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The Nature of Egg Yolk Discoloration Produced by Cottonseed Meal
T h e Nature of Egg Yolk Discoloration Produced by Cottonseed Meal A . D . SWENSEN, E . A . FlEGER, AND C . W . U P P 1 Louisiana State University (Received for publication November 17, 1941)
I
"Assistant in chemistry, professor of chemistry, and professor of poultry research, respectively.
Thompson, Albright, Schnetzler, and Heller (1930) indicated that gossypol plays no part in the production of eggs with olive yolks. Sherwood (1931) on the basis of extensive feeding trials intimated that the substance causing discolored yolks was either in the oil or something closely associated with it. This substance was present in crude and partially refined cottonseed oil but was not present in soap stock, extracted cottonseed meal having a low fat content, nor in highly refined cottonseed oil. The results of Schaible, Moore, and Moore (1934) contradicted the work of Thompson et al. on several points. Schaible and co-workers concluded that either free or bound gossypol was the olive yolk factor. These authors discovered that the time required for the potential olive yolk to develop could be reduced to minutes by breaking out the eggs in an atmosphere of ammonia. They also found that when ferrous sulfate was added to the feed it would prevent the occurrence of olive yolks when cottonseed meal was fed to the extent of 40 percent of the total ration. Schaible et al. did not, however, make any comment as to the action of the iron in preventing olive yolks. No chemical interpretations have been reported of yolk discoloration as caused by cottonseed meal. EXPERIMENTAL
Two methods of experimental approach were used in seeking a solution of this problem. The first involved the separation
[374]
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on May 27, 2015
N THE South where cotton is a major crop, cottonseed meal is abundant and relatively cheap. The protein quality, while not as high as animal protein, satisfies the essential amino acid requirements of poultry to an extent that it is a valuable protein supplement. Except for the rather unfortunate characteristic of producing discolored yolks, cottonseed meal is a good, convenient source of protein for the poultry diet. Research has been done previously in order to ascertain the cause of the egg yolk discoloration brought about by cottonseed meal in the diet. In most cases gossypol, a complex polyhydric naphthalene derivative present in cottonseed meal, has been postulated to be wholly or partially responsible although other workers maintain that gossypol plays no part at all. These conclusions have all been based on feeding trials. No chemical interpretations of the yolk discoloration have been reported. Inasmuch as all biological processes are fundamentally chemical this study was undertaken in order to study the biochemical aspects involved in the formation Of the olive egg yolk produced by the use of cottonseed meal in laying rations. Fifty years ago Roberts and Rice (1891) noted that discolored egg yolks were produced by hens fed rations containing cottonseed meal. However, no real attempt was made to determine the cause'or correction of this discoloration until about 10 years ago.
NATURE OF EGG YOLK DISCOLORATION PRODUCED BY COTTONSEED MEAL
FEEDING TRIALS Ten Single Comb White Leghorn pullets of like maturity and health were placed in five lots with two birds per lot. The rations fed are given in Table 1. The hens in lot 2 developed a violent diarrhea by the end of the second day so the daily dosage of gossypol was reduced immediately to 0.05 percent in lots 2 and 3. The condition then improved but did not disappear entirely. Eggs were collected after the birds had been on the experimental diets for one month. Half of them were placed in storage and the other half were submitted to the ammonia treatment. All of the eggs from hens in lots 2; 3, and 5 developed the characteristic chocolate color in the ammonia atmosphere (the explanation as to why this differs from the olive green color is given in a later section of this paper) and the olive green color in storage. No differences could be detected in the egg yolk colors from the gossypol fed hens and those of the eggs from cottonseed
meal fed hens, for eggs given the same treatment. The yolk colors were normal in the eggs from lots 1 and 4. These results agree with those of Schaible et al. and TABLE 1.—Rations
Ration and how fed 1 2 3 4 5
Commercial laying ration Commercial laying ration and 0.33 gm. gossypol1 per day. Fed in gelatin capsule Commercial laying ration and 0.3 percent gossypol dissolved in Wesson oil Commercial laying ration and cottonseed oil extracted from seeds by petroleum ether Thirty percent cottonseed meal allmash ration 2
Percent gossypol in the diet 0.0 0.33 0.3 0.0 0.32
1 Thirty-three hundredths gram was taken as the approximate weight of gossypol ingested by a hen on a 30 percent diet of cottonseed meal, consuming 0.25 lb. per day when the meal contains 1 percent gossypol by weight. The figures are necessarily averages. 2 The complete ration: 30 percent cottonseed meal' (which analyzed 1.07 percent gossypol by weight); 31 percent cornmeal; 10 percent wheat bran; 10 percent wheat shorts; 10 percent pulverized oats; 3 percent X X X liquor, vitamin concentrate; 5 percent oyster shell flour; 0.5 percent NaCl and 0.5 percent cod liver oil. The oils were added in lots 3 and 4 in the ratio of 2.1 percent by weight of the total feed. This provided these lots with the same cottonseed oil content that lot 5 received.
strongly indicate that gossypol is the olive yolk factor in cottonseed meal. Conclusive evidence that gossypol is the causative agent was obtained by two other tests. A normal fresh egg yolk was allowed to stand overnight in a small beaker of Wesson oil to which a small amount of pure gossypol had been added. As a check a second normal yolk was treated similarly in oil containing no gossypol. These two yolks and one from a cottonseed meal fed hen were placed in an atmosphere of ammonia. The cottonseed meal yolk and one which had been suspended in the gossypol solution changed to the same brown color simultaneously. The yolk that had been in
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on May 27, 2015
and identification of the fraction of cottonseed meal which is responsible for the formation of olive yolks. The second approach was a study of the chemistry involved in the formation of olive colored yolks. Two methods were used in the first section of this investigation. Feeding trials using various fractions of cottonseed meal made up the first method and the second method consisted of the use of the pure suspected component of cottonseed meal to induce the olive color in a normal fresh yolk simply by adding the compound to the yolk and placing the mixture in an atmosphere of ammonia. Gossypol and cottonseed oil were used in the feeding trials to attempt to clear up the uncertainty existing as to the responsibility of these components in the formation of the olive yolk color.
375
376
A. D. SWENSEN, E. A. FIEGER, AND C. W. U P P
EGG YOLK FRACTIONATION
As soon as it was established that gossypol was the factor in cottonseed meal which causes olive yolks, the next problem was to determine which fraction of the egg yolk combined with the gossypol to form the color. Normal fresh yolks were fractionated and each fraction was treated by mixing with an acetone solution of gossypol and placing the system in an atmosphere of ammonia. A brownish black color appeared on testing the protein fraction while no change occurred in the fat fraction. The proteins of the egg yolk were fractionated by separating vitellin, method of Calvery and White (1931), livetin, method of Kay and Marshall (1928), and an albumin, a globulin, and a "myxoprotein" by Piettre's (1936) method. All of these different proteins gave definitely positive tests with gossypol. A protein fraction of egg yolk which is exceedingly rich in iron was separated by Bunge's (1885) procedure. The time required for color formation with gossypol was much less with this fraction than any preceding fraction. The intensity of the color was deeper also, appearing black in only a few seconds after being placed in ammonia.
One of the characteristic properties of gossypol is its affinity for ferrous and ferric ions, forming compounds having a dark olive green color. When these olive green compounds are placed in an atmosphere of ammonia the color changes to a chocolate brown. This is precisely the same color change exhibited by a cottonseed meal egg yolk which had previously developed the olive green color in storage, when it was placed in an ammonia atmosphere. All iron was extracted from fat free egg yolk protein with 6N HC1. The iron free protein was wetted with an acetone solution of gossypol and allowed to stand 48 hours in an atmosphere of ammonia. No color developed during this period. Smythe and Schmidt (1930) showed that proteins may form compounds with ferric iron that are so slightly dissociated that a negative test is obtained with potassium thiocyanate. Experiments in this investigation have shown that moist fresh yolk proteins give negative tests for iron with potassium thiocyanate, thiosalicylic acid, thioglycolic acid, and an acetone solution of gossypol. However, when these systems were placed in an atmosphere of ammonia the characteristic colors of their iron salts were obtained. Apparently ammonia liberates ferric ions from the egg yolk proteins in sufficient concentration to react with qualitative ferric ion color reagents and gossypol. These results indicate that iron is the constituent of egg yolk which combines with gossypol from cottonseed meal in forming olive colored egg yolks. Karrer (1938) points out that iron displaces the hydrogen from the two hydroxyl groups in catechol to form a compound having an olive green color. When a trace of ammonia or alkali is added to the compound thus formed, the ammonium or sodium compound is produced. This salt is reddish brown in color and has the following formula:
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on May 27, 2015
Wesson oil only, retained its normal orange yellow color throughout the ammonia' treatment. The same change in color was produced in a normal yolk when it was broken and well mixed with an acetone solution of pure gossypol and then placed in an ammonia atmosphere. This evidence clearly shows that gossypol is the factor responsible for the formation of olive egg yolks when hens are fed cottonseed meal and indicates that gossypol is absorbed without chemical change from the intestines and is present as such in the egg. This latter point is being investigated in this laboratory.
NATURE OF EGG YOLK DISCOLORATION PRODUCED BY COTTONSEED MEAL
(NH 4 ) 3
feces dropped by the hens in each lot was determined for a three-day period. The quantity of gossypol ingested and the amount in the feces was determined by the (NH ( ), method Smith and Halverson (1933) used
Fe
o \ •o-
377
Fe;
TABLE 2.—Balance trial of gossypol
o-
Gossypol has the following formula, Adams (1938):
IRON SALTS AS A PREVENTIVE OF MOTTLED YOLKS
To maintain that iron is a factor involved in the production as well as the prevention of cottonseed meal egg yolk discoloration seems, at first sight, to be a paradox. It can be shown, however, that this is the case. Five lots of two Single Comb White Leghorn pullets each were fed a 30 percent allmash cottonseed meal diet (same as given in Table 1, lot S). As indicated in Table 2, varying amounts of a ferric chloride solution were added in the several lots. After a three-day preliminary period on these diets, the amount of feed consumed and
in feed
Gossypol ingested
Gossypol returned
Gossypol returned
1 2 3 4 5
percent 0.00 0.05 0.10 0.20 0.50
grams 1.39 1.53 1.18 2.36 2.01
grams 0.545 0.736 0.678 1.640 1.890
percent 39.2 48.2 57.5 69.5 94.0
in determining the total gossypol in cottonseed meal. The use of this procedure was tenable since it was shown that two samples of cottonseed meal gave the same analysis for total gossypol even after one of the samples had been treated with a ferric chloride solution forming the dark colored iron gossypol compound within the meal. These results indicate clearly that the presence of ferric chloride inhibits the absorption of gossypol by the hen. The mechanism for this inhibition is not known, but is probably due to the decreased solubility in the intestines of the iron-gossypol compound as compared to free gossypol. SUMMARY 1. Gossypol has been shown to be the factor in cottonseed meal which is responsible for the formation, in storage, of the olive colored yolks in eggs from hens fed cottonseed meal. 2. The characteristic olive color of the yolks is due to a chemical combination of the gossypol from the cottonseed meal with ferric iron released from the yolk proteins. 3. The iron combination with the yolk proteins of a fresh egg yolk is so slightly dissociated that insufficient ferric ions are
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on May 27, 2015
It contains two of the same functional groups as catechol, namely ortho hydroxyl groups, for which ferric ions have an apparent affinity. It appears then that the brown to black compound produced when a gossypol containing yolk is placed in an atmosphere of ammonia is the ammonium salt of the compound that imparts an olive green color to the yolk in storage.
Ferric
Lot no.
378
A. D . SWENSEN, E . A. FlEGER, AND C. W . UPP
REFERENCES
Adams, R., R. C. Morris, T. A. Geissman, D. J. Butterbough, and E. C. Kirkpatrick, 1938. Structure of gossypol. Journal of the American Chemical Society 60:2193-2204. Bunge, G. von, 1885. Uber die Assimilation des Eisens. Ztschr. f. Physiol. Chem. 9:49-59. Abs. Berichte 18:412. Calvery, H. O., and A. White, 1931. Vitellin of
hen's eggs. Jour. Biol. Chem. 94:635-639. Karrer, P., 1938. Organic chemistry. Nordemann Publishing Co., New York. Translated from the latest German edition by A. J. Mee. Kay, H. D., and P. G. Marshall, 1928. The second protein (livetin) of egg yolk. Biochem. Jour. 22:1264-1269. Piettre, M., 1936. Recherches sur les proteides de l'ovocytc (jaune de l'oeuf la poule). Comp. Rend. 202 :699-702. Roberts, I. P., and J. E. Rice, 1891. The effect on fowls of nitrogenous and carbonaceous rations. Expt. Sta. Rec. 2:506-507. Schaible, P. J., L. A. Moore, and J. M. Moore, 1934. Gossypol, a cause of discoloration in egg yolks. Science 79:372. Sherwood, R. M., 1929. The effect of various rations on the storage quality of eggs. Poultry Sci. 8:99-102. Smith, F. H., and J. O. Halverson, 1933. Estimation of total and bound (D) gossypol in cottonseed meal. A modified method. Indus, and Engin. Chem. (analytical ed.) 5:319. Smythe, C. V., and C. L. A. Schmidt, 1930. Studies on the mode of combination of iron with certain proteins, amino acids, and related compounds. J. Biol. Chem. 88:241-269. Thompson, R. B., W. P. Albright, E. E. Schnetzler, and V. G. Heller, 1933. Feeds and their relation to egg quality. Oklahoma Agr. Expt. Sta. . Rpt. 1930-32 :35.
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on May 27, 2015
present to give a positive test with iron testing reagents. 4. During storage the iron proteins decompose liberating sufficient iron to combine with gossypol to form the olive color. 5. Ammonia accelerates the release of iron from the yolk proteins. 6. The chocolate colored compound formed when iron and gossypol react in the presence of ammonia is believed to be the ammonium salt of the green colored compound present in olive yolks. 7. Addition of soluble ferric salts to the ration containing cottonseed meal prevents the absorption of the gossypol by the hen and the formation of olive yolks in the stored eggs.
I
"Assistant in chemistry, professor of chemistry, and professor of poultry research, respectively.
Thompson, Albright, Schnetzler, and Heller (1930) indicated that gossypol plays no part in the production of eggs with olive yolks. Sherwood (1931) on the basis of extensive feeding trials intimated that the substance causing discolored yolks was either in the oil or something closely associated with it. This substance was present in crude and partially refined cottonseed oil but was not present in soap stock, extracted cottonseed meal having a low fat content, nor in highly refined cottonseed oil. The results of Schaible, Moore, and Moore (1934) contradicted the work of Thompson et al. on several points. Schaible and co-workers concluded that either free or bound gossypol was the olive yolk factor. These authors discovered that the time required for the potential olive yolk to develop could be reduced to minutes by breaking out the eggs in an atmosphere of ammonia. They also found that when ferrous sulfate was added to the feed it would prevent the occurrence of olive yolks when cottonseed meal was fed to the extent of 40 percent of the total ration. Schaible et al. did not, however, make any comment as to the action of the iron in preventing olive yolks. No chemical interpretations have been reported of yolk discoloration as caused by cottonseed meal. EXPERIMENTAL
Two methods of experimental approach were used in seeking a solution of this problem. The first involved the separation
[374]
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on May 27, 2015
N THE South where cotton is a major crop, cottonseed meal is abundant and relatively cheap. The protein quality, while not as high as animal protein, satisfies the essential amino acid requirements of poultry to an extent that it is a valuable protein supplement. Except for the rather unfortunate characteristic of producing discolored yolks, cottonseed meal is a good, convenient source of protein for the poultry diet. Research has been done previously in order to ascertain the cause of the egg yolk discoloration brought about by cottonseed meal in the diet. In most cases gossypol, a complex polyhydric naphthalene derivative present in cottonseed meal, has been postulated to be wholly or partially responsible although other workers maintain that gossypol plays no part at all. These conclusions have all been based on feeding trials. No chemical interpretations of the yolk discoloration have been reported. Inasmuch as all biological processes are fundamentally chemical this study was undertaken in order to study the biochemical aspects involved in the formation Of the olive egg yolk produced by the use of cottonseed meal in laying rations. Fifty years ago Roberts and Rice (1891) noted that discolored egg yolks were produced by hens fed rations containing cottonseed meal. However, no real attempt was made to determine the cause'or correction of this discoloration until about 10 years ago.
NATURE OF EGG YOLK DISCOLORATION PRODUCED BY COTTONSEED MEAL
FEEDING TRIALS Ten Single Comb White Leghorn pullets of like maturity and health were placed in five lots with two birds per lot. The rations fed are given in Table 1. The hens in lot 2 developed a violent diarrhea by the end of the second day so the daily dosage of gossypol was reduced immediately to 0.05 percent in lots 2 and 3. The condition then improved but did not disappear entirely. Eggs were collected after the birds had been on the experimental diets for one month. Half of them were placed in storage and the other half were submitted to the ammonia treatment. All of the eggs from hens in lots 2; 3, and 5 developed the characteristic chocolate color in the ammonia atmosphere (the explanation as to why this differs from the olive green color is given in a later section of this paper) and the olive green color in storage. No differences could be detected in the egg yolk colors from the gossypol fed hens and those of the eggs from cottonseed
meal fed hens, for eggs given the same treatment. The yolk colors were normal in the eggs from lots 1 and 4. These results agree with those of Schaible et al. and TABLE 1.—Rations
Ration and how fed 1 2 3 4 5
Commercial laying ration Commercial laying ration and 0.33 gm. gossypol1 per day. Fed in gelatin capsule Commercial laying ration and 0.3 percent gossypol dissolved in Wesson oil Commercial laying ration and cottonseed oil extracted from seeds by petroleum ether Thirty percent cottonseed meal allmash ration 2
Percent gossypol in the diet 0.0 0.33 0.3 0.0 0.32
1 Thirty-three hundredths gram was taken as the approximate weight of gossypol ingested by a hen on a 30 percent diet of cottonseed meal, consuming 0.25 lb. per day when the meal contains 1 percent gossypol by weight. The figures are necessarily averages. 2 The complete ration: 30 percent cottonseed meal' (which analyzed 1.07 percent gossypol by weight); 31 percent cornmeal; 10 percent wheat bran; 10 percent wheat shorts; 10 percent pulverized oats; 3 percent X X X liquor, vitamin concentrate; 5 percent oyster shell flour; 0.5 percent NaCl and 0.5 percent cod liver oil. The oils were added in lots 3 and 4 in the ratio of 2.1 percent by weight of the total feed. This provided these lots with the same cottonseed oil content that lot 5 received.
strongly indicate that gossypol is the olive yolk factor in cottonseed meal. Conclusive evidence that gossypol is the causative agent was obtained by two other tests. A normal fresh egg yolk was allowed to stand overnight in a small beaker of Wesson oil to which a small amount of pure gossypol had been added. As a check a second normal yolk was treated similarly in oil containing no gossypol. These two yolks and one from a cottonseed meal fed hen were placed in an atmosphere of ammonia. The cottonseed meal yolk and one which had been suspended in the gossypol solution changed to the same brown color simultaneously. The yolk that had been in
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on May 27, 2015
and identification of the fraction of cottonseed meal which is responsible for the formation of olive yolks. The second approach was a study of the chemistry involved in the formation of olive colored yolks. Two methods were used in the first section of this investigation. Feeding trials using various fractions of cottonseed meal made up the first method and the second method consisted of the use of the pure suspected component of cottonseed meal to induce the olive color in a normal fresh yolk simply by adding the compound to the yolk and placing the mixture in an atmosphere of ammonia. Gossypol and cottonseed oil were used in the feeding trials to attempt to clear up the uncertainty existing as to the responsibility of these components in the formation of the olive yolk color.
375
376
A. D. SWENSEN, E. A. FIEGER, AND C. W. U P P
EGG YOLK FRACTIONATION
As soon as it was established that gossypol was the factor in cottonseed meal which causes olive yolks, the next problem was to determine which fraction of the egg yolk combined with the gossypol to form the color. Normal fresh yolks were fractionated and each fraction was treated by mixing with an acetone solution of gossypol and placing the system in an atmosphere of ammonia. A brownish black color appeared on testing the protein fraction while no change occurred in the fat fraction. The proteins of the egg yolk were fractionated by separating vitellin, method of Calvery and White (1931), livetin, method of Kay and Marshall (1928), and an albumin, a globulin, and a "myxoprotein" by Piettre's (1936) method. All of these different proteins gave definitely positive tests with gossypol. A protein fraction of egg yolk which is exceedingly rich in iron was separated by Bunge's (1885) procedure. The time required for color formation with gossypol was much less with this fraction than any preceding fraction. The intensity of the color was deeper also, appearing black in only a few seconds after being placed in ammonia.
One of the characteristic properties of gossypol is its affinity for ferrous and ferric ions, forming compounds having a dark olive green color. When these olive green compounds are placed in an atmosphere of ammonia the color changes to a chocolate brown. This is precisely the same color change exhibited by a cottonseed meal egg yolk which had previously developed the olive green color in storage, when it was placed in an ammonia atmosphere. All iron was extracted from fat free egg yolk protein with 6N HC1. The iron free protein was wetted with an acetone solution of gossypol and allowed to stand 48 hours in an atmosphere of ammonia. No color developed during this period. Smythe and Schmidt (1930) showed that proteins may form compounds with ferric iron that are so slightly dissociated that a negative test is obtained with potassium thiocyanate. Experiments in this investigation have shown that moist fresh yolk proteins give negative tests for iron with potassium thiocyanate, thiosalicylic acid, thioglycolic acid, and an acetone solution of gossypol. However, when these systems were placed in an atmosphere of ammonia the characteristic colors of their iron salts were obtained. Apparently ammonia liberates ferric ions from the egg yolk proteins in sufficient concentration to react with qualitative ferric ion color reagents and gossypol. These results indicate that iron is the constituent of egg yolk which combines with gossypol from cottonseed meal in forming olive colored egg yolks. Karrer (1938) points out that iron displaces the hydrogen from the two hydroxyl groups in catechol to form a compound having an olive green color. When a trace of ammonia or alkali is added to the compound thus formed, the ammonium or sodium compound is produced. This salt is reddish brown in color and has the following formula:
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on May 27, 2015
Wesson oil only, retained its normal orange yellow color throughout the ammonia' treatment. The same change in color was produced in a normal yolk when it was broken and well mixed with an acetone solution of pure gossypol and then placed in an ammonia atmosphere. This evidence clearly shows that gossypol is the factor responsible for the formation of olive egg yolks when hens are fed cottonseed meal and indicates that gossypol is absorbed without chemical change from the intestines and is present as such in the egg. This latter point is being investigated in this laboratory.
NATURE OF EGG YOLK DISCOLORATION PRODUCED BY COTTONSEED MEAL
(NH 4 ) 3
feces dropped by the hens in each lot was determined for a three-day period. The quantity of gossypol ingested and the amount in the feces was determined by the (NH ( ), method Smith and Halverson (1933) used
Fe
o \ •o-
377
Fe;
TABLE 2.—Balance trial of gossypol
o-
Gossypol has the following formula, Adams (1938):
IRON SALTS AS A PREVENTIVE OF MOTTLED YOLKS
To maintain that iron is a factor involved in the production as well as the prevention of cottonseed meal egg yolk discoloration seems, at first sight, to be a paradox. It can be shown, however, that this is the case. Five lots of two Single Comb White Leghorn pullets each were fed a 30 percent allmash cottonseed meal diet (same as given in Table 1, lot S). As indicated in Table 2, varying amounts of a ferric chloride solution were added in the several lots. After a three-day preliminary period on these diets, the amount of feed consumed and
in feed
Gossypol ingested
Gossypol returned
Gossypol returned
1 2 3 4 5
percent 0.00 0.05 0.10 0.20 0.50
grams 1.39 1.53 1.18 2.36 2.01
grams 0.545 0.736 0.678 1.640 1.890
percent 39.2 48.2 57.5 69.5 94.0
in determining the total gossypol in cottonseed meal. The use of this procedure was tenable since it was shown that two samples of cottonseed meal gave the same analysis for total gossypol even after one of the samples had been treated with a ferric chloride solution forming the dark colored iron gossypol compound within the meal. These results indicate clearly that the presence of ferric chloride inhibits the absorption of gossypol by the hen. The mechanism for this inhibition is not known, but is probably due to the decreased solubility in the intestines of the iron-gossypol compound as compared to free gossypol. SUMMARY 1. Gossypol has been shown to be the factor in cottonseed meal which is responsible for the formation, in storage, of the olive colored yolks in eggs from hens fed cottonseed meal. 2. The characteristic olive color of the yolks is due to a chemical combination of the gossypol from the cottonseed meal with ferric iron released from the yolk proteins. 3. The iron combination with the yolk proteins of a fresh egg yolk is so slightly dissociated that insufficient ferric ions are
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on May 27, 2015
It contains two of the same functional groups as catechol, namely ortho hydroxyl groups, for which ferric ions have an apparent affinity. It appears then that the brown to black compound produced when a gossypol containing yolk is placed in an atmosphere of ammonia is the ammonium salt of the compound that imparts an olive green color to the yolk in storage.
Ferric
Lot no.
378
A. D . SWENSEN, E . A. FlEGER, AND C. W . UPP
REFERENCES
Adams, R., R. C. Morris, T. A. Geissman, D. J. Butterbough, and E. C. Kirkpatrick, 1938. Structure of gossypol. Journal of the American Chemical Society 60:2193-2204. Bunge, G. von, 1885. Uber die Assimilation des Eisens. Ztschr. f. Physiol. Chem. 9:49-59. Abs. Berichte 18:412. Calvery, H. O., and A. White, 1931. Vitellin of
hen's eggs. Jour. Biol. Chem. 94:635-639. Karrer, P., 1938. Organic chemistry. Nordemann Publishing Co., New York. Translated from the latest German edition by A. J. Mee. Kay, H. D., and P. G. Marshall, 1928. The second protein (livetin) of egg yolk. Biochem. Jour. 22:1264-1269. Piettre, M., 1936. Recherches sur les proteides de l'ovocytc (jaune de l'oeuf la poule). Comp. Rend. 202 :699-702. Roberts, I. P., and J. E. Rice, 1891. The effect on fowls of nitrogenous and carbonaceous rations. Expt. Sta. Rec. 2:506-507. Schaible, P. J., L. A. Moore, and J. M. Moore, 1934. Gossypol, a cause of discoloration in egg yolks. Science 79:372. Sherwood, R. M., 1929. The effect of various rations on the storage quality of eggs. Poultry Sci. 8:99-102. Smith, F. H., and J. O. Halverson, 1933. Estimation of total and bound (D) gossypol in cottonseed meal. A modified method. Indus, and Engin. Chem. (analytical ed.) 5:319. Smythe, C. V., and C. L. A. Schmidt, 1930. Studies on the mode of combination of iron with certain proteins, amino acids, and related compounds. J. Biol. Chem. 88:241-269. Thompson, R. B., W. P. Albright, E. E. Schnetzler, and V. G. Heller, 1933. Feeds and their relation to egg quality. Oklahoma Agr. Expt. Sta. . Rpt. 1930-32 :35.
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on May 27, 2015
present to give a positive test with iron testing reagents. 4. During storage the iron proteins decompose liberating sufficient iron to combine with gossypol to form the olive color. 5. Ammonia accelerates the release of iron from the yolk proteins. 6. The chocolate colored compound formed when iron and gossypol react in the presence of ammonia is believed to be the ammonium salt of the green colored compound present in olive yolks. 7. Addition of soluble ferric salts to the ration containing cottonseed meal prevents the absorption of the gossypol by the hen and the formation of olive yolks in the stored eggs.