New Insights into the Anti‐Inflammatory and Antioxidant Properties of Nitrated Phospholipids

Abstract

Nitro-fatty acids (NO -FA) have been widely studied with regard to their identification, structural characterization, and biological actions. NO -FA could also be present endogenously esterified to phospholipids (PL), and NO -PL were already detected in cardiac mitochondria from diabetic rats and cardiomyoblasts subjected to starvation. However, the biological actions of NO -PL have been overlooked. In this study, we evaluate the antioxidant and anti-inflammatory potential of the nitrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) formed in vitro by incubation with NO BF , in a well-recognized mimetic model of nitroxidative stress. Nitrated POPC showed anti-radical ability to reduce both 2,2-diphenyl-1-picrylhydrazyl radical (DPPH ) (IC = 225 ± 4 μg/mL; Trolox equivalent (TE) = 86 ± 6 μmol Trolox/g lipid) and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical cation (ABTS ) (IC = 124 ± 2 μg/mL; TE = 152 ± 9 μmol Trolox/g lipid). Also, higher lag times were achieved in oxygen radical absorbance capacity (ORAC) assay for nitrated POPC, indicating a faster reaction with oxygen-derived radicals (TE = 1.03 ± 0.22 and TE = 1.30 ± 0.16 mmol Trolox/g lipid for nonmodified and nitrated POPC, respectively). Nitrated POPC showed the ability to inhibit lipid oxidation induced by the hydroxyl radical generated under Fenton reaction conditions, monitored by electrospray ionization (ESI) mass spectrometry (MS) using phosphatidylcholine (PtdCho) liposomes as a model of cell membrane. Nitrated POPC showed anti-inflammatory potential, as assessed by the inhibition of inducible nitric oxide synthase (iNOS) expression in RAW 264.7 macrophages activated by the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS) in a well-described in vitro model of inflammation. Altogether, this study provides new clues regarding the antioxidant and anti-inflammatory potential of nitrated POPC, which should be explored in depth.

Advancing Target Identification of Nitrated Phospholipids in Biological Systems by HCD Specific Fragmentation Fingerprinting in Orbitrap Platforms

“…Nitrated PLs include many structurally diverse lipid species, and its nitro derivatives (NO 2 -PLs) were the main products identified in biological samples, including the H9c2 cardiomyoblasts [10] and cardiac mitochondria of diabetic rats treated with streptozotocin [12]. These modified lipids showed antioxidant properties thanks to their radical scavenging potential against the 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical cation (ABTS •+ ) and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH • ) [11,32] and can also act as anti-inflammatory agents by inhibiting the expression of inducible nitric oxide synthase (iNOS; at the protein level) in RAW 264.7 macrophages treated with lipopolysaccharide [32]. Moreover, nitrated POPC induced a series of downstream cellular effects in SW13/cl.2 cells, showing nitrated PLs as new potential electrophilic lipid mediators with selective actions [33].…”

Valuing Bioactive Lipids from Green, Red and Brown Macroalgae from Aquaculture, to Foster Functionality and Biotechnological Applications

“…The antioxidant scavenging activity against the 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical cation (ABTS ●+ ) was evaluated using a previously described method [ 94 , 95 ] with some modifications. The ABTS radical solution (3.5 mmol/L) was prepared by mixing 10 mL of ABTS stock solution (7 mmol/L in water) with 10 mL of potassium persulfate, K2S2O8 (2.45 mmol/L in water).…”

“…The antioxidant scavenging activity against the 2,2-diphenyl-1-picrylhydrazy radical (DPPH ● ) was evaluated using a previously described method [ 94 , 95 ] with some modifications. A stock solution of DPPH ● in ethanol (250 μmol/L) was prepared and diluted to provide a working solution with an absorbance value of ~0.9 measured at 517 nm using a UV–vis spectrophotometer (Multiskan GO 1.00.38, Thermo Scientific, Hudson, NH, USA) controlled by the SkanIT software version 3.2 (Thermo Scientific).…”

Characterization of Nitrophospholipid‐Peptide Covalent Adducts by Electrospray Tandem Mass Spectrometry: A First Screening Analysis Using Different Instrumental Platforms

“…NO 2 ‐PL was correlated with beneficial effects in the recovery phase in a cellular model of myocardium infarction in autophagy . Also, NO 2 POPC showed antioxidant properties as scavenging agent and anti‐inflammatory properties by inhibiting the expression of iNOS in Raw 264.7 macrophages stimulated with LPS …”