Bilirubin Metabolism and Its Disorders

Opening of the Heme Ring by Heme Oxygenase

Heme (ferroprotoporphyrin IX) is a ring of four tetrapyrroles connected by methene bridges ( Fig. 58-1 ). The ring is opened by cleavage of the α-methene bridge, catalyzed by microsomal heme oxygenases (HOs). Initially, an electrophilic attack at Fe(II) by a reducing agent, such as the reduced form of nicotinamide adenine dinucleotide phosphate, and oxygen results in the formation of α-oxyheme (see Fig. 58-1 ). Subsequently, the α-methene bridge carbon is eliminated as carbon monoxide (CO) and the porphyrin ring carbons that flank the α-methene bridge are oxidized by two additional oxygen molecules, resulting in the two lactam oxygens of biliverdin and bilirubin. Iron is released from the open tetrapyrrole after addition of electrons, suggesting that conversion of ferric iron to ferrous iron is required. Only a minute fraction of heme is opened at the β, γ, or δ bridges, resulting in the excretion of traces of bilirubin IXβ, IXγ, or IXδ, respectively, in bile.

HO catalyzes physiologic heme degradation. It consists of three structurally related isozymes: HO-1, HO-2, and HO-3. HO-1 is the inducible form, HO-2 is a constitutive isoform, and HO-3 is a minor isoform present in spleen, liver, thymus, heart, kidney, brain, and testis. HO-2 is the isoform in hepatocytes and spleen responsible for biliverdin and CO production under normal physiologic conditions. High levels of HO-2 activity are present in cells involved in the breakdown of hemoproteins, such as cells in the spleen, where senescent erythrocytes are sequestered. In the liver, both hepatocytes and Kupffer cells have HO activity; the activity in the Kupffer cells is as high as that in the spleen. Apart from breakdown of heme from circulating hemoglobin, constitutive HO-2 is important for cellular hemoprotein homeostasis. HO-2 also functions as an oxygen sensor in the lungs. HO-2-deficient mice are severely hypoxic, and data suggest that HO-2 is responsible for matching the ventilation to perfusion. HO-1 is a 32-kDa protein that is induced by lipopolysaccharides, cytokines, heavy metals, reactive oxygen species, protoheme IX, oxidized low-density lipoprotein, hypoxia, and probably also shear stress in endothelial cells in the cirrhotic liver. Nuclear factor κB and p38 mitogen-activated protein kinase signaling pathways mediate the lipopolysaccharide-dependent induction of HO-1 gene expression via DNA sequences in the proximal promoter region. HO-1 acts as a stress-response protein and, by converting prooxidant heme to antioxidant biliverdin and bilirubin, it plays a role in the cellular defense against oxidative injury. A prerequisite for this antioxidant action is that toxic ferrous iron that is released on cleavage of the porphyrin ring is efficiently scavenged by ferritin. HO-1 deficiency in humans is associated with growth retardation, hyperlipidemia, endothelial cell damage with consumption coagulopathy, and microangiopathic hemolytic anemia. Inducible HO-1 is particularly important for cytoprotection in vascular endothelium and renal tubular epithelium. HO-1-deficient mice show early atherosclerosis, particularly when they are also hypercholesterolemic.

In addition to these cellular effects, the products of the HO reaction, CO and bilirubin, have more distant effects. CO is a signaling molecule with vasodilatory effects and effects on intestinal motility and sphincters. Moreover, a possible negative correlation between circulating bilirubin levels and coronary heart disease has been reported. Whether this protective effect of bilirubin also holds for patients with Gilbert syndrome is unclear.

Binding of heme to HO requires the propionic acid substituents in the C-6 and C-7 positions and a metal, such as iron, tin, or zinc. Oxygen binds to ferrous heme and undergoes reductive activation. Noniron metalloprotoporphyrins, such as tin and zinc protoporphyrins, bind HO with even greater affinity but do not activate oxygen and are therefore not degraded by HO. These metalloporphyrins are dead-end inhibitors of heme degradation. Tin and zinc protoporphyrins also disrupt the integrity of HO-2 but not that of HO-1. The loss of integrity of HO-2, the more abundant form of HO, may partly account for the suppression of bilirubin formation by tin protoporphyrin.

Conversion of Biliverdin to Bilirubin

The immediate product of HO-mediated ring opening is the green pigment biliverdin, which is the major bile pigment in many amphibian, avian, and fish species. In most mammals, biliverdin is converted to the orange pigment bilirubin. Being less polar, bilirubin crosses placental membranes more readily than does biliverdin, although some placentate animals, such as nutria and rabbits, excrete biliverdin as the main bile pigment. Conversely, bilirubin formation has been found in early vertebrates, such as teleost and elasmobranch fish, that precede the evolution of the placenta.

Reduction of biliverdin to bilirubin is catalyzed by biliverdin reductase (see Fig. 58-1 ), a family of cytosolic enzymes that use the reduced form of nicotinamide adenine dinucleotide at pH 6.7 and the reduced form of nicotinamide adenine dinucleotide phosphate at pH 8.5 as cofactors. Guinea pig liver biliverdin reductase is a 70-kDa protein. Several interconverting forms of biliverdin reductase found in rat liver and spleen are produced by tissue-specific posttranslational modification of a single gene product. Recently, biliverdin reductase was shown to induce activating transcription factor 2, which controls the HMOX1 transcription, thereby forming a regulatory loop in which HO-1 and biliverdin reductase expression are interdependent.

Physical Conformation and Solubility of Bilirubin IXα

Crystallized bilirubin IXα- Z , with two protonated carboxyl groups, is virtually insoluble in water but is readily soluble in polar solvents, provided the intramolecular hydrogen bonds can be disrupted. Bilirubin and polar ligands, such as sulfonamides, share a binding site in the polar region of albumin with other polar substances, such as sulfonamides. Therefore, despite its insolubility in water at physiologic pH, bilirubin should be considered a relatively polar substance, the mechanism of toxicity of which may differ from that of truly lipophilic toxins, such as dichlorodiphenyltrichloroethane (also known as DDT ).

Insolubility of bilirubin IXα in water despite the presence of two propionic acid side chains, four amino groups, and two lactam oxygens is explained by internal stabilization of the molecule by hydrogen bonding between the carboxyl and the two external pyrrolenone rings ( Fig. 58-2 ). X-ray diffraction studies of crystalline bilirubin confirmed hydrogen bonding between each propionic acid side chain and the pyrrolic and lactam sites in the opposite half of the molecule. These hydrogen bonds constrain the molecule into a ridge-tile conformation (see Fig. 58-2 ) and make it insoluble in water by engaging the polar groups of the bilirubin. The integrity of the hydrogen-bonded structure requires the interpyrrolic bridges at positions 4 and 15 of bilirubin to be in trans or Z configuration. Addition of methanol, ethanol, or 6 mol/L urea interferes with the hydrogen-bonded structure and makes bilirubin more labile, water soluble, and rapidly reactive with diazo reagents. In the liver, conjugation of the propionic acid carboxyls of bilirubin with glucuronic acid moieties disrupts the hydrogen bonds, resulting in the formation of water-soluble conjugates that are readily excreted in bile. Resonance Raman spectroscopic studies of bilirubin-sphingomyelin complexes suggest that the intramolecular hydrogen bonds are disrupted in such complexes, and the propionic acid carboxyls form ion pairs with the quaternary ammonium ion of the choline moiety of sphingomyelin.

Absorption Spectra and Circular Dichroism of Bilirubin IXα

Bilirubin IXα has a main absorption band at 450 nm to 474 nm in most organic solvents with an extinction coefficient of 48.0 mmol/L to 63.4 mmol/L at its absorption maximum for a 1-cm path length. Circular dichroism spectroscopy shows that biliverdin preferentially adopts a minus-helicity conformation when bound to human serum albumin, whereas bilirubin IXα prefers a plus-helicity conformation. Therefore reduction of human serum albumin–bound biliverdin to bilirubin results in a conformational inversion from minus to plus helicity. Such sign inversion also occurs on addition of halothane, chloroform, or other volatile anesthetics to the albumin-bilirubin complex, suggesting that the volatile anesthetics alter the internal topography of receptor sites, influencing the stereoselectivity of ligand binding.

Conformational Isomerization and Cyclization

Normally, bilirubin IXα remains in a ZZ form, in which both of the interpyrrolic bridges at positions 5 and 15 are in the Z ( trans ) configuration. Exposure of circulating bilirubin to light changes the configuration of one or both of the interpyrrolic bridges from Z ( trans ) to E ( cis ) ( Fig. 58-3 ). Blue light is most efficient in mediating the conformational changes. As hydrogen bonding requires the Z configuration, (4 Z ,15 E )-bilirubin IXα and (4 E ,15 Z )-bilirubin IXα lack hydrogen bonds in one half of the molecule, whereas ( E,E )-bilirubin IXα lacks hydrogen bonds in both halves. Of these conformational isomers, (4 Z ,15 E )-bilirubin IXα is more abundant. Following absorption of two photons, the vinyl substituent at position C-3 of (4 E ,15 Z )-bilirubin IXα is cyclized with the methyl substituent on the internal pyrrole ring, forming the structural isomer ( E )-cyclobilirubin, or lumirubin. Although cyclization of bilirubin occurs at a slower rate than formation of configurational isomers, because of the relative stability of cyclobilirubin, this form may be quantitatively more important in phototherapy of neonatal jaundice. The conformational isomers are more polar than is ( Z,Z )-bilirubin IXα and can be excreted in bile without conjugation.

Fluorescence

Although pure bilirubin does not fluoresce, when dissolved in detergents, albumin, or alkaline methanol it exhibits intense fluorescence at 510 nm to 530 nm, which has been used for quantification of blood bilirubin concentrations and the unsaturated bilirubin-binding capacity of albumin.

Photooxidation and Degradation

In the presence of light and oxygen, bilirubin undergoes a self-sensitized reaction involving singlet oxygen, resulting in the formation of colorless fragments, chiefly maleimides and propentdyopent adducts. A small amount of biliverdin is also formed.

Dismutation

Bilirubin IXα is asymmetric because of the difference in the side chains of the two halves of the molecule. On exposure to light, two bilirubin IXα molecules undergo free-radical disproportionation, forming two symmetric nonphysiologic isomers termed bilirubin IIIα and bilirubin XIIIα . The reaction is enhanced in the presence of acid and oxygen, and is inhibited by ascorbate.

Bilirubin Encephalopathy in Gunn Rats

Bilirubin deposition in specific areas of the brain accompanied by structural damage is termed kernicterus . The Gunn rat is the only spontaneous mutant animal model in which bilirubin-induced brain damage has been observed. Normally, albumin binding inhibits bilirubin deposition in the brain. Displacement of bilirubin from albumin binding sites by drugs such as salicylates or sulfonamides increases bilirubin accumulation in the brain and may precipitate kernicterus. Gunn rats that are rendered genetically analbuminemic by crossbreeding with Nagase analbuminemic rats have serum bilirubin levels that are only 25% of those of other Gunn rats, whereas their cerebral bilirubin content is 1.2-fold to 2.7-fold higher. Such hybrid rats die within 3 weeks of birth. Therefore for clinical purposes, it is important to calculate the molar ratio between plasma albumin and bilirubin. However, the plasma free bilirubin level does not correlate well with brain bilirubin concentration, and it is not certain whether unbound bilirubin is the only toxic species of the pigment.

Various degrees of hearing deficiency due to abnormalities of the cochlear nuclei occur commonly as a complication of neonatal hyperbilirubinemia. Brainstem auditory evoked potential studies in Gunn rats indicate functional abnormalities of the central auditory pathways at and rostral to the cochlear nuclei beginning at 17 days of age. Similar changes are found in human neonates with severe hyperbilirubinemia.

Sulfonamides displace bilirubin from albumin binding, thereby promoting the net transfer of bilirubin into neural tissues. Administration of sulfonamides results in reversible abnormalities of brainstem auditory evoked potentials in Gunn rats. Under these conditions, focal bilirubin staining occurs in Purkinje cells of the cerebellum, hippocampus, and basal ganglia. Similar changes occur in human infants with fully developed kernicterus. A large number of Purkinje cells are affected in the cerebellum of Gunn rats at the age of 7 days; most of these cells degenerate and disappear between day 12 and day 30, resulting in cerebellar hypoplasia. The remaining Purkinje cells recover and persist into adult life. However, synapse formation among these Purkinje cells or with other neural cells may remain abnormal. Cerebellar mitochondria are enlarged and distorted in Gunn rats. Increased activities of the lysosomal enzymes arylsulfatase and cathepsin occur in the cerebellum of Gunn rats by the eighth day of life. Cerebellar cyclic GMP concentrations decrease progressively from day 15 to day 30, but cyclic AMP levels remain normal.

Clinical Features of Bilirubin Encephalopathy

Except in patients with severe inherited deficiency of bilirubin glucuronidation (discussed later in this chapter), the occurrence of kernicterus is usually limited to the neonatal period and the first few months of life. Bilirubin encephalopathy may present with a broad spectrum of neurologic features. In the severest cases, overt kernicterus presents between the third and sixth days of life. The normal Moro reflex is lost, the muscles become hypotonic, the cry is high-pitched, athetoid movements appear, and reflex opisthotonos occurs in response to a startling stimulus. This may progress to lethargy, atonia, and death. Occasionally, in some children with Crigler-Najjar syndrome type 1, bilirubin encephalopathy may present late with cerebellar symptoms as the presenting feature. Those who survive acute kernicterus may develop chronic hearing abnormalities, athetoid movements, paralysis of upward gaze, and mental retardation, in various combinations. The cochlear nucleus is commonly affected by hyperbilirubinemia. Cells of the auditory system that receive synaptic input from end-bulbs or calyces appear to be early targets. In Gunn rat pups, these morphologic changes correlate with abnormalities of brainstem auditory evoked potentials. The sensitivity of auditory evoked potential testing can be increased by the recording of binaural difference waves obtained by subtraction of the sum of two monaural brainstem auditory evoked potentials from a binaural brainstem auditory evoked potential.

Bilirubin staining of the hippocampus, basal ganglia, and nuclei of the cerebellum and brainstem occurs in infants dying in the acute phase of bilirubin encephalopathy ; however, such staining is not found in children dying in the chronic stage of the disorder. Clinical manifestations precede histologic evidence of brain damage by approximately 72 hours. Focal necrosis of neurons and glial cells occurs later. Gliosis of the affected areas is seen in chronic cases. As these histologic lesions are not present from the onset of clinical kernicterus, they may not be the initiating pathophysiologic events in bilirubin-induced brain damage. Nonspecific signs of encephalopathy in the neonate may result from other causes, such as cerebral hemorrhage, and therefore kernicterus cannot always be diagnosed without pathologic documentation. Conversely, focal bilirubin staining of the brain may occur in other forms of brain injury. Thus, in the absence of neuronal degeneration, bilirubin staining alone does not establish the diagnosis of classic kernicterus.

Peak serum bilirubin levels of up to 10 mg/dL or 12 mg/dL are usually considered safe. The prognostic significance of a moderate degree of hyperbilirubinemia is not entirely clear. Serum bilirubin levels that are not high enough to cause kernicterus have been reported to result in an increased incidence of neurologic abnormalities or decreased intellectual performance later in life.

The Blood-Brain Barrier and Cerebral Bilirubin Clearance

The equilibration of hydrophilic water-soluble substances and proteins between the blood and the brain is restricted by a functional blood-brain barrier. Tight junctions between capillary endothelial cells and foot processes of astroglial cells represent the structural component of this barrier. Specific transport mechanisms for translocation of ions, water, and nutrients from plasma to brain may provide the functional counterpart of the blood-brain barrier. Conventionally, immaturity of the blood-brain barrier in neonates has been implicated in the high incidence of kernicterus in this age group. However, it has been difficult to confirm a more rapid passage of labeled markers or lipophilic substances into the immature brain. Therefore there is no firm evidence to support the concept of an immature blood-brain barrier in the neonate.

The efficiency of cerebral bilirubin clearance may be inversely related to the cerebral toxicity of bilirubin. Experimentally, the blood-brain barrier can be unilaterally and reversibly opened without causing brain damage by infusion of hypertonic urea or arabinose. The hyperosmolarity-associated shrinkage of the capillary endothelial cells results in temporary opening of the tight junctions. When the blood-brain barrier is opened in newborn rats by this technique, intravenously administered albumin-bound bilirubin rapidly enters the brain. Following the reversal of the blood-brain barrier, bilirubin is rapidly cleared from the brain. The clearance of bilirubin from brain parallels its clearance from plasma, suggesting that bilirubin is cleared by diffusion or active transport back into the general circulation. Slower bilirubin clearance from the injured edematous brain may make the damaged brain more vulnerable to bilirubin toxicity.

Endothelial cells of cerebral microvessels and the choroid plexus together form the blood-brain barrier and cerebrospinal fluid–blood barrier. These endothelial cells strongly express the ATP-binding cassette (ABC) superfamily of transporting proteins. Notably, multidrug resistance protein 1 (MDR1; a P-glycoprotein) and the multidrug resistance–associated proteins (MRPs) MRP1, MRP4, MRP5, and, to a lesser extent, MRP6 are expressed in these tissues. The MRPs located in the microvascular endothelial cells and the basolateral membranes of the choroid plexus epithelial cells act as export pumps for drugs from the brain and the cerebrospinal fluid to the blood. MRP1 is preferentially expressed in the astroglial component of the blood-brain barrier. Recent evidence indicates that bilirubin is a substrate for MRP1 (ABCC1). According to these concepts the blood-brain barrier is not merely a passive anatomic structure but is an active tissue that can pump bilirubin and other metabolites and drugs out of the brain, thus reducing their intracellular concentrations. Substrate competition at the level of these pumps may be another way by which drug exposure can modulate bilirubin brain toxicity.

Biochemical Basis of Bilirubin Toxicity

In cell culture systems, bilirubin shows a very broad range of toxicity. It is not clear which of the toxic effects observed in monotype cell cultures are relevant in bilirubin encephalopathy, and results observed with cultured cells do not always mirror findings in vivo. Although bilirubin is an antioxidant, bilirubin toxicity has been found to be associated with oxidative stress. This is partly explained by proteomic analysis of cerebella of Ugt1a1 -knockout jaundiced mice, which showed reduced levels of several proteins that are involved in the cellular defense against reactive oxygen species. These included protein deglycase DJ-1, superoxide dismutase, and peroxiredoxins 2 and 6. Reduction in the levels of these proteins occurred despite increased messenger RNA (mRNA) levels of nuclear factor erythroid-derived 2–like 2, which regulates the expression of the antioxidant genes. The mechanism of reduced expression of these proteins appears to be posttranscriptional because the corresponding mRNA levels were not altered. Thus a variety of protective mechanisms fail to protect the cells in the presence of excessive amounts of bilirubin, ultimately leading to neurodegeneration. Inhibition of cell division and enhancement of cellular apoptosis by bilirubin have been linked to the induction of a tumor suppressor protein, phosphatase and tensin homolog. In a cell-free system, bilirubin irreversibly inhibits calcium-activated, phospholipid-dependent protein kinase C activity and cAMP-dependent protein kinase activity. Because protein phosphorylation is the final common pathway of neuronal signal transmission, inhibition of protein kinase C by bilirubin may play a role in bilirubin encephalopathy in the newborn.

Physiologic mechanisms of protection against bilirubin-induced injury and cellular mechanisms of bilirubin toxicity are summarized in Table 58-1 .

Bilirubin Nephrotoxicity

Renal medullary deposition of unconjugated bilirubin results in medullary necrosis and formation of bilirubin crystals on the renal papillae in Gunn rats and in hyperbilirubinemic infants. In adult Gunn rats, abnormality of the ascending loop of Henle leads to an impairment of urinary concentration, which is ameliorated by reduction of serum bilirubin levels. The urinary concentration defect has not been found in mature neonates with hyperbilirubinemia or in patients with Crigler-Najjar syndrome type 1 who survive to adult age.

Bilirubin-Binding Sites and Completion With Other Ligands

There is a primary and a secondary binding site on albumin for bilirubin. Affinity labeling studies indicate that the primary binding site of bilirubin is located at residues 124 to 297 and, to a lesser extent, at residues 446 to 547. Lysine 240 in human albumin and lysine 238 in bovine serum albumin appear to be critical in bilirubin binding.

Albumin inhibits the neurotoxic effects of unconjugated bilirubin following intravenous injection in puppies. The role of albumin in preventing bilirubin toxicity is clearly shown in analbuminemic-Gunn hybrid rats, which die of bilirubin encephalopathy shortly after birth. Normally, bilirubin binding to albumin is reversible and can be affected by alternative ligands. Ligands that bind to the bilirubin-binding site of albumin, such as sulfonamides, antiinflammatory drugs, and cholangiographic contrast media, displace bilirubin competitively from albumin, increasing the non–protein-bound fraction of bilirubin. Prophylactic use of sulfonamides in newborns enhances bilirubin encephalopathy, probably by enhancing the dissociation of bilirubin from albumin, thereby increasing the net uptake of bilirubin into neural tissues. On the other hand, binding at non–bilirubin-binding sites may cause configurational changes that can enhance (cooperative binding) or diminish (anticooperative) bilirubin binding.

Determination of Reserve Bilirubin-Binding Capacity of Albumin

Because of the influence of many metabolites and drugs on albumin binding of bilirubin and its transfer from plasma to the central nervous system, measurement of total plasma bilirubin concentration does not accurately estimate the risk of brain damage from unconjugated bilirubin. Unbound bilirubin in serum can be quantified by gel chromatography, peroxidase treatment, electrophoresis on cellulose acetate, and fluorometry of serum with or without detergent treatment. Unbound bilirubin concentration may be roughly estimated as the product of serum bilirubin concentration, the concentration of reserve bilirubin-binding sites on albumin, and the association constant for bilirubin.

Irreversible Binding of Bilirubin to Albumin

During prolonged conjugated hyperbilirubinemia, bilirubin becomes covalently bound to albumin. This fraction of bile pigments is not cleared by the liver or kidneys and persists for a long time in the circulation, reflecting the long half-life of serum albumin.

Bilirubin Metabolism in Inherited Analbuminemia

In view of this discussion it is interesting to note that analbuminemia is compatible with life and that elimination of amphipathic compounds such as bilirubin and bromosulfophthalein (BSP) is disturbed to a relatively minor degree in analbuminemic rats. For example, elimination of a tracer dose of bilirubin was entirely normal, although the biliary recovery after a loading dose was decreased. Other plasma proteins, such as high-density lipoproteins, can assume some of the assigned roles of albumin.

Transporters

The basolateral hepatocyte membrane contains various transporter proteins that function as carriers for the uptake of a multitude of endogenous and exogenous substances. Internalization of organic anions from plasma into the hepatocytes occur by a non–energy-consuming process termed facilitated diffusion . Organic anion–transporting polypeptides (OATPs), encoded by a family of solute carrier anion transporter genes carry bile acids, bilirubin, interleukins, and hormones such as thyroid and steroid hormones into the hepatocyte across the basolateral membrane. OATPs also transport numerous drugs, such as tyrosine kinase inhibitors and statins. The uptake carriers in human hepatocytes are listed in Table 58-2 . Na ⁺ -taurocholate–cotransporting polypeptide (NTCP), encoded by SLC10A1 in humans and Slc10a1 in rodents, has a narrow range of substrate specificity and is a specialized carrier for the Na ⁺ -dependent hepatic uptake of bile salts. Although non–carrier-mediated transmembrane diffusion was proposed as the mechanism of bilirubin influx, because this process is hepatocyte preferred, specific carriers facilitating bilirubin diffusion have been sought. OATP1B1 (encoded by SLCO1B1 ) was reported to transport unconjugated bilirubin but other studies did not confirm this conclusion. Thus the mechanism by which unconjugated bilirubin is transported across the sinusoidal surface membrane of the hepatocytes remains to be conclusively identified. On the other hand, OATP1B1 and OATP1B3 (encoded by SLCO1B3 ) have been shown to transport bilirubin glucuronide into hepatocytes, which is physiologically relevant in reuptake of bilirubin glucuronides that are pumped out into the hepatic sinusoids by the ATP-utilizing pump MRP3 (ABCC3).

Acquired and Inherited Abnormalities of Hepatic Bilirubin Uptake

The expression of hepatic uptake carrier proteins is regulated at both the transcriptional level and the posttranscriptional level. Endotoxin, tumor necrosis factor α, interleukin-1β, and interleukin-6 reduce bile salt and organic anion uptake by down-regulating the expression of NTCP and OATP1B3. The significance of OATP1B3 in bilirubin metabolism is highlighted by the association of polymorphisms of the encoding gene with a statistically significant increase in serum total bilirubin levels in adults, as well as in neonates. OATP1B1 and OATP1B3 are now known to be involved in hepatocellular reuptake of bilirubin glucuronides that are transported into the plasma by hepatocytes. Simultaneous inactivating mutations of SLCO1B1 and SLCO1B3 , the genes that encode these two transporters, result in mild conjugated hyperbilirubinemia, which is seen in Rotor syndrome. Notably, the SLC family of genes is also involved in the transport of numerous organic anions, including hormones, cytokines, endogenous metabolites, and drugs, as highlighted by a high incidence of myopathy, induced by statins in individuals carrying SLCO1B1 variants. High intracellular bile salt concentrations, as found during cholestasis, decrease the expression of NTCP and OATP1B1. Bile salts bind to the farnesoid X receptor (FXR), a cytosolic nuclear hormone receptor. On binding bile salts, this receptor associates with retinoid X receptor (RXR), and the heterodimer then translocates to the nucleus. Here it binds to an FXR-RXR response element that is present in a number of genes, including the NR0B2 gene (also known as SHP ). Thus high bile salt concentration activates small heterodimer partner (SHP) expression and the up-regulated SHP interferes with RAR-RXR binding to the SLC10A1 gene, inhibiting transcription. Also OATP1B1 expression is down-regulated in cholestasis. OATP1B1 expression is under transcriptional control of hepatocyte nuclear factor 1α, which in turn is controlled by hepatocyte nuclear factor 4α. SHP inhibits hepatocyte nuclear factor 4α–mediated transactivation of the hepatocyte nuclear factor 1α promoter in cotransfection assays. In addition, the human hepatocyte nuclear factor 4α gene promoter is repressed by chenodeoxycholic acid through an SHP-independent mechanism. This explains why, both NTCP and OATP1B1are down-regulated in cholestatic liver disease. As discussed later in this chapter, MRP2 (ABCC2), the biliary export pump for conjugated bilirubin, is down-regulated during cholestasis, whereas MRP3 (ABCC3), a transporter with affinity for conjugated bilirubin, is up-regulated in the basolateral membrane. Via this transporter, conjugated bilirubin is pumped from the hepatocyte into blood. As OATP1B1is involved in reuptake of conjugated bilirubin, its down-regulation in cholestatic diseases may be expected to further increase the plasma accumulation of conjugated bilirubin. Subsequent clearance of bilirubin conjugates by the kidneys in cholestasis is facilitated by the up-regulation of renal MRP2 expression.

Separate transport proteins for bilirubin and bile salt internalization by hepatocytes explains the discrepancy between the extent of elevation of plasma bile acid and bilirubin levels in some clinical situations. For example, early in the course of primary biliary cirrhosis, bile acid levels are elevated but bilirubin levels may still be normal. Reduced hepatic bilirubin uptake has been observed in some cases of Gilbert syndrome (discussed later).

ATP-Dependent Organic Anion Transport

ATP-dependent transporters, containing ABCs are important in the canalicular excretion of bilirubin glucuronides. MRP2 (ABCC2) is an organic anion efflux pump present in the canalicular surface of hepatocytes and apical surface of the renal and intestinal epithelia. for organic anions. In the liver, MRP2 functions as the efflux pump for many organic anions, including bilirubin monoglucuronide and bilirubin diglucuronide, most of which are products of phase II drug metabolism. MRP2 also contributes to bile formation by transporting glutathione, a major driving force for bile salt–independent bile flow. Genetic deficiency of MRP2 in humans leads to Dubin-Johnson syndrome, characterized by hyperbilirubinemia. The TR ⁻ rat is an animal model with conjugated bilirubinemia caused by a single nucleotide deletion in the ABCC2 gene. MRP1 and MRP3 are located in the basolateral membrane of hepatocytes. These export pumps are expressed at low levels in normal liver but are greatly induced during cholestasis and hyperbilirubinemia. MRP1 and MRP3 both transport glucuronides, including bilirubin glucuronides, from hepatocytes to blood, whereas MRP1 pumps glutathione S-conjugates. MRP3 normally transports a fraction of bilirubin glucuronides formed in the hepatocyte back to plasma. In conditions where MRP2 expression is down-regulated, such as in cholestasis and genetic MRP2 deficiency, up-regulation of MRP3 at the basolateral surface of the liver results in increased transport of bilirubin glucuronides to the blood.

Constitutive androstane receptor (CAR) is the dominant transcriptional regulator of MRP3. Phenobarbital is the prototypic substrate of CAR. MRP3 expression is enhanced in patients with Dubin-Johnson syndrome and in Eisai hyperbilirubinemic rats, suggesting that conjugated bilirubin may also be a CAR substrate. A dose- and time-dependent induction of Abcc2 expression was observed in isolated rat hepatocytes cultured in the presence of xenobiotics, including vincristine, tamoxifen, or the pregnane X receptor (PXR)-ligand rifampicin, indicating that Abcc2 gene transcription may respond to substrates of MRP2 itself. The promoter regions of the human ABCC2 and rat Abcc2 genes have been identified. Sequence analysis of the human ABCC2 promoter showed a number of putative consensus binding sites for both ubiquitous and liver-enriched transcription factors, including activating protein 1, specificity protein 1, hepatocyte nuclear factors 1 and 4, as well as CAR, PXR, and FXR. An unusual sequence was identified 440 base pairs upstream of the ABCC2 transcription initiation site that binds with high-affinity to PXR, CAR, and FXR as heterodimers with RXR. Human and rodent hepatocytes reacted with a robust induction of ABCC2 mRNA levels on exposure to PXR, CAR, and FXR agonists—rifampicin, dexamethasone, pregnenolone 16α-carbonitrile, and spironolactone (PXR); phenobarbital (CAR); and chenodeoxycholic acid (FXR). Ugt1A1 in rodents has also been reported to be a PXR target gene. Like ABCC2, UGT1A1 contains a multicomponent enhancer element in its promoter region with CAR, PXR, and aryl hydrocarbon receptor motifs. In addition, both glucuronidation and secretion are induced by PXR and CAR agonists. Thus PXR and CAR are major regulators of bilirubin uptake, glucuronidation, and secretion. The various secretion transporters and the substances that they handle are shown in Table 58-3 .

Transport of Bile Acids and Non–Bile Acid Organic Anions

Transport of bile acids and other cholephilic organic anions into the bile canaliculus is important in bile formation and therefore excretion of conjugated bilirubin into the bile. Human MDR1 (ABCB1), multidrug resistance protein 3 (MDR3; ABCB4), ATPase class I type 8B member 1 (ATP8B1), and bile salt export pump (BSEP; ABCB11)—and their rat orthologues MDR1A and MDR1B, multidrug resistance protein 2 (MDR2), ATP8B1, and BSEP respectively—are P-glycoproteins that are constitutively expressed in the canalicular membrane of the hepatocyte. Human MDR3, rat MDR2, and human and rat BSEP are expressed in the liver only, whereas MDR1 and ATP8B1 are also expressed in various nonhepatic secretory epithelia. The canalicular BSEP is of paramount importance for bile formation and liver function. BSEP appears to be the principal driving force for the enterohepatic circulation of bile salts and the bile salt–dependent fraction of bile flow. Bile salts are the major, if not the only, substrates of BSEP. Inherited deficiency of ATP8B1 and BSEP leads to progressive familial intrahepatic cholestasis type I and type II respectively. These conditions both lead to severe life-threatening cholestatic liver disease, associated with both conjugated and unconjugated hyperbilirubinemia. Surprisingly, some mutations of ATP8B1 and BSEP also cause forms of benign intrahepatic cholestasis. MDR3 in humans (MDR2 in mice) is involved in the biliary secretion of phosphatidylcholine, the only phospholipid in bile. Mice with a knockout mutation of the Mdr2 gene produce bile in which phospholipids are absent. These mice and humans with a similar defect develop severe liver disease characterized by bile duct proliferation, portal fibrosis, and eventually cirrhosis. Heterozygosity for MDR3 deficiency is also in part responsible for intrahepatic cholestasis of pregnancy as well as for intrahepatic and gallbladder cholesterol stone formation. Approximately one third of adult patients presenting with unexplained cholestasis have mutations in the coding region of at least one ABCB4 allele. ABCB4 mutations also cause acute recurrent biliary pancreatitis, biliary cirrhosis, and fibrosing cholestatic liver disease in adults with or without biliary symptoms.