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Medical Diagnostics
Chapter 22
Benedict R. capacio, p h d * ; J. RichaRd Smith ; RichaRd K. GoRdon, p h d ; Julian R. haiGh, p h d § ; John
R. BaRR, p h d ¥ ; a n d Gennady e. platoff J r , p h d
* Chief, Medical Diagnostic and Chemical Branch, Analytical Toxicology Division, US Army Medical Research Institute of Chemical Defense, 3100
Rickets Point Road, Aberdeen Proving Ground, Maryland 21010-5400
Chemist, Medical Diagnostic and Chemical Branch, Analytical Toxicology Division, US Army Medical Research Institute of Chemical Defense, 3100
Rickets Point Road, Aberdeen Proving Ground, Maryland 21010-5400
Chief, Department of Biochemical Pharmacology, Biochemistry Division, Walter Reed Army Institute of Research, 503 Robert Grant Road, Silver Spring,
Maryland 20910-7500
§ Research Scientist, Department of Biochemical Pharmacology, Biochemistry Division, Walter Reed Army Institute of Research, 503 Robert Grant Road,
Silver Spring, Maryland 20910-7500
¥ Lead Research Chemist, Centers for Disease Control and Prevention, 4770 Buford Highway, Mailstop F47, Atlanta, Georgia 30341
Colonel, US Army (Retired); Scientific Advisor, Office of Biodefense Research, National Institute of Allergies and Infectious Disease, National Institutes
of Health, 6610 Rockledge Drive, Room 4069, Bethesda, Maryland 20892-6612
Medical Aspects of Chemical Warfare
in the past, issues associated with chemical war-
fare agents, including developing and implementing
medical countermeasures, field detection, verifica-
tion of human exposures, triage, and treatment, have
primarily been a concern of the military community
because most prior experience with chemical war-
fare agents was limited to the battlefield. however,
chemical agents have been increasingly employed
against civilian populations, such as in iraqi attacks
against the Kurds and the attacks organized by the
aum Shinrikyo cult in matsumoto city and the tokyo
subway. the attacks on the World trade center in
new york city and the pentagon in Washington, dc,
in 2001 have increased concern about the potential
large-scale use of chemical warfare agents in a civilian
sector. incidents involving large numbers of civilians
have shown that to facilitate appropriate treatment, it
is critical to identify not only those exposed, but those
who have not been exposed, as well. in addition to
health issues associated with exposure, the political
and legal ramifications of a chemical warfare attack
can be enormous. it is therefore essential that testing
for exposure be accurate, sensitive, and rapid.
for the most part, monitoring for the presence of
chemical warfare agents in humans, or “biomonitor-
ing,” involves examining specimens to determine if
an exposure has occurred. assays that provide de-
finitive evidence of agent exposure commonly target
metabolites, such as hydrolysis products and adducts
formed following binding to biomolecular entities.
unlike drug efficacy studies in which blood/plasma
levels usually focus on the parent compounds, assay
techniques for verifying chemical agent exposure
rarely target the intact agent because of its limited
longevity in vivo. following exposure, many agents
are rapidly converted and appear in the blood as hy-
drolysis products (resulting from reactions with water)
and are excreted in urine. Because of rapid formation
and subsequent urinary excretion, the use of these
products as markers provides a limited window of op-
portunity to collect a sample with measurable product.
more long-lived markers tend to be those that result
from agent interactions with large-molecular–weight
targets, such as proteins and dna. these result from
covalent binding of the agent or agent moiety to form
macromolecular adducts. as such, the protein acts as
a depot for the adducted agent and the residence time
is similar to the half-life of the target molecule.
Blood/blood components, urine, and, in rare in-
stances, tissue specimens, termed “sample matrices” or
“biomedical samples,” can be used to verify chemical
agent exposure. however, any sample obtained from
an exposed individual may be considered as a potential
matrix (eg, blister fluid from sulfur mustard [north
atlantic treaty organization (nato) designation:
hd] vesication). Regardless, analyses of these types of
samples are inherently difficult because of the matrix’s
complex composition and the presence of analyte in
trace quantities.
noninvasive urine collection does not require highly
trained medical personnel or specialized equipment.
although biomarkers present in urine are usually short-
lived (hours to days) metabolites, they can be present
in relatively high concentrations in samples obtained
shortly after exposure. the collection of blood/plasma
should be performed by trained medical personnel.
Blood/plasma samples offer potential benefits because
both metabolites and the more long-lived adducts of a
macromolecular target can be assayed. the effective-
ness of using tissue to verify chemical agent exposure is
generally limited to postmortem sampling. for example,
formalin-fixed brain tissues from fatalities of the tokyo
subway attack were successfully used to verify sarin
(nato designation: GB) as the agent employed in that
attack. 1 other tissue samples can be obtained from the
carcasses of animals at the incident site.
methods that do not directly analyze cholinesterase
(che) activity typically involve detection systems like
mass spectrometry (mS) combined with either gas
chromatography (Gc) or liquid chromatography (lc)
to separate the analyte from other matrix components.
mS detection methods are based upon specific and
characteristic fragmentation patterns of the parent
molecule, making mS detection desirable because it
identifies the analyte fairly reliably. other detection
systems, such as nitrogen-phosphorus detection and
flame photometric detection, have also been used.
Some analytes can be directly assessed, whereas others
may require chemical modification (eg, derivatization)
to enhance detection or make them more volatile in
Gc separations. more sophisticated techniques may
employ Gc or lc with tandem mS (mS-mS) detection
systems, allowing more sensitivity and selectivity.
Validating the performance of an analytical tech-
nique subsequent to initial in-vitro method develop-
ment is usually accomplished with in-vivo animal
exposure models. additional information may be
gleaned from archived human samples from past
exposure incidents. Samples from humans exposed to
sulfur mustard during the iran-iraq War and a limited
number of samples from the Japanese nerve agent at-
tacks have been used to evaluate assay techniques. in
the case of some agents, background marker levels are
known to exist in nonexposed individuals, making it
Medical Diagnostics
difficult to interpret the results of potential incidents.
therefore, in addition to assessing performance in ani-
mal models and archived human samples, it is essential
to determine potential background levels and inci-
dence of markers in nonexposed human populations.
it should be stressed that, for the purpose of exposure
verification, results from laboratory testing must be
considered along with other information, such as the
presentation of symptoms consistent with the agent in
question and results from environmental testing.
in the late 1980s the uS army medical Research
institute of chemical defense was tasked by the de-
partment of defense to develop methods that could
confirm potential chemical warfare agent exposure.
the uS army medical Research institute of chemi-
cal defense had previously published procedures for
verifying exposure to nerve agents and sulfur mustard.
these methods primarily focused on Gc-mS analysis
of hydrolysis products excreted in the urine following
exposure to chemical warfare agents. Subsequently, the
methods using urine or blood samples were compiled
as part of technical Bulletin medical 296, titled “assay
techniques for detection of exposure to Sulfur mus-
tard, cholinesterase inhibitors, Sarin, Soman, Gf, and
cyanide.” 2 the publication was intended to provide
clinicians with laboratory tests to detect exposure to
chemical warfare agents.
in the mid 1990s, after the publication of technical
Bulletin medical 296, the military adapted some of the
laboratory analytical methods for field-forward use.
the concept was demonstrated by the uS army 520th
theater army medical laboratory, which used the
test-mate op Kit (eQm Research inc, cincinnati, oh)
for acetylcholinesterase (ache) assay and a fly-away
Gc-mS system. the lengthy preparation of Gc and mS
samples for analysis in a field environment was one
of the reasons that alternative methods of analysis for
chemical warfare agents were later examined.
preexposure treatments or tests to monitor potential
chemical agent exposure may be warranted for military
personnel and first responders who must enter or oper-
ate in chemically contaminated environments. how-
ever, laboratory testing may not be as useful for large
civilian populations unless there is a clear impending
chemical threat. at the same time, determining the
health effects of chemical exposure is complex because
it can affect the nervous system, respiratory tract, skin,
eyes, and mucous membranes, as well as the gastroin-
testinal, cardiovascular, endocrine, and reproductive
systems. individual susceptibility, preexisting medical
conditions, and age may also contribute to the severity
of a chemically related illness. chronic exposures, even
at low concentrations, are another concern. in addition
to development of diagnostic technologies, strategies
to detect chemical agent exposure have become a
public health issue.
the transition of laboratory-based analytical tech-
niques to a far-forward field setting can generate
valuable information for military or civilian clinicians.
in this transition, problems such as data analysis,
interpreting complex spectra, and instrument trouble-
shooting and repair may need addressing. as analyti-
cal methods are developed, refined, and sent farther
from the laboratory, advanced telecommunication will
be needed to provide a direct link between research
scientists and field operators; telecommunication will
become critical to confirming patient exposure and
tracking patient recovery and treatment.
this chapter provides a basic outline and refer-
ences for state-of-the-art analytical methods presently
described in the literature. methods of verification
for exposure to nerve agents, vesicants, pulmonary
toxicants, metabolic poisons, incapacitating agents,
and riot control agents will be reviewed. Biological
sample collection, handling, storage, shipping, and
submission will be explained.
amounts were produced by the end of the war. 4 five
of the op compounds are generally regarded as nerve
agents: tabun, sarin, soman, cyclosarin (nato des-
ignation: Gf), and Russian VX. 4 these compounds
demonstrated extreme toxicity, which was attributed
to long-lasting binding and inhibition of the enzyme
ache. as a result, the compounds were referred to
as “irreversible” inhibitors. Related, but less toxic
compounds (ie, “reversible” inhibitors), are becoming
widely used therapeutically; for example, in the treat-
ment of alzheimer’s disease. the relative description
as reversible or irreversible refers to the length of the
binding to the enzyme (figure 22-1).
the first organophosphorus (op) nerve agent,
tabun (nato designation: Ga) was developed
shortly before and during World War ii by German
chemist Gerhard Schrader at iG farbenindustrie in
an attempt to develop a commercial insecticide. 3,4,5
Shortly thereafter, sarin was synthesized. Both are
extremely toxic. the German government realized
the compounds had potential as chemical warfare
agents and began producing them and incorporating
them into munitions. Subsequently, soman (nato
designation: Gd) was synthesized, but only small
Medical Aspects of Chemical Warfare
Sarin (GB)
Soman (GD)
Cyclosarin (GF)
H 3 C
OCH(CH 3 ) 2
H 3 C
OCHC(CH 3 ) 3
H 3 C
CH 3
Ta bun (GA)
Russian VX
OCH 2 CH 3
H 3 C
OCH 2 CH 3
H 3 C
OCH 2 CH(CH 3 ) 2
N(CH 3 ) 2
SCH 2 CH 2 N
CH(CH 3 ) 2
SCH 2 CH 2 N
CH 3
CH(CH 3 ) 2
CH 3
Fig. 22-1 . chemical structures of nerve agents. the nerve agents sarin (GB), soman (Gd), and cyclosarin (Gf) lose fluorine
subsequent to binding to cholinesterase. the agents tabun (Ga), VX, and Russian VX lose cyanide and the thiol groups.
many of the assays developed for exposure verifica-
tion are based on the interaction of nerve agents with
che enzymes. nerve agents inhibit che by forming
a covalent bond between the phosphorus atom of the
agent and the serine residue of the enzyme active site.
that interaction results in the displacement or loss of
fluorine from sarin, soman, and cyclosarin. the bind-
ing of tabun, VX, and Russian VX is different in that the
leaving group is cyanide followed by the thiol groups
(see figure 22-1). 6 Spontaneous reactivation of the
enzyme or hydrolysis reactions with water can occur
to produce corresponding alkyl methylphosphonic
acids (mpas). alternatively, the loss of the o-alkyl
group while bound to the enzyme produces a highly
stable organophosphoryl-che bond, a process referred
to as “aging.” once aging has occurred, the enzyme is
considered resistant to reactivation by oximes or other
nucleophilic reagents. 4 the spontaneous reactivation
and aging rates of the agents vary depending on the o-
alkyl group. for example, VX-inhibited red blood cell
(RBc) che reactivates at an approximate rate of 0.5%
to 1% per hour for the first 48 hours, with minimal ag-
ing. on the other hand, soman-inhibited che does not
spontaneously reactivate and has a very rapid aging
rate, with a half-time of approximately 2 minutes. 4
lab-based, non-che analytical methods have been
developed, and several successfully utilized, to verify
nerve agent exposure. for the most part, these employ
mS with Gc or lc separations. the tests are relatively
labor intensive, requiring trained personnel and so-
phisticated instrumentation not usually available in
clinical settings. most experience using these tech-
niques has come from animal exposure models. these
assessments allow for determination of test sensitivity
and biomarker longevity in experimental models. in
humans, there is limited experience from accidental
and terror-related exposures. this chapter will review
assays for chemical warfare agent exposure that have
been published in the literature and how they have
been applied in potential exposure situations
Assay of Parent Compounds
analyzing for parent nerve agents from biomedi-
cal matrices, such as blood or urine, is not a viable
diagnostic technique for retrospective detection of
exposure. 7 parent agents are relatively short-lived be-
cause of rapid hydrolysis and binding to plasma and
tissue proteins, imposing unrealistic time restraints
on sample collection. the short residence time is es-
pecially profound with the G agents (relative to VX).
following the intravenous administration of soman at
2 times the median lethal dose (ld 50 ) results in parent
agent detection at toxicologically relevant levels for
104 and 49 minutes in guinea pigs and marmosets, re-
spectively; rapid elimination was reflected in terminal
General Clinical Tests
With the exception of che analysis, there are no
standard clinical assays that specifically test for nerve
agent exposure. however, over the years numerous
Medical Diagnostics
half-life rates (16.5 min for guinea pigs; 9 min for
marmosets). 8 inhalation experiments using nose-only
exposure of guinea pigs to 0.8 × lc t 50 (the vapor or
aerosol exposure that is lethal to 50% of the exposed
population) agent demonstrate terminal half-lives of
approximately 36 and 9 minutes for sarin and soman,
respectively. 9 in contrast, similar studies with VX in
hairless guinea pigs and marmosets indicate VX is
more persistent than the G agents. 10 these studies
show that VX can be found at acutely toxic levels for
10 to 20 hours following intravenous administration
at a dose one or two times the ld 50 , with terminal
elimination rates of 98 minutes (1 times the ld 50 in
hairless guinea pigs), 165 minutes (2 times the ld 50 ),
and 111 minutes in marmosets (at a dose equivalent
to 1 ld 50 in hairless guinea pigs). percutaneous ad-
ministration of the ld 50 of VX to hairless guinea pigs
demonstrated relatively low blood levels (140 pg/
ml), which reached a maximum after approximately
6 hours. 10 Because the route of human exposure to
VX would most likely occur percutaneously, the time
frame of 6 hours may be the more relevant assessment
of its persistence in blood. this allows very limited
time for sample collection and analysis. others have
demonstrated that VX can be assayed from spiked
rat plasma. 11 these authors noted that 53% of the VX
was lost in spiked plasma specimens after 2 hours.
the disappearance was attributed to the enzyme
action of the op hydrolase splitting or to cleavage
of the sulfur-phosphorus bond to form diisopropyl
aminoethanethiol (daet) and ethyl methylphospho-
nic acid (empa). 11
and VX, respectively (figure 22-2). additionally for
VX, hydrolysis of the sulfur-phosphorus bond occurs,
yielding daet and empa. the formation and assay
of daet has been reported in rat plasma spiked with
VX. 11 furthermore, the presence of diisopropyl amino-
ethyl methyl sulfide, presumably resulting from the
in-vivo methylation of daet, has been reported in hu-
man exposures. 18 to date, numerous variations of the
alkyl mpa assay for biological fluids, such as plasma
and urine, have been developed. these include Gc
separations with mS, 18–20 tandem mS (mS-mS), 18,19,21,22
and flame photometric detection. 23,24 other methods
involving lc with mS-mS 25 and indirect photometric
detection 26 have also been reported (table 22-1).
Application to Human Exposures
the utility of some methodologies has been dem-
onstrated in actual human exposure incidents. most
involve assays of urine and plasma or serum. tsuchi-
hashi et al 18 demonstrated the presence of empa in
the serum of an individual assassinated with VX in
osaka, Japan, in 1994. as mentioned earlier, these
authors also reported the presence of diisopropyl
aminoethyl methyl sulfide, which resulted from the
in-vivo methylation of daet subsequent to cleavage of
the sulfur-phosphorus bond. Reported concentrations
in serum collected 1 hour after exposure were 143 ng/
ml diisopropyl aminoethyl methyl sulfide and 1.25
μg/ml for empa.
the aum Shinrikyo cult attacked citizens twice in
Japan using sarin. the first was in an apartment com-
plex in matsumoto city, where approximately 12 liters
of sarin were released using a heater and fan. accord-
ing to police reports, 600 inhabitants in the surround-
ing area were harmed, including 7 who were killed.
in the second attack, sarin was released into the tokyo
subway, resulting in more than 5,000 casualties and 10
deaths. 27 assay of hydrolysis products as a definitive
marker were used to verify that sarin was the agent em-
ployed in these events. minami et al 23 and nakajima et
al 24 demonstrated the presence of impa or mpa in vic-
tims’ urine following sarin exposure in the tokyo and
matsumoto attacks, respectively. these methods used
Gc separations of the prepared urine matrix coupled
with flame photometric detection. in the matsumoto
incident, urinary concentrations of impa and mpa,
as well as the total dose of the sarin exposure, were
reported. 24 for one victim, mpa concentrations were
0.14 and 0.02 ug/ml on the first and third days after
exposure, and 0.76, 0.08, and 0.01 ug/ml for impa,
respectively, on the first, third, and seventh days after
exposure. 24 in this case, the individual was estimated
to have been exposed to 2.79 mg of sarin.
Assay of Hydrolysis Compounds
Analytical Methods
an alternative approach to direct assay of parent
nerve agents is to measure metabolic or hydrolysis
products in specimens. these compounds are pro-
duced in vivo as a result of hydrolysis or detachment
following spontaneous regeneration of the ache
enzyme. Studies of parent nerve agents with radioiso-
topically labeled phosphorus ( 32 p) or hydrogen ( 3 h) in
animals suggest that agents are rapidly metabolized
and hydrolyzed in the blood and appear in the urine
as their respective alkyl mpas. 12–15 this observation
led to the development of assays for alkyl mpas in
biological samples, 16 the applicability of which was
subsequently demonstrated in animals exposed to
nerve agents. 17 the common products found are
isopropyl methylphosphonic acid (impa), pinacolyl
methylphosphonic acid, cyclohexyl methylphosphonic
acid, and empa derived from sarin, soman, cyclosarin,
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