Abstract
The safety of oats for people with celiac disease remains unresolved. While oats have attractive nutritional properties that can improve the quality and palatability of the restrictive, low fiber gluten-free diet, rigorous feeding studies to address their safety in celiac disease are needed. Assessing the oat prolamin proteins (avenins) in isolation and controlling for gluten contamination and other oat components such as fiber that can cause non-specific effects and symptoms is crucial. Further, the avenin should contain all reported immunogenic T cell epitopes, and be deliverable at a dose that enables biological responses to be correlated with clinical effects. To date, isolation of a purified food-grade avenin in sufficient quantities for feeding studies has not been feasible. Here, we report a new gluten isolation technique that enabled 2 kg of avenin to be extracted from 400 kg of wheat-free oats under rigorous gluten-free and food grade conditions. The extract consisted of 85% protein of which 96% of the protein was avenin. The concentration of starch (1.8% dry weight), β-glucan (0.2% dry weight), and free sugars (1.8% dry weight) were all low in the final avenin preparation. Other sugars including oligosaccharides, small fructans, and other complex sugars were also low at 2.8% dry weight. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the proteins in these preparations showed they consisted only of oat proteins and were uncontaminated by gluten containing cereals including wheat, barley or rye. Proteomic analysis of the avenin enriched samples detected more avenin subtypes and fewer other proteins compared to samples obtained using other extraction procedures. The identified proteins represented five main groups, four containing known immune-stimulatory avenin peptides. All five groups were identified in the 50% (v/v) ethanol extract however the group harboring the epitope DQ2.5-ave-1b was less represented. The avenin-enriched protein fractions were quantitatively collected by reversed phase HPLC and analyzed by MALDI-TOF mass spectrometry. Three reverse phase HPLC peaks, representing ~40% of the protein content, were enriched in proteins containing DQ2.5-ave-1a epitope. The resultant high quality avenin will facilitate controlled and definitive feeding studies to establish the safety of oat consumption by people with celiac disease.
Highlights
Establishing a safe gluten-free diet (GFD) for people with celiac disease (CD) is important for the health of the 1.4% of affected sufferers globally [1]
The cloudy precipitate produced by adding water was extremely difficult to spin down and resisted precipitation at 5,000 g, beyond the capacity of the centrifuge used for the large scale avenin preparation
The chill-induced precipitation of avenin commenced below 15◦C and could be reversed by warming, resulting in a clear solution that could be reproduced at least 10 times
Summary
Establishing a safe gluten-free diet (GFD) for people with celiac disease (CD) is important for the health of the 1.4% of affected sufferers globally [1]. Since the 1950s, after gluten was identified as the causative antigenic trigger, its sole treatment has been strict and lifelong gluten exclusion by removing wheat, barley, and rye In this clinical context, the term gluten encompasses all of the pathogenic prolamin proteins from wheat (gliadin and glutenin), barley (hordein) and rye (secalin) that are immunotoxic in CD. Key avenin peptides that stimulate the pathogenic gluten-specific T cells in CD patients in vivo have been defined [12, 13] These peptides contain the immunodominant T cell epitopes DQ2.5-ave-1a (PYPEQEEPF), DQ2.5-ave-1b (PYPEQEQPF), DQ2.5-ave-1c (PYPEQEQPI), and DQ2.5-ave-2 (PYPEQQPF) with close sequence homology to barley T cell epitopes immunotoxic in CD such as DQ2.5-hor-3 (PIPEQPQPY) [14]. The collective uncertainty of these findings regarding the true clinical safety of oats in CD has translated into different feeding recommendations; while Australia and New Zealand mandate the exclusion of oats from the GFD, most countries do not
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