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T01008:Development of an
in vitro
intestinal cell model to predict bioavailability of food components in humans
Friday 21 January 2005
This research project aimed to establish an
in vitro
intestinal cell model for prediction of in vivo bioavailability of food components from the human diet.
Study Duration
: April 1998 to August 2002
Contractor
: Rowett Research Institute, Aberdeen
Background
There is a need to understand in more detail, the transport of food constituents across intestinal cells and the factors that influence their rate of uptake. Rather than perform such studies in living persons, an
in vitro
model comparable to in vivo conditions can facilitate much easier and reproducible investigations into transport mechanisms in the gut.
Research Approach
Three intestinal cell lines (Caco-2, HT-29 and T84) were used to develop
in vitro
models of passive permeability and active uptake.
These were used to predict the bioavailability of food chemicals from digested foods. Transport of food components across intestinal monolayers (apical to basolateral/serosal) was determined using Transwell
®
(tissue culture treated permeable polyester membrane cell culture inserts).
Native compounds and metabolites were routinely detected by high performance liquid chromatography (HPLC).
In addition, a small range of compounds were radiolabelled in-house with
14
C carbon which is easy to
measured post-uptake.
Emphasis was placed on the optimisation of culture conditions using matrix proteins and soluble factors in the culture of the cells, improving on current procedures by reducing the required culture period prior to experimentation.
Results and findings
This project examined the applicability of
in vitro
intestinal cell models, commonly used in the pharmaceutical industry, to predict bioavailability of food components in humans.
Assay Development:
-
Several intestinal cell lines were evaluated as models for human gut function.
The Caco-2 human colon adenocarcinoma cell line was found to be the most representative.
-
The Biopharmaceutical Classification System was used to assess the performance of the model. This allowed the transport characteristics for food chemicals to be compared to known drug �standards� for which human bioavailability data exists.
-
In this assay, the cells are grown in Transwell
chambers, which allow the transport of chemicals across the cells to be measured.
It is important that the cells are grown for at least 21 days so that they form a continuous sheet of cells (called a confluent differentiated monolayer) that mimics the cells that line the gut.
This study found that a confluent differentiated monolayer could be achieved more quickly if grown upon proteins mimicking the extracellular matrix and in a defined cell culture medium.
-
An additional factor affecting absorption of food components from the gut is the presence of mucus on the gut surface.
Mucus is secreted by goblet cells in the intestinal epithelium.
In order to mimic this aspect of gut physiology, co-culture experiments were attempted.
In these experiments, Caco-2 cells were grown in culture along with HT29-MTX cells, which are enriched for mucus secreting goblet-like cells.
Differences in absorption were observed when Caco-2 cells were cultured alone and in the presence of the HT29-MTX cell line.
However, it could not be determined whether this was due to the presence of mucus on the surface of the monolayer or the alterations to the integrity of the monolayer due to the presence of a foreign cell type.
Absorption of naturally occurring phenolic food chemicals:
-
Cinnamic acid, p-couramic, caffeic and feurlic acids are naturally occurring phenolic food chemicals that are common in the western diet.
These were highly absorbed in this in vitro system, 95%, 88%, 83% and 52% respectively, suggesting that similar uptake might be expected
in vivo
.
-
Food chemicals may be absorbed from the gut by both active and passive transport mechanisms.
Passive transport occurs as food chemicals diffuse across the gut wall, down a concentration gradient.
Active transport mechanisms rely on specific proteins within the membranes of gut cells which transport food chemicals across the gut wall, sometimes against their concentration gradient.
Both active and passive transport mechanisms appeared to be involved in the transport of phenolic food chemicals across the human gut. Caffeic and feurlic acids appeared to be passively transported whereas cinnamic acid and p-couramic acids appeared to be actively transported.
-
The apparent active transport of cinnamic and p-couramic acids is interesting.
Despite similarities in structure these chemicals do not seem to compete with each other for absorption.
Further research would be required to identify the specific transporters that mediate this active uptake.
This approach provides an
in vitro
assay that gives an indication of the uptake of food components from the gut.
This assay may be of use in assessing dietary exposure to chemicals that are present in foods.
Dissemination information
The final report is available from the Agency's Information Centre.
To obtain a copy, please contact the Enquiry Desk, Information Services, Food Standards Agency (tel: 020 7276 8181/8182 or email:
infocentre@foodstandards.gsi.gov.uk
)
Contact
: For any enquiries concerning this research project, please contact the relevant Programme contact or email:
science@foodstandards.gsi.gov.uk
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