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A Chick Vitamin D Assay Method Using Radioactive Calcium (Ca45)
A Chick Vitamin D Assay Method Using Radioactive Calcium (Ca45) H. PATRICK AND G. K. SCHWEITZER University of Tennessee, Knoxville, Tennessee (Received for publication November 22, 1954)
L
EXPERIMENTAL
Preparation of Chicks and Administration of Test Materials. Day-old White Leghorn cockerels were housed in electrically heated battery brooders and given the basal ration shown in Table 1 for 6 days. They were then divided into lots of 8 chicks each and given the basal ration plus supplements of vitamin D as shown in Table 2. After receiving the supplemented rations for 7 days, each chick was given 20 microcuries of Ca46 by pipette.
TABLE 1
Vitamin D Assay Basal Ration: Ground wheat Corn meal Alfalfa meal Soybean oil meal Casein Salt & 5 % manganese sulfate Vitamins per 100 lbs. of ration: Riboflavin Calcium pantothenate Niacin Vitamin B l 2 Aureomycin
25 40 4 25 5 1
pounds pounds pounds pounds pounds pound
200 mg. 200 mg. 1 gram 1 mg. 1 gram
The Ca46 solution contained 20 microcuries of calcium46 in 1.3 milligrams of calcium chloride. Twenty-four hours after Ca46 supplementation the chicks were killed and the left and right tibia removed and cleaned free of soft tissue. Determination of Percentage Ca® Deposited in Tibiae. The left and right tibiae were cut into two parts and placed in a TABLE 2.—Effect of different levels of vitamin D on bone mineralization Supplement per 100 grams of basal ration None 5 I.C.U. Vitamin 10 I.C.U. Vitamin 15 I.C.U. Vitamin 20 I.C.U. Vitamin 25 I.C.U. Vitamin 30 I.C.U. Vitamin 35 I.C.U. Vitamin 40 I.C.U. Vitamin 100 I.C.U. Vitamin 200 I.C.U. Vitamin
D3 D3 D3 D3 D3 D3 D3 D3 D3 D3
Percent C« Bone asl in tibiae* % 5.5 10.0 11.5 12.1 12.6 12.7 12.68 12.67 12.6 13.41 13.40
24 28 29.8 32.8 33.0
* Results show percentage of 20 microcuries of Ca46 found in combined left and right tibia 24 hours after supplementation with Ca45. f Bone ash determinations made on 21 day-old chicks (A.O.A.C. method was used).
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IMITATIONS of the chick bone ash and •^ chemical methods for assay of vitamin D are well established. The chick bone ash method requires approximately 4 weeks and large amounts of labor. Chemical methods used for determination of vitamin D are limited to concentrations of the vitamin D active compound or to chemical reactants which will produce a chemical change such as color and therefore may lack specificity. Vitamin D influences the absorption of calcium from the digestive system of the chick, and also the deposition of Ca46 in the tibiae of the chick can be used as an index of percentage absorption (Migicovsky and Emslie, 1949, 1950). Simplified, our experiments on a chick vitamin D assay method using radioactive calcium, involved a quantitative comparison of influence of graduated amounts of vitamin D on Ca45 bone mineralization values; bone mineralization values being the percentage Ca46 of a single dosage of radioactive calcium deposited in the combined left and right tibiae of the chick.
835
NEWS AND NOTES
RESULTS
Experiment 1: This test was designed to determine the graduated amounts of vitamin D required to establish an assay curve and to compare also the bone ash and Ca46 methods. The results are summarized in Table 2. The results demonstrate that 20 I.C.U. of vitamin D will produce normal bone mineralization; whereas, it requires at least 30 I.C.U. vitamin D 3 to produce a normal bone ash. When based on these data, the unknown sample should be used to supply 5 to 10 I.C.U. of vitamin D when assaying for this vitamin using the Ca46 bone mineralization method. Experiment 2: The determination of vitamin D in a commercial sample of a vitamin D concentrate was made using Ca46 bone mineralization values. The com-
TABLE 3.—Determination of vitamin D content of a sample of vitamin D concentrate Supplement*
Bone mineralization value
None 5 I.C.U. Vitamin D3 10 I.C.U. Vitamin D3 IS I.C.U. Vitamin D3 20 I.C.U. Vitamin D3 Unknown f
9.5 11.0 12.5 12.8 12.7 10.7
* Per 100 grams of ration. f Unknown fed at a level to supply 5 I.C.U. Vitamin D 3 per 100 grams of ration as based on guarantee.
mercial sample was fed at a level calculated to supply 5 I.C.U. vitamin D per 100 grams of ration. The results which are summarized in Table 3 demonstrate that the manufacturer's guaranteed vitamin D content is similar to the value established by the Ca46 vitamin D assay method. SUMMARY A m e t h o d is p r e s e n t e d u s i n g Ca 4 6 b o n e m i n e r a l i z a t i o n v a l u e s for a s s a y of v i t a m i n D . T h e v i t a m i n D c o n t e n t of a c o n c e n t r a t e c a n b e d e t e r m i n e d in less t i m e u s i n g Ca 4 6 t h a n t h e b o n e a s h m e t h o d . REFERENCES Migicovsky, B. B., and A. R. G. Emslie, 1949. Interaction of calcium, phosphorus, and vitamin D. III. Study of mode of action of vitamin D using Ca45. Arch. Biochem. 20: 325-330. Migicovsky, B. B., and A. R. G. Emslie, 1950. Deposition of radiocalcium in rachitic and nonrachitic chick tibia from oral and intramuscular doses of Ca46. Arch. Biochem. 28: 324-328.
NEWS AND NOTES {Continued from page 833) in 1921, and a Ph.D. degree in 1929. He did postgraduate work at Columbia and Harvard Universities. He was appointed Assistant Nutritionist at the Ohio Agricultural Experiment Station in 1920. In 1926 he became Assistant Professor in the Department of Poultry Science at the Ohio State Uni-
versity, was promoted to Associate Professor in 1934, and Professor in 1945. From 1932-35 and 1942-44 he served as part-time Instructor in Bacteriology at Ohio State. From 1917-19 he was a civilian with the U. S. Army and in 1944-45 Dr. Winter, while on leave of absence, was Research Associate at Iowa State
[Continued on page 842)
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crucible and ashed in a muffle furnace until relatively free of organic matter. The ashed bones were dropped into a 100 ml. volumetric flask and 1.5 ml. of dilute HC1 added. After the ash was dissolved the contents were diluted to mark and then shaken until thoroughly mixed. One ml. of this solution was pipetted into a small stainless steel counting pan and dried under a heat lamp. The Ca46 activity was then determined with a thin window counting tube and results compared to a Ca4B standard. The standard was made by using one ml. of a solution containing 20 microcuries of Ca45 in 100 ml. of dilute HC1.
L
EXPERIMENTAL
Preparation of Chicks and Administration of Test Materials. Day-old White Leghorn cockerels were housed in electrically heated battery brooders and given the basal ration shown in Table 1 for 6 days. They were then divided into lots of 8 chicks each and given the basal ration plus supplements of vitamin D as shown in Table 2. After receiving the supplemented rations for 7 days, each chick was given 20 microcuries of Ca46 by pipette.
TABLE 1
Vitamin D Assay Basal Ration: Ground wheat Corn meal Alfalfa meal Soybean oil meal Casein Salt & 5 % manganese sulfate Vitamins per 100 lbs. of ration: Riboflavin Calcium pantothenate Niacin Vitamin B l 2 Aureomycin
25 40 4 25 5 1
pounds pounds pounds pounds pounds pound
200 mg. 200 mg. 1 gram 1 mg. 1 gram
The Ca46 solution contained 20 microcuries of calcium46 in 1.3 milligrams of calcium chloride. Twenty-four hours after Ca46 supplementation the chicks were killed and the left and right tibia removed and cleaned free of soft tissue. Determination of Percentage Ca® Deposited in Tibiae. The left and right tibiae were cut into two parts and placed in a TABLE 2.—Effect of different levels of vitamin D on bone mineralization Supplement per 100 grams of basal ration None 5 I.C.U. Vitamin 10 I.C.U. Vitamin 15 I.C.U. Vitamin 20 I.C.U. Vitamin 25 I.C.U. Vitamin 30 I.C.U. Vitamin 35 I.C.U. Vitamin 40 I.C.U. Vitamin 100 I.C.U. Vitamin 200 I.C.U. Vitamin
D3 D3 D3 D3 D3 D3 D3 D3 D3 D3
Percent C« Bone asl in tibiae* % 5.5 10.0 11.5 12.1 12.6 12.7 12.68 12.67 12.6 13.41 13.40
24 28 29.8 32.8 33.0
* Results show percentage of 20 microcuries of Ca46 found in combined left and right tibia 24 hours after supplementation with Ca45. f Bone ash determinations made on 21 day-old chicks (A.O.A.C. method was used).
834
Downloaded from http://ps.oxfordjournals.org/ at NERL on May 13, 2015
IMITATIONS of the chick bone ash and •^ chemical methods for assay of vitamin D are well established. The chick bone ash method requires approximately 4 weeks and large amounts of labor. Chemical methods used for determination of vitamin D are limited to concentrations of the vitamin D active compound or to chemical reactants which will produce a chemical change such as color and therefore may lack specificity. Vitamin D influences the absorption of calcium from the digestive system of the chick, and also the deposition of Ca46 in the tibiae of the chick can be used as an index of percentage absorption (Migicovsky and Emslie, 1949, 1950). Simplified, our experiments on a chick vitamin D assay method using radioactive calcium, involved a quantitative comparison of influence of graduated amounts of vitamin D on Ca45 bone mineralization values; bone mineralization values being the percentage Ca46 of a single dosage of radioactive calcium deposited in the combined left and right tibiae of the chick.
835
NEWS AND NOTES
RESULTS
Experiment 1: This test was designed to determine the graduated amounts of vitamin D required to establish an assay curve and to compare also the bone ash and Ca46 methods. The results are summarized in Table 2. The results demonstrate that 20 I.C.U. of vitamin D will produce normal bone mineralization; whereas, it requires at least 30 I.C.U. vitamin D 3 to produce a normal bone ash. When based on these data, the unknown sample should be used to supply 5 to 10 I.C.U. of vitamin D when assaying for this vitamin using the Ca46 bone mineralization method. Experiment 2: The determination of vitamin D in a commercial sample of a vitamin D concentrate was made using Ca46 bone mineralization values. The com-
TABLE 3.—Determination of vitamin D content of a sample of vitamin D concentrate Supplement*
Bone mineralization value
None 5 I.C.U. Vitamin D3 10 I.C.U. Vitamin D3 IS I.C.U. Vitamin D3 20 I.C.U. Vitamin D3 Unknown f
9.5 11.0 12.5 12.8 12.7 10.7
* Per 100 grams of ration. f Unknown fed at a level to supply 5 I.C.U. Vitamin D 3 per 100 grams of ration as based on guarantee.
mercial sample was fed at a level calculated to supply 5 I.C.U. vitamin D per 100 grams of ration. The results which are summarized in Table 3 demonstrate that the manufacturer's guaranteed vitamin D content is similar to the value established by the Ca46 vitamin D assay method. SUMMARY A m e t h o d is p r e s e n t e d u s i n g Ca 4 6 b o n e m i n e r a l i z a t i o n v a l u e s for a s s a y of v i t a m i n D . T h e v i t a m i n D c o n t e n t of a c o n c e n t r a t e c a n b e d e t e r m i n e d in less t i m e u s i n g Ca 4 6 t h a n t h e b o n e a s h m e t h o d . REFERENCES Migicovsky, B. B., and A. R. G. Emslie, 1949. Interaction of calcium, phosphorus, and vitamin D. III. Study of mode of action of vitamin D using Ca45. Arch. Biochem. 20: 325-330. Migicovsky, B. B., and A. R. G. Emslie, 1950. Deposition of radiocalcium in rachitic and nonrachitic chick tibia from oral and intramuscular doses of Ca46. Arch. Biochem. 28: 324-328.
NEWS AND NOTES {Continued from page 833) in 1921, and a Ph.D. degree in 1929. He did postgraduate work at Columbia and Harvard Universities. He was appointed Assistant Nutritionist at the Ohio Agricultural Experiment Station in 1920. In 1926 he became Assistant Professor in the Department of Poultry Science at the Ohio State Uni-
versity, was promoted to Associate Professor in 1934, and Professor in 1945. From 1932-35 and 1942-44 he served as part-time Instructor in Bacteriology at Ohio State. From 1917-19 he was a civilian with the U. S. Army and in 1944-45 Dr. Winter, while on leave of absence, was Research Associate at Iowa State
[Continued on page 842)
Downloaded from http://ps.oxfordjournals.org/ at NERL on May 13, 2015
crucible and ashed in a muffle furnace until relatively free of organic matter. The ashed bones were dropped into a 100 ml. volumetric flask and 1.5 ml. of dilute HC1 added. After the ash was dissolved the contents were diluted to mark and then shaken until thoroughly mixed. One ml. of this solution was pipetted into a small stainless steel counting pan and dried under a heat lamp. The Ca46 activity was then determined with a thin window counting tube and results compared to a Ca4B standard. The standard was made by using one ml. of a solution containing 20 microcuries of Ca45 in 100 ml. of dilute HC1.