Introduction:
The liver performs a wide range of metabolic and excretory functions essential for maintaining body homeostasis. Several biochemical tests are available to assess hepatic function, including estimation of serum bilirubin, urine bilirubin, serum proteins, excretory function, and enzymes. These tests help detect and monitor liver diseases such as hepatitis, cirrhosis, hemolytic disorders, and obstructive jaundice.
Serum Bilirubin
Bilirubin is the principal bile pigment produced from the breakdown of hemoglobin. It exists in two major forms in plasma — both bound to albumin:
- Unconjugated (indirect) bilirubin – non-esterified form
- Conjugated (direct) bilirubin – bilirubin diglucuronide (BDG), an esterified form
Routine laboratory tests measure both total bilirubin (conjugated + unconjugated) and direct (conjugated) bilirubin. The unconjugated bilirubin concentration is obtained by subtracting the direct fraction from the total value. This helps in differentiating hemolytic jaundice from hepatocellular and obstructive jaundice.
The classical method of bilirubin estimation involves converting bilirubin into an azo dye through a diazo reaction and measuring absorbance spectrophotometrically. This principle forms the basis of both manual and automated assays.
Manual Determination of Bilirubin
Principle:
- Total bilirubin reacts with diazotized sulfanilic acid in the presence of caffeine and sodium benzoate (accelerators) to form an azobilirubin complex.
- In alkaline solution, the azobilirubin turns blue, and its intensity is measured at 600 nm.
Reagents:
- Caffeine reagent: Acts as an accelerator by releasing bilirubin from albumin.
- Diazo reagent: Formed by mixing sulfanilic acid with sodium nitrite.
- Alkaline tartrate solution: Converts the red azobilirubin to blue form for color measurement.
- Bilirubin standard: Used to prepare standard curves for calibration.
Procedure (Summary):
- Add serum sample, caffeine reagent, and diazo reagent.
- Allow reaction for 10 minutes, then add alkaline tartrate.
- Measure absorbance at 600 nm against a blank.
Calculation: Total and conjugated bilirubin concentrations are derived using standard curves. Unconjugated bilirubin is calculated as the difference between total and conjugated fractions.
Clinical Interpretation
- Total Bilirubin: Mildly elevated in chronic hemolytic disease (up to 5 mg/dL); moderately to severely increased (10–30 mg/dL) in hepatocellular disease; markedly elevated (up to 50–60 mg/dL) in cholestasis.
- Conjugated Bilirubin: Increased in hepatocellular and obstructive jaundice.
- Unconjugated Bilirubin: Increased in hemolytic jaundice and neonatal jaundice.
Decreased bilirubin concentrations are rare and clinically insignificant.
Automated Bilirubin Determination
Automated analyzers use the Jendrassik and Grof method, where bilirubin reacts with diazotized sulfanilic acid in the presence of caffeine-benzoate accelerators. The reaction forms azobilirubin, which is measured spectrophotometrically.
Direct Spectrophotometric Determination (for Neonates):
In newborns, total bilirubin can be estimated directly by measuring absorbance at 455 nm. Because neonatal serum lacks interfering pigments like carotene, this method is suitable. Absorbance at 575 nm is used to correct for hemoglobin interference.
Calculation: Corrected bilirubin = A455 – A575
Urine Bilirubin
Bilirubin is normally absent in urine. Its presence indicates liver or biliary tract pathology. The pigment excreted in urine is bilirubin diglucuronide — a water-soluble conjugated form.
Detection Methods:
- Tablet Method (Ictotest): A diazotized reagent tablet reacts with urinary bilirubin to form a blue or purple color within 30 seconds, indicating a positive result.
- Dipstick Method: A rapid screening test using multi-test strips. A color change on the pad indicates bilirubin presence.
- Oxidation Spot Test (Fouchet’s Test): Involves oxidation of bilirubin to biliverdin (green) or its further products (blue) using Fouchet’s reagent. Color intensity corresponds to bilirubin concentration.
Any detectable bilirubin in urine is abnormal and suggests hepatocellular injury or biliary obstruction.
Urine Urobilinogen
Urobilinogen is a colorless compound derived from intestinal bacterial reduction of bilirubin. A portion is reabsorbed into the portal circulation, re-excreted by the liver, and a small amount (1–4 mg/24 hr) is excreted in urine.
Clinical Significance:
- Increased urobilinogen: Seen in hemolytic diseases, hepatocellular damage, and congestive heart failure.
- Decreased or absent urobilinogen: Suggests complete biliary obstruction.
Detection Methods:
- Dipstick Method: Based on reaction with p-dimethylaminobenzaldehyde (Ehrlich’s reagent), producing a red color proportional to urobilinogen concentration.
- Semi-Quantitative Test: Urobilinogen in a 2-hour urine sample reacts with Ehrlich’s aldehyde reagent. The color intensity is compared spectrophotometrically to a phenol-sulfon-phthalein (PSP) standard and expressed in Ehrlich units.
Reference Range: 0.1 – 1.0 Ehrlich units per 2 hours
Summary Table: Hepatic Function Tests
| Test | Analyte | Clinical Significance |
|---|---|---|
| Serum Bilirubin | Total, Conjugated, Unconjugated Bilirubin | Evaluates hepatic uptake and excretory function |
| Urine Bilirubin | Bilirubin Diglucuronide | Indicates hepatocellular or obstructive jaundice |
| Urine Urobilinogen | Urobilinogen | Increased in hemolysis; decreased in obstruction |
Detailed Notes:
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