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Malodorants are mixtures containing mercaptans, which trigger the flight instinct upon exposure and might thus be deployed in military and civilian defense scenarios. Exposure to mercaptans might lead to unconsciousness, thus representing a possible threat for health. Therefore, we developed and validated a bioanalytical procedure for the simultaneous detection and identification of corresponding biomarkers for the verification of exposure to mercaptans. Disulfide-adducts of ethyl mercaptan (SEt), n -butyl mercaptan (S n Bu), tert -butyl mercaptan (S t Bu) and iso- amyl mercaptan (S i Am) with cysteine (Cys) residues in human serum albumin (HSA) were formed by in vitro incubation of human plasma. After pronase-catalyzed proteolysis, reaction products were identified as adducts of the single amino acid Cys and the dipeptide cysteine-proline (Cys34Pro) detected by a sensitive μLC-ESI MS/MS method working in the scheduled multiple reaction monitoring (sMRM) mode. Dose-response studies showed linearity for the yield of Cys34Pro-adducts in the range from 6 nM to 300 μM of mercaptans in plasma and limits of identification (LOI) were in the range from 60 nM to 6 μM. Cys34-adducts showed stability for at least 6 days in plasma (37 °C). The presented disulfide-biomarkers expand the spectrum for bioanalytical verification procedures and might be helpful to prove exposure to malodorants. [Display omitted] • Generation of mercaptan-adducts with human plasma proteins. • Detection and identification of mercaptan-biomarkers by mass spectrometry. • Development of a sensitive μLC-ESI MS/MS method working in the scheduled MRM mode. • Validation of the method for simultaneous detection of 8 biomarkers. • Analytical characterization of the HSA-derived biomarker peptides. [ABSTRACT FROM AUTHOR]

Sulfur mustard (SM, bis[2‐chloroethyl]‐sulfide) is a banned chemical warfare agent deployed in the violent conflict in the Middle East poisoning humans and animals. For legal reasons, bioanalytical methods are mandatory proving exposure to SM. Reaction products (adducts) of SM with endogenous proteins, for example, serum albumin (SA), are valuable long‐lived targets for analysis. Whereas nearly all methods known so far focus on human proteins, we address for the first time neat chicken SA and avian serum from chicken, duck, and ostrich. After proteolysis, protein precipitation, evaporation of the supernatant, and re‐dissolution analysis were performed by micro‐liquid chromatography‐electrospray ionization tandem‐mass spectrometry in the selected reaction monitoring mode, μLC‐ESI MS/MS (SRM), for detection of the hydroxyethylthioethyl product ion [HETE]+ at m/z 105.0. After in vitro incubation with SM and pronase‐catalyzed proteolysis, the alkylated amino acids Glu(‐HETE) and His(‐HETE) were detected. Both borne the SM‐characteristic HETE‐moiety bound to their side chain. The eightfold deuterated SM analog (d8‐SM) was also applied to support adduct identification. Proteolysis conditions were optimized with respect to pH (8.0), temperature (50°C), and time to maximize the yield of Glu(‐HETE) (30 min) and His(‐HETE) (180 min). Amino acid adducts were stable in the autosampler for at least 24 h. Protein‐adducts were stable in serum at −30°C for at least 33 days and for three freeze‐and‐thaw cycles. At the body temperature of chicken (+40°C), Glu(‐HETE) was degraded in serum (period of half‐change 3 days), whereas His(‐HETE) remained stable. The presented method broadens the toolbox of procedures to document poisoning with SM. [ABSTRACT FROM AUTHOR]

Intoxication by organophosphorus (OP) poisons, like nerve agents and pesticides, is characterized by the life-threatening inhibition of acetylcholinesterase (AChE) caused by covalent reaction with the serine residue of the active site of the enzyme (phosphylation). Similar reactions occur with butyrylcholinesterase (BChE) and serum albumin present in blood as dissolved proteins. For forensic purposes, products (adducts) with the latter proteins are highly valuable long-lived biomarkers of exposure to OP agents that are accessible by diverse mass spectrometric procedures. In addition, the evidence of poison incorporation might also succeed by the detection of remaining traces of the agent itself, but more likely its hydrolysis and/or enzymatic degradation products. These relatively short-lived molecules are distributed in blood and tissue, and excreted via urine. This review presents the mass spectrometry-based methods targeting the different groups of biomarkers in biological samples, which are already internationally accepted by the Organisation for the Prohibition of Chemical Weapons (OPCW), introduces novel approaches in the field of biomedical verification, and outlines the strict quality criteria that must be fulfilled for unambiguous forensic analysis.

Some epidemiological studies show that heme iron consumption, in red meat, is associated to the development of several chronic diseases, including cancers and cardio-metabolic diseases. As heme iron intestinal absorption is finely regulated, we hypothesized that heme iron may act indirectly, through the peroxidation of dietary lipids, in food or in the intestinal lumen during digestion. This heme-iron-induced lipid peroxidation provokes the generation of toxic lipid oxidation products that could be absorbed, such as 4-hydroxynonenal (HNE). In a first experiment, heme iron given to rats by oral gavage together with the linoleic-acid-rich safflower oil induced the formation of HNE in the intestinal lumen. The HNE major urinary metabolite was elevated in the urine of the treated rats, indicating that this compound has been absorbed. In a second experiment, we showed that stable isotope-labeled HNE given orally to rats was able to reach non-intestinal tissues as a bioactive form and to make protein-adducts in heart, liver and skeletal muscle tissues. The presence of HNE-protein adducts in those tissues suggests a putative biological role of diet-originating HNE in extra-intestinal organs. This finding could have major consequences on the onset/development of chronic diseases associated with red meat over-consumption, and more largely to peroxidation-prone food consumption.

Exposure to toxic organophosphorus pesticides (OPP) represents a serious problem in the public healthcare sector and might be forced in terroristic attacks. Therefore, reliable verification procedures for OPP-intoxications are required for forensic, toxicological and clinical reasons. We developed and optimized a toolbox of methods to detect adducts of human serum albumin (HSA) with OPP considered as long-term biomarkers. Human serum was incubated with diethyl-oxono and diethyl-thiono pesticides for adduct formation used as reference. Afterwards serum was subjected to proteolysis using three proteases separately thus yielding phosphorylated tyrosine residues (Y*) detected as single amino acid (pronase), as hexadecapeptide LVRY* 411 TKKVPQVSTPTL (pepsin) and as the tripeptide Y* 411 TK (trypsin), respectively. Adducts were analyzed via microbore liquid chromatography coupled to electrospray ionization (μLC-ESI) and tandem-high-resolution mass spectrometry (MS/HR MS). Using paraoxon-ethyl as model OPP for adduct formation, methods were optimized with respect to MS/HR MS-parameters, protease concentrations and incubation time for proteolysis. HSA-adducts were found to be stable in serum in vitro at +37 °C and −30 °C for at least 27 days and resulting biomarkers were stable in the autosampler at 15 °C for at least 24 h. Limits of identification of adducts varied between 0.25 μM and 4.0 μM with respect to the corresponding pesticide concentrations in serum. Applicability of the methods was proven by successful detection of the adducts in samples of OPP-poisoned patients thus demonstrating the methods as a reliable toolbox for forensic and toxicological analysis. [ABSTRACT FROM AUTHOR]

Recent experimental data suggest that the idiosyncratic nature of drug-induced liver disease (DILD) may be due in part to a deficiency of one or more hepatoprotective factors. In this study we have investigated whether interleukin (IL)-6 may also be one of these factors. Following the induction of liver injury with acetaminophen (APAP), a time-dependent increase in liver mRNA expression of IL-6 and its family members IL-11, leukemia inhibitory factor, and oncostatin M was observed in wild type (WT) mice, suggesting a possible hepatoprotective role played by this cytokine family. Indeed, mice lacking IL-6 (IL-6−/−) were more susceptible than were WT mice to APAP-induced liver injury. The increased susceptibility of the IL-6−/− mice was associated with a deficiency in the expression of hepatic heat shock protein (HSP)25, 32, and 40 as well as inducible HSP70 following APAP treatment. These results suggest that IL-6 and possibly other family members may protect the liver from injury, at least in part, by up-regulating the hepatic expression of several cytoprotective HSPs. [Copyright &y& Elsevier]

Some epidemiological studies show that heme iron consumption, in red meat, is associated to the development of several chronic diseases, including cancers and cardio-metabolic diseases. As heme iron intestinal absorption is finely regulated, we hypothesized that heme iron may act indirectly, through the peroxidation of dietary lipids, in food or in the intestinal lumen during digestion. This heme-iron-induced lipid peroxidation provokes the generation of toxic lipid oxidation products that could be absorbed, such as 4-hydroxynonenal (HNE). In a first experiment, heme iron given to rats by oral gavage together with the linoleic-acid-rich safflower oil induced the formation of HNE in the intestinal lumen. The HNE major urinary metabolite was elevated in the urine of the treated rats, indicating that this compound has been absorbed. In a second experiment, we showed that stable isotope-labeled HNE given orally to rats was able to reach non-intestinal tissues as a bioactive form and to make protein-adducts in heart, liver and skeletal muscle tissues. The presence of HNE-protein adducts in those tissues suggests a putative biological role of diet-originating HNE in extra-intestinal organs. This finding could have major consequences on the onset/development of chronic diseases associated with red meat over-consumption, and more largely to peroxidation-prone food consumption. [ABSTRACT FROM AUTHOR]

Intoxication by organophosphorus (OP) poisons, like nerve agents and pesticides, is characterized by the life-threatening inhibition of acetylcholinesterase (AChE) caused by covalent reaction with the serine residue of the active site of the enzyme (phosphylation). Similar reactions occur with butyrylcholinesterase (BChE) and serum albumin present in blood as dissolved proteins. For forensic purposes, products (adducts) with the latter proteins are highly valuable long-lived biomarkers of exposure to OP agents that are accessible by diverse mass spectrometric procedures. In addition, the evidence of poison incorporation might also succeed by the detection of remaining traces of the agent itself, but more likely its hydrolysis and/or enzymatic degradation products. These relatively short-lived molecules are distributed in blood and tissue, and excreted via urine. This review presents the mass spectrometry-based methods targeting the different groups of biomarkers in biological samples, which are already internationally accepted by the Organisation for the Prohibition of Chemical Weapons (OPCW), introduces novel approaches in the field of biomedical verification, and outlines the strict quality criteria that must be fulfilled for unambiguous forensic analysis., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 THE AUTHORS. Publishing services by ELSEVIER B.V. on behalf of MSACL.)