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Hokkaido Foundation for the Promotion of Scientific and Industrial Technology, Colabo-Hokkaido, Sapporo 001-0021, Japan;
*
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan; and
Research Center, Nippon Beet Sugar Company, Limited, Obihiro 080-0831, Japan
1To whom correspondence should be addressed. E-mail: hara{at}chem.agr hokudai.ac.jp.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • Ca absorption • intestine • indigestible saccharide • Ussing chamber • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,2
), oligosaccharides (2
–5
) and polysaccharides (6
–8
), results in an increase in Ca absorption in rats, as demonstrated by in vivo balance studies. These sugars increase the Ca content (8
,9
) and the breaking forces of bone (10
) in rats. Sugar alcohols also retard bone resorption in rats (11
). Thus, the ingestion of indigestible sugars might play a beneficial role in the absorption of Ca and its retention in the body.
Intraluminal infusion of sorbitol (a monosaccharide sugar alcohol) increases Ca absorption in the ileal loop of rats (12
). Using a tracer technique with 45Ca, maltitol (a disaccharide sugar alcohol) stimulates Ca absorption in small intestine in rats (13
). In in vitro experiments using everted sacs, maltitol (14
,15
), difructose anhydride (DFA)2
III (4
) and DFAIV (all disaccharides) (5
), and polydextrose (PD, a polysaccharide) (8
) stimulated Ca transport from the mucosal side to the serosal side of the rat small intestine. However, there have been no reports concerning whether these indigestible saccharides stimulate the epithelial tissue of the large intestine directly and promote Ca absorption from the luminal wall of the large intestine. Questions such as which types of saccharides have a strong effect, which portion of intestine is important in resistant sugar–induced Ca absorption and the mechanism by which indigestible sugars enhance Ca absorption in the intestine all remain to be clarified.
Thus, we compared the effects of a variety of indigestible saccharides (disaccharide sugar alcohols, disaccharides, oligosaccharides and polysaccharides) on Ca absorption when applied to the luminal side of epithelial tissue from rat jejunum, ileum, cecum and colon in vitro, using the Ussing chamber system (16
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Male Sprague-Dawley rats (6 wk old, Japan SLC, Shizuoka, Japan) were housed in individual stainless steel metabolic cages. The cages were placed in a room with controlled temperature (22–24°C), relative humidity (40–60%) and lighting (light 0800–2000h). The rats had free access to water and a solid laboratory diet (CE-2, Japan Clea, Tokyo, Japan) for >1 wk before the start of the experiments. They were used in the experiments at 7–9 wk of age (220–290 g). This study was approved by the Hokkaido University Animal Committee and the rats were maintained in accordance with the Hokkaido University guidelines for the care and use of laboratory animals.
Tissue preparation.
On the day of the experiment, the rats were anesthetized with pentobarbital sodium (30 mg/kg body). The jejunum (12 cm in length after Trietz), ileum (12 cm before the point 2 cm from the ileocecal junction), cecum (whole sac) or colon (middle and distal parts, 
12 cm) were quickly removed. The outside surface of each specimen was washed with ice-cold (4°C) saline (154 mmol/L NaCl); then each specimen was cut open along the mesenteric border to produce a flat sheet, and rinsed with ice-cold Tris-HCl buffer solution (TBS). The TBS used consisted of 125 mmol/L NaCl, 4 mmol/L KCl, 10 mmol/L glucose, 30 mmol/L Tris (hydroxymethyl) aminomethane, and 1.25 or 10 mmol/L CaCl2 · 2H2O, gassed with 5% CO2 in O2 to maintain a constant pH of 7.4. TBS containing 1.25 mmol/L Ca was used as the bathing solution for the mucosal component during the experiment and as a stabilizing solution for both the mucosal and the serosal sides between experiments. The serosa and muscle layers were removed, and six stripped preparations, consisting of the mucosa and the submucosa, were mounted onto six Ussing chambers (Diffusion chamber system, Corning Costar, Cambridge, UK), which exposed a circular area of epithelium of 0.64 cm2. The serosal and mucosal sides of the segments were bathed in 1 mL of TBS containing 1.25 mmol/L Ca, continuously exposed to 5% CO2 in O2. After a 30-min stabilization period, the medium on both sides of the tissue was removed by aspiration and 1-mL portions of the appropriate solutions were added to the mucosal and serosal sides.
Indigestible saccharides tested.
Maltitol (Wako Chemical, Osaka, Japan) is a hydrogenated derivative of maltose and is used as an additive in food (1
). DFAIII (4
) and DFAIV (5
) are produced on a large scale by the fermentation of inulin and levan, respectively. Raffinose (Wako Chemical, Osaka, Japan) is a trisaccharide composed of galactose, glucose and fructose produced from sugar beets (2
). Fructooligosaccharide (FOS; Meioligo-P, Meiji Seika, Tokyo, Japan), a mixture of 42% 1-ketose, 46% nystose and 9% fructofuranosylnystose, is an indigestible but fermentable oligosaccharide, which has been well characterized (17
). Polydextrose (PD; Litesse, Culter Foods Japan, Tokyo, Japan) is a random-bonded polyglucose resistant to digestive enzymes and has a relative molecular mass of 1500 kDa (8
).
Experimental procedure.
The six segments of the intestine prepared, including segments of the jejunum, ileum, cecum and colon, were used repeatedly for the experiments at all dosages of each of the six indigestible saccharides tested. A preliminary experiment without indigestible saccharides added to the mucosal medium was performed as a control. Fresh TBS containing 1.25 mmol/L Ca was applied to the serosal bath and 10 mmol/L Ca-TBS was applied to the mucosal bath in the experiments using the jejunum, ileum, cecum or colon preparations. In our previous reports using everted sacs of rat small intestine, the luminal application of DFAIII (4
) or DFAIV (5
) increased net Ca absorption under the experimental condition at 10 mmol/L Ca in the mucosal medium. After a 30-min incubation period, a 0.01-mL sample of the serosal solution was transferred to a polyethylene test tube. The inside of each of the chambers was washed 3 times by the repeated addition and removal of TBS by aspiration, followed by the addition of fresh TBS containing 1.25 mmol/L Ca; then the specimens were left to equilibrate for 30 min before the next application. The same procedure (a 30-min equilibration period and a 30-min incubation period) was repeated for each experiment. The concentration of indigestible saccharides in the mucosal solution was increased (0.1, 1, 10, 50, 100 and 200 mmol/L) at each incubation step.
Analyses.
The Ca2+ concentrations were measured by a colorimetric method using a commercial kit (Calcium C-Test, Wako Chemical). The net transepithelial passage of Ca was expressed as nmol Ca transferred per min per square cm of surface area. All results were expressed as means ± SEM. Statistical analyses were performed by one-way or two-way ANOVA followed by Dunnett’s or Duncan’s test. A difference with P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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shows the dose-response relationship between the concentrations of indigestible saccharides in the mucosal medium and the transepithelial Ca transport. Net Ca absorption in the jejunal, ileal, cecal and colonic epithelial preparations increased dose-dependently. The threshold concentrations required to enhance Ca transport differed among the saccharides tested and among the portions of the small and large intestine examined. Because the net Ca absorption was increased significantly (Dunnett’s test) at saccharide concentrations > 100 mmol/L in all of the sugars tested, the percentage of increase in Ca absorption relative to the control value (at 0 mmol/L) was calculated. The values obtained for the six indigestible saccharides in the four portions of the intestine were compared (Fig. 2
). Two-way ANOVA showed significant differences in net Ca absorption among the saccharides tested (P < 0.001) and among the portions of the intestine examined (P < 0.001), but the interaction was not significant (P = 0.6317). DFAIV had the strongest effect (P < 0.05, Duncan’s test) on net Ca transport in the jejunum and the cecum. Differences in the effects of the applied saccharides on net Ca transport were observed in the ileum, but not in the colon.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In in vivo balance tests in rats, ingestion of indigestible carbohydrates has been shown to result in an increase in Ca absorption (1
–8
). Microbial fermentation in the cecum is considered to be important for transepithelial transport of Ca in the large intestine. Some studies have suggested that the decrease in pH caused by organic acids produced from indigestible sugars changes the solubility of Ca and that this is responsible for the enhancement of Ca absorption (7
,18
,19
). Thus, indigestible saccharides can promote Ca absorption directly (through the saccharide itself) and indirectly (through the organic acids produced from the saccharide) in the large intestine.
Under the experimental conditions employed in this study, the Ca (CaCl2) present in the solution inside the chamber is thought to be a soluble ionized form of Ca. Change in the solubility of Ca was not a contributing factor to the increase in the transepithelial passage of Ca under these conditions. Further, an increase in solute concentration, achieved by adding glycerol or PEG400 at concentrations up to 100 mmol/L, did not affect Ca absorption in the rat intestinal preparations examined (20
). These results indicate that the increase in osmolality by applying saccharides is not a factor in stimulating Ca transport in the epithelium of isolated intestine in this experimental condition.
Among the six saccharides tested, DFAIV showed the strongest effect on Ca transport in the jejunum and cecum (P < 0.05) and its effect also tended to be strong in the ileum (P 
0.215). DFAIV is a disaccharide consisting of two fructose residues with a unique bond and it is a structural isomer of DFAIII. There were no differences among the disaccharides (maltitol and DFAIII), oligosaccharides (raffinose and FOS) and polysaccharide (PD) tested in effectiveness in enhancing Ca transport across the epithelium. Thus, molecular structure rather than the size of the sugar residue may be an important factor in increasing Ca absorption in the epithelium of the intestine.
In general, transepithelial Ca transport in the intestine occurs by two routes, a transcellular pathway and a paracellular pathway (21
,22
). Transcellular absorption is dependent on an active transport process driven by metabolic energy. The diffusion of Ca ions across the cytoplasm is the rate-limiting step because this process is dependent on a Ca-binding protein (22
,23
). It is essentially localized in the upper duodenum and it is totally dependent on vitamin D (22
,23
). Paracellular Ca absorption, involving passive transport (diffusion), requires a chemical gradient of Ca concentration between the lumen and the basolateral side of the intestinal mucosa. Evidence has accumulated indicating that tight junctions, located on the luminal side of adjacent epithelial cells, regulate the absorption of various nutrients including Ca (21
). To date, there is no evidence to suggest that indigestible sugars directly affect the mucosal cells of the intestine and enhance active transcellular Ca transport. On the other hand, there have been a few reports supporting the view that indigestible sugars affect passive paracellular Ca transport in the rat intestine in vitro (4
,5
,15
). Under the experimental conditions used in our study, the Ca concentration in the mucosal medium was 10 mmol/L, higher than that in the serosal medium (1.25 mmol/L). Ca transport in the jejunal, ileal, cecal and colonic epithelium increased linearly in relation to the Ca concentrations in the mucosal medium at Ca concentrations in the range of 2.5–40 mmol/L (unpublished observations). Because no saturation of transepithelial Ca transport was observed, the extent of transcellular Ca passage was considered to be very low under the conditions employed in this study.
After a variety of food-derived chemicals or drugs interact with the mucosal tissue, changes in transepithelial permeability of epithelium of the intestine via the activation of tight junctions are reported to occur in cell lines (24
,25
) and isolated intestinal epithelium (26
). The intracellular mechanism proposed suggests that condensation of actin microfilaments induced by myosin light-chain kinase (MLCK) participates in the opening of tight junctions (27
). The maltitol-enhanced Ca transport in everted ileal sacs of rats was completely inhibited by the preapplication of a calmodulin-dependent MLCK antagonist (15
). It seems possible that a common mechanism via the paracellular pathway is involved in saccharide-induced Ca absorption in the epithelium of the gastrointestinal tract. Further studies are required to determine whether enhancement of Ca transport by the paracellular route is regulated via the activity of tight junctions.
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FOOTNOTES |
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Manuscript received June 25, 2001. Initial review completed July 24, 2001. Revision accepted September 7, 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Goda, T., Yamada, M., Takase, S. & Hosoya, N. (1992) Effect of maltitol intake on intestinal calcium absorption in the rat. J. Nutr. Sci. Vitaminol. (Tokyo) 38:277-286.[Medline]
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Goda, T., Kishi, K., Ezawa, I. & Takase, S. (1998) The maltitol-induced increase in intestinal calcium transport increases the calcium content and breaking force of femoral bone in weanling rats. J. Nutr. 128:2028-2031.
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18. Duflos, C., Bellaton, C., Pansu, D. & Bronner, F. (1995) Calcium solubility, intestinal sojourn time and paracellular permeability codetermine passive calcium absorption in rats. J. Nutr. 125:2348-2355.
19. Younes, H., Demigné, C. & Rémésy, C. (1996) Acidic fermentation in the caecum increases absorption of calcium and magnesium in the large intestine of the rat. Br. J. Nutr. 75:301-314.[Medline]
20. Mineo, H, Hara, H. & Tomita, F. (2001) Short-chain fatty acids enhance diffusional Ca transport in the epithelium of the rat cecum and colon. Life Sci 69:517-526.[Medline]
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