Vitamin D Testing Science and Biochemistry

How Vitamin D is Tested Can Make All the Difference

In 1922, Edward Mellanby discovered Vitamin D while researching a disease called rickets, which causes bone deformities in children.

Long known for its role in the prevention of childhood rickets and in the intestinal absorption of dietary calcium, vitamin D has now been found to be important in protecting the body from a wide range of diseases.

Vitamin D Deficiency Diseases
Diseases linked with vitamin D deficiency include stroke, cardiovascular disease, osteoporosis, osteomalacia, several forms of cancer, some autoimmune diseases such as multiple sclerosis, rheumatoid arthritis and type I diabetes, and even type 2 diabetes, depression and schizophrenia.

Common Causes of Vitamin D Deficiency
A major cause of vitamin D deficiency is inadequate sun exposure of the skin, usually for climatic or cultural reasons (even in countries near the equator, women in particular must have much of their skin area covered), and through the popular use of sunscreen.

Vitamin D status is therefore an important screening test, especially for people who spend much of their time indoors, or who live in colder climates. Vitamin D testing may also be used to monitor vitamin D supplementation to ensure that adequate blood levels are achieved.

Why It’s so Important to Measure both Vitamin D2 and D3

Vitamin D3 (cholecalciferol) is technically a prohormone and is produced from the action of ultraviolet light on 7-dehydrocholesterol in the skin. It is also found in cod liver oil and vitamin D supplements that state “cholecalciferol” in the ingredients.

Vitamin D2 (ergocalciferol) is not found in animals, but is manufactured commercially by irradiating ergosterol, a component of fungal cell membranes, with ultraviolet light; it is the predominant form for prescription use in the U.S., especially in high dose preparations.

Both vitamin D2 and D3 are hydroxylated in the liver to form their 25-hydroxy metabolites, the major circulating form of the prohormone. The long half-life of 25-hydroxyvitamin D (> 2 weeks) allows for supplementation with large doses every few months in deficient individuals, since vitamin D is stored by the body in adipose tissue.

The 25-hydroxy metabolites are further hydroxylated, primarily in the kidneys, to form 1,25-dihydroxy vitamin D2 and 1,25-dihydroxy vitamin D3, which are the highly active forms of the hormone that bind to specific vitamin D receptors in target tissues. Formation of 1,25-dihydroxy vitamin D is tightly regulated by the action of parathyroid hormone, and therefore has a very short half-life, making it unsuitable for assessing vitamin D status. In vitamin D deficient states, there is in fact excess production of parathyroid hormone (secondary hyperparathyroidism) stimulating the kidneys to produce even more 1,25-dihydroxyvitamin D, such that levels can appear to be normal or even elevated. The 25-hydroxy metabolite, which reflects total body bioavailability of the prohormone, is therefore the commonly accepted measure of vitamin D status.

Vitamin D2 has been effective when used as a supplement to prevent rickets, and has historically been assumed to be equivalent to vitamin D3. However, more recently it has been found that D2 and D3 have different properties. Research shows that 25-hydroxyvitamin D2 has a lower affinity than 25-hydroxyvitamin D3 for vitamin D binding protein, which carries the metabolites in the bloodstream, as well as a shorter half-life. Because of this, vitamin D2 is significantly less bioactive than D3, and must be given in much larger doses, although when given daily D2 is as effective as D3 in maintaining circulating levels of total 25-hydroxyvitamin D.

Vitamin D assays that measure only a “total” 25-hydroxyvitamin D do not distinguish how much is coming from the exogenous D2 and how much is biologically-identical D3 (whether from D3 supplementation or endogenous production). This is important when assessing vitamin D status in people supplementing with D2, which has a greater potential for toxicity than D3 if supplementation is not properly monitored with an accurate assay.

The bioequivalence of D2 and D3 is an interesting area for research, particularly since trials showing reduced fractures with vitamin D supplementation have employed D3 at adequate levels rather than D2, and many vitamin producers are already switching to D3 in their over-the-counter preparations.

ZRT uses liquid chromatography/tandem mass spectrometry (LC-MS/ MS) and not an immunoassay to measure 25-hydroxyvitamin D. Radioimmunoassays (RIA) typically do not differentiate between D2 and D3, and also don’t respond equally to both. This is a problem because an RIA could seriously underestimate blood levels in people supplementing with D2, and lead to dangerous recommendations for additional supplementation. LC-MS/MS is the gold standard for determining 25-hydroxyvitamin D and differentiating between 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3.

Reprinted with permission of ZRT Laboratory.