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The Nutrient Content of High and Low Quality Fresh Eggs.
T h e Nutrient Content of High and Low Quality Fresh Eggs. II. Vitamins L. W. CHARKEY, ELIZABETH DYAR AND H. S. WILGUS, J R .
Colorado Agricultural Experiment Station, Colorado A. and M. College, Fort Collins (Received for publication June 21, 1947)
I
N AN attempt to relate the quality of eggs to their chemical composition, eggs of high and low albumen quality were assayed comparatively for their content of certain vitamins. Procedures for the selection and handling of samples are detailed in the previous paper, as is also the uniform procedure for digestion and extraction of subsamples for microbiological assays. Nicotinic acid was determined by the method of Krehl, Strong and Elvehjem (1943), using Lactobacillus arabinosus 17-5 as the test organism. Apparent* folic acid and apparent* pyridoxine were measured with Streptococcus faecalis, by modifications, of the methods of Luckey, Briggs and Elvehjem (1944) and of Snell, Guirard and Williams (1942), respectively. In all microbiological assays folic acid added was the pure synthetic material, t Carotene was determined colorimetrically in a washed, dried petroleum ether extract of the saponified yolk samples. Vitamin A was measured colorimetrically also, following addition of the Carr-Price (1926) reagent to the same extract. As recommended by Davies and Moore
Scientific Journal Series Paper 248, Colorado Agricultural Experiment Station. * See discussion for significance attached to this term. t Kindly furnished by Dr. E. L. R. Stokstad of the Lederle Laboratories, Pearl River. New York.
(1939) acetic anhydride was added prior to the Carr-Price reagent in order to prevent hydrolysis, which had been found to be difficult to remove completely in the type of sample being studied. It was established by previous tests that no color developed because of addition of acetic anhydride to the petroleum ether extract. RESULTS AND DISCUSSION
The findings are recorded in Table 1 in terms of averages of values as determined, namely on the wet basis. The numbers in parentheses indicate the number of individual measurements represented by each average shown. The data are presented for the value they may have to those interested in the vitamin content of eggs, per se, and in the distribution of these factors among the egg fractions. It is of interest to note that the yolks are much richer in apparent folic acid and apparent pyridoxine than are the albumen fractions, while they are poorer than are the albumen fractions in nicotinic acid. It will be seen from the table that high quality egg fractions did not differ consistently from low quality fractions in content of any of the vitamins determined. No conclusions appear to be justified from these results regarding a relationship of any of the factors to egg albumen quality, since the differences were not highly significant, statistically, on either the wet or dry basis in any case. After reduction to the dry basis most of the differences indi-
632
633
NUTRIENT CONTENT OF FRESH EGGS
cate a slightly higher content per gram of low quality solids. This follows logically if it is assumed that the vitamins are presented in water solution free of combination with protein, at least in partj so that their content in the egg corresponds roughly to that of water. Nicotinic acid in firm albumen is an exception to this general statement; the statistical odds
(Beadle and Tatum, 1941), indicated a negligible amount of this substance in eggs,, the responses obtained with S. faecalis must have been due largely to pyridoxal and pyridoxamine. Since in this case the amount of the compounds required to cause the response was very small, and since according to Sarma et al. (1946) these compounds are only roughly
TABLE 1.—Content of moisture and of certain vitamins in high and low quality egg fractions Fluid albumen High Moisture—per cent Nicotinic Acid—mcg./g.
Low
86.63 88.10 (23) (25)
Firm albumen
Yolks
Ave.
High
Low
Ave.
High
87.36 (48)
87.50 (24)
88.17 (25)
87.33 (49)
47.08 47.12 (25) (25)
47.10 (50)
1.53 (21)
1.73 (42)
0.71 (20)
0.71 (39)
Low
Ave.
1.67 (21)
1.56 (21)
1.61 (42)
1.94 (21)
Apparent Folic Acid— mmcg./g.
15.0 (25)
14.85 (25)
14.9 (50)
16.4 (24)
14.0 (26)
15.2 (50)
224.6 :227.2 225.9 (23) (44) (21)
Apparent Pyridoxine mcg./g.
43.5 (16)
48.7 (16)
46.1 (32)
46.4 (16)
43.5(17)
44.9 (33)
457.0 453.0 455.0 (14) (29) (15)
0.71 (19)
Vitamin A—I.IT./g.
48.6 (16)
46.1 (16)
47.3 (32)
Carotene—mcg./g.
1.77 (19)
1.97 (19)
1.87 (38)
being about 40:1 that high quality firm albumen contains more nicotinic acid than does low quality firm albumen. Whether or not this has any relationship to the higher 1( —) tryptophane values reported in the previous paper for both fluid and firm high quality albumen fractions is not apparent from the present studies. Apparent Pyridoxine This term is used in recognition of the finding of Snell et al. (1942) that natural materials contain pyridoxal and pyridoxamine, each of which is several thousand times as stimulatory to S. faecalis as is pyridoxine itself (Snell, 1944). Since regulatory assays by the method of Stokes et al. (1943) using Neurospora sitophila 299, which responds only to pyridoxine
equivalent to pyridoxine in the nutrition of higher animals, the data indicate that eggs are a relatively poor source of the "vitamin B6 complex" for animal nutrition unless they contain still other B6active compounds, as yet unrecognized. Apparent Folic Acid The existence of a conjugated form of folic acid unavailable to S. faecalis is now well established (Bird and Robbins, 1946; Pfiffner et al, 1945, 1946); as is also the ability of hog kidney and other preparations to release active folic acid from the conjugated forms (Bird et al., 1945, 1946; Mims and Laskowski, 1945). Accordingly a series of assays was run in which the folic acid activities of egg fractions were measured before and after
634
L. W.
C H A R K E Y , E L I Z A B E T H D Y A R , AND H . S. W I L G U S , J R .
treatment with desiccated hog kidney (Bird et al., 1945). Quite consistent increases were obtained for each egg fraction, the amount of increase obtained being relatively much larger in both fluid and firm albumen t h a n in yolk. From the results of nine assays, hog kidney folic acid conversion factors were computed as follows: For fluid albumen, 21.6 times the original folic acid value For firm albumen, 30.2 times the original folic acid value For yolk, 3.2 times the original folic acid value No significant difference in the amount of increase was found between high and low quality eggs for any of the three fractions. T h e egg whites have a much larger proportion of their " t o t a l " folic acid in conjugated form t h a n do the yolks. By application of the above factors to the average folic acid values in Table 1 it is seen t h a t the yolks contain approximately 0.7 microgram of " t o t a l " folic acid per gram of fresh yolk. Roughly half as much is contained per gram of fresh egg white. SUMMARY AND CONCLUSION 1. The content and distribution in eggs was determined for vitamin A, carotene, nicotinic acid, folic acid (apparent and total), and apparent pyridoxine. 2. Egg albumen quality does not appear to be related to the content of any of the above factors. REFERENCES
Beadle, G. W. and E. L. Tatum, 1941. Genetic Control of Biochemical Reaction in Neurospora. Proc. Nat. Acad. Sci. 27:499-506. Bird, 0. D., B. Bressler, R. A. Brown, C. J. Camp-
bell and A. D. Emmett, 1945. The Microbiological Assay of Vitamin Be Conjugate. J. Biol. Chem. 159: 631-636. Bird, O. D. and Mary Robbins, 1946. The Response of Lactobacillus casei and Streptococcus faecalis to Vitamin Be and Vitamin Be Conjugate. J. Biol. Chem. 163:661-665. Bird, O. D., Mary Robbins, J. M. Vandenbelt and J. J. Pfiffner, 1946. Observations on Vitamin Be Conjugase from Hog Kidney. J. Biol. Chem. 163: 649-659. Carr, F. H. and E. A. Price, 1926. Color Reactions Attributed to Vitamin A. Biochem. J. 20: 497501. Davies, A. W. and T. Moore, 1939. Determination of Vitamin A and Carotene in Organs. Vitaminforsch. 9: 254-255. Krehl, W. A., F. M. Strong and C. A. Elvehjem, 1943. Determination of Nicotinic Acid. Ind. and Eng. Chem., Anal. Ed. 15:471-475. Luckey, T. D., G. M. Briggs, Jr. and C. A. Elvehjem, 1944. The Use of Streptococcus lactis R for the Measurement of "Folic Acid." J. Biol. Chem. 152:157-167. Mims, Virginia and Michael Laskowski, 1945. Studies on Vitamin Be Conjugase from Chicken Pancreas. J. Biol. Chem. 160:493-503. Pfiffner, J. J., D. G. Calkins, B. L. O'Dell, E. S. Bloom, R. A. Brown, C. J. Campbell and O. D. Bird, 1945. Isolation of an Antianemia Factor (Vitamin Be Conjugate) in Crystalline Form from Yeast. Science. 102: 228-230. Pfiffner, J. J., D. G. Calkins, E. S. Bloom and B. L. O'Dell, 1946. On the Peptide Nature of Vitamin Be Conjugate from Yeast. J. Am. Chem. Soc. 68:1392. Sarma, P. S., Esmond E. Snell and C. A. Elvehjem, 1946. The Vitamin B6 Group. VIII. Biological Assay of Pyridoxal, Pyridoxamine and Pyridoxine. J. Biol. Chem. 163:55-63. Snell, Esmond E., Beverly M. Guirard and Roger J. Williams, 1942. Occurrence in Natural Products of a Physiologically Active Metabolite of Pyridoxine. J. Biol. Chem. 143:519-530. Snell, Esmond E., 1944. The Vitamin Activities of Pyridoxal and Pyridoxamine. J. Biol. Chem. 154: 313-316. Stokes, Jacob L., Alma Larsen, Carl R. Woodward, Jr. and Jackson W. Foster, 1943. A Neurospora Assay for Pyridoxine. J. Biol. Chem. 150: 17-24.
Colorado Agricultural Experiment Station, Colorado A. and M. College, Fort Collins (Received for publication June 21, 1947)
I
N AN attempt to relate the quality of eggs to their chemical composition, eggs of high and low albumen quality were assayed comparatively for their content of certain vitamins. Procedures for the selection and handling of samples are detailed in the previous paper, as is also the uniform procedure for digestion and extraction of subsamples for microbiological assays. Nicotinic acid was determined by the method of Krehl, Strong and Elvehjem (1943), using Lactobacillus arabinosus 17-5 as the test organism. Apparent* folic acid and apparent* pyridoxine were measured with Streptococcus faecalis, by modifications, of the methods of Luckey, Briggs and Elvehjem (1944) and of Snell, Guirard and Williams (1942), respectively. In all microbiological assays folic acid added was the pure synthetic material, t Carotene was determined colorimetrically in a washed, dried petroleum ether extract of the saponified yolk samples. Vitamin A was measured colorimetrically also, following addition of the Carr-Price (1926) reagent to the same extract. As recommended by Davies and Moore
Scientific Journal Series Paper 248, Colorado Agricultural Experiment Station. * See discussion for significance attached to this term. t Kindly furnished by Dr. E. L. R. Stokstad of the Lederle Laboratories, Pearl River. New York.
(1939) acetic anhydride was added prior to the Carr-Price reagent in order to prevent hydrolysis, which had been found to be difficult to remove completely in the type of sample being studied. It was established by previous tests that no color developed because of addition of acetic anhydride to the petroleum ether extract. RESULTS AND DISCUSSION
The findings are recorded in Table 1 in terms of averages of values as determined, namely on the wet basis. The numbers in parentheses indicate the number of individual measurements represented by each average shown. The data are presented for the value they may have to those interested in the vitamin content of eggs, per se, and in the distribution of these factors among the egg fractions. It is of interest to note that the yolks are much richer in apparent folic acid and apparent pyridoxine than are the albumen fractions, while they are poorer than are the albumen fractions in nicotinic acid. It will be seen from the table that high quality egg fractions did not differ consistently from low quality fractions in content of any of the vitamins determined. No conclusions appear to be justified from these results regarding a relationship of any of the factors to egg albumen quality, since the differences were not highly significant, statistically, on either the wet or dry basis in any case. After reduction to the dry basis most of the differences indi-
632
633
NUTRIENT CONTENT OF FRESH EGGS
cate a slightly higher content per gram of low quality solids. This follows logically if it is assumed that the vitamins are presented in water solution free of combination with protein, at least in partj so that their content in the egg corresponds roughly to that of water. Nicotinic acid in firm albumen is an exception to this general statement; the statistical odds
(Beadle and Tatum, 1941), indicated a negligible amount of this substance in eggs,, the responses obtained with S. faecalis must have been due largely to pyridoxal and pyridoxamine. Since in this case the amount of the compounds required to cause the response was very small, and since according to Sarma et al. (1946) these compounds are only roughly
TABLE 1.—Content of moisture and of certain vitamins in high and low quality egg fractions Fluid albumen High Moisture—per cent Nicotinic Acid—mcg./g.
Low
86.63 88.10 (23) (25)
Firm albumen
Yolks
Ave.
High
Low
Ave.
High
87.36 (48)
87.50 (24)
88.17 (25)
87.33 (49)
47.08 47.12 (25) (25)
47.10 (50)
1.53 (21)
1.73 (42)
0.71 (20)
0.71 (39)
Low
Ave.
1.67 (21)
1.56 (21)
1.61 (42)
1.94 (21)
Apparent Folic Acid— mmcg./g.
15.0 (25)
14.85 (25)
14.9 (50)
16.4 (24)
14.0 (26)
15.2 (50)
224.6 :227.2 225.9 (23) (44) (21)
Apparent Pyridoxine mcg./g.
43.5 (16)
48.7 (16)
46.1 (32)
46.4 (16)
43.5(17)
44.9 (33)
457.0 453.0 455.0 (14) (29) (15)
0.71 (19)
Vitamin A—I.IT./g.
48.6 (16)
46.1 (16)
47.3 (32)
Carotene—mcg./g.
1.77 (19)
1.97 (19)
1.87 (38)
being about 40:1 that high quality firm albumen contains more nicotinic acid than does low quality firm albumen. Whether or not this has any relationship to the higher 1( —) tryptophane values reported in the previous paper for both fluid and firm high quality albumen fractions is not apparent from the present studies. Apparent Pyridoxine This term is used in recognition of the finding of Snell et al. (1942) that natural materials contain pyridoxal and pyridoxamine, each of which is several thousand times as stimulatory to S. faecalis as is pyridoxine itself (Snell, 1944). Since regulatory assays by the method of Stokes et al. (1943) using Neurospora sitophila 299, which responds only to pyridoxine
equivalent to pyridoxine in the nutrition of higher animals, the data indicate that eggs are a relatively poor source of the "vitamin B6 complex" for animal nutrition unless they contain still other B6active compounds, as yet unrecognized. Apparent Folic Acid The existence of a conjugated form of folic acid unavailable to S. faecalis is now well established (Bird and Robbins, 1946; Pfiffner et al, 1945, 1946); as is also the ability of hog kidney and other preparations to release active folic acid from the conjugated forms (Bird et al., 1945, 1946; Mims and Laskowski, 1945). Accordingly a series of assays was run in which the folic acid activities of egg fractions were measured before and after
634
L. W.
C H A R K E Y , E L I Z A B E T H D Y A R , AND H . S. W I L G U S , J R .
treatment with desiccated hog kidney (Bird et al., 1945). Quite consistent increases were obtained for each egg fraction, the amount of increase obtained being relatively much larger in both fluid and firm albumen t h a n in yolk. From the results of nine assays, hog kidney folic acid conversion factors were computed as follows: For fluid albumen, 21.6 times the original folic acid value For firm albumen, 30.2 times the original folic acid value For yolk, 3.2 times the original folic acid value No significant difference in the amount of increase was found between high and low quality eggs for any of the three fractions. T h e egg whites have a much larger proportion of their " t o t a l " folic acid in conjugated form t h a n do the yolks. By application of the above factors to the average folic acid values in Table 1 it is seen t h a t the yolks contain approximately 0.7 microgram of " t o t a l " folic acid per gram of fresh yolk. Roughly half as much is contained per gram of fresh egg white. SUMMARY AND CONCLUSION 1. The content and distribution in eggs was determined for vitamin A, carotene, nicotinic acid, folic acid (apparent and total), and apparent pyridoxine. 2. Egg albumen quality does not appear to be related to the content of any of the above factors. REFERENCES
Beadle, G. W. and E. L. Tatum, 1941. Genetic Control of Biochemical Reaction in Neurospora. Proc. Nat. Acad. Sci. 27:499-506. Bird, 0. D., B. Bressler, R. A. Brown, C. J. Camp-
bell and A. D. Emmett, 1945. The Microbiological Assay of Vitamin Be Conjugate. J. Biol. Chem. 159: 631-636. Bird, O. D. and Mary Robbins, 1946. The Response of Lactobacillus casei and Streptococcus faecalis to Vitamin Be and Vitamin Be Conjugate. J. Biol. Chem. 163:661-665. Bird, O. D., Mary Robbins, J. M. Vandenbelt and J. J. Pfiffner, 1946. Observations on Vitamin Be Conjugase from Hog Kidney. J. Biol. Chem. 163: 649-659. Carr, F. H. and E. A. Price, 1926. Color Reactions Attributed to Vitamin A. Biochem. J. 20: 497501. Davies, A. W. and T. Moore, 1939. Determination of Vitamin A and Carotene in Organs. Vitaminforsch. 9: 254-255. Krehl, W. A., F. M. Strong and C. A. Elvehjem, 1943. Determination of Nicotinic Acid. Ind. and Eng. Chem., Anal. Ed. 15:471-475. Luckey, T. D., G. M. Briggs, Jr. and C. A. Elvehjem, 1944. The Use of Streptococcus lactis R for the Measurement of "Folic Acid." J. Biol. Chem. 152:157-167. Mims, Virginia and Michael Laskowski, 1945. Studies on Vitamin Be Conjugase from Chicken Pancreas. J. Biol. Chem. 160:493-503. Pfiffner, J. J., D. G. Calkins, B. L. O'Dell, E. S. Bloom, R. A. Brown, C. J. Campbell and O. D. Bird, 1945. Isolation of an Antianemia Factor (Vitamin Be Conjugate) in Crystalline Form from Yeast. Science. 102: 228-230. Pfiffner, J. J., D. G. Calkins, E. S. Bloom and B. L. O'Dell, 1946. On the Peptide Nature of Vitamin Be Conjugate from Yeast. J. Am. Chem. Soc. 68:1392. Sarma, P. S., Esmond E. Snell and C. A. Elvehjem, 1946. The Vitamin B6 Group. VIII. Biological Assay of Pyridoxal, Pyridoxamine and Pyridoxine. J. Biol. Chem. 163:55-63. Snell, Esmond E., Beverly M. Guirard and Roger J. Williams, 1942. Occurrence in Natural Products of a Physiologically Active Metabolite of Pyridoxine. J. Biol. Chem. 143:519-530. Snell, Esmond E., 1944. The Vitamin Activities of Pyridoxal and Pyridoxamine. J. Biol. Chem. 154: 313-316. Stokes, Jacob L., Alma Larsen, Carl R. Woodward, Jr. and Jackson W. Foster, 1943. A Neurospora Assay for Pyridoxine. J. Biol. Chem. 150: 17-24.