pp 1–16 | Cite as

The measurement of vitamin D metabolites: part I—metabolism of vitamin D and the measurement of 25-hydroxyvitamin D

  • Konstantinos MakrisEmail author
  • Christopher Sempos
  • Etienne Cavalier
Review Article


It has been more than 80 years since the discovery of vitamin D and its ability to cure rickets in children. Vitamin D is a secosteroid and comes in two distinct forms, vitamin D2 and vitamin D3. During the last 40 years, the synthesis and metabolism of vitamin D were elucidated and more than 50 metabolites of vitamin D have been discovered, though commercial measurement procedures have been developed for only a few of them. The clinical significance of vitamin D in calcium and phosphorus homeostasis is well appreciated. However, recent epidemiological data have indicated that it has several extra-skeletal physiologic actions which are still a matter of scientific debate. Both research findings and the debate around the interpretation of the research results have created increased interest in more measurements of vitamin D. With the ever growing family of measurable vitamin D metabolites and the measuring techniques comes a question: What metabolic product will provide the right answers and which is the best way to measure it. The right choice of analytical technique is connected with the question of which metabolite we aim to measure, what is its serum concentration, and the purpose of the measurement. The aim of the first part of this review is to provide a brief overview of vitamin D metabolism and a more detailed analysis of the existing methods and the status of standardization for the measurement of 25-hydroxyvitamin D.


Vitamin D 25-hydroxyvitamin D Liquid chromatography Mass spectrometry Immunoassays Standardization Vitamin D standardization program 



dehydrocholesterol or provitamin D3


24,25-dihydroxyvitamin D3


calcidiol or 25-hydroxyvitamin D3


calcitriol or 1,25-dihydroxyvitamin D3


C3-epimer of the 25(OH)D






parathyroid hormone


fibroblast growth factor-23


vitamin D binding protein


vitamin D-dependent rickets


vitamin D receptor


Vitamin D Standardization Program


chronic liver disease


chronic kidney disease


secondary hyperparathyroidism




high-performance liquid chromatography


liquid chromatography coupled with mass spectrometry


competitive protein binding assays




enzyme-linked immunosorbent assays


chemiluminescent assays


reference measurement procedure


primary reference material


National Institute for Standards and Technology


International Federation of Clinical Chemistry


Joint Committee for Traceability in Laboratory Medicine


American Association for Clinical Chemistry


Vitamin D External Quality Assessment Scheme


College of American Pathologists


Center for Disease Control


External Quality Assessment



None of the authors declares any funding or grant related to this article.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


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Copyright information

© Hellenic Endocrine Society 2020

Authors and Affiliations

  1. 1.Clinical Biochemistry DepartmentKAT General HospitalAthensGreece
  2. 2.Vitamin D Standardization Program (VDSP)Havre de GraceUSA
  3. 3.Department of Clinical ChemistryUniversity of LiegeCHU de Liege, LiegeBelgium

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