三田村　邦子 （ミタムラ　クニコ）

コミュニケーション情報 byコメンテータガイド

• コメント

  生体内分子や薬物の代謝物の、信頼性の高い測定法について研究しています。「分子の目で病態をみる」をキーワードに、病因の解明、病気の診断や薬物の適正使用に貢献することを目指しています。

研究者情報

学位

• 博士（薬学）（金沢大学）

ホームページURL

https://jglobal.jst.go.jp/detail?JGLOBAL_ID=200901030376281862

J-Global ID

• 200901030376281862

研究キーワード

• 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   

現在の研究分野（キーワード）

生体内分子や薬物の代謝物の、信頼性の高い測定法について研究しています。「分子の目で病態をみる」をキーワードに、病因の解明、病気の診断や薬物の適正使用に貢献することを目指しています。

研究分野

• ライフサイエンス / 医療薬学
• ライフサイエンス / 薬系分析、物理化学

経歴

• 2009年04月 - 現在  近畿大学薬学部准教授
• 2005年04月 - 2009年03月  近畿大学薬学部講師
• 1995年04月 - 2005年03月  金沢大学薬学部助手
• 1992年04月 - 1995年03月  金沢大学薬学部教務職員
• 1992年  - Kanazawa University

学歴

•         - 1990年   金沢大学   薬学部   製薬化学科
•         - 1990年   金沢大学   Faculty of Pharmaceutical Science

所属学協会

• 日本医用マススペクトル学会   日本臨床化学会   クロマトグラフィー科学会   日本分析化学会   日本薬学会   

研究活動情報

論文

• Protein components of maple syrup as a potential resource for the development of novel anti‑colorectal cancer drugs.

  Tetsushi Yamamoto; Ryota Shiburo; Yoshie Moriyama; Kuniko Mitamura; Atsushi Taga

  Oncology reports 50 4 2023年10月 

  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.
• Correction: Sato et al. Separation of Fructosyl Oligosaccharides in Maple Syrup by Using Charged Aerosol Detection. Foods 2021, 10, 3160.

  Kanta Sato; Tetsushi Yamamoto; Kuniko Mitamura; Atsushi Taga

  Foods (Basel, Switzerland) 12 11 2023年05月 

  In the original publication [...].
• Insulin dysregulation drives mitochondrial cholesterol metabolite accumulation: Initiating hepatic toxicity in NAFLD.

  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年03月 

  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.
• Coffee modulates insulin-HNF-4α-Cyp7b1 pathway and reduces oxysterol driven liver toxicity in a NAFLD mouse model.

  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年10月 

  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.
• Separation of Fructosyl Oligosaccharides in Maple Syrup by Using Charged Aerosol Detection.

  Kanta Sato; Tetsushi Yamamoto; Kuniko Mitamura; Atsushi Taga

  Foods (Basel, Switzerland) 10 12 2021年12月 

  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.
• Development of simultaneous quantitative analysis of tricarboxylic acid cycle metabolites to identify specific metabolites in cancer cells by targeted metabolomic approach.

  Tetsushi Yamamoto; Kanta Sato; Masafumi Yamaguchi; Kuniko Mitamura; Atsushi Taga

  Biochemical and biophysical research communications 584 53 - 59 2021年12月 

  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.
• Correction: Sato, K., et al. Identification of a Novel Oligosaccharide in Maple Syrup as a Potential Alternative Saccharide for Diabetes Mellitus Patients. Int. J. Mol. Sci. 2019, 20, 5041.

  Kanta Sato; Noriaki Nagai; Tetsushi Yamamoto; Kuniko Mitamura; Atsushi Taga

  International journal of molecular sciences 21 14 2020年07月 [査読有り]
   

  The authors wish to make the following corrections to this paper [...].
• A method for detecting tumor cells derived from colorectal cancer by targeting cell surface glycosylation with affinity capillary electrophoresis.

  Tetsushi Yamamoto; Kanta Sato; Shinpei Wakahara; Kuniko Mitamura; Atsushi Taga

  Journal of pharmaceutical and biomedical analysis 182 113138 - 113138 2020年04月 [査読有り]
   

  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.
• Cyclophilin a knokdown inhibits cell migration and invasion through the suppression of epithelial-mesenchymal transition in colorectal cancer cells.

  Tetsushi Yamamoto; Hideki Takakura; Kuniko Mitamura; Atsushi Taga

  Biochemical and biophysical research communications 526 1 55 - 61 2020年03月 [査読有り]
   

  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.
• Identification of a Novel Oligosaccharide in Maple Syrup as a Potential Alternative Saccharide for Diabetes Mellitus Patients.

  Kanta Sato; Noriaki Nagai; Tetsushi Yamamoto; Kuniko Mitamura; Atsushi Taga

  International journal of molecular sciences 20 20 2019年10月 [査読有り]
   

  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.
• A Method for Quantification of Tetrahydroglucocorticoid Glucuronides in Human Urine by LC/MS/MS with Isotope-coded Derivatization.

  Matsumoto T; Yamazaki W; Jo A; Ogawa S; Mitamura K; Ikegawa S; Higashi T

  Analytical Sciences 34 9 1003 - 1009 2018年06月 [査読有り]
  
• Collagen peptides from soft‑shelled turtle induce calpain‑1 expression and regulate inflammatory cytokine expression in HaCaT human skin keratinocytes.

  Yamamoto T; Nakanishi S; Mitamura K; Taga A

  International journal of molecular medicine 42 2 1168 - 1180 2018年05月 [査読有り]
   

  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.
• Proteomic profile of the lens in a streptozotocin-induced diabetic rat model using shotgun proteomics

  Noriaki Nagai; Tetsushi Yamamoto; Kuniko Mitamura; Atsushi Taga

  Biomedical Reports 7 5 445 - 450 2017年11月 [査読有り]
   

  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.
• Shotgun label-free proteomic analysis for identification of proteins in HaCaT human skin keratinocytes regulated by the administration of collagen from soft-shelled turtle.

  Yamamoto T; Nakanishi S; Mitamura K; Taga A

  Journal of biomedical materials research. Part B, Applied biomaterials 106 6 2403 - 2413 2017年11月 [査読有り]
   

  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.
• Effect of dark-colored maple syrup on cell proliferation of human gastrointestinal cancer cell

  Tetsushi Yamamoto; Kanta Sato; Yuika Kubota; Kuniko Mitamura; Atsushi Taga

  Biomedical Reports 7 1 6 - 10 2017年 [査読有り]
   

  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.
• Identification of aldolase A as a potential diagnostic biomarker for colorectal cancer based on proteomic analysis using formalin-fixed paraffin-embedded tissue

  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 37 10 13595 - 13606 2016年10月 [査読有り]
   

  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.
• Two major bile acids in the hornbills, (24R,25S)-3α,7α,24-trihydroxy-5β-cholestan-27-oyl taurine and Its 12α-hydroxy derivative

  Satoh R; Ogata H; Saito T; Zhou B; Omura K; Kurabuchi S; Mitamura K; Ikegawa S; Hagey LR; Hofmann AF; Iida T

  Lipids 51 6 757 - 768 2016年06月 [査読有り]
   

  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.
• Novel, major 2α- and 2β-hydroxy bile alcohols and bile acids in the bile of Arapaima gigas, a large South American river fish.

  Sato Née; Okihara R; Saito T; Ogata H; Nakane N; Namegawa K; Sekiguchi S; Omura K; Kurabuchi S; Mitamura K; Ikegawa S; Raines J; Hagey LR; Hofmann AF; Iida T

  Steroids 107 112 - 120 2016年03月 [査読有り]
   

  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.
• Optimization of Method to Extract Collagen from "Emperor" Tissue of Soft-shelled Turtles

  Tetsushi Yamamoto; Kentaro Uemura; Yuki Sawashi; Kuniko Mitamura; Atsushi Taga

  JOURNAL OF OLEO SCIENCE 65 2 169 - 175 2016年02月 [査読有り]
   

  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.
• Improvement of solid material for affinity resins by application of long PEG spacers to capture the whole target complex of FK506

  Miyuki Mabuchi; Tadashi Shimizu; Masahiro Ueda; Kuniko Mitamura; Shigeo Ikegawa; Akito Tanaka

  BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 25 14 2788 - 2792 2015年07月 [査読有り]
   

  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.
• Inhibitory effect of maple syrup on the cell growth and invasion of human colorectal cancer cells

  Tetsushi Yamamoto; Kentaro Uemura; Kaho Moriyama; Kuniko Mitamura; Atsushi Taga

  ONCOLOGY REPORTS 33 4 1579 - 1584 2015年04月 [査読有り]
   

  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.
• Changes in Plasma Glucose in Otsuka Long-Evans Tokushima Fatty Rats After Oral Administration of Maple Syrup

  Noriaki Nagai; Tetsushi Yamamoto; Wataru Tanabe; Yoshimasa Ito; Satoshi Kurabuchi; Kuniko Mitamura; Atsushi Taga

  JOURNAL OF OLEO SCIENCE 64 3 331 - 335 2015年03月 [査読有り]
   

  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.
• Development and validation of a method for determination of plasma 25-hydroxyvitamin D-3 3-sulfate using liquid chromatography/tandem mass spectrometry

  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 969 230 - 234 2014年10月 [査読有り]
   

  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.
• Simultaneous determination of 18 tetrahydrocorticosteroid sulfates in human urine by liquid chromatography/electrospray ionization-tandem mass spectrometry

  Kuniko Mitamura; Rika Satoh (Nee Okihara); Mami Kamibayashi; Kanta Sato; Takashi Iida; Shigeo Ikegawa

  STEROIDS 85 18 - 29 2014年07月 [査読有り]
   

  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.
• N-Methyltaurine N-acyl amidated bile acids and deoxycholic acid in the bile of angelfish (Pomacanthidae): A novel bile acid profile in Perciform fish

  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 80 15 - 23 2014年02月 [査読有り]
   

  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.
• An efficient synthesis of 7α,12α-dihydroxy-4-cholesten-3-one and its biological precursor 7α-hydroxy-4-cholesten-3-one: Key intermediates in bile acid biosynthesis.

  Ogawa S; Zhou B; Kimoto Y; Omura K; Kobayashi A; Higashi T; Mitamura K; Ikegawa S; Hagey LR; Hofmann AF; Iida T

  Steroids 78 9 927 - 937 2013年09月 [査読有り]
   

  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.
• Clostridium scindens: a human gut microbe with a high potential to convert glucocorticoids into androgens

  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 54 9 2437 - 2449 2013年09月 [査読有り]
   

  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.
• LC/MS/MS of Steroids Having Vicinal Diol as Electrospray-Active Boronates

  Tatsuya Higashi; Katsumi Kawasaki; Nagisa Matsumoto; Shoujiro Ogawa; Kuniko Mitamura; Shigeo Ikegawa

  CHEMICAL & PHARMACEUTICAL BULLETIN 61 3 326 - 332 2013年03月 [査読有り]
   

  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.
• Synthesis of multiply deuterated 3- and 21-monosulfates of allo-tetrahydrocorticosteroids as internal standards for mass spectrometry

  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 77 13 1423 - 1437 2012年11月 [査読有り]
   

  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.
• A novel varanic acid epimer--(24R,25S)-3α,7α,12α,24-tetrahydroxy-5β-cholestan-27-oic acid--is a major biliary bile acid in two varanid lizards and the Gila monster.

  Hagey LR; Ogawa S; Kato N; Satoh née; Okihara R; Une M; Mitamura K; Ikegawa S; Hofmann AF; Iida T

  Steroids 77 13 1510 - 1521 2012年11月 [査読有り]
   

  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.
• Involvement of SIK3 in Glucose and Lipid Homeostasis in Mice

  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 7 5 e37803  2012年05月 [査読有り]
   

  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.
• Synthesis of the 3-sulfates of S-acyl glutathione conjugated bile acids and their biotransformation by a rat liver cytosolic fraction

  Kuniko Mitamura; Naohiro Hori; Shiori Mino; Takashi Iida; Alan F. Hofmann; Shigeo Ikegawa

  CHEMISTRY AND PHYSICS OF LIPIDS 165 3 261 - 269 2012年04月 [査読有り]
   

  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.
• Identification of S-acyl glutathione conjugates of bile acids in human bile by means of LC/ESI-MS.

  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 2011年12月 [査読有り]
   

  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.
• Synthesis of 3- and 21-monosulfates of [2,2,3β,4,4-²H₅]-tetrahydrocorticosteroids in the 5β-series as internal standards for mass spectrometry.

  Ikegawa S; Nagae K; Mabuchi T; Okihara R; Hasegawa M; Minematsu T; Iida T; Mitamura K

  Steroids 76 12 1232 - 1240 2011年11月 [査読有り]
   

  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.
• Chemical synthesis of the (25R)- and (25S)-epimers of 3α,7α,12α-trihydroxy-5α-cholestan-27-oic acid as well as their corresponding glycine and taurine conjugates.

  Ogawa S; Mitamura K; Ikegawa S; Krasowski MD; Hagey LR; Hofmann AF; Iida T

  Chemistry and physics of lipids 164 5 368 - 377 2011年07月 [査読有り]
   

  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.
• Synthesis of the 3-sulfates of N-acetylcysteine conjugated bile acids (BA-NACs) and their transient formation from BA-NACs and subsequent hydrolysis by a rat liver cytosolic fraction as shown by liquid chromatography/electrospray ionization-mass spectrometry

  Kuniko Mitamura; Toshihiro Sakai; Risa Nakai; Tateaki Wakamiya; Takashi Iida; Alan F. Hofmann; Shigeo Ikegawa

  ANALYTICAL AND BIOANALYTICAL CHEMISTRY 400 7 2061 - 2072 2011年06月 [査読有り]
   

  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.
• Characterization of non-enzymatic acylation of amino or thiol groups of bionucleophiles by the acyl-adenylate or acyl-CoA thioester of cholic acid

  Kuniko Mitamura; Eriko Aoyama; Toshihiro Sakai; Takashi Iida; Alan F. Hofmann; Shigeo Ikegawa

  ANALYTICAL AND BIOANALYTICAL CHEMISTRY 400 7 2253 - 2259 2011年06月 [査読有り]
   

  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.
• Characterization of Oxysterols and Their Sulfates in Primary Rat Hepatocytes (Prh) Following Increased Expression of the Mitochondrial Cholesterol Delivery Protein, StarD1

  Genta Kakiyama; Shigeo Ikegawa; Kuniko Mitamura; Douglas M. Heuman; William M. Pandak; Shunlin Ren

  GASTROENTEROLOGY 140 5 S936 - S936 2011年05月 [査読有り]
  
• 液体クロマトグラフィー/質量分析法によるヒト胆汁中グルタチオン抱合型胆汁酸の同定

  池川 繁男; 堀 直宏; 三田村 邦子

  胆膵の病態生理 27 1 23 - 27 日本胆膵病態・生理研究会 2011年01月 [査読有り]
  
• Identification of bile acid S-acyl glutathione conjugates in rat bile by liquid chromatography/electrospray ionization-linear ion trap mass spectrometry

  Kuniko Mitamura; Naohiro Hori; Takashi Iida; Alan F. Hofmann; Shigeo Ikegawa

  STEROIDS 76 1-2 68 - 77 2011年01月 [査読有り]
   

  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.
• Salivary chenodeoxycholic acid and its glycine-conjugate: Their determination method using LC-MS/MS and variation of their concentrations with increased saliva flow rate

  Tatsuya Higashi; Yujin Shibayama; Takuya Ichikawa; Koichi Ito; Toshimasa Toyo'oka; Kazutake Shimada; Kuniko Mitamura; Shigeo Ikegawa; Hitoshi Chiba

  STEROIDS 75 4-5 338 - 345 2010年04月 [査読有り]
   

  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.
• Potential Corticoid Metabolites: Chemical Synthesis of 3- and 21-Monosulfates and Their Double-Conjugates of Tetrahydrocorticosteroids in the 5 alpha- and 5 beta-Series

  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 58 3 344 - 353 2010年03月 [査読有り]
   

  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.
• Simultaneous Determination of Twelve Tetrahydrocorticosteroid Glucuronides in Human Urine by Liquid Chromatography/Electrospray Ionization-Linear Ion Trap Mass Spectrometry

  Shigeo Ikegawa; Maki Hasegawa; Rika Okihara; Chikara Shimidzu; Hitoshi Chiba; Takashi Iida; Kuniko Mitamura

  ANALYTICAL CHEMISTRY 81 24 10124 - 10135 2009年12月 [査読有り]
   

  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.
• Chemical synthesis of N-acetylcysteine conjugates of bile acids and in vivo formation in cholestatic rats as shown by liquid chromatography/electrospray ionization-linear ion trap mass spectrometry

  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 877 25 2630 - 2638 2009年09月 [査読有り]
   

  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.
• Chemical synthesis of bile acid acyl-adenylates and formation by a rat liver microsomal fraction

  Shigeo Ikegawa; Hiromi Ito; Motohiro Ohshima; Masako Maeda; Alan F. Hofmann; Kuniko Mitamura

  STEROIDS 74 9 751 - 757 2009年09月 [査読有り]
   

  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.
• Formation and biliary excretion of glutathione conjugates of bile acids in the rat as shown by liquid chromatography/electrospray ionization-linear ion trap mass spectrometry

  Kuniko Mitamura; Saai Watanabe; Yutaka Mitsumoto; Toshihiro Sakai; Mitsuru Sogabe; Tateaki Wakamiya; Shigeo Ikegawa

  ANALYTICAL BIOCHEMISTRY 384 2 224 - 230 2009年01月 [査読有り]
   

  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
• A facile synthesis of C-24 and C-25 oxysterols by in situ generated ethyl(trifluoroxymethyl)dioxirane

  Shoujiro Ogawa; Genta Kakiyama; Akina Muto; Atsuko Hosoda; Kuniko Mitamura; Shigeo Ikegawa; Alan F. Hofmann; Takashi Iida

  STEROIDS 74 1 81 - 87 2009年01月 [査読有り]
   

  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.
• Immunoprecipitation and MALDI-MS identification of lithocholic acid-tagged proteins in liver of bile duct-ligated rats

  Shigeo Ikegawa; Tetsushi Yamamoto; Hiromi Ito; Shunji Ishiwata; Toshihiro Sakai; Kuniko Mitamura; Masako Maeda

  JOURNAL OF LIPID RESEARCH 49 11 2463 - 2473 2008年11月 [査読有り]
   

  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.
• Production and Characterization of a Monoclonal Antibody to Capture Proteins Tagged with Lithocholic Acid

  Shigeo Ikegawa; Tetsushi Yamamoto; Takahiro Miyashita; Rika Okihara; Shunji Ishmata; Toshihiro Sakai; Rung-Hwa Chong; Masako Maeda; Alan F. Hofmann; Kuniko Mitamura

  ANALYTICAL SCIENCES 24 11 1475 - 1480 2008年11月 [査読有り]
   

  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.
• Isolation and chemical synthesis of a major, novel biliary bile acid in the common wombat (Vombatus ursinus): 15 alpha-hydroxylithocholic acid

  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 48 12 2682 - 2692 2007年12月 [査読有り]
   

  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.
• リトコール酸固定化担体を用いるアフィニティークロマトグラフィーによるラット肝細胞内タンパク質の捕捉と液体クロマトグラフィー質量分析法による同定(若手研究者の初論文特集)

  堺 俊博; 三田村 邦子; 多賀 淳; 本田 進; 池川 繁男

  分析化学 56 9 713 - 720 公益社団法人日本分析化学会 2007年09月 [査読有り]
   

  モノヒドロキシ胆汁酸であるリトコール酸(LCA)の24位カルボキシル基と3位ヒドロキシ基を介して支持体に固定化した2種類のアフィニティー担体を調製し,これらを用いてラット肝細胞内ミトコンドリア画分のLCAに結合親和性を持つタンパク質を捕捉するとともに,電気泳動法によるディファレンシャルディスプレーで特定したタンパク質を液体クロマトグラフィー/エレクトロスプレーイオン化質量分析に付して分子量とヒット率を加味しながらデータベース検索にてタンパク質を同定した.その結果,carbamoyl phosphate synthase I,glutamate dehydrogenase,acyl-CoA dehydrogenase,enoyl-CoA hydratase,acetyl-CoA acyltransferase,aldehyde dehydrogenaseなど,ミトコンドリアに局在する酵素タンパク質のほか,ミトコンドリア画分に混入した血中のalbumin,細胞質画分のglutathione S-transferaseなどのタンパク質であることを明らかにした.
• Determination of digoxin in human serum using stable isotope dilution liquid chromatography/electrospray ionization-tandem mass spectrometry

  Kuniko Mitamura; Aki Horikawa; Yuko Yamane; Yukari Ikeda; Youichi Fuji; Kazutake Shimada

  BIOLOGICAL & PHARMACEUTICAL BULLETIN 30 9 1653 - 1656 2007年09月 [査読有り]
   

  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.
• Analysis of bile acid glutathione thioesters by liquid chromatography/electrospray ionization-tandem mass spectrometry

  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 855 1 88 - 97 2007年08月 [査読有り]
   

  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.
• Oxysterols are substrates for cholesterol sulfotransferase

  Hirotoshi Fuda; Normal B. Javitt; Kuniko Mitamura; Shigeo Ikegawa; Charles A. Strott

  JOURNAL OF LIPID RESEARCH 48 6 1343 - 1352 2007年06月 [査読有り]
   

  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.
• Determination of sulfates of androsterone and epiandrosterone liquid in human serum using isotope diluted chromatography-electrospray ionization-mass spectrometry

  K Mitamura; M Setaka; K Shimada; S Honma; M Namiki; E Koh; A Mizokami

  BIOMEDICAL CHROMATOGRAPHY 19 10 796 - 801 2005年12月 [査読有り]
   

  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.
• Determination method for steroid 5 alpha-reductase activity using liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry

  K Mitamura; C Ogasawara; A Shiozawa; E Terayama; K Shimada

  ANALYTICAL SCIENCES 21 10 1241 - 1244 2005年10月 [査読有り]
   

  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.
• Contribution of glucuronic acid and sulfonic acid moieties during photocatalytic degradation of estrogen conjugates

  T Mizuguchi; Y Shibayama; K Mitamura; K Shimada

  JOURNAL OF HEALTH SCIENCE 51 4 447 - 452 2005年08月 [査読有り]
   

  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.
• Determination of digitoxin in human serum using stable isotope diluted liquid chromatography/electrospray ionization-tandem mass spectrometry

  K Mitamura; A Horikawa; A Nagahama; K Shimada; Y Fujii

  JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 28 18 2839 - 2848 2005年 [査読有り]
   

  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.
• Degradation of estrogen conjugates using titanium dioxide as a photocatalyst

  K Mitamura; H Narukawa; T Mizuguchi; K Shimada

  ANALYTICAL SCIENCES 20 1 3 - 4 2004年01月 [査読有り]
   

  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.
• Simultaneous determination of androstenediol 3-sulfate and dehydroepiandrosterone sulfate in human serum using isotope diluted liquid chromatography-electrospray ionization-mass spectrometry

  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 796 1 121 - 130 2003年10月 [査読有り]
   

  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.
• Determination of estrogens in rat brains using gas chromatography/mass spectrometry/mass spectrometry

  Mitamura K; Mikami N; Kambara Y; Ohno E; Shimada K

  Chromatography 23 2 65 - 71 2002年07月 [査読有り]
  
• Identification of dehydroepiandrosterone metabolites formed from human prostate homogenate using liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry

  K Mitamura; T Nakagawa; K Shimada; M Namiki; E Koh; A Mizokami; S Honma

  JOURNAL OF CHROMATOGRAPHY A 961 1 97 - 105 2002年06月 [査読有り]
   

  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.
• Studies on neurosteroids XII. Determination of enzymatically formed catechol estrogens and guaiacol estrogens by rat brains using liquid chromatography mass spectrometry mass spectrometry

  K Mitamura; M Yatera; K Shimada

  JOURNAL OF CHROMATOGRAPHY B 748 1 89 - 96 2000年10月 [査読有り]
   

  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.
• Studies on neurosteroids XII. Determination of enzymatically formed catechol estrogens and guaiacol estrogens by rat brains using liquid chromatography-mass spectrometry-mass spectrometry

  K. Mitamura; M. Yatera; K. Shimada

  Journal of Chromatography B: Biomedical Sciences and Applications 748 1 89 - 96 2000年10月 [査読有り]
   

  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.
• Studies on neurosteroids. Part XIII. Characterization of catechol estrogens in rat brains using liquid chromatography-mass spectrometry-mass spectrometry

  K Mitamura; M Yatera; K Shimada

  ANALYST 125 5 811 - 814 2000年 [査読有り]
   

  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.
• Quantitative determination of pregnenolone 3-sulfate in rat brains using liquid chromatography/electrospray ionization-mass spectrometry

  K Mitamura; M Yatera; K Shimada

  ANALYTICAL SCIENCES 15 10 951 - 955 1999年10月 [査読有り]
   

  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.
• Studies on neurosteroids IX. Characterization of estrogens in rat brains using gas chromatography-tandem mass spectrometry

  K Shimada; K Mitamura; M Shiroyama; K Yago

  JOURNAL OF CHROMATOGRAPHY A 847 1-2 171 - 178 1999年06月 [査読有り]
   

  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.
• Levels of 24,25-dihydroxyvitamin D-3, 25-hydroxyvitamin D-3 and 25-hydroxyvitamin D-3 3-sulphate in human plasma

  T Higashi; K Mitamura; H Ohmi; N Yamada; K Shimada; K Tanaka; H Honjo

  ANNALS OF CLINICAL BIOCHEMISTRY 36 1 43 - 47 1999年01月 [査読有り]
   

  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.
• High-performance liquid chromatographic separation of vitamin D-3 3-fatty acid esters and their liquid chromatography mass spectrometry

  K Mitamura; Y Nambu; M Tanaka; A Kawanishi; J Kitahori; K Shimada

  JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 22 3 367 - 377 1999年 [査読有り]
   

  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.
• Enzymatic hydrolysis of the conjugate of vitamin D and related compounds

  K Shimada; K Mitamura; K Saito; Y Ohtake; Nakatani, I

  JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS 15 9-10 1207 - 1214 1997年06月 [査読有り]
   

  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.
• In vitro glucuronidation of 25-hydroxyvitamin D-3 and its pro-form

  K Shimada; Y Kamezawa; K Mitamura

  BIOLOGICAL & PHARMACEUTICAL BULLETIN 20 6 596 - 600 1997年06月 [査読有り]
   

  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.
• Characterization of monoglucuronides of vitamin D2 and 25-hydroxyvitamin D2 in rat bile using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

  Kazutake Shimada; Kuniko Mitamura; Ito Nakatani

  Journal of Chromatography B: Biomedical Applications 690 1-2 348 - 354 1997年03月 [査読有り]
   

  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.
• Characterization of monoglucuronides of vitamin D-2 and 25-hydroxyvitamin D-2 in rat bile using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

  K Shimada; K Mitamura; Nakatani, I

  JOURNAL OF CHROMATOGRAPHY B 690 1-2 348 - 354 1997年03月 [査読有り]
   

  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.
• Determination of 25-hydroxyvitamin D3 in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

  Kazutake Shimada; Kuniko Mitamura; Nami Kitama; Michiko Kawasaki

  Journal of Chromatography B: Biomedical Applications 689 2 409 - 414 1997年02月 [査読有り]
   

  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.
• Determination of 25-hydroxyvitamin D-3 in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

  K Shimada; K Mitamura; N Kitama; M Kawasaki

  JOURNAL OF CHROMATOGRAPHY B 689 2 409 - 414 1997年02月 [査読有り]
   

  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.
• Separation and characterization of Monoglucuronides of vitamin D-3 and 25-hydroxyvitamin D-3 in rat bile by high-performance liquid chromatography

  K Shimada; Nakatani, I; K Saito; K Mitamura

  BIOLOGICAL & PHARMACEUTICAL BULLETIN 19 4 491 - 494 1996年04月 [査読有り]
   

  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.
• SYNTHESES AND ENZYMATIC-HYDROLYSIS OF 25-HYDROXYVITAMIN-D MONOGLUCURONIDES

  K SHIMADA; K SUGAYA; H KAJI; NAKATANI, I; K MITAMURA; N TSUTSUMI

  CHEMICAL & PHARMACEUTICAL BULLETIN 43 8 1379 - 1384 1995年08月 

  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.
• SEPARATION AND CHARACTERIZATION OF 25-HYDROXYVITAMIN D-3 3-SULFATE IN HUMAN PLASMA BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

  K SHIMADA; K MITAMURA; M MUKOUYAMA; T OKUMURA; K SUGAYA

  JOURNAL OF CHROMATOGRAPHIC SCIENCE 33 2 82 - 85 1995年02月 [査読有り]
   

  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.
• RETENTION BEHAVIOR OF VITAMIN-D AND RELATED-COMPOUNDS DURING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

  K SHIMADA; K MITAMURA; M MIURA; A MIYAMOTO

  JOURNAL OF LIQUID CHROMATOGRAPHY 18 14 2885 - 2893 1995年 [査読有り]
   

  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.
• Syntheses and Enzymatic Hydrolysis of 25-Hydroxyvitamin D Monoglucuronides

  Kazutake Shimada; Katsuko Sugaya; Hidefumi Kaji; Ito Nakatani; Kuniko Mitamura; Noriko Tsutsumi

  Chemical and Pharmaceutical Bulletin 43 8 1379 - 1384 1995年 [査読有り]
   

  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.
• Quantitative determination of 25‐hydroxyvitamin D3 3‐sulphate in human plasma using high performance liquid chromatography

  Kazutake Shimada; Kuniko Mitamura; Nami Kitama

  Biomedical Chromatography 9 5 229 - 232 1995年 [査読有り]
   

  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.
• RETENTION BEHAVIOR OF CONJUGATED METABOLITES OF VITAMIN-D AND RELATED-COMPOUNDS IN HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

  K SHIMADA; K MITAMURA; H KAJI; M MORITA

  JOURNAL OF CHROMATOGRAPHIC SCIENCE 32 3 107 - 111 1994年03月 [査読有り]
   

  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.
• HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC SEPARATION OF SENSITIVE FLUORESCENT DERIVATIVES OF BILE-ACIDS WITH CYCLODEXTRIN-CONTAINING MOBILE-PHASE

  K SHIMADA; K MITAMURA; S ISHITOYA; K HIRAKATA

  JOURNAL OF LIQUID CHROMATOGRAPHY 16 18 3965 - 3976 1993年 [査読有り]
   

  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.
• SEPARATION OF THE DIASTEREOMERS OF BACLOFEN BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY USING CYCLODEXTRIN AS A MOBILE-PHASE ADDITIVE

  K SHIMADA; K MITAMURA; M MORITA; K HIRAKATA

  JOURNAL OF LIQUID CHROMATOGRAPHY 16 15 3311 - 3320 1993年 [査読有り]
   

  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.
• HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC SEPARATION OF BILE-ACID PYRENACYL ESTERS WITH CYCLODEXTRIN-CONTAINING MOBILE PHASE

  K SHIMADA; Y KOMINE; K MITAMURA

  JOURNAL OF CHROMATOGRAPHY-BIOMEDICAL APPLICATIONS 565 1-2 111 - 118 1991年04月 [査読有り]
   

  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.
• Retention behavior of bile acid derivatives using cyclodextrin in the mobile phase in high-performance liquid chromatography

  Kazutake Shimada; Yoshihiro Komine; Mitamura Kuniko

  Journal of Chromatographic Science 28 6 331 - 335 1990年 [査読有り]
   

  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.

書籍

• 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 (担当:分担執筆範囲:第1章 1-6 c.その他 pp.49-55)南江堂 2015年09月 ISBN: 9784524403196 298 pp.49-55
• 試料分析講座 脂質分析

  池川 繁男; 三田村 邦子 (担当:分担執筆範囲:酸化コレステロールと胆汁酸の分析)丸善出版 2011年12月 ISBN: 9784621084649 238 135-148
• 分析化学便覧 改訂6版

  東 達也; 三田村 邦子 (担当:分担執筆範囲:ステロイド、胆汁酸)丸善 2011年12月 ISBN: 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 (担当:分担執筆範囲:第1章 1-4 c. その他)南江堂 2010年08月 ISBN: 9784524402625 274 41-46
• 薬学分析科学の最前線

  池川 繁男; 三田村 邦子 (担当:分担執筆範囲:LC/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年03月 ISBN: 9784840739658 185 130-131
• ライフサイエンス系の基礎英語テクニカルターム

  飯田隆; 池川繁男; 伊藤順子; 宇根瑞穂; 為我井秀行; 三田村邦子 (担当:分担執筆範囲:)三共出版 2008年11月 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 (担当:分担執筆範囲: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年12月 ISBN: 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 (担当:分担執筆範囲:第4章 ステロイド)丸善 2001年12月 ISBN: 9784621084090 389-394

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  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年03月 ポスター発表
• 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  [通常講演]

  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年03月 ポスター発表 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  [通常講演]

  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年03月 ポスター発表 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  [通常講演]

  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年03月 ポスター発表
• 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  [通常講演]

  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年03月 ポスター発表
• 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  [通常講演]

  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年 ポスター発表 札幌
• Identificaiton of antitumor component in maple syrup to develop novel anti-cancer drugs for colorectal cancer.  [通常講演]

  Yamamoto T; Shiburo R; Mitamura K; Taga A

  第75回日本癌学会学術総会 2016年10月 ポスター発表
• The role of maple syrup on cell proliferation of colorectal cancer cells  [通常講演]

  Kubota C; Yamamoto T; Mitamura K; Taga A

  第75回日本癌学会学術総会 2016年10月 ポスター発表
• Protein component in maple syrup has a potential to develop novel anti-cancer drugs for colorectal cancer  [通常講演]

  amamoto T; Shiburo R; Mitamura K; Taga A

  米国消化器病週間 2016年05月 ポスター発表
• 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.  [通常講演]

  Takakura H; Yamamoto T; Mitamura K; Kudo M; Naito Z; Taga A

  米国消化器病週間 2016年05月 ポスター発表
• 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  [通常講演]

  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年03月 口頭発表（一般）
• スッポンからのコラーゲン抽出法の最適化  [通常講演]

  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年03月 口頭発表（一般）
• 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  [通常講演]

  三田村 邦子

  日本薬学会第136年会 2016年03月 ポスター発表
• 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  [通常講演]

  山本 哲志; 三田村 邦子; 多賀 淳

  日本薬学会第136年会 2016年03月 ポスター発表
• A Novel Bile Acid Synthesis Pathway in Mouse Liver Initiated by 24-Hydroxycholesterol.  [通常講演]

  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年11月 ポスター発表 San Francisco, CA
• 薬学による臨床化学へのアプローチ  [通常講演]

  三田村 邦子

  第55回日本臨床化学会年次学術集会 2015年10月 シンポジウム・ワークショップパネル（指名） 大阪 日本臨床化学会
  
• ホルマリン固定パラフィン包埋大腸癌組織を用いたプロテオーム解析  [通常講演]

  山本哲志; 工藤光洋; 彭為霞; 高田英志; 三田村邦子; 多賀淳; 内藤善哉

  第74回日本癌学会学術総会 2015年10月 ポスター発表
• 大腸癌細胞の増殖・浸潤におけるメープルシロップの効果  [通常講演]

  久保田千晶; 山本哲志; 三田村邦子; 多賀淳

  第74回日本癌学会学術総会 2015年10月 ポスター発表
• Comparison of the sensitivity and selectivity for the CID and PQD mode in the analysis of conjugated steroids  [通常講演]

  Kuniko Mitamura; Satoshi Kurabuchi; Mamiko Ueda; Tetsushi Yamamoto; Atsushi Taga; Shigeo Ikegawa

  63th ASMS Conference on Mass Spectrometry and Allied Topics 2015年06月 ポスター発表 アメリカ、セントルイス 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～  [通常講演]

  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年04月 公開講演，セミナー，チュートリアル，講習，講義等 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  [通常講演]

  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年03月 口頭発表（一般） 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  [通常講演]

  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年03月 ポスター発表 神戸 日本薬学会
  
• High Speed Molecular Weight Measurement of Proteins by Capillary Electrophoresis  [通常講演]

  Atsushi Taga; Yuri Miyazaki; Tetsushi Yamamoto; Kuniko Mitamura

  The 4th International Symposium on Steel Science - ISSS 2014 2014年11月 ポスター発表 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  [通常講演]

  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年10月 ポスター発表 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  [通常講演]

  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年10月 ポスター発表 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  [通常講演]

  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年10月 ポスター発表 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－  [通常講演]

  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年10月 ポスター発表 京都、京都薬科大学 日本薬学会近畿支部
  
• Maple syrup の大腸癌抑制作用  [通常講演]

  上村 健太朗; 山本 哲志; 三田村 邦子; 多賀 淳

  第73回日本癌学会学術総会 2014年09月 ポスター発表 横浜、パシフィコ横浜 日本癌学会
  
• ホルマリン固定パラフィン包埋大腸癌組織を用いた新規治療標的の探索  [通常講演]

  山本 哲志; 工藤 光洋; 彭 為霞; 高田 英志; 三田村 邦子; 多賀 淳; 内藤 善哉

  第73回日本癌学会学術総会 2014年09月 ポスター発表 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  [通常講演]

  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年03月 ポスター発表 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  [通常講演]

  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年03月 ポスター発表 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  [通常講演]

  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年03月 ポスター発表 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  [通常講演]

  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年03月 ポスター発表 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  [通常講演]

  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年03月 ポスター発表 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  [通常講演]

  Atsushi Taga; Yuri Miyazaki; Tetsushi Yamamoto; Kuniko Mitamura

  13th International Symposium on Hyphenated Techniques in Chromatography and Separation Technology (HTC-13) 2014年01月 ポスター発表
• Reversal of NAFL Through Selective Increased Intracellular Hepatic Cholesterol Catabolism  [通常講演]

  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年11月 ポスター発表 Washington, DC, USA.
• Proteomic profiling of formalin-fixed paraffin-embedded colorectal cancer tissue for identification of novel biomarker.  [通常講演]

  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年10月 ポスター発表 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  [通常講演]

  三田村 邦子

  第60回日本臨床検査医学会学術集会 2013年10月 シンポジウム・ワークショップパネル（指名） 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  [通常講演]

  三田村 邦子

  医用質量分析認定士 第1回講習会 2013年09月 公開講演，セミナー，チュートリアル，講習，講義等 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  [通常講演]

  池川繁男; Shijian CHU; 三田村邦子

  日本薬学会第133年会 2013年03月 ポスター発表 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  [通常講演]

  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年03月 ポスター発表 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  [通常講演]

  池川繁男; Shijian Chu; 三田村邦子

  第34回胆汁酸研究会 2012年12月 口頭発表（一般） 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  [通常講演]

  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年10月 ポスター発表 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  [通常講演]

  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年10月 ポスター発表 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  [通常講演]

  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年10月 ポスター発表 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  [通常講演]

  Narimi Kato; Takashi Iida; Shoujiro Ogawa; Mizuho Une; Kuniko Mitamura; Shigeo Ikegawa; Lee R. Hagey; Alan F Hofmann

  XXII Bile Acid Meeting 2012年10月 ポスター発表 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.  [通常講演]

  Kaoru Omura; Yuusuke Kimoto; Takashi Iida; Shoujiro Ogawa; Kuniko Mitamura; Shigeo Ikegawa; Lee R. Hagey; Alan F Hofmann

  XXII Bile Acid Meeting 2012年10月 ポスター発表 Vienna, Austria
• Identification of S-acyl glutathione conjugates of bile acids in human bile by means of LC/ESI-MS  [通常講演]

  Shigeo Ikegawa; Naohiro Hori; Takashi Iida; Alan F. Hofmann; Kuniko Mitamura

  XXII Bile Acid Meeting 2012年10月 ポスター発表 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  [通常講演]

  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年09月 口頭発表（一般） 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  [通常講演]

  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年08月 口頭発表（一般） 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  [通常講演]

  Kuniko Mitamura; Rika Okihara; Maki Hasegawa; Mami Kamibayashi; Takashi Iida; Shigeo Ikegawa

  60th ASMS Conference on Mass Spectrometry and Allied Topics 2012年05月 ポスター発表 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  [通常講演]

  Shigeo Ikegawa; Naohiro Hori; Takashi Iida; Alan F. Hofmann; Kuniko Mitamura

  60th ASMS Conference on Mass Spectrometry and Allied Topics 2012年05月 ポスター発表 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  [通常講演]

  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年03月 ポスター発表 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  [通常講演]

  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年03月 ポスター発表 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  [通常講演]

  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年03月 口頭発表（一般） 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  [通常講演]

  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年02月 口頭発表（一般） 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  [通常講演]

  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年10月 口頭発表（一般） 神戸、神戸大学ﾎﾟｰﾄｱｲﾗﾝﾄﾞｷｬﾝﾊﾟｽ 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  [通常講演]

  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年09月 ポスター発表 大阪、ホテル阪急ｴｷｽﾎﾟﾊﾟｰｸ 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  [通常講演]

  三田村邦子; 池川繁男

  第60回日本分析化学会 2011年09月 口頭発表（一般） 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  [通常講演]

  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年08月 口頭発表（一般） 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  [通常講演]

  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年07月 口頭発表（一般）
• 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  [通常講演]

  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年06月 口頭発表（一般）
• Liquid chromatography-mass spectrometric characterization of sulfation of N-acetylcysteine conjugated bile acids by a rat liver cytosolic fraction  [通常講演]

  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年05月 ポスター発表 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  [通常講演]

  Kuniko Mitamura; Eriko Aoyama; Toshihiro Sakai; Takashi Iida; Alan F. Hofmann; Shigeo Ikegawa

  59th ASMS Conference on Mass Spectrometry and Allied Topics 2011年05月 ポスター発表 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  [通常講演]

  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年03月 ポスター発表 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  [通常講演]

  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年03月 ポスター発表 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  [通常講演]

  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年02月 口頭発表（一般） 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  [通常講演]

  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年11月 口頭発表（一般） 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.  [通常講演]

  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年10月 Freiburg, Germany Folk Foundation
  
• Analysis of glutathione conjugates of bile acids in rat bile by LC/ESI-MS/MS  [通常講演]

  Shigeo Ikegawa; Naohiro Hori; Takashi Iida; Kuniko Mitamura

  XXI International Bile Acid Meeting Bile Acid as Metabolic Integrators and Therapeutics 2010年10月 ポスター発表 Freiburg, Germany Folk Foundation
  
• Simultaneous Determination of Tetrahydrocorticosteroid Glucuronides by Liquid Chromatography/Electrospray Ionization-Mass Spectrometry  [通常講演]

  Kuniko Mitamura; Shigeo Ikegawa

  The 12th Asian-Pacific Congress of Clinical Biochemistry 2010年10月 シンポジウム・ワークショップパネル（指名） 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  [通常講演]

  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年09月 ポスター発表 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  [通常講演]

  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年09月 口頭発表（一般） 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  [通常講演]

  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年07月 口頭発表（一般） 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  [通常講演]

  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年06月 口頭発表（一般） 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.  [通常講演]

  Naohiro Hori; Kuniko Mitamura; Toshihiro Sakai; Takashi Iida; Shigeo Ikegawa

  58th ASMS Conference on Mass Spectrometry 2010年05月 ポスター発表 Salt Lake City, USA ASMS
  
• Quantitative Metabolic Profiling of Twelve Tetrahyrocorticosteroid Glucuronides in Urine by Isotope Dilution LC-ESI-Linear Ion Trap MS/MS  [通常講演]

  Kuniko Mitamura; Maki Hasegawa; Rika Okihara; Chikara Shimidzu; Hitoshi Chiba; Takashi Iida; Shigeo Ikegawa

  58th ASMS Conference on Mass Spectrometry 2010年05月 ポスター発表 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  [通常講演]

  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年03月 口頭発表（一般） 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  [通常講演]

  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年03月 口頭発表（一般） 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  [通常講演]

  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年03月 ポスター発表 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  [通常講演]

  三田村 邦子; 池川 繁男

  日本薬学会第130年会 2010年03月 ポスター発表 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  [通常講演]

  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年02月 口頭発表（一般） 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  [通常講演]

  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年02月 口頭発表（一般） 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  [通常講演]

  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年12月 口頭発表（一般） 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  [通常講演]

  Shigeo Ikegawa; Naohiro Hori; Toshihiro Sakai; Takashi Iida; Kuniko Mitamura

  日本薬物動態学会第24回年会 2009年11月 ポスター発表 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  [通常講演]

  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年11月 口頭発表（一般） 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  [通常講演]

  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年10月 口頭発表（一般） 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  [通常講演]

  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年10月 口頭発表（一般） 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  [通常講演]

  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年09月 ポスター発表 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  [通常講演]

  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年09月 口頭発表（一般） 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  [通常講演]

  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年09月 ポスター発表 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  [通常講演]

  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年09月 ポスター発表 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  [通常講演]

  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年09月 ポスター発表 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  [通常講演]

  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年07月 口頭発表（一般） 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  [通常講演]

  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年07月 ポスター発表 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  [通常講演]

  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年03月 ポスター発表 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  [通常講演]

  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年03月 ポスター発表 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  [通常講演]

  三田村 邦子

  日本薬学会第129年会 2009年03月 シンポジウム・ワークショップパネル（指名） 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  [通常講演]

  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年03月 口頭発表（一般） 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  [通常講演]

  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年02月 口頭発表（一般） 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  [通常講演]

  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年01月 口頭発表（一般） 大阪、大阪医大 日本臨床化学会近畿支部
  

MISC

• 大腸癌に対するメープルシロップに含まれるタンパク質画分による抗腫瘍効果の検討(The effect of protein component in maple syrup as a resource to development pharmaceutical drug for colorectal cancer.)

  山本 哲志; 三田村 邦子; 多賀 淳 日本癌学会総会記事 81回 P -2274 2022年09月
• トマト由来ステロイドアルカロイド トマチンによる膵臓癌細胞に対する抗腫瘍効果の検討

  山本 哲志; 辻本 伊織里; 辻 翔斗; 三田村 邦子; 多賀 淳 日本薬学会年会要旨集 142年会 27PO1 -77 2022年03月
• LC-MS/MSを用いたTCA回路を標的としたターゲットメタボロミクスの構築

  山本 哲志; 三田村 邦子; 多賀 淳 日本薬学会年会要旨集 141年会 29V11 -am06 2021年03月
• Identification of potential diagnostic biomarker for pancreatic cancer based on shotgun proteomic analysis.

  Tetsushi Yamamoto; Kuniko Mitamura; Atsushi Taga CANCER SCIENCE 112 524 -524 2021年02月
• ショットガンプロテオミクスによる新規膵臓癌診断マーカーの同定

  山本 哲志; 三田村 邦子; 多賀 淳 日本癌学会総会記事 79回 OE15 -5 2020年10月
• キャピラリー電気泳動を用いた血中循環腫瘍細胞検出法の検討

  山本 哲志; 三田村 邦子; 多賀 淳 日本薬学会年会要旨集 140年会 28P -am206 2020年03月
• プロテオーム解析を用いたストレプトゾトシン誘発糖尿病モデルラットの角膜における創傷治癒遅延機構の解析

  山本 哲志; 蛭子 小春; 三田村 邦子; 長井 紀章; 多賀 淳 JSBMS Letters 44 (Suppl.) 110 -110 2019年08月
• メープルシロップ中タンパク質成分は新規大腸癌治療薬開発の標的となり得る(Protein components of maple syrup as a potential source to develop novel anti-cancer drugs for colorectal cancer)

  山本 哲志; 森山 由瑛; 三田村 邦子; 多賀 淳 日本癌学会総会記事 77回 2200 -2200 2018年09月
• LC‐MS/MSを用いたTCA回路代謝物の一斉分析

  佐藤完太; 山口真史; 山本哲志; 三田村邦子; 多賀淳 JSBMS Letters 43 (Supplement) 139 2018年08月
• ホルマリン固定パラフィン包埋大腸癌組織を用いた新規大腸癌診断マーカーの探索とその定量法の開発

  山本 哲志; 工藤 光洋; 橋本 知樹; 三田村 邦子; 内藤 善哉; 多賀 淳 JSBMS Letters 43 (Suppl.) 66 -66 2018年08月
• 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

  大竹裕子; 山本哲志; 三田村邦子; 多賀淳; 長井紀章 日本薬学会年会要旨集(CD-ROM) 138th (3) ROMBUNNO.26PA‐pm325 -243 2018年
• 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

  山本哲志; 中西紗緒理; 三田村邦子; 多賀淳 日本薬学会年会要旨集(CD-ROM) 138th ROMBUNNO.27PA‐am384 2018年
• 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

  三田村邦子; 森莉子; 上田麻美子; 亀井美希; 池川繁男 臨床化学 46 294 2017年09月
• 細胞外基質lumicanの膵臓癌特異糖鎖修飾を標的とした新規治療法の開発

  山本哲志; 多賀淳; 三田村邦子 大和証券ヘルス財団研究業績集 40 93‐96 2017年03月
• The effect of cyclophilin A on cell invasion of colorectal cancer cells

  Tetsushi Yamamoto; Kuniko Mitamura; Atsushi Taga INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 40 S52 -S52 2017年 [査読有り]
  
• 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

  松本孝彬; 山崎航; 小川祥二郎; 三田村邦子; 池川繁男; 東達也 日本薬学会年会要旨集(CD-ROM) 137th ROMBUNNO.27PB‐am021 2017年
• 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

  山本哲志; 谷田和香奈; 橋本知樹; 三田村邦子; 多賀淳 日本薬学会年会要旨集(CD-ROM) 137th ROMBUNNO.25PB‐am180 2017年
• 新規大腸癌治療薬開発のためのメープルシロップ中抗腫瘍成分の同定

  山本 哲志; 澁路 龍大; 三田村 邦子; 多賀 淳 日本癌学会総会記事 75回 P -1276 2016年10月
• 大腸がん細胞の細胞増殖におけるメープルシロップの役割について

  久保田 千晶; 山本 哲志; 三田村 邦子; 多賀 淳 日本癌学会総会記事 75回 P -1277 2016年10月
• Protein Component in Maple Syrup Has a Potential to Develop Novel Anti-Cancer Drugs for Colorectal Cancer

  Tetsushi Yamamoto; Ryota Shiburo; Kuniko Mitamura; Atsushi Taga GASTROENTEROLOGY 150 (4) S625 -S625 2016年04月 [査読有り]
  
• 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

  Hideki Takakura; Tetsushi Yamamoto; Kuniko Mitamura; Mitsuhiro Kudo; Zenya Naito; Atsushi Taga GASTROENTEROLOGY 150 (4) S620 -S620 2016年04月 [査読有り]
  
• 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。

  高倉英樹; 山本哲志; 三田村邦子; 工藤光洋; 内藤善哉; 多賀淳 日本薬学会年会要旨集(CD-ROM) 136th ROMBUNNO.28O‐PM03S 2016年
• 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

  山本哲志; 三田村邦子; 多賀淳 日本薬学会年会要旨集(CD-ROM) 136th ROMBUNNO.27AB‐PM272 2016年
• スッポンからのコラーゲン抽出法の最適化

  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 日本薬学会年会要旨集(CD-ROM) 136th ROMBUNNO.28R‐AM07 2016年
• LC/ESI‐MSによる乾燥ろ紙尿中糖質コルチコイド代謝物スクリーニング法の開発

  三田村邦子; 森莉子; 上田麻美子; 亀井美希; 山本哲志; 多賀淳 日本薬学会年会要旨集(CD-ROM) 136th ROMBUNNO.28AB‐AM342 2016年
• A Novel Mitochondrial Pathway to Bile Acids through 24-Hydroxycholesterol in Mouse Liver

  Genta Kakiyama; Dalila M. Marques; Kuniko Mitamura; Hajime Takei; Hiroshi Nittono; Daniel Rodriguez-Agudo; Gregorio Gil; Huiping Zhou; Phillip B. Hylemon; William M. Pandak HEPATOLOGY 62 609A -610A 2015年10月
• ホルマリン固定パラフィン包埋大腸癌組織を用いたプロテオーム解析によるバイオマーカーの探索

  山本 哲志; 工藤 光洋; 彭 為霞; 高田 英志; 三田村 邦子; 多賀 淳; 内藤 善哉 日本癌学会総会記事 74回 P -1358 2015年10月
• 大腸癌細胞の増殖・浸潤におけるメープルシロップの効果

  久保田 千晶; 山本 哲志; 三田村 邦子; 多賀 淳 日本癌学会総会記事 74回 P -1308 2015年10月
• メープルシロップを用いた大腸癌抗腫瘍効果の検討

  山本哲志; 上村健太朗; 三田村邦子; 多賀淳 日本薬学会年会要旨集(CD-ROM) 135th ROMBUNNO.27H-PM14 2015年
• 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

  三田村邦子; 上田麻美子; 山本哲志; 多賀淳; 池川繁男 日本薬学会年会要旨集(CD-ROM) 135th ROMBUNNO.27PA-PM087 2015年
• 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

  三田村邦子; 蔵渕慧; 上田麻美子; 池川繁男; 山本哲志; 多賀淳 JSBMS Lett 39 (Supplement) 62 2014年09月
• ホルマリン固定パラフィン包埋大腸癌組織を用いた新規治療標的の探索(Proteomic analysis of formalin-fixed paraffin-embedded colorectal cancer tissue for identification of therapeutic target)

  山本 哲志; 工藤 光洋; 彭 為霞; 高田 英志; 三田村 邦子; 多賀 淳; 内藤 善哉 日本癌学会総会記事 73回 P -3254 2014年09月
• Maple syrupの大腸癌抑制作用(Anti-cancer Effect of Maple Syrup for Colorectal Cancer)

  上村 健太朗; 山本 哲志; 三田村 邦子; 多賀 淳 日本癌学会総会記事 73回 P -2416 2014年09月
• メープルシュガーにおける血糖値上昇抑制効果

  多賀淳; 長井紀章; 山本哲志; 三田村邦子; 伊藤吉將 日本薬学会年会要旨集(CD-ROM) 134th ROMBUNNO.28AML-101 2014年
• 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

  山本哲志; 木場克斗; 三田村邦子; 工藤光洋; 内藤善哉; 多賀淳 日本薬学会年会要旨集(CD-ROM) 134th ROMBUNNO.30PML-136 2014年
• 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

  三田村邦子; 大橋勇斗; 山本哲志; 多賀淳; 池川繁男 日本薬学会年会要旨集(CD-ROM) 134th ROMBUNNO.29AML-070 2014年
• 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

  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 日本薬学会年会要旨集(CD-ROM) 134th ROMBUNNO.30PML-139S 2014年
• MS解析でわかる胆汁酸の世界

  三田村 邦子; 東 達也; 池川 繁男 分子消化器病 9 (3) 273 -278 2012年03月 [招待有り]
  
• 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

  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 日本薬学会年会要旨集 131年会 (4) 142 -142 2011年03月
• 胆汁酸のグルタチオン抱合体とLC/MS

  三田村 邦子; 堺 俊博; 池川 繁男 Jasco report 51 (1) 7 -13 2009年09月 [招待有り]
  
• LC/ESI-MSによる内分泌・代謝疾患の科学診断へのアプローチ

  三田村 邦子; 長谷川 真紀; 沖原 梨香; 池川 繁男 臨床化学 38 (3) 291 -298 2009年07月 [査読有り]
  
• LC/ESI-MS2によるラット胆汁中グルタチオン抱合型胆汁酸の同定

  池川 繁男; 堀 直宏; 堺 俊博; 飯田 隆; 三田村 邦子 日本薬物動態学会年会講演要旨集 24 (0) 201 -201 2009年
• 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的診断へのアプローチ

  三田村 邦子 臨床化学 37 5 -5 2008年08月
• コレステロール硫酸転移酵素とコレステロール代謝

  布田 博敏; 三田村 邦子; 池川 繁男 臨床化学 37 (2) 148 -160 2008年04月 [査読有り]
  
• 胆汁酸の代謝活性中間体を経るＮ－アセチルシステイン抱合:胆汁酸のＮーアセチルシステイン抱合

  池川 繁男; 渡辺 彩愛; 堺 俊博; 曾我部 充; 若宮 建昭; 三田村 邦子 日本薬物動態学会年会講演要旨集 23 (0) 306 -306 2008年
• Study on the Syntheses and LC/ESI-MS^n Analyses of Glutathione Conjugates of Bile Acids

  SOGABE Mitsuru; MITAMURA Kuniko; WATANABE Saai; IKEGAWA Shigeo; YAMAGUCHI Yoshihiro; WAKAMIYA Tateaki Peptide science : proceedings of the ... Japanese Peptide Symposium 2006 295 -296 2007年03月
• 胆汁酸の代謝活性中間体を経るグルタチオン抱合体の生成

  三田村 邦子; 池川 繁男; 若宮 建昭 臨床化学 36 (3) 181 -188 2007年 [査読有り]
  
• 胆汁酸と疾患関連タンパク質

  三田村 邦子; 堺 俊博; 池川 繁男 日本医用マススペクトル学会サーキュラー 68 4 -8 2006年11月 [招待有り]
  
• ヒト皮膚を大腸菌から防御するタンパク質psoriasin

  三田村 邦子 臨床化学 34 (2) 147 -147 2005年 [招待有り]
  
• 前立腺癌とアンドロゲンの代謝分析

  東 達也; 三田村 邦子; 島田 和武 臨床化学 33 (3) 177 -182 2004年12月 [査読有り]
  
• Development of Analyses of Biological Steroids Using Chromatography : Special Reference to Vitamin D Compounds and Neurosteroids

  Shimada Kazutake; Higashi Tatsuya; Mitamura Kuniko Chromatography : journal of separation and detection sciences = クロマトグラフィー : 分離・検出科学 24 (1) 1 -6 2003年02月
• Development of analyses of biological steroids using chromatography --Special reference or vitamin D compounds and neurosteroids--

  Shimada K; Higashi T; Mitamura K CHROMATOGRAPHY 24 (1) 1 -6 2003年02月 [査読有り]
  
• Determination of Estrogens in Rat Brains Using Gas Chromatography/Mass Spectrometry/Mass Spectrometry

  Mitamura Kuniko; Mikami Nanae; Kambara Yukiko; OHNO Emi; SHIMADA Kazutake Chromatography : journal of separation and detection sciences = クロマトグラフィー : 分離・検出科学 23 (2) 65 -71 2002年07月
• Gas chromatography and high-performance liquid chromatography of natural steroids

  K Shimada; K Mitamura; T Higashi JOURNAL OF CHROMATOGRAPHY A 935 (1-2) 141 -172 2001年11月 [査読有り]
  
• Derivatization in liquid chromatography/mass spectrometric analysis of neurosteroids.

  K. Mitamura; K. Shimada Se pu = Chinese journal of chromatography / Zhongguo hua xue hui 19 508 -512 2001年01月
• 脳内カテコールエストロゲンのLC/MS分析における誘導体化の有用性

  河越 悠; 三田村 邦子; 島田 和武 Chromatography : journal of separation and detection sciences = クロマトグラフィー : 分離・検出科学 21 (4) 336 -337 2000年10月
• LC/MSにおける誘導体化の開発と応用

  三田村 邦子 Chromatography : journal of separation and detection sciences = クロマトグラフィー : 分離・検出科学 21 (4) 254 -255 2000年10月
• 脳内ステロイドホルモン分析法の開発XIII : LC/MS^nによる脳内カテコールエストロゲンの同定

  三田村 邦子; 矢寺 美佐子; 島田 和武 Chromatography : journal of separation and detection sciences = クロマトグラフィー : 分離・検出科学 21 (2) 125 -126 2000年05月
• Derivatization in liquid chromatography/mass spectrometric analysis of neurosteroids

  Mitamura K; Shimada K Chromatography 22 (1) 11 -15 2000年 [査読有り]
  
• アンドロゲンの生合成と代謝

  本間誠次郎; 湊 宏一; 三田村邦子; 島田和武 HORMONE FRONTIER IN GYNECOLOGY 22 (1) 11 -15 2000年 [査読有り]
  
• Determination of vitamin D3 metabolites using high-performance liquid chromatography or immunoaffinity chromatography

  Kazutake Shimada; Kuniko Mitamura; Tatsuya Higashi Journal of the Chinese Chemical Society 47 (2) 285 -289 2000年 [査読有り]
  
• LC/MSにおける抱合型ビタミンD代謝物の誘導体化

  東 達也; 三田村 邦子; 北堀 淳一; 島田 和武 Chromatography : journal of separation and detection sciences = クロマトグラフィー : 分離・検出科学 20 (2) 148 -149 1999年06月
• ステロイドの高速液体クロマトグラフィー/質量分析

  三田村 邦子; 島田 和武 分析化学 48 (4) 401 -411 1999年 [査読有り]
  
• ステロイドのLC/MSにおける誘導体化

  三田村 邦子; 矢後 謙一郎; 島田 和武 Chromatography : journal of separation and detection sciences = クロマトグラフィー : 分離・検出科学 19 (2) 144 -145 1998年08月
• LC/MSにおける誘導体化

  三田村 邦子; 島田 和武 藥學雜誌 118 (6) 206 -215 1998年06月 [査読有り]
  
• Characterization of monoglucuronides of vitamin D_2 and 25-hydroxyvitamin D_2 in rat bile using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

  SHIMADA Kazutake; MITAMURA Kuniko; NAKATANI Ito Journal of chromatography. B, Biomedical sciences and applications 690 (1) 348 -354 1997年03月
• Determination of 25-hydroxyvitamin D_3 in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

  SHIMADA Kazutake; MITAMURA Kuniko; KITAMA Nami; KAWASAKI Michiko Journal of chromatography. B, Biomedical sciences and applications 689 (2) 409 -414 1997年02月
• DERIVATIZATION OF THIOL-CONTAINING COMPOUNDS

  K SHIMADA; K MITAMURA JOURNAL OF CHROMATOGRAPHY B-BIOMEDICAL APPLICATIONS 659 (1-2) 227 -241 1994年09月 [査読有り]
  

受賞

• 2008年 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年 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年 第11回クロマトグラフィー科学会奨励賞
   JPN

共同研究・競争的資金等の研究課題

• ソフトイオン化質量分析法を基盤とする乾燥ろ紙尿中ステロイドの臨床検査法の開発

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  研究期間 : 2014年04月 -2017年03月 

  代表者 : 三田村 邦子

   

  非侵襲的に採取可能な尿中のステロイド代謝物のプロファイルを明らかにし、内分泌・代謝疾患の病態解析、診断指標の一助とすることを目的として、LC/MSによる尿中抱合型オキシステロールおよびテトラヒドロキシコルチコステロイドのプロファイル分析法を開発した。さらに本法が乾燥ろ紙尿に適用可能なことを示した。乾燥ろ紙尿は尿試料の保存や輸送が簡便であり、実施施設が限られた臨床検査用の検体として有用視される。
• 胆汁酸を核化合物とする新規プロドラッグの創製

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  研究期間 : 2011年 -2013年 

  代表者 : 三田村 邦子; 池川 繁男

   

  ウルソデオキシコール酸のN-アセチルシステイン抱合体（UDCA-NAC）が、肝で硫酸抱合を受ける一方、NACが加水分解されてUDCAとして体内を循環することを明らかにした。さらに、UDCA-NACはアセトアミノフェン誘発肝障害ラットの肝疾患マーカー酵素の上昇を抑制することを示した。これらの知見は、UDCA-NACが新規肝・胆・消化機能改善薬のプロドラッグとして有用であることを示している。
• ソフトイオン化質量分析法を基盤とする内分泌・代謝疾患のメタボローム解析

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  研究期間 : 2009年 -2011年 

  代表者 : 池川 繁男; 三田村 邦子

   

  最新のソフトイオン化質量分析法を基盤とするテトラヒドロコルチコステロイド、胆汁酸、オキシステロールなどのグルクロン酸、硫酸、グルタチオン、N-アセチルシステインなどとの抱合体の高感度測定法を開発し、本法が内分泌・代謝疾患の病因・病態解析に有用なことを示した。
• グルタチオン抱合型胆汁酸の生成と代謝機構に関する研究

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  研究期間 : 2008年 -2010年 

  代表者 : 三田村 邦子; 池川 繁男

   

  胆汁酸の代謝活性中間体を経るグルタチオン(GSH)抱合体の生成と代謝機構の解明を目的として、胆汁酸がin vitro、in vivoでGSH並びにN-アセチルシステイン(NAC)との抱合を受けることを明らかにするとともに、ラット及びヒト胆汁中にGSH抱合型胆汁酸が排出されていることを実証し、胆汁酸の新規代謝経路として提示した。また、NAC抱合型ウルソデオキシコール酸が肝機能改善薬のプロドラッグとなる可能性を示す基礎的知見を得た。
• 質量分析法を基盤とする臨床化学の新展開

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  研究期間 : 2007年 -2008年 

  代表者 : 池川 繁男; 三田村 邦子

   

  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。
• 胆汁酸修飾タンパク質を標的分子とする疾患プロテオミクスに関する基礎的研究

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  研究期間 : 2005年 -2006年 

  代表者 : 池川 繁男; 三田村 邦子

   

  本研究では、初めに胆汁うっ滞や大腸癌と関連して重要視されるリトコール酸(LCA)修飾タンパク質の捕捉と構造解析を目的として、まず、LCAのステロイド核に相補的で高い特異性を有する抗体を創出し、これによるラット肝細胞内LCA修飾タンパク質の捕捉とMSによる構造解析を試みた。その結果、LCAとRab関連タンパク質との共有結合付加体が存在し,これが肝疾患と深く関わることを示した。引き続き、ヒト主要胆汁酸5種のグルタチオン(GSH)抱合体標品を化学合成するとともに、これらのLC/ESI-MSnによる高感度直接一斉分析法を構築し、本法によってラット肝における胆汁酸のin vitro並びにin vivo GSH抱合体への変換を実証し、胆汁酸のGSH抱合という新規代謝経路の存在を明らかにした。また、胆汁酸修飾タンパク質の生成とGSH抱合への変換を統合的に解析する上で、鍵を握る代謝活性中間体(アシルアデニレートやCoAチオエステル)の生成を触媒する胆汁酸のCoAリガーゼ(rBAL並びにhBAL)を発現する大腸菌並びにHEK293細胞株の樹立にも成功した。これらの実績と経験を基に肝毒性や大腸癌発症のプロモーターとして知られるLCAを不溶性担体に固定化して用いるアフィニティークロマトグラフィーによって、LCAに親和性を持つラット肝細胞内の疾患関連タンパク質の捕捉と構造解析を試み、LCAに親和性を持つ各種酵素タンパク質の存在を明らかにし、LCAの肝毒性を考える上で極めて興味ある新たな知見を加えることができた。さらに、LCAをリガンドとするGタンパク質共役型受容体の発現亢進が、各種の癌と深く関わることが想定されることから、分子生物学的手法によって調製した組み換え型GPCRを免疫抗原としてモノクローナル抗体を作成し、これを用いるウエスタンブロットによって大腸菌細胞由来WiDrのみならず食道癌細胞にも発現していることを明らかにした。
• 前立腺癌予後診断用バイオマーカーの開発と臨床応用

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2003年 -2005年 

  代表者 : 島田 和武; 東 達也; 三田村 邦子; 本間 誠次郎

   

  現在,前立腺癌予後診断には血中前立腺特異的抗原(PSA)のスクリーニングが汎用されているが,PSAは組織特異的抗原であり,良性の前立腺肥大症などとの区別はなされない.さらに病理組織検査でもこれらの識別は容易ではない. 最近,血中又は前立腺中テストステロン(T)とその活性型である5α-ジヒドロテストステロン(DHT)の比率が前立腺肥大症と前立腺癌患者で大きく異なることが示唆され,バイオマーカーとして有望視されている.本研究は,T/DHTの超高感度分析法を開発し,臨床知見との相関を求めることで,前立腺癌予後診断用バイオマーカーを確立,臨床診断へ応用,キット化,更には新規治療薬の開発を行わんとして計画されたものである. 以上の背景の下にT/DHTのLC/MSによる超高感度分析法の開発に取り組み,それに必要な誘導体化試薬の開発に成功した.本試薬による誘導体化で,誘導体化前に比してTは70倍,DHTは130倍の高感度化がもたらされた.本試薬を基に精度,正確度に優れるLC/ESI-MS分析法を確立した.さらに本法を針生検で得られる程度(10mg)の前立腺中T及びDHTの定量へ適用したところ,前立腺肥大症患者(n=7)のそれではDHTが定量可能であるのに対し,癌患者(n=3)のそれでは定量下限値以下であった.またいずれの検体でもTは定量下限値以下であった.この知見はマススクリーニングへの適用上有用であった. また,DHT生成と深く関係する5α-reductaseの阻害薬開発を目指して,その活性測定法をLC/MSを用いて開発した.これはTを基質として生成するDHT及び5α-androstane-3α,17β-diolを定量して活性測定を行う方法で,放射性物質を基質としない点が優れている.
• 前立腺癌再燃促進物質の超高感度臨床分析法の開発

  日本学術振興会：科学研究費助成事業 若手研究(B)

  研究期間 : 2002年 -2004年 

  代表者 : 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.
• 一分子のキャラクタリゼーションを目指すLC/MS用第2世代誘導体化試薬の開発

  日本学術振興会：科学研究費助成事業 奨励研究(A)

  研究期間 : 2000年 -2001年 

  代表者 : 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.
• 老人性痴呆治療薬の開発を志向した脳内ステロイドホルモンの分析

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 1997年 -1998年 

  代表者 : 島田 和武; 本間 誠次郎; 東 達也; 三田村 邦子

   

  高齢化社会を迎え,老人性痴呆は大きな社会的問題となっており,各種の抗痴呆薬の開発が進んでいる.しかし,認可が取り消されるそれが相次いでおり,開発の困難さを示唆している. ところで最近,ほ乳類の脳内に末梢血中の10倍以上ものステロイドホルモンが見出され多大の注目を集めている.これらはneurosteroidsあるいはneuroactive steroidsと呼ばれ,17-又は20-オキソステロイドの遊離型,各種抱合型(sulfate,fatty acid ester,sulpholipid抱合体など)より構成され脳内で生合成されることが知られている.このような折,pregnenoloneやestrogenの老人性痴呆薬としての有用性が示唆され,上記の知見との関連が注目されている. そこで,HPLC,LC/MSを駆使してラット脳を検索したところ,新たにpregnenolone,dehydroepiandrosteroneの3-stearate,-palmitate(計4種)を同定することに成功した.この際,各種oxime誘導体へ導くことが,分子量関連ピークの検出を容易とし,LC/MSでの同定上極めて有用なことも見出した.さらに,上記脂肪酸抱合体がストレスにより減弱し,逆に遊離型neurosteroidsが増加することを明らかとした.また,脳内におけるclassical estrogen及びguaiacol estrogenの存在をGC/MS/MSで初めて確認した.従来の研究では脳内におけるエストロゲンレセプターの存在は明らかとされているが,estrogenの脳内における存否については不確かであった.Estrogenの投与が老人性痴呆に有用であるとの疫学的知見も得られており,本研究は今後の抗痴呆薬の開発研究に一つの方向を示唆したものとして評価される.
• LC/MS/MSの生体内超微量ステロイド分析への応用

  日本学術振興会：科学研究費助成事業 奨励研究(A)

  研究期間 : 1997年 -1998年 

  代表者 : 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.
• 脳内ステロイドホルモン分析法の開発と脳機能解明への応用

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 1996年 -1997年 

  代表者 : 島田 和武; 三田村 邦子

   

  脳内ステロイドホルモンの化学構造は比較的簡単な17-又は20-オキソステロイドよりなることが知られている.しかし,遊離型のみならず硫酸あるいは脂肪酸抱合型などとしても存在し複雑であるとされているが,詳細については明らかでない. そこで,HPLC,LC/MSを駆使してラット脳を検索したところ,pregnenolone,dehydroepiandrosteroneの3-stearate,-palmitate(計4種)を同定することに成功した.また,脳内におけるpregnenolone,dehydroepiandrostrone,pregnenolone 3-sulfateの存在もLC/MSなどにより改めて確認した.この際,methyloxime誘導体へ導くことが,分子イオンピーク又は関連するピークの検出を容易とし,LC/MSでの同定上極めて有用なことも見出した. 一方,定量法の開発は以下のようにして行った.測定法は簡便性を考慮して蛍光検出HPLCを,測定対象は脳内ステロイドホルモン中で最も多くを占めるとされているpregnenoloneを,蛍光誘導体化試薬には1-anthroylcyanideを選択した,確立した分析法を実試料(ラット脳)へ適用したところ,文献記載値(測定法はRIA又はGC/MS)と符合する個体も見られたが,著しい低値を示す個体もあり,大きな疑問を生じるに至った.この原因がサンプル採取上の問題によるのか,あるいは報告されている分析法に問題があるのかは明らかでないが,いずれにしてもその生理作用と共に解決されるべき重要な課題を提起したことになる.このように,本研究は今後の脳内ステロイドホルモンの研究に一つの方向を示唆したものとして評価される.
• 抱合型25-ヒドロキシビタミンD分析法の開発

  日本学術振興会：科学研究費助成事業 奨励研究(A)

  研究期間 : 1996年 -1996年 

  代表者 : 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

担当経験のある科目

病態検査学近畿大学
臨床検査学近畿大学
日本薬局方近畿大学

その他のリンク

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https://researchmap.jp/read0046446