Tea, derived from the Camellia sinensis plant, is a widely consumed beverage known for its health benefits attributed to dietary flavonoids, particularly polyphenols Green tea contains monomeric catechins such as epicatechin (EC) and epigallocatechin-3-O-gallate (EGCG), while black tea, produced through fermentation, features oligomeric catechins like theaflavins (TFs) Research has extensively highlighted the health benefits of tea polyphenols, demonstrating their potential in preventing cardiovascular diseases, diabetes, and certain cancers.
Understanding the absorption, distribution, metabolism, and excretion (ADME) of polyphenols is crucial for elucidating their bioactive mechanisms and determining effective dosages Generally, polyphenols are absorbed into the bloodstream, distributed to various organs, and excreted through urine and feces Among catechins, epicatechin (EC) and epicatechin gallate (EGC) exhibit higher bioavailability compared to gallate catechins like epicatechin gallate (ECG) and epigallocatechin gallate (EGCG) In human studies, plasma concentrations of EC, EGC, ECG, and EGCG were measured at 174, 145, 50.6, and 20.1 pmol/mL, respectively, after tea catechin consumption Additionally, research indicates that ECG and EGCG are absorbed in their intact forms, while EC and EGC are metabolized into conjugated forms Notably, EGCG demonstrates high stability during the absorption process In Caco-2 cell line experiments, EC showed lower cellular accumulation compared to gallate catechins due to significant efflux back to the apical side, with only ECG and EGCG predominantly accumulating in cells.
Research on the absorption of black tea flavan-3-ols (TFs) indicates that even with high doses of 700 mg, plasma and urine levels remain low at 1 and 2 ng/mL, respectively Furthermore, TFs were undetectable in urine after consuming 1000 mg of tea extract A Caco-2 cell transport study confirmed the non-absorbable nature of TFs, as TF3’G was not found in the basolateral side after 60 minutes of transport Despite their poor absorption and low bioavailability, TFs may play a role in regulating intestinal absorption pathways, potentially exerting physiological effects in the small intestine However, the exact absorption behavior of TFs and their ability to integrate into intestinal membranes remains uncertain.
Polyphenols, once absorbed into the circulation system or organs, undergo phase II metabolism, which includes processes such as methylation, sulfation, and glucuronidation The enzymes responsible for these metabolic reactions are catechol-O-methyltransferase, sulfotransferase, and uridine diphosphate-glucuronosyltransferase These enzymes are present not only in the intestine but also in the liver and kidneys.
Research indicates that more absorbable catechins, specifically EC and EGC, are more prone to metabolic reactions than gallate catechins like ECG and EGCG In the case of EC absorption, a major sulfate conjugate of EC is released from enterocytes into the intestinal perfusate, while the glucuronide conjugate is absorbed into the bloodstream, bile, and urine.
[21] When 500 mL of green tea was given to 10 volunteers, only intact ECG and
EGCG and its metabolites, including glucuronide, methyl-glucuronide, and methyl-sulfate conjugates of EC and EGC, have been detected in human plasma Studies on the absorption of EGCG in mice and ECG in Wistar rats revealed the presence of sulfate and glucuronide conjugates in various tissues, including blood, liver, and kidney These findings indicate the need for further research to enhance our understanding of polyphenol bioavailability.
The low bioavailability of polyphenols is largely attributed to their efflux to the apical compartment and metabolic degradation, primarily involving ATP-binding cassette (ABC) transporters like multidrug resistance protein 2 (MRP2) and P-glycoprotein (P-gp) In vitro studies using Caco-2 cell transport experiments have shown that inhibiting MRP2 with MK-571 significantly decreases the efflux of monomeric catechin (EC) and its sulfate conjugates Furthermore, research on MRP2 and P-gp transfected cells indicates that the cellular accumulation of epicatechin gallate (ECG) is notably enhanced by inhibitors of both transporters, highlighting the role of ECG in the activity of these ABC transporters.
To understand the absorption and metabolism of tea polyphenols, various analytical evaluations have been conducted However, in vivo studies may not fully capture the transport routes of these polyphenols Therefore, to clarify the intestinal absorption and metabolism of polyphenols, the Caco-2 cell model has become a widely utilized in vitro approach.
Caco-2 cells, derived from human colon carcinoma, mimic enterocytes and exhibit transport systems similar to those in the small intestine Extensive research on the transport routes of polyphenols has utilized the Caco-2 transport system in conjunction with transporter inhibitors However, despite the convenience of cell-line experiments, the Caco-2 model has limitations, including differing protease expression compared to actual intestinal membranes As an alternative for absorption studies, the ex vivo Ussing chamber method has been suggested.
The chamber system, equipped with animal intestinal membranes, has been effectively utilized to evaluate the intestinal absorption of drugs with varying membrane permeability, as demonstrated by Miyake et al This ex vivo system serves as a valuable tool for investigating transport mechanisms akin to in vivo intestinal absorption events, making it suitable for the transport of both drugs and peptides.
Analytical assays are essential for monitoring target analytes in absorption studies, despite the availability of suitable absorption systems Liquid chromatography-mass spectrometry (LC-MS) in electrospray ionization (ESI) mode is widely utilized for studying polyphenols, as it allows for the simultaneous detection of both target compounds and their metabolites However, LC-based methods have limitations, including the need for complex pre-treatment processes and the inability to determine the localization of analytes within biological tissues.
On the other hand, matrix-assisted laser desorption/ionization MS (MALDI-MS),
MALDI-MS, known for its "soft" ionization technique, enables the production of intact pseudomolecular ion species without fragmentation, making it ideal for the simultaneous and selective detection of targets in complex matrices, including both low and high molecular compounds This method offers high sensitivity and selectivity, rapid analysis, and tolerance for impurities, allowing for the analysis of various ionizable compounds such as proteins, lipids, and drugs Recently, the development of MALDI-MS imaging techniques has gained significant attention, as it not only detects targets in sample tissues but also provides insights into their distribution and localization This combination of mass spectrometry detection with spatial distribution opens new avenues in the scientific fields of food and drug delivery systems Reports have highlighted the effectiveness of MALDI-MS imaging for analyzing a wide range of peptides, proteins, lipids, drugs, and food compounds, suggesting its potential in elucidating the ADME (Absorption, Distribution, Metabolism, and Excretion) of these substances In practice, target tissues are cryosectioned into thin slides, coated with MALDI matrix reagents, and irradiated with a laser, which facilitates the desorption and ionization of analytes for detection based on their mass-to-charge ratio.
7 value (for 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
This research aimed to utilize the MALDI-MS imaging technique to investigate the intestinal absorption and metabolism of polyphenols that exhibit lower activity in MALDI To accomplish this goal, several studies were conducted.
Chapter II focuses on identifying suitable matrix reagents for the effective MALDI-MS detection of polyphenols, as there have been limited reports on this topic The study targets a subtraction reaction involving the proton of neutral polyphenols, aiming to enhance detection sensitivity Matrix reagents capable of forming a nitrosophenyl pyridine moiety upon UV irradiation during the MALDI process were evaluated The research successfully ionized a variety of polyphenols, including flavonols, flavones, flavanones, chalcone, stilbenoid, and phenolic acids, under optimized MALDI conditions.
In Chapter III, a novel in situ MALDI-MS imaging approach was proposed to investigate the intestinal absorption behavior of polyphenols, utilizing visualization-guided evidence Employing the Ussing Chamber transport system with rat intestinal membranes, ex vivo transport experiments were conducted on both absorbable and non-absorbable polyphenols Following transport, membrane segments were analyzed using established MALDI-MS imaging techniques, successfully detecting and visualizing both types of polyphenols.
Introduction
Polyphenols, naturally found in various fruits and vegetables, may help prevent cardiovascular diseases, diabetes, and cancers Despite their potential health benefits, the absorption, distribution, metabolism, and excretion (ADME) behavior of polyphenols is not well understood due to complex metabolic processes like methylation, sulfation, and glucuronidation Liquid chromatography-mass spectrometry (LC-MS) with electrospray ionization (ESI) is commonly used to study their bioavailability, but its application is limited by the absence of standards for individual metabolites In contrast, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) enables the simultaneous detection of a wide range of compounds and has gained attention for its imaging capabilities, providing valuable insights into the distribution of these compounds in biological samples.
MALDI-MS imaging is increasingly used for visualizing the distribution and metabolism of targets in biological samples, such as the anti-cancer drug erlotinib in the lungs of tumor-bearing mice, without the need for labeling preparation.
Despite the advantages of MALDI-MS, its application is limited by matrix-dependent ionization, necessitating the development of new matrices suitable for target compounds Effective MALDI matrices must exhibit strong absorption at the laser wavelength, compatibility with analyte solvents, vacuum stability, low vapor pressure, and the ability to engage in photochemical reactions like protonation or deprotonation The rational design of MALDI matrices should consider the physicochemical properties relevant to the target compounds For negative MALDI mode, matrices should be strong bases, while those for positive mode require high acidity in the gas phase Common matrices such as α-cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA), and 2,5-dihydrobenzoic acid (DHB) are widely used for detecting proteins, peptides, and lipids However, conventional matrices face limitations, including signal interference at low mass ranges in positive mode and low ionization efficiency in negative mode, prompting ongoing efforts to identify and develop matrices with higher ionization efficiency for negative MALDI-MS.
[46] The use of 9-aminoacridine (9-AA) as a matrix for negative ionization mode
The high gas-phase basicity of certain compounds enables the sensitive detection of low-molecular weight substances, including carboxylic acids, amines, alcohols, and phenols A notable strong base, 1,8-bis(dimethyl-amino)naphthalene (DMAN), facilitates the identification of various low molecular weight compounds, such as fatty acids, amino acids, plant and animal hormones, vitamins, and small peptides in negative MALDI-MS The unique steric arrangement of the two dimethyl-amino groups in DMAN allows it to effectively deprotonate analytes during MALDI ionization Additionally, the matrix 1,5-diaminonaphthalene (1,5-DAN) has been utilized to visualize polyphenols and their metabolites in rat liver and kidney tissues in negative mode, although the exact mechanism of 1,5-DAN as a MALDI matrix remains unclear.
Recent efforts have focused on identifying metal adducts of polyphenols using MALDI-MS in positive mode, employing traditional matrices like DHB and CHCA, as well as alternative matrices such as trans-3-indoleacrylic acid (IAA).
[49] and 1,5-DAN [15] in negative MS detection mode However, poor MALDI-
Detecting neutral polyphenols through mass spectrometry (MS) poses challenges due to their absence of proton-removal or -addition groups This study introduces a novel strategy for screening suitable matrices by utilizing a proton subtraction reaction from the polyphenol molecule Chapter II focuses on the use of a photobase generator, specifically nifedipine, which can generate a proton-acceptor moiety when exposed to UV irradiation To assess the proton subtraction capabilities of the matrices, EGCG and TF3’G, the most prevalent polyphenols in green and black tea, were selected for evaluation under MALDI-irradiation.
Materials and methods
The study utilized various compounds sourced from reputable suppliers, including CHCA, DHB, 2’,4’,6’-trihydroxyacetophenone (THAP), nimodipine, EC, EGCG, naringin, and 5-O-methylnaringenin from Sigma-Aldrich, and 1,5-DAN and resveratrol from Tokyo Chemical Ind Additionally, nitrendipine, amlodipine, TF3’G, hesperidin, sakuranetin, luteolin, acacetin, curcumin, and quercetin were obtained from Nacalai Tesque Co Other compounds such as nifedipine, IAA, kaempferol, and ellagic acid were sourced from Wako Pure Chemical Ind., while 9-AA came from Merck Millipore Procyanidin B2 and isosakuranetin were acquired from Extrasynthese Theasinensin A was synthesized following the method by Tanaka et al., involving the oxidation of EGCG with Japanese pear homogenate and subsequent processing with ascorbic acid and methanol elution Naringenin was purchased from MP Biomedicals, and all other chemicals used were of analytical grade and applied without further purification.
In this study, polyphenols were dissolved in a 50/50 (v/v) methanol/water solution, while matrix reagents were prepared in a 3/1 (v/v) acetonitrile/water solution The polyphenol solution, with concentrations ranging from 0.1 to 200 àmol/L, was combined with an equal volume of matrix solution (2 to 30 mg/mL) A 0.2 àL aliquot, representing 0.01 to 20 pmol of the polyphenol solution, was manually spotted onto an ITO-glass slide from Bruker Daltonics After air-drying at room temperature, optical images of the matrix crystals on the ITO-coated glass slide were captured using a BZ-9000 microscope from KEYENCE.
MALDI-MS analysis was performed using an Autoflex III MS equipped with SmartBeam III (Bruker Daltonics) MS data were acquired in the range of
The mass spectrometry (MS) analysis utilized summed signals from 100 consecutive laser pulses over a sample area, measuring a mass-to-charge ratio (m/z) range of 100–1000 Key MS parameters included ion source voltages of 20.00 kV and 18.80 kV, a lens voltage of 7.50 kV, and a gain factor of 2.51 The laser operated at a frequency of 200 Hz with full power and an offset of 60%, while the analysis range was set to 20% and laser focus was maintained at 100% Signal intensity and signal-to-noise (S/N) ratio were derived from the MS data collected from 100 randomly selected positions across the entire spotted area, employing the batch analysis function in Flexanalysis 3.3 (Bruker Daltonics).
Data on intensity and signal-to-noise (S/N) ratio were reported as mean ± standard deviation (SD) Statistical comparisons between groups were conducted using one-way analysis of variance (ANOVA), followed by Tukey-Kramer’s t-test for post-hoc analysis, with a significance threshold set at P < 0.05 All statistical analyses were carried out using GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA, USA).
Results and discussion
3.1 Screening of matrix reagents for negative MALDI-MS detection of monomeric and condensed catechins
This study investigates suitable matrix reagents for negative MALDI-MS detection of polyphenols, specifically using EGCG (m/z 457.1) and TF3’G (m/z 715.1) as model compounds due to their low ionization efficiency and fragment production during MALDI-ionization Three commonly used matrices—CHCA, DHB, and THAP—were selected, along with IAA, 9-AA, and 1,5-DAN based on prior research indicating enhanced negative MALDI detection Notably, nifedipine was identified as a potential new matrix reagent for polyphenols, as it generates a proton-acceptor photobase upon UV irradiation, potentially acting as a catalyst in UV-mediated cross-linking.
The use of Nd:YAG laser at 355 nm in the MALDI-MS system effectively excites nifedipine, facilitating the formation of a proton-acceptor photobase In the search for suitable matrix reagents for detecting neutral polyphenols, EGCG was evaluated, with results indicating that it was successfully detected in negative MALDI-MS using IAA, 1,5-DAN, and nifedipine, while other reagents were ineffective Notably, the detection of EGCG in IAA yielded a S/N ratio exceeding 70, aligning with previous findings on procyanidin detection However, nifedipine demonstrated a significantly higher detection efficiency, with EGCG intensity measured at 84,035 ± 4,598 (S/N ratio 3,669 ± 103), which is 5.3 times greater than in IAA and comparable to 1,5-DAN Additionally, nifedipine showed enhanced negative MALDI-MS detection of TF3’G, with an intensity of 113,322 ± 2,513 (S/N ratio 3,158 ± 98), outperforming IAA by 2.6 times and demonstrating its potential as a novel MALDI matrix for ionizing neutral polyphenols.
18 being comparable for 1,5-DAN (62,088 ± 1,469; S/N ratio, 995 ± 100) Figure 2-
1 and Figure 2-2 also demonstrated that no multiple-charged ions of EGCG ([M
Nifedipine appears to preferentially remove one proton from its targets, as indicated by the production of TF3’G ([M - 2H] 2-, m/z 357.1) and another compound with m/z 228.1 In the positive mode of MALDI-MS, no peak corresponding to EGCG was detected.
+ H] + of m/z 459.1 in nifedipine as well as other matrices (Figure 2-3), indicating the preferential use of nifedipine for negative-mode MALDI-MS detection by subtracting proton from polyphenols
Matrix-related peaks can hinder the detection of target peaks in low-mass ranges In this study, while 1,5-DAN enabled the detection of EGCG at 20 pmol/spot alongside nifedipine, its effectiveness diminished at lower concentrations of 1 pmol/spot due to overlapping with contaminating matrix peaks Conversely, nifedipine facilitated significant detection of EGCG at 1 pmol/spot without interference from matrix peaks The matrix crystal images showed that 1,5-DAN formed heterogeneous crystals on ITO glass, which negatively impacted MS resolution and intensity, limiting the detection of analytes under 500 Da In contrast, the homogeneous crystal formation of nifedipine on ITO glass enhanced the sensitive and selective detection of EGCG in low-mass ranges.
Figure 2-1 MALDI-MS detection of EGCG at 20 pmol/spot in negative mode by CHCA, DHB, THAP, IAA, 9-AA, 1,5-DAN, and nifedipine as matrix
EGCG spotted onto ITO-glass (20 pmol/spot) was used for MALDI-MS measurements in negative mode Matrix reagents (CHCA, DHB, THAP, IAA, 9-
In this study, 10 mg/mL solutions of AA, 1,5-DAN, and nifedipine in a 3:1 acetonitrile/water mixture were combined with an equal volume of EGCG MALDI-MS analysis was conducted to measure EGCG ([M - H]-, m/z 457.1) across a range of 100–1000 m/z by averaging signals from 100 consecutive random laser pulses on the sample area Results are presented as mean signal-to-noise ratio (S/N) ± standard deviation (SD), with non-detected values marked as N.D.
Figure 2-2 MALDI-MS detection of TF3’G at 20 pmol/spot in negative mode by CHCA, DHB, THAP, IAA, 9-AA, 1,5-DAN, and nifedipine as matrix
TF3’G spotted onto ITO-glass (20 pmol/spot) was used for MALDI-MS measurements in negative mode Matrix reagents (CHCA, DHB, THAP, IAA, 9-
AA, 1,5-DAN, and nifedipine), each at 10 mg/mL in acetonitrile/water (3/1, v/v) was mixed with an equal volume of TF3’G MALDI-MS measurements of
TF3’G ([M - H] -, m/z 715.1) analysis was conducted over a mass range of 100‒1000 m/z, utilizing the cumulative signals from 100 random laser pulses on the sampled area The results are reported as the mean signal-to-noise ratio (S/N) ± standard deviation (SD), with N.D indicating not detected.
Figure 2-3 Detection of EGCG at 20 pmol/spot in positive mode MALDI-
MS by CHCA, DHB, THAP, IAA, and nifedipine as matrix
EGCG was spotted onto ITO-glass at a concentration of 20 pmol/spot for MALDI-MS measurements in positive mode The reagents used, including CHCA, DHB, THAP, IAA, and nifedipine, were prepared at a concentration of 10 mg/mL in a 3:1 acetonitrile/water mixture The matrices were combined with an equal volume of EGCG MALDI-MS measurements of EGCG, identified as [M + H]+ at m/z 459.1, were conducted within the 100–1000 m/z range by averaging signals from 100 consecutive random laser pulses on the sample area Results are reported as mean signal-to-noise ratio (S/N) ± standard deviation (SD), with N.D indicating not detected.
Figure 2-4 MALDI-MS detection of EGCG at 1 pmol/spot in negative mode using IAA, 1,5-DAN, and nifedipine (A), and the crystal images of IAA, 1,5-
EGCG was spotted onto ITO-glass at a concentration of 1 pmol per spot for MALDI-MS measurements in negative mode Matrix reagents, including IAA, 1,5-DAN, and nifedipine, were prepared at a concentration of 10 mg/mL in a 3:1 acetonitrile/water solution and mixed with an equal volume of EGCG Crystal images were captured using a KEYENCE BZ-9000 microscope, while MALDI-MS conditions were maintained as specified in Figure 2-1.
3.2 Effect of concentration of nifedipine on negative MALDI-MS detection of monomeric and condensed catechins
The study investigated the impact of nifedipine concentration on the detection of EGCG and TF3’G using negative MALDI-MS, with concentrations ranging from 1 to 15 mg/mL Results indicated that the MS intensity for both compounds plateaued at concentrations of 5 mg/mL or higher Consequently, subsequent experiments utilized a nifedipine concentration of 5 mg/mL At this optimal concentration, nifedipine-enhanced MALDI-MS successfully detected EGCG at levels exceeding 0.1 pmol/spot with a signal-to-noise ratio of 4.2, and TF3’G at levels above 0.01 pmol/spot with a signal-to-noise ratio of 10.0.
7) Considering the reported detection of EGCG by 1,5-DAN (>5 pmol/spot) [15] , nifedipine seems to show higher detection than 1,5-DAN as shown in Figure 2-
Figure 2-5 Effect of concentration of nifedipine on EGCG (A) and TF3’G (B) detection by MALDI-MS in negative mode
Nifedipine concentrations on EGCG- and TF3’G-spotted ITO-glass were tested at levels of 1, 5, 10, and 15 mg/mL (20 pmol/spot) The MALDI-MS conditions were consistent with those outlined in Figure 2-1 Results are presented as mean intensity ± SD (n = 3), with data lacking common letters indicating significant differences (P < 0.05) determined by Tukey-Kramer’s t-test.
Figure 2-6 Effect of concentration of EGCG on MALDI-MS detection in negative mode
EGCG was successfully detected on ITO-glass at concentrations of 0.1, 0.5, 1, and 5 pmol/spot using nifedipine (5 mg/mL) in negative mode via MALDI-MS The MALDI-MS conditions adhered to the specifications outlined in Figure 2-1.
Figure 2-7 Effect of concentration of TF3’G on MALDI-MS detection in negative mode
TF3’G-spotted ITO-glass was analyzed at concentrations of 0.01, 0.05, 0.5, and 1 pmol/spot using nifedipine (5 mg/mL) in a negative mode MALDI-MS setup, consistent with the conditions outlined in Figure 2-1.
3.3 Photobase reaction of nifedipine as matrix in MALDI
Nifedipine acts as a photobase generator, producing nitrosophenyl pyridine upon UV irradiation To explore its mechanism as a MALDI matrix, dihydropyridin derivatives—nitrendipine, nimodipine, and amlodipine—were tested alongside nifedipine in negative mode MALDI-MS for EGCG and TF3’G detection Results indicated that nitrendipine, nimodipine, and amlodipine were ineffective in enhancing MS detection of these compounds It has been observed that the nitrosophenyl pyridine derivative forms through UV-dehydration, involving intramolecular proton transfer from the pyridine moiety to the nitro group at the ortho-position of the phenyl ring.
Nifedipine, characterized by an ortho-positioned nitro group, uniquely produces the nitrosophenyl pyridine moiety, unlike nitrendipine, nimodipine, and amlodipine, which do not undergo UV-dehydration This highlights the importance of the nitrosophenyl pyridine derivative formed from nifedipine under UV-irradiation, as it significantly enhances the MALDI-MS detection of polyphenols, particularly EGCG and TF3’G, indicating its crucial role in the detection process.
To verify the generation of the active nitrosophenyl pyridine photobase from nifedipine under MALDI irradiation using a Nd:YAG laser at 355 nm, nifedipine was applied to an ITO glass slide at a concentration of 5 mg/mL.
Summary
MALDI-MS is an effective analytical tool for detecting ionizable compounds like peptides, proteins, and lipids, but it has limited capability for neutral compounds such as polyphenols due to their lack of proton-attachment groups Chapter II explores a matrix screening strategy utilizing a proton subtraction reaction with photobase generators, revealing that nifedipine, a photobase generator, enables MALDI-MS detection of polyphenols in negative mode, outperforming other matrices Among the dihydropyridines tested, only nifedipine successfully detected EGCG and TF3’G, indicating that its nitrosophenyl pyridine derivative formed during UV-dehydration under laser irradiation is crucial for deprotonating polyphenols Nifedipine preferentially removes a proton from the 5-position OH group in the A ring of flavonoids, significantly enhancing MS detection across various polyphenols, including flavonols, flavones, flavanones, chalcones, stilbenoids, and phenolic acids Consequently, nifedipine presents a promising novel MALDI matrix reagent for analyzing neutral or less MS-active polyphenols.
Intestinal transport experiments using rat jejunum membrane in the Ussing
Intestinal transport experiments were conducted using jejunum membranes from 9-week-old male SD rats, following established protocols The rats were sourced from Charles River Japan, specifically the SPF/VAF Crj:SD strain.
In a study conducted in Kanagawa, Japan, a segment of the jejunum was excised from the small intestine, washed with Krebs-Bicarbonate Ringer’s (KBR) solution, and prepared for analysis The jejunum was carefully dissected to expose the mucosal side and mounted onto a Dual Channel Ussing Chamber system Following a 15-minute incubation with fresh KBR solution to stabilize the tissue, the apical solution was replaced with KBR buffer containing either 50 µmol/L TF3’G or ECG For inhibitor experiments, various inhibitors were introduced to the apical side, followed by another incubation period Throughout the transport experiment, the solutions were continuously bubbled with an O2/CO2 mixture and maintained at 37˚C After a 60-minute transport period, the solutions were collected for metabolite identification using LC-TOF-MS, and the intestinal membranes were subsequently washed and frozen for further analysis.
Frozen membranes were preserved at -30˚C prior to MALDI-MS imaging analysis All animal experiments adhered to the Faculty of Agriculture's guidelines and complied with Japan's Animal Experimentation Law (No 105, 1973) and Notification (No 6).
1980, of the Prime Minister’s Office) of the Japanese Government All experiments were reviewed and approved by the Animal Care and Use Committee of Kyushu University (permit number: A28-040).
LC-TOF-MS analysis
An aliquot of 4 mL from either the apical or basolateral side after 60 minutes of transport experiments was processed using a Waters Sep-Pak Plus C18 cartridge, followed by elution with a mixture of methanol and formic acid (100/0.1, v/v) The resulting eluate was evaporated to dryness and reconstituted in 100 µL of a methanol/water/formic acid solution (50/50/0.1, v/v/v) A 20 µL injection of this solution was analyzed using an LC-TOF-MS system, which included an Agilent 1200 series HPLC equipped with a micro degasser, binary pump, thermostatically controlled oven, and a Cosmosil 5C18-MS-II column for the separation of TF3'G, ECG, and their metabolites The mobile phase utilized a linear gradient over 30 minutes, transitioning from 0 to 100% solvent B, at a flow rate of 0.2 mL/min and a temperature of 40°C Mass spectrometry was conducted with a micrOTOF-II mass spectrometer to analyze the compounds.
The ESI-MS analysis was conducted in negative mode within the mass range of m/z 100–1000, utilizing a drying gas (nitrogen) at a flow rate of 8.0 L/min and a drying temperature of 200˚C The nebulizing gas pressure was set at 1.6 bar, with a capillary voltage of 3800 V A calibration solution of 10 mmol/L sodium formate in a 1:1 mixture of water and acetonitrile was injected at the start of the analysis Data acquisition and analysis were performed using DataAnalysis 3.2 software from Bruker Daltonics.
Preparation of intestinal membrane section and matrix reagent
Frozen intestinal segments were sliced into 12 µm-thick sections using a CM1100 Leica Cryomicrotome at -20˚C after transport experiments The intestinal sections were then thaw-mounted on ITO-coated conductive glass slides and dried under nitrogen gas These prepared ITO-mounted sections were stored at -30˚C until needed for further analysis.
Matrix spraying was conducted using an ImagePrep automatic matrix sprayer (Bruker Daltonics) with various matrix reagents, including IAA, 1,5-DAN, DHB, and nifedipine at concentrations of 20 mg/mL in a 3:1 acetonitrile/water solution Additionally, 9-AA was used at 10 mg/mL in the same solvent mixture, while nifedipine was also prepared with 5 mmol/L phytic acid These matrix reagents were applied individually onto ITO-mounted jejunum membrane sections, utilizing a spray power setting of 20%.
46 modulation, 20%; spraying time, 1.5 s; incubation time, 10 s; drying time, 60 s; and spraying, 60–70 cycles, until homogeneous matrix layers were established.
MALDI-MS imaging analysis
MALDI-MS imaging was conducted using an Autoflex III mass spectrometer with a SmartBeam III in negative ion-linear mode to analyze TF3’G, ECG, their metabolites, rifampicin, phloretin, and estrone-3-sulfate For cyclosporine A, prazosin, valspodar, and wortmannin detection, positive ion-linear mode was utilized MS data were collected within the m/z range of 100–1300, with ion source 1 set at 20.00 kV, ion source 2 at 18.80 kV, and lens voltage at 7.50 kV Additional parameters included a gain of 12.00, laser frequency of 200 Hz, laser power at 80%, offset at 60%, range at 20%, and laser focus range at 100% The imaging analysis achieved a spatial resolution of 50 μm, and the resulting MS spectra were processed using FlexAnalysis 3.3, combining 100 spectra from the region of interest Visualization of the image data was reconstructed with a mass filter of ± 0.2 m/z.
FlexImaging 2.1 (Bruker Daltonics) Intensity scale was fixed for each compound in all measurements
Optimization of MALDI-MS imaging for visualization of monomeric and
The application of MALDI-MS imaging techniques on biological tissues often encounters challenges due to overlapping MS peaks from matrix and tissue contaminants, which can hinder the selective detection of targets This study focused on evaluating matrix reagents for the selective detection of TF3’G and ECG in SD rat intestinal membranes, utilizing 9-AA, IAA, and 1,5-DAN, along with nifedipine, which enhances the MS detection of neutral polyphenols Each matrix was uniformly sprayed onto membrane sections, and the SD rat jejunum membranes treated with TF3’G or ECG were analyzed using the MALDI-MS imaging system Results indicated that 1,5-DAN and nifedipine effectively visualized both targets in negative MS mode, while the other matrices failed to do so However, the over-detection of peaks by 1,5-DAN on blank sections highlighted its limitations in detecting polyphenols in the jejunum membrane Consequently, further imaging experiments utilized nifedipine as the preferred matrix Additionally, the presence of minerals in tissues significantly impacts MALDI-ionization, and incorporating phytic acid as a matrix additive proved effective in enhancing target detection due to its chelating properties with minerals.
3-1C, the addition of 5 mmol/L phytic acid to nifedipine solution was also applicable for adequate visualization of TF3’G and ECG in the SD rat jejunum membrane section.
In situ visualization of monomeric and condensed catechins in rat jejunum
LC-TOF-MS analysis of the basolateral solution after 50-µmol/L, 60-minute transport of TF3’G indicated that this compound was not transported across the jejunum membranes of SD rats under the tested conditions, aligning with previous reports Conversely, ECG was confirmed as a transportable polyphenol, demonstrating an apparent permeability of 0.32 × 10^-6 cm/s in Caco-2 cell transport experiments Additionally, a significant absorption of 14.8 µg/mL of ECG in plasma was observed 2 hours post single oral administration at a dose of 650 mg/kg The study utilized nifedipine/phytic acid-aided MALDI-MS imaging, which visually confirmed the distribution of ECG throughout the jejunum membrane These findings collectively underscore ECG's absorbability across intestinal membranes and highlight the efficacy of nifedipine/phytic acid-aided MALDI-MS imaging as a novel in situ analytical tool for absorption studies.
The next challenge involved using nifedipine/phytic acid-aided MALDI-MS imaging to provide evidence of the location of non-absorbable TF3’G or TFs in intestinal membranes, as there was no prior report clarifying their incorporation.
MS imaging demonstrated that TF3’G was distinctly localized at the apical region of the rat jejunum membrane after 60 minutes of transport (Figure 3-1C) This initial observation of TF3’G's regional localization, confirmed by the MS imaging technique, suggests that certain compounds may exhibit no intestinal absorption.
TF and saponins [67] ) may be incorporated into membrane, and play a role in the regulation of transport route(s), like TF [14]
Figure 3-1 MALDI-MS imaging-based detection of TF3’G and ECG in rat jejunum membrane
The distribution of TF3’G and ECG in the rat jejunum membrane was analyzed following 50-µmol/L, 60-minute transport experiments conducted in the Ussing Chamber system This study utilized matrix reagents dissolved in acetonitrile/water (3/1, v/v), including 9-AA (10 mg/mL), IAA (20 mg/mL), and 1,5-DAN (20 mg/mL), along with nifedipine (20 mg/mL).
MALDI-MS imaging was conducted at a spatial resolution of 50 µm to visualize TF3’G (m/z 715.1) and ECG (m/z 441.1) in a solution containing 20 mg/mL and 5 mmol/L phytic acid The intensities for TF3’G and ECG were represented using a fixed pseudocolor scale for each target.
Figure 3-2 LC-TOF-MS chromatograms of TF3’G and ECG in apical and basolateral solutions
In a study utilizing the Ussing Chamber system, apical and basolateral solutions of TF3’G and ECG were analyzed after a 60-minute transport experiment at a concentration of 50 µmol/L The liquid chromatography (LC) separations were conducted on a Cosmosil 5C18-MS-II column, using a gradient of methanol and formic acid over 30 minutes The mass spectrometry (MS) conditions were detailed in the Materials and Methods section, with results indicating that certain compounds were not detected (N.D.).
Absorption route(s) of monomeric and condensed catechins in rat jejunum
We aimed to assess the effectiveness of MALDI-MS imaging in studying intestinal transport routes, utilizing transport inhibition methods To investigate the absorption pathways of TF3’G and ECG, we employed phloretin (200 àmol/L), a known inhibitor of MCT, along with estrone-3-sulfate.
In a study examining the transport of TF3’G and ECG, inhibitors of organic anion transporting polypeptides (OATP) and transcytosis pathways were utilized The results indicated that wortmannin did not affect the visualization intensity of TF3’G, suggesting that the transcytosis route was not involved in its incorporation Conversely, the use of phloretin and estrone-3-sulfate significantly reduced the visualized region of TF3’G in the jejunum membrane, confirming the role of MCT and OATP transport routes This was further supported by the distribution of inhibitors at the apical side of the membrane Similar findings were observed with ECG, where MCT and OATP inhibitors also decreased the visualized regions These results strongly suggest that both TF3’G and ECG are incorporated into the intestinal membrane through MCT and OATP transporters, aligning with previous reports on their involvement.
Recent studies have highlighted the role of OATP transporters in the cellular accumulation of ECG in Caco-2 cell monolayers and OATP-transfected cells Specifically, OATP transporters have been shown to facilitate the uptake of TFs, with OATP inhibition resulting in decreased cellular accumulation of TFs in human embryonic kidney cells expressing OATP2B1 While the potential involvement of other intestinal transport routes, such as MCT, cannot be dismissed, these findings suggest a new analytical approach utilizing MALDI-MS imaging combined with various inhibitors.
Figure 3-3 MALDI-MS imaging of TF3’G in 60-min transported jejunum membrane in the absence or presence of influx transporter inhibitors
Influx transporter inhibitors including: phloretin (200 àmol/L), estrone-3-sulfate
(100 àmol/L), and wortmannin (1 àmol/L) were used in intestinal transport experiments TF3’G (m/z 715.1), together with phloretin (m/z 273.1) and estrone- 3-sulfate (m/z 349.1), were visualized in negative ion-linear mode by MALDI-
Mass spectrometry imaging achieved a spatial resolution of 50 µm using a nifedipine/phytic acid matrix reagent To visualize wortmannin (m/z 451.1), 2,5-dihydroxybenzoic acid (DHB) in a solution of acetonitrile and water (3:1, v/v, 20 mg/mL) was employed as the matrix reagent in positive ion-linear mode The visualized intensities for TF3’G, phloretin, estrone-3-sulfate, and wortmannin are represented using a fixed pseudocolor scale for each target compound.
Figure 3-4 MALDI-MS imaging of ECG in 60-min transported jejunum membrane in the absence or presence of influx transporter inhibitors
Influx transporter inhibitors including: phloretin (200 àmol/L), estrone-3-sulfate
(100 àmol/L), and wortmannin (1 àmol/L) were used in intestinal transport experiments ECG (m/z 441.1), together with phloretin (m/z 273.1) and estrone-
3-sulfate (m/z 349.1), were visualized in negative ion-linear mode by MALDI-
MS imaging at a spatial resolution of 50 àm by using nifedipine/phytic acid matrix reagent For the visualization of wortmannin (m/z 451.1), DHB
In positive ion-linear mode, a matrix reagent composed of acetonitrile and water in a 3:1 volume ratio at a concentration of 20 mg/mL was utilized The visualized intensities for ECG, phloretin, estrone-3-sulfate, and wortmannin were displayed using a consistent pseudocolor scale for each target compound.
Efflux route(s) of monomeric and condensed catechins in rat jejunum membrane
Previous findings in Section 3.3 demonstrated the incorporation of TF3’G and ECG into intestinal membranes using the MALDI-MS imaging technique However, it remains uncertain whether these compounds are recognized and transported by efflux transporters, similar to other polyphenols The apical membranes contain efflux ABC transporters, including MRP2, P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP) To investigate the role of these transporters in the intracellular efflux of TF3’G and ECG, further studies utilizing efflux inhibitors in conjunction with MALDI-MS are necessary.
MS imaging utilizing cyclosporine A (20 μmol/L), a non-specific ABC efflux transport inhibitor, revealed that the regions visualized for TF3’G and ECG significantly expanded, indicating recognition and efflux of both polyphenols by ABC transporters This supports previous findings that catechins are extensively effluxed to the apical side during intestinal absorption, with MRP2 and P-gp transporters playing a key role in this process The research demonstrated that absorbable ECG was partially effluxed back to the apical compartment via ABC transporters, as shown in the MALDI analysis.
The MS imaging technique revealed that the accumulation of TF3’G in the intestinal membrane was enhanced by efflux inhibition, marking the first discovery that transport factors (TFs) are absorbed through both influx and efflux absorption processes.
A study investigated the efflux routes of TF3’G using specific inhibitors: prazosin (20 µmol/L, targeting BCRP), rifampicin (20 µmol/L, targeting MRP2), and vanspodar (20 µmol/L, targeting P-gp) The results, illustrated in Figure 3-6, indicated that all inhibitors expanded the region of TF3’G, suggesting its efflux occurred through non-specific routes This research highlights the efficacy of the inhibitor-aided MALDI-MS imaging technique for direct analysis of intestinal absorption of analytes, eliminating the need for pretreatment steps like extraction and separation.
Figure 3-5 MALDI-MS imaging of TF3’G and ECG in 60-min transport jejunum membrane in the absence or presence of an efflux transporter inhibitor
In intestinal transport experiments, the efflux transporter inhibitor cyclosporine A (20 àmol/L) was utilized MALDI-MS imaging, conducted in negative ion-linear mode with a spatial resolution of 50 àm, successfully visualized TF3’G (m/z 715.1) and ECG (m/z 441.1) using a nifedipine/phytic acid matrix reagent For the visualization of cyclosporine A (m/z 1224.6), a DHB matrix reagent (acetonitrile/water, 3/1, v/v, 20 mg/mL) was employed in positive ion-linear mode The visualized intensities of TF3’G, ECG, and cyclosporine A were represented using a fixed pseudocolor scale for each target.
Figure 3-6 MALDI-MS imaging of TF3’G in 60-min transport jejunum membrane in the absence or presence of efflux transport inhibitors
In intestinal transport experiments, specific efflux transporter inhibitors such as prazosin, rifampicin, and valspodar were utilized at a concentration of 20 àmol/L The compounds TF3’G (m/z 715.1) and rifampicin (m/z 821.4) were visualized using MALDI-MS imaging in negative ion-linear mode with a spatial resolution of 50 àm, employing a nifedipine/phytic acid matrix reagent Additionally, the visualization of prazosin (m/z 273.3), valspodar (m/z 1237.4), and cyclosporine A (m/z 1224.6) was achieved using a DHB matrix in a solvent mixture of acetonitrile and water at a ratio of 3:1 (v/v).
In positive ion-linear mode, a matrix reagent concentration of 20 mg/mL was utilized The visualized intensities for the targets TF3’G, prazosin, rifampicin, valspordar, and cyclosporine A are presented using a fixed pseudocolor scale for each compound.
3.5 Visualized detection of metabolites of monomeric and condensed catechins during intestinal absorption
MALDI-MS imaging was utilized to investigate the metabolic behavior of TF3’G and ECG during a 60-minute transport in the jejunum membrane of SD rats, highlighting its advantages for non-targeted analysis To improve the detection of potential metabolites, analyses were conducted in the presence of cyclosporine A, a non-specific efflux inhibitor The results indicated that under the current MALDI-MS conditions, phase II metabolism of TF3’G, including sulfation, methyl-sulfation, and glucuronidation, did not occur Although a slight presence of methylated TF3’G was detected in the apical region, additional LC-TOF-MS analysis suggested that TF3’G, or possibly TFs, exhibited stability and resistance to phase II metabolism during the absorption process.
The study revealed that ECG is prone to phase II metabolism during intestinal absorption, as evidenced by MALDI-MS imaging of the ECG-incorporated intestinal membrane and LC-TOF-MS analysis of the apical solution These analyses demonstrated the formation of various ECG metabolites, including methylated ([Me-ECG - H] -, m/z 455.1), sulfated ([sulfate-ECG - H] -, m/z 521.0), and methyl-sulfated forms.
In the analysis of ECG metabolites, several forms were identified, including glucuronide ECG (m/z 617.1) and sulfated ECG (m/z 521.0384), as well as methyl-sulfate-ECG (m/z 535.0540), indicating the presence of multiple isomers resulting from different sulfation positions on the ECG skeleton Notably, LC-MS results showed that ECG metabolites were primarily detected in the apical solution after 60 minutes of transport, suggesting limited transport of these metabolites following phase II metabolism Additionally, MALDI-MS imaging analysis revealed a rapid metabolism of ECG into sulfated and methyl-sulfated conjugates within just 15 minutes at the apical side, highlighting the dynamic processes involved in ECG absorption.
Polyphenols are known to undergo phase II metabolism in the intestine, liver, and kidney, which contributes to their poor bioavailability in their intact form However, the specific metabolic processes of polyphenols within the intestinal membrane remain unclear Notably, no phase II metabolites of TF have been detected in human urine following the oral administration of 1000 mg of TF extract Additionally, the absence of TF3’G metabolites after a 60-minute transport in the jejunum membrane of SD rats supports the notion that phase II metabolism of TFs is limited in this region In contrast, ECG has shown susceptibility to phase II metabolism, as evidenced by the presence of methylated, sulfated, and glucuronide conjugates in the plasma of Wistar rats after oral administration.
Research indicates that the metabolism of catechins, specifically methylation and sulfation, occurs preferentially in Caco-2 cell monolayers and the human intestine Notably, ECG metabolites were detected on the apical side but absent on the basolateral side, reinforcing previous findings that ECG is primarily present in its free form in plasma following oral administration in humans Additionally, during Caco-2 cell transport experiments, ECG metabolites were identified in the apical compartment.
Figure 3-7 MALDI-MS imaging analyses of TF3’G and its metabolites in 60-min-transported jejunum membrane in the presence of 20 àmol/L cyclosporine A
TF3’G (m/z 715.1) and its metabolites, including Me-TF3’G (m/z 729.1), sulfate-TF3’G (m/z 795.1), Me-sulfate-TF3’G (m/z 809.1), and Glc A-TF3’G (m/z 891.2), were successfully visualized using MALDI-MS imaging in negative ion-linear mode The imaging was conducted at a spatial resolution of 50 µm with a nifedipine/phytic acid matrix reagent, and the intensities of TF3’G and its metabolites were represented using a fixed pseudocolor scale for clarity Notably, some components were not detected (N.D.).
Figure 3-8 LC-TOF-MS chromatograms of TF3’G and its metabolites in apical and basolateral solutions
Apical and basolateral solutions of TF3’G after their 50-àmol/L, 60-min transport experiments in the Ussing Chamber system was subjected to LC-TOF-
The analysis involved liquid chromatography (LC) separations using a Cosmosil 5C18-MS-II column (2.0 mm × 150 mm), with a gradient elution of 0 to 100% methanol/formic acid (100/0.1, v/v) over a duration of 30 minutes Detailed mass spectrometry (MS) conditions are outlined in the Materials and Methods section.
Figure 3-9 MALDI-MS imaging analyses of ECG and its metabolites in 60- min-transported jejunum membrane in the presence of 20 àmol/L cyclosporine A
MALDI-MS imaging in negative ion-linear mode successfully visualized ECG (m/z 441.1) and its metabolites, including Me-ECG (m/z 455.1), sulfate-ECG (m/z 521.0), Me-sulfate-ECG (m/z 535.1), and Glc A-ECG (m/z 617.1), using a nifedipine/phytic acid matrix reagent at a spatial resolution of 50 µm The visualized intensities for ECG and its metabolites were represented with a fixed pseudocolor scale for each target.
Figure 3-10 LC-TOF-MS chromatograms of ECG and its metabolites in apical and basolateral solutions
Tea polyphenols, including monomeric and condensed catechins, are globally consumed for their potential health benefits, such as preventing cardiovascular diseases, diabetes, cancers, and aiding intestinal absorption Despite their advantages, tea polyphenols face challenges with low bioavailability due to poor intestinal absorption, extensive metabolism, and efflux during the absorption process, making the intestine crucial for their bioavailability To assess the bioavailability of tea polyphenols, advanced analytical techniques like LC-MS have been developed, while MALDI-MS is favored for its sensitivity, selectivity, and ability to tolerate impurities The application of MALDI in imaging techniques has gained traction in pharmacological sciences, allowing for the visualization of drugs and food compounds, including proteins, lipids, and polyphenols.
MALDI-MS imaging offers a significant advantage by delivering information on both the presence and precise location of compounds within biological tissues, all without the need for labeling preparations or complex extraction and separation processes This capability enhances the analytical applications of MALDI-MS imaging in various research studies.
This study focuses on enhancing the negative MALDI-MS analysis of tea polyphenols, which are known for their low ionization efficiency in conventional MALDI matrices By utilizing a photobase generator, nifedipine, an effective matrix was developed to facilitate proton subtraction reactions Additionally, the research aims to establish an in situ MALDI-MS imaging technique to explore the intestinal absorption and metabolism of tea polyphenols.
Chapter II: Enhanced matrix-assisted laser desorption/ionization mass spectrometry detection of polyphenols
MALDI-MS is an effective analytical technique for identifying ionizable compounds, thanks to its exceptional sensitivity and ability to handle impurities However, its application is limited for neutral compounds like polyphenols, primarily due to the absence of suitable matrix reagents.
Chapter II explores a novel screening strategy for matrix application in MALDI-MS detection of polyphenols, focusing on the proton subtraction reaction of nifedipine when exposed to UV-irradiation The study evaluates the proton subtraction properties of nifedipine using EGCG and TF3’G, the predominant polyphenols found in green and black tea, respectively The photobase dehydration reaction of nifedipine was examined with dihydropyridine derivatives, while naringenin and its methylnaringenin analogues were assessed for their proton-removal capabilities within the flavonoid structure A diverse range of polyphenols, including flavonols, flavones, flavanones, chalcones, stilbenoids, and phenolic acids, were analyzed to determine the effectiveness of nifedipine as a MALDI-MS matrix This chapter reveals for the first time that nifedipine at 5 mg/mL enables successful MS detection of EGCG and TF3’G, outperforming common matrix reagents in negative MALDI-MS, with detection limits of 0.1 pmol/spot and 0.01 pmol/spot, respectively Notably, only nifedipine, featuring a nitro group at the ortho position of the phenyl ring, facilitated the detection of these polyphenols, indicating the critical role of the nitrosophenyl pyridine photobase generated from nifedipine during the dehydration reaction under 355 nm laser irradiation in promoting deprotonation in negative MALDI-MS.
MS detection of 5-O-methylnaringenin among naringenins, indicated that nifedipine
Nifedipine has been shown to enhance mass spectrometry (MS) detection of various polyphenols, including flavonols, flavones, flavanones, chalcones, stibenoids, and phenolic acids This suggests that nifedipine may serve as a novel matrix reagent for matrix-assisted laser desorption/ionization (MALDI) analysis, particularly by facilitating the removal of a proton from the 5-position OH group in the A ring of the flavonoid skeleton.
Chapter III: Application of matrix-assisted laser desorption/ionization mass spectrometry-imaging technique for intestinal absorption of polyphenols
The MALDI-MS imaging technique is well-established in pharmacological science, enabling simultaneous visualization of targets and their metabolites in target organs, which surpasses the limitations of LC-based methods Chapter III explores the practical application of MALDI-MS imaging for studying the intestinal absorption of polyphenols, specifically targeting ECG, an absorbable polyphenol, and TF3’G, a non-absorbable polyphenol Intestinal transport experiments were conducted using SD rat jejunum membrane in an Ussing Chamber system, with membrane transporter inhibitors employed to clarify the absorption routes of these polyphenols Building on previous findings of nifedipine as a novel matrix for negative MALDI-MS and phytic acid as a matrix additive for enhanced visualization, the nifedipine/phytic acid combination was utilized for MALDI-MS visualization in rat intestinal membrane The results demonstrated effective visualization of TF3’G and ECG, revealing that TF3’G localized specifically in the apical region of the intestinal membrane, while ECG was distributed throughout the membrane This indicates that MALDI-MS imaging serves as a valuable in situ analytical tool for absorption studies, with the visualized regions of TF3’G and ECG being influenced by various compounds.
Research indicates that TF3’G and ECG can be integrated into the intestinal membrane through MCT and OATP transport pathways, with subsequent efflux to the apical compartment via ABC transporters The findings highlight the effectiveness of inhibitor-aided MALDI-MS imaging as a novel analytical method for directly analyzing intestinal absorption routes of compounds The non-targeted visualization of TF3’G and ECG metabolites using MALDI-MS imaging revealed that TF3’G remains stable against phase II metabolism, while ECG is prone to phase II metabolism, resulting in methylation, sulfation, and their combined conjugates within the intestinal membrane.
This study highlights the potential of nifedipine as a photobase generator that forms nitrosophenyl pyridine under UV light, serving as an innovative matrix reagent for negative MALDI-MS detection of polyphenols The combination of nifedipine and phytic acid in MALDI-MS imaging, along with an inhibitor-aided intestinal transport experiment using a rat intestinal membrane in an Ussing Chamber system, presents a novel and effective analytical approach for investigating the intestinal absorption and metabolism of polyphenols This method eliminates the need for staining, labeling, and complex extraction and separation processes.
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I would like to express my heartfelt gratitude to my supervisor, Prof Toshiro Matsui, for his unwavering support throughout my Ph.D studies and research His intellectual guidance and innovative ideas significantly enhanced my academic journey in the food analysis laboratory I am also thankful to my committee members, Prof Mitsuya Shimoda and Prof Takahisa Miyamoto, for their valuable insights and constructive feedback, which were instrumental in the successful completion of my doctoral dissertation.