The Controversy Surrounding Vitamin D Lab Testing

Vitamin D has received a lot of attention in the news recently. Public discussion has focused on the debate over the value of testing for vitamin D levels and monitoring of vitamin D levels for replacement therapies. But what exactly is vitamin D, and why is it clinically important? What is the recent interest generating the dramatic surge in vitamin D orders that laboratories are experiencing? This article will focus on the controversy and laboratory testing for vitamin D.

Vitamin D is a fat-soluble vitamin that plays a role in calcium metabolism and promotes strong bone formation. There are two sources of vitamin D: vitamin D 2 (ergocalciferol) which is derived from plants and fungus, and vitamin D 3 (cholecalciferol) which is converted from 7-dehydrocholesterol by light in the skin of animals. Both forms of vitamin D 2 and vitamin D 3 are biologically important prohormones. These prohormones are converted to 25-hydroxy vitamin D in the liver and then to 1,25-dihydroxy vitamin D in the kidneys. It is the 1,25-dihydroxy vitamin D that is physiologically active and binds to receptor proteins in the nuclei of target cells (particularly intestine, bone, kidney, and parathyroid hormone) that regulate calcium and phosphorus metabolism.

Nutritional sources of vitamin D include vitamin D 2 from plants, primarily fungus, mushrooms, and algae, and vitamin D 3 from fish liver oils (cod liver oil), fatty fishes (herring, catfish, salmon, mackerel, sardines, tuna, and eel), and eggs. However, the presence of vitamin D in fish appears to be due to the ingestion and concentration of vitamin D from ocean algae rather than endogeneous synthesis. Vitamin D 3 is produced in the skin by exposure of 7-dehydrocholesterol to UVB light wavelengths between 270 – 300 nm (peak synthesis occurs at 295-297 nm). This quality of light is found when the UV index is above 3 in the tropical and subtropical climates, but rarely occurs in the artic regions. Exposure of face, arms, or back to 10 – 15 minutes of light without sunscreen only twice a week is sufficient to produce the required amounts of vitamin D. Longer exposure does not lead to toxicity, as the body quickly achieves equilibrium in which the excess vitamin D is metabolized and eliminated as it is produced. Sun exposure does increase the risk of skin cancer, so many foods (milk, yogurt, margarines, cereals, and bread) are fortified with vitamin D. Both vitamin D 2 and vitamin D 3 are utilized as nutritional supplements and for pharmaceutical replacement therapies as the metabolites of both forms of the vitamin have equivalent biological activity.

Vitamin D is required for calcium metabolism. The active form of vitamin D, 1,25-dihydroxy vitamin D, helps maintain serum calcium levels by stimulating the absorption of calcium in the intestine, and by increasing calcium resorption from the bones. The 1,25-dihydroxy vitamin D also affects cell differentiation and proliferation, particularly peripheral mononuclear, pancreas, and skin cells. Vitamin D deficiency is associated with a softening of the bones; rickets in children; and osteomalacia in adults (adult rickets). Daily recommended dietary allowance for vitamin D is 400 IU/day (10 micrograms). This amount is generally consumed in the diet from foods fortified with vitamin D. However, certain groups at higher risk for developing vitamin D deficiency may require higher daily amounts or vitamin D supplementation including the elderly, pregnant women, breast-fed infants, and strict vegetarians abstaining from milk and eggs. The American Pediatrics Association recommends supplementing 400 IU/day (10 micrograms) for all infants from birth, and the Canadian Pediatric Association recommends 400 IU/day (10 micrograms) for breast-fed babies and 800 IU/day (20 micrograms) for babies living above the 55 degrees latitude. Pregnant and breast-feeding women should take 2000 IU/day (45 micrograms) vitamin D supplements.

Acute vitamin D toxicity is rare. Because the production of active 1,25-dihydroxy vitamin D is tightly regulated, excess vitamin D halts further metabolism until the excess can be metabolized or excreted. Skin production reaches maximum in about 20 minutes of exposure, with the excess also metabolized. Even multiple doses of cod liver oil and pharmaceutical supplements rarely cause long-term effects, and the only cases of known acute vitamin D overdose have resulted from industrial accidents in manufacturing. The long-term effects of chronic exposure to high doses of vitamin D are not known. Doses as high as 10,000 IU/day (250 micrograms) are believed to be safe, and all cases of vitamin D hypercalcemia have been due to chronic use of doses above 40,000 IU/day (1000 micrograms). Thus, ordering laboratory testing for vitamin D toxicity should be rarely done, and levels less than 100 ng/mL are generally believed to be safe (normal reference range of 10 – 50 ng/mL dependent on the method).

Besides bone disease, recent research has linked vitamin D deficiency to several diverse chronic diseases, including increased risk of cancer (breast and colon), higher risk of heart attacks in men, multiple sclerosis, psoriasis, and diabetes. Other researchers have reported a role of vitamin D deficiency in high blood pressure, periodontal disease, chronic pain, depression, schizophrenia, seasonal affective disorder, autoimmune diseases, Parkinson’s disease, and even a possible cause of influenza or the flu. This hype has led to the recent controversy regarding monitoring individual blood levels and supplementing with vitamin D to prevent risk of these disorders.

A number of assays have been developed for both 25-hydroxy vitamin D and 1,25-dihydroxy vitamin D, including high-pressure liquid chromatography, mass spectrometry, and immunoassays. The more specific assays, like mass spectrometry, can even resolve vitamin D 2 and its 25-hydroxy and 1,25-dihydroxy metabolites from vitamin D 3 and its metabolites. However, many of the currently marketed immunoassays are sensitive and specific enough to screen for vitamin D deficiency and monitor vitamin D replacement therapy without requiring specialized metabolite differentiation. More than 35 different metabolites of vitamin D 2 and D 3 have been identified, but only the measurement of 25-hydroxy vitamin D and 1,25-dihydroxy vitamin D have proven clinically useful. Separate measurement of the D 2 and D 3 forms does not provide an ability to distinguish dietary from endogenously produced vitamin D, since most food and pharmaceuticals are supplemented with both the D 2 and D 3 forms of the vitamin. Since both the 25-hydroxy and 1,25-dihydroxy metabolites of D 2 and D 3 are equally active, assays that are equally sensitive to both forms of the vitamin are clinically useful.

Many general practitioners and internal medicine physicians are confused by which vitamin D test to order. The 25-hydroxy vitamin D is the major circulating form of vitamin D and the best indicator of vitamin D status. The 25-dihydroxy vitamin D is present in 1000-fold concentration over the 1,25-dihydroxy metabolite and circulates with a half-life of 2 – 3 weeks, while 1,25-dihydroxy vitamin D only has a half-life of a few hours. The 1,25-dihydroxy form of vitamin D is generally only required to assist in the diagnosis of certain cases of rare endocrine disorders (primary hyperparathyroidism, hypothyroidism, pseudohypoparathyroidism), or for diagnosing renal osteodystrophy and vitamin D resistant rickets. Both tests are not needed as a panel for determining a patient’s vitamin D status or to monitor routine vitamin D replacement therapy.

In summary, there has been a recent rise in public interest regarding vitamin D and testing for vitamin D levels. Some of this is sparked by the increased awareness of the risk of skin cancer and exposure to the sun. Greater use of sunscreens and avoidance of direct sunlight has patients concerned over whether they are consuming adequate amounts of dietary vitamin D. In addition, recent research has linked vitamin D deficiency to a wide variety of diseases. Monitoring vitamin D status offers patients one potential option to reduce a possible source of risk for common disorders like depression, cancer, heart attack, and diabetes. Laboratories are seeing a surge in the number of vitamin D tests that are ordered as well as the variety of physicians ordering the test. While vitamin D used to be a rare test, ordered by renal physicians or endocrinologists, general practitioners, internal medicine, and even psychiatrists are starting to routinely order the test. This opens a new market for testing in the chemistry laboratory through either specialized chromatography or immunoassay methods. Some laboratories have even been able to justify the purchase of an immunoassay analyzer just for performing the test in-house over the added costs of sending the testing out to a reference laboratory. Previous tests have been hyped, become the test of the month, and then faded in their ordering patterns. Homocysteine and high sensitivity C-reactive protein are two such tests that experienced a lot of public hype and increased physician demand, followed by the ordering patterns dropping off. While no one is certain if or when this market will peak or if this trend will endure, vitamin D is certainly the test du jour until a new test comes along.