faculty of bioscience engineering academic year 2014...

Faculty of Bioscience Engineering

Academic year 2014 – 2015

Study of vitamin A bioavailability by in vitro models

Hannah Manssens

Promotors: Prof. dr. ir. John Van Camp

Prof. dr. ir. Tom Van de Wiele

Tutors: dr. ir. Charlotte Grootaert

Paulina Escobar

Master’s dissertation submitted in partial fulfilment of the requirements for the

degree of Master of Science in Bioscience Engineering: Cell and Gene

Biotechnology

I

COPYRIGHT

“The author and the promoters give permission to use this master’s dissertation for consulting purposes

and to copy parts of it for personal use. However, every other use is subject to the limitations of copyright

regulations, more specifically the stringent obligation to explicitly mention the source when citing parts

out of this master’s dissertation.”

“De auteur en de promotoren geven de toelating deze masterproef voor consultatie beschikbaar te stellen

en delen ervan te kopiëren voor persoonlijk gebruik. Elk ander gebruik valt echter onder de beperking

van het auteursrecht, in het bijzonder met betrekking tot de verplichting uitdrukkelijk de bron te

vermelden bij het aanhalen van delen uit deze masterproef.”

Ghent, June 2015

The promotor, The co-promotor,

Prof. dr. ir. John Van Camp Prof. dr. ir. Tom Van de Wiele

The author,

Hannah Manssens

II

ABSTRACT

Vitamin A and its derivatives have various crucial functions during human growth and development.

The limited bioavailability of provitamin A carotenoids, more particularly β-carotene, has been shown

to be problematic and causes severe health problems worldwide. More knowledge about the maturation

of the intestinal transport function is still needed to fully explain and control the higher incidence of

deficiencies in children. In this master’s thesis, the hypothesis was explored that luminal molecules are

capable to trigger the development of certain receptors for vitamin A on intestinal cells and thus,

influence the bioavailability.

An in vitro Caco-2 cell model was developed that resembled the intestinal wall and made bioavailability

studies possible. Detection of the carotenoids was herein achieved by optimization of the extraction and

HPLC method. The co-solvent evaporation method was optimized and used in the emulsification of the

hydrophobic β-carotene for delivery to the cells. Prior to the transport experiment, Caco-2 cells were

pretreated with distinct intestinal samples during their differentiation to evaluate if luminal components

can influence the maturation of the transport function of the cells for β-carotene.

In the developed Caco-2 cell model, a LOD of 0.06 µM of β-carotene in cell culture medium was reached

with the applied detection procedure, which made measurements of cellular uptake possible, but

restricted analysis of secreted carotenoids. By use of the co-solvent evaporation method, small micelles

sizes (< 200 nm), a high dispersion stability and a good incorporation of the carotenoids could be

achieved. As such, this emulsion proved to be more suitable for delivery of β-carotene to intestinal Caco-

2 cells than earlier established methods.

After pretreatment of the cells with proximal and distal colon water during their differentiation, a

significant reduction in cellular uptake of β-carotene was found in the distal colon water condition

compared to the condition with proximal colon water. Prior incubation with FOS, stimulating

carbohydrate fermentation, was necessary to obtain a significant distinction between the conditions.

These findings give a first indication that certain luminal components could direct the transport function

of intestinal Caco-2 cells for β-carotene.

Key words: β-carotene, bioavailability, Caco-2, in vitro, digestion, emulsion, functional food

III

ABSTRACT (DUTCH)

Vitamine A en zijn derivaten vervullen verscheidene cruciale functies tijdens de groei en ontwikkeling

van de mens. De beperkte biobeschikbaarheid van provitamine A carotenoïden, en in het bijzonder van

β-caroteen, veroorzaakt wereldwijd ernstige gezondheidsproblemen. Een verruiming van de kennis over

de ontwikkeling van de intestinale transportfunctie is nodig om de hogere incidentie van deficiënties in

kinderen volledig te verklaren en onder controle te krijgen. In deze masterproef werd de hypothese

onderzocht dat luminale moleculen kunnen leiden tot de ontwikkeling van bepaalde vitamine A

receptoren op intestinale cellen en zo de biobeschikbaarheid kunnen beïnvloeden.

Een in vitro Caco-2 cellijn model werd ontwikkeld om de intestinale wand te simuleren en zo

biobeschikbaarheidstudies mogelijk te maken. Door optimalisatie van een extractie en HPLC methode,

kon detectie van carotenoïden in dit model verwezenlijkt worden. Een co-solvent evaporatiemethode

werd geoptimaliseerd en gebruikt bij emulsificatie van het hydrofobe β-caroteen alvorens dit aan de

cellen toe te dienen. Om te evalueren of luminale componenten in staat zijn om de maturatie van de

cellulaire transportfunctie voor β-caroteen te beïnvloeden, werd een voorbehandeling met uiteenlopende

intestinale stalen verricht tijdens de differentiatie van Caco-2 cellen, waarna een transportexperiment

werd uitgevoerd.

In het ontwikkelde Caco-2 cellijn model, werd een detectielimiet van 0.06 µM β-caroteen in

celcultuurmedium bereikt met de detectieprocedure. Dit volstond om de cellulaire opname te meten,

maar analyse van de gesecreteerde carotenoïden was gelimiteerd. De co-solvent evaporatiemethode was

in staat kleine micelgroottes (< 200 nm), een hoge emulsiestabiliteit en een goede incorporatie van de

carotenoïden te produceren. Deze emulsie bleek zo meer geschikt voor gebruik met intestinale Caco-2

cellen dan eerdere gebruikte methoden.

Een voorbehandeling van cellen met distaal colonwater gedurende de differentiatie resulteerde in een

significante vermindering in cellulaire β-caroteen opname, in vergelijking met de conditie waarin

proximaal colonwater werd gebruikt. Een voorafgaande incubatie van de stalen met FOS was nodig om

de carbohydraatfermentatie verder te stimuleren en een significant verschil tussen de condities zichtbaar

te maken. Deze resultaten geven een eerste indicatie dat luminale componenten in staat zijn om de

transportfunctie voor β-caroteen in intestinale Caco-2 cellen te sturen.

Sleutelwoorden: β-caroteen, biobeschikbaarheid, Caco-2, in vitro, digestion, emulsie, functionele

voeding

IV

ACKNOWLEDGEMENTS

The realization of this master’s dissertation would not have been possible without the guidance and the

support of several individuals. For this reason, I would first like to acknowledge their contribution and

assistance during the progression and completion of this work.

Foremost, I would like to offer my sincere gratitude to my main supervisor, dr. ir. Charlotte Grootaert,

for the professional guidance and support throughout the period of this master’s thesis. Her knowledge,

useful critiques and continuous optimism during the research have been invaluable.

I would like to express my great appreciation to Prof. dr. ir. John Van Camp for his enthusiastic

encouragement and for taking the time and effort necessary to provide relevant and constructive

recommendations on this dissertation.

I am also very grateful to Prof. dr. ir. Tom Van de Wiele for providing valuable and useful suggestions

during the planning and development of this research.

Further, I would like to offer my special thanks to Paulina Escobar for her help during the experimental

work and for sharing her expertise in carotenoid research.

Advice and assistance given by Quenten Denon and Prof. dr. ir. Paul Van der Meeren with regard to the

produced dispersions was greatly appreciated.

I would like to extend my thanks to the staff of the Research Group Food Chemistry and Human

Nutrition and especially to everyone working in the cellular lab for their help in handling certain

instruments and advice regarding the experimental work.

Finally, I wish to acknowledge the useful comments and assistance provided by the staff of the

Laboratory of Microbial Ecology and Technology. I am particularly grateful to the technical staff who

helped in performing the gas chromatography.

V

TABLE OF CONTENTS

List of abbreviations ................................................................................................................................ 1

List of tables and figures ......................................................................................................................... 2

Introduction ............................................................................................................................................. 4

I Literature study ................................................................................................................................ 5

I.1 Vitamin A and its importance in human health ....................................................................... 5

I.1.1 Molecular structure and occurrence in food sources ........................................................... 5

I.1.2 Functions in the human body .............................................................................................. 5

I.1.3 Vitamin A deficiency .......................................................................................................... 6

I.1.4 Bioavailability, influencing factors and recommended uptake of vitamin A ...................... 6

I.2 Digestion and metabolism of vitamin A .................................................................................. 8

I.2.1 Biological conversions of vitamin A and its derivatives ..................................................... 8

I.2.2 Solubilization in the gastrointestinal tract ......................................................................... 10

I.2.3 Intestinal absorption of vitamin A molecules .................................................................... 10

I.3 Influences on the intestinal transport function for vitamin A ................................................ 13

I.3.1 Development and regulation of the transport function in the intestine ............................. 13

I.3.2 Disorders causing vitamin A malabsorption ..................................................................... 14

I.3.3 Intestinal components influencing the absorption of vitamin A ........................................ 14

I.4 β-carotene micelle formation as a means to improve absorption .......................................... 18

I.4.1 Barriers for β-carotene absorption in the gastrointestinal tract ......................................... 18

I.4.2 Techniques to obtain emulsions ........................................................................................ 18

I.4.3 Stability of β-carotene emulsions ...................................................................................... 19

I.5 In vitro simulation of the gastrointestinal tract ...................................................................... 20

I.5.1 Caco-2 cell line .................................................................................................................. 20

I.5.2 Cell culture-based simulation model ................................................................................. 21

II Problem statement ......................................................................................................................... 23

III Objectives .................................................................................................................................. 24

VI

IV Material and methods ................................................................................................................ 25

IV.1 Chemicals and products ......................................................................................................... 25

IV.2 Cell culture ............................................................................................................................ 25

IV.2.1 Caco-2 cell line .............................................................................................................. 25

IV.2.2 Transport assay .............................................................................................................. 26

IV.2.3 Cytotoxicity tests ........................................................................................................... 26

IV.3 Method of detection for β-carotene ....................................................................................... 27

IV.3.1 High-Performance Liquid Chromatography .................................................................. 27

IV.3.2 Cell lysis ........................................................................................................................ 27

IV.3.3 Extraction of carotenoids ............................................................................................... 28

IV.4 Emulsification of β-carotene ................................................................................................. 29

IV.4.1 Measurement of micelle characteristics ........................................................................ 29

IV.4.2 Oil stripping ................................................................................................................... 29

IV.4.3 Co-solvent evaporation method ..................................................................................... 30

IV.4.4 Self-emulsification method ........................................................................................... 31

IV.5 Development of an in vitro model to study carotenoid transport .......................................... 31

IV.6 Pretreatment of Caco-2 cells with intestinal water ................................................................ 32

IV.6.1 SHIME incubation ......................................................................................................... 32

IV.6.2 Characteristics of intestinal samples.............................................................................. 33

IV.6.3 Effect of pretreatment on β-carotene transport .............................................................. 33

IV.7 Statistical analysis of data ..................................................................................................... 34

V Results ........................................................................................................................................... 35

V.1 Characteristics of the β-carotene emulsions .......................................................................... 35

V.1.1 Stability of the emulsions .............................................................................................. 35

V.1.2 Incorporation of β-carotene in the micelles ................................................................... 35

V.1.3 Toxicity of the emulsions to Caco-2 cells ..................................................................... 36

V.2 Study of β-carotene transport through Caco-2 cells .............................................................. 39

V.2.1 Detection method for carotenoids.................................................................................. 39

V.2.2 In vitro cell model to study transport............................................................................. 40

VII

V.3 Pretreatment of Caco-2 cells with intestinal water ................................................................ 41

V.3.1 Characteristics of the SHIME samples .......................................................................... 41

V.3.2 Influence on β-carotene transport .................................................................................. 42

VI Discussion ................................................................................................................................. 44

VI.1 Delivery of β-carotene to the Caco-2 cells ............................................................................ 44

VI.2 Development of an in vitro model to study β-carotene transport .......................................... 49

VI.3 Pretreatment of Caco-2 cells with intestinal water ................................................................ 51

Conclusion ............................................................................................................................................. 55

Future perspectives ................................................................................................................................ 56

References ............................................................................................................................................. 57

Addendum: Reflection on Sustainability ............................................................................................... 65

1

LIST OF ABBREVIATIONS

ABC ATP-Binding Cassette

ABCA1 ATP-Binding Cassette subfamily A member 1

ABCG5 ATP-Binding Cassette subfamily G member 5

apoB Apolipoprotein B

ARAT Acyl CoA:retinol acyltransferase

ATCC American Type Culture Collection

AUC Area Under the Curve

BBM Brush Border Membranes

BCO1 β-carotene 15,15’-oxygenase 1

BCO2 β-carotene 15,15’-oxygenase 2

BHT 2,6-di-tert-butyl-4-methyl-phenol

CBP Carotenoid-Binding Protein

CD36 Cluster Determinant 36

CEH Cholesterol Ester Hydrolase

CRBPII Cellular Retinol-Binding Protein II

DGAT1 Acyl CoA:diacylglycerol acyltransferase 1

DMEM Dulbecco's modified eagle medium

DMSO Dimethylsulfoxide

FBS Fetal Bovine Serum

FOS Fructo-oligosaccharide

HDL High-Density Lipoprotein

HPLC High-Performance Liquid Chromatography

IDL Intermediate-Density Lipoprotein

IS Internal Standard

ISX Intestine-Specific Homebox

LDL Low-Density Lipoprotein

LOD Limit Of Detection

LOQ Limit Of Quantification

LRAT Lecithin:retinol acyltransferase

MTT 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide

NEAA Non-essential amino acids

NO Nitric Oxide

NPC1L1 Niemann-Pick C1-like 1

PBS Phosphate-Buffered Saline

RALDH Retinal dehydrogenase

RBP Retinol-Binding Protein

RBPR2 RBP4-receptor 2

RDH Retinol dehydrogenase

RE Retinol Equivalent

SCFA Short Chain Fatty Acid

SHIME Simulator of the Human Intestinal Microbial Ecosystem

SRB Sulforhodamine B

SR-BI Scavenger Receptor class B type I

STRA6 Stimulated by Retinoic Acid gene 6

TCA Trichloroacetic acid

TEER Trans Epithelial Electrical Resistance

VLDL Very-Low-Density Lipoprotein

2

LIST OF TABLES AND FIGURES

Table 1: Molecular structures of the most abundant forms of vitamin A occurring in the human diet and their main

dietary sources. The structures were obtained from ChemSpider [5], while the main food sources were found

on NutritionData [6]. ...................................................................................................................................... 5 Table 2: Different intestinal components able to influence the absorption of vitamin A in the intestine and the

mechanisms that could be involved. PC = phosphatidylcholine; LysoPC = lysophosphatidylcholine......... 15 Table 3: Factors influencing the performance of Caco-2 cells. BB: Brush border; TEER: Trans-epithelial electrical

resistance [183]. ........................................................................................................................................... 21 Table 4: Concentration of the different components in the emulsion made by co-solvent evaporation. ............... 30 Table 5: Concentration of the different components in the emulsion made by self-emulsification. ..................... 31 Table 6: Summary of all performed transport experiments with Caco-2 cells (ATCC) grown onto Transwell®

plates. Changes in composition, performed pretreatments and extra added components in the medium are

mentioned. For each experiment the used cell surface area, the cell passage number and the time of transport

are indicated. Also the used extraction procedure after the experiment is presented. .................................. 32

Figure 1: Diversity of vitamin A recommendations for males (left) and females (right) in Europa expressed in µg

retinol equivalents (RE) per day for selected ages. The boxes indicate the interquartile range (IQR) in which

the median is indicated by a horizontal line. Vertical lines at the boxes indicate values less than 1.5 IQR

below the first quartile or above the third quartile. Outliers are indicated by open dots and stars. DACH =

Austria, Germany and Switzerland [30]. ........................................................................................................ 7 Figure 2: Products of the central and eccentric cleavage pathways of β-carotene [40]. .......................................... 9 Figure 3: Possible structure of a mixed β-carotene micelle in the upper part of the intestines. ............................ 10 Figure 4: Proteins involved in uptake, transport and secretion pathways of vitamin A and carotenoids across the

enterocyte. Vit = vitamin; βC = β-carotene; αC = α-carotene, βC = β-cryptoxanthine, Lut = lutein; Lyc =

lycopene; Car = carotenoids; A = retinol putative specific transporter; B = unidentified apical transporter; C

= passive diffusion; D = unidentified basolateral efflux transporter; ? = putative pathway [2]. .................. 12 Figure 5: Synthesis of bile acids present in human bile. The primary bile acids, cholic acid and chenodeoxycholic

acid, are synthesized from cholesterol in hepatocytes, where they are conjugated with taurine or glycine to

be finally secreted as bile salts by the gallbladder. Secondary bile acids, on the other hand, are formed if

bacterial biotransformation occurs prior to the enterohepatic circulation. This way, deoxycholic acid and

lithocholic acid can be produced, which can also be conjugated in the liver. Gly = glycine; Taur = taurine

[106]. ............................................................................................................................................................ 15 Figure 6: Processes that have to take place to obtain absorption of carotenoids in the gastro-intestinal tract. UWL

= unstirred water layer.tion/Caco-2 cell uptake model to assess carotenoid. CEL = carboxyl ester lipase

bioavailability (based upon Failla et al. [147]). ............................................................................................ 18 Figure 7: Representation of a coupled in vitro digestion/Caco-2 cell uptake model to assess carotenoid

bioavailability. CEL = carboxyl ester lipase (based upon Failla et al. [147]). ............................................. 22 Figure 8: Biochemical vitamin A deficiency (retinol) as a public health problem by country 1995–2005: Preschool-

age children. Countries and areas with survey data and regression-based estimates [191]. ......................... 23 Figure 9: Schematic representation of an insert in a Transwell® plate [194]. ....................................................... 26 Figure 10: Schematic representation of the different stages in a SHIME incubation [206]. ................................. 33 Figure 11: Micelle sizes (left) and zeta-potentials (right) of the emulsions containing different types of oil phases:

palm oil, stripped or non-stripped sunflower oil and oleic acid. (B) Error bars represent the standard

deviations. *: p < 0.05; **: p < 0.01 (t-test compared to the blank). The blank only contains

phosphatidylcholine and sodium cholate, which are present in all other conditions. β-car: β-carotene. ...... 35 Figure 12: The calculated incorporation percentages of β-carotene in the micelles. The results are presented as the

percentage of the area under the curve of β-carotene to internal standard compared to the respective blank.

Blanks represent the same amount of β-carotene dissolved in ethanol before extraction. Error bars represent

the standard deviations. *: p < 0.05; **: p < 0.01 (t-test compared to the respective blank). ....................... 36

3

Figure 13: Results of the MTT and the SRB assay 3 days after treatment of (A) undifferentiated and (B)

differentiated Caco-2 (ATCC) cells. The values are presented as the percentage of the optical density

compared to the untreated cells. Error bars were calculated using the standard deviations. *: p < 0.05; **: p

< 0.01 (t-test compared to untreated cells). .................................................................................................. 36 Figure 14: Results of the MTT and the SRB assay 3 days after treatment of undifferentiated Caco-2 (ATCC) cells

with a dilution series of different emulsions. The values are presented as the percentage of the optical density

compared to the untreated cells. Error bars were calculated using the standard deviations. *: p < 0.05; **: p

< 0.01 (t-test compared to untreated cells). .................................................................................................. 37 Figure 15: The mass percentage of ethanol evaporated from a β-carotene emulsions with a rotary evaporator during

a period of 4 h. Error bars represent the standard deviations........................................................................ 37 Figure 16: Results of the MTT and the SRB assay 3 days after treatment of (A) undifferentiated and (B)

differentiated Caco-2 cells with emulsions containing PBS, whether or not filtered (0.22 µm). The blank only

contains sodium cholate and phosphatidylcholine. The values are presented as the percentage of the optical

density compared to the untreated cells. Error bars were calculated using the standard deviations. *: p<0.05;

**: p<0.01 (t-test compared to untreated cells). ........................................................................................... 38 Figure 17: Results of the MTT and the SRB assay on differentiated Caco-2 cells (A) 1 and 3 days after treatment

with β-carotene emulsions by the self-emulsification method with or without Tween 80, (B) 3 days after

treatment with a dilution series of the amount of Tween 80 present in the self-emulsification method with or

without β-carotene. The values are presented as the percentage of the optical density compared to the

untreated cells. Error bars were calculated using the standard deviations. *: p<0.05; **: p<0.01 (t-test

compared to untreated cells). ........................................................................................................................ 38 Figure 18: Calibration curve of β-carotene in cell culture medium using the second extraction procedure. AUC =

area under curve; IS = internal standard. ...................................................................................................... 39 Figure 19: Recovery of the internal standard (IS) and of different β-carotene concentrations in cell culture medium.

...................................................................................................................................................................... 39 Figure 20: Apical concentrations of β-carotene prepared with the co-solvent evaporation method in function of

transport time. ............................................................................................................................................... 40 Figure 21: Results of the kinetic transport experiment, without (A) and with (B) addition of Tween 80 in the

emulsions prepared by self-emulsification. The percentage of β-carotene in the apical compartment, the basal

compartment and the cells is presented on the y-axis and in the table below for the different time points.

Carotenoids could not be detected if a value of 0 is shown. ......................................................................... 41 Figure 22: Results of the transport experiment, comparing the co-solvent evaporation and self-emulsification

method. On the y-axis, the mass of β-carotene that was found in each compartment is indicated for the

different emulsions. ...................................................................................................................................... 41 Figure 23: Results of the nitric oxide (NO) test on different concentrations of the intestinal samples diluted in cell

culture medium and on cell culture medium after incubation of the samples with undifferentiated Caco-2

(TC-7) cells. Error bars were calculated using the standard deviations. FOS: fructo-oligosacharides. ........ 41 Figure 24: Results of the MTT (A) and SRB (B) assay one day after treatment of undifferentiated Caco-2 (TC-7)

cells with different dilutions of SHIME samples with exposure medium in duplicate. The values are presented

as the percentage of the optical density compared to the untreated cells. Error bars were calculated using the

standard deviations. *: p<0.05; **: p<0.01 (t-test compared to untreated cells). FOS: fructo-oligosacharides.

...................................................................................................................................................................... 42 Figure 25: Short-chain fatty acids (SCFAs) present in the intestinal samples with the specific concentrations of

acetate, propionate and butyrate. The iso-SCFAs, isobutyrate and isovalerate, are also presented. FOS: fructo-

oligosacharides. ............................................................................................................................................ 42 Figure 26: The transepithelial electrical resistance (column charts) and the apparent permeability (Papp) values (line

charts), calculated from the Lucifer Yellow test, of the Caco-2 (TC-7) cells during the pretreatment period

with different intestinal samples. Error bars represent standard deviations. FOS: fructo-oligosacharides. .. 43 Figure 27: Percentage of the total β-carotene amount present in the cells and basal compartment after the transport

experiment on pretreated cells with intestinal samples. Error bars were calculated using the standard

deviations. FOS: fructo-oligosacharides. ...................................................................................................... 43

4

INTRODUCTION

Vitamin A has multiple important functions in the human body, among which its roles in vision and the

immune system are best known. Since the bioavailability of vitamin A molecules is relatively low,

particularly due to their high hydrophobicity, sufficient amounts should be present in the diet.

Considering this seems to be a problem in many developing countries, deficiencies are rather common.

Noteworthy is the higher incidence of vitamin A deficiency in children than in adults, suggesting the

necessity of maturation of the vitamin A transport function in the human intestines during development.

Few studies have however been performed to discover underlying mechanisms for this maturation. More

fundamental research is thus still needed. With this master’s dissertation an attempt was made to

partially eradicate this gap in knowledge.

The hypothesis is investigated that the intestinal matrix is able to influence the development of the

transport function for vitamin A, more particularly the main provitamin A carotenoid β-carotene, in

intestinal cells. In order to reach this final aim, some other objectives have to be completed. Therefore,

a delivery method for β-carotene to intestinal cells is optimized. Further, an in vitro cell line model is

developed to be able to study the absorption and secretion of β-carotene by intestinal cells.

In this master’s thesis, the problem of vitamin A bioavailability is first broadly analyzed in a literature

study. Herein, the digestion and the metabolism of vitamin A is fully explained, potential influences on

these mechanisms are explored and in vitro cell line models to study the bioavailability of vitamin A are

reviewed, with the delivery of β-carotene as an essential aspect. Further, the used techniques in the

performed experiments are described in the material and methods section, after which the obtained

results are presented and thoroughly discussed. Eventually, the most important findings and possible

future perspectives are specified.

5

I LITERATURE STUDY

I.1 Vitamin A and its importance in human health

I.1.1 Molecular structure and occurrence in food sources

Vitamin A refers to a group of organic compounds. Based on solubility, it can be categorized with the

fat-soluble vitamins. Like other vitamins it is essential for the normal functioning of the human body,

since humans are not capable of synthesizing their vitamins in sufficient amounts. Thus, it is essential

to obtain enough vitamin A by the diet [1]. The form in which vitamin A occurs depends mainly on the

type of food source. In foods from animal origin preformed vitamin A is present as retinyl esters

(primarily retinyl palmitate), while in plant-based foods provitamin A carotenoids occur. These

carotenoids can be converted to retinoids in the human body (see I.2.1) [2]. Only a few carotenoids are

present in significant amounts in the human diet and could also be detected in the bloodstream, being β-

carotene, α-carotene and β-cryptoxanthin [3, 4]. In Table 1 the structures of these molecules and their

main dietary sources are listed [5, 6].

Table 1: Molecular structures of the most abundant forms of vitamin A occurring in the human diet and their main

dietary sources. The structures were obtained from ChemSpider [5], while the main food sources were found on

NutritionData [6].

Molecular structure Main food sources

Retinyl palmitate

Liver, kidneys, fatty

fish, margarine, cheese

β-carotene Lettuce, spinach, carrots

α-carotene Carrots, pumpkin

β-cryptoxanthin

Pumpkin, red peppers,

coriander

I.1.2 Functions in the human body

Vitamin A is probably the most multifunctional vitamin in the human body and has been proven to play

a key role from the embryogenesis to adulthood. It is likely that the large extent of cellular activities is

not yet fully known, since new biological functions are constantly being discovered [7]. Some of the

most important functions of vitamin A are summarized below.

6

Vitamin A is particularly known to play an important role in vision. In the eye, retinoids are being

converted to the visual chromophore, 11-cis-retinal, which upon binding to opsins can form functional

visual pigments [8]. Aside from this role in vision, most of the biological functions of vitamin A depend

on its retinoic acid form by regulating nuclear hormone receptors that control gene transcription. This

way, vitamin A is essential for normal cell growth and differentiation and thus has an impact on

reproduction by controlling spermatogenesis and helps improve skin texture by maintaining epithelial

surfaces [9, 10]. In addition, specific functions of vitamin A in embryonic growth and development exist.

Several studies have indicated its particular importance in fetal lung development and maturation [11].

Further, vitamin A fulfills a crucial role in immune competence, for instance by signaling the

differentiation of dendritic cells [12]. The normal functioning of the brain has also been shown to be

dependent upon vitamin A intake. The precise mechanisms are still unclear, but a couple of studies have

indicated a role in sleep, learning and memory [13]. A last property of vitamin A that should be

highlighted is its potential role as an antioxidant in preventing cancer development. Growth of certain

types of tumor cell lines has been proven to be inhibited by interactions of retinoids with DNA [14, 15].

I.1.3 Vitamin A deficiency

Dietary vitamin A deficiency remains a public health problem in most countries, particularly in Africa

and South Asia where meat intake is low. But insufficient vitamin A intake has also been reported in

Western societies [16]. Young children and pregnant women are especially prone to develop this

deficiency [2]. Inadequate intake of vitamin A can lead to severe health problems that are able to cause

blindness, anaemia and increased risk of mortality [17].

Several strategies exist to improve the vitamin A status of which dietary diversification, fortification

and supplementation are the most viable [18]. In the first approach cultivation of more foods rich in

vitamin A is linked with nutritional education to improve the consumer behaviour. Second, fortification

of food can be achieved by genetic engineering of the carotenoid pathway in common crops, like rice,

potatoes and corn [19-21]. In addition, recent agricultural research has also suggested biofortification or

selective breeding as a useful strategy, in which varieties rich in vitamin A are grown, like sweet potato

and manioc with a higher vitamin A content. In the last strategy, supplements with a high vitamin A

concentration in the form of tablets, capsules or injections are administered to populations at high risk

[22]. Yet, β-carotene supplementation has been shown to cause adverse health effects in people at risk

of certain diseases. In several clinical trials the risk of lung cancer and cardiovascular diseases in

smokers was increased by high-dose supplementation [23].

I.1.4 Bioavailability, influencing factors and recommended uptake of vitamin A

The bioavailability of vitamin A can be defined as its absorbed proportion from the diet that is used for

functioning of the body [24]. In contrast, bioaccessibility only refers to the amount of ingested vitamin

7

A that is released from the food matrix in the gastrointestinal tract and turned into a chemical form that

is available for uptake by intestinal cells [25]. Several factors are able to influence the bioavailability of

vitamin A. On the one hand, external factors like the type of food matrix and the chemical form in which

vitamin A is present, can play a role. For instance, vitamin A from plant sources is generally more

difficult to absorb and thus less bioavailable than the preformed vitamin A present in animal products

[26]. On the other hand also internal factors are important, such as gender, age, nutrient status and life

stage (e.g. pregnancy, lactation). It is already known that recommendations for vitamin A will differ

according to these factors and thus be rather host-related [24].

More knowledge about vitamin A bioavailability is still necessary to help translate physiological

requirements into actual dietary needs [27]. Existing dietary recommendations for vitamin A now still

differ between countries and institutions. In Figure 1 the diversity of recommendation in Europe is

presented. Moreover, various terms are used to refer to the levels of requirement, according to the

already available evidence. Mostly, the aim is to define a recommended dietary allowance, which is the

average daily level of intake considered sufficient to meet nutrient requirements of almost all (97-98%)

healthy individuals. But when insufficient evidence is available, the adequate intake can be established

as the derived intake by a defined population group that is assumed to sustain health and ensure

nutritional adequacy [28]. In Europe, European micronutrient Recommendations Aligned (EURRECA),

a network of excellence funded by the European Commission, has been established to address this

problem of differences between countries by developing methodologies to standardize the process of

setting micronutrient recommendations [29, 30]. Further, the amount of vitamin A is most frequently

defined in retinol activity equivalents since the body converts all dietary sources of vitamin A into retinol.

Figure 1: Diversity of vitamin A recommendations for males (left) and females (right) in Europa expressed in µg retinol

equivalents (RE) per day for selected ages. The boxes indicate the interquartile range (IQR) in which the median is

indicated by a horizontal line. Vertical lines at the boxes indicate values less than 1.5 IQR below the first quartile or

above the third quartile. Outliers are indicated by open dots and stars. DACH = Austria, Germany and Switzerland

[30].

8

The consensus has been reached that 1 µg of physiologically available retinol is equivalent to 12 µg of

β-carotene and 24 µg of α-carotene or β-cryptoxanthin. If the type of food source is taken into account,

international units can be defined [28, 31].

I.2 Digestion and metabolism of vitamin A

I.2.1 Biological conversions of vitamin A and its derivatives

It is assumed that only the free forms of vitamin A can be absorbed in the gastrointestinal tract. Thus,

the esterified form of vitamin A released by foods of animal origin cannot be absorbed by the enterocytes

and first, hydrolysis should take place. It has been shown that retinyl esters are essentially hydrolyzed

in the duodenum. Two enzymes are present in pancreatic juice that could perform the hydrolysis:

cholesterol ester hydrolase (CEH) and pancreatic lipase. In in vitro studies, CEH has been shown to be

effective, however in vivo studies using knock-out mice did not show a significant involvement of the

enzyme in this hydrolysis [32-34]. Other in vitro studies provided evidence for the hydrolysis of retinyl

palmitate by lipases without the involvement of CEH. Hence, the assumption could be made that luminal

hydrolysis is achieved by lipase, in association with the pancreatic lipase-related protein 2 [35]. Esters

that have not been hydrolyzed by these enzymes, can still be cleaved by a mucosal or a brush border

esterase near the enterocytes, since studies on homogenates of isolated jejunal rat enterocytes have

shown an esterase activity and similar activities have been found for preparations of human brush border

membranes (BBM) [36, 37]. Finally, a minority of esters could be taken up intactly by the enterocytes

and hydrolyzed intracellularly [38].

Provitamin A carotenoids such as β-carotene can be absorbed intact in the intestine, but about half is

converted to retinol prior to absorption, although this amount can vary broadly among individuals [39].

Next to the intestine, another major site of β-carotene conversion in humans is the liver. Two pathways

have been described that lead to the cleavage of β-carotene to retinoids: central and eccentric (Figure 2)

[40]. The most important pathway is the central cleavage which is catalyzed by the cytosolic enzyme β-

carotene 15,15’-oxygenase 1 (BCO1). β-carotene is being cleaved at its central double bonds and

generates two molecules of retinal [41]. Other provitamin A carotenoids are only able of yielding one

retinal molecule [3, 4]. In the second pathway cleavage occurs at other double bonds of the β-carotene

molecule and in this way β-apo-carotenals can be produced with various chain lengths, which are further

converted to one molecule of retinal by a still unknown mechanism. The existence of this pathway is

still the subject of debate, since the asymmetrical cleavage of β-carotene may also occur non-

enzymatically by auto-oxidation [42]. Nevertheless, in vivo studies with mice were capable of

characterizing an eccentric cleavage enzyme, β-carotene 9’,10’-oxygenase 2 (BCO2), which is able to

convert β-carotene into β-apo-10’-carotenal [43]. After some in vitro studies, it was suggested that this

9

secondary pathway occurred preferentially under oxidative conditions, like smoking and certain diseases,

or in the presence of high concentrations of β-carotene [44].

To obtain retinoic acid, which is responsible for most of the vitamin A functions (see I.1.2.), further

oxidation of the retinal molecule is necessary. The conversion to this biologically active form is

performed by enzymes of the retinal dehydrogenase (RALDH) family [45]. If production of retinoic

acid in tissues surpasses certain levels, more polar compounds, such as 4-oxo retinoic acid, can be

generated through oxidative degradation by enzymes belonging to the family of cytochrome P450. These

compounds are believed to be transcriptionally inactive [46]. Alternatively, retinal can be reduced

reversibly to retinol, which can be catalyzed by various retinol dehydrogenases (RDH). By the action of

acyltransferases on retinal, retinyl esters are generated which are the storage form of vitamin A in various

tissues, under which primarily the liver [45, 47]. Two main enzymes have been proposed to catalyze the

esterification: lecithin:retinol acyltransferase (LRAT) and acyl CoA:retinol acyltransferase (ARAT). In

vivo studies with knock-out mice have demonstrated the major physiological role of LRAT in retinol

esterification [48]. However, it has been shown that the enzyme acyl CoA:diacylglycerol acyltransferase

1 (DGAT1) acts as an ARAT in murine skin and intestine, in the presence of extensive dietary retinol

[49].

Figure 2: Products of the central and eccentric cleavage pathways of β-carotene [40].

10

I.2.2 Solubilization in the gastrointestinal tract

I.2.2.1 Role of dietary fat: micelles

Like other fat-soluble micronutrients, vitamin A is assumed to be incorporated with other lipids into

mixed micelles in the stomach and duodenum [50]. In this first phase of the digestion process,

emulsification of vitamin A and its derivatives into lipid droplets occurs which consist of phospholipids,

cholesterol, bile salts and lipid digestion products, like free fatty acids, monoacylglycerols and

lysophospholipids [51, 52]. Several factors can affect the

transfer of vitamin A into micelles during dietary lipid

lipolysis, such as the molecular structure of vitamin A, pH and

the presence of dietary fat [50, 53]. In Figure 3 the structure

of the micelles obtained is represented. The micelles are

eventually isolated from the other intestinal content in the

unstirred water layer of the glycocalyx area of the enterocytes.

Due to the acidic microclimate in this area, fatty acids will

shift to their protonated form which reduces their solubility in

micelles. Thus, the vitamin A molecules will be released from

the micelles near the BBM [2].

I.2.2.2 Other vehicles involved: vesicles and proteins

Apart from mixed micelles, it cannot be excluded that some other structures when present in the aqueous

fraction of the meal are also capable of incorporating retinol and carotenoids. Lipid structures that

coexist in the gastrointestinal lumen during digestion are vesicles that are assumed to be liposome-like

structures consisting of one or more bilayers of phospholipids. Studies in which incorporation of vitamin

A into phospholipid bilayers was observed, support this hypothesis [54]. Moreover, it was shown that

the presence of vitamin A can enhance the stability of these vesicles [55]. Alternatively, association with

proteins solubilized in the aqueous phase cannot be ruled out either. The protein β-lactoglobulin, present

in cow milk, is capable of binding retinol and carotenoids [56, 57]. Thus, it has to be kept into account

that proteins from the diet or obtained by pancreatic or biliary secretions could also bind vitamin A

molecules and transport them into the enterocytes. The mechanism of vitamin A absorption will most

likely depend on the associated vehicles. But the distribution of vitamin A molecules between the

different vehicles is still unclear [1].

I.2.3 Intestinal absorption of vitamin A molecules

I.2.3.1 General mechanism

A first crucial step in the intestinal absorption of vitamin A is its cellular uptake by intestinal mucosal

cells, which was first studied using rat everted intestinal sacs. In these studies it was hypothesized that

Figure 3: Possible structure of a mixed β-

carotene micelle in the upper part of the

intestines.

11

preformed vitamin A was absorbed via carrier-dependent proteins, while passive diffusion of all

carotenoids occurred [58, 59]. However, posterior findings, such as the high inter-individual variability

of absorption in human studies, the discrimination between isomers for cellular uptake and the

competition for absorption between carotenoids in Caco-2 cells, were not in agreement with this

hypothesis [60, 61]. More recent studies therefore have revisited these assumptions and have shown that

the actual mechanisms are much more complex. It was suggested that passive diffusion of all vitamin A

molecules only occurs at high, pharmacological doses, while at physiological doses, a protein-mediated

transport could be involved [2, 62]. This hypothesis was strengthened by the identification of the

Drosophila gene NinaD encoding for a class B scavenger receptor that was found to be essential for

carotenoid distribution into cells [63].

Second, intracellular transport of the molecules through the enterocytes has to be completed, for which

today little is known yet. However, already various candidate proteins have been proposed to be

involved [1]. A next important step is the secretion of vitamin A from the cells. It is thereby assumed

that the lipophilic vitamin A molecules are mostly incorporated into chylomicrons or other lipoproteins,

which will facilitate their secretion into the lymph and the portal circulation [64-66]. It is hypothesized

that the carotenoids can be exchanged dynamically among lipoproteins, like VLDL, IDL, LDL and HDL,

during their circulation in the body [64, 67]. Finally, vitamin A molecules can be acquired by different

tissues for storage or metabolism [68]. In Figure 4 the most important proteins involved are summarized

[2].

I.2.3.2 Membrane proteins involved in apical uptake and efflux

For the uptake of retinol by enterocytes, it was suggested that the protein Stimulated by Retinoic Acid

gene 6 (STRA6) is involved, since this protein was shown to be a specific receptor for retinol-binding

protein (RBP) [69]. Studies concluded that STRA6 could act as a bidirectional transporter of retinol,

where the polarity is determined by intracellular retinol concentrations [2]. More importantly, a synergy

between STRA6 and LRAT expression has been shown. Enterocytes that express both these proteins

therefore are able to take up more retinol [70]. This gives an indication that the conversion of retinol

into retinyl ester by LRAT in the cell is the driving force for STRA6-mediated retinol uptake. Recently,

it has been discovered that the protein RBP4-receptor 2 (RBPR2), which is structurally related to STRA6,

may also play a role in retinol uptake in the intestine [2].

For the uptake of carotenoids several lipid transporters have been identified on the membranes of

intestinal cells, of which the Scavenger Receptor class B type I (SR-BI), Cluster Determinant 36 (CD36)

and Niemann-Pick C1-like 1 (NPC1L1) are the most important [71, 72]. It has been hypothesized that

the effective role of the first transporter, SR-BI, is to facilitate the uptake of lipids other than cholesterol

[1]. Studies using cell lines have proven the involvement of SR-BI in the intestinal uptake of carotenoids,

like β-carotene [73, 74]. Moreover, it was shown that intestinal lipid absorption by SR-BI is being

12

controlled by retinoid signaling. Retinoic acid is

able to induce the expression of the transcription

factor Intestine-Specific Homebox (ISX) which

represses both SR-BI and BCO1 expression [75].

For another scavenger receptor of interest, CD36,

transfected COS cells and mouse BBM vesicles

have been used to show its role in the uptake of β-

carotene [74]. Finally, an experiment in which the

specific inhibitor of NPC1L1, ezetimibe, was used

on Caco-2 cells, showed decreased α- and β-

carotene uptake by 50% and β-cryptoxanthin

uptake by 20% [76]. However, there is still some

discussion about the actual involvement of

NPC1L1, since a similar study was not able to

reproduce these results [77].

Various proteins on the apical side of enterocytes

are able to efflux molecules back to the intestinal

lumen, this way regulating their absorption. But on

the apical efflux of carotenoids nothing is really

known yet. However, it has already been shown in

Caco-2 cells that SR-BI can efflux vitamin D and

E across the BMM [78, 79]. Therefore, a role of

SR-BI in the efflux of vitamin A cannot be

excluded [2]. Further, it has been hypothesized that

other transporters, like ATP-Binding Cassette (ABC) transporters can serve as efflux pumps. More

particularly, the protein ABCG5 has already been suggested to play a role in the plasma response of

dietary carotenoids [80].

I.2.3.3 Intracellular transporters

As mentioned before, the intracellular transport of vitamin A through the enterocytes is still a black box.

Since these molecules are water-insoluble, it is assumed that they require intracellular proteins or

incorporation into intracellular membranes to finally arrive at the basolateral side. A first possibility for

this transport, are the apical membrane transporters (Figure 4): SR-BI, CD36 and NPC1L1, as they were

already found in different organelles of the cell [81-83]. Upon binding of carotenoids at the apical side,

transfer within the enterocyte to other intracellular transporters or membranes could occur [1]. Further

it has been suggested that more specific transporters are involved, like retinoid-binding proteins that

Figure 4: Proteins involved in uptake, transport and

secretion pathways of vitamin A and carotenoids across

the enterocyte. Vit = vitamin; βC = β-carotene; αC = α-

carotene, βC = β-cryptoxanthine, Lut = lutein; Lyc =

lycopene; Car = carotenoids; A = retinol putative specific

transporter; B = unidentified apical transporter; C =

passive diffusion; D = unidentified basolateral efflux

transporter; ? = putative pathway [2].

13

also play a role in targeting the vitamin A molecules to the right site in the cell. Of special interest for

transport within enterocytes, is the Cellular Retinol-Binding Protein II (CRBPII), as its role in vitamin

A metabolism was shown in CRBPII-deficient mice [49, 84]. Concerning carotenoids, a carotenoid-

binding protein (CBP) has so far only been identified in the midgut of the silkworm Bombyx mori [85].

A last hypothesis is the involvement of non-specific fatty-acid binding proteins in the intracellular

transport, since they have a broad ligand specificity [86].

I.2.3.4 Basolateral secretion

It is assumed that an apoB-dependent route is used to incorporate newly-synthesized retinyl esters and

free forms of carotenoids into chylomicrons that are secreted on the basolateral side of the intestinal

cells [87]. However also another pathway may be involved in retinol absorption, since under some

conditions free retinol could be secreted by Caco-2 cells unassociated with lipoproteins [88]. The

existence of an ABCA1 transporter-dependent pathway has been shown, in which HDL is secreted by

the enterocytes, hence probably facilitating the efflux of free retinol [89, 90]. But studies using ABCA1-

deficient mice could not prove a significant involvement in the intestinal secretion of retinyl esters [91].

I.3 Influences on the intestinal transport function for vitamin A

I.3.1 Development and regulation of the transport function in the intestine

Several factors play a role in the regulation of intestinal transport. First of all, some age-related changes

in nutrient transporters have been shown to be genetically programmed and are thus independent of

external changes [92]. Interindividual variability can in this way be explained by different levels of

expression of genes encoding for crucial proteins in the vitamin A metabolism, such as CD36, BCO1

and SR-BI (see section I.2) [93]. In addition, several hormones are able to modulate postnatal changes

in intestinal transport. For example, aldosterone and glucocorticoids have been correlated with the

maturation of Na+ transport in humans and rats respectively [94, 95]. Also paracrine mediators, such as

histamine and serotonin, can exert both proabsorptive and prosecretory effects. Moreover, the

hypothesis that the epithelium could respond to luminal molecules has been explored. Studies have

confirmed that changing dietary input during ontogeny influences its transport mechanisms, not only by

modulating them, but in some cases they can act as primary signals for the development of transporters

[96]. Despite the potential of many intestinal components to influence vitamin A transport, only few

studies have been performed to investigate some of these effects. A detailed overview of these findings

is given in section I.3.3.

It has been suggested that the transport of vitamins through the intestinal epithelium is subjected to some

distinct developmental changes, which are not following the same pattern for all different vitamins. In

general, it could be concluded that there is a decrease in the rate of passive diffusion processes; while

14

active, saturable transport changes in capacity and affinity [96]. A study on suckling and adult rats found

that the maturation of the carrier-mediated uptake of retinol into enterocytes can be associated with a

decrease in capacity and an increase in the transport affinity. The distribution of the transport along the

small intestine stayed the same during the development [97].

I.3.2 Disorders causing vitamin A malabsorption

Previous studies were able to associate several disorders with the malabsorption of vitamin A. For

instance, in a clinical trial with children suffering from severe protein malnutrition, serum vitamin A

levels could only be elevated when a dietary treatment with milk preceded the administration of vitamin

A [98]. Further, all kind of diseases impairing the liver function have been related to vitamin A

deficiency. It was hypothesized that the reduced intraluminal bile salt concentrations could be an

important factor in this [99-101]. Also the occurrence of pancreatic insufficiency, in which there is an

impaired production of digestive enzymes, can cause malabsorption [102]. A final example of a disorder

able to lower the absorption of vitamin A is small intestinal bacterial overgrowth. It was hypothesized

that the deconjugation of bile acids by intraluminal bacteria is responsible for this by impairing in the

micelle formation [103]. These results confirm that vitamin A absorption is considerably influenced by

the presence of other components in the gastrointestinal tract.

I.3.3 Intestinal components influencing the absorption of vitamin A

A summary of all the components which will be discussed in this section is given in Table 2.

I.3.3.1 Bile acids

The primary function of bile acids is to promote digestion and absorption of dietary lipophilic

compounds by facilitating the formation of micelles in the gastrointestinal tract [104]. Studies have

shown that deletion of bile extract during the small intestinal phase in an in vitro digestion model

inhibited micellarization of carotenoids from a meal. Differences in bile acid secretion could thus

account for variations in vitamin A absorption among individuals [105]. The synthesis of the various

bile acids is presented in Figure 5 [106]. The composition of the bile acid pool varies dependent on diet

and the colonic microflora [107].

It has been shown that bile acids can act as important signaling molecules for regulation of the intestinal

transport function. Dependent upon the physicochemical properties of the bile acid, distinct effects are

exerted on the epithelial cells. The final outcome is however determined by multiple factors, such as the

specific transporters and receptors expressed on the target cells [107]. It has already been shown that

bile acids can decrease apoB secretion in Caco-2 cells by increasing its rate of degradation [108]. Since

the basolateral secretion of vitamin A can be apoB-dependent (see I.2.3.4), bile acids could also this

way be able to influence vitamin A transport.

http://en.wikipedia.org/wiki/Micelle

15

Table 2: Different intestinal components able to influence the absorption of vitamin A in the intestine and the

mechanisms that could be involved. PC = phosphatidylcholine; LysoPC = lysophosphatidylcholine.

Component Possible mechanisms involved Vitamin A absorption References

Bile acids Facilitating the formation of micelles; decrease apoB

secretion

Increased (extent depends

upon several factors such

as type of bile acid and

target cell characteristics)

[104, 107,

108]

Digestible oils Help in micellar solubilization; necessary for

formation of lipoproteins in secretion by intestinal

cells

Increased (extent depends

upon length of fatty acids

and saturation)

[109-113]

Fibers ( e.g. pectin) Interfere with micelle formation in the intestine by

occupying bile salts and fat

Decreased [114-116]

Gut microflora Decrease of intestinal transit time, interfere with the

reabsorption of bile salts

Decreased [117]

Iron Improving micelle stability by formation of a

complex

Decreased [118, 119]

Other carotenoids Competitive mechanisms; synergetic relationships:

by adaptive mechanisms in the body or by an

antioxidant-sparing response in the intestinal tract

Decreased in short-term,

increased in long-term

[120-123]

Phospholipids

Phosphatidylcholine Facilitating the formation of micelles; strong

association of hydrophobic carotenoids with the long-

chain acyl moieties of PC

Decreased [124]

Lysophosphatidylcholine Formation of smaller micelles; increased cellular

level of lipids by uptake of LysoPC results in

increased basolateral secretion

Increased [124, 125]

Sterols Competitive mechanisms between the components,

such as in the micellar incorporation and in the

intestinal uptake into the enterocytes; regulation of

apoB secretion; effects on intestinal transport

properties

Decreased [126-129]

β-lactoglobulin Protecting retinoids and carotenoids from

degradation and oxidation

Increased [57, 130,

131]

Figure 5: Synthesis of bile acids present in human bile. The primary bile acids, cholic acid and

chenodeoxycholic acid, are synthesized from cholesterol in hepatocytes, where they are conjugated with

taurine or glycine to be finally secreted as bile salts by the gallbladder. Secondary bile acids, on the other

hand, are formed if bacterial biotransformation occurs prior to the enterohepatic circulation. This way,

deoxycholic acid and lithocholic acid can be produced, which can also be conjugated in the liver. Gly =

glycine; Taur = taurine [106].

16

I.3.3.2 Phospholipids and dietary lipids

Already a lot of experiments have concluded that the presence of fats can influence the bioavailability

of carotenoids [132, 133]. A first important type of fat present in the gastrointestinal tract are

phospholipids, originating either from the diet or from bile. It has been reported that phosphatidylcholine

can suppress the uptake of β-carotene by Caco-2 cells, whereas its lipolysis product,

lysophosphatidylcholine, is able to enhance the uptake [124]. More recently, it was found that the effect

of phospholipids on β-carotene bioavailability also dependents on the chain length of the phospholipids

[134].

Further, only digestible oils, containing triacylglycerols, can help in the micellar solubilization of β-

carotene and thus, its bioaccessibility in a simulated gastrointestinal tract improves with increasing oil

content [109, 110]. The degree to which the absorption is elevated, depends on the type of triacylglycerol

molecules present. For instance, it could be concluded that co-ingestion of β-carotene with beef tallow

can increase the incorporation into plasma triglyceride-rich lipoproteins to a greater extent than with

sunflower oil [135]. Long chain fatty acids were found to have an increased solubilization capacity for

β-carotene compared to medium chain fatty acids, which results in a greater amount into the chylomicron

fraction of plasma [110, 112, 113]. Finally, also the degree of saturation can have an influence with

lipids rich in unsaturated fatty acids promoting micellarization of carotenoids to a greater extent during

simulated digestion. The uptake of β-carotene by Caco-2 cells seemed to be independent of the fatty

acid composition of the micelles [136]. Nevertheless, when pretreating Caco-2 cells with mixtures

enriched in unsaturated fatty acids, uptake and basolateral secretion of β-carotene could be enhanced

[111].

I.3.3.3 Plant and animal sterols

Plant sterols exhibit an inhibitory effect on carotenoid absorption, according to several in vitro and

human studies. It was suggested that this effect was caused by competitive mechanisms between the

components which could occur at various levels, such as in the micellar incorporation and in the

intestinal uptake into the enterocytes [126-128]. Also indirect effects of sterols on carotenoid absorption

could exist. For instance, in Caco-2 cells newly synthesized cholesterol esters can act as regulators of

apoB secretion, which is important for the basolateral secretion of vitamin A (see I.2.3.4) [129]. In

addition, animal studies have shown effects of low and high cholesterol diets on intestinal transport

properties, both short-term and long-term [137-139].

I.3.3.4 Dietary fibers

Dietary fiber is another dietary component that is able to negatively affect the absorption of carotenoids.

This has been shown in a few human studies [114, 115]. The degree of inhibition of β-carotene

absorption by pectins was found to be related to their structure in chickens, with citrus pectin having the

17

strongest inhibitory effect. It was suggested that fibers interfere with micelle formation in the intestine

by occupying bile salts and fat [116].

I.3.3.5 Gut microflora

In vivo experiments have confirmed that gut microflora affects the bioavailability of carotenoids [140,

141]. When the intestinal microflora was reduced or absent in rats, higher levels of vitamin A could be

stored in the liver. It is suggested that indirect mechanisms are responsible for this effect. The absorption

of carotenoids could be diminished by a decrease in the intestinal transit time caused by the bacteria.

Besides, the microflora can have an indirect effect on the absorption by deconjugation of bile salts,

which will interfere with their reabsorption [117].

In addition, several bacterial factors, such as short chain fatty acids (SCFA) and lipopolysaccharides,

could also be able to influence the bioavailability of vitamin A. However, no studies have been

conducted on this subject. Yet, it was already found that SCFAs have the ability to induce expression of

the vitamin D receptor in intestinal cells in vitro [142].

I.3.3.6 Other dietary components

During growth and development mainly maternal milk, colostrum and amniotic fluid in the uterus will

be able to influence transport in the gastrointestinal tract. Many biologically active substances, such as

hormones and growth factors, have already been detected and evidence for stimulatory effects on the

transport function has been provided by several studies [96, 143]. So far, only the bovine milk protein

β-lactoglobulin was found to promote absorption of β-carotene and retinol both in vivo, using mice, and

in vitro in Caco-2 cells [57]. Due to its structure similarity with RBP, it can bind retinoids and

carotenoids in vitro, this way protecting them from degradation and oxidation [130, 131].

A distinct potential factor for carotenoid bioavailability is the interaction with minerals and trace

elements. Previous studies already confirmed the ability of β-carotene to increase iron absorption in

Caco-2 cells and in humans [144, 145]. It was speculated that by forming a complex, the micelle stability

could be improved. However, no evidence for the exact mechanism was provided yet [119]. The reverse

effect was more recently investigated using Caco-2 cells and it was found that iron inhibits the uptake

of β-carotene [118].

Finally, other carotenoids present in the intestines were shown to cause interactive effects. In most

studies their bioavailability was negatively impacted because of competition between the different

molecules, similar to the sterols described earlier (I.3.3.3) [121-123, 146]. Yet, in a long-term human

study, it was found that synergetic relationships could occur, causing an increase in bioavailability of

the carotenoids. It was hypothesized that these effects can be the result of either adaptive mechanisms

by the body or of an antioxidant-sparing response in the intestinal tract [120].

18

I.4 β-carotene micelle formation as a means to improve absorption

I.4.1 Barriers for β-carotene absorption in the gastrointestinal tract

After uptake of carotenoids with the diet, some processes still have to occur to make absorption by

intestinal cells possible. In Figure 6 these processes are presented [147]. First, transfer of lipophilic

carotenoids from the vegetable matrix into the lipid phase has to appear, after which they have to be

solubilized into mixed micelles in order to obtain an efficient absorption (see I.2.2.1). The presence of

dietary fat during this step has already been shown to improve the bioavailability of β-carotene in

simulated digestion studies [53]. Also the pancreatic lipases and bile salts play an important role during

the micellarization [148]. Another crucial step is diffusion of the micelles towards the microvillus

membrane. The unstirred water layer will hereby be the rate limiting factor, with smaller micelles having

higher diffusion rates, and the pH-change in this area will control the release of the carotenoids from the

micelles [149, 150]. After this, absorption by mucosal cells has to take place, in which the poor

permeation of β-carotene into the cells will form an important barrier. Eventually, the carotenoids have

to be associated with lipoproteins and secreted at the basolateral side. In this final step the uptake of

fatty acids by the enterocyte is important. The loading of β-carotene in micelles has been shown to

improve its absorption in a coupled in vitro digestion/Caco-2 cell model [151].

I.4.2 Techniques to obtain emulsions

In general there are two different approaches for making micelles: high energy and low energy methods.

The high energy methods make use of several forces, such as hydraulic shear, intense turbulence and

cavitation, to obtain small droplets [152]. Micelles are most frequently prepared by techniques including

high-pressure homogenization and ultrasonication [153, 154]. The desired properties of the emulsion

mainly determine the operating conditions and the choice of homogenizer [155]. Nevertheless, some

Figure 6: Processes that have to take place to obtain absorption of carotenoids in the gastro-intestinal tract.

UWL = unstirred water layer.tion/Caco-2 cell uptake model to assess carotenoid. CEL = carboxyl ester lipase

bioavailability (based upon Failla et al. [147]).

19

characteristics of β-carotene, such as its sensitivity to oxygen, heat and light, also have to be taken into

account in the process [156]. Many studies have used high-energy methods to obtain β-carotene

emulsions [109, 113, 156-159].

On the other hand, low energy methods are based on the spontaneous formation of emulsions. This can

be achieved by modulating phase transitions. The specific environmental conditions necessary can be

created by using intrinsic physicochemical properties of surfactants, co-surfactants and oil [160]. Two

main classes of low energy techniques can be distinguished dependent upon the use of solvents. The

methods without the use of any solvents are also called self-nanoemulsification methods. In these,

emulsions are basically formed by the stepwise addition of water to a solution of surfactant and oil,

while gently stirring at room temperature [161]. In the second class, solvents can be applied in different

manners to create micelles. An organic phase is created by dissolving oil in a volatile solvent. After

which this phase is poured into an aqueous phase containing surfactant. If a water miscible solvent, such

as ethanol, is used, the aqueous phase triggers self-assembly, otherwise vigorous shaking is still

necessary [162]. The remaining organic solvent is eventually removed with, for instance, vacuum

evaporation which allows formation of more compact micelles [154, 163]. In only a few studies

nanodispersions containing β-carotene have been prepared with a low-energy method, called the solvent

displacement method [164-166].

I.4.3 Stability of β-carotene emulsions

I.4.3.1 Role of different components

The most important components that influence the stability of emulsions, are emulsifiers or surfactants.

Due to their amphiphilic character, they are able to decrease the surface tension of micelles. Next to the

so-called biosurfactants that naturally occur in the human gastrointestinal tract, such as bile salts and

phospholipids, a large variety of other emulsifiers can be used to stabilize emulsions [159, 167]. The

nature of the emulsifier used to stabilize emulsions can significantly influence their chemical stability

[168]. For instance, it was found that sodium caseinate can give nanodispersions with more resistance

to oxidation compared to other emulsifiers, like Tween 20 [165]. Further, degradation of β-carotene was

shown to be considerably decreased in emulsions using the protein β-lactoglobulin compared to those

with Tween 20 [159]. Besides the direct effect on emulsion stability of emulsifiers, other components

can exhibit an indirect effect, for instance by increasing the particle sizes or by changing the sensitivity

to pH changes.

I.4.3.2 Effect of temperature, pH and ionic strength

The composition of the emulsion will determine to what extent temperature, pH and ionic strength will

influence the stability of the micelles. It could however be concluded from previous studies that in

general β-carotene degradation in micelles is elevated with increased storage temperature [158, 159, 164,

20

169]. In addition, instability of carotenoid emulsions is considerably higher at acidic conditions [170].

When a protein is used as emulsifier, a pH near the isoelectric point will also cause droplet aggregation

in the emulsions. The ionic strength of the emulsion can further also exhibit an effect on the stability. It

has been found that a critical salt level exist above which aggregation occurs, at lower concentrations,

there is little effect [159].

I.4.3.3 Influence of the particle size

In the human gastrointestinal tract a large range of lipid droplet sizes occurs during digestion with a

mean diameter of about 30 µm [53, 171]. Micellar solubilization to particles in the nm-range will occur

and this greatly enhances the transfer across the unstirred water layer [150]. Studies have concluded that

the bioavailability of the incorporated β-carotene dependents on the particle size. When passing

emulsions with various initial particle diameters (23 µm to 0.2 µm) through a simulated gastrointestinal

tract, the proportion of lipid digestion and the β-carotene bioaccessibility noticeably increased with

declining mean droplet size [172]. No studies have however been conducted on the effect of particle

size on the uptake by the intestinal cells. Further, nanoemulsions with a mean droplet diameter smaller

than 200 nm will typically have an improved physical stability [173]. The small micelles are less

sensitive to gravitational separation and particle aggregation, but similar to conventional emulsions, it

are still thermodynamically unstable systems and therefore they can undergo flocculation, coalescence

and Ostwald ripening [174, 175].

I.5 In vitro simulation of the gastrointestinal tract

I.5.1 Caco-2 cell line

I.5.1.1 Origin and characteristics

The intestinal epithelial cell barrier has been simulated in a large amount of in vitro studies by the human

immortalized Caco-2 cell line, which is originally derived from a human colon adenocarcinoma. Due to

intrinsic heterogeneity of the parental American Type Culture Collection (ATCC) line often

spontaneous selection occurs as certain subpopulations of cells with different morphologies become

more prominent in the culture. Therefore, several clonal cell lines, such as TC-7, have been isolated that

show a more homogenous and stable expression of certain characteristics over passages [176, 177]. The

Caco-2 cell line exhibits several structural and functional characteristics of small intestinal enterocytes

upon spontaneous differentiation in long-term culture [178]. After 2 weeks a confluent polarized

monolayer of differentiated cells is formed that is joined by intracellular tight junctions and desmosomes,

which separate the apical side with microvilli from the basolateral membrane [179]. Several cell and

culture related factors are able to influence different features of the cells and thus cause variability.

These factors are listed in Table 3 [176].

21

Despite their colonic origin, the Caco-2 cell line is mainly used to represent the small intestinal

epithelium. During differentiation a change in the gene expression profile has been demonstrated, which

allows a shift in phenotype from tumoral colonic to more ‘normal’ small intestinal [180]. When

confluence is first reached, proteins characteristic for both enterocytes and colonocytes are expressed.

Thereafter, enterocytes-specific proteins are increased, whereas those specific for colonocytes should

diminish. It should however be taken into account that some colonic characteristics can persist during

differentiation due to several factors (see Table 3) [181, 182].

Table 3: Factors influencing the performance of Caco-2 cells. BB: Brush border; TEER: Trans-epithelial electrical

resistance [182].

I.5.1.2 Functions related to vitamin A metabolism

The human intestinal Caco-2 cell line is able to carry out activities necessary in the metabolism of

vitamin A. Firstly, it has been shown that the cells contain the protein CRBPII, which is a specific

intracellular transporter as mentioned before. Also the expression of ABC effluxers and transport

proteins SR-BI and NPC1L1 in Caco-2 cells has been confirmed (see I.2.3). Furthermore, some

important enzymes have also been discovered in Caco-2 cells, specifically retinal reductase, ARAT and

LRAT. Nevertheless, in the parental cell line the BCO1 activity is absent and thus carotenoids are not

cleaved into retinaldehyde or other apo-carotenoids. The presence of BCO1 was though demonstrated

in derived clones, such as the TC-7 cell line [62, 76, 111, 183].

Additionally, Caco-2 cells exhibit an endogenous lipolytic activity mainly in the cytosol, but also in the

apical BBM, allowing the hydrolysis of triglycerides into monoglycerides and free fatty acids [177].

Eventually, synthesis of new triacylglycerols, necessary for assembly and secretion of vitamin A in

lipoproteins, is made possible by the glycerol 3-phosphate pathway present in Caco-2 cells. In contrast,

the monoacylglycerol pathway is used in human enterocytes [184].

I.5.2 Cell culture-based simulation model

I.5.2.1 Coupled in vitro digestion/ Caco-2 cell model

In vitro models can offer useful information about the relative bioavailability of vitamin A. This way,

the effects of various food matrices, different styles of food processing and dietary components on

stability, bioaccessibility, transport and metabolism can be investigated. Since bioaccessibility by

simulated in vitro digestion has been validated as a reliable estimate of the in vivo situation, a coupled

Passage number Medium Support

pH Composition Material Pore size Matrix

BB enzyme activity

Morphology

TEER

Proliferation rate

Cell density

Transporter expression

Motility

Proliferation

Differentiation

Permeability

Permeability

Differentiation

Non-specific adsorption

Cell density

Morphology

TEER

Transport

Dome formation

Growth

Transport

Attachment

Spreading

Cell density

Differentiation

22

model with Caco-2 cells was developed for carotenoids [185]. Some modifications were made to better

reflect physiological conditions and eventually the procedure in Figure 7 has been suggested for further

investigations. It can be seen that after completing in vitro digestion of a meal, the obtained micelles are

isolated, filtered and applied to the Caco-2 cells in medium.

Dependent upon the aim of the experiment, the Caco-2 cells can

be grown on different types of membranes. When grown on a

permeable filter support, access of ions and nutrients to both

sides of the cell monolayer is allowed, which makes transport

of carotenoids possible [147].

I.5.2.2 Extrapolation to the in vivo situation

The coupled in vitro model encompasses some drawbacks if

compared to in vivo models. First off, the in vitro digestion

model is a static, closed system that is not influenced by, for

example, grinding, transit of the matrix through the intestine

and changes in content of digestive enzymes and bile salts due

to composition and quantity of food [147, 186]. Furthermore,

some enzymatic activities are absent in the model. In addition,

the used Caco-2 cultures lack biofilms, mucin and other

epithelial cell types that could affect their activity [147]. The

unstirred water layer above the cells can also become rate-

limiting in the transport of lipophilic compounds, such as

carotenoids [187]. Besides this limitation in transport,

membrane retention and non-specific binding of the

compounds to the filter support can occur. For correct

interpretation of the results, mass balances will be required

[188, 189].

Figure 7: Representation of a coupled in

vitro digestion/Caco-2 cell uptake model to

assess carotenoid bioavailability. CEL =

carboxyl ester lipase (based upon Failla et

al. [147]).

23

II PROBLEM STATEMENT

Vitamin A and its derivatives have multiple essential roles in growth and development and from birth to

adulthood. Since vitamins cannot by synthesized within the human body in sufficient amounts, the intake

of enough vitamin A or provitamin A is crucial to maintain normal functioning. Nevertheless, vitamin

A deficiency remains a public health problem in more than half of all countries, especially in Africa and

South-East Asia. This can lead to some severe consequences and disorders of which the most important

are night blindness, xerophthalmia, impaired immune function and increased risk of mortality [17].

The primary cause for vitamin A deficiency is inadequate consumption of vitamin A. Matrix effects and

the present chemical form of vitamin A herein play an important role, with provitamin A carotenoids

from fruits and vegetables having a much lower bioavailability than preformed vitamin A from animal

and dairy products. Therefore, in countries with low meat intake, deficiencies are more pronounced.

Secondary causes for vitamin A deficiency include chronic malabsorption of lipids, impaired bile

production and chronic exposure to oxidants, such as cigarette smoke [190]. However, these cannot

entirely explain the higher incidence of vitamin A deficiency in children than in adults. According to

the WHO, approximately one third of the children under the age of five around the world is affected and

it was estimated to even claim the lives of 670 000 of these children annually. In Figure 8, the worldwide

spread of this problem is visualized [191].

The high incidence in children indicates an evolution in the vitamin A uptake during human growth and

development. Up until now, the mechanisms behind this maturation and its potential triggers are not

fully understood and thus, more fundamental research is required. This could result in novel approaches

to reduce the prevalence of vitamin A deficiency worldwide.

Figure 8: Biochemical vitamin A deficiency (retinol) as a public health problem by country 1995–2005:

Preschool-age children. Countries and areas with survey data and regression-based estimates [191].

24

III OBJECTIVES

The need for maturation of the intestinal epithelium in order to absorb certain food compounds has been

demonstrated previously. Also for vitamin A, more specifically retinol, this was already proven in rats

[97]. Changes in intestinal transport of other nutrients were found to be genetically programmed.

Nevertheless, some extrinsic factors were able to influence the uptake in the human body too, such as

certain hormones and luminal molecules [96]. However, it is not entirely clear how the development of

the intestinal vitamin A uptake function progresses and more importantly, if this can be influenced by

certain external factors. In this master’s thesis, the main objective is to study the change in bioavailability

of vitamin A, more particularly the provitamin A carotenoid β-carotene, when intestinal cells are

exposed to distinct intestinal matrices during their differentiation.

To reach this final aim, first some subgoals have to be achieved. First of all, a delivery method has to be

realized to administer the very hydrophobic β-carotene to intestinal cells in aqueous medium. Secondly,

an in vitro cell model has to be developed that resembles the intestinal wall and thus could represent the

actual absorption that occurs in the human gastro-intestinal tract. To deliver the β-carotene to intestinal

cells, its loading in micelles could improve the absorption by the cells [151]. The production of the

emulsion should be standardized to avoid variability between different experiments. Further, a high

incorporation of β-carotene has to be achieved, so precipitation can be prevented. The micelles also need

to be quite stable during the experiments, with the release of β-carotene from the micelles occurring

only near the cell surface, similar to the in vivo process. A final crucial characteristic of the emulsion is

its non-toxicity to the cells. In the development of the in vitro cell model, one of the challenges is the

detection of the small amounts of β-carotene in the medium. These physiological concentrations are

necessary to achieve active transport, rather than passive diffusion [2]. Finally, transport of β-carotene

through the cells should occur, analogous to the in vivo mechanism.

When both subgoals are completed, the main objective can be achieved. The intestinal cells have to be

pretreated with intestinal water during their differentiation before performing transport of β-carotene.

Eventually, the hypothesis that changes in the diet of children are able to promote maturation of the

vitamin A transport in intestinal cells can be assessed. This master’s thesis could this way contribute to

new concepts for improving vitamin A bioavailability during human growth and development.

25

IV MATERIAL AND METHODS

IV.1 Chemicals and products

MEM non-essential amino acid (NEAA) solution, β-carotene (type I), triethylamine, sodium cholate

(hydrate, from ox or sheep bile), L-α-Phosphatidylcholine (60 %), TWEEN® 80, oleic acid, sodium

taurocholate hydrate, trichloroacetic acid (TCA), dimethylsulfoxide (DMSO), sulforhodamine B (SRB),

tris(hydroxymethyl)aminomethane, lipase (from porcine pancreas), diethyl ether, trans-β-apo-8′-

carotenal, 2,6-di-tert-butyl-4-methyl-phenol (BHT), aluminium oxide, potassium carbonate (K2CO3),

potassium hydroxide (KOH), silica and Griess reagent (modified) were purchased at Sigma-Aldrich

(Bornem, Belgium). Trypan blue and 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide

(MTT) were obtained from Amresco® (Leuven, Belgium), while sodium sulfate (Na2SO4), sodium

chloride (NaCl) and ethyl acetate were from Chem-Lab (Zedelgem, Belgium). The following solvents

were acquired at Fisher Scientific (Erembodegem, Belgium): dichloromethane, glacial acetic acid,

methanol, ethanol, acetonitrile, petroleum ether and hexane. The cell culture products Dulbecco's

Phosphate-Buffered Saline (PBS-, no calcium and no magnesium), Dulbecco's modified eagle medium

(DMEM), trypsin/EDTA, penicillin/streptomycin and Lucifer Yellow (CH, dipotassium salt) were

purchased at Life Technologies (Merelbeke, Belgium). Further, sunflower oil (Cora) was bought at

Smatch (Belgium) and fetal bovine serum (FBS) at Greiner Bio-One (Vilvoorde, Belgium). Finally,

ultrapure water was obtained by a Milli-Q® water purification system (Merck Millipore).

IV.2 Cell culture

IV.2.1 Caco-2 cell line

For the experimental work, the Caco-2 cell line was used. Besides the parental ATCC cell line, also the

derived TC-7 cell line was used during the research. To feed the cells, Dulbecco's Modified Eagle's

medium (DMEM) with 4.5 g/L glucose and Glutamax was used. This growth medium was supplemented

with fetal bovine serum (FBS, 10 %), MEM non-essential amino acid solution (NEAA, 1 %) and

penicillin-streptomycin (1 %) to further promote cell growth and avoid contamination. During

experiments, exposure medium was used that did not contain FBS.

For maintenance of the cell cultures, a desired amount of cells was passaged to a new flask when almost

reaching confluence. Therefore, phosphate buffered saline (PBS) without calcium and magnesium ions

was first put onto the cells to wash away remaining cell debris, growth medium and divalent cations that

could inhibit trypsin activity. Afterwards, the trypsinization step was performed in which the enzyme

26

trypsin cleaves proteins involved in cell-cell and cell-substratum adhesion, hence allowing adherent cells

to dissociate and detach from the substratum. The trypsin activity was finally inhibited by divalent

cations and FBS present in the added growth medium [192].

When quantification of the cells was needed, cells were counted in a Bürker counting chamber using a

phase-contrast microscope. Trypan blue was used to selectively color dead cells blue. Since viable cells

do not absorb this stain, it is possible to make a distinction between viable and dead cells [193].

IV.2.2 Transport assay

To assess the transport of β-carotene through the

Caco-2 cells, Transwell® plates were used. Caco-

2 cells (ATCC) are grown on a semi-permeable

membrane (0.4 µm, polyester), which allows

transport of compounds from the upper, apical

compartment; through the cells; to the lower,

basal compartment. In Figure 9 an insert of such a

plate is schematically presented [194]. Caco-2 cells were seeded at an initial concentration of

approximately 7.5 x 104 cells per cm2. After 21 days, the transport experiment was performed.

To evaluate and monitor the growth and permeability of the Caco-2 cell monolayer on the semi-

permeable membranes, two non-destructive methods were applied: Trans Epithelial Electrical

Resistance (TEER) measurements and the Lucifer yellow assay. In the first method, the change in TEER

across Caco-2 monolayers is measured using an epithelial voltohmmeter. As junctions between the cells

get stronger, it is more difficult for current to pass and hence the TEER is higher [195, 196]. In the

second assay, the fluorescent dye Lucifer yellow only travels across cell monolayers through passive

paracellular diffusion and has low permeability. It is thus not able to pass monolayers with strong tight

junctions between the cells [196]. The fluorescence in apical and basal medium was measured directly

with a fluorescence plate reader using a 485 nm excitation and an emission filter of 538 nm.

IV.2.3 Cytotoxicity tests

The following assays were carried out in 96 well plates. The Caco-2 cells were seeded at an initial

concentration of at least 6 x 104 cells per cm2. For undifferentiated cells, the treatment of the cells was

performed one to three days after seeding, while differentiated cells were treated after 21 days.

IV.2.3.1 SRB assay

The SRB assay is used to determine the cell density by measuring the cellular protein content [197].

Firstly, the cells were fixed for 1 h (4 °C) with trichloroacetic acid (TCA, 50 % in ultrapure water), after

which extracellular proteins were washed away using tap water. The protein dye sulphurodamine B

Figure 9: Schematic representation of an insert in a

Transwell® plate [194].

27

(0.4 % in 1 % glacial acetic acid) was subsequently added to bind proteins. Cells were then rinsed with

glacial acetic acid (1 % in ultrapure water) to remove unbound dye and the cells were suspended in Tris

buffer (10 mM). To measure the absorbance, a multireader was used at a wavelength of 490 nm.

IV.2.3.2 MTT assay

In the MTT assay a solution of the yellow 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide

(5 mg/mL in PBS) is brought onto the cells, which is reduced to the blue molecule formazan by

mitochondrial activity. An increased purple coloring therefore indicates an increased activity of the cells

[198]. After incubation in the dark (2 h, 37 °C), the medium was gently removed and the cells were

suspended in DMSO. The absorbance was determined at 570 nm with a multireader.

IV.2.3.3 Nitric oxide assay

Another test to study the cytotoxic potential of certain compounds to the cells, is the nitric oxide (NO)

assay. The production of this reactive nitrogen species by NO synthase in the cells is hereby examined.

NO was found to be an inflammatory mediator that helps in modulating the intestinal immune response

[199]. The measurement of NO is indirectly via its breakdown products, nitrite and nitrate, in the cell

culture medium [200]. To this end, 100 µL of cell culture medium (phenol free) and 100 µL of Griess

reagent were added in a 96 well plate, and after 15 min, the absorbance at 540 nm was measured using

a spectrophotometer. Concentrations of NO2 were quantified through a six-point matrix-matched

standard curve of sodium nitrite (NaNO2) (0-20 µM) in cell culture medium.

IV.3 Method of detection for β-carotene

IV.3.1 High-Performance Liquid Chromatography

β-carotene was analyzed by reversed-phase high-performance liquid chromatography (HPLC) with

DAD detection (Ultimate 3000, Dionex, Thermo). A reversed phased YMC-pack C30 column (250 mm

x 4.6 mm i.d., S-5 µm) from YMC (Schermbeck, Germany) was used with methanol:acetonitrile (9:1

v/v) as mobile phase A and ethyl acetate with 0.25 % triethylamine as mobile phase B. The flow rate

was 1 mL/min and the column temperature was 30 °C. The gradient profile was as follows: at 0 min

100 % A, linear gradient to 40 % A and 60 % B over 25 min, isocratic for 10 min, linear gradient to

100 % A over 5 min, isocratic for 5 min.

IV.3.2 Cell lysis

Before extracting the Caco-2 cells, a cell lysis step had to be performed. For collection of the cells, a

method analogous to literature was chosen [201]. The cell monolayers were first washed for two times

with precooled PBS to stop uptake and to wash the surface. Then, 10 % ethanolic PBS was added for

28

two times to dissociate cell monolayers. After mixing and scraping the cells from the surface, the cell

suspension was collected.

IV.3.3 Extraction of carotenoids

IV.3.3.1 Procedure 1

In this procedure, magnesium carbonate (50 mg) and 2,6-di-tert-butyl-4-methyl-phenol (BHT, 25 mg)

are first added to the samples. Magnesium carbonate will neutralize traces of organic acids that could

cause structural transformation or destruction of the β-carotene molecule, while BHT acts as an

antioxidant and prevents oxidation processes. During the whole procedure, aluminium foil prevented

light from reaching the samples. Then an ethanol:hexane (4:3 v/v, 35 mL) solution and an internal

standard solution of trans-β-apo-8′-carotenal dissolved in dichloromethane (0.175 g/L) was put into the

samples to be able to correct for losses during the extraction. This mixture was filtrated (185 mm

diameter) in a separatory funnel. Ethanol:hexane (4:3 v/v) solution, ethanol and hexane were used to

wash the filter. Further, a saponification step of 2 h was performed by incubating with 10 mL of a 10 %

KOH solution. Afterwards, the filtrate was washed with 10 w% NaCl-solution (10 mL) and distilled

water till a pH of 7 was reached. The organic layer was collected and filtrated on Na2SO4 to remove any

remaining water. The solution was evaporated at 40 °C with a rotary evaporator and dried under a

nitrogen flow. The samples were stored in the dark at -18 °C. Before performing the HPLC, the samples

were solved in 3 mL of acetonitrile, filtrated through an HPLC filter (0.45 µm) and brought into a vial.

An injection volume of 25 µL was used for the HPLC.

The protocol was adjusted to smaller sample volumes by reducing the used solvents and products. Only

2 mg of magnesium carbonate, 1 mg of BHT and 10 mL of ethanol:hexane solution were used. The

saponification step was reduced to 15 min after adding 0.5 mL of KOH. Afterwards, the samples were

resolved in 1 mL of acetonitrile. The injection volume in the HPLC was elevated to 100 µL.

IV.3.3.2 Procedure 2

For the extraction of the cell culture medium and the cells, an internal standard solution of trans-beta-

apo-8’-carotenal (0.175 g/L) was added to the samples to take into account any losses during the

procedure. Then, an ethanol:hexane (1:4 v/v, 2.5 mL) solution was mixed with the sample by vortexing.

The samples were centrifuged at 4500 g until phase separation was obtained. Afterwards, the organic

phase above was transferred to a glass vial and the sample was washed for 3 times with hexane (1 mL).

Finally, the organic phase was dried under a nitrogen flow. When necessary, the samples were stored in

the dark at -18 °C. The samples were resolved in 200 µL of acetonitrile for the HPLC and an injection

volume of 100 µL was used.

A calibration curve was obtained by extracting known amounts of β-carotene in cell culture medium.

Exposure medium was therefore incubated with Caco-2 (TC-7) cells for 1 day and after collection, the

http://www.ncbi.nlm.nih.gov/pubmed/3841351

http://www.ncbi.nlm.nih.gov/pubmed/3841351

29

β-carotene (125 µg/mL in dichloromethane) was spiked in 1.5 mL of medium per sample. Amounts

ranging from 15 nmol to 0.01 nmol of β-carotene were used to obtain the curve and to determine the

limit of detection (LOD) and quantification (LOQ). The recovery of β-carotene in the procedure was

assessed by putting some of the used amounts of β-carotene and internal standard immediately into the

HPLC vials and dissolving them in acetonitrile after drying.

IV.4 Emulsification of β-carotene

IV.4.1 Measurement of micelle characteristics

Dynamic light scattering, using a Malvern PCS-100 Spectrometer with 7032 CN digital correlator and

15 mW He–Ne laser, was applied to measure the micelle sizes of the emulsions. This device uses

electrophoretic light scattering technology to determine the z-average diameter as a measure for the

micelle size. The electrophoretic mobility was determined at 25 °C by the Zetasizer IIc (Malvern

instruments, England) and converted into a ζ-potential, which is the voltage difference between the

electrically charged micelle surface and the medium. Hence, it provides a measure of the electrical

repulsive force between the particles. Both characteristics give an indication of the stability of the

dispersion [202, 203].

Another characteristic of the emulsions that was assessed, is their cytotoxicity. Therefore, the earlier

described MTT and SRB assay were used (see IV.2.3). Finally, the incorporation of β-carotene into the

micelles was determined by comparing a certain amount of β-carotene dissolved in ethanol with the

same amount applied in the emulsion (1 mL sample). Both samples were filtered (0.45 µm) and

afterwards extracted and analyzed by a procedure described earlier (IV.3.3).

IV.4.2 Oil stripping

Sunflower oil was stripped to remove any remaining compounds that could interfere in further

experiments; such as tocopherols, pigments, sterols and carotenoids; by using two different columns.

The first column was filled with a mix of silica and hexane. The oil (50 g) was then dissolved in hexane

(50 mL) and added to the column. A layer of hexane was put on top to avoid air. During the entire

procedure the column was wrapped in aluminium foil to prevent light-induced oxidations. Hexane in

the collected oil was evaporated with a rotary evaporator (40 °C), after which the flask was flushed with

nitrogen and stored in the freezer till the second step. Aluminium oxide (100 g) was activated overnight

at 200 °C and then mixed with petroleum ether to pack the second column. Further, the oil from the first

step (40 g) was mixed with petroleum ether (56 mL) and loaded on the column. After this, the column

was rinsed with hexane (100 mL). Again the column was wrapped in aluminium foil during the process.

Hexane in the collected oil was again removed by rotary evaporation.

30

IV.4.3 Co-solvent evaporation method

IV.4.3.1 General procedure

To prepare the emulsion, phosphatidylcholine (60 µL,

170 g/L) and β-carotene (100 µL, 10 mM) dissolved in

dichloromethane were put into a round bottom flask and

dried under a nitrogen flow. Then oil (10 µL) and sodium

cholate (1 mL, 21 g/L in ethanol) were added and

everything was dissolved in ethanol (70 %, 5 mL). The

concentration of each component in the final emulsion is presented in Table 4. For the ratio of sodium

cholate to phosphatidylcholine, the composition of human bile was taken into account (70 % of sodium

cholate and 20 % of phosphatidylcholine). For the final concentrations, the median content of bile after

a meal (8 mM or 3 g/L) was used [204]. After mixing all components in the flask, the solution was dried

by rotary evaporation at 40 °C. Everything was then brought back into solution by adding a smaller

amount of ethanol (1.2 mL). Next, this solution was pipetted drop-wise in ultrapure water (10 mL) while

vortexing. Finally, the ethanol phase was removed by using rotary evaporation (40 °C, 20 min).

IV.4.3.2 Initial characterization experiment: different oil types

The emulsion was made with 4 different oil types in similar concentrations (9.2 x 102 mg/L): sunflower

oil, stripped sunflower oil, palm oil and oleic acid. The stripped sunflower oil was obtained by using the

oil stripping procedure in IV.4.2. The size and ζ-potential of the micelles, the incorporation of β-carotene

into the micelles and their cytotoxicity were determined as described in IV.4.1. For the MTT and SRB

assays, the micelles were 1/10 diluted in exposure medium for treatment of both differentiated and

undifferentiated cells. Controls without β-carotene and oil were included and each condition was applied

in 6-fold.

IV.4.3.3 Reducing the cytotoxicity of the emulsions

To determine the limit of toxicity, different dilutions (1/10, 1/20, 1/100, 1/200) of the emulsions were

made with exposure medium to treat the undifferentiated cells in a MTT and SRB assay (IV.4.1).

Finally, the rate of evaporation of ethanol in the rotary evaporator was monitored during the evaporation

step. In the last step of evaporation, the flasks were weighted at different time points during a period of

4 h. Additionally, the stability of β-carotene was monitored by taking a sample of 1 mL each hour.

Extraction of these samples was performed by the optimized first extraction procedure (IV.3.3.1).

IV.4.3.4 Final optimization steps

The emulsions in IV.4.3.2 were made again with some adjustment to the protocol, taking into account

the conclusions of previous described experiments. During the procedure only absolute ethanol was used.

Component Concentration

(mg/L) (mM)

β-carotene 5.4 x 101 0.1

Sodium cholate 2.1 x 103 5.0

Phosphatidylcholine 1.0 x 103 1.3

Sunflower oil 9.2 x 102 1.0

Table 4: Concentration of the different components

in the emulsion made by co-solvent evaporation.

31

In the last step, 700 µL of this ethanol was added to dissolve all of the components in the flask, which

was subsequently put drop-wise into 10 mL of PBS while vortexing. Finally, an evaporation step of

75 min was performed using a rotary evaporator. These emulsions were used to treat differentiated

(passage 24) and undifferentiated (passage 30) Caco-2 cells for the MTT and SRB assays. In addition,

also the filtered (0.22 µm) emulsions were added to the cells.

IV.4.4 Self-emulsification method

To prepare the emulsion, a standard β-carotene

solution in dichloromethane (125 µg/mL) was put into

a glass vial and dried under a nitrogen flow. Next, oleic

acid and sodium taurocholate were added. Finally, the

exposure medium was brought into the vial and the

solution was vortexed and sonicated until a

homogenized emulsion was obtained. The final concentration of each component in the cell culture

medium is presented in Table 5. The used concentrations are the same as in the established ‘Tween 40’

delivery method for carotenoids in literature [183].

In a first optimization step Tween 80 (polysorbate 80) was added to the β-carotene solution. Similar to

Tween 40, this surfactant has the potential to deliver carotenoids to Caco-2 cells in culture. The used

concentration was proven to be non-toxic to the cells during an exposure time of 24 h [205]. After

mixing, this solution was dried and the procedure was preceded as described above. Secondly, the

emulsion was filtered (0.22 µm) to avoid contamination.

The MTT and SRB toxicity tests were used to examine the toxicity of this emulsion, similar to the co-

solvent evaporation method. Further, also incorporation of β-carotene in the emulsion was checked

(0.45 µm filtration) and micelle sizes were measured (see IV.4.1).

IV.5 Development of an in vitro model to study carotenoid transport

Both emulsions were used to deliver the carotenoids to the cells. In each experiment an initial β-carotene

concentration of 10 µM in the cell culture medium was applied, to be able to obtain active transport.

The emulsions were added in the apical compartment of Transwell® plates, as described in IV.2.2. On

the basal side of the differentiated Caco-2 cells (ATCC), only the exposure medium was added. Several

attempt were made to obtain transport of β-carotene through the intestinal cells and all used conditions

are summarized in Table 6. After the mentioned incubation time, all medium was collected and the cells

were lysed. When necessary, samples were stored at -18 °C prior to extraction. Extraction of the

carotenoids and HPLC were finally performed to obtain the results (IV.3).

Component Concentration

(mg/L) (mM)

β-carotene 5.4 1.0 x 10-2

Sodium taurocholate 2.7 x 102 0.5

Oleic acid 5.0 x 102 1.6

Tween 80 1.1 x 103 0.8

Table 5: Concentration of the different components

in the emulsion made by self-emulsification.

32

Table 6: Summary of all performed transport experiments with Caco-2 cells (ATCC) grown onto Transwell® plates.

Changes in composition, performed pretreatments and extra added components in the medium are mentioned. For each

experiment the used cell surface area, the cell passage number and the time of transport are indicated. Also the used

extraction procedure after the experiment is presented.

Oil phase (mM)1 Pretreatment5 Extra

Cells

(cm2)

Passage

number

Transport

time (h)

Extraction

procedure4

Co-solvent evaporation2

1 Sunflower oil3 (1.0) - - 0.99 + 12 1 – 3 – 6 1

2 a) No extra phase

b) Sunflower oil (5.0)

c) Sunflower oil (5.0)

- a) -

b) -

c) Lipase6

3.36 + 14 17 1

3 Sunflower oil3 (1.0) a) Sodium cholate

+ oil3

b) Sodium cholate

+ oil3 + lipase6

c) Sodium cholate

+ oleic acid

- 4.67 + 15 19 1

4 Sunflower oil (1.0) - a) -

b) OA/TC7

2.24 + 16 18 1

5 a) Sunflower oil (1.0)

b) Oleic acid (0.8)

- TEER8 3.36 + 23 16 2

Self-emulsification9

6 No extra phase - - 2.24 + 16 18 1

7 a) No extra phase

b) Tween 80 (0.8)

- 4.67 + 9-10 2 – 4 – 6 –

8 – 24

2

8 Tween 80 (0.8) - Filtered10 3.36 + 22 2411 2

9 Tween 80 (0.8) - Filtered10 13.44 + 11 7212 2

10 Tween 80 (0.8) - Filtered10

+ TEER8

3.36 + 23 16 2

(1) The final concentration of the component in the cell culture medium is mentioned. (2) The adjusted method was used. Sodium cholate and

phosphatidylcholine were added standard to the emulsions. The emulsions were 1/10 diluted with exposure medium. (3) Stripped sunflower

oil. (4) Optimized procedure. (5) Micelles were first formed with these components using the co-solvent evaporation method. Used concentrations: oil (1.0 mM), sodium cholate (5.0 mM) and oleic acid (0.8 mM). Treatments were performed twice a week from day 7 to day

21. (6) Porcine pancreas lipase was added in the medium preceding to pretreatment to avoid prior hydrolysis of the oil. The applied

concentration (4.5 mg/L) of lipase was calculated taking into account its activity (100 units/mg), so that complete hydrolysis of the oil should be obtained within 30 min after treatment. (7) Oleic acid (OA) and taurocholate (TC) were added in the medium at the same concentrations

as in the self-emulsification method. (8) TEER measures were performed during the experiment. (9) Oleic acid and taurocholate were added standard to the emulsions. (10) Prior to the transport experiment, the emulsion was filtered through a 0.22 µm filter. (11) After 4 h and 8 h the

medium was also collected and new emulsion was supplied apically. (12) After 24 h and 48 h the medium was also collected and new emulsion

was supplied apically.

IV.6 Pretreatment of Caco-2 cells with intestinal water

IV.6.1 SHIME incubation

Samples were taken from the proximal part (colon ascendens) and from the distal part (colon descendens)

of a Simulator of the Human Intestinal Microbial Ecosystem (SHIME) incubation, as indicated in Figure

10. These were 1/2 diluted with PBS buffer and in half of the samples the fructo-oligosaccharide (FOS),

inulin, was added in a concentration of 10 g/L. All conditions were incubated for 3 days at 37 °C, after

which they were centrifuged to obtain only the intestinal water. The samples of intestinal water were

finally filtered (0.22 µm) and stored at -18°C.

33

Figure 10: Schematic representation of the different stages in a SHIME incubation [206].

IV.6.2 Characteristics of intestinal samples

All tests described in IV.2.3 were performed on the SHIME samples to choose the dilution of the

intestinal samples that is used during treatment of the cells. The following dilutions with exposure

medium were prepared for treatment of Caco-2 cells (TC-7): 1/2, 1/5 and 1/10. The cells were

undifferentiated since pretreatment will also start when the cells have just reached confluence. The NO

test was also performed on just the intestinal samples, without treatment of cells to clarify if nitric oxide

was already present in the samples.

Short-chain fatty acids (SCFAs) were extracted from all samples and analyzed as described previously

by De Weirdt et al. [207]. Briefly, 500 µL of intestinal sample was treated with 500 µL of H2SO4 (1:1,

v/v) followed by the addition of 400 µL of 2-methyl hexanoic acid as an internal standard. Subsequently,

0.4 g NaCl and 2 mL of diethyl ether were added and centrifuged at 3000 g for 3 min (Sigma). The

supernatants were injected into a GC-2014 gas chromatograph (Shimadzu, ‘s-Hertogenbosch, the

Netherlands), equipped with a capillary fatty acid-free EC-1000 Econo-Cap column (25 x 0.53 mm,

1.2 mM; Alltech, Laarne, Belgium), a flame ionization detector (FID) and a split injector. The

temperature profile was set from 110 to 160 °C, with a temperature increase of 6 °C per min. Nitrogen

was used as a carrier gas, the injection volume was 1 mL and the temperature of the injector and detector

were maintained at 100 and 220 °C, respectively.

IV.6.3 Effect of pretreatment on β-carotene transport

Caco-2 cells (TC-7, passage 43) were seeded in 6-well Transwell® plates (surface area: 4.67 cm2). After

1 week, confluence was reached and thus pretreatment was started. The samples of intestinal water were

initially 1/30 diluted before treatment of the cells, after 4 days the dilution was decreased to 1/15. Each

condition was present in triplicate. During pretreatment, TEER measurements and Lucifer Yellow tests

34

were performed. The transport experiment was executed after a week of treatment. The co-solvent

evaporation emulsion, containing sunflower oil, was 1/10 diluted with exposure medium to deliver the

carotenoids to the Caco-2 cells during a period of 18 h. Finally, the second extraction and HPLC

procedure (IV.3.3.2) were used to analyze the samples.

IV.7 Statistical analysis of data

All data are expressed as the mean value and its standard deviation. Since all samples sizes are very

small in the performed experiments, statistical analyses were done by two-sample Student’s t-tests for

comparing means between different test groups assuming equal variances, as recommended by de

Winter [208]. RStudio R version 3.1.0 was used to perform all statistical tests. A p-value smaller than

0.05 was considered significant and when a p-value lower than 0.01 was obtained, this is explicitly

mentioned.

35

V RESULTS

V.1 Characteristics of the β-carotene emulsions

V.1.1 Stability of the emulsions

For the emulsions prepared by co-solvent evaporation, the average micelle sizes and ζ-potentials under

the different conditions (see IV.4.3.2) are presented in Figure 11. All micelles sizes were significantly

higher than the blank containing only phosphatidylcholine and sodium cholate (p < 0.01). The size of

the micelles in the β-carotene emulsion without oil is significantly lower than for the other emulsions (p

< 0.01). The β-carotene emulsions with sunflower oil (stripped or unstripped) have the highest ζ-

potential, while the emulsion with palm oil has the lowest one. When no oil is added, the ζ-potential is

significantly smaller than in the conditions with β-carotene and sunflower oil (p < 0.01).

For the self-emulsification method, the average micelle sizes in the emulsions were determined. When

only oleic acid and sodium taurocholate were present, the z-average diameter of the particles was (191.3

± 2.4) nm, while in the presence of β-carotene a value of (555.7 ± 3.1) nm was obtained. If Tween 80

was present in combination with the carotenoids, no measurement was possible, suggesting that average

diameters were close to or higher than 1 µm.

V.1.2 Incorporation of β-carotene in the micelles

In Figure 12, the incorporation percentages for β-carotene are presented. Overall, the emulsions obtained

with the co-solvent evaporation method have significantly higher incorporation percentages (p < 0.01).

When oil is included, the incorporation is significantly improved to 80 % (p < 0.01). By self-

Figure 11: Micelle sizes (left) and zeta-potentials (right) of the emulsions containing different types of oil phases: palm

oil, stripped or non-stripped sunflower oil and oleic acid. (B) Error bars represent the standard deviations. *: p < 0.05;

**: p < 0.01 (t-test compared to the blank). The blank only contains phosphatidylcholine and sodium cholate, which are

present in all other conditions. β-car: β-carotene.

****

**

**

**

**

**

**

0

40

80

120

160

Ave

rage

mic

elle

siz

e (

nm

)

**

****

**

-60

-40

-20

0

Zeta

-po

ten

ial (

mV

)

36

emulsification, incorporation is only about 37 % with the use of Tween 80. Without Tween 80, the

incorporation of β-carotene into the micelles is significantly lowered to 4 % (p < 0.01).

V.1.3 Toxicity of the emulsions to Caco-2 cells

V.1.3.1 Co-solvent evaporation emulsion

The results of the MTT and SRB assay 3 days after treatment with several oil types are presented in

Figure 13. The emulsion was added at 10 % to the exposure medium. For the undifferentiated cells, all

samples that contained phosphatidylcholine and sodium cholate were significantly lower than the

untreated condition in both tests. On differentiated cells, it can be seen that the treatment with just water

in the medium has a significant effect on the cells. Further, the SRB test indicates that the amount of

cells slightly increases when just phosphatidylcholine and sodium cholate or also sunflower oil and palm

oil were added. The MTT however shows a more pronounced effect and most conditions herein seem

to cause stress to the Caco-2 cells.

The results of the dilution series of the different emulsions are represented in Figure 14. For the SRB

assay a clear trend can be seen, in which the number of cells increases towards the untreated level when

**

**

**

**

0

20

40

60

80

100

Blank - oil + oil Blank - Tween + Tween

Inco

rpo

rati

on

%

Co-solvent evaporation Self-emulsification

Figure 12: The calculated incorporation percentages of β-carotene in the

micelles. The results are presented as the percentage of the area under the

curve of β-carotene to internal standard compared to the respective blank.

Blanks represent the same amount of β-carotene dissolved in ethanol before

extraction. Error bars represent the standard deviations. *: p < 0.05; **: p <

0.01 (t-test compared to the respective blank).

Figure 13: Results of the MTT and the SRB assay 3 days after treatment of (A) undifferentiated and (B) differentiated

Caco-2 (ATCC) cells. The values are presented as the percentage of the optical density compared to the untreated cells.

Error bars were calculated using the standard deviations. *: p < 0.05; **: p < 0.01 (t-test compared to untreated cells).

**

**

* ** ***

* * ***

0

50

100

150

200

%

- β-carotene + β-carotene

MTT SRB

A

B

** ****

** ** **

**

****

**** **

**

**

** **

0

50

100

150

200

%

- β-carotene + β-carotene

MTT SRB A

37

less emulsion is added to the cell culture medium. The mitochondrial activity, measured by the MTT,

seems to reach a maximum when a dilution of 5 % with the medium is made. However, for the emulsions

with β-carotene no clear maximum is noticeable. This experiment was repeated and proved to be

reproducible.

In Figure 15, the evaporation of ethanol from the emulsions during the last evaporation step in the co-

solvent evaporation method is visualized. It can be seen that the original mass without addition of

ethanol is reached after about 3.5 hours. The amount of β-carotene was not affected by the evaporation

procedure, since the analyzed emulsion samples gave similar β-carotene contents over the 4 hours.

The results of the toxicity tests of the filtered and non-filtered emulsions made by the optimized protocol

are shown in Figure 16. The graph for the undifferentiated cells shows that all conditions containing β-

carotene cause a reduced cell number and a lower mitochondrial activity. In addition, the same

conclusions can be drawn for the filtered emulsion with only phosphatidylcholine and sodium cholate.

When comparing the non-filtered and the filtered results for each condition, only a significant difference

could be detected for the emulsion with β-carotene in the MTT and for the emulsion with

phosphatidylcholine and sodium cholate in the SRB. These differences could only be detected at the 5 %

***

*** *

**

**

* **

****

******

**

**

**

**

*

**** **

0

20

40

60

80

100

120

Untreated 10 % 5 % 1 % 0,5 % 10 % 5 % 1 % 0,5 % 10 % 5 % 1 % 0,5 % 10 % 5 % 1 % 0,5 %

%

Blank + Oil + Oil + β-carotene + β-carotene

MTT SRB

Figure 14: Results of the MTT and the SRB assay 3 days after treatment of undifferentiated Caco-2 (ATCC) cells with

a dilution series of different emulsions. The values are presented as the percentage of the optical density compared to

the untreated cells. Error bars were calculated using the standard deviations. *: p < 0.05; **: p < 0.01 (t-test compared

to untreated cells).

Figure 15: The mass percentage of ethanol evaporated from a β-carotene emulsions with a rotary evaporator during

a period of 4 h. Error bars represent the standard deviations.

0

20

40

60

80

100

120

0 50 100 150 200 250

% E

than

ol e

vap

ora

ted

Time (min)

38

significance level. Although the mitochondrial activity of the differentiated cells seems to be lower for

all the emulsions, only some of them could be called different from the untreated cells on the 5 %

significance level. However, between the different emulsions no significant difference in MTT result

was found. Concerning the SRB results, only two results are significantly different from the untreated

condition. Though, for the non-filtered emulsion containing oil and β-carotene, this result is not

significantly different from the emulsion with just oil.

V.1.3.2 Self-emulsification method

The results of the cytotoxicity tests performed with the emulsions of the self-emulsification method are

presented in Figure 17. The emulsion with Tween 80 shows a significant reduction in cells after 1 day

and also the mitochondrial activity is diminished after 3 days. In the dilution series of the Tween 80

emulsion a clear trend is visible with the emulsion becoming less toxic with greater dilutions.

Figure 17: Results of the MTT and the SRB assay on differentiated Caco-2 cells (A) 1 and 3 days after treatment with

β-carotene emulsions by the self-emulsification method with or without Tween 80, (B) 3 days after treatment with a

dilution series of the amount of Tween 80 present in the self-emulsification method with or without β-carotene. The

values are presented as the percentage of the optical density compared to the untreated cells. Error bars were calculated

using the standard deviations. *: p<0.05; **: p<0.01 (t-test compared to untreated cells).

**** **

* ***

** ** ** *

0

20

40

60

80

100

120

%

Non-filtered Filtered

MTT SRB A

* * *

* *

0

20

40

60

80

100

120

%

Non-filtered Filtered

MTT SRB

Figure 16: Results of the MTT and the SRB assay 3 days after treatment of (A) undifferentiated and (B) differentiated

Caco-2 cells with emulsions containing PBS, whether or not filtered (0.22 µm). The blank only contains sodium cholate

and phosphatidylcholine. The values are presented as the percentage of the optical density compared to the untreated

cells. Error bars were calculated using the standard deviations. *: p<0.05; **: p<0.01 (t-test compared to untreated

cells).

B

**

**

**

****

**

**

**

0

20

40

60

80

100

120

%

Tween Tween + β-carotene

MTT SRB

**

**

**

0

20

40

60

80

100

120

%

1 day 3 days

MTT SRB A B

39

V.2 Study of β-carotene transport through Caco-2 cells

V.2.1 Detection method for carotenoids

The first extraction procedure did not allow correct quantification of the small amounts of β-carotene

that were spiked in the cell culture medium. Changing the injection volume or the volume of acetonitrile

also did not lower the LOD and LOQ enough to obtain a useful calibration curve for low concentrations.

Therefore, the results are not presented.

For the second extraction procedure, the calibration curve for β-carotene in cell culture medium is shown

in Figure 18. At small concentrations another trend was visible than at higher concentrations, hence two

different trend lines were fitted. An artificial starting point of the higher concentrations was set at an

AUC of β-carotene to the AUC of the internal standard of 0.3, so both curves would have a relatively

good fit. It can be seen that the fit of the curves (R2) is around 0.97. The limits of quantification and

detection were calculated from the signal-to-noise ratio using peak heights of the HPLC results [209].

This way, the LOD and LOQ correspond to 3 and 10 times the noise level, respectively. The LOQ for

β-carotene in cell culture medium is 0.6 µM (0.5 µg/mL), while the LOD is 0.06 µM (0.05 µg/mL).

In Figure 19, the recovery of β-carotene at different concentrations in cell culture medium is visualized.

A trend can be noticed with lower concentrations having a lower recovery percentage. At the LOQ the

recovery is still 58 %, while at the LOD, it has lowered to 39 %.

y = 2,3565x + 1,2906R² = 0,9675

y = 7,4408x + 0,0441R² = 0,9772

0

2

4

6

8

10

0 0,5 1 1,5 2 2,5 3 3,5

c(β

-car

ote

ne

)/c(

IS)

AUC(β-carotene)/AUC(IS)

Linear (High concentrations) Linear (Low concentrations)

Figure 18: Calibration curve of β-carotene in cell culture medium using the second extraction procedure.

AUC = area under curve; IS = internal standard.

Figure 19: Recovery of the internal standard (IS) and of

different β-carotene concentrations in cell culture medium.

0

25

50

75

100

IS 10 µM 2,5 µM 0,6 µM 0,06 µM

Re

cove

ry %

β-carotene concentration

40

V.2.2 In vitro cell model to study transport

In the kinetic transport experiment (Table 6 no. 1)

with the emulsion prepared by co-solvent evaporation

a decrease is visible of the amount of β-carotene in the

apical compartment, which is shown in Figure 20. In

the basal compartment no carotenoids could however

be detected. The cells were not extracted in this

experiment.

In the other transport experiments (Table 6 no. 2-4)

using co-solvent evaporation emulsions in

combination with the first extraction procedure, for the samples of the apical compartment small peaks

representing β-carotene could be seen on the HPLC chromatograms. Quantification was however not

possible. In the 2nd experiment condition b also detection of β-carotene in the cells was possible, while

for condition a in the 4th experiment a peak near the LOD was obtained for carotenoids in the basal

compartment. For the self-emulsification method, in the transport experiment (Table 6 no. 6) analyzed

with the 1st extraction procedure, no carotenoids could be detected.

When the 2nd extraction procedure was used, detection of β-carotene was possible using the emulsion

by the self-emulsification method. The results of the kinetic transport experiment (Table 6 no. 7) with

the emulsion made by self-emulsification are presented in Figure 21. Before transport, the TEER values

were (1768 ± 211) Ω x cm2. During transport, aggregates of β-carotene could be seen microscopically

on the Caco-2 cells when no Tween 80 was used in the emulsion, indicating β-carotene precipitation on

the cells, which has probably interfered with the results. In further experiments, emulsions were filtered

(0.22 µm) to avoid bacterial contamination. To compensate the loss of β-carotene by filtration, some

attempts were made to increase basal secretion by the cells. In experiment 8 (Table 6) more β-carotene

was delivered to the cells. Nevertheless, the amount of β-carotene in the basal compartment was under

the LOQ. In experiment 9 (Table 6), the transport time was extended and a much larger cell surface was

used. After 72 h, flooting cells could be detected and thus, results of cellular transport could not be

trusted anymore. The amount of carotenoids in the basal part was analyzed after 48 h and this was still

under the LOQ.

The emulsions were compared in a last experiment (Table 6 no. 5 and 10). TEER values before the

transport experiment were on average (676 ± 91) Ω x cm2, which is not significantly different from the

values after transport of (853 ± 143) Ω x cm2. Between the distinct conditions, no significant differences

on the 5 % significance level could be detected. In Figure 22 the results are presented. In the basal

compartment, no carotenoids could be detected.

0

20

40

60

80

100

0 1 3 6

β-c

aro

ten

e (

% o

f in

itia

l)

Time (h)

Figure 20: Apical concentrations of β-carotene

prepared with the co-solvent evaporation method in

function of transport time.

41

Figure 22: Results of the transport experiment, comparing the co-

solvent evaporation and self-emulsification method. On the y-axis,

the mass of β-carotene that was found in each compartment is

indicated for the different emulsions.

V.3 Pretreatment of Caco-2 cells with intestinal water

V.3.1 Characteristics of the SHIME samples

In Figure 23 and Figure 24 the results of the cytotoxicity tests on the intestinal samples are shown. The

short-chain fatty acids detected in the SHIME samples are presented in Figure 25.

2 h 4 h 6 h 8 h

Basal 0 0 0 1,3

Cells 35,4 17,3 23,8 47,4

Apical 64,6 82,7 76,2 51,3

0

20

40

60

80

100

%

No Tween

2 h 4 h 6 h 8 h

Basal 0 0 1,6 3,2

Cells 1,4 1,5 1,9 5,2

Apical 98,6 98,5 96,5 91,6

0

20

40

60

80

100

%

With Tween

Figure 21: Results of the kinetic transport experiment, without (A) and with (B) addition of Tween 80 in the emulsions

prepared by self-emulsification. The percentage of β-carotene in the apical compartment, the basal compartment and

the cells is presented on the y-axis and in the table below for the different time points. Carotenoids could not be detected

if a value of 0 is shown.

A B

0

2

4

6

8

Sunflower oil Oleic acid Tween 80

Am

ou

nt

of

β-c

aro

ten

e (

µg)

Co-solvent evaporation Self-emulsification

Apical Cells

***

*

**

*

0

50

100

150

with cells in samples

%

Proximal Proximal Distal Distal Buffer Buffer colon colon + FOS colon colon + FOS + FOS

Figure 23: Results of the nitric oxide (NO) test on different concentrations of the intestinal samples diluted in cell culture

medium and on cell culture medium after incubation of the samples with undifferentiated Caco-2 (TC-7) cells. Error

bars were calculated using the standard deviations. FOS: fructo-oligosacharides.

42

Figure 25: Short-chain fatty acids (SCFAs) present in the intestinal samples with the specific concentrations of acetate,

propionate and butyrate. The iso-SCFAs, isobutyrate and isovalerate, are also presented. FOS: fructo-oligosacharides.

V.3.2 Influence on β-carotene transport

The TEER values and the apparent permeability of the Caco-2 (TC-7) cells, during the pretreatment

period of 1 week, are presented in Figure 26. On the seventh day, just before the transport experiment,

the apparent permeability of the cells pretreated with distal colon intestinal water was found to be

significantly lower on the 5 % significance level from the conditions with distal or proximal colon water

in which FOS was added. When considering the TEER measurements, the condition with just buffer

proved to be significantly (5 %) lower than only FOS or distal colon water. Further, the pretreatment

with proximal colon water and FOS was also statistically (5 %) lower than only FOS, proximal colon

water, distal colon water and distal colon water with FOS.

For the final transport experiment, the cellular and basal percentages of β-carotene found after transport

are presented in Figure 27. Despite the potential of the TC-7 Caco-2 cell line to convert carotenoids into

retinaldehyde or other apo-carotenoids, these could not be detected on the HPLC chromatograms [183].

0

400

800

1200

1600

acetate propionate butyrate iso-SCFA Total SCFA

Co

nce

ntr

atio

n (

mg/

L)

Proximal colon

Proximal colon + FOS

Distal colon

Distal colon + FOS

Figure 24: Results of the MTT (A) and SRB (B) assay one day after treatment of undifferentiated Caco-2 (TC-7) cells

with different dilutions of SHIME samples with exposure medium in duplicate. The values are presented as the

percentage of the optical density compared to the untreated cells. Error bars were calculated using the standard

deviations. *: p<0.05; **: p<0.01 (t-test compared to untreated cells). FOS: fructo-oligosacharides.

*

0

50

100

150A

%


**

**

**

**

* *

0

50

100

150B

%


43

The cellular uptake of β-carotene after pretreatment with distal colon water and FOS was found to be

significantly different on the 5 % significance level from the conditions with just distal or proximal

colon water and on the 1 % significance level from proximal colon intestinal water with FOS.

0

4

8

12

16

20

0 2 4 7

0

400

800

1200

1600

Pap

p(x

10

-6cm

/s)

Time (d)

TEER

(Ω

.cm

2 )

Buffer

0

4

8

12

16

20

0 2 4 7

0

400

800

1200

1600

Pap

p(x

10

-6cm

/s)

Time (d)

TEER

(Ω

.cm

2 )

Buffer + FOS

0

4

8

12

16

20

0 2 4 7

0

400

800

1200

1600P

app

(x 1

0-6

cm

/s)

Time (d)

TEER

(Ω

.cm

2 )

Proximal colon

0

4

8

12

16

20

0 2 4 7

0

400

800

1200

1600

Pap

p(x

10

-6cm

/s)

Time (d)

TEER

(Ω

.cm

2 )

Proximal colon + FOS

0

4

8

12

16

20

0 2 4 7

0

400

800

1200

1600

Pap

p(x

10

-6cm

/s)

Time (d)

TEER

(Ω

.cm

2 )

Distal colon

0

4

8

12

16

20

0 2 4 7

0

400

800

1200

1600

Pap

p(x

10

-6cm

/s)

Time (d)

TEER

(Ω

.cm

2 )

Distal colon + FOS

Figure 26: The transepithelial electrical resistance (column charts) and the apparent permeability (Papp) values (line

charts), calculated from the Lucifer Yellow test, of the Caco-2 (TC-7) cells during the pretreatment period with different

intestinal samples. Error bars represent standard deviations. FOS: fructo-oligosacharides.

Figure 27: Percentage of the total β-carotene amount present in the

cells and basal compartment after the transport experiment on

pretreated cells with intestinal samples. Error bars were calculated

using the standard deviations. FOS: fructo-oligosacharides.

0

2

4

6

%

Uptake cells Basal secretion

44

VI DISCUSSION

From the literature study, it became clear that many factors can have an influence on the bioavailability

of vitamin A and its provitamin A carotenoids. Hence, there are many possible causes for vitamin A

deficiencies. Besides merely insufficient intake of retinoids and carotenoids, also the food matrix can

have a significant influence, with animal sources having the highest bioaccessibility, and genetic factors

have also been found to play a role. Unfortunately, these cannot fully explain the higher incidence of

vitamin A deficiencies in children. In this master’s thesis, the hypothesis was explored that the intestinal

matrix has an impact on the capacity of intestinal absorptive cells to take up the most important

provitamin A carotenoid, β-carotene. Receptors on intestinal cells that have a significant role in vitamin

A transport could be present to a greater or lesser extent, according to the type of food that is eaten

during human growth and development. Moreover, this could impact the site of β-carotene uptake in the

human intestines. In order to investigate this theory, some subgoals needed to be reached. An emulsion

was necessary as a means to deliver the β-carotene and in addition, an in vitro cell line model had to be

developed to study the transport of β-carotene through the cells.

VI.1 Delivery of β-carotene to the Caco-2 cells

To make delivery of the very hydrophobic β-carotene possible in aqueous cell culture medium, it was

decided to incorporate the molecules into an emulsion. Loading of β-carotene in micelles before

administration has earlier been shown to improve the absorption by the cells [151]. Due to practical

reasons, two low energy methods were chosen: self-emulsification and co-solvent evaporation in which

emulsions are spontaneously obtained by phase transitions [160]. The first procedure that was applied,

is derived from the ‘Tween 40’ method, which is substantially used in literature for β-carotene transport

experiments with in vitro cell models [61, 183, 210, 211]. Instead of the detergent Tween 40, in the

experiments Tween 80, also known as polysorbate 80, was used. However, Tween 80 was shown to

have an equal potential as delivery vehicle for carotenoids to Caco-2 cells as Tween 40 [205]. In this

procedure, oleic acid and sodium taurocholate were added to make lipoprotein production by the

intestinal cells possible. The second procedure, called co-solvent evaporation, is well-known in

pharmaceutical research, where it is used as an elegant method for delivery of several lipophilic drugs

[162]. The composition of the emulsion was selected to resemble in vivo conditions, with sodium cholate

and phosphatidylcholine acting as bio-surfactants. Important in deciding which emulsion would be used

as delivery method for β-carotene in the in vitro cell model are the stability of the emulsion, the

incorporation of the carotenoids in the micelles and the cytotoxicity for Caco-2 cells.

45

A first relevant feature of the emulsions that was assessed, is their stability. It is desired that the

dispersion is stable enough, so that during the experiments the release of β-carotene from the micelles

occurs only near the cell surface, similar to the in vivo process. Two characteristics were evaluated to

get an indication of the stability: the mean micelle size and the ζ-potential.

A first indication for the emulsion stability that was measured, is the average micelle size, with small

micelles being less sensitive to gravitational separation and particle aggregation [173]. In addition, with

smaller particle sizes, transfer of carotenoids across the unstirred water layer is promoted, which was

shown to be a rate-limiting step in in vitro cell models [150, 187]. The emulsions prepared by self-

emulsification were in general larger than those by the co-solvent evaporation method. While average

micelle size of the β-carotene emulsion without Tween 80 could still be determined, the limit of the

applied device was reached when Tween 80 was present. These emulsions will most likely have an

inferior stability. However, no extra parameters were measured to confirm this. Nonetheless, the

unstirred water layer above the cells presumably poses an important barrier for these micelles. In contrast,

the average micelle sizes of all the dispersions produced with the co-solvent evaporation method are in

the range of nanometers (Figure 11). Without the use of oil, the size can even be reduced to almost half

of those that contain oil. All nanoemulsions have a mean droplet diameter smaller than 200 nm and thus

should have an improved physical stability [173]. An additional advantage of these small micelle sizes

is the opportunity this creates for removal of bacteria by passage of the emulsion through a filter with

an average pore diameter of 0.22 µm, although viruses and phages can still remain in the dispersions

this way.

Secondly, the ζ-potential of some co-solvent evaporation dispersions was determined as a direct measure

of their potential stability. The general dividing line between stable and unstable suspensions is mostly

taken at ±30 mV [212, 213]. From the results in Figure 11, it can thus be concluded that the β-carotene

emulsions produced by co-solvent evaporation with sunflower oil (stripped or unstripped) have a good

stability with a high ζ-potential of approximately -50 mV. The β-carotene emulsion without oil is

moderate stable, while the one with palm oil has an incipient instability with a quite low ζ-potential of

about -20 mV. The ζ-potential of the palm oil emulsion is not significantly different from the blank

containing only phosphatidylcholine and sodium cholate, which could indicate a different behavior of

this dispersion. This could be due to the solid state of the chosen type of palm oil at room temperature.

Since the micelles have lower ζ-potential values, flocculation of the particles is more likely to happen

than when sunflower oil is used, making the use of palm oil a less suitable option for formation of the

β-carotene emulsion [214]. During a simulated digestion experiment, it was also concluded that

unsaturated fatty acids, present in sunflower oil, can promote micellarization of carotenoids to a greater

extent than saturated fatty acids, such as in palm oil [136]. This confirms sunflower oil to be the better

choice for use in the co-solvent evaporation emulsion. Eventually, it should be noted that important

factors affecting the ζ-potential, and thus also the stability of the dispersion, are the pH and the ionic

46

strength of the aqueous medium [214]. All emulsions were prepared in ultrapure water for the

measurements, while for the in vitro experiments these emulsions are diluted in cell culture medium in

which a sodium bicarbonate buffer system is present (3.7 g/L) to maintain a physiological pH of 7.4

[215]. Thus, the resistance of the micelles to flocculation within cell culture medium is still uncertain.

A high incorporation of β-carotene into the micelles is desired to prevent precipitation during the

experiments and so improve the delivery. The incorporation is clearly higher in the co-solvent

evaporation emulsion than for the self-emulsification method (Figure 12). As expected, the very

hydrophobic β-carotene is even better included when oil is added. For the self-emulsification method,

the results suggest that Tween 80 is necessary as a surfactant to reach a proper incorporation of β-

carotene. In a previous study, it was demonstrated that 0.05 % (v/v) of Tween 80 in solution was

sufficient to moderately disperse β-carotene at a concentration of 10 mg/L [216]. Since a higher Tween

80 concentration of 0.1 % was used with a lower concentration of β-carotene (5.4 mg/L) and moreover

also sodium taurocholate was present to help solubilize the carotenoids, incorporation to some extent

could be expected. Addition of more Tween 80 could have helped in incorporating more β-carotene but,

due to cytotoxic effects, this was not a suitable option. In addition, it should be noted that the calculated

incorporation percentages are an underestimation of the actual values, since larger micelles with a

diameter over 0.45 µm were also withheld by the filter. This was definitely a problem for the self-

emulsification method, especially when Tween 80 was present, by which larger average micelle sizes

were obtained. The mean micelle sizes in the co-solvent evaporation emulsion may have been lower,

this does not rule out the presence of larger micelles in the emulsions in which β-carotene is still included.

The high incorporation percentage is also important to make filtering of the emulsion possible and this

way exclude bacteria, as mentioned before. Since a filter with pore diameter of 0.22 µm is needed for

this, instead of the 0.45 µm filter used in the incorporation experiment, there is a higher chance that

larger micelles will be excluded. Less β-carotene can this way be delivered to the cells, but

contamination problems due to the use of unsterile components could be avoided. In the co-solvent

evaporation procedure, a large amount of ethanol is applied which disinfects all components and thus

helps in generating a more sterile emulsion. Merely a supplement of antibiotics (penicillin-streptomycin)

in the cell culture medium was sufficient to prevent contamination issues. To the contrary, the self-

emulsification method did cause contamination problems, which made filtration necessary. The low

inclusion of β-carotene into the emulsion, therefore became a problem with a diminished concentration

as a result. From this point-of-view, we prefer the co-solvent evaporation method.

Most important for the delivery method is its compatibility with intestinal cells, more specifically the

Caco-2 cell line used for transport experiments. The emulsions thus cannot be harmful for these cells.

This property was evaluated with the SRB and MTT assays, of which the SRB value is a measure for

the amount of cells present, while the MTT value gives an indication for stress cells are experiencing

[197, 198]. For the self-emulsification procedure, no toxicity problems were expected since the used

47

concentration of Tween 80 was claimed to be non-toxic in literature and therefore a good delivery

vehicle for carotenoids [205]. Nevertheless, the performed assays yielded contradicting results (Figure

17). Similar to the performed MTT assay by O'Sullivan et al. [205], after 1 day no difference could be

detected. The SRB assay however did show a significant decrease in cells. After 3 days, the effect is

visible in both assays, since the metabolic activity of most cells could have been restricted. Since only

a cytotoxic effect can be detected when Tween 80 is added, this component is probably accountable.

This was also confirmed by the dilution series in Figure 17, in which a greater dilution showed less toxic

effects. Especially the combination with β-carotene proved to be toxic, this could be due to the larger

retention of Tween 80 by the filter when this was just added to the medium. In several in vitro studies,

Tween 80 is indeed reported to have cytotoxic effects by damaging the plasma and nuclear membranes

of the cells [217-219]. It was stated by Shah et al. [220] that the same concentration of 0.1 % applied to

Caco-2 cells for 1 day caused an even more drastic decrease in cell viability with only 5 % survival. In

addition, even much lower concentrations (0.001 %) were already able to cause membrane damage in

Caco-2 cells and decreased the transepithelial electric resistance [221]. Considering various studies were

not able to find any cytotoxic effects of Tween 80 on Caco-2 cells [205, 222], inter-laboratory or

interexperimental variability can play a role in this with certain Caco-2 cell cultures having a distinct

sensitivity [223]. Nevertheless, also in some in vivo experiments with rodents, negative effects of Tween

80 on the intestinal membrane barrier function were more recently reported [224, 225]. At low

concentrations, polysorbate monomers could be able to incorporate into the membrane and thus, alter

cell membrane integrity, while at higher concentrations, it is hypothesized that the surfactant causes

cytotoxicity because the membrane components are solubilized [219, 226].

Initially, the co-solvent evaporation method also produced somewhat cytotoxic emulsions. In Figure 13,

it can be seen that especially undifferentiated cells are affected by this cytotoxic effect, with all

emulsions significantly lowering the amount of cells. The MTT assay suggested that some dispersions

also caused stress to differentiated cells and in addition, also the condition in which ultrapure water was

added to the cells seemed to distress the cells. Since eventually the emulsions would be used on

differentiated cells, no cytotoxic effects should be caused on these. The effects could be caused by a few

components that are present in the dispersions. For instance, sodium cholate has earlier been reported

as being cytotoxic, while phosphatidylcholine could compensate this by its cytoprotective effect, which

was demonstrated in several studies [227-230]. Further, during the procedure the solvent ethanol is used

that should be evaporated in the end, however some remaining ethanol in the emulsions cannot be

excluded which could influence the cells. Finally, also the ultrapure water in which the emulsion is

prepared, appears to cause stress to the cells. No definite explanation can be provided for this

phenomenon. Yet, it is thought to be due to a slight change of pH that could be induced by the water.

Although a buffer system is active in the cell culture medium, the 10 % dilution with water could have

been enough to disrupt the balance, certainly since sodium bicarbonate is known to have a rather weak

48

buffer capacity [215, 231]. The dilution of buffer solutions with high-purity water was reported to lead

to pH changes. These variations in pH can be explained by variations in ionic strength and buffer

capacity [232]. Pure water should have a pH close to 7 at 25 °C, which is near the physiological pH of

7.4. However, the temperature in the incubator, where the cells are kept, is 37 °C and pure water appears

to be more acidic at these higher temperatures [233]. Although mammalian cells are known to survive

over a quite wide pH range (6.6-7.8), their optimal growth is only obtained at pH 7.2 to 7.4 and thus,

mitochondrial activity can possibly be affected by a small alteration [234]. Nevertheless, no additional

tests were performed to confirm this hypothesis. The dilution series in Figure 14 gives a better indication

of which components could be responsible for the toxicity to undifferentiated Caco-2 cells. The trend of

the SRB results seems rather logical and confirms that the emulsions are toxic to the undifferentiated

Caco-2 cells, as with higher dilutions more cells are able to grow. The MTT results on the other hand

are not that obvious and tend towards a combined effect of several components. Since sodium cholate

and phosphatidylcholine were added to the emulsion to better simulate the in vivo situation, first some

attempts were made to avoid their removal. As such, the possibility of remaining ethanol was further

assessed.

Besides the toxicity problem for undifferentiated Caco-2 tumor cells, the remaining ethanol in the

emulsions could cause additional problems in future experiments [235]. In literature, it is found that

basolateral exposure of differentiated Caco-2 cells on semi-permeable membranes to ethanol

concentrations of 0.05% (v/v) can already increase the paracellular permeability by loss of tight

junctions integrity [236]. Differentiated Caco-2 monolayers are more resistant and only luminal ethanol

concentrations greater than 1 % are able to decrease their paracellular barrier function [237-239]. In

section I.2.3, the intestinal absorption of vitamin A was fully described and it became clear that only

transcellular transport is occuring. Nevertheless, it is uncertain if considerable damage to the tight

junctions is able to change the transport mechanism of carotenoids, such as β-carotene; therefore, this

effect due to remaining ethanol should be restricted. To be able to determine the evaporation time needed

to remove most ethanol, the rate of evaporation of the ethanol-water emulsion was assessed in Figure

15. It can be seen that after 3 to 4 hours the original mass of the emulsion without ethanol is reached.

But this does not mean all of the ethanol will be evaporated since it is known that an azeotropic ethanol-

water mixture (96 % ethanol and 4 % water in volume) has a lower boiling temperature than pure ethanol,

hence, also some water will be simultaneously evaporated [240]. As mentioned in section IV.4.3.4, the

amount of ethanol was decreased, the evaporation time was increased and the ultrapure water was

replaced by PBS in which the same buffer system is present as in the cell culture medium [241]. The

results of the cytotoxicity tests for this optimized emulsion in Figure 16, show that addition of PBS to

the emulsions is not causing any stress to the cells. For the undifferentiated cells, the emulsions

containing β-carotene are still cytotoxic or inhibit cell growth, which could be explained by its pro-

oxidant properties. Various studies have already concluded that β-carotene can act as an inducer of

49

apoptosis in tumor cell lines [242, 243]. When the emulsions are filtered, smaller sizes for the micelles

in solution can be expected. But this did not cause any significant differences in cell number or

mitochondrial activity compared to the non-filtered solutions. The results of the differentiated cells

suggest that the emulsion is not causing any stress or toxicity to the cells. The significant differences

that could be detected are not really convincing and could just be due to cell-to-cell variability [244].

VI.2 Development of an in vitro model to study β-carotene transport

An important aspect in the study of β-carotene transport with in vitro cell models is the ability to detect

physiological concentrations of this carotenoid in the cell culture medium. These low concentrations are

necessary to obtain active transport by Caco-2 cells, instead of passive diffusion [62]. Therefore, a

procedure used for carotenoid extractions from fruits in the laboratory had to be optimized. Initially only

a large reduction of solvents, a decreased final dilution step and a larger injection volume for the HPLC

were implemented. However, the procedure was still not suitable for cell culture medium with amounts

of β-carotene in the µM-range. All transport experiments analyzed with this first procedure, did not give

useful results, since basal and cellular carotenoids could not be detected properly. Due to the still rather

large volume of solvent that is used, the carotenoids become abundantly diluted and a quite extensive

drying step is needed in which some losses could occur. Further, β-carotene can get lost during the

several transfer and filtration steps. Another issue is the long duration of the procedure, especially due

to the saponification step, which creates more opportunity for light or oxidation processes to appear and

hence, degradation of β-carotene to occur [245-247]. Because of this, a switch was needed to an entirely

different procedure in which a minimal number of transfer steps is performed. In addition, the amount

of solvent was further reduced and the time of the procedure was greatly reduced. This second extraction

procedure reached low enough LOD and LOQ values, so use of the method was possible for the cell

culture medium.

The obtained calibration curve in Figure 18 shows different trends for very low and higher β-carotene

concentrations. The difference in trend could be explained by the very hydrophobic character of the β-

carotene molecules. These can partly bind with other molecules or cellular debris present in the medium,

which prevents their extraction. Therefore, when only a small amount of β-carotene is present, a

relatively large percentage of this will be lost and thus a smaller peak than expected can be seen on the

HPLC chromatogram. This hypothesis is strengthened by the recovery graph (Figure 19) as the

percentage of carotenoids that can be extracted, reduces with lower concentrations in the medium. The

LOD for the second extraction procedure was found to be 0.06 µM of β-carotene in cell culture medium,

while the LOQ is 0.6 µM. It is however expected that this limit will be higher when β-carotene is first

emulsified and incubated with the cells, since the molecules will have more time to bind to other particles.

Also, in the experiment that was performed, the β-carotene was added to the medium dissolved in solvent,

50

which could have promoted its extraction. These limits are slightly higher than found in literature when

HPLC analysis is applied. Huang et al. [248] for instance obtained a LOD of 0.04 µM in human serum

and CDC [249] was even able to reach a LOD of 0.006 µM in serum. No problems were reported

concerning the recovery of carotenoids at small concentrations. Further improvements in detection

method can thus still be achieved. During the experiments, merely the extraction procedure was

optimized. In other studies for instance other solvents for carotenoid extractions were applied, such as

various mixtures of methanol/tetrahydrofuran, acetone/hexane, acetone/ethanol/hexane or ethyl

acetate/hexane [250-252]. Also changes in the applied HPLC method, such as different mobile phases,

could have yielded better detection limits. A combination of the standard HPLC method with mass

spectrometry has further been shown to considerably increase sensitivity and hence, make quantification

of β-carotene at physiologically relevant doses in various human samples possible [253, 254].

Transport of β-carotene through differentiated Caco-2 cells, simulating intestinal cells, was evaluated

with both delivery methods. In the kinetic transport experiment that was performed with the co-solvent

evaporation method, only a decrease in apical concentration could be detected (Figure 20). Cellular

uptake or basal secretion were not visualized. However, since the first extraction procedure was used,

these results are not really reliable. The concentrations in the cells and basal compartment could have

been too small for detection. In other studies, used cell surfaces were also at least 4 times larger to obtain

a large enough volume for detection [255]. Further, the decrease of β-carotene in the apical compartment

could indicate that cellular uptake did occur. Other causes for this decrease could be degradation of the

carotenoids or binding to hydrophobic components in the medium. But considering the high stability of

the co-solvent evaporation emulsion and the relatively short duration of the experiment, it is rather

unlikely that such a large decrease in concentration was caused by this. With the self-emulsification

method, transport over time could be visualized (Figure 21). Tween 80 was hereby found to be necessary

to avoid precipitation of carotenoids onto the cell surface. Cell amounts that were found without Tween

80 in the emulsion are most likely incorrect, as precipitates were not washed off the cell surface by PBS.

Due to the cytotoxic effect of Tween 80 reported in some experiments, there is also no certainty that the

carotenoids were indeed transported via a transcellular pathway [219, 221]. The effect of Tween 80 is

thought to be small during the rather short time period of 8 hours, but no TEER measurements were

performed during the experiment to assure this. Emulsions obtained by the self-emulsification method

were filtered (0.22 µm) during further experiments, since contamination issues were occurring. The low

incorporation hereby proved to be problematic and caused lower concentrations of β-carotene in the cell

culture medium, which made it hard to achieve enough basal transport for quantification. When the

comparison between both emulsions was made in a final experiment (Figure 22), no basal transport

could be visualized. Secretion of chylomicrons by Caco-2 cells was previously shown to depend on oleic

acid and phosphatidylcholine and since in each emulsion one or both components were present, the

composition could not have restricted transport [256]. Since the co-solvent evaporation emulsion with

http://en.wikipedia.org/wiki/Mass_spectrometry

http://en.wikipedia.org/wiki/Mass_spectrometry

51

sunflower oil showed the best cellular uptake and β-carotene recovery, this emulsion is preferred over

the other emulsions. For the self-emulsification emulsion, the problem of low incorporation probably

arised, which is partially due to the presence of larger micelles as described in the previous section. The

micelle sizes in the oleic acid emulsion cannot be the cause of the lower recovery compared to the

sunflower oil emulsion. Probably, this emulsion with unsaturated fatty acids has an inferior stability. It

has been reported earlier that oleic acid encourages oxidation of β-carotene and thus, is not suitable to

replace oil [257]. From the results, it is however not clear if the limited lipase activity of the Caco-2

cells restricts the basal secretion of carotenoids in lipoproteins [177].

Although differences between both emulsions could be expected due to the different components present

with the carotenoids during delivery to the cells, is was not possible to detect other kinetics or levels of

uptake [255]. Besides the contribution of analytical problems described above, also Caco-2 cell models

have earlier been reported to suffer from comparability problems between different experiments and this

could definitely be noticed during the experimental work. In literature, this variation is attributed to

diverse factors [255]. Various concentrations of carotenoids were for instance applied, due to the

sometimes preceding filtration step. Since a linear relationship was found between the β-carotene

concentration in micelles and Caco-2 cell uptake, this could partly explain the variable results with the

self-emulsification method [258]. Another factor that could have caused variation, is the incubation

period of the cells with the emulsion [105]. Further, Caco-2 cells are known to express typical transport

proteins that are involved in carotenoid uptake, such as SR-BI, NPC1L1 and ABCA1, but the degree of

expression highly depends on the stage of differentiation, age of the cells and passage number [259].

Although a constant time point after seeding of the cells was chosen to perform experiments, the period

of post-confluency of the cells was yet variable, due to different surface areas that needed to be covered

and differing growth rates of the cells. In Table 6, it can also be noted that passage number and hence

age of the cells differed over the transport experiments. These issues make comparison of bioavailability

over experiments not possible and hinder the repeatability of results. In addition, no study on the

correlation between in vitro experiments and in vivo investigations was performed yet [255]. Since in

the final pretreatment experiment only one delivery method is used and all cells are in the same

differentiation stage, comparison between the different conditions should be possible.

VI.3 Pretreatment of Caco-2 cells with intestinal water

The final aim of this master’s dissertation was to assess the hypothesis that the capacity of intestinal

cells to take up and transport the carotenoid β-carotene is altered by exposure to certain intestinal

components during their maturation. Therefore, a first experiment was performed in which differentiated

Caco-2 cells were treated with distinct intestinal samples during their differentiation. Since the proximal

and distal part of the colon are known to differ in concentration of several components, samples from

52

these contents seemed appropriate to obtain contrasting treatments [260]. By prior incubation with the

FOS inulin, growth of intestinal bacteria was stimulated and the ratio among various SCFAs produced,

could be changed further to attain more variant conditions [261, 262]. The fatty acids analysis is

visualized in Figure 25 and shows a clear distinction in amount and ratio of SCFAs in the different

samples. Hence, the samples could be capable of influencing the intestinal cells, and more specifically

the carotenoid transporters, in different ways.

Significant differences in cellular β-carotene uptake were eventually found between the pretreatment

with distal colon water incubated with FOS and all other treatments with intestinal water (Figure 27).

The smaller uptake of carotenoids after cellular exposure to distal colon components compared to

proximal colon components is according to expectations. In vivo carotenoid absorption is assumed to

occur predominantly by enterocytes in the small intestine [2]. Thus, cellular transport of vitamin A is

presumed to be higher in the proximal part of the colon than further on in the distal colon. The results

suggest that luminal components present during maturation of the intestinal cells could also contribute

to this distribution of vitamin A transport along the gastro-intestinal tract. Since the distal colon

condition with FOS also differs significantly from the other distal colon treatment, the incubation with

FOS seems to be responsible for this pronounced effect and apparently, carbohydrate fermentation

products had a crucial role in the distinct maturation of the cells [263].

Several components present during the pretreatment could be responsible for these final differences in

cellular uptake. For instance, the total SCFAs concentration, and more particularly of acetate, is clearly

increased in the distal colon water with FOS, compared to the other samples (Figure 25). No statistical

analysis could be performed, due to a lack of replicates, however the variance is expected to be low,

analogous to other studies using this method [207]. The ability of SCFAs to induce expression of

transport receptors on eukaryotic cells was already suggested for vitamin D [142]. Nevertheless, a

correlation between acetate and carotenoid transport has not been reported earlier. Other fermentation

products that have been shown to vary along the colon, likely due to a shift in composition and density

of the bacterial population, are hydrogen sulfide, mercaptan, branched chain fatty acids, polyamine and

phenol concentrations [260, 263, 264]. Further, the bile salt concentration is in general higher in the

proximal colon [265]. As described in the literature study (I.3.3.1), these can act as signaling molecules

for regulation of the intestinal transport function. The bile salts present in the samples were however not

characterized. In addition, the NO test revealed a change in nitric oxide concentration due to incubation

in the presence of FOS, with significantly more NO production by bacteria when no FOS is added

(Figure 23). Recently, this down-regulation of the NO production by FOS, more particularly inulin, has

also been demonstrated in mice [266]. Although NO was already found to modulates absorption and

secretion of electrolytes in epithelial cells, the effect on carotenoid transport was not yet investigated

[267].

53

More specific pretreatment and transport experiments will be necessary to conclude which of these

components have the capacity to guide maturation of the carotenoid transport function in intestinal cells.

The variety of components present during the performed pretreatment could have caused opposite

effects and therefore, the impact of the different components is not visible. Nevertheless, the significant

difference that was found between certain intestinal samples does give an indication that maturation of

the intestinal transport function for carotenoids in differentiated Caco-2 cells can be altered by the

luminal content. The smaller variability in cellular uptake between biological replicates pretreated with

intestinal samples and blanks with buffer or FOS, also suggests that luminal components could be

capable of directing the transport function of Caco-2 cells and so reduce variability in carotenoid

transport by cellular noise. This effect is most pronounced in the distal colon water with FOS. A

repetition of the experiment is however necessary to verify if this effect is reproducible.

The intestinal water also caused a change in the paracellular permeability of the Caco-2 cells, visualized

by the TEER and apparent permeability measures in Figure 26. The Lucifer yellow test showed a

significant lower permeability after pretreatment with distal colon water, compared to the intestinal

conditions in which FOS was added. This effect could be attributed to FOS which was shown to impair

the intestinal barrier in rats by increasing permeability [268]. Another component that could be

responsible, is NO which is present in the samples without FOS and has been reported to help in

maintaining tight junction integrity [267]. Another factor that could have supported changes in the cell

monolayer permeability, is the pH of the different conditions. As discussed earlier, a buffer system is

present in the cell culture medium, but small alterations in pH, when dilutions are made with the

intestinal samples, cannot be excluded though. No pH measurements were performed, but it is known

that colon pH varies from a slightly acidic environment proximally to a more neutral pH in the distal

colon. Since more acidic conditions increase permeability, this could partly explain the observed

differences [269]. As can be seen in Figure 26, the TEER values are clearly correlated to the apparent

permeability, with the transepithelial electrical resistance increasing and the permeability decreasing

over time. Nevertheless, according to the TEER measurements, other significant difference in

permeability are present between conditions after the pretreatment period. These differences are less

logical and could be attributed to external factors. The most important influence for TEER values is the

temperature [195]. Since Transwell® plates had to be transferred from the incubator at 37 °C to the

electrode at room temperature for measurements, plate-to-plate variability cannot be completely

excluded. The noticed changes in permeability for both tests are not correlated with the observed

differences in carotenoid transport. This could be expected, because active transport is most important

for carotenoids at physiological concentrations, as mentioned in the literature study (I.2.3).

Although some significant differences in β-carotene transport could be detected, more effects were

expected, considering the distinct cellular pretreatments that were performed. As mentioned before, the

presence of components exhibiting opposing effects, could have hindered a distinct maturation of the

54

cells. Further, the detection of carotenoids in the used in vitro model is not optimal as was discussed in

the previous section and the LOD could have prevented comparison of basolateral secretions, since in

only some replicates β-carotene was found in the basal compartment. Although the TC-7 cell line is able

to convert carotenoids into retinaldehyde or other apo-carotenoids, these could not be detected with the

used method either [239]. In addition, the intestinal samples were highly diluted with cell culture

medium and thus, intestinal components were present in really low concentrations. Since the distal colon

sample still caused a toxic effect to undifferentiated TC-7 cells with a 1/10 dilution after 1 day, a cautious

3 times higher dilution was chosen to start pretreatment (Figure 24). The treatment could be increased

once the cells became more differentiated and thus, more resistant. Probably a lower dilution of the

samples was possible without cytotoxic effect, which could have induced more distinct effects. Finally,

longer pretreatment until full differentiation of the Caco-2 cells should also be able to make a more clear

distinction between the various conditions visible.

It could be concluded that the obtained results already give a first indication of the potential of luminal

components to influence the capacity of Caco-2 intestinal cells for β-carotene transport. It should

however be noted that the used in vitro cell model certainly does not fully simulate the in vivo situation.

It was already discussed in the literature study (I.5.1.1) that the used Caco-2 cells go through a shift in

phenotype from tumoral colonic to small intestinal [180]. Due to the different origin and maturation

process, it is not possible to extrapolate these effects to the in vivo intestinal cells. Hence, the potential

of the Caco-2 cell model to resemble in vivo maturation should still be validated or further in vivo

experiments will eventually be necessary.

55

CONCLUSION

Despite the general recognition of vitamin A as a crucial dietary element to maintain many different

functions in the human body, deficiencies remain a common health problem in most developing

countries with the highest incidence found in children. The limited bioavailability of vitamin A, and

especially of provitamin A carotenoids, is a critical factor contributing to this issue. Hence, more

research still has to be conducted to gain a better understanding of their intestinal absorption.

A first contribution to fundamental research of vitamin A bioavailability was made by the production of

a new type of β-carotene emulsion. The dispersion was created to better resemble mixed micelles in the

human gastrointestinal tract by use of the intestinal components phosphatidylcholine and sodium cholate.

The use of the co-solvent evaporation method was found to be an elegant way for obtaining small

micelles sizes (< 200 nm), a high dispersion stability and a good incorporation of the carotenoids. The

procedure of emulsification was optimized for delivery to intestinal Caco-2 cells in which uptake and

secretion of β-carotene was found to be possible. As such, this new emulsion was shown to be more

suitable for β-carotene delivery in in vitro Caco-2 models than the earlier established ‘Tween’ methods.

An in vitro cell model was further developed to make investigation of β-carotene bioavailability feasible.

By adjusting the extraction and HPLC method, a LOD of 0.06 µM of β-carotene in cell culture medium

was finally achieved. Although this sufficed to measure cellular uptake, detection of basal secretion

proved to be restricted. Cellular variability in the used parental Caco-2 cell line also entailed difficulties

for reproducibility and comparability between experiments, which is a common issue in this type of in

vitro studies.

Previous achievements were eventually used to assess the stated hypothesis. A significant reduction in

cellular uptake of β-carotene was found after pretreating intestinal Caco-2 cells during their

differentiation with distal colon water compared to proximal colon water. Prior incubation with FOS,

stimulating carbohydrate fermentation, proved to be necessary to make a distinction between the

conditions possible. As such, these findings already indicate that dietary components can direct the

transport function of intestinal Caco-2 cells for β-carotene.

In conclusion, a new β-carotene emulsion was developed that could be used in future research to study

bioavailability. Restrictions of in vitro Caco-2 models used in carotenoid bioavailability studies could

further be confirmed. Finally, a first pretreatment experiment suggested that certain luminal components

can act as triggers in the maturation of the intestinal transport function of β-carotene. This master’s

dissertation has this way contributed to fundamental research about the bioavailability of vitamin A.

56

FUTURE PERSPECTIVES

Whereas some relevant findings were achieved by the performed experiments, further fundamental

research will still be necessary to obtain a better understanding about the bioavailability of vitamin A

and its influencing factors. This master’s thesis could only briefly touch on the stated hypothesis and

therefore, future investigations will have to be conducted to assess this subject more thoroughly.

First of all, the developed model to make the study of β-carotene bioavailability possible, can be further

improved by completing some extra experiments. Regarding the produced emulsion with the co-solvent

evaporation method, further stability tests could be performed to evaluate the degradation of β-carotene

and the evolution of micelle sizes and ζ-potential over time. The actual stability of the micelles after

dilution with cell culture medium should also be verified and in addition, reproducibility of the

dispersion is uncertain. In the in vitro cell model, detection proved to be problematic for secreted

carotenoids. Hence, an improvement in LOD is desirable and could be achieved by, for instance,

changing the used solvents or switching to mass spectrometry for detection.

To further investigate the hypothesis that luminal components can influence the maturation of the

vitamin A transport function in intestinal cells, the performed experiment could be repeated to confirm

the obtained results. Screening of specific intestinal components, such as certain SCFAs or bile salts,

will then be necessary to get an idea of which molecules are capable to trigger the development of certain

receptors necessary in β-carotene transport. The same can be done for other carotenoids or retinoids that

contribute to the vitamin A status. The used Caco-2 cell model also has to be validated for its ability to

extrapolate to the in vivo situation. In the long term, results have to be confirmed with in vivo

experiments and human studies.

Eventually, these investigations could lead to the development of new strategies for improving the

vitamin A status of children in developing countries. By intervening during human development, the

overall problem of vitamin A deficiencies worldwide could potentially be diminished.

57

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ADDENDUM: REFLECTION ON SUSTAINABILITY

Worldwide environmental and social problems demonstrate that current human practices become

untenable. To achieve a transition towards a more social and ecological just future, sustainable research

will have to play a crucial part. In this addendum, a critical reflection is given about the sustainability

of this master’s thesis. Besides merely the societal relevance, the sustainability of the performed

experimental work in the laboratory is discussed.

Firstly, it could be stated that the subject of this dissertation already contributes to sustainability by

focusing on human health, rather than on economic profit. Vitamin A deficiency causes severe health

issues in most countries and thus, scientific research is still needed to gain control over this problem.

Although only fundamental research was performed, it was explained how this work eventually could

be able to generate new intervention strategies. The involvement of several research groups further

indicates that multiple disciplines were brought together to accomplish the study objectives, a

transdisciplinary approach, necessary in sustainability issues, was however lacking.

Nevertheless, sustainability goes beyond just choosing appropriate research subjects. Another important

aspect to reflect on, is if the research was conducted in a sustainable way. From this point-of-view,

ecological concerns were mainly overlooked, which is illustrated by the abundant amount of solvents

used in the extraction and HPLC procedure, the large energy consumption of almost continuously

running devices (laminar flow, fume hood, HPLC) and lots of plastic waste due to the frequent use of

pipet tips, cell vials and tubes. Yet, some efforts were made to reduce the environmental impact of these

laboratory practices. The use of solvents was reduced when possible and part of the pipet tips were

cleaned for reuse in the chemical lab. In the cellular lab, where sterility is an absolute must, reuse is

however not that obvious, but an attempt was still made with the valuable Transwell® plates. Rather

surprising is that contamination was not the prime complication, it were changes in membrane structure

after cleaning that prevented attachment of cells and hence, made secondary use impossible. This

specific case highlights a key obstacle in the development phase of current equipment to proceed

towards sustainable laboratory practices.

It could be concluded that despite the social relevant subject, issues were found regarding the overall

sustainability in the laboratory. Although efforts could be made to reduce and reuse materials, these

measures will primary help in raising awareness among the laboratory staff about the lacking

sustainability. In the long term, more radical changes are required, in which revision of the development

of used materials could be important. Energy-efficient and reusable laboratory equipment is currently

not available and thus, research projects dealing with this lack of alternatives should be adopted.

faculty of bioscience engineering academic year 2014...

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