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Monitoring of gluten-free diet compliance in celiac patients by assessment of gliadin 33-mer equivalent epitopes in feces.

Comino I, Real A, Vivas S, Síglez MÁ, Caminero A, Nistal E, Casqueiro J, Rodríguez-Herrera A, Cebolla A, Sousa C - Am. J. Clin. Nutr. (2012)

Bottom Line: The resistance of a significant part of 33EPs to gastrointestinal digestion was shown in vitro and in vivo.Gluten-derived peptides could be sensitively detected in human feces in positive correlation with the amount of gluten intake.These techniques may serve to show GFD compliance or infringement and be used in clinical research in strategies to eliminate gluten immunotoxic peptides during digestion.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, Seville, Spain.

ABSTRACT

Background: Certain immunotoxic peptides from gluten are resistant to gastrointestinal digestion and can interact with celiac-patient factors to trigger an immunologic response. A gluten-free diet (GFD) is the only effective treatment for celiac disease (CD), and its compliance should be monitored to avoid cumulative damage. However, practical methods to monitor diet compliance and to detect the origin of an outbreak of celiac clinical symptoms are not available.

Objective: We assessed the capacity to determine the gluten ingestion and monitor GFD compliance in celiac patients by the detection of gluten and gliadin 33-mer equivalent peptidic epitopes (33EPs) in human feces.

Design: Fecal samples were obtained from healthy subjects, celiac patients, and subjects with other intestinal pathologies with different diet conditions. Gluten and 33EPs were analyzed by using immunochromatography and competitive ELISA with a highly sensitive antigliadin 33-mer monoclonal antibody.

Results: The resistance of a significant part of 33EPs to gastrointestinal digestion was shown in vitro and in vivo. We were able to detect gluten peptides in feces of healthy individuals after consumption of a normal gluten-containing diet, after consumption of a GFD combined with controlled ingestion of a fixed amount of gluten, and after ingestion of <100 mg gluten/d. These methods also allowed us to detect GFD infringement in CD patients.

Conclusions: Gluten-derived peptides could be sensitively detected in human feces in positive correlation with the amount of gluten intake. These techniques may serve to show GFD compliance or infringement and be used in clinical research in strategies to eliminate gluten immunotoxic peptides during digestion. This trial was registered at clinicaltrials.gov as NCT01478867.

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Related in: MedlinePlus

Mean (±SD) resistance of 33-mer peptides to cleavage by gastrointestinal enzymes. A: Amino acid sequences of the 33-mer peptide. The G12 moAb recognition sequence in the 33-mer peptide is in bold-face type. B: Competition assay for the detection of 33-mer peptide by G12 moAb after treatment with pepsin, trypsin, and chymotrypsin. Three assays were performed with samples run in triplicate. C: Western blot analysis of 33-mer peptide by the G12 moAb after treatment with gastrointestinal enzymes. moAb, monoclonal antibody; MW, molecular weight marker.
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fig2: Mean (±SD) resistance of 33-mer peptides to cleavage by gastrointestinal enzymes. A: Amino acid sequences of the 33-mer peptide. The G12 moAb recognition sequence in the 33-mer peptide is in bold-face type. B: Competition assay for the detection of 33-mer peptide by G12 moAb after treatment with pepsin, trypsin, and chymotrypsin. Three assays were performed with samples run in triplicate. C: Western blot analysis of 33-mer peptide by the G12 moAb after treatment with gastrointestinal enzymes. moAb, monoclonal antibody; MW, molecular weight marker.

Mentions: To verify that the 33-mer peptide remained intact after gastric and intestinal proteolysis, the 33-mer peptide was subjected to simulated gastrointestinal digestion, and its concentration was determined after each digestive step by using a G12 competitive ELISA. The 33-mer peptide concentration after gastric digestion (194 μg/mL) or intestinal digestion (169 μg/mL) did not vary significantly from that of nondigested 33-mer peptide (186 μg/mL) (Figure 2, A and B). These results were confirmed by Western blot analysis in which a single band of ~3.5 kDa in size was seen in the undigested and the sequentially digested 33-mer samples (33-mer theoretical molecular weight: 3.9 kDa) (Figure 2C).


Monitoring of gluten-free diet compliance in celiac patients by assessment of gliadin 33-mer equivalent epitopes in feces.

Comino I, Real A, Vivas S, Síglez MÁ, Caminero A, Nistal E, Casqueiro J, Rodríguez-Herrera A, Cebolla A, Sousa C - Am. J. Clin. Nutr. (2012)

Mean (±SD) resistance of 33-mer peptides to cleavage by gastrointestinal enzymes. A: Amino acid sequences of the 33-mer peptide. The G12 moAb recognition sequence in the 33-mer peptide is in bold-face type. B: Competition assay for the detection of 33-mer peptide by G12 moAb after treatment with pepsin, trypsin, and chymotrypsin. Three assays were performed with samples run in triplicate. C: Western blot analysis of 33-mer peptide by the G12 moAb after treatment with gastrointestinal enzymes. moAb, monoclonal antibody; MW, molecular weight marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3278243&req=5

fig2: Mean (±SD) resistance of 33-mer peptides to cleavage by gastrointestinal enzymes. A: Amino acid sequences of the 33-mer peptide. The G12 moAb recognition sequence in the 33-mer peptide is in bold-face type. B: Competition assay for the detection of 33-mer peptide by G12 moAb after treatment with pepsin, trypsin, and chymotrypsin. Three assays were performed with samples run in triplicate. C: Western blot analysis of 33-mer peptide by the G12 moAb after treatment with gastrointestinal enzymes. moAb, monoclonal antibody; MW, molecular weight marker.
Mentions: To verify that the 33-mer peptide remained intact after gastric and intestinal proteolysis, the 33-mer peptide was subjected to simulated gastrointestinal digestion, and its concentration was determined after each digestive step by using a G12 competitive ELISA. The 33-mer peptide concentration after gastric digestion (194 μg/mL) or intestinal digestion (169 μg/mL) did not vary significantly from that of nondigested 33-mer peptide (186 μg/mL) (Figure 2, A and B). These results were confirmed by Western blot analysis in which a single band of ~3.5 kDa in size was seen in the undigested and the sequentially digested 33-mer samples (33-mer theoretical molecular weight: 3.9 kDa) (Figure 2C).

Bottom Line: The resistance of a significant part of 33EPs to gastrointestinal digestion was shown in vitro and in vivo.Gluten-derived peptides could be sensitively detected in human feces in positive correlation with the amount of gluten intake.These techniques may serve to show GFD compliance or infringement and be used in clinical research in strategies to eliminate gluten immunotoxic peptides during digestion.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, Seville, Spain.

ABSTRACT

Background: Certain immunotoxic peptides from gluten are resistant to gastrointestinal digestion and can interact with celiac-patient factors to trigger an immunologic response. A gluten-free diet (GFD) is the only effective treatment for celiac disease (CD), and its compliance should be monitored to avoid cumulative damage. However, practical methods to monitor diet compliance and to detect the origin of an outbreak of celiac clinical symptoms are not available.

Objective: We assessed the capacity to determine the gluten ingestion and monitor GFD compliance in celiac patients by the detection of gluten and gliadin 33-mer equivalent peptidic epitopes (33EPs) in human feces.

Design: Fecal samples were obtained from healthy subjects, celiac patients, and subjects with other intestinal pathologies with different diet conditions. Gluten and 33EPs were analyzed by using immunochromatography and competitive ELISA with a highly sensitive antigliadin 33-mer monoclonal antibody.

Results: The resistance of a significant part of 33EPs to gastrointestinal digestion was shown in vitro and in vivo. We were able to detect gluten peptides in feces of healthy individuals after consumption of a normal gluten-containing diet, after consumption of a GFD combined with controlled ingestion of a fixed amount of gluten, and after ingestion of <100 mg gluten/d. These methods also allowed us to detect GFD infringement in CD patients.

Conclusions: Gluten-derived peptides could be sensitively detected in human feces in positive correlation with the amount of gluten intake. These techniques may serve to show GFD compliance or infringement and be used in clinical research in strategies to eliminate gluten immunotoxic peptides during digestion. This trial was registered at clinicaltrials.gov as NCT01478867.

Show MeSH
Related in: MedlinePlus