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The Complex Nature of the Animal Protein Factor
750
RESEARCH NOTES
(Nutrisoy) 40.0, fish liver oil 1.0, dicalcium phosphate 2.0, limestone 1.0, iodized salt 0.48, and manganese sulfate 0.02, plus supplementary amounts of all B-group vitamins (except B12) and vitamins E and K. TABLE 1.—Betaine, choline and vitamin Bn interrelationships in chick nutrition
Supplement None 0.2% choline CI 0.2% betaine HC1 Purified Bi2* Purified Bi 2 +choline CI Purified B12+betaine HC1 Crystalline B12, injected Crystalline B12, injected+betaine HC1
Number of chicks
Weight 4th week
17 IS 14 16 18 18 S
132 178 170 276 277 278 268
5
276
* Furnished 1.0 microgram of vitamin B12 per 100 grams of diet.
The results are presented in Table 1. They show that the effect of the APF concentrate previously found to reduce the requirement for methylating compounds by the chick is due to vitamin B12 content. The findings are in agreement with those of Schaefer, Salmon and Strength (1949 a, b) who have presented
evidence of an interrelationship between vitamin B12 and choline in chicks, and between vitamin Bj2 and choline and methionine in rats when these species were fed special experimental diets high in fat and low in methionine and choline. Vitamin B12, however, performs other functions necessary for chick growth since the response obtained with this vitamin in our work and that of Schaefer and associates was much greater under the experimental conditions than that which was obtained with methylating agents alone. REFERENCES
Craig, L. C. 1944. Identification of small amounts of organic compounds by distribution studies. II. Separation by counter current distribution. J. Biol. Chem. 155:519-534. Gillis, M. B. and L. C. Norris. 1949. Effect of the animal protein factor on the requirement for methylating compounds. J. Biol. Chem. 179: 487-488. Schaefer, A. E., W. D. Salmon and D. R. Strength. 1949a. Interrelationship of vitamin B12 and choline. I. Effect on hemorrhagic kidney syndrome in the rat. Proc. Soc. Exp. Biol. Med. 71: 193-196. ———. 1949b. Interrelationship of vitamin B12 and choline. I. Effect on growth of the chick. Proc. Soc. Exp. Biol. Med.-71: 202-204.
THE COMPLEX NATURE OF THE ANIMAL PROTEIN FACTOR C. W. CARLSON, R. F. MILLER, H. T. PEELER, L. C. NORRIS AND G. F. HEUSER Agricultural Experiment Station and School of Nutrition, Cornell University, Ithaca, N. Y. (Received for publication July 27, 1949)
Pure vitamin B12 has been reported by several groups of workers to promote growth responses in chicks, under their experimental conditions, comparable to those obtained with crude sources of the animal protein factor. The experiment reported here, however, furnishes evidence for the existence of additional factors in fish meal and in some batches of dried brewers' yeast.
The basal diet fed in this experiment consisted of 23.5% alpha-protein (The Glidden Company), 65.0% cerelose, 2% soybean oil, 3 % cellophane, 0.3% Lcystine, 0.3% D-L methionine, 0.3% glycine, 1% fish liver oil (2000 AOAC units of vitamin A and 400 IU of vitamin D per gram), 1.1% pulverized limestone, 1.7% dicalcium phosphate, 0.8% dibasic potassium phosphate, 0.5% salt, 0.5% mag-
RESEARCH NOTES
nesium sulfate and the trace minerals and vitamins as described by Hill et al. (1944) except that the thiamine HC1 was increased to 1 mg and 50 fig of folic acid and 1.16 ng of vitamin Bi2 were added per 100 grams of diet. The vitamin B12 was supplied by means of a purified preparation made in this laboratory from U.S.P. liver extract according to the counter current distribution procedure of Craig (1944). The concentration of the vitamin was determined by assay with Lactobacillus leichmannii (ATCC 4797) using crystalline vitamin B12 as a reference. This preparation was also shown in studies with chicks fed cereal-soybean meal experimental diets to be an effective substitute for crystalline vitamin B12. The alpha-protein was assayed for all essential amino acids required by the chick except glycine. The results showed that the basal diet was deficient in methionine. This deficiency was corrected by supplementation with methionine and cystine. Supplementation with glycine was also made, although the presence of a deficiency of this amino acid seemed highly improbable in view of the reported glycine content of soybean proteins. White Leghorn day-old chicks of mixed sex, hatched from hens receiving no animal protein, were used in the experiment. Each lot contained 20 chicks. Fish meal and dried brewers' yeast (AnheuserBusch, Strain S) were used as sources of the unidentified growth factors. They were included in the diet in place of cerelose and alpha-protein, so as to keep the protein constant at 20%. The results are presented in Table 1. The results showed that fish meal and dried brewers' yeast greatly improved the rate of growth and prevented mortality in chicks fed a basal diet adequate in vitamin Bi2. Preceding death the chicks in
751
TABLE 1.—Results showing the chick's requirement for an unidentified factor(s) in addition to vitamin Bn Bl 2
Supplement
None Vitamin Bi2 7.5% dried brewers' yeast 3 % fish meal 7.5% dried brewers' yeast + 3 % fish meal
content of diets
Ave. No. wt. chicks at 4 at 4 weeks weeks
7/100 gm. 1.16 2.90 1.24 1.47
gm. 108 128 248 217
4 3 18 16
1.55
253
19
lots receiving only this vitamin showed symptoms of a partial or complete paralysis. A few of the chicks were also afflicted with tremors and had hemorrhages which were not typical of vitamin K deficiency. This high mortality may be the result of a severe deficiency of the factor reported by Hill (1948) to be present in dried whey, since mortality preceded by paralysis has not been experienced here on all-vegetable type of basal diets containing 70% soybean meal. Soybean meal, at 33% of the diet, was shown by Hill (1948) at times to supply an insufficient amount of the whey factor. He also found the factor to be present in fish meal. The chick growth activities of the various soybean meals studied by Hill (1948) have been shown by Daniel et al. (1949) to be directly correlated with "whey factor" activity as measured by Lactobacillus casei. Daniel et al. (1949) also found that L. casei responds to another factor present in fish meal, liver paste (Wilson and Company) and dried brewers' yeast. This unidentified factor cannot be the same as vitamin B12 since L. casei has been shown not to require vitamin B12. Liver paste, however, was found to contain little "whey factor" activity as measured by this organism. The yeast used in the chick work reported herein was found to be, with one exception, richer in both L. casei
752
RESEARCH NOTES
factors than any of the other yeasts studied (unpublished results). The results of work to be reported elsewhere show that liver paste dialysate and fractions prepared from it by counter current distribution contain a factor required for the growth of chicks fed an all-vegetable diet adequate in the whey factor of Hill (1948). The growth response obtained from these fractions cannot be due to vitamin B^, since they were almost entirely free of this vitamin as determined by assay with L. leichmannii. The chick growth factor may be identical with the second L. casei factor since the latter was found to be present in the fractions. Fish meal and a special batch of dried brewers' yeast have been found to contain an unidentified factor(s) required for the growth of chicks fed a purified diet adequate in vitamin B12. In view of work done by Hill (1948), Daniel et al. (1949) and unpublished work done at this laboratory it appears probable that the growth response obtained from fish meal and the dried brewers' yeast was due to two factors rather than one. The animal protein factor complex appears, therefore, to be composed of vitamin B12 and two unidentified factors, all of which are required for chick growth.
REFERENCES •
Craig, L. C. 1944. Identification of small amounts of organic compounds by distribution studies. II. Separation by counter current distribution. J. Biol. Chem. 155: 519-534. Daniel, Louise J., H. T. Peeler, L. C. Norris and M. L. Scott. 1949. Unidentified factors required by Lactobacillus casei. IV. Evidence for two growth factors in purified liver extracts. J . Biol. Chem. 177: 917-926. Hill, F. W., L. C. Norris and G. F. Heuser. 1944. Studies of unidentified vitamins required by the chick. J. Nutr. 28: 175-188. Hill, F. W. 1948. The multiple nature of the deficiency of unidentified nutrients in crude allvegetable protein chick starter rations. Poultry Sci. 27: 536-541. ACKNOWLEDGMENTS
This work was aided by grants to Cornell University from the Nutrition Foundation, New York, the Cerophyl Laboratories, Kansas City, Missouri, Commercial Solvents Corporation, New York, the Cooperative G. L. F. Exchange, Ithaca, New York, and the Western Condensing Company, San Francisco, California. The assistance of Dr. H. H. Williams of the Department of Biochemistry, Cornell University, in making the amino acid analysis of the alpha-protein is appreciated.
ACTIVITY OF A MICROBIAL ANIMAL PROTEIN FACTOR CONCENTRATE IN A HIGH ENERGY BROILER RATION* A. C. WIESE, C. F. PETERSEN AND C. E. LAMPMAN Department of Agricultural Chemistry and Poultry Husbandry, University of Idaho, Moscow (Received for publication August 5, 1949)
The use of high-energy low-fiber broiler rations containing large amounts of animal proteins has come at a time when * Published with the approval of the Director of the Idaho Agricultural Experiment Station as Research Paper Number 305.
animal products for livestock feeding are extremely scarce and costly. Almost simultaneously animal protein factor (Bi2) preparations have become available as supplements for poultry rations. Crystalline vitamin Bi2 was shown by Ott, et al.
RESEARCH NOTES
(Nutrisoy) 40.0, fish liver oil 1.0, dicalcium phosphate 2.0, limestone 1.0, iodized salt 0.48, and manganese sulfate 0.02, plus supplementary amounts of all B-group vitamins (except B12) and vitamins E and K. TABLE 1.—Betaine, choline and vitamin Bn interrelationships in chick nutrition
Supplement None 0.2% choline CI 0.2% betaine HC1 Purified Bi2* Purified Bi 2 +choline CI Purified B12+betaine HC1 Crystalline B12, injected Crystalline B12, injected+betaine HC1
Number of chicks
Weight 4th week
17 IS 14 16 18 18 S
132 178 170 276 277 278 268
5
276
* Furnished 1.0 microgram of vitamin B12 per 100 grams of diet.
The results are presented in Table 1. They show that the effect of the APF concentrate previously found to reduce the requirement for methylating compounds by the chick is due to vitamin B12 content. The findings are in agreement with those of Schaefer, Salmon and Strength (1949 a, b) who have presented
evidence of an interrelationship between vitamin B12 and choline in chicks, and between vitamin Bj2 and choline and methionine in rats when these species were fed special experimental diets high in fat and low in methionine and choline. Vitamin B12, however, performs other functions necessary for chick growth since the response obtained with this vitamin in our work and that of Schaefer and associates was much greater under the experimental conditions than that which was obtained with methylating agents alone. REFERENCES
Craig, L. C. 1944. Identification of small amounts of organic compounds by distribution studies. II. Separation by counter current distribution. J. Biol. Chem. 155:519-534. Gillis, M. B. and L. C. Norris. 1949. Effect of the animal protein factor on the requirement for methylating compounds. J. Biol. Chem. 179: 487-488. Schaefer, A. E., W. D. Salmon and D. R. Strength. 1949a. Interrelationship of vitamin B12 and choline. I. Effect on hemorrhagic kidney syndrome in the rat. Proc. Soc. Exp. Biol. Med. 71: 193-196. ———. 1949b. Interrelationship of vitamin B12 and choline. I. Effect on growth of the chick. Proc. Soc. Exp. Biol. Med.-71: 202-204.
THE COMPLEX NATURE OF THE ANIMAL PROTEIN FACTOR C. W. CARLSON, R. F. MILLER, H. T. PEELER, L. C. NORRIS AND G. F. HEUSER Agricultural Experiment Station and School of Nutrition, Cornell University, Ithaca, N. Y. (Received for publication July 27, 1949)
Pure vitamin B12 has been reported by several groups of workers to promote growth responses in chicks, under their experimental conditions, comparable to those obtained with crude sources of the animal protein factor. The experiment reported here, however, furnishes evidence for the existence of additional factors in fish meal and in some batches of dried brewers' yeast.
The basal diet fed in this experiment consisted of 23.5% alpha-protein (The Glidden Company), 65.0% cerelose, 2% soybean oil, 3 % cellophane, 0.3% Lcystine, 0.3% D-L methionine, 0.3% glycine, 1% fish liver oil (2000 AOAC units of vitamin A and 400 IU of vitamin D per gram), 1.1% pulverized limestone, 1.7% dicalcium phosphate, 0.8% dibasic potassium phosphate, 0.5% salt, 0.5% mag-
RESEARCH NOTES
nesium sulfate and the trace minerals and vitamins as described by Hill et al. (1944) except that the thiamine HC1 was increased to 1 mg and 50 fig of folic acid and 1.16 ng of vitamin Bi2 were added per 100 grams of diet. The vitamin B12 was supplied by means of a purified preparation made in this laboratory from U.S.P. liver extract according to the counter current distribution procedure of Craig (1944). The concentration of the vitamin was determined by assay with Lactobacillus leichmannii (ATCC 4797) using crystalline vitamin B12 as a reference. This preparation was also shown in studies with chicks fed cereal-soybean meal experimental diets to be an effective substitute for crystalline vitamin B12. The alpha-protein was assayed for all essential amino acids required by the chick except glycine. The results showed that the basal diet was deficient in methionine. This deficiency was corrected by supplementation with methionine and cystine. Supplementation with glycine was also made, although the presence of a deficiency of this amino acid seemed highly improbable in view of the reported glycine content of soybean proteins. White Leghorn day-old chicks of mixed sex, hatched from hens receiving no animal protein, were used in the experiment. Each lot contained 20 chicks. Fish meal and dried brewers' yeast (AnheuserBusch, Strain S) were used as sources of the unidentified growth factors. They were included in the diet in place of cerelose and alpha-protein, so as to keep the protein constant at 20%. The results are presented in Table 1. The results showed that fish meal and dried brewers' yeast greatly improved the rate of growth and prevented mortality in chicks fed a basal diet adequate in vitamin Bi2. Preceding death the chicks in
751
TABLE 1.—Results showing the chick's requirement for an unidentified factor(s) in addition to vitamin Bn Bl 2
Supplement
None Vitamin Bi2 7.5% dried brewers' yeast 3 % fish meal 7.5% dried brewers' yeast + 3 % fish meal
content of diets
Ave. No. wt. chicks at 4 at 4 weeks weeks
7/100 gm. 1.16 2.90 1.24 1.47
gm. 108 128 248 217
4 3 18 16
1.55
253
19
lots receiving only this vitamin showed symptoms of a partial or complete paralysis. A few of the chicks were also afflicted with tremors and had hemorrhages which were not typical of vitamin K deficiency. This high mortality may be the result of a severe deficiency of the factor reported by Hill (1948) to be present in dried whey, since mortality preceded by paralysis has not been experienced here on all-vegetable type of basal diets containing 70% soybean meal. Soybean meal, at 33% of the diet, was shown by Hill (1948) at times to supply an insufficient amount of the whey factor. He also found the factor to be present in fish meal. The chick growth activities of the various soybean meals studied by Hill (1948) have been shown by Daniel et al. (1949) to be directly correlated with "whey factor" activity as measured by Lactobacillus casei. Daniel et al. (1949) also found that L. casei responds to another factor present in fish meal, liver paste (Wilson and Company) and dried brewers' yeast. This unidentified factor cannot be the same as vitamin B12 since L. casei has been shown not to require vitamin B12. Liver paste, however, was found to contain little "whey factor" activity as measured by this organism. The yeast used in the chick work reported herein was found to be, with one exception, richer in both L. casei
752
RESEARCH NOTES
factors than any of the other yeasts studied (unpublished results). The results of work to be reported elsewhere show that liver paste dialysate and fractions prepared from it by counter current distribution contain a factor required for the growth of chicks fed an all-vegetable diet adequate in the whey factor of Hill (1948). The growth response obtained from these fractions cannot be due to vitamin B^, since they were almost entirely free of this vitamin as determined by assay with L. leichmannii. The chick growth factor may be identical with the second L. casei factor since the latter was found to be present in the fractions. Fish meal and a special batch of dried brewers' yeast have been found to contain an unidentified factor(s) required for the growth of chicks fed a purified diet adequate in vitamin B12. In view of work done by Hill (1948), Daniel et al. (1949) and unpublished work done at this laboratory it appears probable that the growth response obtained from fish meal and the dried brewers' yeast was due to two factors rather than one. The animal protein factor complex appears, therefore, to be composed of vitamin B12 and two unidentified factors, all of which are required for chick growth.
REFERENCES •
Craig, L. C. 1944. Identification of small amounts of organic compounds by distribution studies. II. Separation by counter current distribution. J. Biol. Chem. 155: 519-534. Daniel, Louise J., H. T. Peeler, L. C. Norris and M. L. Scott. 1949. Unidentified factors required by Lactobacillus casei. IV. Evidence for two growth factors in purified liver extracts. J . Biol. Chem. 177: 917-926. Hill, F. W., L. C. Norris and G. F. Heuser. 1944. Studies of unidentified vitamins required by the chick. J. Nutr. 28: 175-188. Hill, F. W. 1948. The multiple nature of the deficiency of unidentified nutrients in crude allvegetable protein chick starter rations. Poultry Sci. 27: 536-541. ACKNOWLEDGMENTS
This work was aided by grants to Cornell University from the Nutrition Foundation, New York, the Cerophyl Laboratories, Kansas City, Missouri, Commercial Solvents Corporation, New York, the Cooperative G. L. F. Exchange, Ithaca, New York, and the Western Condensing Company, San Francisco, California. The assistance of Dr. H. H. Williams of the Department of Biochemistry, Cornell University, in making the amino acid analysis of the alpha-protein is appreciated.
ACTIVITY OF A MICROBIAL ANIMAL PROTEIN FACTOR CONCENTRATE IN A HIGH ENERGY BROILER RATION* A. C. WIESE, C. F. PETERSEN AND C. E. LAMPMAN Department of Agricultural Chemistry and Poultry Husbandry, University of Idaho, Moscow (Received for publication August 5, 1949)
The use of high-energy low-fiber broiler rations containing large amounts of animal proteins has come at a time when * Published with the approval of the Director of the Idaho Agricultural Experiment Station as Research Paper Number 305.
animal products for livestock feeding are extremely scarce and costly. Almost simultaneously animal protein factor (Bi2) preparations have become available as supplements for poultry rations. Crystalline vitamin Bi2 was shown by Ott, et al.