d-Galactose induced early aging in human erythrocytes: Role of band 3 protein

Abstract

Aging, a time-dependent multifaceted process, affects both cell structure and function and involves oxidative stress as well as glycation. The present investigation focuses on the role of the band 3 protein (B3p), an anion exchanger essential to red cells homeostasis, in a d-galactose ( d-Gal)-induced aging model. Anion exchange capability, measured by the rate constant of SO₄²^- uptake through B3p, levels of lipid peroxidation, oxidation of membrane sulfhydryl groups, B3p expression, methemoglobin, glycated hemoglobin (Hb), and the reduced glutathione/oxidized glutathione ratio were determined after exposure of human erythrocytes to 25, 35, 50, and 100 mmol/L d-Gal for 24 h. Our results show that: (i) in vitro application of d-Gal is useful to model early aging in human erythrocytes; (ii) assessment of B3p ion transport function is a sensitive tool to monitor aging development; (iii) d-Gal leads to Hb glycation and produces substantial changes on the endogenous antioxidant system; (iv) the impact of aging on B3p function proceeds through steps, first involving Hb glycation and then oxidative events at the membrane level. These findings offer a useful tool to understand the mechanisms of aging in human erythrocytes and propose B3p as a possible target for new therapeutic strategies to counteract age-related disturbances.

Keywords: aging; anion exchange; band 3 protein; d-galactose; erythrocytes; glycation; oxidative stress.

Figure 1

Time course of SO₄ ²⁻ uptake in untreated (control, N = 12) and d‐Gal (d‐galactose)‐treated erythrocytes (N = 7) and in the presence of 10 µmol/L DIDS (4,4ʹ‐diisothiocyanatostilbene‐2,2ʹ‐disulfonate) (n = 5). *p < 0.05 and ***p < 0.001 versus control, as determined by one‐way analysis of variance followed by Dunnett's multiple comparisons posthoc test

Figure 2

TBARS levels (µ mmol/L) in untreated erythrocytes (control) and in erythrocytes treated for 24 h with d‐Gal or alternatively with 50 mmol/L 3‐AT (preincubation for 10 min) + 10 mmol/L H₂O₂ for 1 h. d‐Gal, d‐Galactose; ns, not significant versus untreated; TBARS, thiobarbituric‐acid‐reactive substances. *p < 0.05 and ***p < 0.001 versus control, as determined by one‐way analysis of variance followed by Dunnett's multiple comparisons posthoc test (N = 7)

Figure 3

Membrane sulfhydryl group content (µmol TNB/µg protein) in untreated erythrocytes (control) and in erythrocytes treated with d‐Gal for 24 h or NEM for 1 h. d‐Gal, d‐Galactose; NEM, N‐ethylmaleimide; ns, not significant versus control; TNB, 3‐thio‐2‐nitro‐benzoic acid. ***p < 0.001 versus control, as determined by one‐way analysis of variance followed by Dunnett's multiple comparisons posthoc test (N = 6)

Figure 4

Band 3 protein expression levels measured in untreated (control) and in 25, 35, 50, and 100 mol/L d‐Gal (d‐galactose)‐treated (24 h) erythrocytes, detected by Western blot analysis. ns, not significant versus untreated (control), as determined by one‐way analysis of variance followed by Dunnett's multiple comparisons posthoc test (N = 5)

Figure 5

Methemoglobin (MetHb) levels (%) measured in untreated erythrocytes (control) and in erythrocytes treated with d‐Gal (d‐galactose) for 24 h or 4 mmol/L NaNO₂ for 1 h. ns, not significant versus control and 4 mmol/L NaNO₂. ***p < 0.001 versus control as determined by one‐way analysis of variance followed by Dunnett's multiple comparisons posthoc test (N = 8)

Figure 6

Glycated hemoglobin content (% A1c) in erythrocytes incubated for 24 h with different d‐Gal (d‐galactose) concentrations (25, 35, 50, and 100 mmol/L). **p < 0.01 and ***p < 0.001 versus untreated (control), as determined by one‐way analysis of variuance followed by Dunnett's multiple comparisons posthoc test (N = 10)

Figure 7

Estimation of the GSH/GSSG ratio measured in erythrocytes incubated for 24 h with different concentrations (25, 35, 50, and 100 mmol/L) of d‐Gal (d‐galactose). GSH, reduced glutathione; GSSG, oxidized glutathione. ***p < 0.001 versus untreated (control) erythrocytes, determined by one‐way analysis of variance followed by Dunnett's multiple comparisons posthoc test (N = 8)