BACKGROUND:Neonatal hyperbilirubinaemia remains one of the most common clinical conditions requiring therapeutic intervention. Nevertheless, reliable indicators of bilirubin toxicity are still missing. This prompted us to investigate (a) the progression of cytotoxic events produced by increasing concentrations of bilirubin; (b) the relevance of the membrane lipid package on bilirubin binding to erythrocytes; and (c) the reliability of chloroform extraction compared with albumin extraction to evaluate erythrocyte-bound bilirubin and cytotoxicity. MATERIALS AND METHODS:Morphological alterations, free bilirubin, erythrocyte-bound bilirubin (albumin- and chloroform-extractable), haemolysis and membrane-released lipids, were determined in human erythrocytes at 4 degrees C or 37 degrees C, after 4 h incubation at pH 7.4, with increasing molar ratios of bilirubin to albumin (0.5-5). The reversibility of cytotoxicity by albumin washing was assessed by morphological analysis. RESULTS:Decreased free bilirubin, lower erythrocyte-bound bilirubin concentration by albumin extraction (superficial/non-aggregated bilirubin) and higher values by chloroform extraction (deep/aggregated bilirubin) were observed for 37 degrees C vs. 4 degrees C, at molar ratios > 1. Echinocytosis increased with bilirubin concentration and temperature and was not fully reversed by albumin washing. Haemolysis was already significant at a molar ratio of 1, and was enhanced by temperature at molar ratios 3 and 5 (P < 0.01). The loss of membrane lipids was remarkable at molar ratios > or = 0.5, both at 4 degrees C and 37 degrees C (P < 0.01), although correlation with bilirubin concentration was only significant at 37 degrees C (r = 0.971; P < 0.01). CONCLUSIONS:These results suggest that increased lipid fluidity and high bilirubin concentrations promote membrane bilirubin translocation and toxicity. They also show that albumin is not able to displace the bilirubin located deeply or aggregated within the membrane, which in turn is removed by chloroform. Accordingly, chloroform-extractable rather than albumin-extractable bilirubin is a more accurate parameter to assess erythrocyte-bound bilirubin during severe hyperbilirubinaemia.