31.3 Urobilinogen in Urine
31.3.1 Ehrlich’s Test
31.3.1.1 Principle
A red-colored complex is formed when urobilinogen reacts withp-dimethyl amino
benzaldehyde in conc. HCl. Sodium acetate is used to reduce the acidity after the
reaction of urobilinogen with Ehrlich’s reagent.
31.3.1.2 Reagents
Ehrlich’s Reagent Dissolve 0.5 g para-dimethyl amino benzaldehyde in 100 ml of
150:100 ratio of conc. HCl and distilled water.
31.3.2 Saturated Sodium Acetate Solution
31.3.2.1 Procedure
Mix equal volume (5 ml each) of urine and Ehrlich’s reagent, and keep for 10 min.
Then add 5 ml of saturated sodium acetate solution, and observe the color produced.
If red color is produced, urobilinogen is present in the sample. In case no color
developed, warm at 60C.
Use fresh sample of urine; otherwise false negative result may be observed, as
urobilinogen on standing is oxidized to urobilin. Urobilin shows negative result with
Ehrlich’s reagent. A false positive result may be given by natural substances like
porphobilinogen and drugs like para-aminosalicylic acid, sulfonamides, and
sulfonylureas. In order to distinguish between urobilinogen and porphobilinogen,
add 5 ml chloroform to the red color solution formed in Ehrlich’s test. Shake well
and let the layer to separate. Red color in the lower organic layer indicates the
presence of urobilinogen, while the color in the above aqueous layer indicates
porphobilinogen.
31.4 Precautions
- Use fresh urine sample.
- If bilirubin is present in the sample,first remove the bile pigments by Fouchet’s
test or by adding 10% BaCl 2 to 5 volume of urine. Centrifuge the solution and
take the supernatant and detect urobilinogen by Ehrlich’s test.
126 31 Qualitative Test for Bile Pigments and Urobilinogen in Urine