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