Mass spectral similarity mapping applied to fentanyl analogs.

This manuscript outlines a straight-forward procedure for generating a map of similarity between spectra of a set. When applied to a reference set of spectra for Type I fentanyl analogs (molecules differing from fentanyl by a single modification), the map illuminates clustering that is applicable to automated structure assignment of unidentified molecules. An open-source software implementation that generates mass spectral similarity mappings of unknowns against a library of Type I fentanyl analog spectra is available at http://github.com/asm3-nist/FentanylClassifier.

Pairwise alignment of chromatograms using an extended Fisher-Rao metric.

A conceptually new approach for aligning chromatograms is introduced and applied to examples of metabolite identification in human blood plasma by liquid chromatography-mass spectrometry (LC-MS). A square-root representation of the chromatogram's derivative coupled with an extended Fisher-Rao metric enables the computation of relative differences between chromatograms. Minimization of these differences using a common dynamic programming algorithm brings the chromatograms into alignment. Application to a complex sample, National Institute of Standards and Technology (NIST) Standard Reference Material 1950, Metabolites in Human Plasma, analyzed by two different LC-MS methods having significantly different ranges of elution time is described.

Quantitative measurement of the polydispersity in the extent of functionalization of glass-forming calix[4]resorcinarenes.

The polydispersity in the degree of functionalization for two calix[4]resorcinarenes was determined by measuring quantitatively their molecular mass distribution with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A mathematical method for polydisperse materials is described that creates a calibration curve to correct the ion signal intensities in the mass spectrum to give a more reliable molecular mass distribution. Correction is required due to various sample preparation and instrumental effects that may produce a systematic mass bias in the number of oligomers measured. This method employs gravimetric mixtures of analytes with different degrees of functionalization. One calix[4]resorcinarene was found to give accurate molecular mass distributions with little correction, while another, having a very similar molecular structure, was found to exhibit strong over-counting of the oligomers having a high degree of functionalization.

A general method for quantitative measurement of molecular mass distribution by mass spectrometry.

A method is presented to test whether the conversion of the mass spectrum of a polydisperse analyte to its molecular mass distribution is quantitative. Mixtures of samples with different average molecular masses, coupled with a Taylor's expansion mathematical formalism, were used to ascertain the reliability of molecular mass distributions derived from mass spectra. Additionally, the method describes how the molecular mass distributions may be corrected if the degree of mass bias is within certain defined limits. This method was demonstrated on polydisperse samples of C(60) fullerenes functionalized with ethylpyrrolidine groups measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; however, it is applicable to any polydisperse analyte and mass spectrometric method as long as spectrum resolution allows individual oligomers to be identified. Mass spectra of the derivatized fullerenes taken in positive ion mode were shown to give an accurate measurement of the molecular mass distribution while those taken in negative ion mode were not. Differences in the mechanisms for ion formation are used to explain the discrepancy. Official contribution of the National Institute of Standards and Technology; not subject to copyright in the United States of America.

Numerical optimization of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: application to synthetic polymer molecular mass distribution measurement.

A novel approach is described for the selection of optimal instrument parameters that yield a mass spectrum which best replicates the molecular mass distribution of a synthetic polymer. The application of implicit filtering algorithms is shown to be a viable method to find the best instrument settings while simultaneously minimizing the total number of experiments that need to be performed. This includes considerations of when to halt the iterative optimization process at a point when statistically-significant gains can no longer be expected. An algorithm to determine the confidence intervals for each parameter is also given. Details on sample preparation and data analysis that ensure stability of the measurement over the time scale of the optimization experiments are provided. This work represents part of an effort to develop an absolute molecular mass distribution polymer Standard Reference Material.

Risk of silicosis in cohorts of Chinese tin and tungsten miners, and pottery workers (I): an epidemiological study.

BACKGROUND: Epidemiological evaluations of the risk of silicosis in relation to exposure to crystalline silica have raised the question of whether different types of silica dust exposures vary with respect to their ability to cause silicosis. The aim of this study is to compare the risk of silicosis among cohorts of silica dust-exposed Chinese tin miners, tungsten miners, and pottery workers and to assess whether gravimetric measurements of respirable silica dust sufficiently determine the risk of silicosis or whether other factors of exposure may play a significant role. METHODS: Cohorts were selected from 20 Chinese mines and potteries. Inclusion criteria were starting employment after January 1, 1950 and being employed for at least 1 year during 1960-1974 in one of the selected workplaces. Radiological follow-up for silicosis onset was from January 1, 1950 through December 31, 1994. Silicosis was assessed according to the Chinese radiological criteria for diagnosis of pneumoconiosis (as suspect, Stage I, II, or III). Exposure-response relationships were estimated for silicosis of Stage I or higher. Silica dust exposure was estimated in terms of cumulative total dust exposure, calculated from a workplace, job title, and calendar year exposure matrix, and individual occupational histories. Cumulative total dust exposure was converted in two steps into cumulative respirable dust exposure and cumulative respirable silica dust exposure using conversion factors estimated from side-by-side measurements conducted in 1988-89. RESULTS: The male cohorts included 4,028 tin miners, 14,427 tungsten miners, and 4,547 pottery workers who had similar onset of employment and duration of follow-up. For a given exposure level, the risk of silicosis was higher for the tin and tungsten than the pottery workers. CONCLUSION: The observed differences in the risk of silicosis among the three cohorts suggest that silica dust characteristics, in addition to cumulative respirable silica dust exposure, may affect the risk of silicosis.

A direct comparison of surface and bulk chain-relaxation in polystyrene.

Near-edge X-ray absorption fine-structure (NEXAFS) spectroscopy was used to measure simultaneously the relaxation rates of polystyrene (PS) molecules at the free surface and in the bulk. The samples were uniaxially stretched and annealed at temperatures below the bulk glass transition temperature of PS. The surface and bulk chain relaxation was monitored by measuring the partial-electron and the fluorescence NEXAFS yields, respectively, both parallel and perpendicular to the stretching direction. The decay of the optical birefringence was also measured to provide an independent measure of the bulk relaxation. Relaxation of PS chains was found to occur faster on the surface relative to the bulk. The magnitude of the surface glass transition temperature suppression over the bulk was estimated based on the information on the temperature dependence of the rates.

Monte Carlo analysis of the detection of clay occlusion of respirable quartz particles using multiple voltage scanning electron microscopy.

The electron incident-energy dependence of the relative intensities of Al and Si x-rays produced in a respirable-sized quartz particle by scanning electron microscopy is sensitive to the inhomogeneity of the distribution of Al and Si in the particle. Realistic Monte Carlo calculations of this energy dependence validate the proposal to use this effect for the detection of particles in which an aluminosilicate coating occludes the surface of a silica core.

Intraoperative treatment strategy to reduce the incidence of postcardiopulmonary bypass atrial fibrillation.

PURPOSE: Postcardiopulmonary bypass atrial fibrillation remains a constant complication associated with coronary revascularization, the incidence of which occurs from 20% to 35%. Previous studies have addressed this problem in the postoperative setting utilizing pharmacological agents, but the results have been variable. The purpose of this study was to evaluate a novel intraoperative strategy to reduce the incidence of postcardiopulmonary bypass atrial fibrillation. We theorized that leukocyte depletion by filtration with the addition of aprotinin would reduce the systemic inflammatory effects of bypass and reduce the incidence of atrial fibrillation. METHODS: One hundred and twenty-two patients participated in this randomized study. Only isolated primary coronary revascularization procedures on cardiopulmonary bypass were included. The control group (n=55) received standard moderate hypothermic blood cardioplegia cardiopulmonary bypass. The treatment group (n=65) received similar cardiopulmonary bypass with the addition of strategic leukocyte depletion with Pall Biomedical Products (East Hills, NY) leukodepletion filters and full-dose aprotinin. RESULTS: The intraoperative addition of leukocyte depletion by filtration with aprotinin reduced the incidence of postcardiopulmonary bypass atrial fibrillation by 72%. The incidence.of atrial fibrillation in the control group was 27% (15 of 55). In contrast, the occurrence of atrial fibrillation in the treated group was only 7.6% (5 of 65) (p<0.025). CONCLUSIONS: This novel intraoperative treatment strategy of both mechanical (leukocyte filtration) and pharmacological (aprotinin) intervention appears to markedly reduce the incidence of postcardiopulmonary bypass atrial fibrillation. To our knowledge, this is the first study to combine these two treatment strategies. A previous study has noted a decline in atrial fibrillation with aprotinin in the animal model, but not to the extent observed in our study. The beneficial effects of the reduction of atrial fibrillation include reduced risk of emboli formation and the incidence of ischemia in the heart, lung and brain. In addition, a decrease in length of hospital stay, recovery time and overall cost occurred.

Laser desorption ionization and MALDI time-of-flight mass spectrometry for low molecular mass polyethylene analysis.

Polyethylene's inert nature and difficulty to dissolve in conventional solvents at room temperature present special problems for sample preparation and ionization in mass spectrometric analysis. We present a study of ionization behavior of several polyethylene samples with molecular masses up to 4000 Da in laser desorption ionization (LDI) time-of-flight mass spectrometers equipped with a 337 nm laser beam. We demonstrate unequivocally that silver or copper ion attachment to saturated polyethylene can occur in the gas phase during the UV LDI process. In LDI spectra of polyethylene with molecular masses above approximately 1000 Da, low mass ions corresponding to metal-alkene structures are observed in addition to the principal distribution. By interrogating a well-characterized polyethylene sample and a long chain alkane, C94H190, these low mass ions are determined to be the fragmentation products of the intact metal-polyethylene adduct ions. It is further illustrated that fragmentation can be reduced by adding matrix molecules to the sample preparation.

Effects of phospholipid surfactant on apoptosis induction by respirable quartz and kaolin in NR8383 rat pulmonary macrophages.

Apoptosis was measured in rat alveolar macrophage NR8383 cells challenged in vitro with respirable quartz or kaolin dust and with the dusts pretreated with dipalmitoyl phosphatidylcholine (DPPC) to model conditioning of respired dusts by interaction with a primary phospholipid component of pulmonary surfactant. Quartz dust is known to induce apoptosis in vitro and in vivo. For this study, quartz and kaolin were compared as dusts of similar cytotoxicity in some in vitro assays but of differing pathogenic potential: quartz can cause significant pulmonary fibrosis while kaolin generally does not. NR8383 cells exposed to native quartz at concentrations from 50 to 400 microg/ml for 6 h showed a dose-dependent increase in apoptosis measured by the TdT-mediated dUTP-fluorescein nick end labeling (TUNEL), cell death ELISA, and DNA ladder formation assays, while native kaolin induced significant response only at the higher concentrations and only in the TUNEL and ELISA assays. For cell challenge from 6 h to 5 days at 100 microg/ml of dust, quartz was active at all times while kaolin was active only at 5 days. DPPC pre-treatment suppressed quartz activity until 3 days and kaolin activity through 5 days. Cellular release of lactate dehydrogenase, measured in parallel experiments to compare dust apoptotic and necrotic activities, indicated that components of serum as well as surfactant may affect kaolin in vitro expression of those activities.

Exposure to silica and silicosis among tin miners in China: exposure-response analyses and risk assessment.

OBJECTIVES: To investigate the risk of silicosis among tin miners and to investigate the relation between silicosis and cumulative exposure to dust (Chinese total dust and respirable crystalline silica dust). METHODS: A cohort study of 3010 miners exposed to silica dust and employed for at least 1 year during 1960-5 in any of four Chinese tin mines was conducted. Historical total dust data from China were used to create a job exposure matrix for facility, job title, and calendar year. The total dust exposure data from China were converted to estimates of exposure to respirable crystalline silica for comparison with findings from other epidemiological studies of silicosis. Each worker's work history was abstracted from the complete employment records in mine files. Diagnoses of silicosis were based on 1986 Chinese pneumoconiosis Roentgen diagnostic criteria, which classified silicosis as stages I-III-similar to an International Labour Organisation (ILO) classification of 1/1 or greater. RESULTS: There were 1015 (33.7%) miners identified with silicosis, who had a mean age of 48.3 years, with a mean of 21.3 years after first exposure (equivalent to 11.0 net years in a dusty job). Among those who had silicosis, 684 miners (67.4%) developed silicosis after exposure ended (a mean of 3.7 years after). The risk of silicosis was strongly related to cumulative exposure to silica dust and was well fitted by the Weibull distribution, with the risk of silicosis less than 0.1% when the Chinese measure of cumulative exposure to total dust (CTD) was under 10 mg/m(3)-years (or 0.36 mg/m(3)-years of respirable crystalline silica), increasing to 68.7% when CTD exposure was 150 mg/m(3)-years (or 5.4 mg/m(3)-years of respirable crystalline silica). Latency period was not correlated to the risk of silicosis or cumulative dose of exposure. This study predicts about a 36% cumulative risk of silicosis for a 45 year lifetime exposure to these tin mine dusts at the CTD exposure standard of 2 mg/m(3), and a 55% risk at 45 years exposure to the current United States Occupational Safety and Health Administration and Mine Safety and Health Administration standards of 0.1 mg/m(3) 100% respirable crystalline silica dust. CONCLUSIONS: A clear exposure-response relation was detected for silicosis in Chinese tin miners. The study results were similar to most, but not all, findings from other large scale exposure-response studies.

Effects of simulated pulmonary surfactant on the cytotoxicity and DNA-damaging activity of respirable quartz and kaolin.

Respirable-sized quartz and kaolin dusts were pretreated with simulated pulmonary surfactant dispersions of dipalmitoyl phosphatidylcholine (DPPC) in saline to model the conditioning of particles depositing in alveolar regions of the lung. DPPC-treated and untreated dusts were used to challenge lavaged rat pulmonary alveolar macrophages in vitro. Cytotoxicity was determined over a 5-d period using both total and viable cell counts from a fluorescence-based viability assay. DNA damage, as an indication of genotoxicity, was determined over a 7-d period by the single-cell gel electrophoresis assay. Untreated quartz and kaolin both expressed a significant and potent cytotoxicity, which increased with concentration and time. DPPC-surfactant pretreatment delayed significant expression of this cytotoxicity until 3 to 5 d after challenge. Untreated quartz also caused DNA damage, which increased with concentration and time. DPPC-surfactant treatment of quartz delayed most DNA damage expression to 5 and 7 d. Untreated kaolin expressed weaker activity for DNA damage, significant at the highest concentration through 5 d, and at the higher concentrations on d 7. Surfactant treatment delayed most kaolin activity for DNA damage to 7 d after challenge.

Intracellular surfactant removal from phagocytized minerals: development of a fluorescent method using a BODIPY-labeled phospholipid.

Lung surfactant serves as a protective coating when adsorbed on particle surfaces, so its removal or rate of removal in vivo may affect expression of mineral cytotoxicity. Removal of phospholipid surfactant components from the surface of mineral particles ingested by alveolar macrophages (AM) was measured using fluorescence microscopy. Dipalmitoylphosphatidylcholine with a fluorescent label (BODIPY(trade mark)) substituted for C1-C4 on the second acyl chain (DPPC*), was mixed with dioleoylphosphatidylcholine (DOPC) to coat respirable quartz and kaolin particles. Fluorescence from quartz or kaolin particles of 3-4, 5-6 and 8-9 microm size decreased in intensity with increasing ratios of DOPC/DPPC* for the same DOPC concentration of 0.4 mg/ml. There was a direct correlation between fluorescence and residual phospholipid surfactant remaining on particles using phospholipase A2 (PLA(2)) digestion in a cell-free system, indicating that the presence of the fluorophore on DPPC did not hinder enzymatic recognition. Lavaged primary AM obtained from male Fischer rats were challenged in vitro with DOPC/DPPC* (10:1 mol:mol) coated particles at 50 microg particles/10(6) cells. In contrast to the biexponential response seen in cell-free experiments, the rate of fluorescence decay from ingested coated quartz or kaolin particles over 7 days was monoexponential, with the same t(1/2) (41 h) for each dust. This study suggests that the rate of phagolysosomal digestion and removal of the adsorbed surfactant is not a determinant of the different mineral-specific pathogenicities or toxicities of quartz and kaolin, although residual fluorescence remained on particles even after 7-8 days.

A study of the effect of chrysotile fiber surface composition on genotoxicity in vitro.

Chrysotile fibers (NIEHS intermediate length) were treated with ultrapure HCl to alter the fiber surface chemistry without substantially changing fiber morphology or dimensions. The objective of the study was to determine whether fiber surface chemistry is an important variable in fiber genotoxicity in vitro. The modified fibers, along with native chrysotile fibers, were used to challenge Chinese hamster lung fibroblasts (V79) in vitro using the micronucleus induction genotoxicity assay. Fiber dimensions were assessed using scanning electron microscopy by measuring the distribution of fiber lengths in 3 length ranges: less than 3 microm, 3-10 microm, and greater than 10 microm. For both treated and native fiber samples, 500 fibers were examined. Results indicate that acid-treated fibers were about 20% shorter than untreated chrysotile. Surface chemistry alterations were verified by zeta-potential reversal, x-ray photoelectron spectroscopy (XPS), and scanning electron microscopy/energy-dispersive x-ray spectroscopy (SEM-EDS) elemental analysis. Scanning Auger spectrometry indicated the presence of Mg, O, and Si in both treated and native chrysotile samples, which confirmed the surface purity of both fiber samples. Both XPS and SEM-EDS analysis demonstrated substantial depletion of Mg from fiber surfaces. Results of the micronucleus assay showed a positive concentration-related response for both samples, with toxicity evident only at the highest concentration. No significant difference was found for the treated and untreated chrysotile samples. These results indicate that the surface chemistry is not an important variable in the in vitro genotoxicity of chrysotile asbestos in V79 cells as detected by the micronucleus assay under the conditions used in this study, and support a model of chemically nonspecific chromosomal and spindle damage effects.

Phospholipid surfactant adsorption by respirable quartz and in vitro expression of cytotoxicity and DNA damage.

Respirable-sized quartz was treated with a saline dispersion of dipalmitoyl phosphatidylcholine (DPPC), a primary component of pulmonary surfactant, to model the adsorption of phospholipid surfactant onto quartz dust following particle deposition in the bronchoalveolar region of the lung. Control and surfactant-treated dusts were used to challenge lavaged rat pulmonary macrophages in vitro over a 1-week period, to determine the effects of adsorbed surfactant on the expression of quartz cytotoxicity and genotoxicity. DNA damage was determined by the single cell gel electrophoresis 'comet' assay. Untreated quartz induced DNA damage, increasing with dose and with time of incubation of dust with macrophages over a 5 day period. DPPC treatment of quartz suppressed DNA damage through 1 day of macrophage challenge. DNA damage then increased over a 5 day period, to approximately half the positive control (untreated quartz) values. Cytotoxicity was measured by trypan blue dye exclusion and by the Live-Dead fluorescence assay for cell viability. Cytotoxicity of surfactant-treated quartz measured one day after challenge of lavaged macrophages was suppressed to values near those of the negative controls, and then increased over a 1 week incubation period to levels near those expressed by native quartz positive controls. Quartz similarly treated with dioleoyl phosphatidylcholine mixed with DPPC substituted in one acyl group with a boron-containing fluorescent chromophore was used with confocal microscopy to measure particle-associated fluorescent surfactant in cells. Approximately half of the fluorescence intensity was lost over a 1 week period following challenge of lavaged macrophage. Results are discussed in terms of a model of restoration of quartz particle surface toxicity as prophylactic surfactant is removed from particle surface by cellular enzymatic digestion processes.

Micronucleus formation in V79 cells treated with respirable silica dispersed in medium and in simulated pulmonary surfactant.

Chinese hamster lung fibroblasts (V79 cells) were challenged with respirable silica particles using an in vitro genotoxicity assay. Two particle sizes of crystalline quartz and a non-crystalline silica were assayed for induction of micronuclei (MN) in V79 cells. Some of the silica dusts used were pretreated with simulated pulmonary surfactant to model in vivo exposure conditions. The results showed that both crystalline and non-crystalline silica dispersed in medium (MEM) induced MN formation in a dose-dependent manner. Crystalline silica was more active in this assay than non-crystalline silica on a mass basis. The results also show that the frequency of micronucleated cells in cultures treated with surfactant-coated silica was not significantly different from that of the non-treated control cultures. These results seem to indicate that silica can cause chromosomal aberrations and/or aneuploidies in V79 cells; however, pretreatment of silica particles with simulated pulmonary surfactant reduces or delays genotoxicity in this assay.

The enzymatic removal of a surfactant coating from quartz and kaolin by P388D1 cells.

The macrophage-like cell line, P388D1, was exposed to dipalmitoyl lecithin (DPL)-coated respirable quartz and kaolin, and the disappearance of the DPL was monitored for up to 9 days. The coating was removed rapidly at first (about 50% in the first 3 days) and then more slowly over the remaining 6 days, until about 30% remained on day 9. The rate of DPL digestion was independent of the type of dust and the amount of coated dust within the cell, indicating the existence of an extracellular phospholipase activity. This extracellular phospholipase activity was partially characterized. It was sensitive to temperatures above 56 degrees C, the presence of EDTA, the action of the proteases trypsin and proteinase K, and pH, being active at pH 7 but not at pH 5. This is consistent with reports in the literature of the existence of an extralysosomal phospholipase which is active at pH 7 and dependent on the presence of divalent metal ions. There was a dust-dependent difference in the extracellular rate of DPL digestion from quartz and kaolin. The coating was removed more slowly from the kaolin than it was from quartz. The removal of the DPL coating seen in the presence of cells was presumably due to both an intracellular and an extracellular phospholipase.

Morphological transformation induced by glass fibers in BALB/c-3T3 cells.

Studies were conducted to determine whether 1) glass fibers can induce morphological transformation in BALB/c-3T3 cells, 2) the transforming activity of glass fibers is related to fiber size, and 3) transformed cells induced by glass fibers possess neoplastic properties. In the transformation assay, BALB/c-3T3 cells were treated with three different types of glass fibers: Manville code 100 (JM-100, Manville Corp., Denver, CO), Owens-Corning AAA-10 (AAA-10, Owens-Corning Corp., Toledo, OH), and Owens-Corning general building insulation (ISL, Owens-Corning Corp.) fibers. The neoplastic properties were investigated using the soft agar cloning and gene transfection methods. All three different glass fibers were cytotoxic at high concentrations and induced dose-related increases in morphological transformation. The transforming activity was inversely related to fiber size, with AAA-10 showing higher activity than JM-100 and JM-100 showing higher activity than ISL fiber. Transformed cells induced by glass fibers exerted anchorage-independent growth (90%) and DNA transfection-mediated transformation (100%). These results indicate that glass fibers are capable of transforming mammalian (BALB/c-3T3) cells in vitro as a function of their physical properties and that glass fiber-induced transformed cells possess preneoplastic characteristics.