Combination of Gluten-Digesting Enzymes Improved Symptoms of Non-Celiac Gluten Sensitivity: A Randomized Single-blind, Placebo-controlled Crossover Study

Abstract

Introduction: Recently, the population of individuals with non-celiac gluten sensitivity (NCGS) who do not have celiac disease but show improved symptoms with a gluten-free diet, has increased. Enzyme replacement therapy using digestive enzymes is expected to improve the symptoms of NCGS and be sustainable, since gluten-related proteins that are indigestible by the digestive system have been considered triggers of NCGS.

Methods: We selected patients with NCGS by screening demographic interviews, as well as performing medical evaluations, anti-gluten antibody tests, and gluten challenge tests. We performed a single-blind and crossover clinical trial with these subjects using a gluten challenge with the enzyme mixture or a placebo. Our designed enzyme mixture contained peptidase, semi alkaline protease, deuterolysin, and cysteine protease derived from Aspergillus oryzae, Aspergillus melleus, Penicillium citrinum, and Carica papaya L., respectively.

Results: Administration of the enzyme mixture significantly decreased the change in the score of the symptom questionnaire before and after the gluten challenge compared with administration of the placebo in patients with NCGS without adverse events. In particular, the changes in the score of the gluten-induced incomplete evacuation feeling and headaches were significantly improved. The serum levels of interleukin (IL)-8, tumor necrosis factor (TNF)-α, andregulated on activation, normal T cell expressed and secreted (RANTES) in subjects were not significantly changed by gluten, as expected from previous studies, and the enzyme mixture did not affect these inflammatory markers.

Conclusion: In this human clinical study, we demonstrated the efficacy of the enzyme mixture derived from microorganisms and papaya in improving the symptoms of NCGS.

Conflict of interest statement

Guarantor of the article: Satoshi Koikeda is full responsible for the conduct of this study.

Specific author contributions: Hiroki Ido: designing formulation of enzyme mixture. Hirotaka Matsubara: designing formulation of enzyme mixture. Manabu Kuroda: data analysis. Akiko Takahashi: data analysis and drafting manuscript. Yuzo Kojima: conducting clinical study. Satoshi Koikeda: planning and conducting this study. Makoto Sasaki: planning this study and data analysis.

Financial support: This study was contributed by Amano Enzyme Inc.

Potential competing interest: Hiroki Ido, Hirotaka Matsubara, Manabu Kuroda, Akiko Takahasi, Yuzo Kojima, and Satoshi Koikeda are employees of Amano Enzyme, Inc. None.

Figures

Fig. 1. The schematic flowchart describing the procedure of the clinical research.

V1–8, the subjects attended the visit following overnight fasting (at least 8 h) for data collection and commencement of the next steps. C1 and 2, phone check to determine the subjects’ compliance with the diet and assess any adverse events. GC gluten challenge, GC with E gluten challenge with the enzyme mixture, GC with P gluten challenge with placebo

Fig. 2. The effects of addministration of enzyme mixture or placebo to the change in total CSI score by gluten challenge.

a, b The total CSI score before (Fig. 1, V5 or V7) and after (Fig. 1, V6 or V8) gluten challenge (GC) with enzyme mixture (E; (a)) or placebo (P; (b)). Thick lines and the largest symbols indicate average data. c The change in total CSI score from after washout with gluten-free diet for 2 weeks (Fig. 1, V5 or V7) to after gluten challenge with the enzyme mixture or dextrin as placebo for 2 weeks (Fig. 1, V6 or V8). Box with hatch marks indicates enzyme administration (GC with E, gluten challenge with enzyme mixture). White box indicates placebo (GC with P, gluten challenge with placebo). *p < 0.05

Fig. 3. The changes in score of each question from after washout with gluten-free for 2 weeks (Fig. 1, V5 or V7) to after gluten challenge with the enzyme mixture or dextrin as placebo for 2 weeks (Fig. 1, V6 or V8).

Questions are related to; Q1, pain or discomfort to the upper abdomen or the pit of the stomach. Q2, nausea. Q3, rumbling in your stomach. Q4, bloating. Q5, diarrhea. Q6, incomplete evacuation feeling. Q7, hunger pains. Q8, low energy level. Q9, headaches. Q10, food craving. Q11, loss of appetite. Q12, health condition related to CD. Q13, Overall health. Q14, physical pain. Q15, comfortable. Q16, healthy. Boxes with hatch marks indicate enzyme administration (GC with E). White boxes indicate placebo (GC with P). Data are expressed as mean ± SE. *p < 0.05

Fig. 4. The serum levels of inflammatory markers during this clinical study.

The serum levels of IL-8, TNF-α, and RANTES under the condition of the gluten-free diet (Fig. 1, V3) or gluten challenge (Fig. 1, V4) are shown in a, c, and e. The changes in serum levels of IL-8, TNF-α, and RANTES from after washout with gluten-free (Figure1, V3, V5 or V7) to after gluten challenge, gluten challenge with the enzyme mixture, or gluten challenge with dextrin as placebo (Figure1, V4, V6 or V8) are shown in b, d, and f. No significant difference was detected in each component by statistical analysis performed by paired t-test. ◆GFD gluten free diet, ■GC gluten challenge, ●GC with E gluten challenge with the enzyme mixture, ▲GC with P gluten challenge with placebo. Horizontal bars are median values