Dried blood spot lipid mediator profiling during CPET-ESE enables heart failure phenotyping

Introduction Differentiating heart failure with preserved ejection fraction (HFpEF) from reduced ejection fraction (HFrEF) remains challenging, and exercise intolerance is a hallmark of HFpEF. Oxylipins and their polyunsaturated fatty-acid (PUFA) precursors integrate metabolic and immune signalling, but repeated venous sampling during exercise is burdensome. Purpose To develop and validate a minimally invasive dried blood spot (DBS) workflow for targeted oxylipin/PUFA profiling, and to apply it during cardiopulmonary exercise testing combined with exercise stress echocardiography (CPET-ESE) to characterise inflammatory lipid dysregulation across the heart-failure spectrum. Methods A MEPS-UHPLC-MS/MS method quantified 52 oxylipins (isoprostanoids, prostanoids, hydroxy-/epoxy-fatty acids and specialised pro-resolving mediators) and 4 PUFAs (AA, EPA, DHA, DPA n-3) from a single 50 uL DBS. Labelled internal standards were pre-spotted on antioxidant-treated filter paper. Method performance (LOD/LOQ, recovery, precision, matrix effect) and stability were assessed. We enrolled 89 subjects (Stage A-B, n=33; HFpEF, n=33; HFrEF, n=23) who underwent CPET-ESE with DBS collected at rest (t0), peak effort (t1; RER=1.0) and recovery (t2; 2.5 min post-peak). Results Analytical performance was robust: LODs were 10–1260 pg/mL for oxylipins and 90–780 ng/mL for PUFAs, with linearity R²=0.97–0.99. Recovery was 93–107% (intra-day) and 96–107% (inter-day), and precision was ≤12%. Internal standards on antioxidant-treated cards and methanolic MEPS extracts were stable for up to 4 weeks at 4°C and −80°C, supporting DBS-based logistics for exercise testing. DBS-to-plasma concordance was analyte-specific, with AA, DHA and PGE2 showing strong correlations (r>0.5; p<0.001) that were independent of haematocrit (35–50%), supporting reliable DBS quantification of these key targets in clinical settings. HFpEF was older (78±11 vs. 67±15 and 65±13 years; p<0.001) and had lower peak VO2 (12.7±2.6 vs 18.5±4.8 mL/kg/min; p<0.001). HFrEF showed reduced LVEF (36±8%; p<0.001) and the lowest cardiac output at rest and peak (4.1±1.4 and 7.3±2.2 L/min; p≤0.031). RV–PA coupling at peak exercise was most impaired in HFpEF (TAPSE/sPAP 0.43±0.15; p<0.001). At rest, AA differed between HFpEF and HFrEF (p<0.05) and omega-3 PUFAs were lower in HFrEF (p<0.05). During exercise, HFpEF showed decreases in AA and 8,9-EET at peak (p<0.01) with AA remaining altered at recovery (p<0.01), enabling phenotypic discrimination. Conclusions DBS enables robust, minimally invasive targeted profiling of PUFAs and oxylipins during exercise testing. AA and 8,9-EET dynamics, together with omega-3 PUFA depletion in HFrEF, may support earlier and more accurate phenotyping and provide mechanistic insight into metabolic-immune dysregulation across heart-failure stages.Resting EPA(A), DHA(B), AA(C), DPA(D) levels.