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Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo, 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 MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • nondigestible saccharide • calcium transport • intracellular calcium ion • tight junction • Caco-2 cell
Changes in intestinal calcium absorption and bone mineral density are characteristic of aging or menopause. However, together with lower calcium intake, these changes may also lead to bone diseases. Therefore, increasing not only the dietary intake of calcium but also its bioavailability may provide an effective means of avoiding bone diseases.
A diet rich in nondigestible saccharides may protect against cardiovascular disease, several common cancers, and other chronic diseases (1). The average daily consumption of nondigestible saccharides was estimated to be 14–15 g/d in the United States, although an intake of 25–30 g/d is generally recommended (2). Nondigestible saccharides are categorized as water soluble or insoluble; this reflects different physiochemical properties and ability to produce different biological effects. Ingestion of soluble nondigestible saccharides, including sugar alcohols (3,4), oligosaccharides (5–7), and polysaccharides (8–10), increases calcium absorption in rats, as demonstrated by in vivo balance studies. One mechanism responsible for this increase is the solubilization of calcium salts by acids produced through the microbial fermentation of the saccharides in the large intestine (7,9). Another mechanism is the enhancement of paracellular calcium transport by the direct stimulation of the intestinal epithelium by intact nondigestible saccharides (11–13). We showed previously that difructose anhydride III (DFAIII),2 a nondigestible disaccharide, increased calcium absorption in both the small and large intestines, and that the increase was greater than those induced by the other saccharides in the in vivo study (7). The study also showed that DFAIII enhanced calcium absorption by sacs of the small intestine.
Intestinal calcium absorption involves 2 processes, a transcellular pathway and a paracellular pathway (14,15). Transcellular absorption is a saturable, carrier-mediated active transport process, whereas paracellular absorption through tight junctions is nonsaturable and diffusive, and requires a gradient of calcium concentrations between the lumen and the basolateral side. The tight junctions are regulated by intracellular signaling (16,17). It was reported that increases in intracellular calcium ion concentration ([Ca2+]i) can open tight junctions through the phosphorylation of myosin regulatory light chains (18). However, the mechanism by which the nondigestible saccharides regulate paracellular permeability is not yet known. Further, there are no reports on the effects of these saccharides on the active calcium transport process.
Caco-2 cell lines derived from human colon adenocarcinoma are capable of enterocytic differentiation (19) and are regarded as a valid model of the human small intestinal epithelium system (20). The fully differentiated and polarized Caco-2 cell monolayer is characterized by typical brush border membranes and tight junctions (21). Caco-2 cell monolayers grown on permeable membrane filters are used to determine the mechanisms of transepithelial intestinal calcium transport (22,23).
The purposes of this study were to examine the effects of the nondigestible saccharides, DFAIII, DFAIV, fructooligosaccharides, and raffinose, on calcium transport, and to clarify the route of calcium transport that is increased by the nondigestible saccharides in cultured human intestinal cell monolayers. Furthermore, we investigated the effects of the saccharides on intracellular calcium signaling, which presumably leads to the enhancement of paracellular transport. It is important in human nutrition to determine the exact mechanism by which nondigestible saccharides promote calcium transport using a human-derived cell line.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Cell cultures. Caco-2 cells (HTB37, passage 19) were obtained from the American Type Culture Collection, and were propagated and maintained in high-glucose (4.5 g D-glucose/L) DMEM supplemented with 100 mL/L decomplemented fetal bovine serum, 44 mmol/L sodium bicarbonate, 1 mmol/L sodium pyruvate, 50,000 U/L penicillin, and 50 mg/L streptomycin, adjusted to pH 7.4. Cultures were maintained at 37°C in a 5% carbon dioxide atmosphere. For routine passage, cells were seeded into 75-cm2 flasks (Corning Costar) at a density of 9000 cells/cm2, and passaged every 3–4 d when the cultures were 70–90% confluent. For calcium transport experiments, the cells were seeded into collagen-coated permeable polyester membrane filter supports (Snapwell, 12 mm diameter, 0.4-µm pore size, 1.0 cm2 growth area; Corning Costar) at a density of 56,000 cells/cm2. For paracellular permeability experiments, the cells were seeded into collagen-coated permeable polyester membrane filter supports (Transwell, 12 mm diameter, 0.4-µm pore size, 1.0 cm2 growth area; Corning Costar) at the same density (56,000 cells/cm2). For measurement of [Ca2+]i, the cells were seeded onto collagen-coated glass cover slips (13.2-mm diameter, thickness 0.15–0.18 mm; Matsunami Glass Industry) settled in 6-well tissue culture plates (35-mm diameter; Becton Dickinson) at the same density (56,000 cells/cm2). Before all experiments, cultures were maintained for 25–26 d after seeding. The medium was refreshed every 2–3 d. Cultures were used for experiments between passage 35 and 50.
Effect of saccharides on calcium transport. Calcium transport across Caco-2 cell monolayers was determined using a vertical diffusion chamber system (Harvard Apparatus). For experiments, HEPES buffer solution (HBS) containing 125 mmol/L NaCl, 4 mmol/L KCl, 10 mmol/L D-glucose, 4 mmol/L L-glutamine, 30 mmol/L HEPES, and 1.25 or 10 mmol/L CaCl2 · 2H2O, adjusted to pH 7.4, was used after pregassing with 100% O2 for at least 30 min. The permeable membrane filters with cultured Caco-2 cell monolayers were mounted in the diffusion chamber after rinsing with prewarmed HBS containing 1.25 mmol/L Ca. To evaluate net calcium transport with a calcium gradient, the basolateral and apical chambers of the cells were both bathed in 4 mL of prewarmed HBS containing 1.25 and 10 mmol/L Ca, respectively, at 37°C. After a 30-min stabilization period, the transport experiments were initiated by adding 74 MBq/L 45CaCl2 and each of individual nondigestible saccharides at 0–100 mmol/L as final concentrations to the apical chamber. To assess the movement of 45Ca into the basolateral chamber, 50 µL of basolateral solution was taken at 30, 60, 90, and 120 min after the start of incubation. Tight junction permeability was also evaluated by measuring transepithelial electrical resistance (TEER) across the cell monolayers with a commercial apparatus (Millicell-ERS; Millipore). TEER was recorded at 0 (before incubation), 30, 60, 90, and 120 min after the start of incubation.
To determine transcellular calcium transport (active transport) in the cell monolayers incubated with or without each nondigestible saccharide (100 mmol/L) for 2 h, the calcium concentration in the basolateral solution was equalized to that of the apical solution (10 mmol/L). Calcium transport in the apical-to-basolateral (A-to-B) direction was assessed by the addition of 74 MBq/L 45CaCl2 to the apical chamber, and basolateral-to-apical (B-to-A) transport was assessed by the addition of 74 MBq/L 45CaCl2 to the basolateral chamber. Calcium transport via the transcellular pathway was derived from data by subtracting the value for B-to-A transport from that for A-to-B transport for paired wells. Diffusional transport of solutes through the paracellular pathway in the B-to-A direction is the same as that in the A-to-B direction in Caco-2 cell monolayers (22,24). Throughout the experiments, the HBS-bathed basolateral and apical chambers were maintained at 37°C and were oxygenated continuously with 100% O2.
To measure 45Ca radioactivity, the apical or basolateral solutions were mixed with 5 mL of a scintillation cocktail containing 32 mmol/L diphenyl oxide and 1 mmol/L dimethyl-POPOP in toluene:ethylene glycol monoethyl ether (1:1). The radioactivity of 45Ca was measured by means of a liquid scintillation counting system (LSC-5100, Aloka).
Caco-2 cell viability was assessed by measuring the release of lactate dehydrogenase (LDH) in the apical and basolateral sides of the monolayers incubated with or without 100 mmol/L of each nondigestible saccharide for 2 h. LDH activity was determined using a commercially available kit (LDH UV Test Wako, Wako Pure Chemical). The viability of the cells after a 2 h-incubation was also assessed by the trypan blue technique.
Effects of saccharides on paracellular permeability. The paracellular permeability across Caco-2 cell monolayers was estimated by measuring lucifer yellow (LY) transport and/or TEER in a Transwell bicameral cell culture system. We also examined the effects of the saccharides from the basolateral side of the monolayers and the reversibility of the saccharide effects on paracellular permeability. HBSS supplemented with 10 mmol/L D-glucose, 4 mmol/L L-glutamine, and 10 mmol/L HEPES, adjusted to pH 7.4, was used. The Caco-2 cells grown on the permeable membrane filters were rinsed with prewarmed HBSS at 37°C. The basolateral and apical chambers of the cells were bathed in 1.0 and 0.5 mL prewarmed HBSS at 37°C, respectively. After a 30-min stabilization period, the transport experiments were initiated by adding 100 µmol/L LY and 100 mmol/L of each individual nondigestible saccharide at final concentrations to the apical chamber. We also evaluated the effect of 100 mmol/L ethylene glycol to produce the same osmotic pressure of the apical solution as nondigestible saccharides in a separate experiment. To investigate the basolateral effects of the nondigestible saccharides, the saccharides were added to the basolateral chamber. LY transport was assessed as the movement into the basolateral chamber during the 3-h incubation period. The LY concentration in the basolateral solution was measured fluorometrically at 430 nm for excitation and 530 nm for emission (CAF-110; JASCO International). TEER was recorded at the start and end of incubation. To investigate the reversibility of the enhancement of paracellular permeability induced by the apical application of nondigestible saccharides, TEER was measured 24 h after removal of the saccharides (after a 3-h treatment). Throughout the experiments, the HBSS-bathed basolateral and apical chambers were maintained at 37°C.
Measurements of intracellular calcium ion concentration ([Ca2+]i). [Ca2+]i was measured according to a general protocol on the basis of changes in the excitation spectrum of the fluorescent probe fura-2 when complexed with free calcium ions. Caco-2 cells grown on the cover slips were loaded with 10 µmol/L fura-2 acetoxymethyl ester and 0.2 g/L cremophor EL in DMEM for 1 h at 37°C. The fura-2–loaded cell monolayers were rinsed with prewarmed HBS containing 1.25 mmol/L Ca at 37°C to remove the probe in the extracellular space. The cover slips with the cell monolayers were then mounted in a quartz cuvette with 2 mL HBS containing 1.25 mmol/L Ca and introduced into the sample chamber of a thermostatically controlled (37°C) spectrofluorometer (CAF-110) with constant low-speed stirring (200 rpm). After a 30-min stabilization period, the measurements were started. After the measurement of the steady state for at least 5 min, the individual nondigestible saccharides were added into the cuvette at 0 (vehicle, HBS containing 1.25 mmol/L Ca), 50, or 100 mmol/L (final concentrations). Fura-2 fluorescence intensity was monitored at an emission wavelength of 500 nm by alternating the excitation wavelength between 340 and 380 nm with a dual excitation monochromator. The ratio of the signals at 340 and 380 nm was calculated, and the maximal and minimal intracellular probe fluorescences were determined by the addition of 50 µmol/L digitonin and 20 mmol/L EGTA in 18 mmol/L Tris, respectively (final concentrations). The transformation of fluorescence signals into [Ca2+]i was performed by the method of Grynkiewicz et al. (25).
Calculations and statistical analyses.
All values are expressed as means ± SEM. The calcium transport and LY permeability across the Caco-2 cell monolayers were expressed as nmol calcium and LY transferred/cm2 surface area, respectively. Net calcium transport represents the total movement of calcium into the basolateral chamber via both the transcellular and paracellular pathways. Active calcium transport (transcellular pathway) was derived from data by subtracting the value for B-to-A transport from that for A-to-B transport for paired wells. The TEER is expressed as 
/cm2 surface area or as the percentage decrease in TEER relative to the value before incubation. [Ca2+]i is expressed as nmol/L after subtraction from the concentration at the point at which the solutions containing each nondigestible saccharide were added. Statistical analyses were performed by a repeated-measures 2-way ANOVA with subsequent Dunnett multiple comparison test (Figs. 1, 2, and 5) (26) or one-way ANOVA subsequent Tukey-Kramer multiple comparison test (Figs. 4and 6, Tables 1, and 2) (27). The correlation between calcium transport rate and TEER was evaluated by Pearson’s correlation (Fig. 3). A difference with P < 0.05 was considered significant. These statistical analyses were done by the general linear models procedure of the Statistical Analysis Systems program (version 6.07; SAS Institute).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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/cm2. Net calcium transport and TEER. Changes in net calcium transport after the application of DFAIII, DFAIV, fructooligosaccharides, or raffinose in the apical chambers are shown in Figure 1. There were significant effects of incubation time, nondigestible saccharide concentration, and the interaction on the net calcium transport (P < 0.001, repeated measure 2-way ANOVA). Net calcium transport from the apical to basolateral sides gradually increased after the addition of each nondigestible saccharide. Net calcium transport after the addition of 30 or 50 mmol/L of each saccharide did not differ from the values without nondigestible saccharides (control values) at any time point. At 60 min, net calcium transport incubated with 100 mmol/L DFAIII, but not the other saccharides, was higher than the control value. At 90 and 120 min, values with 100 mmol/L of each nondigestible saccharide were higher than the values of the control and lower concentrations groups.
The resistance value in the cell monolayers 120 min after incubation without nondigestible saccharides was >90% (Fig. 2). There were significant effects of incubation time, nondigestible saccharide concentration, and the interaction on the resistance value (P < 0.001, repeated measure two-way ANOVA). The resistance value decreased dose dependently 120 min after incubation for each nondigestible saccharide. The resistance values in the monolayers incubated with 30 mmol/L DFAIII were lower than the control values at 60, 90, and 120 min. The values were lowered by the addition of 50 mmol/L of each nondigestible saccharide compared with the control values at 60, 90, and 120 min, and by the addition of 100 mmol/L of each nondigestible saccharide at all time points. The lowest values of TEER was 
20% that of the initial value at 120 min with the addition of each saccharide.
Net calcium transport rate was closely correlated with TEER (P < 0.001) for each saccharide tested (Fig. 3) in an exponential manner.
Calcium flux for both directions (A-to-B and B-to-A) and active calcium transport evaluated by using bidirected fluxes are shown in Figure 4. Calcium transport in both the A-to-B and B-to-A directions in the cell monolayers incubated with 100 mmol/L of each saccharide was higher than each control value. The groups did not differ in active calcium transport, which was derived from data by subtracting the value for B-to-A transport from that for A-to-B transport for paired wells.
There were no differences in LDH activity, an indicator of cell membrane integrity, in the apical solution after incubation with or without each saccharide tested (Control, 94.8 ± 7.3 mU/0.5 mL; DFAIII, 84.6 ± 2.5; DFAIV, 85.4 ± 6.0; fructooligosaccharides, 86.0 ± 4.6; raffinose, 77.4 ± 7.1). Activity in the basolateral solution was not detected in all groups. Also, exclusion of trypan blue in >95% of the cells was observed in all groups after a 2-h incubation.
Intracellular calcium ion concentration.
The changes in [Ca2+]i in Caco-2 cell monolayers grown on cover slips were recorded before and after stimulation with 0, 50, and 100 mmol/L of each nondigestible saccharide (Fig. 5). Incubation time and nondigestible saccharide concentration influenced the [Ca2+]i, and there were significant interactions between these factors after treatment with the 4 saccharides (P < 0.001, repeated measure two-way ANOVA). [Ca2+]i did not differ among groups before the addition of test solutions. Addition of a vehicle (0 mmol/L) did not influence [Ca2+]i. Treatment with 50 and 100 mmol/L of each nondigestible saccharide caused an immediate rise in [Ca2+]i, and the [Ca2+]i reached a sustained value 10 min after the addition of the saccharides. The increment in the [Ca2+]i induced by the addition of each saccharide was dose dependent. At 1 min, the values of [Ca2+]i were higher in the monolayers treated with 50 mmol/L raffinose, 100 mmol/L DFAIV, and fructooligosaccharides than that in the vehicle group. At later time points, the values were higher in the monolayers treated with 50 and 100 mmol/L of each nondigestible saccharide than those in the vehicle group, except for the 50 mmol/L DFAIV and the fructooligosaccharides groups at 2 min.
Basolateral effects and the reversibility of the effects of saccharides on the paracellular routes. Changes in resistance values and LY transport by application of each of the nondigestible saccharides from both the apical and basolateral sides are shown in Figure 6. Application of the saccharides to the apical chamber clearly decreased the resistance values and enhanced LY transport. In contrast, basolateral application had a much smaller effect on the resistance values, and no effect on LY transport for each saccharide.
There were no differences in TEER among the groups before incubation (Table 1). Application of 100 mmol/L of DFAIII, DFAIV, fructooligosaccharides, or raffinose to the apical chamber decreased TEER values; however, these values recovered completely 24 h after removal of the saccharides.
Osmotic effects on the paracellular routes. There were no differences in TEER among the groups before incubation (Table 2). Application of 100 mmol/L DFAIII into the apical chamber decreased TEER values and increased LY transport. The same concentration of ethylene glycol had no effect on paracellular permeability as evaluated by both TEER and LY transport.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Transepithelial calcium transport in the intestine occurs by 2 routes, a transcellular pathway and a paracellular pathway (14,15). It was shown that the Km value for transcellular calcium absorption was 2.9 mmol/L in Caco-2 cell monolayers (23). The calcium concentrations in the apical and basolateral chamber in our study were 10 and 1.25 mmol/L, respectively. Under these conditions, both the transcellular and paracellular pathways operated sufficiently to absorb calcium. We demonstrated in the present study that there was a negative exponential relationship between net calcium transport and TEER in the cell monolayers incubated with each of the 4 nondigestible saccharides (P < 0.001). This finding suggests that the paracellular pathway contributes to the increase in net calcium transport stimulated by nondigestible saccharides. We separately evaluated transcellular and paracellular calcium transport across the monolayers under conditions in which calcium concentration in the basolateral medium was equalized to that in the apical medium (10 mmol/L). The addition of 100 mmol/L of each nondigestible saccharide enhanced net calcium transport without any change in transcellular transport. In contrast, paracellular calcium transport from the basolateral to the apical side was increased 300–400% by the saccharides. Active transport is essentially localized in the upper duodenum, whereas paracellular transport occurs throughout the small intestine (28). In addition, a few reports suggested that the paracellular route of calcium absorption is predominant in humans when the calcium intake is sufficiently high (14,29). These results indicate the possibility of increasing calcium absorption via the paracellular pathway in humans by supplementing the diet with these saccharides.
Net calcium transport was not increased by low levels of nondigestible saccharides (30 or 50 mmol/L), whereas resistance values in the monolayers were decreased by these low levels of nondigestible saccharides. TEER in the Caco-2 cell monolayer is very high compared with that in the animal epithelium (13,30). This means that the tight junction pathway in the Caco-2 cell monolayer is too narrow for calcium ions to pass through before the large saccharide-induced decrease in TEER.
The present study demonstrates that the nondigestible saccharides induce continuous increases in [Ca2+]i. Some food-derived chemicals or drugs are reported to enhance paracellular permeability after increases in [Ca2+]i in the monolayers (31,32). An increment in [Ca2+]i triggers the activation of calmodulin-dependent myosin light-chain kinase (18,33). It was proposed that condensation of actin microfilaments induced by myosin light-chain kinase plays a role in the opening of tight junctions (34,35). Thus, the increases in [Ca2+]i stimulated by the nondigestible saccharides may lead to the enhancement of paracellular calcium transport. Our current data provide possible evidence of nondigestible saccharides directly stimulating intestinal cells by a putative sensory system for nondigestible saccharides. However, the basolateral application of the nondigestible saccharides seems to have no substantial effect on paracellular permeability. This finding indicates that the intestinal sensory system for nondigestible saccharides locates on the apical side of the Caco-2 cell monolayer because the nondigestible saccharides do not permeate through the phospholipid bilayer of intestinal cells. Some food-derived chemicals affect the paracellular permeability from both sides of cells (36,37). It is reported that sodium caprate, a medium-chain fatty acid, works similarly from both the apical and basolateral sides with increases in [Ca2+]i (37). As with the nondigestible saccharides, decanoylcarnitine has a stronger effect with apical application than with basolateral application, but it does not evoke calcium signaling (37). Thus, the mechanism for enhancing paracellular calcium transport with nondigestible saccharides may be different from that of these chemicals.
The application of each of the 4 nondigestible saccharides equally increased net calcium transport and decreased TEER, and osmotic pressure with an increase in solute concentration, achieved by adding ethylene glycol at a concentration of 100 mmol/L, did not affect TEER and LY transport. These findings indicate that the effect of the nondigestible saccharides is not merely osmotic stimulation and that common structures in these saccharides are recognized by Caco-2 cells and enhance paracellular calcium transport in the monolayers. The numbers and types of monosaccharides composing each of 4 nondigestible saccharides tested are various, and we cannot clarify the relationship between the extent of the promotive effects on calcium transport and the structure of the saccharides. It is reported that DFAIV enhances net calcium absorption more strongly than does DFAIII in the isolated small and large intestinal epithelium of rats (12). However, this is not supported by in vivo (6) and in situ (unpublished results, H. Hara, Hokkaido University) studies. These results suggest that the specificity of the intestinal putative sensory system for the nondigestible saccharide may be low, and the sensory system may recognize various types of nondigestible saccharides.
Our study shows that activation of the paracellular route by nondigestible saccharides in the Caco-2 cell monolayers is reversible and is not accompanied by cell membrane damage. We also showed that nondigestible saccharides induced cellular calcium signals, which may be involved in the increase in paracellular permeability. These results indicate that the promotive effects of saccharide treatment on calcium absorption are controlled, and do not result in the disruption of tight junctions on the monolayer. Uncontrolled increases in intestinal permeability allow the entry into the bloodstream of undesirable molecules that could be either toxic or able to promote allergies. However, a controlled increase in paracellular permeability is useful for increasing calcium absorption. Sufficient information on this mechanism is necessary to fully establish the safety of the nondigestible saccharides for human consumption.
In conclusion, 4 nondigestible saccharides, DFAIII, DFAIV, fructooligosaccharides, and raffinose, increased net calcium absorption in human intestinal Caco-2 cell monolayers by increasing paracellular transport through the physiologic control of tight junctions. The intracellular calcium ion signaling stimulated by these nondigestible saccharides may result in the activation of paracellular transport. Further investigations are warranted to clarify the sensory system for nondigestible saccharides in the intestinal epithelial cells and the intracellular mechanism leading to calcium signaling.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received 11 March 2004. Initial review completed 14 April 2004. Revision accepted 30 April 2004.
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LITERATURE CITED |
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1. Webb, G. P. (2002) Carbohydrate. Nutrition: A Health Promotion Approach 2nd ed. 2002:209-229 Oxford University Press New York, NY.
2. Webb, F. S. & Whitney, E. N. (2000) The carbohydrates: sugar, starch glycogen, and fiber. Nutrition: Concepts and Controversies 8th ed. 2000:93-123 Thompson Learning Belmont, CA.
3. Ammann, P., Rizzoli, R. & Fleisch, H. (1988) Influence of the disaccharide lactitol on intestinal absorption and body retention of calcium in rats. J. Nutr. 118:793-795.
4. Goda, T., Suruga, K., Takase, S., Ezawa, I. & Hosoya, N. (1995) Dietary maltitol increases calcium content and breaking force of femoral bone in ovariectomized rats. J. Nutr. 125:2869-2873.
5. Mitamura, R., Hara, H., Aoyama, Y. & Chiji, H. (2002) Supplemental feeding of difructose anhydride III restores calcium absorption impaired by ovariectomy in rats. J. Nutr. 132:3387-3393.
6. Saito, K., Hira, T., Suzuki, T., Hara, H., Yokota, A. & Tomita, F. (1999) Effects of DFA IV in rats: calcium absorption and metabolism of DFA IV by intestinal microorganisms. Biosci. Biotechnol. Biochem. 63:655-661.[Medline]
7. Suzuki, T., Hara, H., Kasai, T. & Tomita, F. (1998) Effects of difructose anhydride III on calcium absorption in small and large intestines of rats. Biosci. Biotechnol. Biochem. 62:837-841.[Medline]
8. Hara, H., Suzuki, T. & Aoyama, Y. (2000) Ingestion of the soluble dietary fibre, polydextrose, increases calcium absorption and bone mineralization in normal and total-gastrectomized rats. Br. J. Nutr. 84:655-661.[Medline]
9. Hara, H., Suzuki, T., Kasai, T., Aoyama, Y. & Ohta, A. (1999) Ingestion of guar gum hydrolysate, a soluble fiber, increases calcium absorption in totally gastrectomized rats. J. Nutr. 129:39-45.
10. Shiga, K., Hara, H., Takahashi, T., Aoyama, Y., Furuta, H. & Maeda, H. (2002) Ingestion of water-soluble soybean fiber improves gastrectomy-induced calcium malabsorption and osteopenia in rats. Nutrition 18:636-642.[Medline]
11. Kishi, K., Goda, T. & Takase, S. (1996) Maltitol increases transepithelial diffusional transfer of calcium in rat ileum. Life Sci. 59:1133-1140.[Medline]
12. Mineo, H., Hara, H., Kikuchi, H., Sakurai, H. & Tomita, F. (2001) Various indigestible saccharides enhance net calcium transport from the epithelium of the small and large intestine of rats in vitro. J. Nutr. 131:3243-3246.
13. Mineo, H., Hara, H., Shigematsu, N., Okuhara, Y. & Tomita, F. (2002) Melibiose, difructose anhydride III and difructose anhydride IV enhance net calcium absorption in rat small and large intestinal epithelium by increasing the passage of tight junctions in vitro. J. Nutr. 132:3394-3399.
14. Bronner, F. (1998) Calcium absorption—a paradigm for mineral absorption. J. Nutr. 128:917-920.
15. Bronner, F., Pansu, D. & Stein, W. D. (1986) An analysis of intestinal calcium transport across the rat intestine. Am. J. Physiol. 250:G561-G569.
16. Karczewski, J. & Groot, J. (2000) Molecular physiology and pathophysiology of tight junctions III. Tight junction regulation by intracellular messengers: differences in response within and between epithelia. Am. J. Physiol. 279:G660-G665.
17. Mitic, L. L., Van Itallie, C. M. & Anderson, J. M. (2000) Molecular physiology and pathophysiology of tight junctions I. Tight junction structure and function: lessons from mutant animals and proteins. Am. J. Physiol. 279:G250-G254.
18. Kitamura, T., Brauneis, U., Gatmaitan, Z. & Arias, I. M. (1991) Extracellular ATP, intracellular calcium and canalicular contraction in rat hepatocyte doublets. Hepatology 14:640-647.[Medline]
19. Pinto, M., Robine-Leon, S., Appay, M. D., Kedinger, M., Triadou, N., Dussaulx, E., Lacroix, B., Simon-Assmann, P., Haffen, K., Fogh, J. & Zweibaum, A. (1983) Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture. Biol. Cell 47:323-330.
20. Chantret, I., Rodolosse, A., Barbat, A., Dussaulx, E., Brot-Laroche, E., Zweibaum, A. & Rousset, M. (1994) Differential expression of sucrase-isomaltase in clones isolated from early and late passages of the cell line Caco-2: evidence for glucose-dependent negative regulation. J. Cell Sci. 107:213-225.[Abstract]
21. Vachon, P. H. & Beaulieu, J. F. (1992) Transient mosaic patterns of morphological and functional differentiation in the Caco-2 cell line. Gastroenterology 103:414-423.[Medline]
22. Fleet, J. C., Eksir, F., Hance, K. W. & Wood, R. J. (2002) Vitamin D-inducible calcium transport and gene expression in three Caco-2 cell lines. Am. J. Physiol. 283:G618-G625.
23. Giuliano, A. R. & Wood, R. J. (1991) Vitamin D-regulated calcium transport in Caco-2 cells: unique in vitro model. Am. J. Physiol. 260:G207-G212.
24. Watson, C. J., Rowland, M. & Warhurst, G. (2001) Functional modeling of tight junctions in intestinal cell monolayers using polyethylene glycol oligomers. Am. J. Physiol. 281:C388-C397.
25. Grynkiewicz, G., Poenie, M. & Tsien, R. Y. (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260:3440-3450.
26. Dunnett, C. W. (1970) Multiple comparison tests. Biometrics 26:139-141.
27. Tukey, J. W. (1991) The philosophy of multiple comparisons. Stat. Sci. 6:100-116.
28. Pansu, D., Bellaton, C., Roche, C. & Bronner, F. (1983) Duodenal and ileal calcium absorption in the rat and effects of vitamin D. Am. J. Physiol. 244:G695-G700.[Medline]
29. Bronner, F. & Pansu, D. (1999) Nutritional aspects of calcium absorption. J. Nutr. 129:9-12.
30. Yeh, P. Y., Smith, P. L. & Ellens, H. (1994) Effect of medium-chain glycerides on physiological properties of rabbit intestinal epithelium in vitro. Pharm. Res. 11:1148-1154.[Medline]
31. Sawai, T., Lampman, R., Hua, Y., Segura, B., Drongowski, R. A., Coran, A. G. & Harmon, C. M. (2002) Lysophosphatidylcholine alters enterocyte monolayer permeability via a protein kinase C/Ca2+ mechanism. Pediatr. Surg. Int. 18:591-594.[Medline]
32. Tai, Y. H., Flick, J., Levine, S. A., Madara, J. L., Sharp, G. W. & Donowitz, M. (1996) Regulation of tight junction resistance in T84 monolayers by elevation in intracellular Ca2+: a protein kinase C effect. J. Membr. Biol. 149:71-79.[Medline]
33. Vetter, S. W. & Leclerc, E. (2003) Novel aspects of calmodulin target recognition and activation. Eur. J. Biochem. 270:404-414.[Medline]
34. Lindmark, T., Kimura, Y. & Artursson, P. (1998) Absorption enhancement through intracellular regulation of tight junction permeability by medium chain fatty acids in Caco-2 cells. J. Pharmacol. Exp. Ther. 284:362-369.
35. Ma, T. Y., Hoa, N. T., Tran, D. D., Bui, V., Pedram, A., Mills, S. & Merryfield, M. (2000) Cytochalasin B modulation of Caco-2 tight junction barrier: role of myosin light chain kinase. Am. J. Physiol. 279:G875-G885.
36. Liu, D. Z., LeCluyse, E. L. & Thakker, D. R. (1999) Dodecylphosphocholine-mediated enhancement of paracellular permeability and cytotoxicity in Caco-2 cell monolayers. J. Pharm. Sci. 88:1161-1168.[Medline]
37. Tomita, M., Hayashi, M. & Awazu, S. (1996) Absorption-enhancing mechanism of EDTA, caprate, and decanoylcarnitine in Caco-2 cells. J. Pharm. Sci. 85:608-611.[Medline]
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