How do you test for the percentage of direct bilirubin in a patient taking azathioprine (Imuran) with mildly elevated total bilirubin and normal liver enzymes?

Testing for Percentage of Direct Bilirubin

To determine the percentage of direct bilirubin, order a fractionated bilirubin panel from your laboratory that measures both total bilirubin and direct (or conjugated) bilirubin, then calculate the percentage by dividing direct bilirubin by total bilirubin and multiplying by 100. 1

Laboratory Testing Method

• Request a "fractionated bilirubin" or "bilirubin with fractions" test from your clinical laboratory, which will report both total bilirubin and direct-reacting bilirubin values 1
• The percentage is calculated as: (Direct Bilirubin ÷ Total Bilirubin) × 100 1
• Most clinical laboratories use either the direct diazo reaction method or enzymatic methods to measure direct bilirubin 2, 3

Clinical Interpretation Thresholds

• Direct bilirubin <20-30% of total bilirubin suggests Gilbert's syndrome (unconjugated hyperbilirubinemia) in the absence of hemolysis 1, 4
• Direct bilirubin 20-30% of total bilirubin represents a gray zone requiring clinical correlation and further investigation 4
• Direct bilirubin >30-35% of total bilirubin strongly suggests pathologic cholestatic conditions, including drug-induced liver injury 1, 4

Specific Context: Azathioprine with Mildly Elevated Bilirubin

In your patient taking azathioprine with mildly elevated total bilirubin and normal liver enzymes:

• Order the fractionated bilirubin test immediately to determine if this represents unconjugated hyperbilirubinemia (Gilbert's syndrome) versus early cholestatic injury 1, 4
• If direct bilirubin is <20-30% of total, this likely represents Gilbert's syndrome, which is benign and requires no intervention 1, 4
• If direct bilirubin is >30-35% of total bilirubin, this raises concern for azathioprine-induced cholestatic liver injury, even with normal aminotransferases 1, 4
• Obtain GGT to confirm hepatobiliary origin if direct bilirubin is elevated, as alkaline phosphatase may be normal early in drug-induced injury 4

Important Caveats

• The terms "direct" and "conjugated" bilirubin are often used interchangeably but are not identical: direct bilirubin includes both conjugated bilirubin and delta bilirubin (albumin-bound), which has a 21-day half-life 1, 4
• If prolonged hyperbilirubinemia of uncertain etiology persists, consider requesting breakdown of direct bilirubin into conjugated and delta bilirubin fractions 1, 4
• Persistent isolated elevation of direct bilirubin in patients on medications should be closely monitored, as this may indicate drug-induced liver injury even before aminotransferases rise 1, 4
• Azathioprine can rarely cause nodular regenerative hyperplasia with portal hypertension and mild bilirubin elevation, though this typically occurs after prolonged use 5