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Reference Guide
25-OH Vitamin D
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25-OH Vitamin D
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25-Hydroxy Vitamin D

Vitamin D encompasses two primary forms: Vitamin D2 (ergocalciferol) derived from plant sources and fortified foods, and Vitamin D3 (cholecalciferol), which is synthesized in the skin through sunlight exposure and also obtained from certain animal-based foods. 

Both forms, which are also available as supplements, require metabolic activation in the body through a two-step hydroxylation process. The first critical step occurs in the liver, where either form of vitamin D is converted into 25-hydroxyvitamin D (25(OH)D), also known as calcidiol. 

This metabolite is a prohormone and the principal circulating form of vitamin D, serving as a vital precursor to the biologically active hormone, calcitriol (1,25-dihydroxyvitamin D), synthesized in the kidneys.

As the most reliable marker of vitamin D status, measuring serum levels of 25(OH)D is essential for diagnosing deficiencies or excesses and monitoring the efficacy of supplementation. 

This article explains the sources, metabolism, physiological roles, and health implications of 25-hydroxyvitamin D, highlighting its importance in bone health, immune function, and beyond, while also discussing the nuances of vitamin D supplementation and its role in preventing a range of chronic conditions.

Understanding 25-OH Vitamin D

What is Vitamin D?  [8., 21.]

Vitamin D refers to the parent compounds, vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol). These are obtained from dietary sources or produced in the skin upon exposure to sunlight (specifically UVB radiation).

Vitamin D2 and Vitamin D3 are also available as supplements. 

Vitamin D3 is the form naturally synthesized in the human body, while vitamin D2 is derived from plant sources and is used in some supplements and fortified foods.

Both vitamin D3 and D2 are biologically inert and require two hydroxylation steps in the body to become active.

The first hydroxylation step produces 25-OH vitamin D.

What is 25-OH Vitamin D?  [8., 21., 22., 23.]

25-hydroxyvitamin D (25(OH)D), also known as calcidiol, is a prohormone and the major circulating form of vitamin D in the body.  It is produced in the liver by hydroxylation of vitamin D  (as D2, ergocalciferol or D3, cholecalciferol) by the enzyme 25-hydroxylase (CYP2R1).

As a prohormone, 25(OH)D is biologically inactive and must undergo further hydroxylation in the kidneys by the enzyme 1α-hydroxylase (CYP27B1) to form the biologically active form, 1,25-dihydroxyvitamin D (1,25(OH)2D), also known as calcitriol.

This active form is responsible for maintaining calcium and phosphate homeostasis, as well as regulating various other physiological processes.

The total serum level of 25(OH)D, which includes both 25(OH)D3 and 25(OH)D2, is considered the best indicator of vitamin D status in the body.  This is because 25(OH)D has a longer half-life of approximately 2-3 weeks, compared to the shorter half-life of vitamin D and 1,25(OH)2D.

Measuring serum 25(OH)D levels is crucial for diagnosing vitamin D deficiency, insufficiency, or toxicity, as well as monitoring the efficacy of vitamin D supplementation.

Functions of Vitamin D: What Does Vitamin D Do?  [2., 3., 9., 13., 18., 19.]

Vitamin D plays several vital roles in the body:

  • Bone Health: 
  • Calcium Absorption: It's crucial for calcium absorption in the gut, which is necessary for maintaining healthy bones and teeth. This is particularly important in preventing conditions like rickets in children and osteoporosis in adults.  Adequate calcium in the blood is also essential for muscle and nerve function.
  • Phosphate Absorption: vitamin D also regulates calcium absorption in the GI tract
  • Balances bone resorption and remodeling for healthy growth and bone preservation in adulthood
  • Immune System Function: Vitamin D modulates the immune system.  Specifically, vitamin D promotes a healthy immune balance, prioritizing TH1 cellular immunity and the generation of CD4+CD25+ regulatory T cells (Tregs), essential in immune regulation and mitigating autoimmune conditions. 
  • Mood Regulation and Brain Health: There's growing evidence linking Vitamin D levels to mood regulation and a reduced risk of depression. It's also thought to play a key role in brain health and cognitive function.
  • Muscle and Nerve Function: Adequate Vitamin D levels are important for muscle strength, coordination, and proper nerve signaling.
  • Cardiovascular Health: Emerging research suggests Vitamin D might play a role in heart health, although more research is needed to confirm this association and understand optimal vitamin D levels.  [6.]

RDA of Vitamin D

The recommended dietary allowance (RDA) of Vitamin D varies depending on age, gender, and other factors. Generally, these guidelines are:  [9., 15.]

Infants (0-12 months): 400 IU (10 mcg) per day.

Children and Teenagers (1-18 years): 600 IU (15 mcg) per day.

Adults (19-70 years): 600 IU (15 mcg) per day.

Older Adults (over 70 years): 800 IU (20 mcg) per day.

Pregnant and Breastfeeding Women: 600 IU (15 mcg) per day.

Sources of Vitamin D: What Foods Contain Vitamin D?

Dietary Sources of Vitamin D  [15.]

Vitamin D3 is synthesized in the skin in humans, and it can also be obtained through the consumption of animal-based foods in the diet.

  • Fatty fish such as salmon, mackerel, and tuna are rich dietary sources of vitamin D.
  • Egg yolks and fortified foods such as fortified milk, orange juice, and cereals also contribute to dietary intake of vitamin D.
  • Some mushrooms, particularly those exposed to ultraviolet light, contain small amounts of vitamin D.  Mushrooms contain a compound called ergosterol that is converted to vitamin D2, ergocalciferol, in the presence of UV light.  [7.]

Vitamin D Forms and Supplement Indications

Vitamin D is available as vitamin D2, ergocalciferol, and vitamin D3, cholecalciferol.  

Why Do Doctors Prescribe Vitamin D2 Instead of Vitamin D3? 

Doctors may prescribe vitamin D2 (ergocalciferol) instead of vitamin D3 (cholecalciferol) in certain situations based on patient-specific factors and clinical considerations. 

For example, the decision to prescribe vitamin D2 could be due to its availability in different formulations, such as vegetarian or vegan-friendly options, making it suitable for individuals with dietary restrictions or preferences. 

Additionally, some patients may have medical conditions or allergies that preclude the use of vitamin D3 sourced from animal products. 

Furthermore, cost considerations or insurance coverage may influence the choice of vitamin D formulation, as vitamin D2 may be more affordable or covered by insurance plans. 

However, it's essential to note that while vitamin D2 and D3 are both effective at raising serum vitamin D levels, there is ongoing debate regarding their comparative efficacy and bioavailability.  In the absence of contraindications, many experts recommend vitamin D3 supplementation.   [11.]

The decision to prescribe vitamin D2 over D3 should be based on individual patient needs, preferences, and clinical judgment.

Supplement Indications for Vitamin D: What is Vitamin D Good For?  

Deficiency Symptoms 

Vitamin D deficiency can lead to bone pain, muscle weakness, and increased risk of bone fractures. In severe cases, it can cause rickets in children and osteomalacia or osteoporosis in adults.  [3., 9., 15.]

Association with Chronic Conditions 

Deficient levels of Vitamin D have been associated with various chronic conditions such as autoimmune diseases including type 1 diabetes, osteoporosis, neurodegenerative and mood conditions, rickets in children and may be associated with cardiovascular problems.  [1., 2., 3., 6., 8.]

Cardiovascular Health

Adequate Vitamin D levels may be associated with a reduced risk of heart disease, possibly by influencing blood pressure and vascular health.  [6.]

Cancer Prevention 

Some studies suggest a link between adequate Vitamin D levels and a reduced risk of development of certain cancers, particularly of advanced or fatal cancers.  This benefit may be more apparent in non obese people.  [4.]

Testing Vitamin D Levels

Laboratory Testing Methods

Vitamin D measurements are typically done via serum, although they may also be done via plasma or blood spot analysis.  Fasting or other special preparation is not typically required for a vitamin D test, but patients should consult their healthcare provider to confirm.  

Sample Collection and Preparation

Sample collection and preparation for testing calcitriol levels typically involve obtaining blood samples through venipuncture.  It is a relatively simple and easy sample collection, although some people find blood draws unpleasant.  

Alternatively, blood spot testing for vitamin D involves collecting a small sample of blood from a finger prick onto filter paper. 

This method offers convenience for both patients and healthcare providers and can provide accurate measurements of vitamin D levels, making it useful for screening and monitoring vitamin D status.  A blood spot sample can be taken from the comfort of a patient’s home.  

Interpretation of Vitamin D Blood Tests

What is Measured in a Vitamin D Blood Test?

Vitamin D blood testing measures the concentration of specific forms of vitamin D in the blood. 

This typically includes the measurement of 25-hydroxyvitamin D (25(OH)D), which is the major circulating form of vitamin D and is considered the best indicator of overall vitamin D status as it reflects dietary intake and skin synthesis.  [9.]

Serum analysis of 25(OH) vitamin D comprises 25(OH) vitamin D2 and 25(OH) vitamin D3.  [9.]

Some labs may also measure the active form of vitamin D, known as 1,25-dihydroxyvitamin D (1,25(OH)2D), although this is less commonly done as it has a shorter half-life and may not reflect long-term vitamin D status as accurately as 25(OH)D.

Optimal Ranges for Vitamin D in the Blood

Typical recommended ranges for the total 25-OH vitamin D, measured in ng/mL, is 25-80 ng/mL.  [16.]

Results may also be reported as nmol/L, which has a reference range of 75-125 nmol/L.  [14.]

Many experts define vitamin D deficiency according to the following levels, although it is important to note that there is some discrepancy regarding vitamin D insufficiency:  [14.]

  • Severe deficiency: a serum 25-hydroxyvitamin D (25(OH)D) level below 12 ng/mL (30 nmol/L) which requires intervention through diet changes or supplements
  • Deficiency: a serum 25-hydroxyvitamin D (25(OH)D) level between 12-20 ng/mL (30-50 nmol/L), suggesting a need for increased sun exposure, dietary intake, or supplementation.
  • Vitamin D sufficiency:  a serum 25-hydroxyvitamin D (25(OH)D) level above 30 ng/mL (75 nmol/L)
  • Vitamin D toxicity: serum 25(OH)D levels greater than 150 ng/mL (374 nmol/L)  [21.]

Factors Affecting Vitamin D Levels

Various factors can influence the accuracy of Vitamin D testing:

  • Geographical Location and Sun Exposure: Less sun exposure can lead to lower Vitamin D synthesis in the skin.
  • Dietary Habits: Limited intake of Vitamin D-rich foods can affect levels.
  • Age and Skin Pigmentation: Older individuals and those with darker skin synthesize less Vitamin D from sunlight.

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25-OH Vitamin D
25-Hydroxy Vitamin D, commonly known as Vitamin D, is a special nutrient that your body creates when your skin is exposed to sunlight, specifically the sun's ultraviolet B (UVB) rays. You can also get it from certain foods and supplements. This vitamin is changed in the liver into 25-Hydroxy Vitamin D, which is the main form stored in your body and the one that your doctor measures to check your Vitamin D levels. It has a crucial role in keeping your bones and teeth healthy, supporting your immune system, brain, and nervous system. Additionally, it helps regulate insulin levels and promotes lung function and heart health. In short, 25-Hydroxy Vitamin D is essential for your overall health and well-being.
If Your Levels Are High
High levels of 25-OH Vitamin D might mean that your body is taking in more of this nutrient than it needs. This can happen if you're consuming a lot of Vitamin D from supplements, certain medications (like antifungal drugs or antiretroviral medications), or eating a diet rich in Vitamin D. Sometimes, high levels can also be due to less common conditions like sarcoidosis or other diseases that affect how your body processes this vitamin. It's important to know that while Vitamin D is essential for your health, having too much can lead to a condition called hypervitaminosis D, which can cause an excess of calcium in your blood and potentially affect your heart, bones, and kidneys. Keep in mind that high Vitamin D levels are usually not caused by sun exposure or diet alone.
Symptoms of High Levels
Symptoms of high levels of 25-OH Vitamin D could include nausea, vomiting, weakness, frequent urination, and kidney problems. In severe cases, it could also lead to bone pain and heart abnormalities.
If Your Levels are Low
Low levels of 25-OH Vitamin D might mean that you're not getting enough sunlight, which is important for your body to produce this essential nutrient. Your diet could also be lacking in Vitamin D-rich foods like fatty fish, cheese, and egg yolks. Factors like living far from the equator, having darker skin, or using sunscreen often can also contribute to lower Vitamin D levels. Some medications, such as certain anti-seizure drugs, can affect how your body processes Vitamin D. Additionally, low levels could be a sign of underlying conditions like celiac disease, which can make it harder for your body to absorb nutrients from food.
Symptoms of Low Levels
Symptoms of low levels of 25-OH Vitamin D could include fatigue, bone pain, muscle weakness, mood changes, and frequent illness or infections.
See References

[1.] Abugoukh TM, Al Sharaby A, Elshaikh AO, Joda M, Madni A, Ahmed I, Abdalla RS, Ahmed K, Elazrag SE, Abdelrahman N. Does Vitamin D Have a Role in Diabetes? Cureus. 2022 Oct 18;14(10):e30432. doi: 10.7759/cureus.30432. PMID: 36407246; PMCID: PMC9671203. 

[2.] Akpınar Ş, Karadağ MG. Is Vitamin D Important in Anxiety or Depression? What Is the Truth? Curr Nutr Rep. 2022 Dec;11(4):675-681. doi: 10.1007/s13668-022-00441-0. Epub 2022 Sep 13. PMID: 36097104; PMCID: PMC9468237.

[3.] Bhattarai HK, Shrestha S, Rokka K, Shakya R. Vitamin D, Calcium, Parathyroid Hormone, and Sex Steroids in Bone Health and Effects of Aging. J Osteoporos. 2020 Jun 17;2020:9324505. doi: 10.1155/2020/9324505. PMID: 32612801; PMCID: PMC7317615.

[4.] Chandler PD, Chen WY, Ajala ON, et al. Effect of Vitamin D3 Supplements on Development of Advanced Cancer. JAMA Network Open. 2020;3(11):e2025850. doi:‌

[5.] Colak A, Toprak B, Dogan N, Ustuner F. Effect of sample type, centrifugation and storage conditions on vitamin D concentration. Biochem Med (Zagreb). 2013;23(3):321-5. doi: 10.11613/bm.2013.039. PMID: 24266302; PMCID: PMC3900071.

[6.] Cosentino N, Campodonico J, Milazzo V, De Metrio M, Brambilla M, Camera M, Marenzi G. Vitamin D and Cardiovascular Disease: Current Evidence and Future Perspectives. Nutrients. 2021; 13(10):3603. 

[7.] Haytowitz DB. Vitamin D in Mushrooms.; 2009. 

[8.] Hollis BW, Wagner CL, Drezner MK, Binkley NC. Circulating vitamin D3 and 25-hydroxyvitamin D in humans: An important tool to define adequate nutritional vitamin D status. J Steroid Biochem Mol Biol. 2007 Mar;103(3-5):631-4. doi: 10.1016/j.jsbmb.2006.12.066. Epub 2007 Jan 10. PMID: 17218096; PMCID: PMC1868557.

[9.] Institute of Medicine (US) Committee to Review Dietary Reference Intakes for Vitamin D and Calcium; Ross AC, Taylor CL, Yaktine AL, et al., editors. Dietary Reference Intakes for Calcium and Vitamin D. Washington (DC): National Academies Press (US); 2011. 3, Overview of Vitamin D. Available from:

[10.] Kahwati LC, Weber RP, Pan H, et al. Vitamin D, Calcium, or Combined Supplementation for the Primary Prevention of Fractures in Community-Dwelling Adults: An Evidence Review for the U.S. Preventive Services Task Force [Internet]. Rockville (MD): Agency for Healthcare Research and Quality (US); 2018 Apr. (Evidence Synthesis, No. 160.) Appendix A Table 1, Serum Vitamin D Level Reference Ranges. Available from:   

[11.] Kennel KA, Drake MT, Hurley DL. Vitamin D deficiency in adults: when to test and how to treat. Mayo Clin Proc. 2010 Aug;85(8):752-7; quiz 757-8. doi: 10.4065/mcp.2010.0138. PMID: 20675513; PMCID: PMC2912737.

[12.] Krasniqi E, Boshnjaku A, Wagner KH, Wessner B. Association between Polymorphisms in Vitamin D Pathway-Related Genes, Vitamin D Status, Muscle Mass and Function: A Systematic Review. Nutrients. 2021 Sep 4;13(9):3109. doi: 10.3390/nu13093109. PMID: 34578986; PMCID: PMC8465200.

[13.] Latimer CS, Brewer LD, Searcy JL, et al. Vitamin D prevents cognitive decline and enhances hippocampal synaptic function in aging rats. Proceedings of the National Academy of Sciences. 2014;111(41):E4359-E4366. doi:

[14.] Lung BE, Mowery ML, Komatsu DEE. Calcitriol. [Updated 2023 Jul 17]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 Jan-. Available from: 

[15.] National Institutes of Health. Vitamin D. Published September 18, 2023. 

[16.] Pagana KD, Pagana TJ, Pagana TN. Mosby’s Diagnostic & Laboratory Test Reference. 14th ed. St. Louis, Mo: Elsevier; 2019.

[17.] Pike JW, Shevde NK, Hollis BW, Cooke NE, Zella LA. Vitamin D—binding protein influences total circulating levels of 1,25-dihydroxyvitamin D-3 but does not directly modulate the bioactive levels of the hormone in vivo. Endocrinology. 2008;149(7):3656–67.

[18.] PubChem. Calcitriol. 

[19.] Sîrbe C, Rednic S, Grama A, Pop TL. An Update on the Effects of Vitamin D on the Immune System and Autoimmune Diseases. Int J Mol Sci. 2022 Aug 29;23(17):9784. doi: 10.3390/ijms23179784. PMID: 36077185; PMCID: PMC9456003.

[20.] Vieth R. Vitamin D supplementation: cholecalciferol, calcifediol, and calcitriol. Eur J Clin Nutr. 2020 Nov;74(11):1493-1497. doi: 10.1038/s41430-020-0697-1. Epub 2020 Jul 23. PMID: 32704098.

[21.] Vitamin D3 25-Hydroxyvitamin D: Reference Range, Interpretation, Collection and Panels. eMedicine. Published online April 27, 2021. Accessed March 12, 2024.

[22.] Vitamin D3 1,25-Dihydroxyvitamin D: Reference Range, Interpretation, Collection and Panels. eMedicine. Published online May 19, 2021.

[23.] Wootton AM. Improving the measurement of 25-hydroxyvitamin D. Clin Biochem Rev. 2005 Feb;26(1):33-6. PMID: 16278775; PMCID: PMC1240027. 

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25-Hydroxy Vitamin D

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