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Nitro Fatty Acids

While the free radical-catalysed addition of nitric oxide (NO) and nitrogen dioxide (NO2) radicals (reactive nitrogen oxide species or RNOS) 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 both in vitro and in vivo and at concentrations that had the potential to exert biological effects. In recent years, the nature and biology of these fascinating lipids has attracted considerable research interest and some controversy. They have been classified as electrophilic fatty acids, together with related lipids with α,β-unsaturated carbonyl and epoxide substituents, with a propensity to undergo reversible Michael addition reactions with cellular nucleophiles such as cysteine and histidine-containing peptides and proteins. They are also 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 so have therapeutic potential. Most research on the topic relates to animal biochemistry, but it is now recognized that nitro fatty acids have important functions in plants also.

1.  Occurrence in Animal Tissues

It was initially thought that the more abundant 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. Those active in this area prefer the more familiar lipid usage).

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

However, it is now apparent that 'conjugated linoleic acid' (9-cis,11-trans-octadecadienoic acid or CLA) is 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). They 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. 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 derived from linoleate were detected at significant concentrations in some studies, but not in others, in healthy tissues at least, but it is possible that these were mis-identified and that CLA was the true origin. Similarly, nitrated derivatives of palmitoleic, linolenic, arachidonic and eicosapentaenoic acids (together with their nitrohydroxy derivatives) have also been detected at trace levels in human plasma and urine by sensitive analytical mass spectrometric methods.

In plasma, nitro fatty acids occur in the free form, bound reversibly to thiol-containing proteins and glutathione, and as cholesterol esters and triacylglycerols. Free and esterified concentrations of the two regioisomers in plasma and red blood cells were originally estimated to be of the order of 60 to 600nM, but subsequent studies with stable isotope-dilution methodology suggests that these figures were a considerable over-estimate and that the true basal level in plasma of healthy humans is closer to 1 to 3nM, while 9 nM is quoted for urine. Recent reports suggest that even these may be overestimates. 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.

Nitro-hydroxy derivatives of oleate and linoleate have also been characterized and representative structures are illustrated. In essence, they are formed by addition of reactive nitrogen species across one of the double bonds (see below). 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. In general, there is considerable selectivity in terms of which of the various isomers are detected in tissues. For example, the nitro-eicosatetraenoic acids have the NO2 groups in positions 9, 12, 14 and 15 mainly. Similar metabolites have been found from other fatty acids, including eicosapentaenoic and docosahexaenoic acids (n-3 family), some with two nitro groups. Little is yet known of their biological properties.

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.

Structural formulae for nitro-allyl fatty acids

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 particular biological relevance.

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 similarly. 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 lipoprotein lipase, for example, transported into cells and incorporated into the cellular lipids. The esterified form within cells may act as a reservoir of these compounds. Whole body studies of rats treated with radio-labeled 10-nitro-oleic acid demonstrated rapid incorporation into kidney, liver, lungs and heart. Adipocytes may also act as an inert store, since over a longer term (two weeks) there was a preferential accumulation in adipose tissue with unsaturated nitro-fatty acids incorporated mainly into mono- and diacylglycerols, while analogous saturated metabolites were enriched in triacylglycerols. Nitro fatty acid conjugates with coenzyme A have been detected in tissues, so they can presumably be re-esterified after hydrolysis from a lipid-bound form.

2.  Formation of Nitro Fatty Acids in Tissues

Formation of nitro fatty acids occurs in tissues through the non-enzymatic reactions of free radicals such as nitric oxide (NO), and NO-derived oxides of nitrogen (e.g. nitrogen dioxide (NO2)) and peroxynitrite (ONOO)). These operate in conjunction with oxygen-derived inflammatory mediators such as superoxide (O2), hydrogen peroxide (H2O2) and lipid peroxyl radicals (LOO). Many different mechanisms are involved in the production of the secondary radicals and in their subsequent reactions, which are controlled by such factors as the concentration of the NO radicals, the site of their production, oxygen tension, and the concentrations and membrane environment of the target molecules and of any catalysts and antioxidants. As these reactions are non-enzymatic and involve intact lipids rather than the free acids, their formation has something in common with isoprostane formation. Note that reactive nitrogen oxide species react very specifically with γ-tocopherol in vivo to produce a 5-nitro-metabolite.

The NO2 radical is the main reactive intermediate, and it can arise 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 into the circulation. 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. 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 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. NO per se does not participate as a direct nitrating species, but 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.

Detailed mechanistic studies of nitro fatty acid formation in human and other animal tissues are at an early stage, and the biosynthetic mechanisms proposed are largely extrapolated from chemical studies in vitro. The NO2 radicals can react with unsaturated lipids and lipid radicals to form all the types of products found in tissues. Thus 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, while hydrolysis leads to the production of nitro-alcohols. The nitro-conjugated linoleate isomers illustrated in Figure 2 above, which may be the main 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.

Nitro fatty acid formation by free radical reactions

Similarly, as an NO2 radical can also initiate lipid oxidation reactions, yields of nitration versus oxidation will depend on the concentration of oxygen. For example 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 also be formed through the reactions of lipid peroxyl radical (LOO) and NO, of peroxynitrile radicals, and of a lipid hydroperoxide reaction with N2O4 or with HNO2, the last leading to the production of nitro-epoxy fatty acids. Nitro-hydroxy and nitro-oxo oleate derivatives can be produced from conjugated linoleate under these conditions. On the other hand, nitro fatty acid radicals can be produced that lose HNO2 to re-generate 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: Reduction of nitro fatty acids by prostaglandin reductase-1 in the liver to form non-electrophilic nitroalkanes is the main mechanism of deactivation. 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 as a result of the reversible formation of Michael adducts (see below), i.e. deprotonation of the α-carbon, followed by β-elimination of the thiolate group.

A proportion can be subjected to β-oxidation to yield nitro-7-cis-hexadecenoic acid, nitro-5-cis-tetradecenoic acid and nitro-3-cis-dodecenoic acid, and their corresponding coenzyme A derivatives. 10-Nitro-oleate was found to be subjected to ω- and β-oxidation to produce a number of 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. Nitroalkenoic fatty acids decay rapidly in phosphate buffers, and presumably in the cytoplasm of cells because of solvation reactions with the release of nitric oxide radicals. A number of different mechanisms have been proposed for this reaction, but its 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 in particular that 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 response to inflammatory stimuli, they may be formed in situ or released from membrane or adipose tissue stores. It is evident that they have biological activities in their own right and do not act by releasing NO.

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 and importantly 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. Although such post-translational modifications of proteins are non-enzymic in nature, they appear to be remarkably selective. Intact lipids containing nitro fatty acids are the main reactants rather than those in unesterified form.

Michael addition reaction of nitrofatty acids

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, have the ability to oxidize cysteine-adducted nitro fatty acids with the release of free nitroalkenes.

Reaction of 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. Nitro fatty acids may exert many of their biological effects by this means, 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, including inhibition of the expression of pro-inflammatory genes and direct inhibition of pro-inflammatory enzymes. Thus, nitro-oleic acid 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 the inhibitory action. 10-Nitro-oleic acid is also an inhibitor of the epoxyhydrolase, i.e. the enzyme responsible for the hydrolysis of epoxyeicosatrienoic acid, which has protective effects against hypertension. In addition, it is a potent inducer of the expression of antioxidant genes, while inhibiting TLR4 signalling. 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.

Scottish thistleNitro fatty acids are able to bind to all three PPAR isotypes with high affinities, and as signalling activators 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, for example. In relation to cardiovascular disease, nitro-fatty acids have cardioprotective effects by anti-inflammatory and anti-oxidant mechanisms that include adduct formation and thence critical signalling inhibition of the redox-sensitive transcription nuclear factor kappa B (NF-κB) but stimulation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) - potential therapeutic targets. They reduce lipid accumulation and promote plaque stability in atherosclerosis, and have a number of beneficial effects in myocardial infarction. Taken together with the finding of significant levels of protein cysteine adducts of nitro-oleic acid and the free acid in fresh olives, this has interesting implications both for plant biology and human nutrition.

Nitro-linoleate 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 and by such means also have an anti-inflammatory role in cells. Similarly, both nitro-oleate and nitro-linoleate have been shown to inhibit the lipopolysaccharide-induced secretion of pro-inflammatory cytokines in macrophages, 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, for example, by inhibiting prostaglandin endoperoxide H synthases (COX) 1 and 2 and the synthesis of thromboxanes. 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 may have to be re-evaluated.

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. In human trials, they appear to be tolerated well when administered orally. For example, phase II clinical trials of 10-nitro-oleic acid are underway for the treatment of focal segmental glomerulosclerosis, and trials are planned for pulmonary arterial hypertension and obese asthmatics. In relation to cardiovascular diseases, promising results have been observed in vitro and in vivo with rodent models with respect to arterial hypertension, atherosclerosis, platelet aggregation, ischemic heart disease and inflammatory bowel disease, for example. Nitro-fatty acids have been shown to inhibit hepatic triacylglycerol accumulation and protect against fibrosis during the development of nonalcoholic 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. 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.

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 triggering mitochondrial dysfunction and activation of the intrinsic apoptotic pathway. Similarly, 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 also have signalling functions in plants such as Arabidopsis with nitro-linolenic acid being the key metabolite. There seems to be only one isomer involved in vivo as a single peak is seen on GC analysis, but the precise structure does not appear to have been determined as yet. 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 is also involved in plant defence responses against different abiotic-stress conditions, mainly by inducing heat shock proteins. For example, 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, and low temperature. Some of these activities may be mediated by reversible post-translational nitroalkylation. Nitro-linolenic acid is able to release nitric oxide with its manifold signalling properties in aqueous media so it may act as a signalling molecule either directly or indirectly.

5.  Analysis

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. It is therefore necessary to include extensive control experiments to preclude the formation of spurious by-products, for example by adding unsaturated fatty acids labelled with stable isotopes as internal standards. Thus, inclusion of 15NO2 during tissue handling and extractions will result in the formation of 15NO2-fatty acids if there is unwanted nitration during processing. Acidic pHs must be avoided at all critical phases of lipid extraction. It should also 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. The former may be preferable as fewer derivatization steps are required.

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Lipid listings Credits/disclaimer Updated: August 9th, 2019 Author: William W. Christie LipidWeb icon