Laboratory protocol
BLOOD
Blood was collected after an overnight fast and routine biochemical analyses were carried out locally by standard laboratory methods. Quantification of serum 25(OH)D for screening purposes was performed locally with high-performance liquid chromatography mass spectrometry (HPLC–MS) in Malmoe and Gothenburg while a chemiluscent method (Diasorin) was used in Uppsala. Baseline and week-12 measurements of 25(OH)D were all analyzed centrally by HPLC–MS. Measurements of serum HEP-25 were carried out with mass spectrometry that specifically measures HEP-25. This method has a lower limit of quantification of 0,5 nmol/L, an intra-assay coefficient of variation of 6%, and an inter-assay coefficient of variation of 8%. Finally, the quantification of intact fibroblast growth factor 23 (FGF-23) was performed with an enzyme-like immunosorbent method (Kainos, Japan).In healthy individuals it has been shown that serum ferritin explains 60% of the variance in serum levels of hepcidin-25 [