Pattern of Fecal Endogenous Nitrogen Excretion in Rats Fed Leguminous Diets. Semiramis M. A. DOMENE, and Admar C. de OLIVEIRAI 1

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1 J. Nutr. Sci. Vitaminol., 42, 27-33, 1996 Pattern of Fecal Endogenous Nitrogen Excretion in Rats Fed Leguminous Diets Semiramis M. A. DOMENE, and Admar C. de OLIVEIRAI 1 Departamento de Alimentos e Tecnicas de Alimentos, Faculdade de Ciencias Medicas, Pontificia Universidade Catolica de Campinas, Campinas, SP, Brasil 1 Departamento de Planejamento Alimentar e Nutricao, Faculdade de Engenharia de Alimentos, Universidade Estadual de Campinas, Campinas, SP, Brasil (Received May 22, 1995) Summary The main objective of this investigation was to establish the pattern in relation to time of the rat fecal endogenous nitrogen excretion by continuous feeding of balanced diets containing common peas, cow peas or common beans as the protein sources (10% protein), labeled with atoms % of 15N-excess. Nitrogen of endogenous origin was measured by the isotope dilution method in a 6-day experiment. Fecal excretion of endogenous nitrogen of rats fed the leguminous diets was roughly twice that of rats fed the non-protein diet (88mg, 42mg), and the excretion of total fecal nitrogen did not differ among leguminous diets. From the third to the sixth day of the experiment, the endogenous nitrogen excretion, either as a percentage or quantity (mg), attained a statistically non different value (p>0.05). A common pattern of excre tion of fecal endogenous nitrogen as a function of time was expressed by a strongly negative (r<.95) power regression (y=a Ex-B) for the common pea, the cowpea or the common bean diets. Conversely, the excretion of dietary nitrogen did not show a common pattern as a function of time for all experimental diets. Key Words nitrogen excretion, leguminous diets, nitrogen-15, rat The determination of endogenous nitrogen in the feces and urine of animals used as biological models has been studied for a long time, and for many years a protein-free diet was used for this purpose (1). Subsequently, a new methodolog ical approach, i.e., the use of a low-protein diet, was developed to minimize the problems arising from the absence of nitrogen, e. g. lack of growth and the irregularity of the diet ingestion (2). Experimentally, information about the measurement of the obligatory excretion of nitrogen by the use of non-protein or low-protein diets pointed out inaccuracies (3-7). 27

2 28 S. M. A. DOMENE and A. C. de OLIVEIRA Leguminous seeds are a major protein source for the population of most developing countries, but the dynamics of the endogenous nitrogen excretion in man or animals fed with these seeds is not yet clear. It has been demonstrated that diets containing raw leguminous seeds stimulate endogenous nitrogen excretion of rats when compared with non-protein or casein diets. Even in the case of cooked seeds this effect was noted (6, 8, 9) which improved the former concept about the influence of raw legumes in the increase of fecal nitrogen of rats (10). In spite of the large quantity of information available concerning the effect of leguminous seeds on rat excretion of metabolic nitrogen, it is unknown how the excretion of fecal endogenous nitrogen occurs over time. In this investigation, the daily excretion of fecal endogenous nitrogen of growing Wistar rats fed balanced diets with 10% protein from cooked beans, cowpeas or peas, labeled with }0.142 atoms % of 15N-excess, was determined during a 6-day feeding period. The correlation and the regression equation between the fecal endogenous nitrogen and the elapsed time, which represented the pattern of excretion of the endogenous (corporal) nitrogen of the rat, was determined. EXPERIMENTAL Labeled protein sources. Leguminous seeds from pure cultivars of common bean (Phaseolus vulgaris, L., cv. Aroana-80), cowpea (Vigna unguiculata, L., cv. Pitiuba) and common pea (Pisum sativum, L., cv. Torta-de-Flor-Roxa) obtained from the Centro Nacional de Pesquisa de Arroz e Feijao, Empresa Brasileira de Pesquisa Agropecuaria, Goiania, Goias, and the Instituto Agronomico de Campi nas, Campinas, Sao Paulo, Brasil were used in this study. The seeds were planted in experimental plots at the Centro de Energia Nuclear na Agricultura, Universid ade de Sao Paulo, Piracicaba, Sao Paulo, Brasil, cultivated with added (15NH4)2SO4 enriched with 10 atoms % of 15N, was subjected to common agricultural proce dures. At harvest, the leguminous seeds had }0.142 atoms % of 15N-excess After selection, washing and soaking in tap water for 12h, the seeds of the common bean and cowpea were cooked under pressure (14.7 psi) for 20 and 15min,. respectively, and the common pea was cooked in boiling water for 20min at atmospheric pressure. The cooked seeds were freeze-dried and ground to a 40-mesh size, in order to prepare the experimental diets. Diets. The experimental diets were prepared isoproteic by containing 10.7 } 0.4% crude protein (N ~6.25), 8% lipids (11) consisting of the legume lipids plus soy oil, 4.0% salt mixture (12) and 2% vitamin diet fortification mixture (13); the diets also contained 3.2 }0.7% of natural seed crude fiber. All diets were completed to 100% with a mixture of cornstarch and sucrose at a ratio of 3:1 were isocaloric (365.2 }19.3kcal/100g). A non-protein diet was also used, in which the protein was replaced by the same cornstarch and sucrose mixture used in the other diets and 3% of cellulose fiber (Microcel was added for comparative purposes., and, Blanver, pharmaceutical grade) J. Nutr. Sci. Vitaminol.

3 Nitrogen Excretion and Leguminous Diets 29 Biological assay. Twenty-four weaning male Wistar rats weighing 82.7 }6.4 g at the start of the trial, were randomly distributed in 4 groups of six per treatment, with the same weight mean. The rats were maintained in individual metabolic cages, with temperature control (22 }2 Ž) and alternate cycles of light and dark (12h) and received diets and water ad libitum. Prior to the 6-day experimental feeding period, the rats were fasted for 24h to reduce the content of their intestinal tract (14) in order to minimize contamination with unlabeled feed residues. Feces were collected daily and analysed for total nitrogen and nitrogen-15. The nitrogen of endogenous origin was determined by the isotopic dilution method (15), taking into account the 15N in the feces, in the diets and the natural-15n (9). Analytical methods. Nitrogen-15. Nitrogen-15 in the protein sources, diets and feces of rats was determined by the procedure recommended by Bremner (16), using a Varian Matt-230 mass spectrometer. Statistical treatment. The experimental design used was the "Completely Random Design" (17). Comparison between means was effected by the analysis of variance (F-test) and Duncan's test (18) at the 5% level. In order to express fecal endogenous nitrogen excretion during the time course, the best Pearsons's correla tion coefficient (r) was chosen amongst the power (y=a xb), exponential (y =A E ebx), logarithmic (y=a+b lnx) and linear (y=a+bx) equations. RESULTS The quantities (mg) of total fecal nitrogen, as well as the percentages and quantities (mg) of fecal endogenous nitrogen excreted by the rats fed on the different diets during the 6-day experimental period, obtained by isotopic dilution or by the use of a non-protein diet, are shown in Table 1. It is interesting to note that total fecal nitrogen excretion of the three protein sources showed no significant difference (p>0.05), and that the fecal excretion of endogenous nitrogen of rats fed the leguminous diets was roughly twice than that of rats fed the non-protein diet. Table 1. Total fecal nitrogen and fecal nitrogen of endogenous origin excreted by Wistar rats previously fasted for 24h and fed diets containing common peas, cowpeas, or common beans labeled with 15N as the protein sources, and non-protein diet, for a 6 day feeding experiment. Values are the mean and standard deviation of 6 rats. Values not sharing a common superscript are significantly different (p<0.05) by Duncan's multiple range test. 1 Determined by 15N isotopic dilution except for rats fed non-protein diet. 2M }SD. Vol. 42, No. 1, 1996

4 30 S. M. A. DOMENE and A. C. de OLIVEIRA Fig. 1. Time course of percentages and quantities (mg) of fecal endogenous or dietary nitrogen excreted by rats fed common pea (A and B), cowpea (C and D) or common bean (E and F) diets, labeled with 15N. Values represent the mean and standard deviation for six animals per group; * indicates p<0.05 by ANOVA. Power regressions (y=a Ex-B) express fecal endogenous nitrogen excretion per day. J. Nutr. Sci. Vitaminol.

5 Nitrogen Excretion and Leguminous Diets 31 There was a significant difference (p<0.05) between the percentage of endogenous nitrogen in the feces of rats fed the diet containing common pea and those fed the cowpea or common bean diets. The rats maintained on the common pea diet also excreted the largest quantity of nitrogen of endogenous origin, which differed from the excretion of those fed the cowpea diet. Nevertheless, the three quantities, in general, were similar. A common pattern of excretion of fecal endogenous nitrogen for the rats fed the different legume diets was observed, measured by the daily determination of nitrogen-15 in the feces. This pattern was expressed by a strong and negative (r<.95) power regression, which represents the correlation between either the percentage or the quantity of fecal endogenous nitrogen excreted and the days of the experiment, for all legume diets tested (Fig. 1): N (%)=59.9 [day].56 (r=.99) and N (mg)=24.5[day].44 (r=.98); N (%) = 52.1 [day].52 (r=.96) and N (mg)=26.5 [day].63 (r=0.97); N (%)=46.l E[day].50 (r=.97) and N (mg) =23.2 [day].45 (r=.96) for common pea, cowpea and common bean diets, respectively. Both percentages and quantities of endogenous nitrogen excreted decreased rapidly in the first two days of feeding and decreased slowly up to the third day. After this time, no significant differences (p>0.05) were found among the daily excretions. At the end of the experiment, the mean values of the daily excretions of fecal endogenous nitrogen per rat were 22, 19, 21% and 11, 9, 10mg, for the common pea, cowpea and common bean diets, respectively, while the quantities of nitrogen from dietary origin corresponded to 38, 33 and 38mg for the same protein sources. DISCUSSION This investigation showed that diets containing cooked leguminous seeds as protein sources stimulated twice as much fecal excretion of endogenous nitrogen by rats than the protein-free diet, and that the effect on total fecal nitrogen was quantitatively the same among the legumes, as indicated by the insignificant differences (p>0.05). The influence of beans, bean proteins or bean diets on the dry weight of feces, fecal nitrogen, secretion and excretion of endogenous nitrogen has been the subject of research by many investigators (6, 8, 9,15,19, 20). The power equations determined by regression analysis do in fact show the pattern of excretion of the rat corporal nitrogen. This excretion is obviously influenced by the nature of the dietary protein source, i.e., the adaptation of the rat to the diet. Thus, during the first and second days of the experiment, the 15N dilution was high, which resulted in high values for the percentages or quantities of fecal endogenous nitrogen. Nevertheless, these high values are also, to a lesser extent, due to the presence of unlabeled feed residues in the distal portion of the small intestine and in the large intestine of the rats, efen after fasting. This fact was previously observed by the authors in 24h-fasted rats. Indeed, the method for Vol. 42, No. 1, 1996

6 32 S. M. A. DOMENE and A. C. de OLIVEIRA quantification of fecal endogenous nitrogen used, makes the assumption that all unlabeled fecal nitrogen is of endogenous origin. From the third to the sixth day of the experiment, the fecal endogenous nitrogen excretion showed a small reduction, the values not differing significantly (p>0.05), taking into account the adaptation of the rats to the leguminous diets and the previous excretion of the unlabeled feed residues in the first and second days of the experiment. This observation indicates that the fecal excretion of endogenous nitrogen had already attained a steady state. Thus, the experimental period from the third to the sixth day appears to be appropriate for measuring the fecal endogenous nitrogen excretion of rats fed leguminous diets. Besides the fact that the rats had already addapted to the diets and the feces did not contain unlabeled feed residues, which was indicated by the acute fall in the values in the first and second days of the experiment, a 4-day period had already been demon strated to minimize the reutilization of 15N, i.e., marked endogenous nitrogen (21). Recently, Gaudichon et al. (22), studying [15N]leucine incorporation into gastro jejunal secreted proteins of fasting human beings, showed that plasma tracer enrichment stabilized after 2h of intravenous infusion. The authors recommended that post-absorptive endogenous nitrogen flow rates in the gut should be determined after 6-8h of [15N] leucine infusion, or after reaching the tracer plateau. The data obtained in this work indicate that nitrogen balance or growth assays with legume diets that need animal adaptation could be done at the third day of feeding, which reduces the time of the experiment and results in lower costs, especially in the case of labeled protein sources, which are expensive and difficult to obtain. The similarity of the parameters A and B among the negative power equations (y=a Ex-B) of the fecal endogenous nitrogen excretions, in percentage and quanti ty (mg), in relation to time (days), for all three diets (Fig. 1) implies that these excretions of nitrogen in rats fed leguminous diets can be explained by a common pair of power equations. This hypothesis, however, needs further investigation. REFERENCES 1) Mitchell, H. H. (1923/24): A method for determining the biological value of protein. J. Biol. Chem., 58, ) Mitchell, H. H., and Carman, G. G. (1926): The biological value of the nitrogen of mixtures of patent white flour and animal foods. J. Biol. Chem., 68, ) Bosshardt, D. K., and Barnes, R. H. (1946): The determination of metabolic fecal nitrogen and protein digestibility. J. Nutr., 31, ) Geiger, E., Human, L. E., and Middleton, M. J. (1958): Nitrogen content of gastrointestinal tracts of rats during absorptive period. Proc. Soc. Exp. Biol. Med., 97, ) Nasset, S., and Ju, J. S. (1961): Mixture of endogenous and exogenous protein in the alimentary tract. J. Nutr., 74, J. Nutr. Sci. Vitaminol.

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