Bilirubin: Friend or Foe

1
Bilirubin Friend or Foe?
Virtual Free Radical School
James K. Friel, Ph.D., Russell W. Friesen, B.Sc.,
Angela C. Miller, B.Sc
University of Manitoba Department of Human
Nutritional Sciences H511 Duff Roblin
Building Winnipeg, MB R3T 2N2 Canada Tel
204-474-8080 Fax [phone] E-mail
frielj_at_ms.umanitoba.ca
Learning is my home.
2
The Road Ahead

• Function of Bilirubin
• Bilirubin as an Antioxidant
• Bilirubin as a Toxin
• Biliverdin Reductase
• Cardiovascular Disease
• Jaundice
• Hyperbilirubinemia
• The Premature Infant
• Promise for the Future

3
Bilirubin

• Is a bile pigment.
• Results from the degradation of heme, one of
  the breakdown products of red blood cells.
• It is thought to be a toxin because it is
  associated with neonatal jaundice, possibly
  leading to irreversible brain damage due to
  neurotoxicity.

Tomaro ML, Batlle AM del C. (2002). Bilirubin
its role in cytoprotection against oxidative
stress. Int J Biol Cell Biol., 34 216-220
4
Formation of BilirubinOverview
The Bilirubin Pathway Overview
Bilirubin diglucuronide (soluble)
5
Formation of Bilirubin

• Hemoglobin from senescent or hemolyzed red cells
  is broken down, releasing heme.
• Heme is then degraded in humans by the enzyme
  heme oxygenase (HO), which is the rate-limiting
  step in the formation of bilirubin.
• HO converts heme to biliverdin IX.
• Biliverdin is a hydrophilic compound that is
  reduced by biliverdin reductase into the
  hydrophobic compound bilirubin.

6
Formation of Bilirubin
5. HO catalyses an oxidase reaction opening the
heme ring to convert one of the bridge carbons to
carbon monoxide. This step releases iron from
the now linear tetrapyrrole yielding biliverdin.
6. Biliverdin reductase reduces the double bond
on nitrogen inside one of four of the pyrrole
rings leading to the formation of bilirubin.
7
Excretion of Bilirubin

• Bilirubin is bound to albumin and transported in
  plasma from the reticuloendothelial system to the
  liver, as unconjugated bilirubin.
• In the liver, bilirubin is made water soluble by
  hepatocytes which conjugate bilirubin with
  glucuronic acid to form conjugated bilirubin
  (BC). This process requires the enzyme uridine
  diphosphate-glucuronosyltransferase (UPD-GT) and
  produces bilirubin diglucuronide.
• BC is secreted from hepatocytes to the bile
  canaliculi of the liver and is transported from
  the liver via the gall bladder and common bile
  duct to the gastrointestinal tract.

8
Excretion of Bilirubin

• In the ileum and colon, bacteria converts
  bilirubin into stercobilinogen.
• Stercobilinogen is oxidized to stercobilin, which
  is excreted in the feces.
• While most bilirubin is excreted as stercobilin,
  a small amount of stercobilinogen is reabsorbed
  into the blood, modified by the kidneys, and
  excreted as urobilinogen in the urine.

Higgins, Chris. (2002). Neonatal jaundice, breast
milk, and Gilberts syndrome. Biomedical
Scientist, February.
9
Bilirubin as an Antioxidant
As early as 1959, it was suggested that bilirubin
might be an antioxidant. Bilirubin can suppress
oxidation of lysosomes at oxygen concentrations
that are physiologically relevant. Bilirubin can
act as an important cytoprotector of tissues that
are poorly equipped with antioxidant defense
systems, including myocardium and nervous tissue.
Temme EHM, Zhang J, Schouten EG, Kesteloot H.
(2001). Serum bilirubin and 10-year mortality
risk in a Belgian population. Cancer Causes and
Control, 12 887-894.
10
Bilirubin as an Antioxidant

• At concentrations as low as 10nM, Bilirubin
  can protect against 10,000-fold greater
  concentrations of H2O2.
• Under physiologic conditions, bilirubin
  provides more potent protection against lipid
  peroxidation than a-tocopherol, formerly known to
  be most effective in preventing lipid
  peroxidation.
• Recent research indicates that bilirubin may be
  the most abundant endogenous antioxidant in
  mammalian tissues.

Baranano DE, Rao M, Ferris CD, Snyder SH.
(2002). Biliverdin reductase A major physiologic
cytoprotectant. Proc. Natl. Acad. Sci. USA,
99(25) 16093-16098. Dore S, Takahashi M,
Ferris CD, Hester LD, Guastella D, Snyder SH.
(1999). Bilirubin, formed by activation of heme
oxygenase-2, protects neurons against oxidative
stress injury. Proc. Natl. Acad. Sci. USA, 96
2445-2450.
11
Bilirubin as an Antioxidant

• A linear relationship (R2 0.99) has been
  identified between plasma antioxidant capacity
  and unconjugated bilirubin concentration in
  newborn infants.
• This both confirms bilirubins significance as a
  plasma antioxidant and suggests that moderate
  increases in plasma bilirubin might be favourable
  to infants under oxidative stress.

Belanger S, Lavoie J-C, Chessex P. (1997).
Influence of Bilirubin on the Antioxidant
Capacity of Plasma in Newborn Infants. Biology of
the Neonate, 71 233-238.
12
Bilirubin as an Antioxidant
Comparative relative proportions of plasma
antioxidants between premature and term neonates
expressed as a percentage of the total
antioxidants
Trolox Equivalent Antioxidant Capacity
(Gopinathan et al., 1994) Expressing the data as
relative levels in relation to the major
antioxidants of human plasma emphasises the
contribution of bilirubin to the antioxidant
potential at day 5 for both the term and the
pre-term infants.
13
Bilirubin as an Antioxidant
The proposed mechanism is Bilirubin can
scavenge the chain-carrying peroxyl radical by
donating a hydrogen atom attached to the C-10
bridge of the tetrapyrrole molecule to form a
carbon-centered radical Bil? LOO? Bil ?
LOOH Bil? Bil? LOO? ? Bil-OOL Bil? O2
?? Bil-OO? LOO? BV ? LOO-BV?
Stocker R, Yamamoto Y, McDonagh AF, Glazer AN,
Ames BN. (1987). Bilirubin is an antioxidant of
possible physiological importance. Science, 235
1043-1046.
14
Conjugated Unconjugated Bilirubin
Serve as Antioxidants
in Lipid Peroxidation
Both unconjugated bilirubin (BU) and conjugated
bilirubin (BC) can serve as antioxidants,
protecting human LDL from lipid peroxidation in
vitro against peroxyl radicals (generated by
2,2'-azobis 2-amidinopropane dihydrochloride).
Wu T-W, Fung KP, Wu J, Yang C-C, Weisel RD.
(1996). Antioxidation of human low-density
lipoprotein by unconjugated and conjugated
bilirubins. Biochem Pharmacol, 51 859-862.
15
The Toxic Side of Bilirubin
Erythrocyte morphological changes have been seen
with incubation of cells with different molar
ratios of unconjugated bilirubin. These changes
occur as the bilirubin/human serum albumin molar
ratio increases. This indicates that bilirubin
can illicit toxicity in the erythrocyte membrane
in a concentration and temperature-dependent
manner.
Brito MA, Silva R, Tiribelli C Brites D.
(2000). Assessment of bilirubin toxicity to
erythrocytes. Implication in neonatal jaundice
management. European J Clinical Invest, 30
239-247.
16
The Toxic Side of Bilirubin

• Morphological changes have also been observed in
  microglia exposed to 50 µM unconjugated
  bilirubin (BU). These changes are characteristic
  of those that normally occur during the
  activation of these cells. Therefore, BU
  stimulates microglial activation.
• Persistent activation of microglia stimulates the
  production of highly neurotoxic species that may
  be responsible for the neuronal destruction that
  occurs in various neurodegenerative diseases.

• In addition, BU causes microglia to release
  pro-inflammatory cytokines (TNF-a, IL-1ß and
  IL-6) and cytotoxic glutamate in a
  concentration-dependent manner.
• When incubated with 50 µM or 100 µM BU, microglia
  underwent apoptotic and necrotic cell death.

Gordo AC, Falcão AS, Fernandes A, Brito MA, Rui
F.M. Silva, Brites D. (2006). Unconjugated
bilirubin activates and damages microglia.
Journal of Neuroscience Research, April 12.
Reactive morphological changes of microglia
exposed to BU (bottom).
17
The Toxic Side of Bilirubin

• Bilirubin (BR) can bind to DNA and copper ions to
  form a bilirubin-Cu(II)-DNA complex. This complex
  causes oxidative DNA damage through a DNA
  cleavage reaction. Biliverdin (BV) acts similarly
  with DNA.
• Upon binding to Cu(II), BV/BR reduce Cu(II) to
  Cu(I), stimulating the release of reactive oxygen
  species, particularly the hydroxyl radical. Cu(I)
  acts as an essential intermediate in the DNA
  cleavage reaction.
• In the presence of light, bilirubin has been
  shown to generate hydrogen peroxide and other
  peroxides that can cause DNA damage.
• Under certain conditions, many antioxidants are
  known to act as prooxidants, and bilirubin is no
  exception.

Asad SF, Singh S, Ahmad A, Hadi SM. (2002)
Bilirubin/biliverdinCu(II) induced DNA breakage
reaction mechanism and biological significance.
Toxicology Letters, 131 181-189. Asad SF,
Singh S, Ahmad A, Hadi SM. (1999)
Bilirubin-Cu(II) complex degrades DNA. Biochimica
et Biophysica Acta, 1428(2-3) 201-208.
18
The Role of Biliverdin Reductase

• Reduces water-soluble biliverdin to insoluble,
  potentially toxic bilirubin.
• Participates in a catalytic redox cycle which
  functions to regenerate bilirubin and amplify its
  antioxidant activity.
• Bilirubin is oxidized to biliverdin which is then
  immediately reduced back into bilirubin by
  biliverdin reductase.
• Therefore, the principle function of the
  bilirubin generating system is the cytoprotection
  provided by the biliverdin reductase cycle.
• Biliverdin reductase demonstrates potential to
  become a new effective target for the treatment
  of free radical-mediated diseases.

Baranano et al. (2002). Liu Y, Liu J, Tetzlaff W,
Paty DW Cynader M. (2006). Biliverdin
reductase, a major physiologic cytoprotectant,
suppresses experimental autoimmune
encephalomyelitis. Free Radic. Biol. Med., 40(6)
960-967.
19
Heme Oxygenase (HO)

• A cytoprotective enzyme that breaks the
  prooxidant molecule heme into biliverdin
  (immediately converted into bilirubin), iron, and
  carbon monoxide.
• HO-2, the constitutive isoform, is highly active
  in neurons and accounts for most of the HO
  activity in the brain.
• Destroying the HO-2 gene, and thus limiting BR
  production, leads to increased oxidative damage
  following cerebral ischemia.

Namiranian K, Koehler R, Sapirstein A, Dore S.
(2005). Stroke outcomes in mice lacking the genes
for neuronal heme oxygenase-2 and nitric oxide
synthase. Current Neurovascular Research, 2
23-27.
20
Bilirubin and Cardiovascular Disease

• Low serum bilirubin has been shown to be
  strongly correlated with several cardiovascular
  risk factors, including age, cigarette smoking,
  social class, diabetes, serum cholesterol,
  lower FEV1, and lower serum albumin.
• Serum bilirubin was found to have a U-shaped
  relationship with the events of ischemic heart
  disease (IHD)

Serum bilirubin and adjusted relative risk of
major IHD events in 4916 men measured before
1600 c.
a Adjusted for age, body mass index, smoking,
social class, physical activity, alcohol intake,
preexisting IHO, diabetes, use of
antihypertensive treatment. b Adjusted for the
above and in addition for systolic blood
pressure, blood cholesterol, HDL-C, FEV1, blood
glucose, and serum albumin. Complete data on all
covariates were available for 4678 men (444 IHD
cases). c There was little difference in mean
values up to 1600, but after this, the
concentrations decreased steadily. The
explanation for this decrease is uncertain, but
may be the result of food intake, fasting having
been shown to increase bilirubin concentration.
Breimer LH, Wannamethee G, Ebrahim S, Shap AG.
(1995). Serum bilirubin and risk of ischemic
heart disease in middle-aged British men. General
Clinical Chemistry, 41(10) 1504-1508.
21
Bilirubin and Cardiovascular Disease (cont.)

• Bilirubin perfusion was shown to significantly
  decrease infarct damage caused by IHD.
• Serum bilirubin concentrations in the upper range
  of normal values protect against coronary artery
  disease (CAD).
• However, concentrations in the lower range
  increase atherogenic risk and thus risk of IHD.
• Schwertner et al. discovered an unexpected
  inverse association between serum total bilirubin
  and CAD. The strength of the association with CAD
  was similar to that of smoking or of systolic
  blood pressure.

Prevalence of coronary artery disease, according
to concentration of total bilirubin in 877
patients.
Dore, Sylvain. (2002). Decreased activity of the
antioxidant heme oxygenase enzyme implications
in ischemia and in Alzheimer's disease. Free
Radical Biology and Medicine, 32(12) 1276-1282.

Schwertner HA, Jackson WG, ToIan G. (1994).
Association of low serum concentration of
bilirubin with increased risk of coronary artery
disease. Clinical Chemistry, 40(1) 18-23.
22
Bilirubin and Jaundice

• Neonatal jaundice is a yellowing of the skin and
  eyeballs and may lead to deposition of bilirubin
  in brain cells.
• Normally bilirubin is bound (conjugated) by a
  transport molecule and excreted.
• However unconjugated bilirubin can induce a
  loss of neurons and atrophy of involved fiber
  systems (called Kernicterus).
• Jaundice has become one of the most common
  problems in the neonatal period for both full
  term and premature infants (lt37 weeks gestation),
  affecting 50-70 of newborns.

Gurses D, Kilic I, Sahiner T. (2002). Effects
of hyperbilirubinemia on cerebrocortical
electrical activity in newborns. Pediatr Res.,
52 125-130.
23
Bilirubin and JaundiceBreast Milk Jaundice

• A type of neonatal jaundice that is associated
  with breastfeeding. It develops 6-14 days after
  birth, occurring in approximately one third of
  newborn infants, and continues beyond physiologic
  jaundice.
• It is different than breastfeeding jaundice,
  which occurs as a result of caloric deprivation
  in the first few days of life.
• In general, breastfed infants are 3-6 times more
  likely to develop moderate or severe jaundice
  than formula-fed infants.

Porter ML and Dennis BL. (2002).
Hyperbilirubinemia in the term newborn. American
Family Physician, 65(4) 599-606.
24
Bilirubin and JaundiceBreast Milk Jaundice
(cont.)

• The cause is unknown, but it is suspected to be a
  rare compound present in some breast milk that
  inhibits Uridine diphosphoglucuronosyltransferase
  1A1 (UGT1A1), the enzyme required for conjugation
  of bilirubin.
• Various substances identified in human milk, such
  as nonesterified fatty acids and
  ß-glucuronidases, may inhibit normal bilirubin
  metabolism.
• Also, in certain populations, mutation of the
  UGT1A1 gene, glycine to arginine at codon 71
  (G71R), has been identified as a possible genetic
  cause of breast milk jaundice.

Ramer, Timothy. (2005). Retrieved May 17, 2006,
from http//www.emedicine.com/PED/topic282.htm Mar
uo Y and Sato H. (2002). UDP-Glucuronosyltransfera
se. Japanese Journal of Hygiene, 56(4) 629-633.
25
Hyperbilirubinemia Elevated Bilirubin in the
Blood
Neonatal hyperbilirubinemia is defined as a total
serum bilirubin level above 5 mg/dL. It results
from the overproduction of unconjugated bilirubin
in newborn infants, and their limited ability to
conjugate it or excrete it. These limitations
lead to physiologic jaundice, where high serum
bilirubin concentrations in the first few days of
life color the skin yellow.
Porter ML and Dennis BL. (2002).
Hyperbilirubinemia in the term newborn. American
Family Physician, 65(4) 599-606.
26
Hyperbilirubinemia (cont.)

• Hyperbilirubinemia has the potential for
  neurotoxic effects.
• Bilirubin can enter the brain if it is free (not
  bound to albumin), unconjugated, or if there
  has been damage to the blood brain barrier.
• Once inside the brain, precipitation of
  bilirubin at low pH may have toxic effects.
  Neurons undergoing differentiation are
  particularly susceptible to injury from
  bilirubin, suggesting that prematurity
  predisposes infants to bilirubin encaphalopathy.

Dennery PA, Seidman DS, Stevenson DK. (2001).
Neonatal Hyperbilirubinemia. New England Journal
of Medicine, 344 581-590.
27
Hyperbilirubinemia and Plasma Antioxidant Activity

• Under normal conditions, bilirubin accounts for
  2 of plasma antioxidant activity in jaundiced
  infants, bilirubin accounts for 77.
• Elevated levels of plasma bilirubin were shown to
  affect the concentration of other plasma
  antioxidants, such as Vitamin E, which was
  observed at levels as high as those in adult
  blood.

Belanger S, Lavoie J-C, Chessex P. (1997).
Influence of Bilirubin on the Antioxidant
Capacity of Plasma in Newborn Infants. Biology of
the Neonate, 71 233-238.
28
Treatment of Hyperbilirubinemia

• Phototherapy with fluorescent white light or
  fibreoptic blankets to reduce serum bilirubin.
• Exchange blood transfusions to eliminate
  bilirubin from circulation.
• Phenobarbital given to mothers during the last
  week of pregnancy to increase conjugation and
  excretion in high-risk newborns (with some
  success).
• Disadvantages known risks of blood transfusion
  damage to eyes by UV light increased risk of
  neurotoxic effects and fetal abnormalities
  associated with phenobarbital.

29
Bilirubin and Exchange Transfusion
Exchange transfusions used to treat
hyperbilirubinemia decrease plasma unconjugated
bilirubin, and thus total plasma antioxidant
capacity.
Belanger S, Lavoie J-C, Chessex P. (1997).
Influence of Bilirubin on the Antioxidant
Capacity of Plasma in Newborn Infants. Biology of
the Neonate, 71 233-238.
30
Bilirubin and Exchange Transfusion

• The decrease in TEAC after the exchange
  transfusion was not likely caused by the
  transfusion itself.
• Belanger et al. discovered that the decrease
  could not have been caused by hemolysis, as the
  infants with a hemolytic disease showed no
  significant difference from those without. They
  also verified that exchange for adult blood was
  not the cause, as maternal blood was found to
  have higher TEAC than neonatal blood.

Belanger S, Lavoie J-C, Chessex P. (1997).
Influence of Bilirubin on the Antioxidant
Capacity of Plasma in Newborn Infants. Biology of
the Neonate, 71 233-238.
31
Bilirubin and the Premature Infant

• Premature infants have higher rates of
  bilirubin production than do full term infants
  or adults because their red blood cells have a
  higher turnover rate and shorter life span.
• In addition, the liver of a premature infant is
  less mature and therefore, less efficient at
  conjugating bilirubin for excretion.
• Premature infants have fewer blood proteins
  available to bind bilirubin and prevent it from
  crossing the immature blood brain barrier.

Friel JK, Martin SM, Langdon M, Herzberg G,
Buettner GR. (2002). Human milk provides better
antioxidant protection than does infant formula.
Pediatr Res, 51 612-618. Genna, Catherine W.
(2005). Jaundice in the breastfed baby. Retrieved
June 1, 2006, from http//www.medela.com/NEWFILES/
faq/jaundice.html
32
Bilirubin and the Premature Infant

• Premature infants are also at increased risk of
  oxidative stress from hypoxia due to the
  immaturity of the lungs, followed by risk of
  hyperoxia once mechanical ventilation proceeds.
  Premature infants are often exposed to oxygen
  concentrations as high as 95.
• Bilirubin administration provides protection
  against retinopathy in premature infants.

Friel JK et al. (2002). Dore S, Takahashi M,
Ferris CD, Hester LD, Guastella D, Snyder SH.
(1999). Bilirubin, formed by activation of heme
oxygenase-2, protects neurons against oxidative
stress injury. Proc. Natl. Acad. Sci. USA, 96
2445-2450.
33
Oxidative Stress and Prematurity

• Neonates have impaired antioxidant defenses and
  are susceptible to the development of oxygen
  free radical mediated diseases.
• Neonatal blood has low content of glutathione
  peroxidase, superoxide dismutase, ?-carotene,
  riboflavin, ?- proteinase, vitamin E, selenium,
  copper, zinc, ceruloplasmin and other plasma
  factors.
• The premature brain is rich in polyunsaturated
  fatty acids that are easily oxidized compared to
  monounsaturated fatty acids.

Hammerman C. Goldstein R, Kaplan M, Eran M,
Goldschmidt D, Eidelman AI, Gartmer LM. (1998).
Bilirubin in the premature Toxic waste or
natural defense? Clinic Chem, 44
2551-2553. Gitto E, Reiter RJ, Karbownik M, Tan
D, Gitto P, Barberi S, Barberi I. (2002).
Causes of oxidative stress in the pre-and
perinatal period. Biol Neonate, 81 146-157.
34
Oxidative Stress and Prematurity (cont.)
For the premature infant, bilirubin has always
been considered a toxin. More recently
bilirubins antioxidant properties have been
characterized. It is possible therefore, that
elevated bilirubin is an attempt by an immature
fetus to cope with increased exposure to
ROS. Ironically, in an attempt to rid the
premature of bilirubin, we may be eliminating a
powerful antioxidant that could assist the
immature defense system under attack. Is it
possible that jaundice might actually be a
natural defense system that is necessary for
survival of the premature infant?
Hansen TWR. (2001). Bilirubin production,
breast-feeding and neonatal jaundice. Acta
Paediatrica, 90 716-723.
35
Serum Bilirubin in Neonates Correlated with Total
Antioxidant Activity
(Hammerman et al., 1998) In contrast we did not
find a relation between bilirubin and tissue
damage or antioxidant status in small premature
infants in the first month of life. Friel J,
Widness J, Jiang T, Belkhode SL, Rebouche CJ,
Ziegler EE. (2002). Antioxidant status and
oxidant stress are associated with vitamin E
intakes in VLBW infants in early life. Nutr Res,
22 55-64.
36
Hyperbilirubinemia Protects Against Lipid
Peroxidation in Neonatal Gunn Rats Exposed to
Hyperoxia
Serum bilirubin in jaundiced and non-jaundiced
pups exposed to 95 O2 shows a negative
correlation with lipid hydroperoxides at 3 days
of exposure. Higher serum bilirubin
concentrations resulted in lower lipid
hydroperoxide levels.
Dennery PA, McDonagh AF, Spitz DR, Rodgers PA,
Stevenson DK. (1995). Hyperbilirubinemia results
in reduced oxidative injury in neonatal Gunn rats
exposed to hyperoxia. Free Radic Biol Med., 19
395-404.
37
The Effect of Hyperbilirubinemia in Neonatal Gunn
Rats Exposed to Hyperoxia
Dennery et al. also showed that jaundiced rats
exposed to gt95 O2 showed higher mean serum
bilirubin levels than jaundiced rats exposed to
95 O2 and 5 CO2 or room air.
38
It remains unclear as to how the knowledge of
bilirubins antioxidant properties can be used to
assist in defense against oxidative stress
Promise for the Future
Should elevated levels of bilirubin be allowed to
persist for an unknown period of time in order to
protect the infant? Where is the crossover to
irreparable harm? Should bilirubin be promoted
as a supplemental antioxidant? Can this important
molecule provide similar benefits to a larger
population?