Olives and Olive Oil Are Sources of Electrophilic Fatty Acid Nitroalkenes

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.


Introduction
Olive oil is the principal source of lipids in the Mediterranean diet, with ''extra virgin'' olive oil (EVOO) referring to an oil fraction produced via mechanical rather than chemical extraction of olives at temperatures that limit effects on intrinsic properties of the oil [1,2]. The principal components of EVOO are triglycerides (TG, 98-99%) predominantly esterified with monounsaturated oleic acid (OA), and to a lesser extent palmitic, linoleic (LA) and linolenic acids [3][4][5].
Multiple health benefits are linked with diets rich in olive oil and the Mediterranean diet in general, including anti-inflammatory and anti-hypertensive effects that lead to a reduced risk of cardiovascular morbidity and mortality [6][7][8]. Notably, the Mediterranean diet is also linked with the consumption of fruits and vegetables that are rich in the inorganic anions nitrite (NO 2 2 ) and nitrate (NO 3 2 ) [9,10]. These species are also metastable nitric oxide (NO) oxidation products in vivo. Collectively, these oxides of nitrogen undergo further reactions in the blood and tissues via enzymatic and non-enzymatic reductive metabolism and by the oxidizing, nitrosating and nitrating conditions promoted by digestion, mitochondrial respiration and inflammation [11]. In the case of NO 3 2 , the commensal bacteria of the enterosalivary system reduce dietary NO 3 2 to physiologically-significant levels of NO 2 2 , NO and secondary species [12]. There is also an emerging body of evidence from higher plants that NO and other reactive species mediate nitro-oxidative reactions that regulate plant stress perception, signal transduction and senescence responses. Integral to these events is the redox-mediated formation of heme and protein thiol nitrosyl adducts and protein 3-nitrotyrosine adducts [13]. Considering that plants in general, and fresh olives in particular, are abundant in readily-nitrated unsaturated fatty acids, the present study evaluated whether electrophilic fatty acid nitroalkene derivatives (NO 2 -FA) are a) endogenously present in olives, b) extractable into the EVOO fraction and c) generated after consumption of olive lipids by the acidic conditions of digestion.
Fatty acid nitroalkenes are detectable clinically and in rodent models as free, esterified and protein-adducted species [14], but have not been reported in plants. In mammals, these species are present in low basal concentrations and are formed at greater concentrations by the radical addition reaction of nitrogen dioxide (NO 2 ) to unsaturated fatty acids. The endogenous generation of NO 2 occurs via multiple acid-catalyzed and oxidative inflammatory reactions involving NO and NO 2 2 [15]. Biochemical studies and cell models revealed that NO 2 -FA are electrophilic, if the nitro group is adducted to alkenyl carbons. These species rapidly react via Michael addition with thiols and to a lesser extent primary and secondary amines [16].
Once generated, NO 2 -FA signal by reversibly alkylating susceptible thiols of multiple transcriptional regulatory proteins, thus affecting downstream gene expression and the metabolic and inflammatory responses under their regulation. Via this mechanism, NO 2 -FA activate Nrf2-dependent antioxidant gene expression by adduction of critical thiols in the Nrf2 regulatory protein Keap1 [17]. NO 2 -FA also inhibit pro-inflammatory cytokine, adhesion protein and enzyme expression by adduction of the NF-kB p65 subunit and inhibition of DNA binding by p65 [18]. NO 2 -FA are also partial agonists of peroxisome proliferator-activated receptor-c (PPARc), which NO 2 -FA activate via hydrogen bonding interactions and covalent adduction of the ligand binding domain Cys285 [19]. Finally, NO 2 -FA limit inflammatory responses by non-cGMP-dependent inhibition of platelet and neutrophil function [20,21] and by inhibiting the catalytic activity and gene expression of the pro-inflammatory proteins cyclooxygenase-2 and xanthine oxidoreductase [22,23]. Via these pleiotropic mechanisms, murine models reveal that NO 2 -FA limit pathologies linked with obesity, ischemic episodes, bacterial lipopolysaccharide and surgical procedures such as angioplasty [24][25][26][27][28][29].
Herein, we report the endogenous presence of NO 2 -FA in plants, specifically in olives and EVOO and show the additional formation of NO 2 -FA from EVOO under conditions which mimic gastric pH and NO 2 2 concentrations during digestion. It is speculated that the dietary consumption and physiologic generation of electrophilic anti-inflammatory lipids contribute to the physiological benefits of unsaturated fatty acid-rich diets.  [30][31][32]. Pancreatic lipase, cysteine, [ 13 C 3 , 15 N]cysteine and methanesulphonic acid were purchased from Sigma-Aldrich (L3126, C122009, 658057, 471356). Gastric juice artificial was purchased from Fisher Scientific Company (S76772). Strata NH 2 (55 mm, 70A) columns were from Phenomenex (8B-S009-HCH). Hypersep C18 columns were purchased from Thermo Scientific (60108-305). Mass spectrometry quality solvents were purchased from Burdick and Jackson (Muskegon, MI, USA). Extra virgin olive oils and fresh olives were from Jaen, Spain, and came from three different types of cultivars: Arbequina, Frantoio and Picual [33].

Storage of EVOO
To assure no further oxidation of EVOO occurred during storage, a-tocopherol (a-TOH) was determined in fresh samples stored in the dark at either 220uC or room temperature. The levels of a-TOH were determined by reverse phase HPLC of samples (50 ml) mixed with methanol (450 ml) and vortexed twice for 10 s, centrifuged at 10,000 x g for 10 min at 4uC. a-TOH was resolved on a Supelcosil LC-18 column (2560.46 cm, 5 mm), mobile phase of 100% methanol at a flow rate of 1 ml/min. Fluorescence detection (l exc = 295 nm, l em = 330 nm), comparing peak areas with corresponding standards [34]. a-TOH levels were stable for at least two weeks at -20uC in contrast to storage at room temperature.

In vitro gastric digestion of EVOO
Olive oil (10 ml) was incubated for 1 h at 37uC in 1 ml of gastric juice artificial with 5 mM Na[ 15 N]O 2 , under continuous agitation [32,35]. The lipid fraction was extracted by hexane, dried under a stream of nitrogen and 1 ml pancreatic lipase (0.4 mg protein/ml) in 0.5 M phosphate buffer, pH 7.4 was added and the reaction mixture incubated at 37uC for 3 h, under agitation. The lipid fraction was extracted by Bligh and Dyer method [36], dried under a stream of nitrogen and dissolved in chloroform. Lipid classes were further resolved by solid phase extraction (SPE) Strata NH 2 columns. Briefly, columns were pre-conditioned with 6 ml hexane, followed by 6 ml chloroform/isopropanol (2:1, v/v); samples were added and the column was washed with other 6 ml chloroform/isopropanol (2:1, v/v). Then, free fatty acids were eluted with 6 ml diethylether/2% acetic acid. The solvent was evaporated under a stream of nitrogen and lipids were dissolved in methanol for HPLC-ESI-MS/MS and high resolution mass spectrometry analysis.

Detection and characterization of fatty acid nitroalkenes in EVOO
Analysis of NO 2 -FA was performed by HPLC-ESI-MS/MS using a triple quadrupole mass spectrometer (API4000, Applied Biosystems, Framingham, MA) in parallel with a LTQ Orbitrap Velos (Thermo Scientific) in negative ion mode. NO 2 -FA in lipid extracts were separated using a C18 reverse phase column (26150 mm, 3 mm, Phenomenex) eluted at a flow rate of 0.25 ml/min using a solvent system consisting of A (H 2 O/0.1% acetic acid) and B (acetonitrile/0.1% acetic acid), with the following solvent gradient: 45% B (0-0.1 min); 45-80% B (0.1-45 min); 80-100% B (45-46 min); 100% B (46-47 min) and then columns were re-equilibrated to initial conditions for an additional 10 min. The triple quadrupole mass spectrometer was set with the following parameters: declustering potential (DP) of -65 V, collision energy (CE) of -35 eV and a desolvation temperature of 650uC. Detection of NO 2 -FA was performed via MRM scan mode with specific MRM transitions corresponding to the potential nitrated isomers of OA, LA and cLA [30,32]. In all cases, data was acquired, analyzed and processed using Analyst 1.5.1 software (Applied Biosystems, Framingham, MA) as previously [32,35]. High resolution mass spectrometry analysis was performed using the LTQ Orbitrap Velos equipped with a HESI II electrospray source. The following parameters were used: heater temperature 200uC, capillary temperature 200uC, sheath gas flow rate 6, auxiliary gas flow rate 10, sweep gas flow rate 5, source voltage 26 kV, S-lens RF level 65%. The instrument calibration in FT-mode was performed with manufacture calibration solutions. Data were acquired, analyzed and processed using Xcalibur 2.1 software (Thermo Scientific) as previously [37].

Analysis of conjugated linoleic acid in EVOO
cLA was detected by the formation of Diels-Alder adducts with PTAD [32]. EVOO samples were incubated in chloroform with PTAD for 2 min at room temperature and reactions were stopped by the addition of 1,3-hexadiene [32]. Solvent was evaporated and samples washed three times with methanol followed by digestion with pancreatic lipase and SPE as before. Samples were diluted in methanol and analyzed by HPLC-MS/MS by following the specific MRM transition for different PTAD-derivatized cLA isomers [32].  [36], then 1 ml hexane was added and proteins were sedimented by centrifugation at 1890 x g for 10 min, washed with 2 ml methanol/water (4:1, v/v), centrifuged and resuspended in 1 ml water. An equivalent volume of 8 M methanesulphonic acid and internal standard was added and the samples were hydrolyzed for 6 h at 110uC. After hydrolysis, the samples were diluted to 15% methanol and NO 2 -FA-cysteine adducts purified by SPE using Hypersep C18 columns. Columns were pre-conditioned with 6 ml methanol, followed by 6 ml 15% methanol in 1% formic acid; then samples were loaded, washed with 6 ml 15% methanol/ 1% formic acid and columns were dried under vacuum for 30 minutes. NO 2 -FA-cysteine adducts were then eluted with 3 ml methanol in 1% formic acid and the solvent was evaporated at room temperature under a stream of nitrogen. Samples were dissolved in 100 ml methanol and analyzed by HPLC-MS/MS using a triple quadrupole mass spectrometer (API4000, Applied Biosystems, Framingham, MA) in negative ion mode with a DP of -100 V, CE of -25 eV and a desolvation temperature of 650uC. NO 2 -FA-cysteine adducts were separated using one of two different elution schemes on a C18 reverse phase column (26150 mm, 3 mm, Phenomenex). For structural determinations, the mobile phase consisted of solvent A (H 2 O/0.1% acetic acid) and solvent B (acetonitrile/0.1% acetic acid). Chromatography was at 0.25 ml/min with the following solvent gradient: 5% B (0-1 min); 5-35% B (1-8.5 min); 35-100% B (8.51-47 min); 100% B (47-53 min) and re-equilibrated to initial condition for additional 7 min. For quantitative analysis, NO 2 -FA-cysteine adducts were separated using a C18 reverse phase column (2620 mm, 3 mm, Phenomenex); The same mobile phases were used and chromatography was at 0.75 ml/min using the following solvent gradient: 5% B (0-0.1 min); 5-35% B (0.1-0.85 min); 35-100% B (0.86-4.7 min); 100% B (4.7-5.3 min) and then columns were reequilibrated to initial conditions for an additional 1.6 min. Detection of NO 2 -FA-cysteine adducts was performed using the MRM scan mode and acquired data analyzed and processed using Analyst 1.5.1 software, as previously [32,35].

Results
Endogenous NO 2 -cLA and cLA in olive oil  Figure 1C). Analysis by high resolution mass spectrometry and the electrophilic reactivity of this species, determined by thiol reactivity according to [32,38], further confirmed the endogenous presence of NO 2 -cLA ( Figure S1). Nitrite was undetectable in EVOO, purified lipase preparations and all solvents used for extractions and chromatography, discounting the possibility of artifactual fatty acid nitration during sample preparation.

NO 2 -FA generation from olive oil by modeling digestion ex vivo
The acidic milieu of the gastric compartment and the presence of NO 2 2 in food promotes a nitrative environment due to HNO 2 formation (NO 2 2 pK a , 3.4) that in turn can mediate biomolecule nitration [41,42]. To model this, EVOO was incubated in gastric juice with [ 15 N]O 2 2 and the generation of NO 2 -FA was determined (Figures 2 and 3 (Figures 2A and B; ref. [37]). Under these conditions nitro-oleic acid (NO 2 -OA) was also detected (Figure 3). Specifically, [ 15

Discussion
Olive oil, the principal fat in the Mediterranean diet, promotes anti-inflammatory responses and clinical benefit via poorly-defined mechanisms [1,43]. This study examined the endogenous content of NO 2 -FA in fresh olives and EVOO and the potential for their further formation when EVOO was subjected to the gastric milieu [24,26,30,[44][45][46][47][48][49][50]. These analyses were motivated by the detection of fatty acid nitroalkenes in rodents and humans and an appreciation that these electrophilic species induce beneficial metabolic and inflammatory signaling responses.
To avoid ion suppression from native fatty acids in the HPLC-MS/MS identification of the less abundant fatty acid nitroalkenes in EVOO, glycerides were hydrolyzed by triacylglycerol lipase to free fatty acids that were then subjected to chromatographic separation. While monoenoic OA is the most abundant fatty acid in EVOO, it is less susceptible to nitration than polyenoic fatty acids. Free and esterified NO 2 -OA was not detected under basal conditions in the EVOO from three different cultivars. Two predominant nitro-containing fatty acid ions with the MRM transition 324/46 were detected. These species displayed retention times and fragmentation characteristics corresponding to the [ 15 N]O 2 -cLA standard ( Figure 1A,B) and not the bis-allylic NO 2 -[ 13 C 18 ]LA standard ( Figure 1C). cLA consists of positional isomers of linoleic acid having conjugated dienes in the cis and trans configurations [39,51,52]. There are both plant and animalderived sources of cLA in the human diet. In addition, both mammalian and enterosalivary microbiome enzymes can synthesize conjugated diene fatty acids in humans both de novo and by desaturation of monoenoic fatty acid substrates [39,40]. Plant lipids have cLA levels of up to ,1.0 mg cLA/g fat [39], with reported levels of cLA in olive oils of up to 0.2 mg/g fat [39], predominantly the cis9-, trans11and trans10-, cis12isomers [39]. The presence of cLA in the oils from the three olive cultivars studied herein was confirmed by HPLC-MS/MS detection of a Diels Alder reaction of conjugated dienes with 4-phenyl-1,2,4triazoline-3,5-dione (PTAD, m/z 454) ( Figure S2 [32]).
In addition to olives and olive oil, the Mediterranean diet is also rich in NO 2 and NO 3 2 -containing vegetables, suggesting that NO 2 -FA generation could also occur by acidic nitration reactions in the stomach [1,41,43]. NMR analysis of EVOO has suggested that nitro-oxidative modifications of the phenolic and glyceride constituents could yield nitroalkene, nitroalkane and nitro-hydroxy products [53]. Herein, the formation of electrophilic NO 2 -FA species in EVOO exposed to Na[ 15 N]O 2 in gastric fluid was detected with HPLC-MS/MS (Figures 2 and 3). Under these conditions, there was significant NO 2 -OA, NO 2 -LA and NO 2 -  cLA generation, with 9-NO 2 -cLA and 12-NO 2 -cLA the most prevalent ( Figure 2B).
The electrophilic character of NO 2 -FA in EVOO was confirmed by HPLC-MS/MS detection of Michael reaction products in fresh olives (Figure 4) [38]. Notably, NO 2 -OA-cysteine adducts were endogenously present in olives, where there was a stable pool of protein-adducted nitroalkene derivatives in the peel and mesocarp of different olive cultivars ( Figure 5). These results confirm that EVOO and olives are both a source and metabolic reserve of NO 2 -FA.
Electrophilic nitroalkenes exert signaling actions via the modulation of the expression and activity of both anti-inflammatory [32] and pro-inflammatory enzymes [23]. These effects are entirely dependent on the post-translational modification of transcription factors, enzymes and and other protein targets via Michael addition. Conjugated linoleic acid displays immunemodulatory and anti-inflammatory effects [51,52], with the mechanisms accounting for these actions proposed to include the reduction of pro-inflammatory cytokine levels via inhibition of NF-kB-dependent gene expression and activation of PPAR-regulated gene expression [51,52]. Of significance, very high and nonphysiological concentrations of native cLA and other unsaturated fatty acids are required to exert these effects. In contrast, after fatty acid nitration and conferral of electrophilic reactivity, NO 2 -FA potently modulate these same pathways at nM concentrations [46,54]. Additionally, NO 2 -FA activate Nrf2-regulated antiinflammatory gene expression and heat shock factor-regulated heat shock protein expression [17,46,55]. This is explained by the facile and reversible Michael addition of NO 2 -FA with susceptible thiols of specific protein targets, thus requiring only low concentrations and rates of generation of electrophilic lipids to result in the accumulation of target protein adduction and instigation of downstream signaling responses [16,54]. Consequently, electrophilic lipids which are present in plants, generated by digestion of plant lipids, produced by oxidative inflammatory reactions or administered as pure synthetic homologs [24,26,47,56], can regulate metabolism and the resolution of inflammatory processes. In this regard, olives and EVOO serve as both a direct source of and precursors for NO 2 -FA generation.