Reassessment of the unbound concentrations of unconjugated bilirubin in relation to neurotoxicity in vitro

Most studies of the cellular toxicity of unconjugated bilirubin (UCB) have been performed at concentrations of unbound UCB (B-F) that exceed those in the plasma of neonates with bilirubin encephalopathy. We assessed whether UCB could be toxic to neurons and astrocytes at clinically relevant B-F values (less than or equal to 1.0 muM), a range in which spontaneous precipitation of UCB would be unlikely to occur, even though B-F exceeded the aqueous saturation limit of 70 nM. A meta-analysis yielded twelve published studies that had determined the in vitro effects of UCB on the function of cultured neurons or astrocytes at calculable BF values less than or equal to 1.0 muM. B-F values were recalculated from the stated UCB, albumin, and chloride concentrations by applying affinity constants derived from ultrafiltration of comparable solutions containing C-14-UCB and delipidated human serum albumin. At B-F slightly above aqueous solubility, UCB impaired mitochondrial function and viability of astrocytes. Exposure of neuroblastoma and embryonic neuronal cell lines to B-F above 250 nM impaired cellular proliferation and mitochondrial function and increased apoptosis. Purified UCB inhibited the uptake of glutamate into astrocytes at B-F as low as 309 nM and induced apoptosis in brain neurons at B-F as low as 85 nM. UCB can impair various cellular functions of astrocytes and neurons exposed to BF near or modestly above its aqueous solubility limit, at which UCB exists as soluble oligomers and metastable micro-aggregates. The results render doubtful the long-held concept that precipitation of UCB in or on cells is required to produce neurotoxicity.