PMMA Summary The mini-CB MTS / PMMA

Dr. Barbara Price, mCB MTS Co-Chair Hradec Kralove, Czech Republic 26-30 May 1997.

The first mini-Chemical and Biological Medical Treatment Symposium PMMA-I was held at the Purkyne Military Medical Academy in Hradec Kralove and was attended by 54 scientists and medical doctors from 21 countries.

The purpose of a mini-CB MTS is to focus on a particular subject or area that falls within the general theme of the CB MTS series. For the PMMA-I, the focus was standardization and, in particular, standardization of cholinesterase measurements. There were several papers describing different methods to measure cholinesterase and the types of errors that might ensue. Dr. Rudolf Portmann (Switzerland) of the NC Laboratories spearheaded the movement for standardized measurements. A major achievement of the mCB MTS/PMMA-I was the agreement in principle by many of the countries represented to participate in a round robin series of tests for acetylcholinesterase (AChE) based on a standard method. The group quickly achieved close agreement on the conditions for the method to measure AChE from human erythrocytes.

Once a standard test method is established and the sample is made and distributed and the results are in, all of the labs will be able to correlate their data with each other and with whatever test method is their method of choice. As will occur in a group with wide-ranging interests, there were discussions in other areas as well. Is AChE a better measure of OP exposure than butyrylcholinesterase (BuChE, also known as serum cholinesterase)? Exactly how does the reaction of AChE with OPs occur and what happens during the aging reaction? What are the advantages of the different oximes for use as pre treatment, reactivation, and prevention of aging? How can the measures of intoxication be used with clinical observations to determine the extent and seriousness of OP or carbamate intoxication and treatments? Is it practical to make and use antibodies for OPs in immunoassays and how sensitive are detectors based on antibodies?

Cholinesterase measures of intoxication, while a convenient and sensitive measure of OP intoxication, cannot demonstrate the presence of OPs and several other laboratories are improving the detection of OPs in tissues. Dr. DeLong (Netherlands) and his group have been working on measuring OPs and their adducts in blood to identify the OPs that were present. Dr. Price and Col. Moore (USA) both stressed the importance of sample collection and handling, as well as analytical methods, as part of the evidence in analysis of human tissues for chemical warfare agents. Dr. David Lenz (USA) and Dr. Yimin Zhao (China) both lead very interesting discussions on their work with mutant AChEs as bioscavengers to prevent intoxication by OPs and as catalytic hydrolyzers of OPs. This appears to be one of the most interesting areas of future research in both treatment and detection. We also had a discussion, appropriate to the detection of both chemical and biological warfare agents, lead by Dr. M. Hamilton (Canada) with the emphasis on how the use of the information in large part determines the sensitivity and selectivity of the detection systems.

All of these interesting and challenging discussions were garnished with the hospitality of the PMMA and the wonderful social program planned by our hosts and Dr. Bajgar. All left with an appreciation of the challenge of our technical fields and warm feelings of the Czech people and the beauty of the Czech country.

The mCB MTS/PMMA I structure:

Host, Organizer, Sponsor: Purkyne Military Medical Academy, Hradec Kralove, Czech Republic
International Organizer and Coordinator, Sponsor: Applied Science and Analysis, ASA; US
Institutional and Corporate Co-Sponsors/Supporters: Battelle Memorial Institute and the CBIAC, US; Astra Tech, Sweden; BioDynamics International, BDI, US; Nucleus, Czech Republic; Applied Science and Analysis, ASA, US; Arrow International, Czech Republic; NC-Laboraory Spiez, Switzerland; and VojenskŽ stavby, Czech Republic.
Staff:
Chair: Prof. Jiri Bajgar, PMMA, Czech Republic
Co-Chair: Dr. Barbara Price, ASA, US
Organizer/Coordinator: Col. Richard Price, ASA, US
Science Advisor/Schedule: Dr. Rudolf Portmann, NC- Lab Spiez, Switzerland
Session Chairs/CoChairs: As listed in the following session summaries.

Session 1: Methods for AChE Determinations
Prof. Urs Brodbeck, Chair, U. of Bern, Switzerland; Prof. Yimin Zhao, Co-Chair, Inst. of Pharm/Toxicology, China

The first session was highlighted by the excellent presentation of Dr. I.M. Kovach, who reviewed the catalytic mechanism of AChE and its inhibition by OP-compounds. Special attention was given to the mechanism of aging after soman poisoning. Dr. Kovach╦s elegant presentation gave a clear picture of enzyme mediated dealkylation and the amino acid residues involved in it. Dr. Brodbeck╦s studies supplemented Dr. Kovach╦s findings in as much as he presented results indicating that the rate of aging of soman-inhibited AChE is reduced by the oximes HI-6 and HLš-7. The last paper in this session was presented by Dr. R. Portmann who introduced the audience to the difficulties in standardizing ex-vivo measurements of AChE and BuChE activities in healthy and poisoned persons. He elegantly solved the problem by relating enzyme activity to the content of hemoglobin in blood.

Session 2: Methods for BuChE Determination
Dr. Murray Hamilton, Chair, DRES, Canada; Prof. Vladimir Feld, Co-Chair, Inst. for Limnology, Russia

This session dealt not so much with methods for BuChE determination as with the use of BuChE as a prophylaxis against nerve agent poisoning. Dr. David Lenz presented the USAMRICD work to develop a BuChE mutant that acted as both a scavenger for OPs, as well as possessing catalytic or nerve agent hydrolyzing activity. It was found that a histidine mutant, G117H, developed by site directed mutagenesis, was not only resistant to aging, but also capable of hydrolyzing the agents. The catalytic activity was specific for histidine as insertion of a positively charged lysine moiety at the same location was devoid of catalytic activity. A double mutation, glutamate substitution at E197 and histidine at G117H proved to be the most effective mutation to date (being able to catalyze the hydrolysis of GB, VX, and GD). The research will continue with an aim to provide a signfree response to a 5 LD50 challenge from a nerve agent because of the activity of these designer BuChEs.

Session 3: Poster Session
Prof. Ildiko Kovach, Chair, Catholic U., US; Dr. Shabbir Moochhala, Co-Chair, DMRI, Singapore
To be supplied at later date

Session 4: Standardization of ChE Measurements.
Dr. Rudolf Portmann, Chair, NC-Lab, Switzerland; Dr. Fran Crimmins, Co-Chair, Battelle Memorial Institute, US

Prof. V. Feld gave an overview of different possibilities to measure ChE activity. Besides the well known method of Ellman, he mentioned other possibilities and compared these. In general, the methods, if correctly handled, lead to comparable results. Prof. Szinicz reported a standard operation procedure for the determination of ChE in toxicological units. The AChE is normalized to hemoglobin for reducing individual errors. The arterial blood sample is directly diluted twofold in a plastic bottle and frozen to avoid any further inhibition by freely circulating toxicants or reactivation by reversible active toxicant. Then there was a lengthy discussion on standardization of ChE measurements. Dr. Lenz brought up a USEPA Workshop on carbamate and OP insecticides for the purpose of developing a standardized method to measure cholinesterase (both AChE and BuChE). There was interest in taking part in an interlaboratory comparison with many countries agreeing to participate in such a test. Germany, Switzerland, USA, Czech Republic, Netherlands, Bulgaria, Romania, Croatia, Poland, Singapore, Sweden, Canada, China, Russia and Turkey would like to stay informed for making a decision later on. The proposal and first test round are to be worked out by the NC Laboratory Spiez and the Biochemical Department of the University of Bern, Switzerland.

Contact Dr. Rudolf Portmann for details and to participate in this test round at e-mail: rudolf.portmann@x400.gr.admin.ch or fax: 41-33-6-55 25 91

Some of the details that had rather quick agreement were: use of pH of 7.4 (same as blood), a buffer of 100 mM NaHPO4 and NaH2PO4, no salt for extra ionic strength, 0.1% Triton 100X for hemolysis and solubilize the red blood cell membranes, substrate of ASCh 1mM, 30šC, DTNB for fluorescence, normalized to hemoglobin, based on "finger tip" blood, use of a standard (fetal bovine serum was suggested), and a six point minimum curve when using kinetic measurements.

Session 5: Standardization in Toxicity Assessment
Prof. J. Bajgar, Chair, PMMA, Czech Republic; Dr. Leo Laughlin, Co-Chair, Battelle Memorial Institute, US

Dr. Draganov, Military Medical Academy, Sophia, presented a paper describing the advantages of using a response surface model to visualize dose-response relationship between two drugs used in an experimental therapeutic regimen. Using this method, one can compare/estimate optimum drug doses to be used in treatment of OP intoxication. Professor Szinicz of the German Institute of Pharmacology and Toxicology described the use of red blood cell acetylcholinesterase as a monitor to determine when oxime therapy for OP poisoned patients is no longer necessary or therapeutcally indicated.This AChE monitoring allowed carefull control of oxime dose and reduction in overall atropine used. Professor Voicu and Dr. Mircioiu from the Romanian Army Institute for Medical Research discussed the structural and ionic properties and requirements of cholinesterase reactivators. By studying the behavior of reactivators in monolayers, they hoped to determine how to better configure reactivators. The experimental results did not support the experimental hypothesis, although some insights on reactivator-receptor site interactions were obtained.

Session 6: Other Methods for Determining OP Intoxications
Prof. Edmund Lee, Chair, DMRI, Singapore; Prof. Victor Voicu, Co-Chair, Army Inst. Med. Research, Romania

The session included four papers, three of which were directed towards methods for measuring chemical agents in the environment and in body fluids. Dr. Lenz from the US AMRICD started the session with a paper on immunoassays for various chemical warfare agents. Specific monoclonal antibodies developed against soman were tested to sensitivity levels of about 80 ng/mL. Additional antibodies against sarin and VX were also successfully developed. These antibodies were likely to be used to develop field diagnostic kits for the detection of specific OP poisonings. Such information would have an impact on the selection of appropriate management protocols. The next paper by Mr. W.K. Loke, Singapore, introduced a microassay for OPs based on residual activity of eel anticholinesterase activities. While the assay has the drawback of not being able to differentiate the various OPs, it could be used clinically to monitor OP levels and patient╦s serum. It could be a useful tool in the management of OP intoxication. Dr. L. DeJong from the Netherlands then presented his method of analysis of OPs which was tested on old samples obtained from the Tokyo subway attack. This is a GC method based on the removal of OP from its binding on the enzyme. This was achieved using low pH and high fluoride concentrations. Using this method, he was able to examine old samples collected from patients after the Tokyo subway sarin attack, to decide exposure to OP poisoning. The final paper by Dr. Y. Zhao from China, was about his efforts in creating a catalytic antibody capable of hydrolyzing soman. He described the scheme he used to design and construct antibodies capable of hydrolyzing soman which received many favorable comments. Based on these papers, there was much anticipation that a human catalytic antibody could eventually be made for use as a scavenger.

Session 7: Biodetection
Dr. David Lenz, Chair, USAMRICD, US; Prof. Lotfali Haghighi, Co-Chair, U. of Shiraz, Iran

In this session, an attempt was made to identify the major questions facing those who must develop biodetectors. These questions were (1) Should detectors be broad based, e.g., is the purpose to just detect something or is the purpose to detect some specific level; (2) How rapidly should the detector respond (Dr. Portmann raised this question earlier in the meeting); (3) What level of sensitivity is needed, e.g., detect acute or low-level chronic exposures, and (4) How does one transmit the information from the detector to those who need to know. With respect to biological threats, it was felt similar questions are also relevant. However, it was observed that in some cases, the exposure to a biological agent may not have rapid onset of a toxic response. Under those conditions, the response time of the biodetector could be altered. As an example of the current state of the art in biodetectors, Dr. Hamilton from DRES in Canada discussed the Canadian Integrated Biochemical Agent Detector (CIBADS) Program. That program is designed to put a laboratory on a glass chip. For chemical agent detection, the system is based on the bioassay utilizes a cycling probe technique. The goal is to provide a "real time" response. It was agreed that this approach addresses some of the questions raised initially, but it left room for additional discussion and research.

Session 8: Collection, Storing and Shipping of Biological Samples
Dr. David Moore, Chair, USAMRICD, US; Dr. Sergei Kucherenko, Co-Chair, Inst. of Biochemistry, Ukraine

This session was designed to focus attention of the participants to the multitude of small, but often overlooked details and considerations associated with laboratory analysis of clinical samples for indications of chemical warfare agents. The importance of a successful analytical process relies not only on quality, validated and reproducible analytical methods, but also on attention to details regarding record keeping and chain of custody requirements. Col. Moore presented information contained in the recently published US Army Technical Bulletin "Assay Techniques for Detection of Exposure to Sulfur Mustard, Cholinesterase Inhibitors, Sarin, Soman, GF and Cyanide" and a brief review of specific medical considerations for establishing a program for CW agent analysis of clinical samples. Dr. Barbara Price gave a presentation which provided an overview of characteristics of a successful analytical quality control and quality assurance program.

Session 9: Prophylaxis Against OP Intoxications
Dr. BP Doctor, Chair, Walter Reed Army Inst. of Research, US; Prof. Christopher Dishovski, Co-Chair, MMA, Bulgaria

Dr. BP Doctor gave an overview of the development of ChE-based bioscavengers as a pretreatment drug for the OP toxicity. Attempts are being made to further improve the efficacy of bioscavengers by testing the site-specific mutants that are more easily reactivated (essentially non-aging) than the wild-type enzymes. For the large scale preparation of bioscavengers, it is more likely that recombinant biotechnology may be required. Some native and all recombinant ChEs tested have very short mean residence times in mice compared to serum ChEs, limiting their usefulness as stable, efficient, long-lasting bioscavengers. Dr. Murray G. Hamilton of Canada discussed perspectives in nerve agent medical countermeasures.therapeutic strategies against OP never agents challenges require four critical considerations: a) circumstances: exposure characteristics, b) expected outcome: acceptable risks and casualties c) drug-dose/regimen, and d) animal models and extrapolations to humans. Ideas on standardizing experimental protocols with the aim of simplifying inter-laboratory comparison of efficacy results were discussed. Finally, the value of pyridostigmine as a pretreatment against nerve agent poisoning was discussed by Prof. Ladislaus Szinicz. Based on in vitro experiments with isolated AChE and by a number of animal investigations using various species and protocols, pyridostigmine pretreatment can be recommended in the face of an impending soman exposure, but it cannot be recommended for sarin, cyclosarin, tabun or VX.

Session 10: Interlaboratory Comparison of ChE Testing
Dr. Paul Lundy, Chair, DRES, Canada; Prof. Constantine Mircioiu, Co-Chair, Army Inst. for Med Research, Romania

This was a lively discussion of different laboratory conditions and their effects on ChE testing and results. Temperature, buffer strength, ionic strength, Triton X, amount of blood, etc. all play a role in the analyses.

Session 11: Symptomology, Clinical, Physical in OP Intoxications
Prof. Ladislaus Szinicz, Chair, Inst. of Pharm/Tox, FAF, Germany; Prof. Josef Fusek, PMMA, Czech Republic

There were two presentations. Chris Dishovski "Inhibition of ChE with the OP Compounds. Correlations with Physiological, Morphological, and Biochemical Changes." and G. …zyurt. "The Relationship between Serum Cholinesterase Activity and APACHE II Score in Organophosphate Poisoning." The data presented in both papers show that ChE inhibition activity alone is insufficient as a parameter for diagnosis and monitoring of treatment of anticholinesterase poisoning. It does not correlate well with the severity of poisoning. Papers in the earlier sessions have shown the agreement that AChE activity might give better results. It was suggested that monitoring of AChE and antidote concentrations may further improve the knowledge/information on diagnosis, treatment and prognosis.

 

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