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 Journal of Health Laboratory Technology,Jan2008;ld18No1【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,张立2,李艳霞3,王友沛,王贤理3.温州医学院药学院浙江温州35035:4浙江省苍南县人民医院渐江温州35035(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25800;5.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Tms-hCl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100mg/L,批内变异系数(CV)小于28%,批间变异系数(CV)小于39%,平均回收率为998%。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159.1μmo/L、甘油三酯74mmo/L、血红蛋白116gL、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;乙醇脱氨酶法;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)01-007-03An improved method for determination of a small quantity of alcohol in serum with thealcohol dehydrogenaseHu Yun-liang, Wang Hui-yan, Zhang L, Li Yan-xia, Wang You-pei, Wang Xian-di(1. Department of Laboratory Medicine of the Second Affiliated Hospital, Wenzhou Medical College, Wenzho2. School of Laboratory Medicine, Wenzhou Medical College, Wenzhou 325027, China; 3. School of PharmacyCollege, Wenzhou 325027, China; 4. The Peoples Hospital of Cangnan, Cangnan 325800, China; 5. Yilik20Co 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'was9g/L The linearity was up to 1200 me/L The within run CV was lower than 2.8% and the between run CV was lowerthan 3.9%. The average recovery was 99.8%. Comparing this method (Y) with gas chromatography(X), the regression equationwas obtained, Y=0.993X-9 76, r=0. 998. The assays showed no interference from as much as 159. I 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, speIt isKey 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、放射性能索通过改进试剂配方调整反应条件及分析参数使其检测乙醇在准确度、特异性及稳定性方面有一定程度的改善,现报[基金项目]温州市科技局课题(C2002055道如下:[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 Chinese Journal of Health Laboratory Technology,lan2008;vl18Nol1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 EP6-A文件作线性评价”。在200-1200mg/L范选用三(羟甲基)氨基甲烷~盐酸(Tns-HC)缓冲液体围内稀释变异