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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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Detection of gluten in feces of healthy individuals subjected to a controlled gluten-containing diet. A: Semiquantitative analysis of gluten peptides and proteins in feces of healthy individuals by using G12 immunochromatographic strips HL901–HL911 (studied subjects: n = 11). An asterisk indicates that gluten traces were detected. B: One representative immunochromatographic-strip example of the trial performed with the samples collected during the study period for one subject. Blue stripes represent an internal positive control that indicates that the stick worked properly; pink stripes indicate the presence of gluten. C and D: SDS-PAGE and Western blot of gluten peptides and proteins extracted from feces. GFD, gluten-free diet; MW, molecular weight marker; PWG, Prolamin Working Group.
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fig4: Detection of gluten in feces of healthy individuals subjected to a controlled gluten-containing diet. A: Semiquantitative analysis of gluten peptides and proteins in feces of healthy individuals by using G12 immunochromatographic strips HL901–HL911 (studied subjects: n = 11). An asterisk indicates that gluten traces were detected. B: One representative immunochromatographic-strip example of the trial performed with the samples collected during the study period for one subject. Blue stripes represent an internal positive control that indicates that the stick worked properly; pink stripes indicate the presence of gluten. C and D: SDS-PAGE and Western blot of gluten peptides and proteins extracted from feces. GFD, gluten-free diet; MW, molecular weight marker; PWG, Prolamin Working Group.

Mentions: Given the variability shown in the concentration and time to excretion of gluten-reactive peptides across subjects after consumption of a normal gluten-containing diet, the next experiments to monitor the presence of 33EPs in feces were performed with a fixed amount of gluten. Thus, to examine whether the amount of gluten consumption correlated with the amount of gluten excretion, 11 healthy volunteers were subjected to a strict GFD for 1 wk, after which a fixed amount of 9 g of gluten, which was distributed across the main meals, was administered every day for 4 consecutive days followed by 30 g of gluten for 4 additional days. One single type of gluten (without previous heat treatment) was used in all cases to avoid variability because of the administration of gluten from different sources. For quantification by using G12 immunochromatographic strips, the stool samples were serially diluted after collection to cover a range between 6 and 500 ppm. After consumption of a normal gluten-containing diet, all subjects showed gluten excretion in feces, with values that exceeded 500 ppm (Figure 4), whereas all fecal samples collected during the period in which subjects consumed a GFD had gluten amounts below the detection limit of the method. When subjects consumed 9 g gluten/d, the amount of gluten detected in feces was >250 ppm in all but one case, which contained between 6 and 25 ppm, and when subjects consumed 30 g gluten/d, excreted amounts were >500 ppm (Figure 4, A and B), which represented a 100-fold increase over the detection limit. This result showed an approximate correlation between the amount of gluten consumed and the amount of gluten peptides with G12 epitopes excreted in feces. In addition, Western blot analysis showed G12 moAb reactivity for all samples (positive controls, GFD + 9 g gluten, and GFD + 30 g gluten), except for those obtained during the GFD period (Figure 4, C and D). G12 competitive ELISA, which was used to determine the concentration of toxic peptides in these samples, showed the presence of 33EPs in all stool samples collected during the normal gluten containing–diet period, whereas the amounts of toxic peptides were below the quantification limit of the method (5.4 pg 33EPs/mg sample) when the subjects were maintained on a GFD. The range of 33EPs in subjects who consumed 30 g gluten/d was 6.7–28.0 ng 33EPs/mg feces (1240–5185-fold above the detection limit), which was significantly higher (P = 0.018) than in subjects who consumed 9 g gluten/d, in whom the range was 3.5–9.6 ng 33EPs/mg feces (648–1778-fold above detection limit) (Figure 5). These results were in agreement with those obtained with immunochromatographic strips.


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)

Detection of gluten in feces of healthy individuals subjected to a controlled gluten-containing diet. A: Semiquantitative analysis of gluten peptides and proteins in feces of healthy individuals by using G12 immunochromatographic strips HL901–HL911 (studied subjects: n = 11). An asterisk indicates that gluten traces were detected. B: One representative immunochromatographic-strip example of the trial performed with the samples collected during the study period for one subject. Blue stripes represent an internal positive control that indicates that the stick worked properly; pink stripes indicate the presence of gluten. C and D: SDS-PAGE and Western blot of gluten peptides and proteins extracted from feces. GFD, gluten-free diet; MW, molecular weight marker; PWG, Prolamin Working Group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Detection of gluten in feces of healthy individuals subjected to a controlled gluten-containing diet. A: Semiquantitative analysis of gluten peptides and proteins in feces of healthy individuals by using G12 immunochromatographic strips HL901–HL911 (studied subjects: n = 11). An asterisk indicates that gluten traces were detected. B: One representative immunochromatographic-strip example of the trial performed with the samples collected during the study period for one subject. Blue stripes represent an internal positive control that indicates that the stick worked properly; pink stripes indicate the presence of gluten. C and D: SDS-PAGE and Western blot of gluten peptides and proteins extracted from feces. GFD, gluten-free diet; MW, molecular weight marker; PWG, Prolamin Working Group.
Mentions: Given the variability shown in the concentration and time to excretion of gluten-reactive peptides across subjects after consumption of a normal gluten-containing diet, the next experiments to monitor the presence of 33EPs in feces were performed with a fixed amount of gluten. Thus, to examine whether the amount of gluten consumption correlated with the amount of gluten excretion, 11 healthy volunteers were subjected to a strict GFD for 1 wk, after which a fixed amount of 9 g of gluten, which was distributed across the main meals, was administered every day for 4 consecutive days followed by 30 g of gluten for 4 additional days. One single type of gluten (without previous heat treatment) was used in all cases to avoid variability because of the administration of gluten from different sources. For quantification by using G12 immunochromatographic strips, the stool samples were serially diluted after collection to cover a range between 6 and 500 ppm. After consumption of a normal gluten-containing diet, all subjects showed gluten excretion in feces, with values that exceeded 500 ppm (Figure 4), whereas all fecal samples collected during the period in which subjects consumed a GFD had gluten amounts below the detection limit of the method. When subjects consumed 9 g gluten/d, the amount of gluten detected in feces was >250 ppm in all but one case, which contained between 6 and 25 ppm, and when subjects consumed 30 g gluten/d, excreted amounts were >500 ppm (Figure 4, A and B), which represented a 100-fold increase over the detection limit. This result showed an approximate correlation between the amount of gluten consumed and the amount of gluten peptides with G12 epitopes excreted in feces. In addition, Western blot analysis showed G12 moAb reactivity for all samples (positive controls, GFD + 9 g gluten, and GFD + 30 g gluten), except for those obtained during the GFD period (Figure 4, C and D). G12 competitive ELISA, which was used to determine the concentration of toxic peptides in these samples, showed the presence of 33EPs in all stool samples collected during the normal gluten containing–diet period, whereas the amounts of toxic peptides were below the quantification limit of the method (5.4 pg 33EPs/mg sample) when the subjects were maintained on a GFD. The range of 33EPs in subjects who consumed 30 g gluten/d was 6.7–28.0 ng 33EPs/mg feces (1240–5185-fold above the detection limit), which was significantly higher (P = 0.018) than in subjects who consumed 9 g gluten/d, in whom the range was 3.5–9.6 ng 33EPs/mg feces (648–1778-fold above detection limit) (Figure 5). These results were in agreement with those obtained with immunochromatographic strips.

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