Endocrine laboratory pearls

Vitamin D tests

Over the past decade, awareness and interest in vitamin D testing have increased among physicians, patients and researchers. Various new studies have been performed to determine the prevalence of vitamin D deficiency and its potential impact on health.

The interest and research have resulted in large-scale national screening, and millions of vitamin D tests have been performed. Three issues, however, continue to baffle many physicians:

• Which test is best suited to assess a patient's vitamin D status?
• What is the optimal blood level of vitamin D?
• What is the impact of different testing methods on the interpretation of vitamin D test results?

Ravinder J. Singh, Ph.D., in the Division of Clinical Biochemistry and Immunology and the Department of Laboratory Medicine and Pathology at Mayo Clinic in Rochester, Minn., explains: "Vitamin D is either created by UV light exposure in the skin or derived from nutritional sources (food or supplements). In the case of the former, it stems from cholesterol and is termed vitamin D³. Animal-based nutritional sources provide the same, cholesterol-derived vitamin D³.

"In contrast, vitamin D from plants is derived from ergosterol (the plant equivalent of cholesterol) and is termed vitamin D². Despite some debate, the consensus is that both forms of vitamin D are equipotent and the sum of their concentrations correlates with their biological effects."

Distribution of vitamin D metabolites in human circulation

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Dr. Singh continues: "The amount of vitamin D in blood fluctuates with dietary intake and sun exposure, and most of it is rapidly stored in adipose tissue, from which it can be released on demand.

"The circulating vitamin D fraction is converted in the liver to 25-hydroxyvitamin D (25[OH]D, or calcidiol). Serum levels of 25[OH]D are very stable and reflect the tissue-body stores of its precursor, vitamin D.

"As with vitamin D, 25[OH]D is not active biologically but serves as a readily accessible reservoir for on-demand conversion to the active metabolite, 1,25-dihydroxyvitamin D (1,25[OH]2D, or calcitriol). Because of the on-demand nature of 1,25[OH]2D synthesis, circulating levels of this final metabolite also are relatively poor markers of vitamin D body stores. Of the three main vitamin D metabolites that can be measured, 25[OH]D is the preferred one for assessing body stores of vitamin D."

Stefan K. Grebe, M.D., a colleague of Dr. Singh in the Division of Clinical Biochemistry and Immunology and the Department of Laboratory Medicine and Pathology, highlights one of the major challenges with 25[OH]D testing: "What reference ranges should be used for interpreting 25[OH]D results?"

The 25-hydroxyvitamin D (25[OH]D) reference ranges used at Mayo Medical Laboratories

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Mayo Medical Laboratories has been using its listed reference ranges since 2004.

Dr. Grebe continues: "These reference ranges are based on evidence in the medical literature and are in agreement with the clinical assessments of endocrinologists at Mayo Clinic. There is consensus in the literature that 25[OH]D serum concentrations less than 10 ng/mL are associated with a high risk of osteomalacia or rickets and should be considered as severe deficiency.

"The recent comprehensive Institute of Medicine report has recommended a 25[OH]D blood level of 12 ng/mL as the minimum population requirement.

"There is also consensus that blood concentrations of 25(OH)D on the other end of the spectrum, or greater than 80 to 100 ng/mL, are associated with increased risk of toxicity (hypercalcemia) and should be avoided. The middle ground, however, continues to be contested."

Dr. Grebe elaborates: "Studies of skeletal health in institutionalized elderly populations and surrogate markers of vitamin D biological effects (for example, intestinal calcium absorption, suppression of parathyroid hormone levels) suggest that the minimal desirable blood level of 25[OH]D for bone health should be somewhere between 20 and 35 ng/mL. For the multitude of biological effects of vitamin D on other organ systems, there are few, if any, definitive data that could allow us to derive sensible cutoffs.

"The Institute of Medicine report concludes that careful further investigations and interventional trials are needed to determine optimum levels of 25[OH]D for various diseases that have been associated with low vitamin D levels (for example, cancer, cardiovascular disease, diabetes mellitus, various autoimmune diseases, infections) to avoid problems with either undertreatment or overtreatment."

Across clinical laboratories, markedly different methods are used for vitamin D testing. Dr. Singh strongly believes that liquid chromatography tandem mass spectrometry (LC-MS/MS) is superior to most competitive immunoassays and receptor-binding assays:

• LC-MS/MS is capable of quantifying vitamin D² and D³ metabolites separately, which can be invaluable in assessing issues of supplement malabsorption and patient compliance with treatment.
• Furthermore, LC-MS/MS is the recognized analytical criterion standard technology.
• Yet, some LC-MS/MS methods can be laborious and complex compared with automated immunoassays.

Consequently, only 10 to 15 percent of all clinical laboratories use liquid chromatography-based methods at the international level.

Under optimal conditions, LC-MS/MS, immunoassays, and receptor-binding assays should differ little from each other for measuring the total 25[OH]D level (the sum of 25[OH]D² and 25[OH]D³), but whenever a laboratory changes from one assay to another, the differences in results can be important clinically.

Differences that can be observed in various methods against the mean of all the methods

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Differences could be observed in various methods against the mean of all the methods. Although there is excellent agreement between LC-MS/MS and the all-method mean — reemphasizing the criterion-standard nature of LC-MS/MS — the results of individual immunoassays can easily deviate by 20 percent or more from those of LC-MS/MS.

In particular, some immunoassays are reported to have inconsistent recoveries of 25[OH]D² and thus may show large differences from LC-MS/MS and immunoassays methods with good recovery for patients taking vitamin D² therapy. Therefore, it is recommended that serial testing be performed with the same assay whenever possible and that LC-MS/MS be used if vitamin D² is given.