Epicatechin Influence on Biochemical Modification of Human Erythrocyte Metabolism and Membrane Integrity

Abstract

Red blood cells (RBCs) are the main cells of the blood, perform numerous functions within the body and are in continuous contact with endogenous and exogenous molecules. In this context, the study aims to investigate the effect of epicatechin (EC) (flavan-3-ols) on the erythrocytes, analyzing the protective effect of the molecule and the action exerted on metabolism and RBC membrane. The effect of EC on RBC viability has been evaluated through the change in hemolysis and methemoglobin, assessing caspase 3 activity and performing a cytofluorometric analysis. Next, the impact of the molecule on RBC metabolism was assessed by measuring anion flux kinetics, ATP production, and phosphatase activity. Finally, an evaluation of the potential protection against different stressors was performed. Our results show no detrimental effects of EC on RBCs (no change in hemolysis or methemoglobin and no caspase 3 activation recorded); rather, a protective effect was recorded given the reduction in hemolysis induced by hydrogen peroxide treatment and temperature increase. The increase in anion exchange and intracellular ATP values, with the inhibition of phosphatase PTP1B activity, highlights several biochemical alterations induced by EC. The present results contribute to clarifying the influence of EC on RBCs, confirming the beneficial effects of catechins.

Keywords: Band-3 protein; anion exchange; antioxidant systems; epicatechin; erythrocyte metabolism; red blood cells.

Figure 1

Epicatechin ((2R,3R)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-2H-chromene-3,5,7-triol) chemical structure.

Figure 2

Effect of epicatechin 50 μM on erythrocyte membrane fragility after incubation with different NaCl concentrations (from 0.34 to 0.44%). RBCs in the absence of EC (white boxes), pretreated with 50 μM EC for 5 min (light blue boxes) and for 90 min (blue boxes). Hemolysis was measured by spectrophotometric absorbance of the supernatant at 576 nm. * p < 0.05 vs. ctrl, *** p < 0.001 vs. ctrl.

Figure 3

Protective effect against temperature increase exerted by EC on the erythrocyte membrane. RBCs in the absence of EC (light blue boxes), RBCs pretreated with 50 μM of EC for 30 min (blue boxes), RBCs pretreated with 100 μM of EC for 30 min (dark blue boxes). Absorbance value (at 576 nm). * p < 0.01 vs. ctrl, *** p < 0.001 vs. ctrl.

Figure 4

Protective effect of EC against H₂O₂ exposure for 2 and 14 h. RBCs in the absence of EC (white boxes); RBCs pretreated with EC 50 μM for 30 min and then exposed to 300 mM H₂O₂ for 2 h (red boxes); RBCs pretreated with EC 50 μM for 30 min and then exposed to 300 mM H₂O₂ for 14 h. Hemolysis was measured by spectrophotometric absorbance of the supernatant at 576 nm. *** p < 0.01 vs. ctrl, §§ p < 0.01 vs. H₂O₂ 300 mM, §§§ p < 0.001 vs. H₂O₂ 300 mM.

Figure 5

The figure shows the effects exerted on the RBC membrane by EC (50 μM) and H₂O₂ (50 mM) after 6 h of incubation. In detail, in the control (ctrl; RBC in the absence of EC and H₂O₂) it is possible to observe the classic biconcave disk shape typical of the cells. EC 50 μM (effect of EC on the RBC membrane): RBCs show a slight and negligible change in their structure compared to the control; H₂O₂ 50 mM (RBCs treated with H₂O₂ for 6 h): shrinkage and alteration of the erythrocyte membrane structure due to the action of the stressor agent can be observed; EC 50 μM + H₂O₂ 50 mM (RBCs pretreated with EC for 30 min, then incubation with H₂O₂): EC partially inhibited RBC alteration.

Figure 6

Sulphate flux measured in RBCs in the absence (white boxes) and presence of 50 μM EC (blue boxes), at temperature values of 20 °C (Section “(A)”), 30 °C (section “(B)”), and 40 °C (section “(C)”), respectively. ** p < 0.01 vs. ctrl, *** p < 0.001 vs. ctrl.

Figure 7

Section (A) shows the spectra of Hb from 2.5 μM to 10 μM; section (B) shows the spectrum of 50 μM epicatechin; section (C) shows the spectra resulting from titration of EC (50 μM) with Hb until the concentration of 10 μM is reached. As section (C) of the panel shows, there appears to be no interaction between EC and the protein given the absence of isosbestic dots and the lack of shift in the absorption bands.

Figure 8

Caspase 3 activity in RBCs in the absence (white boxes) and presence of 50 μM EC (blue boxes) and in the presence of 100 μM t-BHT (light blue), respectively. ** p < 0.01 vs. ctrl, *** p < 0.001 vs. ctrl.

Figure 9

Cytofluorometry analysis of red blood cells incubated overnight in the absence (section (A)) and presence of 50 μM EC (section (B)).

Figure 10

PTP-1B activity in human RBCs, in the absence (white box) and presence of 25–50–75–100 μM of EC (blue boxes), compared with the effect of 3 mM OV (red box). *** p < 0.001 vs. ctrl, §§§ p < 0.001 vs. EC 75 μM, ^ p < 0.05 vs. EC 25 μM, ^^^ p < 0.001 vs. EC 25 μM, °° p < 0.01 vs. EC 50 μM, °°° p < 0.001 vs. EC 50 μM.

Figure 11

Effect of 50 and 100 μM epicatechin (blue boxes) on intracellular (A) and extracellular (B) ATP values in RBCs incubated for 30 min at 37 °C. RBCs in the absence of EC (white box). * p < 0.05 vs. ctrl, *** p < 0.001 vs. ctrl, §§§ p < 0.001 vs. EC 50 μM.