Total cholesterol and triglycerides

Blood was collected during terminal procedures after fasting (5 h) and spun to isolate serum, then stored at −80 °C. Serum samples were subsequently analyzed for insulin and IL-10 levels using ELISAs (Millipore and R&D Systems, respectively) or total cholesterol and triglycerides (Sigma). For eicosanoid analysis, PGE₂ was extracted from serum in methanol and isolated by C18 solid-phase extraction chromatography. PGE₂-d₄ (Cayman Chemicals, Ann Arbor, MI, USA) was added to the experimental system as an internal standard. PGE₂ was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a TSQ Quantum Ultra triple quadrupole mass spectrometer coupled to an Accela 1250 UHPLC system (Thermo Scientific, Hemel Hempstead, UK) in negative ion mode with a C18 column and methanol-water gradient. Quantification was performed using the multiple reaction monitoring (MRM) mode using the transitions PGE₂ (351 → 271) and PGE₂-d₄ (355 → 193). The concentration of PGE₂ was determined by comparison to a calibration curve run in parallel.

Blood was collected during terminal procedures after fasting (5 h) and spun at 5000×g to isolate serum, then stored at –80°C. Serum samples were subsequently analysed for insulin using ELISA (R&D Systems) or total cholesterol and triglycerides (Sigma).