Neuzil J, Stocker R
Biochemistry Group, Heart Research Institute, Sydney, New South Wales, Australia.
J Biol Chem. 1994 Jun 17;269(24):16712-9.
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.
低密度脂蛋白（LDL）脂质部分的过氧化被认为是动脉粥样硬化发生的早期事件。由于胆红素是一种具有抗氧化活性的生理性还原剂，我们研究了其对自由基介导的LDL和血浆脂质氧化的抑制作用。将新鲜人血浆暴露于由2,2'-偶氮二（2,4-二甲基戊腈）（AMVN）产生的亲脂性过氧自由基中，会导致泛醇-10迅速氧化，随后是抗坏血酸和胆红素的氧化。只要这三种抗氧化剂存在，血浆脂质就能很好地免受过氧化作用，这可通过在此期间形成的胆固醇酯氢过氧化物的量来评估。在这些抗氧化剂消耗殆尽后，且存在α-生育酚的情况下，氢过氧化物的形成速率急剧增加，每个引发过氧化的事件大约会形成2分子胆固醇酯氢过氧化物。在快速脂质过氧化开始时，即内源性泛醇-10、抗坏血酸和胆红素耗尽后，向AMVN氧化的血浆中补充外源性胆红素，会导致氢过氧化物形成停止以及α-生育酚的消耗停止。当分离的LDL与AMVN一起孵育时，每个引发过氧化的事件大约会形成9分子胆固醇酯氢过氧化物，同时α-生育酚被消耗。在氧化开始时向分离的LDL中添加游离或与白蛋白结合的胆红素，会强烈抑制氢过氧化物的形成和α-生育酚的消耗，游离色素的效果更明显。仅添加相应量的白蛋白则没有效果。在存在与白蛋白结合的胆红素的情况下，约30%的色素最初会转化为胆绿素，而游离色素则未观察到这种氧化产物的形成。此外，在不存在白蛋白但存在白蛋白的情况下，胆红素氧化酶的存在部分逆转了胆红素对AMVN诱导的LDL氧化的抑制活性。当将游离或与白蛋白结合的胆红素添加到正在被AMVN氧化且含有α-生育酚的LDL中时，观察到氢过氧化物形成减少以及LDL中α-生育酚浓度暂时增加。相比之下，当在其α-生育酚完全消耗后将与白蛋白结合的色素添加到正在氧化的LDL中时，氢过氧化物的形成没有受到显著抑制。我们的结果表明，胆红素抑制脂蛋白核心内引发的LDL脂质氧化，并表明这种活性是由色素与LDL的α-生育酚相互作用介导的。
