Exposure Sources, Amounts and Time Course of Gluten Ingestion and Excretion in Patients With Coeliac Disease on a Gluten-free Diet

Jocelyn A. Silvester; Isabel Comino; Lisa N. Rigaux; Veronica Segura; Kathy H. Green; Angel Cebolla; Dayna Weiten; Remedios Dominguez; Daniel A. Leffler; Francisco Leon; Charles N. Bernstein; Lesley A. Graff; Ciaran P. Kelly; Carolina Sousa; Donald R. Duerksen

Disclosures

Aliment Pharmacol Ther. 2020;52(9):1469-1479. 

In This Article

Discussion

This study confirms that gluten ingestion occurs frequently despite efforts to follow a strictly gluten-free diet. Our findings likely represent a 'best case' scenario as participants were highly engaged having participated in a research study since diagnosis and had access to highly skilled dietitians with GFD expertise. Gluten exposure was often unsuspected and occurred at home as well as when eating out. Twelve of 18 persons with coeliac disease on an intentional GFD, based upon standardised self-report measures, were exposed to gluten within the 10 day study period. The estimated amount of gluten consumed ranged from 0.2 mg to more than 80 mg. This is orders of magnitude less than the 3–10 g typically utilised in gluten challenge studies, which may partially explain why most gluten exposures were silent (no symptoms reported) and unsuspected by participants. The clinical consequences of chronic intermittent milligram range gluten exposures observed in our study remain unknown. As previously reported, there was discordance between gluten exposures and villous atrophy in our cohort, which may be related to the timing of follow-up biopsy, duration of the sample collection period, and/or high sensitivity assay detecting milligram-range gluten ingestions.[17]

We observed considerable inter-individual variability in the interval between gluten consumption and GIP excretion in urine, a phenomenon previously reported in healthy individuals.[14,23] Some participants absorbed and excreted GIP relatively rapidly, with GIP detectable in urine collected within hours of ingestion. Others displayed a more protracted time course with GIP appearing in urine collected 24 hours after gluten consumption. Renal dysfunction was an exclusion criterion but we did not study gastric emptying or small intestinal transit. Participants were instructed to collect three urine samples per day (preferably first morning, during the day and at bedtime), so we likely did not capture all urine that contained GIP. This complicates evaluation of the relationship between estimated gluten consumption and detection of GIP in urine. However, among individuals with multiple gluten exposures, the interval between gluten consumption and collection of urine with detectable GIP was generally consistent intra-individually (ie for a given participant) and ranged from <4 to >24 hours.

There was also variation in the amount of gluten ingestion that was associated with detection of gluten in urine. The smallest estimated gluten exposure that was detected was 0.2 mg, which is two orders of magnitude less than what was determined to be the limit of detection for healthy individuals.[14] This participant reported that they may have been exposed to gluten transferred from toast prepared in a 4-slice toaster with a shared crumb tray, but dedicated gluten-free toasting slots. Situations in which gluten may be introduced through cross-contact highlight the challenges associated with estimating gluten content through sampling of food, as gluten may not be homogenously distributed and the sampled portion is not consumed.

The kinetics of gluten absorption and excretion may be affected by multiple factors in addition to the amount consumed. Intact dietary proteins, including ovalbumin,[24] lactoglobulin,[25] and gliadin,[23,26] may be detected in serum within minutes of ingestion and the concentration tends to peak within hours. Numerous studies have demonstrated transcellular passage of proteins across the small intestinal epithelium which is likely the predominant mechanism in healthy persons. In a case series of five patients with CD, Husby et al[25] demonstrated that absorption of ovalbumin and lactoglobulin was more rapid and more extensive in untreated patients with villous atrophy than in those who were following a GFD. Absorbed dietary proteins are not bound to immune complexes and serum protein concentration is not related to antigen-specific IgG, IgA or IgM.[24] The phenomenon of wheat dependent exercise-induced anaphylaxis (WDEIA) is instructive. Affected individuals are prone to anaphylaxis if they exercise after wheat consumption, but not with wheat consumption alone or exercise alone. Exercise is associated with increased serum gliadin levels,[27] and detectable serum gliadin is associated with positive response to wheat plus exercise challenge.[26] Non-steroidal anti-inflammatory drugs (NSAIDs) also increase antigen uptake[23] and may induce wheat-dependent exercise-induced anaphylaxis.

Detection of GIP in urine is dependent upon intestinal absorption as well as renal excretion. Circulating gluten is filtered by the kidneys which reabsorb most proteins. Urinary peptides are derived from proteolysis of non-reabsorbed proteins. Although poorly correlated, renal dysfunction is associated with a decrease in the urine peptide-to-protein ratio.[28] Urinary peptide excretion is increased in children with coeliac disease compared to age- and sex-matched controls[29] and decreases following treatment with a GFD.[30]

Many circumstances may contribute to gluten ingestion. Introduction of gluten to inherently gluten-free grains can occur during any stage from planting through harvest and processing.[31] Commercial seed may legally contain off-types (seeds other than the stated variety or strain). Accordingly, commercial 'naturally' gluten-free grain products often contain >20 ppm gluten in Canada (61/640 (9.5%))[32] and the United States (7/22 (32%)),[33] including grain products with a 'gluten-free' label.[34] Food products labelled as gluten-free have also been tested. In Europe, there was negligible gluten content in the 205 commercially available gluten-free foods tested,[35] whereas in a US study approximately 5% of the 158 gluten-free products tested above levels of 20 ppm.[36] Furthermore, these studies might be underestimating gluten content because they typically utilised the R5 antibody sandwich ELISA method which produced high variation of results in interlaboratory proficiency studies[37] and does not detect small hydrolysed gluten fragments or some of the most immunogenic gluten peptides.[38,39] In a systematic review of 24 cross-sectional studies, gluten contamination of both processed foods (13.2%) and raw ingredients (41.3%)[40] was common. Gluten ingestion may also occur as a result of household contamination, such as through use of shared cooking utensils.[41] Finally, patients may inadvertently ingest gluten due to incomplete information about ingredients or misinterpretation of food labels due to misconceptions regarding the gluten-free status of an ingredient. All these situations occurred during the study, and many participants suspected gluten ingestion based upon how food was prepared in a shared kitchen or unclear product label information. Our study was not designed to assess whether gluten exposure is associated with where a food is prepared. This question is difficult to answer definitively because food preparation is complicated and 'self-prepared' meals are themselves sourced using ingredients from outside the home. Several participants commented upon this in the margin of their food records (eg 'told it was gluten-free' in reference to products purchased at a farmer's market). We did not collect sufficiently granular data on the source of all components of meals, or indeed gluten testing data, to discern the extent to which they were derived from pure ingredients vs assembled from partially prepared foods.

Strengths of this study include analysis of food samples in addition to analysis of temporally relevant urine and stool using an assay for the immunodominant gluten peptide over a multi-day period. As well, participants were recruited from a larger longitudinal research study with comprehensive regular follow-up that included review of practices to avoid cross-contact as well as assessments of label-reading skills. These participants were recruited within weeks of initiating a GFD so were all at a similar point in their disease trajectory when they participated in the study.

While this may limit the generalisability of the findings, one might expect that the general population of patients with coeliac disease, many of whom do not receive regular disease-specific follow-up,[42,43] would be less successful at eliminating gluten from their diet. Previous trials of patients with coeliac disease asked to maintain a GFD[44] have shown a Hawthorne Effect, that is observation of behaviour changes behaviour. Our participants may have initially intensified their adherence to the GFD due to their study participation—two-thirds of positive urine samples were collected during the latter half of the observation period. Thus, it is possible that this study under-estimated typical 'real world' gluten exposures. Another potential limitation is that we did not test all foods and beverages consumed during the 10 day study period, which could under-estimate gluten ingestion, and makes it difficult to correlate gluten excretion during the last days of the study with gluten ingestion. Although participants were given clear instructions, we did not monitor the accuracy of sampling from complex meals composed of multiple foods. Prior to this study, there were limited data available regarding the gluten content of food directly consumed by patients with coeliac disease. Therefore, we made the pragmatic decision to maximise the likelihood of capturing gluten exposure by excluding presumably gluten-free unprocessed foods or medications, which are generally thought to be unlikely to contain clinically significant amounts of gluten.[45]

In conclusion, notable aspects of this study include our use of recently developed tests for gluten in food, urine and stool to measure directly gluten ingestion and excretion in individuals with coeliac disease who are endeavouring to follow a GFD. Even in a relatively brief observation period of just 10 days, two-thirds of participants were found to have ingested and/or excreted gluten. Our results suggest that most patients with coeliac disease in actuality can only attain a gluten reduced diet, and the recommended strict GFD may not be possible to maintain fully. While ongoing unrestricted gluten ingestion is known to be a risk for development of complications, the clinical consequences of intermittent low-level gluten exposures are unknown. Given that gluten exposure may be inevitable, it is necessary to understand better what degree of exposure is harmful and who may be more predisposed to persistent disease activity at the relatively low exposure levels observed in this study. Additionally, the potential role of self-monitoring technologies that provide real-time feedback on gluten content of food and on gluten excretion is largely unexplored. Ultimately, less challenging and more feasible treatments are needed for coeliac disease. A viable pharmacologic treatment for coeliac disease must account for frequent sporadic gluten exposures on the order of milligrams which may be silent and unsuspected.

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