J. Nutr. Tolerance to Long-Term in the Rat: Feeding Evidence Sci. Vitaminol., of Isolated Peanut for a Trophic on the Small 36, 599-607, Lectin Effect Intestines Laurie HENNEY,1 Esam M. ARMED,1 Donald E. GEORGE,2 Kuo J. KAO,3 and Harry S`. SITREN1,*1 Food Science and Human Nutrition Department, University of Florida, Gainesville, Florida 32611, USA 2Division of Gastroenterology and Nutrition , Nemours Children's Clinic, Jacksonville, Florida 32207, USA 3Department of Pathology , University of Florida, Gainesville, Florida 32610, USA (Received June 18, 1990) Summary raw Previously peanut enzyme meal activity effects of 0.04, diet and diets. changes mid, altered rats in days. the of distal with 23 Body small mucosal peanut the gain 0.2% of weight, protein, Of altered in normal. bean 0.2% of and is Key Words activity in These lectin the diet apparently any small 3 the does not trophic peanut are lectin, inhibit for all trophic to areas of agent, mucosa * To whom correspondence border should be sent 599 . with the rat the small proximal, showed marked but without studied, microvillus growth no study. enzymes that small three were under reported or all there fed 0.004, phosphatase, that the intake intestines, levels, diet, alkaline in the rats three contents, conclude food the all DNA suggesting We on normal lectin brush similar determine parameters and intestines. To lectin, intestines and the region, results on was peanut the at peanut mucosal regions morphology. and containing alterations Sprague-Dawley lectin 0.04% intestinal consuming growth male, weight and a diet organs. body purified fed exhibited certain weanling, 0.004 the third on in rats protein maltase, ƒÁ-glutamyltranspeptidase, kidney to any villus namely was fed in and increases was for rats in However, out that of of lectin carried 0.2% shown source composition incorporated In have sole peanut were a casein the and isolated experiments we as none integrity actions of peanut lectin of weanling red at up rats intestines. intestines, intestinal 1990 600 L. HENNEY Lectins in most vitro are and thus winged bean, intestinal (1-6). surface of are also and (3). jack is lectins Although is little a diet weeks as the or its as chemistries of chemical the reported it important Several by several of 4 to chemical apparently some In the are work, lectin mucosa and purified of is fed and of the to enzyme several changes. in in lectins, the rat. been 110,000, we reported is composed carbohydrate. It has (1-3) Peanut a high N-acetyl lectins from in which thymocytes. results levels 4 simi-larities have erythrocytes. and for the other lectin weight of of the lectin rats pancreas observed these peanut free and which these there (Arachis meal liver and peanut ABO on at various composition the of rats weanling disaccharide. ƒÀ-D-galactosyl lymphocytes report peanut (10), a molecular and raw known some of the for we was lectins villi, in 7). peanut that for of characterized, or of not lectin desialyzed mitogenic current peanut intestinal subunits, is properties has containing It agglutinates genotypes isolated It It (3, found from accounted some effects (11-13). receptors D-galactosamine. the biological identical for peanut study and investigators specificity of between 9). overgrowth necrosis affected been border malabsorp reported groundnut activities peanut toxicities composition thought raw be gastro brush bacterial also solely the and been in bean, experimental the and has raw enzyme (8, in to previously component and present may We to recently legumes components. blood due for of in activities cells kidney aspects binding found blood red death Ulceration organs different from enzyme has tolerance protein lectin (2). commonly red cause malabsorption several composition factors of submucosa, the the altered antinutritional Because of cases altered Extraintestinal antinutritional altered to are agglutinate different some a result of the toxicity containing be that to disrupt in interaction concerning showed well to evidence (6). information hypogea) fed also able Lectins to and leading reaching bean known lectin-mucosa lesions ingesting are appears proteins are hemagglutinins. growth, intestines the lectins as bean Toxicity There occurring Many poor small from including naturally known cause the resulting of foods. tract, animals tion a group leguminous et al. of two weanling experiments rats to assess growth and activity. EXPERIMENTAL Isolation and was isolated and described variety was and were in Mo., The hydraulic ground, sulfate, raw and dialyzed, of 0.08%. absorption The Purity spectra, appropriate cyanogen was assessed and subunit by peanuts gel were dialyzed weight et the and Sepharose St. lyophilized. These Yield coefficient, properties J. Nutr. (12) Protein affinity Chemical, extinction (14). feeding al. Florunner saline. by activated electrophoresis, molecular for with purified bromide used Lotan of extracted (Sigma fractions lectin from and N-E-aminocaproyl-ƒÀ-D-galactopyranosylamine USA). peanut adapted Briefly, press, consisted The procedures (14). ammonium column lectin. to elsewhere by with of peanut according detail defatted precipitated matography. and purification purified chro 4B Louis, was UV were Sci. Vitaminol. INGESTED found to agree comparable closely to a #L-0881, lot assay utilizing fresh at rocyte for 15% 1% weeks mix to was the fed to lectin was second mass was the This level studying free casein the control as described is of for each compared 36, No. 6, 1990 was were assayed 3.2.1.20], ƒÁ-glutamyl [AP, EC group water were The 3 equal entire segments. formalin remainder of and the protein, each mucosa DNA, transpeptidase 3.1.3.11. level third in buffered for A This and scraped, of study. A into was amount present The everted, showed fasting. fixed found diet (0.04%). divided One we this Food evaluation. saline, the without and fed. This exposure. segment Homogenates the diets out which utilizing was t-test to at range 3, 5,18 high pair-fed experiment, by added the (2, this experiment from and [ƒÁ-GT, Methodologies lectin EC have lectin of 1 showed at 0.004 lectin and A lectin group. only been by other could possibly After 23 were 2,000ƒÊg/g various group a second Rats of control minimal 0.04%, concentration. a concentration employed 20). level the experiment peanut a higher was in lectins Because results containing lectin a magnitude group each corn protein 9). Thus, lectins. and This work, lectin/g sacrificed isolated of 0.9% [EC of legume diet. Within means saline. carried to other added was methionine, (0.004%). 10% in lectin were diet (8, diet 400ƒÊg histologic ice-cold Because ingestion diet casein formula were previous diet. , ad starch. lectin/g 40ƒÊg/g diet diet lectin In arrival casein (16,17). was casein rapidly ends phosphatase 2. from vitro eryth bitartrate, corn providing excessive rats subsequent alkaline experiment the was a without days, proximal with reported of of diet 23 maltase Experiment effects casein in ice-cold previously in Upon 0.2% function containing effects intestines of diet 40ƒÊg meal 15% The peanut pancreatic into a choline reason. concentration casein After rinsed and a the for activities 2.3.2.2], an a 0.75% utilized. fed the of peanut incorporation the from homogenized the were Agglu Agglutination of cellulose, of with and were 0.2% expense following raw at assess small the growth, the glutaraldehyde segment Vol. fed libitum. the and by (12). and 1% (15), the incorporated for lectin to piece estimated experiments. oil, concentrations containing ingested of diet for at dietary intake, was ad 1cm and diet selected (controls) provided 12) Peanut 0.lƒÊg/ml rats corn mix added selected contain utilized was cages the 8% mineral was casein food group was A a in found titer steel commencing Two was alterations (11, Chemical, approximately stainless sucrose, 3.5% 1. fed rats before Lectin concentration that workers erythrocytes Sprague-Dawley in 25% (15), 100%. Experiment group male casein, vitamin other (Sigma desialyzed of individually 1.5 purified starch A 601 (14). housed libitum type Weanling, were by product Hemagglutinin human concentration suspension LECTIN reported derived 82F-9575-1). a lectin Animals. they those commercially tinin, occurred with PEANUT investigators received days, fed (0.2%). the lectin affect food intake rats were treated , 1. data at p<0.05. were subjected to analysis of variance and the 602 L. HENNEY RESULTS Experiment that and with ingested 0.04% rats fed the the groups. group, 121g for mucosal also any did in a the 10% any the region into toxic raw the a casein response. peanut protein Table 1. Experiment 1: on small intestinal mucosa a Mean•}SE; no 1) significant purified any 23-day 125g not the levels the showed diet 40 or was Effects of feeding of rats. differences (p>0 the 400ƒÊg/g was previously among added were similar the control Small or intestinal DNA content maltase, ƒÁ=GT, compared abnormal Thus, resulted to found to levels no AP controls. of peanut in equivalent groups and with appearance cecum. at comparison for group. low and moderate .05) 123g protein, enzymes no in concentration which 0.04% was in gains was groups nor of weight weight, 2 treatment fashion at the in lectin studied study for differ intestines lower peanut parameter Weekly the Likewise, in blinded diet in and did small The which (controls). intestines. in of to over group, changes examination any diet 0.004% of diet gain (Table show incorporated measured DISCUSSION differences Weight region not casein no lectin-free composition Histologic villi the showed among within AND 1 Rats 0.004 et al. observed that growth of peanut lectin J. Nutr. or present induce within the lectin each Sci. area. Vitaminol. in INGESTED PEANUT LECTIN 603 retardation as well as other abnormalities (8, 9). The results of the present study show that the toxicity observed in the earlier study was apparently due not to the presence of lectins but to other antinutrients, probably protease inhibitors. In studies describing toxicity from other lectins, such as from red kidney bean, soybean, and black bean, the concentration of dietary lectin utilized was in the range of 0.2 to 5%. These levels are substantially higher than the concentrations we studied in this experiment. Therefore, a second experiment was carried out in which the amount of peanut lectin in the casein diet was increased to 0.2%. A higher concentration was not considered due to the low yield from the lengthy and time-consuming extraction procedure. Experiment 2 As was also observed in experiment 1, rats ingesting the lectin-containing diet showed no changes in food intake or growth rate. Weight gain was 127g during the 23-day feeding period while the control (lectin-free diet) group gained 128 g. However, unlike the results in experiment 1, there were a number of definite alterations in the composition of the mucosa in all three regions of the small intestines (Table 2). These changes generally involved mucosal weight, protein, and DNA rather than enzymes activities. In all 3 regions of the small intestines, rats ingesting the 0.2% peanut lectin diet showed an approximately 40% increase in mucosal weight and a 50 to 60% increase in protein. DNA increased by 24% in the proximal region, 39% in the mid-region, and 48% in the distal segment. As was also found in experiment 1, histologic evaluation by light microscopy revealed no consistent abnormal findings in villus structure in any of the regions. These results reveal that peanut lectin appears capable of inducing a mild hyperplasia of the small intestines. Mitogenicity of certain cell types, typically lymphocytic, has been reported for certain lectins (10, 21, 22). Recently, Tajiri et al. (23) reported for the first time that feeding of purified red kidney bean lectin at 0.1% of diet can stimulate rat small intestinal mucosal DNA synthesis and crypt cell division. In that study, rats were examined at periods ranging from 1 to 6 days after commencing the diet. At 6 days, the mucosa from the proximal bowel showed increases in weight of 43%, in protein of 32%, and in DNA of 50%. Mucosal thickness and villus height were only slightly altered. Sucrase and enterokinase activities was markedly diminished but leucine aminopeptidase was unchanged. More pronounced changes in these parameters occurred during earlier sampling times, suggesting that animals recover or adapt to some degree. Our results generally support and extend the findings of Tajiri et al. (23) that lectins may be trophic for the small intestines. At low doses (0.004 and 0.04%), as used in experiment 1, no effects on the intestinal mucosa were observed. However, at a higher dose (0.2%), there was a pronounced enhancement of mucosal weight, protein, and DNA throughout the length of the small intestines. This was found even after 23 days of feeding, a duration expected to allow full adaptation. In the Vol. 36, No. 6, 1990 604 L. HENNEY Table 2. intestinal a Mean•}SE Experiment mucosa . 2: Effects of feeding et al. a high level of peanut lectin on small of rats. * Significantly different at p_??_0.05 within each area. work of Tajiri et al. (23), the distal small intestines showed no increase in DNA content whereas we found a trophic effect in all areas of the small bowel. These workers suggest that red kidney bean lectin may become inactivated during passage down the intestines so that there is insufficient material remaining to interact with the distal region. If true, then this suggests that in our work, peanut lectin is either more resistant to luminal inactivation or the amount ingested exceeded any possible inactivation mechanisms. The latter is unlikely since the hyperplastic response was greatest in the distal area. Alternately, peanut lectin may bind more strongly to the distal region and thereby exert a more pronounced trophic effect. The mechanism by which peanut lectin appears to induce a trophic effect is unknown. The mitogenic effect on enterocytes from kidney bean lectin is thought to occur via attachment to specific carbohydrate binding sites of crypt cells which then leads to an increased proliferation rate of the crypt cells (23). In our study, none of the three brush border enzymes representing a disaccha ridase, a peptidase, and a phosphatase, deviated from normal activity even in rats fed 0.2% dietary peanut lectin. This suggests that no microvillus damage occurred. It is possible, however, that enzyme activities were lower at some point in the J. Nutr. Sci. Vitaminol. INGESTED PEANUT LECTIN 605 experiment but then recovered to normal levels by the end of the 23-day feeding period. One might predict that enzyme activities would rise in support of the observed increases in mucosal weight, protein, and DNA. However, hyperplasia can occur in the crypt region rather than the villus, as was previously reported to result from feeding red kidney bean lectin (23). Inasmuch as brush border enzymes are immature on the crypt cells, no increase in microvillus enzyme activity would occur. In any event, it is probably that, notwithstanding the different specificities of the lectins to bind to receptors on the villus-crypt surface (24), the response to lectin ingestion is dependent, in large part, on the amount ingested and on the length of the feeding period. In the work of Tajiri et al. (23), we estimate that daily lectin ingestion amounted to approximately 15-20mg for 6 days. This intake was not toxic and was mitogenic for the proximal small intestines without increasing brush border enzyme activities. Acute challenge by gastric lavage from 300mg of raw kidney bean [calculated to contain about 10.5mg lectin, assuming a lectin concentration of about 3.5% (25)], results in damage to the microvillus but not to other villus structures (20). Moreover, the damage became repaired within 20h. This suggests that the enterocyte brush border may be repeatedly damaged and then repaired after ingestion of red kidney beans. Banwell et al. (2) fed a casein diet adulterated with 0.5% red kidney bean lectin to weanling rats for up to 3 weeks. Food intake was reduced to about one-third normal and rats lost weight. The average amount of lectin ingested was approximately 19mg/day, similar to the intake reported by Tajiri et al. (23). Specific activities of several disaccharidase enzymes were markedly reduced in the proximal small intestine but not in the distal region when compared with pair-fed controls. Further, the histologic appearance evaluated by both light and electron microscopy was normal. The lack of any observable morphologic changes, even of the microvilli, may have been due to the marked diminution of food intake, thereby reducing lectin intake and allowing rapid repair of microvilli (20). Rouanet et al. (19) reported on intestinal changes in rats fed a semi-purified diet containing 0.25% kidney bean lectin for 17 days. Compared with pair-fed controls, lectin-fed rats showed no significant change in jejunal villus length but crypt depth significantly increased. Mucosal protein increased by 60% but activities of sucrase and -glutamyl transpeptidase were unchanged. These results are consistent with those of Tajiri et al. (23) as well as our findings concerning enzyme activities. In summary, the feeding of isolated peanut lectin to weanling rats at low to moderate levels is without effect on growth and small intestinal morphology and biochemistries, whereas at a high dietary concentration of 0.2%, growth remains normal but the small intestine shows a hyperplastic response. The Statz, authors are grateful and Juanita This publication DAN-4049G-SS-2065-00. Vol. 36, No. 6, 1990 for the able technical assistance of John Applewhite, Rebecca Bagnall. was partially supported Recommendations by the Peanut do not CRSP, represent U. S. AID an official grant position number or policy 606 L. HENNEY of U. S. AID. 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