Abstract
<title>Abstract</title> <p>Background Acute-on-chronic liver failure (ACLF) is characterized by severe systemic inflammation, progressive organ failure, and high short-term mortality. Although numerous alterations in circulating lipids have been described in ACLF and other forms of critical illness, it remains unclear whether biologically meaningful information resides primarily in individual lipid species or emerges through coordinated molecular organization associated with disease progression. We hypothesized that reproducible disease-associated biology would become increasingly apparent at higher levels of lipid organization across independent patient cohorts. Methods We performed targeted LC-MS/MS lipidomic profiling of plasma from two temporally independent cohorts comprising 140 patients with ACLF and healthy controls. Cross-cohort reproducibility was evaluated at the levels of individual lipid species, lipid classes, and co-varying lipid modules. Associations with disease severity and organ failure were assessed using clinical correlation analyses and a composite Lipid Severity Score. Independent neutrophil proteomic datasets, published transcriptomic studies, and computational regulatory analyses were subsequently examined as complementary molecular observations to provide biological context for the identified lipid alterations. Results ACLF was characterized by reproducible coordinated alterations in the circulating lipidome across independent cohorts, including depletion of lysophospholipids and plasmalogens with enrichment of glycerophospholipids and triacylglycerols. Although overlap of individual lipid species between cohorts was modest, lipid class-level alterations demonstrated strong concordance, indicating that reproducible disease-associated information emerged more consistently at higher levels of molecular organization. Unsupervised network analysis identified a phospholipid- and plasmalogen-enriched lipid module whose progressive depletion correlated with MELD, MELD-Na, INR, serum creatinine, cumulative organ failure burden, and the Lipid Severity Score (LSS). Cross-disease lipidomic comparison identified a conserved critical illness lipid signature, with three phosphatidylcholine/phosphatidylethanolamine species [PC(36:3), PC(38:4), and PE(36:4)] consistently shared across ACLF, trauma, and COVID-19 cohorts. Independent neutrophil proteomic and published transcriptomic datasets converged on biological processes related to neutrophil activation, degranulation, and innate immune responses. Conclusions ACLF is characterized by reproducible coordinated alterations in the circulating lipidome that are closely associated with progressive organ failure. Our findings suggest that reproducible disease-associated biology is more robustly represented at higher levels of molecular organization, including coordinated lipid classes and co-varying lipid modules, than by individual lipid species alone. This framework provides a biologically grounded approach for interpreting molecular heterogeneity in ACLF and may facilitate the identification of more reproducible molecular signatures of disease severity and progression.</p>