The effect of expectancy versus actual gluten intake on gastrointestinal and extra-intestinal symptoms in non-coeliac gluten sensitivity: a randomised, double-blind, placebo-controlled, international, multicentre study.

BACKGROUND: Many individuals without coeliac disease or wheat allergy reduce their gluten intake because they believe that gluten causes their gastrointestinal symptoms. Symptoms could be affected by negative expectancy. Therefore, we aimed to investigate the effects of expectancy versus actual gluten intake on symptoms in people with non-coeliac gluten sensitivity (NCGS). METHODS: This randomised, double-blind, placebo-controlled, international, multicentre study was done at the University of Leeds (Leeds, UK), Maastricht University (Maastricht, the Netherlands), and Wageningen University and Research (Wageningen, the Netherlands). People aged 18-70 years with self-reported NCGS (ie, gastrointestinal symptoms within 8 h of gluten consumption) without coeliac disease and wheat allergy were recruited. Participants had to follow a gluten-free or gluten-restricted diet for at least 1 week before (and throughout) study participation and had to be asymptomatic or mildly symptomatic (overall gastrointestinal symptom score ≤30 mm on the Visual Analogue Scale [VAS]) while on the diet. Participants were randomly assigned (1:1:1:1; blocks of eight; stratified by site and gender) to one of four groups based on the expectation to consume gluten-containing (E+) or gluten-free (E-) oat bread for breakfast and lunch (two slices each) and actual intake of gluten-containing (G+) or gluten-free (G-) oat bread. Participants, investigators, and those assessing outcomes were masked to the actual gluten assignment, and participants were also masked to the expectancy part of the study. The primary outcome was overall gastrointestinal symptom score on the VAS, which was measured at and corrected for baseline (before breakfast) and hourly for 8 h, with lunch served after 4 h, and analysed per-protocol. Safety analysis included all participants incorporated in the per-protocol analysis. The study is registered at ClinicalTrials.gov, NCT05779358, and has ended. FINDINGS: Between Oct 19, 2018, and Feb 14, 2022, 165 people were screened and 84 were randomly assigned to E+G+ (n=21), E+G- (n=21), E-G+ (n=20), or E-G- (n=22). One person in the E+G+ group was excluded due to not following test day instructions, leaving 83 participants in the per-protocol analysis. Median age was 27·0 years (IQR 21·0-45·0), 71 (86%) of 83 people were women, and 12 (14%) were men. Mean overall gastrointestinal symptom score was significantly higher for E+G+ (16·6 mm [95% CI 13·1 to 20·0]) than for E-G+ (6·9 mm [3·5 to 10·4]; difference 9·6 mm [95% CI 3·0 to 16·2], p=0·0010) and E-G- (7·4 mm [4·2 to 10·7]; difference 9·1 mm [2·7 to 15·6], p=0·0016), but not for E+G- (11·7 mm [8·3 to 15·1]; difference 4·9 mm [-1·7 to 11·5], p=0·28). There was no difference between E+G- and E-G+ (difference 4·7 mm [-1·8 to 11·3], p=0·33), E+G- and E-G- (difference 4·2 mm [-2·2 to 10·7], p=0·47), and E-G+ and E-G- (difference -0·5 mm [-7·0 to 5·9], p=1·0). Adverse events were reported by two participants in the E+G- group (itching jaw [n=1]; feeling lightheaded and stomach rumbling [n=1]) and one participant in the E-G+ group (vomiting). INTERPRETATION: The combination of expectancy and actual gluten intake had the largest effect on gastrointestinal symptoms, reflecting a nocebo effect, although an additional effect of gluten cannot be ruled out. Our results necessitate further research into the possible involvement of the gut-brain interaction in NCGS. FUNDING: Government of the Netherlands Topsector Agri & Food Top Consortium for Knowledge and Innovation, AB Mauri Global Bakery Ingredients, Baking Industry Research Trust, Borgesius-Albert Heijn, CSM Innovation Centre, the International Maize and Wheat Improvement Center (CIMMYT), DSM Food Specialties, Fazer, Healthgrain Forum, the International Association for Cereal Science and Technology, the International Wheat Gluten Association, Lantmännen, Mondelez International, Nederlands Bakkerij Centrum, Nutrition & Santé, Puratos, Rademaker, Sonneveld Group, and Zeelandia HJ Doeleman.

Impact of food processing on the allergenic properties of amylase trypsin inhibitors from wheat.

Amylase trypsin inhibitors (ATIs) play an important role in wheat allergies and potentially in non-coeliac wheat sensitivity. Food processing could be important to mitigate the pathogenic properties of ATIs, e.g., by denaturation, glycation, enzymatic hydrolysis, cross-linking, and oxidation and reduction. These modifications also impact the solubility and extractability. The complex solubility behaviour of ATI isoforms (water and salt soluble, but also chloroform-methanol soluble, solubility depending on the redox state) becomes even more complex upon processing due to denaturation and (bio)chemical modifications. This significantly hinders the feasibility of quantitative extraction. Moreover, changes in biofunctionality may occur during the process of extraction, and the changes in ATI due to food processing will be more difficult to assess. Heat treatment decreases the extractability of ATIs with water, NaCl, and other buffer extracts, and binding of IgE from wheat-allergic persons to ATIs as observed with Western blotting is decreased or absent. IgE binding is reduced with the total extract in chaotropic and reducing agents. However, it can be increased when the proteins are hydrolyzed by proteases. Fermentation involving certain species of Fructolactobacilli (FLB), followed by baking, decreases the amount of ATIs and IgE binding to ATIs. In yeast-fermented bread, the amount of ATIs decreased in a similar manner, but IgE binding was more prominent, indicating that there was a modification of ATIs that affected the epitope recognition. When isolated ATIs are ingested with high ATI degrading FLB, the immune response in mice is less elevated in vivo, when compared with ATI without high ATI degrading FLB. The pathogenic effects on the skin of dogs and one wheat-allergic child are also decreased when soluble proteins or isolated ATIs are reduced with the thioredoxin/thioredoxin reductase NADPH system. Glycation on the other hand has been shown to potentiate the allergenic properties of ATIs as evidenced by the large increase in IgE binding. The impact of food processing on the pathogenic properties of ATIs is hardly studied in vivo in humans. There seem to be opportunities to mitigate the pathogenic properties in vitro, but potentiation of pathogenic properties is also frequently observed. This requires a deeper understanding on the impact of food processing on the pathogenicity of ATIs.

The Future of Bread in View of its Contribution to Nutrient Intake as a Starchy Staple Food.

The growing, ageing, more urbanized and more sedentary global population urges for increased, more nutritious and more secure food supply. To combat undernutrition, adequate nutrient intake is required. Staple foods, especially starch based ones, like (pseudo)cereals, roots and tubers, are by definition contributing for a very large extent to our nutrient intake. It is important therefore that the contribution of ready to consume staple foods to the recommended intakes of nutrients are compared. This can be done in a transparent and simple way by calculating the nutrient contribution of ready to eat staple foods to the recommended daily allowances (RDA) or daily reference intakes (DRI). By using online nutrition tables from the USA and Europe, and DRI by USA and RDA by European food authorities, the potential nutrient contributions of staple foods were calculated. It can be concluded that consumption of ready to eat staple foods from the bread category and lentils overall contribute most to the recommended nutrient intakes. In view of its convenience, bread, especially whole grain bread is the staple food of choice to combat the future food supply challenges ahead.