Details of a Researcher - Soga, Tomoyoshi

Papers - Soga, Tomoyoshi

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Prediction of metabolite identity from accurate mass, migration time prediction and isotopic pattern information in CE-TOFMS data

Sugimoto, M., Hirayama, A., Robert, M., Abe, S., Soga, T., Tomita, M.

Electrophoresis 31 ( 14 ) 2311 - 2318 2010.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 0173-0835

CE-TOFMS is a powerful method for profiling charged metabolites. However, the limited availability of metabolite standards hinders the process of identifying compounds from detected features in CE-TOFMS data sets. To overcome this problem, we developed a method to identify unknown peaks based on the predicted migration time (t(m)) and accurate m/z values. We developed a predictive model using 375 standard cationic metabolites and support vector regression. The model yielded good correlations between the predicted and measured t(m) (R=0.952 and 0.905 using complete and cross-validation data sets, respectively). Using the trained model, we subsequently predicted the t(m) for 2938 metabolites available from the public databases and assigned tentative identities to noise-filtered features in human urine samples. While 38.9% of the peaks were assigned metabolite names by matching with the standard library alone, the proportion increased to 52.2%. The proposed methodology increases the value of metabolomic data sets obtained from CE-TOFMS profiling.

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Unveiling cellular biochemical reactions via metabolomics-driven approaches

Saito, N., Ohashi, Y., Soga, T., Tomita, M.

Curr. Opin. Microbiol. 13 ( 3 ) 358 - 362 2010.06

Research paper (scientific journal), Joint Work, Accepted

Despite the widespread availability of genome sequences and the systematic assignment of individual open reading frames, many of the activities of metabolic enzymes are still veiled. It is difficult to assign biochemical functions to enzymes and uncover the in vivo regulation of their enzymatic activities, but this is crucial to understand cellular functions. The development of metabolomics and metabolite profiling, which can globally analyze intracellular and extracellular metabolites, has provided some valuable tools for systems biology. This report reviews the recent metabolomics approaches to elucidate unidentified metabolic systems while focusing on their enzymatic activities, including their biochemical and biological functions, and their regulation in response to environmental perturbations in microorganisms.

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A systematic survey of in vivo obligate chaperonin‐dependent substrates

Fujiwara, K., Ishihama, Y., Nakahigashi, K., Soga, T., Taguchi, H.,

EMBO J. 29 ( 9 ) 1552 - 1564 2010.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 14602075

Chaperonins are absolutely required for the folding of a subset of proteins in the cell. An earlier proteome-wide analysis of Escherichia coli chaperonin GroEL/GroES (GroE) interactors predicted obligate chaperonin substrates, which were termed Class III substrates. However, the requirement of chaperonins for in vivo folding has not been fully examined. Here, we comprehensively assessed the chaperonin requirement using a conditional GroE expression strain, and concluded that only approximately 60% of Class III substrates are bona fide obligate GroE substrates in vivo. The in vivo obligate substrates, combined with the newly identified obligate substrates, were termed Class IV substrates. Class IV substrates are restricted to proteins with molecular weights that could be encapsulated in the chaperonin cavity, are enriched in alanine/glycine residues, and have a strong structural preference for aggregation-prone folds. Notably, approximately 70% of the Class IV substrates appear to be metabolic enzymes, supporting a hypothetical role of GroE in enzyme evolution.

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Gene knockout and metabolome analysis of carnitine/organic cation transporter OCTN1

Kato, Y., Kubo, Y., Iwata, D., Kato, S., Sudo, T., Sugiura, T., Kagaya, T., Wakayama, T., Hirayama, A., Sugimoto, M., Sugihara, K., Kaneko, S., Soga, T., Asano, M., Tomita, M., Matsui, T., Wada, M., Tsuji, A.

Pharm. Res. 27 ( 5 ) 832 - 840 2010.05

Research paper (scientific journal), Joint Work, Accepted

Purpose: Solute carrier OCTN1 (SLC22A4) is an orphan transporter, the physiologically important substrate of which is still unidentified. The aim of the present study was to examine physiological roles of OCTN1.
Methods: We first constructed octn1 gene knockout (octn1 ( -/- )) mice. Metabolome analysis was then performed to identify substrates in vivo. The possible association of the substrate identified with diseased conditions was further examined.
Results: The metabolome analysis of blood and several organs indicated complete deficiency of a naturally occurring potent antioxidant ergothioneine in octn1 ( -/- ) mice among 112 metabolites examined. Pharmacokinetic analyses after oral administration revealed the highest distribution to small intestines and extensive renal reabsorption of [(3)H]ergothioneine, both of which were much reduced in octn1 ( -/- ) mice. The octn1 ( -/- ) mice exhibited greater susceptibility to intestinal inflammation under the ischemia and reperfusion model. The blood ergothioneine concentration was also much reduced in Japanese patients with Crohn's disease, compared with healthy volunteers and patients with another inflammatory bowel disease, ulcerative colitis.
Conclusions: These results indicate that OCTN1 plays a pivotal role for maintenance of systemic and intestinal exposure of ergothioneine, which could be important for protective effects against intestinal tissue injuries, providing a possible diagnostic tool to distinguish the inflammatory bowel diseases.

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Analysis of Metabolic Remodeling in Compensated Left Ventricular Hypertrophy and Heart Failure

Kato, T., Niizuma, S., Inuzuka, Y., Kawashima, T., Okuda, J., Tamaki, Y., Iwanaga, Y., Narazaki, M., Matsuda, T., Soga, T., Kita, T., Kimura, T., Shioi, T.

Circ. Heart Fail. 3 ( 3 ) 420 - 430 2010.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 1941-3289

Background: Congestive heart failure (CHF) is associated with a change in cardiac energy metabolism. However, the mechanism by which this change is induced and causes the progression of CHF is unclear.
Methods and results: We analyzed the cardiac energy metabolism of Dahl salt-sensitive rats fed a high-salt diet, which showed a distinct transition from compensated left ventricular hypertrophy to CHF. Glucose uptake increased at the left ventricular hypertrophy stage, and glucose uptake further increased and fatty acid uptake decreased at the CHF stage. The gene expression related to glycolysis, fatty acid oxidation, and mitochondrial function was preserved at the left ventricular hypertrophy stage but decreased at the CHF stage and was associated with decreases in levels of transcriptional regulators. In a comprehensive metabolome analysis, the pentose phosphate pathway that regulates the cellular redox state was found to be activated at the CHF stage. Dichloroacetate (DCA), a compound known to enhance glucose oxidation, increased energy reserves and glucose uptake. DCA improved cardiac function and the survival of the animals. DCA activated the pentose phosphate pathway in the rat heart. DCA activated the pentose phosphate pathway, decreased oxidative stress, and prevented cell death of cultured cardiomyocytes.
Conclusions: Left ventricular hypertrophy or CHF is associated with a distinct change in the metabolic profile of the heart. DCA attenuated the transition associated with increased energy reserves, activation of the pentose phosphate pathway, and reduced oxidative stress.

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Chlorogonium complexum sp. nov. (Volvocales, Chlorophyceae), and morphological evolution of Chlorogonium

Nakada, T., Soga, T., Tomita, M., Nozaki, H.

Eur. J. Phycol. 45 ( 1 ) 97 - 107 2010.03

Research paper (scientific journal), Joint Work, Accepted

We isolated strains of a new species of Chlorogonium (Cg.), Cg. complexum Nakada sp. nov. The genus Chlorogonium was recently emended to have several, irregularly distributed contractile vacuoles, two-to-many pyrenoids associated with fragmented starch plates and tubular thylakoid lamellae penetrating their matrices, and stigma composed of a single layer of globules. Chlorogonium complexum has these diagnostic characters of the emended genus. It is distinguished from other species of Chlorogonium, Cg. euchlorum, Cg. elongatum and Cg. capillatum, on the combination of diagnostic characters, such as the number and stability of pyrenoids and the mode of encystment. Based on a dataset combining 18S rRNA, RuBisCO large subunit (rbcL) and P700 chl a-apoprotein A2 (psaB) gene sequences, the phylogenetic position of Chlorogonium was determined. Chlorogonium complexum represented an independent lineage, sister to Cg. euchlorum and Cg. capillatum. The evolution of diagnostic characters among Chlorogonium species is discussed based on the molecular phylogenetic analyses, and a key to species of Chlorogonium and fusiform chlamydomonads with irregularly distributed contractile vacuoles is presented.

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Hypotaurine is an energy-saving hepatoprotective compound against ischemia-reperfusion injury of the rat liver

Sakuragawa, T., Hishiki, T., Ueno, Y., Ikeda, S., Soga, T., Yachie-Kinoshita, A., Kajimura, M., Suematsu, M.

J. Clin. Biochem. Nutr. 46 ( 2 ) 126 - 134 2010.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 0912-0009

Metabolome analyses assisted by capillary electrophoresis-mass spectrometry (CE-MS) have allowed us to systematically grasp changes in small molecular metabolites under disease conditions. We applied CE-MS to mine out biomarkers in hepatic ischemia-reperfusion. Rat livers were exposed to ischemia by clamping of the portal inlet followed by reperfusion. Metabolomic profiling revealed that l contents of taurine in liver and plasma were significantly increased. Of interest is an elevation of hypotaurine, collectively suggesting significance of hypotaurine/taurine in post-ischemic responses. Considering the anti-oxidative capacity of hypotaurine, we examined if supplementation of the compound or its precursor amino acids could affect hepatocellular viability and contents of taurine in liver and plasma. Administration of hypotaurine, N-acetylcysteine or methionine upon reperfusion comparablly attenuated the post-ischemic hepatocellular injury but with different metabolomic profiling among groups: rats treated with methionine or N-acetylcysteine but not those treated with hypotaurine, exhibited significant elevation of hepatic lactate generation without notable recovery of the energy charge. Furthermore, the group treated with hypotaurine exhibited elevation of the plasma taurine, suggesting that the exogenously administered compound was utilized as an antioxidant. These results suggest that taurine serves as a surrogate marker for ischemia-reperfusion indicating effectiveness of hypotaurine as an energy-saving hepatoprotective amino acid.

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Differential Metabolomics Software for Capillary Electrophoresis-Mass Spectrometry Data Analysis

Sugimoto, M., Hirayama, A., Ishikawa, T., Robert, M., Baran, R., Uehara, K., Kawai, K., Soga, T., Tomita, M.

Metabolomics 6 ( 1 ) 27 - 41 2010.03

Research paper (scientific journal), Joint Work, Accepted

In metabolomics, the rapid identification of quantitative differences between multiple biological samples remains a major challenge. While capillary electrophoresis–mass spectrometry (CE–MS) is a powerful tool to simultaneously quantify charged metabolites, reliable and easy-to-use software that is well suited to analyze CE–MS metabolic profiles is still lacking. Optimized software tools for CE–MS are needed because of the sometimes large variation in migration time between runs and the wider variety of peak shapes in CE–MS data compared with LC–MS or GC–MS. Therefore, we implemented a stand-alone application named JDAMP (Java application for Differential Analysis of Metabolite Profiles), which allows users to identify the metabolites that vary between two groups. The main features include fast calculation modules and a file converter using an original compact file format, baseline subtraction, dataset normalization and alignment, visualization on 2D plots (m/z and time axis) with matching metabolite standards, and the detection of significant differences between metabolite profiles. Moreover, it features an easy-to-use graphical user interface that requires only a few mouse-actions to complete the analysis. The interface also enables the analyst to evaluate the semiautomatic processes and interactively tune options and parameters depending on the input datasets. The confirmation of findings is available as a list of overlaid electropherograms, which is ranked using a novel difference-evaluation function that accounts for peak size and distortion as well as statistical criteria for accurate difference-detection. Overall, the JDAMP software complements other metabolomics data processing tools and permits easy and rapid detection of significant differences between multiple complex CE–MS profiles.

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Capillary Electrophoresis Mass Spectrometry-based Saliva Metabolomics Identifies Oral, Breast and Pancreatic Cancer-Specific Profiles

Sugimoto, M., Wong, D.T., Hirayama, A., Soga, T., Tomita, M.

Metabolomics 6 ( 1 ) 78 - 95 2010.03

Research paper (scientific journal), Joint Work, Accepted

Saliva is a readily accessible and informative biofluid, making it ideal for the early detection of a wide range of diseases including cardiovascular, renal, and autoimmune diseases, viral and bacterial infections and, importantly, cancers. Saliva-based diagnostics, particularly those based on metabolomics technology, are emerging and offer a promising clinical strategy, characterizing the association between salivary analytes and a particular disease. Here, we conducted a comprehensive metabolite analysis of saliva samples obtained from 215 individuals (69 oral, 18 pancreatic and 30 breast cancer patients, 11 periodontal disease patients and 87 healthy controls) using capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS). We identified 57 principal metabolites that can be used to accurately predict the probability of being affected by each individual disease. Although small but significant correlations were found between the known patient characteristics and the quantified metabolites, the profiles manifested relatively higher concentrations of most of the metabolites detected in all three cancers in comparison with those in people with periodontal disease and control subjects. This suggests that cancer-specific signatures are embedded in saliva metabolites. Multiple logistic regression models yielded high area under the receiver-operating characteristic curves (AUCs) to discriminate healthy controls from each disease. The AUCs were 0.865 for oral cancer, 0.973 for breast cancer, 0.993 for pancreatic cancer, and 0.969 for periodontal diseases. The accuracy of the models was also high, with cross-validation AUCs of 0.810, 0.881, 0.994, and 0.954, respectively. Quantitative information for these 57 metabolites and their combinations enable us to predict disease susceptibility. These metabolites are promising biomarkers for medical screening. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-009-0178-y) contains supplementary material, which is available to authorized users.

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Correlation between Sensory Evaluation Scores of Japanese Sake and Metabolome Profiles

Sugimoto, M., Koseki, T., Hirayama, A., Abe, S., Sano, T., Tomita, M., Soga, T.

J. Agric. Food Chem. 58 ( 1 ) 374–383 2010.01

Research paper (scientific journal), Joint Work, Accepted

The aim of this study was to explore the association between taste and metabolite profiles of Japanese refined sake. Nontarget metabolome analysis was conducted using capillary electrophoresis mass spectrometry. Zatsumi, an unpleasant not clear flavor, and sweetness, bitterness, and sourness were graded by four experienced panelists. Regression models based on support vector regression (SVR) were used to estimate the relationships among sensory evaluation scores and quantified metabolites and visualized as a nonlinear relationship between sensory scores and metabolite components. The SVR model was highly accurate and versatile: the correlation coefficients for whole training data, cross-validation, and separated validation data were 0.86, 0.73, and 0.73, respectively, for zatsumi. Other sensory scores were also analyzed and modeled by SVR. The methodology demonstrated here carries great potential for predicting the relevant parameters and quantitative relationships between charged metabolites and sensory evaluation in Japanese refined sake.

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Phylogenetic position of a rare loricated green alga, Cephalomonas granulata N. L. Higinb (Volvocales, Chlorophyceae)

Nakada, T., Soga, T., Tomita, M.

Phycol. Res. 58 ( 1 ) 62 - 68 2010.01

Research paper (scientific journal), Joint Work, Accepted

Cephalomonas (Cp.) granulata N. L. Higinb. (Volvocales, Chlorophyceae), a rare volvocalean phytoflagellate, has recently been isolated from a paddy field in Japan and examined by light and fluorescence microscopy. The vegetative cells of Cp. granulata have granulate loricae and a characteristic mushroom‐like shape with a hemispherical to spherical anterior part and narrower posterior part. Cp. granulata has been classified among the Phacotaceae along with other loricated genera. However, its phylogenetic position has not been investigated using molecular phylogeny. To evaluate the phylogenetic position of Cephalomonas, the 18S rRNA gene sequence of the Japanese strain was determined. The phylogenetic analyses revealed that Cp. granulata was not closely related to other loricated taxa with known phylogenetic affinities, such as Phacotaceae sensu stricto (Phacotus, Pteromonas, and Wislouchiella) and Dysmorphococcus globosus. This indicates at least three independent origins of loricae within the Volvocales.

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Cytotoxic effect of amide derivatives of trifluoromethionine against the enteric protozoan parasite Entamoeba histolytica

Sato, D., Kobayashi, S., Yasui, H., Shibata, N., Toru, T., Yamamoto, M., Tokoro, G., Ali, V., Soga, T., Takeuchi, T., Suematsu, M., Nozaki, T.

Int. J. Antimicrob. Agents. 35 ( 1 ) 56-61 2010.01

Research paper (scientific journal), Joint Work, Accepted

Amoebiasis, caused by infection with the enteric protist Entamoeba histolytica, is one of the major parasitic diseases. Although metronidazole and its derivatives are currently employed in therapy, the paucity of effective drugs and potential clinical resistance necessitate the development of a novel drug. Trifluoromethionine (TFM) is a promising lead compound for antiamoebic drugs. To potentiate the antiamoebic effect of TFM, we synthesised various amide derivatives of TFM and evaluated their cytotoxicity. The amide derivatives of TFM were observed to have a superior cytotoxic effect compared with TFM and metronidazole against E. histolytica in vitro. Although TFM showed cytotoxicity following degradation by methionine gamma-lyase, the derivatives were degraded by the enzyme less efficiently compared with TFM. We further demonstrated that a representative derivative was hydrolysed by the amoebic cell lysate to first yield TFM, followed by degradation similar to TFM. Hydrolysis was partially inhibited by protease inhibitors. A single subcutaneous or oral administration of TFM and its amide derivatives also effectively prevented the formation of amoebic liver abscess in a rodent model. These data demonstrate the improved effectiveness of TFM derivatives against E. histolytica infection and elucidate the mechanisms underlining the mode of action of these compounds.

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SLCO4C1 Transporter Eliminates Uremic Toxins and Attenuates Hypertension and Renal Inflammation.

Toyohara, T., Suzuki, T., Morimoto, R., Akiyama, Y., Souma, T., Shiwaku, H.O., Takeuchi, Y., Mishima, E., Abe, M., Tanemoto, M., Masuda, S., Kawano, H., Maemura, K., Nakayama, M., Sato, H., Mikkaichi, T., Yamaguchi, H., Fukui, S., Fukumoto, Y., Shimokawa, H., Inui, K., Terasaki, T., Goto, J., Ito, S., Hishinuma, T., Rubera, I., Tauc, M., Fujii-Kuriyama, Y., Yabuuchi, H., Moriyama, Y., Soga, T., Abe, T.

J. Am. Soc. Nephrol. 20 ( 12 ) 2546 - 2555 2009.12

Research paper (scientific journal), Joint Work, Accepted

Hypertension in patients with chronic kidney disease (CKD) strongly associates with cardiovascular events. Among patients with CKD, reducing the accumulation of uremic toxins may protect against the development of hypertension and progression of renal damage, but there are no established therapies to accomplish this. Here, overexpression of human kidney-specific organic anion transporter SLCO4C1 in rat kidney reduced hypertension, cardiomegaly, and inflammation in the setting of renal failure. In addition, SLCO4C1 overexpression decreased plasma levels of the uremic toxins guanidino succinate, asymmetric dimethylarginine, and the newly identified trans-aconitate. We found that xenobiotic responsive element core motifs regulate SLCO4C1 transcription, and various statins, which act as inducers of nuclear aryl hydrocarbon receptors, upregulate SLCO4C1 transcription. Pravastatin, which is cardioprotective, increased the clearance of asymmetric dimethylarginine and trans-aconitate in renal failure. These data suggest that drugs that upregulate SLCO4C1 may have therapeutic potential for patients with CKD.

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In Silico Modeling and Metabolome Analysis of Long-stored Erythrocytes to Improve Blood Storage Methods

Nishino, T., Yachie-Kinoshita, A., Hirayama, A., Soga, T., Suematsu, M., Tomota, M.

J. Biotechnol. 144 212 - 223 2009.11

Research paper (scientific journal), Joint Work, Accepted

There is currently no effective method for preventing ATP and 2,3-bisphosphoglycerate (2,3-BPG) depletion during long-term erythrocyte storage in the cold, although these metabolites are strongly associated with cell viability and oxygen delivery after transfusion. Metabolite reduction is caused by whole metabolic networks in the cell, which are regulated by various physical or chemical factors. Mathematical modeling is a powerful tool for integrating such complex and dynamic systems. Here, we developed a mathematical model to predict metabolism in erythrocytes preserved with a mannitol-adenine-phosphate solution (MAP) at 4 degrees C, by modifying a published model of large-scale erythrocyte metabolism. Our model successfully reproduced the reported decreases in ATP and 2,3-BPG during storage. Analysis of our model identified several enzymatic reactions and factors related to ATP and 2,3-BPG depletions, which may serve as possible targets for improving blood storage methods. We also performed metabolome analysis of laboratory-made MAP-stored erythrocytes using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), which provided a comprehensive view of the metabolism dynamics. Alterations in the metabolic intermediate concentrations after long storage were qualitatively predicted by the model. Finally, through further systematic analysis, we also discuss the usability of our model.

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HIF-1α is necessary to support gluconeogenesis during liver regeneration

Tajima, T., Goda, N., Fujiki, N., Hishiki, T., Nishiyama, Y., Senoo-Matsuda, N., Shimazu, M., Soga, T., Yoshimura, Y., Johnson, R.S., Suematsu, M.

Biochem. Bioph. Res. Commun. 387 789 - 794 2009.10

Research paper (scientific journal), Joint Work, Accepted

Coordinated recovery of hepatic glucose metabolism is prerequisite for normal liver regeneration. To examine roles of hypoxia inducible factor-1α (HIF-1α) for hepatic glucose homeostasis during the reparative process, we inactivated the gene in hepatocytes in vivo. Following partial hepatectomy (PH), recovery of residual liver weight was initially retarded in the mutant mice by down-regulation of hepatocyte proliferation, but occurred comparably between the mutant and control mice at 72 h after PH. At this time point, the mutant mice showed lowered blood glucose levels with enhanced accumulation of glycogen in the liver. The mutant mice exhibited impairment of hepatic gluconeogenesis as assessed by alanine tolerance test. This appeared to result from reduced expression of PGK-1 and PEPCK since 3-PG, PEP and malate were accumulated to greater extents in the regenerated liver. In conclusion, these findings provide evidence for roles of HIF-1α in the regulation of gluconeogenesis under liver regeneration.

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Systematic phenome analysis of Escherichia coli multiple-knockout mutants reveals hidden reactions in central carbon metabolism

Nakahigashi, K., Toya, Y., Ishii, N., Soga, T., Hasegawa, M., Watanabe, H., Takai, Y., Honma, M., Mori, H., Tomita, M.

Mol. Syst. Biol. 5 306 2009.09

Research paper (scientific journal), Joint Work, Accepted

Central carbon metabolism is a basic and exhaustively analyzed pathway. However, the intrinsic robustness of the pathway might still conceal uncharacterized reactions. To test this hypothesis, we constructed systematic multiple-knockout mutants involved in central carbon catabolism in Escherichia coli and tested their growth under 12 different nutrient conditions. Differences between in silico predictions and experimental growth indicated that unreported reactions existed within this extensively analyzed metabolic network. These putative reactions were then confirmed by metabolome analysis and in vitro enzymatic assays. Novel reactions regarding the breakdown of sedoheptulose-7-phosphate to erythrose-4-phosphate and dihydroxyacetone phosphate were observed in transaldolase-deficient mutants, without any noticeable changes in gene expression. These reactions, triggered by an accumulation of sedoheptulose-7-phosphate, were catalyzed by the universally conserved glycolytic enzymes ATP-dependent phosphofructokinase and aldolase. The emergence of an alternative pathway not requiring any changes in gene expression, but rather relying on the accumulation of an intermediate metabolite may be a novel mechanism mediating the robustness of these metabolic networks.

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Rescue of anaemia and autoimmune responses in SOD1-deficient mice by transgenic expression of human SOD1 in erythrocytes

Iuchi, Y., Okada, F., Takamiya, R., Kibe, N., Tsunoda, S., Nakajima, O., Toyoda, K., Nagae, R., Suematsu, M., Soga, T., Uchida, K., Fujii, J.

Biochem. J. 422 ( 2 ) 313 - 320 2009.08

Research paper (scientific journal), Joint Work, Accepted

Oxidative stress has been implicated as a cause of various diseases such as anaemia. We found that the SOD1 [Cu,Zn-SOD (superoxide dismutase)] gene deficiency causes anaemia, the production of autoantibodies against RBCs (red blood cells) and renal damage. In the present study, to further understand the role of oxidative stress in the autoimmune response triggered by SOD1 deficiency, we generated mice that had the hSOD1 (human SOD1) transgene under regulation of the GATA-1 promoter, and bred the transgene onto the SOD1(-/-) background (SOD1(-/-);hSOD1(tg/+)). The lifespan of RBCs, levels of intracellular reactive oxygen species, and RBC content in SOD1(-/-);hSOD1(tg/+) mice, were approximately equivalent to those of SOD1(+/+) mice. The production of antibodies against lipid peroxidation products, 4-hydroxy-2-nonenal and acrolein, as well as autoantibodies against RBCs and carbonic anhydrase II were elevated in the SOD1(-/-) mice, but were suppressed in the SOD1(-/-);hSOD1(tg/+) mice. Renal function, as judged by blood urea nitrogen, was improved in the transgenic mice. These results rule out the involvement of a defective immune system in the autoimmune response of SOD1-deficient mice, because SOD1(-/-);hSOD1(tg/+) mice carry the hSOD1 protein only in RBCs. Metabolomic analysis indicated a shift in glucose metabolism to the pentose phosphate pathway and a decrease in the energy charge potential of RBCs in SOD1-deficient mice. We conclude that the increase in reactive oxygen species due to SOD1 deficiency accelerates RBC destruction by affecting carbon metabolism and increasing oxidative modification of lipids and proteins. The resulting oxidation products are antigenic and, consequently, trigger autoantibody production, leading to autoimmune responses.

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Metabolomic Profiling of Anionic Metabolites by Capillary Electrophoresis Mass Spectrometry

Soga, T., Igarashi, K., Itoh, C., Mizobuchi, K., Zimmermann, H., Tomita, M.

Anal. Chem. 81 ( 15 ) 6165 - 6174 2009.08

Research paper (scientific journal), Joint Work, Accepted

We describe a sheath flow capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) method in the negative mode using a platinum electrospray ionization (ESI) spray needle, which allows the comprehensive analysis of anionic metabolites. The material of the spray needle had significant effect on the measurement of anions. A stainless steel spray needle was oxidized and corroded at the anodic electrode due to electrolysis. The precipitation of iron oxides (rust) plugged the capillary outlet, resulting in shortened capillary lifetime. Many anionic metabolites also formed complexes with the iron oxides or migrating nickel ion, which was also generated by electrolysis and moved toward the cathode (the capillary inlet). The metal-anion complex formation significantly reduced detection sensitivity of the anionic compounds. The use of a platinum ESI needle prevented both oxidation of the metals and needle corrosion. Sensitivity using the platinum needle increased from several- to 63-fold, with the largest improvements for anions exhibiting high metal chelating properties such as carboxylic acids, nucleotides, and coenzyme A compounds. The detection limits for most anions were between 0.03 and 0.87 micromol/L (0.8 and 24 fmol) at a signal-to-noise ratio of 3. This method is quantitative, sensitive, and robust, and its utility was demonstrated by the analysis of the metabolites in the central metabolic pathways extracted from mouse liver.

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Measurement of Internal Body Time by Blood Metabolomics

Minami, Y., Kasukawa, T., Kakazu, Y., Iigo, M., Sugimoto, M., Ikeda, S., Yasui, A., van der Horst, G.T., Soga, T., Ueda, H.

Proc. Natl. Acad. Sci. USA 106 ( 24 ) 9890 - 9895 2009.06

Research paper (scientific journal), Joint Work, Accepted

Detection of internal body time (BT) via a few-time-point assay has been a longstanding challenge in medicine, because BT information can be exploited to maximize potency and minimize toxicity during drug administration and thus will enable highly optimized medication. To address this challenge, we previously developed the concept, "molecular-timetable method," which was originally inspired by Linné's flower clock. In Linné's flower clock, one can estimate the time of the day by watching the opening and closing pattern of various flowers. Similarly, in the molecular-timetable method, one can measure the BT of the day by profiling the up and down patterns of substances in the molecular timetable. To make this method clinically feasible, we now performed blood metabolome analysis and here report the successful quantification of hundreds of clock-controlled metabolites in mouse plasma. Based on circadian blood metabolomics, we can detect individual BT under various conditions, demonstrating its robustness against genetic background, sex, age, and feeding differences. The power of this method is also demonstrated by the sensitive and accurate detection of circadian rhythm disorder in jet-lagged mice. These results suggest the potential for metabolomics-based detection of BT ("metabolite-timetable method"), which will lead to the realization of chronotherapy and personalized medicine.

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Metabolite profiling reveals YihU as a novel hydroxybutyrate dehydrogenase for alternative succinic semialdehyde metabolism in Escherichia coli

Saito, N., Robert, M., Kochi, H., Matsuo, G., Kakazu, Y., Soga, T., Tomita, M.

J. Biol. Chem 284 ( 24 ) 16442 - 16451 2009.06

Research paper (scientific journal), Joint Work, Accepted

The search for novel enzymes and enzymatic activities is important to map out all metabolic activities and reveal cellular metabolic processes in a more exhaustive manner. Here we present biochemical and physiological evidence for the function of the uncharacterized protein YihU in Escherichia coli using metabolite profiling by capillary electrophoresis time-of-flight mass spectrometry. To detect enzymatic activity and simultaneously identify possible substrates and products of the putative enzyme, we profiled a complex mixture of metabolites in the presence or absence of YihU. In this manner, succinic semialdehyde was identified as a substrate for YihU. The purified YihU protein catalyzed in vitro the NADH-dependent reduction of succinic semialdehyde to gamma-hydroxybutyrate. Moreover, a yihU deletion mutant displayed reduced tolerance to the cytotoxic effects of exogenous addition of succinic semialdehyde. Profiling of intracellular metabolites following treatment of E. coli with succinic semialdehyde supports the existence of a YihU-catalyzed reduction of succinic semialdehyde to gamma-hydroxybutyrate in addition to its known oxidation to succinate and through the tricarboxylic acid cycle. These findings suggest that YihU is a novel gamma-hydroxybutyrate dehydrogenase involved in the metabolism of succinic semialdehyde, and other potentially toxic intermediates that may accumulate under stress conditions in E. coli.

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