Elecsys insulin

Blood samples (about 10 mL) were collected at around 8:00 a.m. after an overnight fast at the beginning of the BWRP at T1 and T2. Total cholesterol (T-C), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), glucose and insulin were measured.
Colorimetric enzymatic-assays (Roche Diagnostics, Monza, Italy) were used to determine serum T-C, LDL-C, HDL-C and TG levels. The sensitivities of the assays were 3.86 mg/dL [1 mg/dL = 0.03 mmol/L], 3.87 mg/dL [1 mg/dL = 0.03 mmol/L], 3.09 mg/dL [1 mg/dL = 0.03 mmol/L] and 8.85 mg/dL [1 mg/dL = 0.01 mmol/L], respectively.
Serum glucose level was measured by the glucose oxidase enzymatic method (Roche Diagnostics, Monza, Italy). The sensitivity of the method was 2 mg/dL [1 mg/dL = 0.06 mmol/L].
Serum insulin concentration was determined by a chemiluminescent immunometric assay, using a commercial kit (Elecsys Insulin, Roche Diagnostics, Monza, Italy). The sensitivity of the method was 0.2 µIU/mL [1 µU/mL = 7.18 pmol/L].
The intra- and inter-assay coefficients of variation (CVs) were the following: 1.1% and 1.6% for T-C, 1.2% and 2.5% for LDL-C, 1.8% and 2.2% for HDL-C, 1.1% and 2.0% for TG, 1.0% and 1.3% for glucose, and 1.5% and 4.9% for insulin.

Blood samples were taken after a 12-h fasting period to measure serum concentrations of total cholesterol, high-density lipoprotein (HDL), triglycerides, glucose, and insulin. Commercial kits, normally used for routine patient examinations, were used for all analyses. [Cobas Roche colorimetric enzymatic cholesterol Gen.2 test, for total cholesterol assay; homogeneous-phase colorimetric enzymatic test HDL cholesterol Gen.4 Cobas Roche, for HDL cholesterol; colorimetric enzymatic test Triglycerides Cobas Roche, for triglyceride assay; hexokinase enzymatic method Glucose HK Gen.3 Cobas Roche, for glucose assay; immunoassay in ElectroChemiLuminescence Elecsys Insulin Cobas Roche, for insulin assay]. The HOMA index was calculated by dividing the product of serum insulin (µU/mL) and serum glucose (mmol/L) by 22.5 [16 (link)].

The laboratory parameters were analyzed by a certified laboratory (Labor 28 GmbH and Labor Berlin GmbH, both Berlin, Germany). Blood samples were drawn from all participants after >8 hours fasting and kept at 4–8 °C until analysis on the same day. An oral glucose tolerance test (OGTT) was performed according to the WHO-guidelines in subjects without self-reported diabetes (T2D)¹⁹ . Glucose levels (fasting and 2-hours post load) were measured using photometric methods and insulin levels were determined by an electrochemiluminescence immunoassay (Elecsys® Insulin, Cobas/Roche). HbA1c was measured using high-performance chromatography (VARIANT II TURBO HbA1c Kit – 2.0, Bio-Rad). Thyroid-stimulating hormone (TSH) was measured by electrochemiluminescence immunoassay (ECLIA). C-reactive protein (CRP) was determined in serum samples by means of an immunoassay.
To estimate dietary intake of total energy participants completed a validated, self-administered 146-item food frequency questionnaire (European Prospective Investigation into Cancer and Nutrition)^{20 (link)}.

Plasma glucose was measured using the hexokinase method (Quailigentglu, Sekisui, Japan), and HbA1c was measured via high-performance liquid chromatography (G8 Elution Buffer, Tosoh, Tokyo, Japan). The plasma insulin concentration was determined via electrochemiluminescence immunoassay (Elecsys Insulin, Roche Diagnostics GmbH, Mannheim, Germany). Insulin resistance (homeostatic model assessment for insulin resistance [HOMA-IR]) and secretion (HOMA-β) were calculated by homeostasis model assessment [30 (link)].