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* Department of Medicine, Cedars-Sinai Burns & Allen Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048; School of Medicine, University of California, Los Angeles, CA 90024; and ** Ross Products Division, Abbott Laboratories, Columbus, OH 43215
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
LITERATURE CITED
ABSTRACT 
Soy protein, in both intact and hydrolyzed forms, is widely used as the nitrogen source in infant and adult formulas. This protein is also consumed in vast quantities worldwide as soybean-based food products. Digestion is the rate-limiting step in the assimilation of proteins from the gut. As a result, intestinal transit must be slowed when a higher load of protein is available or when this nutrient is delivered in the intact rather than hydrolyzed form. However, little information is available on the effect of load and degree of hydrolysis of soy protein on intestinal transit and protein absorption. To test the hypothesis that inhibition of intestinal transit and protein absorption depend on the load of soy protein and the state of hydrolysis of this nutrient, we compared intestinal transit and protein absorption in dogs equipped with duodenal and midintestinal fistulas during intestinal perfusion with 0, 50, 100, or 200 g/L solutions of intact soy protein versus 0, 100, 200, 300, or 400 g/L solutions of hydrolyzed soy protein. We found that intestinal transit was slowed in a load-dependent fashion by intact (P < 0.001) and hydrolyzed (P < 0.05) soy protein. Soy protein inhibited intestinal transit more potently in the intact than hydrolyzed form (P < 0.05). A greater amount of protein was absorbed by the proximal half of the small intestine when soy protein was delivered in the hydrolyzed than intact form (P < 0.05), and the efficiency of protein absorption was maintained at a high and nearly constant level of 82.6 to 87.4% for intact soy protein and 89.0 to 92.3% for hydrolyzed soy protein. We conclude that in dogs intestinal transit and absorption of soy protein depend on the load and the degree of protein hydrolysis.
KEY WORDS: small intestine · gastrointestinal motility · digestion · dogs · soy proteinINTRODUCTION
The small intestine removes protein from the lumen more slowly when the nutrient is delivered in the intact than hydrolyzed form (Sleisenger and Kim 1979
). While more than 70% of partially hydrolyzed protein is absorbed by the proximal small intestine in 30 to 60 min (Davenport 1982), ~40% of intact dietary protein is still undigested and available in the ileum 4 h after a meal (Chung et al. 1979). Since only 2-5% of digested protein escapes transport across the mucosa once the hydrolyzed form of this nutrient is in contact with the absorptive surfaces of the small intestine (Meyer and Kelly 1976), the time-demanding, rate-limiting step of assimilation of protein by the small intestine is digestion rather than absorption. Thus slower intestinal transit (longer residence time in the small intestine) may be needed when protein is delivered at a greater load or in the intact rather than hydrolyzed form.
Both intact and hydrolyzed forms of soy protein have been successfully used as the protein source for infant feeding (Heird 1994) and for preventing or treating protein-energy malnutrition (Abiodun 1991). Because the biologic value of soy protein is high, food products based on this nutrient are widely used as alternatives to animal protein. The recent finding that soy protein lowers serum lipids (Anderson et al. 1995) may encourage even greater use of this nutrient.
Although proteins mixed with other nutrients have been reported to inhibit intestinal motility and transit and, in turn, increase protein absorption (Schemann et al. 1986, Siegle et al. 1986), no information is available on the effect of the load or the degree of hydrolysis of soy protein on intestinal transit and protein absorption. In this study, using a fistulated dog model we tested the hypothesis that inhibition of intestinal transit and protein absorption depended on the load of soy protein and the state of hydrolysis of this nutrient.
MATERIALS AND METHODS
). With the flanges of the cannula resting against the inner surface of the intestinal wall, the cannulas were fixed against rotation. Just distal to the fistula, a length of polyvinyl chloride tubing (Tygon, Norton Performance Plastic Co., Akron, OH) with a diameter of 2 mm was looped around the intestine and fixed by suture through the visceral peritoneum to the intestinal wall. The length of tubing used was individualized to be as short as possible without a tightening effect on the lumen. This provided a stent against which an inflated Foley balloon could be pulled to provide a water tight seal. All dogs were given a recovery period of 4 wk and underwent testing only after normal feeding behaviors were re-established. The twelve dogs remained healthy with stable body weights and unaffected demeanor for > 12 mo of observation.
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Table 1. Intestinal transit in dogs infused with intact soy protein1 |
). This occlusion method allowed for complete but temporary diversion of chyme at the midintestinal fistula. The test solution was perfused at 2 mL/min for 120 min via a blunted needle inserted through the cork placed in the duodenal cannula. This perfusion method allowed the perfusate to be mixed with endogenous biliary and pancreatic secretions. To allow the protein perfusate access to the whole gut, the output of midintestinal fistula was temporarily diverted for collection of test samples then returned to the distal gut via the Foley catheter using a light sensor-driven pumping system that was synchronized to the outflow from the midintestinal fistula (Lin et al. 1989). This sensor-driven system included a pump that was turned on or off by a light sensor attached to an optical chamber that drained the midintestinal cannula. A two-head pump was switched on when the light sensor detected a rise in the fluid level within the optical chamber (Masterflex, Cole-Palmer, Chicago, IL). The chamber was then drained until the fluid level returned to base line. The pump would then switch off until the next surge of fluid. With heads of different pumping capacities, the fistulous output was returned to the distal limb of the fistula by the large pump head, so that the bulk of chyme traversed the bowel as it normally would have, while a second, small pump head pumped a fraction of total flow (~7%) into a collecting tube for the recovery of the transit marker and the measurement of protein concentration.
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Table 2. Intestinal transit in dogs infused with hydrolyzed soy protein1 |
) was delivered as a bolus into the test segment via the duodenal fistula to begin measurement of intestinal transit. Intestinal transit across the proximal half of the small intestine (~150 cm in length) was measured by counting the radioactivity of 1-mL samples collected every 10 min from the output of the midintestinal fistula for 60 min. Using a matched dose of 99m-Tc to represent the original delivered bolus, the radioactivity delivered into the segment (Johansson 1974, Zierler 1958) and the radioactivity in the recovered fistulous output were all measured in a gamma well counter. All data were corrected for radioactive decay. Intestinal transit was calculated as the cumulative percent recovery of the delivered 99m-Tc over the collection period.
Protein assay.
Samples collected from the midintestinal fistulous output were analyzed for protein concentration. Protein concentration was determined by the Peterson's modified Lowry method with deoxycholate and trichloroacetic acid to precipitate protein (Lowry et al. 1951) using a protein assay kit (Sigma, St. Louis, MO). Since the color of the samples and optical density reading were different for intact vs. hydrolyzed protein at the same concentrations, the original intact or hydrolyzed protein solutions (intact: 50, 100 or 200 g/L and hydrolyzed: 100, 200, 300 or 400 g/L) were used as the standards for analysis of protein concentration in each experiment (i.e., samples collected during experiments with intact protein were assayed with reference to standards established with solutions of intact protein). The amount of protein recovered in each collection sample = concentration of protein in 1 mL sample × fistulous output volume in mL. The amount of protein recovered = cumulative amount of protein recovered during the collection period.
Calculation of estimated protein absorption.
Estimated protein absorption was calculated from the following equations: Estimated amount of protein absorbed (g) = maximum amount of protein recoverable (g) 
amount of protein recovered (g). Maximum amount of protein recoverable (g) = amount of protein delivered (g) × cumulative fractional recovery of 99m-Tc.
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Table 3. Infusion of intact soy protein inhibits intestinal transit in dogs more potently than does hydrolyzed soy protein1 |
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Table 4. Absorption by dog intestine of intact soy protein infusion1 |
).
Analysis of data.
Intestinal transit was compared according to the area under the curve (AUC) represented by the cumulative percent recovery of 99m-Tc over 60 min. The distribution of AUC is skewed to the right with larger variances under conditions with higher measures (this is typical of area measurements). Standard statistical tests assume that data are normal in distribution and that variances are homogeneous. The skewing of intestinal transit AUC was, therefore, reduced by taking the square roots of the areas under the curve (sqrt AUC) (Lin et al. 1992) where 0 = no recovery by 60 min, and 74.16 = theoretical, complete and instantaneous recovery by time 0 (rapid transit). Intestinal transit and protein absorption (g) were then compared using one-way or two-way repeated measures ANOVA. One-way repeated measures ANOVA was used to test dose-responsiveness across eight dogs in the intact soy protein group (Tables 1 and 4) and across nine dogs in the hydrolyzed soy protein group (Tables 2 and 5). When the comparison included dose and state of hydrolysis as the two test conditions, two-way repeated measures ANOVA was used on the data from the five dogs that were tested with both intact and hydrolyzed soy protein (Tables 3 and 6). In these comparisons, the results from 100 and 200 g/L solutions (concentrations used in both intact and hydrolyzed groups) were tested. To validate the intestinal transit, data from the two methods of transit measurement (99m-Tc, PEG) were compared using regression analysis. The computer program used for all statistical analysis was BMDP (Dixon 1990).
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Table 5. Absorption of hydrolyzed soy protein by dog small intestine1 |
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Table 6. Protein absorption in dogs depends on the state of hydrolysis1 |
RESULTS
Inhibition of intestinal transit by soy protein: intact vs. hydrolyzed. Intestinal transit depended on the load and the degree of protein hydrolysis since transit was slower (smaller mean value of sqrt AUC) with intact than hydrolyzed soy protein at 200 g/L (mean sqrt AUC values for intact: 30.1 vs. hydrolyzed: 42.9) but not at 100 g/L protein load (P < 0.05, interaction of load and state of hydrolysis) (Table 3). Correspondingly, the mean cumulative recovery of 99m-Tc at the 200 g/L load was 44.6% for intact soy protein and 71.1% for hydrolyzed soy protein. Absorption of soy protein: intact, hydrolyzed. Absorption of intact and hydrolyzed soy protein by the proximal half of the small intestine are shown in Tables 4 and 5. We found that the estimated amount of protein absorbed increased in a load-dependent fashion (P < 0.001) whether the soy protein was delivered in the intact or hydrolyzed form. For hydrolyzed soy protein, maximal protein absorption was reached at the protein load of 72 g (300 g/L)(Table 5). Increasing the load to 96 g (400 g/L) did not significantly increase the estimated amount of protein absorbed (300 vs. 400 g/L: 47.0 ± 5.0 vs. 52.0 ± 5.9 g). Since percent protein absorbed would be 100 if the estimated amount of protein absorbed equaled the maximum amount of protein recoverable (as represented by the line of identity in Fig. 3), percent protein absorbed (estimated amount of protein absorbed/maximum amount of protein recoverable × 100) represented the efficiency of protein absorption (100 = maximum efficiency). When data from both the intact and hydrolyzed protein groups were plotted together (Fig. 3), the estimated amount of protein absorbed was nearly identical to the maximal amount of protein recoverable (closely matching the line of identity). Specifically, we found that the efficiency of protein absorption was independent of the load of protein delivered but dependent on the degree of protein hydrolysis (P < 0.05), so that percent protein absorption was maintained at a high and nearly constant level of 82.6-87.4% for 50-200 g/L solutions of intact soy protein and an even higher level of 89.0-92.3% for 100-400 g/L solutions of hydrolyzed soy protein. These results suggest that a high efficiency of protein absorption may be maintained whether soy protein was delivered in the intact or hydrolyzed form.
0.56, r2 = 0.96). The symbols represent data from individual experiments. The line of identity (dashed line) represents 100% protein absorption.
Absorption of soy protein: intact vs. hydrolyzed. The proximal half of the small intestine absorbed more protein when the nutrient was presented in the hydrolyzed than intact form (P < 0.05). Specifically, as the load of protein increased from 24 g (100 g/L) to 48 g (200 g/L), the mean estimated amount of protein absorbed only increased from 16.0 to 16.7 g when the nutrient was delivered in the intact form, but increased from 18.6 to 35.3 g when the nutrient was delivered in the hydrolyzed form (P < 0.05, Table 6). Correspondingly, a greater amount of soy protein was still unabsorbed by the midintestinal fistula (Table 6) and available to the distal half of the small intestine when soy protein was delivered intact than when delivered in the hydrolyzed form. The relationship between intestinal transit (sqrt AUC), the amount of protein recovered, and the amount of protein delivered (24 or 48 g) is further illustrated in Figure 4. We found that when the protein load was increased from 24 to 48 g, intestinal transit (sqrt AUC) slowed significantly for the intact but not hydrolyzed protein (P < 0.05).
Validation of transit method. Intestinal transit measured by 99m-Tc collected during 60-120 minutes and by PEG collected during 0-120 minutes were compared as the cumulative percent recovery of the two markers. The cumulative percent recovery of PEG closely matched that of 99m-Tc (r2 = 0.97, P < 0.0001) (Fig. 5), confirming the accuracy of the transit method used in this study in measuring movement of luminutesal content during the 120-minute perfusion period.
DISCUSSION
). Although slowing of intestinal transit by proteins mixed with other nutrients delivered to the whole small intestine has also been reported (Schemann and Jarjis 1986, Siegle et al. 1990), there is little information on the effect of the load or the degree of hydrolysis of soy protein on intestinal transit and protein absorption. Even so, formulas based on soy protein are widely used, and elemental diets containing hydrolyzed proteins are commonly prescribed for patients with impaired digestion or absorption (Voitk and Crispin 1975). In this study, by testing soy protein in both intact and hydrolyzed forms, we showed that 1) intestinal transit was slowed in a load-dependent fashion by intact or hydrolyzed soy protein, 2) soy protein inhibited intestinal transit more potently in the intact than hydrolyzed form, 3) estimated protein absorption by the proximal half of the small intestine was greater for hydrolyzed than intact soy protein, and 4) efficiency of protein absorption was independent of the load but dependent on the degree of protein hydrolysis, so that the efficiency of protein absorption was maintained at a high level of 82.6-87.4% (12-48 g) for intact soy protein and 89.0-92.3% (24-96 g) for hydrolyzed soy protein. These results confirmed our hypothesis that intestinal transit and protein absorption depend on the load and degree of hydrolysis of soy protein.
) or intestinal transit (Lin et al. 1994) was shown to be dependent on the length of small intestine exposed to the nutrient. Specifically, early in the course of gastric emptying, nutrients may surge in a load-dependent fashion into the small intestine in excess of the amount allowed under full inhibitory feedback (Lin et al. 1996). After entry of a large load of nutrients into the small intestine, the absorptive capacity of the proximal small intestine may then be overwhelmed to spill nutrients distally along a longer length of the small intestine (Lin et al. 1989). Greater inhibition is then generated as more nutrient sensors are recruited to participate in the feedback. As suggested by the amount of protein recovered at the midintestinal fistula, load-dependent inhibition of intestinal transit by soy protein may also occur when progressively longer lengths of the small intestine become exposed to the nutrient.
), greater inhibition of intestinal transit may depend not only on the length of contact with nutrients, but the amount of protein spilling into the distal half of the small intestine to generate the ileal brake response.
who reported that the absorption of nutrients was increased when intestinal transit was slowed by lipid-induced ileal brake. We found that the slowing of intestinal transit in accordance with the load and degree of hydrolysis of soy protein was also effective in optimizing absorption. Specifically, the small intestine was able to maintain a high and nearly constant level of efficiency of protein absorption across a wide range of loads (Tables 4 and 5). These results suggest that highly efficient absorption may be maintained even by a single nutrient when the rate of intestinal transit is adjusted according to the load and the degree of hydrolysis.
). Our comparison of the response to intact versus hydrolyzed soy protein provides support for the idea that protein absorption may be enhanced when protein is delivered as a hydrolyzed, peptide-based system rather than a polymeric system containing intact proteins. Although the efficiency of protein absorption was high for both intact and hydrolyzed forms of soy protein, the absorption advantage afforded by the hydrolyzed form of soy protein may be important to maximal absorption of oligopeptides and amino acids in the setting of severe maldigestion or malabsorption.
).
, Adibi and Kim 1981), it is possible that amino acids and oligopeptides may inhibit intestinal transit via different control pathways. Currently, information on these control pathways is limited. Afferent vagal pathways that fire in response to luminal perfusion with various amino acids have been reported (Jeanningros 1982). It is unknown, however, whether separate neural pathways fire in response to oligopeptides. Since intact proteins cannot be absorbed directly, we speculate that the difference in the intestinal transit response to intact vs. hydrolyzed protein is related to the rate-limiting requirement for digestion and the longer length of intestinal exposure to proteins rather than to the stimulation of different neural pathways. Specifically, as a result of the spread of intact protein along a longer length of gut, a greater number of protein-sensitive neural pathways (specific for either amino acids or oligopeptides) may be stimulated to generate more intense inhibitory feedback.
FOOTNOTES
Manuscript received 26 March 1997. Initial reviews completed 23 May 1997. Revision accepted 18 July 1997.
LITERATURE CITED
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