Redox state in jaundice:
The anti-/pro-oxidant effects of bilirubin
Libor Vítek
1st Faculty of Medicine,
Charles University in Prague
Czech Republic
Heme catabolic pathway
Bilirubin metabolism in the liver cell
Unconjugated
(premicrosomal)
hyperbilirubinemia
Blood
Albumin
Bilirubin
SLCO1B1
Hepatocyte
UDP-glucuronate
UGT1A1
Bisglucuronosyl bilirubin
ATP
Conjugated (postmicrosomal)
hyperbilirubinemia
Bile
ABCC2
Icterus
Icteros = Greek term for jaundice from ancient times
Yellowbird, golden oriole (Oriolus oriolus)
Icteridae family of birds
Two faces of bilirubin
Oxidative stress
™ defined
as overproduction of reactive oxygen species
(ROS) overcoming antioxidant defense capacity
™ ROS
physiologically important for
™ energy
generation
™ defense
™ cell
against foreign antigens
signalling
™ however,
ROS overproduction implicated in numerous
pathologic conditions including cardiovascular, cancer,
autoimmune and degenerative diseases
Antioxidant defense system
Function
Antioxidant enzymes
Superoxide dismutase
Elimination of superoxide via dismutation to oxygen and
hydrogen peroxide
Glutathione peroxidase
Elimination of peroxide via glutathione reduction
Glutathione transferase
Elimination of lipoperoxides via glutathione binding
Catalase
Elimination of peroxide via dismutation to oxygen and water
Heme oxygenase
Biliverdin production
Biliverdin reductase
Bilirubin production
Antioxidant substrates
Ascorbic acid (vitamin C)
Bilirubin
Glutathione
Albumin
Lipoic acid
Transferin
Uric acid
Lactoferrin
Carotenoids (vitamin A)
Ferritin
α-tocoferol (vitamin E)
Haptoglobin, hemopexine
Ubiquinol (coenzyme Q)
Ceruloplasmin
1776 citations!!!
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HMOX1 as a therapeutic amplification funnel
((Bach,
Bach, FASEB J 2005)
Bilirubin oxidation
A) Oxidation to biliverdin
Bilirubin
Biliverdin
Radical monocation
Radical dication
according to McDonagh, in: Dolphin. The Porphyrins 1979
™
Extreme suprastoichiometric antioxidant effects of bilirubin were shown in
studies on neuronal and HeLa cells – 10 nM bilirubin counteracted the
proxidant action of 10000 times higher H2O2 concentrations (Dore, PNAS 1999,
Baranano, 2002). However, this was not confirmed in recent Stocker (JBC 2009)
and Mc Donagh studies (FRBM 2010)
Bilirubin/biliverdin redox cycle
Sedlak, T. W. et al. JAMA 2006
Bilirubin oxidation
B) Photooxidation
Propendyopents
Monopyrollic oxidation products
Bonnet R. Some recent advances in tetrapyrollic chemistry. Ann NY Acad Sci 1973
Bilirubin oxidation
B) Production of BOXes
4-methyl-5-oxo-3-vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide
3-methyl-5-oxo-4-vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide
Wurster WL. Bilirubin oxidation products (BOXes): synthesis, stability and chemical characteristics. Acta Chir Suppl 2008
Biopyrrins
Vítek L. Urinary excretion of oxidative metabolites of bilirubin in subjects with Gilbert syndrome. J Gastro Hepatol 2007
Bilirubin
Bilirubin:: antioxidant vs. prooxidant
™ efficient
scavenger of peroxyl radicals (PNAS 1988, JBC 1994,
PNAS 1997), especially in the intravascular compartment
™ in vitro almost 30x more potent than vitamin E in
preventing LDL oxidation (Life Sci 1994)
™ more effective protector of human ventricular
monocytes than vitamin C and vitamin E analogs
(Biochem Pharmacol 1996)
™ conversely, bilirubin
was shown to exert proapoptotic
effects against numerous cancer cells (Ollinger, Cell Cycle
2007), as well as CNS and red blood cells (Brito, Eur J Clin
Invest 2000) most likely due to increased generation of
ROS (Oakes, J Biochem Mol Toxicol 2005), especially driven by
heavy metals (Asad, Chem Biol Interact 2001)
Bilirubin
-Cu(II) complex
Bilirubin-Cu(II)
™ High
2001)
free radical generating potential (Asad, Chem Biol Interac
Effects of elevated bilirubin levels in Gunn rats
NEONATAL NEUROTOXICITY vs. BENEFICIAL EFFECTS IN ADULT RATS
1)
Resistence of Gunn rats to develop diabetes after ip. exposure to
streptozocin (Fujii. Kidney Int 2010; Fu. Tohoku J Exp Med 2010)
2)
Attenuation of pressor and pro-oxidant effects of angiotensin II (Pflueger, Am J
Physiol Ren Physiol 2005)
3)
Attenuation of DOCA (deoxycorticosterone acetate) – induced hypertension
(Nath, Am J Physiol Heart Circ Physiol 2007)
4)
Prevention of balloon injury-induced neointima formation (Ollinger, Circulation
2005)
5)
Reduction of oxidative injury of neonatal Gunn rats exposed to hyperoxia
(Dennery, FRBM 1995)
Oxidative stress in experimental cholestasis
(Muchov
á, J Cell Mol Med 2011)
(Muchová,
1)
Bile duct-ligation in both Wistar and Gunn rats resulted in extreme elevation
of bile acids
2)
Taurocholic acid (TCA) was shown to dramatically increase markers of
oxidative stress in the liver tissue
3)
Bilirubin (either arteficially added to the Wistar liver homogenate, or present
in Gunn rat livers) substatntially suppressed lipid peroxidation
The role of bilirubin in the hepatocyte metabolism under
conditions of oxidative stress
(Zelenka, Biochimie
Biochimie,, submitted
submitted))
Influence of exogenous bilirubin on total
antioxidant status
(Atherosclerosis 2002)
1,8
(μM)
24.5*
31.9
40.6
55.8
79.6
133.0
y = 1.31 + 0.0034x
R2 = 0.991
p < 0.00001
TAS
(mM)
1.37
1.42
1.46
1.51
1.57
1.76
* = serum with defined antioxidant capacity
1,7
1,6
TAS [mM]
bilirubin
1,5
1,4
1,3
0
20
40
60
80
bilirubin [uM]
100
120
140
Bilirubin and TAS
bilirubin (μM)
Quartile 1
Quartile 2
Quartile 3
Quartile 4
2.7-9.0
9.1-12.5
12.6-18.6
18.9-55.2
1.90
1.95
1.97
1.98
(1.87-1.95)
(1.89-2.01)
(1.90-2.01)
(1.92-2.06)
0.0644
0.0149
<0.0001
(min-max) (n=223)
TAS (mM)
(median, 25-75%)
P-value
High TAS in subjects with Gilbert syndrome
(benign hyperbilirubinemia) described also in
Australian (Atherosclerosis 2008) and Turkish studies
Conclusion
Serum bilirubin correlates with total
antioxidant status
y=1.9+0.0036x
R2=0.089
2,3
P<0.00001
2,2
2,1
TAS [mmol/L]
(J Gastroenterol Hepatol 2008)
2,4
2,0
1,9
1,8
1,7
1,6
0
10
20
30
bilirubin [μmol/L]
40
50
60
Serum oxysterols and bilirubin
Quartiles of serum bilirubin (n = 368)
Bilirubin (μM)
(min-max)
Q1
Q2
Q3
Q4
2.7 – 7.1
7.2 – 10.3
10.4 - 14.5
14.6 – 48.6
5.3
4.3
4.0
3.3
2.1-99.6
2.1-66.5
1.7-27.3
1.6-31.7
0.004
<10-5
<10-5
7.5
6.6
5.8
5.2
3.2-164.1
3.1-132.5
2.8-49.8
2.3-78.2
0.066
<10-5
<10-5
7keto-Chol (ng/mL)
(median, 25-75%)
P-value
7bOH-Chol (ng/mL)
(median, 25-75%)
P-value
1.6
1.2
1.4
1.1
7-beta-OH-cholesterol
7-keto-cholesterol
7ketoC: 7-keto-cholesterol; 7b-OHC: 7-beta-hydroxycholesterol
1.2
1.0
0.8
0.6
0.9
0.8
0.7
0.6
0.4
y = 1.291 - 0.2036*x, r = -0.3155, p = 0.0000, r
0.2
0.8
1.0
1.0
1.2
1.4
1.6
1.8
serum bilirubin
2.0
2
y = 1.0876 - 0.0949*x, r = -0.3108, p = 0.00000, 2r = 0.0966
= 0.0995
2.2
2.4
2.6
0.5
0.8
1.0
1.2
1.4
1.6
1.8
2.0
serum bilirubin
2.2
2.4
2.6
Bilirubin levels and cardiovascular diseases
™ Due
to its antoxidant and other biological activities, mildly elevated
serum bilirubin levels have been shown to protect from coronary
heart disease
(Novotný L. Exp Biol Med 2003)
as well as carotid atherosclerosis
Relationship between IMT and age in norm o- and hyperbilirubinem ic m en
1,30
non-GS
1,20
GS
1,10
y = 0,01x + 0,332
1,00
2
IMT[mm]
R = 0,32
0,90
(Vítek L. Cerebrovasc Dis 2006)
0,80
y = 0,0036x + 0,5508
R2 = 0,0734
0,70
0,60
0,50
0,40
20
30
40
50
age [years]
60
70
80
Heme catabolic pathway
as a therapeutic target
?
target?
Heme
Protective effects via HMOX1 induction
Ndisang JF et al. Up-regulating the heme oxygenase system with hemin
improves insulin sensitivity and glucose metabolism in adult spontaneously
hypertensive rats. Endocrinology 2010;151:549.
Ndisang JF, Jadhav A. Up-regulating the hemeoxygenase system enhances
insulin sensitivity and improves glucose metabolism in insulin-resistant
diabetes in Goto-Kakizaki rats. Endocrinology 2009;150:2627.
Ndisang JF. Upregulation of the heme oxygenase system ameliorates
postprandial and fasting hyperglycemia in type 2 diabetes. Am J Physiol
Endocrinol Metab 2009;296:E1029.
Chen YS et al. Hemin, a heme oxygenase-1 inducer, improves aortic endothelial
dysfunction in insulin resistant rats. Chin Med J 2008;121:241.
Zhong W et al.. Hemin exerts multiple protective mechanisms and attenuates
dextran sulfate sodium-induced colitis. J Pediatr Gastroenterol Nutr
2010;50:132.
Conclusion
™
Bilirubin is a very strong biomarker of oxidative
stress-mediated diseases
™
Although very high levels of bilirubin are deleterious,
mildly elevated bilirubin concentrations protect from
diabetes, certain types of cancer, neurodegenerative
and autoimmune diseases
™
Attempts to mildly increase systemic bilirubin levels
to increase protection against these diseases are
currenly being explored
Acknowledgement/collaboration
www.hepatocity.org
USA
• Harvey Schwertner (Wilford Hall Med Center)
• JingPing Lin (NHLBI, NIH)
• Don Ostrow (University of Washington)
• Ron Wong, Henk Vreman (Stanford University)
• Mark McCarty (NutriGuard Research, CA)
ITALY
• Claudio Tiribelli (Centro Study Fegato, Trieste)
• Luigi Iuliano (Universita LaSapienza, Rome)
FRANCE
• Roberto Motterlini (INSERM, Paris)
PRAGUE
• Lucie Muchová
• Alena Jirásková
• Martin Leníček
AUSTRIA
• Karl-Heinz Wagner
UK
• Asif Ahmed (University of Edinburgh)
KOREA
• Hyeon Chang Kim (Yonsei University, Seoul)
AUSTRALIA
• Jiří Neužil, Andrew Bulmer (Griffith University)