Analysis of omega-3 and omega-6 fatty acid-derived lipid metabolite formation in human and mouse blood samples

Mass spectrometry techniques have enabled the identification of different lipid metabolites and mediators derived from omega-6 and omega-3 polyunsaturated fatty acids (n-6 and n-3 PUFA) that are implicated in various biological processes. However, the broad-spectrum assessment of physiologically formed lipid metabolites and mediators in blood samples has not been presented so far.Here lipid mediators and metabolites of the n-6 PUFA arachidonic acid as well as the long-chain n-3 PUFA eicosapentaenoic acids (EPA) and docosahexaenoic acid (DHA) were measured in human blood samples as well as in mouse blood. There were detectable but mostly very low amounts of the assayed compounds in human native plasma samples, whereas in vitro activation of whole blood with the calcium ionophore A23187 led to highly significant increases of metabolite formation, with a predominance of the 12-lipoxygenase (12-LOX) products 12-hydroxyeicosatetraenoic acid (12-HETE), 12-hydroxyeicosapentaenoic acid (12-HEPE) and 14-hydroxydocosahexaenoic acid (14-HDHA). A23187 activation also led to significant increases in the formation of 5-LOX products including leukotriene B4 (LTB4), leukotriene B5 (LTB5) as well as of 15-LOX products and prostaglandin E2 (PGE2) and thromboxane B2 (TXB2). Levels were similar or even higher in A23187-activated mouse blood. The approach presented here thus provides a protocol for the comprehensive and concomitant assessment of the generation capacity of n-3 and n-6 PUFA-derived lipid metabolites as well as thromboxanes and prostaglandins in human and murine blood samples. Further studies will now have to evaluate lipid metabolite generation capacity in different physiological and pathophysiological contexts.

Research highlights▶ Low amounts of omega-3 and omega-6 fatty acid-derived lipid metabolites in native human plasma. ▶ Calcium ionophore A23187 activates lipid metabolite formation in blood. ▶ Predominance of 12-lipoxygenase (12-LOX) products. ▶ Significant increases also in the formation of 5-LOX and15-LOX products as well as PGE2 and TXB2.