Oxidation of bilirubin in the brain - Further characterization of a potentially protective mechanism

Bilirubin is a well-known neurotoxin and presents a particular problem in n ewborn infants. This is partly due to the high incidence of unconjugated hy perbilirubinemia in that age group, but may also be due to increased vulner ability to bilirubin toxicity. The brain may be able to protect itself agai nst bilirubin toxicity through a process of oxidation. The responsible enzy me is localized on the inner mitochondrial membrane and appears to be more active in glia than in neurons and to increase in activity with postnatal m aturation. Here we have investigated the possibility that the responsible e nzyme might be a cytochrome oxidase, malate dehydrogenase, or monoamine oxi dase, all enzymes located on the inner mitochondrial membrane. Mitochondria were obtained from rat brains through homogenization and differential cent rifugation in sucrose medium. The ability of mitochondrial membranes to oxi dize bilirubin was measured by following the change in optical density at 4 40 nm of a bilirubin solution to which a membrane suspension had been added . The activity was not, changed by in vitro inhibitors of malate dehydrogen ase or monoamine oxidase, but was moderately inhibited by ketoconazole and clotrimazole, both known inhibitors of hepatic cytochrome P450 oxidases. Ac tivity was inhibited by depletion of cytochrome c in the mitochondria and r econstituted by reintroducing cytochrome c into the reaction mixture. The r eaction was not modified by the addition of a free radical quencher, but wa s inhibited by removal of oxygen from the reaction mixture. The activity wa s significantly inhibited by cyanide. Activity was retained in a 100,000-g pellet and was not influenced by the addition of NAD, NADP, NADH, NADPH, GS H, or GSSH to this pellet. We conclude that the bilirubin-oxidizing activit y in brain mitochondrial membranes is cytochrome c dependent, but does not appear to be unequivocally identifiable as a cytochrome P450 oxidase. (C) 1 999 Academic Press.