APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1992, p. 3330-3336 Vol. 58, No. 100099-2240/92/103330-07$02.00/0Copyright © 1992, American Society for Microbiology

Determination of Fermentable Carbohydrate from the UpperGastrointestinal Tract by Using Colectomized RatsDAVID J. MONSMA, NICHOLAS W. VOLLENDORF, AND JUDITH A. MARLETI*

Department ofNutritional Sciences, University of Wisconsin-Madison,1415 Linden Drive, Madison, Wisconsin 53706

Received 22 May 1992/Accepted 27 July 1992

The primary aim of this study was to characterize the carbohydrate that would be supplied to the colon forfermentation under physiological conditions. Colectomized rats were fed fiber-free diets or diets containing 5%(wt/wt) gum arabic. Four (fucose, galactose, glucosamine, and galactosamine) of 11 analyzed sugars accountedfor 77% of the total sugar in ileal excreta from colectomized rats fed fiber-free diets. The three sugars in gumarabic, rhamnose, arabinose, and galactose, accounted for 84% of the total sugars in gum arabic ileal excreta.Comparisons of the sugar compositions of the ileal excreta, the water-soluble fractions of the excreta, and threegel filtration fractions of the water-soluble material with those of the water-soluble fraction of rat mucosa, theacetone-soluble fraction of pancreas, and pancreatin suggested that the major source of endogenouscarbohydrate is mucin. Gum arabic increased the daily excretion of the four mucin-derived sugars (fucose,galactose, glucosamine, and galactosamine) by the colectomized rats from 473 M.mol per day to 634 ,umol perday. We conclude that mucin is the major endogenous carbohydrate excreted from the upper gut and that gumarabic increases the amount of this endogenous carbohydrate.

Carbohydrate sources available for fermentation are thosefrom the diet and endogenous sources discharged into or bythe small or large intestine. Dietary fiber is well recognizedas a major source of fermentable carbohydrate. Recentresearch (9) indicates that starch may not be completelydigested in the small intestine, which would provide addi-tional diet-derived carbohydrate for fermentation. Endoge-nously secreted mucin is also recognized as a microbialsubstrate, although the quantitative significance of mucin tothe overall carbohydrate supply for the microflora andwhether it is the primary source of endogenous carbohydrateare unknown.

Several different kinds of research suggest that the processof fermentation is important to understanding the role ofdietary fiber in the colon. Although the relationship betweendiet and colon cancer is complicated by many factors, oneobservation from epidemiological studies is that diets higherin fiber are consumed in countries where there is a lowerincidence of colon cancer (21). Studies of experimentallyinduced large bowel cancer in rats indicate that partiallyfermented or nonfermented dietary fiber sources usuallylower tumor incidence (16), whereas completely fermentedfibers either have no effect or increase the incidence ofexperimentally induced tumors (14).

Differential effects of dietary fiber on stool weight are alsorelated to fermentation. Completely fermented fiber sources,such as pectin (15), have a negligible effect on stool weight(5). Both partially fermented and nonfermented fibers in-crease stool weight; however, partially fermented fibersources, such as wheat brain (23), increase stool weight totwice that yielded by comparable amounts of cellulose, anonfermented fiber (30).Our primary aim was to characterize the carbohydrate that

would be supplied to the colon for fermentation underphysiological conditions. The first objective was to charac-terize the microbial substrate supply by determining the

* Corresponding author.

carbohydrate content and composition of gastrointestinaloutput from colectomized rats fed fiber-free (FF) diets. Thesecond objective was to determine whether gum arabic(GA), a water-soluble dietary fiber source, had any effect onthe amount and selected physiochemical characteristics ofthe endogenous carbohydrate supplied by the upper gastro-intestinal tract. The third objective was to explore possiblesources of the carbohydrate present in the ileal excreta fromcolectomized rats fed purified FF diets. The amounts of thefour major sugars in mucin, fucose, galactose, glucosamine,and galactosamine (24), were used to estimate mucin secre-tion.

MATERIALS AND METHODS

Animals and diets. Colectomized male rats (n = 10; HarlanSprague-Dawley, Inc., Indianapolis, Ind.) were successfullyprepared 11 months prior to this experiment (9) by using thesurgical procedure described by Lambert (19) in which boththe cecum and colon are removed. Rats were individuallyhoused in wire-bottom cages; food and water were availablead libitum. The experiment was approved by the College ofAgricultural and Life Sciences Research Animal ResourcesCommittee, University of Wisconsin-Madison. Seven ratswere fed a FF AIN-76A diet (1, 2) in which the fiber source(5% cellulose) was omitted from the diet formulation. Threerats were fed a diet in which the cellulose was replaced withGA (Sigma Chemical Co., St. Louis, Mo.). Dietary carbo-hydrate was provided by equal amounts of cornstarch andsucrose (32.5% [wt/wt] of each). Diet ingredients were fromTeklad Diets (Madison, Wis.). Food intake and body weightswere determined every 2 days. The mean + standard devi-ation (+ SD) body weights at the beginning of the collectionperiod of the rats fed the FF and GA diets were not different(461 + 27 and 457 + 33 g, respectively; P > 0.05); the bodyweights at the end of each collection period also were notsignificantly different (486 + 20 and 465 + 33 g, respectively;P > 0.05). The mean + SD daily food intakes per rat were21.5 + 1.8 and 17.8 + 1.9 g per day in the FF and GA diet

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groups, respectively, and were significantly greater in therats consuming the FF diet (P < 0.05). The food intake ofrats fed the FF diet is probably high and includes spilled dietbrushed from the very gummy ileal excreta that could not beaccurately measured.

Ileal excreta collection. After a 7-day adaptation period, theileal excreta were collected daily for 3 weeks from the ratsfed the FF diet and for 2 weeks from the rats fed the GA diet.The ileal excreta for each individual rat were pooled andfrozen (-5°C) as soon as they were collected. The excretafrom each rat were blended (Waring Blendor model 7011G;Dynamic Corp. of America, New Hartford, Conn.) withsufficient water (-2 ml/g of sample) to form a uniformhomogenate and lyophilized; lyophilized samples wereground with a mortar and pestle and passed through a300-,um-mesh screen.

After analysis of excreta from a subset of four rats fed theFF diet, two proportional composites of ileal excreta, one ofFF excreta and one of GA excreta, were prepared by-combining -10% of the total output from each rat. The mean(± SD) daily ileal excretion (dry weight) was 1.0 + 0.1 g perday for rats fed the FF diet and 2.3 + 0.2 g per day for ratsfed the diet containing GA. Ileal excretion was significantlygreater in the rats fed fiber (P < 0.05).

Analysis of ileal excreta from individual rats. Ileal excretafrom four rats fed FF diet were individually analyzed forsugar content. Eight neutral sugars and three amino sugarswere simultaneously measured in the ileal excreta by gaschromatographic analysis of the alditol acetate derivatives ofthe sugars. Triplicate aliquots (25 mg) were hydrolyzed by aSaeman acid hydrolysis procedure (18). Allose (10 g/liter)and N-methylglucamine (1 g/liter) (Sigma) were the internalstandards for the neutral and amino sugars, respectively.The acid hydrolysates were neutralized with barium carbon-ate. Alditol acetate derivatives were prepared as previouslydescribed (17), with the following modifications. The entireneutralized sample (-3 ml) was used, with a proportionalincrease of reagents. The removal of borate, as methylbo-rate, and water was done under vacuum at a constant 50°C,and methanol without acetic acid was used in the third andfourth drying steps.Samples (0.5 to 1.0 ,ul) were injected onto a gas chromato-

graph (model HP 5890A; Hewlett-Packard Co., Palo Alto,Calif.) fitted with a borosilicate glass column (30 m by 0.5mm [inner diameter]) coated with a 0.2-,um 2340 phase(Supelco, Inc., Bellafonte, Pa.) and a flame ionization detec-tor. Sugars were quantitated with an HP 3392A integrator(Hewlett-Packard). The gas chromatograph temperatureprogram and response factors were similar to those previ-ously described (17). Duplicate aliquots of a mixture ofstandard sugars (Sigma) were analyzed with each batch ofsix sample tubes to maintain the precision and accuracy ofthe hydrolysis and derivatization.

Previous research determined that cornstarch in the AIN-76A FF diet was completely digested by colectomized rats(9). Thus, the starch content of the excreta was used tocorrect the carbohydrate in the excreta for contaminationwith starch and sucrose from spilled diets. Ethanol-insolublefractions of triplicate aliquots (200 mg) of excreta wereanalyzed for starch (12). The glucose contributed fromsucrose was calculated based on the portion of sucrose in thediet. The fructose moiety of sucrose was not detected in thegas chromatographic analysis. The ileal excreta from thefour rats were corrected for total glucose (155 + 17 p.mol/g)contributed by dietary starch and sucrose.

Analysis of composite ileal excreta. Duplicate aliquots of

the ileal excreta composites were analyzed for uronic acids(3), crude fat (4), ash (11), and crude protein contents.Nitrogen was measured by a micro-Kjeldahl method (Buchi-Brinkmann digestion unit model 430 and distillation unitmodel 320; Brinkmann Instruments, Inc., Westbury, N.Y.)and converted into crude protein by using a protein/nitrogenratio of 6.25.The neutral and amino sugar contents in the composites of

ileal excreta from rats fed the FF or GA diet were deter-mined as described above. The composites were correctedfor 161 and 172 ,umol of total glucose per g, respectively,contributed by dietary starch and sucrose. The compositesof ileal excreta were not corrected for the 17-mg/g contam-ination by other diet components, i.e., protein, fat, and ash.Urea contamination from urine was determined by mea-

suring it in the composite ileal excreta by high-performanceliquid chromatography and subtracting the nitrogen portionof urea from the total nitrogen content of the ileal excreta.The 80% ethanol-soluble fraction of the ileal excreta wasdried by rotoevaporation, redissolved in 2.0 ml of water, andpassed through three Sep-Pak C18 cartridges (Waters Asso-ciates, Milford, Mass.) and a 0.2-p.m-pore-size membranefilter (Gelman Sciences, Inc., Ann Arbor, Mich.). Urea wasquantitated by using an Aminex HPX-87P high-performanceliquid chromatography column (Bio-Rad Laboratories, Rich-mond, Calif.) with a refractive index detector. The ilealexcreta from rats fed FF and GA diets contained 1,100 and1,500 ,umol of urea per g, respectively.

Fractionation of composite ileal excreta. Water-solublefractions of the ileal excreta were obtained by extractingaliquots (7.0 g) of each composite three times with water (30ml/g) with stirring (20 min, ambient temperature). The solu-ble fraction was recovered by decanting the centrifuged(30,000 x g, 30 min) samples. The pellet was the insolublefraction. The soluble and insoluble fractions were dried bylyophilization. The gravimetric yields of the water-solublefraction of the ileal excreta from rats fed the FF and GA dietswere 60.5 and 71.9%, respectively. The yields of the water-insoluble fractions were 35.6 and 20.7%, respectively.

Aliquots (500 mg) of the soluble fractions of ileal excretawere rehydrated in a 0.2-g/liter aqueous sodium azide solu-tion (40 ml) and separated by gel filtration chromatographyon a K50/100 column (80 cm by 5 cm [inner diameter];Pharmacia, Inc., Piscataway, N.J.) containing -1,800 ml ofSepharose CL-2B-300 gel (Sigma). The column flow was

maintained at 90 mi/h with a positive displacement pump(LDC/Milton Roy, Riveria Beach, Fla.). Void (460-ml) andbed (-1,800-ml) volumes of the column were determined bymonitoring at 280 nm; the eluent was aqueous 0.2-g/litersodium azide. Molecular mass standards of dextran (2,000,480, 71.2, 42.5, and 9 kDa; Sigma) were chromatographed todetermine the elution positions of different molecularmasses.The eluent was collected, beginning with the void volume,

in 9-ml aliquots; 0.5 ml of every fourth aliquot was analyzedcolorimetrically for total neutral carbohydrate (6). Columnfractions 1 through 40 from the chromatography of thewater-soluble fraction of FF ileal excreta and fractions 1through 25 from chromatography of the soluble fraction ofGA ileal excreta were pooled into a high-molecular-weightpeak that corresponded to the range in which the 2,000-kDadextran standard eluted; fractions 41 through 92 and 26through 109, respectively, were pooled into a medium-molecular-weight peak, which included the 480-kDa dextran;fractions 93 through 173 and 110 through 173, respectively,were pooled into a low-molecular-weight peak that included

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3332 MONSMA ET AL.

a)

E

;. 4-coCt

n

2-

z

a)

E

CD

co

~a

z

FF

20 40 60 80 100120140160180

Fraction

0 20 40 60 80 100120140160180

Fraction

FIG. 1. Gel filtration chromatography of soluble fractions of ileal

digesta from colectomized rats fed a FF or GA diet. Three peakswere identified by colorimetric analysis (24) of every fourth fraction.Each fraction was 9 ml, the void volume was 460 ml, and the bedvolume was -1,800 ml. Molecular mass dextran standards eluted asfollows: 2,000 kDa, fractions 5 through 17; 480 kDa, fractions 53

through 97; 71.2 kDa, fractions 93 through 117; 42.5 kDa, fractions97 through 121; and 9 kDa, fractions 105 through 135.

the 71.2, 42.5, and 9-kDa dextrans (Fig. 1). Fractions wereconcentrated by lyophilization.

Analysis of fractions of composite ileal excreta. Insolubleand soluble fractions of ileal excreta and the fractions

corresponding to the three molecular weight carbohydratepeaks were analyzed for neutral and amino sugar contents.The mean recoveries of the individual sugars that were

present above trace amounts (>2 ,mol/g) were calculated asthe sum of the amounts of each sugar in the soluble andinsoluble fractions of ileal excreta from rats fed FF and GAdiets divided by the amount of the sugar analyzed in theunfractionated excreta. These recoveries were as follows:rhamnose, 96%; fucose, 102%; ribose, 82%; arabinose, 87%;xylose, 106%; mannose, 90%; galactose, 93%; glucose, 97%;glucosamine, 101%; and galactosamine, 101%. Muramic acidwas detected only at trace amounts.The recovery of total sugars as the sum of the sugars in the

soluble and insoluble fractions of the ileal excreta from ratsfed the FF diet was 90.9%; 93.1% of the total ileal excretasugar was recovered as these two fractions of the excretafrom colectomized rats fed GA.The glucose concentrations of the soluble fractions of ileal

excreta from rats fed FF and GA diets were adjusted toaccount for 56 ,mol (dry weight) of glucose per g fromdietary sucrose contamination, and the insoluble fractions ofboth ileal excreta were corrected for 89 ,umol of glucose from

dietary starch per g. Starch was not detected in the solublefraction of either of the ileal excreta. The chromatographicfractions corresponding to the low-molecular-weight carbo-hydrate peak was assumed to contain the sucrose and wascorrected for the glucose moiety of sucrose.Sodium azide in the column eluent affected the acid

hydrolysis losses of neutral and amino sugars. New responsefactors were determined by analyzing aliquots (10 to 25 mg)of soluble fractions of ileal excreta from rats fed FF and GAdiets in the presence of various amounts of sodium azide (5to 30 mg). The recovery of total sugars from gel filtrationchromatography of the soluble fraction of ileal excreta wasdetermined as the sum of sugars in the three fractionsdivided by the amount of sugars applied to the column; therecoveries were 94 and 97% for the soluble fractions ofexcreta from rats fed the FF and GA diets.

Dietary fiber analysis. The dietary fiber composition of GAwas determined in duplicate by a modification (29) of themethod of Theander and Westerlund (32). Sugars weremeasured by gas chromatography as their alditol acetatederivatives. Uronic acids and crude protein were measuredin duplicate as described above.

Analysis of endogenous carbohydrate sources. Hog pancre-atin (catalog no. P7545; Sigma) and the acetone-solublefraction of rat pancreas (catalog no. P2017, Sigma) wereanalyzed in triplicate for neutral and amino sugar composi-tion. Hog gastric mucin (catalog no. M2378; Sigma) wasextracted with 80% ethanol to remove low-molecular-weightcontaminants, dried, and then extracted with water to obtainwater-soluble mucin (24). The original material was 67.7%water soluble. The water-soluble material was analyzed intriplicate for neutral and amino sugar contents.A partially purified mucin source was prepared from the

small intestines of 15 retired-breeder female rats (HarlanSprague-Dawley) (26). After overnight fasting, the animalswere killed and the small intestines were immediately re-moved. The small intestine was opened onto an ice-coldglass plate; the mucosa was scraped off with a glass slide.The scrapings were homogenized in 0.2 g of sodium azideper liter (-10 ml/g) and centrifuged (30,000 x g, 30 min); themucin-containing supernatant (26) was decanted and lyoph-ilized. A 500-mg aliquot was separated by gel filtrationchromatography as described above. The void volume peak(column fractions 1 through 21), which would contain unde-graded mucin (26), was lyophilized and analyzed in triplicatefor neutral and amino sugar contents.

Statistical analysis. Body weight, food intake, and fecaloutput were compared by unpaired, two-tailed t-test byusing the Number Cruncher Statistical System (10). Differ-ences were considered significant at P < 0.05.

RESULTS

Analysis ofGA dietary fiber. GA was 94.8% water soluble;the water-insoluble fiber fraction of GA was <0.2% of thestarting material and was not analyzed. The GA water-soluble fraction was 87.4% soluble dietary fiber consisting of82.7% neutral sugars and 4.7% uronic acids. The amount ofcrude protein in the water-soluble fraction of GA was 4.5%.Only three neutral sugars (rhamnose, arabinose, and galac-tose in a molar ratio of 1.0:2.1:4.3) were detected in thesoluble fraction of GA. By using these data, the concentra-tion of dietary fiber in the GA diet was calculated to be 4.4%.

Analysis of individual rat ileal excreta. Daily excretion ofsugar by the colectomized rats fed the FF or GA diet wasexpressed on the basis of food intake to account for the

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TABLE 1. Sugar composition of ileal excreta fromcolectomized rats fed FF and GA diets

Amt of sugar (>mol/day/g of food intake)in ileal excreta of rats fed:

Sugar FF diet GA diet

Individuala Compositeb compositeb

Rhamnose 0 0 1 0 33 2Fucose 2 1 3 0 6 0Ribose 1 0 1 0 1 0Arabinose TRC TR 84 + 1Xylose TR TR 1 ± 0Mannose 1 ± 1 2 ± 0 4 0Galactose 5 ± 1 6 ± 0 112 4Glucose 1 ± 0 3 ± 0 13 1Muramic acid TR TR TRGlucosamine 6 1 5 ± 0 12 ± 0Galactosamine 4 ± 1 3 ± 0 7 ± 0Total 19 4 22 ± 0 274 ± 13

a Values are means + SDs from individual rats (n = 4). Each sample wasanalyzed in triplicate.

b Values are means ± SDs of triplicate analyses of composite of ilealexcreta from 7 (FF) or 3 (GA) rats.

c TR, trace amount (<1 jmol per day per g of food intake).

difference in food intake between the two groups of animals.Daily excretion of neutral and amino sugars in the ilealexcreta from four rats fed the FF diet was 19 ± 4 ,umol perday per g of food intake (Table 1). Fucose, galactose,glucosamine, and galactosamine accounted for 89.5% of thesugars in the ileal excreta.

Analysis of composite ileal excreta. The composite of ilealexcreta from rats fed a FF diet contained 86 mg of totalsugars per g, measured as the sum of neutral and aminosugars and uronic acids. Crude fat (83 mg/g), crude protein(473 mg/g), and ash (308 mg/g) made up the balance of theileal excreta. The total sugar concentration of the compositeof ileal excreta was nearly fourfold greater when GA wasincluded in the diet; the ileal excreta containing GA were 372mg of total sugar per g, 60 mg of crude fat per g, 322 mg ofcrude protein per g, and 194 mg of ash per g. Uronic acidswere only minor components (3 and 19 mg/g, respectively) ofthe ileal excreta from rats fed the FF and GA diets. Gravi-metric recoveries, calculated as the sums of total sugar,crude protein, crude fat, and ash, were 95.0 and 94.8% of thecomposites of ileal excreta from colectomized rats fed theFF and GA diets, respectively.

Daily excretion of neutral and amino sugars in the com-posite of ileal excreta from rats fed the FF diet was 22 + 0,umol per day per g of food intake, similar to the individualanalysis of ileal excreta from the four rats fed the FF diet(Table 1). Daily excretion of neutral and amino sugars in thecomposite of ileal excreta increased to 274 + 13 p.mol perday per g of food intake when GA was included in the diet(Table 1). Fucose, galactose, glucosamine, and galac-tosamine accounted for 77.3% of the sugars in the ilealexcreta from rats fed the FF diet (Table 1). The three sugarspresent in GA, rhamnose, arabinose, and galactose, ac-counted for 83.6% of the total sugars in ileal excreta con-taining GA.

Fractionation of composite ileal excreta. Most of the GA inthe ileal excreta was extracted into the water-soluble frac-tion, increasing the sugar content of the soluble fraction from372 + 28 ,umol/g for FF excreta to 1,898 + 55 p,mol/g for GAexcreta; GA had little effect on the sugar content of insoluble

TABLE 2. Sugar composition of water-soluble and -insolublefractions of composites of ileal excreta fromcolectomized rats fed FF and GA dietsa

Amt of sugar (p.mollg [dry wt] of excreta)in composite ileal excreta of rats fed:

Sugar FF diet GA diet

Soluble Insoluble Soluble Insoluble

Rhamnose TRb 6 243 5Fucose 44 4 48 3Ribose 5 4 8 3Arabinose TR TR 552 9Xylose TR TR 11 TRMannose 30 3 25 3Galactose 95 17 799 19Glucose 12 5 80 24Muramic acid 0 TR 0 2Glucosamine 109 9 80 6Galactosamine 64 TR 52 3Total + SD 372 + 28 58 + 4 1,898 + 55 76 + 3

a Values are means of triplicate analyses; the standard deviations forindividual sugars were similar to those in Table 1.

b TR, trace amount (<2 p,mol/g of dry weight).

material in ileal excreta (Table 2). Fucose, galactose, glu-cosamine, and galactosamine accounted for 83.9% of thetotal carbohydrate in the soluble fraction of ileal excretafrom rats fed the FF diet. Rhamnose, arabinose, and galac-tose accounted for 84.0% of the sugar in the soluble fractionof GA ileal excreta. Daily output of water-soluble sugar wasover 10-fold greater with GA in the diet (245 ,umol per dayper g of food intake, compared with 17 p,mol per day in ratsfed the FF diet); daily water-insoluble output was alsogreater (10 and 3 p,mol per day per g of food intake for ratsfed the GA and FF diets, respectively).The concentrations of total carbohydrate in the three

chromatographic fractions corresponding to the high-, medi-um-, and low-molecular-weight peaks of the water-solublefraction from FF ileal excreta were 89, 209, and 279 j±mol/gof soluble ileal excreta, respectively (Table 3). Fucose,galactose, glucosamine, and galactosamine accounted for84.3% of the carbohydrate in the fraction corresponding tothe high-molecular-weight peak of the FF ileal excreta and86.1 and 62.7% of the total carbohydrate in the fractionscorresponding to the medium- and low-molecular-weightpeaks, respectively. When GA was included in the diet, 64and 223 ,umol of carbohydrate per g of soluble ileal excretawere in the fractions corresponding to the high- and low-molecular-weight carbohydrate peaks, respectively; 2,284p,mol/g of soluble excreta was in the fraction containing themedium-molecular-weight compounds (Table 3). Fucose,galactose, glucosamine, and galactosamine accounted for68.8 and 49.3% of the total carbohydrates in the fractionscorresponding to the high- and low-molecular-weight peaks,respectively. Rhamnose, arabinose, and galactose ac-counted for 93.1% of the total carbohydrate in the medium-molecular-weight fraction.

Analysis of endogenous carbohydrate sources. The totalneutral and amino sugar contents of commercially preparedpancreatin and pancreas acetone powder were 152 and 217,umol/g, respectively (Table 4). Ribose accounted for 66.4and 72.8% of the total sugars, respectively. Mannose, galac-tose, glucose, and glucosamine were the only other sugarsdetected in these preparations. The water-soluble fraction ofthe commercially prepared hog gastric mucin contained

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TABLE 3. Sugar compositions of molecular weight peaks of soluble fractions of ileal excreta from rats fed FF and GA dietsa

Amt of sugar (p,g/g [dry wt]) in soluble fraction of ileal excreta in rats fed:

Sugar FF diet GA diet

High Medium Low High Medium Low

Rhamnose TRb TR TR TR 339 TRFucose 10 18 27 7 38 15Ribose TR 0 TR TR TR TRArabinose TR 7 24 13 747 22Xylose TR 0 TR TR TR TRMannose TR 8 40 1 10 26Galactose 26 65 59 16 1,041 40Glucose 13 13 39 7 16 65Muramic acid 0 TR 0 TR 0 TRGlucosamine 20 49 69 11 48 40Galactosamine 19 48 20 10 43 15Total + SD 89 + 2 209 ± 12 279 + 7 64 14 2,284 + 87 223 ± 7

a Values are means of triplicate analyses; the standard deviations for individual sugars were similar to those in Table 1. High, medium, and low refer to fractionswith peaks at -2,000, -480, and <71 kDa, respectively.

b TR, trace amount (<7 p.mol/g [dry weight] of soluble fraction).

3,082 ,umol of neutral and amino sugars per g (Table 4).Fucose, galactose, glucosamine, and galactosamine ac-counted for 95.6% of the total carbohydrate. The mucinextracted from rat small intestine scrapings contained 112,umol of total neutral and amino sugars per g (Table 4).Fucose, galactose, glucosamine, and galactosamine ac-counted for 76.2% of the sugar. Glucose was the only othersugar detected.

DISCUSSION

Sources of fermentable carbohydrate in excreta fromcolectomized rats fed a purified FF diet would include theglycoprotein mucin, gastrointestinal secretions (includingenzymes and immunological components), debris fromsloughed cells, and bacteria. The sugar compositions andmolecular weights of the ileal excreta carbohydrates mea-sured in this study indicate that mucin is a major source of

TABLE 4. Sugar compositions of endogenouscarbohydrate sourcesa

Amt of sugar (p.mol/g [dry wt]) in:

Sugar Water-soluble Rat smallSuPna Pancreatic fraction of intestalextract hog gastric itsinb

mucin mucin

Rhamnose TRC 0 TR TRFucose TR TR 393 ± 254 12 + 21Ribose 101 + 5 158 + 6 87 4 TRArabinose TR TR TR TRXylose TR TR TR TRMannose 18 0 16 0 27 0 TRGalactose 12 2 12 2 928 32 34 4Glucose 7 1 12 1 23 3 18 2Muramic acid TR 0 TR 0Glucosamine 15 + 2 19 + 3 1,016 +77 30 2Galactosamine 0 0 608 ± 41 17 ± 1Total 152 + 7 217 + 8 3,082 ± 154 112 ± 8

a Values are means + SDs of triplicate analyses. Sources of carbohydratesare given in Materials and Methods.

b 2,000-kDa fraction of water-soluble component of rat small intestinalscrapings.

c TR, trace amount (<7 Axmolg of dry weight).

endogenous carbohydrate. The five sugars in mucin arefucose, galactose, glucosamine, galactosamine, and sialicacids (24). Wold et al. (33) fed intact, germfree rats achemically defined, FF diet containing glucose as the solecarbohydrate source and measured the sugar composition ofthe water-soluble fraction of feces that had been extractedsequentially with acetone, chloroform-methanol, and etherto remove nonpolar substances. Four mucin-derived sugars,fucose, galactose, glucosamine, and galactosamine, ac-counted for 85% of the total sugars they detected. Thesesame four sugars accounted for 77% of the total sugar wedetected in unextracted ileal excreta and 84% of the totalsugar in the water-soluble fraction from colectomized ratsfed FF diets. The ratios of these sugars in the excretasamples we and Wold et al. (33) studied are similar to thesugar ratios determined in preparations of intestinal mucin(Table 5). The heterogeneity of the carbohydrate composi-tion of mucin extracted from different sites in the gut (Table5), differences in isolating the mucin and in the methods usedfor carbohydrate analysis, and the fact that colectomized ratileal excreta do not include colonic mucin may be responsi-ble for the small differences in sugar ratios from the variouslaboratories.Mucin is defined, in part, as the water-soluble fraction of

mucosa that is 2,000 kDa; it consists of four 500-kDasubunits (26). The fact that -60% of the four mucin-derivedsugars in the water-soluble fraction of the rat FF ileal excretaeluted upon gel filtration chromatography in the 2,000- and500-kDa ranges also supports the conclusion that mucin is amajor source of endogenous carbohydrate. Fucose, galac-tose, glucosamine, and galactosamine that eluted in thelowest-molecular-mass fraction (<71 kDa) may representthe oligosaccharide chains of 2 to 20 sugar residues that arethe major carbohydrate constituent of mucin (24). Themajority (79%) of the other water-soluble sugars in the ilealexcreta, arabinose, mannose, and glucose, eluted in thefraction that contained molecules of .71 kDa. Conceivably,those molecules could reflect the carbohydrate moietiesfrom membrane glycoproteins (13). The low sugar contentsof pancreatin and pancreas acetone powder suggest that theywould not contribute substantially to the carbohydrate poolavailable for fermentation.

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TABLE 5. Molar ratios of the relative proportion of mucinsugars from various gastrointestinal sources

Source of sugar Relative molar amounts of:(reference) Fucose Galactose Glucosamine Galactosamine

Gastric (31) 1.0 2.2 2.4 0.6Gastric' 1.0 2.8 3.1 1.9Small intestine (7) 1.0 2.3 2.0 2.0Small intestineb 1.0 3.3 2.5 1.5Colon (20) 1.0 3.2 3.7 3.7FF feces from germ- 1.0 2.4 2.8 2.5

free rats (33)Composite of FF ileal 1.0 2.2 2.5 1.3

excreta fromcolectomized ratsc

Water-soluble frac- 1.0 2.1 2.4 1.4tion of compositec

Composite of GA ileal 1.0 _d 2.2 1.2excreta fromcolectomized ratsc

Water-soluble frac- 1.0 - 1.7 1.2tion of compositec

a Water-soluble fraction of hog gastric mucin (see Materials and Methods).b 2,000-kDa fraction of water-soluble component of rat small intestinal

scrapings (this study).This study.

d The calculate fucose/galactose ratio could not be calculated because of thegalactose present in mucin and GA.

Galactose in the composite of ileal excreta from thecolectomized rats fed GA arises from both GA and mucin.When the fucose/galactose ratio in the composite of ilealexcreta from rats fed FF diet (1:2.0) was used to determinethe proportion of galactose derived from endogenoussources, 37 ,umol of the four, mucin-derived sugars per dayper g of food intake was present in the GA ileal excreta. Incontrast, 17 ,umol of the total sugars in the ileal excreta fromrats fed FF diet per day per g of food intake were fucose,galactose, glucosamine, and galactosamine. These estimatessuggest that GA, a water-soluble dietary fiber, increased theamount of mucin-derived carbohydrate available for fermen-tation twofold. This extends the findings of Satchithanandamet al. (27), who observed increases in total luminal mucinfrom the stomach and small intestine when rats were fed adiet containing either 5% guar gum or citrus fiber comparedwith that in rats fed a FF diet. Their measurements utilizeda monoclonal antibody and did not include any estimate ofthe fermentable carbohydrate. The fact that fiber increasesmucin secretion indicates that using carbohydrate excretedduring ingestion of FF diets to correct fiber digestibilitydeterminations (25) would lead to an inflated fecal fibervalue.One concern of the colectomized rat model is the potential

for recolonization of the ileum by bacteria and subsequentfermentation of undigested carbohydrates. Bacteria are nor-mally found in the upper gastrointestinal tract (8), which mayexplain the trace amounts of muramic acid (found exclu-sively in bacteria [28]) that were detected in the ileal excreta.The level of muramic acid detected in the ileal excreta wassubstantially less than the 8 pumollg we measured in rat feces(17). Some of the muramic acid that was detected could haveoriginated from bacterial contamination and growth after theileal excreta had been excreted but before they were col-lected and frozen. When raffinose, a nonabsorbable, fer-mentable trisaccharide, was intubated with AIN-76A dietcontaining the nonabsorbable marker chromic oxide into six

colectomized rats, the amounts of chromium and raffinosecollected in the subsequent 24 h of excreta were similar(-70%), suggesting that there is no significant fermentationin the guts of colectomized rats (22).These analyses demonstrate that in the absence of dietary

fiber, the total amount of carbohydrate available daily forfermentation was small (473 p.molday) and mostly watersoluble, with nearly half (45%) of the soluble carbohydrateeluting in the chromatographic fraction that was <71 kDa.The high degree of solubility and relatively low molecularweight of the carbohydrate suggest that such a substratewould be rapidly fermented in the proximal colon. When GAwas included in the diet, the daily amount of total carbohy-drate from the upper gut was 12-fold greater (4,877 pmol),although most of the carbohydrate in the ileal excreta wasstill water soluble. Thirteen percent of the fermentablecarbohydrate was supplied by mucin. Such a substratesupply may stimulate bacterial growth in the proximal colon,resulting in an overall shift in metabolic activity of themicroflora to the proximal colon.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grant DK-21712 from the National Institute of Diabetes and Digestive andKidney Disease, by University of Wisconsin-Madison Hatch Proj-ect 3389, and by the College of Agricultural and Life Sciences,University of Wisconsin-Madison.

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