生化试验观察血清中谷丙转氨酶的活力变化

实验六 观察血清中谷丙转氨酶的活力变化,Experiment 6 Observing Enzyme Activity Changes of Alanine Aminotransferase in Sample,一、目的要求,1、了解血清谷丙转氨酶活力变化的意义2、掌握血清谷丙转氨酶活力的测定方法,二、相关知识点,1. 转氨作用 transamination,2. 氨基转移酶 转氨酶（aminotransferase，transaminase） 催化氨基酸与酮酸之间氨基转移的一类酶,谷丙转氨酶 glutamic-pyruvic transminase GPT 又称丙氨酸转氨酶alanine aminotransferase ALT,谷草转氨酶 glutamic oxaloacetic transaminase GOT又称天冬氨酸转氨酶 aspartate transaminase, AST,3.转氨作用的机制：,4转氨作用的意义正常时转氨酶主要分布在细胞内，血清中酶活力很低GOT以心脏细胞中活力最大，其次为肝脏细胞GPT则以肝脏细胞中活力最大,5.谷丙转氨酶的临床意义如果GPT血清值超过正常上限23倍，并持续两周以上，表明有肝胆疾病存在的可能。ALT的正常上限是40单位，2.5倍为100单位，20倍为800单位。,三、实验原理,SUBSTRATE,Pyruvate dinitrate phenylhydrazone,2,4-dinitrate phenylhydrazine,alanine,-ketoglutarate,Red brown,pyruvic acid,pyrivic acid,glutamic acid,SALT单位的定义是：1ml 血清在37与底物作用30min，每产生2.5g丙酮酸所需酶的量为一个SALT单位。,四、实验操作,1.标准曲线的绘制,丙酮酸微克数-吸光度标准曲线,2 SALT活力测定：洁净干燥试管4支，即测定管、对照管各2支,4根干燥试管的用途是什么？样品测定为什么要使用对照管？样品测定的酶反应怎样终止？血清样品和底物在使用前最好提前放置于水浴中预热，保证反应时的温度的准确性反应温度和计时要准确,五、注意事项,1. 2，4-二硝基苯肼可与有酮基的化合物作用形成苯腙2. 吸量应准确，严格控制反应时间和温度。3. 在测定SALT活力时，应事先将底物、血清在37水浴中恒温4. 溶血标本不宜采用，因血细胞内转氨酶活力较高，影响测定结果。5在测定时，如酶活力较大（大于100单位），应将样品稀释后再进行测定。,六、专业词汇,glutamic-pyruvic transminase GPT alanine aminotransferase ALTglutamic oxaloacetic transaminase GOT aspartate transaminase, ASTenzyme activity,serumAminotransferase/ transaminasecatalyzealanine-ketoglutarateglutamic acidpyruvic acidalkalisubstrate,ABSTRACT,Observing Enzyme Activity Changes of Alanine Aminotransferase in Sample Abstract: Objetcive: To determine the activity of serum ALT in sample. Method: SALT was measured by Reitman-Frankel colorimetric end point method Results: The normal SALT activity is ( ) unit, the abnormal SALT activity is ( ) unit. Conclusion: The activity of SALT in abnormal sample is much higher than that in normal sample Key Words: alanine aminotransferase， aspartate transaminase, enzyme activity, serum, -ketoglutarate, substrate,（1）draw calibration curve,（2）determining the activity of SALT,Normal tubes,Abnormal tubes,sera,SALT substrate solution,sera,_,Water bathing at 37 for 30,Water bathing at 37 for 30,2,4-dinitrate phenylhydrazine,2,4-dinitrate phenylhydrazine,_,SALT substrate solution,Water bathing at 37 for 20,Water bathing at 37 for 20,Add NaOH solution and k