Gel filtration of hyperbilirubinemic sera through Sephadex G-25 and Sephadex LH-20 for the detection of 'free' non-albumin-bound unconjugated bilirubin.

Hyperbilirubinemic model sera with different bilirubin/albumin (Br/Alb) molar ratio, containing unconjugated bilirubin only, and containing various fractions of conjugated and unconjugated bilirubin, were simultaneously run through small columns packed with Sephadex G-25 and Sephadex LH-20. The bilirubin adsorbed by the gel was detected by a diazo-reagent and also recovered with chloroform. The amount of bilirubin adsorbed by the gel was higher using the Sephadex LH-20 when sera containing unconjugated bilirubin were tested. In case of sera containing also conjugated pigment, the bilirubin adsorbed by the two gels was the same at a Br/Alb molar ratio equal to or below 0.89; above this level the bilirubin adsorbed by the Sephadex LH-20 was higher. But with reference to the concentration of the unconjugated bilirubin the chloroform-recovered bilirubin was in greater amount than expected if unconjugated bilirubin were considered in the tested sera. The possible implication of the role played by the conjugated fraction is discussed in relation also to the clinical application of the gel filtration technique.