Sixty seven patients with T2D and PAD were enrolled in a cohort prospective study between 2005 and 2008. Five patients were excluded from the study, while the remaining 62 patients that met the inclusion criteria were followed-up for 36 months. The study was conducted at the vascular laboratory at University Cardiology Clinic Skopje. The study was carried out according to the Helsinki declaration and was approved by the University Clinic Ethics Committee, Skopje.
Type 2 diabetes was defined based on the criteria of the International Diabetes Federation. Patients with PAD, stage Fontaine I, with an established value of ABI < 0. 9 met the inclusion criteria for the study. Those with advanced PAD stage and with high, pathological ABI values, as well with acute inflammatory state (diabetic foot infection, cold, pneumonia) were excluded from the study. In all patients, we measured at baseline and after 36 months, at the completion of the study the ABI value, using a continuous wave Doppler (Hunleigh Healtcare, Cardiff, UK) to determine the lowest ABI values (ratio of ankle to brachial pressure). ABI was measured by vascular physicians with the same person being involved throughout the study. Inter-observer variability was up to 6%, measured previously. Mean values were calculated from the average of two measurements. Following the endpoint measurement of the ABI, a change in ABI was also calculated.
Laboratory tests were conducted at the University Institute for Clinical Biochemistry. Fibrinogen and C-RP were determined using the BNII nephelometer (N High-Sensitivity C-RP and N Antiserum to Human Fibrinogen; Dade Behring). Serum levels of the following laboratory tests were measured at baseline and at 36 weeks in all patients: total cholesterol (enzymatic methods-in the presence of cholesterol oxidize), triglycerides (in the presence of glycerokinase), and the HDL fraction by direct method.
Automatic analyzer Coba Integra 400/700 (ROCHE Diagnostics) was used. The LDL fraction was evaluated using the Friedewald formula. Non-HDL cholesterol was determinate as a value of total cholesterol minus HDL cholesterol. Fasting plasma glucose concentration was evaluated using the enzymatic-photometric method, in the presence of glucoso-dehydrogenase. All analyses were done according recommendation of International Federation of Clinical Chemistry and Laboratory Medicine.
Multiple linear regression analysis was used to define continuous variables with predictive value for the ABI, when adjusted for systolic blood pressure, BMI index, diabetes duration, age, blood glucose, plasma lipid levels (total, LDL-, HDL- cholesterol), C-RP and fibrinogen. The data is expressed as mean ± SD.