Empeiria

25-hydroxyvitamin D

Also known as vitamin D, 25-OH vitamin D, calcidiol, vit D

C₂₇H₄₄O₂ small molecule

LOINC 62292-8 code verified serum ng/mL

unverified These explanations are written to be easy to read and have not yet been checked against a source. The ranges and measurement notes on this page show theirs.

definition unverified

The storage form of vitamin D, and the right one to measure. Your skin makes it from sunlight, and your liver and kidneys then convert it into the active form.

The storage form of vitamin D, and the right one to measure. Your skin makes it from sunlight, and your liver and kidneys then convert it into the active form.

No clinical version written yet.

why measured unverified

Shortage is very common, especially in winter, in darker skin, and in people who spend most of their time indoors.

Shortage is very common, especially in winter, in darker skin, and in people who spend most of their time indoors.

No clinical version written yet.

if high unverified

Essentially only happens from taking large supplement doses. It is worth avoiding, because it pushes blood calcium up.

Essentially only happens from taking large supplement doses. It is worth avoiding, because it pushes blood calcium up.

No clinical version written yet.

if low unverified

Common and usually straightforward to correct. It matters most for bone strength, where a long shortage genuinely weakens bone.

Common and usually straightforward to correct. It matters most for bone strength, where a long shortage genuinely weakens bone.

No clinical version written yet.

caveats unverified

You will see vitamin D linked to many other conditions. Those links come from studies that watch people rather than test treatments. When trials did test it, most of the benefits did not hold up. Bone health is the part that does.

You will see vitamin D linked to many other conditions. Those links come from studies that watch people rather than test treatments. When trials did test it, most of the benefits did not hold up. Bone health is the part that does.

No clinical version written yet.

What this substance is

formula: C₂₇H₄₄O₂
mass: 400.647 g/mol
monoisotopic: 400.33413
charge: 0
InChIKey: JWUBBDSIWDLEOM-DTOXIADCSA-N
ChEBI: CHEBI:17933
CAS: 19356-17-3

Drawn from ChEBI's own curated 2D drawing, accepted only after its InChIKey, formula and charge matched the ChEBI release. Nothing here is hand-entered. Two masses are given because they answer different questions: the average is natural-abundance weighted and drives unit conversion; the monoisotopic value uses only the commonest isotope of each element and is what mass spectrometry measures.

3D model · 25-hydroxyvitamin D

Generated by RDKit from ChEBI's verified structure, and accepted only because its own 3D geometry reproduces ChEBI's InChIKey. A flexible molecule takes many shapes; this is one low-energy one. Drag to rotate, scroll to zoom.

Reference ranges

Checking your own result against this range needs the running application; this is a static copy. Nothing you type anywhere in Empeiria is ever sent to a server.

all

20 50

normal range · ng/mL

Applies to	Normal	Optimal	Critical
all	20–50 ng/mL	—	—

guideline limit Vitamin D Fact Sheet for Health Professionals, NIH Office of Dietary Supplements cited · not yet checked by a person

How it is measured

In practice

This is a blood test. A health worker takes blood from a vein in your arm. Most vitamin D tests measure a form called 25-hydroxyvitamin D, which shows best whether you have enough vitamin D. You usually do not need to fast, but tell your provider about any vitamins and supplements you take.

Venous blood sample. Most vitamin D tests measure 25(OH)D, the hepatic metabolite, as the most accurate indicator of vitamin D status; 1,25-dihydroxyvitamin D ("active vitamin D") is a different test not used to assess sufficiency. Fasting is usually not required but depends on the laboratory and method.

Before the test

Follow any instructions from your lab about not eating before the test.
Tell your provider about all medicines, vitamins and supplements you take.

Where: laboratory

Sources: 25-hydroxy vitamin D test, MedlinePlus Medical Encyclopedia; Vitamin D Test, MedlinePlus (U.S. National Library of Medicine) cited · not yet checked by a person

In the lab

mass spectrometry

LC-MS/MS

The sample is pushed through a column that separates its parts, then weighed by a very precise instrument. Unlike the antibody tests, it can report the D2 and D3 forms separately.

Liquid chromatography separates the D2 and D3 metabolites, which are then quantified by tandem mass spectrometry against a labelled internal standard.

Reference method: NIST and CDC reference measurement procedures
Standardised to: Vitamin D Standardization Program (VDSP)

Isotope-labelled internal standard

d6-25-hydroxyvitamin D3 (alongside deuterium-labelled 25-hydroxyvitamin D2 and 3-epi standards), added to every sample before extraction; each result is read as the ratio of the analyte's peak to its standard's peak.

Why labs disagree: Immunoassays and mass spectrometry often disagree, partly because antibodies do not recognise every form of 25-hydroxyvitamin D equally, including the D2 form. Results from different methods are not interchangeable, so the same method should be used for repeat tests, and the Vitamin D Standardization Program exists to bring methods into line.

Sources: Laboratory Procedure Manual, 25-Hydroxyvitamin D2, D3 and 3-epi-D3 by HPLC-MS/MS, NHANES 2017-2018, CDC/NCHS; Vitamin D Controversies in the Laboratory Medicine: A Review of Clinical Guidelines and Recommendations. EJIFCC 2024 (PMC open access) cited · not yet checked by a person

immunoassay · less common

Competitive chemiluminescent immunoassay

Simpler and faster to run on automated machines, but it cannot tell the two forms of vitamin D apart and reacts partly with related substances.

Competitive-binding antibody assay with a chemiluminescent read-out.

What can throw it off

Cross-reactivity with other vitamin D metabolites

Sources: 25OHD analogues and vacuum blood collection tubes dramatically affect the accuracy of automated immunoassays. Sci Rep 2015 (PMC open access); Analysis of chemiluminescence and liquid chromatography-mass spectrometry in 25-hydroxyvitamin D detection using fuzzy logic. Sci Rep 2026 (PMC open access) cited · not yet checked by a person

What it reports on

liver synthesized by
The liver changes vitamin D into 25-hydroxyvitamin D, the form most vitamin D blood tests measure.
skin of body synthesized by
Skin makes vitamin D in sunlight, which the liver then turns into the 25-hydroxyvitamin D that is measured.

Associated symptoms

When low

low Deep aching in the bones · suggestive · occasional
Defective bone mineralisation in prolonged severe deficiency.
low Muscles feeling weak · non specific · occasional
low Feeling tired all the time · non specific · common
low Feeling low or flat · non specific · occasional

Conditions this marker features in

When low

low Low vitamin D · defining
Vitamin D status is classified by blood 25-hydroxyvitamin D: deficiency is below 30 nmol/L (12 ng/mL), insufficiency 30 to 50 nmol/L.
low Low blood calcium · supportive
Part of the first work-up. Severe vitamin D deficiency is one of the causes.
low Overactive parathyroid gland · supportive
Low vitamin D is common in this condition and is checked. Vitamin D deficiency must be ruled out before diagnosing the normocalcaemic form, because it can raise PTH by itself.

Ordered as part of