Luminol-, isoluminol- or lucigenin-enhanced chemiluminescence (CL) was used to measure the production of reactive oxygen species by rat blood leukocytes. Opsonized zymosan (OZ), phorbol-12-myristate-13-acetate (PMA), calcium ionophore A23187 (Ca-I) or N-formyl-Met-Leu-Phe (fMLP) were used as activators. The CL signal of isolated blood leukocytes decreased in rank order of luminol > isoluminol > lucigenin. The kinetic profiles of luminol- and isoluminol-enhanced CL were similar upon stimulation by each activator tested. The remarkably higher luminol and isoluminol CL responses were obtained after OZ stimulation when compared with other activators. However, when lucigenin was used, the PMA- and OZ-stimulated CL were comparable. The presence of plasma increased OZ-activated CL because of the enhanced phagocytosis of OZ. This was demonstrated by determining the phagocytosis of the fluorescent OZ using a flow cytometer. In contrast, the presence of plasma decreased PMA-activated CL, due to the antioxidant properties of plasma as determined by the CL method. As far as whole blood is concerned, only OZ activated luminol-enhanced CL was reliable. Blood volumes over 5 microL decreased CL activity due to the scavenging ability of erythrocytes. The results suggest that 0.5 microL whole blood is sufficient for routine luminol-enhanced CL analysis of whole blood oxidative burst in rats.

In vitro experiments have demonstrated that polyphenols exhibit antioxidant and anti-inflammatory activities. The present study was designed to test whether dealcoholized red (DRW) and white (DWW) wines can decrease the oxidative stress associated with inflammation in vivo. Rats were fed for 15 d either a control diet or one supplemented with DRW or DWW. Finally, a granuloma was induced by subcutaneous administration of carrageenan. Although DRW showed higher antioxidant activity in vitro than DWW, both wines decreased the number of cells recruited into the granuloma pouch. Malondialdehyde decreased in plasma and inflammatory exudate from rats fed with DRW-and DWW-rich diets. Moreover, the concentration of NO increased in exudate, which correlates with the increase in the citrulline:arginine ratio. Polymorphonuclear leucocytes from the inflammatory exudate of rats fed dealcoholized wines showed decreased superoxide anion (O 2 z2 ) production and increased NO production ex vivo. This change in NO production resulted from increased expression and activity of inducible NO synthase (EC 1.14.13.39). Moreover, the up regulation of cyclo-oxygenase-2 (EC 1.14.99.1) protein expression observed in rats fed the DRW-rich diet was not related to a direct effect of NO. The present results indicate that the non-alcoholic compounds of wines not only improve antioxidant status in an inflammatory situation, but also limit cell infiltration, possibly through a decrease in O 2 z2 and an increase in NO production.Free radicals: Nitric oxide synthase: Polymorphonuclear leucocytes: Polyphenols Most of the beneficial effects associated with the moderate consumption of red wine are related to polyphenols, although wines contain a broad range of non-polyphenolic compounds. In vitro studies have shown that polyphenols have antioxidant 1 -4 and anti-inflammatory activities. The anti-inflammatory activities are a consequence of the inhibition of phospholipase A 2 (EC 3.1.1.4) 5 , cyclo-oxygenase (COX)-2 6 and 5-lipoxygenase 7 (EC 1.13.11.34). However, evidence from the in vitro experiments cannot be extrapolated to in vivo settings, since polyphenols are extensively metabolized in vivo 8 -10 , which can affect their activities.The inflammatory response is characterized by the production of chemical mediators, and the presence of large amounts of exudate, proteins and cells in the inflamed area. Neutrophils are the first blood cells that migrate towards inflammatory lesions and they have the capacity to express NADPH oxidase, inducible NO synthase (iNOS) and COX-2. We have observed, in a carrageenan-induced granuloma model of inflammation, that polymorphonuclear (PMN) leucocytes generate superoxide anion (O 2 z2 ) and NO 11 and it has been described that O 2 z2 , by reacting with NO, generates peroxynitrite 12,13 . Thus, preventing the generation of O 2 z2 by inhibiting the enzymes responsible or scavenging O 2 z2 or avoiding peroxynitrite formation may attenuate inflammation.It is likely that low levels of flavonoids and their metabolites ...

The aim was to study the antioxidant properties of four wine polyphenols (flavonoids catechin, epicatechin, and quercetin, and hydroxystilbene resveratrol). All three flavonoids exerted significant and dose-dependent scavenging effects against peroxyl radical and nitric oxide in chemical systems. The scavenging effect of resveratrol was significantly lower. All polyphenols decreased production of reactive oxygen species (ROS) by RAW264.7 macrophages. Only quercetin quenched ROS produced by lipopolysaccharide-stimulated RAW264.7 macrophages incubated for 24 h with polyphenols. Quercetin and resveratrol decreased the release of nitric oxide by these cells in a dose-dependent manner which corresponded to a decrease in iNOS expression in the case of quercetin. In conclusion, the higher number of hydroxyl substituents is an important structural feature of flavonoids in respect to their scavenging activity against ROS and nitric oxide, while C-2,3 double bond (present in quercetin and resveratrol) might be important for inhibition of ROS and nitric oxide production by RAW 264.7 macrophages.