Monitoring antioxidant effects using methaemoglobin formation in diabetic erythrocytes †

Abstract

Deficiencies in the reducing power of diabetic erythrocytes can be studied using the sulphone metabolite, monoacetyl dapsone hydroxylamine (MADDS-NHOH) to generate methaemoglobin, which is a form of oxidative stress. The effects of three antioxidants, dyhydrolipoic acid (DHLA), lipoic acid and ascorbate were compared using this method in erythrocytes of type 1 diabetic patients. During 2-hour incubations, DHLA and lipoic acid significantly reduced MADDS-NHOH-mediated methaemoglobin formation to the same extent in diabetic and non-diabetic erythrocytes. In contrast, ascorbate markedly increased MADDS-NHOH-mediated methaemoglobin formation at all time points up to 2 hours for diabetic cells and 15—60 minutes in non-diabetic erythrocytes. Ascorbate abolished the deficiency in methaemoglobin sensitivity normally seen in diabetic erythrocytes compared with non-diabetic erythrocytes. During a 1-hour pre-incubation period, neither DHLA, lipoic acid nor ascorbate significantly altered total thiol concentrations. After a 2-hour incubation with MADDS-NHOH, thiol concentrations did not significantly change in the non-diabetic erythrocytes, but they fell significantly in the diabetic cells. Diabetic and non-diabetic erythrocytes pre-incubated with ascorbate, DHLA and lipoic acid showed no changes in thiol levels in the presence of MADDS-NHOH. In summary, ascorbate, DHLA and lipoic acid showed contrasting effects on methaemoglobin generation, although they each abolished the diabetic erythrocytic deficit in total thiol status caused by hydroxylamine-mediated methaemoglobin formation. This work provides evidence for the potential future use of antioxidant supplements in diabetic management.