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Vitamin D Deficiency and Toxicity: Laboratory Support of Diagnosis and Management

Vitamin D Deficiency and Toxicity: Laboratory Support of Diagnosis and Management

Clinical Focus

Vitamin D Deficiency and Toxicity

Laboratory Support of Diagnosis and Management

  

Contents:

Clinical Background  - Table 1 - Table 2

Individuals Suitable for Testing  - Table 3

Test Availability  - Table 4

Test Selection

Test Interpretation

References
 

Clinical Background [return to contents]

Vitamin D-associated Disorders

Vitamin D deficiency is a common condition associated with rickets in children and osteomalacia and osteoporosis in adults. Vitamin D deficiency is also associated with muscle weakness and secondary hyperparathyroidism, both of which exacerbate these bone disorders and/or their clinical impact. For example, muscle weakness increases the risk of falls and bone fracture in the elderly,1 and parathyroid hormone (PTH) increases bone resorption in patients with secondary hyperparathyroidism.1,2

Recent evidence, mostly from observational studies, suggests a link between vitamin D deficiency and non bone diseases as well. These diseases include cancer (especially colorectal and breast cancer), cardiovascular disease, autoimmune diseases, diabetes, depression, and schizophrenia.1,3,4 

In rare cases, ingestion of large doses of vitamin D results in vitamin D toxicity, which manifests as hypercalcemia, hypercalciuria, or kidney stones.1,5 The risk of vitamin D toxicity may be increased in individuals who have hyperparathyroidism, sarcoidosis, tuberculosis, or lymphoma and are taking vitamin D supplements.5

Pathophysiology

Vitamin D is a fat-soluble vitamin that, through its suppression of PTH, promotes bone health by enhancing intestinal absorption of calcium and phosphorus.1,6 More recent discoveries suggest that vitamin D may have additional biological functions. For example, vitamin D receptors have been found  in a number of tissues including colon, breast, prostate, brain, and immune cells. The active form of vitamin D (1,25-dihydroxyvitamin D) is involved in the control of more than 200 genes, including those responsible for the regulation of cell growth and apoptosis.1

Vitamin D occurs in 2 forms: vitamin D3 and vitamin D2. Vitamin D3, the main form in humans, is produced in the skin in response to sunlight and is obtained in small amounts from animal-based foods. Vitamin D2 can be obtained in small amounts from plant-based foods. Vitamins D3 and D2 are rapidly metabolized in the liver to their respective 25-hydroxyvitamin D [25(OH)D] forms, 25(OH)D3 and 25(OH)D2, which are in turn converted in the kidneys to their corresponding active forms (Table 1).1 Experts disagree about the relative potencies of vitamins D2 and D3; some experts claim that the 2 forms are equally effective in raising 25(OH) levels, whereas other experts assert that vitamin D2 is less effective than vitamin D3 in elevating 25(OH)D.7,8

Table 1. Important Vitamin D Forms

Name

Description

Abbreviation

Description

Ergocalciferol Vitamin D2 NA Plant-based form
Cholecalciferol Vitamin D3 NA Animal-based form; form produced in human skin
Ercalcidiol 25-hydroxyvitamin D2 25(OH)D2 Main circulating form of vitamin D2
Calcidiol 25-hydroxyvitamin D3 25(OH)D3 Main circulating form of vitamin D3
Ercalcitriol 1,25-dihydroxyvitamin D2 1,25(OH)2D2 Active form of vitamin D2; has a short half-life in the blood
Calcitriol 1,25-dihydroxyvitamin D3 1,25(OH)2D3 Active form of vitamin D3; has a short half-life in the blood

Recommended Vitamin D Intake

The American Academy of Pediatrics recommends supplementation with vitamin D if needed to prevent rickets.9 Indeed, vitamin D supplementation/therapy is now the standard of care for preventing and treating rickets. Similarly, the American Association of Clinical Endocrinologists (AACE) recommends vitamin D and calcium supplementation to prevent osteoporosis in postmenopausal women.2,9 Studies have shown that vitamin D supplementation, in conjunction with calcium, increases bone mineral density and reduces the risk of fractures and falls in postmenopausal women.2,10

Optimal intake levels, however, remain a subject of some debate. In 2011, the Institute of Medicine (IOM) increased the recommended daily vitamin D intake from 200-600 IU/d to 400-800 IU/d, depending on age.6,11 Other experts believe that these increases were not sufficient; they recommend as much as 1000-2000 IU/d (Table 2).12 These same experts believe that an intake up to 10,000 IU/d is safe.

Table 2. Institute of Medicine (IOM) and Endocrine Society Recommendations for Vitamin D Intake6,12
Life-stage Groupa IOM Recommendations
(IU/d)
  Endocrine Society Recommendations
(IU/d)
b

Intake

Upper Limitc

Intake

Upper Limitc

0-6 months 400d 1000 400-1000 2000
6-12 months 400d 1500 400-1000 2000
1-3 years 600 2500 600-1000 4000
4-8 years 600 3000 600-1000 4000
9-18 years 600 4000 600-1000 4000
19-30 years 600 4000 1500-2000 10,000
31-50 years 600 4000 1500-2000 10,000
51-70 years 600 4000 1500-2000 10,000
71+ years 800 4000 1500-2000 10,000
Pregnant or lactating women (14-18 years) 600 4000 600-1000 4000
Pregnant or lactating women (19-50 years) 600 4000 1500-2000 10,000

a Includes normal healthy individuals of both genders unless otherwise specified.

b Estimated intake needed to maintain blood 25(OH)D levels above 30 ng/mL.

c Maximum level that is expected to have no risk of adverse effects to healthy individuals. 1 μg of vitamin D is equivalent to 400 IU.
d Refers to adequate intake (intake estimated to maintain protective 25(OH)D levels in a group of healthy individuals with limited sun exposure and vitamin D stores) instead of recommended intake, which could not be established because of insufficient evidence.

Obtaining Sufficient Amounts of Vitamin D

Sufficient amounts of vitamin D can be obtained through adequate sunlight exposure and/or a diet containing enough vitamin D-rich foods such as oily fish. Individuals who do not get enough vitamin D by these means can get it in the form of vitamin D-fortified foods, over-the-counter supplements, or prescription supplements. Both vitamins D2 and D3 are available in fortified foods or in over-the-counter supplements that range in dose from 400 to 50,000 IU. Vitamin D2 is also available by prescription in liquid (8000 IU) or high-potency (50,000 IU) capsule formulations. Most adults with vitamin D deficiency can be treated for 8 weeks with either 50,000 IU once a week or 6000 IU once a day, followed by maintenance therapy of 1500-2000 IU/d.12 Similarly, children with vitamin D deficiency can be treated for 6 weeks with either 50,000 IU once a week or 2000 IU once a day, followed by maintenance therapy of 400-1000 IU/d. Either vitamin D2 or D3 can be used. Higher vitamin D doses may be required for obese people, people with malabsorption syndromes, people who have undergone bariatric surgery, or those taking medications that affect vitamin D metabolism.12

Determining Vitamin D Status

Laboratory measurement of the 25(OH)D blood level is the accepted means for determining vitamin D status.1 25(OH)D levels can be used in: 1) diagnosing vitamin D insufficiency or deficiency, thus identifying individuals who may benefit from supplementation; 2) monitoring response to vitamin D supplements; and 3) diagnosing vitamin D toxicity. Common methods for measuring 25(OH)D include radioimmunoassay (RIA), chemiluminescence immunoassay, high performance liquid chromatography, and liquid chromatography tandem mass spectrometry (LC/MS/MS). There is no consensus about the optimal 25(OH)D level, but many experts accept a range ≥30 ng/mL as optimal.1,2,12 Using this definition, approximately 77% of adults and 70% of children in the United States have inadequate serum levels of vitamin D.13,14

Individuals Suitable for Testing [return to contents]

  • Individuals with suspected vitamin D deficiency (eg, those with persistent, nonspecific musculoskeletal pain)

  • Individuals at increased risk for vitamin D deficiency (eg, pregnant, lactating, dark-skinned, elderly, obese, or housebound individuals, and those dwelling in latitudes higher than 33 degrees north or 33 degrees south)

  • Individuals with disorders associated with reduced 25(OH)D levels [see Table 3]

  • Individuals taking certain medications associated with reduced 25(OH)D levels [see Table 3]

  • Individuals with suspected toxicity (eg, those with hypercalcemia of obscure origin)

Table 3. Effect of Various Disorders and Medications on 25-Hydroxyvitamin D and 1,25-Dihydroxyvitamin D Concentrations1,15-22

Disorder 25(OH)D
Concentration
1,25(OH)2D
Concentration
Anti-convulsant, anti-retrovirals, or anti-tuberculosis medications (increased metabolism)
Fat malabsorption disorders, short bowel syndrome
Intestinal diseases causing excessive loss
of vitamins D2 and D3
Lymphoma, granulomatous disorders or N
Nephrotic syndrome
Nutritional rickets or N
Osteomalacia or N
Secondary hyperparathyroidism
Severe parenchymal liver disease (impaired 25-hydroxylation of vitamin D)
Severe renal disease N
Hypercalcemia of cancer N
Hyperparathyroidism or N or N
Vitamin D-dependent rickets, type Ia N
Vitamin D-dependent rickets, type IIb or N
Vitamin D [25(OH)D] toxicity (uncommon) or N

25(OH)D, 25-hydroxyvitamin D; 1,25(OH)2D, 1,25-dihydroxyvitamin D; N, normal.

a Also called pseudo-vitamin D-deficiency rickets.
b Also called hereditary vitamin D-resistant rickets.

Test Availability [return to contents]

Tests offered by Quest Diagnostics to assist in the diagnosis of vitamin D deficiency or toxicity are presented in Table 4. This table is provided for informational purposes only and is not intended as medical advice. A physician’s test selection and interpretation, diagnosis, and patient management decisions should be based on his/her education, clinical expertise, and assessment of the patient.

Table 4. Tests Available to Determine Vitamin D Levels
Test Code Test Name Method Description
17306 Vitamin D, 25-Hydroxy, LC/MS/MS Liquid chromatography tandem mass spectrometry Reference method; accurately quantifies and reports both forms of vitamin D, 25(OH)D2 and 25(OH)D3; total 25(OH)D reported as the sum of the 2 forms; useful for diagnosing vitamin D insufficiency or deficiency and for monitoring vitamin D2 or D3 therapya
16558 Vitamin D, 1,25-Dihydroxy, LC/MS/MS Liquid chromatography tandem mass spectrometry Accurately quantifies 1,25(OH)2D; useful in differential diagnosis of vitamin D-related diseases and for monitoring vitamin D therapy in patients with chronic renal disease
16761 Vitamin D, 25-Hydroxy and 1,25-Dihydroxy, LC/MS/MS Liquid chromatography tandem mass spectrometry See individual analytes

a May also be used to confirm vitamin D toxicity, which occurs rarely.

Test Selection [return to contents]

25(OH)D

Measurement of 25(OH)D, the main form of circulating vitamin D, is used to diagnose vitamin D insufficiency, deficiency, or toxicity and to monitor most patients receiving vitamin D therapy. Several methods have been used, each with its own advantages and disadvantages. Immunoassays are widely used and include RIAs, electrochemiluminometric assays, and immunochemiluminometric assays. Disadvantages of immunoassays include use of radioactive isotopes (RIAs), method-to-method variation, and laboratory-to-laboratory variation.23 Sample matrix effects and cross-reactivity with interfering substances such as inactive vitamin D metabolites account for some of the variability. Moreover, some immunoassays tend to underestimate 25(OH)D2 levels due to lack of equal specificity for 25(OH)D3 and 25(OH)D2.24,25

LC/MS/MS is not as widely available as are the immunoassays, because it requires specially-trained technologists. However, it is the reference method used by the National Institute of Standards and Technology and by the Centers for Disease Control and Prevention, because it accurately and precisely measures 25(OH)D3 and 25(OH)D2 concentrations with equal specificity.26-28 It has an analytical sensitivity greater than that of an electrochemiluminescence assay and comparable to that of a RIA, without the need for radioisotopes.29

Quest Diagnostics uses an LC/MS/MS method to measure 25(OH)D blood levels. Concentrations
of both 25(OH)D forms are independently reported, and the 2 forms are summed to provide the total
25(OH)D concentration. Thus, the LC/MS/MS assay differentiates the separate contributions of vitamins D3 and D2 to vitamin D status. It detects 25(OH)D2 and 25(OH)D3 at an analytical sensitivity of 4 ng/mL, and has no cross-reactivity with other known vitamin D metabolites. This test can be used to diagnose vitamin D insufficiency or deficiency and to monitor patients receiving vitamin D2 or D3 supplementation. The laboratory turnaround time is 3 days.

1,25(OH)2D

Measurement of 1,25(OH)2D is not used to diagnose vitamin D deficiency because its levels are normal
in most individuals due to tight regulation. Furthermore, 1,25(OH)2D may even be elevated in people with secondary hyperparathyroidism, because PTH enhances the conversion of 25(OH)D to 1,25(OH)2D.1
1,25(OH)2D measurement is reserved for distinguishing some cases of primary hyperparathyroidism from hypercalcemia of cancer and in the differential diagnosis of vitamin D-dependent rickets (type I vs type II) (Table 3). 1,25(OH)2D is also useful for monitoring vitamin D therapy in patients with chronic renal disease, who may have normal 25(OH)D levels. As renal disease progresses, the ability of the kidney to produce 1,25(OH)2D decreases. Supplementation with vitamin D is thus no longer effective, and patients require administration of 1,25(OH)2D. Although 1,25(OH)2D may be increased or decreased in a number of other disorders, levels are typically used for confirmation rather than diagnosis of these conditions (Table 3).

Test Interpretation [return to contents]

Optimal 25(OH)D levels are ≥30 ng/mL, while levels of 21 to 29 ng/mL indicate vitamin D insufficiency and levels ≤20 ng/mL indicate deficiency.1,2,12 Vitamin D insufficiency and deficiency may both lead to elevated PTH levels (secondary hyperparathyroidism),1 and the most severe forms of deficiency may be associated with hypocalcemia, hypophosphatemia, and elevated alkaline phosphatase.

Low 25(OH)D levels are most commonly associated with lack of dietary intake and/or sun exposure. They are also associated with disorders that are characterized by decreased absorption or excessive loss in the gastrointestinal tract, increased vitamin D metabolism, or impaired conversion of vitamin D to 25(OH)D.1,2

Table 3 lists expected 25(OH)D and 1,25(OH)2D levels associated with various disorders and medications.

High 25(OH)D levels are suggestive of vitamin D toxicity. Expert opinions vary regarding an appropriate toxicity threshold. Although the Institute of Medicine cites reports of adverse events at 25(OH)D levels ≥50 ng/mL and recommends relatively low vitamin D intake levels, many experts including the authors of the Endocrine Society clinical practice guideline disagree with such a low toxicity threshold and assert that vitamin D toxicity only occurs at 25(OH)D levels ≥150 ng/mL.1,6,12

References [return to contents]

  1. Holick MF. Vitamin D deficiency. N Engl J Med. 2007;357:266-281.

  2. Watts NB, Bilezikian JP, Camacho PM, et al. American Association of Clinical Endocrinologists medical guidelines for clinical practice for the diagnosis and treatment of postmenopausal osteoporosis. Endocr Pract. 2010;16(Suppl 3):1-37.

  3. Peterlik M, Cross HS. Vitamin D and calcium insufficiency-related chronic diseases: an emerging world-wide public health problem. Int J Environ Res Public Health. 2009;6:2585-2607.

  4. Thatcher TD, Clarke BL. Vitamin D insufficiency. Mayo Clin Proc. 2011;86:50-60.

  5. Vieth R. Vitamin D supplementation, 25-hydroxyvitamin D concentrations, and safety. Am J Clin Nutr. 1999;69:842-856.

  6. Ross AC, Manson JE, Abrams SA, et al. The 2011 Report on Dietary Reference Intakes for Calcium and Vitamin D from the Institute of Medicine: What Clinicians Need to Know. J Clin Endocrinol Metab. 2011;96:53-58.

  7. Holick MF, Biancuzzo RM, Chen TC, et al. Vitamin D2 is as effective as vitamin D3 in maintaining circulating concentrations of 25-hydroxyvitamin D. J Clin Endocrinol Metab. 2008;93:677-681.

  8. Armas LAG, Hollis BW, Heaney RP. Vitamin D2 is much less effective than vitamin D3 in humans. J Clin Endocrinol Metab. 2004;89:5387-5391.

  9. Wagner CL, Greer FR; American Academy of Pediatrics Section on Breastfeeding; American Academy of Pediatrics Committee on Nutrition. Prevention of rickets and vitamin D deficiency in infants, children, and adolescents. Pediatrics. 2008;122:1142-1152.

  10. Cranney A, Horsley T, O’Donnell S, et al. Effectiveness and Safety of Vitamin D in Relation to Bone Health. Rockville, MD: Agency for Healthcare Research and Quality (US); 2007. Available at: http://www.ncbi.nlm.
    nih.gov/books/NBK38416. Accessed January 27, 2012.

  11. Standing Committee on the Scientific Evaluation of Dietary Reference Intakes Food and Nutrition Board, Institute of Medicine. Vitamin D. In: Dietary reference intakes for calcium, phosphorus, magnesium, vitamin D, and fluoride. Washington, DC: National Academy Press; 1999:250-287.

  12. Holick MF, Binkley NC, Bischoff-Ferrari HA, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrin Metab. 2011;96:1911-1930.

  13. Ginde AA, Liu MC, Camargo CA, et al. Demographic differences and trends of vitamin D insufficiency in the US population, 1988-2004. Arch Intern Med. 2009;169:626-632.

  14. Kumar J, Muntner P, Kaskel FJ, et al. Prevalence and associations of 25-hydroxyvitamin D deficiency in US children: NHANES 2001-2004. Pediatrics. 2009;124:e362-e370.

  15. Bringhurst FR, Demay MB, Kronenberg HM. Hormones and disorders of mineral metabolism. In: Melmed S, Polonsky KS, Larsen PR, Kronenberg HM, eds. Williams Textbook of Endocrinology. 12th ed. Philadelphia, PA: Elsevier Saunders; 2011:1237-1304.

  16. Lorenzo JA, Canalis E, Raisz LG. Metabolic bone disease. In: Melmed S, Polonsky KS, Larsen PR, Kronenberg HM, eds. Williams Textbook of Endocrinology. 12th ed. Philadelphia, PA: Elsevier Saunders; 2011:1305-1349.

  17. Bouillon R. Vitamin D: from photosynthesis, metabolism, and action to clinical applications. In: Jameson JL, De Groot LJ, et al, eds. Endocrinology. 6th ed. Philadelphia, PA: Saunders; 2010:1089-1110.

  18. Silverberg SJ, Bilezikian JP. Primary hyperparathyroidism. In: Jameson JL, De Groot LJ, et al, eds. Endocrinology. 6th ed. Philadelphia, PA: Saunders; 2010:1176-1197.

  19. Horwitz MJ, Stewart AF. Malignancy-associated hypercalcemia and medical management. In: Jameson JL, De Groot LJ, et al, eds. Endocrinology. 6th ed. Philadelphia, PA: Saunders; 2010:1198-1211.

  20. National Kidney Foundation. KDOQI clinical practice guidelines for bone metabolism and disease in chronic kidney disease. New York, NY: National Kidney Foundation; 2004.

  21. St-Arnaud R, Glorieux FH. Genetic defects in vitamin D metabolism and action. In: Jameson JL, De Groot LJ, et al, eds. Endocrinology. 6th ed. Philadelphia, PA: Saunders; 2010:1236-1249.

  22. Demay MB, Krane SM. Disorders of calcification: osteomalacia and rickets. In: Jameson JL, De Groot LJ, et al, eds. Endocrinology. 6th ed. Philadelphia, PA: Saunders; 2010:1311-1329.

  23. Binkley N, Krueger D, Cowgill CS, et al. Assay variation confounds the diagnosis of hypovitaminosis D: a call for standardization. J Clin Endocrinol Metab. 2004;89:3152-3157.

  24. Hollis BW. Comparison of commercially available 125I-based RIA methods for the determination of circulating 25-hydroxyvitamin D. Clin Chem. 2000;46:1657-1661.

  25. Glendenning P, Taranto M, Noble JM, et al. Current assays overestimate 25-hydroxyvitamin D3 and underestimate 25-hydroxyvitamin D2 compared with HPLC: Need for assay-specific decision limits and metabolite-specific assays. Ann Clin Biochem. 2006;43:23-30.

  26. Maunsell Z, Wright DJ, Rainbow SJ. Routine isotope-dilution liquid chromatography-tandem mass spectrometry assay for simultaneous measurement of the 25-hydroxy metabolites of vitamins D2 and D3. Clin Chem. 2005;51:1683-1690.

  27. Tai SS, Bedner M, Phinney KW. Development of a candidate reference measurement procedure for the determination of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum using isotope-dilution liquid chromatography/tandem mass spectrometry. Anal Chem. 2010;82:1942-1948.

  28. Chen H, McCoy LF, Schleicher RL, et al. Measurement of 25-hydroxyvitamin D3 (25OHD3) and 25-hydroxyvitamin D2 (25OHD2) in human serum using liquid chromatography-tandem mass spectrometry and its comparison to a radioimmunoassay method. Clin Chem Acta. 2008;391:6-12.

  29. Herrmann M, Harwood T, Gaston-Parry O, et al. A new quantitative LC tandem mass spectrometry assay for serum 25-hydroxy vitamin D. Steroids. 2010;75:1106-1112.
     

Content reviewed 12/2012
 
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