Genomic analysis, immunomodulation and deep phenotyping of patients with nodding syndrome.

The aetiology of nodding syndrome remains unclear, and comprehensive genotyping and phenotyping data from patients remain sparse. Our objectives were to characterize the phenotype of patients with nodding syndrome, investigate potential contributors to disease aetiology, and evaluate response to immunotherapy. This cohort study investigated members of a single-family unit from Lamwo District, Uganda. The participants for this study were selected by the Ugandan Ministry of Health as representative for nodding syndrome and with a conducive family structure for genomic analyses. Of the eight family members who participated in the study at the National Institutes of Health (NIH) Clinical Center, three had nodding syndrome. The three affected patients were extensively evaluated with metagenomic sequencing for infectious pathogens, exome sequencing, spinal fluid immune analyses, neurometabolic and toxicology testing, continuous electroencephalography and neuroimaging. Five unaffected family members underwent a subset of testing for comparison. A distinctive interictal pattern of sleep-activated bursts of generalized and multifocal epileptiform discharges and slowing was observed in two patients. Brain imaging showed two patients had mild generalized cerebral atrophy, and both patients and unaffected family members had excessive metal deposition in the basal ganglia. Trace metal biochemical evaluation was normal. CSF was non-inflammatory and one patient had CSF-restricted oligoclonal bands. Onchocerca volvulus-specific antibodies were present in all patients and skin snips were negative for active onchocerciasis. Metagenomic sequencing of serum and CSF revealed hepatitis B virus in the serum of one patient. Vitamin B6 metabolites were borderline low in all family members and CSF pyridoxine metabolites were normal. Mitochondrial DNA testing was normal. Exome sequencing did not identify potentially causal candidate gene variants. Nodding syndrome is characterized by a distinctive pattern of sleep-activated epileptiform activity. The associated growth stunting may be due to hypothalamic dysfunction. Extensive testing years after disease onset did not clarify a causal aetiology. A trial of immunomodulation (plasmapheresis in two patients and intravenous immunoglobulin in one patient) was given without short-term effect, but longer-term follow-up was not possible to fully assess any benefit of this intervention.

Vitamin E Acetate in Bronchoalveolar-Lavage Fluid Associated with EVALI.

BACKGROUND: The causative agents for the current national outbreak of electronic-cigarette, or vaping, product use-associated lung injury (EVALI) have not been established. Detection of toxicants in bronchoalveolar-lavage (BAL) fluid from patients with EVALI can provide direct information on exposure within the lung. METHODS: BAL fluids were collected from 51 patients with EVALI in 16 states and from 99 healthy participants who were part of an ongoing study of smoking involving nonsmokers, exclusive users of e-cigarettes or vaping products, and exclusive cigarette smokers that was initiated in 2015. Using the BAL fluid, we performed isotope dilution mass spectrometry to measure several priority toxicants: vitamin E acetate, plant oils, medium-chain triglyceride oil, coconut oil, petroleum distillates, and diluent terpenes. RESULTS: State and local health departments assigned EVALI case status as confirmed for 25 patients and as probable for 26 patients. Vitamin E acetate was identified in BAL fluid obtained from 48 of 51 case patients (94%) in 16 states but not in such fluid obtained from the healthy comparator group. No other priority toxicants were found in BAL fluid from the case patients or the comparator group, except for coconut oil and limonene, which were found in 1 patient each. Among the case patients for whom laboratory or epidemiologic data were available, 47 of 50 (94%) had detectable tetrahydrocannabinol (THC) or its metabolites in BAL fluid or had reported vaping THC products in the 90 days before the onset of illness. Nicotine or its metabolites were detected in 30 of 47 of the case patients (64%). CONCLUSIONS: Vitamin E acetate was associated with EVALI in a convenience sample of 51 patients in 16 states across the United States. (Funded by the National Cancer Institute and others.).

Acute Kidney Injury Among Children Likely Associated with Diethylene Glycol-Contaminated Medications - The Gambia, June-September 2022.

On July 26, 2022, a pediatric nephrologist alerted The Gambia's Ministry of Health (MoH) to a cluster of cases of acute kidney injury (AKI) among young children at the country's sole teaching hospital, and on August 23, 2022, MoH requested assistance from CDC. CDC epidemiologists arrived in The Gambia, a West African country, on September 16 to assist MoH in characterizing the illness, describing the epidemiology, and identifying potential causal factors and their sources. Investigators reviewed medical records and interviewed caregivers to characterize patients' symptoms and identify exposures. The preliminary investigation suggested that various contaminated syrup-based children's medications contributed to the AKI outbreak. During the investigation, MoH recalled implicated medications from a single international manufacturer. Continued efforts to strengthen pharmaceutical quality control and event-based public health surveillance are needed to help prevent future medication-related outbreaks.

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.

Guidewire type and prior use affects ureteral stent insertion force.

INTRODUCTION: To improve the success rate and safety of ureteral stent insertion, we sought to identify the effect of guidewire type and prior use upon the force needed to advance a 6Fr ureteral stent over various guidewires. MATERIALS AND METHODS: Two-hundred forty stent insertion trials were performed in an ex vivo porcine urinary tract model. Ten trials were randomly performed over 12 new and 12 used guidewires. For each trial, the force required to advance a 6Fr Cook double-pigtail ureteral stent was recorded. Guidewires included the Olympus Glidewire, Cook Fixed Core, and Boston Scientific Amplatz Super Stiff, Sensor, ZIPwire, and Zebra wire. RESULTS: The mean force needed for stent advancement was the lowest for the new Glidewire (0.18N) and ZIPwire (0.22N), with no significant difference to each other (p = 0.90). The following new wires required increasingly higher stent insertion forces compared to the Glidewire, the Zebra (0.60N; p < 0.01), Fixed Core (1.25N; p < 0.01), Sensor (1.43N; p < 0.01), and Amplatz Super Stiff wires (2.03N; p < 0.01). There was no statistical difference between new and used Glidewires (0.18N versus 0.29N; p = 0.14) and Zebra wires (0.59N versus 0.60N; p = 0.88). All other used wires required a significantly greater advancement force than their new counterparts (p < 0.01). CONCLUSIONS: For the same stent, the force required for stent advancement varies greatly between guidewire types. In addition, used guidewires typically required more force compared to new guidewires. In long or difficult cases, switching to a new wire may improve the ease of stent placement and reduce potential complications.

Evaluation of Bronchoalveolar Lavage Fluid from Patients in an Outbreak of E-cigarette, or Vaping, Product Use-Associated Lung Injury - 10 States, August-October 2019.

CDC, the Food and Drug Administration (FDA), state and local health departments, and multiple public health and clinical partners are investigating a national outbreak of e-cigarette, or vaping, product use-associated lung injury (EVALI). Based on data collected as of October 15, 2019, 86% of 867 EVALI patients reported using tetrahydrocannabinol (THC)-containing products in the 3 months preceding symptom onset (1). Analyses of THC-containing product samples by FDA and state public health laboratories have identified potentially harmful constituents in these products, such as vitamin E acetate, medium chain triglyceride oil (MCT oil), and other lipids (2,3) (personal communication, D.T. Heitkemper, FDA Forensic Chemistry Center, November 2019). Vitamin E acetate, in particular, might be used as an additive in the production of e-cigarette, or vaping, products; it also can be used as a thickening agent in THC products (4). Inhalation of vitamin E acetate might impair lung function (5-7).

Severe Pulmonary Disease Associated with Electronic-Cigarette-Product Use - Interim Guidance.

On September 6, 2019, this report was posted as an MMWR Early Release on the MMWR website (https://www.cdc.gov/mmwr). As of August 27, 2019, 215 possible cases of severe pulmonary disease associated with the use of electronic cigarette (e-cigarette) products (e.g., devices, liquids, refill pods, and cartridges) had been reported to CDC by 25 state health departments. E-cigarettes are devices that produce an aerosol by heating a liquid containing various chemicals, including nicotine, flavorings, and other additives (e.g., propellants, solvents, and oils). Users inhale the aerosol, including any additives, into their lungs. Aerosols produced by e-cigarettes can contain harmful or potentially harmful substances, including heavy metals such as lead, volatile organic compounds, ultrafine particles, cancer-causing chemicals, or other agents such as chemicals used for cleaning the device (1). E-cigarettes also can be used to deliver tetrahydrocannabinol (THC), the principal psychoactive component of cannabis, or other drugs; for example, "dabbing" involves superheating substances that contain high concentrations of THC and other plant compounds (e.g., cannabidiol) with the intent of inhaling the aerosol. E-cigarette users could potentially add other substances to the devices. This report summarizes available information and provides interim case definitions and guidance for reporting possible cases of severe pulmonary disease. The guidance in this report reflects data available as of September 6, 2019; guidance will be updated as additional information becomes available.

High-Confidence Qualitative Identification of Organophosphorus Nerve Agent Adducts to Human Butyrylcholinesterase.

In this study, a data-dependent, high-resolution tandem mass spectrometry (ddHRMS/MS) method capable of detecting all organophosphorus nerve agent (OPNA) adducts to human butyrylcholinesterase (BChE) was developed. After an exposure event, immunoprecipitation from blood with a BChE-specific antibody and digestion with pepsin produces a nine amino acid peptide containing the OPNA adduct. Signature product ions of this peptic BChE nonapeptide (FGES*AGAAS) offer a route to broadly screen for OPNA exposure. Taking this approach on an HRMS instrument identifies biomarkers, including unknowns, with high mass accuracy. Using a set of pooled human sera exposed to OPNAs as quality control (QC) materials, the developed method successfully identified precursor ions with <1 ppm and tied them to signature product ions with <5 ppm deviation from their chemical formulas. This high mass accuracy data from precursor and product ions, collected over 23 independent immunoprecipitation preparations, established method operating limits. QC data and experiments with 14 synthetic reference peptides indicated that reliable qualitative identification of biomarkers was possible for analytes >15 ng/mL. The developed method was applied to a convenience set of 96 unexposed serum samples and a blinded set of 80 samples treated with OPNAs. OPNA biomarkers were not observed in convenience set samples and no false positive or negative identifications were observed in blinded samples. All biomarkers in the blinded serum set >15 ng/mL were correctly identified. For the first time, this study reports a ddHRMS/MS method capable of complementing existing quantitative methodologies and suitable for identifying exposure to unknown organophosphorus agents.

Rapid Laboratory Identification of Neisseria meningitidis Serogroup C as the Cause of an Outbreak - Liberia, 2017.

On April 25, 2017, a cluster of unexplained illness and deaths among persons who had attended a funeral during April 21-22 was reported in Sinoe County, Liberia (1). Using a broad initial case definition, 31 cases were identified, including 13 (42%) deaths. Twenty-seven cases were from Sinoe County (1), and two cases each were from Grand Bassa and Monsterrado counties, respectively. On May 5, 2017, initial multipathogen testing of specimens from four fatal cases using the Taqman Array Card (TAC) assay identified Neisseria meningitidis in all specimens. Subsequent testing using direct real-time polymerase chain reaction (PCR) confirmed N. meningitidis in 14 (58%) of 24 patients with available specimens and identified N. meningitidis serogroup C (NmC) in 13 (54%) patients. N. meningitidis was detected in specimens from 11 of the 13 patients who died; no specimens were available from the other two fatal cases. On May 16, 2017, the National Public Health Institute of Liberia and the Ministry of Health of Liberia issued a press release confirming serogroup C meningococcal disease as the cause of this outbreak in Liberia.

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.

A high-throughput UHPLC-MS/MS method for the quantification of five aged butyrylcholinesterase biomarkers from human exposure to organophosphorus nerve agents.

Organophosphorus nerve agents (OPNAs) are toxic compounds that are classified as prohibited Schedule 1 chemical weapons. In the body, OPNAs bind to butyrylcholinesterase (BChE) to form nerve agent adducts (OPNA-BChE). OPNA-BChE adducts can provide a reliable, long-term protein biomarker for assessing human exposure. A major challenge facing OPNA-BChE detection is hydrolysis (aging), which can continue to occur after a clinical specimen has been collected. During aging, the o-alkyl phosphoester bond hydrolyzes, and the specific identity of the nerve agent is lost. To better identify OPNA exposure events, a high-throughput method for the detection of five aged OPNA-BChE adducts was developed. This is the first diagnostic panel to allow for the simultaneous quantification of any Chemical Weapons Convention Schedule 1 OPNA by measuring the aged adducts methyl phosphonate, ethyl phosphonate, propyl phosphonate, ethyl phosphoryl, phosphoryl and unadducted BChE. The calibration range for all analytes is 2.00-250. ng/mL, which is consistent with similar methodologies used to detect unaged OPNA-BChE adducts. Each analytical run is 3 min, making the time to first unknown results, including calibration curve and quality controls, less than 1 h. Analysis of commercially purchased individual serum samples demonstrated no potential interferences with detection of aged OPNA-BChE adducts, and quantitative measurements of endogenous levels of BChE were similar to those previously reported in other OPNA-BChE adduct assays.

Supplemental Learning in the Laboratory: An Innovative Approach for Evaluating Knowledge and Method Transfer.

The Multi-Rule Quality Control System (MRQCS) is a tool currently employed by the Centers for Disease Control and Prevention (CDC) to evaluate and compare laboratory performance. We have applied the MRQCS to a comparison of instructor and computer-led pre-laboratory lectures for a supplemental learning experiment. Students in general chemistry and analytical chemistry from both two- and four-year institutions performed two laboratory experiments as part of their normal laboratory curriculum. The first laboratory experiment was a foundational learning experiment in which all the students were introduced to Beer-Lambert's Law and spectrophotometric light absorbance measurements. The foundational learning experiment was instructor-led only, and participant performance was evaluated against a mean characterized value. The second laboratory experiment was a supplemental learning experiment in which students were asked to build upon the methodology they learned in the foundational learning experiment and apply it to a different analyte. The instruction type was varied randomly into two delivery modes, participants receiving either instructor-led or computer-led pre-laboratory instruction. The MRQCS was applied and determined that no statistical difference was found to exist in the QC (quality control) passing rates between the participants in the instructor-led instruction and the participants in the computer-led instruction. These findings demonstrate the successful application of the MRQCS to evaluate knowledge and technology transfer.

Comparing the Diagnostic Classification Accuracy of iTRAQ, Peak-Area, Spectral-Counting, and emPAI Methods for Relative Quantification in Expression Proteomics.

Diagnostic classification accuracy is critical in expression proteomics to ensure that as many true differences as possible are identified with acceptable false-positive rates. We present a comparison of the diagnostic accuracy of iTRAQ with three label-free methods, peak area, spectral counting, and emPAI, for relative quantification using a spiked proteome standard. We provide the first validation of emPAI for intersample relative quantification and find clear differences among the four quantification approaches that could be considered when designing an experiment. Spectral counting was observed to perform surprisingly well in all regards. Peak area performed best for smaller fold differences and was shown to be capable of discerning a 1.1-fold difference with acceptable specificity and sensitivity. The performance of iTRAQ was dramatically worse than the label-free methods with low abundance proteins. Using the iTRAQ data set for validation, we also demonstrate a novel iTRAQ analysis regime that avoids the use of ratios in significance testing and outperforms a common commercial alternative.

Multisite tyrosine phosphorylation of the N-terminus of Mint1/X11α by Src kinase regulates the trafficking of amyloid precursor protein.

Mint/X11 is one of the four neuronal trafficking adaptors that interact with amyloid precursor protein (APP) and are linked with its cleavage to generate ß-amyloid peptide, a key player in the pathology of Alzheimer's disease. How APP switches between adaptors at different stages of the secretory pathway is poorly understood. Here, we show that tyrosine phosphorylation of Mint1 regulates the destination of APP. A canonical SH2-binding motif ((202) YEEI) was identified in the N-terminus of Mint1 that is phosphorylated on tyrosine by C-Src and recruits the active kinase for sequential phosphorylation of further tyrosines (Y191 and Y187). A single Y202F mutation in the Mint1 N-terminus inhibits C-Src binding and tyrosine phosphorylation. Previous studies observed that co-expression of wild-type Mint1 and APP causes accumulation of APP in the trans-Golgi. Unphosphorylatable Mint1 (Y202F) or pharmacological inhibition of Src reduced the accumulation of APP in the trans-Golgi of heterologous cells. A similar result was observed in cultured rat hippocampal neurons where Mint1(Y202F) permitted the trafficking of APP to more distal neurites than the wild-type protein. These data underline the importance of the tyrosine phosphorylation of Mint1 as a critical switch for determining the destination of APP. The regulation of amyloid precursor protein (APP) trafficking is poorly understood. We have discovered that the APP adapter, Mint1, is phosphorylated by C-Src kinase. Mint1 causes APP accumulation in the trans-Golgi network, whereas inhibition of Src or mutation of Mint1-Y202 permits APP recycling. The phosphorylation status of Mint1 could impact on the pathological trafficking of APP in Alzheimer's disease.

Quantification of proteins by flow cytometry: Quantification of human hepatic transporter P-gp and OATP1B1 using flow cytometry and mass spectrometry.

Flow cytometry is a powerful tool for the quantitation of fluorescence and is proven to be able to correlate the fluorescence intensity to the number of protein on cells surface. Mass spectroscopy can also be used to determine the number of proteins per cell. Here we have developed two methods, using flow cytometry and mass spectroscopy to quantify number of transporters in human cells. These two approaches were then used to analyse the same samples so that a direct comparison could be made. Transporters have a major impact on the behaviour of a diverse number of drugs in human systems. While active uptake studies by transmembrane protein transporters using model substrates are routinely undertaken in human cell lines and hepatocytes as part of drug discovery and development, the interpretation of these results is currently limited by the inability to quantify the number of transporters present in the test samples. Here we provide a flow cytometric method for accurate quantification of transporter levels both on the cell surface and within the cell, and compare this to a quantitative mass spectrometric approach. Two transporters were selected for the study: OATP1B1 (also known as SLCO1B1, LST-1, OATP-C, OATP2) due to its important role in hepatic drug uptake and elimination; P-gp (also known as P-glycoprotein, MDR1, ABCB1) as a well characterised system and due to its potential impact on oral bioavailability, biliary and renal clearance, and brain penetration of drugs that are substrates for this transporter. In all cases the mass spectrometric method gave higher levels than the flow cytometry method. However, the two methods showed very similar trends in the relative ratios of both transporters in the hepatocyte samples investigated. The P-gp antibody allowed quantitative discrimination between externally facing transporters located in the cytoplasmic membrane and the total number of transporters on and in the cell. The proportion of externally facing transporter varied considerably in the four hepatocyte samples analysed, ranging from only 6% to 35% of intact and viable cells. The sample with only 6% externally facing transporter was further analysed by confocal microscopy which qualitatively confirmed the low level of transporter in the membrane and the large internal population. Here we prove that flow cytometry is an important tool for future protein analysis as it can not only quantify the number of proteins that a cell express but also identify the number of proteins on the surface and it is easy to apply for routine assays.

The sodium channel-blocking antiepileptic drug phenytoin inhibits breast tumour growth and metastasis.

BACKGROUND: Voltage-gated Na(+) channels (VGSCs) are heteromeric protein complexes containing pore-forming α subunits and smaller, non-pore-forming ß subunits. VGSCs are classically expressed in electrically excitable cells, e.g. neurons. VGSCs are also expressed in tumour cells, including breast cancer (BCa) cells, where they enhance cellular migration and invasion. However, despite extensive work defining in detail the molecular mechanisms underlying the expression of VGSCs and their pro-invasive role in cancer cells, there has been a notable lack of clinically relevant in vivo data exploring their value as potential therapeutic targets. FINDINGS: We have previously reported that the VGSC-blocking antiepileptic drug phenytoin inhibits the migration and invasion of metastatic MDA-MB-231 cells in vitro. The purpose of the present study was to establish whether VGSCs might be viable therapeutic targets by testing the effect of phenytoin on tumour growth and metastasis in vivo. We found that expression of Nav1.5, previously detected in MDA-MB-231 cells in vitro, was retained on cells in orthotopic xenografts. Treatment with phenytoin, at a dose equivalent to that used to treat epilepsy (60 mg/kg; daily), significantly reduced tumour growth, without affecting animal weight. Phenytoin also reduced cancer cell proliferation in vivo and invasion into surrounding mammary tissue. Finally, phenytoin significantly reduced metastasis to the liver, lungs and spleen. CONCLUSIONS: This is the first study showing that phenytoin reduces breast tumour growth and metastasis in vivo. We propose that pharmacologically targeting VGSCs, by repurposing antiepileptic or antiarrhythmic drugs, should be further studied as a potentially novel anti-cancer therapy.

Enhanced stability of blood matrices using a dried sample spot assay to measure human butyrylcholinesterase activity and nerve agent adducts.

Dried matrix spots are safer to handle and easier to store than wet blood products, but factors such as intraspot variability and unknown sample volumes have limited their appeal as a sampling format for quantitative analyses. In this work, we introduce a dried spot activity assay for quantifying butyrylcholinesterase (BChE) specific activity which is BChE activity normalized to the total protein content in a sample spot. The method was demonstrated with blood, serum, and plasma spotted on specimen collection devices (cards) which were extracted to measure total protein and BChE activity using a modified Ellman assay. Activity recovered from dried spots was ∼80% of the initial spotted activity for blood and >90% for plasma and serum. Measuring total protein in the sample and calculating specific activity substantially improved quantification and reduced intraspot variability. Analyte stability of nerve agent adducts was also evaluated, and the results obtained via BChE-specific activity measurements were confirmed by quantification of BChE adducts using a previously established LC-MS/MS method. The spotted samples were up to 10 times more resistant to degradation compared to unspotted control samples when measuring BChE inhibition by the nerve agents sarin and VX. Using this method, both BChE activity and adducts can be accurately measured from a dried sample spot. This use of a dried sample spot with normalization to total protein is robust, demonstrates decreased intraspot variability without the need to control for initial sample volume, and enhances analyte stability.

Quantification of metabolites for assessing human exposure to soapberry toxins hypoglycin A and methylenecyclopropylglycine.

Ingestion of soapberry fruit toxins hypoglycin A and methylenecyclopropylglycine has been linked to public health challenges worldwide. In 1976, over 100 years after Jamaican vomiting sickness (JVS) was first reported, the cause of JVS was linked to the ingestion of the toxin hypoglycin A produced by ackee fruit. A structural analogue of hypoglycin A, methylenecyclopropylglycine (MCPG), was implicated as the cause of an acute encephalitis syndrome (AES). Much of the evidence linking hypoglycin A and MCPG to these diseases has been largely circumstantial due to the lack of an analytical method for specific metabolites. This study presents an analytical approach to identify and quantify specific urine metabolites for exposure to hypoglycin A and MCPG. The metabolites are excreted in urine as glycine adducts methylenecyclopropylacetyl-glycine (MCPA-Gly) and methylenecyclopropylformyl-glycine (MCPF-Gly). These metabolites were processed by isotope dilution, separated by reverse-phase liquid chromatography, and monitored by electrospray ionization tandem mass spectrometry. The analytical response ratio was linearly proportional to the concentration of MCPF-Gly and MCPA-Gly in urine from 0.10 to 20 µg/mL with a correlation coefficient of r > 0.99. The assay demonstrated accuracy ≥80% and precision ≤20% RSD across the calibration range. This method has been applied to assess exposure to hypoglycin A and MCPG as part of a larger public health initiative and was used to provide the first reported identification of MCPF-Gly and MCPA-Gly in human urine.

Simplified Method for Quantifying Sulfur Mustard Adducts to Blood Proteins by Ultrahigh Pressure Liquid Chromatography−Isotope Dilution Tandem Mass Spectrometry.

Sulfur mustard binds to reactive cysteine residues, forming a stable sulfur-hydroxyethylthioethyl [SHETE]adduct that can be used as a long-term biomarker of sulfur mustard exposure in humans. The digestion of sulfur mustard-exposed blood samples with proteinase K following total protein precipitation with acetone produces the tripeptide biomarker [S-HETE]-Cys-Pro-Phe. The adducted tripeptide is purified by solid phase extraction, separated by ultra high pressure liquid chromatography, and detected by isotope dilution tandem mass spectrometry. This approach was thoroughly validated and characterized in our laboratory. The average interday relative standard deviation was ≤ 9.49%, and the range of accuracy was between 96.1 and 109% over a concentration range of 3.00 to 250. ng/mL with a calculated limit of detection of1.74 ng/mL. A full 96-well plate can be processed and analyzed in 8 h, which is 5 times faster than our previous 96-well plate method and only requires 50 µL of serum, plasma, or whole blood. Extensive ruggedness and stability studies and matrix comparisons were conducted to create a robust, easily transferrable method. As a result, a simple and high-throughput method has been developed and validated for the quantitation of sulfur mustard blood protein adducts in low volume blood specimens which should be readily adaptable for quantifying human exposures to other alkylating agents.