Hepatic Uptake of Bilirubin and Its Conjugates by the Human Organic Anion Transporter SLC21A6 (2001)

Bilirubin, the end product of heme catabolism, is taken up from the blood circulation into the liver. This work identifies a high-affinity transport protein mediating the uptake of bilirubin and its conjugates into human hepatocytes. Human embryonic kidney cells (HEK293) permanently expressing the recombinant organic anion-transporting polypeptide 2 (human OATP2, also known as LST-1 or OATP-C; symbol SLC21A6) showed uptake of [3H]monoglucuronosyl bilirubin, [3H]bisglucuronosyl bilirubin, and [3H]sulfobromophthalein withKm values of 0.10, 0.28, and 0.14 μm, respectively. High-affinity uptake of unconjugated [3H]bilirubin by OATP2 occurred in the presence of albumin and was not mediated by another basolateral hepatic uptake transporter, human OATP8 (symbol SLC21A8). OATP2 and OATP8 differed by their capacity to extract substrates from albumin before transport. In comparison to the high-affinity transport by OATP2, OATP8 transported [3H]sulfobromophthalein and [3H]monoglucuronosyl bilirubin with lower affinity, withKm values of 3.3 and 0.5 μm, respectively. The organic anion indocyanine green potently inhibited transport mediated by OATP2, with a Ki value of 112 nm, but did not inhibit transport mediated by OATP8. Human OATP2 may play a key role in the prevention of hyperbilirubinemia by facilitating the selective entry of unconjugated bilirubin and its glucuronate conjugates into human hepatocytes. Bilirubin, the end product of heme catabolism, is taken up from the blood circulation into the liver. This work identifies a high-affinity transport protein mediating the uptake of bilirubin and its conjugates into human hepatocytes. Human embryonic kidney cells (HEK293) permanently expressing the recombinant organic anion-transporting polypeptide 2 (human OATP2, also known as LST-1 or OATP-C; symbol SLC21A6) showed uptake of [3H]monoglucuronosyl bilirubin, [3H]bisglucuronosyl bilirubin, and [3H]sulfobromophthalein withKm values of 0.10, 0.28, and 0.14 μm, respectively. High-affinity uptake of unconjugated [3H]bilirubin by OATP2 occurred in the presence of albumin and was not mediated by another basolateral hepatic uptake transporter, human OATP8 (symbol SLC21A8). OATP2 and OATP8 differed by their capacity to extract substrates from albumin before transport. In comparison to the high-affinity transport by OATP2, OATP8 transported [3H]sulfobromophthalein and [3H]monoglucuronosyl bilirubin with lower affinity, withKm values of 3.3 and 0.5 μm, respectively. The organic anion indocyanine green potently inhibited transport mediated by OATP2, with a Ki value of 112 nm, but did not inhibit transport mediated by OATP8. Human OATP2 may play a key role in the prevention of hyperbilirubinemia by facilitating the selective entry of unconjugated bilirubin and its glucuronate conjugates into human hepatocytes. sulfobromophthalein 17%-glucuronosyl estradiol human serum albumin indocyanine green organic anion-transporting polypeptide high pressure liquid chromatography Bilirubin, the main bile pigment in most mammals, is the end product of heme catabolism (1Chowdhury J.R. Chowdhury N.R. Wolkoff A.W. Arias I.M. Anas I.M. The Liver: Biology and Pathobiology. 3rd Ed. Raven Press, New York1994: 471-504Google Scholar). In the blood circulation, bilirubin is bound to serum albumin, which prevents its potential toxicity thought to be caused by the free ligand (2Brodersen R. Stern L. Acta Paediatr. Scand. 1990; 79: 12-19Crossref PubMed Scopus (64) Google Scholar). Despite high-affinity binding to albumin, bilirubin is rapidly and selectively taken up into the liver (3Scharschmidt B.F. Waggoner J.G. Berk P.D. J. Clin. Invest. 1975; 56: 1280-1292Crossref PubMed Scopus (202) Google Scholar, 4Arias I.M. Johnson L. Wolfson S. Am. J. Physiol. 1961; 200: 1091-1094Crossref PubMed Scopus (82) Google Scholar), biotransformed upon conjugation with glucuronate (5Senafi S.B. Clarke D.J. Burchell B. Biochem. J. 1994; 303: 233-240Crossref PubMed Scopus (228) Google Scholar), and secreted into bile across the canalicular membrane of hepatocytes by an ATP-dependent conjugate export pump termed multidrug resistance protein 2 (transporter symbol ABCC2) (6Kamisako T. Leier I. Cui Y. König J. Buchholz U. Hummel-Eisenbeiss J. Keppler D. Hepatology. 1999; 30: 485-490Crossref PubMed Scopus (165) Google Scholar, 7Jedlitschky G. Leier I. Buchholz U. Hummel-Eisenbeiss J. Burchell B. Keppler D. Biochem. J. 1997; 327: 305-310Crossref PubMed Scopus (257) Google Scholar). In addition to a reduction of UDP-glucuronosyl transferase activity (8Black M. Billing B.H. N. Engl. J. Med. 1969; 280: 1266-1271Crossref PubMed Scopus (233) Google Scholar), impaired bilirubin uptake from the blood circulation into liver has been suggested to contribute to a subgroup of patients with Gilbert's syndrome (9Martin J.F. Vierling J.M. Wolkoff A.W. Scharschmidt B.F. Vergalla J. Waggoner J.G. Berk P.D. Gastroenterology. 1976; 70: 385-391Abstract Full Text PDF PubMed Scopus (81) Google Scholar), which is characterized by a mild unconjugated hyperbilirubinemia. Uptake of bilirubin by hepatocytes was considered to be a process mediated by specific membrane proteins, although passive diffusion has also been proposed as a possible mechanism (1Chowdhury J.R. Chowdhury N.R. Wolkoff A.W. Arias I.M. Anas I.M. The Liver: Biology and Pathobiology. 3rd Ed. Raven Press, New York1994: 471-504Google Scholar, 3Scharschmidt B.F. Waggoner J.G. Berk P.D. J. Clin. Invest. 1975; 56: 1280-1292Crossref PubMed Scopus (202) Google Scholar,10Mediavilla M.G. Pascolo L. Rodriguez J.V. Guibert E.E. Ostrow J.D. Tiribelli C. FEBS Lett. 1999; 463: 143-145Crossref PubMed Scopus (21) Google Scholar). Because of its instability and low solubility in aqueous solution, hepatic uptake of bilirubin was studied predominantly by use of structurally related anionic substances like sulfobromophthalein (BSP)1 and indocyanine green (ICG) (3Scharschmidt B.F. Waggoner J.G. Berk P.D. J. Clin. Invest. 1975; 56: 1280-1292Crossref PubMed Scopus (202) Google Scholar, 9Martin J.F. Vierling J.M. Wolkoff A.W. Scharschmidt B.F. Vergalla J. Waggoner J.G. Berk P.D. Gastroenterology. 1976; 70: 385-391Abstract Full Text PDF PubMed Scopus (81) Google Scholar, 11Okuda K. Ohkubo H. Musha H. Kotoda K. Abe H. Tanikawa K. Gut. 1976; 17: 588-594Crossref PubMed Scopus (24) Google Scholar, 12Pascolo L. Del Vecchio S. Koehler R.K. Bayon J.E. Webster C.C. Mukerjee P. Ostrow J.D. Tiribelli C. Biochem. J. 1996; 316: 999-1004Crossref PubMed Scopus (24) Google Scholar). A transport protein for BSP with a Michaelis-Menten constant (Km) of 1.5 μm has been cloned from rat liver (13Jacquemin E. Hagenbuch B. Stieger B. Wolkoff A.W. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 133-137Crossref PubMed Scopus (544) Google Scholar) and designated as organic anion-transporting polypeptide 1 (rat OATP1). Rat OATP1 belongs to a family of transport proteins (OATP family, symbol SLC21A) mediating the transport of organic anions including bile salts, steroid conjugates, thyroid hormones, prostaglandins, and BSP (14Meier P.J. Eckhardt U. Schroeder A. Hagenbuch B. Stieger B. Hepatology. 1997; 26: 1667-1677Crossref PubMed Scopus (322) Google Scholar). For human OATP1 (SLC21A3), which is expressed at high levels in brain and kidney and at a low level in human liver, kinetic studies revealed only a moderate affinity for BSP with a Km value of 20 μm (15Kullak-Ublick G.A. Hagenbuch B. Stieger B. Schteingart C.D. Hofmann A.F. Wolkoff A.W. Meier P.J. Gastroenterology. 1995; 109: 1274-1282Abstract Full Text PDF PubMed Scopus (368) Google Scholar). In a search for additional OATP isoforms in human liver, we and other groups have recently cloned a new member of this transporter family, human OATP2 (also known as LST1 or OATP-C, gene symbol SLC21A6) (16König J. Cui Y. Nies A.T. Keppler D. Am. J. Physiol. 2000; 278: G156-G164Crossref PubMed Google Scholar, 17Hsiang B. Zhu Y. Wang Z. Wu Y. Sasseville V. Yang W.P. Kirchgessner T.G. J. Biol. Chem. 1999; 274: 37161-37168Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar, 18Abe T. Kakyo M. Tokui T. Nakagomi R. Nishio T. Nakai D. Nomura H. Unno M. Suzuki M. Naitoh T. Matsuno S. Yawo H. J. Biol. Chem. 1999; 274: 17159-17163Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar). Most recently, we cloned an additional human liver OATP isoform termed OATP8 (gene symbol SLC21A8), which shares 80% identical amino acids with human OATP2 (19König J. Cui Y. Nies A.T. Keppler D. J. Biol. Chem. 2000; 275: 23161-23168Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). Antibodies raised against both transport proteins localized them to the basolateral membrane of human hepatocytes (16König J. Cui Y. Nies A.T. Keppler D. Am. J. Physiol. 2000; 278: G156-G164Crossref PubMed Google Scholar, 19König J. Cui Y. Nies A.T. Keppler D. J. Biol. Chem. 2000; 275: 23161-23168Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). Northern blot analyses demonstrated an apparently exclusive hepatic expression of both transporters (16König J. Cui Y. Nies A.T. Keppler D. Am. J. Physiol. 2000; 278: G156-G164Crossref PubMed Google Scholar, 19König J. Cui Y. Nies A.T. Keppler D. J. Biol. Chem. 2000; 275: 23161-23168Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). The availability of cell lines stably expressing human OATP2 and OATP8 enabled us to answer the question of whether these two major human hepatic OATP family members are capable of transporting bilirubin and its conjugates from blood across the basolateral membrane into hepatocytes.DISCUSSIONWe conclude that uptake of bilirubin into human hepatocytes, the first step of its detoxification, is mediated by OATP2, a major transport protein localized to the basolateral membrane of hepatocytes, but not by the isoform OATP8 localized to the same membrane domain. Our conclusion is based on the following experimental data: (a) the structurally and chemically related lipophilic anionic compounds BSP, monoglucuronosyl bilirubin, and bisglucuronosyl bilirubin were high-affinity substrates for OATP2, with nanomolarKm values, whereas OATP8 transported BSP and monoglucuronosyl bilirubin with markedly lower affinity (Fig. 1 and Table I); (b) OATP2, but not OATP8, was able to extract substrates from albumin (Fig. 2) to which bilirubin binds with high affinity; (c) ICG inhibited OATP2 at nanomolar concentrations but exerted no inhibitory effect on OATP8 at concentrations up to 10 μm (Fig. 3); and (d) [3H]bilirubin uptake by OATP2 was directly demonstrated by uptake studies with OATP2-expressing HEK transfectants (Fig. 4). Together with previous data, we propose the following scheme for the detoxification and elimination pathway of bilirubin in human liver (Fig. 5): bilirubin (B) bound to albumin is taken up across the basolateral membrane by OATP2 and conjugated in the hepatocyte by the UDP-glucuronosyl transferase 1A (UGT1A1), resulting in monoglucuronosyl bilirubin and bisglucuronosyl bilirubin. Bilirubin glucuronides are finally excreted into bile by the apical conjugate export pump multidrug resistance protein 2 localized to the hepatocyte canalicular (apical) membrane (6Kamisako T. Leier I. Cui Y. König J. Buchholz U. Hummel-Eisenbeiss J. Keppler D. Hepatology. 1999; 30: 485-490Crossref PubMed Scopus (165) Google Scholar,7Jedlitschky G. Leier I. Buchholz U. Hummel-Eisenbeiss J. Burchell B. Keppler D. Biochem. J. 1997; 327: 305-310Crossref PubMed Scopus (257) Google Scholar).Our results here establish a carrier-mediated uptake of bilirubin into hepatocytes. However, we do not exclude additional bilirubin uptake through passive diffusion. The differentiation between carrier-mediated and diffusional bilirubin uptake into the liver will be supported by the identification of mutations in the OATP2 (SLC21A6) gene leading to the loss or functional impairment of OATP2 in the basolateral membrane of hepatocytes. Moreover, in view of the fact that current knowledge of the human OATP family is not complete, additional transport proteins may further contribute to the selective uptake of bilirubin from the blood circulation into liver. Bilirubin, the main bile pigment in most mammals, is the end product of heme catabolism (1Chowdhury J.R. Chowdhury N.R. Wolkoff A.W. Arias I.M. Anas I.M. The Liver: Biology and Pathobiology. 3rd Ed. Raven Press, New York1994: 471-504Google Scholar). In the blood circulation, bilirubin is bound to serum albumin, which prevents its potential toxicity thought to be caused by the free ligand (2Brodersen R. Stern L. Acta Paediatr. Scand. 1990; 79: 12-19Crossref PubMed Scopus (64) Google Scholar). Despite high-affinity binding to albumin, bilirubin is rapidly and selectively taken up into the liver (3Scharschmidt B.F. Waggoner J.G. Berk P.D. J. Clin. Invest. 1975; 56: 1280-1292Crossref PubMed Scopus (202) Google Scholar, 4Arias I.M. Johnson L. Wolfson S. Am. J. Physiol. 1961; 200: 1091-1094Crossref PubMed Scopus (82) Google Scholar), biotransformed upon conjugation with glucuronate (5Senafi S.B. Clarke D.J. Burchell B. Biochem. J. 1994; 303: 233-240Crossref PubMed Scopus (228) Google Scholar), and secreted into bile across the canalicular membrane of hepatocytes by an ATP-dependent conjugate export pump termed multidrug resistance protein 2 (transporter symbol ABCC2) (6Kamisako T. Leier I. Cui Y. König J. Buchholz U. Hummel-Eisenbeiss J. Keppler D. Hepatology. 1999; 30: 485-490Crossref PubMed Scopus (165) Google Scholar, 7Jedlitschky G. Leier I. Buchholz U. Hummel-Eisenbeiss J. Burchell B. Keppler D. Biochem. J. 1997; 327: 305-310Crossref PubMed Scopus (257) Google Scholar). In addition to a reduction of UDP-glucuronosyl transferase activity (8Black M. Billing B.H. N. Engl. J. Med. 1969; 280: 1266-1271Crossref PubMed Scopus (233) Google Scholar), impaired bilirubin uptake from the blood circulation into liver has been suggested to contribute to a subgroup of patients with Gilbert's syndrome (9Martin J.F. Vierling J.M. Wolkoff A.W. Scharschmidt B.F. Vergalla J. Waggoner J.G. Berk P.D. Gastroenterology. 1976; 70: 385-391Abstract Full Text PDF PubMed Scopus (81) Google Scholar), which is characterized by a mild unconjugated hyperbilirubinemia. Uptake of bilirubin by hepatocytes was considered to be a process mediated by specific membrane proteins, although passive diffusion has also been proposed as a possible mechanism (1Chowdhury J.R. Chowdhury N.R. Wolkoff A.W. Arias I.M. Anas I.M. The Liver: Biology and Pathobiology. 3rd Ed. Raven Press, New York1994: 471-504Google Scholar, 3Scharschmidt B.F. Waggoner J.G. Berk P.D. J. Clin. Invest. 1975; 56: 1280-1292Crossref PubMed Scopus (202) Google Scholar,10Mediavilla M.G. Pascolo L. Rodriguez J.V. Guibert E.E. Ostrow J.D. Tiribelli C. FEBS Lett. 1999; 463: 143-145Crossref PubMed Scopus (21) Google Scholar). Because of its instability and low solubility in aqueous solution, hepatic uptake of bilirubin was studied predominantly by use of structurally related anionic substances like sulfobromophthalein (BSP)1 and indocyanine green (ICG) (3Scharschmidt B.F. Waggoner J.G. Berk P.D. J. Clin. Invest. 1975; 56: 1280-1292Crossref PubMed Scopus (202) Google Scholar, 9Martin J.F. Vierling J.M. Wolkoff A.W. Scharschmidt B.F. Vergalla J. Waggoner J.G. Berk P.D. Gastroenterology. 1976; 70: 385-391Abstract Full Text PDF PubMed Scopus (81) Google Scholar, 11Okuda K. Ohkubo H. Musha H. Kotoda K. Abe H. Tanikawa K. Gut. 1976; 17: 588-594Crossref PubMed Scopus (24) Google Scholar, 12Pascolo L. Del Vecchio S. Koehler R.K. Bayon J.E. Webster C.C. Mukerjee P. Ostrow J.D. Tiribelli C. Biochem. J. 1996; 316: 999-1004Crossref PubMed Scopus (24) Google Scholar). A transport protein for BSP with a Michaelis-Menten constant (Km) of 1.5 μm has been cloned from rat liver (13Jacquemin E. Hagenbuch B. Stieger B. Wolkoff A.W. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 133-137Crossref PubMed Scopus (544) Google Scholar) and designated as organic anion-transporting polypeptide 1 (rat OATP1). Rat OATP1 belongs to a family of transport proteins (OATP family, symbol SLC21A) mediating the transport of organic anions including bile salts, steroid conjugates, thyroid hormones, prostaglandins, and BSP (14Meier P.J. Eckhardt U. Schroeder A. Hagenbuch B. Stieger B. Hepatology. 1997; 26: 1667-1677Crossref PubMed Scopus (322) Google Scholar). For human OATP1 (SLC21A3), which is expressed at high levels in brain and kidney and at a low level in human liver, kinetic studies revealed only a moderate affinity for BSP with a Km value of 20 μm (15Kullak-Ublick G.A. Hagenbuch B. Stieger B. Schteingart C.D. Hofmann A.F. Wolkoff A.W. Meier P.J. Gastroenterology. 1995; 109: 1274-1282Abstract Full Text PDF PubMed Scopus (368) Google Scholar). In a search for additional OATP isoforms in human liver, we and other groups have recently cloned a new member of this transporter family, human OATP2 (also known as LST1 or OATP-C, gene symbol SLC21A6) (16König J. Cui Y. Nies A.T. Keppler D. Am. J. Physiol. 2000; 278: G156-G164Crossref PubMed Google Scholar, 17Hsiang B. Zhu Y. Wang Z. Wu Y. Sasseville V. Yang W.P. Kirchgessner T.G. J. Biol. Chem. 1999; 274: 37161-37168Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar, 18Abe T. Kakyo M. Tokui T. Nakagomi R. Nishio T. Nakai D. Nomura H. Unno M. Suzuki M. Naitoh T. Matsuno S. Yawo H. J. Biol. Chem. 1999; 274: 17159-17163Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar). Most recently, we cloned an additional human liver OATP isoform termed OATP8 (gene symbol SLC21A8), which shares 80% identical amino acids with human OATP2 (19König J. Cui Y. Nies A.T. Keppler D. J. Biol. Chem. 2000; 275: 23161-23168Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). Antibodies raised against both transport proteins localized them to the basolateral membrane of human hepatocytes (16König J. Cui Y. Nies A.T. Keppler D. Am. J. Physiol. 2000; 278: G156-G164Crossref PubMed Google Scholar, 19König J. Cui Y. Nies A.T. Keppler D. J. Biol. Chem. 2000; 275: 23161-23168Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). Northern blot analyses demonstrated an apparently exclusive hepatic expression of both transporters (16König J. Cui Y. Nies A.T. Keppler D. Am. J. Physiol. 2000; 278: G156-G164Crossref PubMed Google Scholar, 19König J. Cui Y. Nies A.T. Keppler D. J. Biol. Chem. 2000; 275: 23161-23168Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). The availability of cell lines stably expressing human OATP2 and OATP8 enabled us to answer the question of whether these two major human hepatic OATP family members are capable of transporting bilirubin and its conjugates from blood across the basolateral membrane into hepatocytes. DISCUSSIONWe conclude that uptake of bilirubin into human hepatocytes, the first step of its detoxification, is mediated by OATP2, a major transport protein localized to the basolateral membrane of hepatocytes, but not by the isoform OATP8 localized to the same membrane domain. Our conclusion is based on the following experimental data: (a) the structurally and chemically related lipophilic anionic compounds BSP, monoglucuronosyl bilirubin, and bisglucuronosyl bilirubin were high-affinity substrates for OATP2, with nanomolarKm values, whereas OATP8 transported BSP and monoglucuronosyl bilirubin with markedly lower affinity (Fig. 1 and Table I); (b) OATP2, but not OATP8, was able to extract substrates from albumin (Fig. 2) to which bilirubin binds with high affinity; (c) ICG inhibited OATP2 at nanomolar concentrations but exerted no inhibitory effect on OATP8 at concentrations up to 10 μm (Fig. 3); and (d) [3H]bilirubin uptake by OATP2 was directly demonstrated by uptake studies with OATP2-expressing HEK transfectants (Fig. 4). Together with previous data, we propose the following scheme for the detoxification and elimination pathway of bilirubin in human liver (Fig. 5): bilirubin (B) bound to albumin is taken up across the basolateral membrane by OATP2 and conjugated in the hepatocyte by the UDP-glucuronosyl transferase 1A (UGT1A1), resulting in monoglucuronosyl bilirubin and bisglucuronosyl bilirubin. Bilirubin glucuronides are finally excreted into bile by the apical conjugate export pump multidrug resistance protein 2 localized to the hepatocyte canalicular (apical) membrane (6Kamisako T. Leier I. Cui Y. König J. Buchholz U. Hummel-Eisenbeiss J. Keppler D. Hepatology. 1999; 30: 485-490Crossref PubMed Scopus (165) Google Scholar,7Jedlitschky G. Leier I. Buchholz U. Hummel-Eisenbeiss J. Burchell B. Keppler D. Biochem. J. 1997; 327: 305-310Crossref PubMed Scopus (257) Google Scholar).Our results here establish a carrier-mediated uptake of bilirubin into hepatocytes. However, we do not exclude additional bilirubin uptake through passive diffusion. The differentiation between carrier-mediated and diffusional bilirubin uptake into the liver will be supported by the identification of mutations in the OATP2 (SLC21A6) gene leading to the loss or functional impairment of OATP2 in the basolateral membrane of hepatocytes. Moreover, in view of the fact that current knowledge of the human OATP family is not complete, additional transport proteins may further contribute to the selective uptake of bilirubin from the blood circulation into liver. We conclude that uptake of bilirubin into human hepatocytes, the first step of its detoxification, is mediated by OATP2, a major transport protein localized to the basolateral membrane of hepatocytes, but not by the isoform OATP8 localized to the same membrane domain. Our conclusion is based on the following experimental data: (a) the structurally and chemically related lipophilic anionic compounds BSP, monoglucuronosyl bilirubin, and bisglucuronosyl bilirubin were high-affinity substrates for OATP2, with nanomolarKm values, whereas OATP8 transported BSP and monoglucuronosyl bilirubin with markedly lower affinity (Fig. 1 and Table I); (b) OATP2, but not OATP8, was able to extract substrates from albumin (Fig. 2) to which bilirubin binds with high affinity; (c) ICG inhibited OATP2 at nanomolar concentrations but exerted no inhibitory effect on OATP8 at concentrations up to 10 μm (Fig. 3); and (d) [3H]bilirubin uptake by OATP2 was directly demonstrated by uptake studies with OATP2-expressing HEK transfectants (Fig. 4). Together with previous data, we propose the following scheme for the detoxification and elimination pathway of bilirubin in human liver (Fig. 5): bilirubin (B) bound to albumin is taken up across the basolateral membrane by OATP2 and conjugated in the hepatocyte by the UDP-glucuronosyl transferase 1A (UGT1A1), resulting in monoglucuronosyl bilirubin and bisglucuronosyl bilirubin. Bilirubin glucuronides are finally excreted into bile by the apical conjugate export pump multidrug resistance protein 2 localized to the hepatocyte canalicular (apical) membrane (6Kamisako T. Leier I. Cui Y. König J. Buchholz U. Hummel-Eisenbeiss J. Keppler D. Hepatology. 1999; 30: 485-490Crossref PubMed Scopus (165) Google Scholar,7Jedlitschky G. Leier I. Buchholz U. Hummel-Eisenbeiss J. Burchell B. Keppler D. Biochem. J. 1997; 327: 305-310Crossref PubMed Scopus (257) Google Scholar). Our results here establish a carrier-mediated uptake of bilirubin into hepatocytes. However, we do not exclude additional bilirubin uptake through passive diffusion. The differentiation between carrier-mediated and diffusional bilirubin uptake into the liver will be supported by the identification of mutations in the OATP2 (SLC21A6) gene leading to the loss or functional impairment of OATP2 in the basolateral membrane of hepatocytes. Moreover, in view of the fact that current knowledge of the human OATP family is not complete, additional transport proteins may further contribute to the selective uptake of bilirubin from the blood circulation into liver. We thank W. D. Lehmann (Deutsches Krebsforschungszentrum, Spectroscopy, Heidelberg, Germany) for analysis of the labeled and unlabeled BSP by nanoelectrospray mass spectrometry, J. M. Crawford (University of Florida, Department of Pathology, Gainesville, FL) and A. F. McDonagh (University of California, Division of Gastroenterology, San Francisco, CA) for advice on the preparation of [3H]bilirubin, K. Bode and M. Donner (Deutsches Krebsforschungszentrum, Division of Tumor Biochemistry, Heidelberg, Germany) for help during the biosynthesis of [3H]bilirubin, and G. Jedlitschky (Deutsches Krebsforschungszentrum, Division of Tumor Biochemistry, Heidelberg, Germany) for critical reading of the manuscript.