The influence of lectins isolated from pea and soyabean seeds on performance and pancreatic enzyme activity in rats *

Lectin extracts were isolated from pea (PSA) and soyabean (SBA) seeds. In agglutination tests with human erythrocytes, SBA lectins showed higher activity than PSA lectins. The highest haemagglutination activity was observed with blood type AB, 50% lower activity with types A and 0, and the lowest, with type B. The experiment was performed on 30 male Wistar rats fed ad libitum a control diet or the same diet supplemented with PSA or SBA at a rate of 15 mg/day/rat for 14 days. Supplementation with lectins had a positive effect on feed intake and body weight gain, which differed significantly in the PSA group and numerically in the SBA group. PSA decreased the amylase activity in pancreatic juice and significantly decreased plasma glucose concentrations. Ca, P, Na, K, and Cl levels in pancreatic juice were similar in all groups. In rats fed the diets with lectin extracts, total pancreatic trypsin activity was significantly lower than in the control group and numerically lower in pancreatic juice. The results indicate that the effect of agglutinins from pea and soyabean seeds on pancreatic enzyme activity does not correlate with their agglutination activity.


INTRODUCTION
Legume seeds, including soyabean, contain enzyme inhibitors, allergens, lectins and other naturally occurring substances that may influence diet intake, digestibility, absorption and metabolic processes in animals and humans.In animals, the effects of lectins depend on their origin, dose and duration of feeding (Rådberg et al., 2001;Li et al., 2003a,b).
Since some lectins are resistant to digestion, they remain in the digestive tract in the active form for a considerable time.Dietary lectins can interact with nutrients, enzymes, and microflora in the digestive tract and with epithelial cells and can affect metabolic processes upon absorption (Pusztai and Bardocz, 1999).Animals are exposed most often to soyabean lectins, which are present in soyabean meal in amounts ranging from 0.05 mg/g in normally toasted soyabean meal to 10-20 mg/g in raw seeds (Schulze et al., 1995), but also to pea lectins due to the relatively high proportion of pea in some pig diets.The content of pea lectin in raw pea seeds ranges from 0.1 to10 mg/g; Polish pea cultivars contain between 0.27 and 0.75 mg/g (Gelencsér et al., 2000) The destructive effect of high doses of lectins consists in their ability to bind to complementary carbohydrates present on the surface of enterocytes, for example in glycolipids and glycoproteins of the brush border membrane (Bardocz et al., 1999).As a result of enterocyte atrophy, not only is the nutrient absorption surface area reduced, but the transport of nutrients through the epithelium is impeded, which leads to inhibited growth of animals (Pusztai and Bardocz, 1999;Pusztai, 2000).Absorbed lectins may enter the blood stream and therefore affect metabolism in the pancreas and other internal organs.Finally, they can influence the secretion and activity of enzymes.At high doses, lectins can stimulate intestinal and pancreatic overgrowth (Jordinson et al., 1996;Herzig et al., 1997;Grant et al., 2000;Kelsall et al., 2002), diminish enzyme activity (protease, disaccharidase), disturb lipid absorption in the small intestine and modulate insulin, CCK and gastrin secretion (Liener, 1994;Bardocz et al., 1999).
At small doses, lectins can stimulate structural and functional maturation of the gut and pancreatic growth in suckling piglets and rats.This possibility of inducing gut maturation may lead to better adaptation of the piglets to weaning and to an improved growth rate and health status (Rådberg et al., 2001).
The effect of dietary soyabean and bean lectins has been intensively investigated.Only limited data concerning pea lectin is available, however, as only a few studies concerning this lectin have been reported in the literature.Over the past few years, interest in pea lectin has continued to grow.Pea seeds are relatively inexpensive and can substitute soyabean meal in animal nutrition.
In vitro, plant lectins can agglutinate human and animal red blood cells (RBC) (Grant et al., 1995).The strength of lectin adhesion to RBC is similar as to brush border membrane (Koninkx, 1995;Bardocz et al., 1999).The agglutination test is often used to compare the activity of lectins.
The aim of this study was to compare the effect of lectin extracts isolated from pea (PSA) and soyabean (SBA) seeds on the performance, pancreatic juice parameters, pancreatic trypsin activity and plasma amylase activity of growing rats.

Lectin extraction and agglutination test
Lectins were extracted from raw pea (Pisum sativum) and soyabean seeds (Glycine max.) according to Santiago et al. (1993).Lectin activity was estimated by the agglutination test (Liener, 1989) with human red blood cells (RBC) of groups 0, A, B and AB.

Animals and diets
Thirty male Wistar rats weighing 130-140 g were allocated into 3 groups and fed ad libitum a standard diet containing 154 g crude protein/kg (control group) or the same diet with 15 mg/day/rat of a preparation of pea seed agglutinin (PSA) or soyabean agglutinin (SBA) added to the daily ration.The composition of the diet used in the experiment is shown in Table 1.The animals were kept in individual cages in an air-conditioned room at 21-22°C and 12 h light-dark cycle.Feed intake and body weight were measured for 14 days.At the end of the experiment the rats were anaesthetized by an intraperitoneal injection of uretan (1.8 g/kg BW).The gut was dissected and a catheter introduced into the common pancreatic-bile duct.Pancreatic juice was collected for 1 h.Blood samples were taken from the heart and centrifuged at 4000 g for 10 min.The rats were sacrificed, the pancreas excised, and 2 g of pancreas were homogenized with 1 ml physiologic solution and centrifuged at 3000 g for 10 min.Pancreatic juice, blood plasma, and pancreatic supernatant were frozen at -20°C until analysis.

Analytical methods
The pancreatic juice was analysed for protein content by the method of Lowry et al. (1951), trypsin activity by the method Erlanger et al. (1961), α-amylase activity, by enzymatic methods using Pointe Scientific kits, Ca, Na, K and Cl content, by using Pointe Scientific reagent kits, and P content, by using Analco -GBG kits.Blood plasma was analysed for protein and glucose content by enzymatic methods using Alpha Diagnostics kits and α-amylase activity.The protein content and trypsin activity were measured in the pancreatic supernatant.

Statistical analysis
The results were subjected to one-way analysis of variance using Statgraphic Plus 5.1.software.

RESULTS
In the agglutination test with various types of human erythrocytes, the SBA extract showed higher activity than the PSA extract.The highest haemagglutination activity was observed in agglutination of type AB blood, 50% lower activity was recorded with type A and 0 blood, whereas the lowest activity was with type B blood (Table 2).Feed intake and body weight gain (BWG) were higher in animals fed diets containing PSA or SBA lectins than in controls, while feed and protein efficiency ratios did not differ (Table 3).The plasma protein concentrations in group SBA were significantly higher than in the control and PSA groups.The plasma glucose concentration in the PSA group was significantly lower than in the control and SBA groups.In animals from both groups fed lectin-supplemented diets, plasma amylase activity was similar as in animals from the control group (Table 4).In comparison with control rats, the relative weight of the pancreas and protein concentrations were not significantly different in rats fed the diets containing PSA or SBA.Pancreatic trypsin activity was lower in SBA-and PSA-fed rats than in the control group, with the latter difference being significant (P≤0.05)(Table 5).Amylase activity in pancreatic juice was higher in SBA-and lower in PSAfed rats in comparison with the control group.The protein, Ca, P, Na, K, and Cl concentrations and pancreatic juice trypsin activity were similar in all groups (Table 6).

DISCUSSION
In the present study, agglutination activity depended on the blood group and type of lectin.The SBA extract exhibited greater agglutination activity with various types of human erythrocytes than the PSA extract.The highest agglutination activity was found with blood type AB.This is in agreement with our earlier findings (Czerwiński et al., 2005).Also Koninkx (1995) and Bardocz et al. (1999) reported that the strength of SBA lectin adhesion to Caco-2 cells and to brush border membrane was greater than of PSA.
The anti-nutritional effects of agglutinin are dose-dependent.The dose used in the present study, 0.75 mg/g diet, was also used in our previous work (Leontowicz et al., 2000;Czerwiński et al., 2005).In an in vitro experiment (unpublished) it was established that 0.75 mg SBA and PSA preparation/g diet corresponded to 0.37 mg of pure lectins/g diet.The results of the current experiment indicate that adding this dose of lectins to the diet did not significantly affect the feed efficiency ratio or protein utilization.Li et al. (2003a,b) showed that doses from 0.1 to 1.2 mg SBA/g diet had no significant influence on rat performance, which agrees with results of Leontowicz et al. (2004), our previous study (Czerwiński et al., 2005), and the present results.Bardocz et al. (1999) reported that peanut agglutinin was detected in human blood a few minutes after peanut intake, indicating that absorbed lectins can immediately affect metabolism.According to Liener (1994) and Rådberg et al. (2001) lectins can modulate hormone secretion (insulin, CCK, gastrin).At high doses, lectin intake can lead to intestinal and pancreatic overgrowth (Jordinson et al., 1996;Herzig et al., 1997;Grant et al., 2000;Kelsall et al., 2002).Our results showed that the applied doses of PSA and SBA did not result in significant pancreatic overgrowth, which is in agreement with Li et al. (2003a,b) and our previous study (Czerwiński et al., 2005).
In the present study in rats fed diets with lectin extracts, trypsin activity in the pancreas expressed in U/pancreas was lower than in the control group, however, neither lectin significantly decreased trypsin activity in pancreatic juice.Many authors have shown that lectins interacting with brush border membrane can modulate CCK secretion by intestinal endocrine cells (Jordinson et al., 1996;Herzig et al., 1997;Li et al., 2003a,b).Moreover, absorbed lectins can immediately interact with pancreatic cells (Grant et al., 1997;Kelsall et al., 2002).Pancreatic activity can be stimulated in these two different ways.In the present study, SBA increased amylase activity in pancreatic juice, however, the glucose concentration and α-amylase activity in plasma did not change.In an in vitro experiment, Grant et al. (1997) found that kidney bean (Phaseolus vulgaris) agglutinin and SBA stimulated amylase secretion from the pancreas and that this stimulation was dose dependent.In the present study, supplementation of the diet with PSA decreased amylase activity in pancreatic juice and significantly decreased the serum glucose concentration, however, this lectin had no effect on α-amylase activity in plasma.Leontowicz et al. (2004) also found decreased amylase activity in pancreatic juice in rats fed diets supplemented with evening primrose agglutinin.Cl¯ ions are necessary for pancreatic amylase activity.The decrease of amylase activity in pancreatic juice in the PSA group was not accompanied by changes in electrolyte levels.
The effect of lectin on metabolism is not irreversible and eased in the course of time (Bardocz et al., 1999).The level of lectins in diets, especially diets rich in pea and soyabean products, depends on the manner of heat processing (Schulze et al., 1995).Morover, not only the dose of lectins, but also intervals between applications should be taken into consideration, which is a relevant issue applicable to animal as well as human nutrition.

CONCLUSIONS
It can be concluded that agglutinins from pea and soyabean seeds affect feed intake, body weight and enzyme activity.The effect of lectins on pancreatic enzyme activity does not correlate with their agglutination activity.

Table 2 .
Haemagglutination titre of human red blood cells by PSA and SBA extract 1 1the result are the lowest concentration of lectin (µg/ml), which showed agglutination

Table 3 .
The influence of PSA and SBA on performance (14 days) in rats

Table 4 .
The influence of PSA and SBA extracts on amylase activity, protein and glucose level in blood plasma in rats

Table 5 .
The influence of PSA and SBA extracts on pancreatic parameters in rats

Table 6 .
The influence of PSA and SBA extracts on pancreatic juice parameters in rats