Low vitamin D status (serum 25-hydroxyvitamin D concentration <50 nmol/L) is a global health concern,1 as many people do not receive sufficient sun (ultraviolet-B light [UVB]) exposure or have access to sufficient vitamin D from dietary sources to meet their requirement. Due to the essential role of vitamin D in bone health throughout life, and potential links between low vitamin D status and various chronic diseases, strategies to increase vitamin D status in the population are under investigation.
Circulating concentrations of 25-hydroxyvitamin D (25(OH)D) are measured as a biomarker of vitamin D status, rather than the active form, 1,25(OH)D, which has a brief circulating half-life (four to six hours) and 1000-fold lower circulating concentrations than 25(OH)D.2 Measurement of 25(OH)D is challenging as it is largely protein-bound, and there are two circulating forms, 25(OH)D2 and 25(OH)D3. However, 25(OH)D has been shown to reliably reflect vitamin D status.3
A variety of assays may be used to measure serum 25(OH)D; however, accuracy and precision vary.4,5 Liquid chromatography-mass spectrometry methods that follow the standard reference method procedure (RMP) developed by the National Institutes of Standards and Technology, Ghent University, and the US Centers for Disease Control and Prevention are considered the benchmark.5-7 Where other assays are used, certification of a laboratory's method to the RMPs provides the greatest confidence in an accurate measurement of serum 25(OH)D concentrations.5-7
The prevalence of 25(OH)D concentrations <50 nmol/L ranges from 37% to 48% in the US,8 Canada,9 and Europe.10 Even in Australia, where the potential for UVB irradiance is greater, almost one-fourth of adults have low vitamin D status.11 The Institute of Medicine suggests a vitamin D intake of 10 μg/day as an estimated average requirement for all ages, and a recommended dietary allowance of 15 and 20 μg/day for people aged ≤70 and >70 years, respectively.12 Usual vitamin D intakes commonly fall short of these recommendations, with estimates of mean dietary vitamin D intakes of <5 μg/day in Europe13 and <6 μg/day in Canada.14 In the US, even taking into account intake from fortified foods and supplements, the average daily intake of vitamin D was below the estimated average requirement in 70% of individuals aged ≥2 years who participated in the 2003–2006 National Health and Nutrition Examination Survey.15
Potential strategies to improve vitamin D status at the population level are constrained. Increased sun exposure carries increased risk of skin cancer. As a fat-soluble compound, vitamin D is not readily excreted — with persistent overconsumption, its accumulation in the body may cause toxicity. Therefore, supplements, although effective in increasing 25(OH)D among individuals, are not consistently used across all population sectors, and may be risky as a population-wide intervention due to the potential for toxicity.4,16
Diet offers a possible solution; however, vitamin D is found naturally in few foods and in relatively low concentrations. Consumers may limit or avoid vitamin D–containing foods for various reasons, including cost, religious or cultural practices, and personal preference. Fortification (direct addition of vitamin D to foods) may provide cost-effective, population-wide access to modest and safe, but useful, amounts of vitamin D. In the US, a variety of foods, including milk and milk alternatives, cheese, yoghurt, and juice, are routinely, and voluntarily, fortified with vitamin D.12 Although legislation varies between countries, these fortified foods may also be found in some European countries.13 In Canada, milk, milk substitutes, and margarine must be vitamin D fortified; few other foods are voluntarily fortified.12,14 In Australia, it is mandatory for margarine to be fortified and, although certain other foods may be voluntarily fortified, few are fortified in practice. Biofortification (e.g. addition of vitamin D to animal feed,17,18 or exposure of yeast, mushrooms, or livestock to UVB light19-22 in order to increase the vitamin D content of produce) is also emerging as a potential strategy.
Strategic fortification and biofortification of staple foods may reduce the prevalence of vitamin D deficiency; however, modeling of fortification/biofortification strategies is required to ensure that they do not place consumers at risk of excessive intake of vitamin D. As such, it is important to understand the treatment effect of vitamin D food fortification and biofortification across various population groups in order to develop safe and effective strategies that increase the dietary supply of vitamin D. An earlier meta-analysis of 16 randomized controlled trials (RCTs) published prior to 2012, with a mean dose of vitamin D from fortified foods of 11 μg/d, showed a combined treatment effect of 19.4 nmol/L (95% confidence interval [CI]: 13.90, 24.90), and a serum 25(OH)D increase of 1.2 nmol/L (95% CI: 0.72, 1.68) per 1 μg vitamin D in adults.23 Due to the limited number of relevant RCTs at that time, that review did not examine the difference in effect of the different forms of vitamin D, and included studies with no placebo product provided for control groups. In 2015, a systematic review involving 18 studies of either fortified foods or dietary supplements suggested a mean (standard deviation [SD]) serum 25(OH)D increase of 2.19 (0.97) nmol/L per 1 μg of ingested vitamin D in adults; however, only one fortified food study was included.24 A recent meta-analysis that focused on children found that, of fortified foods, supplements, and bolus injections, fortified foods produced the greatest mean change in serum 25(OH)D concentrations.25 To the authors’ knowledge, there are no previous systematic reviews examining the efficacy of vitamin D biofortification (e.g. addition of vitamin D to animal feed or exposure of produce to UVB light) in improving serum 25(OH)D concentrations in humans. In 2016, a Cochrane Library protocol26 was published for a general investigation of crops biofortified with micronutrients; however, serum 25(OH)D concentration was not listed as an outcome measure.
Several new RCTs have been published since the earlier systematic reviews on adults, providing an increased sample size for review. Hence, this updated review aims to capture recent RCTs and expand the scope of previous reviews by including RCTs of both fortification and biofortification in children and adults. Searches of PROSPERO, the Cochrane Database of Systematic Reviews, and the JBI Database of Systematic Reviews and Implementation Reports revealed no recent or registered related reviews.
What is the treatment effect of vitamin D food fortification or biofortification on serum 25(OH)D concentrations in adults and children?
The review will consider studies that include children and/or adults; studies including participants with a health condition that may compromise gut integrity and vitamin D absorption will be excluded.
Studies that evaluate foods or beverages fortified or biofortified with vitamin D (vitamin D2, vitamin D3, or their hydroxylated forms, 25(OH)D2 and 25(OH)D3) will be considered. A daily fortification or biofortification dose of ≥5 μg vitamin D must have been provided for at least four weeks (an additional 5 μg would allow the average person with an intake of ∼5 μg/day13-15 to achieve the Institute of Medicine's12 estimated average requirement of 10 μg/day).
Treatments must be compared to a placebo food or beverage intervention. Studies that use existing/usual diet as a control will be excluded, due to the impact on participant blinding to the intervention.
We will consider studies that include baseline and end point serum 25(OH)D concentrations. This outcome may be measured by one of several biochemical analysis methods commonly used to measure serum 25(OH)D concentrations, namely chemiluminescence immunoassay (CLIA), chemiluminescentmicroparticle immunoassay (CMIA), competitive protein-binding assay (CPBA), enzyme-linked immunosorbent assay (ELISA), high performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS/MS), or radio-immunoassay (RIA).27
Types of studies
This review will only consider RCTs published in English. Trials must be, at minimum, single-blinded. Studies published from database inception will be included in order to capture all relevant data available to date.
The proposed systematic review will be conducted according to the JBI methodology for systematic reviews of effectiveness.28 This protocol has been registered with PROSPERO: CRD42020145497.
In collaboration with a health sciences faculty librarian, initial searches were conducted in EBSCOhost CINAHL and Ovid MEDLINE to identify keywords and terms for RCTs that compared the effect of vitamin D–fortified or –biofortified foods or beverages with an unfortified control food or beverage on serum 25(OH)D concentrations in humans. Database-specific search strategies (Appendix I) were developed for EBSCOhost CINAHL, Ovid MEDLINE, Ovid Embase, Cochrane Central Register of Controlled Trials, and PubMed in December 2018. With the aim of capturing data for unpublished studies with null or negative results, searches will be conducted in the Cochrane Handbook of gray literature databases, ProQuest Dissertations and Theses, U.S. National Library of Medicine database (ClinicalTrials.gov), World Health Organization International Clinical Trials Registry Platform, All Trials (alltrials.net), and Restoring Invisible and Abandoned Trials. Further main keyword searches will be used to explore any remaining gray literature through the Google and Google Scholar search engines. Bibliographies of included studies will also be screened for relevant articles.
Retrieved articles will be exported to EndNote vX7 (Clarivate Analytics, PA, USA) where duplicates will be removed, and titles and abstracts screened. The remaining articles will be imported into the JBI System for the Unified Management, Assessment and Review of Information (JBI SUMARI; JBI, Adelaide, Australia) for independent full-text review by two authors. Any difference in inclusion/exclusion decisions will be resolved through discussion by the two authors. If a consensus is not reached, a third author will be consulted. Reasons for exclusion of articles will be recorded in JBI SUMARI.
Assessment of methodological quality
Two authors will independently critically appraise eligible studies for design and methodological quality using the JBI SUMARI critical appraisal checklist for RCTs. The tool comprises 13 questions related to randomization, blinding, baseline characteristics of participants, follow-up, measurement of outcomes, statistical analysis, and overall design quality. Studies that meet the minimum blinding requirement (single-blinded) and use an appropriate assay, as listed in the inclusion criteria, for serum 25(OH)D concentration analysis will be included; studies that do not meet these will be excluded. Studies will be included regardless of critical appraisal outcomes; however, quality scores (out of 13, relating to whether each of the RCT critical appraisal tool questions are satisfied) will be recorded. Authors will discuss any differing opinions on study quality, consulting a third author in order to reach consensus if necessary.
Two authors will independently extract data; if recorded data differ, the authors will collaborate to refer back to articles, involving a third author if required. Data will be extracted to Microsoft Excel (Redmond, Washington, USA) in order to capture the breadth of data required and to carry out data conversions where necessary. Information on geographical setting (including latitude), study population, age range, study duration, season(s) of intervention, serum 25(OH)D concentration assay, and study quality score will be tabulated (Appendix II). In a second table (Appendix III), the authors will collate data on food/beverage intervention type, measure of compliance, fortified/biofortified, added daily dose of vitamin D (μg), added daily dose calcium (mg) if data are available, mean baseline and end point of serum 25(OH)D concentration (nmol/L), and number of participants per group. Multiple study arms of the same trial will be included where appropriate. Where multiple publications exist for the same study population, the main study, or the study with the most comprehensive baseline and end point data for the greatest number of participants, will be included; all other duplications will be excluded. Where information is missing, or additional data are required, they will be requested from the authors.
Added vitamin D will initially be separated by vitamin D3, 25(OH)D3, vitamin D2, 25(OH)D2 in case there are a sufficient number of studies to compare the effect of different forms of vitamin D, which may differ in terms of molecular structure and bioactivity. Mean end point data for 25(OH)D will be expressed in nmol/L as mean (SD). When required, the following conversions will be used:
Two authors will carry out all calculations independently and collaborate to resolve any errors, with the assistance of a third author if necessary.
Studies will, where possible, be pooled in statistical meta-analysis using JBI SUMARI. Effect sizes will be expressed as weighted final post-intervention mean differences, and their 95% CIs will be calculated for analysis. Heterogeneity will be assessed using χ2 tests (P <0.10 = significant heterogeneity) and the I2 statistic (I2 of 0% = no heterogeneity, I2 of 25%, 50% and 75% = low, moderate, and high heterogeneity, respectively). Statistical analyses will be performed using the random effects model.29 Where statistical pooling is not possible (e.g., due to high unexplained heterogeneity), the findings will be presented in narrative form including tables and figures to aid in data presentation where appropriate. A funnel plot will be generated in STATA 15.1 (StataCorp, LLC, Texas, USA) to assess publication bias if there are 10 or more studies included in a meta-analysis. Statistical tests for funnel plot asymmetry (Egger test, Begg test, Harbord test) will be performed where appropriate. Reasons for heterogeneity will be explored through subgroup and sensitivity analyses with stratification based on mean baseline 25(OH)D concentrations (< or ≥50 nmol/L), latitude (< or ≥40°), dose (< or ≥10 μg/d), and, if sufficient data are available, by age (e.g., adults 18 to 70 and 70+ years, pregnant women, children birth to two and two to 17 years), form of vitamin D, assay, fortification/biofortification, study quality (critical appraisal tool score < or ≥10), study duration (< or ≥6 months), and by men and women in adults.
Assessing certainty in the findings
The Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach to grade the certainty of evidence and a Summary of Findings will be created using GRADEpro GDT 2015 (McMaster University, ON, Canada). The Summary of Findings will present the following information where appropriate: absolute risks for the treatment and control, estimates of relative risk, and a ranking of the quality of the evidence based on the risk of bias, directness, heterogeneity and inconsistency, precision, and risk of publication bias of the review results. The authors will report the outcome of change in serum 25(OH)D concentration in the Summary of Findings.
Curtin University's Health Sciences Faculty librarian, Diana Blackwood, for her guidance in conducting initial database searches and constructing database-specific search strategies.
ED would like to acknowledge the Australian Government Research Training Program Scholarship in supporting this research.
LJB is supported by a Multiple Sclerosis Research Australia Postdoctoral Fellowship and a Curtin University Research Fellowship.
Funding sources had no involvement in the study design, data collection, analysis and interpretation of data, in the writing of the article, or in the decision to submit the article for publication.
Appendix I: Search strategy
Initial search was conducted on 11 Dec 2018.
Cochrane Central Register of Controlled Trials
Appendix II: Data extraction table 1
Appendix III: Data extraction table 2
1. Palacios C, Gonzalez L. Is vitamin D
deficiency a major global public health problem? J Steroid Biochem Mol Bio
2. Holick MF. Vitamin D
status: measurement, interpretation, and clinical application. Ann Epidemiol
2009; 19 (2):73–78.
3. Seamans K, Cashman K. Existing and potentially novel functional markers of vitamin D
status: a systematic review. Am J Clin Nutr
2009; 89 (6):1997S.
4. Lucas R, Neale R. What is the optimal level of vitamin D
?: separating the evidence from the rhetoric. Aust Fam Physician
2014; 43 (3):119–122.
5. Black LJ, Anderson D, Clarke MW, Ponsonby A-L, Lucas RM, Ausimmune Investigator G. Analytical bias in the measurement of serum 25-hydroxyvitamin D concentrations impairs assessment of vitamin d
status in clinical and research settings. PLoS One
2015; 10 (8):e0135478.
6. Sempos CT, Vesper HW, Phinney KW, Thienpont LM, Coates PM, Vitamin DSP. Vitamin D
status as an international issue: national surveys and the problem of standardization. Scand J Clin Lab Invest
7. Mineva E, Schleicher R, Chaudhary-Webb M, Maw K, Botelho J, Vesper H, et al. 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
2015; 407 (19):5615–5624.
8. Liu X, Baylin A, Levy PD. Vitamin D
deficiency and insufficiency among US adults: prevalence, predictors and clinical implications. Br J Nutr
2018; 119 (8):928–936.
9. Sarafin K, Durazo-Arvizu R, Tian L, Phinney K, Tai S, Camara J, et al. Standardizing 25-hydroxyvitamin D values from the Canadian Health Measures Survey. Am J Clin Nutr
2015; 102 (5):1044–1050.
10. Cashman KD, Dowling KG, Škrabáková Z, Gonzalez-Gross M, Valtueña J, De Henauw S, et al. Vitamin D
deficiency in Europe: pandemic? Am J Clin Nutr
2016; 103 (4):1033–1044.
11. Australian Bureau of Statistics. 4364.0.55.006 - Australian Health Survey: biomedical results for nutrients, 2011–2012 [Internet]. Canberra, A.C.T.: ABS; 2014 [cited 2019 Aug 23]. Available from: http://www.abs.gov.au/ausstats/[email protected]/Lookup/4364.0.55.006Chapter2002011-12
12. Institute of Medicine. Dietary reference intakes for calcium and vitamin D
[Internet]. 2011 [cited 2019 Aug 23]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK56070/pdf/Bookshelf_NBK56070.pdf
13. Kiely M, Black LJ. Dietary strategies to maintain adequacy of circulating 25-Hydroxyvitamin D concentrations. Scand J Clin Lab Invest
14. Vatanparast H, Calvo MS, Green TJ, Whiting SJ. Despite mandatory fortification of staple foods, vitamin D
intakes of Canadian children and adults are inadequate. J Steroid Biochem Mol Bio
2010; 121 (1):301–303.
15. Fulgoni VL, Keast DR, Bailey RL, Dwyer J. Foods, fortificants, and supplements: Where do Americans get their nutrients? J Nutr
2011; 141 (10):1847–1854.
16. Pilz S, März W, Cashman KD, Kiely ME, Whiting SJ, Holick MF, et al. Rationale and plan for vitamin D
food fortification: a review and guidance paper. Front Endocrinol
17. Hayes A, Duffy S, O’Grady M, Jakobsen J, Galvin K, Teahan-Dillon J, et al. Vitamin D
-enhanced eggs are protective of wintertime serum 25-hydroxyvitamin D in a randomized controlled trial of adults. Am J Clin Nutr
2016; 104 (3):629–637.
18. Duffy SK, Kelly AK, Rajauria G, Jakobsen J, Clarke LC, Monahan FJ, et al. The use of synthetic and natural vitamin D
sources in pig diets to improve meat quality and vitamin D
content. Meat Sci
19. Barnkob L, Petersen P, Nielsen J, Jakobsen J. Vitamin D
enhanced pork from pigs exposed to artificial UVB light in indoor facilities. Eur Food Res Technol
2018; 245 (2):1–8.
20. Kühn J, Schutkowski A, Hirche F, Baur AC, Mielenz N, Stangl GI. Non-linear increase of vitamin D
content in eggs from chicks treated with increasing exposure times of ultraviolet light. J Steroid Biochem Mol Bio
21. Itkonen ST, Skaffari E, Saaristo P, Saarnio EM, Erkkola M, Jakobsen J, et al. Effects of vitamin D2-fortified bread v. supplementation with vitamin D2 or D3 on serum 25-hydroxyvitamin D metabolites: an 8-week randomised-controlled trial in young adult Finnish women. Br J Nutr
2016; 115 (7):1232–1239.
22. Cardwell G, Bornman JF, James AP, Black LJ. A review of mushrooms as a potential source of dietary vitamin D
2018; 10 (10):
23. Black L, Seamans K, Cashman K, Kiely M. An updated systematic review and meta-analysis of the efficacy of vitamin D
food fortification. J Nutr
2012; 142 (6):1102–1108.
24. Whiting SJ, Bonjour J-P, Payen FD, Rousseau B. Moderate amounts of vitamin D3 in supplements are effective in raising serum 25-hydroxyvitamin D from low baseline levels in adults: a systematic review. Nutrients
2015; 7 (4):2311–2323.
25. Brett NR, Gharibeh N, Weiler HA. Effect of vitamin D
supplementation, food fortification, or bolus injection on vitamin D
status in children aged 2–18 Years: a meta-analysis. Adv Nutr
2018; 9 (4):454–464.
26. Garcia-Casal MN, Peña-Rosas JP, Pachón H, De-Regil LM, Centeno TE, Flores-Urrutia MC. Staple crops biofortified with increased micronutrientcontent: effects on vitamin and mineral status, as well as health and cognitive function in the general population (Protocol). Cochrane Database Syst Rev
27. Arneson W, Arneson DL. Current methods for routine clinical laboratory testing of vitamin D
2013; 44 (1):E38–E42.
28. Aromataris E, Munn Z, editors. JBI Reviewer's Manual [internet]. Adelaide: JBI, 2014 [cited 2019 Aug 23]. Available from: https://reviewersmanual.joannabriggs.org/
29. Tufanaru C, Munn Z, Stephenson M, Aromataris E. Fixed or random effects meta-analysis? Common methodological issues in systematic reviews of effectiveness. Int J Evid Based Healthc
2015; 13 (3):196–207.