Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Yoshihiro Izumi Last modified date:2021.10.28

Associate Professor / Research Center for Transomics Medicine / Medical Institute of Bioregulation


Papers
1. Yoichiro Kashiwagi, Shunsuke Aburaya, Naoyuki Sugiyama, Yuki Narukawa, Yuta Sakamoto, Masatomo Takahashi, Hayato Uemura, Rentaro Yamashita, Shotaro Tominaga, Satoko Hayashi, Takenori Nozaki, Satoru Yamada, Yoshihiro Izumi, Atsunori Kashiwagi, Takeshi Bamba, Yasushi Ishihama, Shinya Murakami*, Porphyromonas gingivalis induces entero-hepatic metabolic derangements with alteration of gut microbiota in a type 2 diabetes mouse model., Scientific Reports, 10.1038/s41598-021-97868-2, 11, 1, Article number 18398, 2021.09, Periodontal infection induces systemic inflammation; therefore, aggravating diabetes. Orally administered periodontal pathogens may directly alter the gut microbiota. We orally treated obese db/db diabetes mice using Porphyromonas gingivalis (Pg). We screened for Pg-specific peptides in the intestinal fecal specimens and examined whether Pg localization influenced the intestinal microbiota profile, in turn altering the levels of the gut metabolites. We evaluated whether the deterioration in fasting hyperglycemia was related to the changes in the intrahepatic glucose metabolism, using proteome and metabolome analyses. Oral Pg treatment aggravated both fasting and postprandial hyperglycemia (P < 0.05), with a significant (P < 0.01) increase in dental alveolar bone resorption. Pg-specific peptides were identified in fecal specimens following oral Pg treatment. The intestinal Pg profoundly altered the gut microbiome profiles at the phylum, family, and genus levels; Prevotella exhibited the largest increase in abundance. In addition, Pg-treatment significantly altered intestinal metabolite levels. Fasting hyperglycemia was associated with the increase in the levels of gluconeogenesis-related enzymes and metabolites without changes in the expression of proinflammatory cytokines and insulin resistance. Oral Pg administration induced gut microbiota changes, leading to entero-hepatic metabolic derangements, thus aggravating hyperglycemia in an obese type 2 diabetes mouse model..
2. Kazuki Ikeda, Masatomo Takahashi, Shunsuke Aburaya, Daiki Harada, Maki Ikeda, Yume Kitagawa, Yuki Soma, Yoshihiro Izumi, Takeshi Bamba, Mitsuhiro Furuse*, Produced β-hydroxybutyrate after β-hydroxy-β-methylbutyrate (HMB) administration may contribute HMB function in mice., Biochemistry and Biophysics Reports, 10.1016/j.bbrep.2021.101097, 27, Article number 101097, 2021.08, β-Hydroxy-β-methylbutyrate (HMB) is an intermediate in the metabolism of the branched-chain amino acid leucine. HMB has several demonstrated effects on skeletal muscle function, some of which are contradictory. In addition, the effect of exogenous HMB intake on the levels of intermediate metabolites is not known. Therefore, we investigated changes in HMB metabolites after oral HMB administration in mice. First, ICR mice were treated with either distilled water or HMB (0.215 g/10 mL/kg). Sampling was performed at 0, 1, 6, 12, and 24 h after administration. Next, ICR mice were given distilled water or HMB (0.215 g/10 mL/kg/d) for 10 d. Mice given HMB shown a significant increase in liver β-methylcrotonyl-CoA and increased β-hydroxybutyrate in plasma and the gastrocnemius muscle 1 h after HMB administration. Mice administered HMB for 10 d showed significantly decreased food intake and body weight; however, the relative weight of the gastrocnemius muscle was significantly increased. These results may be attributed to an increase in β-hydroxybutyrate resulting from exogenous HMB, since β-hydroxybutyrate inhibits food intake and suppresses skeletal muscle catabolism. In conclusion, β-hydroxybutyrate, a metabolite of HMB, was found to play an important role in the function of HMB..
3. Amandine Dispas*, Adrian Clarke, Alexandre Grand-Guillaume Perrenoud, Luca Gioacchino Losacco, Jean-Luc Veuthey, Quentin Gros, Jérémy Molineau, Angéline Noireau, Caroline West, Fabio Salafia, Mariosimone Zoccali, Luigi Mondello, Amber Guillen, Jenny Wang, Kelly Zhang, Philipp Jochems, Gesa Schad, Kosuke Nakajima, Shinnosuke Horie, Jan Joseph, Maria Kristina Parr, Pierre Billemont, Antoni Severino, Sonja Schneider, Edgar Naegele, Daniel Kutscher, Rick Wikfors, Regina Black, Lee Ingvaldson, Jimmy Oliveira Da Silva, Raffeal Bennett, Erik L Regalado, Thi Phuong Thuy Hoang, David Touboul, Yana Nikolova, Mariana Kamenova-Nacheva, Vladimir Dimitrov, Blair K Berger, Kevin A Schug, Solène Kerviel-Guillon, Fabien Mauge, Masatomo Takahashi, Yoshihiro Izumi, Takeshi Bamba, Florent Rouvière, Sabine Heinisch, Davy Guillarme, Philippe Hubert, Interlaboratory study of a supercritical fluid chromatography method for the determination of pharmaceutical impurities: evaluation of multi-systems reproducibility., Journal of Pharmaceutical and Biomedical Analysis, 10.1016/j.jpba.2021.114206, 203, Article number 114206, 2021.09, Modern supercritical fluid chromatography (SFC) is now a well-established technique, especially in the field of pharmaceutical analysis. We recently demonstrated the transferability and the reproducibility of a SFC-UV method for pharmaceutical impurities by means of an inter-laboratory study. However, as this study involved only one brand of SFC instrumentation (Waters®), the present study extends the purpose to multi-instrumentation evaluation. Specifically, three instrument types, namely Agilent®, Shimadzu®, and Waters®, were included through 21 laboratories (n = 7 for each instrument). First, method transfer was performed to assess the separation quality and to set up the specific instrument parameters of Agilent® and Shimadzu® instruments. Second, the inter-laboratory study was performed following a protocol defined by the sending lab. Analytical results were examined regarding consistencies within- and between-laboratories criteria. Afterwards, the method reproducibility was estimated taking into account variances in replicates, between-days and between-laboratories. Reproducibility variance was larger than that observed during the first study involving only one single type of instrumentation. Indeed, we clearly observed an 'instrument type' effect. Moreover, the reproducibility variance was larger when considering all instruments than each type separately which can be attributed to the variability induced by the instrument configuration. Nevertheless, repeatability and reproducibility variances were found to be similar than those described for LC methods; i.e. reproducibility as %RSD was around 15 %. These results highlighted the robustness and the power of modern analytical SFC technologies to deliver accurate results for pharmaceutical quality control analysis..
4. Yuki Soma, Masatomo Takahashi, Yuri Fujiwara, Tamaki Shinohara, Yoshihiro Izumi, Taizo Hanai*, Takeshi Bamba*, Design of synthetic quorum sensing achieving induction timing-independent signal stabilization for dynamic metabolic engineering of E. coli., ACS Synthetic Biology, 10.1021/acssynbio.1c00008, 10, 6, 1384-1393, 2021.06, Dynamic metabolic engineering that harnesses synthetic biological tools is a next-generation strategy for microbial chemical and fuel production. We previously reported a synthetic quorum sensing system combined with a metabolic toggle switch (QS-MTS) in E. coli. It autonomously redirected endogenous metabolic flux toward the synthetic metabolic pathway and improved biofuel production. However, its functions and effects on host metabolism were attenuated by induction timing delay. Here, we redesigned the QS-MTS to stabilize QS signaling efficiency and metabolic regulation. We performed a metabolome analysis to clarify the effects of QS-MTS redesign on host metabolism. We compared the contributions of conventional and redesigned QS-MTS to fed-batch fermentation. The redesigned QS-MTS was more conducive than the conventional QS-MTS to long-term processes such as fed-batch fermentation. Here, we present a circuit redesign for metabolic flux control based on dynamic characteristic evaluation and metabolome analysis..
5. Takeshi Hara, Gino V Baron, Kosuke Hata, Yoshihiro Izumi, Takeshi Bamba*, Gert Desme*, Performance of functionalized monolithic silica capillary columns with different mesopore sizes using radical polymerization of octadecyl methacrylate., Journal of Chromatography A, 10.1016/j.chroma.2021.462282, 1651, Article number 462282, 2021.08, We report on the possibility to enhance the phase ratio and retention factor in silica monoliths. According to pioneering work done by Núñez et al. [1], this enhancement is pursued by applying a stationary phase layer via radical polymerization with octadecyl methacrylate (ODM) as an alternative to the customary octadecylsilylation (C18-derivatization). The difference in band broadening, retention factor and separation selectivity between both approaches was compared. Different hydrothermal treatment temperatures for the column preparation were applied to produce monolithic silica structures with three different mesopore sizes (resp. 10, 13, and 16 nm, as determined by argon physisorption) while maintaining similar domain size (sum of through-pore and skeleton size). It has been found that the columns with the poly(octadecyl methacrylate)-phase (ODM columns) provided a 60 to 80% higher retention factor in methanol-water mixture compared to the octadecylsilylated (ODS) columns produced by starting from similar silica backbone structures. In acetonitrile-water mixture, the enhancement is smaller (15 to 30% times higher), yet significant. By adjusting the fabrication conditions (for both the preparation of the monolithic backbones and the surface functionalization), the achieved retention factors (up k = 4.89 for pentylbenzene in 80:20% (v/v) methanol/water) are obviously higher than obtained in the pioneering study on ODM monoliths of Núñez et al. [1], and column clogging could be completely avoided. In addition, also separation efficiencies were significantly higher than shown in Ref. [1], with plate heights as low as 5.8 μm. These plate heights are however inferior to those observed on the ODS-modified sister columns. The difference can be explained by the slower intra-skeleton diffusion displayed by the ODM-modified columns, in turn caused by the larger obstruction to diffusion originating from the thicker stationary phase layer..
6. Takahiro Onoki, Yoshihiro Izumi, Masatomo Takahashi, Shohei Murakami, Daisuke Matsumaru, Nao Ohta, Sisca Meida Wati, Nozomi Hatanaka, Fumiki Katsuoka, Mitsuharu Okutsu, Yutaka Yabe, Yoshihiro Hagiwara, Makoto Kanzaki, Takeshi Bamba, Eiji Itoi, Hozumi Motohashi*, Skeletal muscle-specific Keap1 disruption modulates fatty acid utilization and enhances exercise capacity in female mice, Redox Biology, 10.1016/j.redox.2021.101966, 43, Article number 101966, 2021.07, Skeletal muscle health is important for the prevention of various age-related diseases. The loss of skeletal muscle mass, which is known as sarcopenia, underlies physical disability, poor quality of life and chronic diseases in elderly people. The transcription factor NRF2 plays important roles in the regulation of the cellular defense against oxidative stress, as well as the metabolism and mitochondrial activity. To determine the contribution of skeletal muscle NRF2 to exercise capacity, we conducted skeletal muscle-specific inhibition of KEAP1, which is a negative regulator of NRF2, and examined the cell-autonomous and non-cell-autonomous effects of NRF2 pathway activation in skeletal muscles. We found that NRF2 activation in skeletal muscles increased slow oxidative muscle fiber type and improved exercise endurance capacity in female mice. We also observed that female mice with NRF2 pathway activation in their skeletal muscles exhibited enhanced exercise-induced mobilization and β-oxidation of fatty acids. These results indicate that NRF2 activation in skeletal muscles promotes communication with adipose tissues via humoral and/or neuronal signaling and facilitates the utilization of fatty acids as an energy source, resulting in increased mitochondrial activity and efficient energy production during exercise, which leads to improved exercise endurance..
7. Yan-Yu Chen, Yuki Soma, Masahito Ishikawa, Masatomo Takahashi, Yoshihiro Izumi, Takeshi Bamba, Katsutoshi Hori*, Metabolic alteration of Methylococcus capsulatus str. Bath during a microbial gas-phase reaction, Bioresource Technology, 10.1016/j.biortech.2021.125002, 330, Article number 125002, 2021.06, This study demonstrates the metabolic alteration of Methylococcus capsulatus (Bath), a representative bacterium among methanotrophs, in microbial gas-phase reactions. For comparative metabolome analysis, a bioreactor was designed to be capable of supplying gaseous substrates and liquid nutrients continuously. Methane degradation by M. capsulatus (Bath) was more efficient in a gas-phase reaction operated in the bioreactor than in an aqueous phase reaction operated in a batch reactor. Metabolome analysis revealed remarkable alterations in the metabolism of cells in the gas-phase reaction; in particular, pyruvate, 2-ketoglutarate, some amino acids, xanthine, and hypoxanthine were accumulated, whereas 2,6-diaminopimelate was decreased. Based on the results of metabolome analysis, cells in the gas-phase reaction seemed to alter their metabolism to reduce the excess ATP and NADH generated upon increased availability of methane and oxygen. Our findings will facilitate the development of efficient processes for methane-based bioproduction with low energy consumption..
8. Kosuke Hata, Yuki Soma, Toshiyuki Yamashita, Masatomo Takahashi, Kuniyo Sugitate, Takeshi Serino, Hiromi Miyagawa, Kenichi Suzuki, Kayoko Yamada, Takatomo Kawamukai, Teruhisa Shiota, Yoshihiro Izumi*, Takeshi Bamba*, Calibration-curve-locking database for semi-quantitative metabolomics by gas chromatography/mass spectrometry, Metabolites, 10.3390/metabo11040207, 11, 4, Article number 207, 2021.03, Calibration-Curve-Locking Databases (CCLDs) have been constructed for automatic compound search and semi-quantitative screening by gas chromatography/mass spectrometry (GC/MS) in several fields. CCLD felicitates the semi-quantification of target compounds without calibration curve preparation because it contains the retention time (RT), calibration curves, and electron ionization (EI) mass spectra, which are obtained under stable apparatus conditions. Despite its usefulness, there is no CCLD for metabolomics. Herein, we developed a novel CCLD and semi-quantification framework for GC/MS-based metabolomics. All analytes were subjected to GC/MS after derivatization under stable apparatus conditions using (1) target tuning, (2) RT locking technique, and (3) automatic derivatization and injection by a robotic platform. The RTs and EI mass spectra were obtained from an existing authorized database. A quantifier ion and one or two qualifier ions were selected for each target metabolite. The calibration curves were obtained as plots of the peak area ratio of the target compounds to an internal standard versus the target compound concentration. These data were registered in a database as a novel CCLD. We examined the applicability of CCLD for analyzing human plasma, resulting in time-saving and labor-saving semi-qualitative screening without the need for standard substances..
9. Takuya Morikawa, Hiroaki Ohishi, Kengo Kosaka, Tomofumi Shimojo, Akihiro Nagano, Itsuki Taniguchi, Ryuta Fujioka, Kosei Moriyama, Motoko Unoki, Masatomo Takahashi, Motonao Nakao, Yoshihiro Izumi, Takeshi Bamba, Hiroyuki Sasaki, Shiroh Miura, Hiroki Shibata*, Ddhd1 knockout mouse as a model of locomotive and physiological abnormality in familial spastic paraplegia, Bioscience Reports, 10.1042/BSR20204171, 41, 2, Article number BSR20204171, 2021.02, We have previously reported a novel homozygous 4-bp deletion in DDHD1 as the responsible variant for spastic paraplegia type 28 (SPG28; OMIM#609340). The variant causes a frameshift, resulting in a functionally null allele in the patient. DDHD1 encodes phospholipase A1 (PLA1) catalyzing phosphatidylinositol to lysophosphatidylinositol (LPI). To clarify the pathogenic mechanism of SPG28, we established Ddhd1 knockout mice (Ddhd1[−/−]) carrying a 5-bp deletion in Ddhd1, resulting in a premature termination of translation at a position similar to that of the patient. We observed a significant decrease in foot–base angle (FBA) in aged Ddhd1(−/−) (24 months of age) and a significant decrease in LPI 20:4 (sn-2) in Ddhd1(−/−) cerebra (26 months of age). These changes in FBA were not observed in 14 months of age. We also observed significant changes of expression levels of 22 genes in the Ddhd1(−/−) cerebra (26 months of age). Gene Ontology (GO) terms relating to the nervous system and cell–cell communications were significantly enriched. We conclude that the reduced signaling of LPI 20:4 (sn-2) by PLA1 dysfunction is responsible for the locomotive abnormality in SPG28, further suggesting that the reduction of downstream signaling such as GPR55 which is agonized by LPI is involved in the pathogenesis of SPG28..
10. Kazuhiro Tanaka*, Takashi Sasayama, Hiroaki Nagashima, Yasuhiro Irino, Masatomo Takahashi, Yoshihiro Izumi, Takiko Uno, Naoko Satoh, Akane Kitta, Katsusuke Kyotani, Yuichi Fujita, Mitsuru Hashiguchi, Tomoaki Nakai, Masaaki Kohta, Yoichi Uozumi, Masakazu Shinohara, Kohkichi Hosoda, Takeshi Bamba, Eiji Kohmura, Glioma cells require one-carbon metabolism to survive glutamine starvation, Acta Neuropathologica Communications, 10.1186/s40478-020-01114-1, 9, Article number 16, 2021.01, Cancer cells optimize nutrient utilization to supply energetic and biosynthetic pathways. This metabolic process also includes redox maintenance and epigenetic regulation through nucleic acid and protein methylation, which enhance tumorigenicity and clinical resistance. However, less is known about how cancer cells exhibit metabolic flexibility to sustain cell growth and survival from nutrient starvation. Here, we find that serine and glycine levels were higher in low-nutrient regions of tumors in glioblastoma multiforme (GBM) patients than they were in other regions. Metabolic and functional studies in GBM cells demonstrated that serine availability and one-carbon metabolism support glioma cell survival following glutamine deprivation. Serine synthesis was mediated through autophagy rather than glycolysis. Gene expression analysis identified upregulation of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) to regulate one-carbon metabolism. In clinical samples, MTHFD2 expression was highest in the nutrient-poor areas around “pseudopalisading necrosis.” Genetic suppression of MTHFD2 and autophagy inhibition caused tumor cell death and growth inhibition of glioma cells upon glutamine deprivation. These results highlight a critical role for serine-dependent one-carbon metabolism in surviving glutamine starvation and suggest new therapeutic targets for glioma cells adapting to a low-nutrient microenvironment..
11. Masahiro Nagata, Kenji Toyonaga, Eri Ishikawa, Shojiro Haji, Nobuyuki Okahashi, Masatomo Takahashi, Yoshihiro Izumi, Akihiro Imamura, Koichi Takato, Hideharu Ishida, Shigenori Nagai, Petr Illarionov, Bridget L Stocker, Mattie S M Timmer, Dylan G M Smith, Spencer J Williams, Takeshi Bamba, Tomofumi Miyamoto, Makoto Arita, Ben J Appelmelk, Sho Yamasaki*, Helicobacter metabolites exacerbate gastritis through C-type lectin receptors, Journal of Experimental Medicine, org/10.1084/jem.20200815, 218, 1, e20200815, 2020.10, Helicobacter pylori causes gastritis, which has been attributed to the development of H. pylori–specific T cells during infection. However, the mechanism underlying innate immune detection leading to the priming of T cells is not fully understood, as H. pylori evades TLR detection. Here, we report that H. pylori metabolites modified from host cholesterol exacerbate gastritis through the interaction with C-type lectin receptors. Cholesteryl acyl α-glucoside (αCAG) and cholesteryl phosphatidyl α-glucoside (αCPG) were identified as noncanonical ligands for Mincle (Clec4e) and DCAR (Clec4b1). During chronic infection, H. pylori–specific T cell responses and gastritis were ameliorated in Mincle-deficient mice, although bacterial burdens remained unchanged. Furthermore, a mutant H. pylori strain lacking αCAG and αCPG exhibited an impaired ability to cause gastritis. Thus H. pylori–specific modification of host cholesterol plays a pathophysiological role that exacerbates gastric
inflammation by triggering C-type lectin receptors..
12. Tatsuya Fushimi, Yoshihiro Izumi*, Masatomo Takahashi, Kosuke Hata, Yoshihiro Murano, and Takeshi Bamba, Dynamic metabolome analysis reveals the metabolic fate of medium-chain fatty acid in AML12 cells, Journal of Agricultural and Food Chemistry, org/10.1021/acs.jafc.0c04723, 68, 43, 11997-12010, 2020.10, Several studies in hepatocyte cell lines reported that medium-chain fatty acids (MCFAs) with 6–12 carbons showed different metabolic properties from long-chain fatty acids (LCFAs). However, these studies reported unclear effects of different fatty acid molecules on hepatocyte metabolism. This study is aimed to capture the metabolic kinetics of MCFA assimilation in AML12 cells treated with octanoic acid (FA 8:0), decanoic acid (FA 10:0), or lauric acid (FA12:0) [LCFA; oleic acid (FA 18:1)] via metabolic profiling and dynamic metabolome analysis with 13C-labeling. The concentrations of total ketone bodies in the media of cells treated with FA 8:0 or FA 10:0 were 3.22- or 3.69-fold higher than those obtained with FA 18:1 treatment, respectively. FA 12:0 treatment did not significantly increase ketone body levels compared to DMSO treatment (control), whereas FA 12:0 treatment increased intracellular triacylglycerol (TG) levels 15.4 times compared to the control. Metabolic profiles of FA 12:0-treated samples differed from those of the FA 8:0-treated and FA 10:0-treated samples, suggesting that metabolic assimilation of MCFAs differed significantly depending on the MCFA type. Furthermore, the dynamic metabolome analysis clearly revealed that FA 8:0 was rapidly and quantitatively oxidized to acetyl-CoA and assimilated into ketone bodies, citrate cycle intermediates, and glucogenic amino acids but not readily into TGs..
13. Yutaka Konya, Yoshihiro Izumi, Takeshi Bamba*, Development of a novel method for polar metabolite profiling by supercritical fluid chromatography/tandem mass spectrometry, Journal of Chromatography A, 10.1016/j.chroma.2020.461587, 1632, Article number 461587, 2020.09, Supercritical carbon dioxide (scCO2), the main fluid in the mobile phase for supercritical fluid chromatography (SFC), is non-polar. The majority of polar compounds are little soluble in scCO2, thereby rendering them poor candidates for achieving separation by carbon dioxide-based SFC. There is no reported method for the comprehensive analysis of hydrophilic metabolites by SFC with mobile phases comprising a high CO2 ratio. In this study, we investigated the effect of additives in the modifier for enabling the application of SFC to profile diverse polar compounds for metabolomics. Eleven types of columns were screened by using proteinogenic amino acids as the model compounds. The addition of water and acids (formic acid and trifluoroacetic acid (TFA)) to the modifier was also investigated to improve the solubility of the polar compounds and mitigate the unfavorable interaction between the stationary phase and the polar compounds. A significant improvement in the peak shapes of the amino acids was observed upon addition of TFA. The CO2/modifier ratio and TFA concentration in the mobile phases were investigated using the CROWNPAK CR-I (+) column, which showed the best performance during the column-screening. The CO2/methanol/water/TFA ratio of 70/27/3/0.15 (v/v/v/v) was determined as the optimized mobile phase composition. Furthermore, the applicability of the optimized analytical method to other polar compounds was examined; 100 cationic and amphoteric compounds with predicted logPow values that ranged from –5.9 to 1.7 could be simultaneously analyzed without derivatization. Anionic compounds such as organic acids, phosphates, and sugars were excluded from the target analytes. Most of the previously reported SFC methods for analyzing polar compounds employ a gradient elution and require the use of high modifier ratios at 40% or more. In the proposed method, the use of water and TFA enabled the rapid and simultaneous analysis under isocratic elution within 10 min, even with a high CO2 ratio of 70%. Additionally, a rat serum extract was analyzed using the optimized conditions, and 43 polar metabolites were successfully detected. This result demonstrates the applicability of the SFC/tandem mass spectrometry method to real samples..
14. Takeshi Yamamoto, Yoshitsugu Takabatake*, Satoshi Minami, Shinsuke Sakai, Ryuta Fujimura, Atsushi Takahashi, Tomoko Namba-Hamano, Jun Matsuda, Tomonori Kimura, Isao Matsui, Jun Ya Kaimori, Hiroaki Takeda, Masatomo Takahashi, Yoshihiro Izumi, Takeshi Bamba, Taiji Matsusaka, Fumio Niimura, Motoko Yanagita, Yoshitaka Isaka, Eicosapentaenoic acid attenuates renal lipotoxicity by restoring autophagic flux, Autophagy, 10.1080/15548627.2020.1782034, 2020.06, Recently, we identified a novel mechanism of lipotoxicity in the kidney proximal tubular cells (PTECs); lipid overload stimulates macroautophagy/autophagy for the renovation of plasma and organelle membranes to maintain the integrity of the PTECs. However, this autophagic activation places a burden on the lysosomal system, leading to a downstream suppression of autophagy, which manifests as phospholipid accumulation and inadequate acidification in lysosomes. Here, we investigated whether pharmacological correction by eicosapentaenoic acid (EPA) supplementation could restore autophagic flux and alleviate renal lipotoxicity. EPA supplementation to high-fat diet (HFD)-fed mice reduced several hallmarks of lipotoxicity in the PTECs, such as phospholipid accumulation in the lysosome, mitochondrial dysfunction, inflammation, and fibrosis. In addition to improving the metabolic syndrome, EPA alleviated renal lipotoxicity via several mechanisms. EPA supplementation to HFD-fed mice or the isolated PTECs cultured in palmitic acid (PA) restored lysosomal function with significant improvements in the autophagic flux. The PA-induced redistribution of phospholipids from cellular membranes into lysosomes and the HFD-induced accumulation of SQSTM1/p62 (sequestosome 1), an autophagy substrate, during the temporal and genetic ablation of autophagy were significantly reduced by EPA, indicating that EPA attenuated the HFD-mediated increases in autophagy demand. Moreover, a fatty acid pulse-chase assay revealed that EPA promoted lipid droplet (LD) formation and transfer from LDs to the mitochondria for beta-oxidation. Noteworthy, the efficacy of EPA on lipotoxicity is autophagy-dependent and cell-intrinsic. In conclusion, EPA counteracts lipotoxicity in the proximal tubule by alleviating autophagic numbness, making it potentially suitable as a novel treatment for obesity-related kidney diseases. Abbreviations:F: 4-HNE: 4-hydroxy-2-nonenal; ACTB: actin beta; ADGRE1/F4/80: adhesion G protein-coupled receptor E1; ATG: autophagy-related; ATP: adenosine triphosphate; BODIPY: boron-dipyrromethene; BSA: bovine serum albumin; cKO: conditional knockout; CML: N-carboxymethyllysine; COL1A1: collagen type I alpha 1 chain; COX: cytochrome c oxidase; CTRL: control; DGAT: diacylglycerol O-acyltransferase; EPA: eicosapentaenoic acid; FA: fatty acid; FFA: free fatty acid; GFP: green fluorescent protein; HFD: high-fat diet; iKO: inducible knockout; IRI: ischemia-reperfusion injury; LAMP1: lysosomal-associated membrane protein 1; LD: lipid droplet; LRP2: low density lipoprotein receptor-related protein 2; MAP1LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; OA: oleic acid; PAS: periodic-acid Schiff; PPAR: peroxisome proliferator activated receptor; PPARGC1/PGC1: peroxisome proliferator activated receptor, gamma, coactivator 1; PTEC: proximal tubular epithelial cell; ROS: reactive oxygen species; RPS6: ribosomal protein S6; SDH: succinate dehydrogenase complex; SFC/MS/MS: supercritical fluid chromatography triple quadrupole mass spectrometry; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TG: triglyceride; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling..
15. Yuta Matsuoka, Yoshihiro Izumi, Masatomo Takahashi, Takeshi Bamba, Ken Ichi Yamada*, Method for Structural Determination of Lipid-Derived Radicals, Analytical chemistry, 10.1021/acs.analchem.0c00053, 92, 10, 6993-7002, 2020.05, Diversified oxidized-lipid molecules are responsible for inflammation and cell death, including ferroptosis. Lipid radicals are the source of these oxidized lipids, which are the initial key molecules in the lipid peroxidation chain reaction. However, owing to their extremely high reactivity and short half-life, an established detection technique is not available. Here, we propose a high-performance liquid chromatography fluorometry and high-resolution tandem mass spectrometry system combined with a fluorescent probe as a structural analysis method for lipid-derived radicals. We detected 132 lipid-derived radicals, including 111 new species, from five polyunsaturated fatty acids. In addition, a database was constructed for which the initial fatty acid could be determined using the radical structure. Further, 12 endogenous lipid-derived radicals were identified in carcinogen-induced liver cancer mouse models. Therefore, this method and its corresponding database will provide novel insights into mechanisms underlying the lipid peroxidation, including the associated inflammation and ferroptosis..
16. Takeshi Hara, Yoshihiro Izumi, Kosuke Hata, V. Gino Baron, Takeshi Bamba, Gert Desmet*, Performance of small-domain monolithic silica columns in nano-liquid chromatography and comparison with commercial packed bed columns with 2 µm particles, Journal of Chromatography A, 10.1016/j.chroma.2019.460804, 1616, Article number 460804, 2020.04, We report on a direct comparison of the separation performance in capillary nano-LC between commercial packed bed columns and the small-domain silica monoliths in applications. Octadecylsilylated monolithic silica capillary columns with a 50 and 100 µm inner diameter (i.d.) were prepared with a procedure providing domain sizes in the sub-2 µm range. The fabricated monolith columns could provide plate heights (H) of 4.0‒4.2 µm for hexylbenzene (retention factor (k) = 3.6) at an optimal linear velocity range under an isocratic condition, while showing a column permeability (Kv0 = 1.6‒1.8 × 10−14 m2) comparable to that of a column packed with 3‒3.5 µm particles. When the peak capacity (np) for a cytochrome C digest was compared for variable gradient times (tG = 15, 30, 60, and 120 min) and constant gradient steepness (b’), the present monolith columns could show a 30‒40% higher np-value than the packed capillary column with 2 µm particles (e.g. np = 180 versus np = 259 at tG = 30 min). The produced monolith columns showed a high chromatographic repeatability for both isocratic and gradient elution (e.g. relative standard deviation (n = 3, RSD (%)) = 0.5% for H, 2,6% for k, and 5.6% for Kv0 in the isocratic mode using the 100 µm i.d.-columns). The present results show that the domain sizes which can now be achieved for capillary silica monoliths are sufficiently small to result in separation efficiencies that can successfully compete with the commercial packed bed columns available for use in nano-LC applications..
17. Manabu Kodama, Kiyotaka Oshikawa, Hideyuki Shimizu, Susumu Yoshioka, Masatomo Takahashi, Yoshihiro Izumi, Takeshi Bamba, Chisa Tateishi, Takeshi Tomonaga, Masaki Matsumoto*, Keiichi I. Nakayama*, A shift in glutamine nitrogen metabolism contributes to the malignant progression of cancer, Nature communications, 10.1038/s41467-020-15136-9, 11, Article number 1320, 2020.03, Glucose metabolism is remodeled in cancer, but the global pattern of cancer-specific metabolic changes remains unclear. Here we show, using the comprehensive measurement of metabolic enzymes by large-scale targeted proteomics, that the metabolism both carbon and nitrogen is altered during the malignant progression of cancer. The fate of glutamine nitrogen is shifted from the anaplerotic pathway into the TCA cycle to nucleotide biosynthesis, with this shift being controlled by glutaminase (GLS1) and phosphoribosyl pyrophosphate amidotransferase (PPAT). Interventions to reduce the PPAT/GLS1 ratio suppresses tumor growth of many types of cancer. A meta-analysis reveals that PPAT shows the strongest correlation with malignancy among all metabolic enzymes, in particular in neuroendocrine cancer including small cell lung cancer (SCLC). PPAT depletion suppresses the growth of SCLC lines. A shift in glutamine fate may thus be required for malignant progression of cancer, with modulation of nitrogen metabolism being a potential approach to SCLC treatment..
18. Toshiaki Yoshioka, Yoshihiro Izumi*, Masatomo Takahashi, Koji Suzuki, Yasuhisa Miyamoto, Yasushi Nagatomi, Takeshi Bamba*, Identification of Acrylamide Adducts Generated during Storage of Canned Milk Coffee, Journal of Agricultural and Food Chemistry, 10.1021/acs.jafc.9b08139, 68, 12, 3859-3867, 2020.03, Since coffee is a significant contributor to the consumption of acrylamide, its reduction is required. Acrylamide is produced during the roasting of coffee beans, but the roasting process is an essential step in determining the taste of coffee. Acrylamide content in coffee has been suggested to decrease by reacting with proteins and/or other substances during storage, but details are unknown. Investigation of acrylamide adducts may contribute to a strategy for acrylamide reduction in coffee. In this study, a stable isotope labeling technique, combined with high-resolution mass spectrometry, allows the identification of acrylamide adducts (3-hydroxypyridine-acrylamide and pyridine-acrylamide) in canned milk coffee. Other acrylamide adducts derived from milk coffee proteins, Lys-acrylic acid and CysSO2-acrylic acid, were identified. During a 4-month storage period, the formation of these four adducts was found to reduce the total content of acrylamide by 75.3% in canned milk coffee. Therefore, endogenous proteins can be used in acrylamide reduction..
19. Hiroaki Takeda, Yoshihiro Izumi*, Shohei Tamura, Tomonari Koike, Yui Koike, Masashi Shiomi, Takeshi Bamba, Lipid Profiling of Serum and Lipoprotein Fractions in Response to Pitavastatin Using an Animal Model of Familial Hypercholesterolemia, Journal of Proteome Research, 10.1021/acs.jproteome.9b00602, 19, 3, 1100-1108, 2020.03, Statins are widely used for the treatment of atherosclerotic cardiovascular diseases. They inhibit cholesterol biosynthesis in the liver and cause pleiotropic effects, including anti-inflammatory and antioxidant effects. To develop novel therapeutic drugs, the effect of blood-borne lipid molecules on the pleiotropic effects of statins must be elucidated. Myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits, an animal model for hypercholesterolemia, are suitable for the determination of lipid molecules in the blood in response to statins because their lipoprotein metabolism is similar to that of humans. Herein, lipid molecules were investigated by lipidome analysis in response to pitavastatin using WHHLMI rabbits. Various lipid molecules in the blood were measured using a supercritical fluid chromatography triple quadrupole mass spectrometry. Cholesterol and cholesterol ester blood concentrations decreased by reducing the secretion of very low density lipoproteins from the liver. Independent of the inhibition effects of cholesterol biosynthesis, the concentrations of some lipids with anti-inflammation and antioxidant effects (phospholipid molecules with n-6 fatty acid side chains, lysophosphatidylcholines, phosphatidylethanolamine plasmalogens, and ceramide molecules) were significantly altered. These findings may lead to further investigation of the mechanism of statin action..
20. Kosuke Hata, Yoshihiro Izumi*, Takeshi Hara, Masaki Matsumoto*, Takeshi Bamba, In-Line Sample Processing System with an Immobilized Trypsin-Packed Fused-Silica Capillary Tube for the Proteomic Analysis of a Small Number of Mammalian Cells, Analytical chemistry, 10.1021/acs.analchem.9b03993, 92, 4, 2997-3005, 2020.02, Omics analysis at single-cell resolution has helped to demonstrate the shaping of cellular heterogeneity on the basis of the expression of various molecules. However, in-depth proteomic analysis of low-quantity samples has remained challenging because of difficulties associated with the measurement of large numbers of proteins by shotgun proteomics using nanoflow liquid chromatography tandem mass spectrometry (nano-LC/MS/MS). To meet such a demand, we developed a method called in-line sample preparation for efficient cellular proteomics (ISPEC) in which cells were captured, directly lysed, and digested with immobilized trypsin within fused-silica capillaries. ISPEC minimized sample loss during the sample preparation processes with a relatively small number of mammalian cells (<1000 cells) and improved the stability and efficiency of digestion by immobilized trypsin, compared to a conventional preparation method. Using our optimized ISPEC method with nano-LC/MS/MS analysis, we identified 1351, 351, and 60 proteins from 100 cells, 10 cells, and single cells, respectively. The linear response of the signal intensity of each peptide to the introduced cell number indicates the quantitative recovery of the proteome from a very small number of cells. Thus, our ISPEC strategy facilitates quantitative proteomic analysis of small cell populations..
21. Nao Nishida-Aoki, Yoshihiro Izumi*, Hiroaki Takeda, Masatomo Takahashi, Takahiro Ochiya*, Takeshi Bamba, Lipidomic analysis of cells and extracellular vesicles from high-and low-metastatic triple-negative breast cancer, Metabolites, 10.3390/metabo10020067, 10, 2, Article number: 67, 2020.02, Extracellular vesicles (EVs) are lipid bilayer nanovesicles secreted from almost all cells including cancer. Cancer-derived EVs contribute to cancer progression and malignancy via educating the surrounding normal cells. In breast cancer, epidemiological and experimental observations indicated that lipids are associated with cancer malignancy. However, lipid compositions of breast cancer EVs and their contributions to cancer progression are unexplored. In this study, we performed a widely targeted quantitative lipidomic analysis in cells and EVs derived from high-and low-metastatic triple-negative breast cancer cell lines, using supercritical fluid chromatography fast-scanning triple-quadrupole mass spectrometry. We demonstrated the differential lipid compositions between EVs and cells of their origin, and between high-and low-metastatic cell lines. Further, we demonstrated EVs from highly metastatic breast cancer accumulated unsaturated diacylglycerols (DGs) compared with EVs from lower-metastatic cells, without increasing the amount in cells. The EVs enriched with DGs could activate the protein kinase D signaling pathway in endothelial cells, which can lead to stimulated angiogenesis. Our results indicate that lipids are selectively loaded into breast cancer EVs to support tumor progression..
22. Shin Nishiumi*, Yoshihiro Izumi, Takashi Kobayashi, Masaru Yoshida*, Possible involvement of lipids in the effectiveness of kombu in individuals with abnormally high serum triglyceride levels, Journal of Nutritional Science and Vitaminology, 10.3177/jnsv.66.185, 66, 2, 185-190, 2020.02, In Japan, Kombu (Laminaria japonica), which is a type of seaweed, is considered to be a foodstuff with health-promoting benefits, and Japanese people actively incorporate Kombu into their diets. Previously, we reported that the frequent intake of Kombu reduced the serum triglyceride levels of subjects with abnormally high serum triglyceride levels. In the current human study, we performed metabolomic analysis of serum lipids, and then the molecular species profiles of phosphatidylcholines (PC), phosphatidylethanolamines (PE), lysophosphatidylcholines (LPC), lysophosphatidylethanolamines (LPE), and free fatty acids (FFA) were evaluated. As a result, it was found that there were no marked differences between the lipid profiles obtained before and after the intake of Kombu for 4 wk in all subjects. In the subjects with abnormal serum triglyceride levels, the intake of Kombu improved the subjects’ molecular species profiles in terms of their serum levels of the diacyl and acyl forms of PC, PE, LPC, and LPE, and FFA. Furthermore, the intake of Kombu also tended to increase the serum levels of both the plasmanyl and plasmenyl forms of PC and PE in these subjects. The lipid alterations observed in our study might be related to the functionality of Kombu. Furthermore, it is important to evaluate the quality of lipids as well as the quantity of lipids in various types of research, including food functionality studies..
23. Kohta Nakatani, Yoshihiro Izumi*, Kosuke Hata, Takeshi Bamba, An analytical system for single-cell metabolomics of typical mammalian cells based on highly sensitive nano-liquid chromatography tandem mass spectrometry, Mass Spectrometry, 10.5702/massspectrometry.A0080, 9, 1, 2020.01, The rapid development of next-generation sequencing techniques has enabled single-cell genomic and transcriptomic analyses, which have revealed the importance of heterogeneity in biological systems. However, analytical methods to accurately identify and quantify comprehensive metabolites from single mammalian cells with a typical diameter of 10–20 µm are still in the process of development. The aim of this study was to develop a single-cell metabolomic analytical system based on highly sensitive nanoliquid chromatography tandem mass spectrometry (nano-LC-MS/MS) with multiple reaction monitoring. A packed nano-LC column (3-µm particle-size pentafluorophenylpropyl Discovery HSF5 of dimensions 100 µm i.d.×180 mm) was prepared using a slurry technique. The optimized nano-LC-MS/MS method showed 3–132-fold (average value, 26-fold) greater sensitivity than semimicro-LC-MS/MS, and the detection limits for several hydrophilic metabolites, including amino acids and nucleic acid related metabolites were in the sub-fmol range. By combining live single-cell sampling and nano-LC-MS/MS, we successfully detected 18 relatively abundant hydrophilic metabolites (16 amino acids and 2 nucleic acid related metabo-lites) from single HeLa cells (n=22). Based on single-cell metabolic profiles, the 22 HeLa cells were classified into three distinct subclasses, suggesting differences in metabolic function in cultured HeLa cell populations. Our single-cell metabolomic analytical system represents a potentially useful tool for in-depth studies focused on cell metabolism and heterogeneity..
24. Jun Matsuda, Atsushi Takahashi, Yoshitsugu Takabatake*, Shinsuke Sakai, Satoshi Minami, Takeshi Yamamoto, Ryuta Fujimura, Tomoko Namba-Hamano, Hiroaki Yonishi, Jun Nakamura, Tomonori Kimura, Jun Ya Kaimori, Isao Matsui, Masatomo Takahashi, Motonao Nakao, Yoshihiro Izumi, Takeshi Bamba, Taiji Matsusaka, Fumio Niimura, Motoko Yanagita, Tamotsu Yoshimori, Yoshitaka Isaka, Metabolic effects of RUBCN/Rubicon deficiency in kidney proximal tubular epithelial cells, Autophagy, 10.1080/15548627.2020.1712107, 2020.01, Macroautophagy/autophagy is a lysosomal degradation system which plays a protective role against kidney injury. RUBCN/Rubicon (RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein) inhibits the fusion of autophagosomes and lysosomes. However, its physiological role in kidney proximal tubular epithelial cells (PTECs) remains uncertain. In the current study, we analyzed the phenotype of newly generated PTEC-specific rubcn-deficient (KO) mice. Additionally, we investigated the role of RUBCN in lipid metabolism using isolated rubcn-deficient PTECs. Although KO mice exhibited sustained high autophagic flux in PTECs, they were not protected from acute ischemic kidney injury. Unexpectedly, KO mice exhibited hallmark features of metabolic syndrome accompanied by expanded lysosomes containing multi-layered phospholipids in PTECs. RUBCN deficiency in cultured PTECs promoted the mobilization of phospholipids from cellular membranes to lysosomes via enhanced autophagy. Treatment of KO PTECs with oleic acid accelerated fatty acids transfer to mitochondria. Furthermore, KO PTECs promoted massive triglyceride accumulation in hepatocytes (BNL-CL2 cells) co-cultured in transwell, suggesting accelerated fatty acids efflux from the PTECs contributes to the metabolic syndrome in KO mice. This study shows that sustained high autophagic flux by RUBCN deficiency in PTECs leads to metabolic syndrome concomitantly with an accelerated mobilization of phospholipids from cellular membranes to lysosomes. Abbreviations: ABC: ATP binding cassette; ACADM: acyl-CoA dehydrogenase medium chain; ACTB: actin, beta; ATG: autophagy related; AUC: area under the curve; Baf: bafilomycin A1; BAT: brown adipose tissue; BODIPY: boron-dipyrromethene; BSA: bovine serum albumin; BW: body weight; CAT: chloramphenicol acetyltransferase; CM: complete medium; CPT1A: carnitine palmitoyltransferase 1a, liver; CQ: chloroquine; CTRL: control; EGFP: enhanced green fluorescent protein; CTSD: cathepsin D; EAT: epididymal adipose tissue; EGFR: epidermal growth factor receptor; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; FA: fatty acid; FBS: fetal bovine serum; GTT: glucose tolerance test; HE: hematoxylin and eosin; HFD: high-fat diet; I/R: ischemia-reperfusion; ITT: insulin tolerance test; KAP: kidney androgen regulated protein; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LD: lipid droplet; LRP2: low density lipoprotein receptor related protein 2; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MAT: mesenteric adipose tissue; MS: mass spectrometry; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; NDRG1: N-myc downstream regulated 1; NDUFB5: NADH:ubiquinone oxidoreductase subunit B5; NEFA: non-esterified fatty acid; OA: oleic acid; OCT: optimal cutting temperature; ORO: Oil Red O; PAS: Periodic-acid Schiff; PFA: paraformaldehyde; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PPARA: peroxisome proliferator activated receptor alpha; PPARGC1A: PPARG coactivator 1 alpha; PTEC: proximal tubular epithelial cell; RAB7A: RAB7A, member RAS oncogene family; RPS6: ribosomal protein S6; RPS6KB1: ribosomal protein S6 kinase B1; RT: reverse transcription; RUBCN: rubicon autophagy regulator; SAT: subcutaneous adipose tissue; SFC: supercritical fluid chromatography; SQSTM1: sequestosome 1; SREBF1: sterol regulatory element binding transcription factor 1; SV-40: simian virus-40; TFEB: transcription factor EB; TG: triglyceride; TS: tissue specific; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; UN: urea nitrogen; UQCRB: ubiquinol-cytochrome c reductase binding protein; UVRAG: UV radiation resistance associated; VPS: vacuolar protein sorting; WAT: white adipose tissue..
25. Shin Nishiumi*, Yoshihiro Izumi, Takashi Kobayashi, Masaru Yoshida*, A pilot study Effects of kombu intake on lifestyle-related diseases -possibility that kombu intake is effective in individuals with abnormally high serum triglyceride levels, Food Science and Technology Research, 10.3136/fstr.25.827, 25, 6, 827-834, 2019.12, In Japan, kombu (Laminaria japonica), a type of seaweed, has been consumed for centuries. It contains a variety of active compounds such as minerals, vitamins, and dietary fiber. The aim of this human pilot study is to investigate the effects of kombu on lifestyle-related diseases. The study had a randomized crossover design, and the subjects (N=48) freely took 6 g of roasted kombu a day for 4 weeks. The subjects' responses to the Gastrointestinal Symptom Rating Scale questionnaire suggested that the frequent intake of kombu may lead to the relief of constipation, diarrhea, and hard stools. In addition, blood tests indicated the possibility that the frequent intake of kombu can decrease the serum triglyceride levels of subjects with abnormally high serum triglyceride levels. Kombu intake might lead to relief from intestinal ailments and improvements in hypertriglyceridemia..
26. Yoshihiro Izumi, Fumio Matsuda*, Akiyoshi Hirayama, Kazutaka Ikeda, Yoshihiro Kita, Kanta Horie, Daisuke Saigusa, Kosuke Saito, Yuji Sawada, Hiroki Nakanishi, Nobuyuki Okahashi, Masatomo Takahashi, Motonao Nakao, Kosuke Hata, Yutaro Hoshi, Motohiko Morihara, Kazuhiro Tanabe, Takeshi Bamba*, Yoshiya Oda, Inter-laboratory comparison of metabolite measurements for metabolomics data integration, Metabolites, 10.3390/metabo9110257, 9, 11, Article number: 257, 2019.11, Background: One of the current problems in the field of metabolomics is the difficulty in integrating data collected using different equipment at different facilities, because many metabolomic methods have been developed independently and are unique to each laboratory. Methods: In this study, we examined whether different analytical methods among 12 different laboratories provided comparable relative quantification data for certain metabolites. Identical samples extracted from two cell lines (HT-29 and AsPc-1) were distributed to each facility, and hydrophilic and hydrophobic metabolite analyses were performed using the daily routine protocols of each laboratory. Results: The results indicate that there was no difference in the relative quantitative data (HT-29/AsPc-1) for about half of the measured metabolites among the laboratories and assay methods. Data review also revealed that errors in relative quantification were derived from issues such as erroneous peak identification, insufficient peak separation, a difference in detection sensitivity, derivatization reactions, and extraction solvent interference. Conclusion: The results indicated that relative quantification data obtained at different facilities and at different times would be integrated and compared by using a reference materials shared for data normalization..
27. Toshiaki Yoshioka, Yoshihiro Izumi, Yasushi Nagatomi, Yasuhisa Miyamoto, Koji Suzuki, Takeshi Bamba*, A highly sensitive determination method for acrylamide in beverages, grains, and confectioneries by supercritical fluid chromatography tandem mass spectrometry, Food Chemistry, 10.1016/j.foodchem.2019.05.033, 294, 486-492, 2019.10, Acrylamide (AA) analysis is an important topic in food safety. However, it is difficult to rapidly and accurately analyze low concentrations of AA with currently available methods. In the present study, we introduce a highly sensitive method that enables the determination of AA in beverages, grains, and confectioneries by supercritical fluid chromatography tandem mass spectrometry (SFC/MS/MS). The sensitivity of the SFC/MS/MS technique is 11-times higher than that obtained by ultra-high performance liquid chromatography tandem mass spectrometry. We demonstrated that the highly sensitive SFC/MS/MS method was able to quantify low concentrations of AA in beverages (i.e., roasted barley tea and coffee) extracts at less than 10 µg kg −1 level without solid-phase purification. Furthermore, the simplification of the sample preparation procedure provided an improvement in data acquisition time (60 samples per 12 h). In conclusion, the developed analytical system is a potentially useful tool for practical AA determination..
28. Masashi Shiomi*, Hiroaki Takeda, Yasuhiro Irino, Norie Kimura, Satoshi Yamada, Nobue Kuniyoshi, Akio Kikumori, Yu Koike, Tomonari Koike, Masaru Yoshida, Yoshihiro Izumi, Masakazu Shinohara, Takeshi Bamba, Tatsuro Ishida, Identification of novel serum markers for the progression of coronary atherosclerosis in WHHLMI rabbits, an animal model of familial hypercholesterolemia, Atherosclerosis, 10.1016/j.atherosclerosis.2019.02.020, 284, 18-23, 2019.05, Background and aims: The development of serum markers specific for coronary lesions is important to prevent coronary events. However, analyses of serum markers in humans are affected by environmental factors and non-target diseases. Using an appropriate model animal can reduce these effects. To identify specific markers for coronary atherosclerosis, we comprehensively analyzed the serum of WHHLMI rabbits, which spontaneously develop coronary atherosclerosis. Methods: Female WHHLMI rabbits were fed standard chow. Serum and plasma were collected under fasting at intervals of 4 months from 4 months old, and a total of 313 lipid molecules, 59 metabolites, lipoprotein lipid levels, and various plasma biochemical parameters were analyzed. The severity of coronary lesions was evaluated with cross-sectional narrowing (CSN) corrected with a frequency of 75%–89% CSN and CSN> 90%. Results: There was a large variation in the severity of coronary lesions in WHHLMI rabbits despite almost no differences in plasma biochemical parameters and aortic lesion area between rabbits with severe and mild coronary lesions. The metabolites and lipid molecules selected as serum markers for coronary atherosclerosis were lysophosphatidylcholine (LPC) 22:4 and diacylglycerol 18:0–18:0 at 4 months old, LPC 20:4 (sn-2), ceramide d18:1–18:2, citric acid plus isocitric acid, and pyroglutamic acid at 8 months old, and phosphatidylethanolamine plasminogen 16:1p-22:2 at 16 months old. Conclusions: These serum markers were coronary lesion-specific markers independent of cholesterol levels and aortic lesions and may be useful to detect patients who develop cardiovascular disease..
29. Tsunehisa Hirose*, Daniel Keck, Yoshihiro Izumi, Takeshi Bamba, Comparison of retention behavior between supercritical fluid chromatography and normal-phase high-performance liquid chromatography with various stationary phases, Molecules, 10.3390/molecules24132425, 24, 13, 2019.01, The retention behavior of a wide variety of stationary phases was compared in supercritical fluid chromatography (SFC) and normal-phase high-performance liquid chromatography (NP-HPLC). We also attempted to elucidate the retention behavior in SFC by investigating the selectivity of the different stationary phases. SFC separation conditions with polar stationary phases, such as silica gel (SL) and diol (Diol) phases, operate via adsorptions that include hydrophilic and ionic interactions similar to those in NP-HPLC. Moreover, non-polar stationary phases, such as pentabromophenyl (PBr), pyrenylethyl (PYE), and octadecyl (C18), could be used despite the non-polar mobile phase conditions, because the dispersion and p-p interactions were stronger in SFC than in HPLC. These results reflect the selectivity of the stationary phase and its retention factor, thus providing useful information for the selection of appropriate stationary phases for particular analytes..
30. Hiroaki Takeda, Masatomo Takahashi, Takeshi Hara, Yoshihiro Izumi, Takeshi Bamba*, Improved quantitation of lipid classes using supercritical fluid chromatography with a charged aerosol detector, Journal of Lipid Research, 10.1194/jlr.D094516, 60, 8, 1465-1474, 2019.01, Quantitatively and rapidly analyzing lipids is necessary to elucidate their biological functions. Herein, we developed a quantitative method for various lipid classes using supercritical fluid chromatography (SFC) coupled with a charged aerosol detector (CAD), providing high-throughput data analysis to detect a large number of molecules in each lipid class as one peak. Applying the CAD was useful for analyzing lipid molecules in the same lipid class with a constant response under the same mobile phase composition. First, we optimized the washing method for the diethylamine column, achieving baseline separation of lipid classes while maintaining good peak shapes. In addition, the CAD conditions (organic solvent evaporation and numerical correction of the CAD data) were optimized to improve the signal-to-noise ratio. We used an internal standard (ceramide phosphoethanolamine d17:1-12:0), which did not coelute with the lipid classes and showed high extraction efficiency. Based on a quantitative analysis of HepG2 cells, the concentration of lipid classes detected by CAD was adequate compared with that obtained by triple-quadrupole MS (QqQMS) in a previous study because the deviations of the concentrations were 0.6- to 2.3-fold. These results also supported the quantitative performance of SFC-QqQMS developed in our previous report.-Takeda, H., M. Takahashi, T. Hara, Y. Izumi, and T. Bamba. Improved quantitation of lipid classes using supercritical fluid chromatography with a charged aerosol detector. J. Lipid Res. 2019. 60: 1465-1474..
31. Masuko Kobori*, Yumiko Takahashi, Hiroaki Takeda, Masatomo Takahashi, Yoshihiro Izumi, Yukari Akimoto, Mutsumi Sakurai, Hideaki Oike, Toshiyuki Nakagawa, Masanori Itoh, Takeshi Bamba, Toshiyuki Kimura, Dietary Intake of Curcumin Improves eIF2 Signaling and Reduces Lipid Levels in the White Adipose Tissue of Obese Mice, Scientific reports, 10.1038/s41598-018-27105-w, 8, 1, 2018.12, White adipose tissue (eWAT) plays a crucial role in preventing metabolic syndrome. We aimed to investigate WAT distribution and gene expression and lipidomic profiles in epididymal WAT (eWAT) in diet-induced obese mice, reflecting a Western-style diet of humans to elucidate the bioactive properties of the dietary antioxidant curcumin in preventing lifestyle-related diseases. For 16 weeks, we fed C57BL/6J mice with a control diet, a high-fat, high-sucrose and high-cholesterol Western diet or Western diet supplemented with 0.1% (w/w) curcumin. Although the dietary intake of curcumin did not affect eWAT weight or plasma lipid levels, it reduced lipid peroxidation markers' levels in eWAT. Curcumin accumulated in eWAT and changed gene expressions related to eukaryotic translation initiation factor 2 (eIF2) signalling. Curcumin suppressed eIF2α phosphorylation, which is induced by endoplasmic reticulum (ER) stress, macrophage accumulation and nuclear factor-κB (NF-κB) p65 and leptin expression, whereas it's anti-inflammatory effect was inadequate to decrease TNF-α and IFN-γ levels. Lipidomic and gene expression analysis revealed that curcumin decreased some diacylglycerols (DAGs) and DAG-derived glycerophospholipids levels by suppressing the glycerol-3-phosphate acyltransferase 1 and adipose triglyceride lipase expression, which are associated with lipogenesis and lipolysis, respectively. Presumably, these intertwined effects contribute to metabolic syndrome prevention by dietary modification..
32. Takeshi Hara, Yoshihiro Izumi, Motonao Nakao, Kosuke Hata, Gino V. Baron, Takeshi Bamba, Gert Desmet*, Silica-based hybrid porous layers to enhance the retention and efficiency of open tubular capillary columns with a 5 μm inner diame9ter, Journal of Chromatography A, 10.1016/j.chroma.2018.10.023, 1580, 63-71, 2018.12, We report on possibility to enhance the hydrophobicity of octadecylsilylated silica-based porous layered open tubular (PLOT) columns with an inner diameter (i.d.) of 5 μm by applying hybrid tetramethoxysilane (TMOS)/methyltrimethoxysilane (MTMS) layers with inserted methyl groups. Due to this higher hydrophobicity, thinner porous layers suffice to achieve similar retention factor (k) as in octadecylsilylated silica-based PLOT columns synthesized using TMOS only. Since thinner layers have a lower intra-layer mass transfer resistance, this in turn allows to obtain superior column efficiencies in comparison with separations carried out with TMOS-based PLOT columns at the same retention. Since layer thickness contributes to the C-term type of band broadening, this is most pronounced at high velocities. Typical gains in column efficiency at a reduced velocity of νi = 30 are on the order of 15%. Preparing the hybrid PLOT columns in 5 μm i.d.-capillaries with a length of 0.4 m using different TMOS/MTMS preparation mixtures leads to different layer thickness in the capillaries. It is shown that column efficiencies for the most retained compound (k = 0.9–1.5) went from N = 101,000 for PLOT columns with a layer thickness (df) of 250 nm, over N = 95,000 for df = 320 nm to N = 89,000 for df = 400 nm, corresponding to plate heights (H) in the order of 3.5–3.9 μm (reduced plate heights (h = 0.8–1.0)). By applying the same preparation mixtures for much longer capillaries of 1.3 m, a high repeatability of the volumetric phase ratio (m) (difference <1%) and the k-values (difference <5%) was observed between the 0.4 m and 1.3 m PLOT columns. In addition, also a very similar band broadening was obtained, as the h-values in the longer columns coincided well (order of a few % difference) with the reduced plate height curves measured in the shorter columns. The effect of the retention factor and layer thickness on these reduced plate height curves furthermore fits well with the Golay-Aris theory. Depending on the layer thickness, plate numbers in the longer capillary columns were varying from N = 282,000 to N = 379,000 for the most retained compound..
33. Govind Kunduri, Daniel Turner-Evans, Yutaka Konya, Yoshihiro Izumi, Kunio Nagashima, Stephen Lockett, Joost Holthuis, Takeshi Bamba, Usha Acharya, Jairaj K. Acharya*, Defective cortex glia plasma membrane structure underlies light-induced epilepsy in cpes mutants, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.1808463115, 115, 38, E8919-E8928, 2018.09, Seizures induced by visual stimulation (photosensitive epilepsy; PSE) represent a common type of epilepsy in humans, but the molecular mechanisms and genetic drivers underlying PSE remain unknown, and no good genetic animal models have been identified as yet. Here, we show an animal model of PSE, in Drosophila, owing to defective cortex glia. The cortex glial membranes are severely compromised in ceramide phosphoethanolamine synthase (cpes)-null mutants and fail to encapsulate the neuronal cell bodies in the Drosophila neuronal cortex. Expression of human sphingomyelin synthase 1, which synthesizes the closely related ceramide phosphocholine (sphingomyelin), rescues the cortex glial abnormalities and PSE, underscoring the evolutionarily conserved role of these lipids in glialmembranes. Further,we show the compromise in plasma membrane structure that underlies the glial cell membrane collapse in cpes mutants and leads to the PSE phenotype..
34. Masatomo Takahashi, Yoshihiro Izumi*, Fukumatsu Iwahashi, Yasumune Nakayama, Mitsuhiko Iwakoshi, Motonao Nakao, Seiji Yamato, Eiichiro Fukusaki, Takeshi Bamba*, Highly Accurate Detection and Identification Methodology of Xenobiotic Metabolites Using Stable Isotope Labeling, Data Mining Techniques, and Time-Dependent Profiling Based on LC/HRMS/MS, Analytical Chemistry, 10.1021/acs.analchem.8b01388, 90, 15, 9068-9076, 2018.08, A generally applicable method to discover xenobiotic metabolites is important to safely and effectively develop xenobiotics. We propose an advanced method to detect and identify comprehensive xenobiotic metabolites using stable isotope labeling, liquid chromatography coupled with benchtop quadrupole Orbitrap high-resolution tandem mass spectrometry (LC/HRMS/MS), data mining techniques (alignment, peak picking, and paired-peaks filtering), in silico metabolism prediction, and time-dependent profiling. The LC/HRMS analysis was carried out using Arabidopsis T87 cultured cells treated with unlabeled, or 13C- or 2H-labeled 2,4-dichlorophenoxyacetic acid (2,4-D). Paired-peak filtering enabled accurate detection of 83 candidates for 2,4-D metabolites without any false positive peaks derived from solvents or the biological matrix. We confirmed 10 previously reported 2,4-D metabolites and identified 16 novel 2,4-D metabolites. Our method provides accurate detection and identification of comprehensive xenobiotic metabolites and represents a potentially useful tool to elucidate xenobiotic metabolism..
35. Shin Nishiumi*, Yoshihiro Izumi, Masaru Yoshida*, Alterations in Docosahexaenoic Acid-Related Lipid Cascades in Inflammatory Bowel Disease Model Mice, Digestive Diseases and Sciences, 10.1007/s10620-018-5025-4, 63, 6, 1485-1496, 2018.06, Background: Inflammatory bowel disease (IBD) is an intestinal disorder, involving chronic and relapsing inflammation of the digestive tract. Dysregulation of the immune system based on genetic, environmental, and other factors seems to be involved in the onset of IBD, but its exact pathogenesis remains unclear. Therefore, radical treatments for ulcerative colitis and Crohn’s disease remain to be found, and IBD is considered to be a refractory disease. Aims: The aim of this study is to obtain novel insights into IBD via metabolite profiling of interleukin (IL)-10 knockout mice (an IBD animal model that exhibits a dysregulated immune system). Methods: In this study, the metabolites in the large intestine and plasma of IL-10 knockout mice were analyzed. In our analytical system, two kinds of analysis (gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry) were used to detect a broader range of metabolites, including both hydrophilic and hydrophobic metabolites. In addition, an analysis of lipid mediators in the large intestine and ascites of IL-10 knockout mice was carried out. Results: The levels of a variety of metabolites, including lipid mediators, were altered in IL-10 knockout mice. For example, high large intestinal and plasma levels of docosahexaenoic acid (DHA) were observed. In addition, arachidonic acid- and DHA-related lipid cascades were upregulated in the ascites of the IL-10 knockout mice. Conclusions: Our findings based on metabolite profiles including lipid mediators must contribute to development of researches about IBD..
36. Yoshinari Obata, Shunbun Kita*, Yoshihisa Koyama, Shiro Fukuda, Hiroaki Takeda, Masatomo Takahashi, Yuya Fujishima, Hirofumi Nagao, Shigeki Masuda, Yoshimitsu Tanaka, Yuto Nakamura, Hitoshi Nishizawa, Tohru Funahashi, Barbara Ranscht, Yoshihiro Izumi, Takeshi Bamba, Eiichiro Fukusaki, Rikinari Hanayama, Shoichi Shimada, Norikazu Maeda, Iichiro Shimomura, Adiponectin/T-cadherin system enhances exosome biogenesis and decreases cellular ceramides by exosomal release, JCI insight, 10.1172/jci.insight.99680, 3, 8, 2018.04, Adiponectin, an adipocyte-derived circulating protein, accumulates in vasculature, heart, and skeletal muscles through interaction with a unique glycosylphosphatidylinositol-anchored cadherin, T-cadherin. Recent studies have demonstrated that such accumulation is essential for adiponectin-mediated cardiovascular protection. Here, we demonstrate that the adiponectin/T-cadherin system enhances exosome biogenesis and secretion, leading to the decrease of cellular ceramides. Adiponectin accumulated inside multivesicular bodies, the site of exosome generation, in cultured cells and in vivo aorta, and also in exosomes in conditioned media and in blood, together with T-cadherin. The systemic level of exosomes in blood was significantly affected by adiponectin or T-cadherin in vivo. Adiponectin increased exosome biogenesis from the cells, dependently on T-cadherin, but not on AdipoR1 or AdipoR2. Such enhancement of exosome release accompanied the reduction of cellular ceramides through ceramide efflux in exosomes. Consistently, the ceramide reduction by adiponectin was found in aortas of WT mice treated with angiotensin II, but not in T-cadherin-knockout mice. Our findings provide insights into adiponectin/T-cadherin-mediated organ protection through exosome biogenesis and secretion..
37. Shohei Tamura, Yui Koike, Hiroaki Takeda, Tomonari Koike, Yoshihiro Izumi, Ryosuke Nagasaka, Tetsuto Tsunoda, Motoo Tori, Kazuo Ogawa, Takeshi Bamba, Masashi Shiomi*, Ameliorating effects of D-47, a newly developed compound, on lipid metabolism in an animal model of familial hypercholesterolemia (WHHLMI rabbits), European Journal of Pharmacology, 10.1016/j.ejphar.2018.01.013, 822, 147-153, 2018.03, Improvements induced in lipid metabolism in the liver by D-47, a newly developed compound, were examined herein. WHHLMI rabbits, an animal model of hypercholesterolemia and coronary atherosclerosis, was fed D-47-supplemented chow for 5 weeks at a dose of 30 mg/kg. Lipid concentration were assayed using enzymatic methods. Plasma lipoproteins were fractionated with an ultracentrifuge. mRNA expression was analyzed with real-time PCR. Lipidome analyses of lipoproteins were performed using supercritical fluid chromatography mass spectrometry. In the D-47-treated group, serum lipid levels decreased by 23% for total cholesterol and by 40% for triglycerides. These reductions were mainly attributed to decreases in the VLDL fraction. Compared with the control, in the D-47 group, lipid contents in the liver were decreased by 22% in cholesterol and by 69% in triglycerides, and fat accumulation was decreased by 57% in pericardial fat and by 17% in mesenteric fat. In lipidome analyses of VLDL fraction, lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylethanolamine plasmalogen, sphingomyelin, and ceramide were decreased by the D-47 treatment. mRNA expression in the liver was 51% lower for FAS and 24% lower for MTP, but 5.9- and 5.1-fold higher for CYP7A1 and CPT-1, respectively, in the D-47 group than in the control. mRNA expression was 72%, 64%, and 36% higher for LPL, CTP-1, and PPARγ, respectively, in mesenteric fat in the D-47 group. D-47 is a potent lipid-lowering compound that uses a different mechanism of action from that of statins. It has potential as a compound in the treatment of steatohepatitis and metabolic syndrome..
38. Hiroaki Takeda, Yoshihiro Izumi, Masatomo Takahashi, Thanai Paxton, Shohei Tamura, Tomonari Koike, Ying Yu, Noriko Kato, Katsutoshi Nagase, Masashi Shiomi, Takeshi Bamba*, Widely-targeted quantitative lipidomics method by supercritical fluid chromatography triple quadrupole mass spectrometry, Journal of Lipid Research, 10.1194/jlr.D083014, 59, 7, 1283-1293, 2018.01, Lipidomics, the mass spectrometry-based comprehensive analysis of lipids, has attracted attention as an analytical approach to provide novel insight into lipid metabolism and to search for biomarkers. However, an ideal method for both comprehensive and quantitative analysis of lipids has not been fully developed. Here, we have proposed a practical methodology for widely targeted quantitative lipidome analysis using supercritical fluid chromatography fast-scanning triple-quadrupole mass spectrometry (SFC/ QqQMS) and theoretically calculated a comprehensive lipid multiple reaction monitoring (MRM) library. Lipid classes can be separated by SFC with a normal-phase diethylaminebonded silica column with high resolution, high throughput, and good repeatability. Structural isomers of phospholipids can be monitored by mass spectrometric separation with fatty acyl-based MRM transitions. SFC/QqQMS analysis with an internal standard-dilution method offers quantitative information for both lipid class and individual lipid molecular species in the same lipid class. Additionally, data acquired using this method has advantages, including reduction of misidentification and acceleration of data analysis. Using the SFC/QqQMS system, alteration of plasma lipid levels in myocardial infarction-prone rabbits to the supplementation of EPA was first observed. Our developed SFC/QqQMS method represents a potentially useful tool for in-depth studies focused on complex lipid metabolism and biomarker discovery..
39. Takaiku Sakamoto, Eiji Sakuradani*, Tomoyo Okuda, Hiroshi Kikukawa, Akinori Ando, Shigenobu Kishino, Yoshihiro Izumi, Takeshi Bamba, Jun Shima, Jun Ogawa, Metabolic engineering of oleaginous fungus Mortierella alpina for high production of oleic and linoleic acids, Bioresource Technology, 10.1016/j.biortech.2017.06.089, 245, 1610-1615, 2017.12, The aim of this work was to study the molecular breeding of oleaginous filamentous Mortierella alpina for high production of linoleic (LA) or oleic acid (OA). Heterologous expression of the Δ12-desaturase (DS) gene derived from Coprinopsis cinerea in the Δ6DS activity-defective mutant of M. alpina increased the LA production rate as to total fatty acid to 5 times that in the wild strain. By suppressing the endogenous Δ6I gene expression by RNAi in the Δ12DS activity-defective mutant of M. alpina, the OA accumulation rate as to total fatty acid reached 68.0%. The production of LA and OA in these transformants reached 1.44 and 2.76 g/L, respectively, on the 5th day. The Δ6I transcriptional levels of the RNAi-treated strains were suppressed to 1/10th that in the parent strain. The amount of Δ6II RNA in the Δ6I RNAi-treated strain increased to 8 times that in the wild strain..
40. Satoshi Minami, Takeshi Yamamoto, Yoshitsugu Takabatake*, Atsushi Takahashi, Tomoko Namba, Jun Matsuda, Tomonori Kimura, Jun ya Kaimori, Isao Matsui, Takayuki Hamano, Hiroaki Takeda, Masatomo Takahashi, Yoshihiro Izumi, Takeshi Bamba, Taiji Matsusaka, Fumio Niimura, Yoshitaka Isaka, Lipophagy maintains energy homeostasis in the kidney proximal tubule during prolonged starvation, Autophagy, 10.1080/15548627.2017.1341464, 13, 10, 1629-1647, 2017.10, Macroautophagy/autophagy is a self-degradation process that combats starvation. Lipids are the main energy source in kidney proximal tubular cells (PTCs). During starvation, PTCs increase fatty acid (FA) uptake, form intracellular lipid droplets (LDs), and hydrolyze them for use. The involvement of autophagy in lipid metabolism in the kidney remains largely unknown. Here, we investigated the autophagy-mediated regulation of renal lipid metabolism during prolonged starvation using PTC-specific Atg5-deficient (atg5-TSKO) mice and an in vitro serum starvation model. Twenty-four h of starvation comparably induced LD formation in the PTCs of control and atg5-TSKO mice; however, additional 24 h of starvation reduced the number of LDs in control mice, whereas increases were observed in atg5-TSKO mice. Autophagic degradation of LDs (lipophagy) in PTCs was demonstrated by electron microscopic observation and biochemical analysis. In vitro pulse-chase assays demonstrated that lipophagy mobilizes FAs from LDs to mitochondria during starvation, whereas impaired LD degradation in autophagy-deficient PTCs led to decreased ATP production and subsequent cell death. In contrast to the in vitro assay, despite impaired LD degradation, kidney ATP content was preserved in 48-h starved atg5-TSKO mice, probably due to increased utilization of ketone bodies. This compensatory mechanism was accompanied by a higher plasma FGF21 (fibroblast growth factor 21) level and its expression in the PTCs; however, this was not essential for the production of ketone bodies in the liver during prolonged starvation. In conclusion, lipophagy combats prolonged starvation in PTCs to avoid cellular energy depletion..
41. Yuka Fujito, Yoshihiro Hayakawa, Yoshihiro Izumi, Takeshi Bamba*, Importance of optimizing chromatographic conditions and mass spectrometric parameters for supercritical fluid chromatography/mass spectrometry, Journal of Chromatography A, 10.1016/j.chroma.2017.05.071, 1508, 138-147, 2017.07, Supercritical fluid chromatography/mass spectrometry (SFC/MS) has great potential in high-throughput and the simultaneous analysis of a wide variety of compounds, and it has been widely used in recent years. The use of MS for detection provides the advantages of high sensitivity and high selectivity. However, the sensitivity of MS detection depends on the chromatographic conditions and MS parameters. Thus, optimization of MS parameters corresponding to the SFC condition is mandatory for maximizing performance when connecting SFC to MS. The aim of this study was to reveal a way to decide the optimum composition of the mobile phase and the flow rate of the make-up solvent for MS detection in a wide range of compounds. Additionally, we also showed the basic concept for determination of the optimum values of the MS parameters focusing on the MS detection sensitivity in SFC/MS analysis. To verify the versatility of these findings, a total of 441 pesticides with a wide polarity range (logPow from −4.21 to 7.70) and pKa (acidic, neutral and basic). In this study, a new SFC-MS interface was used, which can transfer the entire volume of eluate into the MS by directly coupling the SFC with the MS. This enabled us to compare the sensitivity or optimum MS parameters for MS detection between LC/MS and SFC/MS for the same sample volume introduced into the MS. As a result, it was found that the optimum values of some MS parameters were completely different from those of LC/MS, and that SFC/MS-specific optimization of the analytical conditions is required. Lastly, we evaluated the sensitivity of SFC/MS using fully optimized analytical conditions. As a result, we confirmed that SFC/MS showed much higher sensitivity than LC/MS when the analytical conditions were fully optimized for SFC/MS; and the high sensitivity also increase the number of the compounds that can be detected with good repeatability in real sample analysis. This result indicates that SFC/MS has potential for practical use in the multiresidue analysis of a wide range of compounds that requires high sensitivity..
42. Takahiro Ogawa, Yoshihiro Izumi, Kenichi Kusumoto, Eiichiro Fukusaki, Takeshi Bamba*, Wide target analysis of acylglycerols in miso (Japanese fermented soybean paste) by supercritical fluid chromatography coupled with triple quadrupole mass spectrometry and the analysis of the correlation between taste and both acylglycerols and free fatty acids, Rapid Communications in Mass Spectrometry, 10.1002/rcm.7862, 31, 11, 928-936, 2017.06, Rationale: The acylglycerols in miso have not been studied although it is known that they are important to the taste. In order to determine the fatty acid constituents in the acylglycerols and analyze them individually, multiple reaction monitoring (MRM) was performed utilizing a single platform, typically using both gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. Methods: Acylglycerols and fatty acids (FAs) in miso were extracted using the Bligh-Dyer method. Supercritical fluid chromatography (SFC) with a C30 column was conducted for separation, and mass spectrometric (MS) analysis was performed with electrospray ionization using a triple quadrupole mass spectrometer (QqQMS) in the MRM mode. Results: The detection of FAs from the hydrolysis of acylglycerols and individual acylglycerols was achieved using only an SFC/MS platform. From the quality control (QC) sample of miso, we determined the main FA constituents, and then performed wide target analysis using MRM. In total, 23 triacylglycerols, 10 diacylglycerols, two monoacylglycerols, and five FAs were annotated effectively. Furthermore, the important compounds related to taste were determined through the analysis using both the relative quantitative data of acylglycerols and FAs and the quantitative descriptive analysis data of miso. Conclusions: A method for the determination of the FA constituents in acylglycerols after hydrolysis and the comprehensive analysis of acylglycerols and FAs using MRM with SFC/QqQMS was developed. Using the data from the comprehensive analysis of acylglycerols and quantitative descriptive data, the key compounds related to taste were investigated. This type of research on lipids and the taste of food is expected to progress hereafter..
43. Makoto Suzuki, Shin Nishiumi, Takashi Kobayashi, Arata Sakai, Yosuke Iwata, Takato Uchikata, Yoshihiro Izumi, Takeshi Azuma, Takeshi Bamba, Masaru Yoshida*, Use of on-line supercritical fluid extraction-supercritical fluid chromatography/tandem mass spectrometry to analyze disease biomarkers in dried serum spots compared with serum analysis using liquid chromatography/tandem mass spectrometry, Rapid Communications in Mass Spectrometry, 10.1002/rcm.7857, 31, 10, 886-894, 2017.05, Rationale: The analytical stability and throughput of biomarker assays based on dried serum spots (DSS) are strongly dependent on the extraction process and determination method. In the present study, an on-line system based on supercritical fluid extraction-supercritical fluid chromatography coupled with tandem mass spectrometry (SFE-SFC/MS/MS) was established for analyzing the levels of disease biomarkers in DSS. Methods: The chromatographic conditions were investigated using the ODS-EP, diol, and SIL-100A columns. Then, we optimized the SFE-SFC/MS/MS method using the diol column, focusing on candidate biomarkers of oral, colorectal, and pancreatic cancer that were identified using liquid chromatography (LC)/MS/MS. Results: By using this system, four hydrophilic metabolites and 17 hydrophobic metabolites were simultaneously detected within 15 min. In an experiment involving clinical samples, PC 16:0-18:2/16:1-18:1 exhibited 93.8% sensitivity and 64.3% specificity, whereas PC 17:1-18:1/17:0-18:2 showed 81.3% sensitivity and 92.9% specificity for detecting oral cancer. In addition, assessments of the creatine levels demonstrated 92.3% sensitivity and 78.6% specificity for detecting colorectal cancer. Conclusions: The results of this study indicate that our method has great potential for clinical diagnosis and would be suitable for large-scale screening..
44. Masahiro Nagata, Yoshihiro Izumi, Eri Ishikawa, Ryoko Kiyotake, Rieko Doi, Satoru Iwai, Zakaria Omahdi, Toshiyuki Yamaji, Tomofumi Miyamoto, Takeshi Bamba, Shou Yamasaki*, Intracellular metabolite β-glucosylceramide is an endogenous Mincle ligand possessing immunostimulatory activity, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.1618133114, 114, 16, E3285-E3294, 2017.04, Sensing and reacting to tissue damage is a fundamental function of immune systems. Macrophage inducible C-type lectin (Mincle) is an activating C-type lectin receptor that senses damaged cells. Notably, Mincle also recognizes glycolipid ligands on pathogens. To elucidate endogenous glycolipids ligands derived from damaged cells, we fractionated supernatants from damaged cells and identified a lipophilic component that activates reporter cells expressing Mincle. Mass spectrometry and NMR spectroscopy identified the component structure as β-glucosylceramide (GlcCer), which is a ubiquitous intracellularmetabolite. Synthetic β-GlcCer activated myeloid cells and induced production of inflammatory cytokines; this production was abrogated inMincle-deficient cells. Sterile inflammation induced by excessive cell death in the thymus was exacerbated by hematopoieticspecific deletion of degrading enzyme of β-GlcCer (β-glucosylceramidase, GBA1). However, this enhanced inflammation was ameliorated in a Mincle-deficient background. GBA1-deficient dendritic cells (DCs) in which β-GlcCer accumulates triggered antigen-specific T-cell responses more efficiently than WT DCs, whereas these responses were compromised in DCs from GBA1 × Mincle double-deficient mice. These results suggest that β-GlcCer is an endogenous ligand for Mincle and possesses immunostimulatory activity..
45. John A. Bowden*, Alan Heckert, Candice Z. Ulmer, Christina M. Jones, Jeremy P. Koelmel, Laila Abdullah, Linda Ahonen, Yazen Alnouti, Aaron M. Armando, John M. Asara, Takeshi Bamba, John R. Barr, Jonas Bergquist, Christoph H. Borchers, Joost Brandsma, Susanne B. Breitkopf, Tomas Cajka, Amaury Cazenave-Gassiot, Antonio Checa, Michelle A. Cinel, Romain A. Colas, Serge Cremers, Edward A. Dennis, James E. Evans, Alexander Fauland, Oliver Fiehn, Michael S. Gardner, Timothy J. Garrett, Katherine H. Gotlinger, Jun Han, Yingying Huang, Aveline Huipeng Neo, Tuulia Hyötyläinen, Yoshihiro Izumi, Hongfeng Jiang, Houli Jiang, Jiang Jiang, Maureen Kachman, Reiko Kiyonami, Kristaps Klavins, Christian Klose, Harald C. Köfeler, Johan Kolmert, Therese Koal, Grielof Koster, Zsuzsanna Kuklenyik, Irwin J. Kurland, Michael Leadley, Karen Lin, Krishna Rao Maddipati, Danielle McDougall, Peter J. Meikle, Natalie A. Mellett, Cian Monnin, M. Arthur Moseley, Renu Nandakumar, Matej Oresic, Rainey Patterson, David Peake, Jason S. Pierce, Martin Post, Anthony D. Postle, Rebecca Pugh, Yunping Qiu, Oswald Quehenberger, Parsram Ramrup, Jon Rees, Barbara Rembiesa, Denis Reynaud, Mary R. Roth, Susanne Sales, Kai Schuhmann, Michal Laniado Schwartzman, Charles N. Serhan, Andrej Shevchenko, Stephen E. Somerville, Lisa St John-Williams, Michal A. Surma, Hiroaki Takeda, Rhishikesh Thakare, J. Will Thompson, Federico Torta, Alexander Triebl, Martin Trötzmüller, S. J.Kumari Ubhayasekera, Dajana Vuckovic, Jacquelyn M. Weir, Ruth Welti, Markus R. Wenk, Craig E. Wheelock, Libin Yao, Min Yuan, Xueqing Heather Zhao, Senlin Zhou, Harmonizing lipidomics
NIST interlaboratory comparison exercise for lipidomics using SRM 1950-Metabolites in frozen human plasma, Journal of Lipid Research, 10.1194/jlr.M079012, 58, 12, 2275-2288, 2017.01, As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950- Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra-And interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement..
46. Hiroaki Takeda, Yoshihiro Izumi, Atsumi Tomita, Tomonari Koike, Masashi Shiomi, Eiichiro Fukusaki, Fumio Matsuda, Takeshi Bamba*, Lipoprotein profiling methodology based on determination of apolipoprotein concentration, Bioanalysis, 10.4155/bio-2016-0234, 9, 1, 9-19, 2017.01, Aim: Abnormal lipid metabolism results in the alteration of lipid compositions in lipoproteins; therefore an accurate and quantitative analytical approach is required for the detailed structural characterization of lipoproteins. However, the specific lipid composition of each lipoprotein particle is poorly understood. Materials & methods: Lipid composition of very-low-density lipoprotein and low-density lipoprotein particles derived from myocardial infarction-prone rabbits was determined by normalization of lipidomics data using apoB-100 levels. Results: The ratio of lipid levels between very-low-density lipoprotein and low-density lipoprotein particles was different according to not only lipid classes, but also phosphatidylethanolamine subclasses by applying our developed methodology to myocardial infarction-prone rabbits. Conclusion: Our novel analytical approach represents to be a potentially useful tool to obtain particle-specific lipid components of lipoproteins..
47. Hiroaki Takeda, Tomonari Koike, Yoshihiro Izumi, Takayuki Yamada, Masaru Yoshida, Masashi Shiomi, Eiichiro Fukusaki, Takeshi Bamba*, Lipidomic analysis of plasma lipoprotein fractions in myocardial infarction-prone rabbits, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2015.02.015, 120, 4, 476-482, 2015.10, Lipids play important roles in the body and are transported to various tissues via lipoproteins. It is commonly assumed that alteration of lipid levels in lipoproteins leads to dyslipidemia and serious diseases such as coronary artery disease (CAD). However, lipid compositions in each lipoprotein fraction induced by lipoprotein metabolism are poorly understood. Lipidomics, which involves the comprehensive and quantitative analysis of lipids, is expected to provide valuable information regarding the pathogenic mechanism of CAD. Here, we performed a lipidomic analysis of plasma and its lipoprotein fractions in myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits. In total, 172 lipids in plasma obtained from normal and WHHLMI rabbits were quantified with high throughput and accuracy using supercritical fluid chromatography hybrid quadrupole-Orbitrap mass spectrometry (SFC/Q-Orbitrap-MS). Plasma levels of each lipid class (i.e., phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, lysophosphatidylcholine, lysophosphatidylethanolamine, sphingomyelin, ceramide, triacylglycerol, diacylglycerol, and cholesterol ester, except for free fatty acids) in 21-month-old WHHLMI rabbits were significantly higher than those in normal rabbits. High levels of functional lipids, such as alkyl-phosphatidylcholines, phospholipids including ω-6 fatty acids, and plasmalogens, were also observed in WHHLMI rabbit plasma. In addition, high-resolution lipidomic analysis using very low density lipoprotein (VLDL) and low density lipoprotein (LDL) provided information on the specific molecular species of lipids in each lipoprotein fraction. In particular, higher levels of phosphatidylethanolamine plasmalogens were detected in LDL than in VLDL. Our lipidomics approach for plasma lipoprotein fractions will be useful for in-depth studies on the pathogenesis of CAD..
48. Megumi Ishibashi, Yoshihiro Izumi, Miho Sakai, Takashi Ando, Eiichiro Fukusaki, Takeshi Bamba*, High-throughput simultaneous analysis of pesticides by supercritical fluid chromatography coupled with high-resolution mass spectrometry, Journal of Agricultural and Food Chemistry, 10.1021/jf5056248, 63, 18, 4457-4463, 2015.05, Recently, a generally applicable screening method for multiresidue pesticide analysis, which is simple, quick, and accurate and has a reliable performance, is becoming increasingly important for food safety and international trade. This paper proposes a high-throughput screening methodology that enables the detection of multiresidue pesticides using supercritical fluid chromatography coupled to a high-performance benchtop quadrupole Orbitrap mass spectrometry (SFC/Q Exactive) and an automated library-based detection. A total of 444 chemicals covering a wide polarity range (logPow from -4.2 to 7.7) and a wide molecular weight range (from 99.0 to 872.5) were analyzed simultaneously through a combination of high mass resolution (a value of m/Δm = 70000), high mass accuracy (<5 ppm) with positive/negative polarity switching, and highly efficient separation by SFC. A total of 373 pesticides were detected in QuEChERS spinach extracts without dispersive solid phase extraction at the 10 μg kg-1 level (provisional maximum residue limits in Japan). In conclusion, the developed analytical system is a potentially useful tool for practical multiresidue pesticide screening with high throughput (time for data acquisition, 72 samples per day; and time for data processing of 72 samples, approximately 45 min)..
49. Hiroshi Tsugawa*, Erika Ohta, Yoshihiro Izumi, Atsushi Ogiwara, Daichi Yukihira, Takeshi Bamba, Eiichiro Fukusaki, Masanori Arita, MRM-DIFF
Data processing strategy for differential analysis in large scale MRM-based lipidomics studies, Frontiers in Genetics, 10.3389/fgene.2014.00471, 5, JAN, 2015.01, Based on theoretically calculated comprehensive lipid libraries, in lipidomics as many as 1000 multiple reaction monitoring (MRM) transitions can be monitored for each single run. On the other hand, lipid analysis from each MRM chromatogram requires tremendous manual efforts to identify and quantify lipid species. Isotopic peaks differing by up to a few atomic masses further complicate analysis. To accelerate the identification and quantification process we developed novel software, MRM-DIFF, for the differential analysis of large-scale MRM assays. It supports a correlation optimized warping (COW) algorithm to align MRM chromatograms and utilizes quality control (QC) sample datasets to automatically adjust the alignment parameters. Moreover, user-defined reference libraries that include the molecular formula, retention time, and MRM transition can be used to identify target lipids and to correct peak abundances by considering isotopic peaks. Here, we demonstrate the software pipeline and introduce key points for MRM-based lipidomics research to reduce the mis-identification and overestimation of lipid profiles. The MRM-DIFF program, example data set and the tutorials are downloadable at the "Standalone software" section of the PRIMe..
50. Atsuki Matsubara, Yoshihiro Izumi, Shin Nishiumi, Makoto Suzuki, Takeshi Azuma, Eiichiro Fukusaki, Takeshi Bamba*, Masaru Yoshida*, Supercritical fluid extraction as a preparation method for mass spectrometry of dried blood spots, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 10.1016/j.jchromb.2014.08.013, 969, 199-204, 2014.10, The potential of supercritical fluid extraction (SFE) as a preparation method for mass spectrometry of dried blood spots (DBS) was examined. SFE is generally used for the extraction of hydrophobic compounds, but hydrophilic metabolites such as amino acids, amines, and nucleic-acid-related metabolites could be extracted by adding a low level of methanol as a modifier. Under the optimized conditions, over 200 metabolites were detected from a dried serum spot, of which over 160 metabolites could be analyzed stably (RSD <20%). These results show that SFE is an effective extraction method of metabolites with a wide range of polarity in DBS..
51. Yoshihiro Izumi, Kosuke Aritake, Yoshihiro Urade, Eiichiro Fukusaki*, Practical evaluation of liquid chromatography/tandem mass spectrometry and enzyme immunoassay method for the accurate quantitative analysis of prostaglandins, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2013.12.022, 118, 1, 116-118, 2014.01, The accurate and robust measurement of prostaglandins (PG) concentration could help to understand the many physiological functions. The present study revealed that liquid chromatography/tandem mass spectrometry method for the PGs analysis can satisfy the requirements for both qualitative and quantitative performance as compared to competitive enzyme immunoassays..
52. Yoshihiro Izumi, Shimpei Aikawa, Fumio Matsuda, Tomohisa Hasunuma, Akihiko Kondo*, Aqueous size-exclusion chromatographic method for the quantification of cyanobacterial native glycogen, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 10.1016/j.jchromb.2013.04.037, 930, 90-97, 2013.07, Cyanobacterial glycogen has gained interest as a valuable biomass feedstock for biofuel production. However, an ideal method for native glycogen quantification has not been developed. Here, we have proposed a simple methodology that enables the quantitative determination of cyanobacterial glycogen concentration with high repeatability using aqueous size-exclusion chromatography with a differential refractive index detector (SEC/RID). Our SEC/RID system also allows size distributions for native glycogen based on hydrodynamic volumes (Vh), which is proportional to the product of the molecular mass (M) and intrinsic viscosity [η], obtained by universal calibration using linear homopolymers of known M with Mark-Houwink-Sakurada parameters. The universal calibration curve achieved a broad linear range (Vh parameter [η]M=2×102-8×108mLg-1) with a high correlation coefficient (R2=0.9942), because the developed system is equipped with an OHpak SB-806M HQ aqueous column containing four types of polyhydroxy methacrylate-based particles with different particle and pore sizes. Based on the SEC/RID system, response of molecular size distribution of glycogen in microalgae to the cultivation condition was first observed. Our established SEC/RID method has several advantages over conventional techniques, including the simultaneous quantitative and size distribution analyses of glycogen, and represents a potentially useful tool to elucidate the relationship between structural properties and the roles of glycogen in metabolism..
53. Tomohisa Hasunuma*, Fumi Kikuyama, Mami Matsuda, Shimpei Aikawa, Yoshihiro Izumi, Akihiko Kondo, Dynamic metabolic profiling of cyanobacterial glycogen biosynthesis under conditions of nitrate depletion, Journal of Experimental Botany, 10.1093/jxb/ert134, 64, 10, 2943-2954, 2013.07, Cyanobacteria represent a globally important biomass because they are responsible for a substantial proportion of primary production in the hydrosphere. Arthrospira platensis is a fast-growing halophilic cyanobacterium capable of accumulating glycogen and has the potential to serve as a feedstock in the fermentative production of third-generation biofuels. Accordingly, enhancing cyanobacterial glycogen production is a promising biofuel production strategy. However, the regulatory mechanism of glycogen metabolism in cyanobacteria is poorly understood. The aim of the present study was to determine the metabolic flux of glycogen biosynthesis using a dynamic metabolomic approach. Time-course profiling of widely targeted cyanobacterial metabolic intermediates demonstrated a global metabolic reprogramming that involves transient increases in the levels of some amino acids during the glycogen production phase induced by nitrate depletion. Also, in vivo labelling with NaH13CO3 enabled direct measurement of metabolic intermediate turnover in A. platensis, revealing that under conditions of nitrate depletion glycogen is biosynthesized with carbon derived from amino acids released from proteins via gluconeogenesis. This dynamic metabolic profiling approach provided conclusive evidence of temporal alterations in the metabolic profile in cyanobacterial cells..
54. Hideo Ohira*, Yoshio Fujioka, Chikae Katagiri, Rie Mamoto, Michiko Aoyama-Ishikawa, Katsumi Amako, Yoshihiro Izumi, Shin Nishiumi, Masaru Yoshida, Makoto Usami, Masamichi Ikeda, Butyrate attenuates inflammation and lipolysis generated by the interaction of adipocytes and macrophages, Journal of atherosclerosis and thrombosis, 10.5551/jat.15065, 20, 5, 425-442, 2013.06, Aim: Paracrine interaction between macrophages and adipocytes in obese visceral fat tissues is thought to be a trigger of chronic inflammation. The immunomodulatory effect of the short chain fatty acid, butyric acid, has been demonstrated. We hypothesize that sodium butyrate (butyrate) attenuates inflammatory responses and lipolysis generated by the interaction of macrophages and adipocytes. Methods: Using contact or transwell co-culture methods with differentiated 3T3-L1 adipocytes and RAW264.7 macrophages, we investigated the effects of butyrate on the production of tumor necrosis factor alpha (TNF-α), monocyte chemoattractant protein 1 (MCP-1), interleukin 6 (IL-6), and the release of free glycerol, free fatty acids (FFAs) into the medium. We also examined the activity of nuclear factor-kappaB (NF-κB) and the phosphorylation of mitogen-activated protein kinases (MAPKs) in co-cultured macrophages, as well as lipase activity and expression in co-cultured adipocytes. Results: We found increased production of TNF-α, MCP-1, IL-6, and free glycerol, FFAs in the coculture medium, and butyrate significantly reduced them. Butyrate inhibited the phosphorylation of MAPKs, the activity of NF-κB in co-cultured macrophages, and suppressed lipase activity in co-cultured adipocytes. Lipase inhibitors significantly attenuated the production of TNF-α, MCP-1 and IL-6 in the co-culture medium as effectively as butyrate. Butyrate suppressed the protein production of adipose triglyceride lipase, hormone sensitive lipase, and fatty acid-binding protein 4 in co-cultured adipocytes. Pertussis toxin, which is known to block GPR41 completely, inhibited the antilipolysis effect of butyrate. Conclusion: Butyrate suppresses inflammatory responses generated by the interaction of adipocytes and macrophages through reduced lipolysis and inhibition of inflammatory signaling..
55. Shimpei Aikawa, Ancy Joseph, Ryosuke Yamada, Yoshihiro Izumi, Takahiro Yamagishi, Fumio Matsuda, Hiroshi Kawai, Jo Shu Chang, Tomohisa Hasunuma, Akihiko Kondo*, Direct conversion of Spirulina to ethanol without pretreatment or enzymatic hydrolysis processes, Energy and Environmental Science, 10.1039/c3ee40305j, 6, 6, 1844-1849, 2013.06, Oxygenic photosynthetic microorganisms such as cyanobacteria and microalgae have attracted attention as feedstocks for next-generation biofuels. To date, however, there have been no reports on efficient bioethanol production from cyanobacterial glycogen by yeast fermentation. Additionally, multiple pretreatment and enzymatic hydrolysis steps of polysaccharides are required for conventional ethanol production from agricultural crops and microalgae. Here, we investigate direct ethanol production from Arthrospira (Spirulina) platensis, a fast-growing halophilic cyanobacterium that accumulates large amounts of glycogen, using lysozyme and a recombinant amylase-expressing yeast strain to eliminate the need for biomass pretreatment and amylase hydrolysis. In the direct conversion process from A. platensis to ethanol, 6.5 g L-1 (ethanol productivity of 1.08 g per L per day) of ethanol was produced. The total ethanol yield based on glycogen consumption was 86% of theoretical yield, which to our knowledge, is the highest yield of bioethanol from an oxygenic photosynthetic microorganism. The present findings indicate that A. platensis is a remarkable carbohydrate feedstock in the form of glycogen, which is a promising material for the production of bioethanol and various other commercially valuable chemicals..
56. Toshihiko Okada, Shinji Fukuda, Koji Hase, Shin Nishiumi, Yoshihiro Izumi, Masaru Yoshida, Teruki Hagiwara, Rei Kawashima, Motomi Yamazaki, Tomoyuki Oshio, Takeshi Otsubo, Kyoko Inagaki-Ohara, Kazuki Kakimoto, Kazuhide Higuchi, Yuki I. Kawamura, Hiroshi Ohno, Taeko Dohi*, Microbiota-derived lactate accelerates colon epithelial cell turnover in starvation-refed mice, Nature communications, 10.1038/ncomms2668, 4, Article number 1654, 2013.05, Oral food intake influences the morphology and function of intestinal epithelial cells and maintains gastrointestinal cell turnover. However, how exactly these processes are regulated, particularly in the large intestine, remains unclear. Here we identify microbiota-derived lactate as a major factor inducing enterocyte hyperproliferation in starvation-refed mice. Using bromodeoxyuridine staining, we show that colonic epithelial cell turnover arrests during a 12- to 36-h period of starvation and increases 12-24 h after refeeding. Enhanced epithelial cell proliferation depends on the increase in live Lactobacillus murinus, lactate production and dietary fibre content. In the model of colon tumorigenesis, mice exposed to a carcinogen during refeeding develop more aberrant crypt foci than mice fed ad libitum. Furthermore, starvation after carcinogen exposure greatly reduced the incidence of aberrant crypt foci. Our results indicate that the content of food used for refeeding as well as the timing of carcinogen exposure influence the incidence of colon tumorigenesis in mice..
57. Takashi Kobayashi, Shin Nishiumi, Atsuki Ikeda, Tomoo Yoshie, Aya Sakai, Atsuki Matsubara, Yoshihiro Izumi, Hidetaka Tsumura, Masahiro Tsuda, Hogara Nishisaki, Nobuhide Hayashi, Seiji Kawano, Yutaka Fujiwara, Hironobu Minami, Tadaomi Takenawa, Takeshi Azuma, Masaru Yoshida*, Anovel serum metabolomics-based diagnostic approach to pancreatic cancer, Cancer Epidemiology Biomarkers and Prevention, 10.1158/1055-9965.EPI-12-1033, 22, 4, 571-579, 2013.04, Background: To improve the prognosis of patients with pancreatic cancer, more accurate serum diagnostic methods are required. We used serum metabolomics as a diagnostic method for pancreatic cancer. Methods: Sera from patients with pancreatic cancer, healthy volunteers, and chronic pancreatitis were collected at multiple institutions. The pancreatic cancer and healthy volunteers were randomly allocated to the training or the validation set. All of the chronic pancreatitis cases were included in the validation set. In each study, the subjects' serum metabolites were analyzed by gas chromatography mass spectrometry (GC/MS) and a data processing system using an in-house library. The diagnostic model constructed via multiple logistic regression analysis in the training set study was evaluated on the basis of its sensitivity and specificity, and the results were confirmed by the validation set study. Results: In the training set study, which included 43 patients with pancreatic cancer and 42 healthy volunteers, the model possessed high sensitivity (86.0%) and specificity (88.1%) for pancreatic cancer. The use of the model was confirmed in the validation set study, which included 42 pancreatic cancer, 41 healthy volunteers, and 23 chronic pancreatitis; that is, it displayed high sensitivity (71.4%) and specificity (78.1%); and furthermore, it displayed higher sensitivity (77.8%) in resectable pancreatic cancer and lower false-positive rate (17.4%) in chronic pancreatitis than conventional markers. Conclusions: Our model possessed higher accuracy than conventional tumor markers at detecting the resectable patients with pancreatic cancer in cohort including patients with chronic pancreatitis. Impact: It is a promising method for improving the prognosis of pancreatic cancer via its early detection and accurate discrimination from chronic pancreatitis..
58. Kosuke Takebayashi, Kenji Hirose, Yoshihiro Izumi, Takeshi Bamba, Eiichiro Fukusaki*, Application of ion mobility-mass spectrometry to microRNA analysis, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2012.10.006, 115, 3, 332-338, 2013.03, Liquid chromatography/mass spectrometry is widely used for studying sequence determination and modification analysis of small RNAs. However, the efficiency of liquid chromatography-based separation of intact small RNA species is insufficient, since the physiochemical properties among small RNAs are very similar. In this study, we focused on ion mobility-mass spectrometry (IM-MS), which is a gas-phase separation technique coupled with mass spectrometry; we have evaluated the utility of IM-MS for microRNA (miRNA) analysis. A multiply charged deprotonated ion derived from an 18-24-nt-long miRNA was formed by electrospray ionization, and then the time, called the " drift time" , taken by each ion to migrate through a buffer gas was measured. Each multivalent ion was temporally separated on the basis of the charge state and structural formation; 3 types of unique mass-mobility correlation patterns (i.e., chainlike-form, hairpin-form, and dimer-form) were present on the two-dimensional mobility-mass spectrum. Moreover, we found that the ion size (sequence length) and the secondary structures of the small RNAs strongly contributed to the IM-MS-based separation, although solvent conditions such as pH had no effect. Therefore, sequence isomers could also be discerned by the selection of each specific charged ion, i.e., the 6- charged ion reflected a majority among chainlike-, hairpin-, and other structures. We concluded that the IM-MS provides additional capability for separation; thus, this analytical method will be a powerful tool for comprehensive small RNA analysis..
59. Ping Lai, Atsushi Okazawa*, Yoshihiro Izumi, Takeshi Bamba, Eiichiro Fukusaki, Masaaki Yoshikawa, Akio Kobayashi, Effect of gallic acid on peptides released by trypsin digestion of bovine α-casein, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2012.10.003, 115, 3, 259-267, 2013.03, In this study, the effects of gallic acid (GA) on trypsin digestion of commercial α-casein (α-CN), which contains αs1-CN and αs2-CN, and the peptides released during digestion were investigated. Gallic acid showed no effect on the initial rate of digestion. However, the apparent degree of hydrolysis achieved its maximum value after 1 h, then decreased in the presence of GA, suggesting the cross-linking between peptides once released from α-CN during digestion. In the presence of GA, three peaks derived from αs1-CN disappeared and three new peaks appeared in high-performance liquid chromatography (HPLC) analysis. In these peptides, two Met residues corresponding to the Met135 and Met196 in αs1-CN were oxidized to Met sulfoxide residues. The oxidation of Met196 was quicker than that of Met135. The inhibitory activity of TTMPLW (αs1-CN 193-199) against angiotensin I-converting enzyme was reduced slightly by the oxidation of its Met residue..
60. Ping Lai, Atsushi Okazawa*, Yoshihiro Izumi, Takeshi Bamba, Eiichiro Fukusaki, Masaaki Yoshikawa, Akio Kobayashi, Gallic acid oxidizes Met residues in peptides released from bovine β-lactoglobulin by in vitro digestion, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2012.04.019, 114, 3, 297-305, 2012.09, Phenolic compounds (PCs) are frequently present in foods. However, little is known about the effect of PCs on enzymatic digestion process of food proteins and their products. In this study, the effect of gallic acid (GA) on in vitro digestion of β-lactoglobulin (β-LG) was investigated as a model system for analysis of the interaction between PCs and food proteins. GA showed no effect on the initial rate of β-LG digestion. However, after 1.5 h of digestion, the observed degree of hydrolysis of β-LG was lower in the presence than in the absence of GA. The peptides released from β-LG were characterized by LC/IT-TOF-MS and thirty peptides were identified. In particular, four new peaks were obtained following in vitro digestion of β-LG in the presence of GA. Met7, Met24 and Met145 in the peptides corresponding to these peaks were oxidized to methionine sulfoxide residues..
61. Shin Nishiumi, Takashi Kobayashi, Atsuki Ikeda, Tomoo Yoshie, Megumi Kibi, Yoshihiro Izumi, Tatsuya Okuno, Nobuhide Hayashi, Seiji Kawano, Tadaomi Takenawa, Takeshi Azuma, Masaru Yoshida*, A novel serum metabolomics-based diagnostic approach for colorectal cancer, PloS one, 10.1371/journal.pone.0040459, 7, 7, 2012.07, Background: To improve the quality of life of colorectal cancer patients, it is important to establish new screening methods for early diagnosis of colorectal cancer. Methodology/Principal Findings: We performed serum metabolome analysis using gas-chromatography/mass-spectrometry (GC/MS). First, the accuracy of our GC/MS-based serum metabolomic analytical method was evaluated by calculating the RSD% values of serum levels of various metabolites. Second, the intra-day (morning, daytime, and night) and inter-day (among 3 days) variances of serum metabolite levels were examined. Then, serum metabolite levels were compared between colorectal cancer patients (N = 60; N = 12 for each stage from 0 to 4) and age- and sex-matched healthy volunteers (N = 60) as a training set. The metabolites whose levels displayed significant changes were subjected to multiple logistic regression analysis using the stepwise variable selection method, and a colorectal cancer prediction model was established. The prediction model was composed of 2-hydroxybutyrate, aspartic acid, kynurenine, and cystamine, and its AUC, sensitivity, specificity, and accuracy were 0.9097, 85.0%, 85.0%, and 85.0%, respectively, according to the training set data. In contrast, the sensitivity, specificity, and accuracy of CEA were 35.0%, 96.7%, and 65.8%, respectively, and those of CA19-9 were 16.7%, 100%, and 58.3%, respectively. The validity of the prediction model was confirmed using colorectal cancer patients (N = 59) and healthy volunteers (N = 63) as a validation set. At the validation set, the sensitivity, specificity, and accuracy of the prediction model were 83.1%, 81.0%, and 82.0%, respectively, and these values were almost the same as those obtained with the training set. In addition, the model displayed high sensitivity for detecting stage 0-2 colorectal cancer (82.8%). Conclusions/Significance: Our prediction model established via GC/MS-based serum metabolomic analysis is valuable for early detection of colorectal cancer and has the potential to become a novel screening test for colorectal cancer..
62. Aya Sakai, Shin Nishiumi, Yuuki Shiomi, Takashi Kobayashi, Yoshihiro Izumi, Hiromu Kutsumi, Takanobu Hayakumo, Takeshi Azuma, Masaru Yoshida*, Metabolomic analysis to discover candidate therapeutic agents against acute pancreatitis, Archives of Biochemistry and Biophysics, 10.1016/j.abb.2012.03.025, 522, 2, 107-120, 2012.06, Novel and effective drugs against acute pancreatitis are required. Therefore, we examined the changes in the metabolite levels in the serum and pancreatic tissue of mice with cerulein- and arginine-induced pancreatitis using gas-chromatography/mass-spectrometry (GC/MS) and investigated whether these alterations affected the severity of acute pancreatitis. In the cerulein-induced pancreatitis model, 93 and 129 metabolites were detected in the serum and pancreatic tissue, respectively. In the l-arginine-induced acute pancreatitis model, 120 and 133 metabolites were detected in the serum and pancreatic tissue, respectively. Among the metabolites, the concentrations of tricarboxylic acid (TCA) cycle intermediates and amino acids were altered in pancreatitis, and in pancreatic tissue, the levels of the intermediates involved in the initial part of the TCA cycle were increased and those of the intermediates involved in the latter part of the TCA cycle were decreased. Some metabolites exhibited similar changes in both pancreatitis mouse models, e.g., the levels of glutamic acid and O-phosphoethanolamine were significantly decreased in the pancreatic tissue. Supplementation with glutamic acid and O-phosphoethanolamine attenuated the severity of cerulein-induced acute pancreatitis. Our results suggest that GC/MS-based metabolomics is capable of accurately representing the status of acute pancreatitis, leading to the discovery of therapeutic agents for pancreatitis..
63. Tomoo Yoshie, Shin Nishiumi, Yoshihiro Izumi, Aya Sakai, Jun Inoue, Takeshi Azuma, Masaru Yoshida*, Regulation of the metabolite profile by an APC gene mutation in colorectal cancer, Cancer Science, 10.1111/j.1349-7006.2012.02262.x, 103, 6, 1010-1021, 2012.06, Mutation of the APC gene occurs during the early stages of colorectal cancer development. To obtain new insights into the mechanisms underlying the aberrant activation of the Wnt pathway that accompanies APC mutation, we carried out a gas chromatography-mass spectrometry-based semiquantitative metabolome analysis. In vitro experiments comparing SW480 cells expressing normal APC and truncated APC indicated that the levels of metabolites involved in the latter stages of the intracellular tricarboxylic acid cycle, including succinic acid, fumaric acid, and malic acid, were significantly higher in the SW480 cells expressing the truncated APC. In an in vivo study, we found that the levels of most amino acids were higher in the non-polyp tissues of APC
min/+
mice than in the normal tissues of the control mice and the polyp tissues of APC
min/+
mice. Ribitol, the levels of which were decreased in the polyp lesions of the APC
min/+
mice and the SW480 cells expressing the truncated APC, reduced the growth of SW480 cells with the APC mutation, but did not affect the growth of SW480 transfectants expressing full-length APC. The level of sarcosine was found to be significantly higher in the polyp tissues of APC
min/+
mice than in their non-polyp tissues and the normal tissues of the control mice, and the treatment of SW480 cells with 50 μM sarcosine resulted in a significant increase in their growth rate. These findings suggest that APC mutation causes changes in energetic metabolite pathways and that these alterations might be involved in the development of colorectal cancer..
64. Hiroko Kato, Yoshihiro Izumi, Tomohisa Hasunuma, Fumio Matsuda, Akihiko Kondo*, Widely targeted metabolic profiling analysis of yeast central metabolites, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2011.12.013, 113, 5, 665-673, 2012.05, A method for a widely targeted analysis was developed for the metabolic profiling of yeast central metabolism. The widely targeted method consists of 2 analyses, namely, gas chromatography-quadrupole-mass spectrometry (GC-Q-MS) operated in selected ion monitoring mode with 25m/z channels, and liquid chromatography triple-stage quadrupole (LC-QqQ)-MS operated in multiple reaction monitoring mode. This platform was set up to identify and quantify preselected 99 compounds, including sugars, sugar phosphates, organic acids, amino acids, and cofactors. The method showed good sensitivity and a wide dynamic range. For example, limits of detection for lactate and l-phenylalanine were 1.4fmol and 2.0fmol, respectively. The dynamic ranges for GC-Q-MS analysis and LC-QqQ-MS analysis were approximately 102-105 and 103-104, respectively. The metabolite profiles of 2 yeast strains, YPH499 and BY4741, under glucose-fermenting conditions were compared using the developed method. Although YPH499 and BY4741 were derived from an identical experimental strain, the profiling analysis successfully revealed a variation in metabolic phenotypes among experimental yeast strains demonstrating that the widely targeted method could be a robust and useful method for the investigation of metabolic phenotypes of Saccharomyces cerevisiae..
65. Yoshihiro Izumi, Shin Takimura, Shinichi Yamaguchi, Junko Iida, Takeshi Bamba, Eiichiro Fukusaki*, Application of electrospray ionization ion trap/time-of-flight mass spectrometry for chemically-synthesized small RNAs, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2011.11.007, 113, 3, 412-419, 2012.03, In this study, we have demonstrated an accurate and rapid small RNA analytical method with both sequence determination and detailed modification analysis by electrospray ionization-ion trap/time-of-flight mass spectrometry (ESI-IT/TOFMS). To develop this ideal method, we have examined the performance of ESI-IT/TOFMS using various chemically-synthesized model sequences of modified or unmodified microRNAs (miRNAs). The deconvoluted mass of a 22-nucleotide (nt) miRNA was obtained from a multiply charged precursor ion (MS
1
). The ion exhibited high mass accuracy (<7ppm) and high mass resolution (a value of m/δm=10,000) and was therefore very useful in RNA composition assignment. The optimized MS
2
method using ion trap collision-induced dissociation, as well as automatic annotation analysis of product ions based on the accurate mass information, enabled the precise sequencing determination of intact miRNAs. Further, the detailed structural analysis of 3'-terminal modified nucleic acid in intact methylated miRNA was carried out using the MS
3
capability of the hybrid IT/TOFMS. The direct infusion method also provided a high throughput and good sensitivity because the analytical time and sample concentration needed in a series of experiments with reliable data were only 3min and 100nM, respectively. This study provides a novel approach for characterizing the intact chemically-synthesized small RNA without chemical and enzymatic digestions and would be widely applicable for the structural analysis of complicated modified small RNAs..
66. Shimpei Aikawa, Yoshihiro Izumi, Fumio Matsuda, Tomohisa Hasunuma, Jo Shu Chang, Akihiko Kondo*, Synergistic enhancement of glycogen production in Arthrospira platensis by optimization of light intensity and nitrate supply, Bioresource Technology, 10.1016/j.biortech.2012.01.004, 108, 211-215, 2012.03, Arthrospira (Spirulina) platensis, a fast-growing halophilic cyanobacterium able to accumulate glycogen, was investigated for its feasibility to serve as feedstock for fermentative production of biofuels and chemicals. The culture conditions most appropriate for glycogen production were identified. Glycogen production was maximized by the depleting nitrate source under a high light intensity of 700μmol photons m -2s -1. With optimal control of both light intensity and nitrate supply, glycogen production of A. platensis reached nearly 1.03gL -1 (a glycogen productivity of 0.29gL -1d -1), which is, to the best of our knowledge, the highest α-polyglucan (glycogen or starch) production performance ever reported in microalgae. The outcome of this work supports A. platensis as a promising carbohydrate source for biorefinery..
67. Masaru Yoshida*, Naoya Hatano, Shin Nishiumi, Yasuhiro Irino, Yoshihiro Izumi, Tadaomi Takenawa, Takeshi Azuma, Diagnosis of gastroenterological diseases by metabolome analysis using gas chromatography-mass spectrometry, Journal of Gastroenterology, 10.1007/s00535-011-0493-8, 47, 1, 9-20, 2012.01, Recently, metabolome analysis has been increasingly applied to biomarker detection and disease diagnosis in medical studies. Metabolome analysis is a strategy for studying the characteristics and interactions of low molecular weight metabolites under a specific set of conditions and is performed using mass spectrometry and nuclear magnetic resonance spectroscopy. There is a strong possibility that changes in metabolite levels reflect the functional status of a cell because alterations in their levels occur downstream of DNA, RNA, and protein. Therefore, the metabolite profile of a cell is more likely to represent the current status of a cell than DNA, RNA, or protein. Thus, owing to the rapid development of mass spectrometry analytical techniques metabolome analysis is becoming an important experimental method in life sciences including the medical field. Here, we describe metabolome analysis using liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry (GC-MS), capillary electrophoresis-mass spectrometry, and matrix assisted laser desorption ionization-mass spectrometry. Then, the findings of studies about GC-MS-based metabolome analysis of gastroenterological diseases are summarized, and our research results are also introduced. Finally, we discuss the realization of disease diagnosis by metabolome analysis. The development of metabolome analysis using mass spectrometry will aid the discovery of novel biomarkers, hopefully leading to the early detection of various diseases..
68. Takanori Sugimoto, Takeshi Bamba, Yoshihiro Izumi, Hironari Nomura, Takashi Shiina, Eiichiro Fukusaki*, Use of ultra-performance liquid chromatography/time-of-flight mass spectrometry with nozzle-skimmer fragmentation for comprehensive quantitative analysis of secondary metabolites in Arabidopsis thaliana, Journal of Separation Science, 10.1002/jssc.201100552, 34, 24, 3587-3596, 2011.12, This study sought to develop techniques for LC/MS-based metabolomics and to verify that an MS/MS spectral tag (MS2T) could be used in practical secondary metabolite profiling. The retention time (RT), precursor ions, and fragment ions generated by nozzle-skimmer fragmentation were determined using ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOF-MS) and compared with the MS2T. A standard mix was analyzed with UPLC/TOF-MS under the same conditions as were used to construct the MS2T. The difference in RT for the standards was less than 0.15 min and the average RSD was about 2.8%, suggesting that the analysis was highly repeatable. Both precursor ions and fragment ions were observed when the cone voltage was 75 V. Experimental data and fragmentation pattern in the MS2T annotation list were highly similar. Wild-type and cas-1 mutant Arabidopsis thaliana samples treated with an elicitor were analyzed using UPLC/TOF-MS. Sixty-five peaks were successfully annotated. Fragment ions were observed with nozzle-skimmer fragmentation in 50 of 65 (77%) peaks. The reliability of annotation may have increased as a result of fragment ions. Results of multivariate analysis suggested that cas-1 was related to induction of the biosynthesis of these flavonoids. The devised method facilitated practical secondary metabolite profiling..
69. Atsushi Okazawa*, Katsuhito Hori, Yohei Okumura, Yoshihiro Izumi, Naoki Hata, Takeshi Bamba, Eiichiro Fukusaki, Eiichiro Ono, Honoo Satake, Akio Kobayashi, Simultaneous quantification of lignans in Arabidopsis thaliana by highly sensitive capillary liquid chromatography-electrospray ionization-ion trap mass spectrometry, Plant Biotechnology, 10.5511/plantbiotechnology.11.0221a, 28, 3, 287-293, 2011.08, Lignans are phenylpropanoid dimers in which the monomers are linked by the central carbon (C8) atoms. Because many lignans have physiological effects, including antioxidant activity, they are now in demand as components in health foods. However, the lignan biosynthetic pathways in plants are only now being understood. Recently, lariciresinol was detected in Arabidopsis thaliana. This observation indicated the existence of common lignan biosynthetic pathways in A. thaliana, despite a low amount of lignans other than lariciresinol glycosides. In this study, we established a highly sensitive analytical method that enables quantification of both glycoside and aglycone forms of lignans in A. thaliana simultaneously using capillary liquid chromatography-electrospray ionization-ion trap mass spectrometry. Some lignans not previously detected in A. thaliana were quantified in extracts of both roots and shoots. Our method can be used for the comprehensive analysis of lignans in small samples from mutants and transformants. This method will be utilized to elucidate the metabolic pathways and physiological roles of lignans as well as the regulation of their biosynthesis in plants..
70. Yoshihiro Izumi, Atsushi Okazawa, Takeshi Bamba, Akio Kobayashi, Eiichiro Fukusaki*, Development of a method for comprehensive and quantitative analysis of plant hormones by highly sensitive nanoflow liquid chromatography-electrospray ionization-ion trap mass spectrometry, Analytica Chimica Acta, 10.1016/j.aca.2009.07.001, 648, 2, 215-225, 2009.08, In recent plant hormone research, there is an increased demand for a highly sensitive and comprehensive analytical approach to elucidate the hormonal signaling networks, functions, and dynamics. We have demonstrated the high sensitivity of a comprehensive and quantitative analytical method developed with nanoflow liquid chromatography-electrospray ionization-ion trap mass spectrometry (LC-ESI-IT-MS/MS) under multiple-reaction monitoring (MRM) in plant hormone profiling. Unlabeled and deuterium-labeled isotopomers of four classes of plant hormones and their derivatives, auxins, cytokinins (CK), abscisic acid (ABA), and gibberellins (GA), were analyzed by this method. The optimized nanoflow-LC-ESI-IT-MS/MS method showed ca. 5-10-fold greater sensitivity than capillary-LC-ESI-IT-MS/MS, and the detection limits (S/N = 3) of several plant hormones were in the sub-fmol range. The results showed excellent linearity (R2 values of 0.9937-1.0000) and reproducibility of elution times (relative standard deviations, RSDs, <1.1%) and peak areas (RSDs, <10.7%) for all target compounds. Further, sample purification using Oasis HLB and Oasis MCX cartridges significantly decreased the ion-suppressing effects of biological matrix as compared to the purification using only Oasis HLB cartridge. The optimized nanoflow-LC-ESI-IT-MS/MS method was successfully used to analyze endogenous plant hormones in Arabidopsis and tobacco samples. The samples used in this analysis were extracted from only 17 tobacco dry seeds (1 mg DW), indicating that the efficiency of analysis of endogenous plant hormones strongly depends on the detection sensitivity of the method. Our analytical approach will be useful for in-depth studies on complex plant hormonal metabolism..
71. Yoshihiro Izumi, Shin'Ichiro Kajiyama*, Ryosuke Nakamura, Atsushi Ishihara, Atsushi Okazawa, Eiichiro Fukusaki, Yasuo Kanematsu, Akio Kobayashi, High-resolution spatial and temporal analysis of phytoalexin production in oats, Planta, 10.1007/s00425-008-0887-x, 229, 4, 931-943, 2009.03, The production of oat (Avena sativa L.) phytoalexins, avenanthramides, occurs in response to elicitor treatment with oligo-N- acetylchitooligosaccharides. In this study, avenanthramides production was investigated by techniques that provide high spatial and temporal resolution in order to clarify the process of phytoalexin production at the cellular level. The amount of avenanthramides accumulation in a single mesophyll cell was quantified by a combination of laser micro-sampling and low-diffuse nanoflow liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) techniques. Avenanthramides, NAD(P)H and chlorophyll were also visualized in elicitor-treated mesophyll cells using line-scanning fluorescence microscopy. We found that elicitor-treated mesophyll cells could be categorized into three characteristic cell phases, which occurred serially over time. Phase 0 indicated the normal cell state before metabolic or morphological change in response to elicitor, in which the cells contained abundant NAD(P)H. In phase 1, rapid NAD(P)H oxidation and marked movement of chloroplasts occurred, and this phase was the early stage of avenanthramides biosynthesis. In phase 2, avenanthramides accumulation was maximized, and chloroplasts were degraded. Avenanthramides appear to be synthesized in the chloroplast, because a fluorescence signal originating from avenanthramides was localized to the chloroplasts. Moreover, our results indicated that avenanthramides biosynthesis and the hypersensitive response (HR) occurred in identical cells. Thus, the avenanthramides production may be one of sequential events programmed in HR leading to cell death. Furthermore, the phase of the defense response was different among mesophyll cells simultaneously treated with elicitor. These results suggest that individual cells may have different susceptibility to the elicitor..
72. Daisuke Igarashi*, Yoshihiro Izumi, Yuko Dokiya, Kazuhiko Totsuka, Eiichiro Fukusaki, Chieko Ohsumi, Reproductive organs regulate leaf nitrogen metabolism mediated by cytokinin signal, Planta, 10.1007/s00425-008-0858-2, 229, 3, 633-644, 2009.02, The metabolism of vegetative organs in plants changes during the development of the reproductive organs. The regulation of this metabolism is important in the control of crop productivity. However, the complexity of the regulatory systems makes it difficult to elucidate their mechanisms. To examine these mechanisms, we constructed model experiments using Arabidopsis to analyze metabolic and gene expression changes during leaf-stage progression and after removal of the reproductive organs. Leaf gene expression levels and content of major amino acids, both of which decreased during leaf-stage progression, increased after removal of the reproductive organs. In particular, the levels of expression of cytokinin biosynthesis genes and cytokinin-responsive genes and the cytokinin content increased after removal of the reproductive organs. Analysis of plants with knockout of a cytokinin-biosynthetic gene (AtIPT3) and a cytokinin receptor gene (AHK3) indicated that glutamate dehydrogenase genes (GDH3) were regulated by cytokinin signaling. These data suggest that cytokinins regulate communication between reproductive and vegetative organs, and that GDH3 is one target of the cytokinin-mediated regulation of nitrogen metabolism..
73. Ryosuke Nakamura, Yoshihiro Izumi, Shin'Ichiro Kajiyama, Akio Kobayashi, Yasuo Kanematsu*, Line-scanning microscopy for time-gated and spectrally resolved fluorescence imaging, Journal of Biological Physics, 10.1007/s10867-008-9113-0, 34, 1-2 SPEC. ISS., 51-62, 2008.04, Laser-scanning fluorescence microscopy for efficient acquisition of time-gated and spectrally resolved fluorescence images was developed based on line illumination of the laser beam and detection of the fluorescence image through a slit. In this optical arrangement, the fluorescence image was obtained by scanning only one axis perpendicular to the excitation line, and the acquisition time was significantly reduced compared with conventional laser-scanning confocal microscopy. A multidimensional fluorescence dataset consisting of fluorescence intensities as a function of x-position, y-position, fluorescence wavelength, and delay time after photoexcitation was analyzed and decomposed based on the parallel factor analysis model. The performance of the line-scanning microscopy was examined by applying it to the analysis of one of the plant defense responses, accumulation of antimicrobial compounds of phytoalexin in oat (Avena sativa), induced by the elicitor treatment..
74. Shin'ichiro Kajiyama*, Takeshi Shoji, Shinya Okuda, Yoshihiro Izumi, Ei Ichiro Fukusaki, Akio Kobayashi, A novel microsurgery method for intact plant tissue at the single cell level using ArF excimer laser microprojection, Biotechnology and Bioengineering, 10.1002/bit.20709, 93, 2, 325-331, 2006.02, A novel microsurgery technique for the partial removal of rigid cell-walls in intact plant tissue is established. Using a size-variable slit, an ArF excimer laser was microprojected on the surface of the targeted cell, and this method enabled the area- and depth-controllable processing of the cortical structure of plant cells including the cuticle and cell wall layer. In epidermal cells of all tested plants, viabilities of more than 90% were retained 24 h after irradiation. Scanning electron microscope (SEM) observation revealed that the cuticle layer of the irradiated region was completely ablated, and the cellulose microfibrils of the secondary cell wall were partially removed; furthermore, 4 days after laser treatment, the regeneration of cell wall fibrils was observed. As a model experiment, the transient expression of synthetic green fluorescent protein (sGFP) was performed by the microinjection of cauliflower mosaic virus (CMV) 35S promoter-derived sGFP gene through an "aperture" in the treated cell surface. Moreover, micron-sized fluorescent beads were successfully introduced by the same method into the onion cells indicating that this method can be used to introduce foreign materials as large as organelles..