Real-world omega-3 supplementation patterns and whole-blood fatty-acid biomarkers: a cross-sectional analysis of 762,150 dried blood spot samples

Abstract Background Long-chain omega-3 polyunsaturated fatty acids, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), as well as compositional measures such as the arachidonic acid-to-EPA ratio (AA/EPA), have been extensively examined in fatty-acid biomarker research and in relation to cardiometabolic risk. However, large-scale characterisation of how these biomarkers and broader fatty-acid profiles vary across real-world omega-3 supplementation-exposure categories remains limited. This study aimed to characterise whole-blood fatty-acid profiles, with particular emphasis on EPA + DHA, AA/EPA, and their distributions across three pre-existing real-world supplementation-exposure groups in a large direct-to-consumer DBS dataset. Methods A retrospective cross-sectional analysis was performed using 762,150 whole-blood DBS samples submitted to a commercial fatty-acid profiling service between 2012 and 2023. Based on self-reported supplementation exposure, samples were classified as non-user, self-supplemented (≥ 120 days), or program-supplemented (≥ 120 days within a structured program including a standardized fish-oil/extra-virgin olive-oil formulation, weight-based dosing guidance, and biomarker testing/feedback). Whole-blood fatty-acid profiles, EPA + DHA, and ln(AA/EPA) were compared across groups after adjustment for age, sex, BMI, and geographic region. Additional analyses examined AA/EPA across comparable EPA + DHA intervals and regional variation in AA/EPA. Results Supplementation group was associated with differences in the measured relative whole-blood fatty-acid profile, with the largest group-related effect sizes observed for long-chain n-3-related measures and AA/EPA. In primary adjusted models, supplementation group had partial η² values of 0.274 for EPA + DHA and 0.352 for ln(AA/EPA). Adjusted mean EPA + DHA was progressively higher across the non-user, self-supplemented, and program-supplemented groups (3.30%, 4.16%, and 5.61%, respectively), while adjusted geometric mean AA/EPA was progressively lower (16.6, 10.9, and 5.2, respectively). The self-supplemented group showed intermediate but broadly dispersed biomarker values. By contrast, the program-supplemented group showed a more compact AA/EPA distribution, lower AA/EPA at comparable EPA + DHA levels below approximately 10%, and less regional heterogeneity in AA/EPA. Conclusions In this large cross-sectional DBS dataset, pre-existing real-world supplementation-exposure groups were associated with differences in covariate-adjusted EPA + DHA and AA/EPA, and biomarker dispersion. Simple self-reported supplement-use categories captured group-level biomarker differences but masked substantial within-category heterogeneity. AA/EPA provided complementary information on fatty-acid profiling not summarized by measured EPA + DHA alone and may help characterize biomarker patterning across real-world omega-3 supplementation contexts. These observational findings should be interpreted as associations rather than evidence of causal effects of specific supplementation strategies.