Qualitative Determination of Bilirubin in Urine

Introduction

•      Bilirubin is an orange-yellow pigment formed in the liver by the breakdown of haemoglobin and excreted in bile.

•      This occurs during the normal breakdown of red blood cells.

•      High bilirubin levels cause yellow pigmentation of the skin and/or sclera. This condition is called jaundice.

 

·         Conditions associated with bilirubinemia/bilirubinuria include liver diseases (liver cirrhosis, hepatitis,), increased RBC destruction (sickle anemia, severe malaria and other forms of hemolytic anemia) and diseases involving the gall bladder.

·         Clinical findings include; Urine color becomes dark yellow with yellow foam and Elevated serumconjugated bilirubin.

Methods for detection of bilirubin in urine

 

1)      Foam test

2)      Gmelin’s test

3)      Lugol iodine test,

4)      Fouchet’s test

5)      Ictotest tablet test, and

6)      Reagent strip test.

1. Foam test: About 5 ml of urine in a test tube is shaken and observed for development of yellowish foam. Similar result is also obtained with proteins and highly concentrated urine. In normal urine, foam is white.
2. Gmelin’s test: Take 3 ml of concentrated nitric acid in a test tube and slowly place equal quantity of urine over it. The tube is shaken gently; play of colors (yellow, red, violet, blue, and green) indicates positive test (Figure 823.1).
3. Lugol iodine test: Take 4 ml of Lugol iodine solution (Iodine 1 gm, potassium iodide 2 gm, and distilled water to make 100 ml) in a test tube and add 4 drops of urine. Mix by shaking. Development of green color indicates positive test.
4. Reagent strips or tablets impregnated with diazo reagent: These tests are based on reaction of bilirubin with diazo reagent; color change is proportional to the concentration of bilirubin. Tablets (Ictotest) detect 0.05-0.1 mg of bilirubin/dl of urine; reagent strip tests are less sensitive (0.5 mg/dl).

 

Fouchet's Reagent

Ingredients

·         Trichloroacetic acid 25.00 gm

·         Ferric chloride,10% aqueous 10.00 ml

·         Distilled water 100.00 ml

Principle and Interpretation

Barium chloride precipitates the sulphate radicals present in urine to form precipitate of barium sulphate. If bile pigments are present in urine, they adhere to these molecules. Ferric chloride present in Fouchet’s reagent then oxidizes yellow bilirubin, in the presence of trichloroacetic acid to green biliverdin. Therefore the development of green color due to the formation of biliverdin indicates the presence of bilirubin (bile) in urine.

 

Procedure

1.      Take three test tubes, clean with tape water.

2.      Label the test tubes as follows; PN (for control normal), PC (for control positive) and T (for test)

3.      Take 5 ml of fresh urine in a test tube (normal, positive and patient)

4.      Add 2.5 ml of 10% of barium chloride in each test.

5.      Mix well, precipitateof sulphates appears to which bilirubin is bound (barium sulphate-bilirubin complex)

6.      Filter to obtain the precipitate on a filter paper.

7.      Unfold a filter paper on an absorbent paper.

8.      To the precipitate on the filter paper, add 1 drop of Fouchet’s reagent

9.      Immediate development of blue-green color around the drop indicates presence of bilirubin.

10.  Yellowish white indicates the absence of bilirubin.

Results

Development of green color due to formation of biliverdin indicates the presence of bile (bilirubin) in the urine.

NOTE: Bilirubin is converted to non-reactive biliverdin on exposure to light (daylight or fluorescent light) and on standing at room temperature. Biliverdin cannot be detected by tests that detect bilirubin. Therefore fresh sample that is kept protected from light is required.

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