The Influence of Fatty Acid Expression Methods on Associations with Cardiometabolic Biomarkers: Evidence from NHANES 2011–2012

Background/Objectives: Fatty acids (FAs) are critical mediators of human physiology, yet associations between dietary intake, circulating FA levels, and health outcomes remain debated. Circulating FAs are typically reported as a percentage of total FA (% total), whereas absolute concentrations are measured infrequently. Although mathematically related, these metrics provide distinct information. NHANES 2011–2012 uniquely provided serum FA concentrations, enabling direct comparison of concentration and % total within the same individuals. Methods: Serum FAs measured by electron capture negative-ion mass spectrometry in NHANES 2011–2012 were expressed as both absolute concentrations (µmol/L) and % total. Associations of each representation with lipid and non-lipid cardiometabolic biomarkers were evaluated and compared with total FA concentrations. Results: For multiple FAs and biomarkers, the direction of association differed depending on whether FAs were expressed as concentration or % total. These reversals occurred when total FAs varied more strongly with the biomarker than the individual FA concentration, allowing a FA to increase in absolute concentration while decreasing as a percentage of the total FA pool. Reversals were observed for several major FAs, including linoleic acid (LA), arachidonic acid (ARA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), and stearic acid (SA). Total FA concentrations were strongly associated with most biomarkers. Conclusions: FA concentrations and % total are complementary, not interchangeable, measures that capture distinct aspects of circulating FA biology. Associations with outcomes can be in opposite directions when the total plasma FA pool varies. These differences reflect underlying mathematical and biological relationships rather than methodological error. Future studies should routinely evaluate the total FA pool size alongside FA composition. Examining both representations alongside total FA can help distinguish changes in absolute FA abundance from changes in relative composition and provide a more complete interpretation of FA associations.