Vitamin D

Vitamin D
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Measuring Total Vitamin D

Vitamin D levels can be measured separately or as a total value, but not all immunoassays have the same reactivity to vitamin D2 and D3. Some immunoassays only detect one type of vitamin D and others may not fully detect the entire amount. No matter which assay you use, the most important value is the final total value, since it represents the total amount of 25-hydroxy-vitamin D (both D2 and D3) in the blood. This ensures patients have the most accurate result regardless of level and whether or not they are supplemented over-the-counter or by prescription.

Measuring Total Vitamin in Determining Sufficiency

In the case of a true concentration that is just into the sufficiency vitamin D range, if the assay does not detect D2, it is likely that result will be reported in the insufficient range. This could also happen if the assay detects only a fraction of the D2 that is present. To get a true reading of the patient’s vitamin D level, an assay should be used that detects both vitamin D2 and D3 equally.

Measuring Total Vitamin in Determining Toxicity

In the case of a patient that is being treated for malabsorption with a high dose of vitamin D, the different reactivity for D2 and D3 also can cause the patient status to be mis-identified. If the supplement is D2, it is likely to be the largest vitamin D concentration in these patients. Consequently, by not having D2 detected or partially detected, it may result in the under reporting of the total vitamin D concentration. This can result in missing a patient with toxic levels.


Risk of Vitamin D Toxicity1

When serum 25-hydroxy-vitamin D levels are consistently > 150 ng/mL (375 nmol/L), it is potentially toxic. This typically occurs due to vitamin D over-supplementation and is observed in patients taking more than the prescribed 50,000 IU per day. Toxicity due to sunlight overexposure and/or diet is unlikely. When vitamin D levels are this high, calcium concentrations rise as well, which can result in nausea, weight loss and constipation. As a result of increased levels of vitamin D and calcium the patient can develop kidney stones.


C3-epi-25(OH)D3 Can Confound Accurate Measurement

While guidelines specify detection of the primary metabolite 25(OH) vitamin D2 and D3, it is important to know the amount of cross-reactivity an assay has to C3-epimer. The percentage of C3-epimer varies but its presence can be found in both children and adults. Unless an assay reports the specific fraction/quantity of the C3-epimer, it is not possible to determine if vitamin D is overestimated. Overestimation can create therapeutic errors, since patients who are deficient or insufficient may appear sufficient and toxicity may be reported in patients with high-normal levels.


In 2008 Phinney stated "the most widely used indicator of vitamin D status is the measurement of 25-hydroxyvitamin D [25(OH)D] in either serum or plasma. Because circulating 25(OH)D can arise from hydroxylation of either vitamin D2 or vitamin D3, measurement of total 25(OH)D [both 25(OH) D2 and 25(OH) D3] is essential for accurate assessment of vitamin D status."2



1. Dietary Supplemental Fact Sheet: Vitamin D. Office of Dietary Supplements. National Institutes of Health. Updated 11/13/2009. Accessed 08/17/2010.
2. Phinney KW. Development of a standard reference material for vitamin D in serum. Vitamin D and Health in the 21st Century: an Update American Journal of Clinical Nutrition. Vol. 88, No. 2. 511S-512S. August 2008.