caused by protein in diets (independently of trypsin inhibitor activity) used highly purified phaseolin and did not find pancreatic hypertrophy in rats fed with this ...
Alim. Nutr., Araraquara v. 16, n. 1, p. 5-10, jan./mar. 2005
ISSN 0103-4235
PANCREATIC HYPERTROPHY IN RATS CAUSED BY CHICKPEA (Cicer arietinum L.) PROTEIN INTAKE* Olga Luisa TAVANO** Sinézio Inácio da SILVA JÚNIOR** Aureluce DEMONTE** Valdir Augusto NEVES** ABSTRACT: The objectives of this work were demonstrate the occurrence of pancreatic hypertrophy in rats, caused by chickpea protein intake, and the possible relation to the presence of trypsin inhibitors in the protein samples. The principal protein fractions of chickpea were isolated, the effect of heating was also tested (121°C/15 min). The heated chickpea diets did not cause significant pancreatic hypertrophy in rats, in relation to the casein control group. Only unheated chickpea flour and albumin diets caused pancreatic weight increases correlating to the presence of trypsin inhibitors in these samples. Apart from the trypsin inhibitor activity the other chickpea protein components appear not to exert any alteration in pancreatic weight. KEYWORDS: Pancreatic hypertrophy; pancreatic hyperplasy; chickpea; trypsin inhibitors; protein fractions.
Chickpea contains low levels of trypsin inhibitors compared with other crops2,4,5,16. Paiva16 shows values between 36.0 and 63.0 TIU/mg flour in ten varieties of beans and a variation from 37.0 to 132.0 TIU/mg flour sample in eight soybean varieties. Chickpea IAC-Marrocos seeds presented 8.26 TIU/mg sample, and chickpea unheated flour 9.68 TIU/mg sample, supporting the results of Sotelo et al.25 which report a variation from 9.0 to 15.7 TIU/mg flour samples in nine Mexican varieties of chickpea. However, Avancini et al.2 reported 5.57 TIU/mg flour in IAC-Marrocos chickpea. The aim of this work was to study the occurrence of pancreatic hypertrophy in rats caused by intake of chickpea flour and its protein fractions, verifying the possible relation to trypsin inhibitor activities or its correlation with behavior of the chickpea protein; therefore, the principal protein fractions of chickpea were studied separately as well as in its native and heated (121C°/15 min) forms.
Introduction
Material and methods
The presence of antinutritional compounds in legumes, for example trypsin inhibitors, has been associated with abnormal increase in rat pancreas3,5,6,7,12,15,17,19,20,23. The mechanisms by which the trypsin inhibitors cause pancreatic hypertrophy have been demonstrated10,11 . However, some authors suggest that this pancreas alteration can be caused by protein components, independently of trypsin inhibition activities. Liener12 suggests that only 40% of pancreatic hypertrophy and low growth response in rats can be attributed to trypsin inhibitors, considering that other proteins could complex with trypsin causing its inhibition. Santoro et al.20 studying the possible alterations in rat organs caused by protein in diets (independently of trypsin inhibitor activity) used highly purified phaseolin and did not find pancreatic hypertrophy in rats fed with this phaseolin diet in relation to lactalbumin control diet. Rubio et al.19 did not find differences between the pancreas weight of the rats fed with the control diet (lactalbumin) and unheated flour and globulin fraction of chickpea, in contrast with rats fed with fava beans that presented hypertrophy.
Material Chickpea seeds (Cicer arietinum L.) cv. IACMarrocos, were purchased from Instituto Agronômico de Campinas, São Paulo, Brazil. Methods 1. Flour preparation The seeds were soaked in distilled water (4°C/12 h), manually decorticated and powdered to 60-mesh size. The flour was defatted with hexane and used for all extractions and analysis. 2. Isolation of protein fractions Albumin, total globulin, prolamin and glutelin protein fractions were extracted from defatted flour as described by Sathe & Salunkhe22, with some modifications.
*Trabalho elaborado com o auxílio financeiro do Programa de Apoio ao Desenvolvimento Científico da Faculdade de Ciências Farmacêuticas da UNESP e bolsa concedida pela CAPES. **Departamento de Alimentos e Nutrição - Faculdade de Ciências Farmacêuticas - UNESP -14801-902 - Araraquara - SP-Brasil.
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The flour sample was extracted twice with 0.5M NaCl solution (1:10 w/v) by shaking for 1h/4°C and centrifuged at 9000g for 60min. The combined supernatants, containing salt soluble proteins, were save and the residue was extracted with 70% ethanol and reextracted twice with 0.1N NaOH solution to separate prolamin and glutelin fraction, respectively, by shaking for 1h/4°C and centrifuged at 9000g for 60min. Salt soluble proteins, in the supernatant, were separated in albumin and globulin fractions by extensive dialysis against distilled water. Albumins stayed in the supernatant and the globulins were obtained as precipitate. The major globulin was obtained from total globulin precipitate as described by Kumar & Venkataraman9. The total globulin sample was dissolved in 10% NaCl (1:20 w/v) and this solution was diluted 10 times with distilled water. After centrifugation (5000g x 30min) the supernatant containing vicilin-type globulin was separated and the precipitate was again dissolved in 10% NaCl (1:20 w/v) and diluted 20 times with distilled water. The major globulin (legumin-type) precipitate was collected by centrifugation (5000g x 30min). The globulin fractions precipitated, containing salt residues, were resuspended in water and this suspension and the glutelin extract, containing NaOH, were dialyzed against distilled water. All extracts were lyophilized.
7. Biological trials (diets and feedings) Male weanling rats, Wistar, weighting 50 ± 3.0g, were fed with standardized laboratory rat chow for an acclimation period of 2 days. After this period, the animals were randomly divided into 10 experimental groups with 8 rats in each. They were housed in individual metabolic cages in a care room at 24 ± 1°C, 50-60% relative humidity, with 12 hours light/dark cycle. The rats were fed, for 14 days period, with modified diets formulated according to the AIN93 diet for growth, as described by Reeves et al.18. The diets were composed of 8% test protein, 10% sucrose, 7% fat, 1,0% vitamin mix, 3,5% mineral mix, 5% fiber, 0,25% choline bitartrate and cornstarch to make up to 100%. The test protein sources of chickpea were: unheated and heated samples of flour, albumin and total globulin protein fractions, and heated samples of major globulin and glutelin fractions. Casein (Sigma C-7078) was used as a control. Feed and water were provided ad libitum. 8. Organ weights After 14 days the rats were killed by chloroform overdose and the organs (pancreas, liver, spleen, stomach, heart, caecum and kidneys) immediately removed, immersed in 0.9% NaCl, dried on filter paper and weighted. The results were expressed in g/100 g animal body weight.
3. Heat treatment 9. Statistical analysis A portion of defatted flour and protein fractions were suspended in distilled water (1:6 w/v), autoclaved at 121°C for 15 minutes then immediately cooled, frozen and lyophilized. 4. Nitrogen determination Nitrogen was determined by the micro-kjeldahl method1. Protein content was calculated as N x 6.25 for chickpea proteins and N x 6.38 for casein samples. Lowry et al.13 method was also used in some determinations. 5. Chemical composition Moisture, fat and ash content in chickpea seeds and flours were determined by AOAC methods1. Protein was determined by the micro-kjeldahl method as described above. Crude fiber content was estimated as acid detergent fiber fraction. Carbohydrate was estimated by difference. 6. Analysis of the trypsin inhibitors contents Trypsin inhibitors were measured as described by Kakade et al.8, using BAPNA as substrate (TIU = Trypsin inhibited unit).
Analysis of variance were used in results comparison (p 0.05).
Results and discussion The total protein content of chickpea seeds and decorticated and defatted chickpea flour was 17.62 ± 0.90 and 25.01 ± 0.42 %, respectively. These results are in accordance with Singh et al.24 that found a range between 16.1% to 30.3% for protein contents of chickpea grown at different locations. Avancini et al. 2 found 18.77% protein content in the same chickpea variety (IACMarrocos). Like other legumes, the globulin fraction was the major constituent of chickpea flour protein, followed by albumins, glutelins and prolamins (Table 1). Major globulins were about 80% of total globulins. All of the samples were studied in heated form, however, the two more expressive fractions were also tested in their native form: total globulins and albumins (representing about 66% of total protein), and the chickpea flour (total protein). Due to the very low prolamin percentages this was not studied in this work. Table 2 shows the trypsin inhibitor activities in chickpea flour and protein fractions.
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Table 1 - Composition of chickpea protein (Cicer arietinum L.), var. IAC Marrocos.
Protein Protein fractions
% Flour*
% Total Protein
Albumins
4.66
0.80
18.63
3.19
Total Globulin
11.96
0.93
47.85
3.74
Major Globulin
10.63
1.18
42.52
4.72
Prolamin
0.04
0.01
0.17
0.02
Glutelin
2.50
0.86
10.02
3.43
* Decorticated and defatted flour containing 25.01% of total protein. Values are means sd, n=3
Table 2 - Trypsin inhibitor activity in unheated and heated (121°C/15min) chickpea seeds and protein fractions. Residual activity, after Sample TIU*/mg sample TIU*/mg protein heating (%)
Seeds Unheated flour Heated flour* Unheated albumin Heated albumin Unheated total globulin Heated total globulin Unheated major globulin Heated major globulin Unheated glutelin Heated glutelin
8.26 0.12 9.68 0.32 0.53 0.08 179.40 0.57 4.10 0.24 8.63 0.25 1.41 0.27 5.18 0.36 0.43 0.06 3.14 0.09 0.0
46.88 0.67 38.72 1.29 2.31 0.38 239.20 0.65 5.47 0.35 9.46 0.28 1.54 0.29 5.32 0.36 0.54 0.06 4.58 0.13 0.0
5.97 % 2.29 % 16.28% 10.15% 0%
*TIU = trypsin inhibited unit. Values are means sd, n=3.
Unheated albumin showed the greatest values of TIU (239.20 TIU/mg protein), as expected, since trypsin inhibitors are always present in albumin fractions. The other fractions also presented inhibitory activities. Sastry & Murray21 found trypsin inhibitor activities in the globulin fraction of chickpea, and Marquez & Lajolo 14 found activities in globulin and glutelin fractions of Phaseolus vulgaris L. The thermal treatment (121°C/15min) was efficient in reducing trypsin inhibitor activities for all samples. This activity was more drastically reduced in the chickpea flour and the albumin fraction; nevertheless, only the glutelin fraction did not present activity after heating (Table 2). This observation, about the percentage of inhibitor inactivation in these samples, is in accordance with the observed by Ellenrieder et al.4 that suggest that the presence of other components in the sample accelerate the inhibitor inactivation by heating. That is, those chickpea protein samples that present a higher percentage of other non protein substances showed greater inactivation. Table 3 shows the components used to test diet formulations.
The pancreas, liver, spleen, stomach, heart, caecum and kidney weights are in Table 4. They have been corrected to g/100 g of body weight for ease of comparison. The pancreas weights of rats fed with unheated chickpea flour and unheated albumin chickpea diets were significantly heavier than of those rats fed with a casein diet, showing a weight increase of around 47% and 46%, respectively. After heating, the protein fractions and chickpea flour did not cause pancreatic hypertrophy in the rats compared with the casein reference diet. This suggests that there was no heat-stable component in the chickpea samples that could cause pancreas alteration. These results are according to data presented in Table 2, which show a positive correlation between the trypsin inhibitor contents and the pancreatic weight. Moreover, it can be observed that, although the trypsin inhibitor activity in unheated albumin samples was about six times higher than in unheated chickpea flour samples (239,20 UIT/mg protein in albumin fraction and 38,72 UIT/mg protein in unheated flour sample), the rat pancreas weight of these two groups were not different.
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Table 3 - Components of experimental diets.
Experimental diet1
Protein source2
Soybean oil
Fiber
(g / kg diet) CAS
96.46
70.0
50.0
FC
339.15
68.56
44.98
FA
352.42
68.48
44.71
AC
117.90
70.0
50.0
AA
116.99
70.0
50.0
GTC
84.63
70.0
50.0
GTA
86.91
70.0
50.0
GPA
91.44
70.0
50.0
GLUT A
124.64
70.0
50.0
1
CAS (casein control), FC (unheated chickpea flour), FA (autoclaved chickpea flour), AC (unheated albumin), AA (autoclaved albumin), GTC (unheated total globulin), GTA (autoclaved total globulin), GPA (autoclaved major globulin), A Glut (autoclaved glutelin). The autoclaved samples were maintaining at 121°C for 15 minutes. Each diet also contained: sucrose, 10g; AIN-93 mineral mixture, 35g; AIN-93 vitamin mixture, 10g; choline bitartarate, 2.5g; and cornstarch to make up 1kg. 2 Equivalent masses to 80 g protein.
Table 4 - Organs weight of rats fed with chickpea proteins.
Diet1 CAS FC FA AC AA GTC GTA GPA GLUT A
Pancreas 0,3918a 0,0517 0,5772b 0,0725 0,4064a 0,0712 0,5726b 0,0787 0,3944a 0,0831 0,3644a 0,0495 0,3932a 0,0464 0,4379a 0,0566 0,4379a 0,0642
Liver 5,6458ac 0,6423 5,0918ab 0,3493 5,1673ab 0,4238 5,3413ab 0,5229 5,6107abc 1,3374 5,2021ab 0,4517 4,8879b 0,2908 5,9273abc 0,8410 6,1859c 0,6642
Organs weight (g/100g animal body weight) Kidneys Spleen Heart 1,0122a 0,2935a 0,5513a 0,0674 0,0220 0,0423 1,6226b 0,2681a 0,5959bc 0,0968 0,0196 0,0384 1,1746c 0,2715a 0,5414a 0,0581 0,0278 0,0495 1,5716be 0,3046a 0,5251a 0,1459 0,0563 0,0460 1,1837acd 0,2714a 0,5552a 0,2240 0,0430 0,0585 1,3547de 0,2765a 0,5340ab 0,0869 0,0158 0,0546 1,3424de 0,2814 a 0,6141c 0,0580 0,0315 0,0233 1,4377ef 0,2507a 0,5514ab 0,1222 0,0264 0,0375 1,3118df 0,2566a 0,5357a 0,0680 0,0248 0,0319
Cecum 0,2515a 0,0257 0,4507bc 0,0990 0,3951bc 0,0732 0,5049b 0,0930 0,4739b 0,0453 0,3634cd 0,0767 0,3886c 0,0604 0,2961ad 0,0536 0,3812c 0,0522
Stomach 0,7027ad 0,1068 0,8240ab 0,0764 0,7430a 0,0531 0,9314bc 0,1412 0,9856c 0,0405 0,6759d 0,0348 0,8008ab 0,0656 0,6237d 0,0626 0,6930d 0,0347
Values are means sd, n=8, and values with different superscripts are significantly different (p 0.05). 1CAS (casein control), FC (unheated chickpea flour), FA (autoclaved chickpea flour), AC (unheated albumin fraction), AA (autoclaved albumin), GTC (unheated total globulin), GTA (autoclaved total globulin), GPA (autoclaved major globulin), A Glut (autoclaved glutelin). The autoclaved samples were maintaining at 121°C for 15 minutes.
Apart from the results found in the chickpea flour and albumin fraction, Rackis et al.17 found an increase of the pancreas weight (0,70g pancreas weight/ 100g body weight), in comparison with the casein control, when rats were fed with diet rich in trypsin inhibitor (88.2-96.6 UIT/ mg sample), that is, about 20000 UIT/g diet or more. In this work the unheated chickpea albumin diet presented about 19000 UIT/g diet, and the unheated chickpea flour diet presented about 3 000 UIT/g diet. Therefore, the trypsin inhibitor activity found in this unheated flour diet should
not be able to cause rat pancreas hypertrophy. An explanation for this observation could be the presence of other heat-labile toxic and antinutritional components in the unheated flour sample. Their actions, in addition to trypsin inhibitor activity, could be causing these alterations. Sathyamoorthy et al.23 demonstrated that the increase of pancreas secretory activity might be caused not only by higher trypsin secretion, but also by an increase in the secretion of other enzymes, such as arginase, ornithine transcarbamylase, aspartate aminetransferase and alanine
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aminotransferase. It is worth remember that in unheated flour diet, it was necessary 339.15 g of flour sample/kg diet. Under this conditions about 75% of the flour sample is non protein compounds which are increased in the formulated diet. The effects of chickpea samples on the other rat organs apparently have no relation to trypsin inhibitor activity. The alterations observed in rat liver, kidneys, heart and stomach were not conclusive. The spleen weights of the rats fed with test diets were not significantly different from those fed with casein diets. The caecum weights of the rats fed with unheated and heated chickpea flour and albumins were particularly significantly greater than those of the control group, especially the albumin groups. This effect could be caused by the production of volatile components as a result of bacterial fermentation of oligosaccharides present in chickpeas.
Conclusion This study shows that consumption of cooked (121°C/ 15min) chickpea diets did not cause pancreas hypertrophy in rats. Only unheated flour and albumin diets caused pancreas weight increase. This indicated a relation to the presence of trypsin inhibitors in the samples. Except the trypsin inhibitor activity, other chickpea protein components do not appear to exert any alteration in pancreas weight.
TAVANO, O. L.; SILVA JUNIOR, S.I.; DEMONTE, A.; NEVES, V.A. Hipertrofia pancreática em ratos causada por ingestão de proteína de grão-de-bico (Cicer arietinum L.). Alim. Nutr., Araraquara, v. 16, n. 1, p. 5-10, jan./mar. 2005. RESUMO: Os objetivos do trabalho foram verificar a ocorrência de hipertrofia pancreática em ratos causados por ingestão de proteínas de grão-de-bico e sua possível relação à presença de inibidores de tripsina nas amostras protéicas. As principais frações protéicas do grão-debico foram isoladas e também verificado o efeito do aquecimento (121°C/15 min). As dietas contendo proteínas de grão-de-bico aquecidas não causaram aumento significativo no peso dos pancreas dos ratos em relação ao controle contendo caseína. Somente as dietas contendo farinha de grão-de-bico e fração albumina não aquecidas foram capazes de causar aumento de peso no pancreas correlacionando a presença de inibidores nas amostras. Exceto a atividade de inibidor de tripsina os outros componentes protéicos do grão-de-bico não parecem exercer qualquer alteração no peso do pancreas. PALAVRAS-CHAVE: Hipertrofia pancreática; hiperplasia pancreática; grão-de-bico; frações protéicas; inibidor de tripsina.
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