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单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,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,正常组织,异常组织,心脏 肝脏,心脏 肝脏,细胞,中酶活力,GOT,高,GOT/GPT,高,细胞破损酶溢出,血清,中酶活力,GOT,低,GOT/GPT,低,GOT,升高,GOT/GPT,升高,5.,谷丙转氨酶的临床意义,如果,GPT,血清值超过正常上限,2,3,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,试剂 管号,0,1,2,3,4,5,丙酮酸,标准液，,ml,SALT,底物溶液,，,ml,磷酸缓冲液，,ml,0.00,0.50,0.10,0.05,0.45,0.10,0.10,0.40,0.10,0.15,0.35,0.10,0.20,0.30,0.10,0.25,0.25,0.10,37,水浴中保温,10min,2,4-,二硝基苯肼,0.50,0.50,0.50,0.50,0.50,0.50,37,水浴保温,20min,氢氧化钠,ml,5,5,5,5,5,5,室温下静置,10min,蒸馏水调零点，于,520nm,处测吸光度,为什么在制作标准曲线时要加入底物溶液,为什么加入底物溶液的体积不同，对反应没有影响,怎么计算丙酮酸的质量,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,试剂,测定管,对照管,血清，,ml,SALT,底物溶液，,ml,0.1,0.5,0.1,_,37,水浴,30min,2,4-,二硝基苯肼溶液，,ml,SALT,底物溶液，,ml,0.5,_,0.5,0.5,37,水浴,20min,0.4mol/L,氢氧化钠溶液，,ml,5.0,5.0,室温下静置,10min,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,SALT,活力,=,测定管微克数,对照管微克数,2.50.1,（单位）,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 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 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,tubes,Add standard,pyrivic acid,solution,SALT substrate solution and phosphate buffer,Water bathing at,37,for 10,Add 2,4-,dinitrate phenylhydrazine,and water bath at,37,for 20,Add NaOH solution and keep in room temperature for 10,Assay at 520nm,（,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,
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