Architect insulin assay

Twenty-four hours prior to performing the insulin release studies, cells were harvested from routine culture seed 500,000 cells per well/mL media into six-well plates. The six-well plates were returned to the incubator for overnight culture. Prior to the acute tests (insulin release studies), media was removed by inverting the plate and incubating cells in each well with 1.5 mL of Krebs-Ringer bicarbonate buffer (KRBB) (115 mM NaCl, 4.7 mM KCl, 1.28 mM CaCl.6H₂O, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄.7H₂O) containing 1.1 mM glucose for 40 min at 37 °C. The preincubation reagent was removed. Then, pre-warmed, acute test samples in triplicates were used, made up KRBB for 20 min at 37 °C (1.5 mL/well). After 20 min, plates were taken from the incubator and 1 mL of supernatant was carefully removed from each well and placed into pre-labeled tubes. Samples were stored immediately at −20 °C until further analysis. Insulin level was measured using chemiluminescent microparticle immunoassay technology (ARCHITECT Insulin assay, Abbott).

Serum samples of different groups were harvested as mentioned above. The level of fasting insulin was quantified by ARCHITECT® insulin assay (Abbott Laboratories, Abbott Park, IL, United States) on ARCHITECT i2000 fully automated immunoassay analyzer (Abbott Laboratories, Abbott Park, IL, United States). HOMA-IR was used to evaluate insulin resistance [29 (link)].
HOMA-IR = fasting serum insulin (μIU/ml) × fasting plasma glucose (mmol/L) / 22.5.

Peripheral blood samples were drawn at 8:00 a.m. after an overnight fast. Plasma glucose, glycated hemoglobin (HbA1c), alanine transaminase (ALT), aspartate aminotransferase (AST), cholesterol (CH), high density lipoprotein (HDL), low density lipoprotein (LDL), triglycerides (TG), and blood count were measured by routine analysis. Plasma insulin was measured by ARCHITECT Insulin assay (Abbott Laboratories), a chemiluminescent microparticle immunoassay (CMIA)^{11 (link)}.

Blood draws were done at the trial site (Weizmann Institute of Science) or at the central medical laboratory of the trial (AMC Medical Center Laboratory, LTD). All blood specimens were processed and lab tests performed by one lab technician at the central laboratory, who was unaware of the arm assignment or any other characteristics of participants. HbA1c determination was based on the turbidimetric inhibition immunoassay (TINIA) for hemolyzed whole blood, standardized according to IFCC transferable to DCCT/NGSP (Tina-quant HbA1c Gen. 3 assay, cobas, Roche) [26 (link)]. Plasma glucose was measured with the use of a hexokinase method (GLUC2 assay, cobas, Roche). Fructosamine was measured with the use of a colorimetric test by reaction with nitroblue tetrazolium (FRA assay, cobas, Roche) [27 (link)]. Insulin was measured with a one-step immunoassay to determine the presence of human insulin in human serum or plasma, using CMIA technology (ARCHITECT Insulin assay, Abbot). For lipid profile, cholesterol was measured with the use of enzymatic, colorimetric method (CHOL2 test, cobas, Roche), HDL cholesterol was measured with homogeneous enzymatic colorimetric test (HDLC4, cobas, Roche) [28 (link)]. Triglyceride level was measured with enzymatic colorimetric test (TRIGL assay, cobas, Roche/Hitachi).

At the follow-up study visit, height, weight and waist circumference were measured and BMI was calculated as weight (kg)/(height (m²)). Blood samples were drawn following overnight fasting, and serum samples were separated, aliquoted, and stored at −70 °C. The samples were thawed for the first time for the following analyses. Serum triglycerides, total cholesterol, HDL-cholesterol, and serum glucose were analyzed using an AU400 instrument (Olympus, Hamburg, Germany) and applicable system reagents (Beckman Coulter, Brea, CA, USA). LDL cholesterol concentration was estimated using the Friedewald formula [27 (link)]. If triglyceride level was ≥4.5 mmol/L, LDL cholesterol was set to missing. Serum insulin was determined using an ARCHITECT insulin assay (Abbott, Chicago, IL, USA) on an Architect ci8200 analyzer (Abbott, USA), and insulin resistance was estimated using the homeostatic model for assessing insulin resistance (HOMA-IR; fasting insulin × (fasting glucose/22.5)). Sitting blood pressure was measured using an oscillometric device, with an average of three measurements used in the analyses. Data regarding physical activity and smoking habits were collected by questionnaires.