The Journal of Nutrition Vol. 129 No. 1 January 1999, pp. 39-45

Ingestion of Guar Gum Hydrolysate, a Soluble Fiber, Increases Calcium Absorption in Totally Gastrectomized Rats¹

Hiroshi Hara², Takuya Suzuki, Takanori Kasai, Yoritaka Aoyama, and Atsutane Ohta^*

Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University, Sapporo 060-8589 and ^* Bioscience Laboratories, Meiji Seika Kaisha, Ltd., Sakado 350-02, Japan

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Gastrectomy induces osteopenia. We examined the effects of feeding a diet containing soluble dietary fiber, guar gum hydrolysate (GGH, 50 g/kg diet), on intestinal calcium absorption and bone mineralization in totally gastrectomized (Roux-en-Y esophagojejunostomy) rats by comparing them with those in two control groups (laparotomized and bypassed rats). In the bypassed rats, chyme bypassed the duodenum and upper jejunum without gastrectomy. In a second separate experiment, we compared calcium absorption and bone mineralization in the gastrectomized rats fed diets containing soluble and insoluble calcium salts and in bypassed rats fed insoluble calcium. In Experiment 1, apparent absorption of calcium supplied as a water-insoluble salt was more than 50% lower in gastrectomized rats than in the intact (laparotomized) or bypassed rats 3 wk after the start of feeding the test diets (P < 0.05). Calcium absorption was higher (P < 0.05) in gastrectomized rats fed the GGH diet than in those rats fed the GGH-free diet. In Experiment 2, absorption of soluble calcium in the gastrectomized rats did not differ from the absorption of calcium from calcium carbonate by bypassed rats. The soluble calcium pool in the cecal contents was significantly lower in gastrectomized rats (Experiment 1) than in intact or bypassed control rats, and was higher (P < 0.05) in the GGH-fed gastrectomized rats than in those fed the GGH-free diet. However, calcium absorption correlated most closely (r = 0.787, P < 0.01) with cecal propionic acid concentration. The femur calcium content was significantly lower in gastrectomized rats fed insoluble calcium than in bypassed rats fed the same diet, but was partially restored in the rats fed soluble calcium (Experiment 2). Bone calcium was not increased by feeding GGH in gastrectomized rats (Experiment 1). We conclude that the severely diminished calcium absorption following total gastrectomy is totally due to a decrease in calcium solubilization, and feeding GGH partially restores calcium absorption. The decrease in bone calcium that occurs as a result of gastrectomy is mainly due to diminished intestinal calcium absorption.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Gastric resection induces osteopenia (Koga et al. 1979, Nilas et al. 1985), and decreased intestinal calcium absorption may be responsible for the reduction of bone calcium content that occurs as a result of gastrectomy. Dietary fiber was suggested as an inhibitor of calcium absorption (Balasubrananian et al 1987, O'Brien et al. 1993). Recently, it was reported that ingested soluble dietary fiber enhances calcium absorption (Demigné et al. 1989, Younes et al. 1996). We have previously demonstrated that ingestion of guar gum hydrolysate (GGH)³, a highly fermentable dietary fiber, fully restored calcium absorption in the large intestine of rats when it was lowered due to renal failure (Hara et al. 1996). Also, Ohta et al. (1998) showed that fructooligosaccharides, which are indigestible, increased intestinal calcium absorption in rats when it was lowered by total gastrectomy.

Solubilization of calcium salts by acids generated through microbial fermentation in the large intestine was proposed as the mechanism responsible for the increase in calcium absorption observed following the ingestion of highly fermentable, indigestible materials (Younes et al. 1996). Also, the effects of dietary fiber on intestinal calcium absorption and bone mineralization in gastrectomized rats are not known. We adopted Roux-en-Y esophagojejunostomy for reconstruction in rats subjected to total gastric resection. Absence of gastric acid caused by this operation may impair insoluble calcium absorption because the gastric acid is the most important factor for solubilization of insoluble calcium salts. After this operation, chyme does not pass through the duodenum and upper jejunum (bypassed). The effects of such a bypass on calcium absorption are not known.

The aims of the present study were to examine the effects on calcium absorption and bone mineralization of feeding water-soluble dietary fiber to totally gastrectomized rats as compared to intact rats and rats with the duodenum and upper jejunum bypassed, and to examine the effects on calcium absorption and bone mineralization of feeding water-soluble and -insoluble calcium salts to the gastrectomized rats.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Animals and diets.  Male Sprague-Dawley rats (Japan SLC, Hamamatsu, Japan), weighing about 100 g, were given free access to deionized water and the semi-purified stock diet shown in Table 1 for an acclimation period of 4-6 d, and were divided into three groups in Experiment 1 and two groups in Experiment 2. Under anesthesia (40 mg pentobarbital sodium/kg body weight; Abbott, North Chicago, IL), rats of one group were subjected to total gastrectomy with Roux-en-Y reconstruction (Lambert 1965), rats of a second group were subjected to an operation in which the duodenum and upper jejunum were bypassed (bypassed group) and rats of the last group (Experiment 1 only )were subjected to laparotomy (intact group). These are illustrated in Figure 1. In the case of gastrectomized rats, the stomach was removed after ligation of blood vessels, the cut edge of the esophagus was end-to-side anastomosed to the upper jejunum 8 cm distal from the ligament of Treitz, and a 2-cm segment of the duodenum bearing the papilla of Vater was transposed to the jejunum 5 cm from the position of esophagojejunal anastomosis. In the bypassed group, rats were operated on in the same manner as for the gastrectomized group, but without gastric resection. In this group chyme, emptied from the stomach, bypassed the duodenum and upper jejunum. After the operations, the rats were deprived of food and water for 24 h, then were fed cow's milk for 2-3 d, followed by an Fe-free stock diet for 14-16 d.

View larger version (22K): [in this window] [in a new window]   Fig 1. The gastrointestinal tract in intact (laparotomized), bypassed, and totally gastrectomized rats. The duodenal segment was transposed to the jejunum in bypassed and gastrectomized rats, and chyme in these rats bypassed the duodenum and upper jejunum.

In Experiment 1, each of the three groups of rats was divided into two subgroups using a randomized block design based on the rat's body weight after the recovery period. The diet fed to one of the subgroups of each operation group was changed to the test diet containing guar gum hydrolysate (50 g diet/kg, GGH, Guar Fiber, Meiji Seika Kaisha, Ltd., Tokyo, Japan), and the diet fed to the other subgroup was changed to the GGH-free test diet for 3 wk. Guar gum hydrolysate was prepared by digestion with -1,4-mannanase, having an average molecular weight of 15,000. This fiber material was added to the test diet as a source of dietary fiber at the expense of the whole diet. Calcium in the diet (3.0 g Ca /kg diet) was supplied as a water-insoluble calcium salt, calcium carbonate. The calcium content of the test diets was the minimum level required by intact rats of the same strain (Hara et al. 1996). Vitamin B-12 (36 µg/kg body wt · d¹) and Fe (1.8 mg/kg body wt · d¹) as FeCl2 (Wako Pure Chemical Industries, Tokyo, Japan) were supplied subcutaneously every 5 d during the recovery and test periods. Body weight and food intake were measured every day.

In Experiment 2, the group of gastrectomized rats was divided into two subgroups. The diet fed to one of the subgroups of gastrectomized rats and rats of the bypassed group was changed to the GGH-free test diet containing 3.0 g Ca/kg diet, supplied as a water-insoluble calcium salt, calcium carbonate, and the diet fed to the other subgroup of gastrectomized rats was changed to the GGH-free test diet containing 3.0 g Ca/kg diet, supplied as a water-soluble calcium salt, calcium chloride, for 3 wk. Bypassed rats were fed the calcium carbonate diet. Other details were the same as in Experiment 1.

Feces were collected continuously for 3 d from the 11th day (Experiment 1 only) and from the 25th day after the start of feeding the test diets to evaluate calcium excretion and apparent absorption of calcium. The feces collected in the 3 d period were sampled, and freeze-dried.

Rats used in the experiment were housed individually in stainless steel cages with mesh bottoms. The cages were placed in a room with controlled temperature (22-24°C), relative humidity (40-60%) and lighting (lights on: 0800-2000 h).

At the end of the experiment, the rats were killed under pentobarbital anesthesia. The cecum was removed without loss of its contents; the contents were collected quantitatively, frozen immediately with liquid nitrogen and stored at 40°C until subsequent analyses.

This study was approved by the Hokkaido University Animal Committee, and animals were maintained in accordance with the Hokkaido University guidelines for the care and use of laboratory animals.

Analytical methods.  Freeze-dried feces were milled. Powdered feces (about 70 mg) were wet-ashed with a mixture of nitric acid (10 mol/L) and perchloric acid (2.3 mol/L). The cecal contents diluted with 9 volumes of deionized water were homogenized by means of a teflon homogenizer. Amounts of total calcium in the homogenates were measured after the samples had been wet-ashed in the same way as the feces. Soluble calcium was assayed in the supernatant obtained upon centrifugation (30,000 × g for 20 min) of the homogenate. Calcium concentrations in the ashed solutions were measured by atomic absorption spectrophotometry (AA-6400F, Shimadzu, Kyoto, Japan) after adequate dilution with water.

Concentrations of short-chain fatty acids (SCFA) and other organic acids in the homogenate of the cecal contents were evaluated by the previously described method (Hara et al. 1994). Individual SCFA were measured by gas-liquid chromatography (Shimadzu GC-14A with a prepacked glass column [1600 mm × 3 mm, SP-1220 + H₃PO₄ (15% + 1%) on 80-100 mesh Chromosorb W-AW DMCS, Shimadzu Corporation, Kyoto, Japan] after adding phosphoric acid (final concentration 0.67 mol/L.) Succinic and lactic acids were analyzed using a capillary electrophoresis system (Waters capillary ion analyzer, Waters Assoc., Milford, MA).

Calculations and statistical analysis.  Apparent absorption of calcium was calculated as follows:

The results were analyzed by two-way (GGH and operation) ANOVA, in Experiment 1 and one-way ANOVA in Experiment 2. Duncan's multiple range test was used to determine whether mean values were significantly different (Duncan 1995, P < 0.05). Correlation coefficients for the relationships between apparent calcium absorption and several cecal parameters were assessed by the least squares method (Sokal 1973). These statistical analyses were done by the GLM procedure of SAS (SAS version 6.07, SAS Institute Inc., Cary, NC).

	RESULTS

Abstract Introduction Methods Results Discussion References

The initial body weight of gastrectomized rats was lower than that of the intact or bypassed rats, and body weight gain and food intake for the 3-wk test period were lower in the gastrectomized groups than in rats of the other groups in Experiment 1 (Table 2). In Experiment 2, there were no significant difference among groups in these variables.

Apparent calcium absorption in the gastrectomized groups was significantly lower than that in the intact or bypassed groups both at 2 wk (Fig 2A) and at 3 wk (Fig. 2B) after the start of feeding the test diets. Calcium absorption in the gastrectomized groups was significantly higher in rats fed the GGH diet than in rats fed the GGH-free diet at both 2 and 3 wk. Calcium absorption in the GGH-fed bypassed rats was slightly but significantly lower than that in the GGH-fed intact group at 2 wk after the start of feeding the test diets (Fig. 2A), but not after 3 wk of feeding (Fig. 2B). In rats fed the GGH-free diet, bypass of the duodenum and upper jejunum did not influence calcium absorption.

View larger version (43K): [in this window] [in a new window]   Fig 2. Apparent calcium absorption in intact, bypassed and gastrectomized rats fed diets with or without guar gum hydrolysate (50 g GGH/kg diet) at 2 wk (A) and 3 wk (B) after the start of feeding the test diets in Experiment 1. Each value is the means ± SEM, n = 6 (intact or gastrectomized groups) and n = 7 (bypassed groups). P values estimated by two-way ANOVA were 0.059 for GGH and <0.001 for operation (A) and NS for GGH and <0.001 for operation (B). Values at a time not sharing a letter are significantly different, P < 0.05.

Calcium absorption in gastrectomized rats fed the diet containing calcium chloride (Experiment 2) was not different from that in bypassed rats fed the diet containing calcium carbonate (Fig 3). The absorption in gastrectomized rats fed insoluble calcium was significantly lower than that in bypassed rats or gastrectomized rats fed soluble calcium.

View larger version (63K): [in this window] [in a new window]   Fig 3. Apparent calcium absorption in bypassed and gastrectomized rats fed a water-insoluble calcium salt, CaCl2, and in gastrectomized rats fed a water-soluble calcium salt, CaCO3, 3 wk after the start of feeding the test diets in Experiment 2. Each value is the mean ± SEM, n = 9 except for the group of gastrectomized rats fed the CaCO3-containing diet (n = 7). P values estimated by one-way ANOVA were <0.001. Mean values not sharing a letter are significantly different, P < 0.05.

Femur dry weight (g/100 g body wt.) in gastrectomized rats was significantly lower than that in intact or bypassed rats in Experiment 1 (Table 3). The calcium content of the femur was significantly lower in the gastrectomized groups than that in the intact or bypassed groups. The calcium content in bypassed rats fed the GGH diet was significantly lower than in intact rats fed GGH. The calcium concentration in the femur was also lower in the gastrectomized rats than in either the intact or bypassed rats. Ingestion of GGH did not influence femur relative weight or calcium level. In Experiment 2 (Table 3), the femur relative dry weight and femur calcium content were significantly lower in gastrectomized rats fed calcium carbonate compared to bypassed rats fed calcium carbonate, which is consistent with the results of Experiment 1. In gastrectomized rats fed calcium chloride (soluble calcium), the values of these femur variables were higher than those in the gastrectomized rats fed calcium carbonate (insoluble calcium), but lower than those in bypassed rats fed calcium carbonate. The calcium concentration in the femur in gastrectomized rats fed the diet containing calcium chloride did not differ from that in bypassed rats.

The soluble calcium pool and soluble calcium concentration in the cecal contents were significantly lower in gastrectomized rats than in intact or bypassed rats (Table 4). In the gastrectomized rats, the soluble calcium pool was significantly higher in the GGH-fed group than those fed GGH-free diet.

Weights of the cecal wall and cecal contents were significantly higher and the pH of the cecal contents was lower in rats fed the GGH diet than in rats fed the GGH-free diet in all groups (Table 5).

The cecal propionic acid concentration and the sum of the concentrations of three major short-chain fatty acids (total), in gastrectomized rats were significantly higher in the GGH-fed group than in the GGH-free diet fed group (Table 6).

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Apparent absorption of calcium supplied as a water-insoluble salt was markedly decreased as a result of total gastrectomy. However, in gastrectomized rats, the absorption of calcium supplied in a soluble form was the same as that in bypassed rats fed insoluble calcium. Calcium absorption in bypassed rats was not different from that in intact rats after 3 wk. We previously observed that absorption of calcium supplied as a soluble salt, calcium chloride, was very similar to that of calcium carbonate in intact male Sprague-Dawley rats (unpublished data). The results of this study clearly demonstrate that lack of solubilization of insoluble calcium salt by gastric acid is responsible for the impairment of calcium absorption in totally gastrectomized rats.

In rats with calcium absorption lowered by gastrectomy, the apparent absorption was increased by feeding GGH. Because this fiber has very low viscosity, this increment in absorption may occur in the large intestine [viscosity of a fiber solution (10 g/L) was similar to water, data not shown], and may not affect calcium absorption in the proximal intestine. Also, the fiber is highly fermentable in the large intestine (Takahashi et al. 1994). We showed that feeding the GGH diet led to a greater pool of soluble calcium in the cecal contents (Table 4), a lower cecal pH (Table 5) and increased concentrations of total short-chain fatty acids (Table 6) in gastrectomized rats. These findings suggest that in the cecum solubilization of insoluble calcium salts occurs through acidification by organic acids produced in the cecal fermentation of GGH. The capacity of the large intestine to absorb calcium is high (Karbach and Feldmeier 1993, Pitcher and Buffenstein 1994). Also, we have previously demonstrated that the large intestine has sufficient capacity for calcium absorption to supply the daily calcium requirement in rats (Ohta et al. 1997), and lowered calcium absorption in the proximal intestine is fully restored through increased absorption in the large intestine (Hara et al. 1996).

We searched for cecal factors associated with increases in calcium absorption upon GGH feeding in gastrectomized rats. Table 7 shows the correlation coefficients for the relationships between calcium absorption and several cecal factors in Experiment 1. Apparent calcium absorption in gastrectomized rats was positively correlated with the soluble calcium pool and soluble calcium concentration and the propionic acid concentration and the concentration of total short-chain fatty acids in the cecal contents, and negatively correlated with cecal pH. It is likely that the soluble calcium concentration directly affects calcium absorption in the large intestine; however, calcium absorption was most strongly correlated with the concentration of propionic acid. There was a high negative correlation between the propionic acid concentration and cecal pH, and between cecal pH and the soluble calcium concentration. These results suggest that the concentration of propionic acid is a more important factor in calcium absorption in the large intestine than other factors, soluble calcium and cecal pH. These factors may be correlated with calcium absorption indirectly. That is, an increase in propionic acid occurs as a result of enhancement of cecal fermentation, leading to lowered cecal pH and an increase in soluble calcium in the cecal contents. Trinidad et al. (1996) reported that propionate is associated with colonic calcium absorption in humans.

In the present experiments, calcium absorption was not increased by GGH feeding in intact and bypassed rats (Fig. 2). In these rats, decreases in cecal pH were significant but small, and soluble calcium concentration was not increased in the cecum by feeding GGH. However, we found that total short-chain fatty acid concentration did not differ due to GGH consumption in these rats, especially in intact rats. This result supports the hypothesis that short-chain fatty acids are involved in calcium absorption in the large intestine. We previously showed that feeding fructooligosaccharides did not increase the calcium absorption (Ohta et al. 1998). In the intact rats, calcium is absorbed sufficiently when rats are fed a fiber-free diet.

Roux-en-Y esophagojejunostomy employed in the present study is the preferred method of reconstruction for total gastrectomy in human patients (Bozzetti et al. 1996). After this operation, chyme bypasses the duodenum and upper jejunum. The duodenum, but not the ileum, has vitamin D-sensitive, active calcium transport (Armbrecht 1998, Pansu et al. 1983), however, the contribution of the duodenum to whole calcium absorption has not been established. We examined the effects of the duodenum and upper jejunum bypass on calcium absorption. In the bypassed rats, apparent calcium absorption was slightly decreased 2 wk after the start of feeding the test diet, however, there was no difference between intact and bypassed rats after 3 wk of feeding. These findings demonstrate that calcium absorption in vivo is not impaired when there is no absorption via the upper small intestine. Marcus and Lengemann (1962) reported that substantial absorption of calcium occurs in the ileum. The femur calcium content in GGH-fed, bypassed rats was significantly lower than that in GGH-fed, intact rats (Table 3), which agreed with the small decrease in calcium absorption observed in the second week (Fig. 2A). In the present study, the calcium level in the test diets (3.0 g/kg diet) was the minimum required by intact rats (see Materials and Methods). The small decrease in calcium absorption from that in the intact rats possibly affects bone mineralization in the bypassed group in the early test period. Another possibility is that the bypassed segment of the small intestine may be involved in calcium metabolism in bone. Small intestinal resection or bypass is known to reduce bone mass (Hessov et al. 1984, Simmons et al. 1975).

Femur dry weight and the content and concentration of calcium were dramatically reduced by total gastrectomy (Table 3). These results agree with previous reports (Klein et al. 1987, Ohta et al. 1998, Persson et al. 1993). The reduction in bone calcium content in gastrectomized rats was not restored by GGH feeding, in spite of the increase in calcium absorption that occurred as a result of GGH feeding. A possible explanation is that, although calcium absorption was increased by GGH feeding, the increment does not achieve a level allowing calcium to be incorporated into bone. In contrast, bone calcium was largely restored in gastrectomized rats fed the soluble calcium salt, and calcium absorption in these rats was the same as that in the bypassed rats. This finding demonstrates that the increment in calcium absorption is effective in increasing bone mineralization impaired by total gastrectomy. However, the bone calcium pool was still lower in gastrectomized rats fed the soluble calcium than in the bypassed rats, even when calcium absorption was completely restored to normal. These observations show that the levels of bone calcium in gastrectomized rats are not solely determined by the amount of calcium absorbed and suggest that gastric factors other than acid are involved in bone mineralization. The acid-producing gland of the stomach secretes a hormone that regulats bone metabolism, and gastrin is involved in the secretion of this hormone (Persson et al. 1989, Hakanson et al. 1990). Exclusion of the gastric endocrine factors is possibly responsible for the discrepancy between calcium absorption and bone calcium content.

In conclusion, a decrease in calcium solubilization, attributable to the absence of gastric acid, caused a severe reduction in calcium absorption after total gastrectomy, and the lowered absorption was partially restored as a result of GGH feeding. This restoration of calcium absorption may be associated with products derived from fermentation of GGH in the large intestine. Bone calcium levels lowered by gastrectomy were partly restored, but did not fully recover, although soluble calcium feeding led to a sufficient increase in calcium absorption.

	FOOTNOTES

Manuscript received 29 June 1998. Initial reviews completed 4 August 1998. Revision accepted 22 September 1998.

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