title: 5,6-δ-DHTL, a stable metabolite of arachidonic acid, is a potential substrate for paraoxonase 1.
abstract: 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±0.01μM/s and Km=150.99±62.1μM. 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.
authors.affiliation: Laboratory of Human Health and Nutrition Sciences, MIGAL-Galilee Research Institute, Ltd., Kiryat Shmona, Israel; Tel-Hai College, Upper Galilee, Israel.
authors.forename: Suzy
authors.initials: S
authors.lastname: Eryanni-Levin
authors.affiliation: Laboratory of Oxidative Stress and Human Diseases, MIGAL-Galilee Research Institute, Ltd., Kiryat Shmona, Israel; Tel-Hai College, Upper Galilee, Israel.
authors.forename: Soliman
authors.initials: S
authors.lastname: Khatib
authors.affiliation: Laboratory of Human Health and Nutrition Sciences, MIGAL-Galilee Research Institute, Ltd., Kiryat Shmona, Israel; Tel-Hai College, Upper Galilee, Israel.
authors.forename: Reut
authors.initials: R
authors.lastname: Levy-Rosenzvig
authors.affiliation: Laboratory of Human Health and Nutrition Sciences, MIGAL-Galilee Research Institute, Ltd., Kiryat Shmona, Israel; Tel-Hai College, Upper Galilee, Israel.
authors.forename: Snait
authors.initials: S
authors.lastname: Tamir
authors.affiliation: Laboratory of Human Health and Nutrition Sciences, MIGAL-Galilee Research Institute, Ltd., Kiryat Shmona, Israel; Tel-Hai College, Upper Galilee, Israel. Electronic address: Andreas@migal.org.il.
authors.forename: Andrea
authors.initials: A
authors.lastname: Szuchman-Sapir
mesh_terms: D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D043303:Aryldialkylphosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D004851:Epoxide Hydrolases / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism*; D007668:Kidney / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D062105:Molecular Docking Simulation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics; D014664:Vasodilation
chemical_list: C103232:5,6-dihydroxyeicosatrienoate-1,5-lactone; D006893:Hydroxyeicosatetraenoic Acids; D011994:Recombinant Proteins; D016718:Arachidonic Acid; C040776:5,6-epoxy-8,11,14-eicosatrienoic acid; C042783:5,6-dihydroxy-8,11,14-eicosatrienoic acid; D043303:Aryldialkylphosphatase; C560867:PON1 protein, mouse; D004851:Epoxide Hydrolases; D015126:8,11,14-Eicosatrienoic Acid
keywords: 5,6-Dihydroxylactone; 5,6-epoxyeicosatrienoic acid; Arachidonic acid metabolism; Paraoxonase 1