Fluorescence quenching of dipyridamole associated to peroxyl radical scavenging: a versatile probe to measure the chain breaking antioxidant activity of biomolecules

Dypiridamole is a highly efficient chain breaking antioxidant (Iuliano et al., Free Radic. Biol. Med. 18 (1995) 239-247) with an aromatic ring system responsible for an intense absorption band in the 400-480-nm region and for an intense fluorescence. Dipyridamole fluorescence is quantitatively quenched upon reaction with peroxyl radicals. In the presence of a flux of peroxyl radicals generated by thermal dissociation of ate-initiators, dipyridamole fluorescence decays linearly, showing a first-order reaction with respect to peroxyl radicals, and zero-order with respect to dipyridamole. The pH optimum for the fluorescence quenching is in the 7-8 range, from pH 7 to 6, the decay of fluorescence rapidly decreases to became negligible below pH 5.5. Dipyridamole consumption is blocked in the presence of an added chain breaking antioxidant for a time that is proportional to the antioxidant concentration. This effect is shown for ascorbic acid, trolox, vitamin E, uric acid, and N,N'-diphenyl-p-phenylenediamine. The slope of the linear correlation relative to trolox allows calculation of the bimolecular rate constant for a given molecule and peroxyl radicals. Comparison of data obtained by the dipyridamole consumption are comparable to values obtained by the oxygen consumption method. (C) 2000 Elsevier Science B.V. All rights reserved.Dypiridamole is a highly efficient chain breaking antioxidant (Iuliano et al., Free Radic. Biol. Med. 18 (1995) 239^247) with an aromatic ring system responsible for an intense absorption band in the 400^480-nm region and for an intense fluorescence. Dipyridamole fluorescence is quantitatively quenched upon reaction with peroxyl radicals. In the presence of a flux of peroxyl radicals generated by thermal dissociation of azo-initiators, dipyridamole fluorescence decays linearly, showing a first-order reaction with respect to peroxyl radicals, and zero-order with respect to dipyridamole. The pH optimum for the fluorescence quenching is in the 7^8 range, from pH 7 to 6, the decay of fluorescence rapidly decreases to became negligible below pH 5.5. Dipyridamole consumption is blocked in the presence of an added chain breaking antioxidant for a time that is proportional to the antioxidant concentration. This effect is shown for ascorbic acid, trolox, vitamin E, uric acid, and N,NP -diphenyl-p-phenylenediamine. The slope of the linear correlation relative to trolox allows calculation of the bimolecular rate constant for a given molecule and peroxyl radicals. Comparison of data obtained by the dipyridamole consumption are comparable to values obtained by the oxygen consumption method

NOX2-mediated arterial dysfunction in smokers: acute effect of dark chocolate

Background Cocoa seems to exert artery dilatation via oxidative stress inhibition but the mechanism is still unclear. Objectives To investigate whether in smokers, dark chocolate elicits artery dilatation via down-regulation of NOX2, the catalytic core of NADPH oxidase. Methods Flow-mediated dilatation (FMD), oxidative stress (as assessed by urinary isoprostanes excretion), nitric oxide generation (as assessed by serum levels of nitrite/nitrate (NOx)), NOX2 activity (as assessed by blood levels of soluble NOX2 derived peptide (sNOX2-dp)) and serum epicatechin were studied in 20 smokers and 20 healthy subjects (HS) in a crossover, single-blind study. Patients were randomly allocated to 40 g dark chocolate (>85% cocoa) or 40 g of milk chocolate (<= 35% cocoa). FMD, urinary isoprostanes, NOx and sNOX2-dp were assessed at baseline and 2 h after chocolate ingestion. Results Smokers had lower FMD and NOx and higher sNOX2-dp compared to HS. After dark chocolate intake, urinary isoprostanes and sNOX2-dp significantly decreased and FMD and NOx significantly increased in smokers but not in HS. No changes of the above variables were observed after milk chocolate intake. Multiple linear regression analysis showed that in smokers the only independent predictive variable associated with a change in FMD was a change in sNOX2-dp. Serum epicatechin increased in either group only after dark chocolate intake, reaching values higher than 0.1 mu M. Platelets from smokers (n=5), but not from HS (n=5), showed lower p47(phox) translocation to platelet membrane and higher NOx when incubated with 0.1-10 mu M epicatechin. Conclusion Results suggest that in smokers, cocoa enhances artery dilatation by lowering of NOX2 activation

Evaluation of D-dimer and factor VIII in cirrhotic patients with asymptomatic portal venous thrombosis

D-dimer and factor VIII levels raise in advanced cirrhosis. We investigated the behavior and the diagnostic usefulness of D-dimer and factor VIII in cirrhotic patients with asymptomatic portal venous thrombosis. Factor VIII coagulant and D-dimer values were measured in 136 consecutive outpatients with stable cirrhosis divided according to Child-Pugh (CP) classification, who underwent color/power ultrasonography to detect portal thrombosis. Portal thrombosis was found in 33 patients (24.2%). In patients without thrombosis, factor VIII was significantly higher in CP class C compared with class A and B. Conversely, class C patients with portal thrombosis had lower factor VIII levels than those without thrombosis. In both groups, D-dimer was significantly increased in class C compared with class A and B. In class C, thrombotic patients showed higher D-dimer values than did patients without thrombosis. In class C, a D-dimer value >= 0.55 mu g/mL provided a sensitivity and a negative predictive value for portal thrombosis of 100%, and a factor VIII coagulant level <= 80% showed a specificity and a negative predictive value of 76% and 84%, respectively. In class B, a D-dimer value <= 0.225 mu g/mL had a sensitivity of 89% and a negative predictive value of 82%. In conclusion, our study shows that factor VIII values increase in severe cirrhosis but significantly decrease in the presence of concomitant portal thrombosis, likely because of consumption during thrombosis; D-dimer is enhanced by both liver failure and portal thrombosis; in severe cirrhosis, normal D-dimer and factor VIII values may safely exclude the presence of asymptomatic portal thrombosis