Direct Spectrometric Determination of the Concentration of Bilirubins in Serum

Abstract

Earlier reported methods for direct spectrometric determination of the concentration of bilirubins (TB) in human serum are evaluated by inserting spectrometric data for bilirubin (UB), conjugated bilirubin (CB), oxyhemoglobin (HbO₂), methemalbumin, transferrin, carotenoids, and turbid components (T). About 170 new formulas, with the number of reading points varying from two to eight, are constructed and likewise tested. The best of the reported and our own formulas are applied to 179 native and enriched sera from infants and adults and to 10 different commercial comparison materials. The results are compared with those of diazo-determination and nominal values by regression analysis. The best formula is c(TB)=f X (21.6 X A466 - 27.4 X A522) μmol/l, where f is the reciprocal volume fraction of serum in a mixture with dipotassium tetraborate (c=100 mmol/l), pH=9.30; A466 means the absorbance at 466 nm, pathlength=10 mm. It corrects fully for HbO₂, T, and differences in molar absorption coefficients of UB and CB. Carotenoids remain a problem, but even the subgroup of sera from anicteric adults gives results with a better correlation to the diazomethod than previously reported. The analytical procedure is described; it is simple, rapid, and requires little serum; the coefficient of variation of its between-series disturbance is estimated to be 0.03 (for diazo-method, 0.07).

Key Words:

• Bilirubin
• carotenoids
• conjugated bilirubin
• direct spectrometric method
• methemalbumin
• oxyhemoglobin
• transferrin
• turbid components