It has long been recognized that energy metabolism is linked to the production of reactive oxygen species (ROS) and critical enzymes allied to metabolic pathways can be affected by redox reactions. This interplay between energy metabolism and ROS becomes most apparent during the aging process and in the onset and progression of many age-related diseases (i.e. diabetes, metabolic syndrome, atherosclerosis, neurodegenerative diseases). As such, the capacity to identify metabolic pathways involved in ROS formation, as well as specific targets and oxidative modifications is crucial to our understanding of the molecular basis of age-related diseases and for the design of novel therapeutic strategies.Herein we review oxidant formation associated with the cell's energetic metabolism, key antioxidants involved in ROS detoxification, and the principal targets of oxidant species in metabolic routes and discuss their relevance in cell signaling and age-related diseases.

Extra virgin olive oil (EVOO) and olives, key sources of unsaturated fatty acids in the Mediterranean diet, provide health benefits to humans. Nitric oxide (•NO) and nitrite (NO2 −)-dependent reactions of unsaturated fatty acids yield electrophilic nitroalkene derivatives (NO2-FA) that manifest salutary pleiotropic cell signaling responses in mammals. Herein, the endogenous presence of NO2-FA in both EVOO and fresh olives was demonstrated by mass spectrometry. The electrophilic nature of these species was affirmed by the detection of significant levels of protein cysteine adducts of nitro-oleic acid (NO2-OA-cysteine) in fresh olives, especially in the peel. Further nitration of EVOO by NO2 − under acidic gastric digestive conditions revealed that human consumption of olive lipids will produce additional nitro-conjugated linoleic acid (NO2-cLA) and nitro-oleic acid (NO2-OA). The presence of free and protein-adducted NO2-FA in both mammalian and plant lipids further affirm a role for these species as signaling mediators. Since NO2-FA instigate adaptive anti-inflammatory gene expression and metabolic responses, these redox-derived metabolites may contribute to the cardiovascular benefits associated with the Mediterranean diet.

Although the binding of xanthine oxidase (XO) to glycosaminoglycans (GAGs) results in significant alterations in its catalytic properties, the consequence of XO/GAG immobilization on interactions with clinically relevant inhibitors is unknown. Thus, the inhibition kinetics of oxypurinol for XO was determined using saturating concentrations of xanthine. When XO was bound to a prototypical GAG, heparin-Sepharose 6B (HS6B-XO), the rate of inactivation for uric acid formation from xanthine was less than that for XO in solution (k inact ‫؍‬ 0.24 versus 0.39 min ؊1 ). Additionally, the overall inhibition constant (K i ) of oxypurinol for HS6B-XO was 2-5-fold greater than for free XO (451 versus 85 nM). Univalent electron flux (O 2 . formation) was diminished by the binding of XO to heparin from 28.5% for free XO to 18.7% for GAG-immobilized XO. Similar to the results obtained with HS6B-XO, the binding of XO to bovine aortic endothelial cells rendered the enzyme resistant to inhibition by oxypurinol, achieving ϳ50% inhibition. These results reveal that GAG immobilization of XO in both HS6B and cell models substantially limits oxypurinol inhibition of XO, an event that has important relevance for the use of pyrazolo inhibitors of XO in clinical situations where XO and its products may play a pathogenic role.The interactions of reactive oxygen species (ROS) 1 with biomolecules that result in alterations in cell function or overt cellular damage have been proposed as contributing to the pathogenic mechanisms of various disease processes (1-3). Xanthine oxidoreductase is a molybdenum-pterin protein that serves as the rate-limiting enzyme catalyzing the oxidation of hypoxanthine to xanthine and, finally, to urate. Upon sulfhydryl oxidation or limited proteolysis, the dehydrogenase form of xanthine oxidoreductase (xanthine dehydrogenase) is converted to an oxidase (xanthine oxidase or XO), which utilizes O 2 as the terminal e Ϫ acceptor, yielding superoxide (O 2 . ) and hydrogen peroxide (H 2 O 2 ) rather than NADH. Under inflammatory conditions, XO serves as a significant source of O 2 . and H 2 O 2 in the vasculature (4 -8). During such states, it has been demonstrated that xanthine dehydrogenase is released into the circulation, is rapidly (Ͻ1 min) converted to XO, and binds with high affinity (K d ϭ 6 nM) to positively charged glycosaminoglycans (GAGs) on the surface of vascular endothelial cells (9, 10). In this location, XO can generate ROS that, in turn, can modulate the bioavailability of nitric oxide (⅐NO) and, thus, vascular cell signaling (8). Xanthine oxidase displays an affinity for heparin sulfate-containing GAGs on endothelial cells; intravenous administration of heparin results in increases in plasma XO activity, suggesting that XO is bound to the vascular endothelium in both humans and animal models of disease (9,11,12). Sequestration of proteins by GAGs does the following: 1) increases their local concentration by up to an order of magnitude; 2) diminishes their rotational and translational mobility; and ...

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.

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Nitroalkene derivatives of fatty acids act as adaptive, anti-inflammatory signalling mediators, based on their high-affinity PPARγ (peroxisome-proliferator-activated receptor γ ) ligand activity and electrophilic reactivity with proteins, including transcription factors. Although free or esterified lipid nitroalkene derivatives have been detected in human plasma and urine, their generation by inflammatory stimuli has not been reported. In the present study, we show increased nitration of cholesteryl-linoleate by activated murine J774.1 macrophages, yielding the mononitrated nitroalkene CLNO2 (cholesteryl-nitrolinoleate). CLNO2 levels were found to increase ~20-fold 24 h after macrophage activation with Escherichia coli lipopolysaccharide plus interferon-γ ; this response was concurrent with an increase in the expression of NOS2 (inducible nitric oxide synthase) and was inhibited by the •NO (nitric oxide) inhibitor L-NAME (NG-nitro-L-arginine methyl ester). Macrophage (J774.1 and bone-marrow-derived cells) inflammatory responses were suppressed when activated in the presence of CLNO2 or LNO2 (nitrolinoleate). This included: (i) inhibition of NOS2 expression and cytokine secretion through PPARγ and •NO-independent mechanisms; (ii) induction of haem oxygenase-1 expression; and (iii) inhibition of NF-κB (nuclear factor κB) activation. Overall, these results suggest that lipid nitration occurs as part of the response of macrophages to inflammatory stimuli involving NOS2 induction and that these by-products of nitro-oxidative reactions may act as novel adaptive down-regulators of inflammatory responses.

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