Competitive binding of free fatty acids and bilirubin to albumin: differences in HBABA dye versus Sephadex G-25 interpretation of results

Summary 1. The competitive binding of oleate and bilirubin to albumin was evaluated in vitro using three different methods; reserve albumin binding capacity by HBABA dye, Sephadex G-25 gel filtration, and bilirubin spectral curves. 2. In a bilirubin-albumin solution of molar ratio 1.6, the percentage depression of reserve albumin binding capacity was linearly proportional to the amount of oleate added. 3. The Sephadex G-25 technique showed that actual displacement of bilirubin did not occur until the concentration of oleate exceeded 1.5 mEq/L; a finding confirmed by bilirubin spectral curve analysis. At this oleate concentration there was already a 40–50% depression of the reserve albumin binding capacity. 4. These findings suggest that the assessment of reserve albumin binding capacity is non-specific for bilirubin or for any of its competing anions and cannot distinguish between competitive and non-competitive binding to albumin. 5. The Sephadex G-25 method yields more meaningful quantitative data in terms of actual displacement of bilirubin by any other anions in the system under study.