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

  • Charles Coudray1Email author,

    Affiliated with

    • Mathieu Rambeau1,

      Affiliated with

      • Christine Feillet-Coudray1,

        Affiliated with

        • Claude Jean Tressol1,

          Affiliated with

          • Christian Demigne1,

            Affiliated with

            • Elyett Gueux1,

              Affiliated with

              • Andrzej Mazur1 and

                Affiliated with

                • Yves Rayssiguier1

                  Affiliated with

                  Nutrition Journal20054:29

                  DOI: 10.1186/1475-2891-4-29

                  Received: 31 August 2005

                  Accepted: 27 October 2005

                  Published: 27 October 2005

                  Abstract

                  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.

                  Inulin intestinal absorption status calcium magnesium fermentation stable isotope age rat

                  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 [13]. 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 [46]. 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

                  Materials and methods

                  Materials and reagents

                  The enriched Ca isotope (44Ca) as CaCO3 and the enriched Mg isotope (25Mg) as MgO were obtained from Chemgas, (Boulogne, France). The atomic abundances of these enriched isotopes were as follows: 40Ca = 3.41%, 42Ca = 0.09%, 43Ca = 0.03%, 44Ca = 96.45% 46Ca =< 0.01% 48Ca = 0.02% and 24Mg = 1.6%, 25Mg = 97.8%, 26Mg = 0.6%. HNO3 (ultrapure), Mg and beryllium standard solutions (1 g/L) were obtained from Merck (Darmstadt, Germany). All other chemicals were of the highest quality available. Distilled water was used throughout. A Perkin-Elmer 6100DRC system (Perkin-Elmer Instruments, Courteboeuf, France) equipped with a Meinhard nebulizer was used for isotopic measurement, and a Perkin Elmer AA800 (Perkin Elmer Instruments, Courteboeuf, France) was used for total Mg measurement.

                  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-breeding – until 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.
                  Table 1

                  Diet composition (g/kg) during the experiment

                   

                  Control diet

                  Inulin diets

                    

                  3.75%

                  7.5%

                  Wheat starch

                  650

                  612.5

                  575

                  Casein

                  200

                  200

                  200

                  Corn oil

                  50

                  50

                  50

                  Cellulose

                  50

                  50

                  50

                  Mineral mix (AIN 1993)a

                  35

                  35

                  35

                  Vitamin mix (AIN 1993)b

                  10

                  10

                  10

                  DL-Methionine

                  3

                  3

                  3

                  Choline bi-tartrate

                  2

                  2

                  2

                  Inulin

                  0

                  37.5

                  75

                  a: Mineral mix AIN 1993 ensures the following mineral levels in the diets (mg/kg): Na, 1020; K, 3600; P, 4000; Ca, 5000; Mg, 500; Zn, 30; Fe, 35; Cu, 6; Mn, 54; Se, 0.1; I, 0.2; Cr, 2.

                  b: Vitamin mix AIN 1993 ensures the following mineral levels in the diets (mg/kg): thiamine, 6; riboflavine, 6; pyridoxine, 7; nicotinic acid, 30; calcium pantothenate, 16; folic acid, 2; D-biotin: 0.2; and (μg/kg) cyanocobalamine (vitamin B12), 10; vitamin K, 50; and (IU/kg) vitamin A, 4000; vitamin E, 50; vitamin D, 1000.

                  Preparation of stable isotope solution

                  215 mg of the 44Ca (in carbonate form = 508 mg) and 255 mg of the 25Mg (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 44Ca suspension and two ml of HCl 12 N was added to the 25Mg 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 HNO3 and H2O2 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 44Ca and 25Mg determination, samples were appropriately diluted before analysis using 1% HNO3. 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 24Mg, 75 ms for 9Be, 25Mg, 26Mg, and 44Ca, 150 ms for 42Ca and 300 ms for 43Ca, Scanning mode = peak hopping. DRC operating conditions (for 42Ca, 43Ca and 44Ca) were as follows: Cell Gas A Flow Rate = 0.5 L ammonia/min, RPa = 0, and RPq = 0.45.

                  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

                  Ca and Mg each have different stable isotopes with the following natural abundances: 40Ca = 96.941%, 42Ca = 0.647%, 43Ca = 0.135%, 44Ca = 2.086% 46Ca = 0.004% 48Ca = 0.187% and 24Mg = 78.99%, 25Mg = 10.00% and 26Mg = 11.01% [15]. 44Ca and 25Mg isotopic enrichments were obtained, respectively, from the following equations: (44Ca/43Ca measured ratio - 44Ca/43Ca baseline ratio)/(44Ca/43Ca baseline ratio) and (25Mg/26Mg measured ratio - 25Mg/26Mg baseline ratio)/(25Mg/26Mg baseline ratio).

                  Non-absorbed 44Ca and 25Mg isotopes in the fecal or urine samples (coming only from the 44Ca or 25Mg isotope labels) were calculated as follows:

                  for 44Ca (mg) = (total fecal or urine Ca (mg) × (natural abundance 44Ca × enriched 44Ca)/(1 + (natural abundance 44Ca × enriched 44Ca);

                  for 25Mg (mg) = (total fecal or urine Mg (mg) × (natural abundance 25Mg × enriched 25Mg)/(1 + (natural abundance 25Mg × enriched 25Mg)).

                  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].

                  Intestinal absorption of 44Ca and 25Mg was then calculated as administered 44Ca or 25Mg - 44Ca or 25Mg excreted in the feces, and retention of 44Ca and 25Mg was calculated as administered 44Ca or 25Mg - 44Ca or 25Mg excreted in the feces and in the urine.

                  Total cecal SCFA content (μmol/cecum) was calculated as the supernatant SCFA concentration (μmol/ml) × cecal water (ml/cecum).

                  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 2-way 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.

                  Results

                  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)

                  Table 2

                  Effect of age and inulin intake and their interaction on cecum fermentation parameters and cecal Ca and Mg levels in rats

                   

                  Cont 3 Mo

                  Cont 6 Mo

                  Cont 11 Mo

                  Cont 21 Mo

                  Inulin 3 Mo

                  Inulin 6 Mo

                  Inulin 11 Mo

                  Inulin 21 Mo

                  inulin

                  age

                  interaction

                  Cecal content pH

                  6.92 ± 0.24

                  6.87 ± 0.17

                  6.72 ± 0.58

                  6.62 ± 0.31

                  5.71 ± 0.58

                  5.41 ± 0.22

                  5.64 ± 0.37

                  5.57 ± 0.22

                  <0.0001

                  NS

                  NS

                  Cecal content, g

                  2.20 ± 0.35

                  2.34 ± 0.68

                  2.53 ± 0.97

                  2.86 ± 0.74

                  6.18 ± 1.68

                  6.46 ± 1.57

                  7.09 ± 2.31

                  7.10 ± 1.91

                  <0.0001

                  NS

                  NS

                  Cecal wall, g

                  0.87 ± 0.07

                  1.11 ± 0.22

                  1.32 ± 0.29

                  1.25 ± 0.15

                  1.80 ± 0.37

                  2.32 ± 0.29

                  2.51 ± 0.39

                  2.46 ± 0.30

                  <0.0001

                  NS

                  NS

                  Acetate, μmol/cecum

                  22.4 ± 6.3

                  24.5 ± 9.4

                  24.4 ± 11.8

                  28.0 ± 8.5

                  49.2 ± 22.3

                  70.4 ± 16.7

                  54.3 ± 17.3

                  63.5 ± 11.4

                  <0.0001

                  0.033

                  NS

                  Propionate, μmol/cecum

                  5.49 ± 1.48

                  6.31 ± 2.20

                  5.11 ± 2.10

                  6.11 ± 1.72

                  15.56 ± 12.29

                  12.84 ± 5.04

                  9.96 ± 4.86

                  11.26 ± 3.92

                  <0.0001

                  NS

                  NS

                  Butyrate, μmol/cecum

                  9.90 ± 2.70

                  8.63 ± 3.10

                  6.37 ± 2.78

                  8.01 ± 3.38

                  48.13 ± 25.12

                  69.51 ± 32.50

                  39.98 ± 18.42

                  43.15 ± 8.63

                  <0.0001

                  0.018

                  0.037

                  Total SCFA, μmol/cecum

                  37.8 ± 9.1

                  39.5 ± 13.5

                  35.9 ± 15.9

                  42.2 ± 12.6

                  112.9 ± 50.8

                  152.8 ± 40.0

                  104.2 ± 27.7

                  117.9 ± 17.3

                  <0.0001

                  0.024

                  0.049

                  Values are mean ± SD, n = 10 animals.

                  The rats were received inulin (7.5%) for 4 weeks before cecal parameters assessment.

                  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).

                  Intestinal absorption and retention of calcium (table 3)

                  Table 3

                  Effect of age and inulin intake and their interaction on intestinal absorption and retention of Ca in rats

                   

                  Cont 3 Mo

                  Cont 6 Mo

                  Cont 11 Mo

                  Cont 21 Mo

                  Inulin 3 Mo

                  Inulin 6 Mo

                  Inulin 11 Mo

                  Inulin 21 Mo

                  inulin

                  age

                  interaction

                  Administered 44 Ca, μg

                  1637 ± 46

                  1610 ± 14

                  1602 ± 17

                  1605 ± 24

                  1593 ± 19

                  1614 ± 19

                  1621 ± 25

                  1626 ± 22

                  NS

                  NS

                  <0.0005

                  Fecal 44 Ca enrichment, %

                  12.5 ± 3.3

                  17.3 ± 2.4

                  20.2 ± 4.3

                  18.7 ± 2.8

                  10.9 ± 4.2

                  17.8 ± 6.2

                  17.4 ± 2.1

                  21.2 ± 3.1

                  NS

                  <0.0001

                  NS

                  Fecal 44 Ca level, μg/g

                  112 ± 30

                  182 ± 35

                  218 ± 45

                  204 ± 38

                  76 ± 30

                  152 ± 49

                  163 ± 26

                  188 ± 26

                  <0.0001

                  <0.0001

                  NS

                  Fecal 44 Ca excretion, μg

                  856 ± 224

                  1139 ± 153

                  1389 ± 96

                  1366 ± 115

                  541 ± 223

                  926 ± 142

                  1207 ± 195

                  1192 ± 142

                  <0.0001

                  <0.0001

                  NS

                  Intestinal 44 Ca absorption, μg

                  781 ± 206

                  471 ± 153

                  213 ± 90

                  239 ± 117

                  1052 ± 222

                  689 ± 142

                  413 ± 202

                  434 ± 143

                  <0.0001

                  <0.0001

                  NS

                  Intestinal 44 Ca absorption, %

                  47.8 ± 12.9

                  29.3 ± 9.4

                  13.3 ± 5.6

                  14.9 ± 7.3

                  66.1 ± 13.9

                  42.7 ± 8.8

                  25.4 ± 12.4

                  26.7 ± 8.7

                  <0.0001

                  <0.0001

                  NS

                  Urinary 44 Ca enrichment, %

                  17.4 ± 6.5

                  20.6 ± 6.7

                  14.8 ± 3.1

                  16.9 ± 3.9

                  13.7 ± 3.3

                  17.5 ± 6.7

                  18.6 ± 4.2

                  18.5 ± 4.1

                  NS

                  NS

                  0.0569

                  Urinary 44 Ca excretion, μg

                  15.3 ± 5.9

                  14.8 ± 6.0

                  25.8 ± 10.7

                  28.1 ± 7.9

                  23.5 ± 7.2

                  21.8 ± 9.9

                  49.0 ± 11.8

                  40.3 ± 12.5

                  <0.0001

                  <0.0001

                  0.036

                  44 Ca retention, μg

                  765 ± 204

                  456 ± 153

                  188 ± 92

                  212 ± 113

                  1029 ± 219

                  667 ± 135

                  364 ± 203

                  394 ± 145

                  <0.0001

                  <0.0001

                  NS

                  44 Ca retention, %

                  46.9 ± 12.8

                  28.3 ± 9.5

                  11.7 ± 5.7

                  13.2 ± 7.1

                  64.6 ± 13.8

                  41.3 ± 8.3

                  22.4 ± 12.4

                  24.2 ± 8.9

                  <0.0001

                  <0.0001

                  NS

                  Values are mean ± SD, n = 10 animals.

                  The rats were given 44Ca after 14 days of inulin intake (7.5%), and fecal non-absorbed 44Ca isotope was determined in a 4d feces and urine pools.

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

                  Intestinal absorption and retention of magnesium (table 4)

                  Table 4

                  Effect of age and inulin intake and their interaction on intestinal absorption and retention of Mg in rats

                   

                  Cont 3 Mo

                  Cont 6 Mo

                  Cont 11 Mo

                  Cont 21 Mo

                  Inulin 3 Mo

                  Inulin 6 Mo

                  Inulin 11 Mo

                  Inulin 21 Mo

                  inulin

                  age

                  interaction

                  Administered 25 Mg, μg

                  2553 ± 71

                  2511 ± 22

                  2499 ± 26

                  2504 ± 38

                  2485 ± 30

                  2518 ± 30

                  2527 ± 39

                  2536 ± 35

                  NS

                  NS

                  <0.0005

                  Fecal 25 Mg enrichment, %

                  47.9 ± 7.1

                  51.9 ± 7.4

                  54.6 ± 12.3

                  54.1 ± 8.1

                  33.9 ± 20.3

                  41.0 ± 19.9

                  47.4 ± 13.9

                  65.7 ± 17.6

                  NS

                  0.0007

                  0.027

                  Fecal 25 Mg level, μg/g

                  149 ± 26

                  184 ± 34

                  205 ± 44

                  204 ± 38

                  51 ± 33

                  70 ± 35

                  91 ± 31

                  120 ± 35

                  <0.0001

                  <0.0001

                  NS

                  Fecal 25 Mg excretion, μg

                  1138 ± 201

                  1157 ± 229

                  1311 ± 200

                  1366 ± 177

                  358 ± 224

                  430 ± 206

                  673 ± 205

                  757 ± 184

                  <0.0001

                  <0.0001

                  NS

                  Intestinal 25 Mg absorption, μg

                  1415 ± 187

                  1354 ± 225

                  1188 ± 199

                  1137 ± 175

                  2127 ± 224

                  2087 ± 208

                  1855 ± 232

                  1780 ± 186

                  <0.0001

                  <0.0001

                  NS

                  Intestinal 25 Mg absorption %

                  55.5 ± 7.5

                  54.0 ± 9.0

                  47.5 ± 8.0

                  45.4 ± 7.0

                  85.6 ± 8.9

                  82.9 ± 8.2

                  73.3 ± 8.4

                  70.2 ± 7.2

                  <0.0001

                  <0.0001

                  NS

                  Urinary 25 Mg enrichment, %

                  29.3 ± 2.7

                  29.1 ± 3.5

                  28.0 ± 3.7

                  28.2 ± 2.32

                  34.2 ± 3.8

                  35.5 ± 2.4

                  33.5 ± 3.7

                  36.7 ± 6.5

                  <0.0001

                  NS

                  NS

                  Urinary 25 Mg excretion, μg

                  398 ± 64

                  323 ± 36

                  298 ± 80

                  292 ± 88

                  699 ± 91

                  792 ± 167

                  633 ± 146

                  551 ± 164

                  <0.0001

                  0.003

                  NS

                  25 Mg retention, μg

                  1017 ± 189

                  1031 ± 225

                  890 ± 179

                  845 ± 142

                  1428 ± 216

                  1295 ± 192

                  1221 ± 242

                  1228 ± 165

                  <0.0001

                  0.011

                  NS

                  25 Mg retention, %

                  39.8 ± 7.3

                  41.1 ± 9.0

                  35.6 ± 7.1

                  33.7 ± 5.7

                  57.8 ± 8.9

                  51.5 ± 7.8

                  48.3 ± 9.2

                  48.4 ± 6.5

                  <0.0001

                  0.008

                  NS

                  Values are mean ± SD, n = 10 animals.

                  The rats were given 25Mg after 14 days of inulin intake (7.5%), and fecal non-absorbed 25Mg isotope was determined in a 4d feces and urine pools.

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

                  Calcium and magnesium status (table 5)

                  Table 5

                  Effect of age and inulin intake and their interaction on status biomarkers of Ca and Mg in rats

                   

                  Cont 3 M

                  Cont 6 M

                  Cont 11 M

                  Cont 21 M

                  Inulin 3 M

                  Inulin 6 M

                  Inulin 11 M

                  Inulin 21 M

                  inulin

                  age

                  interaction

                  Plasma Ca, mg/L

                  98 ± 4

                  98 ± 5

                  95 ± 6

                  100 ± 5

                  102 ± 5

                  99 ± 4

                  98 ± 3

                  100 ± 4

                  0.0601

                  0.0619

                  NS

                  Tibia weight, mg dw

                  480 ± 42

                  630 ± 80

                  717 ± 89

                  630 ± 93

                  489 ± 66

                  617 ± 66

                  841 ± 92

                  648 ± 47

                  0.042

                  <0.0001

                  0.023

                  Bone Ca, mg/g dw

                  207 ± 21

                  214 ± 15

                  216 ± 15

                  215 ± 21

                  205 ± 13

                  215 ± 18

                  202 ± 14

                  228 ± 7

                  NS

                  0.018

                  0.0639

                  Plasma Mg, mg/L

                  17.9 ± 1.1

                  17.7 ± 1.1

                  17.2 ± 1.0

                  16.9 ± 1.3

                  17.6 ± 1.1

                  17.9 ± 1.3

                  18.2 ± 1.5

                  18.2 ± 1.7

                  0.0570

                  NS

                  NS

                  Erythrocyte Mg, mg/L

                  45.4 ± 3.8

                  44.2 ± 4.7

                  42.5 ± 3.4

                  43.4 ± 3.0

                  44.9 ± 4.9

                  43.8 ± 2.5

                  44.3 ± 3.6

                  43.8 ± 3.9

                  NS

                  NS

                  NS

                  Bone Mg, mg/g dw

                  3.92 ± 0.10

                  3.79 ± 0.08

                  3.72 ± 0.08

                  3.76 ± 0.08

                  3.91 ± 0.10

                  3.72 ± 0.07

                  3.73 ± 0.09

                  3.72 ± 0.09

                  NS

                  <0.0001

                  NS

                  Values are mean ± SD, n = 10 animals.

                  Mean plasma Ca varied from 95 to 102 mg/L, showing a tendency to increase with inulin intake (+2%, p = 0.0601) and to decrease with increasing age (-1%, p = 0.0619). Mean bone Ca varied from 202 to 228 mg/g dry weight, and was unaffected by inulin intake. However, mean bone Ca increased significantly with increasing age. Mean plasma Mg varied from 16.9 to 18.2 mg/L, showing a tendency to increase with inulin intake (+3%, p = 0.0570). However, mean plasma Mg was not modified by age. Plasma Mg increased in the inulin-fed aged rats (+6.7%), whereas there was no increase in the young and adult rats (-0.3%). Mean red blood cell Mg levels varied from 42.5 to 45.4 mg/L and remained unchanged when age increases or under inulin intake. Mean bone Mg levels varied from 3.72 to 3.92 mg/g dry weight, decreasing significantly with aging (p < 0.0001). However, mean bone Mg was unaffected by inulin intake.

                  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, 1719]. 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 [2022] 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.

                  1 – 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. 44Ca absorption ranged from 48% without inulin to 66% 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 [2527] 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 44Ca 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 44Ca 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].

                  2 – 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. 25Mg 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 [3335], 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 25Mg 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 25Mg 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]).

                  3 – 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 44Ca 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 25Mg 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 25Mg absorption was not age-dependent. It is possible that inulin intake may lead to a higher increase in 44Ca absorption in the older rats than in the younger rats, whereas inulin intake leads to a similar increase in 25Mg absorption in young, adult and aged rats, thus confirming the hypothesis we formulated for this study.

                  http://static-content.springer.com/image/art%3A10.1186%2F1475-2891-4-29/MediaObjects/12937_2005_Article_76_Fig1_HTML.jpg
                  Figure 1

                  Stimulatory effect of dietary inulin intake on intestinal absorption of 44 Ca and 25 Mg in rats of different ages. Intestinal 44Ca absorption (A) and ntestinal 25Mg 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 44Ca and 25Mg after 14 days of inulin intake (7.5%), and fecal non-absorbed isotopes were determined in a 4d feces pool.

                  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.

                  Abbreviations

                  Ca: 

                  calcium

                  Mg: 

                  Magnesium

                  ICP/MS: 

                  Inductively coupled plasma/mass spectrometry

                  OS: 

                  oligosaccharides

                  SCFA: 

                  Short-chain fatty acids

                  Declarations

                  Acknowledgements

                  The authors are grateful to ORAFTI (Tienen, Belgium) for providing the inulin for this study. The authors thank Séverine Thien, Lydia Jaffrelo, Claudine Lab and Pierre Lamby for their technical assistance.

                  Authors’ Affiliations

                  (1)
                  Centre de Recherche en Nutrition Humaine d'Auvergne, Unité Maladies Métaboliques et Micro-nutriments

                  References

                  1. Gibson GR, Beatty ER, Wang X, Cummings JH: Selective stimulation of bifidobacteria in the human colon by oligofructose and inulin. Gastroenterology 1995, 108:975–982.View Article
                  2. Jackson KG, Taylor GR, Clohessy AM, Williams CM: The effect of the daily intake of inulin on fasting lipid, insulin and glucose concentrations in middle-aged men and women. Br J Nutr 1999, 82:23–30.
                  3. Kaur N, Gupta AK: Applications of inulin and oligofructose in health and nutrition. J Biosci 2002, 27:703–714.View Article
                  4. Delzenne N, Aertssens J, Verplaetse H, Roccaro M, Roberfroid M: Effect of fermentable fructo-oligosaccharides on mineral, nitrogen and energy digestive balance in the rat. Life Sci 1995, 57:1579–1587.View Article
                  5. Ohta A, Ohtsuki M, Baba S, Adachi T, Sakata T, Sakaguchi E: Calcium and magnesium absorption from the colon and rectum are increased in rats fed fructooligosaccharides. J Nutr 1995, 125:2417–2424.
                  6. Younes H, Demigne C, Remesy C: Acidic fermentation in the caecum increases absorption of calcium and magnesium in the large intestine of the rat. Br J Nutr 1996, 75:301–314.View Article
                  7. Levrat MA, Remesy C, Demigne C: High propionic acid fermentations and mineral accumulation in the cecum of rats adapted to different levels of inulin. J Nutr 1991, 121:1730–1737.
                  8. Lutz T, Scharrer E: Effect of short-chain fatty acids on calcium absorption by the rat colon. Exp Physiol 1991, 76:615–618.
                  9. Trinidad TP, Wolever TM, Thompson LU: Effects of calcium concentration, acetate, and propionate on calcium absorption in the human distal colon. Nutrition 1999, 15:529–533.View Article
                  10. Topping DL, Clifton PM: Short-chain fatty acids and human colonic function: roles of resistant starch and nonstarch polysaccharides. Physiol Rev 2001, 81:1031–1064.
                  11. Remesy C, Levrat MA, Gamet L, Demigne C: Cecal fermentations in rats fed oligosaccharides (inulin) are modulated by dietary calcium level. Am J Physiol 1993, 264:G855–62.
                  12. Coudray C, Tressol JC, Gueux E, Rayssiguier Y: Effects of inulin-type fructans of different chain length and type of branching on intestinal absorption and balance of calcium and magnesium in rats. Eur J Nutr 2003, 42:91–98.View Article
                  13. Coudray C, Demigne C, Rayssiguier Y: Effects of dietary fibers on magnesium absorption in animals and humans. J Nutr 2003, 133:1–4.
                  14. Demigne C, Remesy C, Rayssiguier Y: Effect of fermentable carbohydrates on volatile fatty acids, ammonia and mineral absorption in the rat caecum. Reprod Nutr Dev 1980, 20:1351–1359.View Article
                  15. De Bievre P, Taylor PDP: Table of isotopic composition of the elements. International Journal of Mass Spectrometry Ion Process 123 1993, 123:149–166.View Article
                  16. Coudray C, Pepin D, Tressol JC, Bellanger J, Rayssiguier Y: Study of magnesium bioavailability using stable isotopes and the inductively-coupled plasma mass spectrometry technique in the rat: single and double labelling approaches. Br J Nutr 1997, 77:957–970.View Article
                  17. Coudray C, Bellanger J, Castiglia-Delavaud C, Remesy C, Vermorel M, Rayssignuier Y: Effect of soluble or partly soluble dietary fibres supplementation on absorption and balance of calcium, magnesium, iron and zinc in healthy young men. Eur J Clin Nutr 1997, 51:375–380.View Article
                  18. Lopez HW, Coudray C, Levrat-Verny MA, Feillet-Coudray C, Demigne C, Remesy C: Fructooligosaccharides enhance mineral apparent absorption and counteract the deleterious effects of phytic acid on mineral homeostasis in rats. J Nutr Biochem 2000, 11:500–508.View Article
                  19. Younes H, Coudray C, Bellanger J, Demigne C, Rayssiguier Y, Remesy C: Effects of two fermentable carbohydrates (inulin and resistant starch) and their combination on calcium and magnesium balance in rats. Br J Nutr 2001, 86:479–485.View Article
                  20. Ohta A, Ohtsuki M, Baba S, Hirayama M, Adachi T: Comparison of the nutritional effects of fructo-oligosaccharides of different sugar chain length in rats. Nutr Res 1998, 18:109–120.View Article
                  21. Scholz-Ahrens KE, Schaafsma G, van den Heuvel EG, Schrezenmeir J: Effects of prebiotics on mineral metabolism. Am J Clin Nutr 2001, 73:459S-464S.
                  22. Cashman K: Prebiotics and calcium bioavailability. Curr Issues Intest Microbiol 2003, 4:21–32.
                  23. Kayne LH, Lee DB: Intestinal magnesium absorption. Miner Electrolyte Metab 1993, 19:210–217.
                  24. Schaafsma G: Bioavailability of calcium and magnesium. Eur J Clin Nutr 1997, 51 Suppl 1:S13–6.
                  25. Armbrecht HJ: Effect of age on calcium and phosphate absorption. Role of 1,25-dihydroxyvitamin D. Miner Electrolyte Metab 1990, 16:159–166.
                  26. Chan EL, Lau E, Shek CC, MacDonald D, Woo J, Leung PC, Swaminathan R: Age-related changes in bone density, serum parathyroid hormone, calcium absorption and other indices of bone metabolism in Chinese women. Clin Endocrinol (Oxf) 1992, 36:375–381.View Article
                  27. Kinyamu HK, Gallagher JC, Prahl JM, DeLuca HF, Petranick KM, Lanspa SJ: Association between intestinal vitamin D receptor, calcium absorption, and serum 1,25 dihydroxyvitamin D in normal young and elderly women. J Bone Miner Res 1997, 12:922–928.View Article
                  28. Weaver CM: Age related calcium requirements due to changes in absorption and utilization. J Nutr 1994, 124:1418S-1425S.
                  29. Chonan O, Watanuki M: The effect of 6'-galactooligosaccharides on bone mineralization of rats adapted to different levels of dietary calcium. Int J Vitam Nutr Res 1996, 66:244–249.
                  30. Coudray C, Feillet-Coudray C, Tressol JC, Gueux E, Thien S, Jaffrelo L, Mazur A, Rayssiguier Y: Stimulatory effect of inulin on intestinal absorption of calcium and magnesium in rats is modulated by dietary calcium intakesShort- and long-term balance studies. Eur J Nutr 2004.
                  31. Ohta A, Motohashi Y, Ohtsuki M, Hirayama M, Adachi T, Sakuma K: Dietary fructooligosaccharides change the concentration of calbindin-D9k differently in the mucosa of the small and large intestine of rats. J Nutr 1998, 128:934–939.
                  32. Takasaki M, Inaba H, Ohta A, Motohashi Y, Sakai K, Morris H, Sakuma K: 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 2000, 70:206–213.View Article
                  33. Durlach J, Bac P, Durlach V, Rayssiguier Y, Bara M, Guiet-Bara A: Magnesium status and ageing: an update. Magnes Res 1998, 11:25–42.
                  34. Coudray C, Gaumet N, Bellanger J, Coxam V, Barlet JP, Rayssiguier Y: Influence of age and hormonal treatment on intestinal absorption of magnesium in ovariectomised rats. Magnes Res 1999, 12:109–114.
                  35. Vaquero MP: Magnesium and trace elements in the elderly: intake, status and recommendations. J Nutr Health Aging 2002, 6:147–153.
                  36. Ferment O, Touitou Y: Magnesium: metabolism and hormonal regulation in different species. Comp Biochem Physiol A 1985, 82:753–758.View Article
                  37. Hardwick LL, Jones MR, Brautbar N, Lee DB: Site and mechanism of intestinal magnesium absorption. Miner Electrolyte Metab 1990, 16:174–180.

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                  © Coudray et al. 2005

                  This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.