Improved method for accurate quantitation of total and conjugated bilirubin in serum.

We describe an improved method for the quantitation of total and direct bilirubin in serum in which sodium dodecyl sulfate is used as an accelerator. Viscous and caustic reagents are not needed, and the effects of protein matrix, hemoglobin interference, and turbidity are minimized. For total bilirubin determination we first add the sodium dodecyl sulfate reagent to serum, then a diazo reagent that lowers the pH to 5.6, the effective pI(2 of maleic acid. After 10 mm we add sulfanilic#{149}HCI reagent to bring the pH to 1.55. For determination of conjugated bilirubin, we add the sulfanilic#{149}HCI reagent first, followed by the diazo reagent, an ascorbic acid solution, and finally, the sodium dodecyl sulfate reagent. The same sample blank is used for determination of both total and direct bilirubin. At 565 nm the molar absorptivity of azobilirubin determined with Standard Reference Material 916 in human serum albumin, bovine serum albumin, and pooled serum is 82780 ± 496 L mo11cm1. The absorptivity remains constant to within 0.3% in the pH range of 1.45 to 1.67. The color at both pH 5.6 and 1.55 is stable to within 0.1% for at least 1 h. The method is unaffected by protein concentrations of 2-120 gIL, has a linear standard curve to within 0.5% for bilirubin concentrations of 0-250 mg/L, and correlates well with the Jendrassik-Grof method by linear-regression analysis of 52 samples from patients (r = 0.999; slope, 1.02; yintercept, 0.27 mg/L). We discuss a mechanism to explain the effects of hemolysis on the coupling of the diazo reagent, and discuss the effects of several heme-binding agents that can be used to minimize such interference.