Vitamin D fortification of eggs alone and in combination with milk in women aged 44–65 years: Fortification model and economic evaluation

A cross-sectional study of the Health Interview Survey (HIS) type and a Clinical Health Examination Survey (HES) was conducted between 1. March and 31. May 2021. Three hundred and nineteen healthy women from the Central Slovenian region, aged between 44 and 65, were recruited for the study.

Participants were initially recruited by healthcare workers during preventive health visits at Kamnik and Vrhnika health centres (Figure 1). Since recruitment was hindered by Covid-19 preventive measures, the snowball sampling method was successfully employed (32). This meant that after the participants were informed of their vitamin D status, either by post or email, they were asked to invite suitable peers to the study. They were also given a link to an online contact form through which new participants could join.

Figure 1

All willing women between the ages of 44 and 65 were included in the study. For the final analysis, the exclusion criteria, summarised according to (33) and supplemented, were: 1) confirmed osteoporosis; 2) current oestrogen therapy (prior oestrogen therapy was not an exclusion criterion if >3 months have elapsed since the last dose); 3) treatment with corticosteroids in the previous 12 months;

4) treatment of hyperthyroidism or hypothyroidism in the previous 12 months; 5) any new or unregulated chronic disease (e.g. unregulated diabetes); 6) malignant diseases at any time in life; 7) hysterectomy was not an exclusion criterion; 8) health conditions associated with vitamin D deficiency (malabsorption, chronic inflammatory bowel disease, gastrointestinal resection, liver disease, acute gallbladder disease, chronic kidney disease grade 3 to 5); 9) institutionalisation and poor physical mobility; 10) excessive vitamin D intake (serum 25(OH)D3 >250 nmol/L).

After obtaining signed informed consent forms (ICF), a telephone survey was performed by trained registered nurses (RN) or nutritionists (MNutr.) using a purpose-made questionnaire. The questionnaire was tested in a pilot study carried out in 2019 (34). It contained a modified food frequency questionnaire (FFQ), body weight, height, health status, use of food supplements with vitamin D, food intake, menstruation/menopause, sun exposure status, and socio-economic and socio-demographic status. Blood samples were collected during regular working hours at the participating health centres and Ljubljana University Medical Centre between 1. March and 31. May 2021. All samples were transported to a central laboratory at Ljubljana University Medical Centre, where they were analysed the same day or stored at -80ºC until analysis.

Measurements were performed at the Clinical Institute of Clinical Chemistry and Biochemistry (Ljubljana University Medical Centre). In all participants, S-25(OH)D was measured by an Architect 25-OH D vitamin kit (Abbott Diagnostics, Lake Forest, USA).

There are many studies on the fortification of eggs with vitamin D; the best to date, containing data for both cholecalciferol (D3) and 25(OH)D3, is by Browning et al. (20). The models are made to be safe, cost-effective, simple and feasible.

The input data for the model is: 1) assessment of vitamin D status by 25(OH)D3 determination; 2) assessment of food intake that will be used for vitamin D intake calculation and for the calculation of fortification; 3) food composition data; 4) method for achieving the biofortification of eggs (20); 5) vitamin D ingredient prices.

Vitamin D status was assessed by serum 25(OH)D3 determination. The data for food sources of vitamin D was assessed using an FFQ. Fish composition data was obtained from the Open Platform for Clinical Nutrition (OPEN) database. A mean of 10.01μg/100 g was used for fatty fish and a mean of 1.49 μg/100 g was used for lean fish (35); for eggs, 2.4 μg/1 egg (63.8 g) was used (20). In the fortification model, egg composition data from Browning et al. (20) was used. Eggs were produced using feed containing 250 μg/kg of cholecalciferol and 69 μg/kg of 25(OH)D3. The mean egg weight was 63.8 g and the feed conversion-to-egg ratio was 2.27±0.693) (kg feed/kg egg). For the production of one enriched egg, 144.82 g of feed was used. This amount contained 36.21 μg cholecalciferol and 9.99 μg of 25(OH)D3. Conversion from feed to eggs was 14.32% for cholecalciferol and 13.54% for 25(OH)D3. There were no detrimental effects on animal health or production parameters (20). For the economic analysis, the prices of vitamin D3 and feed grade 25(OH)D3 were provided by DSM Nutritional Products. The milk and yoghurt can be fortified by direct addition during manufacture (16). Milk and yoghurt do not differ significantly in nutrient content and method of vitamin D addition.

We built three models: a no-fortification model, Model 1 (only egg fortification used) and Model 2 (combination of egg fortification and milk and yoghurt fortification). Fortification model input data is presented in Table 1. For the fortification model calculations, vitamin D contents for unenriched milk, yoghurt and eggs were substituted with enriched food vitamin D content. All participants were included in the building of both fortification models even if they did not consume milk, yoghurt and/or eggs.

The variables observed in the analysis were: 1) serum 25(OH)D3 vitamin D (dependent variable); 2) taking (frequency and quantity) of dietary supplements/ medicines with vitamin D; 3) survey questionnaire (eating habits – frequency and quantity, vitamin D content, sun exposure); and 4) body mass index (BMI).

Serum 25(OH)D3 levels were stratified using Endocrine Society cut-off values: Deficiency: <50 nmol/L, Insufficiency: 50–75 nmol/L and target concentration for optimal vitamin D effects 75–125 nmol/L (36, 37).

The data has a normal distribution. The differences between users and non-users of supplements were examined using a two-sample t-test. The statistical analysis was conducted using MS Excel and SPSS, version 27 (IBM, 2020).

The study protocol was approved by the Slovenian National Medical Ethics Committee, identification no KME 0120-68/2019/9 (approval letter ID 0120-68/2019/9, date of approval: 22. March 2019). The study was performed in compliance with the requirements of local authorities. All subjects signed a written informed consent form (ICF) before participating in the study.

Mean vitamin D intake from food without fortification was 2.19±1.34 μg/d. With fortification Model 1, it would be6.49±4.45 μg/d and with Model 2 it would be 10.53±6.49 μg/d. Without fortification, none (0%) of the participants would reach a daily vitamin D intake >10 μg. With fortification Model 1 (egg fortification), 15.3% would reach >10 μg and with Model 2 (egg and milk fortification) 46.2% would reach >10 μg. Vitamin D supplementation was not included.

Figure 3 shows vitamin D intake without fortification, with fortified eggs, and fortified eggs in combination with fortified milk (including yoghurt).

Figure 3

Figure 4 presents the annual cost of fortification compared with vitamin D-containing prescription drugs and food supplements. The annual cost of 10 μg vitamin D/d/person is shown in Table 3.

Figure 4

Food supplements are an effective strategy for preventing vitamin D deficiency on an individual level. However, there are significant limitations. Supplements have a higher risk of excessive intake than food fortification (16, 44). Typically, they do not reach people at the highest risk of deficiency, as supplement intake is positively associated with a healthier lifestyle and higher socioeconomic status (16).

Achieving 25(OH)D3 levels >50 nmol/L in the entire population by fortification is unrealistic and unsafe, as it would mean a very high median intake. The food fortification goal set out in the World Health Organization (WHO) guidelines is ‘to provide most (97.5%) of individuals in the population group(s) at greatest risk of deficiency with an adequate intake of specific micronutrients, without causing a risk of excessive intakes in this or other groups. Inadequate intake is thus defined as intake below Estimated Average Requirements (EAR) and not the recommended intake (RI) value (45). Therefore, based on the WHO guidelines (45), the target intake should be at least EAR (46), which is 10 μg (16). The RI of vitamin D to reach serum 25(OH)D3 levels >50 nmol/L for people over one year of age is set at 20 μg (800 IU) by DACH (45, 46, 47), at 15 μg (600 IU) by the European Food Safety Agency (EFSA) and the Institute of Medicine (IOM) (46, 47), and at 25 μg (1000 IU) by the Endocrine Society (36, 37, 47). It is important to note that supplementation with 10 μg of vitamin D/day is not enough to achieve vitamin D sufficiency (49). As in the Finnish case, food fortification effectively prevented 25(OH)D levels <30 nmol/L, but 13.7% of non-users of supplements still did not achieve levels >50 nmol/L. Importantly, it did not cause a decline in vitamin D supplementation, as supplementation with vitamin D increased almost fourfold (13).

The EFSA report concluded that a long-term daily dose of 250 μg (10,000 IU) is considered to reflect a no observed adverse effect level (NOAEL). Using a safety factor, the upper tolerable intake level (UL) was established at 100 μg (4,000 IU) per day (48). In our fortification models, none of the participants would exceed >30 μg/day even with the maximum modelled fortification.

The effect of the fortification can be difficult to predict as serum 25(OH)D3 levels do not increase proportionally; in populations with lower baseline concentrations of serum 25(OH)D3, there is a higher increase than in populations with higher baseline levels. In Finland, mean serum levels of 65 nmol/L 25(OH)D3 were achieved by systematic vitamin D food fortification and with guidelines suggesting supplementation. This was sufficient to decrease the prevalence of 25(OH)D3 concentrations <30 nmol/L below 1% (15).

Our study is the first to implement fortification modelling to calculate the effects of egg biofortification on vitamin D intake. Previous studies have been concerned only with egg fortification (19–25). The main limitation of this study is that it is based on data about egg vitamin D content from a single study (20), as other studies lacked 25(OH)D3 data (20–26). Before implementation, additional studies on the transfer of vitamin D from feed to eggs that would include 25(OH)D3 would need to be conducted.

Additionally, before biofortified eggs can be put on the EU market, feed legislation will need to be changed to allow higher vitamin D levels (29), as current legislation allows for vitamin D content in eggs to a maximum of 4.54±1.38 μg of vitamin D/egg (16). Currently, such eggs would be illegal in the EU. Since changes were made in 2006 to vitamin D legislation concerning the addition of vitamins and minerals in 2006 (9), feed legislation could also see changes in the future, especially if research provides the basis for doing so.

Enrolment in the study was offered to all women coming for preventive medical examinations; however, those who chose to enrol in this voluntary study had significantly higher education and lower BMI than the general population (50). Additionally, socio-economic factors, such as language barrier, access to transport and flexibility of working hours, influenced withdrawal from the study.

These fortification models are simple and could be readily applied to any country with significant egg intake. In countries with very high egg intake, such as Mexico (>350 eggs/capita/year) or Japan (>325 eggs/capita/year), egg fortification would be viable even with lower (51) legal levels of fortification (16).

The models could also be applied to already-collected data from representative populations older than one year to confirm suitability for the entire population (31). Additionally, simultaneous fortification of eggs with several other nutrients, e.g. iodine, would also be possible (52).