In this article, we describe a novel method to detect and monitor the presence of immunodominant gluten peptides in human feces on the basis of the use of the antigliadin 33-mer G12 antibody. To our knowledge, this was the first study to detect gluten-derived peptides in feces of patients with CD or other intestinal pathologies. The resistance of gluten peptides to gastrointestinal digestion, in particular that of the immunotoxic 33-mer peptide, ensures that a significant part of the ingested gluten peptides are excreted in feces, and thus recovery of measurable amounts of the immunotoxic fraction in feces would indicate that gluten has passed through the digestive tract and, therefore, that gluten has been consumed. In this study, the anti–33-mer G12-based immunoassays showed that >30% of the gliadin-reactive peptides remained intact after hydrolysis during in vitro simulated gastrointestinal digestion. However, the absorption of intact gliadin-reactive peptides along the gastrointestinal tract may vary between individuals. Diversity in the gut microflora (microbiome) and in the accompanying diet could have a dramatic impact on the resultant peptide concentration in feces (20
). Although a low digestion rate of gluten peptides would reduce their absorption, most of the undigested gluten peptides would be excreted, and either rapid immunochromatographic strips or ELISAs could be used to detect gluten ingestion in feces.
A main application of these assays to measure gluten in feces could be the monitoring of GFD compliance in CD patients. In the current study, we observed that, despite a large variation between individuals, there was a rough correlation between the amount of gluten consumed and the amount of gluten excreted. Differences in the amount of ingested gluten could be estimated by using different methods, such as immunochromatographic strips, competitive ELISA, and Western blot. Immunochromatographic strips might be attractive as clinical standard assays in a broad range of laboratories as well as in point-of-care settings. However, the use of a G12 competitive ELISA could be more suitable in situations that require a more quantitative analysis of the sample such as when monitoring a GFD, identifying the source of dietary infringement in CD patients, or assessing the efficacy of novel therapies in the destruction of gluten toxic peptides (4
Some authors (19
) have suggested that prolonged ingestion of 50 mg gluten/d causes significant damage to the architecture of the small intestine in patients who have begun treatment of CD. Furthermore, high consumption of gluten (1–5 g/d), although lower than that of the nonceliac population, causes a relapse of the disease at clinical, serologic, and histologic amounts. As a result, it is important to know the capacity of the analytic method used to detect ingestion of small amounts of gluten. Our results indicated that the ingestion of gluten amounts as low as 50 mg in processed bread could be detected in feces.
Currently, there are no validated methods to monitor adherence to a GFD. Antitissue transglutaminase antibodies have been proposed as markers of GFD compliance; however their effectiveness is still not clear (10
). IgA-class antibodies can take several months to decrease, and IgG-class antibodies can take ≥1 y to decrease, after the antigen source has been eliminated from the diet. Therefore, serology does not predict recovery (8
) and is not useful for follow-up. The use of serial endoscopies is not useful either because it is not considered an ethical practice. Other tests suggested as suitable diet-monitoring markers were the permeability test and fecal calprotectine (12
). We can measure the consequences of dietary transgressions, such as mucosal inflammation, but there is no way to show gluten intake and avoid the harmful aftermaths. Dietary interviews, although shown to be helpful in the determination of diet compliance, are often not possible to standardize, are subjective or rely on a truthful response from the patient, and cannot identify involuntary infringements. Our results showed that a broad range of gluten amounts (from 50 mg to 30 g) could be detected in feces of pediatric patients who were recently diagnosed with CD, were in remission, or were subjected to a gluten challenge; however, future studies that involved adult CD patients should be performed. These studies confirmed the use of G12 immunoassays to more accurately diagnose and monitor the disease and, thus, their value to study nonresponsive patients and to monitor adherence to a GFD.
In this study, we estimated the time of gluten toxic-peptide excretion to be between 2 and 4 d. These results should be taken into account if these methods are used in the diagnosis of gluten intoxication or if acute CD symptoms appear in an individual already treated with GFD.
Patients with RCD do not experience remission of symptoms while consuming a strict GFD. The diagnosis of RCD requires the exclusion of other diseases that can cause diarrhea and villous atrophy and the confirmation of adherence to a strict GFD (7
). A previous study showed that 82–90% of RCD patients referred to 2 large tertiary care centers were consuming gluten and had been improperly diagnosed of RCD (22
). Moreover, there is no effective method to rule out if RCD symptoms are due to hypersensitive to trace amounts of gluten or to involuntary gluten exposure. The results obtained in the current study with patients with active CD patients, in remission, or after a gluten challenge demonstrated that these methods could be used to determine uncontrolled gluten ingestion and might be a useful for studying RCD (7
In a previous study, we showed that the reactivity of antigliadin 33-mer moAbs correlated with the potential immunotoxicity of gluten proteins. T cell–reactivity analysis and enzymatic detoxification of gluten by glutenases showed that the signal of the antigliadin 33-mer moAbs correlated with the sample's potential toxicity for celiac patients (4
), which suggested that glutenases were able to cleave the epitopes detected by these antibodies. Therefore, the determination of the content of 33EPs in feces could be used to monitor gluten detoxification of celiac patients treated with novel enzymatic therapies (3
), proving the utility of these methods in experimental therapies for CD.
In this article, we have shown how to monitor the consumption of gluten by simple immunologic assays in feces and thereby overcome some unresolved scientific and clinical problems in celiac-patient monitoring, including 1) the monitoring of short -term GFD compliance, 2) assessment of the efficacy of enzymatic therapies, and 3) how to diagnose gluten intoxication that is due to involuntary food contamination. Researchers may want to consider the use of these methods when designing clinical trials in CD and to correctly interpret acute or refractory symptoms in celiac patients. In conclusion, the resistance of 33EPs to gastrointestinal digestion and the use of G12 anti–33-mer-based tests may be useful for monitoring dietary compliance in CD patients due to its noninvasiveness, sensitivity, and significant correlation with consumed gluten.