In vitro electron paramagnetic resonance characterization of free radicals: Relevance to exercise-induced lipid peroxidation and implications of ascorbate prophylaxis

Abstract

This study tested the hypothesis that exercise-induced oxidative stress is caused by free radical-mediated damage to polyunsaturated fatty acids (PUFA) which can be prevented following ascorbate prophylaxis. Hyperfine coupling constants (HCC) of α-phenyl-tert-butylnitrone (PBN)-adducts were measured via room temperature electron paramagnetic resonance (EPR) spectroscopy in the venous blood of 12 subjects at rest and following maximal exercise during a randomized double-blind placebo-controlled trial and compared to those observed following room-air incubation (2 h at 37°C) of L-α-phosphatidycholine, linoleic acid, α-linolenic acid and arachidonic acid. All adducts exhibited similar HCC [aN 13.6 Gauss (G) and aβH 1.8 G] with the exception of L-α-phosphatidycholine [aN1=13.4 G, aβH1=1.6 G (37%) and aN2=14.9 G, aβH2=0.3 G (63%)] consistent with the trapping of lipid-derived alkoxyl and oleate radicals, respectively. Ascorbate pre-treatment ablated radical formation in both systems. These findings identify circulating PUFA as a potential source of secondary radicals that are capable of initiating oxidative stress in the exercising human.