W. R. FEATHERSTON ANDJ. C. ROGLER Department of Animal Sciences, Purdue University, West Lafayette, Indiana 47907 ABSTRACT Studies were conducted with chicks on the interrelation ship between cystine and methionine in either crystalline amino acid or wheat-peanut meal diets containing suboptimal levels of these two amino acids. The crystalline amino acid basal diet contained 0.2% DL-methionine and 0.2% L-cystine whereas the wheat-peanut meal diet contained 0.2% methionine and 0.26% cystine. Marked improvements in weight gain and feed efficiency were observed when these diets were supplemented with 0.2 to 0.6% DL-methionine. When similar levels of L-cystÃ-newere added to the basal diets containing 0.2% methionine, chick growth was depressed. When the diets contained 0.4% methionine, the growth depressing effect of cystine was not observed. These results point to the existence of an an tagonism of cystine on methionine utilization when the dietary level of methionine is suboptimal. J. Nutr. 108: 1954-1958, 1978. methionine •cystine •interrelations INDEXING KEY WORDS •chicks Numerous studies, including an extensive series of investigations by Baker and coworkers, are reported in the literature con cerning the percentage of the sulfur amino acid requirement of the chick (1-3) and rat (4) that can be supplied by cystine or cysteine. In these studies, the total sulfur amino acid level was maintained constant, and differing proportions of cystine and methionine were fed to make up a given amino acid level. With this experimental design, not only was cystine increasing but methionine was decreasing at the same time. In other studies, the total sulfur amino acid content has been varied, but the ratio of methionine to cystine was held constant or the level of methionine has been varied while keeping the level of cystine constant (5). Sowers et al. (6) conducted two experi ments with rats in which graded levels of L-methionine and L-cystìnewere fed in a factorial arrangement of treatments. At a suboptimal level of methionine, they noted small decreases in weight gain at the

higher levels of cystine (0.30% in one ex periment and 0.45% in both experiments). When varying levels of methionine and/or cystine were added to isolated soybean protein diets containing 0.17% methionine and 0.13%) cystine, Baker et al. (7) noted a decrease in weight gain of young pigs in one of two experiments when only cystine was added. This report describes the effects on chick weight gain and feed efficiency of supple mental cystine and methionine when added to diets containing suboptimal levels of these two amino acids. EXPERIMENTAL

PROCEDURE

Male White Mountain chicks were ran domly allotted to treatments at 1 day of age. Each chick was weighed, wingbanded, and placed in an electricallyheated battery brooder with raised wire Received for publication June 23, 1978. 1Journal paper No. 7203, Purdue University Agri cultural Experiment Station.

1954

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Methionine-Cystine Interrelations in Chicks Fed Diets Containing Suboptimal Levels oÃ-Methionine

METHIONINE-CYSTINE INTERRELATIONS

TABLE 1 Crystalline amino acid basal diet

TABLE 2 Wheat-peanut meal basal diet Ingredient Wheat Peanut meal L-lysine-HC1 L-threonine L-histidine-HCl-H2O Soybean oil Dicalcium phosphate Limestone Sodium chloride (iodized) Vitamins + trace minerals + BHT1 Variables

75.75 15.00 0.60 0.30 0.10 3.00 2.25 1.00 0.40 1.20 0.40

1The vitamin and mineral premix provided the following ingredients per kilogram of diet: man ganese sulfate-HjO, 0.5 g; zinc oxide, 0.06 g; sodium selenite, 0.22 mg; butylated hydroxy toluene (BHT), 0.125 g; choline chloride, 1.3 g; retinyl palmitate, 5000 IU; cholecalciferol, 2250 ICU; D-alpha tocopheryl acetate, 22 IU; menadione sodium bisulfite, 1.4 mg; riboflavin, 8.8mg; calcium pantothenate, 17.6 mg; niacin, 39.6 mg; vitamin Bi2, 11.1«.

A wheat-peanut meal basal diet supple mented with lysine, threonine, and histidine was used in experiment 4 (table 2). starchCorn Corn The amino acid composition of the wheat oilVitamin premix'Sodium & mineral and peanut meal was determined by ion exchange chromatography.2 The basal diet bicarbonateDL-methionineL-cystineL-arginine contained 18.0% protein, 0.2% methionine, HC1L-histidine-HCl-H2OL-lysine • and 0.26% cystine. Analysis of variance (9) was used to HC1GlycineL-tryptophanL-tyrosineL-phenylalanineL-threonineL-leucineL-isoleucineL-valineL-prolineL-glutamic • statistically analyze the weight gain and feed efficiency data for each experiment. Individual treatment differences were tested by the Newman-Keuls multiple range test (9). In addition, weight gain data of chicks fed the 0.2% DL-methionine and 0.2 and 0.4 % L-cystÃ-nein experiments 1, 2, and 3 were pooled for statistical acidVariables analysis. or starch49.3615.0013.501.000.200.201.150.411.140.600.150.450.500.651.000.600.690.4012.001.00 Ingredient

%

1The vitamin and mineral premix provided the following ingredients per kilogram of diet : cellulose, 30 g; calcium carbonate, 19.1 g; calcium phosphate •H20(monobasic), 21.15 g; potassium phosphate, 11.2 g; sodium chloride, 6.0 g; ferrous sulfate, 0.2 g; zinc oxide, 0.1225 g; cupric sulfate-5H2O, 0.015 g; manganese sulfate- H2O, 0.51 g; potassium iodide, 0.04 g; magnesium carbonate, 2.5 g ; sodium molybdate-2H2O, 1.0 mg; sodium selenite, 0.22 mg; butylated hydroxy toluene, 0.125 g ; choline chloride, 1.3 g ; retinyl palmitate, 5000 IU; cholecalciferol, 2250 ICU; D-alpha tocopheryl acetate, 17.6 IU; menadione sodium bisulfite, 2 mg; inositol, l g; riboflavin, 9 mg; thiamin, 6 mg; calcium pantothenate, 20 mg; niacin, 50 mg; pyridoxine, 8 mg; folie acid, 2 mg; biotin, 0.3 mg; vitamin Bi2, 20 ¿ig-

RESULTS The weight gain and feed efficiency of chicks fed from 0 to 14 days of age in ex periment 1 are shown in table 3. Supple mentation of the basal diet containing 0.2% DL-methionine and 0.2% L-cystìne with 0.2% additional DL-methionine re sulted in a marked improvement in chick growth and feed efficiency. As was true in subsequent studies, additional methionine 2 Technicon York 10502.

Instrumenta

Corporation,

Ardsley,

New

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floors. Five chicks were placed in each pen with four pens (replicates) per treatment in experiments 1, 2, and 3, and seven chicks per pen with four replicates per treatment in experiment 4. No two repli cates of the same treatment appeared in the same battery or the same deck level of another battery. Feed and water were pro vided ad libitum during the 14-day experi mental period of experiments 1, 2, and 3, and for the 21-day experimental period of experiment 4. The crystalline amino acid basal diet used in experiments 1, 2, and 3 is shown in table 1. Crystalline amino acids were added in amounts similar to those used by Sasse and Baker (8) with the exception that DL-methionine and L-cystine were each added at the 0.2% level.

1955

1956

W. R. FEATHERSTON AND J. C. ROGLER TABLE 3

( P < 0.01 ) improved weight gain, whereas cystine supplementation significantly (P < 0.05) depressed weight gain. However, comparisons of all means showed signifi cant differences only between 0.2% me gainTreatmentBasal 14-day wt thionine and the higher methionine levels change)+ regardless of cystine level. Although the differences were not significant, the higher DL-methionine+ (0.2% L-cystine)Basal 0.2% cystine levels resulted in poorer growth DL-methionineBasal + 0.2% 100.4+ rate, particularly at the lowest level of me DL-methionineBasal + 0.4% 1064thionine (0.2%). Averaged across methio 29.5L-cystineBasal + 0.2% nine levels, the 0.6% cystine level was sig L-cystinePooled t- 0.4% 39.6GainFeed0.34°0.47'0.52'0.32°0.31°0.02 SE(8)76.6»153.5°158.1«54.0».'46.3e6.2(% nificantly poorer (P < 0.05) than the 0.2% level, and the difference between 0.2 and 1Means of four replicates of five chicks each. Means not shar 0.4% cystine approached significance ( P > ing a common superscript letter are significantly different 0.05). Other than the response in going (P < 0.05). from 0.2 to 0.4% methionine, no effect was beyond 0.4% DL-methionine with 0.2% noted from the dietary treatments on feed efficiency. In this experiment, feed effi L-cystÃ-nedid not result in any further im provement in weight gain but did result ciency as well as weight gain was as good in some improvement in feed efficiency. In with 0.4%) methionine and 0.2% cystine as contrast to the growth stimulation from with higher levels. In experiment 3, crystalline amino acid methionine, supplementation of the diet diets containing either 0.2 or 0.4% L-cystine with an additional 0.2% L-cystÃ-neresulted and 0.2, 0.25, 0.3, 0.35, and 0.4% DL-methio in a 29% decrease (P < 0.10) in weight gain while the 0.4% supplemental L-cystine nine were fed (table 5). Chicks fed 0.2% resulted in a 39% decrease (P < 0.05) in methionine and 0.4% L-cystine tended to weight gain. The growth depression with grow slower than chicks fed the same level of methionine with 0.2% cystine, but the the additional 0.2% L-cystine occurred even though the total sulfur amino acid difference was not significant. At higher levels of methionine, no differences in content of the diet was only 0.6%. weight gain or feed efficiency were noted In experiment 2, a 3x3 factorial ar rangement of treatments was used with 0.2, due to the different levels of cystine. Since crystalline amino acid diets con 0.4, and 0.6% total L-cystine and the same taining 0.2% DL-methionine with either levels of DL-methionine (table 4). Fac 0.2 or 0.4% L-cystine were fed in experi torial analysis of the data indicated that ments 1, 2, and 3, the results of the three methionine supplementation significantly Response of chicks fed crystalline amino acid diets limiting in sulfur amino acids to supplemental methionine or cystine (experiment I)1

(%)0.468.7' DL-Methionine0.2

0.4 0.6X Pooled SE0.289.2'

(0.51°) 193.3°(0.71') 198.6° (0.71»)160.4°

(0.52°) 188.8° (0.70') 188.0° (0.70')148.5°.'

(0.64°) 5.6 (0.01 )L-Cystine 9.0

(0.51°) 188.1°(0.70') 175.2° (0.69')142.1'

(0.51°) 190.1°(0.706) 187.3°(0.70°)

(0.63°) (0.64°) (0.01 )0.662.9' 7.7 (0.01 )X73.6'

1Weight gain and gain/feed ratio (in parentheses) of four replicates of five chicks each at 14 days of age. Comparisons were made among all nine treatments, methionine treatments across cystine treatment, and cystine treatments across methionine treatment. Within these comparisons, means not sharing a common superscript letter are significantly different (P < 0.05).

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TABLE 4 Response of chicks fed crystalline amino acid diets limiting in sulfur amino acids to supplemental methionine and/or cystine (experiment #)'

METHIONINE-CYSTINE

TABLE 6 Response of chicks fed wheat-peanut meal diets limit ing in sulfur amino acids to supplemental methionine or cystine (experiment 4) gainTreatmentBaaal1

Experiment 4 was conducted to deter mine if the growth depressing effect of sup plemental cystine noted with a crystalline amino acid diet occurred when cystine was added to a diet composed of common feedstuffs (table 2). Supplementation of this diet with 0.2% methionine gave a marked growth response (table 6); 0.4% methio nine resulted in no greater growth re sponse, but feed efficiency was significantly improved. Supplementation of this diet with 0.2 % additional cystine resulted in a significant decrease in weight gain. In creasing the level of supplemental cystine to 0.4% did not result in a further de crease in chick performance, an observa tion which is difficult to explain.

1957

21-day wt change)+

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experiments were pooled for analysis. The treatment x experiment interaction was not significant ( P > 0.05 ) , whereas the growth depressing effect of cystine was significant

INTERRELATIONS

methionine+ (0.2% DL L-cystine)Basal 0.26% DL-methionineBasal + 0.2% 36.9+ DL-methionineBasal + 0.4% 37.2-16.1-15.4GainFeed0.56°0.62*0.64«0.55»0.54 L-cystineBasal + 0.2% L-cystinePooled + 9.4% SE(g)292.5»400.4«401.3«245.4«247.4«10.4(% 1Wheat-peanut meal basal diet. Means of four replicates of seven chicks each. Means not sharing a common superscript letter are significantly different (P < 0.05).

tal sulfur amino acids are required, they are added as either DL-methionine or its hydroxy analogue. Several workers, including Suda (11), Finkelstein and Mudd (12), and Shannon et al. (4), have reported depressed crystaDISCUSSION thionine synthase activity in rats when The results of these studies emphasize cystine is added to low methionine diets the need to consider the methionine con as compared with the values for rats fed tent of the diet as well as considering the unsupplemented diets. However, Stipanuk total sulfur amino acid content. They are and Benevenga ( 13 ) concluded from their not in agreement with the conclusion of studies as well as those of Harney (14) Rose (10) and more recent investigators that there appeared to be little basis for that cystine improves growth only when the speculation that the methionine-sparing methionine is supplied in suboptimal effect of cystine is due to a depression in amounts. From a practical standpoint, most the activity of crystathionine synthase and natural diets contain at least as much me subsequently of methionine oxidation. They thionine as cystine, and when supplemenconcluded that changes in the oxidation of amino acids are secondary to the utiliza TABLE 5 tion of amino acids for protein synthesis. Response of chicks fed crystalline amino acid diets This could explain the effect of cystine limiting in sulfur amino acids to supplemental supplementation of low methionine diets methionine and/or cystine (experiment 3)1 on both growth and methionine oxidation. (%)0.2T}i*»f Dietary L-cystine Graber et al. (2) reported that cystine could replace methionine to the extent of making up 56 to 60% of the total sulfur SITVDL-methionine(%)0.200.250.300.350.40Pooled amino acids for the 2-week old chick. Sub sequent studies by Sasse and Baker (3) using 0.4% total sulfur amino acids, as in the present study, indicated that cystine could provide only about 41% of the total '0.70«0.68°'«0.010.4Weightgain78.8»162.5°165.2°169.9°170.4«6.9GainFeed0.59«0.66».«0.67».«0.69».«0.69».«0.01 sulfur amino acids. The results of the pres ent studies agree with those of Sasse and SEWeightgain92.4'159.7«167.4»179.8«170.0»5.6GainFeed0.56«0.64«0.67°. Baker ( 3 ) in showing that the absolute re quirement for methionine per se by chicks 1Means of four replicates of five chicks each. Means not shar fed the basal diet was greater than that ing a common superscript letter are significantly different (P < 0.05). for cysteine.

1958

W. R. FEATHERSTON

LITERATURE

CITED

1. Baker, D. H. (1976) Nutritional and meta bolic interrelationships among sulfur com pounds in avian nutrition. Federation Proc. 35 1917—1922 2. Graber, G., Scott, H. M. & Baker, D. H. ( 1971 ) Sulfur amino acid nutrition of the growing chick: Effect of age on the capacity of cystine to spare dietary methionine. Poultry Sci. 50, 1450-1455. 3. Sasse, C. E. & Baker, D. H. (1974) Sulfur utilization by the chick with emphasis on the effect of inorganic sulfate on the cystinemethionine interrelationship. J. Nutr. 104, 244-251. 4. Shannon, B. M., Howe, J. M. & Clark, H. E. (1972) Interrelationships between dietary methionine and cystine as reflected by growth, certain hepatic enzymes, and liver composi tion of weanling rats. J. Nutr. 102, 557-562. 5. Soares, J. H., Jr. (1974) Experiments on the requirement of inorganic sulfate by the chick. Poultry Sci. 53, 246-252. 6. Sowers, J. E., Stockland, W. L. & Meade, R. J. ( 1972 ) L-methionine and L-cystine require ments of the growing rat. J. Anim. Sci. 35, 782-788. 7. Baker, D. H., Clausing, W. W., Harmon, B. G., Jensen, A. H. & Becker, D. E. (1969) Replacement value of cystine for methionine for the young pig. J. Anim. Sci. 29, 581-584. 8. Sasse, C. E. & Baker, D. H. (1973) Modi fication of the Illinois reference standard amino acid mixture. Poultry Sci. 52, 19701972. 9. Steel, R. G. D. & Torrie, J. H. (1960) Principles and Procedures of Statistics. Mc Graw-Hill Book Co., Inc., New York. 10. Rose, W. C. (1937) The nutritive signifi cance of the amino acids and certain related compounds. Science 86, 298-300. 11. Suda, M. (1966) A view of the comparison of the regulation of enzymes in mammalian and microbial systems. Adv. Enzyme Regul. 5, 181-209. 12. Finkelstein, J. D. & Mudd, S. H. (1967) Trans-sulfuration in mammals: The methionine-sparing effect of cystine. J. Biol. Chem. 242, 873-880. 13. Stipanuk, M. H. & Benevenga, N. J. (1977) Effect of cystine on the regulation of methio nine in rats. J. Nutr. 107,1455-1467. 14. Harney, M. E., Swick, R. W. & Benevenga, N. J. (1976) Estimation of tissue protein synthesis in rats fed diets labeled with [U-"C]tyrosine. Am. J. Physiol. 23Õ, 10181023. 15. National Research Council ( 1977 ) Nutrient Requirements of Poultry, 7th ed., National Academy of Sciences, Washington, D.C. 16. Lerner, J. & Taylor, M. W. (1967) A com mon step in the intestinal absorption mecha nisms of D- and L-methionine. Biochim. Biophys. Acta 135, 990-999. 17. Hagihira, H., Lin, E. C. C. & Wilson, T. H. ( 1961 ) Active transport of lysine, ornithine, arginine, and cystine by the intestine. Biochem. & Biophys. Res. Comm. 4, 478-481.

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The growth depressions noted in these studies from 0.2% L-cystÃ-nesupplementa tion occurred with both crystalline amino acid diets (0.2% Met; 0.2% Cys) and pro tein diets (0.2% Met; 0.26% Cys). In both diets, the total sulfur amino acid concen tration after 0.2% cystine supplementation was still below the value indicated by the National Research Council (15) or Sasse and Baker ( 8 ) as being the requirement of the chick. It is difficult to envision a meta bolic change with these low levels which would explain the growth depressions. A more plausible explanation for what appears to be an antagonism of cystine on methionine is in relation to absorption of these amino acids. Lerner and Taylor ( 16 ) demonstrated that one site exists on the chick mucosal epithelial membrane which provides for the absorption of both D- and L-methionine. They also demonstrated that L-cystine inhibited the uptake of both isomers by competing with them for the com mon site. A small decrease in the absorp tion of methionine, which is already pres ent in growth limiting quantities, as a result of the diet supplementation with cystine could readily result in a growth depression as compared with that of chicks fed the basal diet. If this effect is medi ated by an inhibition of methionine uptake from the intestine, it is difficult to under stand why the growth depression noted with 0.6% L-cystine was not more severe than that observed with 0.4% L-cystine. Also, the work of Lerner and Taylor ( 16) used a tissue accumulation method in which the competition of different amino acids for a common site on the mucosal epithelial membrane of intestinal tissue segments was studied rather than amino acid transport, which has been used in most studies. Their results do not agree with those of Hagihira et al. ( 17 ) who have shown that cystine and the basic amino acids share the same transport sys tem which is different from the transport systems responsible for the absorption of methionine and other neutral amino acids. Further studies are being conducted to at tempt to explain the deleterious effect of cystine when added to chick diets contain ing suboptimal levels of methionine.

AND J. C. ROGLER

Methionine-cystine interrelations in chicks fed diets containing suboptimal levels of methionine.

W. R. FEATHERSTON ANDJ. C. ROGLER Department of Animal Sciences, Purdue University, West Lafayette, Indiana 47907 ABSTRACT Studies were conducted with...
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