Dietary Sodium Butyrate Decreases Postweaning Diarrhea by Modulating Intestinal Permeability and Changing the Bacterial Communities in Weaned Piglets

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Dietary Sodium Butyrate Decreases Postweaning Diarrhea by Modulating Intestinal Permeability and Changing the Bacterial Communities in Weaned Piglets¹,²,³

+Author Affiliations

⁴State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, China;
⁵Department of Animal and Poultry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; and
⁶Department of Internal Medicine, Center for Autophagy Research, University of Texas Southwestern Medical Center, Dallas, TX

↵*To whom correspondence should be addressed. E-mail: maxi@cau.edu.cn or xi.ma@utsouthwestern.edu.

Abstract

Background: The vast majority of substances used as alternatives to antibiotics produce inconsistent results and rarely equal the effectiveness of in-feed antibiotics.

Objective: This study evaluated the effects of the combined use of sodium butyrate (SB) and reduced antibiotics in a piglet diet in promoting performance and to control weaning diarrhea.

Methods: Piglets weaned at 28 d were randomly assigned to a corn-soybean meal control ration [negative control (NC)]; a similar ration with 50 mg kitasamycin/kg, 20 mg colistin sulfate/kg, and 1000 mg encapsulated SB/kg [reduced antibiotics + SB (ASB)]; or to a ration with 100 mg kitasamycin/kg and 40 mg colistin sulfate/kg [positive control (PC)] for 28 d. Performance, diarrhea incidence, intestinal permeability, and changes in the bacterial communities in the ileum and colon were determined.

Results: Weight gain and the ratio of weight gain to feed intake were significantly greater in the ASB and PC piglets than in the NC piglets (P < 0.05). Diarrhea incidence was lower in the ASB and PC piglets than in the NC piglets (P < 0.05). Urinary lactulose to mannitol ratios were 25% and 30% lower, respectively, whereas jejunal and colonic occludin protein expressions were significantly greater in the ASB and PC piglets compared with the NC piglets (P < 0.05). In the intestinal mucosa, malondialdehyde was lower in the ASB and PC piglets (by 42% and 43%, respectively), whereas tumor necrosis factor α (TNF-α) was 63% lower in the ASB piglets and 59% lower in the PC piglets compared with the NC piglets (P < 0.05). 16S ribosomal RNA gene sequence analysis revealed a higher colonic Shannon index and a lower colonic Simpson index in the ASB and PC piglets than in the NC piglets. In addition, the ASB and PC treatments caused a striking decrease in Lactobacillaceae and a noticeable increase in Clostridiaceae in the ileal and colonic lumen, as well as increases in Ruminococcaceae, Lachnospiraceae, and Bacteroidetes in the colonic lumen.

Conclusion: Collectively, our results support an important role for SB in improving performance and decreasing diarrhea incidence in weaned piglets by modulation of intestinal permeability and the bacterial communities in the ileum and colon.

Keywords:

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Introduction

The period after weaning is characterized by a high incidence of intestinal disturbances with diarrhea and decreased performance in piglets (1). Subtherapeutic doses of antibiotics have been proven to increase growth rate, improve feed utilization, and reduce the incidence of postweaning diarrhea (2). However, indiscriminate use of antibiotics has led to an increasing number of antibiotic-resistant pathogens as well as public concern with regard to their cross-transfer to humans (3). As a result, the use of antibiotics in diets fed to livestock has been banned in the European Union since 2006. Many materials have been investigated as alternatives (4), among which some organic acids and their salts incorporated in diets are known to be helpful in overcoming the postweaning syndrome (3).

Butyrate, an SCFA, is physiologically produced by large bowel microbial fermentation of dietary carbohydrates in mammals (5). It is quickly absorbed and can be utilized as a major energy source by epithelial cells in the terminal ileum and large intestine (6), as well as stimulating growth of the small intestinal epithelium (5). The effects of butyrate on carcinogenesis (5), inflammation (7), oxidative stress (8), and intestinal barrier function (9) have been described, particularly its activities in promoting gut health both in vitro (10) and in vivo (11).

Because it is easier to handle in the feed-manufacturing process, butyrate is often used in the form of sodium salt instead of free acid (5). Accumulated studies have been conducted with regard to the effects of sodium butyrate (SB)⁸ on performance in weaned piglets, including feed intake, weight gain, and feed efficiency. One trial by Piva et al. (12) showed that SB supplementation at 800 mg/kg increased weight gain and feed intake in piglets in the first 2 wk after weaning (P < 0.05). In another feeding trial, weaned piglets fed 1000-mg-SB/kg diets showed improved performance compared with those fed a control diet or 500 mg SB/kg (P < 0.05) (13). However, inconsistent results were obtained in other studies. Performance did not differ between piglets fed a basal diet with SB at 0, 1000, 2000, or 4000 mg/kg in an experiment by Biagi et al. (14). Weber and Kerr (15) reported that there was no effect of dietary SB (500, 1000, 2000, and 4000 mg/kg) on overall feed efficiency. In another trial, dietary SB (1000 mg/kg) significantly decreased diarrhea incidence in weaned piglets (P < 0.05) without improvements in weight gain, feed intake, or feed efficiency (16). A conclusion can be drawn that SB does not consistently provide growth-promoting effects to weaned piglets. Therefore, it would appear that SB cannot be used as the sole alternative to antibiotics in piglet feeding.

Although many other countries are expected to introduce an antibiotic ban in diets fed to livestock, antibiotics are still extensively used in many areas of the world. The entire withdrawal of antibiotics in piglet diets would likely result in considerable production loss in these areas because the vast majority of antibiotic substitutes tested produce inconsistent results and rarely equal the effectiveness of antibiotics (4). An efficient strategy may be to use a partial substitution of antibiotics with other growth-promoting substances, as suggested by a previous study in which supplemental acidifiers appeared to act synergistically with avilamycin (17). However, to our knowledge, few studies have focused on the application of SB inclusion, combined with decreased antibiotics, in piglet feeding.

In the present study, we evaluated the effects of the combined use of SB and reduced antibiotics in promoting performance and to control postweaning diarrhea in piglets. Furthermore, to probe into the possible mechanisms for these effects, some variables with regard to intestinal barrier function and changes in the bacterial communities in the ileum and colon were determined.

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Methods

Piglets, diets, and experimental protocol.

All procedures used in this experiment were approved by the Institutional Animal Care and Use Committee of China Agricultural University. The heaviest 90 crossbred (Duroc × Landrace × Large White) weaned piglets (weaned at 28 d) were selected from a pool of 30 litters. After a 3-d adaptation period during which all piglets were fed the same base diet, the pigs (10.24 ± 1.90 kg body weight) were assigned to 1 of 3 treatments (n = 30) that were homogenous for weight and sex. A basal diet was formulated to meet the nutrient requirements of the pigs according to the NRC (18) (Supplemental Table 1). Dietary treatments included a corn-soybean meal control ration without antibiotics [negative control (NC)]; a similar ration with 50 mg kitasamycin/kg, 20 mg colistin sulfate/kg, and 1000 mg encapsulated SB/kg [Lideshi, Inc.; reduced antibiotics + SB (ASB)]; and a ration with 100 mg kitasamycin/kg and 40 mg colistin sulfate/kg [positive control (PC)]. The encapsulated structure in SB was used to deter prompt absorption and metabolism of butyrate in the duodenum and jejunum, which ensured considerable release and absorption of butyrate in the lower intestine portion (19).

During the 28-d experiment, pigs were fed their respective diets and allowed ad libitum access to feed and water. The piglets were individually weighed on day 28, and feed consumption per pen was recorded weekly. The amount of feed wasted was recorded daily.

Fecal consistency was visually assessed at 0900 and 1600 h each day by observers who were blind to treatments with the use of a modification of the method described by Ma et al. (20). Fresh excreta were graded by using the following scale: 0 = solid, 1 = semisolid, 2 = semiliquid, and 3 = liquid. The occurrence of diarrhea was defined as production of grade 2 or 3 feces for 2 continuous days.

Small intestinal permeability.

Small intestinal permeability was assessed on day 28 before weighing by using the lactulose to mannitol differential absorption test (21). Briefly, urine samples of piglets per treatment (n = 6) were collected after 6 h of feed deprivation for baseline urinary sugar measurement. After the samples were obtained, 5 mL lactulose (0.4 g/mL; Sigma) and 5 mL of mannitol (0.2 g/L; Sigma) were administered intragastrically to the piglets. Piglets were feed-deprived for the 6-h study period but were allowed to drink water after 30 min. Urinary lactulose and mannitol concentrations were determined by an enzymatic technique (22). Mannitol excretion was corrected by subtraction of baseline values, and the lactulose to mannitol excretion ratio was calculated as an index of intestinal permeability.

Sample collection.

On day 28, 6 blood samples per treatment were harvested from the jugular vein by using tubes without anticoagulant (Becton Dickinson). Blood samples were allowed to clot at room temperature for 20 min and centrifuged at 1610 × g for 10 min at 4°C. Serum was then removed and stored at −20°C until assay. Intestinal segments (duodenum, jejunum, ileum, and colon); mucosa in the duodenum, jejunum, ileum, and colon; as well as chyme from the ileum and colon were obtained and stored at −80°C until further assay (23).

Morphologic evaluations.

Villus height and crypt depth in the duodenum, jejunum, and ileum were determined as previously described (23). Briefly, these segments were fixed in 4% paraformaldehyde for 24 h and then embedded in paraffin wax. Sections of 4 μm were cut and stained with hematoxylin and eosin. Measurements for villus height and crypt depth were taken by using the Axioskop-2 microscope (Olympus) and the Image Processing System (Visitron Systems).

Protein extraction and immunoblot analysis.

The expression of the protein occludin was determined due to its crucial roles in tight junction structure and paracellular permeability (24). The total protein amount contained in the jejunal and colonic tissue samples was extracted according to the method described by a ProteoJET Total Protein Extraction Kit (Fermentas). A Bicinchoninic Acid Protein Assay Kit (Applygen Technologies) was used to determine the protein concentration. Equal amounts of protein extracts (20 μg) were fractionated on 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories). The membranes were blocked with a 5% skimmed-milk solution at room temperature for 2 h and then incubated with diluted antibodies against occludin (1:200; Santa Cruz) and GAPDH (1:20,000; Sigma). After incubation with HRP-conjugated secondary antibody, signals were visualized by the Odyssey Infrared Imaging System (LI-COR Biosciences). Blot analysis was carried out at 6 replicates/treatment by Quantity One software (BioRad Laboratories), with subsequent calculation of the ratio between the band intensities of occludin and GAPDH.

Antioxidant variables and immune indexes.

Serum samples were thawed and thoroughly mixed immediately before testing. Equal amounts of mucosa in different intestinal segments from the same piglet were blended to form a single sample. The blended samples (n = 6/treatment) were homogenized in PBS (10 mM; pH 7.4) and centrifuged at 3000 × g for 10 min at 4°C. The supernatant was stored at −80°C until further assays.

Antioxidant variables, including superoxide dismutase, glutathione peroxidase, glutathione, malonaldehyde, as well as the cytokines complement 3, IL-1β, IL-2, IFN-γ, and TNF-α in serum and intestinal mucosa were all determined by using assay kits according to the manufacturer’s instructions. All of the assay kits were purchased from the Nanjing Jiancheng Bioengineering Institute.

Composition and diversity of the bacterial communities.

DNA in each ileal and colonic chyme sample was extracted according to the manufacturer’s protocol (Bocai Biology). The V3 and V4 regions of the 16S ribosomal DNA gene were chosen for PCR. The primers were 338F (5′ACTCCTACGGGAGGCAGCA-3′) and 806R (5′GGACTACHVGGGTWTCTAAT-3′). The procedure to obtain amplicons was previously described, with modifications (25). The products were examined on a 2% (wt:vol) agarose gel. The amplicons from 6 replicates/treatment were blended in equimolar ratios on the basis of concentration. The blended samples were sent out for MiSeq Next-Generation Sequencing System on an Illumina MiSeq PE300 platform at the Majorbio Bio-Pharm Technology Company. Only sequences >50 bp were used for phylotype analysis at the 0.03 operational taxonomic unit level.

Statistical analysis.

Differences in diarrhea incidence between the 3 treatments were tested by the chi-square contingency test. All other data were analyzed by using SAS version 9.1. The results are presented as means ± SEMs. One-factor ANOVA followed by Tukey’s multiple-range tests were used for equal variances. Kruskal-Wallis 1-factor ANOVA was performed to compare means with unequal variances. For all statistical analyses, P values <0.05 were considered significant.

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Results

Performance and diarrhea incidence.

Compared with the NC piglets, weight gain was significantly greater in the ASB and PC piglets (P < 0.05) with feed intake unchanged, resulting in an improved gain to feed ratio in the ASB and PC piglets (P < 0.05) (Table 1). Diarrhea incidence was significantly lower in the ASB and PC piglets than in the NC piglets (P < 0.05) (Table 1). There were no significant differences in performance or diarrhea incidence between the ASB and PC piglets.

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TABLE 1

Performance and diarrhea incidence in weaned piglets fed corn-soybean meal without antibiotics (NC), corn-soybean meal with reduced antibiotics and sodium butyrate (ASB), or corn-soybean meal with antibiotics (PC) for 28 d¹

Small intestinal morphology.

Villus height in the jejunum and crypt depth in the duodenum and jejunum were lower in the ASB piglets than in the NC and PC piglets (P < 0.05). The ASB piglets had lower crypt depth in the ileum than did the NC piglets (P < 0.05), whereas the value for the PC piglets was intermediate to values for the NC and ASB piglets (Table 2).

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TABLE 2

Small intestinal morphology of weaned piglets fed corn-soybean meal without antibiotics (NC), corn-soybean meal with reduced antibiotics and sodium butyrate (ASB), or corn-soybean meal with antibiotics (PC) for 28 d¹

Villus height in the duodenum and ileum was greater in the PC piglets than in the NC and ASB piglets (P < 0.05). Villus height to crypt depth ratio in the ileum in the PC piglets was also greater than in the NC piglets (P < 0.05), whereas the ratio for the ASB piglets was intermediate to that for the NC and PC piglets (Table 2).

Small intestinal permeability and occludin abundance.

Small intestinal permeability was lower in the ASB and PC piglets as indicated by the decreased urinary lactulose to mannitol ratios compared with the NC piglets (P < 0.05) (Figure 1). The expression of the intestinal tight junction protein occludin in the jejunum and colon was significantly higher in the ASB and PC piglets than in the NC piglets (P < 0.05) (Figure 2). These variables in the ASB piglets were comparable to those in the PC piglets.

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FIGURE 1

Small intestinal permeability in weaned piglets fed corn-soybean meal without antibiotics (NC treatment), corn-soybean meal with reduced antibiotics and encapsulated sodium butyrate (ASB treatment), or corn-soybean meal with antibiotics (PC treatment) for 28 d. Values are means ± SEMs, n = 6. Labeled means without a common letter differ, P < 0.05. ASB, reduced antibiotics + sodium butyrate; NC, negative control; PC, positive control.

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FIGURE 2

Immunoblot analysis of occludin protein abundance in the jejunal (A) and colonic (B) tissue of weaned piglets fed corn-soybean meal without antibiotics (NC treatment), corn-soybean meal with reduced antibiotics and encapsulated sodium butyrate (ASB treatment), or corn-soybean meal with antibiotics (PC treatment) for 28 d. Representative Western blots are shown. Values are means ± SEMs, n = 6. Labeled means without a common letter differ, P < 0.05. ASB, reduced antibiotics + sodium butyrate; NC, negative control; PC, positive control.

Antioxidant variables.

The serum content of superoxide dismutase in the ASB piglets was significantly higher compared with that in the PC piglets (P < 0.05), whereas the value for the NC piglets was at an intermediate level. Activities of glutathione peroxidase in the intestinal mucosa were increased in the ASB piglets compared with the NC and PC piglets (P < 0.05). The ASB piglets had decreased malondialdehyde in the serum compared with the NC piglets (P < 0.05), whereas the value for the PC piglets was intermediate to that of the NC and ASB treatments. The malondialdehyde concentration in the intestinal mucosa in the ASB piglets was comparable to that in the PC piglets, both of which were significantly lower than in the NC piglets (P < 0.05) (Table 3).

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TABLE 3

Antioxidant variables in serum and intestinal mucosa of weaned piglets fed corn-soybean meal without antibiotics (NC), corn-soybean meal with reduced antibiotics and sodium butyrate (ASB), or corn-soybean meal with antibiotics (PC) for 28 d¹

Immune indexes.

Serum IFN-γ was higher in the ASB piglets compared with the NC piglets (P < 0.05), whereas the value for the PC piglets was at an intermediate level. The PC piglets had a lower concentration of TNF-α in serum than did the NC piglets (P < 0.05), whereas the value for the ASB piglets was intermediate to those for the NC and PC piglets. In the intestinal mucosa, TNF-α concentrations in the ASB and PC piglets were lower than in the NC piglets (P < 0.05), although no significant difference was observed between the ASB and PC piglets (Table 4).

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TABLE 4

Immune indexes in serum and intestinal mucosa of weaned piglets fed corn-soybean meal without antibiotics (NC), corn-soybean meal with reduced antibiotics and sodium butyrate (ASB), or corn-soybean meal with antibiotics (PC) for 28 d¹

Bacterial composition and diversity.

The ileal lumen harbored decreased bacterial diversity with the ASB and PC treatments, as indicated by the decreased Shannon index and increased Simpson index, whereas the bacterial community in the colonic lumen became more diverse (Table 5). A noticeable increase was observed in Bacteroidetes by the ASB and PC treatments, from 0.7% in the NC piglets to 3.4% in the ASB piglets and 10.3% in the PC piglets (data not shown). The majority of classifiable sequences belonged to Lactobacillaceae (63.8%), Streptococcaceae (18.3%), Pasteurellaceae (7.2%), and Enterobacteriaceae (6.3%) in the ileal lumen, whereas the colonic lumen was dominated by Lactobacillaceae (64.6%), Streptococcaceae (10.1%), Ruminococcaceae (7.7%), Lachnospiraceae (6.0%), and Neisseriaceae (4.0%) in the NC piglets (Figure 3). The decreases in Lactobacillaceae abundance with exposure to the ASB and PC were striking: from 63.8% in the ileal lumen of the NC piglets to 6.8% with the ASB treatment and 8.7% with the PC treatment; the values for the colonic lumen were 64.6%, 44.4%, and 4.4%, respectively. Nonabundant Clostridiaceae became dominant in the ileal lumen, with large increases from 0.3% in the NC piglets to 83.2% in the ASB piglets and 35.0% in the PC piglets (Figure 3). In addition, noticeable increases in Clostridiaceae abundance were also observed in the colonic lumen with the ASB (1.6%) and PC (10.9%) treatments compared with the NC piglets (0.3%) (Figure 3). In addition, certain bacterial families decreased with the ASB and PC treatments, such as Pasteurellaceae and Enterobacteriaceae in the ileal lumen, whereas Ruminococcaceae and Lachnospiraceae were increased in the colonic lumen with the ASB and PC treatments (Figure 3).

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TABLE 5

Bacterial community diversity in the ileal and colonic chyme of weaned piglets fed corn-soybean meal without antibiotics (NC), corn-soybean meal with reduced antibiotics and sodium butyrate (ASB), or corn-soybean meal with antibiotics (PC) for 28 d¹

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FIGURE 3

Family-level distribution of luminal bacteria in the ileal and colonic chyme of weaned piglets fed corn-soybean meal without antibiotics (NC treatment), corn-soybean meal with reduced antibiotics and encapsulated sodium butyrate (ASB treatment), or corn-soybean meal with antibiotics (PC treatment) for 28 d. ASB, reduced antibiotics + sodium butyrate; NC, negative control; PC, positive control.

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Discussion

Kitasamycin is an antibiotic growth promoter that exhibits activity mainly against gram-positive micro-organisms (26), whereas colistin sulfate is a decapeptide with antibacterial activity mainly against gram-negative micro-organisms (27). In the present study, the combined use of SB and reduced antibiotics was introduced given the inconsistent effects of SB fed alone and the still widespread use of antibiotics in many countries. Alterations with regard to intestinal barrier function may explain the improved performance and decreased diarrhea incidence by the ASB and PC treatments.

We first validated that the ASB treatment improved performance in weaned piglets with effects comparable to those for the PC treatment. This observation is similar to that in a previous study (17), which reported that a combination of acidifiers and avilamycin gave better performance-promoting effects than did acidifiers or avilamycin fed alone in piglet diets, although the mechanisms were not well determined. The decreased diarrhea incidences in the ASB and PC piglets were at least partly due to the redefinition of the bacterial communities by the antibiotics. In addition, SB was speculated to influence the intestinal microflora (14) and bacteria virulence (5) in weaned piglets. Our experiment revealed an effective strategy to control postweaning problems with reduced use of antibiotics.

The decreased digestion and absorption of nutrients, fluids, and electrolytes due to villous atrophy and crypt hypertrophy as a result of early weaning may contribute to diarrhea (28). In the present study, the improved villus height in the duodenum and ileum in the PC piglets may partly explain the improved performance and lower diarrhea incidence by the PC treatment. An increase in crypt depth can be used as a predictor of increased crypt cell production rate and overall stimulation of cell turnover in the small intestine, which are generally associated with reduced digestive and absorptive capacity (29). The decreased crypt depth in the duodenum, jejunum, and ileum in the ASB piglets was in agreement with the improved performance.

Increased intestinal secretion or losses of fluid and electrolytes may be considered as a final common pathway of diarrhea production, and many factors interact in subtle ways in the development of diarrhea (28). Increased intestinal permeability is characterized by downregulation of fluid and electrolyte absorption, which leads to fluid and electrolyte accumulation in the bowel (24). In our study, the decreased small intestinal permeability by the ASB treatment may help to confer a decrease in diarrhea incidence. The intercellular tight junction protein complexes play an important role in the mucosal barrier against translocation of intraluminal toxins, antigens, and enteric flora from the lumen into subepithelial tissues and systemic blood circulation (30). Note that the higher expression of occludin in jejunal tissue by the ASB treatment was consistent with the decreased small intestinal permeability observed. The ASB and PC treatments might also decrease paracellular permeability in the hindgut as indicated by the increased expression of occludin in colonic tissue. Previous studies showed that absorption in the large intestine is also involved in the pathophysiology of diarrhea, and a loss of fluid and electrolytes in the small intestine will only cause diarrhea when remedial reabsorption by the large intestine partly or completely fails (31). Collectively, the ASB and PC treatments decreased diarrhea incidence by modulating permeability in both the small intestine and hindgut.

Mucosal oxidative stress has been shown to be sufficient to induce diarrhea and to play an important role during the progression of weaning diarrhea (32). It is likely that the ASB triggered a localized form of attenuation in antioxidant status that was not reflected in the extraintestinal environment due to minor changes in serum antioxidant variables. This discrepancy could be explained by the negligible finding of butyrate in the peripheral blood as a result of rapid metabolism in the gut wall and/or in the liver (5). Glutathione peroxidase is implicated in the protection of gastrointestinal mucosal cells against damage from various insults (32). Malondialdehyde concentrations in tissues and blood are generally used as biomarkers of endogenous lipid peroxidation and free radical-induced damage (33). The increase in glutathione peroxidase in the intestinal mucosa could mainly be attributed to SB, as suggested by the absence of an increase in intestinal mucosal glutathione peroxidase content in the PC piglets, whereas antibiotics are mainly responsible for malondialdehyde decreases in intestinal mucosa. Decreased oxidative stress may lead to reduced damage to the intestinal mucosal barrier, which subsequently leads to reduced intestinal permeability.

Some cytokines are thought to be critical in the predisposition to and exacerbation of some gastrointestinal dysfunctions (34). TNF-α is a central mediator of intestinal inflammatory diseases (34) and has been shown to play a role in the control of intestinal permeability (35). T cell–derived TNF-α inhibits phosphorylation of the myosin light-chain mediated by the myosin light-chain kinase, leading to alternatively disrupted tight junction stability and dysregulation of occludin expression (36). Therefore, the increased occludin expression in the ASB and PC piglets could be partly attributed to decreased TNF-α in the intestinal mucosa. Moreover, TNF-α mediates mast cell degranulation along with some other mediators. TNF-α is, in turn, rapidly released by mast cells after degranulation as are some other inflammatory mediators (24). Mast cell degranulation and the consequent histamine and prostaglandin release profoundly influence intestinal epithelial barrier function (28). The 6-fold decrease in intestinal mucosal TNF-α may indicate an amelioration of mast cell degranulation in the ASB and PC piglets. More research is needed to explain the unexpected increase in serum IFN-γ, whereas the IFN-γ concentration in the intestinal mucosa was not affected by the ASB and PC treatments.

An understanding of how the ASB and PC treatments affect the intestinal bacterial communities may help to reveal the linkages between their changes and improvements in performance and diarrhea incidence. Higher bacterial diversity may be associated with an improved competitive defense against pathogens and decreased vulnerability to environmental perturbations, such as shifts in gut microbial communities, changes in piglet diet, or overt pathogenic challenge (37). Shiga toxin–producing Escherichia coli elicits fluid and electrolyte accumulation in the bowel by disrupting the usual balance of intestinal absorption and secretion toward net secretion (38). The increased bacterial diversity in the colonic lumen and decreased Enterobacteriaceae (mainly Shigella) in both the ileal and colonic lumen may help to decrease diarrhea incidence in ASB and PC piglets.

The colonic lumen harbored a higher number of bacteria associated with degrading complex carbohydrates than did the ileal lumen, which was consistent with previous observations (23, 39). The intestinal bacterial communities may participate in modulation of piglet performance due to the energy derived from hindgut bacterial fermentation. Bacteroidetes is well known for its role in polysaccharide degradation (40). Ruminococcaceae has been consistently found in the hindgut of pigs, and an increase in Ruminococcaceae may improve feed conversion in piglets due to its cellulose-degrading capacity (39). Lachnospiraceae is known to produce an array of bacteriocins and butyrate, and many isolates have shown promise as probiotics (41). In our study, the increases in the relative abundance of Bacteroidetes, Ruminococcaceae, and Lachnospiraceae in the colonic lumen of ASB and PC piglets could help the host obtain more energy from complex polysaccharides that are resistant to the action of digestive enzymes. Whereas the decrease in Lactobacillaceae populations in the ileal lumen may reduce energy losses, previous studies showed that Lactobacilli is the main contributor to microbial bile salt hydrolase activity in the small intestine, which results in impaired lipid absorption by the host animal and consequent dietary energy losses (42). In addition, we propose a hypothesis that the ASB and PC treatments improved the ratio of weight gain to feed via inhibition of the normal microbiota, leading to increased nutrient utilization and a reduction in the maintenance costs of the gastrointestinal system (43). By contrast, an undesirable effect may be the substantially increased Clostridiaceae population in ASB and PC piglets. The persistence of Clostridiaceae in the whole intestinal bacterial communities in ASB and PC piglets and their effect on piglet growth cannot be explained because the possible benefits from intestinal colonization by nonpathogenic Clostridiaceae have not been explored (44).

In summary, the present study showed that the ASB significantly improved weight gain and reduced diarrhea incidence in weaned piglets (P < 0.05). These changes were accompanied by decreased small intestinal permeability, increased intestinal occludin expression, and improvements in some antioxidant variables and immune indexes, as well as considerable alterations in intestinal bacterial communities.

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Acknowledgments

We thank Katrina Mohror, South Dakota State University, for excellent assistance in editing this manuscript. C Huang, PS, and XM designed and conducted the research; C Huang, PF, C Hou, and XM analyzed the data; C Huang and XM wrote the manuscript; PT critically reviewed the manuscript; and XM had primary responsibility for final content. All authors read and approved the final manuscript.

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Footnotes

↵1 Supported in part by the National Basic Research Program of China (973 Program, 2013CB117301), the National Natural Science Foundation of China (31528018, 31272448, and 31472101), the National Department Public Benefit Research Foundation (201403047), and Beijing Nova program (xx2013055).
↵2 Author disclosures: C Huang, P Song, P Fan, C Hou, P Thacker, and X Ma, no conflicts of interest.
↵3 Supplemental Table 1 is available from the “Online Supporting Material” link in the online posting of the article and from the same link in the online table of contents at http://jn.nutrition.org.
↵7 These authors contributed equally to this work.
↵8 Abbreviations used: ASB, reduced antibiotics + sodium butyrate; NC, negative control; PC, positive control; SB, sodium butyrate.

Manuscript received: June 3, 2015.
Initial review completed: June 23, 2015.
Revision accepted: September 29, 2015.

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Sodium butyrate in piglet feed reduces diarrhoea

http://www.pigprogress.net/Piglets/Articles/2014/1/Sodium-butyrate-in-piglet-feed-has-positive-effect-1437516W/

Sodium butyrate in piglet feed reduces diarrhoea

One of the benefits of adding sodium butyrate in the feed of piglets is the reduction of post weaning diarrhoea, which is one of the most frequent explanations for the morbidity and mortality observed at this age.

In a recent paper (J. Anim. Sci. 2012), Ma et al. tried to understand by which mechanism sodium butyrate can decrease the incidence of diarrhoea. In an in-vitro experiment, cultured cells were scratched to induce wound, then were treated with sodium butyrate. The publication shows that addition of sodium butyrate accelerated the process of wound healing, confirming a protecting effect of butyrate on the intestinal mucosa. When compared to a control group, the authors observed that mRNA expression of the intestinal mucosal tight junction proteins occludin and zonula occluden protein-1 was increased.

Besides, in the butyrate-treated cells, the levels of superoxide dismutase and glutathione peroxidase, two of the main antioxidant enzymes, as well as glutathione, one of the nonenzymatic antioxidant components, were significantly higher than in the control group. Also, malondialdehyde, a marker of free radical mediated lipid peroxidation injury, was present at lower levels in the cells treated with sodium butyrate. These results confirm previous research suggesting that one of the benefits of sodium butyrate in the intestine is to maintain the function of the intestinal barrier.

Together with the report of Peng et al. (2009), this study also indicates that adding sodium butyrate in feed can probably play an important role in maintaining the intestinal tight junctions, which are essential to preserve the gut integrity.

Butyrate promotes the recovering of intestinal wound healing

http://www.feedadditive.com/docs/jas-90-Supplement_4-266.pdf

Article (PDF Available) in Journal of Animal Science 90(Supplement 4):266-268 · January 2013 with 198 Reads

X. Ma,*2 P. X. Fan,*L. S. Li,* S. Y. Qiao,* G. L. Zhang,† D. F. Li*
*State Key Lab of Animal Nutrition, China Agricultural University, Beijing, 100193, China; and †Department of Animal
Science, Oklahoma State University, Stillwater, Oklahoma, 74078

Abstract

Postweaning diarrhea is one of the most common causes of morbidity and mortality in weanling piglets. Feeding sodium butyrate to weanling piglets decreased the incidence of diarrhea, but the mechanism has not been fully elucidated. The present study was to evaluate the effect of sodium butyrate on diarrhea in relation to wound healing of intestinal barrier using IPEC-J2 cell model. Cultured cells were scratched to induce wound and then were treated with 4 mM sodium butyrate. The results showed that supplementation of the cells with sodium butyrate significantly promoted the process of wound healing, indicating the protective effects of butyrate on the intestinal mucosa. Butyrate treatment enhanced mRNA expression of the intestinal mucosal tight junction proteins occludin and zonula occluden protein-1 (P < 0.05), which suggested that the promotion of wound healing by butyrate is related to the maintenance of the function of the intestinal barrier. In addition, in the butyrate-treated group, intestinal total superoxide dismutase and glutathione peroxidase (P < 0.05), two of the main antioxidant enzymes, as well as glutathione (P < 0.05), one of the nonenzymatic antioxidant components, were enhanced whereas the malondialdehyde level, a marker of free radical mediated lipid peroxidation injury, was decreased (P < 0.05) compared with the control group. Collectively, these results indicate that dietary sodium butyrate might, at least partly, play an important role in recovering the intestinal tight junctions having a positive effect on maintaining the gut integrity.

Butyrate promotes the recovering of intestinal wound healing through its positive effect on the tight junctions (PDF Download Available).

Oral Butyrate in Treatment of Congenital Chloride Diarrhea

http://www.nature.com/ajg/journal/v103/n1/full/ajg20085050a.html

https://ojrd.biomedcentral.com/articles/10.1186/1750-1172-8-194

The American Journal of Gastroenterology 103, 252-254 (January 2008) | doi:10.1111/j.1572-0241.2007.01562_14.x

Roberto Berni CananiEmail author, Gianluca Terrin, Ausilia Elce, Vincenza Pezzella, Peter Heinz-Erian, Annalisa Pedrolli, Chiara Centenari, Felice Amato, Rossella Tomaiuolo, Antonio Calignano, Riccardo Troncone and Giuseppe Castaldo

Received: 4 June 2013

Accepted: 10 December 2013

Published: 19 December 2013

Abstract

Background

Congenital chloride diarrhea (CLD) is an autosomal recessive disorder characterized by life-long, severe diarrhea with intestinal Cl^– malabsorption. It results from a reduced activity of the down regulated in adenoma exchanger (DRA), due to mutations in the solute carrier family 26, member 3 (SLC26A3) gene. Currently available therapies are not able to limit the severity of diarrhea in CLD. Conflicting results have been reported on the therapeutic efficacy of oral butyrate.

Methods

We investigated the effect of oral butyrate (100 mg/kg/day) in seven CLD children with different SLC26A3 genotypes. Nasal epithelial cells were obtained to assess the effect of butyrate on the expression of the two main Cl^– transporters: DRA and putative anion transporter-1 (PAT-1).

Results

A variable clinical response to butyrate was observed regarding the stool pattern and fecal ion loss. The best response was observed in subjects with missense and deletion mutations. Variable response to butyrate was also observed on SLC26A3 (DRA) and SLC26A6 (PAT1) gene expression in nasal epithelial cells of CLD patients.

Conclusions

We demonstrate a genotype-dependency for butyrate therapeutic efficacy in CLD. The effect of butyrate is related in part on a different modulation of the expression of the two main apical membrane Cl^– exchangers of epithelial cells, members of the SLC26 anion family.

Trial registration

Australian New Zealand Clinical trial Registry ACTRN12613000450718.

Keywords

SLC26A3 SLC26A6 DRA Mutations Short chain fatty acids Pediatrics Children

Introduction

Congenital chloride diarrhea (CLD-OMIM 214700) is an autosomal recessive disorder characterized by life-long, severe diarrhea with intestinal Cl^– malabsorption. It results from a reduced activity of the down-regulated in adenoma exchanger (DRA), due to mutations in the solute carrier family 26, member 3 (SLC26A3) gene [1, 2, 3]. In humans, SLC26A3 encodes for a 764-amino acid protein and is located on chromosome 7 in a head-to-tail arrangement with SLC26A4 (pendrin), indicating ancient gene duplication [1, 2, 3]. Over 50 different SLC26A3 mutations, including founder mutations in Finland, Poland, Saudi Arabia and Kuwait populations, have been identified in CLD patients [4]. Such mutations are heterogeneous (mainly missense, insertion/deletion, nonsense and splicing), spread all over the SLC26A3 gene, and have a different impact on the expression and the activity of DRA [5, 6]. Although no genotype-phenotype correlation attributed to different SLC26A3 mutations has been noted, the overall clinical picture and outcome of CLD patients range from severe neonatal disease, with life threatening hypoelectrolytemia and dehydration, to a relatively mild chronic form, which may remain undiagnosed for long time [7, 8, 9, 10]. Increasing evidences suggest the importance of early diagnosis and treatment, and of other undefined environmental factors, as modulators of the prognosis and clinical severity of CLD [7, 8, 9, 10, 11]. In patients with CLD, supplementation therapy with a combination of Cl^– salts (NaCl and KCl) is essential in preventing episodes of dehydration that could result in mental and psychomotor impairment, and in chronic contraction of the intravascular space that could lead to renal dysfunction and gout [7, 11]. Unfortunately, this therapy is unable to limit the severity of diarrhea, as for other therapeutic approaches, such as omeprazole, acetazolamide and cholestyramine [12, 13, 14, 15].

The role of the amylase-resistant starch has been increasingly recognized for the management of diarrheal diseases [16, 17]. Dietary fibres are fermented by gut microbiota into short-chain fatty acids (SCFAs), including acetate, propionate, and butyrate [18, 19, 20]. Butyrate exerts a powerful pro-absorptive stimulus on intestinal NaCl transport and an anti-secretory effect on Cl^– secretion [2, 19, 20]. In a child affected by CLD, we demonstrated the therapeutic efficacy of oral butyrate, showing a progressive reduction to normal values in the number of bowel movements and stool volume, an improvement in stool consistency, and a reduction of fecal incontinence episodes. A reduction of fecal electrolyte and persistency of normal serum electrolyte concentrations were also demonstrated [18]. Subsequently, Wedenoja et al. evidenced different results in five CLD patients homozygous for a frameshift mutation [21]. These findings suggest that the variable response to butyrate could depend, at least in part, on different SLC26A3 genotype.

The two main transporters involved in Cl^– absorption at intestinal level are DRA and putative anion transporter 1 (PAT-1), encoded by SLC26A6 gene [22]. It has been demonstrated that butyrate is able to regulate DRA gene expression in intestinal epithelial cells [22], but the possible effect of butyrate on SLC26A3 and SLC26A6 expression in CLD patients is still unknown.

In this study we evaluated the therapeutic effect of butyrate in children affected by CLD with different SLC26A3 genotype through a clinical trial and an in vitro investigation.

Methods

Clinical trial

Ethics

The study protocol was approved by the Ethics Committee of the University of Naples Federico II (n. 3469/07) and by the Italian Agency for Drugs (AIFA), and it was registered in the Australian New Zealand Clinical trial Registry (ACTRN12613000450718). All authors had access to the study data and had reviewed and approved the final manuscript.

Population

The Pediatric Gastroenterology Unit at the University of Naples “Federico II” is an International Reference Center for patients with CLD, and served as Coordinator Center of this study. From 2005 to 2010, 35 cases of suspected CLD were referred to the Center, and a definitive diagnosis of CLD was obtained in 25 patients with different ethnicity. Demographic, clinical and laboratory data of all CLD patients were collected in a dedicated data-base. All subjects included in this database were invited to participate in the study with the aim to evaluate at least one patient for each of main mutations (missense, deletion, nonsense and splicing). The physicians of all Centers received by E mail the protocol and any request of information was satisfied by a direct contact with the Coordinator Center. Exclusion criteria were: severe dehydration; concomitant presence of infections; concomitant other chronic diseases; renal insufficiency; use of probiotics/prebiotics, non-steroideal anti-inflammatory drugs (NSAIDs), or antibiotics in the last 4 weeks.

Genotype definition of children enrolled into the clinical trial

Molecular analysis was performed in the laboratory of CEINGE-Biotecnologie Avanzate , the reference Center for molecular diagnosis of inherited diseases in Campania region (about 6 million of inhabitants), located in southern Italy. DNA was extracted from an EDTA blood sample with the Nucleon BACC2 kit (Amersham Biosciences, USA). The primers used are reported elsewhere [23]. The touchdown PCR protocol that enables co-amplification of all exons under the same PCR conditions is available on request. Sequencing analysis was carried out on both strands with an automated procedure (3100 Genetic Analyzer, Applied Biosystem). All PCR fragments were sequenced with the primers used for PCR. Furthermore, we used the Expand Long Template PCR System (Roche, Germany) to verify deletion extension in patient bearing c.2008-151_2061 + 1546 del mutation. We used the forward primer of exon 17 and the reverse primer of exon 19 [23], both known to be intact in exon-specific assays. The expected fragment is about 6300 bp. The PCR conditions are available on request.

Intervention

We used a commercially available sodium butyrate formulation (SOBUTIR®, Promefarm, Milan, Italy). Butyrate was administered orally at 100 mg/kg/day, divided in 2 doses, with a maximal dosage of 4 g/day, for 1 week. Number of tablets of sodium butyrate (1 gr/tablet) consumed by the child during the trial were reported in a specific form by the parents. A good compliance was considered the intake of at least 80% of the prescribed doses. Parents of the enrolled children were advised to avoid co-administration of other treatments, including anti-diarrheal drugs, antibiotics, prebiotics or probiotics during the trial. Children continued their normal diet during the study period. Throughout the study period, all CLD subjects were examined as outpatients and they had free access to the services of referred hospitals.

Trial design and data collection

This was an open trial on subjects with a confirmed diagnosis of CLD. The purposes and the modalities of the study were illustrated to the parents during the first visit (Visit 1), and a written informed consent was obtained from parents or tutors of each enrolled patient. Baseline clinical and laboratory data were collected during the week before butyrate treatment, and were considered representative of the usual pattern of each enrolled patient. In particular, the parents of each patient were instructed to record daily in a specific clinical chart: number of bowel movements, fecal volume, stool consistency (using a scoring system: normal = 0, loose = 1, semi-liquid = 2, liquid =3), and presence of incontinence. At the end of the baseline week of observation, the clinical chart of each child was collected and the patient was re-evaluated (Visit 2). A full clinical evaluation was performed and serum, fecal and urinary ion concentrations were determined, together with serum pH, renin and aldosterone values, as previously described [18]. Fecal electrolyte concentrations were measured on stool samples collected daily during the last 3 days of observational period. The parents of each enrolled subject received a written prescription about the modalities of butyrate administration for 1 week associated with oral NaCl/KCl supplementation, as previously described [18]. In the last 3 days of treatment we collected daily a fecal sample to study the effect of butyrate on fecal Na⁺ and Cl^– concentration. At the end of treatment with butyrate, the patients were re-evaluated (Visit 3), and serum, urinary and fecal electrolyte concentrations were measured again. Primary outcome of the study was the reduction of Cl^– and Na⁺ fecal losses induced by butyrate therapy.

In vitro study

Ex-vivo epithelial cell collection by nasal brushing

Nasal brushing was performed using an endo-brush at the level of the inferior turbinate without anesthetic procedures.

Epithelial cell culture

The sample obtained from each nostril was immediately conserved in a 15 mL tube containing 2.5 mL of RPMI 1640 medium, complemented with 3% ampicillin. Cells were placed on Eppendorf Thermomixer, in agitation at 300 rpm for one hour. Once removed the brush from every sample, cells were centrifuged at 931 Xg (2000 rpm) for 20 minutes, supernatant was discarded and cell pellet was treated with 150 μL of Trypsin-Versene (EDTA) solution (Lonza, SW) for 4 minutes at 37°C, in order to disaggregate possible cell clusters. Trypsin was inactivated by adding 3 mL of serum-free Bronchial Epithelial cell Growth Medium (BEGM Clonetics, USA). After centrifugation at 2000 rpm for 10 minutes, cells were placed in CELL + T 25 flasks (Sarstedt Ltd, UK). At confluence of 60%, cells were passed in new flasks after count using Invitrogen Cell Countess (Invitrogen, UK). Trypan blue exclusion test was used in order to establish total viable cells number and percentage of viability.

Effect of butyrate on epithelial cells

At the confluence of >80%, cells were treated with 5 mM of sodium butyrate for 24 hours. RNA was extracted with TRIZol method (Invitrogen, UK). Total RNA amount was quantified with Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, UK). One microgram of total RNA was retro-transcribed with Quantitect Reverse Transcription kit (Qiagen, Germany), cDNA was diluted for downstream applications like quantitative real-time PCR analysis. Expression levels of either SLC26A3 and SLC26A6 from treated and untreated cells were measured by semi-quantitative real-time PCR on LightCycler 480 Real-Time PCR System (Roche, Germany) with Taqman probe chemistry (experiments were performed in replicate using also SYbr Green chemistry). Results were normalized for housekeeping glyceraldheyde 3-phosphate dehydrogenase (GAPD) gene. Levels of SLC26A3 and SLC26A6 expression before and after treatment were compared in order to establish changes determined by butyrate exposure. Calculation of relative gene expression was performed according to Pfaffl et al. [24], and the expression was calculated using the formula of relative gene expression with DDCt method (where DDCt corresponds to the increase in the threshold cycle of the target gene with respect to the increase in the threshold cycle of the housekeeping gene). Hence, the final quantification value for each condition indicated the relative change of gene expression in the target gene compared to the control, for each sample.

Statistical analysis

The Kolmogorov-Smirnov test was used to determine whether variables were normally distributed. The chi-square test was applied for categorical variables, and for continuous variables, differences between groups were analyzed by Mann- Whitney U test, and Kruskal-Wallis H test. A multivariate analysis was performed to evaluate if the effects of butyrate may depend by clinical or genetic factors. All analyses were conducted on an intention-to-treat (ITT) basis. Statistical analysis was carried out by the SPSS software for Windows 16.0 and by StatDirect 1.7.

Results

Clinical trial

Enrollment of patients was performed from January 2010 to December 2012. Out of 25 eligible children with CLD, 18 subjects were excluded (8 not meeting inclusion criteria, 10 declined to participate because different Ethics Committee regulatory procedures and logistic difficulties), 7 were enrolled in the study (Figure 1). All patients showed a typical clinical picture of CLD with early onset diarrhea. Three subjects received a late diagnosis.

Figure 1

**Flow diagram of the study (according to CONSORT guidelines).**

SLC26A3 genotype was defined for each subjects. Patients 1, 3 and 7 presented previously unreported mutations. Patients 1 and 2 had missense mutations and the protein was expressed on cell membrane. Patient 3 was homozygous for a large deletion that caused the synthesis of a truncated protein and some amount of the protein was present at membrane level. Patients 4 to 6 were homozygous for nonsense mutations with no protein expression at membrane level. Patient 7 was homozygous for a splicing mutation that causes the synthesis of an aberrant mRNA, with no protein expression at membrane level. The genotype and main demographic features of study subjects are reported in Table 1.

Table 1

Main demographic characteristics and genotype of patients with congenital chloride diarrhea

Patient	Age at diagnosis	Age the enrollment	Sex	Ethnic origin	Body weight (kg)	SLC26A3 genotype	Type of mutation
1	6.5 y	16 y	M	Caucasian	70	c.1484A > C*	Missense
1	6.5 y	16 y	M	Caucasian	70	c.1640C > A*	Missense
2	4 m	3 y	M	Caucasian	22	c.386C > T	Missense
2	4 m	3 y	M	Caucasian	22	c.386C > T	Missense
3	6 y	12 y	M	Caucasian	32	c.1008-151_2061 + 1546del*	Deletion
3	6 y	12 y	M	Caucasian	32	c.1008-151_2061 + 1546del*	Deletion
4	3 m	18 y	F	Caucasian	58	c.2132 T > G	Nonsense
4	3 m	18 y	F	Caucasian	58	c.2132 T > G	Nonsense
5	1 m	18 y	M	African	55	c.559G > T	Nonsense
5	1 m	18 y	M	African	55	c.559G > T	Nonsense
6	1 m	15 y	M	African	57	c.559G > T	Nonsense
6	1 m	15 y	M	African	57	c.559G > T	Nonsense
7	10 m	1.5 y	M	Caucasian	10	c.1408-G > C*	Splicing
7	10 m	1.5 y	M	Caucasian	10	c.1408-G > C*	Splicing

*Novel mutations.

All patients were evaluated in stable clinical conditions. Overall, butyrate therapy induced a reduction of Cl^– (136 mmol/l, IQR 13 vs 120 mmol/l, IQR 42; p < 0.001) and Na⁺ (78 mmol/l, IQR 29 vs 50 mmol/l, IQR 49; p = 0.002) fecal losses in CLD patients, but a variable response was observed in children with different SLC26A3 genotype. The more evident reduction of fecal ion losses was observed in patients with missense and deletion mutations (Figure 2).

Figure 2

**Fecal sodium (a) and chloride concentration (b) in children with congenital chloride diarrhea treated with butyrate.** Box and bar represent median and min-max range, respectively.

A variable clinical response was also observed on stool pattern in CLD patients with different mutations. Clinical response (defined by a concomitant significant reduction of ≥2 variables) was observed in patients with missense and deletion mutations (Table 2). A reduction of incontinence episodes was observed in patients 1, 2 and 3. The effect of butyrate on stool pattern became evident within the first 48 hours and remained stable during the following days of treatment.

Table 2

Effects of butyrate on daily stool pattern in patients with congenital chloride diarrhea

Patient	Bowel movements			Stool volume (ml)			Stool consistency (score)
	Basal	On Butyrate	p	Basal	On Butyrate	p	Basal	On Butyrate	p
1	4 (3)	2 (1)	0.003	2100 (500)	1600 (450)	0.047	3 (2)	2 (1)	0.379
1	(3–6)	(2–3)	0.003	(1800–2300)	(1400–1850)	0.047	(1–3)	(2–2)	0.379
2	4 (2)	3 (1)	0.147	1400 (450)	1200 (300)	0.046	3 (0)	2 (1)	0.023
2	(3–5)	(3–4)	0.147	(1200–1650)	(1000–1300)	0.046	(3–3)	(2–3)	0.023
3	3 (2)	2 (0)	0.021	1500 (200)	900 (400)	0.015	3 (0)	2 (1)	0.107
3	(2–4)	(2–2)	0.021	(1400–1600)	(800–1200)	0.015	(3–3)	(2–3)	0.107
4	6 (2)	6 (1)	0.892	2000 (400)	1500 (200)	0.053	3 (0)	2 (1)	0.037
4	(5–7)	(6–7)	0.892	(1800–2200)	(1500–1700)	0.053	(3–3)	(2–3)	0.037
5	2 (2–3)	2 (1)	0.263	1200 (200)	1100 (300)	0.261	2 (1)	3 (1)	0.606
5	(2–3)	(1–2)	0.263	(1100–1300)	(900–1200)	0.261	(2–3)	(2–3)	0.606
6	2 (2)	1 (0)	0.238	900 (200)	800 (400)	0.435	2 (1)	2 (1)	0.872
6	(0–2)	(1–1)	0.238	(800–1000)	(700–1100)	0.435	(1–2)	(1–2)	0.872
7	3 (1)	4 (1)	0.299	1000 (200)	900 (200)	0.289	2 (1)	2 (0)	0.254
7	(3–4)	(3–4)	0.299	(900–1100)	(800–1000)	0.289	(1–2)	(2–2)	0.254

Note. Data are expressed as median (interquartile range). Stool consistency score: normal = 0, loose = 1; semi-liquid = 2; liquid = 3.

The multivariate analysis revealed that only the genotype significantly (p = 0.008) influence the response to butyrate treatment (i.e. missense and deletion mutations that allow the expression of DRA at membrane level). The study procedures and oral butyrate treatment were well accepted by the patients. Serum and urinary electrolyte concentrations, and serum pH, renin and aldosterone levels remained stable within normal ranges during the study period. In Table 3 were summarized the main results of butyrate therapy according to the type of mutations observed in CLD patients.

Table 3

Effects of butyrate on fecal electrolytes loss and on stool pattern, according to variation of genotype in patients with congenital chloride diarrhea

	Genotype		Response to butyrate
Patient	SLC26A3 genotype	Type of mutation	Fecal Cl^–loss	Stool Pattern*
1	c.1484A > C*	Missense	Reduced	Improved
1	c.1640C > A*	Missense	Reduced	Improved
2	c.386C > T	Missense	Reduced	Improved
2	c.386C > T	Missense	Reduced	Improved
3	c.1008-151_2061 + 1546del*	Deletion	Reduced	Improved
3	c.1008-151_2061 + 1546del*	Deletion	Reduced	Improved
4	c.2132 T > G	Nonsense	Unchanged	Unchanged
4	c.2132 T > G	Nonsense	Unchanged	Unchanged
5	c.559G > T	Nonsense	Unchanged	Unchanged
5	c.559G > T	Nonsense	Unchanged	Unchanged
6	c.559G > T	Nonsense	Unchanged	Unchanged
6	c.559G > T	Nonsense	Unchanged	Unchanged
7	c.1408-G > C*	Splicing	Unchanged	Unchanged
7	c.1408-G > C*	Splicing	Unchanged	Unchanged

*defined as a significant reduction of a least two out of three parameters (bowel movements, stool volume, stool consistency).

In vitro study

A variable response to butyrate was observed on SLC26A3 mRNA expression in epithelial nasal cell culture. Butyrate was able to increase the expression of SLC26A3 gene in 5 out of 7 CLD patients (i.e., patients 1, 2, 3, 4 e 7). In two cases (i.e., patients 5 and 6, both homozygous for the G187X nonsense mutation) butyrate significantly inhibited SLC26A3 expression (Figure 3). The SLC26A6 mRNA expression resulted significantly increased by butyrate in epithelial nasal cells from 5 out of 7 CLD patients, i.e., cases 2, 4, 5, 6 and 7, while it was significantly reduced in one case (i.e., patient 1) and remained unchanged in patient 3 (Figure 3).

Figure 3

*SLC26A3/DRA* **mRNA and** *SLC26A6/PAT-1* **mRNA expression in epithelial cells of children enrolled into the trial before and after** *in vitro* **stimulation with butyrate.** *p < 0.05 vs. basal.

Discussion

This is the first study exploring the efficacy of butyrate in a population of CLD subjects with different mutations in SLC26A3 gene. We confirm the efficacy of butyrate on intestinal ion transport in a subset of CLD patients and we demonstrate that the clinical effect of butyrate is at least in part dependent on genotype. A full response to butyrate (defined by a concomitant significant reduction of Na⁺ and Cl^– fecal losses and improvement in stool pattern) was observed only in patients with missense and deletion mutations. On the contrary, a partial response was observed in patients with nonsense or splicing mutations.

It has been previously demonstrated that butyrate is able to modulate transepithelial ion transport through at least 2 mechanisms: i) stimulation of Na⁺/H⁺ exchangers 2 (NHE2) and 3 (NHE3) activity; ii) inhibition of Cl^– secretion by limiting the action of the co-transporter Na-K-2Cl (encoded by NKCC1) on enterocyte baso-lateral membrane [19]. It has been shown that butyrate stimulates DRA expression in LS174T colonic cells [25], whenever data on a possible activity on PAT-1 gene expression were unavailable. Now we demonstrate that, in CLD patients, butyrate is able to modulate the expression of the two main intestinal Cl^– transporters: DRA and PAT1. The effect elicited on these two transporters could be considered a third potential mechanism of action, and it could be involved in the genotype-dependency of butyrate effect in CLD patients. The effect of butyrate on these two transporters seems to be different according to the type of SLC26A3 mutation. Patients with non-sense or splicing mutations showed a lower response in SLC26A3, but a more pronounced increase in SLC26A6 mRNA expression; whereas patients with missense or deletion mutation showed a more pronounced increase in SLC26A3 expression and a lower effects on SLC26A6 mRNA expression in nasal epithelial cell culture. Interestingly, a 3-fold up-regulation of PAT-1 expression was detected in DRA-knockout mice [26]. Altogether, these findings suggest that the up-regulation of PAT-1 in CLD patients may play a compensatory role in electrolyte homeostasis. DRA has been shown to be the major apical Cl^– absorbing isoform in the colon and ileum able to regulate a large amount of water daily [25]. Additionally, studies have shown that DRA-knockout mice show reduction in apical Cl^–/HCO₃ exchange activity and exhibit diarrheal phenotype with significant increased Cl^– and water stool content [25]. On the contrary, although PAT-1 is involved in Cl^– transport, unlike DRA it is not directly coupled to the water movements demonstrated by PAT-1-knockout mice showing a reduction in Cl^– absorption, but not exhibiting a diarrheal phenotype [26, 27]. Thus it is possible to hypothesize that the effect induced by butyrate on fecal ions loss is due at least in part by a regulatory action on PAT-1 expression.

The variable butyrate effect in CLD patients seems to be influenced by SLC26A3 genotype. However, the different diarrhea-reducing responses of butyrate observed among patients with similar mutations strongly suggests the existence of other still unidentified regulatory elements. The hypothesis that butyrate may display a large pattern of biochemical effects on intestinal ion channels with a strong inter-individual variability is also supported by a study on 5 CLD patients, all homozygous for a deletion mutation, showing a variable clinical response to this treatment [21].

We feel that the evidence of an improved clinical outcome by butyrate at least in a subset of CLD patients is of potential importance either for the therapeutic management and for the interpretation of the mechanisms that regulate ion absorption at intestinal level in this condition. The effects of endogenous production of butyrate elicited by different dietary habits could be able to influence the clinical picture in CLD patients with same genotype, as previously reported [19].

Conclusion

We demonstrate that butyrate may act efficiently on either fecal ion loss, and on the severity of diarrhea in a subset of CLD patients. The activity of butyrate seems to be more complex than expected, depending either on the profile of the SLC26A3 gene mutations, but also on other genes, and our study starts to make light on this network.

Author’s contribution

BCR, TG, CA, TR and CG designed the study and wrote the first draft of the paper. EA, TR and AF performed in vitro study. HEP, PA, CC, PV and TR cared for the patients and participated in the writing of the paper. TG performed data analysis. All authors contributed to the final version of the manuscript and approved the content of the paper.

http://www.feedadditive.com/docs/butyrate_diarrhea_Children.pdf

Efficacy of butyrate in the treatment of diarrhoea-predominant irritable bowel syndrome

http://www.sciencedirect.com/science/article/pii/S1594580408600066

E. Scarpellini. .E.C. Lauritano. .A. Lupascu. .C. Petruzzellis. .M.L. Novi. .D. Roccarina. .M. Gabrielli. .M. Serricchio. .G. Gasbarrini. .A. Gasbarrini. .

Abstract

Introduction

Short-chain fatty acids affect enterocyte metabolism and differentiation. Butyric acid in particular is already used in ulcerative rectal colitis, pouchitis and antibiotic-induced diarrhoea.

Aims

To assess the efficacy of butyrate in the treatment of irritable bowel syndrome (IBS).