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. 2010 Aug;30(6):559-65.
doi: 10.1002/jat.1526.

Dichlorvos, chlorpyrifos oxon and Aldicarb adducts of butyrylcholinesterase, detected by mass spectrometry in human plasma following deliberate overdose

Bin Li  1 , Ivan Ricordel, Lawrence M Schopfer, Frédéric Baud, Bruno Mégarbane, Patrick Masson, Oksana Lockridge
Affiliations

Dichlorvos, chlorpyrifos oxon and Aldicarb adducts of butyrylcholinesterase, detected by mass spectrometry in human plasma following deliberate overdose

Bin Li et al. J Appl Toxicol. 2010 Aug.

Abstract

The goal of this study was to develop a method to detect pesticide adducts in tryptic digests of butyrylcholinesterase in human plasma from patients poisoned by pesticides. Adducts to butyrylcholinesterase in human serum may serve as biomarkers of pesticide exposure because organophosphorus and carbamate pesticides make a covalent bond with the active site serine of butyrylcholinesterase. Serum samples from five attempted suicides (with dichlorvos, Aldicarb, Baygon and an unknown pesticide) and from one patient who accidentally inhaled dichlorvos were analyzed. Butyrylcholinesterase was purified from 2 ml serum by ion exchange chromatography at pH 4, followed by procainamide affinity chromatography at pH 7. The purified butyrylcholinesterase was denatured, digested with trypsin and the modified peptide isolated by HPLC. The purified peptide was analyzed by multiple reaction monitoring in a QTRAP 4000 mass spectrometer. This method successfully identified the pesticide-adducted butyrylcholinesterase peptide in four patients whose butyrylcholinesterase was inhibited 60-84%, but not in two patients whose inhibition levels were 8 and 22%. It is expected that low inhibition levels will require analysis of larger serum plasma volumes. In conclusion, a mass spectrometry method for identification of exposure to live toxic pesticides has been developed, based on identification of pesticide adducts on the active site serine of human butyrylcholinesterase.

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Figures

Figure 1
Figure 1
Structures of pesticides and the adducts formed by covalent binding to Serine 198 of human BChE. Dichlorvos and aged chlorpyrifos oxon adducts have the same added mass of +108. Aldicarb and Baygon give the same BChE adduct with an added mass of +57. The unlabeled active site peptide of BChE produced by digestion with trypsin has a monoisotopic mass of 2928.5 amu.
Figure 2
Figure 2
MRM transitions for the aged dichlorvos labeled tryptic BChE peptide. The quadruply-charged parent ion has a mass to charge ratio of 756.8 m/z and yields singly-charged product ions at 1001.5 and 1088.2 amu. Q1 is the mass of the parent ion. Q3 is the mass of the product ion.
Figure 3
Figure 3
MSMS spectrum of the aged dichlorvos labeled tryptic peptide of BChE derived from serum of a dichlorvos poisoned patient. The quadruply charged parent ion has 756.6 m/z. The y9 and y10 transition ions are present. In addition a series of b and y ions are present that fit the masses of the active site tryptic peptide of BChE. The doubly-charged y22+2 ion at 1145.2 m/z carries the aged dichlorvos (monomethoxyphosphate) on Ser 198. The triply and doubly charged ions labeled with the symbol Δ contain dehydroalanine in place of Ser 198. They have lost the OP plus a molecule of water during collision with gas molecules in the mass spectrometer. Facile loss of the organophosphorus agent plus a molecule of water to produce dehydroalanine is a characteristic feature of modified serine. The masses of the ions labeled with the symbol Δ support the conclusion that the modified residue is Ser 198.
Figure 4
Figure 4
MSMS spectrum of the aged chlorpyrifos oxon labeled BChE tryptic peptide from serum taken from the patient exposed to an unknown poison. The quadruply charged parent ion of 760.1 m/z is consistent with aged chlorpyrifos oxon labeled BChE peptide.
Figure 5
Figure 5
MSMS spectrum of the Aldicarb labeled BChE tryptic peptide from serum taken from the Aldicarb poisoned patient. The quadruply charged parent ion has a mass to charge ratio of 747.1 m/z.
Figure 6
Figure 6
Spontaneous reactivation of Aldicarb-inhibited BChE. BChE activity in Aldicarb poisoned sera was measured with 1 mM butyrylthiocholine in 1 ml of 0.1 M potassium phosphate pH 7.0 in the presence of 0.5 mM dithiobisnitrobenzoic acid by recording increase in absorbance at 412nm. BChE activity was 0.07 units/ml at the beginning of the measurement and was 0.34 units/ml at 23 min.
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