2.3. Assays

Fasting venous blood samples were taken from each patient in the early morning hours. Following 15 min at room temperature, blood was allowed to coagulate for 10 min before being centrifuged for 10 min at 3000 rpm. After serum separation, it was transferred to Eppendorf tubes and kept at −80 °C until the assays were carried out. Serum IL-6, IL-10 and IL-1β levels were determined using commercial ELISA sandwich kits purchased from Sunlong Biotech Co., Ltd. (HangZhou, Zhejiang, China) and Melsin Medical Co. (Changchun, Jilin, China) provided the remaining ELISA kits (IL-17, IL-21, IL-22, IL-23, and TNF-α). The kits detected IL-10, IL-17, IL-21, IL-22, IL-23, and TNF-α at 1.0 pg/mL and IL-1β and IL-6 at 0.1 pg/mL. Calcium and magnesium were measured using spectrophotometric kits supplied by Biolabo^® (Maizy, France). The coefficient of variance within the tests was <10.0% for all assays (precision within assay).