Jonathan E. Teitelbaum
EMBRYOLOGY AND STRUCTURE OF THE LIVER
EMBRYOLOGY
At the 18th day of gestation (2.5-mm stage), a thickening of the ventral floor of the distal foregut, corresponding to the future duodenum, heralds the appearance of the hepatic diverticulum. This diverticulum penetrates the adjacent mesoderm and capillary plexus, known as the septum transversum. Cellular interactions between the endoderm and mesoderm result in rapid cell proliferation and the formation of hepatocytes, angioblasts, and sinusoids.1
By the third week of gestation (3- to 4-mm stage), the growing diverticulum enlarges to form a double diverticulum that projects into the septum transversum, then divides into a solid cranial portion and hollow caudal portion is evident by the 5-mm stage. The cranial portion differentiates into proliferating cords of hepatocytes and intrahepatic bile ducts while the smaller caudal portion (pars cystica) forms the primordium of the gallbladder, common bile duct, and cystic duct.
The budding liver sequentially invades the vitelline veins and then the umbilical (placental) veins. The vitelline veins run from the gut–yolk sac complex to the heart. As the liver invades the vitelline veins, the midsection of the veins becomes capillarized. The caudal ends persist as the primitive portal veins, and the cranial ends as the primitive hepatic veins. During the 6- to 7-mm stage part of the left umbilical vein becomes the ductus venosus, which shunts placenta derived arterial blood from the umbilical vein to the inferior vena cava.
The hepatocytes of the hepatic portion grow as thick epithelial sheets intermingling between branching channels of the vitelline veins within the septum transversum to form a system of connecting liver cell plates, and the capillaries become the hepatic sinusoids. The sinusoids, present by 5 weeks of gestation, act as templates for the three-dimensional growth of the hepatic cords. The liver cell plates are initially 3 to 5 cells thick. However, over time they gradually transform to one-cell-thick plates, a process that is not complete until 5 years of age. Intrahepatic bile ducts begin to form at 6 weeks of gestation within the hilum of the liver and gradually spread to the periphery until complete at 3 months.
The pars cystica is initially hollow, but epithelial proliferation obliterates the lumen early in its development. Therefore, both the primitive gallbladder and common bile duct consist of solid chords of epithelial cells directly beneath the developing liver in the 6- to 7-mm embryo. Recanalization of the common bile duct and hepatic duct occurs in the 7- to 8-mm and 10-mm embryo, respectively. At the 16-mm stage the proximal gallbladder and cystic duct are hollow. At the third month the gallbladder is fully hollow, and the intrahepatic and extrahepatic biliary structures are joined. Bile secretion into the duodenum starts by the fourth month.
In the third month the liver begins to store iron, and hematopoietic elements derived from the mesenchyme of the septum transversum localize to the extravascular component of the lobule. The liver thus becomes the major blood-forming organ of the embryo. This function is gradually transferred to the developing bone marrow so that by birth only an occasional focus of hematopoiesis remains in the liver.
MACROSCOPIC STRUCTURE
The adult liver weighs 1200 to 1500 g, representing 2% of the total adult body weight. In neonates and young infants the liver is proportionally even larger, accounting for 5% of the total body weight. It is held in place by the falciform and triangular ligaments. During fetal life the falciform ligament conducts the umbilical vein from the umbilicus to the liver. After birth this vein atrophies to form the ligamentum teres. A thin, firm, and smooth capsule (Glissen capsule) covers the liver and is continuous with the porta hepatis where the portal vein, hepatic artery, and common bile duct enter the liver.
At the porta hepatis, the right and left hepatic ducts coalesce to form a common hepatic duct located to the right of the main hepatic artery, in front of the portal vein. The common hepatic duct is joined by the cystic duct, which drains the gallbladder at its right side to create the common bile duct. The common bile duct joins the pancreatic duct in 85% to 90% of the cases just proximal to the ampulla of Vater, which empties into the duodenum. The ampulla of Vater is encased by the sphincter of Oddi, a complex of smooth muscle fibers that regulates the flow of bile into the intestine.
The liver has a dual blood supply. The portal vein, which is rich in nutrients as it drains the gastrointestinal tract and splenic vascular beds, carries approximately 75% of the blood to the liver. The hepatic artery, rich in highly oxygenated blood, usually arises from the second branch of the celiac artery, although this is variable. In 20% of cases the right hepatic artery arises from the superior mesenteric artery rather than branching off of the common hepatic artery. The hepatic veins that drain the liver into the suprahepatic inferior vena cava are formed by the union of the central veins.
Several approaches are used to demarcate sections of the liver. Traditional anatomic descriptors divide the liver into lobes including the right, left, quadrate, and caudate lobes. Another approach is to divide the liver into functional sections based upon vascular supply rather than gross anatomy. A vascular watershed intersects the gallbladder fossa and the vena cava fossa, dividing the liver into nearly equal halves (the right side representing 60% of the liver volume).
A universal terminology has now been adopted that combines both approaches as shown in eFigure 418.1 .2 The liver is divided into hemilivers along the plane that intersects the gallbladder fossa and the fossa for the inferior vena cava (the midplane of the liver). Further divisions of the liver into segments are based on the internal anatomy of the hepatic artery and bile duct so that the left hemiliver contains segments 2 through 4 and the right hemiliver contains segments 5 through 8. The caudate lobe is composed of segments 1 and 9. This segmentation schema is clinically relevant in the context of pediatric liver transplantation, allowing transplantation of a left lateral section.
MICROSCOPIC STRUCTURE
The microscopic anatomy of the liver had traditionally been defined as lobules with a portal tract (bile duct, branches of the hepatic artery and portal vein, along with nerves and lymphatics) and central vein. The edges of liver cells that encircle each portal tract form the limiting plate. However, in vivo microcircula-tory studies revealed that the functional unit of the liver is the acinus (Fig. 418-1). This is based on the fact that the most oxygenated and nutrient-rich blood is in the portal and periportal areas, whereas the least oxygenated blood is centrilobular. The central vein is designated as the terminal hepatic venule, the portal area zone 1, and the hepatocytes around the terminal hepatic venule zone 3. The acinus is composed of hepatocytes arranged in plates of cells with bile canaliculi between them along with sinusoids on the vascular sides. Zone 1 cells form the most active core of the acinus and are the last to die and the first to regenerate. Zone 3 cells are the most prone to toxic, viral, or anoxic injury.
FIGURE 418-1. Diagram of the liver acinar unit as described by Rappaport. Terminal extensions of a portal venule (TPV), hepatic arteriole (HA), terminal hepatic venules (THV), and bile ductule (BD). The zones of the liver include zone 1 adjacent to the portal area, zone 2 in the middle of the acinus, and zone 3 around the terminal hepatic venule. (Source: Gumucio JJ, Miller DL. Liver cell heterogeneity. In: Arias I, Boyer J, Chisari F, et al, eds. The Liver: Biology and Pathobiology. New York, NY: Raven Press; 1982:647-661.)
The functions of the hepatic sinusoid lining cells include pinocytosis, phagocytosis, erythrophagocytosis, iron metabolism, clearance of immune complexes and antigens, and secretion of endogenous pyrogens, collagenase, lysosomal hydrolases, and erythropoietin.3 The endothelial cells form the wall of the sinusoid, separating the sinusoidal lumen from the subendothelial space of Disse. The sinusoidal endothelial cells lack a basement membrane and are perforated by abundant small fenestrae (average diameter 100 nm) in clusters called sieve plates, which act as blood-hepatocyte barriers. Microvilli of the hepatocytes protrude into the sinusoid through the fenestrae. The endothelial cells also express Fc receptors, suggesting a role in removing immune complexes. The space of Disse is located between the endothelial lining and the hepatocyte. As large blood cells move through the small sinusoids, they push the endothelium closer to the hepatocyte, thus promoting the circulation of plasma along the space of Disse. Lymph flow extends from the space of Disse to portal lymphatic vessels at the hilum of the liver.
Kupffer cells are hepatic macrophages most abundant in zone 1 within the sinusoidal wall, anchored to endothelial cells. These cells endocytose and destroy microorganisms, clear endotoxins and senescent erythrocytes, and can act as antigen-presenting cells. When activated they release IL-1, IL-6, TNFα, TGF-β, LTB4, and interferon. Stellate cells (formerly called Ito cells) are located in the space of Disse and are fat-storing cells. They store vitamin A, participate in retinoid metabolism, produce extracellular matrix proteins such as collagen I, III, IV, V, and VI, laminin, fibronectin, and proteoglycans and are thus responsible for hepatic fibrosis seen with chronic injury. Pit cells are large, granular lymphocytes attached to the sinusoidal wall that have natural killer activity. They are extrahepatic in origin and have a role in immune surveillance and hepatic antitumor defense.
Biliary drainage begins by secretion of fluid into the small biliary canaliculi formed by specialized membranes of adjacent hepatocytes. These small biliary canaliculi form channels continuous with the short duct of Hering that join the cholangioles at the limiting plate of the portal areas. These cholangioles then merge into larger bile ducts.
NORMAL HEPATOBILIARY FUNCTION
ENERGY METABOLISM
The supply of nutrients changes drastically during the transition from fetal to postnatal life. Whereas the fetus is supplied with a continuous flow of high-carbohydrate, low-fat, and high–amino acid nutrients via the placenta, the newborn is fed at intervals with a milk, high-fat, and lower-carbohydrate diet. At weaning the shift to an adult-type diet includes more carbohydrates and less fat. The liver plays a central role in these adaptations through gluconeogenesis and regulation of fat and protein metabolism. Throughout gestation the number of mitochondria and amounts of rough and smooth endoplasmic reticula increase. In addition, there is greater metabolic heterogeneity in the adult hepatocyte as compared with the fetal hepatocyte. The adult pattern develops shortly after birth as the liver’s blood supply changes from one dominated by umbilical venous blood to one in which the hepatic artery plays an equally important role. This allows for the development of the functional zones within the liver, each with unique metabolic demands. Zone 1 (periportal) hepatocytes predominantly perform gluconeogenesis, fatty acid β-oxidation, cholesterol biosynthesis, bile acid secretion, ureogenesis, and sulfation of drugs, whereas zone 3 (pericentral) cells perform glycolysis, lipogenesis, ketogenesis, glutamine synthesis, and glucuronidation of drugs.
CARBOHYDRATE METABOLISM
The liver plays an important role in the handling of dietary starches. This role changes as the child transitions from fetal to postnatal life and again on weaning. The first step in glucose metabolism is the phosphorylation of glucose to glucose-6-phosphate; in the fetal liver this is performed predominantly by hexokinase I, an enzyme without substrate specificity for glucose. Fetal glucose utilization approximately equals umbilical glucose uptake. Hepatic galactokinase, which phosphorylates galactose, rises rapidly in the liver near the end of gestation. Although little is known about hepatic glucose uptake in the neonate, data from the newborn lamb suggest that galactose is preferentially used by the liver for carbohydrate synthesis, whereas glucose is delivered to the peripheral tissues. At weaning two factors allow for the increase of hepatic glucose uptake. First is the presence of a high-capacity, low-affinity glucose transporter, GLUT2 that is insulin independent. The second is glucokinase, which replaces hexokinase as the predominant glucose phosphorylation enzyme within the hepatocyte, allowing for specific action on glucose and induction by insulin.
Gluconeogenesis is the synthesis of glucose from lactate, amino acids, or other small molecules such as glycerol or proprionate. Gluconeogenesis accounts for 64% of glucose production during the first 22 hours of fasting. In the post-absorptive state, 75% of total glucose delivery is to insulin-dependent tissues (50% to brain, 25% to splanchnic/liver). Glucose utilization (average 2mg/kg/min) is exactly balanced with release of glucose from the liver. Hepatic glucose metabolism is altered in various disease states; for example, in type 2 diabetes there is increased postabsorptive glucose production and impaired suppression of hepatic glucose production.4
PROTEIN SYNTHESIS
Protein synthesis is a major function of the liver, even in fetal life, because there is evidence that cells within the hepatic diverticulum produce albumin. The mature liver manufactures and exports most of the major plasma proteins, including albumin, lipoproteins, enzymes, coagulation proteins, and a variety of carrier proteins. Although the fetal liver is capable of synthesizing these proteins after the third month of gestation, their concentrations in fetal plasma are low. Lipoproteins increase in the first week after birth to levels maintained until puberty. Albumin reaches adult levels after several months, and there is a reciprocal decline in the primary fetal plasma protein, α-fetoprotein. Ceruloplasmin and complement factors increase to mature values during the first year. Transferrin levels are present in the low adult range at birth and slowly rise to normal adult levels thereafter.
CHOLESTEROL METABOLISM
The liver is the primary organ involved in regulating cholesterol metabolism.5 Cholesterol is an important component of cell membranes, and a precursor molecule for the synthesis of steroid hormones, vitamin D, and bile salts. Newly formed cholesterol is synthesized in the endoplasmic reticulum from acetyl-coenzyme A (acetyl-CoA) through a sequence of enzymatic steps, with HMG-CoA reductase being the rate-limiting and most regulated reaction. In addition to endogenous cholesterol synthesis, the liver each day takes up several grams of cholesterol associated with all classes of lipoproteins. Chylomicron remnants, low-density lipoproteins (LDLs), and very low-density lipoproteins (VLDLs) are taken up by the LDL receptor and LDL receptor-related protein. High-density lipoproteins (HDLs) are taken up through a scavenger receptor, SR-BI. The storage form of cholesterol in the liver, cholesteryl ester, is produced by the enzymatic action of hepatic acyl-CoA cholesterol transferase on cholesterol. This substance serves as a pool for a constant supply of cholesterol through neutral hydrolase for bile salt and lipoprotein assembly in the endoplasmic reticulum.
The liver exports cholesterol to the tissues in VLDL and in HDL particles. Direct secretion of cholesterol in LDL can occur in pathologic states such as familial hypercholesterolemia. Secretion of VLDL involves the complexing of triglyceride with ApoB100 during translation in the endoplasmic reticulum under the agency of microsomal triglyceride transfer protein (MTP). Homozygous mutations in MTP are the basis for abetalipoproteinemia with absent circulatory VDRL and LDL.
Cholesterol is lost from the body as bile salts and intestinal cholesterol that is not absorbed (40–70%) as well in sebum. Daily looses account for about 900 mg daily (550 mg form fecal loss of bile and desquamated cells, 250 mg unabsorbed bile salts, 100 mg sebum), and must be balanced by dietary intake or synthesis.
FATTY ACID METABOLISM
Normal fatty acid metabolism is shown in Figure 150-1. Fat storage begins during fetal life, with the vast majority being in the form of triacylglycerols that contain mainly palmitic and oleic acids. The supply of fatty acids to the fetal liver is regulated by placental carnitine. Synthesis of fatty acids by the fetal liver occurs despite low levels of acetyl-CoA carboxylase, the rate-limiting step in fatty acid synthesis in the adult liver. The triacylglycerol that accumulated during fetal life is mobilized for local utilization after birth. The breakdown and oxidation of the fat by lysosomal acid lipase provides local ATP and ketone bodies for use by peripheral tissues. This intrahepatic oxidation is regulated by the availability of dietary fatty acids, enzymatic activity within the hepatocyte, and hormonal regulation. Hepatic fatty acid oxidation also plays a role in activating hepatic gluconeogenesis as long and medium chain fatty acids increase the supply of gluconeogenic precursors.
Hepatic steatosis represents an excess accumulation of fat (tiacylglycerols) in hepatocytes and can be seen with fatty liver disease, acute fatty liver of pregnancy, hepatitis C virus, liver transplantation, and so on.6
BILE ACID METABOLISM
Cholic and chenodeoxycholic acids are the two primary bile acids of humans and are synthesized from cholesterol. Studies of fetal bile reveal that bile acids are produced as early as the 10th week of gestation, with chenodeoxycholic acid being predominant.
Bile acid synthesis involves a complex series of reactions involving at least 14 enzymatic steps (Fig. 165-1). Failure to perform any of these reactions blocks bile acid production and results in the accumulation of unusual bile acids and intermediate metabolites. Some of these metabolites can be hepatotoxic. Nine recognized inborn errors of bile acid metabolism have been described.7
The first reaction in bile acid synthesis is catalyzed by a liver-specific microsomal cholesterol 7α-hydroxylase. This enzyme is regulated in part by negative feedback of bile acids returning by way of the portal vein during their enterohepatic recycling. However, different bile acids vary in the strength of this negative feedback. For example, primary bile acids successfully down-regulate synthesis, but those with a 7β-hydroxy group such as ursodeoxycholic acid do not. Factors that influence cholesterol 7α-hydroxylase activity cause concomitant changes in HMG-CoA reductase, the rate-limiting enzyme for cholesterol synthesis. This allows for maintenance of a constant cholesterol pool size. After the synthesis of 7α-hydroxycholesterol, modifications to the steroid nucleus result in oxidoreduction and hydroxylation. The final step is side chain conjugation of cholic and chenodeoxycholic acids to the amino acids glycine and taurine within peroxisomes (ie, amidation). Although in adults glycine is the most common conjugate, in early life more than 80% of the bile acids are taurine conjugated because of an abundance of hepatic taurine stores. Other naturally occurring conjugates to the steroid nucleus include sulfates, glucuronidide ethers and esters, glucosides, N-acetyl-glucosaminides, and conjugates of some drugs. These account for a relatively large amount of urine bile acids because conjugation increases the polarity of the normally hydrophobic molecules, facilitating renal excretion.
The final products, referred to as primary bile acids, are secreted in canalicular bile and stored in gallbladder bile. The gallbladder concentrates the bile and releases it into the duodenum during meals. This raises the intraluminal concentration of bile salts above the critical micellar concentration, allowing formation of micelles (macromolecular aggregates with phospholipids and cholesterol). Micelles promote solubilization of nonpolar dietary constituents and assist in the delivery of lipids to the intestinal absorptive surface. Bile acids are efficiently absorbed in the distal ileum by a carrier-mediated transport mechanism, returning to the liver by the portal vein. The total bile acid pool circulates approximately twice with each meal, or 10 to 12 times each day. Bacterial enzymes metabolize primary bile acids to secondary bile acids with different physicochemical characteristics. 7α-dehydroxylation of cholic and chenodeoxycholic acids results in the formation of the secondary bile acids deoxycholic and lithocholic acids, which are relatively insoluble and thus poorly absorbed. They make up the largest proportion of fecal bile acids. The large portion (95%) that is reabsorbed results in feedback inhibition of new bile acid synthesis.
In infants the total bile acid pool size is a fraction of that of the adult: at 32 weeks of gestation the fetus has a relative pool size one-sixth that of an adult. In a premature infant, the intraluminal bile acid concentrations may fall below the critical micellar concentration (1–2 mmol/L). In addition, there is less effective intestinal reabsorption of bile acids, inadequate hepatic canalicular secretion of bile acids, and inefficient hepatic uptake of bile acids from the systemic circulation. The cumulative effects of the immature bile acid metabolism and homeostasis system in newborns result in relatively inefficient absorption of dietary fats and fat-soluble vitamins and a tendency toward cholestasis.
DRUG AND TOXIN METABOLISM
Hepatic drug metabolism, or biotransformation, is divided into two broad aspects: activation (phase I) and detoxification (phase II). Different families of enzymes are important in each, and the balance between these two processes plays an important role in hepatic toxicity.8 The hemoprotein cytochromes of the P-450 system are associated with most phase I reactions and are particularly important in the liver, although they are found in most body tissues. These cytochromes can be detected in embryonic and fetal tissues at low levels. Approximately 50 cytochrome P450 isoenzymes have been identified in humans with CYP3A4 being overall the most important in drug metabolism. They catalyze diverse reactions including hydroxylation, dealkalinization, and dehalogenation. All reactions involve monooxygenation, in which one oxygen atom is inserted into the substrate. The P-450 system has overlapping substrate specificity. Distinct families of cytochromes of P-450 have been identified, with those in the 1A, 2C, 2D, 2E, and 3A subfamilies being particularly important in drug-xenobiotic metabolism and toxicity in humans.
Phase II detoxifying reactions are performed by different enzymes including glutathione S-transferases, glucuronosyl transferases, epoxide hydrolase, sulfotransferases, and N-acetyltransferases. These catalyze reactions to complete the transformation of hydrophobic compounds to hydrophilic ones that can be excreted into the urine or bile. Some of these enzymes are inducible, and some are polymorphic. An example of a polymorphic form includes arylamine N-acetyltransferase 2 (NAT-2), which allows for individuals to be rapid or slow acetylators. This accounts for differences in ability to metabolize certain drugs (ie, sulfasalazine, trimethoprim-sulfamethoxazole, and isoniazid). This trait has been shown to be inherited among certain ethnic groups.
EXCRETION
Hepatocytes are responsible for the excretion of numerous substances via bile, including bilirubin, drug metabolites, and heavy metals such as zinc and copper. Bile secretion starts at the beginning of the fourth month of gestation, and the presence of bile in the lumen of the intestine is responsible for the dark green color of meconium. Bile formation at the canalicular level occurs as a result of active transport of solutes followed by passive movement of water. Bile is functionally an isosmotic solution because organic constituents such as bile acids are either in mixed lipid micelles or self-aggregates and thus have less osmotic activity. Bile ductules and ducts significantly alter the volume and composition of fluid produced at the canalicular level by reabsorption and secretion of water and electrolytes. Secretin and vasoactive intestinal polypeptide (VIP) produce a bicarbonate-rich choleresis at the ductular level. In contrast, somatostatin inhibits bile flow. Bile can also be altered within the gallbladder, where it can be concentrated up to 10-fold.
Bilirubin is formed from the degradation of heme products, most notably hemoglobin, cytochromes, catalases, tryptophan pyrrolase, and muscle myoglobin. One gram of hemoglobin yields 35 mg of bilirubin. Bilirubin is formed through the cleaving of the tetrapyr-role ring of protoheme (protoporphyrin IX). Microsomal heme oxygenase reduces the iron from Fe3+ to Fe2+ and hydroxylates the α-methine carbon. The cleaved α carbon is excreted as carbon monoxide. The remaining linear tetrapyrrole is biliverdin IXα. The C-10 carbon is then reduced to form bilirubin IXα by biliverdin reductase. In healthy term infants, bilirubin is formed at a rate of 6 to 8 mg/kg/day compared to 3 to 4 mg/kg/day in healthy adults. This difference results from the increased red blood cell mass and shorter red blood cell life span in infants.
Bilirubin is poorly soluble in aqueous solvents because of extensive internal hydrogen bonding and subsequent folding, yielding a nonpolar, lipophilic molecule. The carbon-carbon double bonds at positions 4–5 and 15–16 allow for cis (designated Z for the German zusammen, meaning together) and trans (designated E from the German for entgegen, meaning “opposite forms”). The naturally occurring form of bilirubin is 4Z,15Z-bilirubin IXα. The hydrophobic nature requires a carrier molecule, albumin, for transport from production in the reticuloendothelial system to excretion by the liver. The binding affinity between these two molecules is so high that at normal bilirubin levels all serum bilirubin is bound to albumin. Bilirubin is taken up by hepatocytes from the sinusoids by a plasma membrane-bound carrier called bilitranslocase, but there are likely other transporters (eg, in the OATP family). Once within the aqueous environment of the hepatocyte bilirubin is bound to an intracellular carrier glutathione S-transferase (GST) also called ligandin or the Y protein. Bilirubin is then conjugated with glucuronic acid within the endoplasmic reticulum of the hepatocyte by bilirubin glucuronyl transferase (UDP-GT) to form both mono and diglucuronides. Narcotics anticonvulsants contraceptive steroids and bilirubin itself can increase UDP-GT activity Alternatively its activity may be inhibited by caloric and protein restriction.
Once conjugated, bilirubin is excreted into the bile predominantly as bilirubin diglucuronide. Infants have lower levels of UDP-GT and thus have fewer diglucuronides than adults. The rate-limiting step in bilirubin clearance is its hepatic secretion. Secretion can be enhanced by choleretic agents such as phenobarbital and inhibited by cholestatic agents such as estrogens and anabolic steroids. In pathologic conditions, bilirubin may reflux back into the circulation, causing clinical jaundice. When bilirubin conjugates enter the intestinal lumen, normal bacterial flora can hydrogenate the carbon double bonds to produce urobilinogens. Neonates lack the bacteria Clostridium ramosumand Escherichia coli and are thus more likely to absorb bilirubin from the intestine. Reduction-oxidation reactions also occur to form urobilinoids, which are not reabsorbed by the enterohepatic circulation and are thus excreted in the feces. Bilirubin can also be unconjugated by bacterial or tissue β-glucuronidase and readily absorbed from the intestine.