Serum thioredoxin elucidates the significance of serum ferritin as a marker of oxidative stress in chronic liver diseases.

BACKGROUND/AIMS: Serum thioredoxin (TRX) levels have recently been established as an indicator of oxidative stress in various diseases. The aim of the present study was to clarify the clinical significance of serum ferritin in chronic liver diseases. METHODS: Levels of ferritin, transferrin saturation (TS), aspartate aminotransferase (AST), and TRX were measured in the sera of patients with chronic hepatitis C (CH-C, n=92), chronic hepatitis B (CH-B, n=28), nonalcoholic fatty liver (FL, n=31), or alcoholic liver diseases (ALD, n=17). Serum TRX levels were evaluated with a recently established sandwich enzyme-linked immunosorbent assay kit. RESULTS: Serum TRX levels were significantly higher in CH-C, FL, and ALD than in healthy volunteers. A larger proportion of patients with CH-C, FL, and ALD had elevated levels of serum ferritin than CH-B. Serum ferritin levels were positively correlated with levels of TS, AST, and TRX in CH-C, but were merely correlated with TS values in CH-B. Ferritin levels were also well correlated with AST and TRX, but not with TS in FL and ALD. CONCLUSION: Oxidative stress, which was evaluated by measuring serum TRX, in addition to storage iron and hepatocyte damage is a cause of increasing serum ferritin levels in chronic liver diseases. An elevated serum ferritin level, which was correlated with TS, indicates that iron-induced oxidative stress contributes to CH-C. Elevated ferritin levels in FL and ALD may be mostly due to iron-unrelated stresses.

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. RESULTS: The percentages of improvement in alanine transaminase (ALT) and gamma-glutamyl transpeptidase (gamma-GTP) in CH were almost the same in LC. The rates of improvement in ALT in PBC were negatively correlated with histological stages in the liver. Total serum bile acid levels in LC rose to the same extent as in CH, but the increases in PBC were significantly smaller at stages 3-4 than stages 1-2. The urinary levels of hydroxylated metabolites of UDCA only slightly increased in LC, but they increased significantly at PBC stages 3-4. CONCLUSIONS: The efficacy of UDCA was preserved in LC, but diminished at PBC stages 3-4. The poor enrichment of UDCA in the bile acid pool and extensive biotransformation of UDCA may cause the limited efficacy of UDCA in the cirrhotic stage of PBC.

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.

Thioredoxin levels in the sera of untreated viral hepatitis patients and those treated with glycyrrhizin or ursodeoxycholic acid.

Thioredoxin (TRX), a thiol-containing protein, is induced by various oxidative stresses. Serum TRX levels were measured with a sandwich enzyme-linked immunosorbent assay kit in 210 hepatitis C virus (HCV)-infected patients, 39 hepatitis B virus (HBV)-infected patients, and 17 healthy volunteers. The effects of hepatoprotective drugs on TRX levels were also examined. The median TRX levels were significantly higher in HCV-infected patients than in controls (34.2 vs. 23.5 ng/ml, respectively; p < 0.005), but were not elevated in HBV-infected patients (26.7 ng/ml). The TRX levels were significantly correlated with serum lipid peroxide levels and indocyanine green exclusion test values, and were markedly decreased following treatment with Stronger Neo-Minophagen C or ursodeoxycholic acid. In conclusion serum TRX levels, a marker of oxidative stress, were higher in patients with HCV infection than those with HBV infection and healthy controls. The therapeutic efficacy of hepatoprotective drugs may be connected with the decrease in oxidative stress in hepatitis patients.

Proton magnetic resonance assay of total and taurine-conjugated bile acids in bile.

Biliary bile acids, coexisting with phospholipid and cholesterol, are partly conjugated with taurine. In the present report we show that total and taurine-conjugated bile acids in bile can be simultaneously and quantitatively measured by high-resolution (1)H-nuclear magnetic resonance ((1)H-NMR) spectroscopy. We used a 7.05-Tesla NMR spectrometer to obtain the (1)H-NMR spectra of model and biological biles. Only addition of trimethylsilyl-3-propionic acid sodium salt-D(4) (TSP) to each sample as an internal standard was required in preparation for (1)H-NMR measurement. In (1)H-NMR spectra of rat bile, peaks of C-18 methyl protons of bile acids and of C-25 methylene protons on the taurine moiety of taurine-conjugated bile acids were detected at 0.7 ppm and 3.1 ppm, respectively. Peak areas, of C-18 and C-25 peaks, increased in proportion to the concentrations of bile acids or taurine-conjugated bile acids, even in the presence of phospholipid and cholesterol. The accuracy of NMR measurement of total and taurine-conjugated bile acids was confirmed by comparing the results of NMR with those of enzyme-fluorimetry. The results clearly demonstrate that (1)H-NMR spectroscopy can be applied to the quantitative determination of total and taurine-conjugated bile acids in bile without troublesome preparative steps.

Ursodeoxycholic acid protects hepatocytes against oxidative injury via induction of antioxidants.

The therapeutic efficacy of ursodeoxycholic acid (UDCA) has been widely demonstrated in various liver diseases, suggesting that UDCA might protect hepatocytes against common mechanisms of liver damage. A candidate for such protection is oxidative injury induced by reactive oxygen species. This study was designed to assess the effects of UDCA on oxidative injury and antioxidative systems in cultured rat hepatocytes. The viability of the hepatocytes dose-dependently decreased after hydrogen peroxide or cadmium administration. Pretreatment with UDCA significantly prevented this decrease in viability. The amounts of glutathione (GSH) and protein thiol increased significantly, but the activities of antioxidative enzymes such as superoxide dismutase, glutathione peroxidase and catalase were unchanged in UDCA-treated hepatocytes. The mRNA levels of gamma-glutamylcysteine synthetase and metallothionein (MT) were significantly higher in UDCA-treated hepatocytes than in controls. In conclusion, UDCA increased hepatocyte levels of GSH and thiol-containing proteins such as MT, thereby protecting hepatocytes against oxidative injury. Our results provide a new perspective on the hepatoprotective effect of UDCA.

A paucity of unusual trihydroxy bile acids in the urine of patients with severe liver diseases.

To clarify the relationship between the occurrence of unusual trihydroxy bile acids, namely hyocholic acid, ursocholic acid (UCA), and omega-muricholic acid (omega-MCA) in urine and liver disease severity, urinary bile acids were analyzed by gas-liquid chromatography in acute and late phases of acute hepatitis and before and after ursodeoxycholic acid (UDCA) loading in healthy adults and liver cirrhosis patients. In 11 patients with acute hepatitis, the occurrence rates and amounts of unusual trihydroxy bile acids were increased in the late (recovery) phase, as compared with those in the early phase. In 10 patients with severe acute hepatitis who had prothrombin times exceeding 16 seconds, these bile acids had completely disappeared from the urine in the early phase but reappeared in the late phase in those who had a good outcome, though never in a patient who died. After UDCA administration for a week, the amounts of unusual bile acids, especially UCA and omega-MCA, which are thought to be synthesized through 12 alpha- and 6 alpha-hydroxylations, respectively, from UDCA, were clearly increased in 10 healthy adults but only slightly changed in 10 patients with liver cirrhosis. In conclusion, hepatic hydroxylations of dihydroxy bile acids as a detoxification reaction were impaired in severe liver diseases, which may play a role in the intensification and perpetuation of hepatocellular injuries.

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.

Bile acids influence hepatic chemiluminescence in normal and oxidative-stressed rats.

The aim of the present study was to clarify whether bile acids influence chemiluminescence (CL) in the liver in vivo. Hepatic CL was determined on the surface of the liver of anaesthetized rats by using a photon counter. In normal rats, hepatic CL was significantly decreased 30 min after enteral administration of chenodeoxycholic acid (CDCA) or deoxycholic acid (DCA), but returned to its initial level 3 h later, after part of the CDCA administered was metabolized. Ursodeoxycholic acid (UDCA) and cholic acid had no effect on CL. In contrast, hepatic CL was markedly increased 30 min after CDCA or DCA administration in rats given either buthionine sulphoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase, or diethyldithiocarbamate (DDC), an inhibitor of both superoxide dismutase and glutathione peroxidase. Chenodeoxycholic acid further increased the CL of BSO- or DDC-treated rats during inhalation of oxygen via a tracheal cannula. Coadministration of UDCA eliminated the effects of CDCA on the hepatic CL of normal and BSO- or DDC-treated rats with or without oxygen inhalation. We conclude that cytotoxic bile acids, such as CDCA, increase CL in the antioxidants-depleted or oxidative-stressed liver in vivo, but that UDCA prevents CDCA from developing CL.

Ursodeoxycholic acid enhances glucocorticoid-induced tyrosine aminotransferase-gene expression in cultured rat hepatocytes.

Ursodeoxycholic acid (UDCA) is an effective treatment for immune-mediated liver diseases, suggesting that UDCA is functionally similar to glucocorticoids (GCs). We investigated the effects of UDCA on the enzyme activity and the mRNA levels of tyrosine aminotransferase (TAT), a hepatocyte-specific marker of GC action, in primary cultured rat hepatocytes. Addition of UDCA resulted in a significant increase in TAT activity in the presence of dexamethasone (DEX), compared with DEX alone, and this increase was completely suppressed by sphingosine, a protein kinase C (PKC) inhibitor, or actinomycin D, a transcriptional inhibitor. UDCA could not induce TAT activity in the absence of DEX. UDCA increased the TAT mRNA levels in the presence of DEX. In conclusion, UDCA enhances the GC-induced TAT-gene expression in hepatocytes, and UDCA-activated PKC may play a role in this upregulation.

Hepatic damage influences the decay of nitroxide radicals in mice--an in vivo ESR study.

To determine the role of the liver in the elimination of free radicals from the body, the clearance rate (K) of nitroxide radicals (Tempol) at the hepatic domain was compared with that at the pelvic domain of live mice, using L-band ESR spectroscopy. The reduction of Tempol in biopsy specimens (liver tissue and femoral muscle) and blood obtained from Tempol-treated mice was also monitored using X-band ESR spectroscopy. Results indicated that the reduction of nitroxide radicals was delayed in both the liver and peripheral tissues when the liver was damaged. The decrease in both blood supply and reductants in the damaged liver might be involved in delaying the reduction in the whole body, because the liver can reduce the radicals supplied via the blood from the peripheral tissues, and the reductants such as reduced, glutathione in the peripheral tissues are supplied from the liver.

Evidence of a direct action of taurine and calcium on biological membranes. A combined study of 31P-nuclear magnetic resonance and electron spin resonance.

To determine the actions of taurine and calcium on biological membranes, the effects of these compounds on the mobility of phospholipids of resealed and sonicated ghosts of human erythrocytes were investigated, using 31P-NMR spectroscopy. In addition, the effects of taurine and calcium on lipid fluidity were investigated by ESR spectroscopy, using a spin-labeling method with 5-doxyl stearic acid. The mobility of the membranes decreased following treatment with 10 mM taurine, but coadministration of 2.5 mM calcium blocked this effect. The fluidity of the membranes was not changed following treatment with 10 mM taurine, but decreased following coadministration of 2.5 mM calcium. These actions of taurine and calcium on the dynamics of biological membranes might explain, in part, the observation that most of the pharmacological effects of taurine on mammalian organs occur in the presence of calcium ions.

Taurine in the liver. The function of taurine conjugated with bile acids.

1. Taurine had cytoprotective effects on hepatocytes under hyperoxic or hypoxic condition in the presence of Ca2+. 2. Bile acids had toxic effects on hepatocytes; there was no significant difference in the toxicity between free bile acids and taurine-conjugated bile acids. 3. The mobility of the membranes, examined by 31P-NMR, decreased after treatment with taurine or free bile acids, but increased after treatment with taurine-conjugated bile acids. 4. The fluidity of the membranes, measured by ESR, did not change after treatment with taurine, but decreased after treatment with free bile acids and increased after treatment with taurine-conjugated bile acids. In conclusion, because most bile acids are conjugated with taurine in the liver after administration of taurine, the influence of taurine-conjugated bile acids has to be taken into consideration in estimating the functions of taurine in the liver.

[A clinical trial of SM-4300 against severe infections in surgery].

SM-4300, a newly developed human immunoglobulin for intravenous use, has been evaluated clinically in combination with the antibiotics in the patients with severe bacterial, fungal or viral infections which are resistant to antibiotic therapy in the surgery. Total number of 26 patients affected with various severe infections were treated with SM-4300. Clinical effects of SM-4300 were good in 10 cases, fair in 7 cases, poor in 7 cases and unknown in 2 cases. The efficacy rate was 41.7%. Severe infections in these 26 patients consisted of 14 cases of peritonitis, 5 cases of intrathoracic infections, 4 cases of sepsis suspected and 3 cases others. Twenty-three patients had underlying diseases, in which 15 cases were patients with cancer. Clinically isolated organisms were obtained from 12 cases, of 26 strains were evaluated for bacteriological effects which were eradicated in 10 strains, decreased in 3 and persisted in 13 strains, the eradication rate being 38.5%. The subjective and objective clinical side effects were noted.