Determination of ALT (Alanine aminotransferase) in human serum Theory: Aminotransferases are enzymes that facilitate the conversion of one amino acid to another. This helps maintain a balanced supply of amino acid units needed for protein synthesis. Increased alanine aminotransferase activity is an important indicator of liver, heart and skeletal muscle activity. In practice, transaminases are the body's own substances, which are usually found in cells. ALT transaminase is found mainly in the cells of the liver, heart, skeletal muscles, kidneys, brain and red blood cells. After their breakdown, they pass into the blood serum. Thus, increased ALT means increased cell lysis in these areas. Standard: 0.06 - 0.14 ukat / l Limit value: 0.42 ukat / l Task: To determine ALT in human serum Accessories: eppenndorph stand adjustable pipettes thermal bath at 37^oC ELISA reader with 340 nm filter Principle of the method: alanine aminotransferase (L-alanine: 2-oxoglutarate aminotransferase EC2.6.1.2) catalyzes the reaction between L-alanine and 2-oxoglutarate, which converts to L-glutamate and pyruvic acid in an alkaline environment. Pyruvic acid hydrazone has a higher absorbance. L-alanine + oxoglutarate pyruvate + L-glutamate Pyruvate + NADH + H +  lactate + NAD + The catalytic concentration of ALT is proportional to the decrease in absorbance at 340 nm. Reagents R1. Buffer: Tris buffer pH = 7.5, L-alanine, LD LD ≥ 2.5 cat NADH 21.6 /mol / vial R2. Starter NADH, 2-oxoglutarate 180 mmol / l Sodium azide 0.1% Activator Pyridoxal-5-phosphate 6 µmol / tablet Calibration BIO-LA-TEST LYONORM CALIBRATOR, cat. No. (1,40µkat / l), 3204,3206 Preparation of working solution Initially, the contents of the Reagent 1 vial are dissolved in 100 ml of Reagent 3. After dissolution, 2 tablets of Reagent 4 are added. Adjusted to: 25% by weight of the contents of the Reagent 1 vial are dissolved in 25 ml of Reagent 3 solution. Analysis procedure Samples: non-hemolytic serum, heparinized or EDTA plasma Wavelength: 340 nm ELISA plate Temperature: 37 ° C Sample type amount Working solution 10min inkubation Reagent 2 Serum sample 2x diluted 10 µl 100 µl 10 µl Blank (Fyz. roztok) 10 µl 100 µl 10 µl Standard 2x diluted 10 ml 100 µl 10 µl Standard concentrated 10 ml 100 µl 10 µl Use a blank, use Lyonorm (biochemical) as a standard Mix and incubate at 37 ° C for 10 minutes Reagent 2 is added in an amount of 10µl Mix, incubate for 2 minutes at 37 ° C, measure absorbance at 1 minute intervals for at least 3 minutes. Calculate the average change in absorbance over 1 min (δA). δA = average (A1 + A2 + A3)