Focal fatty change

Steatosis is normally a diffuse process, but a more localized form was first described in 1980. Since then, steatosis has become increasingly recognized, as revealed by imaging techniques such as ultrasound or computed tomography, and may simulate metastatic disease. The lesions may be single or multiple, are typically “geographical” in shape but may sometimes be nodular, and they have occasionally been associated with other hepatic abnormalities such as cirrhosis. However, the basic architecture of the liver is usually preserved, and although some patients have factors predisposing to generalized macrovesicular steatosis, the mechanisms leading to a nonhomogeneous distribution of fat are not clear. Local hypoxia has been postulated to play a role, however, as has a direct, local effect of insulin from an aberrant pancreatic vein. The lesion must be distinguished from other intrahepatic, lipid-rich lesions, such as lipoma, angiolipoma, myelolipoma and coelomic fat ectopia. Rarely, the converse can be observed, with areas of sparing in an otherwise diffuse steatosis ; this generally occurs adjacent to the gallbladder or near the porta hepatis in areas receiving blood from an aberrant gastric vein.

Hepatocellular ballooning

Ballooned hepatocytes are a common feature in steatohepatitis and are currently considered an essential finding for the diagnosis of steatohepatitis. These cells are usually increased in size, have a rounded outline and a rarified, clear, ‘oedematous’ cytoplasm and may have a hyperchromatic nucleus and thickened cell membrane ( Fig. 5.2 ). However, smaller hepatocytes may share similar cytoplasmic alterations and can be considered ‘ballooned’. Ballooned hepatocytes are not specific for fatty liver disease and can also be seen in viral hepatitis and chronic cholestasis. Ballooning may contribute to the development of hepatomegaly and may have a direct functional effect; some studies reported a correlation between hepatocyte enlargement and intrahepatic pressure in alcoholic fatty liver disease, although this was not confirmed by others.

Hepatocyte ballooning. Many of the cells in this field are enlarged with rarefaction of the cytoplasm. Some of the cell swelling is the result of the accumulation of fat, but there is also excess cytoplasmic fluid. (H&E stain.)

Ballooned hepatocytes are recognized most easily when they contain Mallory–Denk bodies, formerly referred to as ‘Mallory’s hyalin’ or ‘alcoholic hyaline’ (see next section). However, not all ballooned hepatocytes contain these intracytoplasmic cytoskeletal aggregates, and some may contain fat droplets. Ultimately, ballooning may be a structural manifestation of microtubular disruption. Whether this feature represents cells undergoing lytic necrosis (as in viral hepatitis), oxidative damage or possibly an adaptive change similar to ‘brown fat’ remains to be resolved. Altered intracytoplasmic cytoskeletal contents of hepatocytes, i.e. keratins 8 (K8) and 18 (K18), are well recognized. On light microscopy, ballooned hepatocytes typically are enlarged and have altered, reticulated cytoplasm. Lackner et al. have proposed the use of the K8/18 immunohistochemical (IHC) stain as a method for verification of hepatocyte ‘clearing’ resulting from loss of diffuse intracytoplasmic keratins in ballooning. This stain also highlights the aggregated K8/18 in Mallory–Denk bodies. This immunostain is routinely available, and its application in practice may be helpful ( Fig. 5.3 ).

Ballooning can be detected by the lack of cytoplasmic keratins 8 and 18 (K8/18) as well as the presence of clumped keratin aggregates (K8/18 immunohistochemical stain).

Mallory–Denk bodies

Mallory first described aggregates of amorphous, eosinophilic material in hepatocytes in alcohol-related liver disease in 1911. These inclusions are now often referred to as Mallory–Denk bodies (MDBs) in recognition of Helmut Denk’s work in this field. MDBs are a feature of both alcoholic and nonalcoholic steatohepatitis but are also seen in association with prolonged cholestasis, which occurs in diseases such as primary biliary cirrhosis, Wilson disease, Indian childhood cirrhosis, focal nodular hyperplasia and hepatocellular carcinoma. MDBs have been produced experimentally using a variety of agents, including griseofulvin, diethylnitrosamine and 3,5-diethoxy carbonyl-1,4 dihydrocollidine (DDC).

Three ultrastructurally distinct forms of MDBs have been described : type 1 has bundles of filaments in parallel arrays, type 2 is seen as clusters of randomly oriented fibrils, and type 3 is identified as granular or amorphous substance containing only scattered fibrils. The filament thickness varies from 5 to 20 nm, depending on the method of measurement. Type 2 MDBs are the type seen most frequently in alcohol-related liver disease ( Fig. 5.4 ). Purified from human liver, MDBs are composed predominantly of protein; five polypeptide bands have been detected by electrophoretic analysis, with molecular weights ranging from 32 to 56 kD. Small amounts of carbohydrate have also been detected. MDB formation is thought to result from derangement of the intermediate filament component of the cytoskeleton of the hepatocyte, but they are not composed solely of intermediate filament proteins. Antibodies to MDBs react with the cytoplasmic filament system of normal hepatocytes (K8, K18), and they also contain a number of unique antigenic determinants, together with K19 and K20. The keratin molecules may be hyperphosphorylated. MDBs from alcoholic and nonalcoholic liver disease have been shown to share a common antigenic determinant.

Mallory–Denk body (MDB) type 2. A, The MDB consists of a mass of randomly orientated filaments. Several mitochondria, of varying sizes and shapes, are also seen. (Magnification ×25,100) B, Higher magnification shows the randomly arranged filaments (×57,000).

Hypotheses concerning the pathogenesis of MDB formation are outlined in a number of excellent reviews. Extensive literature addresses the biology of MDBs in animal models, as well as gender and strain differences in their formation. Rather than reflecting a passive process of collapse of intracytoplasmic structures, MDBs result from concerted, active metabolic pathways that include transamidation, hyperphosphorylation and ubiquitination of abnormally folded intermediate filaments associated with the chaperone heat shock proteins. A protein product of an early-response gene, p62 (sequestosome-SQSTM1) associates with MDBs; this process may represent a hepatoprotective sequestration of the abnormal proteins into biologically inert inclusions.

The possible detrimental effects of alcohol and its metabolic products on proteasome dysfunction have been highlighted. Hirano et al. have designed an in vitro system in which MDBs can be generated; this is likely to lead to further opportunities to establish their pathogenesis. In addition to IHC stain to K8/18, anti-ubiquitin and anti-p62 antibodies can be used to demonstrate the presence of MDBs ( Fig. 5.5 ).

Immunoreactivity of Mallory–Denk bodies to p62.

Necroinflammation

Ballooned hepatocytes in steatohepatitis may be a manifestation of progressive injury leading to lytic necrosis. Such cells, however, including those containing MDBs, are not destined to immediate cell death, and animal studies in particular have shown that these sublethal changes may persist for some time, even months. This has implications in the diagnostic setting, where histological changes of alcoholic hepatitis can remain for such duration despite abstinence. Cell death in steatohepatitis also occurs by apoptosis ( Fig. 5.6 ), and a relationship exists between apoptotic cell numbers and the severity of disease. Serum levels of K18 fragments from apoptotic hepatocytes are now being used as surrogate markers to predict severity. Confluent and bridging necrosis are rarely seen in fatty liver disease but can be observed in severe ALD—so-called central sclerosing hyaline necrosis (see later)—and was seen in some cases of steatohepatitis after jejunoileal bypass surgery. The inflammation that accompanies the cell injury is variable in both intensity and cellular nature. In most fatty liver diseases, lobular inflammation is mixed, with lymphocytes, macrophages and neutrophil polymorphs all being present ( Fig. 5.7 ). The number of neutrophils is generally greater in alcoholic steatohepatitis than in NASH, but in both, they may be seen to encircle ballooned hepatocytes containing MDBs, so-called satellitosis ( Fig. 5.8 ). It has been postulated that this may occur through a chemotactic effect of the inclusions, although current concepts suggest that it more likely is associated with local chemokine release, including interleukin-8 (IL-8). Portal tract inflammation may occur in all forms of steatohepatitis. It is predominantly lymphocytic in NASH ( Fig. 5.9 ) but may be mixed in ALD. Biopsies from a subset of patients with ALD show a predominantly portal-based inflammation, possibly reflecting another mechanism of injury.

Alcoholic steatohepatitis. The hepatocytes show fatty change, and focal necrosis is accompanied by a mixed inflammatory cell infiltrate. An apoptotic cell (centre) is also seen. (H&E stain.)

Steatohepatitis. In this field there is a mixed lobular inflammatory infiltrate. Neutrophil polymorphs are present, but there are also lymphocytes and occasional activated Kupffer cells. (H&E stain.)

Satellitosis. This micrograph shows a single hepatocyte which contains a poorly formed Mallory–Denk body. The cell is surrounded by neutrophil polymorphs. (H&E stain.)

Portal inflammation in nonalcoholic steatohepatitis. This is mild and not accompanied by significant interface hepatitis. (H&E stain.)

Alcohol metabolites and associated biochemical disturbances

Alcohol metabolism and its associated disturbances can at least partly explain some features of alcohol-induced injury. Alcohol is readily absorbed from the gastrointestinal tract and is distributed throughout the body in proportion to the amount of fluid in the tissues. Less than 10% is eliminated through the lungs, kidneys and sweat; the remainder is oxidized predominantly in the liver, which probably explains the marked metabolic disturbances that occur there.

Three pathways for alcohol metabolism exist in the liver. The principal pathway involves cytosolic alcohol dehydrogenase (ADH), which catalyses the oxidative metabolism of alcohol to acetaldehyde. Hydrogen is transferred from alcohol to the cofactor nicotinamide adenine dinucleotide (NAD), converting it to the reduced form, and acetaldehyde is produced. The generation of excess reducing equivalents (NADH) in the cytosol results in a marked shift in the oxidation-reduction (redox) potential, as indicated by the increased lactate/pyruvate ratio. Some of the hydrogen equivalents are transferred from the cytosol to the mitochondria via several shuttle systems.

Acetaldehyde generated by the metabolism of alcohol is oxidized to acetate; again, this occurs predominantly in the liver. It occurs through the action of mitochondrial aldehyde dehydrogenase (ALDH2). Some of the acetaldehyde and most of the acetate are excreted by the liver into the bloodstream and metabolized peripherally. Patients with chronic alcoholism have accelerated alcohol metabolism and higher levels of blood acetaldehyde than nondrinkers. The metabolism of acetaldehyde produces excess NADH in liver mitochondria, which in turn leads to a decrease in β-oxidation of long-chain fatty acids by inhibiting long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) activity.

Chronic alcohol consumption results in high levels of acetaldehyde, which is highly toxic due to a variety of mechanisms, including glutathione depletion, oxidative stress, lipid peroxidation and formation of adducts with proteins.

Chronic alcohol consumption accelerates ADH-related alcohol metabolism to a limited extent. ADH activity does not increase, however, and a hypermetabolic state has been proposed as a possible mechanism. There are multiple isoenzymes of ADH, which are divided into five major classes and encoded by at least seven different gene loci.

The microsomal ethanol-oxidizing system (MEOS) is a cytochrome P-450-dependent pathway and in particular involves the ethanol-inducible cytochrome P-450 2E1 (CYP2E1). CYP2E1 was initially thought to be the only isoenzyme involved in ethanol metabolism, but ethanol can also induce CYP1A1, CYP3A and CYP4A. Increased CYP2E1 activity after chronic alcohol consumption is probably the major mechanism for the increased rates of alcohol clearance from the blood and metabolic tolerance that develop in regular drinkers. The increased xenobiotic toxicity and carcinogenicity seen in association with chronic alcohol consumption, in both humans and various animal models, can be explained largely by the induction of CYP2E1, which leads to enhanced metabolism of a wide variety of agents, e.g. paracetamol (acetaminophen), that are metabolized by this isoenzyme.

Catalase, located in peroxisomes, plays only a minor role in alcohol metabolism, possibly accounting for <2% of overall alcohol oxidation, although there is some evidence for increased metabolism of alcohol by peroxisomes in chronic drinkers.

Alcohol metabolism may cause lactic acidosis, with a reduced renal capacity for uric acid excretion producing a secondary hyper­uricaemia. There is also impaired carbohydrate metabolism with reduced gluconeogenesis from amino acids; this may produce hypoglycaemia. Of particular importance to the development of steatosis is the related impaired fat metabolism: H ⁺ ions replace two-carbon fragments derived from fatty acids as the main energy source in hepatocyte mitochondria and also depress the citric acid cycle. There is decreased fatty acid oxidation; an increase in α-glycerophosphate also occurs, with a consequent increase in trapping of fatty acids, and an accompanying increased synthesis of triglycerides. In longstanding alcohol abuse, protein synthesis is depressed, which together with impaired liver cell secretory function, causes retention of lipoproteins and contributes to the accumulation of fat in hepatocytes.

Molecular mechanisms of alcohol-induced steatosis

The biochemical events associated with alcohol metabolism just outlined are now thought to be insufficient to fully explain the initiation and perpetuation of alcohol-induced steatosis. In animal models, for example, the redox shifts are attenuated over time, but the steatosis remains. A number of alternative pathways have been identified that regulate lipid metabolism and with which alcohol has been found to interact ( Fig. 5.11 ).

Alcoholic metabolism, transcriptional control of lipid metabolism and endoplasmic reticulum (ER) stress; outline of mechanisms. AMPK, Adenosine monophosphate-activated protein kinase; FA, fatty acid; MTP, microsomal triglyceride transfer protein; PPARα, peroxisome proliferator-activated receptor α; SREBP-1, sterol regulatory element-binding protein 1.

Peroxisome proliferator-activated receptor α (PPARα) is a nuclear hormone receptor which regulates the oxidation and transport of fatty acids. PPARα agonists have been shown to reverse steatosis in alcohol-fed mice. Furthermore, PPARα knockout mice display impaired fatty acid oxidation with increased serum free fatty acids and develop steatosis. Liver injury is more severe in alcohol-treated knockout animals compared to wild-type controls. PPARα plays an important role in fatty acid transport through its effects on microsomal triglyceride transfer protein (MTP), a key molecule in the assembly of very-low-density lipoproteins (VLDLs) before export, and alcohol-induced alterations to PPARα activity could contribute to lipid accumulation through this route.

The adenosine monophosphate (AMP)-activated protein kinase (AMPK) enzyme is a master regulator of metabolism that acts as a sensor for various forms of cellular stress, causing increased activity of energy-generating metabolic pathways, including fatty acid oxidation. AMPK has been found to be decreased in alcohol-fed animals, and over time this was associated with accumulation of SREBP. The effect of alcohol on AMPK may therefore contribute to both increased fatty acid synthesis and decreased fatty acid oxidation, contributing to steatosis.

Sterol regulatory element-binding proteins (SREBPs) represent a family of transcription factors that regulate the genes for enzymes responsible for the synthesis of cholesterol, fatty acids and triglyceride. SREBP-1 isoforms are involved in regulating fatty acid synthesis while SREBP-2 controls cholesterol synthesis. Alcohol feeding of mice led to increased intrahepatic levels of SREBP-1 which correlated with triglyceride accumulation. AMPK inhibits SREBP-1c, and therefore reduced AMPK levels caused by alcohol may contribute to elevating SREBP-1 levels and activity in steatosis.

Alcohol also interferes with liver methionine metabolism. Normally, methionine is converted to S -adenosylmethionine and then to S -adenosylhomocysteine and finally to homocysteine; conversion back to methionine involves methionine synthase. Homocysteine and S -adenosylhomocysteine are elevated in alcoholic patients and are thought to induce the endoplasmic reticulum (ER) stress response. This involves the accumulation of unfolded and misfolded proteins triggering the ‘unfolded protein response’. The ER stress response may play an important role in the pathogenesis of alcohol-induced steatosis but may also contribute to hepatocyte cell death through activation of proapoptotic genes.

Sirtuin-1 is another master metabolic regulator. In experimental animals, alcohol exposure led to decreased levels of SIRT-1, blocking the deacetylation of SREBP with the resultant increase in SREBP-1c, and in turn increased fatty acid synthesis.

There is a growing list of additional pathways that have been implicated in the development of alcohol-induced steatosis. This includes factors such as tumor necrosis factor alpha (TNFα), long considered to be involved in necroinflammation but now also implicated in steatosis. Others that may either exacerbate or protect from lipid accumulation include adiponectin, IL-6, plasminogen activator inhibitor 1, endocannabinoids and complement factors.

Mitochondrial abnormalities, oxidant stress and lipid peroxidation

Nassir and Ibdah recently reviewed mitochondrial alterations in alcohol-related liver disease. Impairment of liver mitochondrial function after chronic alcohol exposure is well documented. Isolated mitochondria from experimental animals following chronic alcohol administration are more likely to increase the permeability transition in response to a range of cellular stress. Oxidative phosphorylation is impaired because of a defect in protein synthesis encoded by mtDNA. As noted later, mtDNA mutations may be acquired, and morphological changes may occur in the structure of the organelle itself, manifest histologically by the formation of giant mitochondria ( Fig. 5.12 ). The cytokine IL-6 may play a critical role in mtDNA repair after alcohol-induced injury.

Giant mitochondria containing crystalline inclusions. A number of normal-sized mitochondria are also seen, some with inclusions. (×17,500.)

As noted earlier, chronic ethanol ingestion induces CYP2E1; this results in enhanced lipid peroxidation and increased rates of reactive oxygen species (ROS) production. Mitochondria are both the source and the target of ROS. Other sources of ROS include endotoxin-activated Kupffer cells and neutrophil polymorphs. Chronic ethanol ingestion simultaneously results in enhanced acetaldehyde production, caused by CYP2E1 induction, and depletion of glutathione, which impairs a major defence mechanism against oxidative stress. Comprehensive reviews further discuss the role of oxidative stress.

Mitochondrial dysfunction can also promote both apoptosis and hepatocyte necrosis. Apoptotic cells can be seen in liver biopsies from patients with ALD (see Fig. 5.6 ), and DNA end-nick labelling has been used to confirm the presence of apoptotic bodies. Apoptosis in ALD is probably triggered by oxidative stress as well as by a variety of cytokines. Autophagy may be a defence mechanism within hepatocytes that protects against alcohol-induced apoptosis (and steatosis).

Protein hyperacetylation

Experimental studies have demonstrated that exposure to alcohol can cause global hyperacetylation. This is associated with a reversible, post-translational modification of the ε-amino groups of lysine residues, shown to modulate a diverse range of cellular processes, from transcriptional activation to microtubule stability. The lysine acetylation is controlled by two counteracting enzymes, histone acetyltransferases and histone deacetylases. The precise role of such molecular alterations in ALD is uncertain, but recent work has demonstrated that acetyl-CoA generated from acetate (derived from alcohol metabolism) increases acetylation of proinflammatory gene histones in macrophages, enhancing their inflammatory response.

Innate immunity

Rao et al. reviewed the now considerable evidence supporting the concept that endotoxaemia, resulting from alcohol-induced leakiness of the small intestine and bacterial translocation, plays an important role in the pathogenesis of ALD. The importance of perturbations in the gut–liver axis in the induction of steatosis, necroinflammation and fibrosis is now well accepted, including increasing evidence that altered gut microbiota may be important. Endotoxins, which consist of lipopolysaccharide (LPS) derived from the walls of gram-negative bacteria, normally permeate only to a very limited extent across the mucosa of the gut. Any that is transported in the portal venous system is cleared by hepatocytes and Kupffer cells through a number of important LPS recognition systems that include the binding of LPS to surface receptors, enzymatic degradation of the lipid A moiety and inactivation of LPS itself.

When LPS levels exceed those that can be dealt with by homeostatic mechanisms, they can activate Kupffer cells through Toll-like receptors (TLRs), in particular through binding to TLR4, leading to cytokine production and generation of ROS. Animal studies have demonstrated an increased susceptibility of female rats to alcohol/endotoxin-induced liver injury. Acetaldehyde has been shown to increase intestinal permeability by altering the distribution of tight junction proteins and adherens junction proteins on enterocytes. Alcohol may also exert this effect indirectly through increased levels of microRNA-212 that decreases ZO-1 expression. Numerous human and animal studies of ALD have demonstrated an increased production of proinflammatory cytokines such as TNFα (produced primarily by activated Kupffer cells), IL-1, IL-6 and osteopontin. The effects of TNFα are responsible for many of the clinical features seen in alcoholic hepatitis (e.g. fever, neutrophilia, even hypotension in severe cases).

Kupffer cells that have been activated by endotoxin show enhanced production of macrophage inflammatory protein 2 (MIP2), which is a potent chemotactic agent for polymorphs. Endotoxin also upregulates the expression of intercellular adhesion molecule 1 (ICAM1) on hepatocytes. The alcohol-related influx of endotoxin into the circulation is thought to lead to an enhanced interaction between adhesion molecules on neutrophils and ICAM1 on hepatocytes, thus contributing to liver injury and to the migration of polymorphs into the liver. Furthermore, monocyte secretion of MIP1 and MIP1αI is increased in alcoholic hepatitis.

Disturbances of IgA metabolism, with elevated total serum immunoglobulin A and a characteristic linear deposition of IgA in the hepatic sinusoids, are well described in ALD. IgA has also been shown to trigger the secretion of TNFα by monocytes and synergistically enhance the endotoxin-induced secretion of this cytokine.

The complement system forms an important part of innate immunity, and the possible role of its components in the pathogenesis of alcoholic hepatitis has been studied. The complexities of the interactive role of cytokines and inflammatory mediators is discussed in several comprehensive reviews.

Adaptive immunity and autoimmune phenomena

Many immunological disturbances occur as a result of alcoholic liver injury and may be considered secondary events. Nevertheless, some morphological features suggest that acquired immunological mechanisms sometimes play a primary role in alcohol-related liver injury.

The neutrophil polymorph infiltrate seen in and around hepatocytes, which contain Mallory–Denk bodies, could indicate a local Arthus reaction with humoral sensitization to MDBs. An antibody to MDB antigen has been reported in alcoholic hepatitis but was not demonstrated in patients with fatty liver or inactive cirrhosis. Hypergammaglobulinaemia in ALD is characterized by a polyclonal increase in all classes of immunoglobulins, but with serum IgA levels being particularly increased and correlating closely with the severity of liver damage.

Low titres of autoantibodies have been reported in ALD, with antinuclear factor in 12–17% and anti-smooth muscle autoantibodies in 12–27% of patients. IgG and IgA antibodies to liver cell membrane antigens (LMAs) have been demonstrated in all stages of alcoholic liver injury. IgG class antibodies reacting with ethanol-altered rabbit hepatocytes were detected in more than 70% of patients with various stages of ALD but in only 25% of those with nonalcoholic liver disease.

A number of studies have suggested that an immune response is directed against acetaldehyde–protein adducts formed during the metabolism of alcohol. Different proteins have been detected in these adducts in hepatocytes and include a 37-kD protein in the cytosol and CYP2E1 in microsomes. IHC studies have demonstrated the presence of protein–acetaldehyde adducts in perivenular hepatocytes at an early stage of alcoholic injury. In addition, malondialdehyde and acetaldehyde can react together with proteins to form hybrid adducts termed ‘malondialdehyde–acetaldehyde adducts’ (MAAs). Albano and Vidali have reviewed the complex interactions between lipid peroxidation and the induction of autoantibody formation against adducts.

Hypoxia

Alcohol-induced liver injury selectively affects the perivenular region in the early stages. It has been postulated that a relatively lower oxygen tension in this zone may exaggerate the shift in redox potential that accompanies alcohol metabolism. Hypoxia has been shown to induce perivenular necrosis in rats chronically fed ethanol. Israel et al. likened the acceleration of alcohol metabolism with an associated enhancement of oxygen requirement to a ‘hypermetabolic state’, and this has been the basis for animal and human studies of propylthiouracil.

Lieber et al. suggested that impaired oxygen utilization, rather than lack of oxygen supply, and enhanced alcohol metabolism in this zone are factors in alcohol-associated liver injury. Fluctuations in blood flow have also been suggested as a mechanism for hypoxia-related injury in animal models for ALD; French comprehensively reviewed the role of hypoxia in liver injury

Nutritional factors

A number of reviews have addressed the interaction among nutritional factors, alcohol and liver disease in humans. Primary malnutrition caused by poor diet is well recognized in the chronic drinker. Moreover, secondary malnutrition is now well recognized in drinkers in whom the diet is adequate.

Mezey examined the role of dietary fat in ALD; the severity of alcoholic steatosis is clearly related to the amount of fat in the diet. Patients with biopsy evidence of ALD have lower levels of polyunsaturated fatty acids in their livers than chronic drinkers who have morphologically normal livers.

The recognition that metabolic and nutritional factors contribute to the pathogenesis of NASH (see later) has stimulated renewed interest in the role of nutritional factors in the pathogenesis of ALD. There is now clear evidence that obesity potentiates the severity of alcohol-related hepatic injury, although it is not yet clear whether the effect is additive or synergistic. Naveau et al. have shown that adipose tissue is inflamed in patients with ALD and that the amount correlates with the severity of liver injury, postulating that this may be another source of proinflammatory cytokines. In various animal models, manipulation of the type and amount of fat and carbohydrate in the diet can influence the development and severity of liver injury.

Sclerosing hyaline necrosis

Sclerosing hyaline necrosis, described by Edmondson et al., is not a separate pattern of alcohol-induced liver injury but rather forms part of the morphological spectrum of alcoholic hepatitis, characterized by extensive perivenular liver cell necrosis associated with the deposition of fibrous tissue ( Fig. 5.21 ). The terminal hepatic venules may become occluded, and portal hypertension can occur in the absence of cirrhosis. Initially, fibrosis is seen only in the perivenular region and in association with foci of necrosis (see Fig. 5.10 ).

Sclerosing hyaline necrosis. Mild fatty change, mild alcoholic hepatitis and several Mallory-Denk bodies are apparent in the region of a hepatic venule. Increased amounts of fibrous tissue are seen in the parenchyma adjacent to the venule. (H&E stain.)

With progression, more extensive pericellular fibrosis is seen. More severe bridging necrosis between adjacent terminal hepatic venules or between terminal hepatic venules and portal tracts results in condensation of pericellular fibrosis tissue and the formation of septa. Elastic fibres, which stain with orcein, can be seen in active fibrous septa, but not in areas of passive collapse of the reticulin framework.

Cholestatic syndromes

Alcoholic liver disease may present with clinical and biochemical features that are strongly suggestive of extrahepatic biliary obstruction. Features include jaundice, right upper quadrant pain and tenderness, hepatomegaly and marked elevation of serum bilirubin, and alkaline phosphatase and cholesterol levels. Biliary obstruction caused by gallstones or alcoholic pancreatitis can be excluded by ultrasound, transhepatic cholangiography, endoscopic retrograde cholangiopancreatography (ERCP) or magnetic resonance cholangio­pancreatography (MRCP).

A high index of clinical suspicion of intrahepatic cholestasis is required to avoid unnecessary surgery in patients with ALD, because the risk of postoperative hepatic and renal failure is considerable. Severe cholestasis has been described in association with: fatty liver ( Fig. 5.29 ), alcoholic foamy degeneration, alcoholic hepatitis, decompensated alcoholic cirrhosis and Zieve syndrome, which comprises alcoholic steatosis, jaundice, hyperlipidaemia and haemolytic anaemia.

Fatty liver and cholestasis. The hepatocytes show fatty change, and bile is present in some canaliculi. (H&E stain.)

There is a significant correlation between tissue cholestasis and mortality in ALD. The possibility that jaundice occurring in patients with a history of excess alcohol consumption may be caused by nonalcoholic liver disease, such as viral hepatitis or drug-induced cholestasis, must also be considered.

In patients with a severe fatty liver who develop a cholestatic syndrome, the liver biopsy may show portal tract changes with oedema, increased prominence of marginal bile ducts and a mild to moderate cholangitis with a neutrophil polymorph infiltrate, so-called microscopic cholangitis.

Portal tract changes

Portal fibrosis has not been considered to result from classic alcohol injury. However, it may sometimes be a feature; Morgan et al. found that the presence of portal fibrosis correlated with a previous history of viral hepatitis or episodes of acute pancreatitis. Michalak et al. suggested that portal fibrosis is part of progressive ALD. In their experience, portal and septal fibrosis was more frequent than perivenular fibrosis. Furthermore, portal fibrosis appeared to contribute to a greater proportion of overall fibrosis than did perivenular scarring. These intriguing findings merit further investigation.

Increased numbers of portal tract macrophages are seen frequently in ALD at virtually all stages. Also, macrophages greatly enhanced lysosomal enzyme release compared with normal liver, suggesting that cytotoxic mediators released by these activated cells may be contributing to liver injury. Rakha et al. described ‘more than mild’ portal inflammation in 47% of biopsies from patients with alcoholic fatty liver disease.

Hepatic siderosis

Excess stainable iron is found in both hepatocytes and Kupffer cells in many patients with ALD. In one study, 57% of patients had mild siderosis and 7% had grade 3–4 siderosis. The explanation for the excessive iron accumulation is still unclear but suggests increased iron absorption, attributed to a variety of factors: the high iron content of some alcoholic beverages, in particular home-brewed beer, which in sub-Saharan Africa is made in iron pots ; a direct effect of alcohol on the small intestine ; haemolysis associated with spur cell leading to increased iron absorption ; and upregulation of the transferrin receptor. It is now generally accepted that only mild siderosis, grade 1–2, is alcohol related, so-called alcoholic siderosis. Patients with ALD in whom iron accumulation is massive (grade 3–4) may also have hereditary haemochromatosis (see Fig. 5.15 ). Nevertheless, iron overload can be seen in extrahepatic tissues of patients with end-stage ALD in the absence of HFE mutations.

In alcoholic siderosis, the iron-containing hepatocytes are distributed randomly, and the iron granules are often few in number. This contrasts with the zonal distribution of iron, with a periportal predominance, and often a pericanalicular distribution in the hepatocytes in hereditary haemochromatosis.

Before the identification of genetic abnormalities involved in iron metabolism, alcoholic siderosis and hereditary haemochromatosis were differentiated by biochemical measurement of the hepatic iron concentration and calculation of the hepatic iron index (HII; hepatic iron concentration divided by patient’s age). An HII >2 is virtually diagnostic of hereditary haemochromatosis, but calculation of the HII has been largely replaced by genotyping. If genotyping is not available, however, and when marked iron overload is an unexpected finding in a liver biopsy specimen, the hepatic iron, as demonstrated by the Perls method, can be measured by image analysis, or the remainder of the liver tissue can be removed from the paraffin block and the iron concentration measured biochemically in the dewaxed specimen.

The liver of chronic drinkers who are homozygous or heterozygous for the HFE (C282Y) mutation, or one of the less common mutations involved in iron overload, may show evidence of both hepatic iron overload and alcohol-related liver injury. A biopsy study of 206 patients with HFE -associated haemochromatosis showed that cirrhosis was almost nine times as common in those who drank >60 g of alcohol daily than in those who drank <60 g. In such patients, episodes of alcoholic hepatitis, in which iron is released from dead hepatocytes, can lead to an altered distribution of liver iron, with haemosiderin appearing in Kupffer cells and portal tract macrophages.

Both primary and secondary hepatic iron overload appear to potentiate liver injury in humans. Similarly, dietary iron overload potentiated alcohol/hepatotoxin-mediated fibrosis and cirrhosis in an animal model. Possible mechanisms for this potentiation of hepatic fibrosis included increased amounts of lipid peroxidation, iron-induced activation of hepatic stellate cells and iron-dependent activation of nuclear factor κB (NFκB) in Kupffer cells, leading to the production of proinflammatory cytokines. The effects of alcohol on iron storage diseases of the liver have been reviewed.

Porphyria cutanea tarda

Alcohol is thought to hasten the onset of the hepatic and cutaneous manifestations of porphyria cutanea tarda; alcohol withdrawal is followed by a dramatic clinical and biochemical improvement. The hepatocytes may contain needle-shaped cytoplasmic inclusions of uroporphyrin which show brilliant-red autofluorescence under ultraviolet light; this is specific for porphyria cutanea tarda. Variable degrees of siderosis are usually present in the liver, and there may also be evidence of ALD.

Hepatocellular carcinoma

Hepatocellular carcinoma (HCC) develops in 5–15% of patients with alcoholic cirrhosis, usually in association with macronodular cirrhosis ( Fig. 5.30 ). In Western countries, alcohol may be the most common cause of HCC, accounting for 32–45% of cases. Tumours are often seen in association with a combination of alcoholic and viral liver disease (HBV and HCV related) but are also frequently described in patients with negative markers for viral hepatitis. Some evidence now indicates that alcohol may cause genetic alterations. In addition, alcohol can act as a co-carcinogen because of its ability to induce the hepatic microsomal P-450-dependent biotransformation system, in particular the ethanol-inducible isoenzyme cytochrome P-450, leading to enhanced activation of a variety of procarcinogens present in food, tobacco smoke and alcoholic beverages. A study of American patients with cirrhosis, with and without HCC, showed that alcohol, tobacco and obesity were independent, synergistic risk factors for HCC development.

Hepatocellular carcinoma arising in a liver showing macronodular cirrhosis. This 58-year-old man had a past history of heavy drinking but had abstained for several years. (H&E stain.)

Alcohol is recognized as an important potentiating factor for HCV-associated HCC. Yamauchi et al. reported a 10-year cumulative HCC occurrence rate of 80.7% in patients with alcoholic cirrhosis who were anti-HCV positive and drank >120 g alcohol daily. In contrast, 18.5% of those with alcoholic cirrhosis alone developed HCC over 10 years, and the 10-year rate for tumours in HCV-related cirrhosis in nondrinkers was 56.5%. One review of alcohol and HCC reported that chronic alcohol consumption >80 g/day for >10 years increases the HCC risk almost fivefold, and that alcohol doubles the risk of this tumour in CHC compared with the risk with the virus alone.

Autopsy studies in the past, as well as recent prospective studies of liver biopsies, have suggested that dysplastic nodules, which develop within cirrhotic nodules, especially macroregenerative nodules (>5 mm diameter), may be the precursor lesion for HCC. A prospective liver biopsy study of radiologically identified hepatic nodules concluded that an increased ratio of nuclear density >1.5, clear-cell change and small-cell change are features indicating a high risk for evolution to HCC.

Lee et al. evaluated large-cell change, seen as foci of hepatocellular enlargement, nuclear pleomorphism, hyperchromasia and multinucleation, and concluded that this feature is an independent risk factor for HCC, with an estimated odds ratio of 3.3. However, large-cell change is not thought to be a direct precursor of HCC, but rather an indicator of increased risk of tumour development. A comment about the presence or absence of both small-cell and large-cell change should always be included in reports of cirrhotic livers.

Recent epidemiological studies have demonstrated an increased prevalence and incidence of intrahepatic cholangiocarcinoma and a relationship to the presence of cirrhosis. Interestingly, Wu et al. described biliary intraepithelial neoplasia lesions in the livers of patients with alcoholic cirrhosis, suggesting that these may be precursor lesions for the development of biliary malignancy.

Adult disease

The absolute worldwide prevalence of NAFLD is not known. Estimates vary among populations and are influenced by ethnicity, dietary patterns and method of ascertainment. Arguably the most accurate noninvasive technique to detect NAFLD is ¹ H-magnetic resonance spectroscopy ( H-MRS) quantification of hepatic triglyceride content (HTGC), with NAFLD defined as an HTGC >5.56%. Applying these criteria to a population sample of 2349 North American adults, the Dallas Heart Study reported an overall prevalence of approximately 31% across ethnicities (45% of Hispanics, 33% of Caucasians, 24% of African Americans). More recently, a meta-analysis incorporating >8.5 million individuals across 45 studies determined that NAFLD affects approximately 25% of the adult population worldwide. Although prevalence varied somewhat between continents, it was strikingly similar in the United States and Europe (24.1% and 23.7%, respectively), with higher levels reported in the Middle East, South America and Asia (31.8%, 31.5% and 27.4%, respectively) and lower levels in Africa (13.5%). The prevalence of NAFLD was also influenced by gender (42% white males versus 24% white females).

Because of the reliance on histological assessment for its detection, potentially influencing selection of cases for liver biopsy, estimates of NASH prevalence are subject to greater selection and ascertainment bias. The most robust of the limited data available come from histological assessment of apparently healthy prospective liver donors, in whom the prevalence of NASH was estimated at 3–16% in Europe and 6–15% in North America. In the meta-analysis, 7–30% of histologically characterized NAFLD patients without a clear indication for liver biopsy exhibited features of NASH. This equates to a NASH prevalence of 1.5–6.5% of the global general population.

As expected, prevalence of NAFLD increases substantially in groups with recognized risk factors for metabolic syndrome. In an unselected southern European population sample, 91% of obese patients (BMI ≥30 kg/m ² ), 67% of overweight patients (BMI 25–30 kg/m ² ) and 25% of normal-weight individuals had radiological evidence of NAFLD. Similarly, 73–97% of bariatric patients had NAFL and 25–33% had NASH. Among patients with type 2 diabetes, 40–70% had NAFLD.

Although the prevalence of most liver diseases is stable, the prevalence of NAFLD is rising and placing an increasing burden on already overstretched health care systems worldwide.

Paediatric disease

Paediatric NAFLD is now recognized as the most common cause of chronic liver disease in children worldwide. The increased incidence of NAFL and NASH in children currently is not restricted to Western populations and has paralleled the dramatic increase in obesity and type 2 diabetes mellitus in this age-group. Lifestyle changes, including a shift to more sedentary, less physically demanding activities, as well as dietary exposure to high fats and refined sugars, are implicated. Despite these facts, obesity and obesity-associated liver disease remain under-recognized by primary caregivers.

The prevalence of NAFLD in obese children and adolescents in developed countries based on elevated liver enzymes or ultrasound has been estimated at 10–77%, although ultrasound screening and elevated alanine transaminase (ALT) levels both underestimate the true prevalence of fatty liver. A histological autopsy study showed an overall prevalence of fatty liver of 9.6% in children in southern California, with older age in the paediatric range, obesity and Hispanic ethnicity being major risk factors. Paediatric NAFLD is reported to increase with body weight, up from 13–15% to 30–80% in obesity. Boys are more often affected by NAFLD than girls: 12.4% versus 3.5% in a screening study and 14.4% versus 7.4% in obese children ; both studies used serum ALT. Ethnic susceptibility is also varied in children; as in adults, obese African American children had decreased prevalence of clinical NAFLD in a series from an urban clinic based on ALT elevation. The National Health and Nutrition Examination Survey (NHANES) showed elevated serum ALT in 7.4% of Caucasian children, 11.5% of Mexican American children and 6.0% of African American children. Literature reviews indicate that similar factors and markers of inflammation are present in paediatric NAFLD as in adult disease.

Weiss et al. reported increased incidence of clinical features for cardiovascular risk factors associated with insulin resistance, now identified by a unified term, ‘metabolic syndrome’, and with severity of obesity in 490 children and adolescents, none of whom was diabetic or taking medications to lower blood pressure, lipids or glucose. All the findings were independent of age or pubertal status, confirming that well-recognized features of metabolic syndrome in adults are present in children. In their 2-year follow-up of 77 patients, metabolic syndrome persisted in all affected patients, and eight had progressed to type 2 diabetes. Schwimmer et al. further demonstrated that each feature of metabolic syndrome was more likely to occur in 150 obese children with NAFLD than in 150 obese children without NAFLD.

Beyond the classic metabolic syndrome-related forms of NAFLD, inherited conditions that share the common features of obesity and impaired glucose tolerance, insulin resistance or type 2 diabetes include Bardet–Biedl syndrome, Alström syndrome and Prader–Willi syndrome. A case report of a lean child with an inherited multisystem triglyceride breakdown and storage disorder, Dorfman–Chanarin syndrome, documented steatohepatitis and cirrhosis. Hypothalamic dysfunction with subsequent obesity is a recognized complication of many forms of treated childhood cancer. The rare disorder pseudoneonatal adrenoleukodystrophy, caused by homozygous deficiency of acyl-CoA-oxidase, an enzyme essential for fatty acid oxidation, is associated with hepatic steatosis. NASH has also been described in Werner syndrome, a rare autosomal recessive disease characterized by the premature onset of multiple age-related disorders.

Adult disease

With obesity and insulin resistance rapidly becoming the norm in the general population of many countries, a large proportion of the population have NAFLD. Paradoxically, however, only a minority progress to advanced liver disease characterized by hepatic fibrosis, cirrhosis or hepatocellular carcinoma (HCC) and thus experience liver-related morbidity. NAFLD is therefore characterized by substantial interindividual variation in disease severity and outcome, with liver-related mortality occurring in <5% of patients and being the third most common cause of death after cardiovascular disease and extrahepatic malignancy.

Historically, dogma has dictated that, within the NAFLD spectrum, ‘simple’ steatosis (NAFL) is largely a benign condition with minimal risk of progression or clinical sequelae, and that NASH is the progressive and prognostically relevant form of the disease. Although much remains to be learned about the natural history and prognostic features of NAFLD, several recent ‘serial liver biopsy’ and ‘longitudinal follow-up’ studies have provided new insights into disease natural history that have important implications for the routine management of patients. Biopsy studies provide detailed information on how histological features of NAFL and NASH evolve and the rate at which fibrosis progresses. Follow-up studies describe the clinical disease course in patient cohorts and progression to specific endpoints (e.g. death, transplantation).

A systematic review and meta-analysis of cases with serial liver biopsy data, comprising 411 histologically characterized NAFLD patients (150 NAFL, 261 NASH) drawn from 11 small studies, demonstrated the dynamic nature of hepatic fibrosis in NAFLD. During more than 2145 person-years follow-up, 34% of patients exhibited fibrosis progression, 43% showed stable disease, and disease regression was observed in the remaining 23%. NAFL patients without fibrosis on baseline biopsy exhibited fibrosis progression at a rate of 0.07 stage/year on average, whereas NASH patients progressed more rapidly, at 0.14 stage/year. These rates equate to a one-stage increase in fibrosis over 14 years in NAFL and 7 years in NASH. Of great clinical relevance, disease progression occurred irrespective of whether the index biopsy showed NAFL or NASH; about 20% of patients showed rapid fibrosis, with progression from stage F0 to F3/4, whereas the remaining 80% had minimal progression. These findings were independently validated by a subsequent large, single-centre UK study. In 108 NAFLD patients with serial liver biopsy data covering a median interval of 6.6 years, 42% had fibrosis progression, 40% had stable disease, and disease regression was seen in 18%. No significant difference in the proportion exhibiting fibrosis progression was observed between patients with NAFL (37%) and those with NASH (43%) on index biopsy, confirming that the distinction between NAFL and NASH is of limited prognostic value. However, all patients in whom fibrosis progression was observed also exhibited features of NASH on the follow-up biopsy, suggesting that although NASH may not be present in the early phases of disease, it is a necessary pathogenic driver of fibrosis progression. The development of type 2 diabetes between biopsies was associated with greater fibrosis. Indeed, 80% of the patients with NAFL on initial biopsy who subsequently showed fibrosis progression had also developed type 2 diabetes in the interval between biopsies, compared with 25% of nonprogressors.

Longitudinal cohort studies that have followed patients representing the full spectrum of histological NAFLD at enrollment have provided important insights into long-term disease outcomes, including liver transplantation (LT) and death. Relative to a population reference sample, a cohort of 229 European NAFLD patients showed a 29% increase in all-cause mortality during an average 26 years of follow-up. The presence of NAFLD was associated with a modest increased risk of cardiovascular disease (1.29-fold) but a 3.2-fold increased risk of cirrhosis and a 6.5-fold increased risk of HCC. In multivariate analysis, only fibrosis stage at enrollment was significantly associated with overall long-term mortality, cardiovascular disease or cirrhosis. In a cohort of 619 North American patients with a median follow-up of 12.6 years, the long-term risk of LT or liver-related death was detectable even at mild stages of hepatic fibrosis. This risk increased with fibrosis stage: compared to patients with stage F0 disease (no fibrosis), those exhibiting F1/2 (mild) fibrosis had a 11.2-fold increased risk of LT or death, and those with F4 (advanced fibrosis/cirrhosis) had an 85.8-fold increased risk. Once again, the presence or stage of fibrosis, rather than discrimination of NAFL or NASH at index biopsy, was of greatest prognostic value.

It is believed that the presence or stage of fibrosis is of greater prognostic value than discriminating NAFL and NASH (despite NASH being pathogenetically necessary for fibrogenesis) because there is a greater chance of fibrosis being present in patients with NASH than in those with NAFL, which affects the outcome of multivariate analyses. This is corroborated by two additional studies, which examined 118 patients with biopsy-confirmed NAFLD followed for a median of 21 years and 209 NAFLD patients with a median 12 years of follow-up. In the former, no difference in overall or liver-related mortality was seen between NAFL and NASH patients, but those who died were more likely to have evidence of fibrosis, often stage F2 or greater. In the latter study the presence of NASH only correlated with liver-related mortality if fibrosis was included in the definition. Indeed, when the individual histological features were analyzed, only stage F3/4 was independently associated with liver-related mortality, with a 5.68-fold hazard ratio. Studies showing that noninvasive scoring systems such as the NAFLD Fibrosis Score (discussed later) are capable of predicting liver-related events, LT and death support the prognostic importance of fibrosis in NAFLD.

Based on current evidence, these findings are likely the result of a dynamic bidirectional flux between the states of NAFL and NASH, while changes in fibrosis stage occur much more slowly. Thus at liver biopsy, some patients exhibit only NAFL but will be developing NASH and, if susceptible, will ultimately progress to advanced fibrosis and experience adverse liver outcomes. In contrast, others exhibit NASH but may be regressing to NAFL. Therefore the presence or absence of NASH at biopsy provides limited long-term prognostic information. It does appear, however, that patients with only very mild/moderate steatosis in the absence of inflammation may be at the lowest risk of progression. Also, fibrosis progression in most cases is slow (F0 to F1 in ~8 years), although a subgroup of patients are ‘rapid progressors’ and will quickly progress three or even four stages over 2–6 years. In the most recent study, those with NASH were on average 9 years older than patients with NAFL, and 44% of NAFL patients had developed NASH during a median 8 years of follow-up. NASH may therefore simply reflect longer duration of disease, and NASH usually develops after steatosis.

Hepatocellular carcinoma may develop in a background of NASH-related cirrhosis (see later HCC section). Small series and numerous case reports indicate that HCC may also develop on a background of noncirrhotic NASH, but the extent to which this occurs is not well defined.

Paediatric disease

A long-term follow-up of 66 children over 20 years showed the potential for rapid progression to cirrhosis in NASH (4.75–6.6 years in two of five cases with multiple repeat biopsies), recurrence of ‘aggressive NASH’ in a transplanted liver and lower overall survival of affected children compared with age- and gender-matched children.

Steatosis (nonalcoholic fatty liver)

The accumulation of triglyceride in the liver was once thought to contribute to impaired cellular function and cellular injury. However, data published over the past decade have suggested that triglyceride accumulation is relatively inert, although it does serve as an indicator of an increased flux of fatty acids, a defect in the secretion of triglyceride, or both. Therefore from the standpoint of pathogenesis, the accumulation of triglyceride as steatosis and the development of cellular injury should be considered separately. As a generalization, nonalcoholic fatty liver (NAFL) can be considered a consequence of an oversupply of fatty acids coupled with a contribution from defects in their metabolism ( Fig. 5.33 ).

Central role of lipotoxicity in nonalcoholic steatohepatitis (NASH). The emerging lipotoxicity model of NASH pathogenesis is based on the generation of nontriglyceride fatty acid metabolites primarily responsible for the hepatocellular injury and death that characterize NASH. The primary sources of hepatocellular fatty acids are uptake of circulating free fatty acids and synthesis of new fatty acids ( de novo lipogenesis). Fatty acids are normally eliminated by oxidative and secretory pathways. When these pathways are impaired or overwhelmed, lipotoxic species can be formed. The accumulation of triglyceride in lipid droplets may be an adaptive protective response that occurs when the formation and secretion of triglyceride as very-low-density lipoprotein (VLDL) is insufficient to handle the amount of triglyceride synthesized. Supply-side factors that predispose to NASH include insulin resistance at the level of adipose tissue resulting in failure to suppress lipolysis and excessive de novo lipogenesis in the liver caused by an overabundance of substrate, typically carbohydrates. A minor contributor to the hepatocellular free fatty acid burden is the uptake of circulating lipoprotein remnants (e.g. chylomicron remnants, low-density lipoprotein remnants). A potentially important source of hepatocellular free fatty acids not shown is the turnover of lipid droplets, either through lipolytic enzymes such as adipose triglyceride lipase or lysosomal breakdown of autophagosome contents. Thus, formation of lipid droplets is only a temporizing measure, and the liver must be able to handle the fatty acids released from this pool at some point. Oxidative pathways generate reactive oxygen species that can be associated with oxidant stress, but a universal role of this process in the pathogenesis of NASH has not been demonstrated. ER, Endoplasmic reticulum; SER, smooth endoplasmic reticulum.

Hepatocyte micro- and macrovesicular steatosis represent the accumulation of triglyceride in droplets surrounded by a phospholipid monolayer membrane. Hepatocytes synthesize triglyceride from free fatty acids, a property shared only by small bowel enterocytes and mammary gland epithelia. The fatty acids used to make triglyceride are mostly derived from the uptake of circulating fatty acids, the latter produced by adipocyte lipolysis. A small fraction of fatty acids in hepatocytes are synthesized through the process of de novo lipogenesis (DNL) using excessive carbohydrate and amino acids as precursors for the acetyl-CoA that feeds into the DNL pathway. An elegant study using stable isotope tracers demonstrated that this synthesis of new fatty acids normally contributes only about 5% of the fatty acids used for hepatocyte triglyceride production, but that this fraction is increased to about 25% in NASH patients. Liver biopsies with NAFLD associated with the metabolic syndrome typically show hepatocytes containing a mix of large-droplet fat that pushes the nucleus to the periphery and small-droplet fat. In contrast to this variable mix of large- and small-droplet fat, the presence of a foamy appearance caused by accumulation of microvesicular fat is often associated with mitochondrial dysfunction. Mitochondrial dysfunction may contribute to the pathogenesis of NASH and clearly plays a major role in ALD (see earlier), perhaps explaining why microvesicular fat is more common in ALD than NAFLD.

Steatohepatitis (nonalcoholic steatohepatitis)

In NASH the criteria for establishing the diagnosis of steatohepatitis are based on the presence of abnormalities associated with significant hepatocellular injury in the setting of steatosis. An emerging viewpoint based on in vivo studies and human data is that steatohepatitis is caused by fatty acid metabolites other than triglyceride, and that the accumulation of triglyceride should be considered as a visible marker of an increased metabolic burden as the liver handles excessive fatty acids, either from peripheral lipolysis or excessive DNL (see Fig. 5.33 ). Diversion of fatty acids into the triglyceride lipid-droplet pool may actually represent an adaptive protective pathway that prevents fatty acids from being used by metabolic pathways that produce lipotoxic intermediates. Establishing the identities of the molecular species responsible for lipotoxic liver injury remains an area of ongoing investigation. One possible candidate is lysophosphatidylcholine, the product of removing a fatty acid acyl group from phosphatidylcholine (lecithin). Additional candidates include free fatty acids themselves, ceramides, phosphatidic acids, diacylglycerols and the many downstream metabolites of these molecules. The number of double bonds in fatty acids, their relative positions and whether they are in the natural cis configuration or the synthetically created trans configuration also appear to be important. Fully saturated fatty acids (i.e. those without any double bonds) are lipotoxic in cell culture systems, and trans fats have been shown to cause steatohepatitis in mice, whereas PUFAs such as those found in fish oil are currently being evaluated in clinical trials as possible therapeutic agents for NASH.

Even when lipotoxic fatty acid metabolites capable of causing the phenotype of NASH are found, this mechanism is unlikely to be the sole cause of steatohepatitis in all patients with NASH. The phenotype currently identified as steatohepatitis in the absence of alcohol abuse likely represents multiple underlying disease mechanisms, either present singly or in combination. Pathogenetic factors in addition to, or perhaps caused by, the accumulation of lipotoxic species include ER stress, oxidative stress, mitochondrial dysfunction, membrane cholesterol accumulation, excessive exposure to gut-derived endotoxin and dysregulated adipokinesis. Additional recognized environmental factors that could play a role in some patients include intermittent hypoxia from OSA and changes in gut flora.

Drugs

A variety of drugs have been implicated as causing NAFLD (see Tables 5.1 and 5.2 ). The most compelling data for drug-induced NASH are provided by studies of patients treated with tamoxifen who clearly developed imaging evidence of NAFLD after beginning the drug and on further evaluation were found to have NASH. The causative role of tamoxifen has been further established by the demonstration of improvement when tamoxifen was discontinued. However, several studies have shown that components of the metabolic syndrome are also risk factors for the development of NASH during tamoxifen treatment, suggesting that tamoxifen accentuates pre-existing metabolic abnormalities to cause NASH in some women.

Drug- and toxin-induced fatty liver disease is discussed in more detail later in the section on ‘NAFL and NASH in other clinical settings’.

Imaging

The three most common imaging modalities—ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI)—can each detect steatosis with increasing sensitivity and cost, in that order. US is relatively insensitive, requiring at least 30% fat for consistently detectable increased echogenicity that characterizes steatosis and is qualitative rather than quantitative. Non-contrast-enhanced CT demonstrates steatosis as having a lower liver density than normal, but also lacks sensitivity for lesser amounts of fat. MRI and H-MRS are the most accurate modalities for detection of steatosis and can provide quantitative information, allowing detection of changes in hepatic lipid content. Currently, no routinely available imaging modality has been robustly demonstrated to distinguish NAFL from NASH accurately or to detect early stages of fibrosis.

Noninvasive markers of steatohepatitis and NASH-related fibrosis

Performing liver biopsies on every patient suspected of having NAFLD is neither possible in terms of health care resources nor recommended from a risk/benefit standpoint. A more pragmatic approach initially employs noninvasive strategies, including both serological tests and imaging techniques, reserving liver biopsy for patients in whom it offers clinically relevant additional information. To date, no noninvasive test to reliably distinguish NAFL from NASH has been developed, although this remains an area of active research. Tests for fibrosis stage, both commercial and noncommercial, have been more promising. Serological tests may be divided into ‘indirect’ markers (reflecting alterations in hepatic function, e.g. AST/ALT ratio) and ‘direct’ markers (biologically related to fibrogenesis and extracellular matrix turnover). These noninvasive tests typically perform best at the ends of the spectrum of disease, discriminating normal liver or cirrhosis. The NAFLD Fibrosis Score and the FIB-4 Score are calculated using standard clinical parameters such as age, presence of type 2 diabetes, BMI, AST/ALT ratio, albumin level and platelet count. NAFLD Fibrosis Score values <−1.455 effectively exclude advanced fibrosis (negative predictive value [NPV] 93%) and values >0.676 predict presence of advanced fibrosis (positive predictive value [PPV] 90%). The FIB-4 Score appears to be one of the best simple noninvasive tests for advanced fibrosis and narrowly outperforms the NAFLD Fibrosis Score. In general, simple panels have relatively robust NPV and thus can reliably exclude advanced fibrosis, but have poor PPV. The NAFLD Fibrosis Score has been shown to be predictive of long-term disease outcome and mortality. Noninvasive testing has been recently reviewed.

Elastography

Liver fibrosis reduces tissue elasticity, making measurement of liver stiffness an attractive surrogate for fibrosis severity. Transient elastography (Fibroscan), an imaging technique employing US monitoring of the passage of a low-frequency pressure wave through liver tissue, has been found to be a promising noninvasive technique for the detection of advanced fibrosis caused by chronic viral hepatitis and NASH. Although abdominal obesity may compromise its utility in NAFLD, this technique has an Area Under Receiver Operating Characteristic (AUROC) of 0.84 and 0.93 for the detection of ≥F2 and ≥F3 fibrosis, respectively, and when low liver stiffness measures are obtained, elastography does reliably exclude advanced fibrosis. Novel alternatives, including acoustic radiation force impulse imaging (ARFI) and magnetic resonance elastography (MRE), are emerging but currently are not widely adopted.

Steatosis

In noncirrhotic NAFLD, steatosis is macrovesicular and primarily located in acinar zone 3 or zones 3 and 2 in adults (i.e. sparing zone 1), in contrast to periportal or nonzonal steatosis in children or chronic hepatitis C. Steatosis was present in 100% of 433 cases of NASH from 13 series between 1979 and 2000. Studies that use Oil Red O stain for evaluation of the presence and amount of steatosis often report much greater amounts than those with standard H&E staining, and most pathologists agree that special stains for macrovesicular fat are not required. This type of steatosis is characterized by a single large droplet that occupies all (or nearly all) the hepatocytic cytoplasm, leaving the nucleus eccentrically placed (large-droplet macrovesicular steatosis). There may also be hepatocytes with combinations of single large droplets and multiple smaller droplets that are often clustered around the larger droplet (small-droplet macrovesicular steatosis). Usually, a combination of small- and large-droplet macrovesicular steatosis is seen in NAFLD.

In NAFLD, as in ALD, clusters of hepatocytes with delicate intracytoplasmic septations caused by microvesicular steatosis may occur and are often nonzonally located ( Fig. 5.35 ). Mixed steatosis, when both macro- and microvesicular types coexist, is common in NAFLD, but pure microvesicular steatosis has not been reported in NAFLD to date. The significance of microvesicular steatosis patches in NAFLD has not yet been elucidated, but their presence correlates with increased steatosis severity and progressive disease.

Nonalcoholic fatty liver disease. A portion of the parenchyma shows macrovesicular steatosis, while another area is predominantly microvesicular. (H&E stain.)

Lipid droplets are dynamic and metabolically active organelles with a central core of triacylglycerols and cholesterol esters and a peripheral layer of phospholipids with associated proteins of the perilipin/PAT family. These include perilipin, adipophilin, TIP47, S3-12 and MLDP, newly named as perilipins 1–5 to unify nomenclature and expand for newly identified molecules. Perilipins are sequentially expressed during the formation of intracellular lipid droplets, with TIP47- and MLDP-positive microvesicular steatosis evolving over time to adipophilin- and perilipin 1-positive macro­vesicular steatosis. The differences in lipid-droplet PAT–protein composition may be responsible for the different clinical significance of micro- and macrovesicular steatosis and could aid the distinction between acute and chronic steatosis. The rs738409, I148M sequence polymorphism in patatin-like phospholipid domain-containing protein (PNPLA3) has been correlated with NAFLD development. The inactive PNPLA3 accumulates on the surface of lipid droplets and is associated with an increase in macrovesicular steatosis. In NAFLD, accumulation of triacylglycerols within intracytoplasmic droplets may actually protect hepatocytes from the detrimental effect of non-droplet-bound, lipotoxic, saturated free fatty acids.

To date, the amount of steatosis required to be considered ‘pathological’ is not truly known; sophisticated imaging quantification studies have calculated that 5.6% hepatic lipid content is ‘normal’ in the American population, which approximates a commonly accepted normal value for liver steatosis of 5%, based on lipid content measurements. Steatosis >5% is therefore the minimum criterion for a histopathological diagnosis of NAFLD. Common semiquantitative assessments for steatosis are based on the percentage surface area of a histological section involved or the percentage area of uninvolved parenchyma. Zonal architecture can be a useful guide: zone 3 versus zone 1 predominance. On light microscopy, subdivisions of thirds is relatively straightforward because this incorporates the acinar divisions of the liver into zones 3, 2 and 1; thus the assessments are ≤33% (or 0–5%, 5–33%), 34–66% and >66%. The severity of steatosis may be converted into mild, moderate or severe, respectively. Although a comparison of this approach with stereological point counting suggested that semiquantitative analysis overestimates the area/volume of fat content, especially in severe steatosis, other studies assessing biochemical measurements of triglyceride have shown close association and concluded that the histopathological estimation remains useful. The accuracy of estimating the extent of steatosis with the microscope can be increased with the use of guideline images. Recently, digital image analysis (DIA) has been proposed as an objective method to quantify steatosis in liver tissue.

In patients with extensive steatosis, reticulin loss may be observed independent of the severity of inflammation or stage of fibrosis. Steatosis may ‘burn out’ during NAFLD progression and is frequently absent in NAFLD-related cirrhosis.

Steatohepatitis

Hepatocyte ballooning is considered a manifestation of significant cell injury ( Fig. 5.36 ). This finding remains a ‘required’ lesion of NASH in adults for many investigators. In the seminal study that proposed four types of NAFLD based on histology, clinically significant outcomes (cirrhosis and liver death) correlated with progressive NAFLD, and ballooning was an identified characteristic of the progressive forms of NAFLD types 3 and 4. The underlying pathogenetic mechanisms that result in ballooning in NASH are not fully understood. Recent studies have shown that ballooned hepatocytes in NASH produce the ligand sonic hedgehog (Shh) that can initiate Hedgehog pathway signalling promoting fibrogenesis and can also serve as an autocrine survival factor during lipotoxic stress. Shh-positive ballooned hepatocytes are found in zone 3 and are frequently surrounded by collagen fibres (pericellular fibrosis) ( Fig. 5.36 B and D ). Their number increases with NASH severity and disease progression. Interestingly, in the PIVENS clinical trial, therapy of aggressive NASH with the antioxidant vitamin E, but not the insulin sensitizer pioglitazone, improved steatosis and lobular inflammation and reduced the number of Shh-positive hepatocytes. This reduction also correlated with an improvement in serum ALT and AST values reflecting a decrease in liver injury. In NASH, loss of cytoplasmic K8/18 immunostain and Shh-positive cytoplasmic immunostain may serve as objective markers of ballooned hepatocytes ( Fig. 5.36 C and D ).

Nonalcoholic steatohepatitis. A, Ballooned hepatocytes are more prominent in zone 3 admixed with steatosis; the involved hepatocytes are enlarged and swollen and may or may not contain Mallory–Denk bodies. B, Ballooned hepatocytes are surrounded by collagen fibres (CAB stain). C, Loss of cytoplasmic K8/18 immunostaining in ballooned hepatocytes. D, Ballooned hepatocytes are decorated by antibodies specific for sonic hedgehog (Shh) (immunostain for Shh).

Courtesy of Dr. Carolin Lackner.

Lobular inflammation is common in NASH, usually mild and composed of mixed infiltrates ( Fig. 5.37 ), in which polymorphonuclear leukocytes may be a minor (or even absent) component. In the early reported series of NASH, polymorphonuclear leukocytes were noted in 56–100% of cases in series where the feature had been specifically examined. Satellitosis is less common in NASH than in ALD. However, pathologists disagree about the requirement of polymorphs in NASH.

Nonalcoholic steatohepatitis in metabolic syndrome. There is mixed acute and chronic inflammation in this field, where steatosis and ballooned hepatocytes are seen. (H&E stain.)

Kupffer cells may be aggregated intensely in foci of ballooning and sinusoidal fibrosis and result in potential confusion for a portal tract. On IHC, Kupffer cell enlargement and aggregation in zone 3 have been shown in cases of steatohepatitis (alcoholic or nonalcoholic), whereas in non-NASH steatosis and in normal livers, CD68+ Kupffer cells were diffusely present in the lobules as well as in portal tracts. Kupffer cells play a significant role in NAFLD pathogenesis and progression by regulating hepatic triglyceride storage, mediating inflammation, contributing to hepatocyte injury and initiating fibrosis. Their numbers in NAFL/NASH have been correlated with necroinflammatory activity, progressive injury and stage of fibrosis.

Both a diet-induced steatohepatitis animal model and a study of human liver biopsies have shown a progressive increase in natural killer T cells in liver tissue samples in the progressive forms of NAFLD, although neither described microscopic localization. Both studies indicated that these cells are involved in fibrogenesis.

Portal inflammation is relatively common in NASH, is mixed lymphocytic and mononuclear and typically mild (see Fig. 5.9 ). Neutrophils and a prominent ductular reaction are suggestive of alcoholic hepatitis; numerous plasma cells and eosinophils should suggest another, possibly concurrent disease process. To date, there are four documented settings in which portal inflammation can be noted in NAFL/NASH: (1) paediatric NAFLD, (2) severe NASH in adults and children, (3) post-treatment for NASH and (4) concurrent liver disease in NAFLD. A study of 728 adults and 205 children from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)-sponsored Nonalcoholic Steatohepatitis Clinical Research Network (NASH CRN) documented mild or greater portal inflammation in 84% of adult biopsies and 90% of paediatric biopsies. The study further showed significant correlations of portal chronic inflammation with steatosis, ballooning and advanced fibrosis in adults and portal/periportal fibrosis in children. There were no correlations with lobular inflammation scores, laboratory markers of liver injury or presence of autoantibodies. In adults, greater than mild portal inflammation correlated with BMI, female gender, increased age and markers of insulin resistance. In children, correlations were only with younger age. However, in a recent study of 430 children with biopsy-proven NAFLD, portal inflammation was independently associated with more advanced disease and features of the metabolic syndrome. In adult NAFLD, portal macrophage accumulation has been associated with hepatocyte ballooning and lobular inflammation but not with the extent of steatosis. Portal inflammation may contribute directly to fibrogenesis because it is strongly correlated with fibrosis stage and the ductular reaction. The ductular reaction represents expansion of the hepatic progenitor cell compartment in response to injury and is composed of poorly formed ductular structures within an acutely inflamed fibrous stroma. Portal inflammation may also influence the fate of hepatic progenitor cells related to the ductular reaction, regulating the balance between liver repair and fibrosis.

Increased chronic portal inflammation in NASH has also been shown in studies of treatment efficacy with paired pre- and post­intervention biopsies. Portal inflammation either increased or did not decrease in concert with the other lesions of steatohepatitis after clinical and histopathological reversal of NASH and fibrosis. Other examples include bariatric surgery and medical therapy. Another consideration for increased portal inflammation in fatty liver is in the setting of concurrent liver injury of another origin, such as chronic viral hepatitis or autoimmune liver disease, as discussed later.

Mallory–Denk bodies

In NASH, Mallory–Denk bodies (MDBs) are noted in ballooned hepatocytes, usually in zone 3, and are often described as ‘poorly formed’. In contrast, abundant and well-formed MDBs are more suggestive of alcoholic hepatitis. MDBs may be chemotactic, as described earlier, and affected hepatocytes may be surrounded by polymorphs (satellitosis) ( Fig. 5.38 ). Immunostains for p62 and ubiquitin are useful for the detection of early forms not visible in routinely stained sections (see Fig. 5.5 ). In their prognostic study of NAFLD, Matteoni et al. used MDBs as a lesion to delineate the progressive type 4 from types 1, 2 and 3. Other studies of NASH have concurred that MDBs are more often found in conjunction with higher necroinflammatory grades of steatohepatitis and other markers of progression and fibrosis in NAFLD.

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