5,6-δ-DHTL, a stable metabolite of arachidonic acid, is a potential substrate for paraoxonase 1

Paraoxonase 1 (PON1) is an antiatherogenic high density lipoprotein-associated lactonase. Recent findings revealed that PON1 knockout mice have low blood pressure, which is negatively correlated with the level of 5,6-epoxyeicosatrienoic acid (5,6-EET), a cytochrome P450 -derived arachidonic acid metabolite. 5,6-EET is an endothelium-derived hyperpolarizing factor that causes arterial dilation. Under physiological conditions, 5,6-EET is unstable, transforming to its δ-lactone (5,6-δ-DHTL) that evades the degradation by soluble epoxide hydrolase (sEH), arguing for the existence of yet another enzyme that is responsible specifically for its hydrolysis. We therefore hypothesized that PON1 degrades the 5,6-δ-DHTL, and this specific PON1 lactonase activity thus decreases endothelial vasodilatation. The aim of the present study was to investigate the PON1-5,6-δ-DHTL relationship. A liquid chromatography mass spectrometry based method for 5,6-EET derivatives identification was developed. Tracking the lactonization of 5,6-EET in a physiological solution revealed that 5,6-EET was fully converted into 5,6-δ-DHTL. Incubation of 5,6-δ-DHTL with rePON1 resulted in 85.1 ± 3.4% degradation of the substrate to 5,6 dihydroxytrienoic acid (5,6-DHET), while only 12.0 ± 8.7% hydrolysis was detected in the absence of PON1. Accordingly, the levels of 5,6-DHTL were found to be significantly higher in the PON1KO mice than in the wild type mice. Kinetic analysis revealed values of Vmax = 0.021 ± 0.01 μM/s and Km = 150.99 ± 62.1 μM. Calculation of the docking energy suggested possible interaction of the 5,6-δ-DHTL in the catalytic region of PON1 with free energy of -5.57 Kcal/mol, preferentially for the (S) enantiomer. These findings demonstrate that 5,6-δ-DHTL is a PON1 substrate and imply that the 5,6-EET vasodilation effect may be impaired by PON1.