Indirect markers of lipid peroxidation confined to the peripheral venous circulation and a reliance on unsuitable exercise models has previously complicated interpretation of the source and mechanisms associated with exercise-induced free radical generation. Therefore, the present study combined functionally isolated quadriceps exercise with electron paramagnetic resonance (EPR) and 1H magnetic resonance spectroscopy to directly quantify free radical outflow by contracting skeletal muscle and examine implications of altered intracellular PO2 (iPO2) and O2 flux. Following ethical approval, five apparently healthy males aged 48 ± 25 years old (mean ± S.D.) performed 3 min of single-leg knee extensor exercise in normoxia at 25, 70 and 100 % of their previously established normoxic maximum work rate (WRMAX). Blood flow (Q) was assessed using a thermodilution technique (Andersen & Saltin, 1985) and samples were collected from the femoral artery/vein and immediately mixed ex vivo with the spin trap, α-phenyl-tert-butylnitrone (PBN). Nuclear hyperfine splittings of resultant nitroxide adducts were consistent with the trapping of oxygen or carbon-centred free radical species (aN = 1.38 ± 0.01 mT and aHβ = 0.17 ± 0.01 mT). Exercise per se resulted in a clear venoarterial difference in PBN adduct concentration (Δv-a), thus stimulating a net adduct outflow that was associated with leg VO2 (r2 = 0.53, P < 0.05, Pearson product moment correlation). However, Table 1 demonstrates that the magnitude of increase in Δv-a expressed in absolute and relative (normalised for VO2 ) terms between 70 and 100 % WRMAX (where iPO2 remained invariant despite an increase in VO2) was clearly less marked than that observed between 25 and 70 % WRMAX (where iPO2 was shown to decrease).
These findings provide the first direct, quantitative evidence for free radical outflow from isolated contracting human skeletal muscle. Preliminary indications tentatively suggest that outflow may be regulated by a decrease in intracellular oxygenation and not merely a consequence of ‘electron leakage’ due to a mass action effect initiated by increased mitochondrial O2 flux.
This work was supported by NIH 000964.
All procedures accord with current local guidelines and the Declaration of Helsinki.