Characterization of novel radicals from COX-catalyzed arachidonic acid peroxidation

Abstract

The peroxidation of arachidonic acid (AA) catalyzed by cyclooxygenase (COX) is a well-known free radical-mediated process that forms many bioactive products. Because of a lack of appropriate methodologies, however, no comprehensive structural evidence has been found previously for the formation of COX-mediated and AA-derived free radicals. Here we have used a combination of LC/ESR and LC/MS with a spin trap, alpha-[4-pyridyl-1-oxide]-N-tert-butylnitrone (POBN), to characterize the carbon-centered radicals formed from COX-catalyzed AA peroxidation in vitro, including cellular peroxidation in human prostate cancer cells (PC-3). Three types of radicals with numerous isomers were trapped by POBN as ESR-active peaks and MS-active ions of m/z 296, 448, and 548, all stemming from PGF(2)-type alkoxyl radicals. One of these was a novel radical centered on the carbon-carbon double bond nearest the PGF ring, caused by an unusual beta-scission of PGF(2)-type alkoxyl radicals. The complementary nonradical product was 1-hexanol, another novel beta-scission product, instead of the more common aldehyde. The characterization of these novel products formed from in vitro peroxidation provides a new mechanistic insight into COX-catalyzed AA peroxidation in cancer biology.

Figure 1

Off-line ESR spectra from the complete COX-AA reaction system (incubated 30 min) and its relevant controls. (A) ESR spectrum of complete reaction system (100 mM Tris-Cl buffer, pH 8.0) containing 100 mM POBN, 50 μM hematin, 5 KUnits/mL COX1 or COX2, 5 mM hydroquinone, and 2 mM AA. A six-line ESR signal (a^N ≈ 15.63 G, a^H ≈ 2.53 G) overlapped a five-line signal (benzosemiquinone radicals) at the center of the spectrum; (B) ESR spectrum of the reaction excluding POBN. A five-line signal of benzosemiquinone radical was observed (a^H ≈ 2.35 G); (C) ESR spectrum of the reaction excluding COX. A combination of five- and six-line ESR signals was measured at much lower intensities than that of the complete reaction system; and (D) ESR spectrum of the reaction excluding AA. A five-line benzosemiquinone radical signal dominated a very weak six-line ESR signal (a^N ≈ 15.65 G, a^H ≈ 2.75 G), which represents the POBN adduct of α-hydroxyethyl radical (^•CH(OH)CH₃) formed from ethanol oxidation (1% ethanol, v/v, present in the system because the AA-ethanol stock solution was replaced by ethanol). Note that magnetic field scans were all centered at 3494.4 G, and the low magnetic field for the first line of the six-line POBN adduct spectrum (marked as ‘*’ in Figure 1A) was fixed and used to monitor radical adducts in on-line LC/ESR measurements (Figure 2). However, the field (∼3474 G) for off-line measurements differed slightly from the on-line field in Figure 2 because different ESR cells were used.

Figure 2

On-line LC/ESR and LC/MS chromatograms of complete COX-AA reaction system (incubated 30 min). (A) UV chromatogram at 265 nm using a C₁₈ column and combination of gradient and isocratic elution (details described in Materials and Methods). Peaks of interest are labeled by retention time (min); (B) ESR chromatogram with ESR magnetic field fixed on the maximum of the first line of the six-line signal. There was a nine second offset between the UV and the ESR detection due to the connections. All ESR-active peaks are labeled by number as well as the retention time (min); (C) Projected EIC of m/z 548, m/z 448, and m/z 296 (from on-line LC/MS, full scan from m/z 50 to m/z 600). There was an offset of thirty-five seconds between the UV and MS measurements due to the connection settings. All MS-active peaks are also labeled with their corresponding ESR-active peaks numbers. Note that the first two minutes of LC eluant always bypassed the MS detector and the peaks on the EIC marked with ‘X’ were the false peaks. The fixed field on the first line of the POBN adduct was shifted to ∼3498 G in on-line LC/ESR from ∼3474 G in off-line ESR (asterisked in Figure 1A) due to different resonance frequencies of the flat cell and the Aquax cell.

Figure 3

Individual EIC of complete COX-AA reaction system (incubated 30 min). (A-B) EICs of m/z 296 and m/z 305 from the POBN and D₉-POBN spin trapping experiments, respectively; (C-D) EICs of m/z 448 and m/z 457 from the POBN and D₉-POBN spin trapping experiments, respectively; and (E-F) EICs of m/z 548 and m/z 557 from the POBN and D₉-POBN spin trapping experiments, respectively. The retention time differences between POBN- and D₉-POBN-trapped radicals were inconsistent during isocratic elution (5-25 min). Note that all isomers of each adduct are labeled by their retention times in EICs, while peaks marked by ‘X’ were the false ions because they are ESR-silent and not confirmed in the D₉-POBN experiments.

Figure 4

LC/MS² analysis of spin adduct of the ^•C₂₀H₃₄O₅ radical. (A-B) LC/MS² (positive ion mode) of m/z 548 and m/z 557 from POBN and D₉-POBN spin trapping experiments, respectively; (C-D) LC/MS² (negative ion mode) of m/z 546 and m/z 555 from POBN and D₉-POBN spin trapping experiments, respectively. Fragment ions of m/z 289 and m/z 445 (bold) in the POBN experiment corresponded to those of m/z 298 and m/z 454 in the D₉-POBN experiment, suggesting the radical trapping site of the spin adduct as described in Scheme 1-A. Note that although the same fragmentation pattern was observed in LC/MS² analysis of its 12 isomers, the spectra failed to offer enough information to distinguish differences among the relevant isomers.

Figure 5

LC/MS² analysis of spin adduct of the ^•C₁₄H₂₁O₄ radical. (A) LC/MS² of m/z 448 from the COX-AA reaction in the presence of POBN; (B) LC/MS² of m/z 447 from the COX-AA reaction in the presence of D₉-POBN; (C) LC/MS² of m/z 455 from the COX-AA-D₈ reaction in the presence of POBN; and (inset of C) EIC of m/z 455 from the COX-AA-D₈ reaction in the presence of POBN. Observation of b ions as m/z 343 vs. m/z 361 and b-H₂O ions as m/z 350 vs. m/z 368 supported the proposed structure described as a special β-scission preferentially occurring towards the carbon-carbon double bond, resulting in removal of the H of AA (or the deuterium of AA-D₈) from the carbon (ω-6) bearing oxygen (labeled as β′ in Scheme 1-B). Note that although the same fragmentation pattern was observed in all LC/MS² analyses of its five isomers, the spectra failed to offer enough information to distinguish their differences.

Figure 6

LC/MS² analysis of the spin adduct of the ^•C₆H₁₂O molecule. (A) LC/MS² of m/z 296 from the COX-AA reaction in the presence of POBN; (B) LC/MS² of m/z 305 from the COX-AA reaction in the presence of D₉-POBN; and (C) LC/MS² of m/z 297 from the COX-AA-D₈ reaction in the presence of POBN. Note that observation of same fragmentation pattern, including those from the inset b-ion structures in 3 spin trapping experiments, confirmed the structure assignment. Although the same fragmentation pattern was observed in LC/MS² of its two isomers, the spectra failed to offer enough information to distinguish a difference among the relevant isomers.

Figure 7

Off-line ESR and EIC analysis of radicals formed from PC-3 cellular peroxidation. (A) off-line ESR study of cell-PBS suspension incubated with 30 mM POBN and 0.5 mM AA for 30 min; (B-D) EICs of m/z 296, m/z 305, and m/z 297 projected from the LC/MS² of cellular COX-AA peroxidation in the presence of POBN, D₉-POBN, and POBN-AA-D₈, respectively.

Scheme 1

Proposed mechanisms of formation of spin trapped free radicals from COX-AA peroxidation. (A) Formation of the m/z 548 adduct; (B) Formation of m/z 448 and m/z 296. ‘†’: in the case of the D₉-POBN experiment, two fragment ions of m/z 454 and m/z 298 were measured in the LC/MS² (negative ion mode) of m/z 555 (D₉-POBN/^•C₂₀H₃₃O₅). ‘‡’: a proposed special β′-scission, via 1, 5 intra-molecular H abstraction, to form hexanol and/or hydroxyhexyl radical (^•C₆H₁₁O), rather than forming hexanal: ‘*’: in the case of AA-D₈, formation of the m/z 455 ion as POBN/^•C₁₄H₁₄D₇O₄ as the deuterium of AA-D₈ (or the H of AA) is removed from the ω-6 carbon:

Reaction 1

Reaction 2