Unconjugated bilirubin (UCB), the principal mammalian bile pigment, is the end intravascular product of heme catabolism. Like many weakly polar, poorly soluble compounds, UCB is transported in blood tightly bound to albumin, with less than 0.01% of total bilirubin circulating in an unbound form (free bilirubin [Bf]). This fraction governs UCB tissue flux, and is responsible for its pathophysiological effects on cells and tissues.

The transport mechanisms of different organic anions across the hepatocyte membrane have been the subject of extensive investigation over the last three decades. The primary reason for this interest is the crucial role played by the liver in the biotransformation of several endogenous and exogenous substances and their secretion via the biliary system. UCB is no exception since this endogenous organic anion is taken up rapidly and selectively by the liver and secreted into the bile after metabolic biotransformation (conjugation).

The uptake of UCB across the basolateral membrane of the hepatocyte may be a carrier-mediated process based on experiments performed with different experimental models ranging from isolated basolateral plasma membrane–enriched vesicles to isolated and perfused liver. It is generally accepted that UCB enters the liver cells via a saturable, possibly carrier-mediated mechanism at low concentration (<40–50 nM) while its transport across the basolateral plasma membrane is passive and concentration dependent when the Bf concentration increases above certain levels (≈70 nM). Spontaneous diffusion accounts for the observation that UCB may enter any cell when its plasma concentration reaches a certain threshold. Evidence has accrued that the spontaneous transmembrane UCB diffusion is rapid and efficient, and that the overall transfer is mainly determined by the dissociation rate of UCB from albumin.

Diffusion across the cell plasma membrane is particularly important to account the entry of UCB in cells other than the hepatocyte that has a unique mechanism for conjugating the pigment. As high concentrations of UCB in the cell are toxic, it is important to understand the mechanisms by which UCB diffused into the cell can be eliminated therefrom.

In the 1990s, an extensive debate took place on the nature and the existence of UCB transporter(s) involved in the transmembrane passage of the pigment, particularly in the hepatocyte. At least four different putative transporters were suggested (bilirubin/BSP-binding protein [BBBP], organic anion–binding protein [OABP], bilitranslocase, and organic anion–transporting polypeptide [OATP])1 but the use of surrogate dyes instead of bilirubin limited the conclusive nature of these studies. More recently data have been provided indicating that one member of the OATP family, human SLC01B1 (OATP1B1), also known as SLC21A6, OATP2, OATPC, and LST-1, may mediate hepatic bilirubin transport.2 This conclusion was not confirmed in a subsequent study, however, leaving the issue of OATP1B1-mediated bilirubin membrane transport still unsettled. Data obtained in isolated liver cells, however, do indicate that UCB enters the cells in a saturable, carrier-mediated mechanism at low concentration. However, the molecular species involved in this function requires further investigation.3 The role of another putative transport protein in UCB uptake by the liver, SLC01B3 (OATP8), is not clear. The available studies indicate that when expressed in HEK293 cells, this carrier is able to transport at least monoglucuronosyl bilirubin.2,4 The carrier-mediated transport of UCB was also assessed in placenta with regard to the potential involvement of three members of the SLC21A family of carriers described at the level of the liver cell (SLC01A2, also known as OATP-A; SLC01B1 [OATP1B1], also known as OATP-C; and SLC01B3, also known as OATP8). Results suggested that SLC01B3 (OATP8) may play a role in the carrier-mediated uptake of the fetal UCB by the placental trophoblast, whereas both SLC01B3 (OATP8) and SLC01B1 (OATP1B1), also known as OATP-C, may substantially contribute to UCB uptake by adult hepatocytes.5 Collectively these data suggest the possible involvement of one or more members of the large OATP family (see Figure 4-1) although the nature of the transporter(s) is still unsettled.

By contrast, Zucker et al. have presented evidence that uptake of bilirubin is a carrier-independent (diffusion) process.6 Regarding placental transport McDonagh showed that, if the mother has elevated levels of UCB in her blood (as for Crigler–Najjar patients or Gunn rat model), bilirubin transport is consistent with passive bidirectional diffusion and does not reflect active transport against a concentration gradient.7

The ATP-binding cassette (ABC) superfamily is the largest transporter family reported,8 and one or more of its members have been found in almost all organisms. The ABC proteins bind intracellularly ATP, and use the energy to drive the transport of various molecules across the plasma membrane as well as intracellular membranes of the endoplasmic reticulum (ER), peroxisomes, and mitochondria against a concentration gradient. These proteins translocate a wide variety of substrates including sugars, amino acids, metal ions, peptides, and proteins, and a large number of hydrophobic compounds.8 Based on organization of domains and amino acid homology, ABC genes can be divided into seven families: ABCA, ABCB, ABCC, ABCD, ABCE, ABCF, and ABCG.9 These genes are essential for many processes in the cell. Mutations in some of the ABC genes cause or contribute to many genetic disorders, including cystic fibrosis, anemia, neurological disease, cholesterol and bile acid transport defects, and retinal degeneration.

Multidrug resistance proteins (MRPs), together with the cystic fibrosis conductance transmembrane regulator (CFTR/ABCC7) and the sulfonylurea receptors (SUR1/ABCC8 and SUR2/ABCC9), comprise the 13 members of the human ATP-binding cassette C (ABCC) family. Establishing the substrate specificities of the MRPs has been, and remains, an area of considerable research activity. Many studies have focused on xenobiotic substrates because of the potential role of the MRPs in clinical drug resistance and in protection against a wide range of environmental potentially toxic compounds. Other studies investigated the involvement of MRPs in the transport of endogenous substrates, including UCB, in order to gain insight into possible physiological functions of the proteins.10 It was shown that UCB undergoes ATP-dependent export from trophoblastic BeWo cells and that the transport activity is somehow proportional to the level of expression and activity of ABCC1 (MRP1).11 Studies using membrane vesicles obtained from MRP1-transfected MDCKII kidney cells confirmed that UCB is a substrate for ABCC1.12 The transport was ATP and GSH dependent, with an apparent Km of 10 nM, by the lowest Km for any known substrate of this transporter.12 By contrast, MDCKII cells overexpressing multidrug resistance-related protein 2 (MRP2) did not transport UCB, indicating that MRP2 is not involved in the excretion of UCB.12 These data demonstrate that MRP1 transports UCB suggesting a physiological role of MRP1 in the cellular export of UCB.12

Belonging to the same family, ABCC3 (also known as multidrug resistance protein 3 [MRP3]) shares the highest degree of amino acid homology with MRP1 (58%).13 The presence of MRP3 protein has been confirmed in the liver, kidneys, the intestines, adrenals, pancreas, gallbladder, and spleen.14 In polarized epithelial cells, MRP3 localizes to the basolateral membrane.14 Higuchi et al.15 investigated the expression of MRP3 in the hyperbilirubinemic Gunn rat. The hepatic expression of MRP3 mRNA (RT-PCR) and protein (Western blot and immunohistochemical staining) was significantly higher in Gunn rats than in normal rats, in agreement with findings of Ogawa et al.16 Evidence regarding MRP3 transport of conjugated bilirubin will be discussed in the next paragraph.

Another ABC protein claimed to be involved in transport of endogenous compounds is ABCB1 (MDR/PGP1/P-glycoprotein 1) that belongs to the ABCB subfamily. The ABCB subfamily is unique in that it contains both four full transporters and seven half transporters. PGP1 was the first human ABC transporter cloned and characterized through its ability to confer a multidrug resistance phenotype to cancer cells.9 Watchko et al. suggested that UCB is a substrate for PGP1 based on the observation following an intravenous bilirubin load; mdr1a[–/–], Pgp-deficient mice showed a significantly greater bilirubin content in the brain than wild-type littermates.17 Further support for the concept that PGP1 may be involved in UCB transport is derived from Caco-2 cells overexpressing the protein at the apical membrane. These cells showed basal-to-apical vectorial transport of UCB that was significantly attenuated by the P-glycoprotein 1 inhibitor, verapamil.18

Although PGP1 probably has a lower affinity for the UCB than does MRP1, these data may be interpreted as evidence that UCB may be a substrate for both PGP1 and MRP1, and both may be involved in the extrusion from the cell of UCB thus contributing to prevent bilirubin cellular toxicity.

The naturally occurring UCB in adults is IXα isomer with internal hydrogen bonding engaging all polar groups and giving UCB its hydrophobic properties. These hydrogen bonds can be disrupted by configurational isomerization of bilirubin, which occurs on exposure to light. Unlike UCB, the photoisomers and photodegradation products behave as more polar molecules and are readily secreted into bile without conjugation.

In the liver cell, UCB is transported within the cell bound to a group of cytosolic proteins (Figure 4-2), preferentially to glutathione S-transferase B (ligandin or protein Y) and fatty acid–binding protein 1 (FABP1 or protein Z), which serve as an intracellular storage (sink) for UCB. In addition, almost half of the intracellular UCB may be membrane bound.19 In the ER, UCB is conjugated for efficient elimination as a water-soluble molecule into the bile.