Glucose analyser

All laboratory measurements were performed after at least 12 h of fasting. Glycaemia was determined by the glucose oxidase method (glucose analyser, BeckmanCoulter, Milan). Blood levels of total cholesterol, low‐density lipoprotein (LDL) cholesterol, high‐density lipoprotein (HDL) cholesterol and triglycerides were analysed by enzymatic methods (Roche Diagnostics GmbH, Mannheim, Germany). Creatinine levels were measured using the Jaffe method. The estimation of glomerular filtration rate (eGFR) was based on the new CKD‐EPI (Chronic Kidney Disease Epidemiology Collaboration) equation.²¹ Serum uric acid (UA) levels were assessed using URICASE/POD method (Boehringer Mannheim, Mannheim, Germany). The high‐sensitivity C‐reactive protein (hs‐CRP) was quantified by the immunoturbidimetric method automated system (Cardio Phase hs‐CRP, Milan, Italy).

Oral glucose tolerance test (OGTT) and intravenous glucose tolerance test (IVGTT) with frequent blood sampling (FSIGTT) were conducted on separate occasions in all subjects. Tests were performed after 3 days on a 300 g per day carbohydrate diet and after an overweight fast of 10 hr. Blood samples for plasma glucose and plasma insulin were drawn at baseline and every 30 min for 2 hr, after a 75-g glucose load. The modified IVGTT (FISGTT) was also performed after overnight fasting, according to the previously published procedures [11 (link), 12 (link)]. After an overnight fast, catheters were placed in a forearm vein and a hand vein of the contralateral arm. Basal samples were collected for glucose and insulin at −15, −10, −5, and −1 min. Glucose (300 mg/kg) was injected as a bolus at time 0 over 1 min and flushed with saline to ensure complete delivery. After 20 min, 0.05 IU/kg of short-acting insulin (Actrapid HM, NovoNordisk) was injected. Blood samples were drawn at 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 19, 22, 23, 24, 25, 27, 30, 40, 50, 60, 70, 90, 100, 120, 140, 160, and 180 min for measuring plasma glucose and insulin levels. Glucose was measured in a Beckman glucose analyser, using the glucose oxidase method. Insulin (mU/L) and testosterone (nmol/L) (basal) were measured by radioimmunoassay (RiA INEP, Zemun).

All laboratory measurements were performed after 12 h of fasting. Plasma glucose was determined immediately by the glucose oxidation method [Glucose analyser, Beckman Coulter, Milan; intra-assay coefficient of variation (CV) 2.2%, inter-assay CV 3.8%]. Total, LDL-, and HDL-cholesterol and triglyceride concentrations were measured by enzymatic methods (Roche Diagnostics GmbH, Mannheim, Germany). Uric acid and creatinine were measured using the Jaffe methodology. Values of estimated glomerular filtration rate (e-GFR) (mL/min/1.73 m²) were calculated by using the CKD-EPI equation [43 (link)]. High-sensitivity C-reactive protein (hs-CRP) levels were measured with an automated instrument (Cardio—Phase hs-CRP, Siemens Healthcare, Milano, Italy). Fibrinogen was dosed with a coagulation method on the instrument BCSXP. The complete blood count was performed by flux cytometry.