ERYTHROCYTE CATALASE ACTIVITY IN EVER-SMOKERS AND NO-SMOKERS HEALTHY SUBJECTS: INFLUENCE OF FLAVONOIDS(QUERCETIN AND LUTEOLIN) Background. Oxygen free radicals are highly reactive species that can cause cell damage including lipid peroxidation, enzyme inactivation and DNA damage. Inactivation and removal of highly reactive species depend by the antioxidant defence systems. The catalase (CAT) belongs to the primary antioxidant defence system of the cell which catalyses the decomposition of hydrogen peroxide to water and molecular oxygen. Flavonoids are a group of polyphenolic compounds with different chemical structure and properties. They are widely distributed in fruits, vegetables, nuts, seeds and beverages such as wine and tea. The major flavonoide classes include flavonols, flavone, flavonones, flavanols, anthocyanidins and isoflavones. These compounds may scavenge free radical species and other oxidants. In previous studies, we have observed that some polyphenols are able to cross the erythrocyte’s membrane and this process is influenced by the plasmatic albumin; in fact quercetin intracellular concentrations is albumin dose depending. The influence of flavonoids on catalase activity has been reported in some papers but the results are contradictory. Some authors have found an increase of catalase activity in cell in the presence of flavonoids (1,2). Others have observed any effect or even a decrease of catalase activity (3). The present work is based on a study of Krych's, (4), in which the influence of flavonoids on catalase in model system has been evaluated. Aim of this work was to study the role of red body cells in the antioxidant balance. The primary goal was to evaluate the antioxidant status of no-smokers and ever-smokers healthy subjects by the determination of the plasma antioxidant capacity and of the catalase activity of erythrocyte and then to evaluate if flavonoids (quercetin and luteolin) are able to modify the enzyme activity. Methods. This is a pilot study. Nine healthy subjects, aged 24-55 years, of which six females (3 no-smokers and 3 ever smokers) and 3 males (2 smokers and 1 no-smokers) were recruited. None of the subjects had any pathologies at the time of sampling. We assayed the CAT activity in erythrocytes isolated from whole blood of the subjects by the colorimetric assay and the plasma antioxidant capacity using the spectrophotometric method known “crocin bleaching assay”. The catalase activity was performed in human erythrocytes (control) and after the incubation of them with the flavonoids (quercetin and luteolin). Human venous blood (in heparin) from healthy volunteers was obtained by venepuncture. The blood was centrifuged and then plasma, buffy coat and upper 15% of the packed erythrocytes were removed. The isolated erythrocytes isolated were washed twice with cold PBS and then re-suspended and incubated with flavonoids according Fiorani (5) method. The catalase activity was analysed by a catalase assay kit purchased from Sigma-Aldrich. All results are presented as mean ± SD or mean ±SEM. Correlation statistics between variables were assessed by calculating the Pearson coefficient. Differences in means between groups were analyzed by the unpaired t-test. Differences were considered statistically significant at P<0.05. Multiple regression analysis was used to investigate the influence of different variables on the enzyme activities. Results. The flavonoids were efficiently taken up by human erythrocytes in dose-depending manner. There was no significant difference in the percent accumulation of both molecules(quercetin and luteolin) inside the erythrocytes when incubated at the same concentration of 50μM. In physiologic condition the catalase activity varies from 28.6 mU/g protein to 40,6 mU/g protein. Data have shown that CAT activity of erythrocytes was significantly lower in ever smokers than in no-smokers (Figure 1). It was also found that Quercetin at the concentration of 100μM is able to increase the catalase levels in ever-smokers up to the normal values observed in no smokers (Figure 2). The study on the luteolin has not produced the same effects. In fact, Luteolin is able to reduce the CAT levels in no-smokers subject according with the data from Krych, 2013. The different actions of compounds on catalase can been explained as consequence of flavonoid interaction with enzymatic protein. The inhibiting action of the luteolin can be a consequence of a conformational change which occurs upon the flavonoid binding to catalase. This interaction changes the geometry of the substrate channel and thus inhibits the reaction of H2O2 with the heme center. Plasma antioxidant capacity was lower in no smokers than in smokers. An inverse correlation has been found between age and plasma antioxidant capacity. Conclusion from the results of this study affirms that an oxidative stress condition is present in ever-smokers respect to non-smokers, but the quercetin is able to restore the erythrocyte oxidative stress condition of ever-smokers back to the normality. Further studies are necessary in the future to better investigate the role of luteolin on the catalase activity of human erythrocytes. Wu et al. have warned that microenvironment can shift erythrocytes from friendly to a harmful behaviour. The release of iron-hemoglobin following RBC hemolysis can cause excess accumulation of free iron to catalyze the generation of the highly toxic hydroxyl, peroxyl and alkoxyl radicals. Therefore by virtue of their antioxidant and chelating properties for divalent metals both free and polyphenols bound to RBC might also act to neutralize the toxic effects of ROS. References [1] J.Elavarasan, P.Velusamy, T.Ganesan, S.K. Ramakrishnan, D. Rajasekaran, K.Periandavan. 2012. Titolo. Journal of Pharmacy and Pharmacology 64: 1472-1482; [2] A. Kampkötter, Y. Chovolou, A. Kulawik, E. Röhrdanz, N. Weber, P. Proksch, W. Wätjen. 2008.Titolo. Nutrition Research 28: 620-628 [3] G.Valdameri, M. Trombetta- Lima, P.R. Worfel, A.R.A. Pires, G.R. Martinez, G.R. et al. 2011. Titolo. Chemistry-Biological interactions 193:180-189 [4] J.Krych, L. Gebicka. 2013. Catalase is inhited by flavonoide. International Journal of Biological Macromolecules 58:148-153 [5] M.Fiorani, A . Accorsi. 2005. Dietary flavonoids as intracellular substrates fora n erytrocyte trans-plasma membrane oxidoreductase activity. British Journal of Nutrition 94: 338-345.
Di Majo, D., Giammanco, M., Di Sclafani, E., Flandina, C., Crescimanno, M., Leto, G., et al. (2013). ERYTHROCYTE CATALASE ACTIVITY IN EVER-SMOKERS AND NO-SMOKERS HEALTHY SUBJECTS: INFLUENCE OF FLAVONOIDS (QUERCETIN AND LUTEOLIN) [Altro].
ERYTHROCYTE CATALASE ACTIVITY IN EVER-SMOKERS AND NO-SMOKERS HEALTHY SUBJECTS: INFLUENCE OF FLAVONOIDS (QUERCETIN AND LUTEOLIN)
DI MAJO, Danila;GIAMMANCO, Marco;FLANDINA, Carla;CRESCIMANNO, Marilena;LETO, Gaetano;LA GUARDIA, Maurizio
2013-01-01
Abstract
ERYTHROCYTE CATALASE ACTIVITY IN EVER-SMOKERS AND NO-SMOKERS HEALTHY SUBJECTS: INFLUENCE OF FLAVONOIDS(QUERCETIN AND LUTEOLIN) Background. Oxygen free radicals are highly reactive species that can cause cell damage including lipid peroxidation, enzyme inactivation and DNA damage. Inactivation and removal of highly reactive species depend by the antioxidant defence systems. The catalase (CAT) belongs to the primary antioxidant defence system of the cell which catalyses the decomposition of hydrogen peroxide to water and molecular oxygen. Flavonoids are a group of polyphenolic compounds with different chemical structure and properties. They are widely distributed in fruits, vegetables, nuts, seeds and beverages such as wine and tea. The major flavonoide classes include flavonols, flavone, flavonones, flavanols, anthocyanidins and isoflavones. These compounds may scavenge free radical species and other oxidants. In previous studies, we have observed that some polyphenols are able to cross the erythrocyte’s membrane and this process is influenced by the plasmatic albumin; in fact quercetin intracellular concentrations is albumin dose depending. The influence of flavonoids on catalase activity has been reported in some papers but the results are contradictory. Some authors have found an increase of catalase activity in cell in the presence of flavonoids (1,2). Others have observed any effect or even a decrease of catalase activity (3). The present work is based on a study of Krych's, (4), in which the influence of flavonoids on catalase in model system has been evaluated. Aim of this work was to study the role of red body cells in the antioxidant balance. The primary goal was to evaluate the antioxidant status of no-smokers and ever-smokers healthy subjects by the determination of the plasma antioxidant capacity and of the catalase activity of erythrocyte and then to evaluate if flavonoids (quercetin and luteolin) are able to modify the enzyme activity. Methods. This is a pilot study. Nine healthy subjects, aged 24-55 years, of which six females (3 no-smokers and 3 ever smokers) and 3 males (2 smokers and 1 no-smokers) were recruited. None of the subjects had any pathologies at the time of sampling. We assayed the CAT activity in erythrocytes isolated from whole blood of the subjects by the colorimetric assay and the plasma antioxidant capacity using the spectrophotometric method known “crocin bleaching assay”. The catalase activity was performed in human erythrocytes (control) and after the incubation of them with the flavonoids (quercetin and luteolin). Human venous blood (in heparin) from healthy volunteers was obtained by venepuncture. The blood was centrifuged and then plasma, buffy coat and upper 15% of the packed erythrocytes were removed. The isolated erythrocytes isolated were washed twice with cold PBS and then re-suspended and incubated with flavonoids according Fiorani (5) method. The catalase activity was analysed by a catalase assay kit purchased from Sigma-Aldrich. All results are presented as mean ± SD or mean ±SEM. Correlation statistics between variables were assessed by calculating the Pearson coefficient. Differences in means between groups were analyzed by the unpaired t-test. Differences were considered statistically significant at P<0.05. Multiple regression analysis was used to investigate the influence of different variables on the enzyme activities. Results. The flavonoids were efficiently taken up by human erythrocytes in dose-depending manner. There was no significant difference in the percent accumulation of both molecules(quercetin and luteolin) inside the erythrocytes when incubated at the same concentration of 50μM. In physiologic condition the catalase activity varies from 28.6 mU/g protein to 40,6 mU/g protein. Data have shown that CAT activity of erythrocytes was significantly lower in ever smokers than in no-smokers (Figure 1). It was also found that Quercetin at the concentration of 100μM is able to increase the catalase levels in ever-smokers up to the normal values observed in no smokers (Figure 2). The study on the luteolin has not produced the same effects. In fact, Luteolin is able to reduce the CAT levels in no-smokers subject according with the data from Krych, 2013. The different actions of compounds on catalase can been explained as consequence of flavonoid interaction with enzymatic protein. The inhibiting action of the luteolin can be a consequence of a conformational change which occurs upon the flavonoid binding to catalase. This interaction changes the geometry of the substrate channel and thus inhibits the reaction of H2O2 with the heme center. Plasma antioxidant capacity was lower in no smokers than in smokers. An inverse correlation has been found between age and plasma antioxidant capacity. Conclusion from the results of this study affirms that an oxidative stress condition is present in ever-smokers respect to non-smokers, but the quercetin is able to restore the erythrocyte oxidative stress condition of ever-smokers back to the normality. Further studies are necessary in the future to better investigate the role of luteolin on the catalase activity of human erythrocytes. Wu et al. have warned that microenvironment can shift erythrocytes from friendly to a harmful behaviour. The release of iron-hemoglobin following RBC hemolysis can cause excess accumulation of free iron to catalyze the generation of the highly toxic hydroxyl, peroxyl and alkoxyl radicals. Therefore by virtue of their antioxidant and chelating properties for divalent metals both free and polyphenols bound to RBC might also act to neutralize the toxic effects of ROS. References [1] J.Elavarasan, P.Velusamy, T.Ganesan, S.K. Ramakrishnan, D. Rajasekaran, K.Periandavan. 2012. Titolo. Journal of Pharmacy and Pharmacology 64: 1472-1482; [2] A. Kampkötter, Y. Chovolou, A. Kulawik, E. Röhrdanz, N. Weber, P. Proksch, W. Wätjen. 2008.Titolo. Nutrition Research 28: 620-628 [3] G.Valdameri, M. Trombetta- Lima, P.R. Worfel, A.R.A. Pires, G.R. Martinez, G.R. et al. 2011. Titolo. Chemistry-Biological interactions 193:180-189 [4] J.Krych, L. Gebicka. 2013. Catalase is inhited by flavonoide. International Journal of Biological Macromolecules 58:148-153 [5] M.Fiorani, A . Accorsi. 2005. Dietary flavonoids as intracellular substrates fora n erytrocyte trans-plasma membrane oxidoreductase activity. British Journal of Nutrition 94: 338-345.
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