Fasting Blood Collection and Analysis

Fasting blood samples were collected using sterile, disposable materials by a licensed phlebotomist. Blood was drawn directly into SST vacutainers. SST tubes sat at room temperature for 30 min and were then centrifuged in a refrigerated Centra CL3R (International Equipment Co.) for 10 min at 100× g at 10 °C. Next, 50 uL of serum was inserted into a basic metabolic reagent disk and placed into a Piccolo Xpress Chemistry Analyzer (Abbott, Princeton, NJ, USA) for determination of glucose and CVD risk markers, which included the following: glucose, total cholesterol, HDL, LDL, VLDL, non-HDLc and triglycerides. Ratios of total cholesterol to HDL and LDL to HDL were calculated.
Insulin was measured in duplicate using Meso Scale Delivery (MSD) Multi-plex Assay System and were conducted according to the manufacturer’s instructions. Briefly, 150 uL of Blocker A was added to each well of the MSD plate, which was then sealed, incubated and shook (1000 rpm) for one hour at room temperature. The plate was then washed with phosphate-buffered saline plus 0.05% Tween-20 (PBS-T), and 50 uL of the sample and standard were added to each well. The plate was then sealed, incubated and shook (1000 rpm) for 2 h at room temperature. The plate was washed again with PBS-T and then 25 uL of detection antibody solution was added to each well. The plate was then sealed, incubated and shook (1000 rpm) at room temperature for one hour. The plate was washed for a final time with PBS-T and then 150 uL of Read Buffer T was added to each well. The plate was read on the MSD QuickPlex SQ 120 imager and quantified using an 8-point standard curve. Insulin concentrations were used to calculate HOMA-IR and QUICKI (Equation (3)).