Influence of flavonoids on the transmembrane electron transport:study ex-vivo

The oxidative stress results from a change in the physiological balancebetween oxidant and antioxidant species. This condition induces an chemical change in the redox state of cells.. The function of t-PMET is to cooperate with intracellular redox pairs, like piruvate and lactate, to maintain thecytoplasmatic NAD+/NADH balance. The t-PMET is correlated with the modulation of internal pH and redox homeostasis, as it is able to activate proton release. Thus, t-PMET causes external and internal pH modification, as well as development of an inside negative membrane potential. The aim of this work was evaluate the erythrocytes redox status in a group of health volunteers and then to study whether some of flavonoids, enclosed in sub-class of flavonols (Quercetin and Kaempferol), are able to modify the erythrocytes redox homeostasis.Our attention has been focused on red blood cells (RBC) because a close link between t-PMET and metabolic status of erythrocytes has been reported. The RBCs act as antioxidants to themselves and, inaddition, their mobility carries their antioxidant capabilities to all the plasma accessible parts of the body.The subjects that participating at this work attested no supplement intake or some other substance that could interfere with our tests. Human venous blood from twenty healthy volunteers of both sexes between the ages of 25-50 years were obtained by venipuncture in heparin after an over-night fast and centrifuged.The antioxidant capacity of plasma was analyzed by crocin bleaching assay and FRAP. On the other hand, the reducing activity in erythrocytes that represents the body redox state of the last 120 days was evaluated by FRAP method. After centrifugation at 3000 rpm for 10 minutes at 4°C, plasma was separate from red blood cells. The resultant plasma was transferred to microcentrifuge tubes and used at least in part for ferric-reducing activity power (FRAP) and for Crocin Bleaching Assay (CBA) and the others aliquots was stored -80°C before to use for further analysis. Red blood cells, after removal of buffy coat and upper 15% of the packed red blood cells, were washed two times with cold PBS. A stock solution (20mM) of each flavonoid was prepared in dimethyl sulfoxide and then diluted 1:2 with PBS. Packed RBC (10%v/v) were incubatedin PBS containing 5mM glucose at 37°C for 10. minutes with a 50 μM concentration of each flavonoids. After this time the suspensions was centrifuged, the RBC were washed and then analyzed. The percentage of hemolysis was evaluated in the same sample by measuring the haemoglobin contents.The extent of lysis was not different than the controls and never higher than 0.5%.All the values confirm the data of literature about the antioxidant status of human being in physiological condition. All compounds were taken up by the erythrocytes and displaying significant FIC-reducing activity. Both the analysed compounds (Quercetin and Kaempferol) are able to increase the reducing activity of the erythrocytes of 15% and 13% respectively respect to the value recorded for the control. On the other hand, only the quercetin was able to increment the activity of the tPMET system; not any significant difference has been recorded between the Kaempferol and the control group .This study shows that the flavonoids are able to form stable complexeswith the erythrocytes and to influence the intracellular redox homeostasis.Therefore, it could affirm that the polyphenols are able to increase the defence of erythrocytes against ROS. This work underlines that the RBC plays a pivotal role in the distribution and bioavailability of circulating polyphenols which contribute to the defence against injury induced by ROS in various clinical disorders.