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Hepatobiliary Function
Overview of Liver Physiology
After the skin, the liver and the brain are the largest organs in the human body. The liver weighs between 1200 and 1500 g, representing 2% to 5% of body weight in the adult and ~4% to 5% in the newborn. The liver is strategically situated in the circulatory system to receive the portal blood that drains the stomach, small intestine, large intestine, pancreas, and spleen (see Fig. 24-7 A ). In this position, the liver plays a key role in handling foodstuffs assimilated by the small intestine. However, the liver's role is far more diverse; it serves as a chemical factory, an excretory system, an exocrine gland, and an endocrine gland.
The liver biotransforms and degrades substances taken up from blood and either returns them to the circulation or excretes them into bile
A major function of the liver is to metabolize, detoxify, and inactivate both endogenous compounds (e.g., steroids and other hormones) and exogenous substances (e.g., drugs and toxins). In addition, by virtue of its large vascular capacity and abundance of phagocytes (Kupffer cells), the liver provides an important filtering mechanism for the circulation by removing foreign particulate matter, including bacteria, endotoxins, parasites, and aging red blood cells. Kupffer cells constitute 80% to 90% of the fixed macrophages of the reticuloendothelial system.
The liver has the capacity to convert important hormones and vitamins into a more active form. Examples include the initial hydroxylation of vitamin D and the deiodination of the thyroid hormone thyroxine (T 4 ) to triiodothyronine (T 3 ). Moreover, numerous enzymes in the liver process lipophilic chemicals into more polar, water-soluble metabolites, which are more readily excreted into bile.
Bile is a complex secretory product produced by the liver. Biliary secretion has two principal functions: (1) elimination from the body of many endogenous and exogenous waste products, such as bilirubin and cholesterol; and (2) promotion of digestion and absorption of lipids from the intestine. The composition of bile is modified significantly as a result of the absorptive and secretory properties of epithelial cells that line the intrahepatic and extrahepatic bile ducts. Moreover, bile solutes are further concentrated as bile is stored in the gallbladder.
The liver stores carbohydrates, lipids, vitamins, and minerals; it synthesizes carbohydrates, protein, and intermediary metabolites
The products of digested food, including carbohydrates, peptides, vitamins, and some lipids, are avidly extracted from portal blood by the liver. Depending on the metabolic requirements of the body, these substrates may be stored by the hepatocytes or released into the bloodstream either unbound (e.g., glucose) or associated with a carrier molecule (e.g., a triacylglycerol molecule complexed to a lipoprotein).
The liver also synthesizes—in a highly regulated fashion—many substances that are essential to the metabolic demands of the body. These substances include albumin, coagulation factors, and other plasma proteins; glucose; cholesterol; fatty acids for triacylglycerol biosynthesis; and phospholipids. The liver must provide a supply of substrates as fuels for other organs, particularly in the fasted state. For example, the liver produces ketone bodies, which can be used by the central nervous system during periods of fasting; this use of ketone bodies as fuel spares ~50% of the amount of glucose that would otherwise be used by this tissue. Thus, the liver has a critical and unique role in the energy metabolism of all nonhepatic organs.
Functional Anatomy of the Liver and Biliary Tree
Hepatocytes are secretory epithelial cells separating the lumen of bile canaliculi from the fenestrated endothelium of sinusoids
One way of looking at the organization of the liver is to imagine that a classic lobule is a hexagon in cross section ( Fig. 46-1 A ) with a branch of the hepatic vein at its center and, at each of the six corners, triads composed of branches of the hepatic artery, portal vein, and bile duct. Hepatocytes account for ~80% of the parenchymal volume in human liver. Hepatocytes form an epithelium, one cell thick, that constitutes a functional barrier between two fluid compartments with differing ionic compositions: the tiny canalicular lumen containing bile, and the much larger sinusoid containing blood (see Fig. 46-1 B ). Moreover, hepatocytes significantly alter the composition of these fluids by vectorial transport of solutes across the hepatocyte. This vectorial transport depends critically on the polarized distribution of specific transport mechanisms and receptors that are localized to the apical membrane that faces the canalicular lumen and the basolateral membrane that faces the pericellular space between hepatocytes and the blood-filled sinusoid (see Fig. 46-1 B, C ). As in other epithelia, the apical and basolateral membrane domains of hepatocytes are structurally, biochemically, and physiologically distinct.
The space of Disse, or perisinusoidal space, is the extracellular gap between the endothelial cells lining the sinusoids and the basolateral membranes of the hepatocytes. These basolateral membranes have microvilli that project into the space of Disse to facilitate contact with the solutes in sinusoidal blood. The microvilli greatly amplify the surface of the basolateral membrane, which accounts for ~85% of the total surface area of the hepatocyte.
The bile canaliculi, into which bile is initially secreted, are formed by the apical membranes of adjoining hepatocytes. The apical membrane of the hepatocyte runs as a narrow belt that encircles and grooves into the polygonal hepatocyte (see Fig. 46-1 B, C ). Two adjacent hepatocytes form a canaliculus that is ~1 µm in diameter by juxtaposing their groove-like apical membranes along their common face (i.e., one side of the polygon). Because a hepatocyte has many sides and a different neighbor on each side, the canaliculi form a chicken wire–like pattern along the contiguous surfaces of hepatocytes and communicate to form a three-dimensional tubular network. Although the apical membrane belt is very narrow (i.e., ~1 µm), its extensive microvillous structure amplifies its surface area so that the canalicular membrane constitutes as much as 15% of the total membrane surface area. Because of this high surface-to-volume ratio, the total apical surface area available for the movement of water and solutes in the human liver is in excess of 10.5 m 2 .
The seal that joins the apical membranes of two juxtaposed hepatocytes and that separates the canalicular lumen from the pericellular space—which is contiguous with the space of Disse—comprises several elements, including tight junctions (see Fig. 46-1 D ) and desmosomes (see p. 45 ). By virtue of their permeability and morphology, hepatic junctions can be classified as having an intermediate tightness, somewhere between that of tight epithelia (e.g., toad bladder) and leaky epithelia (e.g., proximal tubule). Specialized structures called gap junctions (see pp. 158–159 ) allow functional communication between adjacent hepatocytes.
Hepatocytes do not have a true basement membrane, but rather they rest on complex scaffolding provided by the extracellular matrix in the space of Disse, which includes several types of collagens (I, III, IV, V, and VI), fibronectin, undulin, laminin, and proteoglycans. Cells are linked to the matrix through specific adhesion proteins on the cell surface. The extracellular matrix not only provides structural support for liver cells but also seems to influence and maintain the phenotypic expression of hepatocytes and sinusoidal lining cells.
The liver contains endothelial cells, macrophages (Kupffer cells), and stellate cells (Ito cells) within the sinusoidal spaces
Slightly more than 6% of the volume of the liver parenchyma is made up of cells other than hepatocytes, including endothelial cells (2.8%), Kupffer cells (2.1%), and stellate cells (fat-storing or Ito cells, 1.4%). The endothelial cells that line the vascular channels or sinusoids form a fenestrated structure with their bodies and cytoplasmic extensions. Plasma solutes, but not blood cells, can move freely into the space of Disse through pores, or fenestrae, in the endothelial cells. Some evidence indicates that the fenestrae may regulate access into the perisinusoidal space of Disse by means of their capacity to contract.
The Kupffer cells are present within the sinusoidal vascular space. 
N46-1 This population of fixed macrophages removes particulate matter from the circulation. Stellate cells are in the space of Disse and are characterized morphologically by the presence of large fat droplets in their cytoplasm. These cells play a central role in the storage of vitamin A, and evidence suggests that they can be transformed into proliferative, fibrogenic, and contractile myofibroblasts. On liver injury, these activated cells participate in fibrogenesis through remodeling of the extracellular matrix, production of cytokines, and deposition of type I collagen, which can lead to cirrhosis.
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Kupffer Cells
Kupffer cells—or Browicz-Kupffer cells —are part of the reticuloendothelial system. They were originally described in 1876 by Karl Wilhelm von Kupffer, who incorrectly thought that they were part of the endothelium of the liver. In 1898, Tadeusz Browicz correctly identified them as macrophages.
The liver has a dual blood supply, but a single venous drainage system
The blood supply to the liver has two sources. The portal vein contributes ~75% of the total circulation to the liver; the hepatic artery contributes the other 25% ( Fig. 46-2 A ). Blood from portal venules and hepatic arterioles combines in a complex network of hepatic sinusoids (see Fig. 46-2 B ). Blood from these sinusoids converges on terminal hepatic venules (or central veins ), which, in turn, join to form the hepatic veins (see Fig. 46-2 C ). Branches of the portal vein, hepatic artery, and a bile duct (i.e., the triad), as well as lymphatics and nerves, travel together as a portal tract.
The arterial supply for the bile ducts arises mainly from the right hepatic artery (see Fig. 46-2 C ). These arterioles give rise to an extraordinarily rich plexus of capillaries that surround the bile ducts as they pass through the portal tracts. Blood flowing through this peribiliary plexus empties into the sinusoids by way of branches of the portal vein so that this blood may pick up solutes from the bile ducts and cycle them back to the hepatocytes. Thus, the peribiliary plexus may provide the means for modifying biliary secretions through the bidirectional exchange of compounds such as proteins, inorganic ions, and bile acids between the bile and blood within the portal tract.
Hepatocytes can be thought of as being arranged as classic hepatic lobules, portal lobules, or acinar units
The complex structure of the liver makes it difficult to define a single unit—something analogous to the nephron in the kidney—that is capable of performing the functions of the entire liver. One way of viewing the organization of the liver is depicted in Figures 46-1 and 46-2 , in which we regard the central vein as the core of the classic hepatic lobule. Thus, the classic hepatic lobule ( Fig. 46-3 A ) includes all hepatocytes drained by a single central vein, and it is bounded by two or more portal triads. Alternatively, we can view the liver as though the triad is the core of a portal lobule (see Fig. 46-3 B ). Thus, the portal lobule includes all hepatocytes drained by a single bile ductule and is bounded by two or more central veins. A third way of viewing the liver is to group the hepatocytes according to their supply of arterial blood (see Fig. 46-3 C ). Thus, the portal acinus is a small three-dimensional mass of hepatocytes that are irregular in size and shape, with one axis formed by a line between two triads (i.e., high 
) and another axis formed by a line between two central veins (i.e., low 
).
Periportal hepatocytes specialize in oxidative metabolism, whereas pericentral hepatocytes detoxify drugs
Rappaport first proposed that a zonal relationship exists between cells that constitute the portal acini and their blood supply (see Fig. 46-3 C ). Hepatocytes close to the vascular core formed by the terminal portal venule and terminal hepatic arteriole are perfused first and thus receive the highest concentrations of oxygen and solutes. These periportal hepatocytes are said to reside in zone I, and as a consequence of their location, they are the most resistant to the effects of circulatory compromise or nutritional deficiency. These cells are also more resistant to other forms of cellular injury and are the first to regenerate. Hepatocytes in the intermediate zone II and the most distal population of pericentral hepatocytes located near the terminal hepatic venule (central vein) in zone III are sequentially perfused with blood that is already modified by the preceding hepatocytes; thus, they are exposed to progressively lower concentrations of nutrients and oxygen. The exact boundaries of these zones are difficult to define.
The concept of zonal heterogeneity of liver function has evolved as a result of these differences in access to substrate. Because of the specialized microenvironments of cells in different zones, some enzymes are preferentially expressed in one zone or another ( Table 46-1 ). For example, in zone I, oxidative energy metabolism with β-oxidation, amino-acid metabolism, ureagenesis, gluconeogenesis, cholesterol synthesis, and bile formation is particularly important. Localized in zone III are glycogen synthesis from glucose, glycolysis, liponeogenesis, ketogenesis, xenobiotic metabolism, and glutamine formation. Molecular techniques have allowed an even more precise definition of which hepatocytes express particular messenger RNA (mRNA) and proteins. For example, the enzyme glutamine synthetase is expressed exclusively in only one or two hepatocytes immediately adjacent to the hepatic venules. Hepatocytes of zone III also seem to be important for general detoxification mechanisms and the biotransformation of drugs. The zonal distribution of drug-induced toxicity manifested as cell necrosis may be attributed to zone III localization of the enzymatic pathways involved in the biotransformation of substrates by oxidation, reduction, or hydrolysis. Although it appears that each hepatocyte is potentially capable of multiple metabolic functions, the predominant enzymatic activity appears to result from adaptation to the microenvironment provided by the hepatic microcirculation. In some cases, it has been possible to reverse the zone I–to–zone III gradient of hepatocyte function by experimentally reversing the direction of blood supply (i.e., nutrient flow).
TABLE 46-1
Zonal Heterogeneity of Preferential Hepatocyte Function
| ZONE I | ZONE III |
|---|---|
| Amino-acid catabolism | Glycolysis |
| Gluconeogenesis | Glycogen synthesis from glucose |
| Glycogen degradation | Liponeogenesis |
| Cholesterol synthesis (HMG-CoA reductase) | Bile acid biosynthesis (Cholesterol 7α-hydroxylase) |
| Ureagenesis (all hepatocytes with the exception of the last one or two rows encircling the hepatic venules) | Ketogenesis |
| Bile acid–dependent canalicular bile flow | Glutamine synthesis |
| Oxidative energy metabolism and probably β-oxidation of fatty acids | Bile acid–independent canalicular bile flow |
| Biotransformation of drugs |
Bile drains from canaliculi into small terminal ductules, then into larger ducts, and eventually, via a single common duct, into the duodenum
The adult human liver has >2 km of bile ductules and ducts, with a volume of ~20 cm 3 and a macroscopic surface area of ~400 cm 2 . Microvilli at the apical surface magnify this area by ~5.5-fold.
As noted above, the canaliculi into which bile is secreted form a three-dimensional polygonal meshwork of tubes between hepatocytes, with many anastomotic interconnections (see Fig. 46-1 ). From the canaliculi, the bile enters the small terminal bile ductules (i.e., canals of Hering ), which have a basement membrane and in cross section are surrounded by three to six ductal epithelial cells or hepatocytes ( Fig. 46-4 A ). The canals of Hering then empty into a system of perilobular ducts, which, in turn, drain into interlobular bile ducts. The interlobular bile ducts form a richly anastomosing network that closely surrounds the branches of the portal vein. These bile ducts are lined by a layer of cuboidal or columnar epithelium that has microvillous architecture on its luminal surface. The cells have a prominent Golgi apparatus and numerous vesicles, which probably participate in the exchange of substances among the cytoplasm, bile, and blood plasma through exocytosis and endocytosis.
The interlobular bile ducts unite to form larger and larger ducts, first the septal ducts and then the lobar ducts, two hepatic ducts, and finally a common hepatic duct (see Fig. 46-4 B ). Along the biliary tree, the biliary epithelial cells, or cholangiocytes, are similar in their fine structure except for size and height. However, as discussed below (see pp. 960–961 ), in terms of their functional properties, cholangiocytes and bile ducts of different sizes are heterogeneous in their expression of enzymes, receptors, and transporters. Increasing emphasis has been placed on the absorptive and secretory properties of the biliary epithelial cells, properties that contribute significantly to the process of bile formation. As with other epithelial cells, cholangiocytes are highly cohesive, with the lateral plasma membranes of contiguous cells forming tortuous interdigitations. Tight junctions seal contacts between cells that are close to the luminal region and thus limit the exchange of water and solutes between plasma and bile.
The common hepatic duct emerges from the porta hepatis after the union of the right and left hepatic ducts. It merges with the cystic duct emanating from the gallbladder to form the common bile duct. In adults, the common bile duct is quite large, ~7 cm in length and ~0.5 to 1.5 cm in diameter. In most individuals, the common bile duct and the pancreatic duct merge before forming a common antrum known as the ampulla of Vater. At the point of transit through the duodenal wall, this common channel is surrounded by a thickening of both the longitudinal and the circular layers of smooth muscle, the so-called sphincter of Oddi. This sphincter constricts the lumen of the bile duct and thus regulates the flow of bile into the duodenum. The hormone cholecystokinin (CCK) relaxes the sphincter of Oddi via a nonadrenergic, noncholinergic neural pathway (see pp. 344–345 ) involving vasoactive intestinal peptide (VIP).
The gallbladder lies in a fossa beneath the right lobe of the liver. This distensible pear-shaped structure has a capacity of 30 to 50 mL in adults. The absorptive surface of the gallbladder is enhanced by numerous prominent folds that are important for concentrative transport activity, as discussed below. The gallbladder is connected at its neck to the cystic duct, which empties into the common bile duct (see Fig. 46-4 B ). The cystic duct maintains continuity with the surface columnar epithelium, lamina propria, muscularis, and serosa of the gallbladder. Instead of a sphincter, the gallbladder has, at its neck, a spiral valve— the valve of Heister —formed by the mucous membrane. This valve regulates flow into and out of the gallbladder.
Uptake, Processing, and Secretion of Compounds by Hepatocytes
The liver metabolizes an enormous variety of compounds that are brought to it by the portal and systemic circulations. These compounds include endogenous molecules (e.g., bile salts and bilirubin, which are key ingredients of bile) and exogenous molecules (e.g., drugs and toxins). The hepatocyte handles these molecules in four major steps ( Fig. 46-5 A ): (1) the hepatocyte imports the compound from the blood across its basolateral (i.e., sinusoidal) membrane, (2) the hepatocyte transports the material within the cell, (3) the hepatocyte may chemically modify or degrade the compound intracellularly, and (4) the hepatocyte excretes the molecule or its product or products into the bile across the apical (i.e., canalicular) membrane. Thus, compounds are secreted in a vectorial manner through the hepatocyte.
An Na-K pump at the basolateral membranes of hepatocytes provides the energy for transporting a wide variety of solutes via channels and transporters
Like other epithelial cells, the hepatocyte is endowed with a host of transporters that are necessary for basic housekeeping functions. N46-2 To the extent that these transporters are restricted to either the apical or basolateral membrane, they have the potential of participating in net transepithelial transport. For example, the Na-K pump (see pp. 115–117 ) at the basolateral membrane of hepatocytes maintains a low [Na + ] i and high [K + ] i (see Fig. 46-5 B ). A basolateral Ca pump (see p. 118 ) maintains [Ca 2+ ] i at an extremely low level, ~100 nM, as in other cells. The hepatocyte uses the inwardly directed Na + gradient to fuel numerous active transporters, such as the Na-H exchanger, Na/HCO 3 cotransporter, and Na + -driven amino-acid transporters. As discussed below, the Na + gradient also drives one of the bile acid transporters. The hepatocyte takes up glucose via the GLUT2 facilitated-diffusion mechanism (see p. 114 ), which is insensitive to regulation by insulin.
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Hepatocyte Housekeeping Functions
As noted in the text, the basolateral membrane of the hepatocyte has both K + and Cl − channels. The basolateral K + conductance is high and is regulated by cAMP, [Ca 2+ ] i , cell volume, and temperature. The basolateral Cl − conductance is under the regulation of hormones and cell volume.
As is the case for most cells, hepatocytes actively regulate their intracellular pH (see pp. 644–645 ) using two acid extruders, the basolateral (i.e., sinusoidal) Na-H exchanger and an electrogenic Na/HCO 3 cotransporter. The apical (i.e., canalicular) Cl-HCO 3 exchanger may contribute as an acid loader. The pH gradient across the canalicular membrane also drives the transport of inorganic solutes (e.g., HCO 3 -SO 4 exchange) and maintains the transmembrane gradients of weak acids and bases that cross the membrane by nonionic diffusion (see p. 784 ).
The basolateral membrane has both K + and Cl − channels. The resting membrane potential ( V m ) of −30 to −40 mV is considerably more positive than the equilibrium potential for K + ( E K ) because of the presence of numerous “leak” pathways, such as the aforementioned electrogenic Na + -driven transporters as well as Cl − channels ( E Cl = V m ).
Hepatocytes take up bile acids, other organic anions, and organic cations across their basolateral (sinusoidal) membranes
Bile Acids and Salts
The primary bile acids are cholic acid and chenodeoxycholic acid, both of which are synthesized by hepatocytes (see p. 959 , below). Other “secondary” bile acids form in the intestinal tract as bacteria dehydroxylate the primary bile acids. Because the p K values of the primary bile acids are near neutrality, most of the bile acid molecules are neutral; that is, they are bile acids (H ⋅ BA) and thus are not very water soluble. Of course, some of these molecules are deprotonated and hence are bile salts (BA − ). The liver may conjugate the primary bile acids and salts to glycine or taurine (Z in Fig. 46-5 C ), as well as to sulfate or glucuronate (Y in Fig. 46-5 C ). Most of the bile acids that the liver secretes into the bile are conjugated, such as taurocholate (the result of conjugating cholic acid to taurine). These conjugated derivatives have a negative charge and hence they, too, are bile salts (BA-Z − and BA-Y − ). Bile salts are far more water soluble than the corresponding bile acids.
Because the small intestine absorbs some bile acids and salts, they appear in the blood plasma, mainly bound to albumin, and are presented to the hepatocytes for re-uptake. This recycling of bile acids, an example of enterohepatic circulation (see p. 962 below). Dissociation from albumin occurs before uptake. Surprisingly, the presence of albumin actually stimulates Na + -dependent taurocholate uptake, perhaps by increasing the affinity of the transporter for taurocholate.
Uptake of bile acids has been studied extensively and is mediated predominantly by an Na + -coupled transporter known as Na/taurocholate cotransporting polypeptide or NTCP (a member of the SLC10A1 family; see Fig. 46-5 C ). This transporter is a 50-kDa glycosylated protein, and it appears to have seven membrane-spanning segments. NTCP handles unconjugated bile acids, but it has a particularly high affinity for conjugated bile acids. In addition, NTCP can also transport other compounds, including neutral steroids (e.g., progesterone, 17β-estradiol sulfate), cyclic oligopeptides (e.g., amantadine and phalloidin), and a wide variety of drugs (e.g., verapamil, furosemide). N46-3
N46-3
Regulation of Na/Taurocholate Cotransport
Bile acid uptake via the Na/taurocholate cotransporting polypeptide (NTCP) is under the regulation of several second messengers. For example, cAMP stimulates taurocholate uptake, whereas this effect is blocked by inhibitors of protein kinase A. This direct stimulation presumably reflects the phosphorylation of the transporter or an essential activator. cAMP also stimulates uptake indirectly by increasing translocation of the transport protein to the membrane.
Certain hormones, such as prolactin, also stimulate bile acid uptake directly.
As is the case for many other transporters, NTCP activity is low in the fetus and neonate and increases with development.
NTCP has now been classified as a member of the SLC10 gene family (see Table 5-4 ) of Na/bile-salt cotransporters. For a detailed discussion of the family members, consult the review by Hagenbuch and Dawson listed below.
Although NTCP also carries unconjugated bile acids, as much as 50% of these unconjugated bile acids may enter the hepatocyte by passive nonionic diffusion (see Fig. 46-5 B ). Because unconjugated bile acids are weak acids of the form
the neutral H ⋅ ΒΑ form can diffuse into the cell. Conjugation of bile acids enhances their hydrophilicity (taurine more so than glycine) and promotes dissociation of the proton from the side chain (i.e., lowers the p K a ), thus raising the concentration of BA − . Both properties decrease the ability of bile acid to traverse membranes via passive nonionic diffusion.
Organic Anions
The organic anion–transporting polypeptides (OATPs) are members of the SLC21 family (see p. 125 ) N46-4 and mediate the Na + -independent uptake of a wide spectrum of endogenous and exogenous amphipathic compounds—including bile acids, bilirubin, eicosanoids, steroid and thyroid hormones, prostaglandins, statin drugs, methotrexate, bromosulfophthalein, and many xenobiotics. Individual OATPs share considerable overlap in substrate specificity and can substantially influence the pharmacokinetics and pharmacological efficacy of drugs they carry. OATPs—predicted to have 12 membrane-spanning segments, with intracellular amino and carboxy termini—appear to exchange organic anions for intracellular 
(see Fig. 46-5 C, D ). Expression of OATPs is under the control, in a cell- and tissue-specific way, of nuclear receptors (FXR, LXR, SXR, CAR; see Table 3-6 ) and hepatocyte nuclear factor 4 (HNF4). OATP1B1, OATP1B3, and OATP2B1 are liver specific and are located on the sinusoidal (basolateral) membrane of hepatocytes.
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Organic Anion Transporters
The organic-anion transporting proteins (OATPs) have now been classified as members of the SLC21 gene family (see Table 5-4 ). For a detailed discussion of the family members, consult the review by Hagenbuch and Meier listed below.
Note that some have inappropriately stated that because this family has an immense number of genes, it really ought to be treated as a superfamily, with the designation SLCO (here the O refers to “organic”). However, the superfamily is large only when one includes genes from all known organisms. The actual number of OATP genes in any given vertebrate organism (humans have 11 such genes) is about the same as for other SLC families. Thus, the OATPs are appropriately described as a “family.”
Thus, the basolateral uptake of bile acids into the hepatocyte is a complex process that involves both an Na + -dependent transporter (NTCP) and Na + - in dependent transporters (OATPs), as well as nonionic diffusion of unconjugated bile acids.
Bilirubin
Senescent erythrocytes are taken up by macrophages in the reticuloendothelial system, where the degradation of hemoglobin leads to the release of bilirubin into the blood ( Fig. 46-6 A and Box 46-1 ). The mechanism by which hepatocytes take up unconjugated bilirubin remains controversial. As evidenced by yellow staining of the sclerae and skin in the jaundiced patient, bilirubin can leave the circulation and enter tissues by diffusion. However, uptake of albumin-bound bilirubin by the isolated, perfused rat liver and isolated rat hepatocytes is faster than can occur by diffusion and is consistent with a carrier-mediated process. Electroneutral, electrogenic, and Cl − -dependent transport have been proposed (see Fig. 46-6 B ).
Box 46-1
Jaundice
J aundice denotes a yellowish discoloration of body tissues, most notable in the skin and sclera of the eyes. The condition is caused by an accumulation of bilirubin in extracellular fluid, either in free form or after conjugation. Bilirubin is a yellow-green pigment that is the principal degradation product of heme (see Fig. 46-6 A ), a prosthetic group in several proteins, including hemoglobin.
The metabolism of hemoglobin (Hb) of senescent red cells accounts for 65% to 80% of total bilirubin production. Hb released into the circulation is phagocytized by macrophages throughout the body, which split Hb into globin and heme. Cleavage of the heme ring releases both free iron, which travels in the blood by transferrin, and a straight chain of 4-pyrrole nuclei called biliverdin (see Fig. 46-6 A ), which the cell rapidly reduces to free bilirubin. This lipophilic form of bilirubin is often referred to as unconjugated bilirubin. After it enters the circulation, unconjugated bilirubin binds reversibly to albumin and travels to the liver, which avidly removes it from the plasma (see Fig. 46-6 B ). After hepatocytes take up bilirubin, they use UGT1A1 to convert the bilirubin to monoglucuronide and diglucuronide conjugates. These two forms of water-soluble, conjugated bilirubin —which make up the direct bilirubin measured in clinical laboratories—enter the bile canaliculus via MRP2 (see Table 5-6 ). Although suitable for excretion into bile, conjugated bilirubin cannot be absorbed by the biliary or intestinal epithelia.
Because of avid extraction and conjugation of bilirubin by the liver, the normal plasma concentration of bilirubin, which is mostly of the unconjugated variety, is ~0.5 mg/dL or lower. The skin or eyes may begin to appear jaundiced when the bilirubin level rises to 1.5 to 3 mg/dL.
Jaundice occurs under several circumstances. Increased destruction of red blood cells or hemolysis may cause unconjugated hyperbilirubinemia. Transient physiological neonatal jaundice results from an increased turnover of red blood cells combined with the immaturity of the pathways for conjugation of bilirubin (exacerbated in premature infants) Pathological conditions that can increase bilirubin production in neonates include isoimmunization, heritable hemolytic disorders, and extravasated blood (e.g., from bruises and cephalhematomas). Genetic disorders of bilirubin conjugation include the mild deficiency of UGT1A1 seen in the common Gilbert syndrome and the near-complete or complete deficiency of UGT1A1 seen in the rare Crigler-Najjar syndrome. Extreme unconjugated hyperbilirubinemia can lead to a form of brain damage called kernicterus (from the Dutch kern [nucleus, as in a brain nucleus] + the Greek icteros [jaundice]). Neonatal hyperbilirubinemia is often treated with phototherapy, which converts bilirubin to photoisomers and colorless oxidation products that are less lipophilic than bilirubin and do not require hepatic conjugation for excretion. Photoisomers are excreted mainly in the bile, and oxidation products, predominantly in the urine.
Jaundice can also result from defects in the secretion of conjugated bilirubin from hepatocytes into bile canaliculi (as with certain types of liver damage) or from defects in transiting the bilirubin to the small intestine (as with obstruction of the bile ducts). In either case, conjugated bilirubin refluxes back into the systemic circulation, where it now accounts for most of the bilirubin in plasma. Because the kidneys can filter the highly soluble conjugated bilirubin—in contrast to the poorly soluble free form of bilirubin mostly bound to albumin—it appears in the urine. Thus, in obstructive jaundice, conjugated bilirubin imparts a dark yellow color to the urine. Measurement of free and conjugated bilirubin in serum serves as a sensitive test for detecting liver disease.
Under normal conditions, approximately half of the bilirubin reaching the intestinal lumen is metabolized by bacteria into the colorless urobilinogen (see Fig. 46-6 A ). The intestinal mucosa reabsorbs ~20% of this soluble compound into the portal circulation. The liver then extracts most of the urobilinogen and re-excretes it into the gastrointestinal tract. The kidneys excrete a small fraction (~20% of daily urobilinogen production) into the urine. Urobilinogen may be detected in urine by using a clinical dipstick test. Oxidation of urobilinogen yields urobilin, which gives urine its yellow color. In the feces, metabolism of urobilinogen yields stercobilin, which contributes to the color of feces. In obstructive jaundice, no bilirubin reaches the intestine for conversion into urobilinogen, and therefore no urobilinogen appears in the blood for excretion by the kidney. As a result, tests for urobilinogen in urine are negative in obstructive jaundice. Because of the lack of stercobilin and other bile pigments in obstructive jaundice, the stool becomes clay colored.
OATP1B1 and OATP1B3 can transport conjugated, and possibly unconjugated, bilirubin in vitro. Indeed, human mutations resulting in the complete deficiency of OATP1B1 or OATP1B3 cause Rotor syndrome, a relatively benign autosomal recessive disorder characterized by conjugated —not unconjugated —hyperbilirubinemia. How did this conjugated bilirubin—made only in hepatocytes—get into the blood? It is now clear that hepatocytes secrete substantial amounts of glucuronidated bilirubin across the sinusoidal membrane into the space of Disse and that OATP1B1/OATP1B3 is responsible for the reuptake of this conjugated bilirubin under physiological conditions. Other hepatic mechanisms may mediate the uptake of unconjugated bilirubin.
Organic Cations
The major organic cations transported by the liver are aromatic and aliphatic amines, including important drugs such as cholinergics, local anesthetics, and antibiotics, as well as endogenous solutes such as choline, thiamine, and nicotinamide ( Fig. 46-7 ). At physiological pH, ~40% of drugs are organic cations, in equilibrium with their respective conjugate weak bases (see p. 628 ). Members of the organic cation transporter (OCT) family mediate the uptake of a variety of structurally diverse lipophilic organic cations of endogenous or xenobiotic origin (see p. 115 ). OCT-mediated transport is electrogenic, independent of an Na + ion or proton gradient, and may occur in either direction across the plasma membrane. Human hepatocytes express only OCT1 (SLC22A1) and OCT3 (SLC22A3), localized to the sinusoidal membrane. OCT1 and OCT3 have partly overlapping substrate specificities. OCT1 is also present in the plasma membrane of cholangiocytes. Acyclovir and lidocaine are examples of OCT1 substrates. The neurotransmitters epinephrine, norepinephrine, and histamine are exclusive OCT3 substrates. In addition to the OCTs, members of the OATP family as well as an electroneutral proton-cation exchanger may contribute to organic cation uptake across the basolateral membrane.
Neutral Organic Compounds
This group of molecules is also taken up by an Na + -independent, energy-dependent process, although the nature of the driving force is not known. The best-characterized substrate is ouabain, uptake of which is inhibited by other neutral steroids, such as cortisol, aldosterone, estradiol, and testosterone. OATP1B1 transports some of these compounds.
We return to Figures 46-5 through 46-7 below, when we discuss the movement of solutes into the bile canaliculus.
Inside the hepatocyte, the basolateral-to-apical movement of many compounds occurs by protein-bound or vesicular routes
Bile Salts
Some compounds traverse the cell while bound to intracellular “binding” proteins (see Fig. 46-5 C ). The binding may serve to trap the molecule within the cell, or it may be involved in intracellular transport. For bile salts, three such proteins have been identified. In humans, the main bile acid–binding protein appears to be the hepatic dihydrodiol dehydrogenase, one of a large family of dehydrogenases, the catalytic and binding properties of which are organ and species specific. The two others are glutathione- S -transferase B and fatty acid–binding protein. Intracellular sequestration of bile salts by these proteins may serve an important role in bile acid transport or regulation of bile acid synthesis. Transcellular diffusion of bile salts bound to proteins can be detected within seconds after bile salts are applied to hepatocytes; this mechanism may be the primary mode of cytoplasmic transport under basal conditions. Free, unbound bile acids may also traverse the hepatocyte by rapid diffusion.
At high sinusoidal concentrations, hydrophobic bile acids may partition into membranes of intracellular vesicles. These conditions may also cause increased targeting of the vesicles to the canalicular membrane—that is, transcellular bile acid transport by a vesicular pathway. Whether transcellular transport occurs by protein-binding or vesicular pathways, it is unknown how bile acids are so efficiently targeted to the canalicular membrane for excretion into bile.
Bilirubin
After uptake at the basolateral membrane, unconjugated bilirubin is transported to the endoplasmic reticulum (ER), where it is conjugated to glucuronic acid (see Fig. 46-6 ). Because the resulting bilirubin glucuronide is markedly hydrophobic, it was thought that intracellular transport was mediated by binding proteins such as glutathione- S -transferase B. However, spontaneous transfer of bilirubin between phospholipid vesicles occurs by rapid movement through the aqueous phase, in the absence of soluble proteins. Thus, direct membrane-to-membrane transfer may be the principal mode of bilirubin transport within the hepatocyte. In addition, the membrane-to-membrane flux of bilirubin is biased toward the membrane with the higher cholesterol/phospholipid ratio. Hence, the inherent gradient for cholesterol from the basolateral membrane to the ER membrane may direct the flux of bilirubin to the ER.
In phase I of the biotransformation of organic anions and other compounds, hepatocytes use mainly cytochrome P-450 enzymes
The liver is responsible for the metabolism and detoxification of many endogenous and exogenous compounds. Some compounds taken up by hepatocytes (e.g., proteins and other ligands) are completely digested within lysosomes. Specific carriers exist for the lysosomal uptake of sialic acid, cysteine, and vitamin B 12 . Clinical syndromes resulting from an absence of these carriers have also been identified. The lysosomal acid hydrolases cleave sulfates, fatty acids, and sugar moieties from larger molecules.
Hepatocytes handle other compounds by biotransformation reactions that usually occur in three phases. Phase I reactions represent oxidation or reduction reactions in large part catalyzed by the P-450 cytochromes. The diverse array of phase I reactions includes hydroxylation, dealkylation, and dehalogenation, among others. The common feature of all of these reactions is that one atom of oxygen is inserted into the substrate. Hence, these monooxygenases make the substrate (RH) a more polar compound, poised for further modification by a phase II reaction. For example, when the phase I reaction creates a hydroxyl group (ROH), the phase II reaction may increase the water solubility of ROH by conjugating it to a highly hydrophilic compound such as glucuronate, sulfate, or glutathione:
Finally, in phase III, the conjugated compound moves out of the liver via transporters on the sinusoidal and canalicular membranes.
The P-450 cytochromes are the major enzymes involved in phase I reactions. Cytochromes are colored proteins that contain heme for use in the transfer of electrons. Some cytochromes—not the P-450 system—are essential for the electron transport events that culminate in oxidative phosphorylation in the mitochondria. The P-450 cytochromes, so named because they absorb light at 450 nm when bound to CO, are a diverse but related group of enzymes that reside mainly in the ER and typically catalyze hydroxylation reactions. Fifty-seven human CYP genes encode hundreds of variants of cytochrome P-450 enzymes (see Table 50-2 ). Genetic polymorphisms exist in the genes encoding all the main P-450 enzymes that contribute to drug and other xenobiotic metabolism, and the distribution and frequency of variant alleles can vary markedly among populations.
In this text, we encounter P-450 oxidases in two sets of organs. In cells that synthesize steroid hormones—the adrenal cortex (see p. 1021 ), testes (see p. 1097 ), and ovary (see p. 1117 ) and placenta (see Table 56-5 )—the P-450 oxidases are localized either in the mitochondria or in the ER, where they catalyze various steps in steroidogenesis. In the liver, these enzymes are located in the ER, where they catalyze a vast array of hydroxylation reactions involving the metabolism of drugs and chemical carcinogens, bile acid synthesis, and the activation and inactivation of vitamins. The same reactions occur in other tissues, such as the intestines and the lungs.
Hepatic microsomal P-450 enzymes have similar molecular weights (48 to 56 kDa). The functional protein is a holoenzyme that consists of an apoprotein and a heme prosthetic group. The apoprotein region confers substrate specificity, which differs among the many P-450 enzymes. These substrates include RH moieties that are as wide ranging as the terminal methyl group of fatty acids, carbons in the rings of steroid molecules, complex heterocyclic compounds, and phenobarbital. In general, phase I processes add or expose a functional group, a hydroxyl group in the case of the P-450 oxidases, which renders the molecule reactive with phase II enzymes. The metabolic products of phase I may be directly excreted, but more commonly, because of only a modest increment in solubility, further metabolism by phase II reactions is required.
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