1. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes with  -cyclodextrin is not inhibited by high-density lipoproteins Elisabet Fernández-García, Irene Carvajal-Lérida, Francisco Rincón, José J. Ríos and Antonio Pérez-Gálvez* *aperez@cica.es Food Biotechnology Department Instituto de la Grasa (CSIC) Av. Padre García Tejero 4, 41012 Sevilla (SPAIN)

2. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… SIGNIFICANCE OF BIOAVAILABILITY STUDIES Interest in the screening of bioavailability has increased for different reasons 1.Existence of undernourished population 2.Epidemiological studies have associated between consumption of fruit and vegetables to a lower risk of developing degenerative diseases 3.Development of food products with added nutritional value 4.Food legislation concerning functional foods

3. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… A MULTIFACTORIAL SYSTEM EFFECTS CAROTENOID ASSIMILATION Carotenoids are fat soluble compounds 1.Liberation from food matrix 2.Incorporation to mixed micelles 3.Absorption by epithelial cells through simple/facilitated diffusion mechanisms Absorption efficiency is relatively low from fruits and vegetables 1.Fiber, kind and amount of fat, interaction among carotenoids 2.Increase of absorption efficiency from processed fruits and vegetables (homogenization and thermal processing)

4. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… Responders group non-Responders group INTER-INDIVIDUAL VARIABILITY AND THE NON-RESPONDER CONCEPT Comparison of the in vivo lutein absorption efficiency: non-responder versus lutein-responders group

5. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… AIM OF THE STUDY Estimation of the bioaccessibility of dietary carotenoids reached when they are delivered as inclusion complexes 1.Dietary carotenoids (  -carotene, lutein and lycopene) were formulated as micellar solutions (control) or inclusion complexes with  -cyclodextrin 2.BBMVs preparations were used as the in vitro model to assay carotenoid uptake from both carotenoid formulations (micellar solution or carotenoid-CyDIC) at three concentration levels 3.Comparison of absorption efficiency under inhibition conditions of membrane protein transporters (BBMVs pre- incubated with HDLs)

6. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… RESULTS Comparison of the carotenoid absorption efficiency in function of the concentration and delivering method Assimilation of  -carotene micellar inclusion complex 1.Saturation versus linear trend 2.Increase of efficiency at 2.5  M

7. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… RESULTS Comparison of the carotenoid absorption efficiency in function of the concentration and delivering method Assimilation of lycopene micellar inclusion complex 1.Saturation versus linear trend 2.Increase of efficiency at 1.0  M

8. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… RESULTS Comparison of the carotenoid absorption efficiency in function of the concentration and delivering method Assimilation of lutein micellar inclusion complex 1.Saturation versus linear trend 2.Increase of efficiency at 2.5  M 3.A lower absorption efficiency was observed versus carotenes

9. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes…  -carotene luteinlycopene Concentration Donor solution type Inhibition 2.085 1.370 720 784 579 -256 2.087 1.502 398 RESULTS Primary effects of the factors concentration, donor solution type and inhibition

10. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… SUMMARY Factors: concentration and donor solution type 1.Association between concentration and assimilation mechanism 2.Structural features (polarity) or different affinity of transporters may explain the absorption efficiency data of carotenes and lutein 3.Significant increase on efficiency of the assimilation is reached when carotenoids were delivered as inclusion complex with CyD

11. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… RESULTS Does delivery of carotenoids as inclusion complex mean an increase on absorption efficiency? Absorption rate in pmol/(mg protein x min) C. E.C. I. C.  -carotene C. E.C. I. C. lutein C. E.C. I. C. lycopene 0.5  M 1.0  M 2.5  M 32.9 65.3 70.7 20.1 36.6 106 9.85 26.9 30.1 14.2 27.8 85.9 19.1 28.9 69.4 11.7 43.9 158

12. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… SUMMARY Factors: concentration and donor solution type 1.At the lowest concentration the carotenoids from micellar solutions were more efficiently assimilated 2.At 1.0  M a heterogeneous behavior was observed 3.Only at the highest concentration, carotenoids from inclusion complex solutions were more efficiently assimilated in comparison with the carotenoid micellar solutions at that concentration (  -Car: 51%; Lut: 185%; Lyc: 128%). What absorption mechanism does apply for inclusion complexes?

13. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… RESULTS Two-stage mechanism for carotenoid assimilation from inclusion complex solutions: release and absorption Lutein inclusion complex at the donor solution Lysate of BBMVs after assimilation procedure with lutein inclusion complex at the donor solution

14. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… K complexation Assimilation: Passive or facilitated diffusion + De-complexation RESULTS Solubility of carotenoids is a rate-limiting step of absorption. Dissolution kinetics of the complex is enhanced at high concentrations and depends on binding constant of the host- guest complex

15. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… RESULTS Comparison of the carotenoid absorption efficiency in function of the concentration and delivering method with inhibitor Assimilation of  -carotene micellar inclusion complex 1.Decrease of 50% (mean value) 2.Saturation versus linear trend 3.Increase of efficiency at 0.5  M

16. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… RESULTS Comparison of the carotenoid absorption efficiency in function of the concentration and delivering method with inhibitor Assimilation of lycopene micellar inclusion complex 1.Decrease of 40% (mean value) 2.Saturation versus linear trend 3.Increase of efficiency at 0.5  M

17. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… RESULTS Comparison of the carotenoid absorption efficiency in function of the concentration and delivering method with inhibitor Assimilation of lutein micellar inclusion complex 1.Decrease of 70% (mean value) 2.Saturation versus linear trend 3.Increase of efficiency at 0.5  M

18. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes…  -carotene luteinlycopene Concentration Donor solution type Inhibition 2.085 1.370 720 784 579 -256 2.087 1.502 398 RESULTS Primary effects of the factors concentration, donor solution type and inhibition

19. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… SUMMARY Factors: concentration, donor solution type and inhibition 1.Assimilation of carotenoids from micellar solutions is significantly inhibited with the use of HDLs 2.Significant decrease of the assimilation level, (70% drop for lutein), although it did not reached 100%. Co-existence of simple diffusion mechanism and work of transporters not totally blocked under the established experimental conditions 3.Carotenes were more efficiently absorbed than lutein even under inhibition conditions. They probably take help of different protein transporters

20. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… SUMMARY Factors: concentration, donor solution type and inhibition 4.Carotenoid-CyDIC were more efficiently absorbed than the carotenoid micellar solutions under inhibition conditions. How did the factor inhibition affect the carotenoid assimilation from CyDIC?

21. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… RESULTS Comparison of the carotene-CyDIC absorption efficiency in function of the inhibition factor Assimilation of  -carotene-CyDIC no inhibition inhibition 1.Increase of efficiency from 0.5  M under inhibited transport conditions 2.Increase of 86% at 1.0  M

22. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… RESULTS Comparison of the carotene-CyDIC absorption efficiency in function of the inhibition factor Assimilation of lycopene-CyDIC no inhibition inhibition 1.Increase of efficiency from 0.5  M under inhibited transport conditions 2.Increase of 165% at 1.0  M

23. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… RESULTS Comparison of the carotene-CyDIC absorption efficiency in function of the inhibition factor

24. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… RESULTS Comparison of the carotene-CyDIC absorption efficiency in function of the inhibition factor

25. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… RESULTS Comparison of the lutein absorption efficiency in function of the inhibition factor Assimilation of lutein at 1.0  M micellar inclusion complex 1.70% drop of micellar lutein under inhibited transport conditions 2.28% drop of lutein-CyDIC under inhibited transport conditions

26. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… SUMMARY Comparison of the carotenoid-CyDIC absorption efficiency in function of the inhibition factor 1.A different effect of HDLs was observed for the assimilation efficiency of carotene-CyDICs or lutein-CyDICs 2.Process of competition between HDLs and lutein-CyDIC may not be efficient enough in comparison with the same process for carotene-CyDIC 3.Inhibition promoted by HDLs affects specific protein transporters

27. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… SUMMARY Comparison of the in vivo lutein absorption efficiency: non-responder versus lutein-responders group non-Responders group Lutein-responders group

28. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… CONCLUSIONS Factors: concentration, donor solution type and inhibition 1.First, inter-individual differences on carotenoid assimilation efficiency should be evaluated, as they are a direct consequence of facilitated diffusion mechanism and expression/location of transporters. Interaction with drugs 2.New strategies to increase carotenoid assimilation to develop food formulae. Interaction with lipoprotein/apoprotein components

29. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… CONCLUSIONS Factors: concentration, donor solution type and inhibition 3.Data point to the existence of different affinity of transporters and even different transporters for carotenes and the xanthophyll lutein. Non-/low-responder effect 4.Bringing pharmaceutical concepts to food technology and nutrition will help to consolidate functional food

30. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… ACKNOWLEGMENTS Financial support from Spanish Government (projects AGL2007-61146; AGR-03025) Scientific and organizing committees of the 6 th International Congress on Pigments in Food - Budapest Dr. Antonio Pérez-Gálvez; aperez@cica.es Food Biotechnology Department Instituto de la Grasa (CSIC) Av. Padre García Tejero 4, 41012 Sevilla (SPAIN) THANKS FOR YOUR ATTENTION!