Urine Analysis: Part 20 – Urine For Bile pigments (Bilirubin, Urobilinogen, and Bile Salts)

Sample

1. The sample is urine.
2. Bile salt can be checked on random urine.
3. Bile pigmetns also include bilirubin and biliverdin.

Indications

1. This is the presence of bile pigments in the urine indicate liver dysfunction.
2. The presence of bile pigments in the urine helps to diagnose the increased production of bilirubin.

Pathophysiology

1. The bile is synthesized by the liver and consists of :
   1. Conjugated bile salts.
   2. Bilirubin diglucuronide.
   3. Cholesterol.
   4. Phospholipids.
   5. Electrolytes.
   6. Water.
   7. Bile is alkaline due to the presence of bicarbonate.
   8. Bile color is golden-brown to greenish-yellow.
      

      Bile acids and bile salt formation and excretion

2. Bile acids are synthesized in the hepatocytes from cholesterol. These are excreted into the bile and then pass into the duodenum.
   1. The primary bile acids are:
      1. Cholic acid.
      2. Chenodeoxycholic acid.
      3. These primary bile acids pass into the duodenum (intestine), by the bacterial action are converted to secondary bile acids.
   2. Secondary bile acids are:
      1. Deoxycholic acid.
      2. Lithocholic acid.
   3. The bile acid can conjugate with glycine and taurine and form bile salts.
   4. Bile salts help in stimulating the bile flow and is a potent antibacterial.
      

      Bile acids and bile salt formation, and enterohepatic circulation

3. The primary bile pigments are due to catabolism of the hemoglobin are:
   2. Bilirubin. is orange or yellow in color.
   3. Biliverdin is green in color.
   4. Urobilinogen (Urobilin).
4. The leading site of the formation of bile pigments in the liver.
5. Ultimately these leave the body in feces and a small amount in the urine.
6. The human bile contains:
   1. Cholate conjugate 38%.
   2. Chenodeoxycholate conjugate = 34%.
   3. Deoxycholate conjugate = 28%.
   4. Lithocholate = 1 to 2%.
7. The hepatic bile contains 5% to15% of the solids, and the major components are bile acids.
8. The following diagrams show how the bilirubin and urobilinogen are excreted.

 

 

Bile salt and bilirubin detection procedure:

1. Bile pigments (Bilirubin) give the urine greenish-yellow,  yellow or brown color.
2. The Fouchet’s test detects bile pigments (Bilirubin).
3. For bile salts, advise sulfur granules test.
4.  Fouchet’s test. It will detect the bilirubin in the urine.
   1. Reagents are:
      1. Barium chloride 10%
      2. Fouchet’s reagents consist of:
         1. Trichloracetic acid = 25 grams.
         2. Ferric chloride (FeCl3) 10% = 10 mL
         3. Distle water = 100 mL
   2. The procedure of Fouchet’s test:
      1. Take 5 ml of the urine, if the urine is alkaline then add one to 2 drops of glacial acetic acid (concentrated).
      2. Take 5 mL of urine and add barium chloride (BaCl2 10%), 2.5 mL.
      3. Mix well.
      4. The sample will become cloudy.
      5. Filter or centrifuge to obtain the precipitate which contains bilirubin.
         1. A precipitate of sulfates appears to which bilirubin is bound (barium-sulfate-bilirubin complex).
      6. To the filter paper precipitate adds one drop of Fouchet’s reagent (Ferric chloride).
      7. When ferric chloride is added to urine in trichloroacetic acid, it oxidizes bilirubin to green color biliverdin.
      8. Immediately blue-green color develops indicating positive for bilirubin.
         

         Fouchet’s reaction and its principle

4. Foam test for bilirubin:  Urine is kept in a small vial and vigorously shaken. The bile pigments will stain the resulting foam.
   1. There is yellowish foam in the case of urine bilirubin.
   2. In normal urine, the foam will be white.
5. Sulfur granule test for bile salts: 
   4. 4. Carefully sprinkle the sulfur granules over the surface of urine.
      5. Also can run water as the control.
      6. In the case of bile salts, sulfur will settle in the bottom.
      7. In the case of negative urine, sulfur granules will remain over the surface.
   

   Urine bile salt test with sulfur granules

Normal

• Normally bile pigments and bile salt are absent in normal urine.
  

  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

   

---