Elisas

Plasma non-esterified fatty acids (NEFAs) were determined via colorimetric assay according to manufacturer’s protocol (Wako Pure Chemical Industries Ltd., Osaka, Japan). Total cholesterol, LDL, HDL, Non-HDL, and triglycerides were determined via Cholestech LDX lipid profile cassettes (Alere Inc., Hayward, CA, USA). Glucose was measured using a bed-side analyzer (YSI Stat, Yellow Springs, USA; ABL, Radiometer, Denmark). Insulin and glucagon were measured via commercially available ELISAs according to manufacturer’s protocol (Crystal Chem Inc., Elk Grove Village, IL, USA). Cystatin-C was measured via ELISA (R&D Systems Inc., Minneapolis, MS, USA). Plasma concentrations of Cystatin-C were subsequently used to calculate estimated glomerular filtration rate via the following equation: ${\mathrm{eGFR}}_{\text{Cystatin-C}}\left(\mathrm{mL}·{\min }^{-1}·1.73{\mathrm{m}}^{-2}\right)=(\frac{84.6}{\mathrm{Cystatin}-\mathrm{C}\left(\frac{\mathrm{mg}}{\mathrm{L}}\right)})-3.2$
This equation was first described by MacIsaac et al. and was later validated against isotopic measurement of GFR [45 (link),46 (link)]. Cortisol, IL-6, and TNF-α were measured via ELISA (R&D Systems Inc., Minneapolis, MS, USA) and have been previously reported along with HbA1c [40 (link),47 (link)].