Bouke Salden

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.

Paracetamol as a Post Prandial Marker for Gastric Emptying, A Food-Drug Interaction on Absorption

The use of paracetamol as tool to determine gastric emptying was evaluated in a cross over study. Twelve healthy volunteers were included and each of them consumed two low and two high caloric meals. Paracetamol was mixed with a liquid meal and administered by a nasogastric feeding tube. The post prandial paracetamol plasma concentration time curve in all participants and the paracetamol concentration in the stomach content in six participants were determined. It was found that after paracetamol has left the stomach, based on analysis of the stomach content, there was still a substantial rise in the plasma paracetamol concentration time curve. Moreover, the difference in gastric emptying between high and low caloric meals was missed using the plasma paracetamol concentration time curve. The latter curves indicate that (i) part of the paracetamol may leave the stomach much quicker than the meal and (ii) part of the paracetamol may be relatively slowly absorbed in the duodenum. This can be explained by the partition of the homogenous paracetamol-meal mixture in the stomach in an aqueous phase and a solid bolus. The aqueous phase leaves the stomach quickly and the paracetamol in this phase is quickly absorbed in the duodenum, giving rise to the relatively steep increase of the paracetamol concentration in the plasma. The bolus leaves the stomach relatively slowly, and encapsulation by the bolus results in relatively slow uptake of paracetamol from the bolus in the duodenum. These findings implicate that paracetamol is not an accurate post prandial marker for gastric emptying. The paracetamol concentration time curve rather illustrates the food-drug interaction on absorption, which is not only governed by gastric emptying. Trial Registration ClinicalTrials.gov NCT01335503 Nederlands Trial Register NTR2780

A Critical Evaluation of In Vitro Hesperidin 2S Bioavailability in a Model Combining Luminal (Microbial) Digestion and Caco-2 Cell Absorption in Comparison to a Randomized Controlled Human Trial

SCOPE Bioavailability strongly determines polyphenol bioactivity, and is strongly influenced by food matrix, enzymatic and microbial degradation, and gastrointestinal absorption. To avoid human trials for pre-screening of polyphenol bioavailability, studies have focused on in vitro model development. Nevertheless, their predictive value for bioavailability can be questioned. METHOD AND RESULTS We used the orange flavonoid hesperidin 2S to validate a model combining digestion in the simulator of the human intestinal microbial ecosystem (SHIME) and Caco-2 cell transport, with a human intervention study. In vitro, hesperidin was resistant to degradation in the stomach and small intestine, but was rapidly deconjugated on reaching the proximal colon. Extensive and colon-region-specific degradation to smaller phenolics was observed. Hydrocaffeic and dihydroisoferulic acid accumulated in proximal, and hydroferulic acid in distal colon. Caco-2 transport was the highest for dihydroisoferulic acid. In humans, plasma and urine hesperetin-glucuronide levels increased significantly, whereas the impact on small phenolics was limited. CONCLUSIONS In the combined in vitro model, smaller phenolics strongly accumulated, whereas in humans, hesperetin conjugates were the main bioavailable compounds. Future in vitro model development should focus on simulating faster polyphenol absorption and elimination of smaller phenolics to improve their predictive value of in vivo polyphenol bioavailability.

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