Albumin, a new biomarker of organophosphorus toxicant exposure, identified by mass spectrometry
Albumin, a new biomarker of organophosphorus toxicant exposure, identified by mass spectrometry
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DOI：
10.1093/toxsci/kfi023
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发表时间：
2005-02-01
影响因子：
3.8
通讯作者：
Lockridge, O
中科院分区：
文献类型：
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作者：
Peeples, ES;Schopfer, LM;Lockridge, O
接触有机磷毒物 (OP) 的经典实验室测试是抑制血液中的乙酰胆碱酯酶 (AChE) 和丁酰胆碱酯酶 (BChE) 活性。在寻找 OP 暴露的新生物标志物时，我们用生物素化有机磷剂 FP-生物素治疗小鼠。通过与亲和素-琼脂糖结合来纯化肌肉中的生物素化蛋白质，通过凝胶电泳分离，用胰蛋白酶消化，并在四极飞行时间质谱仪上根据其碎片模式进行鉴定。发现白蛋白和 ES1 羧酸酯酶 (EC 3.1.1.1) 是 FP-生物素的主要靶标。通过将白蛋白和羧酸酯酶活性染色的非变性凝胶上的条带图案与与链霉亲和素 Alexa-680 杂交的印迹上的条带图案进行比较，还可以在小鼠血浆中鉴定出这些 FP 生物素化蛋白质。通过发现酶活性被抑制 50-80%，在小鼠血浆中鉴定出另外两个 FP-生物素靶标：AChE (EC 3.1.1.7) 和 BChE (EC 3.1.1.8)。小鼠血浆中含有另外 8 个 FP 生物素化条带，其身份尚未确定。人血浆体外实验表明，毒死蜱、乙硫磷、马拉氧磷、对氧磷、甲基对氧磷、重氮磷、二异丙基氟磷酸盐和敌敌畏与 FP-生物素竞争与人白蛋白的结合。尽管纯化白蛋白的实验先前已表明白蛋白与 OP 共价结合。这是 OP 在活体动物中与白蛋白结合的第一份报告。羧酸酯酶不是人类的生物标志物，因为人类血液中没有羧酸酯酶。结论是，与白蛋白结合的 OP 可以作为人类 OP 暴露的新生物标志物。
The classical laboratory tests for exposure to organophosphorus toxicants (OP) are inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity in blood. In a search for new biomarkers of OP exposure, we treated mice with a biotinylated organophosphorus agent, FP-biotin. The biotinylated proteins in muscle were purified by binding to avidin-Sepharose, separated by gel electrophoresis, digested with trypsin, and identified from their fragmentation patterns on a quadrupole time-of-flight mass spectrometer. Albumin and ES1 carboxylesterase (EC 3.1.1.1) were found to be major targets of FP-biotin. These FP-biotinylated proteins were also identified in mouse plasma by comparing band patterns on nondenaturing gels stained for albumin and carboxylesterase activity, with band patterns on blots hybridized with Streptavidin Alexa-680. Two additional FP-biotin targets, AChE (EC 3.1.1.7) and BChE (EC 3.1.1.8), were identified in mouse plasma by finding that enzyme activity was inhibited 50-80%. Mouse plasma contained eight additional FP-biotinylated bands whose identity has not yet been determined. In vitro experiments with human plasma showed that chlorpyrifos oxon, echothiophate, malaoxon, paraoxon, methyl paraoxon, diazoxon, diisopropylfluorophosphate, and dichlorvos competed with FP-biotin for binding to human albumin. Though experiments with purified albumin have previously shown that albumin covalently binds OP. this is the first report of OP binding to albumin in a living animal. Carboxylesterase is not a biomarker in man because humans have no carboxylesterase in blood. It is concluded that OP bound to albumin could serve as a new biomarker of OP exposure in man.