Antioxidant and cytotoxic effects of bilirubin on neonatal erythrocytes

Bilirubin, the breakdown product of heme from erythrocytes, accumulates in the neonate in the first days of life. In recent years, the antioxidant properties of bilirubin have been demonstrated in vitro and in vivo, yet it is clear that bilirubin can be toxic to cells. To study the range in which bilirubin exerts its beneficial effect, we used erythrocytes derived from cord blood and incubated them with 0-60 mg/dL bilirubin combined with 3 g/dL BSA (bilirubin/BSA) to mimic physiologic and pathologic conditions. Oxidative stress was induced by incubating the erythrocytes with a solution of 0.6 mM H2O2 and 0.15 M CuSO4 to generate hydroxyl radical mediated injury. The loss of fluorescence of cis-parinaric acid and the degree of protein oxidation of erythrocyte membranes were assessed. Additionally, we determined erythrocyte membrane integrity, glucose-6 phosphate dehydrogenase activity, and adenosine triphosphatase activity before and after incubation with bilirubin/BSA. Incubation with bilirubin/BSA at concentrations up to 60 mg/dL and a bilirubin/ BSA molar ratio of two was associated with dose-dependent protection of erythrocytes against lipid peroxidation. However, concentrations of bilirubin equal to or exceeding 30 mg/dL and a bilirubin:BSA ratio of one were associated with increased protein oxidation, decreased erythrocyte,glucose-6 phosphate dehydrogenase and adenosine triphosphatase activity, and altered cell membrane integrity. We conclude that bilirubin: at physiologic concentrations, protects neonatal red blood cells against oxidative stress in the presence of physiologic concentrations of BSA but that bilirubin concentrations of 30 mg/dL or higher and a bilirubin:BSA ratio of greater than one are associated with significant cytotoxicity.