Medical Aspects of Chemical Warfare (2008) - The Black Vault

Medical DiagnosticsabcOCH 2CH 2SCH 2CH 2OHNHNH 2NNNNH 2NNNNCH 2 CH 2 SCH 2 CH 2 OHOCH 2 CH 2 SNHNH 2 NNN2Upon release, the adduct (in the form of TDG) wasderivatized and analyzed using negative-ion chemicalionization GC-MS (Figure 22-11; see Exhibit 22-3). Thelower limit of detection for the assay in plasma was 25nM, as determined using in-vitro exposures of sulfurmustard in human plasma. 118The limits of detection or equivalent levels of exposureto sulfur mustard reported for most of the bloodassays are based on in-vitro exposures of whole bloodor plasma. Quantitation of patient samples report theamount of sulfur mustard adducts that are found inthe samples relative to the amount of adducts thatare found from in-vitro exposures of whole bloodor plasma at various known concentrations of sulfurmustard. Choosing whole blood over plasma togenerate the in-vitro standard curves produces verydifferent results because sulfur mustard readily reactswith hemoglobin. Additionally, the technique usedfor generating in-vitro standards can have significanteffects. For example, approximately a 30% differencewas observed for the generation of two in-vitro standardcurves, depending on how the sulfur mustardwas incubated in blood. 119 Higher adduct levels wereobserved when the sulfur mustard was allowed toreact with the blood for 2 hours at 37°C, as opposedto 4 hours at room temperature.Application to Human ExposuredH 2 NNOCH 2 CH 2 SCH 2 CH 2 OHNFig. 22-9. Deoxyribonucleic acid adducts resulting fromreaction with sulfur mustard. (a) N7-HETE-guanine. (b)N3-HETE-adenine. (c) Bis[2-(guanin-7-yl)ethyl]sulfide. (d)O 6 -HETE-guanine.matography rather than the precipitation procedure. 116The modified procedure significantly reduced thesample preparation time.The final method for analyzing blood samples tobe discussed targets blood proteins in a more generalapproach. It was previously shown that sulfur mustardadducts of glutamic and aspartic acids to keratin couldbe cleaved using base. 117 Using a similar approach,precipitated proteins from plasma, whole blood, orRBCs were treated with base to liberate the sulfur mustardadduct, hydroxyethylthioethyl, from the protein.NNHBlood samples following a suspected human exposureto sulfur mustard rarely become available forlaboratory analysis. Three of the five known reportsinvolve the analysis of samples that were taken fromcasualties of the Iran-Iraq War, frozen for several years,then reanalyzed to verify exposure as new methodswere developed. The other two published reports areon the analysis of blood samples obtained from threeindividuals who were casualties of accidental exposuresto World War I munitions.The blood from two Iranian casualties who were believedto have been exposed to sulfur mustard in 1988was analyzed using both the ELISA method for DNAadducts and the GC-MS method for the analysis ofthe N-terminal valine of hemoglobin. 120 Samples werecollected 22 and 26 days following the suspected exposure.One of the casualties had skin injuries that wereconsistent with an exposure to sulfur mustard, butthe second casualty had injuries that were describedas only “vaguely compatible” with sulfur mustardexposure. Both individuals had approximately thesame level of hemoglobin valine adduct, equivalent tothe amount observed from a 900-nM, in-vitro, sulfurmustard exposure in whole blood. ELISA DNA adductlevels observed in the granulocytes were also similar721