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TOXICOLOGY AND APPLIED PHARMACOLOGY ARTICLE NO. 144, 198–203 (1997) TO978110 HIGHLIGHT Definitive Evidence for the Acute Sarin Poisoning Diagnosis in the Tokyo Subway MASATAKA NAGAO, TAKEHIKO TAKATORI, YUKIMASA MATSUDA, MAKOTO NAKAJIMA, HIROTARO IWASE, AND KIMIHARU IWADATE Faculty of Medicine, Department of Forensic Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan Received December 10, 1996; accepted January 9, 1997 Definitive Evidence for the Acute Sarin Poisoning Diagnosis in the Tokyo Subway. NAGAO, M., TAKATORI, T., MATSUDA, Y., NAKAJIMA, M., IWASE, H., AND IWADATE, K. (1997). Toxicol. Appl. Pharmacol. 144, 198–203. A new method was developed to detect sarin hydrolysis products from erythrocytes of four victims of sarin (isopropylmethylphosphonofluoridate) poisoning resulting from the terrorist attack on the Tokyo subway. Sarin-bound acetylcholinesterase (AChE) was solubilized from erythrocyte membranes of sarin victims, digested with trypsin, the sarin hydrolysis products bound to AChE were released by alkaline phosphatase digestion, and the digested sarin hydrolysis products were subjected to trimethylsilyl derivatization and detected by gas chromatography – mass spectrometry. Isopropylmethylphosphonic acid, which is a sarin hydrolysis product, was detected in all sarin poisoning, victims we examined and methylphosphonic acid, which is a sarin and soman hydrolysis product, was determined in all victims. Postmortem examinations revealed no macroscopic and microscopic findings specific to sarin poisoning and sarin and its hydrolysis products were almost undetectable in their blood. We think that the procedure described below will be useful for the forensic diagnosis of acute sarin poisoning. the sera of almost all victims, and isopropylmethylphosphonic acid was not in any samples (data not shown). Therefore, we could not definitely diagnose the cause of death of these victims at this stage. Sarin binds to a hydroxyl group of serine in the active site of the acethylcholinesterase (AChE) molecule and inhibits the enzyme activity strongly, after which, its isopropyl ester is hydrolyzed—a phenomenon called aging. Finally, methylphosphonic acid, which is conjugated to the serine residue in the enzyme molecule, remains, and each AChE molecule bound to sarin contains one molecule of isopropylmethylphosphonoserine or methylphosphonoserine. If isopropylmethylphosphonic and/or methylphosphonic acids conjugated to AChE in the blood and/or tissues of sarin victims are detected, this provides strong evidence of sarin poisoning. In this paper, we describe the development of a new method to detect the sarin hydrolysis products from the erythrocytes of 4 sarin victims of the terrorist attack on the Tokyo subway and describe the diagnosis of the cause of death as acute sarin poisoning. CASE PROFILE q 1997 Academic Press On March 20, 1995, the Tokyo subway system was subjected to a horrifying terrorist attack with sarin gas (isopropylmethylphosphonofluoridate) that left 12 persons dead and over 5000 injured (Suzuki et al., 1995; Masuda et al., 1995; Nozaki et al., 1995). Sarin is a highly toxic organophosphorus agent, and it is easily hydrolyzed especially under alkaline conditions. It is usually difficult to detect sarin itself and/or one of its hydrolysis products, isopropylmethylphosphonic acid, in the blood of sarin victims. We performed judicial autopsies on 4 sarin victims within a few days of the attack. No macroscopic and microscopic findings specific to sarin poisoning were observed in these 4 acute sarin poisoning victims. Methylphosphonic acid was not detected in 0041-008X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved. AID TOX 8110 / 6h17$$$121 The profiles of the victims we examined are summarized in Table 1. Cases 1 and 2 were killed instantly before they could be treated clinically, whereas Cases 3 and 4 were hospitalized and given pralidoxime chloride (2-PAM). When the victims arrived at the hospitals, they all had pinpoint pupils, which disappeared by the time autopsy was performed, and no findings specific to sarin poisoning were observed. The plasma cholinesterase activities of Cases 2, 3, and 4 were examined and found to be extremely low compared with normal levels. METHODS AChE activities in the brain cortices and blood of sarin victims. AChE activity was measured according to the method described by Ellman et al. 198 04-16-97 16:45:55 toxa AP: Tox 199 HIGHLIGHT TABLE 1 Profiles of the Victims of the Terrorist Attack with Sarin on the Tokyo Subway Case Age Sex Plasma ChE activity (normal range) Dosage of 2-PAM Time of death after poisoning 1 2 3 4 29 50 50 64 Male Male Male Male 0.03 D pH (0.70 Ç 1.20) NTa 21 mIU (185 Ç 431) 5 mIU (185 Ç 431) 0 0 / / Instant death Instant death About 20 hours About 2 days a NT, not tested. (1961) and expressed as percentages of the control average. The cortex from external part of the frontal lobe and the whole blood of the sarin victims were used as brain and blood samples, respectively. Brain tissue and blood from males dying from other causes were used as controls (20– 78 years old). Sample preparation. Red blood cell ghosts from sarin poisoning victims were prepared (Hanahan et al., 1974). Briefly, 3 ml hemolyzed blood was mixed with 40 ml 10 mM Tris–HCl buffer (pH 7.4) and centrifuged at 20,000g for 40 min at 47C. The resulting pellet was resuspended in this buffer and centrifuged again at 20,000g for 40 min at 47C. This procedure was carried out a total of four times. The isolated red cell ghosts were resuspended in 40 ml Tris–HCl buffer containing 1% w/v 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps, Pope and Padilla, 1989; Konno et al., 1994), incubated for 30 min at 47C, and then centrifuged at 100,000g for 1 hr at 47C (Wright and Plummer, 1972). The supernatant was collected and over 90% of the acetylcholinesterase in red blood cells was present. The supernatants were concentrated by ultrafiltration using Centriplus 30 (Amicon, Inc., Beverly, MA) and the resulting solubilized proteins were digested with trypsin (20 mg/mg protein) at 377C for 24 hr, after which the digested peptide solutions were alkalinized by adding 1 M Tris–HCl buffer (pH 10.0) and incubated with bovine intestinal alkaline phosphatase (3 U/mg protein) at 377C for 48 hr. The high-molecular-weight peptide digestion products were removed by ultrafiltration using Centriplus 3 (Amicon, Inc.), the water was evaporated, and the residue was dissolved in a small volume of pyridine. An aliquot of this pyridine solution was mixed with an equal volume of the trimethylsilylation (TMS) agent (bistrimethylsilyl)trifluoroacetamide (containing 1% v/v trimethylchlorosilane) and left for 60 min at room temperature (D’Agostino and Provost, 1992). Gas chromatography–mass spectrometry (GC–MS) condition. TMSderivatized specimens were subjected to gas chromatography using a Hewlett Packard gas chromatograph 5890B equipped with a capillary column (length, 30 m; diameter, 0.25 mm i.d.; film thickness, 0.25 mm) HP-5MS, with helium at 10.9 psi, 1.0 ml/min as the carrier gas. The oven was heated to 407C for 2 min, from 40 to 1507C at 87C/min, from 150 to 2807C at 157C/min and held at 2807C for 10 min. GC–MS analysis was carried out using a Hewlett Packard mass spectrometer 5889. One-microliter samples were injected in the splitless mode. The electron impact MS operating conditions were electron energy, 70 eV and source temperature, 2007C, and the methane chemical impact MS operating conditions were electron energy, 230 eV and source temperature, 2007C. RESULTS Inhibitory Effects of Sarin on Blood and Brain AChE Activities Figure 1 shows the AChE activities in the brain cortices and blood of the victims. AChE activities in the control brain AID TOX 8110 / 6h17$$$122 04-16-97 16:45:55 cortices and blood were 110.0 { 8.1 mU/g wet tissue and 5.00 { 1.20 U/ml blood, respectively. The AChE activities in the brains of all the victims were lower than those in the controls. The AChE activities in the blood of Cases 1 and 2 were considerably lower, whereas those of Cases 3 and 4 were only slightly lower than the control values. GC–MS Analyses Figure 2 shows the total ion chromatogram (TIC) obtained for Case 1 and Fig. 3 shows the mass spectrum of the peak observed at about 10.850 min on Fig. 2. Figure 3a is the electron impact–mass spectrum (EI–MS) and Fig. 3b is the chemical impact–mass spectrum (CI–MS). The peaks at m/ z 209 and m/z 153 were observed in the EI–MS and peaks at m/z 211 and m/z 153 were present in the CI–MS. Figure 4 shows the mass spectra of the peak observed at about 11.390 min on Fig. 2. Figures 4a and 4b are the EI–MS and the CI–MS, respectively. In the EI–MS, the peaks at m/z 240 and m/z 225 were observed and peaks at m/z 241 and m/z 269 were present in the CI–MS. The EI–MS of the peaks at about 10.850 and 11.390 min in the TMS-derivatized TICs from Cases 1–4 contained m/ z 153, m/z 240, and m/z 225 fragments and the CI–MS contained m/z 211, m/z 153, and m/z 241 fragments. DISCUSSION Cases 3 and 4 received 2-PAM in the hospital and their plasma cholinesterase activities before 2-PAM administration were extremely low. Therefore, 2-PAM administration reversed the plasma cholinesterase inhibition. However, 2PAM cannot penetrate the blood–brain barrier (Klaassen and Rozman, 1991), which accounts for the low AChE activities in the brains of these two victims. Extreme depression of AChE activity is one of the symptoms of poisoning with organophosphorus agents and is not specific to acute sarin poisoning. Our preliminary experiments on AChE activities in several human organs and blood showed that the AChE activity in erythrocytes was the highest (data not shown). Therefore, toxa AP: Tox 200 HIGHLIGHT FIG. 1. Acetylcholinesterase (AChE) activities in the brain cortices and blood of sarin poisoning victims. in this study, we used erythrocytes from sarin victims in an attempt to detect sarin hydrolysis products. In blood, amphipathic AChE dimers of globular form (G2 , Otto and Brodbeck, 1978; Dutta-Choudhury and Rosenberry, 1984) are anchored to the plasma membrane by a covalently attached glycoinositol phospholipid (Roberts et al., 1987, 1988a,b). In order to purify AChE from blood cells, red blood cell ghosts were prepared from sarin poisoning victims and then AChE was solubilized from the ghosts with the nondenaturing detergent Chaps. In order to separate isopropylmethylphosphonic and/or methylphosphonic acids from solubilized AChE, enzymatic digestion with alkaline phosphatase, which can hydrolyze both phosphoric and methylphosphonic esters (data not shown), was performed. However, the active site of the AChE molecule lies at the base of a narrow gorge 20 angstroms deep (Sussman et al., 1991), which prevents alkaline phosphatase coming into contact with isopropylmethylphosphonoserine and/or methylphosphonoserine at the active site, necessitating some conformational modification of the molecular structure of AChE. In view of the amino acid sequence of human AChE (Soreq et al., 1990), the active site of AChE could be degraded to a 41-residue peptide (about 4.1 kDa) by trypsin and alkaline phosphatase digestion of FIG. 2. Total ion chromatogram for the TMS derivatives of substances digested from the red cells of Case 1. AID TOX 8110 / 6h17$$$122 04-16-97 16:45:55 toxa AP: Tox 201 HIGHLIGHT FIG. 3. EI–MS (a) and CI–MS (b) of the peak observed at about 10.850 min on Fig. 2. solubilized AChE after trypsin digestion was found to be very useful for releasing the sarin hydrolysis products from AChE. After the hydrolysis products had been released, the high-molecular-weight peptide digestion products were removed by ultrafiltration and the water was evaporated. Next, the residues were subjected to TMS derivatization and the derivatives were analyzed by GC–MS. In the chromatograms of the TMS derivative of an authentic isopropylmethylphosphonic acid, the total ion chromatogram has one sharp peak at about 10.850 min and the EI–MS of this sharp peak shows peaks at m/z 209, which is due to [M 0 H]/, and m/ z 153. In the CI–MS, the peaks of m/z 211, m/z 239, and m/z 251, which are due to [M / H]/, [M / C2H5]/, and [M / C3H5]/, respectively, and the peak of m/z 153 were observed. In the chromatograms of the TMS derivative of AID TOX 8110 / 6h17$$$122 04-16-97 16:45:55 an authentic methylphosphonic acid, the total ion chromatogram has one sharp peak at about 11.390 min and the EI– MS of this sharp peak shows peaks at m/z 240, the parent peak, and m/z 225. In the CI–MS, the peaks of m/z 241, m/ z 269, and m/z 281, which are due to [M / H]/, [M / C2H5]/, and [M / C3H5]/, respectively, were observed. Compared with the fragment patterns of the EI–MS and CI–MS of the authentic isopropylmethylphosphonic and methylphosphonic acids, the parent peak and other prominent peaks were also present in the chromatograms of the TMS derivatives obtained from the red blood cells of Case 1 shown in Figs. 3 and 4, respectively. The retention times of these substances shown in Figs. 3 and 4 were almost the same as those of the TMS derivative of the authentic isopropylmethylphosphonic and methylphosphonic acids, re- toxa AP: Tox 202 HIGHLIGHT FIG. 4. EI–MS (a) and CI–MS (b) of the peak observed at about 11.390 min on Fig. 2. spectively, confirming that the former fragments were from the TMS derivative of isopropylmethylphosphonic acid and the latter ones were from that of methylphosphonic acid. The EI–MS and CI–MS patterns of the TMS derivatives from Cases 2, 3, and 4 were also similar to those of the TMS derivatives of isopropylmethylphosphonic and methylphosphonic acids. In normal red blood cells, however, no isopropylmethylphosphonic and/or methylphosphonic acids have been detected after the same procedures as those for the sarin poisoning victims were used. These results prove that isopropylmethylphosphonic acid, a sarin hydrolysis product, and methylphosphonic acid, another sarin hydrolysis product, bound to AChE in the blood of all sarin poisoning victims were found. We believe that the cause of death of all victims was acute sarin poisoning. Although there are AID TOX 8110 / 6h17$$$123 04-16-97 16:45:55 some reports on human exposure to sarin, the victims were all military personnel whose symptoms were mild (Gazzard and Thomas, 1975; Rengstroff, 1989; Rengstroff, 1994). Recently, the residues of chemical warfare agents and their degradation products were detected in soil samples from a Kurdish village (Black et al., 1994). We believe, however, that this is the first report of a new method of detecting isopropylmethylphosphonic and methylphosphonic acids, sarin hydrolysis products, in AChE from the erythrocytes of sarin-poisoning victims of terrorism. We believe this procedure will be useful in the forensic diagnosis of acute sarin poisoning. REFERENCES Black, R. M., Clarke, R. J., Read, R. W., and Reid, M. T. J. (1994). 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