Analytical methods based on liquid chromatography for the analysis of albumin adducts involved in retrospective biomonitoring of exposure to mustard agents.

The objective of the present review is to list, describe, compare, and critically analyze the main procedures developed in the last 20 years for the analysis of digested alkylated peptides, resulting from the adduction of albumin by different mustard agents, and that can be used as biomarkers of exposure to these chemical agents. While many biomarkers of sulfur mustard, its analogues, and nitrogen mustards can easily be collected in urine such as their hydrolysis products, albumin adducts require blood or plasma collection to be analyzed. Nonetheless, albumin adducts offer a wider period of detectability in human exposed patients than urine found biomarkers with detection up to 25 days after exposure to the chemical agent. The detection of these digested alkylated peptides of adducted albumin constitutes unambiguous proof of exposure. However, their determination, especially when they are present at very low concentration levels, can be very difficult due to the complexity of the biological matrices. Therefore, numerous sample preparation procedures to extract albumin and to recover alkylated peptides after a digestion step using enzymes have been proposed prior to the analysis of the targeted peptides by liquid chromatography coupled to mass spectrometry method with or without derivatization step. This review describes and compares the numerous procedures including a number of different steps for the extraction and purification of adducted albumin and its digested peptides described in the literature to achieve detection limits for biological samples exposed to sulfur mustard, its analogues, and nitrogen mustards in the ng/mL range.

Progressive expansion of albumin adducts for organophosphorus nerve agent traceability based on single and group adduct collection.

Protein adducts are important biological targets for traceability of organophosphorus nerve agents (OPNAs). Currently, the recognized biomarkers that can be used in actual samples in the field of chemical forensics only include Y411 in albumin and the active nonapeptide in butyrylcholinesterase (BChE). To explore stable and reliable protein adducts and increase the accuracy of OPNAs traceability further, we gradually expanded OPNAs-albumin adducts based on single and group adduct collection. Several stable peptides were found via LC-MS/MS analysis in human serum albumin (HSA) exposed to OPNAs in a large exposure range. These adducts were present in HSA samples exposed to OPNAs of each concentration, which provided data support for the reliability and stability of using adducts to trace OPNAs. Meanwhile, the formation mechanism of OPNAs-cysteine adduct was clarified via computer simulations. Then, these active sites found and modified peptides were used as raw materials for progressive expansion of albumin adducts. We constructed an OPNAs-HSA adducts group, in which a specific agent is the exposure source, and three or more active peptides constitute data sets for OPNAs traceability. Compared with single or scattered protein adducts, the OPNAs-HSA adduct group improves OPNAs identification by mutual verification using active peptides or by narrowing the identity range of the exposure source. We also determined the minimum detectable concentration of OPNAs for the adduct group. Two or more peptides can be detected when there is an exposure of 50 times the molar excess of OPNAs in relation to HSA. This improved the accuracy of OPNAs exposure and identity confirmation. A collection of OPNAs-albumin adducts was also examined. The collection was established by collecting, classifying, and integrating the existing albumin adducts according to the species to which each albumin belongs, the types of agents, and protease. This method can serve as a reference for discovering new albumin adducts, characteristic phosphonylated peptides, and potential biomarkers. In addition, to avoid a false negative for OPNAs traceability using albumin adducts, we explored OPNAs-cholinesterase adducts because cholinesterase is more reactive with OPNAs than albumin. Seven active peptides in red blood cell acetylcholinesterase (RBC AChE) and serum BChE can assist in OPNAs exposure and identity confirmation.

Quo vadis blood protein adductomics?

Chemicals are measured regularly in air, food, the environment, and the workplace. Biomonitoring of chemicals in biological fluids is a tool to determine the individual exposure. Blood protein adducts of xenobiotics are a marker of both exposure and the biologically effective dose. Urinary metabolites and blood metabolites are short term exposure markers. Stable hemoglobin adducts are exposure markers of up to 120 days. Blood protein adducts are formed with many xenobiotics at different sites of the blood proteins. Newer methods apply the techniques developed in the field of proteomics. Larger adducted peptides with 20 amino acids are used for quantitation. Unfortunately, at present the methods do not reach the limits of detection obtained with the methods looking at single amino acid adducts or at chemically cleaved adducts. Therefore, to progress in the field new approaches are needed.

Aflatoxin Exposure during Early Life Is Associated with Differential DNA Methylation in Two-Year-Old Gambian Children.

Background: DNA methylation is an epigenetic control mechanism that may be altered by environmental exposures. We have previously reported that in utero exposure to the mycotoxin and liver carcinogen aflatoxin B1 from the maternal diet, as measured using biomarkers in the mothers' blood, was associated with differential DNA methylation in white blood cells of 6-month-old infants from The Gambia. Methods: Here we examined aflatoxin B1-associated differential DNA methylation in white blood cells of 24-month-old children from the same population (n = 244), in relation to the child's dietary exposure assessed using aflatoxin albumin biomarkers in blood samples collected at 6, 12 and 18 months of age. HM450 BeadChip arrays were used to assess DNA methylation, with data compared to aflatoxin albumin adduct levels using two approaches; a continuous model comparing aflatoxin adducts measured in samples collected at 18 months to DNA methylation at 24 months, and a categorical time-dose model that took into account aflatoxin adduct levels at 6, 12 and 18 months, for comparison to DNA methylation at 24 months. Results: Geometric mean (95% confidence intervals) for aflatoxin albumin levels were 3.78 (3.29, 4.34) at 6 months, 25.1 (21.67, 29.13) at 12 months and 49.48 (43.34, 56.49) at 18 months of age. A number of differentially methylated CpG positions and regions were associated with aflatoxin exposure, some of which affected gene expression. Pathway analysis highlighted effects on genes involved with with inflammatory, signalling and growth pathways. Conclusions: This study provides further evidence that exposure to aflatoxin in early childhood may impact on DNA methylation.

A sensitive quantification approach for detection of HETE-CP adduct after benzyl chloroformate derivatization using ultra-high-pressure liquid chromatography tandem mass spectrometry.

Sulfur mustard (HD) reacts with human serum albumin (HSA) at Cys34 and produces a long-term biomarker of HD exposure. Here, we present a novel, sensitive, and convenient method for quantification of HD exposure by detection of HD-HSA adducts using pronase digestion, benzyl chloroformate (Cbz-Cl) derivatization, and ultra-high-pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). The HSA in HD-exposed plasma in vitro was precipitated with acetone and digested (2 h, 50 °C) with pronase to form the alkylated dipeptide, S-hydroxyethylthioethyl-CysPro (HETE-CP). The HETE-CP adduct was derivatized with Cbz-Cl to generate N-carbobenzoxy HETE-CP (HETE-C(Cbz)P). The derivatized product was analyzed by UHPLC-MS/MS. HD surrogate, 2-chloroethyl ethyl sulfide (2-CEES), was introduced as a non-isotope internal standard (ISTD) instead of traditional d8-HD for quantification. The method was found to be linear between 1.00 and 200 ng/mL HD exposure (R2 > 0.998) with precision of ≤ 9.0% relative standard deviation (RSD) and accuracy ranged between 97.1 and 111%. The limit of detection (LOD) is 0.500 ng/mL (S/N~5), over 15 times lower than that of the previous method (7.95 ng/mL). Time-consuming affinity purification or solid phase extraction (SPE) is not needed in the experiment and the operation takes less than 5 h. This study provides a new strategy and useful tool for retrospective analysis of HD exposure by HETE-CP biomarker detection. Graphical abstract Flow diagram for quantification of sulfur mustard exposure by detection of HETE-CP dipeptide adduct after benzyl chloroformate derivatization using ultra-high-pressure liquid chromatography tandem mass spectrometry.

Comparison of urinary aflatoxin M1 and aflatoxin albumin adducts as biomarkers for assessing aflatoxin exposure in Tanzanian children.

PURPOSE: To determine levels of urinary aflatoxin M1 (AFM1) in children and correlate the concentrations with previously reported aflatoxin albumin adduct (AF-alb) levels in these children. MATERIALS AND METHODS: Matched urine and blood samples were collected from 84 Tanzanian children aged 6-14 months old. From 31 children in one village (Kigwa), samples were collected at three time points six months apart. Samples were collected from 31 and 22 children from two different regions at the second time point only. Urinary AFM1 was measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit with a modified protocol to improve sensitivity. AF-alb was measured using an established ELISA method. RESULTS: The relative ranking of the three villages for exposure to aflatoxin based on either AFM1 or AF-alb biomarker measurements was the same. In Kigwa village, both AFM1 and AF-alb levels were higher at six months post-harvest compared to baseline. However, at the next visit, the AFM1 levels dropped from a GM (interquartile range) of 71.0 (44.7, 112.6) at visit two to 49.3 (31.5, 77.3) pg/ml urine, whereas AF-alb levels increased from 47.3 (29.7, 75.2) to 52.7 (35.4, 78.3) pg/mg albumin between these two visits, reflecting the fact that AFM1 measures short-term exposure, whereas AF-alb measures longer term exposure. There was a correlation between AFB1 intake and AFM1 excretion (r= 0.442, p ≤ 0.001). CONCLUSIONS: Urinary AFM1 is a good biomarker for AFB1 exposure in Tanzanian children, reflecting geographical and temporal variations in exposure to this foodborne toxin.

Aflatoxin B1 exposure increases the risk of cirrhosis and hepatocellular carcinoma in chronic hepatitis B virus carriers.

The relation between aflatoxin B1 (AFB1 ) and cirrhosis in chronic carriers of hepatitis B virus (HBV) remains inconclusive. This case-control study nested in a large community-based cohort aimed to assess the effect of AFB1 exposure on cirrhosis and HCC in chronic HBV carriers. Serum AFB1 -albumin adduct levels at study entry were measured in 232 cirrhosis cases, 262 HCC cases and 577 controls. Multivariate-adjusted odds ratios (aORs) and 95% confidence intervals (95% CIs) were estimated using logistic regression. Among all chronic HBV carriers, the time intervals between study entry and diagnosis of HCC, cirrhosis, cirrhotic HCC, and non-cirrhotic HCC were all significantly (p < 0.0001) shorter in participants with high serum levels of AFB1 -albumin adducts than those with low/undetectable levels. There were significant dose-response relations with serum AFB1 -albumin adduct level at study entry for cirrhosis (p-trend = 0.0001) and cirrhotic HCC (p-trend < 0.0001) newly diagnosed within 9 years after entry as well as non-cirrhotic HCC (p-trend = 0.021) newly diagnosed within 4 years after entry. The aORs (95% CIs) for high versus undetectable serum AFB1 -albumin adduct levels were 2.45 (1.51-3.98) for cirrhosis (p = 0.0003), 5.47 (2.20-13.63) for cirrhotic HCC (p = 0.0003), and 5.39 (1.11-26.18) for non-cirrhotic (p = 0.0368) HCC, respectively. There remained a significant dose-response relation between serum AFB1 -albumin adduct level and HCC risk (p-trend = 0.0291) in cirrhosis patients, showing an aOR (95% CI) of 3.04 (1.11-8.30) for high versus undetectable serum levels (p = 0.0299). It is concluded that AFB1 exposure may increase the risk of cirrhosis and HCC in a dose-response manner among chronic HBV carriers.

Comparison of biological effects with albumin adducts of 4,4'-methylenediphenyl diisocyanate in workers.

Lung sensitization and asthma are the main health effects of 4,4'-methylenediphenyl diisocyanate (MDI). Albumin adducts (isocyanate-specific adducts) of MDI might be involved in the etiology of sensitization reactions. Albumin adducts of MDI were analyzed in sera of diisocyanate-exposed worker with and without diisocyanate occupational asthma (DA), as well as in exposed workers with and without diisocyanate-specific IgG antibodies. In DA-positive workers and IgG-positive workers, albumin adducts were significantly higher versus workers without DA and those who were specific IgG negative. The odds ratio to be DA-positive was 57 times larger for workers with adduct levels above 230 fmol/mg. The odds ratio to be IgG-positive was 10 times larger for workers with adduct levels above 113 fmol/mg. Therefore, albumin adducts appear to be a good predictor of the biological effects. The albumin-adduct levels in workers without biological effects were in the range of the adduct levels found in previous studies of healthy MDI-factory and construction site workers.

Determination of albumin adducts of 4,4'-methylenediphenyl diisocyanate in workers of a 4,4'-methylenedianiline factory.

Lung sensitization and asthma are the main health effects of 4,4'-methylenediphenyl diisocyanate (MDI). Albumin adducts (isocyanate specific adducts) of MDI might be involved in the etiology of sensitization reactions. Albumin adducts of MDI have been found in subjects classified as 4,4'-methylenedianiline (MDA) workers. The mean adduct levels in these MDA-workers were 1.5 times higher than in MDI-workers of the same company. MDA-specific hemoglobin adducts, were present ten times more in the MDA-workers than in the MDI-workers. MDA-workers with specific work task had significantly higher albumin adduct levels.

Correlations of Aflatoxin Exposure from Cooking Oil and Dietary Foods During Pregnancy with Birth Weight and Gestational Age at Birth in Guangxi, China.

Background: Cooking oil and dietary foods are easily contaminated by aflatoxins (AFs) in Guangxi, China where low birth weight and preterm birth were prevalent. However, there are no data on AF exposure in pregnant women or their impact on newborn birth outcomes. This study aims to measure the levels and correlations of AFs in cooking oil, estimated dietary intake (EDI) of AFs in dietary foods, and serum AFB1 albumin adducts (AFB1-alb) with newborn birthweight and gestational age at birth. Methods: A prospective study was conducted among 126 pregnant women in Guangxi, China. All recruited women were interviewed for demographic data and behavior and obstetric information and then followed up until giving birth. AF measurements were obtained from cooking oil, dietary foods, maternal serum, and cord blood and the correlations of AF levels with newborn birthweight and gestational age at birth were tested using correlation analysis. Results: The median EDI of AFs in cooking oil was 2.61 ng/kg.bw/day and in dietary foods 2.95 ng/kg.bw/day. High positive correlations among EDI of aflatoxin B1 (AFB1) from cooking oil and dietary foods were found (r > 0.7). Low positive correlations of AFB1-alb in maternal serum and cord blood and both EDI of AFB1 in both cooking oil and dietary foods were shown (r ≈0.3). Significant correlations between AF levels in both cooking oil and dietary foods with birth weight were found, but very low negative correlations (r = - 0.244 ~ -0.285). AFB1 levels in foods, maternal serum and cord blood levels were high in pregnant women with newborn low birth weight and preterm birth. Conclusion: The EDIs of AFB1 from both cooking oil and dietary foods were significantly correlated with AFB1-alb in maternal serum and cord blood. Negative correlations of AFs from cooking oils and foods with newborn birth weight should be paid more attention.

Cytotoxicities of Tumor-Seeking Dyes: Impact on Future Clinical Trials.

Heptamethine (Cy7) dyes with meso-Cl substituents injected intravenously (iv) into mice accumulate in tumors and persist there over several days. We believe this occurs via meso-Cl displacement by the only free cysteine residues of albumin; therefore, conjugating tumor-seeking dyes with fragments can increase selective therapeutic delivery to tumors and drug residence. This strategy has elevated significance recently because the first tumor-seeking dye-drug conjugate has moved into clinical trials. Options for further clinical research include modifying the dye, and use of preformed albumin adducts instead of dyes alone. Herein we show correlations of cytotoxicities, lipophilicities, organelle localization, apoptosis, cell-cycle arrest, wound healing/migration assays, and reactivities/affinities with human serum albumin are difficult to observe. However, our studies arrived at an important conclusion: preformed dye-drug-HSA adducts are less cytotoxic, and therefore preferable for subsequent clinical work, relative to direct injection of meso-Cl-containing forms.

Treasure hunt for peptides with undefined chemical modifications: Proteomics identification of differential albumin adducts of 2-nitroimidazole-indocyanine green in hypoxic tumor.

2-Nitroimidazole is a well-known chemical probe targeting hypoxic environments of solid tumors, and its derivatives are widely used as imaging agents to investigate tissue and tumor hypoxia. However, the underlying chemistry for the hypoxia-detection capability of 2-nitroimidazole is still unclear. In this study, we deployed a biotin conjugate of 2-nitroimidazole-indocyanine green (2-nitro-ICG) for the investigation of in vivo hypoxia-probing mechanism of 2-nitro-ICG compounds. By implementing mass spectrometry-based proteomics and exhaustive data mining, we report that 2-nitro-ICG and its fragments modify mouse serum albumin as the primary protein target but at two structurally distinct sites and possibly via two different mechanisms. The identification of probe-modified peptides not only contributes to the understanding of the in vivo metabolism of 2-nitroimidazole compounds but also demonstrates a competent analytical workflow that enables the search for peptides with undefined modifications in complex proteome digests.

Methionine329 in human serum albumin: A novel target for alkylation by sulfur mustard.

Exposure to the vesicant sulfur mustard (SM) may lead to erythema and blistering. Toxicity of SM is hypothesized due to the alkylation of DNA bases and nucleophilic amino acid side chains in proteins (adducts) by forming the hydroxyethylthioethyl (HETE) moiety. Despite its prohibition by the chemical weapons convention, SM still represents a serious threat to military personnel and civilians. Therefore, development and improvement of forensic analytical methods for the verification of SM exposure is of high interest. Protein adducts have been shown to be highly suitable and beneficial biomarkers of poisoning. Herein we present methionine329 in human serum albumin (HSA) as a novel target of SM forming a HETE-methionyl sulfonium ion. The alkylated tetrapeptide LeuGlyMet329 (-HETE)Phe, LGM(-HETE)F, was detected after pepsin-mediated proteolysis and subsequent analysis by microbore liquid chromatography-electrospray ionization-high-resolution tandem-mass spectrometry. Compound identity was confirmed by a synthetic reference. Proteolysis conditions for HSA were optimized towards maximum yield of LGM(-HETE)F and its limit of identification (32.3 nM SM in serum) was similar to those of the established HSA-derived biomarkers HETE-CysPro and HETE-CysProPhe (15.6 nM SM in serum). Stability of the alkylated Met329 in vitro and in vivo was limited to 5 days making this modification a beneficial short-time biomarker. Furthermore, it was found that the HETE-methionyl sulfonium ion can transfer its HETE moiety to the side chain of cysteine and glutamic acid as well as to the N-terminus of peptides and proteins in vitro thus revealing novel insights into the molecular toxicity of SM.

Application of lanthanum stearate monolayers as a metal-affinity sorbent for the selective sorption of soman adducts to human serum albumin.

A new metal-affinity sorbent based on lanthanum stearate monolayers has been developed and characterized. The prospect of its application to specific extraction of organophosphorous compound (OP) adducts of blood proteins was demonstrated. For this, the patterns of soman adducts of human serum albumin (HSA) were comprehensively characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS).

N-Acetylcysteine as a chemical scavenger for sulfur mustard: New insights by mass spectrometry.

The vesicant sulfur mustard (SM) is a banned chemical warfare agent. Although, SM has been used in combat since WWI, there is no causal therapy currently available. Accordingly, development and investigation of antidotes and scavengers targeting SM are of high clinical relevance. N-acetylcysteine (NAC) was shown to mitigate symptoms of SM intoxications in vitro and in vivo. However, it is still unclear whether the beneficial effects of NAC are only due to physiological processes or also due to chemical scavenging of SM. Therefore, in this study, we examined the scavenging potential of NAC toward SM. Co-incubations of SM and different NAC concentrations in human serum were performed to monitor diverse adducts (covalent reaction products) of human serum albumin (HSA), NAC, and SM. After proteolytic cleavage of HSA with proteinase K the alkylated tripeptide hydroxyethylthioethyl-CysProPhe (HETE-CPF) and the disulfide bridged tripeptide NAC-CPF were detected. Samples were analyzed by microbore liquid chromatography-electrospray ionization-high-resolution tandem-mass spectrometry (µLC-ESI MS/HR MS). Furthermore, degradation kinetics of SM in phosphate buffered saline were measured in the presence and absence of NAC. Although NAC-CPF was identified and characterized for the first time by mass spectrometry and reaction products of NAC and SM were detected and identified by MS/HR MS, analyses clearly documented minor reactivity not significantly contributing to reduction of SM concentrations. Therefore, we conclude that chemical scavenging of SM by NAC does not play the key role in NAC therapy of SM poisoning.

Novel cysteine- and albumin-adduct biomarkers to prove human poisoning with the pesticide oxydemeton-S-methyl.

We herein report on the forensic analysis of plasma samples to prove human poisoning with oxydemeton-S-methyl (ODM), S-(2-(ethylsulfinyl)ethyl)-O,O-dimethyl phosphorothioate. This organophosphorus pesticide is the active ingredient of Metasystox®, that was swallowed by a 77-year-old woman to commit suicide. ODM belongs to the class of dimethyl phosphoryl (DMP) pesticides, contains a 2-(ethylsulfinyl)ethanethiol (ESOET) leaving group and undergoes adduct formation with endogenous molecules as elaborated herein with human serum exposed to pesticides in vitro. A novel bioanalytical micro liquid-chromatography-electrospray ionization tandem high-resolution mass spectrometry method (µLC-ESI MS/HR MS) was developed to target multiple biomarkers of exposure. Following pronase-catalyzed proteolysis of patient plasma and subsequent ultrafiltration, the filtrate was analyzed. Diverse reaction products of ODM as well as of its oxidized biotransformation product demeton-S-methyl sulfone (DSMS), that possesses a 2-(ethylsulfonyl)ethanethiol (ESO2ET) leaving group, were simultaneously detected. Phosphorylated tyrosine residues (Tyr-DMP) derived from human serum albumin (HSA) as well as novel dipeptide-adducts containing the Cys34 residue of HSA coupled to ESOET and ESO2ET via a disulfide bond (ESOET-CysPro and ESO2ET-CysPro) were found. In addition, a related disulfide-product was detected comprising the single amino acid cysteine and ESOET (ESOET-Cys). Whereas Tyr-DMP only proved the intake of any DMP pesticide in general, its simultaneous detection with ESOET-CysPro, ESO2ET-CysPro and ESOET-Cys allowed unambiguous identification of the ingested pesticide. Therefore, the novel biomarkers and the method developed expand the possibilities of forensic investigations of ODM poisoning.

Aflatoxin B1 exposure increases the risk of hepatocellular carcinoma associated with hepatitis C virus infection or alcohol consumption.

BACKGROUND: Hepatocarcinogenicity of aflatoxin B1 (AFB1) has rarely been studied in populations with hepatitis C virus (HCV) infection and those without hepatitis B virus (HBV) and HCV infection (non-B-non-C). This case-control study nested in a community-based cohort aimed to investigate the HCC risk associated with AFB1 in HCV-infected and non-B-non-C participants. METHODS: Baseline serum AFB1-albumin adduct levels were measured in 100 HCC cases and 1767 controls seronegative for anti-HCV and HBsAg (non-B-non-C), and another 103 HCC cases and 176 controls who were anti-HCV-seropositive and HBsAg-seronegative. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were estimated using logistic regression. RESULTS: In 20 years of follow-up, the follow-up time to newly developed HCC was significantly shorter in participants with higher serum AFB1-albumin adduct levels in non-B-non-C (p = 0.0162) and HCV-infected participants (p < 0.0001). Within 8 years of follow-up, HCV infection and AFB1 exposure were independent risk factors for HCC. Elevated serum AFB1-albumin adduct levels were significantly associated with an increased risk of HCC newly developed within 8 years of follow-up in non-B-non-C participants with habitual alcohol consumption [crude OR (95% CI) for high vs. low/undetectable levels, 4.22 (1.16-15.37)] and HCV-infected participants [3.39 (1.31-8.77)], but not in non-B-non-C participants without alcohol drinking habit. AFB1 exposure remained an independent risk predictor for HCV-related HCC after adjustment for other HCC predictors (multivariate-adjusted OR [95% CI], 3.65 [1.32-10.10]). CONCLUSIONS: AFB1 exposure contributes to the development of HCC in participants with significant risk factors for cirrhosis including alcohol and HCV infection.

Glutathione as an antidote for sulfur mustard poisoning: Mass spectrometric investigations of its potency as a chemical scavenger.

The banned chemical warfare agent sulfur mustard (SM) still represents a serious threat to civilians and military personnel. Therefore, identification of antidotes and scavengers is of high concern. One promising substance is glutathione (GSH). GSH is known to mitigate symptoms of SM poisoning in vitro and in vivo. However, the mechanism of action remains unclear with respect to physiological impact as well as chemical scavenging by reaction between GSH and SM. Therefore, a novel in vitro method was used to characterize the scavenging potential of GSH. Accordingly, alkylation of human serum albumin (HSA), which represents an established biomarker for SM intoxication, was used as a measure for remaining SM. Coincubation of GSH and SM in human serum was performed, and time-dependent degradation of SM was monitored in the presence and absence of GSH. Protein-derived and small molecular reaction products between GSH, HSA, and SM were analyzed using microbore liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry. Although covalent modification of GSH by SM was observed, measurements clearly documented no significant reduction of SM concentration in the presence of GSH. Accordingly, beneficial therapeutic mechanisms of GSH in the case of SM poisoning would appear to be based on physiological effects than on chemical scavenging.

Albumin adducts and urinary metabolites resulting from occupational exposure to 1,5-naphthalene diisocyanate.

OBJECTIVES: 1,5-Naphthalene diisocyanate (NDI) is used in the plastic industry as a curing agent. 1,5-Naphthalene diisocyanate is classified as a sensitizing agent. The objective of this study has been to develop biomonitoring methods for the evaluation of exposure to NDI. MATERIAL AND METHODS: We obtained blood and urine samples from a group of 20 male workers exposed to NDI. The workers answered a questionnaire about their exposure history, job description, the number of years with the company and the time spent working with NDI over the 10 days of the study. Total plasma, albumin, and urine were analyzed for the presence of 1,5-naphthalenediamine (NDA) after acid hydrolysis using gas chromatography-mass spectrometry (GC-MS). RESULTS: 1,5-Naphthalenediamine was found in about 60% of the samples obtained from the workers. 1,5-Naphthalenediamine was obtained after acid hydrolysis of plasma, albumin, and urine at levels up to 1.5 pmol NDA/mg of plasma proteins, 1.15 pmol NDA/mg of albumin, and 55.3 pmol NDA/ml of urine, respectively. CONCLUSIONS: 1,5-Naphthalenediamine found in urine correlates best with the plasma levels (r = 0.91, p < 0.01). The albumin-adduct levels did not correlate with the NDI-specific immunoglobulin E (IgE) or total IgE present in the workers. The adduct and metabolite levels correlate with the air levels of NDI. Int J Occup Med Environ Health 2017;30(4):579-591.

Determination of albumin adducts of 4,4'-methylenediphenyl diisocyanate after specific inhalative challenge tests in workers.

4,4'-Methylenediphenyl diisocyanate (MDI) is the most important isocyanate used in the industry. Lung sensitization with bronchial asthma is the main disorder in exposed workers. Albumin adducts of MDI might be involved in specific immunological reactions. MDI adducts with lysine (MDI-Lys) of albumin have been found in MDI-workers and construction workers. MDI-Lys is an isocyanate-specific adduct of MDI with albumin. In the present study, we report MDI-adducts in workers undergoing diagnostic MDI challenge tests. The workers were exposed for 2h to 5ppb of MDI. The adduct levels increase significantly after the exposure to MDI in the challenge chamber. About 0.6% of the dose was bound to albumin. So far, only urinary metabolites of MDI were measured to monitor isocyanate workers. However, such urinary metabolites are not isocyanate specific. Therefore, we propose to measure albumin adducts for monitoring MDI exposed subjects.