All patients passed urine immediately before 8 p.m., discarded this sample and recorded the time. All the urine passed until 6 a.m. was collected into containers without a preservative. The urine volume was recorded, and aliquots were stored in glass tubes at - 70° Celsius until analysis. Fasting blood samples were also obtained. The urinary albumin concentration was estimated by solid-phase competitive chemiluminescent enzyme immunoassay (Immulite 1000 Systems, DPC Medlab, Los Angeles, CA, sensitivity of 0.5 ug/ml) with intra-assay and inter-assay coefficients of variation of 4.4% and 6.1%, respectively. Based on the AER in at least two out of three overnight urine specimens, patients were divided into two groups: Microalbuminuria (AER ≥ 20 ug/min and < 200 ug/min in two out of three overnight urine specimens) and normoalbuminuria (AER < 20 ug/min in two out of three overnight urine specimens). Each urine specimen was tested for bacteriuria, and when the latter was present (> 105/mm3), it was discarded.
Blood pressure was measured by the same observer 3 times after a 5-min rest in the supine position using a standard mercury sphygmomanometer. Diastolic blood pressure (dBP) was recorded at the disappearance of Korotkoff sounds (phase 5). The mean of the measurements of systolic and diastolic blood pressure was used. Hypertension was defined as systolic blood pressure (sBP) > 140 mmHg and/or dBP > 90 mm Hg [2
] or any value in patients under antihypertensive treatment. Blood samples were drawn in the morning between 7:30 and 8:30 a.m., after the last urine collection and an overnight fast. After centrifugation at 2500 × g for 15 min at room temperature (19°Celsius), aliquots of plasma and sera were stored at -70° Celsius until analysis. Serum CRP (mg/dl) was measured using a highly sensitive immunonephelometry assay (Behring Nephelometer, Germany) with a detection limit of 0.01 mg/dl and with intra-assay and inter-assay CV of 1% and 5.3%, respectively. Vitamin B12 was determined by an enzyme immunoassay method (reference value: 180-914 pg/mL).
Creatinine (mg/dl), FBG (mg/dl), postprandial plasma glucose (PPG, mg/dl), triglyceride (mg/dl), HDL cholesterol (mg/dl) and total cholesterol (mg/dl) levels were measured using an auto-analyzer (Cobas-Mira Roche) by enzymatic techniques. Postprandial glucose was measured two hours after the usual breakfast. Patients were instructed to take their usual medication.
Body mass index (BMI) was calculated by dividing the weight (kg) by height squared (m2). Anthropometric measurements were taken in a standing position after subjects removed their heavy clothes. WHR was measured twice, and the mean value was used for analysis. Waist and hip circumferences were measured on bare skin at the level of the umbilicus and iliac crest, respectively, during mid-respiration to the nearest 0.5 cm. The WHR was defined according to the average of two duplicate measurements.
Charts were reviewed to verify other relevant medical conditions, including micro and macrovascular complications of diabetes, and the medication history. A simple satisfaction questionnaire comprising four questions (Do you fell better with metforminXR? Do you think that diarrhea, nausea an vomiting are limiting symptoms to use Metformin ? Do you think that compliance is better using Metformin XR ? Do you want to get back to Metformin?) was applied to the patients at the end of the study.
The Student t- test was used for comparisons of continuous dependent (intra-group) and continuous independent variables (inter-group). Chi-Square with Yates correction and Fisher's exact test were used for comparison of categorical variables. These analyses were performed using the statistical package for the social sciences (SPSS, version 13.0). Values were expressed as mean (± SD) or median (minimum/maximum). A two-sided P value of less than 0.05 was considered to be significant.