Virtual Free Radical School Bilirubin Friend or Foe

Virtual Free Radical School Bilirubin: Friend or Foe? James K. Friel, Ph. D. , Russell W. Friesen, B. Sc. , & Angela C. Miller, B. Sc University of Manitoba Department of Human Nutritional Sciences H 511 Duff Roblin Building Winnipeg, MB R 3 T 2 N 2 Canada Tel: 204 -474 -8080 Fax: 204 -474 -7593 E-mail: frielj@ms. umanitoba. ca “Learning is my home. ” Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 1

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 Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 2

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 Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 3

Formation The Bilirubin Pathway: of Bilirubin: Overview Bilirubin diglucuronide (soluble) Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 4

Formation of Bilirubin 1. Hemoglobin from senescent or hemolyzed red cells is broken down, releasing heme. 2. Heme is then degraded in humans by the enzyme heme oxygenase (HO), which is the rate-limiting step in the formation of bilirubin. 3. HO converts heme to biliverdin IX. 4. Biliverdin is a hydrophilic compound that is reduced by biliverdin reductase into the hydrophobic compound bilirubin. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 5

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. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 6

Excretion of Bilirubin 1. Bilirubin is bound to albumin and transported in plasma from the reticuloendothelial system to the liver, as unconjugated bilirubin. 2. 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. 3. 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. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 7

Excretion of Bilirubin 3. In the ileum and colon, bacteria converts bilirubin into stercobilinogen. 4. Stercobilinogen is oxidized to stercobilin, which is excreted in the feces. 5. 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 Gilbert’s syndrome. Biomedical Scientist, February. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 8

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. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 9

Bilirubin as an Antioxidant At concentrations as low as 10 n. M, Bilirubin can protect against 10, 000 -fold greater concentrations of H 2 O 2. Under physiologic conditions, bilirubin provides more potent protection against lipid peroxidation than αtocopherol, 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. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 10

Bilirubin as an Antioxidant A linear relationship (R 2 = 0. 99) has been identified between plasma antioxidant capacity and unconjugated bilirubin concentration in newborn infants. This both confirms bilirubin’s 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. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 11

Bilirubin as an Antioxidant Comparative relative proportions of plasma antioxidants between premature and term neonates expressed as a percentage of the total antioxidants Albumin Urate Ascorbate α-tocopherol Bilirubin 0. 63 1. 02 0. 99 0. 97 1. 5 49 37. 5 9. 5 3 1 Term at birth 34. 5 47. 8 13. 8 0. 5 3. 4 Term - Day 5 41 24. 1 10. 2 1. 6 23 Prem at birth 33. 5 38. 1 22. 7 0. 9 4. 8 Prem - Day 5 30. 8 26. 4 8. 5 1. 2 33. 1 TEAC* value Reference level [9] *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 preterm infants. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 12

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 + LOO Bil-OOL Bil + O 2 Bil-OO LOO + BV LOO-BV Stocker R, Yamamoto Y, Mc. Donagh AF, Glazer AN, & Ames BN. (1987). Bilirubin is an antioxidant of possible physiological importance. Science, 235: 1043 -1046. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 13

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. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 14

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. 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. This indicates that bilirubin can illicit toxicity in the erythrocyte membrane in a concentration and temperature-dependent manner. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 15

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 proinflammatory cytokines (TNF-α, IL-1β and IL-6) and cytotoxic glutamate in a concentrationdependent 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). Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 16

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/biliverdin–Cu(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): 201208. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 17

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 radicalmediated 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. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 18

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. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 19

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 FEV 1, and lower serum albumin. Serum bilirubin and adjusted relative risk of major IHD events in 4916 men measured before 16: 00 c. Relative Risk (95% confidence interval) Bilirubin (µmol/L) Adjusted a Adjusted b <7 1. 00 7 0. 89(0. 66 -1. 21) 0. 92(0. 69 -1. 32) 8 0. 69 (0. 55 -0. 91) 0. 68 (0. 51 -0. 89) 10 0. 78(0. 58 -1. 05) 0. 75 (0. 55 -1. 03) 12 0. 90 (0. 68 -1. 20) 0. 99 (0. 73 -1. 34) a Adjusted for age, body mass index, smoking, social class, physical activity, alcohol intake, preexisting IHO, diabetes, use of antihypertensive treatment. Adjusted for the above and in addition for systolic blood pressure, blood cholesterol, HDL-C, FEV 1, blood glucose, and serum albumin. Complete data on all covariates were available for 4678 men (444 IHD cases). b • Serum bilirubin was found to have a U-shaped relationship with the events of ischemic heart disease (IHD) c There was little difference in mean values up to 16: 00, 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. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 20

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. Prevalence of coronary artery disease, according to concentration of total bilirubin in 877 patients. Schwertner HA, Jackson WG, & To. Ian G. (1994). Association of low serum concentration of bilirubin with increased risk of coronary artery disease. Clinical Chemistry, 40(1): 18 -23. • 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. 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. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 21

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 (<37 weeks gestation), affecting 5070% of newborns. Gurses D, Kilic I, & Sahiner T. (2002). Effects of hyperbilirubinemia on cerebrocortical electrical activity in newborns. Pediatr Res. , 52: 125 -130. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 22

Bilirubin and Jaundice: “Breast 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. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 23

Bilirubin and Jaundice: “Breast 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 1 A 1 (UGT 1 A 1), the enzyme required for conjugation of bilirubin. • Various substances identified in human milk, such as nonesterified fatty acids and β-glucuronidases, may inhibit normal bilirubin metabolism. • Also, in certain populations, mutation of the UGT 1 A 1 gene, glycine to arginine at codon 71 (G 71 R), 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/topic 282. htm Maruo Y and Sato H. (2002). UDP-Glucuronosyltransferase. Japanese Journal of Hygiene, 56(4): 629 -633. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 24

Hyperbilirubinemia: Elevated Bilirubin in the Blood Neonatal hyperbilirubinemia is defined as a total serum bilirubin level above 5 mg/d. L. 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. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 25

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 p. H 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. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 26

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. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 27

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. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 28

Bilirubin and Exchange Transfusion Effect of Exchange Transfusion on Plasma Concentration Before Exchange After Exchange (n = 11) Bilirubin, µmol/L 363 ± 19 171 ± 12* Albumin, g/L 31 ± 2 31 ± 1 Iron, μmol/L 13 ± 3 15 ± 2 Vitamin E, μmol/L 13. 0 ± 2. 9 14. 7 ± 1. 0 Urate, μmol/L 253 ± 21 265 ± 17 Means ± SEM *p < 0. 05 vs. samples before exchange 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. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 29

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. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 30

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 Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 31

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. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 32

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. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 33

Oxidative Stress and Prematurity (cont. ) For the premature infant, bilirubin has always been considered a toxin. More recently bilirubin’s 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. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 34

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. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 35

Hyperbilirubinemia Protects Against Lipid Peroxidation in Neonatal Gunn Rats Exposed to Hyperoxia Serum bilirubin in jaundiced and non-jaundiced pups exposed to 95% O 2 shows a negative correlation with lipid hydroperoxides at 3 days of exposure. Higher serum bilirubin concentrations resulted in lower lipid hydroperoxide levels. Dennery PA, Mc. Donagh 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. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 36

The Effect of Hyperbilirubinemia in Neonatal Gunn Rats Exposed to Hyperoxia Dennery et al. also showed that jaundiced rats exposed to >95% O 2 showed higher mean serum bilirubin levels than jaundiced rats exposed to 95% O 2 and 5% CO 2 or room air. Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 37

Promise for the Future It remains unclear as to how the knowledge of bilirubin’s antioxidant properties can be used to assist in defense against oxidative stress 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? Bilirubin 1/2003 – Updated 6/2006 Society For Free Radical Biology and Medicine Friel, Friesen & Miller 38