Characterization of the cytosolic epoxide hydrolase-catalyzed hydration products from 9,10:12,13-diepoxy stearic esters

Abstract

In a previous report we hypothesized that diepoxy fatty methylesters are metabolized to tetraols and/or tetrahydrofurandiols through an epoxydiol intermediate. In this study, p-nitrophenyldiepoxystearate was incubated with affinity-purified liver cytosolic epoxide hydrolase and product formation was monitored by reverse phase HPLC. The diepoxystearate was converted to the corresponding 9,10,12,13-tetraol using a concentrated enzyme (⩾100 μg/ml). When lower concentration of the enzyme was used, simultaneous elevation of 9,10-epoxy-12,13-dihydroxy and 12,13-epoxy-9,10-dihydroxystearate along with disappearance of tetraol was observed. The epoxydiols were intermediates which could be isolated and cyclized quantitatively to form two chromatographically distinct tetrahydrofurandiols (A with a low R f value and B with a high R f value on TLC). Gas Chromatographic analysis on a cyclodex-β capillary column revealed that each compound was composed of two different isomers. The structure of these isomers was 9(12)-oxy-10,13-dihydroxystearate and 10(13)-oxy-9,12-dihydroxystearate using mass spectrometry. Stereochemistry of the aliphatic chain across the tetrahydrofuran moiety was determined by nuclear Overhauser effect spectroscopy. Chemically and enzymatically generated tetrahydrofurandiols had similar retention time on GC and HPLC, and identical mass spectra using the electron impact mode.