Takaharu Yoh

Serum thioredoxin levels as a predictor of steatohepatitis in patients with nonalcoholic fatty liver disease

BACKGROUND/AIMS Thioredoxin (TRX) is a stress-inducible thiol-containing protein. The aim of this study was to evaluate the clinical significance of serum TRX in patients with nonalcoholic steatohepatitis (NASH) or simple steatosis. METHODS Serum TRX levels were determined using an enzyme-linked immunosorbent assay kit in 25 patients with NASH, 15 patients with simple steatosis, and 17 healthy volunteers. RESULTS Serum TRX levels (medians and (ranges), ng/ml) were significantly elevated in patients with NASH (60.3 (17.6-104.7)), compared to those in patients with simple steatosis (24.6 (16.6-69.7), P=0.0009) and in healthy controls (23.5 (1.3-50.7), P<0.0001). Serum ferritin levels in patients with NASH were also significantly higher than the levels in patients with simple steatosis. The receiver operating characteristic curve confirmed that serum TRX and ferritin levels were predictors for distinguishing NASH from simple steatosis. Higher grades of histological iron staining were observed in NASH than in simple steatosis. Serum TRX tended to increase in accordance with hepatic iron accumulation and the histological severity in patients with NASH. CONCLUSIONS The pathogenesis of NASH may be associated with iron-related oxidative stress. The serum TRX level is a parameter for discriminating NASH from simple steatosis as well as a predictor of the severity of NASH.

Serum thioredoxin levels as an indicator of oxidative stress in patients with hepatitis C virus infection

BACKGROUND/AIM It has recently been suggested that oxidative stress may be associated with hepatitis C virus (HCV) infection. Thioredoxin (TRX) is a stress-inducible thiol-containing protein. The aim of this study was to evaluate the clinical significance of serum TRX levels in patients with HCV-related chronic liver diseases. METHODS Serum TRX levels were determined with a sandwich enzyme-linked immunosorbent assay kit in 174 serum HCV-RNA positive patients, including 6 asymptomatic carriers, 124 chronic hepatitis, 20 liver cirrhosis, and 24 hepatocellular carcinoma, and in 15 healthy volunteers. RESULTS The serum TRX levels (medians and [ranges], ng/ml) were significantly elevated in the HCV-infected patients; 30.9 [20.7-37.7] in asymptomatic carriers, 34.5 [8.6-135.6]* in chronic hepatitis, 42.5 [21.4-97.2]* in liver cirrhosis, and 43.9 [11.7-180.3]** in hepatocellular carcinoma (*p<0.05, **p<0.001, vs. 24.9 [1.3-50.7] in healthy controls). Serum TRX levels were significantly correlated with the serum levels of ferritin and fibrogenesis markers, and with the histological stage of hepatic fibrosis. The serum TRX levels before interferon treatment of patients whose serum HCV-RNA was still positive on day 14 following interferon treatment (42.6 [20.1-90.0]) were significantly higher than those of patients whose serum HCV-RNA was negative on day 14 following interferon treatment (25.8 [7.4-59.8], p<0.05). CONCLUSIONS The serum TRX levels of patients with HCV infection increased with their serum ferritin levels and the progression of liver fibrosis. Patients with higher serum TRX levels exhibited resistance to interferon therapy. Oxidative stress may therefore be responsible for the pathological mechanism of HCV-related liver diseases and be one of the impediments to eradication of HCV during interferon treatment.

Insulin resistance/β‐cell function and serum ferritin level in non‐diabetic patients with hepatitis C virus infection

Background/Aims: Since impaired glucose tolerance and iron overload are frequently demonstrated in hepatitis C virus (HCV)‐related liver diseases, in this study we investigated insulin resistance, pancreatic β‐cell function, i.e., insulin secretion, and serum ferritin levels in patients with HCV infection, especially non‐diabetic patients.

Effects of Glycyrrhizin on Glucocorticoid Signaling Pathway in Hepatocytes

The cytoprotective effects of glycyrrhizin (GL) are similar to glucocorticoids. We investigated the effects of GL on the glucocorticoid receptor (GR) and on the enzyme activity of tyrosine aminotransferase (TAT), an hepatocyte-specific marker of glucocorticoid action, in rat hepatocytes. Pretreatment with GL significantly decreased the affinity of GRs for dexamethasone (DEX) and increased the period of time required for TAT activity to reach a peak after the addition of DEX. GL did not affect the amount of GR, but significantly decreased the amount of heat-shock protein 90 (HSP90) and HSP90-associated GR. Alternatively, TAT activity and TAT mRNA levels increased significantly after the addition of GL to hepatocytes pretreated with DEX. In conclusion, GL reduces the affinity of GRs for ligands through the decreased HSP90 expression, but significantly enhances the glucocorticoid-induced TAT-gene expression at the transcriptional level in rat hepatocytes.

Effects of nitric oxide on the redox status of liver microsomes-electron spin resonance monitoring using nitroxide probes.

Nitroxide radicals (nitroxides) are reduced to the corresponding hydroxylamines and lose their electron spin resonance (ESR) signals, but these hydroxylamines are easily reoxidized to nitroxides and regain the ESR signals. In the present study the effects of nitric oxide (NO) on the reduction/oxidation (redox) status of hepatic microsomes were investigated by ESR spectroscopy using nitroxide probes. Rat hepatic microsomes were treated with an NO donor, NOR3 or NOC7, and then labeled with a water-soluble nitroxide, 2,2,6,6-tetramethyl-4-hydroxy-1-piperidinyloxy (Tempol), or a lipid-soluble nitroxide, 5-doxyl stearic acid (5-DSA). The reduction of Tempol was facilitated under hypoxic conditions in control microsomes. In NOR3 or NOC7-treated microsomes, the reduction of Tempol and the reoxidation of the corresponding hydroxylamine hardly occurred under both normoxic and hypoxic conditions. The ESR signals of 5-DSA changed just as those of Tempol did in control and NO-treated microsomes. The concentrations of total thiol and cytochrome P-450, and the activity of mixed function amine oxidase were reduced in NOR3 or NOC7-treated microsomes. In conclusion, NO affects not only the reduction of nitroxides but also the oxidation of hydroxylamines in hepatic microsomes, suggesting that the microsomal capability of redox regulation was lost by NO.

Non‐prescription supplement‐induced hepatitis with hyperferritinemia and mutation (H63D) in the HFE gene

A 55‐year‐old Japanese woman was hospitalized because liver function tests showed an abnormality. Transaminases and biliary enzymes were markedly elevated with hyperferritinemia. Her imaging tests revealed no significant abnormality. She had been taking various non‐prescription supplements for over approximately 6 months. After the supplements were discontinued her liver function gradually improved. This clinical course was suggestive of supplement‐induced hepatitis. She had no history of taking supplements containing iron, so it was interesting that she had hyperferritinemia. We examined C282Y and H63D, which are important mutations in theiron‐metabolizing gene, HFE. She was found to be heterozygous for the H63D mutation. The interaction between hyperferritinemia and supplements is unknown, but it can be speculated that some interaction between iron overload and supplements may have underlain the pathogenesis of her liver injury.

Differences in the efficacy of ursodeoxycholic acid and bile acid metabolism between viral liver diseases and primary biliary cirrhosis

Aim and Methods: The effects of ursodeoxycholic acid (UDCA, 600 mg/day) on liver function test values, and serum and urinary bile acids levels in hepatitis C virus‐related chronic hepatitis (CH, n = 39) and liver cirrhosis (LC, n = 25), and in primary biliary cirrhosis (PBC, n = 25) were compared.

Transfer of nitric oxide from the liver to erythrocytes — An ESR study using nitroglycerin-treated mice

Nitric oxide (NO) formation in the liver and blood of the mouse following intraperitoneal treatment with nitroglycerin (glycerol trinitrate, GTN) was determined using electron spin resonance (ESR) spectroscopy. ESR signals of heme-NO complexes were detected at maximum levels within 5 min in the liver, but increased to a maximum level about 15-30 min later in the blood. GTN is not metabolized to release NO in vitro in the blood of the mouse. The hepatic microsomes which showed the heme-NO complexes ESR signals were incubated with mouse erythrocytes, with the result that a hemoglobin-NO signal was obtained from the erythrocytes. The activities of microsomal cytochrome P-450, the hepatic level of glutathione, and the reduction rate of nitroxide radicals in the in vivo liver, measured using L-band ESR spectroscopy, were temporarily decreased following GTN administration. In conclusion, NO in the liver could be scavenged by circulating erythrocytes, which might minimize NO-induced liver damage.

Serum thioredoxin as an indicator of iron-induced oxidative stress in chronic viral hepatitis

Aim: Thioredoxin (TRX), a thiol-containing protein, is induced by various oxidative stresses. The aim of the present study is to evaluate the clinical significance of serum TRX levels in patients with chronic viral hepatitis. Methods: TRX levels and other laboratory parameters, including ironmarkers, were measured in the sera of patients with chronic hepatitis type C (CH-C, n= 163) and type B (CH-B, n=35) as well as in healthy controls (n=17). Forty-five of the CH-C patients underwent a liver biopsy. Fifteen of the CH-C patients received biweekly phlebotomies in which 400 ml of blood were drawn. Serum TRX levels were determined using a recently established sandwich ELISA kit (Fujirebio Inc., Japan). Results: I) Serum TRX levels (ng/ml, mean:tSD) were significantly higher in CH-C patients (39.1±21.2) than in CH-B patients (30.9± 16.5)(p<.05) or in controls (20.6± 12.5)(p<.001). No significant differences in TRX levels were found between CH-B patients and controls. 2) TRX levels did not correlate with ALT levels or virus titers in either CH-C or CH-B patients. Serum iron, ferritin, and transferrin saturation values were significantly higher in the CH-C patients than in the CH-B patients (p<.05). Serum TRX levels were significantly correlated with these iron-markers in CH-C patients, but not in CH-B patients. 3) In CH-C patients, significant correlations were observed between TRX levels and the levels of serum markers for hepatic fibrosis, such as hyaluronic acid, IV collagen, and P-III-P. Histological examination also demonstrated that TRX levels were significantly correlated with the degree of hepatic fibrosis (r=0.333, p<.05), but not with that of hepatic activity. Serum ferritin levels tended to increase with the progression of fibrosis. 4) The removal of excess iron by phlebotomy resulted in a reduction of TRX and ferritin serum levels in CH-C patients. Conclusions: The present findings suggest that serum TRX levels may be a useful indicator of oxidative stress in hepatitis patients. The positive correlations between serum TRX levels and the value of iron-markers as well as the histological stages of hepatic fibrosis and the good response of CH-C patients to phlebotomy indicate that iron-induced oxidative stress may contribute to the pathological mechanism of hepatitis C. No significant increases in serum TRX levels were found in CH-B patients, suggesting that hepatitis B may not be influenced by oxidative stress.