Unconjugated Bilirubin Exhibits Spontaneous Diffusion through Model Lipid Bilayers and Native Hepatocyte Membranes

Abstract

The liver is responsible for the clearance and metabolism of unconjugated bilirubin, the hydrophobic endproduct of heme catabolism. Although several putative bilirubin transporters have been described, it has been alternatively proposed that bilirubin enters the hepatocyte by passive diffusion through the plasma membrane. In order to elucidate the mechanism of bilirubin uptake, we measured the rate of bilirubin transmembrane diffusion (flip-flop) using stopped-flow fluorescence techniques. Unconjugated bilirubin rapidly diffuses through model phosphatidylcholine vesicles, with a first-order rate constant of 5.3 s ؊1 (t1 ⁄2 ‫؍‬ 130 ms). The flip-flop rate is independent of membrane cholesterol content, phospholipid acyl saturation, and lipid packing, consistent with thermodynamic analyses demonstrating minimal steric constraint to bilirubin transmembrane diffusion. The coincident decrease in pH of the entrapped vesicle volume supports a mechanism whereby the bilirubin molecule crosses the lipid bilayer as the uncharged diacid. Transport of bilirubin by native rat hepatocyte membranes exhibits kinetics comparable with that in model vesicles, suggesting that unconjugated bilirubin crosses cellular membranes by passive diffusion through the hydrophobic lipid core. In contrast, there is no demonstrable flip-flop of bilirubin diglucuronide or bilirubin ditaurate in phospholipid vesicles, yet these compounds rapidly traverse isolated rat hepatocyte membranes, confirming the presence of a facilitated uptake system(s) for hydrophilic bilirubin conjugates.Unconjugated bilirubin is the principal degradation product of heme metabolism. Although the physiologic isomer, bilirubin IX␣, is a dicarboxylic acid, the molecule has minimal aqueous solubility at physiologic pH (1, 2) due to the formation of intramolecular hydrogen bonds (3). For this reason, bilirubin undergoes biotransformation in the liver to more polar conjugates prior to secretion in the bile. However, while unconjugated bilirubin is not hydrophilic, neither can it be characterized as lipophilic (1), since this compound is equally insoluble in apolar solvents (e.g. n-hexane, 1-pentanol). These unique physical-chemical properties have generated controversy regarding the mechanism of bilirubin uptake by the liver. Based on the spontaneous leakage from multilamellar liposomes (4), it has been proposed that bilirubin is able to diffuse through cellular membranes (5, 6). On the other hand, in vivo (7) and whole organ (8, 9) studies indicate that hepatic bilirubin uptake is saturable and occurs against a concentration gradient, findings that support a protein-mediated transport mechanism. To date, four putative bilirubin transporters have been identified in liver cells: BSP/bilirubin-binding protein, organic anion-transporting polypeptide, bilitranslocase, and organic anion-binding protein. Each of these proteins facilitates uptake of the hydrophilic organic anion, sulfobromophthalein (BSP), 1 a process that is competitively inhibited by bilirubin (10, 11).However, des...

Unconjugated bilirubin induces apoptosis in colon cancer cells by triggering mitochondrial depolarization

“…11, right panel). Moreover, addition of BDT, which we previously showed is incapable of spontaneous transmembrane diffusion, 35 did not cause the hyperpolarization phenomenon observed with bilirubin treatment. As bilirubin and BDT exhibit similar inner filter effects, these findings provide further support that the modulation of rhodamine fluorescence is due to bilirubin-specific induction of changes in mitochondrial potential.…”

“…We 35 and others 79 have shown that unconjugated bilirubin diffuses rapidly through model and native membrane vesicles and that this represents an important mechanism by which bilirubin is taken up into cells. 37 Once inside the cytoplasm, bilirubin likely associates with mitochondrial membranes, as well as other organelles, as a result of high-affinity binding to membrane lipids.…”

“…Furthermore, we employed an alternative method for bilirubin solubilization involving dissolution of bilirubin in 50 mM potassium phosphate (pH 12), as previously described. 35,36 Addition of bilirubin solubilized in this manner in an aliquot comprising Յ1% of the total volume did not alter the pH of the culture medium. 37 Using this alternative dissolution method, viability results were identical to those obtained using DMSO as the bilirubin vehicle.…”

“…60 Based on our previous work demonstrating that bilirubin is capable of transporting protons across model and biologic membranes, 35 we speculated that bilirubin might induce apoptosis by directly depolarizing cellular mitochondria and, hence, may not require activation of the PTP. To test this hypothesis, we examined the effect of bilirubin on the permeability of isolated rat liver mitochondria.…”

“…37 Once inside the cytoplasm, bilirubin likely associates with mitochondrial membranes, as well as other organelles, as a result of high-affinity binding to membrane lipids. 36,74,80 Because bilirubin traverses phospholipid bilayers as the uncharged diacid species, 35 the physiologic bilirubin dianion must acquire 2 protons prior to diffusion across the mitochondrial membrane. These protons are subsequently released from the bilirubin diacid at the opposing bilayer hemileaflet.…”