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Oltipraz, a synthetic dithiolethione, has chemopreventive effect through nuclear factor erythroid 2-related factor 2 (Nrf2) activation. Nrf2 is known to be involved in the development of experimental steatohepatitis in rodents. In this study, to evaluate the effect of oltipraz on lipid and bile acid metabolism, wild-type and Nrf2-null mice were fed the standard diet (containing 4% soybean oil) with or without oltipraz. Based on these results, we examined the effect of oltipraz on the experimental steatohepatitis in high-fat diet (containing 4% soybean oil and 20% lard) fed Nrf2-null mice. Oltipraz induced hepatic mRNA expression of peroxisome proliferator-activated receptor α, carnitine palmityl transferase 1, and bile salt export pump by Nrf2 independent mechanisms. In Nrf2-null mice fed a high-fat diet for 12 weeks, moderate to severe inflammation and fibrosis were observed. Oral administration of oltipraz suppressed the degree of inflammation and fibrosis in Nrf2-null mouse liver fed a high-fat diet. These histopathological findings approximately corresponded to the data of mRNA expression of tumor necrosis factor α, monocyte chemoattractant protein-1, Timp-1, and collagen type 1α1. These results indicated that oltipraz administration ameliorated liver injury by Nrf2 independent manner in a model of steatohepatitis generated by Nrf2-null mice with high-fat diet.

The purpose of this study was to investigate the immunological and physical characteristics of IgM-λ type M-protein from patients who were measured low in the turbidimetric immunoassay (TIA) IgM assay without error codes for high concentration to determine the cause of the false low levels and to clarify the mechanism of their occurrence.Materials were IgM patient samples and 8 serum samples from other IgM M-protein patients as controls. Patient samples were assayed by the TIA method, in which five manufacturers and six models (two reagent manufacturers) share the principle, and the BN ProSpec method (nephelometric method), which has a different principle. Dilution linearity tests, IgG addition experiments, isoelectric point electrophoresis, and hydrophobic chromatography were performed on patients and subjects. In addition, the binding capacity of γ-globulin by BIACORE was also examined.The reaction curve of the patient IgM curved downward when the concentration of IgM exceeded 20 g/L, and no error code was obtained. In the measurement by the TIA method of five manufacturers and six models, patient IgM was measured at a false low level with no error code obtained in undiluted dilution by any of the instruments and reagents, but could be measured without any problem by the nephelometric method. In addition, in the patient IgG addition experiment, only patient IgM showed a false low level under high IgG concentration. Furthermore, the binding capacity of patient IgM to γ-globulin (IgG) by BIACORE was significantly higher than that of the control IgM-type M protein.Patient IgM has an affinity (binding capacity) for IgG and forms an IgM-IgG complex under conditions of high IgG concentration. It was speculated that this complex inhibited the reaction with the anti-IgM antibody and the absorbance of the second reaction did not increase, suggesting a false low.

Introduction The hematology analyzer, Celltac G (Nihon Kohden), designed to improve platelet count (Plt) accuracy, is equipped with new sheath flow control technology. Clinical evaluation of the Celltac G was assessed by comparability with XN-9000 (Sysmex Corporation) and CELL-DYN Sapphire (Abbott Diagnostics). The accuracy of all three analyzers, which use different measuring principles, was compared with the immunoplatelet reference method (FCM-Ref). Methods Repeatability and within-laboratory imprecision were assessed using 10 clinical fresh whole blood samples and three control materials with differing levels. Carryover was evaluated using 6 clinical fresh whole blood samples. For method comparison between the three analyzers, 388 samples were used. Plt accuracy among the three analyzers was evaluated using 54 blood samples, including 42 samples with a platelet count less than 50x109 /L. The International Council for Standardization in Haematology method for Plt was used as the FCM-Ref. Results The Celltac G showed sufficient performance with regard to imprecision, carryover, and comparability. The Analytical Measurement Interval (AMI) and linearity for all parameters of Plt were validated within 4.6 to 809.1 (×109 /L). All hematology analyzers showed some disagreement in Plt when compared with the immunoplatelet reference method. Conclusion The Celltac G hematology analyzer is suitable for clinical use. Platelet count evaluation of the three analyzers suggests the need to determine a reportable measurement interval (RMI) in the clinical laboratory for adequate reporting of a Plt from multiple different values.

Maternal high-fat diet (HFD) consumption during pregnancy and lactation affects metabolic outcomes and lipid metabolism of offspring in later life in a gender-specific manner. However, it is not known whether maternal HFD alters bile acid metabolism in adult mice offspring. The purpose of this study was to elucidate the relationship between maternal HFD-induced metabolic diseases and bile acid metabolism in male and female adult mice offspring. Female mice were fed either standard chow (C) or HFD (H) for 10 weeks pre-pregnancy until lactation. After weaning, offspring were fed a chow diet until 11 weeks of age, then challenged with either C or H diet for 4 weeks, and divided into eight groups in accordance with mother’s and offspring’s diets: male(M) CC, MHC, MCH, MHH, female(F) CC, FHC, FCH, and FHH. MHH showed greater weight gain compared to FHH. Liver weight was higher in MHH than in FHH. Serum total cholesterol levels were higher in MHH than in MHC, and tended to be higher in MHH than in FHH. Serum glucose levels were higher in MHH than in MHC. Hepatic triglyceride levels were higher in MHH than in MHC. Hepatic mRNA expression of bile acid uptake transporters Oatp1a1 and Oatp1b2 was increased in MHH, compared to MCH. Hepatic mRNA expression of HMGCoAR, Cyp7a1, Sult2a1, and Oatp1a4 was increased in FHH, compared to FCH. In conclusion, maternal HFD consumption may promote bile acid synthesis, sulfation and excretion in female offspring fed a HFD, which may confer resistance to HFD-induced metabolic phenotypes.

Ezetimibe and pemafibrate are lipid-lowering drugs and promote reverse cholesterol transport. However, it is unknown whether cholesterol is mainly excreted by hepatobiliary excretion or by non-biliary transintestinal cholesterol efflux (TICE). We evaluated the effects of ezetimibe and pemafibrate on hepatic and intestinal cholesterol transporter regulation in Sham-operated rats, and examined the effects of these drugs on TICE in bile duct-ligated rats. Seven-week-old male Sprague-Dawley rats were treated as follows for two weeks: 1) Sham, Sham operation; 2) BDL, bile duct ligation; 3) E-Sham, Sham + ezetimibe; 4) E-BDL, BDL + ezetimibe; 5) P-Sham, Sham + pemafibrate; and 6) P-BDL, BDL + pemafibrate. Blood, liver, jejunum, and feces were collected 72 h post-surgery. Hepatic cholesterol levels were decreased in P-Sham and E-Sham, and were lower in E-BDL and P-BDL than in BDL. Fecal cholesterol levels increased in E-Sham and P-Sham compared with Sham, and were higher in E-BDL and P-BDL than in BDL. In liver, Abcg5 mRNA showed induction in E-Sham, Abcg5 and Abca1 mRNA were induced in P-Sham, Abcg5 mRNA was reduced in E-BDL, and Abca1 mRNA was increased in P-BDL. In jejunum, Abcg5 mRNA was induced in E-Sham. Abcg8 mRNA was induced in E-Sham and P-Sham. NPC1L1 mRNA showed reduced expression in P-Sham and P-BDL. SR-B1 mRNA was reduced in P-Sham, and the expression decreased in P-BDL. LDL receptor mRNA was induced in BDL and P-BDL. Ezetimibe and pemafibrate may promote TICE by increasing Abcg5/g8, while pemafibrate may inhibit intestinal cholesterol absorption by decreasing SR-B1 and NPC1L1.

Background We experienced a patient with multiple myeloma whose urine contained a considerable amount of Bence Jones protein (BJP), which demonstrated poor thermal reactivity in heat coagulation test. The mechanism for this phenomenon was assessed. Methods Immunoelectrophoretic analyses reveal that a band corresponding to BJP in the urine had 2,600 Dalton by reduction after glycosidase treatment, but not after sialidase treatment. In addition, the glycosidase-treated urine tested positive in heat coagulation test. Conclusions Glycosylation of the immunoglobulin light chain, which has rarely been seen, is the cause of the unexpected behavior of this patent's BJP in heat coagulation tests.

Streptococcus pneumoniae is one of the most common bacteria causing community-acquired pneumonia and meningitis. The use of 7-valent pneumococcal conjugate vaccine (PCV7) has reduced the incidence of pneumococcal disease while changing pneumococcal population through herd immunity and non-vaccine pneumococci replacement. This study investigated molecular epidemiologic characteristics of pneumococcal strains in the Kinki region of Japan from 2008 to 2013. A total of 159 invasive pneumococcal isolates were characterized by serotyping, antibiotic susceptibility testing, PCR analysis of penicillin-binding protein genes, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). In adult populations, pediatric PCV7 introduction decreased isolates expressing PCV7 serotypes via herd immunity and increased isolates expressing non-PCV7 serotypes. The rate of penicillin resistance and isolates with alterations in all three pbp genes was higher in PCV7 type isolates than in non-PCV7 type isolates. In MLST analysis, all of serotype 19F isolates were of the same sequence type, ST236, which is the antimicrobial-resistant clone Taiwan19F-14, and the majority of serotypes 23F and 19A isolates were of ST1437 and ST3111 respectively, which are the predominant clones of antimicrobial-resistant pneumococci in Japan. In PFGE profiles, serotype 6B-ST2224, serotype 19F-ST236, serotype 19A-ST3111, and serotype 23F-ST1437 formed six separate clusters composed of genetically identical strains, and genetically identical serotype 22F-ST433 formed two different clusters between the pre- and post-PCV7 period. The results of molecular analysis suggest the spread and persistence of these identical antimicrobial resistant clones in the Kinki region and genetic changes of epidemic clone serotype 22F-ST433 before and after pediatric PCV7 introduction.

Besides diet therapy, hypolipidemic pharmacological therapy may be a crucial component of nonalcoholic fatty liver disease (NAFLD) treatment. Ezetimibe may be a promising drug for treatment of NAFLD. N-3 polyunsaturated fatty acids, which are abundant in fish oil, reduces serum and hepatic cholesterol and triglycerides in rodents. The aim of this study was to examine the combined effects of dietary fish oil and ezetimibe on lipid metabolism in rats. Seven-week old male SD rats were allocated to four different diets containing 1) 10% soybean oil (C), 2) 10% fish oil (F), 3) 10% soybean oil + 0.005% ezetimibe (E), and 4) 10% fish oil + 0.005% ezetimibe (F+E) for 4 weeks, when the liver, jejunum, blood, and fecal samples were collected. Compared to the C group, the F+E-diet decreased hepatic triglycerides and cholesterol 84% and 86%, but it did not increase fecal cholesterol. In liver, the expression of lipogenic enzymes were decreased in F+E diet, whereas β-oxidation related genes were not increased. Abcg5/g8 mRNA expression were increased 1380%/442% when ezetimibe was added to the F-diet. These gene expression changes are related to the decrease in hepatic lipids. In jejunum, Abcg5/g8 mRNA were increased 244%/841% when ezetimibe was added to the F-diet. Hepatic induction of Abcg5/8 rather than intestinal induction correlates with the marked decrease in liver cholesterol when ezetimibe was added to the F-diet. These data suggest that fish oil diet and ezetimibe in combination may be beneficial therapy for NAFLD by increasing hepatic Abcg5/g8 and decreasing lipogenic genes. SIGNIFICANCE STATEMENT There is currently no single treatment for NAFLD. Thus, lifestyle modifications including dietary regulation and physical activity are also important options. In this study, ezetimibe, a cholesterol absorption inhibitor, was evaluated for the treatment of liver steatosis in rats fed on the different diets. We found that ezetimibe and fish oil in combination markedly improved fatty liver by increasing cholesterol efflux transporters. The combination therapy of fish oil agents and ezetimibe may be effective for NAFLD.

6-Methylsulfinylhexyl isothiocyanate (6-MSITC) is a bioactive ingredient present in Japanese horseradish (wasabi) and has been shown to be an Nrf2 activator. To determine whether 6-MSITC ameliorates hepatic steatosis and iron accumulation, wild-type and Nrf2-null mice were fed the following diets for 12 weeks: 1) control diet, 2) high-fat diet (HFD), 3) HFD plus 6-MSITC (10mg/Kg/day ip), 4) HFD for 6 weeks followed by an iron-supplemented HFD for 6 weeks (HFD/Iron), 5) HFD/Iron plus 6-MSITC. The HFD increased hepatic triglycerides in both genotypes and 6-MSITC suppressed increased hepatic triglycerides in wild-type mice but did not reduce these triglycerides in Nrf2-null mice. Compared to the HFD and HFD/Iron groups, respectively, 6-MSITC did not block hepatic iron accumulation in either genotype. This article is protected by copyright. All rights reserved.

Maternal high-fat diet (HFD) consumption during pregnancy and lactation affects metabolic outcomes and lipid metabolism of offspring in later life in a gender-specific manner. However, it is not known whether maternal HFD alters bile acid metabolism in adult mice offspring. The purpose of this study was to elucidate the relationship between maternal HFDinduced metabolic diseases and bile acid metabolism in male and female adult mice offspring. Female mice were fed either standard chow (C) or HFD (H) for 10 weeks pre-pregnancy until lactation. After weaning, offspring were fed a chow diet until 11 weeks of age, then challenged with either C or H diet for 4 weeks, and divided into eight groups in accordance with mother's and offspring's diets: male(M) CC, MHC, MCH, MHH, female(F) CC, FHC, FCH, and FHH. MHH showed greater weight gain compared to FHH. Liver weight was higher in MHH than in FHH. Serum total cholesterol levels were higher in MHH than in MHC, and tended to be higher in MHH than in FHH. Serum glucose levels were higher in MHH than in MHC. Hepatic triglyceride levels were higher in MHH than in MHC. Hepatic mRNA expression of bile acid uptake transporters Oatp1a1 and Oatp1b2 was increased in MHH, compared to MCH. Hepatic mRNA expression of HMGCoAR, Cyp7a1, Sult2a1, and Oatp1a4 was increased in FHH, compared to FCH. In conclusion, maternal HFD consumption may promote bile acid synthesis, sulfation and excretion in female offspring fed a HFD, which may confer resistance to HFDinduced metabolic phenotypes.

Objective The menstrual cycle-related changes in clinical laboratory values were analysed by use of data obtained in the Asian multicentre study aimed at derivation of common reference intervals for 85 major clinical laboratory tests. Methods Among 1876 healthy female volunteers, 893 had regular menstruation. They were classified into five groups according to dates between sample collection and the start of the last menstrual cycle: early follicular phase (1–6 days), late follicular phase (7–12 days), ovulatory phase (13–16 days), early luteal phase (17–22 days), and late luteal phase (23–31 days). Multiple linear regression analysis was performed to evaluate the menstrual cycle-related changes in test results. The magnitude was expressed as a standard deviation ratio of between-phase standard deviation to between-individual standard deviation based on nested ANOVA. Results Aside from obvious changes for four sex hormones (oestradiol, progesterone, follicle-stimulating hormone, and luteinizing hormone), we observed statistically significant menstrual cycle-related changes in the following tests (standard deviation ratio >0.15): Na, Cl, creatine kinase, C-reactive protein, serum amyloid A, carbohydrate antigen 125, and parathyroid hormone were higher during the early follicular phase, while insulin, total cholesterol, and white blood cell were higher during the luteal phase. Significant associations of those test items with the four sex hormones were revealed. Conclusions The menstrual cycle-related changes in laboratory test results were revealed in some commonly tested items other than sex hormones. The findings are of interest in understanding female physiology in relation to hormonal changes, but the magnitude of changes is rather small and not very relevant in interpreting test results.

The 7-valent pneumococcal conjugate vaccine (PCV7) was introduced among children in Japan in 2010. There are no long-term multicenter surveillance studies of antimicrobial resistance in S. pneumoniae before and after the introduction of PCV7. Therefore, we examined chronological trends in antimicrobial resistance among 4534 strains of S. pneumoniae isolated from both children and adults in the Kinki region of Japan during 2001–2015. High-level penicillin and third-generation cephalosporin resistance in S. pneumoniae increased among both children and adults during the period before the introduction of PCV7 (2001–2010). Besides penicillin and cephalosporin, pneumococcal carbapenem and macrolide resistance increased among children. The rate of resistance to these antibiotics was higher among children than among adults. The introduction of PCV7 decreased the rate of non-susceptibility to β-lactam antibiotics and the rate of multidrug resistant S. pneumoniae among children, but not among adults.

Nuclear factor-E2-related factor 2 (Nrf2) is a regulator of lipid metabolism as well as various cytoprotective enzymes and may be involved in the pathogenesis of non-alcoholic fatty liver disease. Although, bile acids affect lipid metabolism, the role of Nrf2 in bile acid metabolism remains unclear. In this study, it was tested how Nrf2 modulates lipid and bile acid homeostasis in liver in response to changes of cholesterol absorption under high-fat diet using Nrf2-null mice. Eight-week-old male wild-type and Nrf2-null mice (n = 6/group) were divided into three groups fed the following diets: 1) control diet containing 4% soybean oil and 16% lard, 2) control diet plus ezetimibe, 3) control diet plus cholesterol. Blood and livers were removed after 4 weeks feeding. High cholesterol diet increased hepatic expression of liver X receptor α target genes related to fatty acid metabolism (FAS, ACC1, SREBP-1c, SCD-1c and CD36), cholesterol transport (Abcg5/abcg8) and bile acid synthesis (Cyp7a1) in wild type mice. However, these genes were not induced in Nrf2-null mice. These findings suggest that Nrf2 has a relation to liver X receptor α and controls the regulation of gene expressions related to lipid and bile acid metabolism.

Accumulating evidence suggests that ezetimibe may be a promising agent for treatment of nonalcoholic fatty liver disease and steatohepatitis (NAFLD/NASH). Phlebotomy and dietary iron restriction reduce serum transaminase in NAFLD/NASH patients. Recent studies have shown that a mutual effect exists between lipid metabolism and iron metabolism. Accordingly, we examined the effect of ezetimibe on iron metabolism in mice fed a high-fat diet with or without iron. We fed C57BL/6 mice the following diets for 12 weeks. Experiment 1 comprised [1] a control diet (C), [2] C plus ezetimibe (0.3 mg/day; 4 weeks) (CE), [3] a high-fat diet (H), and [4] H plus ezetimibe (HE). Experiment 2 comprised [1] C containing carbonyl iron (average; 22.4 mg/day; 6 weeks) (CI), [2] CI plus ezetimibe (CIE), [3] H containing carbonyl iron (HI), and [4] HI plus ezetimibe (HIE). Blood, livers, and duodenum were removed after 12 weeks. In experiment 1, the hepatic iron levels were higher in HE than H, whereas there was no difference between C and CE. Hepatic mRNA expression of transferrin receptor 1 and 2, ferritins, and hepcidin were increased more in CE than C, and more in HE than H. In the duodenum, divalent metal transporter 1, ferritin H, and hephaestin mRNA levels were increased in CE compared with C. In experiment 2, hepatic iron concentrations were higher in HIE than HI. Hepatic mRNA expression of ferritin L and hepcidin were increased in HIE compared with HI. In duodenum, ferritin L mRNA was increased in HIE compared with CIE. Ezetimibe induced hepatic iron uptake transporter expression in mice fed a high-fat diet, causing increased hepatic iron concentrations.

Background We encountered a rare case of Waldenstrom macroglobulinemia with temporary appearance of 7S IgM half molecule and with monoclonal proteins binding to agarose gel. Methods The patient's serum and urine were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The N-terminal amino acid sequences of the IgM with abnormal mass (68 kDa) were determined and compared with those of known immunoglobulin. Results The 68 kDa IgM consisted of a defective μ chain (36 kDa) and an intact κ chain. N-terminal amino acid sequence analysis demonstrated that the defective μ chain had the variable region of IgM. The agarose gel-binding ability of the IgM-κ M-protein was lost after reduction or alkaline treatment of serum. Conclusions The 7S half molecule IgM in the present case may miss a large part of the constant region of the µ chain.

Background and Aim: Hepatic excessive iron may play a role in the pathogenesis of non-alcoholic steatohepatitis (NASH). Nrf2 is a master regulator of antioxidative responses. However, the role of Nrf2 in lipid and iron homeostasis remains unclear. Accordingly, it was examined how Nrf2 regulates lipid-related and iron-regulatory genes after feeding a high-fat diet (HFD) with iron. Methods: Wild-type and Nrf2-null mice were fed the following diets: (i) control diet (4% soybean oil) for 12 weeks, (ii) control diet for 8 weeks followed by control diet containing 0.5% carbonyl iron for 4 weeks, (iii) HFD (4% soybean oil and 16% lard) for 12 weeks, (iv) HFD for 8 weeks followed by HFD containing 0.5% carbonyl iron for 4 weeks. Blood and livers were removed after 12 weeks. Results: Nrf2-null control mice exhibited a tendency towards higher hepatic triglycerides compared to wild-type control mice. Hepatic malondialdehyde was higher and hepatic iron levels tended to be higher in Nrf2-null mice than wild-type counterparts while on a HFD. The HFD with iron synergistically induced mRNA expression of Pparα targets, including Acox and Cpt1 in wild-type mice, yet the induction was diminished in Nrf2-null mice. Hepatic hepcidin and ferroportin 1 mRNA expression were increased in wild-type mice after feeding a HFD with iron, but were unchanged in any group of Nrf2-null mice. Conclusions: Nrf2 deletion dysregulates hepatic mRNA expression of β-oxidation enzymes and iron-related genes, possibly causing a trend for increased hepatic triglyceride and iron concentrations. Nrf2 may have roles in the progression of NASH.

Aim: Fish oil rich in n-3 polyunsaturated fatty acids is known to affect hepatic lipid metabolism. Several studies have demonstrated that fish oil may affect the bile acid metabolism as well as lipid metabolism, whereas only scarce data are available. The aim of this study was to investigate the effect of fish oil on the gene expression of the transporters and enzymes related to bile acid as well as lipid metabolism in the liver and small intestine. Methods: Seven-week old male C57BL/6 mice were fed diets enriched in 10% soybean oil or 10% fish oil for 4 weeks. After 4 weeks, blood, liver and small intestine were obtained. Results: Hepatic mRNA expression of lipids (Abcg5/8, multidrug resistance gene product 2) and bile acids transporters (bile salt export pump, multidrug resistance associated protein 2 and 3, organic solute transporter α) was induced in fish oil-fed mice. Hepatic Cyp8b1, Cyp27a1 and bile acid CoA : amino acid N-acyltransferase were increased in fish oil-fed mice compared with soybean-oil fed mice. Besides, intestinal cholesterol (Abcg5/8) and bile acid transporters (multidrug resistance associated protein 2 and organic solute transporter α) were induced in fish oil-fed mice. Conclusion: Fish oil induced the expression of cholesterol and bile acid transporters not only in liver but in intestine. The upregulation of Abcg5/g8 by fish oil is caused by an increase in cellular 27-HOC through Cyp27a1 induction. The hepatic induction of bile acid synthesis through Cyp27a1 may upregulate expression of bile acid transporters in both organs.

We isolated three strains of vancomycin intermediate Staphylococcus aureus (VISA) from a blood sample of a patient with infective endocarditis (VISA-1), postoperative pneumonia sputum (VISA-2), and pyogenic spondylitis blood sample (VISA-3). These VISA strains did not carry vanA, vanB, vanC1, or vanC2/C3 genes. Cell wall thickening was observed. VISA-1 and VISA-3 PFGE patterns showed the completely same pattern compared to the PFGE pattern of methicillin-resistant Staphylococcus aureus first isolated from patients 1 and 3. After 10 days on brain heart infusion agar, wall thickening in all three type of VISA was unchanged, but VISA-2 and VISA-3 reversed vancomycin susceptibility. The most suitable use of vancomycin in patients with MRSA infection thus appears to be in reducing the opportunity for cell wall thickening.

We evaluated the diagnostic utility of peripheral blood neutrophil distribution patterns obtained using the CELL-DYN SAPPHIRE hematology analyzer in patients with myelodysplastic syndrome (MDS). Peripheral blood was obtained from 467 individuals including 32 patients with MDS, and the respective neutrophil distribution patterns were observed using two light scatters [7-degree complexity (7D) and 90-degree lobularity (90D)]. These scattering intensities are shown as median (median neutrophil distribution: MND) and coefficient of variation (neutrophil distribution width: NDW). Generally, MDS patients showed lower 7D MND, higher 7D NDW, lower 90D MND and higher 90D NDW than other comparable groups. Whereas 90D parameters were more diagnostically efficient than 7D ones in patients with MDS. The sensitivity and specificity of 90D MND for MDS patients became 78.1 and 78.9%, respectively (cut-off value = 14,514). 90D NDW was most diagnostically effective with 87.5% sensitivity and 91.0% specificity (cut-off value = 21.2%). Both 90D parameters showed no evident correlation with the degree of either leukocytopenia or peripheral blood dysgranulopoiesis. In conclusion, neutrophil distribution parameters, especially 90D NDW, appear to provide convenient and objective markers for the screening of patients with MDS in routine laboratory hematology.

Background and Aim: To evaluate the importance of cholesterol and bile acid concentrations in the intestinal lumen to cholesterol homeostasis, we investigated the effect of cholesterol-, bile salt- or cholestyramine-administration on the regulation of intestinal mRNA related to cholesterol and bile acid metabolism. Methods: Male Wistar rats fed on standard laboratory chow (AIN-93) were allocated into four experimental groups: (i) control group; (ii) cholesterol group (CH), which was fed cholesterol in diet (2% [w/w]) for 14 days; (iii) cholic acid (CA) group, which was fed CA in diet (1% [w/w]) for 14 days; (iv) cholestyramine (CT) group, which was fed CT in diet (5% [w/w]) for 14 days. Blood, liver and small intestine were obtained after 14 days. Serum lipids and bile acids were measured by colorimetric assays, and hepatic and intestinal mRNA relating to lipid and bile acid metabolism was studied by reverse transcription–polymerase chain reaction. Results: Intestinal ABCG8, liver X receptor α, small heterodimer partner (SHP) and sterol regulatory element binding protein 1c (SREBP-1c) mRNA expressions were markedly increased in the CH group. Intestinal multidrug resistance associated protein (MRP) 2 and MRP3 mRNA expressions were markedly increased in the CA group. In the CT group intestinal MRP2, ABCG5, ABCG8, SHP and SREBP-1c mRNA expressions were markedly decreased. Conclusion: The bile acid availability in the intestinal lumen alters the expression of various intestinal mRNA relating to not only bile acid metabolism, but also lipid metabolism. Though the mechanism of the intestinal SHP elevation is unclear, cholesterol feeding may affect the intestinal bile acid metabolism via intestinal SHP expression.

Osthol, a coumarin compound, was isolated from the dried fruits of Cnidium monnieri (Umbelliferae) and the effect of dietary osthol on hypertension and lipid metabolism was examined in stroke-prone spontaneously hypertensive rats (SHRSP). Six-week-old male SHRSP were fed the experimental diet containing 0.05% osthol by weight for 4 weeks with free access to the diet and water. Elevation of systolic blood pressure was significantly suppressed on and after 3 weeks. In addition, significant decreases in cholesterol and triglyceride contents in the liver were recognized without any significant changes in serum lipids profiles. A comparative study on hepatic mRNA expression indicated that osthol induced a significant increase in 3-hydroxy-3-methylglutaryl coenzymeA (HMG-CoA) reductase mRNA expression, which may lead to decrease in hepatic cholesterol pool through inhibition of the enzyme activity. Moreover, osthol induced a significant increase in acyl-CoA oxidase mRNA expression associated with an increase in carnitine palmitoyl transferase 1a mRNA expression, which suggests the acceleration of beta-oxidation of hepatic fatty acids. This may be responsible, at least in part, for the reduction of hepatic triglyceride content in SHRSP. These beneficial effects of osthol could be useful for both prevention of atherosclerosis and suppression of hepatic lipid accumulation.

Hemochromatosis represents primary or secondary deposition of an excessive amount of iron in many tissues and organs in the body. Recent studies have evaluated regional myocardial function by tissue Doppler echocardiography. Myocardial strain imaging facilitates the evaluation of regional myocardial function without being influenced by cardiac rotation and translation. Several studies have reported the usefulness of myocardial strain and myocardial strain gradient imaging in patients with various myocardial diseases. In this paper, we performed myocardial strain imaging in a patient with secondary hemochromatosis.

Background and Aim: Multidrug resistance associated gene product 2 (Mdr2) is believed to have a significant role in biliary cholesterol and phospholipid secretions. Both pravastatin and bezafibrate resulted in Mdr2 induction, but increased cholesterol secretion was observed only in pravastatin treatment. To explore the mechanism, the hepatic expression of genes that are responsible for the metabolism of the lipids was studied. Methods: Rats were divided into three experimental groups: (i) the control group; (ii) the bezafibrate group, which was fed a diet containing 0.45% bezafibrate for 5 days; and (iii) the pravastatin group, which was fed a diet containing 0.1% pravastatin for 5 days. Serum, hepatic and biliary lipids were measured by colorimetric assays and hepatic mRNA related to lipid metabolism was studied by reverse transcription–polymerase chain reaction (RT-PCR). Results: In the bezafibrate group biliary phospholipid secretion was increased although cholesterol secretion was not increased. In the pravastatin group, biliary cholesterol and phospholipid secretions were significantly increased. The biliary cholesterol/phospholipid ratio was decreased in the bezafibrate group, but the ratio did not change in the pravastatin group. Hepatic Mdr2, Abcg5 and Abcg8 mRNA expression was remarkably increased in the pravastatin group in comparison with the control group (184%, 264% and 247% of control value, respectively). In the bezafibrate group the hepatic gene expression of Mdr2 was increased (157% of control value), but there were no significant changes in hepatic Abcg5 and Abcg8 mRNA expression compared with the control group. Conclusions: Compared with Mdr2, Abcg5 and Abcg8 seem to be more essential transporters for biliary secretion of cholesterol. Pravastatin upregulated Abcg5/Abcg8 while bezafibrate did not, which appears to explain the different effects of these compounds on biliary lipid secretion. © 2004 Blackwell Publishing Asia Pty Ltd

Platelet-activating factor acetylhydrolase (PAF-AH) is an enzyme that catalyzes hydrolysis of PAF and is important for regulation of serum PAF concentration. Twenty-seven patients with hyperbililubinemic hepatobiliary diseases (eight patients with liver cirrhosis, five with acute hepatitis and 14 with obstructive cholestasis) were studied. Serum PAF-AH activity was elevated in hyperbilirubinemic condition associated with liver cirrhosis, obstructive cholestasis or acute hepatitis. There was a significant correlation of serum PAF-AH activity with serum cholesterol (r=0.891, P=0.014 in liver cirrhosis, r=0.813, P=0.002 in obstructive cholestasis) or low density lipoprotein (LDL) cholesterol (r=0.771, P=0.007 in obstructive cholestasis). Serum PAF-AH activity/cholesterol ratio and PAF-AH activity/LDL cholesterol ratio were elevated in the three types of hyperbilirubinemic liver disease. LDL cholesterol increased and PAF-AH/LDL cholesterol ratio decreased in acute hepatitis after remission. On the other hand, LDL cholesterol decreased and PAF-AH/LDL cholesterol ratio did not change after treatment in obstructive cholestasis. These findings suggest that, though serum LDL is a factor influencing serum PAF-AH activity in obstructive jaundice, other factors, i.e. reduced biliary PAF-AH excretion, may also influence serum PAF-AH activity in hyperbilirubinemic hepatobiliary diseases.

Background: Despite advances in endoscopic treatment methods for upper GI hemorrhage, hemostasis is often difficult to achieve. This study evaluated the usefulness of endoscopic band ligation for upper GI hemorrhage exclusive of hemorrhage from chronic gastroduodenal ulcer and varices. Methods: This prospective study included 27 patients who underwent endoscopic band ligation and 31 patients in whom bipolar electrocoagulation was performed for upper GI hemorrhage. In both groups, the causes of hemorrhage included Dieulafoy's ulcer, Mallory-Weiss tear, gastric ulcer after polypectomy, and gastric angiodysplasia. Patients with esophageal varices and those with chronic gastroduodenal ulcer were excluded. Results: Hemostasis was achieved in all 27 patients in the endoscopic band ligation group and in 26 of 31 patients (83.9%) in the bipolar electrocoagulation group. The median procedure time required for achieving hemostasis was 17.0 minutes (interquartile range: 11.5-23.5) in the endoscopic band ligation group versus 27.0 minutes (interquartile range: 20.5-40.0) in the electrocoagulation group. No major complications occurred in either group. Conclusion: Endoscopic band ligation is efficient, simple, and safe. Therefore, this technique should be considered as a treatment option for nonvariceal, nonchronic gastroduodenal ulcer upper GI hemorrhage. (Gastrointest Endosc 2002;55:214-8.)

Components of the multidrug resistance-associated protein (mrp) family mediate the adenosine triphosphate (ATP)-dependent transport of conjugated organic anions in the liver. Of these, mrp1 and mrp2 have been shown to have similar substrate specificity and nucleotide sequence. The intracellular localization and distribution of mrp1 under normal condition and cholestasis have not been as yet completely elucidated. To clarify this point, in the present study we evaluated the intracellular localization of mrp1 in rat liver and kidney after bile duct ligation (BDL). Bile duct was ligated in Wistar rats. Sequential staining of mrp1 by immunofluorescence was carried out in rat liver and kidneys 1, 3, and 5 days after bile duct ligation using confocal laser scanning microscopy. Weak granular staining of mrp1 was observed in cytoplasm of control rat hepatocytes. In addition to increased cytoplasm staining of mrp1, belt-and granule-like staining of mrp1 in basolateral membrane of hepatocytes was also shown after BDL. Furthermore, mrp1 immunofluorescence increased over time after BDL. No specific immunoflurescence of mrp1 was detected in control rat kidney. However, mrp1-positive staining was observed in epithelia of some renal tubules after BDL. This study showed that mrp1 immunofluorescence increased in hepatocyte basolateral membrane and cytoplasm and epithelia of some renal tubules after BDL. This increased mrp1 expression may be an adaptive response to impairment of hepato-biliary organic anion transport during obstructive cholestasis.

Human chorionic gonadotropin (hCG) is generally quantified in serum, but spot urine samples are also used to assess hCG levels in Japan. The purpose of the present study was to elucidate whether urinary hCG can be used clinically as a substitute for serum hCG. A total of 189 samples of serum and spot urine were collected from patients, including cases of normal pregnancy (NP) -13, abortion (AB) -21, extrauterine pregnancy (EP) 25, and hydatidiform mole (MOL) -7, during medical treatment and comparisons were made concerning serum and urinary hCG levels. The histogram of relative urinary/serum hCG(U-hCG.act/S-hCG) of the samples showed a wide distribution of values, but tended to converge to a narrow distribution by creatinine correction (U-hCG.cor/S-hCG). U-hCG.cor/S-hCG of the AB, EP, and MOL groups decreased 1 day to 14 days or was no earlier than 15 days postoperatively compared to preoperatively. The alteration of serum Intact/Total tended to be similar to that of U-hCG.cor/S-hCG in clinical course. The presented case indicated that U-hCG.act/S-hCG did not correspond to serum hCG levels. Because urinary hCG levels are inconsistent depending on whether spot urine is concentrated or diluted, the levels of hCG in spot urine do not always correlate with serum levels of hCG. Therefore, the data of urinary hCG should be interpreted after creatinine correction. Overall, it is recommended to determine serum hCG levels rather than creatinine corrected urinary hCG levels, considering that the relative urinary/serum hCG was not constant postoperatively.

Bilirubin is taken up from blood into hepatocytes by sinosuidal membrane transporters and then excreted into bile through the bile canalicular membrane mainly as bilirubin glucuronides. (1) Mechanism of bilirubin uptake into hepatocytes: many organic anions are incorporated into hepatocytes by organic anion transporting polypeptides (rat, oatp 1, oatp2, oatp3; human, OATP), liver-specific transporter (rlst/HLST), and/or by organic anion transporters (OAT2, OAT3). Oatp1 and HLST transport bilirubin monoglucuronide. However, a transporter of unconjugated bilirubin in the sinusoidal membrane has not as yet been identified. Unconjugated bilirubin may also go across the hepatocyte sinusoidal membrane by a diffusion process. (2) Intrahepatic transport and conjugation of bilirubin: ligandin carries bilirubin to the endoplasmic reticulum (ER) of hepatocytes. In the ER, bilirubin is conjugated by bilirubin uridine diphosphate (UDP)-glycosyltransferase (bilirubin UGT; UGT1A1) to form mono- and diglucuronides of bilirubin. (3) Transport mechanism of bilirubin glucuronides across the hepatocyte canalicular membrane: at the canalicular membrane, bilirubin glucuronides are excreted into bile by multidrug resistance-associated protein 2 (MRP2), a member of the ATP-binding cassette transporter family. (4) Regurgitation of bilirubin glucuronides into blood: MRP3, which is located in the lateral membrane, transports bilirubin glucuronides into blood under conditions of impaired biliary bilirubin excretion.

Background & Aims: Multispecific organic anion–transporting polypeptides (Oatps) are involved in the transcellular movement of amphipathic compounds in many tissues including the liver, kidney, and blood–brain barrier. Recently, a high-affinity digoxin transporter (Oatp2) was cloned from rat brain and shown to be also expressed in the liver. Methods: We investigated the cellular and subcellular distribution of Oatp2 in rat liver by in situ hybridization technology and immunofluorescence microscopy and compared its substrate specificity with that of Oatp1 in complementary RNA–injected Xenopus laevis oocytes. Results: The results show a selective basolateral (sinusoidal) expression of Oatp2 in midzonal to perivenous hepatocytes, but not in periportal or the innermost layer of perivenous hepatocytes. Common substrates of both Oatp1 and Oatp2 include bile salts, steroid conjugates, thyroid hormones (T3, T4), ouabain, and the endothelin receptor antagonist BQ-123 (Michaelis constants: Oatp1, ~600 μmol/L; Oatp2, ~30 μmol/L). Other organic anions including sulfolithotaurocholate, bilirubin monoglucuronide, and sulfobromophthalein were transported only by Oatp1. Conclusions: These results provide definite evidence for the partially overlapping and partially selective substrate specificities of Oatp1 and Oatp2. The unique acinar distribution of Oatp2 might indicate that it represents a high-affinity "backup" system for complete hepatocellular removal of certain cholephilic substances from portal blood plasma. GASTROENTEROLOGY 1999;117:688-695

The secretion of bilirubin conjugates from hepatocytes into bile represents a decisive step in the prevention of hyperbilirubinemia. The bilirubin conjugates, monoglucuronosyl bilirubin (MGB) and bisglucuronosyl bilirubin (BGB), were previously suggested to be endogenous substrates for the apical multidrug resistance protein (MRP2), a member of the adenosine triphosphate (ATP)-binding cassette family of transporters (symbol ABCC2), also termed canalicular multispecific organic anion transporter. We have characterized this ATP-dependent transport using membrane vesicles from human embryonic kidney (HEK) cells expressing recombinant rat as well as human MRP2. MGB and BGB, 3H-labeled in the glucuronosyl moiety, were synthesized enzymatically with recombinant UDP-glucuronosyltransferase 1A1, and stabilized with ascorbate. Rates for ATP-dependent transport of MGB and BGB (0.5 μmol/L each) by human MRP2 were 183 and 104 pmol × mg protein−1 × min−1, respectively. Km values were 0.7 and 0.9 μmol/L for human MRP2, and 0.8 and 0.5 μmol/L for rat MRP2, with MGB and BGB as substrates, respectively. Leukotriene C4 and 17β-glucuronosyl estradiol, which are both known high-affinity substrates for human MRP2, inhibited [3H]MGB transport with IC50 values of 2.3 and 30 μmol/L, respectively. Cyclosporin A competitively inhibited human and rat MRP2-mediated transport of [3H]MGB, with Ki values of 21 and 10 μmol/L, respectively. Our results provide direct evidence that recombinant MRP2, cloned from rat as well as human liver, mediates the primary-active ATP-dependent transport of the bilirubin conjugates MGB and BGB.

Background/Aims: No study has so far been conducted to clarify whether the presence of hyperbilirubinemia is detrimental to liver and renal functions. In the present study, the effects of polyethylene glycol-modified bilirubin oxidase (PEG-BOX) therapy on liver and renal function tests, hepatic energy charge and urinary prostaglandin levels were evaluated in a rat model of obstructive jaundice. Methods: Sprague-Dawley rats were used in the experimental model of obstructive jaundice. PEG-BOX or an equivalent amount of PEG alone was intravenously injected into the animals and sampling of blood and urine, and liver harvesting were done sequentially after bile duct ligation. Results: Conventional liver function tests showed no difference between PEG-BOX and control groups. However, bilirubin concentrations in the peripheral blood and liver tissue specimens markedly decreased, and the hepatic energy charge significantly increased in the PEG-BOX group as compared to controls. The blood concentration of bile acid was lower, but its urinary excretion was higher in the PEG-BOX group than in the control group. In vitro incubation of PEG-BOX with serum from rats with obstructive jaundice decreased the concentration of bilirubin but not that of bile acid. The urinary levels of prostaglandin E2 and the thromboxane B2/6-keto-prostaglandin F1 a ratio were significantly lower in the PEG-BOX group than in the control group. Conclusions: The systemic reduction of bilirubin concentration may contribute to normalization of the urinary levels of prostaglandins and thromboxane B2, to decrease in serum bile acid levels, and to improvement of the hepatic energy charge in obstructive jaundice. These findings suggest that preoperative improvement of jaundice may be beneficial to patients with obstructive jaundice.

Gilbert's syndrome is a constitutional mild unconjugated hyperbilirubinemia. In patients with this syndrome, mutation in the promoter and coding region of UDP-glucuronosyltransferase 1 ∗ 1 gene have been reported independently. In the present study, we examined mutations in UGT1 ∗ 1 gene using leukocytes of one male and four female patients with Gilbert's syndrome. In two cases, heterozygous mis-sense mutation of Gly71Arg was detected in exon 1A with simultaneous heterozygous mutation in the promoter region (2-base pair (TA)-insertion in the TATA box; A(TA) 7 TAA, normal: A(TA) 6 TAA). In the other three cases, mutation was not detected in the coding region and the homozygous A(TA) 7 TAA mutation was observed. From these results, it is concluded that either homozygous mutation (inherited recessively) in TATA box of the promoter region or heterozygous mis-sense mutation (inherited dominantly) in the coding region were present in the patients with Gilbert's syndrome.

Background Bilirubin has antioxidant properties and is known to have a role in the prevention of cardiovascular disease. Bilirubin is known to be negatively associated with serum parameters related to atherosclerosis. Triglyceride-rich lipoprotein is known to play an important role in the progression of atherosclerosis. The aim of the present study was to elucidate the relationship between serum bilirubin and serum remnant lipoprotein cholesterol (RLP-c). Methods We examined 270 Japanese males (mean age 64.8 years) without liver dysfunction and anemia from the Outpatient Clinic of Kinki University Hospital. Serum bilirubin, cholesterol, LDL cholesterol, HDL cholesterol, triglyceride and RLP-c levels were measured. Results Serum RLP-c level was significantly lower in patients in the high bilirubin group than those in the low bilirubin group. Conclusions The findings of this study show that serum bilirubin within physiological levels may affect the serum RLP-c and suggest bilirubin may have a role in preventing atherosclerosis.

In systemic IgG4-related disease, an elevation of the serum IgG4 level(IgG4: 135 mg/dl or higher) and IgG4-positive plasma cell infiltration occurs. Since the total IgG and sum of subclasses, IgG1 through IgG4, were markedly different in a patient suspected of having Mikuliez's disease, we investigated the relationship between total IgG and sum of IgG subclasses. The subjects were healthy individuals, and low IgG4, high IgG4, hyper-gamma globulinemia and hypo-y globulinemia groups. Total IgG was measured using 'N-assay TIA IgG-SH' (Nittobo) and Hitachi 7700, and IgG subclasses were measured using BS-NIA reagent (Binding Site) and BN II (Siemens). Designation of total IgG and the sum of IgG subclasses was established in the healthy control subjects. However the total IgG level and sum of IgG1-4 levels were different when the balance among the IgG subclasses was lost. In case such as :1) the IgG4 level was high and 2) IgG1-type M protein was present. These results indicate that the reevaluation of measured data is necessary when the IgG4 concentration is high and the difference between total IgG concentration and the sum of IgG subclasses is large.

Crigler-Najjar syndrome (types I and II) and Gilbert's syndrome are familial disorders associated with severe to mild unconjugated hyperbilirubinemia. In these conditions, the activity of bilirubin UDP-glucuronosyltransferase (UGT1∗1), which is located in the hepatocyte endoplasmic reticulum, is defective, and severely and moderately decreased, respectively. UGT1∗1 is derived from one of the UGT1 genes. It has a promoter containing a TATA box and consists of exon 1A (which is one of the individual first exons) and common exons 2–5. UGT1∗1 mRNA is formed by differential splicing of these exons. In recent years, gene analysis of these syndromes has been carried out, and genetic abnormalities have been clarified. Homozygous nonsense mutations, mis-sense mutations, and other relevant mutations of exons 1A-5 have been reported in almost all of the patients with Crigler-Najjar syndrome type I, while mainly homozygous mis-sense mutations of exons 1A, 2, and 5 have been reported in type II patients. Almost all patients with this syndrome (types I and II) show autosomal recessive inheritance. On the other hand, some patients with Gilbert's syndrome show heterozygous mis-sense mutations in exons 1A, 4, and 5, while others show homozygous 2-base pair-insertion mutation (TA) into the TATA box in the promoter region [A(TA)7TAA; normal: A(TA)6TAA]. The pattern of inheritance can be autosomal recessive or autosomal dominant. It has also been clarified that enzyme activity is lowered to about 30% (rather than 50%) by heterozygous mutations of the coding region, because of the occurrence of dominant negative mutation based on subunit-structure of the enzyme.

Transport of various intrinsic and extrinsic organic anions across the canalicular membrane of hepatocytes has been confirmed to occur via ATP-dependent primary active transport. To investigate these processes in the presence of obstructive jaundice, the uptake of radiolabeled taurocholate and pravastatin, a non-bile acid organic anion, by isolated hepatocyte canalicular membrane vesicles was observed after bile duct ligation in rats. The Mg+ +-ATPase and Na+, K+ -ATPase activity did not differ between membrane vesicle preparations from sham-operated and bile duct-ligated rats. Taurocholate uptake decreased significantly to 45% and 29%, while pravastatin uptake decreased to 35% and 35% of that in sham-operated rats at 2 and 7 days after bile duct ligation. These results indicate that the ATP-dependent transport systems for both bile salts and non-bile acid organic anions are impaired soon after the onset of obstructive jaundice.

In this study, a covalent bilirubin-binding amino acid residue of the albumin molecule in δ bilirubin was identified. Icteric human serum was treated with a diazo reagent and bilirubin was converted to a stable azodipyrrole. Then, an albumin fraction containing an azodipryrrole-albumin covalent conjugate was obtained by affinity chromatography using Blue Sepharose CL-6B, and it was further purified by reverse phase high performance liquid chromatography. To identify the covalently bound azodipyrrole amino acid residue in albumin, the azodipyrrole-albumin conjugate was cleaved to fragmented peptides by chemical and enzymatical methods, and three main peptide fractions which had the absorption characteristics of azodipyrrole at 530 nm were isolated. Their amino acid sequence analysis revealed that all of the peptides corresponded to residues 187–191 in albumin, where lysine residue 190 was identified as the covalent-binding site of albumin to bilirubin in δ bilirubin.

Hepatic excretion of non-bile acid organic anions is reported to be ATP-dependent and a defect of this transport has been reported in congenitally jaundiced rats, animal models of human Dubin-Johnson syndrome. To investigate the effect of the transmembrane pH gradient on hepatocyte canalicular membrane transport of ATP-dependent organic anions, uptake of pravastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase-inhibiting organic anion, by hepatocyte canalicular membrane vesicles was observed in the presence or absence of transmembrane pH gradients. Uptake was assessed by a rapid filtration technique. ATP-dependent pravastatin uptake was stimulated in the presence of a transmembrane pH gradient (in > out) in Sprague-Dawley (SD) rats. Uptake was dependent on both pravastatin and ATP concentrations and showed saturation kinetics. After intravenous injection of [ 14 C]-pravastatin (0.3 μmol), 81% of the dose was excreted in the bile within 35 min in SD rats, whereas only 20% was excreted in the bile in Eisai hyperbilirubinuria rats. ATP and the pH gradient also co-stimulated the uptake of pravastatin in Eisai hyperbilirubinuria rats, although the K m was much higher and V max was much lower than corresponding values in SD rats. This coincided well with the marked reduction in in vivo biliary excretion of pravastatin in jaundiced rats.

Hepatic excessive iron may play a role in the pathogenesis of non-alcoholic steatohepatitis (NASH). Nrf2 is a master regulator of antioxidative responses. However, the role of Nrf2 in lipid and iron homeostasis remains unclear. Accordingly, it was examined how Nrf2 regulates lipid-related and iron-regulatory genes after feeding a high-fat diet (HFD) with iron.Wild-type and Nrf2-null mice were fed the following diets: (i) control diet (4% soybean oil) for 12 weeks, (ii) control diet for 8 weeks followed by control diet containing 0.5% carbonyl iron for 4 weeks, (iii) HFD (4% soybean oil and 16% lard) for 12 weeks, (iv) HFD for 8 weeks followed by HFD containing 0.5% carbonyl iron for 4 weeks. Blood and livers were removed after 12 weeks.Nrf2-null control mice exhibited a tendency towards higher hepatic triglycerides compared to wild-type control mice. Hepatic malondialdehyde was higher and hepatic iron levels tended to be higher in Nrf2-null mice than wild-type counterparts while on a HFD. The HFD with iron synergistically induced mRNA expression of Pparα targets, including Acox and Cpt1 in wild-type mice, yet the induction was diminished in Nrf2-null mice. Hepatic hepcidin and ferroportin 1 mRNA expression were increased in wild-type mice after feeding a HFD with iron, but were unchanged in any group of Nrf2-null mice.Nrf2 deletion dysregulates hepatic mRNA expression of β-oxidation enzymes and iron-related genes, possibly causing a trend for increased hepatic triglyceride and iron concentrations. Nrf2 may have roles in the progression of NASH.

Fish oil rich in n-3 polyunsaturated fatty acids is known to affect hepatic lipid metabolism. Several studies have demonstrated that fish oil may affect the bile acid metabolism as well as lipid metabolism, whereas only scarce data are available. The aim of this study was to investigate the effect of fish oil on the gene expression of the transporters and enzymes related to bile acid as well as lipid metabolism in the liver and small intestine. Seven-week old male C57BL/6 mice were fed diets enriched in 10% soybean oil or 10% fish oil for 4 weeks. After 4 weeks, blood, liver and small intestine were obtained. Hepatic mRNA expression of lipids (Abcg5/8, multidrug resistance gene product 2) and bile acids transporters (bile salt export pump, multidrug resistance associated protein 2 and 3, organic solute transporter α) was induced in fish oil-fed mice. Hepatic Cyp8b1, Cyp27a1 and bile acid CoA : amino acid N-acyltransferase were increased in fish oil-fed mice compared with soybean-oil fed mice. Besides, intestinal cholesterol (Abcg5/8) and bile acid transporters (multidrug resistance associated protein 2 and organic solute transporter α) were induced in fish oil-fed mice. Fish oil induced the expression of cholesterol and bile acid transporters not only in liver but in intestine. The upregulation of Abcg5/g8 by fish oil is caused by an increase in cellular 27-HOC through Cyp27a1 induction. The hepatic induction of bile acid synthesis through Cyp27a1 may upregulate expression of bile acid transporters in both organs.