Vitamin D:The More We Know, the Less We Know

Moderators: Mitchell G. Scott1 and Ann M. Gronowski1*

Experts: Ian R. Reid,2 Michael F. Holick,3 Ravi Thadhani,4 and Karen Phinney5

Over the last 20 years there have been numerous stud-ies, including NHANES III (the National Health andNutrition Examination Survey III), the Women’sHealth Study, and the Nurses’ Health Study, showingan association between decreased 25-hydroxyvitaminD [25(OH)D]6 concentrations in blood and the risks ofcardiovascular disease, stroke, cancer, fractures, andmortality (see the Supplemental Reading List that accom-panies the online version of this Q&A at http://www.clinchem.org/content/vol60/issue12). Approximately 10years ago, these studies led to recommendations frommultiple professional societies that the definition of25(OH)D deficiency be changed from �20 ng/mL (50nmol/L) to �30 ng/mL (75 nmol/L). In the US, we andother institutions saw the volume of 25(OH)D testing in-crease 5–6-fold between 2004 and 2007. Furthermore, theuse of �30 ng/mL (75 nmol/L) to define 25(OH)D defi-ciency results in almost half of the tested population in alarge Midwestern US hospital, such as ours, as being vita-min D deficient.

In late 2010, the Institute of Medicine (IOM) issued areport that vitamin D supplementation was unlikely tobe beneficial for any condition other than bone healthand that blood concentrations of 20 ng/mL (50nmol/L) or greater were sufficient for maintainingbone health. Since then, several metaanalyses havefailed to show that low 25(OH)D concentrations areassociated with risk for any of the above-mentionednonskeletal chronic conditions, with the possible ex-ception of fractures. Complicating the association of25(OH)D blood concentrations with risk has been thepoor agreement among 25(OH)D immunoassays(e.g., differences in recognition of 25(OH)D2 and25(OH)D3, the 2 forms of commercial supplements ofthe vitamin), for which there are ongoing efforts tostandardize 25(OH)D assays. Further complicating

what we know and don’t know is the recent discovery ofgenetic polymorphisms in vitamin D binding proteins(VDBPs) that segregate well between blacks and whites,which may explain the paradox of blacks having lower25(OH)D blood concentrations than whites but higher,or equivalent, bone density. These studies suggest thatperhaps we should be looking at bioavailable 25(OH)Drather than total 25(OH)D. Here, 4 experts who have con-tributed to what we know about 25(OH)D address whatwe don’t know and where we might be headed.

How should we define 25(OH)D reference intervalsor deficiency? Central 95%? Outcomes? Which out-comes? By age? By race? By sex? Season? By biologicdefinitions such as a rise in parathyroid hormone?

Ian Reid: I think it is ap-propriate to move backto defining the referenceinterval for 25(OH)D inthe same way that we de-fine other reference in-tervals, i.e., as the central95% of the values foundin a healthy normal pop-ulation. Such a rangewould vary by race andseason. If it is possible to

reach consensus that adverse health outcomes are caus-ally related to low concentrations of 25(OH)D, thenthese could be substituted for a conventionally derivedinterval. However, that consensus does not exist atpresent, other than for the outcome of osteomalacia,which occurs only in individuals whose 25(OH)D is�10 ng/mL (25 nmol/L). The progressive increasesof the reference interval based on associations of25(OH)D with health outcomes has led to wide-spread use of vitamin D supplements, which is not

1 Division of laboratory and Genomic Medicine, Washington University School ofMedicine, St. Louis, MO; 2 Distinguished Professor of Medicine, University of Auck-land, Auckland, New Zealand; 3 Professor of Medicine, Physiology and Biophysics,Director General Clinical Research Unit, Director of Bone Health Care Clinic atBoston University Medical Center, Boston, MA; 4 Professor of Medicine, HarvardMedical School, Chief, Division of Nephrology, Massachusetts General Hospital,Boston, MA; 5 Group Leader–Bioanalytical Science, Biomolecular MeasurementDivision, National Institute of Standards and Technology, Gaithersburg, MD.

* Address correspondence to this author at: Division of Laboratory and GenomicMedicine, Box 8118, Washington University School of Medicine, 660 S. Euclid,St. Louis, MO 63110. Fax 314-362-1461; email gronowski@wustl.edu.

Dr. Phinney’s statements represent her own opinions and are not official NISTpositions.Received June 4, 2014; accepted June 9, 2014.6 Nonstandard abbreviations: 25(OH)D, 25-hydroxyvitamin D; IOM, Institute of

Medicine; VDBP, vitamin D binding protein; ICU, intensive care unit.

Clinical Chemistry 60:12000 – 000 (2014) Q&A

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http://hwmaint.clinchem.org/cgi/doi/10.1373/clinchem.2014.222521The latest version is at Papers in Press. Published July 23, 2014 as doi:10.1373/clinchem.2014.222521

Copyright (C) 2014 by The American Association for Clinical Chemistry

supported by randomized control trials, and to mas-sive expenditure on 25(OH)D assays, which is notjustified by either the quality of the measurementsproduced or by demonstrable health benefits fromthat investment.

Michael F. Holick: It isgenerally agreed world-wide and supported byboth the IOM and the En-docrine Society’s practiceguidelines that 25(OH)Ddeficiency regarding bonehealth is defined as a25(OH)D �20 ng/mL (50nmol/L). The EndocrineSociety’s practice guide-lines defined 25(OH)D in-

sufficiency as a concentration of 25(OH)D of 21–29ng/mL (52–72 nmol/L). This is based on several lines ofevidence, including the observation by Priemel (J BoneMiner Res 2010;25:305–12), who reported that 25(OH)Ddeficiency osteomalacia was not observed in any of 675adult ileac crest biopsies, obtained at autopsy, when con-centrations of 25(OH)D were �30 ng/mL (75 nmol/L).Furthermore, 24% of these presumed healthy adults with25(OH)D 21–29 ng/mL (52–72 nmol/L) had evidence of25(OH)D deficiency osteomalacia. Several studies relat-ing declining parathyroid hormone (PTH) concentra-tions with 25(OH)D concentrations found that the PTHconcentrations plateaued when the 25(OH)D concentra-tions were at least 30 ng/mL (75 nmol/L). Many associa-tion studies relating 25(OH)D status and chronic illnesshave suggested significant decreases in incidence whenblood concentrations of 25(OH)D were �30 ng/mL (75nmol/L). There is a seasonal variation in serum 25(OH)Dconcentrations. The highest concentrations are at the endof the summer and the lowest concentrations at the end ofthe winter. There is no firm evidence that concentrationsof 25(OH)D should be any different based on age, ethnic-ity, and sex.

Ravi Thadhani: The def-inition of 25(OH)D defi-ciency should be basedon risk for developing agiven disorder and/or ev-idence of bone disease,increased PTH, or de-creased calcium. Age,race, and season are allcritical in determiningexpected concentrations;however, the presence of

the above alterations should take precedence in defin-ing deficiency.

Will differences in VDBP concentration affect themeasurement of 25(OH)D by different immunoas-says or mass spectrometry methods?

Karen Phinney: Inmethods with an extrac-tion step, the 25(OH)D istypically isolated throughthe addition of organicsolvents or other re-agents that denature pro-teins in the sample andrelease protein-boundspecies. When developedand applied properly,such methods are not

likely to be affected by sample-to-sample differences inVDBP concentration. In automated methods, detailsof the process for dissociation of 25(OH)D from theVDBP are generally proprietary to the assay platform.There has been at least one report of VDBP concentra-tion affecting the results of automated assays. Heijboeret al. (N Engl J Med 2013;369:1991–2000) reported aninverse relationship between VDBP concentration anddeviations from the LC-MS/MS comparison methodfor 4 of the 5 automated assays investigated. One factorlimiting the interpretation of these results, however, isthe absence of well-established methods for measure-ment of VDBP concentrations. In addition, geneticvariants of the VDBP are believed to have differentbinding affinities for 25(OH)D. Further study isneeded before additional conclusions can be drawnabout the impact, if any, of VDBP concentrations onthe performance of the many different methods used inclinical laboratories.

Michael F. Holick: Differences in the VDBP concen-trations theoretically should not affect the ability ofimmunoassays to detect total 25(OH)D. However, it iswell documented that these assays are often unable toquantitatively detect 25(OH)D2. This may be due toeither the concentration or binding capacity of theVDBP. This is not observed with LC-MS/MS.

Ravi Thadhani: Approximately 80% of total 25(OH)Dis carried by VDBP. VDBP is a negative acute-phasereactant, with a short half-life (1–2 days). In acute con-ditions such as sepsis, liver production of VDBP goesdown, and one should expect concentrations of total25(OH)D to follow suit. Although most current assayswill correctly measure 25(OH)D concentrations, onewill more than likely find that patients with acute sepsis

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are 25(OH)D deficient. The question is whether thesepatients are truly 25(OH)D deficient, or only appear tobe transiently deficient until liver production returnsto normal. In my experience, total concentrations of25(OH)D return to baseline within 1–3 weeks of leav-ing the intensive care unit (ICU). This is not unlikedefining hypothyroidism in patients in the ICU, fromwhere the term “sick euthyroid” emanated.

Aside from acute changes in VDBP, there is somecontroversy as to the appropriate assay for VDBP. Isuggest developing an assay that directly measuresbioavailable 25(OH)D (albumin-bound and free25(OH)D), which will bypass the need to measureVDBP, especially until the differences between assaysare sorted out. That said, I believe the genetic differ-ences in VDBP are real, and do account for some por-tion of the differences in 25(OH)D between blacks andwhites. In other words, it is difficult to label all blackpatients as deficient, when in general their bone healthon all fronts is better than that of whites. Therefore,how deficiency is defined should be reexamined, as wellas which component of 25(OH)D is the appropriatemeasure to define 25(OH)D status.

Ian Reid: Yes. Current assays measure total 25(OH)D,which is made up of the free component and thatbound to VDBP.

Should we be measuring bioavailable 25(OH)D asopposed to total?

Michael F. Holick: There is not enough information atthis time to suggest that the bioavailable 25(OH)D is abetter marker for 25(OH)D status as it relates to eitherbone health or other associated nonskeletal benefits.

Ian Reid: In theory, this would be preferable. However,there are very few VDBP assays available at present, andeven fewer direct measurements of free 25(OH)D.There appear to be major technical hurdles to be over-come before such assays are freely available andaffordable.

Ravi Thadhani: The concept put forward that bioavail-able 25(OH)D is more physiologically important than to-tal concentrations of 25(OH)D is one “borrowed” fromthe metabolism of other hormones with carrier proteins,such as testosterone and thyroid hormone. While I believeit is the component of 25(OH)D that should be measured,further studies using validated assays (not yet available)are needed before routine use of bioavailable 25(OH)Dmeasurements can be advocated.

Karen Phinney: At the present time, few techniques areavailable for the direct measurement of either free or

bioavailable 25(OH)D, and such techniques are notsuited for routine use, nor has their accuracy been as-sessed. Mathematical approaches to estimation of bio-available 25(OH)D have also been reported. These ap-proaches use measured concentrations of VDBP andalbumin along with published values for affinity con-stants for the 3 primary genetic variants of VDBP. Theaccuracy of methods for the measurement of VDBP hasnot yet been established, and indeed, the work of Poweet al. showed that there was no significant correlationbetween two different commercial assays for VDBP. Inaddition, these computational methods rely upon theavailability of genotyping information that is not rou-tinely available. Additional research is needed to dem-onstrate that measured or estimated bioavailable25(OH)D is actually linked to particular health out-comes. Finally, given that 25(OH)D is also believed toenter cells through megalin-mediated endocytosis,consideration of only “free” 25(OH)D may overlookother mechanisms of action of 25(OH)D.

Should the recent findings about VDBP polymor-phisms affect interpretations about the association of25(OH)D concentrations with cancer, diabetes, andcardiovascular disease mortality? Should such stud-ies be repeated?

Karen Phinney: The body of evidence supporting anassociation between VDBP polymorphisms and con-centrations of circulating 25(OH)D is growing. Thereare 3 major variants of the VDBP (GC-1f, GC-1s, GC-2), which can be combined to form 6 diplotypes. Al-though genetic factors do appear to play a role in ob-served 25(OH)D concentrations, other factors such asvitamin D intake, season, and ancestry are probablyequally important. For example, Gozdzik et al. (J Ste-riod Biochem Mol Biol 2011;127:405–12) examinedyoung Canadian adults of various ancestries and foundthat GC-2 alleles were associated with lower 25(OH)Dconcentrations in East Asians in fall and winter, but noassociation was observed for South Asians. Malik et al.(Crit Rev Clin Lab Sci 2013;50:1–22) recently reviewedthe available evidence linking variants of the VDBPgene with adverse health outcomes including diabetes,cancer, and infectious diseases. Perhaps not surpris-ingly, as described in Malik’s review, conflicting resultshave been obtained in some cases, suggesting that otherfactors such as environment, ethnicity, and lifestylemust be considered. In addition, many of the studieshave lacked sufficient statistical power to draw mean-ingful conclusions. Nevertheless, VDBP polymor-phisms clearly add additional complexity to the inter-pretation of 25(OH)D concentrations and theirrelationship to health and disease.

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Clinical Chemistry 60:12 (2014) 3

Michael F. Holick: There have been several studiessuggesting that the polymorphisms for the VDBP aswell as the vitamin D receptor may be related to bonehealth as well as nonskeletal chronic illnesses, includ-ing some cancers. We need larger and better-designedstudies to determine the importance of these prelimi-nary observations. Furthermore, studies should beconducted to see whether increasing concentrations of25(OH)D to �30 ng/mL (75 nmol/L) can overcomethese polymorphic influences.

Ian Reid: It is highly likely that most of these associa-tions between 25(OH)D concentrations and diseasesimply reflect the fact that people who are unwell, fromwhatever cause, spend less time exercising outdoors.There is also some evidence that inflammatory condi-tions reduce concentrations of VDBP, and this mayalso contribute to some disease associations. The re-duced concentration of 25(OH)D in obesity is a furtherpossible contributor. These factors are probably muchmore important than interracial differences in VDBPpolymorphisms.

Ravi Thadhani: The studies should indeed be repeated.The data on genetic polymorphisms, differentiatingblacks from whites, are important, and other studieshave suggested that polymorphisms in the VDBP geneaccount for �10% of the variation of 25(OH)D con-centrations in population studies. Genetic differences,in conjunction with accounting for acute changes inVDBP (a negative acute-phase reactant), must also beaccounted for in studies of cancer, diabetes, and car-diovascular disease mortality.

Who should have 25(OH)D testing? General popu-lation screening? By certain age or sex groups? Basedupon symptoms?

Michael F. Holick: The Endocrine Society and IOMboth agree that general population screening shouldnot be encouraged. However, children and adults whohave risk factors for 25(OH)D deficiency should bescreened and followed after being treated with vitaminD. Some of the risk factors include obesity, fat malab-sorption syndromes, granulomatous disorders, preg-nancy, and medications such as prednisone, antisei-zure drugs, and HIV medications. Age and sex are notconsidered risk factors for 25(OH)D screening. Peopleof color are at higher risk for 25(OH)D deficiency butdo not need to be screened if provided an adequateamount of vitamin D supplementation. 25(OH)D de-ficiency symptoms are nonspecific and are not helpfulfor screening purposes.

Ian Reid: To provide an answer to this question, weneed to reach agreement regarding concentrations of25(OH)D that exert adverse effects on health. At pres-ent, the only persuasive data are for the prevention ofosteomalacia. If 25(OH)D concentrations are main-tained above 16 ng/mL (40 nmol/L), then osteomalaciais prevented with a considerable margin of safety. Thisis consistent with the recent recommendations fromthe IOM. The low reliability of the assays for 25(OH)Din widespread clinical use does not recommend themfor routine patient assessment. It is probably muchmore cost-effective to target low-dose vitamin D sup-plements (e.g., 400 – 800 U per day) to individuals whohave clinical risk factors for severe 25(OH)D deficiency(such as frailty, seldom venturing outdoors, dark skin,being habitually veiled) and not to measure 25(OH)Deither before or after institution of supplementation.

Ravi Thadhani: Measurement of 25(OH)D should beperformed in individuals at risk for deficiency, whichincludes, among other signs and symptoms, individu-als with poor bone health, history or risk for fractures,muscle pain, and kidney disease.

Moderators

We want to thank the experts for their careful andthoughtful replies to our questions. Clearly, differencesof opinion exist on what defines 25(OH)D deficiency,what should be measured to determine deficiency, andeven whether 25(OH)D measurement is needed. Weawait future studies from the experts and others to elu-cidate what approach is most clinically relevant.

Author Contributions: All authors confirmed they have contributed tothe intellectual content of this paper and have met the following 3 re-quirements: (a) significant contributions to the conception and design,acquisition of data, or analysis and interpretation of data; (b) draftingor revising the article for intellectual content; and (c) final approval ofthe published article.

Authors’ Disclosures or Potential Conflicts of Interest: Upon man-uscript submission, all authors completed the author disclosure form.Disclosures and/or potential conflicts of interest:

Employment or Leadership: M.G. Scott, Clinical Chemistry, AACC;A.M. Gronowski, Clinical Chemistry, AACC.Consultant or Advisory Role: None declared.Stock Ownership: None declared.Honoraria: R. Thadhani, Diasorin.Research Funding: R. Thadhani, NIH.Expert Testimony: None declared.Patents: None declared.

Previously published online at DOI: 10.1373/clinchem.2014.222521

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