Antioxidant vitamins C and E ameliorate hyperglycaemia-induced oxidative stress in coronary endothelial cells

Objective: Vitamins C and E have protective features in many disease states associated with enhanced oxidative stress. The aim of this study was to investigate whether vitamin(s) C and/or E modulate hyperglycaemia-induced oxidative stress by regulating enzymatic activities of prooxidant, i.e. NAD(P)H oxidase and/or antioxidant enzymes, namely endothelial nitric oxide synthase (eNOS), superoxide dismutase, catalase and glutathione peroxidase, using coronary microvascular endothelial cells (CMEC). Methods: CMEC were cultured under normal (5.5 mM) or high glucose (22 mM) concentrations for 7 days. The enzyme activities were determined by specific assays. The levels of O-2(-) and nitrite were measured by cytochrome c reduction and Griess assays respectively. Results: Hyperglycaemia did not alter eNOS activity or overall nitrite generation, an index of NO production. However, it increased NAD(P)H oxidase and antioxidant enzyme activities (p < 0.05). Specific inhibitors of NAD(P)H oxidase, i.e. pherylarsine oxide (0.1-3 mu M) and 4-(2-aminoethyl)benzenesulfonyl fluoride (5-100 mu M) and vitamins C and E (0.1-1 mu M) significantly reduced prooxidant and antioxidant enzyme activities in CMEC exposed to hyperglycaemia (p < 0.01). The differences in enzyme activities were independent of increases in osmolarity generated by high glucose levels as investigated by using equimolar concentrations of mannitol in parallel experiments. Conclusions: Vitamins C and E may protect CMEC against hyperglycaemia-induced oxidative stress by concomitantly regulating prooxidant and antioxidant enzyme activities.