Urine for Urobilinogen and Ehrlich Reagent

Urine for Urobilinogen

Sample of urine for urobilinogen

1. The test is done in the urine.
2. A random sample can be taken.
   • 2 hours fresh urine sample can be used, kept cool, and protect from the light.
3. The sample is stable at 2 to 8 °C for 24 hours when urine is kept in the dark.
4. Don’t expose urine to light or store it at room temperature.
5. 2 to 3 hours postprandial after the noon meal is the best sample.

Purpose of the test (Indications)

1. To assess liver function.
2. This is the earliest sign of liver disease.
3. This is positive in hemolytic anemia.

Precautions for estimation of urobilinogen in urine:

1. False-positive test seen in:
2. Drugs containing azo dyes:
   1. Phenazopyridine.
   2. Nitrofurantoin.
   3. Riboflavin.
   4. p-aminobenzoic acid.
   5. Highly colored pigments and their metabolites.
   6. Methyldopa.
3. False-negative test seen in:
   1. Oxidation of urobilinogen to urobilin.
   2. Formalin preservative.

Pathophysiology of Urobilinogen

5. Hemoglobin breaks into bilirubin enter the intestine, and thereby the action of bacteria is transformed into urobilinogen.
   1. Urobilinogen in the intestine is excreted as urobilin, which gives a yellow color to urine.
6. Urobilinogen in the intestine is reduced to brown color stercobilin, which gives color to stool.
   

   Urobilinogen excretion in the urine

7. Urobilinogen is absorbed into the portal circulation, reaches the liver, is metabolized, and is excreted into the bile.
8. Urobilinogen traces escape removal by the liver carried to the kidney excreted in the urine.
   

   Urobilinogen fate in the body

9. Urobilinogen is colorless.
   1. The intestinal bacteria oxidize urobilinogen to Urobilin, which is a brown pigment.
10. Its absence indicates obstructive jaundice.
    

    Urobilinogen excretion

Normal urobilinogen in urine

• Random sample = < 1 mg/dL.
• 24 hours sample = 0.5 to 4 mg/dL.

Another source

• Urine = 0.5 to 4.0 Ehrlich units/day.
• Feces = 75 to 400 Ehrlich units/day.

Another source

• Urine urobilinogen  =  0.1 to 1.0  Ehrlich units /2 hours.
• One Ehrlich unit = approximately 1 mg of urobilinogen.

Procedure for urobilinogen in urine

1. Add fresh urine sample to Ehrlich’s reagent.
2. Ehrlich’s reagent:
3. Principle:
   1. Urobilinogen + p-dimethylamino benzaldehyde (Erhlich’s reagent) = Form red color.
   2.  Ascorbic acid is added as a reducing agent to keep urobilinogen in a reduced state.
   3. The addition of saturated sodium acetate stops the reaction.
4. Preparation of the reagent Ehrlich’ reagent:
   1. p-Dimthylaminobenzaldehyde (4-dimethylaminobenzaldehyde) =  4 grams.
   2. Hydrochloric acid, concentrated = 40 mL.
   3. Distle water = 160 mL.
      1. Take the 4-dimethylaminobenzaldehyde dissolve in distle water.
      2. Add the conc HCL and mix well.
   4. This is stable at room temperature in a colored bottle, especially in dark brown. Protect from the daylight.
5. The procedure of Ehrlich reaction:
   1. Take two test tubes.
   2. Label one as a test (T)  and the other for comparison (B).
   3. Add 5 ml of freshly voided urine to each test tube.
   4. Add 0.5 mL of Ehrilich reagent to test (T).
   5. Add 0.5 mL of conc HCL to the B test tube and mix.
   6. Leave both test tubes at room temperature for 5 minutes.
   7. Note for the red color in the test (T) tube.
      

      Urobilinogen by Ehrlich’s reagent

6. For feces, the same method can be  used except:
   1. Stool Aquos extract is treated with alkaline ferrous hydroxide to reduce urobilin to urobilinogen.
   2. Now add Ehrlich’s reagent.

Increased Urobilinogen is seen in:

1. Cirrhosis due to:
   1. Alcoholic cirrhosis.
   2. Viral diseases.
   3. Chemicals.
2. Biliary obstruction.
3. Biliary tract infection, cholangitis.
4. Hepatotoxic drugs e.g.aminosalicylic acid, Phenothiazine, and penicillin,
5. Due to increased RBCs destruction:
   1. Hemolytic anemia.
   2. Pernicious anemia.
   3. Malaria.
   4. Drugs.
6. Acute hepatitis.
7. Pulmonary infarction.
8. Hematoma.
9. Excessive ecchymosis.

Decreased Urobilinogen is seen in:

1. Gallstones.
2. Biliary obstruction.
3. Biliary tract infection (severe).
4. Pancreatic cancer (head of the pancreas)
5. Drugs, e.g., Aspirin, Chloramphenicol, Insulin, phenytoin, Streptomycin, tetracycline, Methyldopa, and sulphonamide.

Table showing the presence of urine bilirubin and urobilinogen in various conditions:

Test	Normal person	Hemolytic anemia	Liver disease	biliary obstruction
urine bilirubin	negative	negative	positive/Negative	positive (+++)
urine urobilinogen	negative	positive (+++)	positive (++)	absent (Low)

Normal urine picture:

Physical features	Chemical features	Microscopic findings
Color = Pale yellow or amber Appearance = Clear to slightly hazy pH = 4.5 to 8.0 Specific gravity = 1.015 to 1.025	Blood = Negative Glucose = Negative Ketones= Negative Protein = Negative Bilirubin = Negative Urobilinogen = Negative (±) Leucocyte esterase = Negative Nitrite for bacteria = Negative	RBCs = Rare or Negative WBC = Rare or Negative Epithelial cells = Few Cast = Negative (Occasional hyaline) Crystal = Negative (Depends upon the pH of the urine) Bacteria = Negative

 

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