Strenuous exercise leads to an increase in metabolic rate, increased production of reactive oxygen species, and compromised antioxidant defense systems. To study the effects of oxidative stress during strenuous exercise, a homogeneous group of 31 male subjects participated in a 6-month, 5 days͞week training schedule involving two extreme marches of 50 km and 80 km at sea level, separated by 2 weeks of regular training. Each participant carried 35 kg of extra weight. Blood samples were drawn imediately before and after each march. Twenty-nine subjects completed the 50-km march, and only 16 completed the 80-km march. Plasma levels of reduced ascorbic acid, total ascorbate, and dehydroascorbate did not undergo significant changes during either march. However, the 50-and 80-km marches led to 25% and 37% increases, respectively, in plasma levels of uric acid; due presumably to increases in the metabolic rate and consequent pyrimidine nucleotide metabolism. Both marches led to Ϸ10-fold increase leakage of creatine phosphokinase into the plasma. Likewise, plasma levels of aspartate transaminase, a characteristic marker of liver injury, increased Ϸ4-fold. Plasma levels of bilirubin, creatine, urea, and glucose also increased. Plasma protein carbonyl content, a marker of protein oxidative damage, decreased significantly during each march. These results are discussed with respect to the consideration that elevation of the respiration rate during exercise leads to production of more reactive oxygen species than the antioxidant systems can scavenge. Plausible explanations for leakage of molecules into the plasma are discussed.

Depletion of cellular antioxidant defense mechanisms and the generation of oxygen free radicals by advanced glycation end products (AGEs) have been proposed to play a major role in the pathogenesis of diabetic vascular complications. Here we demonstrate that incubation of cultured bovine aortic endothelial cells (BAECs) with AGE albumin (500 nmol/l) resulted in the impairment of reduced glutathione (GSH) and ascorbic acid levels. As a consequence, increased cellular oxidative stress led to the activation of the transcription factor NF-kappaB and thus promoted the upregulation of various NF-kappaB-controlled genes, including endothelial tissue factor. Supplementation of the cellular antioxidative defense with the natural occurring antioxidant alpha-lipoic acid before AGE albumin induction completely prevented the AGE albumin-dependent depletion of reduced glutathione and ascorbic acid. Electrophoretic mobility shift assays (EMSAs) revealed that AGE albumin-mediated NF-kappaB activation was also reduced in a time- and dose-dependent manner as long as alpha-lipoic acid was added at least 30 min before AGE albumin stimulation. Inhibition was not due to physical interactions with protein DNA binding, since alpha-lipoic acid, directly included into the binding reaction, did not prevent binding activity of recombinant NF-kappaB. Western blots further demonstrated that alpha-lipoic acid inhibited the release and translocation of NF-kappaB from the cytoplasm into the nucleus. As a consequence, alpha-lipoic acid reduced AGE albumin-induced NF-kappaB mediated transcription and expression of endothelial genes relevant in diabetes, such as tissue factor and endothelin-1. Thus, supplementation of cellular antioxidative defense mechanisms by extracellularly administered alpha-lipoic acid reduces AGE albumin-induced endothelial dysfunction in vitro.