Measure Total 25-Hydroxyvitamin D (25-OH-D)
When checking vitamin D levels, you should order total 25-hydroxyvitamin D (25-OH-D), which represents the sum of both 25-OH-D2 and 25-OH-D3. This is the single best biomarker for assessing vitamin D status and is the universally recommended test across all major clinical guidelines 1, 2, 3.
Why Total 25-OH-D is the Correct Test
The most recent ESPEN guideline (2022) explicitly states that "serum/plasma concentrations of total 25-hydroxyvitamin D (25-OHD), the sum of 25-OHD3 and 25-OHD2, is recognized as a valid biomarker for vitamin D status" 1. This recommendation is echoed by:
The Endocrine Society (2011): Recommends using serum 25(OH)D measured by a reliable assay to evaluate vitamin D status, explicitly stating this is the major circulating form with a 2-3 week half-life 3
Cystic Fibrosis Foundation (2012): States that "clinically, the total 25(OH)D should be adequate to assess vitamin D status, which accounts for vitamin D produced from skin and obtained from the diet" 2
Critical Point: What NOT to Order
Do not order 1,25-dihydroxyvitamin D [1,25(OH)2D] to assess vitamin D status 2, 3. This is a common pitfall. The 1,25(OH)2D has:
- An extremely short half-life of only 4 hours (versus 2-3 weeks for 25-OH-D)
- Levels that are tightly regulated by PTH, calcium, and phosphate—not by vitamin D stores
- Concentrations that can be normal or even elevated in vitamin D deficiency due to secondary hyperparathyroidism
- No reflection of actual vitamin D reserves
The 1,25(OH)2D test is only useful for specific inherited or acquired disorders of vitamin D metabolism, chronic kidney disease, or granulomatous diseases like sarcoidosis 3.
Assay Requirements
When ordering the test, ensure your laboratory uses an assay that:
Measures both D2 and D3 forms: This is critical in countries where vitamin D2 (ergocalciferol) is used for supplementation 4. Some assays don't measure both forms with equal sensitivity 2
Reports the total sum: If the lab separates 25-OH-D2 and 25-OH-D3 (using LC-MS/MS), they should report the sum as the main result 4
Uses standardized methodology: LC-MS/MS has become the gold standard with better accuracy than immunoassays, particularly for patients on vitamin D2 supplementation 1, 5
Common Pitfalls to Avoid
Don't order PTH, osteocalcin, or alkaline phosphatase to assess vitamin D status routinely—these don't reflect vitamin D stores and add unnecessary cost 2
Timing considerations: While there's no circadian rhythm for 25-OH-D, there is seasonal variation with lowest levels at the end of winter 1, 2. Ideally test at the end of winter to capture the nadir, ensuring year-round sufficiency
Fasting is not required: There's no evidence supporting the need for fasting before 25-OH-D measurement 2
Avoid gel tubes: Some evidence suggests gel tubes may interfere with LC-MS/MS measurements 4
Target Levels for Clinical Decision-Making
Once you have the total 25-OH-D result:
- Deficiency: <20 ng/mL (50 nmol/L) 3
- Insufficiency: 21-29 ng/mL 3
- Sufficiency: ≥30 ng/mL, with many experts recommending 30-40 ng/mL or higher for optimal health 3, 4
- Upper safety limit: <100 ng/mL 4
The straightforward answer: order total 25-hydroxyvitamin D and nothing else for routine vitamin D status assessment.