Hiroshi Murayama

YAMASA Corporation, Tiba, Chiba, Japan

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Publications (16)49.93 Total impact

  • Hiroshi Murayama, Masaki Ikemoto, Masaru Hamaoki
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    ABSTRACT: As ornithine carbamyltransferase (OCT) has proved to be a sensitive serum marker in the detection of hepatotoxicity in several models, it is important to confirm its application to the diagnosis of non-alcoholic fatty liver disease. C57BL/6, KK-Ta and KK-Ay mice were fed a high-fat diet for 8 weeks and serum enzyme markers were examined. Serum OCT and alanine aminotransferase (ALT) were also measured in diabetic obese ob/ob and db/db mice fed a normal diet. Liver damage in these mice was evaluated by the hepatic content of tumor necrosis factor-alpha. Serum levels of OCT increased in KK-Ay fed a high-fat diet compared with the normal diet-fed group, whereas C57BL/6 and KK-Ta mice were not affected. In ob/ob mice, the relative increase was always greater in OCT than in ALT. In contrast, in db/db mice, the relative increase was always greater in ALT. Hepatic tumor necrosis factor-alpha was significantly elevated in ob/ob mice, but not in db/db mice. Serum OCT seemed to reflect tumor necrosis factor-alpha-mediated hepatic damage when compared with ALT in diabetic obese mice and could be useful in the application for non-alcoholic fatty liver disease with features of metabolic syndrome, such as obesity and diabetes.
    Journal of Gastroenterology and Hepatology 09/2009; 25(2):413-9. · 3.33 Impact Factor
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    ABSTRACT: We previously hypothesized that S100A8/A9 binds with several kinds of proinflammatory cytokines, such as TNF-alpha, IL-6 and IL-1beta, to form the S100A8/A9-proinflammatory cytokine complexes in vivo in acute inflammation, leading to subsidence of inflammatory responses. Our goal was to verify the presence of these complexes in liver tissues of rats with lipopolysaccharide (LPS) induced damage. We firstly prepared two kinds of the full-length cDNA encoding amino acid sequences of human S100A8 and S100A9 proteins, and constructed their pCold-I expression vectors. The recombinant S100A8 and S100A9 were successfully expressed in E. coli, and then purified by Ni-agarose columns, respectively. The S100A8/A9 was noncovalently synthesized in 2.0 mol/1 Tris-NaOH solution (pH 12) using the purified S100A8 and S100A9. After purification, this heterodimer (1 mg) was intraperitoneally injected into a rat 1h after injection of LPS. Two kinds of ELISA systems were used to detect the S100A8/A9-inflammatory cytokine complexes in the rat liver tissue. As determined by the ELISA-A and B, the reaction was apparently positive and quantitative. Immunohistochemistry provided such complexes-positive cells in the liver with damage. The S100A8/A9-positive cells almost corresponded to the cytokines-positive ones morphologically, strongly suggested the presence of the S100A8/A9-proinflammatory cytokine complexes. In conclusion, the possibility that these complexes were formed in vivo and accumulated to the immunological cells, such as macrophages and/or activated neutrophils, was indicated. Our effort is currently addressed to isolate the S100A8/A9-proinflammatory cytokine complexes using biochemical techniques, and to comprehensively resolute their clinical significance in the differential diagnosis of inflammatory diseases.
    Rinsho byori. The Japanese journal of clinical pathology 05/2009; 57(4):324-31.
  • Hiroshi Murayama, Masaki Ikemoto, Masaru Hamaoki
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    ABSTRACT: Although mitochondrion-derived markers such as ornithine carbamyltransferase (OCT) and glutamate dehydrogenase (GLDH) have been reported to be good markers for alcohol-induced hepatic injury, their use has been limited due to the notion that mitochondrial markers are less sensitive than cytosol-derived markers. We determined the clinical importance of mitochondrion-derived markers in the evaluation of alcohol-induced hepatotoxicity. Rats were administered alcohol chronically (5-30% ethanol in drinking water with or without high fat diet feeding for 15 weeks) and hepatic damages were evaluated by serum OCT and GLDH, together with other liver enzymes such as alanine aminotransferase and aspartate aminotransferase. Hepatic content of the enzymes was also evaluated in the chronic ethanol feeding model to confirm whether induction of the enzyme in the liver reflects the serum activity. The serum activities of OCT and GLDH increased significantly by chronic ethanol feeding while other markers did not. Although the hepatic content of OCT and GLDH also increased, the serum activities did not correlate with the hepatic activities and the extent of increase in the liver was much less than in serum. Mitochondrion-derived markers, especially OCT, appeared superior to cytosol-derived markers in the detection of alcohol-induced liver injury.
    Clinica chimica acta; international journal of clinical chemistry 01/2009; 401(1-2):100-4. · 2.54 Impact Factor
  • Hiroshi Murayama, Masaki Ikemoto, Atsuo Nagata
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    ABSTRACT: In order to find sensitive serum markers in non-alcoholic steatohepatitis, liver-specific injury markers were thoroughly examined in mild models of NASH in rats. Wistar and Sprague-Dawley rats were fed a choline-deficient diet for 4 weeks, and serum activities of liver-specific enzyme markers were examined. In the drug-induced steatohepatitis model, tetracycline (0.4 mmol/kg) was given i.p. to rats and the course of hepatotoxicity was evaluated with serum markers, together with the accumulation of total lipid and thiobarbituric acid-reactive substances in the liver. In Wistar rats, serum activities of most enzymes tested were significantly increased. In Sprague-Dawley rats, in contrast, the serum level of ornithine carbamyltransferase and glutamate dehydrogenase were markedly elevated in the choline-deficient diet group compared with the control diet groups, whereas other markers were not significantly increased. In the tetracycline-induced steatohepatitis model, the extent of the increase was much higher in mitochondrial markers and the peak of the increase in these markers corresponded with the increase of hepatic total lipid and thiobarbituric acid-reactive substance. These observations show that serum mitochondrial enzyme markers are potent markers for non-alcoholic steatohepatitis in rats and are possibly applicable to humans.
    Journal of Gastroenterology and Hepatology 10/2008; 24(2):270-7. · 3.33 Impact Factor
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    ABSTRACT: Despite the restricted distribution to mitochondria of hepatocytes in the periportal region, ornithine carbamyltransferase (OCT) have been suggested to be a sensitive marker in addition to type-I arginase (ARG), even in centrilobular damage of the liver. We attempted to confirm the universal advantages of ARG and OCT in the evaluation of hepatotoxicity induced by toxicants, and to clarify whether the character of a marker is a more important factor than its localization in its clinical superiority. Rats were administered carbon tetrachloride, allyl alcohol, D-galactosamine, lipopolysaccharide, and concanavalin A and the course of damage was monitored by serum ARG and OCT, together with alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The significant increase in the serum levels of the markers was faster in ARG and OCT than AST and ALT. Further, the extent of the increase at the peak was always higher in ARG and OCT than in AST and ALT. The superiority of ARG and OCT over AST and ALT in the detection of hepatotoxicity seems universal, at least in toxicant-induced acute liver injuries. The apparent faster appearance of mitochondria-derived enzyme, OCT, in serum than cytosol-derived enzyme, ALT, shows that leakage into the circulation is dependent on the marker rather than its localization.
    Clinica Chimica Acta 06/2008; 391(1-2):31-5. · 2.85 Impact Factor
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    ABSTRACT: We investigated a patient with type 1 diabetes mellitus undergoing pancreatic islets transplantation. In this patient, we evaluated the clinical usefulness of serial measurement of serum S100A8/A9 complex levels for detecting acute inflammatory responses associated with rejection of transplanted pancreatic islets. The serum S100A8/A9 complex was a more sensitive marker for acute inflammation associated with islet transplant rejection than the serum C-reactive protein. Thus, the serial measurement of the serum S100A8/A9 complex concentration is useful for monitoring the patients with pancreatic islet transplantation.
    Annals of Clinical Biochemistry 12/2007; 44(Pt 6):570-2. · 1.92 Impact Factor
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    ABSTRACT: To evaluate the clinical advantage of the ratio of serum ornithine carbamoyltransferase (OCT) to alanine aminotransferase (ALT) in the diagnosis of hepatocellular carcinoma (HCC). Serum levels of hepatic enzyme markers and their combinations were evaluated and compared with those of two other markers for HCC. OCT/ALT was significantly higher in case of HCC than chronic hepatitis or liver cirrhosis. Its sensitivity (64.3%) was higher than those of alpha-fetoprotein and PIVKA-II (21.4% and 42.9%, respectively). Fluctuations of OCT/ALT before and after treatment were similar to those of alpha-fetoprotein. OCT/ALT is a potent indicator for the diagnosis and the prognosis of HCC.
    Clinical Biochemistry 10/2007; 40(13-14):1077-80. · 2.45 Impact Factor
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    ABSTRACT: The ratio of ornithine carbamoyltransferase (OCT) to alanine aminotransferase (ALT) or glutamate dehydrogenase (GDH) in serum has been suggested as an indicator for the diagnosis of hepatocellular carcinoma and alcoholic liver disease, respectively. However, the mechanisms responsible for the increase in these ratios are still unclear. Wistar rats were pretreated with lipopolysaccharide (LPS) or gadolinium chloride (GD) before being administered with thioacetamide (TAA, 200 mg/kg, ip). Serum OCT and ALT levels were compared with control values. Half-lives of the enzymes in circulation were evaluated after the intravenous injection of the purified enzymes into rats with or without the pretreatment. The serum level of OCT at 24 h after the administration of TAA was significantly lower in the LPS-treated group, and not influenced by pretreatment with GD. The half-life of OCT was prolonged from 1.06+/-0.14 to 2.07+/-0.29 h (p<0.05) by the pretreatment with GD, but not influenced by the administration of LPS. No change was observed in the clearance of GDH or ALT among the pretreatments. Leakage into and clearance from the circulation of OCT are influenced by whether Kupffer cells are activated or not. OCT alone or in combination with other markers may be a useful indicator for Kupffer cell activation as well as mitochondrial damage in hepatic cells.
    Clinica Chimica Acta 05/2007; 380(1-2):170-4. · 2.85 Impact Factor
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    ABSTRACT: We hypothesized that the S100A8/A9 complex is effective in the suppression of acute inflammatory changes. To clarify such a functional role of the S100A8/A9 complex in acute inflammatory disorder, the complex purified from human leukocytes (approx. 1 mg) was intraperitoneally injected into rats 1.0 or 3.5 h after an injection of lipopolysaccharide (LPS). The serum concentrations of interleukin-6 (IL-6) and nitric oxide (NOx) were significantly decreased in the treated rats. Conversely, when anti-S100A8/A9 complex IgG was injected into the tail blood vessel of a rat 1.0 h after the injection of LPS, the serum concentration of IL-6 increased slightly, indicating that the antibody immunoregulatorily blocked the activity of the complex as an anti-inflammatory protein in vivo. In addition, the S100A8/A9 complex bound non-specifically with interleukin-1beta (IL-1beta), IL-6 and TNF-alpha in vitro, suggesting that the complex could bind with these cytokines in vivo. A large number of endogenous S100A8/A9 complex-positive cells that accumulated in the inflamed region in the liver 6 h after the injection of LPS were microscopically observed, while apparent inflammatory changes were not found microscopically in other organs, such as the kidney, lung and spleen. In rats treated with the S100A8/A9 complex, neither acute inflammatory changes nor S100A8/A9 complex-positive cells were also observed microscopically in the liver tissue. These findings suggest that the S100A8/A9 complex indirectly suppresses the overproduction of NOx from activated neutrophils and/or macrophages by neutralizing the activity of pro-inflammatory cytokines. Thus, the S100A8/A9 complex may play an important role in the suppression of acute inflammation by modulating the vital activity of pro-inflammatory cytokines in vivo.
    Clinica Chimica Acta 03/2007; 376(1-2):197-204. · 2.85 Impact Factor
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    ABSTRACT: We evaluated the usefulness of serum type-I arginase (ARG) and ornithine carbamoyltransferase (OCT) in thioacetamide (TAA)-induced acute and chronic liver injury in rats. In an acute injury model, we measured the time-courses of serum concentrations of ARG and OCT using ELISA, together with AST and ALT using biochemical enzymatic assays after a single administration of TAA (200 mg/kg, i.p.). In the chronic model, TAA was repeatedly administered (20 mg/kg/day, p.o.) for 16 weeks and serum concentrations of the enzymes were evaluated. In the acute model, the concentrations of the enzymes were increased in a similar manner, peaking 24 h after the administration, and ARG showed the earliest and greatest increase among the enzymes tested. In the chronic model, the serum concentration of OCT was significantly increased only 1 week after oral treatment, while concentrations of the other enzymes were increased at 8 to 12 weeks. In the histological analysis, TAA treatment damaged hepatocytes in both the acute and chronic model. These results clearly show the usefulness of ARG and OCT for the evaluation of acute and chronic liver injury, respectively.
    Clinica Chimica Acta 02/2007; 375(1-2):63-8. · 2.85 Impact Factor
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    ABSTRACT: We developed a new enzyme-linked immunosorbent assay (ELISA) for ornithine carbamoyltransferase (OCT) and evaluated its usefulness. Recombinant human OCT expressed in E. coli was used as an antigen to obtain the monoclonal antibodies for this assay. The reactivity of the antibodies to native OCT as well as recombinant OCT was enhanced at alkaline pH (8.5-10), and the assay's sensitivity was markedly improved. The antibodies react identically with native and recombinant OCT at pH 9.4. The dilution test showed a good linearity between dilution ratios and the concentrations. Different concentrations of OCT added were recovered on average at 90.4%. There was a good correlation between OCT protein levels in the ELISA and OCT enzyme activities (r=0.987, p<0.0001). A significant difference in the serum level of OCT was observed between chronic hepatitis patients (110.7+/-80 ng/ml) and healthy subjects (34.4+/-20.7 ng/ml) (p<0.0001). The serum levels of OCT between sexes differed significantly in the healthy subjects (p<0.0001). Our newly established ELISA for OCT using monoclonal antibodies is sensitive enough for clinical application.
    Clinica Chimica Acta 06/2006; 368(1-2):125-30. · 2.85 Impact Factor
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    ABSTRACT: There has been a lack of consensus among results of assays for insulin autoantibody (IAA) carried out by different laboratories, despite a reduction in the non-specific effect using a cold insulin competitor in radioimmunoassays (RIAs) for IAA in type I diabetes. We speculated that the discrepancies are partly a result of the non-specific binding (NSB) of [125I]insulin to unidentified molecules in serum on polyethylene glycol separation, and tried to improve IAA RIAs. The molecular weight of a candidate for the factor causing NSB was estimated to be about 700 kDa by gel filtration analysis, resembling that of alpha 2-macroglobulin (a2M). Further, the addition of purified a2M to the assay resulted in an increase in NSB. Screening revealed that heterocyclic compounds, such as isothiazolinone derivatives (ProClin300), were greatly effective at reducing NSB in control subjects from 2.904+/-0.909% to 1.347+/-0.254% (n=283, mean+/-SD, p<0.0001). Using our newly developed IAA RIA with ProClin300, the sensitivity for newly diagnosed type I diabetes patients (n=55) was 32.7% and 30.9% with or without insulin competition, respectively, whereas that of the former assay without ProClin300 was only 20.0%.
    Journal of Autoimmunity 03/2006; 26(2):127-32. · 8.15 Impact Factor
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    ABSTRACT: C-reactive protein (CRP), a useful marker for inflammatory diseases, is not always sensitive to inflammatory reaction in the liver or other tissues. The aim of this study was to develop a sensitive and specific method for detecting inflammatory responses associated with transplant rejection. We developed a new, highly sensitive ELISA system for the measurement of serum human myeloid-related protein complex (MRP8/14), using monoclonal antibodies against MRP8/14, and applied it to specimens obtained from patients undergoing small intestine or liver transplantation. This assay could detect MRP8/14 concentrations as low as 2 micro g/L. Within-run CVs were 3.7-6.1% and between-day CVs were 5.6-8.7% for MRP8/14 concentrations of 117-3300 micro g/L. Mean recovery was 104% (range, 80-128%). We observed a marked increase in serum MRP8/14 postoperatively in most recipients of transplants, followed by an increase in CRP 1-7 days after the increase in the complex. The increase in serum MRP8/14 occurred simultaneously with permeation of lymphocytes into the transplanted tissues as a result of rejection of the graft tissues. Accurate measurement of serum MRP8/14 provides a useful clinical diagnostic method tool for detecting inflammation associated with rejection of transplanted tissues.
    Clinical Chemistry 05/2003; 49(4):594-600. · 7.15 Impact Factor
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    ABSTRACT: Insulin-like growth factor-1 (IGF-1) reportedly suppresses the development of type 1 diabetes in NOD mice, suggesting that IGF-1 is possibly a candidate for the autoantigens in type 1 diabetes. We therefore examined the anti-IGF-1 autoantibodies (IGF-1 Ab) in sera from patients with type 1 diabetes using radioimmunoassay. However, we were unable to demonstrate the existence of anti-IGF-1 autoantibodies in type 1 diabetes with our method. Further study is necessary to clarify whether anti-IGF-1 autoantibodies are existent in human type 1 diabetes and to determine the significance of IGF-1 in the pathogensesis of type 1 diabetes.
    Annals of the New York Academy of Sciences 05/2002; 958:267-70. · 4.38 Impact Factor
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    ABSTRACT: To develop a new ELISA system for liver-type arginase using monoclonal antibodies against the enzyme, and to verify the utility of the arginase in diagnosis of hepatic disorders. We have developed an enzyme-linked immunosorbent assay (ELISA), using two kinds of monoclonal antibodies (Mo6G3 and Mo9C5) for human liver-type arginase as the first and second antibodies respectively. We have also developed a new method to eliminate the influence of erythrocyte-derived arginase contamination in hemolytic samples. This ELISA was applied to specimens received from patients with acute and chronic hepatic disease and also patients who had undergone partial hepatectomy. This assay is sensitive and reproducible for the measurement of liver-type arginase in the sera of patients with liver dysfunction, and enabled us to detect enzyme concentrations as low as 27 pmol/L without any processing of the samples. The assay showed within-run coefficients of variation (CV) ranging from 1.9 to 4.1% and between-day CV from 3.6 to 5.1% for arginase concentrations varying from 57.1 to 1200 pmol/L. The recovery was 113% (mean) with a range of 96 to 129%. These antibodies reacted strongly with both recombinant and native liver-type arginases, while, to some extent, with erythrocyte-derived arginase. Correction for erythrocyte-derived arginase contamination in hemolytic samples was, however, easily made by assaying peroxidase-like activity of hemoglobin. From the view of a limited localization of arginase in the liver, the marked increase of the enzyme in serum reflects initiation of liver injury, while the rapid decrease reflects termination of the damage. Such quick normalization in circulating liver-type arginase indicated another merit of the enzyme in diagnosis of liver diseases. The changes in circulating liver-type arginase level could be helpful not only in the diagnosis of liver diseases but also subsequent treatment of the patients with liver damage.
    Clinical Biochemistry 10/2001; 34(6):455-61. · 2.45 Impact Factor
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    ABSTRACT: Objectives: To develop a new ELISA system for liver-type arginase using monoclonal antibodies against the enzyme, and to verify the utility of the arginase in diagnosis of hepatic disorders.Design and Methods: We have developed an enzyme-linked immunosorbent assay (ELISA), using two kinds of monoclonal antibodies (Mo6G3 and Mo9C5) for human liver-type arginase as the first and second antibodies respectively. We have also developed a new method to eliminate the influence of erythrocyte-derived arginase contamination in hemolytic samples. This ELISA was applied to specimens received from patients with acute and chronic hepatic disease and also patients who had undergone partial hepatectomy.Results: This assay is sensitive and reproducible for the measurement of liver-type arginase in the sera of patients with liver dysfunction, and enabled us to detect enzyme concentrations as low as 27 pmol/L without any processing of the samples. The assay showed within-run coefficients of variation (CV) ranging from 1.9 to 4.1% and between-day CV from 3.6 to 5.1% for arginase concentrations varing from 57.1 to 1200 pmol/L. The recovery was 113% (mean) with a range of 96 to 129%. These antibodies reacted strongly with both recombinant and native liver-type arginases, while, to some extent, with erythrocyte-derived arginase. Correction for erythrocyte-derived arginase contamination in hemolytic samples was, however, easily made by assaying peroxidase-like activity of hemoglobin. From the view of a limited localization of arginase in the liver, the marked increase of the enzyme in serum reflects initiation of liver injury, while the rapid decrease reflects termination of the damage. Such quick normalization in circulating liver-type arginase indicated another merit of the enzyme in diagnosis of liver diseases.Conclusions: The changes in circulating liver-type arginase level could be helpful not only in the diagnosis of liver diseases but also subsequent treatment of the patients with liver damage.
    Clinical Biochemistry - CLIN BIOCHEM. 01/2001; 34(6):455-461.