Published papers

Capillary electrophoresis-mass spectrometry based metabolomics exhibits unlimited potentials to elucidate global metabolic profiles of various types of samples including clinical tissues. In addition, non-targeted metabolome analysis by this method holds the promise of discovering novel biomarker candidates. In this commentary, we introduce our strategies for studying cancer-specific metabolism by analyzing metabolome of surgically excised tumor tissues.

Lipidomics is one of the major approaches in metabolomics, and often uses a liquid chromatography-mass spectrometer (LC-MS) to obtain profiling of lipid metabolites and to detect changing molecules statistically. However, previously reported methods have been technically insufficient in the simultaneous analysis of amphiphilic glycosphingolipids, such as gangliosides, and in the global detection of various polar metabolites from complex lipid mixtures. Here, we report on newly targeted and non-targeted methods to measure these lipid metabolites sensitively. In the targeted approach, we were able to detect individual ganglioside molecular species using the combination of reversed-phase or hydrophilic interaction liquid chromatography and ESI-MS with multiple reaction monitoring (MRM). Meanwhile, in a non-targeted approach, global analysis of various polar metabolites was possible by reverse-phased high-resolution (LC) coupled quadrapole time-of-flight MS and sensitive twodimensional (2D) profiling. Our methods will be useful for the elucidation of cancers, metabolic syndrome and nervous-system diseases, etc. ; these diagnostic markers may be detectable.

配合飼料中の8種類の有機酸 (ギ酸, フマル酸, 酒石酸, リンゴ酸, クエン酸, 酢酸, 乳酸及びプロピオン酸) のキャピラリー電気泳動 (CE) による分析法を開発した. 各有機酸は水で同時抽出し, ろ過した後, ろ液を0.02mol/Lの水酸化ナトリウムで希釈し, 限外ろ過フィルターユニット (分画分子量30,000) で除タンパクを行いCEに供した. 3種類の配合飼料に8種類の有機酸を0.05～2.0%の範囲で添加して回収試験を行ったところ, 平均回収率は85.1～107.3%で繰り返し精度 (RSDr) は13.6%以内であった. また, 3試験室において共同試験を行った結果, 平均回収率は87.3～105.0%, 室間再現精度 (RSDR) は16.3%以内であった.

Metabolomics, which can be defined as the measurement of the level of all intracellular metabolites, has become a powerful new tool for gaining insight into functional biology. Intercellular metabolites not only provide metabolic phenotypes but also inducers to gene expression. Thus, metabolome analysis will be as important as genome and proteome research. However, very few methods for a large-scale metabolite analysis have been reported. This paper reports a method for the direct and quantitative analysis of charged metabolite using capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS). Its utility is demonstrated in the determination of basal metabolic intermediates of glycolysis and the TCA cycle pathways in Bacillus subtilis cells, yielding new information about how changes in metabolites are related to sporulation events.

Metabolomics, which can be defined as the measurement of the level of all intracellular metabolites, has become a powerful new tool for gaining insight into cellular and physiological responses. Here we have developed a comprehensive and quantitative analysis method based on capillary electrophoresis mass spectrometry (CE-MS). In this marriage of techniques, CE confers rapid analysis and efficient resolution, and MS provides excellent selectivity and sensitivity. Previous work in our laboratory demonstrated that CE-MS techniques were quite useful for the metabolome analysis. We applied this approach to profile liver metabolites following acetaminophen-induced hepatotoxicity and revealed ophthalmate as a sensitive indicator of reduced glutathione (GSH) depletion. Our results specifically indicate that serum ophthalmate is a sensitive indicator of hepatic GSH depletion, and may be a new biomarker for oxidative stress.

This study aimed to elucidate how sodium-glucose cotransporter 2 (SGLT2) inhibitors and sulfonylureas differentially modulate hepatic metabolism in persons with metabolic dysfunction-associated steatotic liver disease (MASLD) and type 2 diabetes (T2D). In this 48-week randomized, open-label, parallel-group trial, Japanese participants with type 2 diabetes and biopsy-confirmed MASLD received either tofogliflozin or glimepiride. Paired liver biopsy samples and serum were collected at baseline and after 48 weeks. We performed a post hoc metabolomic analysis to assess hepatic and systemic metabolic shifts and their correlations with histological and clinical parameters. Tofogliflozin treatment significantly increased hepatic glucose 1-phosphate and acetyl-CoA levels, reflecting a metabolic shift toward ketogenesis and gluconeogenesis. Regarding TCA cycle intermediates, tofogliflozin elevated serum citrate but reduced serum succinate; notably, the reduction in succinate correlated with reduction in liver enzyme levels. Furthermore, higher baseline serum succinate predicted superior histological improvements (ballooning and inflammation) in the tofogliflozin group. In contrast, glimepiride specifically reduced hepatic branched-chain amino acids (BCAAs), which was associated with decreased steatosis. Tofogliflozin also elevated serum urea cycle intermediates, suggesting a distinct “aestivation-like” response to hypovolemia. The metabolic signature of SGLT2 inhibitors in the human liver is characterized by starvation-induced glucose production and hypovolemia-induced aestivation-like response. Serum succinate emerged as a potential biomarker associated with MASLD severity and treatment-related improvements under SGLT2 inhibition.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that urgently requires effective treatment. Mitochondrial dysfunction underlies ALS pathology and represents a potential therapeutic target. Here, we demonstrated the therapeutic potential of mitochonic acid 5 (MA-5), a novel mitochondria-targeted compound that ameliorated ALS phenotypes by enhancing mitochondrial function. In a Drosophila ALS model expressing a mutant human SOD1 (G85R), MA-5 significantly improved locomotor activity, with a trend toward restoration of mitochondrial integrity. In skin fibroblasts derived from ALS patients and motor neurons derived from induced pluripotent stem cells, MA-5 restored ATP production and increased mitochondrial motility. Multiomics analyses suggested that MA-5 modulated mitochondria-linked gene expression and downregulated the glycerophosphate shuttle, contributing to mitochondrial reactive oxygen species production. Transcriptomic analysis identified C7orf31 as a potential marker for monitoring the therapeutic effects of MA-5 and diagnosing ALS subtypes. These findings support MA-5 as a promising therapeutic candidate for ALS and propose C7orf31 as a potential biomarker for treatment monitoring and for disease subtyping.

Mammalian liver metabolism shifts substantially during fasting, yet the Gibbs free energy change of reaction (ΔrG′), a fundamental thermodynamic quantity that defines reaction driving force and constrains metabolic directionality, remains poorly characterized in intact organs. Here, we mapped the ΔrG′ landscape of mouse liver glucose metabolism during fasting using absolute metabolite concentrations and GLEAM (Gibbs free energy of reaction landscape estimation from metabolome assisted by variance-covariance matrix), a computational method developed here. Despite metabolite fluctuations, ΔrG′ landscapes remained robust, changing only modestly, even for reactions reversing direction between glycolysis and gluconeogenesis. Thermodynamically inferred potential rate-limiting steps were maintained during fasting. Enzyme-cost analysis further suggested a trade-off between thermodynamic robustness and enzyme requirements during glycolytic switching. Obese mouse liver also showed thermodynamic robustness despite metabolic disruption. Our framework provides a novel thermodynamic perspective on intact organ metabolism, revealing preservation of key thermodynamic characteristics across fasting and obesity.