This study's participants comprised patients with IBS (as defined by Rome IV criteria) (20) without any other disorders aged between 18 and 70 years old with body mass index (BMI) ranging from 18.5 to 25. Patients were recruited between June 2020 and December 2020 from two gastroenterology clinics in Tehran, Iran. Patients who did not have the following disorders were eligible to participate in the study: any organic intestinal diseases based on colonoscopy over the past 5 years, intestinal infection, history of colorectal disorders, major intestinal surgery, liver, kidney, psychiatric disease, or any GI disease other than IBS.

Patients were excluded from the study if they (a) were taking any medication with antispasmodics, antibiotics, anti-diarrhea or laxative properties, prokinetics, non-steroidal anti-inflammatory, and immunosuppressive agents or (b) were a smoker, pregnant, or breastfeeding. Patients with known allergies to glutamine or whey protein were excluded, as were those who were taking or had taken supplements containing glutamine or whey protein. Patients were also excluded if they had consumed synthetic sweeteners within 2 days before the study or during the study, as such sweeteners can alter intestinal permeability. Finally, any patients unwilling to adhere to the recommended diet were also excluded.

Patients were sub-classified as either having predominant diarrhea (IBS-D), predominant constipation (IBS-C), mixed or alternating bowel habits (IBS-M), or undetermined categories (IBS-U). All patients provided informed written consent to participate in the study after the study protocol was thoroughly explained.

This study was a randomized, double-blind, placebo-controlled trial. Fifty patients who met the inclusion criteria were randomly assigned to either the experimental (glutamine) or control (placebo) group. Randomization was based on a random table sequence, and all investigators were blind regarding which patients were in which group.

Since receiving 15 g of glutamine or whey does not cause side effects for patients (21), participants received an oral glutamine powder or placebo powder (whey protein) at a dose of 15 g (5 g mixed in water three times per day) for 6 weeks. The powders were similar in color, consistency, and taste. The supplements were concealed as A or B by a third party—for the duration of the study, neither the participants nor investigators knew which group was taking which supplement.

All participants were advised to follow a low FODMAP diet in addition to taking the assigned supplement. Diets were administered by an experienced dietitian according to the guidelines of the National Institute for Health and Care Excellence (NICE) while omitting high FODMAPs foods. All diets contained <5-g FODMAPs per day. Patients' adherence to the diet was evaluated by recording of 3 days (1 weekend and 2 workdays) dietary recalls at week 2 and the end of the study.

Dietary intakes of FODMAPs were assessed using Monash University's low-FODMAP diet database and quantitative reports of FODMAP content in recent studies (22, 23). Patients were followed by phone calls to assess protocol adherence, record any supplement side effects, and answer any study-related questions. Patients' competency was assessed using the measurement of remained supplements.

The primary outcome was a significant reduction in IBS-symptom severity score (IBS-SSS). Secondary outcomes were changes in IBS symptoms, quality of life, and stool consistency and frequency. All clinical outcomes were evaluated at baseline and the end of the study using the IBS-SSSQ (24). This instrument includes five clinically relevant items that determine the severity of abdominal pain, the frequency of pain, abdominal distension, satisfaction with bowel habits, and the interference of IBS with community function as measured on the visual analog scale (VAS) (with an array of 100 mm, with 0 indicating no symptoms and 100 representing extremely severe symptom). The sum of these five items was IBS- symptom severity score (range = 0–500). Scores of 75–175, 175–300, and > 300 indicated mild, moderate, and severe cases, respectively.

Each patient's quality of life (QoL) was assessed at baseline and the end of the study using a 34-item self-report measure specific to IBS (IBS-QoL) (25). Each item was answered on a 5-point Likert scale, and participants' scores were summed to derive the overall score. Scores were subsequently transposed onto a scale from 0 to 100, with higher scores indicating a better quality of life.

Stool consistency was assessed using the validated Bristol Stool Form Scale (BSFS) (26). Stool frequency (i.e., number of stools per day) was recorded at baseline and the end of the study.

The sample size was calculated based on the formula by considering α (type 1 error) = 0.05 and power of 80% according to the published article (11), which was obtained 21. Therefore, according to the formula 21 subjects were needed in each group, considering the probable dropouts, we assigned 25 subjects for each group to meet the adequate power.

All hypothesis tests were 2-tailed, with P < 0.05 denoting statistical significance. The Kolmogorov–Smirnov-test with a significance level of 5% was used to test continuous variables for the normality assumption. The chi-square test or Fisher exact-test was used to determine the differences of categorical variables between groups. Comparison between the variables was performed by paired-samples t-test/Wilcoxon at the beginning and end of the study in each group. To detect differences in continuous variables between the two groups, independent-samples t-test/Mann-Whitney was used. The main effects and interaction effects of the interventions were compared between groups using analysis of covariance (ANCOVA) with baseline measures as a covariate. The data were analyzed according to the intention-to-treat principle. Patients missing the final data were imputed. A multiple imputation procedure was used based on multiple imputations by chained equation. In the multiple imputation procedure, five imputed data sets were generated. The results of the five imputed data sets were pooled to obtain data estimates.

Collected information by food diaries was analyzed in the Nutritionist 4 software modified for Iranian foods (Karen Pharma & Food Supplement Co., Tehran, Iran). Data from the food-record questionnaires were entered and analyzed by an expert dietitian.

The study protocol was approved by Shahid Beheshti Ethics Committee (IR.SBMU.NNFTRI.REC.1398.083), and it was registered at the Iranian Registry of Clinical Trials, with the registration number IRCT20100524004010N28.

Between June 2020 and December 2020, 70 patients were recruited from two gastroenterology clinics and screened for this trial. From this initial group, 20 patients were excluded and the other 50 were enrolled and underwent the randomization process. Among the 50 included patients, 25 were assigned to the glutamine group and 25 were assigned to the placebo group. One patient was excluded from the glutamine group after discontinuing the study protocol, and two were lost to follow-up issues. Meanwhile, three patients from the control group were excluded due to non-compliance with the recommended diet. Therefore, 22 patients in the glutamine group and 22 patients in the control group completed the study protocol (Figure 1).

Table 1 shows the baseline characteristics of participants. The groups were similar in all characteristics except abdominal pain frequency (p = 0.022).

There was no significant difference in the nutritional compositions of the diet between the two groups (Table 2). All participants' dietary recalls demonstrated acceptable adherence to the diet.

Improvement in IBS-severity score of more than 45% was observed in 22 of 25 participants (88%) in the glutamine group, while it was only 15 of 25 participants (60%) in the control group (P = 0.015; Figure 2). The total scores of IBS-SS and the scores for individual items (abdominal pain intensity, abdominal pain frequency, abdominal distension, dissatisfaction with bowel habits, and interference with life), stool frequency, and consistency are shown in Table 3. Significant improvements were observed in these variables in both groups at the end of the study compared with the baseline (p < 0.001 for total scores and individual items of IBS-SSS and P = 0.002, P = 0.003, respectively, for stool frequency and consistency).

Table 4 shows the comparison of pre-post-treatment changes between the glutamine and placebo groups. The glutamine group had significant changes in total IBS-severity score, dissatisfaction of bowel habit and interference with community function (58% reduction; P < 0.001, 57% reduction; P < 0.001, 51% reduction; P = 0.043, respectively). No adverse effect was reported in any group of interventions.

Baseline quality of life scores was not significantly different between the two groups (Table 1). The QOL scores increased in both intervention groups (+13 ± 8.12, P < 0.001 in the glutamine group and +11 ± 5.12, P < 0.001 in the control group) at the end of the study compared with the baseline (Table 3). No significant difference was observed between groups at the end of the study (P = 0.353).