Prospective Analysis of L-arginine Dependent Nitric Oxide Production in the Blood of Patients with Type 2 Diabetes 5 Years after First Clinical Onset

Abstract

Objective: Our study is the first prospective investigation of the L-arginine (L-arg)–nitric oxide (NO) metabolic pathway in the erythrocytes (RBCs) and plasma of subjects with type 2 diabetes. Methods: RBCs and the plasma were collected from 10 patients with type 2 diabetes, at first clinical onset (baseline) and after 5 years of disease evolution (follow-up). L-arg content was assayed by competitive enzyme linked immunoassay. We measured arginase activity and nitrate/nitrite levels using spectrophotometry. Results: When compared with baseline, L-arg content decreased in RBCs and remained similar in the plasma (3.52 ± 0.2 vs. 3.10 ± 0.2 μmol/g Hb, P=0.022, and 0.28 ± 0.04 vs. 0.24 ± 0.03 μmol/g protein, P=ns respectively); NO production decreased either in RBCs (2.60 ± 0.23 vs. 1.64 ± 0.22 μM/g Hb, P=0.007; 3.69 ± 0.36 vs. 1.97 ± 0.25 μM/g Hb, P=0.001, for nitrite and nitrite+nitrate respectively) and the plasma (10.0 ± 0.45 vs. 2.51 ± 0.15 μM, and 31.5 ± 1.38 vs. 11.6 ± 0.83 μM, for nitrite and nitrite+nitrate respectively, P<0.01); arginase activity was lower in RBCs and increased in the plasma (6.15 ± 0.96 vs. 4.56 ± 0.5 μmol urea/g Hb/min, P=0.025, and 0.93 ± 0.05 vs. 1.33 ± 0.14 μM/min, P=0.016 respectively). Similarities were found in the absolute difference in values of arginase activity (3.46 ± 0.61 vs. 3.22 ± 1.1 μmol urea/gHb/min, in RBCs and 0.55 ± 0.14 vs. 0.68 ± 0.23 μM/min in plasma, P=ns) and NO production (1.77 ± 0.32 vs. 2.25 ± 0.4 μM/gHb in RBCs and 17.7 ± 2.4 vs. 21.2 ± 2.8 μM in plasma, P=ns) at baseline and follow-up in the blood of angiotensin converting enzyme inhibitors and statins treated vs. non treated subjects. Conclusions: L-arg NO metabolic pathway decreases in the RBCs of patients with type 2 diabetes 5 years after first clinical onset. The persistent decrease in RBC arginase activity 5 years after first clinical onset fails to compensate for sustained decrease of RBCs NO production in the diabetic environment. This is the first prospective study showing that the RBC L-arg and NO pool are depleted during progression of diabetes.