- Email: [email protected]
Vitamin D measurement standardization: The way out of the chaos
Accepted Manuscript Title: Vitamin D Measurement Standardization: The Way Out of the Chaos Author: N. Binkley B. Dawson-Hughes R. Durazo-Arvizu M. Thamm L. Tian J.M. Merkel J.C. Jones G.D. Carter C.T. Sempos PII: DOI: Reference:
S0960-0760(16)30341-7 http://dx.doi.org/doi:10.1016/j.jsbmb.2016.12.002 SBMB 4836
To appear in:
Journal of Steroid Biochemistry & Molecular Biology
Received date: Accepted date:
13-8-2016 11-12-2016
Please cite this article as: N.Binkley, B.Dawson-Hughes, R.Durazo-Arvizu, M.Thamm, L.Tian, J.M.Merkel, J.C.Jones, G.D.Carter, C.T.Sempos, Vitamin D Measurement Standardization: The Way Out of the Chaos, Journal of Steroid Biochemistry and Molecular Biology http://dx.doi.org/10.1016/j.jsbmb.2016.12.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Vitamin D Measurement Standardization: The Way Out of the Chaos Binkley N1, Dawson‐Hughes B2, Durazo‐Arvizu R3, Thamm M4, Tian L5, Merkel JM6, Jones JC7, Carter GD7, Sempos CT6,8 For the Vitamin D Standardization Program 1
Osteoporosis Clinical Research Program and Institute on Aging, University of Wisconsin‐ Madison, Madison, WI, USA; 2 Bone Metabolism Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts 02111; 3 Department of Public Health Sciences, Loyola University Stritch School of Medicine, Maywood, IL, 60153, USA; 4Robert Koch Institute, 12101 Berlin, Germany; 5Department of Health Research and Policy, Stanford University, Palo Alto, CA, U.S.A.; 6Office of Dietary Supplements, National Institutes of Health, Bethesda MD 20892. 7Vitamin D External Quality Assurance Scheme (DEQAS) Coordination Centre, Imperial College, London, UK; 8Department of Population Health Sciences, University of Wisconsin‐Madison, Madison, WI, 53726, USA. Corresponding Author: Christopher T. Sempos, Ph.D. Coordinator, Vitamin D Standardization Program (VDSP) NIH Office of Dietary Supplements 6100 Executive Blvd., Rm. 3B01 Bethesda, MD 20892‐7517, U.S.A. [FedEx: Rockville, MD 20852, U.S.A.] Tel: +716‐400‐3925 E‐mail: [email protected]
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Highlights The lack of standardized laboratory measurement of serum total 25‐hydroxyvitamin D [25‐ OHD] in vitamin D research impedes development of consensus 25‐OHD values to define stages of vitamin D status. Some 60,000 vitamin D papers have been published since the discovery of 25‐OHD with nearly all the values from non‐standardized assays. We cannot just ignore this old data and focus on standardizing 25‐OHD in current and future research. Vitamin D Standardization Program (VDSP) has developed “retrospective standardization” protocols to address this problem. VDSP retrospective standardization of 25‐OHD values to gold standard reference measurements values has been shown to be accurate. Retrospective standardization results can vary widely depending on if initial 25‐OHD measurements were determined using an assay that was positively or negatively biased and the level of bias in the old assay. An International effort is needed to identify key prior studies with stored samples for re‐ analysis and standardization, initially, to define the 25‐OHD level associated with vitamin D deficiency (rickets/osteomalacia). Subsequent work could focus on defining inadequacy. Finally, levels of assay variation and lack of standardized research data highlight the importance of suspending publication of meta‐analyses based on unstandardized 25(OH)D results.
Abstract Substantial variability is associated with laboratory measurement of serum total 25‐ hydroxyvitamin D [25(OH)D]. The resulting chaos impedes development of consensus 25(OH)D values to define stages of vitamin D status. As resolving this situation requires standardized measurement of 25(OH)D, the Vitamin D Standardization Program (VDSP) developed methodology to standardize 25(OH)D measurement to the gold standard reference measurement procedures of NIST, Ghent University and CDC. Importantly, VDSP developed protocols for standardizing 25(OH)D values from prior research based on availability of stored serum samples. The effect of such retrospective standardization on prevalence of “low” vitamin
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D status in national studies reported here for The Third National Health and Nutrition Examination Survey (NHANES III, 1988‐1994) and the German Health Interview and Examination Survey for Children and Adolescents (KIGGS, 2003‐2006) was such that in NHANES III 25(OH)D values were lower than original values while higher in KIGGS. In NHANES III the percentage with values below 30, 50 and 75 nmol/L increased from 4% to 6%, 22% to 31% and 55% to 71%, respectively. Whereas in KIGGS after standardization the percentage below 30, 50, and 70 nmol/L decreased from 28% to 13%, 64% to 47% and 87% to 85% respectively. Moreover, in a hypothetical example, depending on whether the 25(OH)D assay was positively or negatively biased by 12%, the 25(OH)D concentration which maximally suppressed PTH could vary from 20 – 35ng/mL.. These examples underscore the challenges (perhaps impossibility) of developing vitamin D guidelines using unstandardized 25(OH)D data. Retrospective 25(OH)D standardization can be applied to old studies where stored serum samples exist. As a way forward, we suggest an international effort to identify key prior studies with stored samples for re‐analysis and standardization initially to define the 25(OH)D level associated with vitamin D deficiency ( rickets/osteomalacia). Subsequent work could focus on defining inadequacy. Finally, examples reported here highlight the importance of suspending publication of meta‐ analyses based on unstandardized 25(OH)D results. Abbreviations: 25(OH)D, Serum Total 25‐hydroxyvitamn D; CDC, Centers for Disease Control and Prevention; EIA, enzyme immunoassay; IOM, Institute of Medicine KiGGS, German Health Interview and Examination Survey for Children and Adolescents;
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LC‐MS/MS, Liquid chromatography‐tandem mass spectrometry; NANS, Irish National Health/Nutrition Surveys; NHANES, National Health and Nutrition Examination Survey; NIST, National Institute of Standards and Technology; RMP, reference measurement procedure; UCC, University College Cork, Cork, Ireland; VDSP, Vitamin D Standardization Program; Key Words: 25‐hydroxyvitamin D, CAP, DEQAS, NIST, PT/EQA, VDSP, Vitamin D
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Introduction The vitamin D field is in chaos. As an example, we have two very different sets of guidelines for defining clinically relevant states of vitamin D status – especially vitamin D deficiency and insufficiency; i.e. those of the Institute of Medicine and those released by the Endocrine Society (see Table 1) [1, 2]. In both, serum total 25‐hydroxyvitamin D [25(OH)D] concentration was used to interpret vitamin D status. As these guidelines are not concordant, a common question is: Which set of guidelines is correct? This is obviously a very important question for clinicians and patients, but it may not be the most important question to ask. A more fundamental question, given the multitude of clinical studies and meta‐analyses that have been and continue to be published is: Why has the field not been able to reach consensus? In our opinion, the answer to this question is in large part due to the substantial variability both within and among the various laboratory assays for measuring 25(OH)D [3‐5]. This lack of standardized 25(OH)D research data is a fundamental limitation in establishing consensus clinical and public health vitamin D guidelines [6]. To address this limitation and thus facilitate guideline development, the Vitamin D Standardization Program (VDSP) [7] since 2010 has coordinated an international effort to standardize the laboratory measurement of 25(OH)D in current and future measurement systems to the gold standard reference assays or reference measurement procedures (RMPs) developed by the National Institute for Standards and Technology (NIST), Ghent University and the Centers for Disease Control and Prevention (CDC) [8‐11]. Thus, an approach is in place to produce standardized 25(OH)D results in current and future studies. However, given that some 60,000 papers on vitamin D have been published since 25(OH)D was first isolated and
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chemically identified in 1968 it is essential, where possible, to retrospectively standardize 25(OH)D measurements from key studies conducted in the past [12, 13]. To meet that need the VDSP has developed a robust statistical sampling procedure for selecting a relatively small sample of properly stored serum samples (n ≈ 150) from completed studies for re‐analysis and use in calibrating all of the 25(OH)D values to gold standard reference measurement procedure values [7, 14]. In this paper we demonstrate the potential impact of assay standardization on the distribution of 25(OH)D in national health/nutrition surveys, and in clinical and epidemiological studies. Standardizing 25 (OH)D Measurements from the Past Can old measurements of 25(OH)D from national health/nutrition surveys and clinical and epidemiological studies be calibrated (standardized) to the gold standard reference measurement procedures? Yes – if properly stored serum samples are available – the VDSP has developed and expanded two different protocols for calibrating those old values [14, 15]. In general, the VDSP protocols consist of five basic steps: (1) Estimate the number of serum samples to be re‐measured (n ≈ 150 serum samples in most cases); (2) Select the specific stored samples to be re‐measured from the sorted original 25(OH)D values of the complete data set using the VDSP’s uniform sampling approach [14]; (3) Re‐measure the 25(OH)D concentration in these samples using an assay that is certified to be traceable to the gold standard RMPs; (4) Develop a mathematical model to calibrate (standardize) the “Old” 25(OH)D values to “True” ones; (5) Apply the model to the entire data set to calibrate (standardize) all values to the gold standard RMP values.
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Validity of VDSP Algorithm A study to evaluate the validity of the VDSP protocols was recently conducted by Cashman et al. at University College Cork (UCC), Cork, Ireland [15]. The results clearly support the validity of the VDSP protocols for standardizing 25(OH)D values from old studies (Table 2). In that study, data from the Irish National Health/Nutrition Surveys (NANS) were used to compare the original results (n=1,132) obtained using an Immunodiagnostic Systems Holdings, PLC (IDS) enzyme immunoassay (EIA) with calibrated values using the VDSP protocol. The VDSP protocol was applied by the selection and re‐measurement of 99 serum samples using the UCC’s CDC‐ certified traceable LC‐MS/MS assay, development of a calibration equation and the application of that equation to all 1,132 NANS values to calculate standardized 25(OH)D values for the entire sample. Additionally, UCC’s CDC‐certified assay was used to re‐measure all 1,132 NANS samples to produce an entire data set where all the samples reflected the standardized or true 25(OH)D concentration. As noted in Table 2, the proportion below commonly accepted cutpoints is virtually identical whether the VDSP protocol is applied of when all samples re‐ measured.
Since conducting this proof of principal study, the VDSP protocols has been used to
standardize 25(OH)D values from the Canadian Health Measures Survey [16], Nordic Countries [17], the surveys participating in the ODIN study of vitamin D status in EU member states [18], and the US National Health and Nutrition Examination Surveys (NHANES) [19]. These studies demonstrate the potential for using the VDSP approach to retrospectively standardize 25(OH)D data from past studies and in this way to develop an unbiased research database composed of 25(OH)D values calibrated to the gold standard RMPs.
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Impact of Assay Standardization
Assay standardization can have a profound effect on the interpretation of research data. For
example, prior to standardization, it appeared that there was a decline in mean serum total 25(OH)D from NHANES III (1988‐1994) through NHANES 2005‐2006 (Figure 1). However, after standardizing the results it is clear that there was no change in mean serum total 25(OH)D over that approximately 15 year period [19]. Importantly, the impact of assay standardization may vary from study to study depending on whether the original measurements were measured using an assay that was unbiased, negatively biased or positively biased in the clinical range [15‐19]. For example, in the Irish NANS and in NHANES III the impact of retrospective 25(OH)D assay standardization was to increase the percentage of the population with low vitamin D status [15‐19]. In contrast, in the German KIGGS survey the proportion of the German population with low vitamin D status was greatly reduced (Figures 2A and 2B) [17]. In addition to affecting estimates of the prevalence of low vitamin D status, standardization of 25(OH)D research data may also have an impact of the development of vitamin D intake guidelines. For example, in a reanalysis of data used by the IOM Committee, the 25(OH)D concentration achieved based on a usual intake of 600 IU vitamin D3, could vary from 50‐70 nmol/L depending on the bias of the assays used [12]. In addition, depending on whether the 25(OH)D assay was positively or negatively biased by 12%, the concentration of serum 25(OH)D which maximally suppresses PTH could vary from 20 – 35ng/mL (Figure 3).
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To summarize, failure to utilize standardized 25(OH)D assay data can lead to falsely high or low prevalence estimates for vitamin D inadequacy. Additionally, use of unstandardized data can lead to differing determinations of what 25(OH)D level constitutes “low” vitamin D status. Moving Forward: A proposal In conclusion, unless and until 25(OH)D data from research studies identified as being key sources of information are standardized, efforts to develop consensus guidelines will continue to flounder. The examples reported here highlight the challenges inherent in attempting meta‐ analyses using unstandardized 25(OH)D results; we recommend suspending publication of meta‐analyses based on unstandardized 25(OH)D data. Given this, what is the best course to move forward? If one revisits the IOM [1], Endocrine Society [2], and the recently released UK Scientific Advisory Committee on Nutrition (SACN) guidelines [20] it should be possible to identify the studies that have had an impact on those guidelines. Once identified it can be determined if banked serum samples from these studies exist. If they do, obtaining funding to calibrate those old values to gold standard 25(OH) values is indicated. If banked serum samples do not exist, the original investigators might decide to attempt to repeat the original research using an assay traceable to the gold standard RMPs.
Initially, it seems reasonable to focus on studies of vitamin D deficiency. Specifically, we
propose an international effort to identify key studies evaluating the association between serum total 25(OH)D and the presence/grade of vitamin D related rickets and osteomalacia. That is, what are the specific concentrations of serum 25(OH)D that are associated with established vitamin D rickets in children and osteomalacia in adults? The necessity of this approach was highlighted by a recent evidenced‐base review of data on this question in
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children that states, “While 25(OH)D assays differed across the studies, these results suggest that the serum 25(OH)D concentration associated with rickets may be much higher than previously thought” [21]. Assuming retrospective standardization harmonizes the definition of vitamin D deficiency, the next logical step would be to take a similar course to define what constitutes “vitamin D inadequacy.” The VDSP offers to work with any and all investigators willing to participate in this effort (email: [email protected]).
Disclaimer: The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the National Institutes of Health or the US Department of Health and Human Services.
Conflict of interest: none declared.
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References [1] Institute of Medicine (US) Committee to Review Dietary Reference Intakes for Vitamin D and Calcium, Dietary Reference Intakes for Calcium and Vitamin D, National Academies Press, Washington, DC, 2011. [2] M.F. Holick, N.C. Binkley, H.A. Bischoff‐Ferrari, C.M. Gordon, D.A. Hanley, R.P. Heaney, M.H. Murad, C.M. Weaver, S. Endocrine, Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline, J Clin Endocrinol Metab, 96 (2011) 1911‐1930. [3] N. Binkley, D. Krueger, C.S. Cowgill, L. Plum, E. Lake, K.E. Hansen, H.F. DeLuca, M.K. Drezner, Assay variation confounds the diagnosis of hypovitaminosis D: a call for standardization, J Clin Endocrinol Metab, 89 (2004) 3152‐3157. [4] G.D. Carter, Accuracy of 25‐hydroxyvitamin D assays: confronting the issues, Curr Drug Targets, 12 (2011) 19‐28. [5] J.K. Lai, R.M. Lucas, E. Banks, A.L. Ponsonby, G. Ausimmune Investigator, Variability in vitamin D assays impairs clinical assessment of vitamin D status, Intern Med J, 42 (2012) 43‐50. [6] N. Binkley, C.T. Sempos, D.S.P. Vitamin, Standardizing vitamin D assays: the way forward, J Bone Miner Res, 29 (2014) 1709‐1714. [7] C.T. Sempos, H.W. Vesper, K.W. Phinney, L.M. Thienpont, P.M. Coates, D.S.P. Vitamin, Vitamin D status as an international issue: national surveys and the problem of standardization, Scand J Clin Lab Invest Suppl, 243 (2012) 32‐40.
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[8] S.S. Tai, M. Bedner, K.W. Phinney, 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, 82 (2010) 1942‐1948. [9] H.C. Stepman, A. Vanderroost, K. Van Uytfanghe, L.M. Thienpont, Candidate reference measurement procedures for serum 25‐hydroxyvitamin D3 and 25‐hydroxyvitamin D2 by using isotope‐dilution liquid chromatography‐tandem mass spectrometry, Clin Chem, 57 (2011) 441‐ 448. [10] E.M. Mineva, R.L. Schleicher, M. Chaudhary‐Webb, K.L. Maw, J.C. Botelho, H.W. Vesper, C.M. Pfeiffer, A candidate reference measurement procedure for quantifying serum concentrations of 25‐hydroxyvitamin D(3) and 25‐hydroxyvitamin D(2) using isotope‐dilution liquid chromatography‐tandem mass spectrometry, Anal Bioanal Chem, 407 (2015) 5615‐5624. [11] Bureau International des Poids et Mesures (BIPM), JCTLM Database: Laboratory Medicine and in vitro Diagnostics, http://www.bipm.org/jctlm/, (accessed August 10, 2016). [12] J.W. Blunt, H.F. DeLuca, H.K. Schnoes, 25‐hydroxycholecalciferol. A biologically active metabolite of vitamin D3, Biochemistry, 7 (1968) 3317‐3322. [13] C.T. Sempos, R.A. Durazo‐Arvizu, N. Binkley, J. Jones, J.M. Merkel, G.D. Carter, Developing vitamin D dietary guidelines and the lack of 25‐hydroxyvitamin D assay standardization: The ever‐present past, J Steroid Biochem Mol Biol, (2015).
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[14] L. Tian, R.A. Durazo‐Arvizu, G. Myers, S. Brooks, K. Sarafin, C.T. Sempos, The estimation of calibration equations for variables with heteroscedastic measurement errors, Stat Med, 33 (2014) 4420‐4436. [15] K.D. Cashman, M. Kiely, M. Kinsella, R.A. Durazo‐Arvizu, L. Tian, Y. Zhang, A. Lucey, A. Flynn, M.J. Gibney, H.W. Vesper, K.W. Phinney, P.M. Coates, M.F. Picciano, C.T. Sempos, Evaluation of Vitamin D Standardization Program protocols for standardizing serum 25‐hydroxyvitamin D data: a case study of the program's potential for national nutrition and health surveys, Am J Clin Nutr, 97 (2013) 1235‐1242. [16] K. Sarafin, R. Durazo‐Arvizu, L. Tian, K.W. Phinney, S. Tai, J.E. Camara, J. Merkel, E. Green, C.T. Sempos, S.P. Brooks, Standardizing 25‐hydroxyvitamin D values from the Canadian Health Measures Survey, Am J Clin Nutr, 102 (2015) 1044‐1050. [17] K.D. Cashman, K.G. Dowling, Z. Skrabakova, M. Kiely, C. Lamberg‐Allardt, R.A. Durazo‐ Arvizu, C.T. Sempos, S. Koskinen, A. Lundqvist, J. Sundvall, A. Linneberg, B. Thuesen, L.L. Husemoen, H.E. Meyer, K. Holvik, I.M. Gronborg, I. Tetens, R. Andersen, Standardizing serum 25‐hydroxyvitamin D data from four Nordic population samples using the Vitamin D Standardization Program protocols: Shedding new light on vitamin D status in Nordic individuals, Scand J Clin Lab Invest, 75 (2015) 549‐561. [18] K.D. Cashman, K.G. Dowling, Z. Skrabakova, M. Gonzalez‐Gross, J. Valtuena, S. De Henauw, L. Moreno, C.T. Damsgaard, K.F. Michaelsen, C. Molgaard, R. Jorde, G. Grimnes, G. Moschonis, C. Mavrogianni, Y. Manios, M. Thamm, G.B. Mensink, M. Rabenberg, M.A. Busch, L. Cox, S. Meadows, G. Goldberg, A. Prentice, J.M. Dekker, G. Nijpels, S. Pilz, K.M. Swart, N.M. van Schoor,
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P. Lips, G. Eiriksdottir, V. Gudnason, M.F. Cotch, S. Koskinen, C. Lamberg‐Allardt, R.A. Durazo‐ Arvizu, C.T. Sempos, M. Kiely, Vitamin D deficiency in Europe: pandemic?, Am J Clin Nutr, 103 (2016) 1033‐1044. [19] R.L. Schleicher, M.R. Sternberg, D.A. Lacher, C.T. Sempos, A.C. Looker, R.A. Durazo‐Arvizu, E.A. Yetley, M. Chaudhary‐Webb, K.L. Maw, C.M. Pfeiffer, C.L. Johnson, The vitamin D status of the US population from 1988 to 2010 using standardized serum concentrations of 25‐ hydroxyvitamin D shows recent modest increases, Am J Clin Nutr, 104 (2016) 454‐461. [20] Scientific Advisory Committee on Nutrition (SACN), Vitamin D and Health, Public Health England, 2016, https://www.gov.uk/government/publications/sacn‐vitamin‐d‐and‐health‐ report, (accessed August 10, 2016). [21] A. Cranney, T. Horsley, S. O'Donnell, H. Weiler, L. Puil, D. Ooi, S. Atkinson, L. Ward, D. Moher, D. Hanley, M. Fang, F. Yazdi, C. Garritty, M. Sampson, N. Barrowman, A. Tsertsvadze, V. Mamaladze, Effectiveness and safety of vitamin D in relation to bone health, Evid Rep Technol Assess (Full Rep), (2007) 1‐235.
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Figure Legends: Figure 1. Impact of Assay Standardization on Trends in Serum Total 25‐hydroxyvitamin D from the US National and Nutrition Examination Surveys: NHANES III (1988‐1994), NHANES 2001‐ 2002, 2003‐2004 and 2005‐2006. In these data from NHANES, use of the original 25(OH)D results would lead to the conclusion that mean 25(OH)D declined from 1988‐1994, and potentially further declined in 2005‐2006. In contrast, use of standardized 25(OH)D data clearly demonstrates no change in mean value. Figure 2. Impact of Assay Standardization on the Prevalence of Persons with Serum Total 25‐ hydroxyvitamin D Concentrations < 30, < 50 and < 70 nmol/L. Results from the US NHANES III (1988‐1994) (A) and the German KIGGS (B). The potential effect of retrospective 25(OH)D standardization on vitamin D status prevalence is noted here. In the NHANES data (a), the prevalence of “low” vitamin D status below commonly utilized cutpoints was increased whereas in the KIGGS data (b), the prevalence was reduced. Figure 3. Hypothetical Example of the Effect of Assay Standardization on the Relationship of 25(OH)D with PTH. The 25(OH)D/PTH relationship is often utilized as part of the guideline development approach with adequacy considered to be present when higher 25(OH)D is no longer associated with lower PTH values. In this hypothetical example, the unstandardized 25(OH)D values obtained in this study were arbitrarily reduced 12% (top figure; circles) or increased 12% (bottom figure; squares). Assay variability of this extent is common. It is apparent that the point at which PTH plateaus differs from 20 ng/mL to 35 ng/mL, potentially leading to different conclusions regarding vitamin D “inadequacy.”
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Figure 1
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Figure 2
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Figure 3
A
B
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Table 1. A Comparison of the Institute of Medicine (IOM) and Endocrine Society Guidelines [1, 2] for interpreting the concentration of serum total 25‐hydroxyvitamin D. Which is Correct? Vitamin D Status Interpretation
Institute of Medicine [1]
Endocrine Society [2]
Serum Total 25‐hydroxyvitamin D (ng/mL)*
Deficient
< 12 ng/mL
< 20 ng/mL
Insufficient
12‐20 ng/mL
20‐29 ng/mL
Sufficient
20‐30 ng/mL
30‐100 ng/mL
No Added Benefit
30‐50 g/mL
>50 ng/mL
> 100 ng/mL
Possible Harm *Note: To convert ng/mL to nmol/L multiple by 2.5
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Table 2. Percent of Irish National Health/Nutrition Survey (NANS) with Serum 25‐ hydroxyvitamin D Concentrations Below Cut‐points: A Comparison of Original Results (n=1,132) with VDSP Standardized Results Based on a Re‐Measurement of a Statistical Subsample (n=99), and with Standardized Results Based on a Re‐Measurement of All Study Samples (n=1,132) using University College Cork’s (UCC) CDC Certified Traceable LC‐MS/MS Assay. VDSP Statistical Original IDS/EIA Assay
UCC’s CDC Certified Standardization
Results
LC‐MS/MS *
Protocols Serum Total 25(OH)D (n=1,132)
(n=99)
(n=1,132)
< 30 nmol/L
6.5%
11.4%
11.2%
< 40 nmol/L
21.9%
25.3%
27.2%
< 50 nmol/L
40.0%
43.7%
45.0%
>125 nmol/L
1%
0.3%
0.6%
Cutpoint
*Mathematical model based on the results of 99 serum samples re‐measured using UCC’s CDC Certified Traceable Assay was used to Calibrate/Standardize all NANS 25(OH)D concentrations.
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S0960-0760(16)30341-7 http://dx.doi.org/doi:10.1016/j.jsbmb.2016.12.002 SBMB 4836
To appear in:
Journal of Steroid Biochemistry & Molecular Biology
Received date: Accepted date:
13-8-2016 11-12-2016
Please cite this article as: N.Binkley, B.Dawson-Hughes, R.Durazo-Arvizu, M.Thamm, L.Tian, J.M.Merkel, J.C.Jones, G.D.Carter, C.T.Sempos, Vitamin D Measurement Standardization: The Way Out of the Chaos, Journal of Steroid Biochemistry and Molecular Biology http://dx.doi.org/10.1016/j.jsbmb.2016.12.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Vitamin D Measurement Standardization: The Way Out of the Chaos Binkley N1, Dawson‐Hughes B2, Durazo‐Arvizu R3, Thamm M4, Tian L5, Merkel JM6, Jones JC7, Carter GD7, Sempos CT6,8 For the Vitamin D Standardization Program 1
Osteoporosis Clinical Research Program and Institute on Aging, University of Wisconsin‐ Madison, Madison, WI, USA; 2 Bone Metabolism Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts 02111; 3 Department of Public Health Sciences, Loyola University Stritch School of Medicine, Maywood, IL, 60153, USA; 4Robert Koch Institute, 12101 Berlin, Germany; 5Department of Health Research and Policy, Stanford University, Palo Alto, CA, U.S.A.; 6Office of Dietary Supplements, National Institutes of Health, Bethesda MD 20892. 7Vitamin D External Quality Assurance Scheme (DEQAS) Coordination Centre, Imperial College, London, UK; 8Department of Population Health Sciences, University of Wisconsin‐Madison, Madison, WI, 53726, USA. Corresponding Author: Christopher T. Sempos, Ph.D. Coordinator, Vitamin D Standardization Program (VDSP) NIH Office of Dietary Supplements 6100 Executive Blvd., Rm. 3B01 Bethesda, MD 20892‐7517, U.S.A. [FedEx: Rockville, MD 20852, U.S.A.] Tel: +716‐400‐3925 E‐mail: [email protected]
1
Highlights The lack of standardized laboratory measurement of serum total 25‐hydroxyvitamin D [25‐ OHD] in vitamin D research impedes development of consensus 25‐OHD values to define stages of vitamin D status. Some 60,000 vitamin D papers have been published since the discovery of 25‐OHD with nearly all the values from non‐standardized assays. We cannot just ignore this old data and focus on standardizing 25‐OHD in current and future research. Vitamin D Standardization Program (VDSP) has developed “retrospective standardization” protocols to address this problem. VDSP retrospective standardization of 25‐OHD values to gold standard reference measurements values has been shown to be accurate. Retrospective standardization results can vary widely depending on if initial 25‐OHD measurements were determined using an assay that was positively or negatively biased and the level of bias in the old assay. An International effort is needed to identify key prior studies with stored samples for re‐ analysis and standardization, initially, to define the 25‐OHD level associated with vitamin D deficiency (rickets/osteomalacia). Subsequent work could focus on defining inadequacy. Finally, levels of assay variation and lack of standardized research data highlight the importance of suspending publication of meta‐analyses based on unstandardized 25(OH)D results.
Abstract Substantial variability is associated with laboratory measurement of serum total 25‐ hydroxyvitamin D [25(OH)D]. The resulting chaos impedes development of consensus 25(OH)D values to define stages of vitamin D status. As resolving this situation requires standardized measurement of 25(OH)D, the Vitamin D Standardization Program (VDSP) developed methodology to standardize 25(OH)D measurement to the gold standard reference measurement procedures of NIST, Ghent University and CDC. Importantly, VDSP developed protocols for standardizing 25(OH)D values from prior research based on availability of stored serum samples. The effect of such retrospective standardization on prevalence of “low” vitamin
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D status in national studies reported here for The Third National Health and Nutrition Examination Survey (NHANES III, 1988‐1994) and the German Health Interview and Examination Survey for Children and Adolescents (KIGGS, 2003‐2006) was such that in NHANES III 25(OH)D values were lower than original values while higher in KIGGS. In NHANES III the percentage with values below 30, 50 and 75 nmol/L increased from 4% to 6%, 22% to 31% and 55% to 71%, respectively. Whereas in KIGGS after standardization the percentage below 30, 50, and 70 nmol/L decreased from 28% to 13%, 64% to 47% and 87% to 85% respectively. Moreover, in a hypothetical example, depending on whether the 25(OH)D assay was positively or negatively biased by 12%, the 25(OH)D concentration which maximally suppressed PTH could vary from 20 – 35ng/mL.. These examples underscore the challenges (perhaps impossibility) of developing vitamin D guidelines using unstandardized 25(OH)D data. Retrospective 25(OH)D standardization can be applied to old studies where stored serum samples exist. As a way forward, we suggest an international effort to identify key prior studies with stored samples for re‐analysis and standardization initially to define the 25(OH)D level associated with vitamin D deficiency ( rickets/osteomalacia). Subsequent work could focus on defining inadequacy. Finally, examples reported here highlight the importance of suspending publication of meta‐ analyses based on unstandardized 25(OH)D results. Abbreviations: 25(OH)D, Serum Total 25‐hydroxyvitamn D; CDC, Centers for Disease Control and Prevention; EIA, enzyme immunoassay; IOM, Institute of Medicine KiGGS, German Health Interview and Examination Survey for Children and Adolescents;
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LC‐MS/MS, Liquid chromatography‐tandem mass spectrometry; NANS, Irish National Health/Nutrition Surveys; NHANES, National Health and Nutrition Examination Survey; NIST, National Institute of Standards and Technology; RMP, reference measurement procedure; UCC, University College Cork, Cork, Ireland; VDSP, Vitamin D Standardization Program; Key Words: 25‐hydroxyvitamin D, CAP, DEQAS, NIST, PT/EQA, VDSP, Vitamin D
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Introduction The vitamin D field is in chaos. As an example, we have two very different sets of guidelines for defining clinically relevant states of vitamin D status – especially vitamin D deficiency and insufficiency; i.e. those of the Institute of Medicine and those released by the Endocrine Society (see Table 1) [1, 2]. In both, serum total 25‐hydroxyvitamin D [25(OH)D] concentration was used to interpret vitamin D status. As these guidelines are not concordant, a common question is: Which set of guidelines is correct? This is obviously a very important question for clinicians and patients, but it may not be the most important question to ask. A more fundamental question, given the multitude of clinical studies and meta‐analyses that have been and continue to be published is: Why has the field not been able to reach consensus? In our opinion, the answer to this question is in large part due to the substantial variability both within and among the various laboratory assays for measuring 25(OH)D [3‐5]. This lack of standardized 25(OH)D research data is a fundamental limitation in establishing consensus clinical and public health vitamin D guidelines [6]. To address this limitation and thus facilitate guideline development, the Vitamin D Standardization Program (VDSP) [7] since 2010 has coordinated an international effort to standardize the laboratory measurement of 25(OH)D in current and future measurement systems to the gold standard reference assays or reference measurement procedures (RMPs) developed by the National Institute for Standards and Technology (NIST), Ghent University and the Centers for Disease Control and Prevention (CDC) [8‐11]. Thus, an approach is in place to produce standardized 25(OH)D results in current and future studies. However, given that some 60,000 papers on vitamin D have been published since 25(OH)D was first isolated and
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chemically identified in 1968 it is essential, where possible, to retrospectively standardize 25(OH)D measurements from key studies conducted in the past [12, 13]. To meet that need the VDSP has developed a robust statistical sampling procedure for selecting a relatively small sample of properly stored serum samples (n ≈ 150) from completed studies for re‐analysis and use in calibrating all of the 25(OH)D values to gold standard reference measurement procedure values [7, 14]. In this paper we demonstrate the potential impact of assay standardization on the distribution of 25(OH)D in national health/nutrition surveys, and in clinical and epidemiological studies. Standardizing 25 (OH)D Measurements from the Past Can old measurements of 25(OH)D from national health/nutrition surveys and clinical and epidemiological studies be calibrated (standardized) to the gold standard reference measurement procedures? Yes – if properly stored serum samples are available – the VDSP has developed and expanded two different protocols for calibrating those old values [14, 15]. In general, the VDSP protocols consist of five basic steps: (1) Estimate the number of serum samples to be re‐measured (n ≈ 150 serum samples in most cases); (2) Select the specific stored samples to be re‐measured from the sorted original 25(OH)D values of the complete data set using the VDSP’s uniform sampling approach [14]; (3) Re‐measure the 25(OH)D concentration in these samples using an assay that is certified to be traceable to the gold standard RMPs; (4) Develop a mathematical model to calibrate (standardize) the “Old” 25(OH)D values to “True” ones; (5) Apply the model to the entire data set to calibrate (standardize) all values to the gold standard RMP values.
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Validity of VDSP Algorithm A study to evaluate the validity of the VDSP protocols was recently conducted by Cashman et al. at University College Cork (UCC), Cork, Ireland [15]. The results clearly support the validity of the VDSP protocols for standardizing 25(OH)D values from old studies (Table 2). In that study, data from the Irish National Health/Nutrition Surveys (NANS) were used to compare the original results (n=1,132) obtained using an Immunodiagnostic Systems Holdings, PLC (IDS) enzyme immunoassay (EIA) with calibrated values using the VDSP protocol. The VDSP protocol was applied by the selection and re‐measurement of 99 serum samples using the UCC’s CDC‐ certified traceable LC‐MS/MS assay, development of a calibration equation and the application of that equation to all 1,132 NANS values to calculate standardized 25(OH)D values for the entire sample. Additionally, UCC’s CDC‐certified assay was used to re‐measure all 1,132 NANS samples to produce an entire data set where all the samples reflected the standardized or true 25(OH)D concentration. As noted in Table 2, the proportion below commonly accepted cutpoints is virtually identical whether the VDSP protocol is applied of when all samples re‐ measured.
Since conducting this proof of principal study, the VDSP protocols has been used to
standardize 25(OH)D values from the Canadian Health Measures Survey [16], Nordic Countries [17], the surveys participating in the ODIN study of vitamin D status in EU member states [18], and the US National Health and Nutrition Examination Surveys (NHANES) [19]. These studies demonstrate the potential for using the VDSP approach to retrospectively standardize 25(OH)D data from past studies and in this way to develop an unbiased research database composed of 25(OH)D values calibrated to the gold standard RMPs.
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Impact of Assay Standardization
Assay standardization can have a profound effect on the interpretation of research data. For
example, prior to standardization, it appeared that there was a decline in mean serum total 25(OH)D from NHANES III (1988‐1994) through NHANES 2005‐2006 (Figure 1). However, after standardizing the results it is clear that there was no change in mean serum total 25(OH)D over that approximately 15 year period [19]. Importantly, the impact of assay standardization may vary from study to study depending on whether the original measurements were measured using an assay that was unbiased, negatively biased or positively biased in the clinical range [15‐19]. For example, in the Irish NANS and in NHANES III the impact of retrospective 25(OH)D assay standardization was to increase the percentage of the population with low vitamin D status [15‐19]. In contrast, in the German KIGGS survey the proportion of the German population with low vitamin D status was greatly reduced (Figures 2A and 2B) [17]. In addition to affecting estimates of the prevalence of low vitamin D status, standardization of 25(OH)D research data may also have an impact of the development of vitamin D intake guidelines. For example, in a reanalysis of data used by the IOM Committee, the 25(OH)D concentration achieved based on a usual intake of 600 IU vitamin D3, could vary from 50‐70 nmol/L depending on the bias of the assays used [12]. In addition, depending on whether the 25(OH)D assay was positively or negatively biased by 12%, the concentration of serum 25(OH)D which maximally suppresses PTH could vary from 20 – 35ng/mL (Figure 3).
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To summarize, failure to utilize standardized 25(OH)D assay data can lead to falsely high or low prevalence estimates for vitamin D inadequacy. Additionally, use of unstandardized data can lead to differing determinations of what 25(OH)D level constitutes “low” vitamin D status. Moving Forward: A proposal In conclusion, unless and until 25(OH)D data from research studies identified as being key sources of information are standardized, efforts to develop consensus guidelines will continue to flounder. The examples reported here highlight the challenges inherent in attempting meta‐ analyses using unstandardized 25(OH)D results; we recommend suspending publication of meta‐analyses based on unstandardized 25(OH)D data. Given this, what is the best course to move forward? If one revisits the IOM [1], Endocrine Society [2], and the recently released UK Scientific Advisory Committee on Nutrition (SACN) guidelines [20] it should be possible to identify the studies that have had an impact on those guidelines. Once identified it can be determined if banked serum samples from these studies exist. If they do, obtaining funding to calibrate those old values to gold standard 25(OH) values is indicated. If banked serum samples do not exist, the original investigators might decide to attempt to repeat the original research using an assay traceable to the gold standard RMPs.
Initially, it seems reasonable to focus on studies of vitamin D deficiency. Specifically, we
propose an international effort to identify key studies evaluating the association between serum total 25(OH)D and the presence/grade of vitamin D related rickets and osteomalacia. That is, what are the specific concentrations of serum 25(OH)D that are associated with established vitamin D rickets in children and osteomalacia in adults? The necessity of this approach was highlighted by a recent evidenced‐base review of data on this question in
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children that states, “While 25(OH)D assays differed across the studies, these results suggest that the serum 25(OH)D concentration associated with rickets may be much higher than previously thought” [21]. Assuming retrospective standardization harmonizes the definition of vitamin D deficiency, the next logical step would be to take a similar course to define what constitutes “vitamin D inadequacy.” The VDSP offers to work with any and all investigators willing to participate in this effort (email: [email protected]).
Disclaimer: The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the National Institutes of Health or the US Department of Health and Human Services.
Conflict of interest: none declared.
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References [1] Institute of Medicine (US) Committee to Review Dietary Reference Intakes for Vitamin D and Calcium, Dietary Reference Intakes for Calcium and Vitamin D, National Academies Press, Washington, DC, 2011. [2] M.F. Holick, N.C. Binkley, H.A. Bischoff‐Ferrari, C.M. Gordon, D.A. Hanley, R.P. Heaney, M.H. Murad, C.M. Weaver, S. Endocrine, Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline, J Clin Endocrinol Metab, 96 (2011) 1911‐1930. [3] N. Binkley, D. Krueger, C.S. Cowgill, L. Plum, E. Lake, K.E. Hansen, H.F. DeLuca, M.K. Drezner, Assay variation confounds the diagnosis of hypovitaminosis D: a call for standardization, J Clin Endocrinol Metab, 89 (2004) 3152‐3157. [4] G.D. Carter, Accuracy of 25‐hydroxyvitamin D assays: confronting the issues, Curr Drug Targets, 12 (2011) 19‐28. [5] J.K. Lai, R.M. Lucas, E. Banks, A.L. Ponsonby, G. Ausimmune Investigator, Variability in vitamin D assays impairs clinical assessment of vitamin D status, Intern Med J, 42 (2012) 43‐50. [6] N. Binkley, C.T. Sempos, D.S.P. Vitamin, Standardizing vitamin D assays: the way forward, J Bone Miner Res, 29 (2014) 1709‐1714. [7] C.T. Sempos, H.W. Vesper, K.W. Phinney, L.M. Thienpont, P.M. Coates, D.S.P. Vitamin, Vitamin D status as an international issue: national surveys and the problem of standardization, Scand J Clin Lab Invest Suppl, 243 (2012) 32‐40.
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[8] S.S. Tai, M. Bedner, K.W. Phinney, 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, 82 (2010) 1942‐1948. [9] H.C. Stepman, A. Vanderroost, K. Van Uytfanghe, L.M. Thienpont, Candidate reference measurement procedures for serum 25‐hydroxyvitamin D3 and 25‐hydroxyvitamin D2 by using isotope‐dilution liquid chromatography‐tandem mass spectrometry, Clin Chem, 57 (2011) 441‐ 448. [10] E.M. Mineva, R.L. Schleicher, M. Chaudhary‐Webb, K.L. Maw, J.C. Botelho, H.W. Vesper, C.M. Pfeiffer, A candidate reference measurement procedure for quantifying serum concentrations of 25‐hydroxyvitamin D(3) and 25‐hydroxyvitamin D(2) using isotope‐dilution liquid chromatography‐tandem mass spectrometry, Anal Bioanal Chem, 407 (2015) 5615‐5624. [11] Bureau International des Poids et Mesures (BIPM), JCTLM Database: Laboratory Medicine and in vitro Diagnostics, http://www.bipm.org/jctlm/, (accessed August 10, 2016). [12] J.W. Blunt, H.F. DeLuca, H.K. Schnoes, 25‐hydroxycholecalciferol. A biologically active metabolite of vitamin D3, Biochemistry, 7 (1968) 3317‐3322. [13] C.T. Sempos, R.A. Durazo‐Arvizu, N. Binkley, J. Jones, J.M. Merkel, G.D. Carter, Developing vitamin D dietary guidelines and the lack of 25‐hydroxyvitamin D assay standardization: The ever‐present past, J Steroid Biochem Mol Biol, (2015).
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[14] L. Tian, R.A. Durazo‐Arvizu, G. Myers, S. Brooks, K. Sarafin, C.T. Sempos, The estimation of calibration equations for variables with heteroscedastic measurement errors, Stat Med, 33 (2014) 4420‐4436. [15] K.D. Cashman, M. Kiely, M. Kinsella, R.A. Durazo‐Arvizu, L. Tian, Y. Zhang, A. Lucey, A. Flynn, M.J. Gibney, H.W. Vesper, K.W. Phinney, P.M. Coates, M.F. Picciano, C.T. Sempos, Evaluation of Vitamin D Standardization Program protocols for standardizing serum 25‐hydroxyvitamin D data: a case study of the program's potential for national nutrition and health surveys, Am J Clin Nutr, 97 (2013) 1235‐1242. [16] K. Sarafin, R. Durazo‐Arvizu, L. Tian, K.W. Phinney, S. Tai, J.E. Camara, J. Merkel, E. Green, C.T. Sempos, S.P. Brooks, Standardizing 25‐hydroxyvitamin D values from the Canadian Health Measures Survey, Am J Clin Nutr, 102 (2015) 1044‐1050. [17] K.D. Cashman, K.G. Dowling, Z. Skrabakova, M. Kiely, C. Lamberg‐Allardt, R.A. Durazo‐ Arvizu, C.T. Sempos, S. Koskinen, A. Lundqvist, J. Sundvall, A. Linneberg, B. Thuesen, L.L. Husemoen, H.E. Meyer, K. Holvik, I.M. Gronborg, I. Tetens, R. Andersen, Standardizing serum 25‐hydroxyvitamin D data from four Nordic population samples using the Vitamin D Standardization Program protocols: Shedding new light on vitamin D status in Nordic individuals, Scand J Clin Lab Invest, 75 (2015) 549‐561. [18] K.D. Cashman, K.G. Dowling, Z. Skrabakova, M. Gonzalez‐Gross, J. Valtuena, S. De Henauw, L. Moreno, C.T. Damsgaard, K.F. Michaelsen, C. Molgaard, R. Jorde, G. Grimnes, G. Moschonis, C. Mavrogianni, Y. Manios, M. Thamm, G.B. Mensink, M. Rabenberg, M.A. Busch, L. Cox, S. Meadows, G. Goldberg, A. Prentice, J.M. Dekker, G. Nijpels, S. Pilz, K.M. Swart, N.M. van Schoor,
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P. Lips, G. Eiriksdottir, V. Gudnason, M.F. Cotch, S. Koskinen, C. Lamberg‐Allardt, R.A. Durazo‐ Arvizu, C.T. Sempos, M. Kiely, Vitamin D deficiency in Europe: pandemic?, Am J Clin Nutr, 103 (2016) 1033‐1044. [19] R.L. Schleicher, M.R. Sternberg, D.A. Lacher, C.T. Sempos, A.C. Looker, R.A. Durazo‐Arvizu, E.A. Yetley, M. Chaudhary‐Webb, K.L. Maw, C.M. Pfeiffer, C.L. Johnson, The vitamin D status of the US population from 1988 to 2010 using standardized serum concentrations of 25‐ hydroxyvitamin D shows recent modest increases, Am J Clin Nutr, 104 (2016) 454‐461. [20] Scientific Advisory Committee on Nutrition (SACN), Vitamin D and Health, Public Health England, 2016, https://www.gov.uk/government/publications/sacn‐vitamin‐d‐and‐health‐ report, (accessed August 10, 2016). [21] A. Cranney, T. Horsley, S. O'Donnell, H. Weiler, L. Puil, D. Ooi, S. Atkinson, L. Ward, D. Moher, D. Hanley, M. Fang, F. Yazdi, C. Garritty, M. Sampson, N. Barrowman, A. Tsertsvadze, V. Mamaladze, Effectiveness and safety of vitamin D in relation to bone health, Evid Rep Technol Assess (Full Rep), (2007) 1‐235.
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Figure Legends: Figure 1. Impact of Assay Standardization on Trends in Serum Total 25‐hydroxyvitamin D from the US National and Nutrition Examination Surveys: NHANES III (1988‐1994), NHANES 2001‐ 2002, 2003‐2004 and 2005‐2006. In these data from NHANES, use of the original 25(OH)D results would lead to the conclusion that mean 25(OH)D declined from 1988‐1994, and potentially further declined in 2005‐2006. In contrast, use of standardized 25(OH)D data clearly demonstrates no change in mean value. Figure 2. Impact of Assay Standardization on the Prevalence of Persons with Serum Total 25‐ hydroxyvitamin D Concentrations < 30, < 50 and < 70 nmol/L. Results from the US NHANES III (1988‐1994) (A) and the German KIGGS (B). The potential effect of retrospective 25(OH)D standardization on vitamin D status prevalence is noted here. In the NHANES data (a), the prevalence of “low” vitamin D status below commonly utilized cutpoints was increased whereas in the KIGGS data (b), the prevalence was reduced. Figure 3. Hypothetical Example of the Effect of Assay Standardization on the Relationship of 25(OH)D with PTH. The 25(OH)D/PTH relationship is often utilized as part of the guideline development approach with adequacy considered to be present when higher 25(OH)D is no longer associated with lower PTH values. In this hypothetical example, the unstandardized 25(OH)D values obtained in this study were arbitrarily reduced 12% (top figure; circles) or increased 12% (bottom figure; squares). Assay variability of this extent is common. It is apparent that the point at which PTH plateaus differs from 20 ng/mL to 35 ng/mL, potentially leading to different conclusions regarding vitamin D “inadequacy.”
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Figure 1
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Figure 2
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Figure 3
A
B
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Table 1. A Comparison of the Institute of Medicine (IOM) and Endocrine Society Guidelines [1, 2] for interpreting the concentration of serum total 25‐hydroxyvitamin D. Which is Correct? Vitamin D Status Interpretation
Institute of Medicine [1]
Endocrine Society [2]
Serum Total 25‐hydroxyvitamin D (ng/mL)*
Deficient
< 12 ng/mL
< 20 ng/mL
Insufficient
12‐20 ng/mL
20‐29 ng/mL
Sufficient
20‐30 ng/mL
30‐100 ng/mL
No Added Benefit
30‐50 g/mL
>50 ng/mL
> 100 ng/mL
Possible Harm *Note: To convert ng/mL to nmol/L multiple by 2.5
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Table 2. Percent of Irish National Health/Nutrition Survey (NANS) with Serum 25‐ hydroxyvitamin D Concentrations Below Cut‐points: A Comparison of Original Results (n=1,132) with VDSP Standardized Results Based on a Re‐Measurement of a Statistical Subsample (n=99), and with Standardized Results Based on a Re‐Measurement of All Study Samples (n=1,132) using University College Cork’s (UCC) CDC Certified Traceable LC‐MS/MS Assay. VDSP Statistical Original IDS/EIA Assay
UCC’s CDC Certified Standardization
Results
LC‐MS/MS *
Protocols Serum Total 25(OH)D (n=1,132)
(n=99)
(n=1,132)
< 30 nmol/L
6.5%
11.4%
11.2%
< 40 nmol/L
21.9%
25.3%
27.2%
< 50 nmol/L
40.0%
43.7%
45.0%
>125 nmol/L
1%
0.3%
0.6%
Cutpoint
*Mathematical model based on the results of 99 serum samples re‐measured using UCC’s CDC Certified Traceable Assay was used to Calibrate/Standardize all NANS 25(OH)D concentrations.
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