Table 1.
Enzyme activities of digestive enzyme supplements and purified enzymes.
Table 2.
Protein compositon of digestive enzyme supplements.
Fig 1.
Degradation of gliadin as monitored by R5 ELISA immunoassay.
Degradation of gliadin was monitored as a function of time at pH 4.5 (A) and pH 6.2 (B), using AN-PEP as a control enzyme. To exclude the presence of an interfering substance in digestive enzyme supplements we also analyzed AN-PEP control enzyme in the presence of Supplement C. Error bars represent standard deviation of duplicate experiments. Supplements D and E were not tested since they were not compatible with R5 ELISA immunoassay procedure.
Table 3.
Epitopes contained in the gastrointestinal-enzyme resistant 26-mer and 33-mer gluten peptides.
Fig 2.
Mass spectrometric analysis of the degradation products of 26-mer and 33-mer peptide by digestive enzyme supplements.
Peptide was incubated at 37°C for 30 minutes at pH 6.0 in the presence of 1 capsule equivalent digestive enzyme supplements. As a control, 1/100 capsule equivalent of AN-PEP at pH 5.0 was also incubated. Peptide reaction products were analyzed by Q-TOF LC-MS and their MS spectra shown in this Figure. The identity of the prominent peptides 1–6 and a-g are given in Table 4. In the case of AN-PEP, small amounts of epitope-containing peptides were observed under these conditions, but they disappear at higher enzyme concentration or prolonged incubation (see Fig 3). The digestive enzyme supplements display comparable activities and remove at most two amino acids from the N-terminus of the 26-mer (peptide 1 (26-mer) is degraded to peptide 2, and then to peptide 3), and three amino acids at most from the 33-mer peptide (peptide a (33-mer) is degraded to b, to c, and then to peptide d).
Table 4.
Peptides generated from the 26-mer and 33-mer.
Fig 3.
Digestive enzyme supplements do not neutralize any of the 6 gluten epitopes of the 33-mer, whereas the control enzyme AN-PEP does.
The chart shows the number of epitopes found in the reaction mixture after incubation with digestive enzyme supplements or AN-PEP. An epitope is counted as present irrespective of its concentration, or the form in which it appears, 33-mer or one of its degradation products. Reactions with digestive enzymes were performed at their optimal pH 7.0, and reactions with AN-PEP at pH 5.0. In addition, when tested in the whole pH range 2 to 11, none of the digestive enzyme supplements was able to neutralize a single epitope (see also S4 and S5 Figs). A, No neutralization at a dose of 1 or 10 capsule equivalents digestive enzyme supplements (1/10 capsule was not tested) after 30 minutes at 37°C. As a control, AN-PEP was tested at a dose of 1/10 and 1 capsule. B, No neutralization of epitopes by digestive enzyme supplements, even after 120 minutes of incubation at 37°C. As a control, the time-dependent degradation by AN-PEP is shown.
Fig 4.
Ineffective detoxification by digestive enzyme supplements.
33-mer was incubated for 60 minutes with 1 capsule equivalent of digestive enzyme supplements, or 1/10 capsule equivalent of AN-PEP as a control. The peptide reaction products were deamidated using tissue transglutaminase, purified by C18 solid phase extraction and tested for T-cell activation. As a negative control, T-cells were incubated without the 33-mer peptide or digestive enzyme supplements (blanc). As a positive control, T-cells were incubated with 33-mer peptide. The enzyme preparations, when tested separately, were not toxic for the T-cells (not shown). T-cells stimulated with control peptide (LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF) incorporated 40,000 cpm. T-cell proliferation ([3H]thymidine incorporation in cpm) is shown as a function of the input of 33-mer and its degradation products. Error bars represent standard deviation of triplicate experiments. The result shown is representative for two independent experiments.