Photochemiluminescent detection of antiradical activity .4. Testing of lipid-soluble antioxidants

A new method for quantification of antiradical properties of pure lipid-soluble antioxidants and for measurement of integral antioxidant capacity in the lipid phase (ACL) of polycomponent systems, such as blood plasma or tissue homogenates, is developed. It is based on an antioxidant-sensitive inhibition of a photo-induced, chemiluminescence accompanied autoxidation of luminol. The sensitivity of the photochemiluminescent (PCL) assay lies within nmol quantities of substances, the measuring range for alpha-tocopherol is between 0.1 and 3 nmol. The interassay variability of the method is lower than 5%, the intraassay variability <2%. The antioxidant efficiency of gamma-tocopherol was found to be 43% of alpha-tocopherol. The results of the PCL measurements on pure antioxidants and on lipid extracts from blood plasma were compared with the level of, 'vitamin E' (VE) determined as a sum of alpha- and gamma-tocopherol by HPLC. Very good coincidence of both methods was observed for pure substances (r = 0.998, P<0.001). The ACL of human blood plasma was found to be 27.98 +/- 0.68 mu mol equivalents of alpha-tocopherol/l (mean +/- mean error, n = 142), it is similar to 25% more than the concentration of VE found in the same samples (22.09 +/- 0.59 mu mol/l). In this case, the correlation of both parameters was lower: r = 0.811, P<0.001. The animal experiments showed that synthetic antioxidants may not only increase the value of ACL of blood plasma but in the same time reduce the concentration of biological antioxidants, e.g. VE drastically. The prooxidant activity of synthetic antioxidants in vivo or the replacing of structured alpha-tocopherol from its position can be the cause. This important circumstance has to be considered during the testing of new antioxidants for clinical application.