Total Oxidant Scavenging Capacity Assay Research Articles

Antioxidant capacity and phenolics of Pouteria macrophylla, an under-utilized fruit from Brazilian Amazon

The potential of fruits occurring in the Amazon with regard to their antioxidant capacity is being addressed increasingly during the last years. In this paper, the antioxidant capacity of fruits from Pouteria macrophylla (cutite) and the compounds which are responsible for that functional property were studied. Diphenyl-picrylhydrazyl (DPPH) and total oxidant scavenging capacity (TOSC) assays were used for the evaluation of antioxidant capacity of fruit aqueous extracts. TOSC assay showed inhibitory effects against peroxyl (TOSC50% = 560 mg/L) and peroxynitrite (TOSC50% = 830 mg/L), two important reactive oxygen species, while DPPH showed that cutite (EC50 = 300 ± 3.9 g/g DPPH) has significant antioxidant capacity, greater than many other known Amazon fruits. It was also seen that the cutite fruits are a good alimentary source of polyphenols (867 ± 8 mg GAE/100 g), analyzed by Folin-Ciocalteu assay. Using HPLC-DAD-ESI-MS n and HPLC-DAD, gallic acid (4.72 mg/g fresh fruit pulp) and digalloyl glucose were found as the main phenolic compounds in the cutite fruits. TOSC assays with HPLC fractions of fruit aqueous extracts show that both compounds are predominantly responsible for the antioxidant capacity observed in the cutite.

Anomalies in sperm chromatin packaging: implications for assisted reproduction techniques

Sperm protamine deficiency and DNA damage were analysed employing chromomycin A 3 (CMA 3) staining and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay, respectively, in 132 patients (82 IVF, 50 intracytoplasmic sperm injection [ICSI]). The antioxidant ability of seminal plasma was analysed in 10 men, using the total oxidant scavenging capacity assay. A significant negative correlation was found between abnormal protamination and sperm parameters, including sperm DNA fragmentation ( P < 0.01). A close relationship was found between sperm protamination and fertilization and pregnancy only in IVF ( P = 0.004 and P < 0.04, respectively); in ICSI there was a correlation between DNA fragmentation and pregnancy ( P = 0.031). Finally, there was a negative correlation between chromatin under-protamination and the antioxidant ability of seminal plasma ( P < 0.01). Results of this study underline that, despite sperm abnormal protamination and DNA fragmentation being positively correlated, they affect the reproductive outcome in different ways: in particular there was good prognostic value for CMA 3 analysis only in IVF, whereas DNA fragmentation analysis was prognostic only for ICSI outcome. Data are also provided to support the idea of a relationship between defective antioxidant system activity and impairment of chromatin packaging.

Total oxidant scavenging capacities of Euterpe oleracea Mart. (Açaí) fruits

The antioxidant capacities of 11 commercial and non-commercial samples of Euterpe oleracea Mart. (açaí) fruit pulp were studied with the total oxidant scavenging capacity assay in a modified and automated version against three reactive oxygen species. The antioxidant capacities of all purple açaí samples were found to be excellent against peroxyl radicals, good against peroxynitrite and poor against hydroxyl radicals compared with common European fruit and vegetable juices recently analysed. In all cases the correlation between sample concentration and antioxidant capacities was non-linear. The antioxidant capacities against all three reactive oxygen species of the fruit pulp from one white açaí variety were very low. The phenolic compounds in purple açaí fruit pulp were identified by high-performance liquid chromatography–mass spectrometry, and the two major anthocyanins, cyanidin-3-glucoside and cyanidin-3-rutinoside, were quantified by high-performance liquid chromatography–visible spectrometry. The contributions of the anthocyanins to the overall antioxidant capacities of the fruit were estimated to be only approximately 10%. Obviously, compounds not yet identified are responsible for the major part of the antioxidant capacities of the açaí fruit pulp.

Sex-related responses to oxidative stress in primary cultured hepatocytes of European flounder ( Platichthys flesus L. )

Effects of oxidative stress induced by xenobiotic compounds were studied in primary cultures of isolated hepatocytes of immature European flounder ( Platichthys flesus L.) of both sexes caught in a relatively unpolluted area of the German Bight (North Sea). Cells were exposed to oxidative stressors such as 100 μM hydrogen peroxide (H 2O 2), 100 μM benzo[a]pyrene (B[a]p) and 50 μM nitrofurantoin ( N-(5-nitro-2-furfurylidene)-1-aminohydantoin; NF) for 2 and 24 h. Cell mortality was determined with the use of the fluorescent ethidium homodimer-1 and calcein. Oxidative stress response was assessed by quantitative analysis of (1) intracellular reactive oxygen species (ROS) formation with dihydrorhodamine 123, (2) lipid peroxidation on the basis of concentrations of lipid hydroperoxides and the lipid peroxidation products malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) and (3) cellular total oxidant-scavenging capacity (TOSC) using the TOSC assay ( Winston et al., 1998). An increase in ROS formation was detected as early as 2 h after exposure to H 2O 2, B[a]p and NF. After 24 h, stress responses were lower, except following exposure to NF. The pattern of responses differed with the different oxidative stressors. Lipid peroxidation and the capacity to scavenge ROS were increased significantly in both sexes only after exposure to H 2O 2, whereas B[a]p and NF provoked sex-dependent responses. B[a]p-induced lipid peroxidation and increase in scavenging capacity were observed only in hepatocytes of females, whereas NF initiated these responses only in cells of males. Sex differences in oxidative stress response only after exposure to pro-oxidants that require enzymatic activation infer the importance of biotransformation pathways in stress responses. Because of their sensitivity to oxidative stress, flounder hepatocytes provide a useful model for early risk assessment of xenobiotics.

Quantification of Total Oxidant Scavenging Capacity of Antioxidants for Peroxynitrite, Peroxyl Radicals, and Hydroxyl Radicals

We have extended the application of our previously reported total oxidant scavenging capacity (TOSC) assay (Winstonet al., Free Radical Biol. Med.24, 480–493, 1998) to permit facile quantification of the absorbance capacity of antioxidants toward three potent oxidants, i.e., hydroxyl radicals, peroxyl radicals, and peroxynitrite. Respectively, these oxidants were generated by the iron plus ascorbate-driven Fenton reaction, thermal homolysis of 2,2′-azobis(2-methylpropionamidine) dihydrochloride (ABAP), and 3-morpholinosydnonimineN-ethylcarbamide (SIN-1). Each of these oxidants reacts with α-keto-γ-methiolbutyric acid (KMBA), which is oxidized and yields ethylene. The antioxidant capacity of the compounds tested is quantified from their ability to inhibit ethylene formation relative to a control reaction. Assay conditions were established in which control reactions give comparable yields of ethylene with each of the oxidants studied. Thus, the relative efficiency of various antioxidants could be compared under conditions of quantitatively similar KMBA oxidizing capability by the three oxidants. Reduced glutathione was an efficient scavenger of peroxyl radicals, but scavenged peroxynitrite and hydroxyl radicals relatively poorly. Uric acid, Trolox, and ascorbic acid were comparable scavengers of peroxynitrite and peroxyl radicals. Uric acid and Trolox were approximately an order of magnitude less efficient as scavengers of hydroxyl radicals. The classical hydroxyl radical scavenging agents mannitol, dimethyl sulfoxide, and benzoic acid had much higher TOSC values with hydroxyl than with peroxyl radicals or peroxynitrite. The very different chemical reactivity toward KMBA by the SIN-1- and iron–ascorbate-generated oxidants indicates that hydroxyl radical is not a major oxidant produced by the SIN-1 system. The data show that the TOSC assay is useful and robust in distinguishing the reactivity of various oxidants and the relative capacities of antioxidants to scavenge these oxidants.