Laboratory measurements

Anthropometric and performance measurements were performed at the same time, and blood was collected in the following 4 months. The blood samples were drawn after a 12 hours fast, and creatinine, cystatine C, hemoglobin, albumin, serum 25(OH) vitamin D and intact PTH levels were measured. Serum FGF23 was analyzed by a second generation C-terminal FGF23 two-site Enzyme Linked Immunonosorbent Assay (ELISA; Immutopics, San Clemente, CA, USA). This assay detects two epitopes in the C-terminus of FGF23, and has a sensitivity of 1.5 relative units per mL (RU/mL), and inter- and intra-assay coefficients of variations of less than 5%. Serum creatinine was measured using a modified kinetic Jaffé colorimetric method on an autoanalyzer (AU 400, Beckman Coulter, CA. USA), which was calibrated to isotope dilution mass spectrometry, using a standard reference material (914a) traceable to the National Institutes of Standards and Technology (NIST). Plasma cystatin C levels were determined by an automated particle-enhanced immunoturbidimetric method using a Beckman AU 400 analyzer (Beckman Coulter), and reagents (code number LX002. s2361. X0973. X0974) obtained from DakoCytomation (Glostrup, Denmark), following the procedures recommended by the reagent producer. We estimated glomerular filtration rate (eGFR) by the CKD-EPI creatinine-cystatin C equation. All samples were frozen at −80°C.