Elucidation of in Vitro Chlorinated Tyrosine Adducts in Blood Plasma as Selective Biomarkers of Chlorine Exposure

Abstract

Chlorine is a widely available industrial chemical and involved in a substantial number of cases of poisoning. It has also been used as a chemical warfare agent in military conflicts. To enable forensic verification, the persistent biomarkers 3-chlorotyrosine and 3,5-dichlorotyrosine in biomedical samples could be detected. An important shortfall of these biomarkers, however, is the relatively high incidence of elevated levels of chlorinated tyrosine residues in individuals with inflammatory diseases who have not been exposed to chlorine. Therefore, more reliable biomarkers are necessary to distinguish between endogenous formation and exogeneous exposure. The present study aims to develop a novel diagnostic tool for identifying site-specific chlorinated peptides as a more unambiguous indicator of exogeneous chlorine exposure. Human blood plasma was exposed in vitro to various chlorine concentrations, and the plasma proteins were subsequently digested by pronase, trypsin, or pepsin. After sample preparation, the digests were analyzed by liquid chromatography tandem mass spectrometry (LC–MS/MS) and liquid chromatography high-resolution tandem mass spectrometry (LC–HRMS/MS). In line with other studies, low levels of 3-chlorotyrosine and 3,5-dichlorotyrosine were found in blank plasma samples in this study. Therefore, 50 site-specific biomarkers were identified, which could be used as more unambiguous biomarkers for chlorine exposure. Chlorination of the peptides TY*ETTLEK, Y*KPGQTVK, Y*QQKPGQAPR, HY*EGSTVPEK, and Y*LY*EIAR could already be detected at moderate in vitro chlorine exposure levels. In addition, the latter two peptides were found to have dichlorinated fragments. Especially, Y*LY*EIAR, with a distinct chlorination pattern in the MS spectra, could potentially be used to differentiate exogeneous exposure from endogenous causes as other studies reported that this part of human serum albumin is nitrated rather than chlorinated under physiological conditions. In conclusion, trypsin digestion combined with high-resolution MS analysis of chlorinated peptides could constitute a valuable technique for the forensic verification of exposure to chlorine.

Verification of exposure to chemical warfare agents through analysis of persistent biomarkers in plants

“…The applied method was adapted from protocols originally described for the detection of CWAs in biomedical samples. 4,21,40 For pronase digestion, 400 mL ABC buffer (50 mM) and 100 mL pronase (10 mg mL −1 in 50 mM ABC) were added to 10 mg of dried leaf. The digests aer HD and chlorine exposure were incubated overnight at 37 °C and a rotational speed of 800 rpm.…”

“…For chlorine exposure, the plants were placed in a closed 2 or 20 L vessel and chlorine was generated by the reaction of calcium hypochlorite and HCl, similar to the method applied by de Bruin-Hoegée et al 21 An 1.5 mL Eppendorf tube with 1, 10 and 50 mg calcium hypochlorite was placed in the vessel above the plant. A syringe was put through a septum and slowly, over a period of one minute, 1 mL of a 12 M HCl solution was added to the calcium hypochlorite, after which 50 mg m −3 , 500 mg m −3 , 5 g m −3 or 25 g m −3 chlorine gas was generated.…”

“…The trypsin digests were analyzed on an LC-HRMS/MS instrument based on a previously published method. 21 An Ultimate 3000 RSLCnano system (Thermo Scientific Dionex Softron GmbH, Germany) coupled to an Orbitrap mass spectrometer (Q Exactive plus, Thermo Scientific, Bremen, Germany) was used. A volume of 10 μL was injected onto an Acclaim PepMap 100 C18 μ-precolumn (5 mm × 300 μm I.D., 5 μm, 100 Å, Thermo Fisher Scientific) and subsequently separated on an Acclaim PepMap C18 Analytical column (250 mm × 75 μm I.D., 2 μm, 100 Å, Thermo Fisher Scientific).…”

“…19 In such cases, biomedical samples may provide evidence for a longer period of time, where persistent biomarkers can be used for verication. 20,21 Nonetheless, it may be problematic to get access to human biological samples because of ethical, cultural and safety reasons. In addition, it might be difficult to link the victims to the actual exposure area.…”