Natural Vitamin D in Food: To What Degree Does 25‐Hydroxyvitamin D Contribute to the Vitamin D Activity in Food?

ABSTRACT Vitamin D3, vitamin D2, 25‐hydroxyvitamin D3 [25(OH)D3], and 25‐hydroxyvitamin D2 [25(OH)D2]constitute the vitamin D activity in food. In general, vitamin D activity in food depends on the food's fat content, the feed the animals have been fed, and the animal's exposure to ultraviolet B (UVB) light. There are many gaps in our knowledge of 25‐hydroxyvitamin D in food, including the amount present in different types of food, and the amount we process in our daily dietary intake. We aimed to assess the vitamin D vitamers in food (eggs, milk, dairy products, chicken, veal, beef, and pork) on the Danish market using accredited analytical methods. We then combined these data with existing Danish data, as well as with the information from the Danish Dietary Survey to estimate the dietary intake of vitamin D3 and of 25(OH)D3 by Danes. We report the level of vitamin D in 10% minced pork from free‐range pigs slaughtered in summer as 1.39 μg vitamin D3/100 g and 0.40 μg 25(OH)D3/100 g, which are significantly higher amounts (p < 0.001) than in early spring. The levels of vitamin D2 and 25(OH)D2 are usually <0.05 μg/100 g, though in beef they are up to 0.14 μg/100 g. 25(OH)D3 accounts for up to 100% in veal and 8% in fat from free‐range pigs. In the Danish diet, the share of 25(OH)D3 is 24% for children (4–17 years) and 18% for adults (18‐75 years). Changes in animal‐feeding strategy in the agriculture sector could change the share of 25(OH)D3 to 11% and 12% if extra vitamin D3 is added to the feed, and the animals are exposed to sunlight or UVB lightlight. Replacing vitamin D3 by 25(OH)D3 in the feed may result in a share of 25(OH)D3 of 52% and 40%, respectively, in children and adults. These estimates are based on the assumption that vitamin D3 and 25(OH)D3 contribute equally to the vitamin D activity. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.


Supplementary S1. Detailed information of sampling and storage of foods in subprojects
A study into the market for the food of interest was conducted prior the studies into eggs, milk, dairy products, chicken, veal and beef. Thus we identified the products on the market, the market share of each of the supermarkets, the needed sampling period i.e. is sampling through the year necessary, and we considered if single or a composite sample should be analysed. Then the detailed sampling plan were made.
-Eggs: All samples in all available sizes (S/M/L/XL) were bought in supermarkets from February 2011 to January 2012. All samples were produced in Denmark by Hedegaard or Danaeg.
-Milk and dairy products: The market share of Danish produced milk and dairy products was >90%, except soft cheese. Thus all samples bought were produced in Denmark except soft cheeses, which were imported products produced in France. In each month from August 2013 to July 2014 two samples of each product were bought in one supermarket in the Copenhagen area and in one supermarket in Funen or Jutland. The amounts of samples bought were 1 L milk and yoghurt, 250 g spreadable, 250 mL cream, and 250-500 g cheese. For each product the same amount from each samples was taken into the composite samples, the amount from each sample depended on the content of fat. Thus 10-50 g of milk, 5 g cream, spreadable, and cheese from each sample bought were put into the relevant composite sample of August to October, November to January, February to April, and May to July.
-Chicken: All samples were bought in local supermarket in the Copenhagen area in October and November. Two types of chicken were bought in COOP-supermarkets and two types of chicken in Dansk Supermarked. Two types of chicken at 1200 g was manufactured from the two main Danish producers, one type of chicken was organically farmed (1400 g), and one type of chicken was imported from France (1400 g). For each of the four types, three chickens produced at three different days were bought. The skin was carefully separated from the breast, and one of the thigh was carefully separated into bone, meat and skin. Each separated part i.e. breast without skin, thigh, thigh meat and thigh bone were weighted.
-Veal and beef: The samples were bought in local supermarket in the Copenhagen area in June and July 2018. In total 24 samples of veal, of which 75% originated from Denmark, while 25% was of unknown origin. In total 48 samples of beef, of which 42% was of Danish origin, 5% from Germany, while 1-2% were raised in each of the following countries: Argentina,

Supplementary Information
3 of 11 25-HYDROXYVITAMIN D3 IN FOOD AND DIET Australia, Belgium, Brazil, The Netherlands, Ireland, UK, Uruguay, and US, while the rest 11% was of unknown origin. Each samples of calf liver and minced chicken collected for the composite samples for the two foods, were produced in Denmark. The study into vitamin D in veal and beef, is part of a project that includes quantification of other nutrients and micronutrients, therefore approx. 2 kg of each samples were bought.
-Pork: In August 2019, the 20 samples of the shoulder with rind, were collected directly at the slaughterhouse at August, 7. Twenty free-range pigs from two farmers (10 from each) were selected, and the shoulder with rind, send to DTU Food at 5 °C. At DTU one steak of approx.
200 g was cut at the part of the shoulder closest to the loin. In March 2020 a similar procedure was agreed, but due to Covid-19, an alternative collection was chosen, which made it impossible to include samples from the same farmers. Thus the 20 samples, were from freerange pigs, but slaughtered at the same slaughterhouse. The shoulder with or without rind was ordered by Meny, Søborg, DK, which then cut the steak at approx. 200 g following the procedure used at DTU Food for the samples from August 2019. Within two days each steaks from August 2019 and March 2019 was carefully divided into lean meat (intra-muscular fat was removed), subcutaneaous fat, and skin cut free from any visible fat at our facilities at DTU Food or in the home of the technician due to Covid-19. See Figure S1. The skin was cut into 1 cm 2 pieces, and the lean meat and subcutaneous fat were homogenised.     Scenario 1 i.e. based on levels in foods today. Scenario 2 i.e. levels in foods taking into account research results for feeding vitamin D3 and sun-exposure. Scenario 3 i.e. levels in foods taking into account research results for feeding 25-hydroxyvitamin D3.