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.

Scandinavian Journal of Clinical & Laboratory Investigation, 2015; 75: 549–561

ORIGINAL ARTICLE

Scand J Clin Lab Invest Downloaded from informahealthcare.com by Kungliga Tekniska Hogskolan on 08/24/15 For personal use only.

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

KEVIN D. CASHMAN1,2, KIRSTEN G. DOWLING1, ZUZANA ŠKRABÁKOVÁ1, MAIREAD KIELY1, CHRISTEL LAMBERG-ALLARDT3, RAMON A. DURAZO-ARVIZU4, CHRISTOPHER T. SEMPOS5, SEPPO KOSKINEN6, ANNAMARI LUNDQVIST6, JOUKO SUNDVALL7, ALLAN LINNEBERG8,9,10, BETINA THUESEN8, LISE LOTTE N. HUSEMOEN8, HAAKON E. MEYER11,12, KRISTIN HOLVIK12, IDA M. GRØNBORG13, INGE TETENS13 & RIKKE ANDERSEN13 1Vitamin

D Research Group, School of Food and Nutritional Sciences, and 2Department of Medicine, University College Cork, Cork, Ireland, 3Department of Food and Environmental Sciences, Helsinki University, Finland,4Department of Public Health Sciences, Loyola University Stritch School of Medicine, Chicago, IL, USA, 5Office of Dietary Supplements, National Institutes of Health, Bethesda, MD, USA, 6Department of Health, Functional Capacity and Welfare, National Institute for Health and Welfare (THL), Finland, 7Department of Chronic Disease Prevention, THL, Finland, 8Research Centre for Prevention and Health, the Capital Region of Denmark, Copenhagen, Denmark, 9Department of Clinical Experimental Research, Glostrup University Hospital, Glostrup, Denmark, 10Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark, 11Department of Community Medicine, University of Oslo, Norway, 12Division of Epidemiology, Norwegian Institute of Public Health, Oslo, Norway, and 13Division of Nutrition, National Food Institute, Technical University of Denmark, 2860 Søborg, Denmark Abstract Knowledge about the distributions of serum 25-hydroxyvitamin D (25(OH)D) concentrations in representative population samples is critical for the quantification of vitamin D deficiency as well as for setting dietary reference values and food-based strategies for its prevention. Such data for the European Union are of variable quality making it difficult to estimate the prevalence of vitamin D deficiency across member states. As a consequence of the widespread, methodrelated differences in measurements of serum 25(OH)D concentrations, the Vitamin D Standardization Program (VDSP) developed protocols for standardizing existing serum 25(OH)D data from national surveys around the world. The objective of the present work was to apply the VDSP protocols to existing serum 25(OH)D data from a Danish, a Norwegian, and a Finnish population-based health survey and from a Danish randomized controlled trial. A specifically-selected subset (n 100–150) of bio-banked serum samples from each of the studies were reanalyzed for 25(OH)D by LC-MS/ MS and a calibration equation developed between old and new 25(OH)D data, and this equation was applied to the entire data-sets from each study. Compared to estimates based on the original serum 25(OH)D data, the percentage vitamin D deficiency (⬍ 30 nmol/L) decreased by 21.5% in the Danish health survey but by only 1.4% in the Norwegian health survey; but was relatively unchanged (0% and 0.2%) in the Finish survey or Danish RCT, respectively, following VDSP standardization. In conclusion, standardization of serum 25(OH)D concentrations is absolutely necessary in order to compare serum 25(OH)D concentrations across different study populations, which is needed to quantify and prevent vitamin D deficiency. Key Words: 25-hydroxyvitamin D, standardization, health surveys, Nordic countries

Correspondence: Kevin D. Cashman, School of Food and Nutritional Sciences, and Department of Medicine, University College Cork, Cork, Ireland. Tel: ⫹ 353 21 4901317. Fax: ⫹ 353 21 4270244. E-mail: [email protected] (Received 7 March 2015 ; accepted 31 May 2015) ISSN 0036-5513 print/ISSN 1502-7686 online © 2015 Informa Healthcare DOI: 10.3109/00365513.2015.1057898

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Introduction Knowledge of the distributions of serum 25hydroxyvitamin D [25(OH)D1] concentrations in nationally representative populations is critical for the quantification of vitamin D deficiency as well as for devising dietary reference values and food-based strategies for its prevention [1,2]. Data on the distribution of serum 25(OH)D in nationally representative populations with appropriate consideration of sex, life-stage and ethnicity would enable quantification of vitamin D deficiency in the European Union (EU) population [2]. However, serum 25(OH)D distribution data for the EU are of variable quality making it difficult to estimate the prevalence of vitamin D deficiency across member states. Moreover, even for regions within Europe, significant variability exists in mean serum 25(OH)D concentrations [3]. For example, a recent systematic review has shown that within the Nordic region, mean year-round serum 25(OH)D concentration for Norway (64.5 nmol/L on average; based on data from four study samples) appears to be much higher than that for either Denmark or Finland (44.8 and 42.4 nmol/L on average, respectively; based on data from five study samples for each country) [3]. While there are many likely contributory reasons for differences in prevalence estimates between populations, such as representativeness of study samples, racial and ethnic composition, ambient UVB sun light and sun exposure practices as well as diet, vitamin D food fortification and supplementation practices, differences in analytical methodology for serum total 25(OH)D are also likely to contribute [1]. Several reports have shown that available 25(OH)D assays can yield markedly differing results [4–8]. As a consequence of the widespread, methodrelated differences in results of serum 25(OH)D [5,9], which confounds international efforts to develop evidence-based guidelines, the Vitamin D Standardization Program (VDSP) developed protocols for standardizing 25(OH)D measurement in national health/nutrition surveys around the world, and these have been described in detail elsewhere [1,10]. Application of the VDSP protocols to existing enzyme-immunosorbent assay (ELISA)-derived serum 25(OH)D data from the Irish National Adult Nutrition Survey, recently showed the year-round prevalence rate for serum 25(OH)D concentration ⬍ 30 nmol/L (reflecting vitamin D deficiency [11]) was 6.5% via original ELISA measurement and this increased to 11.4% when the VDSP protocol for standardizing 25(OH)D values from past surveys was applied [12]. Importantly, re-analysis of all serums (n ⫽ 1118) by standardized liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed the prevalence estimate as 11.2% and validated the VDSP protocol for standardizing 25(OH)D values from past surveys [12].

Within the VDSP, nationally representative nutrition and health surveys were prioritized, however some member states in Europe do not have such surveys with nationally representative data on serum 25(OH)D concentrations. Thus, in the absence of such data, well-curated samples from regionally representative health surveys can also achieve some degree of population coverage. The objective of the present work was to apply the VDSP protocols to existing serum 25(OH)D data from a national health survey in Finland, and two population-based health surveys in the Danish and Norwegian capitals, Copenhagen and Oslo, respectively. The distributions of standardized serum 25(OH)D concentrations will be explored in, and compared across, these samples. The determinants of risk of having standardized serum 25(OH)D concentrations ⬍ 50 nmol/L, a threshold identified by the Institute of Medicine (IOM) and the Nordic Council of Ministers (NORDEN) recently in relation to covering the needs of nearly all in the population in terms of bone health [11,13], will also be explored in all three population samples. Such international comparisons can help with the development of guidelines to tackle vitamin D deficiency.

Study samples and methods Subject sampling and recruitment procedures, and methods of data collection for the four study populations Each of the study populations used in the present work are summarized in the following section. The Health 2011 Study (Finland) The Health 2011 health examination survey in Finland is a follow-up study to the Health 2000 Survey, details of which have been reported in detail elsewhere [14]. In brief, the Health 2000 Survey drew 9922 adults from a nationwide population register in Finland, on the basis of dividing the country into five geographical strata defined by the university hospital districts of Helsinki, Turku, Tampere, Kuopio and Oulu. The follow-up Health 2011 examination called all the participants of the Health 2000 Survey, and was a national survey of changes in the health status, functional capacity and welfare of the population. The survey also examines health and welfare inequalities between population groups and changes in them as well as the use of health services. Our analytical sample for the present work included 4102 adults, aged 29–77 years. The study was approved by the Ethical Committee at the Hospital District of Helsinki and Uusimaa, Finland. Written informed consent was given by the participants.

Vitamin D status in Nordic individuals


The Oslo Health Study (Norway) A detailed description of the subject sampling, recruitment procedures, and methods of data collection used in the Oslo Health Study (HUBRO) has been reported elsewhere [15]. Briefly, HUBRO was a cross-sectional population-based multipurpose study conducted in 2000–2001, inviting all individuals in the city of Oslo aged 30, 40, 45, 59, 60, 75 and 76 years. Vitamin D status was measured in a subsample (n ⫽ 1042) during May 2000 to January 2001. The participants recruited for vitamin D status measurement constituted a random sample of individuals born in Norway (quoted as Norwegians) aged 45, 60 and 75 years (n ⫽ 866), and a random sample of individuals born in Pakistan (quoted as Pakistanis) of all age groups (n ⫽ 176). The participants were invited in a random order throughout the study period regardless of ethnic background. The study protocol was evaluated by the Regional Committee for Medical Research Ethics and approved by the Norwegian Data Inspectorate. Written informed consent was given by the participants. The Health2006 Study (Copenhagen) A detailed description of the subject sampling and recruitment procedures and methods of data collection used in the Health2006 study has been reported elsewhere [16]. Briefly, a total of 3471 adults aged 18–69 years (of 7931 invited) from the general population in Copenhagen, all with Danish citizenship and born in Denmark, underwent a 2-h general health examination between June 2006 and May 2008 in Glostrup, Denmark. The survey had a broad health focus. Only Health2006 participants with serum 25(OH)D data (n ⫽ 3409) were included in the present analyses. The Ethics Committee of the Copenhagen County approved the study (KA20060011). Written and verbal informed consent was given by the participants to be enrolled in the study and for their information to be stored in the hospital database and used for research. The VitmaD study A detailed description of the subject sampling and recruitment procedures and methods of data collection used in the randomized controlled trial of ‘Effect of vitamin D fortification in Danish families’ (VitmaD) has been reported elsewhere [17]. Briefly, the study was a double-blind, randomized, placebocontrolled intervention trial with 782 children and adults recruited as families. Families were randomly allocated to either vitamin D3-fortified milk and bread or non-fortified placebo milk and bread for 6 months during the winter (September 2010 to April 2011). Participants were instructed to replace their usual consumption of milk and bread with the

551

products provided in the study. The principle in the study design was to investigate a realistic fortification strategy in a real-life setting. The exclusion criteria were pregnancy, disease or medication that could influence vitamin D metabolism. All eligible adults and custody holders gave their written informed consent on their participation. Adult participants were seen 3 times during the study period (months 0, 3, and 6), and children (4–17 y old) were seen twice (months 0 and 6). Blood samples were analyzed for serum 25(OH)D concentration (primary endpoint) and serum PTH and total calcium (secondary endpoints), but only the baseline samples for all subjects were used in the present work. The study protocol was approved by the Research Ethics Committee of the Capital Region of Denmark (record H-4-2010-020). The key summary demographic characteristics of the four samples (age, gender distribution, and season of blood sampling) as well as the information on the methodology used for the original serum 25(OH)D analysis are shown in Table I. For climatological purposes, seasons are regarded as threemonth periods as follows: ‘winter’ covering the period December to February, ‘spring’ covering the period March to May, ‘summer’ covering the period June to August, and ‘autumn’ covering the period September to November. This is a common grouping in the meteorological practice of many countries in the middle and northern latitudes [18]. Specifically in relation to potential UVB-induced dermal synthesis of vitamin D, the following wider definition was also applied within the present work, where appropriate: An extended winter period – November to March; and an extended summer period – April to October [19]. Blood collection and original analysis of serum 25-hydroxyvitamin D within the four study populations In each study, blood (fasting [Health2006, Health 2011] and non-fasting [HUBRO and VitmaD]) samples, once collected, were processed to serum and stored either directly at ⫺ 70/80°C (or ⫺ 20°C for up to 8 weeks and thereafter at ⫺ 70°C) until required for biochemical analysis. The concentrations of total 25(OH)D in serum samples were measured by the originally used methods as follows: in Health 2011 (Finland), a Chemiluminescent Microparticle Immunoassay (CMIA), Architect ci8200-analyzer was used by the Department of Chronic Disease Prevention, National Institute for Health and Welfare for the analysis and the inter-assay coefficient of variation (CV) for the assay was 3.6%. This assay measures 25(OH)D3 with 102% specificity and 25(OH)D2 with ⬍ 81.5% specificity. In Health2006 (Denmark), a chemiluminescence immunoassay (Cobas e41, Roche Diagnostics, Mannheim, Germany) was used. This assay

10.2 (3.7) [4–17]

40.5 (7.7) [18–60]

26.8 (16.4) [4–60]

49.4 (13.0) [19–72]

58.9 (12.9) [30–76]

55.8 (13.7) [29–77]

Age Mean (SD) [Range] (years)

*n, The number of sera used for standardization purposes and as selected by uniform sampling with quartiles.

345

765 (139)

Gladsaxe Municipality, Denmark (55.7°N)

Effect of vitamin D fortification in Danish families (VitmaD) [2010–2011] Total sample:

Children only:

3409 (147)

Copenhagen, Denmark (55.7°N)

Health2006 [2006–2008]

420

1042 (105)

Oslo, Norway (59.6°N)

The HUBRO study [2000–2001]

Adults only:

4102 (101)

N (n*)

Representative sample/Finland (60–70°N)

Region/Country (latitude)

Health 2011 [2011]

Sample full name (acronym) [Year(s) of study]

52.2:47.8

50.0:50.0

49.0:51.0

55.1:44.9

45.5:54.5

55.2:44.8

Sex [female: male] (%)

Table I. Characteristics of samples for standardization of serum 25-hydroxyvitamin D concentrations in Nordic study populations.

Winter: 12.1 Spring: 0 Summer: 0 Autumn: 87.9 Winter: 9.1 Spring: 13.1 Summer: 27.8 Autumn: 50.0 Winter: 23.2 Spring: 24.1 Summer: 22.0 Autumn: 30.7 Winter: 0 Spring: 0 Summer: 0 Autumn: 100 Winter: 0 Spring: 0 Summer: 0 Autumn: 100 Winter: 0 Spring: 0 Summer: 0 Autumn: 100

Season of sampling (%)


Chemiluminescence immunoassay Cobas e411 (Roche Diagnostics, Germany) [6.9–9.9%] Isotope-dilution liquid chromatography-tandem mass spectrometry (LC/MS-MS) [2.2–7.6%]

Radioimmunoassay (Diasorin, MN, USA) [15%]

Chemiluminescent microparticle immunoassay, Architect ci8200-analyzer [3.6%]

Serum 25(OH)D assay details [Inter-assay CV(%)]

552 K. D. Cashman et al.


Vitamin D status in Nordic individuals measures 25(OH)D3 with 100% specificity and 25(OH)D2 with ⬍ 10% specificity, and the interassay CV was 9.9% (at 63.0 nmol/L) and 6.9% (at 163.7 nmol/L). In HUBRO (Norway), serum 25(OH)D analysis was via a radioimmunoassay (DiaSorin, Stillwater, MN, USA). This assay measures both 25(OH)D3 and 25(OH)D2, (with 100 and 104% specificity, respectively) and the inter-assay CV was 15%. The 25(OH)D levels of the first 191 samples (18%) were measured by an in-house HPLCmethod and calibrated to the DiaSorin method by a cross-calibration study [15]. To compare the two methods used, 64 serum samples were measured with both the DiaSorin method and the HPLCmethod. The HPLC-results were then corrected according to the calculated linear regression (‘Diasorin’ ⫽ ‘HPLC’ ⫻ 0.96 ⫹ 5.79, r ⫽ 0.85. There was no significant deviation from linearity). Only samples analyzed by the Diasorin method were included in the development of the regression calibration equations in the present work. In the VitmaD study (Denmark), all blood samples were analyzed at the Clinical Biochemical Department, Holbæk Hospital, Denmark. Serum 25(OH)D was determined as both 25(OH)D2 and 25(OH)D3 by isotope-dilution liquid chromatography-tandem mass spectrometry (LC/MS-MS). The inter-assay CVs for 25(OH)D3 measured by the LC/ MS-MS method were 2.2% and 2.8% measured at 30 nmol/L and 180 nmol/L, respectively. The interassay CVs for 25(OH)D2 were 7.6% and 4.6% at 43 and 150 nmol/L, respectively. Re-analysis of bio-banked serum for total 25-hydroxyvitamin D by liquid chromatography-tandem mass spectrometry The concentrations of total 25(OH)D (i.e. 25(OH) D2 plus 25(OH)D3) in specifically selected biobanked serum samples from each of the study populations (see below) were measured by the Vitamin D Research Group at University College Cork using a LC-MS/MS method, as has been described in detail elsewhere [12,20]. In brief, the LC-MS/MS method measures 25(OH)D2 and 25(OH)D3 in serum as well as the 3-epimer of 25(OH)D3 (3-epi-25(OH) D3), which is not chromatographically resolved from 25(OH)D3 by most routine LC-MS/MS methods. The presence of 3-epimers of 25(OH)D can pose problems for LC-MS/MS methods because the precursor ion and fragmentation patterns are the same as 25(OH)D, thus failure to account for these metabolites can result in overestimation of 25(OH) D3 in particular as the quantitatively more abundant metabolite. The intra-assay CV of the method was ⬍ 5% for all 25-hydroxyvitamin D metabolites, while the inter-assay CV was ⬍ 6%. The Vitamin D Research Group is a participant in the VDSP [1] and is certified by Centers for Disease Control and

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Prevention’s Vitamin D Standardization Certification Program [21]. The inter-assay CV for total 25(OH)D was 3.6%. Both the VDSP and the certification program reports total 25(OH)D as well as 25(OH)D2 and 25(OH)D3 using the higher order reference laboratories. In addition, the quality and accuracy of serum total 25(OH)D analysis by the LC-MS/MS in our laboratory is monitored on an ongoing basis by participation in the Vitamin D External Quality Assessment Scheme [DEQAS, Charing Cross Hospital, London, UK]. All solvents and mobile phase additives were MS grade and purchased from Sigma-Aldrich (Wicklow, Ireland). Zinc sulphate was sourced from SigmaAldrich while stable isotope labelled d3-25(OH)D2, d3-25(OH)D3 and d3-3-epi-25(OH)D3 were purchased from Isosciences (Trevose, PA, USA). Certified calibrators for 25(OH)D2 and 25(OH)D3 were bought from National Institute of Standards and Technology (NIST), USA (SRM 2972) while a CertiMass reference standard for 3-epi-25(OH)D3 was sourced from Isosciences. Low and High serum QC materials were commercially available from Chromsystems (Munich, Germany). The chromatographic column was a Supelco Ascentis Express F5 available from Sigma-Aldrich. Applying the VDSP protocol for standardization of serum 25(OH)D data from past surveys to the four study populations The VDSP protocol for standardization of serum 25(OH)D data from past surveys, as employed by some of us previously on the Irish national serum 25(OH)D data [12], and again in this study, generally entails three steps (outlined in detail elsewhere [1,10]): [1] use results from the VDSP inter-laboratory comparison study to develop a master equation to convert values based on the current measurement procedure (LC-MS/MS in our case) to the reference measurement procedures at Ghent University and NIST (Protocol 1) [however, this step was not necessary for the present work because the LC-MS/MS method within our Vitamin D Research Group is certified as being standardized to that of the reference measurement procedures; see http://www.cdc.gov/labstandards/pdf/ hs/CDC_Certifi ed_Vitamin_D_Procedures.pdf]; [2] re-analysis of a statistically defined sub-sample of the stored (bio-banked) sera from the study population (see below) and an equation is developed to convert all past serum 25(OH)D values to the current measurement procedure (Protocol 2); and [3] the equation is used to convert the previous serum 25(OH)D values to the Vitamin D Research Group certified procedure. To facilitate Protocol 2, a statistical algorithm for estimating the number of stored samples that need to be re-analyzed was developed within the VDSP,


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and published recently [22]. The sample size of stored serum samples required for this protocol was calculated using procedures for the estimation of the predicted LC-MS/MS-based 25(OH)D value for a given serum 25(OH)D value from the original method of analysis (e.g. immunoassay or LC-MS/ MS) with a pre-defined precision of a 95% confidence interval, which have been described elsewhere [12,22]. This resulted in sample size of 95–140 sera dependent on the study, so we decided to round to 100–150. The respective n 100–150 serum samples within each study population separately were then selected by first dividing the range of the previous methodbased serum 25(OH)D measurements into quartiles, with each quartile being sampled according to a uniform distribution [22]. This method has been shown, via computer simulations, to be statistically more efficient than uniform random sampling in the entire range [22]. The selected serum samples for each population study were retrieved from the respective bio-banks and each set shipped to the Vitamin D Research Group, where they were reanalyzed for serum total 25(OH)D using LC-MS/ MS (as outlined above). Serum total 25(OH)D concentration within each sample were calculated as the sum of respective 25(OH)D2 and 25(OH)D3 concentrations. The relationship between serum 25(OH)D in the statistical algorithm-defined subset of the sera for each of the four studies separately, as measured by the original method and re-analyzed by our traceable LC-MS/MS method, was evaluated using regression analysis, as described elsewhere [12]. Several best fit lines were evaluated for each data set (as defined by R2 and well as consideration of the residuals plots), and the resulting regression equation which provided the best fit was applied to the entire data set for that population study, as per the VDSP Protocol 2. Serum 25-hydroxyvitamin D thresholds Measured serum 25(OH)D concentrations were compared with cut-offs for 25(OH)D as per the IOM dietary reference intake (DRI) committee’s recent definitions: persons are at risk of deficiency at serum 25(OH)D concentrations below 30 nmol/L, while 40 and 50 nmol/L are consistent with the Estimated Average Requirement (EAR)-like and Recommended Dietary Allowance (RDA)-like serum values, respectively [11]. In addition, serum 25(OH)D concentrations ⬎ 125 nmol/L have been suggested by the IOM DRI committee as being possibly some reason for concern [11]. While we included this threshold in the present work, we have previously shown that the number of study participants with serum 25(OH) D ⬎ 125 nmol/L are greatly reduced following standardization of the data, and thus these estimates need

to be interpreted with caution due to the small n values [12]. A serum 25(OH)D concentration ⬍ 25 nmol/L has also been a traditional cut-off used in Europe to define vitamin D deficiency on the basis of metabolic bone disease [23,24], and thus was also included. Finally, as the Task Force for the Clinical Guidelines Subcommittee of The Endocrine Society has recently suggested that to maximize the effect of vitamin D on calcium, bone and muscle metabolism, serum 25(OH)D concentration should exceed 75 nmol/L [25], we also used this cut-off for comparison purposes. Data and statistical analysis Data and statistical analysis was conducted using SPSS© Version 20.0 for Windows™ (SPSS Inc. Chicago, IL, USA), STATA 12 (StataCorp LP, College Station, TX, USA) and CBStat5 (Kristian Linnet, Charlottenlund, Denmark). Descriptive statistics (frequencies, means) were used to characterize serum 25(OH)D concentrations by threshold using original measured values and LC-MS/MS standardized values. Regression models (ordinary least squares (unweighted and weighted), Deming (unweighted and weighted), and Piece-wise) were used to establish the relation between the original serum 25(OH) D measured values and the LC-MS/MS re-analyzed in the subsets. Binary logistic regression models which included season (summer as referent), age and gender as explanatory variables were used to explore determinants of serum 25(OH)D ⬍ 50 nmol/L in Health2006 and HUBRO. A p-value of ⬍ 0.05 was taken as being statistically significant.

Results The relationship between serum 25(OH)D in the statistical algorithm-defined subsets (n ⫽ 101–147) of serum samples from the four study samples (Health 2011, HUBRO, Health2006, and VitmaD), as measured by their original methodologies and re-analyzed by our traceable LC-MS/MS method, is shown in Figure 1. The best fit for Health 2011 was a piecewise linear fit (LC-MS/MS ⫽ 54.402 ⫹ (1.172 ⫺ 50.730) ⫻ X1 ⫹ 0.548 ⫻ (X2 ⫺ 50.730); r2 ⫽ 0.93) which took account of the two linear relationships, one at concentrations below 50.7 nmol/L and another at concentrations of serum 25(OH)D equal to and above this cut-point. The best fit for HUBRO was also a piecewise linear fit (LC-MS/MS ⫽ 81.994 ⫹ (0.898 ⫺ 85.643) ⫻ X1 ⫹ 0.692 ⫻ (X2 ⫺ 85.643); r2 ⫽ 0.85) which took account of the two linear relationships, one at concentrations below 85.6 nmol/L and another at concentrations of serum 25(OH)D equal to and above this cut-point. Similarly, the best fit for Health2006 was again a piecewise linear fit (LCMS/MS ⫽ 86.684 ⫹ (1.136 ⫺ 60.941) ⫻ X1 ⫹ 0.291



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Figure 1. The relationship between serum 25(OH)D (nmol/L) in a sub-sample (n 101–147) of the Health 2011 (A), Oslo Health (B), Health2006 (C), and VitmaD (D) samples measured by original 25(OH)D assay and standardized LC-MS/MS at University College Cork as per Vitamin D Standardization Program protocol.

⫻ (X2 ⫺ 60.941); r2 ⫽ 0.69) which took account of the two linear relationships, one at concentrations below 61.0 nmol/L and another at concentrations of serum 25(OH)D equal to and above this cut-point. The best fit for VitmaD was a weighted Deming fit (LC-MS/ MS ⫽ 0.781 ⫻ [original serum 25(OH)D] ⫹ 3.512; r2 ⫽ 0.96). The Bland-Altman comparisons for originally measured serum 25(OH)D concentrations and the newly measured LC-MS/MS concentrations in each of the four study samples are shown in Supplementary Figure 1, available online at http:// informahealthcare.com/doi/abs/10.3109/00365513. 2015.1057898. Residual plots arising from the initial exploratory regression analysis (linear) of data for Health 2011, HUBRO and Health2006 showed the need for inclusion of a non-linear term which was accommodated by the piecewise regression and for which the resulting Residual plots showed the appropriateness of the models (data not shown). The mean, SD, median, 5th, 25th, 75th and 95th percentile of serum 25(OH)D, as well as prevalence rates for serum 25(OH)D concentration below (or above) variously proposed public health-relevant thresholds, for the original serum 25(OH)D data as well as for those standardized by the VDSP protocol in the four study samples are shown in Table II. The original serum 25(OH)D data from Health 2011

(Finland) was only modestly changed by standardization of the data by the VDSP protocol. The originally measured values had a positive bias (Figure 1a) and adjusting for this led to decreases in the mean and median serum 25(OH)D and both increases and decreases in prevalence estimates for standardized serum 25(OH)D below or above the six selected thresholds compared to pre-standardization estimates (Table II). The original serum 25(OH)D data from HUBRO (Norway) was only very modestly changed by standardization of the data by the VDSP protocol. The originally measured values had a slightly positive bias (Figure 1b) and adjusting for this led to minor decreases in the mean and median serum 25(OH)D and prevalence estimates for serum 25(OH)D below or above the six selected thresholds were very similar pre- and post-standardization (Table II). The original serum 25(OH)D data for Health2006 (Denmark) was dramatically influenced by application of the VDSP protocol, such that when the considerable negative bias (Figure 1c) was adjusted for, the mean and median serum 25(OH)D concentration increased by 20.6 and 23.1 nmol/L, respectively, on average (Table II). The very high prevalences of serum 25(OH)D below thresholds associated with deficiency, inadequacy, and insufficiency (i.e. ⬍ 25 to

75.9 22.2 74.0 44.0 61.0 88.0 115.0 0.1 0.4 2.7 8.9 51.6 2.7

Original

44.4 22.6 41.5 12.3 29.5 55.5 84.5 17.6 25.8 46.4 66.4 91.7 0.8

N Mean SD Median 5th Percentile 25th Percentile 75th Percentile 95th Percentile % ⬍ 25 nmol/L % ⬍ 30 nmol/L % ⬍ 40 nmol/L % ⬍ 50 nmol/L % ⬍ 75 nmol/L % ⬎ 125 nmol/L

Sample Serum 25(OH)D (nmol/L)

n Mean SD Median 5th Percentile 25th Percentile 75th Percentile 95th Percentile % ⬍ 25 nmol/L % ⬍ 30 nmol/L % ⬍ 40 nmol/L % ⬍ 50 nmol/L % ⬍ 75 nmol/L % ⬎ 125 nmol/L

(65.4, 70.1) (13.0, 13.3) (64.9, 69.5) (44.2, 48.8) (57.4, 62.6) (72.8, 76.9) (87.6, 91.7) (0.0, 0.4) (0.2, 0.7) (1.6, 3.1) (5.9, 8.9) (70.0, 80.6) (0.2, 0.2)

3,409 65.0 19.2 64.6 31.4 51.0 80.5 93.5 0 4.3 10.9 23.6 67.7 0.1 (60.9, 69.1) (19.0, 19.3) (61.5, 67.6) (25.6, 37.2) (47.7, 54.3) (75.7, 85.3) (89.0, 98.0) (0, 4.8) (0, 7.3) (6.8, 15.5) (18.3, 28.7) (61.5, 73.7) (0.2, 0.4)

Standardized

Health2006

4,102 67.7 13.2 67.2 46.5 60.0 74.8 89.6 0.2 0.4 2.3 6.6 75.7 0.2

Standardized

Health 2011

74.7 19.8 73.1 44.9 61.5 86.7 107.4 0.5 0.8 2.4 8.8 53.9 1.4

Original

66.2 29.0 68.0 16.0 48.0 85.0 110.0 10.2 13.4 20.1 26.9 61.4 2.2

Original

(57.1, 66.6) (14.8, 16.1) (55.9, 65.2) (34.8, 42.3) (47.2, 55.8) (66.1, 76.3) (81.6, 93.2) (0.4, 0.8) (0.8, 2.0) (3.0, 10.3) (13.1, 32.8) (72.8, 88.8) (0, 0.7)

Standardized 765 61.8 15.5 60.6 38.6 51.5 71.2 87.4 0.8 1.0 6.4 20.5 82.2 0.1

All

(59.8, 67.5) (24.5, 24.7) (62.9, 69.4) (13.9, 25.1) (45.0, 51.2) (76.9, 85.9) (95.3, 102.4) (4.4, 12.0) (8.9, 15.4) (17.0, 21.7) (24.0, 31.3) (59.9, 71.7) (0.5, 1.0)

Standardized 1,042 63.6 24.6 66.1 19.5 48.1 81.4 98.9 8.9 12.0 19.6 27.9 65.6 0.8

All

*Vitamin D Standardization Program (VDSP)-standardized are presented as mean estimates and their 95% CIs.

Original

Sample………. Serum 25(OH)D (nmol/L)

74.9 21.6 74.4 41.5 60.6 88.0 110.0 1.0 1.4 4.0 10.7 51.0 1.9

Original

74.5 23.8 73.0 37.1 58.0 89.0 113.0 0.3 1.8 6.2 13.7 53.6 2.7

Original

(67.3, 74.7) (19.0, 19.9) (67.1, 74.2) (34.7, 42.1) (54.2, 60.1) (80.0, 88.6) (97.3, 104.6) (0.0, 1.3) (0.1, 2.7) (3.7, 7.9) (10.5, 18.6) (51.7, 65.9) (0.6, 1.2)

420 62.0 16.9 61.6 35.9 50.8 72.2 89.4 1.4 1.9 8.8 23.1 81.7 0.2

(57.3, 66.7) (16.2, 17.6) (56.9, 66.3) (32.3, 39.5) (46.6, 55.1) (67.0, 77.3) (83.5, 95.3) (0.7, 1.4) (1.4, 3.3) (5.2, 12.4) (15.2, 33.6) (71.0, 88.1) (0, 1.2)

Standardized

Adults (18–60 y)

VitmaD

866 71.0 19.5 70.6 38.4 57.2 84.3 100.9 0.1 1.3 5.8 14.9 58.7 0.9

Standardized

Norwegian

HUBRO

74.5 17.3 71.4 48.1 62.6 85.2 105.5 0 0 0.3 6.4 57.4 0.9

Original

25.1 13.7 22.0 9.6 15.0 31.0 54.5 58.5 70.5 88.1 91.5 100 0

Original

Table II. Distribution of original and standardized serum 25-hydroxyvitamin D concentrations in Nordic populations, and stratified by ethnic background or age-grouping*.


(22.7, 32.5) (11.3, 13.3) (19.8, 29.8) (7.4, 19.9) (12.8, 24.2) (28.7, 37.1) (51.0, 56.9) (26.1, 64.8) (52.3, 77.8) (82.4, 89.8) (90.3, 93.8) (100, 100) (0, 0)

345 61.6 13.5 59.2 41.1 52.4 70.1 85.9 0 0 3.5 17.4 82.9 0

(56.9, 66.3) (13.0, 14.1) (54.6, 63.9) (37.2, 44.9) (48.1, 56.7) (65.0, 75.1) (80.1, 91.6) (0,0) (0, 0.3) (0.3, 7.8) (10.4, 31.9) (75.1, 89.6) (0,0)

Standardized

Children (4–17 y)

176 27.6 12.3 24.8 13.7 18.5 32.9 54.0 52.3 64.8 87.5 92.0 100 0

Standardized

Pakistani

556 K. D. Cashman et al.


Vitamin D status in Nordic individuals ⬍ 75 nmol/L) decreased considerably following standardization. Likewise, the percentage with serum 25(OH)D ⬎ 125 nmol/L (which the IOM suggest as being possibly some reason for concern [11]) decreased from 0.8–0.1%. The mean and median of the original serum 25(OH)D concentrations from the baseline sampling point from the VitmaD RCT, in autumn, decreased by 12.9 and 12.5 nmol/L, respectively, on average, following standardization (Table II), which was linked to the positive bias in the original analysis (Figure 1d). The prevalence of serum 25(OH)D ⬍ 25 to ⬍ 75 nmol/L increased, whereas the percent with concentrations ⬎ 125 nmol/L decreased from 1.4–0.1% (Table II). Comparison of mean serum 25(OH)D concentrations or prevalence estimates around thresholds between the Danish, Norwegian and Finnish samples using the standardized data requires consideration of key demographic characteristics such as ethnicity profiles, age-categories included and distribution of season in which blood sampling occurred. While Health 2011, Health2006 and VitmaD were in Caucasian white individuals, approximately 17% of the HUBRO sample was of Pakistani ethnicity. The standardized (and original) serum 25(OH)D in HUBRO stratified by ethnicity is shown in Table II. The mean serum 25(OH)D concentration of persons of Norwegian and Pakistani ethnicity in HUBRO was 71.0 and 27.6 nmol/L, respectively. The prevalence of vitamin D deficiency (serum 25(OH)D ⬍ 30 nmol/L [11]) in both population subgroups was 1.3% and 64.8%, respectively, whereas the prevalence of vitamin D inadequacy (serum 25(OH)D ⬍ 50 nmol/L [11]) was 14.9% and 92.0%, respectively. Comparison of the standardized serum 25(OH) D data from the Danish Health2006 with the ethnic Norwegian group in HUBRO would suggest that year-round mean 25(OH)D concentration was modestly higher in Oslo, Norway (71.0 nmol/L) compared to Copenhagen, Denmark (65.0 nmol/L) and consequently the prevalence of vitamin D deficiency (25(OH)D ⬍ 30 nmol/L) at 1.3% was lower than the 4.3%, respectively. Prior to standardization of serum 25(OH)D data, the difference between the two populations appeared much greater (mean: 44.4 nmol/L vs. 74.5 nmol/L, respectively and vitamin D deficiency: 25.8% vs. 1.8%, respectively for Denmark vs. Oslo). In addition, the mean standardized serum 25(OH)D data in the Finnish Health 2011 sample (67.7 nmol/L) was similar to that of Copenhagen and lower than that of the Norwegian component of HUBRO. The standardized (and original) serum 25(OH) D concentrations in VitmaD (Denmark), stratified by age-group is shown in Table II. While the mean standardized serum 25(OH)D in adults and children were similar (∼ 62 nmol/L), the percentage below 30 and 50 nmol/L were slightly higher in adults (1.9% and 23.1%, respectively) than in chil-

557

dren (0 and 17.4%, respectively). Comparison of the prevalence of serum 25(OH)D ⬍ 30 and ⬍ 50 nmol/L, using the standardized data for adults from baseline VitmaD (which was predominantly a September blood sampling) (2.3 and 21.7%, respectively) with those of Danish Health2006 in September only (n 388; 2.1 and 14.2%, respectively) showed relatively good agreement, whereas the original estimates from Health2006 were disparate (16.5 and 59.3%, respectively). Binary logistic regression analysis of the determinants of likelihood of having serum 25(OH)D concentrations ⬍ 50 nmol/L (threshold used by NORDEN [13] and IOM [11] as the estimate of vitamin D requirement covering the needs of 97.5% of individuals from a bone health perspective) in Health2006 (Denmark) and HUBRO (Norwegian adults only) showed that in a model which also included gender and age, season of blood sampling was the main determinant. In both populations, being sampled in winter or spring carried odds ratios (OR) of between 2.0 and 4.6, and 1.9 and 3.8, respectively, relative to being sampled in summer (p ⬍ 0.01; data not shown). Of note, in HUBRO (Pakistani adults only) there was no increase in OR in winter, spring, autumn over summer (p ⬎ 0.7; data not shown). In light of the impact of seasonality and the differences in the distribution of season in which blood sampling occurred in the Finnish, Norwegian and Danish population samples, the mean, SD and median serum 25(OH)D as well as prevalences below 30 and 50 nmol/L during autumn (September to November) and an extended winter period (i.e. November to March) are shown in Table III. As was evident in the year-round data for serum 25(OH)D concentration in adults, limiting the data to the extended winter period only again showed that ethnic Norwegians from Oslo, Norway had a higher mean serum 25(OH)D concentration and lower prevalence of vitamin D deficiency and inadequacy compared to that of adults in Copenhagen, Denmark. Limiting the data to the autumn period only (for which all three populations have representative data) showed that white adults from Finland had a lower prevalence of vitamin D deficiency and inadequacy compared to that of adults in Oslo and which in turn were lower than adults in Copenhagen. Mean serum 25(OH)D concentrations in autumn were slightly higher in Norwegian adults in Oslo compared to adults in either Finland or Copenhagen, which had very similar mean concentrations (Table III). The mean serum 25(OH)D2, 25(OH)D3, epimer of 25(OH)D3 and ratio of epimer of 25(OH)D3 to 25(OH)D3 in the statistical algorithm-defined subsets (n 101–147) of serum samples from the four study samples, as measured by the LC-MS/MS method is shown in Table IV. While the concentrations of vitamin D metabolites are from a sub-sample

D Standardization Program (VDSP)-calibrated are presented as mean estimates and their 95% CIs. *As participants in Health 2011 were sampled in September through December only, data on the basis of an extended winter (November through March) were not generated. 1Vitamin

1,399 (55.9, 64.3) (19.2, 19.4) (56.3, 62.2) (0, 11.6) (26.9, 38.5) 60.1 19.3 59.2 7.2 33.0 –∗ – – – – –

66.4 18.1 67.0 2.1 19.8

329 (62.8, 70.0) (17.8, 18.4) (63.7, 70.3) (0.3, 3.6) (14.0, 25.5)

1,047 (64.5, 72.7) (17.9, 18.8) (65.2, 71.9) (0, 4.1) (11.7, 21.5) 68.6 18.3 68.6 2.1 16.5 430 (67.9, 75.3) (19.1, 19.9) (67.9, 75.2) (0.2, 2.8) (9.5, 18.8) 71.6 19.5 71.5 1.2 13.7 3,040 (64.8, 69.6) (12.7, 13.1) (64.3, 68.9) (0.3, 0.8) (6.3, 9.4) 67.2 12.9 66.6 0.5 6.9

Serum 25(OH)D (nmol/L): Autumn only n Mean SD Median % ⬍ 30 nmol/L % ⬍ 50 nmol/L Extended winter only n Mean SD Median % ⬍ 30 nmol/L % ⬍ 50 nmol/L

Health 2011

HUBRO (ethnic Norwegian)

Health2006


Sample

Table III. Distribution of standardized serum 25-hydroxyvitamin D concentrations during autumn (all three white population samples) and in extended winter in two white population samples1.


558

of the original populations they represent the spread within such populations, as the sera were chosen by a uniform within quartile sampling procedure. In general, the mean concentrations of 25(OH)D2 as well as of ratio of epimer of 25(OH)D3 to 25(OH) D3 are similar across the four study populations (Table IV). Two participants in the HUBRO sample had higher serum 25(OH)D2 (38.3 and 46.9 nmol/L) which contributed to the slightly higher mean and large SD; when excluded the mean (SD) for HUBRO was 1.2 (0.8) nmol/L (data not shown).

Discussion The recent validation of the VDSP protocol for standardization of serum 25(OH)D values from past surveys using data from the national adult nutrition survey in Ireland [12] was an important contribution toward international standardization. The VDSP protocol involves only relatively modest re-analysis by using LC-MS/MS of only about 100–150 specifically selected biobanked sera from the study sample which will allow a prediction of LC-MS/MS-like serum 25(OH)D values for the entire study sample [12]. The standardization of serum 25(OH)D values from past surveys can provide more clearly defined prevalence estimates of vitamin D deficiency within and across continents [1,10]. Application of the VDSP protocol to the existing serum 25(OH)D datasets in the present work eliminated the sizeable gap between the mean serum 25(OH)D concentration of adults in Copenhagen, Denmark and those in either Oslo, Norway or Finland (44.4 vs. 66.2 or 75.9 nmol/L, respectively) such that once all three were standardized they had a mean serum 25(OH)D concentration of around 65 nmol/L. The mean serum 25(OH)D concentration of the HUBRO sample however was brought down by the fact that 17% of the entire population sample were Norwegian adults of Pakistani ethnicity, and who had a mean 25(OH)D concentration of only 27.6 nmol/L compared to the 71.0 nmol/L in the Norwegian population. Thus, the Norwegian Oslo adult population had a modestly higher mean serum 25(OH)D concentration (by ∼ 3–6 nmol/L on average) than their counterparts in Copenhagen or Finland. Using the standardized serum 25(OH)D data, the percentage of the population which had vitamin D deficiency in all three white samples was relatively low at 0.4–4.1%, while 6.6–23.6% had serum 25(OH)D below the NORDEN [13] and IOM [11] suggested 50 nmol/L threshold. Use of the standardized serum 25(OH)D data from the Danish-based Health2006 and VitmaD studies, both of adults in the Copenhagen region, showed that the percentage with serum 25(OH) D ⬍ 30 nmol/L in autumn (which was the baseline sampling point in the VitmaD RCT) was 2.1 and 2.3%, respectively, and agreed better than the 1.5

0.07 ⫾ 0.03 0.07 ⫾ 0.04 0.06 ⫾ 0.03 0.05 ⫾ 0.01 represent means and their standard deviations. 1Values

68.9 ⫾ 33.8 67.3 ⫾ 34.8 66.9 ⫾ 29.7 61.9 ⫾ 22.1 Health 2011 HUBRO Health2006 VitmaD

101 105 147 139

1.7 ⫾ 1.2 2.1 ⫾ 6.1 1.3 ⫾ 0.5 1.3 ⫾ 0.5

4.8 ⫾ 3.3 4.3 ⫾ 2.6 4.5 ⫾ 4.2 3.0 ⫾ 1.7

3-epi-25(OH)D3(nmol/L):25(OH)D3 (nmol/L) 3-epi-25(OH)D3 (nmol/L) 25(OH)D3 (nmol/L) 25(OH)D2 (nmol/L) n Serum vitamin D metabolite

Table IV. Serum 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and 3-epimer of 25-hydroxyvitamin D3 (3-epi-25(OH)D3) concentrations and ratio of C3-epi-25(OH)D3 to 25-hydroxyvitamin D3 in the four Nordic populations1.



559

and 16.5%, respectively, which existed for these two prior to standardization. The very high negative bias exhibited by the cobas Roche assay as used originally in Health2006 relative to the LC-MS/MS used in the present analysis, and which caused the significant disparency in serum 25(OH)D concentrations relative to the other population samples, is in agreement with recent findings by Enko et al. [26]. While the assay only had ⬍ 10% specificity for serum 25(OH) D2 concentrations, this under-detection would not explain the magnitude of the negative bias observed in the present analysis as our LC-MS/MS analysis of the subset of sera suggests serum 25(OH)D2 concentration was low (mean 1.3 nmol/L). Enko et al. have suggested that poor antibody specificity with cross-reactivity to other vitamin D metabolites, incomplete extraction of the 25(OH)D analyte from the vitamin D-binding protein, and confounding matrix substances such as lipids could be potential reasons for the unacceptable performance of the cobas Roche assay [26]. Binary logistic regression analysis showed that being sampled in winter and spring, periods of lower ambient UVB radiation and thus potential for dermal synthesis of vitamin D [27], increased the odds of having serum 25(OH)D ⬍ 50 nmol/L from ∼ 2-fold to ∼ 4.5-fold compared to being sampled in summer. Using the standardized serum 25(OH)D data from an extended winter period (November to March) again showed that the Norwegian adults in the Oslo Health study had on average about 6 nmol/L higher mean concentrations than those in Copenhagen. This was a much smaller increment compared to that which the originally measured serum 25(OH) D data from these population samples had suggested (31 nmol/L). Considering the higher latitude of Oslo compared to Copenhagen, the higher mean serum 25(OH)D concentration of the Oslo population might be linked to the high consumption of salmon and widespread use of cod liver oil and vitamin supplements in Norway relative to Denmark (personal communications from KH and BT in relation to usage in Norway and Denmark, respectively), but this warrants further investigation. Progressive increments in habitual vitamin D intakes in the Finnish population in recent years, arising from enhanced food fortification and supplementation practises, may well underpin the much lower (from about onethird to half, depending on time of the year) prevalence of vitamin D inadequacy compared to that in Copenhagen and Oslo. It should be noted however that the Finnish sample was surveyed in 2011, while the Oslo and Copenhagen surveys were in 2001 and 2006, respectively. The VitmaD sample population was sampled in a limited geographical area. However, the sample provides valuable data on children which are generally scarce in relation to vitamin D status in the Northern countries. The VitmaD standardized serum


560


25(OH)D data showed that the prevalence of vitamin D inadequacy during autumn in children aged 4–17 y was less than that in adults aged 18–60 y. Andersen et al. [28], in their comparison of vitamin D status (measured by a HPLC method) in females in four EU member states, showed that Danish girls (aged 12.5 y) had a higher prevalence of vitamin D inadequacy (serum 25(OH)D ⬍ 50 nmol/L) in winter than postmenopausal women (93% vs. 56%, respectively), and likewise the following winter period (87% vs. 48%, respectively) [29]. The prevalence of vitamin D inadequacy during summer, however, was similar in girls and postmenopausal women (18.5% vs. 19.2%, respectively) [29]. The standardized serum 25(OH)D data in Pakistani adults in HUBRO showed that the prevalence of vitamin D deficiency (⬍ 30 nmol/L) was worryingly excessive at 64.8% and the percentage with serum 25(OH)D ⬍ 50 nmol/L was almost universal at 92.0%, with consequences in terms of risk of osteomalacia and rickets in this population subgroup. The binary regression analysis showed that being sample in winter, spring or autumn relative to Summer carried no increased odds of having serum 25(OH)D inadequacy (⬍ 50 nmol/L) in Pakistanis. In addition, gender was not a significant predictor in the model (p ⬎ 0.5). Andersen et al. [30] showed that 84% and 65% of Danish Pakistani adult women and men had year-round serum 25(OH)D concentrations ⬍ 25 nmol/L. The reasons for the much higher prevalence of vitamin D deficiency and inadequacy in the Pakistani adults in Nordic regions may relate to reduced sun exposure, reduced capacity of cutaneous vitamin D synthesis due to pigmentation, a low intake of the few foods rich in vitamin D, and infrequent use of vitamin D supplements [16]. Based on the standardized serum 25(OH)D data in the present work, the Norwegian and Danish adults had a lower prevalence of vitamin D deficiency during an extended winter period (3–7%, respectively) compared to that in adults (aged 18–84 y) in Ireland (51–54°N), which using the same LC-MS platform as used in the present study, was 18.2% [12]. The reasons for these differences are likely to relate to differences in dietary intakes, patterns of supplement use and UVB sun availability and indeed exposure behavior. The data on serum 25(OH)D2 and 3-epimer of 25(OH)D3 from the subset of four Nordic population samples in the present study provided some insight into the mean concentrations of these vitamin D metabolites and were quite similar to those reported recently for a representative sample of Irish adults, as measured by the same analytical platform [20,31]. The collective standardized findings of these northern European countries highlight the need to explore and devise strategies for prevention of vitamin D inadequacy in the population. Interestingly,

the VitmaD RCT provided experimental evidence of the effects of vitamin D-fortified milk and bread on preventing deficient winter-time serum 25(OH)D concentrations in its 201 families (n 782 children and adults, aged 4–60 y) [17]. The groups randomized to vitamin D unfortified and fortified foods (median intakes of vitamin D of 2.2 and 9.6 μg/d, respectively) had a prevalence at the end of winter serum 25(OH)D concentrations ⬍ 50 nmol/L, of 65% and 16%, respectively [17]. There is also a need to explore food vehicles for ethnic groups also in light of the high prevalence of vitamin D deficiency and inadequacy in these population sub-groups. In conclusion, the present work not only further illustrates the huge benefits of standardizing existing population serum 25(OH)D data so as to allow for more valid comparisons across countries and regions, the data also sheds further light on the vitamin D status and prevalence of deficiency and inadequacy in the Nordic region.

Note 1. The notation 25(OH)D concentration used in this paper refers to the sum of the concentrations of 25(OH)D2 and 25(OH)D3.

Acknowledgements This research was funded in part by the Nordic Council of Ministers (NORDEN) and in part by internal strategic funding within the Vitamin D Research Group, School of Food and Nutritional Sciences, University College Cork. The authors wish to acknowledge support of the Higher Education Authority under its Programme for Research in Third Level Institutions (FoodIreland, R14109) for funding the LC-MS/MS used in this analysis. Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.

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