Phenotyping and relative quantification of vitamin D binding protein in a paediatric population by using liquid chromatography-tandem mass spectrometry.

BACKGROUND: Adequate concentrations of vitamin D are required to ensure bone health and minimize the incidence of multiple extraskeletal diseases. Although total 25-hydroxyvitamin D (25OHD) remains the recommended biomarker for assessing vitamin D status, it has been speculated that free 25OHD correlates better with clinical outcomes. The calculation of free 25OHD depends on the concentrations of vitamin D binding protein (DBP), the determination of which involves different immunoassays and has led to varying results and conclusions. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous identification and relative quantification of DBP isoforms. METHODS: We used serum samples from healthy children ( n = 79), mainly Caucasian (88%). Proteins were denatured, reduced, alkylated and digested with trypsin. Purified peptides were analysed by LC-MS/MS. The DBP phenotype was established by using the combinations of tryptic peptides associated with each of the three isoforms and one peptide common to all of them to perform relative quantification. The genotyping of volunteers ( n = 7) facilitated verification of the ability of our method to correctly identify the DBP phenotype. RESULTS: The DBP phenotype was correctly established in all samples from volunteers, based on the 100% correlation observed with the genotype. The most common DBP phenotype in Caucasian children was 2/1S (34%) and the rarest 1F/1F (2%). The relative quantification of DBP concentrations did not show statistically significant differences between phenotypes ( P = 0.11). CONCLUSIONS: LC-MS/MS enabled simultaneous phenotyping and relative quantification of DBP, while avoiding the analytical limitations of immunoassays and confirming similar concentrations of DBP in all phenotypes.

Can vitamin D status be assessed by serum 25OHD in children?

BACKGROUND: To examine the relationship of serum 25-hydroxyvitamin D (25OHD) concentrations with serum parathyroid hormone (PTH) levels, body mass index (BMI), and environmental factors in a population of Caucasian children living at latitude 43°N. METHODS: Cross-sectional study on 288 children aged 1 month to 13 years who presented to a pediatric emergency unit during a 21-month period. RESULTS: Mean (SD) serum 25OHD concentrations were 40.6 (17.6), 30.9 (12.0), and 26.4 (9.9) ng/ml (1 ng/ml = 2.5 nmol/l), in children aged 0-1, 2-5, and ≥ 6 years, respectively. Serum PTH levels were 26.6 (13.6), 24.3 (11.9), and 32.7 (12.1) pg/ml in the same groups. Infants had 25OHD concentrations significantly higher. PTH levels were significantly higher in children aged ≥ 6 years. There was no significant correlation between serum 25OHD and PTH concentrations. Totals of 15.6 % and 2.1 % of children had 25OHD values less than 20 and 10 ng/ml, respectively, but none had elevated serum PTH or clinical manifestations related with vitamin D deficiency. Age (inverse correlation) and season (higher values in summer), but not BMI, sex, and time spent outdoors, influenced serum 25OHD concentrations. CONCLUSIONS: Our results raise doubt on the assumption of only a serum 25OHD threshold as indicative of vitamin D deficiency in children.