Bilirubin Benefits: Cellular Protection by a Biliverdin Reductase Antioxidant Cycle

Abstract

Bilirubin is widely known as an end product of heme metabolism. Very high levels of serum bilirubin lead to its accumulation in the brain, causing kernicterus.1,2 Almost all newborns display some level of jaundice, and some display high enough serum bilirubin levels that phototherapy or exchange transfusion is considered.What most of the medical profession has not appreciated is that, from a teleologic perspective, biosynthesis of bilirubin as the key catabolite of heme does not seem to make sense. Bilirubin is a secondary degradation product of heme. Heme is best known as a constituent of hemoglobin, which is released in association with the breakdown of aging red blood cells. Heme also is contained in a wide range of enzymes whose turnover also leads to free heme release. Free heme can be toxic, so nature evolved a family of heme oxygenase enzymes to degrade heme,3,4 and their blockade leads to greatly increased excretion of unmetabolized heme in the bile.5 These enzymes cleave the heme ring to form biliverdin, iron, and a 1-carbon fragment as carbon monoxide (CO; Fig 1). CO is increasingly appreciated as a neurotransmitter,6,7 and iron, itself toxic, is excreted from cells by a recently characterized pump.8–11 Biliverdin would seem to be an appropriate end product of the pathway, being readily excreted in the bile to enter the intestine and leave the body in the feces. Indeed, in birds, reptiles, and amphibians, biliverdin is the predominant end product of heme degradation.12 For reasons that until now have seemed obscure, in mammals, biliverdin undergoes additional metabolism, being reduced by biliverdin reductase (BVR) to bilirubin, a step that consumes the energy resource nicotinamide adenine dinucleotide phosphate (NADPH).13 As bilirubin is more hydrophobic and insoluble than biliverdin, it is glucuronidated to facilitate excretion into the bile, costing additional cell resources.Why have mammals evolved an energetically expensive and apparently unnecessary enzymatic step to converting the relatively innocuous biliverdin to the more toxic bilirubin? Moreover, why would nature develop a system that generates “elevated” bilirubin levels in a high proportion of all neonates? Nature may not be altogether foolhardy, as the mildly to moderately elevated levels of bilirubin in neonates are not always toxic. In 1965, in this journal, Wishingrad and associates14,15 argued that hyperbilirubinemia of premature infants is not as deleterious as previously thought. Furthermore, some individuals with the impaired bilirubin glucuronidation system of type 2 Crigler-Najjar syndrome maintain bilirubin levels of 19 mg/dL for 50 years without detectable damage to the nervous system.16Some authors have suggested that unconjugated bilirubin is physiologically useful, because it can cross the placenta, moving from the fetal to the maternal circulation easier that biliverdin.17–19 However, isomer specificities of fetal and maternal BVR differ. The principal isomer of early fetal bilirubin is IXβ, whereas the adult forms bilirubin IXα.20,21 Hence, adult BVR cannot have evolved to service needs of the fetus.One possible physiologic role for bilirubin is as an antioxidant. As early as the 1950s, bilirubin was reported to protect against the oxidation of lipids such as linoleic acid and vitamin A.22–24 In the late 1980s, Ames and colleagues25,26 demonstrated that the antioxidant effect of bilirubin exceeds that of vitamin E toward lipid peroxidation. Serum concentrations of bilirubin are high enough to account for a substantial portion of the total antioxidant capacity of serum.27,28 Thus, bilirubin might alleviate oxidant stress in the blood. However, what matters most is what goes on inside cells. During the oxidant stress associated with myocardial and cerebral infarcts, infection, inflammation, and various causes of ischemia, the intracellular environment is exposed to high concentrations of reactive oxygen species. It has long been assumed that the principal cellular antioxidant is the peptide glutathione (GSH), whose tissue concentrations are millimolar, presumably sufficient to cope with most instances of oxidative stress. By contrast, levels of bilirubin in rodent tissues are only 10 to 50 nanomolar, at least 10000 times lower than concentrations of GSH (D. Baranano and S.H. Snyder, unpublished observations).Insight into a mechanism whereby low nanomolar concentrations of bilirubin can protect cells came from studies of the heme oxygenase system in the brain. Heme oxygenase was first characterized by Schmid and associates in the 1960s.29 Maines and associates30,31 purified and cloned a family of heme oxygenase isozymes. The first known form of heme oxygenase, HO1, is highly concentrated in the spleen, the repository of aging red blood cells. HO1 is a remarkably inducible enzyme, with its synthesis rapidly and profoundly stimulated by more stimuli than almost any other known inducible enzyme.32 Heme itself, released from