25-Hydroxyvitamin D (D2, D3)
The 25-Hydroxyvitamin D (D2, D3) Test measures total 25(OH)D and its ergocalciferol and cholecalciferol fractions by LC-MS/MS, the gold standard for vitamin D status. It diagnoses deficiency and insufficiency, guides repletion therapy, and is interpreted with PTH, calcium, and phosphate in metabolic bone disease, malabsorption, chronic kidney disease, and osteoporosis.
3
Biomarkers
Blood (serum)
Sample
3–5 business days
Turnaround
No
Fasting
What is the 25(OH)D test?
The 25-Hydroxyvitamin D (D2, D3) Test measures the serum concentration of total 25(OH)D and its D2 and D3 fractions by liquid chromatography-tandem mass spectrometry, the gold-standard method that resolves the two isoforms and avoids cross-reactivity with other metabolites. It is the definitive test for vitamin D status, ordered to diagnose deficiency or insufficiency, to guide repletion and maintenance dosing, and to investigate conditions in which vitamin D plays a causal or contributing role. Separate measurement of D2 and D3 adds clinical utility in monitoring adherence to specific supplementation regimens, distinguishing endogenous from exogenous sources, and troubleshooting unexpected total 25(OH)D levels. Practitioners order this test to evaluate bone health, to screen populations at risk of deficiency (malabsorption, minimal sun exposure, chronic kidney disease, obesity), and to monitor patients receiving high-dose or maintenance vitamin D therapy where serial trending informs dose adjustment and guards against toxicity.
What does the 25(OH)D test measure?
Vitamin D from both dietary ergocalciferol (D2) and skin-synthesised or supplemented cholecalciferol (D3) undergoes 25-hydroxylation in the liver to form 25-hydroxyvitamin D, the major circulating form and the best index of vitamin D status. Its half-life is several weeks, making it a stable marker that integrates skin synthesis, diet, and supplementation over time. Further 1-alpha-hydroxylation in the kidney and other tissues produces 1,25-dihydroxyvitamin D, the active hormone, but renal regulation makes 1,25(OH)2D poorly correlated with body stores and unsuitable for assessing sufficiency.
25(OH)D is the standard assay for evaluating vitamin D status. Deficiency impairs intestinal calcium absorption and can lead to secondary hyperparathyroidism, bone loss, osteomalacia in adults, and rickets in children. Insufficiency has been linked to increased fracture risk, muscle weakness, and potentially to immune, cardiovascular, and metabolic outcomes, though causality for extra-skeletal conditions remains contested. The test guides repletion therapy and is essential in the investigation of metabolic bone disease, unexplained hypocalcaemia, and chronic kidney disease-mineral bone disorder.
D2 arises from dietary ergocalciferol (plant-derived vitamin D) and some fortification sources. Its 25-hydroxylated form is measured separately by LC-MS/MS assays. D2 has lower binding affinity for vitamin D-binding protein and a shorter half-life than D3, so its contribution to total 25(OH)D is usually minor unless D2 is being supplemented.
Reporting D2 separately allows the clinician to confirm D2 supplementation or identify very low D2 in patients taking only D3, which can help track adherence and source of vitamin D. It has no independent clinical threshold but contributes to total 25(OH)D.
D3 arises from endogenous synthesis in the skin under ultraviolet B exposure and from cholecalciferol in supplements and some fortified foods. Its 25-hydroxylated metabolite has higher affinity for vitamin D-binding protein and a longer circulating half-life than the D2 form, making it the predominant contributor to total 25(OH)D in most individuals.
Reporting D3 separately confirms the source and relative contribution of cholecalciferol to vitamin D status. Some evidence suggests D3 supplementation raises total 25(OH)D more efficiently than D2, though both forms correct deficiency. Like D2, it has no independent clinical threshold but is summed into total 25(OH)D for interpretation.
Why is the 25(OH)D test ordered?
- Investigation of unexplained hypocalcaemia, secondary hyperparathyroidism, or elevated alkaline phosphatase suggestive of metabolic bone disease
- Screening of patients at high risk of deficiency: malabsorption syndromes (coeliac disease, inflammatory bowel disease, bariatric surgery), chronic kidney disease, liver disease, or obesity
- Baseline and monitoring in osteoporosis or osteopenia, especially before and during bisphosphonate or denosumab therapy
- Assessment of patients with limited sun exposure (institutionalised, veiled, high-latitude), dark skin pigmentation, or strict sun-avoidance behaviour
- Monitoring adherence and response to ergocalciferol (D2) versus cholecalciferol (D3) supplementation, where separate isoform reporting clarifies source
- Evaluation of suspected vitamin D toxicity (though 25(OH)D is ordered, not 1,25(OH)2D) or investigation of granulomatous disease with hypercalcaemia
- Paediatric assessment in rickets or failure to thrive with suspected nutritional deficiency
- Pre-operative optimisation in patients undergoing orthopaedic or bariatric surgery, where sufficiency may improve outcomes
Sample collection and turnaround
Sample type
Blood (serum)
Fasting required
No
Collection method
Venipuncture at patient service centre
Turnaround
3–5 business days
Collection notes
Phlebotomy required; specimen sent to Access Medical Laboratories for LC-MS/MS analysis. No special preparation. Protect from prolonged light exposure during handling, though 25(OH)D is relatively stable.
Specimen requirements
Minimum 1 mL serum. Separate from cells and refrigerate or freeze if not analysed promptly. Stability at room temperature is several hours; refrigerated or frozen specimens are stable for weeks to months.
What can affect 25(OH)D results?
Vitamin D is fat-soluble and sequestered in adipose tissue, lowering circulating 25(OH)D. Obese patients require higher loading doses to achieve sufficiency.
Loss of renal mass reduces 1-alpha-hydroxylase activity, impairing conversion to active 1,25(OH)2D, but 25(OH)D itself may also be low due to proteinuria (loss of vitamin D-binding protein), reduced synthesis, or dietary restriction.
Hepatic 25-hydroxylation is impaired in severe liver disease, lowering 25(OH)D. Cholestasis also reduces fat-soluble vitamin absorption.
Enzyme-inducing drugs accelerate catabolism of vitamin D metabolites via CYP24A1 and related pathways. Glucocorticoids inhibit intestinal calcium absorption and may lower 25(OH)D indirectly.
Extra-renal 1-alpha-hydroxylase in macrophages and granuloma tissue can drive unregulated production of 1,25(OH)2D, causing hypercalcaemia, but 25(OH)D levels may be normal or low; the clinical picture is hypercalcaemia with inappropriately normal or elevated 1,25(OH)2D.
Cutaneous synthesis is highest in late summer and lowest in winter at higher latitudes, so 25(OH)D shows seasonal variation in populations relying on sun exposure rather than supplementation.
What do 25(OH)D results mean?
Elevated
25(OH)D above 250 nmol/L (>100 ng/mL) raises concern for vitamin D toxicity, which can cause hypercalcaemia, hypercalciuria, nephrocalcinosis, and renal impairment. Toxicity is almost always iatrogenic (excessive supplementation) rather than from sun exposure. Levels between 150–250 nmol/L (60–100 ng/mL) are generally safe but lack evidence for additional benefit beyond skeletal health, so the risk-benefit of maintaining such levels should be individualised.
Normal
Sufficiency (≥75 nmol/L or ≥30 ng/mL by Endocrine Society criteria, or ≥50 nmol/L by IOM criteria) supports skeletal health and calcium homeostasis. Some practitioners target higher levels (100–150 nmol/L) for putative extra-skeletal benefits, but evidence is mixed and such targets are not universally endorsed.
Reduced
Deficiency (<50 nmol/L or <20 ng/mL) impairs calcium absorption and can lead to secondary hyperparathyroidism, osteomalacia, muscle weakness, and increased fracture risk. Insufficiency (50–74 nmol/L or 20–29 ng/mL) is associated with suboptimal bone health. Identify the cause — inadequate intake, malabsorption, lack of sun exposure, obesity, liver or kidney disease, or medication — and treat with ergocalciferol or cholecalciferol at doses guided by baseline level and body weight, then recheck to confirm response.
Note: Total 25(OH)D is the sum of D2 and D3; separate reporting clarifies supplementation source and can explain discrepancies. LC-MS/MS is preferred over immunoassay for accuracy and isoform resolution. Vitamin D-binding protein polymorphisms and conditions altering binding-protein concentration (nephrotic syndrome, liver disease, pregnancy) can affect total 25(OH)D without changing free (bioavailable) vitamin D, though free-D assays are not yet routine. Interpret 25(OH)D with PTH, calcium, phosphate, and alkaline phosphatase for a complete metabolic-bone picture, and remember that 1,25(OH)2D is not a marker of vitamin D status.
Frequently asked questions
D2 (ergocalciferol) comes from plant sources and some fortified foods, while D3 (cholecalciferol) is synthesised in the skin under UV light and is also available in supplements. D3 has a longer half-life and higher binding affinity, so it raises total 25(OH)D more efficiently than D2. Separate reporting by LC-MS/MS clarifies which form a patient is taking or producing.
How to access the 25(OH)D test
The 25(OH)D reference page is free and open to all practitioners. Create a free practitioner account to see pricing, save tests to your list, and generate requisition forms. Verification takes 1–2 business days.
Create Free AccountAbout this page
Published: October 7, 2026
Last reviewed: October 7, 2026
- Holick MF, et al. Endocrine Society clinical practice guideline: evaluation, treatment, and prevention of vitamin D deficiency. J Clin Endocrinol Metab. (2011)
- 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)
- Bikle DD, Schwartz J. Vitamin D binding protein, total and free vitamin D levels in different physiological and pathophysiological conditions. Front Endocrinol (Lausanne). (2019)
- Tripkovic L, 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)
- NICE guideline: Vitamin D — increasing supplement use in at-risk groups. (2023)
This content is for licensed healthcare practitioners only and does not constitute medical advice. Clinical decisions should be based on the full clinical picture and not on any single test result.