Assessment of children's bone health: Establishing paediatric reference (PREFER study) values for 1,25 vitamin D

Background: The active form of vitamin D, 1,25(OH)2D, plays a key role in regulating calcium and phosphorus metabolism and bone homeostasis. In paediatrics, maintaining optimal 1,25(OH)2D levels is crucial for supporting musculoskeletal growth. The hormone also serves as a diagnostic indicator for multiple disorders such as vitamin-D dependent rickets. Current Literature lacks comprehensive reporting of age-specific reference ranges in paediatrics. Immunoassays have traditionally been used, but the antibodies have variable affinities to both 1,25(OH)2D2 and 1,25(OH)2D3 and can cross-react with circulating isomers of vitamin D. We have developed an LC–MS/MS method to measure 1,25(OH)2D concentrations with greater specificity than immunoassay and have established age-specific ranges for a healthy paediatric cohort.

Methods: Written informed consent was taken from healthy children aged 0 to 16 years attending a tertiary centre for a planned surgical procedure. Fasting serum samples were collected, and calcium intake and medical history recorded. Serum 1,25(OH)2D concentration was measured by immunoassay (Diasorin LIAISON XL) and LC–MS/MS (Waters Xevo TQ-XS). Assay performance was compared via regression analysis. Parametric tests were used to assess the statistical significance of differences in 1,25(OH)2D levels to inform age and sex partitioning.

Results: 375 individuals (mean age 7.5±4.5 years S.D.) and were included in the analysis. The measurements obtained by immunoassay (mean: 134.8±37.6 pmol/L S.D.) were higher than those by LC–MS/MS (mean: 124.9±37.8 pmol/L S.D.). Regression analysis revealed a moderate linear relationship between both methods (y=0.8x+15.4, r2=0.65). All ranges were reported as 95% confidence intervals (CIs). Reference ranges were independently reported for four age groups: 0 to <3 3 to <7, 7 to <13, and 13 to <15. Sex partitioning was needed for those aged 13 to <15. There was no significant impact of gender at all other ages. 1,25(OH)2D concentrations were found to be affected by seasonal variation.

Conclusion: Diasorin immunoassay measured concentrations were significantly higher than the new LC–MS/MS method. This is believed to be secondary to differences in assay specificity to 1,25(OH)2D. Establishing age-specific normative data for 1,25(OH)2D using LC–MS/MS data will provide more accurate reference ranges for the paediatric population.