Vitamin D Testing

Vitamin D is a fat-soluble prohormone with several forms. In humans, the most important forms are vitamin D3 (also known as cholecalciferol) and vitamin D2 (ergocalciferol).

• Vitamin D3 is the more common form. It is found in food and is also made in sunlight-exposed skin from the conversion of 7-dehydrocholesterol. It is also found in over-the-counter—but not prescription— supplement form.
• Vitamin D2 is derived from fungal and plant sources. It is available in over-the-counter and prescription supplements used to treat vitamin D deficiency.

Both vitamin D2 and vitamin D3 are converted to 25-hydroxyvitamin D [25(OH)D or calcidiol] in the liver.

This is later hydroxylated in the kidneys and other tissues to 1,25-dihydroxyvitamin D [1,25(OH)2D or calcitriol].

• 25(OH)D is the main form of vitamin D circulating in the blood and the best indicator of vitamin D deficiency or excess in patients not suffering from renal disease.
• 1,25(OH)2D is the most metabolically active form of vitamin D; however, serum 1,25(OH)2D does not reflect   vitamin D reserves, and measurement of 1,25(OH)2D is not useful for monitoring the vitamin D status of patients. Serum 1,25(OH)2D is frequently either normal or even elevated in those with vitamin D deficiency, due to secondary hyperparathyroidism.¹

Note: throughout this document, “vitamin D” refers to vitamin D3 or D2 (when discussing intake) or 25(OH)D (when discussing levels in the body) unless otherwise specified.

Vitamin D is critical for maintaining healthy levels of calcium and phosphorus by aiding in their absorption from the gut. This helps the body form and maintain strong bones. Vitamin D also modulates neuromuscular, immune, and other cellular functions.

Vitamin D deficiency can lead to bone diseases, such as rickets and osteomalacia. Deficiency has also been associated with a wide range of medical conditions, including heart disease, hypertension, diabetes, and cancer.

The Endocrine Society provides recommendations for vitamin D requirements and dietary intake.

Obese children and adults and those on certain medications (eg, anticonvulsants, glucocorticoids, AIDS medications, antifungals) may need at least 2 to 3 times the suggested dietary intake for their age group.³

People who are vitamin D insufficient or deficient may need over-the-counter or prescription supplementation.

Refer to the Endocrine Society guidelines¹ for treatment recommendations.

Vitamin D can be obtained from exposure to sunlight and from diet. Dietary sources of vitamin D include:

• Oil-rich fish such as salmon, mackerel, and herring
• Shiitake mushrooms.
• Foods and beverages fortified with vitamin D, such as milk and other dairy products, orange juice, and some grain products
• Multivitamins and other supplements

Note: none of these sources may be adequate for patients with liver or kidney disease; these patients may need supplementation with the active form of vitamin D (1,25-dihydroxyvitamin D [1,25(OH)2D]).

Low vitamin D concentrations can result from:

• Inadequate sunlight (especially common in winter and in people with darker skin color)
• Dietary deficiency
• Poor vitamin D absorption
• Impaired metabolism of sterol in the liver

Vitamin D deficiency, as defined by the Endocrine Society, is common. As much as 68% to 77% of the population is estimated to have suboptimal (<30 ng/mL) levels of vitamin D.^4–6

This seasonal effect is more notable in northern latitudes than in southern latitudes. The common use of sunscreen may also affect patients’ vitamin D levels. Thus, there may be more of a need to supplement, or to supplement with higher doses of vitamin D, in the winter months than in the summer months.

The Endocrine Society recommends regular screening for individuals at risk for deficiency. These include patients who have¹:

• Osteoporosis
• Osteomalacia and rickets
• Chronic kidney disease
• Diseases that require certain medications (anti-seizure medications, glucocorticoids, AIDS medications, antifungals, cholestyramine)
• Malabsorption syndromes, including inflammatory bowel disease, Crohn’s disease, cystic fibrosis, history of bariatric surgery
• Hyperparathyroidism
• Hepatic failure

Screening is also recommended for¹:

• African American and Hispanic children and adults
• Pregnant and lactating women
• Older adults with history of falls
• Older adults with history of nontraumatic fractures
• Obese children
• Obese adults (see Question 11 for information about expanded Medicare coverage)

If a patient is at risk for vitamin D deficiency and has not been tested recently, a test should be ordered to assess levels. Some physicians may wish to monitor people receiving vitamin D therapy to evaluate compliance and expected change in concentration.

Vitamin D tests generally measure the total concentration of 25(OH)D. In addition, vitamin D tests using liquid chromatography with tandem mass spectrometry (LC/MS/MS) can measure the concentrations of vitamin D₂ and D₃ (when added together, these equal the total vitamin D concentration).

25(OH)D is the major circulating form of vitamin D, with a circulating half-life of 2–3 weeks. It is the best indicator to monitor for vitamin D status.¹

The circulating half-life of 1,25(OH)2D, on the other hand, is approximately 4 hours. It circulates at a 1,000-times lower concentration than 25(OH)D, and the blood level is tightly regulated by serum levels of PTH, calcium, and phosphate. Serum 1,25(OH)2D does not reflect vitamin D reserves, and measurement of 1,25(OH)2D is not useful to monitor vitamin D status. Serum 1,25(OH)2D is frequently either normal or even elevated in those with vitamin D deficiency, because of secondary hyperparathyroidism. Thus, 1,25(OH)2D measurement does not reflect vitamin D status.¹

Measurement of 1,25(OH)2D is useful only for patients with disorders of 25(OH)D and phosphate metabolism. These include chronic kidney disease, hereditary phosphate-losing disorders, oncogenic osteomalacia, pseudovitamin D-deficiency rickets, vitamin D-resistant rickets, and chronic granuloma-forming disorders, such as sarcoidosis and some lymphomas.¹

Quest offers 2 methods. Both methods are considered accurate and reliable.

• Immunoassay—suitable for most patients; measures total 25(OH)D concentrations and not its separate components; provides high-quality quantitative results that meet the National Institute of Standards and  Technology (NIST Standard Reference Material [SRM] 972) requirements; certified by the Centers for Disease Control and Prevention Vitamin D Standardization Certification Program.
• Liquid chromatography, tandem mass spectrometry method (also known as LC/MS/MS)—measures 25(OH)D2, 25(OH)D3, and total 25(OH)D concentrations; represents the reference method endorsed by experts at the National Institute of Standards and Technology, the Centers for Disease Control and Prevention, and the National Institutes of Health.

Infants (birth up to 3 years) may have circulating levels of the 25-hydroxyvitamin D3 C3-epimer, a low-activity form of vitamin D. The immunoassay does not detect the C3-epimer; the LC/MS/MS does detect it, but overestimates total vitamin D if the C3-epimer is not accounted for.

To address this, Quest offers a separate LC/MS/MS test for infants, which involves separation of the 25(OH)D3 C3-epimer and exclusion of it from the 25(OH)D3 and total vitamin D results reported.

Immunoassay:

• Provides reliable results suitable for most patients and situations
• Has fast turnaround times, with results available the day after specimen submission
• Can be reported at the same time as many other tests
• Reports total 25(OH)D concentration without differentiating D2 and D3

LC/MS/MS:

• Provides reliable reference method results
• Differentiates D2 and D3 and reports their concentrations along with the total 25(OH)D concentration
• Differentiates the contribution of vitamin D2 and vitamin D3 supplementation

For infants younger than 3 years of age, either the immunoassay or the LC/MS/MS test code that is specifically for infants (test code 91935) is suitable.

All of our vitamin D tests provide the concentration of total 25(OH)D in a patient’s serum, the total 25(OH)D reference range, and suggested cut points to define optimal, insufficient, and deficient vitamin D status.

Tests using the LC/MS/MS method provide the concentration of 25(OH)D₂ and 25(OH)D₃. Neither reference nor interpretative ranges have been established for these components of total 25(OH)D.

No. There are no special patient preparations prior to testing.

In alignment with Endocrine Society guidelines, Medicare includes coverage for vitamin D testing in patients at high risk for deficiency.

At-risk categories for vitamin D deficiency include:

• Osteoporosis
• Osteomalacia and rickets
• Obesity
• Chronic kidney disease
• Diseases that require certain medications (anti-seizure medications, glucocorticoids, AIDS medications, antifungals, cholestyramine)
• Malabsorption syndromes including inflammatory bowel disease, Crohn’s disease, cystic fibrosis, history of bariatric surgery
• Hyperparathyroidism
• Hepatic failure
• Certain lymphomas

In some jurisdictions (eg, Florida, Pennsylvania, the Southwest region), coverage for at-risk patients includes testing up to 3 times per year.

In most jurisdictions, Medicare has expanded coverage for vitamin D testing for obese patients, or those who have a BMI >30. When ordering tests for these patients, be sure to include the appropriate ICD-10 code to support accurate payment and reduce disruptions. For obesity, these include Z68.30–Z68.45. For a list of top diagnosis codes used by ordering physicians, visit QuestDiagnostics.com/MLCP.