25-Hydroxyvitamin D measurement

Blood samples were collected from each participant at a mobile examination centre for measurement of serum 25(OH)D concentrations. The blood samples were immediately frozen at −30°C and then shipped to the National Center for Environmental Health (Centers for Disease Control and Prevention, Atlanta, GA, USA) for analysis.

There are two forms of physiologically important vitamin D: ergocalciferol (vitamin D₂) and cholecalciferol (vitamin D₃). Ergocalciferol is typically obtained from fortified foods and sun-dried mushrooms, while cholecalciferol is synthesized in the skin from 7-dehydrocholesterol upon exposure to UVB (290–320 nm) and is found in oily fish (e.g. salmon, sardines and mackerel), egg yolks and fish oils (e.g. cod-liver oil)^(7,8).

Serum samples were measured using ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS; ThermoElectron Corp., West Palm Beach, FL, USA) for the quantitative detection of 25-hydroxycholecalciferol (25(OH)D₃), 25-hydroxyergocalciferol (25(OH)D₂) and 3-epi-25-hydroxycholecalciferol (3-epi-25(OH)D₃)^(24,25). Total 25(OH)D concentrations were summed from 25(OH)D₃ and 25(OH)D₂ concentrations in SI units of nmol/l; total 25(OH)D did not include 3-epi-25(OH)D₃ concentrations, a predominant form in infants under the age of 1 year⁽²⁶⁾. The CV in NHANES 2007–2010 was 3·0–3·8 % for 25(OH)D₃ and 3·8–6·5 % for 25(OH)D₂. The range of serum concentrations was within 5·41–213 nmol/l for 25(OH)D; 3·02–212 nmol/l for 25(OH)D₃; and 1·45–183 nmol/l for 25(OH)D₂. For 25(OH)D levels >1 ng/g, within-set CV was 0·1–0·5 % and between-set CV was 0·2–0·6 %; for 25(OH)D levels <1 ng/g, within-set CV was 0·9–2·9 % and a between-set CV was within 2·0 %⁽²⁷⁾.

In the statistical analysis, total 25(OH)D and 25(OH)D₃ concentrations were categorized into three groups: <30 nmol/l (deficient), 30–49·99 nmol/l (insufficient) and ≥50 nmol/l (sufficient), based on cut-off points suggested by the US Institute of Medicine⁽²⁸⁾.