UNIT I

Serum Bilirubin

1.1 Formation and excretion of bilirubin

1.2 Conjugated and unconjugated bilirubin

1.3 Principle and procedures of serum bilirubin estimation (Direct & Indirect)

1.4 Clinical significance of serum bilirubin estimation

Serum Bilirubin

Breakdown of red blood cells releases bilirubin in blood. It is processed in the liver and excreted in the bile. Bilirubin is excreted from the body with feces. There are two types of bilirubin. These are conjugated bilirubin (Direct bilirubin) and unconjugated bilrubin (Indirect bilirubin). Unconjugated bilirubin is insoluble in water. It bounds with albumin inside blood and transported to liver. Inside liver, it undergoes conjugation reaction to become water soluble conjugated bilirubin. Conjugated bilirubin is excreted in bile.

Serum bilirubin is measured to assess liver functions. Normal level of serum bilirubin is 1.0 mg/dL in normal adult. Higher level of serum bilirubin indicates liver dysfunction such as jaundice, hepatitis, cirrhosis or biliary obstruction.

1.1 Formation and excretion of bilirubin (Metabolism of bilirubin):

Bilirubin is biproduct of red blood cells breakdown. Bilirubin in blood is formed through following steps:

1. Red Blood Cells Breakdown: Normal life span of RBCs is 120 days. At the end of life span, RBCs breakdown in reticuloendothelial system releases heme (iron) and globin (protein) in the blood. This process is called haemolysis.

2. Biliverdin formation: Non iron portion of Haem is converted into green coloured biliverdin by enzyme haeme oxygenase. .

3. Biliverdin reduction: Enzyme biliverdin reductase converts biliverdin into yellow-orange coloured bilirubin pigment. This bilirubin is Unconjugated bilirubin (Indirect bilirubin). It is water insoluble bilirubin.

4. Transport to liver: Unconjugated bilirubin in blood bonds with blood albumin. Uncojugated bilirubin-albumin complex travel to the liver with the blood stream. It enters into liver cells.

5. Conjugation: Inside liver cells, Conjugated bilirubin- albumin complex dissociates to form free bilirubin and free albumin. Bilirubin undergoes conjugation with glucoronic acid to form water soluble conjugated bilirubin (Direct bilirubin) by enzyme bilirubin UDP glucuronic transferase. .

6. Excretion: Conjugated bilirubin enters into gallbladder with bile. Gall bladder releases bile with bilirubin into small intestine. Microbial flora in the large intestine converts bilirubin into colourless urobilinogen. Small amount of urobilinogen is absorbed and enter into liver. Some part of it is excreted into bile and rest is excreted in urine by kidney. They develop pale yellow colour in urine. Rest urobilinogen is converted into stercobilin inside large intestine. Stercobilin develop brown colour in stool. They are excreted with stool.

1.2 Conjugated and unconjugated bilirubin:

Unconjugated bilirubin is also called indirect bilirubin. Unconjugated bilirubin is a by-product of heme breakdown. Heme is a component of red blood cells released after red blood cells break down. Unconjugated bilirubin is water-insoluble and cannot be excreted from the body. Unconjugated bilirubin binds with albumin in blood and is transported into liver cells with the bloodstream.

Unconjugated bilirubin is toxic at high concentrations. An increase in unconjugated bilirubin level above its normal level is called hyperbilirubinemia. Proper functioning of the liver is a must to maintain a normal level of unconjugated bilirubin in blood. Hyperbilirubinemia indicates liver diseases, hemolytic anemia, or certain genetic disorders.

Conjugated bilirubin: Conjugated bilirubin is also called direct bilirubin. Liver cells convert unconjugated bilirubin by conjugation with glucuronic acid to conjugated bilirubin. This reaction is called glucuronidation. Conjugated bilirubin is water soluble and less toxic than unconjugated bilirubin. Conjugated bilirubin is excreted into the small intestine with bile. Then Conjugated bilirubin is excreted with feces.

1.3 Principle and procedures of serum bilirubin estimation (Direct & Indirect)

There are several methods to estimate direct bilirubin and total bilirubin in the serum sample. The Diazo method to estimate serum direct bilirubin and total bilirubin is the most commonly used method.

Principle: Both direct bilirubin and indirect bilirubin react with diazo reagent in solution form to produce purple-coloured azobilirubin. The intensity of color produced is directly proportional to the amount of bilirubin in the serum sample.

Methanol is used in the reaction because both direct bilirubin and indirect bilirubin are soluble in methanol. Distilled water is used in place of methanol to estimate serum direct bilirubin because direct bilirubin is soluble in water and indirect bilirubin is not soluble in water. The difference between serum indirect bilirubin and serum direct bilirubin values gives the amount of indirect bilirubin in the serum sample.

Normal Range of serum bilirubin: 1.0 mg/dL.

Direct bilirubin: 0.5 mg/dL.

Indirect bilirubin: 0.5 mg/dL.

Reagents

1. Diazo-A: 0.1 gm sulfanilic acid in 100mL of 1.5% v/v hydrochloric acid

2. Diazo-B: 0.5 gm sodium nitate in 100 mL D. Water.

3. Diazo blank: 1.5 ml hydrochloric acid in 100 mL D. Water.

4. Methanol

5. Stock standard: 0.29 mL methyl red in 100 mL glacial acetic acid.

6. Working standard: 0.1 mL stock standard and 1.44 gm sodium nitrate in 100 mL D Water.

Reagents 1,3,4 and 5 are stable at room temperature. Reagent 2 is stable at temperatures of 2 to 8 degrees C in an amber-colored bottle.

Procedure: The following steps are followed to estimate serum bilirubin

1. Sample collection: Collect blood samples from the patient using a syringe or venipuncture from the arm.

2. Sample preparation: Centrifuge blood sample to collect serum.

3. Mixing and incubation: Mix the sample and reagents as per the below details

   1. For total bilirubin estimation:

      1. D. Water: 1.8 mL.

      2. Serum sample: 1.8 mL.

      3. Diazo mixture: 0.5 mL.

      4. Methanol: 2.5 mL

   For total bilirubin blank

   1. D. Water: 1.8 mL.

   2. Serum sample: 1.8 mL.

   3. Diazo blank reagent: 0.5 mL.

   4. Methanol: 2.5 mL

   For direct bilirubin estimation

   1. D. Water: 1.8 mL.

   2. Serum sample: 1.8 mL.

   3. Diazo mixture: 0.5 mL.

   4. D. Water: 2.5 mL

   For direct bilirubin blank

   1. D. Water: 1.8 mL.

   2. Serum sample: 1.8 mL.

   3. Diazo blank reagent: 0.5 mL.

   4. D. Water: 2.5 mL

Incubate the mixture in the dark for 30 minutes Allow the reaction between bilirubin and diazo reagent to develop color in the serum sample.

4. Colour measurement: Read the optical density (O.D.) of serum samples for the total bilirubin test sample and direct the bilirubin test sample in a colorimeter at 540 nm (green filter) to measure the color density. Also, measure the O.D. of the standard solution.

5. Calculation

O.D. of total bilirubin = O.D. of total test- O.D. of total blank.

O.D. of direct bilirubin = O.D. of direct test – O.D. of direct blank.

Total bilirubin in mg/dL = O.D. of total bilirubin/ O.D. of standard X 10.

Direct bilirubin in mg/dL = O.D. of direct bilirubin/O.D. of standard X 10.

Indirect bilirubin in mg/ dL = Total bilirubin in mg/dL - Direct bilirubin in mg/dL.

Author: Dr Pramila Singh

1.4 Clinical significance of serum bilirubin estimation

The following are the clinical significance of serum bilirubin estimation

1. Liver function assessment: Increased level of bilirubin indicates liver damage such as liver cancer, hepatitis, cirrhosis, or jaundice.

2. Hemolytic disorders: Increased level of bilirubin indicates excessive breakdown of red blood cells. It occurs in hemolytic anemia and sickle cell disease.

3. Newborn health: Neonatal Jaundice in newborns is diagnosed by bilirubin estimation.

4. Medication and drug reactions: Side effects of certain drugs affect bilirubin metabolism in the liver. Serum bilirubin estimation detects liver damage due to side effects of drugs.

5. Progress of disease monitoring: A series of bilirubin estimations helps to monitor disease progress and to assess the Effectiveness of disease treatment.