Serum Ubiquitin Levels in Patients With Alcoholic Liver Disease

Serum concentrations of free ubiquitin and multiubiquitin chain as determined by immunoassays were compared between 10 healthy subjects, and 11 patients with alcoholic hepatic fibrosis, 10 with alcoholic cirrhosis, and 6 with viral liver cirrhosis. All measurements of multiubiquitin chains were expressed in terms of a standard multiubiquitin chain reference preparation 1. Serum concentrations (mean ± SD) of free ubiquitin and multiubiquitin chains were significantly higher in patients with alcoholic cirrhosis (63.5 ± 33.7 ng/ml and 7.5 ± 4.6 ng/ml) than in the normal subjects (29.6 ± 6.6 ng/ml, p < 0.05 and 4.1 ±1.7 ng/ml, p < 0.05), and those with alcoholic hepatic fibrosis (34.8 ± 16.3 ng/ml, p < 0.05 and 3.0 ± 0.7 ng/ml, p < 0.05) and viral liver cirrhosis (28.8 ± 7.5 ng/ml, p < 0.05 and 4.2 ± 1.3 ng/ml, p < 0.05). Serum levels of both forms of ubiquitin in six patients with alcoholic cirrhosis showed a tendency to decline after 3 months of abstinence. In a total of 14 patients with alcoholic liver damage, 11 with brain atrophy had significantly higher serum levels of both ubiquitin forms than did three patients without brain atrophy ( p < 0.05). No correlation was seen between serum concentrations of either form of ubiquitin and liver function test results in the patients with alcoholic liver damage. However, serum levels of both forms of ubiquitin levels correlated significantly with cumulative alcohol intake ( p < 0.05). A significant correlation ( p < 0.05) also was observed between serum levels of multiubiquitin chains and mean corpuscular volume, a marker of alcohol consumption. These results suggest that the serum concentrations of ubiquitin, especially multiubiquitin chain is a good marker for the diagnosis of alcoholic cirrhosis.     

Alcoholic Liver Disease: The Gut Microbiome and Liver Cross Talk

Alcoholic liver disease (ALD) is a leading cause of morbidity and mortality worldwide. Alcoholic fatty liver disease can progress to steatohepatitis, alcoholic hepatitis, fibrosis, and cirrhosis. Patients with alcohol abuse show quantitative and qualitative changes in the composition of the intestinal microbiome. Furthermore, patients with ALD have increased intestinal permeability and elevated systemic levels of gut‐derived microbial products. Maintaining eubiosis, stabilizing the mucosal gut barrier, or preventing cellular responses to microbial products protect from experimental ALD. Therefore, intestinal dysbiosis and pathological bacterial translocation appear fundamental for the pathogenesis of ALD. This review highlights causes for intestinal dysbiosis and pathological bacterial translocation, their relationship, and consequences for ALD. We also discuss how the liver affects the intestinal microbiota.     

Histochemical Study of Hyaluronate in Alcoholic Liver Disease

Recently, it has been reported that serum hyaluronate (hyaluronic acid; HA) concentrations increase in various liver diseases, especially in alcoholic liver disease (ALD), and serum HA concentration has been used as a marker for hepatic fibrosis. However, it is unknown whether hepatic HA contents in ALD increase by alcohol or not. In this study, we histochemically stained HA in liver biopsy specimens obtained from ALD patients while actively drinking and after abstinence to clarify the effects of alcohol on hepatic HA contents. Liver biopsy specimens were obtained from 13 patients with ALD and 10 patients with non-ALD. In ALD patients, liver biopsy was performed twice within 3 days, and 4 to 8 weeks after abstinence when serum levels of AST and ALT normalized. HA in biopsy specimens was stained histochemically with biotinylated HA binding protein. Staining intensity of HA in liver tissue was also determined by computer-assisted imaging analyzer. HA staining was clearly observed in sinusoidal wall and fibrous regions around the portal tract and central vein in liver diseases. HA staining intensities in patients actively drinking with ALD increased markedly, compared with those in patients with non-ALD, and these intensities decreased with abstinence. These results clearly suggest that hepatic HA contents in ALD may be increased by alcohol in addition to hepatic fibrosis, and, therefore, increased HA deposition in the liver may be reversible by abstinence of alcohol.     

Microheterogeneity with Concanavalin A Affinity of Serum Transferrin in Patients with Alcoholic Liver Disease

Microheterogeneity of transferrin (Tf) with concanavalin A (ConA) affinity was investigated by sensitive lectin-affinity electrophoresis and antibody-affinity blotting technique of sera obtained from patients with alcoholic liver disease (ALD) and normal subjects. Serum Tf was separated by ConA into three bands–a strongly ConA-reac-tive major band (C1), a weakly reactive minor band (C2), and a non-reactive trace band (C3). The C3 fraction was significantly increased in patients with ALD before alcohol abstinence, compared with normal subjects and patients with ALD after 4 weeks of abstinence. Furthermore, a significant correlation was found between the C3 fraction and serum carbohydrate-deficient transferrin or γ-glutamyl-transpeptidase. These results indicate that the microheterogeneity of serum Tf in patients with ALD may be a more complex abnormality of elongation and processing on the glycans than merely a loss of terminal sialic acids. Determination of the C3 fraction is a useful marker for ALD.     

Circulating Neutrophils and Liver Injury in Rat Models of Experimental Alcoholic Liver Disease

The present study examined the relationship between circulating neutrophils and liver injury in two widely used rat models of chronic ethanol administration. Hematological alterations, liver histopathology, and biochemical indices of liver injury were assessed in rats receiving chronic ethanol by oral liquid diet feeding (Lieber-DeCarli method) or by continuous intragastric infusion (Tsukamoto-French method). Oral administration of ethanol did not affect circulating neutrophil counts, but resulted in minimal liver injury characterized by elevated serum alanine aminotransferase (79%), increased liver mass (15%), and moderate steatosis. In contrast, rats receiving ethanol by continuous intragastric infusion showed an ∼ 2-fold increase in circulating neutrophils, and a moderate degree of liver injury, indicated by a 169% elevation of serum alanine aminotransferase and a 2-fold increase in liver mass. Liver biopsies from these rats showed severe steatosis and scattered necrotic hepatocytes, and some neutrophil infiltrates. To determine whether an increase in the number of circulating neutrophils could potentiate liver injury induced by oral ethanol feeding, rats were treated with human recombinant granulocyte colony-stimulating factor at a dose of 100 μg/kg/day (sc) for 4 days. Treatment with granulocyte colony-stimulating factor resulted in a 6- to 9-fold increase in circulating neutrophil counts. Nevertheless, this change did not enhance the minor degree of ethanol-induced liver injury in this model. Our results indicate that, whereas neutrophil leukocytosis accompanies more severe manifestations of ethanol hepatotoxicity in rats, this condition per se does not directly induce or exacerbate ethanol-induced liver injury.     

Characteristics of Serum Hyaluronate Concentrations in Patients with Alcoholic Liver Disease

It has been reported that serum hyaluronate [hyaluronic acid (HA)] concentrations are increased in liver diseases, especially in alcoholic liver disease (ALD). However, the characteristics of serum HA concentration in patients with ALD have not been studied. In this study, first, we measured serum HA concentrations in patients with different stages of both ALD and non-ALD to clarify the characteristics of serum HA concentration in patients with ALD. Second, we measured serum HA concentrations in patients with ALD sequentially after abstinence. We also measured serum HA concentrations in patients with chronic type C hepatitis before and after treatment with interferon. Finally, we analyzed the relationship between serum HA concentrations and the contents of type IV collagen and laminin in the livers of both ALD and non-ALD patients. Serum HA concentrations in liver disease were higher than the cut-off value, and increased significantly ( p < 0.001) in parallel with the progression of hepatic fibrosis in both ALD and non-ALD patients. Serum HA concentrations in patients actively drinking with ALD were significantly higher ( p < 0.001) than those in non-ALD. After 4 weeks of abstinence, these concentrations fell to the levels of non-ALD. Although serum ALT levels were decreased in 80% of patients treated with interferon, serum HA concentrations were not changed or increased. A significant correlation between serum HA concentrations and hepatic type IV collagen and laminin content was present in ALD, but not in non-ALD. These results clearly suggest that the increase of serum HA concentrations in ALD may be associated with not only hepatic fibrosis, but also alcohol drinking.     

Alcoholic Liver Disease and Apoptosis

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Carol A. Casey and Amin Nanji. The presentations were (1) Mechanisms of apoptosis in alcoholic liver disease, by Amin A. Nanji; (2) Impaired receptor-mediated endocytosis: Its role in alcoholic apoptosis, by Carol A. Casey; (3) Toxicity of ethanol in HepG2 cells that express CYP2E1, by Arthur I. Cederbaum; (4) Mitochondrial regulation of ethanol-induced hepatocyte apoptosis, by M. Adachi; and (5) Apoptosis in alcoholic hepatitis, by T. Takahashi.     

Comparison of Hepatocellular Carcinoma Patients With Alcoholic Liver Disease and Nonalcoholic Steatohepatitis

Background: Alcoholic hepatitis and nonalcoholic steatohepatitis (NASH) show different clinical features with similar liver histology, but both disorders may progress to cirrhosis and hepatocellular carcinoma (HCC). HCC arising in alcoholic liver disease (ALD) or NASH, without hepatitis B or C virus infection, has been a rare observation, and there are no studies comparing the characteristics of ALD and NASH patients with HCC. Therefore, we compared the characteristics of ALD and NASH patients with HCC.
Methods: A total of 1202 patients received a diagnosis of HCC at Tokyo Women's Medical University from 1989 to 2003, and their clinical data were collected prospectively. A clinical diagnosis was made to diagnose ALD, and clinical and histological changes were required to diagnose NASH. Of these patients, 88 received a diagnosis of HCC arising from ALD. Among them, a biopsy specimen was obtained in 50 patients (ALD-HCC group). We compared the clinical and histological characteristics of 50 ALD and 8 NASH patients (NASH-HCC group) associated with HCC. They all were negative for hepatitis virus infection by serological methods.
Results: The most significant difference between these groups was sex. Women were significantly more common in the NASH-HCC group (6% vs. 63%; p < 0.0001). The median age was 65 years in the ALD-HCC group and 68 years in the NASH-HCC group. The risk factors for NASH all were high in the NASH-HCC group. However, liver function tests were similar in these groups. In the ALD-HCC group, 46 (92%) patients showed severe fibrosis; 2 had septal fibrosis and 44 had cirrhosis. All patients in the NASH-HCC group showed severe fibrosis, and seven had cirrhosis.
Conclusions: Severe fibrosis might be an important risk factor for HCC. Patients who have ALD or NASH with cirrhosis may develop HCC. This seems to occur in a sufficient number of cases to warrant regular screening for this complication.     

Contribution of Hepatitis C Virus to the Progression of Alcoholic Liver Disease

Background: We determined hepatitis C virus (HCV) antibody, HCV RNA, and genotype in patients with alcoholic liver disease and studied the involvement of HCV in alcoholic liver disease. Additionally, we used the histological activity index (HAI) to study the influence of HCV on the severity of inflammation. Methods: The subjects were 143 patients with alcoholic liver disease: 7 with fatty liver (FL), 18 with hepatic fibrosis (HF), 24 with alcoholic hepatitis (ALH), 39 with chronic hepatitis (CH), 42 with liver cirrhosis (LC), and 13 with hepatocellular carcinoma (HCC). The HCV RNA positivity rate in each type of disease was 0/7 (0%), 1/18 (6%), 2/24 (8%), 27/39 (69%), 24/42 (57%), and 7/13 (54%), respectively. It was high in the advanced hepatic lesions. Results: Clinically, the serum hepatic function tests after abstinence from drinking improved significantly in the HCV RNA negative patients compared with the positive patients. The proportion of genotype II in each type of disease was 0/0, 0/1 (0%), 1/2 (50%), 18/27 (67%), 18/24 (75%), and 7/7 (100%), respectively. It became high with the advance of pathophysiology. The HCV RNA amount stood at 7.5 ± 0.4 [log (copies/ml)] in CH, 7.9 ± 0.4 in LC, and 8.4 ± 0.8 in HCC, with a statistically significant difference between CH and HCC. However, we found no changes in the HCV RNA amount due to abstinence from drinking. The HAI score was high in the HCV RNA positive patients, but several cases in the HCV RNA negative group showed severe inflammatory changes. Therefore, judging the presence or absence of HCV RNA with the HAI score alone was considered difficult. Conclusions:: These results suggest that HCV, particularly genotype II, plays an important role in the advance of disease to LC and HCC in heavy drinkers.     

Models of Alcoholic Liver Disease in Rodents: A Critical Evaluation

This article represents the proceedings of a workshop at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were J. Christian Bode and Hiroshi Fukui. The presentations were (1) Essentials and the course of the pathological spectrum of alcoholic liver disease in humans, by P. de la M. Hall; (2) Lieber-DeCarli liquid diet for alcohol-induced liver injury in rats, by C. S. Lieber and L. M. DeCarli; (3) Tsukamoto-French model of alcoholic liver injury, by S. W. French; (4) Animal models to study endotoxin-ethanol interactions, by K. O. Lindros and H. Järveläinen; and (5) Jejunoileal bypass operation in rats—A model for alcohol-induced liver injury? by Christiane Bode, Alexandr Parlesak, and J. Christian Bode.     

Clinical Significance of Hepatitis GB Virus C Infection in Alcoholic Liver Disease

Recently, hepatitis GB virus C (HGBV-C) has been recovered from patients with non-A-E hepatitis. However, it has been unclear whether HGBV-C may be related to the development of alcoholic liver disease (ALD) or not. In this study, we determined HGBV-C RNA in sera from alcoholic patients without markers for hepatitis C and B viruses to evaluate the role of HGBV-C in ALD. Serum samples were obtained from 68 patients with ALD and 40 nonalcoholic patients with chronic type C liver disease. HGBV-C RNA was detected in only 3 of 68 (4.4%) patients with ALD, in 2 of 27 patients with hepatic fibrosis, and in 1 of 5 patients with chronic hepatitis. There was no HGBV-C RNA in sera from patients with fatty liver, alcoholic hepatitis, or cirrhosis. Serum levels of AST, ALT, and γ-glutamyltranspeptidase in alcoholic patients with, as well as without, HGBV-C RNA decreased to normal levels after abstinence. In addition, an inflammatory change was not observed in liver biopsy specimens obtained from two HGBV-C-positive patients with alcoholic hepatic fibrosis. Our results clearly suggest that the prevalence of HGBV-C infection in patients with ALD is rare and that HGBV-C may not play an important role in the development of liver disease in alcoholics.     

Sarcopenia in Alcoholic Liver Disease: Clinical and Molecular Advances

Despite advances in treatment of alcohol use disorders that focus on increasing abstinence and reducing recidivism, alcoholic liver disease (ALD) is projected to be the major cause of cirrhosis and its complications. Malnutrition is recognized as the most frequent complication in ALD, and despite the high clinical significance, there are no effective therapies to reverse malnutrition in ALD. Malnutrition is a relatively imprecise term, and sarcopenia or skeletal muscle loss, the major component of malnutrition, is primarily responsible for the adverse clinical consequences in patients with liver disease. It is, therefore, critical to define the specific abnormality (sarcopenia) rather than malnutrition in ALD, so that therapies targeting sarcopenia can be developed. Skeletal muscle mass is maintained by a balance between protein synthesis and proteolysis. Both direct effects of ethanol (EtOH) and its metabolites on the skeletal muscle and the consequences of liver disease result in disturbed proteostasis (protein homeostasis) and consequent sarcopenia. Once cirrhosis develops in patients with ALD, abstinence is unlikely to be effective in completely reversing sarcopenia, as other contributors including hyperammonemia, hormonal, and cytokine abnormalities aggravate sarcopenia and maintain a state of anabolic resistance initiated by EtOH. Cirrhosis is also a state of accelerated starvation, with increased gluconeogenesis that requires amino acid diversion from signaling and substrate functions. Novel therapeutic options are being recognized that are likely to supplant the current “deficiency replacement” approach and instead focus on specific molecular perturbations, given the increasing availability of small molecules that can target specific signaling components. Myostatin antagonists, leucine supplementation, and mitochondrial protective agents are currently in various stages of evaluation in preclinical studies to prevent and reverse sarcopenia, in cirrhosis in general, and ALD, specifically. Translation of these data to human studies and clinical application requires priority for allocation of resources.     

Immunohistochemical Study of Hyaluronate Receptor (CD44) in Alcoholic Liver Disease

Background: It has been suggested that the elevation of serum hyaluronate (HA) levels in liver diseases may be due to increased synthesis of HA by hepatic stellate cells or decreased degradation by sinusoidal endothelial cells. The increase in serum HA levels in patients with cirrhosis is thought to be a response to a reduction in HA receptors (CD44) in hepatic sinusoidal endothelial cells. To learn more about how alcohol affects the number and distribution of HA receptors of hepatic sinusoidal endothelial cells, we immunohistochemically studied CD44 levels in liver biopsy obtained from patients with alcoholic liver disease (ALD patients) and also from patients with nonalcoholic liver disease (non-ALD); ALD patients were evaluated when they were currently drinking and again after they became abstinent. Normal liver tissue obtained from three autopsy cases served as a control. Methods: Liver biopsy specimens were obtained from 18 ALD patients and 12 non-ALD patients. In ALD patients, liver biopsy was performed twice within 3 days and 4 to 8 weeks after abstinence when serum levels of aspartate aminotransferase and alanine aminotransferase became normal. CD44 in liver specimens was stained with anti-CD44 antibody by streptavidine-biotin-peroxidase complex. The intensity of the staining of CD44 in liver tissue was determined by a computer-assisted imaging analyzer. We also measured serum levels of CD44 in both ALD and non-ALD patients. Results: The intensity and the number of CD44 staining increased in both ALD and non-ALD patients compared with those in normal liver, which was negative. The staining intensity of CD44 in liver specimens obtained from patients with ALD who were active in alcohol consumption were significantly higher when compared with patients with ALD after abstinence. Serum levels of CD44 in patients with liver disease increased compared with those of healthy subjects. Conclusions: The results suggest that HA receptors may increase to degrade the increased HA in serum and/or liver.     

Hepatic Veno-Occlusive Lesions in Severe Alcoholic Hepatitis and Alcoholic Liver Cirrhosis: A Comparative Histopathological Study in Autopsy Cases

Clinicopathological features of veno-occlusive lesions in hepatic veins were studied in autopsy cases of severe alcoholic hepatitis (15 cases) and alcoholic liver cirrhosis (15 cases). All the cases were heavy drinkers and died of liver failure or variceal rupture. The frequency and degree of veno-occlusive lesions, and the diameter and number of hepatic veins were studied from stained sections of liver blocks from each case. The hepatic veins observed ranged from 60 to 3000 μm in diameter. The veno-occlusive lesions were found in hepatic veins mainly 60 to 1200 μm in diameter. These findings were recognized in the majority of severe alcoholic hepatitis cases and alcoholic liver cirrhosis cases. Furthermore, more severe veno-occlusive lesions were noted in severe alcoholic hepatitis, compared with alcoholic liver cirrhosis. In the cases with obstruction in hepatic veins of <400 μm, a decrease in the number of hepatic veins and zonal necrosis were noted. In addition, some of the veno-occlusive lesions were recognized focally in the same cases. Clinical findings also indicated that ascites increased with the progression of the veno-occlusive lesions. We conclude that investigation of veno-occlusive lesions in severe alcoholic liver disease has clinicopathological significance.     

Diagnosis and Management of Alcoholic Liver Disease

Alcohol is a leading cause of liver disease and is associated with significant morbidity and mortality. Several factors, including the amount and duration of alcohol consumption, affect the development and progression of alcoholic liver disease (ALD). ALD represents a spectrum of liver pathology ranging from fatty change to fibrosis to cirrhosis. Early diagnosis of ALD is important to encourage alcohol abstinence, minimize the progression of liver fibrosis, and manage cirrhosis-related complications including hepatocellular carcinoma. A number of questionnaires and laboratory tests are available to screen for alcohol intake. Liver biopsy remains the gold-standard diagnostic tool for ALD, but noninvasive accurate alternatives, including a number of biochemical tests as well as liver stiffness measurement, are increasingly being utilized in the evaluation of patients with suspected ALD. The management of ALD depends largely on complete abstinence from alcohol. Supportive care should focus on treating alcohol withdrawal and providing enteral nutrition while managing the complications of liver failure. Alcoholic hepatitis (AH) is a devastating acute form of ALD that requires early recognition and specialized tertiary medical care. Assessment of AH severity using defined scoring systems is important to allocate resources and initiate appropriate therapy. Corticosteroids or pentoxifylline are commonly used in treating AH but provide a limited survival benefit. Liver transplantation represents the ultimate therapy for patients with alcoholic cirrhosis, with most transplant centers mandating a 6 month period of abstinence from alcohol before listing. Early liver transplantation is also emerging as a therapeutic measure in specifically selected patients with severe AH. A number of novel targeted therapies for ALD are currently being evaluated in clinical trials.     

Serum Type I Collagen and N-terminal Peptide of Type III Procollagen in Patients with Alcoholic Liver Disease: Relationship to Liver Histology

This study compared, in patients with alcoholic liver disease, the serum concentration of N-terminal peptide of type III procollagen and of a novel serum marker, type I collagen, with liver histological data and assessed the role of these markers in the diagnosis and follow-up of liver changes. Ninety-six patients (mean age 51 years, 61 men and 35 women) were included. All had alcoholic liver disease diagnosed on usual clinical, biochemical, and histological criteria. Two histological scores, one for alcoholic hepatitis and one for fibrosis, were established. Serum N-terminal peptide of type III procollagen and type I collagen were assayed by liquid phase radioimmunoassay. Significant correlations between serum type I collagen and score of fibrosis (r = 0.34, p less than 0.001) and between serum N-terminal peptide of type III procollagen and score of alcoholic hepatitis (r = 0.60, p less than 0.0001) were noted. There was no significant correlation between serum aminotransferases and the score of alcoholic hepatitis. In 25 patients with alcoholic hepatitis reassessed between 3 and 6 months, serum N-terminal peptide of type III procollagen significantly decreased (p less than 0.05) as did the score of alcoholic hepatitis, but serum type I collagen and the score of fibrosis were not modified. These serum markers of collagen metabolism could be useful for the assessment and follow-up in patients with alcoholic liver disease.     

Human Hepatocyte Growth Factor in Alcoholic Liver Disease: A Comparison with Change in α-Fetoprotein

To evaluate the hepatic regenerative response in patients with alcoholic liver disease, sera from 263 patients with severe alcoholic hepatitis and/or cirrhosis were analyzed for hepatocyte growth factor (HGF) and α-fetoprotein (AFP). HGF concentration was elevated above healthy controls in 95% of the patients (median level = 2.4 ng/ml), whereas AFP tended to be depressed below controls (median level = 4.1 ng/ml). Correlations with parameters of liver injury (i.e., ascites, encephalopathy, AST bilirubin, and protime) all showed a more significant correlation with HGF concentrations than those of AFP. Patients with HGF levels below the mean (4 ng/ml) exhibited significantly better survival (median survival = 35 months vs. 8.5 months for those with HGF ≥4 ng/ml; p = 0.007). Serum HGF levels were associated with various specific histologic features of alcoholic hepatitis that included, but were not exclusively related to, necrosis.