TITLE: DETERMINATION OF BILIRUBIN IN SERUM DATE:4 th March,2025
OBJECTIVES:
1. To differentiate between total, direct (conjugated), and indirect (unconjugated) bilirubin in serum
2. To understand the clinical significance of bilirubin measurement in serum.
3. To interpret the results of bilirubin levels in relation to normal reference ranges and pathological
conditions like jaundice, liver dysfunction, hemolytic anemia
INTRODUCTION
Bilirubin is a yellow-orange pigment formed as a byproduct of the catabolism of heme, primarily from
the breakdown of hemoglobin in senescent red blood cells. Once formed, unconjugated (indirect)
bilirubin is transported in the plasma bound to albumin and delivered to the liver. In the hepatocytes, it
undergoes conjugation with glucuronic acid by the enzyme UDP-glucuronosyltransferase, forming water-
soluble conjugated (direct) bilirubin. This conjugated form is then excreted into bile and eventually
eliminated via the intestines.
The measurement of serum bilirubin levels is a critical biochemical parameter in the clinical evaluation
of hepatic function, biliary obstruction, and hemolytic diseases. Elevated bilirubin levels may result from
increased hemolysis, impaired hepatic uptake or conjugation, or obstruction to bile flow, liver cirrhosis
or drug induced reactions. Clinically, elevated bilirubin manifests as jaundice—a yellow discoloration of
the skin and sclera—and can be categorized as pre-hepatic, hepatic, or post-hepatic jaundice based on
the underlying pathophysiology. Elevated bilirubin is referred to as hyperbilirubinemia
Total bilirubin refers to the sum of conjugated (direct) and unconjugated (indirect) bilirubin. While total
bilirubin is a useful screening tool, distinguishing between direct and indirect fractions is essential for
accurate diagnosis and clinical decision-making. For instance, an increase in indirect bilirubin is often
seen in hemolytic anemias or Gilbert’s syndrome, while direct bilirubin is elevated in hepatocellular
injury or cholestasis.
Differentiation between direct and indirect bilirubin is important in determining elevated bilirubin levels
PRINCIPLE
The determination of serum bilirubin is based on the diazo reaction, also known as the Jendrassik–Grof
method, or in simpler setups, the Malloy-Evelyn method may be used.
In this reaction, bilirubin reacts with diazotized sulfanilic acid (diazo reagent) to form a colored
azobilirubin complex. The unconjugated bilirubin couples with the sulfanilic acid in the presence of a
caffein benzoate accelerator. The intensity of the color formed is directly proportional to the amount of
bilirubin present in the sample.
Bilirubin +diazotized sulfanilic acid azobilirubin complex
To calculate the concentration of the direct or indirect bilirubin use the following formula:
Bilirubin conc = absorbance (test sample) *13mg/dl
Total Bilirubin concentration = Absorbance (Total) *13mg/dl
Indirect Bilirubin concentration = Total Bilirubin – Direct Bilirubin
The measured bilirubin levels fall within the normal reference range or may indicate a pathological
condition depending on the result and clinical context.
Normal Reference Ranges:
Total Bilirubin: 0.2 – 1 mg/dL
Direct (Conjugated): 0.1 – 0.4 mg/dL
Indirect (Unconjugated): 0.2 – 0.8 mg/dL