Shigekazu Takemura

Hepatitis C virus infection as a likely etiology of intrahepatic cholangiocarcinoma

Although hepatitis C virus (HCV)‐related cirrhosis has been suggested as a risk factor for intrahepatic cholangiocarcinoma (ICC), few sizeable studies have tested this hypothesis. We investigated ICC risk factors, with special reference to HCV infection. We conducted a hospital‐based case‐control study including 50 ICC patients and 205 other surgical patients without primary liver cancer. HCV seropositivity was detected in 36% of ICC patients and 3% of controls. By univariate analysis, the odds ratio (OR) for association of anti‐HCV antibodies with development was 16.87 (95% confidence interval (CI), 5.69 to 50.00). History of blood transfusion or diabetes mellitus, elevated serum total bilirubin, elevated aspartate aminotransferase and alanine aminotrans‐ferase, decreased serum albumin and decreased platelet count were identified as other possible ICC risk factors. By multivariate analysis, anti‐HCV antibodies (adjusted OR, 6.02; 95% Cl, 1.51 to 24.1), elevated alanine aminotransferase, decreased serum albumin, and decreased platelet count were found to be independent risk factors for ICC development. As liver status worsened, the adjusted OR for ICC tended to increase. HCV infection is a likely etiology of ICC in Japan.

Incidence and management of Bile leakage after hepatic resection for malignant hepatic tumors

BACKGROUND Bile leakage is one of the frequent and disturbing complications of hepatic resection. STUDY DESIGN Clinical records of the 363 patients who underwent hepatic resections without biliary reconstruction for hepatic cancers between January 1994 and June 2001 were reviewed. Postoperative bile leakage was defined as continuous drainage with a bilirubin concentration of 20 mg/dL or 1,500 mg/d lasting 2 days. Leakage that continued longer than 2 weeks or that required surgical intervention was defined as uncontrollable. Differences in incidence and frequency of uncontrollable leakage for the different types of hepatic resection, tumors, and underlying liver disease were investigated. Outcomes after treatment for uncontrollable bile leakage were also reviewed. RESULTS Postoperative bile leakage occurred in 26 of 363 patients (7.2%). Although the incidence in patients with cholangiocellular carcinoma (3/9 [33%]) was higher (p = 0.03) than in patients with hepatocellular carcinoma, rates of occurrence were similar among the different types of hepatic resection and underlying liver disease. Eight of the 26 patients (31%) had uncontrollable leakage. Two patients required reoperation to control leakage; one of these developed hepatic failure and died 2 months after surgery. Four patients underwent endoscopic nasobiliary drainage 21 to 34 days after hepatectomy, and the leakage resolved within 3 to 21 days. Fibrin glue sealing was effective in two patients whose leaking bile ducts were not connected to the common bile duct. CONCLUSIONS Although meticulous surgical technique can minimize the risk of postoperative bile leakage, some instances of leakage are unavoidable. Nonsurgical treatments, such as nasobiliary drainage or fibrin glue sealing, are preferable to reoperation.

Hepatic cytochrome P450 is directly inactivated by nitric oxide, not by inflammatory cytokines, in the early phase of endotoxemia

BACKGROUND/AIMS Although the activity of the liver in metabolizing and eliminating various drugs decreases in endotoxemia, the mechanism remains to be elucidated. The generation of nitric oxide by the inducible type of nitric oxide synthase increases in endotoxemia. Nitric oxide readily reacts with heme proteins such as cytochrome P450 that metabolize various compounds, including steroids and eicosanoids. The purpose of this study was to determine the effect of nitric oxide on the function of hepatic cytochrome P450 in endotoxemic rats. METHODS To determine the dynamic aspects of nitric oxide metabolism, hepatic levels of the inducible type of nitric oxide synthase and heme-iron nitrosyl complexes, and plasma levels of nitrite and nitrate were determined in rats before and after intravenous administration of lipopolysaccharide. Changes in the levels of P450 isoforms and testosterone hydroxylation activity in hepatic microsomes were also determined. To evaluate in vivo CYP3A2 activity, midazolam sleep time was measured. RESULTS When lipopolysaccharide increased the hepatic inducible type of nitric oxide synthase and plasma levels of nitric oxide metabolites, the intensity of low-spin signal of electron spin resonance responsible for the ferric form of P450 decreased with a concomitant increase in heme-iron nitrosyl complexes in the liver. Lipopolysaccharide-related nitric oxide generation is followed by an early decrease in the levels of cytochrome P450 and of testosterone hydroxylation activity in liver microsomes. Midazolam sleep time was prolonged by lipopolysaccharide. All these early changes were prevented by the inhibitor of nitric oxide synthase, N(G)-iminoethyl-L-ornithine. Moreover, lipopolysaccharide suppressed the gene expression of CYP2C11 and CYP3A2. Decreases in levels of cytochrome P450 and their mRNAs were more pronounced at 24 h after LPS administration, but apparently they are NO-independent. CONCLUSIONS These results suggest that lipopolysaccharide-induced modulation of cytochrome P450 may occur via the interplay of two different mechanisms and that, especially in the early phase, nitric oxide-dependent inhibition is more important.

Effects of lamivudine on outcome after liver resection for hepatocellular carcinoma in patients with active replication of hepatitis B virus

Aim:  Patients with high serum hepatitis B virus (HBV) DNA concentrations are at high risk of tumor recurrence after liver resection for HBV‐related hepatocellular carcinoma (HCC).

Calorie Restriction Improves Cardiovascular Risk Factors via Reduction of Mitochondrial Reactive Oxygen Species in Type II Diabetic Rats

Uncoupling protein 2 (UCP2) is an important regulator of intracellular reactive oxygen species (ROS) production. We determined the effects of calorie restriction (CR) on the dynamic aspects of mitochondrial ROS production, UCP2, and the nitric oxide (NO)-cGMP pathway in the cardiovascular tissues of type II diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Some rats were on restricted diets (30% reduction from free intake) from age 29 to 42 weeks. Blood glucose, hemoglobin A1c, plasma levels of free fatty acid, triacylglycerol, and plasminogen activator inhibitor-1 in OLETF rats were significantly higher than those in nondiabetic control [Long-Evans Tokushima Otsuka (LETO)] rats at 29 weeks. Mitochondrial ROS production and UCP2 expression significantly increased in the heart and aorta of OLETF rats compared with those in LETO rats. A fibrogenic growth factor, transforming growth factor (TGF)-β1 in the coronary vessels, endothelial nitric-oxide synthase, and aortic nitrotyrosine were increased in OLETF rats at 42 weeks. In contrast, an index of the NO-cGMP pathway, phosphorylated vasodilator-stimulated phosphoprotein, and superoxide dismutase activity in the aorta were significantly diminished. The relationship between UCP2 and ROS production in the cardiovascular function of diabetic rats being fed a calorie-restricted diet is unknown. These abnormalities in OLETF rats were reversed to normal levels by CR. CR significantly improved the NO-cGMP pathway via normalizing ROS generation in OLETF rats. A decrease in UCP2 expression by CR may be a compensatory mechanism to counteract decreased intracellular oxidative stress. The data suggest that CR may prevent cardiovascular tissues from oxidative stress provoked by diabetes mellitus.

Risk factors for postoperative delirium after liver resection for hepatocellular carcinoma.

We investigated risk factors for delirium in 100 patients who underwent liver resection for hepatocellular carcinoma. Postoperative delirium developed in 17 (17%). Univariate analysis revealed that advanced age (especially = 70 years old), a history of smoking, a decreased serum albumin concentration (especially < 3.8 g/dl), advanced cancer stage (II–IV), major hepatectomy, prolonged operating time, and large intraoperative blood loss were possible risk factors for postoperative delirium. When patients’ preoperative condition and laboratory test results were subjected to multivariate analysis, only advanced age [odds ratio (OR) 1.201; confidence interval (CI) 1.063–1.357] and a decreased serum albumin concentration (OR 0.151; CI 0.025–0.900) were independent risk factors for the delirium. The percentages of patients with high aspartate and alanine aminotransferase activities, a high indocyanine green retention rate at 15 minutes, a low platelet count, and advanced cancer stage (II–IV) were higher in patients with a low (< 3.8 g/dl), rather than high (= 3.8 g/dl) serum albumin concentration. These findings indicate that multiple factors, including advanced age, impaired liver function, and advanced cancer stage, affect the development of postoperative delirium after liver resection for hepatocellular carcinoma.

Isoforms of cytochrome P450 on organic nitrate-derived nitric oxide release in human heart vessels

Glutathione S‐transferases and the cytochrome P450 system have been proposed for the vascular biotransformation systems in the metabolic activation of organic nitrates. The present study was designed to elucidate the role of human cytochrome P450 isoforms on nitric oxide formation from organic nitrates using lymphoblast microsomes transfected with human CYP isoforms cDNA. CYP3A4‐transfected microsomes had the most effective potential of nitric oxide formation from isosorbide dinitrate. Anti‐CYP3A2 antibody (which cross‐reacts with CYP3A4) or ketoconazole (an inhibitor of the CYP3A superfamily) inhibited nitric oxide formation from isosorbide dinitrate in rat heart microsomes. Immunohistochemistry of human heart also showed intense bindings of CYP3A4 antibody in the endothelium of the endocardium and coronary vessels. These results suggest that the CYP3A4‐NADPH‐cytochrome P450 reductase system specifically participates in nitric oxide formation from isosorbide dinitrate.

Iron restriction improves type 2 diabetes mellitus in Otsuka Long-Evans Tokushima fatty rats

Accumulating evidence suggests that alcohol, hepatitis C virus infection, steatosis with obesity, and insulin resistance are accompanied by iron overload states. Phlebotomy and oral iron chelators are effective treatments for these conditions and for hemochromatosis. However, the mechanisms by which iron depletion improves clinical factors remain unclear. We examined the effect of iron depletion in a model of type 2 diabetes, Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Age-matched Long-Evans Tokushima Otsuka (LETO) rats were used as controls for all experiments. Iron restriction was performed by eliminating iron in the diet from 15 wk of age or by phlebotomy. Phlebotomy was commenced at 29 wk of age by removing 4 and 3 ml of blood from the tail vein every week in OLETF and LETO rats, respectively. Rats were euthanized at 43 wk of age, and detailed analyses were performed. The plasma ferritin concentration was markedly higher in OLETF rats and decreased in iron-deficient (ID) diet and phlebotomy rats. Hemoglobin A(1c) (Hb A(1c)) was decreased significantly in OLETF rats fed the ID diet and in the phlebotomy group. Increased levels of triglycerides, glucose, free fatty acids, and total cholesterol were found in ID OLETF rats. Plasma, liver, and pancreas lipid peroxidation and hepatic superoxide production decreased in both groups. Pancreatic fibrosis and insulin levels improved in both groups of OLETF rats. Pancreatic levels of peroxisome proliferator-activated receptor-beta/delta (PPARbeta/delta) ligands and hypoxia-inducible factor (HIF)-1alpha were decreased significantly in OLETF rats. These factors were normalized in both rats fed ID and phlebotomy groups of OLETF rats. In conclusion, iron depletion improved diabetic complications by inhibition of oxidative stress and TGFbeta signal pathways and the maintenance of pancreatic PPARbeta/delta and HIF-1alpha pathways.

Outcomes of hepatic resection for hepatolithiasis

BACKGROUND Hepatic resection is main approach to treatment of hepatolithiasis, but the long-term follow-up results of hepatic resection for hepatolithiasis are rarely reported. METHODS A retrospective study was conducted of 87 patients with hepatolithiasis who underwent hepatic resection. RESULTS The final stone clearance rates were 95%. There was a significant difference in the incidence of recurrent stones between patients with and without remaining biliary strictures. On multivariate analysis, the presence of residual or recurrent stones was an independent risk factor associated with recurrent cholangitis. The 10-year overall survival rate was 80.3%. On multivariate analysis, the development of cholangiocarcinoma was an independent predictor of survival in patients who underwent hepatic resection for hepatolithiasis. CONCLUSIONS The long-term outcomes after hepatic resection were satisfactory in patients whose intrahepatic stones and strictures were completely removed. Cholangiocarcinoma associated with hepatolithiasis was an independent prognostic factor in patients with hepatolithiasis who underwent hepatic resection.

Simultaneous measurement of F2-isoprostane, hydroxyoctadecadienoic acid, hydroxyeicosatetraenoic acid, and hydroxycholesterols from physiological samples

Oxidative stress induced by various oxidants in a random and destructive manner is considered to play an important role in the pathophysiology of a number of human disorders and diseases. It is important to assess the oxidative injury in vivo accurately and inclusively. We have developed an improved method for the measurement of in vivo lipid peroxidation by using a single plasma or liver sample, where total 8-iso-prostaglandin F(2alpha) (t8-iso-PGF(2alpha)), total hydroxyoctadecadienoic acids (tHODEs), total hydroxyeicosatetraenoic acids (tHETEs), and total 7-hydroxycholesterol (t7-OHCh), as well as their parent molecules linoleic acid (t18:2) and cholesterol (tCh), are determined by LC-MS/MS (for t8-iso-PGF(2alpha), tHODE, and tHETE) and GC-MS (for t7-OHCh, t18:2, and tCh) analyses. The plasma and liver samples from human are reduced with sodium borohydride and saponified by potassium hydroxide after the addition of heavy isotopic standards. After extraction by chloroform/ethyl acetate (CHCl(3)/CH(3)COOC(2)H(5), 4:1), they are analyzed without any further sample processing. We applied this method to hepatitis C virus-infected patients (n=8, plasma and liver), hepatitis B virus-infected patients (n=2, plasma and liver), and controls (virus free, n=8, plasma and liver). It was found that in the plasma of patients and controls, the concentrations of oxidized lipids decreased in the following order: tHODE tHETE t7-OHCh >> t8-iso-PGF(2alpha). As expected, the virus clearly increased these concentrations. The ratio of stereoisomers of HODE [(E,E)-HODE/(E,Z)-HODE], which reflects the antioxidant capacity in vivo, can also be determined by this method. A significant decrease in the stereoisomer ratio for the liver of patients was observed, indicating liver dysfunction. t8-iso-PGF(2alpha), tHODE, tHETE, and t7-OHCh are measured satisfactorily and inclusively by the current method from biological fluids and tissues, and they can account for a large portion of oxidized lipids in vivo.