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1Departments of Anesthesiology and Surgery, Colorado Center for Altitude Medicine and Physiology, University of Colorado Health Sciences Center, Aurora, Colorado 80111; 2Hypoxia Research Unit, Department of Physiology, School of Applied Sciences, University of Glamorgan, South Wales CF37 1DL; 3Department of Medicine, Queens University, Belfast BT12 6BJ, United Kingdom; and 4Department of Medicine, University of California San Diego, La Jolla, California 92093
Submitted 17 February 2004 ; accepted in final form 13 May 2004
Incremental knee extensor (KE) exercise performed at 25, 70, and 100% of single-leg maximal work rate (WRMAX) was combined with ex vivo electron paramagnetic resonance (EPR) spectroscopic detection of 
-phenyl-tert-butylnitrone (PBN) adducts, lipid hydroperoxides (LH), and associated parameters in five males. Blood samples were taken from the femoral arterial and venous circulation that, when combined with measured changes in femoral venous blood flow, permitted a direct examination of oxidant exchange across a functionally isolated contracting muscle bed. KE exercise progressively increased the net outflow of LH and PBN adducts (100% > 70% > 25% WRMAX, P < 0.05) consistent with the generation of secondary, lipid-derived oxygen (O2)-centered alkoxyl and carbon-centered alkyl radicals. Radical outflow appeared to be more intimately associated with predicted decreases in intracellular PO2 (iPO2) as opposed to measured increases in leg O2 uptake, with greater outflow recorded between 25 and 70% WRMAX (P < 0.05 vs. 70–100% WRMAX). This bias was confirmed when radical venoarterial concentration differences were expressed relative to changes in the convective components of O2 extraction and flow (25–70% WRMAX P < 0.05 vs. 70–100% WRMAX, P > 0.05). Exercise also resulted in a net outflow of other potentially related redox-reactive parameters, including hydrogen ions, norepinephrine, myoglobin, lactate dehydrogenase, and uric acid, whereas exchange of lipid/lipoproteins, ascorbic acid, and selected lipid-soluble anti-oxidants was unremarkable. These findings provide direct evidence for an exercise intensity-dependent increase in free radical outflow across an active muscle bed that was associated with an increase in sarcolemmal membrane permeability. In addition to increased mitochondrial electron flux subsequent to an increase in O2 extraction and flow, exercise-induced free radical generation may also be regulated by changes in iPO2, hydrogen ion generation, norepinephrine autoxidation, peroxidation of damaged tissue, and xanthine oxidase activation.
electron paramagnetic resonance; spin-trapping; lipid peroxidation; antioxidants; mitochondrial redox
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