KINDAI UNIVERSITY


*A space between the first name and last name, please enter

MITAMURA Kuniko

Profile

FacultyDepartment of Pharmacy / Graduate School of Medicine
PositionAssociate Professor
Degree
Commentator Guidehttps://www.kindai.ac.jp/meikan/808-mitamura-kuniko.html
URL
Mail
Last Updated :2020/08/10

Education and Career

Education

  •  - 1990 , Kanazawa University, Faculty of Pharmaceutical Sciences
  •  - 1990 , Kanazawa University, Faculty of Pharmaceutical Science

Academic & Professional Experience

  •   2009 04 ,  - 現在, Faculty of Pharmacy, Kindai University
  •   2005 04 ,  - 2009 03 , Faculty of Pharmacy, Kindai University
  •   1995 04 ,  - 2005 03 , Faculty of Pharmaceutical Sciences, Kanazawa University
  •   1992 04 ,  - 1995 03 , Faculty of Pharmaceutical Sciences, Kanazawa University
  •   1992 , - Kanazawa University

Research Activities

Research Areas

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

Research Interests

  • MS, LC, LC-MS, HPLC, Analytical Chemistry

Published Papers

  • 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, Analytical Sciences, 34(9), 1003 - 1009, Jun. 2018 , Refereed
  • 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, International journal of molecular medicine, 42(2), 1168 - 1180, May 2018 , Refereed
  • Proteomic profile of the lens in a streptozotocin-induced diabetic rat model using shotgun proteomics., Nagai N, Yamamoto T, Mitamura K, Taga A, Biomedical reports, Biomedical reports, 7(5), 445 - 450, Nov. 2017 , Refereed
  • 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, Journal of biomedical materials research. Part B, Applied biomaterials, Nov. 2017 , Refereed
  • Effect of dark-colored maple syrup on cell proliferation of human gastrointestinal cancer cell., Yamamoto T, Sato K, Kubota Y, Mitamura K, Taga A, Biomedical reports, Biomedical reports, 7(1), 6 - 10, Jul. 2017 , Refereed
  • Two Major Bile Acids in the Hornbills, (24R,25S)-3 alpha,7 alpha,24-Trihydroxy-5 beta-cholestan-27-oyl Taurine and Its 12 alpha-Hydroxy Derivative, Rika Satoh, Hiroaki Ogata, Tetsuya Saito, Biao Zhou, Kaoru Omura, Satoshi Kurabuchi, Kuniko Mitamura, Shigeo Ikegawa, Lee R. Hagey, Alan F. Hofmann, Takashi Iida, LIPIDS, LIPIDS, 51(6), 757 - 768, Jun. 2016 , Refereed
    Summary: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 2 alpha- and 2 beta-hydroxy bile alcohols and bile acids in the bile of Arapaima gigas, a large South American river fish, Rika Sato (nee Okihara), Tetsuya Saito, Hiroaki Ogata, Naoya Nakane, Kazunari Namegawa, Shoutaro Sekiguchi, Kaoru Omura, Satoshi Kurabuchi, Kuniko Mitamura, Shigeo Ikegawa, Jan Raines, Lee R. Hagey, Alan F. Hofmann, Takashi Iida, STEROIDS, STEROIDS, 107, 112 - 120, Mar. 2016 , Refereed
    Summary: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.
  • An efficient synthesis of 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one and its biological precursor 7 alpha-hydroxy-4-cholesten-3-one: Key intermediates in bile acid biosynthesis, Shoujiro Ogawa, Biao Zhou, Yusuke Kimoto, Kaoru Omura, Akiko Kobayashi, Tatsuya Higashi, Kuniko Mitamura, Shigeo Ikegawa, Lee R. Hagey, Alan F. Hofmann, Takashi Iida, STEROIDS, STEROIDS, 78(9), 927 - 937, Sep. 2013 , Refereed
    Summary: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.
  • A novel varanic acid epimer - (24R,25S)-3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-27-oic acid - is a major biliary bile acid in two varanid lizards and the Gila monster, Lee R. Hagey, Shoujiro Ogawa, Narimi Kato, Rika Satoh (nee Okihara), Mizuho Une, Kuniko Mitamura, Shigeo Ikegawa, Alan F. Hofmann, Takashi Iida, STEROIDS, STEROIDS, 77(13), 1510 - 1521, Nov. 2012 , Refereed
    Summary: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.
  • Synthesis of 3-and 21-monosulfates of [2,2,3 beta,4,4-H-2(5)]etrahydrocorticosteroids in the 5 beta-series as internal standards for mass spectrometry, Shigeo Ikegawa, Kaori Nagae, Takayuki Mabuchi, Rika Okihara, Maki Hasegawa, Toshie Minematsu, Takashi Iida, Kuniko Mitamura, STEROIDS, STEROIDS, 76(12), 1232 - 1240, Nov. 2011 , Refereed
    Summary: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 3 alpha,7 alpha,12 alpha-trihydroxy-5 alpha-cholestan-27-oic acid as well as their corresponding glycine and taurine conjugates, Shoujiro Ogawa, Kuniko Mitamura, Shigeo Ikegawa, Matthew D. Krasowski, Lee R. Hagey, Alan F. Hofmann, Takashi Iida, CHEMISTRY AND PHYSICS OF LIPIDS, CHEMISTRY AND PHYSICS OF LIPIDS, 164(5), 368 - 377, Jul. 2011 , Refereed
    Summary: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.
  • Identification of glutathione conjugates of bile acids in human bile by liquid chromatography/mass spectrometry, 池川 繁男, 堀 直宏, 三田村 邦子, 胆膵の病態生理, 胆膵の病態生理, 27(1), 23 - 27, Jan. 2011 , Refereed
  • Capture of proteins in rat liver by affinity chromatography using immobilized lithocholic acid and identification with liquid chromatography-mass spectrometry, Toshihiro Sakai, Kuniko Mitamura, Atsushi Taga, Susumu Honda, Shigeo Ikega, BUNSEKI KAGAKU, BUNSEKI KAGAKU, 56(9), 713 - 720, Sep. 2007 , Refereed
    Summary:Affinity chromatography is a powerful method for protein separation. It is based on a specific interaction between an immobilized ligand and the target proteins to be separated. Since lithocholic acid (LCA), one of secondary bile acids, has been shown to exert its function as the ligand toward nuclear receptors and a membrane-type G protein-coupled receptor, the abilities of molecular recognition and acquisition of LCA may be applicable for ligands to capture unknown functional proteins by affinity chromatography. In this study, LCA was covalently bound to an activated agarose through a bridge introduced at the C-3 and C-24 positions. The affinity absorbents were applied to capture proteins in a rat mitochondrial fraction. Structure analysis of the captured proteins after SDS-PAGE separation was carried out by liquid chromatography/electrospray ionization-tandem mass spectrometry combination with computer-assisted programs, where carbamoyl phosphate synthase I, glutamate dehydrogenase, acyl-CoA dehydrogenase, enoyl-CoA hydratase, acetyl-CoA acyltransferase and aldehyde dehydrogenase were identified. Serum albumin and cytosolic glutathione S-transferase, which were contaminated in mitochondrial fraction, were also identified.
  • Determination of estrogens in rat brains using gas chromatography/mass spectrometry/mass spectrometry, Mitamura K, Mikami N, Kambara Y, Ohno E, Shimada K, Chromatography, Chromatography, 23(2), 65 - 71, Jul. 2002 , Refereed
  • 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, Journal of Chromatography B: Biomedical Sciences and Applications, 748(1), 89 - 96, Oct. 2000 , Refereed
    Summary: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.
  • 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, Journal of Chromatography B: Biomedical Applications, 690(1-2), 348 - 354, Mar. 1997 , Refereed
    Summary: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.
  • 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, Journal of Chromatography B: Biomedical Applications, 689(2), 409 - 414, Feb. 1997 , Refereed
    Summary: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.
  • Quantitative determination of 25-hydroxyvitamin D3 3-sulphate in human plasma using high performance liquid chromatography, K. Shimada, K. Mitamura, N. Kitama, Biomedical Chromatography, Biomedical Chromatography, 9(5), 229 - 232, Nov. 1995 , Refereed
    Summary:The quantitative determination of 25-hydroxyvitamin D3 3-sulphate in human plasma was completed using reversed phase high performance 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.
  • Syntheses and enzymatic hydrolysis of 25-hydroxyvitamin D monoglucuronides, K. Shimada, K. Sugaya, H. Kaji, I. Nakatani, K. Mitamura, N. Tsutsumi, Chemical and Pharmaceutical Bulletin, Chemical and Pharmaceutical Bulletin, 43(8), 1379 - 1384, Jan. 1995 , Refereed
    Summary: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.
  • Retention behavior of bile acid derivatives using cyclodextrin in the mobile phase in high-performance liquid chromatography, K. Shimada, Y. Komine, K. Mitamura, Journal of Chromatographic Science, Journal of Chromatographic Science, 28(6), 331 - 335, Jul. 1990 , Refereed
    Summary: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.
  • 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, Journal of pharmaceutical and biomedical analysis, 182, 113138 - 113138, Apr. 15 2020 , Refereed
    Summary: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, Biochemical and biophysical research communications, Mar. 16 2020 , Refereed
    Summary: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, International journal of molecular sciences, 20(20), Oct. 11 2019 , Refereed
    Summary: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.
  • 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, TUMOR BIOLOGY, 37(10), 13595 - 13606, Oct. 2016 , Refereed
    Summary: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.
  • 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, JOURNAL OF OLEO SCIENCE, 65(2), 169 - 175, Feb. 2016 , Refereed
    Summary: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, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 25(14), 2788 - 2792, Jul. 2015 , Refereed
    Summary: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, ONCOLOGY REPORTS, 33(4), 1579 - 1584, Apr. 2015 , Refereed
    Summary: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, JOURNAL OF OLEO SCIENCE, 64(3), 331 - 335, Mar. 2015 , Refereed
    Summary: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, JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES, 969, 230 - 234, Oct. 2014 , Refereed
    Summary: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, STEROIDS, 85, 18 - 29, Jul. 2014 , Refereed
    Summary: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, STEROIDS, 80, 15 - 23, Feb. 2014 , Refereed
    Summary: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.
  • 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, JOURNAL OF LIPID RESEARCH, 54(9), 2437 - 2449, Sep. 2013 , Refereed
    Summary: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, CHEMICAL & PHARMACEUTICAL BULLETIN, 61(3), 326 - 332, Mar. 2013 , Refereed
    Summary: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, STEROIDS, 77(13), 1423 - 1437, Nov. 2012 , Refereed
    Summary: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.
  • 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, PLOS ONE, 7(5), e37803, May 2012 , Refereed
    Summary: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, CHEMISTRY AND PHYSICS OF LIPIDS, 165(3), 261 - 269, Apr. 2012 , Refereed
    Summary: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, Steroids, 76(14), 1609 - 14, Dec. 20 2011 , Refereed
    Summary: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 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, ANALYTICAL AND BIOANALYTICAL CHEMISTRY, 400(7), 2061 - 2072, Jun. 2011 , Refereed
    Summary: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, ANALYTICAL AND BIOANALYTICAL CHEMISTRY, 400(7), 2253 - 2259, Jun. 2011 , Refereed
    Summary: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, GASTROENTEROLOGY, 140(5), S936 - S936, May 2011 , Refereed
  • 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, STEROIDS, 76(1-2), 68 - 77, Jan. 2011 , Refereed
    Summary: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, STEROIDS, 75(4-5), 338 - 345, Apr. 2010 , Refereed
    Summary: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, CHEMICAL & PHARMACEUTICAL BULLETIN, 58(3), 344 - 353, Mar. 2010 , Refereed
    Summary: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, ANALYTICAL CHEMISTRY, 81(24), 10124 - 10135, Dec. 2009 , Refereed
    Summary: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, JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES, 877(25), 2630 - 2638, Sep. 2009 , Refereed
    Summary: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, STEROIDS, 74(9), 751 - 757, Sep. 2009 , Refereed
    Summary: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, ANALYTICAL BIOCHEMISTRY, 384(2), 224 - 230, Jan. 2009 , Refereed
    Summary: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, STEROIDS, 74(1), 81 - 87, Jan. 2009 , Refereed
    Summary: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, JOURNAL OF LIPID RESEARCH, 49(11), 2463 - 2473, Nov. 2008 , Refereed
    Summary: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, ANALYTICAL SCIENCES, 24(11), 1475 - 1480, Nov. 2008 , Refereed
    Summary: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, JOURNAL OF LIPID RESEARCH, 48(12), 2682 - 2692, Dec. 2007 , Refereed
    Summary: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.
  • 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, BIOLOGICAL & PHARMACEUTICAL BULLETIN, 30(9), 1653 - 1656, Sep. 2007 , Refereed
    Summary: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, JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES, 855(1), 88 - 97, Aug. 2007 , Refereed
    Summary: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, JOURNAL OF LIPID RESEARCH, 48(6), 1343 - 1352, Jun. 2007 , Refereed
    Summary: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, BIOMEDICAL CHROMATOGRAPHY, 19(10), 796 - 801, Dec. 2005 , Refereed
    Summary: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, ANALYTICAL SCIENCES, 21(10), 1241 - 1244, Oct. 2005 , Refereed
    Summary: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, JOURNAL OF HEALTH SCIENCE, 51(4), 447 - 452, Aug. 2005 , Refereed
    Summary: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, JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES, 28(18), 2839 - 2848, 2005 , Refereed
    Summary: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, ANALYTICAL SCIENCES, 20(1), 3 - 4, Jan. 2004 , Refereed
    Summary: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, JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES, 796(1), 121 - 130, Oct. 2003 , Refereed
    Summary: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.
  • 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, JOURNAL OF CHROMATOGRAPHY A, 961(1), 97 - 105, Jun. 2002 , Refereed
    Summary: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, JOURNAL OF CHROMATOGRAPHY B, 748(1), 89 - 96, Oct. 2000 , Refereed
    Summary: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. Part XIII. Characterization of catechol estrogens in rat brains using liquid chromatography-mass spectrometry-mass spectrometry, K Mitamura, M Yatera, K Shimada, ANALYST, ANALYST, 125(5), 811 - 814, 2000 , Refereed
    Summary: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, ANALYTICAL SCIENCES, 15(10), 951 - 955, Oct. 1999 , Refereed
    Summary: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, JOURNAL OF CHROMATOGRAPHY A, 847(1-2), 171 - 178, Jun. 1999 , Refereed
    Summary: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, ANNALS OF CLINICAL BIOCHEMISTRY, 36(1), 43 - 47, Jan. 1999 , Refereed
    Summary: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, JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES, 22(3), 367 - 377, 1999 , Refereed
    Summary: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, JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS, 15(9-10), 1207 - 1214, Jun. 1997 , Refereed
    Summary: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, BIOLOGICAL & PHARMACEUTICAL BULLETIN, 20(6), 596 - 600, Jun. 1997 , Refereed
    Summary: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 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, JOURNAL OF CHROMATOGRAPHY B, 690(1-2), 348 - 354, Mar. 1997 , Refereed
    Summary: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 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, JOURNAL OF CHROMATOGRAPHY B, 689(2), 409 - 414, Feb. 1997 , Refereed
    Summary: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, BIOLOGICAL & PHARMACEUTICAL BULLETIN, 19(4), 491 - 494, Apr. 1996 , Refereed
    Summary: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, CHEMICAL & PHARMACEUTICAL BULLETIN, 43(8), 1379 - 1384, Aug. 1995
    Summary: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, JOURNAL OF CHROMATOGRAPHIC SCIENCE, 33(2), 82 - 85, Feb. 1995 , Refereed
    Summary: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, JOURNAL OF LIQUID CHROMATOGRAPHY, 18(14), 2885 - 2893, 1995 , Refereed
    Summary: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.
  • 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, JOURNAL OF CHROMATOGRAPHIC SCIENCE, 32(3), 107 - 111, Mar. 1994 , Refereed
    Summary: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, JOURNAL OF LIQUID CHROMATOGRAPHY, 16(18), 3965 - 3976, 1993 , Refereed
    Summary: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, JOURNAL OF LIQUID CHROMATOGRAPHY, 16(15), 3311 - 3320, 1993 , Refereed
    Summary: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, JOURNAL OF CHROMATOGRAPHY-BIOMEDICAL APPLICATIONS, 565(1-2), 111 - 118, Apr. 1991 , Refereed
    Summary: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.

Books etc

  • Technical Terms in Life Sciences, Iida T, Ikegawa S, Ito J, Une M, Tamegai H, Mitamura K, Contributor,   2008 11

Conference Activities & Talks

  • Identificaiton of antitumor component in maple syrup to develop novel anti-cancer drugs for colorectal cancer., Yamamoto T, Shiburo R, Mitamura K, Taga A,   2016 10 07
  • The role of maple syrup on cell proliferation of colorectal cancer cells, Kubota C, Yamamoto T, Mitamura K, Taga A,   2016 10 07
  • Protein component in maple syrup has a potential to develop novel anti-cancer drugs for colorectal cancer, amamoto T, Shiburo R, Mitamura K, Taga A, DDW2016,   2016 05 22
  • 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., Takakura H, Yamamoto T, Mitamura K, Kudo M, Naito Z, Taga A, DDW2016,   2016 05 22
  • A Novel Bile Acid Synthesis Pathway in Mouse Liver Initiated by 24-Hydroxycholesterol., Genta Kakiyama, Dalila Marques, Kuniko Mitamura, Hajime Takei, Hiroshi Nittono, Daniel Rodriguez-Agudo, Gregorio Gil, Huiping Zhou, Phillip Hylemon, William M. Pandak, AASLD The Liver Meeting 2015,   2015 11 13
  • Proteomics using formalin-fixed paraffin-embedded, Tetsushi Yamamoto, Mitsuhiro Kudo, Wei-Xia Peng, Hideyuki Takata, Kuniko Mitamura, Atsushi Taga, Zenya Naito,   2015 10 08
  • The effect of maple syrup on cell proliferation and invasion in, Chiaki Kubota, Tetsushi Yamamoto, Kuniko Mitamura, Atsushi Taga,   2015 10 08
  • Comparison of the sensitivity and selectivity for the CID and PQD mode in the analysis of conjugated steroids, Kuniko Mitamura, Satoshi Kurabuchi, Mamiko Ueda, Tetsushi Yamamoto, Atsushi Taga, Shigeo Ikegawa, 63th ASMS Conference on Mass Spectrometry and Allied Topics,   2015 06 04 , American Society for Mass Spectrometry
  • High Speed Molecular Weight Measurement of Proteins by Capillary Electrophoresis, Atsushi Taga, Yuri Miyazaki, Tetsushi Yamamoto, Kuniko Mitamura, The 4th International Symposium on Steel Science - ISSS 2014,   2014 11 03
  • Anti-cancer Effect of Maple Syrup for Colorectal Cancer, Kentaro Uemura, Tetsushi Yamamoto, Kuniko Mitamura, Atsushi Taga,   2014 09 25
  • Proteomic analysis of formalin-fixed paraffin-embedded colorectal cancer tissue for identification of therputic terget., Tetsushi Yamamoto, Mitsuhiro Kudo, Wei-Xia Peng, Hideyuki Takata, Kuniko Mitamura, Atsushi Taga, Zenya Naito,   2014 09 25
  • High Speed Determination of Protein Molecular Weight by Capillary Electrophoresis, Atsushi Taga, Yuri Miyazaki, Tetsushi Yamamoto, Kuniko Mitamura, 13th International Symposium on Hyphenated Techniques in Chromatography and Separation Technology (HTC-13),   2014 01 28
  • Reversal of NAFL Through Selective Increased Intracellular Hepatic Cholesterol Catabolism, Genta Kakiyama, Dalila Marques, Kuniko Mitamura, Shigeo Ikegawa, Shunlin Ren, Daniel Rodriguez-Agudo, Patricia Copper, Phillip Hylemon, Huiping Zhou, William M. Pandak, The Liver Meeting (AASLD2013),   2013 11 01
  • 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, Narimi Kato, Takashi Iida, Shoujiro Ogawa, Mizuho Une, Kuniko Mitamura, Shigeo Ikegawa, Lee R. Hagey, Alan F Hofmann, XXII Bile Acid Meeting,   2012 10 15
  • 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., Kaoru Omura, Yuusuke Kimoto, Takashi Iida, Shoujiro Ogawa, Kuniko Mitamura, Shigeo Ikegawa, Lee R. Hagey, Alan F Hofmann, XXII Bile Acid Meeting,   2012 10 15
  • Identification of S-acyl glutathione conjugates of bile acids in human bile by means of LC/ESI-MS, Shigeo Ikegawa, Naohiro Hori, Takashi Iida, Alan F. Hofmann, Kuniko Mitamura, XXII Bile Acid Meeting,   2012 10 15
  • Development of isotope dilution LC/ESI-MS/MS analysis of eighteen tetrahydrocorticosteroid sulfates in human urine, Kuniko Mitamura, Rika Okihara, Maki Hasegawa, Mami Kamibayashi, Takashi Iida, Shigeo Ikegawa, 60th ASMS Conference on Mass Spectrometry and Allied Topics,   2012 05 20 , American Society for Mass Spectrometry
  • Liquid chromatography-mass spectrometric characterization of sulfation of glutathione conjugated bile acids by a rat liver cytosolic fraction, Shigeo Ikegawa, Naohiro Hori, Takashi Iida, Alan F. Hofmann, Kuniko Mitamura, 60th ASMS Conference on Mass Spectrometry and Allied Topics,   2012 05 20 , American Society for Mass Spectrometry
  • Liquid chromatography-mass spectrometric characterization of sulfation of N-acetylcysteine conjugated bile acids by a rat liver cytosolic fraction, Shigeo Ikegawa, Toshihiro Sakai, Risa Nakai, Tateaki Wakamiya, Takashi Iida, Alan F. Hofmann, Kuniko Mitamura, 59th ASMS Conference on Mass Spectrometry and Allied Topics,   2011 05 , 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, Kuniko Mitamura, Eriko Aoyama, Toshihiro Sakai, Takashi Iida, Alan F. Hofmann, Shigeo Ikegawa, 59th ASMS Conference on Mass Spectrometry and Allied Topics,   2011 05 , American Society for Mass Spectrometry
  • Chemical synthesis of the (25R)- and (25S)-epimers of 3α,7α,12α-trihydroxy-5α-cholestan-27-oic acid and their glycine and taurine conjugates., XXI International Bile Acid Meeting Bile Acid as Metabolic Integrators and Therapeutics,   2010 10 07 , Folk Foundation
  • Analysis of glutathione conjugates of bile acids in rat bile by LC/ESI-MS/MS, Shigeo Ikegawa, Naohiro Hori, Takashi Iida, Kuniko Mitamura, XXI International Bile Acid Meeting Bile Acid as Metabolic Integrators and Therapeutics,   2010 10 07 , Folk Foundation
  • Simultaneous Determination of Tetrahydrocorticosteroid Glucuronides by Liquid Chromatography/Electrospray Ionization-Mass Spectrometry, Kuniko Mitamura, Shigeo Ikegawa, The 12th Asian-Pacific Congress of Clinical Biochemistry,   2010 10 03
  • Identification of Novel Metabolites of Bile Acids: S-Acyl Glutathione Conjugates in Rat Bile by LC/ESI-Linear Ion Trap MS/MS., Naohiro Hori, Kuniko Mitamura, Toshihiro Sakai, Takashi Iida, Shigeo Ikegawa, 58th ASMS Conference on Mass Spectrometry,   2010 05 , ASMS
  • Quantitative Metabolic Profiling of Twelve Tetrahyrocorticosteroid Glucuronides in Urine by Isotope Dilution LC-ESI-Linear Ion Trap MS/MS, Kuniko Mitamura, Maki Hasegawa, Rika Okihara, Chikara Shimidzu, Hitoshi Chiba, Takashi Iida, Shigeo Ikegawa, 58th ASMS Conference on Mass Spectrometry,   2010 05 , ASMS
  • IDENTIFICATION OF GLUTATHIONE CONJUGATES OF BILE ACIDS IN RAT BILE BY LC/ESI-MS2, Shigeo Ikegawa, Naohiro Hori, Toshihiro Sakai, Takashi Iida, Kuniko Mitamura,   2009 11 27

Misc

  • 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 09
  • 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 08 25 , http://jglobal.jst.go.jp/public/201802251404128818
  • 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 08
  • 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, ROMBUNNO.26PA‐pm325,   2018 , http://jglobal.jst.go.jp/public/201802224797816915
  • 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 , http://jglobal.jst.go.jp/public/201802225198127089
  • 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 09 , http://jglobal.jst.go.jp/public/201702268578829950
  • 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 03 10 , http://jglobal.jst.go.jp/public/201702263534525132
  • 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 , http://jglobal.jst.go.jp/public/201702214926408169
  • 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 , http://jglobal.jst.go.jp/public/201702237981945675
  • 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 10
  • 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 10
  • 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 , http://jglobal.jst.go.jp/public/201602274150482142
  • 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 , http://jglobal.jst.go.jp/public/201602281216293857
  • 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 , http://jglobal.jst.go.jp/public/201602283376938431
  • 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 , http://jglobal.jst.go.jp/public/201602291196662880
  • 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 10
  • 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 10
  • メープルシロップを用いた大腸癌抗腫瘍効果の検討, YAMAMOTO TETSUSHI, UEMURA KENTARO, MITAMURA KUNIKO, TAGA ATSUSHI, 日本薬学会年会要旨集(CD-ROM), 135th, ROMBUNNO.27H-PM14,   2015 , http://jglobal.jst.go.jp/detail.php?from=API&JGLOBAL_ID=201502285760476858
  • 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, MITAMURA KUNIKO, UEDA MAMIKO, YAMAMOTO TETSUSHI, TAGA ATSUSHI, IKEGAWA SHIGEO, 日本薬学会年会要旨集(CD-ROM), 135th, ROMBUNNO.27PA-PM087,   2015 , http://jglobal.jst.go.jp/detail.php?from=API&JGLOBAL_ID=201502295108504314
  • 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 09 01 , http://jglobal.jst.go.jp/detail.php?from=API&JGLOBAL_ID=201402294671380938
  • 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 09
  • 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 09
  • 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 , http://jglobal.jst.go.jp/detail.php?from=API&JGLOBAL_ID=201402251488298822
  • 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 , http://jglobal.jst.go.jp/detail.php?from=API&JGLOBAL_ID=201402282507468940
  • 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 , http://jglobal.jst.go.jp/detail.php?from=API&JGLOBAL_ID=201402292968391588
  • 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 , http://jglobal.jst.go.jp/detail.php?from=API&JGLOBAL_ID=201402203597392796
  • 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 03
  • Analysis of glutathione conjugates of bile acids by LC/MS, 三田村 邦子, 堺 俊博, 池川 繁男, Jasco report, 51, 1, 7, 13,   2009 09 , 招待有り, http://ci.nii.ac.jp/naid/40016867163
  • An approach to endocrine and metabolic diseases by liquid chromatography/mass spectrometry, MITAMURA Kuniko, HASEGAWA Maki, OKIHARA Rika, IKEGAWA Shigeo, Japanese Journal of Clinical Chemistry, 38, 3, 291, 298,   2009 07 31 , Refereed, http://ci.nii.ac.jp/naid/10025633137
  • IDENTIFICATION OF GLUTATHIONE CONJUGATES OF BILE ACIDS IN RAT BILE BY LC/ESI-MS2, Ikegawa Shigeo, Hori Naohiro, Sakai Toshihiro, Iida Takashi, Mitamura Kuniko, Abstracts of Annual meeting of Japanese Society for the Study of Xenobiotics, 24, 0, 201, 201,   2009 , 10.14896/jssxmeeting.24.0.201.0, http://ci.nii.ac.jp/naid/130004639581
  • Cholesterol sulfotransferase and cholesterol metabolism, FUDA Hirotoshi, MITAMURA Kuniko, IKEGAWA Shigeo, 臨床化学, 37, 2, 148, 160,   2008 04 30 , Refereed, 10.14921/jscc1971b.37.2_148, http://ci.nii.ac.jp/naid/10025631871
  • A NEW INSIGHT IN THE FORMATION OF N-ACETYLCYSTEINE CINJUGATES OF BILE ACIDS:N-ACETYLCYSTEINE CINJUGATES OF BILE ACIDS, Ikegawa Shigeo, Watanabe Saai, Sakai Toshihiro, Sogabe Mitsuru, Wakamiya Tateaki, Mitamura kuniko, Abstracts of Annual meeting of Japanese Society for the Study of Xenobiotics, 23, 0, 306, 306,   2008 , 10.14896/jssxmeeting.23.0.306.0, http://ci.nii.ac.jp/naid/130005024402
  • Formation of glutathione conjugates of bile acids via metabolic activation, Mitamura Kuniko, Ikegawa Shigeo, Wakamiya Tateaki, Japanese Journal of Clinical Chemistry, 36, 3, 181, 188,   2007 , Refereed, 10.14921/jscc1971b.36.3_181, http://ci.nii.ac.jp/naid/130003357125
  • Prostate Cancer and Metabolic Analysis of Androgens, HIGASHI Tatsuya, MITAMURA Kuniko, SHIMADA Kazutake, Jap. J. Clin. Chem, 33, 3, 177, 182,   2004 12 , Refereed, 10.14921/jscc1971b.33.3-4_177, http://ci.nii.ac.jp/naid/10014070714
  • 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 02 , Refereed, http://chromsoc.jp/Journal/Vol_24/abs24-1_1.html
  • 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 01 , http://www.scopus.com/inward/record.url?partnerID=HzOxMe3b&scp=0142197324&origin=inward
    Summary:Liquid chromatography/mass spectrometry (LC/MS) is now considered to be the most promising analytical method for the determination of biological substances, especially nonvolatile or highly polar substances. However, some compounds do not show enough sensitivity in LC/MS and soft-ionization methods commonly used in LC/MS, such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), sometimes do not give satisfactory structural information. This report presents an overview of the derivatization methods in the LC/MS analysis of neurosteroids or neuroactive neurosteroids, which are synthesized and accumulated in the nervous system. The derivatization of pregnenolone 3-sulfate, one of these steroids, with 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole gave a satisfactory sensitivity during the quantitative analysis using LC/ESI-MS. The obtained results are much lower than those previously obtained using gas chromatography/mass spectrometry or radioimmunoassay. On the other hand, the derivatization to acetate was useful for the treatment of labile catechol estrogens in rat brains and gave enough structural information in LC/APCI-MS, which confirmed the existence of catechol estrogens in mammalian brains.
  • 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 12 , Refereed, http://www.scopus.com/inward/record.url?partnerID=HzOxMe3b&scp=0038219013&origin=inward
    Summary:Our investigation of the analysis of vitamin D3 metabolites has been reviewed. The development of high-performance liquid chromatographic methods for the quantitative determination of 25-hydroxyvitamin D3 3-sulfate and 25-hydroxyvitamin D3, which are the major circulating metabolites of vitamin D3 in human serum/plasma, has been described. The developed methods were applied to the determination of the correlation between the concentration of the sulfate and its genin in healthy subjects and patients with chronic renal failure. The development of immunoaffinity chromatography immobilizing the highly specific anti-1,25-dihydroxyvitamin D3 antibody for the pretreatment of radioreceptor assay of 1,25-dihydroxyvitamin D3, which is the active metabolite of vitamin D3, is also described.
  • Derivatization in liquid chromatography/mass spectrometric analysis of neuro, Mitamura K, Shimada K, Chromatography, 22, 1, 11, 15,   2000 , Refereed, http://chromsoc.jp/Journal/Vol_22/chrom22-1j.html
  • High-performance liquid chromatography/mass spectrometry of steroids, MITAMURA Kuniko, SHIMADA Kazutake, Japan analyst, 48, 4, 401, 411,   1999 04 , Refereed, 10.2116/bunsekikagaku.48.401, http://ci.nii.ac.jp/naid/110002905702
    Summary:An overview of high-performance liquid chromatography/mass spectrometry (LC/MS) of steroids is presented according to groups of steroids. LC/MS is now considered to be the most promising analytical method for the determination of steroids (especially the conjugated type) in biological fluids without derivatization. However, due to its low sensitivity compared with gas chromatography/MS, some skilful techniques, including derivatization, are necessary to overcome this problem. An overview of the ionization methods of LC/MS is also presented.
  • Derivatization in LC/MS, K Mitamura, K Shimada, YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN, 118, 6, 206, 215,   1998 06 , Refereed, 10.1248/yakushi1947.118.6_206, http://ci.nii.ac.jp/naid/110003649151
    Summary:LC/MS is now considered to be the most promising analytical method for the determination of biological substances, especially nonvolatile or high molecular weight substances. But one of the drawbacks of this method is its low sensitivity in comparison with GC/MS. In order to overcome this problem, the derivatization methods are used in LC/MS. This report overviews the detection-oriented derivatization methods in LC/ MS including our results. The typical examples are as follows. The derivatization of vitamin D with a Cookson-type reagent (analog of 4-phenyl-1,2,4-triazoline-3,5-dione) gave satisfactory sensitivity, which was also observed on the derivatization of oxosteroids with O-methylhydroxylamine. Although the oxosteroid fatty acid esters gave a de-esterified ion as a base peak in atmospheric pressure chemical-ionization MS, the methyloxime of which gave a quasimolecular ion as a base peak, which is helpful to identify the compounds in the brain.
  • 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 , Refereed
  • 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 04 , Refereed, 10.1016/S0016-5085(16)32147-3
  • 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 04 , Refereed, 10.1016/S0016-5085(16)32129-1
  • 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 10
  • 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 11 , Refereed, 10.1016/S0021-9673(01)00943-8, http://www.scopus.com/inward/record.url?partnerID=HzOxMe3b&scp=0035940720&origin=inward
    Summary:This review article underlines the importance of gas chromatography (GC), high-performance liquid chromatography (HPLC) and their hyphenated techniques using mass spectrometry (MS) for the determination of natural steroids, especially in human biological fluids. Steroids are divided into eight categories based on their structures and functions, and recent references using the above methodologies for the analysis of these steroids are cited. GC and GC-MS are commonly used for the determination of volatile steroids. Although HPLC is a widely used analytical method for the determination of steroids including the conjugated type in biological fluids, LC-MS is considered to be the most promising one for this purpose because of its sensitivity, specificity and versatility. (C) 2001 Elsevier Science B.V. All rights reserved.
  • Derivatization in liquid chromatography/mass spectrometric analysis of neurosteroids, Chromatography, 22, 1, 11, 15,   2000
  • DERIVATIZATION OF THIOL-CONTAINING COMPOUNDS, K SHIMADA, K MITAMURA, JOURNAL OF CHROMATOGRAPHY B-BIOMEDICAL APPLICATIONS, 659, 1-2, 227, 241,   1994 09 , Refereed, 10.1016/0378-4347(93)E0444-U, http://www.scopus.com/inward/record.url?partnerID=HzOxMe3b&scp=0028087987&origin=inward
    Summary:The determination of thiol-containing compounds in biological fluids is important in biochemistry and clinical chemistry. In this paper, derivatization reagents for thiols are reviewed with respect to their reactivity, selectivity, spectroscopic characteristics and their applicability especially to high-performance liquid chromatography. Derivatization used in ultraviolet and electrochemical detection. The derivatization reagents contain a functional group, e.g. an N-substituted maleimide, active halogen or aziridine, which react with the thiol group. Derivatization for use in flow injection analysis, thin-layer chromatography or gas chromatography-mass spectrometry is also described.

Research Grants & Projects

  • Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(基盤研究(C)), Studies on biosynthesis and metabolism of the glutathione conjugated bile acids, In order to elucidate the mechanism on the formation and metabolism of glutathione (GSH) conjugated bile acids via their reactive intermediates, we demonstrated that bile acid were tansacylated with GSH and N-acetylcysteine (NAC) by in vitro and in vivo studies. In addition, we clarified that the several kinds of glutathione conjugated bile acids were excreted into the bile of rats and patients with biliary diseases. We also showed the NAC conjugated ursodeoxycholic acid could be a prodrug for hepatobiliary fuction improvement drug.
  • Analytical Chemistry of Steroids