Effect of bilirubin on cytochrome c oxidase activity of mitochondria from mouse brain and liver

Mouse liver mitochondria increase unbound UCB oxidation, consequently increasing the apparent rate constant for unbound UCB oxidation by HRP (Kp), higher than in control and mouse brain mitochondria, emphasizing the importance of determining Kp in complete systems containing the organelles being studied. The in vitro effects of UCB on cytochrome c oxidase activity in mitochondria isolated from mouse brain and liver were studied at Bf ranging from 22 to 150 nM. The results show that UCB at Bf up to 60 nM did not alter mitochondrial cytochrome c oxidase activity, while the higher concentrations significantly inhibited the enzyme activity by 20% in both liver and brain mitochondria.We conclude that it is essential to include the organelles being studied in the medium used in measuring both Kp and Bf. A moderately elevated, pathophysiologically-relevant Bf impaired the cytochrome c oxidase activity modestly in mitochondria from mouse brain and liver.Unconjugated bilirubin (UCB) at low concentrations is a potent antioxidant [1,2] that is neuroprotective [3], while higher levels of UCB may damage neurons and astrocytes [3-6], resulting in bilirubin-induced neurological dysfunction (BIND) in some neonates with severe unconjugated hyperbilirubinemia. It has been shown that the unbound, free concentration (Bf) of unconjugated bilirubin (UCB), and not the total UCB level, correlates with bilirubin cytotoxicity [7], but the key molecular mechanisms accounting for the toxic effects of UCB are largely unknown. Although the primary subcellular targets for the toxic effects of UCB are not fully identified, mitochondria are believed to be particularly vulnerable [4,8-12]. However, with few exceptions [10,13], most prior research was performed at unbound UCB concentrations (Bf) orders of magnitude higher than its hypothesized upper normal limit of 20 nM (1.2 μg/dL) in plasma of term infants [14]. In addition, most studies used unpurified commercial bilirubin, rendering uncertain whe