Nitration of Unsaturated Fatty Acids by Nitric Oxide-Derived Reactive Nitrogen Species Peroxynitrite, Nitrous Acid, Nitrogen Dioxide, and Nitronium Ion

Abstract

Reactive nitrogen species derived from nitric oxide are potent oxidants formed during inflammation that can oxidize membrane and lipoprotein lipids in vivo. Herein, it is demonstrated that several of these species react with unsaturated fatty acid to yield nitrated oxidation products. Using HPLC coupled with both UV detection and electrospray ionization mass spectrometry, products of reaction of ONOO- with linoleic acid displayed mass/charge (m/z) characteristics of LNO2 (at least three products at m/z 324, negative ion mode). Further analysis by MS/MS gave a major fragment at m/z 46. Addition of a NO2 group was confirmed using [15N]ONOO- which gave a product at m/z 325, fragmenting to form a daughter ion at m/z 47. Formation of nitrated lipids was inhibited by bicarbonate, superoxide dismutase (SOD), and Fe3+-EDTA, while the yield of oxidation products was decreased by bicarbonate and SOD, but not by Fe3+-EDTA. Reaction of linoleic acid with both nitrogen dioxide (*NO2) or nitronium tetrafluoroborate (NO2BF4) also yielded nitrated lipid products (m/z 324), with HPLC retention times and MS/MS fragmentation patterns identical to the m/z 324 species formed by reaction of ONOO- with linoleic acid. Finally, reaction of HPODE, but not linoleate, with nitrous acid (HONO) or isobutyl nitrite (BuiONO) yielded a product at m/z 340, or 341 upon reacting with [15N]HONO. MS/MS analysis gave an NO2- fragment, and 15N NMR indicated that the product contained a nitro (RNO2) functional group, suggesting that the product was nitroepoxylinoleic acid [L(O)NO2]. This species could form via homolytic dissociation of LOONO to LO* and *NO2 and rearrangement of LO* to an epoxyallylic radical L(O)* followed by recombination of L(O)* with *NO2. Since unsaturated lipids of membranes and lipoproteins are critical targets of reactive oxygen and nitrogen species, these pathways lend insight into mechanisms for the formation of novel nitrogen-containing lipid products in vivo and provide synthetic strategies for further structural and functional studies.

Red cell membrane and plasma linoleic acid nitration products: Synthesis, clinical identification, and quantitation

“…acidic conditions (pH Ͻ4), documented to result in HNO 2 -dependent fatty acid nitration and oxidation (20). This analytical pitfall raises the question of whether the various reported LNO 2 species were indeed endogenous, and it impairs an ability to determine concentration and structural characteristics.…”

“…This ''molecular lens'' effect that is induced by solvation in hydrophobic cell compartments accelerates the reaction of • NO with O 2 to yield N 2 O 3 and N 2 O 4 . As a consequence of this diversity of • NO reactions with partially reduced oxygen species, a rich spectrum of products is formed that orchestrates target molecule oxidation, nitrosation, and nitration reactions.Multiple mechanisms account for the nitration of fatty acids by• NO-derived species (15)(16)(17)(18)(19)(20). During both basal cell-signaling and tissue-inflammatory conditions, • NO 2 that is generated by the aforementioned reactions can react with membrane and lipoprotein lipids.…”

“…Acid-catalyzed fatty acid nitration is expected during physiological and pathological conditions in which NO 2 Ϫ is exposed to low pH (e.g., pH Ͻ 4.0), such as in the gastric compartment after endosomal or phagolysosomal acidification or in tissues after postischemic reperfusion. Nitrated linoleic acid (LNO 2 ) ‡ displays robust cell-signaling activities that appear to be antiinflammatory (20,(22)(23)(24)(25). LNO 2 inhibits platelet function by means of cAMP-dependent mechanisms (26), and it inhibits neutrophil O 2…”

“…• NO-derived species (15)(16)(17)(18)(19)(20). During both basal cell-signaling and tissue-inflammatory conditions, • NO 2 that is generated by the aforementioned reactions can react with membrane and lipoprotein lipids.…”

Glucocerebrosidase, a new player changing the old rules in Lewy body diseases

“…Changes in lipid composition may occur through oxidative modifications or metabolic alterations ( O ' Donnell et al, 1999 ;Mosca et al , 2011 ). Significant alterations in membrane lipid composition, such as sphingolipidoses, have been reported in lysosomal storage diseases (Walkley , 2004 ).…”