Plasma Amino Acids and Excretion of Protein End Products by Mice Fed 10 or 40% Soybean Protein Diets with or without Dietary 2-Acetylaminofluorene or A/,/V-Dimtrosopiperazme1 PATRICIO HEVIA,2 C. RICHARD TRUEX, PETER B. IMREY, STEVEN K. CLINTON, HEATHER J. MANGIAN AND WILLARD J. VISEK3 University of Illinois College of Medicine at Urbana-Champaign, Urbana, IL 61801 Downloaded from jn.nutrition.org by guest on June 9, 2014 ABSTRACT Studies were conducted with 5- to 8-week-old male and female BeCaFi mice to determine the influence of two carcinogens, 2-acetylaminofluorene (AAF) and N,]V-dinitrosopiperazine (DNP), on plasma amino acid concentrations and on the excretion of lipids and nitrogenous metabolites. The carcinogens, AAF and DNP, were fed at concentrations of 0.25 and 0.05 g/kg of purified diet, respectively. Soybean protein constituted either 10 or 40% of the diet. Nutritional balances were measured over a 7-day period, after 7 days of acclimatization. Females ate less feed, gained less weight during acclimatization and excreted less fecal lipid as a percentage of intake than males. On the average, animals fed 40% protein consumed less total feed than those fed 10% protein. During acclimatization, DNP-fed animals ate and gained significantly less than controls. During week 2 DNP-fed animals gained signifi cantly less than controls, although their feed intake was not significantly different. Fecal lipid excretion as a percentage of intake was significantly lower with carcino gens in the diet. The 40% protein diets increased lipid excretion in total and as a percentage of intake. With the exception of decreasing fecal lipid, AAF caused no consistent changes in feed intake, body weight, nitrogen (N) retention or N excretion. Neither carcinogen significantly influenced total fecal or urinary N, or the relative concentrations of the different forms of urinary N, when expressed as a percentage of N intake Plasma ammonia rose with AAF feeding and plasma histidine rose with DNP feeding. Plasma concentrations of other amino acids were not changed consis tently by either carcinogen. Feeding 40% protein caused a significant rise in plasma branched-chain amino acids, glycine and phenylalanine, and a significant decline in aspartate, threonine, serine, proline, citrulline, lysine and arginine. J. Nutr. 114: 555-564, 1984. INDEXING KEY WORDS plasma amino acids â€¢mice â€¢2-acetylaminofluorene â€¢N,Af-dinitrosopiperazine â€¢soybean protein A large proportion of human cancers are at any of several steps (2). related to environmental factors (1). The Most experimental carcinogenesis models current outlook views cancer as resulting _ from the eluding interaction OÃ• multiple genetic predisposition, factors in- Â©19S4 American Institute of Nutrition. Received for publication chemical 2s,Ju1*' 1983 substances, radiation, infectious agents, and diet. Cancer . ... is a multistage ,., , PrOCeSS , fromi and nutrition may modify the sequence of events . 'Current address: Oepto. Procesos BiolÃ³gicosy BioquÃ-micos,Universidad SimÃ³nBolÃ-var,Caracas, Apartado Postal 80659, Venezuela. 555 ib whom reprint requests should besent. 556 HEVIA ET AL. MATERIALS AND METHODS B6C3Fi 5- to 6-week-old male and female mice, weighing from 17 to 23 g and 18 to 26 g, respectively, were randomly assigned4 to individual hanging wire cages so that ani mals of opposite sex occupied alternate posi tions in the rack. They were fed ad libitum a 20% soybean protein diet containing the American Institute of Nutrition recommended vitamin and mineral mixes (5) (table 1) for 5 days while adapting to their environment. The animals were then transferred to two 1Diet TABLE compositionIngredientsIsolated diet20.00.315.050.05.05.03.51.00.2100.0 of protein'DL-MethionineCornstarchSucroseFiber2Corn soybean oilMineral mix3Vitamin mix4Choline bitartrateTotal% 'Supro 620, Ralston Purina, St. Louis, MO. 2Solka Floe, Brown Co., Berlin, NH. 'Mineral mixture (grams/kilogram mixture): calcium phosphate, dibasic, 500.0; sodium chloride, 74.0; potassium citrate, monohydrate, 220.0; potassium sulfate, 52.0; magnesium ox ide, 24.0; manganous carbonate, 3.5; ferric citrate, 6.0; zinc carbonate, 1.6; cupric carbonate, 0.3; potassium iodate, 0.01; sodium selenite, 0.01; chromium potassium sulfate, 0.55; sucrose, powdered, 118.03. 'Vitamin mixture (in milligrams/kilogram mixture except as noted): thiamin â€¢ HC1, 600; riboflavin, 600; pyridoxine â€¢ HC1, 700; niacin, 3 g; calcium pantothenate, 1.6 g; folie acid, 200; biotin, 20; cyanocobalamin, 1; retinyl palmitate (500,000 lU/g), 800; d/-a-tocopheryl acetate (250 lU/g), 20 g; cholecalciferol (40,000,000 lU/g), 2.5; menaquinone, 5.0; sucrose, finely powdered, 972.9 g. racks of 24 identically arranged metabolism cages (No. AC5462, Acme Metal Products, Inc., Chicago, IL [name changed to Hazeltine]) with four males and four females assigned to each of the following six treat ments: 10% soybean protein, 10% soybean protein + AAF, 10% soybean protein + DNP, 40% soybean protein, 40% soybean protein + AAF, 40% soybean protein + DNP. The 10 and 40% soybean protein diets were iden tical to the basal diet, except that the pro tein was exchanged for sucrose on an equal weight basis. AAF (A-410-9, Aldrich Chemi cal Co., Milwaukee, WI) or DNP (1033, Frinton Laboratories, Vineland, NJ) was mixed into the complete diets by successive dilutions, to final concentrations of 0.25 and 0.05 g/kg, respectively. Dietary carcinogen concentrations (6; Perkins, E. G., Truex, C. R. & Visek, W. J., manuscript in prep aration), total N (7) and crude lipids (8) of *A formal sex-stratified randomized scheme, generated using the Statis tical Analysis System procedure PLAN (16), was employed in the initial caging of animals and maintained (with necessary adjustments due to transfers to and from metabolism cages) throughout the experiment. Downloaded from jn.nutrition.org by guest on June 9, 2014 utilize a small number of exposures to high doses of an initiating agent. Laboratory procedures, facilities and safety precautions necessary to chronically expose animals to low dietary doses of carcinogenic agents are expensive and time consuming. However, models utilizing such long-term low dose feeding of chemical carcinogens are much more analogous to human exposure and offer certain advantages for examining nu tritional influences on carcinogenesis. To understand how dietary protein may influence early stages of chemical carcino genesis, we are investigating the effects on nitrogen (N) metabolism of diets containing carcinogenic agents. JV,N-dinitrosopiperazine (DNP) and 2-acetylaminofluorene (AAF) represent two major classes of environmental carcinogens, the nitrosamines and aromatic amines, respectively. These carcinogens were fed for 2 weeks, at concentrations known to be carcinogenic (3, 4), to groups of male and female adult mice consuming diets differing in the level of protein. Plasma amino acids, fecal nitrogen excretion, and urinary urea, ammonia, creatinine, uric acid and allantoin were measured. The present studies were conducted to obtain information about the effects of car cinogenic agents on nutritional status and the influence of specific dietary treatments on the ability of mice to withstand these chemical insults. BeCsFi mice, used in these studies, show a low incidence of spontane ous tumors, are extensively employed for testing potential carcinogens and have been suggested as highly desirable animals for nutrition and cancer investigations. This report also includes some base-line nutri tional data on this mouse strain. CARCINOGENS AND NITROGEN METABOLISM tiple covariance analysis (15). The three major replicates were treated as statistical blocks, as were the two racks within each replicate Covariates representing horizontal and vertical positions within a rack were also incorporated into the analysis. This was done to account for environmentally related differences associated with cage position that were not fully equalized across diets by the experimental plan or were later unbal anced by death of mice during the experi ment. Main effects and all interactions of carcinogen, protein level and sex were in cluded in the statistical models used, as were interactions of carcinogen with block. The validity of each statistical analysis was examined by residual plotting. For each variable, a few outlying observations were corrected or removed as most reasonable. Residual plots strongly suggested that statis tical analysis of the amino acids and six of the nitrogen-related variables be conducted on the log scale, primarily to equalize vari ances, so this was done All significance test ing of main effects utilized the 5% level, but testing of interactions employed the 1% level to control the effects of testing multiple hypotheses about the same variable Statisti cal computing was accomplished with the Statistical Analysis System procedure GLM (16), employing Types I and III sums of squares. RESULTS Observed means for each sex by diet com bination over all three experimental blocks are shown in table 2 for food, N, carcinogen and lipid intakes, body weight changes, carcass N, N output, overall N recovery and fecal lipid output. Table 2 also summarizes statistically significant main effects of sex and dietary factors. For each experimental factor, the magnitude of a significant effect is reported as a relative difference, obtained by dividing the adjusted mean difference between relevant groups by the adjusted mean of a reference group [eg., (adjusted female mean minus adjusted male mean)/ (adjusted male mean) x 100% for effect of sex]. For instance, females averaged 19% less carcass N than males, although N intake for females averaged only 5% less. Summa rizing sex effects in table 2, female mice con sumed less food and protein, gained less Downloaded from jn.nutrition.org by guest on June 9, 2014 the experimental diets were determined be fore initiation of feeding. Protein (6.25% X Kjeldahl N) ranged between 8.7 and 8.9% for the 10% protein diets and between 35.1 and 35.8% for the 40% protein diets. After the 5-day adaptation period, mice were assigned to the experimental diets and placed in metabolism cages for 14 days. The metabolism cages were modified from the standard design to minimize loss of feed and mixing of excreta with food. Food consump tion and weight change were recorded for each week of this period. After 6 days the mice were placed in their conventional cages for 24 hours while the metabolism cages were cleaned. The mice were then returned to the metabolism cages for a 7-day collec tion period. Urine and feces were collected on days 9, 12 and 14 and subsequently pooled for analysis. To prevent microbial growth, urine was collected in vessels containing 0.7 ml of l M H2SO4. On day 14, the mice were transferred to their conventional cages, fasted for 9 hours, lightly anesthetized with ether, and blood samples were collected by cardiac puncture. Plasma from heparinized blood was immediately deproteinized with sulfosalicylic acid precipitation (9). The depro teinized plasma was quickly frozen in liquid N and stored at -70Â°C (Beckman applica tion DS 561) until analysis (Beckman 119CL Amino Acid Analyzer, Beckman Instruments, Palo Alto, CA). The mice were killed by cervical fracture, and the intestinal contents were removed. The carcasses were analyzed for N content. Fecal and urinary collections were stored at - 18Â°C.Feces were freeze-dried, weighed, ground and analyzed for total N (7) and crude lipids (8). Urine total N (7), urea (10), creatinine (11), ammonia (12), uric acid (13) and allantoin (14) were determined. The ratio of N recovered in the urine to the total in urine and cage washings was used to esti mate losses during the collection process. The entire experiment as described above was replicated three times in sequence, with 48 animals used each time, for a total of 144 animals. The three replicates differed in arrangements of diets across cages, which were chosen so as to minimize the influence of any environmental effects related to cage position. Some adjustments to the feeder were also made during the first run. The observations were interpreted by using mul 557 558 HEVIA ET AL. TABLE 2 Mean intakes, weight changes, carcass nitrogen, nitrogen balance measures, fecal weight and fecal lipid output measures, by sei and treatment, with statistically significant main effects of sex, protein and carcinogen' Males fed 40% protein + 10% protein Measure Week 1 Food intake, g Carcinogen intake,7 mg Body wt change, g Week 2 Food intake, g N intake,' Body wt change, g Carcass N, mg Carcass N, % N gain,9 mg N balance Total N output,8 mg 27.3 1.39 26.4 363 1527 1.10 752 3.01 31.6 16.2 361 26.3 5.7 1.41 25.9 1.5 1.25 25.2 361 25.6 352 1505 5.5 0.82 749 2.96 23.4 31.1 330 1485 1.5 1.01 689 2.85 23.9 30.2 322 AAF 24.9 1.80 24.5 5.4 1.56 DNP 22.5 1.3 -0.90 25.2 1426 25.7 1433 24.8 1406 1565 1680 5.7 1.32 807 3.25 42.7 200.1 1232 1674 1.4 1.00 694 2.87 28.4 184.0 1245 1.27 746 3.08 35.5 152.8 1269 Overall %Fecal N recovery," gFecalwt, outputFecal lipid lipid/lipid Intakes x 1Q-3942.19564922.18858922.18357893.0206132892.8200123882.9192115 'Values are means. Abbreviations: C.V., coefficient of variation; AAF, 2-acetylaminofluorene; DNP, N,Ndinitrosopiperazine; Prot, protein. 'Levels of statistical significance: 'P < 0.001; bP < 0.05; "P < 0.01; NS, P > 0.05. 3For variables analyzed on the logarithmic scale, this "% difference" characterizes the relative ef fect of the involved factor at all levels of the other experimental variables when there is no interaction. For variables not transformed prior to analysis, it is the absolute rather than the relative difference which is constant across levels of the other factors, and relative variation may be higher or lower than reported at particular factor levels associated with low and high average responses, respectively. The reported percent difference is, however, essen tially an average (the harmonic mean) of the true percent differences at the various levels of the other factors, and hence provides a reasonable measure of relative effect. 4Female mice relative to males. 540% soybean protein diet relative to 10% soybean protein diet. "Carcinogen present relative to no carcinogen. weight, showed less total N output and car cass N, and excreted less fecal dry matter, less fecal crude lipids and less fecal lipid per lipid intake than their male counterparts. Mice fed 40% protein diets consumed less food during both experimental weeks, and gained 28% less weight during week 1 than mice fed the 10% protein diets, while con suming almost four times the protein. They also excreted almost four times the N, 37% more fecal dry matter and lipid, and more fecal lipid per lipid intake during the collec tion period (week 2). Average food intake was depressed during the first week in all groups fed DNP, with weight gain depressed by 86% in the first week of carcinogen feed ing and 18% during the collection week. No significant interactions were observed involv ing the above variables. A statistically signifi cant interaction of sex and AAF, relative to N gain, stems from an estimated overall 15% greater N gain associated with AAF in females, but not in males. However, this difference between males and females was not observed consistently across blocks or protein levels and appears to be a statistical artifact. Mice fed AAF or DNP had less fecal lipid per lipid intake. A significance test suggests that AAF affects N balance, but unusual patterns of variation in the data indicate that the test is unreliable and that the results must be viewed as inconclusive. Downloaded from jn.nutrition.org by guest on June 9, 2014 Lipid intake7 Carcinogen intake,7 mg DNP AAF 559 CARCINOGENS AND NITROGEN METABOLISM Females fed 40% protein 10% protein AAF 24.5 0.85 23.5 5.2 0.66 DNP 23.6 1.4 0.41 AAF 22.7 0.73 22.6 5.0 0.88 Overall mean DNP 20.6 1.2 0.01 24.1 0.84 Significant effects2 % difference1 C.V. 11.3 187 Sex4 Prot5 AAF" DNP6 -9a -9- NS -8' -48b -28e NS -86e 25.53551480__1.086002.7927.832.9322912.1835624.735414725.41.136022.8730.048.0306862.0735024.934514361.51.075872.7725.934.8310891.96647 L15261.116672.9230.5101.77366.35 Downloaded from jn.nutrition.org by guest on June 9, 2014 L892.4132.986.410.3â€”1.711.3_6112.910.96.35.4â€”3.217.813.317.214.3-6e-5eNS-1 Proportional to food intake and dose (carcinogen intake) or proportional to food intake and dietary lipids (lipid intake). "L denotes mean natural logarithm, for variables analyzed on that scale. Reported group means for nitrogen intake, output, balance and recovery occasionally appear inconsistent due to partial missing data for particular animals. Each variable was analyzed appropriately by using all reliable data pertaining to it. "N gain represents the change in weight observed during week 2 of the experiment converted to N using the carcass N determined after death. Significant interaction (P < 0.01) of sex and AAF. See text. '"Although a statistical ly significant main effect of AAF was found through formal hypothesis testing, discrepancies between blocks and unusual patterns of within-group variation preclude meaningful inference of an effect of AAF on this variable. "Nitrogen recovery = (Total N output/N intake) x 100. No other significant effects of AAF or DNP were found for this set of variables. Table 3 summarized significant main effects of experimental factors on fecal and urinary N, and on five forms of urine N. Excretion variables were analyzed on two scales: total measured N and fraction of N intake. Use of the latter would be expected to eliminate differences between groups due solely to variations in N intake, if excretion were roughly proportional to intake over the range of intake studied. Female mice excreted significantly more ammonia and uric acid N but less urea N than male counterparts, after adjustment for intake Significant re ductions of total N output, urinary N and urinary creatinine N in females did not per sist after correction for intake. Total and urinary N excretion rose as N intake increased from 10 to 40% soybean protein diets. How ever, the distribution of excretion products varied with protein level. Urea and ammonia N excretion increased for 40% protein-fed animals more than did N intake, and the increase in urea remained significant after correction for intake. Based on either scale of analysis, urinary allantoin, uric acid, creatinine and fecal N outputs increased significantly less than intake Whereas AAF produced small reductions in total urea and allantoin N and DNP reduced total urinary N, allantoin, creatinine, fecal and total N excretion, none of these differences remained significant when corrected for intake No 560 HEVIA ET AL. TABLES Statistically significant main effects of sex, diet and carcinogen on total nitrogen (N) output, total urinary N, colorimetrie N, and fecal N, with and without adjustment for intake, expressed as % diffÃ©rences1 Excretion/ intake Excretion Parameter measured Sex" Significant effects Prot3 AAF4 DNP4 Mean5 C.V. Significant effects Sex2 Prot3 AAF4 DNP4 mg/wk Mean5 C.V. mg excreted/ mg intake % Urinary N TotalUrea L514.05.97 NAmmonia L18.42.61 NAllantoin L14.30.62.098.04.41 NUric NCreatinine acid NFecal KA-72-24NSNSNSNSNSNSNSNSNSNSNSNSNSNS0.7630.5920.0220.0210.0010.004- Lâ€”3.5â€”3.0â€”11.515.419.915.3â€”5.6NS-834NS10NSNSNS16"NS-54_ 'See table 2, footnotes l, 3. !Female mice relative to males. Total urinary N, uric acid and unadjusted ammonia effects are significant at P < 0.05; total N output, creatinine N, and adjusted urea and ammonia N effects are significant at P < 0.01, and other effects at P < 0.001. 3Dietary 40% soybean protein relative to 10% soybean protein. Creatinine and adjusted urea effects are significant at P < 0.01; other effects are significant at P < 0.001. The adjusted urea N effect is the average of effects which differed significantly (P < 0.01) between experimental blocks. 'Carcinogen present relative to no carcinogen. AAF effects are significant at P < 0.05, as are DNP effects on total urinary N, total N output and fecal N. DNP effects on creatinine N and allantoin N are significant at P < 0.01 and P < 0.001, respectively. 5L denotes mean natural logarithm, for variables analyzed on that scale. "Reported effect averaged over significantly different blocks. significant interactions were observed. Observed pooled means of plasma amino acids are shown in table 4 along with statis tically significant main effects of sex and diet. All plasma amino acid concentrations were statistically analyzed after logarithmic transformation. Females showed higher plas ma concentrations of citrulline, tryptophan and lysine but lower concentrations of aspar tate, threonine, serine, asparagine, gluta mate, glycine, valine, leucine, tyrosine and histidine than males. Mice fed 40% protein diets had higher concentrations of glycine, valine, isoleucine, leucine, tyrosine and phenylalanine and lower concentrations of aspartate, threonine, serine, proline, citrul line, lysine and arginine than those fed 10% protein diets. The reduction in lysine associ ated with the 40% protein diet was signifi cantly greater for females (32%) than for males (15%). Dietary AAF was associated with a sig nificant (39%) increase in plasma ammonia concentrations, and the sex difference in aspartate concentration when AAF was fed was slightly greater than in control animals. No other effects of dietary AAF on plasma amino acid concentrations were statistically significant. Dietary DNP increased plasma histidine by 13%, decreased tryptophan by 32% in males and increased tryptophan by 16% in females. DNP was associated with an in crease in plasma phosphoserine of about 60% in females fed 10% protein diets, al though other sex by diet groups showed DNP-linked reductions averaging 23%. This finding was not consistent across blocks, and may be artifactual. Another three-way inter action, involving DNP and phenylalanine, was significant due to large but opposite effects in different blocks, and no reliable conclusion is apparent. The occasional occur rence of statistically significant but possibly random complex interactions is to be expected in a study involving this large a number of variables, in which several types of interac tion are separately tested in relation to each variable. DISCUSSION The mouse and rat are competing animal models for use in long-term cancer studies. Downloaded from jn.nutrition.org by guest on June 9, 2014 L0.12819.615.933.717.021.4â€”2.623.0 5.825 N-10-1322NSNS-14NS28233832684'76*7187NS-10NS-8NSNSNS-9NSNS-14NS-8-11645.06.24 CARCINOGENS AND NITROGEN METABOLISM 561 1'1i 8 .s T! I 1 Q - Â» -Ãˆ-S -s Â£T ible So a 2 a S -S1S Sexâ€¢s f.s -Â» I+ IÂ« ; .B 2 Z. m->,o cH S P Â« _- Si 2 7zz7z+lzzzllz 2 + ZZ + , z co iâ€”co io ^r ' O3 O5 00 IO Ci us CO CO 00 ^f i*- <â€”<l~- oâ€”< COÅ“t^^H^HlO^Ã¯OCNQCOtâ€” t^t^w5co-Â«ro^c7iocooiraco-H^HOj O3CNOÃ•CO CO CM^lâ€”CNtâ€” CD oiiooiinoio l p Â£Â£ 1Â« o* C/3* ts S z â€¢ Â»li iCNOst^tâ€” I 3 i- -H oo co co ' â€¢H ^H ' . ! '-l 135 r- (N sa â€¢Â§, Â«v . S O) <N CM ! 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The effects of the initial actions of chemi cal carcinogens on protein and purine metab olism are unknown. Other studies have shown elevated uric acid and allantoin ex cretion by rats following irradiation or in jection of alkylating agents such as dimethylnitrosoamine (30-33), suggesting some al teration of nucleic acid metabolism during the initiation phase of carcinogenesis. Al though adding DNP to the diets reduced food intake, weight gain and N output, the effects of DNP or AAF on the N in the ex cretory products measured in the present studies were minimal and generally not sig nificant when expressed as a function of N intake This suggests that the depressions in the urinary output of urea and allantoin associated with dietary AAF or of total N, allantoin, creatinine, fecal N and total N output associated with DNP resulted from differences in N intake and were unrelated to direct toxic effects. Even though these concentrations of AAF and DNP are carcino genic in long-term studies, short-term feed ing caused no significant changes in excre tion of protein or purine metabolites. The levels of plasma amino acids in BeCaFi mice used in this study compared well with values reported for other mouse strains (3436). The higher level of lysine in the plasma of female mice compared to male mice has also been well documented by previous re searchers (34). The high concentrations of citrulline, tryptophan and lower levels of threonine, serine, glutamate, glycine, valine, leucine, tyrosine and histidine found in female mice in our study have not been noted previously. The large number of in dividual samples, more modern equipment and procedures, precise control of dietary intake, and powerful statistical analysis undoubtedly increase the sensitivity of the presently reported experiment. There are few studies reporting plasma amino acid levels in mice fed diets differing in protein concentration. The present data provides base-line information on plasma Downloaded from jn.nutrition.org by guest on June 9, 2014 Since mice are smaller and require 75 to 80% less feed than rats, their use offers dis tinct economic advantages. However, although dietary requirements of the two species are known to be qualitatively similar (17, 18), more nutritional data and studies of carcino gen metabolism are available for rats than for mice. Also, mice tend to scatter food, requiring special precautions to adequately measure food intake. Since the volume of excretory products produced by mice is low, small losses of urine or feces introduce pro portionately larger errors into metabolic studies of mice than of rats. The special feeder employed in this study minimized spillage and urination into the feed. Recovery of N in urine and feces av eraged 89% of N intake, estimated from ration weights. Several factors presumably contributed to the departure from total recovery. Skin and hair losses were not esti mated. Subject animals, which gained weight, undoubtedly accumulated N over the study period. This accumulation was estimated, from weight change and carcass N data, as about 30 mg/week. Food scattering and excretory collection errors also contributed. As expected, increasing dietary protein by a factor of four increased excretion of pro tein end products. However, while there was approximately a fourfold increase in the excretion of urea and ammonia associated with increasing protein from 10 to 40% of the diet, there was only a doubling of the purine end products, uric acid and allantoin. Increases in uric acid excretion with increased protein intake have also been shown in hu mans (19). On the other hand, others have seen no difference in urinary allantoin and uric acid in rats fed 8 or 21% protein (20). A doubling of fecal crude lipid excretion was associated with the increase in dietary soybean protein from 10 to 40% (table 2). Since the ratio of lipid excretion to lipid intake was also higher, the higher fecal lipid output was clearly due to the increased in take of soybean protein. The increase in fecal lipids resulting from increased protein intake may influence intestinal carcinogenesis. We have previously reported increased 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis and blood cholesterol in rats fed diets high in protein (21). The association among dietary lipids, fecal lipids, fecal steroid excretion and colon cancer has been CARCINOGENS AND NITROGEN METABOLISM food intake when high protein or amino acid-deficient or imbalanced diets are fed (41, 43). Other than the elevation in serum am monia concentrations by AAF and the eleva tion of histidine by DNP, these carcinogens had no consistent effects on plasma amino acid concentrations. ACKNOWLEDGMENTS The authors are grateful to Joan M. Alster, M.S., for data management and statistical programming support, as well as editorial assistance; Pamela A. Anderson, Ph.D. and Sherry Long, M.S., for assistance with tech nical procedures. LITERATURE CITED 1. Doll, R. & Peto, R. (1981) The causes of cancer: quantitative estimates of avoidable risks of cancer in the United States today. J. Nati. Cancer Inst. 66, 1192-1308. 2. National Research Council (1982) Report of the Committee on Diet, Nutrition and Cancer. National Academy of Sciences, Washington, DC. 3. Clayson, D. B., Lawson, T. A., Santana, S. & Bonser, G. M. (1965) Correlation between the chemical induction of hyperplasia and the malig nancy in the bladder epithelium. Brit. J. Cancer 19, 297. 4. Greenblatt, M., Mirvish, S. & So, B. T. (1971) Nitrosamine studies: induction of lung adenomas by concurrent administration of sodium nitrite and secondary amines in Swiss mice. J. Nati. Cancer Inst. 46, 1029-1034. 5. American Institute of Nutrition (1977) Report of the AIN Ad Hoc Committee on standards for nutritional studies. J. Nutr. 107, 1340-1348. 6. Perkins, E. G., Hendren, D. J., Truex, C. R. & Visek, W. J. (1983) A simple and rapid method for the determination of 2-acetylaminofluorene in laboratory diets. J. Liq. Chrom. 6, 367-373. 7. Shahinian, A. H. & Reinhold, ]. G. (1971) Appli cation of the phenol-hypochlorite reaction to measurement of ammonia concentrations in Kjeldahl digests of serum and various tissues. Clin. Chem. 17, 1077-1079. 8. Blight, E. G. & Dyer, W. J. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911-917. 9. Mondino, A., Bogiovanni, G., FumerÃ²,S. & Rossi, L. (1972) An improved method of plasma deproteination with sulphosalicilic acid for determining amino acids and related compounds. J. Chromatogr. 74, 255-263. 10. Foster, L. B. & Hochholzer, J. M. (1971) A single reagent manual method for directly deter mining urea nitrogen in serum. Clin. Chem. 17, 921-925. 11. Folin, O. & Wu, H. (1919) A system of blood Downloaded from jn.nutrition.org by guest on June 9, 2014 amino acids in BeCsFi mice fed a defined diet under controlled conditions. It should be noted that these mice were fasted for 9 hours prior to plasma sampling, which may have minimized changes in plasma amino acids. Based on current recommendations for weanling mice, the 10% soybean protein diet is slightly deficient, while the 40% soy bean protein diet greatly exceeds require ments (5, 37). Soybean protein is relatively high in lysine and low in methionine. Publi cations concerning relationships between protein level and source (and thus amino acid intake) and concentrations of plasma amino acids tend to vary. For example, Steele et al. (38) found no difference in plas ma amino acids of mice fed 10 or 50% pro tein. Harker et al. (39) reported that when rats were fed diets composed of 115, 110, 85 and 70% of the protein requirements (1) only plasma lysine was positively related to protein intake at all levels, (2) threonine was directly related to protein intake at 100, 85 and 70% and (3) there was no consistent relationship between intakes of nonessential amino acids and plasma amino acids. Young et al. (40) reported that essential amino acid levels in the blood of rats were lower at all times in those fed protein-deficient diets than in controls. In an experiment where rats were offered a self-selected diet (41), neither changes in individual or total essen tial amino acids correlated with the protein selected. Our findings of lower plasma con centration of threonine, serine, proline, citrulline, lysine and arginine and higher glycine, valine, isoleucine, leucine, tyrosine and phenylalanine for animals fed the 40% protein diet compared to the 10% diet does not suggest a previously reported pattern. The large decrease in plasma arginine in mice fed high protein diets may be related to a greater use of arginine for urea synthesis (42). The effects of an increase in dietary protein on plasma amino acids will depend on the physiological and nutritional state of the animal, the strain and species, the amino acid composition of the diet, and the time of plasma sampling, factors that make it diffi cult to compare reported studies (43). Mice fed 40% protein diets had decreased feed intakes during both experimental weeks. This agrees with previous studies and sug gests that changes in plasma amino acid levels may contribute to the regulation of 563 564 HEVIA ET AL. 28. 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