Dietary inulin intake and age can significantly affect intestinal absorption of calcium and magnesium in rats: a stable isotope approach

Background previous studies have shown that non-digestible inulin-type fructan intake can increase intestinal mineral absorption in both humans and animals. However, this stimulatory effect on intestinal absorption may depend on experimental conditions such as duration of fermentable fiber intake, mineral diet levels and animals' physiological status, in particular their age. Objectives the aim of this study was to determine the effect of inulin intake on Ca and Mg absorption in rats at different age stages. Methods eighty male Wistar rats of four different ages (2, 5, 10 and 20 months) were randomized into either a control group or a group receiving 3.75% inulin in their diet for 4 days and then 7.5% inulin for three weeks. The animals were fed fresh food and water ad libitum for the duration of the experiment. Intestinal absorption of Ca and Mg was determined by fecal monitoring using stable isotopic tracers. Ca and Mg status was also assessed. Results absorption of Ca and Mg was significantly lower in the aged rats (10 and 20 mo) than in the young and adult rat groups. As expected, inulin intake increased Ca and Mg absorption in all four rat groups. However, inulin had a numerically greater effect on Ca absorption in aged rats than in younger rats whereas its effect on Mg absorption remained similar across all four rat age groups. Conclusion the extent of the stimulatory effect of inulin on absorption of Ca may differ according to animal ages. Further studies are required to explore this effect over longer inulin intake periods, and to confirm these results in humans.


Introduction
When non-digestible inulin-type fructans reach the large intestine, they are fermented by the local microflora and stimulate the growth of bifidobacteria and lactobacilli, which may have health-promoting functions [1][2][3]. Several studies have demonstrated that rats fed with prebiotic fructans absorbed more Ca and Mg than control rats, despite an increase in total fecal mass [4][5][6]. Indeed, the products of fructan fermentation can influence the intestinal absorption of Ca and Mg in many ways. Short-chain fatty acids (SCFA) are fermentation products that are responsible for lowering the pH of cecal content, which in turn increases mineral solubility, leading to improved mineral absorption [7]. SCFA can directly influence mineral absorption by forming complexes with the minerals, thereby increase their uptake by the intestinal cells [8,9]. It is thought that the bacterial metabolites (e.g. butyrate) can stimulate the intestinal epithelium and increase its absorptive capacity [10]. These various factors are closely linked to the nature of the prebiotic carbohydrates and to experimental conditions [7,11,12]. Inulin has been shown to have generally high and consistent effects on intestinal Mg absorption in both animals and humans [13], but the effects of inulin on calcium (Ca) absorption seem to be dependent on experimental conditions (dose of inulin, dietary Ca content, experiment duration, animal age and mineral requirements). In this study, we investigated the relationship between animal age and the stimulatory effect of inulin on intestinal absorption and retention of Ca and Mg using a stable isotope approach following short-term administration of inulin in rats aged from 2 to 20 months. This is the first time that the effect of inulin is studied in rats using a stable isotope approach

Animals and diets
Eighty male Wistar rats aged 2, 5, 10 or 20 months were purchased from Janvier (Le Genest Saint Ile, France). They were fed a commercial pellet diet (Ssniff R/S-breedinguntil 3 mo, then Ssniff R/S maintenance from 3 to 24 mo age). Two groups were formed for each age bracket to receive either a control diet or a semi-purified diet containing inulin until the end of the experiment. The composition of these two diets is given in Table 1. Tested inulin was purchased from Orafti, Tienen, Belgium (Raftaline ® ). The target Ca and Mg levels in these diets were 5000 mg Ca/kg and 500 mg Mg/Kg diet. Powder diet (100 g) was made up with 100 ml of distilled water to form a kind of semi-liquid food prepared on-site each day. Chemical analysis of the diets offered confirmed the expected Ca and Mg contents in the experimental diets: 5107 mg Ca/kg and 5050 mg Ca/kg, and 495 mg Mg/kg and 514 mg Mg/kg in the control and inulin diets, respectively. Chemical analysis showed that the inulin contained approximately 40 mg Ca/kg and less than 1 mg Mg/kg. Dietary inulin level was maintained at 3.75% during the first 4 days and then 7.5% from day 5 until the end of the experiment. The 8 rat groups were given fresh food and water daily, made available ad libitum. Food consumption and body weight were recorded weekly. Throughout the experiment, the rats were housed two per cage (wire-bottomed to limit coprophagy) in a temperature-controlled room (22°C) with dark period from 08:00 pm to 08:00 am. Total experiment duration was 30 days. All procedures complied with the Institute's ethical guidelines on the care and use of laboratory animals.

Preparation of stable isotope solution
215 mg of the 44 Ca (in carbonate form = 508 mg) and 255 mg of the 25 Mg (in oxide form = 412 mg) were first individually moistened with 2 ml of distilled water. One ml of HCl 12 N (ultrapure) was added to the 44 Ca suspension and two ml of HCl 12 N was added to the 25 Mg suspension to transform the carbonate and the oxide into soluble chlorides of Ca and Mg, respectively. Each solution was then diluted with 50 ml of distilled water, both solutions were then mixed, and pH was adjusted to between 3 and 6 with 1 N sodium hydroxide solution. The resulting study solution was then completed to 150 ml with distilled water and maintained for several days at +4°C until utilization. Total and isotopic Ca and Mg contents were checked before use.
The rats were transferred to metabolic cages and housed individually three days before the beginning of the isotopic balance study to allow them to adapt to their new environment. Animals received by gavage about 1.7 ml of isotopic solution. The urine and faeces of each rat were quantitatively collected for four consecutive days, and excreted isotopes in these two media and in the gavage solution were quantitatively determined by ICP/MS, as described below.

Sampling procedures
The rats were sacrificed just after the dark period (between 08:00 am and 10:00 am), i.e. at a time when cecal fermentation was still very active. After anesthesia (40 mg sodium pentobarbital/kg body weight), blood was withdrawn from the abdominal aorta, placed into tubes containing sodium heparin and centrifuged at 1,000 g for 10 minutes. Plasma samples were stored at 4°C for mineral analysis. The cecum, complete with contents, was removed and weighed (total cecal weight). The cecal wall was flushed clean with ice-cold saline, blotted on filter paper, and weighed (cecal wall weight). For each rat, duplicate samples of cecal contents were collected into 2 ml microfuge tubes and immediately frozen at -20°C until analysis. The pH of cecal content was determined on site using a Sentron pH-system 1001 portable pH-meter (Sentron Europe B.V. Ac Roden, The Netherlands). Supernatants of the digestive contents were obtained by centrifuging one of the two microfuge tubes at 20,000 g for 10 minutes at 4°C, and then frozen until analysis. One tibia was also sampled for Ca and Mg analysis.

Analytical procedures
Ca and Mg concentrations were determined in the plasma and urine after adequate dilution into 0.1% (w/v) lanthanum chloride. Diet aliquots, fecal materials and tibia were dry-ashed (10 hours at 500°C) and dissolved with concentrated HNO 3 and H 2 O 2 on a heating plate until complete decoloration. The resulting mineral solutions were set at 10 ml with water and adequately diluted in 0.1% lanthanum chloride. Mineral concentrations were measured by atomic absorption spectrophotometry (on a Perkin-Elmer AA800) at wavelengths of 422 nm for Ca and 285 nm for Mg.
For isotopic 44 Ca and 25 Mg determination, samples were appropriately diluted before analysis using 1% HNO 3 . Ca and Mg concentration and isotope ratios were determined by ICP-MS using Ca and Mg as external standard and beryllium as internal standard. The instrument operating conditions were set as follows after optimization with a solution of 1 µg indium/l: RF Power = 1050 W, Nebulizer Ar flow rate = 0.79 L/min, Auxiliary Ar flow rate = 1.2 L/ min, Outer Ar flow rate = 15 L/min. Data acquisition parameters were set as follows: Sweeps/reading = 50, Readings/replicate = 1, Number of replicates = 3, Dwell time = 50 ms for 24 Mg, 75 ms for 9 Be, 25 Mg, 26  Cecal SCFA concentrations, including acetic, propionic and butyric acid, were determined by gas-liquid chromatography on portions of supernatant fractions of cecal contents as previously described [14].  Calculations were also made directly from ICP-MS data. The two modes of calculation give the same results when the ICP-MS quantitative procedure is used [16].

Calculations
Intestinal absorption of 44 Ca and 25 Mg was then calculated as administered 44 Ca or 25 Mg -44 Ca or 25 Mg excreted in the feces, and retention of 44 Ca and 25 Mg was calculated as administered 44 Ca or 25 Mg -44 Ca or 25 Mg excreted in the feces and in the urine.
Soluble Ca and Mg levels in the cecal contents were determined on the supernatant concentration (µg/ml), and soluble Ca and Mg contents per cecum were calculated as (µg Ca/ml or µg Mg/ml) × cecal water (ml).

Data analysis
Values are given as means ± SD, and data were tested by 2way ANOVA using the General Linear Models procedure of the Super ANOVA package (Abacus, Berkeley, CA). Post-hoc comparisons were performed using Fisher's least significant difference procedures. Differences of p < 0.05 were considered statistically significant. Simple linear correlation analysis was used to assess the relationships between intestinal absorption of Ca and Mg and other relevant parameters. Values of p < 0.05 were considered statistically significant.

Food intake and growth rate
Inulin intake at the dose of 7.5% showed only a tendency to decrease animal food intake in this study. The slight decrease in food intake in inulin-fed rats led to a non-significantly lower growth rate (p < 0.10) towards the end of the experiment in inulin-fed rats compared to controls. The lower calorific value of the inulin diets (-4%) compared to the control diets may also be responsible for this reduced weight gain. In addition, food intake decreased significantly with increasing age, as expected (data not shown).

Cecal fermentation parameters and total and cecal soluble Ca and Mg levels (table 2)
As expected, inulin intake significantly increased cecal wall weight and cecal content and significantly decreased the pH of cecal content. These variables did not change with rat age. In addition, inulin intake considerably increased the individual and total pools of SCFA in the cecal contents (p < 0.0001). The effect of age on these SCFA pools was less clear. No significant age-related difference was observed amongst the control group rats, whereas in the inulin-fed group, the intestinal bacteria produced higher acetate, butyrate and total SCFA in the rats aged 10 mo than in the three other groups (p < 0.05). Values are mean ± SD, n = 10 animals. The rats were given 44 Ca after 14 days of inulin intake (7.5%), and fecal non-absorbed 44 Ca isotope was determined in a 4d feces and urine pools.

Intestinal absorption and retention of calcium (table 3)
The amount of gavaged 44 Ca was about 1.60 mg/rat, which led to a fecal 44 Ca enrichment of 10% to 20% in the 4-day feces pool. Fecal 44 Ca excretion expressed as mg/g of feces or as mg/day increased significantly with age. Consequently, net (mg) and relative (%) 44 Ca absorption were significantly lower in the aged rats than in the young adult or adult rats. In addition, urinary 44 Ca excretion (mg) increased significantly with age. Consequently, net (mg) and relative (%) 44 Ca retention were considerably lower in the aged rats than in the young adult or adult rats. Inulin intake significantly decreased fecal 44 Ca excretion, expressed as µg/g of feces or as µg, in all groups. Consequently, inulin intake significantly increased net (mg) and relative (%) 44 Ca absorption. Moreover, inulin intake increased urinary 44 Ca excretion (mg). Lastly, inulin intake significantly increased net (mg) and relative (%) 44 Ca retention in the four age-related groups compared to the control diet groups.

Intestinal absorption and retention of magnesium (table 4)
The amount of gavaged 25 Mg was about 2.50 mg/rat, which led to a fecal 25 Mg enrichment of 35% to 65% in the 4-day feces pool. Fecal 25 Mg excretion expressed as mg/g of feces or as mg increased significantly with age. Consequently, net (mg) and relative (%) 25 Mg absorption were significantly lower in the aged rats than in the young adult or adult rats. In addition, urinary 25 Mg excretion (mg) increased significantly with age. Consequently, net (mg) and relative (%) 25 Mg retention were significantly lower in the aged rats than in the young adult or adult rats. As expected, inulin intake significantly decreased fecal 25 Mg excretion, expressed as µg/g of feces or as µg, in all groups. Consequently, inulin intake significantly increased net (mg) and relative (%) 25 Mg absorption. Similarly, inulin intake increased urinary 25 Mg excretion (mg). However, inulin intake led to significantly higher net (mg) and relative (%) 25 Mg retention in all four groups compared to the control diet.

Discussion
Previous studies have repeatedly shown that intake of different inulin-type fructans can variably increase mineral intestinal absorption in humans and animals [4,5,[17][18][19]. Indeed, inulin-type fructans strongly and consistently increase intestinal Mg absorption [12], whereas their effect on Ca absorption seems to be dependent on experimental conditions such as inulin type, dietary Ca levels, duration of fructan intake [20][21][22] and the animals' physiological state, particularly age. It is well known that the absorption mechanisms of Ca and Mg differ considerably [23,24], which may explain the observed differences between these two minerals in terms of inulin effect. In this study, we investigated the enhancing effect of fructan intake on Ca and Mg intestinal absorption and balance in rats of different ages.

-Effect of animal age and inulin intake on Ca absorption
Our results clearly showed that aged rats exhibited less efficient intestinal absorption and retention of Ca. 44 Ca absorption ranged from 48% without inulin to 66% Values are mean ± SD, n = 10 animals. The rats were given 25 Mg after 14 days of inulin intake (7.5%), and fecal non-absorbed 25 Mg isotope was determined in a 4d feces and urine pools.
under inulin intake in the young and adult rats and from 15% without inulin to 27% under inulin intake in the old and very old rats. This decline in Ca absorption with age is not new, and has already been reported in animal and human studies [25][26][27] and is largely confirmed in this study. This decline is primarily due to an energy-and vitamin D-dependent Ca transport component in the elderly [28]. Our results clearly showed that inulin intake increased the efficiency of Ca intestinal absorption and retention. The mean 44 Ca absorption in the four rat control groups was 26.3% compared to 40.2% in the four inulin-fed groups, with an overall increase in 44 Ca absorption of 53%. These results are in agreement with literature data showing that the effect of inulin on Ca absorption seems to be optimal in the early weeks, then decreasing gradually with experiment duration [20,29,30]. One possible explanation for this phenomenon is a down-regulation of the active pathway of intestinal Ca absorption after several weeks of feeding inulin, as previously reported [31,32].

-Effect of animal age and inulin intake on Mg absorption
Our results showed that aged rats exhibited less efficient intestinal absorption and retention of Mg. 25 Mg absorption ranged from 56% without inulin to 86% under inulin intake in the young and adult rats and from 45% without inulin to 70% under inulin intake in the old and very old rats. This decline in Mg absorption with age is not well documented in the literature in either animal or human studies. Few, if any, incomplete studies have reported an age effect on Mg absorption [33][34][35], and the results are inconsistent. Hence, to our knowledge, this is the first robust report to clearly show that Mg absorption decreases with age in the rat. Although Mg absorption is generally described as a passive phenomenon, one component of this absorption remains under hormonal control [36,37], which may explain the observed results. Our results clearly showed that inulin intake considerably increased Mg intestinal absorption and retention efficiency. Mean 25 Mg absorption in the four rat control groups was 50.6%, compared to 78.0% in the four corresponding inulin-fed rat groups, with an overall increase in 25 Mg absorption of 54%. These results are in agreement with literature data showing that inulin intake considerably increases Mg absorption in animals and humans (see recent review [13]).

-Modulation of the stimulatory inulin effect on Ca and Mg absorption with rat age
Since Ca absorption is generally well controlled, the observed absorption increase under inulin intake may be down-regulated (known as a feed-back phenomenon) in adult rats. Thus, given that Ca absorption is low and the adaptative phenomenon less well controlled in aged rats, we hypothesized that inulin intake would lead to a much greater increase in Ca absorption in aged rats than in the young or adult rats. Conversely, since Mg absorption is only weakly controlled with a generally consistent increase under inulin intake in adult rats, we hypothesized that inulin intake would increase Mg absorption in both aged rats and young or adult rats to the same extent.
The relative increase in 44 Ca absorption under inulin intake was 41.5% and 84.5% in the younger (3 and 6 mo old) and older rats (11 and 21 mo old), respectively (figure 1). Although these increase percents are numerically more important in the older rats than in the younger rats, there was no statistically significant interaction between age and inuline. It is highly possible that the number of animals used in this experiment was not enough to reach significant level. Furthermore, the relative increase in 25 Mg absorption under inulin intake was 53.5% and 54.5% in the younger and older rats, respectively ( figure  1). This indicates that the stimulatory effect of inulin on 25 Mg absorption was not age-dependent. It is possible that inulin intake may lead to a higher increase in 44 Ca absorption in the older rats than in the younger rats, whereas inulin intake leads to a similar increase in 25 Mg absorption in young, adult and aged rats, thus confirming the hypothesis we formulated for this study.
In conclusion, as expected, our results confirmed that short-term inulin intake stimulates the absorption of both Ca and Mg. Furthermore, not only these results confirmed that Ca absorption declines considerably with age but also showed for the first time that Mg absorption also declines with age in the rat. Moreover, these results confirmed our hypothesis of a greater stimulatory effect of inulin on Ca absorption in aged rats than in the young or adult rats, and a similar stimulatory effect of inulin on Mg absorption in aged rats and young and adult rats. Further studies are required to explore this effect on longer inulin intake periods and to validate these results on the stimulatory effect of inulin on Ca and Mg absorption in the elderly.  25 Mg absorption (B) in the control groups was normalized to 100% for each age group. The stimulatory effect of inulin (%) for a given age group was calculated as follows: 100* (intestinal absorption in the inulin-fed age group/intestinal absorption in the same age group without inulin). The rats were given 44 Ca and 25 Mg after 14 days of inulin intake (7.5%), and fecal nonabsorbed isotopes were determined in a 4d feces pool.