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Les scientifiques de l’École de Médecine Hopkins résolvent le paradoxe de
la Bilirubine, l’identifiant comme le principal antioxydant cellulaire.
Lire l’article ici:
http://www.hopkinsmedicine.org/press/2002/November/021125B.htm
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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