Inhibition of protein thiol modification in hepatocytes isolated from rats supplemented with vitamin E under oxidative stress.
This study examined the effect of vitamin E on maintaining the protein reactive thiols under oxidative stress. Hepatocytes were prepared from male Sprague-Dawley rats fed diets containing three levels of vitamin E (0,100 and 15,000 mg/kg) for 12 wk. Cells were isolated by collagenase perfusion and treated with 0.5 Him tert-butyl hydroperoxide (t-BuOOH) after 24 hr in culture. Carbonic anhydrase III (CA III) having two reactive thiols that can react with GSH under oxidative stress was chosen as the study subject. CA III S-glutathionation was measured by isoelectric focusing/immunoblotting. Results indicated that thiol modification of CA III was induced by t-BuOOH and the pattern of modification was dependent on the vitamin E status. With t-BuOOH treatment, CA III S-glutathionation was quickly induced and the maximum modification was achieved at 3 min in cells isolated from rats fed high levels of vitamin E; however, modification was continuously increased and reached the maximum at 9 min of vitamin E-normal or -deficient cells. Following the maximum modification, a reversion occurred (dethiolation); the rate of reversion was also related to vitamin E status. As shown by image analysis, twofold more (40 v. 20%) CA III was modified in vitamin E-deficient hepatocytes than in cells from rats fed high vitamin E. Glutathione was also abruptly converted to the oxidized state at 3 min in all cells, then gradually reverted to the reduced state. As with the dethiolation of CA III, the rate of glutathione disulfide reduction was correlated to vitamin E status. The production of thiobarbituric acid-reactive substances corresponded to vitamin E status as well and was significantly inhibited in cells from rats fed high vitamin E. These results suggest that vitamin E not only inhibits lipid peroxidation but also plays a role in maintaining the protein thiols under oxidative stress.
Lii
C K
CK
Department of Nutrition, Chung-Shan Medical College, 113, Sec. 2, Ta-Chien St, Taichung, Taiwan 40203, Republic of China.
Chen
H W
HW
Wang
S T
ST