Search Results (444)

Background/Objectives: This study aimed to explore the association between maternal gut microbiota and metabolic profiles in the first trimester and the subsequent risk of gestational diabetes mellitus (GDM), as well as to characterize association patterns linking gut microbiota, serum metabolites, and metabolic traits. Methods: A nested case–control study was conducted among women with GDM (n = 47) and those without GDM (n = 94). Metagenomic sequencing was applied to analyze fecal microbiota, and liquid chromatography–mass spectrometry (LC–MS) was used for non-targeted plasma metabolomics. Differential microbiota and metabolites between groups were identified, and correlation analyses were conducted to assess their associations with clinical indicators. Results: Women who later developed GDM showed lower alpha diversity and higher beta diversity. Eleven differential species were identified, with Collinsella aerofaciens and Clostridium bartlettii enriched in GDM, while nine species such as Alistipes putredinis and Bacteroidales bacterium ph8 were enriched in controls. Sixty-four plasma metabolites differed between groups, including increased glycerol-3-phosphate, aromatic amino acids, and glycerophosphocholine, and decreased cysteine, tryptophan, niacinamide, and stearic acid. Correlation analyses revealed significant relationships between Alistipes putredinis, Eubacterium eligens, and Bacteroidales bacterium ph8 with metabolic and clinical indicators (e.g., TG, TC, LDL). Conclusions: In this nested case–control study, women who later developed GDM exhibited reduced gut microbial diversity and altered metabolic profiles during the first trimester of pregnancy. Several microbial taxa and microbiota–metabolite associations were observed in relation to subsequent GDM status, highlighting early-pregnancy microbial and metabolic features that may be relevant to GDM-related metabolic changes. Full article

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a peril to public health. Xanthine oxidoreductase (XOR) is implicated in oxidative stress and lipid metabolism, which constitute the pathological basis of MASLD. As a specific XOR inhibitor, febuxostat therefore exhibits considerable potential for mitigating MASLD. However, the efficacy and underlying mechanisms of febuxostat in this context remain to be elucidated. Against this background, the present study aimed to observe the effect of febuxostat on the physiological changes of male MASLD rats and explore the related mechanisms. All rats were assigned to three groups: control, high-fat diet (HF), and high-fat diet with febuxostat (HF + F). After euthanasia, biosamples were immediately harvested to conduct an extensive suite of experiments, encompassing histological examination, assessment of biochemical and oxidative stress markers, serum non-targeted metabolomics, and Western blot analysis. Histological examination showed marked reductions in hepatic lipid accumulation and hepatocellular degeneration in the HF + F group relative to the HF group. Consistently, compared to the HF group, the HF + F group showed significant reductions in the elevated levels of plasma/hepatic lipids, and plasma oxidative stress markers (p < 0.05). Serum metabolomics revealed distinct metabolic profiles among groups, with 51 differential metabolites between HF + F and HF groups, with pathways such as taurine and hypotaurine metabolism and starch and sucrose metabolism being significantly altered (p < 0.05). Western blot analysis showed reduced p-JNK and increased NRF2 and HO-1 expression in the HF + F group (p < 0.05). In summary, we found that inhibiting XOR with febuxostat improved hepatic steatosis, serum metabolic dysregulation and systemic oxidative stress status, and it accompanied by JNK/NRF2/HO-1 pathway key molecule protein alterations in male MASLD rats. Full article

Background: Black spot disease severely constrains Chinese cabbage production. Methods: To elucidate the defence mechanisms underlying this response, transcriptomic and metabolomic profiles were analysed in leaves of the Chinese cabbage line 904B at 24 h post-inoculation (hpi) with Alternaria brassicicola. In parallel, gene silencing and overexpression were conducted for BraPBL, an RLCK family member in Chinese cabbage. Results: The Chinese cabbage line 904B exhibited marked suppression of cytokinin and auxin signalling, coupled with enhanced expression of genes involved in ethylene and jasmonic acid signalling. Multiple secondary metabolites exhibited differential changes, specifically the sterol compound 4,4-dimethyl-5alpha-cholest-7-en-3beta-ol was significantly upregulated in the treatment group. These metabolites were primarily enriched in the indole alkaloid metabolism and glycerolipid metabolism pathways. Concurrently, BraPBL exhibits increasing expression with prolonged infection. BraPBL overexpression enhances resistance to black spot disease, whereas silencing reduces resistance. Subcellular localization confirmed BraPBL at the plasma membrane. Overexpression of BraPBL upregulates the reactive oxygen species-related gene RBOH and the signal transduction-related gene MEKK1, whilst simultaneously activating the JA pathway. Conclusions: Overall, 904B activates defence-related hormones while suppressing growth and development-related hormones during early infection. Secondary metabolites, particularly the sterol compound 4,4-dimethyl-5alpha-cholest-7-en-3beta-ol, play key roles in defence, and BraPBL functions as a black spot disease–related defence gene in Chinese cabbage. Full article

Background: Persistent liver pathology despite a sustained virologic response (SVR) to hepatitis C virus (HCV) therapy is a major clinical concern. This is particularly relevant for people with HIV (PWH) with HCV coinfection, a population prone to accelerated liver disease progression. This study aimed to characterize the plasma miRNA profile in PWH with cirrhosis one year after successful completion of HCV therapy, and to explore their relationship with metabolite alterations. Methods: This cross-sectional study enrolled 47 PWH who achieved HCV clearance with antiviral therapy. Using plasma samples collected approximately one year after completion of HCV therapy, participants were stratified into two groups based on liver stiffness measurement (LSM): compensated cirrhosis (n = 32, LSM ≥ 12.5 kPa) and non-cirrhosis (n = 15, LSM < 12.5 kPa). Plasma miRNAs and metabolites were determined using small RNA sequencing and untargeted capillary electrophoresis-mass spectrometry (CE-MS), respectively. Significantly differentially expressed (SDE) miRNAs were identified using generalized linear models (GLM) with a negative binomial distribution, and their correlation with metabolite levels was quantified using Spearman’s correlation. Results: In the cirrhosis group (n = 32), we identified a distinct signature of 15 SDE miRNAs (9 upregulated, 6 downregulated) compared to the non-cirrhotic group (n = 15), showing hsa-miR-10401-3p, hsa-miR-548ak, hsa-miR-141-3p, and hsa-miR-3940-3p the largest expression changes. miRNA-gene interaction and pathway enrichment analysis suggested that these 15 SDE miRNAs potentially regulate multiple genes involved in immune response and amino acid metabolism. In addition, correlation analyses with our metabolomic data revealed significant associations between specific SDE miRNAs and amino acids and their derivatives. Specifically, the expression of upregulated miRNAs (e.g., hsa-miR-10401-3p and hsa-miR-16-5p) was positively correlated with plasma levels of L-methionine and its derivatives, while downregulated miRNAs (e.g., hsa-miR-625-5p) were inversely correlated with L-tryptophan. Conclusions: In cirrhotic PWH with history of HCV coinfection, a distinct plasma miRNA signature linked to dysregulated amino acid metabolism is found one year after completion of HCV therapy. This underscores that the HCV cure does not equate to complete hepatic recovery, highlighting the critical need for long-term monitoring in this high-risk population. Full article

This study investigated the metabolic mechanisms driving physiological functional remodeling in RFM by analyzing plasma biochemical parameters and metabolomic profiles at key peripartum timepoints (21 and 7 d prepartum and 4 h postpartum), integrated with placental and fetal membrane metabolic characteristics. The results revealed that RFM cows exhibited significant negative energy balance (NEB) as early as 21 days before parturition, characterized by elevated plasma levels of non-esterified fatty acids, β-hydroxybutyrate, and malondialdehyde, alongside reduced activity of antioxidant enzymes (GSH-Px, CAT) (p ≤ 0.05). Metabolomic analysis demonstrated persistent lipid metabolism dysregulation, amino acid imbalance, and nucleotide metabolism disturbances in RFM cows from 21 days prepartum to 4 h postpartum, indicating premature mobilization of adipose and muscle tissues. Further metabolomic analyses of the placenta and fetal membranes confirmed that metabolic dysfunction compromises energy supply during parturition, adversely affecting immune homeostasis and extracellular matrix degradation in the placenta and fetal membranes of RFM dairy cows. These physiological dysfunctions have the potential to impede the timely expulsion of fetal membranes after calving. In conclusion, RFM is closely associated with early-onset metabolic dysfunction during the periparturient period, where insufficient energy supply due to NEB, oxidative stress, and immune-endocrine disruptions collectively impair normal fetal membrane detachment. Full article

Hypertension is a major global health challenge, with excessive dietary salt intake recognized as a key environmental factor contributing to its pathogenesis. However, safe and effective dietary interventions for salt-sensitive hypertension remain limited. Vine tea (Ampelopsis grossedentata), a traditional herbal tea widely consumed for centuries in southern China, has been reported to exhibit antioxidant, anti-inflammatory, and hepatoprotective activities, yet its antihypertensive efficacy and underlying mechanisms remain unclear. In this study, the chemical profile of vine tea aqueous extract (VTE) was characterized by UPLC–Q–TOF–MS, identifying dihydromyricetin, isoquercitrin, and myricetin as the predominant flavonoids. The protective effects of VTE were evaluated in C57BL/6J mice with high-salt-diet (HSD)-induced hypertension. VTE treatment significantly lowered systolic blood pressure and ameliorated cardiac and renal injury, accompanied by reduced inflammation, fibrosis, and cardiac stress-related gene expression. Gut microbiota analysis using 16S rRNA gene sequencing revealed that VTE restored microbial richness and diversity, enriching short-chain fatty acid-producing taxa while suppressing pathogenic Desulfovibrio and Ruminococcus torques. Untargeted plasma metabolomic profiling based on UPLC–Q–TOF–MS further showed that VTE normalized tryptophan, bile acid, and glycerophospholipid metabolism, decreasing the uremic toxin indoxyl sulfate while increasing tauroursodeoxycholic acid. Notably, these protective effects were abolished under antibiotic-induced microbiota depletion, confirming that VTE acts through a gut microbiota-dependent mechanism. Collectively, VTE mitigates salt-induced hypertension and cardiorenal injury by remodeling the gut microbiota–metabolite axis, supporting its potential as a natural dietary intervention for managing hypertension. Full article

Background: Branched-chain amino acids (BCAAs) are popular as sports supplements due to their ability to enhance performance and recovery. However, the full spectrum of metabolic alterations triggered by acute supplementation with BCAAs in conjunction with exercise remains incompletely understood. Methods: A randomized crossover trial was conducted in 8 healthy active young males, who received either BCAA or placebo supplementation for three consecutive days prior to a high-intensity cycling test. Plasma samples were collected pre- and post-exercise and analyzed by ultra-high-performance liquid chromatography–quadrupole time-of-flight mass spectrometry, followed by correlation and enrichment analyses. Results: Acute BCAA supplementation was significantly associated with enhanced fat oxidation and attenuated post-exercise increases in plasma ammonia, creatine kinase, and lactate dehydrogenase, suggesting the potential improvements in energy supply and membrane stability. Metabolomics analysis identified differential metabolites primarily involved in lipid, amino acid, and glucose metabolism. Pathway enrichment revealed coordinated regulation of fatty acid oxidation (FAO) and tryptophan-related pathways. Correlation analysis further showed that changes in metabolite profiles were strongly associated with biochemical outcomes, particularly linking enhanced fat oxidation and ammonia clearance with BCAA intake. Conclusions: Short-term BCAA supplementation could enhance FAO and membrane stability via coordinated regulation of lipid and amino acid metabolism post exercise, supporting its potential role as a precision nutrition strategy. Full article

Background: Existing exercise metabolomics studies have predominantly focused on changes in the type and abundance of metabolites, while rarely addressing the toxicity risk of differential metabolites. Metabolic toxicity refers to the potential of endogenous or exogenous metabolites to induce oxidative stress, cell death, and other forms of biological damage when excessively accumulated and serves as a key driver of metabolic disorders. This study aims to characterize the toxicity risk of plasma differential metabolites before and after a single session of moderate-intensity running, so as to investigate the exercise-induced changes in metabolic toxicity. Methods: A single-group self-pretest–posttest control design was adopted in this study. Participants were recruited from Wuhan Sports University, China, with the inclusion criteria of healthy females aged 22–30 years and BMI 18.5–24.9. Individuals with a history of metabolic diseases or who met other exclusion criteria were excluded, and 5 females were finally enrolled. The exercise protocol consisted of a single 40 min session of moderate-intensity running on a treadmill. We collected plasma samples from five healthy females before and after exercise and performed untargeted LC-MS/MS metabolomic profiling. The gap-Δenergy algorithm was applied to calculate the toxicity scores of differential metabolites, and the proportion of metabolites with high toxic potential (score > 0.6) was compared. Results: Plasma metabolic profiles underwent notable remodeling after exercise. Thirty-two metabolites were upregulated and the phosphosphingolipid SM(d18:2(4E,14Z)/16:0) was the most significant. Meanwhile 32 metabolites were downregulated and the phosphosphingolipid PC(18:1(9Z)/14:0) was the most significant. The 64 differential metabolites were enriched in 9 KEGG pathways including amino acid metabolism and lipid metabolism. Moreover, we systematically evaluated the toxicity of these metabolites using the gap-Δenergy algorithm and found that the downregulated metabolites exhibited a significantly higher toxicity score compared to the upregulated ones. In addition, 37.5% of the downregulated metabolites had a high toxicity score, while the proportion of high toxicity in the upregulated group was only 15.6%. Conclusions: This study demonstrates that moderate-intensity running may confer metabolic health benefits to individuals by reducing metabolic toxicity, specifically through the downregulation of metabolites with high toxic potential. These findings offer novel evidence for exercise’s role in improving metabolic health. They also open a new direction for exercise-based interventions in metabolic disease–toxicity regulation. Full article

Background/Objectives: Longitudinal metabolomics analysis offers valuable insights into how metabolic pathways change according to age and health status. However, metabolite levels can fluctuate due to biological factors (e.g., age, diet, and health status) and technical factors (e.g., sample handling, storage times, and instrument performance), with some metabolites exhibiting greater sensitivity to these sources of variability than others. This study aimed to characterize the longitudinal and technical stability of untargeted plasma and cerebrospinal fluid (CSF) metabolites and to identify a subset that remains reliable over the extended time scales required for epidemiological research. Methods: Untargeted ultrahigh-performance liquid chromatography–mass spectrometry (LC-MS) metabolomic profiles were available from multiple visits in the Wisconsin Registry for Alzheimer’s Prevention (WRAP) and Wisconsin Alzheimer’s Disease Research Center (ADRC) studies. For this analysis, we constructed a subset of generally healthy participants with samples drawn at four time points (~2.5 years apart): two visits analyzed in 2017 and two visits analyzed in 2023, corresponding to two distinct analytical waves. We computed Rothery’s intraclass correlation coefficients (ICCs) to quantify intra-wave and inter-wave stability, evaluated pooled quality-control (QC) variation, classified metabolite stability by established thresholds, and developed a composite score integrating longitudinal stability and susceptibility to technical variance. Results: Across all metabolites, median stability was classified as ‘fair’ (Rothery’s ρ > 0.40 to ≤0.75) for both plasma and CSF. Although analytical batches were bridged using pooled QC samples, inter-wave stability was significantly lower than intra-wave stability, reflecting increased technical variability across waves. Using the composite score, we identified subsets of metabolites with ‘excellent’ stability and low susceptibility to batch effects in plasma and CSF. Stability patterns varied across biochemical super pathways. Conclusions: This work highlights metabolites suitable for long-term epidemiological studies and informs experimental design and analytical strategies for combining data across cohorts and analytical batches. Full article

Background: Metabolomics is recognized as crucial technology for advancing our ability to diagnose, characterize, and monitor treatment of disease. Yet, metabolomics-based diagnostic testing has not been widely adapted into clinical practice because its technical requirements make it generally incompatible with operation at the point of care. One way to expand the reach of metabolomics-based testing, and its clinical benefits, is to utilize dried blood spots (DBS) as a testing sample type. Their easy collection, ambient storage capability, and cost-effective shipment make DBSs ideal for diagnostic tests that require the use of a centralized technology. Methods: To date, relatively few studies have investigated the performance of DBSs at capturing the global metabolome and reporting changes associated with physiological processes. In this study, we investigated those factors by performing global metabolomic profiling on DBSs collected from study volunteers under fasted and postprandial states, with and without dietary fish oil supplementation. Results: DBSs demonstrated broad coverage of metabolic pathways and captured numerous metabolic changes associated with feeding, fasting, and fish oil supplementation that have been reported in plasma and serum. Conclusions: Our findings support the hypothesis that DBSs are a viable sample type for metabolomics-based diagnostic testing and justify follow-up validation studies. Full article

High-yielding dairy cows suffer from a high metabolic load and oxidative stress, which lead to systemic inflammation and metabolic disorders, increasing the susceptibility of these cows to various production diseases. Dihydromyricetin (DMY) has demonstrated potent antioxidant and anti-inflammatory physiological functions; however, research into its application in ruminants remains limited. This study investigated whether DMY supplementation is associated with the maintenance of metabolic homeostasis through the regulation of gut microbiota and metabolite profiles. A total of 14 mid-lactation Holstein dairy cows were randomly divided into two groups (n = 7 per group) and supplemented with DMY at 0 or 0.05% in their basal diet for 60 consecutive days. The effects of DMY on the blood biochemical indicators and the antioxidant capacity of the dairy cows were then determined. Alterations to the gut microbiome and the fecal and plasma metabolome were analyzed through 16S rDNA sequencing and untargeted metabolomics. The results showed that DMY significantly improved the activity of serum glutathione peroxidase (GSH-Px) and presented a trend of increasing the total antioxidant capacity (T-AOC). The abundance of multiple fiber-degrading and beneficial commensal bacteria in the gut, including Fibrobacter_succinogenes, Ruminococcus_albus, and Turicibacter, was significantly elevated by the DMY intervention, accompanied by the upregulation of 8,11,14-eicosatrienoic acid, myricetin, dihydro-3-coumaric acid, PGE1, L-leucine, nicotinuric acid, pantothenic acid, and pyruvate in the feces and plasma. Moreover, DMY supplementation notably reduced the abundance of potential pathogenic microbes, such as Chloroflexi, Deltaproteobacteria, RFP12, and Succinivibrio, and downregulated the levels of 12-hydroxydodecanoic acid, 12,13-DHOME (12,13-dihydroxy-9Z-octadecenoic acid), 16-hydroxyhexadecanoic acid, niacin, and glycerol 3-phosphate. These differential metabolites were principally enriched in the mTOR signaling pathway; pantothenate, nicotinate, and thiamine metabolism; glutathione metabolism; and glycolysis/gluconeogenesis. In summary, dietary supplementation with DMY increased the abundance of intestinal fiber-degrading bacteria and multiple metabolites with known anti-inflammatory and antioxidant properties in the feces and plasma, and was associated with alterations in metabolic pathways involving B-vitamins, amino acids, and glutathione. This suggests a potential role for DMY in supporting metabolic homeostasis in dairy cows. Full article

Rhizomelic chondrodysplasia punctata type 1 (RCDP1) is a peroxisomal disorder characterized by skeletal shortening, intellectual disability, seizures, cataracts, and reduced lifespans. RCDP1 is caused by biallelic loss-of-function variants in PEX7, which encodes a protein required for importing select enzymes into the peroxisome matrix, including those essential for ether lipid synthesis (e.g., plasmalogens) and the branched-chain fatty acid catabolism. Plasmalogen deficiency is a hallmark of RCDP1 and other peroxisomal disorders, including RCDP types 2-5 (RCDP2-5) and Zellweger spectrum disorders (ZSD). Here, we performed comprehensive metabolomic profiling of clinical samples from RCDP patients and Pex7-deficient mouse models. We identified profound neurometabolic disturbances in the cerebral cortex and cerebellum of Pex7-deficient mice involving multiple lipid classes, including phosphatidylethanolamines (PEs), phosphatidylcholines (PCs), acylcarnitines, and sphingomyelins. Notably, many of these neurometabolic alterations were absent in patient and Pex7-deficient mouse plasma, indicating that plasma-based profiling can underrepresent the extent of CNS lipid remodeling. Overall, these findings reveal novel insights into neurometabolic adaptations to plasmalogen deficiency and suggest the potential involvement of additional pathways that may contribute to neurological dysfunction in RCDP. Full article

Understanding the drivers of average daily gain (ADG) is key to enhancing the productivity of Tan sheep. This study employed an integrated multi-omics approach to compare rumen microbial communities (16S rRNA sequencing) and metabolomic profiles between Tan sheep with high (HADG) and low (LADG) ADG. The novelty of this work lies in the systems-level identification of functional linkages between specific rumen bacteria and metabolites that underlie divergent growth phenotypes. The results revealed no significant difference in initial body weight between the two groups (p > 0.05). However, the HADG group showed significantly higher final body weight (p < 0.05), markedly greater ADG and Average Daily Dry Matter Intake (ADFI) (p < 0.01), and a substantially lower FCR (p < 0.01). Plasma Total Antioxidant Capacity (T-AOC) and Superoxide Dismutase (SOD) levels were significantly elevated in the HADG group (p < 0.05), while Malondialdehyde (MDA) concentration was significantly reduced (p < 0.05). In contrast, plasma Globulin (GLB), Glucose (GLU), and Triglycerides (TG) concentrations were significantly lower in HADG sheep (p < 0.05). Rumen metabolomics identified 265 differentially abundant metabolites between groups, with 64 down-regulated and 201 up-regulated in LADG compared to HADG sheep. These metabolites were significantly enriched in tyrosine metabolism, β-alanine metabolism, and thiamine metabolism pathways. Receiver Operating Characteristic (ROC) curve analysis identified 15 key differential metabolites, including succinic acid, 2-hydroxyglutarate, and pyridoxal phosphate. 16S rRNA sequencing indicated significant differences in microbial genera such as UCG-002, Blautia, norank_f__Bacteroidales_UCG-001, and norank_f__norank_o__Rhodospirillales. Correlation analysis revealed that UCG-002 and norank_f__Bacteroidales_UCG-001 were highly negatively correlated with succinic acid (p < 0.01), and significantly negatively correlated with 1-aminocyclopropanecarboxylic acid, pyridoxal phosphate, and 2-hydroxyglutarate (p < 0.05). Conversely, beta-alanine, ureidoacrylic acid, L-proline, and 2′-deoxyguanosine showed a highly significant positive correlation with norank_f__Bacteroidales_UCG-001 (p < 0.01), and a significant positive correlation with UCG-002 (p < 0.05). These findings elucidate the molecular mechanisms behind growth differences in Tan sheep and provide actionable insights for developing targeted nutritional strategies. Full article

Background/Objectives: Chronic Obstructive Pulmonary Disease (COPD) exacerbations affect health and mortality, yet current risk assessments based on previous events have limitations and are less preventative. This study used metabolomics to assess if amino acid profiles are linked to higher COPD exacerbation risk, compared to an amino acid-based panel to standard risk stratification methods, and explored its clinical decision-making and prevention potential. Methods: This prospective cohort study measured plasma concentrations of 19 amino acids in 88 individuals with COPD using ultra-performance liquid chromatography. Participants were observed for 2.5 years to track occurrences of moderate and severe COPD exacerbations. Results: During follow-up, 44 participants (50%) had an exacerbation. Tyrosine and hsCRP were independently linked to exacerbations in multivariable analysis and formed the basis of the “COPDAE score.” This score independently predicted future exacerbations after adjusting for Global Initiative for Chronic Obstructive Lung Disease (GOLD) classifications. A COPDAE score above 1.73 correlated with lower event-free survival in several subgroups: GOLD group A (log-rank = 13.7, p < 0.001), groups A and B (log-rank = 5.0, p = 0.025), and groups A and C (log-rank = 15.2, p < 0.001). Conclusions: Tyrosine and hsCRP together form a biomarker panel to assess and stratify COPD exacerbation risk. This score can help identify stable patients at risk, even with mild symptoms or few prior events, enabling earlier, more personalized interventions. Full article

Background/Objectives: Cytochrome P450 (CYP) enzymes are crucial for drug metabolism, yet inter-individual variability in their activity remains a significant clinical challenge. Current phenotyping methods are often impractical or even impossible, particularly in forensic toxicology and vulnerable populations. This proof-of-concept study investigated the feasibility of using in vitro assays with human liver microsomes (HLM) and recombinant CYP enzymes (isoenzymes), combined with untargeted metabolomics, to identify potential endogenous biomarker candidates indicative of CYP phenotype. Methods: This study uses in vitro incubations of HLM and isoenzymes in tandem with targeted and untargeted LC-(HR)MS and metabolomics techniques as well as statistical processing. Results: We demonstrate that HLM and isoenzymes maintain activity in the presence of complex biological matrices (blood/plasma), enabling metabolomic profiling. Untargeted analysis of assays in plasma revealed numerous potential biomarkers, with several showing significant correlations to enzyme activity. Conclusions: While identification remains the major challenge, this approach offers a promising avenue for developing accessible and efficient methods for indirect CYP phenotyping, potentially facilitating investigations in scenarios where traditional approaches are limited. This work provides a foundation for future studies focused on further developing in vitro assays and validating the proposed biomarkers, as well as establishing their utility in clinical and forensic settings. Full article