基于探针底物法的高效液相色谱-串联质谱法研究蚯蚓体内CYP1A2、CYP2C9与CYP3A4酶活力  

Study of Enzymatic Activities of CYP1A2,CYP2C9 and CYP3A4 in Earthworms by HPLC-MS/MS Based on Probe Substrate Method

作　　者：杨晓霞[1] 龚久平[1] 杨俊英[1] 李典晏 张雪梅[1] 李必全[1] 柴勇[1] YANG Xiaoxia;GONG Jiuping;YANG Junying;LI Dianyan;ZHANG Xuemei;LI Biquan;CHAI Yong(Institute of Agricultural Quality Standard and Testing Technology,Chongqing Academy of Agricultural Sciences,Chongqing 401329,China)

机构地区：[1]重庆市农业科学院农业质量标准与检测技术研究所

出　　处：《理化检验（化学分册）》2019年第10期1117-1125,共9页Physical Testing and Chemical Analysis(Part B:Chemical Analysis)

基　　金：“食品安全研发技术”重大研发项目(2017YFC1602004);重庆市基础研究与前沿探索项目(cstc2018jcyjAX0613,cstc2018jAX0623);重庆市绩效激励引导专项项目(cstc2018jxjl20002);重庆市基本科研项目(2016cstc-jbky-00523)

摘　　要：按规定方法解剖蚯蚓取得其内脏,经清洗后转移至盛有4mL匀浆缓冲液的组织研磨器中均匀破碎,并将匀浆体积定为6.0mL。将此匀浆液通过差速离心法取其微粒体(即第2次离心后的沉淀物)用3mL保存缓冲液重新悬浮,制得蚯蚓微粒体蛋白悬浮液。将此悬浮液100μL加入于含孵育体系650μL及探针底物混合液(含非那西汀100μmmol·L^-1、双氯芬酸钠100μmmol·L^-1和睾酮25μmmol·L^-1)250μL中,升温至37℃,孵化20min,加入甲醇200μL中止反应。离心10min,取其上清液,经0.22μm滤膜过滤。所得滤液流经Acquity HPLC C18色谱柱(50mm×2.1mm,1.8μm),并用不同体积比的(A)每升溶液中含甲酸0.5mL的1mmol·L^-1乙酸铵溶液和(B)甲醇组成的混合液作为流动相进行梯度洗脱,使所加入的3种底物被蚯蚓体内的细胞色素P450亚酶的催化作用而相应产生的3种特异代射物分离。然后在电喷雾正离子源和多反应监测模式条件下,用串联质谱法测得其含量。结果表明:所述3种代谢物(对乙酰氨基酚、4′-羟基双氯芬酸、6β-羟基睾酮)的线性范围依次在0.40,0.20,10.0μg·L^-1以内,检出限(3S/N)依次为0.008,0.005,0.1μg·L^-1。根据所测得3种代谢物的生成量可分别评价蚯蚓体内3种相应的亚酶CYP1A2、CYP2C9和CYP3A4活力。此外,还对受敌敌畏染毒的土壤中培养3d和14d后的蚯蚓样品,按上述方法分析,获得了染毒的土壤对蚯蚓体内上述3种CPY亚酶活力的变化情况。所得结果可作为土壤污染全面诊断的生物标记。Earthworm sample was dissected by the given method and the internal organs obtained were homogenized with 4 mL of homogeneous buffer mixture giving a homogenate having a volume of 6.0 mL.Microsomes(i.e.,the precipitate settled out in the 2 nd centrifugation)were separated from the homogenate by differential centrifugation.The microsomes were re-suspended in 3 mL of preservation buffer mixture to give the microsome protein suspension of earthworm.An aliquot of 100μL of this suspension was taken and incubated in650μL of an incubation system and 250μL of probe substrate solution(containing 100μmol·L^-1 of phenacetin,100μmol·L^-1 of diclofenac sodium and 25μmol·L^-1 of testosterone)at 37 ℃ for 20 min,when 200μL of methanol were added to stop the incubation.After centrifugation for 10 min,the supernatant was taken and filtered through 0.22μm filtering membrane.The filtrate was passed through Acquity HPLC C18 chromatographic column(50 mm×2.1 mm,1.8μm)with the sample solution introduced and using mixtures of various volumic ratios of(A)1 mmol·L^-1 NH4 OAc solution containing 0.5 mL of formic acid perliter,and(B)methanol,as mobile phases in the gradient elution to separate the 3 specific metabolites(i.e.,acetaminophen,4′-hydroxy diclofenac and 6β-hydroxyl testosterone)produced respectively by the catalytic actions of the sub-enzymes(i.e.,CYP1 A2,CYP2 C9 and CYP3 A4)existing in earthworm body on the respective substracts,and then the metabolites were determined by MS/MS under the condition of ESI+and MRM.Linearity ranges of the 3 metabolites were found within 0.40,0.20,10.0μg·L^-1 with detection limits of 0.008,0.005,0.1μg·L^-1 respectively.Based on the amounts of the3 metabolites produced,the activity for the 3 sub-enzymes existing in body of the earthworm could be evaluated.Furthermore,a study on the variation of activity of the 3 CPY sub-enzymes of earthworms which were breeded in soil contaminated by DDVP for 3 dand 14 dwas made by the above method.Beneficial results were obtained,which can be u

关 键 词：高效液相色谱-串联质谱法 酶活力 蚯蚓 对乙酰氨基酚 4′-羟基双氯芬酸 6β-羟基睾酮 

分 类 号：O657.63[理学—分析化学]