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Influence of Long-Term Administration of Lactulose and Saccharomyces Boulardii on the Colonic Generation of Phenolic Compounds in Healthy Human Subjects

Department of Gastrointestinal Research, University Hospital Gasthuisberg, K.U. Leuven, Herestraat 49, B-3000 Leuven, Belgium

Address reprint requests to: Kristin Verbeke, Ph.D., Department of Gastrointestinal Research, University Hospital Leuven, Herestraat 49, 3000 Leuven, BELGIUM. E-mail: Kristin.Verbeke{at}uz.kuleuven.ac.be

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS ACKNOWLEDGMENTS REFERENCES

 
Objective: Proteins are degraded in the colon by bacterial fermentation into potentially toxic metabolites such as phenolic compounds. The aim of the present study was to investigate whether long-term administration of lactulose or Saccharomyces boulardii cells would result in a lower protein degradation. In addition, the influence of a long-term dietary intake on different gastrointestinal parameters was investigated.

Methods: The effect of long-term intervention of the substrates was evaluated in a randomized, cross-over study in 43 healthy volunteers. At the start of the study and at the end of each 4-week treatment period, urine was collected during 48 h in different fractions and faeces during 72 h. Breath test samples and blood samples were taken to study gastrointestinal parameters.

Results: No influence of long-term administration of both substrates was found on GE, OCTT and serum lipids. A significant decrease in small intestinal permeability was found after long-term dietary intervention with lactulose. Long-term administration of lactulose significantly decreased urinary p-cresol excretion, but did not lower fecal p-cresol excretion. No significant effects were observed after S. boulardii intake.

Conclusion: The results obtained in present study have indicated that colonic amino acid fermentation can be reduced by the administration of lactulose as a fermentable carbohydrate.

Key words: p-cresol, lactulose, S. boulardii, gastro-intestinal parameters

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS ACKNOWLEDGMENTS REFERENCES

 
In recent years, several studies have indicated that functional foods, i.e. pre-, pro- and synbiotics, may substantially contribute to the maintenance of health or prevention of disease [1–5]. Probiotics are live micro-organisms which, when administered in adequate amounts, confer a health benefit to the host [6], whereas prebiotics have been introduced as nutrients for a number of colonic bacteria. Prebiotics are defined as non-digestible food ingredients that beneficially affect the host by selectively stimulating growth, and/or activity, of one or a restricted number of bacteria in the colon [7]. A synbiotic is defined as a combination of a pre- and probiotic. As a consequence, an important aim of the administration of pre- and probiotics is to increase the saccharolytic activity and to decrease the proteolytic activity of the colonic microbiota. Proteolytic fermentation results in a number of end products which include phenolic compounds, amines and ammonia, all of which are potentially toxic [8,9]. Like ammonia, phenols have often been used as markers of colonic protein metabolism and as putative markers for cancer risk since they have been shown to be implicated in the pathogenesis of bladder and bowel cancers [8]. p-Cresol and phenol are unique bacterial metabolites of aromatic amino acid-containing proteins, which are either excreted in the faeces or absorbed through the colonic wall and, after sulphate- or glucuronide-conjugation in the mucosa or liver, urinary excreted. As a consequence, the extent of urinary and fecal excretion of phenol and p-cresol may reflect the degree of colonic protein degradation.

A number of studies have investigated the influence of different types of dietary intervention on the generation and accumulation of phenols in the colon. However, conflicting results have been observed. Muir et al. demonstrated that a high starch low fat Chinese diet administered to 12 Australians increased fecal concentrations of phenols as compared to a western-type diet [10], whereas Birkett et al. showed in a randomized cross-over study in 11 subjects that consumption of a high resistant starch (RS) diet (39 g/d) resulted in a significantly lower fecal excretion of phenols as compared to a low RS-diet (5 g/d) [11]. In the two former studies, changes in fecal phenols, rather in urinary phenols, were measured since it was assumed that fecal phenol concentrations more closely reflected changes in the luminal environment. However, in our previous study we demonstrated that, after oral administration of the stable isotope labeled biomarker ²H₄-tyrosine incorporated in egg proteins, pre- and probiotic intervention resulted in a significant reduction in urinary p-²H₄-cresol excretion and these results provided direct evidence that colonic amino acid fermentation is reduced by pre- and probiotic administration [12]. As a consequence, it can be assumed that also the urinary excretion of p-cresol adequately reflects the influence of dietary intervention on amino acid fermentation in the colon. These results are in line with a study of Cummings et al. in which addition of dietary fibre to a high protein diet resulted in a lower urinary excretion of phenols [13]. Also after administration of probiotic Lactobacillus GG cells a decrease in urinary p-cresol excretion was found [14], whereas Spanhaak et al. did not find a significant reduction of the urinary p-cresol excretion after administration of Lactobacillus casei Shirota cells [15].

Therefore, the primary aim of the present study was to investigate in vivo whether long-term intervention with a selected prebiotic (i.e. lactulose) or probiotic organism (i.e. Saccharomyces boulardii) would result in a reduced concentration of protein fermentation metabolites, i.e. phenolic compounds, in the colon. However, when evaluating colonic events after dietary intervention, it is important to monitor as well the possible influences of the administrated substrates on more proximal processes in the gastro-intestinal tract. Especially alterations in motility have to be excluded since they can affect the amount of substrates reaching the colon and in this way, influence the parameters to be measured. It is well known that certain non-digestible carbohydrates may have an influence on transit and digestion in the gastrointestinal tract. Lactulose has previously been shown to delay gastric emptying by increasing viscosity of the gut contents and to accelerate small intestinal transit [16–18]. A shorter small bowel transit can result in maldigestion of nutrients and as a consequence a greater supply to the colonic microbiota can occur. Therefore, also the influences of the administered substrates on gastric emptying (GE) and orocaecal transit time (OCTT) were evaluated. Furthermore, the influence of a long-term dietary intake on intestinal permeability and serum lipids was investigated.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS ACKNOWLEDGMENTS REFERENCES

 
Subjects
Forty-five volunteers (23 women and 22 men) aged 23 ± 2 y (range: 20–26 y) participated in the study. None of the subjects had a history of gastrointestinal or metabolic disease or previous surgery (apart from appendectomy). The subjects were free of antibiotics or any other medical treatment influencing gut transit or intestinal flora for at least 3 months before the start of the study. The Ethical Committee of the University of Leuven approved the study and all subjects gave informed consent.

Experimental Design
Healthy volunteers were randomly assigned to 3 different treatment groups (group 1: n = 15, group 2: n = 15 and group 3: n = 15). A placebo-controlled study was conducted over 16 weeks, consisting of three 4-wk ingestion periods followed by a 4-wk wash-out period. Throughout the study, the volunteers consumed their usual diet, taking care that the diet remained as stable as possible over the four periods. In addition, they were advised to avoid intake of fermented milk products and food components containing high quantities of fermentable carbohydrates. During the ingestion periods, each subject received two different substrates twice a day as indicated in Table 1. Lactulose (Duphalac®, Solvay Pharma & Cie, Brussels, Belgium) and lyophilized Saccharomyces boulardii cells (Perenterol®, Biodiphar, Dübendorf, Switzerland) were chosen as prebiotic [19,20], respectively probiotic [21,22] substrates, whereas the placebo consisted of maltodextrine (AVEBE B.A. Food, Foxhol, The Netherlands).

View larger version (6K): [in this window] [in a new window]   Fig. 1. Schematic representation of the study design: at the start of the study and at the end of each treatment/wash-out period the volunteers performed a test (black arrows: urine/faeces collection and evaluation of gastro-intestinal parameters).

Sample Collection
Urine was collected in specified recipients to which neomycin was added for prevention of bacterial growth. A basal urine sample was collected before consuming the test meal. After intake of the test meal, a 48-h urine collection was performed in different fractions. The 0–24 h urine collection was split into a 0–6 h and 6–24 h fraction to differentiate between effect on small and large intestinal permeability. For the analysis of the phenolic compounds, the combined 0–24 h and the 24–48 h urine collection were studied to evaluate the lingering of the pre- and/or probiotic effect. After measurement of the volume, samples were taken and stored at –20°C until analysis.

Three-day fecal collections were obtained and weighed immediately after voiding. All stools collected on the same day were combined, diluted fivefold with sterile pyrogen-free water and homogenized. The homogenate was ultracentrifuged at 25000 g during 120 min (MR22i, Jouan, St-Herblain, France) and the supernatants was subsequently filtered through a 0.2-µm syringe filter (Supor Acrodisc 32, Gelman Sciences, Ann Arbor, MI, USA) in order to discard the ultimate fecal rests and the bacteria. The final filtrate was used for determination of total phenol and p-cresol. Aliquots were frozen at –20°C.

Analytical Procedures
Performance of Breath Tests.
After an overnight fast, breath samples were taken to determine the basal values of ¹³CO₂ and ¹⁴CO₂. After ingestion of the pancake test meal, breath samples were taken at 15-min intervals for 4 h (for ¹³CO₂ measurement) and 10 h (for ¹⁴CO₂ measurement) to assess gastric emptying and orocaecal transit time, respectively.

Analysis of Breath Samples.
For ¹³CO₂ measurement, breath was collected in exetainers (PDZ, Cheshire, UK). The ¹³C breath content was determined by isotope ratio mass spectrometry (PDZ, Cheshire, UK). Breath samples for measurement of ¹⁴CO₂ were collected by blowing through a pipette into vials containing 2 mmol of hyamine hydroxide until discoloration of the thymolphtaleine indicator, corresponding to the capture of 2 mmol of CO₂. ¹⁴CO₂ was measured by ß-scintillation counting (Packard Tricarb Liquid Scintillation Spectrometer, model 3375, Packard Instruments Inc., Downers Grove, IL, USA) after addition of 10 ml of Hionic fluor (Perkin Elmer, Boston, USA).

Data Analysis: Gastric Emptying Parameters and Orocaecal Transit Time.
CO₂ production was assumed to be 300 mmol/m² of body surface area per hour. The body surface area was calculated by the weight-height formula of Haycock et al. [27]. The result of the ¹³CO₂ breath test was expressed as the percentage of administered dose of ¹³C excreted per hour and the cumulative percentage of the administered dose of ¹³C excreted over 4 h. The ¹³CO₂ excretion data were further analysed by non-linear regression analysis and the gastric half-emptying time (t_1/2) was calculated according to Ghoos et al. [23]. The orocaecal transit time obtained with the inulin-¹⁴C-carboxylic acid breath test was defined as the time at which a significant increase in ¹⁴C from the background was seen in the breath, i.e. 2.5 times the standard deviation of all previous points above the running average of all previous points [23,28].

Analysis of Lipids in Blood.
Serum samples were analysed for cholesterol, triglycerides, HDL and LDL using standard laboratory techniques.

Permeability Test.
Three ml aliquots of the 0–6 h and 6–24 h urine collections as well as a standard solution of the administered ⁵¹Cr-EDTA solution were counted for radioactivity in a NaI(T1)-scintillation counter (1480 Wizard^TH3", Wallac, Turky, Finland) and the results were expressed as percent of administered dose of ⁵¹Cr-EDTA. It was assumed that ⁵¹Cr-EDTA recovered in the 0–6 h collection originated from permeation in the small bowel, whereas the 6–24 h value represents the colonic permeability [29].

Determination of Urinary and Fecal p-Cresol and Phenol Content
p-Cresol and phenol content was measured by gas chromatography-mass spectrometry technology. Therefore samples (urine or fecal supernatants) with a volume of 950 µl were taken and the pH of the samples was adjusted to pH 1 with concentrated H₂SO₄ (Merck KgaA, Darmstadt, Germany). This solution was heated for 30 min. at 90°C to deproteinise and hydrolyse the conjugated phenols. After cooling down to room temperature, 50 µl of 2,6 dimethylphenol (20 mg/100 ml) (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) was added as internal standard. The phenols were extracted with 1 ml of ethyl acetate (Merck KgaA). The ethyl acetate layer was dried and 0.5 µl of this solution was analysed on a GC-MS (Trace GC-MS). The analytical column was a 30 m x 0.32 mm i.d., 1 µm AT5-MS (Alltech Associates, Deerfield, USA). Helium GC grade was used as a carrier gas with a constant flow of 1.3 ml/min. The oven was programmed from 75°C (isothermal for 5 min), with 10°C/min to 160°C and to 280°C with 20°C/min. Mass spectrometric detection was performed in E.I. full scan mode from m/z 59 to m/z 590 at 2 scans/sec. The results were expressed as total p-cresol content (mg) excreted in urine or feces.

Statistical Analysis
Results are expressed as mean ± standard deviation. The statistical analysis was performed with SPSS software (SPSS 12.0 for Windows; SPSS Inc., Chicago, IL, USA). Non-parametric statistical analysis was used, regardless of the distribution of results, because of the low number of subjects in the treatment groups (Friedman analysis of variance (ANOVA), Kruskal-Wallis test, Wilcoxon test and Mann-Whitney test). The level of statistical significance was set at p < 0.05.

	RESULTS

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The characteristics of the subjects who completed the study are summarized in Table 2. One male subject withdrew from the study during the first ingestion period due to necessity of antibiotic intake (group 1) and another female volunteer withdrew from the experiment because of personal reasons (group 2). Data from these subjects were excluded from analysis. All other subjects completed the study. Some volunteers in the second group reported flatulence during the prebiotic ingestion period.

Permeability.
Comparison of the results between the different test conditions showed no significant differences for small intestinal and colon permeability in either of the test groups (Tables 5 and 6), although a tendency to a lower small intestinal permeability was seen after administration of the prebiotic lactulose as compared to baseline conditions. Combination of the results of group 1 and 3 (n = 29; both groups consumed 2 x 10 g lactulose per day during the first ingestion period) resulted in a significant decrease of the small intestinal permeability after lactulose intake (from 0.58 ± 0.33 to 0.43 ± 0.23 (p = 0.006)) as shown in Fig. 2.

View larger version (30K): [in this window] [in a new window]   Fig. 2. Paired scattergram of the 0–6 h 51Cr-EDTA excretion before (baseline) and after 4-weeks administration of lactulose (2 x 10 g/d, n = 29) expressed as % of administered dose.

	DISCUSSION

Although in the present study design, lactulose was not included in the test meal, alterations in motility (gastric emptying and OCTT) have been evaluated in order to exclude the possibility that effects observed in p-cresol and phenol excretion would be due to changes in the amounts of substrates, in particular proteins, reaching the colon. Interestingly, the number of people in whom OCTT could be determined after lactulose administration was lower than in the other test situations where the response rate corresponded to that obtained in previous studies (approximately 75%) [24]. It was unlikely that the OCTT was longer than 600 min or that the microflora did not ferment the substrate after lactulose intake. Therefore, a possible explanation could be that the formed ¹⁴CO₂ was not exhaled, but consumed by the microflora, stimulated by the lactulose.

Most studies described in literature, in which urinary or fecal phenolic compounds have been measured as an indication of the degree of colonic protein fermentation, have investigated the influence of the actual presence of a fermentable carbohydrate [10,11]. The mechanisms leading to reduced protein fermentation have been described as (i) a decrease in protease activity due to a lower pH in the colonic lumen, (ii) a process of so-called catabolite repression which results in an inhibition of the deamination of amino acids and (iii) an enhanced uptake of amino acids and intermediary metabolites for bacterial biosynthesis [30]. To the contrary, in our study long-term dietary intervention with either lactulose and/or Saccharomyces boulardii was performed to find out whether the selected substrates would be able to modify the composition of the colonic microbiota resulting in a modification of the metabolic activity, in particular a decrease in the proteolytic activity. Since these tests were performed after the dietary intervention, i.e. in the absence of either the pre- or probiotic, the observed effects can not be attributed to the actual fermentation of the pre- and/or probiotic substrate.

It is generally known that dietary intervention with lactulose stimulates the growth and activity of bifidobacteria which is mainly a saccharolytic activity [20,31]. The present results have indicated that simultaneously a decrease in proteolytic activity is obtained, which is reflected by a decreased urinary p-cresol concentration in the 0–24 h as well as in the 24–48 h urine collection. Whereas, this decrease was statistically significant in the 0–24 h urine collection, the effect was less pronounced in the 24–48 h urine collection. These data suggest that the modification in the composition of the colonic microbiota induced by lactulose is only temporary and readily disappears once administration of lactulose has been ceased. These observations are in line with previous studies in which a decrease of bifidobacteria, accompanied with a lower saccharolytic activity, was noted once the intervention period with lactulose ended [20,31]. Therefore a continuous stimulation of the microbiota through prebiotic administration might be necessary to maintain beneficial effects.

On the other hand, the influence of long-term dietary intervention with the probiotic yeast Saccharomyces boulardii on the colonic generation of p-cresol was investigated. Administration of S. boulardii has been shown to exert trophic effects on the intestinal mucosa [32,33], to inhibit toxin binding to intestinal receptors [34] and to increase the production of secretory IgA [35], however, to our knowledge, no data is available on the potential to modify the composition of the intestinal microbiota and the effect on the metabolic activity. The present study demonstrated that the higher colonic availability of Saccharomyces boulardii cells did not influence the bacterial amino acid fermentation and therefore it could be hypothesized that the probiotic effects of S. boulardii are probably situated in a different pathway.

It was rather unexpected that the synbiotic combination of lactulose and S. boulardii resulted in less pronounced effects as compared to the administration of lactulose alone. From these observations, it could be hypothesized that S. boulardii is capable to use lactulose as an energy source leaving less lactulose available for the colonic microbiota, although no literature data is available to confirm this hypothesis.

The results of the present study also demonstrate a reduction in the small intestinal permeability after long-term lactulose administration. Animal studies have already shown that dietary administration of non-digestible carbohydrates results in increased height of the villi and depth of the crypts and an increase in absorptive capacity [36,37]. These effects are not caused by lactulose itself but are due to short chain fatty acids (SCFA), generated in the colon by fermentation of lactulose, which have been shown to increase the proglucagon mRNA expression in the intestine [38–41]. Glucagon like peptide 2 (GLP-2), a proglucagon derived peptide, appears to modulate small bowel epithelial proliferation and as a consequence may have an effect on small bowel permeability [42]. Further characterisation of the relation between the SCFAs, a result of prebiotic intake, and the possible GLP-2 mediated effect on small intestinal permeability needs to be done in future evaluations.

In the present study the lipid-lowering properties of lactulose and/or Saccharomyces boulardii cells on serum lipids in normolipidaemic individuals were evaluated as well. A recent in vitro study demonstrated that Saccharomyces boulardii was able to remove various amounts of cholesterol from a growth medium by assimilation of cholesterol into the yeast cell [43]. However, the results of the present study did not demonstrate a hypocholesteric effect of the selected Saccharomyces boulardii strain in vivo. The influence of lactulose on cholesterol levels is expected to be mediated by the SCFA, which have been suggested to decrease the systemic levels of blood lipids in the gut by inhibiting hepatic cholesterol synthesis and/or redistributing cholesterol from plasma to liver [44]. Acetate in the serum seems to increase total cholesterol, while propionate increases blood glucose and tends to lower the hypercholesterolemic response caused by acetate by reducing its utilization by the liver for fatty acid and cholesterol synthesis. As a consequence, sufficient propionate has to be produced to offset the effects of acetate generation as a precursor for lipid synthesis [45]. However, fermentation of several non-digestible carbohydrates, such as lactulose, has been described to enhance production of acetate, but not that of propionate, and as a consequence, lactulose administration might modulate the lipid metabolism in a negative way [46]. A study in rats already demonstrated that dietary intake of lactulose resulted in an increase of liver cholesterol levels [47]. On the other hand, some bacteria may interfere with cholesterol absorption from the gut either by deconjugating bile salts or by directly assimilating cholesterol. A number of bacteria have been reported to hydrolyze conjugated bile acids, such as bifidobacteria, lactobacilli, clostridia, streptococci [48]. It is generally known that dietary intervention with lactulose selectively stimulates the growth of bifidobacteria [20,31]. In this way, lactulose might exert a hypocholesterolemic effect due to the assimilation of cholesterol by bifidobacteria. However, in the present study no changes in lipid levels were noted upon long-term lactulose administration.

In the present study, the effects of the substrates were evaluated under ‘normal’ conditions since pre- and probiotics are often recommended as food supplements for healthy individuals in normal circumstances. Since the volunteers were not able to discriminate effective treatment from placebo, it was considered unlikely that the observed significant effects were due to casual changes in the diet and, as a consequence, in the components reaching the colon.

In conclusion, the results obtained in present study have indicated that colonic amino acid fermentation can be reduced by the administration of lactulose as a fermentable carbohydrate, resulting in a lower concentration of potentially toxic metabolites, but once administration of lactulose ceases, the saccharolytic activity of the colonic microbiota declines slowly.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS ACKNOWLEDGMENTS REFERENCES

 
This work was supported by IWT-Vlaanderen, Brussels, Belgium (GBOU project nr. 010054). The authors also acknowledge the financial support from the Fund for Scientific Research-Flanders and the University Research Councils. The company Biodiphar (Dübendorf, Switserland) was acknowledged for providing the substrates.

L. De Vuyst (Research Group of Industrial Microbiology, Vrije Universiteit Brussel, Brussel, Belgium), G. Huys (Laboratory of Microbiology; Universiteit Gent, Gent, Belgium), J. Swings (BCCM^TM/LMG Bacteria Collection, Universiteit Gent, Gent, Belgium) and B. Pot (Bacteriology of Ecosystems, Institut Pasteur de Lille, Lille Cedex, France), as scientific partners of the GBOU project nr. 010054, are acknowledged for providing scientific comments.

Received August 28, 2005. Accepted July 11, 2006.

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