Synthesis, isomer characterization, and anti-inflammatory properties of nitroarachidonate.
Nitrated fatty acids (nitroalkenes) have been recently detected and quantified in cell membranes and human plasma. However, nitration of arachidonate (AA), that could redirect AA-dependent cell signaling pathways, has not been studied in detail. Herein, we synthesized and determined for the first time the isomer distribution of nitroarachidonate (AANO2) and demonstrate its ability to modulate inflammation. Synthesis of AANO2 was achieved by AA treatment with sodium nitrite in acidic conditions following HPLC separation. Mass spectrometry (MS) analysis showed the characteristic MS/MS transition of AANO2 (m/z 348/301). Moreover, the IR signal at 1378.3 cm(-1) and NMR studies confirmed the presence of mononitrated nitroalkenes. Positional isomer distribution was determined by NMR and MS fragmentation with lithium; four major isomers (9-, 12-, 14-, and 15-AANO2) were identified, which exhibited key anti-inflammatory properties. These include their ability to release biologically relevant amounts of nitric oxide, induce cGMP-dependent vasorelaxation, and down-regulate inducible nitric oxide synthase (NOS2) expression during macrophage activation, providing unique structural evidence and novel regulatory signaling properties of AANO2.
Trostchansky
Andrés
A
Department of Biochemistry and Center for Free Radical and Biomedical Research, Faculty of Medicine, University of the Republic, Montevideo, Uruguay.
Souza
José M
JM
Ferreira
Ana
A
Ferrari
Mariana
M
Blanco
Fabiana
F
Trujillo
Madia
M
Castro
Diego
D
Cerecetto
Hugo
H
Baker
Paul R S
PR
O'Donnell
Valerie B
VB
Rubbo
Homero
H
D000818:Animals; D000893:Anti-Inflammatory Agents / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D016718:Arachidonic Acid / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D007536:Isomerism; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D015394:Molecular Structure; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D009574:Nitro Compounds / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects; D021241:Spectrometry, Mass, Electrospray Ionization; D013055:Spectrophotometry, Infrared; D014664:Vasodilation / Q000187:drug effects
D000893:Anti-Inflammatory Agents; D009574:Nitro Compounds; D016718:Arachidonic Acid; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase