Laboratory Examination in Nerve Agent Intoxication
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REVIEW ARTICLE LABORATORY EXAMINATION IN NERVE AGENT INTOXICATION Jiří Bajgar University of Defence, Faculty of Military Health Sciences, Hradec Králové, Czech Republic: Department of Toxicology; University of South Bohemia České Budějovice, Faculty of Social and Health Studies, Czech Republic: Department of Radiology and Toxicology Summary: Diagnosis of nerve agent intoxication is based on anamnestic data, clinical signs and laboratory examination. For acute poisoning, cholinesterase activity in the blood (erythrocyte AChE, plasma/serum BuChE) is sensitive, simple and most frequent laboratory examination performed in biochemical laboratories. Specialized examinations to precise treatment (reactivation test) or to make retrospective diagnosis (fluoride induced reactivation etc.) can be conducted. Other sophisticated methods are available, too. Keywords: Nerve agents; Metabolites; Diagnosis; Blood; Acetylcholinesterase; Erythrocytes; Butyrylcholinesterase; Plasma; Reactivation Introduction ited to the acute stage of intoxication; usually it is not very sensitive, depending on the dose administered; Chemical weapons (CW) belong to the weapons of – determination of changes caused by the agent (group of mass destruction. Their development, production, use etc. agents) in question; it is mostly less specific indicating are prohibited and controlled by the internatonal coonven- more group of agents. However, it is sensitive and de- tion (Convention on the Prohibition of the Development, tectable long time interval after the intoxication. Production, Stockpiling and Use of Chemical Weapons and Both determinations are realized mostly in the blood on their Destruction, CWC) (16). After ratification with (erythrocytes/serum/plasma) but some other materials are sufficient number of the State Parties, CWC entered into not excluded (e.g. tissues such as the brain, muscle, saliva, force (29 April 1997) and at present, 188 State Parties are cerebrospinal fluid etc.). It can be used for forensic purpos- involved including Czech Republic. In the Czech Repub- es, too. The problem is determination/information on the lic, the State Institute for Nuclear Security (Státní ústav pro normal values in biological sample. For further studies, it jadernou bezpečnost, SÚJB), was established as the exec- can be recommended the monography dealing with many utive and control organ responsible for implementation of aspects of CWA edited by Professor Gupta (25). the Convention in the Czech Republic. Simultaneously, this organ licensed laboratories for ability to work/handle with Nerve agents highly toxic chemicals such as chemical warfare agents (CWA). Without the license it is impossible to work/manip- Nerve agents belong to the highly toxic agents; they are ulate with CWA (e.g. sarin, soman etc.) and this condition the most important group from CWA having high toxicity, is limiting factor for analysis of these agents. Though the rapid action at all routes of administration including inha- use of CWA is in general prohibited by the Convention, it lation and percutaneous exposure. They can be divided into is not excluded their misuse either in military or terroristic two main groups, G-agents and V-agents; between them, actions (57). group of agents combining in their structures G an V group The group of nerve agents is the most important group is observed, called GV (GP) agents. It can be pointed out that of CWA. The stocks of these compounds (weaponized or compounds of the same basic structure (organophosphates, stored) in the Albania, India, South Korea, United States OP) are used in industry, veterinary or human medicine and and Russia (officially declared) represent about 69,429 tons in agriculture, e.g.. Metathion, Malathion, Actellic, Dichlor- (53); it is quite clear that the paper is focused to these agents. vos (DDVP), Paraoxon, Parathion, In-stop etc. (Fig. 1) Laboratory diagnostics following poisoning with nerve In the CWC, examples of nerve agents containing gen- agents is focused mostly to: eral structure are given (Schedule 1, CWC) (16): – determination of the mother compound or metabolites; O-Alkyl (≤C10, incl. cycloalkyl) alkyl (Me, Et, n-Pro or examination is specific for such compound but it is lim- i-Pro) phosphonofluoridates, e.g. sarin, soman; ACTA MEDICA (Hradec Králové) 2013; 56(3):89–96 89 R2 action of nerve agents. Increased level of the neuromediator acetylcholine causes symptomas observed in the course of R1 P R3 poisoning. These symptoms are muscarinic, nicotinic and central as described in detail many times (3, 5, 14, 25, 36, O 44, 58 etc.). However, before action at cholinergic synapses, nerve agent is penetrating into the blood, and, by this trans- Fig. 1: General structure of organophosphates: R1–2 are hy- port system is distributed to the target organs – peripheral drogen, alkyl (including cyclic), aryl and others, alkoxy, and central nervous system. During penetration nerve agents alkylthio and amino groups. R3 is a dissociable group, e.g. react with the blood cholinesterases – erythrocyte AChE and halogens, cyano, alkylthio group, rest of inorganic or organic plasma butyrylcholinesterase (BuChE, EC 3.1.1.8). The part acid. More about the chemistry of organophosphates see e.g. of the agent bound to cholinesterases is excluded from toxic Fest and Schmidt (23); modern trends in the development of action. To the targets, only a part of the dose administered nerve agents was described by Halámek and Kobliha (27) is penetrating and inhibiting AChE at cholinergic synapses. Schematic representation of interaction of AChE with nerve agent/organophosphate is shown in Fig. 3. O-Alkyl (≤C10, incl. cycloalkyl) N,N-dialkyl (Me, Et, AChE (E) is reacting with nerve agent (P) to an in- n-Pro or i-Pro) phosphoramidocyanidates, e.g. tabun; termediate complex (EP) and then to phosphorylated O-Alkyl (≤C10, incl. cycloalkyl) S-2-dialkyl (Me, Et, (phosphonylated) complex EP1. The complex EP1 is reac- n-Pro or i-Pro)-aminoethyl alkyl (Me, Et, n-Pro or i-Pro) tivatable by cholinesterase reactivators (R). EP1 complex phosphonothiolates and corresponding alkylated or proto- is able in some cases to be changed to non-reactivatable nated salts, e.g. VX. complex (EP2) – reactivators are not effective. This reaction Thus, all these chemicals having above mentioned struc- (EP1 → EP2) is called aging of inhibited AChE and, typi- ture are under control of the CWC. cally, it is observed for interaction of AChE with sarin and Mechanism of their action, symptoms, principles of soman (Fig. 4). Relevant rate constants (k+1,−1,+2,+3,+4 and ka) diagnosis and treatment for OP and nerve agents are very for the reactions are indicated. The complex EP is not eas- similar. Structures of some nerve agents and OP are shown ily detectable, EP1 and EP2 complexes can be determined. in Fig. 2. Their distinction is difficult. Non-cholinergic effects are not specific and they are observed in long term intervals (hours– Pharmacodynamics of nerve agents days) following intoxication (3, 5, 32, 36, 44). Spontaneous recovery of AChE activity (reactivation) Irreversible acetylcholinesterase (AChE, EC 3.1.1.7) is long lasting as well as the synthesis of AChE de novo (3, inhibition at cholinergic synapses is basic toxicodynamic 5, 36, 44, 58, 59). Reaction of AChE and nerve agents is of F O F O CH3 CH F O P P 3 CH3 H C O P H3C O 3 H C O CH 3 CH3 3 H3C Sarin Cyclosarin Soman H3CH2CO O H3C N C O P CH3 P H C S H CH CO N CH3 3 N 3 2 CH3 H3C H3C Tabun VX CH3 H C H3C O O 3 P O O H3C O O P CH2CH3 Cl H C S 3 N Cl CH2CH3 Russian VX DDVP Fig. 2: Structural formulae of some nerve agents and DDVP 90 E system) reaches to a small fraction of the dose administered k–1 k+3 (10% and less). Detoxification of nerve agents and organo- E + P EP EP1 EP2 phosphates was described in detail by Jokanovič (29). The k+1 k+2 k+4 (for some OP) binding of nerve agent to cholinesterases is important and E + P advantageous for diagnostic purposes: blood cholinesterases ka R R are easily accessible for laboratory examination. (effective) (uneffective) AChE a BuChE differences Fig. 3: Schematic representation of interaction between AChE (E) and nerve agent/organophosphate (P) AChE and BuChE are similar enzymes differing in their localisation, enzymatic properties, sequence of amino acids and physiological function (3, 5, 13, 17, 37, 45, 52, 61). classic inhibition kinetics: reactions important for laboratory AChE is observed at cholinergic synapses, erythrocytes diagnosis are shown in Fig. 3. and BuChE is contained mostly in plasma/serum and liver. The effect of reactivators is limited by the rate of ag- AChE and BuChE have different sensitivity to substrates ing process; it is chemical reaction when inhibited AChE is and inhibitors; inhibition by substrate is typical for AChE. changed to be resistant to the effect of reactivators. The rate The function of AChE is splitting neuromediator acetylcho- of this reaction is dependent on the time and the structure of line at cholinergic synapses; its function in erythrocytes is inhibitor. The half-lives of aging for soman are reported to not yet known. The function of BuChE is not known, it can be in minutes; for sarin, the half-life is about 10 hours and play a complementary role in cholinergic transmission or AChE inhibited with VX is dealkylated within 24 and more it is involved in non-specific detoxification reactions and hours (e.g. 5, 36). This fact is limiting factor for the treat- neurological diseases (5, 17,18, 29). ment using reactivators. For OP insecticides, this reaction is A qualitative difference between the BuChE of suxam- not practically important (they are not dealkylated) (Fig. 4). ethonium sensitive individuals and that of other patients Pharmacodynamics of nerve agent can be described by were described in detail by Whittaker (60). The biosynthe- u a the simple scheme containing following steps – penetration sis of BuChE is controlled by two allelic genes, E 1 and E 1.