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Nitro Fatty Acids

While the free radical-catalysed addition of nitric oxide (NO) and nitrogen dioxide (NO2) radicals 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 because of their 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 plant systems are now being studied.

1.  Occurrence in Animal Tissues

Sensitive analytical mass spectrometric methods have been used to demonstrate that nitrated derivatives of palmitoleic, oleic, linoleic, linolenic, arachidonic and eicosapentaenoic acids together with their nitrohydroxy derivatives are present in human plasma and urine. Of these, the two most abundant species are derived from oleic acid, i.e. 9- and 10-nitro-9-cis-octadecenoic acids. (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

In plasma, they 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. 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. Inevitably, such reactions compound the analytical difficulties and can lead to underestimates.

Analogous compounds derived from linoleate have been detected at significant concentrations in some studies, but not in others in healthy tissues at least. All the possible nitro-linoleate isomers have been found in tissues, but 10-nitro- and 12-nitro-9-cis,12-cis-octadecadienoic acids are the main ones found; it appears that the 9-isomer is relatively unstable and is rapidly degraded. Nitro fatty acids derived from oleate and linoleate have been detected in olives and virgin olive oil.

On the other hand, it now appears 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). They have been detected in plasma and urine of healthy humans with and without CLA supplementation and are generated during digestion, metabolic stress and inflammation.

Nitration products of conjugated linoleic acid (CLA)

Nitro-hydroxy derivatives of oleate and linolenate have also been characterized and their 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. Such compounds are now receiving particular attention because of their potential to influence eicosanoid metabolism in addition to having biological effects in their own right.

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.

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; reactions take place mainly in the membranes of cells because partitioning of NO/NO2 into this cellular component occurs preferentially. In addition, nitro fatty acid formation can occur in foods, and these could potentially reach tissues via the digestive system.

The NO2 radical can arise from various endogenous and exogenous sources in humans. For example, 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. Meat and other foods may contain appreciable quantities of nitrite (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, and these are absorbed from the intestines and thence enter into the circulation. Under gastric conditions, it has been established that both unesterified fatty acids and those in triacylglycerols can be rapidly nitrated, and they are transported in plasma in these forms. Nitro fatty acids are stored in adipose tissue, but studies in vitro have shown that the unsaturated (electrophilic) forms are esterified primarily to mono- and diacylglycerols, while triacylglycerols contain mainly saturated (non-electrophilic) forms.

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-nitrite intermediates. Loss of nitrous acid (HNO2) from these intermediates results in the formation of nitroalkenes, while hydrolysis leads to the production of nitro-alcohols. 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.

However, 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

Catabolism: Reduction of nitro fatty acids by prostaglandin reductase-1 in the liver to form nitroalkanes is the main mechanism of deactivation. In addition, 9-nitro-oleate infused into mice was partly hydrogenated to nitro-stearate and partly desaturated to a nitro-octadecadienoic acid. Some was subjected to β-oxidation yielding 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.

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, but the potential role of nitro fatty acids in mediating these reactions has only recently become apparent. However, in view of the findings that basal levels of such fatty acids are substantially lower than had earlier been believed, much of the early literature on the topic has had to be re-evaluated. That said, it is well established from experiments both in vitro and in vivo that they elicit metabolic responses that are generally beneficial, and in particular that they are anti-inflammatory mediators.

In plasma, nitro fatty acids are stabilized by incorporation into lipoproteins, while in erythrocytes and other cells the membrane environment is protective and may provide a reservoir of these compounds. Similarly, triacylglycerols in adipocytes may act as an inert store. However, nitroalkenoic fatty acids decay rapidly in phosphate buffers, and presumably in the cytoplasm of cells, due to solvation reactions with release of nitric oxide radicals. A number of different mechanisms have been proposed for this reaction, which may be central to some of the biological functions of nitro acids, but detailed experimental evidence is scarce at present. However, the main mechanism and signalling action of nitro fatty acids is mediated via post-translational modification of proteins by covalent adduction. Nitrated unsaturated fatty acids are powerful electrophiles that mediate 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. Although such post-translational modifications of proteins are non-enzymic in nature, they appear to be remarkably selective and importantly, they are reversible.

Michael addition reaction of nitrofatty acids

As nitro adducts with conjugated linoleate (CLA) are the main nitro fatty acids in serum, mechanistic and kinetic studies have demonstrated that reactions with thiols occur rapidly to form adducts both β and δ to the nitro group. 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.

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. 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. In experimental animals, it has beneficial affects against chronic kidney diseases and it attenuates experimental inflammatory bowel disease. 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 a signalling activator has the potential to regulate the expression of multiple PPAR target genes, though PPARγ is activated most robustly with 12-nitro-linoleate as a particularly potent activator. 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 redox-sensitive transcription factors (NF-κB). They reduce lipid accumulation and promote plaque stability in atherosclerosis, and have a number of beneficial effects in myocardial infarction. Under conditions of stress, where they may confer resistance to myocardial injury. 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; it may explain in part the benefits to cardiovascular health associated with the Mediterranean diet.

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. It is certain that the peroxynitrite per se has profound effects on the enzymes of prostanoid biosynthesis. Similarly, the trans-arachidonate isomers formed as by-products of nitration reactions are emerging as biomarkers that target various biological systems. For example, they inhibit prostaglandin endoperoxide H synthases 1 and 2 and the synthesis of thromboxane B2. On the other hand, the recent 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.

There is obviously considerable therapeutic potential for the use of nitro-fatty acids against inflammatory conditions such as cardiovascular and kidney diseases, and indeed Phase II clinical trials are underway. In human trials, they appear to be tolerated well when administered orally.

4.  Nitro Fatty Acids in Plants

It has now been determined that nitro fatty acids have signalling functions in plants with nitro-linolenic acid being the key metabolite. There seems to be a single isomer involved in vivo as only a single peak is seen on GC analysis, but the precise structure does not appear to have been determined. Although research is still at an early stage, it has been determined that this fatty acid is 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 ascorbate peroxidase, and its concentration increases in response to wounding or exposure to salinity, cadmium, and low temperature. It is able to release nitric oxide in aqueous media so has the potential to act as a signalling molecule.

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. 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: July 11th, 2017 Author: William W. Christie LipidWeb icon