Ph.D Patrick Borel
email: patrick.borel@univ-amu.fr

D Caillaud

N J Cano

Vitamin D bioavailability: State of the art

Keywords: cholecalciferol, ergocalciferol, 25-hydroxyvitamin D, bioaccessibility, absorption, intestine

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Introduction

Vitamin D is the generic name for compounds exhibiting the biological activity of cholecalciferol (vitamin D 3 ). Cholecalciferol is the main dietary source of vitamin D and it is present mostly in foods of animal origin. A significant fraction of vitamin D 3 (estimated at around 80%, but depending on sun exposure) is produced endogenously in the skin from 7dehydrocholesterol by the action of UV light. Diet can also contain 25-hydroxy cholecalciferol (25OHD 3 ) and trace amounts of dihydroxy cholecalciferol (1,25(OH) 2 D 3 ). It is assumed that most people need dietary vitamin D to reach the recommended serum level, i.e. greater than 30 ng/mL (75 nmol/L) [START_REF] Wimalawansa | Vitamin D in the New Millennium[END_REF]. The chemical structures of these vitamin D forms are recalled in Figure 1. Very few naturally-occurring foods contain vitamin D. The flesh of fatty fish and fish liver oils are among the best sources. Small amounts of vitamin D are found in beef liver, dairy products and egg yolk. Vitamin D in these foods occurs primarily as vitamin D 3 and its metabolite 25OHD 3 . Some mushrooms provide vitamin D 2 in variable amounts. In some groups, e.g. infants, elderly persons not sufficiently exposed to sunlight, and patients suffering from fat malabsorption, consumption of vitamin D supplements or vitamin D fortified foods are required to meet the daily need, i.e. approximately 2000 IU/day to maintain serum 25(OH)D levels greater than 30 ng/mL [START_REF] Wimalawansa | Vitamin D in the New Millennium[END_REF], for vitamin D.

In supplements and fortified foods, vitamin D can be available as either vitamin D 2 or vitamin D 3 . Vitamin D 2 (ergocalciferol) is manufactured by the UV irradiation of ergosterol in yeast, and vitamin D 3 by the irradiation of 7-dehydrocholesterol from lanolin and the chemical conversion of cholesterol. The mechanisms involved in vitamin D absorption and the factors assumed to affect this process have been under-researched. Vitamin D is assumed to follow the fate of lipids in the human upper gastrointestinal tract. It is also assumed that it is secreted in the chylomicrons and then transported to the liver. The absorption efficiency of vitamin D is lower than that of triacylglycerols, and varies widely. Some factors suspected of affecting its absorption efficiency have been extensively studied, while there is scant or even no data on others. After a review on the fate of vitamin D in the upper human gastrointestinal tract and on recent discoveries concerning mechanisms involved in its absorption, factors known or thought to affect its bioavailability, which is acknowledged as the fraction of ingested vitamin D that is recovered in the blood during the postprandial period after absorption of a known dose of vitamin D, are presented.

Fate of vitamin D in the human upper gastrointestinal tract

The fate of vitamin D in the human upper gastrointestinal tract consists of several sequential steps including physicochemical and enzymatic events. It is assumed that vitamin D absorption is governed by the same intraluminal factors as those that have been described for major lipids (triacylglycerols, cholesterol and phospholipids) [START_REF] Tso | The absorption and transport of lipids by the small intestine[END_REF]. These include emulsification, solubilisation in mixed micelles, diffusion across the unstirred water layer and permeation through the enterocyte membrane.

Vitamin D metabolism may begin in the stomach, where foods are subjected to acid conditions and gastric enzymes. There is apparently no data on the sensitivity of the main dietary forms of vitamin D (cholecalciferol, ergocalciferol, 25OHD 3 ) to the acidic pH of the stomach. It can be hypothesised that pepsin plays a role in vitamin D absorption by releasing the fraction of vitamin D associated with proteins. Because vitamin D is fat-soluble it can also be hypothesised that a fraction of dietary vitamin D not incorporated in oils, e.g. vitamin D in fish flesh, transfers to the fat phase of the meal during digestion. This might improve vitamin D bioavailability, assuming that vitamin D in oil is more bioaccessible than vitamin D in a complex matrix. It is reasonable to suggest that the transfer efficiency depends on the characteristics of the food matrix and on the presence of fat, but no data is available on this topic. Finally, there is no data on the role of gastric lipase on vitamin D ester hydrolysis, but it can be suggested that vitamin D esters [START_REF] Hollis | Effect of age on the intestinal absorption of vitamin D3-palmitate and nonesterified vitamin D2 in the term human infant[END_REF] are at least partially hydrolysed by this lipase. This could be of importance when vitamin D is ingested as vitamin D esters, and when pancreatic lipase activity is not optimal (e.g. neonates and pancreatic insufficiency).

In the duodenum, digestive enzymes, e.g. proteases, amylases and lipases, continue to release vitamin D from the food matrix. It is assumed that vitamin D not incorporated in oils, i.e. present naturally in dietary fat or transferred from a non-food matrix to the fat phase of the meal in the stomach, is transferred from the food matrix to dietary fat and then into micelles, but it is not known whether there is a direct transfer of vitamin D from the food matrix into micelles. In the duodenum, it is assumed that vitamin D esters, if present, are hydrolysed by bile salt stimulated lipase [START_REF] Lombardo | Studies on the substrate specificity of a carboxyl ester hydrolase from human pancreatic juice. II. Action on cholesterol esters and lipidsoluble vitamin esters[END_REF], also called carboxyl ester lipase, but it is not known whether other hydrolytic enzymes, such as pancreatic lipase (Mathias et al., 1981a), pancreatic lipase related proteins (Reboul et al., 2006a), or enzymes of the brush border (Mathias et al., 1981b) are also involved in their hydrolysis. It is assumed that vitamin D 3 is solubilised in mixed micelles [START_REF] Rautureau | Aqueous solubilisation of vitamin D3 in normal man[END_REF] and absorbed by the lymphatic route, while 25OHD 3 , being more polar, is less dependent on bile acids, and being water-soluble, is mainly absorbed via the portal route [START_REF] Maislos | Intestinal absorption of vitamin D sterols: differential absorption into lymph and portal blood in the rat[END_REF]. This has been confirmed by an Israeli team who studied the effect of absence of bile salts on the absorption of non-hydroxylated and hydroxylated vitamin D forms. Their results showed that 1,25(OH) 2 D 3 absorption was unaffected by the lack of biliary salts. Conversely, absorption of 25OHD 3 was decreased and that of vitamin D 3 was fully impaired [START_REF] Maislos | Bile salt deficiency and the absorption of vitamin D metabolites. In vivo study in the rat[END_REF].

Fat-soluble entities can be theoretically solubilised in structures other than micelles in the gut.

More precisely, as vesicles have been observed in the human duodenal lumen during digestion [START_REF] Staggers | Physical-chemical behavior of dietary and biliary lipids during intestinal digestion and absorption. 1. Phase behavior and aggregation states of model lipid systems patterned after aqueous duodenal contents of healthy adult human beings[END_REF], it is hypothesised that they can incorporate fat-soluble substances in their phospholipid membranes [START_REF] Urano | Membrane Stabilization of Vitamin E; Interactions of a-Tocopherol with Phospholipids in bilayer liposomes[END_REF][START_REF] Kirilenko | Fat soluble vitamins in liposomes: studies on incorporation efficiency and bile salt induced vesicle disintegration[END_REF]. However, it is not known whether a significant proportion of the different forms of vitamin D is incorporated in vesicles, and whether this has any effect on their absorption.

The main site of vitamin D absorption in humans is not accurately known, but in the rat, uptake was observed in both jejunum and ileum [START_REF] Hollander | Vitamin D-3 intestinal absorption in vivo: influence of fatty acids, bile salts, and perfusate pH on absorption[END_REF]Reboul et al., 2011), the ileum being the main site of absorption (Reboul et al., 2011). Absorption efficiency of vitamin D 3 given in peanut oil was found to range between 55 % and 99 % (mean 78%) in healthy subjects [START_REF] Thompson | Absorption of vitamin D3-3 H in control subjects and patients with intestinal malabsorption[END_REF]. These rates are similar to those from animal experiments, where rates between 66% and 75% were reported. Vitamin D is detected in the bile. However, the significance of a conservative enterohepatic circulation of vitamin D is controversial. Figure 2 summarises current knowledge on the fate of vitamin D in the human upper gastrointestinal tract. In addition, although the metabolism of vitamin D lies outside the scope of this review, we need to know how vitamin D metabolism modifies the forms of vitamin D that circulate in the blood; a figure is therefore presented that summarises the current knowledge of vitamin D metabolism in the human body (Figure 3).

Absorption mechanisms of vitamin D

From early studies using everted rat gut sacs [START_REF] Hollander | Mechanism and site of small intestinal uptake of vitamin D3 in pharmacological concentrations[END_REF] and perfusion of rat small intestinal loops [START_REF] Hollander | Vitamin D-3 intestinal absorption in vivo: influence of fatty acids, bile salts, and perfusate pH on absorption[END_REF], the mechanism of absorption of the non-hydroxylated forms of vitamin D, i.e. cholecalciferol and ergocalciferol, had been assumed to be a non-saturable passive diffusion process. However, recent results using the human intestinal cell line Caco-2 and HEK transfected cells have shown that intestinal cell membrane proteins are involved in the uptake of these non-hydroxylated forms at the apical side of the enterocyte (Reboul et al., 2011). These proteins are SR-BI (Scavenger Receptor class B type 1), CD36 (Cluster Determinant 36) and NPC1L1 (Niemann-Pick C1-Like 1), also involved in apical uptake of cholesterol and of other lipidic micronutrients: vitamin E (Reboul et al., 2006b;[START_REF] Narushima | Niemann-pick C1-like 1 mediates alpha-tocopherol transport[END_REF] and carotenoids [START_REF] Reboul | Lutein transport by Caco-2 TC-7 cells occurs partly by a facilitated process involving the scavenger receptor class B type I (SR-BI)[END_REF][START_REF] Van Bennekum | Class B scavenger receptor-mediated intestinal absorption of dietary beta-carotene and cholesterol[END_REF][START_REF] Moussa | Lycopene absorption in human intestinal cells and in mice involves scavenger receptor class B type I but not Niemann-Pick C1-like 1[END_REF]. The results of Reboul et al. (Reboul et al., 2011) also suggest that vitamin D uptake is mainly protein-mediated at low, dietary concentrations of vitamin D, while it is also passive at high, pharmacological concentrations. Finally, the fact that cholecalciferol uptake was higher in the rat jejunum than in the duodenum suggests that another transporter specifically expressed in the jejunum might play an important role in vitamin D uptake (Reboul et al., 2011). There is no data concerning absorption mechanisms of the hydroxylated forms of vitamin D. However, it can be hypothesised that the mechanisms are different, because these entities (25OHD 3 and 1,25(OH) 2 D 3 ) are less dependent on bile acids for absorption, and their absorption efficiency is considerably higher, about three times that of the non-hydroxylated forms [START_REF] Compston | Comparison of the appearance of radiolabelled vitamin D3 and 25-hydroxy-vitamin D3 in the[END_REF]. Figure 4 shows the current knowledge on proteins involved in absorption of vitamin D by human intestinal cells.

Possible factors affecting absorption

Before it can be absorbed, vitamin D has to be released from the food matrix, e.g. fish oil, fish flesh, or mushrooms, in which it is embedded, and be presented to the brushborder in a state such that it can be absorbed by intestinal cells. It is assumed that absorption efficiency depends on an array of variables including: (i) the food matrix, (ii) the composition of the meal, (iii) the activities of digestive enzymes, and (iv) the efficiency of transport across the enterocyte. The mnemonic "SLAMENGHI", listing the factors thought to govern carotenoid absorption (de Pee and West, 1996), was used to review the factors suspected to affect vitamin D absorption. Each letter stands for a factor: Species (molecular forms) of vitamin D, molecular Linkage of vitamin D (e.g. esterification), Amount of vitamin D consumed in a meal, Matrix in which vitamin D is incorporated, Effectors of absorption and bioconversion, Nutrient status of the host, Genetic factors, Host-related factors, and mathematical Interactions. Each SLAMENGHI factor was reviewed by querying the on-line US National Library of Medicine (last review January 31, 2012). It appeared that some factors had been extensively studied, e.g. the effect of ageing or the effect of diseases that lead to fat malabsorption, while there is no data on other factors, e.g. effect of gender or genetic factors.

Results from the available literature are presented here.

a. Species of vitamin D

Although cholecalciferol is the main dietary form of vitamin D, foods can also contain ergocalciferol and 25OHD 3 . Several studies have compared the bioavailabilities of these two vitamin D species with that of vitamin D 3 .

 Ergocalciferol vs. cholecalciferol: In a first study, bioavailability was evaluated by administering single doses of 50,000 IU of either cholecalciferol or ergocalciferol to 20 healthy male subjects. The two forms produced similar initial rises in serum 25OHD over the first 3 days, suggesting equivalent intestinal absorption [START_REF] Armas | Vitamin D2 is much less effective than vitamin D3 in humans[END_REF]. In a second study [START_REF] Holick | Vitamin D-2 is as effective as vitamin D-3 in maintaining circulating concentrations of 25-hydroxyvitamin D[END_REF], healthy subjects received either placebo, or 1000 IU vitamin D 3 , or 1000 IU vitamin D 2 , or a mixture of 500 IU vitamin D 3 and 500 IU vitamin D 2 . 25OHD increased similarly in all the vitamin D supplemented groups, again suggesting similar bioavailabilities of the two forms. A consistent result was obtained recently when vitamin D 2 and vitamin D 3 , incorporated in orange juice or in a supplement, gave similar serum 25OHD responses [START_REF] Biancuzzo | Fortification of orange juice with vitamin D(2) or vitamin D(3) is as effective as an oral supplment in maintaining vitamin D status in adults[END_REF]. However, two clinical studies [START_REF] Romagnoli | Short and long-term variations in serum calciotropic hormones after a single very large dose of ergocalciferol (vitamin D-2) or cholecalciferol (vitamin D-3) in the elderly[END_REF]Khazai et al., 2009a) suggested a higher bioavailability of vitamin D 3 than of vitamin D 2 . However, there was a confounding variable in the study of Khazai et al. (Khazai et al., 2009a): vitamin D 3 was in a powder-based supplement while vitamin D 2 was in oil. Furthermore, the subjects had cystic fibrosis, and the effect of the vehicle in which vitamin D is solubilised can be more critical, especially when it is oil, in subjects with fat malabsorption than in healthy subjects.

The study of [START_REF] Romagnoli | Short and long-term variations in serum calciotropic hormones after a single very large dose of ergocalciferol (vitamin D-2) or cholecalciferol (vitamin D-3) in the elderly[END_REF] found that vitamin D 3 was apparently almost twice as potent as vitamin D 2 in increasing serum 25OHD after 60 d. However, as discussed by the authors themselves, the 25OHD increased in parallel, suggesting a comparative absorption. Thus the difference in 25OHD levels after 60 d was attributed to a more rapid metabolism/clearance of vitamin D 2 than of vitamin D 3 metabolites (see below).

The best way to avoid the effect of liver and kidney metabolism on blood vitamin D concentration is to compare absorption in cultures of human intestinal cell. This was recently done (Reboul et al., 2011) and it was shown that vitamin D 3 and vitamin D 2 were absorbed with similar efficiencies by Caco-2 cells.

The biological equivalence between vitamin D 3 and vitamin D 2 is still a matter of debate [START_REF] Armas | Vitamin D2 is much less effective than vitamin D3 in humans[END_REF]Khazai et al., 2009b;[START_REF] Binkley | Evaluation of ergocalciferol or cholecalciferol dosing, 1,600 IU daily or 50,000 IU monthly in older adults[END_REF][START_REF] Youssef | Differences in outcomes between cholecalciferol and ergocalciferol supplementation in veterans with inflammatory bowel disease[END_REF].

Thus the fact that absorption efficiencies of vitamin D 3 and vitamin D 2 are apparently similar contrasts with the finding in the clinical study by [START_REF] Armas | Vitamin D2 is much less effective than vitamin D3 in humans[END_REF]) that serum 25OHD continued to rise in the vitamin D 3 -treated subjects, peaking at 14 d, while 25OHD fell rapidly in the vitamin D 2 -treated subjects and was not different from baseline at 14 d.

Consequently, area under the curve (AUC) to day 28 was 151 nmol.d/L for vitamin D 2 and 512 for vitamin D 3 . It was concluded that vitamin D 2 potency was less than one third that of vitamin D 3 . This conclusion is supported by three studies from another team who showed that treatment with vitamins D 2 and D 3 in the same doses produced considerably different serum concentrations of vitamin D metabolites (Tjellesen et al., 1985b;[START_REF] Tjellesen | Serum concentration of vitamin D metabolites during treatment with vitamin D2 and D3 in normal premenopausal women[END_REF], and not the same action of the two vitamins on bone metabolism (Tjellesen et al., 1985a), and also by the results of a study with very large doses of vitamin D 2 and D 3 given to elderly subjects [START_REF] Romagnoli | Short and long-term variations in serum calciotropic hormones after a single very large dose of ergocalciferol (vitamin D-2) or cholecalciferol (vitamin D-3) in the elderly[END_REF]. The mechanisms explaining the different biological effects of vitamin D 2 and D 3 are apparently not linked to their first hydroxylation by the liver, as similar increases in serum 25OHD levels were observed after intravenous injection of either vitamin D 2 or D 3 . Other mechanisms have been suggested, e.g. different affinities of 1,25(OH) 2 D 2 and 1,25OHD 3 for the VDR nuclear receptor, but these hypotheses remain to be addressed in dedicated studies.

 25OHD 3 vs. cholecalciferol and ergocalciferol: The relative bioavailability of vitamin D 3 and 25OHD 3 is well-documented. In healthy subjects, the total amount of [3H]vitamin D 3 recovered in plasma 6 h after [3H]25OHD 3 intake was considerably higher after [3H]25OHD 3 than after [3H]cholecalciferol intake. Conversely, a much higher percentage of [3H]vitamin D 3 was found in the chylomicron fraction after ingestion of [3H]vitamin D 3 than after ingestion of [3H]25OHD 3 [START_REF] Compston | Comparison of the appearance of radiolabelled vitamin D3 and 25-hydroxy-vitamin D3 in the[END_REF]. This apparent discrepancy was explained by the fact that vitamin D 3 absorption occurs via chylomicrons, while that of 25OHD 3 occurs via the portal route. However it is also possible that a significant fraction of newly absorbed cholecalciferol and ergocalciferol is quickly stored in adipose tissue leading to lower blood concentrations of these forms as compared to 25OHD 3 . The higher bioavailability of 25OHD 3 than of vitamin D 2 and D 3 was confirmed in another study in which ten times more vitamin D, either as ergocalciferol or as cholecalciferol, than 25OHD 3 was required to produce equivalent plasma 25OHD 3 concentrations [START_REF] Stamp | Comparison of oral 25hydroxycholecalciferol, vitamin D, and ultraviolet light as determinants of circulating 25-hydroxyvitamin D[END_REF]. As patients suffering from diseases that lead to fat malabsorption also malabsorb vitamin D [START_REF] Lo | Vitamin D absorption in healthy subjects and in patients with intestinal malabsorption syndromes[END_REF][START_REF] Heubi | Bone disease in chronic childhood cholestasis. I. Vitamin D absorption and metabolism[END_REF][START_REF] Lark | Diminished and erratic absorption of ergocalciferol in adult cystic fibrosis patients[END_REF], several studies were dedicated to assessing whether 25OHD 3 could be used to improve vitamin D absorption in these patients: the results were positive. The absorption of cholecalciferol and 25OHD 3 was compared in normal subjects and in patients with mild and severe cholestatic liver disease. Results showed that absorption of 25OHD 3 was greater than that of cholecalciferol in all three groups [START_REF] Sitrin | Intestinal absorption of cholecalciferol and 25hydroxycholecalciferol in chronic cholestatic liver disease[END_REF]. In another study in patients with Crohn's disease and resections of the small bowel, it was also observed that 25OHD 3 was better absorbed than cholecalciferol [START_REF] Leichtmann | Intestinal absorption of cholecalciferol and 25-hydroxycholecalciferol in patients with both Crohn's disease and intestinal resection[END_REF].

In conclusion, 25OHD is better absorbed than the non-hydroxylated forms cholecalciferol and ergocalciferol. These forms are apparently absorbed with the same efficiency, but metabolism and biological activities of the hydroxylated metabolites of vitamin D 2 and D 3 are apparently different, and lead to an apparent lower biological activity of vitamin D 2 compared with vitamin D 3 .

b. Molecular Linkage

Dietary vitamin D is non-esterified, but because some supplements may contain vitamin D esters [START_REF] Hollis | Effect of age on the intestinal absorption of vitamin D3-palmitate and nonesterified vitamin D2 in the term human infant[END_REF], the question arises of whether esterification affects vitamin D bioavailability. Only one study is dedicated to comparing the relative absorption efficiency of free vitamin D and vitamin D esters [START_REF] Hollis | Effect of age on the intestinal absorption of vitamin D3-palmitate and nonesterified vitamin D2 in the term human infant[END_REF]. In this study, performed at various postnatal ages, the abilities of vitamin D 3 -palmitate and non-esterified vitamin D 2 to elevate circulating vitamin D 3 and vitamin D 2 , respectively, were compared. It was concluded that the two forms approached equivalence when the gastrointestinal tract was mature.

Conversely, vitamin D 3 -palmitate bioavailability was lower below age 10 days, probably due to the lack of maturation of the digestive tract. This confirms the need for an efficient digestion of lipids to efficiently absorb vitamin D, in particular vitamin D esters.

c. Amount consumed in a meal

It is generally assumed that like other vitamins, the absorption efficiency of vitamin D decreases with increasing dose. The effect of the dose of vitamin D on its absorption efficiency was studied in the rat. In both the jejunum and ileum, a linear relationship was found between the absorption rate of the vitamin and its intraluminal concentration [START_REF] Hollander | Vitamin D-3 intestinal absorption in vivo: influence of fatty acids, bile salts, and perfusate pH on absorption[END_REF]. This suggests that at least in the range of the studied concentrations, vitamin D absorption efficiency does not significantly decrease with dose. This result is supported by the result of a clinical study in which 116 subjects were placed in nine treatment groups that ingested vitamin D 3 (25, 250 or 1250 µg/d for 8 weeks), 25OHD 3 (10, 20 or 50 µg/d for 4 weeks) or 1,25(OH) 2 D 3 (0.5, 1.0 or 2.0 µg/d for 2 weeks). Results showed that serum levels of all three forms increased linearly with the dietary dose, showing a similar efficiency of absorption, at least in the range of doses studied [START_REF] Barger-Lux | Vitamin D and its major metabolites: serum levels after graded oral dosing in healthy men[END_REF]. A recent study, performed in the human intestinal cell line Caco-2, has shown that at low concentrations of vitamin D 3 in micelles, i.e. between 0.1 and 2-3 µmole/L, higher than the physiological concentration in the human duodenum, calculated to be about 0.01 µmole/L (Reboul et al., 2011), the uptake of both vitamin D 3 and D 2 was saturable, while it was linear at high pharmacological concentrations, i.e. up to 10 µmole/L. This result supports the involvement of intestinal proteins in vitamin D absorption at dietary concentrations and a passive diffusion that becomes preponderant at pharmacological concentrations.

d. Matrix in which vitamin D is incorporated

This factor is thought to be important because it is assumed that vitamin D needs to be extracted from its food or supplement matrix to become bioaccessible, i.e. to become solubilised in micelles and available for absorption.

Vitamin D bioavailability in foods was measured in some foods only, i.e. meat, mushrooms, orange juice, milk and fortified cheese. This was done in different studies and it is therefore difficult to compare the values obtained. In a clinical study, the relative bioavailability of vitamin D 2 from pig meat sources was estimated to be about 60% compared with a vitamin D 2 supplement (van den [START_REF] Van Den Berg | Bioavailability of vitamin D[END_REF]. In two studies, performed in the rat, it was concluded that vitamin D 2 from UV-irradiated mushrooms (Agaricus bisporus or Lentinula edodes) was bioavailable [START_REF] Jasinghe | Bioavailability of vitamin D2 from irradiated mushrooms: an in vivo study[END_REF][START_REF] Koyyalamudi | Vitamin D2 formation and bioavailability from Agaricus bisporus button mushrooms treated with ultraviolet irradiation[END_REF], but there was no comparison of the bioavailability with any other matrix. The bioavailability of vitamin D 2 from mushrooms (Cantharellus tubaeformis) was confirmed in a clinical study involving 27 volunteers. The volunteers were randomly divided up into three groups of nine persons.

For 3 weeks, group 1 received mushrooms providing 14 µg ergocalciferol/d with their lunch, group 2 received an ergocalciferol supplement providing 14 µg/d, and group 3 received no supplementation. Serum 25OHD concentrations at 3 weeks were higher in groups 1 and 2 than in group 3, and did not significantly differ between groups 1 and 2, suggesting that the vitamin D 2 in mushrooms had the same bioavailability as the vitamin D 2 in supplement [START_REF] Outila | Bioavailability of vitamin D from wild edible mushrooms (Cantharellus tubaeformis) as measured with a human bioassay[END_REF]. A recent controlled trial confirmed this finding, showing that vitamin D 2 from UV-Birradiated button mushrooms had the same bioavailability as that of a vitamin D 2 supplement [START_REF] Urbain | Bioavailability of vitamin D from UV-B-irradiated button mushrooms in healthy adults deficient in serum 25-hydroxyvitamin D: a randomized controlled trial[END_REF]. A clinical study found similar bioavailabilities of 25000 UI vitamin D 2 ingested with either milk (whole or skimmed) or 0.1 mL corn oil on toast [START_REF] Tangpricha | Fortification of orange juice with vitamin D: a novel approach for enhancing vitamin D nutritional health[END_REF], suggesting no effect of milk nutrients on vitamin D bioavailability. Four years ago, a clinical study compared the bioavailability of vitamin D 3 from fortified wheat bread, fortified rye bread and a supplement [START_REF] Natri | Bread fortified with cholecalciferol increases the serum 25-hydroxyvitamin D concentration in women as effectively as a cholecalciferol supplement[END_REF]. Both fortified breads increased serum 25OHD as effectively as the supplement. It was concluded that bread matrix did not significantly affect vitamin D bioavailability. The bioavailability of vitamin D in bread was confirmed recently in a study that showed that vitamin D2-rich yeast baked into bread was bioavailable and improved bone quality in vitamin D-deficient rats [START_REF] Hohman | Bioavailability and efficacy of vitamin D2 from UV-irradiated yeast in growing, vitamin D-deficient rats[END_REF]. Recently, a clinical study involving 80 subjects was dedicated to comparing the bioavailability of vitamin D 3 between fortified cheeses and a supplement (liquid vitamin D 3 ). It was concluded (from measurement of blood 25OHD) that vitamin D 3 was equally bioavailable from both sources [START_REF] Wagner | The bioavailability of vitamin D from fortified cheeses and supplements is equivalent in adults[END_REF]. Finally, a recent study showed that vitamins D2 and D3 were equally bioavailable in orange juice and in supplement [START_REF] Biancuzzo | Fortification of orange juice with vitamin D(2) or vitamin D(3) is as effective as an oral supplment in maintaining vitamin D status in adults[END_REF].

Concerning vitamin D bioavailability in supplements, the effect of the vehicle substance used in vitamin D supplements on vitamin D bioavailability was recently reviewed (Grossmann and Tangpricha, 2010). It is concluded that although limited studies are available, vitamin D in an oil vehicle is more bioavailable than when incorporated in a powder-based vehicle (cellulose or lactose) or in ethanol. However, a recent clinical trial has concluded that vitamin D is more bioavailable from a lactose capsule than from an oily drop formulation [START_REF] Coelho | Bioavailability of vitamin D3 in non-oily capsules: the role of formulated compounds and implications for intermittent replacement[END_REF].

In summary, most data suggest that food matrix has no marked effect on vitamin D bioavailability. The effect of supplement matrix is less clear.

e. Effectors of absorption

A very recent study found that taking vitamin D supplement with the largest meal of the day improved vitamin D absorption (Mulligan and Licata). This suggests that one or more components of the meal, or gut enzymes secreted after meal intake, improve vitamin D absorption. However, another study did not confirm this finding, showing no significant difference in blood 25OHD levels between subjects consuming a vitamin D supplement with or without food [START_REF] Wagner | The bioavailability of vitamin D from fortified cheeses and supplements is equivalent in adults[END_REF]. As foods contain a multitude of substances (nutrients, micronutrients, fibre, phytochemicals, etc.) it is likely that the different results of these two studies were due to the different composition of the meals. To evaluate whether food components can affect vitamin D bioavailability, it is necessary to perform dedicated experiments to study the effect of each food component. Some studies presented here have used this approach.

I. Dietary lipids

Lipids are assumed to improve absorption of fat-soluble microconstituents (FSM) by several mechanisms. First, they can facilitate the release of FSM from food matrices by providing a hydrophobic phase where FSM can be solubilised. Second, because lipids stimulate biliary secretion, and consequently micelle production, they can increase the proportion of micellarised FSM, i.e. of FSM available for absorption. Third, lipid digestion products, e.g. fatty acids, monoglycerides and lysophospholipids, are micelle components and so the more lipids digested, the more micelles are available to solubilise FSM [START_REF] Hofmann | The function of bile salts in fat absorption. The solvent properties of dilute micellar solutions of conjugated bile salts[END_REF]. Finally, by inducing chylomicron synthesis, lipids can enhance FSM transport outside the enterocytes and thus prevent the accumulation of vitamin D in enterocytes. This in turn will theoretically increase FSM absorption. Several characteristics of dietary lipids are thought to affect FSM absorption: (i) the amount of triacylglycerols ingested with vitamin D, (ii) the species of fatty acids that constitute triacylglycerols (iii) the amounts of phospholipids, d) the species of phospholipids, and (iv) the emulsification of lipids. However, data on the effect of only two of these characteristics is available.



Amount of fat (triacylglycerols)

Surprisingly, adding 2.5 mM fatty acids of varying chain length and degree of saturation resulted in a decrease in the rate of vitamin D 3 absorption in rats [START_REF] Hollander | Vitamin D-3 intestinal absorption in vivo: influence of fatty acids, bile salts, and perfusate pH on absorption[END_REF]. This suggests that conversely to what is observed for other fat-soluble vitamins

(vitamin E and β-carotene), fat may diminish vitamin D absorption. However, four clinical studies did not find any significant effect of fat on vitamin D bioavailability. The first one, mainly designed to compare the bioavailability of vitamin D in two beverages (orange juice and milk), showed that the fat content of milk did not significantly affect vitamin D 2 bioavailability in 18 adults [START_REF] Tangpricha | Fortification of orange juice with vitamin D: a novel approach for enhancing vitamin D nutritional health[END_REF]. The second study did not find any difference between vitamin D 3 fortified cheddar cheese (~33% fat) and vitamin D 3 fortified low-fat cheese (~7% fat) in their ability to increase blood 25OHD [START_REF] Wagner | The bioavailability of vitamin D from fortified cheeses and supplements is equivalent in adults[END_REF]. The third study found no difference between vitamin D in multivitamin tablets and vitamin D in fish oil in their ability to increase serum 25OHD [START_REF] Holvik | A randomised comparison of increase in serum 25-hydroxyvitamin D concentration after 4 weeks o daily oral intake of 10 mu g cholecalciferol from multivitamin tablets or fish oil capsules in healthy young adults[END_REF]. In the fourth trial, consumption of vitamin D with 2 g fish oil once weekly did not improve vitamin D absorption [START_REF] Korkor | Effect of fish oil on vitamin D absorption[END_REF]. We note that in a recent clinical study [START_REF] Raimundo | Effect of High-versus Low-Fat Meal on Serum 25-Hydroxyvitamin D Levels after a Single Oral Dose of Vitamin D: A Single-Blind, Parallel, Randomized Trial[END_REF], it was concluded that a high-fat meal (25.6 g fat/meal) increased vitamin D absorption compared with a low fat meal (1.7 g). However, a close look at the results obtained reveals that there was no effect of either the low fat or high fat meals, which contained 50000 UI vitamin D 3 , on serum 25OHD 7 days after intake. Furthermore, it was not a crossover study and the authors acknowledged that some of the subjects may have had secondary hyperparathyroidism. Thus the available data suggests that the amount of fat ingested with vitamin D has no major effect on vitamin D bioavailability.



Type of fatty acid (within triacylglycerols)

The pioneering work on this topic was performed by Hollander's team [START_REF] Hollander | Vitamin D-3 intestinal absorption in vivo: influence of fatty acids, bile salts, and perfusate pH on absorption[END_REF]. It was shown in rat perfusate intestine that addition of fatty acids of varying chain length and degrees of saturation, i.e. butyric, octanoic, oleic and linoleic, resulted in a decrease in the rate of vitamin D 3 absorption. More precisely, the inhibitory effects of the oleic and linoleic acids were higher than that of octanoic acid in the distal part of the intestine.

The authors suggested that unlike medium-chain fatty acids, which are not incorporated in micelles, long-chain fatty acids hinder vitamin D absorption by causing enlargement of the micellar size, thereby slowing their diffusion towards the enterocyte. However, this result was not confirmed in a clinical trial: absorption of a pharmacological dose of vitamin D 3 was compared when ingested with either medium-chain triacylglycerols or long-chain triacylglycerols (peanut oil). Results showed that the serum levels of vitamin D 3 were significantly higher after administration in peanut oil (long-chain triglycerides) than after administration in medium-chain triglycerides. [START_REF] Holmberg | Absorption of a pharmacological dose of vitamin D3 from two different lipid vehicles in man: comparison of peanut oil and a medium chain triglyceride[END_REF]. Concerning the comparison between different types of fatty acids, a recent clinical study has concluded that diets rich in monounsaturated fatty acids may improve the effectiveness of vitamin D 3 supplements in healthy older adults, while those rich in polyunsaturated fatty acids may reduce it [START_REF] Niramitmahapanya | Type of dietary fat is associated with the 25-hydroxyvitamin D3 increment in response to vitamin D supplementation[END_REF].

Taken together, these results suggest that the type of fatty acid can affect vitamin D bioavailability. However, further studies are required to draw firm conclusions on the compared effects of each type of fatty acid on vitamin D bioavailability.

II. Dietary Fibre

Dietary fibre has been suspected of affecting FSM absorption by several mechanisms:

(i) by affecting micelle formation; (ii) by altering emulsification and triacylglycerol lipolysis [START_REF] Pasquier | Emulsification and lipolysis of triacylglycerols are altered by viscous soluble dietary fibres in acidic gastric medium in vitro[END_REF] and thus affecting the release of FSM embedded in fat droplets, and

(iii) by increasing the viscosity of the chyme and thus limiting the diffusion of FSM containing micelles to the brush border. Compston suggested that the high dietary fibre intake of Asian immigrants caused vitamin D bioavailability to diminish, thus explaining the higher incidence of rickets and osteomalacia in this population [START_REF] Compston | Rickets in Asian immigrants[END_REF]. However, there are only two clinical studies on the effect of fibre on vitamin D absorption. The first study compared the rate of plasma disappearance of radiolabelled 25OHD in healthy volunteers on either normal or high-fibre diets (20 g/d). The authors observed that the mean plasma half-life of 3H-25OHD 3 in the high-fibre group was 19.2 ± 1.7 d, significantly shorter than in the group on normal diet (27.5 ± 2.1 d). They suggested that fibre enhanced elimination of 25OHD by an action in the intestinal lumen [START_REF] Batchelor | Reduced plasma half-life of radio-labelled 25hydroxyvitamin D3 in subjects receiving a high-fibre diet[END_REF]. However, a recent clinical study did not find any significant differences between vitamin D fortified low-fiber wheat bread (3 g/100 g) and high-fiber rye bread (12 g/100 g) consumed for 3 weeks, in their ability to increase blood 25OHD in 41 healthy subjects [START_REF] Natri | Bread fortified with cholecalciferol increases the serum 25-hydroxyvitamin D concentration in women as effectively as a cholecalciferol supplement[END_REF]. However, the authors acknowledged that there was no significant difference in daily total fiber intake among the two groups of subjects, because they were allowed to eat other breads. In conclusion, there is too little data to conclude on the effect of this factor on vitamin D bioavailability.

III. Inhibitors of lipid absorption

Since obesity is a major health problem, several antiobesity drugs have been proposed

to diminish the absorption of triacylglycerols and cholesterol. Because of the closely similar fate of lipids and vitamin D in the gastrointestinal lumen, it has been hypothesised that these drugs can also impair vitamin D absorption. It has been shown that tetrahydrolipstatin (orlistat), a non-absorbed inhibitor of gastric and pancreatic lipases, and olestra, a sucrose polyester used as a fat substitute, reduce absorption of vitamin D [START_REF] Schlagheck | Olestra's effect on vitamins D and E in humans can be offset by increasing dietary levels of these vitamins[END_REF][START_REF] Mcduffie | Effects of orlistat on fat-soluble vitamins in obese adolescents[END_REF]. Phytosterols, the plant sterols used to diminish cholesterol absorption, might also impair vitamin D absorption by impairing its solubilisation in mixed micelles. This hypothesis was supported both by results obtained in rats that showed a decrease in both blood and liver vitamin D levels after dietary levels of stanol esters for 13 wks [START_REF] Turnbull | 13-week oral toxicity study with stanol esters in rats[END_REF], and by recent results obtained in vitro and in mice that showed that phytosterols compete with vitamin D 3 for incorporation into mixed micelles as well as for apical uptake [START_REF] Reboul | Vitamin D intestinal absorption is not a simple passive diffusion: Evidences for involvement of cholesterol transporters[END_REF]. Surprisingly, phytosterols were claimed to have no effect on vitamin D status in different clinical trials [32,33]. However, in all these studies, vitamin D status was evaluated as plasma 25OHD, which is highly variable depending on factors such as season and sun exposure. As a consequence, 25OHD levels can display a high variability between the beginning and end of the study or between the different groups [START_REF] Gylling | The effect of a very high daily plant stanol ester intake on serum lipids, carotenoids, and fat-soluble vitamins[END_REF]. In addition, the intervention periods may have been too short to reveal a significant effect on vitamin D homeostasis. Finally, we note that a close look at one of these studies reveals a significant lowering effect of a sitostanol ester enriched margarine on vitamin D plasma level after a long-term treatment [START_REF] Gylling | Retinol, vitamin D, carotenes and alpha-tocopherol in serum of a moderately hypercholesterolemic population consuming sitostanol ester margarine[END_REF]. We note that the negative effect of inhibitors of lipid absorption on absorption of fat soluble vitamins can be offset by adding vitamin supplements to the diet. This was demonstrated in a clinical study in 102 subjects in whom serum concentrations of vitamin E and 25OHD 2 were restored to control concentrations by adding 2.1 mg d-α-tocopheryl acetate and 0.06 µg ergocalciferol per gram of olestra, respectively, to the diet [START_REF] Schlagheck | Olestra's effect on vitamins D and E in humans can be offset by increasing dietary levels of these vitamins[END_REF].

IV. Microconstituents

As the main dietary source of vitamin D, i.e. cholecalciferol, can be ingested together with other vitamin D species (vitamin D 2 , vitamin D esters, etc.) and other lipid micronutrients (other fat-soluble vitamins, carotenoids and phytosterols), and because common mechanisms of absorption are involved (Reboul and Borel, 2011), it has been hypothesised that interactions can occur that affect their absorption (Reboul and Borel, 2011).

A study performed in our laboratory in Caco-2 cells supports this hypothesis by showing that high concentrations of vitamin D 2 significantly impair cholecalciferol absorption (Reboul et al., 2011). However, this competition should be re-assessed in vivo, as conflicting results have been published: in a first clinical study, oral administration of 400 IU/d of vitamin D 2 for 1 week led to very small variations of plasma vitamin D 3 and its metabolites [START_REF] Matsuoka | Changes in the concentrations of vitamin D and its metabolites in the plasma of healthy subjects orally given physiological doses of vitamin D2 by multivitamin or vitamin D preparations[END_REF]. Furthermore, in a recent study, a mixture of 500 IU of vitamin D 3 and 500 IU of vitamin D 2 led to a similar rise in 25OHD as 1000 IU of vitamin D 3 [START_REF] Holick | Vitamin D-2 is as effective as vitamin D-3 in maintaining circulating concentrations of 25-hydroxyvitamin D[END_REF], suggesting no inhibition of D 3 absorption by D 2 at relatively low doses. Conversely, in another study, vitamin D 2 treatment (4000 IU/d for 8 weeks) decreased blood 25OHD 3 and 1,25(OH) 2 D 3 [START_REF] Tjellesen | Serum concentration of vitamin D metabolites during treatment with vitamin D2 and D3 in normal premenopausal women[END_REF]. Finally, a recent study in cows has shown that vitamin D 3 , given as an oral bolus of 250 mg (1.0 x 10 7 IU) after vitamin D 2 , is less efficient in increasing the plasma status of 25OHD 3 than the same dose of vitamin D 3 given without previous vitamin D 2 administration [START_REF] Hymoller | Vitamin D impairs utilization of vitamin D in highyielding dairy cows in a cross-over supplementation regimen[END_REF]. Thus high doses of vitamin D 2 supplements might decrease vitamin D 3 absorption, but available data are in different species and therefore further studies are needed to definitely conclude on the effect of high doses vitamin D 2 on vitamin D 3 absorption in humans.

V. Milk and milk-derived products

The bioavailability of vitamin D naturally present in milk has never been measured, probably owing to the low concentration of the vitamin in it (about 25 IU of vitamin D/L human milk). By contrast, studies were performed to measure bioavailability of vitamin D in fortified milk or cheeses. In a first study in 18 subjects, it was shown that milk, either whole or skimmed, did not significantly modify vitamin D 2 bioavailability compared with its intake without milk [START_REF] Tangpricha | Fortification of orange juice with vitamin D: a novel approach for enhancing vitamin D nutritional health[END_REF]. In a second study in young and older adults, it was observed that vitamin D bioavailability was higher when incorporated in cheese than when solubilised in water [START_REF] Johnson | Bioavailability of vitamin D from fortified process cheese and effects on vitamin D status in the elderly[END_REF]. In a third study, dedicated to comparing vitamin D bioavailability between fortified cheeses and supplement, it was concluded that vitamin D was equally absorbed between fortified cheese and water [START_REF] Wagner | The bioavailability of vitamin D from fortified cheeses and supplements is equivalent in adults[END_REF]. In conclusion, it appears that milk or cheese do not significantly affect vitamin D bioavailability.

VI. Absorption enhancers

Some substances, e.g. β-cyclodextrine, nanoparticles and compounds able to form micelles, are used to improve absorption efficiency of liposoluble drugs or fat-soluble vitamins. Their ability to enhance absorption efficiency of vitamin D was assessed in some studies. The first absorption enhancer studied was a water-soluble form of vitamin E (tocopheryl succinate polyethylene glycol 1000 (TPGS). This substance was a good candidate for improving vitamin D absorption as it is able to form micelles that can solubilise lipophilic compounds including vitamin D. This was assessed in a clinical study with eight children (aged 5 mo to 19 y) suffering from severe chronic cholestasis. Results showed a significantly higher absorption of vitamin D administered in a mixture with TPGS [START_REF] Argao | d-Alpha-tocopheryl polyethylene glycol-1000 succinate enhances the absorption of vitamin D in chronic cholestatic liver disease of infancy and childhood[END_REF]. The ability of β-cyclodextrine to enhance absorption of vitamin D was studied in rats. Results

showed a 2.5-fold increase in absorption compared with vitamin D alone [START_REF] Szejtli | Improvement of the absorption of 3H-Cholecalciferol by formation of its cyclodextrin complex[END_REF].

f. Nutrient status of the host

As vitamin D has numerous biological effects, and as large amounts of vitamin D can be toxic, it can be hypothesised that as observed for vitamin A [START_REF] Lobo | ISX is a retinoic acid-sensitive gatekeeper that controls intestinal beta,beta-carotene absorption and vitamin A production[END_REF], vitamin D absorption is regulated by vitamin D status. Surprisingly, only one study in rats has been dedicated to testing this hypothesis. In this study it was observed that the intestinal absorption of vitamin D 3 and its hydroxylation in the liver were higher in vitamin D-deficient rats than in vitamin D-treated ones [START_REF] Apukhovskaia | Effect of varying vitamin D status in the body on intestinal absorption and metabolism of vitamin D in the rat liver[END_REF]. This very interesting finding requires support from further experiments.

g. Genetic factors

A line of results has suggested that genetic variants play a key role in modulating 25OHD blood concentrations [START_REF] Orton | Evidence for genetic regulation of vitamin D status in twins with multiple sclerosis[END_REF]Fu et al., 2009;[START_REF] Sinotte | Genetic polymorphisms of the vitamin D binding protein and plasma concentrations of 25hydroxyvitamin D in premenopausal women[END_REF][START_REF] Ahn | Genome-wide association study of circulating vitamin D levels[END_REF][START_REF] Karohl | Heritability and seasonal variability of vitamin D concentrations in male twins[END_REF][START_REF] Mcgrath | A systematic review of the association between common single nucleotide polymorphisms and 25hydroxyvitamin D concentrations[END_REF]. The fact that proteins are involved in cellular uptake of vitamin D raises the question of the impact of modulations in the expression or activity of these proteins on blood and tissue concentrations of vitamin D. The expression and activity of proteins can be modulated by several factors, including genetic ones. Genetic variations in or near genes that encode proteins may affect protein expression, e.g. a genetic variation that affects the binding of a transcription factor in the promoter region of a gene [START_REF] Hernandez-Romano | Polymorphisms in gene regulatory regions and their role in the physiopathology of complex disease in the post-genomic era[END_REF], or protein activity, e.g. a genetic variation that leads to a functional modification in the amino acid sequence of the protein, and in turn the ability of these proteins to accurately perform their function in the metabolism [START_REF] Lindqvist | Loss-of-function mutation in carotenoid 15,15'-monooxygenase identified in a patient with hypercarotenemia and hypovitaminosis A[END_REF][START_REF] Borel | Genetic variations involved in interindividual variability in carotenoid status[END_REF]. However, no result has been published on this exciting topic.

h. Host-related factors

A line of evidence suggests that host-related factors (mainly age and diseases) can have an effect on vitamin D absorption. Thus several studies have been performed to assess their effect in order to optimise RDA as a function of age and disease.

I. Age

Ageing, by affecting numerous physiological processes, may directly or indirectly affect vitamin D absorption. More precisely, age-related alterations of gastrointestinal tract functions [START_REF] Vellas | Effects of aging process on digestive functions[END_REF][START_REF] Russell | Changes in gastrointestinal function attributed to aging[END_REF][START_REF] Ikuma | Effects of aging on the microclimate pH of the rat jejunum[END_REF] may modify the efficiency of vitamin D absorption, as suggested for vitamin E [START_REF] Borel | Postprandial chylomicron and plasma vitamin E responses in healthy older subjects compared with younger ones[END_REF]. As most elderly people have a low vitamin D status, it has been hypothesised that absorption efficiency of vitamin D is less efficient in elderly persons than in young adults.

The first study dedicated to testing this hypothesis was performed in 20 elderly women, most of whom were vitamin D-depleted. Because the plasma [3H]cholecalciferol response after oral ingestion was significantly lower than that of a group of younger female subjects, it was suggested that there was a defect in intestinal absorption of cholecalciferol in the elderly [START_REF] Barragry | Intestinal cholecalciferol absorption in the elderly and in younger adults[END_REF]. However, subsequent studies on this topic did not confirm this finding: a study in rats pointed to a normal absorption of vitamin D 3 in ageing animals [START_REF] Hollander | Influence of aging on vitamin D absorption and unstirred water layer dimensions in the rat[END_REF]. In another study, serum vitamin D 2 concentrations were compared after oral administration of 50,000 IU of vitamin D 2 in both healthy vitamin Dsufficient elderly subjects and young adults. Again, no evidence of malabsorption of vitamin D in the elderly subjects was observed [START_REF] Clemens | Serum vitamin D2 and vitamin D3 metabolite concentrations and absorption of vitamin D2 in elderly subjects[END_REF]. In a third study, involving four young (23 to 50 yr) and four older (72 to 84 yr) subjects, peak serum 25OHD and 25OHD

areas under the curve were not significantly different between the younger and older adults [START_REF] Johnson | Bioavailability of vitamin D from fortified process cheese and effects on vitamin D status in the elderly[END_REF]. Finally, a study by Harris and coworkers involving 25 young men (18 to 35 yr) and 25 older men (62 to 79 yr) supplemented with 800 IU of vitamin D 3 per day [START_REF] Harris | Plasma vitamin D and 25OHD responses of young and old men to supplementation with vitamin D3[END_REF], found that the magnitude of blood 25OHD increase was identical between the two age groups. In conclusion, there is apparently no significant effect of ageing on vitamin D absorption efficiency. The frequent lower vitamin D status in elderly than in young subjects may be due to lower dietary intakes, lower exposure to sunlight, lower efficiency of vitamin D synthesis in the skin or lower hydroxylation of vitamin D by the liver.

II. Diseases/digestive tube surgery

The intestinal absorption of vitamin D requires normal digestive functions, and so subjects with impaired fat absorption caused by any of several diseases (obstructive jaundice, pancreatic insufficiency, cystic fibrosis or adult coeliac disease) have been suspected of impairing vitamin D absorption. This is supported by the results of several studies: serum vitamin D 2 and D 3 were undetectable in infants and children (age 4-22 mo) with extrahepatic biliary atresia, whose portoenterostomy failed to produce bile flow, despite supplements of 2500-5000 IU/day [START_REF] Heubi | Bone disease in chronic childhood cholestasis. II. Better absorption of 25-OH vitamin D than vitamin D in extrahepatic biliary atresia[END_REF]. Also, serum vitamin D 2 concentrations were undetectable despite oral supplementation with 2,500 to 50,000 IU per day of vitamin D 2 in children with cholestasis since infancy [START_REF] Heubi | Bone disease in chronic childhood cholestasis. I. Vitamin D absorption and metabolism[END_REF]. Finally, cystic fibrosis patients absorbed less than one third (Farraye et al.), or one half [START_REF] Lark | Diminished and erratic absorption of ergocalciferol in adult cystic fibrosis patients[END_REF] the amount of oral vitamin D 2 that was absorbed by healthy subjects. However, we note that the negative effect of these diseases on vitamin D bioavailability can be partially corrected by sun exposure (Robberecht et al.) or by using hydroxylated vitamin D. Patients with intestinal fat malabsorption syndromes have a relatively well-preserved absorption of 25OHD 3 , which is absorbed directly via the portal vein [START_REF] Leichtmann | Intestinal absorption of cholecalciferol and 25-hydroxycholecalciferol in patients with both Crohn's disease and intestinal resection[END_REF].

The effect of gastric surgery on vitamin D bioavailability has been studied by Aarts et al. [START_REF] Aarts | Vitamin D absorption: consequences of gastric bypass surgery[END_REF]. This team showed that peak serum cholecalciferol levels were about 30% lower after Roux-en-Y gastric bypass than before.

III. Obesity

As obesity is usually associated with vitamin D insufficiency, a study was dedicated to assessing the effect of obesity on the cutaneous production of vitamin D 3 and on vitamin D bioavailability, evaluated by the blood response in vitamin D after a vitamin D-rich meal.

Obese and matched lean control subjects received either whole-body ultraviolet radiation or a pharmacologic dose of vitamin D 2 orally. Results showed that body mass index was inversely correlated with serum vitamin D 3 concentrations after irradiation, and with peak serum vitamin D 2 concentrations after vitamin D 2 intake. It was concluded that obese subjects had lower blood concentrations of vitamin D (coming either from the skin or the food) because of its deposition in the body fat compartment [START_REF] Wortsman | Decreased bioavailability of vitamin D in obesity[END_REF], and not because of lower skin synthesis or bioavailability. This was supported by a very recent study suggesting that dilution of ingested or cutaneously synthesised vitamin D in the large fat mass of obese patients explains their typically low vitamin D status (Drincic et al., 2012).

i. Mathematical Interactions

These are the interactions that can occur when several factors are involved [START_REF] West | Quantification of the ''SLAMENGHI'' factors for carotenoid bioavailability and bioconversion[END_REF]. One recent study (Khazai et al., 2009a) shows a good example of interactions between factors: in this clinical study, bioavailability of vitamin D 3 was better than that of vitamin D 2 , but this was probably not due to any difference in absorption efficiency of the two forms of vitamin D (see chapter on the S factor), but rather to an effect of the vehicle in which vitamin D was incorporated (M factor), an effect probably exacerbated in patients with fat malabsorption (H factor). [START_REF] Gylling | Retinol, vitamin D, carotenes and alpha-tocopherol in serum of a moderately hypercholesterolemic population consuming sitostanol ester margarine[END_REF]. Cholesterol reduction by different plant stanol mixtures and with variable fat intake. Metabolism. 48: 575-580.

Conclusions

FiguresFigure 1

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Figure 3 29 Figure 4

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