Bilirubin binding by primary sites of human serum albumin, with suppression of secondary-site interference by use of a high salt concentration.

A high (2.0 mol/L) NaCl concentration apparently suppresses the secondary-site binding of bilirubin by human serum albumin. Thus if an excess of bilirubin is added to human serum albumin or to neonatal serum in buffer containing 0.1 mol of tris(hydroxymethyl)aminomethane and 2.0 mol of NaCl per liter (pH 7.5), and the bilirubin not bound to albumin is removed by treatment with calcium carbonate, the bilirubin binding reserve of primary binding sites can be estimated from direct measurement of spectral absorbance at 468 nm. Hemolysis and conjugated bilirubin apparently do not interfere. In a comparison study with serum samples from neonates the method gave results that generally agreed with those obtained by a commercially available Sephadex G-25 column procedure. In serum samples from adults the calculated unbound unconjugated bilirubin (free bilirubin) values derived by using binding reserves determined by the proposed method correlated well with the free bilirubin concentrations measured by a peroxidase method, but were only about one-half the amounts obtained by the peroxidase method.