Fetal bovine serum (FBS) has been used as a universal supplement in cell culture for more than six decades. This includes the investigation of lipid and lipid mediator formation and biology. Little is known about the (polyunsaturated) fatty acid composition and their oxidation products in FBS. Therefore, we analyzed six different FBS purchased from three different companies regarding their fatty acid and oxylipin concentrations. We found pronounced differences in the fatty acid concentrations. Even two batches of "standardized" FBS batches from one company showed drastic differences (e.g., for eicosapentaenoic acid 5 AE 1 μM vs. 11 AE 1 μM). Oxylipin concentrations also markedly differ between the FBS lots. The highest differences were found for 12-lipoxygenase products (e.g., 12-hydroxyeicosatetraenoic acid free 21-87 nM and total 58-108 nM), probably due to inconsistent serum generation procedures. Our results indicate that for cell culture studies dealing with lipid metabolism, researchers should carefully characterize their used FBS to ensure reliability and reproducibility of study outcomes.

Several eicosanoids and other oxylipins are potent lipid mediators, which are involved in the regulation of physiological functions such as inflammation. Therefore, oxylipins have raised strong interest in clinical lipidomics notably to investigate the modulation of oxylipin pattern in vivo in response to dietary supplementation of polyunsaturated fatty acids (PUFA) or to pharmaceutical inhibition of the enzymes of the arachidonic acid cascade. However, the outcome of such clinical trials might be massively influenced by (unsuitable) sample handling during sample generation and sample preparation leading to artificial formation or degradation of oxylipins. Especially the oxylipin stability during the transitory stage between blood collection and freezing of plasma is crucial since samples are often stored for different time lengths before analysis in longitudinal studies and daily clinical routine. We therefore investigated the influence of different storage times and temperatures of whole blood and plasma on the total oxylipin concentration in human plasma. We show that almost all oxylipin classes remain stable up to 24 h at 4°C or 4 h at 20°C before centrifugation of blood collected in EDTA tubes and up to 5 days at -20°C after centrifugation. Only drastic intervention in storage conditions significantly increased concentrations of total oxylipins. Interestingly, no correlation between the affected oxylipin concentration and storage time, temperature or stage of plasma generation could be observed. When plasma samples are stored up to 6 h at 4°C after centrifugation, slightly increased concentrations of hydroxy fatty acidsparticularly 12-lipoxygenase productswere found revealing residual enzymatic activity presumably mediated by resuspended platelets after centrifugation. At these time points, autoxidatively formed isoprostanes were elevated as well.In conclusion, oxylipins are robust against delays during plasma generation, however, samples should be kept cool and centrifugation as well as freezing of the samples should be performed as soon as possible to avoid ex vivo formation or degradation of oxylipins.