The Measurement of Bilirubin Fractions in Serum

Bilirubin fractions are measured by (1) the direct diazo reaction, (2) high-performance liquid chromatography (HPLC), (3) direct spectrophotometry, and (4) enzymatic methods. HPLC, which effects separation and quantitation of the four bilirubin fractions, is the method of choice, but impractical for routine use. A special application of direct spectrophotometry allows the measurement of unconjugated bilirubin and the sum of bilirubin conjugates. This approach, which provides essentially the same information as HPLC, unfortunately is available only in one clinical analyzer. The direct diazo reaction measures bilirubin conjugates plus delta-bilirubin, albeit not very accurately. Direct diazo methods that measure unconjugated bilirubin as direct could obscure the clinical diagnosis. At acid pH, enzymatic methods measure all direct reacting bilirubins, while at pH 10 only conjugated bilirubins are measured. Because the measurement of conjugated bilirubins is clearly more helpful than that of direct bilirubin in the differential diagnosis of jaundice, direct diazo methods should be replaced by methods specific for bilirubin conjugates.