Gas chromatography/mass spectrometry (GC/MS) is widely employed in metabolomics owing to its high resolution, reproducibility, and compatibility with volatile and semi-volatile compounds. While helium has traditionally been used as the carrier gas, increasing costs and global shortages have prompted the exploration of hydrogen gas as an alternative. Hydrogen offers faster analysis times and reduced ion source contamination but presents challenges, including ion source reactions, which can change compound fragmentation patterns. To address this, a hydrogen-deactivated electron ionization (EI) source has been developed to minimize undesired chemical interactions and improve spectral integrity. In this study, we systematically evaluated the effect of extraction lens sizes (3, 6, and 9 mm) under hydrogen carrier gas conditions using n-alkane and human metabolomic standard solutions. The hydrogen-deactivated ion source equipped with a 3 mm extraction lens provided superior peak symmetry and the highest signal intensities, with acceptable relative standard deviation (RSD) values (i.e., below 30%). In addition, the reproducible fragmentation patterns as those observed with helium carrier gas were obtained for phosphorylated metabolites. Using this optimized configuration, we applied hydrogen gas-based GC/MS to metabolomic profiling of the human colon cancer cell line HCT116 with mutant isocitrate dehydrogenase 1 (IDH1). Key metabolic alterations, including increased levels of the known biomarker 2-hydroxyglutaric acid, were detected in IDH1-mutant cells compared with wild-type cells. These findings establish hydrogen gas-based GC/MS with a hydrogen-deactivated ion source as a robust and reliable platform for metabolomics, offering an effective alternative to helium-based systems.

Animals integrate environmental cues with internal physiological states through inter-organ communication to regulate reproduction. However, how environmental microbes are incorporated into these systemic pathways to control reproductive stem cell proliferation remains poorly understood. Here, we demonstrate that environmental microbes colonizing the gut promote stem cell-mediated oogenesis in Drosophila melanogaster by increasing germline stem cell (GSC) number. This process requires microbial activation of gut metabolic pathways, including glycolysis and the pentose phosphate pathway (PPP), linking microbial cues to organismal physiology. We further show that microbial cues modulate circadian clock gene expression in both the gut and brain, and that circadian gene activity in these tissues is required for microbe-induced increases in GSCs. These metabolic changes promote ecdysone and juvenile hormone signaling in ovarian somatic cells, and single-cell transcriptomic analyses further reveal cell type-specific metabolic and hormonal responses across germline and follicle cell populations. Together, our findings establish that microbe-dependent gut-mediated inter-organ communication integrates metabolism, circadian gene expression, and endocrine signaling to regulate stem cell-mediated reproduction in Drosophila.

Phosphoinositides coordinate membrane trafficking and bioenergetic homeostasis, and many tumors rely on elevated phosphoinositide flux to sustain growth. Therapeutic development has largely pursued single-phosphoinositide kinase inhibition, whereas polypharmacologic strategies that perturb the broader network remain underexplored. FTY720 (fingolimod), a clinically approved sphingosine-1-phosphate receptor modulator, shows anti-tumor activity at micromolar concentrations, but its non-canonical mechanisms remain incompletely defined. Building on our work with the structurally related compound KRP203, we show that high-dose FTY720 produces isozyme-divergent modulation across phosphoinositide kinases and biases PIKFYVE activity toward phosphatidylinositol, a pattern we term ASURA (Asymmetric Simultaneous Uncoupling of Related Activities). FTY720 induces vacuolization and endomembrane remodeling in cancer cells, and suppresses macropinocytic ruffling as demonstrated by tracer uptake and scanning ion conductance microscopy analyses. Quantitative metabolomics revealed depletion of intracellular amino acids and ribonucleoside triphosphates, coupled with reduced glycolysis. Concurrently, FTY720 induced extensive rewiring of the hexosamine pathway, nitrogen metabolism, and tricarboxylic acid (TCA)-cycle anaplerosis, along with redox signatures indicating oxidative stress despite a nutrient-replete medium. The metabolites depleted by FTY720 showed extensive, directionally concordant overlap with those depleted by PTEN induction. Patient-derived glioblastoma (GBM) neurospheres were sensitive to FTY720, and co-treatment with a PI3Kα-selective inhibitor augmented growth suppression in U87MG cells. Together, these data support a model in which ASURA-dose FTY720 disrupts phosphoinositide-regulated trafficking and nutrient access, imposing intracellular nutrient stress that culminates in tumor-cell death.

Environmental exposures can influence offspring health through epigenetic alterations in the male germline. Folate deficiency, a dietary perturbation that disrupts one-carbon metabolism and S-adenosylmethionine (SAM) production, has been linked to altered histone methylation and developmental abnormalities in offspring. However, when and how folate availability shapes the germline epigenome during spermatogenesis remains unclear. In this study, unbiased metabolomic profiling of spermatogenic cells uncovers stage-specific metabolic remodeling, including downregulation of serine-glycine-one-carbon (SGOC) metabolism in meiotic spermatocytes. Using a post-weaning folate-deficient mouse model, we investigate how folate availability influences germline epigenome establishment during spermatogenesis. Consistent with this metabolic transition, genome-wide chromatin accessibility profiling demonstrates extensive, stage-dependent remodeling under folate-deficient conditions, particularly in meiotic spermatocytes and post-meiotic spermatids. These accessibility changes display cell-type-specific genomic distributions and preferential localization to repressive chromatin compartments in post-meiotic cells. Histone modification analyses further reveal bidirectional redistribution of the active histone mark H3K4me3 in round spermatids. Although genome-wide distribution of the repressive mark H3K27me3 remains largely stable, folate deficiency alters its nuclear organization. Notably, a subset of H3K4me3 alterations established in post-meiotic cells is retained in mature sperm, providing a mechanistic link between paternal metabolic perturbation and the germline epigenome. Together, these findings demonstrate that folate availability shapes germline epigenome establishment through stage-specific metabolic and chromatin remodeling during spermatogenesis, revealing a metabolic basis for paternal environmental effects on the germline epigenome.

CONTEXT:Previous metabolomics studies suggest potential associations between menopausal changes in lipids and an increased risk of metabolic syndrome (MetS). However, longitudinal data on other key metabolites, such as branched-chain amino acids (BCAAs) and homocysteine, remain limited, and most studies lack long-term follow-up across the menopause transition. OBJECTIVE:This study aimed to investigate longitudinal changes in circulating metabolites during menopause over a mean follow-up of 5 years and assess their associations with subsequent MetS development. METHODS:Premenopausal women from the Tsuruoka Metabolomics Cohort Study who participated in at least one follow-up survey were included. Menopausal status, data on MetS, and plasma metabolites profiled using capillary electrophoresis mass spectrometry were assessed at each visit. Thirty-one metabolites were examined for associations with menopausal status using mixed-effects models. The association of these menopause-related metabolites with MetS development was examined via logistic regression analysis adjusted for follow-up duration. RESULTS:Among 953 women (aged 43.8 ± 5.4 years), 316 (33.2%) reached menopause during follow-up (5.0 ± 1.1 years). Eighteen metabolites changed significantly with menopause, particularly those related to BCAA metabolism, urea cycle, and homocysteine metabolism. Of 695 women without MetS at baseline, 65 (9.4%) developed MetS. Glutamate (odds ratio [95% CI]: 1.95 [1.49-2.57]) was associated with higher MetS risk. Higher levels of glutamate, valine, leucine, and cystine were significantly associated with the development of hyperglycemia. CONCLUSION:Longitudinal changes in charged metabolites occur across the menopausal transition, with specific metabolites such as glutamate possibly contributing to the metabolic alterations underlying increased MetS risk.

Metabolomics investigates an extraordinarily broad chemical space and generates experimental data with substantial potential for reuse in chemistry, biology, and related fields. We developed MB-POST (https://repository.massbank.jp), a new metabolomics mass spectrometry data repository based on a concept distinct from that of existing platforms, to fully realize this potential. MB-POST implements a reanalysis-oriented metadata framework that captures the information essential for data reuse while providing a streamlined submission system that enables the rapid deposition of standardized metabolomics metadata together with experimental data. Since its public pilot release in December 2024, MB-POST has attracted strong community adoption, with more than 170 experimental projects deposited within its first year and a half. Thus, MB-POST provides a practical foundation for the large-scale reuse of metabolomics mass spectrometry data and represents a concrete step toward accelerating open and data-driven scientific research.

Pancreatic ductal adenocarcinoma (PDAC) is the cancer with poorest prognosis, with metabolic reprogramming reported. We investigated metabolic alterations in mice with PDAC using capillary electrophoresis-mass spectrometry (CE-MS) and mass spectrometry imaging (MSI). o-Acetylcarnitine, a metabolite of carnitine, and acetyl-CoA increased during cancer progression in a PDAC mouse model by CE-MS, while MSI revealed that o-acetylcarnitine was mainly localized in PDAC cells. Also, immunohistochemistry showed overexpression of γ-butyrobetaine 2-oxoglutarate dioxygenase 1 (BBOX1), which synthesizes carnitine from γ-butyrobetaine, mainly in PDAC cells. Meldonium, an inhibitor of BBOX1, inhibited PDAC proliferation and cytokine secretion, thereby prolonging the survival of PDAC-bearing mice, accompanied by improved skeletal muscle atrophy. In patients undergoing PDAC resection, BBOX1 expression was determined as an independent poor prognostic factor for overall survival. Therefore, these results suggest that blocking L-carnitine synthesis would improve the prognosis of patients with PDAC.

Age-related disease burden accumulates heterogeneously from later midlife to older age, but the biology underlying these divergent trajectories is poorly understood. We analysed 7199 adults aged 40 years and over in the Tsuruoka Metabolomics Cohort Study, Japan, with baseline fasting plasma metabolomics (94 metabolites measured by capillary electrophoresis–mass spectrometry) and linked health insurance claims. Monthly cumulative Charlson Comorbidity Index scores were constructed from aligned cohort entry to 60 months to capture accumulation of newly documented Charlson conditions after follow-up start. K-means clustering identified six trajectories of claims-recorded disease burden, and ordinal logistic regression related metabolites to ordered trajectory severity with adjustment for demographic and lifestyle factors. Six trajectories ranged from minimal accumulation to rapid progression. Nineteen metabolites were associated with greater trajectory severity after false discovery rate correction. Glutamate showed the strongest positive association (odds ratio, 1.18 per standard deviation; 95

Abstract Workplace stress impacts productivity and health, necessitating non-invasive, rapid, and objective assessment methods. This study investigates the potential of metabolite profiling of mouth-rinsed water (MW)—an oral biofluid collected in just 10 s—as a screening tool for psychophysiological stress among office workers. Thirty-two participants were classified into high-stress and control groups based on the State-Trait Anxiety Inventory and the Brief Job Stress Questionnaire, and objective physiological measures. MW samples were collected at four time points, including before and after brief mental stress tasks. Biochemical features were profiled using capillary electrophoresis–mass spectrometry and liquid chromatography–mass spectrometry. A total of 559 analytes, including 532 water-soluble metabolites, 25 steroids, and 2 salivary proteins, were measured, of which 127 analytes with acceptable analytical precision were selected for subsequent analyses. Statistically significant differences in numerous metabolites indicated stress-associated alterations in metabolic activity. Notably, a predictive model using the ratio of N-acetyl-β-alanine to asymmetric dimethylarginine achieved high accuracy, with an area under the receiver operating characteristic curve (AUC) of 0.845 for identifying high stress. The distinct metabolite dynamics under varying stress conditions suggest multiple stress-response mechanisms. These findings underscore MW metabolite profiling as a promising approach for assessing and managing workplace stress.

Starvation induces complex metabolic adaptations in skeletal muscle, a key tissue for maintaining energy homeostasis; however, these adaptations are largely impaired in obesity. How obesity alters global metabolic adaptations to starvation in skeletal muscle remains unclear. Here, we analyzed the metabolic adaptations on a trans-omics scale during starvation in skeletal muscle from wild-type (WT) and leptin-deficient obese (ob/ob) mice. We measured multi-omics data during starvation and constructed global trans-omics networks in WT and ob/ob mice. We found that starvation induces “responsiveness” in WT mice, characterized by increases or decreases in key regulator metabolites, including ATP and AMP, as well as enzyme proteins, leading to global regulation of metabolic pathways, which was lost in ob/ob mice. In contrast, during starvation, ob/ob mice exhibit “difference” in comparison to WT mice, manifested by the persistently elevated expression of metabolic enzymes. These features were similarly found in liver, another key metabolic organ. Thus, global loss of responsiveness and elevated enzyme proteins are systemic features of metabolic dysregulation in ob/ob mice.

Mycophenolate mofetil (MMF) is an immunosuppressive prodrug whose efficacy depends on conversion to mycophenolic acid (MPA). Quantitative, stoichiometry-ready measurement of MMF-to-MPA conversion in matched extracellular and intracellular matrices remains limited, restricting interpretation of prodrug activation and cellular exposure beyond plasma MPA. A common expectation is that a lipophilic prodrug rapidly equilibrates across membranes; however, extracellular concentrations alone cannot resolve intracellular accumulation, activation, and retention. Here, we developed a common product ion (m/z 207.1) LC-MS/MS workflow that enables stoichiometrically comparable quantification of MMF and MPA across matched extracellular and intracellular matrices from microliter-scale samples and applied it to paired medium/cell sampling in murine Hepa1-6 cells as a proof-of-application system. In Hepa1-6 cells, intracellular MMF transiently exceeded extracellular MMF, peaking at ∼5.5 µM at 15 min (≈1.4-fold above the 4 µM dosing level); in contrast, with direct MPA dosing, intracellular MPA did not exceed extracellular MPA, consistent with equilibration. With carboxylesterase inhibition by bis-nitrophenyl phosphate (BNPP), intracellular MMF reached ∼9.2 µM and exceeded extracellular MMF by ∼2.4-fold at 1 h (∼9.2 vs ∼3.9 µM), consistent with intracellular accumulation and/or retention not readily explained by simple passive equilibration alone. Integrating the data with a mechanistic medium-cell model separated intracellular conversion from bidirectional exchange and parameterized these processes. This compartment-resolved workflow provides a proof-of-application framework for cellular MMF/MPA measurements in matched medium/cell samples.

Microbiota-accessible carbohydrates modulate host immunity by shaping gut microbial composition and metabolism. However, their role in modulating the microbiota to influence allergic responses is unclear. Here we show that a widely used antidiabetic agent, the α-glucosidase inhibitor acarbose, redirects dietary carbohydrate utilization by gut bacteria to suppress mast-cell-dependent anaphylaxis in mice, independently of adaptive immune responses. Enhanced carbohydrate availability promoted the proliferation of Parabacteroides distasonis in the mouse gut, leading to increased succinate abundance and intracellular NAD+ levels, and reduced reliance on b-type cytochrome-dependent anaerobic respiration. Direct administration of succinate suppressed systemic anaphylaxis and mast cell degranulation in vitro, implicating succinate as a key effector. A human cohort analysis revealed that patients treated with α-glucosidase inhibitors showed a lower incidence of anaphylaxis than untreated individuals. These findings uncover a previously unrecognized gut-microbiota-mediated pathway linking dietary carbohydrate metabolism to systemic immune regulation.

BACKGROUND:The role of the apoptosis inhibitor of macrophages (AIM) in human lipid metabolism remains unclear. OBJECTIVE:This study aimed to investigate the cross-sectional associations between serum AIM and plasma metabolites and to determine if baseline AIM concentrations predict incident dyslipidemia. METHODS:This study used a community-based cohort of 3139 participants for cross-sectional metabolomic analysis. Among them, 1292 participants without dyslipidemia at baseline were followed prospectively for up to 8.5 years. Cox proportional hazards models were used to estimate hazard ratios for incident high low-density lipoprotein cholesterol (LDL-C) and metabolic dyslipidemia (high triglycerides and/or low high-density lipoprotein cholesterol [HDL-C]) across AIM tertiles. RESULTS:Cross-sectionally, higher AIM concentrations were associated with elevated acylcarnitines and tricarboxylic acid cycle intermediates. Longitudinally, higher baseline AIM was associated with an increased risk of developing metabolic dyslipidemia in women (highest vs lowest tertile hazard ratio, 1.84) and showed a similar but smaller nonsignificant trend in men. The risk for high LDL-C was significantly increased in women but not in men. These associations remained robust after multivariable adjustment. CONCLUSION:Elevated baseline AIM concentrations were prospectively associated with the development of metabolic dyslipidemia in women, with a similar but nonsignificant trend in men, and with high LDL-C in women. The related metabolomic profile suggests enhanced lipolysis and altered lipid metabolism. Therefore, AIM may serve as a biomarker for future dyslipidemia, particularly for triglyceride and HDL-C dysregulation.

Lower grade gliomas frequently harbor mutations in isocitrate dehydrogenase (IDH), which define biologically distinct tumor subtypes. Although IDH-mutant and IDH-wildtype gliomas share similar histological morphology, they display markedly different metabolic profiles that may be exploited for targeted therapy. In this study, we investigated therapeutic approaches tailored to these metabolic differences. Using capillary electrophoresis–mass spectrometry, we compared the metabolomes of engineered IDH-wildtype and IDH-mutant glioma cell models. IDH-mutant cells exhibited elevated asparagine levels and reduced glutamine and glutamate levels compared with IDH-wildtype cells. These differences were corroborated in vivo by proton magnetic resonance spectroscopy of 130 patients with diffuse gliomas, showing lower glutamine and glutamate in IDH-mutant tumors. Pharmacological depletion of asparagine with L-asparaginase, which converts asparagine to aspartate, preferentially inhibited the growth of IDH-wildtype glioma cells, and this effect was potentiated by inhibition of asparagine synthetase. In contrast, inhibition of glutamate dehydrogenase 1 (GLUD1), the enzyme catalyzing the conversion of glutamate to α-ketoglutarate, selectively suppressed proliferation of IDH-mutant glioma cells by inducing reactive oxygen species accumulation and apoptosis. In vivo, L-asparaginase suppressed tumor growth in xenografted IDH-wildtype gliomas, whereas GLUD1 inhibition significantly reduced tumor growth in IDH-mutant glioma xenografts. These findings reveal distinct amino acid metabolic vulnerabilities defined by IDH mutation status and identify L-asparaginase and GLUD1 inhibition (via R162) as promising, mutation-specific therapeutic strategies. L-asparaginase demonstrated potent antitumor activity against IDH-wildtype gliomas, while GLUD1 inhibition selectively suppressed IDH-mutant gliomas both in vitro and in vivo. These results highlight the clinical potential of targeting amino acid metabolism in gliomas and provide a strong rationale for translating these mutation-specific approaches into future clinical trials.

Supplementary Figure S3. Method to generate macrophages and the phenotype of the macrophages.

Mammalian liver metabolism undergoes a substantial shift during fasting. Thermodynamic principles impose fundamental constraints on metabolism, and the Gibbs free energy change of reaction (Δ r G ′) indicates the reaction’s direction and distance from equilibrium. However, Δ r G ′ landscapes in intact mammalian organs remain largely uncharacterized. Here, we mapped Δ r G ′ profile of glucose metabolism in mouse liver during fasting, using experimentally measured absolute metabolite concentrations and a newly developed computational method, GLEAM. We found that despite large metabolite fluctuations during fasting, the corresponding Δ r G ′s remained robust, even for reactions reversing the direction between glycolysis and gluconeogenesis. A remarkable thermodynamic robustness is found in maintaining potential candidates of rate-limiting steps during fasting. This robustness is achieved by expending substantial costs for enzyme expressions, contributing to efficient switching from glycolysis to gluconeogenesis. Furthermore, obese mouse liver also showed thermodynamic robustness despite obesity-induced metabolic disruption. Our framework provided a novel thermodynamic perspective on intact organ metabolism, demonstrating that the liver robustly maintains thermodynamic characteristics favorable for metabolic control by buffering metabolite concentration differences.

Introduction:Minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) are podocytopathies with varying clinical courses and therapeutic responses. FSGS often leads to end-stage renal disease. Consequently, their heterogeneity requires case stratification and pathophysiological elucidation. The involvement of energy metabolism in FSGS pathogenesis and stratification has not been clarified. Therefore, this study aimed to verify whether evaluating energy kinetics can be a new approach to MCD or FSGS stratification and explore the role of energy metabolism in MCD or FSGS. Methods:Cultured human podocytes were treated with sera from patients with biopsy-confirmed MCD or FSGS. Serum-treated podocytes were analyzed for apoptosis using flow cytometry, metabolomics via mass spectrometry, and real-time adenosine triphosphate (ATP) production rates using an extracellular flux analyzer. Adriamycin-induced nephropathy was induced in podocyte-specific lactate dehydrogenase (LDH) A (LDHA)-deficient and control mice. Results:The sera from patients with FSGS significantly induced apoptosis in human podocytes compared with those from individuals with MCD. Apoptosis severity was associated with segmental obliteration and corticosteroid resistance. Metabolomic analysis revealed differences in anaerobic glycolysis and tricarboxylic acid cycle (TCA)-related metabolites in podocytes exposed to the sera of patients with MCD and FSGS. In the podocytes treated with sera from patients with FSGS, glycolytic ATP production significantly decreased in cases with high apoptosis rates. The sera from patients with FSGS suppressed LDHA activity, suppressed α-actinin 4 (ACTN4) expression, and promoted actin remodeling of podocytes. Segmental sclerosis was more prominent in podocyte-specific LDHA-deficient mice with adriamycin-induced nephropathy than in control mice. Conclusion:FSGS progression was associated with decreased anaerobic glycolysis in podocytes.

Plasma amino acids (AAs) have emerged as promising biomarkers for metabolic disorders, yet their causality remains unclear. We aimed to investigate the genetic determinants of AA levels in a cohort of 10,333 individuals and their causal effects on cardiometabolic traits using Mendelian randomization (MR). Plasma levels of 20 AAs were quantified using capillary electrophoresis mass spectrometry. Genome-wide association studies were conducted using BOLT-LMM and heritability estimation via LDSC analysis. Causal effects of AAs on 11 cardiometabolic traits were examined using two-sample MR analyses. We identified 85 locus-metabolite associations across 43 genes for 18 AAs, including 44 novel loci linked to metabolic genes. Heritability for AAs was estimated at 16%. MR analysis demonstrated cystine to positively associate with systolic blood pressure (SBP) (beta = 0.056, SE = 0.010), while serine indicated protective effects on SBP (beta = - 0.040, SE = 0.011), diastolic BP (beta = - 0.044, SE = 0.010), and coronary artery disease (odds ratio 0.888, SE = 0.028). We identified potentially novel genetic loci associated with AA levels and demonstrated robust causal associations between several AAs and cardiometabolic traits. These findings reinforce the importance of AAs as potential biomarkers and therapeutic targets in cardiometabolic health.