Bilirubin appears to be toxic in vitro to several cellular functions localized 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 μCi [3H]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 separated into aliquots of 2 mL, and the protein content was determined in each aliquot. Radioactivity was determined by scintillation counting, and the content of bilirubin per milligram of protein was calculated. Statistical comparisons were performed with Kruskal-Wallis nonparametric ANOVA. There were highly significant differences in bilirubin content per milligram of protein among subcellular compartments in all groups and at both time points. In all groups there were relatively high concentrations of bilirubin in the myelin fraction, an interesting observation in light of the theory that membranes are the primary target of bilirubin toxicity. The very high concentration of bilirubin relative 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 after hyperosmolar opening of the blood–brain barrier. The present data may be useful in planning in vitro studies of bilirubin toxicity in cell organelles.Abbreviations: P1 P2 P3 P4, first second third and fourth pellets from sequential density centrifugations; S1 S2 S3 S4, first second third and fourth supernatants from sequential density centrifugations
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