Ch22_pg691-752.pdf

Medical Diagnostics

Chapter 22

MEDICAL DIAGNOSTICS

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 ¶

INTRODUCTION

NERVE AGENTS

SULFUR MUSTARD

LEWISITE

CYANIDE

PHOSGENE

3-QUINUCLIDINYL BENZILATE

SAMPLE CONSIDERATIONS

SUMMARY

* 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

691

Medical Aspects of Chemical Warfare

INTRODUCTION

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

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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.

NERVE AGENTS

Background

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

693

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

694

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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