一种氧化应激生物标志物定量分析系统的建立及应用

摘要

OBJECTIVE: To develop a detection method for free radical scavenging capacity in biological samples,based on the oxygen radical absorbance capacity (ORAC) method,which was applied to evaluate the biological antioxidant capacity and to classify the extent of environmental pollution and toxic effects. METHODS:Zebrafish was chosen as the experimental animal,to test the hydroperoxyl free radical (ROO·). The azo compound 2,2´-azobis(2-amidinopropane) dihydrochloride (AAPH) was chosen as the oxygen free radical source. Fluorescein (FL) as the fluorescence indicator and Trolox as the quantitative standard. Then fluorescence intensity decay process was recorded after free radicals reacted with FL,and the ability of antioxidant delay the fluorescence intensity recession was assessed to evaluate the effect of chemicals on ORAC. The established method was applied to study the effect of different concentrations of four heavy metals (copper,cadmium,chromium and lead) on ORAC of zebrafish.RESULTS:The established method showed high specificity and good accuracy (between 97% and 108%) and precision (CV less than 5%);this method also had good reproducibility (CV is 2.92%). The limit of quantitation (LOQ) and the limit of detection 2(LOD) were 3.03 and 1.00μmol Trolox/mg protein,respectively,the correlation coefficient(R) was ≥ 0.99. According to the new established quantitative analysis methods,we determined the physiological normal range (0-50μmol Trolox/mg protein) and classified the heavy metal contamination degree and toxic effects based on the effect of metals on ORAC of zebrafish.CONCLUSION:The established method may be suitable for evaluating biological antioxidant capacity and classifying environmental pollution and toxic effects. It could be effective in enhancing the inter laboratory data recognition and promoting the application of biomarkers for antioxidant protection in environmental pollution monitor and environmental risk assessment.%目的：以抗氧化能力指数(ORAC)检测方法为基础，建立一种机体清除自由基能力的检测方法，并应用于生物抗氧化能力的评价及对环境污染物污染程度和毒性效应进行评级和分类。方法：选用斑马鱼作为试验生物，针对过氧自由基(ROO·)进行检测，以偶氮化合物2，2´-偶氮-双-(2-脒基丙烷)氯化二氢(AAPH)作为氧自由基来源，荧光素钠(FL)为荧光指示剂，并以维生素E类似物Trolox为定量标准，观察自由基与荧光素钠作用后荧光强度的衰减过程，检测各种物质加入反应体系后延缓荧光素钠荧光强度衰减的能力，以此评价反应体系中抗氧化剂的抗氧化能力，并应用此法研究不同浓度的4种重金属(铜，镉，铬，铅)对斑马鱼抗氧化能力指数的影响。结果：所建立的定量分析方法特异性强，精密度高，变异系数小于5%；准确度在97%~108%之间；重现性佳(变异系数是2.92%)；定量检出限和最低检测限分别为3.03和1.00μmol Trolox/mg，可接受相关系数()≥0.99。依据所建立的定量分析方法对斑马鱼ORAC生理正常值进行检测，正常值范围为0~50μmol Trolox/mg，并根据上述4种重金属对斑马鱼ORAC的影响，对其污染程度和毒性效应进行了评级和分类。结论：此法适用于生物抗氧化能力的评价和环境污染程度和毒性效应分级，提高了试验数据互认程度，推动了抗氧化防御系统生物标志物在环境污染监测和环境风险评价中的应用。