Hemoglobin in the brain frontal lobe tissue of patients with Alzheimer’s disease is susceptible to reactive nitrogen species-mediated oxidative damage

Abstract

Brain inflammation in Alzheimer’s disease (AD) involves reactive nitrogen species (RNS) generation. Protein contents of 3-nitrotyrosine, a product of RNS generation, were assessed in frontal lobe brain homogenates from patients with AD, patients with vascular dementia (VaD) and non-dementia (ND) controls. Western blotting revealed a dominant 15 kDa nitrated protein band in both dementia (AD/VaD) and ND frontal lobe brain tissue. Surprisingly, this protein band was identified by mass spectrometry as hemoglobin, an erythrocytic protein. The same band stained positively when western blotted using an anti-hemoglobin antibody. On western blots, the median (IQR) normalized staining intensity for 3-nitrotyrosine in hemoglobin was increased in both AD [1.71 (1.20–3.05) AU] and VaD [1.50 (0.59–3.04) AU] brain tissue compared to ND controls [0.41 (0.09–0.75) AU] (Mann-Whitney U test: AD v ND, P < 0.0005; VaD v ND, P < 0.05; n = 11). The median normalized staining of the nitrated hemoglobin band was higher in advanced AD patients compared with early-stage AD (P < 0.005). The median brain tissue NO₂⁻ levels (nmol/mg protein) were significantly higher in AD samples than in ND controls (P < 0.05). Image analysis of western blots of lysates from peripheral blood erythrocytes suggested that hemoglobin nitration was increased in AD compared to ND (P < 0.05; n = 4 in each group). Total protein-associated 3-nitrotyrosine was measured by an electrochemiluminescence-based immunosorbent assay, but showed no statistically significant differences between AD, VaD and ND. Females showed larger increases in hemoglobin nitration and NO₂⁻ levels between disease and control groups compared to males, although the group sizes in these sub-analyses were small. In conclusion, the extent of hemoglobin nitration was increased in AD and VaD brain frontal lobe tissue compared with ND. We propose that reactive nitrogen species-mediated damage to hemoglobin may be involved in the pathogenesis of AD.