Biochemical analysis
At the study visit, participant fasting blood was drawn between 7-9 AM for measurement of serum ferritin, 25OHD, osteocalcin (OC), C-terminal telopeptide of type-I collagen (CTX) and Hb concentrations in the hospital’s central laboratory. For Dkk-1 measurement, a separate blood sample was collected, centrifuged, and stored at -70 oC within 1 hour until the analysis.
Serum Dkk-1 concentrations were measured using a sandwich enzyme-linked immunosorbent assay kit (Abbexa Ltd., Cambridge, United Kingdom) with the reported intra- and inter-assay coefficient of variation of <10% and the detection range of 31.2-2000 pg/mL. OC, CTX and 25OHD concentrations were measured with electrochemiluminescence immunoassay on an automatic Roche Cobas e601 analyzer (Roches Diagnostics, Mannheim, Germany). 25OHD had a measuring range of 3-70 ng/mL. OC had a measuring range of 0.5-300 ng/mL with the reported repeatability and intermediate precision of <1.4% and <2.4%, respectively. CTX had a measuring range of 0.01-6.0 ng/mL with the reported repeatability and intermediate precision of <4.8% and <5.8%, respectively. OC and CTX concentrations were expressed as z-scores according to the reported reference ranges in children and adolescents ≤18 years of age utilizing an assay similar to ours.20,21 Among participants >18 years of age, we used the mean concentrations at age 18 for z-score conversion.