Hemoglobin sulfur mustard adducts

Figure 4. Persistence of sulfur mustard adduct to N-terminal valine residue of hemoglobin in blood of a marmoset after sulfur mustard administration (4.1 mg/kg, i.v.) at t = 0. At the time points indicated blood samples were collected, globin was isolated and analyzed by using the modified Edman degradation for determination of the N-terminal valine adduct. Globin from human blood exposed to ris-sulfur mustard (10 iM) was used as an internal standard. (Reprinted from Toxicology and Applied Pharmacology, Vol. 184, D. Noort, H.P. Benschop and R.M. Black, Biomonitoring of Exposure to Chemical Warfare Agents A Review, pages 116-126 (2002), with permission from Elsevier Science.)...

Noort and colleagues (2008) investigated the persistence of sulfur mustard adducts to albumin and hemoglobin in rats. The albumin adduct (S -2-hydroxyethylthioethyl)-Cys-Pro-Tyr was detectable up to 7 days after the exposure, while the adduct to the N-terminal valine in hemoglobin was still detected after 28 days. The decrease of adduct concentration corresponded with albumin half-life and the hfetime of the rat erythrocyte, respectively. 

Analysis of Blood Samples. Urinary metabolites undergo relatively rapid elimination from the body, whereas blood components offer biomarkers that have the potential to be used for verification of sulfur mustard exposure long after the exposure incident. Three different approaches have been used for blood biomarker analysis. The intact macromolecule such as protein or DNA with the sulfur mustard adducts still attached can be analyzed. To date, this approach has only been demonstrated for hemoglobin using in vitro experiments. For proteins, an alternate approach is to enzymatically digest them to produce a smaller peptide with the sulfur mustard adduct still attached. Methods of this type have been developed for both hemoglobin and albumin. A third approach has been to cleave the sulfur mustard adduct from the macromolecule and analyze in a fashion similar to that used for free metabolites found in the urine. The later two approaches have both been successfully used to verify human exposure of sulfur mustard. 

Sulfur mustard forms adducts with the blood proteins hemoglobin and albumin. Adducts with histidine residues are the most abundant after exposure of hemoglobin in vitro to sulfur mustard. Analysis of adducted histidine by GC/MS is hampered by poor thermal stability of volatile derivatives. A sensitive method was developed using LC/ESI/MS/MS after... 

Recently, matrix-assisted laser desorption ioniza-tion/time-of-flight/mass spectrometry (MALDI/TOF/ MS) of intact adducted hemoglobin was explored as a diagnostic tool for the confirmation of exposure to sulfur mustard (23). Multiple alkylated species were observed from incubates of hemoglobin with sulfur mustard however, the methodology has not yet been reported for diagnostic purposes. 

A. Fidder, D. Noort, A.L. De Jong, H.C. Trap, L.P.A. De Jong, H.P. Benschop, Monitoring of in vitro and in vivo exposure to sulfur mustard by GC/MS determination of the N-terminal valine adduct in hemoglobin after a modified Edman degradation, Chem. Res. Toxicol, 9, 788-792 (1996). 

Noort, D., Fidder, A., Degenhardt-Langelaan, C.E., Hulst, A.G. (2008). Retrospective detection of sulfur mustard exposure by mass spectrometric analysis of adducts to albumin and hemoglobin an in vivo study. J. Anal Toxicol. 32 25-30. 

The blood from two Iranian casualties that were believed to have been exposed to sulfur mustard in 1988 was analyzed using both the ELISA method for DNA adducts and the GC-MS method for the analysis of the N-terminal valine of hemoglobin (Benschop et al., 1997). Samples were collected 22 and 26 days following the suspected exposure to sulfur mustard. One of the casualties had injuries to the skin that were consistent with an exposure to sulfur mustard, but the second casualty had injuries that were described as only vaguely compatible with sulfur mustard exposure. Both individuals had approximately the same level of hemoglobin valine adduct that was equivalent to the amount observed from a 900 nM in vitro sulfur mustard exposure in whole blood. ELISA DNA adduct levels observed in the granulocytes were also similar for both individuals, 150-160 nM. The individual with the skin injuries consistent with sulfur mustard exposure had observed ELISA DNA adduct levels in the lymphocytes that were only about half that observed in the individual with injuries that were less pronounced, 220 and 430 nM, respectively. 

Black, R.M., Clarke, R.J., Harrison, J.M., and Read, R.W., Biological fate of sulfur mustard Identification of valine and histidine adducts in hemoglobin from casualties of sulphur mustard poisoning, Xenofa ofica, 27(5), 499, 1997. 

The reported findings are in reasonable agreement with those of Benschop et al (1997), who detected the Ai -adduct to guanine and the N-terminal valine adduct of hemoglobin in the blood of two Iranian victims 22-26 days after the alleged exposure. One of the victims showed clear signs of sulfur mustard exposure, ie. skin injuries but no respiratory effects, whereas the other victim showed symptoms that were only vaguely compatible with sulfur mustard. 

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