Epoxyeicosatrienoic acids (EETs) are lipid mediators with beneficial effects on metabolic and cardiovascular (CV) health. EETs and other FFA epoxides are quickly metabolized to biologically less active diols by soluble epoxide hydrolase (sEH). Inhibition of sEH, which increases EETs, improves metabolic and CV health and is proposed as an effective strategy to treat diabetes and CV diseases. Despite its importance to health, how sEH is regulated in vivo is poorly understood, particularly in humans where sEH is difficult to study, largely due to limited access to individual tissues where sEH is expressed or lack of appropriate tools to assess sEH activity. sEH activity is often inferred in human studies by measuring FFA epoxide-diol ratios in the blood. As many as 15 FFA epoxide-diol pairs can be measured in blood. We recently developed a new method to estimate sEH activity in vivo, which combines metabolomic determination and simultaneous analysis of all measurable epoxide-diol pairs. This method⎯Simultaneous Analysis of Multiple Indices (SAMI)⎯markedly increases the power to detect changes (or group differences) in sEH activity. This is important, especially in human studies, as increased power allows detection of treatment effects with smaller sample sizes, reducing cost. One objective of this proposal is to evaluate the performance of our new metabolomic approach, SAMI, in detecting changes in sEH activity or EET metabolism under various conditions in animals, before it can be widely applied to human studies. Using SAMI, our recent study showed that sEH activity decreased profoundly after a meal in rats. Interestingly, this effect was completely abolished in animals with gut bacteria depleted by antibiotic treatment, suggesting that gut bacteria may be involved in the postprandial effect. Our preliminary data suggest the intriguing possibility that lithocholic acid (LCA), a secondary bile acid produced by gut bacteria, may be an endogenous inhibitor of sEH, mediating the postprandial regulation of sEH activity. Another objective of this proposal is to validate this novel mechanism of postprandial sEH regulation involving LCA. Thus, the proposed studies will explore a novel mechanism for sEH regulation in vivo and evaluate the performance of our new method to assess sEH activity based on plasma oxylipins. Successful completion of the proposed studies would facilitate studies of sEH regulation in vivo in animals and humans and lead to new therapeutic targets or strategies in the treatment of diabetes or CV diseases.