16.P20. A new method to evaluate total antioxidant capacity against reactive oxygen and nitrogen species (RONS) in aquatic organisms

AMADO, L.L.; DÍAZ-JARAMILLO, M.; ROCHA, A.M.; FERREIRA, J.L.R.; GARCIA, L.M.; RAMOS, P.B.; URRUTIA, C.; BELTRÁN, C.; MONSERRAT, J.M.

COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY A-MOLECULAR AND INTEGRATIVE PHYSIOLOGY

ELSEVIER SCIENCE INC

Lugar: Amsterdam; Año: 2007 vol. 148 p. 75 - 76

1095-6433

For the understanding of how antioxidants interact with reactive oxygen and nitrogen species (RONS), the determination of total antioxidant capacity is needed, instead of the measurements of limited number of antioxidants. Several techniques exist, including gas chromatography-based methods, although some of them are extremely time-consuming. A new methodology is proposed, based on the detection of RONS by fluorometry (ex/em: 485/520 nm) employing 2′,7′-dichlorofluorescin diacetate (H2DCF-DA) as substrate. Peroxyl radicals were generated by thermal (35 °C) decomposition of 2,2′-azobis(2methylpropionamidine)dihydrochloride (ABAP) in different tissues of several aquatic organisms, including the teleost fish Jenynsia multidentata (Anaplebidae), Pimelodella laticeps (Heptapteridae) and the polychaeta Perinereis gualpensis (Nereididae). Different protein (2, 3.5 and 4 mg/mL) and ABAP (4 and 8 mM) concentrations were assayed in order to get the best signal and curve fitting of fluorescence data over time (30 min). Total antioxidant capacity against peroxyl radicals was estimated as the difference in RONS area with and without ABAP, with low ACAP values meaning high antioxidant competence. The best combination of protein (2 mg of proteins/mL) and ABAP (4 mM) was employed in all the assays. The nektonic fish (J. multidentata) presented statistically lower (p < 0.05) antioxidant competence in liver samples when compared with muscle, brain and gill samples. The benthic fish (P. laticeps) showed lower antioxidant competence in gill samples when compared with muscle (p < 0.05). Polychaete sampled at different regions from Chile showed distinct ACAP values, indicating that this methodology is also suitable for environmental studies.