Generation and esterification of electrophilic fatty acid nitroalkenes in triacylglycerides

Abstract

Electrophilic fatty acid nitroalkenes (NO(2)-FA) are products of nitric oxide and nitrite-mediated unsaturated fatty acid nitration. These electrophilic products induce pleiotropic signaling actions that modulate metabolic and inflammatory responses in cell and animal models. The metabolism of NO(2)-FA includes reduction of the vinyl nitro moiety by prostaglandin reductase-1, mitochondrial β-oxidation, and Michael addition with low molecular weight nucleophilic amino acids. Complex lipid reactions of fatty acid nitroalkenes are not well defined. Herein we report the detection and characterization of NO(2)-FA-containing triacylglycerides (NO(2)-FA-TAG) via mass spectrometry-based methods. In this regard, unsaturated fatty acids of dietary triacylglycerides are targets for nitration reactions during gastric acidification, where NO(2)-FA-TAG can be detected in rat plasma after oral administration of nitro-oleic acid (NO(2)-OA). Furthermore, the characterization and profiling of these species, including the generation of beta oxidation and dehydrogenation products, could be detected in NO(2)-OA-supplemented adipocytes. These data revealed that NO(2)-FA-TAG, formed by either the direct nitration of esterified unsaturated fatty acids or the incorporation of nitrated free fatty acids into triacylglycerides, contribute to the systemic distribution of these reactive electrophilic mediators and may serve as a depot for subsequent mobilization by lipases to in turn impact adipocyte homeostasis and tissue signaling events.

Keywords: Adipocytes; Conjugated linoleic acid; Nitrate; Nitrite; Nitro fatty acids; Nitro-oleic acid; Nitroalkane; Triglycerides.

Fig. 1. Detection and characterization of NO₂-CLA-TAG generation by in vitro gastric conditions

(A) Mass chromatogram of NO₂-CLA-TAG as ammonium adduct (m/z 893.7) [M+NH₄]⁺ and inset with its atomic composition and isotopic abundance. (B) MS² spectral data of NO₂-CLA-TAG. (C) MS³ spectral data of the MS² even fragments m/z 620.4 [M-16:0-NH₃]⁺. (D) MS³ spectral data of the MS² odd fragment m/z 551.5 [M-NO₂-CLA-NH₃]⁺. Representative chemical structures are only shown for the 9-NO₂-CLA-TAG isomer and corresponding product ions.

Fig. 2. Time-dependent changes in extracellular NO₂-FA after supplementation of NO₂-OA in adipocytes

Values of electrophilic nitroalkenes (filled square) and non-electrophilic nitroalkanes (open square) are given as area ratio of (A) nitro- C₁₈-, (B) C₁₆-, (C) C₁₄- and (D) C₁₂- fatty acid/NO₂-[¹³C₁₈]OA over a period of 24 h. Data shown are the means ± SD of 3 independent experiments with 5 and 3 replicates each for NO₂-OA and OA treatment, respectively.

Fig. 3. Metabolism, distribution and esterification of NO₂-FA into TAG in adipocytes supplemented with NO₂-OA

Lipase-hydrolyzed TAG, cell lysate and cell media were analyzed by HPLC-ESI-MS/MS in negative mode to determine (A) esterified into TAG, (B) intracellular and (C) extracellular content of NO₂-FA at 24 h, respectively. A strong correlation (r² = 0.98, P < 0.001) was observed between (D) intracellular and extracellular but not between (E) esterified into TAG and intracellular content of NO₂-FA. Values are given as area ratio of NO₂-FA/NO₂-OA. Data shown are the means ± SD of 3 independent experiments with 5 and 3 replicates each for NO₂-OA and OA treatment, respectively.

Fig. 4. Mass spectrometry analysis of adipocyte NO₂-FA-TAG

Purified TAG fractions from adipocytes treated with 5 μM NO₂-OA for 24 h were subjected to HPLC-HR-MS analysis in positive mode. (A) Mass chromatogram of NO₂-FA-TAG as ammonium adducts (m/z 893.7) [M+NH₄]⁺, inset with atomic composition and isotopic abundance. (B) MS² spectral data of peak 1 in Fig. 4A. (C) MS² spectral data of peak 2 in Fig. 4A. (D) MS³ spectral data of the MS² even fragments m/z 622.5 [M-16:1-NH₃]⁺ in Fig. 4B. (E) MS³ spectral data of the MS² odd fragment m/z 547.4 [M-NO₂-18:0-NH₃]⁺ in Fig. 4B. ^a Order of notation does not connote regiochemistry. * Presence of a shared MS² fragment from a co-eluting NO₂-FA-TAG isomer (NO₂-16:0/17:1/17:1-TAG).

Fig. 5. Characterization of rat plasma NO₂-FA-TAG

Kinetic analysis of (A) nitro- C₁₈-, (B) C₁₆-, (C) C₁₄- fatty acid concentration (nM) in lipase hydrolyzed rat plasma triacylglycerides after supplementation of NO₂-OA. (D, F) MRM chromatograms for NO₂-18:0/16:0/18:2-TAG and NO₂-18:0/18:2/18:2-TAG, as ammonium adducts in positive mode, in (upper panel) untreated and (lower panel) treated rat plasma samples. (E) EPI analysis of peak 1 for m/z 921.8. (G) EPI analysis of peak 2 for m/z 945.8. Data shown are the means ± SD of a pharmakinetic study performed on two treated and one control animal. ^a Order of notation does not connote regiochemistry.