Are there different blood tests for vitamin D and its active form, 1,25-dihydroxyvitamin D (1,25(OH)2D)?

Yes, There Are Two Different Blood Tests for Vitamin D

Yes, there are distinct blood tests for vitamin D: the 25-hydroxyvitamin D [25(OH)D] test measures vitamin D stores and is the standard test for assessing vitamin D status, while the 1,25-dihydroxyvitamin D [1,25(OH)₂D] test measures the active hormonal form but does not reflect vitamin D status. 1

Which Test to Order

Standard Vitamin D Testing

• Order the 25-hydroxyvitamin D [25(OH)D] test for routine vitamin D assessment, as this is the barometer for vitamin D status and the gold standard across all major guidelines 2, 1
• The 25(OH)D test measures both vitamin D₂ (ergocalciferol) and D₃ (cholecalciferol) forms, providing a complete picture of total body vitamin D availability 2
• Serum 25(OH)D is the most reliable biochemical marker for defining vitamin D stores in the body 2

When NOT to Order Active Vitamin D Testing

• Do not order 1,25-dihydroxyvitamin D [1,25(OH)₂D] testing for routine vitamin D status assessment, as it provides no information about vitamin D status 1
• The 1,25(OH)₂D level is often normal or even paradoxically elevated in vitamin D deficiency due to secondary hyperparathyroidism 1
• The active form has a much shorter half-life and does not reflect body stores 3

Key Differences Between the Tests

25-Hydroxyvitamin D [25(OH)D]

• Reflects vitamin D stores from both sun exposure and dietary intake 1
• Has higher affinity for vitamin D binding protein (VDBP) 3
• Longer circulating half-life makes it ideal for status assessment 3
• Target level should be at least 30-40 ng/mL for optimal health 2

1,25-Dihydroxyvitamin D [1,25(OH)₂D]

• The biologically active hormonal form that binds to vitamin D receptors 1
• Has higher affinity for the vitamin D receptor (VDR) than 25(OH)D 3
• Tightly regulated by parathyroid hormone and does not correlate with vitamin D stores 1
• Reserved for specific clinical scenarios like renal disease or calcium metabolism disorders 1

Testing Methodology Considerations

Assay Selection

• Choose an assay that measures both 25(OH)D₂ and 25(OH)D₃ forms, especially where both vitamin D₂ and D₃ supplements are available 2
• High-performance liquid chromatography with mass spectrometry (LC-MS/MS) and immunoassays are the most widely used methods 2, 4
• Be aware that classification of samples as "deficient" or "nondeficient" can vary by 4% to 32% depending on which assay is used 5, 2

Sample Collection

• Serum is the sample of choice; avoid gel tubes until more data are available 2
• No significant difference exists between serum and lithium-heparin plasma samples 6

Interpretation Guidelines

Target Ranges for 25(OH)D

• Deficiency: <20 ng/mL 1
• Insufficiency: 21-29 ng/mL 1
• Optimal: ≥30 ng/mL (30-40 ng/mL preferred) 2, 1
• Upper safety limit: 100 ng/mL 2

Monitoring After Supplementation

• Wait at least 3 months after starting vitamin D supplementation before retesting 25(OH)D levels 2
• Response to treatment varies based on environmental factors and baseline levels 2

Common Pitfalls to Avoid

• Do not confuse the two tests: ordering 1,25(OH)₂D when you need 25(OH)D is a common error that provides no useful information about vitamin D status 1
• Do not interpret normal 1,25(OH)₂D as adequate vitamin D status: this can miss significant vitamin D deficiency 1
• Be aware of significant inter-laboratory and inter-assay variability (10-20%) when interpreting borderline results 2
• Recognize that total serum 25(OH)D may underestimate vitamin D status in African Americans due to differences in vitamin D binding protein, though bioavailable 25(OH)D may be similar across races 5