ESR study of a biological assay on whole blood: antioxidant efficiency of various vitamins

This study deals with the activity of various vitamins against the radical-mediated oxidative damage in human whole blood. We have used a biological method that allows both the evaluation of plasma and that of red blood cell resistance against the free radicals induced by 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH). Spin trapping measures using mainly 5-(diethoxyphosphoryl)-5-methyl-1-pyrolline N-oxide nitrone (DEPMPO) were carried out under several conditions to identify the free radicals implicated in this test. Only the oxygenated-centred radical generated from AAPH was found highly reactive to initiate red blood cell lysis. With DEPMPO only alkoxyl radicals were observed and no evidence was found for alkylperoxyl radicals. The antioxidant activity of several lipid- and water-soluble vitamins has been assessed by the biological assay and through two chemical methods. We have noticed high antioxidant activities for tocopherols (in the order delta>gamma>alpha) in the biological test but not through chemical methods. At 1 microM, the delta-tocopherol efficiency in inhibiting radical-induced red blood cell hemolysis was three times as high as the alpha-tocopherol efficiency. For beta-carotene no significant activity even in whole blood was shown. Highly surprising antioxidant activities were observed for acid folic and pyridoxine, compared to ascorbic acid. At 10 microM, the effectiveness of folic acid was almost three times as high as vitamin C. The biological test seems clinically more relevant than most other common assays because it can detect several classes of antioxidants.