THE ability to methylate homocystine

Transcription

1 Homocystine, Vitamin Bi 2, Choline, and Methionine in the Nutrition of the Laying Fowl B. E. WELCH AND J. R. COUCH Departments of Poultry Husbandry and Biochemistry and Nutrition, Texas Agricultural Experiment Station, College Station, Texas THE ability to methylate homocystine to form methionine has been reported for the chick (Jukes et al., 1950, 1951, 1952; Patrick, 1952) and for the rat (Bennett, 1950; Stekol and Weiss, 1950). These workers have further reported that vitamin Bi 2 enhances this conversion. Vitamin B12 has been shown to influence the response to choline in the case of chicks (Gillis and Norris, 1951; Briggs et al, 1950; Schaeffer et al, 1949) and also in the case of rats (Stekol and Weiss, 1950; Bennett et al, 1951). Abbott and De- Masters (1940) and Lucas et al. (1946) have reported a need for choline in the diet of the mature fowl. It has been suggested that choline can be synthesized by the mature fowl (Lucas et al, 1946; Ringrose and Davis, 1946). The latter workers also observed an apparent need for methionine in the diet to serve as a source of labile methyl groups. Leong and McGinnis (1952) and Ingram et al. (1951) have reported that egg production was decreased by a methionine deficiency. The work reported here was carried out in order to determine (1), if homocystine could be methylated by the mature fowl and if so, could the compound be used in a manner similar to that previously described by other workers; (2), if choline was essential in the diet of the mature fowl; and (3), what effect these treatments would have on the Bi 2 content of the livers of the hens and of the egg yolks, on the choline content of the livers, and on the methionine content of the blood plasma. (Received for publication June 7, 1954) MATERIAL AND METHODS The Single Comb White Leghorn pullets used in this experiment had been reared on range and were placed in individual laying cages at approximately 5 months of age. The birds were fed a practical ration adequate in vitamin B12 and methionine prior to being placed on experiment. After a pre-experimental period of 6 weeks, during which time individual egg production and hatchability records were kept, 12 groups of 6 birds each were selected for the experiment reported herein. The management and experimental procedures with regard to the care and handling of the birds, insemination, and incubation were the same as those previously reported by Olcese et al. (1950). All eggs laid by the hens, except those assayed for vitamin B12, were set and hatchability data ascertained. The diet used consisted of 50% ground green split peas, 37.5% glucose (cerelose), 6.0% gelatin, 5.0% salts IV (Hegsted et al, 1941), 1.5% refined soybean oil. The following were added in the specified number of milligrams per kilogram: thiamine hydrochloride, 10.0; riboflavin, 10.0; pyridoxine hydrochloride, 10.0; niacin, 50.0; calcium pantothenate, 50.0; folic acid, 2.0; biotin, 0.2; 2-methyl-l,4-naphthoquinone, 0.5; alpha-tocopheryl acetate, 4.5; aureomycin hydrochloride, 20.0; inositol, 1,000.0, and tryptophane, 2, Vitamins A and D were supplied in all diets at the level of 10,000 I.U. and 1500 I.C.U. per kilogram, respectively. 217

2 218 B. E. WELCH AND J. R. COUCH The first group received the basal diet unsupplemented. The second and third groups received the basal diet plus homocystine and vitamin B12, respectively. The fourth group received the basal diet plus a combination of homocystine and vitamin B12. The fifth, sixth, and seventh groups received choline, choline plus vitamin Bi 2, and choline plus homocystine, respectively. The eighth group received choline plus B12 plus homocystine. The ninth, tenth, and eleventh groups received methionine, methionine plus B12, and methionine plus choline, respectively. The twelfth group received methionine plus vitamin B i2 plus choline. Vitamin B12 was added to the diet at the level of 50 micrograms per kilogram. Choline was added at the level of 2 grams per kilogram, and homocystine and methionine were added at the level of 6 grams per kilogram. The homocystine used in this experiment was prepared as described by Jukes and Stokstad (1952). This preparation contained less than 1% methionine by microbiological assay. The ground, green split peas were assayed microbiologically for methionine and were found to contain 0.42%. Assuming that gelatin contains 0.8% (Block and Boiling, 1951), this would indicate that the diet contained about 0.26% methionine. The diet also contained approximately 0.145% choline, since, according to Engel (1943), the peas contained approximately 0.29% choline. Two separate experiments were run, each for 7 weeks, using the same experimental hens, with a 4-week readjustment period between them. A practical ration, adequate in vitamin Bi 2 and methionine was fed during the adjustment period. In both experiments, egg production and hatchability data were tabulated and in the last trial, eggs from each group were analyzed weekly for vitamin B12. Results obtained from the separate experiments were so similar that it was believed advisable to combine the data. At the end of the second 7-week test, the hens were sacrificed and analyses made on the livers for vitamin B12 and choline and on the blood plasma for methionine. The B12 analyses were carried out according to the procedure of Skeggs et al. (1950), using Lactobacillus leichmannii 4797 as the test organism. The B^ was released from the egg yolks according to the procedure of Halick and Couch (1951). Since potassium cyanide was not used, the B12 values reported are relative and not exact. Vitamin B12 was released from the liver samples by overnight incubations with pancreatin at 37 C. The choline content of the liver was determined by the method of Horowitz and Beadle (1943), using Neurospora crassa cholineless No , A.T.C.C The choline was released by autoclaving in three normal hydrochloric acid for 2 hours. Methionine was determined by the method of Lyman et al. (1946), using Leuconostoc mesenteroides P-60. The methionine was released by the use of sodium tungstate and sulfuric acid. RESULTS AND DISCUSSION Egg Production. An increase in egg production was obtained when the experimental diet was supplemented with vitamin B12 and homocystine (Table 1). There was a consistent decrease in egg production in all groups for the first 3 weeks of the experiment, indicating a depletion of stored factors. After that period of time, the group fed vitamin B12 and homocystine exhibited a slight increase in egg production. Vitamin B i2 or homocystine alone did not appear to affect egg production. The latter provides evidence for the fact that methionine is probably the most limiting factor in this diet, since an increased egg production was obtained upon addition of methionine to the basal diet

3 HOMOCYSTINE, VlTAMIN B12, CHOLINE AND METHIONINE 219 TABLE 1. The effect of vitamin Bu, homocystine, choline and methionine upon egg production 1 Percent Production Supplement to basal 2 Time in weeks Average 3 None Homocystine B12 Homocystine+Bi 2 Choline Choline+Bis Choline+Homocystine Choline-j-Bi2+Homocystine Methionine Methionine+Bi2 Methionine+Choline Methionine+Bi2+Choline * Combined results of two separate experiments. 2 Level of supplements: Bi*, 50 jug./kg.; choline, 2 gm./kg.; homocystine and methionine, 6 gm./kg. 3 L.S.D. at.01 level was Significant at the 0.01 level. (Table 1). When both Bi 2 and homocystine were added to the diet, some methionine was formed through the methylation of homocystine. The effect of vitamin B12 is presumably due to its influence on the synthesis of methyl groups, since Firth et al. (1954) and Stekol et al. (1953) reported that B i2 was not directly concerned with transmethylation. It should be emphasized that the pullets used in this study were not depleted of B J2, and that a more definite effect might have been obtained if the birds had been depleted of Bi 2 prior to the initiation of this test. Egg production was decidedly increased when choline was added to the basal diet in the presence of homocystine (Table 1). Eg/* production was not further increased by adding Bi 2 to the diet containing choline and homocystine. The egg production obtained by adding B i2 to the diet containing choline and homocystine was almost equivalent to that which was produced by adding methionine (Table 1). This is interpreted to mean that the laying hen is capable of methylating homocystine to form methionine. The data of this study were treated statistically by the analysis of variance (Snedecor, 1946); an F value of 24.5 was obtained while a value of 2.54 was required for significance at the 0.01 level of probability. The addition of vitamin Bi 2 or choline had little if any effect on the egg production. On the other hand, the hens probably synthesized methionine when a precursor of methionine, homocystine, was provided, and when a methyl donor, choline, was made available in an optimal amount. Egg production obtained by supplementation of the basal diet with choline and homocystine, or choline, homocystine and vitamin Bi 2 was significantly increased over that observed by the addition of vitamin B i2 and homocystine (Table 1). Further evidence for the fact that methionine is the first limiting factor in the basal diet is shown in Table 1. The egg production obtained by the addition of methionine alone to the basal diet is equivalent to that which resulted from the the addition of choline and homocystine or choline, homocystine, and vitamin Bi 2. Egg production was not increased to an

4 220 B. E. WELCH AND J. R. COUCH appreciable extent by the addition of B i2 in the presence of methionine. A significant increase did occur when choline was fed in the presence of methionine over the methionine supplemented group. The best egg production was obtained by adding B12, choline, and methionine to the basal diet. The average percentage egg production for the third through the seventh week (first and second weeks not considered) was basal, 13.5; homocystine, 8.5; vitamin B i2, 10.1; homocystine and vitamin Bi 2, 24.2; choline, 13.5; choline and vitamin Bi 2, 19.4; choline and homocystine, 43.5; choline, homocystine, and vitamin Bi 2, 38.3; methionine 43.1; methionine and vitamin Bi 2, 49.7; methionine and choline, 57.4; methionine, vitamin B12, and choline, From these data it is readily discernible that homocystine may be methylated to form methionine, and there is an indication that the laying hen does have a requirement for choline per se over and above the requirements of choline to form methionine, since the best egg production was obtained by adding vitamin B i2, choline, and methionine to the diet. The latter would be in agreement with the work of Abbott and DeMasters (1940), Ringrose and Davis (1946), and Lucas et al. (1946). It should be pointed out that the percentage of egg production was entirely comparable to that which resulted from feeding a practical ration to hens in laying cages kept under conditions identical to those used in the present experiment. The average percent hatchability for each group over both 7-week test periods was basal, 92.3; homocystine, 100.0; vitamin B12, 93.0; homocystine and vitamin Bi 2, 88.5; choline, 94.5; choline and vitamin Bi 2, 97.3; choline and homocystine, 90.5; choline, vitamin Bi 2, and homocystine, 94.0; methionine, 90.4; methionine and vitamin B12, 93.0; methionine and choline, 93.8; methionine, vitamin B 12, and choline, A lack of vitamin Bi 2 in the diet did not influence the hatchability due to the fact that the birds were maintained on a complete ration prior to being placed on the experiment, and were maintained on the Bi2-deficient diet for only 7 weeks. Methionine Content of the Blood Plasma and the B 12 Content of the Livers and Egg Yolks. At the end of the second experiment, the hens were sacrificed and blood and liver samples taken for analyses. The results of these analyses, along with the results of the weekly analyses of the egg yolks, appear in Table 2. The methylation of homocystine to form methionine is evidenced by the increased amount of methionine found in the blood plasma when the basal diet was supplemented with homocystine and vitamin Bi 2. The importance of vitamin B i2 in this reaction is reflected in the methionine content of the blood plasma of those birds receiving homocystine alone and those fed homocystine and vitamin Bi 2. The addition of vitamin Bi 2 alone produced an increase in the Bi 2 content of both the livers and the egg yolks, in agreement with the results of Couch and Olcese (1950) and Halick and Couch (1951). Supplementation of the basal diet with choline and homocystine or choline, homo- TABLE 2. The effect of homocystine, vitamin Bit, choline and methionine on the Bn content of the liver and egg yolks and on the methionine content of the plasma Supplement 1 None Homocystine Bi, Homocystine+Bi2 Choline Choline+B,, Choline+Homocystine Choline+Bja+Homocystine Methionine Methionine+Bu Methionine+Choline Methionine-i-Bia+Choline Bij (nvig./gm.) Liver Egg yolks Methionine fcig./cc.) Level of supplements: B», 50 pg./kg.; choline, 2 gm./kg.; homocystine and methionine, 6 gm./kg.

5 HOMOCYSTINE, VlTAMIN B12, CHOLINE AND METHIONINE 221 cystine, and vitamin B n resulted in an increased amount of methionine being found in the blood plasma. The amount present in the latter-mentioned groups was not as great as that found in the group supplemented with homocystine and vitamin Bi 2. This might have been due to the differences that were observed in the egg production of these birds. The birds fed only homocystine and vitamin B 12 had a lower rate of egg production than did those which received choline and homocystine or choline, homocystine, and vitamin B 12. Consequently, the birds having a higher rate of egg production were under a much greater stress than those with a lower rate of production, and a lower level of methionine in the blood plasma resulted. The methionine content of the plasma was increased when the amino acid was added to the basal diet. This is in agreement with the results of Richardson et al. (1953). A further increase was noted when methionine and choline were fed together. Supplementation of the ration with methionine and vitamin B 12 or methionine, B12, and choline produced a decrease in the methionine content of the plasma as compared to the level of the amino acid found in the plasma when methionine alone was added to the basal diet. The B i2 content of the egg yolk varied to an appreciable extent only when the diet was supplemented with vitamin B 12 alone or in combination with the other afore-mentioned supplements. This is in agreement with the earlier report of Halick and Couch (1951). The choline content of the hen livers varied from 302 to 420 micrograms per gram and did not appear to be related to the dietary supplements. The deficiency symptoms listed by Abbott and DeMasters (1940), i.e., fatty livers and aborted yolks were not observed in hens which were sacrificed in this experiment. SUMMARY Data are presented which show that homocystine can be methylated to form methionine in the body of the mature fowl. There is an indication that B12 is concerned with this methylation. Data are presented which illustrate the essential nature of choline in the diet of the mature fowl. The hen has a definite requirement for methionine. The rate of synthesis of methionine from homocystine apparently is not great enough to satisfy the requirement of the hen for methionine under the conditions of these experiments. ACKNOWLEDGMENTS Cerelose was supplied through the courtesy of the Corn Products Refining Company, Argo, Illinois. Folic acid was supplied by Lederle Laboratories, Pearl River, New York; biotin by Hoffman-La Roche, Inc., Nutley, New Jersey and the rest of the B-vitamins by Merck and Company, Rahway, New Jersey. Methionine was obtained from Dow Chemical Company, Freeport, Texas. B. E. Welch was the Ralston Purina Fellow during a part of the period when this study was carried out. This work was supported in part by Lederle Laboratories Pearl River, New York; Grasseli Chemical Company, Wilmington, Delaware and the TJ. S. Public Health Service, National Institutes of Health, Bethesda, Maryland under grant No. RG REFERENCES Abbott, O. D., and C. V. DeMasters, Choline in the diet of chickens. J. Nutrition, 19: Bennett, M. A., Utilization of homocystine for growth in presence of vitamin B12 and folic acid. J. Biol. Chem. 187: Bennett, M. A., J. Joralemon and P. E. Halpern, The effect of vitamin B12 on rat growth and fat infiltration of the liver. J. Biol. Chem. 193: Block, R. J., and D. Boiling, The Amino Acid Composition of Proteins and Foods. Springfield, 111., second ed.

6 222 NEWS AND NOTES Briggs, G. M., E. G. Hill and M. J. Giles, Vitamin B12 in all-plant rations for chicks and sparing activity of methionine and choline. Poultry Sci. 29: 723. Couch, J. R., and O. Olcese, The vitamin B J2 content of chick tissues as influenced by the diet. J. Nutrition, 42: Engel, R. W., The choline content of animal and plant products. J. Nutrition, 25: Firth, J., S. P. Mistry, M. F. James and B. C. Johnson, Vitamin B12 and transmethylation in the baby pig. Proc. Soc. Exp. Biol. Med. 85: Gillis, M. B., and L. C. Norris, The effect of vitamin B12 on the response of chicks to betaine and choline. J. Nutrition, 43: Halick, J. V., and J. R. Couch, Antibiotics in mature fowl nutrition. Proc. Soc. Exp. Biol. Med. 76: Hegsted, D. M., R. C. Mills, C. A. Elvehjem and E. B. Hart, Choline in the nutrition of chicks. J. Biol. Chem. 138: Horowitz, V. H., and G. W. Beadle, A microbiological method for the determination of choline by use of a mutant of Neurospora. J. Biol. Chem. 150: Ingram, G. R., W. W. Cravens, C. A. Elvehjem and J. G. Halpin, The methionine requirement of the laying hen. Poultry Sci. 30: Jukes, T. H., E. L. R. Stokstad and H. P. Broquist, Effect of vitamin B12 on the response to homocystine in chicks. Arch. Biochem. 25: Jukes, T. H., and E. L. R. Stokstad, Studies of vitamin Bj2, choline and related factors in the diets of chicks. J. Nutrition, 43: Jukes, T. H., and E. L. R. Stokstad, Further observations on the utilization of homocystine, choline and related compounds by chicks. J. Nutrition, 48: Leong, K. C, and J. McGinnis, An estimate of the methionine requirement for egg production , and as an Instructor in Poultry Husbandry at Washington State College , and Since joining the staff of the Agricultural Research Center, Dr. Brant has conducted research on methods of determining interior quality of eggs, physical properties of eggs related to quality, microbiology of shell eggs, and yields and quality of poultry meat as NEWS AND NOTES {Continued from page 209) Poultry Sci. 31: Lucas, H. L., L. C. Norris and G. F. Heuser, Observations on the choline requirements of hens. Poultry Sci. 25: Lyman, C. M., O. Moseley, B. Butler, S. Wood and F. Hale, The microbiological determinations of amino acids. III. Methionine. J. Biol. Chem. 166: Olcese, O., J. R. Couch and C. M. Lyman, Vitamin B12 concentrates in the nutrition of the mature domestic fowl. J. Nutrition, 41: Patrick, H The effects of homocystine, methionine, vitamin Bj2 and antibiotics in a methionine-deficient chick ration. Poultry Sci. 31: Ringrose, R. C, and H. A. Davis, Choline in the nutrition of laying hens. Poultry Sci. 25: Richardson, L. R., L. G. Blaylock and C. M. Lyman, Influence of dietary amino acids on the free amino acids in blood plasma. Federation Proc. 12:427. Schaefer, A. E., W. D. Salmon and D. R. Strength, Interrelationship of vitamin B12 and choline. II. Effect on growth of the chick. Proc. Soc. Exp. Biol. Med. 71: Skeggs, H. R., H. M. Neeple, K. A. Valentik, J. W. Huff and L. D. Wright, Observations on the use of Lactobacillus leichmannii 4797 in the microbiological assay of vitamin B J2. J. Biol. Chem. 184: Snedecor, G. W., Statistical Methods Applied to Experiments in Agriculture and Biology. Iowa State College Press, Ames. Iowa. Stekol, J. A., and K. Weiss, Vitamin B12 and growth of rats on diets free of methionine and choline. J. Biol. Chem. 186: Stekol, J. A., P. T. Hsu, S. Weiss and P. Smith, Labile methyl groups and its synthesis de novo in relation to growth in chicks. J. Biol. Chem. 203: related to breeding, feeding, management, and processing. Under his direction the work of the poultry research section is being devoted to research on breeding, nutrition, physiology, viability, products, and the National Poultry and Turkey Improvement Plans. {Continued on page 233)