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

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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
Medical Aspects of Chemical WarfareEXHIBIT 22-2SAMPLE PREPARATION PROCEDURE FOR SULFUR MUSTARD ADDUCTS TO deoxyribonucleicacid IN BLOODProcedure of van der Schans et al for immunuslotblot analysis:• Collect blood specimen in vacutube containing EDTA.• Isolate and denature DNA using following procedure:- Transfer 0.3 mL of blood to Eppendorf tube.- Add RBC lysis solution, mix, and centrifuge.- Lyse pelleted WBCs with cell lysis solution containing proteinase K.- Shake solution for 1 hour.- Treat solution with RNAse.- Add protein precipitation solution, mix, and centrifuge.- Transfer supernatant to tube containing isopropanol and centrifuge.- Wash pellet with 70% ethanol, centrifuge, and air dry.- Dissolve pellet overnight in Tris buffer containing HCl and EDTA.- Determine DNA concentration using UV-VIS spectrometer.- Prepare DNA solution with formamide, formaldehyde, and Tris buffer.- Incubate solution at 52°C.- Cool rapidly on ice and store at – 20°C.• Immunuslotblot assay procedure:- Dilute denatured DNA samples in PBS.- Spot DNA solution onto nitrocellulose filter.- Wash spotted sample with PBS and air-dry filter.- Cross-link DNA to filter using UV-gene-cross-linker.- Incubate filter with blocking solution.- Wash with PBS/Tween solution.- Incubate filter overnight with monoclonal antibodies that recognize N7-(2-hydroxyethylthioethyl)-2’-deoxyguanosine at 4°C with continuous shaking.- Wash with PBS/Tween solution.- Incubate filter with rabbit-anti-mouse-Ig-horseradish peroxidase antibody for 2 hours at room temperaturewith shaking.- Wash with PBS/Tween solution.- Blot dry filter and transfer into a luminometer cassette.- Measure luminescence.DNA: deoxyribonucleic acidEDTA: ethylenediaminetetraacetic acidHCl: hydrochloric acidIg: immunoglobulinPBS: phosphate-buffered salineRBC: red blood cellRNAse: ribonucleaseTris: trishydroxymethylaminomethaneUV-VIS: ultraviolet-visible spectrophotometryWBC: white blood cellData source: (1) Van der Schans GP, Mars-Groenendijk R, de Jong LP, Benschop HP, Noort D. Standard operating procedure for immunoslotblotassay for analysis of DNA/sulfur mustard adducts in human blood and skin. J Anal Toxicol. 2004;28:316–319.722
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The Coat of Arms1818Medical Departm
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Textbooks of Military MedicinePubli
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MEDICAL ASPECTS o fCHEMICAL WARFARE
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ContentsContributorsForeword by The
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ContributorsMICHAEL ADLER, PhDResea
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DONALD M. MAXWELL, MSResearch Chemi
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ForewordThe US military has been co
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PrefaceA significant concern for th
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PrologueThe original edition of Med
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xxii
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Medical Aspects of Chemical Warfare
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Nerve AgentsChapter 5NERVE AGENTSFR
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Nerve AgentsAbout 10,000 to 30,000
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Nerve Agentsphorofluoridate (DFP) w
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Nerve AgentsFig. 5-2. This schemati
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Nerve AgentsExhibit 5-1 continuedis
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Nerve Agentsactivity but only 15% t
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Nerve AgentsTABLE 5-3CHEMICAL, PHYS
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Nerve AgentsTABLE 5-4EFFECTS OF EXP
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Nerve Agentsaffecting the other eye
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Nerve Agentsmay cause severe rhinor
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Nerve Agentsof jerks and tremor.Cen
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Nerve Agentsand they were decreased
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Nerve Agentsnerve agent toxicity on
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Nerve Agentspatient. Decontaminatio
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Nerve AgentsFig. 5-5. The Mark I ki
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Nerve Agentsadministered intramuscu
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Nerve Agentsthat hydroxamine reacti
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Nerve AgentsOral administration may
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Nerve AgentsTABLE 5-7RECOMMENDED TH
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Nerve Agentsimpairment such as whee
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Nerve Agentssuboptimal, manner. The
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Nerve Agentsthan those who did not.
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Nerve Agentssible for binding nerve
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Nerve Agentsfor comparison was not
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Nerve AgentsTABLE 5-11EFFECTS OF PY
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Nerve Agentstivity of smooth muscle
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Nerve Agents38. Grob D, Lilienthal
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Nerve Agents77. McDonough JH, Shih
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Nerve Agents117. McLeod CG Jr, Sing
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Nerve Agents154. Tryphonas l, Veino
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Nerve Agents193. Funckes AJ. Treatm
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Nerve Agents235. Suzuki T, Morita H
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Nerve Agents274. Pellegrini JE, Bak
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PROCESSMedical Aspects of Chemical
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Triage of Chemical CasualtiesChapte
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Triage of Chemical Casualtiescatego
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Triage of Chemical CasualtiesIn a f
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Triage of Chemical Casualtiesnose)
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Triage of Chemical CasualtiesWith n
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Triage of Chemical Casualties14. Sa
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Decontamination of Chemical Casualt
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Abbreviations and AcronymsAbBreviat
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Abbreviations and AcronymsPADPRP: p
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Medical Aspects of Chemical Warfare (2008) - The Black Vault

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