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 (BF) 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 BF values (<or=1.0 microM), a range in which spontaneous precipitation of UCB would be unlikely to occur, even though BF 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 <or= 1.0 microM. BF values were recalculated from the stated UCB, albumin, and chloride concentrations by applying affinity constants derived from ultrafiltration of comparable solutions containing 14C-UCB and delipidated human serum albumin. At BF slightly above aqueous solubility, UCB impaired mitochondrial function and viability of astrocytes. Exposure of neuroblastoma and embryonic neuronal cell lines to BF above 250 nM impaired cellular proliferation and mitochondrial function and increased apoptosis. Purified UCB inhibited the uptake of glutamate into astrocytes at BF as low as 309 nM and induced apoptosis in brain neurons at BF 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 microaggregates. The results render doubtful the long-held concept that precipitation of UCB in or on cells is required to produce neurotoxicity.