A blood sample was obtained for laboratory analyses from all but one child. Local anaesthetical patches (EMLA R; AstraZeneca AB, Södertälje, Sweden) were used to reduce the discomfort of venipuncture. Dietary intakes were calculated from 3-day food records with Diet32 software (Aivo Oy Finland, Turku, Finland). The OSI-906 nutrient contents of the foods was based

on the Finnish National Food Composition Database, Fineli, version 2001, maintained by the National Public Health Institute of Finland, Nutrition Unit. The total intake of vitamin D included intake from diet and from supplements. Laboratory measurements Serum 25-OHD was measured with an OCTEIA immunoenzymometric assay (IDS, Bolton, UK). The intra-assay coefficient of variation (CV) was less than 3.9% and interassay variation (4.5%). Reproducibility was ensured by adhering to the Vitamin D External Quality Assessment Scheme (DEQAS). EIA selleck chemicals results were compared with HPLC results in order to determine the reliability of EIA in measuring 25-OHD2 concentration. The results were consistent (r = 0.751, p < 0.001, R 2 = 0.495); therefore, the EIA results were used throughout the study. Vitamin D status in children was defined as deficient when S-25-OHD was below 37.5 nmol/l, insufficient when it was between 37.6 and 50 nmol/l,

and sufficient when it was above 50 nmol/l, according to the published pediatric reference values [20]. In adults, a concentration of at least 80 nmol/l is considered optimal for multiple health outcomes [22]. Serum bone-specific alkaline phosphatase (S-BALP) was assayed with an OCTEIA Octase BAP immunoenzymometric assay (IDS) in order to characterize bone formation. Samples were diluted 1:5 to meet the standard curve. Intra- and interassay CVs were 6.1% and 6.7%, respectively. The bone resorption marker, serum active isoform 5b of the tartrate-resistant acid phosphatase (S-TRACP), was CH5183284 supplier determined with a bone TRAP assay (SBA Sciences, Turku, Finland). Intra- and interassay

CVs were 1.2% and 3.0%, respectively. pQCT bone measurement Peripheral bone variables were determined by pQCT from the left tibia. One 2.5-mm slice (voxel size, 0.4 mm) at the 20% site of distal tibia, was measured with a XCT-2000 scanner (Stratec, 5-Fluoracil datasheet Pforzheim, Germany) as described previously [10]. Data was analyzed using version 5.50 of the manufacturer’s software package, in which the bone contour was analyzed with a single threshold of 180 mg/cm3 for the detection of total bone mineral density (BMD), BMC, and CSA. The long-term CVs for the phantom BMD and CSA were 1.9% and 1.1%, 2.7% and 0.79%, and 0.50% and 0.78% in the total, cortical, and trabecular bone, respectively. Short-term precision (CV%) was determined with duplicate measurements of five subjects. CVs for the total bone BMD and CSA were 6.0% and 6.5%, respectively. On this basis, the calculated least significant changes for total bone BMD and CSA were 16.7% and 18.1%, respectively.