Search Results (3,482)

Background/Objectives: Vitamin D is a pleiotropic substance that plays a pivotal role in the human body. Several studies have suggested sex-based differences in vitamin D metabolism. Recently, the vitamin D metabolite ratio (VMR), defined as the ratio of serum 24,25-dihydroxyvitamin D [24,25(OH)₂D] to 25-hydroxyvitamin D [25(OH)D], has been identified as a novel marker that may more accurately reflect vitamin D status than serum 25(OH)D alone. This study aimed to assess whether significant variability exists in the VMR across different demographic groups. Methods: Residual serum samples and laboratory data were collected from healthy adults who underwent health checkups at a health center in Japan. Serum concentrations of 25(OH)D₃ and 24,25(OH)₂D₃ were simultaneously measured using a fully automated liquid chromatography–tandem mass spectrometry (LC-MS/MS) system. Factors affecting the levels of 25(OH)D₃, 24,25(OH)₂D₃, and VMR were analyzed using multiple regression analysis and the stabilized inverse probability weighting method. Results: Tentative reference intervals for 24,25(OH)₂D₃ were found to be 0.3–3.0 ng/mL for males and 0.2–2.3 ng/mL for females. Those for VMR were found to be 3.0–11.8% for males and 2.5–10.4% for females, demonstrating statistically significant but moderate differences between the sexes. These differences remained significant even after adjusting for baseline covariates including 25(OH)D₃. However, the standard deviation ratios were less than 0.5. An estimated glomerular filtration rate and an age of ≥50 years were positively associated with 24,25(OH)₂D₃ and VMR, whereas diabetic status was negatively associated with these markers. Conclusions: This study revealed moderate sex-based differences in markers of vitamin D metabolism. Although further research is needed to determine whether these differences are consistent across other populations, future epidemiology and clinical interventions should take into consideration of the sex-based differences in vitamin D metabolism. Full article

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. Full article

Herbs and spices are traditionally added to food in cuisines around the world. Antioxidant activity and content of bioactive compounds were compared in oregano, sage, basil, rosemary, and herbal mixtures. After optimization of ultrasound-assisted extraction of the ethanol–water extracts, the content of reducing compounds of the herb extracts was tested using the Folin–Ciocalteu method, and antioxidant capacity was evaluated with ABTS (2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt) and DPPH (2,2-diphenyl-1-picrylhydrazyl radical) assays. Oregano showed the highest antioxidant activity in all tests used (2.6 mg of gallic acid equivalent (GAE) per mL in the Folin–Ciocalteu test, 4.6 mg Trolox/mL in the ABTS assay, and 3.3 mg Trolox/mL in the DPPH assay) while rosemary had the lowest antioxidant activity (1.2 mg GAE/mL (Folin–Ciocalteu), 1.5 mg Trolox/mL (ABTS), and 1.3 mg Trolox/mL (DPPH)). Apart from antioxidant properties, the content of bioactive compounds was determined with the use of high-performance liquid chromatography–tandem mass spectrometry (LC-MS/MS). It was found that in all tested Lamiaceae herbs and the herbal mixes, rosmarinic acid widely predominates as a major non-volatile phenolic constituent, and its content varies from 1121 µg/g in rosemary to 10,255 µg/g in herbes de Provence. High concentrations were also observed for quinic acid in both oregano and rosemary. Interestingly, the concentrations of rosmarinic acid in the group of herbs studied are positively correlated with the results obtained in the Folin–Ciocalteu, ABTS, and DPPH tests (Spearman’s correlation coefficient 0.7030, 0.6657, and 0.7188, respectively), whereas no such correlation is observed for quinic acid. Overall, the findings indicate that these herbs share a common hydroxycinnamate-based phytochemical framework but display clear species-specific differences reflecting their intrinsic metabolism. Furthermore, the concentration of 3-caffeoylquinic acid in the Sicilian herbs (1021 µg/g) was approximately 10 times higher compared to the samples of Dalmatian herbs, herbes de Provence, and pure herbs, which demonstrates the unique chemical composition of that mixture, including the presence of dried tomatoes and tarragon, which were not included in other tested herbal mixtures. Full article

Rare surviving examples of late eighteenth-century French dress are exceptional. The robe à la circassienne examined in this study is preserved in an unusually intact state, likely due to its small size, which limited its suitability for later wearers, and the rapid shift in fashion that further reduced incentives for alteration or reuse. Discrepancies between the garment’s present coloration, early cataloguing records, and a non-matching reproduction petticoat prompted an investigation into its original appearance. The dress features a ground fabric woven with a pink warp and white weft, accented by vertical multicolored stripes. Organic colorants were analyzed using liquid chromatography–diode array detection–quadrupole time-of-flight mass spectrometry (LC-DAD-qToF-MS), and elemental composition was determined by X-ray fluorescence (XRF) spectroscopy. The dark pink stripe contained safflower (Carthamus tinctorius), a highly fugitive dye. The blue and green stripes showed mixtures of weld (Reseda luteola), an indigoid dye (Indigofera spp.), and Brazilwood (Caesalpinia or Haematoxylum spp.), consistent with historical recipes for bronze and wood shades. Brazilwood and an orchil dye detected in the ground fabric suggest an original reddish-purple hue rather than the pale pink visible today. Arsenic found in the pink fabric and trim provides the first material evidence for the French orchil-dyeing process known as Parelle d’Auvergne. By integrating analytical results with historical dye recipes and curatorial records, this study proposes a hypothesis for the garment’s original appearance and demonstrates how combined scientific and historical approaches deepen our understanding of fragile textile artifacts and the factors that shaped their creation and survival. Full article

Bacteria sense, respond, and adapt to rapidly changing environments through highly sensitive intracellular networks. Among them, cyclic-di-GMP (c-di-GMP) is a well-characterized second messenger controlling the transition from planktonic to sessile lifestyles, making it a key determinant of bacterial adaptation and survival. Two main approaches have been developed to investigate c-di-GMP dynamics. The first uses biological biosensors for live-cell monitoring of intracellular c-di-GMP through transcriptional reporters, RNA-based sensors, or protein-based sensors. Their main advantage is real-time, spatiotemporal analysis in living cells, although they generally do not provide absolute quantification. The second approach relies on analytical chemistry, particularly liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), which enables sensitive and accurate quantification of intracellular c-di-GMP across diverse bacterial species. However, the lack of standardized extraction protocols and the need for cell lysis prevent real-time measurements, providing only a snapshot of the total c-di-GMP pool. These approaches are complementary: biosensors reveal dynamic, single-cell responses, whereas LC-MS/MS provides precise global quantification. Nevertheless, most studies rely on only one method, limiting a comprehensive understanding of c-di-GMP biology. Combining both approaches would provide a more integrated view of c-di-GMP signaling and its role in bacterial physiology. Full article

Among pathogenic microbes that can produce toxins, Bacillus cereus plays an important role in foodborne disorders. It is responsible for the production of cereulide, a cyclic dodecadepsipeptide toxin, very stable to heat and acidic pH, that can cause emetic syndromes. The study describes the secondary validation of the ISO 18465 method for food matrices in the concentration range 1–100 μg/kg and the in-house validation of a more sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method for infant formula at the lower concentration levels required by the EFSA since 2026. To improve the applicability of cereulide determination in infant formula, the solid matrix was reconstituted before extraction and the analysis was performed on the SCIEX QTRAP 7500 system. The method showed good linearity (0.010–1.000 μg/L, R² = 1000), a limit of quantification equal to the half-maximum level (LM = 0.054 μg/L, LOQ = 0.025 μg/L), good recovery (100–109%) and acceptable within-laboratory reproducibility (RSD% < 15%). This method provides a reliable analytical tool for cereulide monitoring in a broad variety of food commodities not considered before by ISO 18465 and makes it possible to reach the new lower limits established for infant formula. Furthermore, data on 332 samples involved in suspected food poisoning in Italy between 2019 and 2024 are presented in this retrospective study. Full article

This study used locally sourced black, red, and white quinoa from Zhangjiakou as experimental materials to optimize the preparation process of quinoa antioxidant peptides and identify their sequence characteristics. Protein was extracted using the alkali-soluble acid precipitation method. The optimal hydrolytic enzyme was selected by comparing the hydrolysis effects of eight proteases. Enzymatic hydrolysis conditions were optimized using single-factor experiments and response surface methodology. After purification by Sephadex G-15 gel filtration chromatography, peptide sequences were identified by liquid chromatography–tandem mass spectrometry (LC-MS/MS) combined with De novo sequencing technology. The results showed that black quinoa and alcalase were identified as the optimal raw material and hydrolytic enzyme, respectively. The optimal enzymatic hydrolysis conditions were an enzyme dosage of 4000 U·g⁻¹, hydrolysis time of 3.5 h, temperature of 42 °C, and pH 7.6, under which the average DPPH radical scavenging rate reached 86.61%. The purified G-1 fraction exhibited the highest antioxidant activity. A total of 1549 peptide sequences with confidence ≥90% were identified, among which peptides containing two or more hydrophobic amino acids accounted for 60.62%. This compositional feature is consistent with structural characteristics commonly associated with antioxidant peptides. This study provides a reference for the processing of quinoa-based functional ingredients. Full article

Background: COVID-19 vaccines have proven effective in reducing severe disease and mortality from SARS CoV-2 infection. The underlying molecular mechanisms and alterations in the human serum metabolome influencing the effectiveness and development of immunity remain unclear. Methods: Serum samples were collected from 29 healthy individuals at three time points: prior to vaccination (A), post-first dose (B), and post-second dose (C). Untargeted high-resolution (HR) liquid chromatography coupled with mass spectrometry (LC-MS) was performed on these samples. Metabolites showing significant differential abundance at each time point were identified, and both multivariate and univariate statistical analyses were performed to determine changes associated with the pairwise comparisons, priming (A vs. B), booster (B vs. C), and the overall vaccine effect (A vs. C). Vaccination-specific features were determined after excluding metabolites associated with SARS-CoV-2 IgG seropositivity to better isolate vaccine-driven metabolic changes. Bioinformatics, pathway, and network analyses were conducted using Ingenuity Pathway Analysis (IPA) to identify relevant pathways. Results: Our study identified significant metabolic changes across the three time points. A total of 377 metabolites were identified, of which 59 metabolites, including prostaglandins, eicosanoids, and lipids, were shared across all three groups. The majority of these metabolites showed an initial decrease after the first dose, followed by broad upregulation after the second dose. We identified 1 (downregulated), 34 (26 upregulated and 8 downregulated), and 18 (2 upregulated and 16 downregulated) unique metabolites in the priming, booster, and the overall vaccine effect groups, respectively. L-3-hydroxykynurenine was observed to be significantly reduced by the priming dose effect. By contrast, the booster effect showed decreased myo-inositol 1,3,4,5-tetrakisphosphate, while levels of DL-DOPA, 3-methoxytyrosine, and prostaglandin-esterified phospholipids, including PC(P-16:0/PGF1α) and PE(PGF1α/18:0), increased. On the other hand, the overall vaccine effect revealed decreased cyclic AMP and increased 3′-O-methyladenosine levels. These changes were associated with perturbations in arachidonic acid metabolism, glycerophospholipid metabolism, arginine biosynthesis, and steroid hormone biosynthesis. IPA network analysis identified AKT, TP53, EGFR, and cAMP as key dysregulated nodes. Conclusions: Longitudinal metabolomic profiling demonstrated that COVID-19 vaccination induced distinct but interrelated biochemical changes throughout the vaccination course. The priming dose induced a limited set of early metabolic changes, whereas the booster was associated with more significantly changed metabolites that were involved in lipid, bile acid, steroid, amino acid, and nucleotide pathways. Together, these findings indicate that sequential vaccination is associated with dose-dependent systemic metabolic adaptation, with the booster dose having the largest number of dysregulated metabolites. Full article

The current clinical test for ketosis in dairy cows, which is based on measuring blood β-hydroxybutyrate (BHB) concentrations, is an invasive procedure that impacts herd welfare. Alternative biomarkers, especially non-invasive ones, are urgently needed for subclinical ketosis (SCK) diagnosis. To address this, we examined the milk lipidomic profiles of healthy and SCK cows from a research dairy farm over two consecutive years. A total of 125 polar lipid species were quantified in milk samples using a targeted liquid chromatography–mass spectrometry (LC-MS) approach. Chemometric analysis identified four potential biomarkers (PC 28:0, PC 29:0, PC 30:1 and PC 31:0), which met the thresholds for p value (<0.0004) and fold change (FC > 2). Global fatty acid (FA) profiling by gas chromatography–flame ionization detection (GC-FID) after transesterification of all lipids revealed that the levels of C8:0, C10:0, C12:0, C14:0, C15:0 and C18:1t11 were significantly suppressed in the SCK group (15–52% lower compared to the control group, p < 0.01), and C15:0 and C18:1t11 were the most promising markers for SCK in dairy cows at the FA level (43–50% reduction compared to the control group, p < 0.0001). Tandem LC-MS and FA data generated by GC demonstrated that SCK also caused significant changes in regio-isomer and double bond isomer composition of a model phospholipid molecule (PC 34:1) and a model triglyceride molecule TAG 52:2; these fine-structural alterations could also be explored for discovering novel SCK biomarkers in dairy cows. The PLS-DA predictive model built from lipidomic data showed excellent accuracy (>95%) in predicting cows with SCK. All of the promising biomarkers identified and the predictive model built in this study need further validation using larger SCK cohorts from different herds. Full article

Elymus repens (L.) Gould has a long history of use in traditional medicine across the British Isles, particularly among Gaelic and Anglo-Saxon communities, where it has been employed as a diuretic and anti-inflammatory agent. Despite its ethnopharmacological significance, comprehensive insights into its phytochemical composition and biological activities remain limited. The present study aimed to provide an integrated characterization of E. repens through macro- and microscopic analyses, advanced phytochemical profiling, and evaluation of the biological activities of the rhizome part. Microscopic examination revealed distinct anatomical features differentiating rhizome and stem, leaf tissues, supporting accurate identification and pharmacognostic standardization. Chemical profiling using Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (LC-QToF-MS) enabled the tentative identification of 93 metabolites, including amino acids, in aerial and rhizome extracts. These compounds were primarily classified into polyamines (e.g., feruloylputrescine, hydroxycoumaroylagmatine), phenolic acids (gallic, vanillic, and ferulic acids), flavonoids (apigenin, tricin, saponarin analogues), amino acids (arginine, tyrosine, tryptophan), organic acids (malic, succinic acids), nucleosides (adenosine, thymidine), and phospholipids. Biological evaluation demonstrated that the hydroethanolic rhizome extract exhibited notable antifungal activity against Aspergillus fumigatus (IC₅₀ = 34.9 µg/mL). Additionally, hydroethanolic extracts of both the aerial and rhizome parts showed no cytotoxicity in the Artemia salina lethality assay at concentrations up to 10 mg/mL, indicating a favorable preliminary safety profile. Of particular interest is tricin, which possesses anti-inflammatory, antioxidant, antimicrobial, and potential nephroprotective therapeutic effects; its mechanism of action is associated with the suppression of oxidative stress, the inhibition of pro-inflammatory mediators, and the disruption of metabolic processes in microbial cells. Overall, this study provides a comprehensive phytochemical and pharmacognostic characterization of E. repens, highlighting its potential as a source of bioactive compounds. The metabolite profile obtained directly from the biologically active extract, combined with its proven antifungal activity, serves as direct confirmation of the antimicrobial aspect of this plant’s traditional use, while its broader applications in ethnomedicine require targeted pharmacological testing. These findings contribute valuable data for chemotaxonomic classification and future pharmacological investigations. Full article

Glycosylation is a critical determinant of the efficacy, stability, and pharmacological behavior of therapeutic proteins. R27T, an engineered variant of interferon-β1a, contains two N-glycosylation sites (Asn25 and Asn80), increasing its structural complexity and analytical requirements. In this study, we performed comprehensive total and site-specific glycan profiling of R27T using complementary analytical approaches. For total glycan analysis, the released N-glycans were fluorescently labeled with procainamide, providing enhanced sensitivity and broader glycan coverage compared with conventional 2-aminobenzamide labeling. Site-specific glycan profiling was performed by liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based peptide mapping. Protease digestion conditions were optimized to improve recovery of site-specific glycopeptides, with chymotrypsin identified as the most effective enzyme for resolving glycopeptides from individual glycosylation sites. Total glycan distributions reconstructed from peptide-mapping data were compared with fluorescence-based glycan profiling, showing that total and site-specific glycan data can be effectively combined. Minor discrepancies were observed depending on glycan structure, mainly due to differences in ionization efficiency. Distinct glycan distributions were observed between the two N-glycosylation sites of R27T. Molecular modeling further suggested that the additional glycan at Asn25 may enhance structural stability and receptor-binding affinity. These results demonstrate an integrative strategy for accurate glycan characterization in multi-site glycoproteins relevant to biotherapeutic development. Full article

Sulforaphane (SFN), an isothiocyanate abundant in cruciferous vegetables, possesses well-documented anticancer, antioxidant, and anti-inflammatory properties, and has attracted interest as a dietary chemopreventive agent. However, sensitive and fully automated methods for its quantification in foods remain limited. In this study, we developed an automated analytical system coupling in-tube solid-phase microextraction (IT-SPME) with high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) to determine SFN in vegetable samples. Vegetable samples were autohydrolyzed in aqueous solution at 37 °C to generate SFN, which was then extracted with methanol by sonication. The extract was concentrated onto a Carboxen 1006 capillary column by IT-SPME, separated within 5 min on an Inertsil C8 column, and detected in positive electrospray ionization mode using multiple reaction monitoring. The detection limit was 5.6 pg/mL, with good linearity (R² = 0.9995) over 0.1–100 ng/mL, intra- and inter-day precisions below 2.9% and 3.9%, respectively, and accuracy of 98–102%. SFN was detected at concentrations of 600 μg/g or higher in broccoli sprouts, florets, and stems, but was found at less than one-tenth of these levels in other vegetables tested. This method provides a simple, sensitive, and solvent-efficient tool for SFN quantification in vegetables, with potential application in food functionality assessment and chemopreventive research. Full article

Persistently poor glioblastoma (GBM) survival necessitates better elucidation of tumor drug responses. After observing that low curcumin and all-trans retinoic acid (ATRA) doses stimulated cell proliferation and counteracted each other’s high-dose antiproliferative effects in U87 GBM cells, drug influences on cell growth, migration, and death and the antiproliferative interaction proteome were further studied. Cell proliferation and migration were assessed by xCELLigence Real-Time Cell Analysis (RTCA). Cell death was defined using flow cytometry. Drug interactions were determined with CompuSyn software (version 1.0). Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS), High-Performance Liquid Chromatography (HPLC), and SequestHT software (version 1.4) were utilized for peptide generation and identification. ATRA at high doses inhibited cell growth and migration more efficiently. Curcumin was more proliferative and antagonistic against anti-growth effects at low doses. Migration inhibition and apoptosis occurred synergistically at the highest drug doses. ATRA influenced the proteome more remarkably, reducing Transforming Growth Factor Beta Induced (TGFBI), Phosphoglycerate Dehydrogenase (PHGDH), tenascin, and Sequestosome 1 (SQSTM1). These effects were alleviated by curcumin, except for SQSTM1. Uveal Autoantigen With Coiled-Coil Domains And Ankyrin Repeats (UACA) and Sad1 And UNC84 Domain Containing 2 (SUN2) were increased by ATRA and curcumin, and to a lesser extent by the combination. Hexokinase 2 (HXK2) was increased by curcumin and the combination. Heme Oxygenase 1 (HMOX1) was depleted by the combination, but not by the single agents. SQSTM1 and HMOX1 reductions may mediate anticancer synergism, while the remaining changes may indicate ongoing hormetic pathways not reflected in cell counts. Full article

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. Full article

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. Full article