Serum intercellular adhesion molecule-1 in patients with nonalcoholic steatohepatitis: comparison with alcoholic hepatitis.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) includes both nonalcoholic fatty liver (FL) and nonalcoholic steatohepatitis (NASH). It has previously been reported that alcoholic hepatitis, which shows morphological findings similar to that of NASH, leads to the onset of endotoxinemia and to an increase in the production of tumor necrosis factor-alpha (TNF-alpha) and/or interleukin-1 (IL-1) from macrophages. Tumor necrosis factor-alpha and IL-1 induce strong expression of intercellular adhesion molecule-1 (ICAM-1) of the cell membranes of hepatocytes and/or sinusoidal endothelial cells, resulting in increased serum ICAM-1 levels in our previous study. In this study, we clarified the significance of serum ICAM-1 levels in patients with NAFLD, and especially in NASH. METHODS: Thirty-three obese patients of NAFLD (FL: n=14, NASH: n=19) with no habit of drinking, 20 cases of alcoholic liver diseases (alcoholic hepatitis; ASH: n=10, alcoholic hepatic fibrosis; HF: n=10), and 10 healthy individuals were studied. Serum ICAM-1 concentrations were analyzed by enzyme-linked immunoassay in patients with NAFLD and alcoholic liver diseases. Potential factors were assessed for increase in serum ICAM-1 and a diagnostic tool for NASH including ICAM-1 levels, C-reactive protein (CRP), white blood cell count, aspartate aminotransferase, alanine aminotransferase, gamma-gluatmyl transferase, total cholesterol, triglyceride, type-IV collagen, body mass index, homeostasis model assessment (HOMA-IR), and existence of high blood pressure. RESULTS: The serum ICAM-1 level was significantly higher in the patients with NASH than in the patients with FL, and in the normal subjects. The serum ICAM-1 level was also significantly higher in the patients with ASH. The serum ICAM-1 level in the patients with ASH was remarkably high compared with that of the patients with NASH. No significant difference in serum ICAM-1 levels was found between the patients with NASH and those with HF. The serum ICAM-1 level was significantly higher in patients with high blood pressure than in those without high blood pressure in NAFLD. A multivariate analysis using multiple logistic regression showed that high blood pressure and GGTP were the significant factors contributing to high serum ICAM-1 levels, while highly sensitive CRP and ICAM-1 were the significant factors for the diagnosis of NASH. CONCLUSIONS: The serum ICAM-1 concentration is increased in patients with NASH. The serum level of ICAM-1 in patients with NAFLD may be a useful marker for the diagnosis of NASH.

Effect of oral branched chain amino acid supplementation in the late evening on the nutritional state of patients with liver cirrhosis.

Previous studies have shown that nocturnal glucose supplementation and a late evening meal reduced raised protein turnover rates and led to a better nitrogen balance in patients with cirrhosis. In this study, we investigated whether or not oral branched chain amino acid (BCAA) supplementation in the late evening would improve the nutritional state of patients with liver cirrhosis. Fourteen patients with liver cirrhosis spent two 14 day periods in the ward. All the patients received three meals a day and two doses of BCAA supplementation. Meals were given at 0800, 1200, and 1830. BCAA supplementation was given at 0830 and 1900 (after dinner) and at 0830 and 2230 (late evening), respectively. The daily excretion of 3-methylhistidine, the ratio of 3-methyhistidine to creatinine, and serum free fatty acid in the late evening treatment group were significantly lower as compared to the usual treatment group. These results suggest that oral BCAA supplementation in the late evening also may be useful in improving protein catabolism and lypolysis in cirrhotic patients.

[Clinical significance of hepatic and splenic volumes measured by computed tomography in patients with alcoholic liver cirrhosis].

Hepatic and splenic volumes were measured by computed tomography in 43 patients with alcoholic liver cirrhosis (AL-LC), 10 patients with HBs antigen-positive liver cirrhosis (B-LC), 6 patients with HCV associated liver cirrhosis (C-LC) and 6 healthy subjects. Hepatic volume was significantly larger in the patients with AL-LC than in those with B-LC, those with C-LC or healthy subjects. Hepatic volume in patients with AL-LC was also significantly larger in the anti-HBc antibody (anti-HBc)-negative patients than in the anti-HBc-positive patients. This data suggests HBV occult infection may decrease hepatic volume. Hepatic volume showed significantly positive correlations with serum levels of total bilirubin (T. bil), gamma-GTP, type IV collagen levels, continuing alcohol intake and Child-Pugh score, and also showed significant negative correlation with cumulative alcohol intake and prothrombin time (PT). Splenic volume showed significantly positive correlations with serum levels of T. bil and Child-Pugh score, and also showed significantly negative correlations with serum albumin, PT, platelet count and BCAA (branched chain amino acids)/tyrosine ratio. Stepwise logistic regression analysis showed that enlarged hepatic volume, presence of hepatocellular carcinoma and elevated serum gamma-GTP were independently significant risk factors for the development of hepatic failure. Serial determination of hepatic and splenic volume may be useful for the estimation of liver function and prognosis in the patients with AL-LC.

[Effect of type of dietary fat and ethanol on hepatic leukotriene level in experimental alcoholic liver disease].

The purpose of our study is to determine if a relationship exists between the severity of injury in experimental alcoholic liver disease and hepatic levels of leukotriene B4, leukotriene C4 and lipid peroxide. Splague-Dawley rats were fed ethanol (46% of calories) with either safflower oil (SE) or beef oil (BE) (20% of calories) for 12 weeks. Control animals were fed isocaloric amounts of dextrose instead of ethanol with the same diets. The followings were evaluated in each group: hepatic levels of leukotriene B4, C4, lipid peroxide, and collagen-bound hydroxyl-proline, hepatic fatty acid composition, incorporation of 14C-L-proline into hydroxyproline of collagen protein by liver slice. Rats fed SE showed the most abundant accumulation of hepatic hydroxyproline and lipid peroxide. Hepatic leukotriene B4 and C4, hepatic levels of linoleic acid and arachidonic acid were also greater in rat livers from animals fed the SE diet. A strong positive correlation was seen between hepatic levels of leukotrien B4 as well as C4 and lipid peroxide. The hepatic level of lipid peroxide also correlated positively with hepatic levels of linoleic acid and arachidonic acid. Our study shows the importance of leukotriene derived from arachidonic acid cascade in the pathogenesis of experimental alcoholic liver disease.

[Gender difference of clinical features in Japanese patients with alcoholic liver cirrhosis].

Gender difference of alcohol intake and laboratory data was investigated in 165 Japanese patients with alcoholic liver cirrhosis. Mean age of first drinking and habitual drinking were higher in female. Duration of drinking was shorter in female. Although cumulative alcohol intake was larger in male, mean daily alcohol intake did not differ in both gender. Moreover, daily alcohol intake adjusted to body weight was significantly larger in female. Body mass index, serum levels of total protein, albumin and cholinesterase were significantly decreased in female. Platelet counts on admission did not differ in both gender. However, it was significantly increased in female after one month abstinence. C reactive protein, ammonia and serum levels of total bilirubin were significantly higher in female as compared to male. In conclusion, female alcoholics seems to progress to liver cirrhosis earlier because of high daily alcohol intake adjusted to body weight, poor nutritional condition and inflammation caused by endotoxin.

[A case of female alcoholic who developed liver cirrhosis with small amounts of alcohol by the common use of contraceptive agent].

A case of female alcoholic who developed liver cirrhosis with small amounts of alcohol by the common use of contraceptive agent was reported. A case was a 33-year-old female who had complained of systemic edema and jaundice. She had been drinking alcohol, while she had been taking the contraceptive agent from 20-year-old. On admission, she had a large amount of ascites with jaundice. She was diagnosed as liver cirrhosis and hepatic failure by CT scanning of abdomen and laboratory data. Her condition was temporarily improved by the abstinence and the treatment. Since she drank under hospitalization, she had to change the hospital and died after 2 months. She had been drinking for only 10 years. Her cumulative alcohol intake was also very small. She may have developed alcoholic cirrhosis with small amount of alcohol because of common use of contraceptive agent with drinking.

[Two cases of alcoholics associated with rhabdomyolysis and acute renal failure].

Two cases of alcoholics associated with rhabdomyolysis and acute renal failure were reported. Case 1 was a 67-year-old male who had complained of general fatigue and generalized muscle pain. He had drunken and slept outdoor in winter until he was found. Laboratory data on admission showed remarkable elevation of muscle enzymes (AST, LDH, CPK) and serum levels of myoglobin, BUN, and Cr. He was treated with hemodialysis because of acute renal failure caused by rhabdomyolysis and recovered from renal failure. Case 2 was a 50-year-old male who had been unconscious and suffered from muscle weakness. He had drunken and slept in the bed for several days without eating any food until he was found by his sister. Laboratory data on admission showed remarkable elevation of muscle enzymes and serum levels of myoglobin, BUN, and Cr. It also showed hypoglycemia and hyponatremia. He developed into acute renal failure caused by rhabdomyolysis, but had a good clinical course without hemodialysis. The rhabdomyolysis of case 1 might have been caused by alcohol and sleeping outdoor in winter. That of case 2 might have been caused by alcohol and pressure necrosis due to immobility for several days in his bed.

Autoantibody response to microsomal epoxide hydrolase in hepatitis C and A.

Autoimmune responses were observed in a large proportion of hepatitis C cases and are suspected to be part of viral pathogenesis. The AN6520 antigen (AN-Ag) is a normal cellular protein mainly expressed in liver that was found associated with non-A, non-B hepatitis. To elucidate its pathogenic role in hepatitis C, we developed an IgM capture assay using purified AN-Ag and confirmed that the antibody response to AN-Ag is associated almost exclusively with hepatitis C cases (29%). Screening of a human liver expression library revealed that AN-Ag is mainly the microsomal epoxide hydrolase (mEH), a drug-metabolizing enzyme that plays an important role in the metabolism of some mutagenic and carcinogenic epoxides. Using the purified recombinant human mEH as an antigen, we now found that antibodies against this protein are associated with nearly 82% of hepatitis C virus infections and surprisingly with 46% of patients with hepatitis A. The appearance of AN-Ag/mEH in the incubation period of hepatitis C as previously reported and the antibody responses shown here indicate that this enzyme may be a marker for or even a cause of some of the pathology associated with hepatitis C and A.

Serum ubiquitin-protein conjugates in normal subjects and patients with alcoholic liver diseases: immunoaffinity isolation and electrophoretic mobility.

BACKGROUND: We previously reported that levels of multiubiquitin chains, representing ubiquitin-protein conjugates, were significantly higher in sera of patients with alcoholic liver cirrhosis than in normal subjects and patients with other types of alcoholic liver disease. METHODS: To characterize them, ubiquitin-immunoreactive proteins were purified from sera of healthy human volunteers and patients with alcoholic liver diseases by using affinity chromatography on a Sepharose column containing an immobilized monoclonal antibody recognizing conjugated ubiquitin. RESULTS: SDS-PAGE analysis followed by Western blotting revealed that the immunoaffinity-purified proteins mainly contained multiple components with molecular masses greater than 60 kDa, almost all of which were immunostained with the ubiquitin antibody. This size heterogeneity was in agreement with the property of ubiquitin-protein conjugates in all cells examined. These results indicate that the immunoaffinity-purified serum proteins are polyubiquitinated proteins presumably derived from some somatic cells. These ubiquitinated proteins obtained from the alcoholic cirrhosis patients were stained more strongly than those from the normal subjects and patients with other types of alcoholic liver disease, although equal amounts of multiubiquitin chains were analyzed simultaneously. In addition, marked differences were observed in the two-dimensional PAGE pattern of the ubiquitin-protein conjugates purified from the alcoholic cirrhosis patient serum compared with those from the normal human serum: four distinct broad spots (presumably polyubiquitin-protein complexes) were observed only in the former. CONCLUSIONS: This is the first report on isolation of ubiquitin-protein conjugates from human serum, and it indicates that not only their levels, but also their molecular compositions, were greatly affected by alcoholic cirrhosis.

Acetaldehyde-induced growth retardation and micro-heterogeneity of the sugar chain in transferrin synthesized by HepG2 cells.

BACKGROUND: A carbohydrate-deficient transferrin (CDT) is the most useful marker of alcohol abuse; however, the mechanism of production and the pathophysiologic roles of CDT remain obscure. The effects of alcohol and its metabolites on growth and proliferation, transferrin synthesis, and phosphomannomutase enzyme activity in a human hepatoblastoma, HepG2, were examined. METHODS: HepG2 cells were treated with either ethanol at 80 mM or acetaldehyde at 400 microM. Transferrin secreted by the cells was prepared from conditioned culture medium by single-step immunoaffinity column chromatography using a goat-specific antibody against human transferrin. Phosphomannomutase and some related enzyme activities in the cell extracts were determined. Reverse transcription-polymerase chain reaction analysis of phosphomannomutase mRNA expression was also determined in HepG2 cultured with or without acetaldehyde (400 microM). RESULTS: HepG2 cells usually synthesized and secreted transferrin with three separated bands: main broad bands estimated to be 78 to 82 kDa, 75 kDa, and 72 kDa. The last two bands were compatible with part or the entire N-glycans-deficient transferrin (CDT) from alcoholic liver damage. Increased secretion of CDT from HepG2 correlated well with the extent of growth retardation to the level of confluent cell density. The activity of phosphomannomutase also decreased with prolongation of cellular doubling time. Furthermore, acetaldehyde treatment at 400 microM accelerated the inhibitory effect of cell growth compared with nontreated cells, and this condition facilitated CDT secretion from HepG2 cells. Determination of the enzyme activity and mRNA expression indicated that acetaldehyde showed competitive type inhibition of phosphomannomutase activity but not suppression of phosphomannomutase gene expression. CONCLUSIONS: By culturing HepG2 cells with acetaldehyde containing media, growth inhibition-dependent increase of CDT showed good correlation with reduced enzyme activity of phosphomannomutase. Acetaldehyde facilitated growth retardation, inhibition of phosphomannomutase activity, and increased secretion of CDT. The HepG2 cell line is useful as an in vitro model to investigate the pathophysiologic state of alcoholic liver damage and mechanisms of production as well as the physiologic role of CDT.

Experimental studies on the relationship between immune responses and liver damage induced by ethanol after immunization with homologous acetaldehyde adducts.

BACKGROUND: It has been considered that acetaldehyde (AcH) adducts induce liver injury through an immune response. Previous experimental studies showed that hepatic necrosis, inflammatory cell infiltration, and hepatic fibrosis were induced in guinea pigs immunized with heterologous human AcH adducts and ethanol feeding. AcH modification of foreign protein may markedly increase immunogenicity of the protein itself, leading to enhanced formation of immune complex and possible liver injury. The present study investigated whether immune responses and alcoholic liver disease would be induced in mice by immunization with mouse albumin-AcH adducts and ethanol feeding. METHODS: 6B6 mice were divided into six groups with or without immunization and ethanol feeding. Mice were immunized with mouse albumin-AcH adducts three times at 2-week intervals and fed ethanol for 10 weeks. The stimulation index of [(3)H]thymidine uptake into lymphocytes cultured with mouse albumin or mouse albumin-AcH adducts was measured. Histologic findings of the liver were examined, and the plasma levels of aspartate transaminase and alanine aminotransferase were also measured. RESULTS: The stimulation index was increased remarkably in ethanol-fed mice that were immunized with mouse albumin-AcH adducts. However, neither inflammatory cell infiltration nor hepatic necrosis was observed in the liver. There were also no differences in the plasma activities of aspartate transaminase and alanine aminotransferase between the group of mice regardless of ethanol feeding or immunization. CONCLUSION: Although marked immune responses were observed, no liver damage was induced by long-term ethanol feeding in our mouse model using AcH-homologous albumin adducts. These results suggest that homologous protein adducts may not induce liver injury by long-term ethanol feeding or may have lower immunogenicity than heterologous protein adducts. These results also suggest that nonreduced AcH adducts and/or a larger amount of ethanol may be needed for liver injury in this model.

Analysis of risk factors for hepatocellular carcinoma in patients with HBs antigen- and anti-HCV antibody-negative alcoholic cirrhosis: clinical significance of prior hepatitis B virus infection.

BACKGROUND: The hepatitis B virus (HBV) or hepatitis C virus (HCV) markers frequently are detected in alcoholic patients with hepatocellular carcinoma (HCC). However, risk factors for the development of HCC in patients with HBs antigen (Ag)- and anti-HCV antibody (anti-HCV)-negative alcoholic cirrhosis have not been clearly documented. The present study was conducted to elucidate the occurrence rates of HCC in HBs Ag- and anti-HCV-negative male alcoholic cirrhosis and to assess the risk factors for hepatocellular carcinogenesis. METHOD: We prospectively studied 91 consecutive patients with HBs Ag- and anti-HCV-negative alcoholic cirrhosis for 0.5 to 12.5 years (median 5.9 years). Potential risk factors assessed for liver carcinogenesis included the following six variables: age, total alcohol intake, association of continuing alcohol intake after diagnosis, indocyanine green retention rate at 15 min, anti-HB core antibodies (anti-HBc), and association of diabetes mellitus. RESULTS: Cumulative occurrence rates of HCC were 6.4%, 18.9%, and 28.7% at the end of the 5th, 7th and 10th years, respectively. When classified by anti-HBc, the occurrence rates of HCC in 31 patients with anti-HBc and 60 patients without anti-HBc were 15.6% and 2.9% at the 5th year, 28.4% and 13.5% at the 7th year, and 40.4% and 22.1% at the 10th year, respectively. The occurrence rates of HCC were also significantly related to the cumulative alcohol intake. Cox proportional hazard model identified that cumulative alcohol intake (p = 0.0047) and positive anti-HBc antibodies (p = 0.0598) were independently associated with the occurrence rates of HCC. CONCLUSION: These epidemiologic results suggest that heavy cumulative alcohol intake and prior exposure to HBV infection are risk factors for the development of HCC in patients with HBs Ag- and anti-HCV-negative alcoholic cirrhosis.

Pioglitazone prevents acute liver injury induced by ethanol and lipopolysaccharide through the suppression of tumor necrosis factor-alpha.

BACKGROUND: Peroxisome proliferator-activated receptor-gamma (PPAR-gamma), which is a ligand-dependent transcriptional factor, forms a heterodimer with retinoid X receptor (RXR) and controls many genes that are relevant to the regulation of lipid metabolism and insulin sensitization. Recent studies have shown that stimulation of PPAR-gamma inhibits the production of inflammatory cytokines in monocytes and macrophages. Alcohol and lipopolysaccharide (LPS) have already been shown to induce liver injury through the activation of many inflammatory cytokines. Thus, the activation of PPAR-gamma by its ligand may represent a potential effect causing liver injury. In this study, we investigated the effects of pioglitazone, a ligand for PPAR-gamma, on acute liver injury induced by ethanol and LPS. METHODS: Female Sprague-Dawley rats that weighed 300 g were given ethanol (5 g/kg body weight) intragastrically and received an intraperitoneal injection of LPS 24 hr later. Subsequently, pioglitazone (1 mg/kg body weight) or vehicle alone was injected intraperitoneally 10 min and 24 hr after ethanol administration. Plasma levels of aspartate transaminase and alanine aminotransferase were measured by spectrophotometer. Plasma levels of tumor necrosis factor-alpha (TNF-alpha) were also determined using an enzyme-linked immunosorbent assay. Plasma and hepatic levels of lipid peroxide were measured, and the histologic findings of the liver were examined. Reverse transcription-polymerase reaction analysis of TNF-alpha, PPAR-gamma, RXR-alpha, and beta-actin mRNA was performed. Western blot analysis using the p65 subunit of NF-kappaB was also performed. RESULTS: Pioglitazone prevented increase in plasma aspartate transaminase, alanine aminotransferase, and TNF-alpha levels but had no effect on plasma and hepatic levels of lipid peroxide. Pioglitazone also prevented hepatic inflammation and necrosis induced by ethanol and LPS. Ethanol and LPS induction of TNF-alpha mRNA in the liver was blunted by pioglitazone; however, RXR-alpha mRNA was not affected. PPAR-gamma mRNA levels were suppressed by ethanol and LPS but recaptured by pioglitazone. Western blot analysis showed that pioglitazone did not inhibit translocation of NF-kappaB to nuclei. CONCLUSION: These results suggest that pioglitazone may prevent liver injury induced by ethanol and LPS through the suppression of TNF-alpha.