Glucose analyzer

Fasting blood (5 mL) was collected from all participants between the second and fifth day of menstruation, between 08:00 to 10:00, in plain redtop tubes to measure LH, FSH, 17β-estradiol (E2), leptin, insulin and lipid levels in the serum. 2 mL blood was drawn in fluoride tubes (grey top) for glucose estimation. On 20^th or 21^st day of the menstrual cycle, 5 mL blood samples were collected to measure 17OH-progesterone (P), testosterone (T), and sex-hormone binding globulin (SHBG). The collected blood samples were immediately centrifuged, and the serum was stored at -80 °C until further analysis. The LH, FSH, E2, P, T, SHBG, leptin and insulin levels were detected via double antibody sandwich enzyme-linked immunosorbent assay (ELISA). Glucose oxidase method (Beckman Glucose Analyzer (Fullerton, CA)) was used to measure plasma glucose levels. Fasting serum triglyceride (TG), total cholesterol (TC), highdensity lipoprotein (HDL), and low-density lipoprotein (LDL) were detected using enzymatic colourimetric in vitro test on automated clinical chemistry analyzers. HOMA-IR was calculated using the formula: glucose (mmol/L x insulin (mU/L)/22.5.

After 2-weeks wash-out period (e.g., the patients received no B blocker treatment for a period of 2 weeks prior to their inclusion, to eliminate previous B blocker effect before starting carvedilol/nebivolol), our study protocol started including the following both at the beginning of the study as well as at the study end: NYHA class assessment, vitals (pulse/minute, blood pressure), body mass index (BMI), EF% (by Simpson Method), 6-minute walk test [15 (link)], routine laboratory investigations, fasting glucose level, glycosylated hemoglobin (HbA1C%), and fasting insulin of the blood samples collected for measurement of glucose and insulin. Plasma glucose was measured by glucose oxidase method with a Beckman glucose analyzer, and plasma insulin concentrations were determined by radioimmunoassay.

All laboratory measurements were performed after fasting for 12 h. Plasma glucose was determined immediately by the glucose oxidation method [Glucose analyzer, Beckman Coulter, Milan; intra-assay coefficient of variation (CV) 2.2%, inter-assay CV 3.8%]. Total, low-density lipoprotein- (LDL), and high-density lipoprotein- (HDL) cholesterol and triglyceride concentrations were measured using enzymatic methods (Roche Diagnostics GmbH, Mannheim, Germany). Uric acid and creatinine were measured using the Jaffe methodology. Values of estimated glomerular filtration rate (eGFR) (ml/min/1.73 m²) were calculated using the CKD-EPI equation. We preferred this equation because it was developed from a much larger cohort of patients, including both normal individuals and those with chronic kidney disease. High-sensitivity C-reactive protein (hs-CRP) levels were measured with an automated instrument (Cardio- Phase hs-CRP, Siemens Healthcare). Fibrinogen is dosed with a coagulation method on the instrument BCSXP. The complete blood count was performed by flux cytometry.

Fasting glucose was measured using a glucose oxidase method (Glucose Analyzer Beckman Instruments, Irvine, CA, USA), as described in a previous report [30 (link)]. Serum total cholesterol, triglycerides, and low-density lipoprotein (LDL) cholesterol levels were measured using commercially available kits on a Hitachi 7150 Auto-analyzer (Hitachi Ltd., Tokyo, Japan), as described previously [31 (link)]. After precipitating serum chylomicrons, LDL-cholesterol, and very low-density lipoprotein with dextran sulfate-magnesium, the high-density lipoprotein (HDL) cholesterol left in the supernatant was measured using a previously described enzymatic method [31 (link)]. Serum fasting albumin, preabumin and transferrin were measured using commercially available kits on a Hitachi 7150 Auto-analyzer (Hitachi Ltd., Tokyo, Japan). White blood cell (WBC) and absolute lymphocyte (ALC) counts were determined using the HORIBA ABX diagnostics system (HORIBA ABX SAS, Parc Euromedicine, France).

Blood samples were collected in fluoride/heparin microcentrifuge tubes (Sarstedt, Numbrecht, Germany) and centrifuged for 30 s at 13,000 × g. Plasma was separated and stored at −80°C until analysis. Plasma glucose was measured by an automated glucose oxidase procedure (Beckman Glucose Analyzer). Pancreatic and intestinal tissues were extracted using buffer containing 20 mM Tris HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA and 1% Triton × 100 and stored at −80 °C. Biochemical analyses were carried out for insulin by radioimmunoassay [42] (link), total GLP-1 (GLP-1 total ELISA, EZGLP-1T-36K, Millipore), GIP (rat/mouse GIP ELISA, EZRMGIP-55K, Millipore) and glucagon (glucagon chemiluminescent assay, EZGLU-30K, Millipore) by specific enzyme linked immunoassays following the manufacturers' instructions. All commercial assay kits have been shown to exhibit a high degree of specificity.

The body weight, blood pressure, plasma glucose, insulin, cholesterol, and triglyceride levels were measured before and after the 6-month feeding period. Systolic blood pressure was measured with a noninvasive tail-cuff monitor (MK2000; Muromachi Kikai, Tokyo, Japan). Eight-hour fasting plasma glucose was measured with a Beckman Glucose Analyzer. Plasma insulin was determined with a radioimmunoassay kit (Linco Research, Inc., St. Charles, MO). The plasma cholesterol and triglyceride levels were measured using enzymatic methods (Roche, Pleasanton, CA, USA) through an automatic analyzer (Roche).