Superoxide dismutase (SOD), Catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) activities were determined in neutrophils using a microplate reader (Tecan, Salzburg, Austria). CAT activity was measured as described by Aebi (1984) based on the direct decomposition of hydrogen peroxide (H2O2). SOD activity was measured using the method described by Ewing and Janero (1995) which involves the reduction of O2- radicals by nitroblue tetrazolium (NBT) for 3 min. Glutathione Torin 1 peroxidase (Mannervik, 1985) and glutathione reductase (Carlberg and Mannervik, 1985) activities were measured based
on the oxidation of β-NADPH in the presence of tert-butyl hydroperoxide, used as substrate. Reduced (GSH) and oxidized (GSSG) glutathione content in neutrophils were measured as described by Rahman et al. (2006). The method is based on the reaction between reduced thiol groups (such as in GSH) with 5,5´-dithiobis-2-nitrobenzoic acid (DTNB) to form 5-thio-2-nitrobenzoic acid (TNB), which is stoichiometrically detected by absorbance at 412 nm. Purified GSH and GSSG (Sigma-Aldrich) were used as standards. The total protein content of cells was measured by the method of Bradford, using BSA as standard (Bradford, 1976). All data points are presented as the mean values with standard errors of at least three independent experiments, each one performed in triplicate. The data were
analyzed by one-way ANOVA followed by the Tukey’s post-test. The software employed for statistical analysis buy PD-0332991 was GraphPad Prism (version4; GraphPad Software, San Diego, CA, USA). Cell membrane integrity was tested
by using flow cytometer and propidium iodide as a probe. After 24 h of culture, none of the groups showed any significant loss of cell membrane integrity. These results indicate that the concentrations of MGO, glucose, astaxanthin and vitamin C selected to evaluate the functional parameters of neutrophils did not cause cell death (Fig. 2). Additionally, MGO, high glucose, astaxanthin and vitamin C alone did not promote changes in cell viability (data not shown). In order to determine the potential of MGO and glucose to modulate the phagocytic capacity of human neutrophils, we measured Tyrosine-protein kinase BLK the incorporation of opsonized zymosan particles in the cells (Table 1). There was a significant decrease of 30% in the phagocytic capacity of neutrophils after treatment with glucose + methylglyoxal (GM group), whereas there was an increase of 22% in the phagocytic capacity after AV-treatment as compared to the control group. When GM-treated cells were added with antioxidants (AVGM group) we observed a complete restoration in the phagocytic capacity. Neither glucose nor MGO alone promoted the same effect observed when those compounds were combined (data not shown). Vitamin C alone promoted improvement in the phagocytic capacity (data not shown).