*aperez@cica.es Food Biotechnology Department Instituto de la Grasa (CSIC)

1. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes with b-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 • Existence of undernourished population • Epidemiological studies have associated between consumption of fruit and vegetables to a lower risk of developing degenerative diseases • Development of food products with added nutritional value • 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 • Liberation from food matrix • Incorporation to mixed micelles • Absorption by epithelial cells through simple/facilitated diffusion mechanisms • Absorption efficiency is relatively low from fruits and vegetables • Fiber, kind and amount of fat, interaction among carotenoids • 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… • INTER-INDIVIDUAL VARIABILITY AND THE • NON-RESPONDER CONCEPT • Comparison of the in vivo lutein absorption efficiency: non-responder versus lutein-responders group Responders group non-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 • Dietary carotenoids (b-carotene, lutein and lycopene) were formulated as micellar solutions (control) or inclusion complexes with b-cyclodextrin • 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 • Comparison of absorption efficiency under inhibition conditions of membrane protein transporters (BBMVs pre-incubated with HDLs)

6. micellar inclusion complex 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 b-carotene • Saturation versus linear trend • Increase of efficiency at 2.5 mM

7. micellar inclusion complex 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 • Saturation versus linear trend • Increase of efficiency at 1.0 mM

8. micellar inclusion complex 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 • Saturation versus linear trend • Increase of efficiency at 2.5 mM • A lower absorption efficiency • was observed versus carotenes

9. b-carotene lutein lycopene 2.085 784 2.087 Concentration 1.370 579 1.502 Donor solution type 720 -256 398 Inhibition In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… • 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 • Association between concentration and assimilation mechanism • Structural features (polarity) or different affinity of transporters may explain the absorption efficiency data of carotenes and lutein • Significant increase on efficiency of the assimilation is reached when carotenoids were delivered as inclusion complex with CyD

11. b-carotene lutein lycopene C. E. C. I. C. C. E. C. I. C. C. E. C. I. C. 0.5 mM 32.9 20.1 9.85 14.2 19.1 11.7 1.0 mM 65.3 36.6 26.9 27.8 28.9 43.9 2.5 mM 70.7 106 30.1 85.9 69.4 158 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)

12. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… • SUMMARY • Factors: concentration and donor solution type • At the lowest concentration the carotenoids from micellar solutions were more efficiently assimilated • At 1.0 mM a heterogeneous behavior was observed • Only at the highest concentration, carotenoids from inclusion complex solutions were more efficiently assimilated in comparison with the carotenoid micellar solutions at that concentration (b-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 Lysate of BBMVs after assimilation procedure with lutein inclusion complex at the donor solution Lutein inclusion complex at the donor solution

14. In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… • 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 + K complexation De-complexation Assimilation: Passive or facilitated diffusion

15. micellar inclusion complex 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 b-carotene • Decrease of 50% (mean value) • Saturation versus linear trend • Increase of efficiency at 0.5 mM

16. micellar inclusion complex 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 • Decrease of 40% (mean value) • Saturation versus linear trend • Increase of efficiency at 0.5 mM

17. micellar inclusion complex 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 • Decrease of 70% (mean value) • Saturation versus linear trend • Increase of efficiency at 0.5 mM

18. b-carotene lutein lycopene 2.085 784 2.087 Concentration 1.370 579 1.502 Donor solution type 720 -256 398 Inhibition In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes… • 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 • Assimilation of carotenoids from micellar solutions is significantly inhibited with the use of HDLs • 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 • 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 • 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. no inhibition inhibition 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 b-carotene-CyDIC • Increase of efficiency from 0.5 mM • under inhibited transport conditions • Increase of 86% at 1.0 mM

22. no inhibition inhibition 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 • Increase of efficiency from 0.5 mM • under inhibited transport conditions • Increase of 165% at 1.0 mM

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. micellar inclusion complex 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 mM • 70% drop of micellar lutein • under inhibited transport conditions • 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 • A different effect of HDLs was observed for the assimilation efficiency of carotene-CyDICs or lutein-CyDICs • Process of competition between HDLs and lutein-CyDIC may not be efficient enough in comparison with the same process for carotene-CyDIC • 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 • 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 • 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 • Data point to the existence of different affinity of transporters and even different transporters for carotenes and the xanthophyll lutein. Non-/low-responder effect • 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 6th 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!