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The effect of amino acids on the survival of the hyperthyroid rat
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
74, 437-442 (1958)
The Effect of Amino Acids on the Survival of the Hyperthyroid Rat’ R. E. Boldt, A. E. Harper and C. A. Elvehjem From the Department
of Biochemistry, University Madison, Wisconsin
of Wisconsin,
Received July 23, 1957 INTRODUCTION
It is well known that animals in the hyperthyroid state have increased requirements for many nutrients (1). Considerable work has demonstrated increased requirements for many of the vitamins, both water soluble and fat soluble (l-6). Also, fat has been shown to alleviate partially the toxicity of thyroactive materials (7)) and the more unsaturated fatty acids appear to be the most protective (8). Aside from the known essential nutrients, there appears to be at least one unidentified dietary factor, found in various natural products (9-ll), that is required by the hyperthyroid rat. Since several of these natural products are excellent protein sources, it appeared possible that the protective effect might be due to their effect in creating a favorable amino acid balance in the diet. This concept formed the basis of the experiments, reported in this paper, in which various amino acids were examined for their effect on hyperthyroid rats. EXPERIMENTAL Male rats of the Sprague-Dawley strain were used throughout the experiments. Each group consisted of six weanling rats weighing between 40 and 50 g. All rats were housed in individual suspended, mesh-bottom cages and were supplied with feed and water ad Zibitum. The animals were weighed at weekly intervals throughout the 60-day experimental period. The diets contained 5yo Salts IV (12)) 5yo corn oil, vitamins in mg./lOO g. ration 1 Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. Supported in part by a grant from the National Live Stock and Meat Board, Chicago, Illinois. Crystalline vitamins generously provided by Merck, Sharp and Dohme Research Laboratories, Rahway, N. J. 437
438
BOLDT,
HARPER
AND
ELVEHJEM
as follows: thiamine hydrochloride 1.5, riboflavine 2.5, pyridoxine hydrochloride 1.0, calcium pantothenate 10.0, niacin 10.0, biotin 0.05, folic acid 0.125, cyanocobalamin 0.01, i-inositol 50.0, p-aminobenzoic acid 62.5, and choline chloride 500. Fat-solubIe vitamins were given by oral supplementation of 2 drops/week of fortified Haliver Oil supplying 400 I.U. of vitamin A, 4 I.U. of vitamin D, 6.4 mg. of a-tocopherol, and 0.08 mg. of menadione. Twenty-four per cent protein was supplied either by casein or pork. Iodinated casein* was included in each diet at a level of either 0.4 or 0.5% (see table for specific diets), and sucrose was added to make each diet to 100%. The pork used was prepared as follows: Pork butts purchased from a retail store were partially freed of fat by slicing off exterior layers. The butts were then cooked at 260°C. (500°F.) for 4% hr., ground in a meat grinder, and dried in shallow pans in circulating air at 70°C. The pork was then extracted with petroleum ether (Skelly B) and ground to pass a 20.mesh screen. The resulting pork contained 94% protein (6.25 X N). RESULTS
Although casein is known to contain lower levels of certain amino acids than muscle tissue (13), supplementation of the casein basal diet with amino acids to equal the levels in muscle, and inclusion of a high level of the potential -SH compound cystine and the non-essential amino acid glycine did not increase the survival time of hyperthyroid rats. These data are presented in Table I (Expt. I). In agreement with Tappan et al. (ll), pork was shown to support survival of hyperthyroid rats much better than casein. It seemed important to study further the supplementation of the casein basal diet and its effect on survival. Thus, various essential and nonessential amino acids were added to the casein basal diet, and their effect on survival was determined. Table I (Expt. II) shows the results of this experiment. It can be seenthat supplementation with these amino acids markedly increased the survival time of the hyperthyroid rats. In order to determine which amino acids supplemented in Expt. II were responsible for the increased survival, each one was added separately to the casein control. As shown in the results of Expt. III, methionine exerted the greatest protective effect. Phenylalanine, serine, and alanine also showed some protective action, and again the combination of amino acids markedly prolonged survival. It can be seen in Table I (Expt. III) that supplementation of the casein basal diet with 0.6 % methionine either alone or in combination with other amino acids severely inhibited growth. Since the casein basal * Protamone,
Cerophyl
Laboratories,
Kansas City, MO.
E$ect
of Supplements and Survival
TABLE I of Various Amino of Hyperthyroid
Acids Rats Weight gain (2 weeks)
Expt.
on Growth Number surviving 60 days
Average survivala
g.lweeR
days
16
1. Casein 2. Casein + amino acid mix Ad 3. Pork
27.0 28.1 25.9
24.0 f 20.7 f 50.2 f
IIb
1. Casein 2. Casein + amino acid mix Be 3. Pork
29.7 19.4 30.7
21.3 f 3.5 47.5 f 5.5 50.2 f 6.5
0 2 4
25.7 28.8 18.4 28.6 25.0 25.5 23.3 28.8 18.1 29.6
23.5 32.5 45.2 32.5 27.2 28.3 24.5 31.8 52.3 54.2
f 1.1 f 5.3 f 3.7 f 4.2 f 2.8 f 1.5 i 3.2 f 2.0 i 3.5 f 4.7
0 0 1 0 0 0 0 0 1 2
22.5 23.0 24.0 25.0
26.5 35.8 43.0 57.3
f * f f
1.0 3.4 6.6 2.7
0 0 0 5
27.5 22.0 18.0 25.0 24.0 22.0 18.5
27.8 32.0 41.0 33.2 35.8 46.4 34.3
i f f i f f i
2.4 5.6 3.3 1.5 2.6 4.0 1.9
0 0 0 0 0 1 0
IIIf
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
IV’
1. 2. 3. 4.
Vf
1. 2. 3. 4. 5. 6. 7.
Casein Casein Casein Casein Casein Casein Casein Casein Casein Pork
+ + + + + + + +
0.7% 0.6% 1.0% 0.9% 0.5yo 0.7% 1.0% amino
nn-phenylalanine nn-methionine DL-serine L-leucine nn-isoleucine nn-valine nn-alanine acid mix Be
Casein Casein + 0.3% nn-methionine Casein + 0.3% betaine.HCl Pork
Casein Casein Casein Casein Casein Casein Casein 0.3% 8. Pork
-I- 0.3% nn-methionine + 0.6% nn-methionine + 0.3% betaine.HCl + 0.6% betaine.HCl + 1.0% betaine.HCl + 0.3% nn-methionine betaine.HCl
+
26.0
1.4c 0.9 2.6
0 0 2
54.7 f 3.8 4 0 Mean f standard error of the mean for a group of six rats. b All diets of this experiment contained 0.5% iodinated oasein. c Mean ZIZstandard error of the mean for a group of 12 rats. d Amino acid mix A contained 0.84yo L-arginine .HCl, 0.03yo L-histidine. HCl, 0.36yo L-lysine.HCl, 0.03% nr,-tryptophan, 0.49% L-cystine, 0.24% nn-threonine, and 0.70yo glycine. e Amino acid mix B contained a combination of all amino acids fed (at the same levels) in groups 2-8 of Expt. III. f All diets of this experiment contained 0.4% iodinated casein. 439
440
BOLDT,
HARPER
AND
ELVEHJEM
diet itself contains about 0.9 % methionine, this represents a total methionine content of 1.5 %. This level is known to retard the growth of normal rats (14). Thus the argument might be set forth that feeding levels of methionine that retard growth reduces feed intake thereby decreasing the amount of iodinated casein ingested, leading to increased survival. For this reason, a lower level of methionine was supplemented in order to avoid the growth reduction but still demonstrate its protective action. Since methionine is known to act as a methyl donor, it was felt that its effect on survival might be due to this property, and that another methyl donor might also show a protective action toward hyperthyroid rats. For this reason betaine was also tested for its survival-enhancing action. Table I (Expt. IV) shows that a lower level of methionine, supporting growth equal to the casein control, did indeed still enhance the survival of hyperthyroid rats. Betaine, also fed at the level of 0.3%, was observed to prolong survival markedly. The results presented in Table I (Expt. V) show that feeding higher levels of betaine results in improved survival of hyperthyroid rats. By using the relatively less toxic betaine, it was possible to feed higher levels of this methyl donor than was the case with methionine, and it can be seen that the higher levels resulted in still better survival. DISCUSSION
From the data presented in this paper, it appears that certain amino acids, especially methionine, have the ability to prolong the survival time of hyperthyroid rats. That this ability may be due to the labile methyl group of methionine is supported by the evidence that betaine, another labile methyl-containing compound, also exerts a protective action. Ershoff (15) in 1947 also tested various compounds, including methionine, for their effects on the length of survival of the immature thyroidfed rat. Feeding levels of 0.3 and 0.6 % of nn-methionine, he observed no increase in survival time. His diets, however, were deficient in both vitamin Blz and folic acid. A deficiency of folic acid has been shown by Stekol et al. (16) to decrease the utilization of methionine for choline and creatine synthesis. The question as to functions of vitamin B~z aside from its role in the de novo synthesis of methyl groups remains to be resolved. Thus it seems possible that the failure to observe a response to methionine may have been due to the lack of these vitamins in E&off’s diets.
AMINO
ACIDS
AND
HYPERTHYROID
RAT
441
O’Dell et al. (17), although not presenting the details of the experiment, have reported that a mixture of glycine, methionine, and arginine did not stimulate the growth of hyperthyroid rats. In 1942, however, Stetten and Grail (18) showed that additions of guanidoacetic acid to a diet limiting in methionine caused decreased choline in liver lipides and a markedly fatty liver. He explained these results on the basis of a draining of the available methionine for the irreversible methylation of guanidoacetic acid to creatine. Thus under the conditions of O’Dell, it would seem that the concurrent addition of glycine and a&nine creates a demand for the methyl group of methionine for synthesizing creatine, therefore making the added methionine unavailable for other transmethylations. Although the protective effect of casein can be made to approach that of pork by supplementation of the casein with high levels of methionine, this cannot be the basis for the superiority of pork since pork and casein contain approximately the same amount of methionine.3 It has also been shown (19) that the availability of the methionine in pork and casein is approximately equal. Thus the explanation for the action of pork must await further investigation. SUMMARY
Methionine, added to a casein diet which is complete for normal rats, markedly increases the survival time of hyperthyroid rats. That the improved survival is due to the labile methyl group of methionine is supported by the fact that betaine, another labile methyl-containing compound, also increases the survival time of hyperthyroid rats. REFERENCES 1. 2. 3. 4.
5. 6. 7.
8. 9. 10. 11.
DRILL, V. A., Physiol. Revs. !Z3, 355 (1943). DRILL, V. A., Proc. Sot. Ezptl. Biol. Med. 39,313 (1938). DRILL, V. A., AND OVERMAN, R., Am. J. Physiol. 136, 474 (1924). MARTIN, G. J., Am. J. Digest. Diseases 14, 341 (1947). EMERSON, G. A., Proc. Sot. Exptl. Biol. Med. 70,392 (1949). BETHEIL, J. J., AND LARDY, H. A., J. Nutrition 37,495 (1949). ABELIN, I., Klin. Wochschr. 6,367 (1926). ZAIN, H., Arch. exptl. Pathol. Pharmakol. 187,302 (1937). ERSHOFF, B. H., Proc. Sot. Ezptl. Biol. Med. 64, 500 (1947). ERSHOFF, B. H., Proc. Sot. Ezptl. Biol. Med. 74, 391 (1950). TAPPAN, D. V., BOLDT, R. E., AND ELVEHJEM, C. A., Proc. Sot. Med. 83, 135 (1953).
3 M.
L. Orr
and B. K. Watt,
personal
cummunication.
Exptl. Biol.
442
BOLDT,
12. HEGSTED, D.M.,
14. 15. 16. 17. 18.
19.
AND
ELVEHJEM
MILLS, R.C., ELVEHJEM,~. A., AND HART, E.B., J.BioZ. 138, 459 (1941). BLOCK, R. J., AND BOLLINO, D., “The Amino Acid Composition of Proteins and Foods,” 2nd. ed. Charles C Thomas, Springfield, Ill., 1951. WRETLIND, K. A. J., AND ROSE, W. C., J. Biol. Chem. 187, 697 (1950). ERSHOFF, B. H., Arch. Biochem. 16, 365 (1947). STEKOL, J. A., in “Amino Acid Metabolism” (McElroy, W. D., and Glass, B., eds.), p. 540. The Johns Hopkins Press, Baltimore, Md., 1955. O'DELL, B. L., STOLZENBERG, S.J.,BRUEMMER, J. H., AND HOGAN, A. G., Arch. Biochem. Biophys. 64, 232 (1955). STETTEN, D., JR., AND GRAIL, G. F., J. Biol. Chem. 144, 175 (1942). SCHWEIGERT, B. S., AND GUTHNECK, B. T., J. Nutrition 64,333 (1954). Chem.
13.
HARPER
OF
BIOCHEMISTRY
AND
BIOPHYSICS
74, 437-442 (1958)
The Effect of Amino Acids on the Survival of the Hyperthyroid Rat’ R. E. Boldt, A. E. Harper and C. A. Elvehjem From the Department
of Biochemistry, University Madison, Wisconsin
of Wisconsin,
Received July 23, 1957 INTRODUCTION
It is well known that animals in the hyperthyroid state have increased requirements for many nutrients (1). Considerable work has demonstrated increased requirements for many of the vitamins, both water soluble and fat soluble (l-6). Also, fat has been shown to alleviate partially the toxicity of thyroactive materials (7)) and the more unsaturated fatty acids appear to be the most protective (8). Aside from the known essential nutrients, there appears to be at least one unidentified dietary factor, found in various natural products (9-ll), that is required by the hyperthyroid rat. Since several of these natural products are excellent protein sources, it appeared possible that the protective effect might be due to their effect in creating a favorable amino acid balance in the diet. This concept formed the basis of the experiments, reported in this paper, in which various amino acids were examined for their effect on hyperthyroid rats. EXPERIMENTAL Male rats of the Sprague-Dawley strain were used throughout the experiments. Each group consisted of six weanling rats weighing between 40 and 50 g. All rats were housed in individual suspended, mesh-bottom cages and were supplied with feed and water ad Zibitum. The animals were weighed at weekly intervals throughout the 60-day experimental period. The diets contained 5yo Salts IV (12)) 5yo corn oil, vitamins in mg./lOO g. ration 1 Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. Supported in part by a grant from the National Live Stock and Meat Board, Chicago, Illinois. Crystalline vitamins generously provided by Merck, Sharp and Dohme Research Laboratories, Rahway, N. J. 437
438
BOLDT,
HARPER
AND
ELVEHJEM
as follows: thiamine hydrochloride 1.5, riboflavine 2.5, pyridoxine hydrochloride 1.0, calcium pantothenate 10.0, niacin 10.0, biotin 0.05, folic acid 0.125, cyanocobalamin 0.01, i-inositol 50.0, p-aminobenzoic acid 62.5, and choline chloride 500. Fat-solubIe vitamins were given by oral supplementation of 2 drops/week of fortified Haliver Oil supplying 400 I.U. of vitamin A, 4 I.U. of vitamin D, 6.4 mg. of a-tocopherol, and 0.08 mg. of menadione. Twenty-four per cent protein was supplied either by casein or pork. Iodinated casein* was included in each diet at a level of either 0.4 or 0.5% (see table for specific diets), and sucrose was added to make each diet to 100%. The pork used was prepared as follows: Pork butts purchased from a retail store were partially freed of fat by slicing off exterior layers. The butts were then cooked at 260°C. (500°F.) for 4% hr., ground in a meat grinder, and dried in shallow pans in circulating air at 70°C. The pork was then extracted with petroleum ether (Skelly B) and ground to pass a 20.mesh screen. The resulting pork contained 94% protein (6.25 X N). RESULTS
Although casein is known to contain lower levels of certain amino acids than muscle tissue (13), supplementation of the casein basal diet with amino acids to equal the levels in muscle, and inclusion of a high level of the potential -SH compound cystine and the non-essential amino acid glycine did not increase the survival time of hyperthyroid rats. These data are presented in Table I (Expt. I). In agreement with Tappan et al. (ll), pork was shown to support survival of hyperthyroid rats much better than casein. It seemed important to study further the supplementation of the casein basal diet and its effect on survival. Thus, various essential and nonessential amino acids were added to the casein basal diet, and their effect on survival was determined. Table I (Expt. II) shows the results of this experiment. It can be seenthat supplementation with these amino acids markedly increased the survival time of the hyperthyroid rats. In order to determine which amino acids supplemented in Expt. II were responsible for the increased survival, each one was added separately to the casein control. As shown in the results of Expt. III, methionine exerted the greatest protective effect. Phenylalanine, serine, and alanine also showed some protective action, and again the combination of amino acids markedly prolonged survival. It can be seen in Table I (Expt. III) that supplementation of the casein basal diet with 0.6 % methionine either alone or in combination with other amino acids severely inhibited growth. Since the casein basal * Protamone,
Cerophyl
Laboratories,
Kansas City, MO.
E$ect
of Supplements and Survival
TABLE I of Various Amino of Hyperthyroid
Acids Rats Weight gain (2 weeks)
Expt.
on Growth Number surviving 60 days
Average survivala
g.lweeR
days
16
1. Casein 2. Casein + amino acid mix Ad 3. Pork
27.0 28.1 25.9
24.0 f 20.7 f 50.2 f
IIb
1. Casein 2. Casein + amino acid mix Be 3. Pork
29.7 19.4 30.7
21.3 f 3.5 47.5 f 5.5 50.2 f 6.5
0 2 4
25.7 28.8 18.4 28.6 25.0 25.5 23.3 28.8 18.1 29.6
23.5 32.5 45.2 32.5 27.2 28.3 24.5 31.8 52.3 54.2
f 1.1 f 5.3 f 3.7 f 4.2 f 2.8 f 1.5 i 3.2 f 2.0 i 3.5 f 4.7
0 0 1 0 0 0 0 0 1 2
22.5 23.0 24.0 25.0
26.5 35.8 43.0 57.3
f * f f
1.0 3.4 6.6 2.7
0 0 0 5
27.5 22.0 18.0 25.0 24.0 22.0 18.5
27.8 32.0 41.0 33.2 35.8 46.4 34.3
i f f i f f i
2.4 5.6 3.3 1.5 2.6 4.0 1.9
0 0 0 0 0 1 0
IIIf
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
IV’
1. 2. 3. 4.
Vf
1. 2. 3. 4. 5. 6. 7.
Casein Casein Casein Casein Casein Casein Casein Casein Casein Pork
+ + + + + + + +
0.7% 0.6% 1.0% 0.9% 0.5yo 0.7% 1.0% amino
nn-phenylalanine nn-methionine DL-serine L-leucine nn-isoleucine nn-valine nn-alanine acid mix Be
Casein Casein + 0.3% nn-methionine Casein + 0.3% betaine.HCl Pork
Casein Casein Casein Casein Casein Casein Casein 0.3% 8. Pork
-I- 0.3% nn-methionine + 0.6% nn-methionine + 0.3% betaine.HCl + 0.6% betaine.HCl + 1.0% betaine.HCl + 0.3% nn-methionine betaine.HCl
+
26.0
1.4c 0.9 2.6
0 0 2
54.7 f 3.8 4 0 Mean f standard error of the mean for a group of six rats. b All diets of this experiment contained 0.5% iodinated oasein. c Mean ZIZstandard error of the mean for a group of 12 rats. d Amino acid mix A contained 0.84yo L-arginine .HCl, 0.03yo L-histidine. HCl, 0.36yo L-lysine.HCl, 0.03% nr,-tryptophan, 0.49% L-cystine, 0.24% nn-threonine, and 0.70yo glycine. e Amino acid mix B contained a combination of all amino acids fed (at the same levels) in groups 2-8 of Expt. III. f All diets of this experiment contained 0.4% iodinated casein. 439
440
BOLDT,
HARPER
AND
ELVEHJEM
diet itself contains about 0.9 % methionine, this represents a total methionine content of 1.5 %. This level is known to retard the growth of normal rats (14). Thus the argument might be set forth that feeding levels of methionine that retard growth reduces feed intake thereby decreasing the amount of iodinated casein ingested, leading to increased survival. For this reason, a lower level of methionine was supplemented in order to avoid the growth reduction but still demonstrate its protective action. Since methionine is known to act as a methyl donor, it was felt that its effect on survival might be due to this property, and that another methyl donor might also show a protective action toward hyperthyroid rats. For this reason betaine was also tested for its survival-enhancing action. Table I (Expt. IV) shows that a lower level of methionine, supporting growth equal to the casein control, did indeed still enhance the survival of hyperthyroid rats. Betaine, also fed at the level of 0.3%, was observed to prolong survival markedly. The results presented in Table I (Expt. V) show that feeding higher levels of betaine results in improved survival of hyperthyroid rats. By using the relatively less toxic betaine, it was possible to feed higher levels of this methyl donor than was the case with methionine, and it can be seen that the higher levels resulted in still better survival. DISCUSSION
From the data presented in this paper, it appears that certain amino acids, especially methionine, have the ability to prolong the survival time of hyperthyroid rats. That this ability may be due to the labile methyl group of methionine is supported by the evidence that betaine, another labile methyl-containing compound, also exerts a protective action. Ershoff (15) in 1947 also tested various compounds, including methionine, for their effects on the length of survival of the immature thyroidfed rat. Feeding levels of 0.3 and 0.6 % of nn-methionine, he observed no increase in survival time. His diets, however, were deficient in both vitamin Blz and folic acid. A deficiency of folic acid has been shown by Stekol et al. (16) to decrease the utilization of methionine for choline and creatine synthesis. The question as to functions of vitamin B~z aside from its role in the de novo synthesis of methyl groups remains to be resolved. Thus it seems possible that the failure to observe a response to methionine may have been due to the lack of these vitamins in E&off’s diets.
AMINO
ACIDS
AND
HYPERTHYROID
RAT
441
O’Dell et al. (17), although not presenting the details of the experiment, have reported that a mixture of glycine, methionine, and arginine did not stimulate the growth of hyperthyroid rats. In 1942, however, Stetten and Grail (18) showed that additions of guanidoacetic acid to a diet limiting in methionine caused decreased choline in liver lipides and a markedly fatty liver. He explained these results on the basis of a draining of the available methionine for the irreversible methylation of guanidoacetic acid to creatine. Thus under the conditions of O’Dell, it would seem that the concurrent addition of glycine and a&nine creates a demand for the methyl group of methionine for synthesizing creatine, therefore making the added methionine unavailable for other transmethylations. Although the protective effect of casein can be made to approach that of pork by supplementation of the casein with high levels of methionine, this cannot be the basis for the superiority of pork since pork and casein contain approximately the same amount of methionine.3 It has also been shown (19) that the availability of the methionine in pork and casein is approximately equal. Thus the explanation for the action of pork must await further investigation. SUMMARY
Methionine, added to a casein diet which is complete for normal rats, markedly increases the survival time of hyperthyroid rats. That the improved survival is due to the labile methyl group of methionine is supported by the fact that betaine, another labile methyl-containing compound, also increases the survival time of hyperthyroid rats. REFERENCES 1. 2. 3. 4.
5. 6. 7.
8. 9. 10. 11.
DRILL, V. A., Physiol. Revs. !Z3, 355 (1943). DRILL, V. A., Proc. Sot. Ezptl. Biol. Med. 39,313 (1938). DRILL, V. A., AND OVERMAN, R., Am. J. Physiol. 136, 474 (1924). MARTIN, G. J., Am. J. Digest. Diseases 14, 341 (1947). EMERSON, G. A., Proc. Sot. Exptl. Biol. Med. 70,392 (1949). BETHEIL, J. J., AND LARDY, H. A., J. Nutrition 37,495 (1949). ABELIN, I., Klin. Wochschr. 6,367 (1926). ZAIN, H., Arch. exptl. Pathol. Pharmakol. 187,302 (1937). ERSHOFF, B. H., Proc. Sot. Ezptl. Biol. Med. 64, 500 (1947). ERSHOFF, B. H., Proc. Sot. Ezptl. Biol. Med. 74, 391 (1950). TAPPAN, D. V., BOLDT, R. E., AND ELVEHJEM, C. A., Proc. Sot. Med. 83, 135 (1953).
3 M.
L. Orr
and B. K. Watt,
personal
cummunication.
Exptl. Biol.
442
BOLDT,
12. HEGSTED, D.M.,
14. 15. 16. 17. 18.
19.
AND
ELVEHJEM
MILLS, R.C., ELVEHJEM,~. A., AND HART, E.B., J.BioZ. 138, 459 (1941). BLOCK, R. J., AND BOLLINO, D., “The Amino Acid Composition of Proteins and Foods,” 2nd. ed. Charles C Thomas, Springfield, Ill., 1951. WRETLIND, K. A. J., AND ROSE, W. C., J. Biol. Chem. 187, 697 (1950). ERSHOFF, B. H., Arch. Biochem. 16, 365 (1947). STEKOL, J. A., in “Amino Acid Metabolism” (McElroy, W. D., and Glass, B., eds.), p. 540. The Johns Hopkins Press, Baltimore, Md., 1955. O'DELL, B. L., STOLZENBERG, S.J.,BRUEMMER, J. H., AND HOGAN, A. G., Arch. Biochem. Biophys. 64, 232 (1955). STETTEN, D., JR., AND GRAIL, G. F., J. Biol. Chem. 144, 175 (1942). SCHWEIGERT, B. S., AND GUTHNECK, B. T., J. Nutrition 64,333 (1954). Chem.
13.
HARPER