Vitamin D Testing: Is it 25-OH or 1,25-Dihydroxy?

Nam K. Tran, PhD, MS, FACB, Director of Clinical Chemistry and POCT
Dongguang Wei, MD, Pathology Resident

Introduction: Vitamin D represents a group of fat soluble sterols that help with the intestinal absorption of calcium, magnesium, phosphate, and zinc.1,2 These sterols include ergocalciferol D2, and cholecalciferol D3. Vitamin D is rapidly metabolized in the liver to form 25-hydroxy (OH) vitamin D. Further hydroxylation in the kidney under the influence of parathyroid hormone (PTH) produces 1,25-dihydroxyvitamin D.

The role of vitamin D testing in clinical practice stems from the Institute of Medicine (IoM) “Dietary Reference Intakes for Calcium and Vitamin D” report defining deficiency has having total 25-OH vitamin D (D2 + D3) blood concentrations of <12 ng/mL.2 Patients with 25-OH vitamin D levels of 12 to <20 ng/mL are considered “insufficient”, and patients with levels ≥20 ng/mL to 50 ng/mL are considered as being “sufficient”. Vitamin D values >50 ng/mL may cause adverse effects. It must be noted that the IoM criteria are based only on 25-OH vitamin D.

The testing of vitamin D often relies on immunoassay techniques. Immunoassays use antibodies that target both 25-OH vitamin D2 and D3.3 Most immunoassays report “total vitamin D” (i.e., the sum of D2 and D3 levels) from plasma or serum. The use of immunoassays enables relatively accurate results that are fast and cost-effective. Some immunoassays may be able to differentiate 25-OH vitamin D2 versus D3. Unfortunately, some immunoassays may have cross reactivity against vitamin D epimers–causing falsely elevated total 25-OH vitamin D results.4,5 For example, the presence of 3-epi-25-(OH) vitamin D3, which has low calcemic effect, can be present in significant amounts in both adults and infants!6,7 At UC Davis, we use 25-OH assays that have been shown to better discriminate against vitamin D epimers. Nonetheless “gold standard” for testing vitamin D is by liquid chromatography (LC) tandem mass spectrometry (MS/MS). These methods are less influenced by epimers and provide levels for both vitamin D2 and D3.3 However, vitamin D testing by LC-MS/MS is often a send out test and is not cost-effective.

Lab Best Practice: Although IoM recommendations using 25-OH vitamin D are clear, the inappropriate ordering of 1,25 dihydroxyvitamin D is quite common.1,2 The root causes for inappropriate 1,25 dihydroxyvitamin D ordering include: (a) clinicians not understanding the biological role of 25-OH versus 1,25 dihydroxyvitamin D, or (b) 1,25 dihydroxyvitamin D showing up as the “first” orderable test on electronic medical record (EMR) systems. At UC Davis Medical Center, the EMR list for vitamin D has been modified to show 25-OH vitamin D first, rather than 1,25 dihydroxyvitamin D to encourage proper utilization. All 1,25 dihydroxyvitamin D’s are also reviewed for appropriateness. In brief, while 1,25 dihydroxyvitamin D is the most potent vitamin D metabolite, 25-OH vitamin D levels more accurately reflect vitamin D status since it is not influenced by PTH and other hormones. In unique cases such as renal disease, 1,25 dihydroxyvitamin D levels may be used to better determine vitamin D status.

The use of LC-MS/MS for routine vitamin D testing is not recommended. Some institutions may employ LC-MS/MS for routine testing, but the laborious nature of this technique forces many of these facilities to eventually transition to immunoassays. As a send out test at UC Davis Medical Center, vitamin D testing by LC-MS/MS is reviewed for appropriateness and requires pathologist approval. LC-MS/MS vitamin D testing is best used when there is concern over epimer cross reactivity, the presence of interfering heterophilic antibodies, and/or patients that are not responding to therapy.


  1. National Institutes of Health website:, Accessed on August 1, 2017.
  2. Institute of Medicine, Food and Nutrition Board. Dietary Reference Intakes for Calcium and Vitamin D. Washington, DC: National Academy Press, 2010.
  3. Medical Laboratory Observer (MLO) article:, Accessed on August 1, 2017.
  4. Brown AJ, Ritter C, Slatopolsky E, et al. 1Alpha,25-dihydroxy-3-epi-vitamin D3, a natural metabolite of 1alpha, 25-dihydroxy-vitamin D3, is a potent suppressor of parathyroid hormone secretion. J Cell Biochem. 1999;73:106–113.
  5. Fleet JC, Bradley J, Reddy GS, et al. 1 alpha,25-(OH)2-vitamin D3 analogs with minimal in vivo calcemic activity can stimulate significant transepithelial calcium transport and mRNA expression in vitro. Arch Biochem Biophys. 1996;329:228–234.
  6. Strathmann FG, Sadilkova K, Laha TJ, et al. 3-epi-25 hydroxy-vitamin D concentrations are not correlated with age in a cohort of infants and adults. Clin Chim Acta. 2012;413:203–206.
  7. Singh RJ, Taylor RL, Reddy GS, et al. C-3 epimers can account for a significant proportion of total circulating 25-hydroxy-vitamin D in infants, complicating accurate measurement and interpretation of vitamin D status. J Clin Endocrinol Metab. 2006;91:3055–3061.
By | 2017-08-14T12:38:02+00:00 August 15, 2017|0 Comments

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