Download Definitive Evidence for the Acute Sarin Poisoning

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). Application of gas chromatography–mass spectrometry and gas chromatogra-
toxa
AP: Tox
203
HIGHLIGHT
phy–tandem mass spectrometry to the analysis of chemical warfare samples, found to contain residues of the nerve agent sarin, sulphur mustard
and their degradation products. J. Chromatogr. A 662, 301–321.
D’Agostino, P. A., and Provost, R. (1992). Determination of chemical warfare agents, their hydrolysis products and related compounds in soil. J.
Chromatogr. 589, 287–294.
Dutta-Choudhury, T. A., and Rosenberry, T. L. (1984). Human erythrocyte
acetylcholinesterase is an amphipathic protein whose short membranebinding domain is removed by papain digestion. J. Biol. Chem. 259,
5653–5660.
Ellman, G. L., Courtney, K. D., Andres, Jr., V., and Featherstone, R. M.
(1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95.
Gazzard, M. F., and Thomas, D. P. (1975). A comparative study of central
visual field changes induced by sarin vapor and physostigmine eye drops.
Exp. Eye Res. 20, 15–21.
Hanahan, D. J., and Ekholm, J. E. (1974). The preparation of red cell ghosts
(membrane) In Methods in Enzymology (S. Fleischer, L. Packer, and
P. W. Estabrook, Eds.), Vol. 31, pp. 68–72. Academic Press, New York.
Klaassen, C. D., and Rozman, K. (1991). Absorption, distribution, and excretion of toxicans. In Casarett and Doull’s Toxicology The Basic Science
of Poisons (M. O. Amdur, J. Doull, and C. D. Klaassen, Eds.), 4th ed.,
pp. 50–87. Pergamon Press, New York.
Konno, N., Suzuki, N., Horiguchi, H., and Fukushima, M. (1994). Characterization of high-affinity binding sites for diisopropylfluorophosphate
(DFP) from chicken spinal cord membranes. Biochem. Phamacol. 48,
2073–2079.
Masuda, N., Takatsu, M., Morinari, H., and Ozawa, T. (1995). Sarin poisoning in Tokyo subway. Lancet 345, 1446.
Nozaki, H., and Aikawa, N. (1995). Sarin poisoning in Tokyo subway.
Lancet 345, 1446–1447.
Ott, P., and Brodbeck, U. (1978). Multiple molecular forms of acetylcholinesterase from human erythrocyte membranes: Interconversion and subunit
AID
TOX 8110
/
6h17$$$123
04-16-97 16:45:55
composition of forms separated by density gradient centrifugation in a
zonal rotor. Eur. J. Biochem. 88, 119–125.
Pope, C. N., and Padilla, S. S. (1989). Chromatographic characterization of
neurotoxic esterase. Biochem. Pharmacol. 38, 181–188.
Rengstroff, R. H. (1989). Accidental exposure to sarin: vision effect. Arch.
Toxicol. 56, 201–203.
Rengstroff, R. H. (1994). Vision and ocular changes following accidental
exposure to organophosphates. J. Appl. Toxicol. 14, 115–118.
Roberts, W. L., Kim, B. H., and Rosenberry, T. L. (1987). Differences in
the glycolipid membrane anchore of bovine and human erythrocyte acetylcholinesterases. Proc. Natl. Acad. Sci. USA 84, 7817–7821.
Roberts, W. L., Myher, J. J., Kuksis, A., Low, M. G., and Rosenberry,
T. L. (1988a). Lipid analysis of the glycoinositol phospholipid membrane
anchor of human erythrocyte acetylcholinesterase: Palmitoylation of inositol results in resistance to phosphatidylinositol-specific phospholipase
C. J. Biol. Chem. 263, 18766–18775.
Roberts, W. L., Santikarn, S., Reinhold, V. N., and Rosenberry, T. L.
(1988b). Structural characterization of the glycoinositol phospholipid
membrane anchor of human erythrocyte acetylcholinesterase by fast atom
bombardment mass spectrometry. J. Biol. Chem. 263, 18776–18784.
Soreq, H., Ben-Aziz, R., Prody, C. A., Seidman, S., Gnatt, A., Neville, L.,
Lieman-Hurwitz, J., Lev-Lehman, E., Ginzberg, D., Lapidot-Lifson, Y.,
and Zakut, H. (1990). Molecular cloning and construction of the coding
region for human acetylcholinesterase reveals a G/C-rich attenuating
structure. Proc. Natl. Acad. Sci. USA 87, 9688–9692.
Sussman, J. L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L.,
and Silman, I. (1991). Atomic structure of acetylcholinesterase from
Torpedo californica: A prototypic acetylcholine-binding protein. Science
253, 872–879.
Suzuki, T., Morita, H., Ono, K., Maekawa, K., Nagai, R., and Yazaki, Y.
(1995). Sarin poisoning in Tokyo subway. Lancet 345, 980–981.
Wright, D. L., and Plummer, D. T. (1972). Solubilization of acetylcholinesterase from human erythrocytes by triton X-100 in potassium chloride
solution. Biochim. Biophys. Acta 261, 398–401.
toxa
AP: Tox