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Strenuous exercise induces different stress-related physiological changes, potentially including changes in intestinal barrier function. In the Protege Study (ISRCTN14236739; www.isrctn.com) we determined the test-retest repeatability in responses to exercise in well-trained individuals. Eleven well-trained males (27 ± 4 years old) completed an exercise protocol that consisted of intensive cycling intervals, followed by an overnight fast and an additional 90 min cycling phase at 50% Wmax the next morning. The day before (rest), and immediately after the exercise protocol (exercise) a lactulose/rhamnose solution was ingested. Markers of energy metabolism, lactulose/rhamnose ratio, several cytokines and potential stress-related markers were measured at rest and during exercise. In addition, untargeted urine metabolite profiles were obtained. The complete procedure (Test) was repeated one week later (Retest) to assess repeatability. Metabolic effect parameters with regard to energy metabolism and urine metabolomics were similar for both the Test and Retest period, underlining comparable exercise load. Following exercise, intestinal permeability (one hour plasma lactulose/rhamnose ratio), serum interleukin-6, interleukin-10, fibroblast growth factor-21, and muscle creatine kinase levels were only significantly increased compared to rest during the first test and not when the test was repeated. Responses to strenuous exercise in well-trained young men, as indicated by intestinal markers and myokines, show adaptation in Test-Retest outcome. This might be due to a carry-over effect of the defense mechanisms triggered during the Test. This finding has implications for the design of studies aimed at evaluating physiological responses to exercise. This article is protected by copyright. All rights reserved

4th International Conference on Food Digestion -- MAR, 2015 -- Naples, ITALY, WOS: 000384789000006, Within the active field of in vitro digestion in food research, the COST Action INFOGEST aimed to harmonize in vitro protocols simulating human digestion on the basis of physiologically inferred conditions. A harmonized static in vitro digestion (IVD) method was recently published as a primary output from this network. To validate this protocol, inter-laboratory trials were conducted within the INFOGEST network. A first study was performed using skim milk powder (SMP) as a model food and served to compare the different in-house digestion protocols used among the INFOGEST members. In a second inter-laboratory study applying the harmonized protocol, the degree of consistency in protein hydrolysis was investigated. Analysis of the hydrolyzed proteins, after the gastric and intestinal phases, showed that caseins were mainly hydrolyzed during the gastric phase, whereas beta-lactoglobulin was, as previously shown, resistant to pepsin. Moreover, generation of free amino acids occurred mainly during the intestinal phase. The study also showed that a few critical steps were responsible for the remaining inter-laboratory variability. The largest deviations arose from the determination of pepsin activity. Therefore, this step was further clarified, harmonized, and implemented in a third inter-laboratory study. The present work gives an overview of all three inter-laboratory studies, showing that the IVD INFOGEST method has led to an increased consistency that enables a better comparability of in vitro digestion studies in the future. (C) 2015 The Authors. Published by Elsevier Ltd.

Controlled studies on the effect of exercise on intestinal uptake of protein are scarce and underlying mechanisms largely unclear. We studied the uptake of the major allergen Ara h 6 following peanut consumption in an exercise model and compared this with changes in markers of intestinal permeability and integrity. Ten overnight-fasted healthy non-allergic men (n = 4) and women (n = 6) (23 +/- 4 years) ingested 100 g of peanuts together with a lactulose/rhamnose (L/R) solution, followed by rest or by 60 min cycling at 70% of their maximal workload. Significantly higher, though variable, levels of Ara h 6 in serum were found during exercise compared to rest (Peak p = 0.03; area under the curve p = 0.006), with individual fold changes ranging from no increase to an increase of over 150-fold in the uptake of Ara h 6. Similarly, uptake of lactulose (2-18 fold change, p = 0.0009) and L/R ratios (0.4-7.9 fold change, p = 0.04) were significantly increased which indicates an increase in intestinal permeability. Intestinal permeability and uptake of Ara h 6 were strongly correlated (r = 0.77, p

The gut plays a pivotal role in human wellbeing by forming the body’s largest barrier between the external and internal environment. It has a major function in the digestion and absorption of nutrients and at the same time protects the body from the uptake of potentially harmful compounds. An increase in intestinal permeability could affect the uptake of larger fragments of food components such as partly undigested dietary proteins. Endurance exercise is a widely experienced physiological challenge known to increase intestinal permeability, but the subsequent effects on protein uptake are less studied. Furthermore, several underlying mechanisms for exercise-induced permeability have been hypothesized, but have not been substantiated. The aim of this thesis was to examine the effect of exercise intestinal permeability to dietary protein and peptides. Several in vivo proof-of-concept studies have been performed to study the effect of exercise on intestinal permeability towards small inert sugars versus small (casein-derived betacasomorphin-7) and large (peanut-derived allergen Ara h 6) dietary peptides. An in vitro model was developed to study one of the possible underlying mechanisms for an increase in intestinal permeability. Chapter 1 provides background on intestinal integrity and permeability and their role in digestion and absorption of dietary protein. Furthermore, the in vivo and in vitro models for intestinal permeability as applied in the next chapters are introduced. Additionally, the research aim and the outline of this thesis are presented. Chapters 2, 3, 4, and 5 describe in vivo findings on intestinal permeability. Chapters 2 and 3 show findings of the Protege study; this study consisted of 12 well-trained healthy young men who consumed a casein protein solution in rest or directly after completing a strenuous dual exercise protocol. Chapter 2 shows the effect of the strenuous exercise on urinary peptide excretion and markers of intestinal function and recovery, and inflammation. The exercise protocol resulted in increased urinary excretion of the casein-derived peptide betacasomorphin-7, even though intestinal permeability as measured with inert sugars was less pronounced. The exercise protocol also resulted in major changes in post-prandial amino acid profiles in plasma. It was concluded that strenuous exercise could have an effect on the amount of food-derived (bioactive) peptides crossing the epithelial barrier. In chapter 3 the test-retest repeatability of the outcomes of this strenuous exercise intervention is studied, since the complete intervention was performed twice in two separate weeks. It was shown that the metabolic effect parameters, such as glucose, lactate, and energy expenditure showed low test-retest variation, while stress response parameters to the exercise, such as creatine kinase, fibroblast growth factor 21 and intestinal permeability as measured with a lactulose/rhamnose challenge test showed high test-retest variation. It was concluded that even in well-trained young men an adapted response can be seen in exercise-induced stress after only a single repetition of the exercise intervention. This finding has implications for the design of human studies aiming at evaluating physiological responses to exercise. In chapters 4 and 5, we focused on peanut protein instead of casein and compared to the Protege study less trained individuals were included. These individuals reflect the general population level of training. Chapter 4 describes the PEANUTS Pilot study, which was performed to develop an ELISA method to detect the major peanut allergen Ara h 6 in serum after peanut consumption. This protein could not be detected in four out of ten tested individuals. It was shown with spiking experiments that high levels of Ara h 6-specific immunoglobulin G in the blood hampered the detection of this allergen. This may be a broader phenomenon in studies on the uptake of food allergens in the circulation, and may explain why variable levels of food allergen in serum have been reported in literature. In chapter 5 the developed ELISA detection method from chapter 4 is used in the PEANUTS Study. Potential participants were first screened for the ability to detect Ara h 6 in their serum by a spiking experiment. The PEANUTS study consisted of 10 untrained healthy young men and women who consumed a lactulose/rhamnose test solution and 100 grams of mildly-roasted unsalted peanuts. The first week consumption of the peanuts was followed by rest, the second week each of the participants had to cycle for 60 minutes at 70% of their maximal output directly after the consumption of the sugar solution and the peanuts. The endurance exercise significantly increased intestinal permeability (lactulose/rhamnose ratio) as well as the uptake of Ara h 6. The lactulose/rhamnose ratio and the levels of Ara h6 were strongly correlated. From this it was hypothesized that endurance exercise after food consumption leads to increased paracellular intestinal uptake of food proteins. However, increased permeability due to increased transcytosis or decreased cell integrity could also add to the observed increase in the protein uptake. Chapter 6 shows findings on the newly developed in vitro model for intestinal permeability. This chapter describes the development of an in vitro cell model mimicking to a higher extent the in vivo situation with regard to metabolic phenotype. The Caco-2 monolayers grown in galactose medium instead of glucose medium showed a more oxidative phenotype dependent on mitochondrial ATP production. Obstruction of this mitochondrial energy production resulted in decreased cellular ATP levels coinciding with increased monolayer permeability. Gene expression analysis of tight junction proteins was inconclusive, but pointed towards a defence mechanism of the Caco-2 cells during energy stress. It was concluded that mitochondrial functioning may be essential for maintaining a gut barrier function with high integrity. In chapter 7 the main findings of this thesis are discussed. Furthermore, several methodological considerations are made for future design of in vivo studies on intestinal permeability including an exercise challenge and in vitro studies on intestinal permeability with Caco-2 cells. Next, future implications of my research is placed in the context of the broader aim of the IPOP Customized Nutrition research program with regard to the health effects of novel proteins. Lastly, some general conclusions of my thesis research are drawn.

Some studies have suggested that allergens may appear in the circulation after ingestion of allergenic food sources. The reported levels of allergen in serum, however, are low, and conclusions between studies differ. Here, we investigated factors that determine the detection of allergens in serum after consumption of peanuts. Ten healthy volunteers ingested 100g of light-roasted peanuts. Serum samples were taken at regular intervals for six hours. A double monoclonal sandwich ELISA was used to analyse the presence and quantity of the major peanut allergen Ara h 6 in serum. In 4 out of 10 subjects, no Ara h 6 could be detected. Purified Ara h 6 that was digested in vitro was still reactive in the ELISA, rejecting the possibility that digestion leads to small peptides that could not be detected. Spiking of purified Ara h 6 in baseline serum showed that the pre-ingestion serum of these four subjects partially prevented Ara h 6 to react in the ELISA, with a reduction of reactivity of up to 3 orders of magnitude or more. Pre-ingestion serum of the other six subjects did not show such an effect. The reduction of reactivity of Ara h 6 coincided with high titres of IgG and IgG4, and removal of IgG from pre-ingestion serum abolished this effect completely, indicating that IgG and IgG4 inhibited the reactivity of Ara h6 in the ELISA. We conclude that some individuals have IgG and IgG4 against food allergens in their blood, which interferes with detection of such food allergens in serum. Because this effect does not occur for each individual, the possibility of such interference should be taken into consideration when interpreting immunochemical studies on the absorption of food allergens in serum.

What is the central question of this study? Exercise is known to induce stress-related physiological responses, such as changes in intestinal barrier function. Our aim was to determine the test-retest repeatability of these responses in well-trained individuals. What is the main finding and its importance? Responses to strenuous exercise, as indicated by stress-related markers such as intestinal integrity markers and myokines, showed high test-retest variation. Even in well-trained young men an adapted response is seen after a single repetition after 1 week. This finding has implications for the design of studies aimed at evaluating physiological responses to exercise. Strenuous exercise induces different stress-related physiological changes, potentially including changes in intestinal barrier function. In the Protégé Study (ISRCTN14236739; www.isrctn.com), we determined the test-retest repeatability in responses to exercise in well-trained individuals. Eleven well-trained men (27 ± 4 years old) completed an exercise protocol that consisted of intensive cycling intervals, followed by an overnight fast and an additional 90 min cycling phase at 50% of maximal workload the next morning. The day before (rest), and immediately after the exercise protocol (exercise) a lactulose and rhamnose solution was ingested. Markers of energy metabolism, lactulose-to-rhamnose ratio, several cytokines and potential stress-related markers were measured at rest and during exercise. In addition, untargeted urine metabolite profiles were obtained. The complete procedure (Test) was repeated 1 week later (Retest) to assess repeatability. Metabolic effect parameters with regard to energy metabolism and urine metabolomics were similar for both the Test and Retest period, underlining comparable exercise load. Following exercise, intestinal permeability (1 h plasma lactulose-to-rhamnose ratio) and the serum interleukin-6, interleukin-10, fibroblast growth factor-21 and muscle creatine kinase concentrations were significantly increased compared with rest only during the first test and not when the test was repeated. Responses to strenuous exercise in well-trained young men, as indicated by intestinal markers and myokines, show adaptation in Test-Retest outcome. This might be attributable to a carry-over effect of the defense mechanisms triggered during the Test. This finding has implications for the design of studies aimed at evaluating physiological responses to exercise.