Measurement of bilirubin-albumin binding. I. Comparative analysis of four methods and four human serum albumin preparations.

Four methods for the measurement of bilirubin-albumin binding have been compared. Three of these, the fluorescent dye binding (Direct Yellow 7), Sephadex G-25 column chromatography, and the 2-(4-hydroxyazobenzene)benzoic acid (HBABA) dye binding methods demonstrate significant correlations of measured binding capacities for bilirubin over a range of bilirubin/albumin molar ratios. All three methods concurred in the demonstration that fresh adult human sera had a higher molar albumin binding capacity for bilirubin than the purified human serum albumin preparations. The fluorescent dye binding and Sephadex column methods agreed most closely in defining presumed deficiency in binding capacity. The HBABA dye binding method was less consistent and appeared to measure non-bilirubin binding sites on albumin in addition to bilirubin binding sites. The fourth method, the saturation index, yielded highly variable results as compared with the other methods because of an inherent excessive risk of laboratory error.