MITAMURA Kuniko

Department of PharmacyAssociate Professor

Last Updated :2024/06/18

■Researcher basic information

Degree

  • (BLANK)(Kanazawa University)

Research Keyword

  • システイン   MS   LC   胆汁酸   エレクトロスプレーイオン化   グルタチオン   N-アセチルシステイン   前立腺癌   LC-MS   サルフェート   デヒドロエピアンドロステロンサルフェート   ラット   硫酸抱合   ビタミンD   誘導体化   グルタチオン抱合体   脳内ステロイドホルモン   ラット胆汁   ラット脳   プロドラッグ   LC/MS   HPLC   グルクロニド   カルボン酸   イメージング質量分析法   質量分析法   カテコールエストロゲン   ヒト血中   同定   尿   血清中濃度   分析化学   Analytical Chemistry   

Research Field

  • Life sciences / Clinical pharmacy
  • Life sciences / Pharmaceuticals - analytical and physicochemistry

■Career

Career

  • 2009/04 - Today  Kindai UniversityFaculty of Pharmacy准教授
  • 2005/04 - 2009/03  Kindai UniversityFaculty of Pharmacy講師
  • 1995/04 - 2005/03  Kanazawa UniversityFaculty of Pharmaceutical Sciences助手
  • 1992/04 - 1995/03  Kanazawa UniversityFaculty of Pharmaceutical Sciences教務職員
  • 1992  - Kanazawa University

Educational Background

  •        - 1990  Kanazawa University  Faculty of Pharmaceutical Sciences  製薬化学科
  •        - 1990  Kanazawa University  Faculty of Pharmaceutical Science

■Research activity information

Award

  • 2008 日本臨床化学会 日本臨床化学会 学術賞
     液体クロマトグラフィー/質量分析法による内分泌代謝疾患の科学診断へのアプローチ 
    受賞者: 三田村 邦子
  • 2008 日本医用マススペクトル学会 日本医用マススペクトル学会奨励賞
     LC/MSによる胆汁酸のグルタチオン抱合に関する研究 
    受賞者: 三田村 邦子
  • 2000 第11回クロマトグラフィー科学会奨励賞
     JPN

Paper

  • Tetsushi Yamamoto; Ryota Shiburo; Yoshie Moriyama; Kuniko Mitamura; Atsushi Taga
    Oncology reports 50 (4) 2023/10 
    Maple syrup is a natural sweetener consumed worldwide. Active ingredients of maple syrup possess antitumor effects; however, these ingredients are phenolic compounds. The present study aimed to investigate components other than phenolic compounds that may have antitumor effects against colorectal cancer (CRC). Cell proliferation assays demonstrated that treatment with the more than 10,000 molecular weight fraction significantly inhibited viability in DLD‑1 cells. Therefore, we hypothesized that the protein components of maple syrup may be the active ingredients in maple syrup. We obtained protein components from maple syrup by ammonium sulfate precipitation, and treatment with the protein fraction of maple syrup (MSpf) was found to exhibit a potential antitumor effect. MSpf‑treated DLD‑1 colon adenocarcinoma cells exhibited significantly decreased proliferation, migration and invasion. In addition, upregulation of LC3A and E‑cadherin and downregulation of MMP‑9 expression levels were observed following MSpf treatment. Investigation of the components of MSpf suggested that it was primarily formed of advanced glycation end products (AGEs). Therefore, whether AGEs in MSpf affected the STAT3 pathway through the binding to its receptor, receptor of AGE (RAGE), was assessed. MSpf treatment was associated with decreased RAGE expression and STAT3 phosphorylation. Finally, to determine whether autophagy contributed to the inhibitory effect of cell proliferation following MSpf treatment, the effect of MSpf treatment on autophagy induction following bafilomycin A1 treatment, a specific autophagy inhibitor, was assessed. The inhibitory effect of MSpf treatment on cell proliferation was enhanced through the inhibition of autophagy by bafilomycin A1 treatment. These results suggested that AGEs in MSpf suppressed cell proliferation and epithelial‑mesenchymal transition through inhibition of the STAT3 signaling pathway through decreased RAGE expression. Therefore, AGEs in MSpf may be potential compounds for the development of antitumor drugs for the treatment of CRC with fewer adverse effects compared with existing antitumor drugs.
  • Kanta Sato; Tetsushi Yamamoto; Kuniko Mitamura; Atsushi Taga
    Foods (Basel, Switzerland) 12 (11) 2023/05 
    In the original publication [...].
  • Kei Minowa; Daniel Rodriguez-Agudo; Mitsuyoshi Suzuki; Yamato Muto; Saeko Hirai; Yaping Wang; Lianyong Su; Huiping Zhou; Qun Chen; Edward J Lesnefsky; Kuniko Mitamura; Shigeo Ikegawa; Hajime Takei; Hiroshi Nittono; Michael Fuchs; William M Pandak; Genta Kakiyama
    Journal of lipid research 64 (5) 100363 - 100363 2023/03 
    CYP7B1 catalyzes mitochondria-derived cholesterol metabolites such as (25R)26-hydroxycholesterol (26HC) and 3β-hydroxy-5-cholesten-(25R)26-oic acid (3βHCA) and, facilitates their conversion to bile acids. Disruption of 26HC/3βHCA metabolism in the absence of CYP7B1 leads to neonatal liver failure. Disrupted 26HC/3βHCA metabolism with reduced hepatic CYP7B1 expression is also found in non-alcoholic steatohepatitis (NASH). The current study aimed to understand the regulatory mechanism of mitochondria cholesterol metabolites and their contribution to onset of NASH. We used Cyp7b1-/- mice fed a normal diet (ND), western diet (WD), or high-cholesterol diet (HCD). Serum and liver cholesterol metabolites as well as hepatic gene expressions were comprehensively analyzed. Interestingly, 26HC/3βHCA levels were maintained at basal levels in ND fed Cyp7b1-/- mice livers by the reduced cholesterol transport to mitochondria, and the upregulated glucuronidation and sulfation. However, WD fed Cyp7b1-/- mice developed insulin resistance (IR) with subsequent 26HC/3βHCA accumulation due to overwhelmed glucuronidation/sulfation with facilitated mitochondria cholesterol transport. Meanwhile, Cyp7b1-/- mice fed a HCD did not develop IR or subsequent evidence of liver toxicity. HCD-fed mice livers revealed marked cholesterol accumulation, but no 26HC/3βHCA accumulation. The results suggest 26HC/3βHCA induced cytotoxicity occurs when increased cholesterol transport into mitochondria is coupled to decreased 26HC/3βHCA metabolism driven with IR. Supportive evidence for cholesterol metabolite-driven hepatotoxicity is provided in a diet-induced non-alcoholic fatty liver mouse model and by human specimen analyses. This study uncovers an insulin-mediated regulatory pathway that drives the formation and accumulation of toxic cholesterol metabolites within the hepatocyte mitochondria; mechanistically connecting IR to cholesterol metabolite-induced hepatocyte toxicity which drives NAFLD.
  • Genta Kakiyama; Kei Minowa; Daniel Rodriguez-Agudo; Rebecca Martin; Hajime Takei; Kuniko Mitamura; Shigeo Ikegawa; Suzuki Mitsuyoshi; Hiroshi Nittono; Michael Fuchs; Douglas M Heuman; Huiping Zhou; William M Pandak
    American journal of physiology. Gastrointestinal and liver physiology 323 (5) G488-G500  2022/10 
    Oxysterol 7α-hydroxylase (CYP7B1) controls the levels of intracellular regulatory oxysterols generated by the "acidic pathway" of cholesterol metabolism. Previously, we demonstrated an inability to up-regulate CYP7B1 in the setting of insulin resistance leads to accumulation of cholesterol metabolites such as (25)26-hydroxycholesterol (26HC) that initiate and promote hepatocyte injury; followed by an inflammatory response. The current study demonstrates dietary coffee improves insulin resistance and restores Cyp7b1 levels in a well-characterized western diet (WD)-induced non-alcoholic fatty liver disease (NAFLD) mouse model. Ingestion of a WD containing caffeinated (regular) coffee or decaffeinated coffee markedly reduced the serum ALT level and improved insulin resistance. Cyp7b1 mRNA and protein levels were preserved at normal levels in mice fed the coffee containing WD. Additionally, coffee led to upregulated steroid sulfotransferase 2b1 (Sult2b1) mRNA expression. In accordance with the response in these oxysterol metabolic genes, hepatocellular 26HC levels were maintained at physiologically low levels. Moreover, the current study provided evidence that hepatic Cyp7b1 and Sult2b1 responses to insulin signaling can be mediated through a transcriptional factor, hepatocyte nuclear factor (HNF)-4α. We conclude coffee achieves its beneficial effects through modulation of insulin resistance. Both decaffeinated and caffeinated coffee had beneficial effects, demonstrating caffeine is not fundamental to this effect. The effects of coffee feeding on the insulin-HNF4α-Cyp7b1 signaling pathway, whose dysregulation initiates and contributes to the onset and progression of NASH as triggered by insulin resistance, offer mechanistic insight into approaches for the treatment of NAFLD.
  • Kanta Sato; Tetsushi Yamamoto; Kuniko Mitamura; Atsushi Taga
    Foods (Basel, Switzerland) 10 (12) 2021/12 
    Fructosyl oligosaccharides, including fructo-oligosaccharide (FOS), are gaining popularity as functional oligosaccharides and have been found in various natural products. Our previous study suggested that maple syrup contains an unidentified fructosyl oligosaccharide. Because these saccharides cannot be detected with high sensitivity using derivatization methods, they must be detected directly. As a result, an analytical method based on charged aerosol detection (CAD) that can detect saccharides directly was optimized in order to avoid relying on these structures and physical properties to clarify the profile of fructosyl oligosaccharides in maple syrup. This analytical method is simple and can analyze up to hepta-saccharides in 30 min. This analytical method was also reliable and reproducible with high validation values. It was used to determine the content of saccharides in maple syrup, which revealed that it contained not only fructose, glucose, and sucrose but also FOS such as 1-kestose and nystose. Furthermore, we discovered a fructosyl oligosaccharide called neokestose in maple syrup, which has only been found in a few natural foods. These findings help to shed light on the saccharides profile of maple syrup.
  • Tetsushi Yamamoto; Kanta Sato; Masafumi Yamaguchi; Kuniko Mitamura; Atsushi Taga
    Biochemical and biophysical research communications 584 53 - 59 2021/12 
    The tricarboxylic acid (TCA) cycle is one of the most important pathways of energy metabolism, and the profiles of its components are influenced by factors such as diseases and diets. Therefore, the differences in metabolic profile of TCA cycle between healthy and cancer cells have been the focus of studies to understand pathological conditions. In this study, we developed a quantitative method to measure TCA cycle metabolites using LC-MS/MS to obtain useful metabolic profiles for development of diagnostic and therapeutic methods for cancer. We successfully analyzed 11 TCA cycle metabolites by LC MS/MS with high reproducibility by using a PFP column with 0.5% formic acid as a mobile phase. Next, we analyzed the concentration of TCA cycle metabolites in human cell lines (HaCaT: normal skin keratinocytes; A431: skin squamous carcinoma cells; SW480: colorectal cancer cells). We observed reduced concentration of succinate and increased concentration of citrate, 2-hydroxyglutarate, and glutamine in A431 cells as compared with HaCaT cells. On the other hand, decreased concentration of isocitrate, fumarate, and α-ketoglutarate and increased concentration of malate, glutamine, and glutamate in A431 cells were observed in comparison with SW480 cells. These findings suggested the possibility of identifying disease-specific metabolites and/or organ-specific metabolites by using this targeted metabolomic analysis.
  • Kanta Sato; Noriaki Nagai; Tetsushi Yamamoto; Kuniko Mitamura; Atsushi Taga
    International journal of molecular sciences 21 (14) 2020/07 [Refereed]
     
    The authors wish to make the following corrections to this paper [...].
  • Tetsushi Yamamoto; Kanta Sato; Shinpei Wakahara; Kuniko Mitamura; Atsushi Taga
    Journal of pharmaceutical and biomedical analysis 182 113138 - 113138 2020/04 [Refereed]
     
    Circulating tumor cells (CTCs) are involved in metastasis; thus, one of the most important approaches for identifying metastatic cancer is to detect CTCs in blood. In the present study, we examined whether directly analyzing cells with capillary electrophoresis (CE) could distinguish cancer cells from normal cells, based on differences in cell surface glycosylation. We compared human colorectal cancer (CRC) cell lines to a normal colon epithelium cell line. Our results demonstrated that direct CE analysis could successfully distinguish between CRC and normal cells with high reproducibility, based on migration times. We found that the weighted-average migration time was significantly shorter for CRC cells than for normal cells. Next, we observed changes in the electrophoretic behaviors of CRC cells by adding five different types of lectins. When Aleuria aurantia lectin was added, migration delays were observed in CRC cells, but not in normal colon cells. Therefore, by focusing on shifts in migration time after adding specific lectins, we could distinguish cancer cells from normal cells. These findings suggested that this diagnostic method of directly analyzing cells with CE after adding specific lectin(s) could be useful for detecting the difference in the sugar moieties on a surface of normal and cancer cells.
  • Tetsushi Yamamoto; Hideki Takakura; Kuniko Mitamura; Atsushi Taga
    Biochemical and biophysical research communications 526 (1) 55 - 61 2020/03 [Refereed]
     
    Enhanced expression of cyclophilin A (CypA) in colorectal cancer (CRC) was reported; however, how CypA influences CRC progression is not clear. Therefore, we examine the effects of CypA on CRC cell progression. Knockdown of CypA in SW480 cells significantly inhibited cell migration and invasion but had no effect on cell proliferation. In addition, upregulation of E-cadherin and downregulation of N-cadherin and Snail expression were observed by CypA knockdown. These results suggested that CypA knockdown inhibited cell migration and invasion by suppressing epithelial-mesenchymal transition. CypA knockdown was also associated with increased p38 phosphorylation, and the p38 inhibitor treatment led to increase in the number of invasive CypA-knockdown SW480 cells. Therefore, CypA may be a potential therapeutic target in preventing CRC metastasis.
  • Kanta Sato; Noriaki Nagai; Tetsushi Yamamoto; Kuniko Mitamura; Atsushi Taga
    International journal of molecular sciences 20 (20) 2019/10 [Refereed]
     
    The incidence of diabetes mellitus (DM) is increasing rapidly and is associated with changes in dietary habits. Although restrictions in the use of sweeteners may prevent the development of DM, this might reduce the quality of life of patients with DM. Therefore, there has been a great deal of research into alternative sweeteners. In the search for such sweeteners, we analyzed the carbohydrate content of maple syrup and identified a novel oligosaccharide composed of fructose and glucose, linked at the C-4 of glucose and the C-6 of fructose. This oligosaccharide inhibited the release of fructose from sucrose by invertase (IC50: 1.17 mmol/L) and the decomposition of maltose by α-(1-4) glucosidase (IC50: 1.72 mmol/L). In addition, when orally administered together with sucrose to rats with DM, the subsequent plasma glucose concentrations were significantly lower than if the rats had been administered sucrose alone, without having any effect on the insulin concentration. These findings suggest that this novel oligosaccharide might represent a useful alternative sweetener for inclusion in the diet of patients with DM and may also have therapeutic benefits.
  • Matsumoto T; Yamazaki W; Jo A; Ogawa S; Mitamura K; Ikegawa S; Higashi T
    Analytical Sciences Springer Science and Business Media LLC 34 (9) 1003 - 1009 0910-6340 2018/06 [Refereed]
  • Yamamoto T; Nakanishi S; Mitamura K; Taga A
    International journal of molecular medicine 42 (2) 1168 - 1180 1107-3756 2018/05 [Refereed]
     
    Collagen peptides (CPs), derived by hydrolyzing collagen with chemicals or enzymes, are often used as functional materials, due to their various bioactivities and high bioavailability. A previous study by our group reported that collagen from soft‑shelled turtle, Pelodiscus sinensis, induces keratinocytes to undergo epithelial‑mesenchymal transition and facilitates wound healing. Therefore, CPs derived from soft‑shelled turtle collagen may have useful effects on the skin. In the present study, the functional effects of CPs on human skin were examined by analyzing CP‑treated human keratinocytes with a shotgun liquid chromatography/mass spectrometry‑based global proteomic approach. A semi‑quantitative method based on spectral counting was applied and 211 proteins that exhibited >2‑fold changes in expression after CP treatment were successfully identified. Based on a Gene Ontology analysis, the functions of these proteins were indicated to be closely linked with protein processing. In addition, CP treatment significantly increased the expression of calpain‑1, a calcium‑dependent intracellular cysteine protease. Furthermore, CP‑treated keratinocytes exhibited elevated interleukin (IL)‑1α and IL‑8 expression and reduced IL‑6 expression. CPs also induced the expression of proteins implicated in cell‑cell adhesion and the skin barrier. Therefore, CPs from soft‑shelled turtle may provide significant benefits for maintaining the biological environment of the skin, and may be useful as components of pharmaceuticals and medical products.
  • Noriaki Nagai; Tetsushi Yamamoto; Kuniko Mitamura; Atsushi Taga
    Biomedical Reports Spandidos Publications 7 (5) 445 - 450 2049-9442 2017/11 [Refereed]
     
    Streptozotocin (STZ)-induced diabetic rats (STZ rats) were used to investigate diabetic cataracts. In the current study, a shotgun liquid chromatography (LC)/mass spectrom-etry (MS)-based global proteomic analysis method was used to examine the mechanism of lens opacification as a result of hyperglycemia in STZ rats. The 6-week old Wistar rats were injected with STZ for 2 days (100 mg/kg/day, i.p.) and housed for 3 weeks. The plasma glucose levels were identified to be significantly higher when compared with the normal rats and insulin was not detected in the STZ rats. Furthermore, opacification of the cortical epithelium was observed in the lenses of STZ rats. A total of 235 proteins were identified in the lenses of the STZ rats and 229 in the lenses of the normal rats. A label-free semi-quantitative method, based on spectral counting, identified 52 proteins that were differentially expressed in the lenses of STZ rats compared with normal rats. In particular, superoxide dismutase, which is a critical antioxidant enzyme that detoxifies superoxide through redox cycling, was downregulated when analyzed by the semi-quantitative method. In addition, phosphorylated-p38, which is important in the signaling pathway involved in the oxidative stress response, was significantly increased in the lenses of STZ rats when compared with normal rats (P< 0.05). Thus, the changes in protein expression were evaluated in the lenses of STZ rats using a shotgun LC/MS-based global proteomic analysis approach, and a decrease in antioxidant enzymes and an increase in oxidative stress were identified in the lenses of STZ rats. Further studies are required to examine the role of these proteins in the onset or progression of diabetic cataracts.
  • Yamamoto T; Nakanishi S; Mitamura K; Taga A
    Journal of biomedical materials research. Part B, Applied biomaterials 106 (6) 2403 - 2413 1552-4973 2017/11 [Refereed]
     
    Soft-shelled turtles (Pelodiscus sinensis) are widely distributed in some Asian countries, and we previously reported that soft-shelled turtle tissue could be a useful material for collagen. In the present study, we performed shotgun liquid chromatography (LC)/mass spectrometry (MS)-based global proteomic analysis of collagen-administered human keratinocytes to examine the functional effects of collagen from soft-shelled turtle on human skin. Using a semiquantitative method based on spectral counting, we were able to successfully identify 187 proteins with expression levels that were changed more than twofold by the administration of collagen from soft-shelled turtle. Based on Gene Ontology analysis, the functions of these proteins closely correlated with cell-cell adhesion. In addition, epithelial-mesenchymal transition was induced by the administration of collagen from soft-shelled turtle through the down-regulation of E-cadherin expression. Moreover, collagen-administered keratinocytes significantly facilitated wound healing compared with nontreated cells in an in vitro scratch wound healing assay. These findings suggest that collagen from soft-shelled turtle provides significant benefits for skin wound healing and may be a useful material for pharmaceuticals and medical care products. © 2017 The Authors Journal of Biomedical Materials Research Part B: Applied Biomaterials Published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2403-2413, 2018.
  • Tetsushi Yamamoto; Kanta Sato; Yuika Kubota; Kuniko Mitamura; Atsushi Taga
    Biomedical Reports Spandidos Publications 7 (1) 6 - 10 2049-9442 2017 [Refereed]
     
    Maple syrup is a natural sweetener that is commonly consumed worldwide. While maple syrup mainly comprises sucrose, it also contains phytochemicals that present various biological effects. Maple syrup is made by boiling down sap, and its color and composition vary in accordance with the sap collection season. Typically, seasonal progression is associated with darker syrup color, and antioxidant activity is proportional to the increasingly dark color. The authors previously reported that maple syrup demonstrated inhibitory effects on colorectal cancer cell growth and invasion, which correlated with darker maple syrup color. In the present study, they examined the effects of two different grades of maple syrup on gastrointestinal cancer cell proliferation, to investigate whether the dark-color maple syrup was suitable as a phytomedicine for gastrointestinal cancer treatment. Administration of dark-color maple syrup significantly inhibited gastrointestinal cancer cell growth as compared to non-treated cancer cells. Moreover, administration of dark-color maple syrup clearly inhibited protein kinase B (AKT) phosphorylation and did not impact mitogen-associated protein kinase phosphorylation. These data suggested that dark-color maple syrup may inhibit cell proliferation through suppression of AKT activation and, thus, may be suitable as a phytomedicine for gastrointestinal cancer treatment.
  • Tetsushi Yamamoto; Mitsuhiro Kudo; Wei-Xia Peng; Hideyuki Takata; Hideki Takakura; Kiyoshi Teduka; Takenori Fujii; Kuniko Mitamura; Atsushi Taga; Eiji Uchida; Zenya Naito
    TUMOR BIOLOGY SAGE PUBLICATIONS LTD 37 (10) 13595 - 13606 1010-4283 2016/10 [Refereed]
     
    Colorectal cancer (CRC) is one of the most common cancers worldwide, and many patients are already at an advanced stage when they are diagnosed. Therefore, novel biomarkers for early detection of colorectal cancer are required. In this study, we performed a global shotgun proteomic analysis using formalin-fixed and paraffin-embedded (FFPE) CRC tissue. We identified 84 candidate proteins whose expression levels were differentially expressed in cancer and non-cancer regions. A label-free semiquantitative method based on spectral counting and gene ontology (GO) analysis led to a total of 21 candidate proteins that could potentially be detected in blood. Validation studies revealed cyclophilin A, annexin A2, and aldolase A mRNA and protein expression levels were significantly higher in cancer regions than in non-cancer regions. Moreover, an in vitro study showed that secretion of aldolase A into the culture medium was clearly suppressed in CRC cells compared to normal colon epithelium. These findings suggest that decreased aldolase A in blood may be a novel biomarker for the early detection of CRC.
  • Rika Satoh; Hiroaki Ogata; Tetsuya Saito; Biao Zhou; Kaoru Omura; Satoshi Kurabuchi; Kuniko Mitamura; Shigeo Ikegawa; Lee R. Hagey; Alan F. Hofmann; Takashi Iida
    LIPIDS SPRINGER HEIDELBERG 51 (6) 757 - 768 0024-4201 2016/06 [Refereed]
     
    Two major bile acids were isolated from the gallbladder bile of two hornbill species from the Bucerotidae family of the avian order Bucerotiformes Buceros bicornis (great hornbill) and Penelopides panini (Visayan tarictic hornbill). Their structures were determined to be 3 alpha,7 alpha,24-dihydroxy-5 beta-cholestan-27-oic acid and its 12 alpha-hydroxy derivative, 3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-27-oic acid (varanic acid, VA), both present in bile as their corresponding taurine amidates. The four diastereomers of varanic acid were synthesized and their assigned structures were confirmed by X-ray crystallographic analysis. VA and its 12-deoxy derivative were found to have a (24R,25S)-configuration. 13 additional hornbill species were also analyzed by HPLC and showed similar bile acid patterns to B. bicornis and P. panini. The previous stereochemical assignment for (24R,25S)-VA isolated from the bile of varanid lizards and the Gila monster should now be revised to the (24S,25S)-configuration.
  • Rika Sato (nee Okihara); Tetsuya Saito; Hiroaki Ogata; Naoya Nakane; Kazunari Namegawa; Shoutaro Sekiguchi; Kaoru Omura; Satoshi Kurabuchi; Kuniko Mitamura; Shigeo Ikegawa; Jan Raines; Lee R. Hagey; Alan F. Hofmann; Takashi Iida
    STEROIDS ELSEVIER SCIENCE INC 107 112 - 120 0039-128X 2016/03 [Refereed]
     
    Bile alcohols and bile acids from gallbladder bile of the Arapaima gigas, a large South American freshwater fish, were isolated by reversed-phase high-performance liquid chromatography. The structures of the major isolated compounds were determined by electrospray-tandem mass spectrometry and nuclear magnetic resonance using H-1- and C-13-NMR spectra. The novel bile salts identified were six variants of 2-hydroxy bile acids and bile alcohols in the 5 alpha- and 5 beta-series, with 29% of all compounds having hydroxylation at C-2. Three C27 bile alcohols were present (as ester sulfates): (24 xi,25 xi)-5 alpha-cholestan-2 alpha,3 alpha,7 alpha,12 alpha,24,26-hexol; (25 xi)-5 beta-cholestan-2 beta,3 alpha,7 alpha,12 alpha,26,27-hexol, and (25 xi)-5 alpha-cholestan-2 alpha,3 alpha,7 alpha,12 alpha,26,27-hexol. A single C27 bile acid was identified: (25 xi)-2 alpha,3 alpha,7 alpha,12 alpha-tetrahydroxy-5 alpha-cholestan-26-oic acid, present as its taurine conjugate. Two novel C-24 bile acids were identified: the 2 alpha-hydroxy derivative of allochenodeoxycholic acid and the 2 beta-hydroxy derivative of cholic acid, both occurring as taurine conjugates. These studies extend previous work in establishing the natural occurrence of novel 2 alpha- and 2 beta-hydroxy-C-24 and C-27 bile acids as well as C-27 bile alcohols in both the normal (5 beta) as well as the (5 alpha) "alio" A/B-ring juncture. The bile salt profile of A. gigas appears to be unique among vertebrates. (C) 2016 Published by Elsevier Inc.
  • Tetsushi Yamamoto; Kentaro Uemura; Yuki Sawashi; Kuniko Mitamura; Atsushi Taga
    JOURNAL OF OLEO SCIENCE JAPAN OIL CHEMISTS SOC 65 (2) 169 - 175 1345-8957 2016/02 [Refereed]
     
    Soft-shelled turtles (Pelodiscus sinensis) are widely distributed in some Asian countries, and parts of this turtle contain abundant collagen. In this study, we optimized a method for extracting collagen from the soft-shelled turtle. We used three types of solvent and four extraction conditions to determine an effective collagen extraction method, which was extraction at 37 degrees C with acetic acid after hydrochloric acid pretreatment. Next, we extracted collagen from three regions in the soft-shelled turtle: muscle, skin, and an area of soft tissue in the periphery of the turtle shell known in Japan and China as the "emperor." We determined that emperor tissue yielded the highest concentration and purity of collagen. We then optimized the pretreatment method for extraction from emperor tissue by using formic acid instead of hydrochloric acid, and the amount of extracted collagen increased by approximately 1.3-fold. Finally, we identified the optimal solvent out of four types of organic acid for collagen extraction from emperor tissue; the amount of extracted collagen from emperor tissue increased approximately 3-fold when citric acid was used as the extraction solvent instead of acetic acid. Emperor tissue can regenerate; thus, it is possible to obtain collagen from the emperor repeatedly without killing the turtle. Our findings suggest that the emperor tissue of soft-shelled turtles may be a good source of collagen for pharmaceutical and cosmetic applications.
  • Miyuki Mabuchi; Tadashi Shimizu; Masahiro Ueda; Kuniko Mitamura; Shigeo Ikegawa; Akito Tanaka
    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS PERGAMON-ELSEVIER SCIENCE LTD 25 (14) 2788 - 2792 0960-894X 2015/07 [Refereed]
     
    Solid materials for affinity resins bearing long PEG spacers between a functional group used for immobilization of a bio-active compound and the solid surface were synthesized to capture not only small target proteins but also large and/or complex target proteins. Solid materials with PEG1000 or PEG2000 as spacers, which bear a benzenesulfonamide derivative, exhibited excellent selectivity between the specific binding protein carbonic anhydrase type II (CAII) and non-specific ones. These materials also exhibited efficacy in capturing a particular target at a maximal amount. Affinity resins using solid materials with PEG1000 or PEG2000 spacers, bear a FK506 derivative, successfully captured the whole target complex of specific binding proteins at the silver staining level, while all previously known affinity resins with solid materials failed to achieve this objective. These novel affinity resins captured other specific binding proteins such as dynamin and neurocalcin delta as well. (C) 2015 Elsevier Ltd. All rights reserved.
  • Tetsushi Yamamoto; Kentaro Uemura; Kaho Moriyama; Kuniko Mitamura; Atsushi Taga
    ONCOLOGY REPORTS SPANDIDOS PUBL LTD 33 (4) 1579 - 1584 1021-335X 2015/04 [Refereed]
     
    Maple syrup is a natural sweetener consumed by individuals of all ages throughout the world. Maple syrup contains not only carbohydrates such as sucrose but also various components such as organic acids, amino acids, vitamins and phenolic compounds. Recent studies have shown that these phenolic compounds in maple syrup may possess various activities such as decreasing the blood glucose level and an anticancer effect. In this study, we examined the effect of three types of maple syrup, classified by color, on the cell proliferation, migration and invasion of colorectal cancer (CRC) cells in order to investigate whether the maple syrup is suitable as a phytomedicine for cancer treatment. CRC cells that were administered maple syrup showed significantly lower growth rates than cells that were administered sucrose. In addition, administration of maple syrup to CRC cells caused inhibition of cell invasion, while there was no effect on cell migration. Administration of maple syrup clearly inhibited AKT phosphorylation, while there was no effect on ERK phosphorylation. These data suggest that maple syrup might inhibit cell proliferation and invasion through suppression of AKT activation and be suitable as a phytomedicine for CRC treatment, with fewer adverse effects than traditional chemotherapy.
  • Noriaki Nagai; Tetsushi Yamamoto; Wataru Tanabe; Yoshimasa Ito; Satoshi Kurabuchi; Kuniko Mitamura; Atsushi Taga
    JOURNAL OF OLEO SCIENCE JAPAN OIL CHEMISTS SOC 64 (3) 331 - 335 1345-8957 2015/03 [Refereed]
     
    We investigate whether maple syrup is a suitable sweetener in the management of type 2 diabetes using the Otsuka Long-Evans Tokushima Fatty (OLETF) rat. The enhancement in plasma glucose (PG) and glucose absorption in the small intestine were lower after the oral administration of maple syrup than after sucrose administration in OLETF rats, and no significant differences were observed in insulin levels. These data suggested that maple syrup might inhibit the absorption of glucose from the small intestine and preventing the enhancement of PG in OLETF rats. Therefore, maple syrup might help in the prevention of type 2 diabetes.
  • Tatsuya Higashi; Ayaka Goto; Misato Morohashi; Shoujiro Ogawa; Kenji Komatsu; Takahiro Sugiura; Tetsuya Fukuoka; Kuniko Mitamura
    JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES ELSEVIER SCIENCE BV 969 230 - 234 1570-0232 2014/10 [Refereed]
     
    The quantification of plasma 25-hydroxyvitamin D-3 3-sulfate [25(OH)D3S] is expected to be helpful in the assessment of the vitamin D status, especially for infants. In this study, a simple and sensitive method for the quantification of 25(OH)D3S in plasma using liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) has been developed and validated. The plasma was deproteinized with acetonitrile, purified using an Oasis(R) HLB cartridge, and subjected to LC/ESI-MS/MS operating in the negative-ion mode. Quantification was based on the selected reaction monitoring, and deuterated 25(OH)D3S was used as the internal standard. This method enabled the reproducible (intra- and inter-assay relative standard deviations, 7.9% or lower) and accurate (analytical recovery, 95.8-105.3%) quantification of the plasma 25(OH)D3S using a 20-mu L sample, and the limit of quantification was 2.5 ng/mL. The developed method was applied to the determination of plasma 25(OH)D3S in infants; the result revealed that preterm infants have lower plasma 25(OH)D3S concentrations. (C) 2014 Elsevier B.V. All rights reserved.
  • Kuniko Mitamura; Rika Satoh (Nee Okihara); Mami Kamibayashi; Kanta Sato; Takashi Iida; Shigeo Ikegawa
    STEROIDS ELSEVIER SCIENCE INC 85 18 - 29 0039-128X 2014/07 [Refereed]
     
    A liquid chromatography (LC)/electrospray ionization (ESI)-mass spectrometry (MS) method for the direct determination of eighteen tetrahydrocorticosteroid sulfates in human urine has been developed. The analytes were 3- and 21-monosulfates and 3,21-disulfates of tetrahydrocortisol (THF), tetrahydrocortisone (THE), tetrahydro-11-deoxycortisol (THS), and their corresponding 5 alpha-H stereoisomers. The mass spectrometric behavior of these sulfates in negative-ion ESI-MS/MS revealed the production of intense structure specific product ions within the same group of sulfates and permitted distinction between regioisomeric sulfates by collision-induced fragmentation with the MS/MS technique using a linear ion-trap instrument. For the quantitative analysis, selected reaction monitoring analysis in the negative-ion detection mode using triple-stage quadrupole mass spectrometer was performed by monitoring transitions from [M-H](-) to the most abundant product ion of each tetrahydrocorticosteroid sulfate. After addition of 3- and 21-monosulfates of [2,2,3 beta,4,4-d(5)]-THE, -THE, and -THS as internal standards, urine sample was applied to a solid phase extraction using a lipophilic-weak anion exchange cartridge column, and then analyzed by LC/ESI-MS/MS. The method had satisfactory performance in terms of intra- and inter-assay precision (less than 9.7% and 9.6%, respectively), and accuracy (91.2-108.2%). The limit of quantification was lower than 2.5 ng/mL for all sulfates examined. We applied this method to determine the concentration of eighteen tetrahydrocorticosteroid sulfates in the urine of healthy subjects. Thus, we have developed a sensitive, precise and accurate assay for urinary tetrahydrocorticosteroid sulfates that should be useful for clinical and biological studies. (C) 2014 Elsevier Inc. All rights reserved.
  • Rika Satoh (nee Okihara); Tetsuya Saito; Hiroaki Ogata; Ayumi Ohsaki; Takashi Iida; Kiyoshi Asahina; Kuniko Mitamura; Shigeo Ikegawa; Alan F. Hofmann; Lee R. Hagey
    STEROIDS ELSEVIER SCIENCE INC 80 15 - 23 0039-128X 2014/02 [Refereed]
     
    Two novel N-acyl amidated bile acids, N-methyltaurine conjugated cholic acid and N-methyltaurine conjugated deoxycholic acid, were found to be major biliary bile acids in two species of angelfish the regal (Pygoplites diacanthus) and the blue-girdled (Pomacanthus navarchus) angelfish. The identification was based on their having MS and NMR spectra identical to those of synthetic standards. A survey of biliary bile acids of 10 additional species of angelfish found 7 with N-methyltaurine conjugation. In all 12 species, conjugated deoxycholic acid (known to be formed by bacterial 7-dehydroxylation of cholic acid) was a major bile acid. In all previous studies of biliary bile acids in fish, deoxycholic acid has been present in only trace proportions. In addition, bile acid conjugation with N-methyltaurine has not been detected previously in any known vertebrate. N-methyltaurine conjugated bile acids are resistant to bacterial deconjugation and dehydroxylation, and such resistance to bacterial enzymes should aid in the maintenance of high concentrations of bile acids during lipid digestion. Our findings suggest that these species of angelfish have a novel microbiome in their intestine containing anaerobic bacteria, and describe the presence of N-methyltaurine conjugated bile acids that are resistant to bacterial attack. (C) 2013 Elsevier Ltd. All rights reserved.
  • Shoujiro Ogawa; Biao Zhou; Yusuke Kimoto; Kaoru Omura; Akiko Kobayashi; Tatsuya Higashi; Kuniko Mitamura; Shigeo Ikegawa; Lee R. Hagey; Alan F. Hofmann; Takashi Iida
    STEROIDS ELSEVIER SCIENCE INC 78 (9) 927 - 937 0039-128X 2013/09 [Refereed]
     
    This paper describes a method for the chemical synthesis of 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one (la) and its biological precursor, 7 alpha-hydroxy-4-cholesten-3-one (1b), both of which are key intermediates in the major pathway of bile acid biosynthesis from cholesterol. The principal reactions involved were (1) building of the cholesterol (iso-octane) side chain by 3-carbon elongation of the cholane (iso-pentane) one, (2) oxidation sequence to transform the 3 alpha-hydroxy group of the steroidal A/B-ring to the desired 4-en-3-one system, and (3) appropriate protection strategy for hydroxy groups in the positions at C-7 and C-12 in the steroid nucleus. The absolute structure of 1a and 1b were confirmed by NMR and X-ray crystallography. The targeted compounds 1a and 1b, prepared in 11 steps from 2a and 2b respectively, should be useful for biochemical studies of bile acid biosynthesis or clinical studies of bile acid metabolism, as plasma levels of 1b (also termed C4) have been shown to correlate highly with the rate of bile acid biosynthesis in man. (C) 2013 The Authors. Published by Elsevier Inc. All rights reserved.
  • Jason M. Ridlon; Shigeo Ikegawa; Joao M. P. Alves; Biao Zhou; Akiko Kobayashi; Takashi Iida; Kuniko Mitamura; Genzoh Tanabe; Myrna Serrano; Ainee De Guzman; Patsy Cooper; Gregory A. Buck; Phillip B. Hylemon
    JOURNAL OF LIPID RESEARCH AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 54 (9) 2437 - 2449 0022-2275 2013/09 [Refereed]
     
    Clostridium scindens American Type Culture Collection 35704 is capable of converting primary bile acids to toxic secondary bile acids, as well as converting glucocorticoids to androgens by side-chain cleavage. The molecular structure of the side-chain cleavage product of cortisol produced by C. scindens was determined to be 11 beta-hydroxyandrost-4-ene-3,17-dione (11 beta-OHA) by high-resolution mass spectrometry, H-1 and C-13 NMR spectroscopy, and X-ray crystallography. Using RNA-Seq technology, we identified a cortisol-inducible (similar to 1,000-fold) operon (des ABCD) encoding at least one enzyme involved in anaerobic side-chain cleavage. The des C gene was cloned, overexpressed, purified, and found to encode a 20 alpha-hydroxysteroid dehydrogenase (HSDH). This operon also encodes a putative "transketolase" (des AB) hypothesized to have steroid-17,20-desmolase/oxidase activity, and a possible corticosteroid transporter (des D). RNA-Seq data suggests that the two-carbon side chain of glucocorticords may feed into the pentose-phosphate pathway and are used as a carbon source. The 20 alpha-HSDH is hypothesized to function as a metabolic "rheostat" controlling rates of side-chain cleavage. Phylogenetic analysis suggests this operon is rare in nature and the des C gene evolved from a gene encoding threonine dehydrogenase.jlr The physiological effect of 11 beta-OHAD on the host or other gut microbes is currently unknown.
  • Tatsuya Higashi; Katsumi Kawasaki; Nagisa Matsumoto; Shoujiro Ogawa; Kuniko Mitamura; Shigeo Ikegawa
    CHEMICAL & PHARMACEUTICAL BULLETIN PHARMACEUTICAL SOC JAPAN 61 (3) 326 - 332 0009-2363 2013/03 [Refereed]
     
    A derivatization procedure with (3-dimethylaminophenyl)dihydroxyborane (DAPB) was introduced to enhance the detectability of steroids having a vicinal diol in LC/electrospray ionization (ESI)-MS/MS. DAPB reacted with the vicinal diol on the steroids [4 beta-hydroxycholesterol (4-HCh), pregnanetriol (PT) and 20R,22R-dihydroxycholesterol] in pyridine at 50 degrees C within 1h. The resulting DAPB-derivatives were highly responsive in ESI-MS operating in the positive-ion mode and gave characteristic product ions during MS/MS, which enabled sensitive detection using a selected reaction monitoring mode; the detection responses of the DAPB-derivatives were increased by 20-160-fold over those of the intact steroids and the limits of detection were in the low femtomole or attomole range. The derivatization procedure was successfully applied to biological sample analysis; the derivatization followed by LC/ESI-MS/MS enabled the specific detection of trace amounts of 4-HCh in human plasma and PT in human urine with a small sample volume, simple pretreatment and short chromatographic run time.
  • Kuniko Mitamura; Takayuki Mabuchi; Kaori Nagae; Masataka Nakajima; Rina Matsumoto; Sachi Fujioka; Kanta Sato; Rika Satoh (nee Okihara); Takashi Iida; Shoujiro Ogawa; Alan F. Hofmann; Shigeo Ikegawa
    STEROIDS ELSEVIER SCIENCE INC 77 (13) 1423 - 1437 0039-128X 2012/11 [Refereed]
     
    The accurate analysis of trace components in complex biological matrices requires the use of reliable internal standards. For liquid chromatography/mass spectrometry analysis, the stable isotope-labeled analogues of the analyte molecules are the most appropriate internal standards. In this paper the synthesis of the 3- and 21-monosulfates of allo-tetrahydrocorticosteroids labeled with four or five deuterium atoms is described. The principal reactions used were (1) hydrogen-deuterium exchange reaction of active methylene groups adjacent to 3- and 11-oxo group of 17,20;20,21-bismethylenedioxy derivatives of 5 alpha-3-ketosteroids and/or 5 alpha-11-ketosteroids with NaOD in CH3OD followed by reduction with NaBD4, (2) epimerization of the 3 beta-hydroxy group into a 3 alpha configuration, (3) sulfation of hydroxy groups at C-3 or C-21 in the resulting substrates with sulfur trioxide-trimethylamine complex, and (4) removal of 17,20;20,21-bismethylenedioxy groups with hydrogen fluoride in ethanol. Isotopic purity was found to be satisfactory by MS. and NMR properties of the new compounds were tabulated. The labeled compounds can be used as internal standards in liquid chromatography/mass spectrometry assays for clinical and biochemical studies. (C) 2012 Elsevier Inc. All rights reserved.
  • Lee R. Hagey; Shoujiro Ogawa; Narimi Kato; Rika Satoh (nee Okihara); Mizuho Une; Kuniko Mitamura; Shigeo Ikegawa; Alan F. Hofmann; Takashi Iida
    STEROIDS ELSEVIER SCIENCE INC 77 (13) 1510 - 1521 0039-128X 2012/11 [Refereed]
     
    A key intermediate in the biosynthetic pathway by which C-24 bile acids are formed from cholesterol has long been considered to be varanic acid. (24 xi,25 xi)-3 alpha,7 alpha,12 alpha-24-tetrahydroxy-5 beta-cholestan-27-oic acid. The (24R,25R)-epimer of this tetrahydroxy bile acid, in the form of its taurine N-acyl amidate, was thought to be the major biliary bile acid in lizards of the family Varanidae. We report here that a major biliary bile acid of three lizard species - the Komodo dragon (Varanus komodoensis), Gray's monitor (Varanus olivaceus), and the Gila monster (Heloderma suspectum) - is a novel epimer of varanic acid. The epimer was shown to be (24R,25S)-3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-27-oic acid (present in bile as its taurine conjugate). The structure was established by mass spectroscopy and by H-1 and C-13 nuclear magnetic spectroscopy, as well as by synthesis of the compound. (C) 2012 Elsevier Inc. All rights reserved.
  • Tatsuya Uebi; Yumi Itoh; Osamu Hatano; Ayako Kumagai; Masato Sanosaka; Tsutomu Sasaki; Satoru Sasagawa; Junko Doi; Keita Tatsumi; Kuniko Mitamura; Eiichi Morii; Katsuyuki Aozasa; Tomohiro Kawamura; Meinoshin Okumura; Jun Nakae; Hajime Takikawa; Toshio Fukusato; Minako Koura; Mayumi Nish; Anders Hamsten; Angela Silveira; Alejandro M. Bertorello; Kazuo Kitagawa; Yasuo Nagaoka; Hidehisa Kawahara; Takeshi Tomonaga; Tetsuji Naka; Shigeo Ikegawa; Noriyuki Tsumaki; Junichiro Matsuda; Hiroshi Takemori
    PLOS ONE PUBLIC LIBRARY SCIENCE 7 (5) e37803  1932-6203 2012/05 [Refereed]
     
    Salt-inducible kinase 3 (SIK3), an AMP-activated protein kinase-related kinase, is induced in the murine liver after the consumption of a diet rich in fat, sucrose, and cholesterol. To examine whether SIK3 can modulate glucose and lipid metabolism in the liver, we analyzed phenotypes of SIK3-deficent mice. Sik3(-/-) mice have a malnourished the phenotype (i.e., lipodystrophy, hypolipidemia, hypoglycemia, and hyper-insulin sensitivity) accompanied by cholestasis and cholelithiasis. The hypoglycemic and hyper-insulin-sensitive phenotypes may be due to reduced energy storage, which is represented by the low expression levels of mRNA for components of the fatty acid synthesis pathways in the liver. The biliary disorders in Sik3(-/-) mice are associated with the dysregulation of gene expression programs that respond to nutritional stresses and are probably regulated by nuclear receptors. Retinoic acid plays a role in cholesterol and bile acid homeostasis, wheras ALDH1a which produces retinoic acid, is expressed at low levels in Sik3(-/-) mice. Lipid metabolism disorders in Sik3(-/-) mice are ameliorated by the treatment with 9-cis-retinoic acid. In conclusion, SIK3 is a novel energy regulator that modulates cholesterol and bile acid metabolism by coupling with retinoid metabolism, and may alter the size of energy storage in mice.
  • Kuniko Mitamura; Naohiro Hori; Shiori Mino; Takashi Iida; Alan F. Hofmann; Shigeo Ikegawa
    CHEMISTRY AND PHYSICS OF LIPIDS ELSEVIER IRELAND LTD 165 (3) 261 - 269 0009-3084 2012/04 [Refereed]
     
    The 3-sulfates of the S-acyl glutathione (GSH) conjugates of five natural bile acids (cholic, chenodeoxycholic, deoxycholic, ursodeoxycholic, and lithocholic) were synthesized as reference standards in order to investigate their possible formation by a rat liver cytosolic fraction. Their structures were confirmed by proton nuclear magnetic resonance, as well as by means of electrospray ionization-linear ion-trap mass spectrometry with negative-ion detection. Upon collision-induced dissociation, structurally informative product ions were observed. Using a triple-stage quadrupole instrument, selected reaction monitoring analyses by monitoring characteristic transition ions allowed the achievement of a highly sensitive and specific assay. This method was used to determine whether the 3-sulfates of the bile acid-GSH conjugates (BA-GSH) were formed when BA-GSH were incubated with a rat liver cytosolic fraction to which 3'-phosphoadenosine 5'-phosphosulfate had been added. The S-acyl linkage was rapidly hydrolyzed to form the unconjugated bile acid. A little sulfation of the GSH conjugates occurred, but greater sulfation at C-3 of the liberated bile acid occurred. Sulfation was proportional to the hydrophobicity of the unconjugated bile acid. Thus GSH conjugates of bile acids as well as their C-3 sulfates if formed in vivo are rapidly hydrolyzed by cytosolic enzymes. (C) 2012 Elsevier Ireland Ltd. All rights reserved.
  • Kuniko Mitamura; Naohiro Hori; Takashi Iida; Mitsuyoshi Suzuki; Toshiaki Shimizu; Hiroshi Nittono; Kyoichi Takaori; Hajime Takikawa; Alan F Hofmann; Shigeo Ikegawa
    Steroids 76 (14) 1609 - 14 0039-128X 2011/12 [Refereed]
     
    Previous work from this laboratory has reported the biotransformation of bile acids (BA) into the thioester-linked glutathione (GSH) conjugates via the intermediary metabolites formed by BA:CoA ligase and shown that such GSH conjugates are excreted into the bile in healthy rats as well as rats dosed with lithocholic acid or ursodeoxycholic acid. To examine whether such novel BA-GSH conjugates are present in human bile, we determined the concentration of the GSH conjugates of the five BA that predominate in human bile. Bile was obtained from three infants (age 4, 10, and 13 months) and the BA-GSH conjugates quantified by means of liquid chromatography (LC)/electrospray ionization (ESI)-linear ion trap mass spectrometry (MS) in negative-ion scan mode, monitoring characteristic transitions of the analytes. By LC/ESI-MS, only primary BA were present in biliary BA, indicating that the dehydroxylating flora had not yet developed. GSH conjugates of chenodeoxycholic and lithocholic acid were present in concentrations ranging from 27 to 1120 pmol/ml, several orders of magnitude less than those of natural BA N-acylamidates. GSH conjugates were not present, however, in the ductal bile obtained from 10 adults (nine choledocholithiasis, one bile duct cancer). Our results indicate that BA-GSH conjugates are formed and excreted in human bile, at least in infants, although this novel mode of conjugation is a very minor pathway.
  • Shigeo Ikegawa; Kaori Nagae; Takayuki Mabuchi; Rika Okihara; Maki Hasegawa; Toshie Minematsu; Takashi Iida; Kuniko Mitamura
    STEROIDS ELSEVIER SCIENCE INC 76 (12) 1232 - 1240 0039-128X 2011/11 [Refereed]
     
    The 3- and 21-monosulfates of pentadeuterated 5 beta-tetrahydrocorticosteroides were synthesized, starting from cortisol and 11-deoxycotisol. The principal reactions used were (1) perdeuteration of the methylene groups adjacent to the 3-oxo group of 17,20:20,21-bismethylendioxy-5 beta-3-ketosteroids with NaOD in CH3OD followed by stereoselective reduction with NaBD4, (2) sulfation of hydroxy groups with sulfur trioxide-trimethylamine complex, and (3) removal of the 17,20:20,21-bismethylendioxy group with hydrogen fluoride. The labeled compounds can be used as internal standards in liquid chromatography/mass spectrometry assays for clinical and biochemical studies. (C) 2011 Elsevier Inc. All rights reserved.
  • Shoujiro Ogawa; Kuniko Mitamura; Shigeo Ikegawa; Matthew D. Krasowski; Lee R. Hagey; Alan F. Hofmann; Takashi Iida
    CHEMISTRY AND PHYSICS OF LIPIDS ELSEVIER IRELAND LTD 164 (5) 368 - 377 0009-3084 2011/07 [Refereed]
     
    The (25R)- and (25S)-epimers of C-27 3 alpha,7 alpha,12 alpha-trihydroxy-5 alpha-cholestan-27-oic acid as well as their corresponding N-acylamidate conjugates with glycine or taurine were prepared starting from cholic acid in 14 steps. The principal reactions involved were (1) reduction of a key intermediary C-24 allo-cholic acid performate with NaBH4/triethylamine/ethyl chloroformate, (2) iodination of the resulting 3,7,12-triformyloxy-5 alpha-cholan-24-ol with I-2/triphenylphosphine; (3) nucleophilic substitution of the iodo derivative with diethylmethyl malonate/NaH; and (4) hydrolysis of the resulting 3,7,12-triformyloxy-25-methyl-26,27-diethyl ester with KOH, followed by decarboxylation of the geminal dicarboxylic acid with LiCl. N-Acylamidation of the resulting (25R)/(25S)-3 alpha,7 alpha,12 alpha-trihydroxy-5 alpha-cholestan-27-oic acid mixture with glycine or taurine afforded the corresponding epimeric mixtures of the glycine and taurine conjugates. The (25R)- and (25S)-epimers of the three variants of unconjugated and conjugated 3 alpha,7 alpha,12 alpha-trihydroxy-5 alpha-cholestan-27-oic acid were efficiently separated by HPLC on a reversed-phase C-18 column and their structural characteristics, particularly the chiral center at C-25, delineated using I H and C-13 NMR. These synthetic compounds should be useful as authentic reference standards for establishing their presence in bile as well as being useful in studies on the biosynthesis of alto-bile acids from cholesterol. (C) 2011 Elsevier Ireland Ltd. All rights reserved.
  • Kuniko Mitamura; Toshihiro Sakai; Risa Nakai; Tateaki Wakamiya; Takashi Iida; Alan F. Hofmann; Shigeo Ikegawa
    ANALYTICAL AND BIOANALYTICAL CHEMISTRY SPRINGER HEIDELBERG 400 (7) 2061 - 2072 1618-2642 2011/06 [Refereed]
     
    Previous work from this laboratory has reported the chemical synthesis of N-acetylcysteine (NAC) conjugates of natural bile acids (BAs) and shown that such novel conjugates can be formed in vivo in rats to which NAC has been administered. The subsequent fate of such novel conjugates is not known. One possible biotransformation is sulfation, a major pathway for BAs N-acylamidates in patients with cholestatic liver disease. Here, we report the chemical synthesis of the 3-sulfates of the S-acyl NAC conjugates of five natural BAs (cholic, chenodeoxycholic, deoxycholic, ursodeoxycholic, and lithocholic). We also measured the sulfation of N-acetylcysteine-natural bile acid (BA-NAC) conjugates when they were incubated with a rat liver cytosolic fraction. The chemical structures of the BA-NAC 3-sulfates were confirmed by proton nuclear magnetic resonance, as well as by means of electrospray ionization-linear ion trap mass spectrometry with negative-ion detection. Upon collision-induced dissociation of singly and doubly charged deprotonated molecules, structurally informative product ions were observed. Using a triple-stage quadrupole instrument, selected reaction monitoring analyses by monitoring characteristic transition ions allowed the achievement of a highly sensitive and specific assay. When BA-NACs were incubated with a rat liver cytosolic fraction to which 3'-phosphoadenosine 5'-phosphosulfate was added, sulfation occurred, but the dominant reaction was hydrolysis of the S-acyl linkage to form the unconjugated BAs. Subsequent sulfation occurred at C-3 on the unconjugated BAs that had been formed from the BA-NACs. Such sulfation was proportional to the hydrophobicity of the unconjugated bile acid. Thus, NAC conjugates of BAs as well as their C-3 sulfates if formed in vivo are rapidly hydrolyzed by cytosolic enzymes.
  • Kuniko Mitamura; Eriko Aoyama; Toshihiro Sakai; Takashi Iida; Alan F. Hofmann; Shigeo Ikegawa
    ANALYTICAL AND BIOANALYTICAL CHEMISTRY SPRINGER HEIDELBERG 400 (7) 2253 - 2259 1618-2642 2011/06 [Refereed]
     
    Acyl-adenylates and acyl-CoA thioesters of bile acids (BAs) are highly electrophilic acyl-linked metabolites which can undergo transacylation reactions with amino and thiol groups of nucleophilic groups on acceptor molecules such as amino acids, peptides, and proteins. Here, non-enzymatic acylation at pH 7.4 of glycine, taurine, glutathione (GSH), and N-acetylcysteine (NAC) by cholyl-adenylate (CA-AMP) was compared with that mediated by cholyl-CoA thioester (CA-CoA) using a 1:1 mixture of stable isotopically labeled CA-AMP and unlabeled CA-CoA. The transacylation products of these substrates were analyzed by liquid chromatography/electrospray ionization linear ion-trap mass spectrometry in negative-ion detection mode. CA-AMP was more reactive than CA-CoA with the amino group of glycine or taurine than with the thiol group of GSH or NAC. In contrast, CA-CoA was more reactive than CA-AMP with the thiol group of GSH or NAC and was far less reactive with the amino group of glycine or taurine. These differences in the reactivity of CA-AMP as compared with that of CA-CoA towards amino and thiol groups may be attributed to the electrophilicity of the carbonyl carbon of these acyl-linked cholic acid metabolites and the nucleophilicity of the amino and thiol group in the bionucleophiles that were studied.
  • Genta Kakiyama; Shigeo Ikegawa; Kuniko Mitamura; Douglas M. Heuman; William M. Pandak; Shunlin Ren
    GASTROENTEROLOGY W B SAUNDERS CO-ELSEVIER INC 140 (5) S936 - S936 0016-5085 2011/05 [Refereed]
  • 池川 繁男; 堀 直宏; 三田村 邦子
    胆膵の病態生理 日本胆膵病態・生理研究会 27 (1) 23 - 27 2185-4564 2011/01 [Refereed]
  • Kuniko Mitamura; Naohiro Hori; Takashi Iida; Alan F. Hofmann; Shigeo Ikegawa
    STEROIDS ELSEVIER SCIENCE INC 76 (1-2) 68 - 77 0039-128X 2011/01 [Refereed]
     
    Acyl-adenylates and acyl-CoA thioesters of bile acids (BAs) are reactive acyl-linked metabolites that have been shown to acylate the thiol group of glutathione (GSH); the reaction is catalyzed by glutathione S-transferase (GST) and the product is a thioester-linked BA-GSH conjugate. Such GSH conjugates are present in bile in lithocholic acid and ursodeoxycholic acid dosed-rats. To determine whether such novel BA-GSH conjugates are present in the bile of normal rats, we first synthesized the GSH conjugates of the major and minor biliary BAs of the rat and defined their MS and proton NMR properties. We then analyzed the BA-GSH composition in the bile of anesthetized biliary fistula rats by means of liquid chromatographic separation and electrospray ionization-linear ion trap mass spectrometric detection in negative- and positive-ion scan modes, monitoring characteristic transitions of the analytes. GSH conjugates of cholic, omega-muricholic, hyodeoxycholic, deoxycholic. 12-oxolithocholic, and lithocholic acids were present with concentrations in the range of 1.4-2.8 nmol/ml, some four orders of magnitude less than those of natural BA N-acyl amidates. Our results indicate that BA-GSH conjugates are formed and excreted in bile in the healthy rat, although this novel mode of BA conjugation is a very minor pathway. (C) 2010 Elsevier Inc. All rights reserved.
  • Tatsuya Higashi; Yujin Shibayama; Takuya Ichikawa; Koichi Ito; Toshimasa Toyo'oka; Kazutake Shimada; Kuniko Mitamura; Shigeo Ikegawa; Hitoshi Chiba
    STEROIDS ELSEVIER SCIENCE INC 75 (4-5) 338 - 345 0039-128X 2010/04 [Refereed]
     
    Measurement of steroid levels in saliva has been proposed as a new laboratory tool for characterizing steroid metabolism, but it is not known whether the salivary levels of bile acids can be measured with accuracy and if so, whether such measurements provide information that is of clinical value. We developed and validated a sensitive and specific liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) method for the quantification of chenodeoxycholic acid (CDCA) and glycochenodeoxycholic acid (GCDCA), representative primary non-amidated and glycine-conjugated bile acids, in whole saliva. We also examined whether the salivary bile acid concentrations were dependent on the saliva flow rate, because this is a very important aspect in a discussion of the utility of salivary diagnostics. Saliva was deproteinized with ethanol and purified using a Strata-X cartridge. Bile acids were converted to their hydrazide derivatives using 2-hydrazinopyridine, and subjected to LC-MS/MS. Quantification was based on selected reaction monitoring using characteristic transitions, and deuterated CDCA and GCDCA were used as internal standards. This method allowed the reproducible and accurate quantification of the salivary bile acids using a 200-mu l sample and the limits of quantification for CDCA and GCDCA were 25 and 50 pg/ml, respectively. Using this method, the effect of increased saliva flow rate by gum-chewing on the salivary concentrations of CDCA and GCDCA was determined. The salivary level of GCDCA was significantly decreased by gum-chewing, whereas the concentration of CDCA remained constant. These results indicate that there is a good possibility that saliva may be a clinical tool for non-amidated bile acid testing. (C) 2010 Elsevier Inc. All rights reserved.
  • Rika Okihara; Kuniko Mitamura; Maki Hasegawa; Megumi Mori; Akina Muto; Genta Kakiyama; Shoujiro Ogawa; Takashi Iida; Miki Shimada; Nariyasu Mano; Shigeo Ikegawa
    CHEMICAL & PHARMACEUTICAL BULLETIN PHARMACEUTICAL SOC JAPAN 58 (3) 344 - 353 0009-2363 2010/03 [Refereed]
     
    Here, we describe the chemical synthesis of the complete sets of 18 novel 3- and 21-monosulfates and their double-conjugated form of tetrahydrocortisol (THF), tetrahydro-11-deoxycortisol (THS), and tetrahydrocortisone (THE) in the 5 alpha- and 5 beta-series. The principal reactions involved are: (1) selective protection of a specific hydroxy group in substrates; (2) catalytic hydrogenation at C-5 of Delta(4)-3-ketosteroids with 10% Pd(OH)(2)/C to yield 3-oxo-5 beta-steroids and reductive allomerization with 10% Pd/C to yield 3-oxo-5 alpha-isomers; (3) reduction of the resulting 3-oxo-5 beta- and 3-oxo-5 alpha-steroids to the corresponding 3 alpha-hydroxy-compounds with Zn(BH4)(2) and K-Selectride (R), respectively; and (4) sulfation of hydroxy groups at C-3 and/or C-21 in the tetrahydrocortico-steroid derivatives with sulfur trioxide-triethylamine complex.
  • Shigeo Ikegawa; Maki Hasegawa; Rika Okihara; Chikara Shimidzu; Hitoshi Chiba; Takashi Iida; Kuniko Mitamura
    ANALYTICAL CHEMISTRY AMER CHEMICAL SOC 81 (24) 10124 - 10135 0003-2700 2009/12 [Refereed]
     
    A liquid chromatography/electrospray ionization (ESI)-mass spectrometry (MS) method for the direct determination of 12 tetrahydrocorticosteroid glucuronides in human urine has been developed. The analytes were Sand 21-monoglucuronides of tetrahydrocortisol, tetrahydrocortisone, tetrahydro-11-deoxycortisol, and their 5 alpha-stereoisomers. The mass spectrometric behaviors of these glucuronides in negative-ion ESI-MS/MS revealed the production of intense, structure-specific product ions within the same group of glucuronides. Regioisomeric glucuronides could be distinguished by collision-induced dissociation and tandem mass spectrometry. Using a linear ion trap instrument operating in the negative-ion mode and by monitoring the transition ions of [M - H](-) -> [M - H - CH(2)O](-) for 3-monoglucuronides and [M - H](-) -> [M - H - CH(2)OG](-) for 21-monoglucuronides, a sensitive and specific assay was developed. Initial steps in the assay were a simple solid-phase extraction and the addition of [9,12,12,21,21-d(5)]-tetrahydrocortisone-3-glucuronide (prepared by enzyme-assisted synthesis) as an internal standard. The method was applied to determine the 12 tetrahydrocoticosteroid glucuronides in urine from healthy subjects and from patients with excessive cortisol production. The method described here appears to be useful for clinical and biochemical studies.
  • Kuniko Mitamura; Saai Watanabe; Toshihiro Sakai; Rika Okihara; Mitsuru Sogabe; Tateaki Wakamiya; Alan F. Hofmann; Shigeo Ikegawa
    JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES ELSEVIER SCIENCE BV 877 (25) 2630 - 2638 1570-0232 2009/09 [Refereed]
     
    N-Acetylcysteine (NAC) conjugates of the five major bile acids occurring in man were synthesized in order to investigate the possible formation in vivo of these conjugates. Upon collision-induced dissociation, structurally informative daughter ions were observed. The transformation of cholyl-adenylate and cholyl-CoA thioester into a N-acetyl-S-(cholyl)cysteine by rat hepatic glutathione S-transferase was confirmed by liquid chromatography/electrospray ionization-linear ion trap mass spectrometry (LC/ESI-MS(2)). Lithocholic acid was administered orally to bile duct-ligated rats that also received NAC intraperitoneally. The NAC conjugate of lithocholic acid was identified in urine by means of LC/ESI-MS(2). Rapid hydrolysis of the BA-NAC conjugates by rabbit liver carboxylesterase was found, demonstrating the possible labile nature of the NAC conjugates formed in the liver. (C) 2009 Elsevier B.V. All rights reserved.
  • Shigeo Ikegawa; Hiromi Ito; Motohiro Ohshima; Masako Maeda; Alan F. Hofmann; Kuniko Mitamura
    STEROIDS ELSEVIER SCIENCE INC 74 (9) 751 - 757 0039-128X 2009/09 [Refereed]
     
    In mammals, unconjugated bile acids formed in the intestine by bacterial deconjugation are reconjugated (N-acylamidated) with taurine or glycine during hepatocyte transport. Activation of the carboxyl group of bile acids to form acyl-adenylates is a likely key intermediate step in bile acid N-acylamidation. To gain more insight into the process of bile acid adenylate formation, we first synthesized the adenylates of five common, natural bile acids (cholic, deoxycholic, chenodeoxycholic, ursodeoxycholic, and lithocholic acid), and confirmed their structure by proton NMR. We then investigated adenylate formation by subcellular fractions of rat liver (microsomes, mitochondria, cytsol) using a newly developed LC method for quantifying adenylate formation. The highest activity was observed in the microsomal fraction. The reaction required Mg2+ and its optimum pH was about pH 7.0. In term of maximum velocity (V x) and the Michaelis constant (K), the catalytic efficiency of the enzyme under the conditions used was highest with cholic acid of the bile acids tested. The formation of cholyl-adenylate was strongly inhibited by lithocholic and deoxycholic acid, as well as by palmitic acid; ibuprofen and valproic acid were weak inhibitors. In cholestatic disease, such adenylate formation might lead to subsequent bile acid conjugation with glutathione or proteins. (C) 2009 Elsevier Inc. All rights reserved.
  • Kuniko Mitamura; Saai Watanabe; Yutaka Mitsumoto; Toshihiro Sakai; Mitsuru Sogabe; Tateaki Wakamiya; Shigeo Ikegawa
    ANALYTICAL BIOCHEMISTRY ACADEMIC PRESS INC ELSEVIER SCIENCE 384 (2) 224 - 230 0003-2697 2009/01 [Refereed]
     
    Acyl-adenylates and acyl-CoA thioesters of bile acids (BAs) are reactive acyl-linked metabolites that have been shown to undergo transacylation-type reactions with the thiol group of glutathione (GSH), leading to the formation of thioester-linked GSH Conjugates. In the current Study, we examined the transformation of cholyl-adenylate (CA-AMP) and cholyl-coenzyme A thioester (CA-CoA) into a cholyl-S-acyl GSH (CA-GSH) Conjugate by rat hepatic glutathione S-transferase (GST). The reaction product was analyzed by liquid chromatography (LC)/electrospray ionization (ESI)-linear ion trap mass spectrometry (MS). The GST-catalyzed formation of CA-GSH occurred with both CA-AMP and CA-CoA. Ursodeoxycholic acid, lithocholic acid, and 2,2,4,4-(2)H(4)-labeled lithocholic acid were administered orally to biliary fistula rats, and their corresponding GSH conjugates were identified in bile by LC/ESI-MS(2). These in vitro and in vivo studies confirm a new mode of BA Conjugation in which BAs are transformed into their GSH conjugates via their acyl-linked intermediary metabolites by the catalytic action of GST in the liver, and the GSH conjugates are then excreted into the bile. (C) 2008 Elsevier Inc. All rights reserved
  • Shoujiro Ogawa; Genta Kakiyama; Akina Muto; Atsuko Hosoda; Kuniko Mitamura; Shigeo Ikegawa; Alan F. Hofmann; Takashi Iida
    STEROIDS ELSEVIER SCIENCE INC 74 (1) 81 - 87 0039-128X 2009/01 [Refereed]
     
    Experiments were performed to compare the regioselective hydroxylation of the isopropyl C-H bond at. C-25 in 5 alpha-cholestan-3 beta-yl acetate by in situ generated dimethyldioxirane, methyl(trifluoromethyl)dioxirane, hexafluoro(dimethyl)dioxirane or ethyl(trifluoromethyl)dioxirane (ETDO). The dioxiranes were generated from the corresponding ketones and potassium peroxymonosulfate in aq. NaHCO(3), pH 7.5-8.0. Of the four dioxiranes examined, partially fluorinated, sterically bulky ETDO displayed the highest reactivity and regioselectivity. Using in situ generated ETDO, a facile, synthesis was developed for two naturally occurring oxysterols, i.e., 25-hydroxycholesterol, as well as its 3-sulfate (overall yield of the sulfate, 24%) and 24-oxocholesterol (16%), starting from cholesterol. (C) 2008 Published by Elsevier Inc.
  • Shigeo Ikegawa; Tetsushi Yamamoto; Hiromi Ito; Shunji Ishiwata; Toshihiro Sakai; Kuniko Mitamura; Masako Maeda
    JOURNAL OF LIPID RESEARCH AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 49 (11) 2463 - 2473 0022-2275 2008/11 [Refereed]
     
    Formation of covalently bound protein adducts with lithocholic acid (LCA) might explain LCA's known carcinogenic properties and hepatotoxicity. We performed studies aimed at isolating and identifying hepatic proteins tagged with LCA, presumably via the epsilon-amino group of lysine residues. Antibodies recognizing the 3 alpha-hydroxy-5 beta-steroid moiety of LCA were generated by immunizing rabbits with immunogens in which the carboxyl group of LCA was coupled to BSA via a 6-aminohexanoic acid and/or succinic acid spacer. The resulting antibodies reacted with N-alpha (t-butoxycarbonyl)-L-lysine-epsilon-LCA, the amidated and nonamidated forms of LCA, as well as synthetically prepared LCA adducts with ovalbumin and lysozyme. Proteins tagged with LCA in the liver of bile duct-ligated rats were isolated by immunoprecipitation using these antibodies. Proteins were isolated by two-dimensional electrophoresis, and their structure was identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and computer-assisted programs. Proteins labeled with LCA were Rab-3, Rab-12, Rab-16, and M-Ras. Rab proteins are Ras-like small GTP binding proteins that regulate vesicle trafficking pathways. The covalent binding of the Rab proteins with LCA may influence vesicular transport or binding of vesicles to their cognate membrane and may contribute to LCA-induced liver toxicity.
  • Shigeo Ikegawa; Tetsushi Yamamoto; Takahiro Miyashita; Rika Okihara; Shunji Ishmata; Toshihiro Sakai; Rung-Hwa Chong; Masako Maeda; Alan F. Hofmann; Kuniko Mitamura
    ANALYTICAL SCIENCES JAPAN SOC ANALYTICAL CHEMISTRY 24 (11) 1475 - 1480 0910-6340 2008/11 [Refereed]
     
    Reactive metabolic-modified proteins have been proposed to play an important role in the mechanism(s) of the hepatotoxicity and colon cancer of lithocholic acid (LCA). To identify cellular proteins chemically modified with LCA, we have generated a monoclonal antibody that recognizes the 3 alpha-hydroxy-5 beta-steroid moiety of LCA. The spleen cells from a BALB/c mouse, which was immunized with an immunogen in which the side chain of LCA was coupled to bovine serum albumin (BSA) via a succinic acid spacer, was fused with SP2/0 myeloma cells to generate antibody-secreting hybridoma clones. The resulting monoclonal antibody (gamma 2b, kappa) was specific to LCA-N-alpha-BOC-lysine as well as the amidated and nonamidated forms of LCA. The immunoblot enabled the detection of LCA residues anchored on BSA and lysozyme. The antibody will be useful for monitoring the generation, localization, and capture of proteins tagged with LCA, which may be the cause of LCA-induced toxicity.
  • Genta Kakiyama; Hideyuki Tamegai; Takashi Iida; Kuniko Mitamura; Shigeo Ikegawa; Takaaki Goto; Nariyasu Mano; Junichi Goto; Peter Holz; Lee R. Hagey; Alan F. Hofmann
    JOURNAL OF LIPID RESEARCH AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 48 (12) 2682 - 2692 0022-2275 2007/12 [Refereed]
     
    The major bile acids present in the gallbladder bile of the common Australian wombat (Vombatus ursinus) were isolated by preparative HPLC and identified by NMR as the taurine N-acylamidates of chenodeoxycholic acid ( CDCA) and 15 alpha-hydroxylithocholic acid (3 alpha,15 alpha-dihydroxy5 beta-cholan-24-oic acid). Taurine-conjugated CDCA constituted 78% of biliary bile acids, and (taurine-conjugated) 15 alpha-hydroxylithocholic acid constituted 11%. Proof of structure of the latter compound was obtained by its synthesis from CDCA via a Delta(14) intermediate. The synthesis of its C-15 epimer, 15 beta-hydroxylithocholic acid (3 alpha,15 beta-dihydroxy5 beta- cholan-24-oic acid), is also reported. The taurine conjugate of 15 alpha-hydroxylithocholic acid was synthesized and shown to have chromatographic and spectroscopic properties identical to those of the compound isolated frombile. It is likely that 15 alpha-hydroxylithocholic acid is synthesized in the wombat hepatocyte by 15 alpha-hydroxylation of lithocholic acid that was formed by bacterial 7 alpha-dehydroxylation of CDCA in the distal intestine. Thus, the wombat appears to use 15 alpha-hydroxylation as a novel detoxification mechanism for lithocholic acid.
  • Toshihiro Sakai; Kuniko Mitamura; Atsushi Taga; Susumu Honda; Shigeo Ikega
    BUNSEKI KAGAKU JAPAN SOC ANALYTICAL CHEMISTRY 56 (9) 713 - 720 0525-1931 2007/09 [Refereed]
     
    Affinity chromatography is a powerful method for protein separation. It is based on a specific interaction between an immobilized ligand and the target proteins to be separated. Since lithocholic acid (LCA), one of secondary bile acids, has been shown to exert its function as the ligand toward nuclear receptors and a membrane-type G protein-coupled receptor, the abilities of molecular recognition and acquisition of LCA may be applicable for ligands to capture unknown functional proteins by affinity chromatography. In this study, LCA was covalently bound to an activated agarose through a bridge introduced at the C-3 and C-24 positions. The affinity absorbents were applied to capture proteins in a rat mitochondrial fraction. Structure analysis of the captured proteins after SDS-PAGE separation was carried out by liquid chromatography/electrospray ionization-tandem mass spectrometry combination with computer-assisted programs, where carbamoyl phosphate synthase I, glutamate dehydrogenase, acyl-CoA dehydrogenase, enoyl-CoA hydratase, acetyl-CoA acyltransferase and aldehyde dehydrogenase were identified. Serum albumin and cytosolic glutathione S-transferase, which were contaminated in mitochondrial fraction, were also identified.
  • Kuniko Mitamura; Aki Horikawa; Yuko Yamane; Yukari Ikeda; Youichi Fuji; Kazutake Shimada
    BIOLOGICAL & PHARMACEUTICAL BULLETIN PHARMACEUTICAL SOC JAPAN 30 (9) 1653 - 1656 0918-6158 2007/09 [Refereed]
     
    A method for the determination of digoxin in human serum using a liquid ch rom atograp hy/electrosp ray ionization-tandem mass spectrometry (LGESI-MIS/MS) technique is reported. Digoxin and the internal standard, 121,21,22-(2) H-3]digoxin, were extracted from 250 mu l of human serum using a solid phase extraction cartridge (Oasis HLB) and analyzed by LG/ESI-MS/MS in the selected reaction monitoring mode. The intra- and inter-assay reproducibility and accuracy were satisfactory within the quantification range of 0.20-3.20ng/ml. The concentrations of digoxin in the serum samples obtained from digitalized patients (n=19) were in the range of 0.25-2.84 ng/ml, which were compared to those obtained by radioimmunoassay.
  • Kuniko Mitamura; Mitsuru Sogabe; Hironori Sakanashi; Saai Watanabe; Toshihiro Sakai; Yoshihiro Yamaguchi; Tateaki Wakamiya; Shigeo Ikegawa
    JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES ELSEVIER SCIENCE BV 855 (1) 88 - 97 1570-0232 2007/08 [Refereed]
     
    The formation of thioester-linked glutathione (GSH) conjugates of bile acids (BAs) is presumed to occur via trans-acylation reactions between GSH and reactive acyl-linked metabolites of BAs. The present study examines the chemical reactivity of cholyl-adenylate and cholyl-CoA thioester, acyl-linked metabolites of cholic acid (CA), with GSH to form CA-GSH conjugate in vitro. The authentic specimen of CA-GSH was synthesized along with GSH conjugates of four common BAs found in the human body. Their structures were confirmed by proton-nuclear magnetic resonance spectroscopy and electrospray ionization (ESI)-tandem mass spectrometry in positive- and negative-ion modes. Incubation of cholyl-adenylate or cholyl-CoA thioester with GSH was carried out at pH 7.5 and 37 degrees C for 30 min, with analysis of the reaction mixture by liquid chromatography/ESI-tandem mass spectrometry, where CA-GSH was detected on the product ion mass chrornatograms monitored with stable and abundant dehydrated positive-ion [M + H-H2O](+) at m/z 680.3 and fragmented negative-ion [GSH-H](-) at m/z 306.0, and was definitely identified by CID spectra by comparison with those of the authentic sample. The results show that both cholyl-adenylate and cholyl-CoA thioester are able to acylate GSH in vitro. (C) 2007 Elsevier B.V. All rights reserved.
  • Hirotoshi Fuda; Normal B. Javitt; Kuniko Mitamura; Shigeo Ikegawa; Charles A. Strott
    JOURNAL OF LIPID RESEARCH AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 48 (6) 1343 - 1352 0022-2275 2007/06 [Refereed]
     
    Oxysterols constitute a class of cholesterol derivatives that exhibit broad biological effects ranging from cytotoxicity to regulation of nuclear receptors. The role of oxysterols such as 7-ketocholesterol (7-KC) in the development of retinal macular degeneration and atheromatous lesions is of particular interest, but little is known of their metabolic fate. We establish that the steroid/sterol sulfotransferase SULT2B1b, known to efficiently sulfonate cholesterol, also effectively sulfonates a variety of oxysterols, including 7-KC. The cytotoxic effect of 7-KC on 293T cells was attenuated when these cells, which do not express SULT2B1b, were transfected with SULT2B1b cDNA. Importantly, protection from 7-KC-induced loss of cell viability with transfection correlated with the synthesis of SULT2B1b protein and the production of the 7-KC sulfoconjugate (7-KCS). Moreover, when 7-KCS was added to the culture medium of 293T cells in amounts equimolar to 7-KC, no loss of cell viability occurred. Additionally, MCF-7 cells, which highly express SULT2B1b, were significantly more resistant to the cytotoxic effect of 7-KC. We extended the range of oxysterol substrates for SULT2B1b to include 7 alpha/7 beta-hydroxycholesterol and 5 alpha,6 alpha/5 beta,6 beta-epoxycholesterol as well as the 7 alpha-hydroperoxide derivative of cholesterol. Thus, SULT2B1b, by acting on a variety of oxysterols, offers a potential pathway for modulating in vivo the injurious effects of these compounds.
  • K Mitamura; M Setaka; K Shimada; S Honma; M Namiki; E Koh; A Mizokami
    BIOMEDICAL CHROMATOGRAPHY JOHN WILEY & SONS LTD 19 (10) 796 - 801 0269-3879 2005/12 [Refereed]
     
    A promising liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) for analysis of the sulfates of 5 alpha-androgen, androsterone and epiandrosterone (A-S and EpiA-S) in human serum was developed. The method was used to assess one of the markers of 5 alpha-reductase activity of males including patients with prostate cancer (PC). After deproteinization with acetonitrile, the androgen sulfates and the internal standard, [7,7,16,16-H-2(4)]dehydroepiandrosterone-S, were extracted from human serum using a solid-phase extraction cartridge and washed with hexane. The extract was reconstituted and applied to the LC/ESI-MS system operated in the selected ion monitoring mode. The method was validated over the range 0.02-5 mu g/mL (A-S) and 0.005-1.5 mu g/mL (EpiA-S) using 10 mu L of human serum. The method was a concise procedure without chemical or enzymatic hydrolysis of the conjugates, purification by high-performance liquid chromatography and/or derivatization, and proved to be satisfactory in its reproducibility and accuracy. The levels of these androgen sulfates tended to decrease during aging, and the A-S levels in the sera obtained from both healthy males and patients with PC were correlated with their EpiA-S levels. Copyright (c) 2005 John Wiley & Sons, Ltd.
  • K Mitamura; C Ogasawara; A Shiozawa; E Terayama; K Shimada
    ANALYTICAL SCIENCES JAPAN SOC ANALYTICAL CHEMISTRY 21 (10) 1241 - 1244 0910-6340 2005/10 [Refereed]
     
    A determination method for steroid 5 alpha-reductase activity using liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS) in the positive-ion mode has been developed. The rat prostatic enzyme source was used and the enzymatically formed 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol were determined by LC/APCI-MS using absolute calibration curve method. The sum of the formed products was used as a measurement of the enzyme activity. This method was applied to kinetic study of this enzyme and inhibitory experiments using Finasteride (R) as a model inhibitor.
  • T Mizuguchi; Y Shibayama; K Mitamura; K Shimada
    JOURNAL OF HEALTH SCIENCE PHARMACEUTICAL SOC JAPAN 51 (4) 447 - 452 1344-9702 2005/08 [Refereed]
     
    Contribution of glucuronic acid and sulfonic acid moieties during the photocatalytic degradation of estrogen conjugates, one of the endocrine disrupting chemicals, has been investigated. Estrogens were subjected to photocatalytic degradation using titanium dioxide immobilized on glass beads as a catalyst, whose time courses were measured by HPLC or liquid chromatography (LC)/MS/(MS). Estradiol and estrone, which have an unconjugated phenolic hydroxy group at the C-3 position, were gradually degraded by UV irradiation and nearly disappeared within 6 hr. 3-Desoxyestradiol, which does not have a phenolic hydroxy group at C-3 position, was also degraded like estradiol. The corresponding 17- or 3-glucuronide was degraded faster than the respective genin, estradiol or estrone. The double conjugate, estriol 3-sulfate 16-glucuronide, was also easily degraded within 3 hr. On the other hand, the degradation of estrogen 3-sulfate did not start within 2.5 hr but the reaction was completed within 6 hr. These data showed that the glucuronic acid moiety on the estrogen skeleton and sulfonic acid moiety at phenolic hydroxy group play an important role for this degradation reaction.
  • K Mitamura; A Horikawa; A Nagahama; K Shimada; Y Fujii
    JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES TAYLOR & FRANCIS INC 28 (18) 2839 - 2848 1082-6076 2005 [Refereed]
     
    A method for the determination of digitoxin in human serum using a liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) technique is reported. Digitoxin and the internal standard, [21,21,22- H-2(3)]digitoxin, were extracted from 200 mu L of human serum using a solid phase extraction cartridge and analyzed by LC/ESI-MS/MS in the selected reaction monitoring mode. The intra- and inter-assay reproducibility and accuracy were satisfactory within the quantification range of 5-100 ng/mL, which was sensitive enough to measure the digitoxin in real samples. The concentrations of digitoxin in serum samples obtained from digitalized patients (n=19) were in the range of 5.3-24.1 ng/mL, and these correlated well with those obtained by radioimmunoassay using a very specific antiserum.
  • K Mitamura; H Narukawa; T Mizuguchi; K Shimada
    ANALYTICAL SCIENCES JAPAN SOC ANALYTICAL CHEMISTRY 20 (1) 3 - 4 0910-6340 2004/01 [Refereed]
     
    Estrogen conjugates (estradiol-3-glucuronide, -17-glueuronide, estrone-glucuronide and -sulfate) were subjected to photodegradation using titanium dioxide immobilized on glass beads as a catalyst. Their time courses were measured by HPLC and compared with those of the unconjugated estrogens. Estradiol, its 17-glucuronide and estrone, which have an unconjugated phenolic hydroxy group at the C-3 position, were almost completely degraded by UV irradiation within 4 h. On the other hand, significant amounts of estradiol- and estrone-3-glucuronide (ca. 20%, 25%) and estrone sulfate (ca. 90%), which were conjugated at the 3-hydroxy group, remained after a 6.5 It irradiation. These results supported the hypothesis that the photodegradation of estrogens was initiated at the phenolic hydroxy group.
  • K Mitamura; Y Nagaoka; K Shimada; S Honma; M Namiki; E Koh; A Mizokami
    JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES ELSEVIER SCIENCE BV 796 (1) 121 - 130 1570-0232 2003/10 [Refereed]
     
    A simple method for simultaneous determination of androstenediol 3-sulfate (Adiol-3S) and dehydroepiandrosterone sulfate (DHEA-S) in human serum using isotope diluted liquid chromatography-electrospray ionization-ion trap-mass spectrometry (LC-ESI-ion trap-MS) was developed. After addition of deuterated internal standards ([H-2(5)]Adiol-3S and [H-2(4)]DHEA-S), human serum (100 mul) was deproteinized with acetonitrile and then applied to a solid-phase extraction cartridge, Oasis HLB. The obtained steroid sulfates fraction was washed with hexane and then analyzed by LC-ESI-MS operated in the negative ion mode. The quantification ranges of Adiol-3S and DHEA-S were 10-400 ng/ml and 0.05-8 mug/ml, respectively. The method does not require the chemical or enzymatic hydrolysis of the conjugates and purification with high-performance liquid chromatography, and shows satisfactory reproducibility and accuracy. The concentrations of these sulfates in the sera of healthy male volunteers (n = 14) were 19.2-245.3 mg/ml (Adiol-3S) and 0.175-5.16 mug/ml (DHEA-S), and those of patients with prostate cancer (n = 19) were 15.3-182.7 ng/ml (Adiol-3S; four samples, not detectable) and 0.110-2.421 mug/ml (DHEA-S). (C) 2003 Elsevier B.V. All rights reserved.
  • Mitamura K; Mikami N; Kambara Y; Ohno E; Shimada K
    Chromatography 23 (2) 65 - 71 1348-3315 2002/07 [Refereed]
  • K Mitamura; T Nakagawa; K Shimada; M Namiki; E Koh; A Mizokami; S Honma
    JOURNAL OF CHROMATOGRAPHY A ELSEVIER SCIENCE BV 961 (1) 97 - 105 0021-9673 2002/06 [Refereed]
     
    The identification of the in vitro metabolites of dehydroepiandrosterone formed from human prostate homogenate was investigated by hyphenated techniques using the stable-isotope dilution method. A mixture of dehydroepiandrosterone and [H-2(4)]dehydroepiandrosterone was incubated with hypertrophied human prostate tissue homogenate in the presence of NAD, NADH and NADPH. The metabolites were extracted with AcOEt-hexane, purified by solid-phase extraction, and then analyzed by LC-atmospheric pressure chemical ionization MS and/or GC-MS. Androst-5-ene-3beta,17beta-diol (major product), androst-4-ene-3,17-dione, testosterone, 5alpha-dihydrotestosterone, androsterone, and 7alpha-hydroxydehydroepiandrosterone were identified in comparison with authentic samples based on their chromatographic behavior and mass spectra. (C) 2002 Elsevier Science BV All rights reserved.
  • K Mitamura; M Yatera; K Shimada
    JOURNAL OF CHROMATOGRAPHY B ELSEVIER SCIENCE BV 748 (1) 89 - 96 0378-4347 2000/10 [Refereed]
     
    Enzymic formations of catechol- and guaiacol-estrogens by rat brains have been investigated using classical- and catechol-estrogens as substrates, respectively. The incubation mixtures were pretreated with liquid-liquid and/or solid-phase extraction, and the products were identified by comparison with authentic samples using liquid chromatography-mass spectrometry (-mass spectrometry) {LC-MS (-MS)}. The enzymic activities were also determined by measuring the formed products with LC-MS. (C) 2000 Elsevier Science B.V. All rights reserved.
  • K. Mitamura; M. Yatera; K. Shimada
    Journal of Chromatography B: Biomedical Sciences and Applications 748 (1) 89 - 96 1387-2273 2000/10 [Refereed]
     
    Enzymic formations of catechol- and guaiacol-estrogens by rat brains have been investigated using classical- and catechol-estrogens as substrates, respectively. The incubation mixtures were pretreated with liquid-liquid and/or solid-phase extraction, and the products were identified by comparison with authentic samples using liquid chromatography-mass spectrometry (-mass spectrometry) {LC-MS (-MS)}. The enzymic activities were also determined by measuring the formed products with LC-MS. (C) 2000 Elsevier Science B.V.
  • K Mitamura; M Yatera; K Shimada
    ANALYST ROYAL SOC CHEMISTRY 125 (5) 811 - 814 0003-2654 2000 [Refereed]
     
    The existence of catechol estrogens in rat brains was clarified using liquid chromatography-atmospheric pressure chemical ionization-ion trap-mass spectrometry-mass spectrometry (LC-APCI-MS2). The catechol estrogens were extracted in the presence of ascorbic acid and then derivatized to acetates with acetic anhydride and pyridine. After a successive purification with silica gel mini-column chromatography, reversed-phase solid-phase extraction and preparative HPLC, the obtained fractions containing the catechol estrogen acetates were subjected to LC-APCI-MS2. 2-Hydroxyestrone, 2-hydroxyestradiol and their 4-hydroxy isomers were identified as acetates by comparison with authentic samples based on their chromatographic behavior and mass spectral data. The derivatization to acetate was useful for the treatment of labile catechol estrogens.
  • K Mitamura; M Yatera; K Shimada
    ANALYTICAL SCIENCES JAPAN SOC ANALYTICAL CHEM 15 (10) 951 - 955 0910-6340 1999/10 [Refereed]
     
    A method for the quantitative determination of pregnenolone 3-sulfate (PS) in rat brains has been developed using liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS). The PS fraction was obtained from the rat brain homogenate by solid-phase extraction and ion-exchange chromatography. After the derivatization with 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole, PS was determined using LC/ESI-tandem MS along with the standard addition method. This method was applied to the quantitative determination of this steroid in the brains of Wistar strain rats, most of which showed a much lower amount than that previously reported.
  • K Shimada; K Mitamura; M Shiroyama; K Yago
    JOURNAL OF CHROMATOGRAPHY A ELSEVIER SCIENCE BV 847 (1-2) 171 - 178 0021-9673 1999/06 [Refereed]
     
    The characterization of the classical estrogens (estrone, estradiol, estriol) and guaiacol estrogens (2-hydroxyestrone 3-methyl ether, 4-hydroxyestrone 3-methyl ether) in rat brains was performed using gas chromatography-tandem mass spectrometry (GC-MS-MS). Estrogens were purified from Wistar strain rat brains by some chromatographic pre-treatments, such as solid-phase extraction, preparative thin-layer chromatography or preparative high-performance liquid chromatography. After the derivatization with O-methylhydroxylamine and/or N,O-bis(trimethylsilyl)trifluoroacetamide estrogens were identified by comparison of their chromatographic behavior during GC-MS-MS operating in the product ion scan mode and comparison with the product ion MS spectra of an authentic sample. These evidences suggested that estrogens exist in rat brains as neurosteroids or neuroactive steroids. (C) 1999 Elsevier Science BN. All rights reserved.
  • T Higashi; K Mitamura; H Ohmi; N Yamada; K Shimada; K Tanaka; H Honjo
    ANNALS OF CLINICAL BIOCHEMISTRY ROYAL SOC MEDICINE PRESS LTD 36 (1) 43 - 47 0004-5632 1999/01 [Refereed]
     
    The concentrations of (24R)-24,25-dihydroxyvitamin D-3 [24,25(OH)(2)D-3], 25-hydroxyvitamin D-3 [25(OH)D-3] and its 3-sulphate [25(OH)D(3)3S] in the plasma of healthy subjects, patients with chronic renal failure, patients with climacteric syndrome, pregnant women and foetuses were determined using the enzyme-linked immunosorbent assay and high-performance liquid chromatography. 25(OH)D(3)3S was not detected in about one-third of the plasma samples from patients with chronic renal failure (n = 26). The three metabolites in maternal plasma reached the highest levels in the second trimester of pregnancy followed by a decrease to the values obtained in the first trimester. Older healthy women (age range 44-71 years) showed higher levels of 24,25(OH)(2)D-3 and 25(OH)D-3 in the plasma than did young healthy women (age range 21-29 years), whereas no clear difference was observed between the older healthy women and patients with climacteric syndrome. The level of 25(OH)D(3)3S in the plasma was higher in the latter patients than in healthy women.
  • K Mitamura; Y Nambu; M Tanaka; A Kawanishi; J Kitahori; K Shimada
    JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES MARCEL DEKKER INC 22 (3) 367 - 377 1082-6076 1999 [Refereed]
     
    The separation of authentic vitamin D-3 3-stearate, -palmitate, oleate, and -linoleate, possible metabolites of vitamin D3, was carried out using reversed-phase high performance liquid chromatography. Liquid chromatography/atmospheric pressure chemical ionization - mass spectrometry (LC/APCI-MS) of these esters was also examined, and a deesterified peak was detected as a base peak. On the contrary, the fatty acid ester derivatized with a Cookson-type reagent, 4-phenyl-1,2,4-triazoline-3,5-dione, showed a quasi-molecular ion as an intense peak. The LC/APCI-MS data on the adducts of another Cookson-type reagent having an electron-capture substituent were also reported.
  • K Shimada; K Mitamura; K Saito; Y Ohtake; Nakatani, I
    JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS PERGAMON-ELSEVIER SCIENCE LTD 15 (9-10) 1207 - 1214 0731-7085 1997/06 [Refereed]
     
    The monoglucuronides of vitamin D, 25-hydroxtvitamin D and the corresponding pro-forms were subjected to enzymatic hydrolysis using beta-glucuronidase, and substrate specificities were found in the examined enzymes originating from different sources, which were determined using reversed-phase high-performance liquid chromatography with UV detection. The enzymatic hydrolysis of the corresponding monosulfates was also examined using the same system. (C) 1997 Elsevier Science B.V.
  • K Shimada; Y Kamezawa; K Mitamura
    BIOLOGICAL & PHARMACEUTICAL BULLETIN PHARMACEUTICAL SOC JAPAN 20 (6) 596 - 600 0918-6158 1997/06 [Refereed]
     
    In vitro glucuronidation of 25-hydroxyvitamin D-3 and its pro-form has been investigated by means of HPLC with UV detection. Although both substrates gave 3- and 25-glucuronides in the presence of the rat liver microsomal fraction and uridine-5'-diphosphoglucuronic acid, 25-hydroxyvitamin D-3 and its pro-form yielded 3- and 25-glucuronide as the main product, respectively. The latter glucuronide is the one in which the tert-hydroxy group is conjugated. Each glucuronide was identified by its chromatographic behavior in comparison with an authentic sample and data obtained from liquid chromatography/mass spectrometry (LC/MS) using atmospheric pressure chemical ionization.
  • Kazutake Shimada; Kuniko Mitamura; Ito Nakatani
    Journal of Chromatography B: Biomedical Applications 690 (1-2) 348 - 354 1572-6495 1997/03 [Refereed]
     
    The characterization of vitamin D2 3-glucuronide, 25-hydroxyvitamin D2 3-glucuronide and 25-hydroxyvitamin D2 25-glucuronide, biliary metabolites obtained from rats dosed with vitamin D2 and 25-hydroxyvitamin D2 per os, was carried out using HPLC-atmospheric pressure chemical ionization (APCI)-MS. The glucuronide obtained from bile specimens was identified by comparison of its chromatographic behaviour with an authentic sample using HPLC-APCI-MS operating in the negative-ion mode. Methylation of the respective fraction with diazomethane gave the methyl ester, which was also confirmed by HPLC-APCI-MS operating in the positive-ion mode. The (M - H)- and (M + NH4)+ ions were monitored in the selected-ion monitoring mode.
  • K Shimada; K Mitamura; Nakatani, I
    JOURNAL OF CHROMATOGRAPHY B ELSEVIER SCIENCE BV 690 (1-2) 348 - 354 0378-4347 1997/03 [Refereed]
     
    The characterization of vitamin D-2 3-glucuronide, 25-hydroxyvitamin D-2 3-glucuronide and 25-hydroxyvitamin D-2 25-glucuronide, biliary metabolites obtained from rats dosed with vitamin D-2 and 25-hydroxyvitamin D-2 per os, was carried out using HPLC-atmospheric pressure chemical ionization (APCI)-MS. The glucuronide obtained from bile specimens was identified by comparison of its chromatographic behaviour with an authentic sample using HPLC-APCI-MS operating in the negative-ion mode. Methylation of the respective fraction with diazomethane gave the methyl ester, which was also confirmed by HPLC-APCI-MS operating in the positive-ion mode. The (M-H)(-) and (M+NH4)(+) ions were monitored in the selected-ion monitoring mode.
  • Kazutake Shimada; Kuniko Mitamura; Nami Kitama; Michiko Kawasaki
    Journal of Chromatography B: Biomedical Applications 689 (2) 409 - 414 1572-6495 1997/02 [Refereed]
     
    A method for the determination of 25-hydroxyvitamin D3, the major metabolite of vitamin D3 in human plasma, using a non-radioactive internal standard and reversed-phase high-performance liquid chromatography with UV detection (265 nm) has been developed. The method was applied to the determination of the metabolite in plasma from healthy subjects (n=25) and from patients with chronic renal failure (n=12). 25-Hydroxyvitamin D3 3-sulfate, a major conjugated metabolite of 25-hydroxyvitamin D3, was also determined and the correlation between the concentrations of these metabolites was examined. The study showed that almost equal amounts of both compounds were detected in the plasma of healthy subjects, however, in two subjects, the amount of sulfate in the free form was found to be about twice as high as normally detected. In contrast, the free form was predominant in the plasma of patients with chronic renal failure and the sulfate was not detected in four patients.
  • K Shimada; K Mitamura; N Kitama; M Kawasaki
    JOURNAL OF CHROMATOGRAPHY B ELSEVIER SCIENCE BV 689 (2) 409 - 414 0378-4347 1997/02 [Refereed]
     
    A method for the determination of 25-hydroxyvitamin D-3, the major metabolite of vitamin D-3 in human plasma, using a non-radioactive internal standard and reversed-phase high-performance liquid chromatography with UV detection (265 nm) has been developed. The method was applied to the determination of the metabolite in plasma from healthy subjects (n=25) and from patients with chronic renal failure (n=12). 25-Hydroxyvitamin D-3 3-sulfate, a major conjugated metabolite of 25-hydroxyvitamin D-3, was also determined and the correlation between the concentrations of these metabolites was examined. The study showed that almost equal amounts of both compounds were detected in the plasma of healthy subjects, however, in two subjects, the amount of sulfate in the free form was found to be about twice as high as normally detected. In contrast, the free form was predominant in the plasma of patients with chronic renal failure and the sulfate was not detected in four patients.
  • K Shimada; Nakatani, I; K Saito; K Mitamura
    BIOLOGICAL & PHARMACEUTICAL BULLETIN PHARMACEUTICAL SOC JAPAN 19 (4) 491 - 494 0918-6158 1996/04 [Refereed]
     
    The separation and characterization of vitamin D-3- and 25-hydroxyvitamin D-3-monoglucuronides, biliary metabolites obtained from rats dosed with D-3 and 25-hydroxyvitamin D-3 per os, respectively, were carried out by HPLC. The glucuronide fractions were obtained from bile specimens by the combined use of a Bond Elut C18 cartridge, for solid phase extraction, and a lipophilic gel (piperidinohydroxypropyl Sephadex LH-20), for ion-exchange chromatography. Each glucuronide was identified by comparison with an authentic sample in three ways: its chromatographic behavior, that of its fluorescent labeled derivative using 4-[4-(6-methoxy-2-benzoxazolyl)phenyl]-1,2,4-triazoline-3,5-dione and data obtained following enzymatic hydrolysis using beta-glucuronidase.
  • K SHIMADA; K SUGAYA; H KAJI; NAKATANI, I; K MITAMURA; N TSUTSUMI
    CHEMICAL & PHARMACEUTICAL BULLETIN PHARMACEUTICAL SOC JAPAN 43 (8) 1379 - 1384 0009-2363 1995/08 
    25-Hydroxyvitamin D-3 (D-2) 3- and 25-monoglucuronides were synthesized from the corresponding provitamin D or its derivatives with the Koenigs-Knorr reaction using silver carbonate as a catalyst, followed by photochemical reaction, thermal isomerization and then alkali hydrolysis. The obtained glucuronides were subjected to enzymatic hydrolysis using beta-glucuronidase, and substrate specificities were found in the examined enzymes originating from different sources.
  • K SHIMADA; K MITAMURA; M MUKOUYAMA; T OKUMURA; K SUGAYA
    JOURNAL OF CHROMATOGRAPHIC SCIENCE PRESTON PUBLICATIONS INC 33 (2) 82 - 85 0021-9665 1995/02 [Refereed]
     
    The separation and characterization of 25-hydroxyvitamin D3 3-sulfate in human plasma are carried out by high-performance liquid chromatography. The vitamin D sulfate fraction is obtained from a plasma specimen (three volunteers) by the combined use of a Sep-Pak C18 cartridge, for solid-phase extraction, and a lipophilic gel (piperidinohydroxypropyl Sephadex LH-20), for ion-exchange chromatography. 25-Hydroxyvitamin D3 3-sulfate is identified in the examined three specimens in two ways: its chromatographic behavior and that of its fluorescent labeled derivative using 4-[4-(6-methoxy-2-benzoxazolyl)phenyl]-1,2,4-triazoline-3,5- dione; and data obtained from the solvolysis reaction.
  • K SHIMADA; K MITAMURA; M MIURA; A MIYAMOTO
    JOURNAL OF LIQUID CHROMATOGRAPHY MARCEL DEKKER INC 18 (14) 2885 - 2893 0148-3919 1995 [Refereed]
     
    The retention behavior of vitamin D-2-D-5 and provitamin D-2-D-5 are examined using high-performance liquid chromatography. Inclusion chromatography using cyclodextrin as the mobile phase additive in reversed-phase high-performance liquid chromatography is also used for this purpose. Reversed-phase high-performance liquid chromatography is more effective than normal-phase high-performance liquid chromatography in separating these analogs. The addition of methyl-beta-cyclodextrin to the mobile phase is effective in separating the pair of vitamin-D-2 and -D-3 or provitamin-D-2 and -D-3. The separation of the pair of stereoisomeric Cookson-type derivatives of vitamin-D-2 or -D-3 was also examined and found that normal-phase high-performance liquid chromatography is effective for this purpose.
  • Kazutake Shimada; Katsuko Sugaya; Hidefumi Kaji; Ito Nakatani; Kuniko Mitamura; Noriko Tsutsumi
    Chemical and Pharmaceutical Bulletin 43 (8) 1379 - 1384 1347-5223 1995 [Refereed]
     
    25-Hydroxyvitamin D3 (D2) 3- and 25-monoglucuronides were synthesized from the corresponding provitamin D or its derivatives with the Koenigs-Knorr reaction using silver carbonate as a catalyst, followed by photochemical reaction, thermal isomerization and then alkali hydrolysis. The obtained glucuronides were subjected to enzymatic hydrolysis using β-glucuronidase, and substrate specificities were found in the examined enzymes originating from different sources. © 1995, The Pharmaceutical Society of Japan. All rights reserved.
  • Kazutake Shimada; Kuniko Mitamura; Nami Kitama
    Biomedical Chromatography 9 (5) 229 - 232 1099-0801 1995 [Refereed]
     
    The quantitative determination of 25‐hydroxyvitamin D3 3‐sulphate in human plasma was completed using reversed phase high performace liquid chromatography with UV detection (265 nm) and an internal standard method. The vitamin D sulphate fraction was obtained from a plasma specimen with the combined use of a Bond Elut C18 cartridge for solid‐phase extraction and a piperidinohydroxypropyl Sephadex LH‐20 column for lipophilic ion‐exchange chromatography. Separation of the compounds was performed on a YMC‐Pack ODS‐AM column. The limit of quantitation was 5 ng/mL and the assay was linear from 5 to 50 ng/mL. The proposed method is satisfactory in its accuracy and precision. Copyright © 1995 John Wiley & Sons, Ltd.
  • K SHIMADA; K MITAMURA; H KAJI; M MORITA
    JOURNAL OF CHROMATOGRAPHIC SCIENCE PRESTON PUBLICATIONS INC 32 (3) 107 - 111 0021-9665 1994/03 [Refereed]
     
    The retention behavior of sulfates or glucuronides of provitamin D, vitamin D, and 25-hydroxyvitamin D3, together with its fluorescent derivatives, are examined with reversed-phase high-performance liquid chromatography. Inclusion chromatography using cyclodextrin as a mobile-phase additive is also used for this purpose. Conventional reversed-phase high-performance liquid chromatography clearly separates the positionally isomeric conjugates of 25-hydroxyvitamin D3. The addition of the host compound to the mobile phase is effective in separating the pairs of fluorescent derivatives of vitamin-D3 and -D2 conjugates or provitamin-D3 and -D2 conjugates.
  • K SHIMADA; K MITAMURA; S ISHITOYA; K HIRAKATA
    JOURNAL OF LIQUID CHROMATOGRAPHY MARCEL DEKKER INC 16 (18) 3965 - 3976 0148-3919 1993 [Refereed]
     
    The high-performance liquid chromatographic behavior of new bile acid fluorescent derivatives, the 7-methoxy-1,4-benzoxazin-2-one-3-methyl esters, with a cyclodextrin-containing mobile phase is compared to those of the bile acid-pyrenacyl and - anthroyl esters. Compared with conventional methods, inclusion chromatography gives a much more satisfactory separation of the bile acid fluorescent derivatives in a short time, but the chromatographic behavior of these derivatives has not been much influenced by the fluorophore used. The detection limits of the new derivatives are in the range of 10-20 fmol at a signal to noise ratio of 5. The application of this method for the separation of glycine-conjugated bile acids in human bile is also described.
  • K SHIMADA; K MITAMURA; M MORITA; K HIRAKATA
    JOURNAL OF LIQUID CHROMATOGRAPHY MARCEL DEKKER INC 16 (15) 3311 - 3320 0148-3919 1993 [Refereed]
     
    Baclofen (4-amino-3-p-chlorophenylbutyric acid), a skeletal muscle relaxant used in the treatment of spastic disorders, is administered clinically as a racemic mixture. This paper describes the separation of the diastereomers of baclofen, which was derivatized with (+)-1-(9-fluorenyl)ethyl chloroformate, (+)-1-(1-naphthyl)ethyl isocyanate, 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide or 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate, by reversed-phase high-performance liquid chromatography using cyclodextrin as a mobile phase additive.
  • K SHIMADA; Y KOMINE; K MITAMURA
    JOURNAL OF CHROMATOGRAPHY-BIOMEDICAL APPLICATIONS ELSEVIER SCIENCE BV 565 (1-2) 111 - 118 0378-4347 1991/04 [Refereed]
     
    The high-performance liquid chromatographic separation of bile acid pyrenacyl esters with cyclo-dextrin-containing mobile phase is presented. Compared with conventional methods, inclusion chromatography gives much more satisfactory separation of derivatized bile acids in a short time. The application of this method to the separation of glycine-conjugated bile acids in human bile is also described.
  • Kazutake Shimada; Yoshihiro Komine; Mitamura Kuniko
    Journal of Chromatographic Science 28 (6) 331 - 335 1945-239X 1990 [Refereed]
     
    The retention behavior of 3-(1-anthroyl)bile acids together with bile acid glucuronides, sulfates, and 12-dehydro derivatives is examined by the addition of cyclodextrin to the mobile phase in reversed-phase high-performance liquid chromatography. The data suggest that the functional group at the 12 position of the steroid moiety may be the important factor for the formation of the inclusion complex from the solute and cyclodextrin. The separation of these bile acid derivatives is much improved by this inclusion chromatography. © 1990 Oxford University Press.

MISC

Books and other publications

  • 薬学生のための臨床化学 改訂第4版
    池川繁男; 三田村 邦子 (Contributor第1章 1-6 c.その他 pp.49-55)南江堂 2015/09 9784524403196 298 pp.49-55
  • 試料分析講座 脂質分析
    池川 繁男; 三田村 邦子 (Contributor酸化コレステロールと胆汁酸の分析)丸善出版 2011/12 9784621084649 238 135-148
  • 分析化学便覧 改訂6版
    東 達也; 三田村 邦子 (Contributorステロイド、胆汁酸)丸善 2011/12 9784621084090 850 389-392
  • 薬学生のための臨床化学 改定第3版, 第1章 1-4 c. その他
    池川 繁男; 三田村 邦子 (Contributor第1章 1-4 c. その他)南江堂 2010/08 9784524402625 274 41-46
  • 薬学分析科学の最前線
    池川 繁男; 三田村 邦子 (ContributorLC/ESI-MS2によるグルタチオン抱合型胆汁酸の高感度分析法の開発と代謝研究への応用)じほう 2009/03 9784840739658 185 130-131
  • Technical Terms in Life Sciences
    Iida T; Ikegawa S; Ito J; Une M; Tamegai H; Mitamura K (Contributor)三共出版 2008/11 286
  • 先端の分析法-理工学からナノ・バイオまで
    島田和武; 三田村邦子; 東 達也 (Contributor液体クロマトグラフィー/質量分析法(LC/MS)とガスクロマトグラフィー/質量分析法(GC/MS))エヌ・ティー・エス 2004/12 4860430670 372-380
  • 分析化学便覧 改訂5版
    島田和武; 東 達也; 三田村邦子 (Contributor第4章 ステロイド)丸善 2001/12 9784621084090 389-394

Lectures, oral presentations, etc.

  • LC/MSによる玄米米糠麹中グルコシルセラミド同定・定量法の開発  [Not invited]
    三田村 邦子; 上野 光穂; 内海 裕子; 日野 美紀; 村井 勇太; 門出 健次; 山本 哲志; 多賀 淳; 根本 英幸; 池川 繁男
    日本薬学会第139年会  2019/03
  • キャピラリー電気泳動による糖鎖重合度決定法の開発  [Not invited]
    佐藤 完太; 山本 哲志; 三田村 邦子; 多賀 淳
    日本薬学会第139年会  2019/03
  • キャピラリー電気泳動による酸性多糖の分析法の開発  [Not invited]
    奥野 人美; 山本 哲志; 三田村 邦子; 多賀 淳
    日本薬学会第139年会  2019/03
  • メープルシロップに含まれるタンパク質成分による大腸癌細胞に対する抗腫瘍効果の検討  [Not invited]
    山本 哲志; 森山 由瑛; 三田村 邦子; 多賀 淳
    日本薬学会第139年会  2019/03
  • LC-MS/MSを用いたターゲットメタボロミクスによるTCA回路代謝物の分析法の開発  [Not invited]
    山口 真史; 佐藤 完太; 山本 哲志; 三田村 邦子; 多賀 淳
    日本薬学会第139年会  2019/03
  • プロテオーム解析によるすっぽんコラーゲンのヒト皮膚角化細胞への影響の検討  [Not invited]
    山本哲志; 中西紗緒理; 三田村邦子; 多賀淳
    日本薬学会第138年会  2018/03  金沢
  • ショットガンプロテオミクス解析を用いた糖尿病白内障要因の解析  [Not invited]
    大竹裕子; 山本哲志; 三田村邦子; 多賀淳; 長井紀章
    日本薬学会第138年会  2018/03  金沢
  • ICD - LC/ESI-MS/MS による尿中テトラヒドロコルチコステロイドグルクロニ ドの定量法の開発  [Not invited]
    松本 孝彬; 山崎 航; 小川 祥二郎; 三田村 邦子; 池川 繁男; 東 達也
    日本薬学会第137年会  2017/03
  • 細胞外基質 Lumican の発現抑制による新規膵臓癌細胞増殖抑制法の開発  [Not invited]
    山本 哲志; 谷田 和香奈; 橋本 知樹; 三田村 邦子; 多賀 淳
    日本薬学会第137年会  2017/03
  • LC/MS/MSによる乾燥尿ろ紙中抱合型テトラヒドロコルチコステロイド定量法の開発  [Not invited]
    三田村邦子; 森莉子; 上田麻美子; 亀井美希; 池川繁男
    第57回日本臨床化学会年次学術集会  2017  札幌
  • Identificaiton of antitumor component in maple syrup to develop novel anti-cancer drugs for colorectal cancer.  [Not invited]
    Yamamoto T; Shiburo R; Mitamura K; Taga A
    第75回日本癌学会学術総会  2016/10
  • The role of maple syrup on cell proliferation of colorectal cancer cells  [Not invited]
    Kubota C; Yamamoto T; Mitamura K; Taga A
    第75回日本癌学会学術総会  2016/10
  • Protein component in maple syrup has a potential to develop novel anti-cancer drugs for colorectal cancer  [Not invited]
    amamoto T; Shiburo R; Mitamura K; Taga A
    DDW2016  2016/05
  • The role of cyclophilin A as a novel therapeutic target of colorectal cancer based on proteome analysis using formalin–fixed and paraffin embedded colorectal cancer tissue.  [Not invited]
    Takakura H; Yamamoto T; Mitamura K; Kudo M; Naito Z; Taga A
    DDW2016  2016/05
  • ショットガンプロテオミクス解析に基づく、大腸癌における新規治療標的 CyclophilinA の役割  [Not invited]
    高倉 英樹; 山本 哲志; 三田村 邦子; 工藤 光洋; 内藤 善哉; 多賀 淳
    日本薬学会第136年会  2016/03
  • スッポンからのコラーゲン抽出法の最適化  [Not invited]
    久保田 千晶; 山本 哲志; 上村 健太郎; 澤岻 有喜; 三田村 邦子; 多賀 淳
    日本薬学会第136年会  2016/03
  • LC/ESI-MSによる乾燥ろ紙尿中糖質コルチコイド代謝物のスクリーニング法の開発  [Not invited]
    三田村 邦子
    日本薬学会第136年会  2016/03
  • メープルシロップ投与による大腸癌細胞増殖抑制機構の検討  [Not invited]
    山本 哲志; 三田村 邦子; 多賀 淳
    日本薬学会第136年会  2016/03
  • A Novel Bile Acid Synthesis Pathway in Mouse Liver Initiated by 24-Hydroxycholesterol.  [Not invited]
    Genta Kakiyama; Dalila Marques; Kuniko Mitamura; Hajime Takei; Hiroshi Nittono; Daniel Rodriguez-Agudo; Gregorio Gil; Huiping Zhou; Phillip Hylemon; William M. Pandak
    AASLD The Liver Meeting 2015  2015/11  San Francisco, CA
  • 薬学による臨床化学へのアプローチ  [Not invited]
    三田村 邦子
    第55回日本臨床化学会年次学術集会  2015/10  大阪  日本臨床化学会
  • Proteomics using formalin-fixed paraffin-embedded  [Not invited]
    Tetsushi Yamamoto; Mitsuhiro Kudo; Wei-Xia Peng; Hideyuki Takata; Kuniko Mitamura; Atsushi Taga; Zenya Naito
    第74回日本癌学会学術総会  2015/10
  • The effect of maple syrup on cell proliferation and invasion in  [Not invited]
    Chiaki Kubota; Tetsushi Yamamoto; Kuniko Mitamura; Atsushi Taga
    第74回日本癌学会学術総会  2015/10
  • Kuniko Mitamura; Satoshi Kurabuchi; Mamiko Ueda; Tetsushi Yamamoto; Atsushi Taga; Shigeo Ikegawa
    63th ASMS Conference on Mass Spectrometry and Allied Topics  2015/06  America's Center, St. Louis, Missouri  American Society for Mass Spectrometry
  • LC/MSによる抱合型ステロイド代謝物測定~臨床への応用を目指して~  [Not invited]
    三田村 邦子
    日本分析化学会近畿支部2015年度第1回講演会  2015/04  大阪、大阪科学技術センター  日本分析化学会近畿支部
  • メープルシロップを用いた大腸癌抗腫瘍効果の検討  [Not invited]
    山本哲志; 上村健太郎; 三田村邦子; 多賀淳
    日本薬学会第135年会  2015/03  神戸  日本薬学会
  • LC/ESI-MS/MSによる尿中抱合型テトラヒドロコルチコステロイドのプロファイル分析  [Not invited]
    三田村邦子; 上田麻美子; 山本哲志; 多賀淳; 池川繁男
    日本薬学会第135年会  2015/03  神戸  日本薬学会
  • High Speed Molecular Weight Measurement of Proteins by Capillary Electrophoresis  [Not invited]
    Atsushi Taga; Yuri Miyazaki; Tetsushi Yamamoto; Kuniko Mitamura
    The 4th International Symposium on Steel Science - ISSS 2014  2014/11  京都、関西セミナーハウス
  • LC/ESI-LIT-MS/MSにおけるPQD及びCID測定による尿中硫酸抱合型ステロイドの分析  [Not invited]
    三田村邦子; 蔵渕 慧; 上田麻美子; 池川繁男; 山本哲志; 多賀 淳
    第39回日本医用マススペクトル学会年会  2014/10  千葉、三井ガーデンホテル千葉  日本医用マススペクトル学会
  • 同位体希釈LC/ESI-MS/MSによるヒト尿中硫酸抱合型オキシステロールの高感度直接一斉定量法の開発  [Not invited]
    蔵渕 慧; 三田村邦子; 池川繁男; 山本哲志; 多賀淳
    第64回日本薬学会近畿支部総会・大会  2014/10  京都、京都薬科大学  日本薬学会近畿支部
  • HPLC及びLC/MSによるヒト血清中テイコプラニンの定量の試み  [Not invited]
    新 幸子; 三田村邦子; 山本哲志; 多賀淳
    第64回日本薬学会近畿支部総会・大会  2014/10  京都、京都薬科大学  日本薬学会近畿支部
  • LC/ESI-MS/MSによる尿中抱合型テトラヒドロコルチコステロイドのプロファイル分析-硫酸抱合体とグルクロン酸抱合体の分別定量の試み-  [Not invited]
    上田麻美子; 三田村邦子; 山本哲志; 多賀淳
    第64回日本薬学会近畿支部総会・大会  2014/10  京都、京都薬科大学  日本薬学会近畿支部
  • Anti-cancer Effect of Maple Syrup for Colorectal Cancer  [Not invited]
    Kentaro Uemura; Tetsushi Yamamoto; Kuniko Mitamura; Atsushi Taga
    第73回日本癌学会学術総会  2014/09  横浜、パシフィコ横浜  日本癌学会
  • Proteomic analysis of formalin-fixed paraffin-embedded colorectal cancer tissue for identification of therputic terget.  [Not invited]
    Tetsushi Yamamoto; Mitsuhiro Kudo; Wei-Xia Peng; Hideyuki Takata; Kuniko Mitamura; Atsushi Taga; Zenya Naito
    第73回日本癌学会学術総会  2014/09  横浜、パシフィコ横浜  日本癌学会
  • ピラルクー(Arapaima gigas)の胆嚢胆汁から新規胆汁酸タウリン抱合体の単離と構造決定  [Not invited]
    齊藤 徹也; 佐藤 梨香; 関口 匠太郎; 飯田 隆; 池川 繁男; 三田村 邦子
    日本薬学会第134年会  2014/03  熊本、熊本市総合体育館ほか  日本薬学会
  • LC/ESI-MS/MSによるラット胆汁中抱合型フェニル酢酸の同定  [Not invited]
    三田村 邦子; 大橋 勇斗; 山本 哲志; 多賀 淳; 池川 繁男
    日本薬学会第134年会  2014/03  熊本、熊本市総合体育館ほか  日本薬学会
  • プロテオーム解析を用いたlumicanの膵臓癌細胞増殖制御機構の解析  [Not invited]
    山本 哲志; 木場 克斗; 三田村 邦子; 工藤 光洋; 内藤 善哉; 多賀 淳
    日本薬学会第134年会  2014/03  熊本、熊本市総合体育館  日本薬学会
  • ショットガンプロテオミクス解析による大腸癌早期発見のための新規診断マーカーの探索  [Not invited]
    上村 健太朗; 山本 哲志; 三田村 邦子; 工藤 光洋; 内藤 善哉; 多賀 淳
    日本薬学会第134年会  2014/03  熊本、熊本市総合体育館ほか  日本薬学会
  • メープルシュガーにおける血糖値上昇抑制効果  [Not invited]
    多賀 淳; 長井 紀章; 山本 哲志; 三田村 邦子; 伊藤 吉將
    日本薬学会第134年会  2014/03  熊本、熊本市総合体育館ほか  日本薬学会
  • High Speed Determination of Protein Molecular Weight by Capillary Electrophoresis  [Not invited]
    Atsushi Taga; Yuri Miyazaki; Tetsushi Yamamoto; Kuniko Mitamura
    13th International Symposium on Hyphenated Techniques in Chromatography and Separation Technology (HTC-13)  2014/01
  • Reversal of NAFL Through Selective Increased Intracellular Hepatic Cholesterol Catabolism  [Not invited]
    Genta Kakiyama; Dalila Marques; Kuniko Mitamura; Shigeo Ikegawa; Shunlin Ren; Daniel Rodriguez-Agudo; Patricia Copper; Phillip Hylemon; Huiping Zhou; William M. Pandak
    The Liver Meeting (AASLD2013)  2013/11  Washington, DC, USA.
  • Proteomic profiling of formalin-fixed paraffin-embedded colorectal cancer tissue for identification of novel biomarker.  [Not invited]
    山本哲志; 工藤光洋; Peng Wei-Xia; 高田英志; 三田村邦子; 多賀淳; 内藤善哉
    第72回日本癌学会学術総会  2013/10  横浜、パシフィコ横浜  日本癌学会
  • 尿中ステロイド抱合体のLC-MS/MS  [Not invited]
    三田村 邦子
    第60回日本臨床検査医学会学術集会  2013/10  神戸、神戸国際会議場  日本臨床検査医学会
  • 質量分析法の基礎  [Not invited]
    三田村 邦子
    医用質量分析認定士 第1回講習会  2013/09  神戸、神戸市産業振興センター  日本医用マススペクトル学会
  • Barrett食道患者の胃液内胆汁酸の解析  [Not invited]
    池川繁男; Shijian CHU; 三田村邦子
    日本薬学会第133年会  2013/03  横浜、パシフィコ横浜  日本薬学会
  • 同位体希釈LC/ESI-MS/MSによるヒト尿中テトラヒドロコルチコステロイド・サルフェート18種の直接定量法の開発  [Not invited]
    三田村邦子; 上林麻美; 佐藤(沖原)梨香; 飯田 隆; 池川繁男
    日本薬学会第133年会  2013/03  横浜、パシフィコ横浜  日本薬学会
  • LC/MSによるBarrett食道患者の胃液内胆汁酸の解析  [Not invited]
    池川繁男; Shijian Chu; 三田村邦子
    第34回胆汁酸研究会  2012/12  名古屋、サンプラザシーズンズ  胆汁酸研究会
  • LC/MSによるテトラヒドロコルチコステロイドのヒト肝における第II相代謝反応の解析  [Not invited]
    三田村邦子; 上林麻美; 佐藤完太; 藤岡佐知; 中島理陽; 佐藤(沖原)梨香; 飯田隆; 池川繁男
    第37回日本医用マススペクトル学会年会  2012/10  名古屋、ウインクあいち  日本医用マススペクトル学会
  • LC/ESI-MSによるテトラヒドロコルチコステロイドのラット並びにヒト肝サイトゾール画分における硫酸抱合の解析  [Not invited]
    藤岡佐知; 佐藤完太; 三田村邦子; 池川繁男
    第62回日本薬学会近畿支部総会・大会  2012/10  西宮、武庫川女子大学  日本薬学会近畿支部
  • グルタミン抱合型胆汁酸の合成とそれらのESI-MSにおける挙動  [Not invited]
    山根崇郎; 三田村邦子; 池川繁男
    第62回日本薬学会近畿支部総会・大会  2012/10  西宮、武庫川女子大学  日本薬学会近畿支部
  • A novel, major epimer of varanic acid from the bile of monitor lizards: taurine-conjugated (24R, 25S)-3α,7α,12α,24-tetrahydroxy-5β-cholestan-27-oic acid  [Not invited]
    Narimi Kato; Takashi Iida; Shoujiro Ogawa; Mizuho Une; Kuniko Mitamura; Shigeo Ikegawa; Lee R. Hagey; Alan F Hofmann
    XXII Bile Acid Meeting  2012/10  Vienna, Austria
  • Chemical synthesis of 7α,12α-dihydroxy-cholest-4-en-3-one and its 12-deoxy analogue (C4): key intermediates in the biosynthesis of bile acids from cholesterol.  [Not invited]
    Kaoru Omura; Yuusuke Kimoto; Takashi Iida; Shoujiro Ogawa; Kuniko Mitamura; Shigeo Ikegawa; Lee R. Hagey; Alan F Hofmann
    XXII Bile Acid Meeting  2012/10  Vienna, Austria
  • Identification of S-acyl glutathione conjugates of bile acids in human bile by means of LC/ESI-MS  [Not invited]
    Shigeo Ikegawa; Naohiro Hori; Takashi Iida; Alan F. Hofmann; Kuniko Mitamura
    XXII Bile Acid Meeting  2012/10  Vienna, Austria
  • 安定同位体希釈LC/ESI-MS/MSによる尿中グルタミン抱合型胆汁酸の高感度測定法の開発  [Not invited]
    三田村 邦子; 池川 繁男
    第52回日本臨床化学会年次学術集会  2012/09  盛岡、いわて県民情報交流センター  日本臨床化学会
  • ヒトおよびラット肝サイトゾール画分におけるテトラヒドロコルチコステロイドの硫酸抱合  [Not invited]
    三田村 邦子; 佐藤 完太; 藤岡 佐知; 中島 理陽; 佐藤(沖原)梨香; 飯田 隆; 池川 繁男
    第25回バイオメディカル分析科学シンポジウム  2012/08  東京、慶応大薬  日本薬学会(物理系薬学部会)
  • Development of isotope dilution LC/ESI-MS/MS analysis of eighteen tetrahydrocorticosteroid sulfates in human urine  [Not invited]
    Kuniko Mitamura; Rika Okihara; Maki Hasegawa; Mami Kamibayashi; Takashi Iida; Shigeo Ikegawa
    60th ASMS Conference on Mass Spectrometry and Allied Topics  2012/05  Vancouver, Canada  American Society for Mass Spectrometry
  • Liquid chromatography-mass spectrometric characterization of sulfation of glutathione conjugated bile acids by a rat liver cytosolic fraction  [Not invited]
    Shigeo Ikegawa; Naohiro Hori; Takashi Iida; Alan F. Hofmann; Kuniko Mitamura
    60th ASMS Conference on Mass Spectrometry and Allied Topics  2012/05  Vancouver, Canada  American Society for Mass Spectrometry
  • テトラヒドロコルチコステロイドのグルクロン酸抱合に関する検討  [Not invited]
    三田村邦子; 永迫三恵; 上林麻美; 池川繁男
    日本薬学会第132年会  2012/03  札幌、北海道大  日本薬学会
  • オオトカゲ胆嚢胆汁成分から新規胆汁酸の単離・同定: Tauro-(24R,25S)- 3α,7α,12α,24-tetrahydroxy-5-cholestan-27-oic acid  [Not invited]
    小川 祥二郎; 飯田 隆; 三田村 邦子; 池川 繁男; 宇根 瑞穂; Lee; R. Hagey; Alan; F.Hofmann
    日本薬学会第132年会  2012/03  札幌、北海道大  日本薬学会
  • 同位体希釈LC/ESI-MSに用いる多重重水素標識A/B-トランスallo-tetrahydrocorticosteroid 3- 及び 21-monosulfateの合成  [Not invited]
    池川繁男; 中島理陽; 藤岡佐知; 佐藤完太; 小川祥二郎; 飯田 隆; 三田村邦子
    日本薬学会第132年会  2012/03  札幌、北海道大  日本薬学会
  • グルタミン抱合型胆汁酸の高感度測定法開発に関する基礎的検討  [Not invited]
    池川繁男; 山根崇郎; 三田村邦子
    第21回日本小児胆汁酸研究会  2012/02  東京、山の上ホテル  日本小児胆汁酸研究会
  • Allo-tetrahydro-11-deoxycortisol 3-及び21-monosulfateの同位体希釈LC/ESI-MSに用いる多重重水素標識体の合成  [Not invited]
    池川繁男; 中嶋理陽; 藤岡佐知; 佐藤完太; 小川祥二郎; 飯田隆; 三田村邦子
    第61回日本薬学会近畿支部総会・大会  2011/10  神戸、神戸大学ポートアイランドキャンパス  日本薬学会近畿支部
  • ラット胆汁中グルタチオン抱合型フェニル酪酸のLC/ESI-MS/MSによる同定  [Not invited]
    池川繁男; 橋本優希; 大橋勇斗; 三田村邦子
    第36回日本医用マススペクトル学会年会  2011/09  大阪、ホテル阪急エキスポパーク  日本医用マススペクトル学会
  • LC/MSによるグルタチオン抱合型胆汁酸の動態解析  [Not invited]
    三田村邦子; 池川繁男
    第60回日本分析化学会  2011/09  名古屋、名古屋大  日本分析化学会
  • グルタチオン抱合型胆汁酸の胆汁排泄  [Not invited]
    三田村 邦子; 池川 繁男
    第51回日本臨床化学会年次学術集会  2011/08  札幌、札幌医大  日本臨床化学会
  • N-アセチルシステイン抱合型ウルソデオキシコール酸の体内動態と肝機能完全作用に関する基礎的検討  [Not invited]
    三田村邦子; 村上葉月; 佐野拓見; 池川繁男
    第20回西日本臨床胆汁酸研究会  2011/07
  • グルタチオン抱合型胆汁酸3-サルフェートの合成とラット肝サイトゾール画分における生成  [Not invited]
    三田村邦子; 堀直宏; 池川繁男
    第28回日本胆膵病態生理研究会  2011/06
  • Liquid chromatography-mass spectrometric characterization of sulfation of N-acetylcysteine conjugated bile acids by a rat liver cytosolic fraction  [Not invited]
    Shigeo Ikegawa; Toshihiro Sakai; Risa Nakai; Tateaki Wakamiya; Takashi Iida; Alan F. Hofmann; Kuniko Mitamura
    59th ASMS Conference on Mass Spectrometry and Allied Topics  2011/05  Denver、USA  American Society for Mass Spectrometry
  • Liquid chromatography/mass spectrometric characterization of non-enzymatic acylation of amino or thiol groups of bionucleophiles by the cholyl-adenylate or cholyl-CoA thioester  [Not invited]
    Kuniko Mitamura; Eriko Aoyama; Toshihiro Sakai; Takashi Iida; Alan F. Hofmann; Shigeo Ikegawa
    59th ASMS Conference on Mass Spectrometry and Allied Topics  2011/05  Colorado Convention Center, Denver, CO  American Society for Mass Spectrometry
  • LC/ESI-MS/MSによるヒト胆汁中グルタチオン抱合型胆汁酸の同定  [Not invited]
    池川繁男; 堀直宏; 三田村邦子; 飯田隆; 鈴木光幸; 清水俊明; 入戸野博; 高折恭一; 滝川一
    日本薬学会第131年会  2011/03  静岡  日本薬学会
  • グルタチオン抱合型胆汁酸3-サルフェートの合成とラット肝サイトゾール画分における生成  [Not invited]
    三田村邦子; 堀直宏; 美濃詩織; 飯田隆; Alan F. Hofman; 池川繁男
    日本薬学会第131年会  2011/03  静岡  日本薬学会
  • LC/ESI-MS/MSによるヒト胆汁中グルタチオン抱合型胆汁酸の同定  [Not invited]
    三田村邦子; 堀直宏; 飯田隆; 鈴木光幸; 清水俊明; 入戸野博; 高折恭一; 滝川一; 池川繁男
    第21回日本臨床化学会近畿支部総会  2011/02  大阪  日本臨床化学会近畿支部
  • グルタチオン抱合型胆汁酸のラット肝サイトゾール画分における硫酸抱合の解析  [Not invited]
    池川繁男; 堀直宏; 美濃詩織; 飯田隆; Alan F. Hofman; 三田村邦子
    第32回胆汁酸研究会  2010/11  仙台  胆汁酸研究会
  • Chemical synthesis of the (25R)- and (25S)-epimers of 3α,7α,12α-trihydroxy-5α-cholestan-27-oic acid and their glycine and taurine conjugates.  [Not invited]
    池川 繁男; 三田村 邦子; 飯田隆
    XXI International Bile Acid Meeting Bile Acid as Metabolic Integrators and Therapeutics  2010/10  Freiburg, Germany  Folk Foundation
  • Analysis of glutathione conjugates of bile acids in rat bile by LC/ESI-MS/MS  [Not invited]
    Shigeo Ikegawa; Naohiro Hori; Takashi Iida; Kuniko Mitamura
    XXI International Bile Acid Meeting Bile Acid as Metabolic Integrators and Therapeutics  2010/10  Freiburg, Germany  Folk Foundation
  • Simultaneous Determination of Tetrahydrocorticosteroid Glucuronides by Liquid Chromatography/Electrospray Ionization-Mass Spectrometry  [Not invited]
    Kuniko Mitamura; Shigeo Ikegawa
    The 12th Asian-Pacific Congress of Clinical Biochemistry  2010/10  Seoul, Korea
  • イオンクラスター法による胆汁酸代謝活性中間体のチオール基並びにアミの基との反応性の解析  [Not invited]
    三田村邦子; 青山瑛里子; 村上葉月; 堀直宏; 堺俊博; 池川繁男
    第35回日本医用マススペクトル学会年会  2010/09  名古屋、金城学院大学  日本医用マススペクトル学会
  • ヒト血中硫酸抱合型オキシステロールの同位体希釈LC/ESI-MSによる直接一斉分析法の開発  [Not invited]
    坂野理絵; 三田村 邦子; 池川 繁男
    第50回日本臨床化学会年次学術集会  2010/09  甲府、県民文化ホール  日本臨床化学会
  • 内分泌・代謝疾患のLC/MSによるメタボロミクスに用いる多重重水素標識A/B-シス-テトラヒドロコルチコステロイド・サルフェートの合成  [Not invited]
    池川繁男; 沖原梨香; 森愛; 並木理恵; 馬渕賢幸; 長江香織; 三田村邦子
    第23回バイオメディカル分析科学シンポジウム  2010/07  松島  日本薬学会(物理系薬学部会)
  • 胆汁酸のグルタチオン抱合体と胆汁排泄  [Not invited]
    池川繁男; 堀直宏; 三田村邦子; 飯田隆; 鈴木光幸; 清水俊明; 入戸野博; 高折恭一
    第27回日本胆膵病態・生理研究会  2010/06  弘前  日本胆膵病態・生理研究会
  • Identification of Novel Metabolites of Bile Acids: S-Acyl Glutathione Conjugates in Rat Bile by LC/ESI-Linear Ion Trap MS/MS.  [Not invited]
    Naohiro Hori; Kuniko Mitamura; Toshihiro Sakai; Takashi Iida; Shigeo Ikegawa
    58th ASMS Conference on Mass Spectrometry  2010/05  Salt Lake City, USA  ASMS
  • Quantitative Metabolic Profiling of Twelve Tetrahyrocorticosteroid Glucuronides in Urine by Isotope Dilution LC-ESI-Linear Ion Trap MS/MS  [Not invited]
    Kuniko Mitamura; Maki Hasegawa; Rika Okihara; Chikara Shimidzu; Hitoshi Chiba; Takashi Iida; Shigeo Ikegawa
    58th ASMS Conference on Mass Spectrometry  2010/05  Salt Lake City, USA  ASMS
  • LC/ESI-MS/MSによるラット胆汁中重水素標識N-アセチルシステイン抱合型胆汁酸の同定  [Not invited]
    堺 俊博; 中井莉沙; 堀 直宏; 三田村邦子; 若宮建昭; 池川繁男
    日本薬学会第130年会  2010/03  岡山  日本薬学会
  • ラット胆汁中グルタチオン抱合型胆汁酸の合成とLC/MSによる同定  [Not invited]
    堀 直宏; 堺 俊博; 三田村邦子; 飯田 隆; 若宮建昭; 池川繁男
    日本薬学会第130年会  2010/03  岡山  日本薬学会
  • LC/ESI-MS/MSによるN-アセチルシステイン抱合型ウルソデオキシコール酸の体内動態に関する基礎的検討  [Not invited]
    池川 繁男; 堺 俊博; 堀 直宏; 三田村 邦子
    日本薬学会第130年会  2010/03  岡山  日本薬学会
  • LC/ESI-MS/MSによるヒト尿中テトラヒドロ-並びにヘキサヒドロコルチコステロイド・グルクロニド直接一斉分析法の開発  [Not invited]
    三田村 邦子; 池川 繁男
    日本薬学会第130年会  2010/03  岡山  日本薬学会
  • 肝・胆・消化機能改善作用を指向したプロドラッグ開発の基礎的検討:重水素標識N-アセチルシステイン抱合型ウルソデオキシコール酸の体内動態の追跡  [Not invited]
    三田村邦子; 堺 俊博; 堀 直宏; 池川繁男
    第19回日本小児胆汁酸研究会  2010/02  東京  日本小児胆汁酸研究会
  • 重水素標識N-アセチルシステインによる胆汁酸代謝活性中間体のトラッピング  [Not invited]
    池川繁男; 中井莉沙; 堺 俊博; 堀 直宏; 若宮建昭; 三田村邦子
    第19回日本小児胆汁酸研究会  2010/02  東京  日本小児胆汁酸研究会
  • 肝・胆・消化機能改善薬として期待されるN-アセチルシステイン抱合型ウルソデオキシコール酸の体内動態に関する基礎的研究  [Not invited]
    池川繁男; 堀 直宏; 三田村邦子
    第20回日本臨床化学会近畿支部総会  2009/12  大阪  日本臨床化学会近畿支部総会
  • IDENTIFICATION OF GLUTATHIONE CONJUGATES OF BILE ACIDS IN RAT BILE BY LC/ESI-MS2  [Not invited]
    Shigeo Ikegawa; Naohiro Hori; Toshihiro Sakai; Takashi Iida; Kuniko Mitamura
    日本薬物動態学会第24回年会  2009/11  京都  日本薬物動態学会
  • ラット胆汁中グルタチオン抱合体の合成とこれらのLC/MSによる同定  [Not invited]
    堀 直宏; 堺 俊博; 三田村邦子; 飯田 隆; 若宮建昭; 池川繁男
    第31回胆汁酸研究会  2009/11  東京、山上会館  胆汁酸研究会
  • LC/MSによる硫酸抱合型オキシステロール直接一斉分析法の開発  [Not invited]
    坂野理絵; 貴田亜希子; 三田村邦子; 飯田 隆; 村井 毅; 黒澤隆夫; 池川繁男
    第59回日本薬学会近畿支部大会  2009/10  大阪、近畿大  日本薬学会近畿支部
  • 多重重水素標識テトラヒドロコルチコステロイド・サルフェートの合成とESI-MSnにおける挙動  [Not invited]
    三田村邦子; 沖原梨香; 馬渕賢幸; 長江香織; 森 愛; 池川繁男
    第59回日本薬学会近畿支部大会  2009/10  大阪、近畿大  日本薬学会近畿支部
  • LC/ESI-MS/MSによるN-アセチルシステイン抱合型胆汁酸3-サルフェートのトレースアナリシス  [Not invited]
    池川繁男; 堺 俊博; 堀 直宏; 飯田 隆; 三田村邦子
    日本分析化学会第58年会  2009/09  札幌、北大高等教育機能開発総合センター  日本分析化学会
  • 同位体希釈LC/ESI-MS/MSによる尿中テトラヒドロコルチコステロイド・サルフェート定量法の開発  [Not invited]
    三田村邦子; 沖原梨香; 長谷川真紀; 池川繁男
    第49回日本臨床化学会年会  2009/09  長崎、長崎大医  日本臨床化学会
  • 77. ラット胆汁中グルタチオン抱合型胆汁酸の同定  [Not invited]
    堀 直宏; 三田村邦子; 飯田 隆; 若宮建昭; 池川繁男
    第34回日本医用マススペクトル学会年会  2009/09  大阪、近大  日本医用マススペクトル学会
  • LC/ESI-MS/MSによるN-アセチルシステイン抱合型胆汁酸のラット肝サイトゾール画分における硫酸抱合の解析  [Not invited]
    堺 俊博; 堀 直宏; 三田村邦子; 飯田 隆; 池川繁男
    第34回日本医用マススペクトル学会年会  2009/09  大阪、近大  日本医用マススペクトル学会
  • 唾液中胆汁酸:LC-ESI-MS/MS定量法と唾液分泌量亢進に伴う濃度変動  [Not invited]
    東 達也; 柴山優人; 島田和武; 三田村邦子; 池川繁男; 豊岡利正
    第34回日本医用マススペクトル学会年会  2009/09  大阪、近大  日本医用マススペクトル学会
  • LC/ESI-MS/MSによるラット並びにヒト胆汁中グルタチオン抱合型胆汁酸の解析  [Not invited]
    三田村邦子; 堺 俊博; 渡辺彩愛; 田中志穂; 池川繁男; 若宮建昭; 曾我部充; 鈴木光幸; 清水俊明; 入戸野 博; 高折恭一
    第19回西日本臨床胆汁酸研究会  2009/07  大阪、リーガロイヤルホテル
  • 胆汁酸のグルタチオン抱合と胆汁排泄  [Not invited]
    池川繁男; 堀 直宏; 堺 俊博; 渡辺彩愛; 田中志穂; 曾我部充; 若宮建昭
    第22回バイオメディカル分析科学シンポジウム  2009/07  岐阜、内藤記念くすり博物館  日本薬学会(物理系薬学部会)
  • A/B-trans構造を有するテトラヒドロコルチコステロイド・サルフェートの合成とESI-MSにおける挙動  [Not invited]
    沖原梨香; 森 愛; 長谷川真紀; 三田村邦子; 武藤晃奈; 柿山玄太; 飯田 隆; 池川繁男
    日本薬学会第129年会  2009/03  京都、国立京都国際会館  日本薬学会
  • LC/MSによる硫酸抱合型オキシステロールの高感度定量法の開発  [Not invited]
    三田村邦子; 貴田亜希子; 吉岡慎司; 河本里鶴; 武藤晃奈; 柿山玄太; 飯田 隆; 村井 毅; 黒澤隆夫; Hirotoshi Fuda; Charles A. Strott; 池川繁男
    日本薬学会第129年会  2009/03  京都、国立京都国際会館  日本薬学会
  • LC/MSによる臨床化学へのアプローチ  [Not invited]
    三田村 邦子
    日本薬学会第129年会  2009/03  京都、国立京都国際会館  日本薬学会
  • LC/ESI-MS/MSによるラット胆汁中グルタチオン抱合型胆汁酸の解析  [Not invited]
    堺 俊博; 渡辺彩愛; 田中志穂; 三田村邦子; 曾我部充; 若宮建昭; 池川繁男
    フィジカル・ファーマフォーラム2009  2009/03  大阪、大阪薬科大学  日本薬学会(物理系薬学部会)
  • ラット胆汁中アミノ酸並びにグルタチオン抱合型胆汁酸の解析  [Not invited]
    堺 俊博; 渡辺彩愛; 田中志穂; 三田村邦子; 池川繁男
    第18回日本小児胆汁酸研究会  2009/02  東京、山の上ホテル  日本小児胆汁酸研究会
  • テトラヒドロコルチコステロイドのグルクロン酸抱合に関する基礎的検討  [Not invited]
    三田村邦子; 長谷川真紀; 奥村拓朗; 由井晴子; 沖原梨香; 池川繁男
    第19回日本臨床化学会近畿支部総会  2009/01  大阪、大阪医大  日本臨床化学会近畿支部

Affiliated academic society

  • JAPANESE SOCIETY FOR BIOMEDICAL MASS SPECTROMETRY   JAPAN SOCIETY OF CLINICAL CHEMISTRY   クロマトグラフィー科学会   日本分析化学会   日本薬学会   

Research Themes

  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2014/04 -2017/03 
    Author : MITAMURA Kuniko
     
    Profile determination methods for oxysterols sulfate and tetrahydrocorticosteroids glucuronides (THC-G) and/or these sulfates in urine samples by means of LC/MS/MS were developed. The method was applicable to quantitative analysis of THC-G in dried urine spots (DUS) sample. The method was well validated, and the concentration of THC-G in DUS were well correlated with those in liquid urine. DUS samples provides convenient and safe transportation, storage and handling of sample. DUS samples would be a useful alternative for clinical screening tests of endocrine disorders, particularly, when the sample needs to be transported over long distances.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2011 -2013 
    Author : MITAMURA Kuniko; IKEGAWA Shigeo
     
    We demonstrated that N-acetylcysteine conjugate of ursodeoxycholic acid (UDCA-NAC) was hydrolyzed and circulated in body as UDCA after administration by injection via jugular vein, while UDCA-NAC converted to NAC-sulfate di-conjugate by rat liver cytosolic fraction. Furthermore, UDCA-NAC was shown to inhibit the increase of liver disease marker enzymes, AST and ALT, in serum of acetaminophen-induced liver injury rats. These results showed that UDCA-NAC is useful as a prodrug of the new liver, bile, digestive function improving agent.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2009 -2011 
    Author : IKEGAWA Shigeo; MITAMURA Kuniko
     
    Highly sensitive and specific soft ionization mass spectrometry for the determination of tetrahydrocorticosteroids, bile acids, and oxysterols, which are conjugated with glucuronic and sulfuric acids, glutathione, and N-acetylcysteine, have been developed. The developed methods were useful for diagnosis of endocrinological and metabolic diseases.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2008 -2010 
    Author : MITAMURA Kuniko; IKEGAWA Shigeo
     
    In order to elucidate the mechanism on the formation and metabolism of glutathione (GSH) conjugated bile acids via their reactive intermediates, we demonstrated that bile acid were tansacylated with GSH and N-acetylcysteine (NAC) by in vitro and in vivo studies. In addition, we clarified that the several kinds of glutathione conjugated bile acids were excreted into the bile of rats and patients with biliary diseases. We also showed the NAC conjugated ursodeoxycholic acid could be a prodrug for hepatobiliary fuction improvement drug.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2007 -2008 
    Author : IKEGAWA Shigeo; MITAMURA Kuniko
     
    病因・病態を化学の目で把握・理解し、適切な薬物療法を設定する上で、組織の変化や血液、尿などの体液中に含まれる成分の信頼度の高い測定法の確立が強く求められる。本研究では、最新の質量分析法を基盤とする胆汁酸、ステロイドホルモン、オキシステロールなどの硫酸、グルクロン酸との抱合体の体内動態解析法を構築し、本法によって新規代謝排泄経路の存在を世界に先駆けて実証するとともに、内分泌・代謝疾患の診断のみならず病態解析にも有用なことを示した。
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2005 -2006 
    Author : IKEGAWA Shigeo; MITAMURA Kuniko
     
    Formation of covalently bound protein adducts with lithocholic acid (LCA) has been proposed as a possible explanation for hypersensitivity and toxic responses of this bile acid. We therefore carried out to capture of LCA-modified cellular proteins in the bile duct-ligated rat liver by immunoprecipitation using an antibody with high affinity to the 3α-hydroxy-5β-steroid moiety of LCA. Results of the structural analysis of the captured proteins by the basic proteomic technique of 2-DE combined with the use of MALDI-TOFMS and computer assisted programs indicated the presence of LCA bound Rab proteins, which may be the course of LCA-induced liver toxicity. Further studies on the formation of the glutathione (GSH) conjugates via trans-acylation reactions between GSH and reactive acyl-linked metabolites of bile acids have been carried out to demonstrate the presence of detoxification mechanism. For this purpose, the authentic GSH conjugates of five major bile acids were chemically synthesized and their structures were characterized by ESI-tandem mass spectrometry in negative and positive ion modes. Incubation of cholyl-adenylate and cholyl-CoA, reactive metabolites of cholic acid (CA), with GSH in a phosphate buffer (pH 7.5) solution demonstrated that both compounds are able to acylate GSH in vitro. In addition, we cloned and expressed rat and human liver bile acid CoA-ligase to better understand the mechanism for the formation of acyl-adenylate and bile acid bound proteins. Finally, our efforts were directed to explore the proteins having affinity with LCA in rat liver by using affinity adsorbents, in which LCA was covalently bound to an activated agarose through the bridges introduced at the C-3 and C-24 positions of LCA. Results of LC/ESI-MS^n analysis of the captured proteins after separation by SDS-PAGE revealed the presence of functional proteins, i.e., carbamoyl phosphate synthase I, glutamate dehydrogenase, acyl-CoA acyltransferase, glutathione-S-transferase, and catalase, indicating the hypothesis involved in the appearance of the liver toxicity by inhibition of these enzymes by LCA.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2003 -2005 
    Author : SHIMADA Kazutake; HIGASHI Tatsuya
     
    During the last decade, prostate cancer has been a commonly diagnosed malignancy in males. The research focused on steroids [testosterone (T) and its active form dihydrotestosterone (DHT)] in human prostate or serum, which are the potential bio-markers for diagnosing prostate cancer (PC) or benign prostatic hyperplasia (BPH). We have developed a new derivatization reagent (2-hydrazino-1-methylpyridine) having a quarternary pyridinium moiety for the analysis of these androgens using LC/electrospray ionization-MS. The reagent quantitatively reacted with the above oxo-steroids (T and DHT) and the derivatives provided a 70-1600 fold higher sensitivity compared to intact steroids. The developed method allowed the reproducible quantification of nanogram/gram levels of T and DHT with a 10 mg tissue sample of the prostate. The method was applied to the determination of T and DHT in PC and BPH tissues. DHT was determined in BPH tissue, but not detected in PC tissue. T was not detected in both tissues. These data is useful for establishment of mass screening for these diseases.
  • 日本学術振興会:科学研究費助成事業 若手研究(B)
    Date (from‐to) : 2002 -2004 
    Author : 三田村 邦子
     
    5α-レダクターゼは代表的なアンドロゲンであるテストステロン(T)を活性な5α-ジヒドロテストステロン(DHT)に還元する酵素であるが,前立腺癌や前立腺肥大症などのアンドロゲン依存性疾患によって,その発現量や活性が変化することが知られており,有用な5α-レダクターゼ阻害剤の開発が進められている.その評価には5α-レダクターゼ活性の測定が必須であるが,従来のそれは放射性同位体標識基質を用いる方法によるものが主で,それに伴う多くの問題を有している.そこでLC/APCI-MSによる5α-レダクターゼ活性測定法の開発を企て,以下の結果を得た. 1.基質(T),生成物であるDHT及び5α-アンドロスタン-3α,17β-ジオール(3α,5α-diol)に加え,これらの5位又は3位異性体のクロマトグラフ的挙動を精査したところ,従来のカラムより高速分離可能なモノリス型カラムが有用であった. 2.Tをラット前立腺より得た酵素源,NADPHと共に緩衝液中インキュベートし,反応停止後固相抽出により精製後,LC/APCI-MSに付したところ,生成物としてDHT及び3α,5α-diolに対応するピークのみ検出された.これらの前処理操作による回収率は約80%で,5-40ng/tubeでの定量が可能であった. 3.本法を用いてラット前立腺中5α-レダクターゼ活性を測定したところ,K_m=0.91±0.3μM, V_=13.0±3.5nmol/min/mg protein,また代表的な5α-レダクターゼ阻害剤であるフィナステライドの阻害活性を測定したところIC_<50>=237nMであり,いずれも報告値と符合するものであった. 以上,開発した5α-レダクターゼ活性測定法は放射性同位体標識体を用いず,定量性にも優れており,5α-レダクターゼ阻害剤の開発に有用であると期待される.
  • 日本学術振興会:科学研究費助成事業 奨励研究(A)
    Date (from‐to) : 2000 -2001 
    Author : 三田村 邦子
     
    本年度は昨年度の研究を踏まえ,誘導体化LC/MSを各種生体内ステロイドの分析に導入し,以下の成果を得た. 1.アンドロステンジオール(A-diol)は主に3位硫酸抱合体として血中に存在しているが,近年,前立腺癌との関連が注目されている.硫酸抱合型A-diolには3位のほか,17位抱合体の存在も考えられるが,脱抱合を伴う従来の同定法では抱合位置が不明確であった.そこで血中から抽出した本アンドロゲンを直接,あるいは無水酢酸-ピリジンを用いてアセテートへ誘導体化後,LC/ESI-MSにより分析し,誘導体化前後でのクロマトグラフ的挙動及びマススペクトルを標品のそれと比較した.その結果,大量の3位抱合体のほか,17位抱合体も微量ながら存在することを確認した. 2.遊離型A-diolのLC/ESI-MSによる高感度分析を目的としてメチルピリジニウム誘導体化し,正イオンモードでの検出を試みた.その結果,必ずしも高感度化は達せられなかったが,これは誘導体化率にも起因すると考えられることから今後に期待される. 3.先に著者は,ラット脳内にカテコールエストロゲンが存在することを,無水酢酸-ピリジンによりアセテートへ誘導体化後LC/MSを用いて同定した.しかし生体内にエストロゲンがエステル体として存在することが報告されている.そこでラット脳よりエストロゲンを抽出後,重水素標識無水酢酸-ピリジンを用いて誘導体化しLC/MSで分析したところ,カテコールエストロゲンアセテートは検出されなかった.以上のようにして同定したカテコールエストロゲンが内因性のアセテートではなく,カテコール体として脳内に存在していることを確認した.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 1997 -1998 
    Author : SHIMADA Kazutake; HONMA Seijiro; HIGASHI Tatsuya; MITAMURA Kuniko
     
    The term neurosteroids applies to those steroids that are both synthesized in the nervous system, either de novo from cholesterol or from steroid hormone precursors, and that accumulate in the nervous system to levels that are at least in part independent of steroidogenic gland secretion rates. Neurosteroids consist of 17- or 20-oxosteroids and accumulate in brains as unconjugated form and their sulfates, fatty acid esters and sulfolipid conjugates. An assay method for pregnenolone and its 3-stearate in brains has been developed using liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (LC/APCI-MS) operating in the positive ion mode. The method was applied to the determination of these steroids in the gray matters and olfactory bulbs of rat brains. Although pregnenolone in both regions of the rat brains increased more than five times after acute stress, its 3-stearate decreased after the stress. The existence of classical and guaiacol estrogens in rat brains were also clarified using GC/MS^n. The usefulness of estrogen replacement therapy for dementia of the Alzheimer type and the existence of the estrogen receptor in brains have been reported, our data suggested that estrogen exists in brains as neurosteroids or neuroactive steroids.
  • 日本学術振興会:科学研究費助成事業 奨励研究(A)
    Date (from‐to) : 1997 -1998 
    Author : 三田村 邦子
     
    本年度は昨年度の研究を踏まえ,LC/MSにおける誘導体化の有用性をさらに明らかにすると共に,生体試料中微量ステロイド分析に応用した. 1. ビタミンD骨格と選択的に反応するCookson型試薬(4-phenyl-1,2,4-triazoline-3,5-dione等)を用いてビタミンD脂肪酸エステルを誘導体化し,LC/APCI-MSに付したところ,正負両イオンモードで分子量関連イオン([M+H]^+又は[M-H]^-)のみならず特徴的なフラグメントイオンが生成し,抱合位置に関する情報も得られた.これは生体試料中ビタミンD代謝物の同定上有用な知見となり得るものであった. 2. 脳内ステロイドホルモンの一種であるプレグネノロン 3-サルフェート(PS)に着目し,まず標品PSをO-methylhydroxylamine,O-pentafluorobenzylhydroxylamine又は4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole(DBD-H)で誘導体化後,負イオン検出LC-ESI-MSに付し,その諸性質を精査した.その結果,いずれも[M-H]^-が基準イオンとして観察される上に,本イオンを用いる選択イオン検出において,誘導体化前に比し約10倍の高感度な応答を示し,誘導体化の有用性が明らかとなった.また,誘導体化率,後処理の簡便性,実試料に適用した際のクロマトグラフ的挙動を考慮した結果,上記の誘導体化の中でDBD化が最も優れていた. 3. 上記2の誘導体化法及び重水素化内標準物質[^2H_4]PSを用い,LC/MS(/MS)によるラット脳内PS定量法を開発し,実試料へ適用した.その結果,従来法(RIA等)による文献値(21±5 ng/g tissue)に比し,はるかに低値を示すなど,興味ある知見が得られた.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 1996 -1997 
    Author : SHIMADA Kazutake; MITAMURA Kuniko
     
    The term neurosteroids applies to those steroids that are both synthesized in the nervous system, either de novo from cholesterol or from steroid hormone precursors, and that accumulate in the nervous system to levels that are at least in part independent of steroidogenic gland secretion rates. Neurosteroids consist of 17- or 20-oxosteroids and accumulate in the brain as unconjugated form and their sulfates, fatty acid esters and sulfolipid conjugates. The separation and characterization of pregennolone, dehydroepiandrosterone and their 3-fatty acid esters (stearate, palmitate) in the brain are carried out using liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (LC/APCI-MS) operating in the positive ion mode. These obtained from rat brains were identified in comparison with their chromatographic behavior with authentic samples during LC/APCI-MS.Pregnenolone 3-sulfate in rat brains was also identified as above. The quantitative determination of pregnenolone in rat brains was done using HPLC with fluorescence detection. The method was applied to the determination of pregnenolone in rat brains, most of which showed lower amounts than that previously reported.
  • 日本学術振興会:科学研究費助成事業 奨励研究(A)
    Date (from‐to) : 1996 -1996 
    Author : 三田村 邦子
     
    1.先に合成したビタミンD(D)及び25-ヒドロキシビタミンD[25(OH)D]の抱合体(サルフェート及びグルクロニド)のHPLCにおけるクロマトグラフ的挙動を精査し,D_2,D_3系相互,位置異性体相互が良好に分離する条件を設定した. 2.上記の知見を基に,D,25(OH)D及び対応するプロ体のグルクロニドをβ-グルクロニダーゼ水解に付し,残存基質及び成績体をUV検出HPLCにより分析したところ,用いる酵素原により基質特異性が観察された.一方,サルフェートはほとんど酵素水解を受けなかった. 3.ヒト血中25(OH)D_3のUV検出HPLCによる定量法を確立した.本法は内標準物質として非放射性物質を用いるもので,従来法に比し汎用性に優れるものであった.本法と先に開発した25(OH)D_3 3-サルフェート(3S)定量法を駆使し,健常人と慢性腎不全患者血中における25(OH)D_3と25(OH)D_33Sの相関を求めたところ,病態との関連を示唆する興味ある知見が得られた. 4.D_3又は25(OH)D_3投与後の胆管ろうラット胆汁中に各々のモノグルクロニド[D_33G又は25(OH)D_3-3G,-25G]が存在することを確認した.前処理には逆相系固相抽出カートリッジと疎水性陰イオン交換ゲルを用い,グルクロニドを選択的に抽出した.同定は標品を指標とし,UV,フォトダイオードアレイUV及び誘導体化後の蛍光検出HPLCにより行った.また,2の知見を基に酵素水解し,その抱合形式とともにゲニン部の構造を確認した.特に,立体障害の大きい3級水酸基である25位への抱合が観察されたことは,興味深いものであった.
  • ステロイドの分析化学
  • Analytical Chemistry of Steroids