Vitamin D

Vitamin D is produced endogenously when ultraviolet-B (UVB) rays from sunlight strike the skin and trigger vitamin D synthesis. It can also be obtained from the diet in the form of vitamin D3, cholecalciferol, from animal sources (e.g. fatty fishes such as salmon, tuna and mackerel, fish liver oils, beef liver, cheese and egg yolks) or vitamin D2, ergocalciferol, in mushrooms irradiated with UVB light, vitamin D-fortified foods (dairy products, oils, margarine and spreads, and some breakfast cereals) and vitamin D-containing supplements 1,2. Vitamins D2 and D3 are similar compounds except for the structure of their side chains. The conversion of vitamins D2 and D3 into active compounds requires a two-step enzymatic hydroxylation process, although they have different conversion efficacy 3. A meta-analysis indicates supplementation with vitamin D3 had a significant and positive effect in raising serum 1,25-dihydroxyvitamin D (1,25(OH)2D) concentrations, the physiologically active form also known as calcitriol, compared to supplementation with vitamin D2 (P = 0.001) 4. However, vitamin D2 is considered an active substance and is not excluded as a source of dietary vitamin D.

The most widely accepted and used indicator of vitamin D status is plasma or serum 25-hydroxyvitamin D (25(OH)D), which is reflective of exposure to vitamin D from both cutaneous synthesis and dietary intake from food and supplements 5. However, there is no international consensus about the blood concentration associated with optimal status in different population groups 6 and WHO has not yet issued guidance. The United States’ Institute of Medicine (IOM) of the National Academies (now referred to as the Health and Medicine Division of the National Academies of Sciences, Engineering, and Medicine [the National Academies]) established vitamin D recommended nutrient requirements for populations based on preventing serum 25(OH)D concentrations below 30 nmol/L for musculoskeletal outcomes 7. The Endocrine Society, a global organization representing professionals from the field of endocrinology, defines vitamin D deficiency as 25(OH)D concentrations below 50 nmol/L (20 ng/mL) and vitamin D insufficiency as 52.0–72.5 nmol/L (21–29 ng/mL), based on multiple health outcomes, including but not limited to musculoskeletal outcomes 8. The European Food Safety Authority (EFSA) has suggested similar cutoff values 9.

Specimen collection and management: 25-hydroxyvitamin D is usually measured in plasma or serum specimens obtained by centrifugation of whole blood collected by venipuncture. 25-hydroxyvitamin D is very stable, so whole blood processing can be delayed for up to two days. Serum is stable for at least two weeks at 4˚C and for at least one year at -20˚C 10.

Biomarker analysis: Serum 25-hydroxyvitamin D is commonly measured by a competitive protein-binding assay on a fully automated clinical analyser using commercial assay kits, or by using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The latter approach is resource intensive and not suited for laboratories with limited capacity and infrastructure. The required analysis volume is typically >100 µL, although a minimum specimen volume of >300 µL is needed for repeat analysis. The product sheet for the intended assay will specify the specimen matrix requirements and should be consulted before deciding on the method and ordering survey supplies. Serum is the preferred matrix, since not all assays can utilize EDTA or heparin plasma. Appropriate quality control measures must be followed to ensure high quality results. The assay kits include calibration materials and often also include quality control materials. It is nonetheless recommended to establish “in-house” quality control materials that can be tracked over a longer period to verify that the method did not shift over time. The method imprecision is typically 5–10%.

Serum-based certified reference materials are available from NIST (SRM 972a) to verify method accuracy. However, not every assay may be able to use this material because the assay performance may differ between native patient samples and reference materials that have undergone some processing. Because of the variations in results between assays and between laboratories, efforts have been made to improve assay standardization. The United States’ Office of Dietary Supplements of the National Institutes of Health established the Vitamin D Standardization Program to improve the standardization of 25(OH)D assays. Additionally, the United States Centers for Disease Control Vitamin D Standardization Certification Program (VDSCP) provides participating laboratories with one-time sets of 40 different reference materials for bias assessment and calibration, as well as 40 blinded samples per year with assigned values measured by a reference LC-MS/MS method for both 25(OH)D2 and 25(OH)D3, to certify analytical performance such as bias and imprecision 11. Over 20 laboratories and assay manufacturers are currently participating in the CDC programme 12. Additionally, the Vitamin D External Quality Assessment Scheme, from the Charing Cross Hospital, UK, provides participating laboratories with 20 samples per year that have reference values for 25(OH)D2 and 25(OH)D3, for assessment of bias and to allow for inter-assay and between-laboratory comparisons 13.

Approximate budget requirements for analysis: The cost for a clinical analyser can vary widely but is typically around US$ 100 000. The cost for materials and supplies is approximately US$ 10–20 per sample.

Interpretation of results: There are no WHO recommended cutoff values for defining risk of deficiency at the individual or the population level. The United States IOM of the National Academies identified serum 25(OH)D concentrations for determining vitamin D status at the individual level in all age groups 7 (see Table 3.9). These values are similar to those proposed by a group of experts for the context of skeletal mineralization and mineral ion metabolism for the prevention of nutritional rickets 14.

Table 3.9. 25-Hydroxyvitamin D concentrations in serum for determining individual level vitamin D status in all age groupsa

Serum levels (nmol/L) Interpretation
<30 Risk of deficiency
30-<50 Risk of insufficiency
50-75 Likelihood of sufficiency
>75-125 No increased benefit
>125 Risk of excess

a Source: reference 7.

  1. Battault S, Whiting SJ, Peltier SL, Sadrin S, Gerber G, Maixent JM. Vitamin D metabolism, functions and needs: from science to health claims. Eur J Nutr. 2013;52:429–41. 

  2. De-Regil LM, Palacios C, Lombardo LK, Peña-Rosas JP. Vitamin D supplementation for women during pregnancy. Cochrane Database Syst Rev. 2016;(1):CD008873. doi:10.1002/14651858.CD008873.pub3 

  3. Trang HM, Cole DE, Rubin LA, Pierratos A, Siu S, Vieth R. Evidence that vitamin D3 increases serum 25-hydroxyvitamin D more efficiently than does vitamin D2. Am J Clin Nutr. 1998;68:854–8. 

  4. Tripkovic L, Lambert H, Hart K, Smith CP, Bucca G, Penson S et al. Comparison of vitamin D2 and vitamin D3 supplementation in raising serum 25-hydroxyvitamin D status: a systematic review and meta-analysis. Am J Clin Nutr. 2012;95:1357–64. 

  5. Herrmann M, Farrell CL, Pusceddu I, Fabregat-Cabello N, Cavalier E. Assessment of vitamin D status – a changing landscape. Clin Chem Lab Med. 2017;55:3–26. doi:10.1515/cclm-2016-0264. 

  6. Nutritional rickets: a review of disease burden, causes, diagnosis, prevention and treatment. Geneva: World Health Organization; 2019. Licence: CC BY-NC-SA 3.0 IGO (; accessed 14 June 2020). 

  7. Institute of Medicine (US) Committee to Review Dietary Reference Intakes for Vitamin D and Calcium. Dietary reference intakes for calcium and vitamin D. Washington, DC: National Academies Press; 2011.  2

  8. Holick MF, Binkley NC, Bischoff-Ferrari HA, Gordon CM, Hanley DA, Heaney RP et al. Guidelines for preventing and treating vitamin D deficiency. Eur J Nutr. 2013;52:429–41. 

  9. EFSA Panel on Dietetic Products, Nutrition, and Allergies (NDA). Scientific opinion on dietary reference values for vitamin D. EFSA J. 2016;14:4547–4691. doi:10.2903/j.efsa.2016.4547. 

  10. Drammeh BS, Schleicher RL, Pfeiffer CM, Jain RB, Zhang M, Nguyen PH. Effects of delayed specimen processing and freezing on serum concentrations of selected nutritional indicators. Clin Chem. 2008;54:1883–91. doi: 10.1373/clinchem.2008.108761. 

  11. Laboratory Quality Assurance and Standardization Programs. Hormone Standardization (HoST) Program and the Vitamin D Certification Program (VDSCP): standardization of measurement procedures. Atlanta: Centers for Disease Control and Prevention; 2017 (, accessed 17 June 2020). 

  12. CDC Vitamin D Standardization-Certification Program (CDC VDSCP): certified total 25-hydroxyvitamin D procedures (updated 03/2020). Atlanta: Centers for Disease Control and Prevention; 2020 (, accessed 17 June 2020). 

  13. DEQAS (Vitamin D External Quality Assessment Scheme). London: Endocrine Laboratory; 1989 (, accessed 17 June 2020). 

  14. Munns CF, Shaw N, Kiely M, Specker BL, Thacher TD, Ozono K et al. Global consensus recommendations on prevention and management of nutritional rickets. Horm Res Paediatr. 2016;85:83–106.doi:10.1159/000443136.