their Potential Impact on the Risk of Toxicity from Cholinesterase Inhibitors.

Acetylcholinesterase (AChE) is the physiologically important target for organophosphorus toxicants (OP) including nerve agents and pesticides. Butyrylcholinesterase (BChE) in blood serves as a bioscavenger that protects AChE in nerve synapses from inhibition by OP. Mass spectrometry methods can detect exposure to OP by measuring adducts on the active site serine of plasma BChE. Genetic variants of human AChE and BChE do exist, but loss of function mutations have been identified only in the BCHE gene. The most common AChE variant, His353Asn (H322N), also known as the Yt blood group antigen, has normal AChE activity. The most common BChE variant, Ala567Thr (A539T) or the K-variant in honor of Werner Kalow, has 33% reduced plasma BChE activity. The genetic variant most frequently associated with prolonged response to muscle relaxants, Asp98Gly (D70G) or atypical BChE, has reduced activity and reduced enzyme concentration. Early studies in young, healthy males, performed at a time when it was legal to test nerve agents in humans, showed that individuals responded differently to the same low dose of sarin with toxic symptoms ranging in severity from minimal to moderate. Additionally, animal studies indicated that BChE protects from toxicants that have a higher reactivity with AChE than with BChE (e.g., nerve agents) but not from toxicants that have a higher reactivity with BChE than with AChE (e.g., OP pesticides). As a corollary, we hypothesize that individuals with genetic variants of BChE may be at increased risk of toxicity from nerve agents but not from OP pesticides.

A Study of 2-Iodobutane by Rotational Spectroscopy.

Rotational transitions belonging to 2-iodobutane (sec-butyl-iodide, CH3CHICH2CH3) were measured over the frequency range 5.5-16.5 GHz via jet-pulsed Fourier transform microwave spectroscopy. The complete nuclear quadrupole coupling tensor of iodine, χ, was obtained for the gauche (g)-, anti (a)-, and gauche' (g')-conformers as well as the four (13)C isotopologues of the gauche species. Rotational constants, centrifugal distortion constants, quadrupole coupling constants, and nuclear spin-rotation constants were determined for each species. Changes in χ of the iodine nucleus, resulting from conformational and isotopic differences, are discussed. Isotopic substitution of g-2-iodobutane allowed for an rs structure to be determined for the carbon backbone. Additionally, isotopic substitution in conjunction with an ab initio structure allowed for a fit of various r0 structural parameters belonging to g-2-iodobutane.