Anal. Chem. 2010, 82, 6593–6600
Immunomagnetic Separation and Quantification of Butyrylcholinesterase Nerve Agent Adducts in Human Serum Jennifer L. S. Sporty,† Sharon W. Lemire,† Edward M. Jakubowski,‡ Julie A. Renner,‡ Ronald A. Evans,‡ Robert F. Williams,§ Jurgen G. Schmidt,§ Marcel J. van der Schans,| Daan Noort,| and Rudolph C. Johnson*,† Emergency Response and Air Toxicants Branch, Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Highway, MS F44, Chamblee, Georgia 30341, U.S. Army Edgewood Chemical Biological Center E3150 RDCB-DRT-T, 5183 Blackhawk Rd, Aberdeen Proving Ground, Maryland 21010-5424, Bioscience Division, Bio-Security and Public Health, B-7, Los Alamos National Laboratory, MS E529, Los Alamos, New Mexico 87545, and TNO Defense, Security and Safety, Business Unit CBRN Protection, PO Box 45, 2280 AA Rijswijk, The Netherlands A novel method for extracting butyrylcholinesterase (BuChE) from serum as a means of identifying and measuring nerve agent adducts to human BuChE is presented here. Antibutyrylcholinesterase monoclonal antibodies were conjugated to protein-G ferromagnetic particles and mixed with 500 µL serum samples. The particle-antibody-BuChE product was rinsed and directly digested with pepsin. Native and isotopically enriched nonapeptides corresponding to the pepsin digest products for uninhibited BuChE, and sarin, cyclohexylsarin, VX, and Russian VX nerve agent-inhibited BuChE were synthesized for use as calibrators and internal standards, respectively. Internal standards were added to the filtered digest sample, and the samples were quantified via high performance liquid chromatography-isotope dilutiontandem mass spectrometry. The ratio of adducted to total BuChE nonapeptides was calculated for each nerve agentexposed serum sample using data collected in a single chromatogram. Nerve agent-inhibited quality control serum pools were characterized as part of method validation; the method was observed to have extremely low background noise. The measurement of both uninhibited and inhibited BuChE peptides compensated for any variations in the pepsin digestion before the internal standard peptide was added to the sample and may prove useful in individualizing patient results following a nerve agent exposure. Organophosphorus nerve agents (OPNA) are alkylphosphonic ester chemical warfare agents that, upon even low levels of exposure, can cause muscle twitching, miosis, convulsions, hyperglandular secretions, seizures, and death.1,2 Because of their * To whom correspondence should be addressed: E-mail:
[email protected]. † Centers for Disease Control and Prevention. ‡ U.S. Army Edgewood Chemical Biological Center. § Los Alamos National Laboratory. | TNO Defense. (1) Grob, D.; Harvey, J. C. J. Clin. Invest. 1958, 37, 350–368. (2) Schecter, W. P. Anesthesiol. Clin. North Am. 2004, 22, 579–590. 10.1021/ac101024z 2010 American Chemical Society Published on Web 07/09/2010
high degree of toxicity and potential use for chemical attacks, nerve agents are considered weapons of mass destruction. Sarin, a nerve agent, was used in terrorist attacks in Matsumoto, Japan, in 1994 and in a Tokyo subway in 1995 by the Japanese terrorist cult, Aum Shinrikyo.3 These attacks caused seven deaths and 600 poisonings in Matsumoto and 12 deaths and 5500 poisonings in Tokyo.3 Objective analysis of exposure for these victims included laboratory analysis of erythrocyte acetylcholinesterase and serum cholinesterase activity that showed reduced enzymatic activities, although no baseline activity levels for individual victims had been established. These results indicated exposure to a cholinesterase inhibitor; however, no method was immediately available to clinically identify the agent or the extent of exposure. Traditional methods rely on enzyme activity measurements to assess the extent of exposure. By comparing baseline enzyme levels or, in the absence of baseline levels, normal population levels, with postexposure levels, a decrease in enzyme activity can be measured. This overall decrease in activity serves as an indicator of exposure severity. Several methods have been developed to identify and quantify nerve agent exposure by monitoring urinary metabolites. A method initially developed for quantifying nerve agent metabolites in rat urine4 was extended to a human urine sample from the Matsumoto incident.5 Two sarin metabolites, methylphosphonic acid (MPA) and isopropylmethyl-phosphonic acid (iPMPA), were measured via gas chromatography (GC) with a flame photometric phosphorus detector in urine collected from a 19-year-old man who had been exposed. Results indicated that the victim had been exposed to 2.79 mg, or 0.05 mg/kg, of vaporized sarin, a near-lethal dose. The urinary metabolite method was further extended to analyze phosphonic acid metabolites of sarin, soman, VX, cyclohexylsarin,6,7 and Russian VX6 by tandem (3) Yanagisawa, N.; Morita, H.; Nakajima, T. J. Neurol. Sci. 2006, 249, 76–85. (4) Shih, M. L.; McMonagle, J. D.; Dolzine, T. W.; Gresham, V. C. J. Appl. Toxicol. 1994, 14, 195–199. (5) Nakajima, T.; Sasaki, K.; Ozawa, H.; Sekjima, Y.; Morita, H.; Fukushima, Y.; Yanagisawa, N. Arch. Toxicol. 1998, 72, 601–603. (6) Barr, J. R.; Driskell, W. J.; Aston, L. S.; Martinez, R. A. J Anal. Toxicol. 2004, 28, 372–378. (7) Driskell, W. J.; Shih, M.; Needham, L. L.; Barr, D. B. J Anal. Toxicol. 2002, 26, 6–10.
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GC-mass spectrometry (MS). Urinary metabolite methods can be measured for about 3 days post exposure.8 After that time, metabolite concentrations are either too low to measure reliably or completely excreted from the body. Long-lived blood and serum protein adducts can be monitored as long as 16 days or more post exposure for retrospective analysis when urinary metabolites are no long present at detectable levels.9,10 OPNA, like organophosphorus pesticides, adduct to acetylcholinesterase and to butyrylcholinesterase (BuChE) in the body, inhibiting the enzymes and causing many of the symptoms of nerve agent exposure. Acetylcholinesterase has been reported at
