Metabolomics-based Screening of Serum Metabolic Markers in Rats with Acute Insulin Intoxication

Background: Rising insulin poisoning cases demand more reliable forensic biomarkers as current diagnostic methods face accuracy limitations, highlighting metabolomics’ potential for identifying acute intoxication markers. Aims and Objectives: This study aimed to investigate changes in serum metabolites in rats with acute insulin poisoning using untargeted metabolomics and to identify endogenous metabolic signatures highly associated with acute insulin poisoning, thereby providing auxiliary evidence for forensic identification of insulin intoxication. Materials and Methods: Rat models were established for the control group, low-dose insulin group, and high-dose insulin group. Ultra-high-performance liquid chromatography coupled with quadrupole-Orbitrap high-resolution mass spectrometry (UPLC-Q-Exactive Orbitrap MS) was employed to profile endogenous serum metabolites. Bioinformatics methods were applied to screen characteristic metabolites related to acute insulin poisoning based on variable importance in projection values from the orthogonal partial least squares-discriminant analysis model and fold change values. Pathway analysis was then performed on the identified metabolites. Results: A total of 18 differential metabolites unrelated to insulin dosage were screened, among which four metabolites were upregulated and 14 were downregulated in the insulin-treated groups. These small-molecule metabolites were primarily enriched in the sphingolipid metabolism and primary bile acid biosynthesis pathways. Conclusions: Insulin poisoning induces alterations in small-molecule metabolites in vivo . The identified differential metabolites may serve as auxiliary indicators for forensic determination of insulin intoxication.