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.