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Vitamin B12 and choline synthesis from glycine in vivo
Vitamin Blz and Choline Synthesis from Glycine in ho’
1’
B. Connor Johnson, Jay Firth and S. P. Mistry From
the
Division
of Animal
Nutrition, Received
University July
of Illinois,
Urbana,
Illinois
19, 1954
INTRODUCTION In previous work it has been shown that the baby pig requires choline when fed a synthetic diet containing 30 % casein (supplying approximately 0.8 y0 methionine) and adequately supplemented with vitamin Blz (1,2). The baby pig has also been found to develop a severe vitamin Blz deficiency in from 3 to 6 weeks when fed a Blz-free synthetic diet containing 30 y. a-protein3 adequately fortified with methionine, choline, folic acid, etc. (3, 4). In view of the readily reproducible nature of the Blz deficiency syndrome in the baby pig, this species has been used in work on the mechanism of Blz action. It is well established (5-8) that vitamin Blz is related to methyl metabolism, but it seems evident (7, 9, 10) that it is not involved in transmethylation reactions. The present experiment was designed to study in viva the role of vitamin BU in methyl synthesis for choline formation. EXPERIMENTAL Experiment
I
Baby pigs 2-3 days old were allotted to two groups of six each and housed individually in screen-bottom metal cages in an air-conditioned room. They received a basal diet consisting of: m-protein 29.4’%; nL-methionine 0.50/,; cerelose 31.3’%; lard 30.5’%; and minerals 8.3% (16,17) made up as a “milk” containing 13yo solids and fed ad libitum. The following vitamins were added per liter of milk at the time of feeding : thiamine. HC10.65 mg. ; riboflavin 1.30 mg. ; pyridoxine .HCl 1.30 mg. ; 1 Supported in part b.v grants-in-aid from the National Vitamin York, and from Moorman Manufacturing Co., Quincy, Ill. 2 Presented at the Atlantic City meeting of the American Instit,ute Federation Proc. 13, 457 (1954). 3 A soy protein purchased from Glidden Co., Chicago, 111.
Foundation,
New
467
of Nutrition.
468
JOHNSON,
FIRTH
AND
TABLE The Effect
MISTRY
I
of Blz
on Growth
2.2’%glycine,choline-deficient Aminoethanol -Bn
-BIZ
+Bn
~~
No. of pigs Ave. in. wt., kg. Ave. final wt., kg. Ave. tot. gain, kg.
Diiethylaminoethanol
+BIZ
4 1.65 8.66 7.01
4 1.80 12.29 10.49
1 1.20 3.10 1.90
1 1.25 4.10 2.85
-BIZ ~___
1 1.55 9.00 7.45
+BIZ
1 Died
nicotinic acid 2.60 mg.; calcium pantothenate 7.80 mg.; biotin 0.01 mg.; folic acid 0.52 mg.; 2-methyl-1,4-napthoquinone 0.26 mg.; vitamin A 2000 I.U.; vitamin D 200 I.U. Sulfathalidine was added at the rate of 1.2 g./l. to inhibit intestinal synthesis. The diet was further supplemented with 1.0% glycine to give a basal diet, containing 2.2% glycine and 0.80/o methionine while being deficient in choline and vitamin I3lz . One of the two groups of pigs received vitamin BH at the rate of 0.8 pg./kg. body weight/day by weekly injection. One pig in each group received 0.1% aminoethanol, and a second pig in each group received 0.1% dimethylaminoethanol in addition to the basal diet. The weight data are given in Table I. All pigs were killed during the sixth week when they were near death due to the BH deficiency. Liver and kidney samples were examined histologically for evidence of fatty infiltration and renal damage in order to assess the choline status of the two groups of pigs.
Experiment II Because of the possibility that compounds present in a-protein might be serving as methyl precursorsin the presenceof vitamin BH , a secondcontrol experiment, was carried out in which a-protein was used as protein source, vitamin BH was administered, but the added glycine was omitted from the diet. The four pigs in this experiment were killed at 5 weeks and the livers examined grossly, histologically, and by chemical analysis for fat. In this second experiment a positive control (plus choline and plus vitamin Blz) group of two pigs was also included. RESULTS
The vitamin Blz-deficient group of pigs all showed a marked loss of appetite, vomited frequently, and became very weak during the sixth week of the experiment. Liver histology showed distinct differences between the Blz-deficient group and the group which received Blz in Expt. I (Fig. 1). The former group, which received only the basal diet, all had fatty livers while their four litter mates receiving Blz showed no fatty
FIG. 1. Magnification with Harris’s choline. No. 1. No. 2. No. 3. No. 4. No.
Photomicrographs 157.5. All hematoxplin
Liver Liver Liver Liver ethanol. 5. Liver
from from from from from
of liver sections of pigs at t,ermination of cxpcrimcnt. sections were made by the paraffin method and stained and eosin. All animals received 2.2% glycine and no pig receiving pig receiving pig receiving pig receiving pig
receiving
no B,% 0.8 pg. R,Jkg. body wt./day. no BIZ and 0.1% aminoethanol. 0.8 pg. BIQ/kg. body wt./day no
B 12 and 460
0.1%
and
dimethylaminoethanol.
0.1%
amino-
470
Vitamin
JOHNSON,
B,? Content
oj Pig
Livers
FIRTH
AND
TABLE
II
as Assayed malhamensis
MISTRY
by E.
coli
Muiani
and Liver
GKUp
Supplement
1. Basal Aminoethanol Dimethylaminoethanol 2. Basal
+
Blz Aminoethanol Dimethylaminoethanol
E. coli mpg./g. fresh
22 17 23 22 31 35 119 106 131 131 200 156
lieer
Ochromonas BE 0. mdimmensis NWRJR. fresh liver
8.7 6.7 7.3 7.3 16 12 88 88 78 75 135 109
infiltration, which indicated that they were not choline deficient. In Expt. II the pigs which received the same basal diet as in Expt. I but without the added glycine and with added vitamin Blz had an average of 29.2 y0 fat (dry basis) in their livers (they were grossly fat on autopsy) as compared to 5.4% for the livers of the pigs which received choline. Thus, it is clear that the added glycine in the presence of adequate BPJ protected the pigs from the choline deficiency. The two pigs which were fed 0.1% aminoethanol had very fatty livers both with and without BH . The liver of the Blz-deficient pig receiving 0.1% dimethylaminoethanol was quite normal and contained no fat. The litter mate receiving dimethylaminoethanol and Bi2 died from other causes; necrosis prevented interpretation of the amount of fat in the liver. Assay of the livers for vitamin BPJwith Escherichiu coli (11) indicated about six times as much Bit activity in the control group as in the deficient. When assayed with Ochromonas malhamensis* (12) the responsewas approximately ten times as great for the group reciering BH as for the deficient group (Table II). DISCUSSION
Our previous choline experiments (1, 2) with baby pigs established that on a diet adequate in vitamin B1? and containing approximately 4 J. E. Ford,
unpublished.
VITAMIN
B12
AND
CHOLINE
SYNTHESIS
471
0.8 y0 methionine, 2 % serine, and 0.6 % glycine [based on casein containing 6.8 y0 serine and 2.0 y0 glycine (13)], the animals required choline to prevent fatty livers and kidney damage. That is, in the presence of vitamin Bl2 the components of this diet (those mentioned as well as glucose, histidine, threonine, etc.) at the levels fed did not serve as adequate precursors of choline, and the animals had a choline requirement of approximately 0.1%. The second experiment with the a-protein diet confirms this finding. Even though this ration contained more glycine than the casein basal, the glycine level was still insufficient to protect the animals from fatty livers in the presence of adequate vitamin B12 . In previous experiments (9) we have also shown that the baby pig is able to completely dispense with choline on a diet containing 1.6 $$ methionine. Furthermore, normal as well as vitamin Bl2-deficient animals were able to meet the requirement of choline equally efficiently by transmethylation from the high level of methionine present in the diet. The present experiment shows that an a-protein diet containing a total of approximately 0.8 y0 methionine, 2 y0 serine, and 2.2 % glycine will completely protect the animal from a choline deficiency in the presence of vitamin B12 , but not in its absence. This diet supplies essentially the same amount of serine, threonine, and histidine, but considerably more glycine (1.6 %) than our previous casein ration. These results show that vitamin B12 is required for the synthesis of methyl groups from glycine and that glycine will serve as a satisfactory methyl source for choline synthesis in the baby pig, but only in the presence of vitamin B12 . These results do not show, of course, that serine is not capable of replacing choline in the presence of B12 , but only that it will not at the 2 y0 level. When present at the 4 % level (the level of glycine plus serine used), serine may prove equally satisfactory. In experiments with rats on a labile methyl-free diet containing homocystine, Stekol et al. (14) have reported that glycine will stimulate growth only in the presence of vitamin Bl2 . Similar studies (8, 15) with c+glytine-Cl4 have further shown that vitamin Bl2 has a stimulating effect on the synthesis of methyl groups of choline. Apparently dimethylaminoethanol spared the requirement for methyl synthesis to such an extent that the 0.8% methionine content of the diet was able to provide by transmethylation the rest of the methyl groups required for choline formation. On the other hand, aminoethanol apparently drastically increased the methyl group demand resulting in choline deficiency even in the presence of vitamin Blz and glycine. (The
472
JOHNSON, FIRTH AND MISTRY
actual liver fat values were 26 $%for the Blz-deficient pig and 19 y0 for the pig receiving Blz .) It was interesting to us that glycine did protect completely these young (pre-weaning age) very rapidly growing animals from choline deficiency when vitamin Blz was supplied. ACKNOWLEDGMENTS ThiamineeHCI, riboflavin, pyridoxine.HCl, calcium pantothenate, nicotinic acid and choline. Cl were supplied by Merck and Co., Inc., Rahway, New Jersey, through the courtesy of Dr. H. H. Draper; folic acid and dimethylaminoethanol were supplied by Lederle Laboratories Division, American Cyanamid Co., Pearl River, New York, through the courtesy of Dr. T. H. Jukes; sulfathalidine was supplied by Sharp and Dohme, Inc., West Point, Pa., through the courtesy of Dr. S. F. Scheidy; and DL-methionine was supplied by Dow Chemical Co., Midland, Michigan, through the courtesy of Dr. J. E. Johnson. SUMMARY
The effect of vitamin Blz on choline synthesis from glycine was studied in 2%S-day-old baby pigs fed an a-protein synthetic milk ration containing 2.2 y0 glycine, 0.8 y0 methionine, and lessthan 0.01 y0 choline. In the groups receiving vitamin BN , histological examination of the livers revealed no fatty infiltration characteristic of a choline deficiency; whereas, in the BIZ-deficient groups all the animals had markedly fatty livers. These results indicate that vitamin BILEis required for the synthesis of methyl groups from glycine and-that glycine will serve as a methyl source only in the presence of BU . REFERENCES 1. JOHNSON,
B. C., AND JAMES, M. A. L., KRIDER, J. L., tion 38, 195 (1949). 3. JOHNSON, B. C., AND NEUMANN, 4. JOHNSON, B.C., NEUMANN, A. 2. NEUMANN,
5. 6. 7. 8.
F., J. Nutrition 36,339 (1948). JAMES, M. F., AND JOHNSON, B. C., J. Nutri-
A. L., J. BioZ. Chem. 178,lOOl (1949). L., NESHEIM, R. O., JAMES, M.F., KRIDER, J. L., DANA, A. S., AND THIERSCH, J. B., J. Lab. Clin. Med. 36,537 (1950). SCHAEFER, A. E., SALMON, W. D., AND STRENGTH, D. R., Federation Proc. 9, 369 (1959). SCHAEFER, A. E., AND KNOWLES, J. L., Proc. Sot. Exptl. Biol. Med. 77, 655 (1951). STEKOL, J. A., WEISS, S., SMITH, P., AND WEISS, K., J. Biol. Chem. 201, 299 (1953). ARNSTEIN, H. R. V., AND NEUBERGER, A., Biochem. J. 66,259 (1953).
VITAMIN
B12 AND
CHOLINE
SYNTHESIS
473
9. FIRTH, J., MISTRY, S. P., JAMES, M. F., AND JOHNSON, B. C., Proc. Sot. Exptl. Biol. Med. 86, 307 (1954). 10. MISTRY, S. P., VADOPALAITE, I., CHANG, I., FIRTH, J., AND JOHNSON, B. C., Federation Proc. 13, 265 (1954). 11. BURKHOLDER, P. R., Science 114, 459 (1951). 12. HUNTER, S. H., PROVASOLI, L., AND FILFRED, J., Ann. N. Y. Acad. Sci. 68, 852 (1953). 13. BLOCK, H. J., AND BOLLING, D., “The Amino Acid Composition of Proteins and Food&” 2nd ed., pp. 490,360. C. C Thomas, Springfield, ill., 1951. 14. STEKOL, J. A., WEISS, S., AND WEISS, K. Abstracts, Am. Chem. Sot., 120th meeting, p. 21C. New York, September, 1951. 15. STEKOL, J. A., WEISS, S., AND WEISS, K. W., Arch. Biochem. and Biophys. 36, 5 (1952). 16. JOHNSOX, B. C., JAMES, M. F., AND KRIDER, J. L., J. Animal Sci. 7,486 (1948). 17. NEUMANN, A. L., THIERSCH, J. B., KRIDER, J. L., JAMES, M. F., AND JOHNSON, B. C., J. Animal Sci. 9,83 (1950).
1’
B. Connor Johnson, Jay Firth and S. P. Mistry From
the
Division
of Animal
Nutrition, Received
University July
of Illinois,
Urbana,
Illinois
19, 1954
INTRODUCTION In previous work it has been shown that the baby pig requires choline when fed a synthetic diet containing 30 % casein (supplying approximately 0.8 y0 methionine) and adequately supplemented with vitamin Blz (1,2). The baby pig has also been found to develop a severe vitamin Blz deficiency in from 3 to 6 weeks when fed a Blz-free synthetic diet containing 30 y. a-protein3 adequately fortified with methionine, choline, folic acid, etc. (3, 4). In view of the readily reproducible nature of the Blz deficiency syndrome in the baby pig, this species has been used in work on the mechanism of Blz action. It is well established (5-8) that vitamin Blz is related to methyl metabolism, but it seems evident (7, 9, 10) that it is not involved in transmethylation reactions. The present experiment was designed to study in viva the role of vitamin BU in methyl synthesis for choline formation. EXPERIMENTAL Experiment
I
Baby pigs 2-3 days old were allotted to two groups of six each and housed individually in screen-bottom metal cages in an air-conditioned room. They received a basal diet consisting of: m-protein 29.4’%; nL-methionine 0.50/,; cerelose 31.3’%; lard 30.5’%; and minerals 8.3% (16,17) made up as a “milk” containing 13yo solids and fed ad libitum. The following vitamins were added per liter of milk at the time of feeding : thiamine. HC10.65 mg. ; riboflavin 1.30 mg. ; pyridoxine .HCl 1.30 mg. ; 1 Supported in part b.v grants-in-aid from the National Vitamin York, and from Moorman Manufacturing Co., Quincy, Ill. 2 Presented at the Atlantic City meeting of the American Instit,ute Federation Proc. 13, 457 (1954). 3 A soy protein purchased from Glidden Co., Chicago, 111.
Foundation,
New
467
of Nutrition.
468
JOHNSON,
FIRTH
AND
TABLE The Effect
MISTRY
I
of Blz
on Growth
2.2’%glycine,choline-deficient Aminoethanol -Bn
-BIZ
+Bn
~~
No. of pigs Ave. in. wt., kg. Ave. final wt., kg. Ave. tot. gain, kg.
Diiethylaminoethanol
+BIZ
4 1.65 8.66 7.01
4 1.80 12.29 10.49
1 1.20 3.10 1.90
1 1.25 4.10 2.85
-BIZ ~___
1 1.55 9.00 7.45
+BIZ
1 Died
nicotinic acid 2.60 mg.; calcium pantothenate 7.80 mg.; biotin 0.01 mg.; folic acid 0.52 mg.; 2-methyl-1,4-napthoquinone 0.26 mg.; vitamin A 2000 I.U.; vitamin D 200 I.U. Sulfathalidine was added at the rate of 1.2 g./l. to inhibit intestinal synthesis. The diet was further supplemented with 1.0% glycine to give a basal diet, containing 2.2% glycine and 0.80/o methionine while being deficient in choline and vitamin I3lz . One of the two groups of pigs received vitamin BH at the rate of 0.8 pg./kg. body weight/day by weekly injection. One pig in each group received 0.1% aminoethanol, and a second pig in each group received 0.1% dimethylaminoethanol in addition to the basal diet. The weight data are given in Table I. All pigs were killed during the sixth week when they were near death due to the BH deficiency. Liver and kidney samples were examined histologically for evidence of fatty infiltration and renal damage in order to assess the choline status of the two groups of pigs.
Experiment II Because of the possibility that compounds present in a-protein might be serving as methyl precursorsin the presenceof vitamin BH , a secondcontrol experiment, was carried out in which a-protein was used as protein source, vitamin BH was administered, but the added glycine was omitted from the diet. The four pigs in this experiment were killed at 5 weeks and the livers examined grossly, histologically, and by chemical analysis for fat. In this second experiment a positive control (plus choline and plus vitamin Blz) group of two pigs was also included. RESULTS
The vitamin Blz-deficient group of pigs all showed a marked loss of appetite, vomited frequently, and became very weak during the sixth week of the experiment. Liver histology showed distinct differences between the Blz-deficient group and the group which received Blz in Expt. I (Fig. 1). The former group, which received only the basal diet, all had fatty livers while their four litter mates receiving Blz showed no fatty
FIG. 1. Magnification with Harris’s choline. No. 1. No. 2. No. 3. No. 4. No.
Photomicrographs 157.5. All hematoxplin
Liver Liver Liver Liver ethanol. 5. Liver
from from from from from
of liver sections of pigs at t,ermination of cxpcrimcnt. sections were made by the paraffin method and stained and eosin. All animals received 2.2% glycine and no pig receiving pig receiving pig receiving pig receiving pig
receiving
no B,% 0.8 pg. R,Jkg. body wt./day. no BIZ and 0.1% aminoethanol. 0.8 pg. BIQ/kg. body wt./day no
B 12 and 460
0.1%
and
dimethylaminoethanol.
0.1%
amino-
470
Vitamin
JOHNSON,
B,? Content
oj Pig
Livers
FIRTH
AND
TABLE
II
as Assayed malhamensis
MISTRY
by E.
coli
Muiani
and Liver
GKUp
Supplement
1. Basal Aminoethanol Dimethylaminoethanol 2. Basal
+
Blz Aminoethanol Dimethylaminoethanol
E. coli mpg./g. fresh
22 17 23 22 31 35 119 106 131 131 200 156
lieer
Ochromonas BE 0. mdimmensis NWRJR. fresh liver
8.7 6.7 7.3 7.3 16 12 88 88 78 75 135 109
infiltration, which indicated that they were not choline deficient. In Expt. II the pigs which received the same basal diet as in Expt. I but without the added glycine and with added vitamin Blz had an average of 29.2 y0 fat (dry basis) in their livers (they were grossly fat on autopsy) as compared to 5.4% for the livers of the pigs which received choline. Thus, it is clear that the added glycine in the presence of adequate BPJ protected the pigs from the choline deficiency. The two pigs which were fed 0.1% aminoethanol had very fatty livers both with and without BH . The liver of the Blz-deficient pig receiving 0.1% dimethylaminoethanol was quite normal and contained no fat. The litter mate receiving dimethylaminoethanol and Bi2 died from other causes; necrosis prevented interpretation of the amount of fat in the liver. Assay of the livers for vitamin BPJwith Escherichiu coli (11) indicated about six times as much Bit activity in the control group as in the deficient. When assayed with Ochromonas malhamensis* (12) the responsewas approximately ten times as great for the group reciering BH as for the deficient group (Table II). DISCUSSION
Our previous choline experiments (1, 2) with baby pigs established that on a diet adequate in vitamin B1? and containing approximately 4 J. E. Ford,
unpublished.
VITAMIN
B12
AND
CHOLINE
SYNTHESIS
471
0.8 y0 methionine, 2 % serine, and 0.6 % glycine [based on casein containing 6.8 y0 serine and 2.0 y0 glycine (13)], the animals required choline to prevent fatty livers and kidney damage. That is, in the presence of vitamin Bl2 the components of this diet (those mentioned as well as glucose, histidine, threonine, etc.) at the levels fed did not serve as adequate precursors of choline, and the animals had a choline requirement of approximately 0.1%. The second experiment with the a-protein diet confirms this finding. Even though this ration contained more glycine than the casein basal, the glycine level was still insufficient to protect the animals from fatty livers in the presence of adequate vitamin B12 . In previous experiments (9) we have also shown that the baby pig is able to completely dispense with choline on a diet containing 1.6 $$ methionine. Furthermore, normal as well as vitamin Bl2-deficient animals were able to meet the requirement of choline equally efficiently by transmethylation from the high level of methionine present in the diet. The present experiment shows that an a-protein diet containing a total of approximately 0.8 y0 methionine, 2 y0 serine, and 2.2 % glycine will completely protect the animal from a choline deficiency in the presence of vitamin B12 , but not in its absence. This diet supplies essentially the same amount of serine, threonine, and histidine, but considerably more glycine (1.6 %) than our previous casein ration. These results show that vitamin B12 is required for the synthesis of methyl groups from glycine and that glycine will serve as a satisfactory methyl source for choline synthesis in the baby pig, but only in the presence of vitamin B12 . These results do not show, of course, that serine is not capable of replacing choline in the presence of B12 , but only that it will not at the 2 y0 level. When present at the 4 % level (the level of glycine plus serine used), serine may prove equally satisfactory. In experiments with rats on a labile methyl-free diet containing homocystine, Stekol et al. (14) have reported that glycine will stimulate growth only in the presence of vitamin Bl2 . Similar studies (8, 15) with c+glytine-Cl4 have further shown that vitamin Bl2 has a stimulating effect on the synthesis of methyl groups of choline. Apparently dimethylaminoethanol spared the requirement for methyl synthesis to such an extent that the 0.8% methionine content of the diet was able to provide by transmethylation the rest of the methyl groups required for choline formation. On the other hand, aminoethanol apparently drastically increased the methyl group demand resulting in choline deficiency even in the presence of vitamin Blz and glycine. (The
472
JOHNSON, FIRTH AND MISTRY
actual liver fat values were 26 $%for the Blz-deficient pig and 19 y0 for the pig receiving Blz .) It was interesting to us that glycine did protect completely these young (pre-weaning age) very rapidly growing animals from choline deficiency when vitamin Blz was supplied. ACKNOWLEDGMENTS ThiamineeHCI, riboflavin, pyridoxine.HCl, calcium pantothenate, nicotinic acid and choline. Cl were supplied by Merck and Co., Inc., Rahway, New Jersey, through the courtesy of Dr. H. H. Draper; folic acid and dimethylaminoethanol were supplied by Lederle Laboratories Division, American Cyanamid Co., Pearl River, New York, through the courtesy of Dr. T. H. Jukes; sulfathalidine was supplied by Sharp and Dohme, Inc., West Point, Pa., through the courtesy of Dr. S. F. Scheidy; and DL-methionine was supplied by Dow Chemical Co., Midland, Michigan, through the courtesy of Dr. J. E. Johnson. SUMMARY
The effect of vitamin Blz on choline synthesis from glycine was studied in 2%S-day-old baby pigs fed an a-protein synthetic milk ration containing 2.2 y0 glycine, 0.8 y0 methionine, and lessthan 0.01 y0 choline. In the groups receiving vitamin BN , histological examination of the livers revealed no fatty infiltration characteristic of a choline deficiency; whereas, in the BIZ-deficient groups all the animals had markedly fatty livers. These results indicate that vitamin BILEis required for the synthesis of methyl groups from glycine and-that glycine will serve as a methyl source only in the presence of BU . REFERENCES 1. JOHNSON,
B. C., AND JAMES, M. A. L., KRIDER, J. L., tion 38, 195 (1949). 3. JOHNSON, B. C., AND NEUMANN, 4. JOHNSON, B.C., NEUMANN, A. 2. NEUMANN,
5. 6. 7. 8.
F., J. Nutrition 36,339 (1948). JAMES, M. F., AND JOHNSON, B. C., J. Nutri-
A. L., J. BioZ. Chem. 178,lOOl (1949). L., NESHEIM, R. O., JAMES, M.F., KRIDER, J. L., DANA, A. S., AND THIERSCH, J. B., J. Lab. Clin. Med. 36,537 (1950). SCHAEFER, A. E., SALMON, W. D., AND STRENGTH, D. R., Federation Proc. 9, 369 (1959). SCHAEFER, A. E., AND KNOWLES, J. L., Proc. Sot. Exptl. Biol. Med. 77, 655 (1951). STEKOL, J. A., WEISS, S., SMITH, P., AND WEISS, K., J. Biol. Chem. 201, 299 (1953). ARNSTEIN, H. R. V., AND NEUBERGER, A., Biochem. J. 66,259 (1953).
VITAMIN
B12 AND
CHOLINE
SYNTHESIS
473
9. FIRTH, J., MISTRY, S. P., JAMES, M. F., AND JOHNSON, B. C., Proc. Sot. Exptl. Biol. Med. 86, 307 (1954). 10. MISTRY, S. P., VADOPALAITE, I., CHANG, I., FIRTH, J., AND JOHNSON, B. C., Federation Proc. 13, 265 (1954). 11. BURKHOLDER, P. R., Science 114, 459 (1951). 12. HUNTER, S. H., PROVASOLI, L., AND FILFRED, J., Ann. N. Y. Acad. Sci. 68, 852 (1953). 13. BLOCK, H. J., AND BOLLING, D., “The Amino Acid Composition of Proteins and Food&” 2nd ed., pp. 490,360. C. C Thomas, Springfield, ill., 1951. 14. STEKOL, J. A., WEISS, S., AND WEISS, K. Abstracts, Am. Chem. Sot., 120th meeting, p. 21C. New York, September, 1951. 15. STEKOL, J. A., WEISS, S., AND WEISS, K. W., Arch. Biochem. and Biophys. 36, 5 (1952). 16. JOHNSOX, B. C., JAMES, M. F., AND KRIDER, J. L., J. Animal Sci. 7,486 (1948). 17. NEUMANN, A. L., THIERSCH, J. B., KRIDER, J. L., JAMES, M. F., AND JOHNSON, B. C., J. Animal Sci. 9,83 (1950).