Affinity of human serum albumin for bilirubin varies with albumin concentration and buffer composition - Results of a novel ultrafiltration method

Albumin binding is a crucial determinant of bilirubin clearance in health a nd bilirubin toxicity in certain disease states. However, prior attempts to measure the affinity of albumin for bilirubin have yielded highly variable results, reflecting both differing conditions and the confounding influenc e of impurities. We therefore have devised a method based on serial ultrafi ltration that successively removes impurities in [C-14]bilirubin until a st able binding affinity is achieved, and then we used it to assess the effect of albumin concentration and buffer composition on binding. The apparent b inding affinity of human serum albumin for [C-14]bilirubin was strongly dep endent on assay conditions, falling from (5.09 +/- 0.24) X 10(7) liters/mol at lower albumin concentrations (15 muM) to (0.54 +/- 0.05) X 10(7) liters /mol at higher albumin concentrations (300 mum). To determine whether radio active impurities were responsible for this change, we estimated impurities in the stock bilirubin using a novel modeling approach and found them to b e 0.11-0.13%. Formation of new impurities during the study and their affini ty for albumin were also estimated. After correction for impurities, the bi nding affinity remained heavily dependent on the albumin concentration (ran ge (5.37 +/- 0.26) X 10(7) liters/mol to (0.65 +/- 0.03) X 10(7) liters/ mo l). Affinities decreased by about half in the presence of chloride (50 mm). Thus, the affinity of human albumin for bilirubin is not constant, but var ies with both albumin concentration and buffer composition. Binding may be considerably less avid at physiological albumin concentrations than previou sly believed.