Gut 1996; 38: 870-877

Relations between transit time, fermentationproducts, and hydrogen consuming flora inhealthy humans

L El Oufir, B Flourie, S Bruley des Varannes, J L Barry, D Cloarec, F Bornet,J P Galmiche

AbstractBackground/Aims- To investigate whethertransit time could influence H2 consumingflora and certain indices of colonic bac-terial fermentation.Methods-Eight healthy volunteers (fourmethane excretors and four non-methaneexcretors) were studied for three, threeweek periods during which they received acontrolled diet alone (control period), andthen the same diet with cisapride orloperamide. At the end of each period,mean transit time (MTT) was estimated,an H2 lactulose breath test was performed,and stools were analysed.Results-In the control period, transittime was inversely related to faecalweight, sulphate reducing bacteriacounts, concentrations oftotal short chainfatty acids (SCFAs), propionic andbutyric acids, and H2 excreted in breathafter lactulose ingestion. Conversely,transit time was positively related tofaecal pH and tended to be related tomethanogen counts. Methanogenic bac-teria counts were inversely related tothose of sulphate reducing bacteria andmethane excretors had slower MTT andlower sulphate reducing bacteria countsthan non-methane excretors. Comparedwith the control period, MTT was signifi-cantly shortened (p<0.05) by cisaprideand prolonged (p<005) by loperamide (73(11) hours, 47 (5) hours and 147 (12) hoursfor control, cisapride, and loperamide,respectively, mean (SD)). Cisapridereduced transit time was associated with(a) a significant rise in faecal weight,sulphate reducing bacteria, concentra-tions of total SCFAs, and propionic andbutyric acids and breath H2 as well as (b) asignificant fall in faecal pH and breathCH4 excretion, and (c) a non-significantdecrease in the counts of methanogenicbacteria. Reverse relations were roughlythe same during the loperamide periodincluding a significant rise in the countsof methanogenic bacteria and a signifi-cant fall in those of sulphate reducingbacteria.Conclusions-Transit time differencesbetween healthy volunteers are associatedwith differences in H2 consuming flora andcertain indices of colonic fermentation.Considering the effects ofsome fermenta-tion products on intestinal morphologyand finction, these variations may be

relevant to the pathogenesis of colorectaldiseases.(Gut 1996; 38: 870-877)

Keywords: transit time, H2 consuming flora,hydrogen, methane, short chain fatty acids.

Through fermentation the colonic floragenerates short chain fatty acids (SCFAs),carbon dioxide, and hydrogen (H2). H2 releasedin the colon may be consumed by methanogenicbacteria (MB) producing methane and bysulphate reducing bacteria (SRB) and aceto-genic bacteria.'

There are considerable inter-individualvariations in the composition of faecal flora.23As different species make different contribu-tions to the production of individual SCFAs,inter-subject variations in the faecal pro-portions of acetate, propionate, and butyratealso occur,4 5 and in vitro faecal fermentationpatterns of a given substrate differ betweensubjects.6 7 There are also large individualvariations in the activities of flora producing orconsuming H2. The amounts of H2 excretedin breath in response to identical loads offermentable carbohydrates and the quantitiesof methane exhaled, as well as faecal MBconcentrations, differ considerably amongsubjects.1 8The factors affecting the composition and

activities of colonic flora, and which couldaccount for inter-individual variations, arelargely unknown. The factors likely to beimportant include age, diet, colonic pH andmicrobial interactions, colonic supply, andavailability of nutrients, bile acids and sul-phate, and microbial adaptation to metabolisethese substrates.' 2 69-11 Transit time throughthe gut may be expected to affect colonic florabecause it influences both the amount ofsubstrates flowing to the large bowel'2 13 andthe efficiency of colonic fermentation.' 14Consequently, the availability of substrate andenergy for the colonic flora, as well as the pH ofluminal contents, are changed. In addition,some data suggest that transit itself could affectthe composition and activities of colonic flora.Indeed, the faecal bacterial mass is related totransit time in human subjects on identicaldietary intakes.'5 16 Likewise, in in vitro con-tinuous culture in which bacteria are grownwith a constant substrate supply, bacterial cellgrowth depends on turnover time,17-19 andchanges in turnover time also affect the patternof fermentation products.'8 19 Moreover, fast

Centre de Rechercheen Nutrition Humaine,Laboratoire 'FonctionsDigestives etNutrition', CHU Nord,Nantes, France andINRA Nantes, FranceL El OufirS Bruley des VarannesJ L BarryD CloarecJ P Galmiche

INSERM U290,H6pital Saint-Lazare,Paris, FranceB Flourie

Eridania B6ghin-Say,Paris, FranceF Bornet

Correspondence to:Professor J P Galmiche,Laboratoire 'FonctionsDigestives et Nutrition',CHU, 44035 Nantes,France.Accepted for publication7 December 1996

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Transit time and colonic fernentations

transit time through the large bowel couldimpede SCFAs absorption, leading to sus-tained acidification ofthe contents, and modifycolonic stirring, which has been shown tocontrol H2 consumption.20 Conversely, slowtransit time seems to be physiologically associ-ated with methane production by slow growingMB, although findings are not consistent.2' 24

In normal subjects, transit time through thesmall intestine and whole gut varies greatlybetween people.25-28 For example, a threefoldrange in mean transit time (MTT) has beenfound in men consuming identical diets.'629Individual variations in transit time could thusinfluence the composition and activities ofcolonic flora, which could account in partfor its inter-subject variations. To test thishypothesis, we studied the relations betweentransit time, H2 consuming bacteria, and cer-tain indices of bacterial fermentation in healthyvolunteers given a constant diet. As transit maybe the result rather than the cause of colonicevents, we further changed this variable bypharmacological means and measured thechanges in H2 consuming flora and fermenta-tion products.

Methods

SubjectsThe study was performed in eight healthyadults (three males and five females, 26-35years). None had received antibiotics, laxativesor enemas during the three months precedingthe study. As approximately 50% of the Frenchpopulation are methane excretors,30 weenrolled four subjects who were identified asmethane excretors (methane+) on the basis ofbreath methane concentrations that were atleast one part per million (ppm) above roomatmosphere and four who were non-methaneexcretors (methane-). The protocol wasapproved by the local ethics committee, and allinvestigations were undertaken after writteninformed consent was given by the subjects.

Study designWe applied the protocol designed by Stephenet al,6 and divided study time into three, threeweek periods. The first period served as acontrol, whereas subjects received eithercisapride or loperamide in random orderduring the two subsequent three week periods.During each period, subjects consumed thesame controlled diet and collected their stools.Radio-opaque pellets were used for MTTmeasurement.

According to the sex, subjects could feed inrotation on five one day isocaloric menus ofsimilar composition. For women, daily energyintake was 8-36 MJ (consisting in 79 g protein,91 g fat, and 223 g carbohydrate), and formen, it was 10-45 MJ (consisting in 81 gprotein, 92 g fat, and 311 g carbohydrate).Additional carbohydrates in men came fromfoods containing simple sugars. Both dietswere designed to contain twice the usual dailyFrench intake of dietary fibre (that is, 15 gX 2)

to provide sufficiently fast transit time in thecontrol period such that it could be thenchanged during the loperamide treatmentwithout distress to the participants.Throughout the study, subjects took 30

radio-opaque pellets per day (10 per meal) asnon-absorbable markers. Two marker shapeswere used: small squares and circles. The typeof marker was changed at the end of each threeweek period, so that total marker recoveryduring each period could be estimated.Subjects collected stools throughout the studyand for one week after to assess the total recov-ery of ingested markers. Each stool was col-lected separately in a plastic bag suspendedover the toilet and immediately frozen. Thenumber of markers present in each stool wascounted after radiographing. Transit measure-ments were done using the continuous markertechnique described by Cummings et al,3'which permits continuous day by day MTmeasurement. Drug therapy was titrated toapproximately double or halve the spontane-ous transit time of subjects. Cisapride tablets(10 mg tablets) (Prepulsid, Janssen, Boulogne,France) were used to speed up transit. Initiallytwo tablets were given (one with breakfast andthe other with dinner), and the dose wasincreased by one tablet per day up to 60mg/day if the desired effect was not achieved.Transit was slowed down using loperamide(2 mg tablets) (Imodium, Janssen, Boulogne,France). Initially, two tablets were used, andthe dose was increased up to 10 mg/day if suf-ficient slowing in transit time did not occur. Alladjustments were made during the first week ofeach period. The final daily doses of drugswere 40 mg (n=2) and 60 mg (n=6) forcisapride, and 6 mg (n= 1), 8 mg (n=4), and10 mg (n=3) for loperamide. Stools collectedfrom days 14 to 21 of each period wereweighed, and the percentage of dry matter instool aliquot was determined after freezedrying. MTT was calculated as the averagevalue for the last seven days of control, cis-apride, and loperamide periods.

Lactulose H2 breath testsAfter a standardised low residue evening mealand a 12 hour fast, subjects ingested 10 glactulose (Duphalac, Duphar, Villeurbanne,France) alone or with the drug (cisapride orloperamide) on the last day of each three weekperiod. After a thorough mouth rinse with a1% chlorhexidine solution (Givalex, NorganLaboratoires, Paris, France), three basalsamples of end expiratory air were collectedbefore lactulose ingestion. Further sampleswere taken every 10 minutes during the firsttwo hours and every 15 minutes thereafterfor eight hours. During the tests, smokingand eating were not permitted, and the sub-jects were non-ambulant. H2 and methaneconcentrations in breath samples weredetermined simultaneously using a Quintronmodel DP instrument (Quintron Microlyser,Milwaukee, WI, USA) and expressed asparts per million (1 ppm= approximately0.05 pmol/l).

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Analytical methodsFreshly voided stool obtained from days 18 to21 of each period were immediately refrigeratedat 4°C under anaerobic conditions (AnaerocultsA; Merck, Darmstadt, Germany) for no longerthan two hours before processing. Stools werehomogenised and diluted in anaerobic solution(containing (g/l): NaCl, 5; glucose, 2; cysteine.HCI, 0.3 and 5% minerals, 0.5% oligoelements,and 0.5% vitamins) to give a 10-fold dilution(wet weightlvolume). Faecal pH was measuredusing a pH meter (Micro pH 2000, Crison,Paris, France), and serial 10-fold dilutions weremade.

Total anaerobes were measured in Wilkins-Chalgren agar (Difco Laboratories, DetroitMI, USA). For this purpose 1 ml of each 10-fold dilution was inoculated into 13 mlWilkins-Chalgren agar at 45°C. Innoculatedmedium was then poured into 8X400 mmtubes (in duplicate), which were immediatelycooled to ensure prompt solidification.32

Total SRB were counted similarly in themedium of Pfenning et al.33 Lactate was usedas a substrate for dissimilatory sulphate reduc-tion as it serves as a substrate to the major SRBpopulation of the human large intestine' andthat all species characterised to date utilise H2as electron donor.34

Total MB were measured by most probablenumber estimation in Balch 1 broth.35Triplicate 10-fold dilution series were pre-pared in the anaerobic chamber using theanaerobic solution described above (9 ml in 30ml vials) and incubated for each dilutionbetween 10-2 and 10-11. The gas phase ofeach vial was pressurised weekly at 202 kPausing H2/C02 (4:1). Headspace gas sampleswere removed after 15 days to measuremethane concentrations by gas chromato-graphy (N 200, Delsi Nermag Instrument,Argenteuil, France). Vials with methane con-centrations above 200 ppm were consideredpositive.36 Positive responses at each dilutionlevel were counted, and the most probablenumbers calculated.

For the last two subjects studied (onemethane+ and one methane-), acetogenicbacteria were measured by most probable num-ber estimation in AC-1 1 broth as described byGreening and Leedle.37 Triplicate 10-fold dilu-tion series were prepared in the anaerobicchamber using the same anaerobic solution (9ml in 30 ml vials) and incubated for each dilu-tion between 10-1 and 10- 1 l. The gas phase of

TABLE I Mean transit time, stool weight, and counts of total anaerobes, methanogenicbacteria, and sulphate reducing bacteria in stools collected during the control periodfrom eighthealthy volunteers (four CH4 excretors (CH4+) andfour non-CH4 excretors (CH4-))

Mean Stool Methane Sulphatetransit weight excretor Total Methanogenic reducing

Subjects Sex time (h) (giday) status anaerobes* bacteria* bacteria*

1 F 122 107 CH4+ 11-3 9-8 7-32 F 99 92 CH4+ 10-2 9 8 7-43 M 63 198 CH4+ 10-9 8 7 7 94 F 98 100 CH4+ 10.8 9 8 8.15 F 41 180 CH4 115 <2 906 F 44 141 CH4 10.5 4-1 8-47 M 41 233 CH4 10-6 6-7 8-48 M 74 150 CH4 11.0 <2 8-4Mean (SEM) 73 (11) 150 (18) 4 (4-) 10.9 (0 1) 6-1 (1-5) 8-1 (0.2)

*Results are expressed as logl0 viable micro-organisms per g of wet weight.

each vial was pressurised weekly at 202 kPausing H2/C02 (4:1). For each dilution between10- 1 and 10- l, two control vials were pre-pared and pressurised weekly at 151.5 kPausing N2. Samples were removed after 21 daysto measure acetic acid concentrations by gaschromatography (DN 200, Delsi NermagInstrument). Vials with acetic acid concentra-tions above the mean of the two control vialswere considered positive.37An aliquot ofhomogenised stools was mixed

with a solution containing 1% (w/v) mercuricchloride and 0.61% (v/v) orthophosphoric acidin distilled water and then stored at -20°C.Thawed aliquots were centrifuged at 5000 gfor 15 minutes. After addition of internal stan-dard (4-methyl valeric acid), SCFA concentra-tion was determined by gas chromatography(N 200, Delsi Nermag Instrument).38

Calculations and statistical analysisMouth to caecum transit time (MCTT) wasdefined as the period from lactulose intakeuntil the period just before the initial increaseabove fasting levels of 10 ppm or more H2when this increase was sustained. Breathexcretions of H2 and methane were estimatedby integrating the area under the concentrationcurve (AUC) located between 0 and 600minutes.

Stool data collected at the end of eachperiod were expressed as daily mean (SEM).The quantitative results of bacteriologicalculture were expressed as the log10 of colonyforming units per g wet weight faeces. One wayanalysis ofvariance and paired t tests were usedto compare means from the three periods.Because of the curvilinear nature of curvesbetween transit and the parameters studied,log MTT was used to calculate correlationsbetween MTT and each parameter by simpleregression. Student's unpaired t test was usedto compare data between methane and non-methane excretors.

Results

Mean transit time and stool weightSome 99 (3)% of markers ingested during thestudy were recovered in stools, thereby permit-ting accurate MT estimates.

During the control period, with similarintake of dietary fibre, MTT ranged from 41 to122 hours and faecal output from 92 to 233g/day (Table I). Figure 1 shows the individualdaily MTT values after marker equilibriumwas reached during the control period.Although MTT variations occurred from dayto day, on the whole each subject had a ten-dency to maintain a rather constant, either fastor slow, MTT. MTT was inversely related tostool weight and the percentage of faecal water(r=0-79, p<0.02, n=8 and r=-0.76, p<0-02,n= 8 respectively).

Figure 2 shows MIT changes duringcisapride and loperamide administration.Compared with the control period, MTT wassignificantly shortened by cisapride (47 (5) v 73

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(11) hours, p<005) and significantly length-ened by loperamide (147 (12) v 73 (11) hours,p<0.001). Changes in MTT induced bycisapride and loperamide were associated withsignificant variations in daily stool outputs (Fig2).

Over the three periods, MTT was inverselyrelated to faecal weight (r=-0.89, p<0.001,n=24) and percentage of faecal water(r=-0.82, p<0-001, n=24).

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Control Loperamide CisaprideFigure 2: Mean transit time (A) and daily faecal weight (B) during the last seven days ofthe control, loperamide, and cisapride periods. *p<0.05, tp<O0OO5, tp<0001 v controlperiod.

Mean transit time andfaecal bacterial countsDuring the control period, total viable anaer-obes ranged from 10.2 to 11.5 logl/g wetweight (Table I). In methane+, MB rangedfrom 8.7 to 9.8 logl/g wet weight. SRB, whichwere detected in all subjects, ranged from7.3 to 9.0 logl/g wet weight. SRB countsdiffered significantly between methane+ andmethane- (7.7 (0.2) v 8.6 (0G1) logl/g wetweight, p<005), and were negatively relatedto MB counts (r=-0-82, p<0.02, n=8).MTT was significantly higher in methane+than methane- (96 (12) v 50 (8) hours,p<0.05) and was negatively related to SRBcounts (r=- 0.83, p<0.02, n= 8).

Table II shows the changes in the counts oftotal viable anaerobes as well as MB and SRBinduced by cisapride and loperamide adminis-tration. Total viable anaerobes were not signifi-cantly changed by drugs. Compared withthe control period, the administration ofloperamide induced a significant increase inMB counts (p<0 05) and a significant decreasein SRB counts (p<0005). Conversely, withcisapride MB tended to be lower (p=007),whereas SRB were significantly higher(p<0 05). In the two subjects in whom MBwere below the detection level, neither MB norSRB counts were changed by cisapride or lop-eramide administration. In the two subjectstested, acetogenic bacteria counts wereunchanged during the three periods (Table II).

Over the three periods, MTT correlatedwith MB counts (r=0.58, p=0-01, n=24) andwas inversely related to SRB counts (r= -0.78,p<0-001, n=24).

Mean transit time, MCTT, H2, and methaneexcretion in breath after lactulose ingestionDuring the control period, fasting H2 concen-trations were 2.7 (0.4) and 2.8 (0.6) ppm inmethane+ and methane-. Fasting methaneconcentrations were 17 (4) ppm and 1.0 (0-1)ppm in methane+ and methane-. MTT wasclosely related to MCTT (r=0.97, p<0001,n= 8) and was inversely related to AUC of H2exhaled after lactulose ingestion (r=-0-83,p<002, n=8).Compared with the control period, cisapride

administration induced a significant decreasein MCTT (97 (6) v 160 (13) minutes,p<0 05) and a significant increase in H2 excre-tion in breath as assessed by the AUC of H2(p<005) (Fig 3, Table III). In methane+,fasting methane concentration and conse-quently the AUC ofmethane were significantlyreduced during cisapride administration (10(3) v 17 (4) ppm, p<001 and 1-9 (0.2) v 3.4(0.3) 103 ppm/min, p<005, Fig 3). MCTTand the AUC for H2 were not significantlyaffected during loperamide administration(200 (24) v 160 (13) minutes, p=020 and 7.7(0.9) v 9A4 (1-5) 103 ppm/min, p=0.30) (Fig 3,Table III). In methane+, fasting methane con-centration and consequently the AUC ofmethane were significantly increased duringloperamide administration (29 (2) v 17 (4)ppm, p<0-05 and 4.9 (0.5) v 3-4 (0.3) 103ppmnmin, p<005) (Fig 3). Interestingly, one

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El Oufir, Flouri, Bruley des Varannes, Barry, Cloarec, Bornet, Galmiche

TABLE II Counts (mean (SEM) of total anaerobes, methanogenic, sulphate reducing, andacetogenic bacteria in stools collected during the control, loperamide, and cisapride periods

Control Loperamide Cisapride

Total anaerobes* 10-9 (0.1) 10-6 (0.1) 11 0 (0 1)Methanogenic bactena* 6-1 (1-5) 6-9 (1.6)t4 5-4 (1.3)4§Sulphate reducingbacteria* 8-1 (0-2) 7-4 (0.3)411 8.6 (01 )t4Acetogenic bacteria* 7-2 (0.9) 6-9 (0.7) 7-4 (1.1)

*Results are expressed as log10 viable micro-organisms per g wet weight (n=8, except foracetogenic bacteria for which n=2). tp<0 05 v control period, p<0.O005 cisapride vloperamide period, §p=007 v control period, IIp<O005 v control period.

subject (no 7) was a non-methane excretorduring the control period, but became amethane excretor during loperamide adminis-tration.

Over the three periods, MTT was closelyrelated to MCTT (r=0-74, p=0.0001, n=24)and was inversely related to the AUC of H2(r= -062, p<0.01, n=24) (Fig 4).

Mean transit time, faecal pH, and SCFAsDuring the control period, faecal pH rangedfrom 6.5 to 7-2 and was inversely related toMlT (r=-0-87, p<005, n=8). Comparedwith the control period, faecal pH was signifi-cantly reduced during cisapride administration(6&6 (0.1) v 6-9 (0-1), p<0 05) and signifi-cantly increased with loperamide (7.2 (0.1) v6.9 (0.1), p<0001). Over the three periods,MTT was inversely related to faecal pH(r=-0.85,p<0.001, n=24).During the control period, faecal SCFA

concentration ranged from 51 to 90 mM, withmean molar proportions of 60, 17, and 13 foracetic, propionic, and butyric acids respec-tively (Table IV). MTT was inversely related

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TABLE III Breath H2 excretion (area under the curve, 103ppm/min) after ingestion of 10 g lactulose during the control,loperamide, and cisapride periods according to CH4 excretorstatus

Control Loperamide Cisapnde

CH4' (n=4) 7-2 (1-5)t 6-2 (0.6)4 12-4 (2 1)4CH4- (n=4) 11-6 (1.6)t 9.1 (1.5)4 17-8 (2.2)4All subjects (n=8) 9.4 (1-5) 7-7 (0.9)§ 15-1 (2.1)*

Mean (SEM). tp=0-14, *p=0.13 (CH4- v CH4+), §p=030 vcontrol period, *p<0Q05 v control period.

to the concentration of total SCFAs (r= -0 70,p=005) and propionic and butyric acids con-centrations (r=-0-91, p<001 and r=-0.70,p=0.05 respectively).Compared with the control period, total

SCFA concentrations, and the concentrationsand percentages of propionic and butyric acidwere significantly higher in the cisapride periodand significantly lower in the loperamideperiod (Table IV). A significant oppositevariation was seen in the percentage of aceticacid, whereas its concentration remainedunchanged.

Over the three periods, MTT was inverselyrelated to the concentrations of total SCFAs(r=-0 77, p<0001). It was inversely relatedto the concentrations of propionic and butyricacids (r=-0.94, p<0.001 and r=-0.76,p<0001) (Fig 5) and to the percentages ofpropionic and butyric acids (r=-0.81,p<0Q001 and r=-0.67, p<0Q001).

DiscussionTransit changes induced by drugs in healthyvolunteers given on a constant diet not onlyaffect stool weight but also certain bacterialpopulations and fermentation indices. Thesefurther modifications emphasise the role oftran-sit in some colonic events already seen duringthe control period and suggest that transit timecould be an important factor in the individualvariability of colonic fermentations. Whetherthese changes in faecal flora and colonic fer-mentation result primarily from variations intransit or are secondary to the consequences oftransit variations, basally or pharmacologicallyinduced, needs further discussion.

Inter-individual variations in the composi-tion and activities of colonic flora2 3 as well as

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Transit time and colonic fermentations

TABLE IV Faecal concentrations (mean (SEM)) ofshort chain fatty acids (SCFAs) andmolar proportions (percentages) of acetic, propionic, and butyrc acids during the control,loperamide, and cisapride periods in eight healthy volunteers

Control Loperamide Cisapride

mmol/l (%) mmol/l (%) mmol/l (%)

Total SCFAs 75-0 (4.7) (100) 61.4 (4 0)*t (100) 87-4 (4.2)*: (100)Acetic acid 44-6 (2.4) (60) 39.5 (2.7)§ (64) 48-4 (2.5)§ (55)Propionic acid 13-1 (1.1) (17) 8-0 (0.4)t§ (13) 18-2 (1.1)t§ (21)Butyric acid 10-2 (1-2) (13) 6-8 (0Q7)*§ (11) 14-3 (10)§t (16)

*p<0.005, tp<0 001 v control period, 4p<0 001, Sp<0001 cisapride v loperamide period.

transit time'6 25-29 have been widely reported.On the other hand, it is generally assumed thatcolonic flora,2 3 39 faecal fermentation patternsof a given substrate,6 40 methane excretor status,and faecal MB concentrations41 42 remain stablewithin a given subject over an extended periodof time. Likewise, it is generally assumed that incontrolled conditions, and despite a day to dayvariation, subjects with physiologically fast orslow MTT generally maintain this characteristicover a prolonged time period,2943 as was con-finned in our study during the control period(Fig 1). It was therefore tempting to hypothesisethat the rough constancy of intra-individualtransit time could be one factor responsible forthe stability of certain bacterial populations andactivities of colonic flora.

Although a limited number of subjectswas enrolled in this study, clear individual

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Figure 5: Correlation between mean transit time (log MTT) and concentrations of butyric(A) and propionic (B) acids during the control (0), loperamide ( A), and cisapride (-)periods in eight healthy volunteers.

variability in transit time, faecal composition,and breath excretion of gases was observedduring the control period. Indeed, subjectswith spontaneously fast MTT exhaled moreH2 in response to lactulose. MB counts andpH were low in their faeces, whereas SRBcounts and concentrations of SCFAs andpropionic and butyric acids were high in theirfaeces. Because it may be argued that transit isthe result rather than the cause of colonicevents, we used the experimental protocoldesigned by Stephen et al'6 to demonstratethat transit influences events in the humanlarge intestine.Drug induced MTT changes in faecal flora

did not affect the number of viable anaerobesper g of faeces, but resulted in changes in MBand SRB counts except for the two subjects inwhom MB were below the detection level.Indeed, the increase in MTT with loperamideresulted in a significant fall in SRB counts anda significant rise in MB. Accordingly, breathmethane was higher in the fasting state andafter lactulose ingestion in methane excretors(Fig 3). The reverse was roughly true with cisa-pride. The effect of loperamide was evenstrong enough to convert one non-methaneexcretor into a methane excretor. In this sub-ject the MB count was basally higher than inother methane non-excretors, rising from 6.7to 8.1 logl,/g wet weight during loperamideadministration. Our findings are in agreementwith the reported inverse relation between MBand SRB stool counts.1 44 45 Because MB areslow growing bacteria, it is usually assumedthat slow colonic transit may provide a moresuitable environment for their prolifera-tion.2' 22 When colonic transit time isincreased and provided that MB are present,they may proliferate and eventually out com-pete SRB. Alternatively, it has been shown thatsulphate availability and bile acid losses in thecolon may regulate the proportion of SRBand MB populations.' 046 During cisaprideadministration, MCTT was shorter, whichcould have reduced absorption of dietary sul-phate and bile acids in the small intestine.'0 46

Besides modifications in MB and SRBcounts, our data show that transit modifica-tions change the breath excretion of gasesproduced in the colon. However, interpreta-tion of these data should be cautious becausebreath concentrations of H2 and methane arenot a reliable guide of colonic production ofthese gases because their rate of productionsignificantly affects their proportion exhaled inbreath.47 48

In the control period as well as in the studyas a whole, MIT was inversely related to thevolume ofH2 excreted in breath after lactuloseingestion, and this was significantly increasedduring cisapride administration (Fig 3). Therise in breath H2 excretion when transit wasfaster cannot be explained by better diffusionfrom the colonic lumen towards breath gas asfaster transit provides less time for colonic gasabsorption and H2 is rather excreted inflatus.47 48 Although the rate of lactulose entryinto the colon may affect breath H2 excre-tion,49 the rise in H2 when transit was faster

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876 El Oufir, Flourie, Bruley des Varannes, Barry, Cloarec, Bornet, Galmiche

was more probably as a result of reduced con-sumption of H2 produced in the colon, whichcould have resulted from increased colonicmixing or a decrease in some H2 consumingorganisms, or both.20 Strocchi and Levitt20have suggested on the basis of in vitro studiesthat the amount of H2 released from wellstirred faecal contents is many times greaterthan from poorly stirred faeces. It could thusbe speculated that basal variations in colonicmixing or those induced by cisapride accountfor the relation between fast transit and the risein H2 excretion. Alternatively, changes in H2consuming flora may have reduced intra-colonic H2 consumption. Acetogenic bacteriamay grow at low pH values (optimum pH=6)and, as SRB, are competitively displaced byMB in human faeces.50 51 In this study, weassessed acetogenic bacteria only in the lasttwo subjects and found no change in theircounts during the three periods. During cis-apride administration, SRB stool countsincreased, whereas MB counts tended todecrease. H2 excretion increased, while breathmethane was lower in the fasting state and afterlactulose ingestion in methane excretors.Thus, although SRB were increased duringcisapride administration, they did not consumeH2 efficiently and, interestingly, breath H2after lactulose ingestion rose in methaneexcretors but also in non-methane excretors(Table III). These findings seem to supportprevious reports showing that in human faecesMB outcompete other H2 consuming bacteriafor H2.20 45

However, it should be noted that methaneexcretion in breath and MB were at a higherlevel during loperamide administration, whilethe volume of exhaled H2 was hardly reduced.One possible explanation for this is that the siteof lactulose fermentation is physically separatedfrom that of methane formation, especially insituations where the colonic transit time is slow.This concept is supported by studies inmethane excretors showing that methane pro-duction occurs mainly in the left colon,4' 52whereas the right colon should be the primarysite of lactulose fermentation.Our results confirm that MTT is related

to faecal weight, water output, and pH.16 21Moreover, in the control period as well as inthe study as a whole, MTT was inverselyrelated to the faecal concentration of totalSCFAs and propionic and butyric acids.Speeding up transit pharmacologically pro-duced lower faecal pH, while concentrationsofSCFAs and propionic and butyric acids roseby 17, 39, and 40%, respectively. The reversewas true when transit was slowed down byloperamide. Transit variations could haveinduced changes in the faecal amounts andproportions of SCFAs because of changes insubstrates flowing to the colon or changes inthe environmental conditions of the largeintestine (such as pH, or both). Fast MTT wasassociated with fast MCTT in both controland cisapride periods, which may haveincreased the delivery of fermentable sub-strates to the colon, in particular digestiblestarch'2 13 leading to higher production of

propionate or butyric, or both.6 5354 Increasedsubstrate supply to the colon may in turn lowerintraluminal pH, a factor that by itself canchange fermentation end products.55 In addi-tion, changes in transit time may directly affectthe amount and pattern of fermentation endproducts.'8 19 56 57 Fast transit through thecolon could reduce SCFA absorption, andchanges in their faecal excretion pattern mightbe due to differences in SCFA absorption fromthe colonic lumen.58 Lastly, as mentionedabove fast transit was associated with reducedH2 removal, which could exert selective pres-sure on the colonic bacteria changing thebalance between species or between metabolicpathways.59

In conclusion, this study shows that differ-ences in MTT between healthy volunteersgiven a constant diet are associated with differ-ences in H2 consuming flora and certainindices of colonic fermentation, which mayalso be induced by changing transit time bypharmacological means. Factors (constitu-tional or environmental) affecting transit thusmight play an important part in determininginter-individual differences in the compositionand certain activities of colonic flora.Regarding the potential effects ofsome of thesefermentation products (for example, butyrate)on the colonic mucosa,1 it is therefore reason-able to assume that these changes may havesome important implications in the patho-physiology of certain colorectal diseases.We thank C Bonnet for his excellent technical assistance. Thisresearch was supported in part by Eridania Beghin-Say and by agrant from the French Ministry for Research (Aliment Demaincontract No: 92 G 0554).

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