Changes in fecal short‐chain fatty acids following fecal microbiota transplantation in patients with irritable bowel syndrome

Abstract Background Short‐chain fatty acids (SCFAs) may play a role in the pathophysiology of irritable bowel syndrome (IBS). This study analyzed fecal SCFAs after performing fecal microbiota transplantation (FMT) in the IBS patients who were included in our previous study of the efficacy of FMT. Methods This study included 142 of the 164 IBS patients who participated in our previous study. They were belonging to three groups: placebo (own feces), 30‐g (superdonor feces), and 60‐g (superdonor feces) FMT. The patients completed the IBS Severity Scoring System (IBS‐SSS) Birmingham IBS Symptom, Fatigue Assessment Scale (FAS), the IBS Quality of Life (IBS‐QoL) and Short‐Form Nepean Dyspepsia Index (SF‐NDI) questionnaires and delivered fecal samples at the baseline and 1 month after FMT. The SCFA levels were determined by vacuum distillation followed by gas chromatography. Key Results The fecal butyric acid level was significantly increased after FMT in both the 30‐g and 60‐g groups (both P ≤ 0.001). In the 60‐g group, the levels of total SCFAs and isobutyric, isovaleric, and valeric acids increased after FMT. Butyric acid levels in the responders in both the 30‐g and 60‐g FMT groups were significantly inversely correlated with IBS‐SSS and FAS scores (P = 0.001, r = −0.3 and P = 0.0001. r=‐ 0.3, respectively). There were no differences in the SCFA levels in the placebo group after FMT. Conclusion and Inferences FMT increases the fecal SCFA levels in IBS patients. The increase in the butyric acid level is inversely correlated with symptoms in IBS patients following FMT, suggesting that SCFAs might play a role in the pathophysiology of IBS. www.clinicaltrials.gov (NCT03822299).


| INTRODUC TI ON
The intestinal bacterial profile in patients with irritable bowel syndrome (IBS) differs from that in healthy subjects. 2 The intestine of IBS patients has a lower abundance of butyrate-producing bacteria (Erysipelotrichaceae and Ruminococcaceae spp.) and a higher abundance of methane-producing bacteria (Methanobacteriales spp.) compared with healthy subjects. 2 In addition, the intestine of IBS patients has a higher abundance of Lactobacillus and Ruminococcus spp. and a lower abundance of Bifidobacterium, Faecalibacterium, and Erysipelotrichaceae spp. compared with healthy subjects. 2 Moreover, the diversity of intestinal bacteria (dysbiosis) is lower in patients with IBS than in healthy subjects. 2 In experimental animals, alterations in the intestinal bacteria are associated with gut dysmotility, visceral hypersensitivity, and altered intestinal permeability. 2 All of these abnormalities are observed in patients with IBS. 2 Thus, the gut microbiota is believed to play an important role in the pathophysiology of IBS. 2 The gut microbiota is influenced by dietary modifications and nutritional supplements, which might explain the beneficial effects of different dietary regimes on IBS symptoms. 3 Short-chain fatty acids (SCFAs) are the main products of bacterial fermentation of undigested and unabsorbed carbohydrates in the intestine. 3 SCFAs are the main energy source for colonic epithelial cells, regulating gut barrier functions and local immune defenses. 3 Among the SCFAs, butyric acid has anti-inflammatory and intestinal regenerative effects and administration of sodium butyrate to patients with inflammatory bowel disease changes the gut bacterial composition and induce the growth of butyric acid-producing bacterial genera. 3 The fecal SCFA levels differ between IBS patients and healthy subjects, with the fecal propionic acid level being significantly higher in IBS patients. 3 Whereas the levels of propionic and butyric acids were lower in constipation-predominant IBS (IBS-C) patients than in healthy subjects, that of butyric acid in diarrhea-predominant IBS (IBS-D) patients was higher. 3 A recent randomized double-blind placebo-controlled study performed by our group found that using a single superdonor for fecal microbiota transplantation (FMT) reduced IBS symptoms and fatigue and improved the quality of life in patients with IBS. 4 These improvements were accompanied by marked changes in the bacterial profiles of the patients. 4 The present study investigated whether FMT caused alterations in the fecal SCFAs in the same cohort of patients that we investigated in our previous study.

| Study design and randomization of patients
The design of this study has been described in detail previously. 4 In brief, patients provided a fecal sample and completed five questionnaires to assess their symptoms and quality of life at the baseline and provided another fecal sample and completed a new set of questionnaires at 1 month after FMT. Polyethylene glycol and loperamide were allowed as rescue medication during the study. The patients were randomized to placebo (own feces), 30-g (superdonor feces), or 60-g (superdonor feces) FMT. 4

| Patients
This study included 142 of the 164 IBS patients who participated in our previous study. 4 They were belonging to three groups: placebo (48 patients), 30-g (50 patients), and 60-g (44 patients) FMT. The characteristics of these patients are given in Table 1. The patients enrolled in this study have been described in detail previously. 4 In brief, patients who fulfilled the Rome IV criteria for a diagnosis of IBS were recruited from those attending the outpatient clinic at Stord Hospital. None of the patients had previously consumed a low-FODMAPs (fermentable oligo-, di-, monosaccharides, and polyols) diet, and all of the recruited patients had previously adhered to the National Institute for Health and Care Excellence (NICE)-modified diet for at least 3 months without experiencing any marked improvement in symptoms; the patients were therefore considered as non-responders to this diet. 4 They also received a course of IBS treatment that slightly improved their symptoms. 4

Key Points
• Fecal short-chain fatty acid (SCFA) levels differ between patients with irritable bowel syndrome (IBS) and healthy subjects. Abnormalities in SCFAs in patients with IBS might contribute to the pathophysiology of IBS.
• SCFAs increased after fecal microbiota transplantation • This study provides further evidence for the efficacy of FMT in treating IBS. This study suggests that intake of butyrate could be beneficial in the management of IBS.

| Superdonor
The single superdonor used in this study has been described in detail previously. Briefly, he was screened according to the European guidelines for donors for FMT. 6,7 He was a healthy 36-year-old man, non-smoker, not taking any medication regularly, and had a BMI of 23.5 kg/m 2 . He was born via a vaginal delivery, breastfed, and had taken a few courses of antibiotics during his life. He exercised regularly and took sport-specific dietary supplements, which made his diet richer than average in protein, fiber, minerals, and vitamins. 4 The GA-map Dysbiosis Test ® revealed that he was normobiotic, but his fecal bacterial profile deviated from the expected normal abundance in 14 of the 39 bacteria markers examined: 12 bacteria in the Firmicutes phylum and 1 each in the Proteobacteria and Verrucomicrobia phyla. 4 SCFAs were analyzed in donor's fecal samples taken at 0, 3, 6, 12, and 18 months.

| Fecal sample collection, preparation, and administration
Fecal samples were frozen immediately and kept at −20°C until they were delivered frozen to the laboratory, where they were kept at −80°C. The process of FMT has been described in detail previously. 4 In brief, the patients randomized to the placebo received 30 g of their own feces, while those in the 30-g and 60-g FMT groups received 30 and 60 g of the superdonor's feces, respectively. The fecal material was thawed for 2 days at 4°C, mixed with 40 ml of sterile saline, and filtered before administration. The transplant was administered to the distal duodenum via the working channel of a gastroscope, followed by another 40 ml of sterile saline.

| Symptom and quality-of-life assessments
Symptoms were assessed using the IBS-SSS and the Birmingham IBS Symptom (Birmingham IBS-S) questionnaires. 8,9 Fatigue was measured using the Fatigue Assessment Scale (FAS). 10 Quality of life was measured using the IBS Quality of Life (IBS-QoL) and Short-Form Nepean Dyspepsia Index (SF-NDI) questionnaires. [11][12][13] Higher IBS-QoL and lower SF-NDI scores indicate a better quality of life.
Patients who exhibited a decrease of ≥50 points in the total IBS-SSS score after FMT were considered responders.

| Determination of fecal SCFA levels
The fecal samples were weighed and homogenized with a solution containing 3 mmol/L 2-ethylbutyric acid and 0.5 mmol/L H 2 SO 4 .

TA B L E 1 Baseline characteristics of patients
Note: Data are mean ± SD, n, %, or n (%) values.

TA B L E 2 The Birmingham IBS Severity
Scoring System and its three domains in placebo and FMT-treated patients F I G U R E 2 FAS scores: total (A), physical fatigue (B), and mental health (C). * p < 0.05; ** p < 0.01 compared to placebo Agilent J&W GC columns, Agilent). 14,15 Flame ionization was used to determine the levels of total SCFAs, acetic, propionic, isobutyric, n-butyric, isovaleric, n-valeric acid, isocapronic, and ncapronic acids, with the results expressed in units of mmol/kg wet weight.

| Ethics
The study was approved by the Regional Committee for Medical and Health Research Ethics West, Bergen, Norway (approval no. 2017/1197/REK vest). All subjects provided both oral and written consents to participate. The study was registered at www.clini caltr ials.gov (NCT03822299) and www.crist in.no (ID657402).

| Statistical analysis
The sample size required in each arm of the study was 20 patients, as calculated by assuming that a placebo effect was 40% and an effect response was 80% (α = 0.05, 1−β = 0.80). We included 142 patients in order to avoid type 2 statistical errors. Differences between the placebo, 30, and 60-g FMT groups in age, IBS-SSS score, and FAS score were analyzed using one-way ANOVA with Tukey's multiplecomparisons test as a post-test. Differences between the baseline and after 1 month in Birmingham IBS-S, IBS-QoL, and SF-NDI were analyzed using the Mann-Whitney test. Differences between the placebo, 30-g, and 60-g FMT groups in sex, overall responses, numbers of IBS subtypes included in the study, and IBS subtype medications were analyzed using the chi-squared test. The paired t test was used to identify differences in the SCFA levels before and 1 month after FMT in the placebo, 30-g, and 60-g groups and with the IBS subtypes. Control for multiple testing with the SCFA parameters was not done. The correlations between the changes in SCFA levels and the IBS-SSS and FAS scores were analyzed using the non-parametric Spearman test. These analyses were performed using GraphPad Prism (version 8). All of the authors had access to the study data and reviewed and approved the final version of the manuscript.

| Symptom assessment
The response rates to FMT were 18.2%, 74.0%, and 88.6% in the placebo, 30-g, and 60-g groups, respectively. The response differed significantly between the three groups (P < 0.0001) and between the 30-g and 60-g FMT groups (P = 0.01). The total and the subitems IBS-SSS scores decreased significantly after FMT (Figure 1).
Birmingham IBS-S total score and its three domains were also significantly reduced after FMT ( Table 2). The total FAS score was also sig-  were not (Figure 2). The total score of IBS-QoL was significantly increased, and SF-NDI total score was significantly decreased after FMT (Tables 3 and 4).

| Fecal SCFA levels
The levels of total SCFAs and the individual SCFAs studied did not change after FMT in the placebo group ( Table 5). The fecal butyric acid level increased significantly after FMT in the 30-g group as a whole and in the responders, but not in the non-responders (Table 6). In the 60-g group, the fecal levels of total SCFAs, isobutyric acid, and butyric acid were higher after FMT in the whole group and in the responders, but not in the non-responders. Moreover, the fecal levels of isovaleric and valeric acids increased in the responders after FMT (Table 7).
At the baseline, the fecal butyric acid level was significantly higher in IBS-D patients than in IBS-C patients and patients with mixed-diarrhea-and-constipation IBS (IBS-M) ( Table 8). The response to FMT did not differ significantly between the IBS subtypes in the placebo group regarding the levels of the total SCFAs or the other SCFA acids measured (Table 9). In the 30-g group, the fecal butyric acid level increased after FMT in IBS-C and IBS-M patients, but not in IBS-D patients. The response to FMT in terms of the changes in the levels of total SCFAs and other acids investigated did not differ between the IBS subtypes (Table 9).
In the 60-g group, the response to FMT in terms of SCFA lev-   TA B L E 7 Fecal SCFA levels in the 60-g group before and after FMT  * Statistically significant.

| Adverse events
The adverse events were presented in detail previously, shortly about 20% of the patients treated with FMT experienced abdominal pain, cramping, tenderness, diarrhea, or constipation, compared with 2% of those in the placebo group. These adverse events were mild, self-limiting, and occurred during the first 2 days after FMT. 4

| DISCUSS ION
The present study showed that FMT-induced marked changes in the fecal SCFAs levels in patients with IBS. These changes varied with the transplant dose, while the alterations in the SCFAs levels in response to FMT differed with the IBS subtype. In both the 30-g and 60-g groups, the fecal butyric acid level increased significantly in the responders after FMT. However, in the 60-g group, the fecal levels of total SCFAs, isobutyric, isovaleric, and valeric acids increased in the responders following FMT.
At the baseline, the fecal butyric acid level was lower in IBS-C and IBS-M patients than in IBS-D patients. This finding agrees with a previous report of the butyric acid level being lower in IBS-C patients than in healthy subjects. 16 The propionic acid level did not differ significantly between the IBS subtypes in the present study, which disagrees with the previous report of the propionic acid level being lower in IBS-C patients than in healthy subjects. 16  levels. 17 The differences in the fecal levels of different SCFAs between the IBS subtypes may therefore be caused by differences in the intestinal transit time between these subtypes. SCFAs regulate the secretion and absorption of water and electrolytes and intestinal motility. 18,19 SCFAs increase also the secretion and upregulate the gene expression of peptide YY, 20,21 which is a mediator of the ileal brake and stimulates the absorption of water and electrolytes in the large intestine. 18,22,23 It is also possible that the differences in the SCFA levels between the IBS subtypes were due to differences in intestinal secretion, absorption, and motility.   in both the 30-g and 60-g groups after FMT observed in this study.
Butyrate is an important source of energy for colonic epithelial cells, and its lack can cause mucosal atrophy. 19 Butyrate also affects the immune response, modulates the oxidative stress of the host, and decreases intestinal cell permeability and intestinal motility. 22 Furthermore, butyrate appears to modulate colonic hypersensitivity, and treatment with butyrate reduces the abdominal pain in patients with IBS. [26][27][28] In the present study, the butyric acid level was inversely correlated with abdominal symptoms and fatigue, suggesting its role in the pathophysiology of IBS. The role of the increased levels of isobutyric, isovaleric, and valeric acids in the 60-g group after FMT is not clear. Further studies using animal models may shed light on the pathophysiology of SCFAs in IBS. 29 One of the main strengths of this study is that it investigated a relatively large cohort of patients with IBS, included three of the four IBS subtypes, and used a single superdonor. However, the study also had limitations: It did not include the unclassified IBS subtype (IBS-U), it did not investigate the long-term effects of FMT on fecal SCFA levels, and the cohort of included patients were those who did respond to the modified NICE diet; this means that the outcomes cannot be applied to the whole IBS population. Moreover, it is a single-center study whose main findings are yet to be replicated in larger multicenter studies.

CO N FLI C T O F I NTE R E S T
The authors have nothing to disclose.