Increased erythrocytes by-products of arginine catabolism are associated with hyperglycemia and could be involved in the pathogenesis of type 2 diabetes mellitus.

Diabetes mellitus (DM) is a worldwide disease characterized by metabolic disturbances, frequently associated with high risk of atherosclerosis and renal and nervous system damage. Here, we assessed whether metabolites reflecting oxidative redox state, arginine and nitric oxide metabolism, are differentially distributed between serum and red blood cells (RBC), and whether significant metabolism of arginine exists in RBC. In 90 patients with type 2 DM without regular treatment for diabetes and 90 healthy controls, paired by age and gender, we measured serum and RBC levels of malondialdehyde (MDA), nitrites, ornithine, citrulline, and urea. In isolated RBC, metabolism of L-[(14)C]-arginine was also determined. In both groups, nitrites were equally distributed in serum and RBC; citrulline predominated in serum, whereas urea, arginine, and ornithine were found mainly in RBC. DM patients showed hyperglycemia and increased blood HbA1C, and increased levels of these metabolites, except for arginine, significantly correlating with blood glucose levels. RBC were observed to be capable of catabolizing arginine to ornithine, citrulline and urea, which was increased in RBC from DM patients, and correlated with an increased affinity for arginine in the activities of putative RBC arginase (Km = 0.23±0.06 vs. 0.50±0.13 mM, in controls) and nitric oxide synthase (Km = 0.28±0.06 vs. 0.43±0.09 mM, in controls). In conclusion, our results suggest that DM alters metabolite distribution between serum and RBC, demonstrating that RBC regulate serum levels of metabolites which affect nitrogen metabolism, not only by transporting them but also by metabolizing amino acids such as arginine. Moreover, we confirmed that urea can be produced also by human RBC besides hepatocytes, being much more evident in RBC from patients with type 2 DM. These events are probably involved in the specific physiopathology of this disease, i.e., endothelial damage and dysfunction

Serum and RBC levels of MDA, arginine, and nitrites from blood obtained from control subjects and patients with type 2 diabetes mellitus.

<p>The results are expressed as the mean ± SD for levels of blood MDA (panel A), arginine (panel B), or blood nitrites (panel C) in RBC samples from control healthy volunteers (n = 90) and in patients with type 2 diabetes mellitus (n = 90). Symbols indicating each experimental group at the top of the panels. Statistics: *p<0.01 against control values (healthy subjects).</p

Formation and release of (¹⁴C)-citrulline, (¹⁴C)-ornithine and of (¹⁴C)-urea after incubation with (¹⁴C)-arginine in RBC from control subjects and patients with type 2 diabetes mellitus.

<p>The results are expressed as the mean ± SD for levels of produced and released, in nmols per mL, of radio-labeled citrulline, ornithine, and urea, after incubation with (¹⁴C)-arginine, quantified in supernatants, or in the RBC pellets obtained from control healthy volunteers (n = 30) and in patients with type 2 diabetes mellitus (n = 30). Statistics: *p<0.01 as compared to healthy controls.</p

Serum and RBC levels of citrulline, ornithine, and urea from blood obtained from control subjects and patients with type 2 diabetes mellitus.

<p>The results are expressed as the mean ± SD for levels of blood citrulline (panel A), ornithine (panel B), or blood urea (panel C) in RBC samples from control healthy volunteers (n = 90) and in patients with type 2 diabetes mellitus (n = 90). Symbols indicating each experimental group at the top of the panels. Statistics as indicated in Fig. 1.</p

Overview of the possible metabolic pathways for arginine in RBC and their interaction with endothelial cells.

<p>Enzymatic routes that can probably directly use or produce arginine, ornithine, urea, or citrulline in the RBC. Key to abbreviations: Arg: arginase; eNOS: endothelial nitric oxide synthase; NO: nitric oxide; NOS: nitric oxide synthase, and OCT: ornithine carbamoyl transferase.</p

Effect of added arginine on its own release and that of MDA, and rate of RBC hemolysis from control subjects and patients with type 2 diabetes mellitus.

<p>The results are expressed as the mean ± SD for levels of released arginine (panel A) and MDA (panel B), in RBC samples from control healthy volunteers (n = 30) and in patients with type 2 diabetes mellitus (n = 30). Panel C shows the rate of hemolysis obtained in each preparation. Start of the incubation at 37°C in the presence of increasing arginine concentrations is indicated by the upper arrow. Symbols indicating each experimental group at the top of the panels. Statistics: *p<0.01 against control basal values (zero) and **p<0.01 vs. the basal metabolite value (zero) in samples from DM patients.</p

Clinical parameters of control subjects and patients with type 2 DM.

<p>The results are expressed as means ± SD. Abbreviations: BMI, body mass index; TG, triacylglycerols, and hs-CRP, high-sensitive C-reactive protein. Statistics: *p<0.01 as compared to healthy controls.</p

Arginine and its metabolite ratios in blood, serum, and RBC from control subjects and patients with type 2 DM.

<p>The results are expressed as means ± SD. Statistics: *p<0.01 as compared to healthy controls.</p

Pearson’s correlation coefficients (r) matrix for blood metabolites with serum glucose and RBC-glycated hemoglobin.

<p>Statistical significance: *p<0.01;</p>**<p>p<0.005.</p

Production and release of nitrites and urea from RBC from control subjects and patients with type 2 diabetes mellitus after incubation with arginine.

<p>The results are expressed as the mean ± SD for levels of produced and released nitrites (panel A) and urea (panel B), in RBC samples from control healthy volunteers (n = 30) and in patients with type 2 diabetes mellitus (n = 30). Start of the incubation at 37°C in the presence of increasing arginine concentrations is indicated by the upper arrow. Symbols for each experimental group at the top of the panels. Statistics as in Fig. 3.</p