Advanced Alcoholic Liver Disease Research Articles

Heterozygotes for HFE mutations have no increased risk of advanced alcoholic liver disease

Background—Iron overload is common in the livers of alcoholics and may play a role in disease pathogenesis. An MHC like gene, HFE,has recently been identified that is mutated in most...

Histomorphometric assessment of fibrosis in advanced alcoholic liver disease

Histomorphometric assessment of fibrosis in advanced alcoholic liver disease

Spur cell anemia and hepatic iron stores in patients with alcoholic liver disease undergoing orthotopic liver transplantation

Following orthotopic liver transplantation (OLT) histological examination of explant livers from patients with alcoholic liver disease (ALD) sometimes shows extensive iron deposits in a distribution suggestive of homozygous haemochromatosis.To use haemochromatosis gene (HFE) assays to distinguish between ALD with notable siderosis and hereditary haemochromatosis. To evaluate the possible influence of spur cell haemolytic anaemia on hepatic iron loading.Thirty seven patients with ALD were abstinent for at least six months prior to OLT. Twenty three patients had transferrin saturations greater than 55%, 16 also had increased serum ferritin (>350 micrograms/l). Eight of 37 (22%) explant livers had grade 3 or 4 hepatic iron deposition, predominantly in hepatocytes. Of these, four had a hepatic iron index greater than 1. 9 and most seemed to have spur cell haemolytic anaemia.Mutation analysis for C282Y and H63D mutations was performed on DNA extracts from peripheral blood or explant liver. Spur cell haemolytic anaemia was diagnosed when the haemoglobin was 105 g/l in the presence of notable acanthocytosis.None of the eight patients with grade 3 or 4 hepatic iron had evidence of the C282Y mutation. Two of the eight were heterozygous for H63D. None of the remaining 28 patients tested showed homozygous HFE mutations. Spur cell anaemia was present in six of the eight patients with heavy iron deposition and only one of the remaining patients.The HFE mutation was not present in these patients with advanced ALD and heavy iron loading. Spur cell haemolytic anaemia provides an alternative potential mechanism for the heavy iron loading.

Randomised controlled double-blind trial of the calcium channel antagonist amlodipine in the treatment of acute alcoholic hepatitis

Background/Aims: Calcium channel blockers have a hepatoprotective action in animal models of alcohol-induced liver injury but their effect in alcoholic liver disease in humans has not been previously investigated. We have conducted a randomised, placebo-controlled trial to investigate the possible benefit of the calcium channel blocker amlodipine in terms of 4-week survival in hospitalised patients with severe acute alcoholic hepatitis. Methods: Sixty-two patients with acute alcoholic hepatitis were randomised to receive 5–10 mg amlodipine each day for 1 year or an identical capsule containing placebo. In 36 (58%), acute alcoholic hepatitis was confirmed on biopsy and in the remainder on clinical and laboratory criteria. There were no statistically significant differences in clinical characteristics and disease severity in the treated and placebo groups. Results: Of the 32 patients receiving amlodipine, there were six deaths (19%) in the first 4 weeks compared with seven (23%) of the placebo patients ( p=0.329). Causes of death were similar in the amlodipine and control groups, with liver failure predominant. Analysis by the Cox proportional hazards model after adjustment for other prognostic factors showed survival was not significantly influenced by active treatment ( p=0.07). One patient in each group was withdrawn because of the development of hypotension, but this did not recur on reintroduction of the capsules. Conclusions: This study shows that calcium channel blockers are well tolerated with few side effects in advanced alcoholic liver disease, but there is no conclusive evidence from this study that calcium channel blockers are helpful in the treatment of alcoholic hepatitis.

Decreased endotoxin-binding capacity of whole blood in patients with alcoholic liver disease

Background/Aims: The proinflammatory effects of endotoxemia, which is often observed in alcohol-abusing patients with various degrees of liver disease, may be modulated by changes in the concentration of endotoxin binding factors. Therefore, the plasma endotoxin concentration and the overall endotoxin binding capacity of whole blood were measured in these patients. Methods: Patients with minor (A1: n=27), more pronounced (A2; n=13), cirrhotic alcoholic liver disease (A3; n=18), and non-alcoholic cirrhosis (NC: n=6), and 15 healthy control persons (HC) were included in the study. Endotoxin plasma levels were determined using a standardized limulus assay. A modified assay was applied to additionally detect tightly bound endotoxin. To measure the endotoxin-binding capacity, aliquots of whole blood were incubated with serial dilutions of endotoxin, supernatants were obtained, and endotoxin retrieval was estimated by addition of limulus lysate, followed by photometric measurement of the maximal reaction velocity (dOD max). Endotoxin binding capacity equals the endotoxin concentration at which dOD max reaches a predefined threshold. Results: All groups of alcohol abusers had significantly elevated endotoxin plasma levels with a considerable portion of ‘bound’ endotoxin. Conversely, the endotoxin binding capacity was markedly diminished, mainly in patients with more advanced liver disease (A1: 85.8% of the control value [non-significant vs. controls]; A2: 25.4% [ p<0.05]; A3: 43.6% [ p<0.02], NC: 43.2%). Conclusions: The endotoxin-binding capacity is diminished in patients with alcoholic and non-alcoholic cirrhosis, as well as in less advanced alcoholic liver disease. Reduced endotoxin binding may contribute to the adverse effects of endotoxemia.

The use of color doppler flow imaging in the diagnosis of acute alcoholic hepatitis: Evidence of intrahepatic arterial dilatation

Objective: In this article, a color Doppler flow imaging investigation was performed to demonstrate the segmental arterial dilatation within the liver parenchyma of patients with acute alcoholic hepatitis and to compare these findings with those obtained in a group of patients with alcoholic liver disease, but without clinical and biochemical parameters suggesting acute hepatitis. Methods: Color Doppler flow imaging was performed in 59 patients with a previously established diagnosis of alcoholic disease of the liver. Among those patients, 25 fulfilled physical and biochemical criteria of acute alcoholic hepatitis. Ultrasound scans were performed by two independent, equally skilled investigators with a 3.5 MHz transducer. Based on a previous observation, special attention was given to whether a intrahepatic arterial dilatation was present. Results: Of the 59 patients with alcoholic liver disease, intrahepatic arterial dilatation was seen in 29 (49.2%) patients. In 27 of 29 patients (93.1%) with intrahepatic arterial dilatation the arterial nature of one of the vessels was confirmed by pulsed-Doppler flowmetry. Intrahepatic arterial dilatation was observed in 22 of 25 (88.0%) patients with acute alcoholic hepatitis. Among the 34 patients with advanced alcoholic liver disease, but without physical and biochemical evidence of acute hepatitis, the presence of intrahepatic arterial dilatation was found in 7 (20.6%) patients. A systolic hepatic bruit was heard in 19 of 25 (76.0%) patients with acute alcoholic hepatitis, all of whom had intrahepatic arterial dilatation. Comparing patients with acute alcoholic hepatitis who had intrahepatic arterial dilatation with those who had not, significant difference was found with respect to the values of serum bilirubin and aspartate transaminase. For technical reasons an accurate and reproducible measurement of hepatic artery resistive index (RI) was possible in 41 (69.5%) patients (18 patients with and 23 patients without acute alcoholic hepatitis). For the 18 patients with acute alcoholic hepatitis, the mean RI was 0.74 ± 0.08, a level that was significantly lower than that observed in the 23 patients without physical and biochemical findings of acute alcoholic hepatitis (0.85 ± 0.05); P < 0.05). Conclusion: This study provides a reliable assessment of the accuracy of color Doppler flow imaging in diagnosing intrahepatic arterial dilatation as an important and very frequent finding in patients with acute alcoholic hepatitis.

ʼHiddenʼ endotoxin in plasma of patients with alcoholic liver disease

Objective: The results of several earlier studies suggest that previous methods used to determine endotoxin in the plasma of patients with acute or chronic liver disease fail to detect a substantial percentage of lipopolysaccharide (LPS) which is bound to plasma constituents. The aim of this study was to determine this 'hidden' fraction of LPS in the plasma of patients with alcoholic liver disease. Design and methods: LPS was determined in plasma samples from 43 patients with alcoholic liver disease (24 patients with cirrhosis and 14 with fatty liver or alcoholic hepatitis) and nine healthy controls using a chromogenic substrate test. The results obtained following standard pretreatment of the plasma by heating (method A) were compared with those following pretreatment by the addition of perchloric acid and dissolution of the precipitate by triethanolamine, a solvent for LPS, (method B) or addition of a detergent, Tween 80, after heating the plasma (method C). Results: Using the standard method (A), the groups with fatty liver or alcoholic hepatitis and the cirrhotics exhibited distinctly higher plasma LPS concentrations (16.5 ±4.8 and 12.3 ± 2 pg/ml, respectively) compared with those obtained in the controls (2.5 ± 0.5pg/ml; P < 0.01). The addition of Tween 80 (method C) resulted in a pronounced increase (P<0.01) in the concentration of LPS in patients with fatty liver or alcoholic hepatitis (52.1 ±13.3 pg/ml) and in cirrhotics (40.7 ± 8.1 pg/ml), while only a small increase was observed in controls (6.25 ±1.8 pg/ml). Using method B, a virtually identical increase in LPS concentration was observed in the cirrhotics (40.9 ± 4.7 pg/ml). The LPS concentrations revealed a significant negative correlation with the concentrations of HDL cholesterol, albumin and transferrin, three proteins which are capable of binding LPS in plasma. Conclusion: Plasma of patients with both mild and advanced alcoholic liver disease contains distinctly higher LPS concentrations than previously supposed.

Metabolic effects of β-adrenergic receptor blockade in advanced alcoholic cirrhosis

It has been postulated that the adverse metabolic effects of beta-adrenergic blockade with propranolol in cirrhosis may be related to altered delivery and utilisation of oxygen, particularly in patients with advanced alcoholic liver disease (ALD). Consequently, in 10 patients with decompensated ALD, we assessed (a) systemic and hepatic oxygen delivery (DO2), extraction ratio (%O2E) and consumption (VO2), (b) myocardial VO2 (assessed by the rate-pressure product [RPP], together with full systemic and splanchnic haemodynamics) and (c) hepatic redox state (HRS), measured indirectly by the arterial ketone body ratio (KBR i.e. ratio of acetoacetate/beta-hydroxybutyrate), prior to and following intravenous propranolol (0.1-2 mg/kg). Results are expressed as mean +/- S.E.M. Propranolol reduced DO2 (700 +/- 33 vs. 583 +/- 32 ml/min/m2, p < 0.05) and myocardial VO2 (RPP 72 vs. 58, p < 0.05). The %O2E increased however, (18.5 +/- 1.3 vs. 22.6 +/- 1.6%, p < 0.05), resulting in unaltered systemic VO2 (127 +/- 7.3 vs. 131 +/- 6.9 ml/min/m2, p > 0.10). Similarly hepatic VO2 did not change. KBR was not altered (0.44 +/- 0.08 vs. 0.48 +/- 0.07), and in fact improved in two patients (Child C12 and C13) from 0.17 to 0.34 and 0.12 to 0.27, respectively. In conclusion, the results of this study suggest that an underlying O2 debt exists in patients with advanced alcoholic cirrhosis and that beta-adrenergic blockade with propranolol 'normalises' the O2 supply-consumption relationship resulting in more efficient O2 utilisation without adversely affecting HRS. The mechanism of this action may be related to the antagonism of beta 2-mediated arteriovenous shunting resulting in appropriate blood redistribution.

Serum vitronectin receptor in alcoholic liver disease: correlation with fibronectin receptor and morphological features.

In order to clarify the significance of serum level of vitronectin receptor (VNR) in alcoholic liver disease (ALD), we have investigated the relationship with fibronectin receptor (FNR) and histological liver features in 21 ALD patients. Serum level of VNR and FNR was measured by enzyme immunoassay. Liver disease activity was scored based on levels of fibrosis and focal intralobular necrosis and degeneration. The serum level of VNR (micrograms/ml) was significantly higher in the patients with hepatic fibrosis (9.87 +/- 2.51) and liver cirrhosis (10.80 +/- 1.52) than in normal subjects (5.51 +/- 0.52, P < 0.01) and fatty liver subjects (6.58 +/- 0.58, P < 0.05). A positive correlation was found between serum levels of VNR and fibronectin receptor (FNR) (P < 0.05). A positive correlation was observed between the serum level of FNR and the degree of hepatic fibrosis or focal intralobular necrosis and degeneration, while no correlation was found between the serum level of VNR and the degree of histological features. A positive correlation was also noted between the serum level of FNR and N-terminal type III procollagen aminopeptide (PIIIP) (P < 0.001), while no correlation was observed between the serum level of VNR and PIIIP. We conclude that the serum level of VNR is increased in patients with advanced alcoholic liver disease. However, the mechanism by which serum levels of beta subunit of VNR and FNR are increased may be different.

Endoscopic findings in alcoholic liver disease: Does gender make a difference?

Alcohol abuse and alcoholic liver disease are major health problems in many parts of the world. The prevalence of alcoholic liver disease is directly related to both rate and duration of alcohol intake. It is a widely held belief that women are more susceptible to the hepatotoxic effects of ethanol, and develop alcohol-related liver disease more readily than do men. A lower volume of distribution for alcohol, greater immune reactivity and higher activity of alcohol metabolizing enzymes in women are considered, at least in part, to be responsible for the greater susceptibility of women to alcoholic liver disease. Little data exist as to the extent of gastrointestinal involvement in advanced alcoholic liver disease, and even less, as to the relationship of these potential gastrointestinal lesions with gender, and whether or not female patients develop gastrointestinal complications at an earlier time in their liver disease natural history and whether when it occurs it is more severe than that seen in males. To answer these questions, 75 subjects (49 men and 26 women) with alcoholic liver disease underwent elective upper and lower gastrointestinal endoscopy while they were clinically stable. The prevalence of the various gastrointestinal lesions did not differ significantly between male and female alcoholics except for gastric ulcer (GU) and nonspecific inflammatory changes of the colon which were more common in females than in males (23.1% vs. 6.1% and 11.1% vs. 0%, respectively; both p<0.05). Based upon these data, we conclude that unlike the situation with liver disease, women are not at greater risk for the development of the common gastrointestinal complications characteristic of advanced liver disease, except for GU, than are men.

Liver transplantation for alcoholic liver disease: a consideration of reasons for and against.

Orthotopic liver transplantation is a clinical procedure that has been accepted widely as the treatment of choice for individuals with advanced chronic liver disease. As such, its application to the important clinical problem of alcoholic liver disease is inevitable. The arguments for and against liver transplantation for individuals with advanced alcoholic liver disease are presented.

Plasma Human Atrial Natriuretic Factor in Cirrhosis and Ascites With and Without Functional Renal Failure

Functional renal failure of cirrhosis (FRFC) is a usually fatal syndrome of acute renal failure occurring in patients with advanced liver disease. Although not conclusively proven, most evidence suggests that renal arterial and arteriolar vasoconstriction is the cause of the renal failure in these patients. However, the mediators of the vasoconstriction remain unknown. Human atrial natriuretic factor (hANF) is a hormone with potent natriuretic, diuretic, and vasorelaxant properties. A deficiency of hANF could lead to renal arterial vasoconstriction and avid renal sodium retention as seen in FRFC. This study was undertaken to determine if patients with FRFC are deficient in circulating hANF. Seven patients with advanced alcoholic liver disease and renal failure of unknown cause (FRFC) were compared with 7 patients with advanced alcoholic liver disease, ascites, and normal serum creatinine as well as with 14 healthy volunteers. Plasma hANF was measured by radioimmunoassay. Plasma hANF was 742 +/- 227 pg/ml (mean +/- SEM) in patients with FRFC compared with 360 +/- 70 pg/ml in patients with liver disease and normal serum creatinine (p greater than 0.05) and 28 +/- 5.7 pg/ml in healthy volunteers (p less than 0.005 vs. FRFC and chronic liver disease, ascites, and normal serum creatinine). Thus, FRFC is not caused by a deficiency of circulating hANF. The elevated plasma hANF levels in patients with chronic liver disease and continued sodium retention may suggest a renal insensitivity to the natriuretic effects of hANF.

Hepatic zinc content in patients with various stages of alcoholic liver disease and in patients with chronic active and chronic persistent hepatitis.

The hepatic zinc content was determined in liver biopsies of patients with alcoholic and nonalcoholic liver disease using proton-induced X-ray emission. The values obtained in postmortem specimens of the liver from 27 patients with no evidence of acute or chronic liver disease served as controls. The mean value and the range of the zinc content in the controls (75 +/- 24 ppm wet weight) are in good agreement with those reported in the literature. The hepatic zinc content in the control group showed no significant age or sex dependence. The mean zinc content was significantly decreased in all groups of patients with alcoholic liver disease. The decrease was comparable in biopsies from patients with alcoholic fatty liver (-56.7%, n = 12), mild alcoholic hepatitis (-50.5%, n = 6) and alcoholic cirrhosis (-45.6%, n = 10). The hepatic zinc content was also distinctly reduced in patients with chronic active hepatitis (-60.3%, n = 15) and in those with chronic persistent hepatitis (-44.9%, n = 8). The estimation of the zinc content in subcellular fractions of the liver performed in postmortem specimens from seven patients with alcoholic liver cirrhosis and in six controls revealed a significant reduction in the zinc content in the fraction containing cell nuclei and membranes and in the mitochondrial fraction. A similar decrease was seen in the 100,000 g supernatant; however, the difference did not attain statistical difference. The zinc content of the microsomal fraction in the controls was lower than in the other three cell fractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased production, hepatic conjugation, and biliary secretion of bilirubin in the rat after chronic ethanol consumption

Disturbances of bilirubin metabolism such as jaundice or pigment gallstone formation, or both, occur in alcoholic cirrhosis of the liver. We have studied the influence of chronic ethanol consumption on bilirubin metabolism as well as on biliary calcium and bile acids in 16 pair-fed male rats. The animals received nutritionally adequate liquid diets containing 36% of total calories either as ethanol or isocaloric carbohydrates for 4 wk. Bile flow was significantly enhanced after chronic ethanol feeding (p < 0.05 after 90-min bile collection) and was found to be mainly bile acid-independent. The biliary output and concentration of bilirubin monoconjugates, bilirubin diconjugates, and total calcium was significantly increased (p < 0.01) in alcohol-fed rats compared with controls. This was not the case for unconjugated bilirubin and for the calcium/bile acid ratio. Hepatic bilirubin uridine-5′-diphosphate-glucuronosyltransferase activity (p < 0.01), serum total bilirubin (p < 0.01), and serum free hemoglobin (p < 0.001) were significantly increased after ethanol consumption. These data provide evidence for enhanced bilirubin production, probably due to hemolysis, after alcohol ingestion. The enhanced bile production is associated with an increased hepatic conjugation and subsequent biliary secretion of bilirubin conjugates. In advanced alcoholic liver disease, these compensatory mechanisms may fail and contribute to the development of jaundice.

Endotoxin induced hepatic necrosis in rats on an alcohol diet.

The role of endotoxin in the pathogenesis of progressive liver disease is receiving increasing attention, but remains controversial. Similarly, although alcoholic hepatitis is now recognized as the transitional link between alcoholic fatty liver and advanced alcoholic liver disease, the aetiology of liver cell necrosis in alcoholic hepatitis is not known. Rats fed a nutritionally adequate liquid alcohol diet according to the formula of Lieber and DeCarli developed fatty livers. Littermates fed an identical diet and challenged with small IV doses (1 microgram/g body weight) of E. coli lipopolysaccharide endotoxin (LPS) developed focal necrotizing hepatitis. Control littermates fed an identical calorie balanced but alcohol free diet and challenged with identical doses of LPS did not develop any liver lesions. The hepatocyte necrosis with associated inflammatory changes induced by LPS in fatty livers has some features of early human alcoholic hepatitis and suggests that progressive alcohol induced damage may be multifactorial in origin.

Spontaneous and antibody‐dependent cellular immune reactions to ethanol‐altered hepatoma cells

Spontaneous cell-mediated cytotoxicity (SCMC), antibody-dependent cellular cytotoxicity (ADCC) and proliferative lymphocyte stimulation in alcoholic liver disease (ALD) were investigated. Peripheral blood lymphocytes (PBL) from eight patients with advanced ALD and nine normal controls were tested against hepatoma cells (PLC/PRF/5) as targets. Target cells were grown in either normal culture medium or medium supplemented with 1 and 5% ethanol, respectively, for 24 to 48 h. Ethanol-exposed hepatoma cells exhibited profound and characteristic morphological alterations. Ethanol preincubation, however, proved to be without effect on immune reactions. Provided that hepatoma cells are an appropriate model, we assume that the proposed immune reactions in ALD are based on metabolic interactions operative only in vivo but do not parallel morphological alterations of liver cells directly induced by ethanol.

Lymphocyte Subsets Studied with Monoclonal Antibodies in Liver Tissues of Patients with Alcoholic Liver Disease

In order to determine the nature of T lymphocytes accumulating in hepatic tissues of patients with alcoholic liver disease, we phenotyped these cells in situ, using monoclonal antibodies to lymphocyte surface antigens and the avidin-biotin-peroxidase complex (ABC) technique. In portal areas of cirrhotic livers, a significant increase (p less than 0.001) in T lymphocytes was observed as compared to livers showing only fatty changes and normal liver tissues. The T cells accumulating in cirrhotic livers comprised T helper-inducer (T4) and T suppressor-cytotoxic (T8) subsets with the T4/T8 ratio of 1.4 +/- 0.5 (SD, n = 14). In contrast, the T lymphocytes in the cellular projections which extended into the parenchyma consisted mostly of suppressor-cytotoxic cells. The relative enrichment in T8+ lymphocytes among the cells invading the parenchyma suggests that they may be important in the mediation of a cytotoxic injury of hepatocytes in advanced alcoholic liver disease and in regulating an immunologic challenge possibly initiated in response to alcoholic hyaline.

The nature and origin of proliferated bile ductules in alcoholic liver disease.

Proliferation of bile ductules is commonly seen in expanded portal tracts and periportal areas in many conditions, including advanced alcoholic liver disease. Such ductules are usually tortuous and irregular and composed of cuboidal cells. They frequently have poorly defined lumens. The epithelial cells are similar to those of normal bile ductules and small bile ducts; however, cells that appear intermediate between duct epithelial cells and hepatocytes are frequently identified by light and electron microscopy. The origin of the ductular cells from hepatocytes may be confirmed by the demonstration of the markers of hepatocytes, such as glycogen and glucose-6-phosphatase activity in some proliferated bile ductules. In addition, alcoholic hyalin is occasionally recognizable in the epithelial cells of bile ductules. The majority of periportal bile ductules appears to have been derived from transformation of hepatic cords rather than multiplication of preexisting bile ducts. The proliferated bile ductules seem to communicate between the bile canaliculi and the interlobular bile ducts.

Plasma urea and creatinine status in chronic alcoholics

Plasma urea and creatinine concentrations were estimated by Auto-Analyzer in 128 chronic alcoholics (103 males, 25 females; mean age ± SD = 42.73 ± 6.53; age range 20–60 years). Plasma levels of both urea (mean ± SD = 3.46 ± 1.31; normal range 2.0–6.5 mmol/l) and creatinine (mean ± SD = 87.86 ± 15.45; normal range 60–120 μmol/l) were found to be within normal limits. It is concluded from our observations that chronic ethanol ingestion per se is not nephrotoxic. The kidney seems to be the only vital organ generally spared in chronic alcoholics without advanced alcoholic liver disease or hepato-renal syndrome.

Drug Metabolism in Liver Disease: Identification of patients with impaired hepatic drug metabolism

Antipyrine half-life (AP t1/2) was measured in 62 patients with, and 10 control patients without, liver disease to ascertain possible factors which may be useful in identifying patients with abnormal drug metabolism. Antipyrine metabolism was normal or marginally impaired in patients with compensated cirrhosis or acute hepatitis, whereas it was frequently abnormal in those with chronic active hepatitis or advanced alcoholic liver disease. A high degree of correlation was found among AP t1/2 and prothrombin time, hepatic encephalopathy, and ascites. Of patients with severely impaired drug metabolism, 80% had one or more of these features. The severity of histological changes in liver biopsies was of additional help in predicting impaired drug metabolism. Concurrent drug ingestion enhanced antipyrine metabolism in most patients with liver disease as well as in control patients. Inadequate diet was associated with prolongation of AP t1/2, but other environmental factors such as alcohol ingestion, cigarette smoking, and coffee consumption did not affect rates of drug metabolism in patients with liver disease. Consideration of all of the above factors allows qualitative predictions of the rate of hepatic drug metabolism in patients with liver disease, as assessed by the AP t1/2.