Proteins Urine Chemical Examination

PRACTICUM II URINE CHEMICAL EXAMINATION PROTEINS 2.2 Proteins Practice : Protein examination in urine Objective : As a support for diagnosis, examination of liver and kidney function diseases Protein examination is a routine examination. Most routine ways to determine the presence of protein in urine are based on the appearance of turbidity. Because the density or coarseness of the turbidity is a measure of the amount of protein present, using clear urine is an important requirement for protein tests. 2.2.1 Heller method (Nitric Acid, concentrated HNO3) Objective : To indicate the presence of protein in the urine Work principle : Proteins in urine will be denatured by nitric acid, marked by a white ring between the two layers. Sample : Morning urine/morning urine (clear) Tools and materials 1. Test tube + tube rack 2. 5ml measuring pipette 3. Suction rubber 4. Dropper pipette 5. Nitric acid solution, HNO3concentrated Ways of working : 1. Fill the test tube with 1 ml of HNO nitric acid3concentrated. 2. Tilt the tube and slowly pour 1 ml of urine through the wall of the test tube until it forms 2 layers with HNO3that thick. 3. Wait 1 minute. Interpretation of results: Positive (+) : A white layer/ring appears between the two liquids. Negative (-) : No white layer/ring appears between the two liquids. 2.2.2 Method: Ewitz/Sulfosalycilic acid 20% Objective: To state the presence of protein in urine based on turbidity. Principle: Proteins contained in urine will be precipitated by sulfosalycil acid 20% Sample: Urine during/morning Tools and materials 1. 2 test tubes + tube rack 2. Dropper pipette 3. Bunsen / water bath 4. 20% sulfosalicylic acid solution Work procedures : 1. 2 test tubes each filled with 2 ml of clear urine 2. Put 8 drops of 20% sulfosalicylic acid in the first tube and then homogenize 3. Compare the first tube with the second tube - Clarity remains the same test for protein concluded (-) - If the first tube is cloudier than the second tube, heat the first tube over a flame then cool with running water: - The turbidity is still there when heated and is still there after it cools down again so the test for protein is concluded (+), the protein may be albumin, globulin or both - If the turbidity disappears when heated but appears again after cooling, then the test for protein cannot be concluded, perhaps it is caused by Bence Jones protein (a protein that dissolves at boiling temperatures, such as in the diseasemultiple myelomaor several types of bone tumors and leukemia) and requires further examination 2.2.3 Method: Boil (Acetic Acid) Working Principle: Proteins in urine are denatured by heating until boiling, it will form a turbidity and is further confirmed by the addition of dilute acetic acid. If there is still turbidity, then the result is positive. Sample : : Urine during/morning Tools and materials 1. 2 test tubes + tube rack 2. Dropper pipette 3. Bunsen/water bath 4. Acetic acid solution 3 – 6% Ways of working : 1. Put clear urine into the test tube until it is 2/3 full 2. With the help of clamps, the test tube is clamped at the top end of the tube, then the top layer of urine is heated in a Bunsen flame until it boils for 30 seconds 3. Note the occurrence of turbidity in the upper layer of urine by comparing the clarity with the unheated lower layer a. No turbidity occurs, then the protein test is concluded (-) b. If turbidity occurs then: - Drop into the hot urine 3-5 drops of 6% acetic acid solution - The turbidity disappears, meaning the turbidity is caused by crystals (calcium phosphate, calcium carbonate) - If the turbidity persists or is more turbid, the test is considered (+) 2.2.4 Method: Bang Working principle: Proteins in urine are denatured by heating until Boiling, turbidity will form and this will be further confirmed by adding Bang's reagent (PH 4.5 buffer solution). If there is still turbidity, then the result is positive. Sample : Morning urine Tools and materials 1. Test tube + tube rack 2. 5 ml measuring pipette 3. Balm 4. Dropper pipette 5. BANG REAGEN (NAT. ACETATE 11.8g GLACIAL ACETIC ACID 5.65 ml, distilled water @ 100 ml) Ways of working : 1.Put 5 ml of clear urine into a test tube 2.Add 0.5 ml of Bang reagent 3.Heat over boiling water for 5 minutes