Oxidant Stress: The Role of the Glutathione Redox Cycle in Skin Preconditioning

Abstract

The role of the glutathione redox cycle in cellular protection form skin necrosis during the ischemic stress response (preconditioning) is unknown. In this series of experiments, we tested the hypothesis that oxidant stress reduces available total glutathione during injury and contributes to skin necrosis in flaps. Dorsal skin flaps (10 × 4 cm) were raised as acute flaps and skin grafts were obtained from the flaps at 0, 1, 4, 6, 12, or 24 hr. Some flaps were preconditioned as bipedicle flaps for 24, 48, 72, or 96 hr and the distal attachment divided before skin grafts were obtained 24 hr later. Flap survival was measured at 7 days. Total glutathione (GSH) and oxidized GSH (GSSG) were extracted and their levels determined enzymatically. Tissue GSH reductase (GR) activity was assayed with a spectrofluorometer and expressed as μmoles of NADPH oxidized/hr/g. Biochemical data were compared between the proximal and distal ends of the flaps using a two-tailed Student t test while differences between groups were compared using ANOVA. Skin necrosis was 5.4 ± 0.12 cm in the distal ends at 7 days in acute flaps, while there was no skin necrosis in flaps preconditioned for 7 days. In acute flaps, total GSH levels fell precipitously in the distal end at 24 hr (P < 0.05). However, after 72 hr of preconditioning, the GSH levels in the distal end of the flap remained elevated while GSSG levels were undetectable. At 24 hr of ischemia, GR activity was 79 ± 4 in the distal ends of acute flaps, while after preconditioning and 24 hr of ischemia, the GR activity increased to 172 ± 13 in the distal ends (P < 0.05). Activity of GR in nonischemic sham control skin was (101 ± 3) (P < 0.05). The data suggest that oxidative stress reduces GSH levels in ischemic skin and that preconditioning enhances GSH skin levels by increasing glutathione reductase activity, a glutathione-dependent enzyme. Thus, increased GSH redox cycle activity appears to be an important component of the preconditioning phenomenon.