Oxidized low density lipoprotein (LDL) may be of central importance in triggering atherosclerosis. One potential pathway involves the production of nitric oxide (NO) by vascular wall endothelial cells and macrophages. NO reacts with superoxide to form peroxynitrite (ONOO ؊), a potent agent of LDL oxidation in vitro. ONOO ؊ nitrates the aromatic ring of free tyrosine to produce 3-nitrotyrosine, a stable product. To explore the role of reactive nitrogen species such as ONOO ؊ in the pathogenesis of vascular disease, we developed a highly sensitive and specific method involving gas chromatography and mass spectrometry to quantify 3-nitrotyrosine levels in proteins. In vitro studies demonstrated that 3-nitrotyrosine was a highly specific marker for LDL oxidized by ONOO ؊ . LDL isolated from the plasma of healthy subjects had very low levels of 3-nitrotyrosine (9 ؎ 7 mol/mol of tyrosine). In striking contrast, LDL isolated from aortic atherosclerotic intima had 90-fold higher levels (840 ؎ 140 mol/mol of tyrosine). These observations strongly support the hypothesis that reactive nitrogen species such as ONOO ؊ form in the human artery wall and provide direct evidence for a specific reaction pathway that promotes LDL oxidation in vivo. The detection of 3-nitrotyrosine in LDL isolated from vascular lesions raises the possibility that NO, by virtue of its ability to form reactive nitrogen intermediates, may promote atherogenesis, counteracting the well-established anti-atherogenic effects of NO. An elevated level of low density lipoprotein (LDL)1 is a major risk factor for premature atherosclerotic vascular disease. However, a wealth of evidence suggests that LDL must be oxidatively modified to damage the artery wall (1, 2). Pathways that oxidize lipid and protein may thus be pivotal to the development of atherosclerosis. LDL oxidation has been widely studied in vitro, but the mechanisms that promote oxidation within the artery wall remain poorly understood (2). We have described one potential pathway for LDL oxidation that involves oxidants generated by myeloperoxidase, an enzyme secreted by phagocytes (3). Another pathway involves nitrogen monoxide (nitric oxide; NO) generated by vascular wall cells (4). NO is a relatively stable free radical that fails to oxidize LDL at physiological pH (5). However, NO reacts rapidly with superoxide to form peroxynitrite (ONOO Ϫ ; Ref. 6), a reactive nitrogen species that promotes peroxidation of the lipid moiety of LDL in vitro (7). Cultured endothelial cells, macrophages and smooth muscle cells, all components of the atherosclerotic lesion, generate superoxide anion (2), suggesting that ONOO Ϫ or other reactive nitrogen intermediates derived from NO could form in the artery wall.In vitro studies demonstrate that ONOO Ϫ spontaneously reacts with tyrosine residues to yield the stable product 3-nitrotyrosine (Scheme 1; Ref. 8). Macrophages and endothelial cells may play a role because antibodies for 3-nitrotyrosine detect epitopes in human atherosclerotic lesions that are associated ...

The aim of this study was to evaluate whether high-intensity endurance training would alleviate exercise-induced oxidative stress. Nine untrained male subjects (aged 19-21 years) participated in a 12-week training programme, and performed an acute period of exhausting exercise on a cycle ergometer before and after training. The training programme consisted of running at 80% maximal exercise heart rate for 60 min.day-1, 5 days.week-1 for 12 weeks. Blood samples were collected at rest and immediately after exhausting exercise for measurements of indices of oxidative stress, and antioxidant enzyme activities [superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT)] in the erythrocytes. Maximal oxygen uptake (VO2max) increased significantly (P < 0.001) after training, indicating an improvement in aerobic capacity. A period of exhausting exercise caused an increase (P < 0.01) in the ability to produce neutrophil superoxide anion (O2.-) both before and after endurance training, but the magnitude of the increase was smaller after training (P < 0.05). There was a significant increase in lipid peroxidation in the erythrocyte membrane, but not in oxidative protein, after exhausting exercise, however training attenuated this effect. At rest, SOD and GPX activities were increased after training. However, there was no evidence that exhausting exercise enhanced the levels of any antioxidant enzyme activity. The CAT activity was unchanged either by training or by exhausting exercise. These results indicate that high-intensity endurance training can elevate antioxidant enzyme activities in erythrocytes, and decrease neutrophil O2.- production in response to exhausting exercise. Furthermore, this up-regulation in antioxidant defences was accompanied by a reduction in exercise-induced lipid peroxidation in erythrocyte membrane.

The current study was designed to test the hypothesis that endurance training improves the ability of the diaphragm muscle to resist exercise-induced oxidative stress. Twenty-eight male Wistar rats were assigned to either untrained or trained groups. Trained rats were treadmill-trained for 9 wk. Each group was subdivided into acutely exercised or nonexercised groups. Diaphragm muscle from each rat was analyzed to determine the levels of certain antioxidant enzymes: Mn-superoxide dismutase (Mn-SOD), Cu,Zn-superoxide dismutase (Cu,Zn-SOD), glutathione peroxidase, and catalase. In addition, interleukin-1 and myeloperoxidase levels were determined. Endurance training upregulated all of the antioxidant enzymes. Conversely, acute exercise increased glutathione peroxidase and catalase in untrained rats, while it had no overt effect on any antioxidant enzymes in trained rats. Both Mn-SOD and Cu,Zn-SOD contents and activities were increased with endurance training. However, the mRNA expressions of both forms of SOD did not show any significant change with endurance training. Acute exercise also increased the levels of interleukin-1 and myeloperoxidase in untrained rats but not in trained rats. Moreover, acute exercise significantly increased the ability of neutrophils to produce superoxide, especially in untrained rats. The results from this study demonstrate that endurance training can upregulate certain antioxidant enzyme activities in rat diaphragm muscle, indicating the potential for improvement of the resistance to intracellular reactive oxygen species. The results of this study also suggest that acute exercise may cause oxidative damage in rat diaphragm through the activation of the inflammatory pathway and that endurance training may minimize such an extracellular oxidative stress by acute exercise.