Determination of the antioxidant status of plasma from type 2 diabetic patients.

An increase in oxidizing response above a certain threshold produces, in the absence of a concomitant rise in antioxidant/reducing response, oxidative stress that is associated with complications in diabetes. A simple technique involving reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye has been developed in order to determine quantitatively the antioxidant status of plasma. MTT (50microL; 5.0mg/mL in PBS) was incubated with plasma (100microL) in PBS for 30, 60 or 120min at 37 degrees C, the reaction terminated by addition of 1.0mL of 0.04M hydrochloric acid in isopropanol and the absorbance measured at 570nm. The modulation by plasma of the generation of reactive oxygen species (ROS) in 12,13-phorbol dibutyrate (PDB)-stimulated granulocytes was evaluated using a chemiluminescence luminol-dependent assay. Plasma from healthy subjects (n=15) showed significantly higher antioxidant status (p<0.05) over all time periods studied compared with plasma from diabetic patients (n=27). MTT was directly reduced by plasma although platelets were not involved. Moreover, the reduction of MTT by bovine serum albumin at levels equivalent to the concentration of human serum albumin in plasma was much lower. The antioxidant status of plasma, as evaluated by MTT dye reduction, may reflect an antioxidant response since ROS generation in PDB-stimulated granulocytes was rapidly down-regulated by the presence of plasma (3.3-fold in diabetic patients and 5.8-fold in healthy subjects) confirming the lower antioxidant activity of plasma from diabetic patients. The results demonstrate that extracellular reduction of MTT by plasma may occur via enzymatic and non-enzymatic processes.