QUALITATIVE ANALYSIS OF ABNORMAL URINE.pptx

Physical appearance of urine
Test for reducing sugar
Test for protein
Test for ketone bodies
Test for blood

1. QUALITATIVE ANALYSIS OF ABNORMAL URINE

2. QUALITATIVE ANALYSIS OF ABNORMAL URINE Abnormal constituents of urine are proteins, glucose, ketone bodies, bile salts, bile pigments and blood Presence of these substances are analysed usually in 24 hours urine sample Urine on standing undergoes bacterial fermentation it is preserved either by refrigeration or by using preservatives like toluene, chloroform, thymol and Hcl .

3. PHYSICAL CHARACTERISTIC OF ABNORMAL URINE: COLOR: Almost colorless : Dilute urine ( polyuria ) Dark Amber color : Concentrated urine ( oliguria ) Yellow color : Bile pigments (Jaundice) & Increased B complex intake Orange red colour : Rifampicin (Anti tuberculus drug) intake Red to Reddish brown : Hemoglobin (Blood) & increased beet root intake Milky : Presence of fats ( Chyluria ) Cloudy (Turbid) : Pus cells Black on standing : Homogentisic acid ( alkaptonuria ), formic acid poisoning

4. 2. VOLUME: Polyuria : Urine output more than 2 litres / day. Causes for polyuria are excess fluid intake, Diabetes insipidus , Diabetes mellitus etc Oliguria : Urine output between 300 – 500 ml / day. Causes: Less fluid intake, Excess fluid loss like vomiting, diarrhoea , massive sweating, Acute renal failure, burns, nephrotoxic drugs Anuria : Urine output between 30-50 ml. Causes: End stage renal failure, bilateral urinary tract obstruction etc.

5. 3. ODOUR: Smell of acetone : Uncontrolled diabetic ketoacidosis and chronic starvation (fruity odour ) Burnt sugar or maple sugar : Maple syrup urine disease Mousy odour : Phenyl ketonuria Cabbage like odor : Tyrosinemia type 1

6. 4. SPECIFIC GRAVITY: Theoretical extremes of specific gravity are 1.003 to 1.032 Low specific gravity : Diabetes insipidus , during recovery from acute renal failure (diuretic phase) High specific gravity : Diabetes mellitus, Excessive fluid loss as in vomiting & diarrhoea Low fixed specific gravity : Chronic renal failure at 1.010 ( Isothenuria ) 5. pH pH of Normal urine is 4.6 to 8 (average 6) Acidic urine : Metabolic acidosis, ureteric transplantation Alkaline urine : Metabolic alkalosis, Alkali therapy

7. 1.Qualitative test for Proteinuria : EXPERIMENT OBSERVATION INFERENCE A. Heat coagulation test Take a small test tube. Fill ¾ of the test tube with the protein solution. Hold the test tube over the flame in a slanting position. Heat the upper 1/3 of the solution. The lower 2/3 serves as a control. A curdy white coagulum is formed at the heated portion alone. Add few drops of 1% acetic acid to the test tube. If coagulum persist and deepens it indicates the presence of heat coagulable proteins like albumin and globulin. If coagulum disappears it indicates the presence of interfering substances mainly phosphate. Coagulation takes place at the top of the test tube. Presence of albumin.

8. PROTEINURIA Types of Proteinuria could be Functional: eg.Severe exercise,Fever,orthostatic , Pregnancy Glomerular : Any disease causing glomerular damage like Diabetic nephropathy, Systemic lupus erythematosis etc.Protenuria more than 1 gm/day. Tubular : Any disease causing decreased tubular reabsorption or tubular damage like nephrotoxic drugs,heavy metals and anoxia.Protenuria less than 1 gm/day. Over flow Proteinuria : Any disease causing increased blood concentration of freely filtred proteins like Multiple myeloma( Bence Jones protein), myoglogin etc. Normal level of protein in 24 hours urine is less than 150 mg / day. This is mainly constituted by Tamm Horsfall Protein secreted by the Tubular epithelial cells.

9. EXPERIMENT OBSERVATION INFERENCE B)HELLER’S TEST : Take 3ml of of con nitric acid in a test tube.Add 2ml of urine along the side of the test tube C)SULFOSALICYLIC ACID TEST: Take 2ml of urine and add few drops of 20% sulfosalicylic acid. White ring seen at the junction of two liquids White precipitate is observed Con nitric acid denatures proteins and white ring is formed. High urea level in urine will form urea nitrate complex and give a false positive test Sulfosalicylic acid is a alkaloidal agent.It precipitates proteins by neutralizing the positive charges on the proteins.False positive results seen if urine contains radiographic dyes, pencillin,cephalosporins , sulfonamides,hemoglobin , myoglobin,etc.It is very sensitive test and detects even small amount of proteins

10. 2. Test for Reducing sugar EXPERIMENT OBSERVATION INFERENCE Benedict’s test : Take 5 ml of Benedict’s reagent in a test tube. Add exactly 8 drops (0.5 ml) of urine. Mix. Boil for 2 minutes. Green, Yellow or red colored precipitate is obtained On heating in alkaline medium, cupric hydroxide is reduced to cuprous oxide by reducing sugars. On reduction, the color of precipitate depends on the concentration of sugar present in the urine.

11. The approximate concentration of sugar will be 0.5g/100ml (green), 1g % (Yellow) 1.5 g% (Orange) 2% (red) Hence this test is used as a semiquantitative method. Many non carbohydrate reducing substances in urine like Homogentisic acid, Ascorbic acid & Glucuronides give false positive Benedict’s test.

12. 3. Test For Ketone Bodies: EXPERIMENT OBSERVATION INFERENCE Rothera’s Test : (For Acetone and Aceto acetic acid) Take 3 ml of urine in a test tube. Saturate it with solid ammonium sulfate. Add 2 drops of freshly prepared sodium nitro prusside solution. Mix. Add 2 ml of strong ammonia gently along the sides of the test tube. Purple ring is formed at the junction of the two liquids. Sodium nitroprusside reacts with the ketone group in the presence of ammonia to form purple colored complex

13. Ketone bodies Acetone, Aceto acetic acid and β hydroxy butrate are the 3 ketone bodies Ketonuria is the condition when the above ketone bodies appears in the urine Ketonuria occurs when there is excessive lipolysis as in uncontrolled Diabetic mellitus and chronic starvation Other causes are Phenyl ketonuria,maple syrup urine disease,isopropyl alcohol poisoning and salicylate poisoning. β Hydroxy butyrate does not have a ketone group.So it wont answer this test

14. 4. Test for Bile salts EXPERIMENT OBSERVATION INFERENCE Hay’s Test: Take 2 ml of urine in a test tube. Gently sprinkle little quantity of sulphur powder over the surface of urine. Perform the above test taking distilled water instead of urine for control. Sulphur powder sinks to the bottom of the test tube containing urine. Bile salts reduce the surface tension and hence the sulphur powder sinks to the bottom

15. Bile salts In obstructive Jaundice and in obstructive phase of hepatic Jaundice bile salts are seen in urine. Bile salts are Sodium and potassium salts of glycocholate and taurocholates Alcohols and salicylates and soap gives false positive results. Test tube should be free from soap/detergent.

16. 5 . Test for Bile pigments EXPERIMENT OBSERVATION INFERENCE Fouchet’s Test Take 3 ml of urine in a test tube.Add a pinch of Magnesium sulphate . Add 2 ml of 10% Barium chloride solution. Mix. A precipitate of Barium sulphate is formed. Filter. Unfold the filter paper, dry, and add few drops of Fouchet’s reagent. (Ferric chloride in trichloro acetic acid is Fouchet’s reagent) A green or blue color develops on the precipitate. The bile pigment present in the urine gets adsorbed to the precipitated Barium sulphate . Ferric chloride present in Fouchet’s reagent oxidises bilirubin to green colored biliverdin , blue colored bilicyanin or brown colored bilifuchsin

17. Bile pigments Choluric jaundice refers to presence of bile pigments in urine (hepatic jaundice and obstructive jaundice.) Acholuric Jaundice is absence of bile pigments in urine.(Hemolytic jaundice) The bile pigments are bilirubin & biliverdin Nitric acid oxidizes bile pigments During stages of oxidation bile pigments undergo colour change.

18. 4. Test for occult blood EXPERIMENT OBSERVATION INFERENCE Benzidine Test: Take small quantity of Benzidine powder in a knife point.Add 1ml of glacial acetic acid. Add 1 ml of Hydrogen peroxide solution. Mix. Add 1 ml urine.Instead of benzidine powder orthotolidine can also be used in the above test Dark blue green color develops. Then it turns to black colour within few minutes Hb decomposes H2O2 and liberates oxygen which oxidizes benzidine to a coloured compound.This reaction is catalysed by heme . Peroxidase present in pus cells splits H2O2 into nascent oxygen which gives a false positive test. Boiling the urine sample destroys the interfering enzymes and avoids such false positive reactions.

19. Hematuria Hematuria is the condition when the blood appears in the urine Hematuria (blood in urine) occurs in conditions like menstrual periods in women, renal stones, renal carcinomas, urinary tract infection etc.

20. Result: The abnormal constituents of urine are studied. Interpretation of Heat coagulation test: Negative/Trace means no or mild turbidity observed. when the urine column in the tube is placed in front of a typed sheet or paper, printed letters can be clearly read through the tube. + 1 Means definite turbidity observed.Printed leters can be clearly read through the tube . + 2 Means Definite turbidity observed.Printed leters cannot be clearly read through the tube. + 3 Means Definite turbidity observed. Nothing can be observed through the tube . + 4 Means protein clots are seen in the tube.