28 -Alcoholic Liver Disease.pdf

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Chapter 2 8

Alcoholic Liver DiseaseStephen F. Stewart and Chris P. Day

ADH alcohol dehydrogenaseAMPK adenosine monophosphate

activated protein kinase

ALD alcoholic liver diseaseALDHs aldehyde dehydrogenasesALT alanine transaminaseAP-1 activator protein-1poB apolipoprotein BAST aspartate transaminaseATP adenosine triphosphateBax Bcl-2-associated x proteinBid BH3 interacting domain death agonistCOX-2 cyclooxygenase-2CT computed tomographyCTLA-4 cytotoxic T-lymphocyte

antigen-4

CYP2E1 cytochrome P450 2E1ER endoplasmic reticulumDF discriminant functionDISC death-inducing signaling complexECM extracellular matrixFFA free fatty acidsG-3P glycerol-3-phosphateGSH mitochondrial glutathione

HCC hepatocellular cancerHERs hydroxyethyl radicalsHRS hepatorenal syndromeHSC hepatic stellate cellsIgA immunoglobulin AIL-6 interleukin-6iNOS inducible NO synthaseLBP LPS binding proteinLPS lipopolysaccharideLSP liver-specific membrane lipoproteinMAA MDA-acetaldehydeMARS molecular adsorbents recycling

system

MAT methionine adenosyltransferaseMDA malondialdehydeMEOS microsomal ethanol-oxidizing

system

MMPs matrix metalloproteinasesmRNA messenger RNAMS methionine synthaseMTP microsomal triglyceride transfer

protein

NO nitric oxideNK natural killer

NKT natural killer TPAP phosphatidate phosphohydrolasePT prothrombin timePPAR- peroxisome proliferator-activated

receptor-PUFA polyunsaturated fatty acidPTX pentoxifyllineRA retinoic acidROS reactive oxygen speciesSAH S-adenosylhomocysteineSAMe S-adenosylmethionineSOD2 superoxide dismutaseSREBP-1c sterol response element

binding protein-1c

TAG triacylglycerolTGF- transforming growth factor-TLR4 Toll-like receptor 4TNF- tumor necrosis factor-TNFR1 TNF- receptor 1TRAIL tumor necrosis factorrelated

apoptosis-inducing ligand

UPR unfolded protein responseVLDL very-low-density lipoproteins

ABBREVIATIONS

IV

IntroductionAlcohol is consumed by a large percentage of the worlds pop-ulation and is an effective anxiolytic and social lubricant. A small proportion of consumers become dependent, and a moderate proportion of these, and many who are not depen-dent, develop clinically significant liver disease. These prob-lems are not new. Alcohol was recognized to be a cause of liver damage by the ancient Greeks, and is currently a common cause of liver disease in the Western world. The magnitude and range of the health and socioeconomic problems attribut-able to alcohol abuse are enormous. Cirrhosis, predominantly alcoholic, is now the fourth commonest cause of death between the ages 25 and 64 in the United States and alcohol may also make a significant contribution to cardiovascular-related mortality. The overall socioeconomic cost of alcohol abuse in the United States in terms of healthcare, crime, and loss of work capacity has been estimated at more than $180

million per year. It is therefore a significant drain on limited healthcare resources. In common with all alcohol-related disease, abstinence is the cornerstone of management in patients with alcoholic liver disease (ALD). The development of specific therapies has, however, been hampered by a con-tinued lack of a clear understanding of the mechanisms through which ethanol causes liver injury. Intense research efforts have now highlighted several important metabolic and immunologic consequences of excessive alcohol consumption that may contribute to disease pathogenesis, and it is hoped that further defining these disease mechanisms may lead to the development of novel treatment strategies. It has also become increasingly clear that individuals are not all equal in their susceptibility to ALD. Although cumulative alcohol dose undoubtedly plays a role in determining disease risk, only a small proportion of heavy drinkers go on to develop the more advanced forms of ALDhepatitis, fibrosis, and cir-rhosis. Elucidating the genetic and environmental factors

494 Section IVToxinMediatedLiverInjury

capita alcohol intake that has occurred in several countries since the late 1970s (including the United States) and has been reflected in the reduction of deaths due to cirrhosis. More recently, this decline has leveled off and, once again, there has been a rise in ALD mortality rates in some countries.3 This may be associated with a rise in alcohol consumption, but may also be due in part to the increased prevalence of obesity, now recognized to be an important risk factor for the development of ALD.4

In 2005 liver cirrhosis was the twelfth leading cause of death in the United States, and 45.9% of the cases of cirrhosis were alcohol related. These figures translate to around 28,200 deaths from cirrhosis, of which 12,900 are alcohol related.5 These deaths occur in an estimated total population of 2 million individuals with ALD of varying severities that repre-sent approximately 1 in 7 of the estimated 14 million heavy drinkers in the United States. The potential environmental and genetic explanations for this clear interindividual varia-tion in susceptibility to ALD will be discussed later. First it is important to review the putative mechanisms through which this injury occurs. Most of the studies producing the data presented next were driven by the question of how alcohol leads to liver injury. With few exceptions they fail to address the fact that most individuals appear to be remarkably resis-tant to the deleterious effects of ethanol on the liver.

PathogenesisAlthough there is good epidemiologic evidence that heavy ethanol intake can result in liver disease in some individuals, there is still much debate about the main pathogenetic mecha-nisms through which this occurs. Several mechanisms have been proposed, and data from human and animal studies support the fact that more than one is likely to be important. The first and most direct is the effect of ethanol metabolism on liver biochemistry and the resulting steatosis and oxidative stress. The second is the indirect release of cytokines as a result of the increase in gut-derived endotoxin transported to the liver via the portal vein. The third is the liver-directed adaptive immune responses generated as a result of the development of new antigens formed by the reactive intermediates pro-duced by the first two mechanisms. Many of these mecha-nisms have been elucidated using a variety of animal models. The intragastric ethanol-fed rat model designed by Tsuko-moto and French has proven to be the most useful; however, there have also been mouse, guinea pig, hamster, and primate models, each producing their own challenges. As with all animal work, there is often difficulty in interpreting the data with regard to humans. Attempts to minimize this problem lead to studies in the baboon, and, using this model, Lieber and colleagues described lesions resembling alcoholic hepati-tis and cirrhosis.6 Working with animals so closely related to humans has obvious benefits, however, difficulty replicating the experiments and cost and ethical implications have some-what limited the usefulness of this approach.7 Later sections of this chapter attempt to describe the putative mechanisms of ethanol-induced liver injury in detail and offer some theo-ries as to how they may interact. First, however, there is a brief description of the absorption, distribution, metabolism, and elimination of alcohol. Alcohol metabolism will be discussed in terms of the fate of a unit of alcohol following ingestion.

associated with disease progression would be a major step toward disease prevention. This chapter will focus on the pathogenetic mechanisms of alcoholic liver disease and the current treatments available.

EpidemiologySeveral independent studies have demonstrated a close cor-relation between deaths from cirrhosis and per capita alcohol consumption. Perhaps the best example of this is the effect of wine rationing in France during World War II, which was associated with an 80% reduction in cirrhosis deaths, followed by a return to prewar levels when restrictions were removed.1 A similar effect was observed during Prohibition in the United States. Figure 28-1 shows the decline in cases after the act was passed in 1916 and a gradual increase following the repeal of the act in 1932.

The worldwide increase in mortality from cirrhosis observed during the 1950s and 1960s was associated with a similar rise in alcohol consumption, attributed largely to the falling price of alcohol relative to income.2 Conversely, the reduction in per

Fig. 28-1 Age-adjusted death rates from liver cirrhosis by sex.States with death registration 1910-1932 and all United States 1933-2005.(Reproduced with permission from National Institute on Alcohol Abuse and Alcoholism,www.niaaa.gov.)

30

25

20

15

Rat

e pe

r 10

0,00

0 po

pula

tion

10

5

01910 1920 1930 1940 1950 1960 1970 1980 1990 2000

Year

Both sexesMalesFemales

REVISIONS OF THE INTERNATIONAL CLASSIFICATIONOF DISEASES

SecondThird

FourthFifth

SixthSeventh

EighthNinth

Tenth
http://www.niaaa.gov

Chapter 28AlcoholicLiverDisease 495

Furthermore, the higher fat content of female body composi-tion compared with a male has been invoked as part of the explanation for their higher alcohol levels following the inges-tion of similar amounts of alcohol per unit of weight.9 More than 90% of circulating alcohol is oxidatively metabolized, primarily in the liver, and excreted as carbon dioxide and water. The remainder is eliminated unchanged in the urine (


ADH activity is in the order of 1 mmol (4 mg/100 ml), which explains why alcohol follows zero-order kinetics at anything above very low blood levels. Experiments performed both in vivo and in vitro suggest that the principal regulatory mecha-nism for the ADH pathway is the capacity of the mitochondria to reoxidize NADH back to NAD.19

The Microsomal Ethanol-Oxidizing System (MEOS) PathwayIn addition to ADH, alcohol is metabolized by the MEOS; an accessory pathway that principally involves a specific alcohol-inducible form of cytochrome P450 designated CYP2E1.20 The enzyme is located on the endoplasmic reticulum, is present in greater amounts in perivenular than periportal hepatocytes and requires oxygen and NADPH. The CYP2E1 protein has been purified and the human gene cloned, sequenced, and localized to chromosome 10.21 The overall contribution of MEOS to alcohol oxidation in vivo is not yet fully clear. Its Km for alcohol is in the order of 50 to 80 mg/100 ml, so it appears to play an important role at high blood alcohol levels or fol-lowing chronic alcohol abuse, in view of its inducibility. Alcohol induction of CYP2E1, and microsomal enzyme systems in general, has also been implicated in the tolerance to various drugs commonly observed in alcoholics, and may explain their increased susceptibility to hepatotoxicity by other drugs and xenobiotics that are converted to toxic metab-olites by microsomal enzyme systems. An important example of this phenomenon is the increased susceptibility of heavy drinkers to the toxic effects of acetaminophen, where severe liver damage has been reported in alcoholics taking large, but previously considered safe, doses.22,23 More recent data suggests that this effect may, at least in part, be due to an acetaminophen-induced reduction in antioxidants that

although in normal individuals only the alcohol dehydroge-nase enzymes are important.

The Alcohol Dehydrogenase (ADH) PathwayAlcohol dehydrogenase catalyzes the oxidation of a variety of alcohols to aldehydes and ketones. This includes catalyzing the oxidation of ethanol to acetaldehyde and transferring hydro-gen to the cofactor nicotinamide adenine dinucleotide (NAD), which is converted to its reduced form, NADH. The resulting increase in the ratio of NADH/NAD, which is further increased by acetaldehyde oxidation, is partly responsible for the meta-bolic imbalances that occur following alcohol ingestion and has been considered to play a major role in the initial patho-genesis of alcohol-induced fatty liver.

Human ADH exhibits multiple isoenzymes that have been divided into five major classes on the basis of their electro-phoretic mobility, kinetic properties and inhibition by pyr-azole.16 They are encoded by at least seven different gene loci, ADH1 to ADH7, encoding the -, -, -, -, -, -, and - subunits, respectively. The class I isoenzymes are formed by random association of the -, -, and - subunits to form the active homodimeric or heterodimeric isoenzymes, whereas the others are all homodimers. The ADH2 and ADH3 genes are polymorphic, encoding three different and two different subunits with different kinetic properties resulting in isoen-zymes with different rates of alcohol oxidation in vitro.17 Expression of the ADH genes is tissue specific. The liver con-tains the highest levels of class I activity, while class III activity is present equally in all tissues. In humans, the class II isoen-zyme, -ADH has been found only in the liver while the class IV enzyme, -ADH, is present only in the stomach.18 The class I isoenzymes have by far the lowest Km and highest Vmax for alcohol and accordingly are thought to be responsible for the major part of hepatic alcohol oxidation. The overall Km of liver

Fig. 28-3 The three pathways of alcohol oxidation: alcohol dehydrogenase (ADH), microsomal ethanol-oxidizing system (MEOS), and catalase. NADPH,nicotinamide-adeninedinucleotidephosphate.

NAD

NADH

NAD

H2O2

H2O

O2+NADPH

MEOS (CYP2E1)

Acetate

Acetaldehyde

Ethanol

H2O+NADP

H2O+CO2

Alcoholdehydrogenase

Aldehydedehydrogenase

Oxidationperipheral tissues

Peroxisomalcatalase

NADH


of severe liver damage. This increase is due both to alcohol induction of the MEOS and to adaptive changes in the ADH pathway. The basis for the increased activity of the ADH pathway is probably increased mitochondrial reoxidation of NADH to NAD, which, as discussed previously, is the impor-tant rate-limiting step. It has been suggested that the increased mitochondrial NADH-reoxidation rate is secondary to alcohol-induced stimulation of Na+, K+-ATPase activity, leading to enhanced ATP and oxygen consumption.31 This so-called hypermetabolic state of the liver has also been impli-cated in the pathogenesis of alcohol-related liver injury. Alcohol elimination is decreased in jaundiced patients with alcoholic cirrhosis and animals with nonalcohol-related liver disease32; this probably reflects decreased ADH activity.33 An important consequence of the increased rate of alcohol oxida-tion in alcoholics is that, following alcohol ingestion, levels of acetaldehyde in both blood and tissues are higher than those seen after similar ingestion in nonalcoholic controls.34,35 This increase is potentiated by a reduction in the capacity of the mitochondria to oxidize acetaldehyde, at least in alcohol-fed rats, and a reduction in total hepatic ALDH activity, observed in chronic alcoholic patients with and without liver disease.36 As discussed, this may have important implications for disease pathogenesis. Having discussed the pathways of ethanol metabolism, the next sections will review the evidence for how this impacts on disease pathogenesis.

Alcohol Metabolism and the Pathogenesis of ALDMany studies have focused on the downstream effects of ethanol metabolism in an effort to explain the biochemical and histologic features of ALD. These studies have yielded several mechanisms through which this metabolism may result in the generation of fatty liver (steatosis), oxidative stress/lipid peroxidation, and acetaldehyde, all of which are thought to be important in disease pathogenesis.

The Pathogenesis of Fatty LiverThe accumulation of triacylglycerol (TAG), synthesized via the sequential esterification of glycerol-3-phosphate within the liver is an early and reversible effect of alcohol consumption in humans and animal models of ALD. It is the consequence of increased substrate supply (glycerol and free fatty acids [FFA]), increased esterification, and decreased export of TAG from the liver.37 The molecular mechanisms that contribute to these three main effects have recently been elucidated (Fig. 28-4).

The Role of Dietary FatIt is intuitive to suspect that dietary fat will have a role in the development of hepatic steatosis, and indeed rat models of ALD have shown that the rate of development of fatty liver is proportional to the fat content of the diet.38 Further studies in these rats in conjunction with human epidemiologic data have also highlighted the role that different types of dietary fat may play in influencing the severity of the more advanced forms of ALD, such as necroinflammation and fibrosis. These will be discussed in the section on oxidative stress. Although the quantity of dietary fat can increase the supply of fat to the liver, alcohol intake also increases the lipolysis of adipose

subsequently renders the hepatocyte sensitive to apoptosis induced by tumor necrosis factor- (TNF-), a cytokine found at higher concentrations in the livers of heavy drinkers than abstainers.24 Nevertheless, it is likely that increased metabolism plays a significant role in the increased sensitivity of drinkers to acetaminophen. Enhanced microsomal enzyme activity may also lead to an increased rate of testosterone breakdown, contributing to low blood levels of hormone already decreased due to inhibition of testosterone production by the direct toxic effects of alcohol on the testes.25 The methods by which ethanol induces CYP2E1 are not entirely clear but are likely to be multifactorial including increased transcription, increased mRNA stabilization, increased trans-lation and reduced degradation.

The Catalase PathwayThe third pathway for alcohol oxidation is catalyzed by the enzyme, catalase. This enzyme is located in the peroxisomes of most tissues and requires the presence of hydrogen perox-ide. The reaction is limited by the availability of hydrogen peroxide, which in normal circumstances is low and suggests that the catalase pathway accounts for less than 2% of overall in vivo alcohol oxidation.26

Oxidation of Acetaldehyde to AcetateMore than 90% of the acetaldehyde formed from alcohol oxi-dation is further oxidized in the liver to acetate by aldehyde dehydrogenases (ALDHs). ALDH, like ADH, uses NAD as a co-factor and further increases the NADH/NAD ratio. Human ALDHs are encoded at four independent loci on four different chromosomes.27 ALDH2 on chromosome 12 encodes the major mitochondrial enzyme, which has a low Km for acetal-dehyde and is responsible for the majority of acetaldehyde oxidation. The ALDH2 gene exists in at least two allelic forms, ALDH2*1 and ALDH2*2.28 Isoenzymes present in individuals homozygous for the ALDH2*2 allele have little or no catalytic activity, while those present in heterozygotes have measurable although reduced activity compared with the isoenzymes present in ALDH2*l homozygotes.29 The inactive form of ALDH2 is present in about 50% of Asians but has not been found in Caucasian populations. Increased levels of acetalde-hyde are thought to be the mechanism through which homo-zygotes for the ALDH2*2 allele develop the flushing reaction after alcohol. Interestingly, heterozygotes for the allele appear to develop advanced liver disease with lower levels of ethanol consumption.30 This, too, is putatively secondary to increased concentrations of acetaldehyde and adds to the extensive evi-dence that acetaldehyde is centrally involved in the pathogen-esis of ALD. The cytosolic form of ALDH, ALDH1 has a higher Km for acetaldehyde than ALDH2 and may play a role follow-ing the ingestion of large doses of alcohol. ALDH inhibitors such as disulfuram (Antabuse) have been used in the treat-ment of alcoholism to sensitize alcoholics to the unpleasant effects of alcohol intake secondary to high levels of acetaldehyde.

Alterations in Alcohol Metabolism Following Chronic ConsumptionMany studies have shown that chronic alcohol consumption increases the rate of alcohol elimination except in the presence


hepatic fatty acid levels via the mechanisms outlined, would be expected to result in an induction of enzymes in the oxida-tion systems and a subsequent increase in fatty acid oxidation. In fact, ethanol feeding results in a decrease in transcription and activity of many of these enzymes due to an inhibition of the transcriptional and DNA binding activity of PPAR-.41 This effect was replicated with acetaldehyde, and inhibited by inhibiting ethanol metabolism, implicating acetaldehyde as the factor leading to the PPAR- inhibition.41 Treatment with the PPAR- agonists WY14,643 and clofibrate reversed the effects of ethanol-feeding and the resulting abnormalities in hepatic lipid metabolism in rodent models42,43 while alcohol-fed PPAR- null mice develop more steatosis, hepatocyte injury, and fibrosis than their wild-type litter mates.44 It appears, therefore, that PPAR- inhibition plays a critical role in the accumulation of fatty acids in the liver after ethanol feeding, which will then promote TAG synthesis and poten-tially necroinflammation and fibrosis. It also seems likely that acetaldehyde is a key component of this inhibition. The precise mechanism of acetaldehyde-induced PPAR- inhibition is still unclear.

Mechanisms of Altered Triglyceride ExportIn addition to increased substrate supply and esterification resulting in increased levels of TAG in the liver, a decrease in the export of TAG from the liver also appears to contribute to the generation of ethanol-induced steatosis. Usually fat is exported into the circulation in the form of very-low-density lipoproteins (VLDL). With chronic ethanol ingestion, this export mechanism becomes defective for reasons that are not entirely clear but appear to involve the Golgi complex. Acet-aldehyde produced during ethanol metabolism can bind -tubulin45 and disrupt microtubule dynamics.46 Possibly as a result of this, fat accumulates in the Golgi complex and mainly, or at least initially, in the perivenular hepatocytes. This situa-tion is compounded by ethanol-induced down-regulation of microsomal triglyceride transfer protein (MTP).47 MTP is the

tissue, further increasing the concentration of circulating FFA. These are then taken up by the liver and provide the substrate for TAG synthesis. The high concentrations of FFA further promote the synthesis of TAG by increasing the activity of the enzyme phosphatidate phosphohydrolase (PAP), which is the rate limiting step for TAG synthesis catalyzing the dephos-phorylation of phosphatidic acid to diacylglycerol.37,39

Altered Redox StateOf the three well-characterized metabolic pathways discussed previously, two appear to be of clinical and pathogenetic importance; the alcohol dehydrogenase pathway and the MEOS. The first of these involves the oxidation of ethanol to acetaldehyde by cytosolic alcohol dehydrogenases, and subse-quent oxidation to acetate by predominantly mitochondrial aldehyde dehydrogenase. Both of these steps are coupled to the reduction of NAD to NADH.

The increased NADH/NAD ratio has profound effects on the metabolism of carbohydrates and lipids. Gluconeogenesis is impaired and substrate flow through the citric acid cycle is diminished, with acetyl CoA diverted toward ketogenesis and fatty acid synthesis. In addition to increased fatty acid synthe-sis, the altered redox state also directly inhibits fatty acid oxi-dation. Through these two mechanisms, the altered redox state can contribute toward increased substrate supply. The altered NADH/NAD ratio also increases the production of the other key component of TAG, glycerol-3-phosphate (G-3P), thereby again promoting TAG synthesis.

The Role of PPAR- InhibitionRecent evidence suggests that ethanol also has an effect on fatty acid oxidation through the transcriptional factor peroxi-some proliferator-activated receptor- (PPAR-). This ligand-activated receptor/transcription factor is a critical component in the regulation of mitochondrial, microsomal, and peroxi-somal fatty acid oxidation systems in the liver.40 As FFA are ligands for this receptor, ethanol consumption, which increases

Fig. 28-4 The multiple mechanisms by which ethanol metabolism can result in fatty liver.Ethanolmetabolismcontributestofattyliverbyincreasingsubstratesupply,increasingfatesteri-ficationtotriglyceride,andreducingtheexportofvery-low-densitylipoprotein(VLDL)fromtheliver. PPAR, peroxisome proliferator-activatedreceptor; SREBP-1c, sterol regulatory element-bindingprotein1c.

Free fatty acids

Dietary fat intake

Substrate supply

Esterification

Export from the liver

Glycerol-3-phosphate

-oxidation(PPAR- inhibition)

Fatty acid synthesis(SREBP-1c induction)

Lipolysis ofadipose fat

Phosphatidatephosphohydrolase

NADH/NAD

Triglyceride

VLDLIncreased by alcohol

Inhibited by alcohol


homocysteine to methionine, significantly inhibits the devel-opment of steatosis, implying that it is the inhibition of methylation and resulting hyperhomocysteinemia that is responsible.58 It has recently been shown that the effect of ethanol on the development of hyperhomocysteinemia is independent of TNF-, and that these two mechanisms induce steatosis independently and in parallel.59

The Role of Steatosis in the Pathogenesis of Advanced ALDThese described mechanisms all appear to promote the rapid and reproducible accumulation of hepatic TAG. This has been shown in humans and several animal models of alcoholic liver injury. The next question is whether this fat accumulation is harmful or benign. A growing body of evidence suggests that, rather than being an epiphenomenon of excessive alcohol intake, steatosis may play a direct role in progression to more advanced disease.60 In several prospective studies of heavy drinkers the severity and pattern of steatosis on index biopsy predicts the subsequent risk of fibrosis and cirrhosis.61,62 These and other studies have led to steatosis being considered as the first hit, increasing the sensitivity of the liver to a variety of second hits that result in injury and inflammation. These second hits could be gut-derived endotoxin, oxidative stress or, indeed, immune mechanisms. In support, studies in animal models have shown that steatosis increases endotoxin-mediated necroinflammation63 and the degree of lipid peroxi-dation in ethanol- and other drug-induced steatosis.64 Furthermore, genetically obese mice with steatosis have altered proportions of intrahepatic lymphocyte subpopulations. The normal liver contains significant numbers of T cells, B cells, natural killer (NK) cells, and natural killer T (NKT) cells, many of which differ phenotypically and functionally from circulating lymphocytes. Leptin-deficient ob/ob mice have steatotic livers and interestingly a selective reduction in the number of NKT cells. Although these mice are not a model of alcohol-induced steatosis, this finding does raise questions about whether steatosis itself may alter the intrahepatic immune milieu.65

Oxidative Stress and Lipid PeroxidationOxidative stress describes a situation where the generation of prooxidant species within or outside the cell overwhelms the endogenous antioxidant systems. One of the most important consequences of cellular oxidative stress is the peroxidation of the polyunsaturated fatty acid (PUFA) constituents of mem-brane and lipoprotein lipids, which can lead directly to cell death66 and to the release of reactive aldehydes with potent proinflammatory, profibrotic, and proimmune properties.67 An accumulating body of evidence now supports a role for oxidative stress and lipid peroxidation in the pathogenesis of ethanol-induced liver injury that can be summarized as follows: (1) products of lipid peroxidation can be detected in the peripheral blood of heavy drinkers68 and in the livers of patients with ALD,69 and the magnitude of lipid peroxidation correlates with the degree of liver injury70; (2) in patients and animal models of ALD, lipid peroxidation is most prominent in the perivenular region where the liver injury is typically most severe; (3) a variety of sources of oxidative stress have been identified in patients with ALD and in animal models of

principal enzyme responsible for packaging TAG and apoli-poprotein B (apoB) into VLDL particles. In the absence of effective lipidation apoB is degraded in the proteosome.

Role of TNF- in the Pathogenesis of SteatosisThe putative role of TNF- in the pathogenesis of necroin-flammation in ALD is described in the section on endotoxin later. This cytokine has also been linked to the development of hepatic steatosis. Interest in this area stemmed from a study showing that TNF- receptor 1 (TNFR1)-deficient mice developed considerably less steatosis than their wild-type litter mates when fed ethanol.48 Recently, the mechanisms for this TNF- induced steatosis have been better elucidated. They include the down-regulation of MTP (discussed previously), the increased expression of sterol response element binding protein-1c (SREBP-1c),49 a transcription factor critical in con-trolling de novo hepatic lipogenesis, and inhibition of adipo-nectin, an antisteatotic adipocytokine.50 Because one of adiponectins antisteatotic effects is via PPAR-, the inhibition of adiponectin by TNF- provides a potential mechanism for ethanols inhibition of PPAR- activity discussed previously.

Inhibition of the Methionine Cycle and Endoplasmic Reticular StressStudies, predominantly in micropigs, have demonstrated that the inhibition of transmethylation reactions by ethanol is the major mechanism leading to the well-established abnormal methionine metabolism associated with ethanol consump-tion.51 The principal enzyme inhibited by ethanol is methio-nine synthase (MS),52 and a significant effect of this appears to be the development of hyperhomocysteinemia.

Hyperhomocysteinemia has been implicated in the patho-genesis of atherosclerosis and Alzheimer disease through a phenomenon known as endoplasmic reticulum (ER) stress. The ER is the main site of protein synthesis from messenger RNA (mRNA), and is also involved in protein transport and some posttranslational modifications.53 When abnormally folded or unfolded proteins build up in the ER, this acts as a marker to the cell that the quantity of client protein exceeds the ability of the ER to process it and results in a set of responses termed the unfolded protein response (UPR) or ER stress.54 This response, which can be triggered by homocyste-ine, has three main arms. The first results in the increased transcription of ER proteins and chaperone proteins that aid processing. The second is to reduce the biosynthesis of other proteins to reduce the unfolded client load. The third is to up-regulate proapoptotic protein synthesis so that, in extreme circumstances, the cell will apoptose. In addition, ER stress triggered by homocysteine increases the gene expression of SREBP-1c.55 Previous studies have shown that ethanol induces SREBP-1c in rat hepatoma cell lines and mouse liver with a concomitant increase in the expression of lipogenic genes,56 and that this induction may be related to inhibition of AMP-activated protein kinase (AMPK).57 Whether this inhibition of AMPK by ethanol is related to ER stress is unknown, however, studies with the intragastric ethanol-fed mouse have shown that ethanol induces hyperhomocysteinemia, triggers ER stress, and promotes the features of ALD including steatosis. Furthermore, feeding mice betaine, a methyl donor converting


MITOCHONDRIA

The mitochondrial respiratory electron transport chain gen-erates superoxide radicals during the reoxidation of NADH arising during ethanol metabolism. The superoxide anions are subsequently converted to hydrogen peroxide by mitochon-drial SOD2. Although the hydrogen peroxide is further metab-olized to carbon dioxide and water by mitochondrial glutathione peroxidase, the increased rate of production after ethanol consumption results in a significant production of ROS.76 As discussed in detail later, TNF- may play an impor-tant role in the mitochondrial production of ROS through inhibition of electron flow in the respiratory chain. Perhaps the best evidence that mitochondria are involved in the patho-genesis of ALD are the ultrastructural changes that occur in these organelles in human and experimental ALD.77 These changes are almost certainly related to mitochondrial oxida-tive stress and its downstream effects.

INDUCIBLE NITRIC OXIDE SYNTHASENitric oxide (NO) has recently emerged as a critical regulator of certain aspects of mitochondrial function.78 In particular, NO can bind to cytochrome-c oxidase and increase the rate of superoxide production.79 After ethanol consumption, liver mitochondria are much more susceptible to NO-dependent inhibition of respiration. Furthermore, mice deficient in the inducible enzyme that synthesizes NO, inducible nitric oxide synthase (iNOS), develop much milder liver disease after ethanol consumption.80 The fact that these iNOS/ mice have dramatically reduced levels of liver lipid peroxidation end products and reactive nitrogen species provides compelling evidence for the role of this enzyme in oxidative stress-related injury. As with the mitochondrial production of ROS, TNF- is likely to play a critical role in the generation of oxidative stress via iNOS due to its NF-B dependent up-regulation of iNOS gene transcription.

KUPFFER CELLSIn addition to playing a central role in the generation of pro-inflammatory cytokines in response to endotoxin (see later discussion), activated Kupffer cells also act as a rich source of free radicals during ethanol-induced liver injury. Inhibition of Kupffer cells with gadolinium chloride during alcohol expo-sure has a profound inhibitory effect on the magnitude of lipid peroxidation,81 and mice deficient in Kupffer cell NADPH-oxidase have no increase in free radical production and develop no liver pathology after 4 weeks exposure to ethanol.82

Depletion of Antioxidant Defenses in ALDEthanol consumption undoubtedly results in a depletion of endogenous antioxidant capabilities. Consumption of gluta-thione (GSH) during oxidative stress and inhibition of two enzymes involved in the synthesis of its precursor, S-adenosylmethionine (SAMe), methionine synthase, and methionine adenosyltransferase (MAT), contribute to the decreased levels of hepatic SAMe and GSH observed in patients with ALD83 (Fig. 28-5). Depletion of mitochondrial GSH precedes and promotes the progression of alcoholic liver injury in animal models84 with one mechanism of action being an increased sensitivity of hepatocytes to TNF- induced cytotoxicity.85 A recent study has suggested that, rather than reduced GSH levels, a high S-adenosylhomocysteine (SAH)/

disease; (4) ethanol consumption results in the depletion of endogenous antioxidant capabilities and patients with ALD have evidence of antioxidant deficiencies; (5) in animal models of ALD, dietary and genetic manipulations that increase oxi-dative stress increase the severity of liver injury and reducing oxidative stress ameliorates injury (Table 28-1).

Sources of Oxidative Stress in ALDThe principal prooxidant species considered to be important in the pathogenesis of ALD are the reactive oxygen species (ROS), the superoxide anion, hydrogen peroxide and hydroxyl, and hydroxyethyl radicals. Considerable controversy remains over the most important source of these ROS in ALD, but the most likely appear to be microsomal CYP2E1 (the only source of hydroxyethyl radicals71,72), the mitochondrial electron trans-port chain,73 inducible nitric oxide synthase, and Kupffer cells.

CYP2E1In the presence of iron, isolated microsomes can generate suf-ficient oxidizing species to initiate lipid peroxidation. Most of the ROS in this system come from the ethanol-inducible microsomal ethanol oxidizing system (MEOS). The major component of this system is CYP2E1, which can also oxidize alcohol to acetaldehyde and subsequently to acetate, while concomitantly oxidizing NADPH to NADP. It appears that most ROS in the ethanol-fed rat model appear to be generated by CYP2E1. Spin-trapping studies, experiments that allow the identification of highly reactive radicals produced during reactions for very short periods of time to be identified, have revealed that the hydroxyethyl radical is the main radical formed when CYP2E1 metabolizes ethanol. The best in vivo evidence for the important role of CYP2E1 metabolites comes from experiments in the ethanol-fed rat discussed previously, where enzyme inhibition with diallyl sulfide resulted in decreased lipid peroxidation and amelioration of liver injury.74,75

Presenceoflipidperoxidationproducts

FoundinserumandliverofpatientswithALDandcorrelateswithhistologicseverity

Siteoflipidperoxidationproducts

FoundinperivenularregionwhereALDstartsandismostsevere

Multiplepotentialsourcesofreactiveoxygenspeciesinheavydrinkers

Hepatocytemicrosomesandmitochondria,Kupffercells

Depletionofantioxidantdefenses

Depletionofglutathione,selenium,andcoenzymeQisfoundinheavydrinkers

Manipulatingoxidativestressinanimalmodelsinfluencesdiseaseseverity

Prooxidantdietsworsendiseasewhileoverexpressionorunderexpressionofantioxidantenzymesaffectsthedegreeofliverinjury

Table281 Evidence for the Role of Oxidative Stress in the Pathogenesis of ALD


ALD. Cytosolic superoxide dismutase (SOD-1) knockout mice develop more significant liver injury compared with wild-type litter mates following ethanol feeding,97 whereas MAT knockout mice have reduced hepatic glutathione levels and develop spontaneous steatohepatitis even without alcohol.98

Further evidence supporting the role of oxidative stress in ALD comes from studies aimed at reducing its levels during ethanol feeding. Reduction of oxidative stress by supplemen-tation with the GSH precursor and methyl donor SAMe reduced cell and mitochondrial injury in the baboon model of ALD99 and led to the pilot study in humans discussed later. Inhibiting the prooxidant effect of iron with the oral chelator 1,2-dimethyl-3-hydroxypyrid-4-one reduced hepatic-free iron, lipid peroxidation, and fat accumulation in chroni-cally ethanol-fed rats100 and inhibiting the induction of CYP2E1 with diallyl sulfide (DAS) and phenylethyl isothio-cyanate (PIC) resulted in the production of fewer free radicals and end products of lipid peroxidation and ameliorated liver injury in the same model.74,75 Amelioration of liver injury could also be achieved using an adenovirus to deliver the mitochondrial, manganese-dependent superoxide dismutase-2 (SOD2) to rats fed ethanol,101 and, as discussed previously, NADPH-oxidase deficient mice have also been used to show the importance of this source of oxidants in the development of early alcohol-induced hepatitis.82

AcetaldehydeIt is widely considered that acetaldehyde, the first metabolite of ethanol, has a central role in the pathogenesis of ALD, but precisely what role is still unclear. It has been known for some time that acetaldehyde can bind covalently to albumin,102 tubulin,103 hemoglobin,104 plasma proteins,105 collagen,106 and microsomal enzymes107 and results in both stable and unstable adduct formation. These reactions are likely to involve the formation of Schiff bases between the aldehyde and valine, and lysine and tyrosine residues on the carrier protein.104 This binding may affect protein function, and this disruption has been implicated in the pathogenesis of ALD.108 Defects in assembly of microtubules,109 protein excretion,110 and enzy-matic activity108 have all been attributed to acetaldehyde; however, no firm evidence exists for these mechanisms as disease pathways. With regard to disruption of enzymatic activity, an interesting study identified a 37 kDa protein-acetaldehyde adduct in the liver of alcohol-fed rats to be the enzyme 4-3-Ketosteroid 5-reductase.111 This was felt to be important because children having inborn errors in this enzyme develop intrahepatic cholestasis and in some cases

SAMe ratio may be responsible for the ethanol-induced enhanced sensitivity of hepatocytes to TNF- killing, possibly by increasing the activity of caspase-8, a key initiator of the apoptotic cascade.86 Heavy drinkers, including those with ALD, are deficient in the antioxidant trace element selenium,87 which is required for the activity of the antioxidant enzyme GSH peroxidase, the antioxidant vitamins A, C, and E88,89 and coenzyme Q.90 This latter compound is present in plasma and mitochondrial matrix membranes and has emerged as one of the most important, natural free radical scavengers. It is partly derived from the diet, but is also synthesized in the liver. Whether these deficiencies are a cause or an effect of ALD remains unclear, although the fact that antioxidant supple-mentation appears to be of no benefit in patients with ALD is perhaps more in favor of the latter explanation.91

Manipulating Oxidative Stress in Animal Models Influences Disease SeverityDiets that promote oxidative stress increase the severity of ALD in animal models. Perhaps the best example of this comes from studies in which animals were fed a diet supple-mented with different types of dietary fat in addition to alcohol. The degree of liver injury depends on whether the animals had their diet supplemented with beef fat, lard, or corn oil, with beef fat resulting in the least injury and corn oil the most.92 A hypothesis that liver injury correlated with the amount of linoleic acid in the diet was supported by a study in which beef fat was supplemented with linoleic acid. These rats, like the corn oilfed rats, developed severe liver disease when fed ethanol.93 Severe liver injury was also seen when fish oil was used as the source of dietary fat, implying that polyunsaturated fats, which are much more vulnerable to attack from ROS, have a role in promoting liver injury.94 The mechanisms through which polyunsaturated fats promote ethanol-induced liver injury are not entirely clear, but appear to involve up-regulation of CYP2E1 and an increase in lipid peroxidation.94 In addition, polyunsaturated fats are the pre-cursors of eicosanoids, powerful mediators of inflammation. Interestingly, cyclooxygenase-2 (COX-2), the key enzyme involved in the generation of eicosanoids, is inducible by both endotoxin and TNF-.95 Dietary iron supplementation also leads to an increase in the concentration of the aldehyde end products of lipid peroxidation within the liver. The addition of this prooxidant to the diet in the intragastric ethanol infu-sion model exacerbates hepatocyte damage and promotes liver fibrogenesis.96 Studies employing genetic manipulation to increase the degree of oxidative stress provide further support for its role as an important disease mechanism in

Fig. 28-5 The methionine cycle. (1) Methionine adenosyl-transferasecatalyzes the synthesisofS-adenosyl-L-methionine(SAMe) from methionine and adenosine triphosphate. Afterdonation of a methyl group, SAMe becomes S-adenosyl-homocysteine,which,throughhomocysteine,actsasaprecur-sor for glutathione. (2) Methionine synthase regeneratesmethionine from homocysteine in a reaction that requiresnormal levels of folate and vitamin B12, and (3) betaine-homocysteine methyltransferase regenerates methionine fromhomocysteineinareactionthatrequiresbetaine.Ethanolinhib-itsreactions(1)and(2).

Methionine

Methyl groupS-adenosylhomocysteine

S-adenosylmethionineBetaine

Glutathione

Folate/B12

Homocysteine

ATP

123


cytokine production and perpetuation of the inflammatory response appear to be key to the progression of alcoholic hepatitis. As discussed previously, ethanol metabolism and the production of acetate may have an important role in this. Increased translocation and an augmented response to endo-toxin are also central.

EndotoxinEndotoxin, which refers collectively to the lipopolysaccharide (LPS) components of the cell wall of all gram-negative bacte-ria, appears to play a central role in the development of ALD. Ethanol ingestion increases intestinal permeability and increases the translocation of endotoxin from the gut lumen.115 This appears to occur through more than one mechanism. Acetaldehyde can cause redistribution of tight and adherens junctions, and affect epithelial cellcell adhesion. Alcohol also increases the expression of inducible nitric oxide synthase, which can result in the nitration and oxidation of tubulin and result in barrier disruption.116 Interestingly, treatment with lactobacillus can ameliorate alcohol-induced liver injury in a rat mode by improving gut integrity.117

When endotoxin translocates to the portal circulation, it is recognized by intrahepatic macrophages (Kupffer cells). Endotoxinemia has been found in drinkers with varying degrees of liver disease;118 in the ethanol-fed rat, the level of plasma endotoxin correlated with the degree of liver injury.119 When Kupffer cells encounter the LPS component of endo-toxin in conjunction with lipopolysaccharide binding protein (LBP),120 they respond by releasing cytokines and ROS. Although this can be blocked in vitro by the addition of anti-CD14 monoclonal antibodies,121 implying the importance of this constitutively expressed and inducible receptor, CD14 does not have a transmembrane component, and therefore must rely on alternative receptors for signal transduction. Experiments on mice hyporesponsive to LPS have helped to identify the Toll-like receptor 4 (TLR4) as an important con-comitant signal,122 and current data suggest that LPS signaling occurs through activation clusters of these two receptors along with other membrane proteins. The importance of TLRs has been further highlighted recently by the discovery that they control activation of adaptive immune responses as well as innate responses. This occurs through an IL-6 dependent pathway, resulting in suppression of CD4+CD25+T regulatory cells that normally specifically inhibit adaptive immune responses.123 This has profound implications for the role of endotoxin in ALD.

The role of endotoxin and Kupffer cells in the pathogenesis of ALD has been suggested largely by studies in the rodent model of continuous intragastric feeding developed by Tsuka-moto and French.124 As discussed previously, these animals develop lesions similar to human alcoholic hepatitis when fed a diet high in ethanol and fat. The eradication of gram-negative fecal flora with antibiotics reduces endotoxin levels to those of controls not fed alcohol, and prevents liver injury.125 Recent data reveal that this injury is also attenuated in CD14 and TLR4 knockout mice, highlighting the importance of these receptors.126,127 Alcohol can also affect Kupffer cell func-tion directly. Rodents fed alcohol initially become more toler-ant of endotoxin; however, with time, this response converts to one of sensitization.128 An early suppressive effect of alcohol on Kupffer cell function and a later induction of CD14 may

liver failure. Although this may be just one of many proteins that adduct to acetaldehyde, it raises questions about whether the binding of acetaldehyde may indeed alter enzyme activity. Along with potentially disrupting protein function, the forma-tion of protein-acetaldehyde adducts results in the production of immunodominant antigenic determinants. The conse-quence of this is discussed in detail in the Adaptive Immune System section.

More recently, a further and more complex role for acetal-dehyde has been suggested. Hepatocytes are resistant to TNF-induced cytotoxicity unless they have been previously exposed to ethanol.112 As discussed previously, selective deple-tion of mitochondrial glutathione can induce this sensitiza-tion, and is postulated to be the mechanism through which it happens in vivo. When HepG2 cells are exposed to acetalde-hyde, there is a selective reduction in mitochondrial glutathi-one and increased sensitization to TNF-.85 This has been shown to occur even when further metabolism of acetalde-hyde is inhibited, and recent evidence suggests it is due to an inhibition of glutathione transport into the mitochondria sec-ondary to an increased proportion of cholesterol in the mito-chondrial membrane and a resulting increase in viscosity.113 It is postulated that acetaldehyde induces this increase in choles-terol through the unfolded protein and ER stress response via SREBP-1c up-regulating the transcription of cholesterol-synthesizing enzymes. This effect on GSH transport will exac-erbate the GSH depletion resulting from decreased SAMe synthesis and consumption during oxidative stress. In this way, the induction of SREBP-1c by TNF- and by acetalde-hyde and homocysteine-induced ER stress can induce a viscous cycle resulting not only in steatosis, but also in sensi-tization to the cytotoxic effects of TNF-. This is particularly significant in view of the role of endotoxin in ALD, which acts predominantly as a stimulus to the release of TNF- from hepatic Kupffer cells.

AcetateAcetate is the end product of ethanol metabolism in the liver and can be incorporated into acetyl CoA in a reaction cata-lyzed by the enzyme acetyl CoA synthetase. While acetyl CoA is an important donor of carbon atoms to the citric acid cycle for oxidation, it may also have an important role in the control of inflammation. Recent data suggests that acetyl CoA may have a key role in the acetylation of histones, the protein spools in chromatin around which the DNA is wound. The acetylation of histones unwinds the chromatin and allows polymerases to transcribe DNA. Macrophages exposed to ethanol developed an enhanced IL-6, IL-8, and TNF- response to lipopolysaccharide with time-dependent increases in histone acetylation that could be prevented by inhibition of ethanol metabolism and knockout of the acetyl CoA synthe-tase enzyme.114

This novel finding may explain the perpetuation of the inflammatory response in acute alcoholic hepatitis.

The Innate Immune System and the Pathogenesis of ALDA considerable body of evidence supports a role for the innate immune system in the pathogenesis of ALD. Enhancement of


and associates with its analogue Bak, to form channels in the outer mitochondria membrane.135 Increased permeability of the outer mitochondrial membrane releases cytochrome C from the intermembrane space of mitochondria, thus partially blocking the flow of electrons into the respiratory chain and increasing mitochondrial ROS formation.136 The resulting ROS then acts on the same or other mitochondria to open an inner membrane pore, the mitochondrial permeability transi-tion pore. This causes an influx of water into the mitochon-drial matrix, leading to mitochondrial swelling and eventually rupture of the unfolded outer membrane. This leads to the leakage of apoptosis-inducing factors (predominantly cyto-chrome C) into the cytosol, where they can activate caspase-9, which initiates the apoptotic cascade.

Given the role of ROS in this cascade, it is easy to see how the oxidative stress-related mechanisms of alcohol-induced cell injury can synergize with TNF- to induce apoptosis and/or necrosis. TNF-induced apoptosis is increased by alcohol, an effect that is exaggerated in HepG2 cells with high CYP2E1 activity presumably related to increased oxidative stress.112 As discussed, the depletion of glutathione by alcohol will also increase the sensitivity of hepatocytes to the mitochondrial effects of TNF-.24,85 It has recently been shown that the mechanism of TNF-induced cell death, necrosis, or apop-tosis depends on the degree and site of glutathione deple-tion.137 Mitochondrial depletion leads to necrosis due to profound loss of mitochondrial function,85 whereas less severe, predominantly cytosolic, depletion of glutathione leads to apoptosis due to inhibition of the NF-Binduced transacti-vation of survival genes that normally follows TNF- binding to its receptor. As also discussed previously, in addition to TNF- and oxidative stress, the apoptotic cascade may also be initiated in response to ER stress.

Natural Killer CellsA further, less well understood, arm of the innate immune system that appears to be involved in the pathogenesis of ALD is the natural killer (NK) and natural killer T (NKT) cell population. These cells are abundant in the liver138 and both populations have been reported to increase in the peripheral blood of heavy drinkers.139 Recent evidence points to an

partly explain the time course of this response. Acetate-mediated histone acetylation may also contribute. Interest-ingly, when Kupffer cells are inactivated in the ethanol-fed rat model using gadolinium chloride, disease is ameliorated and the steatosis is diminished. This highlights that these cells also have a role in the TAG accumulation, most likely through TNF- production, as discussed previously.

Sinusoidal endothelial cells are also actively involved in the response to endotoxin. These cells constitutively express all the surface molecules necessary for antigen presentation, and may induce tolerance or immunity depending on the local micro-environment. This environment may be dictated by Kupffer cell release of immunomodulatory cytokines in response to varying doses of portal endotoxin, with a high-dose endotoxin resulting in potentially harmful immunity, and low-dose endotoxin resulting in tolerance through the secretion of IL-10.129

The Role of TNF-Kupffer cells are the primary intrahepatic source of TNF-, a cytokine believed to be central to the pathogenesis of ALD. Peripheral blood mononuclear cells produce more basal and lipopolysaccharide induced TNF- than controls,130 and plasma levels are higher in patients with more severe disease.131 Furthermore, mice lacking TNFR1 fail to develop liver injury in the Tsukamoto-French model.48 More recently it has been suggested that these mice develop an ameliorated form of ALD rather than none at all,59 nevertheless, the findings suggest an important role for the cytokine (Fig. 28-6). How TNF- contributes to hepatocyte cytotoxicity in ALD, and whether this is via necrosis or apoptosis is an area of dispute; however, recent studies showing a correlation between the degree of apoptosis and clinical indices of severity in patients with alcoholic hepatitis suggest that apoptosis plays an impor-tant role.132,133 The interaction of TNF- with its receptor TNFR1 (via the death-inducing signaling complex [DISC]) activates procaspase-8 into caspase-8, which cuts Bid (BH3 interacting domain death agonist).134 Truncated Bid can enter the outer mitochondrial membrane to make this membrane leaky, and it also induces a conformational change in Bax (Bcl-2associated x protein), which translocates to mitochondria

Fig. 28-6 Tumor necrosis factor- (TNF-), a central cytokine in alcoholic liver injury.TNF- inducessteatosis throughtheup-regulationofsterolresponseelement-bindingprotein-1c(SREBP1-c),thedown-regulationofmicrosomaltriglyceridetransferprotein(MTP),andtheinhibitionofadiponectin.Dependingonthedegreeofoxidativestressandtheavailabilityofmitochondrialglutathione(GSH),TNF-canalsoinduceeithernecrosisorapoptosisthroughthedeath-inducingsignalingcomplex,withsubsequentactivationofcaspase-8andthemitochondrialdeathpathway.

TNF-

Pro-steatotic throughSREBP-Ic MTP adiponectin

Pro-apoptotic through caspase 8/mitochondrial death pathway

Pro-necrotic through ROSif mitochondrial GSH depleted

EndotoxinCD14 TLR4

Kupffer cell


metabolism154) and aldehydes generated as a result of pro-longed oxidative stress, such a malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE).155 Anti-HER antibodies have been found in significantly higher titer in patients with alcohol-induced cirrhosis than in nonalcohol-induced cir-rhosis or normal controls.156 The third, and final, antigen that has been studied in detail is a combination of acetaldehyde and malondialdehyde called malondialdehyde-acetaldehyde (MAA) adducts. Antibodies to these hybrid conjugates have now been detected in sera from patients with alcohol-induced liver cirrhosis and alcoholic hepatitis but not in heavy drinkers with no liver disease or healthy controls.157

AutoantibodiesWhen ethanol is metabolized by CYP2E1, the hydroxyethyl radical is produced,156 which can form immunogenic adducts with the cytochrome. It has now been demonstrated that a significant proportion of patients with ALD have antibodies reactive with the hydroxyethyl radical complexed with CYP2E1.158 Furthermore, both patients with ALD and rats fed ethanol have been shown to develop anti-CYP2E1 specific autoantibodies.159 Interestingly, in humans with ALD, the presence of these autoantibodies is predicted by the presence of antibodies to hydroxyethyl radicals, suggesting a potential mechanism for tolerance breakdown.160

Whether the humoral immune response is a cause or a consequence of liver disease remains an important question. Antibody dependent cell cytotoxicity (ADCC) has been observed when sera from patients with ALD positive for anti-HER antibodies were co-cultured with ethanol-treated rat hepatocytes and peripheral blood mononuclear cells from healthy controls.161 Further studies are required to provide evidence for the role of this mechanism of disease in vivo.

Cellular Immune Responses in ALDBoth mice and human livers have significant numbers of T cells and some B cells, which recirculate from the peripheral pool; however, in contrast to peripheral blood, there are more CD8+ cells than CD4+ cells.138 In addition there are high fre-quencies of what appear to be resident liver lymphocytes that are found in very low frequencies in peripheral blood. These are natural killer (NK) cells, recognized by CD56 in humans and NK1.1 in mice, and NKT cells that express these proteins along with the T-cell receptor. In normal liver, these cell types are distributed throughout the parenchyma. In liver disease, this situation changes, and in alcoholic cirrhosis expanded portal tracts contain large numbers of classical CD4+ and CD8+ lymphocytes.145 No work has been done to determine the antigen specificity of these intrahepatic lymphocyte popu-lations; however, peripheral blood mononuclear cell responses to malondialdehyde (MDA) have been detected in a signifi-cant proportion of patients.162

A further population of T cells appears to have a role in disease progression. IL-17secreting T cells are found at increased concentration in the liver and peripheral blood of patients with ALD. In chronic liver disease, the numbers cor-relate with the model of end-stage liver disease score, and in acute alcoholic hepatitis they correlate with the discriminant function. These cells can induce chemokine secretion and aid

increase in the number of NKT cells in the livers of mice after alcohol consumption and augmenting their activation using a marine sphingolipid during ethanol feeding results in fatal hepatotoxicity.140 The relevance of this to clinical liver disease is as yet unclear and is made more complicated by the fact that acute ethanol consumption inhibits innate immunity by sup-pressing TLR3 signalling,141 and inhibits NK cell killing of activated stellate cells, which would have marked profibro-genic effects.142

The Adaptive Immune System and the Pathogenesis of ALDSeveral clinical features suggest that adaptive immune mecha-nisms may have a role in the pathogenesis of ALD. Abstinent patients that return to drinking have a rapid and aggressive recurrence of their disease that may imply an immunologic anamnestic response. There is a partial response to immuno-suppressive steroids in selected groups with severe disease.143 Patients receiving interferon therapy for hepatitis C, which can trigger autoimmune thyroid disease, can rapidly develop alco-holic hepatitis with no increase in their daily alcohol intake.144 Lymphocyte infiltration is a well-recognized histologic feature of advanced disease145 and hypergammaglobulinemia is common. These features have led researchers to look at the importance of antigen-specific immune responses and how they might lead to liver injury.

Humoral Immune Responses in ALDAlthough nonspecific liver cell membrane antibodies have been implicated in ALD pathogenesis for some time,146,147 recent studies have focused on determining the antigen speci-ficity of this humoral response.

Antibodies to Acetaldehyde AdductsAcetaldehyde can adduct to host proteins and this can result in the formation of immunogenic epitopes.148 Acetaldehyde-modified proteins have been found in the liver cytosol, mem-branes, and mitochondria of rats chronically fed ethanol, and their decline over several weeks after ethanol withdrawal has been investigated.149 They accumulate particularly in hepato-cytes in the perivenular region, which is also the main area of distribution of the ethanol metabolizing enzyme CYP2E1, the main site of lipid peroxidation and the first area to be injured in ALD.150 Interestingly, extracellular and not intracellular acetaldehyde-adduct staining correlates with fibrosis progres-sion in humans regardless of whether the patients abstained from alcohol or not.151

Antiacetaldehyde (Anti-AcA) adduct antibodies have been found in the sera of heavy drinkers by several groups.151-153 Their relationship to the presence and severity of liver disease is controversial.

Antibodies to the Other Products of Ethanol MetabolismOther targets for the humoral immune response in ALD include the hydroxyethyl radical (HER; a reactive inter-mediate formed by the action of CYP2E1 during ethanol


In ALD, fibrosis starts in the perivenular area and may prog-ress to bridging fibrosis and, ultimately, cirrhosis. This early fibrosis occurs at the site of maximal alcohol-induced hepa-tocyte injury. The principal cell types involved in the activa-tion of HSC in ALD are Kupffer cells and hepatocytes. Kupffer cells are stimulated to release cytokines by endotoxin in ALD, and in turn these cells can lead to the activation and prolifera-tion of stellate cells through the production of transforming growth factor- (TGF-), TNF-, and ROS. Hepatocytes are rich sources of ROS, lipid peroxidation products, and acetal-dehyde during alcohol-induced injury, all of which have been shown to enhance collagen production by HSC.67,77 CYP2E1 may be particularly important in this regard given its induc-ibility by alcohol and a high-fat diet and its perivenular dis-tribution. HSC grown in the presence of hepatocyte cell lines that overexpress CYP2E1 increase their production of colla-gen, an effect that is prevented by antioxidants or a CYP2E1 inhibitor.168 Hepatocyte apoptosis is a notable feature of alco-holic hepatitis,132 and apoptosing hepatocytes express Fas, which can promote stellate cell initiation through the tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL).169 Furthermore, apoptosing hepatocytes may also be ingested by Kupffer cells and HSC, which subsequently release TGF- capable of activating HSC.169,170 Alcohol can also be profibrotic by attenuating the antifibrotic effects of interferon- (IFN-). It does this by reducing expression of IFN- by NK cells and impairing downstream signaling in HSCs (Table 28-2).142

Mechanisms of Hepatocellular CancerEpidemiologic studies reveal that alcohol plays a major con-tributory role in the development of hepatocellular cancer (HCC); however, the primary mechanisms through which this occurs are not clearly defined. Cirrhosis itself is a precancerous condition, and alcohol-related HCC without preexisting cir-rhosis is rare. Nevertheless, three features indicate that alcohol may be a co-carcinogen. The first is that heavy alcohol con-sumption is associated with several extrahepatic cancers (dis-cussed later). The second is that when the incidence of incidental HCCs in liver explants from patients with alcoholic

neutrophil recruitment, and may therefore represent a link between adaptive and innate immune responses.163

Further work is required to determine whether antigen- specific cellular immune responses are an important part of disease progression in ALD.

Mechanisms of Alcohol-Induced FibrosisFibrosis and ultimately cirrhosis is the final common pathway of most chronic liver disease, and the mechanisms underlying it are discussed elsewhere in this book. It is, however, important to briefly review these and to discuss the factors that are particu-larly relevant to the pathogenesis of the fibrosis seen in ALD.

Hepatic stellate cells (HSC) are found in the space of Disse, between hepatocytes and sinusoidal endothelial cells, and are responsible for producing the majority of the extracellular matrix (ECM).164 In a normal liver, the space of Disse contains little collagen; however, when these stellate cells become acti-vated during liver injury by cytokines and ROS, the composi-tion of the ECM changes as more collagen, glycoproteins, proteoglycans, and glycosaminoglycans are produced (Fig. 28-7). In particular, there is a shift in the type of proteoglycans produced and an increase in collagen types I, III, and IV.165 In addition, these changes are associated with an up-regulation in numerous integrins, selectins, and soluble growth factors, which modulate cellcell and cellECM interactions. Sinusoi-dal endothelial cells166 and lymphocytes167 may also play important roles in fibrogenesis. More recently, there has been increasing evidence that fibrosis can be reversible, and the discovery of matrix metalloproteinases (MMPs) that degrade collagen has implications for most liver diseases. These MMPs are activated by proteolytic cleavage and are inactivated by tissue inhibitors of metalloproteinases (TIMPs).

Fibrosis

KupffercellproductionofprofibroticcytokinesKupffercellproductionofprofibroticreactiveoxygenspeciesHepatocyteproductionofprofibroticreactiveoxygenspeciesHepatocyteproductionofacetaldehydeKupffercellandhepaticstellatecellproductionof

transforminggrowthfactorafteringestionofapoptotichepatocytes

Hepatocellular Carcinoma

LipidperoxidationandDNAmutagenesisActivationofcarcinogenicxenobioticsAntiapoptoticeffectoftumornecrosisfactorDNAhypomethylationImmunosuppression

Table282 The Primary Mechanisms Thought to Be Involved in Ethanol-Induced Hepatic Fibrosis and Hepatocellular Carcinoma

Fig. 28-7 Alcohol-induced hepatic fibrosis.


central to tumor surveillance. The primary functional effect appears to be one of suppression.139,183 In addition, there are other, more widespread effects on the innate and adap-tive immune responses that could all have knock-on effects on tumor surveillance (discussed in Associated Conditions and Extrahepatic Manifestations section). A reduction in immunosurveillance, and a subsequent increase in viral replication may also be the mechanism through which alcohol leads to an increased rate of HCC in hepatitis C cirrhosis.184

Susceptibility to Alcoholic Liver DiseaseAlthough the majority of heavy drinkers will develop some degree of steatosis (fatty liver), only around a third go on to develop alcoholic hepatitis and only between 1 in 4 and 1 in 12 ever progress to cirrhosis (Fig. 28-8).185 This leads to the obvious question: what factors determine whether or not a heavy drinker develops advanced ALD?

cirrhosis is compared with that from other causes, it lies between that of immune-mediated liver disease and viral hepatitis.171 It appears therefore that the incidence of HCC is above that of the baseline expected because of pure cirrhotic risk. The third is that there are several plausible mechanisms through which alcohol could promote carcinogenesis.

Lipid Peroxidation and DNA MutagenesisMalondialdehyde (MDA), an end product of lipid peroxida-tion, can bind to DNA and form adducts as it does with other endogenous compounds.172 These adducts were found to be highly mutagenic in E. coli173 and are repaired by nucleotide excision repair. They are also found at significant levels in healthy humans and can induce cell cycle arrest.174 This latter property results in an increase in the number of hepatic pro-genitor cells (oval cells)175 that are more resistant to oxidative stress than fully differentiated hepatocytes. It has been sug-gested that this may promote HCC because the oval cells survive through oxidative damage but remain susceptible to mutagenesis.175

Activation of XenobioticsAnother mechanism proposed for the increased rate of HCC seen in alcoholic cirrhosis is the increased production of car-cinogenic metabolites from other environmental carcinogens (xenobiotics) that are metabolized through the MEOS and other metabolic pathways upregulated in heavy drinkers. This mechanism has been suggested to explain the increased cancer risk seen with tobacco smoking,176 aflatoxin,177 and other chem-icals.178 In addition, CYP2E1 is responsible for the metabolism of retinoic acid (RA) in the liver.179 Up-regulation of CYP2E1 by ethanol therefore synergizes with its inhibition of RA syn-thesis and results in reduced RA levels, increased expression of the AP-1 transcriptional complex, and increased hepatocyte proliferation.180 Supplementation with RA reverses this effect.179

TNF- Induced Survival FactorsAs discussed previously, TNF- has both proapoptotic and antiapoptotic properties and the balance of these appears to depend on the local microenvironment and the disease. Although apoptosis may reduce the risk of HCC, increased cell survival through TNF-induced NF-B activation could have the opposite effect, particularly in combination with the mutagenic effects of lipid peroxidation products.

Reduced DNA MethylationDNA methylation is an important negative regulator of gene expression and hypomethylation of oncogenes has been shown in human and rat HCC.181 Chronic ethanol consump-tion results in reduced concentrations of SAMe, the main methyl donor (as discussed previously) and dietary depletion of SAMe increases the risk of HCC in rats.182

ImmunosuppressionMalnutrition, vitamin deficiencies, and acute ethanol per se can all result in reduced immunosurveillance. Of particular relevance is the effect on natural killer cells, thought to be

Fig. 28-8 Frequency of cirrhotic and precirrhotic liver lesions according to dose and duration of alcohol consumption in 334 drinkers.(Reproduced from Lelbach WK. Cirrhosis in the alcoholic and its relation to the volume of alcohol abuse. Ann N Y Acad Sci 1975;252:85105 with permission of New York Academy of Science.202)

60

40

50

30

10

20

80

70

100

90

00 2 16 206 8 10 124 14 18 22

Years

Mean durationof alcohol abuse

No of cases

Mean dailyalcohol intake(average of minimumand maximum)

3.6 8.3 12.9 21.6(15 y) (610 y) (1115 y) (1>15 y)

163g 177g 192g 227g

73 129 81 51

(130197) (144210) (160224) (197275)

Cirrhosis of the liverCirrhosis and potentially precirrhotic lesions(severe steatofibrosis with inflammatory reactions,chronic alcoholic hepatitis)Moderate to severe fatty infiltration

Ferr

itin

(ng/

ml s

erum

)


Although these studies have provided evidence that dose, pattern, and type of alcohol consumption and dietary (and presumably exercise-related) factors play a role in determin-ing ALD risk, they have also demonstrated that other, endogenous factors are likely to be equally if not more important.

Sex and Risk of ALDThe most obvious endogenous or genetic factor determining ALD risk is female sex. It has long been appreciated that women develop ALD at a lower intake of alcohol than men. The traditional explanation has been that women develop higher blood alcohol concentrations per unit of alcohol con-sumed due to their lower volume of distribution for alcohol. This in turn is attributed to their lower body mass index and to fat constituting a higher percentage of their body mass than in men. Thurman and colleagues have demonstrated in the rat model that estrogen increases gut permeability to endotoxin and accordingly up-regulates endotoxin receptors on Kupffer cells, leading to an increased production of tumor necrosis factor in response to endotoxin.194 These exciting data suggest several new directions for research into human sex-specific susceptibility to ALD.

NonSex-Linked Genetic Factors and Risk of ALDEvidence for nonsex-linked genetic susceptibility to ALD comes principally from a twin study showing that the concor-dance rate for alcoholic cirrhosis was three times higher in monozygotic than in dizygotic twin pairs.195 This difference in concordance rates was not entirely explained by the difference in concordance rates for alcoholism per se. Further indirect evidence of a genetic component to disease risk comes from the observation that the death rate from ALD is subject to wide interethnic variation that is not entirely explained by variations in the prevalence of alcohol abuse.196,197 Hispanics appear to be at particularly high risk, for example. Difficulties in performing family linkage studies in ALD have resulted in almost all of the relevant information thus far coming from classical case-control, candidate gene, allele association studies. Accordingly these studies are subject to all the common pit-falls of this type of study design and must be interpreted with caution.198 Many early reports of positive associations are likely to be subject to type I errors (chance findings), while negative reports may be subject to type II errors (false nega-tives) attributed to small underpowered studies. Given that the most likely mechanisms of hepatocyte injury in excessive drinkers are related to fat accumulation, oxidative stress, endotoxin-mediated release of proinflammatory cytokines, and immunologic damage, the majority of studies reported thus far have focused on genes encoding proteins involved in these various pathways.

Genes Influencing the Severity of SteatosisRecognition of the role played by steatosis in the pathogenesis of more advanced liver disease60 suggests that factors deter-mining its severity may play a key role in determining the risk of cirrhosis. Clearly genetic and environmental factors deter-mining the degree of obesity would fall into this category, as

Dose of EthanolThe observation that only a minority of heavy drinkers develop ALD was first reported 30 years ago by Lelbach and colleagues. They showed that although the risk of disease increased in proportion to the duration of intake, only 20% of consumers of more than 200 g of ethanol (around 20 stan-dard drinks) per day develop cirrhosis after 13 years and fewer than 50% after 20 years.186 Further work from around this time showed that women appeared to develop ALD at lower doses of alcohol consumption than men.187 More detailed studies examining the precise dose-response relation-ship between alcohol intake and risk of ALD, the gender effect and the risk threshold have been reported in the last 10 years.

A large cohort study from Italy involving 6917 subjects between the ages of 12 and 65 reported that the risk of devel-oping ALD begins at 30 g/day of ethanol.185 However, only 5.5% of the individuals drinking this amount showed signs of liver disease. The risk increased according to daily dose, reaching 10% at 60 g per day. The study also reported that the risk is higher among those older than 50 years of age if alcohol is drunk outside mealtimes or consumed in a variety of different beverages rather than one tipple of choice. Inter-estingly, this study showed no sex effect. Further evidence for a dose-response relationship and a risk threshold came from an even larger study from Copenhagen involving 13,285 sub-jects between the ages of 30 and 79.188 A self-administered questionnaire assessed intake, and incidence of disease was taken from death certificates and hospital medical records. This study revealed a dose-dependent increase in risk with women having a significant risk above 7 to 13 units per week, and men 14 to 27 units per week. This group updated their data analysis recently by looking at the type of alcohol con-sumed.189 Their results suggest that the highest risk is seen in drinkers that do not include wine in their drinking repertoire. Furthermore the relative risk of cirrhosis fell as the propor-tion of wine increased. This association between wine intake and ALD risk may be confounded by other factors associated with wine drinking such as a lower prevalence of obesity com-pared with beer and spirit drinkers. One study following the eating habits of beer drinkers compared with wine drinkers has shown that the former tend to buy more unhealthy items at the supermarket.190

Although all these studies have their flaws, with data collec-tion being the most obvious, they do allow a number of con-clusions to be drawn. No dose of alcohol confers a guarantee of developing cirrhosis regardless of the period it is consumed for, and relatively low doses can cause problems.

DietThe data discussed previously from ethanol-fed rats linking a diet high in polyunsaturated fats with an increased risk of alcoholic liver injury are supplemented by an epidemiologic study linking cirrhosis mortality with pork (high in linoleic acid) consumption and dietary intake of unsaturated fats.191 A further case-control study from France has reported that the risk of cirrhosis is increased by diets high in fat and alcohol and low in carbohydrate.192 A more obvious role for diet in ALD risk has been suggested by two studies showing that obesity and associated hyperglycemia increase the incidence of all stages of ALD in heavy drinkers.4,193


identification of promoter polymorphisms in genes encoding endotoxin receptors, cytokines, and cytokine receptors, has recently suggested an alternative set of candidates to explain genetic susceptibility to ALD. CD14, an LPS receptor on monocytes, macrophages, and neutrophils, has no intracellu-lar domain but enhances signaling through another LPS receptor, Toll-like receptor 4 (TLR4). A C/T polymorphism is present at position -159 in the CD14 promoter, with the TT genotype associated with increased levels of soluble and mem-brane CD14.212 A study from Finland has recently reported an association between possession of the TT CD14 genotype and advanced ALD127; however, this has not been observed in a larger study in northeast England.213 This latter study also showed no association between ALD and possession of the Asp299Gly polymorphism in the TLR4 gene, previously reported to be linked to hyporesponsiveness to LPS.214

With respect to polymorphisms in the cytokine genes, the first such association was reported between alcoholic hepatitis and a polymorphism at position -238 in the TNF- promoter region.215 The functional significance of this polymorphism is, however, unclear and the association may well be either spuri-ous or due to linkage disequilibrium with another true disease-associated polymorphism on chromosome 6. An association with ALD has also been reported for a promoter polymorphism in interleukin-10 (IL-10). IL-10 is the classical antiinflammatory cytokine that inhibits: (1) the activation of CD4+ T-helper cells, (2) the function of cytotoxic CD8+ T-cells and macrophages, (3) class II HLA/B7 expression on antigen-presenting cells, and (4) hepatic stellate cell collagen synthesis. A variant CA substitution at position -627 in the IL-10 promoter has been associated with decreased reporter gene transcription, decreased IL-10 secretion by peripheral blood monocytes, and an increased response to -interferon in patients with chronic hepatitis Call consistent with the polymorphism being associated with lower IL-10 production. A strong association between possession of the A allele and ALD has been reported from a study of more than 500 heavy drinkers with and without advanced liver disease.216 This is consistent with low IL-10 favoring inflammatory and immune-mediated mechanisms of disease as well as hepatic stellate cell collagen production.

Immune Response Genes and Risk of ALDIn view of the immunoregulatory functions of IL-10, the asso-ciation between ALD and a low-activity promoter polymor-phism in IL-10 may be considered as further evidence that immune mechanisms are involved in the pathogenesis of ALD. Further evidence supporting a role for immune mechanisms in determining individual susceptibility to ALD has come from a recent study showing that, compared with drinkers with no evidence of ALD, patients with ALD are more likely to have high titres of autoantibodies against CYP2E1160 and to have T-cell responses against oxidative stress-derived adducts.162 Cytotoxic T lymphocyte antigen-4 (CTLA-4) is a T-cell surface molecule that normally acts to damp down the immune response to antigens either directly, by competing with CD28 on the surface of CD4+ Th cells for the antigen-presenting cell costimulatory molecule B7, or indirectly by activating T regulatory cells that act to inhibit CD4+ Th cell function.217 CTLA-4 knockout mice develop lethal autoreac-tive lymphoproliferative disease, and an AG polymorphism

would functional polymorphisms of genes encoding enzymes involved in hepatic lipid metabolism. Most recently a striking association has been demonstrated between a polymorphism in the PNPLA3 gene, encoding adiponutrin, and ALD in Mexican subjects199 and Europeans.200 This rs738409 polymor-phism was identified as a susceptibility gene for nonalcoholic fatty liver disease in two independent genomewide association studies (GWAS).201,202 The precise biologic function of adipo-nutrin is unknown but the polymorphism appears to disrupt triglyceride hydrolysis to cause fatty liver.

Genes Influencing Oxidative Stress and Risk of ALDThe principal class of genes that influences the oxidant load in heavy drinkers are those genes encoding enzymes involved in alcohol metabolism. Polymorphisms have been identified in two of the seven genes encoding alcohol dehy-drogenases (ADH2 and ADH3), in the promoter region of the CYP2E1 gene and in the coding region of the gene encoding the mitochondrial form of aldehyde dehydrogenase (ALDH2). The genes encoding ADH2 and ALDH2 undoubtedly play a role in determining the risk of alcoholism and, to a lesser extent, ALD in Asian populations.203-205 Previously reported associations with ADH3 probably reflect linkage disequilib-rium with ADH2.206 In Caucasians, results from studies reported to date support a role for the ADH2 polymorphism in determining the risk of alcoholism but not ALD.207 Several studies have looked for an association between the c2 pro-moter (Rsa I) polymorphism of the CYP2E1 gene and ALD with no consistent results emerging in any population, although one study did report that the cumulative lifetime alcohol intake of patients with ALD heterozygous for the c2 (more transcriptionally active) allele was almost half that of patients with ALD homozygous for the c1, wild-type allele.208 The HFE gene is another obvious candidate gene for ALD because liver iron promotes oxidative stress and iron deposi-tion is common in ALD. Unfortunately, a case-control study of more than 400 patients and controls found no evidence of an association between ALD and either of the HFE muta-tions associated with hemochromatosis.209 This lack of asso-ciation was explained by the observation that hepatic iron content did not differ between patients with and without the mutations.

The lack of any striking associations between polymor-phisms in genes encoding proteins involved in the generation of reactive oxygen species and ALD has recently turned atten-tion towards polymorphisms in genes encoding proteins involved in the bodys antioxidant defenses. Manganese-dependent SOD2 is the most important mitochondrial anti-oxidant enzyme and a polymorphism altering its mitochondrial targeting sequence has been associated with ALD in a small French study,210 although not confirmed in a larger study from the United Kingdom.211 This and other polymorphisms affect-ing the function of antioxidant defense systems are clearly worthy of further study.

Endotoxin Receptor and Cytokine Genes and Risk of ALDEvidence supporting a role for endotoxin-mediated cytokine release in the pathogenesis of ALD, together with the


HistoryFeatures in the history important for both the confirmation of alcohol abuse and to aid in its subsequent management include the amount and duration of alcohol intake, the pattern of intake, precipitating factors of drinking bouts, and evidence of physical dependence such as early morning tremor, black-outs, and morning drinking. Confirmation of the history should be sought from a family member or close associate. Specific liver-related symptoms, such as jaundice and hematemesis, should be inquired about but are uncommon, even in patients with established disease. In addition, because not all alcohol-dependent patients with liver disease necessar-ily have disease caused by alcohol,222 inquiries should be made concerning other risk factors for liver disease, including family history, foreign travel, blood transfusions, or intravenous drug use.

Clinical ExaminationImportant features to note on examination are the signs of chronic liver disease including hepatomegaly and signs indica-tive of alcohol-related pathology in other organs such as hypertension, atrial fibrillation, and a cushingoid appearance. It is important to understand that many of the classical signs of chronic liver disease, including spider nevi, Dupuytren con-tractures, palmar erythema, and parotid swelling, can occur in alcoholics in the absence of cirrhosis. Clinical signs and history cannot be relied upon to distinguish the various his-tologic subtypes of alcoholic liver disease because patients with cirrhosis can be asymptomatic while patients with hepa-tocellular failure may have only severe fatty change.223

Laboratory InvestigationsBiochemical and hematologic tests can confirm the presence of alcohol abuse and indicate the presence of liver damage, but are not useful in determining the severity of the histologic lesion. Blood alcohol estimations are an often underused method of confirming a suspicion of excess drinking, with levels greater than l00 mg/l00 ml at a morning clinic, or levels greater than 150 mg/100 ml without obvious intoxication, strongly suggestive of alcohol abuse. Elevation of -glutamyl transferase (GT) has been reported in up to 90% of patients abusing alcohol.224 The rise is mainly due to hepatic micro-somal induction and is independent of the presence of liver disease, although hepatocellular necrosis and cholestasis may contribute. It is not specific for alcohol abuse and is raised in other forms of liver injury and in patients taking other enzyme-inducing drugs.225 Its main clinical use is probably in monitoring a period of supposed abstinence because it falls within a week of cessation of drinking. Other biochemical markers of alcohol abuse rather than liver disease include elevated serum uric acid226 hypertriglyceridemia and desi-alylated transferrin.227 The classical hematologic marker of alcohol abuse is a raised mean corpuscular volume (MCV), which has been reported to occur in 80% to 100% of alcohol-ics with and without liver disease,228 and may be more common in alcoholic women. It is due to a direct toxic effect of alcohol on the marrow, although nutritional folate and B12 deficiencies may contribute in some patients. With regard to biochemical markers of alcohol-related liver damage, a rise in serum

in exon 1 leading to a ThrAla substitution has recently been associated with autoimmune liver diseases, insulin-dependent diabetes, and autoimmune thyroid disease. These associations strongly suggest that this polymorphism is associated with impaired CTLA-4 function, although recent data suggest that other tightly linked CTLA-4 polymorphisms may be respon-sible for the functional effect.218 Although the exon 1 polymor-phism has been asso

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28 -Alcoholic Liver Disease.pdf