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)
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
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)
In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes…
INTER-INDIVIDUAL VARIABILITY AND THE
NON-RESPONDER CONCEPT
24
4
2,3
20
1,2
16
12
1
1
8
4
0
0
1
2
3
4
5
6
Time (hours)
Responders group
7
8
TRL
28
3,4
32
total
concentration
(mg/L lipoprotein)
Total
concentration
g/L
TRL
32
total
concentration
(mg/L lipoprotein)
Total
concentration
g/L
 Comparison of the in vivo lutein absorption efficiency:
non-responder versus lutein-responders group
28
24
20
16
12
8
4
0
0
1
2
3
4
5
6
Time (hours)
non-Responders group
7
8
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 (b-carotene, lutein and lycopene) were
formulated as micellar solutions (control) or inclusion
complexes with b-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 preincubated with HDLs)
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
3500
Assimilation of b-carotene
Incorporated amount ( pmol/mg protein )
3000
2500
*
2000
micellar
inclusion complex
1500
*
1000
*
500
0
0.5
1
Concentration
(
M)
Concentration
(mM)
2.5
1. Saturation versus linear trend
2. Increase of efficiency at 2.5 M
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
3500
*
Incorporated amount ( pmol/mg protein )
3000
2500
Assimilation of lycopene
micellar
2000
inclusion complex
1500
*
1000
500
*
0
0.5
1
Concentration
(
M)
Concentration
(mM)
2.5
1. Saturation versus linear trend
2. Increase of efficiency at 1.0 M
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
Absoptionrate
rate(pmol
(mM/minXmg
protein) -1min -1)
Absorption
mg protein
100
Assimilation of lutein
80
micellar
60
inclusion complex
40
20
0
0.0
0.5
1.0
1.5
2.0
Concentration
(
M)
Concentration
(mM)
2.5
3.0
1. Saturation versus linear trend
2. Increase of efficiency at 2.5 M
3. A lower absorption efficiency
was observed versus carotenes
In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes…
RESULTS
 Primary effects of the factors concentration, donor
solution type and inhibition
b-carotene
lutein
lycopene
Concentration
2.085
784
2.087
Donor solution type
1.370
579
1.502
720
-256
398
Inhibition
In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes…
SUMMARY
 Factors: concentration and donor solution type
1. Association
mechanism
between
concentration
and
assimilation
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
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)
b-carotene
lutein
lycopene
C. E.
C. I. C.
C. E.
C. I. C.
C. E.
C. I. C.
0.5 M
32.9
20.1
9.85
14.2
19.1
11.7
1.0 M
65.3
36.6
26.9
27.8
28.9
43.9
2.5 M
70.7
106
30.1
85.9
69.4
158
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 (b-Car: 51%; Lut: 185%; Lyc:
128%). What absorption mechanism does apply for inclusion
complexes?
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
luteinbcdmayo1 #71-80 RT: 17,3-21,0 AV: 10 SB: 1 11,0 NL: 1,70E4
T: - c ESI Full ms [ 900,00-2700,00]
Lysate of BBMVs after assimilation
procedure with lutein inclusion
complex at the donor solution
2269,2
17000
16000
15000
14000
13000
12000
11000
Intensity
10000
1009luteina3 #77-86 RT: 6,0-6,3 AV: 10 NL: 3,18E3
T: - c ESI Full ms [ 229,00-650,00]
1701,0
567,3
9000
3000
8000
2800
7000
2600
6000
2400
5000
2307,6
2200
2326,8
4000
1758,3
2000
3000
1890,0
1905,0
1780,9
1975,9
1190,4
1000
1200
1800
2383,4
1133,5
1000
1400
1600
1800
m/z
2000
2226,4
2200
2398,4
2554,0
2400
Lutein inclusion complex at the
donor solution
Intensity
2000
1600
568,2
1400
1200
1000
800
600
569,2
400
200
246,1
247,0
301,0
250
300
372,6 399,3
321,6
431,4
468,3 492,1
516,7
566,5
622,6
592,9
625,5
594,0
0
350
400
450
m/z
500
550
600
650
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 hostguest complex
K
+
complexation
De-complexation
Assimilation:
Passive or facilitated diffusion
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
6500
6000
Incorporated amount ( pmol/mg protein )
5500
micellar
5000
4500
4000
inclusion complex
3500
3000
2500
2000
1500
1000
500
*
*
*
0
0.5
1
Concentration
( M)
Concentration
(mM)
2.5
1. Decrease of 50% (mean value)
2. Saturation versus linear trend
3. Increase of efficiency at 0.5 M
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
6500
6000
Incorporated amount ( pmol/mg protein )
5500
5000
micellar
4500
4000
inclusion complex
3500
3000
2500
2000
1500
*
1000
500
*
*
0
0.5
1
Concentration
(
M)
Concentration
(mM)
2.5
1. Decrease of 40% (mean value)
2. Saturation versus linear trend
3. Increase of efficiency at 0.5 M
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
6500
6000
Incorporated amount ( pmol/mg protein )
5500
micellar
5000
4500
4000
inclusion complex
3500
3000
2500
2000
1500
1000
500
*
*
*
0
0.5
1
Concentration
( M)
Concentration
(mM)
2.5
1. Decrease of 70% (mean value)
2. Saturation versus linear trend
3. Increase of efficiency at 0.5 M
In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes…
RESULTS
 Primary effects of the factors concentration, donor
solution type and inhibition
b-carotene
lutein
lycopene
Concentration
2.085
784
2.087
Donor solution type
1.370
579
1.502
720
-256
398
Inhibition
In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes…
SUMMARY
 Factors: concentration,
inhibition
donor
solution
type
and
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
In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes…
SUMMARY
 Factors: concentration,
inhibition
donor
solution
type
and
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?
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
6500
6000
Incorporated amount ( pmol/mg protein )
5500
5000
no inhibition
4500
4000
inhibition
3500
3000
*
2500
2000
1500
1000
500
*
*
0
0.5
1
Concentration
(
M)
Concentration
(mM)
2.5
1. Increase of efficiency from 0.5 M
under inhibited transport conditions
2. Increase of 86% at 1.0 M
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
6500
6000
Incorporated amount ( pmol/mg protein )
5500
5000
no inhibition
4500
4000
inhibition
*
3500
3000
2500
2000
1500
*
1000
500
*
0
0.5
1
Concentration
(
M)
Concentration
(mM)
2.5
1. Increase of efficiency from 0.5 M
under inhibited transport conditions
2. Increase of 165% at 1.0 M
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
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
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
Incorporated amount ( pmol/mg protein )
800
micellar
600
inclusion complex
400
200
0
No HDLs
HDLs
PresenceConcentration
of membrane(mM)
protein inhibitors
1. 70% drop of micellar lutein
under inhibited transport conditions
2. 28% drop of lutein-CyDIC under
inhibited transport conditions
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
transporters
by
HDLs
affects
specific
protein
In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes…
SUMMARY
2000
20
16
1500
12
1000
8
500
0
4
1
2
3
4
5
6
7
8
0
2500
32
28
2000
24
20
1500
16
1000
12
8
500
0
4
1
2
3
4
5
6
7
8
Time (hours)
Time (hours)
non-Responders group
Lutein-responders group
0
Total carotenoid
concentration
g/L TRL
Total
concentration
24
TG concentration (TRL)
g/mL TRL
TG concentration
2500
Total
carotenoid
concentrationin
TRLTRL
Total
g/L
concentration
concentration in TRL
g/mL TRL
TG TG
concentration
 Comparison of the in vivo lutein absorption efficiency:
non-responder versus lutein-responders group
In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes…
CONCLUSIONS
 Factors: concentration,
inhibition
donor
solution
type
and
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
In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes…
CONCLUSIONS
 Factors: concentration,
inhibition
donor
solution
type
and
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
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!