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MODEL SYSTEMS, IN VITRO AND IN VIVO, FOR PREDICTING THE BIOAVAILABILITY OF LIPID SOLUBLE COMPONENTS OF FOOD

Deliverables

Carotenoid absorption from food materials is compromised by the food structure. For example, the absorption of the carotenes from raw carrot is significantly lower then that achieved from carrot puree. Little information is available from the current literature on the factors influencing the release of the carotenoids from foods. This impacts on the quality of the advice we are currently able to give consumers on the form carotenoids should be ingested within the diet and the role food processing has on the biaccessibility of the lipid soluble nutrients. The factors influencing carotenoid release from plant tissue during gastric digestion were investigated with a static in vitro model. It was found that the major determinant of release was the degree to which the individual cells had been disrupted. Thus processing of the plant tissue by mashing, pureeing or fine shredding could improve bioaccessibility. Heating of the tissue was not sufficient to cause cell disruption and may well reduce bioaccessibility by enabling cell separation to occur as opposed to cell disruption. Simulated gastric digestion was carried out on spinach (frozen blanched and frozen raw whole leaf), carrot (paste, raw cubes and raw juice) and tomato (fresh and paste). The variables investigated were initial carotenoid concentration, pH(5.5 and 2.5), triglyceride composition (sunflower oil and triolein), triglyceride concentration (20% and 2%w/w), the presence of gastric lipase and the action of pepsin. We have demonstrated that the factors determining the extent of transfer of carotenoids from tissues to an oil phase within a model gastric environment can be ranked: Structure (cell integrity)~tissue particle size>>heating>pH~oil interfacial area oil concentration.
Many minor plant metabolites cannot be practically or economically synthesized so the biosynthetic route is the only practical option. Biosynthesis of stable isotope labeled compound within a food plant does not have the same ethical constraints as the chemically synthesized product where safety is the overriding consideration. The metabolites can be extracted and purified for feeding as a supplement for biochemical studies or can be fed within the plant food matrix to assess the effects of the matrix on bioaccessability and modification of digestion and absorption profiles. From such studies, the kinetics of absorption and disposal can be measured against the endogenous background without the need for dietary intervention that may invalidate the results of conventional studies. The production of batches of food plant material (5kg) has successfully been achieved by pulse labeling the growing plant with 13CO2. The level of label ca. 4% is sufficient to detect the compound at a concentration of 2% in natural abundance background using LC or GC-MS. Sensitivity can be increased by using GC-C-IRMS provided sufficient sample can be obtained. A minimum sample of 500ug is needed for LC and GC-MS although smaller samples would be sufficient for GC-C-IRMS. The method has the potential to enable intrinsic labeling of organic microcomponents of foods for ethically acceptable metabolic and bioavailability studies in man. The result is a scaled up version of known technology and demonstrates the feasibility of the process to produce of using 13CO2 to label working quantities of vegetable and fruit foods for small human feeding studies. The application of the process to human feeding studies depends on the use of high precision mass spectrometry and the availability of LC-MS for non-volatile compounds and GC-MS or GC-C-IRMS for volatile compounds.
We have elaborated an in vitro model which allows study of the transfer of lipid soluble components (LSC) from emulsion lipid droplets to micelles. In the gastrointestinal tract, LSC are trapped in emulsion lipid droplets and they require to be solubilized into mixed micelles to become available for absorption. This step is assumed to be a key step which governs the absorption of LSC. Our in vitro model allows study of the efficiency of transfer of any kind of LSC (fat soluble vitamins or micronutrients, xenobiotics, drugs) and thus to predict their biovailability. Some of the results obtained with this model have been validated by human studies. For example, our model has shown that the transfer of carotenoids from lipid droplets to micelles is inversely related to their hydrophobicity, and the in vivo studies have confirmed that the less hydrophobic carotenoids are better absorbed than the more hydrophobic ones. -This is a new method to estimate the absorption efficiency of lipid soluble components (LSC) of foods: micronutrients, xenobiotics or drugs. -Although the absorption efficiency of LSC is very variable and can be very low, the factors which modulate the absorption efficiency of different LSC are not clearly identified. -This model provides a low cost quick method to estimate the absorption efficiency of a LSC and to identify the key factors which affect absorption of a LSC.
The TNO “simple” in vivo method to study bioavailability of carotenoids comprises measurement of the postprandial responses of carotenoids in the triacylglycerol rich lipoprotein (TRL) fraction of plasma. An optimised protocol was established to maximise sensitivity and to enable application of this method with vegetable sources. This method was subsequently applied with the model foods to compare bioavailability between raw or minimally processed and processed vegetables, as well as comparison with other (in vitro) methods. The results of this study show that bioavailability of lycopene, and ß-carotene from natural sources can be demonstrated using the postprandial TRL response, and compared quite well with results obtained with other methods. For lutein results obtained with the TRL method were lower as compared with the other methods, and the absorption of lutein seems to be underestimated using this approach. An optimized protocol for measuring the postprandial chylomicron response of carotenoids from (vegetable) food sources after a single oral dose (test meal) for assessment of the relative bioavailabilty in man.
A new human model has been elaborated which allows to study the fate of lipid soluble components (LSC) of foods in the upper human gastrointestinal tract. This model allows one to follow the transfer of LSC from the food matrix to the fat phase, or the aqueous phase, of the meal in the human stomach during digestion. It also allows to follow the transfer of LSC from the food matrix to the mixed micelles in the duodenum. The data obtained is very useful in understanding the fate, and thus the absorption efficiency, of various LSC in normal and pathological conditions. - This is a new method to study the fate of lipid soluble components (LSC) of foods in the upper human gastrointestinal tract; - The fate of different LSC in the upper human gastrointestinal tract is not known. Nevertheless these data can help to understand the low absorption efficiency of a LSC with the aim to improve it; - This model provides a new tool to study the factors which affect the absorption of LSC.
Carotenoid absorption from food materials is compromised by the food structure. For example, the absorption of the carotenes from raw carrot is significantly lower than that achieved from carrot puree. Little information is available from the current literature on the factors influencing the release of the carotenoids from foods. This impacts on the quality of the advice we are currently able to give consumers on the form carotenoids, should be ingested within the diet and the role food processing has on the biaccessibility of the lipid soluble nutrients. The factors influencing carotenoid release from plant tissue during gastric digestion were investigated with a static in vitro model. It was found that the major determinant of release was the degree to which the individual cells had been disrupted. The in vitro model was developed to mimic the colloidal and biochemical aspects of gastric digestion. The results of this model were validated against ex vivo gastric contents aspirated from human volunteers who had consumed the same plant tissue. The gastric in vitro model contained a highly controlled lipid phase (emulsion droplets of sunflower oil stabilized by phospholipid to achieve a weight mean droplet diameter of ~10um). Gastric lipase was incorporated to achieve ~10% hydrolysis of the lipid and pepsin was also added. The vegtable tissue was added and the system incubated at 37 degrees Celsius for two hours prior to separation and carotenoid analysis of the lipid phase. The system predicted the release of carotenoids from spinach, carrot and tomato tissue after various processing treatments when compared with the release found in the gastric contents of human volunteers fed the same meals.
A computer controlled model-developed to predict the bioaccessibility of lipolytic compounds in food and medicine-has been validated for fat soluble vitamins from processed vegetables. The system shows similar results as compared to in vivo data obtained with the same meals. The model offers a helpful tool to study the bioaccessibility of lypophilic compound in food, for example in relation to processing and food composition. The TNO gastro-Intestinal Model (TIM) very closely simulates the successive dynamic conditions in the gastrointestinal tract, such as the pH curves and concentrations of (pro-) enzymes in the stomach and small intestine, concentrations of bile salts in the different parts of the gut, and the kinetics of passage of chyme through the stomach and intestine (Havenaar et al., 1996; Minekus et al., 1995; Minekus and Havenaar, 1996, 1998). In this model the digestibility and availability for absorption of nutrients as well as the stability of specific ingredients (e.g. active proteins, peptides) can be studied. A specific system has been developed to remove the bio-accessible mixed-micelles. The system can be used to study the bioaccessibility of fat-soluble (lypophilic) compounds that are incorporated into the mixed micelles prior to absorption, such as fat soluble vitamins, and fat soluble medicines.
The use of human ileostomy volunteers has proved to be a valid approach to the measurement of absorption of carotenoids from various raw and cooked foods of vegetable origin. By avoiding the confounding influence of the large intestinal microflora it is possible to measure the intake and excretion of potentially fermentable nutrients by simple mass balance. The method also has the added advantage that samples of effluent and blood samples can be collected in real time and then compared. The method has the potential to be developed for the measurement of other nutrients where it is difficult to obtain measurements of absorption by the more classical method of measuring plasma concentration excursions following an acute oral dose. It should be particularly useful for micronutrients where plasma measurement are difficult or inappropriate because of complex first pass clearance or rapid dispersal to remote (difficult to sample) tissues. The measurement of absorption of beta-carotene, lutein and lycopene from carrot, spinach and tomato has been successfully carried out in ileostomy volunteers and correlated to absorption measured by the plasma carotenoid concentration excursions. This has confirmed the mathematical model of disposal kinetics.