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Further Bone Phosphatase Studies in Chick Perosis*
Further Bone Phosphatase Studies in Chick Perosis* A. C. WIESE, G. H. BENHAM,! C. A. ELVEHJEM, AND E. B. HART From the Department of Biochemistry, University of Wisconsin, Madison,
Wisconsin
(Received for publication September 9, 1940)
P
EROSIS, an anatomical deformity of
Composition of Ration 604
the leg bones of young chicks, was first Component described by Hunter and Funk (1930). The D e x t r i n • • •; condition is associated with a high intake ^ n e , ee . l n e y
S4
Crude casein
14
Salts 2
Brewers' yeast Ca^PO^ Alcoholic extract of rice bran H a K v e r oil
5 2 3 5
2 d r o p s p e r chick p e r w e e k
This ration when
supplemented with SO S- o f manganese per kilogram of ration produced normal chicks (Wiese et al, 1938). The femurs, tibia, and metatarsal bones were removed as soon as possible after killin S t h e chick > a n d t h e n partitioned as described later. Each bone fraction was preP a r e d a s w e h a v e described in a previous publication (Wiese et al, 1939) and the bone phosphatase activity determined by our modification of the King and Armstrong1 method. EXPERIMENTAL In the first experiment 20 percent of the Day-old White Leghorn chicks were t o t a l l e n 8 t h o f t h e tibia was cut from each divided into groups of five chicks each end > the ends combined, ground, and made and placed in wire-screen-bottom cages UP to volume so that the suspension conequipped with suitable warmers. As a basal t a i n e d 5 percent of bone in water. These diet the chicks were fed ration 604 which preparations were then allowed to autolyze has been described by Clifcorn and associ- f o r 4 8 h o u r s a t room temperature. The ates (1938) for the uniform production of s h a f t s were similarly treated and the perosis. The composition of this ration is as phosphatase activity was determined on eacn follows: part. From the results given in Table 1 it can be seen that practically all the •Published with the approval of the director phosphatase activity is located at the ends m
of the Wisconsin Agricultural Experiment Station. 0f j - n e k 0 n e This work was supported in part by a grant from the Works Progress Administration. * E. J. King and A. R. Armstrong, Canadian t Commonwealth Fund Fellow. Med. Assoc. J. 31:374, 1934. [255]
Downloaded from http://ps.oxfordjournals.org/ at Tamkang University on June 3, 2015
°
of calcium and phosphorus, and a low intake of manganese (Hunter et al, 1931; Milby, 1934; Wilgus et al, 1936-1937; Clifcorn et al, 1938). Later, the work of Wiese et al (1939) showed that the blood and bone phosphatase activity of perotic birds was lower than that of chicks receiving an adequate amount of manganese in their diet. As the leg bones of chicks with perosis are bent and twisted from their normal shape it was thought that there might be an abnormal distribution of the enzyme phosphatase in the bones of these chicks. In this paper we wish to report the results we have observed on the distribution of this enzyme in the leg bones of perotic and normal chicks.
Parts
256
A. C. WIESE, G. H. BENHAM, C. A. ELVEHJEM, AND E. B. HART
In a second experiment 10 percent of the total length of the tibia was removed from each end, and the ends and shafts prepared as above. The results in Table 2 show that the phosphatase activity is very high at the extreme ends of the bone. Increasing the manganese in the diet from 30 to 40 to SO parts per million resulted in a progressive increase in the phosphatase activity in the ends of the bone. A third experiment was set up to deter-
Ration
604 604+50 p.p.m. Mn
No. of birds
6 5
Perosis
+
Units of phosphatase per gm. of End
Shaft
2.4 7.6
0.5 0.9
No. of birds
Ration
604 604+50 p.p.m. Mn
TABLE 2.—Phosphatase partition of tibia (10 percent off each end)
Ration
31 4 5 32
Perosis
++ 1 1
604 604+30 p.p.m. Mn 604+40 p.p.m. Mn 604+50 p.p.m. Mn
No. of birds
Units of phosphatase per gm. of End
Shaft
5.9 8.5 10.7 12.4
0.7 1.2
+
6 7
Left half
Right half
9.9 26.6
10.7 26.9
(Barred rock chicks used in this run.)
in the same proportion to each other. In another experiment a comparison of the phosphatase activity of the two ends of the tibia was made. The results in Table 4 show the proximal end of the bone to be TABLE 4.—Phosphatase partition between top and bottom end of tibia
No. of birds
Ration
mine whether or not there was any difference in the phosphatase activity between the right and left halves of the ends of the bone. The bones were prepared as described above except that the ends were divided into a right and a left half, and the phosphatase activity determined on each half. The results given in Table 3 show that there is no difference in the phosphatase activity between the right and left halves. In this particular experiment Barred Rock chicks were used, hence the absolute value is higher but the differences between perotic and normal chicks are nevertheless
Perosis
Units of phosphatase per gm. of
604 604+50 p.p.m. Mn
Perosis
+
10 10
Units of phosphatase per gm. of Top end
Bottom end
6.3 16.2
4.6 8.6
much higher in the phosphatase activity than the distal end. In the chicks receiving manganese in their diet the differences between the proximal and distal ends manifested themselves even more than in the chicks receiving the manganese low ration. In a fifth experiment a comparison was made between the phosphatase activity of the ends of the femur, tibia, and metatarsal TABLE 5.—Comparison of phosphatase activity between the ends of various bones
Ration
604 604+50 p.p.m. Mn
Units of phosphatase per gm. of No. Peof birds rosis Lower Up- Lower Metaper femur tibia tibia tarsus 5 6
+
4.6
6.0
4.8
8.7
8.6
15.1
8.1
17.8
Downloaded from http://ps.oxfordjournals.org/ at Tamkang University on June 3, 2015
TABLE 1.—Phosphatase partition of tibia (20 percent off each end)
TABLE 3.—Phosphatase partition between left and right lialves of ends of tibia
FURTHER B O N E PHOSPHATASE STUDIES I N C H I C K PEROSIS
257
TABLE 6.—Calcium and phosphorus content of the various parts of the tibia Ration 604 604+50 p.p.m.Mn.
Percent Ca
No. of birds
Perosis
5 5
+
Percent P
Top
Bottom
Shaft
Top
Bottom
Shaft
14.7 16.1
16.1 17.3
25.0 26.9
5.4 5.6
5.9 6.3
6.0 6.0
DISCUSSION
There is a very high concentration of the enzyme phosphatase at the ends of the bone. The ends of bones from normal birds are much higher in phosphatase activity than the ends of bones from birds with perosis. This increase could be due either to an activation of the enzyme by the manganese ion, or to an actual increase in the amount of enzyme present. In a previous paper (Wiese et al., 1939) we reported in vitro studies which showed that it was necessary to add 400 micrograms of manganese per gram of bone to produce as little as a 6 percent activation of the enzyme phosphatase. In in vivo studies an
increase of 2 micrograms of manganese per gram of bone resulted in an increase of bone phosphatase activity of almost 100 percent. From these results it is evident that the increase in phosphatase activity of the normal bones is not due to a mere activation of the enzyme by the manganese ion, but rather to an actual increase in the amount of enzyme present. It has been observed by the various workers in this field that the leg bones of perotic chicks are bent and twisted from their normal shape. This would suggest that there was poor or faulty calcification of these bones. That this is not the case is shown by the fact that the lower bone phosphatase activity of the perotic birds does not result in a poorer calcification of the bones as the calcium and phosphorus content of these bones is normal. Although from Table 6 it can be seen that the calcium content of the tibiae of perotic chicks is somewhat lower than that of non-perotic birds, yet the calcium content is within the normal range. Since there is no unequal distribution of phosphatase between the right and left halves of the bone, the bending and twisting is not due to an unequal distribution of the enzyme which might presumably cause an abnormal calcification in one-half of the bone or the other. The slipping of the tendon actually takes place at the tibio-metatarsal joint. The phosphatase activity of the ends of the bones making up this joint is just as low as the phosphatase activity of the ends of the bones making up the femur-tibia joint. The fact that more strain is put on the tibiometatarsal joint in supporting the weight
Downloaded from http://ps.oxfordjournals.org/ at Tamkang University on June 3, 2015
bones. The bones were prepared and the phosphatase activity determined as has been previously described. The comparison was made between the lower end of the femur and the upper end of the metatarsal. The results in Table 5 show the phosphatase activity in the upper ends of the tibia and metatarsal bones to be higher than the phosphatase activity of the lower ends of the femur and tibia bones, respectively. These findings prompted us to investigate the calcium and phosphorus content of the various parts of such bones. The bones were partitioned as before and the calcium and phosphorus determined by the methods described by Treadwell and Hall (1915). The results in Table 6 show no significant differences in the calcium and phosphorus content of the tibiae of perotic and normal chicks. The manganese content of the same parts of the bone was estimated and again no differences were observed.
258
A. C. WIESE, G. H. BENHAM, C,'. A. ELVEHJEM, AND E. B. HART
SUMMARY
1. Maximum phosphatase activity is located at the extreme end of the bone. 2. The bone phosphatase activity of chicks receiving adequate manganese is much higher than that of perotic birds. 3. The upper end of a given bone has a
much higher phosphatase activity than the lower end. 4. The increase in phosphatase activity is not due to a mere activation of the enzyme by the manganese ion, but rather to an actual increase in the amount of enzyme present. REFERENCES
Clifcorn, L. E., C. A. Elvehjem, and E. B. Hart, 1938. The development of a ration for the study of perosis in chicks. Poultry Sci. 17:28. Heller, V. G., P. Penquite, 1936. Factors producing and preventing perosis. Poultry Sci. 15:425. Hogan, A. G., and L. R. Richardson, 1940. Relation of perosis to unrecognized vitamins. Proc. 7th Ann. Meeting of Amer. Inst, of Nutrition, 14. Hunter, J. E., and E. M. Funk, 1930. The production of slipped tendon in chicks by experimental feeding. Proc. 22nd Ann. Meeting Poultry Sci. Assoc, 45. Hunter, J. E., R. A. Dutcher, and H. C. Kuandel, 1931. Further studies on the production of experimental slipped tendon or hock diseases. Poultry Sci. 10:392. Milby, T. T., 1934. Factors influencing a malformation of the leg bones of growing chickens. Iowa Agr. Exp. Sta. Res. Bull., 172. Treadwell, F. P., and W. T. Hall, 1915. Anal. Chem. II. John Wiley and Sons, Inc., New York. Wiese, A. C , C. A. Elvehjem, E. B. Hart, and J. G. Halpin, 1938. Studies on the prevention of perosis in the chick. Poultry Sci. 17 :33. Wiese, A. C , B. C. Johnson, C. A. Elvehjem, E. B. Hart, and J. G. Halpin, 1939. A study of blood and bone phosphatase in chick perosis. J. Biol. Chem. 127:411. Wilgus, H. S., Jr., L. C. Norris, and G. F. Heuser, 1936. The role of certain inorganic elements in the cause and prevention of perosis. Science 84:252. , 1937. The effect of various calcium and phosphorus salts on the severity of perosis. Poultry Sci. 16:232.
Downloaded from http://ps.oxfordjournals.org/ at Tamkang University on June 3, 2015
of the chick perhaps accounts for the fact that the tendon at this joint slips rather than the tendon at the femur-tibia joint. It was also observed that the phosphatase activity of the lower end of the femur was less than that of the upper end of the tibia. Similar results were obtained for the lower end of the tibia and the upper end of the metatarsus. With the birds receiving manganese in their diet these differences in phosphatase concentrations manifested themselves even more. It may be that calcification goes on more rapidly at the upper end of the bone than at the lower; however, there is no experimental evidence for this latter view. The full significance of the difference in phosphatase activity between adjacent bones is at the present time impossible to explain. In all cases the various parts of perotic bone have a lower phosphatase activity than the corresponding parts of a normal bone. Whether the low phosphatase activity that occurs on low manganese diets is alone responsible for perosis, or whether there is some other factor perhaps organic in nature as suggested by Heller and Penquite (1936), and by Wiese et al. (1938), and later by Hogan (1940), which acts in conjunction with manganese and phosphatase is a matter for further investigation.
Wisconsin
(Received for publication September 9, 1940)
P
EROSIS, an anatomical deformity of
Composition of Ration 604
the leg bones of young chicks, was first Component described by Hunter and Funk (1930). The D e x t r i n • • •; condition is associated with a high intake ^ n e , ee . l n e y
S4
Crude casein
14
Salts 2
Brewers' yeast Ca^PO^ Alcoholic extract of rice bran H a K v e r oil
5 2 3 5
2 d r o p s p e r chick p e r w e e k
This ration when
supplemented with SO S- o f manganese per kilogram of ration produced normal chicks (Wiese et al, 1938). The femurs, tibia, and metatarsal bones were removed as soon as possible after killin S t h e chick > a n d t h e n partitioned as described later. Each bone fraction was preP a r e d a s w e h a v e described in a previous publication (Wiese et al, 1939) and the bone phosphatase activity determined by our modification of the King and Armstrong1 method. EXPERIMENTAL In the first experiment 20 percent of the Day-old White Leghorn chicks were t o t a l l e n 8 t h o f t h e tibia was cut from each divided into groups of five chicks each end > the ends combined, ground, and made and placed in wire-screen-bottom cages UP to volume so that the suspension conequipped with suitable warmers. As a basal t a i n e d 5 percent of bone in water. These diet the chicks were fed ration 604 which preparations were then allowed to autolyze has been described by Clifcorn and associ- f o r 4 8 h o u r s a t room temperature. The ates (1938) for the uniform production of s h a f t s were similarly treated and the perosis. The composition of this ration is as phosphatase activity was determined on eacn follows: part. From the results given in Table 1 it can be seen that practically all the •Published with the approval of the director phosphatase activity is located at the ends m
of the Wisconsin Agricultural Experiment Station. 0f j - n e k 0 n e This work was supported in part by a grant from the Works Progress Administration. * E. J. King and A. R. Armstrong, Canadian t Commonwealth Fund Fellow. Med. Assoc. J. 31:374, 1934. [255]
Downloaded from http://ps.oxfordjournals.org/ at Tamkang University on June 3, 2015
°
of calcium and phosphorus, and a low intake of manganese (Hunter et al, 1931; Milby, 1934; Wilgus et al, 1936-1937; Clifcorn et al, 1938). Later, the work of Wiese et al (1939) showed that the blood and bone phosphatase activity of perotic birds was lower than that of chicks receiving an adequate amount of manganese in their diet. As the leg bones of chicks with perosis are bent and twisted from their normal shape it was thought that there might be an abnormal distribution of the enzyme phosphatase in the bones of these chicks. In this paper we wish to report the results we have observed on the distribution of this enzyme in the leg bones of perotic and normal chicks.
Parts
256
A. C. WIESE, G. H. BENHAM, C. A. ELVEHJEM, AND E. B. HART
In a second experiment 10 percent of the total length of the tibia was removed from each end, and the ends and shafts prepared as above. The results in Table 2 show that the phosphatase activity is very high at the extreme ends of the bone. Increasing the manganese in the diet from 30 to 40 to SO parts per million resulted in a progressive increase in the phosphatase activity in the ends of the bone. A third experiment was set up to deter-
Ration
604 604+50 p.p.m. Mn
No. of birds
6 5
Perosis
+
Units of phosphatase per gm. of End
Shaft
2.4 7.6
0.5 0.9
No. of birds
Ration
604 604+50 p.p.m. Mn
TABLE 2.—Phosphatase partition of tibia (10 percent off each end)
Ration
31 4 5 32
Perosis
++ 1 1
604 604+30 p.p.m. Mn 604+40 p.p.m. Mn 604+50 p.p.m. Mn
No. of birds
Units of phosphatase per gm. of End
Shaft
5.9 8.5 10.7 12.4
0.7 1.2
+
6 7
Left half
Right half
9.9 26.6
10.7 26.9
(Barred rock chicks used in this run.)
in the same proportion to each other. In another experiment a comparison of the phosphatase activity of the two ends of the tibia was made. The results in Table 4 show the proximal end of the bone to be TABLE 4.—Phosphatase partition between top and bottom end of tibia
No. of birds
Ration
mine whether or not there was any difference in the phosphatase activity between the right and left halves of the ends of the bone. The bones were prepared as described above except that the ends were divided into a right and a left half, and the phosphatase activity determined on each half. The results given in Table 3 show that there is no difference in the phosphatase activity between the right and left halves. In this particular experiment Barred Rock chicks were used, hence the absolute value is higher but the differences between perotic and normal chicks are nevertheless
Perosis
Units of phosphatase per gm. of
604 604+50 p.p.m. Mn
Perosis
+
10 10
Units of phosphatase per gm. of Top end
Bottom end
6.3 16.2
4.6 8.6
much higher in the phosphatase activity than the distal end. In the chicks receiving manganese in their diet the differences between the proximal and distal ends manifested themselves even more than in the chicks receiving the manganese low ration. In a fifth experiment a comparison was made between the phosphatase activity of the ends of the femur, tibia, and metatarsal TABLE 5.—Comparison of phosphatase activity between the ends of various bones
Ration
604 604+50 p.p.m. Mn
Units of phosphatase per gm. of No. Peof birds rosis Lower Up- Lower Metaper femur tibia tibia tarsus 5 6
+
4.6
6.0
4.8
8.7
8.6
15.1
8.1
17.8
Downloaded from http://ps.oxfordjournals.org/ at Tamkang University on June 3, 2015
TABLE 1.—Phosphatase partition of tibia (20 percent off each end)
TABLE 3.—Phosphatase partition between left and right lialves of ends of tibia
FURTHER B O N E PHOSPHATASE STUDIES I N C H I C K PEROSIS
257
TABLE 6.—Calcium and phosphorus content of the various parts of the tibia Ration 604 604+50 p.p.m.Mn.
Percent Ca
No. of birds
Perosis
5 5
+
Percent P
Top
Bottom
Shaft
Top
Bottom
Shaft
14.7 16.1
16.1 17.3
25.0 26.9
5.4 5.6
5.9 6.3
6.0 6.0
DISCUSSION
There is a very high concentration of the enzyme phosphatase at the ends of the bone. The ends of bones from normal birds are much higher in phosphatase activity than the ends of bones from birds with perosis. This increase could be due either to an activation of the enzyme by the manganese ion, or to an actual increase in the amount of enzyme present. In a previous paper (Wiese et al., 1939) we reported in vitro studies which showed that it was necessary to add 400 micrograms of manganese per gram of bone to produce as little as a 6 percent activation of the enzyme phosphatase. In in vivo studies an
increase of 2 micrograms of manganese per gram of bone resulted in an increase of bone phosphatase activity of almost 100 percent. From these results it is evident that the increase in phosphatase activity of the normal bones is not due to a mere activation of the enzyme by the manganese ion, but rather to an actual increase in the amount of enzyme present. It has been observed by the various workers in this field that the leg bones of perotic chicks are bent and twisted from their normal shape. This would suggest that there was poor or faulty calcification of these bones. That this is not the case is shown by the fact that the lower bone phosphatase activity of the perotic birds does not result in a poorer calcification of the bones as the calcium and phosphorus content of these bones is normal. Although from Table 6 it can be seen that the calcium content of the tibiae of perotic chicks is somewhat lower than that of non-perotic birds, yet the calcium content is within the normal range. Since there is no unequal distribution of phosphatase between the right and left halves of the bone, the bending and twisting is not due to an unequal distribution of the enzyme which might presumably cause an abnormal calcification in one-half of the bone or the other. The slipping of the tendon actually takes place at the tibio-metatarsal joint. The phosphatase activity of the ends of the bones making up this joint is just as low as the phosphatase activity of the ends of the bones making up the femur-tibia joint. The fact that more strain is put on the tibiometatarsal joint in supporting the weight
Downloaded from http://ps.oxfordjournals.org/ at Tamkang University on June 3, 2015
bones. The bones were prepared and the phosphatase activity determined as has been previously described. The comparison was made between the lower end of the femur and the upper end of the metatarsal. The results in Table 5 show the phosphatase activity in the upper ends of the tibia and metatarsal bones to be higher than the phosphatase activity of the lower ends of the femur and tibia bones, respectively. These findings prompted us to investigate the calcium and phosphorus content of the various parts of such bones. The bones were partitioned as before and the calcium and phosphorus determined by the methods described by Treadwell and Hall (1915). The results in Table 6 show no significant differences in the calcium and phosphorus content of the tibiae of perotic and normal chicks. The manganese content of the same parts of the bone was estimated and again no differences were observed.
258
A. C. WIESE, G. H. BENHAM, C,'. A. ELVEHJEM, AND E. B. HART
SUMMARY
1. Maximum phosphatase activity is located at the extreme end of the bone. 2. The bone phosphatase activity of chicks receiving adequate manganese is much higher than that of perotic birds. 3. The upper end of a given bone has a
much higher phosphatase activity than the lower end. 4. The increase in phosphatase activity is not due to a mere activation of the enzyme by the manganese ion, but rather to an actual increase in the amount of enzyme present. REFERENCES
Clifcorn, L. E., C. A. Elvehjem, and E. B. Hart, 1938. The development of a ration for the study of perosis in chicks. Poultry Sci. 17:28. Heller, V. G., P. Penquite, 1936. Factors producing and preventing perosis. Poultry Sci. 15:425. Hogan, A. G., and L. R. Richardson, 1940. Relation of perosis to unrecognized vitamins. Proc. 7th Ann. Meeting of Amer. Inst, of Nutrition, 14. Hunter, J. E., and E. M. Funk, 1930. The production of slipped tendon in chicks by experimental feeding. Proc. 22nd Ann. Meeting Poultry Sci. Assoc, 45. Hunter, J. E., R. A. Dutcher, and H. C. Kuandel, 1931. Further studies on the production of experimental slipped tendon or hock diseases. Poultry Sci. 10:392. Milby, T. T., 1934. Factors influencing a malformation of the leg bones of growing chickens. Iowa Agr. Exp. Sta. Res. Bull., 172. Treadwell, F. P., and W. T. Hall, 1915. Anal. Chem. II. John Wiley and Sons, Inc., New York. Wiese, A. C , C. A. Elvehjem, E. B. Hart, and J. G. Halpin, 1938. Studies on the prevention of perosis in the chick. Poultry Sci. 17 :33. Wiese, A. C , B. C. Johnson, C. A. Elvehjem, E. B. Hart, and J. G. Halpin, 1939. A study of blood and bone phosphatase in chick perosis. J. Biol. Chem. 127:411. Wilgus, H. S., Jr., L. C. Norris, and G. F. Heuser, 1936. The role of certain inorganic elements in the cause and prevention of perosis. Science 84:252. , 1937. The effect of various calcium and phosphorus salts on the severity of perosis. Poultry Sci. 16:232.
Downloaded from http://ps.oxfordjournals.org/ at Tamkang University on June 3, 2015
of the chick perhaps accounts for the fact that the tendon at this joint slips rather than the tendon at the femur-tibia joint. It was also observed that the phosphatase activity of the lower end of the femur was less than that of the upper end of the tibia. Similar results were obtained for the lower end of the tibia and the upper end of the metatarsus. With the birds receiving manganese in their diet these differences in phosphatase concentrations manifested themselves even more. It may be that calcification goes on more rapidly at the upper end of the bone than at the lower; however, there is no experimental evidence for this latter view. The full significance of the difference in phosphatase activity between adjacent bones is at the present time impossible to explain. In all cases the various parts of perotic bone have a lower phosphatase activity than the corresponding parts of a normal bone. Whether the low phosphatase activity that occurs on low manganese diets is alone responsible for perosis, or whether there is some other factor perhaps organic in nature as suggested by Heller and Penquite (1936), and by Wiese et al. (1938), and later by Hogan (1940), which acts in conjunction with manganese and phosphatase is a matter for further investigation.