Comparing directly measured versus mathematically calculated free serum 25-hydroxy vitamin D level in children

  • Judith Sebestyen VanSickleEmail author
  • Tarak Srivastava
  • Uttam Garg
  • Mohamed H. Rezaiekhaligh
  • Uri S. Alon
Original Article



25-Hydroxy vitamin D (25(OH)D) is essential for calcium homeostasis and bone metabolism. The majority of serum 25(OH)D is bound to vitamin D-binding protein (VDBP) (~ 85%) and to albumin (~ 15%), with only a miniscule amount circulating as free 25(OH)D. Free 25(OH)D can be calculated mathematically by Bikle method from the concentrations of total 25(OH)D, VDBP, and albumin or measured directly by ELISA. A direct head-to-head comparison between the two methods has not been done in children.

Materials and methods

The objective of the study was to compare the mathematically calculated versus directly measured free 25(OH) vitamin D in children. Serum samples from 74 children (ages 1–19 years) were simultaneously analyzed for total 25(OH)D, serum albumin, VDBP, and free 25(OH)D. Pearson correlation analysis and Bland–Altman plot were used to evaluate agreement between the two methods.


The mean age was 9.1 ± 5.1 years, with 61% boys, 76% Caucasians, and 24% African-Americans. The mean ± SD for total 25(OH)D was 38.7 ± 12.8 ng/mL, bioavailable 25(OH)D 3.1 ± 1.1 ng/mL, mathematically calculated free 25(OH)D 8.4 ± 3.2 pg/mL, and directly measured free 25(OH)D 8.9 ± 3.6 pg/mL. Pearson correlation reflected a significant correlation between mathematically calculated and directly measured free 25(OH)D (r = 0.66, p < 0.0005). Bland–Altman plot reflected a tight agreement within a 95% limit of agreement (mean = − 0.026 ± 2SD).


The directly measured and mathematically calculated free 25(OH)D are in close agreement and are interchangeable. Depending on the local availability of instruments and methods, free 25(OH)D can be either directly measured or mathematically calculated.


Vitamin D Free 25(OH)D Bone metabolism 


Compliance with ethical standards

Conflict of interest

All authors have no conflicts of interest.


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Copyright information

© The Japanese Society Bone and Mineral Research and Springer Japan KK, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Division of NephrologyChildren’s Mercy Hospital, University of Missouri at Kansas CityKansas CityUSA
  2. 2.Department of PathologyChildren’s Mercy Hospital, University of Missouri at Kansas CityKansas CityUSA

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