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Little Yellow Molecule Comes Up Big Hopkins scientists solve paradox of bilirubin, identify it as cells’ major antioxidant. See article at: http://www.hopkinsmedicine.org/press/2002/November/021125B.htm
Seasonal Affective Disorder and the role of Bilirubin ABC radio broadcast Monday 8 April 2002 with Norman Swan A study in the U.S. suggest that low nocturnal bilirubin levels may be associated with winter seasonal depression. See article at: http://www.abc.net.au/rn/talks/8.30/helthrpt/stories/s526606.htm
Biological
Properties and Therapeutic Potential of Bilirubin
Ross P. McGeary,1*
Alexander J. Szyczew2 and Istvan Toth1 1 The School of Pharmacy, The University of Queensland,
Brisbane Qld 4072, Australia 2 Bio Pharma Pty Ltd, PO Box 130, Cannon Hill, Qld 4170,
Australia Correspondence:
Dr
Ross P. McGeary The
School of Pharmacy The
University of Queensland Brisbane
Qld 4072, Australia Tel:
+61-7-3365-2082 Fax:
+61-7-3365-1688 Email: r.mcgeary@mailbox.uq.edu.au Abstract
Bilirubin was long considered a useless
metabolite of heme catabolism, responsible for the clinical manifestation of
jaundice, and potentially toxic in high doses, particularly in neonates. In the past two decades the potent
biological properties of bilirubin, particularly as an antioxidant, have been
recognised, and this has prompted a number of investigations into this molecule
concerning its in vitro and in vivo properties. This review summarises that work, as well as more recent investigations
into the potential therapeutic uses of bilirubin. Key
words: bilirubin, heme,
antioxidant,
Introduction Bilirubin is a conjugated tetrapyrroledicarboxylic acid, and is the principle pigment in bile. Bilirubin is the end product of heme catabolism in mammals, and most of the circulating bilirubin derives from senescent erythrocytes. At the end of the normal life span of erythrocytes, the heme dissociates from hemoglobin and is oxidised by the membrane-bound enzyme heme oxygenase (EC 1.14.99.3) to biliverdin, producing carbon monoxide as a by-product. Subsequent metabolism of biliverdin by the cytosolic enzyme biliverdin reductase (EC 1.3.1.24) gives rise to bilirubin (Fig. (1)). Most of the bilirubin produced in the body is mono- or di-glucuronidated in the liver by a glucuronyl transferase enzyme, and these water-soluble products are excreted in the bile. Bilirubin is obtained industrially by extraction of either cattle or pig bile, and it can be isolated as light-sensitive orange-red crystals. In humans, accumulation of bilirubin in the bloodstream causes yellow pigmentation of the plasma, in turn causing the skin and sclerae to become yellow, appearing clinically as jaundice. Normal human serum levels of bilirubin are in the range 5-17 mM, with levels above around 43 mM manifesting as jaundice [1].
Bilirubin exists in the serum in
four major forms: as unconjugated bilirubin, as the monoglucuronide, as the
diglucuronide, and as albumin-bound bilirubin.
Weiss et al. showed that
albumin-bound bilirubin constituted from 8 to 90% of total bilirubin in
patients with jaundice or with Dubin-Johnson syndrome, but could not be
detected in healthy volunteers, indicating a build-up in serum of conjugated
bilirubin when hepatic excretion was impaired [2].
For many years, bilirubin was considered
only as a waste end product of heme catabolism – useless at best and toxic at
worst. During the last few decades,
however, a number of intriguing biochemical properties of bilirubin have been
discovered, and there is now strong evidence for the beneficial role that
bilirubin plays in the body, particularly as an antioxidant. There has also been a long history in
Chinese traditional medicine of the beneficial health properties of ox
gallstones, which consist largely of calcium bilirubinate. In Vitro Studies
Most of the interest in bilirubin as a
potential therapeutic lies in its antioxidant properties. Bilirubin is probably the most abundant
endogenous antioxidant in mammalian tissues [3]. The in vitro antioxidant properties of bilirubin were delineated
largely by Stocker and coworkers in the late 1980s. He found that bilirubin, at micromolar concentrations,
efficiently scavenged peroxyl radicals, either in homogeneous solutions or in
multilamellar liposomes, to a greater extent than a-tocopherol (vitamin E),
which was considered at the time to be the best antioxidant of lipid
peroxidation [4]. In further studies,
Stocker and Ames [5] showed that a water-soluble bilirubin-taurine conjugate
could prevent radical-induced oxidation of phosphatidylcholine in either
micelles or multilamellar liposomes, and that the same conjugate greatly
accelerated Cu2+-catalysed decomposition of linoleic acid
hydroperoxide. As well as describing
the antioxidant effects of this bilirubin conjugate, these workers also showed
that albumin-bound bilirubin, at concentrations comparable to those present in
normal plasma, also had antioxidant activity, and was capable of protecting
albumin-bound linoleic acid against radical-induced oxidation. In competition studies, albumin-conjugated
bilirubin was also found to out-compete an equimolar concentration of uric acid
for peroxyl radicals, but was less efficient in scavenging these radicals than
was ascorbic acid [6]. In later work,
Stocker and Ernst [7] demonstrated the synergistic interaction between
bilirubin and vitamin E in inhibiting the oxidation of phosphatidylcholine
liposomes. Low micromolar
concentrations of bilirubin were able to inhibit oxidation of these liposomes
in a concentration-dependent manner, unlike ascorbic acid or glutathione, which
were ineffective. These authors also
showed that low micromolar concentrations of bilirubin at physiological pH
could efficiently scavenge hypochlorous acid [8]. Frei and coworkers monitored the depletion
of endogenous ascorbate, thiols and bilirubin in plasma after exposure to
aqueous peroxyl radicals, and they found that bilirubin was more effective at
protecting lipids from peroxidative damage than other endogenous antioxidants
[9]. The ability of bilirubin to
scavenge superoxide radical has also been investigated, with bilirubin being
approximately equal in activity with serum albumin, more active than the
water-soluble vitamin E analogue Trolox, but less active than ascorbic acid
[10]. In contrast, bilirubin has been
shown to protect serum albumin itself against oxidation by hydroxyl radicals,
to a greater extent than either ascorbic acid or Trolox [11]. More recently, bilirubin has been shown to
act as an antioxidant of peroxynitrite-mediated protein oxidation in human
blood plasma [12]. Wu et
al. have studied the protective effects of bilirubin against oxidation of
human low-density lipoprotein (LDL).
Oxidation of LDL is implicated in plaque formation in blood vessels
leading to atherogenesis, and there is evidence that prevention of this
oxidation reduces the incidence of coronary heart disease. Bilirubin at a concentration of 17 mM was
found to protect against Cu2+- mediated oxidation of LDL at least 20
times more effectively than Trolox [13]. Cahyana has noted that bilirubin has an
antioxidant effect similar to that of the porphyrins [14], and Dailly has
correlated bilirubin levels in plasma with the plasma’s total peroxyl radical
trapping activity [15]. More recently,
Asad et al. have shown that bilirubin
inhibits L-DOPA-Cu2+-mediated DNA cleavage, and that bilirubin
directly quenches the hydroxyl radicals generated by the L-DOPA-Cu2+
system [16]. Other known biological effects of
bilirubin are its ability to inhibit the mutagenicity of 4-nitroquinoline N-oxide in strain TA100 of Salmonella typhimurium [17], and its
ability to block the complement cascade, especially the C1 step [18]. Cellular studies
Motterlini and coworkers showed that
exogenously applied bilirubin could attenuate hydrogen peroxide-induced damage
in vascular endothelial cells [19].
Later studies by Clark et al.
showed that the addition of bilirubin to the culture medium of vascular smooth
muscle cells could markedly reduce hydrogen peroxide-induced cytotoxicity. These authors further found that hemin-mediated up-regulation of heme oxygenase led to increased levels of bilirubin,
resulting in high resistance to cell injury caused by hydrogen peroxide,
providing strong evidence that bilirubin generated after up-regulation of the
heme oxygenase pathway is cytoprotective against oxidative stress [20]. Doré et
al. have also shown that bilirubin conjugated to human serum albumin is
neuroprotective, reversing the neurotoxic effects of hydrogen peroxide on
hippocampal neuronal cultures, at concentrations as low as 10 nM [21]. Aria et
al. have also shown that bilirubin’s ability to scavenge reactive oxygen
species impairs the bacterial activity of neutrophils in a dose-dependent
manner [22]. Animal and Human
Studies
Yamaguchi and coworkers have investigated
the ischemia-reperfusion of rat liver and found evidence to suggest that
bilirubin acts as an antioxidant in vivo under these conditions, and that
bilirubin biosynthesis is increased by oxidative stress [23]. Other work by these authors with
scurvy-prone ODS-od/od rats treated
with lipopolysaccharide showed that bilirubin acts synergistically as an
antioxidant with ascorbic acid [24].
Hyperbilirubinemia is commonly observed in newborn humans, and the
possible protective role of bilirubin in neonates has long been debated. Evidence for the protective effects of
bilirubin has been provided by Dennery et
al., who showed that bilirubin protects neonatal rats exposed to hyperoxia
against serum oxidative damage in the first few days of life [25]. Clark et
al. [26] have examined the effects of bilirubin on the protection of the
rat heart against postischemic myocardial dysfunction. They found that treatment of the animals
with hemin 24 h before ischemia reduced infarct size on reperfusion of isolated
hearts. Exogenously administered
bilirubin at concentrations as low as 100 nM significantly restored myocardial
function and minimised both infarct size and damage to mitochondria on
reperfusion, providing strong evidence of the cardioprotective effects of
bilirubin against reperfusion injury. Mildly increased serum bilirubin levels
have been suggested to act as a protective factor, reducing the risk of
coronary artery disease (CAD) in humans [27].
Hopkins et al. have tested
this hypothesis on patients with early familial CAD and found that serum
bilirubin was strongly and inversely related to CAD risk [28]. This work has been extended by Madhaven et al., who found an inverse
relationship between bilirubin levels and family history of heart decease. Also found were inverse relationships
between bilirubin levels and both cigarette smoking and adiposity [29]. Toxicity
Bilirubin commonly accumulates in the
serum of neonates, particularly premature babies, causing hyperbilirubinemia
(jaundice). At high concentrations,
particularly in premature or low birth weight babies, bilirubin can deposit in
the brain causing the neurotoxicity associated with kernicterus. Bilirubin has been shown to display some
toxicity towards erythrocytes [30], and Amato has demonstrated a dose-dependent
relationship between bilirubin levels and tyrosine uptake in rat synaptosomes,
providing a plausible mechanism for bilirubin’s neurotoxicity. Hansen and Allen [31] have reported that
neurons are more sensitive to the toxic effects of bilirubin than are glial
cells. Hansen et
al. have shown that bilirubin has widespread inhibitory effects on protein
phosphorylation, inhibiting cAMP-dependent, cGMP-dependent, Ca2+-calmodulin-dependent
and Ca2+-phospholipid-dependent protein kinases, with IC50s
ranging from 20 to 125 mM [32]. Amato
also provided evidence that bilirubin can interfere with surfactant proteins at
the air-liquid interface in the lung, with implications for the treatment of
neonatal respiratory distress syndrome [33]. Ox Gallstones
Traditional Chinese medicine prizes highly
the medicinal properties of ox gallstones (also known as Niu Huang, calculus
bovis, or bezoar). These gallstones are
said to possess calming, antipyretic and antiinflammatory properties. The main constituent of ox gallstone is the
calcium salt of bilirubin, with lesser amounts of cholic acid and deoxycholic
acid [34]. There are a few reports of
in vitro and in vivo examinations of the biological properties of these
products. For example, Takahashi et al. have examined the effects of ox
gallstone extract on the beating pattern of spontaneously contracting cultured
embryonic mouse myocardial cell, and they showed that addition of the gallstone
extract attenuated the cellular response to varying calcium concentration [35]. Rather than attributing this effect to
bilirubin however, these authors suggested a possible role of taurine,
identified in the gallstone extract, for the observed effects on the myocardial
cells. Antiviral activity of ox gallstone has
been identified against encephalitis B in vitro and in mice. Bilirubin also showed in vitro activity, but
not a high as ox gallstone. Antiviral
activity was also observed in mice inoculated with the encephalitis virus, with
the ox gallstone offering higher protection than bilirubin alone. The authors proposed a possible role for
deoxycholic acid in the biological activities of the ox gallstone [36]. The effects of ox gallstone on the humoral
immune response have also been examined in mice injected with sheep red blood
cells. Short-term (1-2 days) daily oral
dosage with ox gallstone stimulated the production of nitric oxide in mouse
macrophages, whereas longer-term (7-14 days) daily oral dosage inhibited nitric
oxide production, as well as decreasing TNF-a and IL-6 production in macrophages
[37]. Li et
al. have examined the antiinflammatory effects of artificial ox gallstone
in mice and rats using both the croton oil-induced mouse ear edema, and the
carrageenan-induced rat hind paw edema.
Ox gallstone was shown to significantly inhibit edema in both
species. The ability of ox gallstone to
inhibit the synthesis of nitric oxide was suggested to explain its
antiinflammatory effects [38]. Finally, Nakashima et al. have investigated the effects of ox gallstone on rats given
i.p. administration of carbon tetrachloride to induce liver toxicity. They found that oral administration of ox
gallstone significantly increased both serum transaminase levels and hepatic
lipid peroxidation, as well as increasing hepatic blood flow. This resulted in ox gallstone exacerbating
carbon tetrachloride-induced hepatic damage through accelerated delivery to the
liver from the peritoneal cavity [39]. Summary
The reputation of bilirubin has been transformed from
that of a toxin responsible for jaundice with no beneficial effects, to that of
a biologically important antioxidant with a wide range of protective
actions. Bilirubin is an effective
radical scavenger at biologically relevant concentrations, more so than most other
endogenous antioxidants. Several biological
effects have been demonstrated in cells, including protective action against
peroxide-induced damage, and neuroprotection of neuronal cultures against
hydrogen peroxide damage. Antioxidant
activity has also been demonstrated in animals, and it has been demonstrated
that bilirubin biosynthesis is increased by oxidative stress. More recently the medicinal properties of ox
gallstone (largely calcium bilirubinate) in rats have been examined, with
interesting effects on the liver, on cytokine production and immune responses
discovered.
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