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Gluten proteins from wheat, rye, barley and, in rare cases, oats, are responsible for triggering hypersensitivity reactions such as celiac disease, non-celiac gluten sensitivity and wheat allergy. Well-defined reference materials (RM) are essential for clinical studies, diagnostics, elucidation of disease mechanisms and food analyses to ensure the safety of gluten-free foods. Various RM are currently used, but a thorough characterization of the gluten source, content and composition is often missing. However, this characterization is essential due to the complexity and heterogeneity of gluten to avoid ambiguous results caused by differences in the RM used. A comprehensive strategy to isolate gluten protein fractions and gluten protein types (GPT) from wheat, rye, barley and oat flours was developed to obtain well-defined RM for clinical assays and gluten-free compliance testing. All isolated GPT (ω5-gliadins, ω1,2-gliadins, α-gliadins, γ-gliadins and high- and low-molecular-weight glutenin subunits from wheat, ω-secalins, γ-75k-secalins, γ-40k-secalins and high-molecular-weight secalins from rye, C-hordeins, γ-hordeins, B-hordeins and D-hordeins from barley and avenins from oats) were fully characterized using analytical reversed-phase high-performance liquid chromatography (RP-HPLC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequencing, electrospray-ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS) and untargeted LC-MS/MS of chymotryptic hydrolyzates of the single GPT. Taken together, the analytical methods confirmed that all GPT were reproducibly isolated in high purity from the flours and were suitable to be used as RM, e.g., for calibration of LC-MS/MS methods or enzyme-linked immunosorbent assays (ELISAs).

Celiac disease (CD) is an inflammatory disorder of the upper small intestine caused by the ingestion of storage proteins (prolamins and glutelins) from wheat, barley, rye, and, in rare cases, oats. CD patients need to follow a gluten-free diet by consuming gluten-free products with gluten contents of less than 20 mg/kg. Currently, the recommended method for the quantitative determination of gluten is an enzyme-linked immunosorbent assay (ELISA) based on the R5 monoclonal antibody. Because the R5 ELISA mostly detects the prolamin fraction of gluten, a new independent method is required to detect prolamins as well as glutelins. This paper presents the development of a method to quantitate 16 wheat marker peptides derived from all wheat gluten protein types by liquid chromatography tandem mass spectrometry (LC-MS/MS) in the multiple reaction monitoring mode. The quantitation of each marker peptide in the chymotryptic digest of a defined amount of the respective reference wheat protein type resulted in peptide-specific yields. This enabled the conversion of peptide into protein type concentrations. Gluten contents were expressed as sum of all determined protein type concentrations. This new method was applied to quantitate gluten in wheat starches and compared to R5 ELISA and gel-permeation high-performance liquid chromatography with fluorescence detection (GP-HPLC-FLD), which resulted in a strong correlation between LC-MS/MS and the other two methods.

Vital gluten is often used in baking to supplement weak wheat flours and improve their baking quality. Even with the same recipe, variable final bread volumes are common, because the functionality differs between vital gluten samples also from the same manufacturer. To understand why, the protein composition of ten vital gluten samples was investigated as well as their performance in a microbaking test depending on the water content in the dough. The gluten content and composition as well the content of free thiols and disulfide bonds of the samples were similar and not related to the specific bread volumes obtained using two dough systems, one based on a baking mixture and one based on a weak wheat flour. Variations of water addition showed that an optimal specific volume of 1.74–2.38 mL/g (baking mixture) and 4.25–5.49 mL/g (weak wheat flour) was reached for each vital gluten sample depending on its specific water absorption capacity.

Hydrolyzed wheat proteins (HWPs) are widely used as functional ingredients in foods and cosmetics, because of their emulsifying and foaming properties. However, in individuals suffering from celiac disease or wheat allergy, HWPs may have a modified immunoreactivity compared to native gluten due to changes in molecular structures. Although a variety of HWPs are commercially available, there are no in-depth comparative studies that characterize the relative molecular mass (Mr) distribution, solubility, and hydrophilicity/hydrophobicity of HWPs compared to native gluten. Therefore, we aimed to fill this gap by studying the above characteristics of different commercial HWP and gluten samples. Up to 100% of the peptides/proteins in the HWP were soluble in aqueous solution, compared to about 3% in native gluten. Analysis of the Mr distribution indicated that HWPs contained high percentages of low-molecular-weight peptides/proteins and also deamidated glutamine residues. We also found considerable differences between the seven HWPs studied, so that each HWP needs to be studied in detail to help explain its potential immunoreactivity.

Background Oral food challenge using gluten and cofactors is the gold standard to diagnose wheat‐dependent exercise‐induced anaphylaxis (WDEIA), but this procedure puts patients at risk of an anaphylactic reaction. Specific IgE to ω5‐gliadins as major allergens and skin prick tests to wheat may yield negative results. Thus, we designed a proof‐of‐principle study to investigate the utility of the basophil activation test (BAT) for WDEIA diagnosis. Methods Different gluten protein types (GPT; α‐, γ‐, ω1,2‐ and ω5‐gliadins, high‐molecular‐weight glutenin subunits [HMW‐GS] and low‐molecular‐weight glutenin subunits [LMW‐GS]) and gluten were used in different concentrations to measure basophil activation in 12 challenge‐confirmed WDEIA patients and 10 control subjects. The results were compared to routine allergy diagnostics. Parameters analyzed include the percentage of CD63+ basophils, the ratio of %CD63+ basophils induced by GPT/gluten to %CD63+ basophils induced by anti‐FcεRI antibody, area under the dose‐response curve and test sensitivity and specificity. Results GPT and gluten induced strong basophil activation for %CD63+ basophils and for %CD63+/anti‐FcɛRI ratio in a dose‐dependent manner in patients, but not in controls (p < 0.001, respectively). BAT performance differed from acceptable (0.73 for LMW‐GS) to excellent (0.91 for ω5‐gliadins) depending on the specific GPT as evaluated by the area under the receiver operating characteristic curve. Patients showed individual sensitization profiles. After determination of the best cut‐off points, ω5‐gliadins and HMW‐GS showed the best discrimination between patients and controls with a sensitivity/specificity of 100/70 and 75/100, respectively. Conclusion This study shows the alternative role of BAT in better defining WDEIA and the causative wheat allergens. The best BAT parameters to distinguish WDEIA patients from controls were %CD63+ basophil values for ω5‐gliadins and HMW‐GS.

The prevalence of hypersensitivities towards wheat has increased in the last decades. Apart from celiac disease these include allergic and other inflammatory reactions summarized under the term non-celiac wheat sensitivity. One suspected trigger is the family of amylase/trypsin-inhibitors (ATIs), non-gluten proteins that are prominent wheat allergens and that activate the toll-like receptor 4 on intestinal immune cells to promote intestinal and extra-intestinal inflammation. We therefore quantified 13 ATIs in 60 German hexaploid winter wheat cultivars originating from 1891 to 2010 and harvested in three years by targeted liquid chromatography-tandem mass spectrometry combined with stable isotope dilution assay using specific marker peptides as internal standards. The total ATI content and that of the two major ATIs 0.19 and CM3 did not change from old cultivars (first registered from 1891 to 1950) to modern cultivars (1951–2010). There were also no significant changes in ATI distribution.

Background In wheat-dependent exercise-induced anaphylaxis (WDEIA), cofactors such as exercise, acetylsalicylic acid (ASA), alcohol or unfavorable climatic conditions are required to elicit a reaction to wheat products. The mechanism of action of these cofactors is unknown, but an increase of gliadin absorption has been speculated. Our objectives were to study gliadin absorption with and without cofactors and to correlate plasma gliadin levels with factors influencing protein absorption in healthy volunteers. Methods Twelve healthy probands (six males, six females; aged 20–56 years) ingested 32 g of gluten without any cofactor or in combination with cofactors aerobic and anaerobic exercise, ASA, alcohol and pantoprazole. Gliadin serum levels were measured up to 120 min afterwards and the intestinal barrier function protein zonulin in stool was collected before and after the procedure; both were measured by ELISA. Stool microbiota profile was obtained by 16S gene sequencing. Results Within 15 min after gluten intake, gliadin concentrations in blood serum increased from baseline in all subjects reaching highly variable peak levels after 15–90 min. Addition of cofactors did not lead to substantially higher gliadin levels, although variability of levels was higher with differences between individuals (p < 0.001) and increased levels at later time points. Zonulin levels in stool were associated neither with addition of cofactors nor with peak gliadin concentrations. There were no differences in gut microbiota between the different interventions, although the composition of microbiota (p < 0.001) and the redundancy discriminant analysis (p < 0.007) differed in probands with low versus high stool zonulin levels. Conclusion The adsorption of gliadin in the gut in healthy volunteers is less dependent on cofactors than has been hypothesized. Patients with WDEIA may have a predisposition needed for the additional effect of cofactors, e.g., hyperresponsive or damaged intestinal epithelium. Alternatively, other mechanisms, such as cofactor-induced blood flow redistribution, increased activity of tissue transglutaminase, or increases in plasma osmolality and acidosis inducing basophil and mast cell histamine release may play the major role in WDEIA.

Due to the ban of certain single-use plastics in the European Union, food contact materials (FCM) from biobased and/or biodegradable polymers are increasingly being used. Some FCM are made from wheat or rye and therefore contain gluten, which is a food allergen and known to cause celiac disease. Legislation currently does not require allergen labelling on FCM and there is only some first information that gluten from FCM can migrate into gluten-free foods. Our aim was to analyze the extent of gluten migration from six different FCM into a variety of liquid and solid foods to assess the risk of exposure for wheat allergy and celiac disease patients. We show that the extent of gluten migration depended on the properties of the material, the liquid or solid food it comes into contact with and contact time. There was no clear effect of temperature or pH value. Of the six FCM studied, wheat bran-based plates had the highest potential to release gluten with concentrations of up to 203.0 mg/L of gluten in deionized water after 30 min. To protect patients, it is important to raise awareness of the risk of gluten migration from such FCM and help patients identify and avoid gluten-containing FCM. Further, legislation needs to be adapted urgently to include mandatory labelling of allergens on these biodegradable FCM.

Wheat gluten is responsible for the unique baking properties of wheat flour, but it also causes wheat-related disorders in predisposed individuals. Different commercially available gluten materials are commonly used for a variety of assays, but a detailed characterization of their composition is missing in many cases. This is why we aimed to provide an in-depth analysis of three commonly used gliadin and gluten materials from two different batches using gel electrophoretic and chromatographic techniques. The gliadin material did not show the typical qualitative and quantitative protein composition and does not appear to be representative of wheat gliadin. The two gluten materials had the expected protein composition, but both showed large batch-to-batch variability regarding total protein content. Since these variations result in different biochemical, immunological, and functional behaviors, it is important to analyze at least the total protein content of each material and each batch.

The safety of gluten-free foods is essential for celiac disease (CD) patients to prevent serious complications. Enzyme-linked immunosorbent assays (ELISAs) are recommended for gluten analysis to monitor the compliance of gluten-free products to the Codex threshold of 20 mg gluten/kg. However, due to the specific features of each gluten ELISA test kit, the results often deviate systematically and largely depend on the characteristics of the antibody. This comprehensive study assessed the specificities and sensitivities of three monoclonal (R5, G12, and Skerritt) and two polyclonal antibodies to the alcohol-soluble prolamin and alcohol-insoluble glutelin fractions of gluten from wheat, rye, and barley, all of which harbor CD-active epitopes. Reversed-phase high-performance liquid chromatography served as independent reference method to quantify gluten protein concentrations and allow comparisons of different gluten fractions within one kit and between kits. Wheat prolamins were detected quite accurately by all antibodies, but high variability between antibody specificities and sensitivities was observed for rye and barley prolamins and rye glutelins, and the largest discrepancies were found for wheat and barley glutelins. The gluten content (sum of prolamins and glutelins) was either overestimated up to six times (rye) or underestimated up to seven times (barley). Overestimation of gluten contents may unnecessarily limit the availability of gluten-free products, but underestimation represents a serious health risk for CD patients. It is important to consider these differences between antibodies used in kits and consider what each kit is capable of measuring, especially with samples where the source of gluten is unknown.