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        25-Hydroxyvitamin D3 affects vitamin D status similar to vitamin D3 in pigs – but the meat produced has a lower content of vitamin D
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        25-Hydroxyvitamin D3 affects vitamin D status similar to vitamin D3 in pigs – but the meat produced has a lower content of vitamin D
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Abstract

In food databases, the specific contents of vitamin D3 and 25-hydroxyvitamin D3 in food have been implemented in the last 10 years. No consensus has yet been established on the relative activity between the components. Therefore, the objective of the present study was to assess the relative activity of 25-hydroxyvitamin D3 compared to vitamin D3. The design was a parallel study in pigs (n 24), which from an age of 12 weeks until slaughter 11 weeks later were fed approximately 55 μg vitamin D/d, as vitamin D3, in a mixture of vitamin D3 and 25-hydroxyvitamin D3, or 25-hydroxyvitamin D3. The end-points measured were plasma 25-hydroxyvitamin D3, and in the liver and loin the content of vitamin D3 and 25-hydroxyvitamin D3. Vitamin D3 and 25-hydroxyvitamin D3 in the feed did not affect 25-hydroxyvitamin D3 in the plasma, liver or loin differently, while a significant effect was shown on vitamin D3 in the liver and loin (P < 0·001). 25-Hydroxyvitamin D3 in the plasma, liver and loin significantly correlates with the sum of vitamin D3 and 25-hydroxyvitamin D3 in the feed (P < 0·05). Therefore, 25-hydroxyvitamin D3 should be regarded as having the same activity as vitamin D3 in food databases. Sole use of 25-hydroxyvitamin D3 as a vitamin D source in pig feed will produce liver and meat with a negligible content of vitamin D3, while an increased content of vitamin D3 in the feed will produce liver and meat with increased content of both vitamin D3 and 25-hydroxyvitamin D3.

Vitamin D deficiency increases the risk of bone fracture due to osteoporosis and decreases muscle strength. Recent investigations show a relationship between vitamin D deficiency and other afflictions such as cancer, reduced immune defence and CVD13. During the summer period, the primary source of vitamin D for man exposed to sunlight is the metabolism of 7-dehydrocholesterol to pre-vitamin D3 in the skin by UV B radiation (290–315 nm), whereas vitamin D in food is the secondary source. In winter oral intake of vitamin D may be the primary source, as absorption through the skin is limited at latitudes above 35°, e.g. for 4 months in Boston, USA (42°N) and for 6 months in Bergen, Norway (61°N)4. Similarly, oral intake of vitamin D is the primary source all year round for people not exposed to sunlight due to confinement indoors or clothing.

Estimation of dietary intake of vitamin D is essential for investigating the influence of vitamin D on health parameters in a population as well as in human intervention studies. Such calculations are based on dietary intake data from dietary surveys combined with the content of nutrients available in food composition tables. Until 10–15 years ago vitamin D data in food composition tables was mainly derived from biological assays, which used the ability of vitamin D to cure rickets in vitamin D-deficient rats5, 6. For the last 10 years, food composition tables have included specific values for vitamin D3 (vitD3) and 25-hydroxyvitamin D3 (25OHD3)712. To calculate the total vitamin D content, the relative activity between 25OHD3 and vitD3 is required.

The studies conducted to assess this factor were performed 30–40 years ago in deficient rats, in which the estimated values were between 1·4 and 51316. However, to date, no consensus has been established17.

The aim of the present study was to investigate the relative activity between vitD3 and 25OHD3 in pigs, as a model for man. The end-points were plasma 25OHD3, and the content of vitD3 and 25OHD3 in the pork loin and liver. Vitamin Dtotal throughout the paper is defined as the sum of vitD3 and 25OHD3.

Materials and methods

Pigs

The twenty-four pigs selected for the present study were a subgroup of 3225 healthy pigs used in a feeding-trial conducted to investigate whether the productivity of the pigs was affected when vitD3 was replaced by 25OHD3. The feeding-trial was conducted at an ordinary Danish farm in stables with partially slatted floors and cover. A computer-controlled system distributed the feed to the separate double pens. Each double pen contained forty-five pigs18. The pigs were fed via a tube feeder with nipple drinks as well as drinking bowls. The pigs were raised without exposure to sunlight. The stable lighting was produced with lamps (F36W/T8/33-630; Osram, Sylvania, MA, USA).

Experimental design

The study was performed as a supplementation study and consisted of a parallel trial with three treatments of vitamin D. Vitamin D was given at equal levels as vitD3, as a mixture of vitD3 and 25OHD3, or as 25OHD3, from weaning at an age of 5 weeks to slaughter at an age of approximately 5½ months. The feedstuff used was produced by DSM Nutritional Products (Copenhagen, Denmark) and DLG (Dansk Landbrugs Grovvareselskab, Copenhagen, Denmark). Detailed information of the content of the feed is given elsewhere18.

Sampling

The amount fed to each pig was calculated per pen for each of the three periods: 6–7 weeks, 8–12 weeks, 13–23 weeks (weight of 32·5 kg, and until slaughter at approximately 100 kg). For the same periods feed was sampled under the principle of the theory of sampling, and analysed for vitD3 and 25OHD319.

The day before slaughter a blood sample was drawn, and processed to EDTA–plasma and stored at − 80°C until analysis. At slaughter the liver was sampled and the next day boneless loin with rind was separated. The loin was subsequently carefully dissected into lean meat without any subcutaneous fat (lean meat), subcutaneous fat without any lean meat (fat), and skin without any subcutaneous fat (skin). All the samples were packed in plastic bags and frozen at − 20°C until analysis, which was performed within 8 months. Before analysis the liver, lean meat and fat were slowly thawed and separately ground in a homogenizer (1094 Homogenizer; Tecator, Paris, France) for 2 min, while the skin was slowly thawed and manually cut into pieces of 10–15 mm2.

Vitamin D3 and 25-hydroxyvitamin D3 in the feed

DSM Laboratory (Basel, Switzerland) carried out analyses of vitD3 and 25OHD3 in the feed. For quantification of 25OHD3, 10 g feed was added to 500 ng d6-hydroxyvitamin D3 (synthesized by Prof. Mourino, University of Santiago di Compostela, Spain20) as the internal standard, and 60 ml water. The sample was gently swirled into a slurry and sonicated at 50°C for 10 min. The vitamins were extracted with 40 ml tert-butyl methyl ether by shaking and sonication for 5 min followed by centrifuging for 3 min at 3000 rpm. Supernatant (10 ml) was evaporated and the residue dissolved in 2 ml mobile phase for preparative HPLC (2-propanol–ethyl acetate–isooctane, 1:10:89). For clean-up 100 μl were injected into a preparative HPLC system equipped with a silica-column (Si60, 3 μm, 150 × 4·6 mm; Hypersil, Shandon Products, Runcorn, UK). The fraction of 25OHD3 and the internal standard with a retention time of 14–16 min was collected. Subsequently, the organic solvent was evaporated and dissolved in 700 μl methanol and 300 μl water. The quantitative determination was performed by injection of 90 μl into the HPLC–atmospheric pressure chemical ionization–MS equipment (Agilent 1946C LC/MSD single-quadrupole mass specific detector equipped with an atmospheric pressure chemical ionization unit; Agilent Technologies AG, Basel, Switzerland). Additionally, the HPLC system consisted of a C18 column (Aquasil C18 (Aquasil, Thermo Fisher Scientific, Waltham, MA, USA), 3 μm, 2·0 × 100 mm) and the mobile phase was a gradient of methanol–water (99·95 : 0·05).

The quantification of vitD3 in the feed was determined by using vitamin D2 (vit D2) as the internal standard according to EN1282121.

Vitamin D3 and 25-hydroxyvitamin D3 in the meat

The analytical method and the equipment used to determine vitD3 and 25OHD3 in the meat are previously described22. Minor modifications were made as 25-hydroxyvitamin D2 (Sigma-Aldrich, Buchs, Switzerland) was used as the internal standard for 25OHD3 similar to the utilization of vitD2 as the internal standard for vitD3. Briefly, the internal standards of vitD2 and 25-hydroxyvitamin D2 were added to the meat samples and saponified with ethanolic potassium hydroxide. The unsaponifiable matter was extracted with diethyl ether–petroleum ether (1 : 1). The solution was then purified on a silica solid-phase extraction column and further cleaned by preparative HPLC equipped with silica and amino columns. Analysis of the liver samples included an extra preparative HPLC procedure, which consisted of a cyano column (Luna, Cyano, 3 μm, 150 × 4·6 mm) from Phenomenex (Torrance, CA, USA), and a mobile phase of 2-propanol–n-heptane (1·5 : 98·5). The fraction of vitD2 and vitD3 co-eluted with a retention time of 5 min at a flow rate of 1 ml/min. This fraction was collected and evaporated to dryness using a gentle stream of nitrogen, and finally dissolved in methanol–acetonitrile (20 : 80). Another fraction containing 25-hydroxyvitamin D2 with retention time at 16 min as well as 25OHD3 with retention time at 21 min was collected in the same vial, evaporated and dissolved in acetonitrile–water (90 : 10). These two fractions were injected into the analytical HPLC system described earlier22.

Content of fat in the meat

Content of fat in the meat was determined by the gravimetric method following a modified Schmid–Bondzynski–Ratslaff method23. Briefly, the sample was boiled with hydrochloric acid followed by the addition of ethanol and extraction of the lipids with diethyl ether–petroleum ether (1 : 1). After evaporation of the solvent, the fat was weighed.

Plasma 25-hydroxyvitamin D

The quantification of 25OHD3 in plasma was performed by the HPLC method described previously24. Briefly, plasma proteins were precipitated with ethanol and the supernatant was cleaned by a MFC18 solid-phase extraction. The 25OHD3 in the solution was separated, detected and measured by analytical HPLC equipped with a diode array detector (220–320 nm) and a UV detector (265 nm) and external calibration.

Data analysis

Based on previously assessed variation of content of vitD3 and 25OHD3 in meat, six pigs should be included in each feeding group to detect a relative activity of 1·5 for 25OHD3 compared to vitD3 with a power of 80 % and a significance level of 5 %7.

To test the effect of the content of 25OHD3 and vitD3 in feed on 25OHD3 in plasma and on 25OHD3 and vitD3 in meat and liver, regression analysis was performed. In the regression model, 25OHD3 in plasma, meat and liver, and vitD3 in meat and liver were dependent variables, and the total content of vitamin D in feed (vitamin Dtotal) and the difference between 25OHD3 and vitD3 (vitamin Ddiff) were independent variables. Furthermore, ANOVA was performed with feed as an independent fixed variable to test and estimate differences between feeding groups. Association between determinants and variables were assessed with Pearson's correlation coefficients. Data are expressed as means and their standard errors. SAS version 9.1 (SAS Institute, Cary, NC, USA) was used for all statistical analyses, with a significance level of 0·05.

Results

Pigs performance

The vitamin D in each of the three diets was given as the same vitamin D source(s) but due to the different feed and consumption levels during the growth period, the mean daily intake differed. vitD3 and 25OHD3 in each of the three feeding periods are presented in Fig. 1. Carcass weights are presented in Table 1. No significant difference was detected between the diets or between the subgroup and the pigs included in the main feeding study (n 3225).

Fig. 1 The mean daily amount of vitamin D (Vitamin Dtotal) in each of the three diet groups (eight pigs in each) and divided into the three different age groups. Vitamin D source in the feed was either 25-hydroxyvitamin D3 (25OHD3, ) or vitamin D3 (vitD3, □). The diet groups A, B and C are vitD3, a mixture of vitD3 and 25OHD3, and 25OHD3, respectively.

Table 1 Weight of the carcass and the effect of the diet groups on 25-hydroxyvitamin D3 (25OHD3) in the serum, liver and separated parts of the loin and vitamin D3 (vitD3) in the liver and separated parts of the loin at slaughter* (Mean values with their standard errors)

a,b,cMean values within a row with unlike superscript were significantly different (P < 0·05).

* Diets A, B and C are vitD3, a mixture of vitD3 and 25OHD3, and 25OHD3, respectively.

Effect of 25-hydroxyvitamin D3 and vitamin D3 as vitamin D source

Results for vitamin D status assessed as plasma 25OHD3, and the contents of 25OHD3 and vitD3 in the liver, and in the three separated parts of the loin including the content of fat are presented in Table 1.

The results show that 25OHD3 in the plasma, in the liver and in the three separated parts of loin did not depend on the vitamin D source, but on the daily intake of vitamin Dtotal, as content of 25OHD3 in the separated cuts was significantly associated with vitamin Dtotal (R 0·42–0·56; P < 0·05).

In contrast, vitD3 in the liver and in the three separated parts of loin depended on the vitamin D source (P < 0·001), and showed no association with vitamin Dtotal. However, content of vitD3 was significantly associated with vitD3 in the feed (R 0·65–0·89; P < 0·001).

Discussion

Previous work has shown that vitD3 and 25OHD3 in meat is positively associated with the content of fat7. Therefore it is essential that comparison of meat derived from pigs fed different diets does not differ in the content of fat. No significant difference was shown for subcutaneous fat, lean meat and skin. The present results show that the intention to produce three similar separated cuts from each pig was fulfilled regarding the content of fat.

The significant effect of the feeding level of vitamin Dtotal on plasma 25OHD3 in these pigs was in line with the positive association between dietary intake of vitamin Dtotal and serum 25OHD3 shown in women and men25, 26.

The present study was originally designed to investigate whether the productivity of the pigs was affected when the vitD3 was replaced by 25OHD3 in the feed. No difference was found in the present study which included 3225 pigs18. As the twenty-four pigs selected for the present nutritional study did not differ from the whole group concerning weight, and growth rate, the mean daily intake of vitamin D calculated from the whole group is applied.

Plasma 25OHD determined as the sum of plasma 25-hydroxyvitamin D2 and plasma 25OHD3 is accepted as the biomarker for vitamin D intake in the absence of sun exposure27. The observed effect that plasma 25OHD achieves a steady state if supplemented at the same level for an adequate time period has previously been used to study differences between natural and synthetic vitD2, vitD3 in fortified bread, juice and supplement, as well as different levels of vitD3 supplement25, 2830. In human intervention studies supplementation levels of 5–10 μg/d for 4 weeks was shown to be adequate to reach a steady state for vitamin D status (J Jakobsen, unpublished results). The half-life of vitamin D in man is 1 month31. Though the differences in the rate of metabolism between pigs and man are unknown for vitamin D, the applied period of 16 weeks to reach a steady state is assumed to be adequate. As the metabolism in pigs and man regarding fat-soluble vitamins is rather similar, it is assumed that the vitamin D status and vitamin D in the liver and in the meat at slaughter was not influenced by the feed given in the earliest stage of growth up to an age of 12 weeks.

To our knowledge, this is the first study investigating the effect of 25OHD3 and vitD3 in healthy mammals.

Thirty to 40 years ago the difference between 25OHD3 and its parent vitD3 was tested in vitamin D-deficient rats either by testing the effect on intestinal calcium absorption measured by the everted gut sac technique, serum calcium and body weight, or by the ability to cure rickets. By the everted gut sac technique an equal effect of the two compounds was shown after 24 h, though 25OHD3 acted more rapidly13, 32, 33. In 1973, 25OHD3 was shown to be five times as active as vitD3 in the maintenance of serum calcium and growth16. In the ability to cure rickets 25OHD3 had an effect 1·4–2 times the activity of vitD3 in three different studies, but in another study the effect was estimated to be 5 times as active1316.

Today, the factor of 5 for the activity between 25OHD3 and vitD3 is widely used in recommended dietary allowances as well as its implementation in food composition tables1012. However, the documentation for the factor of 5 seems limited due to the non-standardized methods used34.

The data obtained for the relative activity between 25OHD3 and vitD3 in pigs need to be verified in a similar study in man, as well as further investigation of the possible difference of the effect of vitamin D derived from pork and from supplements. For mushrooms, no difference was shown between natural vitD2 and vitD2 given as a supplement in the ability to increase vitamin D status, investigated in a human intervention study28. However, the vitamin D activity in meat may not be reflected only by vitD3 and 25OHD3. The content of 1,25-dihydroxyvitamin D3 and other dihydroxyvitamin D3 compounds are unknown, but may contribute to the vitamin D activity of meat.

The effect of 25OHD3 and its parent form vitD3 on plasma 25OHD3 should be regarded equal in the diet for pigs. However, for the nutritional value of pork meat, 25OHD3 in pig feed should be regarded as rather low compared with vitD3, as the content of vitD3 depended on the vitamin D source. The use of 25OHD3 only in the feed instead of vitD3 produced meat and liver with significantly lower content of vitD3. That the pigs fed solely on 25OHD3 did not produce meat and liver with vitD3 is not surprising, as vitD3 is not synthesized in the pigs. The hydroxylation of vitD3 to 25OHD3 by 25-hydroxylase is not a reversible reaction.

Additionally, the present study shows that the concentration of vitD3 as a vitamin D source in the feed determines the concentration of vitD3 in meat and liver even at small differences in the feeding levels, which was previously shown in pigs fed super nutritional levels at 1000 μg vitD3/kg35. Additional feeding trials are necessary to investigate fully the possibility of pork meat bio-fortified with vitamin D.

Presently, a human intervention study is being conducted to evaluate whether human subjects respond to supplements of vitD3 and 25OHD3 in a similar fashion to pigs. However, more research on the relative bioactivity of vitamin Dtotal from animal products compared to supplements of vitD3 is an important issue for the calculation of dietary vitD intake.

Conclusion

The findings of the present study showed that 25OHD3 and vitD3 equally affect 25OHD3 in plasma, meat and liver. However, for the benefit of human nutrition, 25OHD3 in pig feed should be regarded as lower than vitD3, as meat and liver produced by feeding the pigs exclusively 25OHD3 had a significantly lower content of vitD3. Regardless of the vitamin D source the present study identified a dose–response effect between vitamin Dtotal in the meat and in the liver with vitamin Dtotal in the feed, which for meat and liver indicate the possibility to produce meat and liver bio-fortified with vitamin D.

Acknowledgements

The authors thank Kirsten Pinndal, Heddi Koch Jacobsen, Astrid Kvindebjerg from the National Food Institute, Technical University of Denmark for technical skills in the analysis of serum and liver, Peter Hoffman, DSM, Basel for the analysis of feed, Ole Lund Svendsen and Gilbert Weber, DSM Nutritional Product for the approval of this nutritional study attached to the original feeding trial, which were financed by DSM Nutritional Product, and Grethe Andersen, Danish Meat Association for providing finances used for the butchering of the pigs. J. J. designed the nutritional part of the feeding trial and was responsible for the analyses of vitamin D, and wrote the manuscript. H. M. designed, planned and conducted the main feeding trial. A. B. was responsible for the analyses of fat. H. M. S. and O. H. were responsible for the statistical test performed. All authors read and contributed to finalization of the manuscript. The sponsors of the study and the authors had no conflict of interests.

References

Zittermann, A (2003) Vitamin D in preventive medicine: are we ignoring the evidence? Br J Nutr 89, 552572.
Holick, MF (2005) The vitamin D epidemic and its health consequences. J Nutr 135, 2739S2748S.
Giovannucci, E, Liu, Y, Rimm, EB, et al. (2006) Prospective study of predictors of vitamin D status and cancer incidence and mortality in men. J Natl Cancer Inst 98, 451459.
Holick, MF (2004) Vitamin D: importance in the prevention of cancers, type 1 diabetes, heart disease, and osteoporosis. Am J Clin Nutr 79, 362371.
US Pharmacopoeia (1955) Method of assay for vitamin D. In US Pharmacopoeia, 14th ed. Easton, PA: Mack Publishing Co.
The Danish Pharmacopoea Commission (1964) Biological assessment of vitamin D activity. In Pharmacopoea Nordica, vol. IV, pp. 95a100 [Busck, A, editor]. Copenhagen: Nyt Nordisk Forlag.
Clausen, I, Jakobsen, J, Leth, T & Ovesen, L (2003) Vitamin D-3 and 25-hydroxyvitamin D-3 in raw and cooked pork cuts. J Food Comp Anal 16, 575585.
Mattila, P, Lehikoinen, K, Kiiskinen, T & Piironen, V (1999) Cholecalciferol and 25-hydroxycholecalciferol content of chicken egg yolk as affected by the cholecalciferol content of feed. J Agric Food Chem 47, 40894092.
Mattila, PH, Piironen, VI, Uusirauva, EJ & Koivistoinen, PE (1995) Contents of cholecalciferol, ergocalciferol, and their 25-hydroxylated metabolites in milk-products and raw meat and liver as determined by HPLC. J Agric Food Chem 43, 23942399.
Danish Food Composition Databank (2005) http://www.foodcomp.dk/fcdb_useofdata.asp..
Food Standards Agency (2000) McCance and Widdowson's The Composition of Foods. Cambridge: Royal Society of Chemistry.
National Research Council (1989) Recommended Dietary Allowances, 10th ed. Washington, DC: National Academy Press.
Blunt, JW, Tanaka, Y & Deluca, HF (1968) Biological activity of 25-hydroxycholecalciferol – a metabolite of vitamin D3. Proc Natl Acad Sci USA 61, 15031516.
Leerbeck, E (1977) Biologisk aktivitet af 25-hydroxyvitamin D3. (Biological Activity of 25-Hydroxyvitamin D3). Report no. 23. Søborg, Denmark: National Food Agency.
Miravet, L, Redel, J, Carre, M, Queille, ML & Bordier, P (1976) Biological activity of synthetic 25,26-dihydroxycholecalciferol and 24,25-dihydroxycholecalciferol in vitamin-D-deficient rats. Calcif Tiss Res 21, 145152.
Tanaka, Y, Frank, H & Deluca, HF (1973) Biological activity of 1,25-dihydroxyvitamin D3 in rat. Endocrinology 92, 417422.
Ovesen, L, Brot, C & Jakobsen, J (2003) Food contents and biological activity of 25-hydroxyvitamin D: a vitamin D metabolite to be reckoned with? Ann Nutr Metab 47, 107113.
Maribo, H, Nielsen, DH & Jakobsen, J (2007) Vitamin D3 Sources: 25-Hydroxyvitamin D3 as an Alternative to the Traditional Vitamin D3 Source. Report no. 780. Copenhagen: Dansk Svineproduktion, Danish Meat Association.
Gy, P (1998) Sampling for Analytical Purposes. New York: Wiley.
Mascarenas, JL, Perezsestelo, J, Castedo, L & Mourino, A (1991) A short, flexible route to vitamin-D metabolites and their side-chain analogs. Tetrahedron Lett 32, 28132816.
European Standard (2000) Foodstuffs – Determination of Vitamin D by High Performance Liquid Chromatography – Measurement of Cholecalciferol (D3) and Ergocalciferol (D2). EN12821. Brussels: European Committee for Standardization.
Jakobsen, J, Clausen, I, Leth, T & Ovesen, L (2004) A new method for the determination of vitamin D-3 and 25-hydroxyvitamin D-3 in meat. J Food Comp Anal 17, 777787.
Nordic Committee on Food Analysis (1989) Fat. Determination According to SBR (Schmid-Bondynzki-Ratslaff) in Meat and Meat Products. Method no. 131. Oslo: Nordisk Metodik Komitee for Næringsmidler.
Andersen, R, Mølgaard, C, Skovgaard, LT, et al. (2005) Teenage girls and elderly women living in northern Europe have low winter vitamin D status. Eur J Clin Nutr 59, 533541.
Heaney, RP, Davies, KM, Chen, TC, Holick, MF & Barger-Lux, MJ (2003) Human serum 25-hydroxycholecalciferol response to extended oral dosing with cholecalciferol. Am J Clin Nutr 77, 204210.
Viljakainen, HT, Palssa, A, Karkkainen, M, Jakobsen, J & Lamberg-Allardt, C (2006) How much vitamin D-3 do the elderly need? J Am Coll Nutr 25, 429435.
European Commission (2006) Opinion of the Scientific Committee on Food on the Tolerable Upper Intake Level of Vitamin D. Brussels: European Commission, Health and Consumer Protection Dierectorate-General.
Outila, TA, Mattila, PH, Piironen, VI & Lamberg-Allardt, CJ (1999) Bioavailability of vitamin D from wild edible mushrooms (Cantharellus tubaeformis) as measured with a human bioassay. Am J Clin Nutr 69, 9598.
Natri, AM, Salo, P, Vikstedt, T, et al. (2006) Bread fortified with cholecalciferol increases the serum 25-hydroxyvitamin D concentration in women as effectively as a cholecalciferol supplement. J Nutr 136, 123127.
Tangpricha, V, Koutkia, P, Rieke, SM, Chen, TC, Perez, AA & Holick, MF (2003) Fortification of orange juice with vitamin D: a novel approach for enhancing vitamin D nutritional health. Am J Clin Nutr 77, 14781483.
Clements, MR, Davies, M, Hayes, ME, et al. (1992) The role of 1,25-dihydroxyvitamin-D in the mechanism of acquired vitamin-D deficiency. Clin Endocrinol 37, 1727.
Martin, DL & Deluca, HF (1969) Influence of sodium on calcium transport by rat small intestine. Am J Physiol 216, 13511359.
Winter, M, Morava, E, Simon, G & Gyüre, A (1972) The effect of vitaminD3 and 25-hydroxycholecalciferol on intestinal transport of calcium in vivo and in vitro. Experientia 28, 659660.
Jakobsen, J (In the Press) Bioavailability and bioactivity of vitamin D3 active compounds – which potency should be used for 25-hydroxyvitamin D3? In Nutritional Aspects of Osteoporosis. International Congress Series [Burckhardt, P, Dawson-Hughes, B, Heaney, RP, editors]. Amsterdam: Elsevier.
Wilborn, BS, Kerth, CR, Owsley, WF, Jones, WR & Frobish, LT (2004) Improving pork quality by feeding supranutritional concentrations of vitamin D-3. J Anim Sci 82, 218224.