2000 autoanalyzer

Blood was collected on the first day and last day of each intervention period after a 12–hour fast by trained research staff. Specimens were locally processed and stored at −80°C until analyses. Glucose was measured on a Roche Module P chemistry autoanalyzer (Roche Diagnostic Inc., Indianapolis, IN) at the Northwest Lipid Research Laboratories (University of Washington, WA). Insulin was measured using a Tosoh 2000 autoanalyzer (Tosoh Biosciences Inc., South San Francisco, CA) at the Diabetes Endocrinology Research Center Immunoassay Laboratory (University of Washington, WA). The rest of the biomarkers assessments and the genotyping were conducted at the FHCRC Biomarker Core Laboratory and the Molecular Epidemiology Laboratories. Immunoassays were used to measured total adiponectin (Total Adiponectin EIA, Aplco), IGF-1 (Human IGF-I Quantikine ELISA, R&D Systems), IGFBP-3 (Human IGFBP-3 Quantikine ELISA, R&D Systems), and IL-6 (Human IL-6 Quantikine HS ELISA, R&D Systems). CRP was measured using CRP (3 (link))-Wide Range reagent (Kamiya Biomedical Company) on Roche Cobas Mira chemistry analyzer with a high sensitivity protocol. The intra-assay CVs were 0.7%, 7.8%, 1.3%, 1.5%, 1.8%, 2.3%, and 3.3% for glucose, insulin, adiponectin, IGF-1, IGFBP-3, IL-6, and CRP, respectively. The details of specimen collection and analysis have been previously described (14 (link)).

Glucose was measured by the glucose hexokinase method on a c501 autoanalyzer (Roche, Indianapolis, Indiana). C-peptide and insulin were measured by a two-site immune-enzymometric assay performed on the Tosoh 2000 autoanalyzer (Tosoh Bioscience, Inc., South San Francisco, California). Quality control samples were performed at regular intervals with low, medium, medium-high, and high concentrations with inter-assay coefficients of variation ≤2.0% for glucose, ≤4.3% for C-peptide, and ≤3.5% for insulin. Further assay details have been published.^{7 (link)}

Laboratory assessments were performed in a central laboratory at the University of Washington.^{15 (link)–17 (link)} Glucose was measured using the glucose hexokinase method on a Roche c501 autoanalyzer (Roche Diagnostics Inc., Indianapolis, IN). C-peptide and insulin were measured by a two-site immuno-enzymometric assay performed on the TOSOH 2000 autoanalyzer (TOSOH Biosciences, Inc., South San Francisco, CA). Inter-assay CVs on quality control samples with low, medium, medium-high and high concentrations were ≤2.0% for glucose, ≤4.3% for C-peptide and ≤3.5% for insulin. HbA1c was measured on a TOSOH G8 analyzer, under Level 1 NGSP certification. The inter-assay CVs as measured on quality control samples with low and high HbA1c levels were 1.9% and 1.0%, respectively.^{15 (link)–17 (link)}

A 3-hour 75-gram OGTT was performed to determine fasting and 2-hour glucose concentrations, as well as OGTT-derived β-cell response measures, at baseline, month 6 (M06), M12, M15, and M21 (Supplementary Figure S1) [4 (link), 5 (link), 9 ]. The OGTT-derived β-cell response measures included: (1) C-peptide index (CPI, nmol/mmol, ΔC-peptide_30–0/Δglucose_30–0), and (2) insulinogenic index (IGI, pmol/mmol, Δinsulin_30–0/Δglucose_30–0). Inverse fasting insulin (1/fasting insulin (pmol/L) × 10⁻³) was utilized as the estimate of whole-body insulin sensitivity in paired measures with β-cell response measures.
Laboratory assessments were performed in a central laboratory at the University of Washington [4 (link), 5 (link)]. Glucose was measured using the glucose hexokinase method on a Roche c501 autoanalyzer (Roche Diagnostics Inc., Indianapolis, IN). C-peptide and insulin were measured by a two-site immuno-enzymometric assay performed on the TOSOH 2000 autoanalyzer (TOSOH Biosciences, Inc., South San Francisco, CA). Inter-assay CVs on quality control samples with low, medium, medium-high and high concentrations were ≤2.0% for glucose, ≤4.3% for C-peptide and ≤3.5% for insulin. HbA1c was measured on a TOSOH G8 analyzer, under Level 1 NGSP certification. The inter-assay CVs as measured on quality control samples with low and high HbA1c levels were 1.9% and 1.0%, respectively.