Physyological role of t-PMET in erythrocytes redox homeostasis: influence of flavonoids

PHYSYOLOGICAL ROLE OF t-PMET IN ERYTHROCYTES REDOX HOMEOSTASIS:INFLUENCE OF FLAVONOIDS D. Di Majo1, M. La Guardia2, M. Crescimanno1, C. Flandina1, G. Leto1, M. Giammanco1 1Unità Didattico Scientifica di Fisiologia e Farmacologia, Dip. DIGSPO, Università di Palermo; 2Dip. STEBICEF, Università degli Studi di Palermo. Corresponding author: Danila Di Majo, Unità Didattico Scientifica di Fisiologia e Farmacologia, Dip. DIGSPO,Università degli Studi di Palermo. e-mail. danila.dimajo@unipa.it, phone +390916236405 Background In the last decade the trans-Plasma Membrane Electron Transport (t-PMET) has been subject to more research. Ever growing evidence has demonstrated that t-PMET occurs in all types of organisms, including bacteria, yeast plants and animals (1). This system regulates distinct cellular functions and its malfunction relates to some diseases such as cancer, cardiovascular diseases, aging, obesity, neurodegenerative diseases, pulmonary fibrosis, asthma (1). The activity of t-PMET is critical to redox homeostasis in blood. In particular, a close link between t-PMET and metabolic status of erythrocytes has been reported (2). In hypoxic condition the activation of t-PMET may serve to compensate the impaired pentose phosphate pathway, thus ensuring a functional reducing capacity; in this conditions t-PMET may use ascorbic acid or polyphenols as electron donator, since NADH derived from enhanced glycolysis is preferentially utilized by meta-hemoglon reductase (3).Some authors have found a relationship between the dietary flavonoids and trans- plasma membrane oxidoreductase activity, suggesting an additional mechanism whereby dietary flavonoids may exert beneficial effects in human (4). Aim of this work was evaluate whether some of flavonoids, enclosed in sub-class of flavonols (Quercetin and Kaempherol) and phenolic acids, are able to modify the erythrocytes redox homeostasis. Quercetin has been used as control because other authors had previously described its activity to enter erythrocytes and donate electrons to the PMOR system (4). Methods Human venous blood from different healthy volunteers of both sexes between the ages of 25-50 years was obtained by venipuncture in heparin after an over-night fast and centrifuged. The plasma and buffy coat and the upper 15% of the packed red blood cells (RBC) have been removed. The antioxidant capacity of plasma was analyzed by crocin bleaching assay (5) and FRAP. 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 incubated in 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 extracellular concentration of the flavonoids was measured in the medium at the end of the incubation period with the compounds (4). The assay was performed spectrophometrically by measuring the absorbance at the wavelength corresponding to the maximal absorption spectrum. The flavonoids intracellular content was measured on the erythrocytes lysate after extraction three times with ethyl acetate and they have been quantified spectrophometrically. The activity of the erythrocyte plasma membrane redox system (PMRS) was estimated by reduction of ferricyanide according to Fiorani method (4). The catalase activity was measured by catalase assay kit purchased from the SIGMA-ALDRICH. Results All compounds were taken up by the erythrocytes and displaying significant FIC-reducing activity. Their ability to act as intracellular substrates of PMOR is structure-dependence. In physiologic condition the catalase activity varies from 28.6 mU/g protein to 40,6 mU/g protein. Catalase activity decreased with increasing concentration of flavonoid and related compounds. The activity of catalase after incubation in the presence of luteolin is lower by 30% than control (erythrocytes without luteolin). This results is not dose-dependence. In fact, no-significance difference has been observed at both luteolin concentrations (10 and 100μM). Not any correlation has been found between catalase and plasma antioxidant capacity. Conclusions This study shows that the flavonoids are able to form stable complexes with 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. 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