Serum alanine transferase (ALT) measurement assay
Serum alanine transferase (ALT) measurement assay
This experiment enables students to master the principle of ALT determination. The reagent preparation method, this experiment is from Mudanjiang Medical College undergraduate 5-year laboratory guide for laboratory testing.
Operation method
Serum alanine transferase (ALT) measurement assay
Principle
After 30 min reaction, 2,4-dinitrophenylhydrazine was added to terminate the reaction, and the corresponding 2,4-dinitrophenylhydrazone was generated with the two a-keto acids in the reaction solution. Under alkaline conditions, the absorption spectra of the two types of phenylhydrazones differed from each other, with the largest difference at 500-520 nm, and the color rendering temperature of the phenylhydrazone with pyruvic acid was about three times higher than that of phenylhydrazone with a-ketoglutarate at the equimolar concentration. The color development temperature of pyruvate phenylhydrazone was about 3 times higher than that of a-ketoglutarate phenylhydrazone. Calculate the amount of pyruvic acid produced. Move I. Experimental reagents: Caveat 1. The standard curve cannot be extended to 200 calorimetric units due to factors such as feedback inhibition by the concentration of the substrate a-ketoglutarate and the insufficient concentration of 2,4 dinitrophenylhydrazine to react with the product pyruvate. 2. serum ALT can be stored at room temperature (25 ℃) for 2 days, in the refrigerator at 4 ℃ can be stored for a week, below 25 ℃ can be stored for a month. 3. severe lipemia, jaundice or hemolyzed serum may cause an increase in the absorbance of the assay tube; therefore, a serum specimen control should be made when testing such specimens. 4. When the enzyme activity of the serum specimen exceeds 150 kammon units, the serum should be diluted 5-fold or 10-fold with saline before measurement. 5. After addition of 2,4-dinitrophenylhydrazine solution, it should be mixed thoroughly so that the reaction is complete, and sodium hydroxide solution should be added at a consistent rate, as different rates of addition will result in differences in absorbance readings. 6. a-ketoglutaric acid and 2,4-dinitrophenylhydrazine in the substrate are color-developing substances, weighing must be very accurate, the absorbance of the blank tube of each batch of reagents should not fluctuate up and down by more than 0.015A. If it exceeds this range, the reagents and instrumentation should be checked. 7. batch ALT determination of each tube to add serum, the test tube rack should be placed in a 37 ℃ water bath operation, to a certain time interval of each tube to add substrate buffer, each tube instant mixing, to add the first tube to start timing, accurate every reaction for 30 min. immediately according to the same time interval, in turn, to the tubes add 2,4 dinitrosohydrazine to each tube is also mixed instantly, in order to ensure that the accuracy of batch of ATL determination. In this way, the accuracy of ATL determination in batches can be guaranteed. For more product details, please visit Aladdin Scientific website.
l. 0.1 mol/L sodium hydrogen phosphate solution: 17.8 g of sodium dihydrogen phosphate (containing two crystalline waters) was dissolved in water and added to 1000 ml and stored in a refrigerator.
2. 0.1 mol/L potassium dihydrogen phosphate solution: 13.6 g of potassium dihydrogen phosphate was dissolved in water and added to 1000 ml and stored in refrigerator.
3. 0.lmol/L phosphate solution (PH7.4): mix 420mol 0.1L sodium hydrogen phosphate solution 180ml 0.1moL/L potassium dihydrogen phosphate solution, add several drops of chloroform. Keep in refrigerator.
4. Substrate buffer (DL-alanine 200 mmol / L. a-ketoglutarate 2 mmol / L): weigh accurately
Take 1.79g of DL-alanine and 29.2 mg of a-ketoglutaric acid and dissolve them in about 50 m1 of 0.1 mol/L phosphate buffer and adjust them to pH 7.4 with l mol/L sodium hydroxide (about 0.5 ml), add phosphate buffer to 100 ml and store it in the refrigerator. It can be stabilized for 2 weeks. 0.9g of muscimol can be added to each liter of substrate buffer or a few drops of chloroform can be added for preservation, and it can be stored in the refrigerator for at least 1 month, and it can be used for at least 3 months at room temperature after sterilizing the ampoule.
5. 1.0 mmol/L 2,4-dinitrophenylhydrazine solution: weigh 19.8mg, 2,4 nitrophenylhydrazine, dissolved in 10mmol/L hydrochloric acid. After complete dissolution, add distilled water to 100 ml, placed in a brown glass bottle, kept at room temperature if crystals precipitate, should be re-formulated.
6. 0.4 mol / L sodium hydroxide solution, 16.0 g of sodium hydroxide dissolved in water, and add water to 1000 ml, placed in a stoppered plastic reagent bottle, room temperature can be stable for a long time.
7. 2mmol/L pyruvate standard solution: accurately weigh 22.0mg of sodium pyruvate (AR) in 100ml, placed in a volumetric flask and add 0.05mol/L sulfuric acid to the scale.
Second, the experimental operation: before the determination of the appropriate amount of substrate solution in a 37 ℃ water bath box pre-temperature 5min after use, the specific operation of the Table 1. 
After each tube was warmed evenly, it was kept warm at 37℃ for 20min, and then each tube was filled with 0.4mol/L sodium hydroxide solution.
Then each tube was filled with 0.4 mol/L sodium hydroxide solution 5ml, placed at room temperature for 5min, and then the absorbance of each tube was read with a spectrophotometer at 505nm by adjusting the zero point with distilled water, and the absorbance of each tube was measured, and the absorbance of the tube was subtracted from the absorbance of the control tube of the samples, and the unit of ALT activity was found from the calibration curve.
Standard curve
1. Add the corresponding reagents to each tube according to Table 2. 
2. Add 0.5 ml of 2,4 dinitrophenylhydrazine solution to each tube, mix at 37℃ for 20 minutes and then add 0.4 mol/L of 2,4 dinitrophenylhydrazine solution.
5ml of sodium hydroxide solution.
3. Root homogeneous, placed in 5min with a spectrophotometer at a wavelength of 505nm, distilled water to adjust the zero point, read the absorbance, each tube absorbance minus "0" tube absorbance obtained by the difference between the corresponding kamaguchi enzyme activity unit to make a graph.
Reference value: 5-25 kammon units