PARALLEL SESSION A Auditorium Descartes Selected communications Chairs: Glaucia Maria Pastore -...

PARALLEL SESSION A Auditorium Descartes

Selected communications

Chairs: Glaucia Maria Pastore - UNICAMP- Campinas - Brazil Peter Hollman - Wageningen University - The Netherlands

Multiple-approach studies to assess anthocyanin gastric absorption – potential role of GLUT1 Iva Fernandes - University of Porto – Portugal

7th International Conference on Polyphenols and Health

Multiple-approach studies to assess anthocyanin gastric absorption:

potential role of GLUT1

Iva Fernandes

Consumption in Europe:

♂: 19.8 (Netherland) – 64.9 mg/d (Italy)

♀: 18.4 (Spain) – 44.1 mg/d (Italy)

(Heinonen 2007)

(Zamora-Ros, Knaze et al. 2011)

Anthocyanins consumption and human health

AnthocyaninsChange and improve human health!

DiabetesObesity

Cardiovascular

glcO

O+

OH

OH

OR1

OR2

HO

Flavylium cation

Anthocyanins in food and pH equilibrium forms

Cólon

Food

Stomach

Small Intestine

HOOHO

OH

OH

O

O+

OH

OH

OR1

OR2

HO

Flavylium cation

HOOHO

OH

OH

O

O

OH

OH

OR1

OR2

HOOH

Hemiketal Quinoidal base

OH

O

OH

O

R1

R2O

OH

O

OH

OH

R1

R2

-OOH

Anionic pseudobase

HOOHO

OH

OH

O

O

OH

OH

OR1

OR2

HOOH

Hemiketal

HOOHO

OH

OH

O

O

OH

O

OR1

OR2

HO

Neutral Quinoidal bases

HOOHO

OH

OH

O

O

OH

OH

OR1

OR2

HO HO

cis-Chalcone

OH

HO OH

trans-Chalcone

OH

OR1

OR2

HO

OH

O

HOOH

O

O

HOOHO

OH

OH

O

O

OH

OH

OR1

OR2

HO HO

cis-Chalcone

OH

HO OH

trans-Chalcone

OH

OR1

OR2

HO

OH

O

HOOH

O

O

HOOHO

OH

OH

O

O

OH

O

OR1

OR2

-O

Anionic quinoidal bases

pH 1-5 pH 5-7.4 pH 7.4-8

Low concentration in biological samples

Matrix complexity (e. g. EtOH)

Structural diversity

Metabolic disorders (e. g. Obesity)

Cleanup and concentration process

Lack of standards

Analytical methods

pH equilibrium forms

Low concentration in biological samples

Matrix complexity (e. g. EtOH)

Structural diversity

Metabolic disorders (e. g. Obesity)

Cleanup and concentration process

Lack of standards

Analytical methods

pH equilibrium forms

Isofla

vones

Flava

nones

Flavo

nols

Flava

nols

Phenolic

aci

ds

Anthocy

anin

s

0

1

2

3

4

Cm

ax

(

M)

(Manach, Williamson et al. 2005)

Detection of anthocyanins in human samples

Overlooked factors

Small intestine

Intestines

Intestines

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Stomach



Brain and Brain Functions

Sagittal View

6.

8.

5.

7.




Liver and Gallbladder



Kidneys and Urinary Tract



Urinary Tract and BladderBladder (Female)

Urinary Tract and BladderBladder (Female)





Stomach

Enterohepatic recirculation

Mic

rob

iota

Fecal Excretion

Kidneys

Bladder

metabolites/catabolites absorption

Colon

Urinary Excrection

Liver

Systemic circulation

EyesHeart

BrainAdipose tissue

Tissue/organs

MethylSulphate

Glucuronil

Glucose

Anthocyanins

Transporter?

Absorption and metabolism

Human trial

Multiple approach studies...

In vitro / theoretical studies

Human gastric barrier model: MKN-28 cell line

Tight junctions

Hanks mediumpH 5.0

Policarbonate membrane

Transwell insert

Apical side

Basolateral side

STOMACHMKN-28 CELL

LINE

12-well transwell, 0.4-µm pore size, 7 days, TEER > 150 Ω.cm2

HPLC analysis30’60’120’180’

HOOHO

OH

OH

O

O

OH

OH

R1

R2

HO

Anthocyanin

(Fernandes, de Freitas et al. 2012)

http://www.wpclipart.com/science/eppendorf.png

30 60 120 180

0

2

4

6

8

100 % Ethanol

4 % Ethanol

**

Glucose (40 mM)

** ******

Time (min)

% T

ran

sp

ort

Eff

icie

nc

y(T

ota

l a

nth

oc

yan

ins

)

Anthocyanins transport in MKN-28 cell model

30 60 120 180

0

2

4

6

8

10Dp3glc

Cy3glc

Mv3glc

Time (min)

Tra

ns

po

rt e

ffic

ien

cy

(%)

HOOHO

OH

OH

O

O

OH

OH

OH

HO

Cy3glc

HOOHO

OH

OH

O

O

OH

OH

OH

OHHO

Dp3glc

HOOHO

OH

OH

O

O

OH

OH

OCH3

OCH3

HO

Mv3glc

Red Wine0 % alc/vol

Red Wine4 % alc/vol

Red Wine40 mM glucose

• Glucose affects wine anthocyanins

absorption patterns.

~ >

• Individual anthocyanins have similar

absorption efficiencies.

• Wine anthocyanins are absorbed at

gastric level, independent on ethanol.

(Fernandes, de Freitas et al. 2012; Oliveira, Fernandes et al. 2015)

Apical pH effect

30 60 120 1800

2

4

6

8pH 5

pH 7.4

***

Time (minutes)

Tra

ns

po

rt e

ffic

ien

cy

(%)

To

tal r

ed

win

e a

nth

ocy

an

ins

•Higher transport efficiency at lower pH valuesHO

OHO

OH

OH

O

O

OH

OH

R1

R2

-OOH

Anionic Pseudobase

HOOHO

OH

OH

O

O

OH

O

R1

R2

-O

Quinoidal base

Intestine

Intestines

Intestines




1 < pH < 5

6.5 < pH < 8

R3

O+

OH

OH

R1

R2

HO

Flavylium cation

R3

O

OH

OH

R1

R2

HOOH

Hemiketal

Stomach

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Stomach

(Oliveira, Fernandes et al. 2015)

Equilibrium form and possible transporters

qRT-PCR - transporters expression in MKN-28

Apical side

Basolateral sideMKN-28 cell line

GLUT-1

GLUT1 GLUT2 GLUT30

10

20

30

40

RE

L

MCT1 SMCT1 SGLT10.000

0.005

0.010

0.015

0.020

0.025

RE

L


Molecular docking studies: anthocyanins/GLUT1 – extracellular and intracellular sides

anthocyanins (ring B or glucose) – stable complexes with GLUT1

Docking studies – anthocyanin/GLUT1

Gastric barrierGLUT1

pH < 5.0

HOOHO

OH

OH

O

O

OH

OH

R1

R2

HO

Anthocyanin Glucose

A C

B


Molecular docking studies: Asn280, Asn403, Asn407, Trp380, Gln284 in transporter core

Ongoing dynamic studies: transport from extracellular to cytoplasmatic side

Apical side

Basolateral sideMKN-28_SLC2A1 cell line

GLUT-1 GLUT-1

MKN-2

8

MKN-2

8_SLC2A

1

0

5

10

15

20

25***

RE

L G

LU

T1

Stable transfection of GLUT1 in MKN-28 cell line

0

5

10

15

20

25 ***

Red wine anthocyanins

Mv3glc

MKN-28_GLUT1MKN-28

RE

L G

LU

T1

30 60 120 1800

2

4

6

8

10

MKN-28_GLUT1

MKN-28

Time (min)

Tra

ns

po

rt e

ffic

ien

cy

(%)

ConclusionGastric transport of anthocyanins

• Negative feedback after anthocyanins

treatment (3h): GLUT1 levels similar to

MKN-28 cell line

• Slightly higher transport efficiency of Dp3glc

in transfected cell line, without significance

• Passive diffussion/paracellular transport

with similar levels in both cell lines, at 4ºC

• Active transport!

4ºC

Multiple approach studies...

Human trial

In vitro / theoretical studies

Time (min) 0 15 30 60 120

Plasma

Sample clean-up and concentration

Sample analyse by HPLC and qTOF

Blackberry juice

Anthocyanins ~ 650 mg

Overnight fasting

Blackberry juiceName MS/MS2

Cy3glc, 80.7% 449/287

Cy3rut, 10.7% 595/449; 287

Cy3malonylglc, 3.56% 535

Cy3dioxaloylglc, 5.02% 593/187

[Anthocyanins]juice = 2.92 ± 0.66 mg/mL

Human Trial

8 volunteers BMI≤25

0.0 10.0 20.0 30.0 40.0 50.0 60.0Time [min]

120 min

60 min

30 min

0 min

15 min

0.0

Ab

s (5

20

nm

)

Peaks Time(min) MRM Name

1 13.43 625 > 463 Cy3Glc-Glucr

2 16.51 639 > 477 Me-Cy3Glc-Glucr

3 18.09 529 > 287 Cy3glc-Sulp

4 18.94 449 > 287 Cy3glc

5 20.72 463 > 287 Cy-Glucr

6Conjugates 24.29

477 > 301463 > 301463 > 287

Me-Cy-GlucrMe-Cy3glcCy-Glucr

7 24.73 ? ?

8 33.93 ? ?

Plasma samples – HPLC analysis

0 50 100 1500

5.010 - 5

1.010 - 4

1.510 - 4

2.010 - 4

2.510 - 4

3.010 - 4

3.510 - 4

Cy3glc

Conjugates

b

a

c

a

aa

aa

Time (minutes)

Pla

sm

atic

Co

nce

ntr

atio

n(m

g/m

L)

• Cmax (Cy3glc) located within the 2 h, at ~60 min

• Cmax (Conjugates) > 2h

• Conjugates have significantly higher plasmatic levels

Different letters are significantly different

Plasmatic levels of Cy3glc and conjugates

In vitro gastric absorption of anthocyaninsIn vitro gastric absorption of anthocyanins across MKN-28 and MKN-28_GLUT1 cells, at similar levels

Theoretical gastric transporterPutative involvement of glucose transporter 1 in anthocyanin gastric absorption mechanism

Human absorption and metabolismDetection of anthocyanins and their phase II conjugates in human plasma samples

HOOHO

OH

OH

O

O

OH

OH

R1

R2

HO

Anthocyanin

A C

B

GLUT-1

Phase IIenzymes

HOOHO

OH

OH

O

O

OH

OH

R1

R2

HO

Anthocyanin

A C

B

OH

O

OH

OH

OCH3

O

Me-Cy-Glcr

OHO

HOOH

HOOC

Ongoing studies…Silencing GLUT1/GLUT3 expression with small interfering RNA!

First steps…

Research team

Iva Fernandes, Ph.DNatércia Brás, Ph.D.

Helder Oliveira, M.Sc.

Prof. Nuno Mateus

Claudia Marques, M.Sc.

Prof. Victor de Freitas

Prof. Conceição Calhau

Ana Faria, Ph.D.

PARALLEL SESSION A Auditorium Descartes Selected communications Chairs: Glaucia Maria Pastore -...

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