Detection of gluten immunogenic peptides in the urine of patients with coeliac disease reveals transgressions in the gluten-free diet and incomplete mucosal healing

Abstract

Objective: Gluten-free diet (GFD) is the only management for coeliac disease (CD). Available methods to assess GFD compliance are insufficiently sensitive to detect occasional dietary transgressions that may cause gut mucosal damage. We aimed to develop a method to determine gluten intake and monitor GFD compliance in patients with CD and to evaluate its correlation with mucosal damage.

Design: Urine samples of 76 healthy subjects and 58 patients with CD subjected to different gluten dietary conditions were collected. A lateral flow test (LFT) with the highly sensitive and specific G12 monoclonal antibody for the most dominant gluten immunogenic peptides (GIP) and a LFT reader were used to quantify GIP in solid-phase extracted urines.

Results: GIP were detectable in concentrated urines from healthy individuals previously subjected to GFD as early as 4-6 h after single gluten intake, and remained detectable for 1-2 days. The urine assay revealed infringement of the GFD in about 50% of the patients. Analysis of duodenal biopsies revealed that most of patients with CD (89%) with no villous atrophy had no detectable GIP in urine, while all patients with quantifiable GIP in urine showed incomplete intestinal mucosa recovery.

Conclusion: GIP are detected in urine after gluten consumption, enabling a new and non-invasive method to monitor GFD compliance and transgressions. The method was sensitive, specific and simple enough to be convenient for clinical monitoring of patients with CD as well as for basic and clinical research applications including drug development.

Trial registration number: NCT02344758.

Keywords: CELIAC DISEASE; GLUTEN FREE DIET.

Figure 1

Determination of the time to elimination and to appearance of GIP in urines of healthy individuals. Urine samples from healthy individuals, regularly consuming gluten, who were subject to a GFD were collected until reactive peptides became undetectable. Three to six different urine samples per day were collected for 4 days. (A) One representative example of the gluten excretion kinetics from the trial with the representative immunochromatographic strip example of the trial was performed with the samples collected during the study period of one subject. Blue stripes represent an internal positive control that indicates that the stick worked properly; pink stripes indicate the presence of gluten. (B) Kinetics of gluten-derived peptides excreted from four healthy volunteers. GIP, gluten immunogenic peptides; GCD, gluten-containing diet; GFD, gluten-free diet; QL, quantification limit; ND, not detectable.

Figure 2

In vivo monitoring of urinary excretion of gluten peptides in healthy individuals on a GFD with consumption of controlled gluten challenges. Two doses of gluten were administered (25 and 50 mg) to four subjects. Four independent experiments with samples were run in triplicate. GFD, gluten-free diet; GIP, gluten immunogenic peptides; QL, quantification limit; LDT, limit of technique detection; ND, not detectable.

Figure 3

Detection and quantification of GIP in urine samples of healthy and individuals with CD. Patients with CD (n=58) were on GFD and healthy individuals (n=76) on GCD. Each point represents the mean absorbance value of one urine sample from individuals at optical density of 450 nm. According to the QL of technique, individuals with a higher or equal GIP value than QL were considered positive (GIP++) for the presence of GIP, while those with lower GIP content but higher than LDT were considered positive not quantifiable (GIP+) and those with lower GIP content than LDT were considered negative (GIP−); *p<0.0001 (unpaired, two-tailed Student’s t test). CD, coeliac disease; GIP, gluten immunogenic peptides; GFD, gluten-free diet; GCD, gluten containing diet; QL, quantification limit; LDT, limit of technique detection; ND, not detectable.

Figure 4

Correlation between the presence of GIP in urine and small bowel histology in adult patients with CD. Histological appearance determined by the Marsh scale of the severity of mucosal lesion (Marsh I–III). GIP− (white bar), absence of GIP in urine; GIP+ (grey bar), visual presence of GIP not quantifiable in urine (>LDT QL). p=0.0007 (Fisher’s exact test). Values are expressed as percentage of patients. CD, coeliac disease; GIP, gluten immunogenic peptides; LDT, limit of technique detection; QL, quantification limit.