Can dynamic in vitro digestion systems mimic the physiological reality?

During the last decade, there has been a growing interest in understanding the fate of food during digestion in the gastrointestinal tract in order to strengthen the possible effects of food on human health. Ideally, food digestion should be studied in vivo on humans but this is not always ethically and financially possible. Therefore simple static in vitro digestion models mimicking the gastrointestinal tract have been proposed as alternatives to in vivo experiments but these models are quite basic and hardly recreate the complexity of the digestive tract. In contrast, dynamic models that allow pH regulation, flow of the food and injection in real time of digestive enzymes in the different compartments of the gastrointestinal tract are more promising to accurately mimic the digestive process. Most of the systems developed so far have been compared for their performances to in vivo data obtained on animals and/or humans. The objective of this article is to review the validation towards in vivo data of some of the dynamic digestion systems currently available in order to determine what aspects of food digestion they are able to mimic. Eight dynamic digestion systems are presented as well as their validation towards in vivo data. Advantages and limits of each simulator is discussed. This is the result of a cooperative international effort made by some of the scientists involved in Infogest, an international network on food digestion.

Correlation between in vitro and in vivo data on food digestion. What can we predict with static in vitro digestion models?

During the last decade, there has been a growing interest in understanding food's digestive fate in order to strengthen the possible effects of food on human health. Ideally, food digestion should be studied in vivo on humans but this is not always ethically and financially possible. Therefore, simple in vitro digestion models mimicking the gastrointestinal tract have been proposed as alternatives to in vivo experiments. Thus, it is no surprise that these models are increasingly used by the scientific community, although their various limitations to fully mirror the complexity of the digestive tract. Therefore, the objective of this article was to call upon the collective experiences of scientists involved in Infogest (an international network on food digestion) to review and reflect on the applications of in vitro digestion models, the parameters assessed in such studies and the physiological relevance of the data generated when compared to in vivo data. The authors provide a comprehensive review in vitro and in vivo digestion studies investigating the digestion of macronutrients (i.e., proteins, lipids, and carbohydrates) as well as studies of the bioaccessibility and bioavailability of micronutrients and phytochemicals. The main conclusion is that evidences show that despite the simplicity of in vitro models they are often very useful in predicting outcomes of the digestion in vivo. However, this has relies on the complexity of in vitro models and their tuning toward answering specific questions related to human digestion physiology, which leaves a vast room for future studies and improvements.

The EuroPrevall outpatient clinic study on food allergy: background and methodology.

BACKGROUND: The EuroPrevall project aimed to develop effective management strategies in food allergy through a suite of interconnected studies and a multidisciplinary integrated approach. To address some of the gaps in food allergy diagnosis, allergen risk management and socio-economic impact and to complement the EuroPrevall population-based surveys, a cross-sectional study in 12 outpatient clinics across Europe was conducted. We describe the study protocol. METHODS: Patients referred for immediate food adverse reactions underwent a consistent and standardized allergy work-up that comprised collection of medical history; assessment of sensitization to 24 foods, 14 inhalant allergens and 55 allergenic molecules; and confirmation of clinical reactivity and food thresholds by standardized double-blind placebo-controlled food challenges (DBPCFCs) to milk, egg, fish, shrimp, peanut, hazelnut, celeriac, apple and peach. RESULTS: A standardized methodology for a comprehensive evaluation of food allergy was developed and implemented in 12 outpatient clinics across Europe. A total of 2121 patients (22.6% <14 years) reporting 8257 reactions to foods were studied, and 516 DBPCFCs were performed. CONCLUSIONS: This is the largest multicentre European case series in food allergy, in which subjects underwent a comprehensive, uniform and standardized evaluation including DBPCFC, by a methodology which is made available for further studies in food allergy. The analysis of this population will provide information on the different phenotypes of food allergy across Europe, will allow to validate novel in vitro diagnostic tests, to establish threshold values for major allergenic foods and to analyse the socio-economic impact of food allergy.

Development of a standardized low-dose double-blind placebo-controlled challenge vehicle for the EuroPrevall project.

BACKGROUND: Double-blind placebo-controlled food challenge (DBPCFC) is the gold standard for diagnosing food allergy. Standardized materials and protocols are essential for comparing DBPCFC results for multicentre studies such as EuroPrevall. This required the development and piloting of a standardized vehicle and low-dose protocol for confirming food allergy and determination of minimum eliciting doses (MEDs). METHODS: A low-dose DBPCFC protocol was developed, with eight titrated protein doses from 3 µg to 1 g. This was delivered using a simple, microbiologically stable food base incorporating allergenic food ingredients manufactured at three sites and centrally distributed to clinical centres. Allergen blinding was assessed by a professional sensory testing panel using a triangle test. Homogeneity and allergen content were confirmed by ELISA and clinical efficacy was assessed in a pilot study, using celeriac and hazelnut as exemplars. RESULTS: Celeriac and hazelnut ingredients were sufficiently blinded in the dessert. The dessert meals were successfully piloted with hazelnut in allergy clinics in Spain, the Netherlands and Italy and with celeriac and hazelnut in Zurich. The challenges elicited a range of subjective and objective reactions ranging in severity from mild itching of the oral mucosa to bronchospasm. CONCLUSIONS: A standardized challenge vehicle proven to sufficiently blind processed, powdered hazelnut and celeriac ingredients and that can be reproducibly manufactured has been developed. This pilot study shows that the vehicle is promising for the confirmation of food allergy and determination of MEDs in adults and children with body weight >28.8 kg (approximately 7-11 years old).

In vitro digestibility of beta-casein and beta-lactoglobulin under simulated human gastric and duodenal conditions: a multi-laboratory evaluation.

Initially the resistance to digestion of two cow's milk allergens, beta-casein, and beta-lactoglobulin (beta-Lg), was compared using a "high-protease assay" and a "low-protease assay" in a single laboratory. The low-protease assay represents an alternative standardised protocol mimicking conditions found in the gastrointestinal tract. For the high-protease assay, both proteins were incubated with either pepsin or pancreatin and digestion monitored by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and reverse phase-high performance liquid chromatography. The low-protease assay involved gastroduodenal digestion in the presence or absence of phosphatidylcholine (PC). Both beta-casein and beta-Lg were susceptible to hydrolysis by pepsin and pancreatin in the high-protease assay. In contrast, the kinetics of beta-casein digestion in the low-protease assay were slower, beta-Lg being pepsin resistant. During duodenal digestion, beta-Lg was gradually degraded and addition of PC slowed digestion. Subsequently, the reproducibility of the low-protease assay was assessed in 12 independent laboratories by visual assessment of the gels and densitometric analysis: the inter- and intra-laboratory variability was affected by sampling and electrophoresis method employed. The low-protease assay was shown to be reproducible. Future studies will extend these findings using a broader panel of proteins.

Thresholds for food allergens and their value to different stakeholders.

Thresholds constitute a critical piece of information in assessing the risk from allergenic foods at both the individual and population levels. Knowledge of the minimum dose that can elicit a reaction is of great interest to all food allergy stakeholders. For allergic individuals and health professionals, individual threshold data can inform allergy management. Population thresholds can help both the food industry and regulatory authorities assess the public health risk and design appropriate food safety objectives to guide risk management. Considerable experience has been gained with the double-blind placebo-controlled food challenge (DBPCFC), but only recently has the technique been adapted to provide data on thresholds. Available data thus vary greatly in quality, with relatively few studies providing the best quality individual data, using the low-dose DBPCFC. Such high quality individual data also form the foundation for population thresholds, but these also require, in addition to an adequate sample size, a good characterization of the tested population in relation to the whole allergic population. Determination of thresholds at both an individual level and at a population level is influenced by many factors. This review describes a low-dose challenge protocol developed as part of the European Community-funded Integrated Project Europrevall, and strongly recommends its wider use so that data are generated that can readily increase the power of existing studies.

Microcalorimetry of the intestinal mucus: Hydrogen bonding and self-assembly of mucin.

The effect of mucin hydrogen bonding on the structure of intestinal mucus has been studied with micro-differential scanning mirocalorimetry (µ-DSC), supported by spectroscopy. The experiments were performed in water-dimethyl sulfoxide (DMSO) solutions, using either water-DMSO mixtures of an appropriate DMSO content or water as blanks, as to isolate the effects of the solvent to hydrogen bonding. When using matched water-DMSO blanks, thermal events at low temperatures are linked to the negation of mucin-DMSO interactions, while events at higher temperatures are linked to the break-up of hydrogen bonds connecting the sugars of the individual macromolecules. When using a matched solvent as blank, alterations in Cp, such as increases at 10% and 15% DMSO, have been linked to the break-up and creation of quaternary structures. In the case of water as blank, a monotonic but not linear decrease in enthalpy, hence extent of hydrogen bonding, is observed. The above are complemented by UV spectroscopy: A blue shift of the conjugated aminoacids in the presence of DMSO suggests that the inherent stability of mucin is not only due to steric volume exclusions, but also due to extensive hydrogen bonding on behalf of the sugar moieties.

Nullifying drug-induced sensitization: behavioral and electrophysiological evaluations of dopaminergic and serotonergic ligands in methamphetamine-sensitized rats.

Repeated exposure to methamphetamine produces a persistent enhancement of the acute motor effects of the drug, commonly referred to as behavioral sensitization. Behavioral sensitization involves monoaminergic projections to several forebrain nuclei. We recently revealed that the ventral pallidum (VP) may also be involved. In this study, we sought to establish if treatments with antagonists or partial agonists to monoaminergic receptors could "reverse" methamphetamine-induced behavioral and VP neuronal sensitization. Behavioral sensitization was obtained in rats with five once-daily s.c. injections of 2.5mg/kg methamphetamine, an effect that persisted for at least 60 days. After the development of sensitization, 15 once-daily treatments of mirtazapine (a 5-HT(2/3), alpha(2) and H(1) antagonist), SKF38393 (D(1) partial agonist) or SCH23390 (dopamine D(1) antagonist) nullified indices of motor sensitization as assessed by measuring the motoric response to an acute methamphetamine challenge 30 days after the fifth repeated methamphetamine treatment. VP neurons recorded in vivo from methamphetamine-sensitized rats at the 30-day withdrawal time also showed a robust downward shift in the excitatory responses observed to an acute i.v. methamphetamine challenge in non-sensitized rats. This decreased excitatory effect was reversed by mirtazapine, but not by other antagonists that were tested. These data suggest a potential therapeutic benefit for mirtazapine in the treatment of methamphetamine addiction, and point to a possible role for the VP in the sensitization process to methamphetamine.

The manipulation of DNA with restriction enzymes in low water systems.

The cleavage of phage lambda (lambda) DNA by the restriction enzyme HindIII in low water systems has been investigated. Two types of low water systems have been studied--those which contain a surfactant in a reverse micelle environment and a surfactant-free system in which a solid support (celite) is used. The effect of the surfactants themselves in a normal aqueous environment has also been studied. Charged surfactants were found to greatly inhibit HindIII activity in aqueous buffer, while non-ionic surfactants did not affect either the activity or the specificity of the restriction enzyme. The rate of cleavage by HindIII in a reverse micelle system consisting of sodium dioctylsulphosuccinate is very slow, however, in a Triton B system the expected fragments are observed. In a surfactant-free low water environment, cleavage occurs at the expected sites but in a different order to that observed in normal aqueous systems. These results suggest that DNA tertiary structure in low water systems is different to that in aqueous solution and that this influences cleavage by the restriction enzyme HindIII.

Lateral diffusion and conductance properties of a fluorescein-labelled alamethicin in planar lipid bilayers.

In order to follow alamethicin diffusion within membranes under conditions of pore-formation, a fluorescein isothiocyanate (FITC) analogue was synthesized. To test the influence of the fluorescent probe addition on the pore-forming activity of the new analogue, macroscopic and single-channel experiments into planar lipid bilayers were performed. Although the apparent mean number of monomers per conducting aggregate was equivalent, the voltage-dependence of the new analogue was slightly reduced and hysteresses were broader, in agreement with the much longer duration of the open single-channels. Thus, the conducting aggregates seem to be stabilized by the introduction of the probe, presumably through the interaction of the conjugated cycles with the lipid headgroups, while the added steric hindrance may account for the slightly higher conductances of the open substates. Lateral diffusion of the labelled peptide associated with the bilayer was then investigated by the fluorescence recovery after photobleaching technique. Under applied voltage, associated with high conductance, D, the lateral diffusion coefficient, was reduced by 50% when compared to peptide at rest. These results provide new independent experimental evidence for a voltage-driven insertion of the highly mobile surface-associated peptide into the bilayer as a prominent step in pore formation.

Coupling Optical and Electrical Measurements in Artificial Membranes: Lateral Diffusion of Lipids and Channel Forming Peptides in Planar Bilayers.

Planar lipid bilayers (PLB) were prepared by the Montal-Mueller technique in a FRAP system designed to simultaneously measure conductivity across, and lateral diffusion of, the bilayer. In the first stage of the project the FRAP system was used to characterise the lateral dynamics of bilayer lipids with regards to phospholipid composition (headgroup, chain unsaturation etc.), presence of cholesterol and the effect of divalent cations on negatively-charged bilayers. In the second stage of the project, lateral diffusion of two fluorescently-labelled voltage-dependent pore-forming peptides (alamethicin and S4s from Shaker K(+) channel) was determined at rest and in the conducting state. This study demonstrates the feasibility of such experiments with PLBs, amenable to physical constraints, and thus offers new opportunities for systematic studies of structure-function relationships in membrane-associating molecules.

Microgradients in bacterial colonies: use of fluorescence ratio imaging, a non-invasive technique.

Fluorescence ratio imaging is a non-invasive technique for studying the formation of microgradients in immobilised bacterial colonies. These gradients can be quantified easily when combined with the gel cassette system designed at the Institute of Food Research, Norwich, UK. Colonies of Lactobacillus curvatus were observed using this technique and relevant pH gradients were present when the colonies reached a diameter of about 100 microm. These pH gradients were due to production of lactic acid by L. curvatus cells in the colonies. The spatial resolution of the images was about 1.5 microm (scale of bacterial cells) and therefore very suitable for observing local effects in colonies which ranged in sizes from 1 to 500 microm.

Adsorbed protein secondary and tertiary structures by circular dichroism and infrared spectroscopy with refractive index matched emulsions.

The secondary structure of protein adsorbed at the emulsion interface has been studied in refractive index matched emulsions using the techniques of circular dichroism (CD) and Fourier transform infrared spectroscopy. Bovine serum albumin (BSA) and bovine beta-lactoglobulin (betalg) stabilized emulsions were studied, and the refractive index was altered by the addition of glycerol or polyethylene glycol. The effect of additive on the solution and adsorbed protein structure in addition to the effect of adsorption time was considered. Both adsorption and glycerol addition alter protein secondary structure; however, the majority of secondary structure remains. Small changes are observed in the secondary structure of adsorbed protein with time. Near-ultraviolet CD studies showed the effect of glycerol and adsorption on the aromatic groups. BSA showed small changes both upon the addition of glycerol to protein in solution and upon adsorption. betalg showed slightly larger changes upon the addition of glycerol to protein in solution and a larger change upon adsorption.

Effect of calcium ions on in vitro pellicle formation from parotid and whole saliva.

The salivary pellicle is a protein-rich, bacteria-free, self-assembling film that adsorbs to all surfaces within the oral cavity. The pellicle has numerous functions that are vital for maintaining oral health. Currently however, there are no commercially available artificial salivas that accurately mimic the complex film forming properties (i.e. film thickness and viscoelasticity) of human saliva. To understand these properties further we have examined the in vitro formation of the salivary pellicle, by adsorbing stimulated parotid saliva (PS) and whole mouth saliva (WMS) from 14 healthy volunteers, onto oxidised silicon surfaces, using a quartz crystal microbalance with dissipation monitoring (QCMD) and a dual polarisation interferometer (DPI). A dramatic impact on the hydrated mass, polymer mass, thickness and polymer concentration of the pellicle for both WMS and PS was observed when the natural calcium concentration of the respective salivas was increased from 0 mM to 10mM. In addition, QCMD data showed that on addition of 10mM calcium the salivary pellicle formed by both PS and WMS became more predominantly elastic. The results presented here also suggest that calcium can easily diffuse in and out of the pellicle, permitting free calcium exchange between the saliva and the adsorbed pellicle under physiological conditions, which may potentially facilitate the mineralisation of enamel.

Comparison of the orogenic displacement of sodium caseinate with the caseins from the air-water interface by nonionic surfactants.

Displacement of sodium caseinate from the air-water interface by nonionic surfactants Tween 20 and Tween 60 was observed by atomic force microscopy (AFM). The interfacial structure was sampled by Langmuir-Blodgett deposition onto freshly cleaved mica substrates. Protein displacement occurred through an orogenic mechanism: it involved the nucleation and growth of surfactant domains within the protein network, followed by failure of the protein network. The surface pressure at which failure of the protein network occurred was essentially independent of the type of surfactant. The major component of sodium caseinate is beta-casein, and previous studies at the air-water interface have shown that beta-casein networks are weak, failing at surface pressures below that observed for sodium caseinate. The other components of sodium caseinate are alpha(s)- and kappa-caseins. Studies of the displacement of alpha(s)-caseins from air-water interfaces show that these proteins also form weak networks that fail at surface pressures below that observed for sodium caseinate. However, kappa-casein was found to form strong networks that resisted displacement and failed at surface pressures comparable to those observed for sodium caseinate. The AFM images of the displacement suggest that, despite kappa-casein being a minor component, it dominates the failure of sodium caseinate networks: alpha(s)-casein and beta-casein are preferentially desorbed at lower surface pressures, allowing the residual kappa-casein to control the breakdown of the sodium caseinate network at higher surface pressures.

The prevalence, cost and basis of food allergy across Europe.

The development of effective management strategies to optimize the quality of life for allergic patients is currently hampered by a lack of good quality information. Estimates of how many individuals suffer from food allergy and the major foods involved vary widely and inadequacies of in vitro diagnostics make food challenges the only reliable means of diagnosis in many instances. The EuroPrevall project brings together a multidisciplinary partnership to address these issues. Cohorts spanning the main climatic regions of Europe are being developed in infants through a birth cohort, community surveys in school-age children and adults and an outpatient clinic study. Confirmatory double-blind placebo-controlled food challenge diagnosis is being undertaken using foods as they are eaten with titrated doses to allow no-effect and lowest-observable effect levels for allergenic foods to be determined. The cohorts will also facilitate validation of novel in vitro diagnostics through the development of the EuroPrevall Serum Bank. Complementary studies in Ghana, western Siberia, India and China will allow us to gain insights into how different dietary patterns and exposure to microorganisms affect food allergies. New instruments to assess the socioeconomic impact of food allergy are being developed in the project and their application in the clinical cohorts will allow, for the first time, an assessment to be made of the burden this disease places on allergy sufferers and their communities.

Cross-sensitization to morphine in cocaine-sensitized rats: behavioral assessments correlate with enhanced responding of ventral pallidal neurons to morphine and glutamate, with diminished effects of GABA.

Common neurobiological substrates contribute to the progressively increased behavioral effects (i.e., sensitization) that occur with repeated intermittent treatments of cocaine and morphine. Consequently, repeated exposure to cocaine can augment responding to morphine (termed cross-sensitization). Drug-induced sensitization in rats may model aspects of the dysfunction in motivation that are imposed by addiction. The ventral pallidum (VP) is involved in motivated behaviors and its function is altered by acute administration of cocaine and morphine, but the effects of repeated drug exposure remain unknown. Targeting this paucity, the present study evaluated electrophysiological changes in the VP of rats exposed to five once-daily cocaine treatments (15 mg/kg i.p.). This regimen also induced behavioral-sensitization that was expressed 3 days later when the rats received either an acute injection of cocaine (15 mg/kg i.p.) or morphine (10 mg/kg i.p.). VP neurons recorded in vivo 3 days after the repeated cocaine treatment regimen demonstrated increased excitatory responding to microiontophoretic applications of morphine and glutamate. The maximal effect (E(max)) was increased without altering potency, suggesting a change in the functional efficacy of the respective receptor systems. This did not represent a potentiation in transmission in general, for the effects of GABA were diminished. The results provide the first evidence for cellular adaptation in the VP after a sensitizing drug treatment paradigm and reveal that cross-sensitization of drug-induced behaviors temporally correlates with changes in VP neuronal responding. These findings advance an emerging theme that alterations in the VP may contribute to the increased motivation for drug seeking that occurs in drug-withdrawn addicts.

Cheek cell membrane fluidity measured by fluorescence recovery after photobleaching and steady-state fluorescence anisotropy.

Membrane fluidity of human cheek cells was determined using fluorescence recovery after photobleaching (FRAP) and steady-state fluorescence anisotropy. The FRAP data showed that the lateral diffusion coefficient (D) and mobile fraction (%R) of lipid in the plasma membrane of control cells were 2.01 x 10(-9) cm2/sec and 54.25%, respectively. Trypsin treatment increased D and %R to 6.4 x 10(-9) cm2/sec and 72.15%. In contrast, the anisotropy (r) for control cells was 0.270 which remained unchanged by trypsin treatment. The results show that diffusion of lipids in the plane of the membrane is restricted by trypsin-sensitive barriers.

Regionalized lipid diffusion in the plasma membrane of mammalian spermatozoa.

The plasma membrane of mammalian spermatozoa shows pronounced lateral asymmetry with many glycoproteins restricted to specific domains. Some of these antigens are freely diffusing throughout the membrane whereas others appear static in position. It is not clear whether these concepts also apply to membrane lipids. In this investigation we have used fluorescence recovery after photobleaching (FRAP) techniques to spatially resolve lipid dynamics in various surface domains of 5 species of mammalian spermatozoa (bull, boar, ram, mouse, and guinea pig). Sperm plasma membranes were loaded with 5-(N-octadecanoyl)aminofluorescein (ODAF) reporter probe, and its diffusion was measured in various domains by FRAP analysis. Results showed that in live bull, boar, ram, and mouse spermatozoa, diffusion coefficients (D) were significantly higher over the acrosome and postacrosome than on the midpiece and principal piece of the tail. In dead or permeabilized cells, on the other hand, large immobile phases developed, particularly on the sperm tail, that severely reduced D values. ODAF diffusion was also sensitive to temperature and cross-linking of protein components within the membrane with paraformaldehyde. Guinea pig spermatozoa were different in almost all respects from those of the other species tested. It is concluded that lipid diffusion in the plasma membrane of live spermatozoa varies significantly between surface domains, because of either compositional heterogeneity, or differences in bilayer disposition, or the presence of intramembranous barriers that impede free exchange between domains. This study emphasizes the important role of membrane lipids in regulating polarized migration of sperm surface antigens during developmental processes such as maturation and capacitation.

Diffusion barriers in ram and boar sperm plasma membranes: directionality of lipid diffusion across the posterior ring.

The plasma membrane of mammalian spermatozoa, like that of other differentiated cells, is compartmentalized into discrete regions or domains that are biochemically and functionally distinct from one another. Physical structures within the membrane, such as the posterior ring at the juncture of the sperm head and tail, have long been thought to act as diffusion barriers to help segregate important molecules required for fertilization within specific domains and to regulate migration of molecules between domains. In this investigation, we used a quantitative photobleaching technique (video-FRAP) to assess the efficacy of the posterior ring as a barrier to exchange of lipids between the postacrosomal and midpiece plasma membranes. A lipid reporter probe (1,1'-diduodecyl-3,3,3', 3'-tetramethylindocarbocyanine; DiIC(12)) was incorporated into the plasma membrane of live ram and boar spermatozoa, and the directionality of its diffusion across the posterior ring was measured by line-profile analysis. Results showed that DiIC(12) was able to traverse the posterior ring from the direction of the postacrosomal plasma membrane and to diffuse onto the midpiece plasma membrane. These results suggest that the posterior ring is not an immutable barrier to lipid exchange in mature spermatozoa and that there are other mechanisms for maintaining in-plane lipid asymmetry, such as differential phase behavior and interaction with the submembranous cytoskeleton.