Evaluation of oxidative stress markers in pathogenesis of diabetic neuropathy

Abstract

Experimental evidences suggest that hyperglycaemia-induced overproduction of reactive oxygen species and subsequent damage to proteins, lipids and DNA may play a key role in the development of distal symmetric polyneuropathy (DSPN)-the most common complication of diabetes mellitus. The study population consisted of 51 individuals aged 52-82 years classified into 3 groups: 16 patients diagnosed with type 2 diabetes mellitus (T2DM) with DSPN, 16 T2DM patients without DSPN and 19 control subjects without diabetes and neuropathy. The study was conducted to determine the activity of antioxidant enzymes: catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPX) and total antioxidant status (TAS) in the examined groups. An alkaline comet assay was used to determine the extent of DNA damage of oxidized purines as glicosylo-formamidoglicosylase (Fpg) sites, and oxidized pyrimidines as endonuclease III (Nth) sites. A significant decrease of SOD (P < 0.05), GPX (P < 0.05) and nonsignificant decrease of CAT (P > 0.05), and TAS status (P > 0.05) were seen in T2DM patients with neuropathy compared to T2DM patients as well as controls. T2DM patients with or without neuropathy revealed significantly lower (P < 0.05) plasma concentration of nitrous oxide compared to the control subjects. Endogenous level of oxidative DNA damage in T2DM patients with DSPN was significantly higher compared both to the controls and T2DM patients without DSPN (P < 0.001). Moreover, lymphocytes isolated from T2DM patients with DSPN were more susceptible to oxidative DNA lesions induced by hydrogen peroxide than from T2DM patients without DSPN (P < 0.001). Our results confirm hypothesis that oxidative stress may play a substantial role in the development and progression of diabetic distal symmetric polyneuropathy.

Fig. 1

Mean activity of superoxide dismutase (SOD) calculated in adrenaline units (U/g Hb/100 ml) (a), catalase (CAT) calculated in Bergmeyer units (BU/g Hb) (b), glutathione peroxidase calculated in enzymatic units (U/g Hb) (c) and nitric oxide concentration calculated as (μmol/l) (d) measured in T2DM patients with DSPN (T2DM + DSPN), T2DM patients without DSPN (T2DM) and healthy subjects (HS). Each data point represents the mean ± SD. * P < 0.05 as compared with healthy subjects

Fig. 2

Total antioxidant status (TAS) in T2DM patients with DSPN (T2DM + DSPN), T2DM patients without DSPN (T2DM) and healthy subjects (HS) calculated as concentration of antioxidants (mmol/l). Each data point represents the mean ± SD. * P < 0.05 as compared with healthy subjects. ^# P < 0.05 comparison between T2DM patients and T2DM patients with DSPN

Fig. 3

The level of endogenous (a) and H₂O₂-induced (b) DNA damage in lymphocytes of T2DM with DSPN (T2DM + DSPN), T2DM patients without DSPN (T2DM) and healthy subjects (HS). The cells were treated or not with 10 μM H₂O₂ for 10 min at 4 °C with subsequent treatment with endonuclease III (Nth) or formamidopyrimidine-DNA glycosylase (Fpg) at 1 μg/mL. The black bars (control) present DNA strand breaks and alkaline labile sites, the light grey bars (Fpg) present oxidized purines and the dark grey bars (Nth) present oxidized pyrimidines. The DNA damage was measured as the percentage of tail DNA in the alkaline comet assay. Each data point represents the mean ± SEM.*** P < 0.001, ** P < 0.01, * P < 0.05 as compared with healthy subjects. ^# P < 0.05, ^## P < 0.01, ^### P < 0.05 comparison between T2DM patients and T2DM diabetic patients with DSPN