Vitamin D is one of the most commonly ordered laboratory tests in the primary care setting, as well as one of the most widely used forms of dietary supplementation today. While the rationale underlying vitamin D testing and supplementation for deficiency may seem straightforward, in actuality, the metabolism and physiologic functions of vitamin D in the body are quite nuanced and complex, and there remains significant controversy surrounding the appropriate utilization of vitamin D measurement and clinical interpretation of vitamin D test results. In this post, let’s review the basic principles of vitamin D metabolism, its function and mechanisms of regulation in the human body, methods of measurement in the laboratory, and ramifications of vitamin D values on clinical decision-making and management.
Vitamin D Metabolism
Vitamin D is a fat-soluble vitamin and encompasses a group of compounds, all containing a four-ring steroid backbone. The two main forms of vitamin D utilized by humans are vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol). Vitamin D3 is primarily synthesized in the skin from 7-dehydrocholesterol in the presence of sunlight (UVB rays), while vitamin D2 is synthesized in plants from ergosterol and is used to fortify many foods (milk, bread, cereal, etc.).1
Once synthesized in the skin or ingested from the gastrointestinal tract, both vitamin D2 and vitamin D3 travel in the bloodstream (bound to vitamin D-binding protein) to the liver, where both are converted to 25-hydroxyvitamin D [25(OH)D, calcidiol/calcifediol] by the action of 25-hydroxylase.1,2 While 25(OH)D has only limited biologic activity, it has a very long half-life (2-3 weeks) and is therefore the primary form of vitamin D found in the blood.1 Notably, the half-life of 25(OH)D2 is shorter than that of 25(OH)D3 (possibly due to lower affinity to vitamin D-binding protein) and therefore it is present in significantly lower concentrations than 25(OH)3 in the blood.3
25(OH)D is then further converted to 1,25-dihydroxyvitamin D [1,25(OH)2, calcitriol] via the action of 1-α-hydroxylase primarily in the kidney.1 In contrast to 25(OH)D, 1,25(OH)2D is the biologically active form of vitamin D, but it has a much shorter half-life (5-8 hours) and therefore has much lower circulating levels in the blood.1 25(OH)D may alternatively be converted to 24,25-dihydroxyvitamin D [24,25(OH)2D] by 24-α-hydroxylase, also in the kidney. 24,25(OH)2D is an inactive metabolite and thus serves as an end-product in this degradation-type pathway of 25(OH)D.1,4
Vitamin D Physiology
The overall effect of vitamin D in the body is to increase calcium and phosphate levels in the blood. Via binding of 1,25(OH)2D to nuclear receptors within cells, it acts at three main sites: 1) the intestine, where it increases calcium and phosphate absorption, 2) the bones, where it increases calcium resorption by promoting osteoclast maturation, and 3) the kidney, where it increases calcium reabsorption by enhancing the effects of parathyroid hormone (PTH) on the distal convoluted tubule.1
Conversion of 25(OH)D to 1,25(OH)2D by 1-alpha-hydroxylase is tightly regulated by calcium, phosphate, and PTH concentrations in the body. Decreased calcium or phosphate levels, or increased PTH levels in the blood (most commonly resulting from a fall in calcium) will stimulate 1-α-hydroxylase activity and lead to increased production of 1,25(OH)2D, while increased calcium or phosphate levels or decreased PTH levels will suppress 1-α-hydroxylase activity and thus lead to decreased production of 1,25(OH)2D.1,2
From a clinical perspective on vitamin D physiology, there are numerous causes of abnormal vitamin D levels in the body. Here are some common causes of low vitamin D levels:
- Inadequate intake of vitamin D (whether from diet, inadequate sunlight, or malabsorption)
- Decreased PTH (hypoparathyroidism, hyperphosphatemia, hypercalcemia of malignancy)
- End-organ resistance to PTH (pseudohypoparathyroidism)
- Decreased 1-α-hydroxylase activity (renal failure, vitamin D-dependent rickets type 1)5
Conversely, causes of high vitamin D levels are listed below:
- Excessive intake of vitamin D (usually from supplements)
- Increased PTH (primary hyperparathyroidism)
- Increased extrarenal 1-α-hydroxylase activity (seen in granulomatous diseases such as sarcoidosis, as well as some lymphomas)
- End-organ resistance to vitamin D (vitamin D-dependent rickets type 2)5
Vitamin D Measurement in the Laboratory
25(OH)D is the most commonly measured vitamin D metabolite in laboratory assays, since (as mentioned above) it has a longer half-life and a larger concentration in the blood compared to 1,25(OH)2D. In addition, its concentration does not fluctuate as significantly as that of 1,25(OH)2D, since its production from 25-hydroxylase in the liver is not so tightly regulated as 1-α-hydroxylase activity in the kidney.1,6 Nevertheless, 1,25(OH)2D measurement is indicated in a few specific clinical circumstances, including workups for idiopathic hypercalcemia and bone/mineral disorders, and for evaluation of vitamin D status in the setting of renal failure (where 1-α-hydroxylase activity is decreased).1,7
While the gold standard for vitamin D measurement is liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), most laboratories utilize immunoassays (including radioimmunoassays, chemoluminescent immunoassays, and enzyme-linked immunoassays) for vitamin D quantitation.1,7 One significant difference between these two methods is that while LC-MS/MS can differentiate vitamin D3 and vitamin D2 metabolites, immunoassays cannot.6 In addition, the antibodies used in many 25(OH)D immunoassays often have lower cross-reactivities with 25(OH)D2 and therefore may underestimate this form when giving the total 25(OH)D value.6 These antibodies also have varying cross-reactivities with other vitamin D metabolites and so may result in an overestimation of the total 25(OH)D due to positive interference from these metabolites.6
Another advantage of the LC-MS/MS method is that it can detect C3 epimers of 25(OH)D, while immunoassays cannot.8 The physiologic significance of these epimers has not yet been clearly delineated, but recent evidence has shown that while these epimers do not affect calcium concentrations, they do contribute to suppression of PTH levels.8 In addition, while these epimers comprise a low proportion (about 2-3%) of the overall 25(OH)D concentration in adults, they have been found in significantly higher proportions (up to 60%) in infant and pediatric populations.8,9 Thus, the detection of these epimers (and their quantitation, which is possible through high-performance LC-MS/MS) may be more important in these patient populations.
Interpretation of Vitamin D Results
The optimal serum levels of 25(OH)D are not universally established. First of all, levels vary with factors affecting sunlight exposure including latitude, skin pigmentation, and sunscreen use.1 Levels also demonstrate significant seasonal variation, with winter measurements up to 40-50% lower than summer measurements.1 Recommended minimum 25(OH)D levels for optimal bone health differ among various national organizations and generally range from 20 ng/mL to 30 ng/mL; as mentioned above, these thresholds are controversial and there is no established consensus.10-12
Vitamin D deficiency is very common, with the majority of patients exhibiting no clinical symptoms and normal calcium and phosphate concentrations. However, a significant proportion of these asymptomatic patients will have increased PTH levels and concomitant increased risk of osteopenia/osteoporosis and fractures; therefore, all patients with vitamin D deficiency should be treated with repletion.13 If deficiency is severe and persistent, bone demineralization with rickets (in children) and osteomalacia (in adults and children) can develop. In contrast, vitamin D toxicity is very rare and is usually associated with over-supplementation; patients develop hypercalcemia with related symptoms including confusion, muscle weakness, nausea and vomiting, and polydipsia and polyuria.14
Recent studies have linked vitamin D deficiency (usually with residency at higher latitudes) to a wide variety of clinical disorders ranging from autoimmune diseases (multiple sclerosis, rheumatoid arthritis, type I diabetes), to cancers (including colon, breast, and prostate), to psychiatric illnesses (schizophrenia, depression), and cardiovascular disease (including hypertension and congestive heart failure).15 Whether these links possess a causal basis or are merely associative needs to be further investigated. Nevertheless, what is certain is that understanding the functions of vitamin D in the body and methodologies of vitamin D measurement in the laboratory is crucial in appreciating its clinical significance and various, ever-expanding applications in disease pathophysiology and management.
- McPherson RA, Pincus MR. Henry’s Clinical Diagnosis and Management by Laboratory Methods. Elsevier Health Sciences; 2017.
- Brown AJ. Regulation of vitamin D action. Nephrology, dialysis, transplantation: official publication of the European Dialysis and Transplant Association-European Renal Association. 1999 Jan 1;14(1):11-6.
- Armas LA, Hollis BW, Heaney RP. Vitamin D2 is much less effective than vitamin D3 in humans. The Journal of Clinical Endocrinology & Metabolism. 2004 Nov 1;89(11):5387-91.
- Cashman KD, Hayes A, Galvin K, Merkel J, Jones G, Kaufmann M, Hoofnagle AN, Carter GD, Durazo-Arvizu RA, Sempos CT. Significance of serum 24, 25-dihydroxyvitamin D in the assessment of vitamin D status: a double-edged sword?. Clinical chemistry. 2015 Apr 1;61(4):636-45.
- Clarke W. Contemporary practice in clinical chemistry. Amer Assn for Clinical Chemistry; 2016.
- Zerwekh JE. Blood biomarkers of vitamin D status. The American journal of clinical nutrition. 2008 Apr 1;87(4):1087-91.
- Hollis BW. Assessment and interpretation of circulating 25-hydroxyvitamin D and 1, 25-dihydroxyvitamin D in the clinical environment. Endocrinology and Metabolism Clinics. 2010 Jun 1;39(2):271-86.
- Lutsey PL, Eckfeldt JH, Ogagarue ER, Folsom AR, Michos ED, Gross M. The 25-hydroxyvitamin D3 C-3 epimer: distribution, correlates, and reclassification of 25-hydroxyvitamin D status in the population-based Atherosclerosis Risk in Communities Study (ARIC). Clinica chimica acta. 2015 Mar 10;442:75-81.
- Singh RJ, Taylor RL, Reddy GS, Grebe SK. C-3 epimers can account for a significant proportion of total circulating 25-hydroxyvitamin D in infants, complicating accurate measurement and interpretation of vitamin D status. The Journal of Clinical Endocrinology & Metabolism. 2006 Aug 1;91(8):3055-61.
- Del Valle HB, Yaktine AL, Taylor CL, Ross AC, editors. Dietary reference intakes for calcium and vitamin D. National Academies Press; 2011 Apr 30.
- Vieth R. What is the optimal vitamin D status for health?. Progress in biophysics and molecular biology. 2006 Sep 1;92(1):26-32.
- American Geriatrics Society Workgroup on Vitamin D Supplementation for Older Adults. Recommendations abstracted from the American geriatrics society consensus statement on vitamin D for prevention of falls and their consequences. Journal of the American Geriatrics Society. 2014 Jan;62(1):147-52.
- Valcour A, Blocki F, Hawkins DM, Rao SD. Effects of age and serum 25-OH-vitamin D on serum parathyroid hormone levels. The Journal of Clinical Endocrinology & Metabolism. 2012 Nov 1;97(11):3989-95.
- Ozkan B, Hatun S, Bereket A. Vitamin D intoxication. Turk J Pediatr. 2012 Mar 1;54(2):93-8.
- Holick MF. Vitamin D deficiency. New England Journal of Medicine. 2007 Jul 19;357(3):266-81.
-Michelle Lin, MD, is a second-year anatomic and clinical pathology resident at Houston Methodist Hospital in Houston, Texas.