Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950, USA.
J Appl Toxicol. 2010 Aug;30(6):559-65. doi: 10.1002/jat.1526.
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.
本研究旨在开发一种方法，以检测来自因农药中毒的患者血浆中丁酰胆碱酯酶胰蛋白酶消化物中的农药加合物。人血清中丁酰胆碱酯酶的加合物可能作为农药暴露的生物标志物，因为有机磷和氨基甲酸酯类农药与丁酰胆碱酯酶的活性部位丝氨酸形成共价键。分析了 5 例自杀未遂（敌敌畏、涕灭威、拜贡和一种未知农药）和 1 例意外吸入敌敌畏的血清样本。丁酰胆碱酯酶通过 pH4 的离子交换色谱从 2ml 血清中纯化，然后在 pH7 下进行普鲁卡因酰胺亲和色谱。将纯化的丁酰胆碱酯酶变性，用胰蛋白酶消化，并用 HPLC 分离修饰肽。用 QTRAP4000 质谱仪的多重反应监测法分析纯化的肽。该方法成功地鉴定了 4 名丁酰胆碱酯酶抑制率为 60-84%的患者的农药加合丁酰胆碱酯酶肽，但未能鉴定抑制率分别为 8%和 22%的 2 名患者。预计低抑制水平需要分析更大的血清血浆量。总之，已经开发出一种基于鉴定人丁酰胆碱酯酶活性部位丝氨酸上的农药加合物来识别活体有毒农药暴露的质谱方法。
