Primary saturation of α, β-unsaturated carbonyl containing fatty acids does not abolish electrophilicity

Abstract

Metabolism of polyunsaturated fatty acids results in the formation of hydroxylated fatty acids that can be further oxidized by dehydrogenases, often resulting in the formation of electrophilic, α,β-unsaturated ketone containing fatty acids. As electrophiles are associated with redox signaling, we sought to investigate the metabolism of the oxo-fatty acid products in relation to their double bond architecture. Using an untargeted liquid chromatography mass spectrometry approach, we identified mono- and di-saturated products of the arachidonic acid-derived 11-oxoeicosatetraenoic acid (11-oxoETE) and mono-saturated metabolites of 15-oxoETE and docosahexaenoic acid-derived 17-oxodocosahexaenoinc acid (17-oxoDHA) in both human A549 lung carcinoma and umbilical vein endothelial cells. Notably, mono-saturated oxo-fatty acids maintained their electrophilicity as determined by nucleophilic conjugation to glutathione while a second saturation of 11-oxoETE resulted in a loss of electrophilicity. These results would suggest that prostaglandin reductase 1 (PTGR1), known only for its reduction of the α,β-unsaturated double bond, was not responsible for the saturation of oxo-fatty acids at alternative double bonds. Surprisingly, knockdown of PTGR1 expression by shRNA confirmed its participation in the formation of 15-oxoETE and 17-oxoDHA mono-saturated metabolites. Furthermore, overexpression of PTGR1 in A549 cells increased the rate and total amount of oxo-fatty acid saturation. These findings will further facilitate the study of electrophilic fatty acid metabolism and signaling in the context of inflammatory diseases and cancer where they have been shown to have anti-inflammatory and anti-proliferative signaling properties.

Keywords: Dehydrogenase; Eicosanoids; Fatty acid/metabolism; Fatty acid/oxidation; KETE; Mass spectrometry; Prostaglandin reductase; oxoETE.

Figure 1:. Extracted ion chromatograms (XIC) of LC-HRMS analysis of lipid extract from oxoETE treated HUVECs.

Mass spectral features from the discovery experiment were extracted from representative chromatograms with a 5 ppm mass window from the given m/z from positive ion mode LC-HRMS analysis of lipid extracts of HUVECs treated with (A) 11-oxoETE, (B) 15-oxoETE, (C) [¹³C₂₀]-15-oxoETE. Peaks putatively corresponding to double bond saturation were apparent for one and two saturations in 11-oxoETE. Only one saturation was detectable for 15-oxoETE and [¹³C₂₀]-15-oxoETE.

Figure 2:. Time course of oxoETE metabolite accumulation in HUVECs.

Accumulation of putative metabolites after treatment with 10 μM 11-oxoETE or 15-oxoETE. Area under the curve was calculated from the XIC of metabolites. [¹³C₂₀]-15-oxoETE was used as the internal standard and to normalize for extraction and ionization efficiency for relative quantitation. Values are expressed in area under the peak for (Analyte/ISTD) for the (A) mono-saturated 11-oxoETE metabolite, the (B) di-saturated 11-oxoETE metabolite and the (C) mono-saturated 15-oxoETE metabolite.

Figure 3:. LC-MS/HRMS confirmation of 11-oxoETE saturation products.

(A) 11-oxoETE structure and diagnostic fragmentation. Extracted chromatograms and product ion spectrum in negative ion mode for (B) 11-oxoETE (C) the mono-saturated 11-oxoETE metabolite, and (D) the di-saturated 11-oxoETE metabolite.

Figure 4:. LC-MS/HRMS confirmation of the 15-oxoETE saturation product.

(A) 15-oxoETE structure and diagnostic fragmentation. Extracted chromatograms and product ion spectrum in negative ion mode for (B) 15-oxoETE and (C) the mono-saturated 15-oxoETE metabolite. (D) Product ion spectra of 15-oxoETE and the mono-saturated 15-oxoETE metabolite between m/z 150–190 to highlight the fragmentation found at m/z 165.1274 for 15-oxoETE and m/z 167.1068 for the mono-saturated 15-oxoETE metabolite. The m/z of 167.1068 corresponds to fragmentation of the chemical composition C₁₀H₁₅O₂, a fragment adjacent to the C8-C9 double bond that would likely not be present if saturation occurred at this position.

Figure 5:. LC-HRMS chromatograms of GSH-adducts from parent and saturated products of 11-oxo and 15-oxoETEs.

(A) 11-oxoETE, mono-saturated and di-saturated GSH conjugates (B) 15-oxoETE and its mono-saturated product.

Figure 6:. 15-oxoETE metabolism in A549 Cells.

(A) 15-oxoETE metabolism to the mono-saturated metabolite from 0 to 6 hr reported as SEM, n = 9 per condition. Extracted chromatograms of diagnostic product ions for (B) 15-oxoETE and (C) the mono-saturated 15-oxoETE metabolite.

Figure 7:. 17-oxoDHA metabolism in A549 Cells.

(A) 17-oxoDHA metabolism to the mono-saturated metabolite from 0 to 6 h reported as SEM, n = 9 per condition. Extracted chromatograms of diagnostic product ions and product ion spectra for (B) 17-oxoDHA and (C) the mono-saturated 17-oxoDHA metabolite.

Figure 8.. Purified recombinant PTGR1 catalyzes the conversion of oxoFAs to their saturated products.

(A) 15-ketoPGE₂ (B) 11-oxoETE (C) 15-oxoETE (D) 17-oxoDHA (substrates only with no enzyme shown in black) were converted to their saturated metabolites in the presence of PTGR1 and NADH (white) but not in reaction buffer without enzyme. Similar results were obtained with NADPH instead of NADH (gray).

Figure 9:. PTGR1 KD in A549 cells partially abolishes electrophile metabolism.

Mono-saturated metabolite product/substrate ratio in A549 cell lysate for (A) 15-oxoETE, (B) 17-oxoDHA and the positive control (C) 15-ketoPGE₂, n = 3–9 per condition, * p ≤ 0.05, **** p ≤ 0.0001.

Figure 10.. Overexpression of PTGR1 increases intracellular concentration of saturated oxoFAs.

Mono-saturated metabolite product/substrate ratio in cell lysate for (A) 15-oxoETE, (B) 17-oxoDHA and the positive control (C) 15-ketoPGE₂. n = 9 per condition, * p ≤ 0.05, *** p ≤ 0.001.

Scheme 1:. Structures of 11-oxoETE, 15-oxoETE, 17-oxoDHA and proposed saturation products.

11-oxoETE undergoes two rounds of saturation to form mono- and di-saturated metabolites whereas only mono-saturated metabolites were found for 15-oxoETE and 17-oxoDHA. Proposed putative points of saturation based off of the MS² spectra are also shown.