Optimizing the uptake of deuterated docosapentaenoic acid by salmonid liver cells using a uniform shell design and liquid chromatography triple-quadrupole mass spectrometry

Stable isotope-labelled fatty acids are valuable tracers for studying lipid metabolism in cell models. However, published methods rarely provide guidance on selecting the optimal concentration and incubation time to ensure accurate and reproducible measurements of cellular uptake and metabolism that reflect physiological conditions. This study systematically evaluated the uptake of deuterated docosapentaenoic acid (DPA-d₅) in Atlantic salmon (Salmo salar L.) liver cells using a uniform shell design methodology. Cultures were exposed to DPA-d₅ at concentrations ranging from 0 to 100 μM for 24, 48, or 72 h and the uptake was quantified in cell lysates by liquid chromatography-mass spectrometry. Results indicate that the concentration of DPA-d₅ has the strongest positive influence on signal intensity. Incubation time slightly reduces the signal, but when combined with higher concentration, it increases the uptake of DPA-d₅. Modelling of the data allowed selecting 60 μM DPA-d₅ and 48 h as the optimal culturing condition that provides a balance between achieving a strong signal and minimizing the risk of oversaturation or metabolic artifacts. This work provides a methodological foundation for future fatty acid tracer experiments in aquaculture research.