Development of an <i>In Vitro</i> Spiked Lipoprotein Lipase Activity Assay in Serum and Plasma for Evaluation of Substrate and Donor-Specific Lipase Activity

Lipoprotein lipase (LPL) is a membrane-bound, water-soluble enzyme that hydrolyzes triglycerides (TAG) into diacylglycerides (DAG), monoacylglycerides (MAG), and free fatty acids (FFA), enabling lipid uptake and energy distribution across tissues. We developed and validated a mass spectrometry-based in vitro assay to quantify LPL activity and assess interindividual variation in serum and plasma samples. Assay conditions were optimized for LPL concentration, calcium chloride concentration, and incubation time to maximize enzymatic activity. Samples were incubated with and without exogenous LPL and analyzed using targeted lipidomics to quantify changes in TAG, DAG, and FFA species. The assay demonstrated high reproducibility in quality control plasma over 10 days. We observed consistent reductions in TAG species and corresponding increases in DAG and FFA species, which varied according to fatty acid composition. Notably, LPL showed reduced catalytic efficiency for TAG and DAG species containing long-chain polyunsaturated fatty acids (PUFAs). Application of the assay to serum samples from 31 healthy volunteers confirmed these substrate-specific patterns, demonstrating the assay's potential to probe LPL function in physiological and pathophysiological states.