Role of UGT1A1 mutation in fasting hyperbilirubinemia.

BACKGROUND AND AIM: Low-grade fasting hyperbilirubinemia is a common observation in healthy subjects (HS), whereas high-grade fasting hyperbilirubinemia is believed to be a characteristic finding of Gilbert's syndrome. This study was undertaken to assess the role of mutation in bilirubin UDP- glycosyltransferase gene (UGT1A1) on fasting hyperbilirubinemia. METHODS: Analysis of UGT1A1 and a caloric restriction test (400 kcal for 24 h) were performed in 56 healthy subjects (25 males, 31 females), and 28 patients with Gilbert's syndrome (18 males, 10 females). There were 29 healthy subjects with no mutation in UGT1A1, and 27 healthy subjects and 26 Gilbert's syndrome patients with mutations in the coding and/or promoter (TATA box) regions of UGT1A1. RESULTS: The mean increment of serum bilirubin (DeltaSB) was 7.6 micromol/L [corrected] (males) and 4.1 micromol/L (females) in subjects with no UGT1A1 mutation. Subjects with mutation in UGT1A1 showed higher levels of DeltaSB than individuals without mutation. Among healthy subjects, gender difference in DeltaSB values was observed only in individuals with the wild type of UGT1A1, but not in those with mutations in this gene. CONCLUSION: The results of the present study suggest that UGT1A1 mutation has a role in the development of high-grade fasting hyperbilirubinemia after caloric restriction.

Hepatitis C virus core particle detected by immunoelectron microscopy and optical rotation technique.

Hepatitis C Virus (HCV) are 55-65 nm spherical particles, but the internal structure of the virion remains to be clarified. To clarify the morphology of HCV core particles, we performed an immune electron microscopy (IEM) using plasma samples from two blood donors with high HCV RNA titers and a detergent-treated anti-HCV core antibody-free plasma sample with high HCV RNA titer (1.5x10(8) copies/ml). Spherical particles, with 33-40 nm in diameter (an average diameter of 37 nm) were found in 1.22-1.25 g/ml fractions after sucrose density gradient centrifugation by conventional electron microscopy (EM). IEM using rabbit polyclonal antibody (RR8) specific to the putative HCV core protein and goat anti-rabbit IgG colloidal gold particles revealed that these spherical particles specifically reacted with RR8. This finding indicates that the spherical particles are naked HCV core particles. Some of the HCV core particles had an icosahedron-like structure. Optical rotation technique showed that the HCV core particle exhibits six-fold symmetry and that the length of regular hexagon side is approximately 20 nm. These findings showed that HCV core particles are spherical particles of 33-40 nm in diameter and that HCV core particles may possess an icosahedron-like structure and a buoyant density of 1.22-1.25 g/ml.

Paraformaldehyde protects of hepatitis C virus particles during ultracentrifugation.

Divergent buoyant densities of hepatitis C virus (HCV) have been reported. If the destruction of HCV particles occurs during the ultracentrifugation process to separate fractions with different densities, an accurate evaluation of the HCV buoyant density is difficult. To examine this concern, changes were examined in HCV RNA titer of each density fraction after paraformaldehyde fixation of virus particles in the sera of 9 patients with chronic HCV infection. Serum was treated with 4% paraformaldehyde, and the density fractions were then separated by ultracentrifugation. The HCV RNA titer of each fraction was determined by reverse transcription-polymerase chain reaction (RT-PCR) assay, and the results were compared with those obtained from the serum without paraformaldehyde fixation. After fixation, the HCV RNA titer was significantly increased in the 1.11-1.14 g/mL fraction (P=0.0018), and decreased in the 1.14-1.17 and 1.17-1.20 g/mL fractions (P=0.0457 and 0.0003, respectively). Using immunogold electron microscopy, it was found that morphologically destroyed HCV particles are present mainly in the 1.17 g/mL fraction of paraformaldehyde-untreated samples, whereas the intact HCV virion particles are present in the 1.12 and 1.14 g/mL fractions of the paraformaldehyde-treated samples. These results suggest that the destruction of HCV virions occurs during the ultracentrifugation process and that paraformaldehyde treatment protects from destruction. It was also considered that the accurate buoyant density of the HCV virion is 1.11-1.14 g/mL. This study describes a useful method for the purification of HCV virions, and provides new insights for elucidating the physicochemical properties of HCV particles.

Decrease of regional cerebral blood flow in liver cirrhosis.

OBJECTIVE: Alterations of regional cerebral blood flow (rCBF) in subjects with liver cirrhosis have not been fully evaluated. We evaluated quantitative changes in rCBF using single photon emission computed tomography (SPECT). METHODS: Twenty-eight Japanese patients with liver cirrhosis were enrolled in this study. None of them exhibited advanced hepatic encephalopathy at the time of examination. The cause of liver cirrhosis was viral infection in 26 patients; the cause was unknown in two patients. Child-Pugh classification of the patients was as follows: Group A, 12 patients; Group B, 12 patients; and Group C, four patients. The control group consisted of 25 age-matched healthy subjects. Radionuclide angiography was performed by rapid injection of Tc-99m ethyl cysteinate dimer (ECD) (740 MBq) via the right cubital vein, and then SPECT brain images were taken. Using the Patlak graphical method, rCBF values (ml/100 g per min) were calculated in the frontal, parietal, temporal and occipital lobes and cerebellum on SPECT images. RESULTS: The rCBF values were lower in cirrhotic patients than in controls, i.e. by 15% in the frontal lobe, by 12% in the parietal lobe, by 10% in the temporal and occipital lobes, and by 7% in the cerebellum. They decreased concomitantly with the severity of liver disease. A significant negative correlation was noted between rCBF values and Child-Pugh score in the frontal (P<0.01), parietal (P<0.05) and occipital lobes (P<0.01). rCBF values of each region were not correlated with age or with results of neuropsychological test. The degree of association between rCBF values and results of laboratory examination was generally poor. CONCLUSION: Patients with liver cirrhosis without advanced encephalopathy showed widespread reduction in rCBF; this reduction was particularly evident in the frontal lobe. Tc-99m ECD SPECT may be useful for evaluating cerebral functional changes in patients with liver cirrhosis.

Assessment of portosystemic shunt by summation of radioactivity during 201thallium chloride portal scintigraphy in patients with chronic liver disease.

BACKGROUND/AIMS: Portal scintigraphy is a useful non-invasive method for the determination of portosystemic shunts in patients with liver cirrhosis. Several procedures have been reported for its execution in clinical practice but most of them failed to show sufficient sensitivity for the diagnosis of portosystemic shunt. In the present study, we evaluated whether summation of radioisotope counts obtained during intrarectal or intraduodenal administration of 201thalium chloride is useful for increasing the diagnostic yield of porto-systemic shunts in patients with chronic liver disease. METHODOLOGY: Seven patients with chronic viral hepatitis and 8 with liver cirrhosis secondary to viral hepatitis were enrolled in this study. Following the conventional protocol, 201thalium chloride was administered per rectum and the 60-second-heart-to-liver uptake (conv-H/L-R) ratio was calculated after 20 min. Continuous measurement of the radioactivity signals during 20 min were also done and the summated heart-to-liver uptake (sum-H/L-R) ratio from the total radioactivity count were calculated. Measurement of the conventional heart-to-liver uptake (conv-H/L-D) ratio and the summated (sum-H/L-D) ratio were also done as described above after the intraduodenal administration of 201thalium chloride by endoscopy. RESULTS: All ratios (conv-H/L-R, conv-H/L-D, sum-H/D-R, sum-H/L-D) were significantly higher in patients with liver cirrhosis than in those with chronic hepatitis. Among all heart/liver ratios, only the sum-H/L-R ratio was significantly different between patients with and without esophageal varices. Serum hyaluronate level and other liver function tests were found to be significantly correlated with all heart-to-liver ratios, but they were more strongly correlated with the sum-H/D-R and sum-H/L-D ratios than with the conv-H/L-R and conv-H/L-D ratios. CONCLUSIONS: The results of this study showed that the heart-to-liver ratio calculated by summation of radioactivity is better than the conventional method for the diagnosis of portosystemic shunt in patients with chronic liver disease.

Biliary excretion of TT virus (TTV).

A novel DNA virus (TT virus; TTV) was isolated from a patient with post-transfusion hepatitis of unknown etiology. If TTV replicates in the liver, TTV may appear in the bile. In the present study, to clarify whether fecal-oral infection occur via biliary excretion, the presence of TTV DNA was assessed in paired serum and bile samples collected from 28 patients with obstructive jaundice without parenchymal liver disease. TTV DNA was detected by polymerase chain reaction (PCR) using semi-nested primers, and quantified by Real Time Detection PCR (RTD-PCR). The nucleotide sequence of isolates TTV DNAs was also determined and the sequences were compared between serum and bile samples. Among 28 patients, 7 were positive for TTV DNA in both samples, and 3 and 2 were positive in serum and bile respectively. Of 7 patients positive for TTV DNA in both samples, the TTV DNA titer was higher in serum of 4 patients and in bile of 1 patient. Among 7 patients positive for TTV DNA in serum and bile, 6 had the same sequence in both samples. Multiple distinct types of TTV DNA clones were isolated from serum in 2 patients and from bile in 4 patients. In conclusion, TTV DNA is detected frequently in bile from patients with obstructive jaundice, suggesting a fecal-oral route of infection and high prevalence of asymptomatic TTV carriers. TTV DNA was detected only in serum from some patients, suggesting that replication of TTV may occur in other organs as well as in the liver.

High prevalence of transfusion-transmitted virus among patients with non-B, non-C hepatocellular carcinoma.

BACKGROUND: Many patients with hepatocellular carcinoma are positive for hepatitis B surface antigen (HBsAg) or antibodies to hepatitis C virus (anti-HCV). Recently, transfusion-transmitted virus (TTV) DNA was identified in the serum of patients with non-B, non-C posttransfusion hepatitis. In this study, the prevalence of TTV DNA in the serum of patients with non-B, non-C hepatitis-associated hepatocellular carcinoma was evaluated. METHODS: Fifteen patients with hepatocellular carcinoma negative for HBsAg, antibodies to hepatitis B core antigen (anti-HBc), and anti-HCV antibodies were enrolled in this study (non-B, non-C group). Fifteen patients positive for HBsAg and negative for anti-HCV antibody (HBV group) and another group of patients negative for HBsAg but positive for anti-HCV antibody (HCV group) were also enrolled in this study. Data obtained from 27 healthy subjects negative for both HBsAg and anti-HCV antibody and normal levels of serum alanine aminotransferase represented controls. The healthy control group, the non-B, non-C group, and the HCV group were age-matched. TTV DNA was detected by heminested polymerase chain reaction in which specific primers were used. RESULTS: TTV DNA was detected in 10 of 15 patients (67%) in the non-B, non-C group. This prevalence rate in the non-B, non-C group was significantly higher than that in the HBV group (3 of 15 patients, 20%) and the control group (9 of 27 patients, 33%), but it was not significantly different from that in the HCV group (7 of 15 patients, 47%). The noncancerous hepatic tissue samples of 10 TTV-DNA positive patients in the non-B, non-C group included 2 with chronic hepatitis and 8 with cirrhosis. CONCLUSIONS: This study showed that TTV DNA is frequently detected in the serum of patients with non-B, non-C hepatocellular carcinoma. This result suggests a potential pathogenetic association between hepatocellular carcinoma and TTV infection.

Induction of apoptotic cell death in human hepatocellular carcinoma SK-HEP-1 cells by a polyamine synthesis inhibitor, methylglyoxal bis(cyclopentylamidinohydrazone).

The antitumor effects of a polyamine biosynthetic pathway inhibitor methylglyoxal bis(cyclopentylamidinohydrazone) (MGBCP) on the human hepatocellular carcinoma SK-HEP-1 cell line have been investigated. The growth of these cultured hepatocellular carcinoma cells was inhibited by MGBCP in a dose-dependent manner. Spermidine and spermine levels were dose-dependently depressed, and morphological changes due to programmed cell death (apoptosis) were observed in these MGBCP-treated hepatocellular carcinoma cells. These results suggest that in addition to reducing the growth rates, MGBCP can induce apoptotic cell death in this human hepatocellular carcinoma cell line.

Evaluating response to nutritional therapy using the branched-chain amino acid/tyrosine ratio in patients with chronic liver disease.

The branched-chain amino acid (BCAA)/tyrosine (Tyr) ratio (BTR) recently has been reported to be a good indicator of the severity of hepatic parenchymal injury in patients with chronic liver disease. In the present study, sequential changes of BTR after BCAA administration were determined in patients with chronic liver disease to evaluate the value of BTR as a marker of the clinical response to nutritional therapy in these patients. This study comprised 75 patients with chronic hepatitis and 96 with liver cirrhosis. BTR was significantly decreased in patients with cirrhosis and hepatitis compared with healthy subjects. BTR was significantly correlated with the Child-Pugh score and with other liver function tests. BCAA increased significantly 2 hr after BCAA administration and decreased gradually thereafter. Tyr significantly decreased 4 hr after BCAA administration. BTR significantly increased 2 and 4 hr after BCAA therapy. The increase in BTR 3 hr after BCAA administration was low in patients with decreased basal BTR. The results of this study showed that BTR is a good index of the hepatic parenchymal damage and that it may be a useful marker for monitoring response to nutritional therapy in patients with chronic liver disease.

Case report: Alanine aminotransferase deficiency detected in a patient with chronic hepatitis C.

We report a case of alanine aminotransferase (ALT) deficiency in a 68-year-old Japanese female with chronic hepatitis C. The serum was positive for antibody to hepatitis C virus (HCV) and HCV-RNA. Liver biopsy showed histological evidence of chronic active hepatitis. The level of serum aspartate aminotransferase (sAST) was elevated, but sALT was extremely low. The patient was followed up for her serum aminotransferase levels for 1.5 years under the treatment with ursodeoxycholic acid. The low sALT level persisted during all the follow-up period. The ALT activity in liver tissue was also decreased. Based on these findings, ALT deficiency was suspected. sALT activity was also found to be low in her two sons. This latter finding suggests the hereditary character of this abnormality.

[Clinical symptoms of patients with liver cirrhosis].

In patients with liver cirrhosis, there were various symptoms in decompensated state, but not in compensated state. Most of symptoms were due to liver cell dysfunction and portal hypertension. Jaundice, ascites, edema, bleeding tendency and endocrinological symptoms were due to liver cell dysfunction. Hepatic encephalopathy, esophageal varices and splenomegaly were related to portal hypertension. Vascular spiders and palmar erythema were found in patients with alcoholic liver cirrhosis more frequently than in patients with viral liver cirrhosis. Jaundice was a sign of poor prognosis. There were no difference in clinical symptoms between aged patients and young patients. Careful observation of the symptoms is important to care the patients with liver cirrhosis.

[Blood chemistry, hematology of patients with liver cirrhosis].

In patients with liver cirrhosis, there were many abnormalities in laboratory tests. Serum GOT, GPT and LDH were elevated due to the liver cell necrosis. The value of ICG tests reflected the decrease of effective hepatic blood flow and the increase of intrahepatic shunt flow. White blood cell counts and the number of platelet were decreased due to the hypersplenism. Serum albumin and cholineesterase levels were decreased more remarkably in patients with alcoholic liver cirrhosis than in patients with viral liver cirrhosis. Raised GOT and GPT levels were lower in aged patients than in young patients. Serial laboratory tests were important for the management of patients with liver cirrhosis.

Detection of hepatitis C virus RNA by nested polymerase chain reaction in sera of patients with chronic non-A, non-B hepatitis treated with interferon.

To evaluate the effect of interferon-alpha treatment on the levels of serum aminotransferase (sALT) and of hepatitis C virus (HCV)-RNA, we studied 19 patients with chronic non-A, non-B (NANB) hepatitis. Before therapy, 14 patients were positive by nested polymerase chain reaction (PCR) with primers deduced from the 5'-non-coding region of the HCV genome. Serum HCV-RNA had disappeared in 12 (85.7%) of them by the end of therapy, but then reappeared 6 months later in 4 of these 12 patients. A marked improvement in sALT was seen in 5 of the 8 patients with sustained HCV-RNA disappearance, but not in the 4 patients with only transient HCV-RNA negativity. Pre-treatment levels of hepatitis C viremia, analyzed by single PCR and dot blot hybridization, ranged from 2 x 10(3) to 2 x 10(8) copies/ml, and were below 2 x 10(5) copies/ml in patients with a complete response to interferon therapy. These results suggest that this HCV-RNA assay, combined with sALT testing, may be useful for estimation of the long-term efficacy of interferon therapy in hepatitis C.

Prevalence and heterogeneity of serum DNA polymerase activity in patients with non-A, non-B hepatitis and HBsAg-negative blood donors with elevated SGPT.

Serum DNA polymerase activity (DNA-P) was detected in 27.6 per cent of non-A, non-B (NANB) hepatitis patients, 8.7 per cent of patients with alcoholic liver disease (ALD), 8.6 per cent of hepatitis B surface antigen (HBsAg)-positive patients and 19.0 per cent of HBsAg-negative blood donors with elevated serum glutamic-pyruvic transaminase (SGPT) concentrations. In contrast, none of the patients with hepatitis A, drug-induced liver injury or non-alcoholic fatty liver had DNA-P in their sera in the acute phase of the illness. All HBsAg-positive samples with detectable DNA-P were strongly positive for hepatitis B virus (HBV) DNA, but the samples from patients with NANB hepatitis and ALD and HBsAg-negative blood donors had no HBV DNA. Sensitivity to actinomycin D showed the heterogeneity of DNA-Ps in HBsAg-negative blood donors; the enzyme activity of one type was inhibited by 100 micrograms/ml of actinomycin D, whereas the other was not. The preference for exogenous template primers of these DNA-Ps was different to those of HBV and human retroviruses. The results reveal the prevalence of serum DNA-P in NANB hepatitis patients and suggest that two distinct agents are relevant to the aetiology of NANB hepatitis.

Pathology of the liver in "idiopathic portal hypertension" associated with autoimmune disease. The Ministry of Health and Welfare Disorders of Portal Circulation Research Committee.

The histopathology of the liver in idiopathic portal hypertension (IPH) associated with autoimmune disease (15 cases), was examined and compared with that of IPH without autoimmune disease (31 cases). It was found that hepatic histopathology was heterogeneous in the cases with autoimmune disease. That is, the hepatic histopathology in 7 cases was similar to that of classic IPH without autoimmune disease, and the remaining 8 cases disclosed unusual lesions such as focal non-suppurative cholangitis, nodular parenchymal hyperplasia, moderate portal inflammation, and intrahepatic ductopenia. These unusual lesions, which frequently coexisted in the same case, were not typical ones for making other diagnoses such as primary biliary cirrhosis or nodular regenerative hyperplasia of the liver. These findings suggest that unusual histologic lesions in the livers of IPH patients with autoimmune disease may represent an accentuated immunologic reaction inherent in IPH, or that such cases may be an abortive or incomplete form of primary biliary cirrhosis or nodular regenerative hyperplasia of the liver.

Acromegaly and hyperthyroidism associated with McCune-Albright syndrome.

A 36-year-old man is described having McCune-Albright syndrome, acromegaly likely due to somatotroph hyperplasia and hyperthyroidism due to adenomatous goiter. Sexual precocity was not noted. The sella was narrow in size and no mass was seen. The decline of elevated GH by hyperglycemia and increase by GHRH-44(NH2) may support somatotroph hyperplasia, but plasma GHRH-44(NH2) levels were not elevated. A mass in the right lobe and enlargement of the left lobe of the thyroid were noted. Thyroid hormone levels in serum and thyroidal radioiodine uptake values were elevated, while TSH measurements in serum were low. The radioiodine scan showed a cold nodule in the right lobe and a hot area in the left of the thyroid. Thyroidal radioiodine was not suppressed following T3 given orally. These findings are compatible with functioning glands autonomously as the mechanism for the endocrinopathies associated with the McCune-Albright syndrome.