Shohei Sakuda, Masaki Sunaoka, Maho Terada, Ayaka Sakoda, Natsumi Ishijima, Noriko Hakoshima, Kenichi Uchida, Hirofumi Enomoto and Tomohiro Furukawa : Inhibition of Aflatoxin Production in Aspergillus flavus by a Klebsiella sp. and Its Metabolite Cyclo(l-Ala-Gly), Toxins, Vol.16, No.3, 2024.
(要約)
-fold dilution. This strong inhibitory activity suggests that the bacterium is a candidate for an effective biocontrol agent for AF control.
Shudai Sasaki, Tsukasa Mori, Hirofumi Enomoto, Sakiko Nakamura, Hideo Yokota, Hiroshi Yamashita and Naoko Goto-Inoue : Assessing Molecular Localization of Symbiont Microalgae in Coral Branches Through Mass Spectrometry Imaging., Marine Biotechnology, Vol.26, No.2, 223-229, 2024.
(要約)
Reef-building corals are a fundamental pillar of coral reef ecosystems in tropical and subtropical shallow environments. Corals harbor symbiotic dinoflagellates belonging to the family Symbiodiniaceae, commonly known as zooxanthellae. Extensive research has been conducted on this symbiotic relationship, yet the fundamental information about the distribution and localization of Symbiodiniaceae cells in corals is still limited. This information is crucial to understanding the mechanism underlying the metabolite exchange between corals and their algal symbionts, as well as the metabolic flow within holobionts. To examine the distribution of Symbiodiniaceae cells within corals, in this study, we used fluorescence imaging and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MS-Imaging) on branches of the Acropora tenuis coral. We successfully prepared frozen sections of the coral for molecular imaging without fixing or decalcifying the coral branches. By combining the results of MS-Imaging with that of the fluorescence imaging, we determined that the algal Symbiodiniaceae symbionts were not only localized in the tentacle and surface region of the coral branches but also inhabited the in inner parts. Therefore, the molecular imaging technique used in this study could be valuable to further investigate the molecular dynamics between corals and their symbionts.
Abdelrahman Elamin, Hirofumi Enomoto, Maiko Watanabe and Shohei Sakuda : The Mechanism of Ochratoxin Contamination of Artificially Inoculated Licorice Roots., Toxins, Vol.15, No.3, 2023.
(要約)
After incubation for 10 and 20 days, the OT contents of the samples were determined by high-performance liquid chromatography, and microtome sections prepared from the samples were analyzed by desorption electrospray ionization tandem mass spectrometry, to visualize OT localization. The same sections were further examined by light microscopy and scanning electron microscopy, to investigate the path of fungal mycelial penetration of the inner roots. OT concentrations tended to increase from the upper- to the middle-root parts. OTs were located in cut areas and areas of cork layer damage; they were not present in the undamaged cork layer, indicating that the structure of this layer prevents OT contamination of the licorice root.
Tomohiro Furukawa, Masayo Kushiro, Hiroyuki Nakagawa, Hirofumi Enomoto and Shohei Sakuda : Low-dose ethanol increases aflatoxin production due to the adh1-dependent incorporation of ethanol into aflatoxin biosynthesis, iScience, Vol.26, No.2, 2023.
(要約)
indirectly controls aflatoxin production by balancing ethanol production and catabolism.
Hirofumi Enomoto and Nobuhiro Zaima : Desorption electrospray ionization-mass spectrometry imaging of carnitine and imidazole dipeptides in pork chop tissues., Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, Vol.1216, 2023.
(要約)
Carnitine is essential for energy production and lipid metabolism in skeletal muscle. Carnosine and its methylated analogs anserine and balenine are histidine-containing imidazole dipeptides, which are antioxidative compounds. They are major health-related components in meat; however, analytical technique to investigate their distribution among tissues have not fully established. Here, we performed desorption electrospray ionization (DESI)-mass spectrometry imaging (MSI) of pork chop sections containing longissimus thoracis et lumborum muscle (loin), intermuscular fat tissue, transparent tissue, and spinalis muscle to investigate the distributions of carnitine and imidazole dipeptides. Liquid chromatography-MS revealed that the concentrations of carnitine, carnosine, anserine, and balenine were 11.0 ± 0.9, 330.1 ± 15.5, 21.2 ± 1.5, and 9.6 ± 0.5 mg/100 g, respectively. In the mass spectrum obtained by DESI-MSI, peaks corresponding to the chemical formulae of carnitine and imidazole dipeptides were detected. DESI-MSI provided definite identification of carnitine, while DESI-tandem MSI (MS/MSI) was necessary to accurately visualize carnosine, anserine, and balenine. Carnitine and these imidazole dipeptides were mainly distributed in the loin and spinalis muscle, while their distribution was not uniform in both muscle tissues. In addition, the balance between both tissues were different. The concentration of carnitine was higher in the spinalis muscle than that in the loin, while those of imidazole dipeptides were higher in the loin than those in the spinalis muscle. These results were consistent with those obtained by liquid chromatography-MS quantification, suggest that DESI-MSI analysis is useful for the distribution analysis of carnitine and imidazole dipeptides in meat.
Hirona Kugo, Hirofumi Enomoto, Kenichi Yanagimoto, Hiroki Tanaka, Tatsuya Moriyama and Nobuhiro Zaima : Eicosapentaenoic acid is associated with the attenuation of dysfunctions of mesenchymal stem cells in the abdominal aortic aneurysm wall., Food & Function, Vol.13, No.14, 7540-7547, 2022.
(要約)
anti-inflammatory activities. However, relationships between the anti-inflammatory effects and the cells in the AAA wall are poorly understood. In this study, we visualized the distribution of EPA-containing phosphatidylcholine (EPA-PC) in the AAA wall. EPA-PC was not ubiquitously distributed in both animal (hypoperfusion-induced AAA model) and human AAA walls, suggesting the preferential incorporation of EPA into certain cells. In the EPA-PC-high region of both animal and human AAAs, mesenchymal stem cell (MSC) marker positive areas were significantly higher than those in the EPA-PC-low region. Matrix metalloproteinase-positive MSCs were significantly lower in the AAA wall of the animal model which was administered EPA-rich fish oil. These data suggest that EPA is associated with the attenuation of MSC dysfunctions, which result in the suppression of AAA development.
Yiu Shing Tam, Kenichi Uchida, Hirofumi Enomoto, Senji Takahashi, Koichi Makimura and Shohei Sakuda : A new metabolite, mannogeranylnerol, specifically produced at body temperature by Schizophyllum commune, a causative fungus of human mycosis., The Journal of Antibiotics, Vol.75, No.4, 243-246, 2022.
(要約)
Schizophyllum commune is a causative fungus of human mycosis. Its metabolites produced at 27 °C were compared with those produced at 37 °C, to obtain a candidate low-molecular-weight virulence factor related to the pathogenicity of this fungus. We found that S. commune specifically produces two acyclic terpene mannosides at 37 °C. They were identified as nerolidol β-D-mannoside (1) and geranylnerol β-D-mannoside (2) by NMR, MS, and CD analyses. Compound 2, a new compound named mannogeranylnerol, showed weak antibiotic activity that was slightly stronger than that of compound 1.
(キーワード)
Body Temperature / Fungi / Humans / Mannosides / Mycoses / Schizophyllum
Hirofumi Enomoto : Unique distribution of ellagitannins in ripe strawberry fruit revealed by mass spectrometry imaging., Current Research in Food Science, Vol.4, 821-828, 2021.
(要約)
Ellagitannins (ETs) are hydrolysable tannins composed of a polyol core, primarily glucose, which is esterified with hexahydroxydiphenic acid (HHDP), and in some cases, gallic acid. ETs are the major phenolic compounds found in strawberries and may contribute to the health-related properties of strawberries, because of their strong antioxidative activity. However, their distribution in the strawberry fruit remains unclear. In this study, matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) was used to visualize ETs in ripe strawberry fruits. Five peaks, corresponding to the values of ET [M-H] ions detected in the MALDI-MS spectrum of strawberry extracts, were identified as strictinin, pedunculagin, casuarictin, davuriicin M, and an unknown ET using MALDI-tandem MS (MS/MS). In addition, liquid chromatography-electrospray ionization-MS/MS of the extracts revealed the presence of pedunculagin isomers and the unknown ET. Ion images of these five ETs were reconstructed using MALDI-MSI. Strictinin was widely distributed in and around the achene seed coats, while the other ETs were dispersed in and around the seed coats, and at the bottom of the receptacle; pedunculagin was distributed in the epidermis and pith, whereas casuarictin, the unknown ET, and davuriicin M were distributed in the pith. Moreover, MALDI-MSI of a casuarictin standard indicated that in-source fragmentation weakly affected the ion images. The results suggest that the distribution of ETs depends on the presence or absence of their constituents, namely galloyl units, HHDP, and bis-HHDP. To the best of my knowledge, this is the first report on the visualization of ETs in plant tissues using MSI, MALDI-MSI may be a useful tool for analyzing the distribution of ETs in the strawberry fruit.
Hirofumi Enomoto : Distribution analysis of jasmonic acid-related compounds in developing Glycine max L. (soybean) seeds using mass spectrometry imaging and liquid chromatography-mass spectrometry., Phytochemical Analysis : PCA, Vol.33, No.2, 194-203, 2021.
(要約)
Jasmonic acid (JA) and its precursors are oxylipins derived from α-linolenic acid (αLA) and hexadecatrienoic acid, and regulate seed development. However, their spatial distribution in the developing Glycine max L. (soybean) seeds has not been elucidated. To investigate the distribution of JA-related compounds in the developing soybean seeds using desorption electrospray ionisation-mass spectrometry imaging (DESI-MSI) and liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS) analyses. Cryosections of developing seeds were prepared using adhesive films, and subjected to DESI-MSI analysis. Verification of the DESI-MSI ion images were performed using DESI-tandem MSI (MS/MSI), LC-ESI-MS and tandem MS (MS/MS). In the DESI-MSI mass spectrum, peaks matching the chemical formulae of αLA, 12-oxo-phytodienoic acid (OPDA), and 3-oxo-2-(2-(Z)-pentenyl)-cyclopentane-1-octanoic acid (OPC-8:0) were detected. These compounds were mainly distributed in the seed coat, especially near the hilum. This was consistent with the quantitative results obtained by LC-ESI-MS. While, DESI-MS/MSI and LC-ESI-MS/MS suggested the presence of isomers for OPDA and OPC-8:0. The effect of isomers on the DESI-MSI ion images was small for OPDA, and considerable for OPC-8:0. These results demonstrated that free αLA, OPDA, and OPC-8:0 were the abundant JA-related compounds mainly distributed in the seed coat of the developing soybeans. OPDA and OPC-8:0 might exert a biological role in the seed coat. To the best of my knowledge, this is the first report on the accumulation of OPDA and OPC-8:0 in the seed coat. The combination of DESI-MSI and LC-ESI-MS is a useful tool for distribution analysis of JA-related compounds in the developing seeds.
(キーワード)
Chromatography, Liquid / Cyclopentanes / Oxylipins / Seeds / Glycine max / Spectrometry, Mass, Electrospray Ionization / Tandem Mass Spectrometry
Hirofumi Enomoto, Shiro Takeda and Hajime Hatta : Spatial Analysis of Phosphatidylinositol Molecular Species in Pork Chop Tissues Using Matrix-assisted Laser Desorption/ionization-Mass Spectrometry Imaging., Journal of Oleo Science, Vol.70, No.7, 979-987, 2021.
(要約)
Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a powerful technique for visualizing lipids in biological tissues. Phosphatidylinositol (PI), a phospholipid in pork, is a major source of inositol in animal-derived foods believed to be protective against diseases related to pregnancy and cancer. However, the distribution of PI molecular species in pork is not well understood. Here, we performed MALDI-MSI analysis to investigate the distribution and composition of PI molecular species in pork chop comprising Longissimus thoracis et lumborum muscle (loin), intermuscular fat tissue, transparent tissue, and spinalis muscle. Twelve diacyl-PI molecular species were identified using liquid chromatography-electrospray ionization-tandem mass spectrometry (MS/MS) and MALDI-MS/MS analysis and visualized using MALDI-MSI. Spinalis muscle had the highest amount of identified PI molecular species, followed by loin, transparent tissue, and intermuscular fat tissue. The diacyl-PI molecular species containing hexadecadienoic, oleic, linoleic and eicosadienoic acids at the sn-2 position were mainly abundant in the loin and spinalis muscle, whereas those containing mead, arachidonic, docosatetraenoic, and docosapentaenoic acids at the sn-2 position were mainly abundant in both muscles as well as transparent tissues. Notably, the balance of PI molecular species differed among the tissues depending on fatty acid compositions at the sn-2 position. These results suggested that MALDI-MSI is a promising tool for assessing the association between individual pork tissues and the protective effects of PI molecular species against diseases related to pregnancy and cancer. To the best of our knowledge, this is the first report showing tissue-specific distributions of PI molecular species in pork chop using MALDI-MSI.
Hirofumi Enomoto : Adhesive film applications help to prepare strawberry fruit sections for desorption electrospray ionization-mass spectrometry imaging., Bioscience, Biotechnology, and Biochemistry, Vol.85, No.6, 1341-1347, 2021.
(要約)
Desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) is a powerful tool to analyze the distribution of metabolites in biological tissues. Cryosectioning of biological tissues is usually required prior to DESI-MSI, but it can be difficult for tissues that are fragile, hard, and have a high-water content. The Kawamoto method uses transparent adhesive films to prepare cryosections; however, its application for plant tissues, such as strawberry tissues, in DESI-MSI has not been verified. In this study, strawberry cryosections maintained original structures were prepared using adhesive film. Subsequently, numerous peaks were detected for the sections using the positive and negative ion modes of DESI-MSI. Several primary and specialized metabolites, such as amino acids, sugars, organic acids, and flavonoids, were identified and visualized. These results suggest the use of adhesive films when cryosectioning could improve DESI-MSI analysis of the metabolites in strawberry fruits and various tissues of other plant species.
Hirofumi Enomoto and Koji Miyamoto : Unique localization of jasmonic acid-related compounds in developing Phaseolus vulgaris L. (common bean) seeds revealed through desorption electrospray ionization-mass spectrometry imaging., Phytochemistry, Vol.188, 2021.
(要約)
Jasmonic acid (JA) and its precursors are oxylipins derived from α-linolenic acid (αLA). Presumably, they are involved in the regulation of seed embryogenesis, dormancy, and germination. However, their spatial localization in the developing Phaseolus vulgaris L. (common bean) seeds has not been fully elucidated. Therefore, desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) was performed to investigate their localization in the developing seeds. Peaks corresponding to the chemical formulae of αLA and 3-oxo-2-(2-(Z)-pentenyl)-cyclopentane-1-octanoic acid (OPC-8:0) were localized mainly in the radicle and seed coat, while that of 12-oxo-phytodienoic acid (OPDA) in the seed coat. This was consistent with the quantitative results obtained using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis. In contrast, DESI-tandem MSI (MS/MSI) and LC-ESI-MS/MS analyses showed that the effects of isomers on the DESI-MSI ion images were small for αLA and OPDA, but not for OPC-8:0. This indicated that DESI-MSI could accurately visualize αLA and OPDA, while DESI-MS/MSI was necessary to visualize OPC-8:0. The results demonstrated that free αLA and OPC-8:0 were abundant in the radicle and seed coat, while free OPDA was accumulated in the seed coat. Interestingly, the localization pattern of OPDA was similar to that of JA. In addition, compared to the concentrations of OPDA, the concentration of OPC-8:0 was lower in the seed coat and higher in the radicle. These results suggest that OPDA and/or JA play a biological role mainly in the seed coat, while OPC-8:0 is biologically active mainly in the radicle. Therefore, DESI-MSI coupled with LC-ESI-MS is a useful tool for spatial analysis of JA-related compounds in developing common bean seeds.
(キーワード)
Cyclopentanes / Oxylipins / Phaseolus / Seeds / Spectrometry, Mass, Electrospray Ionization / Tandem Mass Spectrometry
Rena Fujishima, Hirona Kugo, Kenichi Yanagimoto, Hirofumi Enomoto, Tatsuya Moriyama and Nobuhiro Zaima : Similar distribution of orally administered eicosapentaenoic acid and M2 macrophage marker in the hypoperfusion-induced abdominal aortic aneurysm wall., Food & Function, Vol.12, No.8, 3469-3475, 2021.
(要約)
Abdominal aortic aneurysm (AAA) is an aortic disease in which the aortic diameter is 3.0 cm; if left untreated, the aortic wall continues to weaken, resulting in progressive dilatation. Effective therapeutic drugs for AAA patients have not been discovered. Eicosapentaenoic acid (EPA) reportedly attenuates the development of AAA in experimental AAA animal models. However, the underlying mechanism of action is still not totally clear. To understand the mechanism, we visualized the distribution of EPA-containing phosphatidylcholine (PC) in the AAA wall by matrix-assisted laser desorption ionization-mass spectrometry imaging. EPA-containing PC was characteristically distributed in the AAA wall, and the positive area for the M2 macrophage marker was significantly higher in the region where EPA-containing PC was highly detected (region 2) than in the region where EPA-containing PC was poorly detected (region 1). The M1 macrophage marker levels were not different between regions 1 and 2. A comparative observation showed a similar distribution of the M2 macrophage marker and EPA-containing PC. These data suggest the preferential incorporation of EPA into M2 macrophages. Positive areas for matrix metalloproteinase 2 and malondialdehyde in region 2 were significantly lower than those in region 1. The reported suppressive effect of EPA on the development of AAA may be partly attributed to the increased anti-inflammatory property of M2 macrophages.
Emi Yumoto, Masaki Sato, Tomoyoshi Kubota, Hirofumi Enomoto, Koji Miyamoto, Hisakazu Yamane and Jinichiro Koga : Direct LC-ESI-MS/MS analysis of plant glucosylceramide and ceramide species with 8E and 8Z isomers of the long chain base., Bioscience, Biotechnology, and Biochemistry, Vol.85, No.2, 205-210, 2021.
(要約)
Glucosylceramides and ceramides with 8E and 8Z isomers of the long chain base are found in plants. These isomers have been difficult to quantify separately using liquid chromatography-tandem mass spectrometry (LC-MS/MS) because the isomers have the same retention time, their precursor and product ions have the same m/z values, and plant ceramide standards are not commercially available. Here we tested trial separations using various ODS columns and prepared plant ceramide standards generated by human glucocerebrosidase (imiglucerase) using commercially available plant glucosylceramide standards as the substrates. Consequently, we were able to quantify the isomers based on differences in retention times in a TSKgel ODS-120A column (Tosoh, Tokyo Japan) using LC-electrospray ionization-MS/MS (LC-ESI-MS/MS).
Hirofumi Enomoto : Mass Spectrometry Imaging of Flavonols and Ellagic Acid Glycosides in Ripe Strawberry Fruit., Molecules, Vol.25, No.20, 2020.
(要約)
Flavonols and ellagic acid glycosides are major phenolic compounds in strawberry fruit. They have antioxidant activity, show protective functions against abiotic and biotic stress, and provide health benefits. However, their spatial distribution in ripe fruit has not been understood. Therefore, matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry imaging (MSI) was performed to investigate their distribution in fruit tissues. Using strawberry extract, five flavonols, namely, three kaempferols and two quercetins, and two ellagic acid glycosides, were tentatively identified by MALDI-tandem MS. To investigate the tentatively identified compounds, MALDI-MSI and tandem MS imaging (MS/MSI) analyses were performed. Kaempferol and quercetin glycosides showed similar distribution patterns. They were mainly found in the epidermis, while ellagic acid glycosides were mainly found in the achene and in the bottom area of the receptacle. These results suggested that the difference in distribution pattern between flavonols and ellagic acid glycosides depends on the difference between their aglycones. Seemingly, flavonols play a role in protective functions in the epidermis, while ellagic acid glycosides play a role in the achene and in the bottom side of the receptacle, respectively. These results demonstrated that MALDI-MSI is useful for distribution analysis of flavonols and ellagic acid glycosides in strawberry fruit.
Yoshihito Shinozaki, P Bertrand Beauvoit, Masaru Takahara, Shuhei Hao, Kentaro Ezura, Marie-Hélène Andrieu, Keiji Nishida, Kazuki Mori, Yutaka Suzuki, Satoshi Kuhara, Hirofumi Enomoto, Miyako Kusano, Atsushi Fukushima, Tetsuya Mori, Mikiko Kojima, Makoto Kobayashi, Hitoshi Sakakibara, Kazuki Saito, Yuya Ohtani, Camille Bénard, Duyen Prodhomme, Yves Gibon, Hiroshi Ezura and Tohru Ariizumi : Fruit setting rewires central metabolism via gibberellin cascades., Proceedings of the National Academy of Sciences of the United States of America, Vol.117, No.38, 23970-23981, 2020.
(要約)
, that coordinated metabolic activation. Furthermore, a kinetic model of sucrose metabolism predicted that the sucrose cycle had high activity levels in unpollinated ovaries, whereas it was shut down when sugars rapidly accumulated in vacuoles in fruit-setting ovaries, in a time-dependent manner via tonoplastic sugar carriers. Moreover, fruit set at least partly required the activity of fructokinase, which may pull fructose out of the vacuole, and this could feed the downstream pathways. Collectively, our results indicate that GA cascades enhance sink capacities, by up-regulating central metabolic enzyme capacities at both transcriptional and posttranscriptional levels. This leads to increased sucrose uptake and carbon fluxes for the production of the constituents of biomass and energy that are essential for rapid ovary growth during the initiation of fruit set.
Hirofumi Enomoto and Takashi Nirasawa : Localization of Flavan-3-ol Species in Peanut Testa by Mass Spectrometry Imaging., Molecules, Vol.25, No.10, 2020.
(要約)
ions were observed in the mass spectrum extracted from the outer epidermis of the peanut testa, using the region of interest function. Catechin and/or epicatechin, five A-type, and one B-type procyanidins were assigned by the fragment ions generated by retro-Diels-Alder, heterocyclic ring fission, and quinone methide reactions detected in MALDI-tandem MS spectra. These flavan-3-ols were localized in the outer epidermis of the peanut testa. This information will contribute to improving the extraction and purification efficiencies of flavan-3-ols from peanut testa. As flavan-3-ols display anti-microbial activity, it is speculated that flavan-3-ols present in the outer epidermis of peanut testa act to prevent pathogen infection.
Hirofumi Enomoto, Masahiro Kotani and Takayuki Ohmura : Novel Blotting Method for Mass Spectrometry Imaging of Metabolites in Strawberry Fruit by Desorption/Ionization Using Through Hole Alumina Membrane., Foods, Vol.9, No.4, 2020.
(要約)
Mass spectrometry imaging (MSI) using matrix-assisted laser desorption/ionization (MALDI) is a powerful technique for visualizing metabolites in the strawberry fruit. During sample preparation for MALDI-MSI, sectioning of the samples is usually required. In general, MALDI-MSI analysis of strawberry fruits that are larger than a single glass slide is difficult because thin sections cannot be prepared. In this study, we attempted to visualize metabolites in large strawberry fruits by MSI, employing a blotting method that uses desorption ionization using a through-hole alumina membrane (DIUTHAME) chip. Large strawberry fruits were cut and a DIUTHAME chip was set on the cross-section to blot the metabolites. After drying the DIUTHAME chip, the metabolites were measured in positive and negative ion modes using a commercial MALDI-type mass spectrometer. Several peaks were detected in both the ion modes. Various metabolites related to food quality, such as sugars, organic acids, and anthocyanins, were detected and successfully visualized by blotting on a DIUTHAME chip in MSI. These results suggest that blotting using a DIUTHAME chip in MSI is useful for visualizing the metabolites present in the strawberry fruit.
Hirofumi Enomoto, Tomohiro Furukawa, Shiro Takeda, Hajime Hatta and Nobuhiro Zaima : Unique Distribution of Diacyl-, Alkylacyl-, and Alkenylacyl-Phosphatidylcholine Species Visualized in Pork Chop Tissues by Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging., Foods, Vol.9, No.2, 2020.
(要約)
Phosphatidylcholine (PC) is the major phospholipid in meat and influences meat qualities, such as healthiness. PC is classified into three groups based on the bond at the position: Diacyl, alkylacyl, and alkenylacyl. To investigate their composition and distribution in pork tissues, including (loin) spinalis muscles, intermuscular fat, and transparent tissues, we performed matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI). Eleven diacyl-, seven alkylacyl-, and six alkenylacyl-PCs were identified using liquid chromatography (LC)-tandem MS (MS/MS) analysis. Despite many alkylacyl- and alkenylacyl-PC species sharing identical / values, we were able to visualize these PC species using MALDI-MSI. Diacyl- and alkylacyl- and/or alkenylacyl-PC species showed unique distribution patterns in the tissues, suggesting that their distribution patterns were dependent on their fatty acid compositions. PCs are a major dietary source of choline in meat, and the amount was significantly higher in the muscle tissues. Consumption of choline mitigates age-related memory decline and neurodegenerative diseases; therefore, the consumption of pork muscle tissues could help to mitigate these diseases. These results support the use of MALDI-MSI analysis for assessing the association between PC species and the quality parameters of meat.
Shiro Takeda, Akiko Harauma, Mariko Okamoto, Hirofumi Enomoto, Toshiji Kudo, Takehito Suzuki, Wataru Mizunoya and Toru Moriguchi : Effects of whey protein hydrolysate on growth promotion and immunomodulation in mouse pups in artificial rearing system., Animal Science Journal, Vol.91, No.1, 2020.
(要約)
This study aimed to investigate the effects of whey protein hydrolysate (WPH) on the growth and immunity of mouse pups in artificial rearing (AR) system. Mouse pups were reared in the AR system with artificial milk including 5% WPH (AR with WPH) or not (AR without WPH), and the remaining pups were reared by their mother (dam) for 14 days after birth. The body weight change and body weight gain rates in the AR with WPH group were significantly higher than those observed in the AR without WPH group and similar to those in the dam group. Moreover the feed and protein efficiencies in the AR with WPH group were significantly higher than those of the AR without WPH group. In addition, the supplement of WPH in the AR system was shown to significantly elevate the number of CD3
Hirofumi Enomoto, Senji Takahashi, Shiro Takeda and Hajime Hatta : Distribution of Flavan-3-ol Species in Ripe Strawberry Fruit Revealed by Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging., Molecules, Vol.25, No.1, 2019.
(要約)
ions were detected in the negative ion mode using 1,5-diaminonaphthalene as matrix. Catechin and/or epicatechin, three B-type procyanidins, and two B-type propelargonidins were identified by MALDI-tandem MS. These flavan-3-ols were mainly distributed in the calyx, in and around the vascular bundles, and in the skin. In-source fragmentation of proanthocyanidins was determined using their standards, suggesting their distribution was mixed ion images of themselves, and fragment ions generated from those had a higher degree of polymerization. B-type procyanidins were predominantly distributed in the vascular bundles than in the skin, whereas B-type propelargonidins were almost equally distributed between the vascular bundles and skin, suggesting that their distribution patterns are different from the type of their flavan-3-ol monomers. Flavan-3-ols, especially B-type procyanidins, may help prevent pathogen infection not only in the skin but also in and around the vascular bundles.
Hirofumi Enomoto, Shiro Takeda, Hajime Hatta and Nobuhiro Zaima : Tissue-Specific Distribution of Sphingomyelin Species in Pork Chop Revealed by Matrix-Assisted Laser Desorption/Ionization-Imaging Mass Spectrometry., Journal of Food Science, Vol.84, No.7, 1758-1763, 2019.
(要約)
Sphingomyelin (SM) species are major sphingolipids in pork meat that affect quality parameters, such as health benefits due to their protective properties against chronic diseases; however, their spatial distribution remains unclear. We used matrix-assisted laser desorption/ionization (MALDI)-imaging mass spectrometry (IMS) to investigate the distribution and composition of SM species in pork chop consisting of longissimus thoracis et lumborum muscle (loin), intermuscular fat tissue, transparent tissue, and spinalis muscle. Four SM species were identified by liquid chromatography-electrospray ionization-tandem MS (MS/MS) and MALDI-MS/MS and visualized using MALDI-IMS. SM species containing stearic acid were predominantly distributed in the loin and spinalis muscle, whereas SM species containing palmitic, lignoceric, and nervonic acids were predominantly distributed in transparent tissue. These results indicated that the distribution of SM species differed among the pork tissues, depending on the tissue-specific fatty acid composition. The total amount including all identified SM species was higher in the loin than in spinalis muscle. Pork is reportedly associated with increased risk for chronic diseases due to the high amount of heme iron. From the observation of color, the amount of heme iron was lower in loin than in spinalis muscle. Thus, the degree of risk for chronic diseases might be lower in the loin than in spinalis muscle. This is the first report on the tissue-specific distribution of SM species in meat at a microscopic resolution using IMS. MALDI-IMS analysis may be useful in assessing the association between SM species and quality parameters of pork meat. PRACTICAL APPLICATION: Sphingomyelin (SM) species are major sphingolipids in pork meat. SM species affect quality parameters such as health benefits due to their protective properties against colon cancer and atherosclerosis. Matrix-assisted laser desorption/ionization-imaging mass spectrometry analysis combined with liquid chromatography-electrospray ionization-tandem mass spectrometry is a suitable method to directly investigate the distribution and composition of SM species at microscopic level among different tissues of pork meat. Therefore, this method is useful to assess the SM species-induced health effect of different tissues of pork meat.
(キーワード)
Animals / Chromatography, Liquid / Meat / Muscles / Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Sphingomyelins / Swine / Tandem Mass Spectrometry / Tissue Distribution
Hirofumi Enomoto, Takuya Sensu, Emi Yumoto, Takao Yokota and Hisakazu Yamane : Derivatization for detection of abscisic acid and 12-oxo-phytodienoic acid using matrix-assisted laser desorption/ionization imaging mass spectrometry., Rapid Communications in Mass Spectrometry: RCM, Vol.32, No.17, 1565-1572, 2018.
(要約)
The optimal TFA concentration and spray volume were 2% and 500 μL, respectively. The OTCD efficiency rates were 61 ± 10% for ABA and 45 ± 5% for OPDA. The peaks corresponding to GirT-derivatized ABA (ABA-GirT) and OPDA (OPDA-GirT) standards were detected on the optimal OTCD-treated seed sections. ABA-GirT was mainly distributed in the embryo, while OPDA-GirT was localized in the external structures. These results are in agreement with our previously published results.
Hirofumi Enomoto, Kei Sato, Koji Miyamoto, Akira Ohtsuka and Hisakazu Yamane : Distribution Analysis of Anthocyanins, Sugars, and Organic Acids in Strawberry Fruits Using Matrix-Assisted Laser Desorption/Ionization-Imaging Mass Spectrometry., Journal of Agricultural and Food Chemistry, Vol.66, No.19, 4958-4965, 2018.
(要約)
Anthocyanins, sugars, and organic acids contribute to the appearance, health benefits, and taste of strawberries. However, their spatial distribution in the ripe fruit has been fully unrevealed. Therefore, we performed matrix-assisted laser desorption/ionization, MALDI-IMS, analysis to investigate their spatial distribution in ripe strawberries. The detection sensitivity was improved by using the TM-Sprayer for matrix application. In the receptacle, pelargonidins were distributed in the skin, cortical, and pith tissues, whereas cyanidins and delphinidins were slightly localized in the skin. In the achene, mainly cyanidins were localized in the outside of the skin. Citric acid was mainly distributed in the upper and bottom side of cortical tissue. Although hexose was distributed almost equally throughout the fruits, sucrose was mainly distributed in the upper side of cortical and pith tissues. These results suggest that using the TM-Sprayer in MALDI-IMS was useful for microscopic distribution analysis of anthocyanins, sugars, and organic acids in strawberries.
Chien-Hsiang Ni, Chih-Shang Lee, Fu-Yuan Chuan, Hirofumi Enomoto, Shiro Takeda, Liang-Chuan Lin and Ryoichi Sakata : Investigation of the Chemical Composition and Functional Proteins of Chicken Gizzard Inner Lining, Food Science and Technology Research, Vol.24, No.5, 893-901, 2018.
Hirofumi Enomoto, Takuya Sensu, Kei Sato, Futoshi Sato, Thanai Paxton, Emi Yumoto, Koji Miyamoto, Masashi Asahina, Takao Yokota and Hisakazu Yamane : Visualisation of abscisic acid and 12-oxo-phytodienoic acid in immature Phaseolus vulgaris L. seeds using desorption electrospray ionisation-imaging mass spectrometry., Scientific Reports, Vol.7, 2017.
(要約)
The plant hormone abscisic acid (ABA) and the jasmonic acid related-compound 12-oxo-phytodienoic acid (OPDA) play crucial roles in seed development, dormancy, and germination. However, a lack of suitable techniques for visualising plant hormones has restricted the investigation of their biological mechanisms. In the present study, desorption electrospray ionisation-imaging mass spectrometry (DESI-IMS), a powerful tool for visualising metabolites in biological tissues, was used to visualise ABA and OPDA in immature Phaseolus vulgaris L. seed sections. The mass spectra, peak values and chemical formulae obtained from the analysis of seed sections were consistent with those determined for ABA and OPDA standards, as were the precursor and major fragment ions observed in tandem mass spectrometry (MS/MS) imaging. Furthermore, the precursor and fragment ion images showed similar distribution patterns. In addition, the localisation of ABA and OPDA using DESI-IMS was confirmed using liquid chromatography-MS/MS (LC-MS/MS). The results indicated that ABA was mainly distributed in the radical and cotyledon of the embryo, whereas OPDA was distributed exclusively in external structures, such as the hilum and seed coat. The present study is the first to report the visualisation of plant hormones using IMS, and demonstrates that DESI-IMS is a promising technique for future plant hormone research.
Jinping Zhao, Hui Zhao, Xiaoyan Wang, Rong Huang, Hirofumi Enomoto, Zhikun He and Can-Peng Li : Characteristics and enhanced antioxidant activity of egg white protein selenized by dry-heating in the presence of selenite., Journal of Agricultural and Food Chemistry, Vol.61, No.12, 3131-3139, 2013.
(要約)
This study reports a new method for the selenization of food proteins. Egg white protein (EWP) was selenized by dry-heating in the presence of selenite, and the physiochemical and functional properties of the selenized EWP were investigated. Selenization was accelerated with a decrease in pH from 7.0 to 3.0, an increase in heating time from 1 to 5 days, and an increase in incubation temperatures from 25 to 80 °C. The electrophoretic mobility of EWP increased with an increase in the level of selenization. Heat-induced polymerization of EWP was promoted by dry-heating in the presence of selenite. The selenite group was acid-stable and base-labile, which suggests that it was bound to the hydroxyl group of an amino acid and formed an -O-SeHO2 linkage. (77)Se NMR spectral data also suggested that the selenite bond (-O-SeHO2) was bound to EWP. The digestibility of EWP was improved by selenization. The antioxidant activities of EWP, including ABTS(+) free radical scavenging capacity, hydroxyl radical scavenging capacity, reducing power, and the Fe(2+) chelating capacity, were remarkably enhanced by selenization. This finding is the first to describe that EWP can be selenized by dry-heating in the presence of selenite and that the antioxidant activities of EWP are markedly enhanced by selenization. This points to a potentially new method for generating antioxidant food protein and a new method for preparing organic Se.
(キーワード)
Antioxidants / Chemical Phenomena / Egg Proteins / Ferrous Compounds / Free Radical Scavengers / Hot Temperature / Hydrogen-Ion Concentration / Hydroxyl Radical / Iron Chelating Agents / Oxidation-Reduction / Selenious Acid / Selenium
Yukihiro Yoshimura, Hirofumi Enomoto, Tatsuya Moriyama, Yukio Kawamura, Mitsutoshi Setou and Nobuhiro Zaima : Visualization of anthocyanin species in rabbiteye blueberry Vaccinium ashei by matrix-assisted laser desorption/ionization imaging mass spectrometry., Analytical and Bioanalytical Chemistry, Vol.403, No.7, 1885-1895, 2012.
(要約)
Anthocyanins are naturally occurring compounds that impart color to fruits, vegetables, and plants, and are believed to have a number of beneficial health effects in both humans and animals. Because of these properties, pharmacokinetic analysis of anthocyanins in tissue has been performed to quantify and identify anthocyanin species although, currently, no methods exist for investigating tissue localization of anthocyanin species or for elucidating the mechanisms of anthocyanin activity. Imaging mass spectrometry (IMS) is powerful tool for determining and visualizing the distribution of a wide range of biomolecules. To investigate whether anthocyanin species could be identified and visualized by IMS, we performed matrix-assisted laser desorption/ionization (MALDI)-IMS analysis, by tandem mass spectrometry (MALDI-IMS-MS), of ten anthocyanin molecular species in rabbiteye blueberry (Vaccinium ashei). The distribution patterns of each anthocyanin species were different in the exocarp and endocarp of blueberry sections. Anthocyanin species composed of delphinidin and petunidin were localized mainly in the exocarp. In contrast, those species composed of cyanidin, peonidin, and malvidin were localized in both the exocarp and the endocarp. Moreover, MALDI-IMS analysis of anthocyanidins in a blueberry section indicated that the distribution patterns of each anthocyanidin species were nearly identical with those of the corresponding anthocyanins. These results suggested that the different distribution patterns of anthocyanin species in the exocarp and endocarp depended on the aglycone rather than on the sugar moieties. This study is the first to visualize anthocyanin molecular species in fruits.
(キーワード)
Anthocyanins / Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Tandem Mass Spectrometry / Vaccinium
The IMS-based histopathologic examination (IbHE) revealed the characteristic peaks of lipid-rich regions, SMCs, and calcified regions in the atherosclerotic lesions. In addition, IbHE revealed the characteristic distribution of lipids in human atherosclerotic lesions. These data indicate that an IMS-based pathologic approach is of considerable value as a new histopathologic examination.
Hirofumi Enomoto, Yuki Sugiura, Mitsutoshi Setou and Nobuhiro Zaima : Visualization of phosphatidylcholine, lysophosphatidylcholine and sphingomyelin in mouse tongue body by matrix-assisted laser desorption/ionization imaging mass spectrometry., Analytical and Bioanalytical Chemistry, Vol.400, No.7, 1913-1921, 2011.
(要約)
The mammalian tongue is one of the most important organs during food uptake because it is helpful for mastication and swallowing. In addition, taste receptors are present on the surface of the tongue. Lipids are the second most abundant biomolecules after water in the tongue. Lipids such as phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphingomyelin (SM) are considered to play fundamental roles in the mediation of cell signaling. Imaging mass spectrometry (IMS) is powerful tool for determining and visualizing the distribution of lipids across sections of dissected tissue. In this study, we identified and visualized the PC, LPC, and SM species in a mouse tongue body section with matrix-assisted laser desorption/ionization (MALDI)-IMS. The ion image constructed from the peaks revealed that docosahexaenoic acid (DHA)-containing PC, LPC, linoleic acid-containing PC and SM (d18:1/16:0), and oleic acid-containing PC were mainly distributed in muscle, connective tissue, stratified epithelium, and the peripheral nerve, respectively. Furthermore, the distribution of SM (d18:1/16:0) corresponded to the distribution of nerve tissue relating to taste in the stratified epithelium. This study represents the first visualization of PC, LPC and SM localization in the mouse tongue body.
Nobuhiro Zaima, Naoko Goto-Inoue, Takahiro Hayasaka, Hirofumi Enomoto and Mitsutoshi Setou : Authenticity assessment of beef origin by principal component analysis of matrix-assisted laser desorption/ionization mass spectrometric data., Analytical and Bioanalytical Chemistry, Vol.400, No.7, 1865-1871, 2011.
(要約)
It has become necessary to assess the authenticity of beef origin because of concerns regarding human health hazards. In this study, we used a metabolomic approach involving matrix-assisted laser desorption/ionization imaging mass spectrometry to assess the authenticity of beef origin. Highly accurate data were obtained for samples of extracted lipids from beef of different origin; the samples were grouped according to their origin. The analysis of extracted lipids in this study ended within 10 min, suggesting this approach can be used as a simple authenticity assessment before a definitive identification by isotope analysis.
(キーワード)
Animals / Cattle / Meat Products / メタボロミクス (metabolomics) / Principal Component Analysis / Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Tandem Mass Spectrometry
Hirofumi Enomoto, Ishimaru Takayuki, Li Can-Peng, Hayashi Yoko, Matsudomi Naotoshi and Aoki Takayoshi : Phosphorylation of ovalbumin by dry-heating in the presence of pyrophosphate : Effect of carbohydrate chain on the phosphorylation level and heat stability, Food Chemistry, Vol.122, No.3, 526-532, 2010.
Hirofumi Enomoto : Improvement of functional properties of whey soy protein phosphorylated by dry-heating in the presence of pyrophosphate, LWT-Food Science and Technology, Vol.43, 919-925, 2010.
Hirofumi Enomoto, Y Hayashi, P C Li, S Ohki, H Ohtomo, M Shiokawa and T Aoki : Glycation and phosphorylation of alpha-lactalbumin by dry heating: effect on protein structure and physiological functions., Journal of Dairy Science, Vol.92, No.7, 3057-3068, 2009.
(要約)
Alpha-lactalbumin (alpha-LA) was glycated with maltopentaose (MP) through the Maillard reaction (MP-alpha-LA) and subsequently phosphorylated by dry heating in the presence of pyrophosphate to investigate its structure and physiological functions. Glycation occurred effectively, and the sugar content of alpha-LA increased by approximately 22.3% through the Maillard reaction. The phosphorylation of MP-alpha-LA was enhanced with an increase in the dry-heating time from 1 to 5 d, and the phosphorous content of MP-alpha-LA increased by approximately 1.01% by dry heating at pH 4.0 and 85 degrees C for 5 d in the presence of pyrophosphate. The electrophoretic mobility of alpha-LA increased with an increase in the phosphorylation level. The circular dichroism spectra showed that the change in the secondary structure of the alpha-LA molecule by glycation and subsequent phosphorylation was slight. However, the Trp fluorescence intensity was increased by phosphorylation after glycation. In addition, the differential scanning calorimetry thermograms of alpha-LA showed that the denaturation temperature of MP-alpha-LA was decreased by phosphorylation. These results indicated that molten (partially unfolded) conformations of alpha-LA were formed by dry heating in the presence of pyrophosphate after glycation. The anti-alpha-LA antibody response was significantly reduced by glycation and subsequent phosphorylation. The suppressive effect of alpha-LA on the production of proinflammatory cytokines such as IL-6 and tumor necrosis factor-alpha from THP-1 cells after stimulation with lipopolysaccharide was significantly enhanced by glycation with MP and was further enhanced by phosphorylation after glycation. The Ca phosphate-solubilizing ability of alpha-LA was enhanced by phosphorylation. The apoptotic activity of alpha-LA was reduced by glycation and subsequent phosphorylation. These results suggest that phosphorylation by dry heating in the presence of pyrophosphate after glycation with MP through the Maillard reaction is a useful method for improvement of the physiological functions of alpha-LA.
Hirofumi Enomoto, C-P Li, K Morizane, R H Ibrahim, Y Sugimoto, S Ohki, H Ohtomo and T Aoki : Improvement of functional properties of bovine serum albumin through phosphorylation by dry-heating in the presence of pyrophosphate., Journal of Food Science, Vol.73, No.2, C84-91, 2008.
(要約)
Bovine serum albumin (BSA) was phosphorylated by 2 methods. One is dry-heating in the presence of pyrophosphate, and the other is conjugation with maltopentaose through the Maillard reaction and subsequent dry-heating in the presence of pyrophosphate. The phosphorus content of BSA was increased to approximately 0.45% by dry-heating at pH 4.0 and 85 degrees C for 5 d in the presence of pyrophosphate, and approximately 0.91% by glycation and subsequent phosphorylation. The circular dichroism spectra showed that the change of secondary structure in the BSA molecule by phosphorylation was mild. However, tryptophan fluorescence intensity of BSA decreased by phosphorylation. The differential scanning calorimetry thermograms of BSA showed a disappearing of the 1st peak and a lowering of the 2nd peak denaturation temperature by phosphorylation. These results indicated molten (partially unfolded) conformations of BSA formed by both phosphorylation methods. The functional properties of BSA such as heat stability and calcium phosphate solubilizing ability were improved by phosphorylation alone and further by phosphorylation after glycation. Transparent gels of BSA with relatively high water-holding capacity were obtained by phosphorylation alone, and the immunogenicity of BSA was reduced significantly by glycation and phosphorylation, respectively.
(キーワード)
Calorimetry, Differential Scanning / Chemical Phenomena / Chemistry, Physical / Circular Dichroism / Diphosphates / Glycosylation / Hot Temperature / Hydrogen-Ion Concentration / Maillard Reaction / Phosphorylation / Protein Denaturation / Protein Structure, Secondary / Serum Albumin, Bovine / Structure-Activity Relationship
Hirofumi Enomoto, Can-Peng Li, Kentaro Morizane, R Hisham Ibrahim, Yasushi Sugimoto, Shinichi Ohki, Hideo Ohtomo and Takayoshi Aoki : Glycation and phosphorylation of beta-lactoglobulin by dry-heating: effect on protein structure and some properties., Journal of Agricultural and Food Chemistry, Vol.55, No.6, 2392-2398, 2007.
(要約)
Beta-lactoglobulin (beta-Lg) was glycated with maltopentaose and subsequently phosphorylated by dry-heating in the presence of pyrophosphate to investigate the structural and functional properties of phosphorylated beta-Lg. The circular dichroism spectra showed that the change of the secondary structure in the beta-Lg molecule by glycation and subsequent phosphorylation was small. The differential scanning calorimetry thermograms of beta-Lg showed that the denaturation temperature of the most stable domain was only slightly affected, whereas the retinol-binding activity of beta-Lg was somewhat reduced by glycation and subsequent phosphorylation. These results indicated that the conformational changes of the beta-Lg molecule by glycation and subsequent phosphorylation were mild. The anti-beta-Lg antibody response was somewhat reduced by glycation, but significant changes were not observed by phosphorylation. Although the stability of beta-Lg against heat-induced insolubility was improved by glycation alone, it was further enhanced by phosphorylation. The calcium phosphate solubilizing ability of beta-Lg was enhanced by phosphorylation following glycation.
(キーワード)
Glycosylation / Hot Temperature / Hydrogen-Ion Concentration / Lactoglobulins / Phosphorus / Phosphorylation / Protein Denaturation / Protein Structure, Secondary / Structure-Activity Relationship
P C Li, Hirofumi Enomoto, S Ohki, H Ohtomo and T Aoki : Improvement of functional properties of whey protein isolate through glycation and phosphorylation by dry heating., Journal of Dairy Science, Vol.88, No.12, 4137-4145, 2005.
(要約)
Whey protein isolate (WPI) was glycated with maltopentaose (MP) through the Maillard reaction, and the MP-conjugated WPI (MP-WPI) was then phosphorylated by dry heating in the presence of pyrophosphate. Glycation occurred efficiently, and the sugar content of WPI increased approximately 19.9% through the Maillard reaction. The phosphorylation of MP-WPI was enhanced with an increase in the dry-heating time from 1 to 5 d, and the phosphorus content of WPI increased approximately 1.05% by dry heating at pH 4.0 and 85 degrees C for 5 d in the presence of pyrophosphate. The electrophoretic mobility of WPI increased with an increase in the phosphorylation level. The stability of WPI against heat-induced insolubility at pH 7.0 was improved by conjugation with MP alone, and further improved by phosphorylation. Although the emulsifying activity of WPI was barely affected by glycation and phosphorylation, the emulsifying stability of phosphorylated MP-WPI (5 d), was 2.2 times higher than that of MP-WPI. Gelling properties such as hardness, resiliency, and water-holding capacity of heat-induced WPI gel were markedly improved, and the gel was rendered transparent by phosphorylation. The calcium phosphate-solubilizing ability of WPI was enhanced by phosphorylation. These results suggested that phosphorylation by dry heating in the presence of pyrophosphate after conjugation with MP is a useful method for improving the functional properties of WPI.
Li Can-Peng, Hirofumi Enomoto, Hayashi Yoko, Zhao Hui and Aoki Takayoshi : Recent advances in phosphorylation of food proteins : A review, LWT-Food Science and Technology, Vol.43, No.9, 1295-1300, 2010.