ABSTRACT
Title
Bilirubin alters Fe(III)/Fe(II) ratio thus reducing lipid peroxidation in rat brain microsomes: a novel mechanism for the antioxidant role of bile pigments.
Authors
C. Mancuso1, R. Siciliano1, E. Barone1, G. Minotti2 and P. Preziosi1.
1Institute of Pharmacology, Catholic University School of Medicine, Rome, Italy;2Center for Integrated Research, Drug Sciences, University Campus Bio-Medico, Rome, Italy.
1Institute of Pharmacology, Catholic University School of Medicine, Rome, Italy;2Center for Integrated Research, Drug Sciences, University Campus Bio-Medico, Rome, Italy.
Abstract
Heme oxygenase (HO) is an ubiquitous microsomal enzyme, which catalyzes the oxidation of the alpha-meso-carbon bridge of heme moieties in hemoproteins yielding equimolar amounts of Fe(II), CO, and biliverdin-IX-alpha (biliverdin). This latter is then reduced by the cytosolic biliverdin reductase into bilirubin-IX-alpha (bilirubin), a powerful free radical scavenger.
The main role of free radical-induced lipid peroxidation in the pathogenesis of neurodegenerative disorders is no longer matter of debate. Reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide and peroxyl radical, have been implicated in the initiation of lipid peroxidation. The hydroxyl radical (·OH) was frequently proposed as the initiating species and the iron-catalyzed Haber-Weiss reaction is described as the sequence through which superoxide anion, hydrogen peroxide and iron, rapidly produce ·OH. In this setting, several research groups proposed that ·OH cannot promote lipid peroxidation,since it exhibits a diffusion-limited reactivity and cannot migrate from the site(s) of generation to the hydrophobic membrane phases where the bis-allylic bonds are buried. On the other hand, lipid peroxidation would occur anytime iron oxidizes incompletely to the ferric form, generating perferryl ions [Fe(II)O2–Fe(III)] or oxygen-bridged Fe(II)–Fe(III) complexes that are more stable and can substitute for hydroxyl radical. This process is favored when iron is chelated by ADP and oxygen serves as the oxidant. Several lines of evidence demonstrated that bilirubin exerts antioxidant properties by scavenging both peroxyl and hydroxyl radicals, but there was not any study in literature demonstrating the interaction of bilirubin with iron as a possible antioxidant mechanism.
The aim of this study was to evaluate whether or not bilirubin alters the Fe(II)/Fe(III) ratio thus reducing lipid peroxidation in rat brain microsomes. As initiator of lipid peroxidation a system formed by NADPH-ADP/Fe(III) was chosen because it generates both perferryl ion and peroxyl radicals involved in the initiation and propagation phases of lipid peroxidation, respectively.
In order to set up the experimental system, preliminary experiments were carried out and demonstrated that, in the presence of 10 mM NADPH, ADP/Fe(III) (5/1, 10/1 and 20/1, 1 μM FeCl3) linearly increased lipid peroxidation which reached the maximum at the ADP/Fe(III) ratio of 20/1. In this experimental system the raise in lipid peroxidation was paralleled by an increase in the formation of Fe(II) which approached to the 50% at the ADP/Fe(III) ratio of 20/1, according to previous results. Bilirubin (0.01-100 μM) significantly inhibited NADPH-ADP/Fe(III)-induced lipid peroxidation in rat brain microsomes and the extent of the inhibition was inversely proportional to the ADP/Fe(III) ratio. Similar results were obtained when bilirubin was complexed with saturating concentration of human serum albumin (200 μM), even if albumin-bound bilirubin displayed a greater efficacy than bilirubin alone. In search for a mechanism for this antioxidant effect, the bilirubin-induced reduction of Fe(III) into Fe(II) was investigated. Bilirubin (0.1-10 μM) dose dependently reduced Fe(III) into Fe(II) approaching to a maximal value of about 80% Fe(II) at 5/1 and 10/1 ADP/Fe(III) ratios. Albumin-bound bilirubin significantly increased Fe(II) at greater extent (maximal value of about 95%) than bilirubin alone and independently from the ADP/Fe(III) ratio. The reduction of Fe(III) into Fe(II) seems to be specific for bilirubin because biliverdin (0.1-10 μM) did not increase Fe(II) formation and maintained the Fe(III)/Fe(II) ratio close to the unit, thus explaining why biliverdin have a low efficacy in reducing NADPH/Fe(III) lipid peroxidation. In conclusion, these results propose a new mechanism through which bilirubin could exert its antioxidant effect in addition to the well-known scavenging activity against ROS.
The main role of free radical-induced lipid peroxidation in the pathogenesis of neurodegenerative disorders is no longer matter of debate. Reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide and peroxyl radical, have been implicated in the initiation of lipid peroxidation. The hydroxyl radical (·OH) was frequently proposed as the initiating species and the iron-catalyzed Haber-Weiss reaction is described as the sequence through which superoxide anion, hydrogen peroxide and iron, rapidly produce ·OH. In this setting, several research groups proposed that ·OH cannot promote lipid peroxidation,since it exhibits a diffusion-limited reactivity and cannot migrate from the site(s) of generation to the hydrophobic membrane phases where the bis-allylic bonds are buried. On the other hand, lipid peroxidation would occur anytime iron oxidizes incompletely to the ferric form, generating perferryl ions [Fe(II)O2–Fe(III)] or oxygen-bridged Fe(II)–Fe(III) complexes that are more stable and can substitute for hydroxyl radical. This process is favored when iron is chelated by ADP and oxygen serves as the oxidant. Several lines of evidence demonstrated that bilirubin exerts antioxidant properties by scavenging both peroxyl and hydroxyl radicals, but there was not any study in literature demonstrating the interaction of bilirubin with iron as a possible antioxidant mechanism.
The aim of this study was to evaluate whether or not bilirubin alters the Fe(II)/Fe(III) ratio thus reducing lipid peroxidation in rat brain microsomes. As initiator of lipid peroxidation a system formed by NADPH-ADP/Fe(III) was chosen because it generates both perferryl ion and peroxyl radicals involved in the initiation and propagation phases of lipid peroxidation, respectively.
In order to set up the experimental system, preliminary experiments were carried out and demonstrated that, in the presence of 10 mM NADPH, ADP/Fe(III) (5/1, 10/1 and 20/1, 1 μM FeCl3) linearly increased lipid peroxidation which reached the maximum at the ADP/Fe(III) ratio of 20/1. In this experimental system the raise in lipid peroxidation was paralleled by an increase in the formation of Fe(II) which approached to the 50% at the ADP/Fe(III) ratio of 20/1, according to previous results. Bilirubin (0.01-100 μM) significantly inhibited NADPH-ADP/Fe(III)-induced lipid peroxidation in rat brain microsomes and the extent of the inhibition was inversely proportional to the ADP/Fe(III) ratio. Similar results were obtained when bilirubin was complexed with saturating concentration of human serum albumin (200 μM), even if albumin-bound bilirubin displayed a greater efficacy than bilirubin alone. In search for a mechanism for this antioxidant effect, the bilirubin-induced reduction of Fe(III) into Fe(II) was investigated. Bilirubin (0.1-10 μM) dose dependently reduced Fe(III) into Fe(II) approaching to a maximal value of about 80% Fe(II) at 5/1 and 10/1 ADP/Fe(III) ratios. Albumin-bound bilirubin significantly increased Fe(II) at greater extent (maximal value of about 95%) than bilirubin alone and independently from the ADP/Fe(III) ratio. The reduction of Fe(III) into Fe(II) seems to be specific for bilirubin because biliverdin (0.1-10 μM) did not increase Fe(II) formation and maintained the Fe(III)/Fe(II) ratio close to the unit, thus explaining why biliverdin have a low efficacy in reducing NADPH/Fe(III) lipid peroxidation. In conclusion, these results propose a new mechanism through which bilirubin could exert its antioxidant effect in addition to the well-known scavenging activity against ROS.