Chemerin

Blood samples were collected after a 12h overnight fast. Biochemical measurements of calcium, glucose, glycated hemoglobin (HbA1c), total cholesterol and fractions (HDLc, LDLc), triglycerides, phosphorous, albumin, alkaline phosphatase, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and creatinine were performed on the day of blood collection. The serum aliquots for the other parameters were stored at −70 °C until the day of the assay. Calcium, phosphorus, alkaline phosphatase, fasting glucose, albumin, AST, ALT and creatinine were determined using an automatic biochemistry analyzer (Wiener lab, CT 600i, Thermo Fisher Scientific, Espoo, Finland). HbA1c and basal insulin levels were measured using an immunoenzymatic assay (Atellica^® CH, Siemens Heathineers, Erlangen, Germany). PTH, 25(OH) vitamin D and osteocalcin (Liaison, Diasorin, Saluggia, Italy) levels were measured using chemiluminescence. Leptin and Chemerin (Biovendor, R&D, Karásek, Czech Republic) were determined using ELISA. Serum insulin levels and HOMA-IR were assessed in 23 controls and 10 patients since DS individuals with type 2 diabetes using insulin therapy were not considered for these parameters. Insulin resistance was calculated using the HOMA-IR index: [fasting glucose (mg/dL) × 0.0555 × fasting insulin (µU/mL)/22.5], with normal values < 2.71 [45 (link)].

The following biomarkers were measured to assess inflammatory profiles in study participants: C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-a), interleukin-10 (IL-10), monocyte chemoattractant protein 1 (MCP-1), chemerin, omentin, leptin, total adiponectin, high molecular weight (HMW) adiponectin, and fetuin-A. Non-HMW adiponectin was estimated based on the difference between total and HMW adiponectin. Venous blood samples were immediately centrifuged and frozen at −80 °C until analysis. CRP concentrations were determined by a highly sensitive immunoturbidimetric assay using ABX Pentra 400 reagents on an ABX Pentra 400 (Horiba ABX, Montpellier, France). Commercially available ELISA kits were used for the measurements of serum leptin (R&D Systems, Minneapolis, MI, USA), total adiponectin, chemerin, omentin, fetuin-A (all from Biovendor, Germany), and high molecular weight adiponectin (Merck Millipore KGaA, Darmstadt, Germany) and U-Plex assay was used to measure IL-6, TNF-a, MCP-1, and IL-10 (MSD, Rockville, ML, USA). Table S4 provides the detection limits of the kits for the measured biomarkers.