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The Journal of Nutrition Vol. 128 No. 7 July 1998,
pp. 1192-1198
Laboratoire Maladies Métaboliques et Micronutriments, Centre de Recherches en Nutrition Humaine d'Auvergne, I.N.R.A., Centre de Recherches Clermont-Ferrand/Theix, F-63122 St-Genès-Champanelle, France
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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The specific effects of phytic acid (PA) and resistant starch (RS) on mineral bioavailability, namely, Ca, Mg, Fe, Zn and Cu, were investigated in rats adapted to semipurified diets. The diets provided either 73 g/100 g digestible wheat starch (DS) alone, or 53 g/100 g DS plus 20 g/100 g crude potato starch (RS) and either 0 or 1.1 g/100 g PA. A period of 3 wk was first planned to adapt the rats to their respective diets. Compared with rats fed the DS diets, those fed the RS diets had significant cecal hypertrophy and an accumulation of short-chain fatty acids, together with greater cecal blood flow. RS enhanced the cecal absorption of Ca and Mg (from 0.15 to 0.55 µmol/min for Ca, and from 0.10 to 0.35 µmol/min for Mg). Mineral balance was enhanced significantly by RS (Ca, +46%; Mg +50%; Fe +20%; Zn, + 33% and Cu, +61%). PA had no significant effect on Ca or Mg solubility and absorption in the cecum, and it failed to alter significantly Ca or Mg balance. The apparent absorption of Fe, Zn and Cu was significantly lower in rats fed the DS + PA diet than in rats fed the DS diet (Fe, 
35%; Zn, 28%; and Cu, 31%). In rats adapted to the RS diet, the inhibitory effects of PA were practically abolished and the mineral balance was restored to the control values. We conclude that the negative effects of PA on mineral balance are relatively minor compared with the stimulatory effect of RS.
The potential health benefits of dietary fibers or fermentable carbohydrates have received increasing attention. However, these complex carbohydrates are able to bind minerals, hence possibly to alter mineral bioavailability. This property is chiefly due to the presence of phytic acid (PA)3 and associated substances (Brune et al. 1992 Animals and diets.
Male Wistar rats, ~7 wk of age, were used. They were from the colony of laboratory animals of the National Institute of Agronomic Research (INRA of Clermont-Ferrand/Theix, France). The animals were housed two per cage (wire-bottomed to limit coprophagy) and maintained in a temperature-controlled room (22°C) with a dark period from 2000 to 0800 h. They were fed one of the experimental semipurified diets for 21 d (Table 1). The animals were allowed free access to fresh food and distilled water. Daily food consumption and body weight were recorded twice a week. Feces were collected over four consecutive days for mineral absorption study. Animals were handled according to the recommendations of the Institutional Ethics Committee (University of ClermontFerrand).
Sampling procedures.
Between 0800 and 0900 h, rats were anesthetized (40 mg/kg sodium pentobarbital), and blood samples were successively taken from the cecal vein (0.5 mL, at the rate of 0.5 mL/min) and then, from the cecal artery, as described previously (Demigné and Rémésy 1985 Analytical procedures.
Ca and Mg were determined on the cecal supernatant fractions (soluble) and on the untreated cecal (total) contents, as well as on the fecal materials after dry-ashing (10 h at 500°C). The resulting ash was redissolved in HCl (6 mol/L) and adjusted to an appropriate volume with lanthanum solution (1 g/L). To determine Fe, Zn and Cu levels, 0.25 g of sample was wet-ashed in HNO3/HClO4 (3:1) for 4 h. After an adequate dilution, mineral concentration was measured by atomic absorption spectrophotometry (Perkin-Elmer 420, Norwalk, CT) at the following wavelengths : 422 (Ca); 285 (Mg); 248 (Fe); 214 (Zn); and 325 nm (Cu) as described previously (Bellanger 1971
Calculations.
The total and soluble Ca and Mg cecal pools and total cecal SCFA pools were calculated as follows: total pool (µmol) = cecal concentration (µmol/g) × cecal fresh content weight (g); soluble pool (µmol) = cecal supernatant concentration (µmol/g) × cecal water (g). The rate of cecal absorption (at the time of the measurement) was determined from the following formula: rate cecal absorption (µmol/min) = [cecal vein concentration Statistical analysis.
Values are given as the means ± SEM and, where appropriate, data were tested by two-way ANOVA using the General Linear Models procedure of the SuperANOVA package (Abacus, Berkeley, CA). Post-hoc comparisons were done by using Fisher's least significant difference procedures. Differences of P Effects of dietary conditions on physiological variables.
Diets containing phytic acid (PA) and/or resistant starch (RS) were well tolerated by rats. There were no significant differences among the diet groups in daily food intake and daily weight gain (Table 2). Fecal excretion was 0.8 g/d in rats fed fiber-free diets (DS and DS + PA groups), but it was much greater ( 2.2-2.7 g/d ) in the case of RS-rich diets (P < 0.001). A significant enlargement of the cecum occurred in rats fed the RS diets, together with hypertrophy of the cecal wall. Cecal blood flow was 75% greater in rats adapted to the RS diets than in rats fed DS diets (P < 0.01). The cecal SCFA pool was significantly larger (P < 0.001) in rats fed the RS diets than in those fed DS diets (Fig. 1). Dietary RS led to a striking rise (P < 0.001) in the molar proportions of propionic and butyric acids. The cecal pH was close to neutrality in rats fed fiber-free diets, whereas it was more acidic in rats adapted to the RS or the RS + PA diets (6.0 and 5.7, respectively).
Effects of dietary conditions on the cecal accumulation and the solubility of Ca and Mg.
The concentration of total Ca was ~600 mmol/L in rats fed the DS diets; this concentration was 58% lower in rats consuming RS in the diet (Table 3). In rats adapted to the DS diets, the cecal concentrations of soluble CA were much lower than those for total CA (i.e., 1.4% of total Ca). In rats adapted to RS diets, the soluble Ca concentrations were higher (20-40 mmol/L), but they represented only 15% (RS group) or 9% (RS + PA) of total Ca. In rats fed RS, there was a significant effect of PA on this variable (P < 0.05) but there was no significant RS × PA interaction. The total Ca pool was significantly enlarged in rats fed RS (P < 0.001); furthermore, the presence of PA significantly increased this pool in rats fed both types of starch (P < 0.001). The soluble Ca pool was largely augmented by dietary RS (13-15 times); in the presence of PA, this augmentation was significantly less.
Effects of dietary conditions on the absorption of Ca and Mg.
The Ca balance, i.e., the difference between intake and fecal excretion (I-FE difference), was positive and was significantly greater in rats adapted to the RS diets (P < 0.01); it is noteworthy that this balance was not significantly modified by dietary PA (Table 4). Mg was very efficiently absorbed, and its balance was also significantly improved by RS ingestion (P < 0.01) but was also unaltered by PA ingestion. For Ca and Mg, the cecal absorption was strongly and significantly stimulated by the presence of RS in foods (Fig. 2). On the other hand, dietary PA did not influence this absorption, whatever the type of dietary starch.
Effects of dietary conditions on the absorption of Fe, Zn and Cu.
In rats fed the DS diet, ~45% of dietary Fe was apparently absorbed, and up to 54% in rats fed RS alone (Table 5). This balance was significantly reduced by dietary PA (P < 0.05) in rats fed the DS (
Several previous studies showed that PA is an efficient inhibitor of absorption of essential dietary minerals such as calcium, zinc and iron (Brune et al. 1992
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
, Hallberg et al. 1987, Harland and Morris 1995, McCance and Widdowson 1942, Roberts and Yudkin 1960, Torre et al. 1991). Myo-inositol hexaphosphate is the major storage form of phosphorus in cereals, legumes and oil seeds. This molecule is highly charged with six phosphate groups extending from the central inositol ring and serves as an excellent chelator of mineral ions such as Ca, Zn and Fe. The phytate content of some foods (whole wheat products, wheat bran, soy products) was reported to be responsible for the decrease in calcium and zinc balance in rats and humans (Heavey et al. 1991, Lönnerdal et al. 1989, Miyazawa and Yoshida 1991, Sandström et al. 1990, Simpson and Wise 1990). In these circumstances, some authors (Reinhold et al. 1976, Sandstead 1992) questioned the advantage of recommending high consumption of PA-rich dietary products. However, less purified products, such as whole wheat flour, provide four- to fivefold more minerals than white wheat flour. In fact, minerals are strongly associated with plant cell walls, and can be released by the microbial breakdown of these complex polysaccharides in the large intestine. It is thus important to consider the contribution of the colon in the overall absorption of minerals in the presence of these substances. In fact, the effects of fermentable carbohydrates and PA on mineral bioavailability are controversial partially because the digestive microflora can express a phytase activity (Miyazawa et al. 1996, Wise and Gilburt 1982, Yoshida et al. 1985) and microbial fermentations can increase the solubility of divalent cations in the large intestine, which may improve their absorption in situ across the cecal wall (Delzenne et al. 1995, Schulz et al. 1993, Trinidad et al. 1993). Thus, there is a possible shift of absorptive sites from the small intestine toward the large intestine, with a potential enhancement of their availability for absorption, especially for Ca and Mg (Younes et al. 1996). Such a shift can overcome the negative chelating effects of fiber and PA. Phytic acid effects would differ in a complex diet containing various fermentable carbohydrates. The aim of this experiment was thus to compare the effects of PA in the presence and in the absence of fermentable carbohydrates on the utilization of major divalent cations (Ca, Mg) and of trace elements (Fe, Zn, Cu) in rats.
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
View this table:
Table 1.
Composition of diets1
). For blood flow measurement, bromosulfophtalein in saline (5 mmol/L) was infused into a small vein on the internal curvature of the cecum at a rate of 50 µL/min: determination of the marker dilution in the vein draining the whole cecum (without collateral circulation to ileum or colon) was used to calculate the cecal blood flow. After blood sampling, the cecum (complete with contents) was removed and weighed. Duplicate samples of cecal contents were placed in 2-mL microfuge tubes and immediately stored at 20°C. Then, the cecal wall was flushed clean with ice-cold saline, blotted on filter paper and weighed (cecal wall weight). Cecal water was determined as the difference between wet weight and dry weight on aliquots of cecal contents that were dried to constant weight. Supernatant fractions of the cecal contents were obtained by centrifuging the microfuge tubes at 20,000 × g for 10 min at 4°C.
). Short-chain fatty acids (SCFA), namely, acetic, propionic and butyric acid, were determined by GLC of portions of the supernatant fractions of cecal contents (Demigné et al. 1980).
View larger version (20K):
[in a new window]
Fig 1.
Respective effects of resistant starch (RS) and phytic acid (PA) on cecal short-chain fatty acid (SCFA) pools in rats adapted to diets containing digestible starch (DS) or RS and with or without phytic acid. Values are means ± SEM, n = 8. Those not sharing a letter are significantly different (P 
0.05).
cecal artery concentration (µmol/mL)] × cecal plasma flow (mL/min). For the determination of mineral balance, food and fecal samples from each pair of rats were homogenized dried, powdered and mineralized before mineral analysis.
0.05 were considered significant.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
View this table:
Table 2.
Respective effects of resistant starch (RS) and phytic acid (PA) on food intake, daily weight gain,
daily fecal excretion and cecal development of rats1,2
View this table:
Table 3.
Respective effects of resistant starch (RS) and phytic acid (PA) on the cecal accumulation
and the solubility of calcium and magnesium in rats1,2
View this table:
Table 4.
Respective effects of resistant starch (RS) and phytic acid (PA) on the daily intake
and fecal excretion of calcium and magnesium in rats1,2
View larger version (12K):
[in a new window]
Fig 2.
Changes in calcium or magnesium absorption rates from the cecum of rats adapted to diets containing digestible starch (DS) or resistant starch (RS) and with or without phytic acid (PA). Values are means ± SEM, n = 8. Those not sharing a letter are significantly different (P 0.05).
33%) or the RS diets (26%). Apparently, only 18% of dietary Zn was absorbed in rats fed the DS diets, and this absorption was slightly but significantly enhanced in the presence of RS in the diet (P < 0.05). Phytic acid significantly depressed Zn absorption in rats fed the DS diet, but not when the diet contained RS. About 13% of Cu intake was absorbed in rats fed the DS diet, and this absorption increased significantly when DS was replaced by RS in the diet (P < 0.01). Phytic acid significantly reduced Cu absorption in rats fed the DS and the RS diets. Nevertheless, it must be noted that the stimulatory effect of RS compensated for the inhibitory effect of PA in the RS + PA group.
View this table:
Table 5.
Respective effects of resistant starch (RS) and phytic acid (PA) on the daily intake and fecal excretion of iron, zinc and copper in rats1,2
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
, Hallberg et al. 1987, Harland et al. 1995, McCance and Widdowson 1942, Roberts and Yudkin 1960, Torre et al. 1991). However, PA does not occur alone in foods and is often consumed with various complex carbohydrates. Thus, these carbohydrates could influence the inhibitory effect of phytate on mineral availability for absorption. The main result of this study clearly shows that the fermentation of complex carbohydrates counterbalanced the inhibitory effect of PA on mineral absorption in rats. This result could be largely attributed to an increase in mineral solubility due to the fermentation process.
, Rémésy et al. 1993). In rats fed the RS diet, the cecal weight was twofold greater than that in the DS group. This hypertrophy resulted in an elevation of cecal wall weight, greater crypt column height and an increase in cell number per crypt, thus leading to a greater exchange surface area. In the presence of dietary RS, the large production of short-chain fatty acids (SCFA) can induce the thickening of cecal mucosa by a hyperplastic process (Rémésy et al. 1992). To neutralize the high organic acid concentration and to maintain the cecal pH at a moderately acidic value (nearly 6.0), a sufficient amount of minerals should reach the cecum. In this regard, Rémésy et al. (1993) showed that the accumulation of insoluble calcium salts, mainly phosphate, plays an important role in the control of cecal pH.
). The total number of these sites could increase due to cecal hypertrophy. It is also conceivable that SCFA may directly influence Ca absorption by modifying electrolyte exchanges (Ca-H); Trinidad et al. (1993) proposed that Ca could pass through the cell membrane more readily in the form of a less-charged complex (Ca acetate)+ by a passive pathway. Lutz and Scharrer (1991) also reported a stimulatory effect of SCFA on Ca absorption in rat large intestine. The stimulatory effect of RS could be related to the acceleration of the transcellular route of Ca absorption, which could involve induction of Ca binding protein. In fact, the Ca absorption rate is highly regulated. A high rate of Ca absorption in the large intestine could trigger a feedback mechanism involving an inhibition of Ca proximal intestine absorption, because there is a control of Ca balance by endocrine factors (Bronner et al. 1986). In spite of this feedback, an improvement of Ca digestibility was observed in the presence of RS.
). Although the literature has frequently reported an inhibitory effect of PA on Ca absorption (Lönnerdal et al. 1989, Rimbach et al.1995, Sandström et al. 1990), some researchers failed to observe any effect of PA on Ca absorption (Likuski and Forbes 1965, Miyazawa et al. 1996, Nickel et al. 1997).
, Karbach and Rummel 1990), and it was previously shown that various types of RS stimulated Mg absorption in rats (Ohta et al. 1995, Schulz et al. 1993). As for Ca, fermentable carbohydrates may also raise the soluble Mg pool in the large intestine as a consequence of acidifying digestive tract contents (Mg solubility is generally higher than that of Ca). The potent effects of RS on cecal Mg absorption result from the cecal hypertrophy, the increase in Mg solubility and probably from a specific effect of SCFA on passive Mg absorption (Scharrer and Lutz 1992, Younes et al. 1996). Indeed, SCFA are predominantly absorbed in an undissociated form in the large intestine, although they occur mainly as anions in the lumen (Rechkemmer et al. 1988). Protons needed for SCFA absorption may be delivered by various ion exchangers (including Mg-H); in return, SCFA absorption at acidic pH would supply more protons to the exchangers, resulting in a higher transport rate (Lutz and Scharrer 1991). In this study, feeding rats diets with RS had a comparable effect on the apparent digestive balance of Mg and Ca. Thus, increasing divalent cation absorption in the large intestine was not accompanied by a lower absorption of these cations in the small intestine. As for Ca, PA did not exert any significant effect on Mg apparent absorption. Some authors found an inhibitory effect of PA on Mg absorption in rats (Likuski and Forbes 1965, Miyazama and Yoshida 1991, Roberts and Yudkin 1960). This contrasts with the fact that cereal products rich in PA are considered excellent sources of Mg. Therefore, it is difficult to ascribe an antinutritional effect to PA on Mg present in complex carbohydrates, particularly when the fibers are fermentable.
, Hallberg et al. 1987, Hurrell et al. 1992). Other studies showed that PA hydrolysis by endogenous phytase increases Fe solubility in cereal products (Sandberg et al. 1996). On the other hand, chelation effects of PA on zinc absorption have often been reported (Lönnerdal et al. 1988, Saha et al. 1994, Wise 1995). The inhibitory effect of phytate on Zn absorption was defined by the ratio of phytate to Zn in the diet (O'Dell 1983). More recently, because of the chelating effects of calcium phytate on zinc, some authors (Fordyce et al. 1987) proposed that dietary ratios of Ca × phytate to Zn would be a better predictor of Zn absorption than is the phytate to Zn ratio alone. Finally, the negative effect of PA on Fe and Zn absorption is well documented, but this is not the case for Cu. Some studies reported an inhibitory effect or no effect of PA on Cu absorption in humans and in animal models (Davies and Nighingale 1975, Morris and Ellis 1985), but others noted a positive effect of PA on Cu absorption in rats (Lee et al. 1988). The effect of PA on Cu absorption seems to be modulated by several dietary factors, especially the zinc level in the diet. PA can indeed enhance Cu absorption due to its ability to bind zinc, thus counteracting its capacity to compete with Cu at the intestinal absorption sites (Champagne and Hinojosa 1987).
reported a significant increase in Fe and Zn absorption in rats fed inulin. However, Coudray et al. (1997) failed to observe any effect of inulin on Fe or Zn absorption in humans. In contrast to RS, cereal products are often partially fermentable, so that the expected pH decrease in the large intestine will not be sufficient to offset the inhibitory effects of PA on mineral absorption.
, Cossack et al. 1992, Lei et al. 1980, Rossander et al. 1992). With fiber-related compounds such as RS or oligosaccharides, there are results from animal models supporting the view that they may enhance mineral absorption, especially Mg, Ca and some trace elements (Delzenne et al. 1995, Schulz et al 1993, Younes et al. 1996). However, data in humans remain scarce. It is also important to keep in mind that fiber and PA occur together in fiber-rich diets, and it is difficult to separate the effects of fiber and PA in the utilization of most essential minerals. In fact, healthy meals that include fiber may be provided by vegetal products (e.g., cereals, fruits or vegetable) that promote intestinal fermentation and mineral utilization. Under such conditions, the consequences of fermentable fiber ingestion (cecal enlargement, cecal pH lowering and SCFA production) can be more important than the PA chelating effect on the soluble mineral pool in the large intestine, and thus on their absorption. Therefore, fiber-rich foods may shift the mineral absorptive sites toward the distal parts of digestive tract without impairing their apparent absorption. In humans, only 20% of Ca is absorbed in the small intestine; thus the accumulation of calcium phosphate in the large intestine is very important (Van Der Meer et al. 1990). The Ca solubility enhancement by organic acid production may be important when Ca absorption in the small intestine is defective, such as in elderly subjects. The consumption of products of vegetal origin can allow increased mineral intake and compensate for the negative effects of food processing. The negative influence of PA on mineral bioavailability depends largely on the presence of fermentable carbohydrates in the diet. In conclusion, this study shows that intestinal fermentation has a very positive effect on mineral utilization. Although PA may exert adverse effects on the absorption of minerals, the simultaneous large intestine fermentation may compensate for this effect.
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FOOTNOTES |
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Manuscript received 10 December 1997. Initial reviews completed 9 February 1998. Revision accepted 23 March 1998.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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A Component of Food (Schweizer, T. F. & Edwards C. A., eds.), pp. 197-216. Springer-Verlag, London, UK. This article has been cited by other articles:
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