itamin D (vit D) belongs to the class of fat-soluble steroids that is categorized as two forms; cholecalciferol (vit D
3) and ergocalciferol (vit D
2) which are derived from animal and plant sources, respectively (Holick, 2007, Jolfaie
et al., 2016). Although sunlight exposure is the main source of vit D (Baeke
et al., 2007, Singh
et al., 2011), access to other sources of vit D through diet is also possible (Holick, 2007, Spiro and Buttriss, 2014).
Natural Sunlight exposure (UV light of wavelength 290-315 nm) can penetrate the epidermis and photolysis 7-dehydrocholesterol to previtamin D
3, which is metabolized in the liver after its entrance. Vit D
3 is then converted to 25-hydroxy vitamin D [25(OH)D] by 25-hydroxylases in the liver. Subsequently, 1, 25-dihydroxy vitamin D [1,25(OH)2D] is made in the kidneys by 1-hydroxylation of 25(OH)D (Bauer
et al., 2013, Spiro and Buttriss, 2014). Although 1,25(OH)2D is the active metabolite of vit D, the plasma concentration of 25(OH)D is the best clinical indicator of vit D status (Runia
et al., 2012).
Vit D deficiency is a worldwide concern and presents when serum 25(OH)D levels are below 50 nmol/L (20 ng/ml) (Christodoulou
et al.). Dark pigmentation, lack of sufficient sun exposure, wearing clothes covering most of the body, sunscreen use, air pollution, and aging are known as common causes of vit D deficiency (Holick, 2002, Nicolaidou
et al., 2006, O’Riordan
et al., 2008, Sloka
et al., 2009, van der Meer
et al., 2006). Additionally, vegetarians and lactose intolerant people are highly at the risk of vit D deficiency (Alharbi and El-Sohemy, 2017, Outila, 2001).
Vit D deficiency adversely affects the health, leading to increased risk for several diseases including rickets in children or osteoporosis in adults, cardiovascular diseases, diabetes, cancer, and autoimmune diseases such as multiple sclerosis (Garland
et al., 2006, Garland
et al., 2009, Kriegel
et al., 2011, Norman
et al., 1974, Papandreou and Hamid, 2015, Pierrot-Deseilligny and Souberbielle, 2013, Wagner and Greer, 2008). Therefore, sufficient vit D status plays a major role in the prevention and treatment of various diseases (Holick, 2004, Holick
et al., 2011, Jolfaie
et al., 2016).
Several studies have suggested that vit D supplementation can increase circulating serum 25(OH)D levels (Björkman
et al., 2009, Havens
et al., 2012, Todd
et al., 2015). In addition to vit D supplementation, food fortification would be advisable to compensate vit D deficiency. Today, food fortification is considered to be a common strategy to improve and combat vit D deficiency. Vit D food fortification is an important and inexpensive strategy to certify the adequacy of vit D intake among populations (Biancuzzo
et al., 2010a, Calvo
et al., 2004, Upreti
et al., 2002).
Vit D3 is typically added to foods such as cheese and yogurt (Al-Khalidi
et al., 2015, Green
et al., 2010, Johnson
et al., 2005, Levinson
et al., 2016, Madsen
et al., 2013, Neyestani
et al., 2012, Rich-Edwards
et al., 2011, Wagner
et al., 2008), orange juice (Biancuzzo
et al., 2010b, Tangpricha
et al., 2003, Tripkovic
et al., 2017), bread (Itkonen
et al., 2016, Natri
et al., 2006, Nikooyeh
et al., 2016) and mushrooms (Mehrotra
et al., 2014, Urbain
et al., 2011). The efficiency of vit D2 as a fortificant in raising and maintaining blood concentrations of 25(OH)D is not yet definitively proven (Itkonen
et al., 2016, Mehrotra
et al., 2014, Urbain
et al., 2011). Due to the concern about the changes in cooking and preparation of foods, studies have used several fortification methods including the use of ultraviolet radiation and water-soluble vit D (Biancuzzo
et al., 2010b, Mehrotra
et al., 2014, Natri
et al., 2006, Urbain
et al., 2011).
The main purpose of the present systematic review was to evaluate the findings from available studies regarding the effect of food fortification with vit D on serum 25(OH)D levels.
Materials and Methods
Search strategy: A systematic search was performed among the published studies in the following electronic databases: PubMed (www.pubmed.com), Scopus (www.scopus.com), ISI Web of Science (www.isiknowledge.com) and Google Scholar (www.scholar.google.com) from inception up to August 2017. The search was done without restriction on language or publication year,
using the combined MeSH and non-MesSH terms
as follows: (vitamin D, vitamin D2, vitamin D3, vitamin-D2, vitamin-D3, 25-hydroxy vitamin D, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 1,25-dihydroxy vitamin D, 1,25-dihydroxy vitamin D2, 1,25-dihydroxy vitamin D3, 25(OH)D, 25(OH)D2, 25(OH)D3, 1,25(OH)2D, 1,25(OH) 2D2, 1,25(OH) 2D3, hydroxycalcidol, 7-dehydrocholesterol, ergosterol, calcitriol, 25-hydroxy cholecalciferol, calcidiol, cholecalciferol, ergocalciferol) AND (“fortified food”, “fortified foods”, “Enriched Food”, “Enriched Foods”, “enriched food”, “enriching food”, “enhanced foods”, “complementary food”).
Broad screening of titles and abstracts were carried out by Talenezhad N and Mohammadi M to exclude irrelevant studies. Discrepancies between reviewers were resolved by the third author (Ramezani-Jolfaie N). In order to look for potential eligible articles and to increase the sensitivity of the search; the references of the retrieved studies were also checked.
Eligibility criteria: The literature that met the following criteria were considered for inclusion in the present systematic review: 1) original articles; 2) randomized controlled clinical trials (RCTs); 3) studies evaluating the effect of fortified foods with vit D on serum 25(OH)D. The studies that did not measure vit D levels, were non-human (in vitro and animal), used fortified food with vit D and another nutrient were excluded from the research. Moreover, the studies that besides vit D food enrichment, had used vit D supplementation were also excluded from the present research since the effect of enrichment could not be controlled separately.
Data extraction: Two reviewers (Talenezhad N, Ramezani-Jolfaie N) independently extracted the data in each study and participant characteristics including the author's last name, publication date, the country in which study was conducted, study design, study duration, number of participants, gender (female/male/both), age (mean or range), type of fortified food, dose, type of vit D, baseline, endpoint and changes in 25(OH)D levels. Data were then tabulated for further evaluations.
Quality assessment: The Cochrane Collaboration's tool for assessing the risk of bias was employed to describe the methodological quality of eligible trials. This tool consists of six domains including random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, and selective reporting. The included studies were classified into three categories: “yes” (low risk of bias), “no” (high risk of bias) or “unclear” (uncertain risk of bias) (Higgins and Green, 2011)
. The quality of each study was evaluated as good (low risk for more than two domains), fair (low risk for two domains), and weak (low risk for less than two domains).
Results
Study selection and characteristics: A total of 791 articles were retrieved based on electronic data search. After further reading of title and abstract, deduplication and exclusion of irrelevant articles, 44 studies were totally remained for the full-text screening, and 30 articles that met exclusion criteria were dropped out. Eventually, after including two papers through hand search, 16 studies were selected for the present systematic review. The description of the study selection is illustrated in
Figure 1.
Table 1 presents the characteristics of the sixteen chosen articles which were published between 2003 and 2017 (Al-Khalidi
et al., 2015, Biancuzzo
et al., 2010b, Green
et al., 2010, Itkonen
et al., 2016, Johnson
et al., 2005, Levinson
et al., 2016, Madsen
et al., 2013, Mehrotra
et al., 2014, Natri
et al., 2006, Neyestani
et al., 2012, Nikooyeh
et al., 2016, Rich-Edwards
et al., 2011, Tangpricha
et al., 2003, Tripkovic
et al., 2017, Urbain
et al., 2011, Wagner
et al., 2008). From these articles, eight studies had examined the effect of dairy products fortifications on 25(OH)D (Al-Khalidi
et al., 2015, Green
et al., 2010, Johnson
et al., 2005, Levinson
et al., 2016, Madsen
et al., 2013, Neyestani
et al., 2012, Rich-Edwards
et al., 2011, Wagner
et al., 2008), three on bread (Itkonen
et al., 2016, Natri
et al., 2006, Nikooyeh
et al., 2016), two on mushrooms (Mehrotra
et al., 2014, Urbain
et al., 2011) and three on orange juice (Biancuzzo
et al., 2010b, Tangpricha
et al., 2003, Tripkovic
et al., 2017). Four studies were carried out in the United States (Biancuzzo
et al., 2010b, Johnson
et al., 2005, Mehrotra
et al., 2014, Tangpricha
et al., 2003), two studies in Canada (Al-Khalidi
et al., 2015, Wagner
et al., 2008), two in Iran (Neyestani
et al., 2012, Nikooyeh
et al., 2016), two in Finland (Itkonen
et al., 2016, Natri
et al., 2006) and one study was conducted in Germany (Urbain
et al., 2011), one in Israel (Levinson
et al., 2016), one in United Kingdom (Tripkovic
et al., 2017), one in Denmark (Madsen
et al., 2013), one in Mongolia (Rich-Edwards
et al., 2011), and one in New Zealand (Green
et al., 2010). All trials were designed as parallel-group studies, and duration of their intervention ranged from two weeks to sixteen weeks.
Risk of bias assessment: The quality assessment of the included articles for all domains of Cochrane risk of bias tool is summarized in
Table 2. Most of the studies (n=12) had good quality (low risk for more than 2 items) (Biancuzzo
et al., 2010b, Green
et al., 2010, Johnson
et al., 2005, Madsen
et al., 2013, Mehrotra
et al., 2014, Neyestani
et al., 2012, Nikooyeh
et al., 2016, Rich-Edwards
et al., 2011, Tangpricha
et al., 2003, Tripkovic
et al., 2017, Urbain
et al., 2011, Wagner
et al., 2008), two of them were fair (low risk for 2 items) (Itkonen
et al., 2016, Levinson
et al., 2016) and others (n=2) were classified as poor quality (low risk for less than 2 items) (Al-Khalidi
et al., 2015, Natri
et al., 2006). The articles having weak quality score did not report blinding of outcome assessment, allocation concealment, and random sequence generation.
The effect of bread fortification with vit D on 25(OH) D levels: Three studies investigated the effect of fortified bread with vit D on serum 25(OH)D concentrations (Itkonen
et al., 2016, Natri
et al., 2006, Nikooyeh
et al., 2016). Natri et al. (Natri
et al., 2006) observed that bioavailability of vit D from both wheat and rye bread is equal, although rye bread contains more fiber. Their three-week investigation emphasized that the consumption of either fortified wheat or rye bread with vit D (12µg/100g) can significantly improve serum 25(OH)D levels in 41 healthy women. In line with their study, Nikooyeh et al. (Nikooyeh
et al., 2016) revealed that over longer intervention among 90 healthy men and women who consumed 50 g bread fortified with 25µg vit D for eight weeks, there was a significant raising in the circulation of 25(OH)D concentrations.
Another study assessed the bioavailability of vit D
2 from UV-irradiated yeast present in bread during eight-week intervention among 33 young adult females (Itkonen
et al., 2016). Participants were divided into four groups with different interventions: placebo pill and regular bread; D
2 supplement and regular bread; D
3 supplement and regular bread and placebo pill and D
2-biofortified bread. The results represented that vit D
2 fortified bread (25µg D
2/d) modestly increased serum 25(OH)D
2, but not as much as vit D supplement (6.4 vs. 31.3 nmol/l, respectively).
The effect of mushroom fortification with vit D on 25(OH)D levels: Two studies assessed the effect of consuming fortified mushrooms with vit D on 25(OH)D changes in the body (Mehrotra
et al., 2014, Urbain
et al., 2011). Twenty-six young adults with low serum 25
)OH
(D (≤ 50 nmol/l) and normal serum calcium concentration (2.2-2.7 nmol/l) were enrolled in a randomized, placebo-controlled study to determine the bioavailability of vit D
2 from vit D
2-enhanced mushrooms by ultraviolet irradiation (Urbain
et al., 2011). An experimental soup was made by fresh mushrooms irradiated with a UV-B dose of 1.5 J/cm
2, increasing vit D
2 content from <1 to 491µg/100g. It was observed that serum 25(OH)D concentrations rose significantly after ingesting 28000 IU of D
2/week for four weeks via experimental soup (3.9 nmol/l) vs. supplement (4.7 nmol/l).
Another research was conducted among pre-diabetic individuals, included 43 nonsmoking participants, with at least two features of metabolic syndrome (Mehrotra
et al., 2014). Two groups of participants who consumed entrees made by 100 g of UVB-treated mushrooms containing 600 IU D
2 or 4000 IU D
2 for sixteen weeks had modest or no increase in 25(OH)D
2 or total 25(OH)D.
The effect of orange juice fortification with vit D on 25(OH)D levels: Tangpricha et al. (Tangpricha
et al., 2003) carried out a study to determine the bioavailability of vit D
3 in orange juice and vit D
2 in whole milk, skim milk, and corn oil on toast. Eighteen healthy adults were asked to drink 240 ml of whole milk or skim milk that contained 25000 IU ergocalciferol or 25000 IU vit D
2 that had been dissolved in 0.1 ml corn oil and applied to toast. After ingestion of fortified whole milk, skim milk and corn oil on toast, there were not any significant changes in blood vit D
2 concentrations. However, remarkable differences appeared in blood vit D
3 among the participants who consumed fortified orange juice.
In addition, another study was conducted on fortified orange juice with vit D
2 and vit D
3 (Biancuzzo
et al., 2010b). Eighty-six healthy participants were randomly assigned into one of the five groups: 1) placebo capsule + orange juice without vit D, 2) placebo capsule + orange juice containing 1000 IU vit D
3, 3) placebo capsule + orange juice containing 1000 IU vit D
2, 4)1000 IU vit D
3 capsule + placebo orange juice, and 5) 1000 IU vit D
2 capsule + placebo orange juice. After eleven weeks of intervention, analysis of the area under the curved demonstrated that the bioavailability of vit D
2 and vit D
3 in orange juice and capsules were similar and raised 25(OH)D effectively. Also, there were no considerable changes in serum 25(OH)D
2 and 25(OH)D
3 in the placebo groups, which means that sunlight exposure and diet could not significantly affect the vit D status.
Another research was carried out in 2017 by Tripkovic et al. (Tripkovic
et al., 2017) indicating that whether vit D
2 or vit D
3 added to juice or biscuit is effective in increasing serum total 25(OH)D. The results showed that 335 women who received 15 µg vit D2 or vit D3 added to juice and biscuit for twelve weeks had improvement in their vit D status.
The effect of dairy products fortification with vit D on 25(OH)D levels: Neyestani et al. (Neyestani
et al., 2012) carried out a study to determine the effect of vit D fortification either with or without calcium on certain inflammatory markers among 90 Iranian individuals with type 2 diabetes. All subjects were randomly divided into three groups of 1) receiving two 250-ml bottles of Doogh (Persian yogurt drink) per day (PD, containing 150 mg calcium and no detectable vit D
3/250ml); 2) vit D-fortified Doogh (DD, containing 500 IU vit D
3 and 150 mg calcium/250ml); and 3) calcium + vit D
3-fortified Doogh (CDD, containing 500 IU vit D
3 and 250 mg calcium/250ml). There was a detectable improvement in vit D status of DD and CDD groups following twelve weeks of intervention. Moreover, inflammatory markers and retinol binding protein-4 concentrations significantly decreased in those groups.
Green et al. also demonstrated that 73 women who consumed fortified milk (5µg vit D
3) for twelve weeks had 10 nmol/L higher serum 25(OH)D concentrations compared those consumed the placebo (Green
et al., 2010).
In the two researches conducted by Johnson, the bioavailability of vit D from fortified process cheese and its effects on 25(OH)D status in the elderly subjects was assessed (Johnson
et al., 2005). One hundred older men and women randomly received 85 g of 600 IU vit D fortified cheese, non-fortified cheese or no cheese during two months of intervention. Unexpectedly, a greater decrease in serum 25(OH)D was observed among the vit D fortified cheese group. The researchers speculate that this decrement may be related to higher baseline serum 25(OH)D concentrations. An additional randomized cross over the trial was also conducted to determine the bioavailability of vit D
2 and its absorption from process cheese and fortified water dilution (Johnson
et al., 2005). A total sample of eight people, divided into two groups of young and old, randomly received either vit D-fortified cheese or water. Consistent with the results obtained from serial blood sampling collected during 24-hours after the intervention, there was a similar peak in the serum vit D of younger (23 to 50 yr) and older (72 to 84 yr) adults, and vit D
2 absorbed more significantly from cheese than from water.
Another study was done by Wanger et al. (Wagner
et al., 2008) also investigated the bioavailability of vit D from fortified cheese. Eighty adults were randomly assigned to one of six weekly interventions of: 1) fortified cheddar cheese (DC) (34g, n=20); 2) fortified low-fat cheese (DLF) (41g, n=10); liquid vit D supplement (1ml), taken with food (DS+) (n=20) or without food (DS-) (n=10); placebo cheddar cheese (n=10); or placebo supplement (n=10). Over eight weeks of intervention, in the placebo groups, baseline 25(OH) D levels of 55.0±25.3 nmol/l declined to 50.7±24.2 nmol/l. In the vit D-treated groups, the mean increases in 25(OH)D values were as follows: 65.3±24.1 (DC), 69.4±21.7 (DLF), 59.3±23.3 (DS+) and 59.3 ±19.6 nmol/l (DS-) which presents that fortified cheese boost vit D status as adequately as a supplement, making it a proper choice for vit D fortification.
The other research was performed to assess the bioavailability and safety of vit D
3 from fortified mozzarella cheese baked on pizza (Al-Khalidi
et al., 2015). The research demonstrated that ingesting 200 IU or 28000 IU vit D
3-fortified mozzarella cheese for eight weeks could increase 25(OH)D levels by 5.1± 11 nmol/l in the low-dose group (n=47; P-value=0.003) and by 73±22nmol/l in the high-dose group (n=49; P-value<0.0001).
Levinson et al. (Levinson
et al., 2016) also assessed the bioavailability of vit D
3 from fat-free yogurt, in re-assembled casein micelles (rCMs) compared to polysorbate-80 (PS80/Tween80) which is commonly used a synthetic emulsifier. Serum 25(OH)D status of participants consuming fat-free yogurt with 50000 IU of either VD
3-rCM, VD
3-PS8 showed increases of ~ 8 ng/ml after two weeks, and no significant differences were found between mean changes of 25(OH)D among the individuals who consumed rCM yogurt versus PS80.
On the other hand, Madsen et al. (Madsen
et al., 2013) investigated the association of fortified milk and bread with vit D status of 782 children and adults. Participants were recruited from 201 families and were randomly assigned to vit D-fortified or non-fortified milk and bread for six months. When comparing from baseline to completion of the intervention, a greater serum 25(OH)D level was seen in the fortification group (67.6 nmol/L) than in the control group (41.7 nmol/L). Similar finding was also reported by Rich-Edwards et al. (Rich-Edwards
et al., 2011). There were improvements in 25(OH)D concentrations among 579 children in Mongolia who received 300 IU vit D via fortified milk.