C peptide

Blood samples were freshly collected through cardiac puncture, and the samples were stored in plain SST tubes for serum biochemical assay. The blood samples were centrifuged at 3000 rpm at 4°C for 15 minutes. The clear serum obtained was separated and labeled for liver function tests (LFT), namely, aspartate aminotransferase (AST) and alanine aminotransaminase (ALT), renal function test (RFT), namely, urea and creatinine, fasting blood sugar (FBS) and serum lipid profile (LP), namely, triglycerides (TG) and total cholesterol. Biochemical tests were performed using ILAB 300 Plus Clinical Chemistry Analyzer, Milano, Italy. Serum leptin, resistin, insulin hormone (USCN Life Science and Technology, Wuhan, China), C-peptide (Mercodia AB, Uppsala, Sweden), and serum levels were determined through ELISA method by referring to the respective manufacturer's instructions.

Commercial ELISA kits were used for measuring insulin concentration, C-peptide, (Mercodia, Uppsala, Sweden) and inflammatory cytokines including IL-1β, TNF-α, IL-17A, MCP-1, and VEGF (R&D Systems, Minneapolis, USA) following the manufacturers’ instructions. Quality control sera were used to measure accuracy and precision of the assays.

Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used for detection of plasma insulin, C-peptide levels (Mercodia, Uppsala, Sweden) as well as circulatory levels of inflammatory cytokines/chemokines or factors including IL-1β, TNF-α, IL-17A, MCP-1, and VEGF (R&D Systems, Minneapolis, MN, USA), following instructions from the manufacturers.