Title: Mammalian soluble epoxide hydrolase is identical to liver hepoxilin hydrolase Cronin A, Decker M, Arand M Ref: J Lipid Res, 52:712, 2011 : PubMed
Hepoxilins are lipid signaling molecules derived from arachidonic acid through the 12-lipoxygenase pathway. These trans-epoxy hydroxy eicosanoids play a role in a variety of physiological processes, including inflammation, neurotransmission, and formation of skin barrier function. Mammalian hepoxilin hydrolase, partly purified from rat liver, has earlier been reported to degrade hepoxilins to trioxilins. Here, we report that hepoxilin hydrolysis in liver is mainly catalyzed by soluble epoxide hydrolase (sEH): i) purified mammalian sEH hydrolyses hepoxilin A(3) and B(3) with a V(max) of 0.4-2.5 mumol/mg/min; ii) the highly selective sEH inhibitors N-adamantyl-N'-cyclohexyl urea and 12-(3-adamantan-1-yl-ureido) dodecanoic acid greatly reduced hepoxilin hydrolysis in mouse liver preparations; iii) hepoxilin hydrolase activity was abolished in liver preparations from sEH(-/-) mice; and iv) liver homogenates of sEH(-/-) mice show elevated basal levels of hepoxilins but lowered levels of trioxilins compared with wild-type animals. We conclude that sEH is identical to previously reported hepoxilin hydrolase. This is of particular physiological relevance because sEH is emerging as a novel drug target due to its major role in the hydrolysis of important lipid signaling molecules such as epoxyeicosatrienoic acids. sEH inhibitors might have undesired side effects on hepoxilin signaling.
Inhibition of the soluble epoxide hydrolase (sEH) has beneficial effects on vascular inflammation and hypertension indicating that the enzyme may be a promising target for drug development. As the enzymatic core of the hydrolase domain of the human sEH contains two tyrosine residues (Tyr(383) and Tyr(466)) that are theoretically crucial for enzymatic activity, we addressed the hypothesis that the activity of the sEH may be affected by nitrosative stress. Epoxide hydrolase activity was detected in human and murine endothelial cells as well in HEK293 cells and could be inhibited by either authentic peroxynitrite (ONOO(-)) or the ONOO(-) generator 3-morpholino-sydnonimine (SIN-1). Protection of the enzymatic core with 1-adamantyl-3-cyclohexylurea in vitro decreased sensitivity to SIN-1. Both ONOO(-) and SIN-1 elicited the tyrosine nitration of the sEH protein and mass spectrometry analysis of tryptic fragments revealed nitration on several tyrosine residues including Tyr(383) and Tyr(466). Mutation of the latter residues to phenylalanine was sufficient to abrogate epoxide hydrolase activity. In vivo, streptozotocin-induced diabetes resulted in the tyrosine nitration of the sEH in murine lungs and a significant decrease in its activity. Taken together, these data indicate that the activity of the sEH can be regulated by the tyrosine nitration of the protein. Moreover, nitrosative stress would be expected to potentiate the physiological actions of arachidonic acid epoxides by preventing their metabolism to the corresponding diols.
        
Title: Rapid determination of soluble epoxide hydrolase inhibitors in rat hepatic microsomes by high-performance liquid chromatography with electrospray tandem mass spectrometry Watanabe T, Hammock BD Ref: Analytical Biochemistry, 299:227, 2001 : PubMed
A rapid and reliable electrospray tandem mass spectrometric method for soluble epoxide hydrolase (sEH) inhibitors in rat hepatic microsomes is described. Four synthesized sEH inhibitors were extracted from rat hepatic microsomes with ethyl acetate and were determined by HPLC using positive ion electrospray tandem mass spectrometry within 7 min. The relationship between signal intensity and concentration of sEH inhibitors was linear over the concentration range of 2.0 to 500 ng/mL per 5-microL injection with the use of a noncoeluting internal standard with a similar chemical structure. The intraassay precision was less than 12.4% relative standard deviation and accuracy ranged from -7.0 to 11.3% deviation from the theoretical values with five duplicate assays. The recovery of sEH inhibitors from rat hepatic microsomes, fortified at levels of 50, 100, and 250 ng/mL, averaged 74.2-107.7% with a RSD of 2.1-7.6%. This method was successfully applied to the quantification of residual sEH inhibitors in rat hepatic microsomes without interference.