Effects on the monkey, pig and rat pancreas of soy products with varying levels of trypsin inhibitor and comparison with the administration of cholecystokinin

Raw and heated soy flour and casein diets were compared in rats, pigs, and monkeys with respect to growth, pancreatic changes, fecal trypsin, and nitrogen digestibility. Cholecystokinin injection was compared to feeding raw soy flour and casein in rats and casein in pigs. Several soy protein preparations were fed to rats and monkeys. Neither raw soy flour nor any other soy product produced pancreatic enlargement in pigs or monkeys. Casein and heated soy flour performed similarly. By comparison, other effects of raw soy flour were as follows. Growth was depressed 60% in rats and 84% in pigs, but not at all in monkeys. Nitrogen digestibility was depressed 5, 45, and 9% in rats, pigs, and monkeys, respectively. Pancreatic DNA, RNA, and protein levels were unchanged in monkeys fed raw soy flour. In rats, RNA per milligram pancreas was increased 40%, in pigs 20%. Pancreatic protein was decreased 7% in pigs and increased 47% in rats. Changes in pancreatic trypsin, chymotrypsin, lipase, and amylase were dissimilar in the three species. Fecal trypsin was elevated 300-400% in rats, and decreased approximately 50% in pigs and monkeys. Cholecystokinin injections in pigs and rats produced changes both quantitatively and qualitatively different from those seen with raw soy flour. Feeding of heated soy flour or soy protein isolates was comparable to feeding casein in all three species, and produced no deleterious effects.     

Effects of dietary proteins and trypsin inhibitor on growth and lipid metabolism in hamsters

The purpose of this study was to determine trypsin inhibitor (TI) activity in sweetpotato and soy flour diets and their effects on the growth and lipid metabolism of hamsters. Male Golden Syrian hamsters were fed different types of dietary protein containing casein, soyprotein, transgenic sweetpotato plus soy flour (TSPF+SF), nontransgenic sweetpotato (NTSPF) plus soy flour (NTSPF+SF), transgenic sweetpotato (TSPF) and nontransgenic sweetpotato flour for 28 days. The TI activity was highest in TSPF+SF (19.30 TIU/mg) and NTSPF+SF (17.20 TIU/mg) diets that induced growth retardation in animals, lowest in TSPF (5.80 TIU/mg) and NTSPF (5.50 TIU/mg) diets, which did not affect the growth of the animals, and negligible in casein (<1.00 TIU/mg) and soyprotein (2.00 TIU/mg) diets. Plasma total cholesterol (TC), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C) and triglyceride (TG) concentrations were significantly higher in hamsters fed the casein diet compared to those fed the soy protein, TSPF+SF, NTSPF+SF, TSPF and NTSPF diets. A positive correlation was observed between plasma TC concentrations of hamsters and dietary methionine, lysine, leucine content and methionine/glycine ratios. Liver TC and TG concentrations of hamsters fed casein were significantly higher than those of all other diet groups. The supplementation of sweetpotato flour with soy flour increased both protein and TI activity in the diets and the lipid metabolism of hamsters were unaffected by TI activity.     

Growth, digestibility, and enzyme activities in the pancreas and intestines of guinea-pigs fed on raw and heated soya-bean flour

The nutritional effects of giving raw (RSF) or heated (HSF) soya-bean flour to young guinea-pigs were investigated in trials 1 and 2, in which the levels of dietary protein were 120 and 190 g/kg diet respectively. The growth rate of animals fed on RSF was lower than that of those fed on HSF. Growth retardation of guinea-pigs fed on RSF was accompanied by a lower apparent digestibility of the protein (0.49-0.53) compared with HSF (0.67-0.76) and lower food conversion efficiency. In RSF-fed animals, increasing dietary protein affected growth and food conversion efficiency negatively. The pancreas of animals fed on RSF and HSF was similar in weight but secreted less trypsin, chymotrypsin and amylase, in RFS-fed animals. It was concluded that the mechanism by which raw soya-bean negatively affects the growth rate of guinea-pigs by reducing the activity of intestinal enzymes, differs from that suggested for rats and chicks, but is similar to that of pigs and calves.     

Pancreatic response in rats and mice to trypsin inhibitors from soy and potato after short- and long-term dietary exposure

The effects on the pancreas of chronic (95 wk) dietary exposure to protease inhibitors from soy and potato were compared in rats and mice. Soy and potato trypsin inhibitor (TI) concentrates were prepared from defatted raw soy flour and potato juice, respectively, by selective precipitation and ultrafiltration. Animals were fed a diet in which casein supplied approximately 20% protein. Each concentrate (less than 1% of the diet) was added to provide 100 and 200 mg of trypsin inhibitor activity per 100 g of diet. In short-term (28 d) experiments in rats, both sources of TI decreased the apparent nutritional quality of casein and produced pancreatic hypertrophy consistent with a hormonally mediated feedback mechanism for pancreatic adaptation to diet that is interactive with the nutritional status of the animal. After long-term feeding (95 wk), soy and potato TI produced dose-related pancreatic pathology in rats consisting of nodular hyperplasia and acinar adenoma, which was typical of that associated with raw soy flour. Although mice responded similarly to rats to soy TI in short-term (28-d) feeding experiments, they were resistant to the formation of these lesions following long-term feeding. This considerable species variation in propensity to develop preneoplastic and neoplastic lesions of the pancreas is not predicted by the short-term hypertrophic and hyperplastic response of the pancreas to TI.     

Effect of sulfur amino acid supplementation of raw soy flour on the growth and pancreatic weights of rats

Rat feeding studies showed that L-cystine, L-cysteine, N-acetyl-L-cysteine and L-methionine enhance the nutritional quality of raw (unheated) soy flour as measured in rats by the protein efficiency ratio (PER). Pancreas weights of rats fed diets supplemented with sulfur amino acids were higher than those of rats fed unsupplemented diets. In some instances this increase resulting from nutritional improvement of the diet was relatively greater than that in total body weight. Explanations are offered for these effects in terms of the proposed biofeedback mechanism involving the gastrointestinal endocrine system and the hormone cholecystokinin. The observed stimulation of pancreatic growth by added sulfur amino acids may be closely associated with this pancreatic regulatory mechanism when activated in response to dietary trypsin inhibitors present in unheated soy protein. The extent to which pancreatic hypertrophy develops as a result of a given exposure to trypsin inhibitors may be limited when the total amount or quality of the protein in the diet is less than optimal.     

The failure of long-term feeding of raw soy flour, in the presence or absence of azaserine, to induce carcinogenic changes in the mouse pancreas

The effects of feeding mice raw or heated soy flours or casein in the presence and absence of injected azaserine were investigated over a period of 18 months. Although the feeding of raw soy flour (compared with heated soy flour or casein) caused a significant inhibition of growth and an enlargement of the pancreas, there was no macroscopic evidence of pancreatic nodules in any of the six experimental groups. Microscopic examination of the pancreas revealed a somewhat higher (not significant) incidence of atypical acinar cell nodules in all animals injected with azaserine, but this difference was little influenced by the diets themselves. We concluded that raw soy flour itself has no carcinogenic effect on the mouse pancreas and does not enhance the sensitivity of the mouse pancreas to azaserine. Thus, it cannot be assumed that the appearance of pancreatic nodules constitutes an obligatory sequela of pancreatic hypertrophy and/or hyperplasia in all species of animals.     

The failure of long‐term feeding of raw soy flour,in the presence or absence of Azaserine,to induce carcinogenic changes in the mouse pancreas

The effects of feeding mice raw or heated soy flours or casein in the presence and absence of injected azaserine were investigated over a peiod of 18 months. Although the feeding of raw soy flour (compared with heated soy flour or casein) caused a significant inhibition of growth and an enlargement of the pancreas, there was no macroscopic evidence of pancreatic nodules in any of the six experimental groups. Microscopic examination of the pancreas revealed a somewhat higher (not significant) incidence of atypical acinar cell nodules in all animals injected with azaserine, but this difference was little influenced by the diets themselves. We concluded that raw soy flour itself has no carcinogenic effect on the mouse pancreas and does not enhance the sensitivity of the mouse pancreas to azaserine. Thus, it cannot be assumed that the appearance of pancreatic nodules constitutes an obligatory sequela of pancreatic hypertrophy and/or hyperplasia in all species of animals.     

Nutritional and antinutritional aspects of an Argentinian soy flour assessed on weanling rats

Inactivation of antinutritional factors on an Argentinian deffated soy flour was evaluated in relation to its corresponding original soybeans. Residual urease and trypsin inhibitors activities, protein solubility, available lysine and phytic acid showed acceptable levels. The nutritional value of a diet based on the same soy flour was compared with that of a casein-based diet by allowing them to each one of two groups of weanling IIMBeta rats during 28 days. Non-significant differences were obtained between groups comparing both body weight gain per gram of protein intake as well as the results of histometric studies performed on the small intestine. Thus, this soy flour contained appropriately inactivated antinutritional factors and its protein content and quality were nutritionally suitable. Large intestine hypertrophy was revealed within the soy-fed group suggesting increased microbial proliferation attributable to non-digestible oligosaccharides and fiber-like compounds. These compounds, employed by gut microflora as fermentation substrates, would be also responsible for an increased nitrogen excretion in the soy-fed group in relation to controls. This increment of nitrogen excretion significantly lowered the value of apparent nitrogen digestibility in the soy-fed group in relation to the casein-fed control one. Nevertheless, nitrogen apparent digestibility in the soy-fed group was equal or higher than other values reported from distinct treated soy meal-based diets.     

Raw soy and purified proteinase inhibitors induce the appearance of inhibitor-resistant trypsin and chymotrypsin activities in Wistar rat duodenal juice

Rats were fed raw soybeans or purified soybean proteinase inhibitors by tube to see whether they were able to produce inhibitor-resistant trypsin, as previously demonstrated in humans. Their duodenal chyme contained only 20-50% of the enzymatic activities of animals fed bovine serum albumin (BSA) as test protein. However, both tryptic and chymotryptic activities had considerable resistance to low- and high-molecular-weight inhibitors of serine proteinases. In particular, the tryptic activity demonstrated a high degree of inhibitor resistance. Human alpha 1-antitrypsin and lima bean inhibitor in amounts that inhibited bovine serum albumin-induced trypsin completely caused only 2-12% inhibition of the raw soybean-induced tryptic activity. The inhibitor-resistant tryptic and chymotryptic activities after raw soybean instillation might be caused by the Bowman-Birk and Kunitz trypsin inhibitors. The physiologic significance of an inhibitor-resistant trypsin might be to assure activation of other pancreatic proenzymes. The results of the present rat experiments confirm the previous findings of inhibitor-resistant trypsin in humans.     

Growth,protein utilization,and secretion of pancreatic enzymes by rats in response to elevated levels of dietary protein

Rats were fed diets containing 15, 30, 45, or 60% casein for 30 days. The weight gains of rats fed diet containing 30% casein were higher (P<0.05) than that of rats fed 15, 45 or 60% casein diets. Food intake was similar in the 15% and 30% casein diets and lower in the 45% and 60% casein diets. Blood plasma urea level increased in parallel to the increase in dietary protein and was inversely related to PER. Water intake increased with the increase in dietary protein level. The relative weights of the pancreas and kidneys increased with the increase in protein level as did the activities of the digestive enzymes. However, while trypsin and chymotrypsin activities were increased both in the pancreas and in the intestine, lipase was increased only in the intestine and amylase only in the pancreas. The response to elevated dietary protein, as shown by the levels of digestive enzymes excreted in the feces, was very marked in the first week and was smaller and inconsistent later on. The digestibility of casein was related to trypsin and chymotrypsin activities in the pancreas and small intestinal contents.     

Comparative inhibition of trypsins from several species by soybean trypsin inhibitors

Inhibition by soybean trypsin inhibitor (SBTI) preparations of trypsins and of total proteolytic activity from several species was compared. Rat, monkey, human, bovine, porcine and mink trypsins were all inhibited 90-100% by SBTI, although there were species differences at low SBTI concentrations. Extent of inhibition of trypsin from the various species did not correlate with SBTI-induced pancreatic enlargement. Total proteolytic activity was inhibited up to 40% in rat, monkey, and human preparations by SBTI, and inhibition curves were similar. SBTIs extracted from unheated soy protein isolate (USPI) or raw soy flour (RSF) were approximately equal in their ability to inhibit human trypsin. SBTI extracted from heated soy protein isolate (SPI) was slightly less effective in inhibiting human trypsin than SBTI from either RSF or USPI.     

Protein digestibility and quality in products containing antinutritional factors are adversely affected by old age in rats

The protein digestibility-corrected amino acid score (PDCAAS) has been recommended to be the most suitable method for routine evaluation of protein quality of foods by FAO/WHO. The PDCAAS method includes the use of young rats for predicting protein digestibility of foods for all ages including the elderly. To assess the usefulness of protein digestibility in old rats in the calculation of PDCAAS for the elderly, the influence of age on the digestibility of protein in 5-wk-old and 20-mo-old rats by the balance method was studied. Fifteen protein products were tested. Each protein product was fed as the sole source of 10% dietary protein. A protein-free diet was also included to obtain an estimate of metabolic fecal protein. Protein digestibility values (corrected for metabolic fecal protein loss) in old rats were significantly (P < 0.05) lower than in young rats for most products; however, these differences were small (up to 3%) for properly processed animal products (casein, whey protein concentrate, whey protein hydrolysate, lactalbumin and skim milk powder). Similarly, the differences attributed to age were not large (up to 5%) for properly processed vegetable protein products (soy protein isolate and autoclaved soybean meal, black beans and fava beans). However, digestibility values in old rats were considerably lower (7-17%) than in young rats when fed products containing antinutritional factors, that is, mustard flour containing glucosinolates; alkaline/heat-treated soy protein isolate and lactalbumin-containing lysinoalanine; raw soybean meal and black beans containing trypsin inhibitors; and heated skim milk powder containing Maillard compounds. Therefore, the inclusion of protein digestibility data obtained using young rats in the calculations of PDCAAS may overestimate protein digestibility and quality of these products for the elderly. For products specifically intended for the elderly, protein digestibility should be determined using old rats.     

Oral toxicity of Kintoki bean (Phaseolus vulgaris) lectin

There are several antinutritive factors in the Kintoki bean such as lectin, trypsin inhibitor, lack of methionine etc. The present experiment has revealed that lectin is mainly responsible for the growth impairment of experimental animals orally fed raw Kintoki bean. Mice fed raw Kintoki bean as the only protein source lost their body weight and died in 8 days, while mice fed the heated bean grew normally. When mice on a 10% albumin diet ingested 20 mg or 40 mg or 60 mg Kintoki bean lectin by daily stomach-feeding, their body weights were reduced to 84%, 74%, 71% of the control group after 5 days respectively and some of them could not live to complete the experiment. The apparent rates of the intestinal absorption of carbohydrate, lipid, and protein were considerably reduced, when rats were fed a diet containing 0.4% lectin. Especially, the rate of protein absorption was decreased to 26.3% from 55.5% of the control rate. The main tissues of mice that had ingested Kintoki bean lectin by stomach-feeding were subjected to microscopic observation. No changes were observed in the liver, kidney, spleen and pancreas. But in the small intestine, the epithelial cells lining the villi were considerably disordered and conspicuously disrupted. These results indicate that the Kintoki bean lectin is one of the most promoting factors for growth impairment in experimental animals and that the first target organ in the case of oral feeding is the small intestine.     

Rapid changes in pancreatic DNA, RNA and protein in the rat during pancreatic enlargement and involution

Pancreatic weight, nucleic acid content and protein content were measured on the involuting rat pancreas after its initial enlargement from a raw soy bean flour (RSF) diet. Rats were fed an RSF diet for four weeks and then changed to an inhibitor-free diet of either heated soy bean flour (HSF) or rat cubes for 1, 2, 7 or 14 days. Control rats were fed cubes continuously from weaning or cubes followed by HSF for two weeks. There was no significant difference in the body weights of rats in any of the test or control groups. After four weeks on RSF the wet weight of the pancreas was double that of the control animals and the protein content and nucleic acid content were also significantly greater. After RSF-fed animals were changed to cube or HSF diets, pancreatic parameters rapidly reverted to normal. The most striking change was in pancreatic DNA which returned to control values within 2 days of changing to cubes or HSF. Pancreatic weight, RNA and protein reverted to control values within 7 days of the change of diet. The rapid increase and decrease in the size of the rat pancreas after diet alterations suggest that pancreatic growth may be more labile than previously thought. If rapid changes in pancreatic cell number occur in rats fed alternating cube and RSF diets, then these animals may be more susceptible to pancreatic carcinogens.     

Effect of ingested winged bean lectin on gastrointestinal function in the rat

The present study was undertaken to provide further evidence for mechanisms proposed for the toxicity of ingested winged bean lectin in animals: to determine its effect on activities of some hydrolases localized in the brush border membrane of the small intestine. An adaptive increase in sucrase activity of rats given a high-sucrose diet (HSD) was restrained by the addition to HSD of a lectin fraction (WBLF) isolated from raw winged beans but not by that of heated WBLF or soybean trypsin inhibitor. Restraining effects of WBLF added to HSD on time-course changes in activities of sucrase, alkaline phosphatase and leucine aminopeptidase of rats after giving HSD were similar to those of concanavalin A, which had been observed in the previous study. These results substantiate that the mechanism of the toxicity of ingested winged bean lectin involves its binding to the luminal surface of the small intestine and in turn disturbing the functional formation of the brush border membrane.     

The effect of fasting on enzyme levels in the enlarged and involuting rat pancreas

1. The effect on pancreatic digestive enzyme levels of fasting and changes from a diet containing trypsin inhibitor (raw soya-bean flour, RSF) to diets free of trypsin inhibitor (heated soya-bean flour, HSF, or commercial rat chow) was studied in rats for up to 7 d. 2. In RSF-fed rats killed without fasting, enzyme levels were low, but after fasting for 24 h before killing there was a marked increase in all enzyme levels. Histological studies showed that pancreatic acinar cells from RSF-fed rats killed without fasting were devoid of zymogen granules, but following a 24 h fast there was a marked accumulation of zymogen granules which extend into the basal cytoplasm. Fasting either produced no change or a fall in enzyme levels in rats fasted after feeding HSF or chow continuously. 3. If animals fed on RSF were changed to HSF and either fed or fasted for 24 h up to the time of killing there was an increase in amylase (EC 3.2.1.1), trypsin (EC 3.4.21.4), lipase (triacylglycerol lipase; EC 3.1.1.3) and protein 1 d after the change, followed by a fall over the next 6 d to levels similar to those seen in rats fed on HSF continuously. 4. Animals changed from RSF to chow showed similar effects as far as trypsin, lipase and protein were concerned, but amylase rose, to reach the level seen in rats fed on chow continuously (about ten times that seen in soya-bean-fed rats), after 2 d. 5. These results suggest that in the rats fed on RSF, pancreatic enzyme synthesis is rapid but secretion is equally rapid and intracellular enzyme levels are low. When these animals are fasted or changed to a diet free of trypsin inhibitor the rate of secretion falls but the high rate of synthesis continues for at least 24 h and enzymes accumulate in the pancreas. In studies of pancreatic enzyme levels in rats fed on trypsin inhibitor the extent of fasting before killing the animal is therefore an important variable. Such animals should probably not be fasted before study.     

Binding of navy bean (Phaseolus vulgaris) lectin to the intestinal cells of the rat and its effect on the absorption of glucose

The main objectives of this investigation were to study the binding of a lectin from navy beans with the epithelial cells of the rat intestine and to assess the effect of such binding on the ability of the intestine to absorb glucose. A Scatchard plot, based on the binding of 125I-labeled lectin to isolated intestinal epithelial cells, was used to calculate an association constant (Ka) of 15 x 10(6)M-1 and the number of binding sites per cell, 12 x 10(6). Metabolic studies were conducted over a period of 5 d on groups of rats fed raw or autoclaved navy bean flour and casein with or without the purified lectin. Growth, protein digestibility, biological value and net protein utilization were significantly lower in animals that had been fed raw navy bean flour or casein plus lectin than in control groups fed diets containing autoclaved navy bean flour or casein alone. Vascular perfusion was used to measure the rate of uptake of glucose by the intestines of rats that had received the various dietary treatments. The rate of absorption of [14C]glucose by intestines from rats fed raw navy bean flour or casein plus lectin was approximately one-half that of their counterparts fed the autoclaved flour or casein alone. These results provide evidence that the lectin, by virtue of its interference with intestinal absorption, is responsible, at least in part, for the nutritional inferiority of raw navy beans.     

Soy induces phase II enzymes but does not inhibit dimethylbenz[a]anthracene-induced carcinogenesis in female rats.

Isoflavones in soy may play a role in the prevention of cancer through their capacity to affect antioxidant or protective phase II enzyme activities. This study evaluated the effects of dietary isoflavone levels on the induction of antioxidant and phase II enzyme activities and inhibition of breast carcinogenesis. Female Sprague-Dawley rats (36 d) were fed one of four purified diets with casein, or with soy containing three levels of isoflavonoids (0.03, 0.4 or 0.81 mg/g diet; low, middle and high level of isoflavones, respectively). After 2 wk, enzyme activity was determined of rats (n = 6-7) from each diet group. Liver glutathione peroxidase and glutathione reductase activities, blood glutathione levels, kidney glutathione S-transferase and colon quinone reductase (QR) activities were greater in rats consuming the high isoflavone diet compared to rats consuming the casein diet. Kidney QR and liver, kidney, small intestine, and colon UDP-glucuronosyltransferase activities were greater in rats fed the high isoflavone diet compared to rats fed the casein and low-isoflavone diets. Liver and blood oxidized glutathione were lower in rats fed the high-isoflavone diet compared to those fed the low-isoflavone diet. A subset of rats (n = 86) was fed the purified diets for 2 wk and intubated with dimethylbenz[a]anthracene or peanut oil and palpated weekly for tumors. At 13 wk, there was an inverse relationship (R(2) = 0.911, P < 0.09) between tumor incidence and increasing isoflavone intake. These data support the mechanism of soy and soy isoflavones as antioxidant and phase II enzyme inducers, but not as tumor inhibitors.     

Effect of dietary raw soybean and soybean trypsin inhibitor on trypsin and chymotrypsin activities in the pancreas and in small intestinal juice of growing swine

Sixty-eight growing gilts with a 12 kg average initial weight were used in seven trials to study the effect of dietary raw soybean (Harosov) and SBTI (Kunitz soybean trypsin inhibitor) on pancreatic and small intestinal trypsin and chymotrypsin activities. A solvent-extracted, heated soybean meal (SBM) was used, cause reduced growth. Both a single-meal and continuous feeding of the raw soybean diet caused a decrease in the pancreatic trypsin and chymotrypsin activities. In contrast, to the rat and the chick, the pancreas of the pigs did not enlarge subsequent to consumption of the raw soybean or SBTI diets. Raw soybean feeding also resulted in an inhibition of the intestinal trypsin and chymotrypsin activities. This inhibiting effect was greater than that of the SBTI, especially the chymotrypsin-inhibiting effect. This suggested that soybean constituents other than the SBTI, such as the Bowman-Birk inhibitor, caused inhibition. In the pig the inhibition of the intestinal proteolysis may be a major cause of reduced growth when raw soybean is fed.     

Functional properties of raw and processed pigeonpea (Cajanus cajan) flour

Emulsification capacity, foam capacity, foam stability, fat and water absorption of raw and processed pigeonpea flour were investigated and compared with those of raw soy flour. The processing methods included soaking in water, soaking and blanching in water and soaking and blanching with trona, a sodium sesquicarbonate salt. Soaking in water was found to reduce the water absorption capacity of pigeonpea flour while soy flour had a slightly higher water absorption capacity than the other processed flours. All the processing treatments increased the fat absorption capacity of pigeonpea flour and the values were also higher than the value obtained for raw soy flour. Data on the emulsifying activities showed that the trona-treated pigeonpea flour had the highest emulsification capacity and was even higher than that of raw soy flour. Soy flour was found to have higher foaming capacity than all the pigeonpea flour samples.