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© 2004 The American Society for Nutritional Sciences J. Nutr. 134:399-402, February 2004

Nutrient Metabolism
Research Communication

Nondigestible Oligosaccharides Increase Calcium Absorption and Suppress Bone Resorption in Ovariectomized Rats1

Tasleem A. Zafar, Connie M. Weaver2, Yongdong Zhao, Berdine R. Martin and Meryl E. Wastney*

Foods and Nutrition, Purdue University, West Lafayette, IN 47907 and * Metabolic Modeling Services, Hamilton, New Zealand

2To whom correspondence should be addressed. E-mail: weavercm{at}cfs.purdue.edu.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Nondigestible oligosaccharides (NDO) including inulin and fructooligosaccharides (FOS) have been reported to stimulate calcium absorption. Here we report the effect of a mixture of inulin and FOS (Raftilose Synergy 1, Orafti) on calcium and bone metabolism in ovariectomized (OVX) rats. OVX rats (6 mo old) were fed a semipurified diet for 3 mo in our animal care laboratory for stabilization after ovariectomy. They were then divided into two groups (n = 13/group) and fed either a control or a NDO-supplemented diet (55 g/kg) for 21 d. Catheters were placed in their jugular veins. After 2 d, a tracer (45Ca) was administered by gavage or i.v. and blood was sampled for up to 300 min. Urine and fecal samples were collected for 4 d after 45Ca administration. Femurs were measured for bone mineral density (BMD), breaking strength, and total calcium. Calcium absorption, femoral calcium content, BMD, and bone balance (Vbal) were significantly increased (P < 0.05) by NDO, whereas the bone resorption rate relative to the bone formation rate was significantly depressed by NDO. We conclude that feeding NDO at 5.5 g/100 g for 21 d has a positive effect on calcium absorption and retention in ovariectomized rats.

KEY WORDS: • calcium • kinetics • ovariectomized rats • nondigestible oligosaccharides • bone turnover

Nondigestible dietary oligosaccharides (NDO)3 including inulin, fructooligosaccharides (FOS), lactulose, and resistant starches increase calcium absorption in animals (1–3). Conflicting results exist in humans. Some studies have shown increased calcium retention (4,5), whereas others do not (6,7). The mechanism for increased calcium retention is not known, but has been related to enhanced absorption efficiency. It is hypothesized by some (8,9) that when soluble fiber reaches the colon, it is fermented by the intestinal microflora and converted into SCFA. These organic acids lower the luminal pH. The insoluble, unabsorbed calcium coming from the upper part of the intestine is converted to the ionic form in the lower pH medium. Both low pH and SCFAs result in the hypertrophy of the mucosal cells, leading to an enlargement of the surface area of the intestine and enhanced calcium absorption. Others (10) have proposed increased paracellular calcium diffusion in the ileum due to an enhanced permeability of the tight junctions in the presence of NDO. An increase in the ceco-colorectal calbindin-D9K protein expression in the presence of inulin has also been reported (11,12).

In the present study, we studied more thoroughly the effect of NDO on calcium metabolism using an ovariectomized rat model to mimic postmenopausal women. We measured absorption, excretion, bone formation, and bone resorption by combining a metabolic balance study with oral and i.v. isotope tracer and compartmental analysis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Animals and diets. Sprague-Dawley virgin ovariectomized (OVX) rats (n = 26; 6 mo old) were purchased from Harlan. Rats were housed individually in stainless steel cages with free access to deionized water and a semipurified AIN 93 mol/L diet (13) for 3 mo for stabilization after ovariectomy. Rats were then randomly assigned to control and experimental groups (n = 13/group). The control group continued the AIN 93 mol/L diet for 21 d. The experimental diet was modified to include NDO (Raftilose Synergy 1, Orafti), 55 g/kg, replacing equal amounts of cornstarch and sucrose. Food intake was recorded and adjusted for spillage. Body weights were recorded every 6 d. All procedures were approved by the Purdue Animal Care and Use Committee, Purdue University, West Lafayette, IN.

    Surgical procedure. For kinetic analysis, rats were catheterized to allow for multiple blood draws. Catheters (0.51 mm i.d., 0.94 mm o.d., VWR) were placed in the rats’ jugular veins by surgical procedure after anesthetization with a mixture of ketamine:xylazine (90:10 mg/kg). Rats were placed in dorsal recumbency, a 1-cm incision was made over the right jugular area, and blunt dissection was used to identify and isolate the vein. A curved hemostat was placed under the vein. A small incision was made in the vein using small iris scissors. A catheter was inserted into the vein toward the heart with the help of a plastic introducer, and ligated around the vein and at both sides of the enlargement with 4/0 suture. The catheters were filled with heparin to prevent any entry of air into the circulatory system. The free ends of the catheters were externalized between the ears. Both skin incisions were closed using 4/0 nylon sutures. The catheters were trimmed to a suitable length, flushed with heparin and then plugged with a stopper. Rats were monitored during their recovery and had free access to food and water immediately after recovery from surgery. Catheters were flushed with heparin once every day to prevent clotting.

    Tracer administration and sampling. After they regained presurgery weight, 6 rats from each treatment group were gavaged with 20 µCi 45Ca and 25 mg calcium as calcium acetate. Three rats from each group received an i.v. injection of 20 µCi 45Ca in 0.5 mL saline while receiving 25 mg calcium as calcium acetate orally. Sample sizes were determined from our previous studies (14). Five rats per group were sufficient to detect differences in oral 45Ca absorption efficiency of >7%. Because there was little individual variation in 45Ca serum levels after i.v. administration, three rats per group were sufficient to detect differences.

However, to estimate changes in calcium absorption capacity after chronic feeding of NDO, NDO was not present in the gavage solution. Blood samples (0.3 mL) were collected at 0, 5, 10, 15, 20, 30, 40, 60, 80, 130, 180, 240, and 300 min after dosing, and serum was obtained for 45Ca assay. Total blood loss was replaced with saline:dextrose (1:1) during sampling. In our previous studies (14), 45Ca peaked in the serum 80–130 min after oral dosing. Thus, a 5-h serum collection is sufficient to calculate absorption of a 25-mg Ca load. Rats were placed in individual metabolic cages immediately after dosing. Urine and feces were collected every 12 h for the first 2 d and then every 24 h for the next 2 d. Four rats were used for determining metabolic mass balance only. Rats were killed by CO2 overdose and right femurs were stored at 4°C in normal saline for dual energy X-ray absorptiometry (DXA) measurement. Left femurs were measured for wet weight and length and stored at refrigerated temperature in normal saline for measuring breaking strength and femur total calcium.

    Sample analyses. The amount of food consumed was measured during the 4-d metabolic balance study. The amount of calcium in the food was calculated including the 25 mg calcium given in the gavage. Feces were ashed for 3 d at 600°C. The ash was dissolved in a few drops of concentrated HCl, transferred to 25-mL acid-washed volumetric flasks, and brought to volume. One milliliter was analyzed for 45Ca by ß-scintillation counting (LS 6500, Beckman Coulter, Fullerton, CA). Total calcium in diet, urine, and feces was measured by atomic absorption spectrophotometry (A Analyst 300, Perkin Elmer).

For serum 45Ca analysis, 100 µL was bleached with 25 µL of 3 mol/L KOH and 100 µL H2O2 (30%, wt/v). After 30 min, 25 µL of 3mol/L HCl was added to neutralize the pH before liquid scintillation counting.

    Data analysis. Calcium retention was calculated as:

Tracer data from serum, urine and feces were analyzed using WinSAAM and a compartmental model (Fig. 1). The modeling approach was as described previously (15) except that in the current study, tracer was given either i.v. or orally.



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  		FIGURE 1 Kinetic model for calcium metabolism in rats. Circles represent compartments. Compartment 1: blood, 2 and 3: exchangeable calcium pools, 8: small intestine, 10: large intestine, Va = absorption, Vf = endogenous fecal excretion, VF = Fecal excretion, Vi = intake, Vu = urinary excretion, Vo+ = bone formation and Vo- = bone resorption. Arrows represent movement of calcium between compartments, and entry of calcium via diet. Tracer (45Ca) was administered to either compartment 8 (Oral) or compartment 1 (i.v.).

 
In brief, serum data obtained after intravenous 45Ca administration without NDO were fitted by three compartments (Compartments 1, 2, and 3) with loss into urine and feces. Data from each rat were fitted simultaneously by the model. Tracer absorption was calculated as the ratio of the fraction of compartment 8 moving into compartment 1 vs. compartment 10 (Fig. 1). Fractional transfer coefficients (/min) were combined with data on serum calcium concentration and dietary calcium intake to determine calcium transport (mg/d). Calcium transferred from compartment 3 to bone was treated as bone formation (Vo+), whereas the inflow of calcium to compartment 1, which maintains the steady state of calcium metabolism, was determined as bone resorption (Vo-). Urinary calcium excretion (Vu) and fecal endogenous excretion (Vf) were also determined. Because the oral dose was not given with NDO, calcium absorption capacity in the acute absence of NDO was estimated using the model and all tracer data from both oral and intravenous administration. True calcium absorption (Abs±NDO) was determined from tracee, or total calcium data, but corrected for the endogenous calcium excretion in both groups. Bone balance was calculated as the difference between bone formation and bone resorption (Vo+ - Vo-).

    Bone analysis. Femurs were brought to room temperature before breaking in the exact center by a three point bending test on a TA-XT2 Texture Analyzer (Texture Technologies) using a test speed of 1 mm/s. Data were expressed as breaking strength (area under curve, kg x m), stiffness (slope kg/m), and peak force (kg at the peak). Broken femurs were dissolved in 3mL 70% (wt/v) HNO3 individually. The diluted femur solution was analyzed for calcium by atomic absorption spectrophotometry (A Analyst 300, Perkin Elmer).

Bone mineral density (BMD) and bone mineral content (BMC) of the distal and proximal region, defined as 25% of the whole length of the femur and central region as 50% of the femur, were determined using DXA.

    Statistical analysis. ANOVA was performed with Tukey’s analysis using SAS to compare means (SAS Institute). A P-value of <0.05 was considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    Balance study. Both groups (control vs. NDO) had similar body weight, food intake, and total calcium intake (Table 1). Fecal calcium excretion was lower in the NDO group, and they absorbed and retained more calcium than the control group (Table 1).


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  		TABLE 1 Effect of feeding NDO on body weight, calcium balance, and femur measurements in OVX rats1

 
    Kinetics study. No difference was found in 45Ca fractional absorption capacity from the test meal that did not contain NDO between the two groups fed chronically, whereas true calcium absorption (Abs±NDO) and Vbal were significantly higher and VF was lower in the NDO group. Vu and Vf did not differ between groups. Both bone formation and bone resorption (Vo+ and Vo-) were significantly lower in the NDO group, showing suppression of rapid turnover in rats supplemented with NDO (Table 2). Vo+ was reduced by ~50%, but Vo- was reduced to essentially zero. Vo- is not literally negative because this would not be physiologically plausible, but appears so within the error of the measurement. Overall, there was a significant increase in bone retention.


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  		TABLE 2 Effect of feeding NDO on calcium absorption and rates of calcium transport in rats1

 
    Total femoral calcium and density. There was an increase in total femoral calcium in the NDO group, which translated into higher femoral BMD at the distal femurs. There were no differences in the weight, length, or breaking strength of the femurs due to NDO (Table 1). However, when femoral calcium content was adjusted by femur weight, the group difference disappeared.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Menopause is a time when estrogen deficiency leads to accelerated bone resorption and negative bone balance. During this life stage, bone balance and calcium absorption were increased and bone turnover decreased by NDO supplementation in the ovariectomized rat model. This resulted in significantly increased calcium content and BMD. Increased calcium retention in the NDO group increased BMD in the distal part of the femurs. The treatment effect was size dependent because femoral calcium content adjusted for femur weight did not differ. BMD in proximal and midshaft did not differ, perhaps due to the relatively short feeding period.

Others have shown enhanced calcium absorption with FOS, although the results depended on the Ca:FOS ratio in the diet. Absorption was increased with 5% FOS and double the calcium (1%) used in our study or with the same level of calcium (0.5%) and a higher (10%) level of FOS in ovariectomized rats (16). However, calcium absorption was unaffected by 5% FOS and 0.5% dietary calcium although trabecular bone was increased (17). In another study, FOS and 0.5% calcium did not alter trabecular bone of ovariectomized rats (16), but feeding FOS and 1% calcium resulted in decreased trabecular bone loss after 8 and 16 wk. Whole body BMC and BMD were increased with NDO at 5 or 10% at three different concentrations of dietary calcium, i.e., 0.2, 0.5, or 1%, up to 22 wk (18).

A shorter kinetic study in growing male rats adapted to FOS for a shorter period (2) than that in our study also showed improved calcium retention with FOS through increased true calcium absorption. However, important differences were also observed. In their study of young, male rats, there were no effects of FOS on bone formation, or bone resorption rates in contrast to our study in OVX rats in which NDO suppressed bone turnover. Differences between the two studies could be related to gender, age, the state of ovariectomy, or possibly differences in the nature of FOS.

The mechanism of enhanced calcium absorption with NDO supplementation has been debated. Significantly more calcium was absorbed, which suppressed bone resorption, by the NDO group. When calcium absorption capacity was estimated from tracer calcium, which was administered without NDO, there was no significant effect of chronic NDO feeding. This lack of change in calcium absorption capacity with NDO does not support mucosal cell proliferation or increased absorptive surface area in the colon as was proposed (8,9). Rather, it supports a mechanism that involves calcium and NDO in the gut at the same time such as would occur with lower gut fermentation. Further studies are warranted to extend our results. Previously, we reported that with higher calcium loads, serum 45Ca peaked much later and the curve was much flatter, which suggests multiple sites of calcium absorption after a high calcium challenge (14). This approach should be used to resolve the site of calcium absorption through kinetic modeling when different loads of calcium are given to rats with and without NDO.

Kinetic modeling allows determination of the point of perturbation of calcium metabolism by an enhancer such as NDO. From the results of our study, the protective effect of NDO on bone in a rat model to mimic menopausal women was established through the following: 1) increased calcium absorption, 2) increased calcium balance, 3) increased bone mineralization, and 4) decreased bone turnover rate.


   ACKNOWLEDGMENTS
 
Nondigestible oligosaccharides were provided by Orafti, Tienen, Belgium.


   FOOTNOTES
 
1 Presented in part at Experimental Biology 03, April 2003, San Diego, CA [Zafar, T. A. & Weaver, C. M. (2003) Inulin and calcium metabolism in ovariectomized (OVX) rats. FASEB J. 17: A721 (abs.)]. Back

3 Abbreviations used: Abs±NDO, true calcium absorption from the experimental diets; BMD, bone mineral density; BMC, bone mineral content; DXA, dual energy X-ray absorptiometry; FOS, fructooligosaccharides; NDO, nondigestible oligosaccharides; OVX, ovariectomized; Vbal, bone balance; Vf, endogenous fecal calcium excretion; VF, fecal calcium excretion; Vo+, bone formation; Vo-, bone resorption; Vu, urinary calcium excretion. Back

Manuscript received 7 October 2003. Initial review completed 20 October 2003. Revision accepted 12 November 2003.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

1. Younes, H., Coudray, C., Bellanger, J., Demigné, C., Rayssiguier, Y & Rémésy, C. (2001) Effects of two fermentable carbohydrates (inulin and resistant starch) and their combination on calcium and magnesium balance in rats. Br. J. Nutr. 86:479-485.[Medline]

2. Morohashi, T., Sano, T., Ohta, A. & Yamada, S. (1998) True calcium absorption in intestine is enhanced by fructooligosaccharide feeding in rats. J. Nutr. 128:1815-1818.[Abstract/Free Full Text]

3. Takahara, S., Morohashi, T., Sano, T., Ohta, A., Yamada, S. & Sasa, R. (2000) Fructooligosaccharide consumption enhances femoral bone volume and mineral concentration in rats. J. Nutr. 130:1792-1795.[Abstract/Free Full Text]

4. van den Heuvel, E. G., Muys, T., van Dokkum, W. & Schaafsma, G. (1999) Oligofructose stimulates calcium absorption in adolescents. Am. J. Clin. Nutr. 69:544-548.[Abstract/Free Full Text]

5. Griffin, I. J., Davila, P. M. & Abrams, S. A. (2002) Non-digestible oligosaccharides and calcium absorption in girls with adequate calcium intakes. Br. J. Nutr. 87:S187-S191.

6. Martin, B., Braun, M., Wigertz, K., Wood, O., Bryant, R. & Weaver, C. M. (2002) The effect of inulin on calcium absorption in adolescent females. J. Am. Coll. Nutr. 21:485 (abs.).

7. Tahiri, M., Tressol, J. C., Arnaud, Y., Bornet, F.R.J., Bouteloup-Demange, C., Feillet-Coudray, C., Brandolini, M., Ducros, V. & Pepin, D., et al (2003) Effect of short chain fructooligosaccharides on intestinal calcium absorption and calcium status in postmenopausal women: a stable-isotope study. Am. J. Clin. Nutr. 77:449-457.[Abstract/Free Full Text]

8. Younes, H., Demigné, C. & Rémésy, C. (1996) Acidic fermentation in the cecum increases absorption of calcium and magnesium in the large intestine of the rat. Br. J. Nutr. 75:301-314.[Medline]

9. Rémésy, C., Levrat, M. A., Gamet, L. & Demigné, C. (1993) Cecal fermentations in rats fed oligosaccharides (inulin) are modulated by dietary calcium level. Am. J. Physiol. 264:G855-G862.

10. Kishi, K., Goda, T. & Takase, S. (1996) Maltitol increases transepithelial diffusional transfer of calcium in rat ileum. Life Sci 59:1133-1140.[Medline]

11. Ohta, A., Motohaschi, Y., Ohtsuki, M., Hirayama, M., Adachi, T. & Sakuma, K. (1998) Dietary fructooligosaccharide change the concentration of calbindin-D9K differently in the mucosa of the small and large intestine of rats. J. Nutr. 128:934-939.[Abstract/Free Full Text]

12. Takasaki, M., Inaba, H., Ohta, A., Motohashi, Y., Sakai, K., Morris, H. & Sakuma, K. (2000) Dietary short chain fructooligosaccharides increase calbindin-D9K levels only in the large intestine in rats independent of dietary calcium deficiency or serum 1, 25 dihydroxy vitamin D levels. Int. J. Vitam. Nutr. Res. 70:206-213.[Medline]

13. Reeves, P. G., Nielsen, F. H. & Fahey, G. C., Jr (1993) AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123:1939-1951.

14. Hanes, D. A., Weaver, C. M. & Wastney, M. E. (1999) Calcium and oxalic acid kinetics differ in rats. J. Nutr. 129:165-169.[Abstract/Free Full Text]

15. Wastney, M. E., Ng, J., Smith, D., Martin, B. R., Peacock, M. & Weaver, C. M. (1996) Differences in calcium kinetics between adolescent girls and young women. Am. J. Physiol. 271:R208-R216.

16. Scholz-Ahrens, K. E. & Schrezenmeir, J. (2002) Inulin, oligofructose and mineral metabolism—experimental data and mechanism. Br. J. Nutr. 87:S179-S186.

17. Taguchi, A., Ohta, A., Abe, M., Baba, S., Ohtsuki, M., Takizawa, T., Yuda, Y. & Adachi, T. (1994) The influence of fructooligosaccharides on the bone of model rats with ovariectomized osteoporosis. Sci. Rep. Meiji Seika Kaisha 33:37-44.

18. Roberfroid, M. B., Cumps, J. & Devogelaer, J. P. (2002) Dietary chicory inulin increases whole body bone mineral density in growing male rats. J. Nutr. 132:3599-3602.[Abstract/Free Full Text]




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