Selectra 2 auto analyzer

A blood sample was taken after 12-14 h of overnight fasting in a sitting position according to the standard protocol and centrifuged within 30-45 min of collection. All blood analyses were performed at the TLGS research laboratory on the day of blood collection. The samples were analyzed using the Selectra 2 auto-analyzer (Vital Scientific, Spankeren, The Netherlands). An enzymatic colorimetric method with glucose oxidase was used to determine fasting plasma glucose (FPG). Both inter-and intra-assay coefficient variations were 2.2 % for FPG. Triglyceride (TGs) level was measured using an enzymatic colorimetric analysis with glycerol phosphate oxidase. Total cholesterol (TC) was measured with cholesterol esterase and cholesterol oxidase, using the enzymatic colorimetric method. High-density lipoprotein cholesterol was measured after precipitation of the apolipoprotein B-containing lipoproteins with phosphotungistic acid. Analyses were performed using commercial kits (Pars Azmoon Inc., Tehran, Iran) and a Selectra 2 auto-analyzer (Vital Scientific, Spankeren, Netherlands). Inter-assay and intra-assay coefficients of variations were 1.6 % and 0.6 % for TGs, 2 % and 0.5 % for HDL-C, and 2 % and 0.5 % for TC, respectively. Low-density lipoprotein cholesterol (LDL-C) was calculated from the serum TC, TGs, and HDL-C concentrations and expressed in mg/dl using the Friedewald formula.

A venous blood sample (10 mL) was obtained from each patient before dialysis and after 12–14 h of fasting. Then, blood samples were centrifuged at 2000 rpm for 10 min to separate serum and subsequently, the extracted serum was transferred to sterile microtubes and stored at −70°C until the time of biochemical analysis. Serum albumin, urea, and creatinine were assessed using Selectra 2 Autoanalyzer (Vital Scientific, Spankeren, the Netherlands) employing commercial kits (Pars-Azmoon, Tehran, Iran) with the intra- and inter-assay coefficients of variation (CV) < 3%. The concentration of serum endothelin-1 was examined via ELISA kits (Biomedica, Vienna, Austria), with an intra- and inter-assay CV of 8.5%. Serum concentrations of malondialdehyde (MDA) and nitric oxide (NO) were measured using a colorimetric approach via commercial kits (Cayman Chemical, Ann Arbor, MI, United States), with the intra- and inter-assay CV of 4.6% and 7.8%, respectively. The serum concentrations of sE-selectin, sVCAM-1, and sICAM-1 were measured via ELISA kits (Diaclone, Besancon, France) with the intra- and inter-assay CV of 6.7, 6.3, and 3.5%, respectively. The serum concentration of hs-CRP was determined using ELISA kits (Diagnostics Biochem Canada, London, Canada) with an intra- and inter-assay CV of 4.6%.

A sample of 5 ml blood was collected from all participants after a 12 to 14 h fast, at baseline and at the end of week 15. These samples were centrifuged at 4000 rpm for 15 min. The samples of serum were separated into small aliquots and were frozen at − 80 °C. For Zn analysis, all tubes were washed by acid and rinsed with distilled water, then atomic absorption spectrometry (variant Chemthech Analytical 2000) was used to determine serum Zn concentration [39 (link), 40 ]. Serum concentration of high-sensitivity C-reactive protein (hs-CRP) was determined by enzyme-linked immunosorbent assay (ELISA) kits (Diagnostics Biochem Canada, Ontario, Canada) with an intra-assay coefficient of variation (CV) of 7.2%. Serum TNF-α was measured by ELISA kits (Diaclone, Besancon, France). Intra-assay CV for serum TNF-αwas 6.5%. Serum apelin concentration was assessed by ELISA kits (ZellBio GmbH, Ulm, Germany), with an intra-assay CV of 7.2%. Serum insulin was determined by ELISA kits (Monobind, USA), with an intra-assay CV of 7.4%. Serum glucosewas measured by commercial kits (Pars Azemoon, Tehran, Iran) with the aid of a Selectra 2 Autoanalyzer (Vital Scientific, Spankeren, The Netherlands). Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) was determined using the following equation: $$\text{HOMA-IR}=\left[\text{Fasting serum glucose}\left(\text{mg}/\text{dL}\right)\times \text{Insulin}\left(\mathrm{\mu }\text{U}/\text{L}\right)\right]/405$$