A method to measure the oxidizability of both the aqueous and lipid compartments of plasma

The lipophilic radical initiator (MeO-AMVN) and the fluorescent probe C11BO DIPY581/591 (BODIPY) were used to measure the lipid compartment oxidizabili ty of human plasma. Aqueous plasma oxidizability was initiated by the aqueo us peroxyl radical generator, AAPH, and 2',7'-dichlorodihydrofluorescein (D CFH) was employed as the marker of the oxidative reaction. The distribution in aqueous and lipid compartments of the two radical initiators was determ ined by measuring the rate of consumption of the plasma hydrophilic and lip ophilic endogenous antioxidants. In the presence of AAPH (20 mM), the order of consumption was: ascorbic acid > alpha -tocopherol > uric acid > beta - carotene, indicating a gradient of peroxyl radicals from the aqueous to the lipid phase. When MeO-AMVN was used (2mM), beta -carotene was consumed ear lier than uric acid and almost at the same time as alpha -tocopherol, refle cting the diffusion and activation of MeO-AMVN in the lipophilic phase. The rate of BODIPY oxidation (increase in green fluorescence) significantly in creased after the depletion of endogenous alpha -tocopherol and beta -carot ene, whereas it was delayed for 180 min when AAPH was used instead of MeO-A MVN. The measurement of lipid oxidation in plasma was validated by adding t o plasma the two lipophilic antioxidants, alpha -tocopherol and beta -carot ene, whose inhibitory effects on BODIPY oxidation were dependent on the dur ation of the preincubation period and hence to their lipid diffusion. DCFH oxidation induced by AAPH only began after uric acid, the main hydrophilic plasma antioxidant, was consumed. In contrast, when MeO-AMVN was used, DCFH oxidation was delayed for 120 min, indicating its localization in the aque ous domain. In summary, the selective fluorescence method reported here is capable of distinguishing the lipophilic and hydrophilic components of the total antioxidant capacity of plasma. (C) 2001 Elsevier Science Inc.