Fatty Acid Transduction of Nitric Oxide Signaling

The aqueous decay and concomitant release of nitric oxide (·NO) by nitrolinoleic acid (10-nitro-9,12-octadecadienoic acid and 12-nitro-9,12-octadecadienoic acid; LNO2) are reported. Mass spectrometric analysis of reaction products supports a modified Nef reaction as the mechanism accounting for the generation of ·NO by the aqueous reactions of fatty acid nitroalkene derivatives. Nitrolinoleic acid is stabilized by an aprotic milieu, with LNO2 decay and ·NO release strongly inhibited by phosphatidylcholine/cholesterol liposome membranes and detergents when present at levels above their critical micellar concentrations. The release of ·NO from LNO2 was induced by UV photolysis and triiodide-based ozone chemiluminescence reactions currently used to quantify putative protein nitrosothiol and N-nitrosamine derivatives. This reactivity of LNO2 complicates the qualitative and quantitative analysis of biological oxides of nitrogen when applying UV photolysis and triiodide-based analytical systems to biological preparations typically abundant in nitrated fatty acids. The results reveal that nitroalkene derivatives of linoleic acid are pluripotent signaling mediators that act not only via receptor-dependent mechanisms, but also by transducing the signaling actions of ·NO via pathways subject to regulation by the relative distribution of LNO2 to hydrophobic versus aqueous microenvironments.

lipoproteins and red blood cell membranes at concentrations of ϳ500 nM, rendering this species the most quantitatively abundant, biologically active oxide of nitrogen in the human vascular compartment (1). Nitrolinoleic acid is a product of nitric oxide ( ⅐ NO)-dependent linoleic acid nitration reactions that predominantly occur at the C-10 and C-12 alkene carbons. The positional isomer distribution of the LNO 2 alkenyl nitro group indicates that in vivo fatty acid nitration is a consequence of nucleophilic (nitronium group (NO 2 ϩ )) and/or radical (nitrogen dioxide ( ⅐ NO 2 )) addition reactions with olefinic carbons.
Recent observations reveal that LNO 2 is a pluripotent signaling mediator that acts via both receptor-dependent and receptor-independent pathways. Nitrated fatty acids are specific and high affinity endogenous ligands for peroxisome proliferator-activated receptors (2) and serve to activate receptordependent gene expression at physiological concentrations. LNO 2 also activates cAMP-dependent protein kinase signaling pathways in neutrophils and platelets, serving to down-regulate the activation of these inflammatory cells (3,4). Finally, LNO 2 induces vessel relaxation in an endothelium-independent manner (5). This LNO 2 -mediated relaxation of phenylephrine-preconstricted aortic rings is 1) a consequence of LNO 2induced stimulation of smooth muscle cell and aortic segment cGMP content, 2) inhibitable by the ⅐ NO scavenger oxyhemoglobin, and 3) 1H- [1,2,4]oxadiazole [4,3-a]quinoxalin-1-one (ODQ)-inhibitable (e.g. guanylate cyclase-dependent). Although these vessel responses to LNO 2 suggest ⅐ NO as the mediator of guanylate cyclase activation, the identity of the proximal LNO 2 -derived, cGMP-dependent signaling molecule was not directly identified (5).
Nitric oxide, synthesized by three different nitric-oxide synthase isoforms, was first shown to mediate endothelium-dependent relaxation via reaction with the heme iron of guanylate cyclase and subsequent activation of cGMP-dependent protein kinases (6). Subsequent to this discovery, there has been a growing appreciation that the cell signaling actions of ⅐ NO are also transduced by secondary products derived from redox reactions of ⅐ NO. These redox reactions yield a variety of oxides of nitrogen displaying both unique and overlapping reactivities that can regulate differentiated cell function via both cGMP-and non-cGMP-dependent mechanisms. These products include nitrite (NO 2 Ϫ ), ⅐ NO 2 , peroxynitrite (ONOO Ϫ ), nitrosothiols (RSNO), and dinitrogen trioxide (N 2 O 3 ). These reactive species serve to transduce the cell signaling actions of ⅐ NO by inducing changes in target molecule structure and function via oxidation, nitration, or nitrosation reactions (7,8).
The lipophilicity and intrinsic chemical reactivities of ⅐ NO facilitate multiple interactions with lipids that impact both cellular redox and ⅐ NO signaling reactions. For example, ⅐ NO concentrates in membranes and lipoproteins, where it more readily reacts with oxygen to yield oxidizing, nitrosating, and nitrating species such as N 2 O 3 and N 2 O 4 (9 -11). In these lipophilic compartments, ⅐ NO can react with lipid peroxyl radicals (LOO ⅐ ) at diffusion-limited rates, readily out-competing tocopherols and ascorbate for the scavenging of intermediates that would otherwise propagate lipid oxidation. In this regard, ⅐ NO displays an oxidant-protective, anti-inflammatory role (12,13). Of relevance to inflammatory signaling, heme-and nonheme-containing peroxidases and oxygenases that catalyze physiological and pathological fatty acid oxygenation reactions also catalytically consume ⅐ NO during enzyme turnover, e.g. lipoxygenases (14,15), cyclooxygenase (16), and myeloperoxidase (17). The reaction of ⅐ NO with these enzymatic catalysts and free radical intermediates of fatty acid oxygenation in turn inhibits rates of fatty acid oxygenation product formation. The convergence of ⅐ NO and fatty acid oxygenation reactions can thus influence the steady-state concentration of both ⅐ NO and eicosanoids in a concerted fashion.
Redox reactions of ⅐ NO frequently induce the chemical modification of target molecules, including the nitrosylation (addition of ⅐ NO) of heme proteins (18), the nitrosation (addition of the nitroso group NO) of thiol substituents (7), and the nitration (addition of the nitro group NO 2 ) of protein tyrosine residues and DNA bases (8). Herein, we report that LNO 2 , a product of ⅐ NO-dependent unsaturated fatty acid nitration reactions that is abundant in red cells and plasma, decays in aqueous solvents to release ⅐ NO. This generation of ⅐ NO by LNO 2 is inhibited by aprotic environments, a milieu that concomitantly stabilizes LNO 2 . Moreover, we show that UV photolysis and triiodide (I 3 Ϫ )-based chemiluminescence approaches, currently used to quantify ⅐ NO derived from protein hemenitrosyl, RSNO, and N-nitrosamine (RNNO) derivatives, also facilitate ⅐ NO release from LNO 2 . This complicates the interpretation of quantitative and qualitative results from the application of these analytical systems in biological preparations. Together, these results reveal that nitroalkene derivatives of fatty acids serve to transduce the signaling actions of ⅐ NO via pathways subject to regulation by the relative distribution of LNO 2 to hydrophobic versus aqueous microenvironments.
Electron Paramagnetic Resonance-EPR measurements were performed at room temperature using a Bruker Elexsys E-500 spectrometer equipped with an ER049X microwave bridge and an AquaX liquid sample cell. The following instrument settings were used: modulation frequency, 100 kHz; modulation amplitude, 0.05 G; receiver gain, 60 dB; time constant, 1.28 ms; sweep time, 5.24 s; center field, 3510 G; sweep width, 100 G; power, 20 milliwatts; and scan parameter, 16 scans.
Spectrophotometry-The UV spectrum of LNO 2 and repetitive scans of LNO 2 decay kinetics were collected using a Hitachi UV 2401 PC spectrophotometer. The apparent ⅐ NO formation was calculated from the extent of oxymyoglobin oxidation in the visible wavelength range (spectrum) and at 580 nm (kinetic mode). Initial oxymyoglobin oxidation was calculated using UV Probe Version 1.10 (⑀ 580 ϭ 14.4 mM Ϫ1 cm Ϫ1 ). Decomposition of the NO 2 group was followed at 268 nm, and the appearance of oxidized products was followed at 320 nm.
Liposome Preparation-Reverse-phase evaporation liposomes were formed from dipalmitoylphosphatidylcholine, cholesterol, and stearylamine (4:2:1 mol ratio) following an established procedure (19). Briefly, dipalmitoylphosphatidylcholine, cholesterol, and stearylamine were dissolved in CHCl 3 and sonicated with 10 mM potassium P i buffer (2:1, v/v). The organic solvent was then removed by evaporation under re-duced pressure at 45°C. The liposomes were allowed to anneal for 12 h at room temperature and then centrifuged, and the pellet was resuspended in the experimental buffer.
Chemiluminescence and UV Photolysis Analyses-For direct detection of ⅐ NO release, LNO 2 (75 M) was incubated directly or with different additions (1.5% (w/v) sulfanilamide in 2 M HCl for 5 min at 25°C with or without 50 mM HgCl 2 ) under aerobic conditions in a capped vial for 3 min. The gas phase was then injected into a chemiluminescence detector (ANTEK Instruments, Houston, TX). Additionally, known concentrations of diethylammonium (Z)-1-(N,N,-diethylamino)diazen-1-ium-1,2-diolate (DEA NONOate) (in 10 mM NaOH) were added to a capped vial containing 0.5 M HCl. NO x concentration profiles of plasma samples were performed by ⅐ NO chemiluminescence analysis. Measurement of putative NO 2 Ϫ , RSNO, and other ⅐ NO derivatives present in plasma was performed using an I 3 Ϫ -based reducing system as described previously (20,21). Rats were treated by intraperitoneal injection of 50 mg/kg Escherichia coli lipopolysaccharide; and 5 h later, blood was collected in EDTA anticoagulation tubes following cardiac puncture. Following removal of red cells by centrifugation at 500 ϫ g for 10 min, plasma samples were pretreated with sulfanilamide (1.5% (w/v) final concentration in 2 M HCl for 5 min at 25°C) with or without 50 mM HgCl 2 prior to injection into the chemiluminescence detector to measure NO 2 Ϫ and HgCl 2 -resistant NO x derivatives, respectively. For UV photolysis studies, a water-cooled reaction chamber was filled with 1 ml of 50 mM phosphate buffer (pH 7.4) containing 10 M DTPA and continuously bubbled with argon. The chamber was illuminated using an ILC PS300 -1A xenon arc source (ILC Technology, Sunnyvale, CA). Samples were injected into the reaction chamber through an air-tight septum, and released ⅐ NO was passed to the reaction chamber of a Sievers NOA 280 NO analyzer and detected by chemiluminescence after reaction with ozone (O 3 ).
Mass Spectrometric Analysis-LNO 2 was extracted using the method of Bligh and Dyer (22). During extraction, [ 13 C]LNO 2 was added as an internal standard, and the LNO 2 content of samples was quantified by liquid chromatography-tandem mass spectrometry (1). Qualitative and quantitative analysis of LNO 2 by electrospray ionization tandem mass spectrometry was performed using a hybrid triple quadrupole/linear ion trap mass spectrometer (4000 Q TRAP, Applied Biosystems/MDS SCIEX) as described (1). For the detection and characterization of nitrohydroxylinoleic acid (L(OH)NO 2 ), the hydration product of LNO 2 generated by a Michael-like addition between H 2 O and the nitroalkene LNO 2 (3 M) was incubated at 25°C for 60 min in 100 mM phosphate buffer (pH 7.4) containing 100 M DTPA and extracted following the method of Bligh and Dyer (22). L(OH)NO 2 was detected using a multiple reaction monitoring scan mode by reporting molecules that undergo an m/z 342 to 295 mass transition. This method selects m/z 342 in the first quadrupole, consistent with the precursor ion, and, following collision-induced dissociation, yields in third quadrupole a species (m/z 295) consistent with loss of the nitro group ([M Ϫ HNO 2 ] Ϫ ). The presence of the nitrohydroxy adduct was confirmed by product ion analysis of m/z 342. The degradation of LNO 2 to secondary products was followed in negative ion mode after chloroform extraction and direct injection into an ion trap mass spectrometer with electrospray ionization (LCQ Deca, Thermo Electron Corp.). (23), with the product of this reaction (cPTI) displaying a characteristic EPR spectrum. To determine whether ⅐ NO is released from LNO 2 , it was incubated at 25°C for different times in 100 mM phosphate buffer (pH 7.4) containing 100 M DTPA in the presence of cPTIO. This resulted in a time-dependent decrease in the characteristic five-peak cPTIO signal and the appearance of a new signal ascribed to cPTI (Fig. 1A). This release of ⅐ NO by LNO 2 was concentration-dependent and followed first-order decay kinetics for LNO 2 . Due to limitations of the ⅐ NO/cPTIO reaction for quantitating yields of ⅐ NO, we utilized oxymyoglobin to measure ⅐ NO release rates (24). LNO 2 was incubated with oxymyoglobin in 100 mM phosphate buffer (pH 7.4) containing 100 M DTPA, and ⅐ NO-dependent oxymyoglobin oxidation was followed spectrophotometrically. LNO 2 oxidized oxymyoglobin in a dose-and time-dependent fashion, yielding metmyoglobin as indicated by the spectral changes depicted in Fig. 1B. The apparent rate constant for ⅐ NO release by LNO 2 , calculated from the oxidation of oxymyoglobin to metmyoglobin, was k ϭ 9.67 ϫ 10 Ϫ6 s Ϫ1 (Fig. 1C). To monitor the concomitant decomposition of the parent LNO 2 molecule, its UV spectrum was first analyzed. LNO 2 displayed a characteristic absorbance spectrum with a peak at 268 nm, ascribed to the electrons of the NO 2 group. During aqueous LNO 2 decay, this maximum decreased, and a new maximum appeared at 320 nm, corresponding to a mixture of vicinal nitrohydroxy, oxygen, and conjugated diene-containing products not yet fully characterized by mass spectrometry (Fig. 1D). The decrease in absorbance at 268 nm paralleled ⅐ NO release, as detected by both EPR and oxymyoglobin oxidation.

Characterization of ⅐ NO Release from LNO
Nitrite Formation during LNO 2 Decomposition-During LNO 2 -dependent ⅐ NO formation, measured via oxymyoglobin oxidation and mass spectrometric analysis of LNO 2 parent molecule loss in aqueous buffers, the stable ⅐ NO oxidation product NO 2 Ϫ accumulated with time ( Fig. 2). The release of ⅐ NO from LNO 2 was maximal at pH 7.4 ( Fig. 3), suggesting a role for protonation and deprotonation reactions in ⅐ NO formation from LNO 2 .
Chemiluminescence Analysis of LNO 2 -derived ⅐ NO-Gasphase O 3 -mediated chemiluminescence detection of ⅐ NO is a highly sensitive and specific method for detecting ⅐ NO. LNO 2 was incubated in capped vials in 100 mM phosphate buffer (pH 7.4) containing 100 M DTPA in air, and the gas phase was directly injected into the detector. The ⅐ NO-dependent chemiluminescence yield was a function of diethylammonium (Z)-1-(N,N-diethylamino)-diazen-1-ium-1,2-diolate (DEA NONOate) and LNO 2 concentrations, studied separately (Fig. 4A). Chemiluminescence was also time-dependent, increasing with time of LNO 2 decay prior to gas sampling from vials (data not shown).
UV photolysis has been used to quantitate RSNO derivatives of proteins and other NO-containing biomolecules (25,26).
When LNO 2 (4 nmol) was subjected to UV photolysis in concert with ⅐ NO chemiluminescence detection, UV light exposure stimulated ⅐ NO release from LNO 2 (Fig. 4B). The ⅐ NO chemiluminescence response to NO 2 Ϫ (4 nmol) added to samples being subjected to UV photolysis and repetitive LNO 2 addition was also examined to address the possibility that LNO 2 -derived NO 2 Ϫ formed during decay reactions might have accounted for some fraction of net chemiluminescence yield; it did not.
Appreciating that nitroalkene derivatives of red cell membrane and plasma fatty acids are present in human blood, we examined whether LNO 2 -derived ⅐ NO has the potential to interfere with the chemiluminescence detection of NO 2 Ϫ , RSNO, RNNO, or NO-heme compounds in plasma when also analyzed via an I 3 Ϫ -based reaction system (21). Plasma from lipopolysaccharide-treated rats was used to exemplify this reaction system because lipopolysaccharide treatment of rodents induces a robust elevation in plasma biomolecule ⅐ NO adduct levels (27). First, plasma was directly injected into the detector chamber, and I 3 Ϫ reagent was added, yielding a signal indicative of net plasma NO 2 Ϫ , RSNO, RNNO, and NO-heme compounds ( Fig.  4C, peak 1). Then, plasma treated with acidic sulfanilamide (which removes NO 2 Ϫ ) was injected, giving a peak of lower intensity after I 3 Ϫ reagent addition, indicative of RSNO and putative RNNO derivatives (20,28). Finally, a plasma sample treated with acidic sulfanilamide and HgCl 2 was injected, which resulted in an even smaller peak following I 3 Ϫ reagent addition (Fig. 4C, peak 3). This latter peak has been referred to as mercury-resistant RNNO derivatives (20,28). Using this strategy and combination of reagents, LNO 2 pretreated with acidic sulfanilamide and HgCl 2 also generated ⅐ NO chemiluminescence for extended periods of time following I 3 Ϫ addition (Fig. 4C, LNO 2

inset).
Hydrophobic Stabilization of LNO 2 -The observation that LNO 2 is stable in organic solvents such as n-octanol, undergoing decay only after solvation in aqueous solutions, led us to analyze the rates of ⅐ NO formation from LNO 2 in the presence of non-ionic detergents. The formation of ⅐ NO was followed by EPR (measuring cPTI formation) in the presence of different concentrations of OG and OTG. The rate of ⅐ NO release was constant and not influenced by these detergents until the critical micellar concentration (CMC) for each was achieved, after which ⅐ NO formation was inhibited as the volume of the hydrophobic environment increased (Fig. 5A). Similar results were obtained when measuring ⅐ NO formation via conversion of oxy-myoglobin to metmyoglobin. The apparent ⅐ NO release rates remained constant until the OG concentration reached ϳ2.8 mg/ml (CMC ϭ 2.77 mg/ml) (11). For OTG, inhibition of LNO 2dependent ⅐ NO release occurred at ϳ7 mg/ml (CMC ϭ 7.8 mg/ml) (11) (Fig. 5B). To further confirm that LNO 2 was protected in lipophilic environments, LNO 2 decomposition was followed by UV absorbance at 268 and 320 nm (Fig. 5, C and D). Inhibition of the NO 2 group loss at 268 nm was paralleled by inhibition of the formation of oxidation products at 320 nm, similarly paralleling the detergent-induced inhibition of ⅐ NO release observed by EPR and oxymyoglobin-based detection. The micellar stabilization of LNO 2 was also documented in OTG-containing buffers by mass spectrometry-based quantification of LNO 2 after extraction (Fig. 6A) following the method of Bligh and Dyer (22). Assuming rapid partitioning of LNO 2 between the aqueous and hydrophobic compartments and that LNO 2 decay occurs only in the aqueous compartment, it can be shown that the rate of the reaction () is given by the following relationship: ϭ k[LNO 2 ]/(1 ϩ ␣(K Ϫ 1)), where k is the rate constant for aqueous breakdown, ␣ is the fraction of total volume that is the hydrophobic volume, and K (hydrophobic/ aqueous concentration ratio) is the partition constant for LNO 2 . Thus, a plot of 1/ versus ␣ will yield a linear plot with the slope divided by the y axis intercept equal to K Ϫ 1. Fig. 6B shows this plot for OG and OTG, yielding K values of 1580 and 1320, respectively.
To evaluate the stability of LNO 2 in bilayers rather than micelles, dipalmitoylphosphatidylcholine/cholesterol/stearylamine (4:2:1 mol ratio) prepared by reverse-phase evaporation were utilized. This alternative hydrophobic bilayer environment also resulted in a dose-dependent inhibition of the release of ⅐ NO from LNO 2 , as detected by EPR analysis of cPTI formation from cPTIO (Fig. 6C).
The decay of LNO 2 in aqueous solutions results in the formation of multiple secondary fatty acid-derived products as well as ⅐ NO. One pathway that may be involved in aqueous LNO 2 decay is the Michael-like addition reaction with H 2 O at the ␣-carbon of the nitroalkene moiety. To test this possibility and the influence of micellar stabilization of LNO 2 , the formation of L(OH)NO 2 (m/z 342) was analyzed by mass spectrometry. LNO 2 -derived L(OH)NO 2 was evident after a 60-min incubation in aqueous buffer at pH 7.4, with a concomitant decrease in the levels of LNO 2 (m/z 324). Addition of OTG at a concen- 4 nmol in 20 l of MeOH, two additions made before and after sodium nitrite addition), and sodium nitrite (4 nmol in 20 l of phosphate buffer). C, blood was obtained by cardiac puncture of lipopolysaccharide-treated rats; red cells were removed by centrifugation; and plasma samples were treated as described under "Experimental Procedures." The following conditions were studied in C: I 3 Ϫ alone (peak 1); I 3 Ϫ plus sulfanilamide (SA) (peak 2); and I 3 Ϫ plus sulfanilamide and HgCl 2 (peak 3), with 75 M of LNO 2 treated with I 3 Ϫ plus sulfanilamide and HgCl 2 as for the corresponding plasma sample (LNO 2 inset). Derived ⅐ NO was measured by ⅐ NO chemiluminescence analysis. Traces are representative of three different experiments.
The release of ⅐ NO by LNO 2 via a modified Nef reaction mechanism was further supported by detecting an aqueous degradation product at m/z 293 (Fig. 8). This mass/charge ratio is consistent with the formation of a conjugated ketone (see Scheme 2, Stage 2). Also present in the mass spectrum is a peak for the vicinal nitrohydroxy adduct (m/z 342) and minor peaks corresponding to the hydroxy and peroxy derivatives of LNO 2 (m/z 340 and 356, respectively).

DISCUSSION
Nitrolinoleic acid is a pluripotent signaling molecule that exerts its bioactivity by acting as a high affinity ligand for peroxisome proliferator-activated receptor (PPAR)-␥ (2); by activating protein kinase signaling cascades; and as shown herein, by serving as a hydrophobically stabilized reserve for ⅐ NO. The activation of PPAR␥-dependent gene expression by LNO 2 requires this ligand to be stabilized and transported as the intact nitroalkene to the nuclear receptor (2). The mechanism(s) involved in protein kinase activation by LNO 2 remain unclear, but can include direct ligation of receptors at the plasma membrane and/or covalent modification and activation of signaling mediators via Michael addition reactions. Current data reveal that the signaling actions of LNO 2 are multifaceted, with the activation of protein kinases and/or PPAR activation not fully explaining observed cellular responses, such as the stimulation of cGMP-dependent vessel relaxation (5). Our observations that LNO 2 decay yields ⅐ NO and that LNO 2 is subject to hydrophobic stabilization thus lend additional perspective to our understanding of how compartmentalization will influence the nature of cell signaling reactions mediated by fatty acid nitroalkene derivatives.
A central challenge in detecting ⅐ NO generation by relatively slow releasing compounds (e.g. RSNO and organic nitrate derivatives) is the risk of lack of specificity and sensitivity. This is especially the case when concurrent oxygen-, heme-, lipid-, protein-, and probe-related redox reactions are possible. Quantitative rigor is also always a concern. To circumvent these problems, multiple approaches for the qualitative and quanti-tative detection of ⅐ NO generation by LNO 2 were employed herein. The release of ⅐ NO by LNO 2 was assessed quantitatively by spectrophotometric analysis of oxymyoglobin oxidation. Additional qualitative proof of LNO 2 -derived ⅐ NO release came from EPR analysis of ⅐ NO-dependent cPTI formation, ⅐ NO-dependent chemiluminescence following reaction with O 3 , and mass spectroscopic detection of anticipated decay products of LNO 2 . Also, in aqueous solutions and in the absence of alternative reaction pathways, 4 mol of ⅐ NO reacted with 1 mol of O 2 to ultimately yield 4 mol of NO 2 Ϫ . Thus, formation of NO 2 Ϫ was used as additional evidence for ⅐ NO formation. The yield of NO 2 Ϫ during LNO 2 decay was 3.5-fold lower than predicted from more direct ⅐ NO measurements based on oxymyoglobin oxidation. Several explanations can account for this apparent discrepancy. First, in the absence of ⅐ NO scavengers, ⅐ NO rapidly equilibrates with the gas phase, thus decreasing ⅐ NO available for oxidation to NO 2 Ϫ . Second, ⅐ NO reactions with carbonyl, hydroxyl, and peroxyl radicals are extremely fast (k Ͼ ϳ1 ϫ 10 10 M Ϫ1 s Ϫ1 ) (29). These free radical intermediates are likely formed during LNO 2 decomposition, as evidenced by products with mass/charge ratios of 340 and 356 (Fig. 8). Thus, the products of the reaction of these species with ⅐ NO may not contribute to NO 2 Ϫ formation. Overall, multiple independent criteria support the capacity of LNO 2 to release ⅐ NO.
The gas-phase chemiluminescence reaction of ⅐ NO with O 3 is a highly sensitive and specific method for detecting ⅐ NO and nitroso derivatives of biomolecules. One widely utilized analytical strategy relies on the reductive cleavage of NO 2 Ϫ and nitroso derivatives by I 3 Ϫ . Treatment of samples with acidic sul- fanilamide and HgCl 2 permits additional discrimination between NO-heme, NO 2 Ϫ , and putative RSNO and RNNO derivatives (20,21,(25)(26)(27)(28). The latter HgCl 2 -resistant species (proposed as RNNO) (20) may best be termed RNO x at this juncture because LNO 2 also yields O 3 chemiluminescence following reaction with acidified sulfanilamide and HgCl 2 prior to injection into iodine/triiodide mixtures and the detection chamber. These data reveal that a contribution of fatty acid nitroalkene derivatives to the measurement of various tissue biomolecule ⅐ NO derivatives must additionally be considered. Of additional interest, the UV photolysis approach for NO x detection in biological samples directly stimulates decay of LNO 2 to yield ⅐ NO. This new insight thus raises significant concern about the accuracy of reported concentrations for ⅐ NO-derived species using UV photolysis because nitrated fatty acids are the most prevalent bioactive oxides of nitrogen yet found in vivo (1). Protein fractionation via solvent extraction (e.g. acetone) prior to analysis of ⅐ NO derivatives in biological samples does not eliminate the possibility that nitrated fatty acids are a source of "detectable" or RSNO-like ⅐ NO formation by UV photolysis, as LNO 2 and other nitroalkenes readily partition into the polar phase of many extraction strategies, including those employing acetone.
The observation that LNO 2 undergoes decay reactions to yield ⅐ NO in aqueous solution initially raised concern regarding how a significant and consistent LNO 2 content in plasma and red cells of healthy humans could be detected at near-micromolar concentrations (1). Appreciating that synthetic LNO 2 is stable in methanol suggested that the ionic microenvironment in which LNO 2 was solvated would significantly modulate stability. To first address the possibility that LNO 2 is stabilized by hydrophobic environments reminiscent of membranes and lipoproteins, it was observed that ⅐ NO release from LNO 2 was inhibited upon LNO 2 solvation in n-octanol (data not shown). Further analysis using non-ionic detergent micelles, in which the relatively hydrophobic NO 2 group of LNO 2 is expected to partition into non-polar microenvironments, revealed that LNO 2 decomposition and ⅐ NO release were inhibited (Fig. 5). Importantly, this occurred at and above the CMC of each detergent and lipid studied. Similar results were obtained using dipalmitoylphosphatidylcholine/cholesterol/stearylamine liposomes (4:2:1 mol ratio), also revealing that LNO 2 is readily incorporated into and stabilized by lipid bilayers (Fig. 6C). This stabilizing influence of liposomes, which have a very low CMC, occurred at low hydrophobic phase volumes. These data reveal that LNO 2 will be stable in hydrophobic environments and that cell membranes and lipoproteins can serve as an endogenous reserve for LNO 2 and its downstream cell signaling capabilities. Indeed, ϳ80% of LNO 2 is esterified to complex lipids in blood, including phospholipids derived from red cell membrane lipid bilayers (1). This further suggests that, during inflammatory responses, esterases and A 2 -type phospholipases may hydrolyze and mobilize membrane-stabilized LNO 2 for mediating cell signaling actions. This regulated disposition of LNO 2 in lipophilic versus aqueous environments thus represents a "hydrophobic switch" that will control the nature of LNO 2 signaling activity (Scheme 1).
The mechanisms accounting for ⅐ NO release from organic nitrites and nitrates are controversial, appear to be multifaceted, and remain to be incisively defined. For example, the nitrate ester derivative nitroglycerin has been used as a vasodilator for more than a century in the treatment of angina pectoris. Nitroglycerin does not directly decay to yield ⅐ NO or an ⅐ NO-like species that will activate soluble guanylate cyclase; rather, cellular metabolism is required to yield a species that induces ⅐ NO-like activation of soluble guanylate cyclase. Although several enzymes have been identified as competent to mediate the denitration and "bioactivation" of nitroglycerin (e.g. xanthine oxidoreductase, cytochrome P450 oxidase and reductase, old yellow protein, and mitochondrial aldehyde dehydrogenase-2), detailed insight is lacking as to unified redox chemistry, enzymatic and cellular mechanisms accounting for (a) the 3e Ϫ reduction of organonitrates to an ⅐ NO-like species and (b) the attenuated nitroglycerin metabolism that occurs during nitrate tolerance (30).
Our report of nonenzymatic release of ⅐ NO from endogenous fatty acid nitroalkene derivatives (e.g. LNO 2 ) lends additional perspective to how nitric-oxide synthase-dependent ⅐ NO signal- into different cell compartments is governed in part by its partition coefficient (K ϳ 1500). LNO 2 may also be stabilized and placed in "reserve," in terms of ⅐ NO-mediated cell signaling capabilities, by esterification into complex lipids of membranes or lipoproteins. Alternatively, LNO 2 derivatives of complex lipids can be formed by direct nitration of esterified unsaturated fatty acids. During inflammatory conditions or in response to other stimuli, LNO 2 may be released from complex lipids by A 2 -type phospholipases (PLA2) or esterases, thus mobilizing "free" LNO 2 , which can in turn diffuse to exert receptor-dependent signaling actions or undergo decay reactions to release ⅐ NO.
ing can be transduced. We have shown via three different analytical approaches that the product of LNO 2 decay is unambiguously ⅐ NO. The mass spectrometric analysis and LNO 2 decay studies reported herein, in concert with the previous understanding of the chemical reactivity of nitroalkenes, reveal a viable mechanism for how nitrated fatty acids can serve to transduce tissue ⅐ NO signaling capacity (Schemes 1 and 2).
The release of ⅐ NO by a vicinal nitrohydroxyarachidonic acid derivative detected in cardiac lipid extracts has been proposed (31). These derivatives induce vasorelaxation of rat aortic rings via possible ⅐ NO-dependent activation of guanylate cyclase. The intermediate formation of an analogous hydroxy derivative of nitrolinoleic acid, L(OH)NO 2 , has been documented in this study to occur during LNO 2 decay in an aqueous milieu (Fig. 8). Fatty acid nitroalkene derivatives are clinically abundant with both nitro and nitrohydroxy derivatives of all principal unsaturated fatty acids present in healthy human blood plasma and urine. 2 Our results indicate that hydroxy derivatives of fatty acid nitroalkenes represent the accumulation of Michael addition-like reaction products with H 2 O that are in equilibrium with the parent nitroalkene and are not a direct precursor to ⅐ NO release.
A more viable mechanism accounting for ⅐ NO release by nitroalkenes is supported by 1) mass spectroscopic detection of the expected decay products and 2) the aqueous and pH dependence of this process (Fig. 3), with LNO 2 decay and the consequent ⅐ NO release involving protonation and deprotonation events. The mechanism accounting for ⅐ NO release by nitroalkenes is based on the Nef reaction (33, 34), a standard reaction of organic nitro derivatives first described in 1894 (35).
The original Nef reaction entails complete deprotonation of an alkyl nitro compound with a base to yield the nitroanion, followed by quenching with an aqueous acid to cause hydrolysis to the corresponding carbonyl compound and oxides of nitrogen. Most Nef reactions are now performed using additional oxidants or reductants, rather than the simple acid/base chemistry of the original reaction (32, 36 -43). There are a few noteworthy points about this proposed mechanism that relate to how ⅐ NO can be ultimately produced. The nitrogen-containing product of the original Nef reaction is N 2 O, a stable oxide of nitrogen that would not be a precursor to ⅐ NO under the neutral aqueous conditions used herein to model biologically relevant LNO 2 decay. The initial oxide of nitrogen formed, HNO, is unstable and quickly disproportionates to form N 2 O as shown in Scheme 2 (Stage 2). Although HNO (or the NO Ϫ anion) might conceivably yield one electron and be oxidized to ⅐ NO, this is not expected under neutral aqueous conditions. Alternatively, a nitroso intermediate formed during LNO 2 decay provides a plausible pathway to yield ⅐ NO. This nitroso intermediate is expected to have an especially weak C-N bond, easily forming ⅐ NO and a radical stabilized by conjugation with the alkene and stabilized by the OH group, a moiety known to stabilize adjacent radicals.
In Scheme 2 (Stage 1), the vicinal nitrohydroxy fatty acid derivative is in equilibrium with the nitroalkene. This is pos-  sible for two reasons. First, the nitro group in the vicinal nitrohydroxy fatty acid makes the adjacent hydrogen very acid (pK a ϳ7-8), thus facilitating formation of a significant amount of the nitronate anion at physiological pH. The anion can then release hydroxide, which, when neutralized with the proton removed in the first step, results in the net loss of neutral water. Second, the fatty acid nitroalkene is a strong electrophile and can readily undergo Michael-like conjugate addition reaction with the small amounts of hydroxide anion that are always present in aqueous solution under physiological pH conditions, explaining the facile equilibrium of vicinal nitrohydroxy fatty acids with their corresponding nitroalkene derivatives. In Scheme 2 (Stage 2), the lipid nitroalkene forms ⅐ NO as described above.
These proposed mechanisms for ⅐ NO formation from LNO 2 provided the testable hypothesis for how nitrated fatty acids can serve as a source of ⅐ NO using simple acid/base chemistry with no additional oxidants or reductants. Mass spectrometric detection of the expected oxidized fatty acid products and direct detection of ⅐ NO formation supported this pathway of nitroalkene decay. This acid/base chemistry may also be employed by as yet undescribed enzymes that could catalyze physiologically significant amounts of ⅐ NO release from the multiple lipid nitroalkene derivatives now being observed. 2 Therapeutic agents that release ⅐ NO are a rapidly expanding area of drug design. Dual-acting nitro and nitroso derivatives of existing drugs have been synthesized and are being studied for efficacy in treating diabetes, metabolic syndrome, hypertension, and atherosclerosis. These include ⅐ NO-releasing statin derivatives and NO-nonsteroidal anti-inflammatory derivatives such as NO-acetylsalicylic acid, NO-ibuprofen, and NOpiroxicam. These adducts were devised based on the precept that an ⅐ NO donor moiety will augment therapeutic breadth and value. This class of pharmaceuticals is of particular relevance when alterations in endogenous ⅐ NO signaling contribute to tissue pathogenesis. In this regard, LNO 2 shares similarities with "chimeric" inflammatory-regulating compounds, as LNO 2 is a potent endogenous PPAR␥ agonist that rivals the extent of PPAR␥ activation induced by similar concentrations of thiazolidinediones (2). In this work, we have shown that LNO 2 also has the capability to release ⅐ NO in a regulated manner. Thus, the signaling actions of LNO 2 are pluripotent in nature.
In summary, ⅐ NO-mediated oxidative reactions with unsaturated fatty acids yield nitroalkene derivatives. Once formed, nitrated fatty acids are hydrophobically stabilized by lipid bilayers and lipoproteins or, alternatively, can be redistributed to aqueous environments to release ⅐ NO via a Nef-like reaction. In its native form, LNO 2 activates nuclear PPAR-mediated regulation of gene expression. These combined actions are expected to transduce the salutary inflammatory signaling reactions that have been described for both ⅐ NO and LNO 2 . Because LNO 2 production is increased by oxidative inflammatory reactions, this species thus represents an adaptive mediator that regulates potentially pathogenic tissue responses to inflammation.