DETECT PORPHOBILINOGEN in urine

PORPHOBILINOGEN:_Porphobilinogen is an intermediate by-product in the biosynthesis of haem. It is absent in normal urine but may be present in some errors of metabolism of porphyrin. The Watson-Schwartz test can detect both urobilinogen and porphobilinogen in urine; where as Hoesch test is specific for porphobilinogen.

Name of mathod: Watson-Schwartz Test for Urobilinogen and Porphobilinogen

Principle --- The Watson-Schwartz test provides differentiation between urobilinogen, porphobilinogen and other intermediate Ehrlich's reagent reacting compounds. All the three compounds give the characteristic cherry-red colour with Ehrlich's reagent. The colour is enhanced in the presence of saturated sodium acetate. They can be differentiated from each other by their solubility in organic solvents such as chloroform and butanol. Urobilinogen is soluble in both the solvents, intermediate Ehrlich-reactive compounds are soluble only in butanol where as porphobilinogen is soluble in neither of the solvents.

Note The urine specimen must be examined while it is very fresh.

Reagents 

1. Ehrlich's reagent (modified)

p-dimethyl aminobenzaldehyde    0.7 g

Hydrochloric acid (conc.)     150 ml

Distilled water                                  100 ml

2. Sodium acetate, saturated solution in distilled water

3. Chloroform

4. Butanol

Procedure 1. Place approximately 3 ml of urine in a test tube

2. Add equal volume of Ehrlich's reagent,mix.

3. Add 6.0 ml of saturated sodium acetate and mix.

4. Development of cherry-red colour indicates the presence of either urobilinogen, porphobilinogen or intermediate Ehrlich reactive compounds.

5. If the test is positive in Step 4, divide the coloured solution into two parts.

6. To one portion, add 2-3 ml of chloroform and shake vigorously. Check whether the lower chloroform layer shows a pink or red colour. If the colour is extracted in the chloroform layer, the urine specimen contains urobilinogen.

7. If the colour is not extracted in chloroform, add 2-3 ml of butanol to the second portion from Step 5. Shake again

Procedure2 1. Place approximately 2.0 ml of Hoeschreagent in a test tube.

2. Add 2 drops of urine on top

3. Appearance of a cherry red colour on top of the solution indicates a positive test for porphobilinogen.

Clinical significance:- Porphobilinogen appears in urine in some metabolic disorders such as acute intermittent porphyria.