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Centre de Recherche en Nutrition Humaine d'Auvergne, Unité Maladies Métaboliques et Micro-nutriments, INRA, Theix, France
1 To whom correspondence should be addressed. E-mail: coudray{at}clermont.inra.fr
ABSTRACT
Nondigestible inulin-type fructan intake can stimulate intestinal mineral absorption in both humans and animals. However, this stimulatory effect may depend on experimental conditions such as the duration of the experience, mineral levels in the diet, and the animal's physiological status. The aim of this study was to determine the effect of inulin intake on Zn and Cu absorption in rats at different ages. Male Wistar rats (n = 80) of 4 different ages (2,5, 10, and 20 mo) were randomly assigned to a control group or a group administered 3.75% inulin in their diet for 4 d followed by 7.5% inulin for 26 d. Absorption of Zn67 and Cu65 was determined on d 21 of the experiment by fecal monitoring using Zn67 and Cu65 isotopes. Zn and Cu status was also assessed. Absorption of Zn67 and Cu65 was significantly lower in 11- and 21-mo-old rats than in 3- and 6 mo-old-rats. Moreover, inulin intake significantly increased Zn67 and Cu65 absorption. In conclusion, age and dietary inulin intake can significantly affect intestinal absorption of zinc and copper in rats. Further studies are required to explore this effect over longer periods of inulin intake and to test the effects of inulin in humans.
KEY WORDS: • inulin • intestinal absorption • trace elements • stable isotope • aging
When nondigestible 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 (1–3). Previous studies showed repeatedly that intake of different fructans can increase the intestinal absorption of minerals in humans and animals to varying degrees (4–12), in particular magnesium, and often calcium, despite an increase in total fecal mass. Indeed, products of fructan fermentation can influence the intestinal absorption of minerals in many ways. Short-chain fatty acids (SCFA) are fermentation products that are responsible for lowering the pH of the cecal contents; this in turn increases mineral solubility, leading to improved mineral absorption (13,14). SCFA can directly influence mineral absorption by forming complexes with the minerals, thereby increasing their uptake by the intestinal cells (15,16). It is also thought that the bacterial metabolites (e.g., butyrate) can stimulate the intestinal epithelium and increase its absorptive capacity (17). Thus, the effect of the prebiotic carbohydrates on mineral absorption is closely linked to the nature of these carbohydrates, to the mineral homeostasis mechanisms, and to the experimental conditions (6,7,12). Indeed, inulin-type fructans strongly and consistently increase intestinal Mg absorption (18), whereas their effect on calcium and on trace element absorption seems to be dependent on experimental conditions such as inulin type and dose, the duration of fructans intake, and the animal's physiological status, particularly age (6,14,19–21). However, little is known about the effect of these compounds on intestinal absorption of trace elements (8,11,22). In this study, we investigated the stimulatory effect of inulin on intestinal absorption and retention of Zn and Cu using a stable isotope approach after short-term administration of inulin in rats 2–20 mo old.
MATERIALS AND METHODS
Materials and reagents. The enriched Zn isotope (Zn67) as ZnO and the enriched Cu isotope (Cu65) as elemental Cu were obtained from Chemgas. The atomic abundances of these isotopes were as follows: 64Zn = 1.00%, Zn66 = 1.55%, Zn67 = 93.30%, 68Zn = 4.10%, 70Zn = 0.05%, and Cu65 = 0.80%, Cu65 = 99.2%. HNO3 (ultrapure), Zn, Cu, and indium standard solutions (1 g/L) were obtained from Merck. All other chemicals were of the highest quality available. Distilled water was used throughout. A Perkin-Elmer 6100DRC system (Perkin-Elmer Instruments) equipped with a Meinhard nebulizer was used for isotopic measurement, and a Perkin Elmer AA800 (Perkin Elmer Instruments) was used for total Zn and Cu measurement.
Animals and diets. Male Wistar rats (n = 80) aged 2, 5, 10, or 20 mo were purchased from Janvier. They were fed a commercial pellet diet (Ssniff R/S-breeding) up to 12 wk of age, then Ssniff R/S-maintenance from wk 13 until they were 2 y old. Two groups were formed from each age bracket to be fed either a control diet or a semipurified diet containing inulin until the end of the experiment. The composition of these diets is given in Table 1. The inulin tested was donated by Orafti (Raftaline®). The target Zn and Cu concentrations in these diets were 40 mg Zn/kg and 7 mg Cu/kg diet. A powdered diet (100 g) was made up with 100 mL distilled water to form a kind of semiliquid food prepared on-site each day. Chemical analysis of the diets offered confirmed the expected Zn and Cu concentrations in the experimental diets: 37.8 mg Zn/kg and 37.2 mg Zn/kg, and 7.3 mg Cu/kg and 7.1 mg Cu/kg in the control and 7.5% inulin diets, respectively. Dietary inulin concentration was maintained at 3.75% for the first 4 d and then increased to 7.5% from d 5 until the end of the experiment to allow rats to adapt gradually. The total duration of the experiment was 30 d. The 8 rat groups were given fresh food and water daily, which they consumed ad libitum. Food consumption and body weight were recorded weekly. Throughout the experiment, the rats were housed 2/cage (wire-bottomed to limit coprophagy) in a temperature-controlled room (22°C) with a dark period from 2000 to 0800. All procedures complied with the INRA Institute's ethical guidelines on the care and use of laboratory animals. At the end of the experiment, when absorption and status of Zn and Cu were measured, the rats were 3,6, 11, and 21 mo; results at these ages are reported.
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1.7 mL of isotopic solution by gavage between 0800 and 1000 and then given access to their food. The urine and feces of each rat were collected quantitatively for 4 consecutive days, and excreted isotopes in these 2 media and in the gavage solution were determined quantitatively by inductively coupled plasma (ICP)-MS, as described below. Sampling procedures. The rats were killed just after the dark period (between 0800 and 1000), i.e., at a time when cecal fermentation was still 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 1000 x g for 10 min. 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 placed at –20°C until analysis. The pH of the cecal contents was determined on site using a Sentron pH-system 1001 portative pH-meter (Sentron Europe B.V.). Supernatants of the digestive contents were obtained by centrifuging 1 of the 2 microfuge tubes at 20,000 x g for 10 min at 4°C; they were then frozen until analysis. One tibia and a sample of the liver were also collected for Zn and Cu analysis.
Analytical procedures. Zn and Cu concentrations were determined in the plasma and urine after adequate dilution in HCl (0.12 mol/L). Aliquots of diet, fecal materials, and tibia were dry-ashed (10 h at 500°C) and dissolved with concentrated HNO3 (14 mol/L) and H2O2 (30%) on a heating plate until there was complete decoloration. The resulting mineral solutions were set at 10 mL with water and adequately diluted in HCl (0.12 mol/L). Mineral concentrations were measured on a atomic absorption spectrophotometer (Perkin-Elmer AA800) at wavelengths of 214 nm for Zn and 325 nm for Cu.
For isotopic Zn67 and Cu65 determination, samples were appropriately diluted before analysis using HNO3 (0.14 mol/L). Zn and Cu concentration and isotope ratios were determined by ICP-MS using Zn and Cu as external standards and indium as an 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 conditions were as follows: sweeps/reading: 50; readings/replicate: 1; number of replicates: 3; dwell time: 100 ms; scanning mode: peak hopping.
Cecal SCFA concentrations, including acetic, propionic, and butyric acids, were determined by GLC on portions of supernatant fractions of cecal contents as described previously (23).
Calculations. Zn and Cu each have different stable isotopes with the following natural abundances: 64Zn = 48.63%; Zn66 = 27.90%; Zn67 = 4.10%; 68Zn = 18.75%; 70Zn = 0.62%; Cu63 = 69.18%; Cu65 = 30.82% (24). Measured Zn67 and Cu65 isotopic enrichments were obtained from the following equations: Zn67 enrichment = (Zn67/Zn66 measured ratio – Zn67/Zn66 baseline ratio)/(Zn67/Zn66 baseline ratio) and Cu65 enrichment = (Cu65/Cu63 measured ratio – Cu65/Cu63 baseline ratio)/(Cu65/Cu63 baseline ratio).
Nonabsorbed Zn67 and Cu65 isotopes in the fecal or urine samples (coming only from the Zn67 or Cu65 isotope labels) were calculated as follows: Zn67 (mg) = [total fecal or urine Zn (mg)] x (natural abundance Zn67 x enriched Zn67)/[1 + (natural abundance Zn67 x enriched Zn67)] and Cu65 = [total fecal or urine Cu (mg)] x (natural abundance Cu65 x enriched Cu65)/[1 + (natural abundance Cu65 x enriched Cu65)]. Calculations were also made directly from the ICP-MS data. The 2 modes of calculation gave the same results when the ICP-MS quantitative procedure was used (25).
Intestinal absorption of Zn67 and Cu65 was then calculated as administered Zn67 or Cu65 – Zn67 or Cu65 excreted in the feces, and retention of Zn67 and Cu65 was calculated as administered Zn67 or Cu65 – Zn67 or Cu65 excreted in the 4-d feces and urine pools.
Total cecal SCFA content (µmol/cecum) was calculated as the supernatant SCFA concentration (µmol/mL) x cecal water (mL/cecum).
Statistical 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). ANOVA assumes that the data are sampled from populations with identical SDs. This assumption was tested using the method of Bartlett. When Bartlett's test suggested that the difference among the SDs was significant, the data were log-transformed before statistical analysis. ANOVA assumes also that the data are sampled from populations that follow Gaussian distributions. This assumption was tested using the method of Kolmogorov and Smirnov. Post hoc comparisons were performed using Fisher's least significant difference procedures. When there was no evidence in the ANOVA of a significant interaction between age and inulin, the comparison between age groups was done on the mean response for both control and inulin groups combined. However, when there was an interaction, Fisher's post hoc test between ages was done, testing separately within the control and inulin groups. Differences were considered significant at P < 0.05.
RESULTS
Food intake and growth rate. Rats fed 7.5% inulin tended to consume less food (P = 0.075) than control rats. This lower food consumption tended to decrease the growth rate (P = 0.084) toward the end of the experiment in inulin-fed rats compared with control rats. The lower energy density of the inulin diets (–4%) compared with the control diets may also be responsible for this lower weight gain. In addition, food consumption decreased significantly with increasing age (data not shown).
Cecal fermentation variables. Rats fed 7.5% inulin had higher cecal wall and cecal content weights and lower cecal content pH (Table 2) than control rats (P < 0.0001). These variables were not affected by age, except that the cecal wall weight increased with age (P < 0.0001). In addition, rats fed 7.5% inulin had higher cecal SCFA production (P < 0.0001) than the control rats. Inulin affected cecal butyrate production (P < 0.05). The 11-mo-old rats produced less butyrate than the 3- and 6-mo-old rats (P < 0.05).
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1 µmol Zn67/rat led to a large fecal Zn67 enrichment of 40–60% in the 4-d feces pool. Fecal Zn67 excretion increased significantly with age (Table 3); thus, Zn67 absorption decreased with age. The Zn67 absorption decreased significantly in the 11- and 21-mo-old rats compared with the 3- and 6-mo-old rats (Fig. 1A). In addition, urinary Zn67 excretion decreased with age (P < 0.0001). Consequently, Zn67 retention decreased considerably with age (P < 0.0001). Rats fed 7.5% inulin had lower fecal Zn67 excretion (P < 0.0001) compared with the control rats; thus inulin intake increased Zn67 absorption (P < 0.0001). Zn67 absorption in the 4 rat control groups was 22.4%, compared with 35.5% in the 4 inulin-fed groups, with an overall increase in Zn67 absorption of 58%. Indeed, Zn67 absorption was positively correlated with the cecal amount (µmol/cecum) of acetate (r = 0.241, P = 0.0370), propionate (r = 0.327, P = 0.0042), butyrate (r = 0.406, P = 0.0003), and total SCFA (r = 0.368, P = 0.0012). Moreover, rats fed 7.5% inulin had higher urinary Zn67 excretion (P < 0.005) than the control rats. Last, inulin intake increased Zn67 retention in the 4 groups (P < 0.0001) compared with the control groups. The relative increases in Zn67 absorption in rats fed 7.5% inulin compared with controls were 56, 24, 64, and 68% in those aged 3, 6, 11, and 21 mo, respectively.
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1 µmol Cu65/rat led to a fecal Cu65 enrichment of 30–40% in the 4-d feces pool. Fecal Cu65 excretion increased significantly with age, particularly in the groups administered inulin. Consequently, Cu65 absorption decreased significantly with age (Table 4). The Cu65 absorption decreased significantly in 11- and 21-mo-old rats compared with 3-mo-old rats (Fig. 1B). In addition, urinary Cu65 excretion decreased with age (P < 0.01); consequently, Cu65 retention decreased with age (P < 0.05). In addition, rats fed 7.5% inulin had lower fecal Cu65 excretion (P < 0.0001) than the control rats. Thus, inulin intake increased Cu65 absorption (P < 0.0001). Cu65 absorption ranged from 17.7% without inulin to 35.6% with inulin consumption in adult rats and from 14.2% without inulin to 26.1% with inulin consumption in old rats. Cu65 absorption in the 4 rat control groups was 16.0% compared with 30.9% in the 4 corresponding inulin-fed rat groups, with an overall increase in Cu65 absorption of 93%. Cu65 absorption was positively correlated with the cecal amount (µmol/cecum) of acetate (r = 0.377, P = 0.0008), propionate (r = 0.307, P = 0.0075), butyrate (r = 0.446, P < 0.0001), and total SCFA (r = 0.445, P < 0.0001). However, urinary Cu65 excretion was not affected by inulin intake. Rats fed 7.5% inulin had higher Cu65 retention compared with the control rats (P < 0.0001). The relative increases in Cu65 absorption with inulin consumption were 125, 46, 50, and 97% in the 4 rat groups (3,6, 11, and 21 mo, respectively) compared with the same age rats without inulin.
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DISCUSSION
Our results clearly showed that aged rats (11 and 21 mo old) exhibited less efficient intestinal absorption and retention of Zn than young rats (3 and 6 mo old). In rats that did not consume inulin Zn67 absorption was 30% in adult rats and 14% in old rats; with inulin consumption, it was 46% in adult rats and 24% in old rats. This decline in Zn absorption with age has not been reported in animal studies. Some human studies did report a decrease in Zn absorption with age (26,27), whereas others did not reproduce such an effect (28,29). Many dietary factors may influence zinc absorption, in particular, phytate and fiber (30). Our results clearly showed that inulin intake increased the efficiency of Zn intestinal absorption and retention. The positive correlation between Zn67 absorption and cecal amounts of acetate, propionate, and butyrate confirms the involvement of inulin-fermentation products in intestinal Zn absorption. These robust results confirmed the rare results reported in the literature showing that the intake of inulin or other fermentable fibers enhances Zn absorption within a few weeks (8,11). The improvement in Zn absorption with inulin intake was not accompanied by any significant modification of Zn status. Such a result agrees with previous observations concerning an increase in Ca absorption after inulin intake without modification in the status of this micronutrient (14). The duration of inulin intake was perhaps insufficient to induce a modification in Zn status. It would be interesting to investigate the effect of a longer duration of inulin intake on Zn status.
Our results showed that aged rats (11 and 21 mo old) exhibited less efficient intestinal absorption and retention of Cu than young rats (3 and 6 mo old). This decrease in Cu absorption with age is not well documented in the literature in either animal or human studies. Only a few studies reported an age effect on Cu absorption (27,31,32), and the results are inconsistent. Hence, to our knowledge, this is the first report to clearly show that Cu absorption decreases with age in rats. However, this decrease in Cu absorption with age was accompanied by a significant increase in plasma Cu concentrations. The positive relation between plasma Cu concentration and age was reported previously (33–35). Our results clearly showed that inulin intake significantly increased Cu intestinal absorption and retention. The positive correlation between Cu65 absorption and cecal amounts of acetate, propionate, and butyrate confirms the high involvement of SCFA in intestinal Cu absorption. These results are in agreement with the few literature data showing that inulin intake increased Cu absorption in animals and humans (8,20). The improvement in Cu absorption with inulin consumption was accompanied by a significant increase in plasma Cu concentrations, but other Cu status biomarkers were not affected. The duration of inulin intake was perhaps insufficient to induce a modification in the other Cu status biomarkers. It would be interesting to investigate the effect of inulin on Cu status in a longer study.
In conclusion, our results showed clearly that older rats (11 and 21 mo) absorb less Zn and Cu than younger rats (6 mo and 3 mo) and that inulin intake stimulates intestinal absorption of both Zn and Cu. Further studies are required to explore this effect with longer periods of inulin intake and to validate these results on the stimulatory effect of inulin on Zn and Cu absorption in the elderly.
ACKNOWLEDGMENTS
The authors are grateful to ORAFTI (Tienen, Belgium) for providing the inulin product for this study. The authors thank M. Rambeau, J. C. Tressol, S. Thien, L. Jaffrelo, C. Lab, and P. Lamby for their technical assistance.
Manuscript received 30 May 2005. Initial review completed 30 June 2005. Revision accepted 23 September 2005.
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