Hemoglobin Glycation and Oxidative Stress Induce Functional Modification of Band 3 Protein in D‐Galactose‐Treated Erythrocytes: Double Effect of Quercetin

Abstract

D‐Galactose (D‐Gal) is a reducing sugar whose concentration in the blood is very low (between 0.00008 and 0.00018 mmol/L). An abnormal accumulation of D‐Gal, which occurs in pathological conditions such as galactosemia or to reproduce experimental models such as aging, leads to an increase in oxidative events and the formation of advanced glycation end products (AGEs), known to alter erythrocyte homeostasis. Specifically, increased levels of glycated hemoglobin (A1c) and oxidative markers have a negative impact on the exchange capability of Band 3 protein (B3p), responsible for the chloride‐bicarbonate exchange, essential for erythrocyte function. Among the natural compounds that can counteract these glycation and oxidation phenomena, quercetin (Q), a flavonoid found in many fruits and vegetables, shows strong anti‐glycation and antioxidant power at low doses, both in vivo and in vitro.Based on this, the hypothesis of the present work is that Q counteracts A1c formation and increased oxidative stress induced by exposure to different doses of D‐Gal in two in vitro experimental models: galactosemia (low doses) and aging (high doses).Therefore, the aim of this study is to verify whether the anti‐glycation and antioxidant action of Q results in a restoration of B3p functionality, measured by the rate constant for SO4²⁻ uptake, slower and better detectable than Cl‐or HCO3‐.In this regard, rate constant of SO4²⁻ uptake, A1c levels, oxidative stress assessment (TBARS levels, ‐SH groups levels, GSH/GSSG ratio), were determined after 1h incubation of erythrocytes with or without Q (10 µmol / L) at 37°C, followed by 24 h exposure to low (0.1‐10 mmol/L) or high (50‐100 mmol/L) doses of D‐Gal at 25°C.Our results show that low doses of D‐Gal reduce the rate constant of SO4²⁻ uptake and increase A1c levels, but do not induce oxidative stress. In contrast, high D‐Gal doses accelerate the rate constant, increase A1c levels and oxidative stress. Furthermore, pre‐treatment with Q reduces A1c levels and oxidative stress, bringing the rate constant back to control values.In conclusion, we can confirm that: i) anion exchange through B3p is a useful tool to monitor the homeostasis of erythrocytes exposed to different concentrations of D‐Gal; ii) all selected doses of D‐Gal affect B3p function via different mechanisms, including glycation events (low doses) and oxidative events (high doses); iii) Q attenuates the D‐Gal effect, impairing the rate constant alterations by preventing A1c formation and reducing oxidative stress.This evidence suggests that Q could be a useful dietary supplement to prevent oxidative events and AGE‐mediated processes linked to disease.