Shujiro Takase

Clinical backgrounds of the patients having different types of hepatitis C virus genomes

Hepatitis C virus (HCV) genomes were recently detected in biological materials, and variations of nucleotide sequences were reported. In the present study, typing of the HCV genomes was performed in 91 HCV-RNA-positive patients and the clinical features of patients with different types of HCV were compared. From the nucleotide sequences of the cDNA fragments, HCV can be divided into at least two types: HCV-K1-PT and HCV-K2. All cDNAs amplified from 91 patients were hybridized with cDNA probes of either HCV-K1-PT or HCV-K2. HCV-K1-PT was found in about 80% of the patients, and HCV-K2 was found in about 20% of the patients. These results indicate that types of HCV are limited to two types, i.e., K1-PT and K2, and the major type is HCV-K1-PT, at least in Japan. Detection rate of antibodies to C-100-3 protein were not different between the patients having HCV-K1-PT and HCV-K2, indicating that the antibodies may develop in HCV-related patients without relation to the types of the HCV genomes. Prevalence of the two types of HCV were nearly the same in various forms of NANB-related liver disease. However, the prevalence was somewhat different in alcoholic liver disease. HCV-K2 was found in patients younger than the patients with HCV-K1-PT. Frequency of a history of blood transfusion tended to be lower and the initial response to interferon treatment was clearly better in patients having HCV-K2 versus patients having HCV-K1-PT. These results suggest the possibility that clinical features due to HCV-K1 may be somewhat different from those due to HCV-K1-PT. However, the number of patients examined was too small to allow a definite conclusion, indicating a necessity for further study with a larger number of patients.

Differences in the hepatitis C virus genotypes in different countries

We recently classified the hepatitis C virus (HCV) into 4 types (HCV-PT, -K1, -K2a and -K2b) according to differences in nucleotide sequences. It was found that HCV-PT, the prototype reported from the U.S.A., was rare in Japan, suggesting that distribution of HCV genotypes may be different in various countries. The prevalence of HCV genotypes was therefore compared in different countries. Genotyping of HCV was performed by slot-blot hybridization analysis using cDNA probes specific to each type of HCV or by restriction fragment length polymorphism analysis. In 121 Japanese non-cancer patients, the prevalence of HCV genotypes was 77.7% for HCV-K1, 16.5% for HCV-K2a and 5.0% for HCV-K2b. HCV-PT was detected in only 1 patient (0.8%). The prevalence in 43 Japanese hepatocellular carcinoma (HCC) patients was 74.4% for HCV-K1, 18.6% for HCV-K2a and 4.7% for HCV-K2b. HCV-PT was found in only 1 sample. In 19 European non-cancer patients, HCV-PT was found in 42.1% and HCV-K1 was found in 52.6%. HCV-K2 was not found. All 7 samples from European HCC patients were HCV-K1, indicating a significantly higher prevalence than in non-cancer patients. In 13 Brazilian non-cancer patients, the distribution pattern was similar to that of the Europeans. In 10 samples from the U.S.A., HCV-PT was found in 70% and HCV-K2 was found in 1 sample. In 18 Chinese non-cancer patients, HCV-K1 was found in 44.4%, HCV-K2a in 50.0% and HCV-K2b in 5.6% HCV-PT was not found. Two samples from Chinese HCC patients were HCV-K1.(ABSTRACT TRUNCATED AT 250 WORDS)

Usefulness of a Combined Evaluation of the Serum Adiponectin Level, HOMA-IR, and Serum Type IV Collagen 7S Level to Predict the Early Stage of Nonalcoholic Steatohepatitis

OBJECTIVE:Since nonalcoholic steatohepatitis (NASH) may progress to cirrhosis, it is important to differentiate NASH from simple steatosis, especially in its early stages. However, a liver biopsy cannot be performed in all patients with nonalcoholic fatty liver disease (NAFLD). We herein investigated whether serum biochemical markers are useful for predicting early-stage NASH.METHOD:Nineteen patients with simple steatosis and 66 patients with early-stage NASH (stage 1–2 in Brunts criteria) were studied. The area under the receiver operating characteristic curve (AUC) was used to illustrate the diagnostic ability of serum biochemical parameters to distinguish between simple steatosis and early-stage NASH.RESULTS: The serum adiponectin level was found to be significantly lower with early-stage NASH group (3.6 μg/mL) than in the simple steatosis group (6.0 μg/mL) (P < 0.001). The AUC was high (0.765) in the early-stage NASH group, and it was also the highest among all other markers. The sensitivity of the serum adiponectin level in the diagnosis of early-stage NASH was 68%, which was higher than for any other factors, while its specificity was 79%. The corresponding sensitivity and specificity of HOMA-IR were 51% and 95%, respectively. For type IV collagen 7S, sensitivity was 41% and specificity 95%. The sensitivity of the combination of three markers was 94%, with a specificity of 74%.CONCLUSION: Approximately 90% of the patients with early-stage NASH can be predicted by a combined evaluation of the serum adiponectin level, HOMA-IR, and serum type IV collagen 7S level.

Biochemical markers of chronic alcoholism

Usefulness of several biochemical markers for the monitoring of chronic alcoholism were studied. Among generally used markers, only gamma-GTP showed a significant difference between alcoholic and non-alcoholic liver diseases. Serum glutamate dehydrogenase (GDH) activity was significantly high in alcoholic liver disease. When the ratios of GDH to ornithine carbamyl transferase (OCT) were calculated, differences between alcoholic and non-alcoholic liver diseases became clearer without overlapping of any value. Serum desialo-transferrin was found in about 60% of the alcoholics, and disappeared by abstinence. Microheterogeneity of serum protein was also found in other glycoproteins. Serum prealbumin level was significantly high in alcoholics without severe liver disease. Acetaldehyde dehydrogenase (ALDH) activity of erythrocytes was significantly low in alcoholics, and gradually increased after abstinence. These results indicate that microheterogeneity of glycoproteins, serum prealbumin level and erythrocyte ALDH activity are good markers of alcohol abuse, and serum GDH/OCT ratio is the most sensitive marker of alcoholic liver injury. Serum gamma-GTP activity is a good marker of both conditions.

Expression of hyaluronic acid in N-nitrosodimethylamine induced hepatic fibrosis in rats

Hyaluronic acid (HA) plays prominent role in the pathogenesis of liver fibrosis. The mechanism of increased serum and liver HA during hepatic fibrosis was studied in rats. Liver injury was induced by intraperitoneal injections of N-nitrosodimethylamine (NDMA) for 7 consecutive days. A group of animals were sacrificed on everyday during injection and also on days 14 and 21 after the start of NDMA administration. The alpha-smooth muscle actin (alpha-SMA) was stained as a marker for activated stellate cells. Liver HA was studied by histochemical methods and serum HA was monitored by HA binding protein assay. CD44 was stained immunohistochemically. After the start of NDMA administration, necrosis was initiated on day 3 and massive necrosis was observed on days 5 and 7. Fibrosis was developed on day 14 and early cirrhosis was present on day 21. Staining of alpha-SMA demonstrated activated stellate cells from day 3 onwards. Serum HA peaked on day 7 and reduced afterwards. Serial liver sections stained for HA revealed excessive accumulation of HA during NDMA administration. On days 14 and 21, alpha-SMA and HA staining was remarkable in fibrotic and cirrhotic areas. CD44 staining was negative except during necrosis. It is concluded that the early elevation of serum HA is due to the increased synthesis and simultaneous release from the necrotic liver. In latter stages the increase of both serum and liver HA is contributed by the increased synthesis by the activated stellate cells and reduced clearance by the impaired sinusoidal endothelial cells.

Changes of hepatic microtubules and secretory proteins in human alcoholic liver disease.

It has been shown that alcohol consumption disrupts liver microtubules, impairs protein secretion and leads to ballooning of the hepatocytes in rats. Ethanol-induced hepatomegaly was accounted for by an increase of the hepatocytes volume. To study whether these changes occur in human alcoholic liver disease, hepatic tubular protein and export protein content were measured in 29 cases of alcoholic liver disease and were compared with those of 37 cases of non-alcoholic liver disease and 5 cases of non-hepatobiliary disease. Hepatic polymerized tubulin was significantly decreased in alcoholic liver disease compared to non-alcoholic liver disease (p less than 0.01), while free tubulin was increased in alcoholic liver disease. Hepatic transferrin (one of the export proteins) content was significantly higher (p less than 0.01) and serum transferrin level was significantly lower (p less than 0.05) in alcoholic liver disease than in non-alcoholic liver disease. These findings indicated that even in humans, chronic alcohol consumption decreased hepatic microtubules by impairing polymerization of tubular protein and increased hepatic export protein content. This decrease in hepatic microtubules by chronic alcohol consumption may play an important role in the development of human alcoholic liver disease.

Characteristic features of alcoholic liver disease in Japan: A review

SummaryThe characteristics of alcoholic liver disease (ALD) in Japanese patients were reviewed and compared with those in Western countries. From the study in Japanese cases, it became clear that alcoholic fibrosis and chronic hepatitis induced by alcohol were types of ALD other than the traditional 3 types. Liver injury in Japanese cases was clearly milder than that in American cases. In American cases, the injury may be fully developed, because of greater alcohol and fat intake. This may be one reason why the two above types of ALD have not been mentioned in the literature of Western countries. In Japanese patients, hepatitis C virus (HCV) infection is not related to alcoholic fibrosis and alcoholic hepatitis. On the other hand, the prevalence of HCV markers was high in chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC) patients. Alcoholic hepatitis and chronic hepatitis are the high risk groups for the development of cirrhosis and the chronic hepatitis group is at high risk for the development of HCC. Although the risk is low in alcoholic fibrosis, some patients also develop cirrhosis. About half of the cases of cirrhosis may develop from alcoholic hepatitis and alcoholic fibrosis, and the remaining half cases may develop from chronic hepatitis. Over 80% of HCC cases may develop from chronic hepatitis in Japan. Chronic alcoholism enhanced the development of HCV-related HCC. Recent increase of HCC in alcoholic cirrhosis in Japan may be related to the increase of alcohol consumption, the increase of blood transfusions, and longer survival of cirrhosis patients.

Effects of genotypes of hepatitis C virus on interferon treatment for chronic type C hepatitis

SummaryInterferon is commonly used for treatment of type C hepatitis, but the effects are variable and many factors may be responsible. Hepatitis C virus (HCV) can be classified into 4 types, PT, Kl, K2a and K2b. Therefore, the responses to interferon treatment in patients with the different HCV genotypes were analyzed. Twenty-four patients with type C hepatitis were treated with 3 to 10 million units of various types of interferon for more than 8 weeks. HCV-RNA encoding the NS5 region (HCV-NS5) was positive in these 24 patients, 16 of which were classified with the Kl type and 8 with the K2 type of HCV. In all patients except for 2, HCV-NS5 became negative within 3 weeks of treatment without relation to the HCV genotypes. Serum alanine aminotransferase levels were normalized in 7 out of 8 patients in the K2 group and in 4 out of 16 patients in the Kl group at the end of 8 weeks. At the 24th week, ALT levels were normalized in 5 out of 6 patients in the K2 group, and in one out of 9 patients in the K1 group. The percentage of patients exhibiting a good response was significantly higher in the K2 group than in the K1 group at both observation periods. During the post-treatment periods, relapse following complete response was found in 3 patients in the K2 group and in one patient in the K1 group. The final effects of interferon were significantly better in the K2 group than in the K1 group. These results indicate that the effects of interferon were significantly better in patients in the K2 group than in patients in the K1 group. Normalization of serum ALT levels was found in two patients with cirrhosis in the K2 group, but in none of the cirrhosis patients in the Kl group, suggesting that effects of interferon may not be related to the progression of liver disease in patients in the K2 group. These results suggest that genotypes of HCV are one of the major determinants of the effect of interferon treatment.

Effect of dietary fat upon ethanol metabolism in rats

The effect of dietary fat upon ethanol metabolism was studied in rats. Wistar strain male rats were divided into four groups according to diet, namely alcohol-high fat, alcohol-low fat, control-high fat, and control-low fat. After 4 weeks of feeding, blood ethanol levels following an intraperitoneal injection of 0.2 g ethanol/100 g of body weight were measured. The disappearance rate of blood ethanol was faster and the metabolic rate of ethanol was significantly greater in the alcohol-high fat group compared to the alcohol-low fat or non-alcoholic groups. Microsomal enzymes, such as the microsomal ethanol-oxidizing system, aniline hydroxylase, and glucose-6-phosphatase, were significantly higher in the alcohol-high fat group than in the alcohol-low fat or non-alcoholic groups. The ethanol uptake rate of the isolated perfused liver was increased significantly in the alcoholic groups. In the alcoholic rats, the high fat group showed significantly higher uptake than the low fat group. Although the ethanol uptake rate after 4-methylpyrazole treatment was not significantly different among the four groups, its fraction of the total ethanol uptake was increased significantly in the alcohol-high fat group. These results suggest that high fat diets accelerate ethanol metabolism through the microsomal ethanol-oxidizing system.

Hepatic aldehyde dehydrogenase isoenzymes: Differences with respect to species

Although changes in the acetaldehyde (Ac-CHO) oxidizing system in the liver are important for understanding the pathogenesis of alcoholic liver injury, interspecies differences of hepatic aldehyde dehydrogenase (ALDH: 1.2.1.3) isozymes have not yet been sufficiently studied. In the present study, the character and subcellular distribution of hepatic ALDH isozymes in male animals such as the Rhesus monkey, domestic cow, albino rabbit and Wistar strain rat were analyzed and compared with those in humans. The optimal pH for ALDH isozymes in human liver was 9.5, while those of monkey, cow, rabbit and rat were 9.0, 9.0, 8.5 and 8.5, respectively. In human liver, low Km ALDH activity was distributed mainly in the cytosol, while the corresponding activity was selectively distributed in the mitochondria in rat liver. The distribution patterns of low Km ALDH in the other animals were similar to those of the rat. In microsomes, low Km ALDH activity was very low or almost negligible in the livers of all species. These results indicate that Ac-CHO degrades mainly in the cytosol in the human liver, whereas, in the other species, it occurs in the mitochondria. This suggests that results obtained with experimental animals cannot be applied directly to humans. It is also suggested that degradation of the Ac-CHO produced in the microsomes may be slow in all species.