Superoxide Anion Radical Scavenging Activity of Serum in Diabetic Patients

In order to evaluate oxidative stress in biological systems, the antioxidative activity of serum (plasma) is often used. The superoxide anion radical (0₂^・-） is an important species which causes oxidative stress in biological systems and several methods have been proposed to evaluate scavenging activity. Of these, the ESR spin-trap method is excellent and is unaffected by other factors as it is highly selective detecting only 0₂^・-.
When the 0₂^・- scavenging activity of serum (plasma) is to be measure d, the difficulty in evaluating haemolysis samples using a photo-absorption techniques such as cytochrome-C, is generally recognised. There are other methods which can be influenced by other factors. Here, we show the comparison against the nitroblue tetrazolium (NBT) method.
Under acidic condition, NBT is reduced by 0₂^・- to form azan. If a sample has 02^・- scavenging activity, production of formazan (560nm) is suppressed, therefore, it is possible to evaluate the antioxidation activity.

The following shows the result of 0₂^・- scavenging activity of serum from diabetic patients by both ESR and NBT methods. (Refer to ER-070002 for details of the ESR method.) Calibration curves for both methods were made using superoxide dismutase (SOD) as a standard material for quantification. As shown in the figure, there was a significant difference in the results. When we measured the amount of Cu,Zn-SOD and Mn-SOD in these samples by the enzyme immunoassay method, the total volume of SOD was up to 2.5 times greater. The possibility is that with the NBT method, antioxidation activity is over-estimated because of interference from other factors. Here we demonstrate that measurement by ESR, because of its higher selectivity for 0₂^・- provides more reliable data.

Difference of 0₂^・- scavenging activity in Diabetic Patient's Serum

Reference

Nakai, Ohta, Sakurai, Aoyama, Kamata, Magnetic Resonance in Medicine, 9, 56・59, Nihon Igakukan (1998)