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The binding of bilirubin to tissue was studied using adult human erythrocytes and rat liver mitochondria. Tissues were incubated with varying bilirubin-albumin molar ratios, varying albumin concentrations of a given bilirubin-albumin molar ratio, and varying pH. Bilirubin binding by tissue was reversible and stoichiometric with the concentration of the free (nonalbumin bound) bilirubin acid salt (bilirubin monovalent anion). Minimal binding of the bilirubin dianion, the predominant state of bilirubin in plasma, was also suggested. The observations support the "free bilirubin theory" where tissue and albumin compete for binding the body's bilirubin pool. Binding to tissue, however, is not determined by the free bilirubin concentration, but by the concentration of the pH dependent subfraction, the free bilirubin acid salt. Tissue binding and toxicity of bilirubin may result from the surfactant properties of the monovalent anion.

The Importance of Free Bilirubin Acid Salt in Bilirubin Uptake by Erythrocytes and Mitochondria