Vitamin D Testing

February 2009

Background

Vitamin D is vital for strong bones. It also has important, emerging roles in immune function and cancer prevention. Deficiencies at any stage of life can have devastating consequences. Similarly, vitamin D toxicity resulting from overmedication can cause serious hypercalcemia. Vitamin D consists of two bioequivalent forms:

• Vitamin D2, which is obtained from vegetable sources (dietary sources, supplements); and
• Vitamin D3, which is derived from both endogenous (synthesized from cholesterol through sun exposure) and exogenous (animal diet) sources.

Until recently, vitamin D replacement in the United States consisted exclusively of 25-hydroxy-vitamin D2. During the last 1-2 years this has started to change, with an increasing proportion of 25-hydroxy-vitamin D3 being used, in particular in high dose prescription formulations.

Vitamin D deficiency is more common than previously believed, especially among adolescents, women, and the elderly. For example, studies have shown that more than 50% of the institutionalized elderly and an equal proportion of women of any age undergoing treatment for osteoporosis have inadequate levels of vitamin D.¹ While treatment with vitamin supplementation is easy and inexpensive, many affected individuals go undiagnosed and untreated.

The total 25-hydroxyvitamin D (25OH Vit D) level (the sum of 25OH vitamin D2 and 25OH vitamin D3) is the appropriate indicator of vitamin D body stores. Although there is no universal consensus about a treatment cut point, studies suggest 25 to 35 ng/mL as the minimal concentration of 25OH Vit D needed to avoid the adverse effects of deficiency.^2,3,4 By contrast, population reference ranges do not correspond with healthy ranges. In northern latitude locations in particular, one third of the population may have levels below 25 ng/mL at the end of winter.

Patient Care

Who Should Be Tested?

The following should be tested for Vitamin D deficiency:

• Individuals receiving therapy to prevent or treat osteoporosis.
• Elderly people, especially those with minimal exposure to sunlight.
• Patients with signs and symptoms of hypocalcemia or hypercalcemia.
• Children and adults with suspected rickets and osteomalacia, respectively.
• Patients receiving vitamin D therapy who do not demonstrate clinical improvement.

Patient Impact

Vitamin D deficiency testing results in potential impacts:

• Establishes the need for replacement therapy and guides appropriate treatment, avoiding undermedicating the patient.
• Overtreatment can also be detected, but it is rare--occurring only with long-term replacement at doses of almost 10,000 U/day or more.

Benefits of Testing

Vitamin D definiency testing provides:

• Reliable assessment of vitamin D stores.
  
  

Technical Comparison

Ligand-Binding Assays

Technical aspects of ligand-binding assays (ie, radioimmunoassay, competitive protein-binding assays) include:

• Fundamentally difficult because such assays work best in an aqueous environment and 25OH Vit D2 and 25OH Vit D3 are poorly soluble in water.
  • Manual extraction assays can overcome solubility problems, but suffer from increased imprecision.
• Lack of result comparability between different assays, and often even between the same assays performed in different laboratories.⁵
  • One comparison of six laboratories found that “whether a patient is considered as being vitamin D insufficient depends, in large measure, on the laboratory used.”⁴
• Some assays significantly overestimate vitamin D levels, others significantly underestimate them.
  • A study conducted by the Vitamin D External Quality Assessment Scheme (DEQAS) showed a 31% overestimation by one immunoassay method.⁶
  • More typically, underestimation is observed. This is particularly the case in patients on vitamin D2 therapy; some immunoassays cannot detect 25 OH Vit D2.
• Ligand-binding assays cannot distinguish between 25OH VitD2 and 25OH VitD3. This makes it difficult to judge whether patients are compliant with therapies or suffer from vitamin D malabsorption.

High-performance liquid chromatography (HPLC)

Technical aspects of HPLC include:

• Overcomes most of the problems of the ligand-binding assays, and it was previously seen as the “gold standard.”
• Requires a relatively large sample volume (≥1.0 mL).
• Labor-intensive and time-consuming
  

Liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Technical aspects of LC-MS/MS include:

• Accurate and precise, due to internal standard and physico-chemical methodology.
  • “Gold standard” method.
  • Uses standards of defined concentrations.
  • Facilitates lab-to-lab and assay-to-assay comparisons with other MS/MS and HPLC assays.
  • Results may be used to determine if target therapeutic levels (25-80 ng/mL) have been obtained.
• Not subject to lot-to-lot variation, reagent availability issues, or licensing fees associated with proprietary kits.
• Faster than HPLC.
• Separates 25OH VitD2 and 25OH VitD3. In patients who show no improvement on replacement therapy (VitD2), absence of rise in 25OH VitD2 may indicate noncompliance or malabsorption, whereas rise in 25OH VitD2 and total vitamin D levels may indicate vitamin D resistance.
• Uses a smaller specimen volume than HPLC (0.25 mL).

How Does the Mayo Test Differ?

The Mayo Vitamin D deficiency test offers key differentiators:

• Mayo-developed and Mayo-validated LC-MS/MS assay.
• Requires fewer repeat analyses as a result of a wider reportable range.
• Provides 25OH VitD2 and 25 Vit D3 levels in addition to the total vitamin D level. This allows assessment of the source of the deficiency and also facilitates treatment monitoring.
• The total 25OH Vit D measured with the new assay correlates well with our previous methodology, which in itself has been shown to outperform other laboratories and tests.
• Improves on accuracy and precision of ligand assays, including our previous assay: ~10% CV at low 25OH Vit D levels (10 ng/mL)⁷

References

1. Holick MF, Siris ES, Binkley N, et al: Prevalence of vitamin D inadequacy among postmenopausal North American women receiving osteoporosis therapy. J Clin Endocrinol Metab 2005;90(6):3215-3224

2. Heaney RP: Vitamin D: how much do we need, and how much is too much? Osteoporos Int 2000;11:553–555

3. Chapuy MC, Preziosi P, Maamer M, et al: Prevalence of vitamin D insufficiency in an adult normal population. Osteoporos Int 1997;7:439–443

4. Binkley N, Krueger D, Cowgill CS, et al: Assay variation confounds the diagnosis of hypo-vitaminosis D: a call for standardization. J Clin Endocrinol Metab2004;89(7):3152-3157

5. Ravinder J. Singh: Are clinical laboratories prepared for accurate testing of 25-hydroxy vitamin D? Clin Chem 2008;54(1):222-223

6. Graham DC, Carter R, Jones J, Berry J: How accurate are assays for 25-Hydroxyvitamin D? Data from the International Vitamin D External Quality Assessment Scheme. Clin Chem 2004;50:2195-2197

7. Mayo unpublished quality control data

View Test

The following test is referenced:

#83670 25-Hydroxyvitamin D2 and D3, Serum

Useful for:

• Diagnosis of vitamin D deficiency
• Differential diagnosis of causes of rickets and osteomalacia
• Monitoring vitamin D replacement therapy
• Diagnosis of hypervitaminosis ...more>>