**Background:** Non-alcoholic fatty liver disease (NAFLD) affects 20–30% of adults in the Western world and can progress to non-alcoholic steatohepatitis (NASH), cirrhosis, and liver failure. The pathophysiology involves insulin resistance and oxidative stress, with lipotoxicity playing a key role. Oxylipins are bioactive lipid peroxidation products of polyunsaturated (PUFA) and saturated (SFA) fatty acids with inflammatory and immune regulatory properties. This study aimed to measure plasma levels of PUFA and SFA oxylipins in NAFLD patients across progressive stages of severity and evaluate their potential as biomarkers for steatosis grading.
**Methods:** Ninety adults (40–60 years) with metabolic syndrome and NAFLD diagnosed by magnetic resonance imaging–estimated proton density fat fraction (MI-PDFF) were recruited in the Balearic Islands, Spain. Participants were grouped by intrahepatic fat content (IFC): IFC0 (<6.4%, n=19), IFC1 (6.4–17.4%, n=42), IFC2 (17.4–22.1%, n=19), and IFC3 (≥22.1%, n=10). Plasma oxylipins and free fatty acids were measured using solid phase extraction and HPLC-MS/MS. Malondialdehyde (MDA) was measured colorimetrically. Statistical analysis used one-way ANOVA with Bonferroni post hoc tests, Pearson correlations, and receiver operating characteristic (ROC) curve analysis.
**Key Results:** No significant differences in BMI were observed between groups (all ~32–34 kg/m²). The IFC3 group had significantly higher glucose (141 vs 102–109 mg/dL, p<0.001), HbA1c (6.6% vs 5.7–5.9%, p=0.002), and triglycerides (218 vs 130–199 mg/dL, p=0.010) compared to other groups. HDL-c was significantly lower in IFC3 (40.2 mg/dL) vs IFC0 (48.7 mg/dL, p=0.004). ALT was increased in IFC2 (42.0 U/L) and IFC3 (49.0 U/L) vs IFC0 (27.1 U/L) and IFC1 (28.3 U/L) (p=0.002). GGT was higher in IFC3 (51.0 U/L) vs IFC0 (26.5 U/L) (p=0.010). MDA levels were significantly increased in IFC2 (1.98 nM) vs IFC0 (1.21 nM) (p=0.038).
Plasma levels of AA, EPA, ETA, MaR1, LXB4, 3HMYR, 16HPAL, 12HEST, and PGF2α were significantly higher in IFC3 compared to all other groups (ANOVA p values: 0.002, 0.006, 0.008, 0.030, 0.018, 0.035, 0.001, 0.002, and 0.023 respectively). For example, AA was 765 nM in IFC3 vs 67.7 nM in IFC0; 12HEST was 345 nM in IFC3 vs 59.3 nM in IFC0. IFC was significantly correlated with 12HEST (r=0.438, p=0.002), 17HDoHE (r=0.488, p<0.001), 15HETE (r=0.442, p=0.001), AA (r=0.572, p<0.001), EPA (r=0.424, p=0.002), 16HPAL (r=0.239, p=0.050), MaR1 (r=0.307, p=0.032), ETA (r=0.513, p=0.003), 3HMYR (r=0.286, p=0.046), LXB4 (r=0.289, p=0.044), PGF2α (r=0.241, p=0.047), and MDA (r=0.222, p=0.023).
ROC analysis showed that PGF2α plasma levels >0.675 nM distinguished IFC3 from IFC0–IFC2 with an AUCROC of 0.748 (95% CI: 0.537–0.958), 80% true positive rate, and only 14% false positive rate. 12HEST >30 nM distinguished IFC3 with AUCROC 0.694 (95% CI: 0.477–0.910), 80% sensitivity, but 53% false positives. 15HETE >0.675 nM also distinguished IFC3 with AUCROC 0.689 (95% CI: 0.484–0.895), 80% sensitivity, 14% false positives. Free ETA >0.42 nM distinguished any steatosis (IFC1–IFC3) from no steatosis (IFC0) with AUCROC 0.647 (95% CI: 0.525–0.768), 82% sensitivity, but 79% false positives.
**Clinical Implications:** The study demonstrates that NAFLD progression, particularly to the severe stage (IFC3), is associated with marked increases in plasma free PUFAs and both polyunsaturated and saturated oxylipins, reflecting enhanced insulin resistance, oxidative stress, and dysregulation of mitochondrial β-oxidation and peroxisomal ω-oxidation pathways. The coexistence of pro-inflammatory (PGF2α) and pro-resolving (MaR1, LXB4) oxylipins in severe NAFLD suggests simultaneous activation of inflammation and resolution processes. Plasma 12HEST and PGF2α show promise as novel biomarkers for non-invasive grading of NAFLD steatosis, though with limited specificity for 12HEST. These findings may aid in developing blood-based tests to monitor NAFLD progression without reliance on liver biopsy.