The rise and rise of vitamin D

With research demonstrating the impact of vitamin D on a range of conditions, the importance of adequate levels of vitamin D is often in the spotlight. This has resulted in increased rates of testing from both GPs and self-referring individuals.

by Robyn Shea and Dr Jonathan Berg

Background
Vitamin D is an essential nutrient required for bone health and calcium homeostasis. It is also described as a pro-hormone as it is the biologically inactive precursor to the active secosteroid hormone 1,25-dihydroxyvitamin D [1,25(OH)2D, also known as calcitriol] [1].
Vitamin D is found in two forms: vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Both forms are available as supplements or in a small number of foodstuffs, naturally occurring or fortified. However, the main source of vitamin D, around 90%, is through endogenous synthesis in the skin with the conversion of 7-dehydrocholesterol, via UVB radiation from the sun, into vitamin D3. Vitamin D2 or D3 are hydroxylated, first in the liver to form 25-hydroxyvitamin D2 [25(OH)D2] and  25-hydroxyvitamin D3 [25(OH)D3] respectively, and secondly in the kidney to form the active hormone 1,25(OH)2D. The first hydroxylation step is unregulated and 25(OH)D levels therefore depend on the availability of the vitamin D substrate. The second hydroxylation step is tightly controlled via parathyroid hormone and through a variety of negative feedback mechanisms including calcium and phosphate levels as well as 1,25(OH)2D itself [2]. Comprehensive discussion about sources and metabolism of vitamin D are covered elsewhere in the literature [3, 4].

How is vitamin D measured?
25(OH)D is the best marker of vitamin D status. It is a difficult analyte to measure – it is very hydrophobic, is present in two forms [25(OH)D3 and 25(OH)D2] and is bound to vitamin D binding protein or albumin. Despite the difficulty in measuring it, a number of analytical platforms are available for 25(OH)D measurement:

• enzyme immunoassay (EIA)
• high pressure liquid chromatography (HPLC)
• radioimmunoassay (RIA)
• enzyme-linked immunosorbent assay (ELISA)
• liquid chromatography-tandem mass spectrometry (LC-MS/MS).

The agreement between the different methodologies is often poor and there can be quite large variation even within method groups, again highlighting how difficult it is to measure 25(OH)D. This has improved over time with the introduction of a NIST Standard Reference Material and the introduction of commercially available calibrators for chromatographic techniques [5].
Historically, manual techniques such as RIA and ELISA were used to measure 25(OH)D. Many laboratories have moved over to more automated random access immunoassays – either standalone automated analysers specifically for 25(OH)D analysis or as part of a large scale automated laboratory where 25(OH)D is just one test in a large repertoire – in order to cope with the ever increasing work load [6]. However, immunoassays, automated or not, have the disadvantages of performance changes over time, e.g. due to changes in kit reagents or antibody, and a large number of the assays are not able to detect both 25(OH)D3 and 25(OH)D2 and therefore may underestimate total 25(OH)D. Some of the immunoassays are also subject to interference from heterophilic antibodies and 24,25-dihydroxyvitamin D [5].
Direct detection methods such as HPLC and LC/MS-MS do not suffer from the disadvantages mentioned above; however, they can suffer from interference from the inactive 3-epi-25(OH)D isomer, which is often seen in high concentrations in infants. The equipment is expensive and specialist knowledge is required to set up and maintain a clinical service. However, the advantages of low consumables costs, potential for automation and high throughput, and the ability to detect both 25(OH)D2 and 25(OH)D3 explains its growing popularity with clinical laboratories [5].

Reasons for testing
Even though vitamin D can be made endogenously and is available through dietary sources, vitamin D deficiency is extremely common, verging on pandemic [7]. In inner-city Birmingham, 24% of the study population was found to be deficient [defined as having 25(OH)D levels <25 nmol/L], with 43% of Asian women deficient [8]. If vitamin D insufficiency is defined as having levels <75 nmol/L, then it has been estimated that 1 billion people worldwide are vitamin D deficient or insufficient [3]. Deficiency of vitamin D is most commonly linked with rickets in children and osteomalacia in adults, but in the last few years it has been associated with a range of non-musculoskeletal conditions such as diabetes, immune function, cardiovascular disease and cancer. As well as being deficient in vitamin D, it is possible to become intoxicated with vitamin D, often as the result of patients taking supraphysiological doses of vitamin D for a variety of reasons. Hypervitaminosis D can lead to hypercalcaemia as a result of increased intestinal calcium absorption and bone resorption, which can ultimately lead to kidney injury [9].
It is very hard to predict what a person’s vitamin D concentration is, even if you take into account factors such as age, supplement use, season, sun exposure, race and body mass intake [8]. However there are a range of risk factors and these can often help to identify people who are vitamin D deficient (Table 1) [7, 11–13].

Routine population screening is not advocated by most of the literature but there is still considerable debate as to when and who to test. The guidelines from the Endocrine Society suggest screening for vitamin D deficiency in individuals at risk for deficiency [7]. The National Osteoporosis Society guidelines suggest that vitamin D deficiency should be corrected before certain drug regimens begin, e.g. before starting treatment with a potent antiresorptive agent (zoledronate or denosumab) implying that 25(OH)D should be checked in these patients. They also recommend testing patients who have symptoms suggestive of osteomalacia or who have chronic widespread pain. They do not, however, recommend routinely testing 25(OH)D in asymptomatic individuals who may be at higher risk of vitamin D deficiency, but suggest that these individuals should have a higher intake of vitamin D [13]. The drawback of this approach is that the recommended daily allowance will not be enough to correct severe deficiency, and giving higher loading doses to those already replete may put them at risk of vitamin D intoxication.

Rate of testing
Many things in the area of vitamin D lack consensus, such as what is the optimal level of serum 25(OH)D; however, as far as the rate of testing goes the evidence is unequivocal: 25(OH)D testing is growing at a staggering rate. The author’s laboratory at City Hospital, Birmingham, UK, has seen a 75% increase in testing since 2012 and will measure around 80,000 serum samples this year for 25-hydroxy vitamin D (Fig. 1). This is even after demand management was introduced in 2010, whereby GPs could only have one 25(OH)D test per patient per year, unless there were strong clinical indications for measurement. Other laboratories have noticed an annual 80–90% growth in 25(OH)D test rates [14]. One report suggested that Quest laboratories in the USA were receiving 500,000 requests for 25(OH)D analysis per month [15].

Self-referral testing
Growth has come from testing in the GP population as well as hospital-based testing. In addition to this, our Dried Blood Spot (DBS) Vitamin D service, which is a direct-to-the-public service, has also grown steadily since its introduction in 2011. We have analysed close to 10,000 samples since the service inception and requests have been received from all over the world as well as the UK (Fig. 2).

The reasons why health professionals want to test 25(OH)D have been discussed but we have found that the reasons the general public want to test for 25(OH)D are very different. Many are knowledgeable about vitamin D and want to check their and their family’s levels are adequate. Many are taking supplements and they want to be sure that they are taking enough. Some have certain conditions, e.g. psoriasis, multiple sclerosis, cancer, and have read about connections between vitamin D and these conditions and want to make sure they have achieved recommended levels of 25(OH)D. Some are not able to get 25(OH)D tested through their GP. A small but significant proportion suspect that they have over-supplemented and they use the DBS service to check how high their levels have got [16].

The different reasons behind testing, as well as population differences such as ethnic distribution, have led to a significantly different distribution of 25(OH)D statuses in the two populations. During 2012, 54.7% of self-referred samples showerd adequate levels, compared to only 20.1% of GP samples were [25(OH)D >50 nmol/L, P<0.001, 95% confidence interval]. Only 0.1% of GP samples were high to toxic [25(OH)D >220 nmol/L] compared with 1% of the DBS samples (P<0.001, 95% confidence interval). For people who want to be pro-active about their health, the self-referral service is giving them this opportunity. It is also allowing a proportion of the population who may have been inadvertently over-supplementing to realise this and act before any long-term harm has occurred. Clinical laboratories are struggling to cope with the increase in demand for vitamin D testing, not just with physically performing the work but also because many are not paid appropriately for the analysis and this has led to financial difficulties for some laboratories. However, the demand from the public is not going to abate, especially as the media and public awareness of vitamin D grows. Self-referral vitamin D testing gives people a viable alternative when they are not able to access the testing through their health care system. Into the future…
Vitamin D testing is going to continue to rise and clinical laboratories are going to have to find strategies to cope. This may mean putting demand management strategies in place, negotiating prices with commissioners in order to be paid appropriately and looking at the methods used for measuring 25(OH)D to see if savings and efficiencies can be made.

As vitamin D remains in the media spotlight, and while vast numbers of papers relating to vitamin D continue to be published, hopefully the awareness of the importance of having adequate levels of vitamin D will continue to rise amongst healthcare professionals, and advice on what to do to achieve adequate levels will improve. One day we will have a national strategy on who to test, when and how often, but for now it seems that some of the population will continue to try to figure it out for themselves.

References
1. Pearce SHS, Cheetham TD. Diagnosis and management of vitamin D deficiency. BMJ 2010; 340: b5664.
2. Norman AW. From vitamin D to hormone D: fundamentals of the vitamin D endocrine system essential for good health. Am J Clin Nutr. 2008; 88(suppl): 491S-499S.
3. Holick MF. Vitamin D Deficiency. N Engl J Med. 2007; 357: 266-281.
4. Zerwekh JE. Blood biomarkers of vitamin D status. Am J Clin Nutr. 2008; 87(suppl): 1087S-1091S.
5. Carter GD. 25-Hydroxyvitamin D: a difficult analyte. Clin Chem. 2012; 58; 486-488.
6. Hollis BW. Measuring 25-hydroxyvitamin D in a clinical environment: challenges and needs. Am J Clin Nutr. 2008; 88(suppl): 507S-510S.
7. Holick MF, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an endocrine society clinical practice guideline. J Clin Endocrinol Metab. 2011; 96: 1911-1930.
8. Ford L, et al. Vitamin D concentrations in an UK inner-city multicultural outpatient population. Ann Clin Biochem. 2006; 43: 468-473.
9. Vogiatzi MG, et al. Vitamin D supplementation and risk of toxicity in pediatrics: a review of current literature [Epub ahead of print]. J Clin Endocrinol Metab. 2014; 99: doi 10.1210/jc.2013-3655.
10. Freedman DM, et al. Sunlight and other determinants of circulating 25-hydroxyvitamin D levels in black and white participants in a nationwide US study. Am J Epidemiol. 2013; 177: 180-192.
11. Harrison M, et al. The Royal College of Pathologists of Australia Position Statement on the use and interpretation of vitamin D testing. 2013.
12. Hull S, Anastasiadis T. Vitamin D guidance. Barts and the London Clinical Effectiveness Group. 2011.
13. Francis R, et al. Vitamin D and bone health: a practical clinical guideline for patient management. National Osteoporosis Society. 2013.
14. Singh RJ. Are clinical laboratories prepared for accurate testing of 25-hydroxy vitamin D? Clin Chem. 2008; 54: 221-223.
15. Carter GD. 25-Hydroxyvitamin D assays: the quest for accuracy. Clin Chem. 2009; 55: 1300-1302.
16. Shea RL, Berg JD. Self referral vitamin D testing: are we just testing the worried well or making an important contribution to healthcare? Ann Clin Biochem. 2013; 50(suppl): 34.

The authors
Robyn Shea* BSc, MSc, MSc, DipRCPath and Dr Jonathan Berg BSc, PhD, MCB, FRCPath, MBA
Department of Clinical Biochemistry, Sandwell and West Birmingham Trust Hospitals NHS Trust, Birmingham, UK

*Corresponding author
E-mail: robyn.shea@nhs.net