Harmonization of acronyms for volatile organic compound metabolites using a standardized naming system.

Increased interest in volatile organic compound (VOC) exposure has led to an increased need for consistent, systematic, and informative naming of VOC metabolites. As analytical methods have expanded to include many metabolites in a single assay, the number of acronyms in use for a single metabolite has expanded in an unplanned and inconsistent manner due to a lack of guidance or group consensus. Even though the measurement of VOC metabolites is a well-established means to investigate exposure to VOCs, a formal attempt to harmonize acronyms amongst investigators has not been published. The aim of this work is to establish a system of acronym naming that provides consistency in current acronym usage and a foundation for creating acronyms for future VOC metabolites.

Large Differences in Urinary Benzene Metabolite S-Phenylmercapturic Acid Quantitation: A Comparison of Five LC-MS-MS Methods.

Benzene is a known genotoxic carcinogen linked to many hematological abnormalities. S-phenylmercapturic acid (PHMA, N-acetyl-S-(phenyl)-L-cysteine, CAS# 4775-80-8) is a urinary metabolite of benzene and is used as a biomarker to assess benzene exposure. Pre-S-phenylmercapturic acid (pre-PHMA) is a PHMA precursor that dehydrates to PHMA at acidic pH. Published analytical methods that measure urinary PHMA adjust urine samples to a wide range of pH values using several types of acid, potentially leading to highly variable results depending on the concentration of pre-PHMA in a sample. Information is lacking on the variation in sample preparation among laboratories regularly measuring PHMA and the effect of those differences on PHMA quantitation in human urine samples. To investigate the differences in PHMA quantitation, we conducted an inter-laboratory comparison that included the analysis of 50 anonymous human urine samples (25 self-identified smokers and 25 self-identified non-smokers), quality control samples and commercially available reference samples in five laboratories using different analytical methods. Observed urinary PHMA concentrations were proportionally higher at lower pH, and results for anonymous urine samples varied widely among the methods. The method with the neutral preparation pH yielded results about 60% lower than the method using the most acidic conditions. Samples spiked with PHMA showed little variation, suggesting that the variability in results in human urine samples across methods is driven by the acid-mediated conversion of pre-PHMA to PHMA.

Development of a pregnancy-specific reference material for thyroid biomarkers, vitamin D, and nutritional trace elements in serum.

OBJECTIVES: Matrix differences among serum samples from non-pregnant and pregnant patients could bias measurements. Standard Reference Material 1949, Frozen Human Prenatal Serum, was developed to provide a quality assurance material for the measurement of hormones and nutritional elements throughout pregnancy. METHODS: Serum from non-pregnant women and women in each trimester were bottled into four levels based on pregnancy status and trimester. Liquid chromatography tandem mass spectrometry (LC-MS/MS) methods were developed and applied to the measurement of thyroid hormones, vitamin D metabolites, and vitamin D-binding protein (VDBP). Copper, selenium, and zinc measurements were conducted by inductively coupled plasma dynamic reaction cell MS. Thyroid stimulating hormone (TSH), thyroglobulin (Tg), and thyroglobulin antibody concentrations were analyzed using immunoassays and LC-MS/MS (Tg only). RESULTS: Certified values for thyroxine and triiodothyronine, reference values for vitamin D metabolites, VDBP, selenium, copper, and zinc, and information values for reverse triiodothyronine, TSH, Tg, and Tg antibodies were assigned. Significant differences in serum concentrations were evident for all analytes across the four levels (p≤0.003). TSH measurements were significantly different (p<0.0001) among research-only immunoassays. Tg concentrations were elevated in research-only immunoassays vs. Federal Drug Administration-approved automated immunoassay and LC-MS/MS. Presence of Tg antibodies increased differences between automated immunoassay and LC-MS/MS. CONCLUSIONS: The analyte concentrations' changes consistent with the literature and the demonstration of matrix interferences in immunoassay Tg measurements indicate the functionality of this material by providing a relevant matrix-matched reference material for the different stages of pregnancy.

Urinary Biomarkers of Exposure to Volatile Organic Compounds from the Population Assessment of Tobacco and Health Study Wave 1 (2013-2014).

Volatile organic compounds (VOCs) are ubiquitous in the environment. In the United States (U.S.), tobacco smoke is the major non-occupational source of exposure to many harmful VOCs. Exposure to VOCs can be assessed by measuring their urinary metabolites (VOCMs). The Population Assessment of Tobacco and Health (PATH) Study is a U.S. national longitudinal study of tobacco use in the adult and youth civilian non-institutionalized population. We measured 20 VOCMs in urine specimens from a subsample of adults in Wave 1 (W1) (2013-2014) to characterize VOC exposures among tobacco product users and non-users. We calculated weighted geometric means (GMs) and percentiles of each VOCM for exclusive combustible product users (smokers), exclusive electronic cigarette (e-cigarette) users, exclusive smokeless product users, and tobacco product never users. We produced linear regression models for six VOCMs with sex, age, race, and tobacco user group as predictor variables. Creatinine-ratioed levels of VOCMs from exposure to acrolein, crotonaldehyde, isoprene, acrylonitrile, and 1,3-butadiene were significantly higher in smokers than in never users. Small differences of VOCM levels among exclusive e-cigarette users and smokeless users were observed when compared to never users. Smokers showed higher VOCM concentrations than e-cigarette, smokeless, and never users. Urinary VOC metabolites are useful biomarkers of exposure to harmful VOCs.

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.).

Assessing the stability of Cd, Mn, Pb, Se, and total Hg in whole human blood by ICP-DRC-MS as a function of temperature and time.

BACKGROUND: Comprehensive information on the effect of time and temperature storage on the measurement of elements in human, whole blood (WB) by inductively coupled plasma-dynamic reaction cell-mass spectrometry (ICP-DRC-MS) is lacking, particularly for Mn and Se. METHODS: Human WB was spiked at 3 concentration levels, dispensed, and then stored at 5 different temperatures: -70 °C, -20 °C, 4 °C, 23 °C, and 37 °C. At 3 and 5 weeks, and at 2, 4, 6, 8, 10, 12, 36 months, samples were analyzed for Pb, Cd, Mn, Se and total Hg, using ICP-DRC-MS. We used a multiple linear regression model including time and temperature as covariates to fit the data with the measurement value as the outcome. We used an equivalence test using ratios to determine if results from the test storage conditions, warmer temperature and longer time, were comparable to the reference storage condition of 3 weeks storage time at -70 °C. RESULTS: Model estimates for all elements in human WB samples stored in polypropylene cryovials at -70 °C were equivalent to estimates from samples stored at 37 °C for up to 2 months, 23 °C up to 10 months, and -20 °C and 4 °C for up to 36 months. Model estimates for samples stored for 3 weeks at -70 °C were equivalent to estimates from samples stored for 2 months at -20 °C, 4 °C, 23 °C and 37 °C; 10 months at -20 °C, 4 °C, and 23 °C; and 36 months at -20 °C and 4 °C. This equivalence was true for all elements and pools except for the low concentration blood pool for Cd. CONCLUSIONS: Storage temperatures of -20 °C and 4 °C are equivalent to -70 °C for stability of Cd, Mn, Pb, Se, and Hg in human whole blood for at least 36 months when blood is stored in sealed polypropylene vials. Increasing the sample storage temperature from -70 °C to -20 °C or 4 °C can lead to large energy savings. The best analytical results are obtained when storage time at higher temperature conditions (e.g. 23 °C and 37 °C) is minimized because recovery of Se and Hg is reduced. Blood samples stored in polypropylene cryovials also lose volume over time and develop clots at higher temperature conditions (e.g., 23 °C and 37 °C), making them unacceptable for elemental testing after 10 months and 2 months, respectively.

Analysis of whole human blood for Pb, Cd, Hg, Se, and Mn by ICP-DRC-MS for biomonitoring and acute exposures.

We improved our inductively coupled plasma mass spectrometry (ICP-MS) whole blood method [1] for determination of lead (Pb), cadmium (Cd), and mercury (Hg) by including manganese (Mn) and selenium (Se), and expanding the calibration range of all analytes. The method is validated on a PerkinElmer (PE) ELAN® DRC II ICP-MS (ICP-DRC-MS) and uses the Dynamic Reaction Cell (DRC) technology to attenuate interfering background ion signals via ion-molecule reactions. Methane gas (CH4) eliminates background signal from 40Ar2+ to permit determination of 80Se+, and oxygen gas (O2) eliminates several polyatomic interferences (e.g. 40Ar15N+, 54Fe1H+) on 55Mn+. Hg sensitivity in DRC mode is a factor of two higher than vented mode when measured under the same DRC conditions as Mn due to collisional focusing of the ion beam. To compensate for the expanded method's longer analysis time (due to DRC mode pause delays), we implemented an SC4-FAST autosampler (ESI Scientific, Omaha, NE), which vacuum loads the sample onto a loop, to keep the sample-to-sample measurement time to less than 5min, allowing for preparation and analysis of 60 samples in an 8-h work shift. The longer analysis time also resulted in faster breakdown of the hydrocarbon oil in the interface roughing pump. The replacement of the standard roughing pump with a pump using a fluorinated lubricant, Fomblin®, extended the time between pump maintenance. We optimized the diluent and rinse solution components to reduce carryover from high concentration samples and prevent the formation of precipitates. We performed a robust calculation to determine the following limits of detection (LOD) in whole blood: 0.07µgdL-1 for Pb, 0.10µgL-1 for Cd, 0.28µgL-1 for Hg, 0.99µgL-1 for Mn, and 24.5µgL-1 for Se.

Induced topological changes in DNA complexes: influence of DNA sequences and small molecule structures.

Heterocyclic diamidines are compounds with antiparasitic properties that target the minor groove of kinetoplast DNA. The mechanism of action of these compounds is unknown, but topological changes to DNA structures are likely to be involved. In this study, we have developed a polyacrylamide gel electrophoresis-based screening method to determine topological effects of heterocyclic diamidines on four minor groove target sequences: AAAAA, TTTAA, AAATT and ATATA. The AAAAA and AAATT sequences have the largest intrinsic bend, whereas the TTTAA and ATATA sequences are relatively straight. The changes caused by binding of the compounds are sequence dependent, but generally the topological effects on AAAAA and AAATT are similar as are the effects on TTTAA and ATATA. A total of 13 compounds with a variety of structural differences were evaluated for topological changes to DNA. All compounds decrease the mobility of the ATATA sequence that is consistent with decreased minor groove width and bending of the relatively straight DNA into the minor groove. Similar, but generally smaller, effects are seen with TTTAA. The intrinsically bent AAAAA and AAATT sequences, which have more narrow minor grooves, have smaller mobility changes on binding that are consistent with increased or decreased bending depending on compound structure.

Large, sequence-dependent effects on DNA conformation by minor groove binding compounds.

To determine what topological changes antiparasitic heterocyclic dications can have on kinetoplast DNA, we have constructed ligation ladders, with phased A5 and ATATA sequences in the same flanking sequence context, as models. Bending by the A5 tract is observed, as expected, while the ATATA sequence bends DNA very little. Complexes of these DNAs with three diamidines containing either furan, thiophene or selenophene groups flanked by phenylamidines were investigated along with netropsin. With the bent A5 ladder the compounds caused either a slight increase or decrease in the bending angle. Surprisingly, however, with ATATA all of the compounds caused significant bending, to values close to or even greater than the A5 bend angle. Results with a mixed cis sequence, which has one A5 and one ATATA, show that the compounds bend ATATA in the same direction as a reference A5 tract, that is, into the minor groove. These results are interpreted in terms of a groove structure for A5 which is largely pre-organized for a fit to the heterocyclic amidines. With ATATA the groove is intrinsically wider and must close to bind the compounds tightly. The conformational change at the binding site then leads to significant bending of the alternating DNA sequence.

Antiparasitic compounds that target DNA.

Designed, synthetic heterocyclic diamidines have excellent activity against eukaryotic parasites that cause diseases such as sleeping sickness and leishmania and adversely affect millions of people each year. The most active compounds bind specifically and strongly in the DNA minor groove at AT sequences. The compounds enter parasite cells rapidly and appear first in the kinetoplast that contains the mitochondrial DNA of the parasite. With time the compounds are also generally seen in the cell nucleus but are not significantly observed in the cytoplasm. The kinetoplast decays over time and disappears from the mitochondria of treated cells. At this point the compounds begin to be observed in other regions of the cell, such as the acidocalcisomes. The cells typically die in 24-48h after treatment. Active compounds appear to selectively target extended AT sequences and induce changes in kinetoplast DNA minicircles that cause a synergistic destruction of the catenated kinetoplast DNA network and cell death.