Low level exposure to sulfur mustard: Development of a SOP for analysis of albumin adducts and of a system for non-invasive diagnosis on skin

In the second year of the grant period, the persistence of the various sulfur mustard adducts has been studied, in particular the adduct to the Cys-34 residue in albumin. After exposure of rats 0.3 mgkg, i.v., the tripeptide adduct S-HETECys-Pro-Tyr could be determined until 7 days after the exposure the observed half-life time of sulfur mustard - alkylated rat albumin was 2 days, which is in accordance with literature values. In the corresponding globin samples of these animals, the N-terminal valine adduct could still be determined after 28 days after the exposure. Remarkably, the maximum adduct level was reached after 2-3 days, implicating the presence of intact sulfur mustard in the animal during the first 2-3 days. With regard to the most abundant adduct, i.e., the histidine adduct, various derivatization methods have been explored and several fluorescent derivatives of the histidine adducts have been prepared in the first year of the grant period. In the second year of the grant period it turned out that the 56-carboxyfluoresceine FAM group gives the best results with regard to sensitivity. The FAM derivatives could be determined at a level of approximately 25 pgml. Results obtained thus far indicate that the albumin - tripeptide assay will become the method of choice for development into an SOP. A tentative Standard Operating Procedure has been drafted for this assay, that incorporates the earlier developed method for rapid isolation of albumin by affinity chromatography and the use of a deuterated internal standard. An immunoslotblot assay was developed for detection of sulfur mustard adducts to keratin in the first year of the agreement, enabling the detection of in vitro exposure of human callus to 0.2 muM sulfur mustard. Problems were encountered regarding the reproducibility of the assay when isolated keratin was used this is currently under investigation.