Plasma concentrations of asymmetric dimethylarginine are increased in patients with type 2 diabetes mellitus

C heart disease (CHD), the major cause of morbidity and mortality in patients with type 2 diabetes, cannot be entirely explained by the presence of conventional risk factors. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase. Plasma ADMA concentrations have been shown to be elevated in animals and patients with hypercholesterolemia and atherosclerosis, and intracellular concentrations of ADMA are increased in regenerated endothelial cells after balloon injury in rabbits with alloxan-induced hyperglycemia. Thus, we hypothesized that increased plasma concentrations of ADMA, by inhibiting NO synthase, could play a role in the depressed endothelial cell-dependent vasodilator responses that have been described in patients with type 2 diabetes. Because endothelial dysfunction is an early event in the process of atherogenesis, we also hypothesized that plasma ADMA concentrations are elevated in hyperglycemic patients with type 2 diabetes, and could contribute to the accelerated atherogenesis in these persons. To begin evaluation of these hypotheses, we compared plasma ADMA concentrations in normal volunteers with those in patients with type 2 diabetes. • • • The study was approved by the Stanford Human Subjects Committee, and volunteers gave informed consent before entering the clinical research center. The study group consisted of 18 nondiabetic subjects and 16 patients with type 2 diabetes. No patient with type 2 diabetes had received any pharmacologic treatment for type 2 diabetes within the past 4 weeks, and had no apparent diabetic complication. All participants had a normal physical examination, blood count, and chemical screening battery. Blood was drawn after an overnight fast, and plasma frozen at –70°C until thawed for measurement of plasma glucose and lipid concentrations as described previously. Plasma concentrations of ADMA and symmetric dimethylarginine (SDMA) in plasma were measured by high-performance liquid chromatography with precolumn derivatization with o-phthaldialdehyde using a modification of a previously described method. ADMA concentrations were calculated by comparing the ADMA/homoarginine ratio with standards of known concentrations. The recovery rate for ADMA was 85% and the intrasample variation was 4%. The detection limit of the assay was 0.1 M. Results are expressed as mean SE, and the statistical significance of differences between the 2 groups estimated by Student’s t test. Results in Table 1 show that the 2 groups to be compared were similar in terms of age, gender distribution, body mass index, and total and low-density lipoprotein cholesterol concentrations. By selection, plasma glucose concentrations were significantly (p 0.001) higher in patients with type 2 diabetes. In addition, plasma triglyceride concentrations were higher (p 0.02) and high-density lipoprotein cholesterol concentrations lower (p 0.005) in patients with type 2 diabetes. Importantly, low-density lipoprotein cholesterol concentrations were similar in the first 2 groups. Figure 1 shows the individual and mean ADMA concentrations of the 2 groups, and it can be seen that the ADMA concentrations were significantly higher (p 0. 01) in patients with type 2 diabetes (1.59 0.22 vs 0.69 0.04 mol/L, p 0.001). Also, the separation of the 2 groups was almost complete, with 14 of the 16 patients with type 2 diabetes having ADMA concentrations higher than all 18 normal volunteers. From the Stanford University School of Medicine, Stanford, California. This report was supported by Research Grants HL-08506, HL-58638, and RR-00070 from the National Institutes of Health, Bethesda, Maryland. Dr. Reaven’s address is: Division of Cardiovascular Medicine, Falk CVRC, Stanford Medical Center, 300 Pasteur Drive, Stanford, California 94305. E-mail: greaven@cvmed.stanford.edu. Manuscript received April 30, 2001; revised manuscript received and accepted July 17, 2001. TABLE 1 Baseline Characteristics of Normal Volunteers and Patients With Type 2 Diabetes