Application of water-soluble radical initiator, 2,2′-azobis-[2-(2-imidazolin-2-yl)propane] dihydrochloride, to a study of oxidative stress

It is essential to generate free radicals at a controled and constant rate for specific duration and at specific site to study the dynamics of oxidation and also antioxidation. Both hydrophilic and lipophilic azo compounds have been used for such purpose. In the present work, the action of 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH) was examined and compared with those of 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) and 2,2′-azobis[2-methyl-N-(2-hydroxyethyl)-propionamide] (AMHP). The rate constant of free radical formation (ek_d) for AIPH was 2.6 × 10^-6/s at 37°C in PBS (pH 7.4) solution, indicating that AIPH gives 3.8 times more free radicals than AAPH under the same conditions. It was found that the dynamics of oxidation and antioxidation induced by AIPH can be studied satisfactorily in the oxidation in micelles, LDL and erythrocyte suspensions, plasma, and cultured cells. The extent of cell death induced by AIPH and AAPH was directly proportional to the total free radicals formed. Interestingly, it was found that rats would not drink water containing AAPH, but they drank water containing AIPH. The levels of 8-iso-prostaglandin F2α (8-isoPs), 7-hydroxycholesterol (FCOH), lysophosphatidylcholine in the plasma of rats given water containing 50 mM AIPH for 1 month increased compared with those of control rats which drank water without AIPH. It may be concluded that AIPH is useful for kinetic and mechanistic studies on oxidative stress to membranes, lipoproteins, cells, and even animal models.