Study on the analytical application of matrix-assisted laser desorption/ionization mass spectrometry-imaging technique for visualization of polyphenols Nguyen Huu Nghi Kyushu University 2018 List of contents Chapter I Introduction Chapter II 11 Enhanced matrix-assisted laser desorption/ionization mass spectrometry detection of polyphenols 11 Introduction 11 Materials and methods 14 2.1 Materials .14 2.2 Sample and matrix preparations 14 2.3 MALDI-MS analyses 15 2.4 Statistical Analyses .15 Results and discussion 16 3.1 Screening of matrix reagents for negative MALDI-MS detection of monomeric and condensed catechins 16 3.2 Effect of concentration of nifedipine on negative MALDI-MS detection of monomeric and condensed catechins 23 3.3 Photobase reaction of nifedipine as matrix in MALDI .27 3.4 Proton-abstractive reaction of nifedipine in flavonol skeleton .32 3.5 Potential of nifedipine as matrix reagent for polyphenol detection 34 i Summary 39 Chapter III 40 Application of matrix-assisted laser desorption/ionization mass spectrometryimaging technique for intestinal absorption of polyphenols 40 Introduction 40 Materials and methods 42 2.1 Materials .42 2.2 Intestinal transport experiments using rat jejunum membrane in the Ussing Chamber system .42 2.3 LC-TOF-MS analysis 44 2.4 Preparation of intestinal membrane section and matrix reagent 45 2.5 MALDI-MS imaging analysis 46 Results and discussion 46 3.1 Optimization of MALDI-MS imaging for visualization of monomeric and condensed catechins in rat jejunum membrane 46 3.2 In situ visualization of monomeric and condensed catechins in rat jejunum membrane by MALDI-MS imaging 48 3.3 Absorption route(s) of monomeric and condensed catechins in rat jejunum membrane 52 3.4 Efflux route(s) of monomeric and condensed catechins in rat jejunum membrane .56 ii 3.5 Visualized detection of metabolites of monomeric and condensed catechins during intestinal absorption 60 Summary 68 Chapter IV 71 Conclusion 71 References 77 Acknowledgement 88 Abbreviations  1,5-DAN, 1,5-diaminonaphthalene  9-AA, 9-aminoacridine desorption/ionization  ABC, ATP-binding cassette spectrometry  ADME, absorption, distribution,  MCT, monocarboxylic transporter metabolism, and excretion  MeOH, methanol AMPK, adenosine monophosphate  MRP2, multidrug resistance protein   activated-protein kinase ANOVA, analysis of variance  BCRP, breast cancer resistance              Nd:YAG, neodymium-doped yttrium aluminum garnet  protein CHCA, OATP, organic anion transporting polypeptides α-cyano-4- hydroxycinnamic acid  PA, proton affinity  DHB, 2,5-dihydroxybenzoic acid  PepT1, peptide transporter  DMAN,  P-gp, P-glycoprotein a m in o) na p ht le ne ga ll at F A, I A IT O, in L C, li 1,8-bis(dimethyl-  S/ N,  si g na  lto n  oi T      mass   MALDI-MS, matrix-assisted laser F3 T F T H tri h y T O F, Chapter I Introduction A popular beverage of tea, derived from the leaves of the Camellia sinensis plant, has been consumed worldwide, and to date, it is considered that the tea intake would be of health-benefit owing to dietary flavonoids (polyphenols) In green or non-fermented tea, major components are monomeric catechins, e.g., epicatechin (EC), epicatechin-3-O-gallate (ECG), epigallocatechin (EGC), and epigallocatechin-3-O-gallate (EGCG) On the other hands, by fermentation of tea leaves to produce black tea, oxidation and polymerization reactions occur in leaves to form oligomeric catechins, such as theasinensins and theaflavins (TFs) including theaflavin (TF), theaflavin-3-Ogallate (TF3G), theaflavin-3’-O-gallate (TF3’G), and theaflavin-3-3’-di-Ogallate (TF-33’diG) [1] To date, extensive studies have been performed on health-benefits of tea polyphenols, and showed their potential in preventing cardiovascular diseases [2], diabetes [3], and cancers [4] Irrespective to the evidences on their preventive effects, it must be essential to know absorption, distribution, metabolism, and excretion (ADME) behavior, since the understanding of ADME is indispensable for elucidating the bioactive mechanism(s) and effective dosage of polyphenols in our body In general, polyphenols are thought to be absorbed into the circulation system, following distribution at organs, and/or excretion into urine and fecal via metabolism [1] Among catechins, EC and EGC have been reported to be highly bioavailable, compared to gallate catechins such as ECG and EGCG [5] In human study, EC, EGC, ECG, and EGCG were detected in plasma to be 174, 145, 50.6, and 20.1 pmol/mL, respectively, after the consumption of tea catechins (EC, 36.54 mg; EGC, 15.48 mg; ECG, 31.14 mg; EGCG, 16.74 mg) [6] Another human study also revealed the absorption of not only catechins, but also their conjugates in plasma at >50 ng/mL [7] They also clarified that ECG and EGCG were absorbed in their intact form, while EC and EGC were susceptible to metabolism to produce conjugated forms [7] Another research group reported high stability of EGCG during absorption process in human [8] In cell-line experiments using Caco-2 cell monolayers, non-gallate catechin, EC, was found to show lower cellular accumulation than gallate ECG, due to high efflux back of EC to apical side [9] After 50-µmol/L, 60-min, Caco-2 transport experiments of EC, ECG, and EGCG, only gallate catechins (ECG and EGCG) were predominantly accumulated in cells at 3037 ± 311 and 2335 ± 446 pmol/mg protein, respectively [10] There were few researches on absorption of black tea TFs In human study, even at high dose intake of 700 mg TFs, plasma and urine levels of TFs were as low as and ng/mL, respectively [11] In urine, TFs were not detected after consumption of 1000 mg of TF extract [12] Non-absorbable property of TFs was also confirmed by Caco-2 cell transport study, in which TF3’G was not detected in basolateral side after 60-min transport [13] Irrespective to poor absorption or low bioavailability of TFs, it was reported that they have potential in the regulation of intestinal absorption route(s); in turn, TFs may exert physiological function at the small intestine [14] However, the absorption behavior of TFs still remains unclear whether they could be incorporated into intestinal membrane or not Once being absorbed into the circulation system or organs, polyphenols undergo phase glucuronidation II [15][16] metabolism, namely, methylation, sulfation, and Phase II enzymes catalyzing the methylation, sulfation, and glucuronidation are catechol-O-methyltransferase, sulfotransferase, and uridine diphosphate-glucuronosyltransferase, respectively [17] These metabolic enzymes were found not only in the intestine, but also in the liver and the kidneys [18][19][20] It has been reported that higher absorbable catechins such as EC and EGC were more susceptible to such metabolic reactions, compared to gallate catechins (ECG and EGCG) [7] For EC absorption, a predominant sulfate conjugate of EC were effluxed from the enterocytes 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e0163498 10 Acknowledgement First and foremost, I would like to express my sincere gratitude to my supervisor, Prof Toshiro Matsui for his continuous support during my Ph.D study and related research His intellectual guidance, innovative ideas, and patience efficiently helped me in all the time of my research It was my great honor and privilege to be a PhD student in laboratory of food analysis under his supervision My academic background and research experience have been improved significantly throughout the time studying in his laboratory I also would like to thank my committee members, Prof Mitsuya Shimoda and Prof Takahisa Miyamoto for their time, consideration, and valuable comments Their advices and suggestions have been a great help in completion of my doctoral dissertation I would like to offer my special thanks to Assistant Prof Mitsuru Tanaka for his technical training, advices, and motivation throughout my period of research His supports and caring is also valuable for my personal life in Japan I am also grateful to Ms Kaori Miyazaki for her technical and secretarial support Without her kind caring and support, my life in Japan would not be smooth as it was I would like to take this opportunity to thank all members of laboratory of food analysis for their help, support, and sharing during my research and my life in Japan A special gratefulness I would like to give to Seong-Min-Hong, Riho Koyanagi, Naoto Hirasaki, Vu Thi Hanh, Toshihiko Fukuda, and Kumrungsee Thanutchporn 10 I highly appreciated the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) for the financial support during my PhD research I also appreciated all Kyushu university staffs for their kind support Last, I express my deep appreciation to my parents, my wife, my children and my friends for their support, encouragement and always behind me during my journey in Japan 10 ... MALDI-MS imaging application, they are visualized with ion density image) Figure 1-1 Schematic workflow of matrix- assisted laser desorption/ ionization mass spectrometry imaging According to the aforementioned... Potential of nifedipine as matrix reagent for polyphenol detection 34 i Summary 39 Chapter III 40 Application of matrix- assisted laser desorption/ ionization mass spectrometryimaging... such as preparation and extraction steps, and could not obtain the localization of analytes in biological tissues [33] On the other hand, matrix- assisted laser desorption/ ionization MS (MALDI-MS),