Diagnosis and dosimetry of exposure to sulfur mustard: Development of a standard operating procedure for hemoglobin adducts: Exploratory research on albumin and keratin adducts

A standard operating procedure (SOP) for determination of the sulfur mustard adduct to the N-terminal valine in hemoglobin was developed. By using this SOP, it was found that the Nterminal valine adduct in globin of hairless guinea pigs and marmosets which had been exposed to sulfur mustard (0.5 LD50) is persistent for at least 56 and 94 days, respectively. The SOP could be properly set up and carried out at another institute (MRICD) within one working day.
Upon pronase digestion of albumin from human blood exposed to sulfur mustard, the major adduct fragment was identified by LC-tandem MS analysis as the tripeptide (S-2-hydroxyethylthioethyl)Cys-Pro-Phe, arising from alkylation at cysteine-34. An LC-tandem MS method was developed for analysis of this peptide, which enabled us to detect exposure to ≥ 10 nM of sulfur mustard, using only 3 mg of albumin. The method was successfully applied to analysis of blood samples of Iranian casualties from the Iran-Iraq conflict. Presently, this is by far the most sensitive marker for exposure of human blood to sulfur mustard.
Upon incubation with alkali (pH 13) of keratin isolated from callus which had been exposed to [14C]sulfur mustard, 80% of the bound radioactivity was split off as [14C]thiodiglycol. This procedure could be used for GC-MS analysis of thiodiglycol (after derivatization with pentafluorobenzoyl chloride) that was released by alkaline treatment from keratin, isolated from human skin which had been exposed to saturated sulfur mustard vapor for ≥ 2 min.
This work was supported by the U.S. Army Medical Research and Materiel Command under Cooperative Agreement DAMD17-97-2-7002 and by the Directorate of Military Medical Services of the Ministry of Defense, The Hague, The Netherlands.