Cobas bio

The hormonal analyses for progesterone, estrogen, insulin, cortisol, and human growth hormone were performed by ELISA using the Anthos HTII microplate reader and employing appropriate standardised kits from DRG Instruments GmbH (Germany). The intra-assay CV for the assays were 3.0% for insulin, 3.6% for human growth hormone, 2.2% for cortisol, 5.7% for estrogen, and 6.8% for progesterone. All hormonal analysis was measured in duplicate with mean value provided.
Plasma NEFA values were determined by an enzymatic spectrophotometric method, while a portion of the plasma was deproteinized with perchloric acid (7% wt/vol) before assay for lactate and glycerol using enzymatic methods. Analyses were performed on a Cobas-Bio centrifugal analyser (Roche Products, Welwyn Garden City, Herts, UK). Plasma insulin was determined using radioimmunoassay (RIA) (IM.78, Amersham International, Amersham, UK).

Fasting blood samples were collected at the baseline exam and stored at −70°C for a few weeks prior to lipid measurement. The ARIC Central Lipid Laboratory measured plasma cholesterol and triglycerides using a Cobas-Bio centrifugal analyzer (Roche Diagnostics, Montclair, NJ) with enzymatic kits (Boehringer Mannheim Diagnostics, Indianapolis, IN).[17 , 18 (link)] HDL cholesterol was estimated by the method of Warnick et al,[19 (link)] and LDL cholesterol was calculated using the Friedewald formula.[20 (link)] If the level of triglycerides was over 400 mg/dL, then LDL cholesterol was not determined. The coefficients of variation within the laboratory for TC, triglycerides, HDL cholesterol, and LDL cholesterol were 2.5%, 2.7%, 3.7%, and 5.2%, respectively.[21 (link)]

Blood samples were collected in fasting state from the antecubital vein (BD Vacutainer^® vacuum system, Franklin Lakes, NJ, USA), after approximately 8 h of sleep (last meal no more than 2 h before bedtime). The following were determined in the blood: glucose concentration (GLU), ketone bodies (KB) (β-hydroxybutyrate—BHB), triglycerides (TG) and free fatty acids (FFA). GLU concentration was determined in the plasma (EDTA and glycolysis inhibitors: sodium fluoride and potassium oxalate). Triglycerides (TG), FFA and BHB, were determined in the serum (clotting activator). The assays were carried out using commercial reagent kits according to the procedure indicated by the manufacturers.
The detection range was, respectively: 0.11–41.6 mmol/L for glucose (GLUC3 Roche Diagnostics International Ltd., Germany), 0.07–2.24 mmol/L for FFA (NEFA FA 115, Randox Laboratories Ltd., UK), 0.1–5.75 mmol/L for 3-hydroxybutyrate (Ranbut, Randox Laboratories Ltd., UK) and 0.11–5.93 mmol/L for triglycerides (TRIG, Ortho-Clinical Diagnostics, France). The determinations were performed using the Cobas c 701/702 (glucose) and Cobas Bio (FFA, BHB) analysers by Roche Diagnostics International Ltd. (Germany) and Vitros 5.1 FS (TG) by Ortho-Clinical Diagnostics (France). The intra-assay % coefficients of variation were: KB 5.2%, TG 1.1%, FFA 4.3%, GLU 1.1%.