Sharon W. Lemire

Immunomagnetic Separation and Quantification of Butyrylcholinesterase Nerve Agent Adducts in Human Serum

A novel method for extracting butyrylcholinesterase (BuChE) from serum as a means of identifying and measuring nerve agent adducts to human BuChE is presented here. Antibutyrylcholinesterase monoclonal antibodies were conjugated to protein-G ferromagnetic particles and mixed with 500 microL serum samples. The particle-antibody-BuChE product was rinsed and directly digested with pepsin. Native and isotopically enriched nonapeptides corresponding to the pepsin digest products for uninhibited BuChE, and sarin, cyclohexylsarin, VX, and Russian VX nerve agent-inhibited BuChE were synthesized for use as calibrators and internal standards, respectively. Internal standards were added to the filtered digest sample, and the samples were quantified via high performance liquid chromatography-isotope dilution-tandem mass spectrometry. The ratio of adducted to total BuChE nonapeptides was calculated for each nerve agent-exposed serum sample using data collected in a single chromatogram. Nerve agent-inhibited quality control serum pools were characterized as part of method validation; the method was observed to have extremely low background noise. The measurement of both uninhibited and inhibited BuChE peptides compensated for any variations in the pepsin digestion before the internal standard peptide was added to the sample and may prove useful in individualizing patient results following a nerve agent exposure.

A high-throughput diagnostic method for measuring human exposure to organophosphorus nerve agents.

An automated high-throughput immunomagnetic separation (IMS) method for diagnosing exposure to the organophosphorus nerve agents (OPNAs) sarin (GB), cyclohexylsarin (GF), VX, and Russian VX (RVX) was developed to increase sample processing capacity for emergency response applications. Diagnosis of exposure to OPNAs was based on the formation of OPNA adducts to butyrylcholinesterase (BuChE). Data reported with this method represent a ratio of the agent-specific BuChE adduct concentration, relative to the total BuChE peptide concentration that provides a nonactivity measurement expressed as percent adducted. All magnetic bead transfer steps and washes were performed using instrumentation in a 96-well format allowing for simultaneous extraction of 86 clinical samples plus reference materials. Automating extractions increased sample throughput 50-fold, as compared to a previously reported manual method. The limits of detection, determined using synthetic peptides, were 1 ng/mL for unadducted BuChE and GB-, GF-, VX-, and RVX-adducted BuChE. The automated method was characterized using unexposed serum and serum pools exposed to GB, GF, VX, or RVX. Variation for the measurement of percent adducted was <12% for all characterized quality control serum pools. Twenty-six (26) serum samples from individuals asymptomatic for cholinesterase inhibitor exposure were analyzed using this method, and no background levels of OPNA exposure were observed. Unexposed BuChE serum concentrations measured using this method ranged from 2.8 μg/mL to 10.6 μg/mL, with an average concentration of 6.4 μg/mL.

Environmental exposure of commuters in Mexico City to volatile organic compounds as assessed by blood concentrations, 1998

OBJECTIVE To assess the extent of exposure for Volatile Organic Compounds (VOCs) among nonoccupationally exposed commuters in Mexico City. MATERIAL AND METHODS Blood concentrations of benzene, toluene, ethylbenzene, m-/p-xylene, o-xylene and methyl tert-butyl ether were determined on samples collected from participants after the morning commute. RESULTS Median blood concentrations of benzene (0.11 microg/l), ethylbenzene (0.081 microg/l), m-/p-xylene (0.32 microg/l) and toluene (0.56 microg/l) in the Mexico City participants were all approximately two times higher than in a nonsmoking subset of the Third National Health and Nutrition Examination Survey population of the United States. On the other hand, median VOC blood levels were similar to medians observed in other studies involving commuters in specific U.S. cities, despite the fact that only half the Mexico City study participants commuted by personal vehicles compared with all U.S. commuters. CONCLUSIONS These results reflect the extent of the air pollution problem in Mexico City. The surrounding topography exacerbates the problems caused by heavy vehicular traffic, poor emission-control devices on older vehicles, and poor maintenance practices. Elevated levels of gasoline components in the blood of nonoccupationally exposed commuters emphasize the need for regulatory initiatives and mass-transit options to reduce hydrocarbon emissions and thus reduce the risk for nonoccupational exposure for the residents of Mexico City.

Development and validation of a high-throughput online solid-phase extraction-liquid chromatography-tandem mass spectrometry method for the detection of gonyautoxins 1&4 and gonyautoxins 2&3 in human urine

Paralytic shellfish toxins (PSTs), including gonyautoxins and saxitoxins, are produced by multiple species of microalgae and dinoflagellates, and are bioaccumulated by shellfish and other animals. Human exposure to PSTs typically occurs through ingestion of recreationally harvested contaminated shellfish and results in nonspecific symptomology. Confirmation of exposure to PSTs has often relied on the measurement of saxitoxin, the most toxic congener; however, gonyautoxins (GTXs), the sulfated carbamate derivatives of saxitoxin, may be present in shellfish at higher concentrations. To improve identification of PST exposures, our group has developed an online solid phase extraction hydrophilic interaction liquid chromatography method to identify GTX1-4 in human urine with tandem mass spectrometry. The reportable range varied for each analyte, with all falling within 0.899 and 250 ng/mL in urine with precision <15% and >85% accuracy as determined for all quality control samples. This new online method quantitates GTX1-4 following exposures to PSTs, supporting the work of public health authorities.