Increased intracellular generation of reactive oxygen species in mononuclear leukocytes from patients with diabetes mellitus type 2.

Since increased cellular production of reactive oxygen species is a source of oxidative stress and thus may contribute to the development of diabetic complications, the baseline and stimulated concentrations of intracellular reactive oxygen species were measured in 16 patients with diabetes mellitus type 2 and 19 healthy control subjects. Reactive oxygen species and cytosolic calcium were monitored spectrophotometrically using dihydrorhodamine-123 and fura-2, respectively, in a suspension of mononuclear leukocytes. Measurements were made in the presence or absence of superoxide dismutase, sodium azide, genistein, or bisindolylmaleimide 1. Baseline reactive oxygen species concentrations were significantly higher in diabetic patients compared with control (p<0.001). Activation of mononuclear leukocytes by formyl-Met-Leu-Phenylalanine and phytohemagglutinin significantly increased reactive oxygen species in diabetic patients compared with control (p<0.05). The formyl-Met-Leu-Phenylalanine-induced increases were unchanged in the presence of superoxide dismutase, but dropped significantly in the presence of sodium azide by 80% and 73% in diabetic patients and control, respectively (each p<0.01). The formyl-Met-Leu-Phenylalanine-induced responses were significantly inhibited by genistein and bisindolylmaleimide 1 (p<0.01), suggesting the involvement of tyrosine kinase and protein kinase C. Resting calcium (p<0.05) and stimulated calcium were significantly greater in diabetic patients than in control. The results show that patients with diabetes mellitus type 2 generate increased reactive oxygen species under stimulated conditions, suggesting increased risk for oxidative stress and associated complications.