*[email protected] Food Biotechnology Department Instituto de la Grasa (CSIC)
description
Transcript of *[email protected] Food Biotechnology Department Instituto de la Grasa (CSIC)
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*
*[email protected] 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 matrix2. Incorporation to mixed micelles3. 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…
Time (hours)
tota
l co
nce
ntr
atio
n (
mg
/L l
ipo
pro
tein
)0
4
8
12
16
20
24
28
32
0 1 2 3 4 5 6 7 8To
tal concentr
ati
on
g/L
TR
L
Time (hours)
tota
l co
nce
ntr
atio
n (
mg
/L l
ipo
pro
tein
)
0
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8
12
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20
24
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32
0 1 2 3 4 5 6 7 8
Tota
l concentr
ati
on
g/L
TR
L
1 1
1,2
2,3
3,44
Responders groupResponders group non-Responders groupnon-Responders group
INTER-INDIVIDUAL VARIABILITY AND THENON-RESPONDER CONCEPT
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…
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)
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
Concentration (mM)
Inco
rpor
ated
am
ount
( p
mol
/mg
prot
ein
)
0
500
1000
1500
2000
2500
3000
3500
0.5 1 2.5Concentration ( M)
*
*
*
Assimilation of -carotene
micellar
inclusion complex
1. Saturation versus linear trend2. 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
Concentration (mM)
Inco
rpor
ated
am
ount
( p
mol
/mg
prot
ein
)
0
500
1000
1500
2000
2500
3000
3500
0.5 1 2.5Concentration ( M)
*
*
*Assimilation of lycopene
micellar
inclusion complex
1. Saturation versus linear trend2. Increase of efficiency at 1.0 M
In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes…
Concentration (mM)
Abs
optio
n ra
te (
mM
/min
Xm
g pr
otei
n)
0
20
40
60
80
100
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Concentration ( M)
Abs
orpt
ion
rate
(pm
ol m
g pr
otei
n-1 m
in-1
)
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 trend2. Increase of efficiency at 2.5 M3. A lower absorption efficiencywas observed versus carotenes
In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes…
-carotene lutein lycopene
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
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
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
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?
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
1009luteina3 #77-86 RT: 6,0-6,3 AV: 10 NL: 3,18E3T: - c ESI Full ms [ 229,00-650,00]
250 300 350 400 450 500 550 600 650m/z
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
Inte
nsity
567,3
568,2
569,2
622,6468,3246,1 492,1301,0 592,9566,5431,4399,3321,6 516,7 625,5372,6247,0 594,0
luteinbcdmayo1 #71-80 RT: 17,3-21,0 AV: 10 SB: 1 11,0 NL: 1,70E4T: - c ESI Full ms [ 900,00-2700,00]
1000 1200 1400 1600 1800 2000 2200 2400m/z
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000
13000
14000
15000
16000
17000
Inte
nsity
2269,2
1701,0
2307,6
2326,8
1758,3
2383,41133,5
1890,0
1905,01780,9 2554,01190,4 2398,41975,9 2226,4
Lutein inclusion complex at thedonor solution
Lysate of BBMVs after assimilation procedure with lutein inclusion complex at the donor solution
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
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 trend3. Increase of efficiency at 0.5 MConcentration (mM)
Inco
rpor
ated
am
ount
( p
mol
/mg
prot
ein
)
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
0.5 1 2.5Concentration ( 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
micellar
inclusion complex
1. Decrease of 40% (mean value)2. Saturation versus linear trend3. Increase of efficiency at 0.5 MConcentration (mM)
Inco
rpor
ated
am
ount
( p
mol
/mg
prot
ein
)
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
0.5 1 2.5Concentration ( 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
micellar
inclusion complex
1. Decrease of 70% (mean value)2. Saturation versus linear trend3. Increase of efficiency at 0.5 MConcentration (mM)
Inco
rpor
ated
am
ount
( p
mol
/mg
prot
ein
)
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
0.5 1 2.5Concentration ( M)
* * *
In vitro intestinal absorption of carotenoids delivered as molecular inclusion complexes…
-carotene lutein lycopene
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
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
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?
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 Munder inhibited transport conditions2. Increase of 86% at 1.0 MConcentration (mM)
Inco
rpor
ated
am
ount
( p
mol
/mg
prot
ein
)
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
0.5 1 2.5Concentration ( 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
no inhibition
inhibition
1. Increase of efficiency from 0.5 Munder inhibited transport conditions2. Increase of 165% at 1.0 MConcentration (mM)
Inco
rpor
ated
am
ount
( p
mol
/mg
prot
ein
)
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
0.5 1 2.5Concentration ( 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
micellar
inclusion complex
1. 70% drop of micellar luteinunder inhibited transport conditions2. 28% drop of lutein-CyDIC underinhibited transport conditions
Concentration (mM)
Inco
rpor
ated
am
ount
( p
mol
/mg
prot
ein
)
0
200
400
600
800
No HDLs HDLs
Presence of membrane protein inhibitors
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
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
Time (hours)
TG
con
cent
rati
on in
TR
L
Tota
l car
oten
oid
conc
entr
atio
n in
TR
L
0
4
8
12
16
20
24
0
500
1000
1500
2000
2500
1 2 3 4 5 6 7 8
Tota
l co
nce
ntr
ati
on g
/L T
RL
TG
conce
ntr
ati
on g
/mL
TR
L
non-Responders groupnon-Responders group
Time (hours)T
G c
once
ntra
tion
(T
RL
)
Tota
l car
oten
oid
conc
entr
atio
n
0
4
8
12
16
20
24
28
32
0
500
1000
1500
2000
2500
1 2 3 4 5 6 7 8
TG
conce
ntr
ati
on g
/mL
TR
L
Tota
l co
nce
ntr
ati
on g
/L T
RL
Lutein-responders groupLutein-responders group
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
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
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; [email protected] Biotechnology Department
Instituto de la Grasa (CSIC)Av. Padre García Tejero 4,
41012 Sevilla (SPAIN)
THANKS FOR YOUR ATTENTION!