The majority of pesticide poisoning cases in Turkey are suicide attempts (Yurumez et al., 2007
). The most frequent clinical signs are miosis, respiratory distress, tachycardia, loss of consciousness, and hypertension. Out of 220 cases treated in hospital emergency rooms in Afyonkarahisar and Kayseri, Turkey, 20 patients died. Sometimes the diagnosis of poisoning is difficult. Cases have been misdiagnosed initially as brainstem stroke (Aygun, 2004
), opioid overdose (Baydin et al., 2008
), and foreign body aspiration (Caksen et al., 2005
). Laboratory assays showing that plasma butyrylcholinesterase activity was below normal, were helpful for achieving the correct diagnosis of OP poisoning. Treatment strategies depend on a correct diagnosis.
The acute toxicity of organophosphorus pesticides is due to inhibition of acetylcholinesterase in nerve synapses. Inhibition of butyrylcholinesterase has no clinical sequelae. Butyrylcholinesterase is a good marker for OP exposure because BChE reacts rapidly with OP to form covalent adducts that have no enzyme activity, and BChE is 3000-fold more abundant in human plasma than acetylcholinesterase (Brimijoin and Hammond, 1988
). It is standard practice to look for inhibition of plasma BChE activity to aid in diagnosis of OP pesticide intoxication.
Though BChE activity assays are helpful for diagnosis, they do not identify the poison. Mass spectrometry can distinguish between classes of poison. Our mass spectrometry study of pure human BChE modified by four OP pesticides will serve as an aid for future work that aims to analyze plasma samples from poisoned individuals.
The pesticides in the present report have not previously been tested by mass spectrometry of adducts with human BChE. The reaction of methamidophos with human and Torpedo californica
acetylcholinesterase has been studied by mass spectrometry, and has been found to yield adducts on the active site serine similar to the major adduct we found for BChE (Elhanany et al., 2001
). A study with radiolabeled methamidophos showed that thiomethyl was the leaving group in the reaction of methamidophos with electric eel acetylcholinesterase (Thompson and Fukuto, 1982
), a result consistent with our result for human BChE. Aging of the acetylcholinesterase adducts was not reported, consistent with our result for BChE where aging was not observed for samples treated with methamidophos for 7 h.
Diazinon is widely used as a pesticide in sheep dip formulations, spray pesticides for household use, and cat flea collars (de Blaquiere et al., 2000
; Garfitt et al., 2002
). Diazinon is a poor inhibitor of acetylcholinesterase and BChE until it is activated to the oxon. Activation to the oxon is mediated by liver microsomal cytochrome P450 (Mutch and Williams, 2006
). A second route to the oxon is a thiono-thiolo rearrangement. In the present work the thiono-thiolo rearrangement occurred slowly in aqueous solution to produce an oxon that inhibited the activity of BChE. A full description of the rearrangement products of diazinon is provided by Barr et al. (Barr et al., 2005
A mass spectrometry method for detection of OP exposure is not expected to be useful to the clinician, who will treat patients based on their symptoms. Knowing the identity of the poison is, however, likely to be useful to forensic toxicologists. The method distinguishes classes of poisons, but does not distinguish between poisons that add an identical mass. For example, dichlorvos and O-methoate both add a mass of 108 for dimethoxyphosphate, or a mass of 94 for monomethoxyphosphate. Another limitation of the method is the requirement for highly purified BChE. Purifying 4 micrograms of BChE from a milliliter of plasma that contains 50,000 micrograms of other proteins is a difficult task at this time. A one step method is needed that selectively extracts BChE from human serum or plasma.
The information provided in the present work provides the masses of possible adducts and how they fragment in the mass spectrometer. This information is needed to set up sensitive multiple reaction monitoring mass spectrometry methods to analyze exposure in real life samples. As of this date, the major application will be for forensic cases, where it is important to distinguish between classes of pesticides, and between nerve agents and pesticides.
It has been estimated that mass spectrometry could potentially detect OP-BChE adducts in human plasma where the BChE was inhibited as little 1% (Tsuge and Seto, 2006
). The multiple reaction monitoring method would require purification of BChE from 5 ml of plasma.