Assessment of bilirubin toxicity to erythrocytes. Implication in neonatal jaundice management.

Background Neonatal hyperbilirubinaemia remains one of the most common clin ical conditions requiring therapeutic intervention. Nevertheless, reliable indicators of bilirubin toxicity are still missing. This prompted us to inv estigate (a) the progression of cytotoxic events produced by increasing con centrations of bilirubin; (b) the relevance of the membrane lipid package o n bilirubin binding to erythrocytes; and (c) the reliability of chloroform extraction compared with albumin extraction to evaluate erythrocyte-bound b ilirubin and cytotoxicity. Materials and methods Morphological alterations, free bilirubin, erythrocyt e-bound bilirubin (albumin- and chloroform-extractable), haemolysis and mem brane-released lipids, were determined in human erythrocytes at 4 degrees C or 37 degrees C, after 4h incubation at pH7.4, with increasing molar ratio s of bilirubin to albumin (0.5-5). The reversibility of cytotoxicity by alb umin washing was assessed by morphological analysis. Results Decreased free bilirubin, lower erythrocyte-bound bilirubin concent ration by albumin extraction (superficial/non-aggregated bilirubin) and hig her values by chloroform extraction (deep/aggregated bilirubin) were observ ed for 37 degrees C vs. 4 degrees C, at molar ratios >1. Echinocytosis incr eased with bilirubin concentration and temperature and was not fully revers ed 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 greater than or equ al to 0.5, both at 4 degrees C and 37 degrees C (P<0.01), although correlat ion 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 bi lirubin concentrations promote membrane bilirubin translocation and toxicit y. They also show that albumin is not able to displace the bilirubin locate d deeply or aggregated within the membrane, which in turn is removed by chl oroform. Accordingly, chloroform-extractable rather than albumin-extractabl e bilirubin is a more accurate parameter to assess erythrocyte-bound biliru bin during severe hyperbilirubinaemia.