SummaryIn 35 neonatal sera, comparisons were made of bilirubin binding capacity determined by Sephadex gel filtration and by peroxidase oxidation techniques. The serum indirect bilirubin concentration was 11.1 ± 4.6 mg/100 ml (190 ± 79 μM/liter); the serum concentration of unbound bilirubin was 50 ± 60 μg/100 ml (0.85 ± 1.03 μM/liter) by gel filtration and 0.77 ± 0.66 μg/100 ml (0.013 ± 0.011 μM/liter) by peroxidase oxidation. Binding capacity averaged 22.9 ± 5.6 mg/100 ml (392 ± 96 μM/liter) by gel filtration and 22.1 ± 6.1 mg/100 ml (378 ± 104 μM/liter) by peroxidase oxidation. The binding capacity, expressed as moles of bilirubin bound per mole of albumin, was 0.80 mol bilirubin/mol albumin by the Sephadex method and 0.77 by the peroxidase method. Individual values for binding capacity were in close agreement (r = 0.961,P< 0.001). At saturation of the first bilirubin binding site, the unbound bilirubin concentration was 190 ± 80 μg/100 ml (3.25 ± 1.37 μM/liter) by gel filtration and 1.87 ± 0.54 μg/100 ml (0.032 ± 0.009 μM/liter) by peroxidase oxidation. There was no correlation between unbound bilirubin concentrations estimated by the two methods. The peroxidase method requires a smaller volume of serum than the Sephadex method and is more sensitive to small changes in free bilirubin concentration.SpeculationBoth Sephadex gel filtration and peroxidase oxidation techniques appear capable of predicting the indirect bilirubin concentration at which the first or “tight” bilirubin binding site will be saturated, and above which free bilirubin concentration will be greatly increased. In neonatal sera, the two methods showed close agreement for individual determinations of binding capacity over a wide range of molar ratios. The lower limit for the neurotoxic concentration of unbound or “free” bilirubin is not precisely known and may vary in the face of multiple clinical factors. However, it seems likely that free bilirubin concentrations in excess of those measured at saturation of the first binding site would be potentially neurotoxic, and that bilirubin bound to secondary binding sites would be more readily dissociable.