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. 2017 Aug 17;187(4):879–890. doi: 10.1093/aje/kwx297

Table 1.

Biomarkers of Vitamin D Metabolism and Status

Biomarker Biospecimen Measurement Options Additional Considerations
Total 25(OH)D Serum, plasma, whole blood, or blood spots LC-MS/MS, immunoassay (such as an ELISA or chemiluminescent immunoassay)

  • Stable in specimens stored long-term

  • Sum of the metabolites 25(OH)D2 and 25(OH)D3

  • Can be expressed in ng/mL or nmol/L (1 ng/mL = 2.496 nmol/L)

  • In direct comparisons, immunoassay methods show bias and increased  variability relative to LC-MS/MS

  • Immunoassay shortcomings: variable reactivity toward 25(OH)D2 and  25(OH)D3 and unable to quantify each independently (however, the need for distinction is infrequent in epidemiologic investigations); susceptible to cross-reactivity with 24,25(OH)2D; and susceptible to unidentified sample-specific matrix effects (interferences)

  • LC-MS/MS shortcomings: unless using advanced platforms, the epimer will  be measured as 25(OH)D; requires expensive equipment and expert staff
Free 25(OH)D Serum, plasma Immunoassay Calculated

  • May be most relevant in populations that show variation in VDBP  concentrations (e.g., pregnant women, women using estrogens, or those with liver or kidney disease)

  • Present at a very low concentration, making direct measurement challenging

  • No gold standard measurement method

  • An immunoassay exists to quantify free 25(OH)D, but it has not been  rigorously validated

  • Can be calculated using an equation which incorporates total 25(OH)D,  VDBP, albumin, and (possibly) genotypic differences in VDBP; however, the validity of these equations has been questioned
Bioavailable 25(OH)D Serum, plasma Calculated

  • Sum of free and albumin-bound 25(OH)D

  • Can be calculated using an equation which incorporates total 25(OH)D,  VDBP, albumin, and (possibly) genotypic differences in VDBP; however, there is some discussion regarding the validity of these equations
Vitamin D-binding protein (VDBP) Serum, plasma Immunoassay
LC-MS/MS

  • Monoclonal ELISA: The commercial assay that was most frequently used is  no longer sold due to concerns about differential binding by genotype. Be skeptical of published literature using this assay.

  • Polyclonal ELISAs: Are not biased by genotype. Several assays are now  commercially available. It will be important to validate these against LC-MS/MS.

  • LC-MS/MS: Unlikely to be biased by genotype. Has been rigorously  validated; however, each laboratory must conduct its own validation processes. Presently being used by very few laboratories to measure VDBP.
3-epi-25(OH)D Serum, plasma LC-MS/MS

  • May be more relevant for research in infants and children; in adults, the  absolute quantity of 3-epi-25(OH)D is small

  • Under typical chromatographic conditions, the epimeric form of 25(OH)D3 is  not resolved from the native form

  • Can be quantified via LS-MS/MS if a longer chromatographic separation or  more selective column is used

  • Cannot be detected with an immunoassay
1,25(OH)2D Serum, plasma Immunoassay
LC-MS/MS

  • Not an ideal epidemiologic biomarker given its short half-life, low  concentration, and tight regulation by serum calcium, phosphate, and PTH

  • Different concentrations by race/ethnicity

  • Reduced concentration in kidney disease
24,25(OH)2D3 Serum, plasma LC-MS/MS

  • Biomarker of 25(OH)D and 1,25(OH)2D catabolism

  • May serve as an indicator of tissue-level 1,25(OH)2D activity

  • Significantly reduced kidney disease

Abbreviations: ELISA, enzyme-linked immunosorbent assay; 3-epi-25(OH)D, 3-epi-25-hydroxyvitamin D; LC-MS/MS, liquid chromatography–tandem mass spectrometry; 1,25(OH)2D, 1,25-dihydroxyvitamin D; 24,25(OH)2D, 24,25-dihydroxyvitamin D; 25(OH)D, 25-hydroxyvitamin D; VDBP, vitamin D binding protein.