Paraoxonase 1 hydrolysis of EPA-derived lactone impairs endothelial-mediated vasodilation.

Human serum paraoxonase-1 (PON1) is a lactonase that plays a significant role in anti-atherosclerotic high-density lipoprotein (HDL) activity. PON1 is also localized in endothelial cell membranes, where it is enzymatically active and regulates endothelial signals. PON1 has a high specificity for lipophilic lactones and has been shown to hydrolyze and regulate lactone lipid mediators derived from arachidonic polyunsaturated fatty acids (PUFA). Previously, we showed that an arachidonic acid lactone metabolite (AA-L) dose-dependently dilates PON1 gene deletion (PON1KO) mouse mesenteric arteries significantly more than wild-type arteries. In contrast, preincubation with HDL or rePON1 reduced AA-L-dependent vasodilation. Recently we showed that an additional δ-lactone metabolite derived from the eicosapentaenoic acid lactone, 5,6-δ-DiHETE lactone (EPA-L) reduced blood pressure by dilating microvessels of hypertensive rats. However, whether PON1 regulates the activity of the EPA-L lipid mediator is unknown. AIM: To demonstrate that PON1 hydrolyzes EPA-L and to reveal the effect of this hydrolysis on endothelial-dependent vascular dilation. METHODS AND RESULTS: In vascular reactivity experiments, EPA-L dose-dependently dilated PON1KO mouse mesenteric arteries significantly more than wild-type mesenteric arteries. This dilation was not affected by nitric oxide inhibition. PON1 impaired the cellular calcium increase mediated by EPA-L in endothelial cells, though this impairment decreased with PON1 internalization to the cell. CONCLUSION: These findings support that PUFA-lactones are physiological substrates of PON1, and that PON1 activity in the endothelial membrane affects the dilation of microvessels that is induced by these endothelial-derived hyperpolarizing PUFA-lactones.

Fishing for lipid lactones using selective reaction and characteristic fragmentation pattern.

Extensive research has been invested in developing sensitive methods to identify lipid mediators (LMs) from multiple biological matrices. Previous studies point to the existence of a potential family of lactone-containing metabolites generated from eicosanoid families, isoprostanes, and prostanoid-like compounds that may function as LMs. However, targeted lipidomic studies do not routinely include lactone-containing lipids due to their low ionizability and instability under some common sample preparation conditions. Thus, the discovery of lactone-containing LM is limited. Herein we describe a method for selective identification of lipid lactones from within biological matrices. This method is based on a selective reaction of lactones with 1-(3-aminopropyl)imidazole, followed by cation exchange solid phase extraction and the identification of characteristic fragmentation patterns unique to reaction products of lactones in LC/MS/MS. NMR and LC/MS results indicated that saturated and unsaturated aliphatic ɣ and δ lactone model compounds mixed with human serum were successfully detected. MS/MS analyses of the reaction products revealed a unique pattern for the lactones, resulting from common neutral losses and fragmentation. When applied to esters and free fatty acids, some reaction products were observed. However, these reaction products' MS/MS fragmentation did not match the specific fragmentation of the lactones' reaction products. Confirming that lactones can be detected in a highly selective manner from within complex biological matrices when using the presented method. Thus, the presented method can selectively analyze lactones and may further complement existing lipidomic approaches to discover new LMs.