22 APPLICATION BIOPHARMA Analyzing cholinesterase in organophosphate poisoning The German army automates the determination of AChE activity in whole blood Bodo Pfeiffer, Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany Agrochemicals belonging to the organophosphate group are used extensively around the world, due to their efficacy and comparatively low environmental persistence. They are, however, highly toxic and present a monitor levels of the affected enzyme parameters used previously could almost considerable risk to humans, as within the body. Muscular AChE is not all be translated and known incubation demonstrated by the estimated 3 million easily accessed in patients, so erythrocyte times, wavelengths and measuring times acute pesticide poisonings and 200,000 AChE and plasma cholinesterase were not altered. This application can be fatalities globally every year. A few (butyrylcholinesterase; BChE) are automated on other Tecan liquid particularly poisonous organophosphates, analyzed instead. handling workstations, including from the Freedom EVO® series. including soman, sarin, tabun and VX The Bundeswehr Institute of Pharmacology (known as nerve agents), were developed and Toxicology co-operates with the Cholinesterase activity is measured in a as chemical weapons and have even been South Asian Clinical Toxicology Research whole blood sample collected from the used recently, notably in the Iran-Iraq war Collaboration Centre for Tropical patient and immediately diluted and and the Tokyo underground in 1995, Medicine, University of Oxford, England3 cooled to slow down any ex vivo reactions despite being outlawed around the in a prospective cohort clinical trial in Sri that can rapidly take place between world. Lanka. Blood samples from patients are AChE, organophosphates and oximes. Organophosphates inhibit serine analyzed in the Institute for AChE activity, Cholinesterases convert specific substrates esterases, particularly acetylcholinesterase BChE activity and hemoglobin. Until and, in the conversion process, thiocholine (AChE), by covalently binding to the recently, the parameters have been is formed as a product (figure 1b). enzyme and disturbing its physiological analyzed manually by measuring a 3 ml This product reacts with DTNB (5-5'dithio function – i.e. preventing cleavage of the sample using a standard commercial bis-(2-nitrobenzoic acid)) present in the neurotransmitter, acetylcholine (ACh), by spectrophotometer with a temperature- sample to give a yellow dye that can be AChE. This leads to an accumulation of controlled cuvette holder and water bath measured with a spectrophotometer; the ACh in cholinergic synapses of both the at 37ºC. However, this manual procedure color development over time serves as a central and peripheral nervous systems quickly reached its limits when analyzing direct measure of the enzyme’s activity. and at the neuromuscular junction, large numbers of samples and, to solve The hemoglobin levels in the dilutions resulting in hyperexcitation and sub- this problem, an automated microplate are also measured, using Zijlstra’s 4 sequent paralysis of the target organs method was developed to process method , to allow correction of any and, eventually, death due to central and multiple samples in parallel. Tecan’s diluting errors in preparation of the peripheral respiratory paralysis1,2. Genesis 150 workstation was introduced, samples; the AChE activity can be with automated pipetting and a robotic correlated with the hemoglobin levels5,6. Organophosphate poisoning can be arm as well as an integrated spectro- To validate this set-up, extensive treated by immediate administration of photometer, a 37°C incubator, temperature- comparative measurements were made atropine and AChE reactivators (oximes; controlled microplates racks and cooled using both the automated and manual Obidoxime in Germany, pralidoxime in sample preparation (figure 1a). Adapting methods. AChE activity was analyzed in the UK and USA) but it is necessary the protocol for automation was aliquots of native donor whole blood that throughout treatment to measure and relatively straightforward because the Tecan Journal 2/2006 APPLICATION BIOPHARMA 23 were inhibited with 100 nM soman; BChE Acetylcholinesterase activity in plasma harvested from soman- Acetylthiocholine + H2O thiocholine + acetate OR: inhibited blood samples was measured and hemoglobin levels were determined Butyrylcholinesterase in dilutions of whole blood samples with Butyrylthiocholine + H2O thiocholine + butyrate different proportions of erythrocytes Thiocholine + 5,5´dithio-bis(2-nitrobenzoate) → and added plasma. In addition, the 5-mercapto-2-nitrobenzoate + 5-mercaptothiocholine nitrobenzoate [yellow] reactivation capability of whole blood (TNB) inhibited with either 100 nM soman, Figure 1a: Tecan GENESIS 150 workstation. Figure 1b: Formation of DTNB produces a yellow which does not allow AChE reactivation, The workstation is an open source system dye that serves as a direct measure of or 200 nM paraoxon-ethyl (PxE), which and can also be used now for kinetic cholinesterase activity readily allows AChE reactivation, was samples. It has an integrated Sunrise™ analyzed and the residual inhibitor was spectrophotometer and uses TOPS™ 4.0 also measured in donor plasma samples with Magellan inhibited with five different concen- trations of paraoxon-ethyl. The results obtained with both methods correlated Quinalphos poisoning Fenthion poisoning very strongly in all measurements. 600 600 500 500 400 n n u 400 Once validated, the automated method n u n n u AChE u AChE 300 u u 300 n Reac n Reac was used to analyze and monitor AChE 200 200 u 100 100 and BChE activity in samples from the Sri n un n nnnn AChE activity (mU/µmolHb) 0 AChE activity (mU/µmolHb) 0 u u u uu Lanka patients who had attempted 0244872 04824 72 96 suicide by oral ingestion of pesticides t=h t=h (quinalphos or fenthion), (figures 2 and 3). Quinalphos poisoning Fenthion poisoning Whole blood samples were collected 100 100 from the patients (t = 0) and oxime 75 75 therapy was started with pralidoxime 50 50 % Inhibition (bolus dose and infusion therapy); further % Inhibition 25 25 u uu u u u samples were collected within a 0 u u uu 0 uu 0244872 02448 72 96 predefined time window. Quinalphos t=h t=h results in diethylphosphorylation of AChE, this enzyme species can be readily Quinalphos poisoning Fenthion poisoning reactivated (figure 2) and so, four hours 3000 3000 2500 2500 after admission to the hospital, no 2000 2000 residual inhibitor remained in those 1500 1500 1000 1000 AChE activity (mU/ml) 500 u u AChE activity (mU/ml) affected patients. Fenthion results in a u u 500 u 0 0 uu u uuuu dimethylphosphorylated enzyme that 0244872 024487296 ages quickly and cannot be reactivated t=h t=h (figure 3), meaning that in patients who Figure 2: Example of Quinalphos poisoning. Figure 3: Example of Fenthion poisoning. had ingested this pesticide, no significant The reactivation capability shows the Due to ageing, reactivation is almost increase could be recorded in AChE maximum achievable AChE activity. The per impossible (top). In spite of a decline in activity, and BChE activity remained cent inhibition shows that no residual residual inhibitor (middle) no significant inhibitor remains after 4 hours increase in AChE activity is recorded (top). completely inhibited. BChE activity is completely inhibited (bottom) The determination of cholinesterase status is primarily intended for analyzing patients poisoned with organophosphates (accidental or suicidal poisoning), but it can also be applied after the use of nerve References agents in military conflicts or terrorist 1. Wiener SW, Hoffman RS. (2004) Nerve agents: a 4. van Kampen EJ, Zijlstra WG (1961) Standardization of attacks, as well as in the context of comprehensive review. J. Intensive Care Med. 19, 22-37 hemoglobinometry, II. The hemoglobincyanide method. occupational medical examinations. Clin Chim Acta 6: 538-544 2. Koelle GB. (1992) Pharmacology and toxicology of organophosphates. In: B. Ballantyne and T. C. Marrs 5. Thiermann H, Szinicz L, Eyer F,Worek F, Eyer P, (Eds.), Clinical and experimental toxicology of Felgenhauer N, Zilker T. (1999) Modern strategies in organophosphates and carbamates. Butterworth & therapy of organophosphate poisoning. Toxicol. Lett. Heinemann, Oxford, pp. 35-39 107, 233-239 3. Eddleston et al. (2005) Differences between 6. Worek et al. (1999) Improved determination of organophosphorous insecticides in human self- acetylcholinesterase in human whole blood. Clin Chim poisoning: a prospective cohort study. Lancet 366: Acta 288: 73-90 1452-1459 Tecan Journal 2/2006.