Subcellular localization of bilirubin in rat brain after in vivo i.v. administration of [H-3]bilirubin

Bilirubin appears to be toxic in vitro to several cellular functions locali zed to different subcellular compartments. It would therefore be useful to know what concentrations of bilirubin might be found in cell organelles in vivo. Rats were anesthetized and allocated to one of three groups: control, hypercarbia, and hyperosmolality. Each rat received a 5-min bolus dose of bilirubin 50 mg/kg i.v. (containing approximately 200 mu Ci [H-3]bilirubin) . Rats were killed 10 or 30 min after the start of the bilirubin infusion. Each brain was homogenized, and subcellular fractions were isolated by high -speed gradient centrifugation in sucrose media. The gradients were separat ed into aliquots of 2 mt, and the protein content was determined in each al iqnot. Radioactivity was determined by scintillation counting, and the cont ent of bilirubin per milligram of protein was calculated. Statistical compa risons were performed with Kruskal-Wallis. Statistical comparisons were: pe rformed with Wallis nonparametric ANOVA. There were highly significant diff erences in bilirubin content per milligram of protein among subcellular com partments in all groups and at both time points. In all groups there were r elatively high concentrations of bilirubin in the myelin fraction, an inter esting observation in light of the theory that membranes are the primary ta rget of bilirubin toxicity. The very high concentration of bilirubin relati ve to protein in cytoplasm, ribosomes, and mitochondria in the hyperosmolar group are also notable in light of data from hyperbilirubinemic animals in which changes in electrophysiology or energy metabolism only appeared afte r hyperosmolar opening of the blood-brain barrier. The present data may be useful in planning in vitro studies of bilirubin toxicity in cell organelle s.