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© 2004 The American Society for Nutritional Sciences J. Nutr. 134:493-500, March 2004

Biochemical and Molecular Actions of Nutrients

Resistant Starch Modulates In Vivo Colonic Butyrate Uptake and Its Oxidation in Rats with Dextran Sulfate Sodium-Induced Colitis

Noëlle M. Moreau*,{ddagger}, Martine M. Champ{dagger},{ddagger}, Stéphane M. Goupry*,{ddagger}, Bruno J. Le Bizec**, Michel Krempf{ddagger}, Patrick G. Nguyen*,{ddagger}, Henri J. Dumon*,{ddagger} and Lucile J. Martin*,{ddagger},1

* Unité de Nutrition et d’Endocrinologie, Ecole Nationale Vétérinaire, Nantes, France; {dagger} Laboratoire des Fonctions Digestives et Nutrition Humaine, Institut National de la Recherche Agronomique, Nantes, France; ** Laboratoire d’Etudes des Résidus et des Contaminants dans les Aliments, Ecole Nationale Vétérinaire, Nantes, France; et {ddagger} Centre de Recherche en Nutrition Humaine, Nantes, France

1To whom correspondence should be addressed. E-mail: lucile.martin{at}vet-nantes.fr.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
We previously demonstrated improvements of colonic lesions due to dextran sulfate sodium (DSS) in rats after 7 d of supplementation with resistant starch (RS) type 3, a substrate yielding high levels of butyrate (C4), a colonic cell fuel source. In the present study, we hypothesized that if inflammation is related to decreased C4 utilization by the colonic mucosa, RS supplementation should restore C4 use simultaneously with an increase in the amount of C4 present in the digestive tract. Hence, we compared, in vivo, the cecocolonic uptake of C4 and its oxidation into CO2 and ketone bodies in control and DSS-treated rats fed a fiber-free basal diet (BD) or a RS-supplemented diet. Sprague-Dawley rats (n = 60) were used. DSS treatment was performed to induce acute colitis and then to maintain chronic colitis. After cecal infusion of [1-13C]-C4 (20 µmol in 1 h), concentrations and 13C-enrichment of C4, ketone bodies, and CO2 were quantified in the abdominal aorta and portal vein. Portal blood flow was recorded. During acute colitis, 13C4 uptake and 13CO2 production were lower in DSS rats than in controls. During chronic colitis, DSS rats did not differ from controls. After 7 d of chronic colitis, RS-DSS rats exhibited the same C4 uptake as BD-DSS rats in spite of higher C4 cecocolonic disposal. After 14 d, C4 uptake was higher in RS-DSS than in BD-DSS rats. Thus, the increased utilization of C4 by the mucosa is subsequent to evidence of healing and appears to be a consequence rather than a cause of this RS healing effect.

KEY WORDS: • butyrate • metabolism • colitis • rat • resistant starch

As end products of the anaerobic colonic fermentation of carbohydrates, the SCFA, mainly acetate, propionate, and butyrate, play a vital role in maintenance of colonic integrity and metabolism. Among them, butyrate (C4)2 apparently has trophic properties for the healthy or injured colonic epithelium (1–3), although some data are conflicting (4). In vitro studies demonstrated that C4 also is the preferred energy-providing substrate for colonic cells (5–7). The pool of acetylCoA, resulting from the ß-oxidation pathway of C4 in the colonocyte, is oxidized into CO2 and ketone bodies (KB), acetoacetate (AA) and ß-hydroxybutyrate (BHB), via the hydroxy-methyl-glutaryl CoA pathway (Fig. 1). Lipogenesis (9), histone acetylation (10,11), detoxification of xenobiotics (12,13), and mucus synthesis (14) depend upon C4 metabolism [and to a lesser degree, upon propionate metabolism (10,14)]. Moreover, C4 is able to prevent the development of abnormal colonocytes more efficiently than propionate and acetate (15).



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  		FIGURE 1 ß-Oxidation pathway of butyrate to acetylCoA, CO2, and ketone bodies in colonocytes [adapted from (8)].

 
Butyrate is of great importance for colonic epithelial health because a lack of C4 can result in intestinal inflammation (16). Rectal instillation of a specific chemical inhibitor of ß-oxidation induces an acute colitis in rats (17). It was suggested that abnormalities in C4 oxidation may be involved in the pathogenesis of mucosal inflammation such as ulcerative colitis (UC) (18). The effects of the inflammation process on C4 utilization are well documented in vitro (18,19–22) but not frequently in vivo (23,24). The decrease of C4 oxidation into CO2 observed during acute inflammatory lesions (16,20,22) tends to regress (18,21,23) or even disappear (19,24) during chronic phases. Nevertheless, in vivo data obtained by measurements of 13CO2 (24) or 14CO2 (23) in exhaled breath after 13C4or 14C4 enemas in patients suffering from UC, give a relatively indirect estimation of C4 colonic oxidation because part of the labeled exhaled CO2 could originate from C4 hepatic oxidation. The few results available on C4 oxidation into KB (18,22) indicated a decreased oxidation in vitro in inflamed colonocytes (18,22)

Consequently, in the present study, we examined the in vivo C4 oxidation into CO2 and KB by the colonic mucosa in a previously validated (25) experimental model of colitis and after [1-13C]-C4 cecal infusions. The colitis was induced by continuous administration of dextran sulfate sodium (DSS) in water and characterized by macroscopical and histological observations (25). This model allowed us to show that a diet containing resistant starch (RS) type 3 induced significant improvements of cecocolonic injuries (26). The selected RS source (Novelose 330, by National Starch) was a pure nongranular retrograded starch devoid of any other macroconstituents with 50 g/100 g of total dietary fiber as measured by the AOAC method (27) and a small intestinal digestibility of ~50% in humans (28). The healing effect of RS observed on DSS colitis seemed to be linked in part to SCFA and particularly C4 production (24). Therefore, we hypothesized that the greater the amount of available C4, the more it would be used by the inflamed mucosa for its energy requirements (29). We also hypothesized that acute and chronic DSS inflammation would impair C4 uptake and both of its oxidation pathways, and that the healing effect of RS is a consequence of an increase in C4 disposal that should restore C4 use for the colonic mucosa.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Animals, diets, and housing. This experiment was approved by the Ethics Committee for Laboratory Animals (hosted by the National Veterinary School of Nantes) and conducted according to the French animal welfare rules (Decree no. 87–848, 19 October 1987). Male Sprague-Dawley rats (n = 60; Harlan), weighing 310 ± 10 g at arrival were used. They were housed individually in cages and had free access to food and water until killed. A light:dark cycle from 0800–2000 h and 2000–0800 h, respectively, was used. Two different diets were specially formulated for the experiment (INRA) (Table 1). The basal diet (BD) was a low-fiber diet (only 2 g/100 g cellulose), whereas the RS-supplemented diet was formulated by substituting 6.0 g/100 g of RS type 3 (retrograded Hylon 7, high amylo-cornstarch; Novelose 330, National Starch and Chemical) for pregelatinized cornstarch included in the basal diet. Diets were formulated to provide 6.0 g/100 g of indigestible carbohydrates in addition to cellulose on a dry matter basis. Body weight, water, and food intakes were recorded daily.


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  		TABLE 1 Composition of the two experimental diets

 
    Induction of colitis. After 7 d of adaptation to both the diet and housing conditions, rats were randomly divided into two groups. The first group (DSS group) was administered a 50 g/L solution of DSS (MW = 36–44 kDa, ICN Pharmaceuticals) in water for 7 d to rapidly induce acute colitis (DSS-acute group). As described in a previous paper (25), injuries were characterized histologically and typical lesions were noted, i.e., ulcerations, infiltrate rich in neutrophils and eosinophils, edema and crypt abscesses, vascular congestion, loss of goblet cells, and focal ischemic lesions (25). The dose of DSS was then reduced to 30 g/L for 7 (DSS-chronic 7) or 14 d (DSS-chronic 14) to maintain the inflammation into a stable chronic active phase. These types of lesions were described previously, and histological analysis revealed severe disruption of tissue architecture, exulcerations, shifting of the submucosal edema in muscularis mucosae localization associated with an inflammatory infiltrate rich in lymphocytes (25). The control group was not administered DSS at any point.

During the adaptation period and the 7 d of 50 g/L DSS treatment (induction of acute colitis), rats were fed BD (Fig. 2). Then, during the 30 g/L DSS treatment period, rats were fed either BD or RS for 7 or 14 d creating 5 DSS groups: DSS-acute, BD-DSS chronic 7, RS-DSS chronic 7, BD-DSS chronic 14, and RS-DSS chronic 14 (n = 6 in each group of rats). Corresponding control groups of 6 rats were also killed at each point of the protocol.



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  		FIGURE 2 Experimental protocol. Control and treated rats were fed a basal diet (BD) or a resistant starch diet (RS) diet. Treated rats were administered 5% DSS for 7 d (acute colitis) then 3% DSS for 7 d (chronic 7) or 14 d (chronic 14).

 
Cecal, proximal, and distal inflammatory injuries were assessed without knowledge of the treatment according to a macroscopic score described by Appleyard and Wallace (30). The macroscopic score (maximum score 16) was the sum of partial scores assessing tissue adhesions, cecal mucosa, ulceration, diarrhea, blood in contents, and the colonic mucosa thickening. We showed previously that this score represented a good marker of the severity of the inflammation as well as mucosal healing (25).

    13C4 infusion and collection of samples. After anesthetization by i.p. injection of 1.5 mg/100 g xylazine (Bayer Pharma) and i.m. injection of 5.5 mg/100 g ketamine (Mérial), rats were laparotomized. A 20 mmol/L infusion of [1-13C] butyric acid (sodium salt, Mass Trace) was performed in the cecum for 1 h with a continuous flow of 1 mL/h delivered by a peristaltic pump (Ismatec SA). Infusion parameters (tracer concentration and infusion duration) were defined to establish an isotopic equilibrium in the portal blood before isotopic measurements. The infusion solution was prepared with sterile water for medical irrigation (B Braun) and osmolarity was adjusted to 300 mOsmol/L. The pH was adjusted to 6.0 using hydrochloric acid (Sigma-Aldrich) before infusion. During the cecal infusion, rats were kept under thermic covers. Portal blood flows were recorded continuously from 30 to 60 min of the infusion with a 2-mm Transonic blood-flow probe (Transonic Systems) around the portal vein. The mean portal blood flow was then calculated for at least 20 min.

After 1 h of infusion, blood samples were collected by venipuncture in the portal vein and in the abdominal aorta with an infusion set (0.4 mm o.d., 27-gauge, 17 mm length; Microflex). Rats were then killed with a 2-mL lethal intracardiac injection of sodium pentobarbital (Vetoquinol).

    Measurements of butyrate and ketone bodies. Isotopic 13C-enrichment and concentrations of butyrate, AA and BHB were measured simultaneously by GC-MS using an analytical procedure we described previously (31). Ketone bodies (AA and BHB) originating from [1-13C] butyric acid could be enriched on none, one (position 1 or 3) or two (position 1 and 3) carbon atoms. Therefore we quantified only ketone bodies (AA and BHB) enriched on none, one or two carbon atoms.

    Measurement of isotopic enrichment of 13CO2 in blood samples. Portal and arterial blood (300 µL) was transferred into a 10-mL evacuated blood collection tube, and atmospheric pressure was reestablished with the addition of helium before slight agitation for 2 h. The gaseous phase was then transferred into a Labco Exetainer system (Labcolimited) and the 13CO2 enrichment was determined by GC/isotope ratio MS (BreathMat, Finnigan). Total blood CO2 concentration was measured using an IRMA Blood Analysis System (Diametrics Medical).

    SCFA measurements in digestive contents. When rats were killed, representative fractions of the cecocolonic contents were removed and processed as previously described (26). Acetate, propionate, and butyrate were quantified by GC (32) (GC-6890, Hewlett-Packard) using 2-ethylbutyric acid as the internal standard.

Calculations and statistics

    C4 uptake. The uptake of digestive C4 by the cecocolonic mucosa was as follows:

It was also expressed as a ratio: C4 uptake ratio

The portal {Delta} 12C4 + 13C4 flux, expressed in µmol/min, was calculated by multiplying the arteriovenous difference ({Delta}) of the 12C4 and 13C4 concentrations (µmol/mL) by the mean portal blood flow (mL/min).

The infused 13C4 flux (µmol/min) was obtained by dividing the amount of infused 13C4 (µmol) by the infusion duration (min). Tracer purity was theoretically 99%. Nevertheless, 13C-enrichment of each infusion solutions was systematically checked and, infused 13C4 flux was corrected in case of 12C4 contamination as necessary.

For digestive 12C4 flux calculations, we supposed that, in the lumen, X represented the digestive 12C4 flux, and Y the digestive 13C4 flux (corresponding to 13C4 natural abundance); A represented the infused 12C4 flux (due to eventual tracer contamination), and B the infused 13C4 flux.

Before and after the 13C4 infusion, the M/[M + (M + 1)] ratio [M is the intensity of the 12C molecular ion and (M + 1) the intensity of the 13C isotopic molecular ion] given by the MS detector for the butyrate corresponded to the ratio (R) of 13C4 flux to the sum of 13C4 and 12C4 fluxes. Calculated with a pool of noninfused rats (6 fed the BD and 6 fed the RS diet), R1 was determined as follows before the infusion: R1 = Y/(X + Y) and Y = R1 · X/(1 - R1).

After the 13C4 infusion, R2 = (Y + B)/(X + Y + A + B)

    Isotopic enrichment of CO2. Based on the enrichment of blood samples ({delta}sa {per thousand}) given by the isotope ratio MS and on the ratio of 13C to 12C of the international PDB standard ({delta}PDB = 0.0112372), the isotopic enrichment of CO2 was expressed in terms of atom % (AP) using the following formula (33):

Then, the isotopic enrichments of CO2 in blood samples were calculated in AP excess (APE) as below: APE = APsample - APbasal

Because of the low blood quantity available in rats, no blood sample representing time 0 was performed before infusion. Basal values were taken from the pool of healthy rats (6 fed the BD and 6 fed the RS diet). Arteriovenous difference ({Delta}) of APE were determined as follows:

All results were expressed as means ± SEM, n = the number of rats. Data were analyzed by 3-way ANOVA with treatment (DSS and control), diet (BD and RS), and time (chronic 7 and chronic 14) as main factors. To analyze the acute colitis data, a 1-way ANOVA was used with treatment (DSS or control) as the factor tested. When differences between means were significant, they were compared by contrasts analysis (Super Anova V.1.11, Abacus Concepts). Differences were considered significant at P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    Food and water consumption and relative body weight gain. During the first 7 d of the experiment (corresponding to the induction of the acute phase of the colitis for the DSS-treated rats), food and water intake were 12.6 ± 0.6 and 14.3 ± 0.9 g/d, respectively, for DSS rats and 15.9 ± 0.5 and 17.1 ± 1.0 g/d, respectively, for control rats. Food and water intakes for the BD-DSS chronic 7, RS-DSS chronic 7, BD-DSS chronic 14 and RS-DSS chronic 14 rats are detailed in Table 2. The water to food intake ratio did not differ between the DSS and control groups (data not shown).


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  		TABLE 2 Food and water intake and relative body weight gain (RBWG) of control and treated rats fed a basal diet (BD) or a resistant starch diet (RS) diet. Treated rats were administered 5% DSS for 7 d (acute colitis) then 3% DSS for 7 d (chronic 7) or 14 d (chronic 14)1, 2

 
Calculated from the beginning of the study to the time of killing, the relative body weight gain (RBWG) was –0.08 ± 0.02 for DSS-acute rats and 0.04 ± 0.01 for the corresponding controls (P = 0.002). The RBWG of the control and DSS chronic 7 and chronic 14 rats are included in Table 2.

    Macroscopic score. The macroscopic scores of all of the control groups were zero. Values were 11.7 ± 1.1 in DSS-acute rats. Macroscopic scores of DSS rats fed the RS diet were significantly lower than BD-DSS rats: 5.2 ± 1.3 in RS-DSS rats vs. 10.8 ± 0.9 in BD-DSS rats after 7 d of supplementation (P = 0.0004) and 5.5 ± 0.8 in RS-DSS rats vs. 8.7 ± 0.6 in BD-DSS-chronic 14 rats (P = 0.0286) (Fig. 3).



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  		FIGURE 3 Macroscopic score of rats fed the basal diet (BD) or the resistant starch diet (RS) and previously treated with DSS [5% DSS for 7 d (acute colitis) then 3% DSS for 7 d (chronic 7) or 14 d (chronic 14)]. Values are means ± SEM, n = 6. Means without a common letter differ, P < 0.05.

 
    SCFA measurements in digestive contents. The dietary treatment affected the total SCFA contents in DSS-rats (RS-DSS chronic 7 vs. BD-DSS chronic 7: P = 0.0001; RS-DSS chronic 14 vs. BD-DSS chronic 14: P = 0.0046). The contents of C4 (as well as total SCFA) were greater in rats fed the RS diet than in those fed the BD diet (Table 3).


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  		TABLE 3 SCFA and butyrate contents of the cecocolon of control and treated rats fed a basal diet (BD) or a resistant starch diet (RS) diet. Treated rats were administered 5% DSS for 7 d (acute colitis) then 3% DSS for 7 d (chronic 7) or 14 d (chronic 14)1, 2

 
    Cecocolonic uptake and oxidation of C4. In the chronic study, regardless of the diet, the treatment, and the time of killing, portal blood flows and 13C4 infused flux did not differ between groups: 7.87 ± 0.55 and 7.65 ± 0.55 mL/min for portal blood flows, and, 0.38 ± 0.02 and 0.35 ± 0.02 µmol/min for 13C4 infused flux for controls and DSS rats, respectively (n = 30 rats per group).

    Acute colitis. The calculated digestive 12C4 flux did not differ between the DSS-acute group (0.48 ± 0.07 µmol/min) and control rats (0.45 ± 0.06 µmol/min). Nevertheless, the colonic butyrate metabolism was altered, as shown in Table 4, i.e., the {Delta}13C4 portal flux was greater in the DSS-acute group compared with the control group. Simultaneously, the parameters associated with the uptake of C4 by the mucosa (C4 uptake and C4 uptake ratio) were significantly lower in the DSS-acute rats than in the control group. Moreover, the acute inflamed mucosa seemed to utilize the digestive C4 differently than the healthy one because the {Delta}13CO2 enrichment (APE x 100) was significantly lower in DSS-acute rats than in controls (Table 4). Portal flux of 12CO2 was not modified between DSS-acute (187.58 ± 17.79 µmol/min) and control rats (167.65 ± 28.42 µmol/min).


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  		TABLE 4 {Delta} 13C4 portal flux, C4 uptake, C4 uptake ratio, and {Delta} 13CO2 enrichment in control rats fed the basal diet and in rats fed that diet and treated with 5% DSS for 7 d1

 
    Chronic colitis. The calculated digestive 12C4 flux did not differ among the groups by time of killing. However, values of digestive 12C4 flux were higher in rats fed the RS diet than in rats fed the BD diet (as expected) regardless of the treatment: 1.80 ± 0.60 and 0.77 ± 0.16 µmol/min) in RS and BD control groups (P = 0.048), 2.65 ± 0.58 and 1.32 ± 0.31 µmol/min) in RS-DSS and BD-DSS groups, respectively (P = 0.011).

Contrary to what occurred during acute colitis, C4 metabolism did not differ between controls and chronically inflamed rats. Results of {Delta}13C4 portal flux did not differ between DSS-chronic 7 (and 14) groups and corresponding control groups with either the BD or the RS diet (Table 5). In all cases, the colonic mucosa utilized between 67 and 85 g/100 g of the available C4. The C4 uptake values (which refer to the uptake of C4 independently of the amount of available C4) tended to be higher in RS-DSS chronic 14 rats than in corresponding controls (P = 0.069). Interestingly, C4 uptake values were significantly higher in RS-DSS chronic 14 than in BD-DSS chronic 14 rats (Table 5).


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  		TABLE 5 {Delta} 13C4 portal flux, C4 uptake, C4 uptake ratio, and {Delta} 13CO2 enrichment in control and treated rats fed a basal diet (BD) or a resistant starch diet (RS) diet. Treated rats were administered 5% DSS for 7 d (acute colitis) then 3% DSS for 7 d (chronic 7) or 14 d (chronic 14)1

 
During the phase of chronic colitis, the oxidation of C4 into CO2 did not differ between the DSS and control groups (Table 5) except for the RS-DSS chronic 14 groups in which {Delta}13CO2 tended to be higher in inflamed rats (P = 0.061).

Substantial heterogeneity was observed in the KB arteriovenous differences within groups. Nevertheless, the ratio of portal 13C-enriched AA to 13C-enriched BHB (singly and doubly labeled molecules) was nearly 3 and did not differ between DSS rats (2.93 ± 0.46) and control rats (3.65 ± 0.56) or between rats fed the BD (3.48 ± 0.48) and the RS diet (3.01 ± 0.54).

The ratio of 13C-enriched KB (the sum of singly and doubly labeled KB) to 13CO2 portal concentrations in DSS-acute rats (1.52 ± 0.28) did not differ from control rats (1.08 ± 0.11). It also did not differ between the RS-DSS (1.45 ± 0.23) and RS-control (2.55 ± 0.67) or between the BD-DSS (1.77 ± 0.60) and BD-control (2.54 ± 0.52) groups.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Many factors are involved in the pathogenesis of UC. Among them, an inadequate uptake or an insufficient utilization of C4 could be implicated. The effect of inflammation on the cecocolonic metabolism of C4 is poorly documented in vivo. Consequently, we hypothesized that acute and chronic inflammatory lesions due to DSS would impair this metabolism. To confirm this, we infused [1-13C] C4 into the cecum of rats, and 13CO2 and 13C-enriched ketone bodies were quantified in portal blood to estimate C4 colonic metabolism in DSS-treated or control rats.

During the study, at each instant, unlabeled butyrate was produced from bacterial fermentation in the intestinal lumen and rapidly absorbed by the mucosa. In our protocol, this system was not interrupted because cecum and blood vessels were not ligated.

The amount of infused tracer was determined according to the previously published values of the pool of cecal C4 (26). In that report, the mean amount of C4 measured at the time of killing was in the range of 10–15 µmol for rats fed the basal diet and 30–74 µmol for rats fed the RS diet. Consequently, the [1-13C]-C4 flux was fixed at 0.33 µmol/min (that is, 20 µmol of tracer infused in 1 h) to follow its utilization by the digestive mucosa and to obtain sufficient concentrations of labeled ketone bodies in the portal blood. Any isotope effect discriminating against labeled butyrate during butyrate transport across the mucosa was very unlikely. Thus, the ratio of 13C-butyrate/12C-butyrate measured in portal blood must reflect the 13C-butyrate/12C-butyrate in the cecal lumen. Because 13C-butyrate/12C-butyrate reached a steady state in portal blood, we therefore assumed that such a steady state also prevailed in the cecal lumen.

During the acute phase of the colitis, our results indicated a decrease in C4 cecocolonic uptake. These data also agree with several in vitro studies, i.e., the mucosal metabolic flux of C4 was decreased in an UC colonic biopsy (21), and the absorption of C4 by the proximal colon was inhibited during acute colitis in rabbits (34). We also showed that {Delta}13CO2 enrichment was lower in DSS-acute rats than in controls. Because portal flux of 12CO2 was not modified between DSS-acute and control rats, we speculated that the decrease in APE was related more to a diminution of 13C4 oxidation to 13CO2 than to an effect of dilution by a higher production of portal 12CO2 in DSS rats. These results of a decrease in C4 oxidation to CO2 are similar to those obtained in isolated colonocytes of mice treated with DSS (22), and in colonocytes isolated from acute UC patients (18). The exhaled 14CO2 measured in acute UC patients after 14C4 enemas was also lower than that in healthy subjects (23).

During the chronic colitis phase, C4 uptake and CO2 production did not differ between rats suffering from chronic colitis lesions and control rats. In the same way, there is no difference in the rate of rectal absorption of C4 from dialysis bags in quiescent UC patients (35). Conversely, a decrease in C4 oxidation to CO2 was observed in the colonic biopsy (20) and isolated colonocytes (18) from quiescent UC patients. This discrepancy between in vivo and in vitro studies could be explained by the difficulty in mimicking the physiologic conditions in in vitro models.

The KB study did not allow us to characterize the possible effect of the inflammatory lesions on KB production with C4 origin. The ratio of 13C-enriched AA to 13C-enriched ß-hydroxy-butyrate (BHB) (singly and doubly labeled molecules) was nearly 3 regardless of the rat group. As observed in ruminants (36), the AA to BHB ratio does not vary with the diet. The addition of C4 to the cell culture medium of colonocytes isolated from healthy rats induces a higher in vitro production of AA than BHB (6,37–39) and the ratio of AA to BHB can vary from 3 (22,40) to 5.5 (38). Ratios of AA to BHB with C4 origin of 1.9, 2.2, and 4.6 were observed in colonocytes isolated from food-deprived rats (38), malnourished rats (38), and DSS-inflamed mice (22). KB production of C4 origin was investigated in vivo in healthy rats after C4 infusions into a cannulated colonic segment cleared of excess mucus and any remaining digesta (41,42). The authors noted that >50% of metabolized C4 was incorporated into BHB, but AA was undetectable (41). Our results refuted this statement and were in accordance with the in vitro AA:BHB ratio. The lack of AA detection by Fitch and Fleming (41) could be due to analytical reasons.

Our results showed a ratio of 13C-labeled KB to 13CO2 of 2.25 ± 0.35 in healthy rats (n = 30). It was slightly higher than the ratios of 14C-labeled KB:14CO2 of 14C4 origin in the above-mentioned studies: 0.5, 0.8, and 1.4 using a 2 mmol/L, 10 mmol/L, or 40 mmol/L 14C4 perfusion (40–60 min) solutions, respectively (41). Increasing perfusion duration (60–90 min) induced a 75% higher ratio using a 10 mmol/L 14C4 solution (42). In our study, a ratio of ~2.25 was obtained using a 20 mmol/L13C4 infusion solution for 60 min, which agrees with a similar 75% increase in the ratio obtained with the 40 mmol/L solution during an infusion of shorter duration (41). Additionally, under our experimental conditions, neither acute nor chronic DSS inflammation affected the ratio of 13C-labeled KB to 13CO2.

In the present study, we showed that C4 colonic uptake decreased during acute colitis, thus reducing the energy contribution available for the colonocytes. This can lead to cellular effects such as cell maturation impairment (18), decrease of mucus synthesis (18), lipogenesis, and cell membrane assembly (9), consequently disturbing the integrity of the mucosa.

By supplementing the diet with RS for 14 d, the uptake of C4 by the inflamed mucosa was higher than in rats fed BD, providing a priori a higher energy supply to the mucosa. Our data also indicated that the digestive pool of C4 measured at the time of killing was significantly higher in DSS-chronic 7 and chronic 14 rats fed the RS diet than in DSS rats fed the basal diet. These results agree with those observed in our previous study (26). In spite of a higher C4 cecocolonic disposal in RS-DSS rats compared with BD-DSS rats, this study showed that the C4 uptake stimulation was observed only after 14 d.

Moreover, using macroscopic markers associated with double-blind histological analysis, we showed in a previous study the healing properties of the RS supplementation on the DSS injuries (26). Major healing signs were present macroscopically and histologically in DSS rats after as little as 7 d of RS supplementation, with confirmation of these improvements after 14 d (26). The RS healing effect was confirmed in the present study using the macroscopic score (Fig. 3).

The improvement of the C4 uptake by the inflamed mucosa appeared after 14 d of RS supplementation, whereas the first signs of mucosal healing were observed after as little as 7 d of RS treatment. First, this seems to indicate that the inflamed colonic epithelium has to be sufficiently healed to utilize the available digestive C4 efficiently. Second, this suggests that the improvement of the C4 uptake by the inflamed mucosa could not be a primary event but a consequence of the healing effect of RS. Consequently, the beneficial properties of dietary fiber yielding large amount of C4 after fermentation, such as RS, may not be obviously related to a C4 direct action via its colonic metabolization. The role of C4 on the immune system (inhibition of nuclear fraction-{kappa}B binding activity, for example) as well as the interactions between bacterial population and dietary substrate appear to be two more convincing hypotheses to explain the anti-inflammatory properties of those dietary fibers (43,44). Interactions between bacterial populations and dietary substrates appear to comprise another potential hypothesis. Indeed, RS ingestion stimulates the growth of lactic-acid bacteria (45,46), which were reported to inhibit sulfate-reducing bacteria particularly involved in the pathogenesis of UC (39,47). Sulfate-reducing bacteria generate high levels of sulfides, sulfites, and mercaptans, and some of these compounds are known to inhibit in vitro C4 oxidation (48) and C4 uptake by isolated colonocytes (49,50). Interactions between RS and lactic-acid bacteria could therefore be one of the mechanisms involved in the healing action of RS.

In conclusion, because the consequences of cecocolonic inflammation on C4 metabolism cannot be predicted from conflicting in vitro data, we developed a model for this study under in vivo conditions, using stable isotopes infusion methods. Our results indicated a decrease in colonic C4 uptake and lower oxidation of C4 to CO2 in rats suffering from DSS acute colitis compared with control rats. During chronic colitis, a normalization of both uptake of C4 and its oxidation to CO2 was observed. After 7 d of RS supplementation, in spite of a higher C4 cecocolonic disposal, chronically inflamed rats fed the RS diet had the same C4 uptake as rats fed the basal diet, whereas this uptake was higher after 14 d of RS supplementation. However, this increase in uptake was observed only after 14 d of RS treatment, which is later than the appearance of the histological evidence of the RS healing effect. The improvement of the colonic ability to utilize C4 appeared to be a consequence of rather than an explanation for the RS action.


   ACKNOWLEDGMENTS
 
The authors are grateful to Professor F. André (LABERCA, National Veterinary School of Nantes) for access to his laboratory. The authors thank Pascale Maugère, Thérèse Frégier, and Anthony Pierre for their excellent technical assistance. The authors acknowledge National Starch (Manchester, UK) for providing the Novelose 330.


   FOOTNOTES
 
2 Abbreviations used: AA, acetoacetate; APE, atom % excess; BD, basal diet; BHB, ß-hydroxybutyrate; C4, butyrate; DSS, dextran sulfate sodium; RBWG, relative body weight gain; RS, resistant starch; UC, ulcerative colitis. Back

Manuscript received 15 April 2003. Initial review completed 16 May 2003. Revision accepted 19 November 2003.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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