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1、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 a
2、minotransferase 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 di
3、nitrate 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
4、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 ALT glutamic oxaloacetic transaminase
5、GOT aspartate transaminase, AST enzyme activity,serum Aminotransferase/ transaminase catalyze alanine -ketoglutarate glutamic acid pyruvic acid alkali substrate,ABSTRACT,Observing Enzyme Activity Changes of Alanine Aminotransferase in Sample Abstract: Objetcive: To determine the activity of serum AL
6、T 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 aminotransfe
7、rase， 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 keep in room temperature for 10,Add NaOH solution and keep in room temperature for 10,Assay at 520nm,Assay at 520nm,