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


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

In This Article


As described previously,[17] participants were recruited from the Manitoba Celiac Disease Inception Cohort study. This prospective longitudinal study enrolled adults (16 years or older) with biopsy-confirmed coeliac disease. Diagnosis of coeliac disease was based upon villous atrophy and intraepithelial lymphocytosis (Marsh 3 lesions) on intestinal biopsy and elevated serum tissue transglutaminase (TTG IgA) and/or endomysial (EMA IgA) antibody levels.[17] In Manitoba, a central laboratory performs all serum TTG IgA testing which facilitated a population-based approach to reduce recruitment bias. Specifically, a list of physicians with patients with elevated serum TTG was generated weekly throughout the enrolment period. These physicians were contacted to inform them of the study, and provided with recruitment materials for their patients. Secondary recruitment methods included referral by the endoscopist, and advertisements at retailers of gluten-free products and in the newsletter of the Manitoba Chapter of the Canadian Celiac Association. Participants were recruited within 6 weeks of GFD initiation, with additional study visits at 6, 12, and 24 months. Per usual practice, participants were referred at diagnosis to a Registered Dietitian with GFD expertise and given information about the Canadian Celiac Association patient support group.

For the DOGGIE BAG sub-study, coeliac disease participants on a strict GFD completed an additional diary, food record, and food, urine and stool sample collection during the 10 days immediately prior to per-protocol follow-up biopsy 24 months after initiation of a GFD (Figure 1). Healthy persons without coeliac disease or a dietary gluten restriction were studied as positive controls for gluten ingestion. Exclusion criteria (applicable to both groups): (a) inability to complete diary and/or collect samples; (b) baseline stool frequency <3/week; or (c) concurrent digestive or malabsorptive disorder other than disaccharide intolerance (eg viral gastroenteritis, small intestinal bacterial overgrowth, inflammatory bowel disease). Participants on a 'strict GFD' were trying to follow a strict gluten-free diet and did not report intentional gluten exposure.

Figure 1.

Study design. Participants completed a detailed food diary and collected three urine samples daily for three days. During the first 7 d, a 'doggie bag' containing 25% of all food and some beverages was saved. Stool (minimum 4 samples on different days) was collected starting at day 4 to allow for washout of any gluten consumed prior to the start of the study period

All DOGGIE BAG study participants were instructed to follow their usual dietary patterns including any packaged/processed and restaurant or take-out meals. To reduce participant burden and account for intestinal transit, food samples were collected for the first 7 days and stools were collected on days 4–10. Participants were instructed to collect their first morning urine (50 mL) plus two additional samples daily (preferably midday and at bedtime) throughout the study period. The participants provided with specific instructions to prevent contamination with gluten during sample collection. Sterile collection containers were provided as well as a −20°C freezer (Whynter LLC) in which to store all food and body waste samples. GIP are stable for >72 hours at room temperature in urine and stool; nevertheless, a thermometer was also provided to monitor freezer temperature.

The study protocol was reviewed by the University of Manitoba Health Research Ethics Board.

Participant Diary and Standardised Measures of Coeliac Disease Activity and Gluten-free Diet Adherence

Participants logged sample collection times and kept a food record. Each day, participants reported whether they thought that they may have ingested gluten, and described any symptoms of suspected gluten exposure. Responses were independent, so participants could suspect gluten exposure without symptoms or vice versa. If gluten exposure was suspected, then participants indicated which food(s) they suspected contained gluten. At the end of the study, participants completed standardised coeliac disease-specific self-report measures, including: Coeliac Symptom Index (CSI),[18] a 16-item measure with possible scores ranging from 16 to 80 on which scores of 30 are less are associated with better quality of life and GFD adherence; Coeliac Diet Assessment Tool (CDAT),[19] a seven-item self-report measure with questions about symptoms and personal traits, with possible scores ranging from 7 to 35 on which scores of 12 or less are associated with adequate adherence; and Gluten-Free Eating Assessment Tool Short (GF-EATs), a single-item self-reported adherence scale with five categorical responses ranging from frequent gluten (more than once per week) to no gluten.[17] In addition, GFD adherence was assessed by a Registered Dietitian with expertise in GFD based using the Dietitian Integrated Evaluation Tool for Gluten-Free Diets (GFD) which incorporates assessment of: 3-day food records; food frequency questionnaire including gluten-free and gluten-containing items; assessment of risk behaviours and precautions to avoid gluten at home, work/school, restaurants and other people's homes; and label reading skills.[20] This was reported using a 10-point ordinal descriptive scale on which higher scores correspond to greater adherence with a score of 10 reserved for individuals living in gluten-free homes who eat out only a few times per year. Scores for the Manitoba Coeliac Disease Inception Cohort at 24 months range from 1 to 10 with a median of 8 (IQR 6–8).

Food Collection

Thawed food samples were batch processed for each participant and pooled based upon time consumed (04.01–10.00, 10.10–16.00, 16.00–04.00). Each pooled sample was homogenised using a 1000W triple blade stainless steel blender (Breville) with deionised water added when required to facilitate mixing. The blender was thoroughly cleaned and rinsed between each sample.

Quantification of Gluten in Food Samples

Gluten content of food (ppm) was determined using gliadin as a standard according to the recommendations of the Working Group on Prolamine Analysis and Toxicity[21] using an A1-G12 antibody sandwich enzyme-linked immunosorbent assay[9] (ELISA; GlutenTox ELISA Sandwich A1-G12, Hygiena Diagnostica) that is specific and sensitive for the most gluten immunogenic peptides in food.[22] Gluten concentration in food samples was determined according to the following formula to account for sample dilution:

The amount of gluten consumed was estimated using the following formula to convert from ppm to mg and account for sampling of ¼ of food as plated (the participant ate the other ¾):

Following sample dilution, the working limit of quantification of this method is 1.6 ppm gluten.

Quantification of GIP in Stool Samples

Stool samples were thawed and the entire collection was manually homogenised. Stool GIP concentration was determined by sandwich ELISA (iVYLISA GIP Stool kit, iVYDAL In Vitro Diagnostics®, Biomedal SL) according to the manufacturer's protocol. Each sample was run in duplicate with ≥4 different aliquots tested on different days using α-gliadin 33-mer as GIP reference material. The accuracy of this method in detecting gluten in stool has been reported previously: analytical sensitivity having limits of detection and quantification of 60 and 160 ng GIP per gram stool respectively; and diagnostic sensitivity and specificity of 98.5% and 100% respectively.[12] GIP are stable in stool stored between −20°C and −80°C for more than 12 months, and through multiple freeze-thaw cycles.

Quantification of GIP in Urine Samples

Urine samples were processed for testing by the iVYCHECK GIP Urine (iVYDAL In Vitro Diagnostics®, Biomedal SL) according to the manufacturer's recommendations. This immunochromatographic test uses G12/A1 antibodies and provides a visual result as a red line in the test zone.[14] Samples with positive qualitative results were quantified using the iVYCHECK Reader. The validity of this method in detecting GFD transgressions was determined by the analytical sensitivity using α-gliadin 33mer peptide (limits of detection and quantification 2.2 and 6.3 ng per mL urine respectively). Each sample was run in duplicate and at least two different aliquots of each sample were tested on different days.

Tissue Transglutaminase Antibody Levels

Serum TTG IgA levels were determined by the Immunology Lab at St Boniface Hospital, Winnipeg, Canada. In May 2015, the assay was changed from ImmuLisa (Immco Diagnostics Inc) to Bioplex 2200 (Bio-Rad Laboratories [Canada] Inc). All results are reported as multiples of the upper limit of normal (ULN) for the assay used (ImmuLisa 20, Bioplex 15).