Urine for Bilirubin in Urine (Bilirubinuria)

Sample

1. The sample is urine.
2. A random sample can be taken.

Precautions

1. A fresh urine sample is needed.
2. Store at 2 to 8 °C for no longer than 24 hours.
3. Avoid urine to light.
4. Exposure to light urine will decrease the bilirubin.
5. The false-positive result is seen in the metabolites of drugs like phenazopyridine.
   1. The substances which color the urine red or turn red in the acid medium like:
      1. Chlorpromazine.
      2. Phenothiazine.
      3. Indol.
      4. Pyridium
6. False-negative results are seen:
   1. With the ascorbic acid intake.
   2. Oxidation of bilirubin to biliverdin.
   3. Increased nitrite concentration.
   4. Hydrolyzation of bilirubin diglucuronide to free bilirubin.

Indications

1. Diagnose hepatitis.
2. Diagnose any liver damage.
3. Monitor the treatment of hepatitis.

Pathophysiology

1. The detectable amount of bilirubin is not found in the urine.
2. Breakdown of hemoglobin leads to bilirubin formation, which goes to the liver.
   1. From the liver excreted into the bile.
   2. Excretion of bilirubin is seen in obstructive jaundice and not in hemolytic jaundice unless there is liver damage.
3. Following the diagram explains bilirubin metabolism and its excretion.
   

   Bilirubin metabolism summary

   

   Bilirubin excretion in the urine

4. Urine bilirubin is the early sign of :
   1. Hepatocellular disease.
   2. Intra or extrahepatic biliary obstruction.
5. Bilirubin appears in the urine before other signs of hepatitis appear.
6. Bilirubin’s presence or absence in the urine is helpful for clinical jaundice.
7. Bilirubin is the major component of bile.
8. If there is an obstruction to the bile, that will lead to conjugated hyperbilirubinemia.

9. Conjugated bilirubin is water-soluble so that it will appear in the urine.
10. So the presence of bilirubin in urine indicates defects after conjugation and defects in excretion.
11. Bilirubin is a yellowish pigment in the bile which is produced by the liver.
12. Bilirubin in urine color urine = dark yellow or orange.
    1. This test measures the amount of bilirubin excreted in the urine.
    2. The presence of bilirubin in the urine indicates jaundice.

Normal

• Normally bilirubin is absent in the urine (0 to 0.02 mg/dL).
• Even traces indicate liver disease like hepatitis.

Procedure for bilirubin in the urine:

1. Fouchet’s or Ferric chloride reaction:
   1. Add urine to barium chloride, and it will form barium sulfate precipitate containing bilirubin.
   2. Barium sulfate + bilirubin filter and get the precipitate.
   3. Add ferric chloride (Fouchet’s solution) to the above precipitate.
   4. Bilirubin oxidized to biliverdin, which gives green-blue color.
      

      Fouchet’s reaction and its principle

      

      Fouchet’s reaction for bilirubin in the urine

Comparison of the Fouchet’s reaction and Diazo reaction:

Characteristics	Fouchet’s reaction (FeCl3)	Diazo reaction
The sensitivity of the tests	Many other compounds give a positive reaction with FeCL3 with different colors.	A tablet test is more sensitive than a dipstick Tablet test = 0.2 to 0.4 mg/dL Dipstick test = 0.05 to 0.1 mg/dL
False-negative result	Oxidation of the bilirubin in case of delayed testing	Vitamin C (ascorbic acid) The increased amount of nitrite Oxidation of bilirubin in case of delay testing >4 hours
False-positive result	Urobilinogen urobilin Aspirin metabolites	Chlorpromazine

2. Dipstick method:
   1. There is a dipstick impregnated with a diazo reagent.
   2. this method detect the concentration of 0.5 mg/dL or greater.
      1. Insert the strip in the urine no longer than one second.
      2. After 60 seconds, the Diazo reagent reacts with bilirubin at an acidic pH.
         1. A pink to red-violet color is produced.
      3. The intensity of the color depends upon the concentration of the bilirubin in the urine.
         The  various strips detection level:

         Type of the strip	The detection level of bilirubin
         Ictotest	0.05 to 0.1 mg/dL
         Multistix	0.4 to 0.8 mg/dL
         Chemstrip	0.5 mg/dL

Increased urine bilirubin is seen in:

1. Hepatitis and liver diseases are caused by infection or exposure to toxic agents ( cirrhosis ).
2. Obstructive biliary tract disease.
3. Liver or biliary tract tumor.
4. Septicemia.
5. Hyperthyroidism.
6. Gallstones.
7. Metastatic tumor in the liver.
8. Drugs
9. Dubin-Johnson syndrome.
10. Rotor syndrome.

Urine Bilirubin/urobilinogen differential diagnosis

Test	Normal	Conjugated (Direct) hyperbilirubinemia	Unconjugated (Indirect) hyperbilirubinemia	Conjugated (Direct) hyperbilirubinemia
Etiology		Biliary obstruction	Hemolysis	Hepatitis (liver injury)
Urine bilirubin	negative	positive (Increased)	Usually absent	positive
Urine urobilinogen	positive / negative	Absent	Positive (Increased)	positive (+++)

• Urine bilirubin is negative in hemolytic disease.

Bilirubin (urine and blood) in various types of jaundice:

Type of the jaundice	Etiology	Urine bilirubin	Urine urobilinogen	Blood bilirubin	Fecal appearance
Hemolytic or prehepatic	Hemolytic anemia Hemolytic anemia of the newborn	Negative (Usually absent)	Increased	Raised	Dark brown
Hepatic or hepatocellular	Physiologic jaundice in neonates Viral hepatitis Toxic hepatitis Cirrhosis	Increased (variable) (+ or -)	Increased (++)	An increased, variable amount	Normal or pale
Post-hepatic or obstructive	Gall stones Malignancy	Increased (+++)	Absent (or very low)	Direct increased Indirect normal or slightly increased	Clay-colored, acholic

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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