Nonenzymatic free radical-catalyzed generation of 15-deoxy-Δ(12,14)-prostaglandin J₂-like compounds (deoxy-J₂-isoprostanes) in vivo

Abstract

15-Deoxy-Δ(12,14)-prostaglandin J₂ (15-d-PGJ₂) is a reactive cyclopentenone eicosanoid generated from the dehydration of cyclooxygenase-derived prostaglandin D₂ (PGD₂). This compound possesses an α,β-unsaturated carbonyl moiety that can readily adduct thiol-containing biomolecules such as glutathione and cysteine residues of proteins via the Michael addition. Due to its reactivity, 15-d-PGJ₂ is thought to modulate inflammatory and apoptotic processes and is believed to be an endogenous ligand for peroxisome proliferator-activated receptor-γ. However, the extent to which 15-d-PGJ₂ is formed in vivo and the mechanisms that regulate its formation are unknown. Previously, we have reported the formation of PGD₂ and PGJ₂-like compounds, termed D₂/J₂-isoprostanes (D₂/J₂-IsoPs), produced in vivo by the free radical-catalyzed peroxidation of arachidonic acid (AA). Based on these findings, we investigated whether 15-d-PGJ₂-like compounds are also formed via this nonenzymatic pathway. Here we report the generation of novel 15-d-PGJ₂-like compounds, termed deoxy-J₂-isoprostanes (deoxy-J₂-IsoPs), in vivo, via the nonenzymatic peroxidation of AA. Levels of deoxy-J₂-IsoPs increased 12-fold (6.4 ± 1.1 ng/g liver) in rats after oxidant insult by CCl₄ treatment, compared with basal levels (0.55 ± 0.21 ng/g liver). These compounds may have important bioactivities in vivo under conditions associated with oxidant stress.

Fig. 1.

15-d-PGJ₂ is formed from PGD₂ through a sequence of dehydration and isomerization reactions.

Fig. 2.

Proposed pathway for the formation of the deoxy-J₂ -IsoPs via the nonenzymatic peroxidation of AA. For simplicity, the stereochemistry of both the lipid peroxidation intermediates and fi nal IsoPs are not shown.

Fig. 3.

Structures of rac-15-d-PGJ₂, consisting of 15-d-PGJ₂ and its enantiomer.

Fig. 4.

LC/ESI-MS/MS analysis of 15-d-PGJ₂ (A) and a mixture of 15-d-PGJ₂-like compounds (deoxy-J₂-IsoPs) obtained from the nonenzymatic oxidation of AA in vitro (B). A: LC/ESI-MS/MS analysis of a chemically synthesized 15-d-PGJ₂ employing SIM (upper chromatogram) and CID (lower product ion spectrum). B: LC/ESI-MS/MS analysis of 15-d-PGJ₂-like compounds obtained from the in vitro oxidation of AA employing SIM for the precursor ion m/z 315 (upper chromatogram) and CID (lower product ion spectrum) indicative of putative 15-d-PGJ₂.

Fig. 5.

LC/ESI-MS/MS analysis of multiple regioisomers of deoxy-J₂-IsoPs generated from the nonenzymatic oxidation of AA in vitro. A: Structurally specific fragmentation of the different deoxy-J₂-IsoP regioisomers. B: SIM chromatogram of the precursor ion at m/z 315 (upper) and CID spectrum (lower) obtained by summing scans over the chromatographic peaks, revealing structurally specific fragmentation of the different deoxy-J₂-IsoP regioisomers.

Fig. 6.

Normal phase LC/APCI-MS/MS analysis of multiple regioisomers of deoxy-J₂ -IsoPs generated from the nonenzymatic peroxidation of AA in vitro. SIM chromatogram of the parent ion at m/z 315 (upper) and CID spectrum (lower) obtained by summing scans over the series of chromatographic peaks reveals structurally specific fragmentation of the different deoxy-J₂ -IsoP regioisomers.

Fig. 7.

Analysis of putative rac-15-d-PGJ₂ in vitro by LC/MS/MS using SRM. LC/MS/MS analysis of rac-15-d-PGJ₂ (A) from the nonenzymatic oxidation of AA in vitro and chemically synthesized [²H₄]15-d-PGJ₂ (d₄-15-d-PGJ₂) (B). The mass spectrometer was operated in negative ion mode using SRM to monitor the precursor-to-product transition of m/z 315–203 for rac-15-d-PGJ₂ (chromatogram A) and the precursor-to-product transition of m/z 319–203 for d₄-15-d-PGJ₂ (chromatogram B).

Fig. 8.

Analysis of GSH adducts of deoxy-J₂ -IsoPs and 15-d-PGJ₂ by LC/ESI-MS/MS. A: SIM chromatogram of putative deoxy-J₂ -IsoP-GSH adducts with the precursor ion m/z 622. B: Composite CID spectrum obtained by summing scans over the series of chromatographic peaks representing deoxy-J₂ -IsoP-GSH adducts. C: CID spectrum of the synthesized 15-d-PGJ₂ -GSH adduct.

Fig. 9.

Analysis of putative deoxy-J₂-IsoPs in vivo by LC/MS/MS using SRM. LC/MS/MS analysis of (A) deoxy-J₂-IsoPs regioisomers and (B) the 15-series deoxy-J₂-IsoPs (rac-15-d-PGJ₂) generated in vivo esterified in liver phospholipids in rats after treatment with CCl₄ and (C) chemically synthesized [²H₄]15-d-PGJ₂ (d₄-15-d-PGJ₂). The mass spectrometer was operated in the negative ion mode with SRM for the precursor-to-product transitions of m/z 315–271 for all deoxy-J₂-IsoPs regioisomers (chromatogram A), m/z 315–203 for rac-15-d-PGJ₂ (chromatogram B), and m/z 319–275 for d₄-15-d-PGJ₂ (chromatogram C).