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Blood AST, ALT and UREA/BUN Level Analysis
血液中AST、ALT和UREA/BUN含量的分析   

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Abstract

AST (aspartate aminotransferase; GOT, glutamate oxalacetate transaminase) and ALT (alanine aminotransferase; GPT, glutamate pyruvate transaminase) are sensitive indicators to monitor the liver function under drugs treatment or with acute viral hepatitis. The elevated AST and ALT values in the blood sample indicate liver damage or injury. The determination of urea is the most widely used for the evaluation of kidney function. This protocol is for the quantitative determination of AST, ALT and UREA/BUN in serum and plasma on Roche automated clinical chemistry analyzers. The principle is shown below:

For AST:
α-ketoglutarate + L-aspartate  L-glutamate + oxaloacetate (AST catalyzes this equilibrium reaction)
oxaloacetate + NADH + H+  L-malate + NAD+ (malate dehydrogenase catalyzes this equilibrium reaction)
The rate of the photometrically determined NADH decrease is directly proportional to the rate of formation of oxaloacetate and thus the AST activity. The above reactions were carried out at 37 °C and measured at a wavelength of 340 nm.

For ALT:
α-ketoglutarate + L-alanine L-glutamate + pyruvate (ALT catalyzes this equilibrium reaction)
Pyruvate + NADH + H+ L-lactate + NAD+ (lactate dehydrogenase catalyzes this equilibrium reaction)
The rate of the photometrically determined NADH decrease is directly proportional to the rate of formation of pyruvate and thus the ALT activity. The above reactions were carried out at 37 °C and measured at a wavelength of 340 nm.

For UREA/BUN:
Urea + H2O → 2 NH4+ + CO2 (urea is hydrolyzed by urease)
α-ketoglutarate + NH4+ + NADH → L-glutamate + NAD+ + H2O (the presence of GLDH yields glutamate and NAD+)
The decrease in absorbance due to consumption of NADH is measured kinetically. The above reactions were carried out at 37 °C and measured at a wavelength of 340 nm.

Keywords: Liver Function(肝功能), AST(AST), ALT(中高音), Urea/BUN(尿素/面包)

Materials and Reagents

  1. Blood
  2. AST (GOT) detection kit (Roche, catalog number: 11876848216 )
  3. ALT (GPT) detection kit (Roche, catalog number: 11876805216 )
  4. UREA/BUN detection kit (Roche, catalog number: 11729691216 )
  5. Cuvette (Roche, catalog number: TA28 ) and sample cups (Roche, catalog number: 1105 )
  6. Calibrator for automated system (Roche, catalog number: 10759350 )
  7. Normal saline (0.9% w/v of NaCl) (see Recipes)

Equipment

  1. Microhematocrit blood tube (heparinized) (Assistant®, catalog number: 563 )
  2. Dropper (3 ml)
  3. Centrifuge (Eppendorf)
  4. Polypropylene test tube (Corning Incorporated, Axygen®, catalog number: MCT-150-C )
  5. Chemistry Analyzer (Roche, Cobas Mira Plus)

Procedure

  1. Collect blood from the orbital sinus with a microhematocrit blood tube (heparinized). Use dropper to push out the blood in the heparinized blood tube and collect about 300 μl blood in the 1.5 ml polypropylene test tube.
  2. Centrifuge at 1500 x g, 4 °C for 15 min. Carefully take the cell-free supernatant plasma (about half volume of blood) and place it in a properly labeled polypropylene test tube and transfer about 150 μl plasma into the sample cups.
  3. Use fresh plasma for blood AST, ALT and UREA/BUN level analysis.
  4. For analysis, it needs 100-150 μl sample volume in the sample cups. Put the sample cups containing plasma in the sample place and empty cuvettes in the detection place of Chemistry Analyzer. If the sample volume is not enough, it can be diluted with normal saline.
  5. Prepare the detection reagent according to the manufacturer’s instruction and transfer the prepared detection reagent into the reagent container for Chemistry Analyzer. It is easy to prepare the detection reagent by mixing R1 and R2 solution provided in the kit. Briefly, in AST and ALT reagent preparation, connect one bottle 1 to one bottle 1a to prepare R1 solution and then combine the volume of R1 and R2 in R1:R2 = 5:1 to get the detection reagent. For UREA/BUN detection reagent preparation, combine the volume of R1 and R2 in R1:R2 = 5:3. These mixed detection reagents are stable for 7 days at 4 °C.
  6. It needs to correct the Chemistry Analyzer with commercial calibrator before sample detection. The calibrator is as positive control and normal saline as negative control.
  7. After correction, it is ready to analyze AST, ALT and BUN levels of sample in Chemistry Analyzer.
    1. The automatic analysis steps for AST and ALT are:
      1. Aspirate 10 μl of plasma sample from sample cups into cuvette.
      2. Dilute sample with 40 μl of normal saline.
      3. Add 300 μl of detection reagent into cuvette.
      4. Begin to detect the values at 340 nm wavelength at different time points.
      5. Report the measured results.
    2. For UREA/BUN analysis, the steps are:
      1. Aspirate 4 μl of plasma sample from sample cups into cuvette.
      2. Dilute sample with 20 μl of normal saline.
      3. Add 400 μl of detection reagent into cuvette.
      4. Begin to detect the values at 340 nm wavelength at different time points.
      5. Report the measured results.

Notes

  1. The sensitivity of AST, ALT and UREA/BUN is 4 U/L, 4 U/L and 0.83 mmol/L, respectively. The measurement range of AST, ALT and UREA/BUN is 4-800 U/L, 4-600 U/L and 0.83-40.00 mmol/L, respectively.
  2. It is not necessary to repeatedly detect the values from the same sample because the program can monitor the values at different time points. If the variation of measured values in the sample is over 5%, the machine will automatically detect the sample again.
  3. In normal mice, the level of ALT, AST and UREA/BUN in plasma is ALT: 25-60 U/L; AST: 50-100 U/L; UREA/BUN: 16-30 mmol/L. The elevated values of ALT and AST indicate liver injury and high level of UREA/BUN indicates loss of kidney function.

Recipes

  1. Normal Saline (0.9% w/v of NaCl)
    1. Make sure all apparatus are clean, dry, and sterile.
    2. Weight 0.9 grams of sodium chloride. Dissolve the powder in about 80 ml distilled water by mixing gently with a stirring bar until all the powder has been dissolved. Add distilled water to a final volume of 100 ml.
    3. Transfer the prepared 0.9% sodium chloride solution (normal saline) in a storage bottle and label properly. Autoclave the solution.

Acknowledgments

This protocol was adapted from the previously published paper, Sher et al. (2009). This work was supported by Grants from NRPGM in DOH97-TD-G-111-041 (to M-C Hung) and DOH97-TD-111-TM003 (to L-Y. Li). YP Sher was also supported by a postdoctoral fellowship award from the National Health Research Institutes, Taiwan (PD9602).

References

  1. Sher, Y. P., Tzeng, T. F., Kan, S. F., Hsu, J., Xie, X., Han, Z., Lin, W. C., Li, L. Y. and Hung, M. C. (2009). Cancer targeted gene therapy of BikDD inhibits orthotopic lung cancer growth and improves long-term survival. Oncogene 28(37): 3286-3295.

简介

AST(天冬氨酸氨基转移酶; GOT,谷氨酸草乙酸转氨酶)和ALT(丙氨酸氨基转移酶; GPT,谷氨酸丙酮酸转氨酶)是监测药物治疗或急性病毒性肝炎的肝功能的敏感指标。血液样品中升高的AST和ALT值指示肝损伤或损伤。尿素的测定是最广泛地用于评价肾功能的。该方案用于在Roche自动化临床化学分析仪上定量测定血清和血浆中的AST,ALT和UREA/BUN。原则如下所示:

对于AST:
alpha-ketoglutarate + L-天冬氨酸  L-苹果酸+ NAD +苹果酸脱氢酶催化这种平衡反应)
光度测定的NADH降低的速率与草酰乙酸盐的形成速率成正比,因此与AST活性成正比。上述反应在37℃下进行,在340nm的波长下测量。

对于ALT:
α-酮戊二酸+ L-丙氨酸丙酮酸+ NADH +谷氨酸+丙酮酸+ H + L-lactate + NAD sup +(乳酸脱氢酶催化这种平衡反应)
光度测定的NADH降低的速率与丙酮酸盐的形成速率成正比,因此与ALT活性成正比。上述反应在37℃下进行,在波长340nm下测量。

对于UREA/BUN:
Urea + H 2 O→2 NH 4+(脲被脲酶水解)
α-酮戊二酸+ NH 4 (GLDH的存在产生谷氨酸和NAD +谷氨酸)+ NADH→L-谷氨酸+ NAD +/sup>)
由动力学测量由于消耗NADH而引起的吸光度的降低。上述反应在37℃下进行,并在340nm的波长下测量。

关键字:肝功能, AST, 中高音, 尿素/面包

材料和试剂


  1. AST(GOT)检测试剂盒(Roche,目录号:11876848216)
  2. ALT(GPT)检测试剂盒(Roche,目录号:11876805216)
  3. UREA/BUN检测试剂盒(Roche,目录号:11729691216)
  4. Cuvette(Roche,目录号:TA28)和样品杯(Roche,目录号:1105)
  5. 自动化系统校准器(Roche,目录号:10759350)
  6. 生理盐水(0.9%w/v NaCl)(见配方)

设备

  1. 微血细胞比容血管(肝素化)(助手,目录号:563)
  2. 滴管(3 ml)
  3. 离心机(Eppendorf)
  4. 聚丙烯试管(Corning Incorporated,Axygen ,目录号:MCT-150-C)
  5. 化学分析仪(Roche,Cobas Mira Plus)

程序

  1. 用微血细胞比容血液管(肝素化)从眶窦收集血液。 使用滴管推出肝素化血液管中的血液,并在1.5ml聚丙烯试管中收集约300μl血液。
  2. 在1500×g离心,4℃15分钟。 小心取无细胞上清液血浆(约一半体积的血液),并将其放在一个正确标记的聚丙烯试管,并将约150μl血浆转移到样品杯。
  3. 使用新鲜血浆进行血液AST,ALT和UREA/BUN水平分析
  4. 对于分析,样品杯中需要100-150μl样品体积。将含有血浆的样品杯放在样品位置,并将空比色杯放在化学分析仪的检测位置。如果样品体积不足,可以用生理盐水稀释
  5. 根据制造商的说明准备检测试剂,将准备好的检测试剂转移到化学分析仪的试剂容器中。通过混合试剂盒中提供的R1和R2溶液,容易制备检测试剂。简单地说,在AST和ALT试剂制备中,将一个瓶子1与一个瓶子1a连接以制备R1溶液,然后将R1和R2的体积在R1:R2 = 5:1中混合以得到检测试剂。对于UREA/BUN检测试剂制备,将R1和R2的体积结合在R1:R2 = 5:3中。这些混合的检测试剂在4℃下稳定7天
  6. 在样品检测之前,需要使用商用校准器校正化学分析仪。校准物作为阳性对照,生理盐水作为阴性对照。
  7. 校正后,准备分析化学分析仪中的样品的AST,ALT和BUN水平
    1. AST和ALT的自动分析步骤为:
      1. 从样品杯吸取10μl血浆样品到比色杯中
      2. 用40μl生理盐水稀释样品。
      3. 将300μl检测试剂加入比色杯中。
      4. 开始在不同时间点检测340 nm波长的值。
      5. 报告测量结果。
    2. 对于UREA/BUN分析,步骤是:
      1. 从样品杯吸取4μl血浆样品到比色皿中。
      2. 用20μl生理盐水稀释样品。
      3. 将400μl检测试剂加入比色杯中。
      4. 开始检测在不同时间点在340nm波长的值。
      5. 报告测量结果。

笔记

  1. AST,ALT和UREA/BUN的灵敏度分别为4U/L,4U/L和0.83mmol/L。 AST,ALT和UREA/BUN的测量范围分别为4-800U/L,4-600U/L和0.83-40.00mmol/L。
  2. 没有必要重复检测来自同一样品的值,因为程序可以监测不同时间点的值。 如果样品中测量值的变化超过5%,机器将自动检测样品。
  3. 在正常小鼠中,血浆中ALT,AST和UREA/BUN的水平为ALT:25-60U/L; AST:50-100U/L; UREA/BUN:16-30mmol/L。 ALT和AST的升高值表示肝损伤,高水平的UREA/BUN表示肾功能的损失

食谱

  1. 正常盐水(0.9%w/v NaCl)
    1. 确保所有设备清洁,干燥,无菌。
    2. 重量0.9克氯化钠。 通过用搅拌棒轻轻地混合将粉末溶解在约80ml蒸馏水中直至所有粉末溶解。 加入蒸馏水至终体积为100ml。
    3. 将制备的0.9%氯化钠溶液(生理盐水)转移到储存瓶中,并适当标记。 高压灭菌溶液。

致谢

该协议改编自先前发表的论文Sher等人。(2009)。 这项工作得到了来自NRPGM的DOH97-TD-G-111-041(到M-C Hung)和DOH97-TD-111-TM003(到L-Y.Li)的Grants的支持。 YP Sher还获得了台湾国家健康研究所博士后研究奖(PD9602)的支持。

参考文献

  1. Sher,Y.P.,Tzeng,T.F.,Kan,S.F.,Hsu,J.,Xie,X.,Han,Z.,Lin,W.C.Li,L.Y.and Hung,M.C。(2009)。 BikDD的癌症靶向基因治疗抑制原位肺癌生长并改善长期生存率。 Oncogene 28(37):3286-3295。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sher, Y. and Hung, M. (2013). Blood AST, ALT and UREA/BUN Level Analysis. Bio-protocol 3(19): e931. DOI: 10.21769/BioProtoc.931.
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