September 2011

Electrophilic fatty acids: redox-derived signaling mediators

Recent advances from a variety of laboratories reveal that free radical-induced lipid oxidation products can mediate potent anti-inflammatory and beneficial metabolic reactions. Fatty acids, at first glance, are among the simplest of biological molecules: straight chain hydrocarbons with a single polar terminus and at most a few cis-double bonds in either a methylene-skipped or conjugated diene pattern. In polyunsaturated fatty acids, the low bond dissociation energy of the doubly allylic methylene hydrogens renders them relatively reactive, making radical abstraction a viable reaction manifold. Fatty acid ligation to proteins is typically accomplished by the formation of esters or thioesters by the carboxylic acid, which increases lipophilicity, can modify protein function and alters the anatomic distribution of modified proteins. Fatty acids also are packaged and distributed for structural and energy storage needs, with the lipid compositions, lengths and number of double bonds determining the fluidity of the resulting lipid rafts.

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In the context of cell signaling, the stereospecific oxygenation of not only arachidonic acid but also other polyunsaturated fatty acids can be catalyzed by the heme and non-heme iron-containing proteins cyclooxygenase-1 and -2 as well as 5-, 12- and 12/15-lipoxygenases. These products are typically viewed as ligands for specific G-protein-coupled receptors that propagate inflammatory responses. Notably, by virtue of their reactive dienes, fatty acids also are susceptible to more random oxidation by the adventitious oxidants produced during mitochondrial respiration, environmental oxidants, and the inflammatory activation of cellular nitric oxide (NO) synthase and superoxide (O2.-) and hydrogen peroxide (H2O2)-generating NAD(P)H oxidase activities. These reactive species are not exclusively toxic, because recent advances show they also can serve as salutary signaling mediators by virtue of an ability to confer post-translational protein modifications. 

Polyunsaturated fatty acids are easily oxidized to hydroperoxyl and hydroxyl derivatives, with multiple enzymatic mechanisms having evolved to further metabolize these intermediates. In the process of oxidation, double bond rearrangments occur, such as from cis- to trans- and into conjugation with a neighboring double bond. This latter species is potentially reactive via the electron-rich conjugated diene, and may be subject to further modification by electron-deficient or unpaired reactive species. Alternatively, the hydroxyl may be directly oxidized via enzymatic or nonenzymatic pathways to an aldehyde or ketone. The α,β-unsaturated carbonyl, now conjugated to the diene, forms a powerful electron-withdrawing group. This moiety is labile to reaction with available nucleophiles, such as protein thiol or histidine residues. Similarly, NO-derived reactive species can instead nitrate the fatty acid by nitrogen dioxide-mediated free radical addition mechanisms. The resulting nitro-fatty acids are even more electron-deficient and thus are a strongly electrophilic and kinetically reactive species. The resulting oxidized or nitrated fatty acids, whether free or esterified, exhibit distinct structural and biochemical properties and can modulate the activity of multiple cell signaling pathways.

The electrophilic reactivity of both α,β-unsaturated carbonyl- and nitro-fatty acid derivatives is the basis for their signaling capacity. Double bonds conjugated to a strong electron withdrawing group are susceptible to Michael addition. This is the reversible addition of a nucleophile to the electrophile, accompanied by bond-rearrangement and proton transfer, to yield adducted (or alkylated) products. In the decidedly reducing cytosolic milieu, most biological functional groups are electron rich, and some are potentially nucleophilic. This redox balance is maintained by glutathione, which is a prevalent nucleophile as well as antioxidant. The adduction of electrophilic fatty acids to thiol- or amine-containing proteins via Michael addition introduces a post-translational modification of proteins, conferring altered cellular distribution, conformation and catalytic activity.

It is the combination of electrophilic adduction with the reversibility of this reaction that may be the most important aspect of this reactivity of soft electrophiles. An irreversible reaction is essentially toxic, rendering useful molecules useless or deleterious. Electrophile adduction to glutathione and subsequent extracellular transport is a common detoxification mechanism. A reversible modification conversely is a typical event; molecules are modified and remodified in continuous succession throughout the cell as a normal aspect of metabolism.

Detection of these fatty-acid products must consider their small proportion among free fatty acids while also accounting for the bioavailable pools that are already adducted to proteins. The reversible electrophilicity of these molecules has been exploited as an analytical methodology designed to take advantage of their intrinsic properties. To distinguish electron-rich fatty acids from oxidized or nitrated electrophilic species, simple methods for exposing the analyte to competing thiol-containing nucleophiles have been developed. Then trans-alkylation to low molecular weight or immobilized nucleophiles permits capture of electrophilic species that are either free or already adducted via Michael addition to GSH or nucleophilic amino acids of proteins. In this regard, β-mercaptoethanol serves as an effective electrophile trapping agent. After chromatographically separating β-mercaptoethanol-adducted electrophiles, these adducts can be identified and analyzed by loss-of-mass observations as they are cleaved under ionizing mass-spectrometric conditions. Through this methodology, new nitrated and oxidized fatty acid species are being detected in both animal models and clinically (1, 2).



Why are electrophilic fatty acids relevant in biology? Multiple transcriptional regulation events and nuclear lipid receptors such as PPARγ are potently modulated by these species, resulting in predominantly anti-inflammatory and beneficial gene expression and metabolic responses (3). One can envision that the redox-dependent generation of electrophilic lipid products thus links gene expression to the metabolic and inflammatory status of tissues.


1. Groeger A. L., Cipollina, C., Cole, M. P., Woodcock, S. R., Bonacci, G., Rudolph, T. K., et al. Cyclooxygenase-2 generates anti-inflammatory mediators from omega-3 fatty acids. (2010) Nat. Chem. Biol. 6, 433 – 41.
2. Schopfer, F. J., Batthyany, C., Baker, P. R., Bonacci, G., Cole, M. P., Rudolph, V., et al. Detection and quantification of protein adduction by electrophilic fatty acids: mitochondrial generation of fatty acid nitroalkene derivatives. (2009) Free Radic. Biol. Med. 46, 1250 – 9.
3. Rudolph, T. K., Freeman, B. A. Transduction of redox signaling by electrophile-protein reactions. (2009) Science Signaling. 2.

Steven Woodcock. Bruce Freeman Steven R. Woodcock ( is an instructor, and Bruce A. Freeman ( is the Irwin Fridovich professor and chair of pharmacology and chemical biology at the University of Pittsburgh School of Medicine.

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