Experimental results and theoretical validation for the antioxidant mechanism of bilirubin

The present study demonstrates that hydroxyl, 1-hydroxy ethyl and glutathiyl radicals abstract hydrogen atom from bilirubin and forms a carbon-centered radical. The bimolecular rate constant for the reaction of center dot OH, 1-hydroxy ethyl and glutathiyl radicals with bilirubin are 1.0 x 10(10), 2.0 x 10(8) and 6.0 x 108 dm(3) mol(-1) s(-1), respectively. The transient absorption spectra obtained in these reactions have two peaks at 350 nm and 540 nm with bleaching in absorption at 450 nm. Both the absorption peaks form and decay with same rate constants showing that they are due to the same species. The bilirubin radical thus produced, decays with first order rate constant of 1.13 x 10(4) S-1. In presence of oxygen the decay becomes faster confirming it to be a carbon-centered radical. Linoleic acid peroxyl radical also reacts with bilirubin via hydrogen abstraction. In the case of NO2 center dot radical reaction, bilirubin produces absorption peak at 590 nm assigned to be the radical cation formed via single electron transfer from bilirubin. The bimolecular rate constant for this reaction is 2.0 x 10(9) dm(3) mol(-1) s(-1). Corrected absorption spectra have been shown in few cases of these oxidation reactions. These results show that the oxidation of bilirubin can occur in two ways, either via hydrogen atom transfer or via single electron transfer depending on the nature of the oxidizing radical. Ab initio molecular orbital calculations have been performed to investigate the structure and energetics of radicals and radical cation produced by removal of hydrogen and electron, respectively from bilirubin molecule. It has been found that hydrogen abstraction from central methylenic group is most probable whereas pyrrole radical cation is most stable.