Luppo Edens

Consumption of gluten with gluten-degrading enzyme by celiac patients: a pilot-study.

AIM To assesses the safety and efficacy of Aspergillus niger prolyl endoprotease (AN-PEP) to mitigate the immunogenic effects of gluten in celiac patients. METHODS Patients with initial diagnosis of celiac disease as confirmed by positive serology with subtotal or total villous atrophy on duodenal biopsies who adhere to a strict gluten-free diet (GFD) resulting in normalised antibodies and mucosal healing classified as Marsh 0 or I were included. In a randomised double-blind placebo-controlled pilot study, patients consumed toast (approximately 7 g/d gluten) with AN-PEP for 2 wk (safety phase). After a 2-wk washout period with adherence of the usual GFD, 14 patients were randomised to gluten intake with either AN-PEP or placebo for 2 wk (efficacy phase). Measurements at baseline included complaints, quality-of-life, serum antibodies, immunophenotyping of T-cells and duodenal mucosa immunohistology. Furthermore, serum and quality of life questionnaires were collected during and after the safety, washout and efficacy phase. Duodenal biopsies were collected after the safety phase and after the efficacy phase. A change in histological evaluation according to the modified Marsh classification was the primary endpoint. RESULTS In total, 16 adults were enrolled in the study. No serious adverse events occurred during the trial and no patients withdrew during the trial. The mean score for the gastrointestinal subcategory of the celiac disease quality (CDQ) was relatively high throughout the study, indicating that AN-PEP was well tolerated. In the efficacy phase, the CDQ scores of patients consuming gluten with placebo or gluten with AN-PEP did not significantly deteriorate and moreover no differences between the groups were observed. During the efficacy phase, neither the placebo nor the AN-PEP group developed significant antibody titers. The IgA-EM concentrations remained negative in both groups. Two patients were excluded from entering the efficacy phase as their mucosa showed an increase of two Marsh steps after the safety phase, yet with undetectable serum antibodies, while 14 patients were considered histologically stable on gluten with AN-PEP. Also after the efficacy phase, no significant deterioration was observed regarding immunohistological and flow cytometric evaluation in the group consuming placebo compared to the group receiving AN-PEP. Furthermore, IgA-tTG deposit staining increased after 2 wk of gluten compared to baseline in four out of seven patients on placebo. In the seven patients receiving AN-PEP, one patient showed increased and one showed decreased IgA-tTG deposits. CONCLUSION AN-PEP appears to be well tolerated. However, the primary endpoint was not met due to lack of clinical deterioration upon placebo, impeding an effect of AN-PEP.

Randomised clinical study: Aspergillus niger-derived enzyme digests gluten in the stomach of healthy volunteers.

Aspergillus niger prolyl endoprotease (AN‐PEP) efficiently degrades gluten molecules into non‐immunogenic peptides in vitro.

Ineffective Degradation of Immunogenic Gluten Epitopes by Currently Available Digestive Enzyme Supplements

Background Due to the high proline content of gluten molecules, gastrointestinal proteases are unable to fully degrade them leaving large proline-rich gluten fragments intact, including an immunogenic 33-mer from α-gliadin and a 26-mer from γ-gliadin. These latter peptides can trigger pro-inflammatory T cell responses resulting in tissue remodeling, malnutrition and a variety of other complications. A strict lifelong gluten-free diet is currently the only available treatment to cope with gluten intolerance. Post-proline cutting enzymes have been shown to effectively degrade the immunogenic gluten peptides and have been proposed as oral supplements. Several existing digestive enzyme supplements also claim to aid in gluten degradation. Here we investigate the effectiveness of such existing enzyme supplements in comparison with a well characterized post-proline cutting enzyme, Prolyl EndoPeptidase from Aspergillus niger (AN-PEP). Methods Five commercially available digestive enzyme supplements along with purified digestive enzymes were subjected to 1) enzyme assays and 2) mass spectrometric identification. Gluten epitope degradation was monitored by 1) R5 ELISA, 2) mass spectrometric analysis of the degradation products and 3) T cell proliferation assays. Findings The digestive enzyme supplements showed comparable proteolytic activities with near neutral pH optima and modest gluten detoxification properties as determined by ELISA. Mass spectrometric analysis revealed the presence of many different enzymes including amylases and a variety of different proteases with aminopeptidase and carboxypeptidase activity. The enzyme supplements leave the nine immunogenic epitopes of the 26-mer and 33-mer gliadin fragments largely intact. In contrast, the pure enzyme AN-PEP effectively degraded all nine epitopes in the pH range of the stomach at much lower dose. T cell proliferation assays confirmed the mass spectrometric data. Conclusion Currently available digestive enzyme supplements are ineffective in degrading immunogenic gluten epitopes.

Influence of dietary components on Aspergillus niger prolyl endoprotease mediated gluten degradation.

Celiac disease (CD) is caused by intolerance to gluten. Oral supplementation with enzymes like Aspergillus niger propyl-endoprotease (AN-PEP), which can hydrolyse gluten, has been proposed to prevent the harmful effects of ingestion of gluten. The influence of meal composition on AN-PEP activity was investigated using an in vitro model that simulates stomach-like conditions. AN-PEP optimal dosage was 20 proline protease units (PPU)/g gluten. The addition of a carbonated drink strongly enhanced AN-PEP activity because of its acidifying effect. While fat did not affect gluten degradation by AN-PEP, the presence of food proteins slowed down gluten detoxification. Moreover, raw gluten was degraded more efficiently by AN-PEP than baked gluten. We conclude that the meal composition influences the amount of AN-PEP needed for gluten elimination. Therefore, AN-PEP should not be used to replace a gluten free diet, but rather to support digestion of occasional and/or inadvertent gluten consumption.

Properties of Hemoglobin Decolorized with a Histidine-Specific Protease.

This study investigated the application of Aspergilloglutamic peptidase (AGP) on porcine hemoglobin decolorization. AGP from fungus Aspergillus niger is identified to possess a high preference towards the histidine residues. As histidine residues in hemoglobin are known to coordinate the heme group within the globin molecule, we therefore hypothesized that incubating hemoglobin with a histidine-specific protease would efficiently separate the non-heme peptides from the heme-enriched peptides with a minimum degree of hydrolysis. AGP-decolored porcine hemoglobin hydrolysates were assessed on their functional (for example, color, emulsification, foaming, and water binding) and sensory properties. The results were compared with commercially available blood-derived proteins (subtilisin-decolored hemoglobin hydrolysates and plasma protein). It was observed that AGP is able to effectively decolor hemoglobin. The degree of hydrolysis (DH) increased less than 3% using AGP to achieve 90% color reduction of hemoglobin, whereas a DH increase of more than 20% is needed using subtilisin. The AGP-decolored hemoglobin hydrolysates (AGP-Hb) possess good emulsification, foaming, and water binding properties, which are better or comparable with the plasma protein, and much better than the subtilisin-decolored hemoglobin hydrolysates (subtilisin-Hb). The model canned meat with addition of AGP-Hb showed the highest value in hardness, springiness, and chewiness from the texture analysis. Furthermore, the canned meat with AGP-Hb was found to have a better sensory profile than the ones with addition of subtilisin-Hb and plasma protein.

Letter: gluten digestion in the stomach and duodenum by Aspergillus niger-derived enzyme – things to ponder. Authors’ reply

SIRS, Due to the absence of proline hydrolysing enzymes in the gastrointestinal tract, gluten-containing diets are not digestible in humans and this may precipitate an abnormal immune response in patients suffering from coeliac disease. 3 Therefore, the ability to digest gluten is an important intervention in such patients, who are restricted to gluten-free diet for their entire life. In this context, Salden et al. have unambiguously demonstrated the ability of Aspergillus niger-derived enzyme (AN-PEP) to efficiently digest gluten, and the stomach was identified as the primary site of action for digestion of gluten. The use of acetaminophen, an established marker of gastric emptying, further demonstrated that increased gastric residence was inconsequential on the ability of AN-PEP to digest gluten from meals of either lowor high-caloric contents. Examination of the literature suggests scanty clinical data on the ability of AN-PEP to digest gluten and, therefore, the work of Salden et al. assumes high importance. Because the study was conducted in a controlled fashion in healthy volunteers using intragastically delivered meals containing gluten, it may be critical to consider the applicability of AN-PEP during in vivo conditions where a gluten-containing meal is consumed. Also, it is important to provide some directions and guidance for future clinical research to optimise AN-PEP for clinical applications. Based on the data generated by Salden et al., there appears to be no lag time needed for the digestion process and the plots of a-gliadin concentration in the stomach suggest almost instantaneous digestion of the gluten content regardless of the meal type. Was this because there was a higher availability of AN-PEP as compared to the gluten content of the meal? If a higher gluten content was tested, would there be a saturation of hydrolytic cleavage? In such situations, would the longer residence of gluten in the stomach render it more amenable to AN-PEP related activity? Some additional thoughts that may be crucial to understand the role of AN-PEP would be whether AN-PEP be ingested before or during the gluten meal, and whether the gluten meal should be enriched with fatty substrates to retard gastric emptying, and therefore improve the efficiency of the hydrolytic cleavage of gluten by AN-PEP.

Editorial: enhancing gluten digestion in the stomach- a further help to minimise unintentional ingestion? Authors' reply

*Division of Gastroenterology-Hepatology, Department of Internal Medicine, NUTRIM, Maastricht University Medical Center (MUMC+), Maastricht, The Netherlands. Department of Immunohematology and Blood Transfusion, Leiden University Medical Centre (LUMC), Leiden, The Netherlands. DSM Biotechnology Centre, Delft, The Netherlands. Department of Pharmacology and Toxicology, CARIM, Maastricht University, Maastricht, The Netherlands. Department of Methodology and Statistics, CAPHRI, Maastricht University Medical Center (MUMC+), Maastricht, The Netherlands. E-mail: bouke.salden@maastrichtuniversity.nl

Su2096 Gluten Degrading Enzyme Effectively Digests Gluten in the Stomach and Small Intestine of Healthy Volunteers

A significant proportion of the population does not tolerate gluten. Aspergillus niger prolyl endopeptidase (AN-PEP) enzyme efficiently degrades gluten molecules into non-immunogenic peptides in a dynamic, multi-compartmental gastrointestinal simulation model but its efficacy in vivo remains to be established. Aim of our study was to assess the efficacy of ANPEP on gastrointestinal breakdown of gluten in healthy subjects, as well as the effect of meal caloric density on AN-PEP efficiency. Methods: In this double-blind, randomized, placebo-controlled cross-over study 12 healthy subjects attended to four test days in random order. On each occasion they received a low (143 kCal) or high caloric (405 kCal) meal, containing 4g gluten, with AN-PEP or placebo via a triple-lumen nasoduodenal catheter. Acetaminophen was added to measure gastric emptying rate. One lumen, positioned in the stomach, was used for administration of the test meal and collection of gastric fluid. A second lumen was used for the continuous injection of the inert dilution marker polyethylene glycol 3350 (PEG3350) to enable the calculation of meal dilution by endogenous secretions, with the injection port positioned just distal to the pylorus. A third lumen, located at the tube tip 10 cm distal to the second port, was used for collection of duodenal content. Fluid samples were taken regularly during 4 hours after meal infusion. The presence of the DQ2.5glia-α3 epitopes was quantified using the Gluten-Tec® ELISA assay, which provides an accurate estimate of the actual α-gliadin content of the samples. Degradation of intact gluten proteins was measured by Western-Blot analysis. Duodenal PEG3350 concentrations and gastric acetaminophen concentrations were determined by high-performance liquid chromatography. The effect of AN-PEP on gluten degradation was measured by the difference in 240-minute Area Under the Curve (AUC) of relative and absolute gluten exposure between AN-PEP and placebo. Differences between combinations of treatment and meal were assessed using linear mixed models. Results: AN-PEP significantly reduced gliadin concentration and absolute output in the stomach and duodenum (as 240-min AUCs) in both low and high caloric meals, compared to placebo. Furthermore, in the high caloric meal with placebo the duodenal gliadin concentration was lower, compared to the low caloric meal with placebo. No differences were observed between gliadin concentrations of a low or high caloric meal containing AN-PEP (Table). The gastric emptying time of the high caloric meal was longer compared to the low caloric meal, both in the presence of placebo (p=0.014) and AN-PEP (p=0.100). Conclusion: AN-PEP addition to a gluten containing meal significantly enhances gluten digestion in healthy volunteers. Meal caloric density does not affect the efficacy of AN-PEP on gluten degradation. Gliadin concentration and absolute output in stomach and duodenum