Chemical examination of urine
Reducing Sugars
Even normal urine contains less than 100 mg of glucose and glucuronides in 24 hrs urine samples, but their amount is too small to cause a reduction in Benedict’s test. A sample of urine that reduces Benedict’s reagent under the conditions laid down for the test may contain reducing sugar unless otherwise proven. A positive Benedict’s test does not necessarily indicate the presence of glucose in urine. However, a positive Benedict’s test is usually taken for glucose unless other conditions are suspected and proven. If mucin is suspected, Benedict’s test should be repeated after removing mucin with kaolin.
Glucose
Benedict’s Test
Principle:
Reducing sugars in urine under hot, alkaline conditions tautomerizes and forms enediols, powerful reducing agents. They reduce the cupric ions of Benedict’s reagent to red cuprous oxide. The cupric hydroxide formed during the reaction is kept in solution by metal chelators like citrate
Procedure:
- To take 5 ml of Benedict’s reagent in a test tube, add 8 drops of urine.
- Shake well and boil for 1 or 2 minutes, or keep it in a water bath for 5 minutes.
Observation:
A colloidal precipitate forms, a colour which may be green, yellow, orange or red, depending on the urine. Concentration of sugar.
Interpretation:
- Blue – absence of reducing sugar
- Green – up to 0.5 gm%
- Yellow – 5 to 1.0 gm%
- Orange – >1.0 to 2.0 gm%
- Brick Red – ≥ 2 gm%
Protein
Heat Coagulation Test
Procedure:
- Fill 3/4th of the test tube with urine. Heat the upper 1/3rd of the urine column with a small flame so the lower 2/3rd will serve as a control.
- Add 30 % (v/v) acetic acid to it.
Observation:
White turbidity or coagulum.
Interpretation:
White turbidity, if it disappears with the addition of acetic acid, indicates the presence of phosphates or carbonates. If the white turbidity formed remains or appears or intensifies upon adding acetic acid points towards the presence of albumin.
Sulphosalicylic Acid
Principle:
Proteins are amphoteric, i.e., they behave as acids in an alkaline medium and as bases in an acidic medium. In the presence of alkaloid reagents, like sulphosalicylic acid, they act as bases and react with the acid to form an insoluble salt of protein sulphosalicylate.
Procedure:
- To 2 ml of clear urine (filter, if not clear), add a few drops of 20% sulphosalicylic acid.
- Turbidity of protein sulphosalicylate indicates the presence of a protein.
Clinical Interpretation
- Conditions of Proteinuria:
- Nephrosis /Nephritis
- Tuberculosis
- Neoplasm
- Diabetic nephropathy
- Bence-Jones proteins in multiple myeloma
- Congestive heart failure
- Hypertension
- Strenuous exercise
- Pregnancy
Ketone bodies
Rothera’s Test
Principle:
Acetone and acetoacetic acid react with sodium nitroprusside (nitroferricyanide) in the presence of alkali to produce a purple colour.
Procedure:
- Saturate 5 ml of urine with ammonium sulfate crystals.
- Add 2 drops of freshly prepared 2% sodium nitroprusside solution or a little sodium nitroprusside powder.
- Shake well. Add 1 ml of liquor ammonia through the sides of the test tube.
Observation:
A reddish-violet ring at the junction of two liquids.
Β-hydroxybutyrate
Procedure:
- Saturate 1 ml of urine with solid ammonium Sulfate until some remains undissolved.
- Add 2 drops of freshly prepared solution of 5% Sodium Nitroprusside (or a small crystal of Sodium Nitroprusside and shake to dissolve in the urine) and mix.
- Then, gently layer 2 ml of ammonia over the urine. A purple ring appears if acetone bodies are at the junction of urine and ammonia layers.
Gerhardt’s Test (Acetoacetic Acid)
Principle:
Acetoacetate reacts with ferric chloride to form a red-brown precipitate.
Procedure:
- To add 5 ml of urine in a test tube, add 5% ferric chloride solution, drop by drop, till no precipitate of ferric phosphate is formed.
- Filter and to the filtrate add some more ferric chloride solution.
- A port wine colour indicates the presence of acetoacetate.
Clinical Interpretation
Normal levels: Less than 1 mg/day
Increased ketone body excretion:
- Starvation
- A diet rich in fat but restricted proteins and carbohydrates
- Diabetic ketoacidosis
- Fevers and severe anaemia
- Phosphorus poisoning
- Recurrent vomiting in children.
Occult Blood
Benzidine Test
Principle:
Heme of haemoglobin decomposes hydrogen peroxide to nascent oxygen, oxidising benzidine to the bluish green product.
Procedure:
- In a dry test tube, dissolve a pinch of benzidine in about 1 ml of glacial acetic acid.
- Add 1 ml of hydrogen peroxide to it.
- Then, add 5 – 10 drops of urine to the test tube.
Observation
The benzidine solution will turn deep blue. If blood pigment is present in the urine.
Clinical Interpretation
Conditions of hematuria:
- Injury to the urinary tract or kidney
- Urinary tract infection
- Benign or malignant tumours of the kidney or urinary tract
- Urinary calculus
- Ruptured venous plexus of enlarged prostate.
Bile salt
Hay’s Test
Principle:
Bile salts reduce the surface tension. Hence, the sulfur powder sinks to the bottom.
Procedure:
Take 5 ml of urine in a test tube and sprinkle sulfur powder on the surface of the urine.
Observation:
Sulfur powder sinks to the bottom.
Inference:
Bile salts are present in urine; otherwise, the sulfur powder would have remained on the surface of the urine column.
Bile Pigment
Fouchet’s Test
Principle:
The precipitate obtained is BaSO4, to which bile pigment, if any, would have adsorbed. When Fouchet’s reagent to the precipitate (Ferric chloride in Trichloroacetic acid) is added, FeCl3 oxidizes bilirubin to biliverdin and Fe3+ (ferric ions) is converted to Fe2+ (ferrous ions). This gives the colour.
Procedure:
- To 10 ml urine, add 1 ml MgSO4 and boil.
- While boiling, add 10% BaCl2 drop by drop till maximum precipitate is obtained.
- Filter and discard the filtrate.
- Dry the filter paper from the funnel by moping it over another paper.
- After drying, add 2 drops of Fouchet’s reagent to the precipitate.
Observation:
A blue/green colour in the presence of bile pigments.
Ehrlich’s Test
Principle:
Urobilinogen forms a coloured adduct with Para dimethyl amino benzaldehyde.
Procedure:
- Add 1 ml of Ehrlich’s reagent (2% Para dimethyl amino benzaldehyde in 20% HCl) to 10 ml of freshly voided urine.
- Shake well and keep it in the rack for 5 minutes for colour development.
Observation:
Normal urine gives only a faint red colour.
Interpretation:
- The intensity of the red colour is related to the concentration of urobilinogen in the following manner.
- No red colour: Urobilinogen absent.
- Faint pink colour: Urobilinogen is present in normal amounts.
- Distinctly red colour: Urobilinogen present in increased amounts