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Hypomagnesemic Effect of Avian Calcitonin
446
V. W. FISCHER, J. S. NELSON AND P. A. YOUNG
SUMMARY
A modified hydrogen peroxide method is utilized to determine differences in susceptibility to lysis of red cells from chickens on normal or vitamin E-deficient diets. Results indicate that erythrocytes from young chicks on a vitamin E-deficient diet are more prone to lysis in dilute
hydrogen peroxide solutions than red cells from chicks on a balanced diet. Reversal of exposure to a normal or vitamin E-deficient diet results in an inverse relationship of hemolysis levels. These data support the view that plasma tocopherol is a major factor in the determination of erythrocyte susceptibility to peroxide lysis. REFERENCES Gyorgy, P., and C. S. Rose, 1949. Tocopherol and hemolysis in vivo and in vitro. Annals New York Acad. Science, 52: 231. Horwitt, M. K., C. C. Harvey, G. D. Duncan and W. C. Wilson, 1956. Effects of limited tocopherol intake in man with relationships to erythrocyte hemolysis and lipid oxidations. Nutrition Symposium Series, No. 12: 100-110. Christensen, F., H. Dam, R. A. Gortner, Jr. and E. Sondergaard, 1955. "In vitro" hemolysis of erythrocytes from vitamin E deficient rats and chicks. Actaphysiol. scand. 35: 215-224. Young, P. A., and L. L. Tureen, 1966. Histological observations on the evolution of nutritional encephalomalacia in chick cerebellum. Acta Neuropathologica, 6: 279-289. Horwitt, M. K., C. C. Harvey and E. M. Harmon, 1968. Lipids, a-tocopherol and erythrocyte hemolysis. Vitamins and Hormones, 26: 487-499
Hypomagnesemic Effect of Avian Calcitonin J. W. LLOYD AND W. E.
COLLINS
Department of Biology, West Virginia University, Morgantown, West Virginia 26506 (Received for publication October 7, 1969)
P
LASMA magnesium levels are maintained within very narrow limits in most organisms. The factors regulating the concentration of magnesium, however, are obscure (Arkawa, 1960). Thyroid status has been implicated in the control of plasma magnesium levels in human beings (Jones et al., 1966) and Care (1965) has stated that thyroxine infusion produced hypomagnesemia in the thyro-
parathyroidectomized sheep, but did not alter net absorption from the ileum. Further, Inskeep and Kenny (1968) reported that thyroidectomy in sheep was accompanied by hypermagnesemia. Most of the published evidence indicates that calcitonin does not modify the concentration of plasma magnesium in mammalian species (Foster et al., 1964, 1966; and Bell et al., 1966). Chausmer et
Downloaded from http://ps.oxfordjournals.org/ by guest on May 16, 2015
normal feed, after initial exposure to dietary deficiency, is at present unclear. An explanation for this phenomenon may be found in Horwitt's observation of the effects of dietary variation on the fatty acid composition of chick erythrocytes (Horwitt et al., 1968) The hydrogen peroxide test has been utilized in animals and humans in the bioassay for vitamin E. Our modified hemolysis test cannot be used consistently to identify all members of a particular group; however, it can be applied successfully to demonstrate an overall increased susceptibility to hemolysis of erythrocytes from vitamin E-deficient chickens.
CALCITONIN AND HYPOMAGNESEMIA
MATERIALS AND METHODS
Acid extracts of ultimobranchial (UB) and thyroid glands were prepared by the method of Copp et al. (1967). The solutions were adjusted to pH 4 and kept on ice during the experiment. Two hundred forty, 8-day-old Leghorn cockerels, which had been fasted twentyfour hours, were used in this experiment. Due to the number of birds to be injected and bled, the experiment was run in two trials. The chicks in each trial were divided randomly into groups of 30 birds treated with: 1) 0.25 ml. of 0.9% saline, pH 4; 2) 1 gland-equivalent (3 mg. dry wt.) UB extract in 0.25 ml. saline; 3) 1/2 gland-equivalent (1.5 mg. dry wt.) UB extract in 0.25 ml. saline; or 4) an extract
of 3 mg. thyroid tissue in 0.25 ml. saline. Birds in each treatment-group were assigned randomly to one of three timesubgroups for sacrifice after injection (i.e. 30, 60, 120 minutes) with 10 birds per time-subgroup. The birds in each of these subgroups were numbered from 1 to 10. Each of 4 injectors was assigned one treatmentrgroup. The three birds numbered 1 within each treatment-group were injected subcutaneously within a 2 minute interval giving a total of 12 birds (4 injectors X 3 birds) injected in the first two minutes. An additional 12 birds were injected in each subsequent 2 minute interval until all 120 birds in the first trial were injected. Another group of 8 workers bled the birds via the right jugular vein at the appropriate intervals after injection. Simultaneous injections and blood collections were used to minimize any effects of time of day on plasma magnesium levels. Two hours after the beginning of the first trial, the second trial was begun using the same procedures. Blood samples were centrifuged at room temperature. Plasma was collected and stored at — 15°C. Magnesium levels were determined by atomic absorption spectrophotometry using a Jarrel-Ash apparatus, model 82-362. The data were analyzed by analysis of variance and differences between treatment means were evaluated by the least significant difference (LSD) test. RESULTS AND DISCUSSION
The effects of UB and thyroid extracts on plasma magnesium levels are presented in Table 1. Since no difference between trials was found by analysis of variance, the trials were combined for the LSD test. Plasma magnesium levels did not differ significantly among the four groups at 60 minutes postinjection. Chicks
Downloaded from http://ps.oxfordjournals.org/ by guest on May 16, 2015
al. (1966) found variable responses of plasma magnesium levels to porcine calcitonin in the dog. Recently, Palmeri et al. (1969), using a more sensitive assay for calcitonin, demonstrated a slight but significant hypomagnesemic effect of calcitonin when it was administered to calcium-deficient rats. In mammals, there is an intimate spatial relationship between cells of the thyroid gland which produce calcitonin (parafollicular " C " cells) and those which produce thyroxine (follicle cells). This relationship leads to difficulties in attempting to delineate the relative importance of thyroxine and calcitonin in magnesium homeostasis. The sources of thyroxine (thyroid gland) and calcitonin (ultimobranchial gland) are separate structural entities in birds. Therefore, birds appeared to lend themselves ideally to the study of this problem. Preliminary data from this laboratory suggested that the ultimobranchial gland of the domestic fowl may play a role in magnesium homeostasis in the young bird (Lloyd eial., 1969).
447
448
J. W. LLOYD AND W. E. COLLINS
TABLE 1.—The effects of idlimobranchial and thyroid extracts on plasma magnesium levels (mg./lOO ml.) of eight-day-old chicks" Treatment Salineb Thyroid 0 ,d 1/2 UBG 1 UBGe
No. chicks per time interval 20 20 20 20
Minutes postinfection 30
60
3.1±0.2 ff 3.1±0.1 £ 3.2±0.1 3.2±0.2' 3.1±0.1 f 2.9±0.1»f 2.7±0.ie 2 . 9 + 0 . l
UB gland may play a role in magnesium homeostasis in the young chick. ACKNOWLEDGMENT
120 3.3±0.1 £f 2.9+0.2 2.4±0.2« 2.4±0.1«
a b c
Mean ± standard error. 0.25 ml. of 0.9% saline at pH 4. The amount of thyroid tissue equivalent to the average weight 1 ultimobranchial gland (3 mg. dry weight) in 0.25 ml. saline at pH 4. *> 1/2 Gland-equivalent (1.5 mg. dry weight) in 0.25 ml. saline at pH 4. e One gland-equivalent (3 mg. dry weight) in 0.25 ml. saline at pH 4. {,s Values in vertical column with the same superscript are not statistically different, p<0.05.
A hypomagnesemic effect of thyroid extracts was not found during the times of sampling. A trend toward a lowering of the plasma magnesium levels with thyroid extracts is suggested at 120 minutes postinjection, but this was not significant. The data demonstrate that UB extract injected into young chicks lowers plasma magnesium levels and suggest that the
REFERENCES Arkawa, J., 1960. Effect of thyroxine and propylthiouracil on Mg metabolism in the rabbit. Proc. Soc. Exp. Biol. Med. 104:594-597. Bell, N. H., R. J. Bennett and R. Patterson, 1966. Effects of porcine thyrocalcitonin on serum calcium, phosphorus and magnesium in the monkey and in man. Proc. Soc. Exp. Biol. Med. 123: 114. Care, A. D., 1965. Secretion of thyrocalcitonin. Nature, 205: 1289. Chausmer, A., R. Mittleman and S. Wallace, 1966. Studies of thyrocalcitonin action. Endocrinology, 29: 131-136. Copp, D. H., D. W. Cockcroft, K. Kueh and A. Yankoon, 1967. Calcitonin from ultimobranchial glands of dogfish and chickens. Sicence, 158: 196-197. Foster, G. V., A. Baghdiantz, M. A. Kumer, E. Slack, H. A. Soliman and I. Maclntyre, 1964. Thyroid origin of thyrocalcitonin. Nature, 202: 1303. Foster, G. V., F. F. Joplin, I. Maclntyre, K. E. W. Melvin and E. Slack, 1966. Effects of thyrocalcitonin in man. Lancet, 1: 107. Inskeep, E. K., and A. D. Kenny, 1968. Calcium homeostasis in thyroidectomized sheep. Endocrinology, 83: 183-185. Jones, J. E., P. C. Desper, S. R. Shane and E. B. Flink, 1966. Magnesium metabolism in hyperthyroidism and hypothyroidism. J. Clin. Invest. 45: 891. Lloyd, J. W., R. A. Peterson and W. E. Collins, 1969. Effects of ultimobranchial gland extract in the chicken. Poultry Sci. 48: 1835. Palmeri, G. M. A., J. S. Thompson and L. P. Eliel, 1969. Hypomagnesemic effect of thyrocalcitonin. Endocrinology, 84:1509.
Downloaded from http://ps.oxfordjournals.org/ by guest on May 16, 2015
treated with both 1/2 and 1 gland-equivalents of UB extract had lower (p<0.01) plasma magnesium levels at 120 minutes postinjection when compared to either the group injected with saline or thyroid extract. These data are in agreement with previous observations (Lloyd et al., 1969) in which 1/2 gland-equivalent of UB extract significantly lowered plasma magnesium levels in 7-day-old Leghorn cockerels at 120 minutes postinjection as compared to saline-injected controls. In the present study 1/2 gland-equivalent of UB extract appeared to elicit a similar degree of hypomagnesemia at 120 minutes. However, the response varied with dose level at 30 minutes and neither dose level produced an effect at 60 minutes. These phases of the response cannot be explained.
The authors thank Drs. R. A. Peterson, E. K. Inskeep and R. L. Butcher and the students in the Reproductive Physiology Graduate Program for their assistance in the experiment. This investigation was supported in part by the American Cancer Society Institutional Research Grant, No. 76G.
V. W. FISCHER, J. S. NELSON AND P. A. YOUNG
SUMMARY
A modified hydrogen peroxide method is utilized to determine differences in susceptibility to lysis of red cells from chickens on normal or vitamin E-deficient diets. Results indicate that erythrocytes from young chicks on a vitamin E-deficient diet are more prone to lysis in dilute
hydrogen peroxide solutions than red cells from chicks on a balanced diet. Reversal of exposure to a normal or vitamin E-deficient diet results in an inverse relationship of hemolysis levels. These data support the view that plasma tocopherol is a major factor in the determination of erythrocyte susceptibility to peroxide lysis. REFERENCES Gyorgy, P., and C. S. Rose, 1949. Tocopherol and hemolysis in vivo and in vitro. Annals New York Acad. Science, 52: 231. Horwitt, M. K., C. C. Harvey, G. D. Duncan and W. C. Wilson, 1956. Effects of limited tocopherol intake in man with relationships to erythrocyte hemolysis and lipid oxidations. Nutrition Symposium Series, No. 12: 100-110. Christensen, F., H. Dam, R. A. Gortner, Jr. and E. Sondergaard, 1955. "In vitro" hemolysis of erythrocytes from vitamin E deficient rats and chicks. Actaphysiol. scand. 35: 215-224. Young, P. A., and L. L. Tureen, 1966. Histological observations on the evolution of nutritional encephalomalacia in chick cerebellum. Acta Neuropathologica, 6: 279-289. Horwitt, M. K., C. C. Harvey and E. M. Harmon, 1968. Lipids, a-tocopherol and erythrocyte hemolysis. Vitamins and Hormones, 26: 487-499
Hypomagnesemic Effect of Avian Calcitonin J. W. LLOYD AND W. E.
COLLINS
Department of Biology, West Virginia University, Morgantown, West Virginia 26506 (Received for publication October 7, 1969)
P
LASMA magnesium levels are maintained within very narrow limits in most organisms. The factors regulating the concentration of magnesium, however, are obscure (Arkawa, 1960). Thyroid status has been implicated in the control of plasma magnesium levels in human beings (Jones et al., 1966) and Care (1965) has stated that thyroxine infusion produced hypomagnesemia in the thyro-
parathyroidectomized sheep, but did not alter net absorption from the ileum. Further, Inskeep and Kenny (1968) reported that thyroidectomy in sheep was accompanied by hypermagnesemia. Most of the published evidence indicates that calcitonin does not modify the concentration of plasma magnesium in mammalian species (Foster et al., 1964, 1966; and Bell et al., 1966). Chausmer et
Downloaded from http://ps.oxfordjournals.org/ by guest on May 16, 2015
normal feed, after initial exposure to dietary deficiency, is at present unclear. An explanation for this phenomenon may be found in Horwitt's observation of the effects of dietary variation on the fatty acid composition of chick erythrocytes (Horwitt et al., 1968) The hydrogen peroxide test has been utilized in animals and humans in the bioassay for vitamin E. Our modified hemolysis test cannot be used consistently to identify all members of a particular group; however, it can be applied successfully to demonstrate an overall increased susceptibility to hemolysis of erythrocytes from vitamin E-deficient chickens.
CALCITONIN AND HYPOMAGNESEMIA
MATERIALS AND METHODS
Acid extracts of ultimobranchial (UB) and thyroid glands were prepared by the method of Copp et al. (1967). The solutions were adjusted to pH 4 and kept on ice during the experiment. Two hundred forty, 8-day-old Leghorn cockerels, which had been fasted twentyfour hours, were used in this experiment. Due to the number of birds to be injected and bled, the experiment was run in two trials. The chicks in each trial were divided randomly into groups of 30 birds treated with: 1) 0.25 ml. of 0.9% saline, pH 4; 2) 1 gland-equivalent (3 mg. dry wt.) UB extract in 0.25 ml. saline; 3) 1/2 gland-equivalent (1.5 mg. dry wt.) UB extract in 0.25 ml. saline; or 4) an extract
of 3 mg. thyroid tissue in 0.25 ml. saline. Birds in each treatment-group were assigned randomly to one of three timesubgroups for sacrifice after injection (i.e. 30, 60, 120 minutes) with 10 birds per time-subgroup. The birds in each of these subgroups were numbered from 1 to 10. Each of 4 injectors was assigned one treatmentrgroup. The three birds numbered 1 within each treatment-group were injected subcutaneously within a 2 minute interval giving a total of 12 birds (4 injectors X 3 birds) injected in the first two minutes. An additional 12 birds were injected in each subsequent 2 minute interval until all 120 birds in the first trial were injected. Another group of 8 workers bled the birds via the right jugular vein at the appropriate intervals after injection. Simultaneous injections and blood collections were used to minimize any effects of time of day on plasma magnesium levels. Two hours after the beginning of the first trial, the second trial was begun using the same procedures. Blood samples were centrifuged at room temperature. Plasma was collected and stored at — 15°C. Magnesium levels were determined by atomic absorption spectrophotometry using a Jarrel-Ash apparatus, model 82-362. The data were analyzed by analysis of variance and differences between treatment means were evaluated by the least significant difference (LSD) test. RESULTS AND DISCUSSION
The effects of UB and thyroid extracts on plasma magnesium levels are presented in Table 1. Since no difference between trials was found by analysis of variance, the trials were combined for the LSD test. Plasma magnesium levels did not differ significantly among the four groups at 60 minutes postinjection. Chicks
Downloaded from http://ps.oxfordjournals.org/ by guest on May 16, 2015
al. (1966) found variable responses of plasma magnesium levels to porcine calcitonin in the dog. Recently, Palmeri et al. (1969), using a more sensitive assay for calcitonin, demonstrated a slight but significant hypomagnesemic effect of calcitonin when it was administered to calcium-deficient rats. In mammals, there is an intimate spatial relationship between cells of the thyroid gland which produce calcitonin (parafollicular " C " cells) and those which produce thyroxine (follicle cells). This relationship leads to difficulties in attempting to delineate the relative importance of thyroxine and calcitonin in magnesium homeostasis. The sources of thyroxine (thyroid gland) and calcitonin (ultimobranchial gland) are separate structural entities in birds. Therefore, birds appeared to lend themselves ideally to the study of this problem. Preliminary data from this laboratory suggested that the ultimobranchial gland of the domestic fowl may play a role in magnesium homeostasis in the young bird (Lloyd eial., 1969).
447
448
J. W. LLOYD AND W. E. COLLINS
TABLE 1.—The effects of idlimobranchial and thyroid extracts on plasma magnesium levels (mg./lOO ml.) of eight-day-old chicks" Treatment Salineb Thyroid 0 ,d 1/2 UBG 1 UBGe
No. chicks per time interval 20 20 20 20
Minutes postinfection 30
60
3.1±0.2 ff 3.1±0.1 £ 3.2±0.1 3.2±0.2' 3.1±0.1 f 2.9±0.1»f 2.7±0.ie 2 . 9 + 0 . l
UB gland may play a role in magnesium homeostasis in the young chick. ACKNOWLEDGMENT
120 3.3±0.1 £f 2.9+0.2 2.4±0.2« 2.4±0.1«
a b c
Mean ± standard error. 0.25 ml. of 0.9% saline at pH 4. The amount of thyroid tissue equivalent to the average weight 1 ultimobranchial gland (3 mg. dry weight) in 0.25 ml. saline at pH 4. *> 1/2 Gland-equivalent (1.5 mg. dry weight) in 0.25 ml. saline at pH 4. e One gland-equivalent (3 mg. dry weight) in 0.25 ml. saline at pH 4. {,s Values in vertical column with the same superscript are not statistically different, p<0.05.
A hypomagnesemic effect of thyroid extracts was not found during the times of sampling. A trend toward a lowering of the plasma magnesium levels with thyroid extracts is suggested at 120 minutes postinjection, but this was not significant. The data demonstrate that UB extract injected into young chicks lowers plasma magnesium levels and suggest that the
REFERENCES Arkawa, J., 1960. Effect of thyroxine and propylthiouracil on Mg metabolism in the rabbit. Proc. Soc. Exp. Biol. Med. 104:594-597. Bell, N. H., R. J. Bennett and R. Patterson, 1966. Effects of porcine thyrocalcitonin on serum calcium, phosphorus and magnesium in the monkey and in man. Proc. Soc. Exp. Biol. Med. 123: 114. Care, A. D., 1965. Secretion of thyrocalcitonin. Nature, 205: 1289. Chausmer, A., R. Mittleman and S. Wallace, 1966. Studies of thyrocalcitonin action. Endocrinology, 29: 131-136. Copp, D. H., D. W. Cockcroft, K. Kueh and A. Yankoon, 1967. Calcitonin from ultimobranchial glands of dogfish and chickens. Sicence, 158: 196-197. Foster, G. V., A. Baghdiantz, M. A. Kumer, E. Slack, H. A. Soliman and I. Maclntyre, 1964. Thyroid origin of thyrocalcitonin. Nature, 202: 1303. Foster, G. V., F. F. Joplin, I. Maclntyre, K. E. W. Melvin and E. Slack, 1966. Effects of thyrocalcitonin in man. Lancet, 1: 107. Inskeep, E. K., and A. D. Kenny, 1968. Calcium homeostasis in thyroidectomized sheep. Endocrinology, 83: 183-185. Jones, J. E., P. C. Desper, S. R. Shane and E. B. Flink, 1966. Magnesium metabolism in hyperthyroidism and hypothyroidism. J. Clin. Invest. 45: 891. Lloyd, J. W., R. A. Peterson and W. E. Collins, 1969. Effects of ultimobranchial gland extract in the chicken. Poultry Sci. 48: 1835. Palmeri, G. M. A., J. S. Thompson and L. P. Eliel, 1969. Hypomagnesemic effect of thyrocalcitonin. Endocrinology, 84:1509.
Downloaded from http://ps.oxfordjournals.org/ by guest on May 16, 2015
treated with both 1/2 and 1 gland-equivalents of UB extract had lower (p<0.01) plasma magnesium levels at 120 minutes postinjection when compared to either the group injected with saline or thyroid extract. These data are in agreement with previous observations (Lloyd et al., 1969) in which 1/2 gland-equivalent of UB extract significantly lowered plasma magnesium levels in 7-day-old Leghorn cockerels at 120 minutes postinjection as compared to saline-injected controls. In the present study 1/2 gland-equivalent of UB extract appeared to elicit a similar degree of hypomagnesemia at 120 minutes. However, the response varied with dose level at 30 minutes and neither dose level produced an effect at 60 minutes. These phases of the response cannot be explained.
The authors thank Drs. R. A. Peterson, E. K. Inskeep and R. L. Butcher and the students in the Reproductive Physiology Graduate Program for their assistance in the experiment. This investigation was supported in part by the American Cancer Society Institutional Research Grant, No. 76G.