BILIRUBIN IS AN EFFECTIVE ANTIOXIDANT OF PEROXYNITRITE-MEDICATED PROTEIN OXIDATION IN HUMAN BLOOD-PLASMA

Bilirubin is a bile pigment that may have an important role as an anti oxidant. Its antioxidant potential is attributed mainly to the scaveng ing of peroxyl radicals. We investigated the reaction of bilirubin wit h peroxynitrite in phosphate buffer and in blood plasma. In phosphate buffer bilirubin was rapidly oxidized by micromolar concentrations of peroxynitrite, and its oxidation yield was higher at alkaline pH with an apparent pK(a) = 6.9. In contrast, the major oxidation product of b ilirubin in plasma was biliverdin, and the pH profile of its oxidation yield showed a slightly increased oxidation at acidic pH without a cl ear inflection point. The addition of NaHCO3 to bilirubin decreased th e peroxynitrite-dependent oxidation, suggesting that the reactive inte rmediates formed in the reaction between CO2 and peroxynitrite are les s efficient oxidants of bilirubin. The antioxidant role of bilirubin w as investigated in some peroxynitrite-mediated plasma protein modifica tions that are enhanced by CO2 (tryptophan oxidation and protein tyros ine nitration) or slightly decreased by CO2 (protein carbonyl groups). Bilirubin in the micromolar concentration range afforded a significan t protection against all these oxidative modifications and, notably, p rotected plasma proteins even when the pigment was added 5 s after per oxynitrite (i.e., when peroxynitrite is completely decomposed). The lo ss of tryptophan fluorescence triggered by peroxynitrite was a relativ ely slow process fulfilled only after a few minutes. After this time, bilirubin was unable to reduce the tryptophan loss, and it was unable to reduce previously formed nitrated albumin or previously formed carb onyls. We deduce that bilirubin in plasma cannot react to a significan t extent with peroxynitrite, and we suggest that bilirubin, through a hydrogen donation mechanism, participates as a scavenger of secondary oxidants formed in the oxidative process. (C) 1998 Academic Press.