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Extraction of Calcium from Blood Fibrin with a Solution of Tetrasodium-Ethylenediaminetetraacetate (Na4 EDTA)1
Extraction of Calcium from Blood Fibrin with a Solution of Tetrasodium-Ethylenediaminetetraacetate (Na4EDTA) 1 H. J. LIN, L. C. N O R M S AND F. H. KRATZER Department of Avian Sciences and School of Medicine, University of California, Davis, California 95616 (Received for publication June 12, 1972)
POULTKY SCIENCE 52: 540-542, 1973
I
N AN experiment of 28 weeks duration evidence was obtained that a purified basal diet containing 14.5% blood fibrin as the protein source, supplied almost, but not quite enough calcium to meet the needs of mature White Leghorn male chickens (Norris et al., 1972). Most of the males were 11 months of age at the start of the experiment but a few were somewhat older. The basal diet was found, when assayed by the atomic absorption procedure to contain 0.03% calcium. Using the same procedure, the blood fibrin was shown to have 0.1881% calcium. The blood fibrin, therefore, proved to be the major source of calcium in the basal diet. Because of the failure to obtain striking evidence of calcium deficiency in male chickens fed the basal diet, an attempt was made to reduce the calcium content of the blood fibrin. In order to do this, recourse was made to the chelating agent, tetrasodium-ethylenediaminetetraacetate (Na 4 EDTA), and a procedure for removing the calcium from the blood fibrin by washing with a Na 4 EDTA solution was 1
Supported in part by NASA Contract NAS25345.
developed. This procedure was an adaptation of the procedure developed by Davis et al. (1963) for reducing the trace mineral content of isolated soybean protein. The use of Na 4 EDTA to prevent interference from calcium by combination with the chelate to form an aqueous system has been employed successfully in a variety of ways (Chaberek and Mart ell, 1959). The procedure has been used by Hurley and Swenerton (1966) to reduce the zinc content of soybean protein in studies on zinc deficiency in adult female rats and a modification of the procedure has been made use of by Swenerton and Hurley (1968) to lower the zinc content of soybean protein in similar studies on weanling rats. EXPERIMENTAL
To remove calcium from blood fibrin, 10 kg. of the protein were suspended in 100 liters of deionized water containing 500 gm. Na 4 EDTA. The ratio of blood fibrin to the chelate was, therefore, one kg. protein per 50 gm. Na 4 EDTA. The procedure was carried out in a doublewalled, stainless steel, steam kettle, similar to those frequently used in largescale cookery. After adding the protein
540
Downloaded from http://ps.oxfordjournals.org/ at New York University on April 22, 2015
ABSTRACT A modification of a procedure, developed previously for reducing the trace mineral content of isolated soybean protein, has been made for the purpose of removing calcium from blood fibrin and thereby lowering the calcium content of a purified diet containing it. The properties of the chelate, tetrasodium ethylenediaminetetraacetate (Na4EDTA), of combining with cations and keeping them in solution so that they could be washed away at the isoelectric point of the proteins were utilized in developing the procedure. The original blood fibrin was found to contain 0.1881% calcium. After purification by the procedure described the calcium content was reduced to 0.0078%. This amounted to approximately 4.15% of the original quantity.
541
BLOOD CALCIUM EXTRACTION
T h e last traces of Na4EDTA were removed from the blood fibrin by washing four times with deionized water after making u p to original volume, stirring for 15 minutes, allowing the protein to settle, and siphoning off the water. After the last washing, 5 ml. of a saturated solution of ammonium oxalate were added to 10 ml. of the supernatant fluid to test for removal of Na4EDTA. Then t h e test solution was adjusted t o p H 11 with N a O H and one drop of a saturated solution of CaCU was added. T h e formation of a precipitate of white calcium oxalate showed t h a t all the Na^EDTA had been removed b y repeated washing with deionized water. Finally approximately 1/4 liter of acetone per kg. of the original blood fibrin was added to the settled protein and the
TABLE 1.—Results of extracting calcium from blood fibrin with Nai EDTA
Expt. 1 Expt. 2 Calcium in purified diet, % 0.03 0.0035 Calcium content crude 0.1881 blood fibrin, % Calcium content purified 0.0078 blood fibrin, % Calcium in diet from 14.5% 0.0273 crude blood fibrin, % Calcium in diet from 14.5% 0.0011 purified blood fibrin, % Calcium in diet from other 0.0027 0.0024 ingredients, % Feed intake per White Leghorn 61.03 75.00 male per day, gm. Calcium intake per White Leg- 18.31 2.63 horn male per day, mg. acetone-water mixture was decanted. T h e acetone aided greatly in removing residual water from the blood fibrin and made it possible to dry the protein quickly. This procedure was repeated, and the washed protein was removed from the tank, placed in thin layers on trays, and dried in a warm room. To prevent contamination by dust in the air, the protein was covered with cheese cloth during drying. RESULTS AND DISCUSSION I n the first a t t e m p t at extracting calcium from blood fibrin b y washing with a N a 4 E D T A solution, the calcium was reduced from 0.1881% to 0.0118%, and in the present more refined procedure, the calcium content was reduced to 0.0078%. The basal diet containing the washed blood fibrin was found b y assay to contain 0.0035% calcium. This diet was fed in a further investigation of the calcium requirement of adult White Leghorn male chickens. Some typical results of applying the extraction procedure in the investigation are given in Table 1. T h e results are compared with those of the first experiment in which t h e basal diet contained crude blood fibrin. T h e calcium content of the ingredients other t h a n
Downloaded from http://ps.oxfordjournals.org/ at New York University on April 22, 2015
to the N a 4 E D T A solution, the p H was adjusted to 11 with 2N N a O H , the solution was stirred for one hour with compressed air, and the p H was readjusted to 5.2, the isoelectric point of blood fibrin, with approximately 2N HC1. After settling, the supernatant fluid was siphoned off. Deionized water was now added to the t a n k containing the treated protein in an amount equal to the previous volume of solution, the p H was adjusted to 5.2 and the mixture was stirred for 15 minutes before allowing it to settle. T h e supernatant fluid was siphoned off and sufficient deionized water was again added to the t a n k to bring the total volume of protein and solution to the original. One hundred gm. of N a 4 E D T A were then added to the protein-water mixture. The ratio of blood fibrin to chelate therefore was one kg. protein per 10 gm. of N a 4 E D T A . After adjustment of the p H to 5.2 the mixture was stirred for 15 minutes after which t h e protein was allowed to settle and the supernatant fluid siphoned off.
542
H. J. LIN, L. C. NORRIS AND F. H. KRATZER
61.0 gm. per day). Normal food intake in experiment 2 was obtained in the groups, supplied 0.025%, 0.05% and 0.10% calcium (66.7 + 1.0 gm., food per day). REFERENCES Chaberek, S., and A. E. Martell, 1959. Organic Sequestering Agents. John Wiley and Sons, Inc., New York. Davis, P. N., L. C. Norris and F. H. Kratzer, 1962. Iron deficiency studies in chicks using treated isolated soybean protein diets. J. Nutrition, 78: 445-453. Hurley, L. S., and H. Swenerton, 1966. Congenital malformations resulting from zinc deficiency in rats. Proc. Soc. Exp. Biol. Med. 123: 692-696. Norris, L. C , F. H. Kratzer, H. J. Lin, A. B. Hellewell and J. R. Beljan, 1972. Effect of quantity of dietary calcium on maintenance of bone integrity in mature White Leghorn male chickens. J. Nutrition, 102: 1085-1091. Swenerton, H., and S. Hurley, 1968. Severe zinc deficiency in male and female rats. J. Nutrition, 95: 8-18.
NEWS AND NOTES {Continued from page 539) sludge handling, design practice, recovery and recycling. Further information may be obtained by contacting F. E. Junkin, Associate Director, Water Resources Research Institute, North Carolina State University, 124 Reddiek Building, Raleigh, North Carolina 27607. POULTRY MEAT SYMPOSIUM A Symposium on Poultry Meat will be held in Roskilde, Denmark, July 30 to August 3. This Symposium is arranged under the auspices of the Subcommittee on Meat Quality of the European Federation of the World Poultry Science Association. The Symposium will be held at the Danish Meat Trade School in Roskilde, where all participants will be housed. Roskilde is 31 kilometers (20 miles) (25 minutes by train) west of Copenhagen. The following is the preliminary program: Monday, July 30, evening: Informal get-together party. Tuesday, July 31, morning, afternoon: Session on chilling and transport of poultry meats, introduced by Prof. Dr. S. Scholtyssek.
Wednesday, August 1, morning: Session on fresh and frozen poultry, introduced by Dr. Ir. B. Erdtsieck. afternoon: Session on yields and conformation of carcasses, introduced by Prof. JjzSrgen Fris Jensen. Thursday, August 2, morning, afternoon: Excursion to poultry slaughtering plant and the Danish Research Institute for Poultry Processing. evening: Banquet. Friday, August 3: Departure. In conjunction with the Symposium, the Subcommittee on Egg Quality of the European Federation of the W.P.S.A. will meet Wednesday afternoon, August 1, and Friday morning, August 3. A social program for accompanying family members will be arranged. The language of the Symposium will be English. There will be no translation arrangements. The Registration Fee for the Symposium before May 31, is D.kr. 200.00 (approximately D.M. 93.00, B.F. 1,283.00, Eng. £12.45, F.F. 147.40, Hfldr. 93.90, It. lire 17,000, and U.S. $29.15). Room (with shower and toilet) and meals at the
{Continued on page 591)
Downloaded from http://ps.oxfordjournals.org/ at New York University on April 22, 2015
blood fibrin, calculated by difference, were very similar in the two experiments, indicating that the calcium assays by the atomic absorption procedure were consistent. Thus, by washing blood fibrin with a Na 4 EDTA solution, it became possible to reduce the daily calcium intake from approximately 18 mg., in experiment 1, to 2.6 mg. in experiment 2. The difference in daily calcium intake became even more striking when it was calculated from the normal feed intake of the males of experiment 2 and the calcium content of the crude blood fibrin basal diet fed in experiment 1. This value was 20.01 mg. calcium per male per day. This calculation is justified since in experiment 1 the daily food intake of the males fed the crude blood fibrin basal diet was the same as that of the positive control (61.04 gm. vs.
POULTKY SCIENCE 52: 540-542, 1973
I
N AN experiment of 28 weeks duration evidence was obtained that a purified basal diet containing 14.5% blood fibrin as the protein source, supplied almost, but not quite enough calcium to meet the needs of mature White Leghorn male chickens (Norris et al., 1972). Most of the males were 11 months of age at the start of the experiment but a few were somewhat older. The basal diet was found, when assayed by the atomic absorption procedure to contain 0.03% calcium. Using the same procedure, the blood fibrin was shown to have 0.1881% calcium. The blood fibrin, therefore, proved to be the major source of calcium in the basal diet. Because of the failure to obtain striking evidence of calcium deficiency in male chickens fed the basal diet, an attempt was made to reduce the calcium content of the blood fibrin. In order to do this, recourse was made to the chelating agent, tetrasodium-ethylenediaminetetraacetate (Na 4 EDTA), and a procedure for removing the calcium from the blood fibrin by washing with a Na 4 EDTA solution was 1
Supported in part by NASA Contract NAS25345.
developed. This procedure was an adaptation of the procedure developed by Davis et al. (1963) for reducing the trace mineral content of isolated soybean protein. The use of Na 4 EDTA to prevent interference from calcium by combination with the chelate to form an aqueous system has been employed successfully in a variety of ways (Chaberek and Mart ell, 1959). The procedure has been used by Hurley and Swenerton (1966) to reduce the zinc content of soybean protein in studies on zinc deficiency in adult female rats and a modification of the procedure has been made use of by Swenerton and Hurley (1968) to lower the zinc content of soybean protein in similar studies on weanling rats. EXPERIMENTAL
To remove calcium from blood fibrin, 10 kg. of the protein were suspended in 100 liters of deionized water containing 500 gm. Na 4 EDTA. The ratio of blood fibrin to the chelate was, therefore, one kg. protein per 50 gm. Na 4 EDTA. The procedure was carried out in a doublewalled, stainless steel, steam kettle, similar to those frequently used in largescale cookery. After adding the protein
540
Downloaded from http://ps.oxfordjournals.org/ at New York University on April 22, 2015
ABSTRACT A modification of a procedure, developed previously for reducing the trace mineral content of isolated soybean protein, has been made for the purpose of removing calcium from blood fibrin and thereby lowering the calcium content of a purified diet containing it. The properties of the chelate, tetrasodium ethylenediaminetetraacetate (Na4EDTA), of combining with cations and keeping them in solution so that they could be washed away at the isoelectric point of the proteins were utilized in developing the procedure. The original blood fibrin was found to contain 0.1881% calcium. After purification by the procedure described the calcium content was reduced to 0.0078%. This amounted to approximately 4.15% of the original quantity.
541
BLOOD CALCIUM EXTRACTION
T h e last traces of Na4EDTA were removed from the blood fibrin by washing four times with deionized water after making u p to original volume, stirring for 15 minutes, allowing the protein to settle, and siphoning off the water. After the last washing, 5 ml. of a saturated solution of ammonium oxalate were added to 10 ml. of the supernatant fluid to test for removal of Na4EDTA. Then t h e test solution was adjusted t o p H 11 with N a O H and one drop of a saturated solution of CaCU was added. T h e formation of a precipitate of white calcium oxalate showed t h a t all the Na^EDTA had been removed b y repeated washing with deionized water. Finally approximately 1/4 liter of acetone per kg. of the original blood fibrin was added to the settled protein and the
TABLE 1.—Results of extracting calcium from blood fibrin with Nai EDTA
Expt. 1 Expt. 2 Calcium in purified diet, % 0.03 0.0035 Calcium content crude 0.1881 blood fibrin, % Calcium content purified 0.0078 blood fibrin, % Calcium in diet from 14.5% 0.0273 crude blood fibrin, % Calcium in diet from 14.5% 0.0011 purified blood fibrin, % Calcium in diet from other 0.0027 0.0024 ingredients, % Feed intake per White Leghorn 61.03 75.00 male per day, gm. Calcium intake per White Leg- 18.31 2.63 horn male per day, mg. acetone-water mixture was decanted. T h e acetone aided greatly in removing residual water from the blood fibrin and made it possible to dry the protein quickly. This procedure was repeated, and the washed protein was removed from the tank, placed in thin layers on trays, and dried in a warm room. To prevent contamination by dust in the air, the protein was covered with cheese cloth during drying. RESULTS AND DISCUSSION I n the first a t t e m p t at extracting calcium from blood fibrin b y washing with a N a 4 E D T A solution, the calcium was reduced from 0.1881% to 0.0118%, and in the present more refined procedure, the calcium content was reduced to 0.0078%. The basal diet containing the washed blood fibrin was found b y assay to contain 0.0035% calcium. This diet was fed in a further investigation of the calcium requirement of adult White Leghorn male chickens. Some typical results of applying the extraction procedure in the investigation are given in Table 1. T h e results are compared with those of the first experiment in which t h e basal diet contained crude blood fibrin. T h e calcium content of the ingredients other t h a n
Downloaded from http://ps.oxfordjournals.org/ at New York University on April 22, 2015
to the N a 4 E D T A solution, the p H was adjusted to 11 with 2N N a O H , the solution was stirred for one hour with compressed air, and the p H was readjusted to 5.2, the isoelectric point of blood fibrin, with approximately 2N HC1. After settling, the supernatant fluid was siphoned off. Deionized water was now added to the t a n k containing the treated protein in an amount equal to the previous volume of solution, the p H was adjusted to 5.2 and the mixture was stirred for 15 minutes before allowing it to settle. T h e supernatant fluid was siphoned off and sufficient deionized water was again added to the t a n k to bring the total volume of protein and solution to the original. One hundred gm. of N a 4 E D T A were then added to the protein-water mixture. The ratio of blood fibrin to chelate therefore was one kg. protein per 10 gm. of N a 4 E D T A . After adjustment of the p H to 5.2 the mixture was stirred for 15 minutes after which t h e protein was allowed to settle and the supernatant fluid siphoned off.
542
H. J. LIN, L. C. NORRIS AND F. H. KRATZER
61.0 gm. per day). Normal food intake in experiment 2 was obtained in the groups, supplied 0.025%, 0.05% and 0.10% calcium (66.7 + 1.0 gm., food per day). REFERENCES Chaberek, S., and A. E. Martell, 1959. Organic Sequestering Agents. John Wiley and Sons, Inc., New York. Davis, P. N., L. C. Norris and F. H. Kratzer, 1962. Iron deficiency studies in chicks using treated isolated soybean protein diets. J. Nutrition, 78: 445-453. Hurley, L. S., and H. Swenerton, 1966. Congenital malformations resulting from zinc deficiency in rats. Proc. Soc. Exp. Biol. Med. 123: 692-696. Norris, L. C , F. H. Kratzer, H. J. Lin, A. B. Hellewell and J. R. Beljan, 1972. Effect of quantity of dietary calcium on maintenance of bone integrity in mature White Leghorn male chickens. J. Nutrition, 102: 1085-1091. Swenerton, H., and S. Hurley, 1968. Severe zinc deficiency in male and female rats. J. Nutrition, 95: 8-18.
NEWS AND NOTES {Continued from page 539) sludge handling, design practice, recovery and recycling. Further information may be obtained by contacting F. E. Junkin, Associate Director, Water Resources Research Institute, North Carolina State University, 124 Reddiek Building, Raleigh, North Carolina 27607. POULTRY MEAT SYMPOSIUM A Symposium on Poultry Meat will be held in Roskilde, Denmark, July 30 to August 3. This Symposium is arranged under the auspices of the Subcommittee on Meat Quality of the European Federation of the World Poultry Science Association. The Symposium will be held at the Danish Meat Trade School in Roskilde, where all participants will be housed. Roskilde is 31 kilometers (20 miles) (25 minutes by train) west of Copenhagen. The following is the preliminary program: Monday, July 30, evening: Informal get-together party. Tuesday, July 31, morning, afternoon: Session on chilling and transport of poultry meats, introduced by Prof. Dr. S. Scholtyssek.
Wednesday, August 1, morning: Session on fresh and frozen poultry, introduced by Dr. Ir. B. Erdtsieck. afternoon: Session on yields and conformation of carcasses, introduced by Prof. JjzSrgen Fris Jensen. Thursday, August 2, morning, afternoon: Excursion to poultry slaughtering plant and the Danish Research Institute for Poultry Processing. evening: Banquet. Friday, August 3: Departure. In conjunction with the Symposium, the Subcommittee on Egg Quality of the European Federation of the W.P.S.A. will meet Wednesday afternoon, August 1, and Friday morning, August 3. A social program for accompanying family members will be arranged. The language of the Symposium will be English. There will be no translation arrangements. The Registration Fee for the Symposium before May 31, is D.kr. 200.00 (approximately D.M. 93.00, B.F. 1,283.00, Eng. £12.45, F.F. 147.40, Hfldr. 93.90, It. lire 17,000, and U.S. $29.15). Room (with shower and toilet) and meals at the
{Continued on page 591)
Downloaded from http://ps.oxfordjournals.org/ at New York University on April 22, 2015
blood fibrin, calculated by difference, were very similar in the two experiments, indicating that the calcium assays by the atomic absorption procedure were consistent. Thus, by washing blood fibrin with a Na 4 EDTA solution, it became possible to reduce the daily calcium intake from approximately 18 mg., in experiment 1, to 2.6 mg. in experiment 2. The difference in daily calcium intake became even more striking when it was calculated from the normal feed intake of the males of experiment 2 and the calcium content of the crude blood fibrin basal diet fed in experiment 1. This value was 20.01 mg. calcium per male per day. This calculation is justified since in experiment 1 the daily food intake of the males fed the crude blood fibrin basal diet was the same as that of the positive control (61.04 gm. vs.