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Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto-shi 606-01, Japan
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
LITERATURE CITED
ABSTRACT 
Calcium distribution was studied in the small intestine of piglets fed skim milk powder (SMP) or defatted soybean flour (DSF ) to investigate the relationship between calcium availability and its forms. Ionized calcium in duodenal and ileal digesta was measured with a selective calcium electrode that was not affected by changes in pH or sodium, potassium and magnesium concentrations, which simulated the liquid phases of digesta. Eight piglets were fed DSF-based diet or SMP-based diet for 30 d, and duodenal and ileal digesta were collected. Soluble calcium concentrations in the ileum were higher in the SMP-fed group than in the DSF-fed group. The proportion of soluble calcium in higher-molecular-weight fraction (MW > 3000) was significantly greater in the ileum than in the duodenum of the SMP group, but did not differ between these intestinal segments within the DSF group. This proportion was significantly higher in the ileum of the SMP-fed group than in that of the DSF-fed group. In the ileum, ionized calcium concentration was significantly greater in the SMP-fed group than in the DSF-fed group. These results suggest that the increase of calcium in the higher-molecular-weight fraction raises soluble calcium concentration and changes the distribution of calcium in the ileum of the SMP-fed group. The complexes of calcium with higher-molecular-weight ligands may be easily exchangeable with ionized calcium, and the increase in these calcium complexes may consequently enhance the recruitment of ionized calcium, which then can be absorbed.
KEY WORDS: ionized calcium · absorption · intestinal digesta · pigs · bioavailabilityINTRODUCTION
Calcium bioavailability in rats is greater from a diet consisting of dairy products than from a diet consisting of soybean products (Brink et al. 1992
, Yuan et al. 1991). Many researchers have shown that casein in milk enhances calcium absorption. Dietary casein can be hydrolyzed to phosphopeptides in the digestive tract of rats, and these phosphopeptides increase the solubility of calcium in the small intestine (Lee et al. 1980). Furthermore, Sato et al. (1986) showed that a casein phosphopeptide prepared from 
-casein stimulated calcium solubility and absorption in the small intestine of rats. These researchers suggested that the production of casein phosphopeptides caused higher availability of calcium in milk through increasing calcium solubility in the digesta. There are other possible stimulants for calcium absorption in milk such as lactose and citric acid. It is not clear how lactose stimulates calcium absorption. Citric acid may increase calcium absorption by increasing the solubility of calcium in the digestive tract (Pak et al. 1989), although the action of citric acid is still controversial.
Soybean products are good protein sources. However, soy bean products interfere with calcium absorption in rats (Brink et al. 1992, Yuan et al. 1991). Lee et al. (1980) showed that a soy protein-based diet lowered solubility of calcium in the small intestine of rats compared to egg albumin- or amino acid mixture- based diets.
Dissolution of calcium in the digestive tract is essential for calcium absorption. However, the solubility of calcium might not completely reflect its absorption. Weaver and Heaney (1991) suggested that in women, exchange among some forms of calcium in the liquid phase of digesta was incomplete, and the forms differently affected calcium absorption. Calcium is considered to be absorbed mainly in its ionized form (Allen 1982). Therefore, it is important to determine ionized calcium concentration in the digesta of the small intestine, which is the major site of calcium absorption. Few investigators have tried to measure ionized calcium in digesta. Laszlo et al. (1992) and Dintzis et al. (1995) estimated free calcium ion concentrations in digesta of pigs. However, in these studies, free calcium ion was defined as calcium that passed through a 1000 MW cut-off ultrafilter, although they suggested that the filtrate contained many soluble calcium complexes. In the experiments reported here, we used a calcium-selective electrode to measure ionized calcium in digesta and studied calcium distribution in duodenal and ileal digesta of piglets fed skim milk powder (SMP)4-based or defatted soybean flour (DSF )-based diets.
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Table 1. Composition of diets |
MATERIALS AND METHODS
, and Schwedt and Konecke (1993) applied the electrode to determine ionized calcium in milk and in extracts of some foods, respectively. To our knowledge, however, there has been no report of measuring ionized calcium in digesta using the electrode. We examined the validity of an ionized calcium analyzer equipped with a calcium-selective electrode (SERA-252, Horiba, Kyoto, Japan) for determining ionized calcium in digesta because ionic strength, pH and ionized magnesium were shown to affect the measurement of ionized calcium in milk (Allen and Neville 1983). Therefore, to confirm the accuracy of the ionized calcium analyzer, ionized calcium concentrations were measured with 5 replications in calcium chloride solutions (0.5 and 1 mmol/L) of differing ionic strength, pH and magnesium concentration, which simulated the liquid phase of digesta. To change ionic strength, sodium chloride (40 to 240 mmol/L) and potassium chloride (20 to 120 mmol/L) were added to the calcium solutions (pH 7.3) because sodium and potassium are major electrolytes in digesta. The pH of calcium solutions containing 150 mmol sodium chloride/L and 60 mmol potassium chloride/L was adjusted between 4.9 and 7.3 by bubbling carbon dioxide gas. Magnesium chloride (12 mmol/L) was added to calcium solutions (pH 7.3) containing 150 mmol/L sodium chloride and 60 mmol potassium chloride/L to test the accuracy of the calcium electrode in the presence of magnesium.
Animals, housing and feeding.
Eight crossbred piglets (Yorkshire × Landrace) were purchased from Shiga Prefecture Livestock Station (Ohtsu, Japan) just before weaning. The animals were weaned at 27 to 30 d of age. The piglets were housed individually in metabolism cages under 12-h light:dark cycle and cared for according to the Guide for the Care and Use of Laboratory Animals (Kyoto University Animal Care Committee). Piglets were divided into 2 groups. One group was fed a diet containing SMP, and the other group was fed a diet containing DSF (Table 1). The piglets were fed 400 g of diet twice daily for 30 d and were given free access to deionized water.
Sampling and sample preparation.
Feces were collected during the last 5 d of the feeding trial. At the end of the feeding trial, piglets were anesthetized with ethyl ether 3 h after the morning feeding. They were then killed by exsanguination from the jugular vein and carotid arteries. Duodenum (first 1 m of the small intestine from the pylorus) and ileum (between 1 m and 2 m anterior to the ileocecal valve) were removed from the abdominal cavity. Digesta was collected from each segment within 30 min after killing. The digesta samples were mixed, and approximately 5 g was stored at 
30°C. The remainder of digesta was centrifuged at 8000 × g for 30 min. Ionized calcium concentration and pH were immediately determined in the supernatant. Approximately 2 mL of the supernatants were ultrafiltered through a 3000 MW cut-off membrane (Centricon-3, Amicon, Cambridge, MA). The supernatants and the ultrafiltered supernatants were also stored at 30°C.
Analyses.
Calcium concentrations were measured in diets, feces, whole digesta, the supernatants of digesta and the ultrafiltered supernatants using an atomic absorption spectrophotometer (AA-6600F, Shimazu, Kyoto, Japan) after digestion with concentrated nitric acid and perchloric acid (6 mol/L). Ionized calcium concentrations and pH of supernatants were measured with the ionized calcium analyzer. Soluble calcium was defined as calcium in supernatant. Calcium solubility was defined as the ratio of soluble calcium to total calcium. Calcium in lower-molecular-weight (LMW) fraction and calcium in higher-molecular-weight (HMW) fraction were calculated by the following equations:
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. Bovine serum albumin solutions were used as standards.
Statistical analyses.
Data are expressed as means ± SD. The differences between known concentrations of ionized calcium and their readings with the electrode were tested by one-sample t-test. Prior to comparisons between the two dietary groups, the equality of two variances was tested by the method of Snedecor and Cochran (1967). When the variances were significantly different, the data were transformed by logarithmic calculation to equalize their variances, and then non-paired Student's t-tests were used. Non-paired Student's t-tests were used to compare the two dietary groups without the transformation when the variances were not significantly different. The data expressed as percentages were transformed by arcsin calculation before the analyses to normalize their distributions. For comparisons between duodenal and ileal digesta in each group, paired t-tests were employed. Differences were considered significant at P < 0.05.
RESULTS
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Table 2. Effect of ionic strength on electrode response to calcium standards1,2 |
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Table 3. Effect of pH on electrode response to calcium standards1,2 |
Table 4.
Apparent calcium absorption and distribution of calcium in duodenal and ileal digesta of pigs fed skim milk powder (SMP)-based diet or defatted soybean flour (DSF)-based diets for 30 d1
Table 5.
Electrolyte concentrations in liquid phases of duodenal and ileal digesta of pigs fed skim milk powder (SMP)-based diet or defatted soybean flour (DSF)-based diets for 30 d1
DISCUSSION
, Yuan et al. 1991). Lee et al. (1980) reported that dietary casein stimulated calcium absorption in rats compared to dietary egg albumin or to an amino acid mixture simulating casein as protein source, and they suggested that calcium absorption was enhanced by the higher solubility of calcium in the ileal digesta of rats fed casein. They also showed that soybean protein reduced calcium absorption and suggested that the interference with calcium absorption was due to the lower solubility of calcium in the digestive tract. The results presented here show that soluble calcium concentration, calcium solubility and apparent absorption of calcium were higher in the SMP group than in the DSF group. These results support the positive relationship between calcium solubility and its absorption. Dairy products contain a large amount of lactose, which may increase calcium absorption. Because lactose was added to the DSF diet, factors other than lactose caused the differences between the two groups.
), the SMP diet contained a considerable amount of calcium citrate. Since the LMW fraction in the SMP group probably included calcium citrate, calcium citrate may contribute to the higher calcium concentration in the LMW fraction of pigs fed the SMP diet than in that of pigs fed the DSF diet.
). On the other hand, calcium is actively absorbed in the duodenum where its absorption is strongly regulated by endocrine factors. In an experiment using everted duodenal sac of rats, Roche et al. (1986) showed that the Michaelis constant for the saturable transfer of calcium was approximately 0.35 mmol/L. This value was less than ionized calcium concentration in the duodenum of each group. These results suggest that calcium can be easily absorbed by active transport in the duodenum, irrespective of the dietary treatments.
and Meisel and Frister (1988) reported that casein phosphopeptides, which bind calcium, were formed in the small intestine of rats and pigs fed diets containing casein. Naito and Suzuki (1974) indicated that a casein phosphopeptide isolated from ileal digesta of rats was slightly smaller than an artificial casein phosphopeptide prepared by tryptic digestion having a calculated MW of approximately 3000, based on amino acid sequence (Manson and Annan, 1971). In this experiment, calcium in the HMW fraction was defined as soluble calcium, which was not ultrafiltered through the 3000 MW cut-off membrane. Casein phosphopeptides formed in the digestive tract would not be ultrafiltered through the membrane because the artificial casein phosphopeptides did not pass the membrane. Thus, in the ileal digesta of the SMP group, calcium in the HMW fraction would include calcium bound to casein phosphopeptides. The soluble calcium concentration and calcium solubility in the ileum were higher in the SMP group than in the DSF group. The production of casein phosphopeptides may be involved in the higher solubility of calcium in the ileal digesta of the SMP group because casein phosphopeptides have been shown to enhance calcium solubility in the ileum of rats (Sato et al. 1986).
have suggested that calcium exchange between an added salt and a food source was complete for milk, calcium exchange is considered to occur easily between ionized calcium and its complexes in the SMP group. The increasing HMW ligands for calcium such as casein phosphopeptides may shift calcium equilibrium from ionized calcium and calcium complexes with LMW ligands to calcium bound to HMW ligands in the ileum of the SMP group. Furthermore, the increase in pH might differently change the affinities of ligands for calcium in the HMW and LMW fractions of the SMP group, which might shift the calcium equilibrium to calcium complexes with HMW ligands. These changes in calcium equilibrium might lower the ratios of ionized calcium and calcium in the LMW fraction to soluble calcium. Additionally, calcium citrate was postulated to be absorbed without dissociation (Pak et al. 1989). The lower calcium in the LMW fraction might be partly due to the absorption of calcium citrate in the SMP group.
). Because the passive absorption depends on ionized calcium concentration, higher ionized calcium concentration increases calcium absorption. Compared to the DSF group, ionized calcium concentration was higher in the ileal digesta of the SMP group. The higher apparent absorption of calcium is probably due to the higher concentration of ionized calcium in the ileum of the SMP group.
suggested that the infusion of casein phosphopeptides into the ileal lumen of rats increased calcium absorption. Li et al. (1989) reported that calcium bound to casein phosphopeptides was not directly absorbed by rats. These reports suggested that calcium bound to casein phosphopeptides was easily exchanged with ionized calcium. Thus ionized calcium may be recruited from its complexes with casein phosphopeptides. This experiment suggests that calcium bound to HMW ligands which likely include casein phosphopeptides, are produced in the ileum of pigs fed the SMP diet. The formation of calcium complexes with HMW ligands and increasing pH change the distribution of calcium in the ileum of the SMP group compared to the DSF group. Complexes of calcium with HMW ligands may be easily exchangeable with ionized calcium, and the increase in these calcium complexes possibly enhances the recruitment of ionized calcium, which results in the higher concentration of ionized calcium in the ileum of the SMP group compared to the DSF group.
FOOTNOTES
Manuscript received 23 September 1996. Initial reviews completed 27 November 1996. Revision accepted 4 March 1997.
LITERATURE CITED
casein.
Arch. Biochem. Biophys.
1971;
145:16-26
[Medline]
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