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. 2014 Jan 14;9(1):e84884.
doi: 10.1371/journal.pone.0084884. eCollection 2014.

Olives and olive oil are sources of electrophilic fatty acid nitroalkenes

Marco Fazzari  1 , Andrés Trostchansky  2 , Francisco J Schopfer  3 , Sonia R Salvatore  3 , Beatriz Sánchez-Calvo  4 , Dario Vitturi  3 , Raquel Valderrama  4 , Juan B Barroso  4 , Rafael Radi  2 , Bruce A Freeman  3 , Homero Rubbo  2
Affiliations

Olives and olive oil are sources of electrophilic fatty acid nitroalkenes

Marco Fazzari et al. PLoS One. .

Abstract

Extra virgin olive oil (EVOO) and olives, key sources of unsaturated fatty acids in the Mediterranean diet, provide health benefits to humans. Nitric oxide (•NO) and nitrite (NO2 (-))-dependent reactions of unsaturated fatty acids yield electrophilic nitroalkene derivatives (NO2-FA) that manifest salutary pleiotropic cell signaling responses in mammals. Herein, the endogenous presence of NO2-FA in both EVOO and fresh olives was demonstrated by mass spectrometry. The electrophilic nature of these species was affirmed by the detection of significant levels of protein cysteine adducts of nitro-oleic acid (NO2-OA-cysteine) in fresh olives, especially in the peel. Further nitration of EVOO by NO2 (-) under acidic gastric digestive conditions revealed that human consumption of olive lipids will produce additional nitro-conjugated linoleic acid (NO2-cLA) and nitro-oleic acid (NO2-OA). The presence of free and protein-adducted NO2-FA in both mammalian and plant lipids further affirm a role for these species as signaling mediators. Since NO2-FA instigate adaptive anti-inflammatory gene expression and metabolic responses, these redox-derived metabolites may contribute to the cardiovascular benefits associated with the Mediterranean diet.

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Conflict of interest statement

Competing Interests: The authors have read the journal's policy and have the following conflicts: BAF and FJS acknowledge financial interest in Complexa, Inc. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Detection of NO2-cLA in EVOO.
Extra virgin olive oil was washed four times with methanol and treated with pancreatic lipase for 2-MS/MS. Before extraction, internal standards [15N]O2-cLA and NO2-[13C18]LA were added. The following MRM transitions were analyzed: 324/46 for NO2-LA, 325/47 for [15N]O2-cLA and 342/46 for NO2-[13C18]LA. The elution profile of (A) lipids from EVOO; (B) [15N]O2-cLA and (C) NO2-[13C18]LA are shown. Neither NO2-LA nor NO2-OA (MRM 326/46) was detected. Data is representative of at least 3 independent experiments.
Figure 2
Figure 2. Identification of specific NO2-LA and NO2-cLA regio-isomers in EVOO by in vitro digestion modeling.
Olive oil (10 µl) was incubated in gastric juice artificial with 5 mM Na[15N]O2. The lipid fraction was extracted, dried and incubated with pancreatic lipase (0.4 mg protein/ml) in phosphate buffer, pH 7.4 at 37°C for 3 h. The lipid fraction was extracted, dried, dissolved in chloroform, then solid phase extraction was performed and lipids analyzed by HPLC-MS/MS. The presence of NO2-LA and NO2-cLA in Picual EVOO gastric fluid was determined following the MRM transition m/z 325/47 compared to (A) the internal standard NO2-[13C18]LA (m/z 342/46) and (B) the standard [15N]O2-cLA (m/z 325/47). Similar results were obtained for the other two EVOO tested (Arbequina and Frantoio oils). The corresponding peaks were also observed when EVOO was nitrated with NaNO2 (data not shown). Data shown are representative of at least 3 independent experiments. (A) Orbitrap Velos analysis confirmed the elemental composition (C18H30O4 15N) and mass accuracy (−2.0443 ppm) for the peaks labeled A, B, C, and related MS2 analysis of each [15N]O2-LA regio-isomers. # and * represents specific fragments for the 9-NO2 and 12-NO2 isomers, respectively. (B) For peaks labeled E to H, Orbitrap Velos analysis confirmed the elemental composition (C18H30O4 15N) and mass accuracy (−2.0923 ppm) and related MS2 analysis of [15N]O2-cLA regio-isomers.
Figure 3
Figure 3. NO2-OA generation from EVOO by in vitro digestion modeling.
Olive oil was nitrated, extracted and treated with Na[15N]O2 as in Figure 2. (A) The presence of NO2-OA in Picual EVOO gastric fluid was determined following the MRM transition m/z 327/47 compared to (B) the internal standard NO2-[13C18]OA (m/z 344/46). Similar results were obtained for the other EVOOs tested. The corresponding peaks were also observed when EVOO was nitrated with NaNO2. Data shown is representative of at least 3 independent experiments. Orbitrap Velos analysis confirmed the elementalcomposition (C18H32O4 15N) and mass accuracy (−2.8007 ppm) and related MS2 analysis of [15N]O2-OA regio-isomers.
Figure 4
Figure 4. Detection of NO2-OA-cysteine adducts in fresh olives.
Whole olives, mesocarp and peel were homogenized and protein fraction was extracted and hydrolyzed with methanesulphonic acid for 6°C. Then, solid phase extraction was performed and NO2-OA-cysteine adducts were analyzed by HPLC-MS/MS. (A) The presence of NO2-OA-cysteine adducts in Picual peel after protein hydrolysis was determined following the MRM transition m/z 447/120 compared to (B) the internal standard NO2-OA-[13C3,15N]cysteine adducts (m/z 451/124). Data shown is representative of at least 3 independent experiments.
Figure 5
Figure 5. Endogenous NO2-OA-cysteine adducts in fresh olives.
Whole olive as well as peel and mesocarp fractions were processed as before, and the presence of the NO2-OA-cysteine adducts were quantitated by HPLC-MS/MS. (A) NO2-OA-cysteine adducts were quantitated in the whole fruit of the three olive cultivars tested. (B) Distribution of NO2-OA-cysteine adducts in whole fruit, peel and mesocarp of Picual olive cultivar is shown. Data plotted correspond to the mean ± SD (n = 3) and is representative of at least 3 independent experiments. Statistical analysis was performed by one-way ANOVA with Bonferroni post hoc comparisons. a, p <0.01.
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