- Email: [email protected]
Stability of Choline in Feed Mixtures
Research Notes STABILITY OF CHOLINE IN FEED MIXTURES M . L. COOLEY* AND J. B . CHRISTIANSEN General Mills, Inc., Larro Research Farm, Detroit, Michigan (Received for publication July 16, 1948)
* Address: General Mills, Inc., Larrowe Division, Rossford, Ohio.
saponified solution with glacial acetic acid to thymolphthalein indicator (4 drops of 1% solution of thymolphthalein in alcohol). Wash the neutralized solution into a 50 ml. volumetric flask with distilled water and dilute to volume. Place a 5 to 10 ml. aliquot in a 15 ml. graduated centrifuge tube. Dilute to the 10 ml. mark with water if necessary. Add 2 ml. of freshly made 2% Ammonium Reineckate in methanol. Mix thoroughly and place in a refrigerator for 2 hours. Centrifuge at 2000 r.p.m. for 5 minutes. Pour off the supernatant liquid. Wash the choline Reineckate precipitate with 3 ml. of Npropanol from a pipette. Centrifuge for 5 minutes. Repeat washing and centrifuging once. Drain after last washing by placing the mouth of the centrifuge tube on paper toweling. Dissolve the precipitate in acetone (warm if necessary) and dilute to 10 ml. with acetone. Mix (if solution is cloudy, centrifuge) and measure the color in a spectrophotometer (a Coleman, Model 11 Universal Spectrophotometer was used) using wavelength of light 520 millimicrons. Results are shown in Table 1. The feeds used ranged from 20% to 27% in protein and represent considerable variation in formula. It is evident that both naturally-occurring' and synthetic choline are quite stable in feeds under normal conditions of storage. This observation is in agreement with the extensive work reported by Lardinois, Elvehjem, and Hart, indicating the general stability of members of the
822
Downloaded from http://ps.oxfordjournals.org/ at McMaster University Library on April 8, 2015
The possibility was suggested to the authors that a lack of stability of choline might explain the occurrence of perosis in chicks fed naturally-occurring or synthetic choline providing supposedly adequate amounts of the factor. Accordingly a series of experimental mixtures representing several different types of practical poultry feed mixtures was prepared. Two 100 pound bags of each of six mashes were made. One bag of each mash contained an added 0.15% Merck choline chloride. Homogeneous distribu-' tion of this choline chloride was assured by use of a Simpson Intensive Laboratory Mixer. The choline chloride was added in water solution to further promote homogeneity. The bags of feed (12 in all) were stored at room temperature (70 F). At the beginning, and at 3 month intervals, samples were taken with a sampling rod and analyzed for choline. The method used was a modification of that outlined by Engel (1942). The procedure was as follows : Weigh 10-15 grams of finely ground feed and mix with about 3 grams JohnsManville Hyflo Super Cel. Place the mixture in an extraction thimble and extract on a fat extractor over night with 60 ml. of methanol. Remove receiving flask and evaporate all but a few ml. of the methanol and add 30 ml. 10% aqueous KOH solution. Saponify for 45 minutes under reflux condenser. Cool and neutralize the
823
RESEARCH NOTES TABLE 1.—Stability of naturally-occurring choline and added choline in mixed feeds Percent choline* Sample
Chloride
Chloride
Chloride
Chloride
Chloride
Chloride
3 months
6 months
0.12
0.10
0.11
0.23
0.22
0.23
0.12
0.12
0.12
0.22
0.22
0.23
0.13
0.12
0.14
0.24
0.25
0.24
0.10
0.11
0.12
0.22
0.22
0.24
0.13
0.14
0.14
0.25
0.25
0.24
0.15
0.15
0.13
0.26
0.24
0.24
* All samples were analyzed in duplicate.
vitamin B complex with respect to storage (Lardinois, et al. 1944). REFERENCES'
Engel, R. W., 1942. Modified methods for the chemi-
cal and biological determination of choline. J. Biol. Chem. 144: 701-710. Lardinois, C. C , C. A. Elvehjem, and E. B. Hart, 1944. The effect of storage on the retention of certain members of the vitamin B complex. J. Dairy Sci. 27: 875-880.
DAILY UPTAKE OF BIOTIN BY THE HEN'S EGG* J. R. CoucH,f W. W.
CRAVENS,
C. A.
ELVEHJEM AND
J. G.
HALPIN
Departments of Poultry Husbandry and Biochemistry, University of Wisconsin, Madison, Wisconsin (Received for publication July 17, 1948)
During the course of studies on the relation of biotin to congenital deformities in the chick by Couch, et al. (1948), it was observed that the hatchability of eggs from hens fed a low biotin diet would de* Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. Supported in part by a grant from the Western Condensing Co., San Francisco, California. We are
crease to zero in four to six weeks. When these birds were switched to a practical indebted to the Western Condensing Co., for the lactose and dried whey; to Wilson Laboratories,. Chicago, Illinois, for the Liver Fraction " L " ; to Merck and Co., Rahway, N. J., for the biotin. t General Education Board Fellow, present address: Poultry Department, Texas A. & M. College, College Station, Texas.
Downloaded from http://ps.oxfordjournals.org/ at McMaster University Library on April 8, 2015
Mash I No added Choline Mash I Plus 0.15% Choline Mash II No added Choline Mash II Plus 0.15% Choline Mash I I I No added Choline Mash I I I Plus 0.15% Choline Mash IV No added Choline Mash IV Plus 0.15% Choline MashV No added Choline Mash V Plus 0.15% Choline Mash VI No added Choline Mash VI Plus 0.15% Choline
Initial
* Address: General Mills, Inc., Larrowe Division, Rossford, Ohio.
saponified solution with glacial acetic acid to thymolphthalein indicator (4 drops of 1% solution of thymolphthalein in alcohol). Wash the neutralized solution into a 50 ml. volumetric flask with distilled water and dilute to volume. Place a 5 to 10 ml. aliquot in a 15 ml. graduated centrifuge tube. Dilute to the 10 ml. mark with water if necessary. Add 2 ml. of freshly made 2% Ammonium Reineckate in methanol. Mix thoroughly and place in a refrigerator for 2 hours. Centrifuge at 2000 r.p.m. for 5 minutes. Pour off the supernatant liquid. Wash the choline Reineckate precipitate with 3 ml. of Npropanol from a pipette. Centrifuge for 5 minutes. Repeat washing and centrifuging once. Drain after last washing by placing the mouth of the centrifuge tube on paper toweling. Dissolve the precipitate in acetone (warm if necessary) and dilute to 10 ml. with acetone. Mix (if solution is cloudy, centrifuge) and measure the color in a spectrophotometer (a Coleman, Model 11 Universal Spectrophotometer was used) using wavelength of light 520 millimicrons. Results are shown in Table 1. The feeds used ranged from 20% to 27% in protein and represent considerable variation in formula. It is evident that both naturally-occurring' and synthetic choline are quite stable in feeds under normal conditions of storage. This observation is in agreement with the extensive work reported by Lardinois, Elvehjem, and Hart, indicating the general stability of members of the
822
Downloaded from http://ps.oxfordjournals.org/ at McMaster University Library on April 8, 2015
The possibility was suggested to the authors that a lack of stability of choline might explain the occurrence of perosis in chicks fed naturally-occurring or synthetic choline providing supposedly adequate amounts of the factor. Accordingly a series of experimental mixtures representing several different types of practical poultry feed mixtures was prepared. Two 100 pound bags of each of six mashes were made. One bag of each mash contained an added 0.15% Merck choline chloride. Homogeneous distribu-' tion of this choline chloride was assured by use of a Simpson Intensive Laboratory Mixer. The choline chloride was added in water solution to further promote homogeneity. The bags of feed (12 in all) were stored at room temperature (70 F). At the beginning, and at 3 month intervals, samples were taken with a sampling rod and analyzed for choline. The method used was a modification of that outlined by Engel (1942). The procedure was as follows : Weigh 10-15 grams of finely ground feed and mix with about 3 grams JohnsManville Hyflo Super Cel. Place the mixture in an extraction thimble and extract on a fat extractor over night with 60 ml. of methanol. Remove receiving flask and evaporate all but a few ml. of the methanol and add 30 ml. 10% aqueous KOH solution. Saponify for 45 minutes under reflux condenser. Cool and neutralize the
823
RESEARCH NOTES TABLE 1.—Stability of naturally-occurring choline and added choline in mixed feeds Percent choline* Sample
Chloride
Chloride
Chloride
Chloride
Chloride
Chloride
3 months
6 months
0.12
0.10
0.11
0.23
0.22
0.23
0.12
0.12
0.12
0.22
0.22
0.23
0.13
0.12
0.14
0.24
0.25
0.24
0.10
0.11
0.12
0.22
0.22
0.24
0.13
0.14
0.14
0.25
0.25
0.24
0.15
0.15
0.13
0.26
0.24
0.24
* All samples were analyzed in duplicate.
vitamin B complex with respect to storage (Lardinois, et al. 1944). REFERENCES'
Engel, R. W., 1942. Modified methods for the chemi-
cal and biological determination of choline. J. Biol. Chem. 144: 701-710. Lardinois, C. C , C. A. Elvehjem, and E. B. Hart, 1944. The effect of storage on the retention of certain members of the vitamin B complex. J. Dairy Sci. 27: 875-880.
DAILY UPTAKE OF BIOTIN BY THE HEN'S EGG* J. R. CoucH,f W. W.
CRAVENS,
C. A.
ELVEHJEM AND
J. G.
HALPIN
Departments of Poultry Husbandry and Biochemistry, University of Wisconsin, Madison, Wisconsin (Received for publication July 17, 1948)
During the course of studies on the relation of biotin to congenital deformities in the chick by Couch, et al. (1948), it was observed that the hatchability of eggs from hens fed a low biotin diet would de* Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. Supported in part by a grant from the Western Condensing Co., San Francisco, California. We are
crease to zero in four to six weeks. When these birds were switched to a practical indebted to the Western Condensing Co., for the lactose and dried whey; to Wilson Laboratories,. Chicago, Illinois, for the Liver Fraction " L " ; to Merck and Co., Rahway, N. J., for the biotin. t General Education Board Fellow, present address: Poultry Department, Texas A. & M. College, College Station, Texas.
Downloaded from http://ps.oxfordjournals.org/ at McMaster University Library on April 8, 2015
Mash I No added Choline Mash I Plus 0.15% Choline Mash II No added Choline Mash II Plus 0.15% Choline Mash I I I No added Choline Mash I I I Plus 0.15% Choline Mash IV No added Choline Mash IV Plus 0.15% Choline MashV No added Choline Mash V Plus 0.15% Choline Mash VI No added Choline Mash VI Plus 0.15% Choline
Initial