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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_at_cica.es Food Biotechnology
Department Instituto de la Grasa (CSIC) Av. Padre
García Tejero 4, 41012 Sevilla (SPAIN)
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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

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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)

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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
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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)

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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

1. Saturation versus linear trend
2. Increase of efficiency at 2.5 mM

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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

1. Saturation versus linear trend
2. Increase of efficiency at 1.0 mM

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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

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In vitro intestinal absorption of carotenoids
delivered as molecular inclusion complexes

• RESULTS
• Primary effects of the factors concentration,
  donor solution type and inhibition

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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

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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)

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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?

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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
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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
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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

1. Decrease of 50 (mean value)
2. Saturation versus linear trend
3. Increase of efficiency at 0.5 mM

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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

1. Decrease of 40 (mean value)
2. Saturation versus linear trend
3. Increase of efficiency at 0.5 mM

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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

1. Decrease of 70 (mean value)
2. Saturation versus linear trend
3. Increase of efficiency at 0.5 mM

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In vitro intestinal absorption of carotenoids
delivered as molecular inclusion complexes

• RESULTS
• Primary effects of the factors concentration,
  donor solution type and inhibition

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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

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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?

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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

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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

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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

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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

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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

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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

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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
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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

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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

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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_at_cica.es
Food Biotechnology Department Instituto de la
Grasa (CSIC) Av. Padre García Tejero 4, 41012
Sevilla (SPAIN)
THANKS FOR YOUR ATTENTION!