改良乙醇脱氢酶法测定血清中微量乙醇

中国卫生检验杂志2008年1月第18卷第1期 Chinese Joumal of Health Laboratory Technology,Jan2008;l18No1【论著】改良乙醇脱氢酶法测定血清中微量乙醇胡云良2,王慧燕1,张立2,李艳霞3,王友沛,王贤理(1.温州医学院附属第二医院检验科浙江温州325027;2.温州医学院检验医学院浙江温州3250353.温州医学院药学院浙江温州325035;4.浙江省苍南县人民医院浙江苍南3258005.温州伊利康生物技术有限公司,浙江温州325011)[摘要]目的建立一个简便灵敏特异试剂稳定的乙醇脱氢酶法检测血清中微量乙醇浓度。方法选择Tis-HC缓冲液作为乙醇脱氢酶法测定乙醇的反应条件通过优化反应体系及自动生化分析仪分析参数采用两点终点法对乙醇进行检测并评价了方法学性能。结果乙醇脱氢酶(ADH)最适用量为1.2g/L,氧化型辅酶I(NAD·)最适浓度为9g/L。线性范围可达1200mg/L,批内变异系数(CV)小于28%,批间变异系数(Cv)小于3.9%,平均回收率为9.8%。本法(Y与气相色谱法(X)比较,具有良好的相关性,Y=0.993x-9.76,r=0.998。胆红素159.1umoL、甘油三酯7.4mmo/L、血红蛋白11.6g/L、乙醛0.3g/L、异丙醇0.2g/L、甲醇0.7g/L、甲醛0.6g/L以下对本法测定乙醇结果无明显干扰。结论:本法测定血清中乙醇无需除蛋白,具有快速、简便、灵敏特异等优点适合于自动生化分析仪检测。[关键词]乙醇;乙醇脱氢酶法;两点终点法;自动生化分析[中图分类号]R446.1文献标识码]B[文章编号]1004-8685(2008)01-0047-03An improved method for determination of a small quantity of alcohol in serum with thealcohol dehydrogenaseHu Yun-liang", Wang Hui- yan, Zhang Li, Li Yan-xia, Wang You-pei, Wang Xian-li(1. Department of Laboratory Medicine of the Second Affiliated Hospital, Wenzhou Medical College, Wenzhou 325027, China;2. School of Laboratory Medicine, Wenzhou Medical College, Wenzhou 325027, China; 3. School of Pharmacy, Wenzhou MedicalCollege, Wenzhou 325027, China; 4. The People's Hospital of Cangnan, Cangnan 325800, China; 5. Yilikang BiotechnologyCo Ltd, Wenzhou 325011, China)Abstract] Objective: To establish a simple, sensitive, specific method of determination of alcohol in serum with stable rea-gents by catalysis of alcohol dehydrogenase. Methods: Tris-HCI buffer was used in this study. In the condition of alkalescencealcohol dehydrogenase(ADH)catalyzed the substance of alcohol to acetaldehyde, with the simultaneous production of deoxidizednicotinamide adenine dinucleotide(NADH). To analyze alcohol by two point end assay after optimizing reaction system and assayparameter of automatic analyzer. Results: The most adaptive quantity of ADH was 1. 2 g/L, and the best concentration of theNAD.*was g/L The linearity was up to 1200 mg/L The within run CV was lower than 2. 8% and the between run CV was lowerthan3.9%.Theaveragerecoverywas99.8%.comparingthismethod(y)withgaschromatography(x),theregressionequationwas obtained, Y=0.993X-9.76, r=0.998. The assays showed no interference from as much as 159. 1 umoV/L of bilirubin7.4 mmol/L of triglyceride, 11.6 g/L of hemoglobin, 0. 3 g/L of acetaldehyde, 0. 2 g/L of isopropanol, 0.7 g/L of methanoland 0.6 g/L of maldehyde. Conclusion: The method established for determining alcohol in serum is a rapid, simple, sensitive, spe-without being deproteinized. It is suiKey words] Alcohol; Alcohol dehydrogenase Two point end assay; Automatic analysis体液中乙醇含量测定是酒精中毒的重要判断依据,通过量衰竭技术及气相色谱法等。其中气相色谱法是测定乙醇的对摄入酒精后血中乙醇含量的测定,可方便迅速地监测和研参考方法,具有较高的灵敏度与特异性,但设备昂贵操作复究乙醇在体内的代谢过程,从而为预防和减轻酒精中毒的研杂而其它方法在稳定性、灵敏度特异性等方面都存在不同究提供相应的指标和依据。测定乙醇的方法主要有化学比色程度的缺陷。我们对乙醇脱氢酶法测定乙醇进行了方法学探法呼出气乙醇分析、渗透压测定法酶试剂分析法放射性能索通中国煤化工分析参数使其检测乙醇有定程度的改善,现报[基金项目]温州市科技局课题(C2002055道如CNMHG[作者简介]胡云良(1966-),男,硕士,副教授,主要从事临床生化教学与医院检验科工作中国卫生检验杂志2008年1月第18卷第1期Chin" e Journal of Health Laboratory Technolog,Jan200g;val18N11材料与方法7170型自动生化分析仪上测定,观察反应动力学,结果见图1。1.1原理按 NCCLS EP-A文件作线性评价。在200-100mL范选用三(羟甲基)氨基甲烷-盐酸(Tni-HC)缓冲液体围内稀释变异