By Carrie Decker, ND
Founder and Medical Director, Blessed Thistle, Madison, WI
Dr. Decker reports no financial relationships relevant to this field of study.
Synopsis: A prolyl endoprotease derived from Aspergillus niger has been shown to be capable of degrading gluten at the level of the stomach from both low- and high-calorie meals, minimizing duodenal and small intestine exposure to gluten. The use the prolyl endoprotease enzyme lowered α-gliadin concentration in the stomach and duodenum and absolute α-gliadin exposure in the duodenum over 240 minutes after consumption of a gluten-containing meal.
Source: Salden BN, et al. Randomised clinical study: Aspergillus niger-derived enzyme digests gluten in the stomach of healthy volunteers. Aliment Pharmacol Ther 2015;42:273-285.
Summary Points
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An Aspergillus niger-derived prolyl endoprotease (AN-PEP) enzyme has been shown to lower α-gliadin concentration in the stomach and duodenum after intake of standardized low- and high-calorie meals compared to placebo.
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The α-gliadin concentration area under the curve over 240 minutes (AUC240) at the duodenum was 7 mcg x min/mL when consumed with AN-PEP vs 168 mcg x min/mL with placebo after exposure to a low-calorie meal containing 4.0 g of gluten protein.
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Similar reductions were observed in the stomach with an AUC240 concentration of 35 mcg x min/mL when consumed with AN-PEP vs 389 mcg x min/mL with placebo.
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The effect of increasing meal caloric density was observed to prolong transit time and reduce AUC240 duodenal gluten concentration with the placebo, and no incremental effect was observed with the use of the AN-PEP enzyme.
Exposures to gluten are difficult to control for individuals with celiac disease. Currently available enzyme preparations specific for gluten digestion have been shown to degrade immunogenic gluten peptides such as α-gliadin incompletely.1 In this double-blind, placebo-controlled, crossover study, an Aspergillus niger-derived prolyl endoprotease (AN-PEP) enzyme was investigated for the purpose of degrading α-gliadin peptides from gluten consumed with a low- or high-calorie meal. The α-gliadin peptide has been suggested to be the primary initiator of the inflammatory response to gluten in individuals with celiac disease.2 The AN-PEP enzyme has been investigated previously and has been shown to be optimally active at a pH between 3 and 5.3 It was shown in a dynamic in vitro model to degrade almost all immunogenic gluten-derived proteins into non-immunogenic fragments during passage through a stimulated stomach.4
The study included 12 healthy volunteers, ranging in age between 18-45 years. Individuals were excluded if they had a history of gastrointestinal disorders or surgery interfering with gastrointestinal function, history of major disease, use of medications other than oral contraceptives, or were pregnant, lactating, dieting, smoking, or engaged in excessive alcohol consumption (> 20 drinks per week). Participants were subject to four total test days, with a week washout period between two test days. Participants were randomized in double-blind fashion to the various interventions of a high- or low-calorie gluten meal and supplementation with AN-PEP or placebo. The primary outcome of the study was the concentration of α-gliadin epitope from 240 minute area under the curve (AUC240) measurements from duodenal and stomach aspirates. In addition to this, the absolute AUC240 level of α-gliadin in the duodenum was evaluated and compared.
After an overnight fast, meals were introduced through a nasogastic tube. Meals consisted of 5.2 g of gluten powder, which contained 4.0 g of gluten protein combined with refined olive oil powder, sodium caseinate, and maltodextrin (high-calorie meal only). The low- and high-calorie meals contained 143.1 kcal and 405.1 kcal per 300 g portion, respectfully. The AN-PEP enzyme or a placebo solution in the amount of 6.1 mL was combined with 100 mL of water and added to the test meals immediately before infusion. The meals, mixed with the AN-PEP or placebo solution and 1 g of acetaminophen, were administered into the stomach over a 5-minute period. Negligible absorption of acetaminophen occurs in the stomach,5 and thus the concentration of acetaminophen in the stomach was utilized to evaluate gastric emptying.
Measurements were made via aspiration of the stomach and duodenal contents from a port in the stomach and a port 15 cm distal to the pylorus. The catheter position was secured by radiology and regularly evaluated during the tests. For the purpose of volumetric measurements of gluten degradation, an inert marker polyethylene glycol in 0.9% saline was also introduced via a port into the duodenum at a continuous rate, beginning 60 minutes prior to meals to achieve steady state and continued until 240 minutes. Measurements of gastric and duodenal contents were sampled at baseline and at times of 15, 30, 45, 60, 75, 90, 120, 150, 180, 210, and 240 minutes. Gastric contents were also measured at 5 and 10 minutes. For the purpose of neutralizing enzyme activity, samples were immediately frozen in liquid nitrogen and stored at -80° C until analysis. Upon thawing, they were treated with NaOH to increase the pH to 11-12, heated to 85° C for 10 minutes, and the pH was then neutralized with HCl. Quantification of α-gliadin content was performed with the Gluten-Tec ELISA assay6 and confirmed by western blot.
After consumption of low-calorie meals, the duodenal α-gliadin AUC240 concentration when consumed with AN-PEP was 7 mcg x min/mL (95% confidence interval [CI], 3-14 mcg x min/mL) vs 168 mcg x min/mL (95% CI, 80-352 mcg x min/mL) with placebo, while the stomach AUC240 concentration was 35 mcg x min/mL (95% CI, 17-73 mcg x min/mL) when consumed with AN-PEP vs 389 mcg x min/mL (95% CI, 180-840 mcg x min/mL) with placebo (P < 0.001). The absolute duodenal α-gliadin AUC240 level was 2813 mcg x min (95% CI, 1206-6555 mcg x min) with the AN-PEP enzyme vs 31,952 mcg x min (95% CI, 12,670-80,579 mcg x min) with placebo after consumption of a low-calorie meal (P < 0.001). Increasing meal caloric density was observed to prolong transit time and reduce duodenal α-gliadin AUC240 concentration and absolute amounts with the placebo, and no incremental effects were observed with the use of the AN-PEP enzyme. Mild gastrointestinal symptoms were reported during testing with no correlation with meal type or intervention.
COMMENTARY
Adverse reactions to gluten are common among the population and include celiac disease and non-celiac gluten sensitivity (NCGS).7,8 Celiac disease is a multifactorial disease characterized by an inflammatory response to ingested gluten in the small intestine. Proteolytically resistant, proline- and glutamine-rich gluten peptides from wheat, rye, and barley persist in the intestinal lumen and elicit an immune response only in genetically susceptible persons.9 The immune response to gluten proteins and polypeptide regions varies, but may involve the innate immune system, class I or class II mediated reactions, and antibody recognition.10
NCGS is characterized by symptoms that occur after gluten ingestion, with improvements within hours or days after elimination and relapse following gluten re-introduction. A distinct manner of testing for NCGS currently does not exist, but the possibility of celiac disease and wheat allergy must be excluded. It has been suggested that testing, including immunoglobulin G (IgG) anti-gliadin antibodies and flow cytometric basophil activation test, with duodenal and/or ileum-colon intraepithelial and lamina propria eosinophil counts, a personal history of food allergy, and atopy in infancy, could be useful to identify NCGS patients.11 Interest in the topic of NCGS is evident by the recent increase in publications pertaining to NCGS relative to celiac disease from1:438 in the period of 1950-1970 to 1:10 in the period of 2010-2013.8 Prevalence of NCGS has been estimated to be up six to 10 times higher than celiac disease;12 however, results of studies widely vary, ranging from 0.5-13%.13
For individuals with these conditions, the primary recommendation is gluten avoidance. However, many individuals may be poorly compliant with a gluten-free diet, and absolute avoidance of gluten is often difficult, particularly in social settings. Inadvertent trace exposures are common in settings such as restaurants or other food preparation facilities, where strict policies to avoid gluten cross-contamination are not imposed.
Enzyme preparations containing the exo-peptidase enzyme dipeptidyl peptidase IV (DDP IV) are advertised to degrade gluten and minimize exposure when gluten is inadvertently consumed. However, it has been observed that gluten-digestion specific preparations containing high amounts of DDP IV with other proteases could only partly neutralize gluten proteins and left the immunogenic gluten fragments of both the α- and γ-gliadins largely intact.1 The AN-PEP enzyme has been shown in vitro to efficiently degrade the immunogenic gliadin fragments in this same investigation,1 as well as a stimulated dynamic gastrointestinal model.4
In this study, the possible effect of meal calorie density on gluten degradation by the AN-PEP enzyme was investigated by the usage of high- vs low-caloric meals. It may have been more informative to investigate the amount of α-gliadin degradation with high vs low gluten content meals, as this would have demonstrated the amount of enzyme necessary for effective degradation of gluten.
Tolerability and safety of the AN-PEP enzyme has been demonstrated in patients with celiac disease in an earlier randomized, double-blind, placebo-controlled pilot study.14 In this investigation, 14 individuals with celiac disease (Marsh scores 0 or I) consumed 7 g of gluten daily for a 2-week period, along with supplementation of the AN-PEP enzyme or a placebo. Duodenal biopsies and celiac disease quality scores were obtained at baseline and subsequent to the intervention. It was observed that increased tissue transglutaminase immunoglobulin A deposits were seen in four of seven patients in the placebo group compared with one of seven patients in the group supplemented with the AN-PEP enzyme. Increased transglutaminase expression has been documented elsewhere in individuals with untreated celiac disease.15 No significant deterioration of duodenal mucosal biopsy or celiac disease quality scores was observed in either group, so further conclusions could not be drawn. There were no serious adverse events reported during this study as well as no patient withdrawals.
Although this study was a controlled intervention far different from typical clinical trials, it provides useful and necessary scientific information pertaining to the in vivo degradation of immugenic epitopes of gluten. The previous clinical study demonstrating tolerability and safety of the AN-PEP enzyme may have been too short in duration, as it was only conducted for a period of 2 weeks. A period of 8 weeks of gluten consumption for individuals previously adhering to a gluten-free diet is recommended prior to duodenal biopsy in the diagnosis of celiac disease, and a similar duration should be applied to future studies. As individuals with NCGS have negative celiac serologies, normal or near normal duodenal biopsies, and a wide variety of symptomologies, studies of the use of the AN-PEP enzyme with this population should include comprehensive evaluation of symptoms and quality of life measurements. As more definitive means of assessment for NCGS with immunochemistry, biopsy, or other means are defined, evaluation also should include these parameters.
Although definitive benefits have not yet been shown with the use of the AN-PEP enzyme in individuals with celiac disease or NCGS, the positive findings of this clinical study and previous in vitro investigations do support its use for individuals with celiac disease. As this study was industry-sponsored to investigate this proprietary enzyme, this must be duly noted. There are multiple preparations of gluten digestion-specific enzymes now on the market containing the Aspergillus niger-derived endoprotease, and for individuals with celiac disease who are concerned with possible gluten exposure, these enzyme preparations may be helpful.
REFERENCES
- Janssen G, et al. Ineffective degradation of immunogenic gluten epitopes by currently available digestive enzyme supplements. PLoS One 2015;10:e0128065.
- Shan L, et al. Structural basis for gluten intolerance in celiac sprue. Science 2002;297:2275-2279.
- Stepniak D, et al. Highly efficient gluten degradation with a newly identified prolyl endoprotease: implications for celiac disease. Am J Physiol Gastrointest Liver Physiol 2006;291:G621-629.
- Mitea C, et al. Efficient degradation of gluten by a prolyl endoprotease in a gastrointestinal model: Implications for coeliac disease. Gut 2008;57:25-32.
- Clements JA, et al. Kinetics of acetaminophen absorption and gastric emptying in man. Clin Pharmacol Ther 1978;24:420-431.
- Mujico JR, et al. Validation of a new enzyme-linked immunosorbent assay to detect the triggering proteins and peptides for celiac disease: Interlaboratory study. J AOAC Int 2012;95:206-215.
- Fasano A, et al. Prevalence of celiac disease in at-risk and not-at-risk groups in the United States: A large multicenter study. Arch Intern Med 2003;163:286-292.
- Catassi C, et al. Non-celiac gluten sensitivity: The new frontier of gluten related disorders. Nutrients 2013;5:3839-3853.
- Kagnoff MF. Celiac disease. A gastrointestinal disease with environmental, genetic, and immunologic components. Gastroenterol Clin North Am 1992;21:405-425.
- Ciccocioppo R, et al. The immune recognition of gluten in coeliac disease. Clin Exp Immunol 2005;140:408-416.
- Mansueto P, et al. Non-celiac gluten sensitivity: Literature review. J Am Coll Nutr 2014;33:39-54.
- Molina-Infante J, et al. [Non-celiac gluten sensitivity: A critical review of current evidence]. Gastroenterol Hepatol 2014;37:362-371.
- Molina-Infante J, et al. Systematic review: Noncoeliac gluten sensitivity. Aliment Pharmacol Ther 2015;41:807-820.
- Tack GJ, et al. Consumption of gluten with gluten-degrading enzyme by celiac patients: A pilot-study. World J Gastroenterol 2013;19:5837-5847.
- Gorgun J, et al. Tissue transglutaminase expression in celiac mucosa: An immunohistochemical study. Virchows Arch 2009;455:363-373.
A prolyl endoprotease derived from Aspergillus niger has been shown to be capable of degrading gluten at the level of the stomach from both low- and high-calorie meals, minimizing duodenal and small intestine exposure to gluten. The use the prolyl endoprotease enzyme lowered α-gliadin concentration in the stomach and duodenum and absolute α-gliadin exposure in the duodenum over 240 minutes after consumption of a gluten-containing meal.
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