Validated bioanalysis of oxylipins confirms specialized pro-resolving mediator formation in vitro and in vivo.

Specialized pro-resolving mediators (SPM) are di- or trihydroxylated polyunsaturated fatty acids with potent inflammation-resolving features. SPM typically occur at lower concentrations than classical pro-inflammatory eicosanoids, making their detection highly challenging. Thus, reporting of biological SPM levels has been discrepant and not always transparent, sparking uncertainty concerning the formation capacity for di- and especially for trihydroxylated SPM in vitro and in vivo. Here, we create common ground by providing a systematic and comprehensive assessment of SPM formation across a broad range of commonly employed in vitro, ex vivo, and in vivo matrices. For this purpose, a quantitative UHPLC-MS/MS method targeting 72 oxylipins (including 19 SPM) was validated in accordance with recommendations by the International Lipidomics Society. By including sample preparation (solid phase extraction) in all validation processes, a conservative lower limit of quantification (10-100 pg/ml matrix) was found to meet clearly defined criteria for signal/noise, accuracy, precision, selectivity, specificity, matrix effects, recovery, and carry-over for 19 SPM. Our results offer analytically defensible reference data to demonstrate (i) the absence of relevant SPM levels in stimulated whole blood and unstimulated cell models; (ii) only trace SPM formation in stimulated peripheral blood mononuclear cells, M1-macrophages, neutrophils, and platelet incubations; but (iii) significant SPM formation in stimulated M2a-macrophages and neutrophil/platelet co-incubations. In healthy C57BL/6JRj mice, SPM formation was low yet organ-specific, with relevant amounts of dihydroxylated SPM detected in spleen. Taken together, we demonstrate SPM formation to be robust, matrix- and stimulus-dependent, but confined to specific di- and trihydroxylated SPM.