Is L-Glutamic Acid Nutritionally a Dispensable Amino Acid for the Young Chick? K. MARUYAMA, M. L . SUNDE AND A . E . HARPER
Departments of Poultry Science and Nutritional Sciences, University of Wisconsin, Wisconsin 53706
Madison,
(Received for publication March 10, 1975)
POULTRY SCIENCE 55: 45-60, 1976
1964). Hepburn et al. (1960), and Hepburn and Bradley (1964) suggested the inclusion of glutamic acid and arginine in order to produce growth of rats fed amino acid diets equivalent to that obtained with intact protein diets. Glutamine was again reported to be effective (Hepburn and Bradley, 1964) and, in addition, to alleviate the toxic effect of excess glycine and serine (Hepburn and Bradley, 1968). Similarly, chick growth on an amino acid diet was improved by the addition of glutamic acid (Klain et al., 1960). When the effectiveness of glutamine or asparagine and glutamic acid or aspartic acid was compared on the basis of nitrogen retention, a-amino nitrogen was more effective than amide nitrogen in growing rats (Womack and Wilson, 1969). Rogers et al. (1970) sug-
INTRODUCTION
I
N the course of development of amino acid diets the importance of nitrogen for the synthesis of dispensable amino acids has been recognized (Frost and Sandy, 1951; Rechcigl et al., 1957; Sauberlich, 1961; Stucki and Harper, 1961, 1962). Greenstein et al. (1956) suggested a role for asparagine and glutamine and to a lesser degree proline.
The omission of asparagine from a mixture of dispensable amino acids resulted in less than maximum growth of rats (Breuer et al.,
Research supported in part by the College of Agricultural and Life Sciences, University of Wisconsin, and the National Institute of Arthritis, Metabolism and Digestive Diseases, project number AM 10748. 45
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ABSTRACT The importance of dispensable amino acids for the chick was reinvestigated. Two-week chick weights were 75.7 g. and 143.7 g. when dietary nitrogen was provided solely by indispensable amino acids and by indispensable amino acids and 10% L-glutamic acid, respectively. Weight gain increased four-fold when L-glutamic acid was added to the mixture of indispensable amino acids. Plasma free amino acid concentrations were considerably decreased and total indispensable amino acid concentration (including cystine and tyrosine) was reduced to approximately a half or less with L-glutamic acid supplementation. Increments of L-glutamic acid as the sole nitrogen source for dispensable amino acids in the diet increased two-week weight gain proportionately over a range from 0 to 10%. When 10% of L-glutamic acid was included in the amino acid diet, growth rate was equivalent to that with a practical diet up to two weeks of age. The mixture of leucine, isoleucine, valine, lysine and arginine produced little growth promotion as a nitrogen source for dispensable amino acids. L-aspartic acid, L-alanine and the mixture of dispensable amino acids devoid of L-glutamic acid were found not as effective as L-glutamic acid. The estimate of effectiveness of L-aspartic acid, L-alanine and the mixture was approximately 80%, 60%, and 80%, respectively, of that of L-glutamic acid during first two weeks. Utilization of diammonium citrate for growth promotion varied with the amount used and the age of chicks. A combination of dispensable amino acids plus nominal levels of DAC was not as effective as glutamic acid during the first week but shortly after that, produced good gains. Even during the 3rd week or 4th week high levels of DAC alone did not produce normal gains of chicks. The activities and the subcellular distribution of glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) in the liver of young chicks were measured. GOT activity was considerably higher than GPT activity. Initially, GOT activity was found primarily in cytosol, but the mitochondrial activity increased to 57% of total activity by day 10, whereas GPT activity was exclusively in mitochondria regardless of the age.
46
K. MARUYAMA, M. L. SUNDE AND A. E. HARPER
EXPERIMENTAL PROCEDURES Day-old New Hampshire x Single Comb White Leghorn chicks of both sexes were
wing-banded, weighed and placed in conventional, electrically heated chick starting batteries. One group of 10 chicks was randomly assigned to each treatment. Feed and water were provided ad libitum. Unless specified, the chicks were fed their respective experimental diets for 14 days and weighed individually at weekly intervals. Weekly feed consumption was recorded for each treatment. The amino acid basal diet (Table 1) contained essential components for the growth of the chicks but contained minimum amounts of indispensable amino acids and no nonspecific nitrogen source for dispensable amino acids. It provided nitrogen equivalent to 10.9% protein. Addition of other amino acids or diammonium citrate were made at the expense of dextrin. Body weight and weight gain data were analyzed statistically by Duncan's multiple range test (Steel and Torrie, 1960). The 0.05
TABLE 1.—Composition of the basal amino acid diet Ingredients Basal amino acid mixture Corn oil Vitamin mixture' Mineral mixture2 Choline chloride ot-tocopheryl acetate (10 mg./g.) Cellulose NaHC0 3 Antiacid3 Dextrin
g./kg. diet 111.25 90.0 5.0 60.0 2.0 1.0 30.0
10.0 10.0 up to 1 kg.
Basal amino acid mixture ./kg. diet g./kg. diet L-Lysine • HC1 14.0 L-Valine 10.0 4.5 L-Histidine • HC1 L-Leucine 14.0 8.0 L-Arginine • HC1 L-Isoleucine 14.0 7.0 L-Phenylalanine L-Threonine 7.0 7.0 Glycine L-Tyrosine 10.0 2.25 L-Methionine L-Tryptophan 7.0 3.0 L-Cystine 3.5 L-Proline 'Vitamin mixture supplied the following in mg. per kg. diet: thiamine HC1, 100; niacin, 100; inositol, 100; Ca pantothenate, 20; riboflavin, 16; pyridoxine-HCl, 6; menadione, 5; folic acid, 4; biotin, 0.6; cyanocobalamin, 0.02; vitamin A palmitate, 10,000 I.U.; vitamin D 3 , 1,000 I.C.U. 2 Mineral mixture supplied the following in mg. per kg. diet: CaC0 3 , 15,000; Ca 3 (P0 4 ) 2 , 13,950; KH 2 P0 4 , 12,000; Na 2 HP0 4 , 7,200; NaCl, 6,000; MgS0 4 -7H 2 0, 5,000; KCl, 3,000; MnSO 4 -2H 2 0, 420; FeC 6 H 5 0 7 , 400; ZnC03, 200; KI, 52; CuS0 4 , 20; NaBr, 10; NaMo04, 10. 3 3 parts Mg 2 Si 3 0 8 and 1 part Al(OH)3.
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gested that it was necessary to include glutamic acid, proline and arginine as a part of the dispensable nitrogen component. Utilization of non-protein nitrogen was reported by various workers with varying results (Sullivan and Bird, 1957; Featherston et at, 1962; Sugahara and Ariyoshi, 1967; Blair et al., 1972; Allen and Baker, 1974). The extent of utilization of non-protein nitrogen is also dependent on the dietary carbohydrate supply (Renner, 1969). These experiments were conducted to investigate the importance of dispensable amino acids, the effect of glutamic acid on growth of chicks and utilization of non-protein nitrogen in growing chicks.
GLUTAMIC ACID FOR CHICKS
The enzyme assays, in duplicate, were carried out by the following procedures: glutamic dehydrogenase (GDH) (Corman and Inamdar, 1970) and lactic dehydrogenase
1. Joel Ltd., Tokyo, Japan.
(LDH) (Straub, 1940) were measured by determination of NADH oxidation at 340 nm. GPT and GOT were measured by an interrupted assay (Swick et al., 1965) in which the amount of products (pyruvate or oxaloacetate) formed during four minutes of incubation at 30° C. or 37° C. was determined by NADH oxidation at 340 nm. in the presence of excess lactic dehydrogenase or malic dehydrogenase, respectively. 2 RESULTS AND DISCUSSION The effect of isonitrogenous supplementation of the basal amino acid mixture diet with more of the mixture of indispensable amino acids (IAA) or with L-glutamic acid is shown in Table 2. The weight gain in the two week period on the negative control diet (the amino acid basal diet) indicated that either additional nitrogen containing compounds are required, or that one or more of the requirement levels of other indispensable amino acids was not reached. Adding more of the IAA mixture (6.8%) improved growth rate significantly, but weight gain was much less than that obtained with 10% of L-glutamic acid. Weight gains in the 2 week period were 35.5 g. and 103.4 g., respectively, for the IAA and glutamic acid groups compared to the 13 g. for the negative control group. Growth improvement with the additional nitrogen was four times as much with L-glutamic acid as with the IAA mixture. Thus indispensable amino acids were not utilized as efficiently as L-glutamic acid. Considerably better growth was reported by Featherston et al. (1962), Blair et al. (1972) and Allen and Baker (1974) with the diets containing only indispensable amino acids which merely met the requirement. The difference could have been accounted for by the experimental conditions. In the present
2. Sigma Chemical Co., St. Louis, MO.
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level of probability was accepted as the criterion for statistically significant differences. At the end of each experiment, blood samples were collected from three chicks of average weight within a treatment by heart puncture using heparinized needles and syringes. The samples were centrifuged for 20 minutes at 28,700 x g and equal volumes of plasma within a treatment were pooled. Deproteinization of plasma was done with 15% sulfosalicylic acid to make the concentration of protein-free supernatant solution 3% in sulfosalicylic acid. Deproteinized plasma was kept at -20° C. until amino acids were analyzed with an automated amino acid analyzer. 1 To study the activity and the subcellular distribution of glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT), the livers from five young chicks fed a practical chick starter diet (Bhargava et al., 1970) were obtained at one, three, five and ten days of age. After cervical dislocation, the liver was immediately excised and blotted dry. Then it was homogenized in 9 volumes of chilled medium composed of 0.225 M mannitol,0.075 M sucrose,0.01 M Tris buffer (pH 7.8) and 0.05 mM EDTA (Tyler and Gonze, 1967) with the use of a Potter-Elvehjem homogenizer with a Teflon pestle. Another portion of the liver was homogenized in a medium composed of 0.05 M potassium phosphate buffer (pH 7.8), 0.025 M L-alanine, 0.002 M L-cysteine and 50% glycerol (v./v.) (Swick et al., 1965) and was used to estimate the total enzyme activity. The mitochondrial transaminase activity was corrected by the fraction glutamic dehydrogenase solublized.
47
48
K. MARUYAMA, M. L. SUNDE AND A. E. HARPER
TABLE 2.—Growth of chicks fed either excess indispensable amino acids or L-glutamic acid as the source of nitrogen for dispensable amino acids Addition to basal amino acid diet' None (neg cont) 6.8% IAA 5 10% L-G1U
Average weight 2 1 week
2 week
g50.0 ± 1.3a4 62.2 ± 2.9b 85.0 ± 2.5c
g53.1 ± 1.6a 75.7 ± 5.0b 143.7 ± 2.6c
g22.6 90.6
an isonitrogenous basis.
Increase in two-week weight accounted for by additional nitrogen to the amino acid basal diet. "Values without a common letter are significantly different (P < 0.05). 5 The mixture of indispensable amino acids used in the amino acid basal diet. study, day-old chicks were used instead of one week-old chicks previously fed an intact protein diet. Hence, it may be desirable to use day-old chicks which seem to be more sensitive to dietary amino acid supply. The influence of the addition of amino acids on the concentration of plasma free amino acids is shown in Table 3. The concentrations of plasma free amino acids were relatively high when the chicks were fed the amino
acid basal diet (the negative control diet). Highest concentrations were observed for threonine, serine and lysine. A marked reduction in plasma free indispensable amino acids was observed when L-glutamic acid was supplemented. The concentrations of indispensable amino acids, with the exception of proline, were reduced by approximately half or to less than half those observed for chicks fed the amino acid
TABLE 3.—Influence of source of nitrogen for dispensable amino acids on the concentration of plasma free amino acids Addition to the basal amino acid diet None
6.8% IAA
10% L-G1U
JJ, mole amino acid/100 ml. plasma Dispensable Glutamic acid Aspartic acid Alanine Serine Subtotal Indispensable1 Threonine Glycine Proline Lysine Histidine Arginine Valine Leucine Isoleucine Methionine + 1 / 2 cystine Phenylalanine + tyrosine Subtotal Total 1
69 6 78 161 314
31 5 81 110 227
28 5 51 63 147
238 61 23 140 15 17 27 19 12 19 35 606 920
136 47 44 157 12 15 22 16 10 17 29 505
64 26 20 73 5 7 11 11 7 10 17 251 398
732
Indispensable amino acids plus cystine and tyrosine were provided in the amino acid basal diet.
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1 Addition of amino acids was made on 2 Average weight ± SEM of 10 chicks. 3
Growth promotion 3
49
GLUTAMIC ACID FOR CHICKS
No improvement in weight gain was observed with (Table 4) the addition of a mixture of L-leucine, L-isoleucine, L-valine, L-lysine and L-arginine but substantial increases in growth rate occurred when glutamic acid was included in the diet. When this mixture of indispensable amino acids was replaced isonitrogenously with 5% of L-glutamic acid the weight gain was twice that of the basal amino acid mixture. Isonitrogenous replacement of lysine and arginine with 2% L-glutamic acid resulted in a 40% increase in weight gain and replacement of leucine, and valine with 3% L-glutamic acid resulted in a 48% increase in weight gain. Thus it appears that
TABLE 4.—Efficiency
of conversion of
indispensable amino acids to dispensable amino acids and effect of the addition of L-glutamic acid on growth of chicks Addition to basal amino acid diet' None (negative control) Leu + He + Val + Lys + Arg 4 Leu + He + Val 4 + 2% L-G1U Lys + Arg 4 + 3% L-GIU
5% L-G1U
Weight gain 1 to 2 week
(%)
822.4a 2
(100)3
22.4a
(100)
31.4b
(140)
33.1b 45.2c
(148) (201)
1 Addition of amino acids was made on an isonitrogenous basis. 2 Values without a common letter were significantly different (P < 0.05). 3 Values within parentheses were computed as a percent of the weight gain of the chicks fed the basal amino acid diet. 4 L-leucine 0.9%, L-isoleucine 0.9%, L-valine 0.8%, L-lysine 0.6%, and L-arginine 0.4%, used as indicated above.
the addition of glutamic acid was the most important factor for the promotion of chick growth. Other dispensable amino acids were next tested to determine if glutamic acid would again be more effective than other dispensable amino acids on an isonitrogenous basis (Table 5). Of the three amino acids used, L-alanine was least effective (approximately 60% of the response to L-glutamic acid) in promoting growth of the chicks. Again, Lglutamic acid was found most effective. L-aspartic acid was consistently less effective than L-glutamic acid and its relative efficiency was approximately 80% of that of L-glutamic acid at two different nitrogen levels. Renner (1969) reported that there was no significant difference either in the utilization of glutamic acid and aspartic acid or in the utilization of aspartic acid and alanine with an abundant supply of dietary carbohydrate. However, the present experiments demonstrated that either L-glutamic acid should be considered an indispensable amino acid or that it is more easily
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basal diet. When the mixture of IAA was increased, plasma free indispensable amino acids were slightly decreased with the exceptions of lysine and proline. The plasma free lysine remained rather high. It is possible that the high level of lysine in the plasma was due to the relatively slow rate of degradation of a surplus of this amino acid (Grove and Roghair, 1971; Wang and Nesheim, 1972; Wang et al., 1973). Although dispensable amino acids were not included in the basal amino acid mixture diet, concentrations of dispensable amino acids were elevated in the plasma. This elevation was particularly noticeable for serine and glutamic acid. Additional nitrogen in the diet reduced the plasma concentrations of the dispensable amino acids. The extent of reduction was greater when L-glutamic acid was used. These observations appeared somewhat similar to those by Swendseid et al. (1963) who observed that increased plasma dispensable amino acids were accompanied with a decrease in growth rate. In the present study the increased growth rate was associated with a decrease in plasma concentrations of dispensable and also indispensable amino acids. L-glutamic acid supplementation improved the chick growth, and reduced the concentrations of plasma indispensable amino acids as well as dispensable amino acids.
50
K. MARUYAMA, M . L . SUNDE AND A . E . HARPER
TABLE 5.—Comparison of effectiveness of dispensable amino acids for growth of the chicks' Addition to amino acid mixture 2
Avg. 2 wk. weight
Wt. gain 1 to 2 wk. 3
Growth promotion 4
ggg(%) 9.3 58.4 — 21.8 31.1 (62) 81.4 27.0 36.3 (78) 100.5 4.5% L-Asp 5%L- •Glu 34.8 44.1 (100) 113.7 8.7 58.0 2 None 9%L- •Asp 46.4 37.7 (79) 118.8 56.4 47.7 (100) 131.0 10% iL-GIU 1 Average of three experiments with 10 chicks per treatment. 2 Addition of amino acids was made on an isonitrogenous basis; 5% glutamic acid nitrogen basis in group 1 and 10% glutamic acid nitrogen basis in group 2. 3 All treatments in either group 1 or group 2 produced a significantly different weight gain from others in three separate experiments except in one experiment where weight gains of 3% L-Ala and 4.5% L-Asp were not significantly different. 4 Increase in weight gain accounted for by dispensable amino acid. 5 Values within parentheses were computed as a percent of growth promotion obtained with L-glutamic acid. 1
None (neg cont)
3%L-•Ala
TABLE 6.-
also observed in these experiments that as the growth rate improved, the total concentration of plasma free amino acids decreased. Increases in the plasma concentrations of alanine and aspartic acid were observed when either of the respective amino
-Influence of additional dispensable amino acids on the concentrations of plasma free amino acids Addition to the basal amino acid mixture 3% Ala
Dispensable Glutamic acid Aspartic acid Alanine Serine Subtotal Indispensable1 Threonine Glycine Proline Lysine Histidine Arginine Valine Leucine Isoleucine Methionine + 1 / 2 cystine Phenylalanine + tyrosine Subtotal Total
4.5% Asp 5% Glu H, mole amino acid/100 ml. plasma
39 6 114 107 ^66~
39 12 83 98 ~232~
34 4 56 75 W
181 62 22 134 17 28 43 22 15 21 51 ~59
GLUTAMIC ACID FOR CHICKS
acid was more effective in promoting growth than a combination of DAC with four dispensable amino acids. After that glutamic acid was not superior to a mixture of other dispensable amino acids. ACKNOWLEDGEMENT
REFERENCES Allen, N. K., and D. H. Baker, 1974. Quantitative evaluation of nonspecific nitrogen sources for the growing chick. Poultry Sci. 53: 258-264. Bhargava, K. K., R. P. Hanson and M. L. Sunde,, 1970. Effects of methionine and valine on antibody production in chicks infected with Newcastle disease virus. J. Nutr. 100: 241-248. Bhargava, K. K., T. F. Shen, H. R. Bird and M. L. Sunde, 1971. Effect of glutamic acid on chick's proline requirement. Poultry Sci. 50: 726-731. Blair, R., D. W. F. Shannon, J. M. McNab and D. J. W. Lee, 1972. Effects on chick growth of adding glycine, proline, glutamic acid or diammonium citrate to diets containing crystalline essential amino acids. Br. Poultry Sci. 13: 215-228. Bradford, N. M., and J. D. McGivan, 1973. Quantitative characteristics of glutamate transport in rat liver mitochondria. Biochem. J. 134: 1023-1029. Breuer, L. H., Jr., W. G. Pond, R. G. Warner and J. K. Loosli, 1964. The role of dispensable amino acids in the nutrition of the rat. J. Nutr. 82: 499-506. Corman, L., and A. Inamdar, 1970. In: Methods of Enzymology, H. Tabor and C. W. Tabor, Eds., Academic Press, New York. 17: 844-850. DeRosa, G., and R. W. Swick, 1975. J. Biol. Chem. in press. Featherston, W. R., H. R. Bird and A. E. Harper, 1962. Effectiveness of urea and ammonium nitrogen for the synthesis of dispensable amino acids by the chick. J. Nutr. 78: 198-206. Featherstqn, W. R., and C. W. Horn, 1973. Dietary influences of the activities of enzymes involved in branched-chain amino acid catabolism in the chick. J. Nutr. 103: 757-765. Featherston, W. R., and C. W. Horn, 1974. Studies
on the utilization of the a-hydroxy acid of methionine by chicks fed crystalline amino acid diets. Poultry Sci. 53: 680-686. Felig, P., T. Pozefsky, E. Marliss and G. F. Cahill, Jr., 1970. Alanine: Key role in gluconeogenesis. Science, 167: 1003-1004. Freedland, R. A., K. D. Martin and L. Z. McFarland, 1966. A survey of glutamic dehydrogenase activity in four tissues of normal and starved coturnix. Poultry Sci. 45: 985-991. Frost, D. V., and H. R. Sandy, 1951. Utilization of non-specific nitrogen sources by the adult proteindepleted rat. J. Biol. Chem. 189: 249-260. Greenstein, J. P., S. M. Birnbaum and M. Winitz, 1956. A water-soluble synthetic diet. Arch. Biochem. Biophys. 63: 266-268. Grove, J. A., and H. G. Roghair, 1971. The metabolism of D- and L-lysine in the chicken. Arch. Biochem. Biophys. 144: 230-236. Hepburn, F.N., and W.B. Bradley, 1964. The glutamic acid and arginine requirement for high growth rate of rats fed amino acid diets. J. Nutr. 84: 305-312. Hepburn, F. N., and W. B. Bradley, 1968. Effect of glutamine on inhibition of rat growth by glycine and serine. J. Nutr. 94: 504-510. Hepburn, F. N., W. K. Calhoun and W. B. Bradley, 1960. A growth response of rats to glutamic acid when fed an amino acid diet. J. Nutr. 72: 163-168. Jo, J-S., N. Ishihara and G. Kikuchi, 1974. Occurrence and properties of four forms of phosphoenolpyruvate carboxykinase in the chicken liver. Arch. Biochem. Biophys. 160: 246-254. Klain, G. J., H. M. Scott and B. C. Johnson, 1960. The amino acid requirement of the growing chick fed a crystalline amino acid diet. Poultry Sci. 39: 39-44. Okuno, G., T. A. I. Grillo, S. Price and P. P. Foa, 1964. Development of hepatic phosphorylase in the chick embryo. Proc. Soc. Exp. Biol. Med. 117: 524-526. Peng, Y-S., M. Brooks, C. Elson and E. Shrago, 1973. Contribution of the cytosol and mitochondrial pathways to phosphoenolpyruvate formation during gluconeogenesis. J. Nutr. 103: 1489-1495. Rechcigl, M., Jr., J. K. Loosli and H. H. Williams, 1957. The net utilization of non-specific nitrogen sources for the synthesis of non-essential amino acids. I. Growth and nitrogen utilization. J. Nutr. 63: 177-192. Renner, R., 1969. Effectiveness of various sources of non-essential nitrogen in promoting growth of chicks fed carbohydrate-containing and "carbohydrate-free" diets. J. Nutr. 98: 297-302. Rogers, Q. R., D. M. Y. Chen and A. E. Harper, 1970. The importance of dispensable amino acids
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The authors wish to acknowledge the gift of the dextrin from the Corn Products Co., Argo, Illinois, through the courtesy of Dr. J. D. Shroder, and Dr. Robert Miller of Merck and Co., Rahway, New Jersey, for some of the vitamins used.
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K. MARUYAMA, M. L. SUNDE AND A. E. HARPER
and glycine by the chicks. J. Nutr. 62: 143-150. Swendseid, M. E., J. Villalobos and B. Friedrich, 1963. Ratios of essential-to-nonessential amino acids in plasma from rats fed different kinds and amounts of proteins and amino acids. J. Nutr. 80: 99-102. Swick, R. W., P. L. Barnstein and J. L. Stange, 1965. The metabolism of mitochondrial proteins. I. Distribution and characterization of the isozymes of alanine aminotransferase in rat liver. J. Biol. Chem. 240: 3334-3340. Tyler, D. D., and J. Gonze, 1967. In: Methods in Enzymology, R. W. Estabrook and M. E. Pullman, Eds., Academic Press, New York. 10: 75-77. Womack, M., and J. E. Wilson, Jr., 1969. Utilization of amide nitrogen by the young rat. J. Food Sci. 34: 430-433. Yoshida, T., and G. Kikuchi, 1971. Significance of the glycine cleavage system in glycine and serine catabolism in avian liver. Arch. Biochem. Biophys. 145: 658-668. Wang, S-H., and M. C. Nesheim, 1972. Degradation of lysine in chicks. J. Nutr. 102: 583-596. Wang, S-H., L. O. Crosby and M. C. Nesheim, 1973. Effect of dietary excesses of lysine and arginine on the degradation of lysine by chicks. J. Nutr. 103:384-391.
Thyroxine and Triiodothyronine in Blood After Ingestion of Iodinated Casein by Chicks W.
S. NEWCOMER
Department of Physiological Sciences, Oklahoma State University, Stillwater, Oklahoma 74074 (Received for publication March 10, 1975)
ABSTRACT White Leghorn cockerels were fed a broiler ration containing 0 (controls), 0.02 or 0.04% Protamone (iodinated casein) either continuously or for 10-14 days after which the 0.02% Protamone ration was switched to control or vice versa. Body growth rate was temporarily suppressed during the first 14 days of feeding 0.02% Protamone. Thyroid weight was promptly depressed during feeding of Protamone and increased more slowly when Protamone was removed from the feed. Only a fleeting increase in oxygen consumption rate was detectable in birds fed either 0.02 or 0.04% Protamone. Serum T4 and T3 promptly rose when Protamone was fed; continued feeding resulted in compensatory adjustments in secretion and perhaps peripheral metabolism of iodohormones within 2-3 days so that a level close to 200 ng. T3 or 2.5 |xg. T 4 /100 ml. serum was subsequently maintained. POULTRY SCIENCE 55: 60-69, 1976
T
HE effects of iodinated casein in the various species to which it has been administered are qualitatively similar to those effects following administration of thyroxine
(T4) and/or triiodothyronine (T3) by any of a variety of routes (Srivastava and Turner, 1967). Iodinated casein is routinely given orally; the presumption is that the physiologi-
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for maximal growth. Proc. Soc. Expt. Biol. Med. 134: 517-522. Sauberlich, H. E., 1961. Growth of rats fed proteinfree diets supplemented with purified amino acid mixtures. J. Nutr. 74: 298-306. Sheid, B., and E. Hirschberg, 1967. Glutamic dehydrogenase and aspartic alanine aminotransferase activities in chick embryo liver. Am. J. Physiol. 213: 1173-1176. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Company, New York. Straub, F. B., 1940. Crystalline lactic dehydrogenase from heart muscle. Biochem. J. 34: 483-486. Stucki, W. P., and A. E. Harper, 1961. Importance of dispensable amino acids for normal growth of chicks. J. Nutr. 74: 377-383. Stucki, W. P., and A. E. Harper, 1962. Effects of altering the ratio of indispensable to dispensable amino acid in diets for rats. J. Nutr. 78: 278-286. Sugahara, M., and S. Ariyoshi, 1967. The nutritional value of the individual nonessential amino acid as the nitrogen source in the chick nutrition. Agr. Biol. Chem. 31: 1270-1275. Sullivan, T. W., and H. R. Bird, 1957. Effect of quantity and source of dietary nitrogen on the utilization of the hydroxyanalogues of methionine
Departments of Poultry Science and Nutritional Sciences, University of Wisconsin, Wisconsin 53706
Madison,
(Received for publication March 10, 1975)
POULTRY SCIENCE 55: 45-60, 1976
1964). Hepburn et al. (1960), and Hepburn and Bradley (1964) suggested the inclusion of glutamic acid and arginine in order to produce growth of rats fed amino acid diets equivalent to that obtained with intact protein diets. Glutamine was again reported to be effective (Hepburn and Bradley, 1964) and, in addition, to alleviate the toxic effect of excess glycine and serine (Hepburn and Bradley, 1968). Similarly, chick growth on an amino acid diet was improved by the addition of glutamic acid (Klain et al., 1960). When the effectiveness of glutamine or asparagine and glutamic acid or aspartic acid was compared on the basis of nitrogen retention, a-amino nitrogen was more effective than amide nitrogen in growing rats (Womack and Wilson, 1969). Rogers et al. (1970) sug-
INTRODUCTION
I
N the course of development of amino acid diets the importance of nitrogen for the synthesis of dispensable amino acids has been recognized (Frost and Sandy, 1951; Rechcigl et al., 1957; Sauberlich, 1961; Stucki and Harper, 1961, 1962). Greenstein et al. (1956) suggested a role for asparagine and glutamine and to a lesser degree proline.
The omission of asparagine from a mixture of dispensable amino acids resulted in less than maximum growth of rats (Breuer et al.,
Research supported in part by the College of Agricultural and Life Sciences, University of Wisconsin, and the National Institute of Arthritis, Metabolism and Digestive Diseases, project number AM 10748. 45
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ABSTRACT The importance of dispensable amino acids for the chick was reinvestigated. Two-week chick weights were 75.7 g. and 143.7 g. when dietary nitrogen was provided solely by indispensable amino acids and by indispensable amino acids and 10% L-glutamic acid, respectively. Weight gain increased four-fold when L-glutamic acid was added to the mixture of indispensable amino acids. Plasma free amino acid concentrations were considerably decreased and total indispensable amino acid concentration (including cystine and tyrosine) was reduced to approximately a half or less with L-glutamic acid supplementation. Increments of L-glutamic acid as the sole nitrogen source for dispensable amino acids in the diet increased two-week weight gain proportionately over a range from 0 to 10%. When 10% of L-glutamic acid was included in the amino acid diet, growth rate was equivalent to that with a practical diet up to two weeks of age. The mixture of leucine, isoleucine, valine, lysine and arginine produced little growth promotion as a nitrogen source for dispensable amino acids. L-aspartic acid, L-alanine and the mixture of dispensable amino acids devoid of L-glutamic acid were found not as effective as L-glutamic acid. The estimate of effectiveness of L-aspartic acid, L-alanine and the mixture was approximately 80%, 60%, and 80%, respectively, of that of L-glutamic acid during first two weeks. Utilization of diammonium citrate for growth promotion varied with the amount used and the age of chicks. A combination of dispensable amino acids plus nominal levels of DAC was not as effective as glutamic acid during the first week but shortly after that, produced good gains. Even during the 3rd week or 4th week high levels of DAC alone did not produce normal gains of chicks. The activities and the subcellular distribution of glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) in the liver of young chicks were measured. GOT activity was considerably higher than GPT activity. Initially, GOT activity was found primarily in cytosol, but the mitochondrial activity increased to 57% of total activity by day 10, whereas GPT activity was exclusively in mitochondria regardless of the age.
46
K. MARUYAMA, M. L. SUNDE AND A. E. HARPER
EXPERIMENTAL PROCEDURES Day-old New Hampshire x Single Comb White Leghorn chicks of both sexes were
wing-banded, weighed and placed in conventional, electrically heated chick starting batteries. One group of 10 chicks was randomly assigned to each treatment. Feed and water were provided ad libitum. Unless specified, the chicks were fed their respective experimental diets for 14 days and weighed individually at weekly intervals. Weekly feed consumption was recorded for each treatment. The amino acid basal diet (Table 1) contained essential components for the growth of the chicks but contained minimum amounts of indispensable amino acids and no nonspecific nitrogen source for dispensable amino acids. It provided nitrogen equivalent to 10.9% protein. Addition of other amino acids or diammonium citrate were made at the expense of dextrin. Body weight and weight gain data were analyzed statistically by Duncan's multiple range test (Steel and Torrie, 1960). The 0.05
TABLE 1.—Composition of the basal amino acid diet Ingredients Basal amino acid mixture Corn oil Vitamin mixture' Mineral mixture2 Choline chloride ot-tocopheryl acetate (10 mg./g.) Cellulose NaHC0 3 Antiacid3 Dextrin
g./kg. diet 111.25 90.0 5.0 60.0 2.0 1.0 30.0
10.0 10.0 up to 1 kg.
Basal amino acid mixture ./kg. diet g./kg. diet L-Lysine • HC1 14.0 L-Valine 10.0 4.5 L-Histidine • HC1 L-Leucine 14.0 8.0 L-Arginine • HC1 L-Isoleucine 14.0 7.0 L-Phenylalanine L-Threonine 7.0 7.0 Glycine L-Tyrosine 10.0 2.25 L-Methionine L-Tryptophan 7.0 3.0 L-Cystine 3.5 L-Proline 'Vitamin mixture supplied the following in mg. per kg. diet: thiamine HC1, 100; niacin, 100; inositol, 100; Ca pantothenate, 20; riboflavin, 16; pyridoxine-HCl, 6; menadione, 5; folic acid, 4; biotin, 0.6; cyanocobalamin, 0.02; vitamin A palmitate, 10,000 I.U.; vitamin D 3 , 1,000 I.C.U. 2 Mineral mixture supplied the following in mg. per kg. diet: CaC0 3 , 15,000; Ca 3 (P0 4 ) 2 , 13,950; KH 2 P0 4 , 12,000; Na 2 HP0 4 , 7,200; NaCl, 6,000; MgS0 4 -7H 2 0, 5,000; KCl, 3,000; MnSO 4 -2H 2 0, 420; FeC 6 H 5 0 7 , 400; ZnC03, 200; KI, 52; CuS0 4 , 20; NaBr, 10; NaMo04, 10. 3 3 parts Mg 2 Si 3 0 8 and 1 part Al(OH)3.
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gested that it was necessary to include glutamic acid, proline and arginine as a part of the dispensable nitrogen component. Utilization of non-protein nitrogen was reported by various workers with varying results (Sullivan and Bird, 1957; Featherston et at, 1962; Sugahara and Ariyoshi, 1967; Blair et al., 1972; Allen and Baker, 1974). The extent of utilization of non-protein nitrogen is also dependent on the dietary carbohydrate supply (Renner, 1969). These experiments were conducted to investigate the importance of dispensable amino acids, the effect of glutamic acid on growth of chicks and utilization of non-protein nitrogen in growing chicks.
GLUTAMIC ACID FOR CHICKS
The enzyme assays, in duplicate, were carried out by the following procedures: glutamic dehydrogenase (GDH) (Corman and Inamdar, 1970) and lactic dehydrogenase
1. Joel Ltd., Tokyo, Japan.
(LDH) (Straub, 1940) were measured by determination of NADH oxidation at 340 nm. GPT and GOT were measured by an interrupted assay (Swick et al., 1965) in which the amount of products (pyruvate or oxaloacetate) formed during four minutes of incubation at 30° C. or 37° C. was determined by NADH oxidation at 340 nm. in the presence of excess lactic dehydrogenase or malic dehydrogenase, respectively. 2 RESULTS AND DISCUSSION The effect of isonitrogenous supplementation of the basal amino acid mixture diet with more of the mixture of indispensable amino acids (IAA) or with L-glutamic acid is shown in Table 2. The weight gain in the two week period on the negative control diet (the amino acid basal diet) indicated that either additional nitrogen containing compounds are required, or that one or more of the requirement levels of other indispensable amino acids was not reached. Adding more of the IAA mixture (6.8%) improved growth rate significantly, but weight gain was much less than that obtained with 10% of L-glutamic acid. Weight gains in the 2 week period were 35.5 g. and 103.4 g., respectively, for the IAA and glutamic acid groups compared to the 13 g. for the negative control group. Growth improvement with the additional nitrogen was four times as much with L-glutamic acid as with the IAA mixture. Thus indispensable amino acids were not utilized as efficiently as L-glutamic acid. Considerably better growth was reported by Featherston et al. (1962), Blair et al. (1972) and Allen and Baker (1974) with the diets containing only indispensable amino acids which merely met the requirement. The difference could have been accounted for by the experimental conditions. In the present
2. Sigma Chemical Co., St. Louis, MO.
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level of probability was accepted as the criterion for statistically significant differences. At the end of each experiment, blood samples were collected from three chicks of average weight within a treatment by heart puncture using heparinized needles and syringes. The samples were centrifuged for 20 minutes at 28,700 x g and equal volumes of plasma within a treatment were pooled. Deproteinization of plasma was done with 15% sulfosalicylic acid to make the concentration of protein-free supernatant solution 3% in sulfosalicylic acid. Deproteinized plasma was kept at -20° C. until amino acids were analyzed with an automated amino acid analyzer. 1 To study the activity and the subcellular distribution of glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT), the livers from five young chicks fed a practical chick starter diet (Bhargava et al., 1970) were obtained at one, three, five and ten days of age. After cervical dislocation, the liver was immediately excised and blotted dry. Then it was homogenized in 9 volumes of chilled medium composed of 0.225 M mannitol,0.075 M sucrose,0.01 M Tris buffer (pH 7.8) and 0.05 mM EDTA (Tyler and Gonze, 1967) with the use of a Potter-Elvehjem homogenizer with a Teflon pestle. Another portion of the liver was homogenized in a medium composed of 0.05 M potassium phosphate buffer (pH 7.8), 0.025 M L-alanine, 0.002 M L-cysteine and 50% glycerol (v./v.) (Swick et al., 1965) and was used to estimate the total enzyme activity. The mitochondrial transaminase activity was corrected by the fraction glutamic dehydrogenase solublized.
47
48
K. MARUYAMA, M. L. SUNDE AND A. E. HARPER
TABLE 2.—Growth of chicks fed either excess indispensable amino acids or L-glutamic acid as the source of nitrogen for dispensable amino acids Addition to basal amino acid diet' None (neg cont) 6.8% IAA 5 10% L-G1U
Average weight 2 1 week
2 week
g50.0 ± 1.3a4 62.2 ± 2.9b 85.0 ± 2.5c
g53.1 ± 1.6a 75.7 ± 5.0b 143.7 ± 2.6c
g22.6 90.6
an isonitrogenous basis.
Increase in two-week weight accounted for by additional nitrogen to the amino acid basal diet. "Values without a common letter are significantly different (P < 0.05). 5 The mixture of indispensable amino acids used in the amino acid basal diet. study, day-old chicks were used instead of one week-old chicks previously fed an intact protein diet. Hence, it may be desirable to use day-old chicks which seem to be more sensitive to dietary amino acid supply. The influence of the addition of amino acids on the concentration of plasma free amino acids is shown in Table 3. The concentrations of plasma free amino acids were relatively high when the chicks were fed the amino
acid basal diet (the negative control diet). Highest concentrations were observed for threonine, serine and lysine. A marked reduction in plasma free indispensable amino acids was observed when L-glutamic acid was supplemented. The concentrations of indispensable amino acids, with the exception of proline, were reduced by approximately half or to less than half those observed for chicks fed the amino acid
TABLE 3.—Influence of source of nitrogen for dispensable amino acids on the concentration of plasma free amino acids Addition to the basal amino acid diet None
6.8% IAA
10% L-G1U
JJ, mole amino acid/100 ml. plasma Dispensable Glutamic acid Aspartic acid Alanine Serine Subtotal Indispensable1 Threonine Glycine Proline Lysine Histidine Arginine Valine Leucine Isoleucine Methionine + 1 / 2 cystine Phenylalanine + tyrosine Subtotal Total 1
69 6 78 161 314
31 5 81 110 227
28 5 51 63 147
238 61 23 140 15 17 27 19 12 19 35 606 920
136 47 44 157 12 15 22 16 10 17 29 505
64 26 20 73 5 7 11 11 7 10 17 251 398
732
Indispensable amino acids plus cystine and tyrosine were provided in the amino acid basal diet.
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1 Addition of amino acids was made on 2 Average weight ± SEM of 10 chicks. 3
Growth promotion 3
49
GLUTAMIC ACID FOR CHICKS
No improvement in weight gain was observed with (Table 4) the addition of a mixture of L-leucine, L-isoleucine, L-valine, L-lysine and L-arginine but substantial increases in growth rate occurred when glutamic acid was included in the diet. When this mixture of indispensable amino acids was replaced isonitrogenously with 5% of L-glutamic acid the weight gain was twice that of the basal amino acid mixture. Isonitrogenous replacement of lysine and arginine with 2% L-glutamic acid resulted in a 40% increase in weight gain and replacement of leucine, and valine with 3% L-glutamic acid resulted in a 48% increase in weight gain. Thus it appears that
TABLE 4.—Efficiency
of conversion of
indispensable amino acids to dispensable amino acids and effect of the addition of L-glutamic acid on growth of chicks Addition to basal amino acid diet' None (negative control) Leu + He + Val + Lys + Arg 4 Leu + He + Val 4 + 2% L-G1U Lys + Arg 4 + 3% L-GIU
5% L-G1U
Weight gain 1 to 2 week
(%)
822.4a 2
(100)3
22.4a
(100)
31.4b
(140)
33.1b 45.2c
(148) (201)
1 Addition of amino acids was made on an isonitrogenous basis. 2 Values without a common letter were significantly different (P < 0.05). 3 Values within parentheses were computed as a percent of the weight gain of the chicks fed the basal amino acid diet. 4 L-leucine 0.9%, L-isoleucine 0.9%, L-valine 0.8%, L-lysine 0.6%, and L-arginine 0.4%, used as indicated above.
the addition of glutamic acid was the most important factor for the promotion of chick growth. Other dispensable amino acids were next tested to determine if glutamic acid would again be more effective than other dispensable amino acids on an isonitrogenous basis (Table 5). Of the three amino acids used, L-alanine was least effective (approximately 60% of the response to L-glutamic acid) in promoting growth of the chicks. Again, Lglutamic acid was found most effective. L-aspartic acid was consistently less effective than L-glutamic acid and its relative efficiency was approximately 80% of that of L-glutamic acid at two different nitrogen levels. Renner (1969) reported that there was no significant difference either in the utilization of glutamic acid and aspartic acid or in the utilization of aspartic acid and alanine with an abundant supply of dietary carbohydrate. However, the present experiments demonstrated that either L-glutamic acid should be considered an indispensable amino acid or that it is more easily
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basal diet. When the mixture of IAA was increased, plasma free indispensable amino acids were slightly decreased with the exceptions of lysine and proline. The plasma free lysine remained rather high. It is possible that the high level of lysine in the plasma was due to the relatively slow rate of degradation of a surplus of this amino acid (Grove and Roghair, 1971; Wang and Nesheim, 1972; Wang et al., 1973). Although dispensable amino acids were not included in the basal amino acid mixture diet, concentrations of dispensable amino acids were elevated in the plasma. This elevation was particularly noticeable for serine and glutamic acid. Additional nitrogen in the diet reduced the plasma concentrations of the dispensable amino acids. The extent of reduction was greater when L-glutamic acid was used. These observations appeared somewhat similar to those by Swendseid et al. (1963) who observed that increased plasma dispensable amino acids were accompanied with a decrease in growth rate. In the present study the increased growth rate was associated with a decrease in plasma concentrations of dispensable and also indispensable amino acids. L-glutamic acid supplementation improved the chick growth, and reduced the concentrations of plasma indispensable amino acids as well as dispensable amino acids.
50
K. MARUYAMA, M . L . SUNDE AND A . E . HARPER
TABLE 5.—Comparison of effectiveness of dispensable amino acids for growth of the chicks' Addition to amino acid mixture 2
Avg. 2 wk. weight
Wt. gain 1 to 2 wk. 3
Growth promotion 4
ggg(%) 9.3 58.4 — 21.8 31.1 (62) 81.4 27.0 36.3 (78) 100.5 4.5% L-Asp 5%L- •Glu 34.8 44.1 (100) 113.7 8.7 58.0 2 None 9%L- •Asp 46.4 37.7 (79) 118.8 56.4 47.7 (100) 131.0 10% iL-GIU 1 Average of three experiments with 10 chicks per treatment. 2 Addition of amino acids was made on an isonitrogenous basis; 5% glutamic acid nitrogen basis in group 1 and 10% glutamic acid nitrogen basis in group 2. 3 All treatments in either group 1 or group 2 produced a significantly different weight gain from others in three separate experiments except in one experiment where weight gains of 3% L-Ala and 4.5% L-Asp were not significantly different. 4 Increase in weight gain accounted for by dispensable amino acid. 5 Values within parentheses were computed as a percent of growth promotion obtained with L-glutamic acid. 1
None (neg cont)
3%L-•Ala
TABLE 6.-
also observed in these experiments that as the growth rate improved, the total concentration of plasma free amino acids decreased. Increases in the plasma concentrations of alanine and aspartic acid were observed when either of the respective amino
-Influence of additional dispensable amino acids on the concentrations of plasma free amino acids Addition to the basal amino acid mixture 3% Ala
Dispensable Glutamic acid Aspartic acid Alanine Serine Subtotal Indispensable1 Threonine Glycine Proline Lysine Histidine Arginine Valine Leucine Isoleucine Methionine + 1 / 2 cystine Phenylalanine + tyrosine Subtotal Total
4.5% Asp 5% Glu H, mole amino acid/100 ml. plasma
39 6 114 107 ^66~
39 12 83 98 ~232~
34 4 56 75 W
181 62 22 134 17 28 43 22 15 21 51 ~59
GLUTAMIC ACID FOR CHICKS
acid was more effective in promoting growth than a combination of DAC with four dispensable amino acids. After that glutamic acid was not superior to a mixture of other dispensable amino acids. ACKNOWLEDGEMENT
REFERENCES Allen, N. K., and D. H. Baker, 1974. Quantitative evaluation of nonspecific nitrogen sources for the growing chick. Poultry Sci. 53: 258-264. Bhargava, K. K., R. P. Hanson and M. L. Sunde,, 1970. Effects of methionine and valine on antibody production in chicks infected with Newcastle disease virus. J. Nutr. 100: 241-248. Bhargava, K. K., T. F. Shen, H. R. Bird and M. L. Sunde, 1971. Effect of glutamic acid on chick's proline requirement. Poultry Sci. 50: 726-731. Blair, R., D. W. F. Shannon, J. M. McNab and D. J. W. Lee, 1972. Effects on chick growth of adding glycine, proline, glutamic acid or diammonium citrate to diets containing crystalline essential amino acids. Br. Poultry Sci. 13: 215-228. Bradford, N. M., and J. D. McGivan, 1973. Quantitative characteristics of glutamate transport in rat liver mitochondria. Biochem. J. 134: 1023-1029. Breuer, L. H., Jr., W. G. Pond, R. G. Warner and J. K. Loosli, 1964. The role of dispensable amino acids in the nutrition of the rat. J. Nutr. 82: 499-506. Corman, L., and A. Inamdar, 1970. In: Methods of Enzymology, H. Tabor and C. W. Tabor, Eds., Academic Press, New York. 17: 844-850. DeRosa, G., and R. W. Swick, 1975. J. Biol. Chem. in press. Featherston, W. R., H. R. Bird and A. E. Harper, 1962. Effectiveness of urea and ammonium nitrogen for the synthesis of dispensable amino acids by the chick. J. Nutr. 78: 198-206. Featherstqn, W. R., and C. W. Horn, 1973. Dietary influences of the activities of enzymes involved in branched-chain amino acid catabolism in the chick. J. Nutr. 103: 757-765. Featherston, W. R., and C. W. Horn, 1974. Studies
on the utilization of the a-hydroxy acid of methionine by chicks fed crystalline amino acid diets. Poultry Sci. 53: 680-686. Felig, P., T. Pozefsky, E. Marliss and G. F. Cahill, Jr., 1970. Alanine: Key role in gluconeogenesis. Science, 167: 1003-1004. Freedland, R. A., K. D. Martin and L. Z. McFarland, 1966. A survey of glutamic dehydrogenase activity in four tissues of normal and starved coturnix. Poultry Sci. 45: 985-991. Frost, D. V., and H. R. Sandy, 1951. Utilization of non-specific nitrogen sources by the adult proteindepleted rat. J. Biol. Chem. 189: 249-260. Greenstein, J. P., S. M. Birnbaum and M. Winitz, 1956. A water-soluble synthetic diet. Arch. Biochem. Biophys. 63: 266-268. Grove, J. A., and H. G. Roghair, 1971. The metabolism of D- and L-lysine in the chicken. Arch. Biochem. Biophys. 144: 230-236. Hepburn, F.N., and W.B. Bradley, 1964. The glutamic acid and arginine requirement for high growth rate of rats fed amino acid diets. J. Nutr. 84: 305-312. Hepburn, F. N., and W. B. Bradley, 1968. Effect of glutamine on inhibition of rat growth by glycine and serine. J. Nutr. 94: 504-510. Hepburn, F. N., W. K. Calhoun and W. B. Bradley, 1960. A growth response of rats to glutamic acid when fed an amino acid diet. J. Nutr. 72: 163-168. Jo, J-S., N. Ishihara and G. Kikuchi, 1974. Occurrence and properties of four forms of phosphoenolpyruvate carboxykinase in the chicken liver. Arch. Biochem. Biophys. 160: 246-254. Klain, G. J., H. M. Scott and B. C. Johnson, 1960. The amino acid requirement of the growing chick fed a crystalline amino acid diet. Poultry Sci. 39: 39-44. Okuno, G., T. A. I. Grillo, S. Price and P. P. Foa, 1964. Development of hepatic phosphorylase in the chick embryo. Proc. Soc. Exp. Biol. Med. 117: 524-526. Peng, Y-S., M. Brooks, C. Elson and E. Shrago, 1973. Contribution of the cytosol and mitochondrial pathways to phosphoenolpyruvate formation during gluconeogenesis. J. Nutr. 103: 1489-1495. Rechcigl, M., Jr., J. K. Loosli and H. H. Williams, 1957. The net utilization of non-specific nitrogen sources for the synthesis of non-essential amino acids. I. Growth and nitrogen utilization. J. Nutr. 63: 177-192. Renner, R., 1969. Effectiveness of various sources of non-essential nitrogen in promoting growth of chicks fed carbohydrate-containing and "carbohydrate-free" diets. J. Nutr. 98: 297-302. Rogers, Q. R., D. M. Y. Chen and A. E. Harper, 1970. The importance of dispensable amino acids
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The authors wish to acknowledge the gift of the dextrin from the Corn Products Co., Argo, Illinois, through the courtesy of Dr. J. D. Shroder, and Dr. Robert Miller of Merck and Co., Rahway, New Jersey, for some of the vitamins used.
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K. MARUYAMA, M. L. SUNDE AND A. E. HARPER
and glycine by the chicks. J. Nutr. 62: 143-150. Swendseid, M. E., J. Villalobos and B. Friedrich, 1963. Ratios of essential-to-nonessential amino acids in plasma from rats fed different kinds and amounts of proteins and amino acids. J. Nutr. 80: 99-102. Swick, R. W., P. L. Barnstein and J. L. Stange, 1965. The metabolism of mitochondrial proteins. I. Distribution and characterization of the isozymes of alanine aminotransferase in rat liver. J. Biol. Chem. 240: 3334-3340. Tyler, D. D., and J. Gonze, 1967. In: Methods in Enzymology, R. W. Estabrook and M. E. Pullman, Eds., Academic Press, New York. 10: 75-77. Womack, M., and J. E. Wilson, Jr., 1969. Utilization of amide nitrogen by the young rat. J. Food Sci. 34: 430-433. Yoshida, T., and G. Kikuchi, 1971. Significance of the glycine cleavage system in glycine and serine catabolism in avian liver. Arch. Biochem. Biophys. 145: 658-668. Wang, S-H., and M. C. Nesheim, 1972. Degradation of lysine in chicks. J. Nutr. 102: 583-596. Wang, S-H., L. O. Crosby and M. C. Nesheim, 1973. Effect of dietary excesses of lysine and arginine on the degradation of lysine by chicks. J. Nutr. 103:384-391.
Thyroxine and Triiodothyronine in Blood After Ingestion of Iodinated Casein by Chicks W.
S. NEWCOMER
Department of Physiological Sciences, Oklahoma State University, Stillwater, Oklahoma 74074 (Received for publication March 10, 1975)
ABSTRACT White Leghorn cockerels were fed a broiler ration containing 0 (controls), 0.02 or 0.04% Protamone (iodinated casein) either continuously or for 10-14 days after which the 0.02% Protamone ration was switched to control or vice versa. Body growth rate was temporarily suppressed during the first 14 days of feeding 0.02% Protamone. Thyroid weight was promptly depressed during feeding of Protamone and increased more slowly when Protamone was removed from the feed. Only a fleeting increase in oxygen consumption rate was detectable in birds fed either 0.02 or 0.04% Protamone. Serum T4 and T3 promptly rose when Protamone was fed; continued feeding resulted in compensatory adjustments in secretion and perhaps peripheral metabolism of iodohormones within 2-3 days so that a level close to 200 ng. T3 or 2.5 |xg. T 4 /100 ml. serum was subsequently maintained. POULTRY SCIENCE 55: 60-69, 1976
T
HE effects of iodinated casein in the various species to which it has been administered are qualitatively similar to those effects following administration of thyroxine
(T4) and/or triiodothyronine (T3) by any of a variety of routes (Srivastava and Turner, 1967). Iodinated casein is routinely given orally; the presumption is that the physiologi-
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for maximal growth. Proc. Soc. Expt. Biol. Med. 134: 517-522. Sauberlich, H. E., 1961. Growth of rats fed proteinfree diets supplemented with purified amino acid mixtures. J. Nutr. 74: 298-306. Sheid, B., and E. Hirschberg, 1967. Glutamic dehydrogenase and aspartic alanine aminotransferase activities in chick embryo liver. Am. J. Physiol. 213: 1173-1176. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Company, New York. Straub, F. B., 1940. Crystalline lactic dehydrogenase from heart muscle. Biochem. J. 34: 483-486. Stucki, W. P., and A. E. Harper, 1961. Importance of dispensable amino acids for normal growth of chicks. J. Nutr. 74: 377-383. Stucki, W. P., and A. E. Harper, 1962. Effects of altering the ratio of indispensable to dispensable amino acid in diets for rats. J. Nutr. 78: 278-286. Sugahara, M., and S. Ariyoshi, 1967. The nutritional value of the individual nonessential amino acid as the nitrogen source in the chick nutrition. Agr. Biol. Chem. 31: 1270-1275. Sullivan, T. W., and H. R. Bird, 1957. Effect of quantity and source of dietary nitrogen on the utilization of the hydroxyanalogues of methionine
