A combination study of spin-trapping, LC/ESR and LC/MS on carbon-centred radicals formed from lipoxygenase-catalysed peroxidation of eicosapentaenoic acid

Abstract

Increased evidence from animal and in vitro cellular research indicates that the metabolism of eicosapentaenoic acid (EPA) can inhibit carcinogenesis in many cancers. Free radical-mediated peroxidation is one of many possible mechanisms to which EPA's anti-cancer activity has been attributed. However, no direct evidence has been obtained for the formation of any EPA-derived radicals. In this study, a combination of LC/ESR and LC/MS was used with α-[4-pyridyl 1-oxide]-N-tert-butyl nitrone to identify the carbon-centred radicals that are formed in lipoxygenasecatalysed EPA peroxidation. Of the numerous EPA-derived radicals observed, the major products were those stemming from β-scission of 5-, 15-and 18-EPA-alkoxyl radicals. By means of an internal standard in LC/MS, this study also quantified each radical adduct in all its redox forms, including an ESR-active form and two ESR-silent forms. The comprehensive profile of EPA's radical formation provides a starting point for ongoing research in defining the biological effects of radicals generated from EPA peroxidation.

The first characterization of free radicals formed from cellular COX-catalyzed peroxidation

“…Other MS settings were the same as the scan mode listed above except the number of scans, 5. For quantification of hydroxylamines, a small amount D 9 -POBN (internal standard as described elsewhere [30–31]) was added to cells and culture media with a stop solution (ACN), and SPE extraction as well as LC/MS analysis was then conducted. The amount of hydroxylamine generated in cellular peroxidation and the spin trapping reaction was normalized to the numbers of cells at different incubation time points, in which total live cells could be significantly different.…”

Characterization of novel radicals from COX-catalyzed arachidonic acid peroxidation

“…This complete reaction mixture (∼1% ethanol, v/v , from an AA stock solution) was incubated at 37°C and 600 rpm on a Thermo-Shaker in the absence of light. After a 30 min incubation, the COX-catalyzed peroxidation was directly analyzed by off-line ESR or immediately stopped by mixing with ACN for the analysis of on-line LC/ESR and LC/MS as described elsewhere [17-18]. Free radicals from cellular COX-mediated peroxidation were generated with PC-3 cells grown in RPMI 1640 medium supplemented with 10% FBS in an incubator containing a humidified atmosphere of 5% CO 2 at 37°C.…”

“…POBN and AA were then added to the cell suspension (final concentrations of 20 mM and 0.5 mM, respectively) to start the AA peroxidation and POBN spin trapping reaction. After a 30-min incubation, the PC-3 cell suspension was used to directly measure radical formation via off-line ESR or was processed for LC/ESR and LC/MS analysis with enzyme denaturation (mixing with 1:1 ACN, v/v , to stop the cellular peroxidation), supernatant collection (centrifuging the ACN-suspension mixture at 10,000 rpm for 10 min), and sample condensation (in Vacufuge™ 5301 concentrator to evaporate the added ACN) [17-18]. …”

“…After precipitating the COX enzyme and removing ACN as described elsewhere [17-18], the enzyme-free condensed samples were analyzed immediately using on-line LC/ESR and LC/MS for the characterization of POBN adduct structures, or stored at -80°C for later analysis. The online LC/ESR system consisted of an Agilent 1200 series HPLC system and the same Bruker EMX ESR system.…”

“…Our results demonstrate that three types of spin trapped carbon-centered radicals are formed from either β-scission or structural rearrangement of PGF 2 -type alkoxyl radicals. Unlike the radical reaction we observed in lipoxygenase (LOX)-catalyzed peroxidation of many types of PUFAs [15-18], a special β-scission during COX-catalyzed AA peroxidation leads to formation of free radicals centered on the C=C bond nearest the PGF ring and formation of a novel non-radical product, 1-hexanol, rather than an aldehyde (a common β-scission product). The characterization of these novel products formed from peroxidation in vitro , including cellular peroxidation of a human prostate cancer cell line (PC-3), improves our knowledge of COX-mediated peroxidation and will assist in further studying of the role of COX peroxidation in cancer biology.…”