Identification of the reactive glutathione conjugate S-(2-chloroethyl)glutathione in the bile of 1-bromo-2-chloroethane-treated rats by high-pressure liquid chromatography and precolumn derivatization with o-phthalaldehyde.

The conjugation of glutathione with 1,2-dihaloethanes leads to the formation of S-(2-haloethyl)glutathione which, following intramolecular cyclization, produces an electrophilic thiiranium ion. The extent to which the formation of the thiiranium ion is responsible for the toxicity associated with 1,2-dihaloethanes has been difficult to determine because of the inherent instability of the compound under physiological conditions. The goal of this study was to attempt to identify a putative precursor of the thiiranium ion, S-(2-chloroethyl)glutathione (CEG), in the bile of rats treated with 1,2-dihaloethanes such as 1-bromo-2-chloroethane (BCE). In order to detect the presence of CEG, a precolumn procedure for derivatizing the amine of CEG with o-phthalaldehyde/2-mercaptoethanol (OPA/MCE) was developed. Studies with a model compound, S-ethylglutathione, indicated that the derivatization reaction between S-ethylglutathione and OPA/MCE proceeded rapidly and under mild conditions. The resulting fluorescent adduct of S-ethylglutathione was detected at low concentrations following separation by reverse-phase HPLC. Derivatization of CEG with OPA/MCE followed by preparative HPLC and mass spectral analysis revealed that the major fluorescent adduct in the reaction mixture was the expected 1-[(2-hydroxyethyl)thio]-2-substituted-isoindole derivative of CEG. Also present in the derivatization reaction mixture were small quantities of S-(2-hydroxyethyl)glutathione, the product of CEG hydrolysis, and a product involving the addition of MCE to CEG. Analysis of the bile samples obtained from bile-cannulated rats treated with BCE showed the presence of a peak corresponding to CEG. Over a 3-h interval, 2% of the BCE administered was excreted into the bile as CEG.