CLINICS 2012;67(7):711-717 their total calories as carbohydrates, 20% as protein, and 30% as fat, and the meals were provided by the Metabolic Unit of the Hospital das Cl´ınicas. Cardiovascular evaluation The study was performed in a laboratory setting at a temperature of 23˚C and in low luminosity, with the patients in the supine position. A catheter was inserted into the forearm vein 30 minutes before blood sample collection for catecholamine, insulin, sodium, and potassium determi- nations and maintained with a saline infusion. During the study, the heart rate and systolic, diastolic, and mean blood pressure were registered using a non- invasive, automatic oscillometric device (Dinamap 1486, Critikon, Inc., Tampa, FL, USA). The images for flow velocity and diameters of the arteries were first established after a 10-minute equilibration period using a transducer (Apogee-800 Plus, ATL Inc., Bothell, WA, USA). Intima- media thickness (IMT), the compliance and distensibility of the carotid artery, and the total and systolic blood flow indices (TFI and SFI) were obtained. Flow-mediated vasodilation of the brachial artery was measured using a method reported elsewhere (20). A cuff placed on the left forearm was inflated to 200 mm Hg for 5 minutes. Imaging of the artery was performed before cuff inflation (baseline; B) and at 60 seconds after cuff deflation (reactive hyperemia; RH) to obtain measurements of flow velocity and the diameter of the artery. Fifteen minutes after acquisition of the post-occlusion image, the baseline image was reobtained (rebase; RE), after which nitroglycerin was administered sublingually; 3 and 5 minutes later, another image was acquired (N3 and N5, respectively) to establish endothe- lium-independent vasodilation. The total and systolic flow indices (TFI and SFI) of the brachial artery were also calculated based on these images. Biochemical and hormonal analyses Glucose was determined by the glucose oxidase method (Labtest, Sa˜o Paulo, Brazil) (21), and HbA1 (normal values: 4 to 8.5%) was determined by ionic chromatography (Labtest, Sa˜o Paulo, Brazil) (22). Total cholesterol was measured by employing the cholesterol oxidase/peroxidase method; HDL cholesterol was separated using the phosphotungstic acid/ Mg2+ method and measured using the oxidase/peroxidase method, while triglycerides were measured by the lipase/ glycerol kinase method (Labtest, Sao Paulo, Brazil) (23). LDL was estimated using the Friedewald equation (LDL choles- terol = total cholesterol minus HDL cholesterol minus 0.2 6 triglycerides). The intra-assay and inter-assay coefficients of variation (CVs) for the glucose and lipid determinations were ,3% and 0%, respectively. Insulin, proinsulin and glucagon were quantified by a double-antibody radioimmunoassay (Linco Research, St. Louis, MO, USA) (24). Catecholamines were measured by high-performance liquid chromatography (25). The intra-assay and inter-assay CVs for the hormonal analyses were 6.8% and 9.6% for insulin, 4.4% and 6.5% for glucagon, 5.5% and 6.8% for catecholamines, and 5% and 5.3% for proinsulin, respectively. Hemostatic factors were measured using the same assay. Plasminogen was measured by chromogenic assay (Plasminogen Accucolor TM Sigma Diagnostic, ST Louis, MO, USA), and the intra-assay coefficient of variation was ,3.0%. Fibrinogen was determined by the CLAUSS method (26) with Fibriquick Assay (Sigma Diagnostics, ST Louis, USA). The 713 Metabolic and vascular effects of antidiabetic drugs Machado HA et al. intra-assay CV was 8%. Platelet aggregation was performed using a method described by Born (27). The activities of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1) were determined by quantitative assays (ChromolizeTM t-PA and ChromolizeTM PAI-1, respectively, Biopool, Umea, Sweden); the intra-assay CVs were 3.9% for t- PA and 3.7% for PAI-1. PAI-1 and t-PA antigens were determined by Imulyse and Tint-Elize (Biopool, Umea, Sweden), respectively. The intra-assay CVs were 5% for PAI-1 and 5.5% for t-PA. All of the analyses were performed in duplicate. Statistical methods Numerical data are reported as means and standard deviations, and nominal data are reported as proportions. Differences (95% CI) between the treatment groups were initially tested for treatment-time interaction (28) and then compared by an analysis of variance for repeated measure- ments and a Tukey’s post-test or Student’s two-tailed test, with p,0.05 considered statistically significant. RESULTS A significant treatment period interaction effect was demonstrated for triglycerides, VLDL cholesterol, plasmi- nogen and norepinephrine levels; hence, only the values from the first treatment period were analyzed. Anthropometric, biochemical, hormonal and hemostatic factor measurements Weight, waist-to-hip ratio, and systolic and diastolic blood pressure did not change after glimepiride or metformin treatment (Table 1). Fasting plasma HbA1 (p = 0.000009) and glucose levels (p = 0.00009) decreased by equal amounts in both treatment groups (Table 2). Fasting insulin levels were greater in the G group (p = 0.009) compared with the M group. Similar decreases in VLDL cholesterol (p = 0.007), triglycerides (p = 0.023) and norepinephrine (p = 0.042) levels were obtained in both groups. There was an increase in plasminogen levels (p = 0.025) after the initial four months of M (118.2¡8.26142.4¡32.0) or G (128.4¡8.66130.2¡8.1) therapy, which although persistent did not remain significant when the groups switched to the alternative drugs. LDL and HDL cholesterol and epinephrine levels were unchanged. Both therapies decreased t-PA activity (p = 0.024). There was no significant effect of either of the therapies on the other hemostatic factors measured (PAI-1 antigen and activity, fibrinogen levels and platelet aggregation — data not shown). 12-h metabolic profile Only the areas under the curve during glimepiride and metformin therapy were analyzed (Figure 1). The 12-h integrated areas for glucose (M:87.7¡11.03 vs. G:104.61¡ 36.63 mmol/L/h) and triglycerides (M:20.47¡9.68 vs. G:22.4 ¡11,03 mmol/L/h) were similar for both therapies. Treatment with metformin was associated with higher glucagon (M:1361.69¡473.25 vs. G:1044.22¡326.90 ng/L/h; p = 0.0046) and lower insulin-integrated (M:1076.61¡389.02 vs. G:1718.69¡837.03 pmol/L/h — p = 0.02) and proinsulin- integrated (M:565.38¡279.11 vs. G:834.71¡299.96 pmol/L/ h — p = 0.0016) areas compared with the G group (Figure 1). The proinsulin-to-insulin molar ratios during the M (0.58¡0.32) and G (0.60¡0.37) therapies did not differ.