Quantitation of acrolein-protein adducts: Potential biomarker of acrolein exposure
Quantitation of acrolein-protein adducts: Potential biomarker of acrolein exposure
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DOI：
10.1080/15287390490276539
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发表时间：
2004-03-26
影响因子：
2.6
通讯作者：
Khan, MF
中科院分区：
文献类型：
--
作者：
Li, H;Wang, JL;Khan, MF
丙烯醛是一种α，β-不饱和醛，是一种普遍存在的环境有毒污染物。由于潜在的人体暴露，需要一种灵敏、可靠和特异的方法来监测丙烯醛暴露。丙烯醛是一种有效的亲电试剂，可与蛋白质反应。主要通过Michael加成反应，生成丙烯醛-蛋白质加合物（阿帕）。本研究旨在建立一种定量生物样品中阿帕的竞争性酶联免疫吸附测定（ELISA）方法。用丙烯醛-匙孔血蓝蛋白加合物（AAA）和天然白蛋白作为抗原，用ELISA法检测兔抗丙烯醛-匙孔血蓝蛋白加合物抗体的特异性。AAA观察到剂量依赖性反应，但与天然白蛋白无免疫反应性。此外，抗丙烯醛抗体与甲醛-、丙二醛-或4-羟基壬烯醛-白蛋白加合物的交叉反应性的缺乏表明其对丙烯醛的特异性。对于竞争性ELISA，1：16，000稀释的抗血清与不同浓度的AAA一起使用，其提供了250和10，000 pg之间的线性检测范围。为了测试该方法可能用作丙烯醛暴露的生物标志物的功效，SD大鼠口服给予I或7个剂量的9.2 mg/kg/d丙烯醛。阿帕水平，定量血清中，显示显着更大的形成（32%和58%后，1和7剂量，分别）丙烯醛处理的大鼠相比，对照组。阿帕蛋白质印迹分析表明，阿帕带（特别是29，31，和100 kD）与对照组相比，具有更大的强度，进一步支持我们的ELISA结果从丙烯醛处理的大鼠血清中。这些结果表明，阿帕的定量有可能被用作丙烯醛暴露的生物标志物，并最终用于分子剂量学和风险评估。
Acrolein, an alpha,beta-unsaturated aldehyde, is a ubiquitous environmental toxic pollutant. Because of potential human exposure, there is a need for a sensitive, reliable, and specific method to monitor acrolein exposure. Acrolein is a potent electrophile and reacts with proteins. mainly through Michael addition reaction, leading to acrolein-protein adducts (APA). The present study aimed to develop, a competitive enzyme-linked immunosorbent assay (ELISA) method for the quantitation of APA in biological samples. Antibody to acrolein-keyhole limpet hemocyanin adduct was raised in rabbits, and the specificity of the antibody was determined by ELISA using acrolein-albumin adduct (AAA) or native albumin. A dose-dependent response was observed with AAA, but no immunoreactivity with native albumin. Further, lack of cross-reactivity of anti-acrolein antibody with formaldehyde-, malondialdehyde- or 4-hydroxynonenal-albumin adducts indicates its specificity for acrolein. For the competitive ELISA, 1:16,000 diluted antisera was used with varying concentrations of AAA, which provided a linear detection range between 250 and 10,000 pg. To test the efficacy of the method for possible use as a biomarker of acrolein exposure, SD rats were orally administered I or 7 doses of 9.2 mg/kg/d acrolein. APA levels, quantitated in the serum, showed significantly greater formation (32% and 58% after 1 and 7 doses, respectively) in acrolein-treated rats as compared to the controls. Western blot analyses of APA in the sera from acrolein-treated rats showed APA bands (especially 29, 31, and 100 kD) with greater intensity in comparison to controls, further supporting our ELISA results. These results suggest that quantitation of APA has potential to be used as biomarker of acrolein exposure and eventually for molecular dosimetry and risk assessment.