Unbound DHA causes a high blank value in β‐oxidation assay: a concern for in vitro studies

The information on binding capacity of different fatty acids (FAs) to albumin is incomplete, however, in the majority of in vitro experiments, FAs and albumin were simply mixed and their affinity believed to be complete. In this study, seven 1‐¹⁴C] FAs were mixed with albumin and assayed for β‐oxidation in rat liver homogenates. In the process of identifying the radioactive background of control assay by LCMS/MS, the results indicated different binding capacity of FAs to albumin. The percentage of unbound FAs recovered in clarified acidic solution was lower than 2% with 16:0 and 18:1n‐9, nearly 5% with EPA, 7% with 18:2n‐6, 18:3n‐3 and 20:4n‐6, and surprisingly high to 41% with DHA. Various FA/albumin molar ratios as well as different types of albumin only marginally affected these data. Thus, the big mass of unbound free DHA led to a high blank value, which is 60 times higher than the real value in the procedure of β‐oxidation measurement in vitro. In the design of future FA research in vitro, the binding capacity of FA to albumin or other proteins must be considered, especially for DHA research.