Metabolites doi: 10.3390/metabo16090694

Authors: Peng Shi Lei Wang Xuxiong Huang Biao Yun Xueqiao Qian

Purpose: This study evaluated the effects of replacing dietary fish meal (FM) with poultry by-product meal (PBM) on growth, physiological status, disease resistance, and hepatic transcriptomic responses in hybrid yellow catfish (Pelteobagrus fulvidraco ♀ × Pelteobagrus vachelli ♂). Methods: Six isonitrogenous and isolipidic diets were formulated by replacing 0%, 12%, 24%, 36%, 48%, or 60% of dietary FM with PBM. Juveniles (initial weight 5.18 ± 0.02 g) were fed the experimental diets for 8 weeks. Results: Weight gain was maximized and the feed conversion ratio minimized in fish fed the FM19 diet, with broken-line regression analysis of weight gain estimating the optimal FM replacement level at 27.4%. Hepatic and intestinal superoxide dismutase activities, as well as intestinal trypsin activity, tended to decline as the level of FM replacement with PBM increased. Following Edwardsiella ictaluri challenge, 96-h cumulative survival was lower in the FM10 group than in all other groups. Principal component analysis of hepatic transcriptomes clearly separated the FM10 group from the FM25 and FM19 groups. Gene Ontology analysis associated differentially expressed genes with responses to hormones and endogenous stimuli (FM10 vs. FM25), protein folding and chaperone-mediated complex assembly (FM19 vs. FM25), and catabolic processes and mitochondrial function (FM10 vs. FM19). Kyoto Encyclopedia of Genes and Genomes analysis identified pathways involving mitogen-activated protein kinase signaling, protein processing in the endoplasmic reticulum, autophagy, apoptosis, and inflammatory responses. Moreover, genes associated with oxidative stress and inflammation showed higher expression in the FM10 group compared with the FM19 and FM25 groups. Conclusions: Moderate FM replacement with PBM improved growth and feed utilization, whereas excessive replacement was associated with lower antioxidant and digestive enzyme activities, altered metabolic responses, and enhanced expression of inflammation-related genes, supporting PBM as a partial FM substitute in low-FM diets for hybrid yellow catfish.

Metabolites doi: 10.3390/metabo16090693

Authors: Guole Qin Daizu Xie Changqin Zhou Delong Guan Jing Song

Background: Cibotium barometz (L.) J. Sm. is a medicinal fern whose rhizome is the sole officinal part, while roots, stems, leaves and young shoots are discarded as agricultural residues. The chemical rationale for this restriction, and the value of the discarded organs, remain undefined. Materials and methods: Here, untargeted LC–MS/MS metabolomics (UHPLC–Orbitrap) of methanol/acetonitrile/water extracts was applied to five organs of C. barometz (three pooled biological replicates per organ), annotating 2765 polar-to-moderately-polar metabolites. Results: Organ identity dominated the metabolic architecture of the sampled population: the first two principal components separated all five organs, with leaf and root as the two mutually opposed metabolic extremes and stem, young shoot and rhizome forming a contiguous module. Exploratory pairwise OPLS-DA combined with FDR-controlled univariate criteria yielded 193–1223 differentially accumulated metabolites per comparison, and tissue-specificity analysis assigned 2031 metabolites (73.45%) predominantly to a single organ. Discussion: Metabolite accumulation patterns along the phenylpropanoid–flavonoid pathway was spatially partitioned, with flavan-3-ols and isoflavones accumulating in root, flavone/flavonol glycosides in leaf and predominantly hydroxycinnamic acids in rhizome and young shoot. Of 22 previously reported or candidate quality control constituents, putatively annotated terpenoid lactones were detected almost exclusively in the rhizome, whereas several phenolics showed markedly higher relative abundance in root or leaf. Conclusions: These relative-abundance data indicate that the non-officinal organs are chemically complementary potential resources; absolute quantification, bioactivity and safety evaluation are prerequisites before any medicinal valorisation.

Metabolites doi: 10.3390/metabo16090692

Authors: Özkul Yılmaz Çolak Melda İşevi Tuğçehan Sezer Akman Özgür Kılıç Neslihan Ünal Akdemir

Background/Objectives: We compared three albumin-anchored ratios calculated from measurements obtained during the first 24 h, C-reactive protein (CRP)-to-albumin (CAR), procalcitonin-to-albumin (PAR) and lactate-to-albumin (LAR), as predictors of intensive care unit (ICU) mortality beyond an established severity model. Methods: Eligibility required an ICU stay reaching 24 h and a complete early work-up; 372 of 1352 screened admissions qualified, with each ratio using the worst component values in that window. Incremental value over a base model (Sequential Organ Failure Assessment (SOFA) score, sex, age-adjusted Charlson index) was assessed by change in the area under the curve (AUC; DeLong test), reclassification metrics (NRI, IDI) and calibration, with alternative baselines and outcomes, multiplicity adjustment, and selection weights from the excluded admissions. Results: Of 372 patients, 248 (66.7%) died. As standalone predictors, CAR (0.671), PAR (0.689) and LAR (0.642) were indistinguishable and inferior to SOFA (0.730). Only CAR significantly improved AUC discrimination (ΔAUC +0.030, p = 0.024; NRI +0.39; IDI +0.042); PAR and LAR did not significantly improve AUC discrimination. Entered together, CAR remained independent (aOR 1.52, 1.15–2.03), while PAR did not (1.20, 0.89–1.63). The cohort was sicker than the 626 patients excluded for incomplete work-up; after weighting, CAR stayed independent (aOR 1.47) but ΔAUC fell to +0.018. The increment was significant against SOFA alone (+0.033) but not against baselines including admission diagnosis or organ support; it remained significant after Benjamini–Hochberg but not Bonferroni adjustment. With in-hospital mortality it was larger (+0.044), but PAR then also retained independence. Conclusions: The first 24 h CAR provided a well calibrated improvement beyond a parsimonious SOFA-based model, but the increment was small and not robust to richer baselines or to multiplicity correction, and no subgroup-specific claim is supported. These hypothesis-generating findings need prospective validation.

Metabolites doi: 10.3390/metabo16090691

Authors: Jiaxin Liu Likun Zheng Zhengri Cong Zehao Liu Chen Hu Yuxin Liu Chong Zhang Mingjun Liu

Background: Obesity is a chronic metabolic disease characterized by lipid overload, low-grade inflammation, oxidative stress, and progressive organ dysfunction. Ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation, has been increasingly implicated in obesity-related complications. However, an acute cell death-centered model does not fully explain the chronic senescence-like decline observed in metabolic tissues. The recently proposed concept of ferro-aging provides a potential framework for linking iron-dependent lipid peroxidation to cellular senescence and metabolic aging. Methods: In this review, we combined bibliometric mapping with mechanistic synthesis to characterize the evolving research landscape of ferroptosis in obesity and integrate the emerging concept of ferro-aging into this field. Bibliometric analysis was performed to screen qualified literature, and keyword co-occurrence and co-citation analyses were conducted to extract mainstream research hotspots. We further systematically summarized the pathological microenvironment constructed by obesity and the core molecular mediator connecting ferroptosis and ferro-aging, and proposed a pathological continuum hypothesis for obesity-mediated lipid peroxidation injury. Results: A total of 564 eligible publications were identified, including 455 original articles and 109 reviews, with a marked increase in publication output after 2020. Keyword and co-citation analyses highlighted oxidative stress, lipid peroxidation, iron metabolism, GPX4/Nrf2-mediated antioxidant defense, mitochondrial dysfunction, gut microbiota, NAFLD, and DCM as major research themes. Mechanistically, obesity creates a pro-ferroperoxidative microenvironment through iron dysregulation, polyunsaturated fatty acid enrichment, chronic inflammation, mitochondrial stress, and impaired antioxidant capacity. Within this context, ACSL4 is proposed as a candidate molecular hub that may link acute ferroptotic injury with chronic iron-lipid peroxidation-driven cellular senescence. We proposed a hypothesis-generating framework. In this framework, acute lipid peroxidation may promote ferroptotic cell death. Persistent sublethal iron-lipid peroxidation stress may contribute to ferro-aging-like senescence. Conclusions: To our knowledge, this is the first review to place ferro-aging within the obesity-ferroptosis framework. Targeting the ACSL4-lipid peroxidation axis and restoring antioxidant defense, particularly through Nrf2-GPX4-related pathways, may provide new translational opportunities for stratifying and managing obesity-related complications. However, clinical validation of ferro-aging biomarkers and intervention strategies remains urgently needed.

Metabolites doi: 10.3390/metabo16090690

Authors: Jayson M. Antonio Shubha Priyamvada Arivarasu N. Anbazhagan Harris J. B. Nathan Calzadilla Rengul C. Atalay Reena Sethi Seema Saksena Ravinder K. Gill Gokhan M. Mutlu Waddah A. Alrefai Ece A. Mutlu Anoop Kumar Pradeep K. Dudeja

Background/Objectives: Down-regulated in adenoma (DRA/SLC26A3), an IBD (Inflammatory Bowel Diseases) susceptibility gene and major colonic Cl−/HCO3− exchanger, is reduced in ulcerative colitis (UC). Whether reduced DRA is linked to broader metabolic remodeling during intestinal inflammation remains unclear. We therefore examined DRA-associated metabolic pathways in human UC datasets and metabolic changes across mucosal, luminal, and systemic compartments in DRA-deficient mice. Methods: Six publicly available human colonic transcriptomic cohorts were analyzed to infer KEGG metabolic pathway activity, compare UC with healthy mucosa, and assess associations with DRA expression using random-effects meta-analysis. DRA knockout mice were evaluated by colonic mucosal RNA sequencing, untargeted LC–MS metabolomics of colonic mucosa, feces, and serum, targeted bile acid profiling, MetOrigin analysis, and fecal 16S rRNA sequencing. Results: In the human cohorts, DRA expression was consistently lower in UC. Of the 75 KEGG metabolic pathways evaluated, 48 were altered in UC relative to healthy mucosa, and 45 of these were also associated with DRA expression levels among UC samples. In DRA knockout mice, colonic RNA sequencing identified broad metabolic transcriptional remodeling across 39 KEGG pathways, with metabolic themes that overlapped with the human UC findings. Multi-compartment metabolomics further revealed alterations involving amino acid, nitrogen, redox/cofactor, polyamine, and bile acid metabolism, together with inferred host-microbiota co-metabolic signatures. Conclusions: DRA deficiency was accompanied by broad metabolic remodeling across mucosal, luminal, and systemic compartments. These changes overlapped with DRA-associated signatures in human UC, supporting a role for DRA in epithelial–luminal metabolic homeostasis beyond ion transport.

Metabolites doi: 10.3390/metabo16090689

Authors: Fangliang He Xianrui Wang Jiating Zhang Lizhi Wan Haonan Wu Xianlong Cheng Jia Chen Xiangri Li Wenguang Jing Feng Wei

Background: Saposhnikoviae radix (SR), a valuable Chinese medicinal plant with high medicinal and edible value, suffers from inconsistent quality caused by growth patterns, cultivation duration, and geographical origin. Methods: In this study, comprehensive volatile metabolite profiling using HS-GC-MS/MS combined with multivariate chemometrics was performed to characterize these quality-related differences. Results: In total, 7, 13, and 11 differential volatile compounds were identified for wild versus cultivated SR, cultivation duration, and geographical origin, respectively. By integrating the three comparisons, 11 recurrent differential compounds were selected for network pharmacology and molecular docking analyses. trans-3-Nonen-2-one, benzaldehyde, and Octanoic acid were predicted to interact with key targets, including TNF, IL1B, ALB, EGFR, and CASP3, which were mainly enriched in inflammation- and pain-related pathways, including the PI3K-Akt signaling pathway, apoptosis, and the HIF-1 signaling pathway. Molecular docking further supported favorable predicted interactions between the selected compounds and core targets. Conclusions: Overall, this study reveals volatile metabolite variation associated with major quality-related factors of SR and identifies candidate bioactive and quality-associated volatile compounds. The integration of volatile metabolite profiling, chemometrics, network pharmacology, and molecular docking provides a complementary strategy for the comprehensive quality evaluation of SR and offers a reliable reference for quality control research on other medicinal Apiaceae species.

Metabolites doi: 10.3390/metabo16090688

Authors: Özgür Eken Halil Uçar Ahmet Kurtoğlu Murat Ozan Yusuf Buzdağlı Mehmet Söyler Monira I. Aldhahi

Background/Objectives: Creatine monohydrate (CR) and L-arginine (LA) are widely used ergogenic supplements, but whether acute co-ingestion provides additional performance or task-specific cognitive benefits beyond either supplement alone remains uncertain. This study compared placebo, CR, LA, and LA+CR on repeated-sprint power, countermovement jump (CMJ) height, and Stroop reaction time (RT). Methods: In this randomized, double-blind, four-period crossover trial, 18 recreationally active men completed placebo (CG), CR (0.3 g·kg−1), LA (6 g), and LA+CR conditions separated by at least 72 h (prospectively registered at ClinicalTrials.gov: NCT07548541). Testing began 60 min after ingestion with the Stroop test, followed by CMJ and the Running-Based Anaerobic Sprint Test (RAST). Co-primary outcomes were RAST average power (AP), peak power (PP), CMJ height, and incongruent Stroop median RT. Results: Condition effects were observed for AP, PP, minimum power, CMJ height, and Stroop RT (all p ≤ 0.011), but not fatigue index or Stroop accuracy. All four co-primary omnibus effects remained significant after Holm correction (all pHolm < 0.001). LA+CR produced higher PP than CG (MD 99.01 W; p < 0.001), LA (MD 51.29 W; p = 0.002), and CR (MD 46.71 W; p < 0.001), and greater CMJ height than all comparison conditions (all p < 0.001). LA+CR also produced lower Stroop RT than CG (MD 0.089 s; p < 0.001) and CR (p = 0.041), but not LA (p = 0.271). For AP, LA+CR exceeded CG and CR, but not LA after Bonferroni adjustment. In a secondary 2 × 2 factorial decomposition, no LA×CR interaction for a co-primary outcome survived Holm correction (all pHolm ≥ 0.104). Conclusions: Acute LA+CR produced outcome-specific improvements in peak sprint power and CMJ and selectively reduced Stroop RT, but did not establish biological synergy. Interpretation should remain exploratory given the small male-only sample, unmodelled period/sequence effects, and possible creatine-related carryover.

Metabolites doi: 10.3390/metabo16090687

Authors: Olivia M. McCarthy Clara Solà Chloe Nicholas Sandra Tawfik Merete Bechmann Christensen Signe Schmidt Kirsten Nørgaard Richard M. Bracken Marietta Kokla

Purpose: To characterize plasma metabolomic responses to maximal exercise and their associations with cardiorespiratory fitness (CRF) in adults with type 1 diabetes. Methods: In this exploratory pooled secondary analysis, 21 adults (67% female; age, 51.6 ± 13.2 years; HbA1c, 67.5 ± 7.0 mmol/mol) completed a cycle-ergometer test to exhaustion; metabolomic data were available for 20. Plasma collected at rest, anaerobic threshold (AT), peak exercise, and recovery underwent targeted and untargeted gas chromatography–mass spectrometry profiling. Peak oxygen uptake (V̇O2peak) quantified CRF. Linear mixed-effects models adjusted for age, sex, HbA1c, and diabetes duration. Results: At the AT, L-(+)-lactic acid and 2-hydroxyisobutyric acid increased relative to rest (both q ≤ 0.010). Peak exercise produced changes in glycolytic, tricarboxylic acid (TCA)-cycle, and purine metabolites, with enrichment of the TCA cycle and nucleotide metabolism. TCA-cycle enrichment persisted during recovery. In exploratory interaction analyses, higher V̇O2peak was associated with increases in L-(+)-lactic acid at AT (β = 0.032, q = 0.040) and peak exercise (β = 0.031, q = 0.024), and in malic acid at peak exercise (β = 0.063, q = 0.010) and recovery (β = 0.061, q = 0.015). Conclusions: These preliminary findings suggest the presence of intensity-dependent and fitness-associated metabolic responses to exercise in adults with type 1 diabetes. Given the small sample size, particularly for interaction analyses, these findings require confirmation in larger independent cohorts.

Metabolites doi: 10.3390/metabo16090686

Authors: Julie A. Spratt Neil Tanday Dearbhla M. McGinn Chidiebere D. Chukwu Victor A. Gault Nigel Irwin

Background/Objective: Kisspeptin peptides are being increasingly recognised as regulators of metabolism, but whether these actions differ depending on peptide isoform remains unclear. Methods: The current study compared the enzymatic stability and biological effects of kisspeptin-14 (KP-14), kisspeptin-13 (KP-13) and kisspeptin-10 (KP-10) on beta-cell health and function, glucose homeostasis and appetite. Peptide stability was assessed in murine plasma and, following confirmation of KISS1R expression in BRIN-BD11 beta-cells, actions on insulin secretion, beta-cell proliferation and apoptosis were investigated, with additional secretory studies in isolated murine islets. Metabolic effects of KP-14, KP-13 and KP-10 were subsequently evaluated in healthy male and female mice. Results: KP-14 was resistant to plasma degradation, whereas KP-13 and KP-10 underwent N-terminal proteolysis, with KP-13 degradation generating KP-10 amongst other fragment peptides. The kisspeptin peptides exerted modest direct effects on insulin secretion from BRIN-BD11 cells, with KP-13 being the most efficacious, a bioactivity profile that was confirmed in islets. In addition, KP-13 significantly enhanced beta-cell proliferation and protected against cytokine-induced apoptosis, producing superior beta-cell proliferative effects compared to exenatide. Acute administration of all kisspeptin peptides elevated circulating glucose concentrations, and while KP-14 and KP-10 impaired glucose tolerance, KP-13 did not. Interestingly, KP-14 enhanced glucose-stimulated insulin secretion, with KP-13 and KP-10 being devoid of such actions. All kisspeptin isoforms suppressed food intake, although only KP-13, and especially KP-10 administration, led to an inhibition of feeding. Conclusions: Taken together, these data identify KP-13 as possessing the most favourable overall metabolic actions, based on a combination of beneficial actions on beta-cell health and function together with inhibition of feeding and lack of prominent glucose-elevating actions, suggesting possible therapeutic application for type 2 diabetes.

Metabolites doi: 10.3390/metabo16090685

Authors: Xiaoyu Xiao Siyi Qin Zijian Jiang Yuhui Huo Junlin Li Minlan Yang

Folate, which is also known as folic acid, vitamin B9, is a key coenzyme in one-carbon metabolism (OCM) and participates in nucleic acid synthesis, methylation, and redox regulation. In recent years, numerous studies have demonstrated its close association with type 2 diabetes mellitus (T2DM), gestational diabetes mellitus (GDM), and diabetic complications. Serum folate levels are decreased in patients with T2DM, which is associated with increased urinary excretion and metformin-mediated inhibition of folate absorption. The onset of GDM is characterized by a folate dose-dependent imbalance characteristic, high folate combined with vitamin B12 deficiency significantly increases the risk of GDM. Maternal folate status can also modulate offspring insulin sensitivity through epigenetic modifications. The mechanisms by which folate regulates diabetes mainly include lowering homocysteine (Hcy) concentrations, suppressing oxidative stress and inflammatory responses, modulating OCM, and mediating DNA methylation modifications. Cumulative evidence-based data indicate that appropriate folic acid supplementation can improve insulin resistance, restore vascular endothelial function, reduce the risk of cardiovascular diseases (CVDs), diabetic peripheral neuropathy, retinopathy, cognitive impairment, and other complications, and alleviate damage induced by high glucose. This review systematically summarizes the roles and molecular mechanisms of folate in diabetes and its complications, highlighting folate as a potential intervention target for diabetes.

Metabolites doi: 10.3390/metabo16090684

Authors: María Clara Donadelli Estefanía Bernay Gricel Alejandra Cagliardi Gustavo Martínez Victoria Casajus

Background: Kale is a vegetable of the Brassicaceae family recognized for its high nutritional value. After harvest, kale leaves undergo senescence, characterized by the loss of green color due to chlorophyll degradation. This deterioration affects organoleptic quality and consumer acceptance. Objective: The present study investigated the effect of leaf developmental stage at the moment of harvest on postharvest senescence and parameters related to the nutritional quality of kale during postharvest storage at 20 °C. Material and Methods: Kale leaves were harvested at three developmental stages—young inner leaves (~20 cm), middle leaves (central position, 20–30 cm), and mature outer leaves (35–40 cm)—and stored at 20 °C for six days in darkness with an RH of 90–95%. At harvest and after two, four, and six days of storage, superficial color, weight loss, total phenolic and flavonoid contents, antioxidant capacity, total and reducing sugars, and total and soluble proteins were evaluated. Because yellowing is the main postharvest symptom in kale leaves, the relative expressions of chlorophyll catabolic genes such as NYC, NOL, SGR, PPH and PaO were also analyzed. Results: The leaf development stage strongly influenced postharvest life and the biochemical composition of the leaves. Inner leaves showed a lower rate of chlorophyll degradation during postharvest storage and retained a more intense green color, consistent with a reduced expression of chlorophyll catabolic genes. In contrast, middle and outer leaves exhibited greater chlorophyll loss and more pronounced yellowing. Inner leaves maintained the best overall visual quality at the end of storage and showed higher phenolic as well as greater antioxidant capacity compared with middle and outer leaves. Inner leaves also contained higher levels of reducing sugars and soluble proteins. Conclusions: Taken together, these findings indicate that harvesting kale at earlier developmental stages might represent an effective strategy to extend shelf-life and provide a product with improved values in the parameters actually measured.

Metabolites doi: 10.3390/metabo16090683

Authors: Junru Zeng Hong Wang Fengrui Shi

Background/Objectives: Triglyceride-rich lipoproteins contribute to residual atherosclerotic cardiovascular risk, but the comparative and dose–response effects of exercise modalities on triglycerides (TGs) remain uncertain. We compared aerobic exercise (AE), resistance training (RT), combined exercise (CE), and high-intensity interval training (HIIT) and characterized modality-specific dose–response relationships. Methods: Five databases were searched from inception through January 2026. Randomized trials in adults were synthesized using contrast-based random-effects network meta-analysis (NMA) and model-based network meta-analysis (MBNMA), with dose harmonized as MET·min/week. Results: Seventy-six independent trials included 4483 participants in 208 randomized arms. The 122 active arms generated 122 exercise–control contrasts; one CE contrast with zero reported sampling variance was excluded from both network models, leaving 121 analyzed contrasts in 86 independent comparator blocks. Exercise reduced TG by 0.149 mmol/L, on average (95% CI 0.099–0.199), although the prediction interval crossed the null. CE had the largest average network estimate (MD −0.255 mmol/L), followed by HIIT (−0.225) and AE (−0.130); RT was inconclusive. The modality-specific linear model had the lowest AICc. Per 1000 MET·min/week, slopes were −0.128 mmol/L for AE, −0.307 for CE, −0.081 for RT, and −0.239 for HIIT; the RT slope crossed the null. No supported interior optimum was identified. Conclusions: AE, CE, and HIIT were associated with lower TGs, but certainty was low, and 50 trials were at high risk of bias. CE produced the largest average estimate within the available network, not definitive evidence of universal superiority. The reported lowest and upper observed doses are descriptive boundaries rather than efficacy thresholds or prescription targets.

Metabolites doi: 10.3390/metabo16090682

Authors: Nesrullah Ayşin Süheyla Altuğ Özsoy Zübeyir Huyut

Objective: Traffic-related air pollution is recognised as a significant environmental risk factor associated with obesity and metabolic dysfunction. Although the positive effects of regular exercise on metabolic health are well established, its protective role against traffic-related air-pollution-induced metabolic dysfunction has not been fully elucidated. This study was conducted to investigate the effects of diesel exhaust exposure and moderate exercise on the development of obesity, metabolic changes, adipokine profile, and inflammatory response in rats. Materials and Methods: In this randomised controlled experimental study, 48 female Wistar Albino rats were randomised into six experimental groups. The animals were exposed to diesel exhaust simulating traffic-related air pollution (average 300 μg PM2.5/m3) for 2 or 4 h daily over an eight-week period. Rats in the exercise groups underwent moderate-intensity treadmill exercise for 30 min at a speed of 15 m/min, five days a week, over the same period. At the end of the study, obesity indicators, serum lipid profile, glucose metabolism parameters, adipokines and inflammation markers were assessed. Results: Diesel exhaust exposure caused a significant increase in final body weight, BMI, Lee index and VAI values (p < 0.001). Furthermore, levels of LDL cholesterol, total cholesterol, triglycerides, glucose, adiponectin, leptin, CRP, IL-37, IL-1β, IL-6 and TNF-α increased significantly, whilst HDL cholesterol and insulin levels decreased significantly (p < 0.001). The most pronounced metabolic and inflammatory changes were observed in the 4 h exhaust gas exposure group. Elevations in pro-inflammatory cytokines, accompanied by increased visceral adiposity, supported the development of systemic metabolic inflammation. Exercise significantly reduced weight gain, visceral adiposity, dyslipidaemia, hyperglycaemia, adipokine imbalance and the inflammatory response (p < 0.001). However, exercise could not fully reverse all the changes associated with long-term diesel exhaust exposure. Conclusions: Traffic-related diesel exhaust exposure led to metabolic disorders in rats, characterised by visceral adiposity, dyslipidaemia, impaired glucose homeostasis, adipokine imbalance and chronic low-grade inflammation. Regular moderate-intensity exercise significantly reduced these adverse effects but could not eliminate them entirely. The findings suggest that traffic-related air pollution may contribute to the development of obesity via inflammation-mediated metabolic mechanisms, and that regular physical activity may serve as an important protective strategy in limiting these effects.

Metabolites doi: 10.3390/metabo16090681

Authors: Hasan Esat Yücel Tufan Ulcay Murat Doğan Saliha Demir Gizem Çolak Ruken Oncu Ebru Ceylan Muhammed Fırat Aladag Emre Uguz Birgül Deniz Doğan Cahit Ucar

Background: Metabolic age (Met-Age), a bioelectrical impedance analysis (BIA)-derived index based on basal metabolic rate (BMR) and body composition, has been associated with cardiometabolic risk. However, its relationship with body mass index (BMI)-defined obesity severity in individuals with type 2 diabetes mellitus (T2DM) remains unclear. This study investigated the association between obesity severity and Met-Age and identified factors associated with Met-Age. Methods: In this retrospective observational study, 683 adults with T2DM were classified into five BMI categories: normal weight, overweight, and class I, II, and III obesity. Anthropometric, laboratory, and BIA-derived body composition data were analyzed using correlation and sex-stratified multivariable linear regression analyses. Results: Although chronological age did not differ significantly across groups, Met-Age increased progressively with increasing obesity severity (p < 0.001). The Jonckheere–Terpstra test confirmed a significant ordered increase in Met-Age across BMI categories. BMI, fat mass, and waist-to-height ratio (WHtR) also increased progressively with increasing obesity severity. In the multivariable regression analyses, chronological age and WHtR were positively associated with Met-Age in both sexes, whereas muscle mass showed a weak inverse association with Met-Age only in female participants. No significant correlations were found between Met-Age and fasting blood glucose (FBG) or hemoglobin A1c (HbA1c) levels (all p > 0.05). Conclusions: In individuals with T2DM, greater obesity severity was associated with higher BIA-derived Met-Age. WHtR was positively associated with Met-Age in both sexes, whereas muscle mass showed a weak inverse association among female participants. Met-Age may serve as a descriptive index reflecting body composition and metabolic characteristics; however, its incremental clinical value beyond BMI and WHtR remains unproven.

Metabolites doi: 10.3390/metabo16090680

Authors: Lei Yang Chaowei Wang Liangyu Cui Yongxiang Zhu Xi Wang

Coffee is one of the most widely consumed beverages worldwide and a major dietary source of bioactive compounds, including caffeine, chlorogenic acids (CGAs), trigonelline, non-digestible polysaccharides, and roasting-derived melanoidins. Habitual coffee consumption has been associated with a lower risk of type 2 diabetes (T2D) and more favorable liver-related outcomes, whereas evidence regarding obesity and metabolic dysfunction-associated steatotic liver disease (MASLD) remains heterogeneous. These associations may reflect interactions among coffee constituents, the gut microbiota, and host metabolic phenotypes rather than the effects of individual compounds alone. In this focused narrative review, we integrate evidence on the gastrointestinal fate, microbial transformation, and systemic availability of major coffee constituents, with particular emphasis on short-chain fatty acids, CGA-derived phenolic acids, and microbiota-dependent bile acid metabolism. We examine how these pathways may influence intestinal barrier integrity, mucosal immunity, enteroendocrine signaling, and gut–liver communication then consider their potential relevance to the regulation of energy, glucose, and lipid metabolism. We evaluate the relevance of these pathways to obesity, T2D, and MASLD, as well as major sources of inter-individual variability, including host genetics, baseline microbiota, metabolic phenotypes, background diet, and coffee composition, processing, brewing, filtration, dose, and beverage additives. Finally, we discuss physiologically relevant exposure levels, candidate biomarkers of individual responses, and risk–benefit considerations. Although mechanistic and preclinical evidence is accumulating, human studies directly demonstrating that coffee-induced microbiota changes mediate beneficial metabolic outcomes remain limited. Future randomized and mechanistic studies should integrate chemically characterized coffee exposures with longitudinal microbiome, metabolomic, physiological, and clinically relevant outcome measurements.

Metabolites doi: 10.3390/metabo16090679

Authors: Fumito Hamada Hikaru Hori Hiroki Kumagai Muneaki Ogata Hiroyuki Yokoyama Yuko Tomiyama Hiroko Sugawara Ryo Asada Akito Hatanaka Masato Masuda Yuta Okamoto Ryusei Hatae Hitoshi Iida Yoshimi Yamaguchi Tetsuya Yoshida Rika Yano Leo Gotoh

Background/Objectives: Bipolar depression impairs psychosocial functioning, cognition, and quality of life. Evidence regarding adjunctive exercise remains limited. This pilot study evaluated the clinical and biological effects of adjunctive exercise in patients with bipolar depression. Methods: In this multicenter cluster-randomized controlled trial, 26 hospitalized patients were assigned by facility to treatment as usual plus supervised exercise (TAU + Ex) or supervised stretching (TAU + SC) for 6 weeks. Twenty-four completed the study (TAU + Ex, n = 14; TAU + SC, n = 10). Clinical outcomes and plasma tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-1β, IL-2, brain-derived neurotrophic factor (BDNF), and vascular endothelial growth factor-A (VEGF-A) were assessed at baseline and week 6. The trial was registered with the University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR; UMIN000045877; registered on 1 December 2021). Results: FAST and QIDS-SR scores improved significantly in both the exercise and stretching groups after the 6-week intervention, but no significant group × time interactions were observed. No significant group, time, or group × time effects were observed for any plasma biomarker. Before correction for multiple comparisons, nominal correlations were observed between changes in IL-1β and FAST, BDNF and FAST, and VEGF-A and Spotter performance. None remained statistically significant after Benjamini–Hochberg FDR correction. Conclusions: Adjunctive exercise therapy was not superior to active stretching. The exploratory biomarker–outcome correlations did not remain significant after correction for multiple comparisons and should be considered hypothesis-generating. Larger, adequately powered randomized controlled trials are warranted.

Metabolites doi: 10.3390/metabo16090678

Authors: Marzhan Rakhimzhanova Bikadisha Bimurat Aimira Jangashkarova Assel Issabayeva Aigul Durmanova Alina Alzhaxina Tatyana Ivanova-Razumova Gulmira Rapilbekova

Background: Lipodystrophy syndromes are a group of rare disorders characterized by partial or generalized loss of adipose tissue, severe insulin resistance, and multiple metabolic complications. Despite increasing global awareness, lipodystrophy remains underrepresented in the published literature from Central Asia, and its true regional prevalence remains unknown. This study presents a case series of patients from Central Asia with congenital and acquired generalized lipodystrophy, reviews the existing regional literature, and determines challenges and future directions to improve lipodystrophy care in this region. Methods: We conducted a targeted regional literature review focusing on published reports from five Central Asian countries: Kazakhstan, Kyrgyzstan, Tajikistan, Turkmenistan, and Uzbekistan. A literature search was performed in electronic databases, including PubMed/MEDLINE, Scopus, and Web of Science, from the earliest available records to 20 July 2026. Results: Reported cases of lipodystrophy from Central Asia were extremely scarce. Our cases also highlighted important barriers to effective care, including limited disease awareness and access to specialized therapies. Discussion: These findings indicate that lipodystrophy is underrepresented in the literature, and the true disease burden and extent of underdiagnosis in Central Asia remain unknown. Improving clinical awareness, developing international collaborations, widening access to genetic testing, and establishing multidisciplinary referral systems could significantly enhance patient outcomes. Conclusions: Lipodystrophy remains an underreported, rare condition in Central Asia. Our findings emphasize the urgent need for improved awareness, regional cooperation, development of expert clinics, and better access to specialized diagnostic and therapeutic resources to optimize care for affected patients.

Metabolites doi: 10.3390/metabo16090677

Authors: Fangyu Zhao Yuanjiang Xu Xiaozhong Lan Zhen Li

Background/Objectives: Tropane alkaloids (TAs) and phenylpropanoid polyamine alkaloids (PPAs) are major alkaloid constituents of solanaceous plants; however, the tissue-specific distribution of these compounds in Anisodus luridus, a medicinal plant native to southeastern Xizang, remains incompletely characterized. Methods: A biosynthesis-informed putative annotation workflow was developed using ultra-performance liquid chromatography–high-resolution mass spectrometry (UPLC-HRMS) to characterize alkaloid profiles across seven tissues (root, stem, leaf, calyx, corolla, ovary, and seed). Putative annotations were guided by substrate–product structural relationships, predicted enzyme-catalyzed modifications, and diagnostic tandem mass spectrometry (MS/MS) fragmentation patterns. Results: Eighteen alkaloid features were characterized, comprising 6 TAs and 12 PPAs. Hyoscyamine and scopolamine were identified using authentic standards, and the remaining 16 features were putatively annotated, including dihydroanisodamine, hexosylhyoscyamine, and several PPAs not previously reported in A. luridus. TAs were predominantly enriched in roots and ovaries, whereas PPAs accumulated in floral tissues and leaves. Orthogonal partial least squares discriminant analysis (OPLS-DA) identified scopolamine (variable importance in projection (VIP) = 1.30) as a principal variable associated with separation among tissue metabolic profiles. Conclusions: This workflow supports the putative annotation of alkaloids in non-model medicinal plants. The observed tissue-specific distribution patterns provide a basis for future comparative studies with A. belladonna and for evaluating cultivation, tissue-selective harvesting, and extraction strategies.

Metabolites doi: 10.3390/metabo16090676

Authors: Luciana Jorge Bianca Castino Junqueira Silva Marcia Bastos Convento Clara Versolato Rasvisckas Maria Aparecida da Glória Cassiane Dezoti da Fonseca Eloiza de Oliveira Silva Maria de Fatima Fernandes Vattimo Fernanda Teixeira Borges

Background: Mechanical ventilation (MV) may be associated with extrapulmonary responses shaped by the host inflammatory-metabolic background. Methods: This exploratory study characterized renal changes during 1 h of reduced-tidal-volume mechanical ventilation in male Wistar rats with diet-induced obesity or lipopolysaccharide (LPS)-induced endotoxemia, together with concurrent respiratory, acid–base, metabolic, and hemodynamic alterations. Arterial blood gas, biochemical, hemodynamic, and urinary variables were assessed at the pre-MV and post-MV time points. Renal protein expression was evaluated only at the post-MV time point. Results: The arterial partial pressure of carbon dioxide (PaCO2) decreased significantly in both groups. In the Obese group, metabolic acid–base abnormalities persisted and were accompanied by increases in serum urea and the urinary protein-to-creatinine ratio. In the LPS group, lactate increased, bicarbonate decreased, and mean arterial pressure and serum creatinine increased. At the post-MV time point, renal interleukin-6 expression was lower in the LPS group than in the Obese group, whereas renal tumor necrosis factor-alpha expression was higher in the LPS group than in the Obese group. Conclusions: These results support interpreting the physiological and renal changes observed in association with the integrated experimental protocol, including mechanical ventilation, in relation to the host’s underlying pathophysiology and inflammatory-metabolic context.

Metabolites doi: 10.3390/metabo16090675

Authors: Hyang Yeon Kim Jung Dae Lee Ho-Seong Lee Ji-Suk Chang Suhkmann Kim Gi-Wook Hwang Kyu-Bong Kim

Background/Objectives: Female soccer players differ from males in body composition, muscular strength, and hormonal fluctuations, which may influence performance, fatigue, recovery, and injury risk. This study aimed to characterize the time course of urinary metabolic perturbation and its persistence during a 20-day winter training season (WTS) using urinary metabolomics. Methods: Urinary metabolites in female soccer players were analyzed before (Bef) and 1 (After_1D) and 7 days after completion (After_7D) of the 20-day WTS using nuclear magnetic resonance (NMR) spectroscopy combined with multivariate analysis. Results: A total of 84 metabolites were identified in urine samples, and distinct group separation was observed by partial least squares discriminant analysis (PLS-DA) in targeted profiling. Among these, 13 metabolites, including adenine, alanine, citrate, creatine, creatine phosphate (PCr), formate, glutamine, glycine, malonate, mannitol, taurine, trimethylamine N-oxide (TMAO), and urea, showed significant changes in the three groups (Bef, After_1D and After_7D). Conclusions: Of the identified metabolites, six metabolites—alanine, PCr, formate, glutamine, glycine, and malonate—were significantly increased at 1 day post-WTS (After_1D) compared with pre-WTS (Bef) levels. These metabolites were lower at 7 days post-WTS (After_7D) than at 1 day post-WTS (After_1D). However, the metabolic profile had not fully returned to pre-WTS levels by day 7, suggesting that recovery following WTS may require longer than at least 7 days after a 20-day WTS.

Metabolites doi: 10.3390/metabo16090674

Authors: Serdar Şerare Serkan Paçacı Necip Arman Ahmet Karadağ Anıl Şahin

Background/Objective: Insufficient research has examined the muscle damage and inflammatory effects of training performed in a fasted state with reduced (low) glycogen reserves. Methods: This study aimed to investigate the acute effects of exercise sessions performed under fasting (FST) and liquid nutrient-supplemented postprandial (PPD) conditions, as well as with full and reduced body glycogen stores, on muscle damage markers creatine kinase (CK) and lactate dehydrogenase (LDH), and the inflammatory marker interleukin-6 (IL-6). Eleven male amateur football players (mean age 19.91 ± 1.64 years) were included in the study. Participants performed a single 60 min aerobic endurance exercise session at 70% VO2max under the liquid nutrient-supplemented PPD condition, and two 60 min sessions with a 60 min recovery interval under 10–12 h FST conditions. Data were analyzed using paired samples t-test, ANOVA, Wilcoxon, and Friedman tests, depending on parametric and nonparametric assumptions. Results: In pre-exercise (PRE-FE) measurements, no significant differences were observed between the FST and PPD conditions for GLU, CK, LDH, and IL-6 levels. In postexercise (POST-FE) measurements, GLU levels were significantly higher in the PPD condition than in the FST condition (p < 0.05), whereas LA, CK, LDH, and IL-6 levels did not differ significantly between the two conditions. In the FST condition, following the second exercise session performed with default reduced glycogen availability (POST-SE), CK levels significantly increased from 279.36 ± 16.7 U/L to 335.63 ± 20.3 U/L, LDH levels from 178.72 ± 38.1 U/L to 208.09 ± 50.7 U/L, and IL-6 levels from 2.60 ± 2.4 pg/mL to 8.69 ± 5.8 pg/mL (p < 0.05). In the PPD condition, no significant changes were observed in any of the parameters following a single exercise session (p > 0.05). Conclusions: The two-a-day training approach performed with default reduced glycogen availability may increase muscle damage and inflammatory responses. These findings are considered as an inference that low-glycogen training should not be implemented during the pre-competition period because of the risk of inducing muscle damage.

Metabolites doi: 10.3390/metabo16090673

Authors: Shouhe Zhu Chuanzhi Tang Weiguo Ding Junxia Wen Xinwei Xu Xinhui Wang Weixing Xu

Background: Metabolic disturbances have been implicated in intervertebral disc degeneration (IVDD) and low back pain, but the contribution of circulating metabolites remains uncertain. Objectives: We examined associations of genetically proxied plasma metabolites and metabolite ratios with IVDD outcomes defined at different anatomical locations and with a related pain phenotype. Methods: Summary statistics for metabolite exposures were obtained from the Canadian Longitudinal Study on Ageing (CLSA) plasma metabolome genome-wide association study (GWAS). The outcomes were Intervertebral Disc Degeneration of Cervical Spine (IVDD_Cervical), Intervertebral Disc Degeneration of Thoracic, Thoracolumbar, and Lumbosacral Spine (IVDD_TTL), and Low Back Pain (Pain_Lowback). IVDD_Cervical and IVDD_TTL represented IVDD outcomes defined at different anatomical locations of the spine, whereas Pain_Lowback was analysed separately as an IVDD-related pain phenotype. One genome-wide significant sentinel single-nucleotide variant (SNV) was used for each exposure, and Mendelian randomization (MR) estimates were calculated with the Wald ratio. The within-outcome Bonferroni threshold was 6.25 × 10−4. Results: Eighty metabolites and metabolite ratios were evaluated. Two associations were significant, both for IVDD_TTL. Higher genetically proxied dimethylarginine (SDMA + ADMA), mapped to DDAH1, was associated with lower IVDD_TTL risk (odds ratio [OR] = 0.80; 95% confidence interval [CI]: 0.72–0.90; p = 2.5 × 10−4), whereas higher alpha-hydroxyisovalerate, mapped to LDHA, was associated with higher risk (OR = 1.08; 95% CI: 1.04–1.13; p = 4.6 × 10−4). No significant association was identified for IVDD_Cervical or Pain_Lowback; 23 associations were classified as suggestive significant. DrugBank annotation identified compounds interacting with DDAH1 and LDHA. Conclusions: The results highlight DDAH1-linked methylarginine metabolism and LDHA-linked glycolytic and lactate-related biology as candidate pathways for IVDD_TTL. Findings for IVDD_Cervical, Pain_Lowback, and cross-outcome overlap remain exploratory.

Metabolites doi: 10.3390/metabo16090672

Authors: Cheng Cui Junhao Chen Haodi Chai Dongyang Liu

Introduction: Altered lipid metabolism is implicated in atrial fibrillation, but longitudinal lipidomic changes during anticoagulation remain poorly characterized. Objectives: To characterize plasma lipidomic changes during 12 weeks of edoxaban treatment and explore associated metabolic pathways in Chinese patients with nonvalvular atrial fibrillation (NVAF). Methods: This prespecified substudy of a prospective multicenter study analyzed fasting predose plasma collected at baseline and week 12 from 74 patients receiving protocol-assigned edoxaban doses (15 mg, n = 3; 30 mg, n = 23; 60 mg, n = 48). Widely targeted UPLC-MS/MS lipid profiling, with laboratory quality control (QC CV < 30%), yielded 1099 lipid features retained for statistical analysis. Differential lipids were defined by paired testing with Benjamini–Hochberg correction (|log2FC| > 0.58 and q < 0.05). Results: The overall, 30 mg, and 60 mg analyses identified 25, 15, and 35 FDR-significant features, respectively; none was significant in the exploratory 15 mg group. Twelve features were shared across the primary analyses and increased at week 12; they comprised mainly acylcarnitines, together with docosadienoic acid and DG (21:0/16:0/0:0), and four lacked HMDB identifiers. The corresponding FDR panels yielded participant-grouped out-of-fold AUCs of 0.918 (95% CI 0.890–0.963), 0.786 (0.701–0.890), and 0.891 (0.835–0.950) for discriminating baseline from week 12, respectively. Pathway mapping identified no multi-compound enriched pathways (q < 0.05). Observed annotations represented single-metabolite signals—most notably DG (21:0/16:0/0:0) (HMDB0094301) and octanoylcarnitine (HMDB0000791)—rather than coordinated, pathway-level alterations. Conclusions: Paired analyses identified consistent longitudinal plasma lipidomic changes in patients receiving edoxaban. The 12 shared features provide focused candidates for targeted validation. Controlled studies are needed to determine whether these changes are specific to edoxaban and to establish their biological and clinical relevance.

Metabolites doi: 10.3390/metabo16090671

Authors: Buket Yilmaz Bulbul Burak Andac Mehmet Celik

Background/Objectives: Postprandial glycemic responses vary markedly among individuals, but it remains unclear whether the glycemic effect of incremental carbohydrate exposure is systematically modified by insulin resistance. We examined whether insulin resistance alters the carbohydrate–postprandial glucose dose-response across distinct meal contexts. Methods: We performed a secondary repeated-measures analysis of the publicly available CGMacros cohort. Forty-five adults spanning normoglycemia, prediabetes, and type 2 diabetes wore blinded FreeStyle Libre Pro and Dexcom G6 Pro continuous glucose monitors while recording meals for approximately 10 days. The primary outcome was 2-h incremental area under the glucose curve (iAUC). Mixed-effects models tested carbohydrate × HOMA-IR interactions, adjusted for protein, fat, fiber, premeal glucose, age, sex, and BMI. Breakfast was the primary structured context, lunch provided within-cohort contextual confirmation, dinner was a free-living contrast, and Dexcom analyses assessed cross-device robustness. Results: The primary Libre analysis included 423 breakfasts and 414 lunches from 44 participants. Higher HOMA-IR significantly amplified the iAUC associated with each 10-g increment in carbohydrate during breakfast (interaction β = 178.5 mg·min/dL per 1-SD higher HOMA-IR; 95% CI 79.5–277.5; p < 0.001) and lunch (β = 109.8; 95% CI 34.2–185.3; p = 0.004). The direction was supported with Dexcom during breakfast (β = 165.3; 95% CI 45.0–285.6; p = 0.007) and lunch (β = 87.9; 95% CI 2.6–173.1; p = 0.043) and remained robust in GEE, participant fixed-effects, random-slope, activity-adjusted, and other sensitivity analyses. No positive interaction was observed during dinner. Conclusions: In this observational secondary analysis, insulin resistance modified the glycemic impact of carbohydrate across two structured meal contexts, with supportive cross-device evidence. These findings are hypothesis-generating and do not establish HOMA-IR-based dietary prescriptions; prospective external validation is required.

Metabolites doi: 10.3390/metabo16090670

Authors: Ayako Oda Yosuke Suzuki Teruhide Koyama Jun Negami Sakura Suzuki Koudai Iino Nao Yamagishi Natsuki Kamio Etsuko Ozaki Yasuyuki Yamamoto Masahiro Nakatochi Yukihide Momozawa Ryota Tanaka Hiroyuki Ono Takahiro Sumimoto Ryosuke Tatsuta Hiroki Itoh Naoyuki Takashima Keitaro Matsuo Keiko Ohno

Background/Objectives: Cytochrome P450(CYP)2C19 activity varies widely among individuals. As genetic factors, CYP2C19*2 and CYP2C19*3 alleles reduce CYP2C19 activity, while the CYP2C19*17 allele increases CYP2C19 activity. However, environmental and physiological factors can also influence individual CYP2C19 activity. In this study, we searched for novel endogenous biomarkers for CYP2C19 activity using CYP2C19 gene polymorphism data combined with results of untargeted metabolomic analysis. Methods: 431 general adults analyzed in the Kyoto J-MICC Study and 255 patients who visited Oita University Hospital were studied. Plasma samples were pretreated by solid-phase and liquid-liquid extraction and subjected to untargeted metabolomic analysis using ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. Based on CYP2C19 gene polymorphism data, participants were classified into extensive metabolizers (EM), intermediate metabolizers (IM), and poor metabolizers (PM). Compounds showing significant differences in abundance among the three groups were considered candidate compounds for predicting CYP2C19 activity. The predictive performance of candidate compounds for CYP2C19 PM status was evaluated using covariate-adjusted receiver operating characteristic (ROC) analysis. Results: The normalized abundance of compounds with m/z 160.1342, 303.2319 (a fatty acyl or prenol lipid), 314.2309, 449.3238, 653.3021, 792.5744, and 811.5988 (a glycerophospholipid or sphingolipid), and 902.5404 (a fatty acyl) differed significantly among CYP2C19 EM, IM, and PM groups (p < 0.05), and these eight compounds were considered candidate compounds. Covariate-adjusted ROC analysis showed that none of the candidate compounds significantly improved the discrimination of CYP2C19 PM status. Conclusions: Untargeted metabolomics combined with CYP2C19 gene polymorphism data yielded eight compounds associated with CYP2C19 phenotype. Further studies are needed to evaluate the usefulness of these compounds as biomarkers of CYP2C19 activity.

Metabolites doi: 10.3390/metabo16090669

Authors: Panpan Li Yaojia Zou Yinghao Mao Fan Ye Tao Luo

Background: Euryale ferox Salisb. seed (Euryales Semen) is a traditional Chinese medicine whose therapeutic application is modulated by wheat bran stir-frying, a process that enhances its effects on tonifying the spleen and kidney. The molecular mechanisms and alterations in bioactive components underlying this processing-enhanced efficacy remain to be fully elucidated. This study aimed to systematically investigate the impact of wheat bran stir-frying on the metabolite profile of Euryales Semen and to reveal its potential pharmacological mechanisms. Methods: An integrated strategy combining UPLC-MS/MS-based metabolomics, network pharmacology, and molecular docking was employed. First, differential metabolites between raw and processed Euryales Semen were identified and quantified. Second, a “metabolite-target-disease” network was constructed to predict core targets and pathways. The docking protocol was validated by re-docking of co-crystallized ligands, with all RMSD values below 2.0 Å. Finally, molecular docking was used to predict the binding interactions between key metabolites and potential protein targets. Results: A total of 1597 metabolites were identified, and 244 were defined as differentially expressed (VIP > 1, |log2(FC)| ≥ 1). Processing significantly upregulated 19 of the 22 key metabolites identified, with Urolithin B showing the most dramatic increase (168-fold), which was identified with Level 1 confidence using an authentic standard. Network pharmacology analysis identified 10 core targets (e.g., MMP2, MMP9, AKR1B1) primarily associated with tumors, diabetes, and other chronic diseases. Molecular docking predicted strong binding affinities (<−9.0 kcal·mol−1) between these core targets and key upregulated metabolites, including Urolithin B, Tangeretin, and Syringetin. Conclusions: This study suggests that wheat bran stir-frying fundamentally reshapes the metabolic profile of Euryales Semen, leading to the enrichment of bioactive metabolites (e.g., Urolithin B) and suggesting a potential for enhanced tonifying, anti-tumor, and anti-diabetic activities that warrants further experimental investigation. The dramatic increase in the content of Urolithin B, a metabolite not commonly found in plants, raises the hypothesis of a processing-induced chemical transformation, which warrants further experimental validation, and highlights its potential as a critical component for the herb’s efficacy. The findings offer a preliminary computational basis for understanding traditional processing practices, linking traditional knowledge with modern scientific evidence.

Metabolites doi: 10.3390/metabo16090668

Authors: Zvonko Antunović Lucija Bronić Željka Klir Šalavardić Mislav Đidara Luka Šramek Josip Novoselec

Background/Objectives: Determination of blood metabolic profile and hemogram are particularly valuable in animals kept in extensive and semi-intensive farming systems, as within those systems, it is not possible to properly monitor animals. This research investigated changes in the hemogram indicators and blood metabolic profile of Dalmatian Pramenka ewes by comparing different lactation stage and litter size. Methods: This study investigated changes in the hemogram indicators and blood metabolic profile of 33 Dalmatian Pramenka ewes during lactation as influenced by the lactation stage (taken on the 40th, 70th and 100th day of lactation) and the litter size (1 or 2 lambs in litter). Referring to the litter size, 11 ewes had twins, and 22 ewes had one lamb in litter. Results: This study confirmed the significant influence of stage of lactation on the blood metabolic profile (minerals: Ca, P-inorganic, Mg, Fe, metabolites: urea, glucose, triglycerides, ALB, GLOB and A/G ratio, and ALT enzyme) and most of the hemogram indicators (RBC and WBC content of HGB and HCT and portion of monocytes) in the blood of lactating ewes. The influence of litter size on the hemogram and blood metabolic profile was insignificant, yet its only statistically significant effect was determined for urea concentrations in the blood of ewes. Conclusions: When planning quality monitoring procedures of a herd, a blood metabolic profile and hemogram of the animals should be implemented as routine herd-monitoring tools for indigenous sheep breeds raised under extensive production systems.

Metabolites doi: 10.3390/metabo16090667

Authors: Candost Ege Satilmis Bulent Akyuz Serpil Demir

Background/Objectives: The association between vitamin D status and neuropathic pain in musculoskeletal disorders remains uncertain. This study examined the relationship between serum 25-hydroxyvitamin D [25(OH)D] levels and neuropathic pain features in adults with musculoskeletal pain. Methods: This single-center analytical cross-sectional study included 300 adults aged 18–79 years who presented between June and December 2024. Participants were classified as vitamin D deficient (<20 ng/mL; n = 105), insufficient (20–<30 ng/mL; n = 117), or sufficient (≥30 ng/mL; n = 78). Neuropathic pain was assessed using DN4, LANSS, painDETECT, and the Short-Form McGill Pain Questionnaire. Multivariable logistic regression adjusted for age, sex, body mass index, diabetes mellitus, inflammatory rheumatic disease, cervical/lumbar discopathy, and fibromyalgia. Results: Neuropathic pain scores increased as 25(OH)D levels decreased. DN4 positivity occurred in 53.3%, 43.6%, and 25.6% of the deficient, insufficient, and sufficient groups, respectively (p < 0.001). Compared with sufficiency, vitamin D deficiency and insufficiency were associated with DN4 positivity (adjusted odds ratio [aOR] 3.53, 95% confidence interval [CI] 1.76–7.08; and aOR 2.38, 95% CI 1.22–4.64, respectively). Each 10 ng/mL decrease in 25(OH)D was associated with 46% higher odds of DN4 positivity (aOR 1.46, 95% CI 1.14–1.86). Similar associations were observed for LANSS and painDETECT ≥13 and persisted in sensitivity analyses. Conclusions: Lower 25(OH)D levels were associated with higher neuropathic pain scores and more frequent positive screens. The cross-sectional design, modest effect sizes, and unmeasured supplementation and calcium–parathyroid hormone variables preclude conclusions regarding causality, diagnostic utility, or treatment efficacy.

Metabolites doi: 10.3390/metabo16090666

Authors: Jing Xu Kai Sang Junjun Zhang

Background: Alcohol-associated liver disease (ALD) involves ethanol-related metabolic injury, inflammation, gut–liver dysfunction, and progressive loss of skeletal muscle and functional reserve. Exercise could influence several of these pathways, but ALD-specific evidence remains limited. We therefore evaluated exercise as an adjunct, not a substitute for established care, across the ALD spectrum. Methods: We conducted a structured narrative review of PubMed/MEDLINE, Google Scholar, and publisher records updated through 20 August 2026. Evidence was classified as direct human ALD/MetALD evidence, direct preclinical alcohol-injury evidence, indirect clinical evidence from MASLD or mixed-etiology cirrhosis, or mechanistic evidence from broader exercise biology. Results: Direct human evidence is predominantly observational. Leisure-time physical activity is associated with lower liver mortality across drinking patterns and with lower odds of at-risk advanced fibrosis in MetALD/ALD. These associations do not establish the efficacy of structured exercise training. Preclinical alcohol-injury models support redox- and Nrf2-mediated hepatoprotection, whereas proposed effects on AMPK-SIRT1-PGC-1α signaling, mitochondrial quality, gut microbial metabolites, myokines, and ammonia handling remain largely extrapolated. Metabolomic studies identify disturbances in redox balance, lipid intermediates, acylcarnitines, bile acids, and amino acid and tryptophan metabolism. These alterations provide candidate endpoints for future trials. Myostatin and decorin are associated with ALD severity, but their responsiveness to exercise is unknown. We therefore propose a stage-specific research and practice framework with explicit safety considerations. Conclusions: Exercise is a promising but unproven multisystem adjunct for ALD. It should complement abstinence support, alcohol use disorder care, nutrition, and standard medical management. ALD-specific trials should integrate metabolomics, liver outcomes, functional measures, sex, alcohol exposure, disease stage, and adverse events.

Metabolites doi: 10.3390/metabo16090665

Authors: Xiangyu Hou Peng Huang Jun Wang Yangxin Chen Benjin Li Feng Chen

Background/Objectives: Stem base rot caused by Fusarium oxysporum is a devastating soil-borne disease, yet long-term chemical pesticide application has raised serious environmental and ecological concerns. Thymol, a plant-derived fungicide, demonstrates a favorable safety profile for humans and environmental systems while exhibiting broad-spectrum antifungal efficacy. This study aimed to evaluate the inhibitory effects of thymol against F. oxysporum and to elucidate its underlying mechanisms of action. Methods: The antifungal activity of thymol was assessed in vitro against F. oxysporum by measuring colony and mycelial growth inhibition, sporulation, and spore germination at varying concentrations. Microscopic examination was performed to observe morphological changes in hyphae. Biochemical assays were conducted to quantify soluble protein content, DNA synthesis, ergosterol levels, and fusaric acid production in treated mycelia. Dose–response data were used to calculate median effective concentrations (EC50) for relevant parameters. Results: Thymol significantly inhibited F. oxysporum growth, with EC50 values of 51.02 μg/mL for colony growth and 57.30 μg/mL for mycelial growth. Microscopy revealed abnormal, shorter, thicker hyphae with enlarged vacuoles. Sporulation and spore germination were also effectively suppressed, with EC50 of 39.37 μg/mL and 53.55 μg/mL, respectively. At 100 μg/mL, thymol reduced soluble protein content by 36.73%, inhibited DNA synthesis by 47.29%, decreased ergosterol content by 48.11%, and reduced fusaric acid production by 76.86%. Conclusions: Thymol exerts multi-targeted inhibitory effects against F. oxysporum through disruption of cellular structure, impairment of macromolecular synthesis, interference with ergosterol biosynthesis, and suppression of toxin production. These findings provide a strong theoretical foundation for developing thymol as a novel biopesticide for sustainable management of stem base rot.

Metabolites doi: 10.3390/metabo16090664

Authors: Ghazal Mohammad Abeer S. Alzaben Yazeed Alshuweishi Sarah F. Faludah Meshal Al-Sharafa Shaima Alothman Arwa S. Altalhi Doaa S. Aljasser Dalal F. ALShamri Saeed Al Shlwi Abdullah Aldriee Latifah Alanzi Fayzah S. Alotaibi Hanaa A. Yamani Ali A. Alshatwi Alaa A. Al-Masud

Background/Objectives: Sleeve gastrectomy induces substantial changes in body composition, energy metabolism, and systemic inflammation, but their temporal patterns during early postoperative follow-up remain incompletely characterized. This pilot study primarily characterized longitudinal changes in body composition, resting energy metabolism, and inflammatory biomarkers during the first nine months following sleeve gastrectomy. A secondary exploratory objective examined associations between the dietary omega-6/omega-3 ratio and these outcomes. Methods: Thirty-seven adults (67.6% female; mean age 35.1 ± 11.3 years) undergoing sleeve gastrectomy were prospectively assessed at baseline and 3, 6, and 9 months postoperatively. Dietary intake was assessed using three-day food records. Body composition, resting metabolic rate (RMR), respiratory quotient, and inflammatory biomarkers (CRP, IL-6, and IL-10) were evaluated longitudinally. Linear mixed models adjusted for age, sex, and comorbidity were used to assess temporal changes and dietary associations. Results: Distinct postoperative trajectories were observed across outcomes. BMI declined progressively from 43.76 ± 6.44 to 28.49 ± 3.77 kg/m2 and total body fat from 52.32 ± 5.76% to 39.62 ± 5.71% (both p < 0.001 for time). In contrast, RMR showed a marked early decline by 3 months, followed by relative stabilization (p < 0.001). CRP progressively decreased from 12.84 ± 12.60 to 2.96 ± 2.95 mg/L (p = 0.001), whereas IL-6 and IL-10 showed no significant temporal changes. Notably, no consistent associations were observed between the dietary PUFA measures and the evaluated outcomes. Conclusions: Postoperative adaptation following sleeve gastrectomy differed across physiological domains, with progressive reductions in adiposity and CRP, an early decline followed by relative stabilization of RMR, and no detectable longitudinal changes in IL-6 or IL-10. Dietary PUFA measures showed no significant associations with postoperative outcomes.

Metabolites doi: 10.3390/metabo16090663

Authors: Chao Li Zhe Su Yiqian Li Huili Liu Mengyi Zheng Hanjing Zhou Cheng Wei Feng Zhou Cuiyu Yang Chen Tang Bin Chen

Background/Objectives: Polyendocrine metabolic ovarian syndrome (PMOS) is characterized by hyperandrogenism, particularly excessive testosterone, as a core clinical feature and a key pathogenic metabolite, yet its molecular mechanisms remain incompletely understood. Methods: This study integrated multi-omics data from Gene Expression Omnibus (GEO) databases with network toxicology, weighted gene co-expression network analysis (WGCNA), and machine learning to identify testosterone-associated core genes in PMOS. Results: Differential expression analysis and WGCNA yielded 42 candidate genes, from which five core genes, including GK5, CYP3A5, EGLN3, VCAM1, and AGTR1, were prioritized as top predictive features through ensemble modeling (RF + XGBoost). Molecular docking predicted favorable testosterone binding conformations. Regulatory network and drug enrichment analysis additionally predicted several upstream transcription factors, hub miRNAs, and potential repurposable drugs. Conclusions: These findings proposed a computational framework for a multi-target molecular landscape linking testosterone to PMOS. The identified genes, regulatory networks, and candidate drugs provided prioritized hypotheses for mechanistic exploration and future evaluation of potential diagnostic and therapeutic applications in hyperandrogenism-related PMOS.

Metabolites doi: 10.3390/metabo16090662

Authors: Mehmet Aslan

Objective: To determine whether systemic inflammation in peripheral artery disease is primarily associated with necrosis or a secondary response, we evaluated the association of tissue oxygenation (red blood cells), acute metabolic stress (admission glucose, albumin), systemic inflammation (C-reactive protein), and the red blood cell-to-albumin ratio. Materials and Methods: We retrospectively analyzed 375 patients undergoing endovascular interventions (58 with necrosis, 317 without). C-reactive protein-to-albumin and red blood cell-to-albumin ratios were calculated. Associations were assessed using parallel multivariate logistic regression models. Results: The necrosis group had significantly higher admission glucose and C-reactive protein-to-albumin ratio, but a lower red blood cell count and red blood cell-to-albumin ratio. In multivariate models of fundamental biomarkers, C-reactive protein lost independent significance; low red blood cells (Odds Ratio: 0.348, p = 0.0003) and elevated admission glucose (Odds Ratio: 1.008, p = 0.0018) were the strongest independently associated variables. In composite index models, the C-reactive protein-to-albumin ratio became a dominant associated factor, attenuating the red blood cell-to-albumin ratio’s statistical significance. Conclusions: Anemia and admission hyperglycemia are strong correlates of tissue necrosis. Systemic inflammation appears to act as a secondary consequence of ischemic injury but profoundly exacerbates tissue loss when combined with nutritional depletion. Comprehensive medical management, including anemia correction and metabolic optimization, is fundamental for limb salvage.

Metabolites doi: 10.3390/metabo16090661

Authors: Hancheng Li Jinwei Gan Yuting Huang Yangkai Wu Chaohua Luo Wenhua Liu Hongwu Wang Zhixian Mo

Background/Objectives: Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease, and ginger contains phenolic constituents with anti-inflammatory potential. This study evaluated the antiarthritic efficacy of ginger ethanolic extract (GE) and explored its potential mechanisms through integrated pharmacological and computational approaches. Methods: GE was prepared by 70% ethanol reflux extraction and administered to adjuvant-induced arthritis (AIA) rats, with methotrexate as a positive control. Body weight; paw swelling; arthritis index; spleen index; and serum interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and prostaglandin E2 (PGE2) were measured. Chemical profiling was performed by ultra-performance liquid chromatography–quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS), followed by network pharmacology, molecular docking, and molecular dynamics (MD) simulation. Results: GE attenuated paw edema, arthritis index elevation, splenic enlargement, and elevated serum inflammatory mediators in AIA rats. UPLC-Q-TOF-MS/MS annotated 38 constituents, mainly gingerols, shogaols, gingerdiones, gingerdiols, and related phenolic derivatives. Network analysis identified 217 overlapping ginger- and RA-associated targets, with TNF, IL6, and matrix metalloproteinase 9 (MMP9) prioritized among inflammatory and matrix-remodeling nodes. Docking and MD simulations supported stable predicted interactions for 8-gingerol-TNF and 6-gingerol-MMP9. Conclusions: GE showed antiarthritic activity in AIA rats. The integrated chemical, in vivo, and computational data suggest that ginger phenolics may modulate inflammatory mediators and candidate RA-related pathways; however, these mechanisms remain exploratory and require histological, tissue-level, and molecular validation.

Metabolites doi: 10.3390/metabo16090660

Authors: Ziyi Qiu Hao Zhang Mengqing Ma Binbin Pan Changchun Cao

Acute kidney injury (AKI) is a common critical syndrome with high morbidity and mortality, and a subset of patients may progress to chronic kidney disease. Recent studies have revealed that gut microbiota and their metabolites play pivotal roles in the pathogenesis of AKI. Under AKI conditions, the gut microbiota composition undergoes significant alterations, characterized by decreased beneficial bacteria and expansion of opportunistic pathogens, accompanied by impaired intestinal barrier and disordered microbial metabolism. Gut microbiota metabolites can be classified into protective metabolites (short-chain fatty acids, secondary bile acids, tryptophan metabolites, D-amino acids, and polyamines) and toxic metabolites (indoxyl sulfate, p-cresyl sulfate, trimethylamine N-oxide, and endotoxin). The former exert renoprotective effects through anti-inflammatory, antioxidant, and barrier-maintaining mechanisms, while the latter aggravate kidney injury via oxidative stress, inflammation activation, and hemodynamic disturbance. Based on the gut–kidney axis theory, interventions targeting gut microbiota (probiotics, prebiotics, fecal microbiota transplantation) and those targeting metabolites (supplementation of protective metabolites, removal of toxic metabolites) have shown promising prospects. This narrative review summarizes the characteristics of gut microbiota changes, classification and function of key metabolites, core mechanisms driving AKI, and microbiota-based intervention strategies, aiming to provide novel insights for early recognition and precision prevention of AKI.

Metabolites doi: 10.3390/metabo16090657

Authors: Qian Li Lutao Xu Mingyu Zhu Gaoge Wang Yu Bai Huan Chen Hongwei Hou

Background: Traditional bulk tissue analyses obscure the precise spatial compartmentalization of nicotine and its molecular effects within individual anatomical regions. This study aimed to develop and apply a high-resolution spatial multi-omics framework to characterize the localized disposition and functional responses induced by an acute nicotine challenge. Methods: We established a spatial multi-omics framework integrating matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-TOF MSI), air-flow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI), laser microdissection (LMD)-based microscale data-independent acquisition (microDIA) proteomics, and targeted LC-MS/MS. This platform was used to analyze the kidney and five brain regions in rats subjected to an acute nicotine challenge following an adaptation regimen. Results: Spatial mapping revealed distinct peripheral and central distribution patterns: nicotine, cotinine, and nornicotine accumulated predominantly in the renal cortex and medulla, whereas their distribution in the brain is region-dependent, with a prominent 3-hydroxycotinine signal in the olfactory bulb. Avoiding tissue homogenization enabled these spatial distributions to be linked to localized functional responses. The striatal dopamine/DOPAC axis showed the strongest acute neurochemical response, consistent with increased dopamine turnover. Spatial metabolomics further demonstrated robust, region-specific metabolic reprogramming, with the hippocampus showing the greatest metabolic variance. LMD-resolved proteomics identified protein-level changes, particularly in the olfactory bulb and thalamus. Cross-omics revealed coordinated alterations in purine, pyrimidine, glycerophospholipid, and alanine/aspartate/glutamate metabolism, with the thalamus showing the greatest extensive metabolite–protein concordance. Conclusions: These findings characterize acute nicotine exposure as a spatially compartmentalized process involving renal handling, region-specific brain distribution, and localized molecular response programs.

Metabolites doi: 10.3390/metabo16090659

Authors: Ambrin Farizah Babu

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common chronic liver disease worldwide, encompassing a spectrum from simple steatosis to metabolic dysfunction-associated steatohepatitis (MASH), fibrosis, cirrhosis and hepatocellular carcinoma. Increasing evidence indicates that disease progression is driven not by hepatic triglyceride accumulation alone but by the metabolic partitioning of excess fatty acids between adaptive and maladaptive pathways. Ketogenesis, traditionally viewed as a fasting-induced mechanism for disposing of excess acetyl-CoA, is now recognized as a key regulator of hepatic metabolic homeostasis, coordinating mitochondrial substrate utilization, carbon flux and systemic metabolic adaptation. In addition to serving as oxidative fuels, ketone bodies, particularly β-hydroxybutyrate, function as signalling metabolites that modulate inflammation, oxidative stress, mitochondrial function and epigenetic regulation. Despite increased fatty acid delivery in obesity and insulin resistance, ketogenic capacity becomes progressively impaired during MASLD, promoting mitochondrial acetyl-CoA accumulation, oxidative stress and diversion of carbon toward lipotoxic lipid synthesis while reducing protective β-hydroxybutyrate signalling. This review examines ketogenesis as an integrative metabolic checkpoint linking fatty acid oxidation, lipid metabolism, mitochondrial function and immune signalling in MASLD. We discuss how impaired ketogenic flux contributes to hepatocellular injury, fibrosis and metabolic inflexibility, and evaluate the therapeutic potential of restoring ketogenesis to prevent disease progression.

Metabolites doi: 10.3390/metabo16090658

Authors: Shenghui Liu Xiaohan Fan Tianyu Song Jinfa Gu Jian Zhu Xing Zhang Hansen Li Hengzhi Deng

Background: Caffeine and sodium bicarbonate (SB) are widely used ergogenic supplements that influence exercise performance through distinct physiological mechanisms and may differentially modulate exercise-related metabolic responses. Whether their co-ingestion provides benefits beyond either supplement alone remains uncertain. This study evaluated the incremental effects of caffeine and SB co-ingestion within trials specifically designed to investigate the combined supplementation strategy. Methods: A systematic review and Bayesian arm-based multilevel network meta-analysis of randomized controlled trials published up to May 2026 was conducted. Eligible studies investigated acute caffeine–SB co-supplementation and, where available, included caffeine-only and SB-only arms, with placebo as the common reference. Exercise performance was the primary outcome; blood lactate, blood pH, blood bicarbonate, and rating of perceived exertion were secondary outcomes. Effects were estimated using Hedges’ g, Bayesian credible intervals, and SUCRA rankings. Results: Thirteen studies involving 181 participants met the eligibility criteria. Within this co-supplementation trial network, caffeine (g = 0.21), SB (g = 0.21), and co-ingestion (g = 0.28) produced small but credible improvements in exercise performance relative to placebo. However, comparisons among active conditions provided no clear evidence that co-ingestion was superior to either supplement alone. SB increased blood pH (g = 0.95) and blood lactate (g = 1.13), whereas caffeine reduced blood bicarbonate (g = −0.89). Co-ingestion elicited the largest increase in blood lactate (g = 1.75), but this response was not accompanied by a proportionally greater improvement in performance. Exploratory analyses suggested possible variation in supplementation responses according to training status. Conclusions: Within trials designed to evaluate caffeine–SB co-supplementation, the caffeine-only, SB-only, and combined conditions showed small improvements in exercise performance relative to placebo. However, co-ingestion elicited more pronounced metabolic responses without a clear performance advantage over either component alone, and the mechanisms underlying this divergence remain uncertain.

Metabolites doi: 10.3390/metabo16090656

Authors: Urszula Bernatowicz-Łojko Elena Sinkiewicz-Darol Maria Wilińska Barbara Baranowska Liliana Pięta Renata Gadzała-Kopciuch

Background/Objectives: Hypothyroidism may influence lactation physiology, but evidence regarding its association with mature human milk composition remains limited. This cross-sectional study evaluated macronutrient composition and apparent thyroid-stimulating hormone (TSH) concentration in 24 h composite mature milk samples from women with a documented history/diagnosis of treated or clinically managed hypothyroidism attending lactation counseling and human milk-bank consultation, compared with controls without known thyroid disorders. Methods: Sixty-six lactating women attending the Human Milk Bank in Toruń, Poland, were enrolled (31 with hypothyroidism, including six with Hashimoto’s disease; 35 controls). Thyroid characterization was based on diagnoses and laboratory results documented during routine preconception or early-pregnancy/perinatal care; the study did not include contemporaneous maternal serum TSH/FT4 testing at milk collection in either group. Composite 24 h milk samples were analyzed using the MIRIS Human Milk Analyzer for fat, crude protein, true protein, carbohydrates, total solids, and energy. Milk TSH was measured using a third-generation ELFA assay. Because this serum/plasma assay was not formally validated for the human milk matrix, values are reported as exploratory apparent milk TSH concentrations. Results: No statistically significant between-group differences were observed in macronutrient composition in the analyses performed. For example, the HG-CG mean difference for energy content was −3.47 kcal/100 mL (95% CI: −8.45 to 1.52; p = 0.169; q = 0.814), and all confidence intervals for milk-composition outcomes included zero. Median apparent milk TSH concentration was 0.0180 [IQR 0.0130–0.0208] µIU/mL in controls and 0.0155 [IQR 0.0093–0.0210] µIU/mL in the hypothyroidism group (Mann–Whitney U = 459.0, p = 0.286). Hyperlactation was frequent in this selected cohort and was more common in controls (27/35; 77.1%) than in the hypothyroidism group (16/31; 51.6%). Exploratory analyses of pre-pregnancy BMI and Hashimoto’s disease were limited by small subgroup sizes. Conclusions: In this small selected cohort, a documented diagnosis/history of treated or clinically managed hypothyroidism was not associated with statistically significant differences in mature milk macronutrient composition or apparent milk TSH concentration. These findings are exploratory and should not be interpreted as evidence of equivalence, confirmed euthyroidism at sampling, or exclusion of smaller effects.

Metabolites doi: 10.3390/metabo16090655

Authors: Ruoke Ma Liuming Wei Pingchuan Zhu Heng Liu Boling Liu Jianhua Qi

Background: Flavonoid secondary metabolites accumulate in xylem during heartwood formation and critically influence wood color and economic value. However, the distribution patterns of individual flavonoids across sapwood, transition zone, and heartwood remain poorly characterized in Castanopsis hystrix, limiting mechanistic understanding of heartwood formation and rational resource utilization. Methods: Sapwood, transition zone, and heartwood samples were collected and analyzed using ultra-performance liquid chromatography coupled with Q-Exactive Orbitrap mass spectrometry (UPLC-QE-MS) for qualitative and quantitative flavonoid determination. Multivariate statistical approaches, including hierarchical clustering, principal component analysis (PCA), and orthogonal partial least-squares discriminant analysis (OPLS-DA), were applied to compare regional metabolic profiles and to identify characteristic differential markers. Results: A total of 26 flavonoids were identified, covering flavones, flavonols, flavanones, dihydroflavonols, and flavanols. Quantitative and cluster analyses revealed distinct region-preferential accumulation: dihydrorobinetin, dihydromyricetin, morin, and kaempferol were highly enriched in heartwood; fustin, taxifolin, and cyanidin predominated in the transition zone, whereas afzelin and astilbin were more abundant in sapwood. PCA and OPLS-DA further selected four characteristic markers, namely dihydromyricetin, dihydrorobinetin, kaempferol and (−)-epicatechin, that effectively discriminated the three regions. Conclusions: This study establishes a clear radial gradient of flavonoid accumulation in C. hystrix xylem, demonstrating selective enrichment of specific metabolites from sapwood to heartwood. These findings provide essential data for deciphering the biochemical mechanisms of heartwood formation and offer a metabolic basis for quality assessment and high-value utilization of this timber species.

Metabolites doi: 10.3390/metabo16090654

Authors: Xintong Hu Qinxue Wang Yingying Lin Jingjing Wan

Small biomolecules provide important information on cellular metabolism, signaling and disease-associated molecular changes. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) enables rapid, high-throughput and spatially resolved molecular analysis, but its application to small biomolecules is often limited by poor ionization, low-mass background interference, ion suppression and insufficient structural information. To address these analytical challenges, this review summarizes chemical derivatization strategies that improve MALDI-MS analysis of small biomolecules. The representative strategies are discussed according to reaction mode and target functional group, including solution-phase, on-target, on-tissue, reactive-matrix-assisted and photo-/in-source approaches, with emphasis on amine-, carbonyl-, carboxyl- and double-bond-containing biomolecules. Chemical derivatization improves MALDI-MS performance by selectively modifying target functional groups, introducing charged or ionizable tags, enhancing molecular discrimination and providing additional structural information. These strategies have expanded the detection and annotation of neuroactive amines, carbonyl compounds, glycans, carboxylic acid metabolites and lipid double-bond isomers, while also extending MALDI-MS analysis to other specific functional groups, natural products and drug-related molecules. Overall, chemical derivatization is an important approach for improving the sensitivity, selectivity, structural annotation and analytical coverage of MALDI-MS-based small biomolecule analysis. Future developments should further address reaction selectivity, spatial fidelity, quantitative reliability and confident identification of derivatization products.

Metabolites doi: 10.3390/metabo16090653

Authors: Shanju Wen Shijuan Xiong Weimo Wu Jinhu Zhi Weiyang Liu Chunming Chi Lu Kang Xiaohong Tian

Background/Objectives: Reductive soil disinfestation (RSD) is increasingly viewed as a viable alternative to chemical fumigation, yet the role of organic amendment quality in regulating greenhouse gas (GHG) fluxes, soil organic carbon (SOC) dynamics, and net climate forcing remains poorly understood. Methodology: In a 30-day anaerobic incubation experiment, we set up three treatments—a flooded control (CK), soil amended with wheat straw (WS, C/N = 55.5), and soil amended with kiwifruit branches (KB, C/N = 110.8)—each replicated three times under identical conditions. Results: Both WS and KB additions strongly stimulated CO2 and CH4 production, while suppressing N2O emissions by over 85% relative to CK. The WS treatment exhibited a substantially higher global warming potential (GWP, 823.52 t ha−1) than KB (636.23 t ha−1), with CH4 accounting for more than 99% of total GWP. Although WS surpassed KB in short-term carbon sequestration efficiency (25.79% vs. 20.11%) and showed greater hydrolytic enzyme activities (βG, CBH, and XYL), the two organic amendments diverged clearly in carbon fraction distribution: Cmic was 17.9% higher under WS, whereas Cmin was 16.2% higher under KB. When factoring in the CO2 equivalent benefit derived from carbon sequestration, the net GWP (NGWP) indicated that both RSD treatments remained net GHG sources. Notably, KB yielded a markedly lower NGWP (611.60 t CO2-eq ha−1) than WS (798.19 t CO2-eq ha−1), highlighting a fundamental trade-off: WS favored rapid SOC accumulation at the expense of elevated methane emissions, whereas KB achieved a smaller climatic footprint despite more moderate carbon retention. Conclusions: These findings underscore that selecting organic amendments for field RSD requires balancing the competing goals of carbon sequestration and GHG mitigation.

Metabolites doi: 10.3390/metabo16090652

Authors: Yirui Chen Hongxin Gui Kai Ma Ruochen Cao Zhijian Zhang Mengyang Wang Rongrong Yang

Background/Objectives: Aflatoxin B1 (AFB1) contamination of maize and peanut is influenced by fungal responses to environmental and food-matrix cues, but the transcriptional coordination of redox adaptation, lipid metabolism, and aflatoxin biosynthesis remains unresolved. This study examined whether an expression-defined redox–lipid state is associated with high relative aflatoxin biosynthetic activity in Aspergillus flavus. Methods: A cross-dataset secondary analysis integrated 18 analytical datasets (247 samples) derived from eight public source records spanning defined-medium, maize, and peanut systems. Harmonized expression profiles were used to derive an aflatoxin biosynthetic activity score (ABAS), oxidative-stress adaptation score (OSAS), and lipid metabolic reprogramming score (LMRS). ABAS is an expression-derived relative score, not a direct measure of biosynthetic flux or accumulated toxin. Dataset-aware mixed-effects models evaluated the OSAS–ABAS association and the linear OSAS × LMRS interaction. Results: ABAS correlated with matched AFB1 measurements in 54 samples (Pearson r=0.734, p=2.70×10−10). The quadratic OSAS term was negative (β=−0.434, 95% CI −0.502 to −0.366; p<0.001), with maximum predicted ABAS near 0.81 SD on the pooled within-dataset-standardized OSAS scale. Separately, the linear OSAS × LMRS interaction was positive (β=0.284, 95% CI 0.198–0.370; p<0.001), and the high-OSAS/high-LMRS quadrant had the greatest adjusted mean ABAS (0.789, 95% CI 0.661–0.917). Candidate prioritization recovered established regulators and nominated redox- and lipid-associated nodes. Conclusions: The findings identify an associative transcriptional signature across heterogeneous food-relevant conditions and provide focused hypotheses for prospective validation using direct toxin, redox, lipid, flux, and functional measurements.

Metabolites doi: 10.3390/metabo16090651

Authors: Ugur Fahri Yurekli Ozlem Orer Beginoglu

Background: This cross-sectional study compared immunoassay urine drug screening with LC–MS/MS confirmation in individuals with substance use disorder undergoing probation monitoring, focusing on samples with results close to assay cutoff values. Methods: The study was conducted between January and March 2026 in Şanlıurfa, Türkiye, and included 110 individuals followed at an Addiction Treatment Center (AMATEM). Urine samples with at least one analyte result within ±20% of the cutoff value were analyzed. Immunoassay screening was performed using a Beckman Coulter AU480 analyzer, whereas confirmation was performed by LC–MS/MS using a SCIEX Triple Quad 5500+ system. Agreement was evaluated using Cohen’s kappa and McNemar’s test, while diagnostic performance was assessed using 2 × 2 contingency tables. Results: Agreement varied substantially among analyte groups. Buprenorphine showed the highest agreement (κ = 0.47), followed by amphetamines (κ = 0.33), whereas opioids showed very low agreement (κ = 0.081). Benzodiazepines, cocaine, and cannabinoids showed no meaningful agreement; however, benzodiazepine and cocaine findings were limited by very low numbers of positive cases. Immunoassay screening produced notable false-positive results for amphetamines and opioids and false-negative results for cannabinoids and opioids. Conclusions: Immunoassay performance was analyte-dependent and appeared limited in samples with results close to cutoff values. LC–MS/MS confirmation remains important for reliable toxicological interpretation in probation monitoring settings.

Metabolites doi: 10.3390/metabo16090650

Authors: Chengdi Shi Wei Gao Zhenglu Yang Lijuan Han Shengzhen Hou Chao Yang Zenghai Luo Linsheng Gui

Background: Intramuscular fat (IMF) is a key determinant of meat sensory quality, but the effects of rumen-protected betaine (RPB) on IMF deposition in Tibetan sheep remain unclear. Methods: Sixty male, 3-month-old Tibetan lambs (Initial weight 17.72 ± 1.36 kg) were randomly assigned to a control (basal diet, n = 30) or RPB supplementation (basal diet supplemented with 0.08% RPB, n = 30) for 90 d. Results: Transcriptomic and lipidomic analyses revealed that RPB modulated glycerophospholipid metabolism-related genes (DGKI and AGPAT1) and altered lipid profiles, including PC (16:0/20:5) and PC (16:0/16:0). RPB increased T-AOC and SOD activities and decreased MDA content (p < 0.05). Intramuscular adipocyte area and diameter increased, while density decreased (p < 0.05). LC-MS/MS analysis showed that unsaturated fatty acids C14:1, C17:1, and C22:4 were significantly increased (p < 0.05). Muscle fiber density in the longissimus dorsi increased, whereas shear force, cooking loss, hardness, and chewiness were reduced (p < 0.05). MYH1, MYH2, and MYH7 expression were upregulated by 2.05-, 1.89-, and 2.73-fold, respectively. Conclusions: In summary, dietary RPB supplementation was associated with increased IMF deposition, which may contribute to improved meat quality, potentially through modulation of glycerophospholipid metabolism.

Metabolites doi: 10.3390/metabo16090649

Authors: Qi Zhang Fanyu Meng Haoxuan Sun Yajie Yin Guoling Ren

Objective: Saline–alkali stress (composed of neutral and alkaline salts) severely constrains soybean productivity. Although nicotinamide can enhance plant salt tolerance, its most effective concentration and molecular basis in soybean remain unclear. This study aimed to systematically investigate the regulatory effect of exogenous nicotinamide on soybean saline–alkaline tolerance and to identify its candidate genes and putative mechanisms under salt-stressed conditions. Methods: Soybean cultivar ‘Hefeng 25’ seedlings were exposed to 200 mmol/L mixed salt stress and treated with nicotinamide at 0 (CK), 10, 50, and 200 mg/L. Growth parameters, ROS levels, ion contents, photosynthetic pigments, osmoregulatory substances, and antioxidant enzyme activities were measured. Transcriptome sequencing and RT-qPCR were performed to identify differentially expressed genes (DEGs), followed by enrichment analysis. Results: Exogenous nicotinamide significantly improved soybean saline–alkaline tolerance relative to the salt-stressed CK group, with 50 mg/L (SA2) showing the most pronounced physiological improvements among all tested concentrations. SA2 treatment enhanced plant height, biomass, K+ content, and antioxidant enzyme activities while reducing Na+ accumulation and ROS levels compared with CK. Transcriptome analysis identified PM1 as the shared gene across the five non-most effective comparisons. Enrichment analysis implicated DEGs primarily in ko03110 (chaperones and folding catalysts), ko00199 (cytochrome P450), and ko04141 (protein processing in the endoplasmic reticulum). RT-qPCR confirmed that PM1, P450 genes (CYP74A1, CYP83D1), HSPs (HSP23, HSP18), and ROS scavengers (NAC1, GSTU43) were upregulated under non-most effective doses (SA1/SA3) but downregulated to levels comparable to the stressed CK in SA2; conversely, SA2 specifically restored the expression levels of stress-repressed WRKY57 and LBD15. Transcriptomically, SA2 exhibited a profile most similar to the saline–alkali-stressed CK group, with the fewest number of differentially expressed genes among all treatments, whereas non-optimal nicotinamide concentrations were associated with extensive transcriptional changes in ER protein processing, detoxification-related, and oxidative stress-related pathways. The limited transcriptional changes in SA2 indicate greater transcriptional similarity to the stressed control, which paralleled its superior physiological performance. Conclusions: Nicotinamide effectively alleviates soybean saline–alkaline stress at 50 mg/L, with the most significant physiological benefits and the fewest transcriptional changes relative to the stressed control. The non-most effective nicotinamide concentrations show extensive stress-related transcriptional reprogramming in protein folding, detoxification, and stress-response pathways relative to the salt-stressed control, putatively implicating ER stress, detoxification, and oxidative stress-related responses at the transcript level, with PM1 acting as a potential candidate transcriptional marker of dose-dependent transcriptional regulation under saline–alkali stress.

Metabolites doi: 10.3390/metabo16090648

Authors: Ekaterina Balaian Iryna Kovtun Fabian Springer Denise Medeiros Selegato Sophie Jonas Uta Oelschlaegel Manja Wobus Michael Wulfert Corinna Strupp Ulrich Germing Michael Zimmermann Martin Bornhäuser Triantafyllos Chavakis Katja Sockel Alexander Funk

Background: Myelodysplastic neoplasms (MDSs) are clonal hematopoietic stem cell disorders associated with ineffective hematopoiesis, chronic inflammation, and increased cardiovascular morbidity. Although metabolic dysregulation has been implicated in MDS pathogenesis, systemic metabolic alterations remain incompletely characterized. Methods: Plasma samples from treatment-naïve patients with MDS or chronic myelomonocytic leukemia (CMML) and age-matched healthy controls were analyzed using quantitative nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry (LC-MS). Metabolomic profiles were compared using unsupervised and supervised multivariate analyses, validated in an independent external MDS cohort, and integrated with re-analysis of publicly available RNA-sequencing datasets from purified CD14+ CMML monocytes. Results: Patients with MDS and CMML exhibited broad reductions in circulating lipoprotein-associated metabolites, including HDL-, LDL-, IDL-, and apolipoprotein-associated fractions, indicating disturbed systemic lipoprotein homeostasis. Within the discovery cohort, CMML samples showed higher concentrations of the ketone bodies 3-hydroxybutyrate and acetoacetate, as well as succinate. LC-MS analysis demonstrated selective increases in C18:1 acylcarnitine, oleic and isopalmitic acids, whereas free carnitine abundance remained unchanged. Elevated 3-hydroxybutyrate levels were not associated with mutational burden, hematologic parameters, disease risk, or immunophenotypic features. Re-analysis of public CMML monocyte transcriptomes demonstrated increased expression of genes involved in lipid uptake and intracellular lipid trafficking, including FABP5, APOE, LPL, and SLC27A2, without coordinated activation of fatty acid oxidation pathways. External cohort analysis confirmed the overall MDS-associated plasma metabolomic profile. Conclusions: MDSs and CMML are associated with reproducible alterations in systemic lipid metabolism characterized by reduced circulating lipoprotein-associated metabolites, while CMML showed more pronounced ketone body- and acylcarnitine-associated metabolic phenotype accompanied by changes in lipid-handling transcriptional programs. These findings support altered systemic lipid metabolism and carnitine-dependent fatty acid handling as characteristic features of myeloid neoplasms and provide a rationale for future functional studies investigating lipid metabolism in disease pathogenesis.

Metabolites doi: 10.3390/metabo16090647

Authors: Alejandro Silva-Palacios Gabriela Navarrete-Anastasio Elizabeth Lira-Silva Leonardo del Valle-Mondragón Willy Ramsés García-Niño Angélica Ruiz-Ramírez Francisco Correa Francisco Javier Roldán-Gómez Jesús Vargas-Barrón Alberto Aranda Salvador Uribe-Carvajal Natalia Pavón

Background: For a long time, it was thought that estrogen degradation metabolites (EDMs) had no role in the female body, but this view has changed. Despite the progress, there are still unknowns about EDMs and the heart. We found that EDMs affect isolated cardiac mitochondria, which may impact heart function. Therefore, our objective was to explore the effect of two EDMs, Met A (2-hydroxyestrone) and Met D (17β-estradiol-3-methyl-ether), on two experimental models: rat H9c2 cardiomyoblast cells exposed for increasing times to different concentrations of each EDM, and, also, the effects of each metabolite on heart function in intact and oophorectomized (Oopho) rats. Methods: The cell viability, mitochondrial potential, and oxidative stress were assayed in H9c2 cells. Echocardiographic analyses, histological analyses, and an assessment of cardiac damage and oxidative stress in intact and Oopho rats were tested. Results: The strongest negative effects were seen with Met D, where antioxidant capacity decreased and oxidative stress increased significantly. Upon further analysis of the effects of each metabolite on heart function, echocardiographic analysis revealed that cardiac function had not changed in either group, although clear signs of cardiac hypertrophy and fibrosis were observed. Conclusions: Our results suggest EDMs have specific effects on female rats. This study emphasizes the need to thoroughly investigate EDMs’ side effects and impact on the heart.

Metabolites doi: 10.3390/metabo16090646

Authors: Dariga Dairbekova Nina V. Terletskaya Aigerim Mamirova Nazym K. Korbozova Elvira A. Shadenova Aizhan S. Mussayeva

Backgrounds: The present study investigated temperature-associated responses of microclones of two halophytic shrubs, Nitraria sibirica Pall. and Nitraria schoberi L., exposed to low positive (+3 °C) and elevated (+30 °C) temperatures for 72 h. Methods: Temperature-associated responses were investigated by evaluation of leaf anatomy, chlorophyll fluorescence, and GC–MS-based metabolite profiling. Results: The results revealed shared and species-specific responses, including changes in leaf anatomical dimensions, maintenance or adjustment of PSII-related fluorescence parameters, and treatment-dependent shifts in the relative composition and diversity of putatively annotated metabolite classes. Metabolomic profiling was performed in both in vitro microclones and soil-acclimatised microclones. In vitro microclones showed more pronounced shifts in the number and relative representation of detected compound classes under temperature exposure, whereas soil-acclimatised microclones demonstrated comparatively greater compositional stability. Comparative analysis of the datasets revealed temperature-associated patterns across anatomical, photosynthetic, and relative metabolomic responses; however, these patterns were interpreted descriptively rather than as direct causal mechanisms. Some detected compounds or compound classes have been associated with biological relevance in previous studies; however, their identity, abundance, and activity require targeted validation. Conclusions: Overall, the findings provide a descriptive experimental dataset on temperature-associated anatomical, chlorophyll fluorescence, and relative GC–MS profile changes in Nitraria microclones and identify candidate metabolite classes for future targeted validation.

Metabolites doi: 10.3390/metabo16090645

Authors: Yujin Natori Dai Mizuno Masaru Doshi Masahiro Kawahara Akira Ishii

Background: Cannabis contains Δ9-tetrahydrocannabinol (THC) together with numerous other constituents, whereas HU210 (HU) is a synthetic full agonist at cannabinoid receptors. We applied integrated metabolomics and proteomics to compare Cannabis extract (CS) and HU in GT1-7 neuronal cells. Methods: Cytotoxicity was evaluated using a lactate dehydrogenase assay. Metabolites and proteins were extracted sequentially from single-cell pellets and analyzed using gas chromatography–tandem mass spectrometry and nano-liquid chromatography–quadrupole–Orbitrap mass spectrometry, respectively. Cannabinoids in the exposure medium were quantified using liquid chromatography–tandem mass spectrometry. Results: The lowest CS and HU concentrations that were cytotoxic at 24 h and non-cytotoxic at 3 h were 2000 µg/mL and 20 µM, respectively; CS delivered 166 nM THC, 800 nM tetrahydrocannabinolic acid, and 11.3 nM cannabidiol. After 3 h, HU altered both the metabolome and the proteome, whereas CS altered the metabolome with minimal proteome change (8 of 2720 proteins). Six pathways differed, including the citrate cycle and pentose phosphate pathway. Relative to both the control and CS, HU decreased citrate; increased 2-ketoglutarate, fumarate, malate, gluconate, ribose-5-phosphate and ribose; and increased phosphoenolpyruvate carboxykinase 2, isocitrate dehydrogenase 3 subunit α, 2-oxoglutarate dehydrogenase complex component E1, succinate dehydrogenase flavoprotein subunit, malate dehydrogenase 1, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase and phosphoglucomutase-1. CS showed the opposite metabolite changes without corresponding enzyme changes. Conclusions: At a pre-lethal time point, CS and HU produced metabolic changes in opposite directions—broad suppression versus coordinated mobilisation—despite comparable cytotoxicity at 24 h.

Metabolites doi: 10.3390/metabo16090644

Authors: Jiayu Liu Yunshu Zhang Xingchi Jiang Haocheng Xue Peiyuan Yin

Background: Hyperlipidemic acute pancreatitis (HAP) is a severe disease driven by systemic lipid overload. While free fatty acids (FFAs) are known to mediate pancreatic lipotoxicity, the intracellular enzymatic mechanisms generating these toxic lipid mediators remain unclear. We aimed to identify the core metabolic drivers linking systemic hyperlipidemia to local pancreatic injury and evaluate targeted prophylactic strategies for HAP. Methods: We integrated public transcriptomic datasets of severe AP and obesity/hyperlipidemia. Three machine learning algorithms were employed to identify comorbidity-associated signature genes. The underlying mechanisms were explored via gene set variation analysis, immune infiltration profiling, and single-cell in silico knockout. In vivo validation was performed using a P-407/caerulein-induced HAP mouse model treated with WWL113, followed by comprehensive histological, biochemical, and lipidomic analyses. Results: A robust three-gene signature (FASN, CES1, IL10) was identified with excellent diagnostic accuracy. Notably, within this signature, the triglyceride hydrolase CES1 was aberrantly upregulated, serving as the primary driver of a maladaptive lipolytic shift. CES1 overexpression was strongly correlated with neutrophil infiltration. Single-cell virtual knockout suggested a potential association between Ces1d and markers of endothelial barrier disruption and neutrophil chemotaxis. In vivo, WWL113 significantly attenuated HAP-induced pancreatic necrosis and systemic inflammation. Crucially, lipidomics confirmed that WWL113 sequestered exogenous lipids in inert triglyceride states, drastically reducing toxic FFAs. Conclusions: This study highlights CES1 as a critical intracellular mediator of lipotoxicity in HAP. Pharmacological inhibition of CES1 effectively halts maladaptive lipolysis, providing proof-of-mechanism for a metabolism-directed prophylactic strategy for HAP.

Metabolites doi: 10.3390/metabo16090643

Authors: Xiaoxu Ren Wu Ying Lihua Chen Bingbin Zheng Shouzeng Xu Le Zhang Xianbo Wang Luxia Chen Bowei He Songlin Ruan

Background/Objectives: To elucidate how flower retention regulates leaf quality formation in Dendrobium officinale and to provide a theoretical basis for high-quality leaf cultivation and high-value utilization of byproducts, D. officinale leaves were subjected to two treatments: flower retention (FR) and flower removal (FT). Methods: Quantitative analysis revealed significant differences in quality-related compounds between the two treatments. Specifically, the polysaccharide content in the FR group (8.72%) was significantly lower than that in the FT group (9.37%). Conversely, the FT group exhibited a significantly higher total flavonoid content (1.74%) compared to the FR group (1.41%). Additionally, the total phenol content in the FR group (0.90%) was significantly higher than that in the FT group (0.69%) (n = 3). Results: Compared with the FR group, the FT group exhibited significantly higher levels of total flavonoids, polysaccharides, tyrosine (Tyr), hydroxylysine (Hylys), lysine (Lys), histidine (His), and arginine (Arg). A total of 1999 (39.5%) of the 5062 annotated metabolites were accumulated significantly differently. The FT group was mainly characterized by upregulated secondary metabolites, including flavonoids. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that nucleotide metabolism, purine metabolism, aminoacyl-tRNA biosynthesis, and flavonoid biosynthesis were the core differential pathways. Furthermore, the differential metabolites exhibited a metabolic trade-off of “upregulated secondary metabolism and downregulated primary metabolism”. Conclusions: Our findings indicated that flower removal can significantly increase the active flavonoid components and amino acids in D. officinale leaves and optimize the quality of this medicinal leaf by regulating the source–sink relationship, carbon–nitrogen partitioning, and key metabolic pathways.

Metabolites doi: 10.3390/metabo16090642

Authors: Zhuangxiu Kang Ran Meng Meng Nie Tianqi Wang

Gut microbial metabolism links intestinal ecology with systemic physiology and neural pathology, but its effects vary across disease stage, tissue compartment, and host background. This review uses Alzheimer’s disease (AD) as the principal model and compares selected features with Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS). Across the AD continuum, fermentation-related changes appear in prodromal cohorts, whereas broader alterations in amino acid products, host–microbial co-metabolites, bile acids, and lipids accompany mild cognitive impairment and dementia. These group-level patterns do not constitute a fixed patient trajectory. Microbial production, intestinal absorption, hepatic conversion, renal clearance, barrier integrity, and tissue-specific receptors jointly determine biological exposure. Experimental studies connect short-chain fatty acids and indole derivatives with epithelial and neuroimmune homeostasis, while imidazole propionate, trimethylamine N-oxide, selected kynurenine products, and remodeled bile acid pools engage vascular, inflammatory, amyloid, or tau-related pathways. Cerebral pathology can also remodel the intestinal ecosystem, creating reciprocal feedback. Apolipoprotein E4 modifies lipid handling, vascular permeability, and immune responses, helping to explain why comparable metabolic profiles may carry different consequences among individuals. Translation therefore requires more than a change in community composition. Trials must verify microbial function, metabolite target engagement, AD biomarker response, and clinical benefit in appropriately stratified participants. Shared pathways in PD and ALS provide comparison points, but disease-specific cells, proteinopathies, and treatment exposures constrain direct transfer of AD-derived targets.

Metabolites doi: 10.3390/metabo16090641

Authors: Janaina Silveira da Silva Rafaela Scalise Xavier de Freitas Jaqueline Fernandes Bruno Guilherme Pegoraro Rissi Eduardo Solano Pina dos Santos Vinicius Laerte Silva Herreira Nara Regina Brandão Cônsolo Gabriel Henrique Ribeiro Luiz Alberto Colnago Ives Cláudio da Silva Bueno

Background: Phytogenic feed additives are being explored as alternatives to conventional rumen modulators, yet their effects on the ruminal ecosystem remain insufficiently characterized when assessed only through conventional fermentation parameters. This study evaluated the effects of ethanolic and hydroethanolic extracts of Urochloa brizantha on ruminal fermentation, methanogenic archaeal diversity, and the ruminal metabolome of sheep. Methods: Eight rumen-cannulated wethers were assigned to a replicated 4 × 4 Latin square design with four 28-day periods. Treatments consisted of no additive (CTL), ethanolic extract of U. brizantha (EE; 50 mL/day), hydroethanolic extract (HE; 80 mL/day), and monensin (MON; 25 mg/kg of DMI). Ruminal fluid was evaluated for fermentation traits, archaeal 16S rRNA gene diversity, and untargeted metabolomic profiles. Results: Dry matter intake and fermentation parameters were unaffected by treatment. Archaeal alpha diversity showed a contrast-dependent response, with lower Shannon diversity in sheep receiving plant extracts than in those receiving monensin (p = 0.034), and Methanimicrococcus was selectively enriched in the EE group (p = 0.012). Untargeted metabolomics revealed the clearest response: despite substantial overlap in global PCA profiles, PLS-DA indicated treatment-specific temporal separation between day 0 and day 21 only in EE-treated sheep, supported by permutation testing based on classification error rate (p = 0.027), although predictive performance should be interpreted cautiously. This EE-specific response was characterized by a significant shift in the carboxylic acid subclass (FDR = 0.022). Top VIP-ranked discriminants (aspartate, succinate, glutamate, phenylalanine, leucine, and choline) decreased nominally but failed multiple-testing correction (FDR = 0.0602–0.2580). CTL, HE, or MON showed no temporal separation. Conclusions: Urochloa brizantha extracts produced limited effects on conventional ruminal fermentation parameters but were associated with selective archaeal responses. In particular, the ethanolic extract was associated with a treatment-specific temporal metabolomic pattern, including a significant shift in the carboxylic acid subclass, while individual metabolite changes remained exploratory and warrant further investigation.

Metabolites doi: 10.3390/metabo16090640

Authors: Shengxu Li Dave Watson Alissa Jorgenson Zainab Adelekan Kathleen Garland Benjamin Deonovic Leah Burns Weihong Tang David M. Overman Marnie T. Huntley

Background: Coagulation markers are used to guide clinical anticoagulation decisions. We aimed to identify circulating metabolites that are associated with coagulation markers in children with congenital heart disease (CHD). Methods: Plasma samples were separated from whole blood under consistent conditions. Untargeted metabolomic data were measured in plasma from up to 203 young patients (age range: 0 days–24 years) with CHD before cardiac surgery. Coagulation markers included activated partial thromboplastin time (aPTT), prothrombin time (PT), and activated clotting time (ACT). Weighted Gene Co-expression Network Analysis (WGCNA) was performed to explore metabolite modules (clusters). Associations of metabolites with the coagulation markers were assessed cross-sectionally with regression models, with false discovery rate (FDR) correction for multiple comparison. Associations between coagulation markers and “eigenmetabolites” from WGCNA modules were assessed by correlation analysis. Results: A total of 776 metabolites were included in the final analysis. Among these, 20 metabolites were associated with PT and one (valine) with ACT (FDR q value < 0.05). Among the metabolites associated with PT, the top three were retinol, 1-palmitoyl-GPI (16:0), and X-25371 (identity unknown). One module from WGCNA with metabolites from the lipid super pathway was correlated with PT (p = 0.004). Conclusions: In this first attempt to identify novel metabolites for coagulation markers, we report 21 metabolites associated with PT or ACT in children with CHD. Future studies are needed to replicate these findings in independent cohorts and to elucidate the biological mechanisms linking these metabolites to hemostatic regulation.

Metabolites doi: 10.3390/metabo16090639

Authors: Ayça Tuzcu Cem Kaba Arzu Ateş Kenan Yörük Mustafa Yılmaz Göksel Tuzcu

Background/Objectives: The extent to which inhibin B, a marker of Sertoli cell function, is affected by cardiometabolic disturbances remains insufficiently understood. The aim of this study was to evaluate the relationships between serum inhibin B levels and the atherogenic index of plasma (AIP), triglyceride–glucose (TyG) index, HOMA-IR, METS-IR, and C-reactive protein (CRP) in adult men aged 40–65 years and to secondarily investigate a possible association with coronary artery calcium score (CACS). Methods: This cross-sectional study included 67 men aged 40–65 years referred for coronary computed tomography angiography, sampled in a balanced manner across coronary artery calcium score (CACS) categories. Serum inhibin B levels were measured by a commercial sandwich ELISA; AIP, TyG index, HOMA-IR, and METS-IR were calculated from fasting biochemical measurements. Associations between inhibin B and cardiometabolic parameters were assessed using Spearman correlation analyses and age-adjusted partial correlations. Significant associations were further examined using multivariable linear regression. Results: The mean serum inhibin B level was 224 ± 35 pg/mL. The TyG index (ρ = −0.308, p = 0.011) and BMI (ρ = −0.348, p = 0.004) showed significant negative correlations with inhibin B, and both associations persisted after age adjustment. No significant associations were found with HOMA-IR, METS-IR, AIP, CRP, or CACS. Although BMI and the TyG index were not significantly correlated with each other (ρ = −0.084, p = 0.564), both variables remained independently associated with inhibin B in the multivariable model (TyG: β = −0.283, p = 0.015; BMI: β = −0.313, p = 0.007). The model explained 18.0% of the variance in inhibin B (R2 = 0.180). Conclusions: In adult men, serum inhibin B levels were independently and inversely associated with BMI and the TyG index, whereas no significant relationship was observed with other metabolic, inflammatory, atherogenic, or coronary calcification markers. These findings suggest that the relationship between inhibin B and cardiometabolic health is not generalizable across all cardiometabolic indices and may be specifically related to the metabolic profile represented by adiposity and the TyG index.

Metabolites doi: 10.3390/metabo16090637

Authors: Xin Chen Yuetong Zhu Qingping Shi Jianing He Siyu Chen Jieru Han

Objectives: Despite the central role of metabolic–immune crosstalk in pulmonary arterial hypertension (PAH), integrative quantitative tools remain lacking. We aimed to construct and validate a metabolic–immune molecular classification score (MIRS) as a quantitative metric to capture the inflammation–metabolism balance and to assess its discriminatory performance for molecular subtyping, cross-cohort applicability, and biological implications. Methods: We integrated five PAH lung tissue transcriptomic datasets from the Gene Expression Omnibus (GEO). A training set was used to identify differentially expressed genes and to derive MIRS as the first principal component of 20 core genes. MIRS was projected onto independent validation cohorts. Immune microenvironment, pathway activities, protein–protein interaction, and drug repositioning analyses were performed. Results: MIRS significantly distinguished Non-IPAH from IPAH in GSE117261 (AUC = 0.718, p = 0.010) and revealed internal heterogeneity in SSc-PAH. MIRS-related gene expression patterns showed significant differences across COPD and ILD lung tissues in independent datasets. A unified fixed PCA projection pipeline with mean imputation for missing core genes was applied to all pulmonary disease datasets, enabling consistent numerical quantification of MIRS across cohorts under the same mathematical framework. MIRS failed to discriminate PAH from controls in PBMCs, indicating that this tissue-derived signature is not readily detectable in peripheral blood—a limitation that restricts its applicability to lung tissue specimens and highlights challenges for blood-based biomarker development in PAH. Higher MIRS correlated with increased immune scores and myeloid cell infiltration in Non-IPAH. Drug prediction identified sirolimus and tocilizumab as top candidates, with MIRS-stratified sensitivity patterns aligning with pathway loading directions. Conclusions: MIRS is a quantitative, tissue-restricted metric that captures metabolic–immune activation in PAH, with exploratory observations in other pulmonary diseases that warrant further validation. It provides a molecular stratification basis for subtype discrimination and a hypothesis-generating framework for future therapeutic investigations.

Metabolites doi: 10.3390/metabo16090638

Authors: Tao Pang Xinyi Zhou Zhe Ge

The gut microbiota can influence tumor immunity and treatment responses through microbial metabolism, intestinal barrier regulation, and immune–inflammatory signaling, but whether exercise engages these mechanisms through the muscle–gut axis remains uncertain. This review integrates evidence from exercise physiology, microbial metabolism, and tumor immunology to examine how exercise-associated host signals may reshape the intestinal ecological niche and microbial function. Three candidate muscle–gut axes are proposed: the myokine–enteroendocrine–substrate delivery–short-chain fatty acid (SCFA) axis, the exercise-associated lactate–microbial cross-feeding–propionate axis, and the muscle-derived endocrine signaling–intestinal epithelial repair–hypoxic niche axis. Separate studies support exercise-associated IL-6/GLP-1/PYY regulation and gastrointestinal transit, lactate entry into the intestinal lumen and lactate-associated microbial remodeling, Veillonella atypica-mediated propionate production, and irisin/apelin-related epithelial repair. Together, these pathways may influence microbial metabolism, barrier homeostasis, and immune–tumor interactions. However, tumor-related links remain incomplete, and none has been validated as a complete causal chain in a single tumor-bearing exercise model. Relatively complete preclinical evidence comes from mouse melanoma, where endurance exercise enhanced microbial folate-dependent one-carbon metabolism and formate output, with microbiota-derived formate promoting CD8+ T-cell antitumor activity and immune checkpoint inhibitor efficacy. Regular exercise with an appropriate load and adequate recovery may support microbial and intestinal barrier homeostasis, whereas excessive or prolonged exercise with inadequate recovery may impair barrier integrity. Human evidence remains limited and largely associative and does not establish microbiota-mediated improvements in tumor immunity or treatment responses. These candidate axes therefore require causal validation in tumor-bearing exercise models and prospective human studies.

Metabolites doi: 10.3390/metabo16090636

Authors: Violeta Kalemi Luca Chiodaroli Simona Rimoldi Genciana Terova

Background/Objectives: This study presents a gas chromatography–mass spectrometry (GC–MS) method for the derivatization-free quantification of the three major short-chain fatty acids (SCFAs)—acetate, propionate, and butyrate—in gilthead sea bream (Sparus aurata) fecal samples. SCFAs are key metabolites involved in intestinal physiology and are widely recognized as indicators of gut microbiota activity and host health across animal species. In aquaculture research, SCFA profiling can provide valuable insight into diet–microbiota interactions and gut functional status. Methods: The method was developed and optimized using gut-content samples collected from 105 gilthead sea bream at the end of two independent feeding trials, yielding 42 pooled samples (18 from FT1 and 24 from FT2). An extraction protocol based on acidified aqueous extraction with water followed by liquid–liquid extraction using methyl tert-butyl ether was developed (ExA) and subsequently optimized (ExB) to improve extraction consistency and efficiency. SCFA separation and quantification were performed by GC–MS using external calibration. Method validation included evaluation of linearity, matrix effects, and partition coefficients between water and methyl tert-butyl ether. Results: The optimized extraction method (ExB) demonstrated high analytical performance, with linearity coefficients exceeding 0.992 and matrix effects below 17.4%. Conclusions: The proposed method represents a practical and reliable approach for routine SCFA analysis in fish fecal samples. It provides a useful analytical tool for studies investigating diet–microbiota interactions, gut health, and functional responses to nutritional interventions in aquaculture species.

Metabolites doi: 10.3390/metabo16090635

Authors: Chuyuan Qiao Chuwei Yang Shenglei Yang Yan Wang

Background/Objectives: Exercise snacks—brief, purposeful bouts of physical activity distributed throughout the day—may modify postprandial metabolic regulation, particularly glucose handling and insulin responses, while reducing time barriers to structured exercise. However, repeated primary studies and variable definitions may overstate evidence maturity. We reappraised evidence on exercise snacks and acute postprandial metabolism using review-level evaluation and independent study families. Methods: We conducted an umbrella review with de novo meta-analyses when evidence was sufficiently comparable. Six bibliographic databases and three supplementary literature-discovery sources were searched through 5 August 2026. Exercise snacks were defined as active bouts ≤ 15 min, performed at least twice per intervention day, and distributed across the day. AMSTAR 2, ROBIS, and corrected covered area (CCA) assessed review quality, risk of bias, and overlap. Random-effects models used restricted maximum likelihood with Hartung–Knapp adjustment; paired crossover effects were Hedges’ gz. RoB 2 and GRADE were applied. Results: Twenty-one systematic reviews were included; outcome-specific CCA ranged from 11.2% to 30.2%. After deduplication, only four study families contributed to acute plasma glucose iAUC, three to plasma insulin iAUC, and two to exploratory acute CGM glucose iAUC. Given these small evidence sets and the conservative Hartung–Knapp adjustment, the analyses had limited precision and statistical power to detect small-to-moderate effects. No statistically clear pooled effect was observed for plasma glucose (Hedges’ gz = −0.127, 95% CI −1.111 to 0.857; I2 = 77.9%) or insulin iAUC (Hedges’ gz = −0.520, 95% CI −1.263 to 0.222; I2 = 15.3%). The two-study CGM synthesis was descriptive and hypothesis-generating and neither supported nor refuted an acute glucose effect. Conclusions: Review-level abundance exceeded the independent quantitative evidence available for robust inference. The current analyses were underpowered to establish small-to-moderate pooled effects of exercise snacks on acute postprandial glucose or insulin metabolism; therefore, the findings indicate limited and statistically unstable evidence rather than evidence of no metabolic benefit.

Metabolites doi: 10.3390/metabo16090634

Authors: Wei Ma Yufei Chen Yaxin Feng Long Yuan Haoyu Liu Yangzi Sun Zixiang Xu Demin Cai Haiyong Sun

Background/Objectives: House dust mites (HDMs) are significant allergens that cause damage to nasal epithelial cells and have a role in the pathophysiology of allergic rhinitis. Lactobacillus johnsonii N5 supernatant (N5sup), a probiotic strain, exhibits possible protective properties; nevertheless, the molecular processes involved remain unclear. Methods: RPMI-2650 human nasal epithelial carcinoma cells were subjected to treatment with HDM, N5sup, or a combination of both. Results: The HDM stimulation induced a certain degree of transcriptional alterations. Compared with the control group, a total of 221 differentially expressed genes were identified in HDM-treated cells, which were mainly enriched in the IRE1–XBP1 branch of the unfolded protein response (UPR) pathway. Comprehensive investigation demonstrated that HDM increased the NR4A1–IRE1–XBP1 signaling pathway, characterized by elevated expression levels of NR4A1, ERN1 (IRE1), and XBP1. Both qRT-PCR and Western blotting verified that N5sup strongly inhibited the HDM-induced increase in NR4A1, IRE1, and XBP1. Analysis of the protein–protein interaction network further confirmed a central hub comprising NR4A1, IRE1, and XBP1. Conclusions: N5sup supernatant protects RPMI-2650 cells from HDM-induced injury, and this protective effect is associated with the NR4A1–IRE1–XBP1 pathway. These findings suggest that this signaling axis may serve as a potential therapeutic target for probiotic intervention in allergic airway diseases.

Metabolites doi: 10.3390/metabo16090633

Authors: Menghu Wang Xinyang Wang Xue Jiang Yafeng Zuo Dong Liu Qianqian Jiang Xiangsong Meng

Background/Objectives: Vitex negundo L. is a multi-part medicinal and aromatic plant, but organ-resolved chemical information for its aerial organs remains incomplete. This study generated an exploratory paired untargeted liquid chromatography–tandem mass spectrometry (LC-MS/MS) dataset for flowers, stems, and leaves and prioritized organ-associated putative metabolite families for subsequent validation. Methods: To reduce inter-individual background variation, flower, stem, and leaf tissues were harvested from the same three V. negundo individuals to form n = 3 paired plant blocks. LC-MS/MS feature data were filtered using pooled quality-control relative standard deviation (QC RSD ≤ 30%), median-normalized and log2-transformed. Principal component analysis (PCA) provided an unsupervised overview, and paired feature-level comparisons were performed using paired t-tests with Benjamini−Hochberg false discovery rate (FDR) correction. Results: A total of 42,125 QC-filtered LC-MS features were retained. PCA showed organ-associated separation among flowers, stems, and leaves. Using FDR < 0.05 and |mean paired log2 difference| ≥ 1, paired screening yielded 13,043, 22,836, and 13,393 FDR-supported LC-MS features for flower vs. stem, flower vs. leaf, and stem vs. leaf comparisons, respectively. Higher-scoring putative annotations suggested organ-associated putative family-level patterns, including flavonoid-related, hydroxycinnamic-acid derivative, phenolamide-related, triterpenoid-related, iridoid-related, and lignan-related signals. Conclusions: This organ-resolved dataset provides a chemical-feature framework for V. negundo aerial organs and putative family-level patterns for future structural confirmation, targeted quantification, bioactivity evaluation, and quality-marker development. Feature counts are not equivalent to unique metabolite counts, and compound labels require validation before being interpreted as confirmed structures.

Metabolites doi: 10.3390/metabo16090632

Authors: Fabiola Welter Ribeiro Emilton Lima Junior

Background/Objectives: Psoriasis and atopic dermatitis have been conceived as opposite immunological poles, yet molecular overlap and clinically recognised coexistence challenge this dichotomy. We sought to adjudicate among three architectures: overlap with no discriminating systemic signature, a shared inflammatory and metabolic continuum with additive coexistence, and distinct phenotypes with synergy. Methods: Cross-sectional analysis of a selected UK Biobank subsample of 160,782 participants, classified by International Classification of Diseases, 10th revision codes L40 and L20 as control, isolated atopic dermatitis (n = 12,859), isolated psoriasis (n = 15,146) and coexistence (n = 1068). Haematological indices, a biochemical and metabolomic panel including glycoprotein acetyls, and circulating inflammatory proteins were evaluated through converging evidence from ordinal effect sizes, equivalence testing at a margin equivalent to an area under the receiver operating characteristic curve of 0.574, quantile regression with interaction terms, permutational multivariate analysis of variance, supervised classification and mixture modelling. Results: Effect sizes were negligible for 46 of 48 contrasts against a comorbidity-free comparator, and 38 of 48 met formal equivalence. None of the 16 interaction terms survived false discovery rate control. Multivariate separation was detectable but explained 0.6% of dispersion, classification reached an area under the curve of 0.57, and latent classes bore no correspondence to clinical diagnosis. When cardiometabolic exclusions were applied symmetrically to all four groups, every contrast met equivalence, and the ordering of the dominant factor did not persist. Polygenic risk scores confirmed that the coded groups carry the expected genetic architecture. Conclusions: Within the systemic biomarkers evaluated these conditions are not discriminable, and coexistence is not an emergent phenotype.

Metabolites doi: 10.3390/metabo16090631

Authors: Michał Biały Małgorzata Inglot

Background: Metabolic complications and weight gain have emerged as primary concerns among virologically suppressed and immunologically competent people living with HIV. Particularly integrase strand transfer inhibitors (INSTIs) and tenofovir alafenamide (TAF) are being connected with this issue. Objectives: This study aimed to evaluate the prevalence of metabolic syndrome (MetS) and its components within a highly homogeneous, virologically suppressed cohort, assessing the impact of antiretroviral therapy (ART) and HIV-unrelated factors. Methods: We conducted a single-center cross-sectional analysis of 93 HIV-positive patients receiving stable ART for >2 years, in Wrocław, Poland. To minimize confounding, individuals with HBV/HCV co-infections or significant alcohol use were excluded. Participants were stratified primarily by regimen, INSTI/TAF-based (n = 50) vs. PI/TAF-based (n = 28), and secondarily by the particular drugs taken. Clinical assessment included anthropometric parameters, lipid profiles, and glucose metabolism. Results: The prevalence of MetS was 10.8%. No statistically significant differences were observed in the incidence of MetS, insulin resistance, or dyslipidemia between the INSTI/TAF and PI/TAF groups. A marginally significant trend toward lower total cholesterol was identified in patients receiving dolutegravir/TAF compared to those on darunavir/TAF. Conclusions: In this stable Caucasian cohort with long-term viral suppression, we found no statistically significant differences in metabolic outcomes between modern INSTI- and PI-based regimens. However, due to the limited sample size and low statistical power, these findings do not prove clinical equivalence. Our results suggest that in particular tightly selected settings of well-controlled patients, traditional risk factors and lifestyle may remain the primary drivers of metabolic health, although modest drug-related effects cannot be ruled out.

Metabolites doi: 10.3390/metabo16090630

Authors: Yuqing Zhang Yunpeng Xie Xinyu Liu Zhen Ning Guowang Xu Yunlong Xia Xinjie Zhao

Background: Atrial fibrillation (AF) is the most prevalent clinical arrhythmia with severe cardiovascular complications, yet its metabolic molecular mechanisms remain poorly defined. Omics-based metabolic profiling provides a powerful strategy to systematically decode AF-associated metabolic disorders. Methods: In this work, high-coverage targeted liquid chromatography–tandem mass spectrometry (LC-MS/MS) metabolomics and lipidomics were applied to absolutely quantify 746 serum metabolites from AF patients and healthy controls. Results: We systematically characterized global metabolic perturbations in AF serum, including impaired fatty acid metabolism, suppressed mitochondrial β-oxidation, myocardial lipotoxic lipid accumulation, and systemic depletion of glycerophospholipids. Global multiscale embedded correlation network analysis (MECNA) further identified 11 AF-specific dysregulated metabolic modules and core hub metabolites driving metabolic remodeling. Leveraging binary logistic regression, we constructed and independently validated a two-molecule diagnostic biomarker panel to distinguish AF patients from healthy subjects. The combined biomarkers Phe-Trp and FA 22:5 achieved outstanding diagnostic performance, with area under the curve (AUC) values of 0.964 in the discovery cohort and 0.993 in the validation cohort. Conclusions: Collectively, this study adopts high-depth targeted quantitative omics to comprehensively map AF metabolic signatures, dissect disease-relevant metabolic networks, and establish a robust serum biomarker panel with great translational potential for non-invasive AF clinical diagnosis.

Metabolites doi: 10.3390/metabo16090629

Authors: Lin Guo Yimin Yin Yanyan Sun Hongwen Zhou Yingyun Gong

Background/Objectives: Insulin clearance is a key determinant of circulating insulin availability, but its diurnal variation and relationship with circadian metabolic programs in metabolic dysfunction associated steatotic liver disease (MASLD) remain unclear. This study aimed to explore diurnal insulin clearance in humans and examine associated metabolic gene signatures in MASLD. Methods: A single-subject pilot assessment was performed to explore daytime-nighttime differences in insulin clearance rate (ICR) surrogate index, followed by evaluation using public hyperinsulinemic-euglycemic clamp datasets from healthy individuals and patients with MASLD. Public circadian transcriptomic datasets, spatial transcriptomic data, and a time course high-fat diet (HFD)-induced mouse dataset were integrated. A predefined panel of insulin clearance-related and circadian genes, including carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), insulin receptor (INSR), insulin-degrading enzyme (IDE), clock circadian regulator (CLOCK), basic helix-loop-helix ARNT like 1 (BMAL1), nuclear receptor subfamily 1 group D member 1/2 (NR1D1/2), period circadian regulator 1/2 (PER1/2), and cryptochrome 1/2 (CRY1/2), was analyzed. Results: The pilot assessment showed higher nighttime than daytime ICR, and independent clamp datasets showed a similar pattern in healthy individuals. In MASLD, nighttime ICR remained relatively higher, whereas overall insulin clearance was reduced compared with controls. Human blood-based circadian transcriptomic datasets identified rhythmic expression patterns of selected genes involved in insulin clearance and circadian regulation, including CEACAM1, CLOCK, NR1D1, CRY1, PER1, and PER2. MASLD liver datasets showed reduced expression of insulin clearance-related and circadian genes, while spatial transcriptomics suggested altered lobular distribution of these signatures. In HFD mouse model, rhythmic expression of selected genes was attenuated. Conclusions: These integrative findings suggest that insulin clearance may exhibit diurnal variation and may be linked to circadian metabolic gene signatures across systemic and hepatic datasets in MASLD. Larger controlled human studies are needed to validate the temporal regulation of insulin clearance and its metabolic relevance.

Metabolites doi: 10.3390/metabo16090628

Authors: Yesim Oztekin Buket Gonen-Colak Selin Tekin Incilay Lay Tomris Erbas Selcuk Dagdelen Zehra Buyuktuncer

Background/Objectives: The study aimed to assess cardiometabolic effects of two pseudocereals, quinoa and buckwheat, comparing them to the effects of rice and bulgur in adults with overweight or obesity. Methods: This study was conducted on 148 adults with overweight and obesity, aged between 25 and 45 years, consuming 40 g/d quinoa, buckwheat, rice, or bulgur for 4 weeks. Anthropometrical measurements were taken, and dietary intake was assessed; blood pressure and biochemical parameters were examined. Incremental areas under the curve (iAUC) for glucose and insulin were calculated. Subgroup analyses were conducted considering sex. Results: The changes in biochemical and anthropometrical measurements did not differ significantly between groups. However, within-group analysis showed significant changes in many parameters. Among anthropometrical measurements, fat mass declined significantly within quinoa and buckwheat groups (p < 0.05). Blood pressure decreased significantly in the buckwheat group, mainly in women. Fasting glucose decreased significantly only in men in the quinoa group. An increase in iAUC glucose within the bulgur group but a significant decrease in iAUC insulin among men within the buckwheat group was recorded. Total cholesterol (Total-C) significantly decreased in men but increased in women following quinoa consumption. On the other hand, a decrease was reported in high-density lipoprotein cholesterol (HDL-C) in women following quinoa and buckwheat consumption (p < 0.05). Conclusions: Compared with the baseline, the most pronounced changes were observed in blood pressure, lipid profile, and body fat mass, suggesting a potential cardiometabolic benefit of quinoa and buckwheat. However, sex-specific cardiometabolic differences may occur following the consumption of pseudocereal or cereal. Further studies are needed for more robust results.

Metabolites doi: 10.3390/metabo16090627

Authors: Olivia Z. B. Ginnard Maria Morales Gabrielle Phillips Mary L. Brandt Sridevi Devaraj Alexis Wood Stephanie R. Sisley

Background/Objectives: Vitamin D deficiency is highly prevalent among children with obesity, but the mechanisms underlying the effectiveness of vitamin D supplementation remain poorly understood. This study examined the expression patterns of VDR-target genes across metabolically diverse tissues and compared these molecular measures with circulating serum 25-hydroxyvitamin D (25OHD), the current clinical marker of vitamin D status. We hypothesized that VDR-pathway gene expression would correlate within metabolically relevant tissues but would not be significantly associated with circulating serum 25OHD levels. Methods: A secondary analysis was performed on blood, intestinal, and visceral and subcutaneous adipose tissue (VAT and SAT, respectively) samples obtained from adolescents with obesity. Subject data included age, gender, race/ethnicity, and BMI. The tissues were analyzed via real-time qPCR to obtain quantitative levels of VDR-target gene expression, which included TLR4, THBD, and VDR in SAT and VAT and TRPV6, S100G, and VDR in intestinal tissue. Blood samples were analyzed for serum 25OHD. Results: Gene expression of THBD, VDR, and TLR4 in SAT and VAT significantly correlated with each other. In intestinal tissue, there was significant correlation between TRPV6, S100G, and VDR. No statistically significant associations were identified between the gene expression levels and serum 25OHD levels. Conclusions: VDR-target gene expression levels correlated with each other across diverse tissues but not with serum 25OHD levels. This discrepancy suggests that circulating 25OHD concentrations may not fully reflect vitamin D action.

Metabolites doi: 10.3390/metabo16090626

Authors: Lin Xu Chang Li Yufei Liu Shijie Wang

Background/Objectives: Aerobic exercise is widely recognized as an effective non-pharmacological strategy for metabolic dysfunction-associated steatotic liver disease (MASLD). However, the effects of different aerobic exercise loads on MASLD-associated hepatic iron dyshomeostasis and ferroptosis, as well as the underlying mechanisms, remain unclear. This study aimed to compare the effects of different aerobic exercise loads on HFD-induced MASLD and to explore whether exercise-induced improvements in hepatic lipid accumulation are associated with changes in iron homeostasis and ferroptosis-related markers. Methods: Forty male Sprague-Dawley rats were randomly assigned to a normal-fat diet group (NFD, n = 8) or a high-fat diet group (HFD, n = 32). After 8 weeks of HFD feeding, HFD-fed rats were further allocated to four groups: HFD control, low-load aerobic exercise (LEH), moderate-load aerobic exercise (MEH), and high-load aerobic exercise (HEH) (n = 8 per group). The exercise intervention lasted for 8 weeks. At the end of the intervention, blood and liver samples were collected to assess metabolic parameters, hepatic steatosis, iron homeostasis, oxidative stress, ferroptosis-related markers, and the IL-6/JAK2/STAT3-hepcidin pathway. Results: All three exercise regimens partially improved hepatic lipid metabolic abnormalities, iron accumulation, and oxidative stress-related liver injury in MASLD rats. These effects were accompanied by suppression of the IL-6/JAK2/STAT3-hepcidin pathway, increased hepatic FPN1 mRNA expression and decreased DMT1 mRNA expression, increased GPX4 expression, reduced PTGS2 expression, and improved antioxidant capacity. MEH and HEH generally produced larger changes than LEH, with HEH showing the most consistent overall response across the measured outcomes. Conclusions: Aerobic exercise attenuated HFD-induced MASLD in association with improved hepatic iron handling and a lower burden of ferroptosis-related molecular alterations. Because ferroptosis-specific rescue experiments and pathway inhibitors were not used, these findings are associative. Within the tested conditions, the 90-min protocol produced the most consistent response, but further dose–response studies are required before translation to humans.

Metabolites doi: 10.3390/metabo16090625

Authors: Federica Armeli Emily Schifano Beatrice Mengoni Arianna Montanari Martina Menin Laura Pompa Maria Luisa Crudeli Thomas Lenz Trevor Archer Daniela Uccelletti Rita Businaro

Background: Oxidative stress, neuroinflammation, impaired autophagy, and mitochondrial dysfunction are major contributors to ageing and neurodegenerative diseases. This study investigated whether AgeViva could counteract these processes by modulating redox balance, inflammation, autophagy, and mitochondrial function. Methods: The effects of AgeViva were evaluated using two complementary experimental models: lipopolysaccharide (LPS)-stimulated BV2 microglial cells and Caenorhabditis elegans (C. elegans) nematodes. In BV2 cells, the expression of inflammatory, autophagy-related, and antioxidant response genes, including NRF2, SOD, and GPX, was evaluated by RT-qPCR, while cell viability was assessed by Trypan Blue exclusion assay. In C. elegans, lifespan, healthspan parameters, ROS accumulation, mitochondrial integrity, membrane potential, and the expression of stress-response, longevity, and autophagy-related genes were analyzed following AgeViva supplementation. Results: In LPS-stimulated BV2 cells, AgeViva significantly reduced the expression of mRNA the pro-inflammatory cytokines Interleukin-1 beta (IL-1β) and Tumor Necrosis Factor alpha (TNF-α) while increasing Interleukin-10 (IL-10) levels, AgeViva also induced changes in autophagy-related transcripts, such as modulation of microtubule-associated protein 1a/1b-Light Chain (LC3) and Sequestosome 1 (p62) expression, activated antioxidant-related gene expression, increasing the expression of Superoxide Dismutase 1(SOD1) and Glutathione Peroxidase (GPX). In C. elegans, AgeViva supplementation extended lifespan and improved healthspan parameters, including locomotor activity and pharyngeal pumping. Treated nematodes showed reduced cytosolic and mitochondrial ROS accumulation, preservation of mitochondrial network integrity, and maintenance of mitochondrial-associated fluorescence, reflecting mitochondrial content and/or membrane potential during ageing. Molecular analyses revealed modulation of key pathways involved in stress resistance and longevity, including Insulin-like Growth Factor 1 (Insulin/IGF-1) signaling Dauer Formation-2 and 16 (DAF-2/DAF-16), Skinhead-1 (SKN-1/Nrf2) signaling, and autophagy-related genes, like Ligating (lgg-1), Autophagy-Related-7 (atg-7), Autophagy Related-18 (atg-18), uncoordinated-51 (unc-51), and ectopic p-granules autophagy protein 5 (epg-5). Conclusions: AgeViva promotes healthy ageing by modulating oxidative stress, inflammation, autophagy, and mitochondrial homeostasis.

Metabolites doi: 10.3390/metabo16090624

Authors: Sinenhlanhla Nonhle Nsele Maropeng Vellry Raletsena Udoka Vitus Ogugua Pierre Adriaanse

Background/Objectives: This study examined the effect of three different cultural media—coconut coir, peat moss, and deep-water culture—on the metabolomic profiles of two tomato cultivars, Heinz and Roma. Tomato fruit quality is influenced by intricate interactions between the genotype and root-zone environment; nevertheless, little is known about how substrate-based systems compare metabolically to deep-water hydroponics. Methods: Fruit samples from both cultivars grown under controlled greenhouse conditions were analyzed using proton nuclear magnetic resonance (1H-NMR) spectroscopy to identify treatment-dependent biochemical changes. Polar metabolites were extracted using a methanol–water solvent solution and examined using a 600 MHz NMR spectrometer. Spectral datasets were processed and analyzed with multivariate statistical tools such as Principal Component Analysis (PCA), Partial Least Squares Discriminant Analysis (PLS-DA), and Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA). Results: Distinct clustering patterns were observed, indicating both cultivar-specific and cultivation-system-dependent metabolic differentiation. The PCA model displayed excellent explanatory and predictive capacity, while supervised OPLS-DA improved group discrimination, demonstrating that both genotype and growing medium significantly influenced the chemical composition of the fruit. Soluble sugars (glucose, fructose, and sucrose), sugar alcohols, organic acids such as citric and malic acids, and a variety of amino acids involved in nitrogen metabolism and stress reactions were among the key distinguishing factors. Carbohydrate-related spectral areas (3.0–5.5 ppm) were highly associated with treatment separation, indicating that the cultivation system had a significant impact on carbon allocation and energy metabolism. Conclusions: Fruits grown in deep water culture have distinct metabolic fingerprints from those grown on coconut coir and peat moss, implying that root-zone oxygen availability and nutrient dynamics may influence primary and secondary metabolism.

Metabolites doi: 10.3390/metabo16090623

Authors: Katelyn Dunigan-Russell Matthew Ryan Smith Hua Zhong ViLinh Tran Dean P. Jones Lynette K. Rogers Trent Tipple

Background: Supraphysiological levels of oxygen are often used as therapy for acute respiratory distress and severe pulmonary morbidities but can cause excessive generation of reactive oxygen species resulting in oxidative and inflammatory injury. Aurothioglucose (ATG), an FDA-approved, gold-containing pharmaceutical, potently and irreversibly inhibits thioredoxin reductase 1, and ATG treatment preserves reduced glutathione levels and attenuates hyperoxic lung injury. Methods: Adult C3H mice were treated with saline or ATG and exposed to room air (RA) or >95% O2. All mice had succumbed or were euthanized at 200 h of >95% O2. In a separate cohort euthanized at 72 h, prior to the appearance of oxygen toxicity, lung tissues were collected for metabolomic analyses. Comparisons were performed between RA and >95% O2 exposure, saline and ATG treatment, and male and female. Results: In <95% O2, differences in survival between the sexes with and without ATG treatment were observed. ATG-treated females survived longer than all other hyperoxia-exposed groups. Features in the glutathione and selenoprotein pathways were significantly different. Metabolomic analysis revealed keratan sulfate (KS) biosynthesis and glycosphingolipid (GSL) biosynthesis as the primary pathways affected in the saline O2 vs. ATG O2 comparison. Carnitine shuttle was identified as the primary pathway between the sexes both with ATG and O2. Conclusions: The current data suggest that the improved survival of hyperoxia-exposed, ATG-treated, female C3H mice may be driven by alterations in glutathione synthesis, energy production, and metabolism resulting in decreased lung injury. These findings may provide direction for further research to improve outcomes after hyperoxia exposure.

Metabolites doi: 10.3390/metabo16090622

Authors: Oleksii Ivashyn Volodymyr Gryshchuk Vita Ivashyna Maryna Miasnykova Heorhii Yurlov Svitlana Stepanenko Dmytro Zhaboiedov

Background: Optical neuropathy is a severe complication in patients with type 2 diabetes mellitus (T2DM) undergoing vitreoretinal surgeries. Early diagnostic indicators are critical to preventing irreversible vision loss. This study aimed to evaluate the alterations in blood serum amino acid profiles, with a specific focus on serine levels, and their potential clinical association with the severity of neuroretinal impairment. Methods: The clinical cohort included 28 patients aged 32 to 77 years with T2DM following vitreoretinal interventions. Visual field functional status was assessed using Humphrey automated perimetry, with mean deviation (MD), pattern standard deviation (PSD), and visual field index (VFI). Structural changes were quantified using optical coherence tomography (OCT) to measure peripapillary retinal nerve fiber layer (RNFL) thickness. Serum amino acid profiles were determined using an ion-exchange chromatography amino acid analyzer, alongside a reference group (n = 12). Results: All patients presented with moderate-to-severe visual field impairment. A profound decline in serum serine levels was observed in diabetic patients compared to the reference group (p < 0.001). The reduction in serine content closely mirrored both the degree of retinal sensitivity loss (MD and PSD values) and the number of thinned peripapillary RNFL sectors. Notably, the compensatory synthesis of serine via glycine cleavage appeared insufficient, as evidenced by a concomitant statistical decrease in glycine content (p < 0.05). No significant differences in serine levels were found between the subgroups when stratified strictly by age or isolated severity stages. Conclusions: It was demonstrated that the decline in serum serine levels in patients with T2DM following vitreoretinal interventions is significantly associated with the severity of optic neuropathy as assessed by optical coherence tomography (OCT). Consequently, serum serine may serve as a valuable dual biomarker, acting as a prognostic indicator for retinopathy progression and a predictive marker for early-stage neuropathy development.

Metabolites doi: 10.3390/metabo16090621

Authors: Xin Tao Shangbo Yan Ranran Chen Fangfang Ren Hongjie Yang Wenheng Long Nan Gao

Background: γ-Valerolactone (GVL), a green volatile platform compound derived from lignocellulosic biomass, has recently been identified as one volatile organic compound produced by the plant growth-promoting rhizobacterium Stutzerimonas stutzeri NRCB010. Although our previous transcriptomic and phenotypic studies have verified that exogenous GVL can promote the growth of tomato seedlings, the global metabolic reprogramming behind this promoting effect remains unclear. Methods: Tomato seedlings were subjected to exogenous GVL treatments at three concentrations (0, 0.25, 0.5 g/L) for 24 h and 48 h separately. Combined with physiological growth measurements, ultra-high-performance liquid chromatography–tandem mass spectrometry-based widely targeted metabolomics was applied to systematically characterize GVL-mediated metabolic reprogramming. Results: GVL remarkably improved seedling height, biomass and root development, with 0.25 g/L GVL showing the strongest promoting effect. A total of 703 metabolites including lipids, flavonoids and alkaloids were identified. Principal component analysis and orthogonal partial least squares discriminant analysis revealed distinct metabolic separation between control and treated groups, verifying time and concentration dependent metabolic shifts. After 24 h, GVL activated primary pathways (the tricarboxylic acid cycle, amino acid and purine metabolism) to supply growth energy; after 48 h, central carbon and secondary biosynthetic pathways were enriched, and 0.5 g/L GVL specifically triggered defensive isoquinoline and indole alkaloid biosynthesis. Conclusions: GVL coordinately remodels primary and secondary metabolic networks to accelerate tomato seedling growth, which provides sufficient metabolomic evidence for developing GVL as a novel bio-based plant biostimulant.

Metabolites doi: 10.3390/metabo16090620

Authors: Aleksandra Radecka Waldemar Pluta Michał Lubkowski Anna Lubkowska

Background: The stability of the musculoskeletal axis is crucial for maintaining mobility in women, yet the impact of controlled thyroid dysfunction on this system remains ambiguous. Objectives: The aim of this cross-sectional study was to compare the body composition and functional performance of women with Hashimoto’s thyroiditis, women with hypothyroidism, and healthy controls in the context of the role of parathyroid hormone (PTH) as a potential comorbid factor. Methods: The study included 70 women divided into three groups: Hashimoto’s thyroiditis (n = 27), hypothyroidism (n = 15), and control (n = 28). Anthropometric parameters, including limb muscle mass (ALM) and bone mineral density (BMD), were assessed using X-ray absorptiometry (DXA). Physical performance was assessed using handgrip strength (HGP) and Timed Up and Go (TUG) tests, supplemented by ELISAs for metabolic and neurotrophic biomarkers (BDNF, irisin, GDF-15, P3NP). Results: The results showed no statistically significant differences between groups in muscle mass (ALM: p = 0.13; ALMI: p = 0.27) or bone density (BMD: p = 0.82). Functional performance outcomes were also comparable between groups (HGP: p = 0.71; TUG: p = 0.143). The levels of the analyzed biomarkers did not differ significantly by thyroid status (BDNF: p = 0.91; irisin: p = 0.85; GDF-15: p = 0.96). Furthermore, correlation analysis revealed that PTH showed a moderate, negative correlation with P3NP in the total population (r = −0.44, p = 0.003). In contrast, its association with muscle mass index (ALM) was attenuated after FDR correction. Conclusions: These preliminary results indicate no detectable deterioration in musculoskeletal and functional parameters in women with managed thyroid dysfunction. However, due to the small sample size of the hypothyroidism subgroup and low statistical power, these findings cannot establish physiological equivalence and must be interpreted with caution, as they do not confirm a direct protective effect of levothyroxine therapy against muscle mass decline.

Metabolites doi: 10.3390/metabo16090619

Authors: Mingli Ye Yukai Ba Zhengrui Chen Boya Liu Xin Li Xiaoya Yu Yonggang Zhao Ban Cao Chao Lei Yu Liu

Background/Objectives: The whole plant of Tetrastigma hemsleyanum is used medicinally, but the tuberous root is by far the most commonly used part, and the way in which its metabolites are partitioned among organs and among geographic origins underpins both the rational choice of the medicinal part and the evaluation of herb quality. Methods: Here, untargeted metabolomics based on ultra-high-performance liquid chromatography coupled to high-resolution Orbitrap mass spectrometry was used to compare roots, stems and leaves collected from nine regions of southern China (81 samples) with tuberous roots collected from 17 sites in seven provinces (51 samples). Annotations were graded according to the Metabolomics Standards Initiative (MSI), curated with explicit plausibility rules, and every conclusion was re-tested across five nested annotation subsets. Results: Organ identity was the dominant source of metabolic variation: the three organs were completely separable (random forest out-of-bag accuracy 100%), the organ effect was about four times larger than that of sampling region (PERMANOVA pseudo-F 16.7 versus 4.1), and this contrast was essentially unchanged from the complete set of 615 annotations down to the most stringent subset of 19 flavonoids and phenolic acids. The organ-level pattern was chemically coherent: amino acids and lipids were relatively enriched in the tuberous root, soluble sugars and phenolic acids in the stem, and flavonoids and alkaloids in the leaf, matching the contrasting roles of a storage, a transport and a photosynthetic organ. Geographic differences among tuberous roots were, by contrast, weak and largely local: although provinces could be separated with 88.9% out-of-bag accuracy, accuracy fell to 42.2% when an entire, previously unseen collection site was held out (chance level 20%), and to 52.9% for a coarse macro-geographic zone (chance level 25%), whereas holding out an entire sampling region left organ classification unaffected (100%). Conclusions: The present data therefore document a strong, generalisable and chemically interpretable organ division of labour, but do not support the use of this metabolome for origin authentication. Because most annotations remain at MSI Level 3, individual compounds are reported as putative throughout, and all conclusions rest on multivariate and class-level evidence.

Metabolites doi: 10.3390/metabo16090618

Authors: Jungyong Lee Taeho Kim Deunsol Hwang Sunghwan Kyun Inkwon Jang Nayeon Kim Kiwon Lim Hun-Young Park Insu Kwon Sung-Woo Kim Jisu Kim

Background/Objectives: Alternate-day fasting (ADF) combined with exercise has been suggested as an effective lifestyle strategy for reducing fat mass while preserving lean mass. However, whether exercise preconditioning before ADF plus concurrent exercise affects subsequent body-composition and substrate-utilization responses remains unclear. This study aimed to evaluate the effects of exercise preconditioning during a subsequent ADF plus concurrent exercise intervention. Methods: Seven-week-old male ICR mice were assigned to the non-exercise preconditioning (Non-ExPC) or exercise preconditioning (ExPC) group (n = 8/group). After the 4-week preconditioning period, both groups underwent 7 days of ADF plus concurrent exercise. Resting energy metabolism was measured during the intervention, and body composition, exercise energy metabolism, muscle and motor function, blood biomarker concentrations, and plasma hormone levels were assessed before and after the intervention. Results: Both groups showed significant reductions in body weight, body fat percentage, and fat mass after the intervention. Lean mass decreased in both groups without between-group differences. The ExPC group maintained significantly lower body fat percentage and fat mass than the Non-ExPC group and showed significantly higher resting oxygen uptake, carbon dioxide production, respiratory exchange ratio, carbohydrate oxidation, and energy expenditure during the intervention. During exercise, the respiratory exchange ratio and carbohydrate oxidation significantly increased in both groups, whereas fat oxidation significantly decreased only in the ExPC group. Conclusions: Exercise preconditioning was associated with the maintenance of lower adiposity during short-term ADF combined with concurrent exercise, while differences in substrate utilization were observed under some conditions.

Metabolites doi: 10.3390/metabo16090617

Authors: İsa Temur Mehmet Özsan Katibe Tuğçe Temur Andaç Batur Çolak

Background/Objectives: This pilot study aimed to develop and compare advanced machine learning models, specifically a Bayesian-regularized artificial neural network (ANN) and a transformer-enhanced physics-informed neural network (PINN), by integrating periodontal health indices and hematological inflammatory markers for the prediction of GDM. Methods: Utilizing a prospective case–control study design, a clinical dataset comprising 80 pregnant women (40 with GDM and 40 healthy controls) was evaluated to develop and compare advanced machine learning models. Clinical, periodontal, and complete blood count-derived inflammatory parameters were integrated into two predictive models: a Bayesian regularization-based artificial neural network (ANN) and a transformer-enhanced physics-informed neural network (PINN). Model performance was evaluated using the coefficient of determination (R2), mean squared error (MSE), root mean squared error (RMSE), mean absolute error (MAE), and residual error analyses. Results: The ANN demonstrated superior predictive performance, achieving an R2 of 0.9872 and an MSE of 3.38 × 10−3, compared with an R2 of 0.9833 for the PINN model. Residual analysis showed that the ANN provided greater prediction stability, with a mean deviation of 0.5691 and a standard deviation of 3.6400. The findings demonstrate that the integrated analysis of periodontal and hematological markers through advanced neural architectures, particularly the Bayesian-regularized ANN, provides a high-fidelity diagnostic signal for GDM prediction. This integrated feature set, when processed by the proposed machine learning framework, enables the identification of complex biological patterns with remarkable precision. Conclusions: The proposed machine learning framework provides an accurate, non-invasive, and clinically applicable approach for predicting GDM. Integrating routinely available periodontal and hematological data may improve early risk stratification and support personalized prenatal care. Further validation in larger and more diverse populations is warranted before clinical implementation.

Metabolites doi: 10.3390/metabo16090616

Authors: Jiangyong An Shaoyan Zhang Yuxin Wang Fuqiang Du Tao Zhang

Background: Persicaria capitata is a perennial herb and one of the top ten authentic Miao medicinal plants in Guizhou province, China, with flavonoids serving as its primary bioactive components. Agricultural production practices have shown that nitrogen fertilizer application can reduce flavonoid accumulation in P. capitata, although the underlying molecular regulatory mechanism remains unclear. Methods: In this study, a pot experiment was performed to investigate the effects of different nitrogen treatments on the growth performance and yield of P. capitata. Moreover, the related changes in transcriptomic characteristics and metabolic profiles were also systematically analyzed. Results: Nitrogen fertilization significantly promoted stem elongation, branch number, and yield formation. The maximum stem length, branch number, and single-plant yield were recorded in the 0.3 g N/kg treatment group as reaching 80.74 cm, 37.30, and 123.64 g/plant, respectively. Transcriptomic screening yielded 10,880 differentially expressed genes (DEGs), and the number of downregulated DEGs was consistently higher than upregulated DEGs across all comparison groups. Metabolomic analysis identified a total of 3356 differentially accumulated metabolites, including 168 differential flavonoid metabolites. With the increase in nitrogen application rate, most flavonoid compounds, such as chlorogenic acid, 5-hydroxyferulic acid, and 4-hydroxycinnamic acid, exhibited a significantly downregulated accumulation pattern. An integrated transcriptomic and metabolomic analysis revealed that the differential flavonoid metabolites and functional genes were predominantly enriched in the phenylpropanoid biosynthesis, flavonoid biosynthesis, and flavone and flavonol metabolism pathways. The decreased expression of key structural genes, including PAL, 4CL, CHS, FLS, F3H, and F3′H, inhibited the biosynthesis and accumulation of core flavonoid components (e.g., chlorogenic acid, naringenin, and luteolin), thereby deteriorating the medicinal quality of P. capitata. Conclusions: Nitrogen supplementation increases the yield of P. capitata but inhibits flavonoid biosynthesis. Downregulated expression of key genes in flavonoid metabolism is responsible for the decreased flavonoid content. This study provides theoretical support for optimized nitrogen management to coordinate yield formation and quality improvement in planting practices for P. capitata.

Metabolites doi: 10.3390/metabo16090615

Authors: Fangming Liu Xingyue Xiong Yanmei Wang Li Dai Zhi Li Xiaodong Geng Juan Wang Yongyu Ren Qifei Cai Zhen Liu

Background/Objectives: Idesia polycarpa Maxim. possesses conspicuous glandular protuberances on its petioles, but the chemical composition and potential biological significance of these structures remain largely unexplored. This study aimed to characterize the metabolic profile of I. polycarpa petiole glands and to determine their organ-specific accumulation patterns of secondary metabolites compared with leaves and shoot tips. Methods: A widely targeted metabolomics approach based on ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was applied to analyze petiole glands, leaves, and shoot tips collected from the same I. polycarpa individuals. Multivariate statistical analyses and differential metabolite profiling were performed to compare metabolic characteristics among the three organs. Results: Petiole glands contained 700 detected metabolites, representing the highest metabolic diversity among the three examined organs, and 39 metabolites were exclusively detected in petiole glands. Principal component analysis clearly separated petiole glands from leaves and shoot tips, demonstrating their distinct metabolic profile. Compared with other organs, petiole glands showed preferential accumulation of several secondary metabolite classes, including flavonoids, tannins, alkaloids, and phenolic acids. The gland-exclusive metabolites included flavonoids such as kaempferide, kaempferitrin, and tectoridin, as well as diverse proanthocyanidins and phenolic acid derivatives. Conclusions: This study provides the first systematic metabolomic characterization of I. polycarpa petiole glands and reveals their organ-specific accumulation of secondary metabolites. The unique metabolic profile of petiole glands suggests potential ecological relevance and provides a foundation for further investigations into the biological functions of gland-associated metabolites.

Metabolites doi: 10.3390/metabo16090614

Authors: Jinghui Zhang Hongchuan Liu Qingrong Qiu Kongcai Zhu Xian Ding Rui Zhao Ting Hu Zhuoling An

Background/Objectives: Drug-induced liver injury (DILI) remains a leading contributor to acute liver failure, yet phenotypic classification still relies on the R-value, leaving the mixed-pattern (MD) subtype a heterogeneous category between hepatocellular (HD) and cholestatic (CD) injury. We aimed to identify a metabolic signature that helps resolve this heterogeneity. Methods: Targeted LC-MS/MS metabolomics was performed on 79 patients with DILI (HD = 54, CD = 7, MD = 18) and 221 healthy controls. A 32-metabolite panel was used to train an XGBoost classifier with pre-specified, fold-invariant hyperparameters, evaluated via Leave-One-Out Cross-Validation and interpreted by TreeExplainer-based SHAP attribution. Results: SHAP identified 5′-methylthioadenosine (MTA) as the dominant contributor. A four-parameter logistic fit of its SHAP dependence curve gave an exploratory, model-derived transition point at a relative abundance of 93. Applying this value to the 18 held-out MD patients assigned 16 to a hepatocellular and 2 to a cholestatic trajectory. MTA correlated with total bilirubin (r = 0.53, p < 0.001) but not ALT (r = −0.19, p = 0.10), dissociating it from hepatocyte leakage. Conclusions: MTA emerges as an exploratory biomarker that uncovers the distinct metabolic trajectories hidden within mixed-pattern DILI. Furthermore, these findings highlight the MTA-methylthioadenosine phosphorylase (MTAP) axis as a potential mechanistic driver of cholestatic injury.

Metabolites doi: 10.3390/metabo16090613

Authors: He N. Xu Jack Kollmar Allison Podsednik Mihiar Wannousse Alexander Shestov Roddy S. O’Connor Joseph A. Baur Julia Tchou Rong Zhou Lin Z. Li

Background/Objectives: Triple-negative breast cancer (TNBC) exhibits substantial metabolic heterogeneity and plasticity, contributing to variable therapeutic responses. We investigated whether optical redox imaging (ORI) could characterize metabolic phenotypes, monitor responses to metabolic perturbation, and relate these responses to functional outcomes in TNBC cells. Methods: Four TNBC cell lines were treated with the lactate dehydrogenase A inhibitor FX11 or the glutaminase inhibitor CB-839, alone or in combination with paclitaxel. Intensity-based label-free ORI was performed and followed by imaging of fluorescent probes to assess mitochondrial membrane potential (MMP), re-active oxygen species (ROS), and cell number in the same dishes. Seahorse assays were used to evaluate mitochondrial respiration and glycolytic flux. Results: TNBC cell lines exhibited distinct basal redox phenotypes and differential responses to acute glycolytic and glutaminolytic perturbation. HCC1806 cells showed the strongest acute ORI responses to both FX11 and CB-839. Acute FX11 treatment induced a rapid reductive shift accompanied by ROS accumulation and loss of MMP, whereas CB-839 produced a more modest early reductive response without detectable ROS accumulation or MMP loss. Prolonged treatment revealed distinct temporal redox trajectories in HCC1806 cells: FX11-treated cells evolved from an acute reductive response toward a more oxidized state, while CB-839-treated cells transitioned from an early reductive shift to a sustained oxidized redox state accompanied by marked reductions in OCR and ECAR. Functionally, in two representative models (HCC1806 and MDA-MB-231), CB-839 reduced cell numbers and enhanced the anti-proliferative effect of paclitaxel, whereas FX11 had no significant effect on cell number despite inducing pronounced acute redox perturbations. Conclusions: Integrating ORI with metabolic flux assays and imaging-based functional measurements enables characterization of multiple dimensions of metabolic behavior, including basal phenotype, pathway-specific responsiveness, temporal redox responses, and treatment-associated outcomes. These findings support intensity-based wide-field ORI as a practical and accessible tool for probing metabolic heterogeneity and characterizing context-dependent metabolic responses in TNBC cells.

Metabolites doi: 10.3390/metabo16090612

Authors: Peipei Wei Binbin Du Xingen Zhang Guohui Li Fang Li Ping Zhang Jiayi Dou Li Zou Junjie Yang Yujuan Wang Jun Dai

Background: Dendrobium huoshanense flowers are a potentially valuable medicinal resource, but metabolic variation among different germplasm materials remains incompletely characterized. This study aimed to characterize metabolic and transcriptional differences among three D. huoshanense germplasm materials and to explore gene–metabolite associations related to phenylpropanoid metabolism. Methods: Untargeted LC–MS metabolomics and transcriptome sequencing were used to comparatively profile flowers of three D. huoshanense germplasm materials (DH-1, DH-2, and DH-3). Results: Metabolomic profiling detected 4357 metabolic features putatively assigned to 12 chemical classes and revealed clear separation among the three materials. A total of 1292, 1861, and 2035 differentially accumulated metabolic features were identified in DH-1 vs. DH-2, DH-1 vs. DH-3, and DH-2 vs. DH-3, respectively. Transcriptome analysis identified 33,665 expressed genes, including 3365, 5244, and 5964 DEGs in the respective pairwise comparisons. Phenylpropanoid biosynthesis was recurrently enriched across all three DEG comparisons. Exploratory gene–metabolite analysis identified associations involving phenylpropanoid–related candidate genes, including PAL, C4H, 4CL/4CL–like, HCT–like, CSE–like, COMT/OMT–like, and CAD, with the DH-2 vs. DH-3 comparison showing the most extensive molecular differences. Conclusions: The three D. huoshanense germplasm materials exhibited distinct metabolic and transcriptional profiles, providing candidate genes and metabolic features for subsequent targeted validation.

Metabolites doi: 10.3390/metabo16090611

Authors: Zhijiao Song Guixiang Li Wenhua Chen Qing Liu Yantong Teng

Background: Bauhiniavariegata is a plant with considerable application potential owing to its combined ornamental, edible, aromatic, and medicinal values. However, research on this species remains limited and superficial both domestically and internationally, and systematic investigation of floral volatile organic compounds (VOCs) is still lacking. Methods: Through integrated metabolome and transcriptome analyses. Results: This study first comprehensively characterizes the VOC composition, floral scent profile, key aroma components, and the molecular mechanisms underlying VOC variation during anthesis in floral buds and flowers of B. variegata. A total of 1214 volatile compounds were identified across buds and flowers, including 239 odor-active compounds and 35 differential odor-active compounds. Flavor statistics revealed that the floral scent profile of B. variegata is dominated by fruity, sweet, floral, green, woody, herbal, citrus, phenol, fresh, and spicy notes. Compared to floral buds, most differential odor-active compounds were markedly upregulated in flowers, including key floral aroma constituents such as phenylacetaldehyde, rose oxide, (Z)-β-ocimene, 2-methylbenzaldehyde, and melon heptenal. Conversely, (R)-(+)-citronellal, which possesses defensive functions, and the bitter-tasting compound 1-methyl-4-nitro-benzene were significantly downregulated in flowers, reflecting a shift from a defense-oriented mode at the bud stage to an attraction-oriented mode at anthesis. Upregulation of phenylalanine/histidine ammonia-lyase, acyl-CoA synthetase, and squalene synthetase genes and downregulation of copper amine oxidase, O-methyltransferase, and aldo–keto reductase genes synergistically promoted accumulation of floral aroma compounds such as phenylacetaldehyde and facilitated the floral transition. Conclusions: This study provides important data support for understanding the ecological interactions between B. variegata floral scent and its pollinators, as well as the molecular mechanisms governing floral scent formation. Furthermore, it contributes to the application of B. variegata in landscaping, edible flower utilization, and fragrance development.

Metabolites doi: 10.3390/metabo16090610

Authors: Mete Hakan Karalök Bağnu Dündar Ayhan Parmaksız Tugba Elgün Sevgi Koçyiğit Sevinç Asiye Gök Yurttaş

Objective: Anti-Müllerian hormone (AMH) is the most widely used biochemical marker of ovarian reserve, but its associations with other reproductive hormones are usually examined one hormone at a time and without accounting for values reported at the assay floor. This study examined the associations between serum AMH and age, follicle-stimulating hormone (FSH), luteinising hormone (LH), estradiol, progesterone and prolactin in women attending a tertiary gynaecology clinic. Materials and Methods: In this retrospective, cross-sectional, single-centre study, the records of women who underwent serum AMH testing together with a reproductive hormone panel between 1 January 2020 and 31 December 2025 were reviewed. Women with polycystic ovary syndrome, primary ovarian insufficiency, pregnancy or previous ovarian surgery were excluded. Bivariate associations were assessed with Pearson and Spearman correlation coefficients on raw and Box–Cox-transformed variables. Because 12.82% (n = 15) of AMH results were left-censored at the analytical reporting floor of 0.02 ng/mL, a multivariable Tobit model with the censoring limit specified on the Box–Cox-transformed scale (λ = 0.309, threshold = −2.270) was fitted, with predictors selected a priori on clinical grounds. Sensitivity analyses examined the influence of high AMH values, an alternative transformation of AMH, and flexible modelling of age. Results: The analysis included 117 women aged 18–45 years. Mean AMH was 2.22 ± 2.24 ng/mL [median 1.61 ng/mL (Q1–Q3, 0.36–3.20)]. In bivariate correlation analysis, AMH showed statistically significant inverse associations with age (r = −0.376, p < 0.001) and FSH (r = −0.445, p < 0.001), whereas LH, estradiol, progesterone, and prolactin showed no statistically significant correlations with AMH (all p > 0.10). In the multivariable Tobit model, only age (β = −0.068; 95% CI [−0.117, −0.018]; p = 0.007) and transformed FSH (β = −3.582; 95% CI [−4.846, −2.317]; p < 0.001) remained independently associated with transformed AMH. LH, estradiol, progesterone, and prolactin were not independently associated with AMH in the multivariable model (all p > 0.05). The standardised association was greater for transformed FSH (β* = −0.559) than for age (β* = −0.234). The associations of age and FSH were consistent in direction and magnitude across all sensitivity analyses. Conclusions: In this selected clinical sample, age and FSH were the only variables independently associated with serum AMH after mutual adjustment. LH, estradiol, progesterone, and prolactin showed no statistically significant associations with AMH in either the unadjusted bivariate analyses or the multivariable model. The absence of independent associations for these hormones should be interpreted cautiously because hormone sampling was not standardised to menstrual cycle day, which may have introduced measurement variability. These findings describe associations within the study population and do not establish causal relationships, reference intervals, or a basis for modifying testing strategies. The analysis also highlights the importance of accounting for left-censoring when modelling AMH data containing a substantial proportion of results at the assay reporting floor.

Metabolites doi: 10.3390/metabo16090609

Authors: Sara Greenfield C. Titus Brown Oliver Fiehn

Objective: Despite most often used in research and medicine, it is unclear how well blood metabolite levels can reflect metabolic changes in tissues. Methods: We used untargeted metabolomic data from plasma, liver, gastrocnemius muscle, kidney, inguinal fat, and gonadal fat tissues from mice treated with lifespan-extending interventions: caloric restriction, rapamycin, canagliflozin, 17-α estradiol, and acarbose. Results: For each tissue, about 37% of the identified metabolites were also found in plasma and used for comparative analyses. We found that plasma metabolites do not primarily reflect the metabolome of a single tissue type. Caloric restriction caused the highest proportion of significantly altered metabolites detected in both organs and blood. Unsaturated triacylglycerols, diacylglycerols, amino acid-derivatives, and sphingomyelins had the most concordant overlaps between four tissues and plasma, followed by carbohydrates with three tissue/plasma intersections. Concordant plasma/tissue changes were found more pronounced in male mice than in female mice. Surprisingly, many compounds that responded similarly to lifespan-extending treatments in tissues and in plasma originated from dietary compounds, specifically, ergothioneine, DHA-containing fats, and diacylglycerides. Ergothioneine, previously associated with healthy aging in humans, showed concordant treatment responses in blood and all tissues except gonadal fat. DHA-containing fats showed consistent regulation between blood and inguinal and gonadal adipose tissue. The kidneys showed coherent trends with blood metabolite levels for LPC 15:0, LPC 17:0, and pinitol. Under treatments with strong life-extending effects, 1,5-anhydro-glucitol blood levels were co-regulated with both liver and kidney levels. Conclusions: Overall, plasma can only serve as limited proxy for tissue metabolism. Yet, several potential blood biomarkers were discovered as concordant in plasma and tissues in lifespan-extending interventions.

Metabolites doi: 10.3390/metabo16090608

Authors: Miriam Longo Marica Meroni Erika Paolini Paola Dongiovanni

With the advent of the new definition, fatty liver disorders have been reframed into metabolic dysfunction-associated steatotic liver disease (MASLD), alcohol-related liver disease (ALD), and the mixed phenotype referred to as MetALD (MASLD and increased alcohol intake). This change reflects the real-world clinical practice, where metabolic dysfunction and alcohol frequently coexist and synergize to increase risks of steatohepatitis, fibrosis, and hepatocellular carcinoma (HCC). While conventional non-invasive tests (NITs) remain the backbone of risk stratification, lipidomics and metabolomics can capture biological information on disease mechanisms and may improve early detection and prognosis. Here, we summarize the current evidence on circulating and tissue lipidomic and metabolomic signatures across MASLD, ALD and MetALD, discuss how the new definitions affect clinical risk assessment, and highlight recent studies which partially distinguish molecular fingerprints for mixed etiology disease.

Metabolites doi: 10.3390/metabo16090607

Authors: Jiahua Liu Xiqiao Gao Hao Liang Jiahao Ye

Objectives: Emotional tears are generated in a distinct neurophysiological context from reflex tears, but their metabolic composition remains poorly understood. Methods: This exploratory paired targeted-metabolomics study compared emotional and reflex tears collected from 22 healthy volunteers using a 600-multiple-reaction-monitoring platform. Among 412 detected metabolites, 344 were retained after data preprocessing. Paired statistical analysis prioritized 23 candidate metabolites based on the combined criteria of unadjusted p-value, fold change, and consistency of within-subject change. Results: Seven candidates were lower, and 16 were higher in emotional tears. Salicylic acid was retained in the descriptive candidate set but excluded from the primary machine-learning analysis because a contribution from the reflex-tear induction procedure could not be ruled out. Among the remaining 22 candidates, 4-hydroxy-3-methylbenzoic acid, vanillic acid, cytidine-5′-monophosphate, and hydroxyphenyllactic acid were consistently ranked among the leading features. A fixed four-metabolite combination achieved an area under the receiver operating characteristic curve of 0.864 (95% CI, 0.756–0.957) under ordinary leave-one-subject-out cross-validation. When candidate screening, feature selection, and model fitting were repeated within each training fold, the best nested pipeline achieved an area under the curve of 0.725 (95% CI, 0.603–0.843). Pathway mapping further linked the candidate metabolites to histidine, tyrosine, fatty-acid, ether-lipid, and ubiquinone-related metabolism. Conclusions: These findings demonstrate measurable within-subject metabolic differences between emotional and reflex tears and identify a focused set of candidate metabolites for future validation. Because no individual metabolite remained significant after false discovery rate correction and no independent validation cohort was available, the candidate signals and discrimination models should be confirmed in larger, independently collected cohorts.

Metabolites doi: 10.3390/metabo16090606

Authors: Phato Avheani Matsheketsheke Nakisani Babra Moyo Bono Nethathe

Background/Objectives: Traditional medicinal plants play a crucial role in infants’ healthcare in underdeveloped countries and communities where access to modern medicine is limited. In Limpopo, the Vhavenda tribe has a custom of feeding babies as young as a day old with tshiunza, a very light and warm, soft porridge prepared in medicinal plant extracts, believed to help treat various ailments and boost the immune and digestive systems. However, reports of negative side effects, including vomiting, haematuria, renal inflammation and hospitalisations, highlight safety concerns of these traditional practices. This study aimed to profile the chemical composition of soft porridge samples prepared in aqueous extracts of medicinal plants reported to be commonly used in infants’ food in the Venda region of Limpopo; Annona senegalensis (roots), Piliostigma thonningii (leaves), Carissa edulis (roots) and Bauhinia galpinii (leaves). Methods: Ultrahigh-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-qTOF-MS) was used for metabolic profiling, followed by putative compound identification using feature-based molecular networking and a compound fragmentation predictor tool, SIRIUS. To evaluate the toxicity of these plants, a cytotoxicity assay was conducted using plant extracts on African monkey kidney cells. Results: Different classes of polyphenols, including flavonoids (flavan-3-ols and flavonols) and hydroxycinnamoyl amides, were among the dominant compounds putatively identified in the methanolic extracts of soft porridge prepared from the four plant extracts. A. senegalensis aqueous extracts were inactive (IC50 > 500 µg/mL, 55.57% viability at 500 µg/mL), while P. thonningii was non-cytotoxic (105–132% viability within the tested concentration range). Conclusions: Although no toxic compounds were identified and no cytotoxic potential was observed in the tested plant extracts, this does not translate to the safe use of medicinal plants in infants’ food. Therefore, future studies should conduct pharmacological and toxicological evaluations to better understand the potential risks of these plant extracts to infant health.

Metabolites doi: 10.3390/metabo16090605

Authors: Onyedikachi Ubani Veronica M. Ngole-Jeme

Background: Crude oil refinery waste sludge (COWS) ranks among the most compositionally complex and ecotoxicologically hazardous industrial residues. Although bulk total petroleum hydrocarbon (TPH) and summed polycyclic aromatic hydrocarbon (PAH) removal are routinely reported, the metabolite-level biochemical fate of individual petrogenic compounds, spanning ring dihydroxylation, catechol cleavage, and entry into central carbon metabolism, remains largely unmapped under co-composting with diverse animal manures. Objectives: This study aimed to construct a metabolite-resolved, microbially anchored biochemical fate map of crude oil sludge during co-composting. Methods: Aerobic microcosms combining crude oil sludge, garden soil, and a wood-chip bulking agent were amended separately with poultry, horse, cow, or swine/pig manure alongside an unamended control and then incubated at 22 °C for 300 days. Analyses integrated untargeted gas chromatography-mass spectrometry (GC-MS) metabolomics, targeted PAH quantification (EPA Methods 3541/8270), 16S rRNA gene amplicon sequencing (Illumina MiSeq, V1–V3, paired-end 300 bp), physicochemical monitoring, and culture-dependent isolation, with National Institute of Standards and Technology (NIST) Mass Spectral library annotation. Results: GC-MS resolved 1169 metabolite features across 17 samples, comprising 538 annotated compounds within 11 chemical classes and 631 unknowns, of which 151 recurred in at least 10 samples. Petrogenic markers (n-alkanes C14–C36, hopanoids, steranes, and alkylated dibenzothiophenes) and ring-cleavage intermediates (2-hydroxyfluorene, 1,4-naphthoquinone, phenanthrene-methanol, benzenediols, butanedioic acid, fatty alcohols C16–C20) elucidated a four-stage degradation cascade consistent with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways map01220 and map00624. PAH mean-removal ranked swine/pig (88.0%) > horse (87.0%) > poultry (80.5%) > cow (79.1%) > control (68.2%). Sequencing recovered 2969 operational taxonomic units (OTUs) enriched in Pseudomonas, Achromobacter, Stutzerimonas, Dietzia, Gordonia, and Mycobacterium, with Pseudomonas dominating high-removal systems; respiration peaked at 18.7 mg CO2-C g−1 in poultry treatments. Conclusions: This work establishes a metabolite-resolved map linking hydrocarbonoclastic taxa to separate degradation steps. The co-occurrence of oxygenated PAH intermediates with decreasing parent PAH concentrations serves as an indicator of transformation processes and may assist in identifying potential residual-risk signals, thereby supporting remediation evaluation and process optimization.

Metabolites doi: 10.3390/metabo16090604

Authors: Tobias Schmidt Maximilian Sombke Helena U. Zacharias Peter J. Oefner Rainer Spang Wolfram Gronwald

Background: In one-dimensional NMR spectra of complex biofluids such as urine and plasma, extensive signal overlap obscures individual metabolite signals. Resolving this overlap by deconvolution is only the first step: turning a set of spectra into a table for subsequent statistical analysis also requires the alignment of signals across samples and their integration into a single feature matrix. Methods: Here, metabodeconplus is presented, an R package that unifies this entire path into a single reproducible end-to-end workflow. From raw one-dimensional spectra, it deconvolutes overlapping signals, aligns resulting signals across samples, and integrates them into a data matrix for built-in sample classification or downstream statistical analysis. Automated parameter optimization removes manual tuning, and a Rust computational backend with parallelization leads to fast runtimes. Results: On the simulated Sim3 spectra, a combined score of correctly identified signals and reconstruction accuracy (maximum 1) rose from 0.712 for the predecessor package to 0.801 for metabodeconplus. For the urinary AKI dataset, metabodeconplus reached a classification accuracy of 73.7 ± 2.20% and an AUC=0.827±0.025, which is comparable to the binning baseline. An advantage is the potential unambiguous metabolite assignment of the deconvoluted signals. Conclusions: The package is freely available as open source on GitHub and on CRAN.

Metabolites doi: 10.3390/metabo16090603

Authors: Adrianna Dylik Mikołaj Musiał Michał Radke Dominika Tuzimek Dominika Rogoża Bartosz Czekała Anna Wawrzyniak Nadzeya Zhuk Katarzyna Skrypnik Damian Skrypnik

Background: Statins are among the most frequently prescribed medications worldwide, with proven benefits in reducing all-cause and cardiovascular mortality, preventing major adverse cardiovascular events (MACEs), and lowering healthcare costs. Despite their well-established safety and effectiveness, discontinuation of statin therapy remains a common problem in both primary and secondary prevention. Methods: A systematic literature review was conducted in accordance with PRISMA guidelines and registered in the PROSPERO database (CRD420261295172). Original studies published since 1 January 2014 were identified through searches of PubMed (MEDLINE) and Google Scholar using predefined keywords. Methodological quality and risk of bias of the included observational studies were evaluated using the Newcastle-–Ottawa Scale (NOS) and Joanna Briggs Institute (JBI) Critical Appraisal Tools. Results: Synthesized data from 29 included studies indicate that up to half of patients discontinue statin therapy within the first year (discontinuation rates ranging from 27.1% to 47.0% in primary prevention and 18.5% to 32.4% in secondary prevention), with rates increasing over time. Major factors contributing to non-adherence include fear of side effects, particularly statin-associated muscle symptoms (SAMSs), misinformation, limited patient–physician communication, socioeconomic barriers, and polypharmacy. Discontinuation is associated with significantly higher all-cause and cardiovascular mortality (Hazard Ratios ranging from 1.30 to 4.65), higher rates of cardiovascular and cerebrovascular events, worsening metabolic outcomes, reduced quality of life, and greater healthcare expenditures. Conclusions: Improving adherence requires better patient education, addressing misconceptions, strengthening patient–physician relationships, and optimising treatment regimens. A multidisciplinary approach is needed to prevent unjustified discontinuation and improve long-term clinical outcomes.

Metabolites doi: 10.3390/metabo16090602

Authors: Guilherme Araújo Sara Domingues Gabriela Jorge da Silva Tiago Lima

Marked interindividual variability in drug response remains a major challenge in clinical pharmacology and cannot be fully explained by host genetics alone. Increasing evidence indicates that the gut microbiota constitutes an additional determinant of drug efficacy and toxicity through bidirectional interactions with pharmacological therapies. This recognition has led to the emergence of pharmacomicrobiomics, a field that investigates how microbial communities influence drug disposition and response, and how drugs, in turn, alter the microbiome. Microbiome-mediated effects on drug efficacy and toxicity have been described across several therapeutic areas, including oncology, multiple sclerosis, and type 2 diabetes mellitus. Although these findings are promising, most mechanistic evidence derives from preclinical and animal studies, with relatively limited validation in controlled clinical trials. Strategies to modulate the gut microbiota, including prebiotics, probiotics, and faecal microbiota transplantation, have shown preliminary promise in optimising drug efficacy and reducing adverse effects, although methodological heterogeneity and incomplete mechanistic understanding limit their current clinical application. The identification of robust microbiome-derived biomarkers and the integration of multi-omics approaches, particularly metabolomics, are expected to accelerate the translation of pharmacomicrobiomics into precision medicine. This review summarises current evidence regarding microbiome–drug interactions, the mechanisms underlying microbiome-mediated modulation of pharmacokinetics and pharmacodynamics, emerging therapeutic strategies, and the challenges that remain before pharmacomicrobiomics can be implemented in clinical practice.

Metabolites doi: 10.3390/metabo16090601

Authors: Huijun Wang Miaoyuan Zhang Kangkang Gao Yaping Zhou Aoxing Xiao Jinyi Liu Xiangjun Qiu

Background: Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) is a progressive condition predisposing to cirrhosis and hepatocellular carcinoma, with prevalence rising globally. Yet effective therapies remain limited. Wuzhi Dripping Pills (WZDP), derived from Schisandra sphenanthera, possess hepatoprotective and anti-inflammatory properties, though their efficacy against MASLD remains unexplored. Methods: Network pharmacology predicted active ingredients and targets, followed by KEGG enrichment analysis. A high-fat diet MASLD rat model was established to evaluate WZDP effects on weight, liver index, and serum markers. Serum metabolomic profiling via UPLC-MS/MS, combined with multivariate statistics and KEGG annotation, identified perturbed pathways. Results: Twelve bioactive compounds and 434 WZDP targets were retrieved, of which 74 intersected with 838 MASLD-associated genes. Enrichment implicated AGE-RAGE, insulin resistance, and HIF-1 signaling. In vivo, WZDP significantly improved anthropometric and biochemical parameters. Metabolomic analysis distinctly separated WZDP-treated from model groups, highlighting phospholipase D signaling as a key differential pathway. Conclusions: This integrative approach confirms that WZDP confers therapeutic benefits in MASLD through coordinated regulation of neuroactive ligand–receptor interaction, bile acid biosynthesis, phospholipase D signaling, and arginine–proline metabolism. Our findings furnish a molecular and metabolic rationale for further mechanistic studies and drug discovery efforts.

Metabolites doi: 10.3390/metabo16080600

Authors: Pan Li Jing Mao Xianglin Hu Yujiao Hu Xiaoke Zhang Qian Zheng Xiaoying Hou Yuchen Liu Min Huang

Machine learning (ML) integrated with metabolomics has emerged as a promising strategy to advance precision pharmacotherapy, enabling data-driven prediction of drug response. This review provides an overview of commonly applied ML methodologies in metabolomics-based pharmacological studies, including supervised models (Random Forest, Extreme Gradient Boosting, Support Vector Machine, Logistic Regression, K-Nearest Neighbors), unsupervised models (K-Means Clustering, Principal Component Analysis), and deep learning approaches. We summarize recent progress in the application of metabolomics-driven ML to personalized medication, with a focus on drug dosage optimization, therapeutic efficacy prediction, and adverse drug reaction assessment. Despite these advances, significant challenges remain, including limited explainability, insufficient prospective clinical validation, lack of standardization and reproducibility, and data dimensionality and quality issues. Addressing these issues will be essential for the clinical translation of ML-metabolomics integration. Looking ahead, continued methodological innovation, large-scale multi-center prospective validation, and integration with other omics platforms will be key to unlocking the full potential of metabolomics combined with ML in precision healthcare.

Metabolites doi: 10.3390/metabo16080599

Authors: Vladislav Aksenov Angelina V. Kurchatova Alexey Golovanov Veronika Ulasenko Olga Zubkova Alexander Zhuravlev Ekaterina Makishvili Nikolay E. Kushlinskii Hartmut Kuhn Igor Ivanov

Background: Mammalian arachidonic acid lipoxygenases (ALOXs) are non-heme iron-containing enzymes that oxygenate polyunsaturated fatty acids (PUFAs) with at least two isolated double bonds to hydroperoxy derivatives. The patho-physiological roles of these enzymes in inflammatory, hyperproliferative, and neurological diseases have made them promising targets for pharmacological interventions. Unfortunately, the expression levels of ALOX isoforms in mammalian cells are very low, which makes functional characterization of native enzymes and the development of isoform-specific inhibitors challenging. Methods: Here, we developed a unifying experimental protocol for fed-batch fermentation of mammalian ALOX isoforms in a bioreactor, followed by purification of the recombinant proteins and their functional characterization. Results: Our methodological protocol allowed the preparation of mg amounts of catalytically active human ALOX15, mouse Alox15 and human ALOX15B. The purified proteins are suitable for high-throughput inhibitor screening assays but can also be used as antigens for the preparation of isoform-specific antibodies and for direct structural analyses. Antibodies cross-reacting with human ALOX15 and ALOX15B have been detected in the blood of patients suffering from colorectal cancer. Conclusions: Our functional ALOX data stresses the catalytic differences between mouse and human ALOX15 orthologs, and these catalytic peculiarities need to be considered when the results of mechanistic studies obtained in mouse models of human diseases are transferred to the human situation.

Metabolites doi: 10.3390/metabo16080598

Authors: Ruibing Xu Qingshan Li Gengrui Zhu Yi Chen Gaoyang Zhang

Background: Wampee (Clausena lansium (Lour.) Skeels) is an understudied Rutaceae crop native to southern China, whose fruit features a complex aroma profile with simultaneous sour, sweet, bitter and astringent notes. Although bioactive compounds including flavonoids, alkaloids and volatile oils in wampee fruit have been partially characterized, the tissue-specific metabolic and transcriptional basis underlying its distinctive aroma formation remains largely unclear. Methods: We performed an integrated volatile metabolomic and transcriptomic analysis on the pericarp (peel) and flesh of wampee fruit across three cultivars (Shanyellowpi, Heijingang, Bingtangxin). Volatile metabolites were profiled via headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS), and transcriptome profiles were generated by RNA-Seq. Multi-omics integration was conducted using Procrustes analysis, gene–metabolite correlation network construction and weighted gene co-expression network analysis (WGCNA). Results: A total of 288 volatile metabolites were identified, representing the most comprehensive volatile inventory for C. lansium reported to date. Principal component analysis and partial least squares discriminant analysis revealed distinct volatile profiles between pericarp and flesh; terpenoids were the dominant chemical class, accounting for 71.56–91.48% of total volatiles in pericarp and 61.88–67.22% in flesh. Notably, organoheterocyclic compounds were significantly enriched in Shanyellowpi flesh (40.45%), forming a cultivar-specific metabolic signature absent in the other two cultivars. Transcriptomic analysis showed that phenylpropanoid biosynthesis was the most significantly enriched pathway among differentially expressed genes, followed by monoterpene biosynthesis and sesquiterpenoid biosynthesis. Procrustes analysis demonstrated a strong global concordance between the two omics layers (M2 = 0.535, p < 0.001). Gene–metabolite correlation networks identified terpene synthase (TPS) genes (HP075360, HP217350) and oxidoreductase genes (SOD1, GST, 10HGO) as candidate co-regulators of terpenoid biosynthesis. WGCNA further prioritized TPS genes, cytochrome P450 genes and MYB transcription factor genes as key regulators driving volatile metabolic divergence between tissues. Conclusion: This study provides a comprehensive volatile and transcriptomic atlas of wampee fruit, and identifies tissue-specific and cultivar-specific metabolic signatures as well as their candidate regulatory genes. These findings advance our understanding of quality differentiation in Rutaceae fruits and lay a foundation for molecular breeding and flavor improvement of wampee.

Metabolites doi: 10.3390/metabo16080597

Authors: You Sun Bowei Li Zhengjiang Qian

Nicotinamide adenine dinucleotide (NAD+) is a fundamental metabolic cofactor and signaling molecule that supports redox reactions, DNA repair, chromatin regulation, stress adaptation, inflammation, and neuronal maintenance. Age-associated NAD+ decline has been implicated in brain aging and neurodegenerative disorders, but the causal node and limiting compartment differ across tissues and disease states. Nicotinamide mononucleotide adenylyltransferase 1 (NMNAT-1) catalyzes the final step in NAD+ biosynthesis and represents the major nuclear isoform of the mammalian NMNAT family. Direct human genetic evidence establishes NMNAT-1 as a causal gene in inherited retinal degeneration, whereas evidence linking endogenous NMNAT-1 to broader brain aging or sporadic neurodegeneration is mainly convergent preclinical, preliminary, or indirect. Beyond NAD+ synthesis, biochemical and Drosophila studies suggest possible chaperone-like and proteostasis-supporting functions, but a separable NAD+-independent function of endogenous mammalian NMNAT-1 has not yet been established in vivo. Here, we review the molecular structure, localization, and regulation of NMNAT-1, emphasizing calibrated distinctions among catalytic nuclear NAD+ supply, engineered axonal protection, pathway-adjacent NAD+ interventions, and putative non-catalytic protection. We further discuss how NMNAT-1 dysfunction may contribute to aging-associated genomic instability, neuroinflammation, synaptic impairment, retinal degeneration, selected neurodegenerative models, and glioma biology. Finally, we evaluate therapeutic strategies targeting NMNAT-1 and NAD+ pathways, noting that no human trial has yet established efficacy for an NMNAT-1-directed neurological therapy. A compartment-aware and evidence-stratified view is therefore essential for translating NMNAT-1 biology into interventions for age-related neural disease.

Metabolites doi: 10.3390/metabo16080596

Authors: Alice S. Ryan Guoyan Li Colleen Lynch Sausan M. Jaber Galya Bigman

Background/Objectives: Ciliary neurotrophic factor (CNTF), a pleiotropic cytokine with central and peripheral actions, has been implicated in obesity, insulin resistance, and aging, yet its role in skeletal muscle and cardiometabolic health remains unclear. This study examined skeletal muscle CNTF mRNA expression in relation to adiposity and glucose tolerance in older adults and assessed the effects of six-month weight loss (WL) and aerobic exercise training (AEX) interventions on its expression. Methods: Sixty-seven adults (age, 60.75 ± 7.58) underwent a vastus lateralis muscle biopsy, VO2max testing, dual-energy X-ray absorptiometry (DXA) scan, a resting metabolic rate (RMR) and substrate oxidation assessment, and a 3-h oral glucose tolerance test with glucose and insulin area under the curve (AUC) calculation. A subset completed 6 months of either a WL program (n = 21) or a supervised 3 times per week AEX intervention (n = 20). Results: At baseline, skeletal muscle CNTF expression was inversely associated with RMR, fat oxidation, 3-h glucose AUC, and insulin AUC, but not with BMI, percent body fat, or VO2max. WL reduced body weight, fat mass, glucose AUC, insulin AUC, and basal muscle CNTF expression, whereas AEX increased VO2max and reduced fat mass without altering body weight, glucose AUC, insulin AUC, or basal CNTF. Insulin-stimulated CNTF expression increased after both WL and AEX, and the intervention-induced change in glucose disposal (M) was positively associated with the change in insulin-stimulated CNTF. Conclusions: In older adults, skeletal muscle CNTF is associated with glucose intolerance but is not associated with body composition or aerobic fitness. Improvements in insulin sensitivity are associated with increases in muscle CNTF during hyperinsulinemia. Further work is needed to determine the mechanism for muscle CNTF change during hyperinsulinemia after weight loss and exercise training, and its role in glucose metabolism and obesity in older adults.

Metabolites doi: 10.3390/metabo16080595

Authors: Yasin Karadag Ozgur Ozdemır Gulseren İpek Karadag Erinç Tekin

Background/Objectives: Gestational diabetes mellitus (GDM) is one of the most common metabolic disorders during pregnancy and is associated with adverse maternal and neonatal outcomes. Xenopsin-Related Peptide-1 (XRP-1) has recently emerged as a potential regulator of glucose metabolism and insulin homeostasis. This study evaluated serum XRP-1 levels and assessed their diagnostic performance in women with GDM. Methods: In this prospective observational study, 120 pregnant women between 24 and 28 weeks of gestation were enrolled, including 60 women with GDM and 60 healthy controls. Serum XRP-1 concentrations were measured using an enzyme-linked immunosorbent assay (ELISA). Clinical, biochemical, and obstetric characteristics were compared between groups. Receiver operating characteristic (ROC) curve analysis and multivariable logistic regression were performed to evaluate the diagnostic performance and independent association of XRP-1 with GDM. Results: Serum XRP-1 levels were significantly higher in women with GDM than in healthy controls (3.45 ± 0.47 vs. 2.87 ± 0.46 ng/mL, p = 0.003). ROC analysis demonstrated moderate discriminatory performance for XRP-1 in identifying GDM (AUC = 0.658, 95% CI: 0.560–0.750), with an optimal cut-off value of 3.12 ng/mL, corresponding to 63.3% sensitivity and 61.7% specificity. Multivariable logistic regression demonstrated that serum XRP-1 remained independently associated with GDM after adjustment for maternal age, pre-pregnancy body mass index, gestational weight gain, and parity (adjusted OR 2.74, 95% CI 1.31–5.73; p = 0.007). HbA1c and C-reactive protein levels were also significantly higher in the GDM group. Conclusions: Serum XRP-1 concentrations were independently associated with GDM and demonstrated moderate diagnostic accuracy. Although XRP-1 cannot replace established diagnostic methods, it may represent a complementary biomarker for identifying gestational dysglycemia. Larger multicenter studies are required to validate its clinical utility.