What is 25-Hydroxyvitamin D (25(OH)D)?
25-hydroxyvitamin D (25(OH)D) is the primary circulating form of vitamin D in the blood and serves as the most accurate biomarker for assessing total body vitamin D status. 1, 2
Biochemical Formation and Function
25(OH)D is formed in the liver through the first hydroxylation of vitamin D (either D2 or D3) on carbon 25, creating a biologically inactive but stable metabolite that reflects vitamin D stores 2, 3
This metabolite requires a second hydroxylation in the kidney (on carbon 1) to form 1,25-dihydroxyvitamin D [1,25(OH)₂D], which is the biologically active form of vitamin D 2, 3
Serum 25(OH)D concentration is the gold standard for determining vitamin D status because it has a longer half-life (2-3 weeks) compared to the active form and accurately reflects both dietary intake and cutaneous synthesis 1, 2
Why 25(OH)D is Measured Instead of Active Vitamin D
Measuring 1,25(OH)₂D (the active form) provides no useful information about vitamin D status because it is often normal or even elevated in vitamin D deficiency due to compensatory secondary hyperparathyroidism 1, 2
In chronic kidney disease, the relationship changes: 1,25(OH)₂D levels become more dependent on available 25(OH)D substrate as kidney function declines 1
Clinical Significance of 25(OH)D Levels
Deficiency thresholds vary by guideline, but most experts agree on the following ranges:
- Severe deficiency: <20 ng/mL (50 nmol/L) 1, 2
- Insufficiency: 20-30 ng/mL (50-75 nmol/L) 1, 2
- Sufficiency: ≥30 ng/mL (75 nmol/L) for optimal health benefits 1, 2, 4
- Upper safety limit: 100 ng/mL 1
Levels below 30 ng/mL are associated with increased parathyroid hormone secretion, reduced bone mineral density, and elevated fracture risk 1, 5, 2
Measurement Methodology
LC-MS/MS (liquid chromatography-tandem mass spectrometry) is the gold standard analytical method for measuring total 25(OH)D, which includes both 25(OH)D2 and 25(OH)D3 1
The assay should measure both vitamin D2 (ergocalciferol) and D3 (cholecalciferol) forms to capture total vitamin D status 1
There is no circadian rhythm for 25(OH)D, but there is a strong seasonal variation with lowest levels after winter (March in Northern Hemisphere) and highest after summer 1
Important Clinical Caveats
Conditions affecting vitamin D-binding protein (DBP) or albumin synthesis can alter total 25(OH)D measurements without changing biologically active free vitamin D levels 6
Liver disease, pregnancy (due to estrogen-stimulated DBP synthesis), and nephrotic syndrome with urinary protein losses all affect total 25(OH)D concentrations 6
Genetic polymorphisms in the DBP gene vary by race and can influence circulating 25(OH)D levels independent of true vitamin D status 6
In patients with chronic kidney disease, 25(OH)D levels are commonly low due to reduced sun exposure, dietary restrictions, decreased cutaneous synthesis, and urinary losses of vitamin D-binding protein in nephrotic-range proteinuria 1