Background/Objectives: Vitamin B12 deficiency is a common but often underdiagnosed complication in patients with type 2 diabetes (T2D) undergoing long-term metformin therapy. Accurate early prediction could enable targeted screening and timely intervention. This study aimed to develop and interpret a machine learning model for predicting vitamin B12 deficiency in metformin-treated patients with T2D, using eXtreme Gradient Boosting (XGBoost). Methods: A retrospective cross-sectional study was conducted at a single endocrinology centre (La Rabta University Hospital, Tunis, Tunisia). Patients with T2D treated with metformin for at least three years were included (n = 257); those with conditions independently affecting vitamin B12 metabolism were excluded. Vitamin B12 deficiency was defined as a serum B12 level below 150 pmol/L or a borderline level (150–221 pmol/L) with concurrent hyperhomocysteinemia (>15 μmol/L). XGBoost was selected after comparison with Logistic Regression (L2), Random Forest, and Support Vector Machine on the same 5-fold stratified cross-validated pipeline. Hyperparameters were optimized via Bayesian search (100 iterations × 5-fold stratified cross-validation), with the Matthews correlation coefficient (MCC) as the primary optimization metric to account for class imbalance. Model interpretability was achieved using SHapley Additive exPlanations (SHAP). Discrimination and calibration were assessed on an independent test set using bootstrap 95% confidence intervals (2000 resamples). Results: Of 257 patients, 95 (37.0%) presented with vitamin B12 deficiency. On the independent test set (n = 52), the optimized XGBoost model achieved an ROC-AUC of 0.671 [95% CI: 0.514–0.818], sensitivity of 0.737 [95% CI: 0.533–0.938], specificity of 0.545 [95% CI: 0.375–0.710], MCC of 0.273 [95% CI: 0.018–0.517], and a Brier Score of 0.259. SHAP analysis identified HbA1c, microalbuminuria, autonomic neuropathy, BMI, DN4 score, and fasting glucose as the most influential predictors. Nonlinear SHAP interaction plots revealed an increased predicted risk in patients with low HbA1c combined with a high cumulative metformin dose. Conclusions: The XGBoost–SHAP framework provided interpretable predictions of vitamin B12 deficiency in patients with T2D on metformin, identifying key clinical profiles for targeted screening. External multi-centre validation is required before clinical deployment.

Although the genus Rhododendron is globally distributed and rich in bioactive constituents, the metabolomic landscapes of most species remain unexplored, hampering elucidation of their adaptive strategies and pharmaceutical potential. Objectives: This study sought to construct comprehensive metabolic atlases of four representative yet understudied Rhododendron species—R. triflorum, R. faucium, R. nivale, and R. strigillosum—and to quantify inter-specific metabolic divergence by UPLC-MS/MS-based, widely targeted metabolomics. Methods: The petals of four Rhododendron species were freeze-dried, pulverised, and extracted with 70% methanol (containing an internal standard). Metabolites were separated on an SB-C18 column (2.1 × 100 mm, 1.8 µm) using a 0–95% acetonitrile gradient (flow rate 0.35 mL min−1, 40 °C) and analysed by tandem mass spectrometry. Reliable quantification was ensured by molecular weight database matching, ion source standardisation, and quality control (QC), achieving a coefficient of variation (CV) < 15%. Principal component analysis (PCA) and optimised partial least squares discriminant analysis (OPLS-DA) were performed on standardised data with unit variance. Results: A total of 3705 metabolites were confidently identified, dominated by flavonoids (870), terpenoids (572), phenolic acids (394), and amino-acid derivatives (332). PCA and OPLS-DA models revealed clear species-specific clustering (R2Y ≥ 0.98, Q2 ≥ 0.95; permutation test p < 0.01). Comparative analysis yielded 1495 significantly differential metabolites; R. triflorum exhibited the highest cumulative abundance, followed by R. faucium, R. nivale, and R. strigillosum. KEGG enrichment highlighted “metabolic pathways” as the most significantly over-represented, together with flavonoid biosynthesis, phenylpropanoid metabolism, and terpenoid backbone biosynthesis. Conclusions: The study delivers the first high-coverage metabolomic reference for four neglected Rhododendron species, evidencing profound inter-specific metabolic differentiation centred on flavonoids, terpenoids, and phenolic acids. The data provide a robust foundation for understanding molecular adaptation to alpine environments and for accelerating targeted drug discovery from Rhododendron resources.

Background: Gastrodia elata Blume (GE) is a valuable traditional Chinese medicine with a wide range of clinical applications, yet the relationship between its developmental stages, phytochemical profiles, and functional properties remains poorly characterized. Methods: In this study, an integrated approach combining chemical assays and UHPLC–Orbitrap–MS/MS-based untargeted metabolomics was employed to characterize three growth stages: Mima (MT, seed tubers), Baima (BT, immature tubers), and Jianma (JT, mature tubers). Results: Multivariate statistical analyses demonstrated clear stage-dependent discrimination in metabolic profiles. A total of 31 differential metabolites were identified, including parishin derivatives, phenolics, amino acids, and organic acids. Specifically, Parishin E, Parishin G, total phenolics, and total flavonoids predominated in the early stage (MT), whereas gastrodin and Parishin C progressively accumulated and peaked in the mature stage (JT). Bioactivity assays revealed that GE extracts exhibited significant antioxidant and hypoglycemic (α-glucosidase and α-amylase inhibitory) effects, which followed an initial decrease followed by a subsequent recovery during development. Correlation analysis further established a strong relationship between phenolic-associated metabolites (particularly Parishin E/G/H) and the observed bioactivities. Conclusions: In summary, these findings elucidate the metabolic dynamics of GE across developmental stages and provide a scientific basis for optimizing harvest timing and raw-material grading to enhance the functional properties of GE-derived products.

Growth, reproduction, and survival are fundamental biological priorities that animals balance by evaluating dietary cues. Cholesterol occupies a unique position among nutrients, serving both as a structural component of cellular membranes and as a precursor for steroid hormones, yet its regulation differs fundamentally across taxa. In mammals, cholesterol availability is buffered by endogenous synthesis and post-ingestive metabolic control. In contrast, insects have evolutionarily lost sterol biosynthesis and are therefore sterol auxotrophs that rely entirely on dietary sources. Here, we synthesize current understanding of cholesterol biology in Drosophila melanogaster, with a focus on sterol auxotrophy, life-stage–specific allocation, and endocrine and lifespan outcomes in a comparative framework. We highlight cholesterol not only as a metabolic substrate but also as a sensory-encoded nutrient that shapes feeding behavior. We further examine how lipophorin (Lpp)-mediated transport, Niemann–Pick type C proteins, ATP-binding cassette transporters, and the nuclear receptor DHR96 coordinate systemic sterol distribution and endocrine output in the absence of endogenous synthesis. By integrating these mechanisms across development, we illustrate how cholesterol availability governs larval growth, ecdysteroid production, adult reproduction, neural function, and lifespan through coupled endocrine and nutrient-signaling networks. This review positions cholesterol as a multifunctional signal linking sensory perception, metabolic regulation, and life-history strategy in sterol-auxotrophic insects, offering a framework for understanding how evolutionary loss of biosynthetic capacity reshapes nutrient sensing and homeostatic control.

Background/Objectives: Low-fishmeal diets are widely adopted to improve sustainability in shrimp aquaculture, yet reduced palatability and metabolic stress frequently suppress feed intake and growth. We evaluated whether a crayfish (Procambarus clarkii) by-product protein hydrolysate (CBPH) could mitigate low-fishmeal-induced performance losses by modulating feeding-related metabolic signaling and gut microbiota features in Pacific white shrimp (Litopenaeus vannamei). Methods: In an 8-week feeding trial, 360 juveniles (initial body weight 0.46 g) were assigned to three diets (four replicates per diet): a commercial control (CON), a low-fishmeal diet (LFM), and LFM supplemented with 2% CBPH (CBPH). Growth, feed utilization, whole-body composition, hemolymph biochemical indices (TP, TG, GLU, AST, ALT), intestinal appetite-related gene expression (5-HTR, CART, CCK1R, D2-like, NPY), and intestinal microbiota profiles (full-length 16S rRNA sequencing, V1–V9, PacBio) were assessed. Results: Compared with the LFM group, CBPH supplementation increased feed intake and improved feed conversion, restoring final body weight and growth rates to levels comparable to CON. CBPH also alleviated low-fishmeal-associated metabolic stress, including reduced AST and ALT activities and lower glucose levels. The LFM diet induced upregulation of anorexigenic genes (5-HTR, CART, D2-like) and downregulation of NPY in the shrimp intestine, whereas CBPH supplementation reversed these transcriptional changes. In addition, microbiota richness indices (ACE and Chao1) were elevated by CBPH relative to LFM, accompanied by compositional shifts at the phylum and genus levels. Conclusions: CBPH effectively alleviated low-fishmeal-induced reductions in feeding and growth, accompanied by coordinated changes in feeding-related gene expression, systemic biochemical markers, and gut microbiota composition, supporting its potential as a functional ingredient to stabilize metabolic responses in low-fishmeal shrimp feeds.

Background: Refugees arriving from conflict zones often continue to experience trauma and are at increased risk of anxiety and depression. Those seeking asylum form a group at higher risk of suffering adverse mental health outcomes, with higher needs for psychosocial and therapeutic care. This study aimed to determine metabolic changes potentially associated with psychological distress in refugees from Syria, using a saliva-based metabolomics approach via proton nuclear magnetic resonance (1H NMR) spectroscopy. Methods: Participants were recruited from Lethbridge Family Services and categorized into high and low stress burden groups using questionnaires assessing depression (PHQ-9) and generalized anxiety (GAD-7). Salivary metabolomic profiles from 26 female and 32 male participants were analyzed using supervised and unsupervised multivariate statistical methods to identify metabolic differences linked to composite stress, depression, and anxiety. Results: Salivary metabolic profiles showed the most prominent differences associated with anxiety in female participants and depression in male participants. Multivariate statistical analyses identified 31 metabolites and 13 biological pathways that were significantly altered according to mental health status, with the greatest changes observed in glycolysis/gluconeogenesis, sphingolipid metabolism, and taurine/hypotaurine metabolism. Conclusions: These findings indicate that salivary 1H NMR metabolomic profiling can identify a quantifiable “metabolic fingerprint” related to impaired mental health and psychological distress in a cost-effective, objective, and non-invasive way. This analytical strategy shows potential as a screening tool to support effective decision-making, enabling early identification of individuals at highest risk who require timely emotional and medical support.

Background/Objectives: Folic acid (FA) intake impacts one-carbon metabolism (OCM), which is crucial for fetal development and epigenetic regulation. While FA supplementation is known to lower homocysteine levels, its comprehensive effects on OCM-related metabolites in maternal and cord blood remain unclear. This study aimed to investigate the association between FA use and serum OCM-related metabolite profiles in Japanese pregnant women. Methods: We analyzed 146 mother-infant pairs from the Chiba study of Mother and Child Health (C-MACH) cohort. Blood samples were collected in early pregnancy, late pregnancy, and at delivery (maternal and cord blood). FA use was assessed via self-administered questionnaires. Serum concentrations of 18 OCM-related metabolites, including 5-methyltetrahydrofolate (5-MTHF) and homocysteine, were measured using LC-MS/MS. Results: FA users exhibited significantly higher 5-MTHF and lower total homocysteine concentrations in maternal blood at all time points and in cord blood compared to non-users. Compared to non-users, FA users exhibited a lower serine/glycine ratio in early pregnancy, a higher betaine/DMG ratio in maternal blood at delivery, and higher S-adenosylmethionine and total cysteine concentrations in maternal blood during late pregnancy. In cord blood, unmetabolized folic acid concentrations did not differ significantly between FA users and non-users. Furthermore, the cord-to-maternal 5-MTHF ratio was significantly lower in FA users. Conclusions: Our findings suggest that FA use during pregnancy may contribute to the optimization of OCM in both the mother and fetus.

Background: Glutaric aciduria type-1 (GA-1) is a genetic disorder caused by glutaryl-coenzyme A dehydrogenase deficiency, leading to the accumulation of glutaryl-CoA and its derivatives. Clinical manifestations include neurological abnormalities; however, the underlying pathological mechanisms remain unclear. Early diagnosis and intervention are crucial for minimizing adverse outcomes. To date, diagnostic methods have certain limitations, and there is a critical need for a sensitive biomarker for diagnosis. We aimed to characterize metabolic dysregulation and identify candidate biomarkers associated with GA-1 in biochemically confirmed patients compared to age- and sex-matched control subjects. Methodology: Untargeted metabolomics profiling of GA-1 patients (n = 29) was compared to matched control subjects by age and sex. Multivariate and univariate statistical analyses were performed to identify dysregulated metabolites. Results: Our findings revealed 220 endogenous human metabolites. Notably, there was a strong enrichment in carboxylic acids and derivatives, including amino acids and derivatives, hydroxy and keto acids, fatty acyls, sphingolipids, phosphatidylcholines, and nucleotides and nucleosides. Pathway analysis indicates alterations in the biosynthesis of cardiolipin and phosphatidylcholine, as well as in pyrimidine metabolism, the urea cycle, and amino sugar metabolism. We demonstrated a robust performance model for 6-Methylnonanoyl-CoA, displaying strong discriminative power. Conclusions: We identified broad dysregulation across various biochemical classes, reflecting an imbalance in energy metabolism that involves carbohydrate and lipid pathways. The results also highlight dysregulation in sphingolipids, phospholipids, and nucleotide metabolism. These findings are preliminary and the clinical relevance of these findings in patients with GA-1 requires further investigation. We identified candidate biomarkers capable of distinguishing GA-1 patients from controls; however, these findings require validation in independent cohorts.

Mandarin orange peel (MOP) is rich in bioactive polymethoxyflavones, including hesperidin and nobiletin, which have shown neuroprotective effects in rodent models. However, comprehensive safety data in dogs are required to support its development as a therapeutic intervention for canine cognitive dysfunction syndrome. In this study, the safety profile of a standardized MOP formulation was evaluated in four healthy Beagle dogs. Initially, compositional analysis was performed, and 202 pesticide residues and psoralens were screened to ensure compliance with Japanese pet food safety standards. Subsequently, a dose-escalation study was conducted in which dogs received oral MOP at 2, 6, and 10 g/head/day for 3-4 weeks at each dose level. Clinical signs, hematology, and serum biochemistry were monitored throughout the study period. The MOP powder composition and residue levels remained within regulatory safety limits. In the dose-escalation study, no significant dose-dependent abnormalities were observed in physical or clinicopathological parameters. One dog exhibited transient loose stools at higher doses and a temporary elevation in alkaline phosphatase levels at 2 g/head/day; however, these symptoms resolved spontaneously despite continued administration. MOP was safe and well tolerated in dogs even at 10 g/head/day (787-952 mg/kg/day), which is approximately five times the anticipated clinical dose. The observed fluctuations in active ingredient concentrations remained within the acceptable range for natural products and did not affect overall safety. Combined with comprehensive screening for residues, these results indicate that MOP is a high-quality and safe dietary intervention for older dogs.

Background/Objectives: Clopidogrel is a widely prescribed antiplatelet prodrug that requires bioactivation, primarily by the polymorphic CYP2C19 enzyme. Genetic variation in this enzyme leads to differences in active metabolite formation and has prompted the development of pharmacogenetics-guided prescribing. However, current pharmacogenetic strategies are grounded in drug metabolism knowledge derived from mass balance studies conducted in small groups of healthy volunteers. This narrow evidence base may limit the data’s applicability to real-world settings, where factors like polypharmacy or altered organ function may influence drug response. Methods: Pharmacogenetics could benefit from real-world drug metabolism and excretion studies, which we conducted for clopidogrel in 38 kidney and 16 liver transplant recipients from the TransplantLines Biobank and Cohort Study (NCT03272841), utilizing existing LC-SWATH/MS pharmacometabolomic data. Clopidogrel-associated metabolic signals were identified using xenobiotic metabolism knowledge and literature-reported pathways. Results: Across both transplant groups, 26 clopidogrel-associated features were prioritized, of which some matched previously reported urinary metabolites, had previously been observed in plasma, or represented previously unreported metabolites. Clopidogrel carboxylic acid predominated in kidney transplant recipients, whereas its glucuronide form was most abundant in liver transplant recipients. Notably, unmetabolized clopidogrel was consistently detected across all patients. Moreover, our data support a thiol desulfurization route, aligning with emerging evidence of clopidogrel’s role as a hydrogen sulfide-releasing drug. Conclusions: More (putative) clopidogrel metabolites were detected than previously reported, demonstrating the value of pharmacometabolomics in expanding our understanding of drug metabolism. This approach provides novel data that may complement pharmacogenetics research to understand clopidogrel response variability among treated patients.