Beatriz Silva^1,2, Dragana Rajcic², Flavia Meotti¹, Tim Hendrikx²

Alcoholic liver disease (ALD) is increasingly understood as a metabolic disorder driven by disruptions in redox and energy homeostasis, leading to lipogenesis, suppressed β-oxidation, and hepatic triglyceride accumulation. A key shared mechanism is reductive stress—excess NADH relative to NAD⁺—exacerbated by alcohol metabolism via ADH and ALDH, which disrupts critical pathways and aggravates clinical outcomes. Recent studies have linked NADH accumulation to dysregulated PRPS2-mediated purine biosynthesis, intensifying energy stress and liver injury. Additional metabolomic analyses also highlight purine metabolism as a key pathway altered in ALD; however, these reports often lack detailed information on metabolite concentrations or the directionality of pathway changes (i.e., up- or down-regulation), limiting their interpretability. Building on this, our study aims to build a robust metabolite database using plasma samples employing the NIAAA chronic-plus-binge ALD model, further investigating purine degradation products as metabolic endpoints connecting reductive stress, ALD, and purine metabolism. C57BL/6J female mice were fed a Lieber–DeCarli diet for 15 days, gavaged with ethanol (5 g/kg BW) or isocaloric control on day 16 and sacrificed 9 h later (Ethics approval: 335/19). Targeted plasma metabolomics was performed via LC–MS/MS in dynamic MRM mode following protein precipitation and isotope-labeled standard spiking. Data preprocessing, univariate (fold-change analysis, t-test, volcano plot), and multivariate (PCA with PERMANOVA, PLS-DA, hierarchical clustering) analyses, as well as pathway enrichment (over-representation and quantitative enrichment analysis using KEGG), were conducted in MetaboAnalyst 5.0, with significance set at p < 0.05 and FDR < 0.05 for enrichment. Plasma ALT and hepatic triglycerides were quantified enzymatically, while liver uric acid, uricase, and xanthine oxidase activities were measured using commercial assays. Hepatic mRNA levels were assessed by qPCR, and protein expression by Western blot. In our targeted metabolomic analysis, we quantified 138 metabolites across plasma samples. Univariate statistics identified 73 metabolites significantly regulated (p < 0.05), with 50 exhibiting a fold-change > 1.5. Enrichment analysis highlighted disrupted amino acid metabolism, altered energy pathways (such as TCA cycle and gluconeogenesis), and increased NAD⁺ biosynthesis, consistent with hallmarks of metabolic disorder induced by reductive stress. Given the modulation of purine biosynthesis in the liver under reductive stress, we quantified hepatic uric acid and allantoin in ethanol-exposed mice. Our data demonstrated no significant change in allantoin, while uric acid concentration decreased. This reduction appears to result from several interlinked factors: 1. Reduced hepatic xanthine oxidoreductase (XOR) protein content and activity, limiting conversion of hypoxanthine and xanthine to uric acid; 2. Diminished renal reabsorption due to decreased expression of urate transporters; 3. Development of a metabolic disturbance associated with decreased plasma pyruvate and lactate. The excessive oxidation of ethanol led to a redox imbalance increasing the NADH/NAD⁺ ratio. This more reductive environment could also contribute to a lower oxidation of hypoxanthine to uric acid, since XOR depends on NAD⁺ as a cofactor, contributing to reduced uric acid synthesis.

Agradecimentos: FAPESP (2022/05623-5; 2019/26473-9); FAPESP JP2 (2018/14898-2); CEPID Redoxoma (2013/07937-8)