Abstract

Peroxidation of the lipid moieties of low density lipoproteins (LDL) is regarded as an early event in atherogenesis. Because bilirubin is a physiological reductant with antioxidant activities, we investigated its inhibitory action on the radical-mediated oxidation of LDL and plasma lipids. Exposing fresh human blood plasma to lipophilic peroxyl radicals generated from 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) resulted in rapid oxidation of ubiquinol-10, followed by that of ascorbate and bilirubin. Plasma lipids were well protected from peroxidation as long as these three antioxidants were present, as assessed by the amounts of cholesterylester hydroperoxides formed during this period. Following consumption of these antioxidants, and in the presence of alpha-tocopherol, the rate of hydroperoxide formation increased sharply with roughly 2 molecules of cholesterylester hydroperoxides being formed for each peroxidation initiating event. Supplementation of AMVN-oxidizing plasma with exogenous bilirubin at the onset of rapid lipid peroxidation, i.e. after depletion of endogenous ubiquinol-10, ascorbate, and bilirubin, led to a halt in both hydroperoxide formation and consumption of alpha-tocopherol. When isolated LDL was incubated with AMVN, approximately 9 molecules of cholesterylester hydroperoxides were formed per peroxidation initiating event and while alpha-tocopherol was consumed. Addition of free or albumin-bound bilirubin to isolated LDL at the onset of oxidation resulted in a strong inhibition of hydroperoxide formation and alpha-tocopherol consumption, the effect being more pronounced with the free pigment. Addition of the corresponding amounts of albumin alone was without effect. In the presence of albumin-bound bilirubin, some 30% of the pigment was initially converted into biliverdin, whereas formation of this oxidation product was not observed with the free pigment. Also, the presence of bilirubin oxidase partially reversed the inhibitory activity of bilirubin on AMVN-induced LDL oxidation in the absence but not presence of albumin. An attenuation of hydroperoxide formation and a temporary increase in LDL's alpha-tocopherol concentration were observed when free- or albumin-bound bilirubin were added to AMVN-oxidizing, alpha-tocopherol-containing LDL. In contrast, hydroperoxide formation was not inhibited significantly when the albumin-bound pigment was added to oxidizing LDL after complete consumption of its alpha-tocopherol. Our results show that bilirubin inhibits oxidation of LDL lipids initiated within the lipoprotein core and indicate that this activity is mediated by interaction of the pigment with LDL's alpha-tocopherol.