The first nonmilk foods that are given to infants contain high levels of starch, a fraction of which is resistant to enzyme hydrolysis. Incomplete digestion of starch may interfere with the absorption of certain minerals. A fraction of dietary starch which is resistant to in vitro enzymatic hydrolysis has been termed resistant starch. The aim of this study was to compare the intestinal apparent absorption of calcium, phosphorus, iron, and zinc in the presence of either resistant or digestible starch. Twelve 7-10-d-old piglets were fitted with a T-tube inserted into the intestine approximately 3 m distal to the duodenum. Animals received in random order 200 mL of a test meal of cooked, cooled, high amylose corn starch (16.4% resistant starch), or cooked rice starch (digestible starch) administered by an orogastric tube. Both meals contained the same amount of calcium, phosphorus, iron, and zinc. The test meal also contained tracer amounts of 59Fe and65 Zn, as well as polyethylene glycol 3350, as a nonabsorbable marker. Intestinal apparent absorption of starch was greater after the meal with digestible starch (71.0 ± 17.0%) than after the meal with resistant starch (49.2 ± 10.3) (p < 0.001). After feeding the meals with resistant and digestible starch, mineral apparent absorption was, respectively: calcium, 40.2 ± 11.8% versus 28.1 ± 16.4% (p < 0.05); phosphorus, 73.2 ± 14.0%versus 67.8 ± 18% (NS); iron, 24.1 ± 12.2%versus 12.6 ± 10.6% (p < 0.01), and zinc, 35.0± 13.0% versus 30.6 ± 8.22% (NS). In conclusion, a meal containing 16.4% resistant starch resulted in a greater apparent absorption of calcium and iron compared with a completely digestible starch meal. If this finding holds true for the whole bowel, administration of resistant starches could have a positive effect on intestinal calcium and iron absorption.
From a very early age, infants in both developed and developing countries are fed starch-containing foods. The starch granule is a semicrystalline, macromolecular complex comprised of linear chains of polysaccharides(e.g. amylose) and branched chains (e.g. amylopectin). Evidence from in vivo intestinal perfusion studies in adult volunteers(1), analysis of material collected in ileostomized patients(2, 3), and detection of hydrogen in expired air(4), indicates that a fraction of the ingested starch remains undigested and enters the colon. These findings confirm previous in vitro observations of the presence of a fraction of starch that was resistant to enzymatic hydrolysis. This fraction, termed RS, is defined as the fraction of starch that, in vitro, is resistant to digestion by amylolytic enzymes, unless previously dispersed with 2 M KOH(5).
Carbohydrates can affect intestinal absorption of minerals in humans(6, 7) and in animals(8, 9, 19). There is little information regarding the effect that RS formed during food preparation and storage may have on mineral absorption(10). It could be hypothesized that incompletely hydrolyzed and thus unabsorbed starch may interfere with the absorption of certain minerals, a matter of relevance to mineral homeostasis in infants, particularly those with borderline nutritional status and who receive large amounts of starch early in life. The aim of this study was to compare the effect of resistant and completely DS on intestinal apparent absorption of calcium, phosphorus, iron, and zinc.
The protocol was approved by the Animal Protocol Review Committee for Baylor College of Medicine.
Animals. Seven- to 10-d-old pigs were the terminal crossbred offspring of crossbred Yorkshire-Landrace sows with crossbred Hampshire-Duroc boars, purchased from Texas A&M University (College Station, TX). Animals were housed in individual cages in a room with an ambient temperature of 28°C and a light/dark cycle of 12 h and had arrived to our facility at least 72 h before surgery. Piglets were fed ad libitum a liquid commercial swine weaning formula (Soweena, Merrick).
Diet preparation. High amylose maize starch (Sigma Chemical Co., St. Louis, MO) was chosen as the RS and rice starch (Sigma Chemical Co.) was selected as the DS. Test meals containing RS (17.5 g) or DS (17.5 g), 350 mg of calcium carbonate (Sigma Chemical Co.), and 350 mg of potassium phosphate monobasic (Sigma Chemical Co.) were dispersed in 250 mL of a solution containing 208 mg of ferrous sulfate heptahydrate (ICN Biomedicals, 44 mg of zinc sulfate, ZnSO4·7H2O (Fisher Scientific), 5 μCi of 59Fe (ferrous sulfate; DuPont NEN), 5.0 μCi of 65Zn (zinc sulfate, DuPont NEN. Boston, MA), and 10 g of PEG-3350 (Sigma Chemical Co.) per liter of deionized Milli-Q water.
The RS meal was cooked in a boiling waterbath with continuous stirring for 1 h and after cooling, stored at 4°C for 48 h before it was administered to the animals. DS was cooked for 30 min at 80°C and was administered to the pigs on the same day in which it was prepared, without a storage period. The final volume and weight of the test meals was 273 mL and 280 g, respectively, and had a pap consistency.
Total starch and RS, and the degree of hydrolysis after 4 h of in vitro enzymatic action were determined in five replicates in aliquots from two of each of the test meals. For total starch determination, 1 mL of NaOH 2 N was added to 500 mg of the above preparation. The mix was shaken for 30 min in a multivortex. The pH was adjusted to 5.0 using 200 μL of glacial acetic acid. One mL of sodium acetate 0.25 N, pH 5.0, was added to the mix before the addition of 356 U of porcine pancreatic α-amylase type I-A EC 18.104.22.168(Sigma Chemical Co.) and 6 U of Aspergillus niger amyloglucosidase EC 22.214.171.124 (Boehringer Mannheim, Indianapolis, IN). Samples were incubated for 24 h at 37°C, after which the enzymes were inactivated by heating at 100°C for 10 min. The volume of each sample was brought to 10 mL by the addition of distilled water, and glucose was determined using the glucose-oxidase method (Glucose Statzyme, Worthington, Freehold, NJ) by a Cobas-Fara II automatic analyzer. The mass of polysaccharide was calculated as 0.9 of the glucose content.
For determination of RS, we followed the same method described for total starch except for the exclusion of the steps involving NaOH and glacial acetic acid. The degree of hydrolysis in 4 h was determined by the same method used for RS, except for the reduction of the duration of the incubation time.
Samples of both test meals were analyzed for phytic acid (tri-, tetra-, penta-, hexaphosphates) by reverse phase HPLC (courtesy of ALKO Biotechnology, Helsinki, Finland, through Enzyme Development Corp., New York). Iron and zinc contents in ash samples of meals were determined by an atomic spectrophotometric method.
Animal studies. Under general anesthesia, a 16 French, rubber T-tube was inserted into the small bowel, 3.0 m distal of the animals' duodenum (approximately half way the distance from the duodenum to the terminal ileum), whereas the other end of the tube was exteriorized through the skin and remained closed. Animals were allowed to recover for 7 d. All infant pigs had been gaining weight for at least 3 d before the studies and did not show any signs of surgical complications. Animals were randomly assigned to receive first either the diet containing RS or the one with DS, and the selected diet was administered for 3 d before the testing day. After the experiment was completed, animals received the other diet for 3 d and the test was repeated. After an overnight fast, the T-tube was opened, flushed with 3 mL of water, and connected to an ostomy bag. A baseline sample of intestinal contents was collected, and 200 mL of one of the selected test meals was administered via an orogastric tube as a bolus. Digesta was collected through the T-tube for the next 3 h, and the volume was recorded and frozen until analysis. After the second experiment, animals were killed, and the small bowel was removed and measured.
Before determination of calcium, phosphorus, 59Fe, 65Zn, and PEG-3350, samples of each test meal and digesta were lyophilized. The dry weight was recorded. For determination of calcium and phosphorus, the lyophilized samples were ashed for 16 h at 440°C. The ash material was mixed with 0.1 mL of water to which 0.5 mL of concentrated nitric acid was added. Samples were heated for 5 min at 80°C. The volume was made to 10 mL(test meal) or 2-5 mL (digesta) with water. Determinations were performed by a colorimetric method using an automatic analyzer (Cobas Fara II) with reagents purchased from Sigma Chemical Co. (360-3, 587-A).
59Fe and 65Zn were analyzed by a Beckman LS3801 liquid scintillation counter using a dual label program. A quench curve was prepared using CCl4 as a quench agent(11). The channel numbers of windows were set up in accordance with the spectrum analysis of the radioisotopes: zinc, 0-390, and iron, 391-1000. Both test meals and digesta(50 mg) were dissolved in 2 mL of 6 N HCl. Cocktail 3a70 (Research Products International, Mt. Prospect, IL; RBI 111152) was added, and samples were shaken until the acquisition of a gel form. Samples contained 4000-8000 dpm for zinc and 2700-7500 for iron. Counting efficiency was between 35-65% and 80-85%, respectively.
PEG-3350 was determined by a spectrophotometric method employing acetone and trichloroacetic acid(12).
Determination of free glucose and total amount of carbohydrates was performed on 20 mg of lyophilized digesta. Free glucose was determined by the glucose-oxidase method, after suspension in 5 mL of water. The total amount of carbohydrate was determined by the same method used for total starch.
Statistical analysis. Apparent intestinal absorption of starch and minerals was calculated using the following equation: where: total in meal or digesta is either the amount of starch, calcium, phosphorus, dpm of 59Fe, or of 65Zn; PEGd is total amount of PEG recovered in digesta; PEGm is total amount of PEG in test meal.
Results are expressed as mean and SD of the mean. The ratio between the length of intestine proximal to the T-tube and the total length of the small intestine (rP/T) was correlated with nutrient absorption using the Pearson coefficient. The comparison between apparent absorption after feeding meals with and without RS starch was performed by paired t test.
Table 1 shows the composition of both meals which contained the same amount of total starch, Ca, P, Fe, Zn, and PEG. The only difference was the amount of RS which, after 24 h of enzymatic hydrolysis, was 16.4% in the RS and 0% in DS. The degree of carbohydrate hydrolysis achievedin vitro after 4 h of enzymatic incubation was approximately the same in both meals. No phytic acid was detected in either meal.
Fourteen piglets underwent surgery and the study was completed successfully in 12; the T-tube was inadvertently dislodged in two. Five piglets received the RS meal first. There was no statistical difference between the mean age and weight of the animals on the day they received the RS mealversus the day they received the DS meal (23.1 ± 2.8 d and 4152 ± 687 g; 22.6 ± 2.5 d and 4028 ± 801 g, respectively).
The mean ratio (rP/T) between the length of intestine proximal to the T-tube and the total length of the small intestine was 0.53 ± 0.11. The volume of digesta collected presented a tendency to be lower with the RS meal(38.3 ± 20.8 mL) than with the DS meal (54.6 ± 39.6 mL)(p = 0.06). As expected, there was a statistically significant negative correlation between the rP/T and the volume of digesta collected for both the RS (r = -0.85, p = 0.000) and the DS (r= -0.90, p = 0.000) meals.
Table 2 presents the apparent intestinal absorption of starch, Ca, P, Fe, and Zn. The correlation between starch apparent absorption and rP/T was 0.54 (p = 0.135) after the RS meal and 0.86(p = 0.000) after the DS meal. The total amount of carbohydrate recovered through the T-tube was composed of free glucose and glucose polymers. The amount of free glucose recovered was not significantly different after administration of the RS meal (0.48 ± 0.35 mg/g of PEG) than after the DS (0.78 ± 0.73 g/g of PEG).
Apparent absorption of Ca and Fe was greater after the RS than after the DS meal. Apparent absorption of P and Zn did not differ between the two meals. There was no correlation between rP/T and Ca, P, Fe, or Zn absorption.
Complex carbohydrates are introduced early in life, sometimes even before infants acquire a mature pattern of digestion(13). Because the addition of complex carbohydrates to the diet usually results in a decrease of milk intake, the major source of minerals for infants, we were interested in determining whether nondigestible carbohydrates interfere with mineral absorption, specifically Ca, P, Fe, and Zn, in comparison with a DS.
Three-week-old pigs were used because their gastrointestinal tract function is similar to that of infants of an age at which starch is normally introduced into their diet(14). Pilot studies were performed to determine the optimal type of T-tube to be used as well as the duration of the period for collection of digesta. Because the amount of undigested carbohydrate arriving in the terminal ileum has a large interindividual variation and may be fermented by bacteria to various degrees, to sample the digesta we arbitrarily chose a distance of 3 m from the angle of Treitzequivalent, representing half the length of the small intestine. Reproducibility of the location of the T-tube is indicated by the fact that the mean rP/T on 12 animals studied was 0.53 ± 0.11.
The mineral composition of both meals tested was designed to reflect the concentration present in commercial infant formulas and cereals. Protein, fat, vitamins, and other minerals were not added to the test meals to avoid any confounding effects and to be able to determine exclusively the effect of RS on mineral absorption. High amylose maize starch was used because, after cooking and cooling, it produces high amounts of RS mostly in the form of retrograded amylose, whereas rice starch is easily hydrolyzed(3, 10). Two definitions can be used for RS(3): one is the chemical definition based on thein vitro enzymatic hydrolysis and another one is the physiologic, which defines RS as the sum of starch and the products of starch hydrolysis not absorbed in the small intestine and recovered in the terminal ileum. We analyzed the degree of starch hydrolysis in vitro during 4 h because that is the approximate intestinal transit time in infant pigs. Our results indicated that most of the starch of both meals was hydrolyzed in that time; however, the duration of enzymatic incubation did not result in any difference in the proportion of starch digested between the meal with (72.5%) or without(68.9%) RS. On the other hand, starch incubation with enzymes for 24 h, revealed the presence of RS (16.4%) in the study meal. This value was close to the difference of the means (21.8%) of apparent starch absorption observed in the animal experiments: 71% after RS feeding minus 49.2% after meal with DS. There is a theoretical possibility that we could have overestimated the amount of RS, as product inhibition is known to limit the ability of amylases to digest starch(16). However, Faisant et al.(3) obtained 49% of RS from their preparation of retrograded starch, whereas our modification to the method, using pure amylose, yielded only 16.7%. Therefore our method may have rather under- than overestimated the amount of RS.
The amount of free glucose recovered in digesta was not significantly different after feeding the meals with and without RS, indicating that the limiting factor for RS absorption is related more to starch hydrolysis than glucose uptake. The strong correlation between rP/T and apparent starch absorption after the meal without RS suggested that digestion and absorption took place progressively during its transit through the small intestine. After feeding the meal with RS this correlation was no longer significant, again suggesting that the decrease in starch absorption was a consequence of inefficiency of enzymatic hydrolysis and not insufficient absorptive surface.
Our results indicate that absorption of Ca and Fe was greater when minerals were administered with RS compared with what occurred when given concurrently with DS. The apparent absorption of P and Zn, however, was not influenced by the presence of RS. There was no correlation between rP/T and mineral apparent absorption, in agreement with the fact that Ca, P, Fe, and Zn are absorbed mostly in the duodenum and proximal jejunum(17).
In human diets, the most important factor that determines the amount of RS formed is the way that starch-containing foods are prepared(18). Gelatinization of starch, which occurs during food processing, renders starch more susceptible to enzymatic hydrolysis. Gelatinization entails the disruption of hydrogen bonds between amylose molecules followed by hydrogen bond formation between amylose molecules and water. However, in some foods, starch granules are located in rigid or very densely packed structures, rendering the granules inaccessible for gelatinization and enzymatic digestion. When these starch granules are heated beyond the gelatinization temperature, the granules are disrupted and amylose molecules are released. Upon cooling, hydrogen bonds re-form between amylose molecules, forming retrograded amylose, which is also resistant to hydrolysis by α-amylase. Therefore, resistant starch may be classified into three categories: physically inaccessible starch, nongelatinized granules and retrograded amylose(19).
From the chemical view point, we could speculate that the greater absorption of Fe and Ca after feeding the meal with resistant starch could be related to the formation of retrograded amylose. The hydrogen bonds reformed during the formation of retrograded amylose may decrease the number of potential binding sites for Ca and Fe on starch molecule, thereby increasing their solubility in water and facilitating their intestinal absorption(20). On the other hand, as in the meal composed only by digestible starch, the retrogradation process did not occur and the starch molecule presented more free sites for Ca and Fe chelation.
Another explanation could be that the resistant starch, as it is nondigestible, remains in the lumen of the bowel increasing lumenal osmolality and delaying gastric emptying. This slows flow in the small bowel and provides more time for minerals to be absorbed.
The unstirred water layer overlies the intestinal epithelium and affects nutrient absorption by raising the kinetic parameters in the active transport of nutrients such as glucose(21). Alterations of the unstirred water layer by the starch preparations could have affected mineral absorption. Bamba et al.(22), however, demonstrated that the addition of 2.5 or 5% polydextrose, a polymer of glucose which is not absorbable by the intestine, did not increase significantly the thickness of the water layer nor affect glucose absorption compared with the polydextrose-free solution.
It remains debatable whether the carbohydrate, the phytic acid, or both are responsible for the decreased absorption of minerals from whole-grain cereal products as a consequence of their chelating properties(23, 24). In our study, phytic acid was not detectable in either of the test meals. Therefore, it cannot be responsible for the differences observed. The effect of uncooked, high amylose corn starch on the intestinal absorption of Ca and magnesium has been previously analyzed in rats; conflicting results were observed(8, 9). It was recently(10) observed in rats that Ca and magnesium were absorbed to a significantly greater degree when administered with uncooked resistant starch than with retrograded amylose or the DS diet. In our study, the meals were cooked and stored at refrigerator temperature to reflect human food preparation and storage.
In conclusion, we have found that an incompletely digestible starch meal containing 16.4% resistant starch, probably retrograded amylose, results in a greater apparent absorption of Ca and Fe compared with a completely digestible starch meal. Further investigation is required to analyze the effect of prolonged use of resistant starch food on Ca and Fe nutritional status and to determine whether this finding is reproducible in human infants. This will allow to make appropriate nutrition recommendations which will result in enhanced mineral absorption.
The authors thank ALKO Biotechnology, Helsinki, Finland, and the Enzyme Development Corporation, New York, for sample analysis for phytate; Gerber Products Company for providing the mineral-free and mineral-fortified rice cereal; Peter Reeds, Ph.D., for editorial assistance; and Iftikhar Khan for technical assistance.
ratio between the length of intestine proximal to the T-tube and the total length of the small intestine
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Journal of Food Science and Technology (2010)