Mechanisms of iron-induced oxidative modifications of creatine kinase in rat brain in vitro. possible involvement of HNE

The model of oxidative stress induced by Fe/ascorbate in rat brain in vitro was used to compare the antioxidant capacity of known antioxidants. Creatine kinase (CK) was selected as a marker of protein injury in such studies. Of the antioxidant enzymes (catalase, superoxide dismutase), oxygen radical scavengers (mannitol, glutathione), and the chelator (EDTA) tested in this work and this system, only catalase and glutathione prevented the injury induced by oxidative stress, indicating that H2O2 and the glutathione peroxidase reaction were involved in the preventive effect. Additionally, the preventive effect of glutathione may be caused also by the fact that glutathione easily reacts with 4-hydroxynonenal (HNE), generated in rat brain homogenate, thus protecting CK from inactivation by this aldehyde. To find out whether and if at which concentrations CK may be oxidatively modified by HNE, pure CK was incubated in the presence of 10 and 64 micromol/l HNE for 30 min at 37 degrees C. The activity of CK incubated with HNE decreased significantly. Simultaneously, the protein carbonyls, determined by electrophoresis and immunoblotting increased at 10 micromol/l HNE or disappeared probably due to crosslinking of CK at 64 micromol/l HNE. The concentration of HNE in rat brain homogenates after oxidative stress was determined by HPLC and was in the range of 10-16 nmol/mg prot., corresponding to a concentration of 10-16 micromol/l HNE. This indicates that CK of rat brain homogenates oxidized by Fe/ascorbate may be impaired not only directly by oxygen radicals but also secondarily by HNE.