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University of Helsinki, Department of Applied Chemistry and Microbiology; * University of Helsinki, Department of Food Technology, Division of Cereal Technology; and The Danish Veterinary and Food Administration, Institute of Food Safety and Nutrition, Division of Food Chemistry
2 To whom correspondence should be addressed. E-mail: christel.lamberg-allardt{at}helsinki.fi.
ABSTRACT
Fortification of foods is a feasible way of preventing low vitamin D status. Bread could be a suitable vehicle for fortification because it is a common part of diets worldwide. The bioavailability of cholecalciferol from bread is not known. We studied cholecalciferol stability, the concentration of the added cholecalciferol, the dispersion of cholecalciferol in bread, and the bioavailability of cholecalciferol from fortified bread. Three batches of fortified low-fiber wheat and high-fiber rye breads were baked; from each batch, 3 samples of dough and bread were analyzed for their cholecalciferol content. In a single-blind bioavailability study, 41 healthy women, 25–45 y old, with mean serum 25-hydroxyvitamin D concentration 29 nmol/L (range 12–45 nmol/L), were randomly assigned to 4 study groups. Each group consumed fortified wheat bread, fortified rye bread, regular wheat bread (control), or regular wheat bread and a cholecalciferol supplement (vitamin D control) daily for 3 wk. The daily dose of vitamin D was 10 µg in all groups except the control group. The vitamin dispersed evenly in the breads and was stable. Both fortified breads increased serum 25-hydroxyvitamin D concentration as effectively as the cholecalciferol supplement. Supplementation or fortification did not affect serum intact parathyroid hormone concentration or urinary calcium excretion. In conclusion, fortified bread is a safe and feasible way to improve vitamin D nutrition.
KEY WORDS: • vitamin D • fortification • bread • bioavailability • vitamin D status
Low serum 25-hydroxyvitamin D concentrations, indicating vitamin D insufficiency are found at high latitudes all over the world in all age groups, especially during the winter months (1–9). Vitamin D is essential for skeletal health and mineralization. In addition, there is growing evidence of association between vitamin D insufficiency and chronic diseases, such as cardiovascular diseases, multiple sclerosis, diabetes, and cancer [for review, see (10)].
When sunlight exposure is scarce, oral intake of vitamin D, either dietary or supplementary, becomes essential. However, natural dietary sources of vitamin D are limited. Cholecalciferol is found in fish and egg yolk and ergocalciferol in some wild mushrooms. In many European countries and in the United States, fortification with vitamin D of certain food stuffs has been accepted as a strategy to improve the vitamin D status of the population. Milk, margarine. and breakfast cereals are commonly fortified. Recently, fortified orange juice was introduced in the United States (11).There are still people, e.g., vegetarians and persons with lactose intolerance who are not reached by natural dietary sources or the current fortification policy, and are thus in danger of poor vitamin D nutrition. Thus, we wanted to introduce a way of fortifying a low-fat food consumed widely in all population groups, namely, bread. We designed the experiment to study cholecalciferol stability, the concentration of added cholecalciferol and its dispersion in the bread, and whether the fortification of bread was technically possible in 2 kinds of commonly used breads, low-fiber wheat bread and high-fiber rye bread; we also examined the bioavailability of cholecalciferol from these breads. We wanted to study cholecalciferol in particular rather than ergocalciferol, which is not as well absorbed and might be metabolized differently (12).
SUBJECTS AND METHODS
Developing and baking of the fortified breads. The developing and baking of the fortified breads occurred in 2 phases. In the first phase, we developed 2 different kinds of recipes for fortified breads to test cholecalciferol stability, the concentration of added cholecalciferol, the dispersion of cholecalciferol in bread, and repeatability of baking. In the second phase we tested the bioavailability. We developed both low-fiber wheat bread and traditional high-fiber sour dough baked rye bread.
The cholecalciferol preparation used was a powder "Dry vitamin D3 type 100, CWS (cold water soluble)," (Roche). The vitamin is incorporated with edible fats finely dispersed in a starch-coated matrix of gelatin and sucrose. It is meant for pharmaceutical and food products. The cholecalciferol concentration of the preparation was 2500 µg/g; our goal was to produce a baked bread with a cholecalciferol concentration of 
10 µg/100 g. Three batches of both types of bread were baked in the first phase. The cholecalciferol powder was dispersed into the liquid of the dough. Samples of dough and bread from each of the 3 batches were stored at –20°C until analysis.
Bioavailability of cholecalciferol from fortified bread. Because the fortification of bread proved technically possible, the bioavailability study was conducted. Women volunteers, who presumably had low vitamin D status, were recruited from the Helsinki University campus area through advertisements. Inclusion criteria were good general health and age between 25 and 45 y. Exclusion criteria were any disease or medication known to affect calcium metabolism, a holiday in a sunny place during the past few months, regular use of vitamin D supplements, and consumption of fish more often than once a week. The study protocol was approved by the Ethics Committee for Studies in Healthy Subjects and Primary Care in the Hospital District of Helsinki and Uusimaa. The protocol was carefully explained to the subjects and their written informed consent was obtained. Eligible subjects (n = 59) were screened preexperimentally for serum 25-hydroxyvitain D (25-OHD) concentration; 44 subjects with the lowest serum 25-OHD concentrations (<58.1 nmol/L) were accepted. The habitual calcium and vitamin D intakes of the subjects were assessed by a semiquantitative FFQ before the start of the study.
The subjects were randomly assigned to 4 study groups, with 11 subjects in each group. Three subjects withdrew during the study because they could not comply with the protocol. Baseline characteristics of the 41 subjects who completed the study are shown in Table 1. The groups were given fortified wheat bread, fortified rye bread, regular wheat bread (control), and regular wheat bread and a daily cholecalciferol supplement of 10 µg (vitamin D control). In a single-blind design, each group received bread and tablets, with the first 3 groups administered placebo tablets. Vitamin D and supplement tablets were both manufactured by Scanpharm A/S. Fortified wheat and rye breads given to subjects were both baked in 3 batches at our department, and stored at –20 °C. Subjects were given the entire amount of frozen bread at the beginning of the study, and they were advised to consume it on a daily basis. The average daily portion of bread was 85 g (4 thin slices), with a mean daily cholecalciferol intake goal of 10 µg from fortified breads, the same amount as from the supplement. The subjects were advised to consume all of the bread they were given. Vitamin D intake from other dietary sources was restricted by asking the subjects not to eat fish more often than once a week, and not to drink milk fortified with vitamin D. With these exceptions, the subject consumed their habitual diets. They were also able to eat freely other breads than those provided by the researchers.
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Laboratory methods. From each of the 3 batches of the breads baked in the first phase, 3 samples of both dough (n = 9) and bread were taken for the analyses (n = 9). Similarly, from the breads given to the subjects in the bioavailability study, 2 samples of each of 3 batches of dough (n = 6) and bread were taken (n = 6). Samples were freeze-dried before analysis and stored at –20°C. Fortified cholecalciferol in dough and bread was determined using a reverse phase HPLC method published by Mattila et al. (13,14). Homogenized freeze dried sample (2 g) was weighed into a Pyrex tube, and ergocalciferol was added as an internal standard. The hot saponification procedure with KOH and the extraction step with n-hexane:ethyl acetate were carried out according to the method published by Salo-Väänänen et al. (15), except that the amounts of sample and all solutions used were doubled. The fraction containing vitamins cholecalciferol and ergocalciferol was purified with a normal phase HPLC method developed by Mattila et al. (13). Cholecalciferol was quantified using reverse phase HPLC with UV detection (264 nm) according to Mattila et al. (13) with a mobile phase containing 7% water in methanol (both HPLC grade). The analysis method, which was tested with a pooled rye bread sample on 3 separate days, had good repeatability (CV = 8%, n = 6).
Serum 25-OHD concentration in the screening samples was analyzed by an immunoradiometric method (DiaSorin). The intra- and interassay CV were <10%. To increase the comparability of the results with other studies, serum 25-OHD concentrations at the beginning and at the end of the study were analyzed at the Danish Veterinary and Food Administration, Department of the Institute of Food Safety and Nutrition, Copenhagen, Denmark, by an in-house HPLC method (Waters): straight-phase with dual wavelength detection (Jakobsen, unpublished). The method has intra- and interassay CV of 4.3 and 6.3%, respectively. Both methods are monitored by participation in the Vitamin D External Quality Assessment Scheme (DEQAS, Charing Cross Hospital). Serum intact parathyroid hormone (iPTH 1–84) was analyzed by immunoenzymometric assay (Octeia® Intact PTH, IDS). The reference range for serum iPTH was 0.8–3.9 pmol/L). The assay has an intra-assay CV of 2.3% and an interassay CV of 4.0%. Serum and urinary calcium concentrations and urinary creatinine concentration were analyzed with an automatic analyzer (Konelab 20; Thermo Clinical Labsystems Oy).
Statistical methods. The data are expressed as means ± SEM. The distribution of the variables was checked for normality. The changes in serum 25-OHD, serum iPTH, serum calcium concentration, and urinary calcium excretion in the groups during the study were compared by paired t test. The effect of supplementation on serum 25-OHD concentration was analyzed by repeated-measures ANOVA. When the treatment effect was significant, contrast analysis was used to compare the responses among the groups. The differences in other variables among the study groups were analyzed by ANOVA. Pearson correlation was applied to study the relations between initial serum 25-OHD concentration and the effect of supplementation. Analyses were made using SPSS 10.0 statistical software. Differences were considered significant at P < 0.05.
RESULTS
Fortification of bread. The mean cholecalciferol concentration of baked wheat and rye bread was 12 µg/100 g (Tables 2 and 3). Recoveries of cholecalciferol in bread samples varied between 79 and 109%, indicating that cholecalciferol was stable during baking and analysis (Tables 2 and 3). Cholecalciferol dispersed evenly in the breads (data not shown). The day-to-day repeatability of separate baking batches was also good, with CV of 9 and 12% for rye and wheat bread, respectively.
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The serum iPTH concentration did not differ among the groups at the beginning or end of the study. The changes in serum iPTH tended to differ among the groups (P = 0.05) with the concentration tending to decline in the vitamin D supplementation group (P = 0.08) (Table 4). Serum calcium concentrations decreased slightly in the fortified rye and control groups (P = 0.05) but concentrations in the 4 groups did not differ. Urinary calcium excretion did not change in any of the groups.
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DISCUSSION
Vitamin D status is largely dependent on dietary vitamin D intake at high latitudes during winter (1,16,17). In a study of healthy young subjects, 26% of the women and 29% of the men were vitamin D deficient (serum 25OHD < 25 nmol/L); based on the inverse relation between serum 25-OHD and PTH concentrations, 86% of the women (serum 25OHD < 80 nmol/L) and 56% of the men (serum 25OHD < 40 nmol/L) had inadequate vitamin D status during the winter (6).
In a recent study it was estimated that 12.5 µg vitamin D is needed from the diet daily to sustain the serum 25 -OHD concentration at 70.3 nmol/L throughout the winter (18). Thus, it is not unexpected that low serum 25-OHD concentrations were found even in people with a vitamin D intake above the recommended 5 µg/d (6,19). Unfortunately, vitamin D occurs naturally in only a few food sources. Extending the current selection of foods fortified with vitamin D might be an effective way of maintaining adequate vitamin D status during the winter months.
We fortified wheat and rye bread with a water-soluble cholecalciferol preparation. On the basis of the analyses of doughs and ready-made breads, cholecalciferol distributed evenly into the doughs and breads and was stable during the baking process.
Little is known about the bioavailability of vitamin D from food. The relative availability of ergocalciferol from the meat of pigs fed a semisynthetic feed containing ergocalciferol was only 60% compared with an ergocalciferol supplement [for review, see (20)]. In contrast, 10 µg/d of ergocalciferol from wild mushrooms increased serum 25-OHD concentrations as effectively as an ergocalciferol supplement in a semicontrolled setting over 3 wk (21). Our results agree well with that study (21). In a 12-wk, double-blind study of 27 subjects, daily ingestion of orange juice, fortified with the same cholecalciferol preparation (25 µg/d) we used in the present study, increased serum 25-OHD and decreased serum iPTH concentrations compared with the control group (11).
The results of this study showed that cholecalciferol was absorbed equally from both wheat and rye bread. This is interesting because rye bread contains more fiber (12 g/100 g) than wheat bread (3 g/100 g). This could have decreased the absorption of cholecalciferol because dietary fiber may contribute directly to vitamin D metabolism by increasing the fecal excretion of bile acids, which may combine with vitamin D and fiber (22). The addition of 20 g of dietary fiber to the habitual diet of 7 healthy adults decreased the plasma half-life of radiolabeled 25-OHD 30% compared with the control group (23). There are also epidemiological data on the association between a high fiber intake and rickets (22). However, the daily total fiber intake did not differ among the groups in the present study because the subjects were allowed to eat other breads in addition to he experimental breads, possibly explaining the lack of difference in fiber intake.
Bread is a primary food in many countries in the world, making it a good candidate for fortification. In this experimental setting, we used 10 µg/100 g bread to study the bioavailability of cholecalciferol. However, if the fortified bread were to be introduced as a commercial product, the vitamin concentration should be reduced. Our present studies of fortified wheat and rye breads showed clearly that the added cholecalciferol dispersed evenly in the bread and was stable and bioavailable. Fortified bread is a feasible way of improving vitamin D nutrition.
FOOTNOTES
1 Supported by grant no. QLK1-CT-2000-00623 from the European Commission. 
Manuscript received 7 July 2005. Initial review completed 16 August 2005. Revision accepted 7 October 2005.
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