Romain Bott

Vitamin D intestinal absorption is not a simple passive diffusion: Evidences for involvement of cholesterol transporters

SCOPE It is assumed that vitamin D is absorbed by passive diffusion. However, since cholecalciferol (vitamin D(3) ) and cholesterol display similar structures, we hypothesized that common absorption pathways may exist. METHODS AND RESULTS Cholecalciferol apical transport was first examined in human Caco-2 and transfected Human embryonic kidney (HEK) cells. Cholecalciferol uptake was then valuated ex vivo and in vivo, using either wild-type mice, mice overexpressing Scavenger Receptor class B type I (SR-BI) at the intestinal level or mice treated or not with ezetimibe. Cholecalciferol uptake was concentration-, temperature- and direction-dependent, and was significantly impaired by a co-incubation with cholesterol or tocopherol in Caco-2 cells. Moreover Block Lipid Transport-1 (SR-BI inhibitor) and ezetimibe glucuronide (Niemann-Pick C1 Like 1 inhibitor) significantly decreased cholecalciferol transport. Transfection of HEK cells with SR-BI, Cluster Determinant 36 and Niemann-Pick C1 Like 1 significantly enhanced vitamin D uptake, which was significantly decreased by the addition of Block Lipid Transport-1, sulfo-N-succinimidyl oleate (Cluster Determinant 36 inhibitor) or ezetimibe glucuronide, respectively. Similar results were obtained in mouse intestinal explants. In vivo, cholecalciferol uptake in proximal intestinal fragments was 60% higher in mice overexpressing SR-BI than in wild-type mice (p<0.05), while ezetimibe effect remained non-significant. CONCLUSION These data show for the first time that vitamin D intestinal absorption is not passive only but involves, at least partly, some cholesterol transporters.

Interindividual variability of lutein bioavailability in healthy men: characterization, genetic variants involved, and relation with fasting plasma lutein concentration

BACKGROUND Lutein accumulates in the macula and brain, where it is assumed to play physiologic roles. The bioavailability of lutein is assumed to display a high interindividual variability that has been hypothesized to be attributable, at least partly, to genetic polymorphisms. OBJECTIVES We characterized the interindividual variability in lutein bioavailability in humans, assessed the relation between this variability and the fasting blood lutein concentration, and identified single nucleotide polymorphisms (SNPs) involved in this phenomenon. DESIGN In a randomized, 2-way crossover study, 39 healthy men consumed a meal that contained a lutein supplement or the same meal for which lutein was provided through a tomato puree. The lutein concentration was measured in plasma chylomicrons isolated at regular time intervals over 8 h postprandially. Multivariate statistical analyses were used to identify a combination of SNPs associated with the postprandial chylomicron lutein response (0-8-h area under the curve). A total of 1785 SNPs in 51 candidate genes were selected. RESULTS Postprandial chylomicron lutein responses to meals were very variable (CV of 75% and 137% for the lutein-supplement meal and the meal with tomato-sourced lutein, respectively). Postprandial chylomicron lutein responses measured after the 2 meals were positively correlated (r = 0.68, P < 0.0001) and positively correlated to the fasting plasma lutein concentration (r = 0.51, P < 0.005 for the lutein-supplement-containing meal). A significant (P = 1.9 × 10(-4)) and validated partial least-squares regression model, which included 29 SNPs in 15 genes, explained most of the variance in the postprandial chylomicron lutein response. CONCLUSIONS The ability to respond to lutein appears to be, at least in part, genetically determined. The ability is explained, in large part, by a combination of SNPs in 15 genes related to both lutein and chylomicron metabolism. Finally, our results suggest that the ability to respond to lutein and blood lutein status are related. This trial was registered at clinicaltrials.gov as NCT02100774.

Phytosterols can impair vitamin D intestinal absorption in vitro and in mice

SCOPE Adequate vitamin D status is necessary and beneficial for health, although deficiency and insufficiency are very common. As cholecalciferol (vitamin D(3) ) structure is close to cholesterol structure, we hypothesized that phytosterols, frequently used to decrease cholesterol, intestinal absorption and consequently to reduce hypercholesterolemia, may also interact with cholecalciferol absorption. METHODS AND RESULTS β-Sitosterol effect on cholecalciferol postprandial response was first assessed in mice. We then evaluated the effect of different sterols on (i) cholecalciferol micellar incorporation, (ii) cholecalciferol apical uptake and (iii) basolateral efflux in vitro or ex vivo. In mice, cholecalciferol bioavailability was 15-fold lower in the presence of β-sitosterol (p<0.05). This can partly be explained by the fact that phytosterols significantly impaired cholecalciferol incorporation into mixed micelles (from -16 to -36% depending on sterol micellar composition). This can also be due to the fact that in Caco-2 cells and mouse intestinal explants, phytosterols significantly lowered cholecalciferol apical uptake (from -13 to -39%). Conversely, phytosterols had no effect on cholecalciferol secretion at the basolateral side of Caco-2 cells. CONCLUSION The present data suggest for the first time that phytosterols can interact with vitamin D(3) intestinal absorption. This interaction can be explained by a competition for micellar incorporation and for apical uptake.

Lycopene bioavailability is associated with a combination of genetic variants.

The intake of tomatoes and tomato products, which constitute the main dietary source of the red pigment lycopene (LYC), has been associated with a reduced risk of prostate cancer and cardiovascular disease, suggesting a protective role of this carotenoid. However, LYC bioavailability displays high interindividual variability. This variability may lead to varying biological effects following LYC consumption. Based on recent results obtained with two other carotenoids, we assumed that this variability was due, at least in part, to several single nucleotide polymorphisms (SNPs) in genes involved in LYC and lipid metabolism. Thus, we aimed at identifying a combination of SNPs significantly associated with the variability in LYC bioavailability. In a postprandial study, 33 healthy male volunteers consumed a test meal containing 100g tomato puree, which provided 9.7 mg all-trans LYC. LYC concentrations were measured in plasma chylomicrons (CM) isolated at regular time intervals over 8 h postprandially. For the study 1885 SNPs in 49 candidate genes, i.e., genes assumed to play a role in LYC bioavailability, were selected. Multivariate statistical analysis (partial least squares regression) was used to identify and validate the combination of SNPs most closely associated with postprandial CM LYC response. The postprandial CM LYC response to the meal was notably variable with a CV of 70%. A significant (P=0.037) and validated partial least squares regression model, which included 28 SNPs in 16 genes, explained 72% of the variance in the postprandial CM LYC response. The postprandial CM LYC response was also positively correlated to fasting plasma LYC concentrations (r=0.37, P<0.05). The ability to respond to LYC is explained, at least partly, by a combination of 28 SNPs in 16 genes. Interindividual variability in bioavailability apparently affects the long-term blood LYC status, which could ultimately modulate the biological response following LYC supplementation.

Dihydroactinidiolide, a High Light-Induced β-Carotene Derivative that Can Regulate Gene Expression and Photoacclimation in Arabidopsis

Dear Editor, The physiological functions of carotenoids in plants go beyond their traditional roles as accessory light-harvesting pigments, natural colorants, and quenchers of triplet chlorophyll and singlet oxygen (O2). Recent studies have indeed emphasized the functional role of molecules derived from carotenoids as phytohormones (Ruyter-Spira et al., 2013) or messengers in stress signaling pathways (Havaux, 2014). In particular, chemical quenching of O2 by carotenoids within the photosystems involves oxidation of the carotenoid molecules, generating a variety of oxidized products (Ramel et al., 2012). β-Cyclocitral, a volatile C7 derivative of β-carotene, is one such molecule produced during high light stress, which was found to induce changes in the expression of O2-responsive genes (Ramel et al., 2012). Moreover, the β-cyclocitral-dependent gene reprogramming was associated with an increased tolerance of the plants to photooxidative stress. These effects appeared to be specific to β-cyclocitral since they were not observed with β-ionone, a C9-oxidized derivative of β-carotene, which was not able to induce or repress the expression of O2 gene markers. Based on those results, it was proposed that β-cyclocitral is a plastid messenger involved in the chloroplast-to-nucleus O2 signaling pathway leading to acclimation to high light stress (Ramel et al., 2012). However, in vitro O2 oxidation of β-carotene is known to produce other volatile compounds besides β-cyclocitral and β-ionone, such as dihydroactinidiolide (dhA, Figure 1A) and α-ionene (Ramel et al., 2012). The dhA molecule is a lactone (cyclic ester) resulting from the secondary oxidation of β-ionone through the intermediate 5,6-epoxy-β-ionone (Havaux, 2014). Both dhA and α-ionene were detected in plant leaves and fruits (e.g. Del Mar Caja et al., 2009; Ramel et al., 2012). Interestingly, dhA, but not α-ionene, was reported to accumulate in Arabidopsis leaves under high light stress (Ramel et al., 2012). The dhA molecule contains a carbonyl group that can react with nucleophilic structures in macromolecules, providing this compound with a high potential reactivity. Actually, dhA is known to be a bioactive molecule in animals. It is a component of pheromones in insects, such as red fire ants (Rocca et al., 1983) and in mammals such as the Cat and the Red Fox (Albone, 1975). dhA was also found to exhibit cytotoxic effects against cancer cell lines (Malek et al., 2009). In contrast, much less is known on the actions of dhA in vascular plants. Nevertheless, dhA was identified as a major component of ethyl acetate extracts of cyanobacteria or aquatic macrophytes which inhibit seed germination and seedling growth (Stevens and Merrill, 1980). This compound has also been identified in wheat glumes where it was suggested to act as a germination inhibitor (Kato et al., 2003). Figure 1B shows that dhA, quantified by GC–MS (Supplementary Data) is present in control Arabidopsis leaves at a concentration of around 3 ng g–1 fresh weight. When Arabidopsis plants were exposed to photooxidative stress conditions (high light and low temperature), dhA was found to rapidly accumulate in leaves, reaching a concentration of ~45 ng g–1 after 8 h (Figure 1B). To check the possible involvement of dhA in gene regulation, we exposed Arabidopsis plants to volatile dhA in an airtight Plexiglass chamber, using a protocole that has been described previously (Ramel et al., 2012) (Supplementary Data). Two different volumes (100 and 200 μl) of pure dhA were applied on cotton wicks placed in the closed chambers, increasing the dhA concentration in the atmosphere from 0 to 0.18 and 0.23 p.p.m., respectively, after 4 h. These treatments induced a rise in the internal dhA content of Arabidopsis leaves to levels (~40 ng g–1) in the concentration range measured in high light-treated plants (Figure 1C). In Figure 1D and 1E, we examined the effect of volatile dhA on genes whose expression is known to be affected by O2. At3g25250 (encoding the kinase OXIDATIVE SIGNAL INDUCIBLE 1), At2g33380 (encoding RESPONSIVE TO DESSICATION 20), At2g29450 (coding for a glutathione transferase GSTU5), At2g15490 (encoding the UDP-glycosyltransferase 73B4), and At3g50970 (encoding LOW TEMPERATURE INDUCED 30) (Figure 1D) were shown to be induced by O2 in the Arabidopsis flu and ch1 mutants (op den Camp et al., 2003; Ramel et al., 2013). Conversely, the genes At1g44446 (encoding CHLORINA 1) and At3g14210 (encoding EPITHIOSPECIFIER MODIFIER 1) (Figure 1E) have been reported to be noticeably repressed by O2. In this study, gene expressions

Phytosterol ester processing in the small intestine: impact on cholesterol availability for absorption and chylomicron cholesterol incorporation in healthy humans

Phytosterols (plant sterols and stanols) can lower intestinal cholesterol absorption, but the complex dynamics of the lipid digestion process in the presence of phytosterol esters (PEs) are not fully understood. We performed a clinical experiment in intubated healthy subjects to study the time course of changes in the distribution of all lipid moieties present in duodenal phases during 4 h of digestion of meals with 3.2 g PE (PE meal) or without (control meal) PE. In vitro experiments under simulated gastrointestinal conditions were also performed. The addition of PE did not alter triglyceride (TG) hydrolysis in the duodenum or subsequent chylomicron TG occurrence in the circulation. In contrast, cholesterol accumulation in the duodenum aqueous phase was markedly reduced in the presence of PE (−32%, P < 0.10). In vitro experiments confirmed that PE reduces cholesterol transfer into the aqueous phase. The addition of PE resulted in a markedly reduced presence of meal-derived hepta-deuterated cholesterol in the circulation, i.e., in chylomicrons (−43%, PE meal vs. control; P < 0.0001) and plasma (−54%, PE meal vs. control; P < 0.0001). The present data show that addition of PE to a meal does not alter TG hydrolysis but displaces cholesterol from the intestinal aqueous phase and lowers chylomicron cholesterol occurrence in humans.

Respective contributions of intestinal Niemann-Pick C1-like 1 and scavenger receptor class B type I to cholesterol and tocopherol uptake: in vivo v. in vitro studies.

The intestinal absorption of cholesterol and lipid micronutrients such as vitamin E has been shown to share some common pathways. The present study aims to further compare the uptake of cholesterol ([3H]cholesterol v. 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3-ol (NBD-cholesterol)) and tocopherol in Caco-2 TC-7 cells and in mouse intestine, with special focus on the respective roles of scavenger receptor class B type I (SR-BI) and Niemann-Pick C1-like 1 (NPC1L1). Conversely to NBD-cholesterol, the uptakes of [3H]cholesterol and tocopherol by Caco-2 cells were impaired by both block lipid transport-1 and ezetimibe, which inhibit SR-BI and NPC1L1, respectively. These inhibitions occurred only when cholesterol or tocopherol was delivered to cells included in micelles that contained biliary acid and at least oleic acid as a lipid. In vivo, after 2 h of digestion in mice, the uptake of the two cholesterol analogues and of tocopherol all showed distinct patterns along the duodenum-jejunum axis. [3H]Cholesterol uptake, which correlated closely to NPC1L1 mRNA expression in wild-type (wt) mice, was strongly inhibited by ezetimibe. Intestinal SR-BI overexpression did not change NPC1L1 expression and led to a significant increase in [3H]cholesterol uptake in the distal jejunum. Conversely, neither ezetimibe treatment nor SR-BI overexpression had an effect on NBD-cholesterol uptake. However, in contrast with SR-BI mRNA expression, tocopherol absorption increased strongly up to the distal jejunum in wt mice where it was specifically inhibited by ezetimibe, and was increased in the proximal intestine of intestinal SR-BI-overexpressing mice. Thus, cholesterol and tocopherol uptakes share common pathways in cell culture models, but display different in vivo absorption patterns associated with distinct contributions of SR-BI and NPC1L1.

A Combination of Single-Nucleotide Polymorphisms Is Associated with Interindividual Variability in Dietary β-Carotene Bioavailability in Healthy Men

BACKGROUND The bioavailability of β-carotene, the main dietary provitamin A carotenoid, varies among individuals. It is not known whether this variability can affect long-term β-carotene, and hence vitamin A, status. OBJECTIVES We hypothesized that variations in genes involved in β-carotene absorption and postprandial metabolism could at least partially explain the high interindividual variability in β-carotene bioavailability. Thus, the main objectives of this study were to identify associated single-nucleotide polymorphisms (SNPs), and to estimate whether populations with different allele frequencies at these SNPs could have different abilities to absorb provitamin A carotenoids. METHODS In this single-group design, 33 healthy, nonobese adult men were genotyped with the use of whole-genome microarrays. After an overnight fast, they consumed a test meal containing 100 g tomato puree providing 0.4 mg β-carotene. The postprandial plasma chylomicron β-carotene concentration was then measured at regular time intervals over 8 h. Partial least squares (PLS) regression was used to identify the best combination of SNPs in or near candidate genes (54 genes representing 2172 SNPs) that was associated with the postprandial chylomicron β-carotene response (incremental β-carotene area-under-the-curve concentration over 8 h in chylomicrons). RESULTS The postprandial chylomicron β-carotene response was highly variable (CV = 105%) and was positively correlated with the fasting plasma β-carotene concentration (r = 0.78; P < 0.0001). A significant (P = 6.54 × 10(-3)) multivalidated PLS regression model, which included 25 SNPs in 12 genes, explained 69% of the variance in the postprandial chylomicron β-carotene response, i.e., β-carotene bioavailability. CONCLUSIONS Interindividual variability in β-carotene bioavailability appears to be partially modulated by a combination of SNPs in 12 genes. This variability likely affects the long-term blood β-carotene status. A theoretic calculation of β-carotene bioavailability in 4 populations of the international HapMap project suggests that populations with different allele frequencies in these SNPs might exhibit a different ability to absorb dietary β-carotene. This trial was registered at clinicaltrials.gov as NCT02100774.

Impact of canning and storage on apricot carotenoids and polyphenols

Apricot polyphenols and carotenoids were monitored after industrial and domestic cooking, and after 2months of storage for industrial processing. The main apricot polyphenols were flavan-3-ols, flavan-3-ol monomers and oligomers, with an average degree of polymerization between 4.7 and 10.7 and caffeoylquinic acids. Flavonols and anthocyanins were minor phenolic compounds. Upon processing procyanidins were retained in apricot tissue. Hydroxycinnamic acids, flavan-3-ol monomers, flavonols and anthocyanins leached in the syrup. Flavonol concentrations on per-can basis were significantly increased after processing. Industrial processing effects were higher than domestic cooking probably due to higher temperature and longer duration. After 2months of storage, among polyphenols only hydroxycinnamic acids, flavan-3-ol monomers and anthocyanins were reduced. Whichever the processing method, no significant reductions of total carotenoids were observed after processing. The cis-β-carotene isomer was significantly increased after processing but with a lower extent in domestic cooking. Significant decreased in total carotenoid compounds occurred during storage.

Increased diffusivity of lycopene in hot break vs. cold break purees may be due to bioconversion of associated phospholipids rather than differential destruction of fruit tissues or cell structures

Lycopene bioaccessibility is enhanced by processing, as explained by the destructuration of plant tissues, making diffusion easier. However, in tomato, the relationship between grinding intensity and lycopene release from purees suffers from uncertainty. In particular, hot break puree exhibited twice as much diffusible lycopene as compared to cold break, while both were processed with the same grinding intensity. To explain the difference, we systematically studied the diffusivity of particles according to their size and integrity, and used microscopic and physical analyses to reveal structural differences. Neither particle size distribution, nor cell destruction, nor plastid transformation exhibited any correlation to the differences in diffusivity. However, Raman microspectroscopy combined with a chemometric analysis revealed significant changes in lycopene spectra and a putative linkage to phospholipid transformation. Phospholipid profiling of five pairs of contrasted purees revealed that, during the cold break, a transition from complex phospholipids to more simple phosphatidic acid molecules systematically occurred.