PRACTICUM II URINE CHEMICAL EXAMINATION BILIRUBIN 2.4 Bilirubin Practice : Determination of Bilirubin Objective : To detect disturbances in liver function Bilirubin is a yellow pigment compound which is a product of the enzymatic catabolism of biliverdin by biliverdin reductase. Oxidation of bilirubin produces biliverdin again, giving this compound antioxidant attributes in cellular physiology. This substance is what gives feces and urine a yellow color. 2.4.1 Horizon Method Objective : To determine bilirubin in urine. Working Principle: Forfat in urine will be precipitated by BaCl², then The bilirubin attached to the precipitate with Fouchet's reagent will be oxidized by FeCl³ in an acidic environment to produce green biliverdin. Tools and Materials: 1. Urine sample 2. Test tube. 3. Test tube rack. 4. Filter Paper 5. Funnel 6. Dropper pipette 7. BaCl2 10 8. Fouchet's reagent, with composition: a. Trichloroacetic acid (TCA) 25g b. Aquadest ad 100 ml c. Ferric chloride solution 10 ml (10 g FeC13 in 100 ml distilled water) Work procedures : 1. 5 ml of urine is put into a test tube. 2. Add 5 ml of 10% barium chloride solution, homogenize then filter. 3. The filter paper containing the precipitate is removed from the funnel, unfolded and laid flat. Leave it for a while until it dries a bit. 4. Drop 2 – 3 drops of Fouchet reagent onto the precipitate on the filter paper. 5. The appearance of a green color indicates the presence of bilirubin. Interpretation of Results: Negative (-) there is no change in color to green on the filter paper. Positive (+) occurs as a green color on the filter paper which becomes clearer over time. Normal Value: Negative (-) 2.4.2 Foam Test Method (FROTH) Objective : To determine the presence of bilirubin in the urine as an examination support liver disease. Work principle : The presence of bilirubin in the urine will form colored foam yellow if the urine is shaken. Tools and materials : 1. Test tube with rubber cap/stopper + rack 2. Urine sample Work procedures : 1. Shake approximately 5 ml of fresh urine in the tube vigorously. 2. If yellow foam occurs, it is a sign that bilirubin is very likely present Interpretation of results: Negative (-): Clear/white foam. Positive (+): Yellow foam Method: Smith-Rossin Principle: The presence of bilirubin in the urine will be oxidized by iodine, so the color appears greenish. Work procedures : 1. Put 5 ml of urine into a test tube and add 1 ml of iodine solution through the tube wall to form 2 layers. 2. Then leave it for approximately 3 minutes. 3. Then observe the color that forms between the two layers Interpretation of results: Negative (-): There is no green color between the 2 layers. Positive (+): There is green color between the 2 layers.