Nitro Fatty Acids

Scottish thistle Nitric oxide is a signalling molecule that is involved in the regulation of many metabolic pathways in animals that include vasodilation, inflammatory cell function and neuronal function, but many related reactive nitrogen species (RNS) are produced non-enzymatically. While the involvement of free radical-catalysed addition of nitric oxide radicals (NO^•) and secondary products of nitrite anions like nitrogen dioxide radicals (NO₂^•) to unsaturated and hydroperoxy fatty acids in vitro has been known for many years, it was only in 1999 that the first paper appeared to show that nitro fatty acids were present in the membrane phospholipids of human tissues in vivo and at concentrations that had the potential to exert biological effects. Since then, the nature and biology of these fascinating lipids have attracted increasing research interest. RNS are produced continuously under normal physiological conditions, and they are present in foods and the environment so can generate nitro-lipids even in healthy populations, but especially during stress conditions such as inflammation and disease when RNS can be overproduced.

These nitro metabolites are classified as electrophilic fatty acids, together with related lipids with α,β‑unsaturated carbonyl and epoxide substituents, which have a propensity to undergo reversible Michael addition reactions with cellular nucleophiles such as cysteine and histidine-containing peptides and proteins. In part through these reactions, they are involved in diverse signalling events, which including triggering peroxisome proliferator-activated receptor (PPAR)-dependent gene expression, inhibiting oxidative stress, increasing endothelial nitric oxide synthesis and suppressing inflammation induced by cytokines. In these reactions, it has been demonstrated that they afford protection from inflammatory injury in several experimental models and have therapeutic potential that is under investigation in clinical trials.

Most research on the topic relates to animal biochemistry, but it is now recognized that nitro fatty acids have important functions in plants. While the main emphasis has been on nitro fatty acids per se, it is increasingly being realized that they be active biologically in the intact lipids in which they are formed.

1. Occurrence in Animal Tissues

It was initially thought that the main nitrated species of fatty acids in animal tissues were derived from oleic acid, i.e., 9- and 10-nitro-9-cis-octadecenoic acids, and these are still the most studied because of their relative availability by chemical synthesis. Note that under the official IUPAC rules of nomenclature these should strictly speaking be designated as trans isomers to reflect the orientation of the nitro group relative to the alkyl substituent on the adjacent carbon atom, but those active in this research topic prefer the more familiar usage based upon the precursors.

Structural formulae for the 9- and 10-nitro-9-cis-octadecenoic acids

It is now apparent that 'conjugated linoleic acid' (9-cis,11-trans-octadecadienoic acid or CLA) may be the primary endogenous substrate for fatty acid nitration in vitro and in vivo, yielding up to 10⁵ more nitration products (mainly 9- and 12-nitro-octadeca-9,11-dienoic acids) than linoleic acid per se, presumably because of resonance stabilization of the radical intermediates formed during biosynthesis (see below). These have been detected in plasma and urine of healthy humans with and without CLA supplementation, and they are generated during digestion, metabolic stress and inflammation, and indeed may often be the only isomers detectable. Red meat and dairy products are the main dietary source of CLA, although some is produced by isomerization of linoleate by microorganisms in the intestines and some by desaturation of vaccenic acid (trans‑11‑18:1) in the liver.

Nitration products of conjugated linoleic acid (CLA)

Analogous compounds reportedly derived from linoleate were detected at significant concentrations in some studies, but not in others, but it is possible that these were misidentified and that CLA was the true origin. Similarly, nitrated derivatives of palmitoleic, linolenic, arachidonic and eicosapentaenoic acids have been detected at trace levels in human plasma and urine by sensitive analytical mass spectrometric methods. In general, there is considerable selectivity in terms of which of the various isomers are detected in tissues, and for example, the nitro-eicosatetraenoic acids have the NO₂ groups in positions 9, 12, 14 and 15 mainly. Comparable metabolites have been described from other fatty acids, including eicosapentaenoic and docosahexaenoic acids (n-3 family), some with two nitro groups. Such findings are controversial, and they were contradicted in one recent study, which reported that nitro derivatives of conjugated linoleate and linoleate were the only isomers detected in urine.

In plasma, nitro fatty acids occur in the free form and esterified in cholesterol esters and triacylglycerols, while in adipose tissue they have been detected in triacylglycerols and membrane phospholipids, but they are also present bound reversibly to thiol-containing proteins and glutathione in tissues. Free and esterified concentrations of nitro fatty acids in plasma and red blood cells were originally greatly overestimated, and subsequent studies with stable isotope-dilution methodology suggests that the true basal level in plasma of healthy humans is 1 to 3nM, while a figure of 9 nM for total nitro-isomers has been quoted recently for urine. Other reports suggest that even these may be overestimates, although concentrations do increase significantly under inflammatory conditions such as vascular injury and myocardial ischemia and reperfusion. In any case, cellular nitro fatty acids would be expected to have a short half-life because of the ease with which they undergo non-enzymatic Michael addition reactions with thiol-containing compounds (see below). Inevitably, these reactions compound the analytical difficulties and can lead to underestimates of the rates of formation of such fatty acids, since as much as 99% of nitro fatty acids may be covalently bound to cysteine in proteins.

Nitro fatty acids can be precursors of further nitrated and nitroxidized species such as nitroso, dinitroso, nitronitroso, and di- and trinitro species, or they can be oxidized to generate various nitroxidized forms such as nitro-hydroxy, nitro-hydroperoxy, nitro-epoxy and nitro-keto species; representative structures of nitro-hydroxy derivatives of oleate and linoleate are illustrated. Subsequently, nitro-eicosatetraenoic, α,β‑nitrohydroxy-eicosatrienoic and trans-arachidonic acids, derived from arachidonic acid via such reactions, were characterized both in vitro and in vivo. Little is yet known of the biological properties of these oxidation products.

Structural formulae for the nitrohydroxy derivatives of oleate and linoleate

Further related metabolites, which have been characterized and are presumed to be formed by comparable mechanisms, include nitro-allyl derivatives of various fatty acids, including oleate, in which both the position and configuration of the double bond is changed. In addition, simple (non-nitrated) geometrical (trans) isomers of unsaturated fatty acids can be produced as a by-product of a nitration reaction, and those derived from arachidonate are of special biological relevance.

Structural formulae for nitro-allyl fatty acids

For historical reasons, most metabolic studies to date have concentrated on nitro-oleate isomers, which are easily characterized and are available from chemical synthesis, but the evidence to date is that nitro-CLA isomers are metabolized in the same way. When 10-nitro-oleic acid was administered orally to dogs, it was efficiently absorbed and esterified to position sn-2 mainly of triacylglycerols more rapidly than its metabolite 10‑nitro-stearic acid and incorporated into lipoproteins (chylomicrons) for distribution in plasma to other tissues. Esterification into triacylglycerols and other lipids is believed to shield the electrophilic nature of these mediators from deactivation in plasma and the liver and so enables efficient distribution to target organs, where they can be released by lipases and thence transported into cells and incorporated into the cellular lipids. Whole body studies of rats treated with radio-labelled 10‑nitro-oleic acid demonstrated rapid incorporation into kidney, liver, lungs and heart. Adipocytes may act as an inert store since over a longer term (two weeks), with unsaturated nitro-fatty acids incorporated mainly into mono- and diacylglycerols, while analogous saturated metabolites were enriched in triacylglycerols. As nitro fatty acid conjugates with coenzyme A have been detected in tissues, they can presumably be re-esterified after hydrolysis from a lipid-bound form.

2. Formation of Nitro Fatty Acids in Tissues

The nitric oxide radical is produced endogenously in animal tissues from L-arginine and oxygen by various nitric oxide synthases, but formation of nitro fatty acids occurs in tissues through the non-enzymatic reactions of many reactive nitrogen species, i.e., free radicals and their precursors, such as nitrite, nitric oxide (NO^•) and NO^•‑derived oxides of nitrogen, (nitrogen dioxide (NO₂^•), peroxynitrite (ONOO^•), nitrosoperoxocarbonate (ONOOCO₂) and dinitrogen trioxide (N₂O₃)), which can give rise to different types of reaction, including nitration, nitrosation and nitro-oxidation. Of these, the NO₂^• radical is the main reactive intermediate, and it can arise spontaneously from various endogenous and exogenous sources in humans. Meat and other foods may contain appreciable quantities of nitrite (NO₂^-), which may be added as a preservative, and nitrate can be reduced to nitrite by aerobic bacteria in the mouth. In the stomach, dietary nitrite decomposes rapidly in the acidic environment to form NO^• and NO₂^• and other bioactive nitrogen oxides, probably via N₂O₃ as an intermediate, and these are absorbed from the intestines and thence enter the circulation.

Formation of the nitrogen dioxide radical

These radicals operate in conjunction with oxygen-derived inflammatory mediators such as superoxide (O₂^•), hydrogen peroxide (H₂O₂) and lipid peroxyl radicals (LOO^•) in tissues. Many different mechanisms are involved in the production of secondary radicals and in their subsequent reactions, which are controlled by such factors as the concentration of the NO₂^• radicals, the site of their production, oxygen tension, and the concentrations and membrane environment of the target molecules and of any catalysts and antioxidants. In addition, immune responses to inflammatory stimuli induce nitric oxide synthase in certain cells that form NO^•, which is then oxidized to NO₂^•. As NO₂^• is a common air pollutant, it can be absorbed via the lungs. Although NO^• per se does not participate as a direct nitrating species, its presence in tissues may be required for nitration to occur. Reactions would then be expected to take place mainly in the membranes of cells because partitioning of NO^•/NO₂^• into this cellular component occurs preferentially.

Mechanistic studies of nitro fatty acid formation in human and other animal tissues support biosynthetic mechanisms proposed first from chemical studies in vitro, though whether all of these occur to a significant extent in vivo is doubtful. The NO₂^• radicals can react with unsaturated lipids and lipid radicals to form all the types of products found in tissues, and at low oxygen tensions, homolytic attack to the double bond yields nitroalkyl radicals, which combine with other NO₂^• radicals to form nitro-nitrito intermediates. Loss of nitrous acid (HNO₂) from these intermediates results in the formation of nitroalkenes - the main products, while hydrolysis leads to the production of nitro-alcohols. The nitro-conjugated linoleate isomers illustrated in Figure 2 above, which are the most abundant products in vivo, are formed by this mechanism. In an alternative reaction, abstraction of a hydrogen atom from the nitroalkyl radicals leads to the formation of nitro-allyl derivatives. As these reactions are non-enzymatic and involve intact lipids rather than the free acids, their formation has much in common with isoprostane formation.

Nitro fatty acid formation by free radical reactions

Under the acid conditions of the stomach, it has been established that both unesterified fatty acids and those in triacylglycerols can be rapidly nitrated, with conjugated linoleic acid produced by microbial fermentation or in foods as a major reactant, and the nitro fatty acids produced are taken up by the intestinal tissue and incorporated into chylomicrons for transport in plasma to the liver and other tissues. In the rat stomach, it has been determined that nitration with conjugated linoleic acid proceeds by generation of nitro-nitrate intermediates (NO₂‑ONO₂‑FA) via reactions dependent upon oxygen and nitrite, and these represent ~70% of all nitrated lipids in the stomach. These metabolites decay in vitro at neutral or basic pH by the loss of the nitrate ester group (-ONO₂) from the carbon backbone to yield an electrophilic fatty acid nitroalkene product (NO₂‑FA) together with nitrate, nitrite and nitrosative species.

Nitration of conjugated linoleate in rat stomach

As an NO₂^• radical can initiate lipid oxidation reactions, yields of nitration versus oxidation products will depend on the concentration of oxygen. At elevated oxygen levels, the NO₂^• radical can interact with an unsaturated fatty acid to form a carbon-centred radical, which can interact with oxygen to form a lipid hydroperoxide. Unstable alkyl peroxynitrite intermediates can be formed through the reactions of lipid peroxyl radical (LOO^•) and NO^•, of peroxynitrite radicals, and of a lipid hydroperoxide reaction with N₂O₄ or with HNO₂, the last leading to the production of nitro-epoxy fatty acids, and under these conditions, nitro-hydroxy and nitro-oxo oleate derivatives can be formed from conjugated linoleate. On the other hand, nitro fatty acid radicals can be produced that lose HNO₂ to regenerate the unsaturated fatty acid but with one of the double bonds isomerized from the cis to the trans configuration.

Nitration reactions under high oxygen tension

A further mechanism for nitroalkene formation is addition of a nitronium ion (NO₂⁺), which can be formed by reaction of a transition metal with peroxynitrite, by electrophilic substitution at the double bond.

Nitro fatty acid formation by electrophilic substitution at the double bond

Deactivation and catabolism: Reactive nitrogen oxide species react very specifically with γ-tocopherol in vivo to produce a 5-nitro-metabolite and not with α-tocopherol. Deactivation of nitro fatty acids is achieved mainly by reduction by prostaglandin reductase-1 in the liver with formation of non-electrophilic nitroalkanes, and for example, 9-nitro-oleate infused into mice and 10‑nitro‑oleate given orally to dogs were hydrogenated in part to nitro-stearates, which are not electrophilic, although some of the 9-isomer was desaturated to a nitro-octadecadienoic acid. In addition, 10-nitro-oleate was isomerized to 10‑nitro-8-trans-octadecenoate, possibly because of reversible formation of Michael adducts (see below), i.e., deprotonation of the α‑carbon followed by β‑elimination of the thiolate group.

A proportion of the 9-nitro-oleate can be subjected to β-oxidation to yield nitro-7-cis-hexadecenoic, nitro-5-cis-tetradecenoic and nitro-3-cis-dodecenoic acids, and their corresponding coenzyme A derivatives. 10‑Nitro‑oleate was found to be subjected to ω- and β-oxidation to produce several oxidized metabolites with 4‑nitro-octanedioic acid as a major urinary product, and these were excreted in part as N‑acetylcysteine, taurine and sulfo-conjugates. In phosphate buffers and presumably in the cytoplasm of cells, nitroalkenoic fatty acids decay rapidly because of solvation reactions with the release of nitric oxide radicals, and while various reaction mechanisms have been proposed, their biological relevance in vivo is uncertain.

3. Biological Effects of Nitro Fatty Acids

It has long been known that nitric oxide per se is involved in innumerable biological processes in tissues, and in contrast to the prevailing dogma, experimental evidence was obtained in the 1990s that NO^• inhibited the oxidation of membranes and plasma lipoproteins more potently than α‑tocopherol and in general had anti-inflammatory, antioxidant and tissue-protective effects. Subsequently, the role of nitro fatty acids in mediating these reactions has become apparent. It is now well established from experiments both in vitro and in vivo that nitro fatty acids elicit metabolic responses that are generally beneficial, and they are anti-inflammatory mediators with appreciable therapeutic potential. They are known to modulate the expression of more than 300 genes crucial for cytoprotective, metabolic and anti-inflammatory responses in addition to direct effects upon enzymes. In response to inflammatory stimuli, they may be formed in situ or released from membrane or adipose tissue stores.

While it is evident that they have biological activities of their own, nitro fatty acids can act by releasing NO^•, for which there are at least two hypothetical mechanisms although confirmation of the exact mechanism is required. This is an important aspect of their metabolism and function and may be a means of delivering NO^• to remote tissues.

Reaction with thiols: The main mechanisms and signalling actions of nitro fatty acids are mediated by post-translational modification of proteins by covalent adduction. Nitrated unsaturated fatty acids are powerful electrophiles, i.e., the nitro-alkene moiety has strong electron-withdrawing properties that render the β‑carbon electron deficient and thus susceptible to attack by nucleophiles. This favours reversible nitroalkylation reactions (Michael reaction) with deprotonated thiolate anions, such as the thiol groups of glutathione and thio-amino acids, as well as the imidazole moiety of histidine and the ε-amino group of lysine residues of proteins, thereby regulating the structure and function of the latter. The kinetically rapid but reversible nature of these reactions differentiates nitro fatty acids from other endogenous signalling electrophiles such as the cyclopentanone prostaglandins and aldehydes derived from lipid oxidation, e.g., 4-hydroxy-2-nonenal, that react more slowly and often irreversibly with nucleophiles. Although such post-translational modifications of proteins are non-enzymic in nature, and 184 potential protein targets for nitro-alkylation have been identified in macrophages alone, these reactions appear to be more selective than might be expected. Transmembrane proteins in the endoplasmic reticulum and nuclear membranes are most susceptible, and then intact lipids containing nitro fatty acids are the main reactants rather than those in unesterified form.

Michael addition reaction of nitrofatty acids

Again, 10-nitro-oleate has been used in most studies, although nitro adducts of conjugated linoleate isomers are particularly potent electrophiles, and they are the main nitro fatty acids in serum. With these, mechanistic and kinetic studies have demonstrated that reactions with thiols occur rapidly to form adducts both β and δ to the nitro group, although the δ-adducts are formed nearly 10 times as quickly as β-adducts. Indeed, the cysteine-δ-adducts have been detected in human urine. In human serum in addition to non-covalent binding with albumin, nitro-CLA has been shown to form covalent adducts at Cys-34, suggesting that this may be a means of systemic distribution. The effective concentrations of nitro fatty acids in tissues are reduced by this means, but under conditions of oxidative stress in vitro in plants at least, reactive oxygen and nitrogen species, as represented by hydrogen peroxide and peroxynitrite, respectively, can oxidize cysteine-adducted nitro fatty acids with the release of free nitroalkenes.

Reaction of nitro-CLA with thiols

It has been determined both in vitro and in vivo that nitroalkylation can result in changes in protein structure, function and subcellular distribution, and this is now considered to be a significant post-translational modification. By acting on signal transduction proteins or on key regulatory proteins to cause either up- or down-regulation, nitro fatty acids may exert many of their biological effects, and this may explain their cytoprotective and anti-inflammatory properties.

Anti-inflammatory reactions: Fatty acid nitro-alkenes are potent anti-inflammatory agents, and there is abundant evidence that nitro lipids promote cyto-protective and anti-inflammatory responses by a variety of mechanisms that include direct effects upon specific enzymes. As both nitric oxide formation and lipoxygenase-mediated pathways involve highly reactive free-radical species and act on signalling cascades in relation to inflammation and the immune response especially, interactions between the two are to be expected. Electrophilic nitro fatty acids may exert anti-inflammatory effects by interacting with nucleophilic amino acids in lipoxygenases, and an irreversible inhibition of ALOX5 has been observed. 10‑Nitro-oleic acid is an inhibitor of the epoxyhydrolase, i.e., the enzyme responsible for the hydrolysis of epoxyeicosatrienoic acid, which has protective effects against hypertension, and it is an irreversible inhibitor of the enzyme xanthine oxidoreductase, which generates pro-inflammatory oxidants and secondary nitrating species. In this instance, it has been established that the carboxyl group, nitration at the 9 or 10 olefinic carbons and the double bond are all required for this action, and comparable mechanisms operate with prostaglandin endoperoxide H synthase and NADPH-oxidase. One further example is increased activation brought about by covalent addition of nitro fatty acids to an N‑terminal cysteine residue of SIRT6, an enzyme that is critical for both glucose and lipid homeostasis.

Scottish thistle Nitro-CLA isomers in red cells and plasma may constitute the single largest pool of bioactive oxides of nitrogen in the vasculature, where they bring about vasorelaxation. In neutrophils and platelets, nitro fatty acids activate cAMP-dependent protein kinase signalling pathways, which have an anti-inflammatory role in cells, while both nitro-oleate and nitro-linoleate have been shown to inhibit the lipopolysaccharide-induced secretion of pro-inflammatory cytokines in macrophages, for example during pulmonary injury, actions that are independent of nitric oxide formation or of activation of PPARs. They complement the activities of the pro-resolving eicosanoids and docosanoids such as the lipoxins, resolvins, protectins and maresins in this way. While studies are still at an early stage, it would not be surprising if there were significant influences upon the eicosanoid cascades through redirection of arachidonic acid metabolism and signalling. Arachidonate isomers formed as by-products of nitration reactions are emerging as biomarkers that target various biological systems, such as inhibition of prostaglandin endoperoxide H synthases (COX) 1 and 2 and the synthesis of thromboxanes. Nitro-arachidonic acid inhibits superoxide production by NADPH oxidase by a reversible covalent adduct formation with critical cysteines on a supportive enzyme protein disulfide isomerase. On the other hand, the discovery of the potent effects of nitro-conjugated octadecadienoic acids as signalling mediators may mean that the relative importance of many isomers in vivo may have to be re-evaluated.

Nitro fatty acids can inhibit the expression of pro-inflammatory genes and the activity of other pro-inflammatory enzymes, and for this reason, protein targets such as peroxisome proliferator-activated receptor gamma (PPARγ) and nuclear factor kappa B (NF-κB) have received most attention. Nitro fatty acids can bind to all three PPAR isotypes with high affinities, and as signalling activators, they have the potential to regulate the expression of multiple PPAR target genes in the immune system or relevant to inflammatory diseases. PPARγ is activated most robustly with 12‑nitro-linoleate as a particularly potent agonist, which binds covalently with high specificity to Cys 285 via Michael addition. By this means, it can induce the expression and activity of endothelial nitrous oxide synthase. Nitro fatty acids form covalent adducts with cysteine residues of Kelch-like ECH-associated protein (Keap-1) with the result that activation of nuclear factor erythroid 2‑related factor 2 (Nrf2) occurs to in turn affect gene expression of anti-inflammatory enzymes. By nitroalkylating specific cysteines in calcineurin, a protein phosphatase which activates the T cells of the immune system, it exerts further anti-inflammatory effects. Taken together with the finding of significant levels of protein cysteine adducts of nitro-oleic acid and the free acid in fresh olives, there are interesting implications both for plant biology and human nutrition.

Intact lipids: Although it is the unesterified forms that have attracted most attention, nitro fatty acids are produced while in esterified form in lipids, and they have been detected in all glycerophospholipid classes where they have the potential at the very least to be disruptive to membranes. However, it is not at all surprising that intact lipids containing such fatty acids have biological activities as well, as has become increasingly evident for oxidized phospholipids, and improved analytical methodologies with mass spectrometry are now facilitating study of complex lipid metabolites of this kind. Phosphatidylcholine containing nitro-oleate has been shown to induce cellular changes such as cytoskeletal rearrangement and cell shrinkage with eventually loss of cell adhesion or impaired cell attachment in various cell types in culture, effects not seen with the free nitro fatty acid, while nitrated phosphatidylcholine and phosphatidylethanolamine have been identified in cardiac mitochondria from the heart of an animal model of type 1 diabetes mellitus. Similarly, nitrosylated forms of cardiolipin have been characterized and might be expected to interfere with mitochondrial metabolism. In models of inflammation in vitro, other nitrated phospholipids have been identified and shown to have antioxidant and anti-inflammatory properties, and such studies are in progress in living systems.

Pharmaceutical properties: There is obviously considerable therapeutic potential for the use of naturally occurring nitro-fatty acids against inflammatory conditions such as cardiovascular and kidney diseases and inflammatory bowel disease. In human trials, they appear to be tolerated well when administered orally, and phase II clinical trials of 10-nitro-oleic acid are underway for the treatment of focal segmental glomerulosclerosis, pulmonary arterial hypertension and asthma in obese patients. Nitro-fatty acids have been shown to inhibit hepatic triacylglycerol accumulation and protect against fibrosis during the development of non-alcoholic fatty liver disease in mice. Also in mice, subcutaneous injections of nitro-oleic acid have been shown to inhibit allergic contact dermatitis by inducing immunosuppressive responses. Administration of this nitro fatty acid reportedly preserves the integrity of the blood-brain barrier and confers neurovascular protection in ischemic brain damage, and there is a preliminary report that it may be neuroprotective in Parkinson's disease. On the other hand, a substantial increase in the concentration of nitro-conjugated linoleic acid was found to occur naturally in lesional skin in inflammatory skin diseases, and topical applications of nitro-oleic acid were found to exacerbate the problem.

In relation to cardiovascular disease, promising results have been observed in vitro and in vivo with rodent models with respect to arterial hypertension, atherosclerosis, platelet aggregation and ischemic heart disease. Nitro-fatty acids transported in low-density lipoproteins (LDL) have cardioprotective effects by anti-inflammatory and antioxidant mechanisms that include adduct formation and thence critical signalling inhibition of the redox-sensitive transcription nuclear factor kappa B (NF-κB), a potential therapeutic target. They reduce lipid accumulation and promote plaque stability in atherosclerosis and have beneficial effects in relation to myocardial infarction.

9-Nitro-oleate been shown to possess anti-tumorigenic effects in rodent models of colorectal cancer in cell culture in vitro and in a murine xenograft model of the human disease. In contrast to their well-known anti-oxidative properties, the nitro fatty acid reduced tumour growth by mediating the generation of mitochondrial oxidative stress in the cancer cells to trigger mitochondrial dysfunction and activation of the intrinsic apoptotic pathway. 10-Nitro-oleic acid in combination with antineoplastic DNA-damaging agents may have beneficial effects in relation to triple-negative breast cancer.

4. Nitro Fatty Acids in Plants

Nitro fatty acids derived from oleate and conjugated linoleate in free form and as protein cysteine adducts have been detected in olives and virgin olive oil, and it has been suggested that they may constitute one of the beneficial effects of the Mediterranean diet. It has now been determined that nitro fatty acids have signalling functions in plants such as Arabidopsis with nitro-linolenic acid being the key metabolite. As a single peak is seen on GC analysis, there seems to be only one isomer involved in vivo, but the precise structure does not appear to have been determined. Although research is still at an early stage, it has been shown that this fatty acid acts as a signalling molecule during seed and plant development, and it is involved in plant defence responses against different abiotic-stress conditions, mainly by inducing heat shock proteins. It reacts to oxidative stress conditions by inducing high levels of the antioxidant protein ascorbate peroxidase, the concentration of which increases in response to wounding or exposure to salinity, cadmium ions and low temperatures.

As nitro-linolenic acid can release nitric oxide with its manifold signalling properties in aqueous media, it may act as a signalling molecule indirectly as well as directly, for example by transferring nitric oxide to glutathione to form S-nitrosoglutathione, believed to be a major mobile reservoir for nitric oxide bioactivity and signal transduction. With tomato cell suspensions, exogenous application of nitro-oleate was found to induce the production of reactive oxygen species (ROS) via activation of NADPH oxidases, requiring calcium entry from the extracellular compartment and protein kinase activation.

5. Analysis

Aside from the multiplicity of different products that can be formed at low levels in tissues, a major difficulty in the analysis of nitrated lipids is that they are easily generated artefactually via adventitious nitrite anions during sample work-up and chromatographic analysis under acidic conditions, and acidic pHs must be avoided at all critical phases of lipid extraction. It is therefore necessary to include extensive control experiments to preclude the formation of spurious by-products by adding unsaturated fatty acids labelled with stable isotopes as internal standards, while inclusion of ¹⁵NO₂⁻ during tissue handling and extractions will result in the formation of ¹⁵NO₂-fatty acids if there is unwanted nitration during processing. It should be noted that nitrated lipids are sensitive to light and are thermally unstable. Thereafter, modern mass spectrometric techniques, HPLC with electrospray ionization MS or GC-MS of pentafluorobenzyl esters with electron-capture negative-ion chemical ionization, provide the enhanced sensitivity and resolution required for analysis of nitro fatty acids. As fewer derivatization steps are required, the former may be preferable. Analysis of intact nitrated lipid classes or protein adducts adds to the challenge.