Prostaglandin endoperoxide H synthase (PGHS) catalyzes the oxidation of arachidonate to prostaglandin H2. We have previously synthesized and chemically characterized nitroarachidonic acid (AANO2), a novel anti-inflammatory signaling mediator. Herein, the interaction of AANO2 with PGHS was analyzed. AANO2 inhibited oxygenase activity of PGHS-1 but not PGHS-2. AANO2 exhibited time- and concentration-dependent inhibition of peroxidase activity in both PGHS-1 and -2. The plot of kobs versus AANO2 concentrations showed a hyperbolic function with kinact = 0.045 s−1 and Ki*app = 0.019 μm for PGHS-1 and kinact = 0.057 s−1 and Ki*app = 0.020 μm for PGHS-2. Kinetic analysis suggests that inactivation of PGHS by AANO2 involves two sequential steps: an initial reversible binding event (described by Ki) followed by a practically irreversible event (Ki*app) leading to an inactivated enzyme. Inactivation was associated with irreversible disruption of heme binding to the protein. The inhibitory effects of AANO2 were selective because other nitro-fatty acids tested, such as nitrooleic acid and nitrolinoleic acid, were unable to inhibit enzyme activity. In activated human platelets, AANO2 significantly decreased PGHS-1-dependent thromboxane B2 formation in parallel with a decrease in platelet aggregation, thus confirming the biological relevance of this novel inhibitory pathway.

Based on this information, we analyze in vivo data supporting nitro-fatty acids as promising pharmacological tools to prevent inflammatory diseases associated with oxidative and nitrative stress conditions. A key future issue is to evaluate whether nitro-fatty acid supplementation would be useful for human diseases linked to inflammation as well as their potential toxicity when administered by long periods of time.

Nitro-fatty acids represent endogenously occurring products of oxidant-induced nitration reactions. We have previously synthesized a mixture of four isomers of nitroarachidonic acid, a novel anti-inflammatory signaling mediator. In this study, we synthesized and chemically and biologically characterized for the first time an esterified nitroalkene derived from the nitration of methylarachidonate (AAMet): 6-methylnitroarachidonate (6-AAMetNO(2)). Synthesis was performed by reacting AAMet with sodium nitrite under acidic conditions. Analysis by mass spectrometry (positive-ion ESI-MS) showed an [M+H](+) ion of m/z 364, characteristic of AAMetNO(2). Fragmentation of this ion yielded a daughter ion at m/z 317, corresponding to the neutral loss of the nitro group ([M+H-HNO(2)](+)). Furthermore, IR signal at 1378 cm(-1) and NMR data confirmed the structure of a 6-nitro-positional isomer. This novel esterified nitroalkene was capable of promoting vascular protective actions including: (a) the induction of vasorelaxation via endothelium-independent mechanisms, associated with an increase in smooth muscle cell cGMP levels, and (b) a potent dose-dependent inhibition of human platelet aggregation. We postulate that 6-AAMetNO(2) could be a potential drug for the prevention of vascular and inflammatory diseases, and the presence of the methyl group may increase its pharmacological potential.

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