1 1 MOLECULAR PHYSIOLOGY AND PATHOPHYSIOLOGY OF BILIRUBIN HANDLING BY THE BLOOD, LIVER, 2 INTESTINE, AND BRAIN IN THE NEWBORN 3 THOR W.R. HANSEN1, RONALD J. WONG2, DAVID K. STEVENSON2 4 1Division of Paediatric and Adolescent Medicine, Institute of Clinical Medicine, Faculty of Medicine, 5 University of Oslo, Norway 6 2Department of Pediatrics, Stanford University School of Medicine, Stanford CA, USA 7 __________________________________________________________________________________ 8 2 9 I. Introduction 10 II. Bilirubin in the Body 11 A. Bilirubin Chemistry 12 1. Bilirubin structure 13 2. Bilirubin solubility 14 3. Bilirubin isomers 15 4. Heme degradation 16 5. Biliverdin and biliverdin reductase (BVR) 17 B. Bilirubin as an Antioxidant 18 C. Bilirubin as a Toxin 19 1. Bilirubin effects on enzyme activity 20 2. Toxicity of bilirubin conjugates and isomers 21 D. Other Functions/Roles 22 1. Drug displacement by bilirubin 23 2. Bilirubin interactions with the immune system and 24 inflammatory/infectious mechanisms 25 III. The Production of Bilirubin in the Body 26 A. Heme Catabolism and Its Regulation 27 1. Genetic variants in bilirubin production 28 B. The Effect of Hemolysis 29 1. Disorders associated with increased bilirubin production 30 IV. Bilirubin Binding and Transport in Blood 31 V. Bilirubin in the Liver 32 A. Hepatocellular Uptake and Intracellular Processing 33 B. Bilirubin Conjugation 34 1. Genetic variants in bilirubin conjugation 3 35 a. Crigler-Najjar syndrome type I 36 b. Crigler-Najjar syndrome type II 37 c. Gilbert syndrome 38 2. Genetic variants in transporter proteins 39 C. Bilirubin Excretion 40 VI. Bilirubin in the Intestines 41 A. Excretion into the Intestine 42 B. Role/Function of Bilirubin in Intestines 43 C. Re-Uptake/Enterohepatic Circulation of Bilirubin 44 1. Breast milk jaundice 45 2. Effects of perturbed intestinal transit 46 D. Metabolism of Bilirubin in the Intestine 47 E. Fecal Excretion 48 VII. Bilirubin in the Brain 49 A. History and Clinical Picture of Kernicterus 50 B. The role of the BBB 51 1. Permeability and its modulation 52 2. Transport mechanisms 53 a. The role of ‘flippases’ 54 b. Other BBB molecules with relevance for brain B uptake and excretion 55 C. Brain Blood Flow 56 D. Excretion 57 1. The CSF “sink” 58 2. The BBB 59 E. Bilirubin Metabolism in the Brain 60 F. Regional and Subcellular Localization 4 61 G. Mechanism(s) of Bilirubin Neurotoxicity 62 1. Transient versus permanent effects 63 2. Inhibition of cell respiration 64 3. Membrane effects 65 4. Neurotransmitter metabolism 66 5. Enzyme induction 67 6. Apoptosis and necrosis 68 7. Cell metabolism 69 8. Infection and immunology 70 9. Differential sensitivity 71 10. Neuroprotection 72 11. Hemolysis 73 12. Bilirubin binding 74 13. A common mechanism? 75 14. A note of caution 76 __________________________________________________________________________________ 77 5 78 Hansen TWR, Wong RJ, Stevenson DK. Molecular Physiology and Pathophysiology of Bilirubin 79 Handling by the Blood, Liver, Intestine, and Brain in the Newborn. Physiol Rev XX: XX, 2020. – 80 Bilirubin is the end-product of heme catabolism formed during a process that involves oxidation- 81 reduction reactions and conserves iron body stores. Unconjugated hyperbilirubinemia is common in 82 newborn infants, but rare later in life. The basic physiology of bilirubin metabolism, such as 83 production, transport, and excretion, has been well described. However, in the neonate numerous 84 variables related to nutrition, ethnicity, and genetic variants at several metabolic steps may be 85 superimposed on the normal physiologic hyperbilirubinemia that occurs in the first week of life and 86 results in bilirubin levels that may be toxic to the brain. Bilirubin exists in several isomeric forms that 87 differ in their polarities and is considered a physiologically important antioxidant. Here we review the 88 chemistry of the bilirubin molecule and its metabolism in the body with a particular focus on the 89 processes that impact the newborn infant, and how differences relative to older children and adults 90 contribute to the risk of developing both acute and long-term, neurologic sequelae in the newborn 91 infant. The final section deals with the interplay between the brain and bilirubin, and its entry, 92 clearance, and accumulation. We conclude with a discussion of the current state of knowledge 93 regarding the mechanism(s) of bilirubin neurotoxicity. 6 94 I. INTRODUCTION 95 Among the many transitional processes that take place in newborn infants, jaundice is arguably the 96 most visible, and also the most common cause for diagnostic and therapeutic intervention during the 97 first days of life (306, 329, 440, 491, 627). Neonatal jaundice (NNJ) is caused by the accumulation of 98 unconjugated bilirubin (UCB) in blood and tissues. The normal physiology of bilirubin production, 99 transport, and excretion has been well described (69-71, 710). However, the neonatal period is in 100 many respects unique in regard to bilirubin metabolism, as very significant elevations of UCB 101 concentrations in serum occur only exceptionally after this age. Because the actions of bilirubin 102 present something akin to a ‘Janus face’, being not only a physiologically important antioxidant; but 103 also, a toxin, particularly in the brain, it is important to understand the factors that distinguish the 104 physiology and pathophysiology of NNJ from that in the more mature organism (445). Although NNJ 105 has been described in medical literature for centuries, the recognition that severely jaundiced infants 106 are at risk for neurotoxicity is more recent. In 1904, the German pathologist Georg Schmorl described 107 the bilirubin-staining pattern and neuropathological findings in brains from infants who had died 108 with severe jaundice and coined the term ‘kernicterus’ (German for ‘jaundice of the basal ganglia’) 109 (588). Descriptions of infants who survived severe NNJ with neurologic sequelae soon followed (44, 110 262). 111 Here we review our current understanding of bilirubin chemistry and 112 physiology/pathophysiology with particular reference to NNJ. Many phenomena which may appear 113 less interesting for the mature organism turn out to be important for an infant with significant NNJ. 114 Thus, bilirubin structure, solubility, and isomerization are all important in the pathophysiology of 115 kernicterus as well as in the therapies we employ to treat jaundiced infants. The balance between 116 the dual roles of bilirubin as an antioxidant and toxin is imperfectly understood (445). Because 117 bilirubin production is a potential target for therapeutic intervention, a more detailed understanding 118 of its molecular processes is needed (221, 487, 610, 713). The genetics of hepatic processing and 119 excretion of bilirubin as well as the molecular mechanisms of intestinal handling, may hold the keys 7 120 to predicting an infant’s risk for developing significant NNJ (420, 689, 692, 732). There are many 121 theories that attempt to explain the mechanisms for bilirubin entry into and processing by the brain, 122 the differential sensitivity to bilirubin neurotoxicity both on the individual and cellular levels, and the 123 ‘basic mechanism of bilirubin neurotoxicity’, if indeed there is only one (305). Neuroprotection has in 124 recent years been developed for asphyxia-related brain damage in the newborn and may be a 125 promising area for NNJ research. Drug treatment has been promising both in vitro as well as in in vivo 126 animal experiments, but is held back by concerns for toxicity (168, 233, 418, 557). Theoretically, the 127 polar bilirubin photoisomers should be less toxic and perhaps also cross the blood-brain barrier (BBB) 128 less easily than the predominant IXα(Z,Z) isomer, but experimental support is needed (304). Thus, the 129 challenges involved in NNJ research remain a fertile field for the inquisitive mind. 130 II. BILIRUBIN IN THE BODY (FIGURE 1) 131 A. Bilirubin Chemistry 132 Bilirubin is formed in the reticuloendothelial system through the catabolism of heme. Hemoglobin 133 (Hb) is the main source (80%–85%), but other heme-containing molecules (myoglobin, cytochrome, 134 peroxidase, catalase) also contribute (70, 522). In hemolytic anemias erythropoiesis increases 135 several-fold, and an even higher proportion of heme is derived from senescing red blood cells (RBCs) 136 (71). Liver production of heme was estimated to contribute 13%–23% to the body’s total production 137 (84). The relative fraction of non-Hb heme may increase in conditions such as porphyria, 138 protoporphyria, and lead poisoning (71). 139 In healthy humans, bilirubin production was estimated at 3.5–4.0 mg/kg body weight 140 (BW)/day (69, 353), but in newborn infants it is twice as high – 8.5±2.3 mg/kg BW/day (444). 141 Increased bilirubin levels or ‘hyperbilirubinemia’ in the body are measured as total serum or plasma 142 bilirubin (TSB) by spectrophotometry or co-oximetry, or in skin by transcutaneous bilirubinometry 143 (TcB). Once bilirubin concentrations exceed certain levels, it can be visually detected as ‘jaundice’. In 144 humans, jaundice predominantly develops during the first week of life (transitional period) in 60%– 145 80% of healthy newborn infants when hepatic bilirubin metabolism mechanisms are not fully mature 8 146 (75). The risk for bilirubin neurotoxicity explains why treatment to reduce TSB levels is so important 147 in neonatal medicine (491). After the first 2 weeks of life, jaundice can sometimes be due to hepatic 148 or bile duct disease, which causes accumulation of conjugated bilirubin (glucuronic acid-bound 149 bilirubin). Rare inherited variants of bilirubin excretion and conjugation, such as Gilbert syndrome, 150 Crigler-Najjar syndrome types I and II, Rotor syndrome, Dubin-Johnson syndrome, Aagenæs 151 syndrome, and several other rare inherited and/or metabolic disorders, may cause jaundice after the 152 newborn period. 153 1. Bilirubin structure 154 The chemical structure of bilirubin was defined in 1937 by Fischer and Orth (215) as a tetrapyrrol 155 with a close relationship to Hb and its successful synthesis was reported in 1942 (FIGURE 2A) (216).