Direct measurement of oxygen free radicals during in utero hypoxia in the fetal guinea pig brain

The present study tested the hypothesis that maternal hypoxia induces oxygen free radical generation in the fetal guinea pig brain utilizing techniques of electron spin resonance spectroscopy and alpha-phenyl-tert-butyl nitrone (PBN) spin trapping. Pregnant guinea pigs of 60 days gestation were divided into normoxic and hypoxic groups and exposed to 21% or 7% oxygen for 60 min. Free radical generation was documented by measuring the signal of PEN spin adducts. Fluorescent compounds were determined as an index of lipid peroxidation and the activity of Na+,K+-ATPase was determined as an index of brain cell membrane function. Hypoxic fetal cerebral cortical tissue showed a significant increase in spin adducts (normoxic: 33.8 +/- 9.3 units/g tissue vs. hypoxic: 57.9 +/- 9.2 units/g tissue, p < 0.01) and fluorescent compounds (normoxic: 0.639 +/- 0.054 mu g quinine sulfate/g brain vs. 0.810 +/- 0.102 mu g quinine sulfate/g brain, p < 0.01) and a decrease in Na+,KC-ATPase activity (normoxic: 43.04 +/- 2.50 mu mol Pi/mg protein/h vs. hypoxic: 33.80 +/- 3.51 mu mol Pi/mg protein/h, p < 0.001). These results demonstrate an increased free radical generation during hypoxia in the fetal guinea pig brain. The spectral characteristics of the radicals were consistent with those of alkoxyl radicals. The increased level of fluorescent compounds and decreased activity of Na+,K+-ATPase indicated hypoxia induced brain cell membrane lipid peroxidation and dysfunction, respectively. These results directly demonstrate an increased oxygen free radical generation during hypoxia and suggest that hypoxia-induced increase in lipid peroxidation and decrease in membrane function, as indicated by a decrease in Na+,K+-ATPase activity, are consequences of increased free radicals. The nature of predominantly present alkoxyl radical indicates ongoing lipid peroxidation during hypoxia. The direct demonstration of oxygen free radical generation during hypoxia is the critical missing link in the mechanism of hypoxia-induced brain cell membrane dysfunction and damage. (C) 1998 Elsevier Science B.V. All rights reserved.