膦酰化肟对乙酰胆碱酯酶的抑制作用及中毒酶重活化特性研究--- 基于效应标志物质谱定量分析技术

doi:10.7503/cjcu20160945

高等学校化学学报 ›› 2017, Vol. 38 ›› Issue (5): 758.doi: 10.7503/cjcu20160945

膦酰化肟对乙酰胆碱酯酶的抑制作用及中毒酶重活化特性研究--- 基于效应标志物质谱定量分析技术

谢琰, 陈佳, 徐斌, 闫珑, 唐吉军(), 谢剑炜()

军事医学科学院毒物药物研究所, 北京100850

收稿日期:2016-12-27 出版日期:2017-05-10 发布日期:2017-04-13
作者简介:联系人简介: 唐吉军, 男, 博士, 副研究员, 主要从事毒物筛查及生物毒素分析方面的研究. E-mail:tangjijun2012@163.com;谢剑炜, 男, 博士, 研究员, 博士生导师, 主要从事毒物药物分析、质谱分析和适配体技术方面的研究. E-mail:xiejw@bmi.ac.cn
基金资助:
国家自然科学基金(批准号: 81202248, 81302473)资助

Studies on Poisoning Characteristics of Phosphylated Oxime on AChE and Efficacy of Reactivators Based on Quantitative Determination of Effect Biomarkers by Liquid Chromatograph-mass Spectrometry^†

XIE Yan, CHEN Jia, XU Bin, YAN Long, TANG Jijun^*(), XIE Jianwei^*()

Institute of Pharmacology Toxicology, Academy of Military Medical Sciences, Beijing 100850, China

Received:2016-12-27 Online:2017-05-10 Published:2017-04-13
Contact: TANG Jijun,XIE Jianwei E-mail:tangjijun2012@163.com;xiejw@bmi.ac.cn
Supported by:
† Supported by the National Natural Science Foundation of China(Nos.81202248, 81302473)

摘要/Abstract

摘要：

将对氧磷(Paraoxon)与肟类重活化剂双复磷(Obidoxime)反应制得膦酰化肟(DEP-obidoxime); 利用基于效应标志物的质谱定量技术研究了膦酰化肟对乙酰胆碱酯酶(AChE)的抑制作用及中毒酶的重活化特性. 结果表明, 膦酰化肟具有极强的AChE抑制毒性, 但因膦酰化肟中毒酶与原有机磷毒物中毒酶结构相同, 故其膦酰化AChE仍可被经典重活化剂[如氯解磷定(Pralidoxime)、双复磷(Obidoxime)及酰胺磷定(HI-6)]重活化, 根据EC₅₀表征结果, 这3种重活化剂的重活化效果强弱顺序依次为氯解磷定>HI-6>双复磷.

关键词: 有机磷农药, 肟类化合物, 膦酰化肟, 生物标志物, 液相色谱-质谱联用

Abstract:

Organophosphorus compounds(OPs) have been extensively utilized throughout the world for many years, however, the security problems are always concerned due to the high toxicity. OPs primarily inhibit acetylcholinesterase(AChE) in nerve system and will cause cholinergic crisis in the synaptic cleft. Enzyme reactivatorsd representing oxime derivatives of quarternary pyridinium compounds are considered as specific medications. Recently, the exact clinical effectiveness of oxime reactivators is a matter of controversy. Although oxime reactivators can recover the activity of AChE and improve neuromuscular transmission, some study reports found oximes could also react with OPs and formmore potent inhibitors of AChE during the process of reactivation, which may intoxicate the reactivated AChE once again, even worsen the poisoning of enzyme due to the high reactivity. In this paper, paraoxon as an example of OPs and obidoxime representing a typical oxime were chosen to demonstrate the reactivation efficacy of oximes in the treatment of OPs poisoning. The reaction product diethylphosphoryl obidoxime(DEP-obidoxime) was first prepared. Then, the poisoning characteristics of phosphylated oxime on AChE and efficacy of reactivators were studied via quantitative determination of the effect biomarkers formed between paraoxon and AChE. The experimental results confirmed that DEP-obidoxime can intoxicate AChE in extremely short time, fortunately, the poisoned AChE by DEP-obidoxime can still be reactivated by typical oximes[pralidoxime, pyramidoxime monohydrate(HI-6) and obidoxime]. EC₅₀ values of each oxime were determined and indicated that the reactivation effectiveness of the above oximes as the following ranking: pralidoxime>HI-6>obidoxime.

Key words: Organophosphorus compound, Oxime, Phosphoryl oxime, Biomarker, Liquid chromatograph-mass spectrometry(LC-MS)

中图分类号:

O627.51

TrendMD:

谢琰, 陈佳, 徐斌, 闫珑, 唐吉军, 谢剑炜. 膦酰化肟对乙酰胆碱酯酶的抑制作用及中毒酶重活化特性研究--- 基于效应标志物质谱定量分析技术. 高等学校化学学报, 2017, 38(5): 758.

XIE Yan, CHEN Jia, XU Bin, YAN Long, TANG Jijun, XIE Jianwei. Studies on Poisoning Characteristics of Phosphylated Oxime on AChE and Efficacy of Reactivators Based on Quantitative Determination of Effect Biomarkers by Liquid Chromatograph-mass Spectrometry^†. Chem. J. Chinese Universities, 2017, 38(5): 758.

图/表 5

Scheme 1 Formation mechanism of DEP-obidoxime by reaction of paraoxon with obidoxime

Fig.1 HPLC separation chromatogram of the mixture of obidoxime and paraoxon(λ=260 nm)(A) and QTOF-MS spectrum of DEP-obidoxime(B)

Fig.2 Product ion spectrum of the diethylphosphoryl adducted peptides containing active site serine residue(X: diethylphosphoryl)

Fig.3 Inhibition degree of AChE by 200 nmol/L paraoxon(a) and 20 nmol/L DEP-obidoxime(b) over time

Table 1 EC50 values of oximes in reactivating paraoxon-inhibited AChE

Oxime	EC₅₀/(μmol·L^-1)	95% Confidence intervals of EC₅₀/(μmol·L^-1)
Pralidoxime	7.94	6.817—9.245
HI-6	37.39	31.65—44.17
Obidoxime	61.13	49.64—75.27

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膦酰化肟对乙酰胆碱酯酶的抑制作用及中毒酶重活化特性研究--- 基于效应标志物质谱定量分析技术

Studies on Poisoning Characteristics of Phosphylated Oxime on AChE and Efficacy of Reactivators Based on Quantitative Determination of Effect Biomarkers by Liquid Chromatograph-mass Spectrometry^†

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