Blood samples were separated into plasma or serum at the time of collection and stored at −80°C until analysis. Plasma glucose was measured by the glucose oxidase method (2300STAT Plus, Yellow Springs Instruments, Yellow Springs, OH). Serum insulin and C-peptide were measured using commercial ELISA kits (#EZHIASF-14K and #EZHCP-20K, respectively, Millipore, St. Louis, MO). Serum triglycerides, total cholesterol, and HDL-cholesterol were measured at the Clinical Chemistry Laboratory of the Oklahoma Veterans Administration Hospital (Oklahoma City) using validated enzymatic assays (Synchron Systems, Beckman Coulter, Brea, CA). Serum non-esterified fatty acids (NEFA) were measured in with an enzymatic colorimetric assay (NEFA-HR2, Wako Chemicals, Richmond, VA). All assays were performed with appropriate standards and quality controls according to manufacturer’s directions.
The fasting concentrations of each analyte was calculated as the average of two blood samples collected at 10 and 2 minutes prior to meal ingestion. The concentrations of glucose, insulin, C-peptide, and NEFA from 0–180 minutes after the meal were used to calculate the total area under the curve (AUC) using the trapezoidal method, and the oral glucose insulin sensitivity index (15 ).
The fasting concentrations of each analyte was calculated as the average of two blood samples collected at 10 and 2 minutes prior to meal ingestion. The concentrations of glucose, insulin, C-peptide, and NEFA from 0–180 minutes after the meal were used to calculate the total area under the curve (AUC) using the trapezoidal method, and the oral glucose insulin sensitivity index (15 ).