生化試驗(yàn)觀察血清中谷丙轉(zhuǎn)氨酶的活力變化

1、實(shí)驗(yàn)六 觀察血清中谷丙轉(zhuǎn)氨酶的活力變化Experiment 6 Observing Enzyme Activity Changes of Alanine Aminotransferase in Sample1一、目的要求1、了解血清谷丙轉(zhuǎn)氨酶活力變化的意義2、掌握血清谷丙轉(zhuǎn)氨酶活力的測(cè)定方法2二、相關(guān)知識(shí)點(diǎn)1. 轉(zhuǎn)氨作用 transamination32. 氨基轉(zhuǎn)移酶 轉(zhuǎn)氨酶（aminotransferase，transaminase） 催化氨基酸與酮酸之間氨基轉(zhuǎn)移的一類酶 4谷丙轉(zhuǎn)氨酶 glutamic-pyruvic transminase GPT 又稱丙氨酸轉(zhuǎn)氨酶alanine amin

2、otransferase ALT5谷草轉(zhuǎn)氨酶 glutamic oxaloacetic transaminase GOT又稱天冬氨酸轉(zhuǎn)氨酶 aspartate transaminase, AST63.轉(zhuǎn)氨作用的機(jī)制：74轉(zhuǎn)氨作用的意義正常時(shí)轉(zhuǎn)氨酶主要分布在細(xì)胞內(nèi)，血清中酶活力很低GOT以心臟細(xì)胞中活力最大，其次為肝臟細(xì)胞GPT則以肝臟細(xì)胞中活力最大895.谷丙轉(zhuǎn)氨酶的臨床意義如果GPT血清值超過(guò)正常上限23倍，并持續(xù)兩周以上，表明有肝膽疾病存在的可能。ALT的正常上限是40單位，2.5倍為100單位，20倍為800單位。10三、實(shí)驗(yàn)原理SUBSTRATEPyruvate dinitrate p

3、henylhydrazone2,4-dinitrate phenylhydrazinealanine-ketoglutarateRed brownpyruvic acidpyrivic acid glutamic acid11SALT單位的定義是：1ml 血清在37與底物作用30min，每產(chǎn)生2.5g丙酮酸所需酶的量為一個(gè)SALT單位。12四、實(shí)驗(yàn)操作1.標(biāo)準(zhǔn)曲線的繪制試劑 管號(hào)012345丙酮酸標(biāo)準(zhǔn)液，mlSALT底物溶液，ml磷酸緩沖液，ml0.000.500.100.050.450.100.100.400.100.150.350.100.200.300.100.250.250.1037水

4、浴中保溫10min2,4-二硝基苯肼 0.500.500.500.500.500.5037水浴保溫20min 氫氧化鈉ml555555室溫下靜置10min 蒸餾水調(diào)零點(diǎn)，于520nm處測(cè)吸光度 為什么在制作標(biāo)準(zhǔn)曲線時(shí)要加入底物溶液為什么加入底物溶液的體積不同，對(duì)反應(yīng)沒有影響怎么計(jì)算丙酮酸的質(zhì)量13丙酮酸微克數(shù)-吸光度標(biāo)準(zhǔn)曲線142 SALT活力測(cè)定：潔凈干燥試管4支，即測(cè)定管、對(duì)照管各2支 試劑測(cè)定管對(duì)照管血清， mlSALT底物溶液， ml0.10.50.1_37水浴30min2,4-二硝基苯肼溶液， mlSALT底物溶液， ml0.5_0.50.537水浴20min0.4mol/L氫氧化鈉

5、溶液， ml5.05.0室溫下靜置10min 4根干燥試管的用途是什么？樣品測(cè)定為什么要使用對(duì)照管？樣品測(cè)定的酶反應(yīng)怎樣終止？血清樣品和底物在使用前最好提前放置于水浴中預(yù)熱，保證反應(yīng)時(shí)的溫度的準(zhǔn)確性反應(yīng)溫度和計(jì)時(shí)要準(zhǔn)確15SALT活力=測(cè)定管微克數(shù)對(duì)照管微克數(shù) 2.50.1 （單位） 16五、注意事項(xiàng)1. 2，4-二硝基苯肼可與有酮基的化合物作用形成苯腙2. 吸量應(yīng)準(zhǔn)確，嚴(yán)格控制反應(yīng)時(shí)間和溫度。3. 在測(cè)定SALT活力時(shí)，應(yīng)事先將底物、血清在37水浴中恒溫4. 溶血標(biāo)本不宜采用，因血細(xì)胞內(nèi)轉(zhuǎn)氨酶活力較高，影響測(cè)定結(jié)果。5在測(cè)定時(shí)，如酶活力較大（大于100單位），應(yīng)將樣品稀釋后再進(jìn)行測(cè)定。17六

6、、專業(yè)詞匯 glutamic-pyruvic transminase GPT alanine aminotransferase ALTglutamic oxaloacetic transaminase GOT aspartate transaminase, ASTenzyme activityserumAminotransferase/ transaminasecatalyzealanine-ketoglutarateglutamic acidpyruvic acidalkalisubstrate18ABSTRACT Observing Enzyme Activity Changes of A

7、lanine 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

8、in abnormal sample is much higher than that in normal sample Key Words: alanine aminotransferase， aspartate transaminase, enzyme activity, serum, -ketoglutarate, substrate,19（1）draw calibration curvetubesAdd standard pyrivic acid solution, SALT substrate solution and phosphate bufferWater bathing at

9、 37 for 10Add 2,4-dinitrate phenylhydrazine and water bath at 37 for 20Add NaOH solution and keep in room temperature for 10Assay at 520nm20（2）determining the activity of SALTNormal tubesAbnormal tubesseraSALT substrate solutionsera_Water bathing at 37 for 30Water bathing at 37 for 302,4-dinitrate phenylhydrazine2,4-dinitrate phenylhydrazine_SALT substrate solutionWater bathing at 37 for 20Water bathing a