*[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


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)


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

4

8

12

16

20

24

28

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


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)


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




Concentration (mM)

Inco

rpor

ated

am

ount

( p

mol

/mg

prot

ein

)

0

500

1000

1500

2000

2500

3000

3500


*

*

*Assimilation of lycopene

micellar

inclusion complex

1. Saturation versus linear trend2. Increase of efficiency at 1.0 M


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

)



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


-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


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


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


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?


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


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



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


* **




Assimilation of lycopene

micellar

inclusion complex


Inco

rpor

ated

am

ount

( p

mol

/mg

prot

ein

)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500


**

*




Assimilation of lutein

micellar

inclusion complex


Inco

rpor

ated

am

ount

( p

mol

/mg

prot

ein

)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500


* * *


-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


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


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?


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


**

*




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


*

*

*








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


SUMMARY Comparison of the carotenoid-CyDIC absorption


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


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


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


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


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!

Documents

*[email protected] Food Biotechnology Department Instituto de la Grasa (CSIC)