Improved Radiolabeled Substrates for Soluble Epoxide Hydrolase

doi:10.1006/abio.1995.1520

Analytical Biochemistry

Volume 231, Issue 1, October 1995, Pages 188-200

https://doi.org/10.1006/abio.1995.1520 Get rights and content

Abstract

Two rapid assays for the soluble epoxide hydrolase (sEH) are described. First, a sensitive radiometric assay based on thin-layer chromatography of [¹⁴C]-cis-9,10-epoxystearic acid and its corresponding diol ([¹⁴C]-9,10-dihydroxystearic acid) is described. The cis fatty acid oxide exhibits higher specific activity of hydration with sEH from mouse, rat, human, and potato compared to trans-stilbene oxide (TSO). The K_m and V_max obtained for [¹⁴C]-cis-9,10-epoxystearic acid with mouse sEH are 11.0 μM and 3460 nmol/min/mg protein, respectively. [¹⁴C]-cis-9,10-Epoxystearic acid might more closely mimic the structures of natural substrates for sEH. Second, [2-³H]-trans-1,3-diphenylpropene oxide ([³H]-tDPPO) and [2-³H]-cis-1,3-diphenylpropene oxide ([³H]-cDPPO) were synthesized and rapid radiometric assays for epoxide hydrolases (EHs) were developed by differential partitioning of the epoxide into iso-octane and its corresponding diol into aqueous phase containing methanol. It was shown that sEHs from mouse, rat, human, and potato rapidly hydrolyze [³H]-tDPPO and in comparison to TSO have 20-, 49-, 28-, and 7-fold higher rates, respectively. Mouse sEH hydrates [³H]-tDPPO at 26,200 nmol/min/mg protein, and a K_m of 2.80 μM is observed.

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This research was conducted to evaluate the hypothesis that gastric ulcers caused by the NSAID diclofenac sodium (DCF) can be prevented by the soluble epoxide hydrolase inhibitor TPPU.
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Citation Excerpt :
The linear region of the plot of the standards shown in Supplementary data Table 1 was used for calculating the products formed. Enzyme assays such as hydrolysis of [3H]t-DPPO, c-SO, CMNA and octanoyl-MP were performed to determine the effect of treatment on hydrolases such as sEH (Borhan et al., 1995), microsomal epoxide hydrolase (mEH) (Gill et al., 1983; Morisseau et al., 2008), carboxylesterase 1 (CES1) (Morisseau et al., 2009) and fatty acid amide hydrolase (FAAH) (Morisseau et al., 2009), respectively. Briefly, the S9 fractions were diluted in 0.1 M of sodium phosphate or Tris/HCl buffer containing 0.1 mg/mL of BSA.
Epoxyeicosatrienoic acids (EETs) are potent endogenous analgesic metabolites produced from arachidonic acid by cytochrome P450s (P450s). Metabolism of EETs by soluble epoxide hydrolase (sEH) reduces their activity, while their stabilization by sEH inhibition decreases both inflammatory and neuropathic pain. Here, we tested the complementary hypothesis that increasing the level of EETs through induction of P450s by omeprazole (OME), can influence pain related signaling by itself, and potentiate the anti-hyperalgesic effect of sEH inhibitor. Rats were treated with OME (100 mg/kg/day, p.o., 7 days), sEH inhibitor TPPU (3 mg/kg/day, p.o.) and OME (100 mg/kg/day, p.o., 7 days) + TPPU (3 mg/kg/day, p.o., last 3 days of OME dose) dissolved in vehicle PEG400, and their effect on hyperalgesia (increased sensitivity to pain) induced by PGE₂ was monitored. While OME treatment by itself exhibited variable effects on PGE₂ induced hyperalgesia, it strongly potentiated the effect of TPPU in the same assay. The significant decrease in pain with OME + TPPU treatment correlated with the increased levels of EETs in plasma and increased activities of P450 1A1 and P450 1A2 in liver microsomes. The results show that reducing catabolism of EETs with a sEH inhibitor yielded a stronger analgesic effect than increasing generation of EETs by OME, and combination of both yielded the strongest pain reducing effect under the condition of this study.
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Regular ArticleImproved Radiolabeled Substrates for Soluble Epoxide Hydrolase

Abstract

Regular Article
Improved Radiolabeled Substrates for Soluble Epoxide Hydrolase