Methods in Free Radical Biology and MedicineNitrated fatty acids: synthesis and measurement
Graphical abstract
Highlights
► Multiple synthetic protocols for nitrated fatty acids are provided. ► Mass spectrometry and chromatographic conditions are optimized. ► An analysis of results and artifact avoidance is given.
Introduction
The enzymatic and free radical-induced oxidation of unsaturated fatty acids generates bioactive molecules that participate in cell signaling [1], [2], [3]. These signaling actions include the activation of G-protein-coupled receptors [4] and alkylation of both thiol-containing small molecules and cysteine residues in proteins [3]. In addition to oxidative stress, nitrative stress is characterized by sustained nitration through the formation of the nitrogen dioxide (•NO2) radical. The biomolecules that are targets of nitration include tyrosine residues [5], nucleic acids (guanine, cGMP, GTP) [6], and unsaturated fatty acids [7]. In particular, the nitration of unsaturated fatty acids results in the formation of electrophilic species that contain a conjugated nitroalkene moiety. The electrophilicity of nitrated fatty acids (NO2-FA), mainly represented by nitro-oleic (NO2-OA), nitro-linoleic (NO2-LA), and nitro-arachidonic acids, promotes reaction with nucleophiles to generate Michael addition products [8]. The targeting of specific cysteine residues by lipid-derived electrophiles is central to modulating enzymatic activity and signaling pathways. Nitrated fatty acids have been shown to potently activate the Nrf2/Keap1 pathway, chaperone heat shock pathways, and inhibit inflammatory responses through multilevel inhibition of NF-κB [3]. These actions result in protective effects in various animal models ranging from metabolic disorders (diabetes) and atherosclerosis to sepsis and ischemia/reperfusion [3]. The data stemming from the pharmacological actions of NO2-FA is in stark contrast to their characterization and quantification in vivo. This is partially due to synthetic challenges of obtaining pure regioisomers, sensitivity to alkaline conditions, the electrophilic nature of the nitrated fatty acid, and the reversible binding to cysteines, all of which result in additional challenges for accurate quantification.
Section snippets
Principles
Nitrated fatty acids form upon exposure of unsaturated fatty acids to nitrating species. In particular, •NO2 plays a central role in the formation of these fatty acid nitroalkenes [9]. The type and characteristics of the precursor fatty acid define the formation of various products. Two main mechanisms have been proposed for the formation of NO2-FA (Fig. 1). The first involves hydrogen atom abstraction from the bis-allylic carbon of a polyunsaturated fatty acid, yielding a delocalized
Reagents for synthetic protocols
Note. In general, use caution as most of the reagents in these protocols are toxic or dangerous in quantity. Please read the associated MSDS for specific precautions. For all synthetic procedures, ensure adequate ventilation, and preferably handle chemicals in well-ventilated fume hoods. Use gloves and safety goggles during all procedures.
Principles
Mass spectrometry has played a central role in the determination of nitrated biomolecules. One of the best characterized nitrated biomolecules is nitrotyrosine [30]. Unlike the single, well-defined product of tyrosine nitration, the multiplicity of substrates and reaction mechanisms involved in the nitration of fatty acids results in a diversity of products. Nitrotyrosine is chemically stable in aqueous solutions, organic solvents, a wide range of pH values, and biological environments in which
Summary
Our methods employ a combination of synthetic approaches and high-resolution analytical techniques to identify and quantify nitrated lipids in biological samples. These methods are appropriate for detecting previously described NO2-FAs, but can also detect a range of other nitrated and nonnitrated electrophilic lipids produced biologically. These approaches demonstrate the synergy between synthetic techniques and powerful analytical methods. Optimizing both components has allowed for improved
Acknowledgments
We thank Bruce Freeman for insightful comments and helpful discussion. This work was supported by National Institutes of Health Grants R01 AT006822-01 (to F.J.S.) and R01-HL058115, R01-HL64937, and P01-HL103455 (to Bruce Freeman). F.J.S. declares a financial interest in Complexa, Inc.
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2021, Redox BiologyCitation Excerpt :NO2–ONO2-CLA species are non-electrophilic and decompose at physiological pH to the electrophilic nitroalkene NO2-CLA in concert with the generation of secondary reactive nitrogen oxide species. 9- and 12-nitro-octadeca-9,11-dienoic acid (9-NO2-CLA and 12-NO2-CLA), and the corresponding isotopically labeled internal standard ([15N]O2-CLA), and 10-nitro-stearic acid (NO2-SA) standard were synthesized and quantitated as previously described [25–27]. The abbreviation NO2-CLA refers to a mixture of the above-mentioned positional isomers.