Diminished Prepartal Plasma Calcitonin Concentration in Cows Developing Parturient Hypocalcemia1 G. P. MAYER,2 J. W. BLUM,3 AND L. J. DEFTOS 4 University of Pennsylvania, School of Veterinary Medicine, New Bolton Center, Kennett Square, Pennsylvania 19348 and Endocrine Umt, Massachusetts General Hospital, Boston, Massachusetts 02114 severe hypocalcemia in the parturient paresis and nonparetic hypocalcernic groups was not accompanied by an increase in plasma calcitonin concentration. Furthermore, plasma calcitonin concentration of these 2 groups was less than that of control cows during the parturient period as well as during the month before and the month after parturition. The plasma calcium nadir at parturition was positively related to the mean prepartal (encompassing the period from 30 days until 60 h before parturition) plasma calcitonin concentration (r = 0.57, t = 3.14, P < 0.005); i.e., the lower the prepartal plasma calcitonin concentration the more severe the hypocalcemia at parturition. These observations suggest that the development of hypocalcemia at parturition is not due to an increased secretion of calcitonin, but instead they suggest that parturient hypocalcemia may be associated with a diminished prepartal secretion of calcitonin. (Endocrinology 96: 1478, 1975)
ABSTRACT. Immunoreactive calcitonin and calcium concentrations were determined on 581 plasma samples collected during 23 studies on 20 cows. Sample collection in each study was begun approximately 1 month prior to parturition and continued for about 1 month after parturition. The cows were grouped according to the degree of hypocalcemia encountered at parturition. The parturient paresis group consisted of 10 cows which developed severe hypocalcemia (3.91 ± 0.22 mg/100 ml, mean ± SE) accompanied by paresis; the nonparetic hypocal-
cemic group consisted of 5 cows which developed severe hypocalcemia (5.70 ± 0.30 mg/100 ml) but not paresis; and the control group consisted of 8 cows which experienced only a mild hypocalcemia (8.50 ± 0.27 mg/100 ml) at parturition. In the prepartal period prior to the onset of hypocalcemia, the respective mean plasma calcium concentrations (± SE) of the 3 groups were 10.1 ± 0.11, 9.95 ± 0.20, and 10.2 ± 0.17 mg/100 ml. The development of
C
APEN and Young (1) first proposed an etiological role in parturient paresis for the hypocalcemic hormone, calcitonin. They found a diminished calcitonin content in thyroid glands of cows with parturient paresis and suggested that an abrupt release of calcitonin near parturition may be related to the development of the hypocalcemia of the syndrome. Bailet induced hypocalcemia in young (2) and mature cows (3) by intravenous administration Received November 4, 1974. 1 Supported in part by NIH research grant No. AM-16, 983 from NIAMDD and USPHS Research Career Development Award No. AM-14874. 2 Present address: Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma, 74074. Send requests for reprints to G. P. Mayer at this address. 3 President address: Institute for Physiology and Hygiene of Farm Animals, Federal Institute of Technology, Universitatstrasse 2, 8006 Zurich, Switzerland. 4 Present address: Veterans Administration Hospital, 3350 La Jolla Village Drive, La Jolla, California 92161.
of porcine calcitonin and also concluded that calcitonin release contributed to the hypocalcemia of parturient paresis. Littledike et al. measured plasma calcitonin concentration by radiommunoassay in cows before parturition and reported in an abstract of their work (4) that, "The degree of severity of the hypocalcemia was often associated with extended periods of high CT prior to parturition." We have measured plasma calcitonin concentration in cows before, during, and after parturition. Our results do not appear to substantiate the concept that parturient hypocalcemia is due to an increased secretion of calcitonin. On the contrary, our data suggest that parturient hypocalcemia may be preceded by a diminished secretion of calcitonin. Materials and Methods Experimental Studies Twenty-three studies were performed on 20 Jersey cows from the same herd. Three cows
1478
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CALCITONIN AND PARTURIENT HYPOCALCEMIA were studied during 2 consecutive parturitions. A total of 581 samples were collected for determination of plasma calcitonin and calcium concentrations. Heparinized jugular blood (100 IU Heparin/30 ml blood) was collected at 2-3-day intervals during the 3-4 weeks before the expected date of parturition. When parturition was only a few days away, blood sampling was more frequent, especially during the onset of hypocalcemia and after intravenous treatment with calcium. Hypocalcemia in cows was allowed to progress until the onset of paresis. Paretic cows were treated by intravenous administration of 500 ml of a 26% solution of calcium borogluconate during an interval of 10 min. After treatment blood sampling was continued for another 3 to 4 weeks. Due to the lability of calcitonin in plasma (5) all samples were handled in a manner designed to minimize degradation prior to analysis. Reduction of sample temperature to 4 C retards but does not completely inhibit degradation (5); therefore, blood samples were immersed in an ice bath immediately after collection and kept on ice until centrifugation was carred out at 4 C. To minimize the effect of slow degradation at 4 C on plasma hormone concentration, we constrained the interval of time between blood collection and freezing to a maximum of 1 h. Degradation of calcitonin during storage at —20 C was assessed at various intervals after freezing by assaying freshly thawed aliquots of a batch of plasma collected from cow 071 during hypercalcemia. Loss of immunoreactivity of this plasma was not observed during storage for a period of 19 months following collection. Since the longest interval between sample collection and assay in these studies was 19 months (samples were collected between Sept. 1969 and April 1971 and assays were done between February and July of 1971), it seems unlikely that degradation during storage can account for differences in plasma calcitonin concentration observed in our studies. Laboratory procedures Plasma calcitonin concentrations were determined by radioimmunoassay (6). The antiserum (B-l-1) used in the assay was obtained by immunizing a guinea pig with bovine calcitonin. A highly purified preparation of bovine calcitonin was used for standards but ovine calcitonin was used for preparation of 125I-
1479
labelled hormone since it could be radioiodinated to a higher specific activity and was immunologically indistinguishable from the bovine hormone (6). Hormone was labelled with radioactive iodine using principally the chloramine-T oxidation method reported by Hunter and Greenwood (7). Purification of labelled hormone was achieved by adsorption on finely powdered silica (Quso obtained from Philadelphia Quartz Co., Philadelphia) followed by washing and subsequent elution with a solution containing 20% acetone and 1% acetic acid (8). Further purification prior to use in the assay was provided by filtration on a column of polyacrylamide gel (Bio-gel, P-10 obtained from Bio Rad Laboratories, Richmond, California). Incubations of hormone standards or unknowns with labelled hormone and antiserum were carred out at 4 C in a diluent containing 7 parts 0.05M sodium phosphate buffer (pH 6.0) and 1 part human plasma obtained from outdated blood at a hospital blood bank. The phosphate buffer also contained 0.005% merthiolate and 0.001M disodium ethylenediamine tetraacetate. Improved sensitivity was achieved by preincubation of hormone standards and unknown samples with antiserum for 48 to 72 h prior to the addition of labelled hormone. Following the addition of tracer, incubation was continued for an additional 48 to 72 h after which labelled hormone was partitioned into bound (combined with antibody) and free (not combined with antibody) fractions by the addition of dioxane (9). Each fraction was counted in a well type gamma scintillation counter to a preset count of 4000. When incubations were conducted in the absence of antiserum, a portion of the labelled hormone behaved as though it were bound to antibody in the process of phase separation. Accordingly, a correction factor (9) was derived from results obtained by incubating corresponding aliquots of each standard and each sample in the absence of antiserum. At least 2 and sometimes 3 aliquots of each freshly thawed sample were assayed in duplicate so that the concentration reported for a sample is the mean of 4 or 6 determinations. Additional standardization was provided by periodically assaying a freshly thawed aliquot of a batch of plasma obtained from a hypercalcemic cow (cow 071) along with samples from the other cows. Plasma calcium concentrations were determined by atomic absorption sepctrophotometry (10).
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1480
Endo • 1975 Vol 96 • No 6
MAYER, BLUM AND DEFTOS TABLE 1.
Plasma calcium and ca!'.citonin concentrationsi of cows before and after parturition
No. of cows
Range of Ca nadirs at parturition mg/100 ml
Controls
8
Nonparetic hypocalcemic
Group
Parturient paresis
Plasma CT, pg/ml (mean ± SE)
Plasma Ca, mg/100 ml (mean ± SE)
Prepartum8
Postpartum6
Prepartum11
Postpartumb
7.10 to 9.70
341 ± 30 (45)
368 ± 17 (64)
10.2 ± 0.17 (45)
9.96 ± 0.13 (64)
5
4.69 to 6.39
239 ± 18 (25)
276 ± 16 (33)
9.95 ± 0.20 (24)
9.71 ±0.11 (33)
10
3.23 to 5.J52
185 ± 13 (64)
198 ± 10 (85)
10.1 ± 0.11 (63)
10.0 ± 0.09 (83)
a Includes all samples collected during interval from 30 days to 60 h prior to parturition. Number of samples is indicated in parenthesis. b Includes all samples collected between 48 h and 30 days after parturition. Number of samples is indicated in parenthesis.
Results
Parturition was associated with varying degrees of hypocalcemia and the cows were grouped accordingly (Table 1). The decline in plasma calcium concentration usually began in the 24 h preceding parturition (Fig. 1) and in most cases returned to the normal range within 72 h following parturition. The nadir of plasma calcium concentration at parturition was determined in each cow from blood sam-
ples collected at least every 8 h during the parturient period. In 8 of the cows, plasma calcium concentration did not decline below 7 mg/100 ml. These cows did not exhibit any clinical signs suggestive of hypocalcemia. This degree of hypocalcemia during the 3rd and subsequent parturitions is generally accepted as normal (11); therefore, these cows were used as controls. The parturient paresis group was comprised of 10 cows which developed
20
1800 Cow 099
18
1600
16
1400 1200 »
. 12
1000
Calcium 3
i2 10
800 >—Q
w
i»
ABSTRACT. Immunoreactive calcitonin and calcium concentrations were determined on 581 plasma samples collected during 23 studies on 20 cows. Sample collection in each study was begun approximately 1 month prior to parturition and continued for about 1 month after parturition. The cows were grouped according to the degree of hypocalcemia encountered at parturition. The parturient paresis group consisted of 10 cows which developed severe hypocalcemia (3.91 ± 0.22 mg/100 ml, mean ± SE) accompanied by paresis; the nonparetic hypocal-
cemic group consisted of 5 cows which developed severe hypocalcemia (5.70 ± 0.30 mg/100 ml) but not paresis; and the control group consisted of 8 cows which experienced only a mild hypocalcemia (8.50 ± 0.27 mg/100 ml) at parturition. In the prepartal period prior to the onset of hypocalcemia, the respective mean plasma calcium concentrations (± SE) of the 3 groups were 10.1 ± 0.11, 9.95 ± 0.20, and 10.2 ± 0.17 mg/100 ml. The development of
C
APEN and Young (1) first proposed an etiological role in parturient paresis for the hypocalcemic hormone, calcitonin. They found a diminished calcitonin content in thyroid glands of cows with parturient paresis and suggested that an abrupt release of calcitonin near parturition may be related to the development of the hypocalcemia of the syndrome. Bailet induced hypocalcemia in young (2) and mature cows (3) by intravenous administration Received November 4, 1974. 1 Supported in part by NIH research grant No. AM-16, 983 from NIAMDD and USPHS Research Career Development Award No. AM-14874. 2 Present address: Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma, 74074. Send requests for reprints to G. P. Mayer at this address. 3 President address: Institute for Physiology and Hygiene of Farm Animals, Federal Institute of Technology, Universitatstrasse 2, 8006 Zurich, Switzerland. 4 Present address: Veterans Administration Hospital, 3350 La Jolla Village Drive, La Jolla, California 92161.
of porcine calcitonin and also concluded that calcitonin release contributed to the hypocalcemia of parturient paresis. Littledike et al. measured plasma calcitonin concentration by radiommunoassay in cows before parturition and reported in an abstract of their work (4) that, "The degree of severity of the hypocalcemia was often associated with extended periods of high CT prior to parturition." We have measured plasma calcitonin concentration in cows before, during, and after parturition. Our results do not appear to substantiate the concept that parturient hypocalcemia is due to an increased secretion of calcitonin. On the contrary, our data suggest that parturient hypocalcemia may be preceded by a diminished secretion of calcitonin. Materials and Methods Experimental Studies Twenty-three studies were performed on 20 Jersey cows from the same herd. Three cows
1478
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CALCITONIN AND PARTURIENT HYPOCALCEMIA were studied during 2 consecutive parturitions. A total of 581 samples were collected for determination of plasma calcitonin and calcium concentrations. Heparinized jugular blood (100 IU Heparin/30 ml blood) was collected at 2-3-day intervals during the 3-4 weeks before the expected date of parturition. When parturition was only a few days away, blood sampling was more frequent, especially during the onset of hypocalcemia and after intravenous treatment with calcium. Hypocalcemia in cows was allowed to progress until the onset of paresis. Paretic cows were treated by intravenous administration of 500 ml of a 26% solution of calcium borogluconate during an interval of 10 min. After treatment blood sampling was continued for another 3 to 4 weeks. Due to the lability of calcitonin in plasma (5) all samples were handled in a manner designed to minimize degradation prior to analysis. Reduction of sample temperature to 4 C retards but does not completely inhibit degradation (5); therefore, blood samples were immersed in an ice bath immediately after collection and kept on ice until centrifugation was carred out at 4 C. To minimize the effect of slow degradation at 4 C on plasma hormone concentration, we constrained the interval of time between blood collection and freezing to a maximum of 1 h. Degradation of calcitonin during storage at —20 C was assessed at various intervals after freezing by assaying freshly thawed aliquots of a batch of plasma collected from cow 071 during hypercalcemia. Loss of immunoreactivity of this plasma was not observed during storage for a period of 19 months following collection. Since the longest interval between sample collection and assay in these studies was 19 months (samples were collected between Sept. 1969 and April 1971 and assays were done between February and July of 1971), it seems unlikely that degradation during storage can account for differences in plasma calcitonin concentration observed in our studies. Laboratory procedures Plasma calcitonin concentrations were determined by radioimmunoassay (6). The antiserum (B-l-1) used in the assay was obtained by immunizing a guinea pig with bovine calcitonin. A highly purified preparation of bovine calcitonin was used for standards but ovine calcitonin was used for preparation of 125I-
1479
labelled hormone since it could be radioiodinated to a higher specific activity and was immunologically indistinguishable from the bovine hormone (6). Hormone was labelled with radioactive iodine using principally the chloramine-T oxidation method reported by Hunter and Greenwood (7). Purification of labelled hormone was achieved by adsorption on finely powdered silica (Quso obtained from Philadelphia Quartz Co., Philadelphia) followed by washing and subsequent elution with a solution containing 20% acetone and 1% acetic acid (8). Further purification prior to use in the assay was provided by filtration on a column of polyacrylamide gel (Bio-gel, P-10 obtained from Bio Rad Laboratories, Richmond, California). Incubations of hormone standards or unknowns with labelled hormone and antiserum were carred out at 4 C in a diluent containing 7 parts 0.05M sodium phosphate buffer (pH 6.0) and 1 part human plasma obtained from outdated blood at a hospital blood bank. The phosphate buffer also contained 0.005% merthiolate and 0.001M disodium ethylenediamine tetraacetate. Improved sensitivity was achieved by preincubation of hormone standards and unknown samples with antiserum for 48 to 72 h prior to the addition of labelled hormone. Following the addition of tracer, incubation was continued for an additional 48 to 72 h after which labelled hormone was partitioned into bound (combined with antibody) and free (not combined with antibody) fractions by the addition of dioxane (9). Each fraction was counted in a well type gamma scintillation counter to a preset count of 4000. When incubations were conducted in the absence of antiserum, a portion of the labelled hormone behaved as though it were bound to antibody in the process of phase separation. Accordingly, a correction factor (9) was derived from results obtained by incubating corresponding aliquots of each standard and each sample in the absence of antiserum. At least 2 and sometimes 3 aliquots of each freshly thawed sample were assayed in duplicate so that the concentration reported for a sample is the mean of 4 or 6 determinations. Additional standardization was provided by periodically assaying a freshly thawed aliquot of a batch of plasma obtained from a hypercalcemic cow (cow 071) along with samples from the other cows. Plasma calcium concentrations were determined by atomic absorption sepctrophotometry (10).
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1480
Endo • 1975 Vol 96 • No 6
MAYER, BLUM AND DEFTOS TABLE 1.
Plasma calcium and ca!'.citonin concentrationsi of cows before and after parturition
No. of cows
Range of Ca nadirs at parturition mg/100 ml
Controls
8
Nonparetic hypocalcemic
Group
Parturient paresis
Plasma CT, pg/ml (mean ± SE)
Plasma Ca, mg/100 ml (mean ± SE)
Prepartum8
Postpartum6
Prepartum11
Postpartumb
7.10 to 9.70
341 ± 30 (45)
368 ± 17 (64)
10.2 ± 0.17 (45)
9.96 ± 0.13 (64)
5
4.69 to 6.39
239 ± 18 (25)
276 ± 16 (33)
9.95 ± 0.20 (24)
9.71 ±0.11 (33)
10
3.23 to 5.J52
185 ± 13 (64)
198 ± 10 (85)
10.1 ± 0.11 (63)
10.0 ± 0.09 (83)
a Includes all samples collected during interval from 30 days to 60 h prior to parturition. Number of samples is indicated in parenthesis. b Includes all samples collected between 48 h and 30 days after parturition. Number of samples is indicated in parenthesis.
Results
Parturition was associated with varying degrees of hypocalcemia and the cows were grouped accordingly (Table 1). The decline in plasma calcium concentration usually began in the 24 h preceding parturition (Fig. 1) and in most cases returned to the normal range within 72 h following parturition. The nadir of plasma calcium concentration at parturition was determined in each cow from blood sam-
ples collected at least every 8 h during the parturient period. In 8 of the cows, plasma calcium concentration did not decline below 7 mg/100 ml. These cows did not exhibit any clinical signs suggestive of hypocalcemia. This degree of hypocalcemia during the 3rd and subsequent parturitions is generally accepted as normal (11); therefore, these cows were used as controls. The parturient paresis group was comprised of 10 cows which developed
20
1800 Cow 099
18
1600
16
1400 1200 »
. 12
1000
Calcium 3
i2 10
800 >—Q
w
i»
